Split (1)

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https://openalex.org/W2212693758	https://europepmc.org/articles/pmc4695491?pdf=render	Latin	null	Parametrized inequality of Hermite-Hadamard type for functions whose third derivative absolute values are quasi-convex	SpringerPlus	2,015	cc-by	4,410	© 2015 Wu et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. RESEARCH Open Access Abstract In this paper we present some inequalities of Hermite-Hadamard type for functions whose third derivative absolute values are quasi-convex. Moreover, an application to special means of real numbers is also considered. Keywords: Hermite-Hadamard type inequality, Parameter, Quasi-convex, Special means Mathematics Subject Classification: 26D10, 26A51, 26E60 Parametrized inequality of Hermite‑Hadamard type for functions whose third derivative absolute values are quasi‑convex Shan‑He Wu1, Banyat Sroysang2, Jin‑Shan Xie1 and Yu‑Ming Chu3* Correspondence: chuyuming@aliyun.com; chuyuming2005@126.com 3 School of Mathematics and Computation Science, Hunan City University, Yiyang 413000, Hunan, People’s Republic of China Full list of author information is available at the end of the article Wu et al. SpringerPlus (2015) 4:831 DOI 10.1186/s40064-015-1633-z for all a, b ∈I. A function f : I ⊆R →R is said to be quasi-convex on I, if Correspondence: chuyuming@aliyun.com; chuyuming2005@126.com 3 School of Mathematics and Computation Science, Hunan City University, Yiyang 413000, Hunan, People’s Republic of China Full list of author information is available at the end of the article A function f : I ⊆R →R is said to be quasi-convex on I, if f (x + (1 −)y) ≤max f (x), f (y) f (x + (1 −)y) ≤max f (x), f (y) f (x + (1 −)y) ≤max f (x), f (y) Background A real-valued function f defined on an interval I ⊆R is said to be convex on I, if f (x + (1 −)y) ≤f (x) + (1 −)f (y) f (x + (1 −)y) ≤f (x) + (1 −)f (y) for all x, y ∈I and ∈[0, 1]. for all x, y ∈I and ∈[0, 1]. for all x, y ∈I and ∈[0, 1]. If f is convex on I, then we have the Hermite-Hadamard inequality (see Mitrinović et al. 1993) (1) f a + b 2 ≤ 1 b −a b a f (x)dx ≤f (a) + f (b) 2 (1) all a, b ∈I. for all a, b ∈I. for all a, b ∈I. for all x, y ∈I and ∈[0, 1]. for all x, y ∈I and ∈[0, 1]. Clearly, any convex function is a quasi-convex function. Furthermore, there exist quasi-convex functions which are not convex. In 2007, Ion (2007) presented an inequality of Hermite-Hadamard type for functions whose derivatives in absolute values are quasi-convex functions, as follows: Page 2 of 9 SpringerPlus (2015) 4:831 Page 2 of 9 Wu et al. SpringerPlus (2015) 4:831 Theorem 1.1 Let f : I ⊂R →R be a differentiable mapping on Io, a, b ∈I with a < b. If f ′ is quasi-convex on [a, b], then the following inequality holds: Theorem 1.1 Let f : I ⊂R →R be a differentiable mapping on Io, a, b ∈I with a < b. If f ′ is quasi-convex on [a, b], then the following inequality holds: Theorem 1.1 Let f : I ⊂R →R be a differentiable mapping on Io, a, b ∈I with a < b. If f ′ is quasi-convex on [a, b], then the following inequality holds: (2) f (a) + f (b) 2 − 1 b −a b a f (x)dx ≤b −a 4 max f ′(a) , f ′(b) . (2) In 2010, Alomari et al. (2010a) established an analogous version of inequality (1), which is asserted by Theorem 1.2 below: Theorem 1.2 Let f : I ⊂R →R be twice differentiable mapping on Io, a, b ∈I with a < b and f ′′ is integrable on [a, b]. If f ′′ is quasi-convex on [a, b], then the following inequality holds: (3) f (a) + f (b) 2 − 1 b −a b a f (x)dx ≤(b −a)2 12 max f ′′(a) , f ′′(b) . (3) Recently, Guo et al. (2015) investigated Hermite-Hadamard type inequalities for geo- metrically quasi-convex functions. Xi and Qi (2014, 2015) and Xi et al. (2012, 2014) showed some new Hermite-Hadamard type inequalities for s-convex functions. For more results relating to refinements, counterparts, generalizations of Hadamard type inequalities, we refer interested readers to Alomari et al. (2010b), Chen (2015), Niculescu and Persson (2006), Pečarić et al. (1992), Sroysang (2014), Sroysang (2013) and Wu (2009). The main purpose of this paper is to present a parametrized inequality of Hermite- Hadamard type for functions whose third derivative absolute values are quasi-convex. As applications, some new inequalities for special means of real numbers are established. Lemmas In order to prove our main results, we need the following lemmas. Lemma 2.1 Let ǫ ∈R and let f: I ⊂R →R be three times differentiable on I◦ and a, b ∈I with a < b. Assume that f ′′′ is integrable on [a, b]. Then (4 −ǫ)f (a) + (2 + ǫ)f (b) 6 − 1 b −a b a f (x)dx −b −a 12 ǫf ′(b) −(2 −ǫ)f ′(a) = (b −a)3 12 1 0 (1 −)(2 −ǫ)f ′′′(a + (1 −)b)d. Wu et al. Lemmas SpringerPlus (2015) 4:831 Page 3 of 9 Proof Integrating by parts, we have Proof Integrating by parts, we have Proof Integrating by parts, we have Proof Integrating by parts, we have roof Integrating by parts, we have 1 0 (1 −)(2 −ǫ)f ′′′(a + (1 −)b)d = 1 a −b =1 =0 (1 −)(2 −ǫ)df ′′(a + (1 −)b) = 1 a −b (1 −)(2 −ǫ)f ′′(a + (1 −)b) =1 =0 − 1 a −b =1 =0 f ′′(a + (1 −)b)d((1 −)(2 −ǫ)) = − 1 a −b =1 =0 f ′′(a + (1 −)b)d((1 −)(2 −ǫ)) = 1 b −a =1 =0 −62 + 2(2 + ǫ) −ǫ f ′′(a + (1 −)b)d = 1 (b −a)2 =1 =0 62 −2(2 + ǫ) + ǫ df ′(a + (1 −)b) = 1 (b −a)2 62 −2(2 + ǫ) + ǫ f ′(a + (1 −)b) =1 =0 − 1 (b −a)2 =1 =0 f ′(a + (1 −)b)d 62 −2(2 + ǫ) + ǫ = −1 (b −a)2 ǫf ′(b) −(2 −ǫ)f ′(a) − 1 (b −a)2 =1 =0 (12 −2(2 + ǫ))f ′(a + (1 −)b)d = −1 (b −a)2 ǫf ′(b) −(2 −ǫ)f ′(a) + 2 (b −a)3 =1 =0 (6 −2 −ǫ)df (a + (1 −)b) = −1 (b −a)2 ǫf ′(b) −(2 −ǫ)f ′(a) + 2 (b −a)3 [(6 −2 −ǫ)f (a + (1 −)b)]=1 =0 − 2 (b −a)3 =1 =0 f (a + (1 −)b)d(6 −2 −ǫ) = −1 (b −a)2 ǫf ′(b) −(2 −ǫ)f ′(a) + 2 (b −a)3 (4 −ǫ)f (a) + (2 + ǫ)f (b) − 12 (b −a)3 =1 =0 f (a + (1 −)b)d. Lemmas 1 0 (1 −)(2 −ǫ)f ′′′(a + (1 −)b)d = 1 a −b =1 =0 (1 −)(2 −ǫ)df ′′(a + (1 −)b) = 1 a −b (1 −)(2 −ǫ)f ′′(a + (1 −)b) =1 =0 − 1 a −b =1 =0 f ′′(a + (1 −)b)d((1 −)(2 −ǫ)) = − 1 a −b =1 =0 f ′′(a + (1 −)b)d((1 −)(2 −ǫ)) = 1 b −a =1 =0 −62 + 2(2 + ǫ) −ǫ f ′′(a + (1 −)b)d = 1 (b −a)2 =1 =0 62 −2(2 + ǫ) + ǫ df ′(a + (1 −)b) = 1 (b −a)2 62 −2(2 + ǫ) + ǫ f ′(a + (1 −)b) =1 =0 − 1 (b −a)2 =1 =0 f ′(a + (1 −)b)d 62 −2(2 + ǫ) + ǫ = −1 b 2 ǫf ′(b) −(2 −ǫ)f ′(a) Page 4 of 9 Wu et al. SpringerPlus (2015) 4:831 Changing variable x = a + (1 −)b, it follows that Changing variable x = a + (1 −)b, it follows that 1 0 (1 −)(2 −ǫ)f ′′′(a + (1 −)b)d = −1 (b −a)2 ǫf ′(b) −(2 −ǫ)f ′(a) + 2 (b −a)3 (4 −ǫ)f (a) + (2 + ǫ)f (b) − 12 (b −a)4 x=b x=a f (x)dx. Thus, (b −a)3 12 1 0 (1 −)(2 −ǫ)f ′′′(a + (1 −)b)d = (4 −ǫ)f (a) + (2 + ǫ)f (b) 6 − 1 b −a b a f (x)dx −b −a 12 ǫf ′(b) −(2 −ǫ)f ′(a) . The proof of Lemma 2.1 is completed. □ The proof of Lemma 2.1 is completed. □ □ The proof of Lemma 2.1 is completed. □ Lemma 2.2 Let ǫ be a real number. Then Lemma 2.2 Let ǫ be a real number. Then Lemma 2.2 Let ǫ be a real number. Then Lemma 2.2 Let ǫ be a real number. Lemmas Then 1 0 (1 −)\|2 −ǫ\|d =            ǫ −1 6 if ǫ ≥2 4ǫ3 −ǫ4 48 + 1 −ǫ 6 if 0 < ǫ < 2 1 −ǫ 6 if ǫ ≤0. Proof We distinguish three cases Proof We distinguish three cases Proof We distinguish three cases Case 1 If ǫ ≥2, then 1 0 (1 −)\|2 −ǫ\|d = 1 0 (1 −)(ǫ −2)d = ǫ −1 6 . Case 2 If 0 < ǫ < 2, then Case 2 If 0 < ǫ < 2, then 1 0 (1 −)\|2 −ǫ\|d = ǫ/2 0 (1 −)(ǫ −2)d + 1 ǫ/2 (1 −)(2 −ǫ)d = 4ǫ3 −ǫ4 48 + 1 −ǫ 6 . Case 3 If ǫ ≤0, then Case 3 If ǫ ≤0, then 1 0 (1 −)\|2 −ǫ\|d = 1 0 (1 −)(2 −ǫ)d = 1 −ǫ 6 . Case 3 If ǫ ≤0, then 1 0 (1 −)\|2 −ǫ\|d = 1 0 (1 −)(2 −ǫ)d = 1 −ǫ 6 . the proof of Lemma 2.2. □ This completes the proof of Lemma 2.2. □ This completes the proof of Lemma 2.2. □ This completes the proof of Lemma 2.2. Page 5 of 9 Wu et al. SpringerPlus (2015) 4:831 Main results Our main results are stated in the following theorems. Our main results are stated in the following theorems. Theorem 3.1 Let q ≥1 and ǫ ∈R, and let f : I ⊂R →R be three times differentiable on I◦ and a, b ∈I with a < b. Assume that f ′′′ is integrable on [a, b], and f ′′′q is quasi- convex on [a, b]. Then (4 −ǫ)f (a) + (2 + ǫ)f (b) 6 − 1 b −a b a f (x)dx −b −a 12 ǫf ′(b) −(2 −ǫ)f ′(a) ≤                (b −a)3 12 ǫ −1 6 max f ′′′(a) q, f ′′′(b) q1/q if ǫ ≥2 (b −a)3 12 4ǫ3 −ǫ4 48 + 1 −ǫ 6 max f ′′′(a) q, f ′′′(b) q1/q if 0 < ǫ < 2 (b −a)3 12 1 −ǫ 6 max f ′′′(a) q, f ′′′(b) q1/q if ǫ ≤0. if ǫ ≥2 Proof Using Lemma 2.1 and Hölder’s inequality gives Proof Using Lemma 2.1 and Hölder’s inequality gives Proof Using Lemma 2.1 and Hölder’s inequality gives Proof Using Lemma 2.1 and Hölder’s inequality gives (4 −ǫ)f (a) + (2 + ǫ)f (b) 6 − 1 b −a b a f (x)dx −b −a 12 ǫf ′(b) −(2 −ǫ)f ′(a) ≤(b −a)3 12 1 0 (1 −)\|2 −ǫ\| f ′′′(a + (1 −)b) d = (b −a)3 12 1 0 ((1 −)\|2 −ǫ\|)1−1/q (1 )\|2 \| f ′′′( + (1 )b) q1/qd (4 −ǫ)f (a) + (2 + ǫ)f (b) 6 − 1 b −a b a f (x)dx −b −a 12 ǫf ′(b) −(2 −ǫ)f ′(a) (4 −ǫ)f (a) + (2 + ǫ)f (b) 6 − 1 b −a b a f (x)dx −b −a 12 ǫf ′(b) −(2 −ǫ)f ′(a ≤(b −a)3 12 1 0 (1 −)\|2 −ǫ\| f ′′′(a + (1 −)b) d = (b −a)3 12 1 0 ((1 −)\|2 −ǫ\|)1−1/q × (1 −)\|2 −ǫ\| f ′′′(a + (1 −)b) q1/qd ≤(b −a)3 12 1 0 (1 −)\|2 −ǫ\|d 1−1/q × 1 0 (1 −)\|2 −ǫ\| f ′′′(a + (1 −)b) qd 1/q . Main results × 1 0 (1 −)\|2 −ǫ\| f ′′′(a + (1 −)b) qd 1/q . By the quasi-convexity of f ′′′q, we obtain By the quasi-convexity of f ′′′q, we obtain (4 −ǫ)f (a) + (2 + ǫ)f (b) 6 − 1 b −a b a f (x)dx −b −a 12 ǫf ′(b) −(2 −ǫ)f ′(a) ≤(b −a)3 12 1 0 (1 −)\|2 −ǫ\|d 1−1/q (4 −ǫ)f (a) + (2 + ǫ)f (b) 6 − 1 b −a b a f (x)dx −b −a 12 ǫf ′(b) −(2 −ǫ)f ′(a) ≤(b −a)3 12 1 0 (1 −)\|2 −ǫ\|d 1−1/q × 1 0 (1 −)\|2 −ǫ\| max f ′′′(a) q, f ′′′(b) q d 1/q = (b −a)3 12 1 0 (1 −)\|2 −ǫ\|d max f ′′′(a) q, f ′′′(b) q1/q. Wu et al. SpringerPlus (2015) 4:831 Page 6 of 9 Page 6 of 9 Utilizing Lemma 2.2 leads to the desired inequality in Theorem 3.1. □ □ Utilizing Lemma 2.2 leads to the desired inequality in Theorem 3.1. □ Remark 3.2 It is worth noticing that if we use a substitution a →b, b →a and ǫ →2 −ǫ in Theorem 3.1, we have the following further generalization of Theorem 3.1. Remark 3.2 It is worth noticing that if we use a substitution a →b, b →a and ǫ →2 −ǫ in Theorem 3.1, we have the following further generalization of Theorem 3.1. Theorem 3.3 Let q ≥1 and ǫ ∈R, and let f : I ⊂R →R be three times differentiable on I◦, a, b ∈I with a = b. Assume that f ′′′ is integrable on [a, b], and f ′′′q is quasi-con- vex on the closed interval formed by the points a and b. Main results Then (4 −ǫ)f (a) + (2 + ǫ)f (b) 6 − 1 b −a b a f (x)dx −b −a 12 ǫf ′(b) −(2 −ǫ)f ′(a) ≤                \|b −a\|3 12 ǫ −1 6 max f ′′′(a) q, f ′′′(b) q1/q if ǫ ≥2 \|b −a\|3 12 4ǫ3 −ǫ4 48 + 1 −ǫ 6 max f ′′′(a) q, f ′′′(b) q1/q if 0 < ǫ < 2 \|b −a\|3 12 1 −ǫ 6 max f ′′′(a) q, f ′′′(b) q1/q if ǫ ≤0. if ǫ ≥2 As a direct consequence, choosing ǫ = 1 in Theorem 3.3, we get the following inequality: Corollary 3.4 Let q ≥1 and ǫ ∈R, and let f : I ⊂R →R be three times differentiable on I◦, a, b ∈I with a = b. Assume that f ′′′ is integrable on [a, b], and f ′′′q is quasi-con- vex on the closed interval formed by the points a and b. Then f (a) + f (b) 2 − 1 b −a b a f (x)dx −b −a 12 f ′(b) −f ′(a) ≤\|b −a\|3 192 max f ′′′(a) q, f ′′′(b) q1/q. In addition, if we utilize Theorem 3.1 with a substitution of ǫ = 0, 0.5, 3, −2, −3, −5, respectively, then we obtain the following results: Corollary 3.5 Let q ≥1 and ǫ ∈R, and let f : I ⊂R →R be three times differentiable on I◦, a, b ∈I with a = b. Assume that f ′′′ is integrable on [a, b], and f ′′′q is quasi-con- vex on the closed interval formed by the points a and b. Main results Then 2f (a) + f (b) 3 − 1 b −a b a f (x)dx + b −a 6 f ′(a) ≤\|b −a\|3 72 max f ′′′(a) q, f ′′′(b) q1/q, 2f (a) + f (b) 3 − 1 b −a b a f (x)dx + b −a 6 f ′(a) ≤\|b −a\|3 72 max f ′′′(a) q, f ′′′(b) q1/q, 7f (a) + 5f (b) 12 − 1 b −a b a f (x)dx −b −a 24 f ′(b) −3f ′(a) ≤71\|b −a\|3 9216 max f ′′′(a) q, f ′′′(b) q1/q, 7f (a) + 5f (b) 12 − 1 b −a b a f (x)dx −b −a 24 f ′(b) −3f ′(a) ≤71\|b −a\|3 9216 max f ′′′(a) q, f ′′′(b) q1/q, Page 7 of 9 Wu et al. SpringerPlus (2015) 4:831 f (a) + 5f (b) 6 − 1 b −a b a f (x)dx −b −a 12 3f ′(b) + f ′(a) ≤\|b −a\|3 36 max f ′′′(a) q, f ′′′(b) q1/q, f (a) − 1 b −a b a f (x)dx + b −a 6 f ′(b) + 2f ′(a) ≤\|b −a\|3 max f ′′′(a) q, f ′′′(b) q1/q, f (a) + 5f (b) 6 − 1 b −a b a f (x)dx −b −a 12 3f ′(b) + f ′(a) ≤\|b −a\|3 36 max f ′′′(a) q, f ′′′(b) q1/q, f (a) − 1 b −a b a f (x)dx + b −a 6 f ′(b) + 2f ′(a) ≤\|b −a\|3 24 max f ′′′(a) q, f ′′′(b) q1/q, 7f (a) −f (b) 6 − 1 b −a b a f (x)dx + b −a 12 3f ′(b) + 5f ′(a) ≤\|b −a\|3 18 max f ′′′(a) q, f ′′′(b) q1/q, 3f (a) −f (b) 2 − 1 b −a b a f (x)dx + b −a 12 5f ′(b) + 7f ′(a) ≤\|b −a\|3 12 max f ′′′(a) q, f ′′′(b) q1/q. Applications to special means We now consider the applications of our results to the special means of real numbers We now consider the applications of our results to the special means of real numbers. The weighted arithmetic mean of real numbers a, b with weight {wa, wb} is defined by A(a, b; wa, wb) = waa + wbb wa + wb , where a, b, wa, wb ∈R with wa + wb = 0. In particularly where a, b, wa, wb ∈R with wa + wb = 0. In particularly where a, b, wa, wb ∈R with wa + wb = 0. In particularly where a, b, wa, wb ∈R with wa + wb = 0. In particularly A(a, b; 1, 1) = A(a, b) = a + b 2 , which is called the arithmetic means. which is called the arithmetic means. The generalized logarithmic mean of real numbers a, b is defined by The generalized logarithmic mean of real numbers a, b is defined by The generalized logarithmic mean of real numbers a, b is defined by Ln(a, b) = bn+1 −an+1 (n + 1)(b −a) 1 n , where a, b, ∈R, n ∈Z with n = 0, −1, a = b. where a, b, ∈R, n ∈Z with n = 0, −1, a = b. Page 8 of 9 Wu et al. SpringerPlus (2015) 4:831 Page 8 of 9 Proposition 4.1 Let a, b, ǫ ∈R, a = b, ǫ = 1 and n ∈N, n ≥3. Then, we have Proposition 4.1 Let a, b, ǫ ∈R, a = b, ǫ = 1 and n ∈N, n ≥3. Then, we have Proposition 4.1 Let a, b, ǫ ∈R, a = b, ǫ = 1 and n ∈N, n ≥3. Then, we have A(an, bn; 4 −ǫ, 2 + ǫ) −(b −a)(ǫ −1)n 6 A(an−1, bn−1; ǫ −2, ǫ) −Ln n(a, b) ≤              \|b −a\|3 72 (ǫ −1)n(n −1)(n −2) max \|a\|n−3, \|b\|n−3 if ǫ ≥2 \|b −a\|3 576 8 −8ǫ + 4ǫ3 −ǫ4 n(n −1)(n −2) max \|a\|n−3, \|b\|n−3 if 0 < ǫ < 2 \|b −a\|3 72 (1 −ǫ)n(n −1)(n −2) max \|a\|n−3, \|b\|n−3 if ǫ ≤0. Proof We consider the function f (x) = xn, x ∈R, n ≥3. Applications to special means It is easy to verify that the function f ′′′(x) = n(n −1)(n −2)xn−3 is quasi-convex on (−∞, +∞) (see Alomari et al. 2010b). The assertion follows from Theorem 3.3 with q = 1. □ Remark 4.2 In a similar way as the proof of the Proposition 4.1, one can easily deduce from Corollary 3.4 the following inequality. Proposition 4.3 Let a, b ∈R, a = b and n ∈N, n ≥3. Then, we have A(an, bn) −(b −a)2 12 n(n −1)Ln−2 n−2(a, b) −Ln n(a, b) ≤\|b −a\|3 192 n(n −1)(n −2) max \|a\|n−3, \|b\|n−3 . Author details 1 1 Department of Mathematics, Longyan University, Longyan 364012, Fujian, People’s Republic of China. 2 Department of Mathematics and Statistics, Faculty of Science and Technology, Thammasat University, Pathumthani 12121, Thailand. 3 School of Mathematics and Computation Science, Hunan City University, Yiyang 413000, Hunan, People’s Republic of China. Acknowledgements Acknowledgements The authors would like to express their hearty thanks to anonymous referees for their valuable comments on this article. This research was supported by the Natural Science Foundation of Fujian province under Grant 2015J05010 and the Out‑ standing Young Incubation Programme and Key Project of Fujian Province Education Department under Grant JA14299. Authors’ contributionsi SW finished the proof and the writing work. BS and JX gave SW some advice on the proof and writing. YC gave SW lots of help in revising the paper. All authors read and approved the final manuscript. Conclusions This paper provides some new results related to the Hermite-Hadamard type inequali- ties. Firstly, we present a parametrized inequality of Hermite-Hadamard type for func- tions whose third derivative absolute values are quasi-convex, the main results are given in Theorems 3.1 and 3.3. As special cases, by assigning special value to the parameter, one can obtain several new and previously known results for Hermite-Hadamard type inequality (Corollaries 3.4 and 3.5). Secondly, as applications of the obtained results, we establish two new inequalities involving special means of real numbers by using the par- ametrized Hermite-Hadamard type inequality (see Propositions 4.1 and 4.3). References Alomari M, Darus M, Dragomir SS (2010) New inequalities of Hermite-Hadamard type for functions whose second deriva tives absolute values are quasi-convex. Tamkang J Math 41(4):353–359 Alomari M, Darus M, Kirmaci US (2010) Refinements of Hadamard-type inequalities for quasi-convex functions with Alomari M, Darus M, Kirmaci US (2010) Refinements of Hadamard-type inequalities for quasi-co applications to trapezoidal formula and to special means Comput Math Appl 59:225 232 Alomari M, Darus M, Kirmaci US (2010) Refinements of Hadamard-type inequalities for quasi-con applications to trapezoidal formula and to special means. Comput Math Appl 59:225–232 Alomari M, Darus M, Kirmaci US (2010) Refinements of Hadamard type inequalities for quasi con applications to trapezoidal formula and to special means. Comput Math Appl 59:225–232 Chen FX (2015) Extensions of the Hermite-Hadamard inequality for harmonically convex functions via fractional integrals. Appl Math Comput 268(1):121–128 Guo XY, Qi F, Xi BY (2015) Some new Hermite-Hadamard type inequalities for geometrically quasi-convex functions on co-ordinates. J Nonlinear Sci Appl 8(5):740–749 Ion DA (2007) Some estimates on the Hermite-Hadamard inequality through quasi-convex functions, Annals of Univer‑ sity of Craiova. Math Comp Sci Ser 34:82–87 Ion DA (2007) Some estimates on the Hermite-Had sity of Craiova. Math Comp Sci Ser 34:82–87 Mitrinović DS, Pečarić JE, Fink AM (1993) Classical and New Inequalities in Analysis. Kluwer Academic Publishers, Dordrecht Niculescu CP, Persson LE (2006) Convex functions and their applications: a contemporary approach. In: CMS Books in Mathematics, vol. 23. Springer, New York Pečarić JE, Proschan F, Tong YL (1992) Convex functions, partial orderings, and statistical applications. Academic Press, New York Pečarić JE, Proschan F, Tong YL (1992) Convex functions, partial orderings, and statistical applications. Academic Press, New York Sroysang B (2013) On the Hermite-Hadamard inequality and other integral inequalities involving several functions. J l i l Sroysang B (2013) On the Hermite-Hadamard inequality and other integral inequalities involving several functio Funct Spaces Appl (Article ID 921828)f Sroysang B (2014) Generalizations on some Hermite-Hadamard type ine applications to weighted means. Sci World J (Article ID 717164) y g ( ) yp qf applications to weighted means. Sci World J (Article ID 717164) y g ( ) yp q applications to weighted means. Sci World J (Article ID 717164) y g yp applications to weighted means. Sci World J (Article ID 71716 g Wu S (2009) On the weighted generalization of the Hermite-Hadamard inequality and its applications. Competing interests The authors declare that they have no competing interests. Page 9 of 9 Wu et al. SpringerPlus (2015) 4:831 Received: 8 October 2015 Accepted: 17 December 2015 Received: 8 October 2015 Accepted: 17 December 2015 References Rocky Mt J Math 39(5):1741–1749 Xi BY, Bai RF, Qi F (2012) Hermite-Hadamard type inequalities for the m- and (α; m)-geometrically convex functions. Aequ Math 84(3):261–269 Xi BY, Qi F (2014) Hermite-Hadamard type inequalities for geometrically r-convex functions. Studia Sci Math Hungarica 51(4):530–546 Xi BY, Qi F (2015) Inequalities of Hermite-Hadamard type for extended s-convex functions and applications to means. J Nonlinear Convex Anal 16(5):873–890 Xi BY, Wang SH, Qi F (2014) Some inequalities for (h, m)-convex functions. J Inequal Appl (Article ID 100)
https://openalex.org/W3122091697	http://publikationen.ub.uni-frankfurt.de/files/55733/1219-Article_Text-5309-1-10-20210128.pdf	English	null	Checklist and a new species of Lippia (Verbenaceae) from the Diamantina Plateau, Minas Gerais, Brazil	European Journal of Taxonomy	2,021	cc-by	5,935	ISSN 2118-9773 www.europeanjournaloftaxonomy.eu 2021 · Cardoso P.H. et al. ISSN 2118-9773 www.europeanjournaloftaxonomy.eu 2021 · Cardoso P.H. et al. European Journal of Taxonomy 733: 42–55 https://doi.org/10.5852/ejt.2021.733.1219 Cardoso P.H., Menini Neto L., Trovó M. & Salimena F.R.G. 2021. Checklist and a new species of Lippia (Verbenaceae) from the Diamantina Plateau, Minas Gerais, Brazil. European Journal of Taxonomy 733: 42–55. https://doi.org/10.5852/ejt.2021.733.1219 Keywords. Endangered species, endemism, Espinhaço Range, Lamiales, Lantaneae. Cardoso P.H., Menini Neto L., Trovó M. & Salimena F.R.G. 2021. Checklist and a new species of Lippia (Verbenaceae) from the Diamantina Plateau, Minas Gerais, Brazil. European Journal of Taxonomy 733: 42–55. https://doi.org/10.5852/ejt.2021.733.1219 Introduction The ﬂ owering plant genus Lippia L. is a member of the tribe Lantaneae Endl. in the vervain family (Verbenaceae J.St.-Hil.). It contains approximately 120 species of perennial herbs, shrubs and subshrubs, often aromatic, native to tropical regions of the Americas and Africa (Atkins 2004; Marx et al. 2010). The genus is diﬃ cult to distinguish from Lantana L., given that the two genera are segregated by the fruit morphology only. Chamisso (1832) characterized Lippia by the presence of schizocarpic fruits bearing two cluses or drupaceous fruits with two pyrenes. Schauer (1847) assigned species with drupes to Lantana and species with schizocarps to Lippia. Finally, Silva & Salimena (2002) redelimited Lantana and reestablished the circumscription proposed by Chamisso (1832) to include only species with monopyrenous drupes. The relationship between Lippia and Lantana is subject to a continuous taxonomic discussion and several delimitations have previously been proposed (Chamisso 1832; Schauer 1847; Troncoso 1974; Silva 1999; Sanders 2001; Silva & Salimena 2002; Atkins 2004). Phylogenetic analyses indicate that neither genus is monophyletic and both must be recircumscribed (Lu-Irving & Olmstead 2013), but an updated classiﬁ cation is yet to be published. The highest species richness in Lippia is concentrated in Brazil, with ca 90 species (Salimena & Cardoso 2020; Cardoso et al. 2019b, 2019c, 2020b). The majority of these occur in the Espinhaço Range (Salimena et al. 2009), which extends for over 1000 km, north to south, from the State of Bahia to the State of Minas Gerais. The region is characterized primarily by its quartzite rocky outcrops (i.e., campos rupestres), with elevations reaching 2000 m (Almeida-Abreu 1995; Giulietti et al. 1997; Gontijo 2008). The Diamantina Plateau, located in the mid-southern region of the Espinhaço Range, Minas Gerais State, is bounded north from the Serra do Cipó and south from Grão Mogol (Gonçalves et al. 2017). It represents the highest plane surface (King 1956), and the watershed between the São Francisco and Jequitinhonha River basins along the N / S axis. The Diamantina Plateau is considered one of the most important centres of plant diversity and endemism in the Espinhaço Range (Giulietti et al. 1997; Echternacht et al. 2011). It is included in the Espinhaço Range Biosphere Reserve (UNESCO 2005), situated between the Cerrado and the Atlantic Forest domains, two global hotspots of biodiversity and conservation priorities (Myers et al. 2000). Checklist and a new species of Lippia (Verbenaceae) from the Diamantina Plateau, Minas Gerais, Brazil Pedro Henrique CARDOSO 1,, Luiz MENINI NETO 2, Marcelo TROVÓ 3 & Fátima Regina Gonçalves SALIMENA 4 1 Universidade Federal do Rio de Janeiro, Museu Nacional, Departamento de Botânica, Quinta da Boa Vista, CEP 20940-040, Rio de Janeiro, RJ, Brazil. 2,4 Universidade Federal de Juiz de Fora, Instituto de Ciências Biológicas, Departamento de Botânica, Herbário Leopoldo Krieger, CEP 36036-900, Juiz de Fora, MG, Brazil. 3 Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Botânica, Av. Carlos Chagas Filho 373, CEP 21941-590, Rio de Janeiro, RJ, Brazil. Correspon ding author: pedrocardoso@ufrj.br 2 Email: menini.neto@gmail.com 3 Email: martrovo@gmail.com 4 Email: frsalimena@gmail.com 1 https://orcid.org/0000-0002-6198-6729 2 https://orcid.org/0000-0001-8750-2422 3 https://orcid.org/0000-0002-0478-2332 4 https://orcid.org/0000-0002-9000-4683 * Correspon ding author: pedrocardoso@ufrj.br 2 Email: menini.neto@gmail.com 3 Email: martrovo@gmail.com 4 Email: frsalimena@gmail.com * Correspon ding author: pedrocardoso@ufrj.br 2 Email: menini.neto@gmail.com 3 Email: martrovo@gmail.com 4 Email: frsalimena@gmail.com 1 https://orcid.org/0000-0002-6198-6729 2 https://orcid.org/0000-0001-8750-2422 3 https://orcid.org/0000-0002-0478-2332 4 https://orcid.org/0000-0002-9000-4683 Abstract. The Diamantina Plateau is located in the central region of the Espinhaço Range, in the State of Minas Gerais, which is dominated by campo rupestre formations. We describe a new species of Lippia L., endemic to the Diamantina Plateau, and provide an annotated checklist and identiﬁ cation key for the 17 species of the genus occurring in the area. Lippia raoniana P.H.Cardoso & Salimena sp. nov. is mainly distinguished by its ovate leaves with adaxial and abaxial surfaces densely covered by sessile glandular trichomes, and drupaceous fruits with two pyrenes. It is known only from two populations, and thus can be provisionally considered as Critically Endangered. Details on the species’ ecology, as well as a comparison with morphologically similar species, a distribution map, and ﬁ eld photographs, are provided. In this work, we also present pictures of the 17 species in their habitats, and we hope that these illustrations will help in the identiﬁ cation and conservation of these taxa. Keywords. Endangered species, endemism, Espinhaço Range, Lamiales, Lantaneae. 42 CARDOSO P.H. et al., Lippia from Diamantina Plateau, Brazil Introduction Ongoing taxonomic studies on Verbenaceae for the “Flora do Brasil 2020” project (http://ﬂ oradobrasil.jbrj.gov.br/), with extensive analyses of herbarium specimens and ﬁ eldwork, allowed the recognition of a new species of Lippia. This new species appears endemic to the Diamantina Plateau in the Espinhaço Range, State of Minas Gerais, and is described and illustrated in the present study. Furthermore, we provide an annotated checklist, identiﬁ cation key, and pictures of the species of Lippia in the Diamantina Plateau, with the aim of increasing our knowledge of the local ﬂ ora, which is essential for conservation purposes. Material and methods Collections of the new species deposited in the herbaria CESJ, ICN, RB and SPF (acronyms of herbaria follow Index Herbariorum (Thiers, continuously updated)) were examined and ﬁ eld expeditions were conducted to observe individuals in their natural habitat. Morphological terminology follows Radford et al. (1974), Harris & Harris (2003), and Gonçalves & Lorenzi (2007). Conservation status is in accordance with the IUCN (2019) criteria; the area of occupancy (AOO) is based on 2 × 2 km grids. The geographical distribution map was prepared using QGIS ver. 3.8 (QGIS Development Team 2018). An identiﬁ cation key to the species of Lippia from the Diamantina Plateau was elaborated by consulting the type specimen images available at Global Plants on JSTOR (https://plants.jstor.org/)and collections 43 European Journal of Taxonomy 733: 42–55 (2021) available from BR, CESJ, F, G, HAL, K, M, MBML, P, R, RB, SP, SPF, VIC and W as well as several herbaria at Herbário Virtual Reﬂ ora (reﬂ ora.jbrj.gov.br/). Species’ distribution records were obtained from the herbaria consulted and followed the delimitation of the Diamantina Plateau proposed by Gonçalves et al. (2017). This delimitation diﬀ ers from the one proposed by Rapini et al. (2002) since it excludes the Serra do Cabral. This location is separated from the remaining Espinhaço Range by the geological depression, which is drained by the Jequitaí and Curimataí Rivers (Saadi 1995). available from BR, CESJ, F, G, HAL, K, M, MBML, P, R, RB, SP, SPF, VIC and W as well as several herbaria at Herbário Virtual Reﬂ ora (reﬂ ora.jbrj.gov.br/). Species’ distribution records were obtained from the herbaria consulted and followed the delimitation of the Diamantina Plateau proposed by Gonçalves et al. (2017). This delimitation diﬀ ers from the one proposed by Rapini et al. (2002) since it excludes the Serra do Cabral. This location is separated from the remaining Espinhaço Range by the geological depression, which is drained by the Jequitaí and Curimataí Rivers (Saadi 1995). Etymology The speciﬁ c epithet is in homage to Raoni Metuktire, an important indigenous leader of Brazil, internationally known as a symbol of environmental preservation. Raoni is dedicated to the ﬁ ght for the rights of native indigenous people and conservation of the tropical forest, mainly Amazonia. His legacy is a symbol of the daily struggle for Brazilian biodiversity, constantly threatened. Lippia raoniana P.H.Cardoso & Salimena sp. nov. urn:lsid:ipni.org:names:77214745-1 Figs 1, 4 B–C Lippia raoniana P.H.Cardoso & Salimena sp. nov. urn:lsid:ipni.org:names:77214745-1 Figs 1, 4 B–C Material examined Type BRAZIL – Minas Gerais • Serro, “próximo ao Km 1 do distrito de Milho Verde” [near to Km 1 of the Milho Verde district];18°27′35.1″ S, 43°29′24.4″ W; 13 Feb. 2020; ﬂ / fr; F.R.G. Salimena & P.H. Nobre 4057; holotype: CESJ; isotypes: HUEFS, RB, SPF. Paratypes BRAZIL – Minas Gerais • Felício dos Santos, “APA Municipal Felício” [Municipal Environmental Protection Area Felício]; 10 Jun. 2006; ﬂ / fr; F.R.G. Salimena et al. 1382; CESJ • Serro, “estrada Diamantina para o distrito de Milho Verde” [road from Diamantina to the Milho Verde district]; 16 Nov. 2010; ﬂ / fr; V. Thode, P. Lu-Irving, N. Mota, M. Toledo 386; CESJ, ICN. Paratypes BRAZIL – Minas Gerais • Felício dos Santos, “APA Municipal Felício” [Municipal Environmental Protection Area Felício]; 10 Jun. 2006; ﬂ / fr; F.R.G. Salimena et al. 1382; CESJ • Serro, “estrada Diamantina para o distrito de Milho Verde” [road from Diamantina to the Milho Verde district]; 16 Nov. 2010; ﬂ / fr; V. Thode, P. Lu-Irving, N. Mota, M. Toledo 386; CESJ, ICN. Diagnosis The new species is similar to Lippia spiraeastrum (Mart. & Schauer) T.R.S.Silva, but diﬀ ers by ovate leaves (vs oblong or oblong-elliptic), slightly discolorous (vs strongly discolorous), matte adaxial surface (vs shiny), glandular abaxial surface (vs tomentose-glandular), shorter peduncle (0.8‒1.3 vs 3.2‒10.5 cm long) and slender (vs thick), ovate bracts (vs lanceolate). Results Taxonomic treatment Taxonomic treatment Taxonomic treatment Class Magnoliopsida Brongn. Order Lamiales Bromhead Family Verbenaceae J.St.-Hil. Genus Lippia L. Description Shrub 1.5 m tall, aromatic, branched, branches erect, tetragonal, sulcate, glabrescent to strigose, with sessile glandular trichomes, nodes conspicuous. Leaves decussate, patent, congested at the stem apices, petioles 2.2‒6 mm long, cylindrical, strigose, with abundant sessile glandular trichomes; blades 1.5‒3 × 0.8‒1.5 cm, chartaceous, ovate, slightly discolorous, apex acute to obtuse, base cuneate, decurrent into the petiole, margin basally entire, crenate to serrate toward the apex, ciliate, slightly revolute, adaxial 44 CARDOSO P.H. et al., Lippia from Diamantina Plateau, Brazil surface matte, sparsely strigose, densely covered by sessile glandular trichomes, abaxial surface densely covered by sessile glandular trichomes, strigose along the veins. Inﬂ orescence one per axil, 0.5‒1 cm long, capituliform, hemispherical, rachis not elongated in the infructescence, peduncle 0.8‒1.3 cm long, cylindrical, slender, strigose, with abundant sessile glandular trichomes; bracts 3‒5 mm long, green, spirally arranged, ovate, apex acute to obtuse, margin ciliate, adaxial surface strigose, covered by sessile Fig. 1. Lippia raoniana P.H.Cardoso & Salimena sp. nov. A. Habit. B–C. Branch with inﬂ orescence. D. Inﬂ orescence frontal view. E-G. Drupaceous fruit. E. Overview. F. Longitudinal section showing t wo pyren es. G. Cross section showing two pyrenes and seeds. Scale bars: E–G = 1 mm. Photos: A–D by Pedro Henrique Nobre; E–G by Nádia Silvia Somavilla. Fig. 1. Lippia raoniana P.H.Cardoso & Salimena sp. nov. A. Habit. B–C. Branch with inﬂ orescence. D. Inﬂ orescence frontal view. E-G. Drupaceous fruit. E. Overview. F. Longitudinal section showing two pyrenes G Cross section showing two pyrenes and seeds Scale bars: E–G = 1 mm Photos: A–D Fig. 1. Lippia raoniana P.H.Cardoso & Salimena sp. nov. A. Habit. B–C. Branch with inﬂ orescence. D. Inﬂ orescence frontal view. E-G. Drupaceous fruit. E. Overview. F. Longitudinal section showing t wo pyren es. G. Cross section showing two pyrenes and seeds. Scale bars: E–G = 1 mm. Photos: A–D by Pedro Henrique Nobre; E–G by Nádia Silvia Somavilla. 45 European Journal of Taxonomy 733: 42–55 (2021) glandular trichomes, abaxial surface densely covered by sessile glandular trichomes; calyx 1‒2 mm long, tubular, 2-lobed, apex 4-toothed, externally hirsute, ciliate, with abundant glandular sessile trichomes; corolla tube 6‒8 mm long, limb 2-labiate, lilac, hypocrateriform, externally pubescent, with abundant glandular sessile trichomes, throat yellow, pubescent, 4 didynamous stamens, inserted at the middle of the corolla tube, included, thecae parallel; ovary 1 mm long, ovoid, glabrous, 2-locular, 1 ovule per locule, stigma oblique, lateral. Distribution, habitat and phenology Lippia raoniana P.H.Cardoso & Salimena sp. nov. is known only from two localities in the Diamantina Plateau (Felício dos Santos and Serro), on the Espinhaço Range, Minas Gerais, Brazil, where it seems to be endemic (Fig. 2). The populations are quite small and grow on quartzitic rock outcrops (campos rupestres), at an elevation of 1200‒1400 m. Plants were collected with ﬂ owers and fruits in January, June, and November. Description Fruit drupaceous, mesocarp dry, 2-pyrenate, 2 mm long, spherical, light- brown, dorsal surface smooth to slightly striated, surrounded by the persistent calyx. Prelimiminary conservation status Lippia raoniana P.H.Cardoso & Salimena sp. nov. is only known from two localities, presenting an estimated Area of Occupation (AOO) smaller than 10 km². The ﬁ rst known population is small and located in the municipality of Serro, along an unpaved road, near to a residential area under urban pressure. The other population is recorded from the municipality of Felício dos Santos, inside a conservation unit. The Diamantina Plateau is now witnessing increasing, uncontrolled tourism, exposing the vulnerability of Fig. 2. Map of the Diamantina Plateau, Minas Gerais, Brazil, showing the known distribution of Lippia raoniana P.H.Cardoso & Salimena sp. nov. Fig. 2. Map of the Diamantina Plateau, Minas Gerais, Brazil, showing the known distribution of Lippia raoniana P.H.Cardoso & Salimena sp. nov. 46 CARDOSO P.H. et al., Lippia from Diamantina Plateau, Brazil its soils and vegetation (Schaefer et al. 2002). Thus, L. raoniana sp. nov. may be considered Critically Endangered (CR) based on criteria and sub-criteria B2ab(ii, iii, iv) of IUCN (2019), due to its restricted AOO, number of known locations, and the continuous decline in the quality of its habitat. its soils and vegetation (Schaefer et al. 2002). Thus, L. raoniana sp. nov. may be considered Critically Endangered (CR) based on criteria and sub-criteria B2ab(ii, iii, iv) of IUCN (2019), due to its restricted AOO, number of known locations, and the continuous decline in the quality of its habitat. Notes Lippia raoniana P.H.Cardoso & Salimena sp. nov. most closely resembles L. spiraeastrum, which is also endemic to the Espinhaço Range, occurring in Grão Mogol, Minas Gerais State (Salimena & Cardoso 2020), due to the shrubby habit, conspicuous nodes, leaves concentrated in the upper portion of the stem, adaxial surface glandular, axillary inﬂ orescences, capituliform, hemispherical, corolla lilac, and fruit with dry mesocarp, 2-pyrenate. However, several characters of the leaves, peduncles and bracts can be used to distinguish them. The new species is mainly characterized by the combination of ovate leaves with adaxial and abaxial surfaces densely covered by sessile glandular trichomes, peduncles 0.8‒1.3 cm long, and ovate bracts with adaxial and abaxial surfaces densely covered by sessile glandular trichomes. Identiﬁ cation key to the species of Lippia L. from the Diamantina Plateau, Minas Gerais, Brazil Identiﬁ cation key to the species of Lippia L. from the Diamantina Plateau, Minas Gerais, Brazil ‒ Leaves evenly distributed along the branch, with several tector trichomes; schizocarp with 2 cluses ............................................................................................................................................... 14 14. Leaves obovate to suborbicular; inﬂ orescences few-ﬂ owered ...................L. hermannioides Cham. ‒ Leaves ovate to ovate-elliptic; inﬂ orescences many-ﬂ owered ....................................................... 15 15. Branches sulcate, nodes conspicuous; bracts elliptic ................................... L. subracemosa Mansf. ‒ Branches not sulcate, nodes inconspicuous; bracts ovate or lanceolate ......................................... 16 16. Branches villous-sericeous; leaves ovate-deltate, abaxially villous-sericeous; bracts lanceolate ...... ...................................................................... L. krenakiana P.H.Cardoso, V.I.R.Valério & Salimena ‒ Branches strigose; leaves elliptic to ovate-elliptic, adaxially strigose; bracts broadly ovate ............. .............................................................................................................................L. rosella Moldenke Identiﬁ cation key to the species of Lippia L. from the Diamantina Plateau, Minas Gerais, Brazil tiﬁ cation key to the species of Lippia L. from the Diamantina Plateau, Minas Gerais, 47 Identiﬁ cation key to the species of Lippia L. from the Diamantina Plateau, Minas Gerais, Brazil 1. Inﬂ orescences more than 2 per leaf axil; bracts tetrastichous, the basal ones connate ..................... 2 ‒ Inﬂ orescence 1 per leaf axil; bracts spiraled, all free ....................................................................... 3 2. Plants with frondose-bracteaose inﬂ orescences ................................................L. stachyoides Cham. ‒ Plants with frondose inﬂ orescences ..................................................................L. origanoides Kunth 3. Calyx laterally ﬂ attened, 2-winged. ......................... L. rubella (Moldenke) T.R.S.Silva & Salimena ‒ Calyx tubular-cylindric, not winged. ................................................................................................ 4 4. Leaves with hyphodromous venation; corolla yellow ............................ L. ﬁ lifolia Mart. & Schauer ‒ Leaves with pinnate venation; corolla pink, lilac, rarely white ........................................................ 5 5. Bracts membranous, pink to magenta, rarely green at base and vinaceous at apex, longer than the corolla tube ....................................................................................................................................... 6 ‒ Bracts foliaceous, green, rarely vinaceous at apex, shorter than the corolla tube .......................... 10 6. Leaf-blades abaxially densely pubescent with pedunculate glandular trichomes; bracts green at base, vinaceous at apex ........................................................................L. pseudothea (A.St.-Hil.) Schauer ‒ Leaf-blades abaxially lacking pedunculate glandular trichomes; bracts pink to magenta ............... 7 7. Leaves ovate-deltoid to suborbicular, margin dentate-lobate ........... L. hederifolia Mart. & Schauer ‒ Leaves ovate, broadly ovate or elliptic, margin crenate ................................................................... 8 8. Branches densely velutinous, pedunculate glandular trichomes present; leaves ovate-elliptic to oblong-elliptic ................................................................................L. rhodocnemis Mart. & Schauer ‒ Branches hirsute or pubescent, pedunculate glandular trichomes absent; leaves ovate to broadly ovate .................................................................................................................................................. 9 9. Branches and peduncles pubescent; inﬂ orescences hemispheric or cylindric .................................... ................................................................................................ L. diamantinensis Glaz. ex Moldenke ‒ Branches and peduncles hirsute; inﬂ orescences globose ...................................... L. lupulina Cham. 10. Corymbs axillary, densely congested at the apex of the branches ...................................................11 ‒ Spikes axillary, not congested at the apex of the branches ............................................................. 13 47 European Journal of Taxonomy 733: 42–55 (2021) 11. Leaf-blades abaxially hirsute, adaxially bullate; bracts elliptic, apex reﬂ exed .................................. ............................................................................................................................L. corymbosa Cham. ‒ Leaf-blades abaxially tomentose, adaxially slightly bullate; bracts ovate-lanceolate, apex straight . ......................................................................................................................................................... 12 12. Leaves ovate, base cordate .................................................................. L. lacunosa Mart. & Schauer ‒ Leaves ovate-orbicular or orbicular, base obtuse to cuneate ...........................L. rotundifolia Cham. 13. Leaves congested at the apex of the branch, with few tector trichomes; drupe with 2 pyrenes ......... ....................................................................................L. raoniana P.H.Cardoso & Salimena sp. nov. Discussion Our ﬂ oristic knowledge of Lippia in the State of Minas Gerais has gradually increased in recent years thanks to studies focusing on the Verbenaceae of small areas in the state (e.g., Salimena-Pires & Giulietti 1998; Salimena & Silva 2009; Cruz & Salimena 2017; Cardoso et al. 2018, 2019a, 2019d, 2020a; Santiago et al. 2020). These studies have revealed new distribution records (Cardoso et al. 2019a), and in some cases, new species (Cardoso et al. 2019c, 2020b). It is not surprising that the Diamantina Plateau harbors some yet undescribed species of Lippia, since the region is ultimately considered one of the main diversity and endemism centres in the Espinhaço Range (Giulietti et al. 1997; Echternacht et al. 2011). The recent discovery of Lippia raoniana P.H.Cardoso & Salimena sp. nov. and L. krenakiana P.H.Cardoso, V.I.R.Valério & Salimena in the Diamantina Plateau (Cardoso et al. 2020b) added to the understanding of ongoing threats to which the ﬂ ora of the Espinhaço Range is subjected (Martinelli & Moraes 2013; Martinelli et al. 2014), emphasizing the need of further taxonomic studies in Lippia. Thus, we present an annotated checklist of the species of Lippia from the area (Table 1). It is important to emphasize that all are found in campos rupestres. In addition, we also provide photographs and an identiﬁ cation key for the 17 species currently recognized in the region (Figs 3–4). This study improves our understanding of species diversity and distributions, and will aid the proposal of new conservation measures for the Espinhaço Range. 48 CARDOSO P.H. et al., Lippia from Diamantina Plateau, Brazil CARDOSO P.H. et al., Lippia from Diamantina Plateau, Braz Fig. 3. A. Lippia corymbosa Cham. B. L. diamantinensis Glaz. ex Moldenke. C. L. ﬁ lifolia Mart. & Schauer. D. L. hederifolia Mart. & Schauer. E. L. hermannioides Cham. F. L. krenakiana P.H.Cardos V.I.R.Valério & Salimena. G. L. lacunosa Mart. & Schauer. H. L. lupulina Cham. I. L. orig anoide Kunth. Photos: A by Daniela Zappi; B, D, F–G, I by Pedro Henrique Nobre; C, E by Luiz Menini Neto H by Vinicius Dittrich. Fig. 3. A. Lippia corymbosa Cham. B. L. diamantinensis Glaz. ex Moldenke. C. L. ﬁ lifolia Mart. & Schauer. D. L. hederifolia Mart. & Schauer. E. L. hermannioides Cham. F. L. krenakiana P.H.Cardoso, V.I.R.Valério & Salimena. G. L. lacunosa Mart. & Schauer. H. L. lupulina Cham. I. L. orig anoides Kunth. Discussion Photos: A by Daniela Zappi; B, D, F–G, I by Pedro Henrique Nobre; C, E by Luiz Menini Neto; H by Vinicius Dittrich. Fig. 3. A. Lippia corymbosa Cham. B. L. diamantinensis Glaz. ex Moldenke. C. L. ﬁ lifolia Mart. & Schauer. D. L. hederifolia Mart. & Schauer. E. L. hermannioides Cham. F. L. krenakiana P.H.Cardoso, V.I.R.Valério & Salimena. G. L. lacunosa Mart. & Schauer. H. L. lupulina Cham. I. L. orig anoides Kunth. Photos: A by Daniela Zappi; B, D, F–G, I by Pedro Henrique Nobre; C, E by Luiz Menini Neto; H by Vinicius Dittrich. 49 European Journal of Taxonomy 733: 42–55 (2021) p f y ( ) ig. 4. A. Lippia pseudothea (A.St.-Hil.) Schauer. B–C. L. raoniana P.H.Cardoso & Salimena sp. nov . L. rhodocnemis Mart. & Schauer. E. L. rosella Moldenke. F. L. rotundifolia Cham. G. L. rubell Moldenke) T.R.S.Silva & Salimena. H. L. stachyoides Cham. I. L. subracemosa Mansf. Photos: A–C G–H by Pedro Henrique Nobre; D by Luiz Menini Neto; F by Maurício Mercadante; I by Luca Marinho. Fig. 4. A. Lippia pseudothea (A.St.-Hil.) Schauer. B–C. L. raoniana P.H.Cardoso & Salimena sp. nov. D. L. rhodocnemis Mart. & Schauer. E. L. rosella Moldenke. F. L. rotundifolia Cham. G. L. rubella (Moldenke) T.R.S.Silva & Salimena. H. L. stachyoides Cham. I. L. subracemosa Mansf. Photos: A–C, E, G–H by Pedro Henrique Nobre; D by Luiz Menini Neto; F by Maurício Mercadante; I by Lucas Marinho. 50 CARDOSO P.H. et al., Lippia from Diamantina Plateau, Brazil Taxon; collection number (herbarium) Distinguishing morphological characters Distribution in the Diamantina Plateau and oﬃ cial conservation status (if available) Lippia corymbosa Cham.; W.R. Anderson 8843 (NY) Branches and leaves hirsute, leaves ternate, subsessile, ovate, margin revolute, adaxially bullate, inﬂ orescences corymbose, short-pedunculate, corolla pink Diamantina, Gouveia, Presidente Juscelino Lippia diamantinensis Glaz. ex Moldenke †; T.B. Cavalcanti 2294 (CEN) Branches pubescent, leaves patent, ovate to orbicular, apex round, base truncate to cordate, inﬂ orescences hemispheric to cylindric, bracts membranous, pink, corolla pink Couto de Magalhães de Minas, Diamantina, Gouveia Lippia ﬁ lifolia Mart. & Schauer; J.R. Pirani 5279 (SPF) Branches viscous, leaves sessile, ﬁ liform, margin entire, hyphodromous venation, corolla yellow Diamantina, Presidente Juscelino Lippia hederifolia Mart. & Schauer; Hatschbach et al. Datas, Diamantina, Gouveia, São Gonçalo do Rio Preto, Serro Couto de Magalhães de Minas, Diamantina, Gouveia, Presidente Juscelino, Serro Discussion 64657 (MBM) Leaves subsessile, congested at the apex of the branches, ovate-deltoid to suborbicular, margin dentate-lobate, adaxially nitid, bracts and corolla pink to magenta Diamantina, Felício dos Santos, São Gonçalo do Rio Preto, Serro Lippia hermannioides Cham.; I. Cordeiro CFCR 9395 (SPF) Leaves obovate to suborbicular, margin entire up to mid-length, crenate-dentate at apex, adaxially scabrid, bullate, inﬂ orescences few- ﬂ owered, corolla white turning pink Diamantina, Felício dos Santos, Gouveia, Rio Vermelho, São Gonçalo do Rio Preto, Serro Lippia krenakiana P.H.Cardoso, V.I.R.Valério & Salimena †,⁑; J.R. Pirani 5673 (SPF) Leaves ovate-deltoid, sericeous-lanate with dense sessile and pedunculate glandular trichomes, inﬂ orescences short-pedunculate, bracts lanceolate, corolla pink Diamantina Lippia lacunosa Mart. & Schauer; P.T. Sano 1015 (SPF) Leaves patent, subsessile, coriaceous, ovate, abaxially foveolate, inﬂ orescences corymbose-paniculate, at the apex of the branches, bracts sericeous, corolla pink or lilac Datas, Diamantina, Gouveia, São Gonçalo do Rio Preto, Serro Lippia lupulina Cham.; J.R. Pirani 6309 (SPF) Branches and peduncles hirsute, leaves ovate to orbicular, apex obtuse, base obtuse to cordate, inﬂ orescences globose, bracts membranous, pink, corolla pink Couto de Magalhães de Minas, Diamantina, Gouveia, Presidente Juscelino, Serro Lippia origanoides Kunth; V.C. Souza CFCT 8783 (SPF) Leaves opposite, inﬂ orescence frondose, many per leaf axil, bracts tetrastichous, imbricate, the basal ones connate, the apical ones free, corolla white Couto de Magalhães de Minas, Diamantina, Gouveia, Monjolos, Serro Lippia pseudothea (A.St.-Hil.) Schauer †; V.C. Souza 20998 (HUEFS) Leaves sessile, spathulate, adaxially with dense pedunculate glandular trichomes, bracts membranous, greenish-pink with vinaceous apex, corolla pink Datas, Diamantina, Felício dos Santos, Gouveia, Presidente Juscelino, São Gonçalo do Rio Preto, Serro Table 1 (continued on the next page). List of species of Lippia L. from the Diamantina Plateau. † = endemic to Minas Gerais State (BFG 2018); ⁑ = Endemic to the Diamantina Plateau. Datas, Diamantina, Gouveia, São Gonçalo do Rio Preto, Serro Couto de Magalhães de Minas, Diamantina, Gouveia, Monjolos, Serro Datas, Diamantina, Felício dos Santos, Gouveia, Presidente Juscelino, São Gonçalo do Rio Preto, Serro Datas, Diamantina, Felício dos Santos, Gouveia, Presidente Juscelino, São Gonçalo do Rio Preto, Serro 51 European Journal of Taxonomy 733: 42–55 (2021) Taxon; collection number (herbarium) Distinguishing morphological characters Distribution in the Diamantina Plateau and oﬃ cial conservation status (if available) Lippia raoniana P.H.Cardoso & Salimena sp. nov.†,⁑; Salimena et al. Discussion 1382 (CESJ) Leaves congested at the apex of the branches, ovate, densely covered by sessile glandular trichomes, bracts ovate, corolla lilac, drupe with 2 pyrenes Felício dos Santos, Serro Lippia rhodocnemis Mart. & Schauer †,⁑; N.L. Menezes CFCR 7703 (SPF) Branches velutine, leaves petiolate, ovate-elliptic to oblong-elliptic, adaxially velutine, bracts membranous, pink to magenta, corolla pink Felício dos Santos, Rio Vermelho, São Gonçalo do Rio Preto, Serro EN B1ab(i, iii, iv) (Salimena et al. 2013) Lippia rosella Moldenke †,⁑; J.R. Pirani CFCR 8707 (NY) Branches strigose, leaves opposite or ternate, adaxially scabrid, abaxially glandular- strigose, bracts broadly ovate, green, vinaceous at apex, corolla pink Diamantina Lippia rotundifolia Cham.; N. Roque 226 (SPF) Branches tomentose-velutine, leaves ovate-orbicular to orbicular, coriaceous, base obtuse to cuneate, inﬂ orescences consisting of many-ﬂ owered corymbs, bracts hirsute, corolla lilac or magenta Datas, Diamantina, Gouveia, Rio Vermelho, São Gonçalo do Rio Preto, Serro Lippia rubella (Moldenke) T.R.S.Silva & Salimena †,⁑; F.R.G. Salimena 3684 (CESJ) Leaves elliptic, margin entire up to mid-length, serrate towards the apex, inﬂ orescences lax, calyx laterally compressed, 2-winged, corolla pink Diamantina EN B2ab(i, ii, iii) (Salimena et al. 2014) Lippia stachyoides Cham.; P.T. Sano 754 (SPF) Leaves opposite, inﬂ orescences frondose-bracteose, many per leaf axil, bracts tetrastichous, imbricate, corolla white or magenta Datas, Diamantina, Gouveia, Rio Vermelho, São Gonçalo do Rio Preto Lippia subracemosa Mansf.; J.R. Pirani 4416 (SPF) Internodes long, nodes conspicuous, inﬂ orescences capituliform, long- pedunculate, bracts elliptic, congested, apex acute to obtuse, corolla pink Monjolos Table 1 (continued). List of species of Lippia L. from the Diamantina Plateau. † = endemic to Minas Gerais State (BFG 2018); ⁑ = Endemic to the Diamantina Plateau. Distribution in the Diamantina Plateau and oﬃ cial conservation status (if available) Felício dos Santos, Rio Vermelho, São Gonçalo do Rio Preto, Serro EN B1ab(i, iii, iv) (Salimena et al. 2013) Diamantina Lippia rotundifolia Cham.; N. Roque 226 (SPF) Branches tomentose-velutine, leaves ovate-orbicular to orbicular, coriaceous, base obtuse to cuneate, inﬂ orescences consisting of many-ﬂ owered corymbs, bracts hirsute, corolla lilac or magenta Datas, Diamantina, Gouveia, Rio Vermelho, São Gonçalo do Rio Preto, Serro Diamantina EN B2ab(i, ii, iii) (Salimena et al. Discussion 2014) Leaves elliptic, margin entire up to mid-length, serrate towards the apex, inﬂ orescences lax, calyx laterally compressed, 2-winged, corolla pink Datas, Diamantina, Gouveia, Rio Vermelho, São Gonçalo do Rio Preto Internodes long, nodes conspicuous, inﬂ orescences capituliform, long- pedunculate, bracts elliptic, congested, apex acute to obtuse, corolla pink 52 CARDOSO P.H. et al., Lippia from Diamantina Plateau, Brazil Acknowledgments We thank Prof. Dr Pedro Henrique Nobre for facilitating the expedition to collect Lippia raoniana sp. nov. and Dr Nádia Silvia Somavilla for sectioning the fruits. We also thank Daniela Zappi, Lucas Marinho, Maurício Mercadante, Pedro Henrique Nobre and Vinicius Dittrich for the photos of the species. The ﬁ rst author also thanks CNPq, Conselho Nacional de Desenvolvimento Cientíﬁ co e Tecnológico – Brasil, for the doctoral grant (process 141837/2020-9). Marcelo Trovó thanks the Alexander von Humboldt Foundation (BRA/1139098), FAPERJ (E26/203.269/2016 and E-26/202.708/2019—JCNE), and CNPq (470349/2013-1—Universal, 306758/2019-9—Pq2) for ﬁ nancial support. The last author thanks the CNPq (REFLORA/563560/2010-0) for the ﬁ nancial support to review the Verbenaceae types in Europe. References Almeida-Abreu P.A. 1995. O supergrupo Espinhaço da Serra do Espinhaço Meridional (Minas Gerais): o rifte, a bacia e o orógeno. Geo nomos 3: 1–18. https://doi.org/10.18285/geonomos.v3i1.211 Almeida-Abreu P.A. 1995. O supergrupo Espinhaço da Serra do Espinhaço Meridional (Minas Gerais): o rifte, a bacia e o orógeno. Geo nomos 3: 1–18. https://doi.org/10.18285/geonomos.v3i1.211 Atkins S. 2004. Verbenaceae. In: Kubitzki K. & Kadereit J.W. (eds) The Families and Genera of Vascular Plants: 449–468. Springer-Verlag, Berlin. https://doi.org/10.1007/978-3-642-18617-2_25 Atkins S. 2004. Verbenaceae. In: Kubitzki K. & Kadereit J.W. (eds) The Families and Genera of Vascular Plants: 449–468. Springer-Verlag, Berlin. https://doi.org/10.1007/978-3-642-18617-2_25 Cardoso P.H., Cabral A., Valério V.I.R. & Salimena F.R.G. 2018. Verbenaceae na Serra Negra, Minas Gerais, Brasil. Rodriguésia 69: 777–786. https://doi.org/10.1590/2175-7860201869235 Cardoso P.H., Cabral A., Santos-Silva F. & Salimena F.R.G. 2019a. Verbenaceae no Parque Estadual da Serra do Papagaio, Minas Gerais, Brasil. Rodriguésia 70: e02932017. https://doi.org/10.1590/2175-7860201970056 Cardoso P.H., Menini Neto L. & Salimena F.R.G. 2019b. A new species of Lippia (Verbenaceae) from the inselbergs of Brazilian Atlantic Forest. Phyotaxa 406: 243–249. https://doi.org/10.11646/phytotaxa.406.4.3 Cardoso P.H., Menini Neto L. & Salimena F.R.G. 2019c. Lippia mantiqueirae (Verbenaceae), a new species from Minas Gerais, Brazil. Phytotaxa 420: 249–254. https://doi.org/10.11646/phytotaxa.420.3.5 Cardoso P.H., Santos-Silva F., Menini Neto L. & Salimena F.R.G. 2019d. Verbenaceae no Parque Nacional do Caparaó, Serra da Mantiqueira, Brasil. Hoehnea 46: e652019. https://doi.org/10.1590/2236-8906-65/2019 Cardoso P.H., Cabral A., Santos-Silva F. & Salimena F.R.G. 2020a. Verbenaceae no Parque Nacional da Serra da Canastra, Minas Gerais, Brasil. Rodriguésia 71: e03072018. https://doi.org/10.1590/2175-7860202071032 Cardoso P.H., Valério V.I.R., Menini Neto L., Trovó M. & Salimena F.R.G. 2020b. Novelties in Lippia (Verbenaceae) from Minas Gerais State, Brazil. Phytotaxa 455: 47–52. https://doi.org/10.11646/phytotaxa.455.1.6 Chamisso L.K.A. 1832. Verbenaceae. Linnaea 7: 105–723. Chamisso L.K.A. 1832. Verbenaceae. Linnaea 7: 105–723. Cruz L.V.V. & Salimena F.R.G. 2017. Verbenaceae J.St.-Hil. do Parque Estadual do Ibitipoca, Minas Gerais, Brasil. Boletim de Botânica da Universidade de São Paulo 35: 65–74. https://doi.org/10.11606/issn.2316-9052.v35i0p65-74 Echternacht L., Trovó M., Oliveira C.T. & Pirani J.R. 2011. Areas of endemism in the Espinhaço Range in Minas Gerais, Brazil. Flora – Morphology, Distribution, Functional Ecology of Plants 206: 782–791. https://doi.org/10.1016/j.ﬂ ora.2011.04.003 Giulietti A.M., Pirani J.R. & Harley R. 1997. Espinhaço Range region, eastern Brazil. In: Davis S.D., Heywood V.H., Herrera-MacBryde O., Villa-Lobos J. &. Hamilton A.C. (eds) Centres of Plant Diversity: 397–404. IUCN publications, Cambrige. 53 European Journal of Taxonomy 733: 42–55 (2021) Gonçalves D.J.P., Shimizu G.H., Yamamoto K. & Semir J. 2017. References Vochysiaceae na região do Planalto de Diamantina, Minas Gerais, Brasil. Rodriguésia 68: 159–193. https://doi.org/10.1590/2175-7860201768124 Gonçalves E.G. & Lorenzi H. 2007. Morfologia vegetal: Organograﬁ a e Dicionário ilustrado de Morfologia das Plantas vasculares. Instituto Plantarum, Nova Odessa. Gontijo B.M. 2008. Uma geograﬁ a para a Cadeia do Espinhaço. Megadiversidade 4: 7– 14. Harris J.G. & Harris M.W. 2003. Plant Identiﬁ cation Terminology: an Illustrated Glossary. 2nd Edition. Spring Lake Publ., Spring Lake. IUCN Standards and Petitions Committee. 2019. Guidelines for Using the IUCN Red List Categories and Criteria. Version 14. Available from http://www.iucnredlist.org/documents/RedListGuidelines.pdf [accessed 30 Jun. 2020]. King L. 1956. Geomorfologia do Brasil Oriental. Revista Brasileira de Geograﬁ a 18: 1–147. Lu-Irving P. & Olmstead R.G. 2013. Investigating the evolution of Lantaneae (Verbenaceae) using multiple loci. Botanical Journal of the Linnean Society 171: 103–119. https://doi.org/10.1111/j.1095-8339.2012.01305.x Martinelli G. & Moraes M.A. 2013. Livro vermelho da Flora do Brasil. Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rio de Janeiro. Martinelli G., Messina T. & Santos Filho L. 2014. Livro vermelho da Flora do Brasil – Plantas raras do Cerrado. Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rio de Janeiro. Marx H.E., O’Leary N., Yuan Y.-W., Lu-Irving P., Tank D.C., Múlgura M.E. & Olmstead R.G. 2010. A molecular phylogeny and classiﬁ cation of Verbenaceae. American Journal of Botany 97: 1647–1663. https://doi.org/10.3732/ajb.1000144 Myers N., Mittermeier R.A., Mittermeier C.G., Fonseca G.A.B. & Kent J. 2000. Biodiversity hotspots for conservation priorities. Nature 403: 853–858. https://doi.org/10.1038/35002501 QGIS Development Team. 2018. QGIS Geographic Information System. ver. 3.8. Open Source Geospatial Foundation Project. Available from https://www.qgis.org [accessed 14 May 2020]. Radford A.E., Dickinson W.C., Massey J.R. & Bell C.R. 1974. Vascular Plant Systematics. Harper Collins, New York. Rapini A., Mello-Silva R. & Kawasaki M.L. 2002. Richness and endemism in Asclepiadoideae (Apocynaceae) from the Espinhaço Range of Minas Gerais, Brazil – a conservationist view. Biodiversity & Conservation 11: 1733–1746. https://doi.org/10.1023/A:1020346616185 Saadi A. 1995. A geomorfologia da Serra do Espinhaço em Minas Gerais e de suas Margens. Geonomos 3: 41–63. https://doi.org/10.18285/geonomos.v3i1.215 Salimena-Pires F.R.G. & Giulietti A.M. 1998. Flora da Serra do Cipó, Minas Gerais: Verbenaceae. Boletim de Botânica da Universidade de São Paulo 17: 155–186. https://doi.org/10.11606/issn.2316-9052.v17i0p155-186 Salimena F.R.G. & Cardoso P.H. 2020. Lippia in Flora do Brasil 2020 under construction. Jardim Botânico do Rio de Janeiro. Available from http://ﬂ oradobrasil.jbrj.gov.br/reﬂ ora/ﬂ oradobrasil/FB15170 [accessed 25 Jan. 2021]. Salimena F.R.G. & Silva T.R.S. 2009. Flora de Grão-Mogol, Minas Gerais: Verbenaceae. References Boletim de Botânica da Universidade de São Paulo 27: 119–126. https://doi.org/10.11606/issn.2316-9052.v27i1p119-120 54 CARDOSO P.H. et al., Lippia from Diamantina Plateau, Brazil Salimena F.R.G., França F. & Silva T.R.S. 2009. Verbenaceae. In: Giulietti A.M., Rapini A., Andrade M.J.G., Queiroz L.P. & Silva J.M.C. (eds) Plantas raras do Brasil: 399–405. Conservação Internacional, Belo Horizonte. Salimena F.R.G., Kutschenko D.C., Monteiro N.P. & Mynssen C. 2013 Verbenaceae. In: Martinelli G. & Moraes M.A. (eds) Livro vermelho da Flora do Brasil: 1010–1016. Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rio de Janeiro. Salimena F.R.G., Moraes L., Kutschenko D.C. & Novaes L. 2014. Verbenaceae. In: Martinelli G., Messina T. & Santos-Filho L. (eds) Livro vermelho da Flora do Brasil – Plantas raras do Cerrado: 266–273. Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rio de Janeiro. Sanders R.W. 2001. The genera of Verbenaceae in the southeastern United States. Harvard Papers in Botany 5: 303–358. Santiago A.O., Cardoso P.H., Salimena F.R.G. & Trovó M. 2020. Verbenaceae no Parque Nacional do Itatiaia, Brasil. Rodriguésia 71: e02462018. https://doi.org/10.1590/2175-7860202071066 Schaefer C.E.R., Ker J.C., Gilkes R.J. Campos J.C., Costa L.M. & Saadi A. 2002. Pedogenesis on the uplands of the Diamantina Plateau, Minas Gerais, Brazil: a chemical and micropedological study. Geoderma 107: 243–269. https://doi.org/10.1016/S0016-7061(01)00151-3 Schauer J.C. 1847. Verbenaceae. In: Candolle A.P. de (ed.) Prodromus Systematis naturalis Regni vegetabilis 11: 522–700. Treuttel et Wü rtz, Paris. Silva T.R.S. 1999. Redelimitação e revisão do gênero Lantana L. (Verbenaceae) no Brasil. PhD thesis, Universidade de São Paulo, São Paulo. Silva T.R.S. & Salimena F.R.G. 2002. Novas combinações e novos sinônimos em Lippia e Lantana (Verbenaceae). Darwiniana 40: 57−59. Thiers B. continuously updated. Index Herbariorum: A Global Directory of Public Herbaria and Associated Staﬀ . New York Botanical Garden’s Virtual Herbarium. il bl f h // b / i /ih/ d Associated Staﬀ . New York Botanical Garden s Virtual Herbarium. Available from http://sweetgum.nybg.org/science/ih/ [accessed 5 Jun. 2020]. Available from http://sweetgum.nybg.org/science/ih/ [accessed 5 Jun. 2020]. Troncoso N.S. 1974. Los géneros de verbenáceas de Sudamérica extratropical. Darwiniana 18: 295–412. UNESCO – United Nations Educational, Scientiﬁ c and Cultural Organization. 2005. Espinhaço Range Biosphere Reserve, Brazil. Available from: https://en.unesco.org/biosphere/lac/espinhaco [accessed 30 Jun. 2020]. References Manuscript received: 29 August 2020 Manuscript accepted: 10 November 2020 Published on: 28 January 2021 Topic editor: Frederik Leliaert Desk editor: Radka Rosenbaumová Manuscript received: 29 August 2020 Printed versions of all papers are also deposited in the libraries of the institutes that are members of the EJT consortium: Muséum national d’histoire naturelle, Paris, France; Meise Botanic Garden, Belgium; Royal Museum for Central Africa, Tervuren, Belgium; Royal Belgian Institute of Natural Sciences, Brussels, Belgium; Natural History Museum of Denmark, Copenhagen, Denmark; Naturalis Biodiversity Center, Leiden, the Netherlands; Museo Nacional de Ciencias Naturales-CSIC, Madrid, Spain; Real Jardín Botánico de Madrid CSIC, Spain; Zoological Research Museum Alexander Koenig, Bonn, Germany; National Museum, Prague, Czech Republic. 55
https://openalex.org/W2099644703	https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.1005239&type=printable	English	null	Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model	PLOS genetics	2,015	cc-by	12,119	OPEN ACCESS Citation: Rahman SH, Kuehle J, Reimann C, Mlambo T, Alzubi J, Maeder ML, et al. (2015) Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model. PLoS Genet 11(5): e1005239. doi:10.1371/journal.pgen.1005239 ☯These authors contributed equally to this work. ‡ AS and TC also contributed equally to this work. * Schambach.Axel@mh-hannover.de (AS); Toni.Cathomen@uniklinik-freiburg.de (TC) ☯These authors contributed equally to this work. ‡ AS and TC also contributed equally to this work. * Schambach.Axel@mh-hannover.de (AS); Toni.Cathomen@uniklinik-freiburg.de (TC) ☯These authors contributed equally to this work. ‡ AS and TC also contributed equally to this work. Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model Shamim H. Rahman1,2,3☯, Johannes Kuehle3☯, Christian Reimann1,2, Tafadzwa Mlambo1,2,4, Jamal Alzubi1,2,3, Morgan L. Maeder5,6, Heimo Riedel3,7, Paul Fisch8, Tobias Cantz9, Cornelia Rudolph10, Claudio Mussolino1,2, J. Keith Joung5,6, Axel Schambach3‡, Toni Cathomen1,2,3‡ 1 Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg, Germany, 2 Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany, 3 Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany, 4 Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany, 5 Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America, 6 Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America, 7 Department of Biochemistry and Mary Babb Randolph Cancer Center, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia, United States of America, 8 Institute of Pathology, University Medical Center Freiburg, Freiburg, Germany, 9 Translational Hepatology and Stem Cell Biology, REBIRTH cluster of excellence, Hannover Medical School, Hannover, Germany, 10 Institute for Cellular and Molecular Pathology, Hannover Medical School, Hannover, Germany Editor: Derry C. Roopenian, The Jackson Laboratory, UNITED STATES Editor: Derry C. Roopenian, The Jackson Laboratory, UNITED STATES Received: November 3, 2014 Accepted: April 26, 2015 Published: May 22, 2015 In vitro disease modeling based on induced pluripotent stem cells (iPSCs) provides a pow- erful system to study cellular pathophysiology, especially in combination with targeted ge- nome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS- SCID), a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK) activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signal- ing to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease caus- ing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(D)J recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected trans- plants for various clinical applications. Copyright: © 2015 Rahman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. RESEARCH ARTICLE Abstract Editor: Derry C. Roopenian, The Jackson Laboratory, UNITED STATES Received: November 3, 2014 Accepted: April 26, 2015 Published: May 22, 2015 Copyright: © 2015 Rahman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. SCID Disease Modeling Institutes of Health [R01 GM088040 to JKJ], and the National Science Foundation [Graduate Research Fellowship to MLM]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Summary Due to the limited availability and lifespan of some primary cells, in vitro disease modeling with induced pluripotent stem cells (iPSCs) offers a valuable complementation to in vivo studies. The goal of our study was to establish an in vitro disease model for severe com- bined immunodeficiency (SCID), a group of inherited disorders of the immune system characterized by the lack of T-lymphocytes. To this end, we generated iPSCs from fibro- blasts of a radiosensitive SCID (RS-SCID) mouse model and established a protocol to reca- pitulate T-lymphopoiesis from iPSCs in vitro. We used designer nucleases to edit the underlying mutation in prkdc, the gene encoding DNA-PKcs, and demonstrated that ge- netic correction of the disease locus rescued DNA-PK dependent signaling, restored nor- mal radiosensitivity, and enabled T-cell maturation and polyclonal T-cell receptor recombination. We hence provide proof that the combination of two promising technolo- gy platforms, iPSCs and designer nucleases, with a protocol to generate T-cells in vitro, represents a powerful paradigm for SCID disease modeling and the evaluation of thera- peutic gene editing strategies. Furthermore, our system provides a basis for further devel- opment of iPSC-derived cell products with the potential for various clinical applications, including infusions of in vitro derived autologous T-cells to stabilize patients after hemato- poietic stem cell transplantation. Competing Interests: TCat is a consultant for TRACR Hematology Ltd. JKJ is a consultant for Horizon Discovery. JKJ has financial interests in Editas Medicine, Hera Testing Laboratories, Poseida Therapeutics, and Transposagen Biopharmaceuticals. JKJ’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. Following the completion of her work for this project, MLM has become an employee of Editas Medicine. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the German Research Foundation [SPP1230Ca311/2 to TCat; SFB 738C9 to AS and TCat; the REBIRTH Cluster of Excellence to AS and TCan (EXC 62/1) and JA and JK (fellowships)], the German Federal Ministry of Education and Research [BMBF 01EO0803 to TCat; ReGene-01GN1003D to TCat and AS], the German Academic Exchange Service [DAAD fellowship to TM], the European Commission¹s 7th Framework Program [PERSIST-222878 to TCat], the National 1 / 21 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 SCID Disease Modeling [8]. Hypomorphic mutations in PRKDC, the locus encoding DNA-PKcs, have recently been described for radiosensitive T and B deficient SCID patients [9]. Hence, DNA-PK dependent signaling is a paradigmatic example of how a single molecule can be simultaneously involved in both, DNA repair and T- and B-cell development, and of how such a process can be dis- turbed by a single point mutation. These particularities make PRKDC an optimal target for novel site-specific gene therapy approaches, such as designer nuclease mediated genome editing. [8]. Hypomorphic mutations in PRKDC, the locus encoding DNA-PKcs, have recently been described for radiosensitive T and B deficient SCID patients [9]. Hence, DNA-PK dependent signaling is a paradigmatic example of how a single molecule can be simultaneously involved in both, DNA repair and T- and B-cell development, and of how such a process can be dis- turbed by a single point mutation. These particularities make PRKDC an optimal target for novel site-specific gene therapy approaches, such as designer nuclease mediated genome editing. For disease modeling, iPSCs can be generated from affected somatic cells by expression of four transcription factors Oct4, Sox2, Klf4 and c-Myc [10,11]. Similar to pluripotent embryonic stem cells, iPSCs have the capacity for unlimited self-renewal, are permissive for transfection with foreign DNA, and importantly, can be expanded in a clonal fashion for characterization. Thus far, iPSCs have been derived from several patients suffering from different hematopoietic and immunological disorders and have been used for disease modeling and gene targeting ap- proaches [12]. Several protocols for in vitro [13–21] and in vivo [22,23] differentiation of iPSCs to hematopoietic cells have been reported. The availability of Notch ligand based cell culture systems, such as the murine stromal cell line OP9-DL1, allows for further differentiation of he- matopoietic stem cells into T-cells in vitro [24,25] Targeted genome modification in iPSCs is an essential tool in disease modeling [12], and gene editing with designer nucleases has developed into a powerful instrument, which has been successfully applied to generate various genetically modified model organisms or human cells to study gene function or the pathophysiology of disease causing mutations. Designer nucle- ases, like meganucleases [26], zinc-finger nucleases (ZFNs) [27], transcription activator-like ef- fector nucleases (TALEN) [28], or the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system [29], induce site-specific DNA double strand breaks (DSBs) at chosen sites. These DSBs activate one of two major DNA repair mechanisms, NHEJ or homol- ogy directed repair (HDR), which can be employed to disrupt genes or to target the integration of exogenous donor DNA sequences to a specific site in the genome, respectively [30]. The goal of this study was to establish an in vitro disease model for T-cell deficiencies and to employ this model to evaluate a designer nuclease-based genome editing strategy. To this end, we generated iPSCs from adult ear fibroblasts of NOD.SCID mice, a model for RS-SCID [31], and established a protocol to recapitulate T-lymphopoiesis from iPSCs in vitro. We used ZFNs to edit DNA-PK deficient fibroblasts and iPSCs and demonstrated that designer nuclease me- diated gene correction led to rescue of DNA-PK dependent signaling, normal radiosensitivity, restoration of T-cell maturation, and polyclonal TCR recombination. We hence provide proof that the combination of two promising technology platforms, iPSCs and designer nucleases, with a protocol to generate T-cells in vitro represents a powerful paradigm for SCID disease modeling and the evaluation of therapeutic gene editing strategies. Introduction Studying the molecular pathology of human disease is often difficult due to the limited avail- ability of particular primary cells, their limited lifespan, or because complex developmental differentiation procedures cannot be easily followed in vivo. In vitro disease modeling with in- duced pluripotent stem cells (iPSCs) provides a practical alternative, and the study of several disorders has benefitted enormously from the convergence of three key technologies: modern genomics that links genetic variants to disease phenotypes, the ability to generate patient-spe- cific iPSCs that can be differentiated into cell types affected by disease, and powerful tools for editing complex genomes [1,2]. T lymphocytes play an important role in adaptive immunity against invading pathogens or in fighting tumor cells. A natural microenvironment for T-cell lymphopoiesis is provided by the thymus. Inherited defects in T-cell function or in T-cell development can lead to severe combined immunodeficiency (SCID), a group of life threatening disorders of the immune sys- tem [3]. Radiosensitive SCID (RS-SCID; OMIM #602450) is characterized on the molecular level by dysfunctional non-homologous end-joining (NHEJ), the most important pathway to repair DNA double strand breaks (DSBs). In human patients, defective DNA repair can lead to a cellular hypersensitivity to ionizing radiation. Moreover NHEJ is essential for physio- logical B- and T-lymphocyte development as it plays an important role in the B-cell receptor (BCR) and T-cell receptor (TCR) recombination process [4]. The diversity of BCRs and TCRs results from the multitude of variable (V), divers (D) and joining (J) gene segments that are al- most randomly reassembled in a process called V(D)J recombination. During V(D)J recombi- nation, specific enzymes cleave at specific recombination signal sequences flanking these gene segments and NHEJ factors play a crucial role in reassembly and final ligation of these gene segments [5,6]. The NHEJ process involves a number of different enzymes, including DNA-de- pendent protein kinase (DNA-PK). DNA-PK is a polyprotein complex, formed by the Ku70/ Ku80 heterodimer and the DNA-PK catalytic subunit (DNA-PKcs) [7], that binds to DNA end structures and serves as a docking site for additional NHEJ factors that mediate DNA repair PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 2 / 21 Restoring DNA-PK activity in SCID fibroblasts by targeted genome editing In the murine disease model RS-SCID is caused by a T-to-A transversion mutation in exon 85 of the prkdc locus. The introduced premature stop codon (Y4046) leads to an 83 aa long C-ter- minal truncation of the encoded DNA-PKcs protein, leading to decreased protein stability and low kinase activity [31]. ZFNs targeted to intron 84 of prkdc were generated using the OPEN platform [32] and their activity verified by in vitro cleavage assays and plasmid-based recombi- nation assays (S1 Fig). To restore function of DNA-PK, we generated a donor DNA encom- passing the wild-type cDNA sequence of prkdc exons 85 and 86, preceded by a splice acceptor 3 / 21 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 SCID Disease Modeling site and followed by a poly(A) signal (Fig 1A). Targeting an intron allowed us to co-introduce a neomycin selection marker cassette to enrich for cells that underwent correct gene targeting. Fig 1. Targeted genome editing in RS-SCID fibroblasts. (A) Schematic of genome editing strategy. Homology-directed repair (HDR) between the prkdc locus and the donor DNA is promoted by ZFN cleavage in intron 84 (BS, binding site). The HDR donor consists of flanking homology arms (dashed lines), splice acceptor (SA), cDNA encoding prkdc exons 85 and 86, polyadenylation signal (pA), neomycin resistance cassette (NeoR). The SCID underlying mutation in exon 85 (mut), and primer binding sites for PCR analysis (5’-junction J5-F/J5-R; 3’-junction J3-F/J3-R; allelic discrimination AD-F/AD-R; mRNA expression RT-F/ RT-R) are indicated. (B) Genome editing. After transfection of SCID fibroblasts with various ratios of donor DNA to ZFN expression plasmids, successful gene targeting in polyclonal samples was detected by an inside-out PCR amplification of the genome–donor 5´-junction (J5-F/J5-R). (C) Expression of corrected prkdc mRNA. After transfection of SCID fibroblasts, successful splicing from exon 83 to cDNA was detected with an inside-out RT-PCR strategy using primers RT-F/RT-R. doi:10.1371/journal.pgen.1005239.g001 Fig 1. Targeted genome editing in RS-SCID fibroblasts. (A) Schematic of genome editing strategy. Homology-directed repair (HDR) between the prkdc locus and the donor DNA is promoted by ZFN cleavage in intron 84 (BS, binding site). The HDR donor consists of flanking homology arms (dashed lines), splice acceptor (SA), cDNA encoding prkdc exons 85 and 86, polyadenylation signal (pA), neomycin resistance cassette (NeoR). The SCID underlying mutation in exon 85 (mut), and primer binding sites for PCR analysi (5’-junction J5-F/J5-R; 3’-junction J3-F/J3-R; allelic discrimination AD-F/AD-R; mRNA expression RT-F/ RT-R) are indicated. (B) Genome editing. Restoring DNA-PK activity in SCID fibroblasts by targeted genome editing After transfection of SCID fibroblasts with various ratios of donor DNA to ZFN expression plasmids, successful gene targeting in polyclonal samples was detected by an inside-out PCR amplification of the genome–donor 5´-junction (J5-F/J5-R). (C) Expression of corrected prkd mRNA. After transfection of SCID fibroblasts, successful splicing from exon 83 to cDNA was detected with a inside-out RT-PCR strategy using primers RT-F/RT-R. Fig 1. Targeted genome editing in RS-SCID fibroblasts. (A) Schematic of genome editing strategy. Fig 1. Targeted genome editing in RS-SCID fibroblasts. (A) Schematic of genome editing strategy. Homology-directed repair (HDR) between the prkdc locus and the donor DNA is promoted by ZFN cleavage in intron 84 (BS, binding site). The HDR donor consists of flanking homology arms (dashed lines), splice acceptor (SA), cDNA encoding prkdc exons 85 and 86, polyadenylation signal (pA), neomycin resistance cassette (NeoR). The SCID underlying mutation in exon 85 (mut), and primer binding sites for PCR analysis (5’-junction J5-F/J5-R; 3’-junction J3-F/J3-R; allelic discrimination AD-F/AD-R; mRNA expression RT-F/ RT-R) are indicated. (B) Genome editing. After transfection of SCID fibroblasts with various ratios of donor DNA to ZFN expression plasmids, successful gene targeting in polyclonal samples was detected by an inside-out PCR amplification of the genome–donor 5´-junction (J5-F/J5-R). (C) Expression of corrected prkdc mRNA. After transfection of SCID fibroblasts, successful splicing from exon 83 to cDNA was detected with an inside-out RT-PCR strategy using primers RT-F/RT-R. d i 10 1371/j l 1005239 001 Fig 1. Targeted genome editing in RS-SCID fibroblasts. (A) Schematic of genome editing strategy. Homology-directed repair (HDR) between the prkdc locus and the donor DNA is promoted by ZFN cleavage in intron 84 (BS, binding site). The HDR donor consists of flanking homology arms (dashed lines), splice acceptor (SA), cDNA encoding prkdc exons 85 and 86, polyadenylation signal (pA), neomycin resistance cassette (NeoR). The SCID underlying mutation in exon 85 (mut), and primer binding sites for PCR analysis (5’-junction J5-F/J5-R; 3’-junction J3-F/J3-R; allelic discrimination AD-F/AD-R; mRNA expression RT-F/ RT-R) are indicated. (B) Genome editing. After transfection of SCID fibroblasts with various ratios of donor DNA to ZFN expression plasmids, successful gene targeting in polyclonal samples was detected by an inside-out PCR amplification of the genome–donor 5´-junction (J5-F/J5-R). (C) Expression of corrected prkdc mRNA. After transfection of SCID fibroblasts, successful splicing from exon 83 to cDNA was detected with an inside-out RT-PCR strategy using primers RT-F/RT-R. PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 SCID Disease Modeling Fig 2. Functional correction of RS-SCID fibroblasts. (A) DNA-PK dependent phosphorylation of RPA2. Treatment of fibroblasts with camptothecin (CPT) induces DNA-PK dependent phosphorylation of RPA2, which was detected by Western blot analysis using an RPA2 specific antibody. Detection of ß-actin served as a loading control. Positions of RPA2 and its phosphorylated form, RPA2-P, are indicated on the right. (B) Rescue of radiosensitivity. Fibroblasts were cultured with increasing amounts of the radiomimetic drug bleomycin. Cellular sensitivity to the drug was quantified by counting number of surviving colonies relative to untreated samples. Data are represented as mean ± SD (N = 3). 3T3, NIH-3T3 fibroblasts; Fib.S, SCID fibroblasts; Fib.T, gene targeted SCID fibroblasts; Fib.D, fibroblasts treated with randomly integrated donor. doi:10.1371/journal.pgen.1005239.g002 Fig 2. Functional correction of RS-SCID fibroblasts. (A) DNA-PK dependent phosphorylation of RPA2. Fig 2. Functional correction of RS-SCID fibroblasts. (A) DNA-PK dependent phosphorylation of RPA2. Treatment of fibroblasts with camptothecin (CPT) induces DNA-PK dependent phosphorylation of RPA2, which was detected by Western blot analysis using an RPA2 specific antibody. Detection of ß-actin served as a loading control. Positions of RPA2 and its phosphorylated form, RPA2-P, are indicated on the right. (B) Rescue of radiosensitivity. Fibroblasts were cultured with increasing amounts of the radiomimetic drug bleomycin. Cellular sensitivity to the drug was quantified by counting number of surviving colonies relative to untreated samples. Data are represented as mean ± SD (N = 3). 3T3, NIH-3T3 fibroblasts; Fib.S, SCID fibroblasts; Fib.T, gene targeted SCID fibroblasts; Fib.D, fibroblasts treated with randomly integrated donor. doi:10.1371/journal.pgen.1005239.g002 doi:10.1371/journal.pgen.1005239.g002 To determine the efficiency of the gene targeting approach, cell clones were generated by single cell dilution. Six out of 20 analyzed clones showed correct targeting. To confirm that re-routed splicing of exon 84 to the artificial exon 85/86 restored DNA-PK activity, cells were treated with camptothecin (CPT), a compound known to induce DSBs dur- ing DNA replication by blocking topoisomerase I. Under these experimental conditions, RPA2 is exclusively phosphorylated by DNA-PK at the stalled replication forks [33]. Upon CPT treat- ment of SCID fibroblasts (Fib.S), a gene edited fibroblast clone (Fib.T) and a donor-containing clone (Fib.D), phosphorylation of RPA2 was detected in Fib.T cells, but not in Fib.S and Fib.D cells (Fig 2A). The fibroblast cell line NIH-3T3 served as a positive control. Fibroblasts of RS-SCID mice are sensitive to gamma-irradiation or the radiomimetic drug bleomycin [34]. PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 Restoring DNA-PK activity in SCID fibroblasts by targeted genome editing doi:10.1371/journal.pgen.1005239.g001 doi:10.1371/journal.pgen.1005239.g001 doi:10.1371/journal.pgen.1005239.g001 site and followed by a poly(A) signal (Fig 1A). Targeting an intron allowed us to co-introduce a neomycin selection marker cassette to enrich for cells that underwent correct gene targeting. To validate our targeting strategy, fibroblasts from a 12-week old male NOD.SCID mouse, in which the SCID mutation in prkdc was confirmed by sequencing, were isolated. Upon cul- turing in vitro these cells transformed spontaneously, probably due to their intrinsic DNA re- pair deficiency. The fibroblasts were transfected with various ratios of donor DNA to ZFN expression plasmids before G418 selection was applied. An inside-out PCR strategy was used to verify correct gene targeting in polyclonal cell populations (Fig 1B). All samples transfected with ZFN expression plasmids and donor revealed successful gene targeting. Splicing of exon 84 to the integrated cDNA was verified by inside-out reverse transcription (RT)-PCR (Fig 1C). PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 4 / 21 Genetic correction of SCID iPSCs While fibroblasts served as an important model to evaluate DNA-PK dependent signaling, the full therapeutic potential of genome editing at the prkdc locus can only be assessed in lymphoid cells. iPSCs have the capacity for unlimited self-renewal, allowing long-term in vitro culture and generation of single-cell derived subclones. As iPSCs can be differentiated into hematopoi- etic cells, including T lymphocytes, they are an ideal platform for disease modeling and the evaluation of gene therapeutic approaches. We generated iPSCs from fibroblasts isolated from a 6-week-old NOD.SCID mouse by transduction with a polycistronic lentiviral vector express- ing the reprogramming factors Oct4, Klf4, Sox2 and c-Myc [35]. Since the DNA repair-defi- cient phenotype interferes with efficient reprogramming [36], we conducted the experiment under hypoxic conditions and added ascorbic acid to reduce damage by reactive oxygen species (ROS) [37]. In addition, small molecule inhibitors for MAP kinase (MEK), glycogen synthase kinase 3 (GSK3) and TGF-beta were used, which have been reported to permit derivation of iPSCs of NOD-derived mouse strains and enhance the reprogramming progress [38,39]. All analyzed iPSC clones expressed pluripotent stem cell markers (S2 Fig), and RT-PCR demon- strated expression of the embryonic stem cell-specific genes in a NOD.SCID iPSC clone (iPS. S6; Fig 3A). In addition, cells from ectodermal (neural rosette-like structures), endodermal (gut-like structures) and mesodermal (smooth muscle patches) origin were detected in terato- ma derived from clone iPS.S6 (Fig 3B). Genome integrity was assessed before and after ZFN mediated genome engineering by spectral karyotyping (Figs 3C and S2). The NOD.SCID iPSC clone iPS.S6 displayed no gross genetic abnormalities and was used for subsequent gene target- ing experiments. In summary, we showed that DNA-repair deficient NOD.SCID fibroblasts could be reprogrammed into iPSCs that display pluripotent behavior and characteristics simi- lar to murine embryonic stem cells. For targeted genome editing, cells of iPSC clone iPS.S6 were nucleofected with donor and ZFN expression plasmids. Following selection and clonal expansion, inside-out PCR amplifica- tion was applied on genomic DNA to detect correct targeting. Of note, 41 out of 46 analyzed clones (89%) showed correct integration of the artificial exon 85/86. Extended PCR analysis of five targeted iPSC clones verified correct 5´- and 3´-junctions between genomic and donor DNA, respectively. An allelic discrimination PCR confirmed mono-allelic targeting in all cases (Fig 4A). Furthermore, expression of the DNA-PKcs encoding mRNA and re-routed splicing to artificial exon 85/86 was validated by inside-out RT-PCR (Fig 4B). SCID Disease Modeling Genetic correction of SCID iPSCs All targeted iPSC clones were positive for expression of pluripotency markers (Fig 3A), formed all three germ layers in teratoma assays (Figs 3B and S2), had an intact karyotype (Figs 3C and S2), and did not show any signs of NHEJ-mediated mutagenesis at the top 15 predicted off-target sites in the mouse genome (S1 Table, S1 Text). The polycistronic lentiviral vector used for generation of iPSCs contained Flp recognition target (FRT) sites in the U3 region of the long-terminal repeats, which allowed us to excise the reprogramming cassette using retroviral-mediated transfer of Flp recombinase [40]. Southern blot analysis confirmed successful removal of the lentiviral vector genome (S3 Fig) and targeted integration of the artificial exon 85/86 into intron 84 of the prkdc locus (S3 Fig). To verify that successful gene targeting could abrogate radiosensitivity, colony survival assays with bleomycin were conducted. We found that the corrected cell line Fib.T dis- played similar resistance to the drug as NIH-3T3 cells, while both Fib.D and Fib.S cells were highly sensitive to bleomycin (Fig 2B). In conclusion, successful ZFN-mediated genome editing restored activity of DNA-PK, which was able to phosphorylate downstream target proteins and to rescue the radiosensitive phenotype of RS-SCID cells. 5 / 21 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 Differentiation of iPSCs to TCR-positive T lymphocytes Since DNA-PK is essential for V(D)J recombination, the RS-SCID immunophenotype is char- acterized by a lack of T and B-lymphocytes [41]. The stromal cell line OP9-DL1 leads to activa- tion of the DL1-mediated Notch signaling in co-cultured cells, which in turn is a prerequisite to induce the T-lymphoid program in multipotent hematopoietic progenitors [24]. Initial ex- periments showed that OP9-DL1 co-cultivation of C57BL/6-derived lineage negative bone 6 / 21 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 SCID Disease Modeling Fig 3. Evaluation of pluripotency of generated iPSCs. (A) Pluripotent stem cell marker gene expression. Oct3/4, Nanog, Zfp42, Esg1, Eras, and fgf4 mRNA expression was determined by qualitative RT-PCR (see S2 Table). Housekeeping gene 36B4 (+/- reverse transcriptase) were included as controls. ES.CCE, murine embryonic stem cell line; Fib.S, SCID fibroblasts; iPS.S6, SCID iPSC clone; iPS.T8, iPS.T25, iPS.T44, gene targeted SCID iPSC clones; iPS.WT, wild-type iPSC clone. (B) In vivo differentiation analysis. Teratoma formation was induced by subcutaneous injection of iPSCs into mice. Hematoxylin/eosin-stained sections of teratoma-derived from clones iPS.S6 and iPS.T25 are shown. (C) Karyotype analysis. Spectral karyotyping (SKY) was performed to detect microscopic genomic abnormalities, translocations and aneuploidies in untreated or genetically corrected SCID iPSC clones. SKY analysis of clone iPSC.T25 is shown (see also S2 Fig). doi:10.1371/journal.pgen.1005239.g003 marrow cells enabled the differentiation of these multipotent stem cells through all (CD4-/ CD8-) double-negative (DN) thymocyte stages, as determined by CD25 and CD44 surface ex- pression. Further culturing of these T-cell precursors on OP9-DL1 led to the generation of CD4+/CD8+ double-positive (DP) T lymphocytes, which expressed the beta chain of the T-cell receptor (TCRß), indicating that these cells have successfully undergone V(D)J recombination and beta-selection in vitro (Fig 5). Since V(D)J recombination is initiated at the DN2 (CD44+/CD25+) stage and beta-selection occurs at the DN3 (CD44-/CD25+) stage, we hypothesized that corrected RS-SCID iPSC-de- rived hematopoietic progenitor cells (HPCs) should be able to differentiate to CD4+/CD8+ double-positive T lymphocytes, while T-cells derived from uncorrected SCID-derived iPSCs would stop at the DN2 thymocyte stage due to their defect in V(D)J recombination (Fig 5A). Fig 4. Targeted genome editing in SCID-derived iPSCs. (A) Verification of gene targeting. Inside-out PCR strategies (see Fig 1A) were used to verify correct 5´ (J5) and 3´-junctions (J3) of the integrated donor. Allelic discrimination (AD) PCR was used to assess mono- vs. bi-allelic integration. Targeted allele runs at 2.99 kb. Sizes of all expected PCR amplicons are indicated on the right. iPS.WT, wild-type iPSC; iPS.S6, SCID iPSC clone; iPS.T8, iPS.T25, iPS.T44, iPS.T45 and iPS.T60, targeted SCID iPSC clones. (B) Expression of corrected prkdc mRNA. Successful splicing from exon 83 to cDNA encompassing exons 84/85 was detected by an inside-out RT-PCR strategy using primers RT-F/RT-R. doi:10.1371/journal.pgen.1005239.g004 Fig 4. Targeted genome editing in SCID-derived iPSCs. (A) Verification of gene targeting. SCID Disease Modeling .pgen.1005239 May 22, 2015 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 7 / 21 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 Inside-out PCR strategies (see Fig 1A) were used to verify correct 5´ (J5) and 3´-junctions (J3) of the integrated donor. Allelic discrimination (AD) PCR was used to assess mono- vs. bi-allelic integration. Targeted allele runs at 2.99 kb. Sizes of all expected PCR amplicons are indicated on the right. iPS.WT, wild-type iPSC; iPS.S6, SCID iPSC clone; iPS.T8, iPS.T25, iPS.T44, iPS.T45 and iPS.T60, targeted SCID iPSC clones. (B) Expression of corrected prkdc mRNA. Successful splicing from exon 83 to cDNA encompassing exons 84/85 was detected by an inside-out RT-PCR strategy using primers RT-F/RT-R. doi:10.1371/journal.pgen.1005239.g004 doi:10.1371/journal.pgen.1005239.g004 8 / 21 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 SCID Disease Modeling PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 9 / 21 After two weeks, thymocyte maturation of iPSC-derived HPCs was measured by flow cytometry, re- vealing the presence of CD44+/CD25- (DN1), CD44+/CD25+ (DN2), CD44-/CD25+ (DN3), CD44-/CD25- (DN4), and CD4+/CD8+ (DP) cells from wild-type iPSCs (iPS.WTX; Fig 5). As hypothesized, T-cell differentiation of NOD.SCID iPSCs (iPS.S6X) was blocked in early DN1 and DN2 thymocyte stages and these T-cell precursors showed neither expression of CD4/ CD8 nor TCRß. In contrast, differentiation from genetically corrected iPSC clones (iPS.T25X) reached DN3 and DN4 stages as well as the CD4+/CD8+ DP T-cell stage, with a fraction of cells expressing TCRß (Fig 5B). Although the same experimental conditions were applied, the abso- lute numbers of generated T-cells varied in between different experiments. To confirm T-cell receptor recombination on the genome level, V(D)J recombination was verified by spectratyping. Control T-cells isolated from the thymus and in vitro generated T- cells from bone marrow lineage negative cells showed a polyclonal T-cell repertoire at Vß chains 1, 6, 8.1, 8.3, 10, 12, 14 and 20 (Figs 6 and S5). While V(D)J recombination was unde- tectable in T-cell precursors derived from SCID iPSCs, T lymphocytes derived from WT or gene targeted iPSCs underwent V(D)J recombination and revealed a polyclonal T- cell repertoire. In summary, we developed a protocol, which allowed us to model T-cell differentiation in vitro. We showed that iPSCs can be differentiated into hematopoietic progenitors and further to various stages of thymocyte development. While wild-type and corrected NOD.SCID iPSCs could be maturated into CD45+ CD4+/CD8+ DP T-cells that express TCRß, differentiation of DNA-PK-deficient cells stopped at the DN2 thymocyte stage. These results provide a proof of concept that iPSC-based in vitro disease modeling is able to reflect in vivo thymocyte matura- tion and that such modeling can be used for both to investigate T-cell maturation defects and to validate gene therapy strategies. SCID Disease Modeling Fig 5. In vitro differentiation of iPSCs to proT-cells and T-cells. (A) Schematic of in vitro T-cell differentiation from iPSCs. Differentiation of iPSCs starts with formation of embryoid bodies that are dissociated to give rise to hematopoietic stem and progenitor cells (HPC). DL-1 mediated Notch signaling coaxes HPC development towards early proT-cells (DN2), which undergo DNA-PK dependent V(D)J recombination. After passing through DN3 and DN4 stages, preT-cells mature into double-positive (DP) T-cells that express the beta chain of the T-cell receptor (TCRß). Dashed lines indicate to what stage iPSC clones are expected to differentiate. (B) Assessment of T-cell differentiation. In vitro T-cell differentiation was analyzed by flow cytometry after two weeks of co- cultivation on OP9-DL1. Gating (indicated on top of each column) was applied in the following order: FSC/SSC and CD45+/DAPI—to assess CD4/CD8 expression; CD8–/CD4—to gate for DN1-DN4 stage cells; CD8–/CD4– (DN) or CD8+/CD4+ (DP) to assess TCRß expression. Numbers indicate percentage of cells in each quadrant. HPC, lineage-negative bone marrow cells; iPS.WTX, wild-type iPSC; iPS.S6X, SCID iPSC clone; iPS.T25, gene targeted SCID iPSC clone. doi:10.1371/journal.pgen.1005239.g005 To this end, we established an embryoid body (EB)-based differentiation protocol for the gen- eration of HPCs from iPSCs. Differentiated and dissociated EBs from all iPSC clones contained cells carrying the early hematopoietic surface markers CD41 and cKit (S4 Fig). Co-cultivation of these cells on OP9-DL1 stroma cells induced differentiation towards T-lymphocytes. After two weeks, thymocyte maturation of iPSC-derived HPCs was measured by flow cytometry, re- vealing the presence of CD44+/CD25- (DN1), CD44+/CD25+ (DN2), CD44-/CD25+ (DN3), CD44-/CD25- (DN4), and CD4+/CD8+ (DP) cells from wild-type iPSCs (iPS.WTX; Fig 5). As hypothesized, T-cell differentiation of NOD.SCID iPSCs (iPS.S6X) was blocked in early DN1 and DN2 thymocyte stages and these T-cell precursors showed neither expression of CD4/ CD8 nor TCRß. In contrast, differentiation from genetically corrected iPSC clones (iPS.T25X) reached DN3 and DN4 stages as well as the CD4+/CD8+ DP T-cell stage, with a fraction of cells expressing TCRß (Fig 5B). Although the same experimental conditions were applied, the abso- lute numbers of generated T-cells varied in between different experiments. To this end, we established an embryoid body (EB)-based differentiation protocol for the gen- eration of HPCs from iPSCs. Differentiated and dissociated EBs from all iPSC clones contained cells carrying the early hematopoietic surface markers CD41 and cKit (S4 Fig). Co-cultivation of these cells on OP9-DL1 stroma cells induced differentiation towards T-lymphocytes. PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 SCID Disease Modeling Fig 6. Polyclonal T-cell receptor recombination. In vitro generated T-cells were analyzed by spectratyping, i.e. quantitative RT-PCR expression analysis of the variable beta chains. Exemplarily shown are results for Vß1, Vß8.3 and Vß10 (see also S5 Fig). X-axis depicts detected PCR fragment size in bp, Y- axis depicts counts of obtained PCR fragments. Thymus, T-cells isolated from thymus as a positive control; HSC, in vitro generated T-cells from lineage- negative bone marrow cells; iPS.WTX, wild-type iPSCs; iPS.S6X, SCID iPSC clone; iPS.T25X, gene targeted SCID iPSC clone. Fig 6. Polyclonal T-cell receptor recombination. In vitro generated T-cells were analyzed by spectratyping, i.e. quantitative RT-PCR expression analysis of the variable beta chains. Exemplarily shown are results for Vß1, Vß8.3 and Vß10 (see also S5 Fig). X-axis depicts detected PCR fragment size in bp, Y- axis depicts counts of obtained PCR fragments. Thymus, T-cells isolated from thymus as a positive control; HSC, in vitro generated T-cells from lineage- negative bone marrow cells; iPS.WTX, wild-type iPSCs; iPS.S6X, SCID iPSC clone; iPS.T25X, gene targeted SCID iPSC clone. doi:10.1371/journal.pgen.1005239.g006 doi:10.1371/journal.pgen.1005239.g006 lifespan, or do not develop due to a differentiation block. Designer nuclease-based gene editing in iPSCs makes this instrument even more attractive because it enables scientists to correlate genotype to phenotype in an isogenic background, either by creating disease models through the insertion of disease specific mutations in normal cells [45] or by correcting the underlying genetic mutation back to wild-type in patient-derived iPSCs [16,46]. Particularly in combina- tion with genetic engineering, iPSCs are preferred over fibroblasts because of their unlimited proliferative potential and their ability of clonal expansion. lifespan, or do not develop due to a differentiation block. Designer nuclease-based gene editing in iPSCs makes this instrument even more attractive because it enables scientists to correlate genotype to phenotype in an isogenic background, either by creating disease models through the insertion of disease specific mutations in normal cells [45] or by correcting the underlying genetic mutation back to wild-type in patient-derived iPSCs [16,46]. Particularly in combina- tion with genetic engineering, iPSCs are preferred over fibroblasts because of their unlimited proliferative potential and their ability of clonal expansion. Hematopoietic differentiation protocols offer the possibility to investigate maturation of various blood lineages in vitro, e. g. Discussion SCID is a group of monogenetic disorders of the immune system characterized by the absence of T-cells, sometimes in combination with a lack of functional B-lymphocytes and/or natural killer cells. RS-SCID is a special form of SCID disorders and serves as a paradigm for radiosen- sitivity and immunodeficiency. On top of the absence of T- and B-lymphocytes, the pathophys- iology of RS-SCID is characterized by a strong sensitivity of all somatic cells to radiation and DNA damaging agents due to a defective DNA repair pathway. The underlying mutations are found in genes coding for NHEJ factors, including LIG4 [42], Artemis [43], XLF [44] and DNA-PKcs [9]. Disease modeling based on patient-derived iPSCs is particularly valuable when studying rare disorders, like RS-SCID, for which patient cells are not easily accessible, have a limited PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 10 / 21 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 SCID Disease Modeling correlations or to characterize the consequence of newly identified genetic mutations on T- lymphopoiesis and/or T-lymphocyte function in more detail. For instance, as compared to in vivo models, individual effects of the microenvironment, cytokines and/or small molecules af- fecting T-cell maturation and expansion, like IL-7 or IL-2, can be analyzed by simple addition to the culture medium. Moreover, existing stroma-free models can be further developed [50] to identify factors downstream of Delta-like Notch ligands that promote T-cell development. Fi- nally, the efficiency of T-cell related gene therapy approaches can be assessed in vitro, without the need of hematopoietic stem cells of the patients. In our study we applied ZFNs for genetic modification of RS-SCID iPSCs. The generation of highly specific ZFNs can be rather challenging and several studies have described off-target cleavage activity of ZFNs [51,52]. While the specificity of ZFNs can be improved, e.g. by opti- mizing the DNA binding properties of the zinc-finger arrays [32], selecting appropriate linker domains [53,54] and employing obligate heterodimeric FokI nuclease domains [55,56], alterna- tive designer nucleases, such as TALENs [28] and CRISPR/Cas9 based nucleases [29], are easier to engineer. Our system provides a basis for further development of iPSC-derived cell products with the potential for various clinical applications. However, although we have tried to transplant iPSC- derived hematopoietic stem/precursor cells into NOD.SCID mice, we did not observe any en- graftment of these cells. This is in line with published data showing that transplantation worked only with iPSC-derived hematopoietic stem/precursor cells that were produced in vivo [22,23]. Further studies will be needed to establish optimal culture conditions to generate trans- plantable stem cells in vitro. Hence, combining in vitro protocols with physiologic in vivo dif- ferentiation seems more promising. For example, transplantation of iPSC-derived early thymocyte progenitor populations could allow for thymic reconstitution and maturation to create polyclonal T-cell effector populations [50]. Infusions of in vitro derived autologous T- cells could be used to stabilize patients suffering from primary immunodeficiencies, like SCID or hemophagocytic lymphohistiocytosis, or after conventional hematopoietic stem cell trans- plantation to close the gap until graft-derived lymphocytes arise. Moreover, given the clinical success of autologous T-cells expressing tumor specific chimeric antigen receptors (CARs) [57], iPSC-derived autologous CAR-T-cells represent an interesting alternative to current pro- tocols, as recently shown [19]. Finally, autologous, CCR5 knockout iPSC lines could present a source to provide HIV patients with HIV-resistant T-cells to reconstitute the adaptive immune system [58]. However, before iPSC-based cell therapies can enter clinical practice, safety con- cerns, especially with regard to the generation of iPSC-derived teratoma, have to be addressed and full functionality of iPSC-derived cells proven. In conclusion, our study describes an iPSC-based disease model for RS-SCID. Our in vitro protocol allowed us to differentiate iPSCs to T-cells and to analyze the influence of NHEJ defi- ciency on V(D)J recombination. Moreover, it emphasizes the significance of designer nucleases as a tool in generating isogenic disease models and their future role in producing iPSC-based, patient-specific, genetically corrected autologous transplants for various applications in the clinic. to study the impact of genomic mutations on protein func- tion in mature blood cells or where specific mutations lead to a block in lymphopoiesis, myelo- poiesis, or erythropoiesis [17,18,46]. While designer nuclease-based gene editing in iPSCs has been established in several labs, differentiation of genetically modified iPSCs to mature im- mune cells has remained challenging. Differentiation of iPSCs derived from a patient suffering from X-linked chronic granulomatous disease (X-CGD) to granulocytes was the first example to show functional correction of a genetic defect by targeted integration of a gp91phox expres- sion cassette into the putative safe harbor site AAVS1 [15]. Myeloid differentiation from pa- tient-derived iPSCs for disease modeling and/or drug development has also been established e.g. for severe congenital neutropenia [47] and pulmonary alveolar proteinosis [20]. Differentiation of iPSCs to lymphocytes, on the other hand, has been reported only from a few labs [18,19]. In the present study, we describe an improved in vitro differentiation proce- dure for iPSCs to T-cells that is based on previously published protocols [18,48,49], and, to our knowledge, use this protocol for the first time to model the functional defects of an immunode- ficiency in vitro and to investigate the effect of genetic engineering of disease iPSCs on T-cell maturation. Because the generated hematopoietic progenitor cells supported the maturation through all early stages of thymocyte differentiation, including V(D)J recombination and beta- selection, we were able to reproduce the stage-specific block induced by the point mutation in the prkdc locus in vitro. This setup can also be used to screen for genotype-phenotype 11 / 21 Characterization of ZFN For expression analysis, ZFNs were expressed in HEK293T for immunoblotting as previously described [59]. The in vitro cleavage assay was basically performed as defined before [61]. Briefly, a target DNA was amplified by PCR from Fib.S gDNA using primers IV-F/IV-R (S2 Table). ZFNs were in vitro transcribed/translated with the TNT SP6 Coupled Reticulocytes Ly- sate System (Promega), 150 ng of target DNA was mixed with the reticulocyte lysates, incubat- ed for 1.5 h at 37°C, and analyzed on a 1.5% agarose gel. The plasmid-based gene targeting assay was conducted as described before [59]. Flow cytometry to determine the percentage of EGFP and REX positive cells was performed on FACSCalibur with CellQuestPro software (BD Biosciences). Plasmids Prkdc-specific zinc-finger arrays (S1 Fig) were generated with the OPEN protocol [32]. To gen- erate ZFNs, the zinc-finger arrays were codon-optimized (GeneArt) and cloned into pRK5 vec- tors, with and without NLS [59], containing the cleavage domains of wild-type FokI or the obligate heterodimeric FokI variant KV/EA [55] and the LRGS linker [54]. The target plasmid pCMV.LacZsPK@GFP was generated by replacing the “31” target site of pCMV.LacZs31@GFP [59] by the ZFN target site aGTTTGCGCCtaactGAAGGTGACa (capital letters indicate target site for ZFN). The donor plasmid pJet.SAE8586Neo (Fig 1A) consists of (i) a splice acceptor (SA) [60]; (ii) a cDNA consisting of prkdc exons 85 and 86, which was PCR amplified from pMEPK7 (kindly provided by Masumi Abe) with primers PRK-F/PRK-R (S2 Table); (iii) an SV40 polyadenylation signal (pA); (iv) a NeoR cassette comprise the aminoglycoside phospho- transferase coding sequence flanked by the HSV thymidine kinase promoter and an SV40 pA (kindly provided by Stefan Weger); (v) left and right homology arms, which were PCR ampli- fied from Fib.S gDNA. SCID Disease Modeling L-glutamine]. Primary mouse ear fibroblasts were cultured in MEF medium [DMEM low glu- cose (PAA) with 15% FCS, L-glutamine, nonessential amino acids (NEAA; Gibco), P/S, 100 μM of ß-mercaptoethanol (Sigma-Aldrich), sodium pyruvate and 50 μg/μl phospho-ascor- bic acid (P-VitC, Sigma-Aldrich)]. ES.CCE cells were cultivated in ES medium [Knockout- DMEM (Gibco) with 15% ES-tested FCS (PAA), P/S, L-glutamine, NEAA, 150 mM monothio- glycerol (MTG, Sigma-Aldrich) and ESGRO mouse LiF (Millipore)]. iPSCs were cultivated in iPS medium [Knockout-DMEM supplemented with 15% ES-tested FCS, NEAA, P/S, L-Gluta- mine, 100 μM of ß-mercaptoethanol and ESGRO mouse LiF, 50 μg/μl of P-VitC, 4 μM of SB431542, 1 μM of PD0325901 and 3 μM of CHIR99021 (all Axon Medchem, together termed 3i) and passaged with Accutase (Gibco). ES.CCE cells and iPSCs were cultivated either on irra- diated C3H or CF-1 MEF feeders on gelatin-coated plates or feeder-free in vented flasks (Sar- stedt). Lineage negative cells (HSC) were isolated by flushing the tibiae and femurs of C57BL/ 6N mice (Charles River) and purified by magnetic cell sorting (MACS) with the Lineage Cell Depletion Kit (MACS Miltenyi) according to the manufacturer’s protocol. Cells were stained with Trypan Blue (Sigma-Aldrich) and counted at 100x microscope magnification prior to in vitro T-cell differentiation. Cell clones were generated either by limiting dilution (fibroblasts) or colony picking (iPSCs). All but HEK293T cells were cultivated under hypoxic conditions (7% CO2 / 5% O2). Materials and Methods Cells and cell culture NIH.3T3 and HEK293T cells were cultured in DMEM (Biochrom) supplemented with 10% FCS (PAA), penicillin/streptomycin (P/S; PAA), L-glutamine (Biochrom) and sodium pyruvate (PAA). OP9 and OP9-DL1 cells (obtained from Juan Carlos Zúñiga-Pflücker) were expanded in OP9 medium [alpha-MEM (Gibco), 20% OP9-tested FCS (PAA), P/S and PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 12 / 21 SCID Disease Modeling 4.8 μl of transfection reagent in 200 μl OptiMEM (Gibco). The ZFN expression plasmids were co-transfected with the donor pJet.SAE8586Neo at different ratios and filled up with pUC118 to 1.6 μg. Selection with 500 μg/ml of G418 (Sigma-Aldrich) was applied 5 days after transfec- tion for 7 days. iPSCs were grown feeder-free before and after transfection. 3x106 cells were nucleofected with 10 μg of pJET.SAE8586Neo and 5 μg of each ZFN expression plasmid using the Mouse ES Cell Nucleofector Kit (LONZA) and Nucleofector II with program A-030. After 5 days of recovery, G418 selection was applied for 7 days at a concentration of 400 μg/ml. After 1 week, iPSC clones were isolated and cultivated on feeders. Functional tests to assay DNA-PK activity To measure DNA-PK dependent RPA2 phosphorylation, 8x105 fibroblasts were treated with 1 μM of camptothecin (Sigma-Aldrich) for 1 h. Cells were harvested in RIPA buffer supple- mented with Complete Protease Inhibitor and PhosSTOP phosphatase inhibitor cocktails (both Roche). Western blot was basically performed as described before [63,64]. RPA2 and ß- actin were detected with rat anti-RPA32 (1:1000, 4E4, Cell Signaling) and rabbit anti-ß-actin (1:1000, Cell Signaling), respectively, and visualized with HRP-conjugated anti-rat and anti- rabbit antibodies (1:20,000, Dianova) and West Pico Chemiluminescence substrate (Thermo Scientific). For the colony survival assay, 1x105 fibroblasts were treated 1 day after seeding with the indicated amounts of bleomycin (Sigma-Aldrich) for 2 h. Cells were washed with PBS, tryp- sinized and 5,000 cells seeded into a 10-cm plate (N = 3). After 4 days the plates were stained with 0.5% (w/MeOH) crystal violet (Sigma-Aldrich) and colonies counted. Genotyping by PCR or Southern blotting Genomic DNA was extracted with the QIAamp DNA Blood Mini Kit (QIAGEN). G418 select- ed fibroblast and iPSC clones were analyzed for legitimate targeted integration by inside-out PCR using Phire Hot Start II DNA polymerase kit (Thermo Scientific). RNA was isolated with TRIzol (Life Technologies), and all RT-PCR reactions performed with the QuantiTect Reverse Transcription Kit (QIAGEN). All used primers are listed in S2 Table. For Southern blot analy- sis [62], genomic DNA was digested with EcoRV or BamHI, separated on a 0.8% agarose gel and transferred to Biodyne B nylon membrane (PALL Life Sciences). DNA was hybridized with a 32P-labeled fragment of PRE (for detection of the reprogramming vector) or NeoR (for detection of donor copies) using the DecaLabel DNA Labeling Kit (Fermentas). Labeled Hin- dIII digested Lambda DNA was used as a marker. PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 Gene targeting in RS-SCID cells For targeted integration into Fib.S fibroblasts, 1x105 cells were transfected 24 h after seeding with Lipofectamine 2000 (Life Technologies). 1.6 μg of endotoxin-free DNA was mixed with PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 13 / 21 SCID Disease Modeling days, emerging iPSC colonies were isolated and expanded for characterization. A total of 12 iPSC clones derived from NOD.CB17-Prkdc scid/J (iPS.S) were initially characterized by as- sessing expression of SSEA-1 by flow cytometry and staining of alkaline phosphatase (Milli- pore) followed by documentation with the Olympus IX71 system. Determination of the vector copy number (VCN), teratoma formation, Flp recombinase-mediated excision, fluorescence in situ hybridization (FISH) and pluripotency factors RT-PCR analysis have been described previ- ously [35,62,66]. Clone iPS.S6 was used for gene targeting and three out of 41 corrected clones (iPS.T8, iPS.T25, iPS.T44) were characterized in detail. The parental uncorrected clone iPS.S6 was included as a negative control, a wild-type NOD/ShiLtJ derived clone (iPS.WT) as a positive control. In vitro T-cell differentiation The protocol was adapted from previously published work [48,49]. For embryoid body (EB) formation, iPSCs were split with Collagenase IV (Gibco) and 5x104 cells were cultured in sus- pension plates in 2 ml of EB medium [IMDM (Biochrom AG) with 15% ES cult FCS (Stem Cell Technologies), 5% PFHM II (Gibco), P/S, L-Glutamine, 50 μg/ml P-VitC, 150 mM MTG, 200 μg/ml human transferrin (Sigma-Aldrich)] in a normoxic incubator on a shaker at 60 rpm. At day 2.5, 0.5 ml of EB medium plus cytokines rhBMP-4, activinA, rhVEGF165 and rhFGF-2 at 5 ng/ml final concentration each (all R&D Systems) was added. At day 8, EBs were har- vested, washed with PBS and collected in Trypsin-EDTA, diluted 1:15 in Collagenase IV. After 30 min, 2.5 ml of cell dissociation buffer (Gibco) was added and cells transferred through a 70- μm mesh. Hematopoietic progenitor cells (HPCs) were washed with PBS and analyzed for CD41/cKit expression by flow cytometry prior to hematopoietic expansion. To this end, 106 EB-derived HPCs were cultivated for 3 days under hypoxic conditions in STFV medium [IMDM, 10% OP9-tested FCS, P/S, L-glutamine, 10 ng/ml mSCF, 20 ng/ml mTPO, 100 ng/ml rhFlt3-L (all Peprotech), and 40 ng/ml rhVEGF165 (R&D Systems) (final concentration each)]. At day 3, cells were harvested through a 100-μm mesh and washed with PBS prior to in vitro T-cell differentiation. To this end, up to 3x105 expanded HPCs or 0.5-1x105 HSCs were added in T-cell differentiation medium [OP9 medium, supplemented with 1 ng/ml mIL-7 (Peprotech) and 5 ng/ml rhFlt3-L]. After 3 days, 2 ml medium was added and cultivation con- tinued for up to 4 weeks. Every 7 days cells were harvested through a 100-μm mesh, washed with PBS, transferred to a new OP9-DL1 cell layer, and analyzed for T-cell differentiation by flow cytometry. PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 Generation, excision and characterization of iPSC Murine adult fibroblasts were extracted from ears of 6-week old NOD/ShiLtJ and NOD. CB17-Prkdc scid/J male mice as described before [62]. Fibroblast from 12-week old NOD. CB17-Prkdc scid/J mouse gave rise to spontaneously transformed Fib.S. The “4-in-1” repro- gramming vector pRRL.PPT.SF.mOKSMco.idTom.PRE, co-expressing the transcription fac- tors Oct4, Klf4, Sox2 and c-Myc with the fluorescent marker tdTomato, has been previously described [35]. To generate versions that allow for Flp recombinas-mediated excision (pRRL. PPT.SF.mOKSMco.idTom.PRE.FRT), FRT sites were introduced into the promoter-deprived U3 region. Virus production has been described elsewhere [65]. The reprogramming was conducted as described before [35]. Briefly, NOD.CB17-Prkdc scid/J or NOD/ShiLtJ-derived fibroblasts were seeded in MEF medium on gelatin-coated 6-well-plates at 8x104/well for trans- duction. After 2 days, cells were transduced with an MOI of 5 and incubated for 8 h, following 2 times washing with PBS. MEF medium with 2 mM VPA (Sigma Aldrich) was added. After 4 days medium was changed to iPS medium with VPA, and after 7 days 3i was added. After 14 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 14 / 21 S1 Text. Supplementary Methods. (DOCX) S1 Fig. ZFNs targeting prkdc locus. (A) Schematic view of ZFN binding site (ZFN BS) in prkdc. The position and sequence of the ZFN BS in intron 84 is shown. F1, F2, F3 indicate tar- get triplets for each binding half-sites. The spacer is highlighted in italics, mut indicates the position of the SCID mutation. (B) Sequence of prkdc-specific ZF modules. Amino acid se- quences of the ZF modules that recognize F1, F2, or F3 target triplets for the left and right tar- get half-sites (5’ to 3’ orientation). Three ZFs for the left binding half-site (L1, L2, L3) and two for the right target half-site (R1, R2) have been selected and tested for their ability to activate a beta-galactosidase (ßgal) reporter [32]. (C) Expression analysis of ZFNs. ZFN-encoding plas- mids were transfected in 293T cells and protein levels detected by immunoblotting using Odys- sey IRDye antibodies. L1, L2, L3, ZFN left subunits 1, 2 or 3 with WT FokI domain; L1H, L2H, L3H, ZFN left subunits 1, 2 or 3 with “EA” obligate heterodimeric FokI domain [55]; L2N, S1 Fig. ZFNs targeting prkdc locus. (A) Schematic view of ZFN binding site (ZFN BS) in prkdc. The position and sequence of the ZFN BS in intron 84 is shown. F1, F2, F3 indicate tar- get triplets for each binding half-sites. The spacer is highlighted in italics, mut indicates the position of the SCID mutation. (B) Sequence of prkdc-specific ZF modules. Amino acid se- quences of the ZF modules that recognize F1, F2, or F3 target triplets for the left and right tar- get half-sites (5’ to 3’ orientation). Three ZFs for the left binding half-site (L1, L2, L3) and two for the right target half-site (R1, R2) have been selected and tested for their ability to activate a beta-galactosidase (ßgal) reporter [32]. (C) Expression analysis of ZFNs. ZFN-encoding plas- mids were transfected in 293T cells and protein levels detected by immunoblotting using Odys- sey IRDye antibodies. Statistics All experiments were performed at least three times. Error bars represent standard deviation (SD). Statistical significance was determined with a two-sided Student's t-test with unequal variance. SCID Disease Modeling analyzed with FlowJo software (Tree Star). T-cell receptor diversity was analyzed by CDR3 spectratyping as previously described [67]. Accession numbers The National Center for Biotechnology Information (NCBI) Nucleotide database (http://www. ncbi.nlm.nih.gov/nuccore) accession number for the ZFN target site in intron 84 of the prkdc gene on mouse chromosome 16 is AB030754: 189732. Analysis of T-cell phenotype and genotype For flow cytometric analysis, cells were resuspended in FACS buffer [PBS supplemented with 2% FCS, 1 mM EDTA and 0.1% sodium azide (both Sigma-Aldrich)]. To stain for pluripotency marker SSEA-1, iPSCs were rinsed with PBS and stained with biotinylated anti-SSEA-1 anti- body (eBioscience) diluted in FACS buffer for 20 min at 4°C. After rinsing the secondary stain- ing was performed with a streptavidin-APC antibody (eBioscience). Hematopoietic cells were pretreated with Mouse BD Fc block (BD Biosciences) before antibody staining. Antibody stain- ing was performed for 20 min at 4°C. EB-derived HPCs were stained with CD41-PE, cKit- APC, or respective isotype controls (all eBioscience). iPSC-derived T-cells were stained with CD44-PE and CD25-APC, or CD4-PE and CD8-APC. Viability staining with 7-AAD was per- formed for 2 min during the last rinsing, before samples were measured on a FACSCalibur. Al- ternatively, iPSC-derived T-cells were stained with CD45-APC-Cy7, CD4-PerCPR-Cy5.5, CD8-PE-Cy7 (all BD Biosciences), CD44-PE, CD25-APC, TCRß-FITC (eBioscience) and DAPI, before analysis on a FACSCanto II with FACSDiva (BD Biosciences). All samples were PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 15 / 21 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 (TIF) S5 Fig. Analysis of T cell receptor diversity of in vitro generated T cells by spectratyping. Quantitative PCR was performed on genomic DNA isolated from in vitro generated T cells. Ex- emplarily shown are PCR analyses of the variable beta chains Vß1, Vß6, Vß8.1, Vß8.3, Vß10, Vß12, Vß14 and Vß20. X axis indicates PCR fragment size in bp, Y axis shows quantity of PCR amplicons. Thymus, control DNA of cells isolated from thymus; HSC, in vitro generated T cells; iPS.WTX, wild-type iPSC clone; iPS.S6 and iPS.S6X, SCID iPSC clones; iPS.T25, iPS. T25X and iPS.T44, targeted iPSC clones; X indicates clones with excised reprogramming cas- sette. (TIF) SCID Disease Modeling S2 Fig. Cellular characterization of iPSC clones. (A) Ssea-1 expression. Surface expression of pluripotency marker Ssea-1 was measured by flow cytometry. Fib.S, NOD.SCID-derived ear fi- broblasts; ES.CCE, murine ES cell line; iPS.S1, iPS.S6 and iPS.S12, NOD.SCID-derived iPSC clones. (B) Alkaline phosphatase staining. NOD.SCID-derived iPSCs were stained using the Alkaline Phosphatase Detection Kit and analyzed by microscopy. (C) Spectral karyotyping (SKY). Multicolor fluorescent in situ hybridization (FISH) based karyotyping was used to as- sess genome integrity. iPS.T8 and iPS.T44, gene targeted iPSC clones. (D) Teratoma Assay. He- matoxylin/eosin-stained sections of teratoma isolated 8 weeks after injection of iPSCs into NSG mice. Detection of ectodermal, endodermal and mesodermal tissues. iPS.T8 and iPS.T44, gene targeted iPSC clones. S3 Fig. Molecular characterization of iPSC clones. (A) Excision of the reprogramming cas- sette. iPSCs have been treated with Flp recombinase to remove the lentiviral reprogramming cas- sette. Excision was confirmed by detection of the viral PRE elements via Southern blot. Genomic DNA was digested with EcoRV or BamHI. Positions of restriction sites in reprogramming cassette and the expected minimal band sizes are indicated on top. M, DNA size marker; S6 and S6X, SCID iPSC clones; T25 and T25X, targeted iPSC clones; WT and WTX, wild-type iPSC clones; X indicates clones with excised reprogramming cassette. (B) Random integration of donor. The NeoR cassette, as an indicator of donor DNA, was detected by Southern blot to confirm targeted integration in prkdc intron 84. Genomic DNA was digested with EcoRV or NdeI. Positions of re- striction sites in the modified intron 84 and the expected band sizes are indicated on top. (C) De- termination of vector copy number (VCN). Copy number of the lentiviral reprogramming vector was assessed by quantitative PCR [62], and is indicated as PRE per endogenous Flk3 copy. Fib, murine ear fibroblasts; iPS.1X and iPS.2X, iPSC clones with excised reprogramming cassette; iPS. S1, iPS.S2, iPS.S3, iPS.S4, iPS.S6, iPS.S8, iPS.S9 and iPS.S12, NOD.SCID-derived iPSC clones. (TIF) S4 Fig. Control of in vitro differentiation. Samples were stained with CD41-PE, cKit-APC or their corresponding isotype controls, and 7-AAD after dissociation of 8 d matured embryoid bodies (EBs). All plots were pre-gated on FSC/SSC and 7-AAD-negativity. Red numbers indi- cate percentage of cells in each quadrant. iPS.WT and iPS.WT X, wild-type iPSC clones; iPS.S6 and iPS.S6 X, SCID iPSC clones; iPS.T25, iPS.T25X and iPS.T44, targeted iPSC clones; X indi- cates iPSC clones with excised reprogramming cassette. S1 Text. Supplementary Methods. (DOCX) L1, L2, L3, ZFN left subunits 1, 2 or 3 with WT FokI domain; L1H, L2H, L3H, ZFN left subunits 1, 2 or 3 with “EA” obligate heterodimeric FokI domain [55]; L2N, ZFN left subunit L2 with “EA” obligate heterodimeric FokI domain without NLS signal; R1, R2, ZFN right subunits 1 or 2 with WT FokI domain; R1H, R2H, ZFN right subunits 1 or 2 with “QK” obligate heterodimeric FokI domain [55]; R1N, R2N, ZFN right subunits 1 or 2 with “QK” obligate heterodimeric FokI domain without NLS signal. GFP served as transfection and loading control. (D) In vitro cleavage assay. ZFN pair L2N and R1N was in vitro tran- scribed/translated and mixed with a ZFN BS-containing PCR product. Cleavage reaction [61] was analyzed on a 1.5% agarose gel. Size markers are indicated. (E) Plasmid-based gene correc- tion assay. 293T cells were transfected with target plasmid, repair matrix and ZFN or SceI plas- mids in order to induce episomal homology-directed repair [59]. Y axis shows the percentage of relative gene correction frequency, which is calculated as GFP-positive cells (target plasmid corrected) REX-positive cells (transfected cells). (+), positive control SceI; (-), negative control SceIΔ; WT, L2 + R1 with homodimeric FokI domain; OH, L2 + R1 with obligate heterodimeric FokI domain;-NLS, L2 + R1 with OH FokI domain without NLS signal. (TIF) 16 / 21 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 References 1. Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, et al. (2008) Disease-specific induced pluripotent stem cells. Cell 134: 877–886. doi: 10.1016/j.cell.2008.07.041 PMID: 18691744 2. Merkle FT, Eggan K (2013) Modeling human disease with pluripotent stem cells: from genome associa- tion to function. Cell Stem Cell 12: 656–668. doi: 10.1016/j.stem.2013.05.016 PMID: 23746975 3. Cavazzana-Calvo M, Andre-Schmutz I, Fischer A (2013) Haematopoietic stem cell transplantation for SCID patients: where do we stand? Br J Haematol 160: 146–152. doi: 10.1111/bjh.12119 PMID: 23167301 4. Slatter MA, Gennery AR (2010) Primary immunodeficiencies associated with DNA-repair disorders. Ex- pert Rev Mol Med 12: e9. doi: 10.1017/S1462399410001419 PMID: 20298636 5. Komori T, Okada A, Stewart V, Alt FW (1993) Lack of N regions in antigen receptor variable region genes of TdT-deficient lymphocytes. Science 261: 1171–1175. PMID: 8356451 6. Harrington J, Hsieh CL, Gerton J, Bosma G, Lieber MR (1992) Analysis of the defect in DNA end joining in the murine scid mutation. Mol Cell Biol 12: 4758–4768. PMID: 1406659 7. Gottlieb TM, Jackson SP (1993) The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 72: 131–142. PMID: 8422676 8. Shih HY, Krangel MS (2013) Chromatin architecture, CCCTC-binding factor, and V(D)J recombination: managing long-distance relationships at antigen receptor loci. J Immunol 190: 4915–4921. doi: 10. 4049/jimmunol.1300218 PMID: 23645930 9. van der Burg M, Ijspeert H, Verkaik NS, Turul T, Wiegant WW, et al. (2009) A DNA-PKcs mutation in a radiosensitive T-B- SCID patient inhibits Artemis activation and nonhomologous end-joining. J Clin In- vest 119: 91–98. doi: 10.1172/JCI37141 PMID: 19075392 10. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663–676. PMID: 16904174 11. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861–872. PMID: 18035408 12. Garate Z, Davis BR, Quintana-Bustamante O, Segovia JC (2013) New frontier in regenerative medi- cine: site-specific gene correction in patient-specific induced pluripotent stem cells. Hum Gene Ther 24: 571–583. doi: 10.1089/hum.2012.251 PMID: 23675640 13. Choi KD, Yu J, Smuga-Otto K, Salvagiotto G, Rehrauer W, et al. (2009) Hematopoietic and endothelial differentiation of human induced pluripotent stem cells. Stem Cells 27: 559–567. doi: 10.1634/ stemcells.2008-0922 PMID: 19259936 14. Irion S, Clarke RL, Luche H, Kim I, Morrison SJ, et al. Author Contributions Conceived and designed the experiments: SHR JK CM JKJ AS TCat. Performed the experi- ments: SHR JK HR CRe TM JA PF MLM TCan CRu. Analyzed the data: SHR JK HR CRe TM JA PF MLM TCan CRu CM JKJ AS TCat. Wrote the paper: SHR JK TCat. Conceived and designed the experiments: SHR JK CM JKJ AS TCat. Performed the experi- ments: SHR JK HR CRe TM JA PF MLM TCan CRu. Analyzed the data: SHR JK HR CRe TM JA PF MLM TCan CRu CM JKJ AS TCat. Wrote the paper: SHR JK TCat. SCID Disease Modeling Krüger, Nico Lachmann, Dietrich Lesinski, Ute Modlich and Bernd Schiedlmeier for experi- mental advice and critical discussions. PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 Acknowledgments We thank Fabienne Lütge and Jessica Wenzl for technical support, Masumi Abe and Stephan Weger for plasmids, Juan Carlos Zúñiga-Pflücker for cells, and Nasreen Hoque, Andreas 17 / 21 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 SCID Disease Modeling 19. Themeli M, Kloss CC, Ciriello G, Fedorov VD, Perna F, et al. (2013) Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat Biotechnol 31: 928– 933. doi: 10.1038/nbt.2678 PMID: 23934177 20. Lachmann N, Happle C, Ackermann M, Luttge D, Wetzke M, et al. (2014) Gene correction of human in- duced pluripotent stem cells repairs the cellular phenotype in pulmonary alveolar proteinosis. Am J Respir Crit Care Med 189: 167–182. doi: 10.1164/rccm.201306-1012OC PMID: 24279725 21. Liu GH, Suzuki K, Li M, Qu J, Montserrat N, et al. (2014) Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs. Nat Commun 5: 4330. doi: 10.1038/ ncomms5330 PMID: 24999918 22. Amabile G, Welner RS, Nombela-Arrieta C, D'Alise AM, Di Ruscio A, et al. (2013) In vivo generation of transplantable human hematopoietic cells from induced pluripotent stem cells. Blood 121: 1255–1264. doi: 10.1182/blood-2012-06-434407 PMID: 23212524 23. Suzuki N, Yamazaki S, Yamaguchi T, Okabe M, Masaki H, et al. (2013) Generation of engraftable he- matopoietic stem cells from induced pluripotent stem cells by way of teratoma formation. Mol Ther 21: 1424–1431. doi: 10.1038/mt.2013.71 PMID: 23670574 24. Schmitt TM, Zuniga-Pflucker JC (2002) Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17: 749–756. PMID: 12479821 25. Schmitt TM, de Pooter RF, Gronski MA, Cho SK, Ohashi PS, et al. (2004) Induction of T cell develop- ment and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat Immunol 5: 410–417. PMID: 15034575 26. Silva G, Poirot L, Galetto R, Smith J, Montoya G, et al. (2011) Meganucleases and other tools for tar- geted genome engineering: perspectives and challenges for gene therapy. Curr Gene Ther 11: 11–27. PMID: 21182466 27. Rahman SH, Maeder ML, Joung JK, Cathomen T (2011) Zinc-finger nucleases for somatic gene thera- py: the next frontier. Hum Gene Ther 22: 925–933. doi: 10.1089/hum.2011.087 PMID: 21631241 28. Joung JK, Sander JD (2013) TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 14: 49–55. doi: 10.1038/nrm3486 PMID: 23169466 29. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816–821. doi: 10.1126/science. 1225829 PMID: 22745249 30. Carroll D (2011) Genome engineering with zinc-finger nucleases. Genetics 188: 773–782. doi: 10. 1534/genetics.111.131433 PMID: 21828278 31. PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 References (2010) Temporal specification of blood progeni- tors from mouse embryonic stem cells and induced pluripotent stem cells. Development 137: 2829– 2839. doi: 10.1242/dev.042119 PMID: 20659975 15. Zou J, Sweeney CL, Chou BK, Choi U, Pan J, et al. (2011) Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting. Blood 117: 5561–5572. doi: 10.1182/blood-2010-12-328161 PMID: 21411759 16. Zou J, Mali P, Huang X, Dowey SN, Cheng L (2011) Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease. Blood 118: 4599–4608. doi: 10. 1182/blood-2011-02-335554 PMID: 21881051 17. Tolar J, Park IH, Xia L, Lees CJ, Peacock B, et al. (2011) Hematopoietic differentiation of induced plu- ripotent stem cells from patients with mucopolysaccharidosis type I (Hurler syndrome). Blood 117: 839–847. doi: 10.1182/blood-2010-05-287607 PMID: 21037085 18. Kennedy M, Awong G, Sturgeon CM, Ditadi A, LaMotte-Mohs R, et al. (2012) T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentia- tion cultures. Cell Rep 2: 1722–1735. doi: 10.1016/j.celrep.2012.11.003 PMID: 23219550 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 18 / 21 SCID Disease Modeling 40. Voelkel C, Galla M, Maetzig T, Warlich E, Kuehle J, et al. (2010) Protein transduction from retroviral Gag precursors. Proc Natl Acad Sci U S A 107: 7805–7810. doi: 10.1073/pnas.0914517107 PMID: 20385817 41. Kienker LJ, Shin EK, Meek K (2000) Both V(D)J recombination and radioresistance require DNA-PK ki- nase activity, though minimal levels suffice for V(D)J recombination. Nucleic Acids Res 28: 2752– 2761. PMID: 10908332 42. van der Burg M, van Veelen LR, Verkaik NS, Wiegant WW, Hartwig NG, et al. (2006) A new type of ra- diosensitive T-B-NK+ severe combined immunodeficiency caused by a LIG4 mutation. J Clin Invest 116: 137–145. PMID: 16357942 43. Moshous D, Callebaut I, de Chasseval R, Corneo B, Cavazzana-Calvo M, et al. (2001) Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency. Cell 105: 177–186. PMID: 11336668 44. Cagdas D, Ozgur TT, Asal GT, Revy P, De Villartay JP, et al. (2012) Two SCID cases with Cernunnos- XLF deficiency successfully treated by hematopoietic stem cell transplantation. Pediatr Transplant 16: E167–171. doi: 10.1111/j.1399-3046.2011.01491.x PMID: 21535335 45. Soldner F, Laganiere J, Cheng AW, Hockemeyer D, Gao Q, et al. (2011) Generation of isogenic pluripo- tent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 146: 318–331. doi: 10.1016/j.cell.2011.06.019 PMID: 21757228 46. Wang Y, Zheng CG, Jiang Y, Zhang J, Chen J, et al. (2012) Genetic correction of beta-thalassemia pa- tient-specific iPS cells and its use in improving hemoglobin production in irradiated SCID mice. Cell Res 22: 637–648. doi: 10.1038/cr.2012.23 PMID: 22310243 47. Morishima T, Watanabe K, Niwa A, Hirai H, Saida S, et al. (2014) Genetic correction of HAX1 in induced pluripotent stem cells from a patient with severe congenital neutropenia improves defective granulopoi- esis. Haematologica 99: 19–27. doi: 10.3324/haematol.2013.083873 PMID: 23975175 48. Lesinski DA, Heinz N, Pilat-Carotta S, Rudolph C, Jacobs R, et al. (2012) Serum- and stromal cell-free hypoxic generation of embryonic stem cell-derived hematopoietic cells in vitro, capable of multilineage repopulation of immunocompetent mice. Stem Cells Transl Med 1: 581–591. doi: 10.5966/sctm.2012- 0020 PMID: 23197864 49. Holmes R, Zuniga-Pflucker JC (2009) The OP9-DL1 system: generation of T-lymphocytes from embry- onic or hematopoietic stem cells in vitro. Cold Spring Harb Protoc 2009: pdb prot5156. 50. Reimann C, Six E, Dal-Cortivo L, Schiavo A, Appourchaux K, et al. PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 Araki R, Fujimori A, Hamatani K, Mita K, Saito T, et al. (1997) Nonsense mutation at Tyr-4046 in the DNA-dependent protein kinase catalytic subunit of severe combined immune deficiency mice. Proc Natl Acad Sci U S A 94: 2438–2443. PMID: 9122213 32. Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, et al. (2008) Rapid "open- source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31: 294–301. doi: 10.1016/j.molcel.2008.06.016 PMID: 18657511 33. Shao RG, Cao CX, Zhang H, Kohn KW, Wold MS, et al. (1999) Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes. EMBO J 18: 1397–1406. PMID: 10064605 34. Biedermann KA, Sun JR, Giaccia AJ, Tosto LM, Brown JM (1991) scid mutation in mice confers hyper- sensitivity to ionizing radiation and a deficiency in DNA double-strand break repair. Proc Natl Acad Sci U S A 88: 1394–1397. PMID: 1996340 35. Warlich E, Kuehle J, Cantz T, Brugman MH, Maetzig T, et al. (2011) Lentiviral vector design and imag- ing approaches to visualize the early stages of cellular reprogramming. Mol Ther 19: 782–789. doi: 10. 1038/mt.2010.314 PMID: 21285961 36. Molina-Estevez FJ, Lozano ML, Navarro S, Torres Y, Grabundzija I, et al. (2013) Brief report: impaired cell reprogramming in nonhomologous end joining deficient cells. Stem Cells 31: 1726–1730. doi: 10. 1002/stem.1406 PMID: 23630174 37. Muller LU, Milsom MD, Harris CE, Vyas R, Brumme KM, et al. (2012) Overcoming reprogramming resis- tance of Fanconi anemia cells. Blood 119: 5449–5457. doi: 10.1182/blood-2012-02-408674 PMID: 22371882 38. Maherali N, Hochedlinger K (2009) Tgfbeta signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc. Curr Biol 19: 1718–1723. doi: 10.1016/j.cub.2009.08.025 PMID: 19765992 39. Hanna J, Markoulaki S, Mitalipova M, Cheng AW, Cassady JP, et al. (2009) Metastable pluripotent states in NOD-mouse-derived ESCs. Cell Stem Cell 4: 513–524. doi: 10.1016/j.stem.2009.04.015 PMID: 19427283 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 19 / 21 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 (2012) Human T-lymphoid progeni- tors generated in a feeder-cell-free Delta-like-4 culture system promote T-cell reconstitution in NOD/ SCID/gammac(-/-) mice. Stem Cells 30: 1771–1780. doi: 10.1002/stem.1145 PMID: 22689616 51. Gabriel R, Lombardo A, Arens A, Miller JC, Genovese P, et al. (2011) An unbiased genome-wide analy- sis of zinc-finger nuclease specificity. Nat Biotechnol 29: 816–823. doi: 10.1038/nbt.1948 PMID: 21822255 52. Pattanayak V, Ramirez CL, Joung JK, Liu DR (2011) Revealing off-target cleavage specificities of zinc- finger nucleases by in vitro selection. Nat Methods 8: 765–770. doi: 10.1038/nmeth.1670 PMID: 21822273 53. Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, et al. (2001) Stimulation of homologous recom- bination through targeted cleavage by chimeric nucleases. Mol Cell Biol 21: 289–297. PMID: 11113203 54. Handel EM, Alwin S, Cathomen T (2009) Expanding or restricting the target site repertoire of zinc-finger nucleases: the inter-domain linker as a major determinant of target site selectivity. Mol Ther 17: 104– 111. doi: 10.1038/mt.2008.233 PMID: 19002164 55. Szczepek M, Brondani V, Buchel J, Serrano L, Segal DJ, et al. (2007) Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol 25: 786–793. PMID: 17603476 56. Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, et al. (2007) An improved zinc-finger nuclease ar- chitecture for highly specific genome editing. Nat Biotechnol 25: 778–785. PMID: 17603475 57. Porter DL, Levine BL, Kalos M, Bagg A, June CH (2011) Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 365: 725–733. doi: 10.1056/NEJMoa1103849 PMID: 21830940 58. Tebas P, Stein D, Tang WW, Frank I, Wang SQ, et al. (2014) Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med 370: 901–910. doi: 10.1056/NEJMoa1300662 PMID: 24597865 59. Alwin S, Gere MB, Guhl E, Effertz K, Barbas CF 3rd, et al. (2005) Custom zinc-finger nucleases for use in human cells. Mol Ther 12: 610–617. PMID: 16039907 20 / 21 PLOS Genetics \| DOI:10.1371/journal.pgen.1005239 May 22, 2015 SCID Disease Modeling 60. Puttaraju M, DiPasquale J, Baker CC, Mitchell LG, Garcia-Blanco MA (2001) Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing. Mol Ther 4: 105–114. PMID: 11482981 61. Söllü C, Pars K, Cornu TI, Thibodeau-Beganny S, Maeder ML, et al. (2010) Autonomous zinc-finger nu- clease pairs for targeted chromosomal deletion. Nucleic Acids Res 38: 8269–8276. doi: 10.1093/nar/ gkq720 PMID: 20716517 62. Kuehle J, Turan S, Cantz T, Hoffmann D, Suerth JD, et al. (2014) Modified lentiviral LTRs allow Flp recombinase-mediated cassette exchange and in vivo tracing of "factor-free" induced pluripotent stem cells. Mol Ther 22: 919–928. doi: 10.1038/mt.2014.4 PMID: 24434935 63. Cathomen T, Collete D, Weitzman MD (2000) A chimeric protein containing the N terminus of the adeno-associated virus Rep protein recognizes its target site in an in vivo assay. J Virol 74: 2372– 2382. PMID: 10666268 64. Mussolino C, Alzubi J, Fine EJ, Morbitzer R, Cradick TJ, et al. (2014) TALENs facilitate targeted ge- nome editing in human cells with high specificity and low cytotoxicity. Nucleic Acids Res 42: 6762– 6773. doi: 10.1093/nar/gku305 PMID: 24792154 65. Schambach A, Bohne J, Chandra S, Will E, Margison GP, et al. (2006) Equal potency of gammaretro- viral and lentiviral SIN vectors for expression of O6-methylguanine-DNA methyltransferase in hemato- poietic cells. Mol Ther 13: 391–400. PMID: 16226060 66. Rudolph C, Schlegelberger B (2009) Spectral karyotyping and fluorescence in situ hybridization of mu- rine cells. Methods Mol Biol 506: 453–466. doi: 10.1007/978-1-59745-409-4_30 PMID: 19110644 67. Hechinger AK, Maas K, Durr C, Leonhardt F, Prinz G, et al. (2013) Inhibition of protein geranylgeranyla- tion and farnesylation protects against graft-versus-host disease via effects on CD4 effector T cells. Haematologica 98: 31–40. doi: 10.3324/haematol.2012.065789 PMID: 22801964 21 / 21
https://openalex.org/W4235204946	https://zenodo.org/records/1556817/files/article.pdf	German	null	Venusphase	Astronomische Nachrichten	1,915	public-domain	605	I n h a l t zu .Nr. 4848. Th. Epstein. Rotationselemente der Sonne. 401. - C. V. L. CharZiey. Die galaktische Verteilung der Helium-Sterne. 403. - M. WoZJ Eigenbewegungen aus der Umgebung von -004132 Aquarii. 407. - A. A6etti. Corneta 1915 e (TayZor). 407. - 7. SedZkici.K. Beobachtungen von Meteoren 1915. 409. - Mitteilungen iiber Kleine Planeten. 411. - H. GeeZmuyden. Occultation des Plkiades 1916 Mars 9. 413. - Mitteilungen iiber Kometen. 415. - 172. JVo& Veranderlicher 10.1916 Comae und S Comae. 415. - Ph. Fuath. Venusphase. 41s. - Berichtipunpen. AI<. hl b H b ld C i Geschlossen 1916 Juni 13. Heraurgeber: H. Kobold. Druck YOU C . Schaidt. Expedition: Kiel, Moltkestr. 80. Postsch 4848 4848 415 1917 U '8 logr logA H Merid. Jan. 1.5 1 6 ~ 5" -. 601 2.68 3.32 6 21h6 Febr.o.5 17 2 t. 5 . 2 2.42 2.79 10 20.3 Marz 2.5 18 11 - 1.8 2 . 1 7 2.29 19 19.5 April 1.5 19 21 +. 4.4' 1.96 1.78 35 18.7 Mai 1.5 2 0 35 + r 2 . 5 1.79 1.54 60 18.0 Juni 0.5 2 1 43 +19.9 1.70 1.33 90 1 7 . 1 Juli 0.5 2 2 48 +24.3 1.70 1.15 120 16.3 1917 a 8 log r log A H Merid: Juli 30.5 ~ 3 ~ 2 8 ~ +2203 1.79 1.03 130 15ho Aug. 29.5 2 3 56 +1.3.1 1-95 0.98 12.0 13.4 Sept. 28.5 23 38 + 0.9 2.16 1.17 7 0 ' 11.2 Okt. 28.5 23 40 - 6.7 2.41 1.58 32 9.3 Nov. 27.5 o I - 8.7 2.67 2.15 14 7.6 Dez. 27.5 o 18 - 7.6 2.95 2.80 7 6.0 , Bei Untersuchung von Eigenbewegungen in Coma stieD ich auf zwei Veranderliche, von denen der eine sich als S Comae erwies, wahrend der andere unbekannt zu sein scheint. Nach ungefahrer Schatzung hatten die beiden Veranderlichen die Helligkeiten: S 10.1916 1892 Marz 26.5 1om3 14* 1901 Jan. 17.6 9.3 11.5 1903 Marz 23.4 9.3 11.5 S 10.1916, 1906 Marz 20.4 I O ? ~ r r m 5 1916 April 3.4 10.3 14 . Der Variable 10.19 16 steht I' nordnordwestlich von einem Stern 10. Groi3e und hat den Ort: wahrend ich fur S Comae ablese: 1855.0 12h14m15S 3-29' 2!2 1855.0 1 2 25 33 f 2 7 49.7 . Bei Untersuchung von Eigenbewegungen in Coma stieD ich auf zwei Veranderliche, von denen der eine sich als S Comae erwies, wahrend der andere unbekannt zu sein scheint. Nach ungefahrer Schatzung hatten die beiden Veranderlichen die Helligkeiten: S 10.1916 Der Variable 10.19 16 steht I' nordnordwestlich von Der Variable 10.19 16 steht I' nordnordwestlich von einem Stern 10. Groi3e und hat den Ort: keiten: S 10.1916 1892 Marz 26.5 1om3 14* 1901 Jan. 17.6 9.3 11.5 1903 Marz 23.4 9.3 11.5 wahrend ich fur S Comae ablese: 1855.0 12h14m15S 3-29' 2!2 1855.0 1 2 25 33 f 2 7 49.7 . I n h a l t zu .Nr. 4848. Th. Epstein. Rotationselemente der Sonne. 401. - C. V. L. CharZiey. Die galaktische Verteilung der Helium-Sterne. 403. - M. WoZJ Eigenbewegungen aus der Umgebung von -004132 Aquarii. 407. - A. A6etti. Corneta 1915 e (TayZor). 407. - 7. SedZkici.K. Beobachtungen von Meteoren 1915. 409. - Mitteilungen iiber Kleine Planeten. 411. - H. GeeZmuyden. Occultation des Plkiades 1916 Mars 9. 413. - Mitteilungen iiber Kometen. 415. - 172. JVo& Veranderlicher 10.1916 Comae und S Comae. 415. - Ph. Fuath. Venusphase. 41s. - Berichtipunpen. AI<. Geschlossen 1916 Juni 13. Heraurgeber: H. Kobold. Druck YOU C . Schaidt. Expedition: Kiel, Moltkestr. 80. Postscheck-Konto Nr. 6238 Hamburg 11.
https://openalex.org/W3016223762	https://art.torvergata.it/bitstream/2108/187559/2/jhep.08.2017.006.pdf	English	null	Search for direct top squark pair production in events with a Higgs or Z boson, and missing transverse momentum in s = 13 $$ \sqrt{s}=13 $$ TeV pp collisions with the ATLAS detector	The Journal of high energy physics/The journal of high energy physics	2,017	cc-by	28,500	Published for SISSA by Springer Received: June 14, 2017 Accepted: July 20, 2017 Published: August 1, 2017 Received: June 14, 2017 Accepted: July 20, 2017 Published: August 1, 2017 Search for direct top squark pair production in events with a Higgs or Z boson, and missing transverse momentum in √s = 13 TeV pp collisions with the ATLAS detector Search for direct top squark pair production in events with a Higgs or Z boson, and missing transverse momentum in √s = 13 TeV pp collisions with the ATLAS detector JHEP08(2017)006 Contents 1 Introduction 2 ATLAS detector 3 Data set and simulated event samples 4 Event selection 4.1 3ℓ1b selection 4.2 1ℓ4b selection JHEP08(2017)006 6 Systematic uncertainties 7 Results 8 Conclusion The ATLAS collaboration 29 E-mail: atlas.publications@cern.ch Contents 1 Introduction 1 2 ATLAS detector 3 3 Data set and simulated event samples 3 4 Event selection 5 4.1 3ℓ1b selection 7 4.2 1ℓ4b selection 7 5 Background estimation 8 5.1 Background estimation in the 3ℓ1b selection 9 5.2 Background estimation in the 1ℓ4b selection 10 6 Systematic uncertainties 14 7 Results 15 8 Conclusion 21 The ATLAS collaboration 29 Contents 1 Introduction 1 2 ATLAS detector 3 3 Data set and simulated event samples 3 4 Event selection 5 4.1 3ℓ1b selection 7 4.2 1ℓ4b selection 7 5 Background estimation 8 5.1 Background estimation in the 3ℓ1b selection 9 5.2 Background estimation in the 1ℓ4b selection 10 6 Systematic uncertainties 14 7 Results 15 8 Conclusion 21 The ATLAS collaboration 29 Contents 1 Introduction 1 2 ATLAS detector 3 3 Data set and simulated event samples 3 4 Event selection 5 4.1 3ℓ1b selection 7 4.2 1ℓ4b selection 7 5 Background estimation 8 5.1 Background estimation in the 3ℓ1b selection 9 5.2 Background estimation in the 1ℓ4b selection 10 6 Systematic uncertainties 14 7 Results 15 8 Conclusion 21 The ATLAS collaboration 29 E-mail: atlas.publications@cern.ch Abstract: A search for direct top squark pair production resulting in events with either a same-ﬂavour opposite-sign dilepton pair with invariant mass compatible with a Z boson or a pair of jets compatible with a Standard Model (SM) Higgs boson (h) is presented. Requirements on the missing transverse momentum, together with additional selections on leptons, jets, jets identiﬁed as originating from b-quarks are imposed to target the other decay products of the top squark pair. The analysis is performed using proton-proton collision data at √s = 13 TeV collected with the ATLAS detector at the LHC in 2015– 2016, corresponding to an integrated luminosity of 36.1 fb−1. No excess is observed in the data with respect to the SM predictions. The results are interpreted in two sets of models. In the ﬁrst set, direct production of pairs of lighter top squarks (˜t1) with long decay chains involving Z or Higgs bosons is considered. The second set includes direct pair production of the heavier top squark pairs (˜t2) decaying via ˜t2 →Z˜t1 or ˜t2 →h˜t1. The results exclude at 95% conﬁdence level ˜t2 and ˜t1 masses up to about 800 GeV, extending the exclusion region of supersymmetric parameter space covered by previous LHC searches. Keywords: Beyond Standard Model, Hadron-Hadron scattering (experiments), Higgs physics ArXiv ePrint: 1706.03986 ArXiv ePrint: 1706.03986 https://doi.org/10.1007/JHEP08(2017)006 Open Access, Copyright CERN, for the beneﬁt of the ATLAS Collaboration. Article funded by SCOAP3. Open Access, Copyright CERN, for the beneﬁt of the ATLAS Collaboration. Article funded by SCOAP3. 1 Introduction Supersymmetry (SUSY) [1–6] is one of the most studied extensions of the Standard Model (SM). It predicts new bosonic partners for the existing fermions and fermionic partners for the known bosons. If R-parity is conserved [7], SUSY particles are produced in pairs and the lightest supersymmetric particle (LSP) is stable, providing a possible dark-matter candidate. The SUSY partners of the charged (neutral) Higgs bosons and electroweak gauge bosons mix to form the mass eigenstates known as charginos (˜χ± k , k = 1, 2) and neutralinos (˜χ0 m, m = 1, . . . , 4), where the increasing index denotes increasing mass. The scalar partners of right-handed and left-handed quarks, ˜qR and ˜qL, mix to form two mass eigenstates, ˜q1 and ˜q2, with ˜q1 deﬁned to be the lighter of the two. To address the SM hierarchy problem [8–11], TeV-scale masses are required [12, 13] for the supersymmetric partners of the gluons (gluinos, ˜g) and the top squarks [14, 15]. Furthermore, the higgsino is required not to be heavier than a few hundred GeV. Top squark production with Higgs (h) or Z bosons in the decay chain can appear either in production of the lighter top squark mass eigenstate (˜t1) decaying via ˜t1 → t˜χ0 2 with ˜χ0 2 →h/Z ˜χ0 1, or in production of the heavier top squark mass eigenstate (˜t2) – 1 – ˜t1 ˜t1 ˜χ0 2 ˜χ0 2 p p t ˜χ0 1 h t ˜χ0 1 Z (a) ˜t2 ˜t2 ˜t1 ˜t1 p p h ˜χ0 1 t Z ˜χ0 1 t (b) Figure 1. Diagrams for the top squark pair production processes considered in this analysis: (a) ˜t1 →t˜χ0 2 and ˜χ0 2 →h/Z ˜χ0 1 decays, and (b) ˜t2 →h/Z˜t1 and ˜t1 →t˜χ0 1 decays. ˜t2 ˜t2 ˜t1 ˜t1 p p h ˜χ0 1 t Z ˜χ0 1 t (b) ˜t1 ˜t1 ˜χ0 2 ˜χ0 2 p p t ˜χ0 1 h t ˜χ0 1 Z (a) h h JHEP08(2017)006 (a) (b) (a) Figure 1. Diagrams for the top squark pair production processes considered in this analysis: (a) ˜t1 →t˜χ0 2 and ˜χ0 2 →h/Z ˜χ0 1 decays, and (b) ˜t2 →h/Z˜t1 and ˜t1 →t˜χ0 1 decays. decaying via ˜t2 →h/Z˜t1, as illustrated in ﬁgure 1. 1ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) in the centre of the detector and the z-axis along the beam pipe. The x-axis points from the IP to the centre of the LHC ring, and the y-axis points upward. Cylindrical coordinates (r, φ) are used in the transverse plane, φ being the azimuthal angle around the beam pipe. The pseudorapidity is deﬁned in terms of the polar angle θ as η = −ln tan(θ/2). Rapidity is deﬁned as y = 0.5 ln [(E + pz)/(E −pz)] where E denotes the energy and pz is the component of the momentum along the beam direction. 1 Introduction Such signals can be discriminated from the SM top quark pair production (t¯t) background by requiring a pair of b-tagged jets originating from the h →b¯b decay or a same-ﬂavour opposite-sign lepton pair originating from the Z →ℓ+ℓ−decay. Although the pair production of ˜t1 has a cross-section larger than that of the ˜t2, and their decay properties can be similar, searches for the latter can provide additional sensitivity in regions where the ˜t1 falls in a phase space diﬃcult to experimentally discriminate from the background due to the similarities in kinematics with t¯t pair production, such as scenarios where the lighter top squark is only slightly heavier than the sum of the masses of the top quark and the lightest neutralino (˜χ0 1). Simpliﬁed models [16–18] are used for the analysis optimisation and interpretation of the results. In these models, direct top squark pair production is considered and all SUSY particles are decoupled except for the top squarks and the neutralinos involved in their decay. In all cases the ˜χ0 1 is assumed to be the LSP. Simpliﬁed models featuring direct ˜t1 production with ˜t1 →t˜χ0 2 and either ˜χ0 2 →Z ˜χ0 1 or ˜χ0 2 →h˜χ0 1 are considered. Additional simpliﬁed models featuring direct ˜t2 production with ˜t2 →Z˜t1 or ˜t2 →h˜t1 decays and ˜t1 →t˜χ0 1 are also considered, where the mass diﬀerence between the lighter top squark and the neutralino is set to 180 GeV, a region of the mass parameter space not excluded by previous searches for ˜t1 with mass greater than 191 GeV [19]. This paper presents the results of a search for top squarks in ﬁnal states with h or Z bosons at √s = 13 TeV using the data collected by the ATLAS experiment [20] in proton- proton (pp) collisions during 2015 and 2016, corresponding to 36.1 fb−1. Searches for direct ˜t1 pair production have been performed by the ATLAS Collaboration at √s = 7, 8 TeV using LHC Run-1 data [19, 21] and √s = 13 TeV with 2015 data [22] and by the CMS Collaboration at √s = 8 TeV [23–28], searches for direct ˜t2 production were performed at √s = 8 TeV by both collaborations [19, 29, 30]. 2 2 2 ATLAS detector 2 The ATLAS experiment [20] is a multi-purpose particle detector with a forward-backward symmetric cylindrical geometry and nearly 4π coverage in solid angle.1 The interaction point is surrounded by an inner detector (ID) for tracking, a calorimeter system, and a muon spectrometer. The ID provides precision tracking of charged particles for pseudorapidities \|η\| < 2.5 and is surrounded by a superconducting solenoid providing a 2 T axial magnetic ﬁeld. It consists of silicon pixel and microstrip detectors inside a transition radiation tracker. One signiﬁcant upgrade for the running period at √s = 13 TeV is the presence of the insertable B-layer [31], an additional pixel layer close to the interaction point, which provides high- resolution hits at small radius to improve the tracking performance. JHEP08(2017)006 In the pseudorapidity region \|η\| < 3.2, high-granularity lead/liquid-argon (LAr) elec- tromagnetic (EM) sampling calorimeters are used. A steel/scintillator tile calorimeter measures hadron energies for \|η\| < 1.7. The endcap and forward regions, spanning 1.5 < \|η\| < 4.9, are instrumented with LAr calorimeters for both the EM and hadronic energy measurements. The muon spectrometer consists of three large superconducting toroids with eight coils each, and a system of trigger and precision-tracking chambers, which provide triggering and tracking capabilities in the ranges \|η\| < 2.4 and \|η\| < 2.7, respectively. A two-level trigger system is used to select events [32]. The ﬁrst-level trigger is imple- mented in hardware and uses a subset of the detector information. This is followed by the software-based high-level trigger stage, which runs oﬄine reconstruction and calibration software, reducing the event rate to about 1 kHz. 3 Data set and simulated event samples The data were collected by the ATLAS detector during 2015 with a peak instantaneous luminosity of L = 5.2 × 1033 cm−2s−1, and during 2016 with a peak instantaneous lumi- nosity of L = 1.4 × 1034 cm−2s−1, resulting in a mean number of additional pp interactions per bunch crossing (pile-up) of ⟨µ⟩= 14 in 2015 and ⟨µ⟩= 24 in 2016. Data quality requirements are applied to ensure that all subdetectors were operating at nominal con- ditions, and that LHC beams were in stable-collision mode. The integrated luminosity of the resulting data set is 36.1 fb−1 with an uncertainty of ±3.2%. The luminosity and its uncertainty are derived following a methodology similar to that detailed in ref. [33] from a preliminary calibration of the luminosity scale using a pair of x-y beam-separation scans performed in August 2015 and May 2016. 3 Physics process Generator Parton shower Cross-section PDF set Tune normalisation SUSY Signals MadGraph5 aMC@NLO 2.2.3 [39] Pythia 8.186 [40] NLO+NLL [41–45] NNPDF2.3LO [46] A14 [47] W(→ℓν) + jets Sherpa 2.2.1 [48] Sherpa 2.2.1 NNLO [49] NLO CT10 [46] Sherpa default Z/γ∗(→ℓℓ) + jets Sherpa 2.2.1 Sherpa 2.2.1 NNLO [49] NLO CT10 Sherpa default t¯t powheg-box v2 [50] Pythia 6.428 [51] NNLO+NNLL [52–57] NLO CT10 Perugia2012 [58] Single-top (t-channel) powheg-box v1 Pythia 6.428 NNLO+NNLL [59] NLO CT10f4 Perugia2012 Single-top (s- and Wt-channel) powheg-box v2 Pythia 6.428 NNLO+NNLL [60, 61] NLO CT10 Perugia2012 t¯tW/Z/γ∗ MadGraph5 aMC@NLO 2.2.2 Pythia 8.186 NLO [39] NNPDF2.3LO A14 Diboson Sherpa 2.2.1 Sherpa 2.2.1 Generator NLO CT10 Sherpa default t¯th MadGraph5 aMC@NLO 2.2.2 Herwig 2.7.1 [62] NLO [63] CTEQ6L1 A14 Wh, Zh MadGraph5 aMC@NLO 2.2.2 Pythia 8.186 NLO [63] NNPDF2.3LO A14 t¯tWW, t¯tt¯t MadGraph5 aMC@NLO 2.2.2 Pythia 8.186 NLO [39] NNPDF2.3LO A14 tZ, tWZ, t¯tt MadGraph5 aMC@NLO 2.2.2 Pythia 8.186 LO NNPDF2.3LO A14 Triboson Sherpa 2.2.1 Sherpa 2.2.1 Generator LO, NLO CT10 Sherpa default Table 1. Simulated signal and background event samples: the corresponding event generator, the parton shower, the cross-section normalisation, the PDF set and the underlying-event tune are shown. JHEP08(2017)006 Table 1. Simulated signal and background event samples: the corresponding event generator, the parton shower, the cross-section normalisation, the PDF set and the underlying-event tune are shown. Monte Carlo (MC) simulated event samples are used to aid in the estimation of the background from SM processes and to model the SUSY signal. 3 Data set and simulated event samples The choices of MC event generator, parton shower and hadronisation, the cross-section normalisation, the parton distribution function (PDF) set and the set of tuned parameters (tune) for the underlying event of these samples are summarised in table 1, and more details of the event generator conﬁgurations can be found in refs. [34–37]. Cross-sections calculated at next-to-next-to- leading order (NNLO) in quantum chromodynamics (QCD) including resummation of next- to-next-to-leading logarithmic (NNLL) soft-gluon terms are used for top quark production processes. For production of top quark pairs in association with vector and Higgs bosons, cross-sections calculated at next-to-leading order (NLO) are used, and the event generator cross-sections from Sherpa (at NLO for most of the processes) are used when normalising the multi-boson backgrounds. In all MC samples, except those produced by Sherpa, the EvtGen v1.2.0 program [38] is used to model the properties of the bottom and charm hadron decays. SUSY signal samples are generated from leading-order (LO) matrix elements with up to two extra partons, using the MadGraph5 aMC@NLO v2.2.3 event generator interfaced to Pythia 8.186 with the A14 tune for the modelling of the SUSY decay chain, parton showering, hadronisation and the description of the underlying event. Parton luminosities are provided by the NNPDF23LO PDF set. Jet-parton matching is realised following the CKKW-L prescription [64], with a matching scale set to one quarter of the pair-produced superpartner mass. In all cases, the mass of the top quark is ﬁxed at 172.5 GeV. Signal cross-sections are calculated to NLO in the strong coupling constant, adding the resumma- tion of soft-gluon emission at next-to-leading-logarithmic accuracy (NLO+NLL) [45, 65, 66]. The nominal cross-section and the uncertainty are based on predictions using diﬀerent PDF sets and factorisation and renormalisation scales, as described in ref. [67]. 4 To simulate the eﬀects of additional pp collisions in the same and nearby bunch cross- ings, additional interactions are generated using the soft QCD processes as provided by Pythia 8.186 with the A2 tune [68] and the MSTW2008LO PDF set [69], and overlaid onto each simulated hard-scatter event. The MC samples are reweighted so that the pile-up distribution matches the one observed in the data. 4 Event selection Candidate events are required to have a reconstructed vertex [73] with at least two associ- ated tracks with transverse momentum (pT) larger than 400 MeV which are consistent with originating from the beam collision region in the x-y plane. The vertex with the highest scalar sum of the squared transverse momentum of the associated tracks is considered to be the primary vertex of the event. Two categories of leptons (electrons and muons) are deﬁned: “candidate” and “signal” (the latter being a subset of the “candidate” leptons satisfying tighter selection criteria). Electron candidates are reconstructed from isolated electromagnetic calorimeter energy deposits matched to ID tracks and are required to have \|η\| < 2.47, a transverse momentum pT > 10 GeV, and to pass a “loose” likelihood-based identiﬁcation requirement [74, 75]. The likelihood input variables include measurements of shower shapes in the calorimeter and track properties in the ID. Muon candidates are reconstructed in the region \|η\| < 2.5 from muon spectrometer tracks matching ID tracks. Candidate muons must have pT > 10 GeV and pass the medium identiﬁcation requirements deﬁned in ref. [76], based on the number of hits in the diﬀerent ID and muon spectrometer subsystems, and on the signiﬁcance of the charge to momentum ratio q/p. Jets are reconstructed from three-dimensional energy clusters in the calorimeter [77] using the anti-kt jet clustering algorithm [78] with a radius parameter R = 0.4. Only jet candidates with pT > 30 GeV and \|η\| < 2.5 are considered as selected jets in the analysis. Jets are calibrated as described in refs. [79, 80], and the expected average energy contribution from pile-up clusters is subtracted according to the jet area [79]. In order to reduce the eﬀects of pile-up, for jets with pT < 60 GeV and \|η\| < 2.4 a signiﬁcant fraction of the tracks associated with each jet must have an origin compatible with the primary vertex, as deﬁned by the jet vertex tagger [81]. Events are discarded if they contain any jet with pT > 20 GeV not satisfying basic qual- ity selection criteria designed to reject detector noise and non-collision backgrounds [82]. Identiﬁcation of jets containing b-hadrons is performed with a multivariate discrim- inant that makes use of track impact parameters and reconstructed secondary vertices (b-tagging) [83, 84]. A requirement is chosen corresponding to a 77% average eﬃciency obtained for b-quark jets in simulated t¯t events. 3 Data set and simulated event samples The MC samples are processed through an ATLAS detector simulation [70] based on Geant4 [71] or, in the case of t¯tt and the SUSY signal samples, a fast simulation using a parameterisation of the calorimeter response and Geant4 for the other parts of the detector [72]. All MC samples are reconstructed in the same manner as the data. JHEP08(2017)006 4 Event selection The rejection factors for light-quark and 5 gluon jets, c-quark jets and τ →hadrons + ν decays in simulated t¯t events are approxi- mately 380, 12 and 54, respectively. To compensate for diﬀerences between data and MC simulation in the b-tagging eﬃciencies and mis-tag rates, correction factors are applied to the simulated samples [84]. Jet candidates within ∆R = p (∆y)2 + (∆φ)2 = 0.2 of a lepton candidate are dis- carded, unless the jet has a value of the b-tagging discriminant larger than the value corresponding to approximately 85% b-tagging eﬃciency, in which case the lepton is dis- carded since it probably originated from a semileptonic b-hadron decay. Any remaining electron candidate within ∆R = 0.4 of a non-pile-up jet, and any muon candidate within ∆R = min{0.4, 0.04 + pT(µ)/10 GeV} of a non-pile-up jet is discarded. In the latter case, if the jet has fewer than three associated tracks or the muon pT is larger than half of the jet pT, the muon is retained and the jet is discarded instead to avoid ineﬃciencies for high- energy muons undergoing signiﬁcant energy loss in the calorimeter. Any muon candidate reconstructed with ID and calorimeter information only which shares an ID track with an electron candidate is removed. Finally, any electron candidate sharing an ID track with a remaining muon candidate is also removed. JHEP08(2017)006 Tighter requirements on the lepton candidates are imposed, which are then referred to as “signal” electrons or muons. Signal electrons must satisfy the “medium” likelihood- based identiﬁcation requirement as deﬁned in refs. [74, 75]. Signal leptons must have pT > 20 GeV. The associated tracks must have a signiﬁcance of the transverse impact parameter with respect to the reconstructed primary vertex, d0, of \|d0\|/σ(d0) < 5 for elec- trons and \|d0\|/σ(d0) < 3 for muons, and a longitudinal impact parameter with respect to the reconstructed primary vertex, z0, satisfying \|z0 sin θ\| < 0.5 mm. Isolation requirements are applied to both the signal electrons and muons. The scalar sum of the pT of tracks within a variable-size cone around the lepton, excluding its own track, must be less than 6% of the lepton pT. 4 Event selection The size of the track isolation cone for electrons (muons) is given by the smaller of ∆R = 10 GeV/pT and ∆R = 0.2 (0.3), that is, a cone of size 0.2 (0.3) at low pT but narrower for high-pT leptons. In addition, in the case of electrons the energy of calorimeter energy clusters in a cone of ∆Rη = p (∆η)2 + (∆φ)2 = 0.2 around the electron (excluding the deposition from the electron itself) must be less than 6% of the electron pT. Simulated events are corrected to account for minor diﬀerences in the signal lepton trigger, reconstruction, identiﬁcation and isolation eﬃciencies between data and MC sim- ulation. The missing transverse momentum vector, whose magnitude is denoted by Emiss T , is deﬁned as the negative vector sum of the transverse momenta of all identiﬁed electrons, photons, muons and jets, and an additional soft term. The soft term is constructed from all tracks originating from the primary vertex which are not associated with any identiﬁed particle or jet. In this way, the Emiss T is adjusted for the best calibration of particles and jets listed above, while maintaining pile-up independence in the soft term [85, 86]. The events are required to have a Emiss T value above 100 GeV and are classiﬁed in a further step into two exclusive categories: at least three leptons plus a b-tagged jet (3ℓ1b selection, aimed at top squark decays involving Z bosons), or at least four b-tagged jets and one or two leptons (1ℓ4b selection, aimed at top squark decays involving Higgs bosons). 6 Requirement / Region SR3ℓ1b A SR3ℓ1b B SR3ℓ1b C Number of leptons ≥3 ≥3 ≥3 nb−tagged jets ≥1 ≥1 ≥1 \|mℓℓ−mZ\| [GeV] < 15 < 15 < 15 Leading lepton pT [GeV] > 40 > 40 > 40 Leading jet pT [GeV] > 250 > 80 > 60 Leading b-tagged jet pT [GeV] > 40 > 40 > 30 njets (pT > 30 GeV) ≥6 ≥6 ≥5 Emiss T [GeV] > 100 > 180 > 140 pℓℓ T [GeV] > 150 — < 80 Table 2. Deﬁnition of the signal regions used in the 3ℓ1b selection (see text for details). JHEP08(2017)006 Table 2. Deﬁnition of the signal regions used in the 3ℓ1b selection (see text for details). 4.1 3ℓ1b selection Events of interest are selected if they contain at least three signal leptons (electrons or muons), with at least one same-ﬂavour opposite-sign lepton pair whose invariant mass is compatible with the Z boson mass (\|mℓℓ−mZ\| < 15 GeV, with mZ = 91.2 GeV). To maximise the sensitivity in diﬀerent regions of the mass parameter space, three overlapping signal regions (SRs) are deﬁned as shown in table 2. Signal region SR3ℓ1b A is optimised for large ˜t2 −˜χ0 1 mass splitting, where the Z boson in the ˜t2 →Z˜t1 decay is boosted, and large pℓℓ T and leading-jet pT are required. Signal region SR3ℓ1b B covers the intermediate case, featuring slightly softer kinematic requirements than in SR3ℓ1b A . Signal region SR3ℓ1b C is designed to improve the sensitivity for compressed spectra (m˜t2 ≳m˜χ0 1 + mt + mZ) with softer jet-pT requirements and an upper bound on pℓℓ T. 4 Event selection In the 3ℓ1b selection, events are accepted if they pass a trigger requiring either two electrons, two muons or an electron and a muon. In the 1ℓ4b selection, events are accepted if they pass a trigger requiring an isolated electron or muon. The trigger-level requirements on the pT, identiﬁcation and isolation of the leptons involved in the trigger decision are looser than those applied oﬄine to ensure that trigger eﬃciencies are constant in the relevant phase space [32]. Additional requirements are applied depending on the ﬁnal state, as described in the following. These requirements are optimised for the best discovery signiﬁcance using the simpliﬁed models featuring ˜t2 production with ˜t2 →Z˜t1 or ˜t2 →h˜t1 decays. 4.2 1ℓ4b selection Similarly to the 3ℓ1b case, three overlapping SRs are deﬁned in the 1ℓ4b selection to have a good sensitivity in diﬀerent regions of the mass parameter space. Only events with one or two signal leptons are selected to ensure orthogonality with the SRs in the 3ℓ1b selection, with at least one lepton having pT > 30 GeV, and the electron candidates are also required 7 Requirement / Region SR1ℓ4b A SR1ℓ4b B SR1ℓ4b C Number of leptons 1–2 1–2 1–2 nb−tagged jets ≥4 ≥4 ≥4 mT [GeV] — >150 >125 HT [GeV] > 1000 — — Emiss T [GeV] > 120 > 150 > 150 Leading b-tagged jet pT [GeV] — — <140 mbb[GeV] 95–155 — — pbb T[GeV] > 300 — — njets (pT > 60 GeV) ≥6 ≥5 — njets (pT > 30 GeV) — — ≥7 Table 3. Deﬁnition of the signal regions used in the 1ℓ4b selection (see text for details). JHEP08(2017)006 Table 3. Deﬁnition of the signal regions used in the 1ℓ4b selection (see text for details). Table 3. Deﬁnition of the signal regions used in the 1ℓ4b selection (see text for details). to satisfy the tight likelihood-based identiﬁcation requirement as deﬁned in refs. [74, 75]. These SRs are deﬁned as shown in table 3. 0 Signal region SR1ℓ4b A is optimised for large ˜t2−˜χ0 1 mass splitting, where the Higgs boson in the ˜t2 →h˜t1 decay is boosted. In this signal region, the pair of b-tagged jets with the smallest ∆Rbb is required to have an invariant mass consistent with the Higgs boson mass (\|mbb −mh\| < 15 GeV, with mh = 125 GeV), and the transverse momentum of the system formed by these two b-tagged jets (pbb T) is required to be above 300 GeV. Signal region SR1ℓ4b B covers the intermediate case, featuring slightly harder kinematic requirements than SR1ℓ4b A . Finally, signal region SR1ℓ4b C is designed to be sensitive to compressed spectra (m˜t2 ≳ m˜χ0 1 +mt +mh). This region has softer jet pT requirements and an upper bound on the pT of the leading b-tagged jet. 4.2 1ℓ4b selection Signal region SR1ℓ4b A includes requirements on HT (computed as the scalar sum of the pT of all the jets in the event), while both signal regions SR1ℓ4b B and SR1ℓ4b C include requirements on the transverse mass mT computed using the missing- momentum and lepton-momentum vectors: mT = q 2pℓ TEmiss T 1 −cos[∆φ(ℓ, Emiss T )] . 5 Background estimation The main SM background processes satisfying the SR requirements are estimated by sim- ulation, which is normalised and veriﬁed (whenever possible) with data events in separate statistically independent regions of the phase space. Dedicated control regions (CRs) en- hanced in a particular background component, such as the production of top quark pairs in association with a Z boson (t¯tZ) and multi-boson production in the 3ℓ1b selection, and t¯t in the 1ℓ4b selection, are used for the normalisation. For each signal region, a simultaneous “background ﬁt” is performed to the numbers of events found in the CRs, using a minimi- sation based on likelihoods with the HistFitter package [87]. In each ﬁt, the normalisations of the background contributions having dedicated CRs are allowed to ﬂoat freely, while the other backgrounds are determined directly using simulation or from additional independent 8 studies in data. This way the total post-ﬁt prediction is forced to be equal to the number of data events in the CR and its total uncertainty is given by the data statistical uncertainty. When setting 95% conﬁdence level (CL) upper limits on the cross-section of speciﬁc SUSY models, the simultaneous ﬁts also include the observed yields in the SR. Systematic uncertainties in the MC simulation aﬀect the ratio of the expected yields in the diﬀerent regions and are taken into account to determine the uncertainty in the back- ground prediction. Each uncertainty source is described by a single nuisance parameter, and correlations between background processes and selections are taken into account. The ﬁt aﬀects neither the uncertainty nor the central value of these nuisance parameters. The systematic uncertainties considered in the ﬁt are described in section 6. JHEP08(2017)006 Whenever possible, the level of agreement of the background prediction with data is compared in dedicated validation regions (VRs), which are not used to constrain the background normalisation or nuisance parameters in the ﬁt. 5.1 Background estimation in the 3ℓ1b selection The dominant SM background contribution to the SRs in the 3ℓ1b selection is expected to be from t¯tZ, with minor contribution from multi-boson production (mainly WZ) and back- grounds containing jets misidentiﬁed as leptons (hereafter referred to as “fake” leptons) or non-prompt leptons from decays of hadrons (mainly in t¯t events). The normalisation of the main backgrounds (t¯tZ, multi-boson) is obtained by ﬁtting the yield to the ob- served data in two control regions, then extrapolating this yield to the SRs as described above. Backgrounds from other sources (t¯tW, t¯th and rare SM processes), which provide a subdominant contribution to the SRs, are determined from MC simulation only. The background from fake or non-prompt leptons is estimated from data with a method similar to that described in refs. [88, 89]. Two types of lepton identiﬁcation criteria are deﬁned for this evaluation: “tight” and “loose”, corresponding to the signal and candidate electrons and muons described in section 4. The leading lepton is considered to be prompt, which is a valid assumption in more than 95% of the cases according to simulations. The method makes use of the number of observed events with the second and third leading leptons being loose-loose, loose-tight, tight-loose and tight-tight in each region. The prob- ability for prompt leptons satisfying the loose selection criteria to also satisfy the tight selection is measured using a data sample enriched in Z →ℓ+ℓ−(ℓ= e, µ) decays. The equivalent probability for fake or non-prompt leptons is measured using events with one electron and one muon with the same charge. The number of events with one or two fake or non-prompt leptons is calculated from these probabilities and the number of observed events with loose and tight leptons. The modelling of the background from fake or non- prompt leptons is validated in events passing a selection similar to the SRs, but removing the Emiss T requirements and inverting the mℓℓrequirements. The two dedicated control regions used for the t¯tZ (CR3ℓ1b t¯tZ ) and multi-boson (CR3ℓ1b V V ) background estimation in this selection are deﬁned as shown in table 4. To ensure orthog- onality with the SRs, an upper bound on Emiss T < 100 GeV is required in CR3ℓ1b t¯tZ , while a b-jet veto is applied in CR3ℓ1b V V . 5.1 Background estimation in the 3ℓ1b selection Events 20 40 60 80 100 120 ATLAS -1 = 13 TeV, 36.1 fb s VV 3l1b CR Data Total SM background Fake and non-prompt leptons Multi-boson Z tt tZ, tWZ Others > 30 GeV) T Jet multiplicity (p 4 5 6 7 8 9 Data / SM 0 1 2 (b) Events 10 20 30 40 50 60 70 ATLAS -1 = 13 TeV, 36.1 fb s Z tt 3l1b CR Data Total SM background Fake and non-prompt leptons Multi-boson Z tt tZ, tWZ Others > 30 GeV) T Jet multiplicity (p 4 5 6 7 8 9 Data / SM 0 1 2 (a) (b) (a) Figure 2. Jet multiplicity distributions in control regions (a) CR3ℓ1b t¯tZ and (b) CR3ℓ1b V V , after normal- ising the t¯tZ and multi-boson background processes via the simultaneous ﬁt described in section 5. The contributions from all SM backgrounds are shown as a histogram stack; the bands represent the total uncertainty in the background prediction. The “Others” category contains the contributions from t¯th, t¯tWW, t¯tt, t¯tt¯t, Wh, and Zh production. The last bin in each ﬁgure contains the overﬂow. The lower panels show the ratio of the observed data to the total SM background prediction, with bands representing the total uncertainty in the background prediction. Table 5 shows the observed and expected yields in the two CRs for each background source, and ﬁgure 2 shows the njet distribution in these regions after the background ﬁt. The normalisation factors for the t¯tZ and multi-boson backgrounds do not diﬀer from unity by more than 30% and the post-ﬁt MC-simulated jet multiplicity distributions agree well with the data. 5.1 Background estimation in the 3ℓ1b selection 9 Requirement / Region CR3ℓ1b t¯tZ CR3ℓ1b V V Number of leptons ≥3 ≥3 \|mℓℓ−mZ\| [GeV] < 15 < 15 Leading lepton pT [GeV] > 40 > 40 Leading jet pT [GeV] > 60 > 30 nb−tagged jets ≥1 0 njets(pT > 30 GeV) ≥4 ≥4 Emiss T [GeV] < 100 — pℓℓ T [GeV] — — Table 4. Deﬁnition of the control regions used in the 3ℓ1b selection. JHEP08(2017)006 Table 4. Deﬁnition of the control regions used in the 3ℓ1b selection. Events 10 20 30 40 50 60 70 ATLAS -1 = 13 TeV, 36.1 fb s Z tt 3l1b CR Data Total SM background Fake and non-prompt leptons Multi-boson Z tt tZ, tWZ Others > 30 GeV) T Jet multiplicity (p 4 5 6 7 8 9 Data / SM 0 1 2 (a) Events 20 40 60 80 100 120 ATLAS -1 = 13 TeV, 36.1 fb s VV 3l1b CR Data Total SM background Fake and non-prompt leptons Multi-boson Z tt tZ, tWZ Others > 30 GeV) T Jet multiplicity (p 4 5 6 7 8 9 Data / SM 0 1 2 (b) Figure 2. Jet multiplicity distributions in control regions (a) CR3ℓ1b t¯tZ and (b) CR3ℓ1b V V , after normal- ising the t¯tZ and multi-boson background processes via the simultaneous ﬁt described in section 5. The contributions from all SM backgrounds are shown as a histogram stack; the bands represent the total uncertainty in the background prediction. The “Others” category contains the contributions from t¯th, t¯tWW, t¯tt, t¯tt¯t, Wh, and Zh production. The last bin in each ﬁgure contains the overﬂow. The lower panels show the ratio of the observed data to the total SM background prediction, with bands representing the total uncertainty in the background prediction. 5.2 Background estimation in the 1ℓ4b selection The dominant SM background contribution to the SRs in the 1ℓ4b selection is expected to be top quark pair (t¯t) production, amounting to more than 80% of the total background. 10 CR3ℓ1b t¯tZ CR3ℓ1b V V Observed events 109 131 Total (post-ﬁt) SM events 109 ± 10 131 ± 11 Fit output, multi-boson 14.5 ± 2.7 105 ± 13 Fit output, t¯tZ 66 ± 14 10.2 ± 2.7 Fake or non-prompt leptons 14 ± 6 12 ± 7 tZ, tWZ 11 ± 6 2.7 ± 1.4 Others 3.2 ± 0.5 1.0 ± 0.3 Fit input, multi-boson 19 137 Fit input, t¯tZ 73 11.2 JHEP08(2017)006 Table 5. Background ﬁt results for the control regions in the 3ℓ1b selection. The nominal predic- tions from MC simulation are given for comparison for those backgrounds (t¯tZ, multi-boson) that are normalised to data. The “Others” category contains the contributions from t¯th, t¯tWW, t¯tt, t¯tt¯t, Wh, and Zh production. Combined statistical and systematic uncertainties are given. The individual uncertainties can be correlated and do not necessarily add in quadrature to the total systematic uncertainty. The number of events with fake or non-prompt leptons is estimated with the data-driven technique described in section 5. The normalisation of the t¯t background for each of the three SRs is obtained by ﬁtting the yield to the observed data in a dedicated CR, then extrapolating this yield to the SRs as described above. Other background sources (single top, t¯th and rare SM processes), which provide a subdominant contribution to the SRs, are determined from MC simulation only. The contribution from events with fake or non-prompt leptons is found to be negligible in this selection. The three t¯t CRs (named CR1ℓ4b t¯t,A, CR1ℓ4b t¯t,B and CR1ℓ4b t¯t,C) are described in table 6. They are designed to have kinematic properties resembling as closely as possible those of each of the three SRs (SR1ℓ4b A , SR1ℓ4b B and SR1ℓ4b C , respectively), while having a high purity in t¯t background and only a small contamination from signal. The CRs are built by inverting the SR requirements on Emiss T and relaxing or inverting those on mbb or mT. Figure 3 shows the jet multiplicity distributions in these CRs after the background ﬁt. 5.2 Background estimation in the 1ℓ4b selection CR1ℓ4b t¯t,A VR1ℓ4b A CR1ℓ4b t¯t,B VR1ℓ4b B CR1ℓ4b t¯t,C VR1ℓ4b C Observed events 863 258 340 86 963 84 Total (post-ﬁt) SM events 863 ± 29 266 ± 34 340 ± 18 96 ± 13 963 ± 31 90 ± 11 Fit output, t¯t 783 ± 33 235 ± 33 307 ± 19 88 ± 12 891 ± 33 82 ± 10 Single top 16 ± 5 9.0 ± 2.1 5.5 ± 1.8 1.7 ± 0.9 12.2 ± 2.4 2.5 ± 1.3 V +jets, multi-boson 11.8 ± 2.9 3.1 ± 1.1 4.7 ± 1.4 0.15+0.20 −0.15 9.8 ± 2.1 1.0 ± 0.4 t¯th, V h 27 ± 4 7.9 ± 1.3 12.7 ± 2.0 3.9 ± 0.7 31 ± 5 2.7 ± 0.6 t¯tW, t¯tZ 19 ± 4 7.2 ± 1.6 7.1 ± 1.7 2.2 ± 0.5 15.6 ± 3.1 1.5 ± 0.5 Others 5.0 ± 2.6 3.6 ± 1.9 3.2 ± 1.7 0.57 ± 0.31 2.7 ± 1.4 0.62 ± 0.32 Fit input, t¯t 495 148 175 50 578 53 Table 7. Background ﬁt results for the control and validation regions in the 1ℓ4b selection. The nominal predictions from MC simulation are given for comparison for the t¯t background, which is normalised to data. The “Others” category contains the contributions from t¯tWW, t¯tt, t¯tt¯t, tZ, and tWZ production. Combined statistical and systematic uncertainties are given. The individual uncertainties can be correlated and do not necessarily add in quadrature to the total systematic uncertainty. able 7. Background ﬁt results for the control and validation regions in the 1ℓ4b selection. The Table 7. Background ﬁt results for the control and validation regions in the 1ℓ4b selection. The nominal predictions from MC simulation are given for comparison for the t¯t background, which is normalised to data. The “Others” category contains the contributions from t¯tWW, t¯tt, t¯tt¯t, tZ, and tWZ production. Combined statistical and systematic uncertainties are given. The individual uncertainties can be correlated and do not necessarily add in quadrature to the total systematic uncertainty. 5.2 Background estimation in the 1ℓ4b selection In a similar manner, three validation regions (named VR1ℓ4b A , VR1ℓ4b B and VR1ℓ4b C ) are deﬁned, each of them corresponding to a diﬀerent CR, with the same requirements on Emiss T as the SR and relaxing or inverting the requirements on mbb, mT or jet multiplicity, as shown in table 6. These VRs are used to provide a statistically independent cross-check of the extrapolation in a selection close to that of the SR but with small signal contamination. Table 7 shows the observed and expected yields in the CRs and VRs for each background source. The large correction to t¯t normalisation after the background ﬁt has also been observed in other analyses [90] and is due to a mismodelling of the t¯t+b¯b, c¯c component in the MC simulation. The background prediction is in agreement with the observed data in all VRs. 11 Requirement / Region CR1ℓ4b t¯t,A VR1ℓ4b A CR1ℓ4b t¯t,B VR1ℓ4b B CR1ℓ4b t¯t,C VR1ℓ4b C Number of leptons 1–2 1–2 1–2 1–2 1–2 1–2 nb-tagged jets ≥4 ≥4 ≥4 ≥4 ≥4 ≥4 mT [GeV] — — >100 >150 <125 <125 Emiss T [GeV] <120 >120 < 150 >150 < 150 >150 Leading b-tagged jet pT [GeV] — — — — <140 <140 mbb[GeV] 95–155 /∈[95,155] — — — — njets (pT > 60 GeV) ≥5 ≥5 ≥5 ≤4 — — njets (pT > 30 GeV) — — — — ≥7 ≥7 Table 6. Summary of selection criteria for the control and validation regions in the 1ℓ4b selection. JHEP08(2017)006 able 6. Summary of selection criteria for the control and validation regions in the 1ℓ4b selection. Table 6. Summary of selection criteria for the control and validation regions in the 1ℓ4b selection. 5.2 Background estimation in the 1ℓ4b selection CR1ℓ4b t¯t,A VR1ℓ4b A CR1ℓ4b t¯t,B VR1ℓ4b B CR1ℓ4b t¯t,C VR1ℓ4b C Observed events 863 258 340 86 963 84 Total (post-ﬁt) SM events 863 ± 29 266 ± 34 340 ± 18 96 ± 13 963 ± 31 90 ± 11 Fit output, t¯t 783 ± 33 235 ± 33 307 ± 19 88 ± 12 891 ± 33 82 ± 10 Single top 16 ± 5 9.0 ± 2.1 5.5 ± 1.8 1.7 ± 0.9 12.2 ± 2.4 2.5 ± 1.3 V +jets, multi-boson 11.8 ± 2.9 3.1 ± 1.1 4.7 ± 1.4 0.15+0.20 −0.15 9.8 ± 2.1 1.0 ± 0.4 t¯th, V h 27 ± 4 7.9 ± 1.3 12.7 ± 2.0 3.9 ± 0.7 31 ± 5 2.7 ± 0.6 t¯tW, t¯tZ 19 ± 4 7.2 ± 1.6 7.1 ± 1.7 2.2 ± 0.5 15.6 ± 3.1 1.5 ± 0.5 Others 5.0 ± 2.6 3.6 ± 1.9 3.2 ± 1.7 0.57 ± 0.31 2.7 ± 1.4 0.62 ± 0.32 Fit input, t¯t 495 148 175 50 578 53 Table 7. Background ﬁt results for the control and validation regions in the 1ℓ4b selection. The nominal predictions from MC simulation are given for comparison for the t¯t background, which is normalised to data. The “Others” category contains the contributions from t¯tWW, t¯tt, t¯tt¯t, tZ, and tWZ production. Combined statistical and systematic uncertainties are given. The individual uncertainties can be correlated and do not necessarily add in quadrature to the total systematic uncertainty. 5.2 Background estimation in the 1ℓ4b selection 12 Events 200 400 600 800 1000 1200 1400 1600 ATLAS -1 = 13 TeV, 36.1 fb s , A tt 1l4b CR Data Total SM Background tt Single top h, Vh tt V+jets, multi-boson Z t W, t tt Others > 60 GeV) T Jet multiplicity (p 4 5 6 7 8 9 10 11 Data / SM 0 1 2 (a) Events 100 200 300 400 500 ATLAS -1 = 13 TeV, 36.1 fb s , B tt 1l4b CR Data Total SM Background tt Single top h, Vh tt V+jets, multi-boson Z t W, t tt Others > 60 GeV) T Jet multiplicity (p 4 5 6 7 8 9 10 11 Data / SM 0 1 2 (b) Events 200 400 600 800 1000 1200 1400 1600 1800 2000 ATLAS -1 = 13 TeV, 36.1 fb s , C tt 1l4b CR Data Total SM Background tt Single top h, Vh tt V+jets, multi-boson Z t W, t tt Others > 30 GeV) T Jet multiplicity (p 4 5 6 7 8 9 10 11 Data / SM 0 1 2 (c) Figure 3. Jet multiplicity distributions in control regions (a) CR1ℓ4b t¯t,A (b) CR1ℓ4b t¯t,B and (c) CR1ℓ4b t¯t,C after normalising the t¯t background process via the simultaneous ﬁt described in section 5. The t¯t background normalisation is constrained to the data observation for jet multiplicity values above the requirements shown in table 6. The contributions from all SM backgrounds are shown as a histogram stack; the bands represent the total uncertainty. The “Others” category contains the contributions from t¯th, t¯tWW, t¯tt, t¯tt¯t, Wh, and Zh production. The last bin in each ﬁgure contains the overﬂow. The lower panels show the ratio of the observed data to the total SM background prediction, with the bands representing the total uncertainty in the background prediction. 6 Systematic uncertainties 6 The primary sources of systematic uncertainty are related to the jet energy scale, the jet energy resolution, the theoretical and the MC modelling uncertainties in the background determined using CRs (t¯tZ and multi-bosons in the 3ℓ1b selection, as well as t¯t in the 1ℓ4b selection). The statistical uncertainty of the simulated event samples is taken into account as well. The eﬀects of the systematic uncertainties are evaluated for all signal samples and background processes. Since the normalisation of the dominant background processes is extracted in dedicated CRs, the systematic uncertainties only aﬀect the extrapolation to the SRs in these cases. JHEP08(2017)006 The jet energy scale and resolution uncertainties are derived as a function of the pT and η of the jet, as well as of the pile-up conditions and the jet ﬂavour composition (more quark- like or gluon-like) of the selected jet sample. They are determined using a combination of simulated and data samples, through measurements of the jet response asymmetry in dijet, Z+jet and γ+jet events [91]. Uncertainties associated with the modelling of the b-tagging eﬃciencies for b-jets, c-jets and light-ﬂavour jets [92, 93] are also considered. The systematic uncertainties related to the modelling of Emiss T in the simulation are estimated by propagating the uncertainties in the energy and momentum scale of all iden- tiﬁed electrons, photons, muons and jets, as well as the uncertainties in the soft-term scale and resolution [85]. Other detector-related systematic uncertainties, such as those in the lepton reconstruc- tion eﬃciency, energy scale and energy resolution, and in the modelling of the trigger [76], are found to have a small impact on the results. The uncertainties in the modelling of the t¯t and single-top backgrounds in simulation in the 1ℓ4b selection are estimated by varying the renormalisation and factorisation scales, as well as the amount of initial- and ﬁnal-state radiation used to generate the samples [34]. Additional uncertainties in the parton-shower modelling are assessed as the diﬀerence be- tween the predictions from Powheg showered with Pythia and Herwig, and due to the event generator choice by comparing Powheg and MadGraph5 aMC@NLO [34], in both cases showered with Pythia. The diboson background MC modelling uncertainties are estimated by varying the renormalisation, factorisation and resummation scales used to generate the samples [94]. 5.2 Background estimation in the 1ℓ4b selection Events 100 200 300 400 500 ATLAS -1 = 13 TeV, 36.1 fb s , B tt 1l4b CR Data Total SM Background tt Single top h, Vh tt V+jets, multi-boson Z t W, t tt Others > 60 GeV) T Jet multiplicity (p 4 5 6 7 8 9 10 11 Data / SM 0 1 2 Events 200 400 600 800 1000 1200 1400 1600 ATLAS -1 = 13 TeV, 36.1 fb s , A tt 1l4b CR Data Total SM Background tt Single top h, Vh tt V+jets, multi-boson Z t W, t tt Others > 60 GeV) T Jet multiplicity (p 4 5 6 7 8 9 10 11 Data / SM 0 1 2 JHEP08(2017)006 (a) (b) Events 200 400 600 800 1000 1200 1400 1600 1800 2000 ATLAS -1 = 13 TeV, 36.1 fb s , C tt 1l4b CR Data Total SM Background tt Single top h, Vh tt V+jets, multi-boson Z t W, t tt Others > 30 GeV) T Jet multiplicity (p 4 5 6 7 8 9 10 11 Data / SM 0 1 2 (c) (b) (a) Events 200 400 600 800 1000 1200 1400 1600 1800 2000 ATLAS -1 = 13 TeV, 36.1 fb s , C tt 1l4b CR Data Total SM Background tt Single top h, Vh tt V+jets, multi-boson Z t W, t tt Others > 30 GeV) T Jet multiplicity (p 4 5 6 7 8 9 10 11 Data / SM 0 1 2 (c) (c) Figure 3. Jet multiplicity distributions in control regions (a) CR1ℓ4b t¯t,A (b) CR1ℓ4b t¯t,B and (c) CR1ℓ4b t¯t,C after normalising the t¯t background process via the simultaneous ﬁt described in section 5. The t¯t background normalisation is constrained to the data observation for jet multiplicity values above the requirements shown in table 6. The contributions from all SM backgrounds are shown as a histogram stack; the bands represent the total uncertainty. The “Others” category contains the contributions from t¯th, t¯tWW, t¯tt, t¯tt¯t, Wh, and Zh production. The last bin in each ﬁgure contains the overﬂow. The lower panels show the ratio of the observed data to the total SM background prediction, with the bands representing the total uncertainty in the background prediction. 13 6 Systematic uncertainties The total systematic uncertainty can be diﬀerent from the sum in quadrature of individual sources due to the correlations between them resulting from the ﬁt to the data. The quoted theoretical uncertainties include modelling and cross-section uncertainties. contamination from prompt leptons in the regions used to measure the probabilities for loose fake or non-prompt leptons to pass the tight signal criteria. contamination from prompt leptons in the regions used to measure the probabilities for loose fake or non-prompt leptons to pass the tight signal criteria. Table 8 summarises the contributions of the diﬀerent sources of systematic uncertainty in the total SM background predictions in the signal regions. The dominant systematic uncertainties in the 3ℓ1b SRs are due to the limited number of events in CR3ℓ1b t¯tZ and theo- retical uncertainties in t¯tZ production, while in the 1ℓ4b SRs the dominant uncertainties are due to t¯t modelling. 6 Systematic uncertainties For t¯tZ, the predictions from the MadGraph5 aMC@NLO and Sherpa event generators are compared, and the uncertainties related to the choice of renormalisation and factorisa- tion scales are assessed by varying the corresponding event generator parameters up and down by a factor of two around their nominal values [95]. The cross-sections used to normalise the MC samples are varied according to the uncertainty in the cross-section calculation, i.e., 6% for diboson, 13% for t¯tW and 12% t¯tZ production [39]. For t¯tWW, tZ, tWZ, t¯th, t¯tt, t¯tt¯t, and triboson production processes, which constitute a small background, a 50% uncertainty in the event yields is assumed. Systematic uncertainties are assigned to the estimated background from fake or non- prompt leptons in the 3ℓ1b selection to account for potentially diﬀerent compositions (heavy ﬂavour, light ﬂavour or conversions) between the signal and control regions, as well as the 14 SR3ℓ1b A SR3ℓ1b B SR3ℓ1b C SR1ℓ4b A SR1ℓ4b B SR1ℓ4b C Total systematic uncertainty (%) 20 24 15 22 17 30 Diboson theoretical uncertainties (%) 6.7 5.5 2.2 <1 <1 <1 t¯tZ theoretical uncertainties (%) 10 10 4.4 <1 <1 <1 t¯t theoretical uncertainties (%) — — — 17 14 22 Other theoretical uncertainties (%) 9.0 6.8 5.4 1.6 2.4 1.7 MC statistical uncertainties (%) 8.5 18 6 7.3 5.2 13 Diboson ﬁtted normalisation (%) 4.6 3.5 3.8 <1 <1 <1 t¯tZ ﬁtted normalisation (%) 12 11 13 <1 <1 <1 t¯t ﬁtted normalisation (%) — — — 3.4 5.1 3.3 Fake or non-prompt leptons (%) — 6.5 — — — — Pile-up (%) 4.7 2.8 0.6 <1 1.4 <1 Jet energy resolution (%) 2.0 2.7 3.0 5.3 <1 13 Jet energy scale (%) 1.0 2.7 3.5 3.2 5.3 6.1 Emiss T resolution (%) 5.3 2.6 1.6 6.8 6.5 4.0 b-tagging (%) 2.4 1.5 3.0 6.8 2.9 3.5 Table 8. Summary of the main systematic uncertainties and their impact (in %) on the total SM background prediction in each of the signal regions studied. The total systematic uncertainty can be diﬀerent from the sum in quadrature of individual sources due to the correlations between them resulting from the ﬁt to the data. The quoted theoretical uncertainties include modelling and cross-section uncertainties. JHEP08(2017)006 Table 8. Summary of the main systematic uncertainties and their impact (in %) on the total SM background prediction in each of the signal regions studied. 7 Results Distribution of Emiss T for events passing all the signal candidate selection requirements except that on Emiss T , for (a) SR3ℓ1b A , (c) SR3ℓ1b B , (e) SR3ℓ1b C and (b) SR1ℓ4b A , (d) SR1ℓ4b B , (f) SR1ℓ4 C after the background ﬁt described in section 5. The contributions from all SM backgrounds are shown; the bands represent the total uncertainty. The expected distributions for signal models with m(˜t2) = 700 GeV and m(˜χ0 1) = 0 GeV, and m(˜t2) = 650 GeV and m(˜χ0 1) = 250 GeV are also shown as dashed lines. The last bin in each ﬁgure contains the overﬂow. The lower panels show the ratio of the observed data to the total SM background prediction, with the bands representing the tota uncertainty in the background prediction. Events / 20 GeV 1 2 3 4 5 6 ATLAS -1 = 13 TeV, 36.1 fb s A 3l1b SR Data Total SM background Fake and non-prompt leptons Multi-boson Z tt tZ, tWZ Others )=(800, 0) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 20 40 60 80 100 120 140 160 180 200 Data / SM 0 1 2 (a) Events / 20 GeV 2 4 6 8 10 12 14 16 18 20 ATLAS -1 = 13 TeV, 36.1 fb s B 3l1b SR Data Total SM background Fake and non-prompt leptons Multi-boson Z tt tZ, tWZ Others )=(700, 200) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 20 40 60 80 100 120 140 160 180 200 Data / SM 0 1 2 (c) Events / 20 GeV 2 4 6 8 10 12 14 16 ATLAS -1 = 13 TeV, 36.1 fb s C 3l1b SR Data Total SM background Fake and non-prompt leptons Multi-boson Z tt tZ, tWZ Others )=(650, 300) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 20 40 60 80 100 120 140 160 180 200 Data / SM 0 1 2 (e) Figure 4. 7 Results The observed number of events and expected yields are shown in table 9 for each of the six SRs. The SM backgrounds are estimated as described in section 5. Data agree with the SM background prediction within uncertainties and thus exclusion limits for several beyond-the-SM (BSM) scenarios are extracted. Figure 4 shows the Emiss T distributions after applying all the SR selection requirements except those on Emiss T . The HistFitter framework, which utilises a proﬁle-likelihood-ratio-test statistic [96], is used to estimate 95% conﬁdence intervals using the CLs prescription [97]. 7 Results The likelihood is built as the product of a probability density function describing the observed number of events in the SR and the associated CR(s) and, to constrain the nuisance parameters 15 Events / 20 GeV 1 2 3 4 5 6 ATLAS -1 = 13 TeV, 36.1 fb s A 3l1b SR Data Total SM background Fake and non-prompt leptons Multi-boson Z tt tZ, tWZ Others )=(800, 0) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 20 40 60 80 100 120 140 160 180 200 Data / SM 0 1 2 (a) Events / 40 GeV 2 4 6 8 10 12 14 16 ATLAS -1 = 13 TeV, 36.1 fb s A 1l4b SR Data Total SM Background tt Single top h, Vh tt V+jets, multi-boson Z t W, t tt Others )=(800, 0) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 50 100 150 200 250 300 350 400 Data / SM 0 1 2 (b) Events / 20 GeV 2 4 6 8 10 12 14 16 18 20 ATLAS -1 = 13 TeV, 36.1 fb s B 3l1b SR Data Total SM background Fake and non-prompt leptons Multi-boson Z tt tZ, tWZ Others )=(700, 200) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 20 40 60 80 100 120 140 160 180 200 Data / SM 0 1 2 (c) Events / 50 GeV 10 20 30 40 50 60 ATLAS -1 = 13 TeV, 36.1 fb s B 1l4b SR Data Total SM Background tt Single top h, Vh tt V+jets, multi-boson Z t W, t tt Others )=(650, 200) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 50 100 150 200 250 300 350 400 450 500 Data / SM 0 1 2 (d) Events / 20 GeV 2 4 6 8 10 12 14 16 ATLAS -1 = 13 TeV, 36.1 fb s C 3l1b SR Data Total SM background Fake and non-prompt leptons Multi-boson Z tt tZ, tWZ Others )=(650, 300) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 20 40 60 80 100 120 140 160 180 200 Data / SM 0 1 2 (e) Events / 50 GeV 10 20 30 40 50 60 ATLAS -1 = 13 TeV, 36.1 fb s C 1l4b SR Data Total SM Background tt Single top h, Vh tt V+jets, multi-boson Z t W, t tt Others )=(550, 250) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 50 100 150 200 250 300 350 400 450 500 Data / SM 0 1 2 (f) Figure 4. 7 Results Distribution of Emiss T for events pas except that on Emiss T , for (a) SR3ℓ1b A , (c) SR3ℓ1 B after the background ﬁt described in section 5 shown; the bands represent the total uncertaint m(˜t2) = 700 GeV and m(˜χ0 1) = 0 GeV, and m(˜t Events / 20 GeV 1 2 3 4 5 6 ATLAS -1 = 13 TeV, 36.1 fb s A 3l1b SR Data Total SM background Fake and non-prompt leptons Multi-boson Z tt tZ, tWZ Others )=(800, 0) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 20 40 60 80 100 120 140 160 180 200 Data / SM 0 1 2 (a) Events / 40 GeV 2 4 6 8 10 12 14 16 ATLAS -1 = 13 TeV, 36.1 fb s A 1l4b SR Data Total SM Background tt Single top h, Vh tt V+jets, multi-boson Z t W, t tt Others )=(800, 0) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 50 100 150 200 250 300 350 400 Data / SM 0 1 2 (b) JHEP08(2017)006 (b) (a) (a) Events / 20 GeV 2 4 6 8 10 12 14 16 18 20 ATLAS -1 = 13 TeV, 36.1 fb s B 3l1b SR Data Total SM background Fake and non-prompt leptons Multi-boson Z tt tZ, tWZ Others )=(700, 200) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 20 40 60 80 100 120 140 160 180 200 Data / SM 0 1 2 (c) (b) Events / 50 GeV 10 20 30 40 50 60 ATLAS -1 = 13 TeV, 36.1 fb s B 1l4b SR Data Total SM Background tt Single top h, Vh tt V+jets, multi-boson Z t W, t tt Others )=(650, 200) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 50 100 150 200 250 300 350 400 450 500 Data / SM 0 1 2 [ ] T (d) Events / 50 GeV 10 20 30 40 50 60 ATLAS -1 = 13 TeV, 36.1 fb s C 1l4b SR Data Total SM Background tt Single top h, Vh tt V+jets, multi-boson Z t W, t tt Others )=(550, 250) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 50 100 150 200 250 300 350 400 450 500 Data / SM 0 1 2 (f) (d) (c) (c) Events / 20 GeV 2 4 6 8 10 12 14 16 ATLAS -1 = 13 TeV, 36.1 fb s C 3l1b SR Data Total SM background Fake and non-prompt leptons Multi-boson Z tt tZ, tWZ Others )=(650, 300) GeV 0 1 χ∼ , 2t~ , m( 2t~ 2t~ [GeV] miss T E 0 20 40 60 80 100 120 140 160 180 200 Data / SM 0 1 2 (e) (f) (e) Figure 4. 7 Results Distribution of Emiss T for events passing all the signal candidate selection requirements, except that on Emiss T , for (a) SR3ℓ1b A , (c) SR3ℓ1b B , (e) SR3ℓ1b C and (b) SR1ℓ4b A , (d) SR1ℓ4b B , (f) SR1ℓ4b C after the background ﬁt described in section 5. The contributions from all SM backgrounds are shown; the bands represent the total uncertainty. The expected distributions for signal models with m(˜t2) = 700 GeV and m(˜χ0 1) = 0 GeV, and m(˜t2) = 650 GeV and m(˜χ0 1) = 250 GeV are also shown as dashed lines. The last bin in each ﬁgure contains the overﬂow. The lower panels show the ratio of the observed data to the total SM background prediction, with the bands representing the total uncertainty in the background prediction. 16 associated with the systematic uncertainties, Gaussian distributions whose widths corre- spond to the sizes of these uncertainties; Poisson distributions are used instead to model statistical uncertainties aﬀecting the observed and predicted yields in the CRs. Table 9 also shows upper limits (at the 95% CL) on the visible BSM cross-section σvis = S95 obs/Ldt, deﬁned as the product of the production cross-section, acceptance and eﬃciency. Model-dependent limits are also set in speciﬁc classes of SUSY models. For each signal hypothesis, the background ﬁt is redone taking into account the signal contamination in the CRs, which is found to be below 15% for signal models close to the Run-1 exclusion limits. All uncertainties in the SM prediction are considered, including those that are correlated between signal and background (for instance, jet energy scale uncertainties), as well as all uncertainties in the predicted signal, excluding PDF- and scale-induced uncertainties in the theoretical cross-section. Since the three SRs are not orthogonal, only the SR with best expected sensitivity is used for each signal point. “Observed limits” are calculated from the observed event yields in the SRs. “Expected limits” are calculated by setting the nominal event yield in each SR to the corresponding mean expected background. JHEP08(2017)006 Figure 5 shows the limits on simpliﬁed models in which pair-produced ˜t1 decay with 100% branching ratio into the ˜χ0 2 and a top quark, assuming B(˜χ0 2 →Z ˜χ0 1) = 0.5 and B(˜χ0 2 →h˜χ0 1) = 0.5. 7 Results A massless LSP and a minimum mass diﬀerence between the ˜χ0 2 and ˜χ0 1 of 130 GeV, needed to have on-shell decays for both the Higgs and Z bosons, are assumed in this model. Limits are presented in the ˜t1-˜χ0 2 mass plane. The two SRs with best expected sensitivity from the 3ℓ1b and 1ℓ4b selections are statistically combined to derive the limits on this model. For a ˜χ0 2 mass above 200 GeV, ˜t1 masses up to about 800 GeV are excluded at 95% CL. Limits for simpliﬁed models, in which pair-produced ˜t2 decay with 100% branching ratio into the ˜t1 and either a Z or a h boson, with ˜t1 →t˜χ0 1, in the ˜t2-˜χ0 1 mass plane are shown in ﬁgure 6. When considering the ˜t2 decays via a Z boson, probed by the 3ℓ1b selection, ˜t2 masses up to 800 GeV are excluded at 95% CL for a ˜χ0 1 of about 50 GeV and ˜χ0 1 masses up to 350 GeV are excluded for ˜t2 masses below 650 GeV. Assuming 100% branching ratio into ˜t1 and a h boson, probed by the 1ℓ4b selection, ˜t2 masses up to 880 GeV are excluded at 95% CL for a ˜χ0 1 of about 50 GeV, and ˜χ0 1 masses up to 260 GeV are excluded for ˜t2 masses between 650 and 710 GeV. These results extend the previous limits on the ˜t2 mass from ATLAS √s = 8 TeV analyses [19, 29] by up to 250 GeV depending on the ˜χ0 1 mass. Exclusion limits as a function of the ˜t2 branching ratios are shown in ﬁgure 7 for representative values of the masses of ˜t2 and ˜χ0 1. For ˜t2 mass of 600 GeV, SUSY models with B(˜t2 →Z˜t1) above 58% are excluded. For higher top squark mass (m˜t2 = 650 GeV), models with B(˜t2 →Z˜t1) above 50% or B(˜t2 →h˜t1) above 80% are excluded. The region with large B(˜t2 →t˜χ0 1) can be probed by searches targeting direct ˜t1 pair production [19]. Table 9. Observed and expected numbers of events in the six signal regions. The nominal pre- dictions from MC simulation are given for comparison for those backgrounds (t¯tZ, multi-boson for the 3ℓ1b selection and t¯t for the 1ℓ4b selection) that are normalised to data in dedicated control regions. For SR3ℓ1b A , SR3ℓ1b B and SR3ℓ1b C , the “Others” category contains the contributions from t¯th, t¯tWW, t¯tt, t¯tt¯t, Wh, and Zh production. For SR1ℓ4b A , SR1ℓ4b B and SR1ℓ4b C , the “Others” category contains the contributions from t¯tWW, t¯tt, t¯tt¯t, tZ, and tWZ production. Combined statistical and systematic uncertainties are given. Signal model-independent 95% CL upper limits on the visible BSM cross-section (σvis), the visible number of signal events (S95 obs), the number of signal events (S95 exp) given the expected number of background events (and ±1σ variations of the expected background), and the discovery p-value (p(s = 0)), all calculated with pseudo-experiments, are also shown for each signal region. 7 Results 17 17 SR3ℓ1b A SR3ℓ1b B SR3ℓ1b C Observed events 2 1 3 Total (post-ﬁt) SM events 1.9 ± 0.4 2.7 ± 0.6 2.0 ± 0.3 Fit output, multi-boson 0.26 ± 0.08 0.28 ± 0.10 0.23 ± 0.05 Fit output, t¯tZ 1.1 ± 0.3 1.4 ± 0.5 1.2 ± 0.3 tZ, tWZ 0.43 ± 0.23 0.36 ± 0.19 0.19 ± 0.10 Fake or non-prompt leptons 0.00+0.30 −0.00 0.45 ± 0.19 0.00+0.30 −0.00 Others 0.09 ± 0.02 0.23 ± 0.06 0.36 ± 0.06 Fit input, multi-boson 0.35 0.37 0.30 Fit input, t¯tZ 1.2 1.5 1.4 S95 obs 4.5 3.8 5.8 S95 exp 4.2+1.9 −0.4 4.9+1.5 −1.1 4.4+1.8 −0.5 σvis [fb] 0.13 0.10 0.16 p(s = 0) 0.42 0.93 0.23 SR1ℓ4b A SR1ℓ4b B SR1ℓ4b C Observed events 10 28 16 Total (post-ﬁt) SM events 13.6 ± 3.0 29 ± 5 10.5 ± 3.2 Fit output, t¯t 11.3 ± 2.9 24 ± 5 9.3 ± 3.1 Single top 0.50 ± 0.18 1.7 ± 0.4 0.24 ± 0.07 V +jets, multi-boson 0.20 ± 0.15 0.23 ± 0.10 0.01 ± 0.01 t¯th, V h 0.89 ± 0.16 1.19 ± 0.35 0.56 ± 0.13 t¯tW, t¯tZ 0.36 ± 0.21 1.09 ± 0.31 0.10 ± 0.10 Others 0.37 ± 0.20 1.33 ± 0.69 0.34 ± 0.18 Fit input, t¯t 7.1 14 6.0 S95 obs 7.8 14.6 15.6 S95 exp 9.6+4.1 −2.3 15.5+5.6 −4.4 10.4+4.2 −2.6 σvis [fb] 0.21 0.40 0.43 p(s = 0) 0.63 0.82 0.11 JHEP08(2017)006 Table 9. Observed and expected numbers of events in the six signal regions. The nominal pre- dictions from MC simulation are given for comparison for those backgrounds (t¯tZ, multi-boson for the 3ℓ1b selection and t¯t for the 1ℓ4b selection) that are normalised to data in dedicated control regions. For SR3ℓ1b A , SR3ℓ1b B and SR3ℓ1b C , the “Others” category contains the contributions from t¯th, t¯tWW, t¯tt, t¯tt¯t, Wh, and Zh production. For SR1ℓ4b A , SR1ℓ4b B and SR1ℓ4b C , the “Others” category contains the contributions from t¯tWW, t¯tt, t¯tt¯t, tZ, and tWZ production. Combined statistical and systematic uncertainties are given. 7 Results Signal model-independent 95% CL upper limits on the visible BSM cross-section (σvis), the visible number of signal events (S95 obs), the number of signal events (S95 exp) given the expected number of background events (and ±1σ variations of the expected background), and the discovery p-value (p(s = 0)), all calculated with pseudo-experiments, are also shown for each signal region. 18 JHEP08(2017)006 ) [GeV] 1t~ m( 500 550 600 650 700 750 800 850 900 950 ) [GeV] 0 2 χ∼ m( 200 300 400 500 600 700 800 ) = 0 GeV 0 1 χ∼ , m( 0 1 χ∼ Z/h + → 2 0 χ∼ , 2 0 χ∼ t + → 1t~ production, 1t~ 1t~ t ) < m 2 0 χ∼ , 1t~ m( ∆ ATLAS -1 =13 TeV, 36.1 fb s All limits at 95% CL ) theory SUSY σ 1 ± Observed limit ( ) exp σ 1 ± Expected limit ( Figure 5. Exclusion limits at 95% CL from the analysis of 36.1 fb−1 of 13 TeV pp collision data on the masses of the ˜t1 and ˜χ0 2, for a ﬁxed m(˜χ0 1) = 0 GeV, assuming B(˜χ0 2 →Z ˜χ0 1) = 0.5 and B(˜χ0 2 →h˜χ0 1) = 0.5. The dashed line and the shaded band are the expected limit and its ±1σ uncertainty, respectively. The thick solid line is the observed limit for the central value of the signal cross-section. The expected and observed limits do not include the eﬀect of the theoretical uncertainties in the signal cross-section. The dotted lines show the eﬀect on the observed limit when varying the signal cross-section by ±1σ of the theoretical uncertainty. ) [GeV] 1t~ m( 500 550 600 650 700 750 800 850 900 950 ) [GeV] 0 2 χ∼ m( 200 300 400 500 600 700 800 ) = 0 GeV 0 1 χ∼ , m( 0 1 χ∼ Z/h + → 2 0 χ∼ , 2 0 χ∼ t + → 1t~ production, 1t~ 1t~ t ) < m 2 0 χ∼ , 1t~ m( ∆ ATLAS -1 =13 TeV, 36.1 fb s All limits at 95% CL ) theory SUSY σ 1 ± Observed limit ( ) exp σ 1 ± Expected limit ( ATLAS Figure 5. 7 Results Exclusion limits at 95% CL from the analysis of 36.1 fb−1 of 13 TeV pp collision data on the masses of the ˜t1 and ˜χ0 2, for a ﬁxed m(˜χ0 1) = 0 GeV, assuming B(˜χ0 2 →Z ˜χ0 1) = 0.5 and B(˜χ0 2 →h˜χ0 1) = 0.5. The dashed line and the shaded band are the expected limit and its ±1σ uncertainty, respectively. The thick solid line is the observed limit for the central value of the signal cross-section. The expected and observed limits do not include the eﬀect of the theoretical uncertainties in the signal cross-section. The dotted lines show the eﬀect on the observed limit when varying the signal cross-section by ±1σ of the theoretical uncertainty. 19 ) [GeV] 2t~ m( 500 550 600 650 700 750 800 850 900 950 1000 ) [GeV] 0 1 χ∼ m( 50 100 150 200 250 300 350 400 450 500 ) = 180 GeV 0 1 χ∼ ) - m( 1t~ , m( 0 1 χ∼ t + → 1t~ + Z, 1t~ → 2t~ production, 2t~ 2t~ -1 =13 TeV, 36.1 fb s All limits at 95% CL Z ) < m 1t~ , 2t~ m( ∆ ATLAS ) theory SUSY σ 1 ± Observed limit ( ) exp σ 1 ± Expected limit ( -1 ATLAS 8 TeV, 20.3 fb (a) ) [GeV] 2t~ m( 500 550 600 650 700 750 800 850 900 950 1000 ) [GeV] 0 1 χ∼ m( 50 100 150 200 250 300 350 400 450 500 ) = 180 GeV 0 1 χ∼ ) - m( 1t~ , m( 0 1 χ∼ t + → 1t~ + h, 1t~ → 2t~ production, 2t~ 2t~ -1 =13 TeV, 36.1 fb s All limits at 95% CL h ) < m 1t~ , 2t~ m( ∆ ATLAS ) theory SUSY σ 1 ± Observed limit ( ) exp σ 1 ± Expected limit ( -1 ATLAS 8 TeV, 20.3 fb (b) ure 6. Exclusion limits at 95% CL from the analysis of 36.1 fb−1 of 13 TeV pp collision data on masses of the ˜t2 and ˜χ0 1, for a ﬁxed m(˜t1)−m(˜χ0 1) = 180 GeV and assuming (a) B(˜t2 →Z˜t1) = 1 b) B(˜t2 →h˜t1) = 1. The dashed line and the shaded band are the expected limit and its ±1σ ertainty, respectively. 7 Results The thick solid line is the observed limit for the central value of the al cross-section. The expected and observed limits do not include the eﬀect of the theoretical ertainties in the signal cross-section. The dotted lines show the eﬀect on the observed limit n varying the signal cross-section by ±1σ of the theoretical uncertainty. The shaded area in lower-left corner shows the observed exclusion from the ATLAS √s = 8 TeV analysis [19, 29]. ) [GeV] t~ m( 500 550 600 650 700 750 800 850 900 950 1000 ) [GeV] 0 1 χ∼ m( 50 100 150 200 250 300 350 400 450 500 ) = 180 GeV 0 1 χ∼ ) - m( 1t~ , m( 0 1 χ∼ t + → 1t~ + Z, 1t~ → 2t~ production, 2t~ 2t~ -1 =13 TeV, 36.1 fb s All limits at 95% CL Z ) < m 1t~ , 2t~ m( ∆ ATLAS ) theory SUSY σ 1 ± Observed limit ( ) exp σ 1 ± Expected limit ( -1 ATLAS 8 TeV, 20.3 fb JHEP08(2017)006 (a) ) [GeV] 2t~ m( 500 550 600 650 700 750 800 850 900 950 1000 ) [GeV] 0 1 χ∼ m( 50 100 150 200 250 300 350 400 450 500 ) = 180 GeV 0 1 χ∼ ) - m( 1t~ , m( 0 1 χ∼ t + → 1t~ + h, 1t~ → 2t~ production, 2t~ 2t~ -1 =13 TeV, 36.1 fb s All limits at 95% CL h ) < m 1t~ , 2t~ m( ∆ ATLAS ) theory SUSY σ 1 ± Observed limit ( ) exp σ 1 ± Expected limit ( -1 ATLAS 8 TeV, 20.3 fb (b) Figure 6. Exclusion limits at 95% CL from the analysis of 36.1 fb−1 of 13 TeV pp collision data on the masses of the ˜t2 and ˜χ0 1, for a ﬁxed m(˜t1)−m(˜χ0 1) = 180 GeV and assuming (a) B(˜t2 →Z˜t1) = 1 or (b) B(˜t2 →h˜t1) = 1. The dashed line and the shaded band are the expected limit and its ±1σ uncertainty, respectively. The thick solid line is the observed limit for the central value of the signal cross-section. The expected and observed limits do not include the eﬀect of the theoretical uncertainties in the signal cross-section. 8 Conclusion This paper reports a search for direct top squark pair production resulting in events with either a leptonically decaying Z boson or a pair of b-tagged jets from a Higgs boson decay, based on 36.1 fb−1 of proton-proton collisions at √s = 13 TeV recorded by the ATLAS experiment at the LHC in 2015 and 2016. Good agreement is found between the yield of observed events and the SM predictions. Model-independent limits are presented, which allow the results to be reinterpreted in generic models that also predict similar ﬁnal states in association with invisible particles. The limits exclude, at 95% conﬁdence level, beyond-the- SM processes with visible cross-sections above 0.11 (0.21) fb for the 3ℓ1b (1ℓ4b) selections. Results are also interpreted in the context of simpliﬁed models characterised by the decay chain ˜t1 →t˜χ0 2 with ˜χ0 2 →Z/h˜χ0 1, or ˜t2 →Z/h˜t1 with ˜t1 →t˜χ0 1. The results exclude at 95% conﬁdence level ˜t2 and ˜t1 masses up to about 800 GeV, extending the region of supersymmetric parameter space covered by previous LHC searches. 7 Results 0.2 0.4 0.6 0.8 0 1 0 1 0 1 ) 0 1 χ∼ t → 2t~( B ) 1t~ h → 2t~( B ) 1t~ Z → 2t~( B = 650 GeV 2t~ m = 200 GeV 1 0 χ∼ m ATLAS -1 = 13 TeV, 36.1 fb s 1 0 χ∼ t → 1t~ ; 1 0 χ∼ , t 1t~ , h 1t~ Z → 2t~ production, 2t~- 2t~ + 180 GeV 1 0 χ∼ = m 1t~ m All limits at 95% CL Observed 3l1b Expected 3l1b Observed 1l4b Expected 1l4b 0.2 0.4 0.6 0.8 0 1 0 1 0 1 ) 0 1 χ∼ t → 2t~( B ) 1t~ h → 2t~( B ) 1t~ Z → 2t~( B = 650 GeV 2t~ m = 200 GeV 1 0 χ∼ m 0.2 0.4 0.6 0.8 0 1 0 1 0 1 ) 0 1 χ∼ t → 2t~( B ) 1t~ h → 2t~( B ) 1t~ Z → 2t~( B = 600 GeV 2t~ m = 250 GeV 1 0 χ∼ m ATLAS -1 = 13 TeV, 36.1 fb s Figure 7. Exclusion limits at 95% CL from the analysis of 36.1 fb−1 of 13 TeV pp collision data as a function of the ˜t2 branching ratio for ˜t2 →˜t1Z, ˜t2 →˜t1h and ˜t2 →t˜χ0 1. The blue and red exclusion regions correspond to the 3ℓ1b and 1ℓ4b selections respectively. The limits are given for two diﬀerent values of the ˜t2 and ˜χ0 1 masses. The dashed lines are the expected limit and the solid lines are the observed limit for the central value of the signal cross-section. JHEP08(2017)006 7 Results The dotted lines show the eﬀect on the observed limit when varying the signal cross-section by ±1σ of the theoretical uncertainty. The shaded area in the lower-left corner shows the observed exclusion from the ATLAS √s = 8 TeV analysis [19, 29]. 20 0.2 0.4 0.6 0.8 0 1 0 1 0 1 ) 0 1 χ∼ t → 2t~( B ) 1t~ h → 2t~( B ) 1t~ Z → 2t~( B = 600 GeV 2t~ m = 250 GeV 1 0 χ∼ m 0.2 0.4 0.6 0.8 0 1 0 1 0 1 ) 0 1 χ∼ t → 2t~( B ) 1t~ h → 2t~( B ) 1t~ Z → 2t~( B = 650 GeV 2t~ m = 200 GeV 1 0 χ∼ m ATLAS -1 = 13 TeV, 36.1 fb s 1 0 χ∼ t → 1t~ ; 1 0 χ∼ , t 1t~ , h 1t~ Z → 2t~ production, 2t~- 2t~ + 180 GeV 1 0 χ∼ = m 1t~ m All limits at 95% CL Observed 3l1b Expected 3l1b Observed 1l4b Expected 1l4b Figure 7. Exclusion limits at 95% CL from the analysis of 36.1 fb−1 of 13 TeV pp collision data as a function of the ˜t2 branching ratio for ˜t2 →˜t1Z, ˜t2 →˜t1h and ˜t2 →t˜χ0 1. The blue and red exclusion regions correspond to the 3ℓ1b and 1ℓ4b selections respectively. The limits are given for two diﬀerent values of the ˜t2 and ˜χ0 1 masses. The dashed lines are the expected limit and the solid lines are the observed limit for the central value of the signal cross-section. Acknowledgments This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. Acknowledgments We thank CERN for the very successful operation of the LHC, as well as the support staﬀ from our institutions without whom ATLAS could not be operated eﬃciently. We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Aus- tralia; BMWFW and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS, CEA-DSM/IRFU, France; SRNSF, Georgia; BMBF, HGF, and MPG, Germany; GSRT, Greece; RGC, Hong Kong SAR, China; ISF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; NWO, Netherlands; RCN, Norway; MNiSW and NCN, Poland; FCT, Portugal; MNE/IFA, Romania; MES of Russia and NRC KI, Russian Federation; JINR; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZˇS, Slovenia; DST/NRF, South 21 Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SERI, SNSF and Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, United Kingdom; DOE and NSF, United States of America. In addition, individual groups and members have received support from BCKDF, the Canada Council, CANARIE, CRC, Compute Canada, FQRNT, and the Ontario Innovation Trust, Canada; EPLANET, ERC, ERDF, FP7, Horizon 2020 and Marie Sk lodowska-Curie Actions, European Union; In- vestissements d’Avenir Labex and Idex, ANR, R´egion Auvergne and Fondation Partager le Savoir, France; DFG and AvH Foundation, Germany; Herakleitos, Thales and Aristeia programmes co-ﬁnanced by EU-ESF and the Greek NSRF; BSF, GIF and Minerva, Israel; BRF, Norway; CERCA Programme Generalitat de Catalunya, Generalitat Valenciana, Spain; the Royal Society and Leverhulme Trust, United Kingdom. JHEP08(2017)006 The crucial computing support from all WLCG partners is acknowledged gratefully, in particular from CERN, the ATLAS Tier-1 facilities at TRIUMF (Canada), NDGF (Denmark, Norway, Sweden), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF (Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Taiwan), RAL (U.K.) and BNL (U.S.A.), the Tier-2 facilities worldwide and large non-WLCG resource providers. Ma- jor contributors of computing resources are listed in ref. [98]. Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. Open Access. References [1] Yu. A. Golfand and E.P. Likhtman, Extension of the algebra of Poincar´e group generators and violation of p invariance, JETP Lett. 13 (1971) 323 [Pisma Zh. Eksp. Teor. Fiz. 13 (1971) 452] [INSPIRE]. [2] D.V. Volkov and V.P. Akulov, Is the neutrino a Goldstone particle?, Phys. Lett. 46B (1973) 109 [INSPIRE]. [3] J. Wess and B. Zumino, Supergauge transformations in four-dimensions, Nucl. Phys. B 70 (1974) 39 [INSPIRE]. [4] J. Wess and B. Zumino, Supergauge invariant extension of quantum electrodynamics, Nucl. Phys. B 78 (1974) 1 [INSPIRE]. [4] J. Wess and B. Zumino, Supergauge invariant extension of quantum electrodynamics, Nucl. Phys. B 78 (1974) 1 [INSPIRE]. [5] S. Ferrara and B. Zumino, Supergauge invariant Yang-Mills theories, Nucl. Phys. B 79 (1974) 413 [INSPIRE]. [5] S. Ferrara and B. Zumino, Supergauge invariant Yang-Mills theories, Nucl. Phys. B 79 (1974) 413 [INSPIRE]. [6] A. Salam and J.A. Strathdee, Supersymmetry and non-Abelian gauges, Phys. Lett. 51B (1974) 353 [INSPIRE]. [6] A. Salam and J.A. Strathdee, Supersymmetry and non-Abelian gauges, Phys. Lett. 51B (1974) 353 [INSPIRE]. [7] G.R. Farrar and P. Fayet, Phenomenology of the production, decay and detection of new hadronic states associated with supersymmetry, Phys. Lett. 76B (1978) 575 [INSPIRE]. [8] N. Sakai, Naturalness in supersymmetric GUTs, Z. Phys. C 11 (1981) 153 [INSPIRE]. [9] S. Dimopoulos, S. Raby and F. Wilczek, Supersymmetry and the scale of uniﬁcation, Phys. Rev. D 24 (1981) 1681 [INSPIRE]. 22 [10] L.E. Ib´a˜nez and G.G. Ross, Low-energy predictions in supersymmetric grand uniﬁed theories, Phys. Lett. 105B (1981) 439 [INSPIRE]. [11] S. Dimopoulos and H. Georgi, Softly broken supersymmetry and SU(5), Nucl. Phys. B 193 (1981) 150 [INSPIRE]. [12] R. Barbieri and G.F. Giudice, Upper bounds on supersymmetric particle masses, Nucl. Phys. B 306 (1988) 63 [INSPIRE]. [13] B. de Carlos and J.A. Casas, One loop analysis of the electroweak breaking in supersymmetric models and the ﬁne tuning problem, Phys. Lett. B 309 (1993) 320 [hep-ph/9303291] [INSPIRE]. JHEP08(2017)006 [14] K. Inoue, A. Kakuto, H. Komatsu and S. Takeshita, Aspects of grand uniﬁed models with softly broken supersymmetry, Prog. Theor. Phys. 68 (1982) 927 [Erratum ibid. 70 (1983) 330] [INSPIRE]. [15] J.R. Ellis and S. Rudaz, Search for supersymmetry in toponium decays, Phys. Lett. 128B (1983) 248 [INSPIRE]. [16] J. Alwall, M.-P. Le, M. Lisanti and J.G. Wacker, Searching for directly decaying gluinos at the Tevatron, Phys. Lett. B 666 (2008) 34 [arXiv:0803.0019] [INSPIRE]. [17] J. References Alwall, P. Schuster and N. Toro, Simpliﬁed models for a ﬁrst characterization of new physics at the LHC, Phys. Rev. D 79 (2009) 075020 [arXiv:0810.3921] [INSPIRE]. [18] LHC New Physics Working Group collaboration, D. Alves, Simpliﬁed models for LHC new physics searches, J. Phys. G 39 (2012) 105005 [arXiv:1105.2838] [INSPIRE]. [19] ATLAS collaboration, ATLAS run 1 searches for direct pair production of third-generation squarks at the Large Hadron Collider, Eur. Phys. J. C 75 (2015) 510 [Erratum ibid. C 76 (2016) 153] [arXiv:1506.08616] [INSPIRE]. [20] ATLAS collaboration, The ATLAS experiment at the CERN Large Hadron Collider, 2008 JINST 3 S08003 [INSPIRE]. [21] ATLAS collaboration, Search for scalar top quark pair production in natural gauge mediated supersymmetry models with the ATLAS detector in pp collisions at √s = 7 TeV, Phys. Lett. B 715 (2012) 44 [arXiv:1204.6736] [INSPIRE]. [22] ATLAS collaboration, Search for top squarks in ﬁnal states with one isolated lepton, jets and missing transverse momentum in √s = 13 TeV pp collisions with the ATLAS detector, Phys. Rev. D 94 (2016) 052009 [arXiv:1606.03903] [INSPIRE]. [23] CMS collaboration, Search for top-squark pair production in the single-lepton ﬁnal state in pp collisions at √s = 8 TeV, Eur. Phys. J. C 73 (2013) 2677 [arXiv:1308.1586] [INSPIRE]. [24] CMS collaboration, Search for supersymmetry using razor variables in events with b-tagged jets in pp collisions at √s = 8 TeV, Phys. Rev. D 91 (2015) 052018 [arXiv:1502.00300] [INSPIRE]. [25] CMS collaboration, Searches for supersymmetry using the MT 2 variable in hadronic events produced in pp collisions at 8 TeV, JHEP 05 (2015) 078 [arXiv:1502.04358] [INSPIRE]. [26] CMS collaboration, Search for supersymmetry in events with soft leptons, low jet multiplicity and missing transverse energy in proton-proton collisions at √s = 8 TeV, Phys. Lett. B 759 (2016) 9 [arXiv:1512.08002] [INSPIRE]. 23 [27] CMS collaboration, Search for direct pair production of scalar top quarks in the single- and dilepton channels in proton-proton collisions at √s = 8 TeV, JHEP 07 (2016) 027 [Erratum ibid. 09 (2016) 056] [arXiv:1602.03169] [INSPIRE]. [28] CMS collaboration, Search for direct pair production of supersymmetric top quarks decaying to all-hadronic ﬁnal states in pp collisions at √s = 8 TeV, Eur. Phys. J. C 76 (2016) 460 [arXiv:1603.00765] [INSPIRE]. References [29] ATLAS collaboration, Search for direct top squark pair production in events with a Z boson, b-jets and missing transverse momentum in √s = 8 TeV pp collisions with the ATLAS detector, Eur. Phys. J. C 74 (2014) 2883 [arXiv:1403.5222] [INSPIRE]. [30] CMS collaboration, Search for top-squark pairs decaying into Higgs or Z bosons in pp collisions at √s = 8 TeV, Phys. Lett. B 736 (2014) 371 [arXiv:1405.3886] [INSPIRE]. JHEP08(2017)006 JHEP08(2017)006 [31] ATLAS collaboration, ATLAS insertable B-layer technical design report, CERN-LHCC-2010-013, CERN, Geneva Switzerland, (2010). [32] ATLAS collaboration, Performance of the ATLAS trigger system in 2015, Eur. Phys. J. C 77 (2017) 317 [arXiv:1611.09661] [INSPIRE]. [33] ATLAS collaboration, Improved luminosity determination in pp collisions at √s = 7 TeV using the ATLAS detector at the LHC, Eur. Phys. J. C 73 (2013) 2518 [arXiv:1302.4393] [INSPIRE]. [34] ATLAS collaboration, Simulation of top quark production for the ATLAS experiment at √s = 13 TeV, ATL-PHYS-PUB-2016-004, CERN, Geneva Switzerland, (2016). [35] ATLAS collaboration, Monte Carlo generators for the production of a W or Z/γ∗boson in association with jets at ATLAS in run 2, ATL-PHYS-PUB-2016-003, CERN, Geneva Switzerland, (2016). [36] ATLAS collaboration, Multi-boson simulation for 13 TeV ATLAS analyses, ATL-PHYS-PUB-2016-002, CERN, Geneva Switzerland, (2016). [37] ATLAS collaboration, Modelling of the t¯tH and t¯tV (V = W, Z) processes for √s = 13 TeV ATLAS analyses, ATL-PHYS-PUB-2016-005, CERN, Geneva Switzerland, (2016). [38] D.J. Lange, The EvtGen particle decay simulation package, Nucl. Instrum. Meth. A 462 (2001) 152 [INSPIRE]. [39] J. Alwall et al., The automated computation of tree-level and next-to-leading order diﬀerential cross sections and their matching to parton shower simulations, JHEP 07 (2014) 079 [arXiv:1405.0301] [INSPIRE]. [40] T. Sj¨ostrand, S. Mrenna and P.Z. Skands, A brief introduction to PYTHIA 8.1, Comput. Phys. Commun. 178 (2008) 852 [arXiv:0710.3820] [INSPIRE]. [41] W. Beenakker, R. Hopker, M. Spira and P.M. Zerwas, Squark and gluino production at hadron colliders, Nucl. Phys. B 492 (1997) 51 [hep-ph/9610490] [INSPIRE]. [42] A. Kulesza and L. Motyka, Threshold resummation for squark-antisquark and gluino-pair production at the LHC, Phys. Rev. Lett. 102 (2009) 111802 [arXiv:0807.2405] [INSPIRE]. [43] A. Kulesza and L. Motyka, Soft gluon resummation for the production of gluino-gluino and squark-antisquark pairs at the LHC, Phys. Rev. D 80 (2009) 095004 [arXiv:0905.4749] [INSPIRE]. 24 [44] W. Beenakker, S. Brensing, M. Kr¨amer, A. Kulesza, E. Laenen and I. Niessen, Soft-gluon resummation for squark and gluino hadroproduction, JHEP 12 (2009) 041 [arXiv:0909.4418] [INSPIRE]. References [45] W. Beenakker et al., Squark and gluino hadroproduction, Int. J. Mod. Phys. A 26 (2011) 2637 [arXiv:1105.1110] [INSPIRE]. [46] H.-L. Lai et al., New parton distributions for collider physics, Phys. Rev. D 82 (2010) 074024 [arXiv:1007.2241] [INSPIRE]. [47] ATLAS collaboration, ATLAS run 1 PYTHIA8 tunes, ATL-PHYS-PUB-2014-021, CERN, Geneva Switzerland, (2014). JHEP08(2017)006 [48] T. Gleisberg et al., Event generation with SHERPA 1.1, JHEP 02 (2009) 007 [arXiv:0811.4622] [INSPIRE]. [49] S. Catani, L. Cieri, G. Ferrera, D. de Florian and M. Grazzini, Vector boson production at hadron colliders: a fully exclusive QCD calculation at NNLO, Phys. Rev. Lett. 103 (2009) 082001 [arXiv:0903.2120] [INSPIRE]. [50] S. Alioli, P. Nason, C. Oleari and E. Re, A general framework for implementing NLO calculations in shower Monte Carlo programs: the POWHEG BOX, JHEP 06 (2010) 043 [arXiv:1002.2581] [INSPIRE]. [51] T. Sj¨ostrand, S. Mrenna and P.Z. Skands, PYTHIA 6.4 physics and manual, JHEP 05 (2006) 026 [hep-ph/0603175] [INSPIRE]. [52] M. Czakon, P. Fiedler and A. Mitov, Total top-quark pair-production cross section at hadron colliders through O(α4 S), Phys. Rev. Lett. 110 (2013) 252004 [arXiv:1303.6254] [INSPIRE]. [53] M. Czakon and A. Mitov, NNLO corrections to top pair production at hadron colliders: the quark-gluon reaction, JHEP 01 (2013) 080 [arXiv:1210.6832] [INSPIRE]. [54] M. Czakon and A. Mitov, NNLO corrections to top-pair production at hadron colliders: the all-fermionic scattering channels, JHEP 12 (2012) 054 [arXiv:1207.0236] [INSPIRE]. [55] P. B¨arnreuther, M. Czakon and A. Mitov, Percent level precision physics at the Tevatron: ﬁrst genuine NNLO QCD corrections to q¯q →t¯t + X, Phys. Rev. Lett. 109 (2012) 132001 [arXiv:1204.5201] [INSPIRE]. [56] M. Cacciari, M. Czakon, M. Mangano, A. Mitov and P. Nason, Top-pair production at hadron colliders with next-to-next-to-leading logarithmic soft-gluon resummation, Phys. Lett. B 710 (2012) 612 [arXiv:1111.5869] [INSPIRE]. [57] M. Czakon and A. Mitov, Top++: a program for the calculation of the top-pair cross-section at hadron colliders, Comput. Phys. Commun. 185 (2014) 2930 [arXiv:1112.5675] [INSPIRE]. [58] P.Z. Skands, Tuning Monte Carlo generators: the Perugia tunes, Phys. Rev. D 82 (2010) 074018 [arXiv:1005.3457] [INSPIRE]. [59] N. Kidonakis, Next-to-next-to-leading-order collinear and soft gluon corrections for t-channel single top quark production, Phys. Rev. D 83 (2011) 091503 [arXiv:1103.2792] [INSPIRE]. [60] N. Kidonakis, Two-loop soft anomalous dimensions for single top quark associated production with a W −or H−, Phys. Rev. D 82 (2010) 054018 [arXiv:1005.4451] [INSPIRE]. [61] N. Kidonakis, NNLL resummation for s-channel single top quark production, Phys. Rev. References D 81 (2010) 054028 [arXiv:1001.5034] [INSPIRE]. 25 [62] G. Corcella et al., HERWIG 6: an event generator for hadron emission reactions with interfering gluons (including supersymmetric processes), JHEP 01 (2001) 010 [hep-ph/0011363] [INSPIRE]. [63] S. Dittmaier et al., Handbook of LHC Higgs cross sections: 2. Diﬀerential distributions, arXiv:1201.3084 [INSPIRE]. [64] L. L¨onnblad and S. Prestel, Matching tree-level matrix elements with interleaved showers, JHEP 03 (2012) 019 [arXiv:1109.4829] [INSPIRE]. [65] W. Beenakker, M. Kr¨amer, T. Plehn, M. Spira and P.M. Zerwas, Stop production at hadron colliders, Nucl. Phys. B 515 (1998) 3 [hep-ph/9710451] [INSPIRE]. JHEP08(2017)006 JHEP08(2017)006 [66] W. Beenakker, S. Brensing, M. Kr¨amer, A. Kulesza, E. Laenen and I. Niessen, Supersymmetric top and bottom squark production at hadron colliders, JHEP 08 (2010) 098 [arXiv:1006.4771] [INSPIRE]. [67] C. Borschensky et al., Squark and gluino production cross sections in pp collisions at √s = 13, 14, 33 and 100 TeV, Eur. Phys. J. C 74 (2014) 3174 [arXiv:1407.5066] [INSPIRE]. [68] ATLAS collaboration, Summary of ATLAS PYTHIA 8 tunes, ATL-PHYS-PUB-2012-003, CERN, Geneva Switzerland, (2012). [69] A.D. Martin, W.J. Stirling, R.S. Thorne and G. Watt, Parton distributions for the LHC, Eur. Phys. J. C 63 (2009) 189 [arXiv:0901.0002] [INSPIRE]. [70] ATLAS collaboration, The ATLAS simulation infrastructure, Eur. Phys. J. C 70 (2010) 823 [arXiv:1005.4568] [INSPIRE]. [71] GEANT4 collaboration, S. Agostinelli et al., GEANT4: a simulation toolkit, Nucl. Instrum. Meth. A 506 (2003) 250 [INSPIRE]. [72] ATLAS collaboration, The simulation principle and performance of the ATLAS fast calorimeter simulation FastCaloSim, ATL-PHYS-PUB-2010-013, CERN, Geneva Switzerland, (2010). [73] ATLAS collaboration, Vertex reconstruction performance of the ATLAS detector at √s = 13 TeV, ATL-PHYS-PUB-2015-026, CERN, Geneva Switzerland, (2015). [74] ATLAS collaboration, Electron eﬃciency measurements with the ATLAS detector using 2012 LHC proton-proton collision data, Eur. Phys. J. C 77 (2017) 195 [arXiv:1612.01456] [INSPIRE]. [75] ATLAS collaboration, Electron identiﬁcation measurements in ATLAS using √s = 13 TeV data with 50 ns bunch spacing, ATL-PHYS-PUB-2015-041, CERN, Geneva Switzerland, (2015). [76] ATLAS collaboration, Muon reconstruction performance of the ATLAS detector in proton-proton collision data at √s = 13 TeV, Eur. Phys. J. C 76 (2016) 292 [arXiv:1603.05598] [INSPIRE]. [77] ATLAS collaboration, Properties of jets and inputs to jet reconstruction and calibration with the ATLAS detector using proton-proton collisions at √s = 13 TeV, ATL-PHYS-PUB-2015-036, CERN, Geneva Switzerland, (2015). [78] M. Cacciari, G.P. Salam and G. Soyez, The anti-kt jet clustering algorithm, JHEP 04 (2008) 063 [arXiv:0802.1189] [INSPIRE]. References 26 [79] ATLAS collaboration, Jet calibration and systematic uncertainties for jets reconstructed in the ATLAS detector at √s = 13 TeV, ATL-PHYS-PUB-2015-015, CERN, Geneva Switzerland, (2015). [80] ATLAS collaboration, Jet global sequential corrections with the ATLAS detector in proton-proton collisions at √s = 8 TeV, ATLAS-CONF-2015-002, CERN, Geneva Switzerland, (2015). [81] ATLAS collaboration, Performance of pile-up mitigation techniques for jets in pp collisions at √s = 8 TeV using the ATLAS detector, Eur. Phys. J. C 76 (2016) 581 [arXiv:1510.03823] [INSPIRE]. [82] ATLAS collaboration, Data-quality requirements and event cleaning for jets and missing transverse energy reconstruction with the ATLAS detector in proton-proton collisions at a center-of-mass energy of √s = 7 TeV, ATLAS-CONF-2010-038, CERN, Geneva Switzerland, (2010). JHEP08(2017)006 JHEP08(2017)006 [83] ATLAS collaboration, Performance of b-jet identiﬁcation in the ATLAS experiment, 2016 JINST 11 P04008 [arXiv:1512.01094] [INSPIRE]. [84] ATLAS collaboration, Optimisation of the ATLAS b-tagging performance for the 2016 LHC run, ATL-PHYS-PUB-2016-012, CERN, Geneva Switzerland, (2016). [85] ATLAS collaboration, Expected performance of missing transverse momentum reconstruction for the ATLAS detector at √s = 13 TeV, ATL-PHYS-PUB-2015-023, CERN, Geneva Switzerland, (2015). [86] ATLAS collaboration, Performance of missing transverse momentum reconstruction for the ATLAS detector in the ﬁrst proton-proton collisions at √s = 13 TeV, ATL-PHYS-PUB-2015-027, CERN, Geneva Switzerland, (2015). [87] M. Baak, G.J. Besjes, D. Cˆote, A. Koutsman, J. Lorenz and D. Short, HistFitter software framework for statistical data analysis, Eur. Phys. J. C 75 (2015) 153 [arXiv:1410.1280] [INSPIRE]. [88] ATLAS collaboration, Measurement of the top quark-pair production cross section with ATLAS in pp collisions at √s = 7 TeV, Eur. Phys. J. C 71 (2011) 1577 [arXiv:1012.1792] [INSPIRE]. [89] ATLAS collaboration, Measurement of the top quark pair production cross section in pp collisions at √s = 7 TeV in dilepton ﬁnal states with ATLAS, Phys. Lett. B 707 (2012) 459 [arXiv:1108.3699] [INSPIRE]. [90] ATLAS collaboration, Search for the Standard Model Higgs boson decaying into b¯b produced in association with top quarks decaying hadronically in pp collisions at √s = 8 TeV with the ATLAS detector, JHEP 05 (2016) 160 [arXiv:1604.03812] [INSPIRE]. [91] ATLAS collaboration, Jet calibration and systematic uncertainties for jets reconstructed in the ATLAS detector at √s = 13 TeV, ATL-PHYS-PUB-2015-015, CERN, Geneva Switzerland, (2015). [92] ATLAS collaboration, Calibration of b-tagging using dileptonic top pair events in a combinatorial likelihood approach with the ATLAS experiment, ATLAS-CONF-2014-004, CERN, Geneva Switzerland, (2014). References [93] ATLAS collaboration, Calibration of the performance of b-tagging for c and light-ﬂavour jets in the 2012 ATLAS data, ATLAS-CONF-2014-046, CERN, Geneva Switzerland, (2014). 27 [94] ATLAS collaboration, Multi-boson simulation for 13 TeV ATLAS analyses, ATL-PHYS-PUB-2016-002, CERN, Geneva Switzerland, (2016). [95] ATLAS collaboration, Expected performance of the ATLAS b-tagging algorithms in run-2, ATL-PHYS-PUB-2015-022, CERN, Geneva Switzerland, (2015). [96] G. Cowan, K. Cranmer, E. Gross and O. Vitells, Asymptotic formulae for likelihood-based tests of new physics, Eur. Phys. J. C 71 (2011) 1554 [Erratum ibid. C 73 (2013) 2501] [arXiv:1007.1727] [INSPIRE]. [97] A.L. Read, Presentation of search results: the CLs technique, J. Phys. G 28 (2002) 2693 [INSPIRE]. JHEP08(2017)006 [98] ATLAS collaboration, ATLAS computing acknowledgements 2016–2017, ATL-GEN-PUB-2016-002, CERN, Geneva Switzerland, (2016). 28 The ATLAS collaboration M. Aaboud137d, G. Aad88, B. Abbott115, O. Abdinov12,∗, B. Abeloos119, S.H. Abidi161, O.S. AbouZeid139, N.L. Abraham151, H. Abramowicz155, H. Abreu154, R. Abreu118, Y. Abulaiti148a,148b, B.S. Acharya167a,167b,a, S. Adachi157, L. Adamczyk41a, J. Adelman1 Y. Abulaiti , , B.S. Acharya , , , S. Adachi , L. Adamczyk , J. Adelman , M. Adersberger102, T. Adye133, A.A. Aﬀolder139, T. Agatonovic-Jovin14, C. Agheorghiesei28c, J.A. Aguilar-Saavedra128a,128f, S.P. Ahlen24, F. Ahmadov68,b, G. Aielli135a,135b, S. Akatsuka71, H Akerstedt148a,148b T P A ˚Akesson84 E Akilli52 A V Akimov98 G L Alberghi22a,22b M. Adersberger102, T. Adye133, A.A. Aﬀolder139, T. Agatonovic-Jovin14, C. Agheorghiesei28c, J.A. Aguilar-Saavedra128a,128f, S.P. Ahlen24, F. Ahmadov68,b, G. Aielli135a,135b, S. Akatsuka71, b ˚ b J.A. Aguilar-Saavedra128a,128f, S.P. Ahlen24, F. Ahmadov68,b, G. Aielli135a,135b, S. Akatsuka71, H. Akerstedt148a,148b, T.P.A. ˚Akesson84, E. Akilli52, A.V. Akimov98, G.L. Alberghi22a,22b, J. Albert172, P. Albicocco50, M.J. Alconada Verzini74, S.C. Alderweireldt108, M. Aleksa32, J. Albert172, P. Albicocco50, M.J. Alconada Verzini74, S.C. Alderweireldt108, M. Aleksa32, I N Al k d 68 C Al 28b G Al d 155 T Al l 10 M Alh b115 B Ali130 I.N. Aleksandrov68, C. Alexa28b, G. Alexander155, T. Alexopoulos10, M. Alhroob115, B. Ali130, JHEP08(2017)006 JHEP08(2017)006 M. Aliev76a,76b, G. Alimonti94a, J. Alison33, S.P. Alkire38, B.M.M. Allbrooke151, B.W. Allen1 P.P. Allport19, A. Aloisio106a,106b, A. Alonso39, F. Alonso74, C. Alpigiani140, A.A. Alshehri56 M.I. Alstaty88, B. Alvarez Gonzalez32, D. ´Alvarez Piqueras170, M.G. Alviggi106a,106b, B.T. Amadio16, Y. Amaral Coutinho26a, C. Amelung25, D. Amidei92, S.P. Amor Dos Santos128a,128c, A. Amorim128a,128b, S. Amoroso32, G. Amundsen25, C. Anastopoulos141, L.S. Ancu52, N. Andari19, T. Andeen11, C.F. Anders60b, J.K. Anders77, K.J. Anderson33, A. Andreazza94a,94b, V. Andrei60a, S. Angelidakis9, I. Angelozzi109, A. Angerami38, A.V. Anisenkov111,c, N. Anjos13, A. Annovi126a,126b, C. Antel60a, M Y. Arai69, J.P. Araque128a, V. Araujo Ferraz26a, A.T.H. Arce48, R.E. Ardell80, F.A. Arduh74, J-F. Arguin97, S. Argyropoulos66, M. Arik20a, A.J. Armbruster32, L.J. Armitage79, O. Arnaez1 H. Arnold51, M. Arratia30, O. Arslan23, A. Artamonov99, G. Artoni122, S. Artz86, S. Asai157, N. Asbah45, A. Ashkenazi155, L. Asquith151, K. Assamagan27, R. Astalos146a, M. Atkinson16 N.B. Atlay143, K. Augsten130, G. Avolio32, B. Axen16, M.K. Ayoub119, G. Azuelos97,d, A.E. Baas60a, M.J. Baca19, H. Bachacou138, K. Bachas76a,76b, M. Backes122, M. Backhaus32, P. Bagnaia134a,134b, M. Bahmani42, H. Bahrasemani144, J.T. Baines133, M. Bajic39, ag a a , a a , a ase a , J a es , aj c , O K Baker179 E M Baldin111,c P Balek175 F Balli138 W K Balunas124 E Banas42 O.K. Baker179, E.M. Baldin111,c, P. Balek175, F. Balli138, W.K. Balunas124, E. The ATLAS collaboration Banas42, K. Baker179, E.M. Baldin111,c, P. Balek175, F. Balli138, W.K. Balunas124, E. Banas42, A. Bandyopadhyay23, Sw. Banerjee176,e, A.A.E. Bannoura178, L. Barak32, E.L. Barberio91, Bandyopadhyay23, Sw. Banerjee176,e, A.A.E. Bannoura178, L. Barak32, E.L. Barberio91, Barberis53a,53b, M. Barbero88, T. Barillari103, M-S Barisits32, J.T. Barkeloo118, T. Barklow145, D. Barberis53a,53b, M. Barbero88, T. Barillari103, M-S Barisits32, J.T. Barkeloo118, T. Barkl N. Barlow30, S.L. Barnes36c, B.M. Barnett133, R.M. Barnett16, Z. Barnovska-Blenessy36a, Barlow30, S.L. Barnes36c, B.M. Barnett133, R.M. Barnett16, Z. Barnovska-Blenessy36a, A. Baroncelli136a, G. Barone25, A.J. Barr122, L. Barranco Navarro170, F. Barreiro85, Baroncelli136a, G. Barone25, A.J. Barr122, L. Barranco Navarro170, F. Barreiro85, J. Barreiro Guimar˜aes da Costa35a, R. Bartoldus145, A.E. Barton75, P. Bartos146a, A. Basala Barreiro Guimar˜aes da Costa35a, R. Bartoldus145, A.E. Barton75, P. Bartos146a, A. Basalaev125, A. Bassalat119,f, R.L. Bates56, S.J. Batista161, J.R. Batley30, M. Battaglia139, M. Bauce134a,134b, A. Bassalat119,f, R.L. Bates56, S.J. Batista161, J.R. Batley30, M. Battaglia139, M. Bauce134a,134b, P. Bechtle23, H.P. Beck18,h, H.C. Beck57, K. Becker122, M. Becker86, M. Beckingham173, P. Bechtle23, H.P. Beck18,h, H.C. Beck57, K. Becker122, M. Becker86, M. Beckingham173, C. Becot112, A.J. Beddall20e, A. Beddall20b, V.A. Bednyakov68, M. Bedognetti109, C.P A. Beermann32, M. Begalli26a, M. Begel27, J.K. Behr45, A.S. Bell81, G. Bella155, T.A. Beermann32, M. Begalli26a, M. Begel27, J.K. Behr45, A.S. Bell81, G. Bella155, L. Bellagamba22a, A. Bellerive31, M. Bellomo154, K. Belotskiy100, O. Beltramello32 N.L. Belyaev100, O. Benary155,∗, D. Benchekroun137a, M. Bender102, K. Bendtz148a,148b, L. Belyaev100, O. Benary155,∗, D. Benchekroun137a, M. Bender102, K. Bendtz148a,148b, N. Benekos10, Y. Benhammou155, E. Benhar Noccioli179, J. Benitez66, D.P. Benjamin48, Benekos10, Y. Benhammou155, E. Benhar Noccioli179, J. Benitez66, D.P. Benjamin48, t52, J.R. Bensinger25, S. Bentvelsen109, L. Beresford122, M. Beretta50, D. Berge109, M. Benoit52, J.R. Bensinger25, S. Bentvelsen109, L. Beresford122, M. Beretta50, D. Berge E. Bergeaas Kuutmann168, N. Berger5, J. Beringer16, S. Berlendis58, N.R. Bernard89, aas Kuutmann168, N. Berger5, J. Beringer16, S. Berlendis58, N.R. Bernard89, rdi83, C. Bernius145, F.U. Bernlochner23, T. Berry80, P. Berta131, C. Bertella35a, G. Bernardi83, C. Bernius145, F.U. Bernlochner23, T. Berry80, P. Berta131, C. Bertella35a Bertoli148a,148b, F. Bertolucci126a,126b, I.A. Bertram75, C. Bertsche45, D. Bertsche115, G. Bertoli148a,148b, F. Bertolucci126a,126b, I.A. Bertram75, C. Bertsche45, D. Bertsche115, J. Besjes39, O. Bessidskaia Bylund148a,148b, M. Bessner45, N. Besson138, C. Betancourt51, G.J. Besjes39, O. Bessidskaia Bylund148a,148b, M. Bessner45, N. Besson138, C. Betancour A. Bethani87, S. Bethke103, A.J. Bevan79, J. Beyer103, R.M. Bianchi127, O. Biebel102, D. Biedermann17, R. Bielski87, K. Bierwagen86, N.V. Biesuz126a,126b, M. Biglietti136a, T.R.V. Billoud97, H. Bilokon50, M. Bindi57, A. Bingul20b, C. Bini134a,134b, S. Biondi22a,22b, T.R.V. Billoud97, H. The ATLAS collaboration Bilokon50, M. Bindi57, A. Bingul20b, C. Bini134a,134b, S. Biondi22a,22b 29 Bisanz57, C. Bittrich47, D.M. Bjergaard48, C.W. Black152, J.E. Black145, K.M. Black24, T. Bisanz57, C. Bittrich47, D.M. Bjergaard48, C.W. Black152, J.E. Black145, K.M. Black24, Blair6, T. Blazek146a, I. Bloch45, C. Blocker25, A. Blue56, W. Blum86,∗, U. Blumenschein79, R.E. Blair6, T. Blazek146a, I. Bloch45, C. Blocker25, A. Blue56, W. Blum86,∗, U. Blumensche unier34a, G.J. Bobbink109, V.S. Bobrovnikov111,c, S.S. Bocchetta84, A. Bocci48, C. Bock102, S. Blunier34a, G.J. Bobbink109, V.S. Bobrovnikov111,c, S.S. Bocchetta84, A. Bocci48, C. Bock102 ler51, D. Boerner178, D. Bogavac102, A.G. Bogdanchikov111, C. Bohm148a, V. Boisvert80, M. Boehler51, D. Boerner178, D. Bogavac102, A.G. Bogdanchikov111, C. Bohm148a, V. Boisvert80, Bokan168,i, T. Bold41a, A.S. Boldyrev101, A.E. Bolz60b, M. Bomben83, M. Bona79, P. Bokan168,i, T. Bold41a, A.S. Boldyrev101, A.E. Bolz60b, M. Bomben83, M. Bona79, Boonekamp138, A. Borisov132, G. Borissov75, J. Bortfeldt32, D. Bortoletto122, M. Boonekamp138, A. Borisov132, G. Borissov75, J. Bortfeldt32, D. Bortoletto122, V. Bortolotto62a,62b,62c, D. Boscherini22a, M. Bosman13, J.D. Bossio Sola29, J. Boudreau127, Bortolotto62a,62b,62c, D. Boscherini22a, M. Bosman13, J.D. Bossio Sola29, J. Boudreau127, J. Bouﬀard2, E.V. Bouhova-Thacker75, D. Boumediene37, C. Bourdarios119, S.K. Boutle56, J. Bouﬀard2, E.V. Bouhova-Thacker75, D. Boumediene37, C. Bourdarios119, S.K. Boutle56, U. Bratzler158, B. Brau89, J.E. Brau118, W.D. Breaden Madden56, K. Brendlinger45, U. Bratzler158, B. Brau89, J.E. Brau118, W.D. Breaden Madden56, K. Brendlinger45, A.J. Brennan91, L. Brenner109, R. Brenner168, S. Bressler175, D.L. Briglin19, T.M. Bristow49 JHEP08(2017)006 JHEP08(2017)006 D. Britton56, D. Britzger45, F.M. Brochu30, I. Brock23, R. Brock93, G. Brooijmans38, T. Brooks80 D. Britton56, D. Britzger45, F.M. Brochu30, I. Brock23, R. Brock93, G. Brooijmans38, T. Brooks Brooks34b, J. Brosamer16, E. Brost110, J.H Broughton19, P.A. Bruckman de Renstrom42, W.K. Brooks34b, J. Brosamer16, E. Brost110, J.H Broughton19, P.A. Bruckman de Renstrom uncko146b, A. Bruni22a, G. Bruni22a, L.S. Bruni109, BH Brunt30, M. Bruschi22a, D. Bruncko146b, A. Bruni22a, G. Bruni22a, L.S. Bruni109, BH Brunt30, M. Bruschi22a, uscino23, P. Bryant33, L. Bryngemark45, T. Buanes15, Q. Buat144, P. Buchholz143, N. Bruscino23, P. Bryant33, L. Bryngemark45, T. Buanes15, Q. Buat144, P. Buchholz143, Buckley56, I.A. Budagov68, F. Buehrer51, M.K. Bugge121, O. Bulekov100, D. Bullock8, A.G. Buckley56, I.A. Budagov68, F. Buehrer51, M.K. Bugge121, O. Bulekov100, D. Bullock8, Burch110, S. Burdin77, C.D. Burgard51, A.M. Burger5, B. Burghgrave110, K. Burka42, T.J. Burch110, S. Burdin77, C.D. Burgard51, A.M. Burger5, B. Burghgrave110, K. Burka42, rke133, I. Burmeister46, J.T.P. Burr122, E. Busato37, D. B¨uscher51, V. B¨uscher86, S. Burke133, I. Burmeister46, J.T.P. Burr122, E. Busato37, D. B¨uscher51, V. B¨uscher86, ssey56, J.M. Butler24, C.M. Buttar56, J.M. Butterworth81, P. Constantinescu28b, G. Conti32, F. Conventi106a,n, M. Cooke16, A.M. Cooper-Sarkar122, The ATLAS collaboration Butti32, W. Buttinger27, P. Bussey56, J.M. Butler24, C.M. Buttar56, J.M. Butterworth81, P. Butti32, W. Buttinger27, A. Buzatu35c, A.R. Buzykaev111,c, S. Cabrera Urb´an170, D. Caforio130, V.M. Cairo40a,40b, uzatu35c, A.R. Buzykaev111,c, S. Cabrera Urb´an170, D. Caforio130, V.M. Cairo40a,40b, O. Cakir4a, N. Calace52, P. Calaﬁura16, A. Calandri88, G. Calderini83, P. Calfayan64, G. Callea40a,40b, L.P. Caloba26a, S. Calvente Lopez85, D. Calvet37, S. Calvet37, T.P. Calvet8 R. Camacho Toro33, S. Camarda32, P. Camarri135a,135b, D. Cameron121, R. Caminal Armadans169, C. Camincher58, S. Campana32, M. Campanelli81, A. Camplani9 A. Campoverde143, V. Canale106a,106b, M. Cano Bret36c, J. Cantero116, T. Cao155, A. Campoverde143, V. Canale106a,106b, M. Cano Bret36c, J. Cantero116, T. Cao155, A. Campoverde143, V. Canale106a,106b, M. Cano Bret36c, J. Cantero116, T. Cao155, M D M Capeans Garrido32 I Caprini28b M Caprini28b M Capua40a,40b R M Car p , , , , , M.D.M. Capeans Garrido32, I. Caprini28b, M. Caprini28b, M. Capua40a,40b, R.M. Carb R. Cardarelli135a, F. Cardillo51, I. Carli131, T. Carli32, G. Carlino106a, B.T. Carlson127, R. Cardarelli135a, F. Cardillo51, I. Carli131, T. Carli32, G. Carlino106a, B.T. Carlson127, L. Carminati94a,94b, R.M.D. Carney148a,148b, S. Caron108, E. Carquin34b, S. Carr´a94a,94b, G.D. Carrillo-Montoya32, J. Carvalho128a,128c, D. Casadei19, M.P. Casado13,j, M. Casolino13 G.D. Carrillo-Montoya32, J. Carvalho128a,128c, D. Casadei19, M.P. Casado13,j, M. Casolino13, D.W. Casper166, R. Castelijn109, V. Castillo Gimenez170, N.F. Castro128a,k, A. Catinaccio32, Casper166, R. Castelijn109, V. Castillo Gimenez170, N.F. Castro128a,k, A. Catinaccio32, J.R. Catmore121, A. Cattai32, J. Caudron23, V. Cavaliere169, E. Cavallaro13, D. Cavalli94a, Catmore121, A. Cattai32, J. Caudron23, V. Cavaliere169, E. Cavallaro13, D. Cavalli94a, avalli-Sforza13, V. Cavasinni126a,126b, E. Celebi20d, F. Ceradini136a,136b, L. Cerda Alberich170, M. Cavalli-Sforza13, V. Cavasinni126a,126b, E. Celebi20d, F. Ceradini136a,136b, L. Cerda Alberic Cerqueira26b, A. Cerri151, L. Cerrito135a,135b, F. Cerutti16, A. Cervelli18, S.A. Cetin20d, A.S. Cerqueira26b, A. Cerri151, L. Cerrito135a,135b, F. Cerutti16, A. Cervelli18, S.A. Cetin20d, hafaq137a, D. Chakraborty110, S.K. Chan59, W.S. Chan109, Y.L. Chan62a, P. Chang169, A. Chafaq137a, D. Chakraborty110, S.K. Chan59, W.S. Chan109, Y.L. Chan62a, P. Chang169, J.D. Chapman30, D.G. Charlton19, C.C. Chau161, C.A. Chavez Barajas151, S. Che113, D. Chapman30, D.G. Charlton19, C.C. Chau161, C.A. Chavez Barajas151, S. Che113, S. Cheatham167a,167c, A. Chegwidden93, S. Chekanov6, S.V. Chekulaev163a, G.A. Chelkov68,l S. Cheatham167a,167c, A. Chegwidden93, S. Chekanov6, S.V. Chekulaev163a, G.A. Chelkov68,l A. Chelstowska32, C. Chen67, H. Chen27, J. Chen36a, S. Chen35b, S. Chen157, X. Chen35c,m, M.A. Chelstowska32, C. Chen67, H. Chen27, J. Chen36a, S. Chen35b, S. Chen157, X. Chen35c, Y. Chen70, H.C. Cheng92, H.J. Cheng35a, A. Cheplakov68, E. Cheremushkina132, erkaoui El Moursli137e, E. Cheu7, K. Cheung63, L. Chevalier138, V. The ATLAS collaboration Delmastro5, C. Delporte119, P.A. Delsart58, D.A. DeMarco161, S. Demers179, D. della Volpe52, M. Delmastro5, C. Delporte119, P.A. Delsart58, D.A. DeMarco161, S. Demers17 emichev68, A. Demilly83, S.P. Denisov132, D. Denysiuk138, D. Derendarz42, M. Demichev68, A. Demilly83, S.P. Denisov132, D. Denysiuk138, D. Derendarz42, Derkaoui137d, F. Derue83, P. Dervan77, K. Desch23, C. Deterre45, K. Dette46, M.R. Devesa29, J.E. Derkaoui137d, F. Derue83, P. Dervan77, K. Desch23, C. Deterre45, K. Dette46, M.R. Deves P.O. Deviveiros32, A. Dewhurst133, S. Dhaliwal25, F.A. Di Bello52, A. Di Ciaccio135a,135b, L. Di Ciaccio5, W.K. Di Clemente124, C. Di Donato106a,106b, A. Di Girolamo32, B. Di Girol B. Di Micco136a,136b, R. Di Nardo32, K.F. Di Petrillo59, A. Di Simone51, R. Di Sipio161, B. Di Micco136a,136b, R. Di Nardo32, K.F. Di Petrillo59, A. Di Simone51, R. Di Sipio161, D. Di Valentino31, C. Diaconu88, M. Diamond161, F.A. Dias39, M.A. Diaz34a, E.B. Diehl92, J. Dietrich17, S. D´ıez Cornell45, A. Dimitrievska14, J. Dingfelder23, P. Dita28b, S. Dita28b, J. Dietrich17, S. D´ıez Cornell45, A. Dimitrievska14, J. Dingfelder23, P. Dita28b, S. Dita28b, M. Dobre28b, C. Doglioni84, J. Dolejsi131, Z. Dolezal131, M. Donadelli26d, S. Donati126a,126b, M. Dobre28b, C. Doglioni84, J. Dolejsi131, Z. Dolezal131, M. Donadelli26d, S. Donati126a,126b ero123a,123b, J. Donini37, J. Dopke133, A. Doria106a, M.T. Dova74, A.T. Doyle56, P. Dondero123a,123b, J. Donini37, J. Dopke133, A. Doria106a, M.T. Dova74, A.T. Doyle56, E. Drechsler57, M. Dris10, Y. Du36b, J. Duarte-Campderros155, A. Dubreuil52, E. Duchovni175, E. Drechsler57, M. Dris10, Y. Du36b, J. Duarte-Campderros155, A. Dubreuil52, E. Duchovn ck102, A. Ducourthial83, O.A. Ducu97,p, D. Duda109, A. Dudarev32, A.Chr. Dudder86, G. Duckeck102, A. Ducourthial83, O.A. Ducu97,p, D. Duda109, A. Dudarev32, A.Chr. Dudder ﬃeld16, L. Duﬂot119, M. D¨uhrssen32, M. Dumancic175, A.E. Dumitriu28b, E.M. Duﬃeld16, L. Duﬂot119, M. D¨uhrssen32, M. Dumancic175, A.E. Dumitriu28b, Duncan56, M. Dunford60a, H. Duran Yildiz4a, M. D¨uren55, A. Durglishvili54b, A.K. Duncan56, M. Dunford60a, H. Duran Yildiz4a, M. D¨uren55, A. Durglishvili54b, uschinger47, B. Dutta45, D. Duvnjak1, M. Dyndal45, B.S. Dziedzic42, C. Eckardt45, D. Duschinger47, B. Dutta45, D. Duvnjak1, M. Dyndal45, B.S. Dziedzic42, C. Eckardt45, Ecker103, R.C. Edgar92, T. Eifert32, G. Eigen15, K. Einsweiler16, T. Ekelof168, K.M. Ecker103, R.C. Edgar92, T. Eifert32, G. Eigen15, K. Einsweiler16, T. Ekelof168, M. El Kacimi137c, R. El Kosseiﬁ88, V. Ellajosyula88, M. Ellert168, S. Elles5, F. Ellinghaus178 Kacimi137c, R. El Kosseiﬁ88, V. Ellajosyula88, M. Ellert168, S. Elles5, F. Ellinghaus178, A.A. Elliot172, N. Ellis32, J. Elmsheuser27, M. Elsing32, D. Emeliyanov133, Y. Enari157, Elliot172, N. Ellis32, J. Elmsheuser27, M. Elsing32, D. Emeliyanov133, Y. Enari157, O.C. Endner86, J.S. Ennis173, J. Erdmann46, A. The ATLAS collaboration Chiarella50, R. Cherkaoui El Moursli137e, E. Cheu7, K. Cheung63, L. Chevalier138, V. Chiarella50, G. Chiarelli126a,126b, G. Chiodini76a, A.S. Chisholm32, A. Chitan28b, Y.H. Chiu172, G. Chiarelli126a,126b, G. Chiodini76a, A.S. Chisholm32, A. Chitan28b, Y.H. Chiu172, D. Chromek-Burckhart32, M.C. Chu62a, J. Chudoba129, A.J. Chuinard90, J.J. Chwastowski42, D. Chromek-Burckhart32, M.C. Chu62a, J. Chudoba129, A.J. Chuinard90, J.J. Chwastowski42, D. Chromek Burckhart , M.C. Chu , J. Chudoba , A.J. Chuinard , J.J. Chwastowski , L. Chytka117, A.K. Ciftci4a, D. Cinca46, V. Cindro78, I.A. Cioara23, C. Ciocca22a,22b, A. Ciocio1 L. Chytka117, A.K. Ciftci4a, D. Cinca46, V. Cindro78, I.A. Cioara23, C. Ciocca22a,22 F. Cirotto106a,106b, Z.H. Citron175, M. Citterio94a, M. Ciubancan28b, A. Clark52, B.L. Clark59, M.R. Clark38, P.J. Clark49, R.N. Clarke16, C. Clement148a,148b, Y. Coadou88, M. C A. Coccaro52, J. Cochran67, L. Colasurdo108, B. Cole38, A.P. Colijn109, J. Collot58, T. Colombo166, P. Conde Mui˜no128a,128b, E. Coniavitis51, S.H. Connell147b, I.A. Co b, E. Coniavitis51, S.H. Connell147b, I.A. C S. Constantinescu28b, G. Conti32, F. Conventi106a,n, M. Cooke16, A.M. Cooper-Sarkar122, 30 F. Cormier171, K.J.R. Cormier161, M. Corradi134a,134b, F. Corriveau90,o, A. Cortes-Gonzalez32, G. Cortiana103, G. Costa94a, M.J. Costa170, D. Costanzo141, G. Cottin30, G. Cowan80, B.E. Cox87, K. Cranmer112, S.J. Crawley56, R.A. Creager124, G. Cree31, S. Cr´ep´e-Renaudin58, F. Crescioli83, W.A. Cribbs148a,148b, M. Cristinziani23, V. Croft108, G. Crosetti40a,40b, A. Cueto8 T. Cuhadar Donszelmann141, A.R. Cukierman145, J. Cummings179, M. Curatolo50, J. C´uth86, S. Czekierda42, P. Czodrowski32, G. D’amen22a,22b, S. D’Auria56, L. D’eramo83, M. D’Onofrio77, 128 128b 87 41 146 M.J. Da Cunha Sargedas De Sousa128a,128b, C. Da Via87, W. Dabrowski41a, T. Dado146a, T. Dai92, O. Dale15, F. Dallaire97, C. Dallapiccola89, M. Dam39, J.R. Dandoy124, M.F. Daneri29, G. Darbo53a, S. Darmora8, J. Dassoulas3, A. Dattagupta118, T. Daubney45, W. Davey23, C. David45, T. Davidek131, D.R. Davis48, P. Davison81, E. Dawe91, I. Dawson141, K. De8, JHEP08(2017)006 JHEP08(2017)006 R. de Asmundis106a, A. De Benedetti115, S. De Castro22a,22b, S. De Cecco83, N. De Groot1 P. de Jong109, H. De la Torre93, F. De Lorenzi67, A. De Maria57, D. De Pedis134a, A. De Salvo134a, U. De Sanctis135a,135b, A. De Santo151, K. De Vasconcelos Corga88, J.B. De Vivie De Regie119, W.J. Dearnaley75, R. Debbe27, C. Debenedetti139, D.V. Dedovich68, N. Dehghanian3, I. Deigaard109, M. Del Gaudio40a,40b, J. Del Peso85, D. Delgove119, F. Deliot13 N. Dehghanian3, I. Deigaard109, M. Del Gaudio40a,40b, J. Del Peso85, D. Delgove119, F. Deliot138, b b Delitzsch7, A. Dell’Acqua32, L. Dell’Asta24, M. Dell’Orso126a,126b, M. Della Pietra106a,106b, C.M. Delitzsch7, A. Dell’Acqua32, L. Dell’Asta24, M. Dell’Orso126a,126b, M. Della Pietra106a,106b lla Volpe52, M. The ATLAS collaboration Giordani167a,167c, F.M. Giorgi22a, P.F. Giraud138, P. Giromini59, G. Giugliarelli167a,167c, D. Giugni94a, F. Giuli122, C. Giuliani103, M. Giulini60b, B.K. Gjelst Giugliarelli167a,167c, D. Giugni94a, F. Giuli122, C. Giuliani103, M. Giulini60b, B.K. Gjelsten121, S. Gkaitatzis156, I. Gkialas9,s, E.L. Gkougkousis139, P. Gkountoumis10, L.K. Gladilin101, Gkaitatzis156, I. Gkialas9,s, E.L. Gkougkousis139, P. Gkountoumis10, L.K. Gladilin101, C. Glasman85, J. Glatzer13, P.C.F. Glaysher45, A. Glazov45, M. Goblirsch-Kolb25, J. Go Glasman85, J. Glatzer13, P.C.F. Glaysher45, A. Glazov45, M. Goblirsch-Kolb25, J. Godlewski42, Goldfarb91, T. Golling52, D. Golubkov132, A. Gomes128a,128b,128d, R. Gon¸calo128a, S. Goldfarb91, T. Golling52, D. Golubkov132, A. Gomes128a,128b,128d, R. Gon¸calo128a, R. Goncalves Gama26a, J. Goncalves Pinto Firmino Da Costa138, G. Gonella51, L. Gonella19, R. Goncalves Gama26a, J. Goncalves Pinto Firmino Da Costa138, G. Gonella51, L. Gonella19 A. Gongadze68, S. Gonz´alez de la Hoz170, S. Gonzalez-Sevilla52, L. Goossens32, Gongadze68, S. Gonz´alez de la Hoz170, S. Gonzalez-Sevilla52, L. Goossens32, P.A. Gorbounov99, H.A. Gordon27, I. Gorelov107, B. Gorini32, E. Gorini76a,76b, A. Goriˇsek78, P.A. Gorbounov99, H.A. Gordon27, I. Gorelov107, B. Gorini32, E. Gorini76a,76b, A. Goriˇsek78, D. Goujdami137c, A.G. Goussiou140, N. Govender147b,t, E. Gozani154, L. Graber57, D. Goujdami137c, A.G. Goussiou140, N. Govender147b,t, E. Gozani154, L. Graber57, I. Grabowska-Bold41a, P.O.J. Gradin168, J. Gramling166, E. Gramstad121, S. Grancagnolo17, abowska-Bold41a, P.O.J. Gradin168, J. Gramling166, E. Gramstad121, S. Grancagnolo17, V. Gratchev125, P.M. Gravila28f, C. Gray56, H.M. Gray16, Z.D. Greenwood82,u, C. Grefe23, V. Gratchev125, P.M. Gravila28f, C. Gray56, H.M. Gray16, Z.D. Greenwood82,u, C. Grefe23, K. Gregersen81, I.M. Gregor45, P. Grenier145, K. Grevtsov5, J. Griﬃths8, A.A. Grillo139 Gregersen81, I.M. Gregor45, P. Grenier145, K. Grevtsov5, J. Griﬃths8, A.A. Grillo139, Grimm75, S. Grinstein13,v, Ph. Gris37, J.-F. Grivaz119, S. Groh86, E. Gross175, K. Grimm75, S. Grinstein13,v, Ph. Gris37, J.-F. Grivaz119, S. Groh86, E. Gross175, osse-Knetter57, G.C. Grossi82, Z.J. Grout81, A. Grummer107, L. Guan92, W. Guan176, J. Grosse-Knetter57, G.C. Grossi82, Z.J. Grout81, A. Grummer107, L. Guan92, W. Guan J. Guenther65, F. Guescini163a, D. Guest166, O. Gueta155, B. Gui113, E. Guido53a,53b, Guenther65, F. Guescini163a, D. Guest166, O. Gueta155, B. Gui113, E. Guido53a,53b, Guillemin5, S. Guindon2, U. Gul56, C. Gumpert32, J. Guo36c, W. Guo92, Y. Guo36a, T. Guillemin5, S. Guindon2, U. Gul56, C. Gumpert32, J. Guo36c, W. Guo92, Y. Guo36a, Gupta43, S. Gupta122, G. Gustavino134a,134b, P. Gutierrez115, N.G. Gutierrez Ortiz81, R. Gupta43, S. Gupta122, G. Gustavino134a,134b, P. Gutierrez115, N.G. Gutierrez Ortiz81, C. Gutschow81, C. Guyot138, M.P. Guzik41a, C. Gwenlan122, C.B. Gwilliam77, A. Haas112 C. Gutschow81, C. Guyot138, M.P. Guzik41a, C. Gwenlan122, C.B. Gwilliam77, A. Haas112, C. Haber16, H.K. Hadavand8, N. Haddad137e, A. Hadef88, S. Hageb¨ock23, M. The ATLAS collaboration Ereditato18, M. Ernst27, S. Errede169, C. Endner86, J.S. Ennis173, J. Erdmann46, A. Ereditato18, M. Ernst27, S. Errede169, M. Escalier119, C. Escobar170, B. Esposito50, O. Estrada Pastor170, A.I. Etienvre138, E. Etzion M. Escalier119, C. Escobar170, B. Esposito50, O. Estrada Pastor170, A.I. Etienvre138, E. E H. Evans64, A. Ezhilov125, M. Ezzi137e, F. Fabbri22a,22b, L. Fabbri22a,22b, V. Fabiani108, Evans64, A. Ezhilov125, M. Ezzi137e, F. Fabbri22a,22b, L. Fabbri22a,22b, V. Fabiani108, G. Facini81, R.M. Fakhrutdinov132, S. Falciano134a, R.J. Falla81, J. Faltova32, Y. Fang35a, G. Facini81, R.M. Fakhrutdinov132, S. Falciano134a, R.J. Falla81, J. Faltova32, Y. Fang35a, S. Farrell16, S.M. Farrington173, P. Farthouat32, F. Fassi137e, P. Fassnacht32, D. Fassouliotis S. Farrell16, S.M. Farrington173, P. Farthouat32, F. Fassi137e, P. Fassnacht32, D. Fassouliotis9, , g , , , , M. Faucci Giannelli80, A. Favareto53a,53b, W.J. Fawcett122, L. Fayard119, O.L. Fedin125,q, W. Fedorko171, S. Feigl121, L. Feligioni88, C. Feng36b, E.J. Feng32, H. Feng92, M.J. Fenton56, y g A. Ferrari168, P. Ferrari109, R. Ferrari123a, D.E. Ferreira de Lima60b, A. Ferrer170, D. Ferrere52, C. Ferretti92, F. Fiedler86, A. Filipˇciˇc78, M. Filipuzzi45, F. Filthaut108, M. Fincke-Keeler172, K.D. Finelli152, M.C.N. Fiolhais128a,128c,r, L. Fiorini170, A. Fischer2, C. Fischer13, J. Fischer178, 31 W.C. Fisher93, N. Flaschel45, I. Fleck143, P. Fleischmann92, R.R.M. Fletcher124, T. Flick178, , , , , , , B.M. Flierl102, L.R. Flores Castillo62a, M.J. Flowerdew103, G.T. Forcolin87, A. Formica138, , , , , F.A. F¨orster13, A. Forti87, A.G. Foster19, D. Fournier119, H. Fox75, S. Fracchia141, P. Francavilla83, M. Franchini22a,22b, S. Franchino60a, D. Francis32, L. Franconi121, M. Frank M. Frate166, M. Fraternali123a,123b, D. Freeborn81, S.M. Fressard-Batraneanu32, B. Freund97, D. Froidevaux32, J.A. Frost122, C. Fukunaga158, T. Fusayasu104, J. Fuster170, C. Gabaldon58, O. Gabizon154, A. Gabrielli22a,22b, A. Gabrielli16, G.P. Gach41a, S. Gadatsch32, S. Gadomski G. Gagliardi53a,53b, L.G. Gagnon97, C. Galea108, B. Galhardo128a,128c, E.J. Gallas122, F.M. Garay Walls49, C. Garc´ıa170, J.E. Garc´ıa Navarro170, J.A. Garc´ıa Pascual35 M. Garcia-Sciveres16, R.W. Gardner33, N. Garelli145, V. Garonne121, A. Gascon Bravo45, JHEP08(2017)006 JHEP08(2017)006 K. Gasnikova45, C. Gatti50, A. Gaudiello53a,53b, G. Gaudio123a, I.L. Gavrilenko98, C. Gay171, G. Gaycken23, E.N. Gazis10, C.N.P. Gee133, J. Geisen57, M. Geisen86, M.P. Geisler60a, K. Gellerstedt148a,148b, C. Gemme53a, M.H. Genest58, C. Geng92, S. Gentile134a,134b, C. Gentsos156, S. George80, D. Gerbaudo13, A. Gershon155, G. Geßner46, S. Ghasemi143, hneimat23, B. Giacobbe22a, S. Giagu134a,134b, N. Giangiacomi22a,22b, P. Giannetti126a,126b, M. Ghneimat23, B. Giacobbe22a, S. Giagu134a,134b, N. Giangiacomi22a,22b, P. Giannetti126a,12 S.M. Gibson80, M. Gignac171, M. Gilchriese16, D. Gillberg31, G. Gilles178, D.M. Gingrich3,d, N. Giokaris9,∗, M.P. Giordani167a,167c, F.M. Giorgi22a, P.F. Giraud138, P. Giromini59, okaris9,∗, M.P. The ATLAS collaboration Jeske173, S. J´ez´equel5, H. Ji176, J. Jia150, H. Jiang67, Y. Jiang36a, Z Jiang145 S Jiggins81 J Jimenez Pena170 S Jin35a A Jinaru28b O Jinnouchi159 H Jivan147c A. Jelinskas , P. Jenni , C. Jeske , S. Jezequel , H. Ji , J. Jia , H. Jiang , Y. Jiang , Z. Jiang145, S. Jiggins81, J. Jimenez Pena170, S. Jin35a, A. Jinaru28b, O. Jinnouchi159, H. Jivan147c, P. Johansson141, K.A. Johns7, C.A. Johnson64, W.J. Johnson140, K. Jon-And148a,148b, Z. Jiang145, S. Jiggins81, J. Jimenez Pena170, S. Jin35a, A. Jinaru28b, O. Jinnouchi159, H. Jivan14 Z. Jiang145, S. Jiggins81, J. Jimenez Pena170, S. Jin35a, A. Jinaru28b, O. Jinnouchi159, H. Jivan147c, P. Johansson141, K.A. Johns7, C.A. Johnson64, W.J. Johnson140, K. Jon-And148a,148b, P. Johansson141, K.A. Johns7, C.A. Johnson64, W.J. Johnson140, K. Jon-And148a,148b, R.W.L. Jones75, S.D. Jones151, S. Jones7, T.J. Jones77, J. Jongmanns60a, P.M. Jorge128a,128b, g g J. Jovicevic163a, X. Ju176, A. Juste Rozas13,v, M.K. K¨ohler175, A. Kaczmarska42, M. Kado119, A. Kaluza86, S. Kama43, A. Kamenshchikov132, N. Kanaya157, L. Kanjir78, V.A. Kantserov100, J. Kanzaki69, B. Kaplan112, L.S. Kaplan176, D. Kar147c, K. Karakostas10, N. Karastathis10 M.J. Kareem57, E. Karentzos10, S.N. Karpov68, Z.M. Karpova68, K. Karthik112, V. Kartvelishvili75, A.N. Karyukhin132, K. Kasahara164, L. Kashif176, R.D. Kass113, A. Kastanas149, Y. Kataoka157, C. Kato157, A. Katre52, J. Katzy45, K. Kawade70, K. Kawagoe73, nas149, Y. Kataoka157, C. Kato157, A. Katre52, J. Katzy45, K. Kawade70, K. Kawagoe73, T. Kawamoto157, G. Kawamura57, E.F. Kay77, V.F. Kazanin111,c, R. Keeler172, R. Kehoe43, awamoto157, G. Kawamura57, E.F. Kay77, V.F. Kazanin111,c, R. Keeler172, R. Kehoe43, Keller31, J.J. Kempster80, J Kendrick19, H. Keoshkerian161, O. Kepka129, B.P. Kerˇsevan78, J.S. Keller31, J.J. Kempster80, J Kendrick19, H. Keoshkerian161, O. Kepka129, B.P. Kerˇsevan S. Kersten178, R.A. Keyes90, M. Khader169, F. Khalil-zada12, A. Khanov116, A.G. Kharlamo Kharlamova111,c, A. Khodinov160, T.J. Khoo52, V. Khovanskiy99,∗, E. Khramov68, T. Kharlamova111,c, A. Khodinov160, T.J. Khoo52, V. Khovanskiy99,∗, E. Khramov68, Khubua54b,ac, S. Kido70, C.R. Kilby80, H.Y. Kim8, S.H. Kim164, Y.K. Kim33, N. Kimura156, J. Khubua54b,ac, S. Kido70, C.R. Kilby80, H.Y. Kim8, S.H. Kim164, Y.K. Kim33, N. Kimura1 O.M. Kind17, B.T. King77, D. Kirchmeier47, J. Kirk133, A.E. Kiryunin103, T. Kishimoto157, M. Kind17, B.T. King77, D. Kirchmeier47, J. Kirk133, A.E. Kiryunin103, T. Kishimoto157, D. Kisielewska41a, V. Kitali45, K. Kiuchi164, O. Kivernyk5, E. Kladiva146b, Kisielewska41a, V. Kitali45, K. Kiuchi164, O. Kivernyk5, E. Kladiva146b, Klapdor-Kleingrothaus51, M.H. Klein92, M. Klein77, U. Klein77, K. Kleinknecht86, T. Klapdor-Kleingrothaus51, M.H. Klein92, M. Klein77, U. Klein77, K. Kleinknecht86, P. Klimek110, A. Klimentov27, R. Klingenberg46, T. Klingl23, T. Klioutchnikova32, E.-E. The ATLAS collaboration Hagihara164 C. Haber16, H.K. Hadavand8, N. Haddad137e, A. Hadef88, S. Hageb¨ock23, M. Hagihara164, H. Hakobyan180,∗, M. Haleem45, J. Haley116, G. Halladjian93, G.D. Hallewell88, K. Hamach H. Hakobyan180,∗, M. Haleem45, J. Haley116, G. Halladjian93, G.D. Hallewell88, K. Hamacher Hamal117, K. Hamano172, A. Hamilton147a, G.N. Hamity141, P.G. Hamnett45, L. Han36a, P. Hamal117, K. Hamano172, A. Hamilton147a, G.N. Hamity141, P.G. Hamnett45, L. Han36a, S. Han35a, K. Hanagaki69,w, K. Hanawa157, M. Hance139, B. Haney124, P. Hanke60a, S. Han35a, K. Hanagaki69,w, K. Hanawa157, M. Hance139, B. Haney124, P. Hanke60a, T. Harenberg178, F. Hariri119, S. Harkusha95, R.D. Harrington49, P.F. Harrison173, T. Harenberg178, F. Hariri119, S. Harkusha95, R.D. Harrington49, P.F. Harrison173, N.M. Hartmann102, M. Hasegawa70, Y. Hasegawa142, A. Hasib49, S. Hassani138, S. L. Helary32, A. Held171, S. Hellman148a,148b, C. Helsens32, R.C.W. Henderson75, Y. Heng176, 32 S. Henkelmann171, A.M. Henriques Correia32, S. Henrot-Versille119, G.H. Herbert17, H. Herde25 q V. Herget177, Y. Hern´andez Jim´enez147c, H. Herr86, G. Herten51, R. Hertenberger102, L. Her g , , , , g T.C. Herwig124, G.G. Hesketh81, N.P. Hessey163a, J.W. Hetherly43, S. Higashino69, E. Hig´on-Rodriguez170, K. Hildebrand33, E. Hill172, J.C. Hill30, K.H. Hiller45, S.J. Hillier19, M. Hils47, I. Hinchliﬀe16, M. Hirose51, D. Hirschbuehl178, B. Hiti78, O. Hladik129, X. Hoad49, J. Hobbs150, N. Hod163a, M.C. Hodgkinson141, P. Hodgson141, A. Hoecker32, M.R. Hoeferkamp107, F. Hoenig102, D. Hohn23, T.R. Holmes33, M. Homann46, S. Honda164, T. Honda69, T.M. Hong1 B.H. Hooberman169, W.H. Hopkins118, Y. Horii105, A.J. Horton144, J-Y. Hostachy58, S. Hou153, J. Hrivnac119, T. Hryn’ova5, A. Hrynevich96, P.J. Hsu63, S.-C. Hsu140, Q. Hu36a, S. Hu36c, Y. Huang35a, Z. Hubacek130, F. Hubaut88, F. Huegging23, T.B. Huﬀman122, E.W. Hughes38, JHEP08(2017)006 JHEP08(2017)006 G. Hughes75, M. Huhtinen32, P. Huo150, N. Huseynov68,b, J. Huston93, J. Huth59, G. Iacobu G. Iakovidis27, I. Ibragimov143, L. Iconomidou-Fayard119, Z. Idrissi137e, P. Iengo32, O. Igonkina109,y, T. Iizawa174, Y. Ikegami69, M. Ikeno69, Y. Ilchenko11,z, D. Iliadis15 G. Introzzi123a,123b, P. Ioannou9,∗, M. Iodice136a, K. Iordanidou38, V. Ippolito59, M.F. Isacson16 N. Ishijima120, M. Ishino157, M. Ishitsuka159, C. Issever122, S. Istin20a, F. Ito164, J.M. Iturbe Ponce62a, R. Iuppa162a,162b, H. Iwasaki69, J.M. Izen44, V. Izzo106a, S. Jabbar3, J.M. Iturbe Ponce62a, R. Iuppa1 P. Jackson1, R.M. Jacobs23, V. Jain2, K.B. Jakobi86, K. Jakobs51, S. Jakobsen65, T. Jakoubek1 D.O. Jamin116, D.K. Jana82, R. Jansky52, J. Janssen23, M. Janus57, P.A. Janus41a, G. Jarlskog N. Javadov68,b, T. Jav˚urek51, M. Javurkova51, F. Jeanneau138, L. Jeanty16, J. Jejelava54a,aa, A. Jelinskas173, P. Jenni51,ab, C. Jeske173, S. J´ez´equel5, H. Ji176, J. Jia150, H. Jiang67, Y. Jiang36a, A. Jelinskas173, P. Jenni51,ab, C. The ATLAS collaboration Klu P. Klimek110, A. Klimentov27, R. Klingenberg46, T. Klingl23, T. Klioutchnikova32, E.-E. Klug Kluit109, S. Kluth103, E. Kneringer65, E.B.F.G. D. Kobayashi159, T. Kobayashi157, M. Kobel47, M. Kocian145, P. Kodys131, T. Koﬀas31, D. Kobayashi159, T. Kobayashi157, M. Kobel47, M. Kocian145, P. Kodys131, T. Koﬀas31, y y y E. Koﬀeman109, N.M. K¨ohler103, T. Koi145, M. Kolb60b, I. Koletsou5, A.A. Komar98,∗, Y. Komori157, T. Kondo69, N. Kondrashova36c, K. K¨oneke51, A.C. K¨onig108, T. Kono69,ad 112 81 64 41 51 R. Konoplich112,ae, N. Konstantinidis81, R. Kopeliansky64, S. Koperny41a, A.K. Kopp51, K. Korcyl42, K. Kordas156, A. Korn81, A.A. Korol111,c, I. Korolkov13, E.V. Korolkova141 O. Kortner103, S. Kortner103, T. Kosek131, V.V. Kostyukhin23, A. Kotwal48, A. Koulouris A. Kourkoumeli-Charalampidi123a,123b, C. Kourkoumelis9, E. Kourlitis141, V. Kouskoura27, 33 A.B. Kowalewska42, R. Kowalewski172, T.Z. Kowalski41a, C. Kozakai157, W. Kozanecki138, A.S. Kozhin132, V.A. Kramarenko101, G. Kramberger78, D. Krasnopevtsev100, M.W. Krasn , , g , p , A. Krasznahorkay32, D. Krauss103, J.A. Kremer41a, J. Kretzschmar77, K. Kreutzfeldt55, P. Krieger161, K. Krizka33, K. Kroeninger46, H. Kroha103, J. Kroll129, J. Kroll124, J. Krose J. Krstic14, U. Kruchonak68, H. Kr¨uger23, N. Krumnack67, M.C. Kruse48, T. Kubota91, H. Kucuk81, S. Kuday4b, J.T. Kuechler178, S. Kuehn32, A. Kugel60a, F. Kuger177, T. Kuhl45, V. Kukhtin68, R. Kukla88, Y. Kulchitsky95, S. Kuleshov34b, Y.P. Kulinich169, M. Kuna134a,134b, T. Kunigo71, A. Kupco129, T. Kupfer46, O. Kuprash155, H. Kurashige70, L.L. Kurchaninov163a, T. Kunigo71, A. Kupco129, T. Kupfer46, O. Kuprash155, H. Kurashige70, L.L. Kurchaninov163a, T. Kwan172, D. Kyriazopoulos141, A. La Rosa103, J.L. La Rosa Navarro26d, L. La Rotonda40a,40b T. Kwan172, D. Kyriazopoulos141, A. La Rosa103, J.L. La Rosa Navarro26d, L. La Rotonda40a,40b F. La Ruﬀa40a,40b, C. Lacasta170, F. Lacava134a,134b, J. Lacey45, H. Lacker17, D. Lacour83, JHEP08(2017)006 JHEP08(2017)006 E. Ladygin68, R. Lafaye5, B. Laforge83, T. Lagouri179, S. Lai57, S. Lammers64, W. Lampl7, E. Ladygin68, R. Lafaye5, B. Laforge83, T. Lagouri179, S. Lai57, S. Lammers64, W. Lampl7, E. Lan¸con27, U. Landgraf51, M.P.J. Landon79, M.C. Lanfermann52, V.S. Lang60a, J.C. Lange R.J. Langenberg32, A.J. Lankford166, F. Lanni27, K. Lantzsch23, A. Lanza123a, A. Lapertosa53a,53b, S. Laplace83, J.F. Laporte138, T. Lari94a, F. Lasagni Manghi22a,22b, M. Lassnig32, P. Laurelli50, W. Lavrijsen16, A.T. Law139, P. Laycock77, T. Lazovich59, assnig32, P. Laurelli50, W. Lavrijsen16, A.T. Law139, P. Laycock77, T. Lazovich59, azzaroni94a,94b, B. Le91, O. Le Dortz83, E. Le Guirriec88, E.P. Le Quilleuc138, M. LeBlanc172, M. Lazzaroni94a,94b, B. Le91, O. Le Dortz83, E. Le Guirriec88, E.P. Le Quilleuc138, M. LeB Compte6, F. Ledroit-Guillon58, C.A. Lee27, G.R. Lee133,af, S.C. Lee153, L. Lee59, T. LeCompte6, F. The ATLAS collaboration Ledroit-Guillon58, C.A. Lee27, G.R. Lee133,af, S.C. Lee153, L. Lee59, febvre90, G. Lefebvre83, M. Lefebvre172, F. Legger102, C. Leggett16, G. Lehmann Miotto32, B. Lefebvre90, G. Lefebvre83, M. Lefebvre172, F. Legger102, C. Leggett16, G. Lehmann Miott Lei7, W.A. Leight45, M.A.L. Leite26d, R. Leitner131, D. Lellouch175, B. Lemmer57, X. Lei7, W.A. Leight45, M.A.L. Leite26d, R. Leitner131, D. Lellouch175, B. Lemmer57, J.C. Leney81, T. Lenz23, B. Lenzi32, R. Leone7, S. Leone126a,126b, C. Leonidopoulos49, K.J.C. Leney81, T. Lenz23, B. Lenzi32, R. Leone7, S. Leone126a,126b, C. Leonidopoulos49, Lerner151, C. Leroy97, A.A.J. Lesage138, C.G. Lester30, M. Levchenko125, J. Levˆeque5, G. Lerner151, C. Leroy97, A.A.J. Lesage138, C.G. Lester30, M. Levchenko125, J. Levˆeque5, D. Levin92, L.J. Levinson175, M. Levy19, D. Lewis79, B. Li36a,ag, Changqiao Li36a, H. Li150, D. Levin92, L.J. Levinson175, M. Levy19, D. Lewis79, B. Li36a,ag, Changqiao Li36a, H. Li150 L. Li36c, Q. Li35a, S. Li48, X. Li36c, Y. Li143, Z. Liang35a, B. Liberti135a, A. Liblong161, K. Li 6c, Q. Li35a, S. Li48, X. Li36c, Y. Li143, Z. Liang35a, B. Liberti135a, A. Liblong161, K. Lie62c, J. Liebal23, W. Liebig15, A. Limosani152, S.C. Lin182, T.H. Lin86, R.A. Linck64, B.E. Lindquis J. Liebal23, W. Liebig15, A. Limosani152, S.C. Lin182, T.H. Lin86, R.A. Linck64, B.E. Lindquist150 A.M. Litke139, B. Liu153,ai, H. Liu92, H. Liu27, J.K.K. Liu122, J. Liu36b, J.B. Liu36a, K. Liu88, A.M. Litke139, B. Liu153,ai, H. Liu92, H. Liu27, J.K.K. Liu122, J. Liu36b, J.B. Liu36a, K. Liu88 Liu169, M. Liu36a, Y.L. Liu36a, Y. Liu36a, M. Livan123a,123b, A. Lleres58, J. Llorente Merino35a, L. Liu169, M. Liu36a, Y.L. Liu36a, Y. Liu36a, M. Livan123a,123b, A. Lleres58, J. Llorente Merino35 S.L. Lloyd79, C.Y. Lo62b, F. Lo Sterzo153, E.M. Lobodzinska45, P. Loch7, F.K. Loebinger87, S.L. Lloyd79, C.Y. Lo62b, F. Lo Sterzo153, E.M. Lobodzinska45, P. Loch7, F.K. Loebinger87, Loesle51, K.M. Loew25, A. Loginov179,∗, T. Lohse17, K. Lohwasser141, M. Lokajicek129, A. Loesle51, K.M. Loew25, A. Loginov179,∗, T. Lohse17, K. Lohwasser141, M. Lokajicek129 A. Long24, J.D. Long169, R.E. Long75, L. Longo76a,76b, K.A. Looper113, J.A. Lopez34b, B.A. Long24, J.D. Long169, R.E. Long75, L. Longo76a,76b, K.A. Looper113, J.A. Lopez34b, Lopez Mateos59, I. Lopez Paz13, A. Lopez Solis83, J. Lorenz102, N. Lorenzo Martinez5, D. Lopez Mateos59, I. Lopez Paz13, A. Lopez Solis83, J. Lorenz102, N. Lorenzo Martinez5 Losada21, P.J. L¨osel102, X. Lou35a, A. Lounis119, J. Love6, P.A. Love75, H. Lu62a, N. Lu92, M. Losada21, P.J. L¨osel102, X. Lou35a, A. Lounis119, J. Love6, P.A. Love75, H. Lu62a, N. L Y.J. Lu63, H.J. Lubatti140, C. Luci134a,134b, A. Lucotte58, C. Luedtke51, F. Luehring64, J. The ATLAS collaboration Meier60a, B. Meirose44, D. Melini170,aj, cia147c, J.D. Mellenthin57, M. Melo146a, F. Melon S.B. Menary87, L. Meng77, X.T. Meng92, A. Mengarelli22a,22b, S. Menke103, E. Meoni40a,40b, S.B. Menary87, L. Meng77, X.T. Meng92, A. Mengarelli22a,22b, S. Menke103, E. Meoni40a,40b, S. Mergelmeyer17, C. Merlassino18, P. Mermod52, L. Merola106a,106b, C. Meroni94a, F.S. Merritt3 JHEP08(2017)006 JHEP08(2017)006 A. Messina134a,134b, J. Metcalfe6, A.S. Mete166, C. Meyer124, J-P. Meyer138, J. Meyer109, H. Meyer Zu Theenhausen60a, F. Miano151, R.P. Middleton133, S. Miglioranzi53a,53b, L. Mij nberg175, M. Mikestikova129, M. Mikuˇz78, M. Milesi91, A. Milic161, D.W. Miller33, G. Mikenberg175, M. Mikestikova129, M. Mikuˇz78, M. Milesi91, A. Milic161, D.W. Miller33, C. Mills49, A. Milov175, D.A. Milstead148a,148b, A.A. Minaenko132, Y. Minami157, I.A. Minashvili68, A.I. Mincer112, B. Mindur41a, M. Mineev68, Y. Minegishi157, Y. Ming176, I.A. Minashvili68, A.I. Mincer112, B. Mindur41a, M. Mineev68, Y. Minegishi157, Y. Ming176, L M Mi 13 K P Mi 124 T Mi i174 J Mi ki102 V A Mi 170 A Mi i18 Mir13, K.P. Mistry124, T. Mitani174, J. Mitrevski102, V.A. Mitsou170, A. Miucci18, L.M. Mir13, K.P. Mistry124, T. Mitani174, J. Mitrevski102, V.A. Mitsou170, A. Miucci18, P.S. Miyagawa141, A. Mizukami69, J.U. Mj¨ornmark84, T. Mkrtchyan180, M. Mlynarikova131, agawa141, A. Mizukami69, J.U. Mj¨ornmark84, T. Mkrtchyan180, M. Mlynarikova131, T. Moa148a,148b, K. Mochizuki97, P. Mogg51, S. Mohapatra38, S. Molander148a,148b, oa148a,148b, K. Mochizuki97, P. Mogg51, S. Mohapatra38, S. Molander148a,148b, R. Moles-Valls23, R. Monden71, M.C. Mondragon93, K. M¨onig45, J. Monk39, E. Monnier88, A. Montalbano150, J. Montejo Berlingen32, F. Monticelli74, S. Monzani94a,94b, R.W. Moore3, N. Morange119, D. Moreno21, M. Moreno Ll´acer32, P. Morettini53a, S. Morgenstern32, D. Mor T. Mori157, M. Morii59, M. Morinaga157, V. Morisbak121, A.K. Morley32, G. Mornacchi32, J.D. Morris79, L. Morvaj150, P. Moschovakos10, M. Mosidze54b, H.J. Moss141, J. Moss145,ak, K. Motohashi159, R. Mount145, E. Mountricha27, E.J.W. Moyse89, S. Muanza88, F. Mueller103, K. Motohashi159, R. Mount145, E. Mountricha27, E.J.W. Moyse89, S. Muanza88, F. Mueller103, F.J. Munoz Sanchez87, W.J. Murray173,133, H. Musheghyan32, M. Muˇskinja78, A.G. Myagkov132,al, M. Myska130, B.P. Nachman16, O. Nackenhorst52, K. Nagai122, R. Nagai69,ad, K. Nagano69, Y. Nagasaka61, K. Nagata164, M. Nagel51, E. Nagy88, A.M. Nairz32, R. Nagai69,ad, K. Nagano69, Y. Nagasaka61, K. Nagata164, M. Nagel51, E. Nagy88, A.M. Nairz3 Y. Nakahama105, K. Nakamura69, T. Nakamura157, I. Nakano114, R.F. Naranjo Garcia45, R. Narayan11, D.I. Narrias Villar60a, I. Naryshkin125, T. Naumann45, G. Navarro21, R. Nayyar7, arayan11, D.I. Narrias Villar60a, I. Naryshkin125, T. Naumann45, G. Navarro21, R. Nayyar7, H.A. Neal92, P.Yu. Nechaeva98, T.J. Neep138, A. Negri123a,123b, M. Negrini22a, S. Nektarijev Neal92, P.Yu. Nechaeva98, T.J. Neep138, A. Negri123a,123b, M. The ATLAS collaboration Lu63, H.J. Lubatti140, C. Luci134a,134b, A. Lucotte58, C. Luedtke51, F. Luehring64, ci134a,134b, A. Lucotte58, C. Luedtke51, F. Luehri . Lukas65, L. Luminari134a, O. Lundberg148a,148b, B. Lund-Jensen149, M.S. Lutz89, P.M. Luzi83, W. Lukas65, L. Luminari134a, O. Lundberg148a,148b, B. Lund-Jensen149, M.S. Lutz89, P.M D. Lynn27, R. Lysak129, E. Lytken84, F. Lyu35a, V. Lyubushkin68, H. Ma27, L.L. Ma36b, Lynn27, R. Lysak129, E. Lytken84, F. Lyu35a, V. Lyubushkin68, H. Ma27, L.L. Ma36b, Y. Ma36b, G. Maccarrone50, A. Macchiolo103, C.M. Macdonald141, B. Maˇcek78, Ma36b, G. Maccarrone50, A. Macchiolo103, C.M. Macdonald141, B. Maˇcek78, Machado Miguens124,128b, D. Madaﬀari170, R. Madar37, W.F. Mader47, A. Madsen45, J. Machado Miguens124,128b, D. Madaﬀari170, R. Madar37, W.F. Mader47, A. Madsen45, J. Maeda70, S. Maeland15, T. Maeno27, A.S. Maevskiy101, V. Magerl51, J. Mahlstedt109, Maeda70, S. Maeland15, T. Maeno27, A.S. Maevskiy101, V. Magerl51, J. Mahlstedt109, C. Maiani119, C. Maidantchik26a, A.A. Maier103, T. Maier102, A. Maio128a C. Maiani119, C. Maidantchik26a, A.A. Maier103, T. Maier102, A. Maio128a,128b,128d, V.P. Maleev125, F. Malek58, U. Mallik66, D. Malon6, C. Malone30, S. Maltezos10, S. Malyukov V.P. Maleev125, F. Malek58, U. Mallik66, D. Malon6, C. Malone30, S. Maltezos10, S. Malyukov32, J. Mamuzic170, G. Mancini50, I. Mandi´c78, J. Maneira128a,128b, L. Manhaes de Andrade Filho26b, J. Manjarres Ramos47, K.H. Mankinen84, A. Mann102, A. Manousos32, B. Mansoulie138, J.D. Mansour35a, R. Mantifel90, M. Mantoani57, S. Manzoni94a,94b, L. Mapelli32, G. Marceca29, L. March52, L. Marchese122, G. Marchiori83, M. Marcisovsky129, M. Marjanovic37, D.E. Marley92, F. Marroquim26a, S.P. Marsden87, Z. Marshall16, M.U.F Martensson168, S. Marti-Garcia170, C B Martin113 T A Martin173 V J Martin49 B Martin dit Latour15 M Martinez13,v V.P. Maleev125, F. Malek58, U. Mallik66, D. Malon6, C. Malone30, S. Maltezos10, S. Malyukov32, J. Mamuzic170, G. Mancini50, I. Mandi´c78, J. Maneira128a,128b, L. Manhaes de Andrade Filho26b, J. Manjarres Ramos47, K.H. Mankinen84, A. Mann102, A. Manousos32, B. Mansoulie138, J.D. Mansour35a, R. Mantifel90, M. Mantoani57, S. Manzoni94a,94b, L. Mapelli32, G. Marceca29, 34 V.I. Martinez Outschoorn169, S. Martin-Haugh133, V.S. Martoiu28b, A.C. Martyniuk81, g y A. Marzin32, L. Masetti86, T. Mashimo157, R. Mashinistov98, J. Masik87, A.L. Maslennikov111,c L. Massa135a,135b, P. Mastrandrea5, A. Mastroberardino40a,40b, T. Masubuchi157, P. M¨attig178, J. Maurer28b, S.J. Maxﬁeld77, D.A. Maximov111,c, R. Mazini153, I. Maznas156, S.M. Mazza94a,94 N.C. Mc Fadden107, G. Mc Goldrick161, S.P. Mc Kee92, A. McCarn92, R.L. McCarthy150, T.G. McCarthy103, L.I. McClymont81, E.F. McDonald91, J.A. Mcfayden81, G. Mchedlidze57, S.J. McMahon133, P.C. McNamara91, R.A. McPherson172,o, S. Meehan140, T.J. Megy51, S. Mehlhase102, A. Mehta77, T. Meideck58, K. Meier60a, B. Meirose44, D. Melini170,aj, S. Mehlhase102, A. Mehta77, T. Meideck58, K. The ATLAS collaboration Negrini22a, S. Nektarijevic108, llist119, A. Nelson166, M.E. Nelson122, S. Nemecek129, P. Nemethy112, M. Nessi32,am, C. Nellist119, A. Nelson166, M.E. Nelson122, S. Nemecek129, P. Nemethy112, M. Nessi32,am , , , , y , , M.S. Neubauer169, M. Neumann178, P.R. Newman19, T.Y. Ng62c, T. Nguyen Manh97, Neubauer169, M. Neumann178, P.R. Newman19, T.Y. Ng62c, T. Nguyen Manh97, M.S. Neubauer169, M. Neumann178, P.R. Newman19, T.Y. Ng62c, T. Nguyen Manh97, R.B. Nickerson122, R. Nicolaidou138, J. Nielsen139, V. Nikolaenko132,al, I. Nikolic-Audit83, Nickerson122, R. Nicolaidou138, J. Nielsen139, V. Nikolaenko132,al, I. Nikolic-Audit83, K. Nikolopoulos19, J.K. Nilsen121, P. Nilsson27, Y. Ninomiya157, A. Nisati134a, N. Nishu35c, A.A. O’Rourke45, V. O’Shea56, F.G. Oakham31,d, H. Oberlack103, T. Obermann23, J. Ocariz83, C.C. Ohm16, H. Ohman168, H. Oide53a,53b, H. Okawa164, Y. Okumura157, T. Okuyama69, A. Olariu28b, L.F. Oleiro Seabra128a, S.A. Olivares Pino34a, D. Oliveira Damazio27, A. Olszewski42, J. Olszowska42, A. Onofre128a,128e, K. Onogi105, P.U.E. Onyisi11,z, H. Oppen121 M.J. Oreglia33, Y. Oren155, D. Orestano136a,136b, N. Orlando62b, R.S. Orr161, B. Osculati53 R. Ospanov36a, G. Otero y Garzon29, H. Otono73, M. Ouchrif137d, F. Ould-Saada121, A. Ouraou138, K.P. Oussoren109, Q. Ouyang35a, M. Owen56, R.E. Owen19, V.E. Ozcan20a, N. Ozturk8, K. Pachal144, A. Pacheco Pages13, L. Pacheco Rodriguez138, C. Padilla Aranda1 35 S. Pagan Griso16, M. Paganini179, F. Paige27, G. Palacino64, S. Palazzo40a,40b, S. Palestini32, M. Palka41b, D. Pallin37, E.St. Panagiotopoulou10, I. Panagoulias10, C.E. Pandini126a,126b, J.G. Panduro Vazquez80, P. Pani32, S. Panitkin27, D. Pantea28b, L. Paolozzi52, Th.D. Papadopoulou10, K. Papageorgiou9,s, A. Paramonov6, D. Paredes Hernandez179, A.J. Parker75, M.A. Parker30, K.A. Parker45, F. Parodi53a,53b, J.A. Parsons38, U. Parzefall51, V.R. Pascuzzi161, J.M. Pasner139, E. Pasqualucci134a, S. Passaggio53a, Fr. Pastore80, S. Pataraia86, J.R. Pater87, T. Pauly32, B. Pearson103, S. Pedraza Lopez170, R. Pedro128a,128b, S.V. Peleganchuk111,c, O. Penc129, C. Peng35a, H. Peng36a, J. Penwell64, B.S. Peralva26b, S. Perrella106a,106b, R. Peschke45, V.D. Peshekhonov68,∗, K. Peters45, R.F.Y. Peters8 B.A. Petersen32, T.C. Petersen39, E. Petit58, A. Petridis1, C. Petridou156, P. Petroﬀ119, JHEP08(2017)006 JHEP08(2017)006 E. Petrolo134a, M. Petrov122, F. Petrucci136a,136b, N.E. Pettersson89, A. Peyaud138, R. Pezoa34b F.H. Phillips93, P.W. Phillips133, G. Piacquadio150, E. Pianori173, A. Picazio89, E. Piccaro79, M.A. Pickering122, R. Piegaia29, J.E. Pilcher33, A.D. Pilkington87, A.W.J. Pin87, M. Pinamonti135a,135b, J.L. Pinfold3, H. Pirumov45, M. Pitt175, L. Plazak146a, M.-A. Pleier2 V. Pleskot86, E. Plotnikova68, D. Pluth67, P. Podberezko111, R. Poettgen148a,148b, R. Poggi123a,123b, L. Poggioli119, D. Pohl23, G. Polesello123a, A. Poley45, A. Policicchio40a,40b, R. Polifka32, A. Polini22a, C.S. Pollard56, V. Polychronakos27, K. Pomm`es32, D. Ponomarenko10 L. Pontecorvo134a, G.A. Popeneciu28d, A. Poppleton32, S. Pospisil130, K. Potamianos16, I.N. Potrap68, C.J. The ATLAS collaboration Potter30, G. Poulard32, T. Poulsen84, J. Poveda32, M.E. Pozo Astigarraga32, P. Pralavorio88, A. Pranko16, S. Prell67, D. Price87, M. Primavera76a, S. Prince90, N. Proklova10 K. Prokoﬁev62c, F. Prokoshin34b, S. Protopopescu27, J. Proudfoot6, M. Przybycien41a, A. Puri16 P. Puzo119, J. Qian92, G. Qin56, Y. Qin87, A. Quadt57, M. Queitsch-Maitland45, D. Quilty56, S. Raddum121, V. Radeka27, V. Radescu122, S.K. Radhakrishnan150, P. Radloﬀ118, P. Rados91, F. Ragusa94a,94b, G. Rahal181, J.A. Raine87, S. Rajagopalan27, C. Rangel-Smith168, T. Rashid119, J.H. Rawling87, M. Raymond32, A.L. Read121, N.P. Readioﬀ58, M. Reale76a,76b, D.M. Rebuzzi123a,123b, A. Redelbach177, G. Redlinger27, R. Reece139, R.G. Reed147c L. Rehnisch17, J. Reichert124, A. Reiss86, C. Rembser32, H. Ren35a, M. Rescigno134a, S. Resconi94a, E.D. Resseguie124, S. Rettie171, E. Reynolds19, O.L. Rezanova111,c, P. Reznicek1 S. Resconi , E.D. Resseguie , S. Rettie , E. Reynolds , O.L. Rezanova , , P. Reznicek , R. Rezvani97, R. Richter103, S. Richter81, E. Richter-Was41b, O. Ricken23, M. Ridel83, P. Rieck103, C.J. Riegel178, J. Rieger57, O. Rifki115, M. Rijssenbeek150, A. Rimoldi123a,123b, M. Rimoldi18, , g , , y , , , R. Rezvani97, R. Richter103, S. Richter81, E. Richter-Was41b, O. Ricken23, M. Ridel83, P. Rieck103, C J Ri l178 J Ri 57 O Rifki115 M Rij b k150 A Ri ldi123a 123b M Ri ldi18 R. Rezvani97, R. Richter103, S. Richter81, E. Richter-Was41b, O. Ricken23, M. Ridel83, P. Rieck10 C.J. Riegel178, J. Rieger57, O. Rifki115, M. Rijssenbeek150, A. Rimoldi123a,123b, M. Rimoldi18, L. Rinaldi22a, G. Ripellino149, B. Risti´c32, E. Ritsch32, I. Riu13, F. Rizatdinova116, E. Rizvi79, C. Rizzi13, R.T. Roberts87, S.H. Robertson90,o, A. Robichaud-Veronneau90, D. Robinson30, J.E.M. Robinson45, A. Robson56, E. Rocco86, C. Roda126a,126b, Y. Rodina88,an, S. Rodriguez Bosca170, A. Rodriguez Perez13, D. Rodriguez Rodriguez170, S. Roe32, C.S. Rogan O. Røhne121, J. Roloﬀ59, A. Romaniouk100, M. Romano22a,22b, S.M. Romano Saez37, E. Romero Adam170, N. Rompotis77, M. Ronzani51, L. Roos83, S. Rosati134a, K. Rosbach51, P. Rose139, N.-A. Rosien57, E. Rossi106a,106b, L.P. Rossi53a, J.H.N. Rosten30, R. Rosten140, M. Rotaru28b, J. Rothberg140, D. Rousseau119, A. Rozanov88, Y. Rozen154, X. Ruan147c, M. Rotaru28b, J. Rothberg140, D. Rousseau119, A. Rozanov88, Y. Rozen154, X. Ruan147c, J.P. Rutherfoord7, N. Ruthmann32, Y.F. Ryabov125, M. Rybar169, G. Rybkin119, S. Ryu6, A. Ryzhov132, G.F. Rzehorz57, A.F. Saavedra152, G. Sabato109, S. Sacerdoti29, H.F-W. Sadrozinski139, R. Sadykov68, F. Safai Tehrani134a, P. Saha110, M. Sahinsoy60a, M. Saimpert45, M. Saito157, T. Saito157, H. Sakamoto157, Y. Sakurai174, G. Salamanna136a,136b, J.E. Salazar Loyola34b, D. Salek109, P.H. Sales De Bruin168, D. Salihagic103, A. The ATLAS collaboration Salnikov145, 170 40 40b 151 62 62b 62 32 J.E. Salazar Loyola , D. Salek , P.H. Sales De Bruin , D. Salihagic , A. Salnikov , J. Salt170, D. Salvatore40a,40b, F. Salvatore151, A. Salvucci62a,62b,62c, A. Salzburger32, D. Sammel51, D. Sampsonidis156, D. Sampsonidou156, J. S´anchez170, V. Sanchez Martinez170, 36 A. Sanchez Pineda167a,167c, H. Sandaker121, R.L. Sandbach79, C.O. Sander45, M. Sandhoﬀ178 C. Sandoval21, D.P.C. Sankey133, M. Sannino53a,53b, Y. Sano105, A. Sansoni50, C. Santoni37, , y , , , , , H. Santos128a, I. Santoyo Castillo151, A. Sapronov68, J.G. Saraiva128a,128d, B. Sarrazin23, O. Sasaki69, K. Sato164, E. Sauvan5, G. Savage80, P. Savard161,d, N. Savic103, C. Sawyer133, Sawyer82,u, J. Saxon33, C. Sbarra22a, A. Sbrizzi22a,22b, T. Scanlon81, D.A. Scannicchio166, L. Sawyer82,u, J. Saxon33, C. Sbarra22a, A. Sbrizzi22a,22b, T. Scanlon81, D.A. Scannicchio166 M. Scarcella152, J. Schaarschmidt140, P. Schacht103, B.M. Schachtner102, D. Schaefer32, Scarcella152, J. Schaarschmidt140, P. Schacht103, B.M. Schachtner102, D. Schaefer32, L. Schaefer124, R. Schaefer45, J. Schaeﬀer86, S. Schaepe23, S. Schaetzel60b, U. Sch¨afer86, A.C. Schaﬀer119, D. Schaile102, R.D. Schamberger150, V.A. Schegelsky125, D. Scheirich131, A.C. Schaﬀer119, D. Schaile102, R.D. Schamberger150, V.A. Schegelsky125, D. Scheirich131, S. Schlenker32, K.R. Schmidt-Sommerfeld103, K. Schmieden32, C. Schmitt86, S. Schmitt45, S. Schlenker32, K.R. Schmidt-Sommerfeld103, K. Schmieden32, C. Schmitt86, S. Schmitt45, , , , , , S. Schmitz86, U. Schnoor51, L. Schoeﬀel138, A. Schoening60b, B.D. Schoenrock93, E. Schopf23, JHEP08(2017)006 JHEP08(2017)006 M. Schott86, J.F.P. Schouwenberg108, J. Schovancova32, S. Schramm52, N. Schuh86, A. Schulte8 M. Schott86, J.F.P. Schouwenberg108, J. Schovancova32, S. Schramm52, N. Schuh86, A. Schulte86 M.J. Schultens23, H.-C. Schultz-Coulon60a, H. Schulz17, M. Schumacher51, B.A. Schumm139, Ph. Schune138, A. Schwartzman145, T.A. Schwarz92, H. Schweiger87, Ph. Schwemling138, 38, A. Schwartzman145, T.A. Schwarz92, H. Schweiger87, Ph. Schwemling138, R. Schwienhorst93, J. Schwindling138, A. Sciandra23, G. Sciolla25, M. Scornajenghi40a,40b, orst93, J. Schwindling138, A. Sciandra23, G. Sciolla25, M. Scornajenghi40a,40b, 126b, F. Scutti91, J. Searcy92, P. Seema23, S.C. Seidel107, A. Seiden139, J.M. Seixas26a, F. Scuri126a,126b, F. Scutti91, J. Searcy92, P. Seema23, S.C. Seidel107, A. Seiden139, J.M. Seixas G. Sekhniaidze106a, K. Sekhon92, S.J. Sekula43, N. Semprini-Cesari22a,22b, S. Senkin37, ze106a, K. Sekhon92, S.J. Sekula43, N. Semprini-Cesari22a,22b, S. Senkin37, C. Serfon121, L. Serin119, L. Serkin167a,167b, M. Sessa136a,136b, R. Seuster172, H. Severini115, , L. Serin119, L. Serkin167a,167b, M. Sessa136a,136b, R. Seuster172, H. Severini115, Sﬁligoj78, F. Sforza32, A. Sfyrla52, E. Shabalina57, N.W. Shaikh148a,148b, L.Y. Shan35a, T. Sﬁligoj78, F. Sforza32, A. Sfyrla52, E. Shabalina57, N.W. Shaikh148a,148b, L.Y. Shan35a, Shang169, J.T. Shank24, M. Shapiro16, P.B. Shatalov99, K. Shaw167a,167b, S.M. Shaw87, R. Shang169, J.T. Shank24, M. Shapiro16, P.B. The ATLAS collaboration Shatalov99, K. Shaw167a,167b, S.M. Shaw87, A. Shcherbakova148a,148b, C.Y. Shehu151, Y. Shen115, N. Sherafati31, P. Sherwood81, L. Shi153,ao A. Shcherbakova148a,148b, C.Y. Shehu151, Y. Shen115, N. Sherafati31, P. Sherwood81, L. Shi15 S. Shimizu70, C.O. Shimmin179, M. Shimojima104, I.P.J. Shipsey122, S. Shirabe73, M. Shiyakova68,ap, J. Shlomi175, A. Shmeleva98, D. Shoaleh Saadi97, M.J. Shochet33, S. Shojai M. Shiyakova68,ap, J. Shlomi175, A. Shmeleva98, D. Shoaleh Saadi97, M.J. Shochet33, S. Sho D.R. Shope115, S. Shrestha113, E. Shulga100, M.A. Shupe7, P. Sicho129, A.M. Sickles169, P.E. Sidebo149, E. Sideras Haddad147c, O. Sidiropoulou177, A. Sidoti22a,22b, F. Siegert47, P.E. Sidebo149, E. Sideras Haddad147c, O. Sidiropoulou177, A. Sidoti22a,22b, F. Siegert47 P.E. Sidebo149, E. Sideras Haddad147c, O. Sidiropoulou177, A. Sidoti22a,22b, F. Siegert47, Dj Sijacki14 J Sil a128a,128d S B Sil e stei 148a V Si ak130 Lj Si ic14 S Si io 119 E. Simioni86, B. Simmons81, M. Simon86, P. Sinervo161, N.B. Sinev118, M. Sioli22a,22b, E. Simioni86, B. Simmons81, M. Simon86, P. Sinervo161, N.B. Sinev118, M. Sioli22a,22b, G. Siragusa177, I. Siral92, S.Yu. Sivoklokov101, J. Sj¨olin148a,148b, M.B. Skinner75, P. Skubic115, M. Slater19, T. Slavicek130, M. Slawinska42, K. Sliwa165, R. Slovak131, V. Smakhtin175, B.H. Smart5, J. Smiesko146a, N. Smirnov100, S.Yu. Smirnov100, Y. Smirnov100, L.N. Smirnova101,aq, O. Smirnova84, J.W. Smith57, M.N.K. Smith38, R.W. Smith38, M. Smizanska75, K. Smolek130, A.A. Snesarev98, I.M. Snyder118, S. Snyder27, R. Sobie172,o, F. Socher47, A. Soﬀer155, A. Søgaard49, D.A. Soh153, G. Sokhrannyi78, C.A. Solans Sanchez32, M. Solar130, E.Yu. Soldatov100, U. Soldevila170, A.A. Solodkov132, A. Soloshenko68, O.V. Solovyanov132, V. Solovyev125, P. Sommer51, H. Son165, A. Sopczak130, D. Sosa60b, ovyanov132, V. Solovyev125, P. Sommer51, H. Son165, A. Sopczak130, D. Sosa60b, C.L. Sotiropoulou126a,126b, R. Soualah167a,167c, A.M. Soukharev111,c, D. South45, B.C. So C.L. Sotiropoulou126a,126b, R. Soualah167a,167c, A.M. Soukharev111,c, D. South45, B.C. Sowden8 7c, A.M. Soukharev111,c, D. South45, B. S. Spagnolo76a,76b, M. Spalla126a,126b, M. Spangenberg173, F. Span`o80, D. Sperlich17, Spagnolo76a,76b, M. Spalla126a,126b, M. Spangenberg173, F. Span`o80, D. Sperlich17, Spettel103, T.M. Spieker60a, R. Spighi22a, G. Spigo32, L.A. Spiller91, M. Spousta131, F. Spettel103, T.M. Spieker60a, R. Spighi22a, G. Spigo32, L.A. Spiller91, M. Spousta131, D. St. Denis56,∗, A. Stabile94a, R. Stamen60a, S. Stamm17, E. Stanecka42, R.W. Stanek6, R.D. St. Denis56,∗, A. Stabile94a, R. Stamen60a, S. Stamm17, E. Stanecka42, R.W. Stanek6, C. Stanescu136a, M.M. Stanitzki45, B.S. Stapf109, S. Stapnes121, E.A. Starchenko132, G.H. Sta C. Stanescu136a, M.M. Stanitzki45, B.S. Stapf109, S. Stapnes121, E.A. Starchenko132, G.H. Stark P. Steinberg27, B. Stelzer144, H.J. Stelzer32, O. Stelzer-Chilton163a, H. Stenzel55, G.A. Stewart P. Steinberg27, B. Stelzer144, H.J. Stelzer32, O. Stelzer-Chilton163a, H. Stenzel55, G.A. Stewart56 M.C. The ATLAS collaboration Tsukerman99, V. Tsulaia16, S. Tsuno69, D. Tsybychev150, Y. Tu62b, A. Tudorache28b, V. Tudorache28b, T.T. Tulbure28a, A.N. Tuna59, S.A. Tupputi22a,22b, S. Turchikhin68, Tudorache28b, T.T. Tulbure28a, A.N. Tuna59, S.A. Tupputi22a,22b, S. Turchikhin68, D. Turgeman175, I. Turk Cakir4b,au, R. Turra94a, P.M. Tuts38, G. Ucchielli22a,22b, I. Ueda69, Turgeman175, I. Turk Cakir4b,au, R. Turra94a, P.M. Tuts38, G. Ucchielli22a,22b, I. Ueda69, M. Ughetto148a,148b, F. Ukegawa164, G. Unal32, A. Undrus27, G. Unel166, F.C. Ungaro91, M. Ughetto148a,148b, F. Ukegawa164, G. Unal32, A. Undrus27, G. Unel166, F.C. Ungaro91, Y. Unno69, C. Unverdorben102, J. Urban146b, P. Urquijo91, P. Urrejola86, G. Usai8, J. Usui69 L. Vacavant88, V. Vacek130, B. Vachon90, K.O.H. Vadla121, A. Vaidya81, C. Valderanis102, L. Vacavant88, V. Vacek130, B. Vachon90, K.O.H. Vadla121, A. Vaidya81, C. Valderanis102, A. Vallier5, J.A. Valls Ferrer170, W. Van Den Wollenberg109, H. van der Graaf109, P. van Gemmeren6, J. Van Nieuwkoop144, I. van Vulpen109, M.C. van Woerden109, M. Vanadia135a,135b, W. Vandelli32, A. Vaniachine160, P. Vankov109, G. Vardanyan180, R. Vari134a, M. Vanadia135a,135b, W. Vandelli32, A. Vaniachine160, P. Vankov109, G. Vardanyan180, R. Vari134a, E.W. Varnes7, C. Varni53a,53b, T. Varol43, D. Varouchas119, A. Vartapetian8, K.E. Varvell152, J.G. Vasquez179, G.A. Vasquez34b, F. Vazeille37, T. Vazquez Schroeder90, J. Veatch57, quez179, G.A. Vasquez34b, F. Vazeille37, T. Vazquez Schroeder90, J. Veatch57, raghavan7, L.M. Veloce161, F. Veloso128a,128c, S. Veneziano134a, A. Ventura76a,76b, V. Veeraraghavan7, L.M. Veloce161, F. Veloso128a,128c, S. Veneziano134a, A. Ventura76a,76b, uri172, N. Venturi32, A. Venturini25, V. Vercesi123a, M. Verducci136a,136b, W. Verkerke109, M. Venturi172, N. Venturi32, A. Venturini25, V. Vercesi123a, M. Verducci136a,136b, W. Verkerke10 M. Venturi , N. Venturi , A. Venturini , V. Vercesi , M. Verducci , W. Verkerke A.T. Vermeulen109, J.C. Vermeulen109, M.C. Vetterli144,d, N. Viaux Maira34b, O. Viazlo84, A.T. Vermeulen109, J.C. Vermeulen109, M.C. Vetterli144,d, N. Viaux Maira34b, O. Viazlo84, I. Vichou169,∗, T. Vickey141, O.E. Vickey Boeriu141, G.H.A. Viehhauser122, S. Viel16, L. Vigani1 M. Villa22a,22b, M. Villaplana Perez94a,94b, E. Vilucchi50, M.G. Vincter31, V.B. Vinogradov68, A. Vishwakarma45, C. Vittori22a,22b, I. Vivarelli151, S. Vlachos10, M. Vogel178, P. Vokac130, G. Volpi126a,126b, H. von der Schmitt103, E. von Toerne23, V. Vorobel131, K. Vorobev100, M. Vos170, R. Voss32, J.H. Vossebeld77, N. Vranjes14, M. Vranjes Milosavljevic14, V. Vrba130, M. Vreeswijk109, R. Vuillermet32, I. Vukotic33, P. Wagner23, W. Wagner178, J. Wagner-Kuhr102, M. Vreeswijk109, R. Vuillermet32, I. Vukotic33, P. Wagner23, W. Wagner178, J. Wagner-Kuhr102, H. Wahlberg74, S. Wahrmund47, J. Wakabayashi105, J. Walder75, R. Walker102, W. Walkow V. Wallangen148a,148b, C. Wang35b, C. Wang36b,av, F. Wang176, H. Wang16, H. Wang3, V. Wallangen148a,148b, C. Wang35b, C. Wang36b,av, F. Wang176, H. The ATLAS collaboration Stockton118, M. Stoebe90, G. Stoicea28b, P. Stolte57, S. Stonjek103, A.R. Stradling8, A. Straessner47, M.E. Stramaglia18, J. Strandberg149, S. Strandberg148a,148b, M. Strauss115, P. Strizenec146b, R. Str¨ohmer177, D.M. Strom118, R. Stroynowski43, A. Strubig49, S.A. Stucci B. Stugu15, N.A. Styles45, D. Su145, J. Su127, S. Suchek60a, Y. Sugaya120, M. Suk130, V.V. Sulin98, DMS Sultan162a,162b, S. Sultansoy4c, T. Sumida71, S. Sun59, X. Sun3, K. Su C.J.E. Suster152, M.R. Sutton151, S. Suzuki69, M. Svatos129, M. Swiatlowski33, S.P. Swift2, 37 I. Sykora146a, T. Sykora131, D. Ta51, K. Tackmann45, J. Taenzer155, A. Taﬀard166, R. Taﬁrout163a, E. Tahirovic79, N. Taiblum155, H. Takai27, R. Takashima72, E.H. Takasugi103 T. Takeshita142, Y. Takubo69, M. Talby88, A.A. Talyshev111,c, J. Tanaka157, M. Tanaka159, R. Tanaka119, S. Tanaka69, R. Tanioka70, B.B. Tannenwald113, S. Tapia Araya34b, S. Tapprogge S. Tarem154, G.F. Tartarelli94a, P. Tas131, M. Tasevsky129, T. Tashiro71, E. Tassi40a,40b, A. Tavares Delgado128a,128b, Y. Tayalati137e, A.C. Taylor107, G.N. Taylor91, P.T.E. Taylor91, W. Taylor163b, P. Teixeira-Dias80, D. Temple144, H. Ten Kate32, P.K. Teng153, J.J. Teoh120, F. Tepel178, S. Terada69, K. Terashi157, J. Terron85, S. Terzo13, M. Testa50, R.J. Teuscher161,o, F. Tepel178, S. Terada69, K. Terashi157, J. Terron85, S. Terzo13, M. Testa50, R.J. Teuscher161,o, A.S. Thompson56, L.A. Thomsen179, E. Thomson124, M.J. Tibbetts16, R.E. Ticse Torres88, A.S. Thompson56, L.A. Thomsen179, E. Thomson124, M.J. Tibbetts16, R.E. Ticse Torres88, p , , , , , V.O. Tikhomirov98,ar, Yu.A. Tikhonov111,c, S. Timoshenko100, P. Tipton179, S. Tisserant88, JHEP08(2017)006 JHEP08(2017)006 K. Todome159, S. Todorova-Nova5, S. Todt47, J. Tojo73, S. Tok´ar146a, K. Tokushuku69, Tolley59, L. Tomlinson87, M. Tomoto105, L. Tompkins145,as, K. Toms107, B. Tong59, E. Tolley59, L. Tomlinson87, M. Tomoto105, L. Tompkins145,as, K. Toms107, B. Tong59, Tornambe51, E. Torrence118, H. Torres144, E. Torr´o Pastor140, J. Toth88,at, F. Touchard88, P. Tornambe51, E. Torrence118, H. Torres144, E. Torr´o Pastor140, J. Toth88,at, F. Touchard88 R. Tovey141, C.J. Treado112, T. Trefzger177, F. Tresoldi151, A. Tricoli27, I.M. Trigger163a, D.R. Tovey141, C.J. Treado112, T. Trefzger177, F. Tresoldi151, A. Tricoli27, I.M. Trigger163a, S. Trincaz-Duvoid83, M.F. Tripiana13, W. Trischuk161, B. Trocm´e58, A. Trofymov45, Trincaz-Duvoid83, M.F. Tripiana13, W. Trischuk161, B. Trocm´e58, A. Trofymov45, Troncon94a, M. Trottier-McDonald16, M. Trovatelli172, L. Truong147b, M. Trzebinski42, C. Troncon94a, M. Trottier-McDonald16, M. Trovatelli172, L. Truong147b, M. Trzebinski42 A. Trzupek42, K.W. Tsang62a, J.C-L. Tseng122, P.V. Tsiareshka95, G. Tsipolitis10, Trzupek42, K.W. Tsang62a, J.C-L. Tseng122, P.V. Tsiareshka95, G. Tsipolitis10, N. Tsirintanis9, S. Tsiskaridze13, V. Tsiskaridze51, E.G. Tskhadadze54a, K.M. Tsui62a, Tsirintanis9, S. Tsiskaridze13, V. Tsiskaridze51, E.G. Tskhadadze54a, K.M. Tsui62a, I.I. Tsukerman99, V. Tsulaia16, S. Tsuno69, D. Tsybychev150, Y. Tu62b, A. Tudorache28b . Weits109, P.S. Wells32, T. Wenaus27, T. Wengler32, S. Wenig32, N. Wermes23, M.D. Werner67, The ATLAS collaboration Wang16, H. Wang3, J. Wang45, J. Wang152, Q. Wang115, R. Wang6, S.M. Wang153, T. Wang38, W. Wang153,a W. Wang36a, Z. Wang36c, C. Wanotayaroj118, A. Warburton90, C.P. Ward30, D.R. Wardrope81, A. Washbrook49, P.M. Watkins19, A.T. Watson19, M.F. Watson19, G. Watts140, S. Watts87, B.M. Waugh81, A.F. Webb11, S. Webb86, M.S. Weber18, S.W. Weber177, S.A. Weber31, J.S. Webster6, A.R. Weidberg122, B. Weinert64, J. Weingarten57, M. Weirich86, C. Weiser51, H. Weits109, P.S. Wells32, T. Wenaus27, T. Wengler32, S. Wenig32, N. Wermes23, M.D. Werner67 38 P. Werner32, M. Wessels60a, K. Whalen118, N.L. Whallon140, A.M. Wharton75, A.S. White92 A. White8, M.J. White1, R. White34b, D. Whiteson166, B.W. Whitmore75, F.J. Wickens13 , , , , , , W. Wiedenmann176, M. Wielers133, C. Wiglesworth39, L.A.M. Wiik-Fuchs51, A. Wildauer103 g F. Wilk87, H.G. Wilkens32, H.H. Williams124, S. Williams109, C. Willis93, S. Willocq89, J.A. Wilson19, I. Wingerter-Seez5, E. Winkels151, F. Winklmeier118, O.J. Winston151, B.T. Winter23, M. Wittgen145, M. Wobisch82,u, T.M.H. Wolf109, R. Wolﬀ88, M.W. Wolter42, B.T. Winter23, M. Wittgen145, M. Wobisch82,u, T.M.H. Wolf109, K.W. Wozniak42, M. Wu33, S.L. Wu176, X. Wu52, Y. Wu92, T.R. Wyat JHEP08(2017)006 Zhu36a, X. Zhuang35a, K. Zhukov98, A. Zibell177, D. Zieminska64, N.I. Zimine68, Zimmermann86, S. Zimmermann51, Z. Zinonos103, M. Zinser86, M. Ziolkowski143, L. ˇZivkovi´c14, G. Zobernig176, A. Zoccoli22a,22b, R. Zou33, M. zur Nedden17 and L. The ATLAS collaboration Zwalinski32 1 Department of Physics, University of Adelaide, Adelaide, Australia 1 Department of Physics, University of Adelaide, Adelaide, Australia 2 Physics Department, SUNY Albany, Albany NY, United States of America 2 Physics Department, SUNY Albany, Albany NY, United States of America 3 Department of Physics, University of Alberta, Edmonton AB, Canada 3 Department of Physics, University of Alberta, Edmonton AB, Canada 4 (a) Department of Physics, Ankara University, Ankara; (b) Istanbul Aydin University, Istanbul; (c) Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey 6 High Energy Physics Division, Argonne National Laboratory, Argonne IL, United Stat 6 High Energy Physics Division, Argonne National Laboratory, Argonne IL, United States 7 Department of Physics, University of Arizona, Tucson AZ, United States of America 7 Department of Physics, University of Arizona, Tucson AZ, United States of America 8 Department of Physics, The University of Texas at Arlington, Arlington TX, United States of America 8 Department of Physics, The University of Texas at Arlington, Arlington TX, United States of America 9 Physics Department, National and Kapodistrian University of Athens, Athens, Greece 9 Physics Department, National and Kapodistrian University of Athens, Athens, Greece 10 Physics Department, National Technical University of Athens, Zografou, Greece 10 Physics Department, National Technical University of Athens, Zografou, Greece 12 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 12 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 13 Institut de F´ısica d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain 14 Institute of Physics, University of Belgrade, Belgrade, Serbia 15 14 Institute of Physics, University of Belgrade, Belgrade, Serbia 15 15 Department for Physics and Technology, University of Bergen, Bergen, Norway 15 Department for Physics and Technology, University of Bergen, Bergen, Norway 16 16 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United States of America 16 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United States of America 17 Department of Physics, Humboldt University, Berlin, Germany 17 Department of Physics, Humboldt University, Berlin, Germany 18 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 18 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 19 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 19 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 20 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics Engineering, Gaziantep University, Gaziantep; (d) Istanbul Bilgi University, Faculty of Engineering and Natural 20 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics Engineering, Gaziantep University, Gaziantep; (d) Istanbul Bilgi University, Faculty of Engineering and Natural 20 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics Engineering, Gaziantep University, Gaziantep; (d) Istanbul Bilgi University, Faculty of Engineering and Natural 39 Sciences, Istanbul; (e) Bahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey 21 Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia 22 (a) INFN Sezione di Bologna; (b) Dipartimento di Fisica e Astronomia, Universit`a di Bologna, Bologna, Italy 23 Physikalisches Institut, University of Bonn, Bonn, Germany 24 Department of Physics, Boston University, Boston MA, United States of America 25 Department of Physics, Brandeis University, Waltham MA, United States of America 26 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Electrical Circuits Department, Federal University of Juiz de Fora (UFJF), Juiz de Fora; (c) Federal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil JHEP08(2017)006 27 Physics Department, Brookhaven National Laboratory, Upton NY, United States of America 28 (a) Transilvania University of Brasov, Brasov; (b) Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest; (c) Department of Physics, Alexandru Ioan Cuza University of Iasi, Iasi; (d) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca; (e) University Politehnica Bucharest, Bucharest; (f) West University in Timisoara, Timisoara, Romania ) West University in Timisoara, Timisoara, Romania epartamento de F´ısica, Universidad de Buenos Aires 30 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 31 Department of Physics, Carleton University, Ottawa ON, Canada 32 CERN, Geneva, Switzerland 32 CERN, Geneva, Switzerland 33 Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America , y f g , g , f 34 (a) Departamento de F´ısica, Pontiﬁcia Universidad Cat´olica de Chile, Santiago; (b) Departamento de F´ısica, Universidad T´ecnica Federico Santa Mar´ıa, Valpara´ıso, Chile Departamento de Fısica, Pontiﬁcia Universidad Catolica de Chile, Sa de F´ısica, Universidad T´ecnica Federico Santa Mar´ıa, Valpara´ıso, Chile 35 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Physics, Nanjing University, Jiangsu; (c) Physics Department, Tsinghua University, Beijing 100084, China 36 (a) Department of Modern Physics and State Key Laboratory of Particle Detection and Electronics, University of Science and Technology of China, Anhui; (b) School of Physics, Shandong University, Shandong; (c) Department of Physics and Astronomy, Key Laboratory for Particle Physics, Astrophysics and Cosmology, Ministry of Education; Shanghai Key Laboratory for Particle Physics and Cosmology, Shanghai Jiao Tong University, Shanghai(also at PKU-CHEP), China 37 Universit´e Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France 38 Nevis Laboratory, Columbia University, Irvington NY, United States of America 39 Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 40 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b) Dipartimento di 40 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; ( Fisica, Universit`a della Calabria, Rende, Italy 40 (a) INFN Gruppo Collegato di Cosenza, Laborat Fisica, Universit`a della Calabria, Rende, Italy INFN Gruppo Collegato di Cosenza, Laborat Fisica, Universit`a della Calabria, Rende, Italy Fisica, Universit`a della Calabria, Rende, Italy 41 (a) AGH University of Science and Technology, Faculty of Physics and Applied Computer Science Krakow; (b) Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 41 (a) AGH University of Science and Technology, Faculty of Physics and Applied Computer Scienc 42 Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland Physics Polish Academy of Sciences, Krakow, Polan 43 Physics Department, Southern Methodist University, Dallas TX, United States of America 44 Physics Department, University of Texas at Dallas, Richardson TX, United States of America Physics Department, University of Texas at Dallas, Richardson TX, United States of America DESY Hamburg and Zeuthen Germany Physics Department, University of Texas at Dallas, Richardson TX, United States of America 45 DESY, Hamburg and Zeuthen, Germany 45 DESY, Hamburg and Zeuthen, Germany 45 DESY, Hamburg and Zeuthen, Germany 46 Lehrstuhl f¨ur Experimentelle Physik IV, Technische Universit¨at Dortmund, Dortmund, Germany 47 Institut f¨ur Kern- und Teilchenphysik, Technische Universit¨at Dresden, Dresden, Germany 48 Department of Physics, Duke University, Durham NC, United States of America 49 SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom UPA - School of Physics and Astronomy, University 49 SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 50 INFN e Laboratori Nazionali di Frascati Frascati Italy 50 INFN e Laboratori Nazionali di Frascati, Frascati, Italy 51 Fakult¨at f¨ur Mathematik und Physik, Albert-Ludwigs-Universit¨at, Freiburg, Germany 52 Departement de Physique Nucleaire et Corpusculaire, Universit´e de Gen`eve, Geneva, Switzerland p y q p , , , 53 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Universit`a di Genova, Genova, Italy 53 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Universit`a di Genova, Genova, Italy 40 54 (a) E. The ATLAS collaboration Andronikashvili Institute of Physics, Iv. The ATLAS collaboration Javakhishvili Tbilisi State University, Tbilisi; (b) High Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 55 II Physikalisches Institut, Justus-Liebig-Universit¨at Giessen, Giessen, Germany 56 SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United K 58 Laboratoire de Physique Subatomique et de Cosmologie, Universit´e Grenoble-Alpes, CNRS/IN2P3, Grenoble, France 59 Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge MA, United States of America 60 (a) Kirchhoﬀ-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, Germany JHEP08(2017)006 62 (a) Department of Physics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong; (b) Department of Physics, The University of Hong Kong, Hong Kong; (c) Department of Physics and Institute for Advanced Study, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China 63 Department of Physics, National Tsing Hua University, Taiwan, Taiwan 64 Department of Physics, Indiana University, Bloomington IN, United States of America stitut f¨ur Astro- und Teilchenphysik, Leopold-Franzen 66 University of Iowa, Iowa City IA, United States of America 66 University of Iowa, Iowa City IA, United States of America 67 Department of Physics and Astronomy, Iowa State University, Ames IA, United States of Ameri 67 Department of Physics and Astronomy, Iowa State University, 68 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 69 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan KEK, High Energy Accelerator Research Organizat 70 Graduate School of Science, Kobe University, Kobe, Japan 71 Faculty of Science, Kyoto University, Kyoto, Japan 72 Kyoto University of Education, Kyoto, Japan 73 Research Center for Advanced Particle Physics and Department of Physics, Kyushu University, Fukuoka, Japan 74 Instituto de F´ısica La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argenti 75 75 Physics Department, Lancaster University, Lancaster, United Kingdom ( ) (b) 76 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Universit`a del Salento, Lecce, Italy 77 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom Oliver Lodge Laboratory, University of Liverpool, L 78 Department of Experimental Particle Physics, University of Ljubljana, Ljubljana, Slovenia 78 Department of Experimental Particle Physics, Joˇzef Stefan Institute and Department of Ph U i it f Lj blj Lj blj Sl i 78 Department of Experimental Particle Physics, Joˇzef Stefan Institute and Department of Physics, University of Ljubljana, Ljubljana, Slovenia 78 Department of Experimental Particle Physics, Jo University of Ljubljana, Ljubljana, Slovenia 79 School of Physics and Astronomy, Queen Mary University of London, Lo 79 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 79 School of Physics and Astronomy, Queen Ma 80 Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 81 Department of Physics and Astronomy, University College London, London, United Kingdom 82 Louisiana Tech University, Ruston LA, United States of America 83 Laboratoire de Physique Nucl´eaire et de Hautes Energies, UPMC and Universit´e Paris-Diderot and CNRS/IN2P3, Paris, France 84 Fysiska institutionen, Lunds universitet, Lund, Sweden 85 Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 85 Departamento de Fisica Teorica C-15, Universidad Auton 86 Institut f¨ur Physik, Universit¨at Mainz, Mainz, Germany 87 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom chool of Physics and Astronomy, University of Manch 88 CPPM, Aix-Marseille Universit´e and CNRS/IN2P3, Marseille, France 89 Department of Physics, University of Massachusetts, Amherst MA, United States of America p f y , y f , , f 90 Department of Physics, McGill University, Montreal QC, Canada 90 Department of Physics, McGill University, Montreal QC, Canada partment of Physics, McGill University, Montreal QC 91 School of Physics, University of Melbourne, Victoria, Australia 92 Department of Physics, The University of Michigan, Ann Arbor MI, United States of America 92 Department of Physics, The University of Michigan, Ann Arbor 92 Department of Physics, The University of Michigan, Ann Arbor MI, United States of America 93 Department of Physics and Astronomy Michigan State University East Lansing MI United States 93 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United Stat of America 41 94 (a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Universit`a di Milano, Milano, Italy 95 B.I. The ATLAS collaboration Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of Belarus 96 Research Institute for Nuclear Problems of Byelorussian State University, Minsk, Republic of Belarus 97 Group of Particle Physics, University of Montreal, Montreal QC, Canada 98 P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Rus 99 Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 100 National Research Nuclear University MEPhI, Moscow, Russia 101 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russia JHEP08(2017)006 JHEP08(2017)006 103 Max-Planck-Institut f¨ur Physik (Werner-Heisenberg-Institut), M¨unchen, Germany 104 Nagasaki Institute of Applied Science, Nagasaki, Japan 105 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan 106 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Universit`a di Napoli, Napoli, Italy 107 Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United Stat of America 108 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands 109 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 110 Department of Physics, Northern Illinois University, DeKalb IL, United States of America 111 Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 112 Department of Physics, New York University, New York NY, United States of America te University, Columbus OH, United States of Ameri 113 Ohio State University, Columbus OH, United States of Americ 114 Faculty of Science, Okayama University, Okayama, Japan 115 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman OK, United States of America 116 Department of Physics, Oklahoma State University, Stillwater OK, United States of America 117 Palack´y University, RCPTM, Olomouc, Czech Republic 117 Palack´y University, RCPTM, Olomouc, Czech Republic 118 Center for High Energy Physics, University of Oregon, Eugene OR, United States of America 118 Center for High Energy Physics, University of Oregon, Eugene OR, United States of America 119 LAL Univ Paris Sud CNRS/IN2P3 Universit´e Paris Saclay Orsay France 120 Graduate School of Science, Osaka University, Osaka, Japan 120 Graduate School of Science, Osaka University, Osaka, Japan 121 Department of Physics, University of Oslo, Oslo, Norway 122 Department of Physics, Oxford University, Oxford, United Kingdom 123 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Universit`a di Pavia, Pavia, Italy 124 Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America 125 National Research Centre “Kurchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics Institute, St. The ATLAS collaboration Petersburg, Russia 126 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy 127 126 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy 127 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of 126 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. America 167 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento di Chimica, Fisica e Ambiente, Universit`a di Udine, Udine, Italy Dipartimento di Chimica, Fisica e Ambiente, Universit`a di Udine, Udine, Italy 168 Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden nt of Physics, University of Illinois, Urbana IL, Unite 170 Instituto de Fisica Corpuscular (IFIC), Centro Mixto Universidad de Valencia - CSIC, Spain 171 Department of Physics, University of British Columbia, Vancouver BC, Canada 172 Department of Physics and Astronomy, University of Victoria, Victoria BC, Canada 173 173 Department of Physics, University of Warwick, Coventry, United Kingdom 174 Waseda University, Tokyo, Japan 174 Waseda University, Tokyo, Japan 175 175 Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel JHEP08(2017)006 JHEP08(2017)006 177 Fakult¨at f¨ur Physik und Astronomie, Julius-Maximilians-Universit¨at, W¨urzburg, Germany 178 Fakult¨at f¨ur Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universit¨at Wuppertal, Wuppertal, Germany 179 Department of Physics, Yale University, New Haven CT, United States of America Physics, Yale University, New Haven CT, United S 180 Yerevan Physics Institute, Yerevan, Armenia 180 Yerevan Physics Institute, Yerevan, Armenia 181 Centre de Calcul de l’Institut National de Physique Nucl´eaire et de Physique des Particules (IN2P3), Villeurbanne, France 182 Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan a Also at Department of Physics, King’s College London, London, United Kingdom b Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan c Also at Novosibirsk State University, Novosibirsk, Russia d Also at TRIUMF, Vancouver BC, Canada e Also at Department of Physics & Astronomy, University of Louisville, Louisville, KY, United States of America f Also at Physics Department, An-Najah National University, Nablus, Palestine g Also at Department of Physics, California State University, Fresno CA, United States of America h Also at Department of Physics, University of Fribourg, Fribourg, Switzerland i Also at II Physikalisches Institut, Georg-August-Universit¨at, G¨ottingen, Germany j Also at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain k Also at Departamento de Fisica e Astronomia, Faculdade de Ciencias, Universidade do Porto, Portugal l Also at Tomsk State University, Tomsk, and Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia University, Dolgoprudny, Russia m Also at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing, China n Also at Universita di Napoli Parthenope, Napoli, Italy o Also at Institute of Particle Physics (IPP), Canada p Also at Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania p Also at Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania q Also at Department of Physics, St. The ATLAS collaboration Fermi, Universit`a di Pisa, Pisa, Italy 127 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of America 127 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of America 128 (a) Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas - LIP, Lisboa; (b) Faculdade de Ciˆencias, Universidade de Lisboa, Lisboa; (c) Department of Physics, University of Coimbra, Coimbra; (d) Centro de F´ısica Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Fisica, Universidade do Minho, Braga; (f) Departamento de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada; (g) Dep Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 129 Institute of Physics, Academy of Sciences of the Czech Republic, Praha, Czech Republic 130 Czech Technical University in Prague, Praha, Czech Republic 131 Charles University, Faculty of Mathematics and Physics, Prague, Czech Republic 42 132 State Research Center Institute for High Energy Physics (Protvino), NRC KI, Russia f g gy y ( ), , 133 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 133 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 134 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy 135 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Universit`a di Roma Tor Vergata, Roma, Italy 136 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Universit`a Roma Tre, Roma, Italy 137 (a) Facult´e des Sciences Ain Chock, R´eseau Universitaire de Physique des Hautes Energies - Universit´e Hassan II, Casablanca; (b) Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c) Facult´e des Sciences Semlalia, Universit´e Cadi Ayyad, LPHEA-Marrakech; (d) Facult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda; (e) Facult´e des sciences, Universit´e Mohammed V, Rabat, Morocco 137 (a) Facult´e des Sciences Ain Chock, R´eseau Universitaire de Physique des Hautes Energies - Universit´e Hassan II, Casablanca; (b) Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c) Facult´e des Sciences Semlalia, Universit´e Cadi Ayyad, LPHEA-Marrakech; (d) Facult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda; (e) Facult´e des Nucleaires, Rabat; (c) Facult´e des Sciences Semlalia, Universit´e Cadi Ayyad, LPHEA-Marrakech; (d) Facult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda; (e) Facult´e des sciences, Universit´e Mohammed V, Rabat, Morocco sciences, Universit´e Mohammed V, Rabat, Morocco JHEP08(2017)006 JHEP08(2017)006 138 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat `a l’Energie Atomique et aux Energies Alternatives), Gif-sur-Yvette, France 139 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz CA, United States of America 140 Department of Physics, University of Washington, Seattle WA, United States of America 141 Department of Physics and Astronomy, University of Sheﬃeld, Sheﬃeld, United Kingdom 143 Department Physik, Universit¨at Siegen, Siegen, Germany 144 Department of Physics, Simon Fraser University, Burnaby BC, Canada 145 SLAC National Accelerator Laboratory, Stanford CA, United States of America 146 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academ 146 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic , , p 147 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics, University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand Johannesburg, South Africa University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand, Johannesburg, South Africa 148 (a) Department of Physics, Stockholm University; (b) The Oskar Klein Centre, Stockholm, Sweden 149 Physics Department, Royal Institute of Technology, Stockholm, Sweden 150 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook NY, United States of America 151 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 152 School of Physics, University of Sydney, Sydney, Australia ool of Physics, University of Sydney, Sydney, Austra 153 Institute of Physics, Academia Sinica, Taipei, Taiwan 154 Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel 155 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 156 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 157 International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan 158 Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 159 Department of Physics, Tokyo Institute o 159 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 160 Tomsk State University, Tomsk, Russia 160 Tomsk State University, Tomsk, Russia 160 Tomsk State University, Tomsk, Russia 161 Department of Physics, University of Toronto, Toronto ON, Canada ( ) ( ) 162 (a) INFN-TIFPA; (b) University of Trento, Trento, Italy ( ) ( ) 162 (a) INFN-TIFPA; (b) University of Trento, Trento, Italy 163 (a) TRIUMF, Vancouver BC; (b) Department of Physics and Astronomy, York University, Toronto ON, Canada 164 Faculty of Pure and Applied Sciences, and Center for Integrated Research in Fundamental Scienc and Engineering, University of Tsukuba, Tsukuba, Japan 165 Department of Physics and Astronomy, Tufts University, Medford MA, United States of America 166 Department of Physics and Astronomy, University of California Irvine, Irvine CA, United States of 43 America Petersburg State Polytechnical University, St. Petersburg, p Also at Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania q Al D f Ph S P b S P l h l U S P b Also at Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania q Also at Department of Physics, St. Petersburg State Polytechnical University, St. America Petersburg, Russia r Also at Borough of Manhattan Community College, City University of New York, New York City, United States of America s Also at Department of Financial and Management Engineering, University of the Aegean, Chios, Greece t Also at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South Africa u Also at Louisiana Tech University, Ruston LA, United States of America v Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain w Also at Graduate School of Science, Osaka University, Osaka, Japan x Also at Fakult¨at f¨ur Mathematik und Physik, Albert-Ludwigs-Universit¨at, Freiburg, Germany y Also at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University 44 Nijmegen/Nikhef, Nijmegen, Netherlands z Also at Department of Physics, The University of Texas at Austin, Austin TX, United States of America aa Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia Also at Institute of Theoretical Physics, Ilia State ab Also at CERN, Geneva, Switzerland ac Also at Georgian Technical University (GTU),Tbilisi, Georgia ad Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan ae Also at Manhattan College, New York NY, United States of America af Also at Departamento de F´ısica, Pontiﬁcia Universidad Cat´olica de Chile, Santiago, Chile ag Also at Department of Physics, The University of Michigan, Ann Arbor MI, United States of America lso at The City College of New York, New York NY, JHEP08(2017)006 JHEP08(2017)006 ai Also at School of Physics, Shandong University, Shandong, China aj Also at Departamento de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada, Portugal ak Also at Department of Physics, California State University, Sacramento CA, United States of America al Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia al Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia am Also at Departement de Physique Nucleaire et Corpusculaire Universit´e de Gen`eve Geneva am Also at Departement de Physique Nucleaire et Corpusculaire, Universit´e de Gen`eve, Geneva, Switzerland an Also at Institut de F´ısica d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain ao Also at School of Physics, Sun Yat-sen University, Guangzhou, China ap Also at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of Sciences, Soﬁa, Bulgaria aq Also at Faculty of Physics, M.V.Lomonosov Moscow State University, Moscow, Russia ar Also at National Research Nuclear University MEPhI, Moscow, Russia l Research Nuclear University MEPhI, Moscow, Russ as Also at Department of Physics, Stanford University, Stanford CA, United States of America at Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary au Also at Giresun University, Faculty of Engineering, Turkey av Also at CPPM, Aix-Marseille Universit´e and CNRS/IN2P3, Marseille, France ax Also at University of Malaya, Department of Physics, Kuala Lu ax Also at University of Malaya, Department of Physics, Kuala Lumpur, Malaysia University of Malaya, Department of Physics, Kuala ay Also at Institute of Physics, Academia Sinica, Taipei, Taiwan az Also at LAL, Univ. America Paris-Sud, CNRS/IN2P3, Universit´e Paris-Saclay, Orsay, France 45
https://openalex.org/W4389671900	https://www.qeios.com/read/T01C5D/pdf	English	null	Review of: "Impact of Environmental Education on the Knowledge and Attitude of University of Benin Students towards Waste Segregation"	null	2,023	cc-by	633	Qeios, CC-BY 4.0 · Review, December 13, 2023 Qeios ID: T01C5D · https://doi.org/10.32388/T01C5D Review of: "Impact of Environmental Education on the Knowledge and Attitude of University of Benin Students towards Waste Segregation" Mayra Liuviana Vega Chica1 Potential competing interests: No potential competing interests to declare. Qeios ID: T01C5D · https://doi.org/10.32388/T01C5D Potential competing interests: No potential competing interests to declare. Norris Igbinosa Erhabor's research focuses on the significant intersection between environmental education and solid waste management, with a particular emphasis on waste segregation. The study, set within the academic context of the University of Benin, Benin City, adopts a quasi-experimental research design to analyze how environmental education influences the awareness and practices of waste segregation among students. Waste segregation is a critical component in solid waste management, and its effective practice can result in improved environmental hygiene and a reduction in the use of landfills. Environmental education is postulated as a powerful tool to foster this sustainable practice, especially in university contexts where students can be catalysts for change. With a sample of 200 students from the departments of Health, Safety, and Environmental Education (HSE) and Human Kinetics (HKS), Erhabor evaluates the premise that environmental education can enhance knowledge and change attitudes towards waste segregation. The methodology includes the use of validated instruments and robust statistical techniques, such as the t-test, to examine the proposed hypotheses. The study's findings are revealing: students exposed to environmental education show a significant difference in their knowledge and attitudes towards waste segregation compared to those who did not receive the same training. This finding emphasizes the importance of environmental education and suggests that it can be a determining factor in improving waste management practices among youth. Erhabor's research highlights the importance of waste segregation as an essential part of waste management, which includes the collection of waste in different types and colors of containers and the separation of useful and useless items at the point of collection. The practical implication of these findings is clear: solid and well-integrated environmental education in the university curriculum can lead to more sustainable behaviors and better solid waste management. Furthermore, the study underscores the need for public awareness programs and the provision of adequate infrastructure to facilitate waste segregation. These initiatives must be complemented by community motivation and participation to achieve effective management of urban waste. Potential competing interests: No potential competing interests to declare. This review of the study not only highlights its contribution to the academic body in the field of environmental education This review of the study not only highlights its contribution to the academic body in the field of environmental education Qeios ID: T01C5D · https://doi.org/10.32388/T01C5D 1/2 and management but also acts as a call to action for policymakers, educators, and environmental health professionals to implement effective educational strategies that promote sustainable waste management practices. In conclusion, E h b ' h id lli id th t i t l d ti h ld b ti l t i Qeios, CC-BY 4.0 · Review, December 13, 2023 Qeios, CC-BY 4.0 · Review, December 13, 2023 and management but also acts as a call to action for policymakers, educators, and environmental health professionals to implement effective educational strategies that promote sustainable waste management practices. In conclusion, Erhabor's research provides compelling evidence that environmental education should be an essential component in university education to foster a proactive and responsible attitude towards waste management and the promotion of a healthy environment. This summary provides an overview of the study's structure and key findings. For a complete review, one could delve deeper into each section, analyzing and discussing in detail the methods, results, and recommendations of the study, and reflecting on its applicability and future implications for research and practice in the field of environmental management. Qeios ID: T01C5D · https://doi.org/10.32388/T01C5D 2/2
W4205533539.txt	https://www.e3s-conferences.org/articles/e3sconf/pdf/2022/05/e3sconf_esma2021_01040.pdf	en		Study on the Single Toxicity of Five Azole Fungicides for Green Algae	E3S web of conferences	2,022	cc-by	2,913	E3S Web of Conferences 338, 01040 (2022) ESMA 2021 https://doi.org/10.1051/e3sconf/202233801040 Study on the Single Toxicity of Five Azole Fungicides for Green Algae Yilun Dai 1, 2, 3, 4, 5, , Jianglong Shen 1, 2, 3, 4, 5, Yutong Sun 1,2,3,4,5 and Yanan Li 1, 2, 3, 4, 5 Shaanxi Provincial Land Engineering Construction Group Co., Ltd. China Institute of Land Engineering and Technology, Shaanxi Provincial Land Engineering Construction Group Co., Ltd. China 3 Key Laboratory of Degraded and Unused Land Consolidation Engineering, Ministry of Natural Resources China 4 Shaanxi Provincial Land Consolidation Engineering Technology Research Center China 5 Land Engineering Technology Innovation Center, Ministry of Natural Resources China 1 2 Abstract. Azole fungicides are widely used to control diseases and insect pests of vegetables, fruits and flowers because of their good antibacterial effect and regulating plant growth. However, their extensive use inevitably enters the water environment with rainfall and surface runoff, which leads to potential harm to aquatic organisms. At present, the research on the biological toxicity of azole fungicides mainly focuses on single azole compounds, such as Paclobutrazol, hexaconazole, propiconazole and Difenoconazole. However, the systematic study on the toxic effects of multi-component mixtures of azole fungicides is still very lacking, and it is unable to accurately assess the ecological risk. Therefore, in this paper, five common azole fungicides (imidazole, tebuconazole, triadimefon, tricyclazole and hymexazol) in the environment were taken as target pollutants. Scenedesmus obliquus was used as the toxicity indicator. The mixture system was designed by the equal effect concentration ratio method and linear average ray method. Study the 96h toxicity and interaction of Scenedesmus obliquus under combined stress of azole fungicides. The main research results are as follows:The 96 h toxicity test of five typical azole fungicides to Scenedesmus obliquus was determined by microplate toxicity analysis method. Fitting with Weibull function and taking EC50 as the criterion,the results show that the single toxicity order of the five tested fungicides was:tebuconazole > Triadimefon > oxacillin > Tricyclazole > imidazole. Key words: azole fungicides, Scenedesmus obliquus, toxicity. 1. Introduction Fungicides usually refer to chemical preparations that can effectively control or kill microorganisms such as bacteria, fungi and algae in the water system environment [1], and generally in the international arena, can be used as agents for the control of various pathogenic microorganisms, known as as a fungicide. The early fungicides were mainly inorganic fungicides, such as Bordeaux mixture, the world's earliest inorganic fungicides. In 1914, Germany took the lead in using organic mercury compounds to control wheat smut [2,3], which marked the beginning of the development of organic fungicides [4]. In 1944, Woolley successfully synthesized the first azole fungicide, benzimidazole. During this period, after more than 70 years of development [5], azole fungicides have gradually become the protagonist of the sterilization market, and are widely used in agricultural production and personal skin care products. and medical drugs. In agriculture, the production of a large number of food crops is reduced every year due to fungal infection, and sometimes even threatens human health. For example, Fusarium can produce mycotoxins when plants grow. The use of azole fungicides can not only prevent the infection of crops, but also can prevent the infection of crops. Infected crops can be effectively treated [6-11]. By 2017, more than 60 azole pesticides have been sold worldwide, accounting for 25% of the global application of fungicides [12]. In terms of personal skin care products, azole fungicides are widely used in household products such as shampoos, skin care products, soaps, toothpastes and shower gels due to their low toxicity, broad spectrum and high efficiency [13], such as ketoconazole It is often used in anti-dandruff hair care formulations [14]. Cribazole is not only used as an anti-dandruff active ingredient, but also as an anti-fungal preservative and anti-aging agent, and its content is up to 2% in washing products. The highest in washing products is 0.5%, and the highest in cosmetic products is 0.5% [14]; the annual sales volume in my country exceeds 3,800 tons [15]. In medical terms, of the 2 million fungi known to date, about 600 are known as human fungal pathogens, of which 3% to 4% can cause invasive fungal infections [16]. Over the past 30 years, annual Invasive fungal infections kill between 1 and 2 Corresponding author: 373289543@qq.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 338, 01040 (2022) ESMA 2021 https://doi.org/10.1051/e3sconf/202233801040 million people, and the mortality rate far exceeds that of malaria and tuberculosis [17]. Among the more than 40 brands of antifungal drugs clinically used on the market, azole fungicides are considered to be the most It has the best curative effect and the most widely used fungicides [18-21]. Chemical pollutants in the actual natural environment often do not exist alone, but in the form of mixtures. With the vigorous development of industry, agriculture, and manufacturing, the types of pollutant mixtures are increasing, and new types of pollutants are increasing. The ecological and environmental problems caused by them are not optimistic. Therefore, in the past half century, the research on the combined toxicity of mixtures has become more and more Having attention. For example, on the issue of industrial sewage treatment, the possibility that there are still unclean pollutants in the treated industrial sewage cannot be excluded. Various new types of pollutants are increasing day by day, and their toxicity is rare, and their detection cannot be analyzed and expressed by a single physical and chemical index. On the other hand, biological monitoring can more vividly reflect the pollution and toxicity of pollutants in different situations in a certain area. In order to reveal the ecotoxicity of azole fungicides exposed in the natural environment, this study took five azole fungicides as target pollutants, and investigated the effects of azole fungicides at different concentrations on the growth of the toxicity indicator organism, Scenedesmus obliquus. To clarify the single toxicity of these five typical azole fungicides to Scenedesmus obliquus, and to provide basic toxicity data for the risk assessment of mixed pollutants in the water environment. 2. Figure 1. Chemical Structures of Five Azole Fungicides Matrials and methods 2.1.2 Algae Species 2.1 Experimental Materials Scenedesmusobliquus were purchased from the Freshwater Algae Seed Bank (FACHB) of the Type Culture Collection Committee of the Chinese Academy of Sciences, and the serial numbers were FACHB-12. After receiving the algae seed, transfer the algae seed to the prepared medium for cultivation in the clean bench. 2.1.1 Experimental Reagents Five typical azole fungicides were selected in this paper as the research target pollutants. The specific physical and chemical properties are shown in Table 1, and the chemical structure is shown in Figure 1. 2.1.3 Cultivation of Algae Table 1 Physicochemical properties of 5 target pollutants Serial Number Compound Short Name CAS 1 Imidazole IDZ 28832-4 8044341-0 2 Tebuconazole CDT 3 Triadimefon TDF 4 Tricyclazole TCL 5 Oxymetholone MHZ 4312143-3 4181478-2 1000444-1 Relative molecular mass Purity （%） Source 68.08 99.50 Accustandard 307.82 98.70 Dr. Ehrenstorfer GmbH 293.75 99.60 Accustandard 189.24 99.50 Accustandard 99.09 99.00 Dr. Ehrenstorfer GmbH The BG11 medium was used to culture Scenedesmus obliquus, and the medium was sterilized in a sterilizing pot at 121 °C and 101 KPa for 20 min. After cooling, green algae were aseptically inoculated in the ultra-clean workbench, and then placed in an artificial constant temperature light incubator for cultivation. The set temperature was 22ºC, the illuminance was about 2000lux, and the light-dark cycle was 12h:12h. Expand the culture every 5-7d, that is, dilute the transfer algae at a ratio of 1:2, so that the algae can fully enter the logarithmic growth phase. The most vigorous period of algal cell metabolism is at 8-10 am, so it is the best time for algal species transfer [66]. The algae should be cultivated in a sterile environment throughout the process. Before the 2 E3S Web of Conferences 338, 01040 (2022) ESMA 2021 https://doi.org/10.1051/e3sconf/202233801040 toxicological test, the algal seeds in the logarithmic growth phase were transferred to fresh medium for cultivation, and the experiment was carried out after culturing for 2 days until the initial light absorption value of the algal seeds was 0.22-0.25. In order to avoid the experimental error caused by the volatilization of the drug, a transparent cover plate should be added during the experimental exposure period. derive effect sizes and effect concentrations for individual compounds and mixtures, a nonlinear fit to the observed concentration-response data must be performed. For classical S-type CRC, Logit or Weibull nonlinear function is generally used for fitting. The function expression is shown in Table 2., where I represents the effect, c represents the concentration of a single compound or mixture, α is the position parameter, and β is the slope parameter [23]. The Logit function and the Weibull function are classical two-parameter functions. The Logit function is based on the integral statistics of logistic distribution and takes half effect (50%) as the center symmetry, so it is suitable for fitting a symmetrical curve. The Weibull function, originally proposed by Gompertz, is based on extreme value distribution integral statistics and is suitable for most asymmetric concentration-effect curves. The MTA method used in this study has only 12 concentration gradients, and the two-parameter Logit and Weibull functions should be used for nonlinear fitting [24]. 2.2 Toxicity Experiment 2.2.1 Green Algae Acute Microplate Experiment The algal microplate toxicity test uses a standard transparent 96-well microplate, and the microplate sample loading design method is as follows. First, add 200 μL of ultrapure water to the four sides of the 96-well microplate (36 wells in total) to prevent edge effects; 100 μL of ultrapure water was added to the 24 wells in columns 2, 6, 7 and 11 respectively as blank control; A total of 6 wells in the 3rd column and 6 wells in the 8th column were respectively added with different concentrations of pesticide pollutants designed according to the dilution factor (see Equation 2.1, and supplemented with ultrapure water to make the volume 100 μL; The parallel trials for column 3 are columns 4 and 5, and the parallel trials for column 8 are columns 9 and 10; Finally, 100 μL of algae liquid with an optical density value of about 0.15 was added to the middle 60 wells, so that the total volume of each well was 200 μL; After adding samples to the microplate, cover with a transparent cover, and place it in a biochemical incubator with a temperature of 22 °C, an illuminance of 2000 lux, and a light-dark ratio of 12h: 12h. At 96h, the microplate was placed in a spectrophotometer to measure the optical density (OD) at a wavelength of 681 nm. The microplate was repeated at least 3 times. � � �� , n=1,2….. Function type Logit Weibull 3. Analysing way I= 1⁄�1+exp�-α-βlgc�� I=1-exp�-exp�α+βlgc�� Results Table 3. Concentration-effect fitting curve parameters of five azole fungicides on Scenedesmus obliquus Compound Fitfunction α β R2 RMSE IDZ CDT TDF TCL HMZ Weibull Weibull Weibull Weibull Weibull 5.706 6.446 4.966 3.174 5.827 2.200 1.509 1.331 1.175 1.708 0.987 0.991 0.975 0.977 0.992 0.0393 0.0242 0.0339 0.0287 0.0299 120 IDZ 100 (1) Inhibition(%) � � � � �� Table 2. Two Common S-Type Concentration-Effect Curve Fitting Models Among them, CH and CL are the highest and lowest concentrations of the experiment, respectively, and n represents the number of concentration gradients in the range of CH and CL. Then the twelve concentration points are Cn=C0*Fn, n=1,2... Among them, C0 is the concentration of the stock solution, and Cn is the set concentration. In this paper, the growth inhibition rate (I) of pollutants to Scenedesmus obliquus is used as the toxicity index of the microplate test, and the calculation formula of I is as follows: I=(ODt,i-ODt,0)/(OD0,iOD0,0)，where ODti is the OD value of the pollutant experimental group at the i-th time, and the OD value of the OD0i blank control group. 80 60 40 20 0 -20 1E-4 Inhibition(%) 100 80 0.001 0.01 Concentration(mol/L) CDT 60 40 20 0 2.2.2 Data Processing 1E-6 The experimental toxicity data were processed by the MATLAB program written by Qin Litang, and the graph of the fitted curve was drawn by the Origin software. To 3 1E-5 1E-4 Concentration(mol/L) EC50 /(molꞏL1) 2.20E-03 3.22E-05 9.81E-05 8.86E-04 2.55E-04 Inhibition(%) Inhibition(%) E3S Web of Conferences 338, 01040 (2022) ESMA 2021 80 70 60 50 40 30 20 10 0 70 60 50 40 30 20 10 0 https://doi.org/10.1051/e3sconf/202233801040 2. TDF 3. 4. 1E-5 1E-4 Concentration(mol/L) 5. TCL 6. 7. 1E-4 0.001 Concentration(mol/L) Inhibition(%) 100 HMZ 8. 80 60 40 9. 20 0 1E-5 1E-4 0.001 Concentration(mol/L) 10. Figure 2 Concentration-response curves (CRC) of five azole fungicides on Scenedesmus obliquus The biological toxicity of azole fungicides was tested with Scenedesmus obliquus as the indicator organism, and the toxicity of azole fungicides was expressed by the inhibition rate of Scenedesmus obliquus at 96 hours. The concentration-effect data of the five azole fungicides on Scenedesmus obliquus can be fitted by the Weibull function or the Logit function. The fitting model parameters (regression parameters α and β values, statistical parameters R2 and RMSE) are shown in Table 2. The R2 values of the five azole fungicides on the toxicity data of Scenedesmus obliquus were between 0.975 and 0.992, all greater than 0.95; The RMSE values of the fitted curves ranged from 0.024 to 0.0339, all less than 0.028, which indicated that the fitting results of the Weibull model were accurate and reliable. Figure 2. shows the concentration-response number fitted curves (CRCs) of the five azole fungicides, all showing a classic S shape. From Table 3, the toxicity under 50% effect can be obtained as: tebuconazole > Triadimefon > oxacillin > Tricyclazole > imidazole. 11. 12. 13. 14. 15. References 1. Liuchen Zhao. Bactericidal properties of bisdecyl dimethyl ammonium formate and its composite system[D]. China Daily Chemical Industry Research Institute,2017. 16. 4 Large, E. C. Control of Potato Blight (Phytophthora infestans) by Spraying with Suspensions of Metallic Copper[J]. Nature, 1943, 151(3820): 195-206. Melnikov N N, Gunther F A, Gunther J D. Chemistry of pesticides[M]. Residue Reviews, 1971, 36(1). Klittich C J. Milestones in Fungicide Discovery: Chemistry that Changed Agriculture[J]. Plant Health Progress, 2008, 9(1):1-8. Woolley D W, Pringle A. Relationship of chemical structure to antibacterial activity among analogues of dimethyldiaminobenzene[J]. Journal of Biological Chemistry, 1952, 194(2): 729-746. Janna H, Scrimshaw M D, Williams R J, et al. From Dishwasher to Tap Xenobiotic Substances Benzotriazole and Tolyltriazole in the Environment[J]. Environmental ence & Technology, 2011, 45(9): 3858-3864. H., Dooley, M., et al. Effect of azole fungicide mixtures, alternations and dose on azole sensitivity in the wheat pathogenZymoseptoria tritici[J]. Plant Pathology, 2015.65(1):124-136. Giavini E, Menegola E. Are azole fungicides a teratogenic risk for human conceptus[J].Toxicology Letters, 2010, 198(2): 106-111. Brauer V S, Rezende C P, Pessoni A M, et al. Antifungal Agents in Agriculture: Friends and Foes of Public Health[J]. Biomolecules, 2019, 9(10): 521534. Escher B I, Baumgartner R, Koller M, et al. Environmental toxicology and risk assessment of pharmaceuticals from hospital wastewater[J]. Water Research, 2011, 45(1): 75-92. Thomas K V, Hilton M J. The occurrence of selected human pharmaceutical compounds in UK estuaries[J]. Marine Pollution Bulletin, 2004, 49(5/6): 436-444. Hof H. Critical Annotations to the Use of Azole Antifungals for Plant Protection[J]. Antimicrobial Agents & Chemotherapy, 2001, 45(11): 87-90. David, Allen, Dustin, et al. Azole antifungals: 35 years of invasive fungal infection management: Expert Review of Anti-infective Therapy: Vol 13, No 6[J]. Expert Review of Anti Infective Therapy, 2015.13(6):787-798. Wishart D S, Knox C, Guo A C, et al. DrugBank: a knowledgebase for drugs, drug actions and drug targets[J]. Nucleic Acids Research, 2007, 36(Database): D901-D906. Autrup H. SCCP (Scientific Committee on Consumer Products) / SCHER (Scientific Committee on Health& Environment Risks) / SCENIHR (Scientific Committee on Emerging and Newly-Identified Health Risks) opinion on:Risk assessment methodologies and approaches for genotoxic and carcinogenic substances[J].Radiographics A Review Publication of the Radiological Society of North America Inc, 2009, 22(2): e4. Gouin T, Egmond R V, Price O R, et al. Prioritising chemicals used in personal care products in China for E3S Web of Conferences 338, 01040 (2022) ESMA 2021 17. 18. 19. 20. 21. 22. 23. 24. https://doi.org/10.1051/e3sconf/202233801040 environmental risk assessment: Application of the RAIDAR model[J]. Environmental Pollution, 2012, 165(Jun.): 208-214. Kathiravan M K, Salake A B, Chothe A S, et al. The biology and chemistry of antifungal agents: a review[J]. Bioorg Med Chem, 2012, 20(19): 56785698. Roemer T, Krysan D J. Antifungal Drug Development: Challenges, Unmet Clinical Needs, and New Approaches[J]. Cold Spring Harbor Perspectives in Medicine, 2014, 4(5): a019703a019703. Perlroth J, Choi B, Spellberg B. Nosocomial fungal infections: epidemiology, diagnosis, and treatment[J]. Medical Mycology, 2007, 45(4): 321-346. Wróbel T M, Kosikowska U, Kaczor A A, et al. Synthesis, Structural Studies and Molecular Modelling of a Novel Imidazoline Derivative with Antifungal Activity[J]. Molecules, 2015, 20(8): 6176. Shafiei M, Peyton L, Hashemzadeh M, et al. History of the development of Antifungal azoles: A review on structures, SAR, and mechanism of action[J]. Bioorganic Chemistry, 2020: 104-240. Jingxiang Zhou, Tao Yun, Quan Huang,et al. Comparative study on digestive enzyme activities of common carp, yellow croaker, mulberry and walleye[J]. Journal of Jilin Agricultural University, 2001, 23(1): 94-96. Zhjiqiang Bian, Jin Zhang, Tao Wang, et al. Characteristics and mechanism of combined toxicity of carbamate pesticides to Chlorella pyrenoidosa [J]. Acta Ecotoxicology, 2019, 14(4): 150-162. Renlong Cheng. Two-stage model predicts timedependent mixed toxicity of antibiotics to Chlorella pyrenoidea [D]. Anhui University of Architecture,2016. 5
https://openalex.org/W4321475009	https://zenodo.org/records/7661425/files/GSCBPS-2023-0032.pdf	English	null	Green synthesis of copper oxide nanoparticles using the outer layer of Allium cepa L. and evaluation of its antimicrobial properties	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	5,782	Abstract The recent antimicrobial resistant to the present antibacterial agents has been the biggest problem in the treatment of antimicrobial infection. Over the years, various strategies has been implemented to overcome the resistance the resistance to the available antibiotic agents. In regards to this, phytochemical found in plants like Allium cepa, which has exhibited potent antimicrobial activity has been used by researchers as natural products to fight against bacterial resistance. Allium cepa also known commonly as onion, is an herbaceous biennial plant in the amaryllis family (Amaryllidaceae) grown for its edible bulb. Copper oxide nanoparticles are one of the most vital nanomaterials used in biomedical application. The phytochemicals in the plant extract were used as reducing and capping agent. The aim of the study is to determine the antimicrobial activity of synthesized copper oxide nanoparticles using Allium cepa (onion) aqueous extracts. The dried outer layer of the Allium cepa was collected and boiled. 150 ml of the plant extract was mixed with 250 ml of the copper nitrate solution, for the purpose of green synthesis. After the reaction was completed, the synthesized nanoparticles was centrifuged, decanted and dried. The dried copper oxide nanoparticles were characterized and accessed for antimicrobial activity. The results of UV-Vis Spectroscopy, FTIR, DLS particles size analysis, TEM and XRD, were obtained and evaluated. Keywords: Phytochemicals; Nanoparticles; Antimicrobial; Allium cepa Ezealisiji, Kenneth M 1, 2, * and Chiamaka Ruth Nwodo 2 Ezealisiji, Kenneth M 1, 2, * and Chiamaka Ruth Nwodo 2 1 Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Port Harcourt, East/West Road, PMB 5323 Choba Rivers State, 500001 Nigeria. 2 Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Madonna University Elele, Rivers State, Nigeria. 1 Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Port Harcourt, East/West Road, PMB 5323 Choba Rivers State, 500001 Nigeria. 2 Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Madonna University Elele, Rivers State, Nigeria. GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 Publication history: Received on 04 December 2022; revised on 20 January 2023; accepted on 23 January 2023 ublication history: Received on 04 December 2022; revised on 20 January 2023; accepted on 23 Jan Article DOI: https://doi.org/10.30574/gscbps.2023.22.1.0032 * Corresponding author: Ezealisiji, Kenneth M Copyright © 2023 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4 Green synthesis of copper oxide nanoparticles using the outer layer of Allium cepa L. and evaluation of its antimicrobial properties 1. Introduction Nanotechnology can simply be defined as a technology performed at a Nano scale, which is at a scale less than 100 nanometer. The word nano is an SI prefix and comes from the Greek word nanos meaning dwarf or something really small. Nanotechnology involves the use of various materials having dimensions of the order of a billionth of a meter. Although modern nanotechnology is relatively new, nanosize or nanoscale had been in existence, with their continuous use for centuries. Nanotechnology advancements and its application in various fields have revolutionized the world. The various fields that have potential application of nanotechnology includes; engineering, water purification, biomedical field, medicine, pharmacy etc. Copper is a semiconductor material considered to be an excellent candidate for the synthesis of metal-based nanoparticles. Besides being highly resistant to heat, it is also robust, stable, cheap and easily synthesized. [1, 2]. CuONPs can be synthesized by various processes. Among all, biocompatible processes emerged as the most investigated in the past few years. Independently of the selected method, during synthesis, CuSO4, CuCl2 · 2H2O, Cu (NO3) 2 or Cu (CH3COO)2 are the most frequently used copper precursors. The two main approaches used in the synthesis of nanoparticles are the top down approach and the bottom up approach. In top-down approaches, bulk materials are broken down produce nano structured materials. Top-down methods include mechanical milling, laser ablation, etching, sputtering, and electro-explosion. Bottom-up, or self-assembly, approaches to nanofabrication * Corresponding author: Ezealisiji, Kenneth M GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 use chemical or physical forces operating at the nanoscale to assemble basic units into larger structures. This approach includes supercritical fluid synthesis, spinning, sol-gel process, laser process, laser pyrolysis, molecular condensation, chemical reduction. Green synthesis using plant extracts such as Lemon grass [3], Euphorbia tirualli [4], Gloriosa superba [5] and Cinnamomum camphora [6] have been reported. Allium cepa also known as onion, is an herbaceous biennial plant in the amaryllis family (Amaryllidaceae) that is grown for its edible bulb. The onion is mostly native to southwestern Asia but is now grown throughout the world, chiefly in the temperate zones. Onions are low in nutrients but are valued for their flavour and are used widely in cooking. They add flavour to such dishes as stews, roasts, soups, and salads and are also served as a cooked vegetable. Allium cepa L. 2.1. Materials Copper Sulphate, and other analytical grade reagents were product of Merck, Germany, and Oxoid, Hampshire, UK. The bulb of Allium cepa L. (Onion) was collected from the southern part of Nigeria, Elele, Rivers state, and were further identified by Mr Boniface of the Department of pharmacognosy, Madonna University. The voucher specimen was deposited in the herbarium. 1. Introduction extracts were reported to inhibit the growth of both Gram-positive and Gram-negative bacteria, such as Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, and Klebsiella pnuemoniae [7, 8, 9, 10, 11]. Quercetin has been a most extensively studied flavonoid. Figure 1 Chemical structure of quercetin Figure 1 Chemical structure of quercetin CuONPs have shown antimicrobial effects. The mechanism of antibacterial activity of CuONPs is not well elucidated yet, but it is believed that it involves bacterial cell wall adhesion triggered by electrostatic interactions. Dissociation of Cu2+ induces the generation of ROS that contact with cellular membranes. These ions also have the capacity to enter the cell, causing membrane damage which is associated with disruption of cells internal content and bacterial cell leakage [12, 13]. The study was conducted in order to evaluate the antimicrobial activities of cupper nanoparticles using the outer layer Allium cepa L., for its antimicrobial properties. Most researches on the antimicrobial effect of Allium cepa L. has been focused on the biological activity of Allium cepa L. alone against microorganisms. Very few researches has tried to explore and investigate into the antimicrobial (antibacterial and antifungal) properties of cupper nanoparticles, hence there are insufficient literature in that regard. Copper oxide nanoparticles combined with various antimicrobial agents has a better antimicrobial effect than cupper nanoparticles or the antimicrobial agent alone. Thus, this study might help discover more medicinal properties of cupper oxide nanoparticles synthesized the outer layer of Allium cepa L., especially its antimicrobial strength and properties, for further development in the field of medicine. 2.2. Extraction The bulb of Allium cepa L. obtained from Elele, Rivers state, was used for the extraction process. The outer petals were separated from the inner portion of the allium cepa bulb, and placed in a 1000ml (1 Liter) beaker. 400ml distilled water was added to the beaker containing the outer petals of Allium cepa and the mixture was heated for 30 minutes at 100 0C . The mixture was filtered while hot, using a filter paper. 2.6.3. Preparation of Sabouraud dextrose agar All aseptic techniques were observed during the experiment. A 3.25 g quantity of sabouraud dextrose agar was weighed and transferred into a 500 ml conical flask. A 50 ml volume of water was added to the powder gradually while shaking to ensure a complete dissolution of the powder in water. The mixture was autoclaved at 1210C for 15minutes. The valve of the autoclave was opened and the pressure was allowed to return to 0 before opening the autoclave. The molten agar formed was then poured into petri dishes, covered and allowed to solidify. 2.5. Instrumentation The formation of CuONPs using the outer layer of Allium cepa L. was monitored by visual colour change The copper oxide nanoparticles (CuONPs) was scanned using the UV-VIS spectrophotometer (JENWAY, 6705) at a wavelength ranging from 180-700 nm.The presence of functional group was detected using the Fourier transform infrared spectrophotometer (FTIR). The Shimadzu FTIR Spectrophotometer (FTIR-8400S) was used to carry out the analysis, with a wave number ranging from 4000-5000 cm-.The particle size distribution analysis was performed to determine the size of the copper nanoparticles. Dynamic light scattering (DLS) and Zeta potential of the synthesized nanoparticles was analyzed to know the average size and stability of particles using DLS-Nano 2s model, UK.The particle size and surface features of the CuONPs were determined by transmission electron microscopy (TEM) using a ZEISS LIBRA 120 KV-UK, at different magnification. The crystalline nature and the average size of the CuONPs was analyzed and calculated using Bruker: D8 Discover, Japan for the X-ray diffraction analysis. 2.6.2. Preparation of Nutrient agar (Mueller Hinton) All aseptic techniques were observed during the experiment. A 2.8 g quantity of the agar powder was weighed into a 500ml conical flask using an electric weighing balance. A 100 ml volume of water was added to the powder gradually while shaking to ensure the complete dissolution of the powder in water. The mixture was autoclaved at a temperature of 121 0C for 15 minutes. The valve of the autoclave was opened and the pressure was allowed to return to 0 before the autoclave was opened. The molten agar formed, was then poured into petri dishes, two-third full. The petri dish was covered and allowed to solidify. 2.3. Green synthesis of cupper nanoparticles using Allium cepa L. A 10 g quantity of copper sulphate were weighed using an electric weighing balance, and dissolved using a 150 ml volume of distilled water in a 50 ml beaker. It was then transferred into a 1 liter volumetric flask, where the volume was made up to the 1 liter mark of the volumetric flask. The copper sulphate solution afforded the copper ion required for 303 GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 the reaction. 150 ml of the plant extract was mixed with 250 ml of the copper nitrate solution, while the plant extract was still hot. The reaction was incubated at a temperature of 27oC, in the dark in order to avoid photochemical activation of the copper sulphate. A dark brown colour was observed after three hours, indicating the formation of copper oxide nanoparticles (CuONPs). the reaction. 150 ml of the plant extract was mixed with 250 ml of the copper nitrate solution, while the plant extract was still hot. The reaction was incubated at a temperature of 27oC, in the dark in order to avoid photochemical activation of the copper sulphate. A dark brown colour was observed after three hours, indicating the formation of copper oxide nanoparticles (CuONPs). 2.6.1. Collection of bacterial isolate The microorganisms were obtained from the diagnostic Labouratory of Madonna University Teaching hospital, Elele, Rivers State. 2.4. Isolation procedure The synthesized copper oxide nanoparticles were centrifuged for 30minutes at 5000 rpm. The pallet containing copper oxide nanoparticles were rinsed using a small volume of distilled water. The nanoparticles was poured into a porcelain plate and placed in the oven at 115 0C to evaporate the solvent for 20minutes. The copper oxide nanoparticles were scrapped off the plate using a spatula and placed in sample tubes. 2.6.4. Preparation of nutrient broth All aseptic techniques was observed during the experiment. A 0.65 g quantity of nutrient broth powder was weighed using an electric weighing balance and transferred into a 500 ml beaker. A 50 ml volume of distilled water was added gradually while shaking the mixture in order to ensure the complete dissolution of the agar powder in water. The mixture was then autoclaved at a temperature of 1210C for 15 minutes. The valve of the autoclave was opened and the pressure was allowed to return to 0 before opening the autoclave. 2.8. Minimum inhibitory concentration (MIC) calculation The agar well diffusion method was used in the determination of minimum inhibitory concentration. Serial dilutions of the reconstituted copper nanoparticles was prepared at different concentrations of 0.3125 – 20 mg/ml in separate test tubes. The Mueller Hinton agar for bacteria and sabouraud dextrose agar for the fungi were separately prepared, autoclaved and poured into the 7 petri dishes. The Mueller Hinton agar was added to the first 4 petri dishes, the sabouraud dextrose agar for the fungi was added to 1 petri dish and the 6th and 7th petri dishes contained only the Mueller Hinton agar and sabouraud dextrose agar respectively. The agar was allowed to solidify. The agar plates were divided into seven quadrants with a marker and labelled 1-7 which represents the different concentrations of copper nanoparticles, plant extracts and the antimicrobial agent used (quadrant 1= 20 mg/ml, quadrant 2= 10mg/ml, quadrant 3= 5 mg/ml, quadrant 4= 2.5 mg/ml, quadrant 5= 1.25 mg/ml, quadrant 6 = 0.625 mg/ml, quadrant 7 = 0.3125 mg/ml). The surface of the agar plate were streaked with the microorganism using a cotton swab and a cock borer was used to bore holes in the individual quadrants. The different concentrations of copper oxide nanoparticles, plant extracts and the antimicrobial agent used were added to their respective holes in the eight quadrants, after which, the agar plates were incubated at 37OC for 24 hours. The agar plates were also observed for the one with the least observable growth, in order to determine the minimum concentration of the copper oxide nanoparticles that inhibit the growth of the microorganisms. 2.7. Antimicrobial sensitivity screening The solidified agar was dried in a hot air oven at a temperature of 450C for 30 minutes, after drying, a suspension of the organisms required for the experiment was swabbed on the agar plates using sterile swab sticks. Each agar plate contains a single organism. After the inoculation of the organisms, on the media, the filter paper were cut at a dimension 304 GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 of 5 mm by 5mm. The filter papers were autoclaved at a temperature of 1210C for 15minutes, and it was allowed to cool after removing it from the autoclave. The agar plates containing the different microorganisms were divided into four quadrants (first quadrant =40mg, second quadrant= 20mg, third quadrant= copper sulphate (CuSO4) solution, forth quadrant= standard (antimicrobial agent)). The copper oxide nanoparticles were reconstituted and impregnated into the autoclaved filter paper, by picking up the sterile filer paper using a pair of sterile forceps and dipping them into the reconstituted nanoparticles that contains 20 and 40 mg of the copper nanoparticles. The filter papers were then placed on the inoculated agar plate. The agar plates were incubated for 24 hours and the minimum inhibitory concentration (MIC) was calculated. 3. Results and discussion The reaction between copper sulphate solution and aqueous extract of the outer layer of Allium cepa produced a colour change from bluish to dark brown, due to the reduction of the copper ions by the phytochemicals present in Allium cepa followed by the capping and stabilization of the nanoparticles formed, clearly indicates the presence of copper oxide nanoparticles (CuONPs). The mechanisms of the synthesis of copper nanoparticles follows a stepwise chemical reaction of copper (II) sulphate in water to dissociate into copper (II) ion (Cu2+) and SO42-. Copper (II) ion further reacts with Phyto-polyphenols of Allium cepa and reduced to copper (I) oxide. The subsequent aggregation and reduction of copper (I) oxide gave CuO nanoparticles as shown below. Figure 2 Reduction of Cu2+ to CuO Figure 2 Reduction of Cu2+ to CuO The CuO nanoparticles formed are stabilized and capped by phytochemicals, which imparts stab copper nanoparticles. Figure 2 Reduction of Cu2+ to CuO Figure 2 Reduction of Cu2+ to CuO The CuO nanoparticles formed are stabilized and capped by phytochemicals, which imparts stability to the synthesized copper nanoparticles. 305 GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 3.1. Ultraviolet-visible spectrophotometry (UV-Vis) The sample analysis using UV- Vis spectrophotometer at a wavelength ranging between 180 – 700 nm gave a surface Plasmon resonance (SPR) spectra. The spectrum in figure 3 had a fairly broad peak at 280 nm, which corresponds to the copper oxide nanoparticles. This result was similar to the findings confirmed from copper oxide nanoparticles fabricated with the aid of Syzygium alternifolium stem bark aqueous extract [14] Figure 3 UV-Vis Absorbance of the synthesized CuONPs Figure 3 UV-Vis Absorbance of the synthesized CuONPs 3.2. Fourier-transform infrared spectroscopy (FTIR) The FTIR spectrum of the synthesized copper nanoparticles (figure 4) shows a sharp narrow band at 3432 cm-1, which corresponds to the N-H vibration mode. The peak at 1631cm-1 represents the N-H bending and the signal at 1072 cm-1 corresponds to the C-N bond stretching. Keyword: T = Transmittance Figure 4 FT-IR of the synthesize CuONPs 3.3. Dynamic light scattering (DLS) Particle size distribution The DLS study (Fig: 5) of the synthesized Copper oxide nanoparticle (CuONPs) gave an average size distribution of 396±1.64 nm. It is expected that the size of CuONPs obtained from the DLS study could be larger than those obtained from XRD and TEM. The increased size could be due to the fact that the DLS measures not only the apparent size (hydrodynamic radius or diameter) of a particle but also the hydrodynamic layers that could be formed around hydrophobic particles. The result could also be attributed to the influence of salvation layer effect of electrolyte dispersion and the restrictions in bond and rotation angles, which leads to larger sizes in colloidal water media than the `geometric` sizes obtained from TEM and XRD. The synthesized copper nanoparticles are positively charged and evenly dispersed in the medium and the Zeta potential was estimated to be 48.3 mV. Keyword: T = Transmittance Figure 4 FT-IR of the synthesize CuONPs Figure 4 FT-IR of the synthesize CuONPs Figure 4 FT-IR of the synthesize CuONPs 3.3. Dynamic light scattering (DLS) Particle size distribution 3.3. Dynamic light scattering (DLS) Particle size distribution The DLS study (Fig: 5) of the synthesized Copper oxide nanoparticle (CuONPs) gave an average size distribution of 396±1.64 nm. It is expected that the size of CuONPs obtained from the DLS study could be larger than those obtained from XRD and TEM. The increased size could be due to the fact that the DLS measures not only the apparent size (hydrodynamic radius or diameter) of a particle but also the hydrodynamic layers that could be formed around hydrophobic particles. The result could also be attributed to the influence of salvation layer effect of electrolyte dispersion and the restrictions in bond and rotation angles, which leads to larger sizes in colloidal water media than the `geometric` sizes obtained from TEM and XRD. The synthesized copper nanoparticles are positively charged and evenly dispersed in the medium and the Zeta potential was estimated to be 48.3 mV. The DLS study (Fig: 5) of the synthesized Copper oxide nanoparticle (CuONPs) gave an average size distribution of 396±1.64 nm. It is expected that the size of CuONPs obtained from the DLS study could be larger than those obtained from XRD and TEM. The increased size could be due to the fact that the DLS measures not only the apparent size (hydrodynamic radius or diameter) of a particle but also the hydrodynamic layers that could be formed around hydrophobic particles. The result could also be attributed to the influence of salvation layer effect of electrolyte dispersion and the restrictions in bond and rotation angles, which leads to larger sizes in colloidal water media than the `geometric` sizes obtained from TEM and XRD. The synthesized copper nanoparticles are positively charged and evenly dispersed in the medium and the Zeta potential was estimated to be 48.3 mV. 306 GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 Figure 5 Dynamic light scattering (DLS) Particle size distribution of synthesized CuONPs Figure 5 Dynamic light scattering (DLS) Particle size distribution of synthesized CuONPs 3.4. Transmission electron microscope (TEM) Figure 6 TEM image of synthesized CuNPs Transmission electron microscopy (TEM) were used to reveal the particle size and morphology of the synthesized CuONPs. The S\synthesized CuONPs were spherical in shape, non-clustering, well dispersed with a size ranging between 28 and 45 nm with a mean particle size of 32±0.25 nm (Figure 6). GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 The X-ray diffraction (XRD) pattern (figure 7) of the biosynthesized CuONPs was analyzed using the Bruker d8 Advanced X-ray diffractometer, using CuKα radiation (λ=1.5406 Ӓ) 40 Kv, 2 Ө/Ө scanning mode. Data was obtained for the 2 Ө range of 10 – 120 degree in step proceeding of 0.0206 degree. Four prominent peaks at 2 Ө values of 18.406o, 22.102o, 23.672o, and 30.902o corresponding to, (020), (022), (022), and (111) integer on the `hkl` planes respectively. This may be recorded as bands for face-centred and crystalline particulate matter which is spherical in nature. Present result showed an excellent correlation with data obtained from International Centre for Diffraction Data (ICDD). Using Debby-Scherrer equation 𝐷= 𝐾𝜆 ᵦ 𝐶𝑜𝑠 Ө Where D = the crystallite size of CuNPs Where D = the crystallite size of CuNPs λ = the wavelength of x-ray source (1.54059 nm) used in XRD β = the full width at half maximum of the diffraction peak K = the Scherrer’s constant with value from 0.9 to 1 θ = the Bragg angle Where D = the crystallite size of CuNPs λ = the wavelength of x-ray source (1.54059 nm) used in XRD β = the full width at half maximum of the diffraction peak K = the Scherrer’s constant with value from 0.9 to 1 θ = the Bragg angle K = the Scherrer’s constant with value from 0.9 to 1 θ = the Bragg angle The average particle size of the CuONPs was calculated to be 22.76 nm at an operational Optimum Bragg reflection obtained at 2Ө of 18.204o. This was confirmed by the TEM study as well. 3.6. 3.3. Dynamic light scattering (DLS) Particle size distribution Figure 5 Dynamic light scattering (DLS) Particle size distribution of synthesized CuONPs 3.4. Transmission electron microscope (TEM) Figure 6 TEM image of synthesized CuNPs Transmission electron microscopy (TEM) were used to reveal the particle size and morphology of the synthesized CuONPs. The S\synthesized CuONPs were spherical in shape, non-clustering, well dispersed with a size ranging between 28 and 45 nm with a mean particle size of 32±0.25 nm (Figure 6). 3.4. Transmission electron microscope (TEM) 3.4. Transmission electron microscope (TEM) microscope (TEM) Figure 6 TEM image of synthesized CuNPs Figure 6 TEM image of synthesized CuNPs Transmission electron microscopy (TEM) were used to reveal the particle size and morphology of the synthesized CuONPs. The S\synthesized CuONPs were spherical in shape, non-clustering, well dispersed with a size ranging between 28 and 45 nm with a mean particle size of 32±0.25 nm (Figure 6). Transmission electron microscopy (TEM) were used to reveal the particle size and morphology of the synthesized CuONPs. The S\synthesized CuONPs were spherical in shape, non-clustering, well dispersed with a size ranging between 28 and 45 nm with a mean particle size of 32±0.25 nm (Figure 6). 3.5. X-ray diffraction analysis (XRD) 3.5. X-ray diffraction analysis (XRD) Figure 7 XRD image of synthesized CuONPs Figure 7 XRD image of synthesized CuONPs 307 GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 Antimicrobial screening results Table 1 Inhibition zone diameter (mm) result S/N Concentration Staphylococcus aureus Escherichia coli Bacillus subtilis Salmonella typhi Candida albicans 1 CuNPs (40mg) 7mm 4 mm 9 mm 6 mm 4 mm 2 CuNPs (20mg) 5 mm 4 mm 7mm 5 mm 2 mm 3 Copper sulphate solution NZ NZ NZ NZ NZ 4 Gentamicin (10µg) 24 mm 22 mm 23 mm 19 mm - 5 Fluconazole - - - - 19 Well size= 8mm; Keyword: NZ = No zone Figure 7a Comparative chart of antimicrobial activity of CuONPs against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Salmonella typhi The average particle size of the CuONPs was calculated to be 22.76 nm at an operational Optimum Bragg reflection obtained at 2Ө of 18.204o. This was confirmed by the TEM study as well. Table 1 Inhibition zone diameter (mm) result S/N Concentration Staphylococcus aureus Escherichia coli Bacillus subtilis Salmonella typhi Candida albicans 1 CuNPs (40mg) 7mm 4 mm 9 mm 6 mm 4 mm 2 CuNPs (20mg) 5 mm 4 mm 7mm 5 mm 2 mm 3 Copper sulphate solution NZ NZ NZ NZ NZ 4 Gentamicin (10µg) 24 mm 22 mm 23 mm 19 mm - 5 Fluconazole - - - - 19 Well size= 8mm; Keyword: NZ = No zone Figure 7a Comparative chart of antimicrobial activity of CuONPs against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Salmonella typhi Figure 7a Comparative chart of antimicrobial activity of CuONPs against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Salmonella typhi Figure 7a Comparative chart of antimicrobial activity of CuONPs against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Salmonella typhi 308 GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 Figure 7b Chart of antifungal activity of CuONPs against Candida albicans Figure 7b Chart of antifungal activity of CuONPs against Candida albicans The zones of inhibition of the synthesized copper oxide nanoparticles against the selected microorganism (Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Salmonella typhi and Candida albicans) at concentrations of 40mg/ml, 20mg/ml, copper sulphate and control which includes gentamycin and fluconazole were shown in table 3.1. The highest zone of inhibition against the test organisms was observed at the control (gentamycin and fluconazole). In copper sulphate solution, there was no zone of inhibition at any of the test organisms. GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 y y pp p g (Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Salmonella typhi and Candida albican ranging between 20mg/ml-0.3125mg/ml (diluted using the two fold serial dilution) were shown a 3.2 the minimum concentration of the synthesized copper oxide nanoparticles that would inhibit t microorganism was 20mg/ml, although the concentration had no effect against candida albicans. GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 It has been reported that nanoparticles combined with various antibiotics have better antimicrobial properties, than the effects of the nanoparticles alone (Li et al, 2005). Even though there would be no zone of inhibition by the copper nanoparticles at the concentration similar to that of the control, there was zone of inhibition in concentrations of 40mg/ml and 20mg/ml, as shown in table 1. Table 2 Minimum inhibitory concentration result Table 2 Minimum inhibitory concentration result S/N Microorganism 20 mg/ml 10 mg/ml 5 mg/ml 2.5 mg/ml 1.25 mg/ml 0.625 mg/ml 0.3125 mg/ml 1 Staphylococcus aureus + _ _ _ _ _ _ 2 Escherichia coli + _ _ _ _ _ _ 3 Bacillus subtilis + _ _ _ _ _ _ 4 Salmonella typhi + _ _ _ _ _ _ 5 Candida albicans _ _ _ _ _ _ _ Keyword: + = Activity; _ = No activity The minimum inhibitory concentration of the synthesized copper oxide nanoparticles against selected microorganisms (Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Salmonella typhi and Candida albicans) at concentrations ranging between 20mg/ml-0.3125mg/ml (diluted using the two fold serial dilution) were shown at table 3.2. From table 3.2 the minimum concentration of the synthesized copper oxide nanoparticles that would inhibit the growth of the test microorganism was 20mg/ml, although the concentration had no effect against candida albicans. he minimum inhibitory concentration of the synthesized copper oxide nanoparticles against selected The minimum inhibitory concentration of the synthesized copper oxide nanoparticles against selected microorganisms (Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Salmonella typhi and Candida albicans) at concentrations ranging between 20mg/ml-0.3125mg/ml (diluted using the two fold serial dilution) were shown at table 3.2. From table 3.2 the minimum concentration of the synthesized copper oxide nanoparticles that would inhibit the growth of the test microorganism was 20mg/ml, although the concentration had no effect against candida albicans. The minimum inhibitory concentration of the synthesized copper oxide nanoparticles against selected microorganisms (Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Salmonella typhi and Candida albicans) at concentrations ranging between 20mg/ml-0.3125mg/ml (diluted using the two fold serial dilution) were shown at table 3.2. From table 3.2 the minimum concentration of the synthesized copper oxide nanoparticles that would inhibit the growth of the test microorganism was 20mg/ml, although the concentration had no effect against candida albicans. References [1] Losasso C., Belluco S., Cibin V., Zavagnin P., Mičetić I., Gallocchio F., Zanella M., Bregoli L., Biancotto G., and Ricci A. (2014). Antibacterial activity of silver nanoparticles: Sensitivity of different Salmonella serovars. Frontiers in Microbiology, 5, 227. [1] Losasso C., Belluco S., Cibin V., Zavagnin P., Mičetić I., Gallocchio F., Zanella M., Bregoli L., Biancotto G., and Ricci A. (2014). Antibacterial activity of silver nanoparticles: Sensitivity of different Salmonella serovars. Frontiers in Microbiology, 5, 227. [2] Qing Y., Cheng L., Li R., Liu G., Zhang Y., Tang X., Wang J., Liu H., and Qin Y. (2018). Potential antibacterial mechanism of silver nanoparticles and the optimization of orthopedic implants by advanced modification technologies. International Journal of Nanomedicine, 13(1), 3311–3327. [2] Qing Y., Cheng L., Li R., Liu G., Zhang Y., Tang X., Wang J., Liu H., and Qin Y. (2018). Potential antibacterial mechanism of silver nanoparticles and the optimization of orthopedic implants by advanced modification technologies. International Journal of Nanomedicine, 13(1), 3311–3327. [3] Iravani, S. (2011) Green Synthesis of metal nanoparticles using plants. Green Chemistry, 13(10); 2638-2650. [4] Lee, H.J., Lee, G., Jang, N.R., Yum, J.H., Song, J.Y., and Kim, B.S. (2011). Biological Synthesis of Copper nanoparticles using plant extract. Nanotechnology, 128(1-2); 83-89. [5] Naika, R.H., Lingaraju. K., Manjunath, K., Kumar, D., Nagaraju, G., Suresh, D., and Nagabhushana, H. (2015). Green Synthesis of Copper Oxide (CuO) Nanoparticles using Gloriosa superb L. extract and their antimicrobial activity; Journal of Taibah University for Science, 9(1); 7-12. [6] Huang, J., Li, Q., and Sun, D. (2007). Biosynthesis of silver and gold nanoparticles by Novel sundried Cinnamomum camphora leaf. Nanotecnology, 18(10); 105104. [7] Eltaweel, M. (2013). Assessment of antimicrobial activity of onion extract (Allium cepa) on Staphylococcus aureus; in vitro study. International Conference on Chemical, Agricultural and Medical Sciences, 12, 29– 30. [8] Induja, M. P., and Geetha, R. V. (2018). Antimicrobial activity of Allium cepa against bacteria causing enteric infection. Drug Invention Today, 10(12), 35– 38 [9] Loredana, L., Giuseppina, A., Filomena, N., Florinda, F., & Donatella, A. (2019). Biochemical, antioxidant properties and antimicrobial activity of different onion varieties in the Mediterranean area. Journal of Food Measurement and Characterization, 13(2), 1232– 1241. [10] Sharma, K., Ko, E. Y., Assefa, A. D., Ha, S., Nile, S. H., Lee, E. T., and Park, S. W. (2015). Temperature-dependent studies on the total phenolics, flavonoids, antioxidant activities, and sugar content in six onion varieties. 4. Conclusion This study provided scientific proof that copper oxide nanoparticles synthesized using the outer layer of Allium cepa has antimicrobial properties against both gram positive and gram negative organisms. It also has activity against fungi, although not as effective as the standard antimicrobial agents. The synthesized CuONPs showed remarkable stability in the characterization using different techniques such as UV-Visible spectroscopy which was used in noting the rate reaction at a given time, FTIR which was used to determine the different functional groups, DLS particle size which was used to determine the size distribution of the particles, Transmission electron microscopy (TEM) which was used to reveal the particle size and morphology of the synthesized CuONPs and finally the X-ray diffraction analysis which was used to analyze The crystalline nature and the average size of the CuONPs. 309 GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 Disclosure of conflict of interest The authors declare no conflict of interest. The authors declare no conflict of interest. Acknowledgments Acknowledgments The authors wish to acknowledge the Chemistry Department, Rhodes University, PO Box 94, Grahamstown, 6140, South Africa for their unprecedented permission to access their state of the art Nanotechnology Laboratories. [16] Bahram-Parvar, M., and Lim, L. T. (2018). Fresh-cut onion: A review on processing, health benefits, and shelf-life. Comprehensive Reviews in Food Science and Food Safety, 17(2), 290– 308. References Journal of Food and Drug Analysis, 23(2), 243– 252. [11] Sharma, K., Mahato, N., and Lee, Y. R. (2018). Systematic study on active compounds as antibacterial and antibiofilm agent in aging onions. Journal of Food and Drug Analysis, 26(2), 518–528. [12] Slavin, Y. N., Asnis, J., Häfeli, U. O., and Bach, H. (2017). Metal nanoparticles: understanding the mechanisms behind antibacterial activity. Journal of nanobiotechnology, 15(1), 1-20. [13] Lara, H.H., Ayala-Nuñez, N.V., Ixtepan-Turrent, L., and Rodriguez-Padilla, C. (2010). Mode of antiviral action of silver nanoparticles against HIV-1. Journal of Nanobiotechnology, 8(1), 1–10. [14] Pulicheria, Y., Thirumalandhuni, V., Yagani, J.R., Palempalli, U. M. D., Golla, N., and Nataru, S. (2018). Cost Effective, Green Synthesis of Copper Oxide Nanoparticles Using Fruit Extract of Syzygium alterniflium (Wt.) Walp., Characterization and Evaluation of Antiviral Activity. Journal of Cluster Science, 28(4), 743-755. [15] Khanna, P., Kaur, A., and Goyal, D. (2019). Algae-based metallic nanoparticles: Synthesis, characterization and applications. Journal of microbiological methods, 163, 105656. [16] Bahram-Parvar, M., and Lim, L. T. (2018). Fresh-cut onion: A review on processing, health benefits, and shelf-life. Comprehensive Reviews in Food Science and Food Safety, 17(2), 290– 308. 310 GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 GSC Biological and Pharmaceutical Sciences, 2023, 22(01), 302–311 [17] Chakraborty, A.J., Uddin, T.M., Zidan, M., Redwan, B.M., Mitra, S., Das, R., Nainu, F., Dhama, K., Roy, A., Hossain, M. and Khusro, A. (2022). Allium cepa: A Treasure of Bioactive Phytochemicals with Prospective Health Benefits. Evidence-Based Complementary and Alternative Medicine, 2022. [18] Charles P. Poole, Jr. and Frank J. Owens. (2003). Introduction to nanotechnology, John Wiley and sons Inc., United States, pp: 1-7. [19] Gao, W., Thamphiwatana, S., Angsantikul, P., and Zhang, L. (2014). Nanoparticle approaches against bacterial infections. Wiley Interdisciplinary Reviews-Nanomedicine and Nanobiotechnology 6(6), 532–547. [20] Kumar, K. S., Bhowmik, D., Chiranjib, B., and Tiwari, P. (2010). Allium cepa: A traditional medicinal herb and its health benefits. Journal of Chemical and Pharmaceutical Research, 2(1), 283-291. [21] Mirhosseini, M. (2015). Synergistic antibacterial effect of metal oxide nanoparticles and ultrasound stimulation. Journal of Biology and Today’s World, 4(6), 138–144. [22] Ramos, F. A., Takaishi, Y., Shirotori, M., Kawaguchi, Y., Tsuchiya, K., Shibata, H., and Takeuchi, M. (2006). Antibacterial and antioxidant activities of quercetin oxidation products from yellow onion (Allium cepa) skin. Journal of Agricultural and Food Chemistry, 54(10), 3551– 3557. References [23] Sánchez-López, E., Gomes, D., Esteruelas, G., Bonilla, L., Lopez-Machado, A.L., Galindo, R., Cano, A., Espina, M., Ettcheto, M., Camins, A. and Silva, A.M. (2020). Metal-based nanoparticles as antimicrobial agents: an overview. Nanomaterials, 10(2), 292. 311
https://openalex.org/W2052181487	https://zenodo.org/records/2138728/files/article.pdf	English	null	SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?	Annals of surgery	1,908	public-domain	7,064	* Read before the Rochester Academy of Medicine, May 3, 1907, 222 SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. My attention was recently called to the views apparently held concerning cholecystitis and gall-stones by the rank and file of the profession, by a remark of my chief of clinic, “ that the general profession at present seems to occupy the same position towards biliary surgery that it did ten years ago towards appendicitis.” I am satisfied that this is largely true, and that the real importance of certain biliary condi¬ tions and the impossibility of successfully dealing with them otherwise than by the knife, is not widely enough recog¬ nized. The idea is certainly too prevalent in the profession that to warrant a diagnosis of cholecystitis some jaundice should be present, and that a painful, tender tumor in the region of the gall-bladder should be demonstrable with pos¬ sibly chill, but certainly marked fever, while the failure to detect jaundice seems to many, unavowedly, to unsettle their tentative diagnosis as to the possibility of cholecystitis. If asked, point blank, whether uncomplicated inflammation of the gall-bladder could produce jaundice, the majority would answer correctly, no, but practically the absence of this symp¬ tom staggers them. My first postulate is that cholecystitis is an infective process which precedes the' formation of calculi, and that either with or without stone formation this disease of the gall-bladder implies certain potential dangers. It is true that the most common form of cholecystitis is produced by germs 222 CHOLECYSTITIS AND CHOLELITHIASIS. 223 of low virulence, but what warrant exists for the belief that secondary infection with virulent organisms will not take place, causing infectious cholangeitis—often a most fatal condition—or suppuration or gangrene of the gall¬ bladder with fatal peritonitis? What certainty is there that crippling .adhesions involving the stomach and intestines will not form with persistent ill health, or even hopeless gastric dilatation? The absence of gall-stones at an operation in chronic cholecystitis does not prove that none have been passed or preclude the probability of their new formation. Let me illustrate my contentions by- reading the notes of a few cases. 1. Mrs. F., age 76 years. A few days previous to entrance to the University of Michigan Hospital had chills followed by high fever and severe pain in the right hypochondriac region. She claimed never to have had any biliary trouble and believed this to be her first attack. Operation revealed a ruptured gall¬ bladder with old inflammatory trouble. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. Five days later a single, noil-faceted stone was removed from the cavity formed among the adherent bowels. 2. Mr. G., age 72 years. ITad never recognized pain or fever and had never vomited; very slight jaundice was present. Operation revealed suppurative cholecystitis with stones blocking the cystic duct. 3. Mrs. C., age 48 years. Vomiting, pain radiating to right shoulder, chills, fever and clay-colored stools were noted. Oper¬ ation showed old adhesions around gall-bladder and common duct; no stones; cure followed loosening of adhesions and drainage. 4. Mrs. D., age 41 years. Severe jaundice, no vomiting, chills or fever, pain described as intestinal cramping, marked diarrhoea. At operation gall-bladder much distended, no stones. Cholecystitis with cholangeitis causing jaundice. 5. Mr. X., age 47 years, rapidly developed after an attack of ptomaine poisoning gastric and hepatic pain, irregular attacks of chills, fever and sweating, acholic stools, intense jaundice, and rapid loss of flesh and strength. An obscure thickening in the region of the pancreas was detected. The family physician’s CHARLES B. G. DE NANCREDE. 224 diagnosis was not concurred in of common duct stone, but infec¬ tive cliolangeitis was believed to be present. His condition for¬ bade operation. Post-mortem.—General suppuration cliolangeitis was found with obstruction of the common duct from some undetermined condition of the pancreas. Early hepatic drainage would have saved this patient. 6. Mr. M., age 56 years, had gradually developed severe jaundice, commencing about seven weeks before admission to the hospital. Neither pain, vomiting, chills, clay-colored stools nor stones in the stools were noted. Operation showed gall-bladder containing a pint of bile, cystic duct kinked by weight; no stones, ante- or post-mortem, the patient dying from hemorrhage in thirty-six hours, having hematesis, bloody stools and free bleeding from the gall-bladder. This case must have had long standing cholecystitis, judging from the conditions found. Disease of the gall-bladder was present in all the cases quoted. No extended argument is needed beyond the his¬ tories of these patients to demonstrate that cholecystitis with or without stone may present few of the ordinary symptoms expected, that it may prove a menace to life when least sus¬ pected, and that the symptoms in some instances closely simulate those of common duct stone, while suppurative and gangrenous cliolangeitis is seen to be a most dangerous con¬ dition which may develop at any time from a chronic cliolangeitis. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. Although the natural resistance of the hepatic and somatic tissues may prevent the most dangerous complica¬ tions related, or even gain the victory so far as life is con¬ cerned, if the assistance of art is invoked, it must be admitted that these conditions of the gall-tracts can only be efficiently dealt with by proper operative intervention, and that early hepatic drainage will often prevent a lifetime of invalidism, or avert death. Moreover, the deteriorating effects of chronic jaundice, cholemia, and infection account for those unrcstrainable capillary hemorrhages that not uncommonly destroy life after otherwise successful operations. Listen to these notes. CHOLECYSTITIS AND CHOLELITHIASIS. 225 7. Mrs. P., age 50 years. Had been deeply jaundiced for many months past. The stools had been clay-colored since the commencement of the jaundice, except when tar-colored from altered blood. Patient had frequent epistaxis, and there were areas of subcutaneous hemorrhage. At operation one stone was removed from the common duct and one from the gall-bladder. Death resulted from steady capillary hemorrhage in thirty-six hours. 8. Mrs. C., age 28 years. Had had attacks of pain for the past seven weeks, located in the liver region, but radiating to the epigastrium. Slight jaundice with repeated chills and fever were noted. Numerous old adhesions were found at operation; the pancreas was two or three times its normal volume. A single loose stone was found in tlie gall-bladder. On the third day the drainage from the gall-bladder was largely blood and quanti¬ ties of it soaked the dressings. The hemoglobin and number of red cells rapidly diminished, every evidence of severe loss of blood with profound shock being present. After the seventh day the hemorrhage ceased and the patient recovered; here the presence of old adhesions proved that chronic cholecystitis had hcen overlooked, and that the nearly fatal hemorrhage resulting from the acute jaundice due to cholangcitis could have been averted by timely hepatic drainage. 9. Mrs. 13., age 55 years. Severe jaundice of long stand¬ ing was present. No chills, fever or pain. At operation, malig¬ nant disease encircling the common duct with liver secondaries was found. Severe hemorrhage took place from the wound on the second day, but this finally ceased and the patient recovered. 9. Mrs. 13., age 55 years. Severe jaundice of long stand¬ ing was present. No chills, fever or pain. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. At operation, malig¬ nant disease encircling the common duct with liver secondaries was found. Severe hemorrhage took place from the wound on the second day, but this finally ceased and the patient recovered. Case 6, as you recall, also died after profuse bleeding. Arc not these cases adequate proof of the dangers of hemor¬ rhage. after chronic cholcmia and jaundice? The tendency to serious capillary hemorrhage usually occurs only in cases of pronounced and prolonged jaundice and cholcmia, but an undetected, mild grade of cholemia preceding a comparatively short and slight jaundice may provide the necessary conditions, as exemplified by Case 8. The constant presence of bile salts in the blood vessels leads to such destruction of the red cells that an impov- 8 CHARLES B. G. DE NANCREDE. 226 erished, imperfectly elaborated pabulum is supplied to the minute vessels, while at the same time these salts attack and compromise the integrity of their intimal coat, of which, in¬ deed the capillaries arc alone formed. An answer to the question “ what causes jaundice in hepatic ailments ” will clarify our ideas. Obstruction of the common duct will compel back pressure and resorption. Adhesions; pressure from without by a tumor; or inflam¬ mation of the pancreas; distortion or narrowing of the duct orifice by traction on the duodenum (as is sometimes caused by a loose kidney) can produce choledoch obstructive jaun¬ dice as well as a calculus. Infective cholangeitis causes ob¬ struction of the intra-hcpatic ducts from swelling of their lining membrane, interfering with or arresting the exit of bile, thus favoring its resorption. When the jaundice is not due to common duct obstruction it can only be produced in this way, if wc except a hematogenous origin. Thus the presence or absence of jaundice in cholecystitis, cystic duct kinks or obstruction of this by adhesions, gall-stones or cystic duct stones is explicable, as well as the absence or presence of acholic stools, because the jaundice is due to a compli¬ cating cholangitis, and not directly to any of the conditions mentioned. Again, the illusory improvement occasionally seen in carcinoma of the liver, from lessening or disappear¬ ance of the jaundice, simply means a lessening or complete subsidence of the swelling of the intra-hepatic duct linings, not to a change in the carcinomatous disease. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. A rather blind acceptance of the group of symptoms supposed to indicate the presence and passage of gall-stones is too prevalent in the profession, viz., pain starting in the right hypochondrium radiating to the back and preferably to the right shoulder; violent vomiting; a sudden cessation of the pain; jaundice; clay-colored stools; and calculi to be found in the stools, if careful enough search is made. Again, many practitioners, having seen chills, fever and sweating with marked jaundice and acholic stools in some cases of common duct obstruction expect to find it in all such cases, CHOLECYSTITIS AND CHOLELITHIASIS. 227 and are also surprised when no common duct obstruction is found after such symptoms. Still further, jaundice means with many as a complement, acholic stools, and when the latter are absent, doubt arises as to any gall tract disease being present. A moment’s reflection upon certain anatomical facts should modify any such views. Why should there be jaun¬ dice even if the cystic duct is blocked by a stone, if the common duct is patent? There is no reason, unless cholan- geitis be present, which in a certain number of cases of cholecystitis and cholelithiasis does obtain, because organisms of more than usual virulence arc being excreted with the bile and set up inflammation in the intra-hcpatic ducts. Why should gall-bladder disease be accompanied by chills followed by fever and sweats, unless suppurative or gan¬ grenous cholecystitis is present? Certainly no reason exists. Excluding these two conditions why should aguish attacks and acholic stools be viewed as produced only by an oc¬ cluding choledoch stone, instead of being mere evidences of common duct obstruction by adhesions, tumor pressure, kinking from over-distension of the gall-bladder, or enlarge¬ ment or disease of the pancreas. Fever, chills, and sweats occur because the lymphatic and vascular arrangement of the common duct favors a rapid absorption of infective products; while, if the cystic duct be blocked, absorption is slow and difficult on account of the scanty lymphatic net¬ work of the gall-bladder. As instances of the uncertainty of the significance of jaundice other than as a symptom of cholangcitis, and be¬ cause these cases present other features of interest, let me run over abstracts of the histories of a score or more of cases, asking you to bear in mind the points emphasized in the cases previously mentioned. 10. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. Female, age 47 years.—Had typhoid fever when a child. Vomits during attacks; pain in right hypochondrium which extends up to the right shoulder; slight jaundice present; CHARLES B. G. DE NANCREDE. 228 no chills or fever; stools normal; no stones detected in stools; calculi found in gall-bladder. no chills or fever; stools normal; no stones detected in stools; calculi found in gall-bladder. 11. Woman, age 47 years. Had typhoid fever as a child; vomiting during attacks following sharp pain in epigastrium; no jaundice; stools normal; no stones were found; calculus in cystic duct. 11. Woman, age 47 years. Had typhoid fever as a child; vomiting during attacks following sharp pain in epigastrium; no jaundice; stools normal; no stones were found; calculus in cystic duct. y 12. Man, age 46 years. Had typhoid fever ten years pre¬ vious to commencement of present trouble which is of a number of years duration. Vomiting lasts from one to twelve hours in each attack; pain referred to epigastrium; continuous jaundice for the past three months; stools negative; calculi in gall¬ bladder. 13. Man age 57 years. Had typhoid fever five years be¬ fore onset of present trouble. Localized pain; jaundice; .acholic stools. Common duct obstructed by enlarged pancreas. 14. Man, age 45 years. Had typhoid fever some years ago during which he had jaundice; vomiting during attacks with pain radiating upwards from the right hypochondrium; has had frequent attacks of jaundice and his stools are frequently acholic; occasional blood in stools and vomitus; calculus in common duct. 15. Man, age 62 years. Severe vomiting during attacks; pain over liver extending to epigastrium; marked jaundice; stools negative, according to patient; occasional chills, fever and sweat; calculi in both gall-bladder and common duct. 16. Man, age 45 years. Had history of typhoid fever preceding the gall-stone trouble. During the course of the enteric fever he was jaundiced and has been much of the time since then. The stools are clay-colored; blood is sometimes seen in the stools, and in the vomitus; vomiting with the exacerbations of pain over the liver radiating upwards; stone in common duct. 17. Woman, age 55 years. Has had no vomiting, jaundice or acholic stools; neither chills nor fever; stools negative. At operation gall-bladder contained many small stones. 18. Woman, age 39 years. Vomiting absent; markedly jaundiced; pain over liver radiating upwards; 184 gall-stones in gall-bladder. 18. Woman, age 39 years. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. Vomiting absent; markedly jaundiced; pain over liver radiating upwards; 184 gall-stones in gall-bladder. 19. Woman, age 33 years. Occasional jaundice; stools negative; pain over liver radiating upwards into right chest; solitary stone in gall-bladder. CHOLECYSTITIS AND CHOLELITHIASIS. 229 20. Woman, age 51 years. Vomiting was present with chills and fever; stools negative; calculi in gall-bladder. 20. Woman, age 51 years. Vomiting was present with chills and fever; stools negative; calculi in gall-bladder. 20. Woman, age 51 years. Vomiting was present with chills and fever; stools negative; calculi in gall-bladder. 21. Mrs. C., age 47 years. Occasional vomiting with slight jaundice noted; stools negative; pain over liver region radiating into epigastrium and up beneath the sternum; neither fever nor chills; stone in common duct. 22. Woman, age 50 years. Vomiting; jaundice; acholic stools containing gall-stones; hepatic pain; neither chills nor fever; numerous calculi in gall-bladder; a sinus persisted and a subsequent exploration revealed carcinoma of the gall-bladder, but no calculi. 23. Woman, age 66 years. Occasional vomiting, with slight jaundice during attacks; chills and fever; acholic stools; stone removed from common duct. 24. Woman, age 34 years. Severe jaundice; chills with fever and acholic stools were noted; pain was located in lower part of the abdomen; gall-bladder filled with stones; persistent fistula. Returned two years later. Stone found in common duct. This patient died one year later, probably from malignant disease. 25. Man, age 56 years. Early and severe jaundice de¬ veloped with repeated attacks of chills, fever and vomiting, acholic stools and calculi in the dejecta, the pain commencing in the appendix region, thence passing up to the liver. Very numerous adhesions with small stone in the gall-bladder. 26. Woman, age 30 years. Vomiting; jaundice; chills and fever; acholic stools with pain in right side extending up into right shoulder; many calculi in gall-bladder; pelvic abscess formed and was operated; parotid abscess also developed; recovery. 27. Man, age 48 years. Vomiting; jaundice; pain over liver; normal stools; gall-bladder much dilated and diseased; no stones anywhere; mass in the head of the pancreas. 28. Woman, age 30 years. Patient operated on elsewhere two years previously for gall-stones, but eight months later recov¬ ered twenty calculi from the stools. She had had neither jaun¬ dice, acholic stools nor aguish attacks. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. She had daily attacks of colicky pain in the right side, but at operation nothing but exten¬ sive adhesions were found, especially between the small intestines and gall-bladder. CHARLES n. G. DE NANCREDE. 230 29. Woman, age 62 years. No jaundice or acholic stools; pain felt in right side; stone in gall-bladder. 30. Woman, age 47 years. Has had neither vomiting nor jaundice; pain radiated from liver to the left side; calculi in gall-bladder. 31. Woman, age 53 years. Jaundice, vomiting, acholic stools, chills and fever all present, with pain in right side. Stones oidy in gall-bladder, not in common duct. 32. Woman, age 55 years. Directly following convales¬ cence from typhoid fever somewhat over two years before admis¬ sion to the hospital, the patient had repeated attacks of severe pain radiating to the centre of the epigastrium with jaundice; neither vomiting nor acholic stools were present; stones in gall¬ bladder. 33. Woman, age 48 years. Typhoid fever one year before the onset of the gall-bladder trouble; vomiting, marked jaundice, chills and fever; stools negative; gall-bladder fdled with stones. 34. Man. Vomiting, slight jaundice, epigastric pain, chills and fever; stones only in gall-bladder and cystic duct. 35. Man. Slight jaundice; vomiting only in first attack; pain over liver extending to the left thigh and also upward; stones in gall-bladder. 36. Woman. Slight jaundice; severe vomiting; pain in right side shooting upwards; calculi in gall-bladder and cystic duct. 37. Woman, age 48 years. Doubtful history of jaun¬ dice; movable kidney; operation for fixation of same revealed through the peritoneum a goodly sized fluctuating swelling at and in front of the lower pole of the kidney, closely simulating a dis¬ tended renal pelvis. Opening the peritoneum, a much dilated gall-bladder was found closely connected with the kidney and filled with stones. 38. Man, age 43 years. Slight vomiting and jaundice with normal colored stools containing numerous calculi. Attacks of severe pain were experienced radiating upwards. A rapidly increasing infiltrating tumor was found on the right side involv¬ ing the abdominal parietes. At operation an ovoidal segment of the abdominal wall was excised to gain safe access to the cavity. Enormous mass of adhesions involving all the neighboring parts. One large imbedded calculus was removed with innumerable 38. Man, age 43 years. Slight vomiting and jaundice with normal colored stools containing numerous calculi. Attacks of severe pain were experienced radiating upwards. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. A rapidly increasing infiltrating tumor was found on the right side involv¬ ing the abdominal parietes. At operation an ovoidal segment of the abdominal wall was excised to gain safe access to the cavity. Enormous mass of adhesions involving all the neighboring parts. One large imbedded calculus was removed with innumerable CHOLECYSTITIS AND CHOLELITHIASIS. 231 minute ones scattered among the adhesions. By microscope no malignancy. 39. Man. Obstructive jaundice due to stones which had ulcerated into the stomach and had been vomited before operation, while others were found in this viscus post-mortem. Enormous dilatation of the stomach existed for which a gastro-enterostomy was done. 40. Woman, age about 50 years. Came to me with a correct diagnosis of eidargcd stomach due to gastric ulcer, or possibly malignant disease, owing to the detection of a resisting mass in the right hypochondrium. There was an obscure history of what might have been gall-bladder trouble. Operation showed a pylorus and duodenum almost occluded by the adhesions, and an enlarged gall-bladder crammed with stones. Owing to the feebleness of the patient and the primary demand for relief of the pyloric stenosis only a gastroenterostomy was done. 40. Woman, age about 50 years. Came to me with a correct diagnosis of eidargcd stomach due to gastric ulcer, or possibly malignant disease, owing to the detection of a resisting mass in the right hypochondrium. There was an obscure history of what might have been gall-bladder trouble. Operation showed a pylorus and duodenum almost occluded by the adhesions, and an enlarged gall-bladder crammed with stones. Owing to the feebleness of the patient and the primary demand for relief of the pyloric stenosis only a gastroenterostomy was done. 41. Man, age 65 years. Showed symptoms of intestinal obstruction for only forty-eight hours before operation. Opera¬ tion by a colleague showed that the obstruction was due to a biliary calculus two inches in diameter. He was never supposed to have had biliary disease but had had “ stomach trouble ” for some undetermined period before this fatal illness. 41. Man, age 65 years. Showed symptoms of intestinal obstruction for only forty-eight hours before operation. Opera¬ tion by a colleague showed that the obstruction was due to a biliary calculus two inches in diameter. He was never supposed to have had biliary disease but had had “ stomach trouble ” for some undetermined period before this fatal illness. 42. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. Woman, age 47 years. She absolutely denied, after repeated questioning, that she had had any form of illness before the attack initiating her present illness. She was a cultured woman and denied jaundice, abnormal stools, pain or discomfort, until about one year previous to the time when sbe came under my care, when an abscess rapidly formed one inch to the left of the umbilicus which had been opened by her attendant, evacuat¬ ing plain pus. The resultant sinus suggested a small fecal fistula due to ulceration of the bowel in a possible umbilical hernia, but nothing except pus was ever detected. Operation showed a sinus tract extending upwards for about three inches, directed towards the gall-bladder, in which were found a number of biliary calculi. No evidences of bile were found during the operation or the course of her rapid convalescence. 43. Man, age 45 years. Had passed gall-stones on sev¬ eral occasions after attacks of biliary colic, but still bad repeated attacks of pain, vomiting, etc. Operation showed that bis last attacks could not have been due to the passage of gall-stones, CHARLES B. G. DE NANCREDE. 232 because the cystic duct was obliterated. Among the dense mass of adhesions a medium sized calculus was found firmly grasped by a shrunken gall-bladder which contained no bile. The gall¬ stones, which had been previously evacuated in the stools, had evidently ulcerated into the colon, as shown by tbe conditions found at the operation. 44. Woman, age 47 years. Twenty-nine years ago had severe pain in the right side, in hepatic region, lasting two hours, which radiated to the region of the stomach and into the back. Patient had had similar attacks ever since at intervals of six months; sometimes these attacks will recur daily. Was entirely free for a period of five years. Has never been jaundiced. Stools normal. Never vomited. At operation the fundus was found to be thickened and was removed with a large portion of the gall-bladder, after extracting numerous stones. Pathological report carcinoma. I11 the notes of the cases mentioned, one must be struck by the absence of many symptoms usually deemed to be almost universally present in the classes of cases described. Roughly analyzing the symptoms presented by these, with those noted in other cases taken at random from old hos¬ pital and private records I have found the following state¬ ments warranted. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. As was to be expected from the prob¬ ability of infection attacking the smaller bile ducts, because of the passage through them of infected bile at some time during the numerous recurrences of the trouble, jaundice was present in seventy-five per cent, of the cases, but in about one-third of these no calculi existed anywhere in the biliary apparatus. In about one-third the jaundice was prac¬ tically continuous, but of this one-third more than half were not cases of biliary lithiasis. The evidence of the actual presence of jaundice in a certain number of cases was doubt¬ ful, resting solely on the alleged yellow staining of the con¬ junctiva, which was in some cases declared to be still present by the medical attendant when it was patently absent to my own eyes, and no biliary constituents could be detected in the urine. I11 this connection too much emphasis cannot be laid CHOLECYSTITIS AND CHOLELITHIASIS. 233 upon tlie fact that a gall-bladder crammed with stones, pro¬ vided catarrhal or infective cholangeitis does not occur, need never throughout the lifetime of a patient give rise to the slightest jaundice, hence the absence of this symptom does not exclude the presence of gall-stones, etc. In about one-sixth of the cases vomiting occurred dur¬ ing the majority of the attacks, while in one-third of the cases studied emesis was only occasional, in some being only present during the first attack. The number of cases whose notes were sufficiently full to draw any definite conclusions from arc too small to lay any great stress upon the percentages given, but they do serve to show the actual happenings in the practice of one surgeon during a given period. Acholic stools were determined in a little more than one-fourth of the cases, and only in one-eighth of these acholic cases were calculi ever detected in the stools. Chills, fever and sweats occurred in almost one-third of the cases, while in the remaining two-thirds these symp¬ toms were positively excluded, or had not been recognized by the patient as pronounced enough to be recalled. Less than half of those suffering from these aguish paroxysms (so commonly thought to be due to common duct stone) had calculi so located, or even duct obstruction from other causes. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. No attempt is here made to discredit the value of these symptoms as usually indicative of choledoch obstruc¬ tion, most often from stone, but numerous cases in my prac¬ tice illustrate the undoubted fact that these symptoms are merely evidences of an infectious process so located that its products can be readily absorbed, so that severe cholecystitis or gangrene of the gall-bladder with cholangeitis may pro¬ vide the necessary amount of toxic substances and also the jaundice and acholic stools. The location of the pain experienced during an attack of gall-stone colic is an interesting study. While this point has not been rigorously determined in all the cases upon which this paper is founded, you will recall that in the notes CHARLES B. G. DE NANCREDE. 234 of a number of those read the pain has been located as fol¬ lows; over the liver; over the liver and epigastric region; over the liver and abdomen; the appendix region; the right hypochondriutn; on the opposite side of the abdomen; on the right side; on the right side not passing beyond the median line; extending from the hepatic region down into the right thigh as well as somewhat upwards; in the epi¬ gastrium alone; over the right side and extending upwards; while in only a few cases did the patient describe the pain as commencing in the hepatic region and extending up to the right shoulder, or back of the neck, hence, the absence of the “ characteristic pain ” believed in by the laity and by many of the profession is of little moment. Again, an interesting query arises in connection with the uncertain location and character of the pain. What then is hepatic colic? Is it always due to the passage of a cal¬ culus? This question is sometimes difficult to determine. In a considerable proportion of these cases, frequent, even daily attacks of pain, perhaps attended with colic were ex¬ perienced, where no stones were present, or where stones were absolutely fixed by the contracted gall-bladder walls and dense adhesions. Sometimes these attacks were what might be called atypical, but I am convinced from my whole experience that during an attack of biliary colic, it is vastly more likely that a stone does not pass than that one does. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. Distension of the gall-bladder or common duct from temporary obstruction due to kinking, or ball- valve action of a calculus; slight adhesions or strictures of the ducts; the passage of a small aggregation of choles- terinc crystals or biliary sand; all these inducing spasms of the muscular coats of the bladder or ducts; slight attacks of localized peritonitis; gastric tenesmus induced by adhe¬ sions; intestinal colic from the same causes; distension of the stomach because of spasmodic pyloric obstruction alone, or this with actual dilatation of the stomach; exacerbations of the ulcerating process in the colon, duodenum, or stomach, whereby large stones are often evacuated from the gall- CHOLECYSTITIS AND CHOLELITHIASIS. 235 bladder; all are conditions that are frequently called attacks of gall-bladder colic in addition to the actual expulsion from the gall-bladder, hepatic or common ducts of a calculus. One of two recent cases of supposed gall-bladder disease experi¬ enced frequent attacks of what were considered to be typical biliary colic due to the passage of stones, the attacks some¬ times recurring daily from considerable periods, the pain being located in the right hypochondrium and the vomiting being both violent and prolonged. Abdominal section re¬ vealed a partial intestinal obstruction produced by a dense band of omentum passing from the hepatic flexure of the colon to the abdominal wall, in its passage downwards being firmly adherent to and constricting the ascending colon. Enormous dilatation of the caput coli and appendix had re¬ sulted. The second case on admission had fever, leucocytosis and a painful tumor somewhat above McBumey’s point, but there was tympany between its upper border and the liver, yet upon the strength of an alleged slight jaundice, with severe vomiting early in the attack, commencing as soon as the pain became pronounced, the condition was viewed by some of my assistants as one of cholecystitis with calculi. I operated for appendicitis and found nothing but this. A point of great etiological interest is the fact that seven at least of these patients, perhaps more, had bad very recently, or antedating their biliary trouble by a greater interval, typhoid fever. All know that the gall-bladder is not infrequently inflamed in this disease, and also that typhoid bacilli have been cultivated from the gall-bladder of patients who have had enteric fever many years before—in one instance seventeen years, in another twenty years had elapsed. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. Moreover, nou-viablc typhoid bacilli are not uncom¬ monly found in the substance of gall-stones. I shall now ask certain questions warranted by the facts given in the cases cited and confirmed by many others not mentioned. Why should pain be always felt which radiates to the epigastrium, to the right shoulder, or indeed in any special direction, when operation reveals in many instances CHARLES B. G. DE NANCREDE. 236 that owing to adhesions and perhaps serious ulceration from the pressure of gall-stones, the sources of pain are really not in the biliary apparatus at all, but in the subhepatic and py¬ loric regions? Vomiting is in proportion to the pain, the amount of irritation of the splanchnic system, and the consequent vaso¬ motor paresis resembling shock, and to individual idiosyn- cracy; why then need this be in any way indicative of any¬ thing except the severity of the pain ? If the pain must stop suddenly, and this is a proof of the passage of a calculus, why are certain facts easily demon¬ strable, viz., that in the majority of attacks of so-called biliary colic, the pain subsides rather slowly, no stones are found in the stools, even temporary obstruction of the com¬ mon duct cannot he demonstrated, and at operation it is often patent that none of the stones, or the stone present, could have even commenced to enter the cystic or common duct? It must then be clear that to demand the so-called “ char¬ acteristic pain,” the vomiting, the jaundice, the clay-colored stools, the sudden cessation of the pain and the recovery of the stones from the stools, is unreasonable, and resembles the demand for high fever, sweats, generalized abdominal distension, obstinate constipation, marked vomiting, pain and tumor in a definite locality, and inability to extend the right thigh, which a decade ago many practitioners seemed to think must all be readily detectable, before a diagnosis of appendicitis should be made. Dangerous Sequelae. I have, when reading the notes of cases, or formulating the statements founded on them, called attention to the dangers of cholecystitis and cholangeitis, but for the purposes of emphasis I desire to restate them, premis¬ ing however, that the probabilities of each complication can neither be stated in figures, nor foreseen in any given case. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. Chronic inflammation of the gall-bladder precedes and predisposes to the formation or reformation of gall-stones, with all their dangers. Adhesions to the duodenum, stomach CHOLECYSTITIS AND CHOLELITHIASIS. 237 and colon are common. Compression and obstruction of the common duct may by adhesions lead to chronic cholemia and infective cholangeitis. Gastric adhesions originated by cholecystitis and its sequela; more often produce so-called stomach disease and serious dilatation of this organ than is commonly believed. Should virulent infective organisms reach the chronically diseased gall-bladder, acute suppura¬ tion with rupture, or even gangrene, may result with all its possible sequences. Carcinoma of the gall-bladder is not an uncommon sequence of cholelithiasis. See cases 22, 24, 44. These direful results have nearly all been exemplified in the histories of my cases. Once gall-stones are formed, all the dangers attending cholangeitis are present, with the greater risks of suppura¬ tion, rupture, gangrene, stomachic and intestinal crippling, and ulceration involving the stomach or colon, with all its dangers; dilatation of the stomach; carcinoma; intestinal obstruction from a large stone, perhaps increased in bulk by calcareous intestinal accretions; and fatal hemorrhage, even without operation.1 A greater refinement in analysis might; perchance reveal some other obscure dangers, but what has been already said really embraces all of importance. Diagnosis.—When temporary obstruction of the common duct has occurred on more than one occasion with jaundice, pain—characteristic ( ?) or not—the stools being acholic, aguish paroxysms having been noted with sudden cessation of pain, rapid clearing up of symptoms, and the recovery of calculi from the stools, he who runs may read, but with the irregular symptoms which many cases present the matter is not so easy. A diagnosis can best be made by exclusion. 1 More than one such case has occurred recently at the University Hospital. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. Stomach and duodenal ulcer, gastric carcinoma, neoplastic obstruction of the bowels, acute or chronic pancreatic dis¬ ease, intestinal obstruction by bands, dilated stomach, renal calculi or disease, a dislocated kidney which is sometimes accompanied with jaundice, appendicitis, the gastric crises 1 M h h h d l h U i i Once gall-stones are formed, all the dangers attending cholangeitis are present, with the greater risks of suppura¬ tion, rupture, gangrene, stomachic and intestinal crippling, and ulceration involving the stomach or colon, with all its dangers; dilatation of the stomach; carcinoma; intestinal obstruction from a large stone, perhaps increased in bulk by calcareous intestinal accretions; and fatal hemorrhage, even without operation.1 A greater refinement in analysis might; perchance reveal some other obscure dangers, but what has been already said really embraces all of importance. Diagnosis.—When temporary obstruction of the common duct has occurred on more than one occasion with jaundice, pain—characteristic ( ?) or not—the stools being acholic, aguish paroxysms having been noted with sudden cessation of pain, rapid clearing up of symptoms, and the recovery of calculi from the stools, he who runs may read, but with the irregular symptoms which many cases present the matter is not so easy. A diagnosis can best be made by exclusion. Stomach and duodenal ulcer, gastric carcinoma, neoplastic obstruction of the bowels, acute or chronic pancreatic dis¬ ease, intestinal obstruction by bands, dilated stomach, renal calculi or disease, a dislocated kidney which is sometimes accompanied with jaundice, appendicitis, the gastric crises CHARLES B. G. DE NANCREDE. 238 of locomotor ataxia, and spinal caries must all be consid¬ ered, while examination of the urine often gives valuable information by revealing the unsuspected presence of biliary substances when jaundice has passed away or is too slight for a certain opinion. The association of appendicitis and cholecystitis must not be overlooked. Without going into the differential diagnosis of the affections mentioned, you will readily understand that during these investigations, it is hardly possible not to recognize such conditions as will lead you to investigate the biliary apparatus so rigidly as to arrive at correct conclusions in the absence of all sup¬ posedly pathognomonic symptoms. Again, despite all care, doubt may still exist between gastric and duodenal ulcer, dis¬ ease of the pancreas, and appendicitis, or even a neoplasm. SHOULD CHOLECYSTITIS AND CHOLELITHIASIS BE ANY LONGER CONSIDERED MEDICAL AFFECTIONS, AND WHAT ARE THE USUAL CONSEQUENCES OF SO TREATING THEM?* BY CHARLES B. G. DE NANCREDE, M.D., OF ANN ARBOR, MICII., Professor of Surgery in (he University! of Mlchigaifaml in Dartmouth Medical College. In such cases, because an operation can alone afford relief for any of these conditions, and when one incision will enable the surgeon to deal with any or all of these ailments, if present, an exploration should be made. In conclusion let me again urge, that this paper simply represents the average conditions presented by cases, gath¬ ered at random, not that another scries in my own practice would necessarily present exactly similar complications or symptoms. My object will have been secured, if I shall have induced some of my readers to recognize the existence of many serious hepatic and biliary conditions, which can only be properly met by use of the knife. If these conditions must be recognized early to secure the best results, a diagnosis must be often reached by discarding the group of symptoms usually relied upon, and reaching correct conclusions by a careful analysis of the symptoms, viewed in the light of anatomy, physiology and modern pathology. In certain rare cases, as already said, only an exploration will decide the question.
https://openalex.org/W4285341333	https://ageconsearch.umn.edu/record/317030/files/Geoghegan_eyt_al_BAE_2_2021.pdf	English	null	The effect of farmer attitudes on openness to land transactions: evidence for Ireland	Bio-based and applied economics	2,021	cc-by	12,527	Bio-based and Applied Economics BAE Bio-based and Applied Economics BAE Bio-based and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 Copyright: © 2021 C. Geoghegan, A. Kinsella, C. O’Donoghue. Open access, article published by Firenze University Press under CC-BY-4.0 License. Firenze University Press \| www.fupress.com/bae Citation: C. Geoghegan, A. Kinsel- la, C. O’Donoghue (2021). The effect of farmer attitudes on openness to land transactions: evidence for Ireland. Bio- based and Applied Economics 10(2): 153-168. doi: 10.36253/bae-9746 Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue Cathal Geoghegan, Anne Kinsella, Cathal O Donoghue Teagasc, the Irish Agriculture and Food Development Authority; National University of Ireland, Galway Teagasc, the Irish Agriculture and Food Development Authority; National University of Ireland, Galway C d h l h l h l Teagasc, the Irish Agriculture and Food Development Authority; National University of Ireland, Galway Received: September 16, 2020 Accepted: June 23, 2021 Published: October 28, 2021 Corresponding author. E-mail: cathal.c.geoghegan@nuigalway.ie Abstract. Ireland suffers from very low levels of farmland mobility by European stand- ards. This paper examines the role of attitudes in farmers’ openness toward land trans- actions using a nationally representative survey of Irish farmers across the major farm systems. The results show that attitudinal factors are a significant predictor of open- ness to land mobility, both on the supply and demand side of the market. Additionally, there appears to be a greater demand amongst farmers for temporary land transactions such as land leasing arrangements than is currently seen in at market level. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Competing Interests: The Author(s) declare(s) no conflict of interest. Keywords: land markets, attitudes, Ireland. ORCID CG: 0000-0002-7023-6313 AK: 0000-0001-5670-9025 COD: 0000-0003-3713-5366 Give to AgEcon Search The World’s Largest Open Access Agricultural & Applied Economics Digital Library This document is discoverable and free to researchers across the globe due to the work of AgEcon Search. Help ensure our sustainability. AgEcon Search http://ageconsearch.umn.edu aesearch@umn.edu Papers downloaded from AgEcon Search may be used for non-commercial purposes and personal study only. No other use, including posting to another Internet site, is permitted without permission from the copyright owner (not AgEcon Search) or as allowed under the provisions of Fair Use U S Copyright Act Title 17 U S C AgEcon Search http://ageconsearch.umn.edu aesearch@umn.edu Papers downloaded from AgEcon Search may be used for non-commercial purposes and personal study only. No other use, including posting to another Internet site, is permitted without permission from the copyright owner (not AgEcon Search), or as allowed under the provisions of Fair Use, U.S. Copyright Act, Title 17 U.S.C. Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue nomically viable size and protecting the family farming model which accounts for 97 per cent of European farms (Davidova & Thomson, 2014; Hennessy, 2014). Enabling land to change hands but also maintaining local connec- tions to rural areas requires a nuanced policy response. Individual member states have tried to balance these policy priorities by giving young, local farmers first refusal when land becomes available locally or by pro- viding brokerage type services between young and retir- ing farmers (Ingram & Kirwan, 2011; Piet et al., 2012). has concentrated on the use of stated intentions sur- veys to accomplish this task by asking what the farmer will do in the future (Breen et al., 2005; Lobley & But- ler, 2010). However, given the static history of the Irish farmland market and stable policy conditions, it is not anticipated that many Irish farmers outside the dairy sector intend to change their current land allocation. Therefore, a more exploratory analysis is required to identify farmers who would be open to land market par- ticipation. h In this context, the abolition of the quota on milk production in 2015 presents an opportunity for structur- al change in European agriculture (Dervillé et al., 2016; Groenveld et al., 2016). The abolition of quota means that for farmers looking to increase production, land rather than quota rights will be the scarcest production factor (Boere et la., 2015). The potential for farmers to adapt to a post-quota landscape depends on many fac- tors including demographics, socio-economic charac- teristics and the availability of inputs (Chevalier et al., 2012; Kempen et al., 2011). Land is an input of particu- lar importance, especially in an Irish context. Irish dairy farming depends upon a grass-based rather than feed- based production system, meaning a sufficient supply of land is necessary to increase dairy production (Dil- lon et al., 2008). However, Ireland suffers from very low levels of land mobility by European standards (Ciaian et al., 2010). This means that accessing extra land for dairy farming may prove difficult. Despite efforts from poli- cymakers to encourage long-term leasing arrangements, most rented land in Ireland is accessed through short- term, 11-month “conacre” contracts, unsuitable for the long-term infrastructural provisions that are required by dairy farmers (O’Neill & Hanrahan, 2012). This study therefore aims to ask three main ques- tions: • Are farmers open to entering the land market? Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue Generally speaking, farmers can either supply or demand land. This study will focus on farmers’ openness to selling or leasing out land on the supply side and buying or leasing in land on the demand side. This contributes to the literature by quantifying land demand and supply in a context where market information is either missing or incomplete. • What distinguishes famers who are open to entering the land market from those that are not? In addi- tion to agronomic and socioeconomic differences, do farmers interested in land transactions approach farming from a different attitudinal standpoint than farmers uninterested in land mobility? Given the desire of policymakers to encourage long- term leasing amongst Irish farmers, are there dif- ferences between farmers interested in leasing and those interested in permanent transactions such as buying and selling?h • Given the desire of policymakers to encourage long- term leasing amongst Irish farmers, are there dif- ferences between farmers interested in leasing and those interested in permanent transactions such as buying and selling?h This paper is structured as follows. Section 2 looks at the policy context of land mobility in greater detail, paying specific attention to the Irish situation. Section 3 deals with the methodology and data used in the study. Section 4 looks at the results of logistic regressions examining farmers’ openness to entering the land mar- ket while Section 5 provides a discussion of the results and their impact on policy. Irish land markets have traditionally been quite static, with land rarely changing hands. The dominant means of transfer of ownership is through non-market arrangements, usually inheritance, which is often attrib- uted to the strong emotional attachment to land in Ire- land (Donnellan et al., 2008). Rented land (both conacre and long-term leasing) only accounts for 18% of Utilis- able Agricultural Area (UAA) in Ireland (Geoghegan & O’Donoghue, 2018). Due to the illiquid land market in Ireland, little information exists about what drives agricultural land transactions. This is especially true in relation to the supply of land. This study attempts to fill this information gap by examining the attitudes of Irish farmers to agricultural land mobility. 1. INTRODUCTION Land mobility is becoming an increasingly important issue for European agriculture. The enhanced market orientation of European agriculture and reduced reliance on subsidies requires farmers to be more efficient in their use of factors of production. This is coupled with European farmers getting older on average and the need amongst young European farmers for access to land (Davidova & Thomson, 2014; Zondag, 2015). Access issues are further complicated by the increasing land concentration in Europe, with more land being held by fewer farmers (Kay et al., 2015; van der Ploeg et al., 2015). Agricultural land transactions in Europe occur within a range of nation- al institutional and regulatory environments (Ciaian et al., 2010; Ciaian et al., 2012; Needham et al., 2011). One consequence of these diverse land gov- ernance frameworks is that land sales and land rental markets may operate uniquely from country to country. Despite an integrated agricultural market and the longstanding Common Agricultural Policy, the share of rented land varies between 20 and 80 per cent across the EU (Ciaian et al., 2010). Prefer- ences for land ownership over land rental or vice versa have been linked to capital market imperfections, farm profitability and government regulations (Swinnen et al., 2016). At an EU policy level, tension exists between encouraging land mobil- ity so as to enable the structural change required for farms to reach an eco- 154 Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue 2. POLICY CONTEXT AND RELATED LITERATURE Only dairy farming has been consistently profitable, on aver- age, over recent years. Land rental market simulation modelling by Loughrey and Hennessy (2019) suggests that a land market based solely on farm profit maximisa- tion would lead to significant increases in farm size con- centration with dairy and tillage farms growing at the expense of cattle and tillage. Bogue (2013) found that three-quarters of farmers with no successor would consider renting out land on either a long or short-term basis when they themselves were no longer able to farm at their current level. This compared with 28% of farmers who would consider sell- ing their land in the same situation. Banovic et al. (2015) found general support for policy measures incentivising land mobility amongst Irish farmers but also found that surveyed farmers were reluctant to take advantage of the policy measures themselves. g Most Irish farms are owner-occupied, with the land owner generally being the farm operator (Don- nellan et al., 2008). Farm ownership generally transfers through inheritance, with a single family member usu- ally inheriting the intact farm structure. As a result, farmland rarely comes onto the open market. Land mobility outside of intra-family transfer is depend- ent upon land rental and sales markets. Attempts have been made at a policy level to increase land mobility in Irish agriculture, most notably the introduction of tax exemptions to incentivise the long-term leasing of land. Traditionally, land has been rented in Ireland on a short- term, 11-month basis. To encourage longer term leas- ing agreements, tax incentives were first introduced in 1985. These incentives allowed income derived from the long-term leasing of land (minimum of five years) to be exempt from income tax up to specified limits. Over time, these exemption limits have increased, with higher limits being added for leases of longer periods. By 2015, up to €40,000 per year can be earned free of income tax for leases of 15 years or longer. Other policy measures to encourage land mobility have been introduced includ- ing stamp duty exemptions for young farmers acquir- ing land, the promotion of farm partnerships and capital gains relief to encourage land consolidation (Macra na Feirme, 2015; DAFM, 2018). O’Neill and Hanrahan (2012) examined Irish farm- ers’ land market decisions from the perspective of the decoupling of agricultural support payments from agri- cultural production. 2. POLICY CONTEXT AND RELATED LITERATURE A lack of land mobility has long been seen as an impediment to structural change in Irish agriculture (Commins, 2001; Inter-Departmental Committee on Land Structure Reform, 1978; Maguire, 1983). Currently, the issue of land mobility is of interest to policymakers in light of public policy commitments to increase the output of Irish agriculture in the coming years (DAFF, 2010; DAFM 2015). One particular commitment is to increase dairy output by 50% by the year 2020, with Given the lack of information about the characteris- tics of farmers who participate in land markets, an ex- ante approach is proposed to determine which types of farmers are open to land transactions. Previous research Bio-based and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 155 The effect of farmer attitudes on openness to land transactions: evidence for Ireland of land transfer (Hennessy & Rehman, 2007; Kennedy, 1991). It has been found that policy instruments incen- tivising either the early retirement of older farmers or the installation of younger farmers on farms have had limited success in increasing the level of land mobility (Bika, 2007; Gillmor, 1999). Land mobility studies in Ire- land outside succession and inheritance processes have been relatively rare. Conway (1986) studied land leasing practices in the west of Ireland and found that although potential lessees were willing the pay more for land than the prevailing rate, potential lessors were generally not interested in leasing out land as long as they were able to continue farming the land themselves. Jenkins (1997) found in a study of leasing activity in the south-east of Ireland that commercial tillage2 and non-local3 farmers were predominant in the rental market, with land being supplied by older farmers operating smaller farms. sectoral growth expected to continue beyond that date1. Achieving this target, as well as future growth, will require the acquisition of additional land by dairy farm- ers (Dillon et al., 2008; Geoghegan & O’Donoghue, 2018; Läpple & Hennessy, 2012). Currently, cattle farming is the dominant form of agriculture in Ireland, accounting for 57% of land (Geoghegan & O’Donoghue, 2018). Dairy farming accounts for 14.9% of agricultural land, with sheep farming taking place on 12.3% of land. Despite making up the largest share of farmland usage in Ireland, the average cattle farm has consistently returned negative market incomes over recent years and is dependent upon subsidies for survival (Hennessy & Moran, 2016). 1 The 50% increase is compared to the output of the average of total production between 2008 and 2010. This target was achieved in 2018, two years ahead of schedule. 2 Specialist tillage farmers for who tillage accounts for at least two-thirds of the farm’s total standard gross margin. 3 Greater than 10 kilometres away. Bio-based and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 2. POLICY CONTEXT AND RELATED LITERATURE Following decoupling, Irish farmers are required to maintain the area of land on which they claim their single payment in a state fit for agricultural production although actual production is not required. The authors found that decoupling led to a modest reduction in net land rental on average but a lack of information on consolidation, where famers whose land rental agreements had expired could transfer payments from areas where they no longer rented to land that they still possessed, made the true impact of decoupling on land decisions difficult to assess. Due to the low number of transactions in Irish land markets, as well as the lack of literature in the area, little information exists about the characteristics and attitudes of Irish farmers who enter the land market. Therefore, this study includes a wide range of factors which may drive willingness to enter the land market. As well as structural and socio-economic factors, farmer attitudes are considered. Farmer behaviour has been shown to be Studies concerning land mobility in Ireland have mostly focused on the succession and inheritance aspect and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue 156 affected by a multiplicity of farming goals and attitudes (Willock et al., 1999a). There is a large literature on the attitudes and objectives of farmers and the impact of these on farming behaviour with farming attitudes being identified as important to risk aversion, innova- tion, diversification, off-farm work, environment, pro- duction, management, legislation, stress, pessimism and satisfaction toward farming (Willock, 1999b provides a review of the literature). Non-economic objectives such as farmer lifestyle have also been shown to strongly affect farmer decisions (Howley et al., 2015; Marr et al., 2019). Studies related to attitudes of farmers toward land have tended to focus on land use, especially in terms of environmental issues (Mills et al., 2013; Wilson, 1996). Given the absence of information about farmers in the Irish land market, these factors will provide a sense of what drives farmers in their consideration of land trans- actions. another, orthogonal (uncorrelated) linear combination that explains the maximum amount of remaining vari- ance (“second principal component”), and so on until all variance is explained (Hamilton, 2013). PCA thus serves as a data reduction technique, allowing the anal- ysis of the attitudinal statements to be simplified. 3. CASE STUDY AND METHODOLOGY This paper uses a nationally representative survey to examine the willingness of Irish farmers to engage in a land transaction. Given the binary nature of this propo- sition (the farmer either does or does not want to engage in the transaction), a logit model is utilised. Four logit models are used to examine the willingness of farmers to lease land in, lease land out, buy land and sell land. One difficulty with interpreting non-linear models such as the logit is that unlike linear models, an explanatory variable’s coefficient does not equal its marginal effect. A given change in an explanatory variable x will usually have less effect when the response probability P(y = 1\|x) is near the extreme values of zero or one as compared with middle values. Therefore, this study uses odds ratios to interpret the marginal effects of the explanatory variables. Odds ratios in logit models can be interpreted as the effect of a one unit change in x in the predicted odds ratio with the other variables in the model held constant. The odds of P(y = 1\|x) increase multiplicatively by eβfor a one unit increase in x, holding all other vari- ables constant. fi The respondents were asked questions based on three different areas: current farm characteristics; atti- tudes to land, farming and future plans; and knowledge about land-based policy initiatives. The survey also con- tained 15 attitudinal questions using a four-level Lik- ert scale ranging from “strongly disagree” to “strongly agree”. The respondents could also choose a “don’t know” option. There is little consensus regarding the correct number of response options or whether an odd number of response options should be used in order to allow a neutral, midpoint response (Sturgis et al., 2014). In the context of this study, it has been found that 4-point scales (as used here) yield similar levels of reli- ability compared to 5-pont scales which would contain a midpoint (Alwin, 2007). Neutral, midpoint responses can also represent hidden “don‘t know” answers (Stur- gis et al., 2014). Therefore, it was decided to use a 4-point scale with an additional “don‘t know” option. In order to determine the attitudinal orientation associated with farmers in the sample, a set of attitudi- nal statements was included as part of the survey ques- tionnaire. Principal Component Analysis (PCA) is used to identify underlying structural relationships between farmer responses to these attitudinal statements. 2. POLICY CONTEXT AND RELATED LITERATURE Each principal component has an eigenvalue, which repre- sents the standardised variance explained by the com- ponent. Principal components with values of less than one eigenvalue explain less than the equivalent of one variable’s variance so are set aside for purpose of analy- sis (Abdi & Williams, 2010). Following the PCA, vari- max orthogonal rotation is used to further simplify the factor structure. 4. DATA This paper’s analysis of farmer attitudes to land mobility is based on a survey of 837 Irish farmers in 2014 and 2015. The survey used random probability sampling to survey a representative number of farmers from each county in Ireland. In order to achieve a repre- sentative geographical spread, a starting point was ran- domly selected in each county with every third farmer being selected to participate in the study. The survey continued in each county until a quota of respondents in each county was reached. Quota sampling set demo- graphic quotas on the sample based on known popula- tion distribution figures. The quotas used here were based on known population distribution figures in rela- tion to specific farm systems (dairy, cattle rearing, cattle other, sheep, tillage and mixed) taken from Central Sta- tistics Office data (CSO, 2012) The effect of farmer attitudes on openness to land transactions: evidence for Ireland 157 The effect of farmer attitudes on openness to land transactions: evidence for Ireland The effect of farmer attitudes on openness to land transactions: evidence for Ireland Table 1. Mean scores and percentage agreement with attitudinal statements. Mean scores Percentage completely agreeing It is important not to leave farm land idle 1.50 55.4 It is important for me to pass on my land in as good a shape or better than I received it 1.46 53.4 I enjoy farming much more than I would other potential sources of employment 1.32 48.1 Farming is a more rewarding job in terms of quality of life, independence and lifestyle than it is in terms of money 1.20 44.6 It is important not to be afraid of adopting new farming practices 1.15 32.4 I have to keep my farm running to ensure I have something to pass on to my children/next generation 1.13 42.1 To be successful in farming it is important for me to adapt and use new technologies (whether agri or non-agri technologies) 1.10 33.5 It is important to visit other farms to look at their methods 1.03 36.2 I don’t think it is a good idea to take too many risks when it comes to farming 1.00 30.5 It is important for me to be respected by other farmers 1.00 30.6 I am good at finding different types of information to help me run my business 0.97 28.5 Agricultural land in Ireland is under-utilised 0.48 20.2 I am cautious about adopting new ideas and farm practices 0.37 16.6 My economic future on this present farm is bright 0.36 16.9 It makes more sense for me to join an agricultural scheme if my neighbours are also joining 0.13 17.4 Table 1. Mean scores and percentage agreement with attitudinal statements. It is important not to leave farm land idle form of transaction. Of the 837 farmers surveyed, 47% were willing to buy land, while 26% were willing to sell. In terms of leasing, 51% were willing to lease in land, while 29% were willing to lease out land. A logis- tic regression model is used to examine the probability of a farmer being open to each land transaction. The dependent variable is the willingness to engage in the land transaction (purchase land, sell land, lease land in, lease land out). The effect of farmer attitudes on openness to land transactions: evidence for Ireland The statements drew on previous work examin- ing Irish farmers’ attitudes toward farming decisions (Howley & Dillon, 2012; Howley et al., 2015), as well as input from experts. The attitudinal statements are listed in Table 1, as well as the mean scores and percentage of respondents completely agreeing with of each statement. Mean scores were computed by assigning a score to each level of agreement (2 for “strongly agree”, 1 for “agree”, -1 for “disagree” and -2 for “strongly disagree”) and averag- ing the scores of the participants for each statement. Besides the attitudinal variables described previ- ously, explanatory variables utilised in the model include variables representing the farmer’s age, plans for future farm production, whether the farmer has children or not, the presence/absence of a successor to take over the farm business, whether the farmer has an off-farm job or not, market farm income, value of entitlements, land prices, the percentage of household income derived from the farm business. Farm structure and agronomic vari- ables such as farm size, farm system, soil type and stock- ing rate are also included (see Table 4). The final specifi- cations of the regression models are shown in Tables 5 and 6. In most cases, respondents chose from a range of values rather than state exact values, so variables based on instances where respondents chose from a range of variables are treated as categorical variables. For cate- gorical variables such as age and farm size, the reference categories are the categories most frequently chosen by respondents. For age, this is the 51-64 years category and for farm size, the 20-49 ha category. Four principal components with eigenvalues above one were generated by the PCA and rotation process, representing different attitudes toward farming among the respondents. These attitudes are related to the importance of innovation in farming, optimism about the future of agriculture, the non-economic benefits of farm work and conservatism regarding the farm busi- ness. A description of the attitudinal variables is avail- able in Table 2. The factor loadings for each attitudinal statement onto the four principal components can be found in Table 3. Respondents were asked about their openness to four forms of land transaction: i. Land purchase; ii. Land sale; iii. Land lease in; iv. Land lease out. 3. CASE STUDY AND METHODOLOGY PCA finds the linear combination that explains the maxi- mum amount of variance among the observed variables – called the “first principal component”. It also finds Bio-based and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 157 The effect of farmer attitudes on openness to land transactions: evidence for Ireland Statement Pleasure of farming Innovative Agri- optimistic Conservative Agricultural land in Ireland is under-utilised 0.1666 0.3447 0.2091 0.0107 I enjoy farming much more than I would other potential sources of employment 0.4027 0.0196 0.5615 0.0332 I am good at finding different types of information to help me run my business 0.1075 0.4731 0.5335 0.0634 My economic future on this present farm is bright 0.0619 0.285 0.662 0.0321 To be successful in farming it is important for me to adapt and use new technologies (whether agri or non-agri technologies) 0.1398 0.5872 0.2708 0.036 I have to keep my farm running to ensure I have something to pass on to my children/ next generation 0.5969 0.2275 0.2029 0.0479 I am cautious about adopting new ideas and farm practices 0.0407 0.0712 0.1231 0.7262 It is important for me to be respected by other farmers 0.0744 0.2416 0.3484 0.5341 It makes more sense for me to join an agricultural scheme if my neighbours are also joining 0.0508 0.2087 0.4206 0.4901 Farming is a more rewarding job in terms of quality of life, independence and lifestyle than it is in terms of money 0.5927 -0.119 0.3865 0.0128 It is important for me to pass on my land in as good a shape or better than I received it 0.6827 0.2444 0.1274 0.0055 It is important to visit other farms to look at their methods 0.1252 0.7124 0.1445 0.1457 It is important not to be afraid of adopting new farming practices 0.1569 0.7693 0.0685 0.0285 It is important not to leave farm land idle 0.6557 0.3514 0.0997 0.0135 I don’t think it is a good idea to take too many risks when it comes to farming 0.2029 0.0144 0.1156 0.6099 Initial eigenvalues 2.29 2.23 1.73 1.55 tare CAP payments by farm system from the 2014 NFS in place of the missing values. Since per hectare CAP payments are closely related to farm system in Ireland, this seemed the most appropriate solution. Average land value and rental prices were sourced for the time period from the Irish Central Statistics Office (CSO) for land prices and Eurostat for land rental values, which are based on data from FADN. The data was available at the NUTS3 regional level (8 regions in Ireland) for land val- ues and NUTS2 level (3 regions) for rental prices. The effect of farmer attitudes on openness to land transactions: evidence for Ireland They agree strongly with statements such as “Farming is a more rewarding job in terms of quality of life, independence and lifestyle than it is in terms of money” and “It is important for me to pass on my land in as good a shape or better than I received it”. Farmers with a high ranking in this variable prefer to rely on traditional farming practices and dislike change. They agree strongly with statements such as “I don’t think it is a good idea to take too many risks when it comes to farming” and “I am cautious about adopting new ideas and farming practices”. Farmers with a high ranking in this variable are optimistic about the future of agriculture and enjoy being farmers. They agree strongly with statements such as “My economic future on this present farm is bright” and “I enjoy farming much more than other potential sources of employment”. Table 3. Factor loadings of attitudinal statements. The effect of farmer attitudes on openness to land transactions: evidence for Ireland Statement Pleasure of farming Innovative Agri- optimistic Conservative Agricultural land in Ireland is under-utilised 0.1666 0.3447 0.2091 0.0107 I enjoy farming much more than I would other potential sources of employment 0.4027 0.0196 0.5615 0.0332 I am good at finding different types of information to help me run my business 0.1075 0.4731 0.5335 0.0634 My economic future on this present farm is bright 0.0619 0.285 0.662 0.0321 To be successful in farming it is important for me to adapt and use new technologies (whether agri or non-agri technologies) 0.1398 0.5872 0.2708 0.036 I have to keep my farm running to ensure I have something to pass on to my children/ next generation 0.5969 0.2275 0.2029 0.0479 I am cautious about adopting new ideas and farm practices 0.0407 0.0712 0.1231 0.7262 It is important for me to be respected by other farmers 0.0744 0.2416 0.3484 0.5341 It makes more sense for me to join an agricultural scheme if my neighbours are also joining 0.0508 0.2087 0.4206 0.4901 Farming is a more rewarding job in terms of quality of life, independence and lifestyle than it is in terms of money 0.5927 -0.119 0.3865 0.0128 It is important for me to pass on my land in as good a shape or better than I received it 0.6827 0.2444 0.1274 0.0055 It is important to visit other farms to look at their methods 0.1252 0.7124 0.1445 0.1457 It is important not to be afraid of adopting new farming practices 0.1569 0.7693 0.0685 0.0285 It is important not to leave farm land idle 0.6557 0.3514 0.0997 0.0135 I don’t think it is a good idea to take too many risks when it comes to farming 0.2029 0.0144 0.1156 0.6099 Initial eigenvalues 2.29 2.23 1.73 1.55 Table 2. Description of attitudinal variables. Description Farmers with a high ranking in this variable acknowledge the importance of technology and new ideas with regard to farming. They agree strongly with statements such as “It is important not to be afraid of adopting new farming practices” and “I am good at finding different types of information to help me run my business”. Farmers with a high ranking in this variable emphasise the non-economic benefits of farming, especially compared to non-farming employment. They are also concerned with their farming legacy. The effect of farmer attitudes on openness to land transactions: evidence for Ireland Respondents replied either “Yes” or “No” in terms of whether they were ever willing to engage in each Bio-based and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 158 Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue Farm income and CAP entitlements payment data was collected as part of the survey but about 30% of the sample decided not to answer. In order to include farm income data, the missing information was replaced tare CAP payments by farm system from the 2014 NFS in place of the missing values. Since per hectare CAP payments are closely related to farm system in Ireland, this seemed the most appropriate solution. Average land Table 2. Description of attitudinal variables. Attitudinal variable Description Innovative orientation Farmers with a high ranking in this variable acknowledge the importance of technology and new ideas with regard to farming. They agree strongly with statements such as “It is important not to be afraid of adopting new farming practices” and “I am good at finding different types of information to help me run my business”. Pleasure of farming orientation Farmers with a high ranking in this variable emphasise the non-economic benefits of farming, especially compared to non-farming employment. They are also concerned with their farming legacy. They agree strongly with statements such as “Farming is a more rewarding job in terms of quality of life, independence and lifestyle than it is in terms of money” and “It is important for me to pass on my land in as good a shape or better than I received it”. Conservative orientation Farmers with a high ranking in this variable prefer to rely on traditional farming practices and dislike change. They agree strongly with statements such as “I don’t think it is a good idea to take too many risks when it comes to farming” and “I am cautious about adopting new ideas and farming practices”. Agri-optimistic orientation Farmers with a high ranking in this variable are optimistic about the future of agriculture and enjoy being farmers. They agree strongly with statements such as “My economic future on this present farm is bright” and “I enjoy farming much more than other potential sources of employment”. Table 3. Factor loadings of attitudinal statements. Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue The effect of farmer attitudes on openness to land transactions: evidence for Ireland Farm income and CAP entitlements payment data was collected as part of the survey but about 30% of the sample decided not to answer. In order to include farm income data, the missing information was replaced using farm income information from the 2014 Teagasc National Farm Survey (Hennessy & Moran, 2014), a yearly, nationally representative survey of Irish farmers which is Ireland’s contribution to the Farm Accountan- cy Data Network (FADN). For CAP payments data, the missing data was replaced by assigning average per hec- Bio-based and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 The effect of farmer attitudes on openness to land transactions: evidence for Ireland 159 Table 4. Independent variables of land mobility model. Variable Description Mean/ Mode1 Standard Deviation Innovative orientation (Innovative)2 Factor variable measuring degree to which farmer feels technology and new ideas are important. 0 1 Pleasure of farming orientation (Pleasure of Farming)2 Factor variable measuring degree to which farmer enjoys farming as opposed to other occupations. 0 1 Agri-optimistic orientation (Agri Optimistic)2 Factor variable measuring degree to which farmer feels optimistic about the future of their farm. 0 1 Conservative orientation (Conservative)2 Factor variable measuring degree to which farmer is cautious about risk-taking and new ideas. Table 4. Independent variables of land mobility model. Average regional per hectare farmland price ave mean zero as each variable is standardised to mean zero as part of the PCA process. p p 3 Farm size share by percentage: <10ha – 6%, 10-19ha – 20%, 20-49ha – 44%, 50-74ha – 15%, 75-99ha – 9%, 100-149ha – 4%, 150+ha – 2%. 4 Soil quality is self-reported but definitions of each soil type were provided to aid respondents. p p 3 Farm size share by percentage: <10ha – 6%, 10-19ha – 20%, 20-49ha – 44%, 50-74ha – 15%, 75-99ha – 9%, 100-149ha – 4%, 150+ha – 2%. 4 Soil quality is self-reported but definitions of each soil type were provided to aid respondents. 5 Farm system share by percentage: dairy – 22%, cattle rearing – 14%, cattle other – 29%, tillage – 11%, sheep – 15%, mixed – 5%, other – 3%. 6 Cattle other refers to cattle finishing farms where cattle are fattened up in preparation for slaughter. 7 Mixed refers to farms that combine grazing livestock and field crops. q y pi yp p p 5 Farm system share by percentage: dairy – 22%, cattle rearing – 14%, cattle other – 29%, tillage – 11%, sheep – 15%, mixed – 5%, other – 3%. 6 Cattle other refers to cattle finishing farms where cattle are fattened up in preparation for slaughter. 7 Mixed refers to farms that combine grazing livestock and field crops. The effect of farmer attitudes on openness to land transactions: evidence for Ireland 0 1 Farm Size3 Number of hectares farmed in 2014 (<10 ha, 10-19 ha, 20-49 ha, 50-74 ha, 75–99 ha, 100-149 ha, 150+ ha) 20-50ha1 N/A Soil Quality4 Description of soil type on land (good soil, medium soil, poor soil) Good1 N/A Increase Future Production Plans for farming over the next five years (aim to increase production, maintain current levels of production) 0.17 0.38 Decrease Future Production Plans for farming over the next five years (aim to decrease production, maintain current levels of production) 0.09 0.28 Diversify Future Production Plans for farming over the next five years (aim to increase diversification, maintain current levels of production) 0.09 0.28 Stocking Rate Number of livestock units (LUs) per hectare 1.5 1.76 Farm System5 Main farm activity (dairy, cattle rearing, cattle other6, tillage, sheep, mixed7, other) Cattle other1 N/A Age Age in years (<35 years, 35-44 years, 45-50 years, 51-64 years, 65+ years) 51-64* N/A Children Does the farmer have any children (Yes, No) 0.71 0.45 Successor Has the farmer identified a successor (Yes, No) 0.43 0.50 Household Income from Farming Percentage of overall yearly household income derived from farming (0-25%, 26-50%, 51-75%, 76-100%) 76-100%1 N/A Market Farm Income Farm income after costs minus subsidies 2,996 10.680 Off-farm Job Does the farmer have an off-farm job (Yes, No) 0.33 0.47 Entitlements Value Value of farm CAP entitlement payments 10,282 14,212 Rent Price Average regional per hectare farmland rental price 252 35 Land Price Average regional per hectare farmland price 16099 4221 1 d Table 4. Independent variables of land mobility model. Attitudinal variables The attitudinal variables derived from the PCA analysis were found to have a statistically significant impact on a farmer’s willingness to enter the land market. The attitudinal orientation “Innovative” has a positive impact on a farmer’s willingness to lease land. For every one unit increase in the orientation, the probability of leasing out land increases by a factor of 1.29 and of leasing in land by a factor of 1.21. The “Pleasure of Farming” variable has a negative impact on willingness to sell land. Additionally, it is positively correlated with a willingness to buy and lease in land. Four logistic regression models were created using the available data. The dependent variable in each case was willingness to engage in the stated land transaction. Two models analysed farmers’ attitudes to the leasing of land. 417 farmers were open to leasing in land while 245 farmers were willing to lease out land. The results from these two models are presented in Table 5. Two models analysed farmers’ attitude to permanent land transac- tions. 447 farmers were open to buying land while 218 farmers were willing to sell land. The results from these two models are presented in Table 6. and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue 160 The “Conservative” variable is negatively related to the willingness to buy and lease in land while the “Agri- Optimistic” orientation is positively related to leasing in and buying land while negatively related to selling land. Farm structure and agronomic variables Of the farm system variables employed in the anal- ysis, cattle rearing, cattle other, and mixed and dairy enterprises proved to be significantly related to willing- Table 5. Factors related to the probability farmers are open to leasing land. Lease Out Coef. Std. Err. Odds Ratio Lease In Coef. Std. Err. Attitudinal variables Odds Ratio Innovative 0.25* 0.09 1.29 0.19 0.09 1.21 Pleasure of Farming -0.13 0.08 0.88 0.18 0.08 1.20 Conservative -0.03 0.09 0.97 -0.21 0.08 0.81 Agri-Optimistic -0.13 0.09 0.88 0.21** 0.09 1.24 Good Soil 0.43 0.35 1.53 -0.45 0.32 0.64 Medium Soil 0.65* 0.35 1.92 -0.35 0.32 0.70 Cattle Rearing 0.18 0.25 1.20 0.25 0.26 1.28 Dairy -0.79 0.33 0.45 0.39 0.32 1.47 Tillage -0.63 0.30 0.53 0.29 0.29 1.34 Sheep -0.70* 0.27 0.49 0.02 0.26 1.02 Mixed -0.96 0.44 0.38 -0.91** 0.41 0.40 Farm Size <10ha 0.08 0.40 1.08 -0.69 0.44 0.50 Farm Size 10-19ha 0.42* 0.23 1.52 -0.20 0.24 0.82 Farm Size 50-74ha 0.37 0.27 1.44 0.27 0.27 1.31 Farm Size 75-99ha -0.11 0.41 0.89 0.31 0.40 1.36 Farm Size 100-149ha 0.59 0.54 1.80 -0.17 0.55 0.85 Farm Size >150ha -0.23 0.96 0.79 0.22 0.98 1.24 Stocking Rate -0.02 0.05 0.98 0.38* 0.09 1.46 Age <35 -1.11 0.46 0.33 0.93 0.40 2.52 Age 35-44 -0.03 0.25 0.97 0.01 0.25 1.01 Age 45-50 -0.28 0.27 0.75 -0.27 0.25 0.76 Age >65 0.21 0.21 1.23 -0.64* 0.21 0.53 Children 0.14 0.20 1.15 0.40 0.20 1.49 Successor -0.40 0.18 0.67 -0.44 0.18 0.64 Increase Future Production -0.18 0.24 0.84 0.77* 0.24 2.16 Decrease Future Production 0.81*** 0.28 2.26 -0.54* 0.33 0.58 Off-Farm Job -0.11 0.21 0.89 0.53** 0.22 1.70 Household Income from Farming ≤ 25% 0.31 0.29 1.36 0.16 0.30 1.17 Household Income from Farming 26-50% -0.04 0.25 0.96 -0.35 0.25 0.70 Household Income from Farming 51-75% 0.36 0.28 1.43 -0.52* 0.29 0.59 Farm Income 0.02* 0.01 1.02 0.02 0.01 1.02 Entitlements Value 0.01 0.02 1.01 0.02 0.02 1.02 Rent Price 0.00 0.00 1.00 -0.01* 0.00 0.99 Constant -1.86 0.78 0.16 2.83* 0.76 16.94 Pseudo R2 0.08* 0.19* AIC 999.13 1007.94 BIC 1159.95 1168.75 Observations 837 837 Note: * p<0.01, ** p<0.05, * p<0.1. Table 5. Factors related to the probability farmers are open to leasing land. Farm structure and agronomic variables The “Conservative” variable is negatively related to the willingness to buy and lease in land while the “Agri- Optimistic” orientation is positively related to leasing in and buying land while negatively related to selling land. Of the farm system variables employed in the anal- ysis, cattle rearing, cattle other, and mixed and dairy enterprises proved to be significantly related to willing- ness to enter the land market. In the “Lease Out” regres- sion, dairy, tillage, sheep and mixed enterprise farmers were significantly less likely to be willing to lease out Bio-based and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 161 The effect of farmer attitudes on openness to land transactions: evidence for Ireland Table 6. Factors related to the probability farmers are open to selling/buying land. Sell Land Buy Land Coef. Std. Err. Odds Ratio Coef. Std. Err. Farm structure and agronomic variables Odds Ratio Innovative 0.12 0.09 1.12 0.01 0.09 1.01 Pleasure of Farming -0.25* 0.08 0.78 0.24* 0.08 1.27 Conservative 0.00 0.09 1.00 -0.23* 0.08 0.80 Agri-Optimistic -0.19 0.09 0.83 0.16* 0.09 1.17 Good Soil -0.05 0.33 0.95 -0.05 0.32 0.95 Medium Soil 0.28 0.33 1.32 0.01 0.32 1.01 Cattle Rearing -0.01 0.27 0.99 0.44* 0.26 1.55 Dairy 0.10 0.33 1.10 0.53* 0.31 1.70 Tillage -0.14 0.32 0.87 0.33 0.30 1.39 Sheep -0.18 0.27 0.83 -0.10 0.25 0.90 Mixed -1.67* 0.59 0.19 -0.08 0.38 0.92 Farm Size <10ha -0.04 0.41 0.96 -1.36* 0.47 0.26 Farm Size 10-19ha 0.03 0.25 1.03 -0.50** 0.24 0.60 Farm Size 50-74ha 0.04 0.28 1.04 -0.04 0.27 0.96 Farm Size 75-99ha -0.04 0.41 0.96 0.23 0.40 1.25 Farm Size 100-149ha 0.04 0.54 1.04 -0.40 0.56 0.67 Farm Size >150ha -2.04* 1.22 0.13 0.07 1.00 1.07 Stocking Rate 0.05 0.05 1.05 0.18* 0.07 1.20 Age <35 -0.28 0.41 0.75 1.37* 0.41 3.94 Age 35-44 0.32 0.25 1.38 0.56 0.25 1.75 Age 45-50 0.56 0.26 1.75 0.25 0.25 1.29 Age >65 -0.15 0.23 0.86 -0.50 0.21 0.61 Children 0.14 0.21 1.15 0.63* 0.20 1.88 Successor -0.50*** 0.19 0.61 0.32* 0.17 1.37 Increase Future Production -0.04 0.24 0.96 0.68* 0.25 1.98 Decrease Future Production 0.62 0.30 1.86 -0.49 0.32 0.61 Off-Farm Job -0.15 0.22 0.86 0.52** 0.22 1.69 Household Income from Farming ≤ 25% 0.22 0.30 1.25 0.17 0.29 1.19 Household Income from Farming 26-50% 0.43* 0.26 1.53 -0.08 0.25 0.92 Household Income from Farming 51-75% 0.41 0.30 1.51 -0.01 0.28 0.99 Farm Income 0.00 0.01 1.00 0.00 0.01 1.00 Entitlements Value 0.03* 0.02 1.03 0.03 0.02 1.03 Land Price -0.00* 0.00 1.00 0.00 0.00 1.00 Constant -0.72 0.54 0.49 -1.24 0.51 0.29 Pseudo R2 0.08* 0.18* AIC 950.86 1018.68 BIC 1111.68 1179.49 Observations 837 837 Note: * p<0.01, p<0.05, * p<0.1. Table 6. Factors related to the probability farmers are open to selling/buying land. Note: * p<0.01, p<0.05, * p<0.1. land than the reference category of cattle other farmers. Stated differently, cattle rearing and cattle other farmers were significantly more willing to lease out land com- pared to all other farm systems. However, farm system was not significantly related to willingness to lease in land. Mixed farmers were significantly less willing to sell land than other farmers while dairy and tillage farmers land than the reference category of cattle other farmers. Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue divided into four categories: 0-25%, 26-50%, 51-75% and 76-100% of household income coming from farm income. The reference category was farmers for whom 76-100% of household income came from their farm (the most common response). Farmers in the 51-75% farm income category were less likely to be open to leasing in land than those who rely on farm revenues for over 75% of household income, while those in the 26-50% farm income category were more open to selling land. than the most common farm size category. Stocking rate was a significantly related to land demand. In terms of leasing in land, an increase of one LU/ha increased the probability of being willing to lease in land by a factor 1.46. An increase of one LU/ha increased the probability of the farmer being open to buying land by a factor of 1.20. Soil quality was also included as an explanatory var- iable in each regression model. Three soil categories were used: good, medium and poor. The good and medium categories were included as dummy variables, with poor quality soil acting as the reference category. Soil qual- ity was a significant explainer of willingness to enter the land market in the “Lease Out” model, with farmers on medium quality soil being significantly more open to leasing out land than those in the reference category. Farmers with an off-farm job were significantly more likely to be open to leasing in and buying land than farmers without off-farm employment. Farmers with off-farm jobs were more likely by a factor of 1.70 to be open to leasing in land and by a factor of 1.69 to buy- ing land than those without off-farm jobs. Market farm income is positively associated with willingness to rent out land while subsidy income from entitlements was positively correlated with selling land at the 10% signifi- cance level. Agricultural land prices and land rents at the regional level are also modelled. Regional farmland prices are significantly negatively correlated with a will- ingness to sell land while regional land rent prices have a negative relationship to willingness to rent land in. 6. DISCUSSION This study examined the extent to which Irish farm- ers would be willing to enter the agricultural land mar- ket. The results show that about half of farmers in the sample are open to buying or leasing in land while about a quarter of farmers sampled are open to selling or leasing out land. The results also show distinct profiles emerging for farmers demanding land, through either leasing in or purchase and farmers open to supplying land, whether through leasing out or sale. The presence of a farm successor was a significant explanatory variable in all four models. Having a succes- sor was associated with farmers being significantly less likely to be willing to lease out or sell land, compared with farmers without a successor. Having a successor decreased the likelihood of being willing to lease out land by a factor of 0.67 and sell land by a factor of 0.61, compared to farmers without a successor. Interestingly, farmers with a successor were more likely to be willing to buy land but significantly less likely to be willing to lease in land than those without a successor.i Farmers demanding land are more likely to have a high ranking on the “Pleasure of Farming” and “Agri- Optimistic” attitudinal orientations and a low ranking on the “Conservative” orientation. They are also more likely to have children, be planning to increase farming activ- ity in the next five years and have an off-farm job. They are more likely to be under 35 years of age and a have a high stocking rate. They are less likely to have farms of less than 10 hectares and be over 65 years of age. h Farmers with children were significantly more will- ing to demand land than farmers without children. Farmers with children were more likely to be willing to lease in land by a factor 1.49 and more likely to be will- ing to buy land by a factor of 1.88, compared with farm- ers without children. The issue of agricultural land demand, especially in how it relates to young farmers, has arisen in recent years in the context of increasing farmland concen- tration in Europe (Conway et al., 2020; van der Ploeg, 2015). A reduction in farm numbers by approximately 3.8 million and an increase in farm size by about 36% was seen in the EU between 2005 and 2015 (Eurostat, Demographic variables Age effects relating to willingness to enter the land market can be seen amongst the youngest and oldest categories of farmers. The youngest category of farmers (those under the age of 35) was significantly more will- ing to lease in and buy land compared to older farmers. Additionally, the youngest farmers were significantly less willing to lease out land. Farmers in the oldest age cat- egory (65 years and older) were significantly less likely to demand land either through leasing or purchase. Farm- ers in the 45-50 years were more likely to be willing to sell land than any other age category. hi Farm structure and agronomic variables Stated differently, cattle rearing and cattle other farmers were significantly more willing to lease out land com- pared to all other farm systems. However, farm system was not significantly related to willingness to lease in land. Mixed farmers were significantly less willing to sell land than other farmers while dairy and tillage farmers were significantly more willing to buy land, albeit at the 10% significance level.if gi In general, significant effects for farm size were lim- ited to either very small or very large farms. Farms under 20 hectares were the least likely to be willing to buy land compared with the most common farm size category. Farms of over 150 hectares were less willing to sell land and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue 162 Financial variables Farmers have been found to have a multiplicity of motivations for why they farm, many of which are non- economic in nature (Howley et al., 2015; Key & Rob- erts, 2009). For these farmers, land may not be seen as an economic resource but as a source of utility in and of itself. Therefore, it is not surprising that farmers with a high ranking in this orientation are opposed to releasing land and are open to increasing their land stock. hi Regional farmland prices were negatively related to willingness to sell, a finding contrary to standard eco- nomic theory. It must be stated that Irish farmland mar- kets are extremely local so prevailing regional prices would not be as significant to farmers’ decision-making regarding land as the local market. Farmers may also be anticipating increasing land prices in the future. Irish farmland prices are heavily influenced by non-agri- cultural factors (Geoghegan & O’Donoghue, 2018), so increasing property prices in Ireland following the 2008 economic crash may be influencing farmers not to sell land until prices peak. h The finding that farmers with a high ranking in the “Innovative” orientation are more open to supply- ing land, both through leasing and sale, suggests that these farmers are less constrained by traditions of keep- ing land “in the family name”. They may see land as just another input in the agricultural production process. Innovative farmers in the Irish context may be thought about as generating new combinations of existing resources (Bender & Laestadius, 2005).hf The positive effect of having children and having a designated successor on willingness to buy land (and lease in land for the “Children” variable) fits in with the farm life cycle concept (Calus et al., 2008; Potter & Lobley, 1992). This farm life cycle concept suggests that a farm can be in one of three stages: growth, matu- rity or decline. Younger farmers are expected to grow, while older farmers are expected to be in the maturity or decline stages. However, farmers with a successor do not enter the decline stage but rather are more likely to want to grow the farm in order to leave a legacy for their successor (Calus et al., 2008; Inwood & Sharp, 2012). Financial variables Farmers were asked what percentage of household income is made up of farm income. Responses were Bio-based and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 163 The effect of farmer attitudes on openness to land transactions: evidence for Ireland 2017). These results confirm the desire of young, opti- mistic farmers to access land but whose ability to do so may be hampered in a competitive land market (Zagata et al, 2017). farmers who are older are significantly less likely to want to increase their farm size (Gale, 1994; Lobley & But- ler, 2010; Katchova & Ahearn, 2015; Weiss, 1999). Older farmers were not significantly more likely to want to lease out or sell land than average aged farmers, sup- porting the theory that older Irish farmers want to maintain land within the family unit rather than sell or lease it out to others (Banovic et al., 2015). f Farmers open to supplying land are more likely to rank high on the “Innovative” orientation for leasing out and rank low on the “Pleasure of Farming” orientation for selling. They are more likely to be intent on decreas- ing farming in the next five years and are less likely to have a successor. They are also more likely to be only somewhat dependant on farm income, receiving greater than 25% but less than 50% of total household income from farming. Farmers with off-farm jobs are more likely to want to add land through lease or purchase than full-time farmers. There is evidence in the literature that off- farm income may help to prevent farm exit by stabilis- ing income (Breustedt & Glauben, 2007; Kimhi, 2000). Farmers in the study with off-farm employment are younger than full-time farmers (45% aged 50 and young- er vs. 31% for full-time farmers) and may have difficulty accessing land in a manner similar to other young farm- ers across the EU (Zondag et al., 2015). Therefore, they may be aiming to increase land holdings going forward. Also, there may be a wealth effect for farmers with off- farm jobs with farmers using their off-farm income in order to acquire more land through increased credit capacity or ability to pay higher rents. Farmers ranking high in the “Pleasure of Farm- ing” orientation value the lifestyle benefits of farming over any pecuniary benefits associated with the profes- sion. 4 496 of the study’s participants were surveyed in winter 2014, of which 196 were cattle farmers. Bio-based and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 7. CONCLUSIONS AND POLICY RECOMMENDATIONS The aim of the study was to explore Irish farm- ers’ attitudes towards land mobility and to build a pro- file of farmers who would be open to partaking in land transactions. Despite previous evidence that Irish farm- ers are reluctant to enter the land market (especially to supply land), this study shows that a considerable num- ber of farmers are open to the possibility. It is important to understand what motivates farmers who are open to trading land. This is especially true in the absence of market data, as is the case in Ireland. This paper sug- gests that farmer attitudes are an important motivating force behind farmers’ willingness to enter land markets. Farmers are not motivated solely by profit maximisa- tion, as evidenced by the significance of the “Pleasure of Farming” and “Conservative” variables. Additionally, there appear to be a group of farmers amongst whom the traditional attachment to land is not as prevalent, as evidenced by the willingness of “Innovative” farmers to supply land through leasing. Therefore, policymak- ers must take account of these attitudes when design- ing policies to enable a more dynamic land market. Such policies should not just focus on economic incentives to encourage land mobility but also on encouraging dis- cussion between farmers, successors, potential farmers, policymakers and agricultural professionals (farm advi- sors, solicitors, accountants etc.) so as to take less tangi- ble factors such as attitudes and motivation into account. i Although numerous farm and farmer characteristics are examined in relation to openness to land transac- tions in this study, factors related to the socio-economic environment around the farm are considered outside of the scope of this paper. Such factors include social and identity pressures (Ní Laoire, 2005), local labour mar- ket conditions (Cavicchioli et al., 2019) and gender (Bal- aine, 2019). Further research examining the intersec- tion between farm characteristics, farmer attributes and socio-economic conditions is required. It should be noted that being open to land transac- tions does not necessarily mean that farmers will par- take in a transaction in the future. This study does not examine the prices farmers are willing to pay and will- ing to accept for land. Although farmers may be willing to engage in land transactions, a mismatch between the prices farmers are willing to pay and willing to accept for land will prevent transactions from taking place. Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue Land Mobility Service that facilitate land mobility (Mac- ra na Feirme, 2019). Additionally, the promotion of joint farm ventures (JFVs) such as cooperatives, farm partner- ships, share farming and contract rearing must be main- tained (Cush & Macken-Walsh, 2016).h in land. Cattle farmers’ openness to leasing out may, as previously stated, have reflected particularly poor eco- nomic conditions at the time the survey was conducted. Their willingness to lease out rather than sell land may reflect a desire to reclaim the land for their own farming purposes once economic conditions for cattle farming improved. There is a similar level of openness amongst farmers to both permanent and temporary land transfer options. This is contrary to conventional thinking that Irish farmers are reluctant to take part in temporary land transactions such as land leasing. This shows that there may be greater demand amongst farmers for land leasing arrangements than is currently thought by policymak- ers. As a result, policies that can promote and facilitate such leasing arrangements should be encouraged. Since financial incentives in the form of tax breaks already exist, institutional solutions such as the establishment of intermediary entities to connect potential lessors and lessees or informational campaigns advertising the ben- efits of leasing may be appropriate. 7. CONCLUSIONS AND POLICY RECOMMENDATIONS Therefore, further research is required to examine the extent of price mismatches and how they affect land markets in Ireland. Together with the economic and socio-demograph- ic information presented here, a picture emerges of the types of farmer policymakers can target with land mobil- ity policies. Young, optimistic farmers with higher than average stocking rates and plans for increasing produc- tion in the near future appear to be most likely to demand land. Innovative cattle farmers who are somewhat but not totally dependent on farm income and are planning to decrease farm activity in the near future are most likely to supply land. Policies that can both identify and medi- ate between these groups should be considered by policy- makers. This can be done by policymakers engaging with farming organisations, through the organisation of infor- mation events and by aiding organisations such as the Financial variables The finding that farmers with successors are more willing to buy land but are significantly less likely than farm- ers without successors to want to lease in land may be related to the lack of trust amongst Irish farmers in the leasing system (Banovic et al., 2015; Bogue, 2013). f The openness of cattle farmers to leasing out land may be related to the difficult financial conditions facing cattle farmers in Ireland. Widespread protests amongst cattle farmers over low beef prices broke out during the collection of the survey which may have led to cattle farmers being particularly pessimistic when surveyed.4 As a result, cattle farmers may have felt more open to leasing out land at this time. The factors that significantly influence farmers’ openness to temporary land transactions such as leasing also seem to significantly influence permanent transac- tions such as buying and selling. However, there are some notable exceptions to this finding. Cattle farmers are open to leasing out land but not selling land while dairy farmers are open to buying land but not leasing Farmer age effects align with previous studies, with younger farmers most likely to want to add land, while and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 164 REFERENCES Abdi, H., & Williams, L. J. (2010). Principal component analysis. Wiley Interdisciplinary Reviews: Computa- tional Statistics, 2, 433–459. DOI: 10.1002/wics.101 Alwin, D.F. (2007). Margins of error: A study of reliability in survey measurement. Hoboken, NJ: Wiley Balaine, L. (2019). Gender and the preservation of family farming in Ireland. EuroChoices, 18(3), 33-37. DOI: 10.1111/1746-692X.12242 Bio-based and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 The effect of farmer attitudes on openness to land transactions: evidence for Ireland The effect of farmer attitudes on openness to land transactions: evidence for Ireland 165 Banovic, M., Duesberg, S., Renwick, A., Keane, M. T., & Bogue, P. (2014, April). The field: Land mobility measures as seen through the eyes of Irish farmers. In The Agricultural Economics Society’s 89th Annual Conference, University of Warwick, United Kingdom, 13-15 April 2014. Retrieved from: http://hdl.handle. net/10197/6730 Centre for European Policy Studies. Retrieved from: http://aei.pitt.edu/32642/1/65._EU_Land_Markets_ and_the_CAP.pdf Ciaian, P., Kancs, d’A., Swinnen, J., Van Herck, K, & Vranken, L. (2012). Institutional factors affecting agricultural land markets (Factor Markets Working Paper 16). Brussels: Centre for European Policy Stud- ies. Retrieved from: http://ageconsearch.umn.edu/ record/120251/files/FM_WP16%20CEPS%20on%20 Institutional%20Factors%20Affecting%20Ag%20 Land%20Markets_D15.3_final.pdf Bender, G., & Laestadius, S. (2005). Non-science based innovativeness. On capabilities relevant to generate profitable novelty. Journal of Mental Changes, 11(1-2), 123-170. Retrieved from: http://cat.inist.fr/?aModele= afficheN&cpsidt=17868457h i Commins, P. (2001). Future perspectives on rural areas. Athenry: Rural Economy Research Centre.i fi Bika, Z. (2007). The territorial impact of the farmers’ ear- ly retirement scheme. Sociologia Ruralis, 47(3), 246- 272. DOI: 10.1111/j.1467-9523.2007.00436.x Conway, A.G. (1986). Land leasing – findings of a study in the west region of the Republic of Ireland. Irish Journal of Agricultural Economics and Rural Sociol- ogy, 11, 1-18. Retrieved from: http://www.jstor.org/ stable/25556545 Boere, E., Peerlings, J., Reinhard, S., & Heijman, W. (2015). The dynamics of dairy land use change with respect to the milk quota regime. European Review of Agricultural Economics, 42(4), 651-674. DOI: 10.1093/erae/jbv002 Conway, S.F., Farrell, M., McDonagh, J., & Kinsella, A. (2020). Mobilising land mobility in the European Union: An under-researched phenomenon. Interna- tional Journal of Agricultural Management, 9, 7-11. DOI: 10.5836/ijam/2020-09-7h Bogue, P. (2013). Land mobility and succession in Ireland. Dublin: Macra na Feirme. hh Breen, J. P., Hennessy, T. C., & Thorne, F. S. (2005). The effect of decoupling on the decision to produce: An Irish case study. Food Policy, 30(2), 129-144. DOI: 10.1016/j.foodpol.2005.03.001 Cush, P., & Macken-Walsh, Á. (2016). REFERENCES The potential for joint farming ventures in Irish agriculture: A socio- logical review. European Countryside, 8(1), 33-48. DOI: 10.1515/euco-2016-0003h Breustedt, G., & Glauben, T. (2007). Driving forces behind exiting from farming in Western Europe. Journal of Agricultural Economics, 58(1), 115-127. DOI: 10.1111/j.1477-9552.2007.00082.x Davidova, S., & Thomson, K. (2014). Family farming in Europe: challenges and prospects (Directorate General for Internal Policies, Policy Department B: Structural and Cohesion Policies). Brussels: European Parlia- ment. Retrieved from: http://www.europarl.europa. eu/RegData/etudes/note/join/2014/529047/IPOL- AGRI_NT(2014)529047_EN.pdf Calus, M., Van Huylenbroeck, G., & Van Lierde, D. (2008). The relationship between farm succession and farm assets on Belgian farms. Sociologia Ruralis, 48(1), 38-56. DOI: 10.1111/j.1467-9523.2008.00448.x Cavicchioli, D., Bertoni, D., Frisio, D. G., & Pretolani, R. (2019). Does the future of a farm depend on its neighbourhood? Evidence on intra-family succession among fruit and vegetable farms in Italy. Agricul- tural and Food Economics, 7(1), 1-17. DOI: 10.1186/ s40100-019-0129-5 Davis, J., Caskie, P., & Wallace, M. (2009). Economics of farmer early retirement policy. Applied Economics, 41(1), 35-43. DOI: 10.1080/00036840600994211 Department of Agriculture, Fisheries & Food (2010). Food Harvest 2020: A vision for Irish agri-food and fisheries. Dublin: DAFF. Central Statistics Office (2012). Census of agriculture 2010. Dublin: CSO. Retrieved from: https://www.cso. ie/en/media/csoie/releasespublications/documents/ agriculture/2010/full2010.pdf i Department of Agriculture, Fisheries & the Marine (2015). Food Wise 2025. Dublin: DAFM. Department of Agriculture, Fisheries & the Marine (2018). Farm restructuring guidelines. Dublin: DAFM. Retrieved from: https://web.archive.org/ web/20190219233855/https://www.agriculture.gov. ie/media/migration/foodindustrydevelopmenttrade- markets/agri-foodandtheeconomy/CapitalGainsTaxS- tampDutyReliefGuidelines270718.pdf Chevalier, F., Veyssiere, L., Buccellato, T., Jicquello, J., & de Oteyza, C. (2012). Analysis on future developments in the milk sector. Prepared for the European Com- mission-DG Agriculture and Rural Development. Retrieved from: https://ec.europa.eu/agriculture/ sites/agriculture/files/events/2013/milk- conference/ ernst-and-young-report_en.pdf Dervillé, M., Allaire, G., Maigné, É., & Cahuzac, É. (2017). Internal and contextual drivers of dairy restructuring: evidence from French mountainous Ciaian, P., Kancs, d’A., & Swinnen, J. (2010). EU land markets and the common agricultural policy. Brussels: Bio-based and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue 166 areas and post‐quota prospects. Agricultural Econom- ics, 48(1), 91-103. DOI: 10.1111/agec.12297h nominated farm heirs in Ireland. Journal of Agricul- tural Economics, 58(1), 61-75. DOI: 10.1111/j.1477- 9552.2007.00078.x Dillon, P. A. T., Hennessy, T., Shalloo, L., Thorne, F., & Horan, B. (2008). Future outlook for the Irish dairy industry: A study of international competitive- ness, influence of international trade reform and requirement for change. International Journal of Dairy Technology, 61(1), 16-29. REFERENCES DOI: 10.1111/j.1471- 0307.2008.00374.x Howley, P., Buckley, C., Donoghue, C. O., & Ryan, M. (2015). Explaining the economic ‘irrationality’of farmers’ land use behaviour: The role of produc- tivist attitudes and non-pecuniary benefits. Eco- logical Economics, 109, 186-193. DOI: 10.1016/j. ecolecon.2014.11.015 Donnellan, T., Hanrahan, K., & Hennessy, T. (2008). Study on the functioning of land markets in the EU member states under the influence of measures applied under the Common Agricultural Policy (End of Pro- ject Report RMIS 5881). Athenry: Galway. Retrieved from: http://hdl.handle.net/11019/868 Howley, P., & Dillon, E. (2012). Modelling the effect of farming attitudes on farm credit use: a case study from Ireland. Agricultural Finance Review, 72(3), 456- 470. DOI: 10.1108/00021461211277286 Ingram, J., & Kirwan, J. (2011). Matching new entrants and retiring farmers through farm joint ventures: Insights from the Fresh Start Initiative in Cornwall, UK. Land Use Policy, 28(4), 917-927. DOI: 10.1016/j. landusepol.2011.04.001 Eurostat (2017). Farm indicators by agricultural area, type of farm, standard output, sex and age of the manager and NUTS 2 regions [Data set]. Eurostat. Retrieved from: https://data.europa.eu/euodp/en/data/dataset/ liNJaTm9RYIzdRdCJKuU2w Inter-Departmental Committee on Land Structure Reform (1978). Final report. Dublin: Stationery Office. Gale Jr., H. F. (1994). Longitudinal analysis of farm size over the farmer’s life cycle. Review of Agricultural Economics, 16(1), 113-123. Retrieved from: http:// www.jstor.org/stable/1349526 fi Inwood, S. M., & Sharp, J. S. (2012). Farm persistence and adaptation at the rural–urban interface: Succes- sion and farm adjustment. Journal of Rural Studies, 28(1), 107-117. DOI: 10.1016/j.jrurstud.2011.07.005 Geoghegan, C., & O’Donoghue, C. (2018). Socioeconom- ic drivers of land mobility in Irish agriculture. Inter- national Journal of Agricultural Management, 7(2), 26-34. Jenkins, W. (1997). Land Leasing in Rural Ireland-some empirical findings from the south-east. Irish Geogra- phy, 30(2), 99-105. DOI: 10.1080/00750779709478637 Kay, C., Peuch, J., & Franco, J. (2015). Extent of farm- land grabbing in the EU. (Directorate-General for Internal Policies. Policy Department B: Structural and Cohesion Policies). Brussels: European Parlia- ment. Retrieved from: http://www.europarl.europa. eu/RegData/etudes/STUD/2015/540369/IPOL_ STU%282015%29540369_EN.pdf Gillmor, D. A. (1999). The scheme of early retire- ment from farming in the Republic of Ire- land. Irish Geography, 32(2), 78-86. DOI: 10.1080/00750779909478602 Groeneveld, A., Peerlings, J., Bakker, M., & Heijman, W. (2016). The effect of milk quota abolishment on farm intensity: shifts and stability. NJAS-Wageningen Journal of Life Sciences, 77, 25-37. DOI: 10.1016/j. njas.2016.03.003 Katchova, A. L., & Ahearn, M. C. (2015). Dynamics of farmland ownership and leasing: implications for young and beginning farmers. REFERENCES DOI: 10.1177/079160350501400206 Lobley, M., & Butler, A. (2010). The impact of CAP reform on farmers’ plans for the future: Some evi- dence from South West England. Food Policy, 35(4), 341-348. DOI: 10.1016/j.foodpol.2010.04.001 O’Neill, S., & Hanrahan, K. (2012). Decoupling of agri- cultural support payments: The impact on land mar- ket participation decisions. European Review of Agri- cultural Economics, 39(4), 639-659. DOI: 10.1093/ erae/jbr064f Loughrey, J., & Hennessy, T. (2019). A microsimulation model for the land rental market in Irish farming. In The International Microsimulation Association’s 7th World Conference, National University of Ireland, Galway, Ireland, 19-21 June 2019. DOI: 10.2139/ ssrn.3630993 Piet, L., Latruffe, L., Le Mouël, C., & Desjeux, Y. (2012). How do agricultural policies influence farm size ine- quality? The example of France. European Review of Agricultural Economics, 39(1), 5-28. DOI: 10.1093/ erae/jbr035 Macra na Feirme (2015). Land mobility service end of pilot programme report 2014-2016. Macra na Feirme: Dublin. Retrieved from: https://web.archive.org/ web/20190219233554/http://landmobility.ie/wp-con- tent/uploads/2017/04/LAND_mobility_2017_finalfi- nal.pdf Potter, C., & Lobley, M. (1992). Ageing and succession on family farms: The impact on decision‐making and land use. Sociologia Ruralis, 32(2‐3), 317-334. DOI: 10.1111/j.1467-9523.1992.tb00935.x Sturgis, P., Roberts, C., & Smith, P. (2014). Middle alter- natives revisited: how the neither/nor response acts as a way of saying “I don‘t know”?. Socio- logical Methods & Research, 43(1), 15-38. DOI: 10.1177/0049124112452527h Macra na Feirme (2019). Land mobility 2019 report. Mac- ra na Feirme: Dublin. Retrieved from: http://web. archive.org/web/20190711230808/http://landmobil- ity.ie/wp-content/uploads/2019/07/LAND-Mobility- 2019-Report.pdf Swinnen, J., Van Herck, K., & Vranken, L. (2016). The diver- sity of land markets and regulations in Europe, and (some of) its causes. The Journal of Development Studies, 52(2), 186-205. DOI: 10.1080/00220388.2015.1060318 Maguire, D. (1983). The land problem. Dublin: Agri- Books. h Marr, E. J., & Howley, P. (2019). The accidental environ- mentalists: Factors affecting farmers’ adoption of pro-environmental activities in England and Ontario. Journal of Rural Studies, 68, 100-111. DOI: 10.1016/j. jrurstud.2019.01.013 Van der Ploeg, J. D., Franco, J. C., & Borras Jr, S. M. (2015). Land concentration and land grabbing in Europe: a preliminary analysis. Canadian Jour- nal of Development Studies/Revue canadienne d’études du développement, 36(2), 147-162. DOI: 10.1080/02255189.2015.1027673 Mazorra, A. P. (2000). Analysis of the evolution of farm- ers’ early retirement policy in Spain. The case of Cas- tille and Leon. Land Use Policy, 17(2), 113-120. DOI: 10.1016/S0264-8377(00)00006-5 Van Passel, S., Nevens, F., Mathijs, E., & Van Huylen- broeck, G. (2007). Measuring farm sustainability and explaining differences in sustainable efficiency. REFERENCES Applied Economic Per- spectives and Policy, 38(2), 334-350. DOI: 10.1093/ aepp/ppv024 Hamilton, L. C. (2012). Statistics with Stata: Version 12. Cengage Learning. Hennessy, T. (2014). CAP 2014–2020 Tools to enhance family farming: opportunities and limits. (Directorate- General for Internal Policies. Policy Department B: Structural and Cohesion Policies). Brussels: European Parliament. Retrieved from: http://www.europarl. europa.eu/RegData/etudes/note/join/2014/529051/ IPOL-AGRI_NT(2014)529051_EN.pdf Kempen, M., Witzke, P., Domínguez, I. P., Jansson, T., & Sckokai, P. (2011). Economic and environmental impacts of milk quota reform in Europe. Journal of Policy Modeling, 33(1), 29-52. DOI: 10.1016/j.jpolm- od.2010.10.007 Kennedy, L. (1991). Farm succession in modern Ireland: Elements of a theory of inheritance. The Economic History Review, 44(3), 477-499. DOI: 10.1111/j.1468- 0289.1991.tb01275.x Hennessy, T., & Moran, B. (2015), Teagasc national farm survey results 2014. Teagasc: Athenry. Hennessy, T., & Moran, B. (2016), Teagasc national farm survey results 2015. Teagasc: Athenry. Key, N., & Roberts, M. J. (2009). Nonpecuniary ben- efits to farming: Implications for supply response to decoupled payments. American Journal of Agricul- Hennessy, T. C., & Rehman, T. (2007). An investigation into factors affecting the occupational choices of Bio-based and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 The effect of farmer attitudes on openness to land transactions: evidence for Ireland 167 tural Economics, 91(1), 1-18. DOI: 10.1111/j.1467- 8276.2008.01180.x tural Economics, 91(1), 1-18. DOI: 10.1111/j.1467- 8276.2008.01180.x Rural Affairs (Defra). CCRI: Gloucester. Retrieved from: www.fslra.ac.uk/media/universityofexeter/ research/microsites/centreforruralpolicyresearch/ pdfs/10570_FarmerAttitudesFinalReportIF01114.pdf Kimhi, A. (2000). Is part-time farming really a step in the way out of agriculture? American Journal of Agricul- tural Economics, 82(1), 38-48. DOI: 10.1111/0002- 9092.00004h Morris, C., & Potter, C. (1995). Recruiting the new con- servationists: Farmers’ adoption of agri-environ- mental schemes in the UK. Journal of Rural Studies, 11(1), 51-63. DOI: 10.1016/0743-0167(94)00037-A Läpple, D., & Hennessy, T. (2012). The capacity to expand milk production in Ireland following the removal of milk quotas. Irish Journal of Agricultural and Food Research, 51, 1-11. Retrieved from: http://www.jstor. org/stable/41756842 Needham, B., Segeren, A., & Buitelaar, E. (2011). Institu- tions in theories of land markets: illustrated by the Dutch market for agricultural land. Urban Studies, 48(1), 161-176. DOI: 10.1177/0042098009360682 Le Mouël, C. (2004). Agricultural land markets: Main issues in the recent literature. Working Paper Series 2 of the IDEMA Project. Retrieved from: http://www. agrifood.se/idema/WPs/IDEMA_deliverable_2.pdf Laoire, C. N. (2005). ‘You’re not a man at all!’: Mascu- linity, responsibility, and staying on the land in con- temporary Ireland. Irish Journal of Sociology, 14(2), 94-114. REFERENCES Eco- logical Economics, 62(1), 149-161. DOI: 10.1016/j. ecolecon.2006.06.008 Mills, J., Gaskell, P., Reed, M., Short, C., Ingram, J., Boat- man, N., Jones, N., Conyers, S., Carey, P., Winter, M., & Lobley, M. (2013). Farmer attitudes and evaluation of outcomes to on-farm environmental management. Report to Department for Environment, Food and Weiss, C. R. (1999). Farm growth and survival: econo- metric evidence for individual farms in Upper Aus- Bio-based and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746 168 Cathal Geoghegan, Anne Kinsella, Cathal O’Donoghue tria. American Journal of Agricultural Economics, 81(1), 103-116. DOI: 10.2307/1244454 Willock, J., Deary, I.J., Edwards‐Jones, G., Gibson, G.J., McGregor, M.J., Sutherland, A., Dent, J.B., Morgan, O. & Grieve, R. (1999a). The role of attitudes and objectives in farmer decision making: business and environmentally‐oriented behaviour in Scotland. Journal of Agricultural Economics, 50(2), 286-303. DOI: 10.1111/j.1477-9552.1999.tb00814.x Willock, J., Deary, I.J., McGregor, M.M., Sutherland, A., Edwards-Jones, G., Morgan, O., Dent, B., Grieve, R., Gibson, G., & Austin, E. (1999b). Farmers’ attitudes, objectives, behaviors, and personality traits: The Edinburgh study of decision making on farms. Jour- nal of Vocational Behavior, 54(1), 5-36. DOI: 10.1006/ jvbe.1998.1642 Wilson, G. A. (1996). Farmer environmental attitudes and ESA participation. Geoforum, 27(2), 115-131. DOI: 10.1016/0016-7185(96)00010-3 Zagata, L., Hrabak, J., Lošťak, M., Bavorová, M., Ratinger, T., Sutherland, L. A., & McKee, A. (2017). Research for AGRI Committee–Young farmers–policy implemen- tation after the 2013 CAP reform. Brussels: European Parliament, Policy Department for Structural and Cohesion Policies. Retrieved from: https://www.euro- parl.europa.eu/RegData/etudes/STUD/2017/602006/ IPOL_STU(2017)602006_EN.pdf Zagata, L., & Sutherland, L. A. (2015). Deconstructing the ‘young farmer problem in Europe’: Towards a research agenda. Journal of Rural Studies, 38, 39-51. DOI: 10.1016/j.jrurstud.2015.01.003 Zondag, M. J., Koppert, S., de Lauwere, C., Sloot, P., & Pauer, A. (2015). Needs of young farmers. Luxem- bourg: Publications Office of the European Union. Retrieved from: https://ec.europa.eu/agriculture/sites/ agriculture/files/external-studies/2015/young-farm- ers/final-report-1_en.pdf ed and Applied Economics 10(2): 153-168, 2021 \| e-ISSN 2280-6e172 \| DOI: 10.36253/bae-9746
https://openalex.org/W3043787286	https://ieeexplore.ieee.org/ielx7/6287639/8948470/09143118.pdf	English	null	Order-Preserving Languages for the Supervisory Control of Automated Manufacturing Systems	IEEE access	2,020	cc-by	21,619	Received June 15, 2020, accepted July 10, 2020, date of publication July 17, 2020, date of current version July 29, 2020. Received June 15, 2020, accepted July 10, 2020, date of publication July 17, 2020, date of current version July 29, 2020. Received June 15, 2020, accepted July 10, 2020, date of publication July 17, 2020, date of current version July 29, 2020. Digital Object Identifier 10.1109/ACCESS.2020.3010030 Digital Object Identifier 10.1109/ACCESS.2020.3010030 ANAS NOORULDEEN1 AND KLAUS WERNER SCHMIDT 2 1 This work was supported by the program 2215 of the Scientiﬁc and Technological Research Council ABSTRACT Automated manufacturing systems (AMSs) consist of computer-controlled interconnected manufacturing components (MCs) that are used to transport and process different product types. Each product type requires a certain sequence of processing steps in different MCs. Hereby, multiple product types can share processing steps on the same MC and the paths of different products types can overlap. In this paper we consider the modeling of AMSs in the scope of supervisory control for discrete event systems (DES). On the one hand, a suitable AMS model must allow the representation of sequential and concurrent processing steps in MCs. On the other hand, such model must be able to track different product types traveling through the AMS so as to process the products correctly. While previous work is commonly concerned with the ﬁrst requirement, this paper identiﬁes that the existing literature lacks a general treatment of the second requirement. Accordingly, we ﬁrst introduce order-preserving (OP) languages that preserve the order of different product types in MCs and we propose a suitable ﬁnite state automaton model for OP languages. Then, we show that the composition of OP languages again leads to an OP language. That is, modeling MCs by OP languages, an OP model of a complete AMS that is suitable for supervisory control is obtained. In addition, it is possible to use both OP models and non-OP models for general AMSs, where MCs have different properties. We demonstrate the applicability of the proposed modeling technique by a ﬂexible manufacturing system example. INDEX TERMS Discrete-event systems, automated manufacturing systems, supervisory control, order- preserving models. is work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ Order-Preserving Languages for the Supervisory Control of Automated Manufacturing Systems ANAS NOORULDEEN1 AND KLAUS WERNER SCHMIDT 2 1Department of Electronics and Communication Engineering, Çankaya University, 06790 Ankara, Turkey 2Department of Electrical and Electronics Engineering, Middle East Technical University, 06800 Ankara, Turkey Corresponding author: Klaus Werner Schmidt (schmidt@metu.edu.tr) This work was supported by the program 2215 of the Scientiﬁc and Technological Research Council of Turkey (TÜBİTAK). VOLUME 8, 2020 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons I. INTRODUCTION We further introduce the notion of capacity for OP languages. The capacity denotes the maximum dif- ference of input and output events in any string of an OP language. Using OP languages for modeling MCs of an AMS, the capacity represents the maximum number of products that can be present in a MC. As the second contribution of the paper, we show that there is a supremal OP language that is recognized by a ﬁnite state automaton, whose state count depends on the capacity and number of input events. As the main contribution of the paper, we prove that composing two supremal OP languages and projecting the resulting language to the input and output events of the ﬁrst and second language, respectively, again leads to a supremal OP language. The capacity of this OP language is the sum of the capacities of the ﬁrst and second language and the projection turns out to be a natural observer. Hence, it is possible to obtain an efﬁcient representation of AMSs with OP MCs that is suitable for nonblocking supervisory control of large-scale AMSs [14], [15], [34]. Since the OP model is general, it can be used together with non-OP models for AMS, whose MCs have different properties. We demonstrate the practicability of the proposed modeling method by an FMS example with multiple products, OP and non-OP MCs and overlapping product paths. In this paper, we use a particular model of MCs with input events and output events that characterize the arrival and departure of products, respectively. We note that such model is also used in existing work such as [47] but in a different context. We further remark that automata models that preserve the order of certain events are also used in the recent papers [48], [49]. Different from this paper, our previ- ous work in [48] focuses on the the algorithmic construction of models for MCs including MCs that preserve the order but without a theoretical analysis. Likewise, the work in [49] uses automata models that preserve the order of message transmis- sions and receptions for FIFO channels in networked DES without a comprehensive analysis and without considering the composition of such models. The remainder of the paper is organized as follows. In the scope of this paper, we consider DES models based on ﬁnite state automata. I. INTRODUCTION Here, the literature generally adopts models of plant and speciﬁcation that are composed of multi- ple components to address the state space explosion problem [33], [36]–[40]. It has to be emphasized that the process of obtaining accurate DES models is not trivial [38], [40]. In particular, tools for supporting the modeling process of both plant and speciﬁcation including the level of abstraction and the modeling of standard operations are highly desirable. This topic is explored by providing templates for basic DES operations in [41]. Furthermore, recent work such as [16], [42] proposes the concept of distinguishers in order to obtain smaller and more easily understandable speciﬁcations for DES. In particular, distinguishers allow identifying different instances of the same event. Moreover, [43] provides re- usable components for the supervisory control of modular production systems and [40] suggests modeling guidelines for large-scale systems. g y In view of the previous discussion, this paper is concerned with the formal deﬁnition, analysis and application of a gen- eral model for an AMS property that has not been investi- gated in the existing literature. In particular, we consider the practical scenario, where multiple products that are processed sequentially can be present in a MC. This is for example the case for a conveyor belt that can hold multiple products. If all the products have the same type, such system can be modeled as a simple buffer since all products will be processed in the same way and follow the same path after leaving the MC. However, for advanced AMSs such as FMSs or RMSs, it is possible that products with different types can enter such MC. That is, different manufacturing processes might be applied to the product on the MC or on the subsequent path through the AMS. Therefore, it is necessary to keep track of the different product types entering and leaving such MC. To the best of our knowledge, the described scenario is not considered in the literature. On the one hand, there is existing work that incorporates the product order in the system model. Nevertheless, these works either use example systems where different product types follow independent paths through the AMS [11], [16], [44] or they deﬁne the product paths such that it is not necessary to remember the product order for MCs that can hold more than one product [9], [14], [15], [34], [35], [45], [46]. I. INTRODUCTION tiple products can share (parts of) the same path through the AMS, while requiring different processing steps. In order to process products correctly, it is hence important to keep track of the type of products traveling through such AMS. The main focus of this paper is the modeling and supervisory control of AMSs with different product types and overlapping product paths. Discrete event system (DES) models are suitable for rep- resenting sequential and concurrent processes in technical systems such as automated manufacturing systems (AMSs) [1]–[8]. Such systems consist of a given hardware setup with various manufacturing components (MCs) such as machines, robots and conveyor belts [9]–[13]. It is generally desired to move products along pre-deﬁned paths and to use pre-speciﬁed MCs for processing these products in a given sequence while avoiding deadlock situations [14]–[16]. Hereby, it is the case that multiple products are present in different parts of such AMS simultaneously and are processed concurrently. Moreover, advanced AMSs such as ﬂexible manufacturing systems (FMSs) or reconﬁgurable manufac- turing systems (RMSs) are able to process different product types using the same MCs [9], [17]–[20]. Accordingly, mul- Using DES models, the logic control of AMSs can be carried out in the framework of supervisory control for DES [21], [22]. That is, a supervisor is designed based on a formal model of the AMS and a formal speciﬁcation of the desired manufacturing sequences. Hereby, achieving the desired system behavior depends both on the adequate formulation of model and speciﬁcation and the applica- tion of a computationally efﬁcient supervisor design pro- cedure. Regarding supervisor design, the existing literature considers various aspects of the logic control of AMSs using different modeling frameworks. The avoidance of deadlocks or forbidden states in Petri Nets is addressed The associate editor coordinating the review of this manuscript and approving it for publication was Yue Zhang . 131901 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs in [5], [23]–[28], the enforcement of linear constraints on Petri Net markings is pursued in [29]–[32] and [14], [15], [33]–[35] develop modular and hierarchical methods for the efﬁcient computation of supervisors for AMSs based on ﬁnite state automata models. order-preserving (OP) languages over an alphabet with input events and corresponding output events based on this dis- cussion. As the distinctive property, the order of input and corresponding output events in any string of an OP language is the same. I. INTRODUCTION On the other hand, [4], [10], [23], [25] do not take into account the product order and implicitly assume that each MC knows which product is currently transported. Practically, this assumption requires a sensor that detects the product type at each MC. The remainder of the paper is organized as follows. Section II summarizes the necessary background information on the supervisory control of DES and provides a prob- lem statement based on a motivating example. Section III introduces the notion of OP languages, analyzes their most important properties and discusses the usage of OP languages for supervisory control. An FMS example in Section IV demonstrates the applicability of the deﬁned OP models and illustrates the advantages when applying the composition of OP languages. Conclusions and ideas for future work are presented in Section V. In order to address the described issue, we ﬁrst discuss scenarios where the model of a MC needs to keep track of the product type and the order of products entering and leaving. As the ﬁrst contribution of the paper, we deﬁne C. PROBLEM STATEMENT The work in this paper is motivated by an observation from modeling complex AMSs, where (i) different product types can be processed on the same MC and (ii) MCs might have different capacities in the sense of being able to hold several products that are then processed sequentially. In order to illustrate this observation, we consider different conveyor belts (CBs) as shown in Fig. 1. We next discuss the cases in the different parts of Fig. 1 together with possible models. Hereby, we introduce events such as inP1, inP2, . . . for products entering the CB and outP1, outP2, . . . for products leaving the CB. Part (a) and (b) show the case of a CB with one and two product types and a capacity of one product. In addition, part (c) considers the case of one product type and a capacity of two. These cases can be straightforwardly modeled by the automata in Fig. 2 (a), (b) and (c). In particular, it can be directly seen that the order of product types entering and leaving the CB is A formal language over 6 is a subset L ⊆6⋆. L := {s1 ∈ 6⋆\| ∃s ∈L s.t. s1 ≤s} deﬁnes the preﬁx closure of L, and L is called preﬁx closed if L = L. Consider two alphabets 61, 62 and their union 6 = 61 ∪62. Then, the natural projection pi : 6⋆→6⋆ i , i = 1, 2, is deﬁned iteratively such that (1) pi(ϵ) := ϵ; (2) for s ∈6⋆, σ ∈6: pi(sσ) := pi(s)σ if σ ∈6i, or pi(sσ) := pi(s) otherwise. The set-valued inverse of pi is written as p−1 i : 6⋆ i →26⋆, p−1 i (t) := {s ∈6⋆\| pi(s) = t}. Using the natural projection, the synchronous composition L1\|\|L2 ⊆6⋆of two languages Li ⊆6⋆ i is computed as L1\|\|L2 = p−1 1 (L1) ∩p−1 2 (L2) ⊆6⋆. In addition, we say that L1 and L2 are non-conﬂicting if it holds that FIGURE 1. CBs with different product types and capacities: (a) one product type and capacity one; (b) two product types and capacity one; (c) one product type and capacity two; (d) two product types and capacity two; (e) two product types and capacity three; (f) three product types and capacity two; (g) two connected CBs with different product types. 131903 L1\|\|L2 = L1\|\|L2. II. PRELIMINARIES This section provides the necessary notation for the for- mal developments in the paper. Section II-A introduces VOLUME 8, 2020 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs sσ ∈L(S). Then, L(G)\|\|L(S) and Lm(G)\|\|Lm(S) represent the closed and marked behavior of the closed-loop system G\|\|S, respectively. sσ ∈L(S). Then, L(G)\|\|L(S) and Lm(G)\|\|Lm(S) represent the closed and marked behavior of the closed-loop system G\|\|S, respectively. the notions related to discrete event systems (DES) and Section II-B gives background information on the super- visory control theory. In addition, Section II-C states the problem considered in this paper based on a motivating example. A language K ⊆Lm(G) is said to be controllable for L(G) and 6u if K6u ∩L(G) ⊆K. There exists a supervisor S such that Lm(G\|\|S) = K if and only if K is controllable for L(G) and 6u [21]. In case, K is not controllable for L(G) and 6u, the supervisor will implement the supremal controllable sub- language of K. We write Lm(S\|\|G) = SupC(K, G, 6u). It is ensured that such supervisor is nonblocking and maximally permissive if SupC(K, G, 6u) ̸= ∅[50]. C. PROBLEM STATEMENT (1) (1) We model a DES as a ﬁnite state automaton G = (X, 6, δ, x0, Xm), with the ﬁnite set of states X; the ﬁnite alphabet of events 6; the partial transition function δ : X × 6 →X; the initial state x0 ∈X and the set of marked states Xm ⊆X. Hereby, δ(x, σ)! ís written if δ is deﬁned at (x, σ) and we extend the transition function δ to a partial function on X ×6⋆in the usual way. The behavior of G is given by its closed language L(G) := {s ∈6⋆\|δ(x0, s)!} and its marked language Lm(G) := {s ∈L(G)\|δ(x0, s) ∈Xm}. We say that G is nonblocking if L(G) = Lm(G). The synchronous compo- sition G1\|\|G2 of two automata G1, G2 is deﬁned in the usual way [22], whereby it holds that L(G1\|\|G1) = L(G1)\|\|L(G2) and Lm(G1\|\|G2) = Lm(G1)\|\|Lm(G2). A. DISCRETE EVENT SYSTEMS (DES) We characterize the behavior of a DES by formal languages over ﬁnite alphabets 6. Each element σ ∈6 is denoted as an event, 6⋆is the set of all ﬁnite strings over 6 and s1 s2 ∈6⋆ deﬁnes the concatenation of two strings s1, s2 ∈6⋆. s1 ≤s indicates that s1 is a preﬁx of s and \|s\| is the length of s, that is, the number of events in s. ϵ ∈6⋆is the empty string with \|ϵ\| = 0 and it holds that sϵ = ϵs = s for all s ∈6⋆. For any string s = σ1, . . . , σ\|s\| ∈6⋆with σi ∈6 for i = 1, . . . , \|s\|, we further write prek(s) = σ1 . . . σk for the preﬁx of s with the ﬁrst k events and sufk(s) = σk+1 · · · σ\|s\| for the sufﬁx of s after the ﬁrst k events. In particular, s = prek(s)sufk(s) for any k ≤\|s\|, pre0(s) = ϵ and suf0(s) = s for any string s ∈6⋆. B. SUPERVISORY CONTROL In supervisory control, we write 6 = 6c ˙∪6u for controllable (6c) and uncontrollable (6u) events. We say that an automa- ton S = (Q, 6, ν, q0, Qm) is a supervisor for a plant G if S can only disable events in 6c. In particular, it must hold for all s ∈L(G) ∩L(S) and σ ∈6u with sσ ∈L(G) that also FIGURE 1. CBs with different product types and capacities: (a) one product type and capacity one; (b) two product types and capacity one; (c) one product type and capacity two; (d) two product types and capacity two; (e) two product types and capacity three; (f) three product types and capacity two; (g) two connected CBs with different product types. FIGURE 1. CBs with different product types and capacities: (a) one product type and capacity one; (b) two product types and capacity one; (c) one product type and capacity two; (d) two product types and capacity two; (e) two product types and capacity three; (f) three product types and capacity two; (g) two connected CBs with different product types. FIGURE 1. CBs with different product types and capacities: (a) one product type and capacity one; (b) two product types and capacity one; (c) one product type and capacity two; (d) two product types and capacity two; (e) two product types and capacity three; (f) three product types and capacity two; (g) two connected CBs with different product types. VOLUME 8, 2020 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs FIGURE 2. CB models for different cases: (a) one product type and capacity one; (b) two product types and capacity one; (c) one product type and capacity two; (d-1) two product types and capacity two with arbitrary order; (d-2) two product types, capacity two and preserving the product order. In this case, products need not be distinguished and models as in Fig. 2 (a) and (c) are suitable. In the second case, it is possible that the paths of different products intersect [9], [10], [14], [15], [23], [25], [34], [35], [45], [46]. Although this implies that the same MC is passed by multiple products, these research works again make simplifying assumptions. One the one hand, [9], [14], [15], [34], [35], [45], [46] choose the control speciﬁcation such that remembering the product order is straightforward. Speciﬁcally, this is achieved by using MCs, whose capacity does not exceed one. On the other hand, [10], [23], [25] allow for MCs with multiple product types and a capacity that is greater than one. However, these papers use models as in Fig. 2 (d-1) for such MCs. That is, the order of products entering and leaving the MC is not taken into account. This means that knowledge about which product leaves such MC must be implicitly known. In a real application this implies that sensors need to be installed to detect the product type leaving such MC. In addition, using a model as in Fig. 2 (d-1) for an OP MC means that the model contains unnecessary behavior. FIGURE 2. CB models for different cases: (a) one product type and capacity one; (b) two product types and capacity one; (c) one product type and capacity two; (d-1) two product types and capacity two with arbitrary order; (d-2) two product types, capacity two and preserving the product order. either irrelevant or is preserved by these models. Differently, the modeling becomes more involved in part (d) of Fig. 1. Here, the CB has a capacity of two products and there are two different product types. That is, products of different types will leave the CB in the same order as entering the CB. In this case, the model in Fig. 2 (d-1), which is commonly used to model the scenario with two product types is not suitable. III. ORDER-PRESERVING LANGUAGES This section deﬁnes and analyzes OP DES models as dis- cussed in the previous section. First, Section III-A introduces the required notation for the deﬁnition of OP languages and determines their basic properties. Then, Section III-B proves the existence of a supremal OP language and Section III-C studies the composition of OP languages. Finally, Section III-D shows that reduced OP models that are suitable for supervisory control can be obtained after composition. In view of the previous discussion, the main aim of this paper is the construction of order-preserving (OP) models for AMSs that keep the order of product types entering and leaving each MC. Instead of constructing a separate model for each scenario, we develop a general OP DES model that is parametrized by the possible product types and the capacity of the MC. In addition, we show that reduced models can be constructed when composing such OP models from multiple MCs. VOLUME 8, 2020 Speciﬁcally, such model suggests that product type P2 can leave the CB before type P1 even P1 enters the CB ﬁrst (string inP1 inP2 outP2). A model that captures the correct order of product types entering and leaving the CB is shown in Fig. 2 (d-2). Here, only outP1 is possible in state 5 (after inP1inP2) and only outP2 is possible in state 6 (after inP2inP1). More complicated scenarios can easily be envisaged by increasing the capacity (as in Fig. 1 (e)) and/or the number of product types (as in Fig. 1 (f)). Moreover, it is possible to consider the connection of multiple CBs (or other MCs) that keep the order of product types as in Fig. 1 (g). Accordingly, this paper focuses on the problem of devel- oping a formal framework for OP DES models of MCs with multiple product types and capacities that are greater than one. Although an initial discussion of OP DES models is given in [48], that paper is restricted to the usage of such model for example applications. Differently, this paper pro- vides a comprehensive formal analysis including the con- struction of supremal OP DES models and the composition of OP DES models. A. NOTATION AND DEFINITIONS We consider an alphabet 6 that is divided into disjoint sets of input events 6in and output events 6out such that 6in ∪ 6out = 6 and 6in ∩6out = ∅. Moreover, we introduce the natural projections to the respective alphabets as pin : 6⋆→ (6in)⋆and pout : 6⋆→(6out)⋆. In addition, we deﬁne a bijective input/output mapping m : 6in →6out such that for each input event α ∈6in, m(α) ∈6out denotes the corresponding output event. We further write m−1 : 6out → 6in for the inverse mapping. It is interesting to note that, although the existing literature considers the control of AMSs with different product types [9]–[11], [14]–[16], [23], [25], [34], [35], [44]–[46], none of the existing works develops DES models that preserve the order of products. In order to avoid the need for OP DES models, existing work restricts the general case by means of the control speciﬁcation. In particular, two special cases can be observed. Relating this deﬁnition to the discussion about AMSs in the previous section, an input event α ∈6in and output event m(α) represent a product entering and leaving a MC, respectively. The ﬁrst special case is restricted to AMSs, where dif- ferent product types use disjoint paths through the system and are hence processed by different MCs [11], [16], [44]. 131904 VOLUME 8, 2020 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs Extending this notion to languages, the capacity of any OP language L ∈LOP(6in, 6out, m) is written as Extending this notion to languages, the capacity of any OP language L ∈LOP(6in, 6out, m) is written as Given the above notation, we introduce the new notion of an OP language, whose strings preserve the order of input events and their corresponding output events. c(L) = max s∈L {c(s)}. (5) (5) Deﬁnition 1: Consider 6, 6in, 6out, pin, pout and m as introduced above. A string s ∈6⋆is denoted as OP if That is, the capacity of an OP string s ∈6⋆captures the maximum difference between the number of input events and the number of output events in any preﬁx s′ of s. Analogously, the capacity of an OP language is given by the maximum capacity of any string s ∈L. B. SUPREMAL ORDER-PRESERVING LANGUAGE B. SUPREMAL ORDER-PRESERVING LANGUAGE In order to analyze properties of OP languages, we introduce In order to analyze properties of OP languages, we introduce Referring to Deﬁnition 1, we write (6) LOP(6in, 6out, m) = {L ⊆6⋆\|L is OP} (3) (3) as the set of OP languages with alphabet 6 = 6in ∪6out and capacity C. Our ﬁrst result shows that OP languages of a given capacity C are closed under arbitrary union with a supremal element Lsup C (6in, 6out, m). for the set of all OP languages with alphabet 6 = 6in ∪6out and the map m. In addition, we introduce the notion of the capacity of an OP language. p C ( ) Theorem 1: Let LOP C (6in, 6out, m) be as deﬁned in (6). Then, LOP C (6in, 6out, m) is closed under arbitrary union and contains a supremal element Deﬁnition 2: Assume 6in, 6out, 6, pin and pout are given as above. Consider an OP string s ∈6⋆. Then, the capacity of s is deﬁned by the function c : 6⋆→N as Lsup C (6in, 6out, m) = [ L∈LOP C (6in,6out,m) L. (7) c(s) = max s′≤s {\|pin(s′)\| −\|pout(s′)}. c(s) = max s′≤s {\|pin(s′)\| −\|pout(s′)}. (4) (7) (4) We note that, for ease of notation, we write LOP, LOP C and Lsup C whenever 6in, 6out and m are clear from the context. C Proof: We ﬁrst show that LOP C is closed under arbitrary union. To this end, let L1, L2 ∈LOP C . We have to show that L1 ∪L2 ∈LOP C . Take an arbitrary string s ∈L1 ∪L2. Then, s ∈L1 or s ∈L2. In both cases, it holds that s is OP since L1 and L2 are both OP. Hence, it follows that L1 ∪L2 is OP. Moreover, c(s) ≤C since c(L1) = c(L2) = C. It remains to show that there exists at least one string s′ ∈L1 ∪L2 such that c(s′) = C. Without loss of generality, we can take some s′ ∈L1 ⊆L1 ∪L2 such that c(s′) = C since c(L1) = C. Together, we conclude that L1 ∪L2 is OP and c(L1 ∪L2) = C. Hence, L1 ∪L2 ∈LOP C . Now, consider Lsup C = S L∈LOP C L. Since LOP C is closed under arbitrary union, it must hold that FIGURE 3. Example automata for OP languages. A language L ⊆6⋆is denoted as OP if it holds for all s ∈L that s is OP. That is, each OP string has the same order of input events and corresponding output events. In particular, it must be the case that each OP string s ∈6⋆has the same number of input and output events: \|pin(s)\| = \|pout(s)\| and each preﬁx s′ ≤s of an OP string s must fulﬁll Consider the OP languages L2 = Lm(G2) and L3 = Lm(G3) of the automata G2 and G3 in Fig. 3. It holds that c(L2) = 2 since the largest difference between the number of input and output events is obtained for strings s ∈L2 that contain the substring a1a2. Differently, c(L3) = 1 since each input event ai, i = 1, 2, is directly followed by the corresponding output event bi. pout(s′) ≤m(pin(s′)). In order to relate the notion of capacity to practical systems, we consider AMSs as discussed in the previous section. Here, input events could be identiﬁed with products of certain types entering a MC, whereas output events can characterize the corresponding products exiting a MC. Therefore, modeling an AMS by an OP language L ensures that products leave the system in the order of entering the system. In that case, the capacity c(L) indicates the maximum number of products that can be in the system simultaneously. This means that an output event can only occur after its corresponding input event. For illustration, we consider the languages L1 = Lm(G1) and L2 = Lm(G2) of the automata G1 and G2 in Fig. 3. Here, we assume that 6 = {a1, a2, b1, b2}, 6in = {a1, a2}, 6out = {b1, b2} and m(a1) = b1, m(a2) = b2. For example, it holds that s1 = a1a2b1b2 ∈L1 and s2 = a2b2a1a2b1b2 ∈ L2 are OP strings. Speciﬁcally, m(pin(s1)) = m(a1a2) = b1b2 = pout(s1) and m(pin(s2)) = m(a2a1a2) = b2b1b2 = pout(s2). It can be further veriﬁed that L2 is an OP language. Nevertheless, it is the case that L1 is not OP. This can be seen by looking at the string s3 = a1a2b2b1 ∈L1 with m(pin(s3)) = b1b2 ̸= pout(s3) = b2b1. A. NOTATION AND DEFINITIONS That is, the capacity of an OP string s ∈6⋆captures the maximum difference between the number of input events and the number of output events in any preﬁx s′ of s. Analogously, the capacity of an OP language is given by the maximum capacity of any string s ∈L. m(pin(s)) = pout(s). (2) (2) A language L ⊆6⋆is denoted as OP if it holds for all s ∈L that s is OP. ( ) = S L∈LOP C L. FIGURE 3. Example automata for OP languages. , αj], αj+1) = [α1, . . . , αjαj+1] for all [α1, . . . , αj] ∈Xsup C and αj+1 ∈6in, j = 1, . . . , C −1, R4 δsup C ([α1, α2, . . . , αj], ω1) = [α2, . . . , αj] for ω1 = m(α1) and for all [α1, α2, . . . , αj] ∈Xsup C , j = 2, . . . , C. C C Finally, we consider the state set Xsup C . There are \|6in\|j combinations for each equivalence class [α1 · · · αj], j = 1, . . . , C and there is one equivalence class [ϵ]. Hence, it fol- lows that Gsup C has a ﬁnite number of C That is, δsup C is deﬁned such that the state of Gsup C keeps track of the sequence of input events, whose corresponding output events did not occur, yet. Speciﬁcally, R1 and R3 address the case where an input event occurs. This input event is hence appended to the current sequence of input events. Conversely, the ﬁrst input event α1 is removed from the sequence of input events in R2 and R4 if its corresponding output event ω1 = m(α1) occurs. Here, it is important to note that only the output event of the ﬁrst input event in the sequence can occur so as to preserve the order of input and output events. In addition, the marked state [ϵ] corresponds to the case where the sequence of input events, whose corresponding output events did not occur, yet, is empty. That is, strings s ∈Lm(Gsup C ) have the property that m(pin(s)) = pout(s) and hence belong to Lsup C . \|Xsup C \| = C X j=0 \|6in\|j states. This directly implies that Lsup C is a regular language. □ According to Theorem 2, Lsup C can be realized by a canon- ical recognizer Gsup C with a ﬁnite number of states as given in (10) that depends on the number of input events \|6in\| and the capacity C. Hereby, each state of Gsup C represents an equivalence class of ≡OP as deﬁned in (9) and the tran- sition relation of Gsup C is deﬁned by the rules (R1) to (R4). It is hence straightforward to compute Gsup C . FIGURE 3. Example automata for OP languages. VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs (i) s ∈Lm(Gsup C ) ⇒s is OP and c(s) ≤C, (ii) s ∈Lsup C ⇒s ∈Lm(Gsup C ). Lsup C ∈LOP C . Hence, Lsup C is indeed the supremal OP language with capacity C. □ (i) s ∈Lm(Gsup C ) ⇒s is OP and c(s) ≤C, C (ii) s ∈Lsup C ⇒s ∈Lm(Gsup C ). h d il d f f 1 In order to further characterize Lsup C , we ﬁrst deﬁne the function The detailed proof of Lemma 1 is provided in Appendix A. The lemma indicates that (i) any string in the marked lan- guage of Gsup C is OP and its capacity is bounded by C and (ii) any OP string, whose capacity is bounded by C belongs to the marked language of Gsup C . Using Lemma 1, Theorem 2 shows that Gsup C has a ﬁnite number of states and Lm(Gsup C ) = Lsup C . f : Lsup C →(6in)⋆: f (s) = suf\|pout(s)\|(pin(s)) (8) (8) for any given s ∈Lsup C . That is, f (s) represents the string of input events in s, whose corresponding output events did not occur, yet. Consider for example the string s = a1a2b1b2a2b2a1a2 ∈L2 = L(G2) in Fig. 3 with f (s) = suf\|b1b2b2\|(pin(s)) = suf3(a1a2a2a1a2) = a1a2. Then, we introduce the equivalence relation ≡OP as the equivalence kernel of f such that for any two strings s, s′ ∈Lsup C , C C C Theorem 2: Consider Lsup C as deﬁned in (7) and Gsup C as introduced above. Then, it holds that Gsup C is a canonical recognizer of Lsup C and \|Xsup C \| = C X i=0 \|6in\|i. (10) (10) s ≡OP s′ ⇐⇒f (s) = f (s′). (9) (9) In particular, Lsup C is a regular language. sup Using ≡OP, we construct the automaton Gsup C = (Xsup C , 6, δsup C , xsup 0,C , Xsup m,C ) as a recognizer of Lsup C . The state set of Gsup C is given by the quotient set Xsup C = Lsup C / ≡OP of Lsup C . Since any string s ∈Lsup C has a capacity c(s) ≤C, it must be the case that \|f (s)\| = \|pin(s)\| −\|pout(s)\| ≤C for any s ∈Lsup C . FIGURE 3. Example automata for OP languages. Hence, the quotient set Proof: In order to prove that Gsup C is a canonical rec- ognizer of Lsup C , we ﬁrst show that Lm(Gsup C ) = Lsup C . That is, Lm(Gsup C ) ⊆Lsup C and Lsup C ⊆Lm(Gsup C ). For the ﬁrst inclusion, let s ∈Lm(Gsup C ). Then, it follows from Lemma 1 (i) that s is OP and c(s) ≤C. That is, s ∈Lsup C . The second inclusion is directly implied by (ii) in Lemma 1. Xsup C = {[ϵ]} ∪{[α1, . . . , αj]\|j ∈{1, . . . , C} In addition, consider an arbitrary state x = [α1 · · · αj] ∈ Xsup C for 1 ≤j ≤C and αi ∈6in for 1 ≤i ≤j. Applying R4 iteratively, it holds that δsup C (x, ω1 · · · ωj−1) = [αj] ∈Xsup C with ωi = m(αi), i = 1, . . . , j −1. Furthermore, δsup C ([αj], ωj) = [ϵ] ∈Xsup m,C for ωj = m(αj) and with R2. Together, we have that δsup C (x, ω1 · · · ωj) = [ϵ] ∈Xsup m,C. Nevertheless, for any state ˆx ∈Xsup C such that ˆx ̸= x, it must be the case that ¬δsup C (ˆx, ω1 · · · ωj)! or δsup C (ˆx, ω1 · · · ωj) ̸= [ϵ] (otherwise ˆx = x when applying R4 and R2). Hence, none of the states in Xsup C can be equivalent according to the Nerode equivalence [22]. Since Lm(Gsup C ) = Lsup C , this implies that Gsup C is a canonical recognizer of Lsup C . ∧αi ∈6in, ∀i = 1, . . . , j} consists of the equivalence class [ϵ] and one equivalence class [α1 · · · αj] for each possible sequence α1 · · · αj of j arbitrary input events αi ∈6in, 1 ≤i ≤j, whereby 1 ≤j ≤C. The initial state xsup 0,C = [ϵ] corresponds to s = ϵ ∈Lsup C and the set of marked states is Xsup m,C = {[ϵ]}. Furthermore, the transition relation of Gsup C is deﬁned with the following rules: C R1 δsup C ([ϵ], α1) = [α1] for all α1 ∈6in, C ( ) R2 δsup C ([α1], ω1) = [ϵ] for ω1 = m(α1), C R3 δsup C ([α1, . . . FIGURE 3. Example automata for OP languages. In words, we consider that the order of the ﬁrst k input events in s is equal to the order of the ﬁrst k output events, whereas the order of the ﬁrst k + 1 input events of s is different from the order of the ﬁrst k + 1 output events. Since m(pin(u1)) = pout(u1αu2), this implies that m(α) ̸= ω. FIGURE 4. OP DES model of a CB with three product types P1, P2, P3 and capacity C = 2. We further know that s ∈L = p(L1\|\|L2) implies that there are s1 ∈L1 and s2 ∈L2 such that s = p(s1\|\|s2). Since Li is OP for i = 1, 2, also si is OP for i = 1, 2. Considering that 6in 1 = 6in, 6out 2 = 6out, 61 ∩6out 2 = ∅and 62 ∩6in 1 = ∅, we further conclude that pin 1 (s1) = pin(s), pout 2 (s2) = pout(s) and pout 1 (s1) = pin 2 (s2). FIGURE 4. OP DES model of a CB with three product types P1, P2, P3 and capacity C = 2. 1 2 Accordingly, we determine prek+1m1(pin 1 (s1)) = prek+1 m1(pin(s)) = m1(pin(u1α)) = m1(pin(u1))α and prek+1m−1 2 (pout 2 (s2)) = prek+1m−1 2 (pout(s)) = m−1 2 (pout(u1αu2ω)) = m−1 2 (pout(u1αu2))m−1 2 (ω). Since m(pin(u1)) = m2(m1(pin(u1))) = pout(u1αu2) and 6out 1 = 6in 2 , we conclude that prekm1(pin 1 (s1)) = prekm−1 2 (pout 2 (s2)). However, FIGURE 3. Example automata for OP languages. As an example, consider 6 = 6in ∪6out = {a1, a2} ∪{b1, b2} with m(a1) = b1 and m(a2) = b2. Then, Fig. 3 shows G3 = Gsup 1 and G4 = Gsup 2 . We next state Lemma 1, which formalizes the relationship between Lsup C and Gsup C . We next state Lemma 1, which formalizes the relationship between Lsup C and Gsup C . We next state Lemma 1, which formalizes the relationship between Lsup C and Gsup C . C C Lemma 1: Consider Lsup C as deﬁned in (7) and Gsup C as introduced above. It holds that Lemma 1: Consider Lsup C as deﬁned in (7) and Gsup C as introduced above. It holds that Moreover, as a more practical example, we consider a CB in an AMS that can transport 3 different product types P1, 131906 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs We ﬁrst show that s is OP. Assume the contrary. That is s ∈L and s is not OP. Then, we can write s = u1αu2ωu3 with u1, u2, u3 ∈6⋆, α ∈6in and ω ∈6out, such that for some k < \|pin(s)\|\| P2 and P3 and that can hold up to 2 products simultaneously. It is further the case that products leave the CB in the same sequence as entering the CB. Hence, the behavior of such CB should be modeled by a supremal OP language with the input events 6in = {iP1, iP2, iP3} and the output events 6out = {oP1.oP2, oP3}. The mapping m is deﬁned by m(iPi) = oPi for i = 1, 2, 3 and the capacity is C = 2. According to (10), the canonical recognizer GCB for the CB as shown in Fig. 4 has 13 states. Hereby, there is one product on the CB in the states shaded in light gray, whereas there are two products on the CB in the states shaded in dark gray. prekm(pin(s)) = m(pin(u1)) = prekpout(s) = pout(u1αu2) prekm(pin(s)) = m(pin(u1)) = prekpout(s) = pout(u1αu2) but prek+1m(pin(s)) = m(pin(u1α)) ̸= prek+1pout(s) = pout(u1αu2ω) = pout(u1αu2)ω. C. COMPOSITION OF ORDER-PRESERVING LANGUAGES Practical AMSs are commonly composed of various modular system components. That is, we next investigate the com- position of multiple MCs with OP languages. To this end, we ﬁrst consider the case of two OP languages Li ⊆6⋆ i over the alphabet 6i with given capacities c(Li) = Ci and maps mi : 6in i →6out i for i = 1, 2. In addition, we assume that the output events of the ﬁrst language are the input events of the second language: 6out 1 = 6in 2 . In an AMS, such languages could represent two MCs, whereby the ﬁrst MC delivers products to the second MC. Deﬁning the alphabets 6in = 6in 1 , 6out = 6out 2 , 6 = 6in ∪6out, the natural projections p : (61∪62)⋆→6⋆, pi : (61∪62)⋆→6⋆ i , i = 1, 2 and the mapping m : 6in →6out : m(s) = m2(m1(s)), we determine the composed language prek+1m1(pin 1 (s1)) = m1(pin(u1))m1(α)̸=prek+1m−1 2 (pout 2 (s2)) = m−1 2 (pout(u1αu2))m−1 2 (ω) prek+1m1(pin 1 (s1)) = m1(pin(u1))m1(α)̸=prek+1m−1 2 (pout 2 (s2)) = m−1 2 (pout(u1αu2))m−1 2 (ω) because m1(α) ̸= m−1 2 (ω) (which follows from m(α) ̸= ω). Hence, pout 1 (s1) ̸= pin 2 (s2) such that p− 1 1(s1) ∩p− 2 1(s2) = ∅, which implies that s ̸∈p(p−1 1 (s1)∩p−1 2 (s2)) ⊆p(L1\|\|L2) = L. This is a contradiction, because we assumed that s ∈L. It remains to show that c(L) = C ≤C1 + C2. To this end, let s ∈p(L1\|\|L2) and s1 ∈L1, s2 ∈L2 such that s = p(s1\|\|s2). From the previous discussion, we know that for each s′ ≤s, pin(s′) = pin 1 (s′ 1), pout(s′) = pout 2 (s′ 2) and pout 1 (s′ 1) = pin 2 (s′ 2) for some s′ 1 ≤s1, s′ 2 ≤s2. That is, we compute L = p(L1\|\|L2). (11) (11) c(s) = max s′≤s {\|pin(s′)\| −\|pout(s′)\|} i c(s) = max s′≤s {\|pin(s′)\| −\|pout(s′)\|} = max s′ 1≤s1,s′ 2≤s2,s′=p(s′ 1\|\|s′ 2){\|pin 1 (s′ 1)\| −\|pout 2 (s′ 2)\|} = max s′ 1≤s1,s′ 2≤s2,s′=p(s′ 1\|\|s′ 2){\|pin 1 (s′ 1)\| −\|pout 1 (s′ 1)\| \| {z } ≤C1 + \|pin 2 (s′ 2)\| −\|pout 2 (s′ 2)\| \| {z } ≤C2 } ≤C1 + C2. That is, we project L1\|\|L2 to the alphabet 6, which only contains the input and output events of the composed lan- guage. C. COMPOSITION OF ORDER-PRESERVING LANGUAGES We next show that L is an OP language with capacity C ≤C1 + C2. Theorem 3: Assume that 6in 1 , 6out 1 , 6in 2 , 6out 2 , m1, m2, 6, m and p are as introduced above with 6in = 6in 1 , 6out = 6out 2 and 6out 1 = 6in 2 . Let L1 ∈LOP C1 (6in 1 , 6out 1 , m1), L2 ∈LOP C2 (6in 2 , 6out 2 , m2) and suppose that L1\|\|L2 ̸= ∅. Then {z ≤C2 Since s ∈L was arbitrary, it follows that c(L) = C ≤C1+C2. That is, L ∈LOP C (6in, 6out, m) for some C ≤C1 + C2. □ Since s ∈L was arbitrary, it follows that c(L) = C ≤C1+C2. That is, L ∈LOP C (6in, 6out, m) for some C ≤C1 + C2. □ L = p(L1\|\|L2) ∈LOP C (6in, 6out, m) (12) L = p(L1\|\|L2) ∈LOP C (6in, 6out, m) (12) for some C ≤C1 + C2. be deﬁned as in (8). Then, it holds that p(t)σ ∈p(L1\|\|L2) ⇒∃v ∈(61 ∪62)⋆s.t. p(v) = σ and tv ∈L1\|\|L2. (14) p(t)σ ∈p(L1\|\|L2) ⇒∃v ∈(61 ∪62)⋆s.t. (14) The proof of Lemma 4 is given in Appendix D. Using Lemma 2 to 4, we next prove Theorem 4. Proof: In order to show that L = p(L1\|\|L2) = Lsup C1+C2, we show that (i) L ⊆Lsup C1+C2 and (ii) L ⊇Lsup C1+C2. Proof: In order to show that L = p(L1\|\|L2) = Lsup C1+C2, we show that (i) L ⊆Lsup C1+C2 and (ii) L ⊇Lsup C1+C2. Proof: In order to show that L = p(L1\|\|L2) = Lsup C1+C2, we show that (i) L ⊆Lsup C1+C2 and (ii) L ⊇Lsup C1+C2. C1+C2 C1+C2 (i) Since L1 = Lsup C1 and L2 = Lsup C2 for C1, C2 > 0, it holds that ϵ ∈L1\|\|L2 ̸= ∅. Hence, we know from Theorem 3 that L is OP and Lemma 2 implies that c(L) = C = C1 + C2. Hence, L ⊆Lsup C1+C2. C1+C2 C1+C2 (i) Since L1 = Lsup C1 and L2 = Lsup C2 for C1, C2 > 0, it holds that ϵ ∈L1\|\|L2 ̸= ∅. Hence, we know from Theorem 3 that L is OP and Lemma 2 implies that c(L) = C = C1 + C2. Hence, L ⊆Lsup C1+C2. 1 2 1 2 (i) Since L1 = Lsup C1 and L2 = Lsup C2 for C1, C2 > 0, it holds that ϵ ∈L1\|\|L2 ̸= ∅. Hence, we know from Theorem 3 that L is OP and Lemma 2 implies that c(L) = C = C1 + C2. Hence, L ⊆Lsup C1+C2. 1+ 2 (ii) In order to show that L ⊇Lsup C , we take an arbitrary OP string s ∈Lsup C . First, we show by induction that s ∈ p(L1\|\|L2). To this end, we write s = σ1 · · · σ\|s\| with σi ∈6 for i = 1, . . . , \|s\|. For the initialization, consider s0 = pre0 s = ϵ. Since ϵ ∈ L1 and ϵ ∈L2, it follows that ϵ ∈L1\|\|L2 ⊆L1\|\|L2. Hence, we have that t0 = ϵ ∈L1\|\|L2 and s0 = p(t0) ∈p(L1\|\|L2). For the induction step, we assume that sk−1 = prek−1s = σ1 · · · σk−1 for some 1 ≤k ≤\|s\| and there is a tk−1 ∈L1\|\|L2 such that p(tk−1) = sk−1. Now consider σk ∈6. be deﬁned as in (8). Then, it holds that In addition, L3 and L7 as well as L4 and L7 are non-conﬂicting. In the sequel, we formalize the observations from this example in Theorem 4 and 5. recognizer G25 (excluding the states shaded in gray) with L25 = Lm(G25) = p(L2\|\|L5) and 6in = {a1, a2} and 6out = {c1, c2} is shown in Fig. 6. It is readily observed that L25 is OP and C25 = c(L25) = 3 = C2 + C5. For illustration, the states of G25 are colored such that orange, blue and green states correspond to strings with capacity 1, 2 and 3, respectively. Moreover, G25 shows two blocking states 2 and 6 that are shaded in gray. In particular, it holds that L2\|\|L5 ̸= L2\|\|L5 such that L2 and L5 are conﬂicting. That is, even Theorem 3 shows that any string s ∈Lm(G25) is OP, there might be strings in L(G25) that cannot be extended to an OP string. Next, we consider L2 = Lm(G2) with C2 = 2 in Fig. 3 and L6 = Lm(G6) with C6 = 2 in Fig. 5. A canonical recognizer G26 for L26 = Lm(G26) = p(L2\|\|L6) with 6in = {a1, a2} and 6out = {c1, c2} is shown in Fig. 6. Here, it turns out that C26 = 3 ̸= C1 + C2 = 4. That is, the capacity of L26 is smaller than the accumulated capacity of L2 and L6. This is possible according to Theorem 3 and occurs since L2 ⊂Lsup 2 and L6 ⊂Lsup 2 . We ﬁnally consider the composition of L3 = Lm(G3) = Lsup 1 and L4 = Lm(G4) = Lsup 2 in Fig. 3 with L7 = Lm(G7) = Lsup 1 in Fig. 5. The resulting OP languages L37 = Lm(G37) and L47 = Lm(G47) are shown in Fig. 6. Hereby, it is interesting to note that L37 = Lsup 2 and L47 = Lsup 3 . In addition, L3 and L7 as well as L4 and L7 are non-conﬂicting. In the sequel, we formalize the observations from this example in Theorem 4 and 5. Lemma 4: Consider the setting in Theorem 4. Then, it holds for any t ∈L1\|\|L2 and σ ∈6 that p(t)σ ∈p(L1\|\|L2) ⇒∃v ∈(61 ∪62)⋆s.t. p(v) = σ and tv ∈L1\|\|L2. (14 The proof of Lemma 4 is given in Appendix D. Using Lemma 2 to 4, we next prove Theorem 4. be deﬁned as in (8). Then, it holds that Then, it holds that tk−1 ∈L1\|\|L2 and sk−1 ∈p(L1\|\|L2) and σk ∈6 and p(tk−1)σk = sk−1σk = preks ∈p(L1\|\|L2). According to Lemma 4, this implies that there is a vk ∈(61 ∪62)⋆ such that p(vk) = σk and tk−1vk ∈L1\|\|L2. In particular, for k = \|s\|, it follows that t = t0v1 · · · v\|s\| ∈L1\|\|L2 and s = pre\|s\|s = p(t) ∈p(L1\|\|L2). Theorem 4: Assume that 6in 1 , 6out 1 , 6in 2 , 6out 2 , m1, m2, 6, m and p are as introduced above with 6in = 6in 1 , 6out = 6out 2 and 6out 1 = 6in 2 . Further, let L1 = Lsup C1 (6in 1 , 6out 1 , m1) and L2 = Lsup C2 (6in 2 , 6out 2 , m2) for C1, C2 > 0. Then, L = p(L1\|\|L2) = Lsup C1+C2(6in, 6out, m). C1+C2 Theorem 4 states that the composition of two supremal OP languages again yields a supremal OP language, whose capacity is the sum of the capacities of the composed lan- guages. \| \| This concludes the induction step and we found that s ∈ p(L1\|\|L2). It remains to show that s ∈p(L1\|\|L2). To this end, we ﬁrst conclude that f (s) = ϵ since s ∈Lsup C and t ∈s1\|\|s2 for some s1 ∈L1 and s2 ∈L2 since t ∈L1\|\|L2. Considering that f (s) = m−1 1 (f2(s2))f1(s1) according to Lemma 3, it must be the case that \|f2(s2))\| = \|f1(s1)\| = \|f (s)\| = 0. Hence, s1 ∈ L1 and s2 ∈L2, which implies that t ∈L1\|\|L2. Therefore, s = p(t) ∈p(L1\|\|L2). Since s ∈Lsup C was arbitrary, it follows that Lsup C ⊆p(L1\|\|L2), which concludes the proof of Theorem 4. □ In order to prove Theorem 4, we establish three lemmas that show general properties of L = p(L1\|\|L2). Lemma 2 determines that the capacity of L is c(L) = C = C1 + C2 if both languages Li, i = 1, 2 are supremal OP languages with capacity Ci. Lemma 2: Consider the setting in Theorem 4. Then, c(L) = C = C1 + C2. The proof of Lemma 2 is given in Appendix B. The proof of Lemma 2 is given in Appendix B. The next theorem conﬁrms the observation from the pre- vious example. It holds that the composition of two supremal OP languages is non-conﬂicting. for some C ≤C1 + C2. for some C ≤C1 + C2. C We demonstrate the implications of Theorem 3 using the example automata in Fig. 3 and 5. Proof: Since L1\|\|L2 ̸= ∅, there is at least one string s ∈ p(L1\|\|L2). Now consider any arbitrary string s ∈p(L1\|\|L2). We need to show that s is OP and c(s) ≤C1 + C2. First consider the languages L2 = Lm(G2) with C2 = 2 in Fig. 3 and L5 = Lm(G5) with C5 = 1 in Fig. 5. A canonical VOLUME 8, 2020 131907 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs FIGURE 5. Example automata for the composition of OP languages. be deﬁned as in (8). Then, it holds that f (s) = m−1 1 (f2(s2)) f1(s1). (13) (13) That is, for any string s = p(s1\|\|s2) ∈p(L1\|\|L2) the excess input events f (s), whose corresponding output events did not occur, yet, are either excess input events f1(s1) ∈(6in 1 )⋆or correspond to excess input events f2(s2) ∈(6in 2 )⋆. The proof of Lemma 3 is given in Appendix C. FIGURE 5. Example automata for the composition of OP languages. Lemma 4 shows that any extension sσ ∈p(L1\|\|L2) of a string s ∈p(L1\|\|L2) with an event σ ∈6 has a corresponding extension of any string t ∈L1\|\|L2 that has the projection p(t) = s. recognizer G25 (excluding the states shaded in gray) with L25 = Lm(G25) = p(L2\|\|L5) and 6in = {a1, a2} and 6out = {c1, c2} is shown in Fig. 6. It is readily observed that L25 is OP and C25 = c(L25) = 3 = C2 + C5. For illustration, the states of G25 are colored such that orange, blue and green states correspond to strings with capacity 1, 2 and 3, respectively. Moreover, G25 shows two blocking states 2 and 6 that are shaded in gray. In particular, it holds that L2\|\|L5 ̸= L2\|\|L5 such that L2 and L5 are conﬂicting. That is, even Theorem 3 shows that any string s ∈Lm(G25) is OP, there might be strings in L(G25) that cannot be extended to an OP string. Next, we consider L2 = Lm(G2) with C2 = 2 in Fig. 3 and L6 = Lm(G6) with C6 = 2 in Fig. 5. A canonical recognizer G26 for L26 = Lm(G26) = p(L2\|\|L6) with 6in = {a1, a2} and 6out = {c1, c2} is shown in Fig. 6. Here, it turns out that C26 = 3 ̸= C1 + C2 = 4. That is, the capacity of L26 is smaller than the accumulated capacity of L2 and L6. This is possible according to Theorem 3 and occurs since L2 ⊂Lsup 2 and L6 ⊂Lsup 2 . We ﬁnally consider the composition of L3 = Lm(G3) = Lsup 1 and L4 = Lm(G4) = Lsup 2 in Fig. 3 with L7 = Lm(G7) = Lsup 1 in Fig. 5. The resulting OP languages L37 = Lm(G37) and L47 = Lm(G47) are shown in Fig. 6. Hereby, it is interesting to note that L37 = Lsup 2 and L47 = Lsup 3 . Theorem 5: Consider the setting in Theorem 4. Then, L1 and L2 are non-conﬂicting. be deﬁned as in (8). Then, it holds that Lemma 3 introduces a decomposition of f (s) in (8) for any string s ∈L = p(L1\|\|L2). Lemma 3 introduces a decomposition of f (s) in (8) for any string s ∈L = p(L1\|\|L2). Lemma 3: Consider the setting in Theorem 4 and let s1 ∈ L1, s2 ∈L2 and s ∈L1\|\|L2 such that s = p(s1\|\|s2). Further, let f : 6⋆→(6in)⋆, f1 : 6⋆ 1 →(6in 1 )⋆and f2 : 6⋆ 2 →(6in 2 )⋆ Theorem 5: Consider the setting in Theorem 4. Then, L1 and L2 are non-conﬂicting. Theorem 5: Consider the setting in Theorem 4. Then, L1 and L2 are non-conﬂicting. 131908 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs nguages: Conflict in G25 and G26; Supremal OP languages in G37 and G47. rary string t ∈L1\|\|L2. Then, e s1 ∈L1, s2 ∈L2 and f (s) = or s = p(t) and some 0 ≤j ≤ e m−1 1 (f2(s2)) = α1 · · · αl and der ωi = m(αi) ∈6out for 1 ≤ ω1 · · · ωlγl+1ωl+1 · · · γjωj ∈ = m−1 2 (ωi), i = l + 1, . . . , j. 1(v) = γl+1 · · · γj and v2 = αl+1 · · · αj = m−1(v1) and FIGURE 7. Composition of OP languages. mal OP languages in G37 and G47. FIGURE 7. Composition of OP languages. FIGURE 6. Composition of OP languages: Conflict in G25 and G26; Supremal OP languages in G37 and G47. FIGURE 6. Composition of OP languages: Conflict in G25 and G26; Supremal OP languages in G37 and G47. Proof: Consider an arbitrary string t ∈L1\|\|L2. Then, it holds that t ∈s1\|\|s2 for some s1 ∈L1, s2 ∈L2 and f (s) = α1 · · · αj = m−1 1 (f2(s2))f1(s1) for s = p(t) and some 0 ≤j ≤ C = C1 + C2. We further write m−1 1 (f2(s2)) = α1 · · · αl and f1(s1) = αl+1 · · · αj. We consider ωi = m(αi) ∈6out for 1 ≤ i ≤j and deﬁne the string v = ω1 · · · ωlγl+1ωl+1 · · · γjωj ∈ (61 ∪62)⋆for γi = m1(αi) = m−1 2 (ωi), i = l + 1, . . . , j. be deﬁned as in (8). Then, it holds that In addition, we write v1 = p1(v) = γl+1 · · · γj and v2 = p2(v) = v. Since f1(s1) = αl+1 · · · αj = m−1 1 (v1) and L1 = Lsup C1 , it holds that s1v1 ∈L1. Furthermore, we ﬁrst con- clude that m−1 1 (f2(s2ω1 · · · ωl)) = ϵ since m(m−1 1 (f2(s2))) = m2(f2(s2)) = ω1 · · · ωl. That is, f2(s2ω1 · · · ωl) = ϵ. Then, it directly follows that f2(s2ω1 · · · ωlγl+1ωl+1 · · · γiωi) = ϵ for all i = l + 1, . . . , j. Hence, also s2v2 ∈L2. Together, we have FIGURE 7. Composition of OP languages. again a supremal OP language and these languages are further nonconﬂicting. Corollary 1: Consider alphabets 6in i , 6out i , 6i such that 6i = 6in i ∪6out i and 6in i ∩6out i = ∅for i = 1, . . . , n and assume that 6out i = 6in i+1 for i = 1, . . . , n −1. Further, let mi : 6in i →6out i be deﬁned as in Section III-A. Assume that Li = Lsup Ci (6in i , 6out i , mi) with Ci > 0 for i = 1, . . . , n. Write 6in = 6in 1 , 6out = 6out n , 6 = 6in ∪6out and let p : (Sn i=1 6i)⋆→6⋆as well as m : 6in →6out such that m(s) = mn(· · · m1(s)) for any s ∈(6in)⋆. Then, L = p(\|\|n i=1Li) = Lsup C1+···+Cn(6in, 6out, m) and L1, . . . , Ln are nonconﬂicting. VOLUME 8, 2020 tv ∈(s1\|\|s2)v ⊆s1v1\|\|s2v2 ⊆L1\|\|L2, tv ∈(s1\|\|s2)v ⊆s1v1\|\|s2v2 ⊆L1\|\|L2, which implies that t ∈L1\|\|L2. Since t was arbitrary, it follows that L1 and L2 are nonconﬂicting. □ The implications of Theorem 4 and 5 can be observed from G37 and G47 in Fig. 6 and the related automata in Fig. 3 and 5. According to the previous discussion, it is the case that Lm(G37) = Lsup C = p(Lm(G3)\|\|Lm(G7)) = p(Lsup C1 \|\|Lsup C2 ) with C1 = 1, C2 = 1 and C = C1 + C2 = 2. Further- more, G37 is nonblocking, which indicates that Lm(G3) and Lm(G7) are nonconﬂicting. Similarly, Lm(G47) = Lsup C = p(Lm(G4)\|\|Lm(G7)) = p(Lsup C1 \|\|Lsup C2 ) with C1 = 2, C2 = 1 and C = C1 + C2 = 3 and G47 is nonblocking, Differently, Lm(G26) ̸= Lsup 3 since Lm(G2) ̸= Lsup 2 and Lm(G6) ̸= Lsup 2 . In addition, G26 is blocking, that is, Lm(G2) and Lm(G6) are conﬂicting. Here, we further note that the conditions in Theorem 4 and 5 are sufﬁcient but not necessary. Consider for example the OP languages Lm(G8) in Fig. 7 and Lm(G7) in Fig. 5. Then, it holds that Lm(G8) ̸= Lsup 1 but Lm(G87) = Lm(G8)\|\|Lm(G7) = Lsup 2 and Lm(G8) and Lm(G7) are noncon- ﬂicting. Proof: We prove the corollary by induction. To this end, we introduce the alphabets 0k = 6in 1 ∪6out k , 3k = Sk i=1 6i and 5k = 6in 1 ∪6k for k = 2, . . . , n. We further use the natural projections p3k,0k : 3⋆ k →0⋆ k, p5k,0k : 5⋆ k → 0⋆ k and the languages L1,k = p3k,0k(L1\|\| · · · \|\|Lk) for k = 2, . . . , n. For the initialization, we know that L1,2 = p32,02(L1\|\|L2) = Lsup C1+C2 from Theorem 4 and L1, L2 are nonconﬂicting from Theorem 5. For the induction step, we assume that L1,k = Lsup C1+···+Ck and L1, . . . , Lk are nonconﬂicting. Since L1,k = Lsup C1+···+Ck and Lk+1 = Lsup Ck+1, it directly follows from Theorem 4 that p3k+1,0k+1(L1\|\| · · · \|\|Lk\|\|Lk+1) = p5k+1,0k+1(p3k,0k(L1\|\| · · · \|\|Lk)\|\|Lk+1) = p5k+1,0k+1(L1,k\|\|Lk+1) = p5k+1,0k+1(Lsup C1+···+Ck\|\|Lsup Ck+1) = Lsup C1+···+Ck+1. p3k+1,0k+1(L1\|\| · · · \|\|Lk\|\|Lk+1) = p5k+1,0k+1(p3k,0k(L1\|\| · · · \|\|Lk)\|\|Lk+1) = p5k+1,0k+1(L1,k\|\|Lk+1) = p5k+1,0k+1(Lsup C1+···+Ck\|\|Lsup Ck+1) = Lsup C1+···+Ck+1. D. USAGE OF COMPOSED ORDER-PRESERVING MODELS IN SUPERVISORY CONTROL The previous section indicates that supremal OP languages are nonconﬂicting and their composition again leads to a supremal OP language. In this section, we determine a further important property of composed supremal OP languages in order to clarify their usability for the nonblocking supervisory control of large-scale DES. We ﬁrst consider the case where a subsystem model of a larger DES is OP and is composed of two supremal OP languages L1 = Lsup C1 and L2 = Lsup C2 with the alphabets 61 = 6in 1 ∪6out 1 , 62 = 6in 2 ∪6out 2 and 6out 1 = 6in 2 as in Theorem 4. Then, the composed supremal OP language L = p(L1\|\|L2) is computed using p : ˆ6⋆→6⋆ with ˆ6 = 61 ∪62 and 6 = 6in 1 ∪6out 2 . This computation is illustrated in Fig. 8 using automata models G1, G2 and G such that Lm(G1) = L1, Lm(G2) = L2 and Lm(G) = L. Theorem 6: Assume that 6in 1 , 6out 1 , 6in 2 , 6out 2 , m1, m2, 6, m and p are as introduced above with 6in = 6in 1 , 6out = 6out 2 and 6out 1 = 6in 2 . Further, let L1 = Lsup C1 and L2 = Lsup C2 for C1, C2 > 0. Then, p : (61 ∪62)⋆→6⋆is a natural observer for L1\|\|L2. f 1\|\| 2 Proof: We ﬁrst recall that L1\|\|L2 = L1\|\|L2 from Theorem 5. In order to show that p is a natural observer for L1\|\|L2, we take an arbitrary string t ∈L1\|\|L2 = L1\|\|L2 and u ∈6⋆such that su = p(t)u ∈p(L1\|\|L2). We next show by induction that there is a v ∈(61∪62)⋆such that p(v) = u and tv ∈L1\|\|L2. To this end, we write u = σ1 · · · σ\|u\| with σk ∈6 for k = 1, . . . , \|u\|. For the initialization, we note that there is a v1 ∈(61 ∪62)⋆such that tv1 ∈L1\|\|L2 and p(v1) = σ1 because of Lemma 4. For the induction step, we consider that tk−1 = tv1 · · · vk−1 ∈L1\|\|L2 such that p(v1 · · · vk−1) = σ1 · · · σk−1. D. USAGE OF COMPOSED ORDER-PRESERVING MODELS IN SUPERVISORY CONTROL Applying Lemma 4, it directly follows that there exists a vk ∈(61 ∪62)⋆with p(vk) = σk and such that tv1 · · · vk ∈L1\|\|L2 and p(v1 · · · vk) = σ1 · · · σk. Speciﬁcally, for k = \|u\|, it holds with v = v1 · · · v\|u\| that tv ∈L1\|\|L2 and p(v) = σ1 · · · σ\|u\|. It remains to show that tv ∈L1\|\|L2. Since su ∈p(L1\|\|L2), it must be the case that \|f (su)\| = 0. In addition, tv ∈L1\|\|L2 implies that tv ∈s1\|\|s2 for some s1 ∈L1, s2 ∈L2. Then, according to Lemma 3, we have that f (s) = m−1 1 (f2(s2))f1(s1) and hence, \|f2(s2)\| + \|f1(s1)\| = 0. Accordingly, f2(s2) = f1(s1) = ϵ, which shows that s1 ∈L1 and s2 ∈L2. Therefore, tv ∈s1\|\|s2 ⊆L1\|\|L2, which concludes the proof. □ FIGURE 8. Subsystem with composition of two OP models. FIGURE 8. Subsystem with composition of two OP models. Since the composed supremal OP language L is differ- ent from the composition of the original languages L1\|\|L2, it needs to be veriﬁed if L can/should be used instead of L1\|\|L2 when designing supervisors for the overall DES. In this context, it is important to note that the setting in Fig. 8 is directly related to the setting of abstraction-based supervisory control of large-scale modular DES [14], [15], [34]. Here, ˆG = G1\|\|G2 with the alphabet ˆ6 represents the original model of a subsystem of a larger DES and G with the alpha- bet 6 is the abstracted subsystem model. The abstraction alphabet 6 = 6in 1 ∪6out 2 is chosen such that it contains all the (external) events that are shared with other subsystems, whereas the internal events in 6out 1 = 6in 2 are projected away. In this setting, the existing literature [15], [34] shows that a nonblocking supervisor computed for the system with the abstracted model G is as well nonblocking for the system with the original model ˆG if the projection p fulﬁlls certain sufﬁcient conditions such as the natural observer condition in Deﬁnition 3 [51]. According to the previous discussion, Theorem 6 implies that the abstracted model G with the marked language Lm(G) = L can be used for the nonblocking supervisor synthesis instead of the original model ˆG = G1\|\|G2. tv ∈(s1\|\|s2)v ⊆s1v1\|\|s2v2 ⊆L1\|\|L2, p3k+1,0k+1(L1\|\| · · · \|\|Lk\|\|Lk+1) = p5k+1,0k+1(p3k,0k(L1\|\| · · · \|\|Lk)\|\|Lk+1) = p5k+1,0k+1(L1,k\|\|Lk+1) = p5k+1,0k+1(Lsup C1+···+Ck\|\|Lsup Ck+1) = Lsup C1+···+Ck+1. An important outcome of Theorem 4 is that the composi- tion of two supremal OP languages is again a supremal OP language. That is, the result of the composition operation has the same properties as its arguments. Using this fact, we next state the following corollary, which shows that the compo- sition of an arbitrary number of supremal OP languages is With the same argument, Theorem 5 implies that L1\|\| · · · \|\|Lk and Lk+1 are non-conﬂicting. Hence, L1, · · · , Lk+1 are non- conﬂicting. Since k was arbitrary, it follows for k = n −1 131909 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs that L = p(\|\|n i=1Li) = Lsup C1+···+Cn and L1, . . . , Ln are noncon- ﬂicting. □ that L = p(\|\|n i=1Li) = Lsup C1+···+Cn and L1, . . . , Ln are noncon- ﬂicting. □ Deﬁnition 3: Let L ⊆ ˆ6⋆be a language, and let p : ˆ6∗→6 be the natural projection for 6 ⊆ˆ6. p is a natural observer for L if it holds for all t ∈L and u ∈6⋆that In order to discuss the practical implication of Corollary 1, we consider an AMS with an arbitrary number of MCs that exchange products and that are modeled by supremal OP languages. Then, it holds that the composition of the MCs is again represented by the supremal OP language and the joint operation of the MCs is nonblocking. p(t)u ∈p(L) ⇒∃v ∈ˆ6⋆ s.t. tv ∈L ∧p(v) = u. In words, p is a natural observer for L if any string t that belongs to the preﬁx-closure L of L can be extended to a string in L whenever its projection p(t) can be extended to a string in p(L). We next show that the projection p : (61∪62)⋆→6⋆, which is used for computing the composed supremal OP language p(L1\|\|L2) in the previous section, is a natural observer for L1\|\|L2. IV. FLEXIBLE MANUFACTURING SYSTEM APPLICATION In this section, we apply the proposed modeling framework to the FMS example in [10], [23]. This example is chosen since it offers different product types that share robots and machines with a capacity greater than one. We describe the FMS and provide OP models of the relevant MCs and a super- visor design in Section IV-A. Section IV-B and IV-C perform modiﬁcations of the FMS that illustrate the advantage of composing OP models and that demonstrate the supervisor design for AMSs with sequential operations and assembly operations, respectively. Finally, Section IV-D elaborates on several practical considerations when using OP models. According to the FMS setup, it is readily observed that several MCs share different product types. For example, R1 moves P2 and P3, R2 transports P1, P2, P3 and R3 delivers P2, P3. In addition, M2 processes P1, P3, M3 processes P2, P3 and M4 processes P2, P3. Hence, it is clear that OP models are needed for R1, R2, R3, M2, M3 and M4. Following the description in [10], the robots can hold a single product, whereas the machines are able to hold up to two products simultaneously. That is, a simple OP model with capacity 1 as in Fig. 12 is suitable to represent the robots. Hereby, we use the following convention for the event names: each event name captures the name of the MC, where the product comes from, the name of the MC that holds the product after delivery and the product type. For example, the name for the event that characterizes moving a product of type P3 from I3 to R1 is written as I3-R1P3. D. USAGE OF COMPOSED ORDER-PRESERVING MODELS IN SUPERVISORY CONTROL Hereby, the main advantage is that G cannot have more states (and generally has fewer states) than ˆG since p is a natural observer for L1\|\|L2 [51]. In summary, when computing nonblocking supervisors for modular DES with OP subsystems, it is possi- ble to use the composed OP model with a smaller state count than the composition of the original OP models in order to reduce the computational effort. We illustrate the discussed features by the example automaton G47 in Fig. 6 that is computed such that Lm(G47) = p(Lm(G4\|\|G7)) with G4 in Fig. 3 and G7 in Fig. 5. Assuming that G4 and G7 are components of a modular system, it is not required to use the automaton G4\|\|G7 with 21 states for the 131910 VOLUME 8, 2020 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs FIGURE 10. FMS overview. FIGURE 11. Product paths through the FMS. FIGURE 10. FMS overview. nonblocking supervisor synthesis. Instead, it is possible to use G47 in Fig. 6 with only 15 states. We note that the result in Theorem 6 was stated for the case of two OP languages. We next extend this result to the case of an arbitrary number of supremal OP languages similar to Corollary 1. The extended setting is illustrated in Fig. 9 with the automata Gi such that Lm(Gi) = Li for i = 1, . . . , n. FIGURE 9 Subsystem with n OP models FIGURE 10. FMS overview. FIGURE 9. Subsystem with n OP models. Corollary 2: Assume that 6in i , 6out i , mi, i = 1, . . . , n are introduced as above with 6out i = 6in i+1 for i = 1, . . . , n −1. Further, deﬁne m = mn(· · · m2(m1)) and 6 = 6in 1 ∪6out n . Then, p : (61 ∪· · · ∪6n)⋆→6⋆is a natural observer for L1\|\| · · · \|\|Ln. We prove Corollary 2 in Appendix E. As the main implica- tion of Corollary 2, it is possible to use the automaton ˆG with Lm( ˆG) = p(L1\|\| · · · \|\|Ln) with a smaller state count than the automaton G with Lm(G) = L1\|\| · · · \|\|L2 for the nonblocking supervisor computation. FIGURE 11. Product paths through the FMS. in Fig. 11 (b). The third product type has two alternative paths that start from I3. On the ﬁrst path, R1 moves the product to M3, R2 moves the product to M4 and R3 delivers the product to O3. On the second path, R1 moves the product to M1, R2 moves the product to M2 and R3 delivers the product to O3 as indicated in Fig. 11 (c). A. FMS DESCRIPTION The outline of the FMS in [10], [23] is shown in Fig. 10. It consists of 3 robots R1, R2, R3 and 4 machines M1, M2, M3, M4. The robots are able to transport products from/to the machines. In addition, the robots can take products from the input buffers I1, I2, I3 and deliver them to the output buffers O1, O2, O3. In principle, it is desired to process 3 different product types using the FMS in Fig. 10. For the ﬁrst product type, R2 takes a product from I1 and moves it to M2. Then, R2 delivers the product from M2 to O1. The product path is shown in Fig. 11 (a). The second product type is taken from I2 by R3 and transported to M4. Then, R2 moves the product to M3 and R1 delivers the product to O2 as can be seen Different from the robots, an OP model with capacity 2 is needed to capture the behavior of M2, M3 and M4 as is shown in Fig. 13. Since there are two different product types on each of these machines, the resulting model has 7 states. Note that no OP model is needed for M1 since this machine only processes a single product type. 131911 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs FIGURE 14. Buffer models. FIGURE 15. Machine models. In addition, it holds that the product paths are already encoded in the MC models such that no additional speciﬁcation is required. That is, we compute the overall plant as FIGURE 12. Robot models. FIGURE 13. OP machine models. Finally, [10] provides models for the input and output buffers that limit the numbers of products of a certain type that can enter the FMS to 3 for P1, 7 for P2 and 11 for P3. These buffer models are shown in Fig. 14. In this context, we note that [10] does not employ OP models for M2, M3, M4. The respective models in [10] are shown in Fig. 15. That is, these models assume that, even a certain product type (such as P1 on M2) enters M2 before FIGURE 14. Buffer models. FIGURE 15. Machine models. FIGURE 12. Robot models. FIGURE 13. OP machine models. FIGURE 14. Buffer models. FIGURE 15. Machine models. A. FMS DESCRIPTION FIGURE 13. OP machine models. Finally, [10] provides models for the input and output buffers that limit the numbers of products of a certain type that can enter the FMS to 3 for P1, 7 for P2 and 11 for P3. These buffer models are shown in Fig. 14. In this context, we note that [10] does not employ OP models for M2, M3, M4. The respective models in [10] are shown in Fig. 15. That is, these models assume that, even a certain product type (such as P1 on M2) enters M2 before P3, it is possible that P3 leaves M2 ﬁrst. In practice this means that a product can overtake another product when moving through the FMS. Although this might be possible, it is then necessary to determine the type of each product leaving such machine (since the event indicating the depar- ture of a product is directly related to the product type). This requires additional sensor information and implementation effort. Differently, the OP models in Fig. 13 naturally keep track of the products entering and leaving each machine. FIGURE 15. Machine models. In addition, it holds that the product paths are already encoded in the MC models such that no additional speciﬁcation is required. That is, we compute the overall plant as C. COMBINATION WITH ASSEMBLY OPERATIONS The previous discussion focuses on sequential operations in AMSs in order to illustrate the concept of OP models. In general, it is the case that AMSs comprise both sequential operations, which characterize the ﬂow of products through the AMS, and assembly operations, where parts of a product are combined in order to obtain a ﬁnal product. The existing literature considers the supervisory control for AMSs with the combination of sequential and assembly operations both using automata models [9], [52], [53] and Petri Net models [4], [54] but without including the order of products in the model. We next illustrate the usage of OP models as intro- duced in this paper for AMSs that both include assembly operations and sequential operations that preserve the product order. To this end, we employ a modiﬁcation of the product paths in Fig. 11 as shown in Fig. 18. Here, P1 and P2 are considered as parts of a ﬁnal product that enter from the input buffers I1 and I2, respectively. After following their respec- tive production paths, these two product parts are assem- bled in the output assembly station OA. That is, the product order is preserved during the sequential operations along the product paths, whereas P1 and P2 are combined when arriving at OA. FIGURE 17. Workcell models. in [10] (that is not OP) has 27 770 states. In addition, it can be veriﬁed that Lm(S\|\|G) ⊆Lm(˜S\|\| ˜G). This is due to the fact that the machine models in [10] do not preserve the order of products and hence model unnecessary behavior. in [10] (that is not OP) has 27 770 states. In addition, it can be veriﬁed that Lm(S\|\|G) ⊆Lm(˜S\|\| ˜G). This is due to the fact that the machine models in [10] do not preserve the order of products and hence model unnecessary behavior. G=GR1∥GR2∥GR3∥GM1∥GM2∥GM3∥GM4∥GB1∥GB2∥GB3. Then, we compute a nonblocking supervisor S such that Lm(S∥G) = SupC(Lm(G), G, 6u) with 6u = ∅with 30 692 states. In comparison, the nonblocking supervisor ˜S for the original model We next discuss the supervisor computation for the FMS according to Section II-B. To this end, we ﬁrst note that the aim of this paper is not an efﬁcient supervisor design method but the usage of OP models as introduced in Section III. ˜G=GR1∥GR2∥GR3∥˜GM1∥˜GM2∥˜GM3∥˜GM4∥GB1∥GB2∥GB3. ˜G=GR1∥GR2∥GR3∥˜GM1∥˜GM2∥˜GM3∥˜GM4∥GB1∥GB2∥GB3. ˜G=GR1∥GR2∥GR3∥˜GM1∥˜GM2∥˜GM3∥˜GM4∥GB1∥GB2∥GB3. 131912 131912 VOLUME 8, 2020 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs Then, we ﬁrst determine the state sizes of the original and reduced workcell models. We write ˆGW2 = GCB2i\|\|GM2\|\|GCB2o, ˆGW3 = GCB3i\|\|GM3\|\|GCB3o, ˆGW4 = GCB4i\|\|GM4\|\|GCB4o ˆGW2 = GCB2i\|\|GM2\|\|GCB2o, ˆGW3 = GCB3i\|\|GM3\|\|GCB3o, ˆGW4 = GCB4i\|\|GM4\|\|GCB4o ˆGW2 = GCB2i\|\|GM2\|\|GCB2o, ˆGW3 = GCB3i\|\|GM3\|\|GCB3o, ˆGW4 = GCB4i\|\|GM4\|\|GCB4o FIGURE 16. Workcell outline. FIGURE 17. Workcell models. for the original workcell models with the alphabets ˆ6W2 = 6CB2i ∪6M2 ∪6CB2o, ˆ6W3 = 6CB3i ∪6M3 ∪6CB3o and ˆ6W4 = 6CB4i ∪6M4 ∪6CB4o. According to Section III-D, we also introduce the alphabets of the reduced OP models for the workcells as 6W2 = 6W2 \6M2, 6W3 = 6W3 \6M3 and 6W4 = 6W4 \6M4 with the related natural projections pW2 : ˆ6⋆ W2 →6⋆ W2, pW3 : ˆ6⋆ W3 →6⋆ W3 and pW4 : ˆ6⋆ W4 →6⋆ W4. That is, the reduced models only contain the external events of the workcells that are shared with the robots. Then, we write GW2, GW3 and GW4 for the reduced OP models such that Lm(GW2) = pW2(Lm( ˆGW2)), Lm(GW3) = pW3(Lm( ˆGW3)) and Lm(GW4) = pW4(Lm( ˆGW4)). ˆ ˆ ˆ It holds that ˆGW2, ˆGW3 and ˆGW4 have 27 states, whereas GW2, GW3 and GW4 have only 15 states (which complies with (10)). Accordingly, a nonblocking supervisor for the overall system with the original workcell models ˆGW2, ˆGW3 and ˆGW4 has 2 444 288 states, whereas a nonblocking supervisor for the overall system with the reduced workcell models GW2, GW3 and GW4 has 643 100 states. That is, in addition to correctly modeling the order of products traveling to the FMS, it is possible to reduce the size of the required models using the particular properties of composed OP languages. FIGURE 18. Modified FMS with assembly operation. FIGURE 19. Modified MC models including an assembly operation. y g This paper is concerned with the development of OP mod- els and the analysis of their properties. Due to this reason, the previous sections consider medium-scale AMSs and per- form a monolithic supervisor design in order to demonstrate the usability of OP models. In practice, large-scale AMSs require the application of efﬁcient supervisor design meth- ods [14], [15], [24], [29]–[34], [55]. Since the proposed OP model is represented by a ﬁnite state automaton, any of the automata-based supervisor design methods for large-scale systems including for example the compositional synthesis in [33] and the abstraction-based design in [14], [15], [34], [55] can be applied to AMSs including OP models. In addition, an interesting direction for future research is the development of OP models for Petri Nets in order to beneﬁt from recent efﬁcient design methods and simpliﬁed supervisor represen- tations as in [24], [29]–[32]. The OP models proposed in this paper are developed for the supervisory control of AMSs, which ensures the correct logical system behavior based on a given behavioral speciﬁcation. In practice, the temporal system behavior also needs to be taken into account in order to achieve a satis- factory system performance such as small cycle time and high throughput [56]–[60]. Since the proposed OP model is represented by a ﬁnite state automaton, any of the automata- based performance-oriented design methods such as the directed control in [56], the time-optimal control based on sequential abstraction in [58] and the time-optimal control using compositional optimization in [59] can be applied to AMSs including OP models. In addition, developing OP models for Petri Nets will make it possible to apply Petri Net-based supervisor synthesis methods for performance improvement such as [57]. FIGURE 19. Modified MC models including an assembly operation. by replacing the events R1-O2_P2 and R2-O1_P1 by R1-OA_P2 and R2-OA_P1, respectively, to indicate that the product parts P1 and P2 are delivered to OA (see Fig. 19). In addition, the model GOA of OA in Fig. 19 captures that the product parts P1 and P2 can enter OA in an arbitrary order (events R1-OA_P2 and R2-OA_P1). When both product parts are present, their assembly and discharge from the AMS is modeled by the event P1-P2. In analogy to Section IV-A, we computed a nonblocking supervisor S for the modiﬁed FMS including the assem- bly operation in Fig. 18. V. CONCLUSION The subject of this paper is the development of discrete event system (DES) models for a particular property of automated manufacturing systems (AMSs). The model accounts for the fact that AMSs are able to manufacture different product types that potentially share various manufacturing compo- nents (MCs) such as machines and robots. Different from the existing literature, the proposed model captures the general case, where MCs can hold multiple products and then process these products sequentially. Speciﬁcally, if the capacity of a FIGURE 18. Modified FMS with assembly operation. The supervisor has 120 697 states addresses both the OP sequential operations and the assembly operation of the FMS. B. COMPOSED ORDER-PRESERVING MODELS FOR THE FMS In order to illustrate the beneﬁts of composing OP models as described in Section III-D, we consider a slightly modiﬁed version of the FMS in Section IV-A. To this end, we replace the machines M2, M3, M4 by corresponding workcells W2, W3, W4 that consist of two CBs and one machine. The outline of these workcells is shown in Fig. 16. That is, products enter the workcell from the input CB (CB2i, CB3i, CB4i), are then processed by the respective machine (M2, M3, M4) and leave the workcell from the output CB (CB2o, CB3o, CB4o). Assuming a capacity of 1 for each of the MCs in the workcells, we use the following OP models in Fig. 17. The same automata models as in Section IV-A are used for the MCs I3, O3, R3, M1, M2, M3 and M4. The robot models GR1, GR2 and the buffer models GB1, GB2 are modiﬁed 131913 131913 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs FIGURE 18. Modified FMS with assembly operation. The main focus of this paper is the development of a general automaton-based OP model for sequential processes in AMSs. This model can be used for any MC of an AMS, where the order of products entering and leaving the MC has to be preserved. In this context, we point out that we do not require that all MCs of an AMS are OP. Since the proposed OP model is represented by a ﬁnite state automaton, it can be used together with automata-based models of MCs that are not OP. This is for example the case in Section IV-C, where the modiﬁed FMS comprises both sequential operations and assembly operations. That is, our OP model is complemen- tary to existing models for MCs. FIGURE 18. Modified FMS with assembly operation. D. PRACTICAL CONSIDERATIONS In this section, we further clarify the main contribution of this paper. To this end, we identify several important design problems, where the proposed OP model can be used and we discuss possible directions for future research. ≤m(pre\|pout(sk)\|(pin(sk)))m(α1α2 · · · αC) = m pre\|pout(sk)\|(pin(sk) suf\|pout(sk)(pin(sk))) = m(pin(sk)) = m(pin(skω1)) = m(pin(sk+1)). MC is greater than one, products will leave the MC in the order of entering the MC. That is, the MC needs to keep track of the product order if different products require different processing steps after leaving the MC. As the main contribution, we formalize this scenario by introducing the new class of order-preserving (OP) lan- guages. We show that a supremal OP language exists and can be recognized by a ﬁnite state automaton. In addition, we prove that it is possible to compose supremal OP lan- guages to again obtain a supremal OP language. We further show that reduced models of composed OP languages that are suitable for nonblocking supervisory control can be obtained. We demonstrate the practicability of the proposed model by applying it to the supervisory control of a ﬂexible manufac- turing system. We further show that OP models can be used together with non-OP models for AMSs with both assembly operations and sequential operations that preserve the product order. Furthermore, f (sk+1) = f (skω1) = α2 · · · αC and accordingly δsup C (xsup 0,C, sk+1) = δsup C ([α1α2 · · · αC], ω1) = [α2 · · · αC] = [f (sk+1)] with (R4). Finally, since c(sk) ≤ C and c(sk+1) ≤ c(sk), it follows that c(sk+1) ≤ C. In the third case, 0 < \|f (sk)\| < C. That is, [f (sk)] = [α1 · · · αj] with j < C. Then, there are two possible tran- sitions. First, let σ = αj+1 ∈ 6in according to (R3). Then, the same argument as in the ﬁrst case shows that pout(sk+1) = pout(skαj+1) = pout(sk) ≤m(pin(sk+1)) = m(pin(skαj+1)) = m(pin(sk))m(αj+1), c(sk+1) = c(skαj+1) = max{c(sk), \|f (sk)\| + 1} ≤ C and δsup C (xsup 0,C, sk+1) = δsup C ([α1 · · · αj], αj+1) = [α1 · · · αjαj+1] = [f (sk+1)]. Sec- ond, let σ = ω1 = m(α1) ∈6out according to (R2) or (R4). Then, the same argument as in the second case shows that pout(sk+1) ≤m(pin(sk+1)), f (sk+1) = α2 · · · αj and accordingly δsup C (xsup 0,C, sk+1) = δsup C ([α1α2 · · · αj], ω1) = [α2 · · · αj] = [f (sk+1)]. In future work we will study nonblocking modular system behavior of OP AMSs depending on the connectivity of MCs. APPENDIX A PROOF OF LEMMA 1 We provide the proof of Lemma 1. For (ii), we consider an arbitrary string s ∈Lsup C and we show by induction that it holds for each k ≤\|s\| that sk ∈L(Gsup C ) and δsup C (xsup 0,C, sk) = [f (sk)] in order to show that s ∈Lm(Gsup C ). For initialization, we consider s0 = ϵ. Then, it holds that δsup C (xsup 0,C, ϵ) = [ϵ] = [f (s0)]. For the induction step, we let k < \|s\| and assume that δsup C (xsup 0,C, sk) = [f (sk)]. Then, we show that δsup C (xsup 0,C, sk+1) = [f (sk+1)]. In general, sk+1 = skσ for σ ∈6 and there are different cases for σ. First, consider that f (sk) = ϵ. Since s is OP, this implies that pout(sk) = m(pin(sk)). Hence, it must be the case that σ = α1 ∈6in. But then, (R1) ensures that δsup C ([ϵ], α1) = δsup C (xsup 0,C, skα1) = δsup C (xsup 0,C, sk+1) = [α1] = [f (sk+1)]. Second, consider that f (sk) = α1α2 · · · αC. Since the capacity of s is c(s) = C and s is OP, it must be the case that σ = ω1 for ω1 = m(α1). But then, (R4) ensures that δsup C ([α1α2 · · · αC], ω1) = δsup C (xsup 0,C, skω1) = δsup C (xsup 0,C, sk+1) = [α2 · · · αC] = [f (sk+1)]. Third, consider that f (sk) = α1α2 · · · αj for 0 < j < C. Since 0 < \|f (sk)\| < C and s is OP, it is possible that σ = αj+1 for some αj+1 ∈6in or σ = ω1 = m(α1) ∈6out. If σ = αj+1, (R3) ensures that δsup C ([α1 · · · αj], αj+1) = δsup C (xsup 0,C, skαj+1) = δsup C (xsup 0.C, sk+1) = [α1 · · · αjαj+1] = [f (sk+1)]. If σ = ω1, (R2) or (R4) ensure that δsup C ([α1α2 · · · αj], ω1) = δsup C (xsup 0,C, skω1) = δsup C (xsup 0,C, sk+1) = [α2 · · · αj] = [f (sk+1)]. sup sup p p Proof: We have to show that (i) s ∈Lm(Gsup C ) ⇒s is OP and c(s) ≤C, (ii) s ∈Lsup C ⇒s ∈Lm(Gsup C ). In particular, for k + 1 = \|s\|, we have that δsup C (xsup 0,C, s) = [f (s)] = [ϵ] = xsup 0,C since s is OP and, hence, \|pin(s)\| = \|pout(s)\|. That is, s ∈Lm(Gsup C ). □ 131914 131914 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs ≤m(pre\|pout(sk)\|(pin(sk)))m(α1α2 · · · αC) = m pre\|pout(sk)\|(pin(sk) suf\|pout(sk)(pin(sk))) = m(pin(sk)) = m(pin(skω1)) = m(pin(sk+1)). ≤m(pre\|pout(sk)\|(pin(sk)))m(α1α2 · · · αC) = m pre\|pout(sk)\|(pin(sk) suf\|pout(sk)(pin(sk))) = m(pin(sk)) = m(pin(skω1)) = m(pin(sk+1)). ≤m(pre\|pout(sk)\|(pin(sk)))m(α1α2 · · · αC) = m pre\|pout(sk)\|(pin(sk) suf\|pout(sk)(pin(sk))) = m(pin(sk)) = m(pin(skω1)) = m(pin(sk+1)). ≤m(pre\|pout(sk)\|(pin(sk)))m(α1α2 · · · αC) = m pre\|pout(sk)\|(pin(sk) suf\|pout(sk)(pin(sk))) = m(pin(sk)) = m(pin(skω1)) = m(pin(sk+1)). In addition, we intend to investigate the development of OP models for Petri Nets in order to beneﬁt from recent advances in the efﬁcient supervisor synthesis. Since k was arbitrary, it follows for k + 1 = \|s\| that pout(s) ≤m(pin(s)), c(s) ≤C and δsup C (xsup 0,C, s) = [f (s)]. Considering that s ∈Lm(Gsup C ), it is further the case that δsup C (xsup 0,C, s) = [ϵ]. Hence, it is the case that m(pin(s)) = pout(s). That is, s is OP according to Deﬁnition 1. APPENDIX A PROOF OF LEMMA 1 Since the capacity of L2 is c(L2) = C2 and L2 = Lsup C2 , it holds that APPENDIX B PROOF OF LEMMA 2 This section provides the proof of Lemma 2. For brevity, we write Lsup C1 and Lsup C2 instead of Lsup C1 (6in 1 , 6out 1 , m1) and Lsup C2 (6in 2 , 6out 2 , m2). s2 = α2 · · · α2 \| {z } C1 α2 · · · α2 \| {z } C2−C1 ω2 · · · ω2 \| {z } C2 α2 · · · α2 \| {z } C1 ω2 · · · ω2 \| {z } C1 = ω1 · · · ω1 \| {z } C1 ω1 · · · ω1 \| {z } C2−C1 ω2 · · · ω2 \| {z } C2 ω1 · · · ω1 \| {z } C1 ω2 · · · ω2 \| {z } C1 ∈L2 and c(s2) = C2. Since ω1 ∈61∩62 = 6out 1 = 6in 2 , it follows that s2 = α2 · · · α2 \| {z } C1 α2 · · · α2 \| {z } C2−C1 ω2 · · · ω2 \| {z } C2 α2 · · · α2 \| {z } C1 ω2 · · · ω2 \| {z } C1 = ω1 · · · ω1 \| {z } C1 ω1 · · · ω1 \| {z } C2−C1 ω2 · · · ω2 \| {z } C2 ω1 · · · ω1 \| {z } C1 ω2 · · · ω2 \| {z } C1 ∈L2 C2 2 2 Proof: We have to show that c(L) = C = C1 + C2. Since L1 = Lsup C1 and L2 = Lsup C2 for C1, C2 > 0, it holds that ϵ ∈L1\|\|L2 ̸= ∅. Hence, we already know from Theorem 3 that C = c(L) ≤C1+C2. In order to show that C = C1+C2, it remains to show that C ≥C1 + C2. and c(s2) = C2. Since ω1 ∈61∩62 = 6out 1 = 6in 2 , it follows that and c(s2) = C2. Since ω1 ∈61∩62 = 6out 1 = 6in 2 , it follows that We consider two cases. In the ﬁrst case C2 ≤C1. In addi- tion, for simplicity, we assume that C1 ≤2 C2. APPENDIX C PROOF OF LEMMA 3 We next prove Lemma 3. Again, we write Lsup C1 and Lsup C2 instead of Lsup C1 (6in 1 , 6out 1 , m1) and Lsup C2 (6in 2 , 6out 2 , m2). C2 holds that, C2 holds that, 1 2 Proof: We show that f (s) = m−1 1 (f2(s2)) f1(s1) by induction on the length of preﬁxes of sk = prek(s). For the initialization, we start from k = 0. It holds that s0 = ϵ and s0 = p(s1,0\|\|s2,0) and f (s0) = m−1 1 (f2(s2,0)) f1(s1,0) = ϵ for s1,0 = ϵ ≤s1, s2,0 = ϵ ≤s2. s = α1 · · · α1 \| {z } C2 α1 · · · α1 \| {z } C1 ω2 · · · ω2 \| {z } C2 ω2 · · · ω2 \| {z } C2 ω2 · · · ω2 \| {z } C1−C2 = p(α1 · · · α1 \| {z } C2 ω1 · · · ω1 \| {z } C2 α1 · · · α1 \| {z } C1 ω2 · · · ω2 \| {z } C2 ω1 · · · ω1 \| {z } C2 × ω2 · · · ω2 \| {z } C2 ω1 · · · ω1 \| {z } C1−C2 ω2 · · · ω2 \| {z } C1−C2 ) = p(s1\|\|s2) ∈p(L1\|\|L2) = L and c(s) = \|pin(α1 . . . α1 \| {z } C2 α1 . . . α1 \| {z } C1 )\| −\|pout(α1 . . . α1 \| {z } C2 α1 . . . α1 \| {z } C1 )\| × C1 + C2 −0 = C1 + C2. APPENDIX A PROOF OF LEMMA 1 That is, indeed, c(L) = C = C1 + C2 □ = α2 · · · α2 \| {z } C2 ω2 · · · ω2 \| {z } C2 α2 · · · α2 \| {z } C2 ω2 · · · ω2 \| {z } C2 α2 · · · α2 \| {z } C1−C2 ω2 · · · ω2 \| {z } C1−C2 = ω1 · · · ω1 \| {z } C2 ω2 · · · ω2 \| {z } C2 ω1 · · · ω1 \| {z } C2 ω2 · · · ω2 \| {z } C2 ω1 · · · ω1 \| {z } C1−C2 ω2 · · · ω2 \| {z } C1−C2 ∈L2 □ and c(s2) = C2 since C1 −C2 ≤C2 by assumption (if C1 −C2 > C2, it is sufﬁcient to add additional substrings ω1 · · · ω1 \| {z } C2 ω2 · · · ω2 \| {z } C2 ). Since 61 ∩62 = 6out 1 = 6in 2 , it further holds that APPENDIX A PROOF OF LEMMA 1 The extension for the case C1 > 2 C2 is conceptually straightforward. Since the capacity of L1 is c(L1) = C1 and L1 = Lsup C1 , it holds for an arbitrary α1 ∈6in 1 and ω1 = m1(α1) ∈6out 1 that s = α1 · · · α1 \| {z } C1 α1 · · · α1 \| {z } C2−C1 α1 · · · α1 \| {z } C1 ω2 · · · ω2 \| {z } C2 ω2 · · · ω2 \| {z } C1 = p(α1 · · · α1 \| {z } C1 ω1 · · · ω1 \| {z } C1 α1 · · · α1 \| {z } C2−C1 ω1 · · · ω1 \| {z } C2−C1 α1 · · · α1 \| {z } C1 × ω2 · · · ω2 \| {z } C2 ω1 · · · ω1 \| {z } C1 ω2 · · · ω2 \| {z } C1 ) ∈p(s1\|\|s2) ⊆p(L1\|\|L2) = L. s1 = α1 · · · α1 \| {z } C2 ω1 · · · ω1 \| {z } C2 α1 · · · α1 \| {z } C1 ω1 · · · ω1 \| {z } C2 ω1 · · · ω1 \| {z } C1−C2 ∈L1 and c(s1) = C1. Now consider that α2 = ω1 ∈6in 2 = 6out 1 and ω2 = m2(α2) ∈6out 2 . Since the capacity of L2 is c(L2) = C2 and L2 = Lsup C2 , it holds that and and c(s) = \|pin(α1 . . . α1 \| {z } C1 α1 . . . α1 \| {z } C2−C1 α1 . . . α1 \| {z } C1 )\| −\|pout(α1 . . . α1 \| {z } C1 α1 . . . α1 \| {z } C2−C1 α1 . . . α1 \| {z } C1 )\| = C1 + C2. APPENDIX A PROOF OF LEMMA 1 C C For (i), we use induction on the string length to show that for each preﬁx sk := prek(s) ≤s, pout(sk) ≤m(pin(sk)), c(sk) ≤C and δsup C (xsup 0,C, sk) = [f (sk)]. C 0,C Let s ∈ Lm(Gsup C ) be arbitrary. For the initialization, we consider s0 = ϵ ≤s. Then, it holds that pout(s0) = m(pin(s0)) = ϵ and c(s0) = 0 ≤ C. Furthermore, δsup C (x0, s0) = [f (s0)] = [ϵ]. C Now assume that for some k < \|s\|, we have that pout(sk) ≤ m(pin(sk)), c(sk) ≤C and δsup C (x0, sk) = [f (sk)]. We show that also pout(sk+1) ≤ m(pin(sk+1)), c(sk+1) ≤ C and δsup C (x0, sk+1) = [f (sk+1)]. There are three cases. In the ﬁrst case, let \|f (sk)\| = 0. That is, [f (sk)] = [ϵ]. Then, sk+1 = sk α1 with α1 ∈6in according to (R1). Furthermore, it holds that pout(sk+1) = pout(skα1) = pout(sk) ≤m(pin(sk+1)) = m(pin(skα1)) = m(pin(sk))m(α1), c(sk+1) = c(skα1) = max{c(sk), 1} ≤C and δsup C (xsup 0,C, sk+1) = δsup C ([ϵ], α1) = [α1] = [f (sk+1)]. In the second case, let \|f (sk)\| = C. That is, [f (sk)] = [α1α2 · · · αC] for some α1, . . . , αC ∈6in. Then, sk+1 = sk ω1 with ω1 = m(α1) ∈6out according to (R4). Noting that f (sk) = α1α2 · · · αC, we further conclude that pout(sk+1) = pout(skω1) = pout(sk)ω1 = m(pre\|pout(sk)\|(pin(sk)))ω1 = m(pre\|pout(sk)\|(pin(sk)))m(α1) pout(sk+1) = pout(skω1) = pout(sk)ω1 = m(pre\|pout(sk)\|(pin(sk)))ω1 = m(pre\|pout(sk)\|(pin(sk)))m(α1) In particular, for k + 1 = \|s\|, we have that δsup C (xsup 0,C, s) = [f (s)] = [ϵ] = xsup 0,C since s is OP and, hence, \|pin(s)\| = \|pout(s)\|. That is, s ∈Lm(Gsup C ). □ 131915 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs APPENDIX B PROOF OF LEMMA 2 This section provides the proof of Lemma 2. For brevity, we write Lsup C1 and Lsup C2 instead of Lsup C1 (6in 1 , 6out 1 , m1) and Lsup C2 (6in 2 , 6out 2 , m2). and c(s1) = C1. Now consider that α2 = ω1 ∈6in 2 = 6out 1 and ω2 = m2(α2) ∈6out 2 . APPENDIX C PROOF OF LEMMA 3 s = α1 · · · α1 \| {z } C2 α1 · · · α1 \| {z } C1 ω2 · · · ω2 \| {z } C2 ω2 · · · ω2 \| {z } C2 ω2 · · · ω2 \| {z } C1−C2 = p(α1 · · · α1 \| {z } C2 ω1 · · · ω1 \| {z } C2 α1 · · · α1 \| {z } C1 ω2 · · · ω2 \| {z } C2 ω1 · · · ω1 \| {z } C2 × ω2 · · · ω2 \| {z } C2 ω1 · · · ω1 \| {z } C1−C2 ω2 · · · ω2 \| {z } C1−C2 ) = p(s1\|\|s2) ∈p(L1\|\|L2) = L and c(s) = \|pin(α1 . . . α1 \| {z } C2 α1 . . . α1 \| {z } C1 )\| −\|pout(α1 . . . α1 \| {z } C2 α1 . . . α1 \| {z } C1 )\| × C1 + C2 −0 = C1 + C2. For the induction step, assume for some k < \|s\|, s1,k ≤ s1, s2,k ≤ s2 that sk = p(s1,k\|\|s2,k) and f (sk) = m−1 1 (f2(s2,k)) f1(s1,k). We have to show that for sk+1 = skσ ≤ s, there are s1,k+1 ≤s1, s2,k+1 ≤s2 such that sk+1 = p(s1,k+1\|\|s2,k+1) and f (sk+1) = m−1 1 (f2(s2,k+1)) f1(s1,k+1). Consider sk+1 = skσ ≤s and write f (sk) = α1 · · · αj for some 0 ≤j ≤C with α1, . . . , αj ∈6in (with a slight abuse of notation, we deﬁne f (sk) = ϵ if j = 0). Then, it is possible that (i) σ ∈6in or (ii) σ ∈6out. For the induction step, assume for some k < \|s\|, s1,k ≤ s1, s2,k ≤ s2 that sk = p(s1,k\|\|s2,k) and f (sk) = m−1 1 (f2(s2,k)) f1(s1,k). We have to show that for sk+1 = skσ ≤ s, there are s1,k+1 ≤s1, s2,k+1 ≤s2 such that sk+1 = p(s1,k+1\|\|s2,k+1) and f (sk+1) = m−1 1 (f2(s2,k+1)) f1(s1,k+1). and Consider sk+1 = skσ ≤s and write f (sk) = α1 · · · αj for some 0 ≤j ≤C with α1, . . . , αj ∈6in (with a slight abuse of notation, we deﬁne f (sk) = ϵ if j = 0). APPENDIX C PROOF OF LEMMA 3 Since 6in 1 ∩62 = ∅, it follows that tσ ∈ s1σ\|\|s2 ⊆L1\|\|L2 and p(tσ) = p(t)σ = sσ. That is, (14) is fulﬁlled with v = σ ∈(61 ∪62)⋆. In the second case, \|f1(s1)\| = C1 and hence \|f2(s2)\| < C2. We write γl+1 = m1(αl+1) ∈6out 1 = 6in 2 . Since L1 = Lsup C1 and L2 = Lsup C2 , it is the case that s1γl+1 ∈L1 and s2γl+1 ∈L2. Furthermore, γl+1 ∈6out 1 , implies \|f1(s1γl+1)\| = \|αl+2 · · · αj\| = C1 −1 < C1. That is, s1γl+1σ ∈L1 since L1 = Lsup C1 . Together, we have that tγl+1σ ∈s1γl+1σ\|\|s2γl+1 ⊆L1\|\|L2 and p(tγl+1σ) = p(t)σ = sσ. That is, (14) is fulﬁlled with v = γl+1σ. (ii) if σ ∈6out = 6out 2 , it must be the case that j > 0. Otherwise, \|f (sσ)\| = j −1 < 0. There are two cases. In the ﬁrst case, \|f2(s2)\| > 0. Since L2 = Lsup C2 , it holds that s2σ ∈L2. Since 6in 2 ∩61 = ∅, it follows that tσ ∈ s1\|\|s2σ ⊆L1\|\|L2 and p(tσ) = p(t)σ = sσ. That is, (14) is fulﬁlled with v = σ ∈(61 ∪62)⋆. In the second case, \|f2(s2)\| = 0 and hence \|f1(s1)\| > 0. In addition, it must hold that σ = ω1 = m(α1) since f (s) = α1 · · · αj. We write γ1 = m1(αl) ∈6out 1 = 6in 2 . Since L1 = Lsup C1 and L2 = Lsup C2 , it is the case that s1γ1 ∈L1 and s2γ1 ∈L2. Furthermore, γ1 ∈6in 2 , implies \|m−1 1 (f2(s2γ1))\| = \|α1\| = 1 > 0. That is, s1γ1ω1 ∈L1 since m2(γ1) = m(α1) = ω1 and L2 = Lsup C2 . Together, we have that tγ1σ = tγ1ω1 ∈s1γl+1\|\|s2γl+1ω1 ⊆ L1\|\|L2 and p(tγl+1σ) = p(t)σ = sσ. That is, (14) is fulﬁlled with v = γ1σ. 2 , , (ii) If σ ∈6out, it must be the case that j > 0 (if j = 0, \|f (skσ)\| = −1 < 0). Accordingly, \|f2(s2,k)\| > 0 or \|f2(s2,k)\| = 0 and \|f1(s1,k)\| > 0. If \|f2(s2,k)\| > 0, we know that f1(s1,k) = αl+1 · · · αj and m−1 1 (f2(s2,k)) = α1 · · · αl for some l > 0. APPENDIX E PROOF OF COROLLARY 2 We next prove Corollary 2. To this end, we ﬁrst state the following result from the existing literature [50], [61]. Lemma 5: Consider alphabets 61, 62 and write 6∩= 61 ∩62, 3 = 61 ∪62. Assume that 6∩⊆5 ⊆3 for some alphabet 5 and deﬁne the projections pi : 6⋆ i →(6i ∩5)⋆ for i = 1, 2 and p : 3⋆→5⋆. Then, it holds for any L1 ⊆6⋆ 1 and L2 ⊆6⋆ 2 that Since k < \|s\| was chosen arbitrary, the proven relation also holds for k = \|s\| −1. That is, □ f (s) = f (sk+1) = m−1 1 (f2(s2,k+1))f1(s1,k+1) = m−1 1 (f2(s2))f1(s2). p(L1\|\|L2) = p1(L1)\|\|p2(L2). (15) (15) APPENDIX C PROOF OF LEMMA 3 Then, it is possible that (i) σ ∈6in or (ii) σ ∈6out. 2 1 × C1 + C2 −0 = C1 + C2. 2 1 × C1 + C2 −0 = C1 + C2. (i) Assume that σ = αj+1 ∈6in. Then, it must be the case that j < C. Consider the contrary, that is, j = C = C1 + C2. Since \|f (sk)\| = \|m−1 1 (f2(s2,k))f1(s1,k)\| and c(si,k) ≤Ci for i = 1, 2, this implies that \|fi(si,k)\| = Ci. Since sk+1 ∈ p(L1\|\|L2), sk = p(s1,k\|\|s2,k) and αj+1 ̸∈62, it must further be the case that sk+1 = p(s1,kuαj+1\|\|s2,ku) for some u ∈6out 1 = 6in 2 . Considering that \|f2(s2,k)\| = C2 it follows that u = ϵ (otherwise, \|f2(s2,ku)\| > C2. But then, \|f1(s1,kuαj+1)\| = \|f1(s1,kαj+1)\| = C1 + 1, which implies that s1,kαj+1 ̸∈L1 In the second case, C1 < C2. In addition, for simplicity, we assume that C2 ≤2 C1. The extension for the case C2 > 2 C1 is conceptually straightforward. Since the capacity of L2 is c(L2) = C2 and L2 = Lsup C2 , it holds for an arbitrary α1 ∈ 6in 1 and ω1 = m1(α1) ∈6out 1 that s1= α1 · · · α1 \| {z } C1 ω1 · · · ω1 \| {z } C1 α1 · · · α1 \| {z } C2−C1 ω1 · · · ω1 \| {z } C2−C1 α1 · · · α1 \| {z } C1 ω1 · · · ω1 \| {z } C1 L s1= α1 · · · α1 \| {z } C1 ω1 · · · ω1 \| {z } C1 α1 · · · α1 \| {z } C2−C1 ω1 · · · ω1 \| {z } C2−C1 α1 · · · α1 \| {z } C1 ω1 · · · ω1 \| {z } C1 ∈L1 131916 s1= α1 · · · α1 \| {z } C1 ω1 · · · ω1 \| {z } C1 α1 · · · α1 \| {z } C2−C1 ω1 · · · ω1 \| {z } C2−C1 α1 · · · α1 \| {z } C1 ω1 · · · ω1 \| {z } C1 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs and hence sk+1 ̸∈p(L1\|\|L2). This contradicts the assumption that s ∈p(L1\|\|L2). APPENDIX C PROOF OF LEMMA 3 Then, it holds that s2,kω1 ∈ L2 since L2 = Lsup C2 and L2 is OP. That is, σ = ω1. Furthermore, sk+1 = skω1 = p(s1,k+1\|\|s2,k+1) for s1,k+1 = sk and s2,k+1 = s2,kω1 and f (sk+1) = α2 · · · αj = m−1 1 (f2(s2,k+1))f1(s1,k+1). If \|f2(s2,k)\| = 0 and \|f1(s1,k)\| > 0, we have that f1(s1,k) = α1 · · · αj and m−1 1 (f2(s2,k)) = ϵ. Since L1 = Lsup C1 , it holds that s1,kγ1 ∈L1 for γ1 = m1(α1). Since L2 = Lsup C2 , γ1 ∈6out 1 = 6in 2 and ω1 = m2(γ1) = m(α1) ∈6out 2 , it is further the case that s2,kγ1ω1 ∈L2. That is, sk+1 = skω1 = p(s1,k+1\|\|s2,k+1) for s1,k+1 = s1,kγ1 and s2,k+1 = s2,kγ1ω1. In addition, it holds that f (sk+1) = α2 · · · αj = m−1 2 (f2(s2,k+1))f1(s1,k+1) since m−1 2 (f2(s2,k+1)) = m−1 2 (f2(s2,kγ1ω1)) = m−1 2 (f2(s2,k)) = ϵ and f1(s1,k+1) = f1(s1,kγ1) = α2 · · · αj. In summary, (14) holds in all possible cases, which proves the lemma. □ □ APPENDIX C PROOF OF LEMMA 3 Hence, indeed j < C. Accordingly, \|f1(s1,k)\| < C1 or \|f1(s1,k)\| = C1 and \|f2(s2,k)\| < C2. If \|f1(s1,k)\| < C1, we know that m−1 1 (f2(s2,k)) = α1 · · · αl and f1(s1,k) = αl+1 · · · αj for l ≤C2 and j −l < C1. Then, it holds that s1,kαj+1 ∈L1 since L1 = Lsup C1 . Furthermore, sk+1 = skαj+1 = p(s1,k+1\|\|s2,k+1) for s1,k+1 = s1,kαj+1 and s2,k+1 = s2,k and f (sk+1) = α1 · · · αlαl+1 · · · αjαj+1 = m−1 2 (f2(s2,k+1))f1(s1,k+1). If \|f1(s1,k)\| = C1 and \|f2(s2,k)\| < C2, we have that m−1 1 (f2(s2,k)) = α1 · · · αl and f1(s1,k) = αl+1 · · · αj for l < C2 and j −l = C1. Consider γl+1 = m1(αl+1). Since L1 = Lsup C1 , L2 = LC2 and γl+1 ∈6out 1 = 6in 2 , it must be the case that s1,kγl+1 ∈L1 and s2,kγl+1 ∈L2. But then, also s1,kγl+1αj+1 ∈L1 since \|f1(s1,kγl+1)\| = C1 −1 < C1. That is, deﬁning s1,k+1 = s1,kγl+1αj+1 and s2,k+1 = s2,kγl+1, we have that sk+1 = skαj+1 = p(s1,k+1\|\|s2,k+1) since αj+1 ̸∈62. In addition, it holds that f (sk+1) = α1 · · · αjαj+1, f1(s1,k+1) = αl+2 · · · αj+1 and m−1 1 (f2(s2,k+1) = α1 · · · αlαl+1 with \|f1(s1,k+1)\| = j + 1 −(l + 1) = j −l = C1 and \|f2(s2,k+1)\| = l + 1 ≤C2. Moreover, f (sk+1) = α1 · · · αlαl+1 · · · αjαj+1 = m−1 2 (f2(s2,k+1))f1(s1,k+1). \|m−1 1 (f2(s2))\| = \|α1 · · · αl\| = l and \|f1(s1)\| = \|f1(αl+1 · · · αj)\| = j −l and 0 ≤j ≤C = C1 + C2. \|m−1 1 (f2(s2))\| = \|α1 · · · αl\| = l and \|f1(s1)\| = \|f1(αl+1 · · · αj)\| = j −l and 0 ≤j ≤C = C1 + C2. j We now consider that (i) σ ∈6in or (ii) σ ∈6out. ( ) ( ) (i) If σ ∈6in = 6in 1 , it must be the case that j < C. Otherwise, \|f (sσ)\| = \|f (s)σ\| = j + 1 > C. There are two cases. In the ﬁrst case, \|f1(s1)\| < C1. Since L1 = Lsup C1 , it holds that s1σ ∈L1. APPENDIX D PROOF OF LEMMA 4 Wang, ‘‘Nonblocking supervisory control of ﬂexible manufacturing systems based on state tree structures,’’ in Formal Methods in Manufacturing Systems: Recent Advances. Hershey, PA, USA: IGI Global, 2013, pp. 1–19. p3k+1,0k+1(L1\|\| · · · \|\|Lk+1) = p5k+1,0k+1 p3k+1,5k+1(L1\|\| · · · \|\|Lk\|\|Lk+1) = p5k+1,0k+1 p3k,0k(L1\|\| · · · \|\|Lk)\|\|Lk+1 p3k+1,0k+1(L1\|\| · · · \|\|Lk+1) [11] P. N. Pena, T. A. Costa, R. S. Silva, and R. H. C. Takahashi, ‘‘Control of ﬂexible manufacturing systems under model uncertainty using supervisory control theory and evolutionary computation schedule synthesis,’’ Inf. Sci., vol. 329, pp. 491–502, Feb. 2016. = p5k+1,0k+1 p3k,0k(L1\|\| · · · \|\|Lk)\|\|Lk+1 [12] T. Sprock, C. Bock, and L. F. McGinnis, ‘‘Survey and classiﬁca- tion of operational control problems in discrete event logistics sys- tems (DELS),’’ Int. J. Prod. Res., vol. 57, nos. 15–16, pp. 5215–5238, Aug. 2019. because of Lemma 5. Here, we already know that p3k,0k is a natural observer for L1\|\| · · · \|\|Lk due to the induction assumption. In order to show that p3k+1,0k+1 is a natu- ral observer for L1\|\| · · · \|\|Lk+1, we take an arbitrary string t ∈ L1\|\| · · · \|\|Lk+1 and u ∈ 0⋆ k+1 and assume that p3k+1,0k+1(t)u ∈p3k+1,0k+1(L1\|\| · · · \|\|Lk+1). We ﬁrst observe that because of Lemma 5. Here, we already know that p3k,0k is a natural observer for L1\|\| · · · \|\|Lk due to the induction assumption. In order to show that p3k+1,0k+1 is a natu- ral observer for L1\|\| · · · \|\|Lk+1, we take an arbitrary string t ∈ L1\|\| · · · \|\|Lk+1 and u ∈ 0⋆ k+1 and assume that p3k+1,0k+1(t)u ∈p3k+1,0k+1(L1\|\| · · · \|\|Lk+1). We ﬁrst observe that [13] R. El-Khalil and Z. Darwish, ‘‘Flexible manufacturing systems perfor- mance in U.S. automotive manufacturing plants: A case study,’’ Prod. Planning Control, vol. 30, no. 1, pp. 48–59, Jan. 2019. [14] K. Schmidt, T. Moor, and S. Perk, ‘‘Nonblocking hierarchical control of decentralized discrete event systems,’’ IEEE Trans. Autom. Control, vol. 53, no. 10, pp. 2252–2265, Nov. 2008. [15] L. Feng and W. M. Wonham, ‘‘Supervisory control architecture for discrete-event systems,’’ IEEE Trans. Autom. Control, vol. 53, no. 6, pp. 1449–1461, Jul. 2008. p3k+1,0k+1(t)u = p5k+1,0k+1p3k+1,5k+1(t)u ∈p5k+1,0k+1p3k+1,5k+1(L1\|\| · · · \|\|Lk+1) [16] J. E. R. Cury, M. H. de Queiroz, G. Bouzon, and M. Teixeira, ‘‘Supervisory control of discrete event systems with distinguishers,’’ Automatica, vol. 56, pp. APPENDIX D PROOF OF LEMMA 4 93–104, Jun. 2015. That is, there must be a w ∈5⋆ k+1 such that p5k+1,0k+1(w) = u and That is, there must be a w ∈5⋆ k+1 such that p5k+1,0k+1(w) = u and [17] K. Andersson, B. Lennartson, and M. Fabian, ‘‘Restarting manufacturing systems; restart states and restartability,’’ IEEE Trans. Autom. Sci. Eng., vol. 7, no. 3, pp. 486–499, Jul. 2010. p3k+1,5k+1(t)w ∈p3k+1,5k+1(L1\|\| · · · \|\|Lk+1) = p3k,0k(L1\|\| · · · \|\|Lk)\|\|Lk+1. p3k+1,5k+1(t)w ∈p3k+1,5k+1(L1\|\| · · · \|\|Lk+1) = p3k,0k(L1\|\| · · · \|\|Lk)\|\|Lk+1. p3k+1,5k+1(t)w ∈p3k+1,5k+1(L1\|\| · · · \|\|Lk+1) = p3k,0k(L1\|\| · · · \|\|Lk)\|\|Lk+1. [18] J. Zhang, G. Frey, A. Al-Ahmari, T. Qu, N. Wu, and Z. Li, ‘‘Analysis and control of dynamic reconﬁguration processes of manufacturing systems,’’ IEEE Access, vol. 6, pp. 28028–28040, 2018. Here, we know that Lk+1 = Lsup Ck+1 by assumption and p3k,0k(L1\|\| · · · \|\|Lk) = Lsup C1+···+Ck because of Theorem 1. Then, Theorem 6 implies that p3k+1,5k+1 is a natural observer for L1\|\| · · · \|\|Lk+1. Accordingly, there is a v ∈3⋆ k+1 such that p3k+1,5k+1(v) = w and tw ∈L1\|\| · · · \|\|Lk+1. Recalling that p5k+1,0k+1(w) = u, we found v ∈3⋆ k+1 such that p3k+1,0k+1(v) = u and tv ∈L1\|\| · · · \|\|Lk+1. Hence, p3k+1,0k+1 is indeed a natural observer. Here, we know that Lk+1 = Lsup Ck+1 by assumption and p3k,0k(L1\|\| · · · \|\|Lk) = Lsup C1+···+Ck because of Theorem 1. Then, Theorem 6 implies that p3k+1,5k+1 is a natural observer for L1\|\| · · · \|\|Lk+1. Accordingly, there is a v ∈3⋆ k+1 such that p3k+1,5k+1(v) = w and tw ∈L1\|\| · · · \|\|Lk+1. Recalling that p5k+1,0k+1(w) = u, we found v ∈3⋆ k+1 such that p3k+1,0k+1(v) = u and tv ∈L1\|\| · · · \|\|Lk+1. Hence, p3k+1,0k+1 is indeed a natural observer. [19] Y. Koren, X. Gu, and W. Guo, ‘‘Reconﬁgurable manufacturing systems: Principles, design, and future trends,’’ Frontiers Mech. Eng., vol. 13, no. 2, pp. 121–136, Jun. 2018. [20] M. Khalgui, O. Mosbahi, and Z. Li, ‘‘On reconﬁguration theory of discrete- event systems: From initial speciﬁcation until ﬁnal deployment,’’ IEEE Access, vol. 7, pp. 18219–18233, 2019. [21] P. J. Ramadge and W. M. Wonham, ‘‘Supervisory control of a class of discrete event processes,’’ SIAM J. Control Optim., vol. 25, no. 1, pp. 206–230, Jan. 1987. [22] C. G. Cassandras and S. Lafortune, Introduction to Discrete Event Systems. APPENDIX D PROOF OF LEMMA 4 We next provide the proof of Corollary 2. We next provide the proof of Corollary 2. Proof: We use the same notation as in the proof or Corol- lary 1 with 0k = 6in 1 ∪6out k , 3k = Sk i=1 6i, 5k = 6in 1 ∪6k for k = 2, . . . , n, p3k,0k : 3⋆ k →0⋆ k, p5k,0k : 5⋆ k →0⋆ k and the languages L1,k = p3k,0k(L1\|\| · · · \|\|Lk) for k = 2, . . . , n. We next prove Lemma 4. As before, we write Lsup C1 and Lsup C2 instead of Lsup C1 (6in 1 , 6out 1 , m1) and Lsup C2 (6in 2 , 6out 2 , m2). 1 2 Proof: Consider an arbitrary string t ∈L1\|\|L2 and let σ ∈ 6 such that p(t)σ ∈ p(L1\|\|L2). We write s = p(t). According to Lemma 3, it holds that s = p(s1\|\|s2) for s1 ∈ L1 and s2 ∈ L2. In addition, f (s) = m−1 1 (f2(s2))f1(s1) = α1 · · · αlαl+1 · · · αj with Next, we prove the assertion in Corollary 2 by induc- tion. For the initialization, we know that p32,02 is a natural observer for L1\|\|L2 from Theorem 6 and L1,2 = Lsup C1+C2 from Theorem 4. 131917 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs For the induction step, we assume that p3k,0k is a natural observer for L1\|\| · · · \|\|Lk. Then, we show that also p3k+1,0k+1 is a natural observer for L1\|\| · · · \|\|Lk\|\|Lk+1. To this end, we ﬁrst note that 3k = 61 ∪· · · ∪6k = 6in 1 ∪6out 1 ∪ 62 · · · 6in k ∪6out k and 6k+1 = 6in k+1 ∪6out k+1 such that 3k ∩6k+1 = 6out k = 6in k+1. Then, we compute [8] X. Guo, S. Wang, D. You, Z. Li, and X. Jiang, ‘‘A siphon-based deadlock prevention strategy for S3PR,’’ IEEE Access, vol. 7, pp. 86863–86873, 2019. [9] M. H. de Queiroz, J. E. R. Cury, and W. M. Wonham, ‘‘Multitasking supervisory control of discrete-event systems,’’ Discrete Event Dyn. Syst., vol. 15, no. 4, pp. 375–395, Dec. 2005. [10] W. Chao, Y. Gan, W. Wonham, and Z. APPENDIX D PROOF OF LEMMA 4 Boston, MA, USA: Springer, 2009. In particular, the obtained result is true for k + 1 = n. That is, p = p3n,0n in Corollary 2 is a natural observer for L1\|\| · · · \|\|Ln. □ [23] Z. Wu Li, M. Chu Zhou, and N. Qi Wu, ‘‘A survey and comparison of Petri net-based deadlock prevention policies for ﬂexible manufacturing systems,’’ IEEE Trans. Syst., Man, Cybern. C, Appl. Rev., vol. 38, no. 2, pp. 173–188, Mar. 2008. [24] Y. Chen, Z. Li, and M. Zhou, ‘‘Behaviorally optimal and structurally simple liveness-enforcing supervisors of ﬂexible manufacturing systems,’’ IEEE Trans. Syst., Man, Cybern. A, Syst. Humans, vol. 42, no. 3, pp. 615–629, May 2012. REFERENCES [1] A. A. Desrochers, Modeling and Control of Automated Manufacturing Systems. Washington, DC, USA: IEEE Computer Society Press, 1989. [25] M. Zhao, M. Uzam, and Y. Hou, ‘‘Near-optimal supervisory control of ﬂex- ible manufacturing systems using divide-and-conquer iterative method,’’ Adv. Mech. Eng., vol. 8, no. 3, 2016, Art. no. 1687814016639823. [2] P. M. Swamidass, ‘‘Automated manufacturing system,’’ in Encyclopedia of Production and Manufacturing Management. Boston, MA, USA: Springer, 2000. [26] Y. Hou and K. Barkaoui, ‘‘Deadlock analysis and control based on Petri nets: A siphon approach review,’’ Adv. Mech. Eng., vol. 9, no. 5, 2017, Art. no. 1687814017693542. [3] D. Y. Chao, ‘‘Automated manufacturing system: Virtual-nets or non- virtual-nets?’’ IET Control Theory Appl., vol. 3, no. 6, pp. 671–680, Jun. 2009. [27] Y. Li, L. Yin, Y. Chen, Z. Yu, and N. Wu, ‘‘Optimal Petri net supervisor synthesis for forbidden state problems using marking mask,’’ Inf. Sci., vol. 505, pp. 183–197, Dec. 2019. [4] H. Hu and M. Zhou, ‘‘A Petri net-based discrete-event control of automated manufacturing systems with assembly operations,’’ IEEE Trans. Control Syst. Technol., vol. 23, no. 2, pp. 513–524, Mar. 2015. [28] Y. Feng, K. Xing, M. Zhou, and H. Liu, ‘‘Liveness analysis and dead- lock control for automated manufacturing systems with multiple resource requirements,’’ IEEE Trans. Syst., Man, Cybern., Syst., vol. 50, no. 2, pp. 525–538, Feb. 2020. [5] N. Du and H. Hu, ‘‘A robust prevention method for automated manufac- turing systems with unreliable resources using Petri nets,’’ IEEE Access, vol. 6, pp. 78598–78608, 2018. [29] H. Hu and Y. Liu, ‘‘Supervisor simpliﬁcation for AMS based on Petri nets and inequality analysis,’’ IEEE Trans. Autom. Sci. Eng., vol. 11, no. 1, pp. 66–77, Jan. 2014. [6] S. Lafortune, ‘‘Discrete event systems: Modeling, observation, and con- trol,’’ Annu. Rev. Control, Robot., Auto. Syst., vol. 2, no. 1, pp. 141–159, May 2019. [7] B. S. Kim, Y. Jin, and S. Nam, ‘‘An integrative user-level customized modeling and simulation environment for smart manufacturing,’’ IEEE Access, vol. 7, pp. 186637–186645, 2019. [30] H. Hu, Y. Liu, and L. Yuan, ‘‘Supervisor simpliﬁcation in FMSs: Compar- ative studies and new results using Petri nets,’’ IEEE Trans. Control Syst. Technol., vol. 24, no. 1, pp. 81–95, Jan. 2016. 131918 VOLUME 8, 2020 A. Nooruldeen, K. W. Schmidt: OP Languages for the Supervisory Control of AMSs [31] J. Luo and M. REFERENCES Zhou, ‘‘Petri-net controller synthesis for partially control- lable and observable discrete event systems,’’ IEEE Trans. Autom. Control, vol. 62, no. 3, pp. 1301–1313, Mar. 2017. [55] R. Su, J. H. van Schuppen, and J. E. Rooda, ‘‘Aggregative synthesis of distributed supervisors based on automaton abstraction,’’ IEEE Trans. Autom. Control, vol. 55, no. 7, pp. 1627–1640, Jul. 2010. [56] J. Huang and R. Kumar, ‘‘Optimal nonblocking directed control of discrete event systems,’’ IEEE Trans. Autom. Control, vol. 53, no. 7, pp. 1592–1603, Aug. 2008. [32] C. Chen and H. Hu, ‘‘Static and dynamic partitions of inequalities: A uni- ﬁed methodology for supervisor simpliﬁcation,’’ IEEE Trans. Autom. Con- trol, vol. 64, no. 11, pp. 4748–4755, Nov. 2019. [57] H. Hu and Y. Liu, ‘‘Supervisor synthesis and performance improvement for automated manufacturing systems by using Petri nets,’’ IEEE Trans. Ind. Informat., vol. 11, no. 2, pp. 450–458, Apr. 2015. [33] H. Flordal, R. Malik, M. Fabian, and K. Åkesson, ‘‘Compositional synthe- sis of maximally permissive supervisors using supervision equivalence,’’ Discrete Event Dyn. Syst., vol. 17, no. 4, pp. 475–504, Nov. 2007. [58] S. Ware and R. Su, ‘‘Time optimal synthesis based upon sequential abstrac- tion and its application to cluster tools,’’ IEEE Trans. Autom. Sci. Eng., vol. 14, no. 2, pp. 772–784, Apr. 2017. [34] K. Schmidt and C. Breindl, ‘‘Maximally permissive hierarchical control of decentralized discrete event systems,’’ IEEE Trans. Autom. Control, vol. 56, no. 4, pp. 723–737, Apr. 2011. [59] F. Hagebring and B. Lennartson, ‘‘Time-optimal control of large-scale systems of systems using compositional optimization,’’ Discrete Event Dyn. Syst., vol. 29, no. 3, pp. 411–443, Sep. 2019. [35] K. Cai and W. M. Wonham, ‘‘Supervisor localization: A top-down approach to distributed control of discrete-event systems,’’ IEEE Trans. Autom. Control, vol. 55, no. 3, pp. 605–618, Mar. 2010. [60] M. Silva, ‘‘Analysis and control of discrete event systems as Petri nets,’’ in Encyclopedia of Systems and Control. London, U.K.: Springer, 2020. [36] M. H. De Queiroz and J. E. Cury, ‘‘Modular supervisory control of large scale discrete event systems,’’ in Discrete Event Systems. Boston, MA, USA: Springer, 2000, pp. 103–110. [61] K. Schmidt, J. Reger, and T. Moor, ‘‘Hierarchical control for structural decentralized DES,’’ in Proc. Discrete Event Syst., 2004, p. 279. [37] R. J. Leduc, M. Lawford, and W. M. Wonham, ‘‘Hierarchical interface- based supervisory control—Part II: Parallel case,’’ IEEE Trans. Autom. Control, vol. 50, no. 9, pp. 1336–1348, Sep. 2005. REFERENCES [38] J. Zaytoon and B. Riera, ‘‘Synthesis and implementation of logic controllers—A review,’’ Annu. Rev. Control, vol. 43, pp. 152–168, Jan. 2017. [39] S. Kumar, R. Devaraj, and A. Sarkar, ‘‘A hybrid ofﬂine-online approach to adaptive downlink resource allocation over LTE,’’ IEEE/CAA J. Automat- ica Sinica, vol. 6, no. 3, pp. 766–777, May 2019. [40] M. Goorden, J. van de Mortel-Fronczak, M. Reniers, W. Fokkink, and J. Rooda, ‘‘Modeling guidelines for component-based supervisory control synthesis,’’ in Proc. Int. Conf. Formal Aspects Compon. Softw. Amsterdam, The Netherlands: Springer, 2019, pp. 3–24. ANAS NOORULDEEN received the B.S. degree in electronic and control engineering techniques from the College of Technology, Kirkuk, Iraq, in 2010, and the M.S. and Ph.D. degrees in electronic and communication engineering from Çankaya University, Ankara, Turkey, in 2012 and 2020, respectively. ANAS NOORULDEEN received the B.S. degree in electronic and control engineering techniques from the College of Technology, Kirkuk, Iraq, in 2010, and the M.S. and Ph.D. degrees in electronic and communication engineering from Çankaya University, Ankara, Turkey, in 2012 and 2020, respectively. [41] L. Grigorov, B. E. Butler, J. E. R. Cury, and K. Rudie, ‘‘Conceptual design of discrete-event systems using templates,’’ Discrete Event Dyn. Syst., vol. 21, no. 2, pp. 257–303, Jun. 2011. [42] M. Teixeira, J. E. R. Cury, and M. H. de Queiroz, ‘‘Exploiting distin- guishers in local modular control of discrete-event systems,’’ IEEE Trans. Autom. Sci. Eng., vol. 15, no. 3, pp. 1431–1437, Jul. 2018. [43] D. Côté and R. St-Denis, ‘‘Component-based method for the modeling and control of modular production systems,’’ IEEE Trans. Control Syst. Technol., vol. 21, no. 5, pp. 1570–1585, Sep. 2013. From 2013 to 2015, he was a Research Assistant with the Department of Electrical Engineering, Kirkuk University, Iraq. His research interests include supervisory control of discrete event systems, automata, order- preserving models, and ﬂexible and reconﬁgurable manufacturing systems. He received the M.S. Research Grant and a Full Ph.D. Scholarship from the Scientiﬁc and Technological Research Council of Turkey (TÜBİTAK), in December 2011 and February 2016, respectively. [44] R. A. Williams, B. Benhabib, and K. C. Smith, ‘‘A hybrid supervisory control system for ﬂexible manufacturing workcells,’’ in Proc. IEEE Int. Conf. Robot. Autom., May 1994, pp. 2551–2556. [45] A. Nooruldeen and K. W. Schmidt, ‘‘State attraction under language spec- iﬁcation for the reconﬁguration of discrete event systems,’’ IEEE Trans. Autom. Control, vol. 60, no. 6, pp. 1630–1634, Jun. 2015. REFERENCES [46] K. W. Schmidt, ‘‘Reconﬁgurability of behavioural speciﬁcations for man- ufacturing systems,’’ Int. J. Control, vol. 90, no. 12, pp. 2605–2617, Dec. 2017. [47] T. Jiao, Y. Gan, G. Xiao, and W. M. Wonham, ‘‘Exploiting symmetry of discrete-event systems by relabeling and reconﬁguration,’’ IEEE Trans. Syst., Man, Cybern., Syst., vol. 50, no. 6, pp. 2056–2067, Jun. 2020. [48] A. Nooruldeen and K. W. Schmidt, ‘‘Order-preserving models for the supervisory control of ﬂexible manufacturing systems,’’ Çankaya Univ. J. Sci. Eng., vol. 16, no. 2, pp. 70–86, 2019. [49] F. Lin, W. Wang, L. Han, and B. Shen, ‘‘State estimation of multichannel networked discrete event systems,’’ IEEE Trans. Control Netw. Syst., vol. 7, no. 1, pp. 53–63, Mar. 2020. KLAUS WERNER SCHMIDT received the Diploma and Ph.D. degrees in electrical, elec- tronic, and communication engineering from the University of Erlangen-Nürnberg, Germany, in 2002 and 2005, respectively. KLAUS WERNER SCHMIDT received the Diploma and Ph.D. degrees in electrical, elec- tronic, and communication engineering from the University of Erlangen-Nürnberg, Germany, in 2002 and 2005, respectively. [50] W. Wonham, Supervisory Control Discrete Event Systems (Systems Con- trol Group). Toronto, ON, Canada: Univ. of Toronto, 2010. [51] K. C. Wong and W. M. Wonham, ‘‘Hierarchical control of discrete-event systems,’’ Discrete Event Dyn. Syst., vol. 6, no. 3, pp. 241–273, Jul. 1996. He is currently a Professor with the Depart- ment of Electrical and Electronics Engineering, Middle East Technical University, Ankara. His research interests include supervisory control for discrete event systems, industrial automation sys- tems, industrial communication networks, intelligent transportation systems, and industrial project control. He serves as an Associate Editor for Discrete Event Dynamic Systems and Turkish Journal of Electrical Engineering and Computer Sciences. [52] S.-J. Park and J.-T. Lim, ‘‘Robust and nonblocking supervisory control of nondeterministic discrete event systems using trajectory models,’’ IEEE Trans. Autom. Control, vol. 47, no. 4, pp. 655–658, Apr. 2002. [53] R. Malik and R. Leduc, ‘‘Hierarchical interface-based supervisory control using the conﬂict preorder,’’ in Proc. 11th Int. Workshop Discrete Event Syst., vol. 45, 2012, pp. 163–168. [54] H. Hu, M. Zhou, Z. Li, and Y. Tang, ‘‘Deadlock-free control of auto- mated manufacturing systems with ﬂexible routes and assembly operations using Petri nets,’’ IEEE Trans. Ind. Informat., vol. 9, no. 1, pp. 109–121, Feb. 2013. 131919 VOLUME 8, 2020 VOLUME 8, 2020
W3127435040.txt	https://pubs.rsc.org/en/content/articlepdf/2021/ra/d0ra08191d	en		Microwave-assisted synthesis of titania–amorphous carbon nanotubes/amorphous nitrogen-doped carbon nanotubes nanohybrids for photocatalytic degradation of textile wastewater	RSC advances	2,021	cc-by	10,675	RSC Advances View Article Online Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. PAPER Cite this: RSC Adv., 2021, 11, 6748 View Journal \| View Issue Microwave-assisted synthesis of titania– amorphous carbon nanotubes/amorphous nitrogen-doped carbon nanotubes nanohybrids for photocatalytic degradation of textile wastewater Sithembela A. Zikalala, a Mandla B. Chabalala,a Nozipho N. Gumbi, Neil J. Coville, b Bhekie B. Mamba, ac Bridget K. Mutuma b and Edward N. Nxumalo *a a The synthesis of TiO2 nanohybrids fabricated using amorphous carbon nanotubes (aCNTs) and amorphous nitrogen doped carbon nanotubes (aNCNTs) via a microwave-assisted hydrothermal method is reported. The photocatalytic removal of Reactive Red 120 (RR 120) and organics from industrial textile wastewater using these nanohybrids is discussed. The synthesis process was shown to promote the removal of nano graphitic ﬂakes from the outer walls of the aNCNTs and aCNTs and subsequent incorporation of these carbonaceous materials into TiO2 nanocrystals as such enabling a stronger interaction between the TiO2 and the carbonaceous material. This enabled the production of a surface plasmon resonance on the TiO2 and NTiO2 nanocrystals. The carbon residue was conﬁrmed to be aCNTs and aNCNTs by TGA and DTA analyses. XPS analysis for the TiO2–aNCNT nanohybrids conﬁrmed the C and N doping of TiO2 due to the amorphous residues from the aNCNTs. In addition, XPS and FTIR spectroscopic analysis conﬁrmed the presence of surface oxygen-based groups. TEM micrograph analysis showed that aCNTs Received 25th September 2020 Accepted 4th January 2021 and aNCNTs promote the formation of monodispersed and small TiO2 particles; all below 7.4 nm. The NTiO2–aNCNT nanohybrids have the lowest energy band gap at 2.97 eV and the lowest PL intensity. The TiO2–aNCNT nanohybrids had superior adsorptive (98.2%) and photocatalytic (99%) removal for 20 ppm RR 120 dye solution at k1app 3.44 102 min1. Lastly, all the nanohybrids demonstrate the formation of DOI: 10.1039/d0ra08191d visible-light absorbing intermediates from VAT-dyed textile wastewater. The work demonstrates the rsc.li/rsc-advances possibility of the use of these nanohybrids to derive new products through photocatalytic nanohybrids. 1. Introduction Carbonaceous materials have received much attention due to their potential in enhancing the photocatalytic properties of TiO2 and its doped forms. Among many carbonaceous materials, carbon nanotubes (CNTs), carbon nanobers (CNFs) and graphene are most widely used as support materials for TiO2 since they enhance the photocatalytic activity of TiO2 and its doped forms in several ways.1 First, their electron-rich surface enhances the absorption of light through photosensitization.2 Second, the high electrical conductivity of these carbonaceous a Institute for Nanotechnology and Water Sustainability, College of Science, Engineering and Technology, University of South Africa, Florida, 1709, Johannesburg, South Africa. E-mail: nxumaen@unisa.ac.za; Tel: +27 11 670 9498 b DSI-NRF Centre of Excellence in Strong Materials and Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, Johannesburg 2050, South Africa c State Key Laboratory of Separation Membranes and Membrane Processes, National Centre for International Joint Research on Membrane Science and Technology, Tianjin, 300387, People's Republic of China 6748 \| RSC Adv., 2021, 11, 6748–6763 supports decreases the electron–hole recombination (also known as charge recombination) in TiO2 hence making both photo-generated electrons (e) and positive holes (h+) available to take part in the photocatalytic processes. In addition, these carbonaceous materials narrow the energy band gap (Eg) in TiO2 thus enabling the absorption of visible light by TiO2.3,4 CNTs are cylindrical, tubular graphitic sheets that range from a single ring (single walled CNTs) to multiple concentric rings (multiwalled CNTs).5 The cylindrical morphology imputes CNTs with a high length to diameter ratio; a characteristic that makes them to have a high surface area.6 CNTs are renowned for their high tensile strength and high electrical conductivity; properties that are primarily due to the sp2 hybridization of the lattice carbons resulting in s–s, C–C bonds and p–p bonding resulting from the lone pair electrons on the carbons within the graphitic layer.7 Defects on these graphitic walls are characterized by sp3 hybridized carbons and as such decrease mechanical strength of CNTs. The introduction of heteroatoms into the graphitic lattice induces strain on the walls due to unequal bond strengths and lengths between the –C–C– and the carbon– © 2021 The Author(s). Published by the Royal Society of Chemistry View Article Online Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Paper heteroatom bond.8 The eﬀect of heteroatom doping on the electrical conductivity, however, is dependent on electronegativity of the non-carbon atom relative to carbon. Electronegative and electron rich elements typically increase electrical conductivity.9 The quality of carbon nanotubes is also aﬀected by the synthesis reaction environment in that low quality, thick-walled and less crystalline carbon nanotubes are produced when the synthesis times are longer temperatures, and carbon source concentrations are higher.10,11 Previous research has shown that chemical vapor deposition synthesis of CNTs at times longer than 1 h, high precursor and carrier gas ow rates and temperatures higher than 800 C, results in the formation of CNTs with wide inner and outer diameters due to thick, but weak walls.10 While the inner layers of the walls may be graphitic, the outer layers generally consist of weakly held nano graphitic sheets and some amorphous residue.12,13 This makes the synthesis of such low-quality carbon nanotubes cheaper and as such appealing for the modication of semiconductors for catalytic applications. These types of CNTs are called amorphous carbon nanotubes (aCNTs).13,14 aCNTs have unique properties that make them nd a diﬀerent niche in several applications that crystalline CNTs do not occupy. The discontinuity of the walls imputes aCNTs with electrical and thermal properties that enable them to be good absorbers of electromagnetic radiation, have a large surface area, and an energy band gap (Eg) that is inversely proportional to the diameter. aCNTs also exhibit relatively the same electronic properties regardless of the chirality. This suggests aCNTs are suited for the attachment of nanoparticles; a feature attributed to the high density of structural defects on the outer surface of aCNTs.15 Additionally aCNTs are resistant to oxidation in air at temperatures below 300 C.16 These properties have made aCNTs to be widely used in such processes as adsorption,17 lithium-ion storage cells,18 space technology.19 The unique electrical properties of aCNTs also make them suitable for use in photocatalytic degradation experiments.16,20 As such, aCNTs and the amorphous residue, have been explored in the modication of semiconductor photocatalysts and found to demonstrate exceptional photocatalytic performances.12,21,22 For example, the degradation of methylene blue (MB) using amorphous carbon (aC) coated TiO2 was found to follow the rst order kinetics at rate constant of 16.4 h1 as opposed to that of bare TiO2 (4.14 h1).23 The synergistic eﬀect of doping TiO2 with N and hybridizing it with amorphous C increased visible light photosensitivity and extended the band edge of the nanohybrid up to 600 nm (which corresponds to Eg 2.36 eV).21 aCNTs have also been reported in the fabrication of Ag–aCNT and MnO–aCNT nanohybrids formed by supporting Ag and MnO nanoparticle onto aCNTs.24,25 The Ag–aCNT nanohybrids had a lower energy Eg than the Ag.24 In the latter case, aCNTs were shown to photosensitize MnO in visible light and extend its absorption edge into the visible range.25 A mechanically weak form of nitrogen doped carbon was used to form a composite with AgPO4 and found to enhance the charge © 2021 The Author(s). Published by the Royal Society of Chemistry RSC Advances separation and hence the photocatalytic performance of the nanocomposite.26 Due to the bulk of semiconductor–crystalline CNT nanohybrids, concerns on these nanohybrids compromising the ltration eﬃciency of polymer membranes through the formation of macropores at the polymer/nanoparticle interface have been raised.27 This has led to the carbonaceous material–crystalline CNT nanohybrids having seen less transition into membrane technology.27 As such, more uniform and smaller carbonaceous material–crystalline CNT material nanohybrids are desirable to impute photocatalytic properties onto polymers while ensuring the sustainable use of semiconductor-based photocatalysts without compromising the ltration capabilities. aCNTs are promising candidates for the tuning of the optical properties of semiconductor photocatalysts with a possibility of maintaining a strong interaction between the TiO2 and the carbonaceous material. The aCNTs and aNCNTs used in this work were tailored to have loosely held graphitic nano akes to investigate the contribution of the amorphous nature of aCNTs on the optical, chemical and mechanical properties of TiO2 and NTiO2. The nanotubes used in this study are characterized by thick walls in the range 40–100 nm, wider inner and outer diameters in the range 100–300 nm and bolus capping or open ends (Fig. 3(g)). Furthermore, addition of surface groups and doping with noncarbon elements into the CNT layers, makes the CNTs more amorphous and more prone to breaking down to shorter fragments and under extreme mechanical strain and extreme oxidizing environments to a mix of graphitic and amorphous residue.28 As such, it is expected that aNCNT would be fragmented to smaller graphitic and amorphous carbon residues which would be incorporated into or onto the NTiO2 nanoparticles. In this study, an investigation of the inuence of aCNT and aNCNTs on the properties of TiO2 and NTiO2 was conducted to study the eﬀects of the amorphous nature of a CNT wall on its interaction with TiO2. In situ polymeric condensation of TiO2 and NTiO2 in an aCNT/aNCNT suspension was carried out using the microwave assisted hydrothermal method. These resultant photocatalytic nanohybrids were then evaluated for the degradation of RR 120 as a model dye. Finally, the nanohybrids were evaluated for the removal of color and organics in industrial textile eﬄuent. 2. Materials and methods 2.1. Materials Titanium butoxide (TiOBut), propanol, butanol, ethanol (Et$OH), acetylacetonate, FeCl$6H2O, Co(NO3)$6H2O and NH4OH, CaCO3 were all supplied by Sigma Aldrich South Africa. C2H2 and N2 gases supplied by Afrox South Africa, a tube furnace with an eﬀective heating length of 80 cm equipped with a thermostat and a 1 m quartz tube were employed for the synthesis of aCNTs and aNCNTs. The synthesis of the TiO2– aCNT, TiO2–aNCNT and NTiO2–aNCNT nanohybrids was carried out in an MDS-6G synthesis/digestion microwave (SineoChina) equipped with a reactor system that consists of Teon- RSC Adv., 2021, 11, 6748–6763 \| 6749 View Article Online RSC Advances Paper lined reactor vessels each covered with a SiC composite. Each vessel is held in a blast safety casing to enable pressure build up during synthesis. Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 2.2. Synthesis of aCNTs and aNCNTs The Fe–Co/CaCO3 bimetallic catalyst was synthesized using the wet impregnation technique adapted from previous procedures using FeCl$6H2O and Co(NO3)$6H2O as precursors.10,29 For the synthesis of aCNTs about 0.85 g of the Fe–Co/CaCO3 bimetallic catalyst powder in a quartz boat, was inserted into the quartz tube and the quartz tube placed into the furnace. The quartz tube was purged with N2 at a ow rate of 240 mL min1 while the temperature of the furnace was being ramped up at 10 C min1 to 800 C. C2H2 was then passed through the tube at 100 mL min1 together with N2 kept at 240 mL min1 for 2 h before cooling the furnace to room temperature in an inert atmosphere. A similar procedure was followed for the synthesis of aNCNTs except that vapor made from 14.5 M NH4OH was passed into the quartz tube with the C2H2 and N2. The aCNTs and aNCNTs were sonicated in aqua regia at 80 C for 5 h and then rinsed with DI until the pH was neutral. The resultant aCNTs and aNCNTs were dried in an electric furnace at 70 C overnight. 2.3. Synthesis of TiO2–aCNT, TiO2–aNCNT and NTiO2– aNCNT nanohybrids The sequence in the addition of the reagents up to aging through stirring was adapted from our previous work due to the degree of control on particle size distribution that the method showed.22 A weighed amount of aNCNTs was sonicated in 50 mL ethanol (Et$OH) at 40 C for 1 h while keeping the beaker covered with paralm to reduce evaporation of the ethanol. The amount of aNCNTs and aCNTs was calculated based on the stoichiometric mass of Ti in the 10 mL titanium butoxide (TiOBut) and was determined to be of a mass equal to 5 wt% of the Ti. A solution of TiOBut (Solution A) was prepared by dissolving 10 mL of TiOBut in a mixture consisting of 100 mL butanol and 10 mL acetylacetone. Another solution (Solution B) of 97% NH4OH dissolved in propanol : DI mixture of ratio 1 : 1 was prepared and the Solutions A and B were simultaneously added dropwise into the suspension of aNCNTs under continuous sonication and stirring to form a gray precursor gel. The precursor gel was then removed from the ultrasonicator but stirring continued on a magnetic stirrer for 5 h. The precursor gel was then transferred into silicon carbide-coated Teon reactor vessels, clamped into a blast-safety casing, and subjected to 30 min of microwave heating at 180 C and 1 MPa Table 1 using an MDS-6G synthesis/digestion microwave reactor. The resultant gray precipitate was sequentially washed in propanol, ethanol followed by a nal rinse in a 1 : 1 DI : Et$OH solution. Aer vacuum drying, the precipitate was further dried in an oven at 100 C for 48 h and crushed using a mortar and pestle. TiO2–aNCNT and TiO2–aCNT nanohybrids with 5 wt% CNT wt loading were synthesized in a similar way except that TiOBut was hydrolyzed using propanol : DI (1 : 1 v/v) instead of NH4OH (Table 1). 2.4. Characterization of nanohybrids Fourier transform infrared (FTIR) spectroscopic analysis (PerkinElmer Frontier FTIR spectrometer) was carried out to investigate the presence of surface functional groups on the nanohybrids. For transmittance measurements, each sample was ground with KBr and pressed to be a semi-transparent pellet and scanned in the range 450–4000 cm1. Elemental analysis was carried out using an Axs TM® X-ray photoelectron spectrophotometer. Each powdered sample was pressed into a pellet before analysis. Particle size measurements and morphology investigations were carried out using a Jeol TEM 2010 (200 kV). Approximately 5 mg of nanohybrid powder was dispersed in 10 mL ethanol by ultrasonicating at room temperature. Two drops of the colloidal suspension were spread onto a Cu grid that had been previously sputter coated with 5 nm carbon. To determine the crystalline phase and to investigate the bonding conguration between NTiO2 and aCNTs/aNCNTs, Raman spectroscopic analysis was carried out using an Alpha 300RA AFM/Raman combined system (Witec). A double-sided, transparent tape was spread onto a microscope glass slide and ca. 5 mg of the nanohybrid powder was pasted onto the tape. Measurements were taken using the Raman 532 module at an integration time of 0.6 s. Thermogravimetric analysis (TGA) and diﬀerential thermal analysis (DTA) were carried out using a Trios TGA 5500 equipped with a high temperature furnace. For these analyses, ca. 10 mg of each nanohybrid was spread onto a platinum high temperature pan before inserting into the high temperature furnace. A ramp rate of 10 C min1 under a 25 mL min1 N2 and air ow were used separately to determine the thermal stability prole in the temperature range 30–850 C. The TGA and DTA data were analyzed using the Trios TGA analyzer soware. UV-vis absorbance was determined using a PerkinElmer Lambda 650 S UV-vis spectrometer equipped with a tungsten and deuterium lamp with a combined scan range of 180– 800 nm. All the scans were done at a rate of 266 nm min1 and A description of the composition of the powdered nanohybrids and their coloration Nanohybrid Description Color TiO2–aCNT TiO2–aNCNT NTiO2–aNCNT Titania–amorphous carbon nanotubes Titania–amorphous nitrogen doped carbon nanotubes Nitrogen doped titania–amorphous nitrogen doped carbon nanotubes Light gray Light gray Yellow gray 6750 \| RSC Adv., 2021, 11, 6748–6763 © 2021 The Author(s). Published by the Royal Society of Chemistry View Article Online Paper Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. all samples were scanned as powders. Photoluminescence spectroscopy was carried out using a Horiba Fluorolog FL3 spectrophotometer equipped with a xenon lamp with scan range of 200–750 nm. The nanohybrids were excited at 350 nm and the emission spectra were collected in the range 270– 600 nm. All data, unless specied, was plotted and analyzed using Origin 8.5 Pro. Version. 2.5. Photocatalytic performance evaluation The photocatalytic performance of the nanohybrids was rst evaluated for the removal of RR 120 by dispersing 5 mg of each nanohybrid photocatalyst in 200 mL of a 20 ppm solution of the dye. The suspension was sonicated for 30 min at room temperature to achieve adsorption–desorption equilibrium between the dye and the nanohybrid photocatalysts. In the analysis of the eﬃciency of the nanohybrids, dye removal eﬃciency was considered during the adsorption–desorption stage and at 30 min aer photocatalytic degradation and at the end of contact time i.e. 300 min. The nanohybrids were further evaluated for the removal of color from vat-dye laden industrial textile eﬄuent under the similar conditions as in the case of RR 120. 3. Results and discussion 3.1. Chemical composition analysis of the titania– amorphous carbon nanotube nanohybrids 3.1.1 Analysis of nanohybrids by surface functional groups composition. FTIR spectroscopic analysis revealed that the aCNTs and aNCNTs showed free and intermolecular bonded alcohol –OH group with peak center at 3450 cm1. However, the nanohybrids have a broad and strong alcohol and carboxylic acid –OH stretch peak in the range 3600–2600 cm1 and a corresponding –OH bending peak at 1637 cm1 (Fig. 1). The FTIR spectra of the titania–amorphous carbon nanotube nanohybrids further conrmed the presence of graphitic forms of carbon residue because of the presence of the alkane C–H FTIR spectra of the titania–amorphous carbon nanotubes nanohybrids1 and amorphous nitrogen doped carbon nanotubes. Fig. 1 © 2021 The Author(s). Published by the Royal Society of Chemistry RSC Advances stretch peaks at 2980, 2930 and 2879 cm1. In addition, a broad peak in the range 1739–1576 cm1 occurs in all the nanohybrids and the aNCNTs; a region indexed to –C]C–, alkane –C]O and amine –N–H bending. There also occurs a primary alcohol –C–O stretching peak at 1059 cm1 for all the nanohybrids and the aNCNTs. It is also expected that the molecular residue from the alcohol and acetylacetone used during the synthesis process also leave traces of these organic groups on the surfaces of the nanohybrids. FTIR peaks due to CO and COO are observed for all nanohybrids at 1115 and 1059 cm1 suggesting that the nanohybrids are capable of adsorbing organic molecules. The peak at 2348 cm1 is assigned to –C–N– and gaseous CO2 bonds and it occurs in the aNCNTs and NTiO2–aNCNT nanohybrids.21 This conrms the doping of N into amorphous C and that the N plays a role in the formation of the TiO2/aNCNT interface. 3.1.2 Elemental composition, oxidation state and bonding conguration analysis of the TiO2–aNCNT nanohybrids. The wide scan spectrum of the TiO2–aNCNT nanohybrids conrmed the exclusive presence of the Ti, O, C and N elements in the nanohybrid at 19.96, 53.87, 25.23 and 0.94 at%, respectively (Fig. 2(a) and Table 2). The N1s spectrum conrmed the incorporation of N into aNCNTs with the occurrence of a broad peak in the range 395–399 eV and centered at 397 eV (Fig. 2(b)). The peak range is indexed to sp2 hybridized N bonded to sp3 hybridized C, i.e. a typical case of N at the periphery of graphitic sheets. The same range is also indexed to binding energy of the –Ti–N– bond30 hence suggesting that some of the N atoms on the aNCNT residue are the centers on which TiO2 nanocrystals were built. The range is also indexed to C]N which could suggest surface N groups.26 The Ti2p spectra has the Ti2p3/2 peak center at 455.75 and its Ti2p1/2 at 461.47 eV indicating a peak separation of the 6 eV and hence indexed to Ti4+ (Fig. 2(c)). These peaks indicate a blue shi of the Ti2p3/2 and Ti2p1/2 in Ti2+ which typically occur at 455.03 and 460.85 eV respectively.31 A blue shi in binding energies suggests that the elemental species is bonded to a more electronegative element; of which, in the case of TiO2–aNCNT nanohybrids, C is the most electronegative on the Pauling scale.32 In order of decreasing electronegativity, the elements in the nanohybrid are O (3.44) > N (3.04) > C (2.55) > Ti (1.54). Therefore, the blue shi of the binding energies for Ti2p3/2 and Ti2p1/2 in Ti2+ suggest the occurrence of the Ti–C dative bond. The occurrence of the –Ti–C– bond is further conrmed by the C1s peaks at 281.92 and 284.41 eV which are indexed to the –Ti–C– bond (Fig. 2(d)). The peak at 283.41 eV has also been found in nanohybrids of TiO2–amorphous carbon and has been attributed to the –Ti–C– bond in the conguration –O– Ti–C–.33 The C1s spectrum also has a peak in the binding energy range 284.01–286.80 eV, a range in which the binding energy for C in the bonding congurations –C–OH, –C–O–C–, –C–C–, –C–C–H and –C–N falls.34,35 The O1s spectrum shows that the nanohybrid consists of both lattice and surface oxygen of which the lattice oxygen is all the oxygen that is found in the TiO2 crystalline matrix (Fig. 2(e)). The surface O accounts for 38.7% of all the oxygen and is typically indexed to the functional –OH and –COOH groups. These groups are negatively RSC Adv., 2021, 11, 6748–6763 \| 6751 View Article Online Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. RSC Advances Fig. 2 Paper XPS analysis for the nanohybrid TiO2–aNCNT showing (a) wide scan, (b) N1s, (c) Ti2p, (d) C1s and (e) O1s spectra. Table 2 Atomic% and elemental mass% concentration of the TiO2– aNCNT nanohybrids O1s C1s N1s Ti2p Atomic [%] Error [%] Elemental mass [%] Error [%] 53.87 25.23 0.94 19.96 0.96 1.20 0.46 0.40 40.39 14.20 0.62 44.79 0.54 0.77 0.30 0.57 charged, hence increasing the aﬃnity of the nanohybrids for positively charged species in adoption and photocatalytic processes. 3.2. Particle morphology analysis of the titania–amorphous carbon nanotube nanohybrids An analysis of the TEM micrographs shows that all the nanohybrids have relatively good particle dispersion hence demonstrating that the synthesis method resulted is well dispersed TiO2 nanoparticles. The role of doping either TiO2 (to form NTiO2) and aCNTs (to form aNCNTs) is revealed in the morphology, particle size and particle size variation of the of TiO2/NTiO2 in the nanohybrids. TiO2 nanoparticles are generally quasi spherical and have small particles with small diﬀerences in average particle size and particle size variation (Fig. 3(a) and (b)). The average particle size for the nanohybrid 6752 \| RSC Adv., 2021, 11, 6748–6763 TiO2–aCNT is 7.35 1.59 nm and that of the TiO2–aNCNT nanohybrids is 7.08 1.60 nm (Fig. 3(d)). The NTiO2–aNCNT nanohybrids, on the other hand, has larger NTiO2 nanoparticles (16.90 4.11 nm) with various shapes including cuboidal, rhomboid, quasi spherical and rod-shaped and hence a wide particle size distribution Fig. 3(c). Unlike the other nanohybrids, the NTiO2 nanoparticles all have dened crystal shapes with distinct edges. These features for NTiO2 are consistent with our previous observations and work by other researchers.36,37 As such, it can be established that the hydrothermal synthesis of N doped TiO2 leads to the formation of cuboidal NTiO2 nanoparticles. The small size of the TiO2–aCNT and TiO2–aNCNT nanohybrids make them better suited for applications where adsorption is a key step since smaller sized nanoparticles oﬀer a higher surface area for adsorption.38 The TiO2–aCNT and TiO2–aNCNT nanohybrids show traces of aCNTs and aNCNTs, respectively (indicated by red arrows on the (Fig. 3(a) and (b)). In the former, the aCNT is densely covered by TiO2 nanoparticles resulting in the general tubular alignment of the TiO2 nanoparticle clusters whereas in the latter, the aNCNT is sparsely covered. The nanohybrid NTiO2– aNCNT, on the other hand, shows no traces of aNCNT structures although alkane and alkene peaks attributed to graphitic sheets and CNTs are observed in the FTIR spectra of the nanohybrid (Fig. 1). These observations are consistent with the Raman spectra of the nanohybrids, where D-and G-bands could be deconvoluted in the TiO2 based nanohybrids but not in NTiO2–aNCNT (Fig. 4 inset (b)). The absence of the aCNTs and the aNCNTs is due to their degradation, which results in their © 2021 The Author(s). Published by the Royal Society of Chemistry View Article Online RSC Advances Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Paper Fig. 3 Particle morphology analysis of nanohybrids showing; TEM micrographs for (a) TiO2–aCNT, (b) TiO2–aNCNT and (c) NTiO2–aNCNT nanohybrids; corresponding particle size distribution analysis for each of the nanohybrids (e and f) and (g) TEM micrographs for amorphous carbon nanotubes (aCNTs). © 2021 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2021, 11, 6748–6763 \| 6753 View Article Online Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. RSC Advances Paper Fig. 4 Raman spectra for the nanohybrids and commercial TiO2 with insets showing (a) the shifts in wavenumber due to hybridization (b) D- and G-bands for NTiO2–N-CNTs after functionalization. graphitic and amorphous residue being incorporated into the TiO2/NTiO2 nanocrystals. Lastly, selected area diﬀraction of the NTiO2 nanoparticles agrees with the Raman peak intensities in indicating good crystallinity of the nanohybrids (Fig. 3(c) inset) and Fig. 4). Therefore, the microwave assisted hydrothermal synthesis method is shown to be eﬃcient in tailoring of doped and undoped TiO2–aCNT/aNCNT nanohybrids. 3.3. Raman spectroscopy analysis of nanohybrids All the nanohybrids are of the anatase phase as indicated by the anatase Eg, A1g and B1g peaks arising from the symmetric stretch, antisymmetric bending and symmetric bending of the –O–Ti–O– bond respectively.39 The weak intensity of the Eg peaks indicate that the hydrothermally synthesized nanohybrids have lower crystallinity than commercial anatase TiO2. All the nanohybrids show a red shi of the Eg anatase peak compared to the Eg peak of commercial anatase TiO2. The anatase Eg peak for commercial TiO2 appears at 152 cm1 while the nanohybrids appear at higher wavelengths in the decreasing order TiO2–aNCNT (163 cm1) > TiO2–aCNT (160 cm1) > NTiO2–aNCNT (157 cm1) (Fig. 4 inset (a)). The degree of the shi of the wavenumber denotes the degree to which the electronic structure of TiO2 has been altered and thus, in the case of the nanohybrids, the strength of the bonds between TiO2/NTiO2 6754 \| RSC Adv., 2021, 11, 6748–6763 nanoparticles and the aCNTs/aNCNTs.33,34 Therefore, results indicate that the bond strength is in the decreasing order TiO2– aNCNT (163 cm1) > TiO2–aCNT (160 cm1) > NTiO2–aNCNT (157 cm1); making the aNCNT to be the most suitable for forming a more stable TiO2–CNT interface. The stronger bonding between aNCNTs and TiO2 can be attributed to the introduction of partial positive charges (d+) over C atoms and partial negative charges (d) over N atoms in the N-CNT skeleton. These charges reduce the surface energy of the NCNTs compared to CNT and thus making it energy favorable for the dative covalent attachment of TiO2 and NTiO2 seed crystals during the initial polymeric condensation of NTiO2 onto the CNTs. The D- and G-bands of the nanohybrids were of low intensity due to the disintegration of the carbon nanotubes and its integration into the TiO2/NTiO2 crystals and hence curve tting was done to determine their exact peak position and intensities (Fig. 4 inset (b)). The disappearance of the D- and G-bands due to aCNTs is in agreement with previous observations where MnO was embedded onto aCNTs.40 Curve tting of the Raman spectra of the nanohybrids showed weak D- and G-peaks for TiO2–aCNT and TiO2–aNCNT nanohybrids such that analysis of these peaks was not possible. An analysis of peak intensities and D- and G-band band positions was carried out to determine © 2021 The Author(s). Published by the Royal Society of Chemistry View Article Online Paper RSC Advances An analysis of changes in band position and intensity changes for the D-and G-bands Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Table 3 Nanohybrid Properties D-band G-band aNCNTs Wavelength pos. (cm1) Band area (a.u.) FWHM (cm1) IG/ID Wavelength pos. (cm1) Band area (a.u.) FWHM (cm1) IG/ID Wavelength pos. (cm1) Band area (a.u.) IG/ID N/A N/A 1367 1589 4748 317 0.272 1357 1293 100 1596 greater change in the wavenumber for the anatase Eg band due to the nanohybrid TiO2–aNCNT. This is despite the dative bond between Ti4+ and the N-CNTs withdraws electrons from the NCNTs. The extra electrons due to the presence of N in the NCNTs and the presence of N in NTiO2 alters the electronic behavior of the nanohybrid and hence its response to the light, including the exciting laser during Raman spectroscopic analysis. This points to the degree by which doping both TiO2 and CNTs alters the optical properties of the nanohybrid. 9303 234.2 0.458 1382 4269 96.8 3.4. Thermogravimetric analysis of nanohybrids aCNTs NTiO2–aNCNT TiO2–aCNT NTiO2–aCNT 392 0.359 1676 1092 the degree of bonding between TiO2/NTiO2 nanoparticles with aCNTs and the aNCNTs. Previous studies have shown that the modication of CNTs with heteroatoms and heteroatomic groups alters the electronic properties of the CNTs thus interfering with both the position and intensity of the graphitic Dand G-bands.7,41,42 An attachment of electron acceptor groups results in a blue shi of the D- and G-bands while electron donor groups produce a red shi.41,42 The D- and G-bands of the nanohybrid NTiO2/TiO2–aCNT/aNCNT had red shi of both the D- and G-bands relative to those of aNCNTs from 1397 to 1382 cm1 for the D-band and 1589 to 1676 cm1 for the graphitic band (Table 3). This could suggest that the bonding between NTiO2/TiO2 and aCNT/aNCNT occurs through the –OH groups on the Ti(OH)4 intermediate donating their lone pair of electrons to the surface –OH and –COOH groups of the aNCNTs and aCNTs. For the aNCNTs, the –OH groups on the Ti(OH)4 intermediate could also donate their lone pair of electrons to the d+ surface C atoms to form a dative covalent bond. This additional bonding conguration is expected to make the bonding between TiO2 and aNCNTs to be stronger as suggested by the Fig. 5 TGA and DTA thermograms for aCNTs and aNCNT conrm the coexistence of an amorphous carbon and the crystalline forms in each sample. It is further revealed that both amorphous and crystalline forms of aNCNTs degrade at lower temperatures compared to those of aNCNTs. Furthermore, the amorphous nature of aNCNTs relative to aCNTs is demonstrated by a lower intensity of the diﬀerential peak corresponding to crystalline aNCNTs being lower that corresponding to crystalline aCNTs (Fig. 5(b)). DTA and TGA thermograms of the nanohybrids show that all the nanohybrids have a dual composition of amorphous carbon and crystalline CNTs and NCNT, with the latter having the highest wt% composition in each hybrid (Fig. 6(a) and (b)). The increase in the DTA peak due to amorphous residue from both the aNCNTs and aCNTs relative to the DTA peak corresponding to the crystalline forms suggest that the nanohybrid synthesis process resulted in the breaking down of part of the crystalline forms into amorphous carbon residue and hence the aim of this work achieved. Previous reports have demonstrated that CNTs and aNCNTs synthesized at high temperatures, at long reaction times, high ow rates of the carbon source are prone to having thick aky walls whose outer wall is weakly held to the inner crystalline form.10 The nanohybrids also demonstrate high adsorption for water and polar organics because of the wt% loss in the temp range 30–250 C (Table 4 and Fig. 6b). This suggest that they are suited for applications where adsorption is part of the mechanism e.g. photocatalytic applications and sensing with NTiO2–aNCNT showing a greater adsorptive capability. The occurrence of these surface groups is also corroborated by the TGA (a) and DTA (b) thermograms for aNCNT and aCNTs. © 2021 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2021, 11, 6748–6763 \| 6755 View Article Online RSC Advances Paper Table 4 Thermogravimetric analysis of wt% loss for nanohybrids at selected temperature ranges Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. wt% loss per temperature range (%) Nanohybrid <250 C 25 < 520 C >520 C TiO2–aCNT TiO2–aNCNT NTiO2–aNCNT 1.27 1.50 2.56 4.73 5.03 2.52 0.377 0.299 0.681 broad –OH vibration peak in all the nanohybrids in the FTIR spectra for the nanohybrids (Fig. 1). For the nanohybrid TiO2– aNCNT the occurrence of the surface groups is further conrmed by the occurrence of adsorbed oxygen peaks in the O1s spectra (Fig. 2(f)). The degradation temperature peaks for the crystalline forms of the aNCNTs and aCNTs occur at slightly lower temperatures in the nanohybrids compared to the aNCNTs and aCNTs. This is attributed to the TiO2 and NTiO2 trapping more heat at any given temperature and hence delivering more heat to the carbonaceous forms compared to the case where the carbonaceous forms are without TiO2.42 The amorphous form of aCNTs and aNCNTs are thermally degraded at a lower temperature range in the nanohybrids (224–448 C) than in the aCNTs (550–650 C) (Fig. 5(b)). However, these amorphous forms in the nanohybrids degrade in the same temperature for all the nanohybrids and over a wider range. In addition, the DTA thermograms have a shoulder peak occurring at 380 C. The longer degradation range and the occurrence of the shoulder peak suggests that the two components are being degraded in the temperature range 224–448 C. We reason that the shoulder peak at 380 C is due to graphitic akes peeling oﬀ from outer walls of the thick aNCNT and aCNTs during the synthesis of the nanohybrids while the lower temperature range is due to the amorphous form and towards the extreme low of this DTA peak, it is the carbonaceous fragments and atoms intercalated into the TiO2 lattice. In retrospect, the DTA and TGA thermograms of the nanohybrids suggest that their chemical and mechanical integrity in air has its upper threshold at 200 C. At higher temperatures, the doped C and the amorphous carbon residue is oxidized yet Fig. 6 these are essential for the maintenance of visible light absorption, and low charge recombination. Also, the changes in the thermal degradation temperatures in the region for degradation for aNCNTs and aCNTs lead to the inference of the strength of the interfacial bond between the TiO2 or NTiO2 nanoparticles and the aCNTs. The greater the shi from the thermal degradation of the aCNTs, the stronger the interaction between the aCNTs and the TiO2 nanoparticles. The idea works on the assumption that the chemical interaction at the interface between the CNTs and the TiO2 introduces a new set of bonds with unique bond strengths and enthalpies. Therefore, these deviations of the thermal degradation temperatures suggest that the nanohybrid between NTiO2/TiO2 and aNCNTs/aCNTs has been formed.34,42 3.5. Optical response of the nanohybrids 3.5.1 UV-vis spectroscopic analysis. The synergistic eﬀect of doping TiO2 with N and making its composite with aNCNT enhanced the energy band-gap reduction better than hybridizing TiO2–aCNTs. This is because the nanohybrid NTiO2– aNCNT has a lower energy band gap of 2.97 eV while the nanohybrids TiO2–aNCNT and TiO2–aCNT have equal energy band gaps of 3.00 eV. A reduction in the energy band gap is the main indicator of the absorption edge of semiconductor photocatalysts and a low Eg indicates a higher absorption edge. In the case of these nanohybrids, the energy band gap for all the nanohybrids indicates that all the nanohybrids absorb in the visible region i.e. beyond 390 nm which is the typical band edge for undoped and perfectly crystalline TiO2. The band gaps for the nanohybrids NTiO2–aNCNT, TiO2–aNCNT and TiO2–aCNT correspond to the absorption edges 430, 424 and 424 nm, respectively; hence increased absorption of visible light compared to commercial TiO2 (with absorption edge at 390 nm). Hybridizing TiO2 with aNCNTs or aCNTs produced nanohybrids with increased photosensitization in the visible range as shown by an increase in absorbance from 0 to the range 0.23– 0.303 for the nanohybrids. aNCNTs have a higher photosensitization eﬀect on TiO2 than aCNTs and doping of TiO2 with N has insignicant contribution into photosensitization of the A comparison of TGA (a) and DTA (b) proﬁles for nanohybrids. 6756 \| RSC Adv., 2021, 11, 6748–6763 © 2021 The Author(s). Published by the Royal Society of Chemistry View Article Online Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Paper RSC Advances Photoresponse of the nanohybrids as depicted by the (a) UV-vis spectrum and (b) Tauc plots showing examples of how the energy band gap is determined by the tangential lines and (c)–(e) Urbach energy (Eu) plots for the nanohybrids. Fig. 7 nanohybrids as shown by the insignicant diﬀerence in the visible light absorbance for the nanohybrids TiO2–aNCNT (0.293 a.u.) and NTiO2–aNCNT (0.303) (Fig. 7(a)). Photosensitization of the nanohybrids is further enhanced by the Urbach energies of the nanohybrids. Urbach energy (Eu) is evidenced by the occurrence of Urbach tails at the band edge of semiconductor UV-vis spectra.37,43 At constant temperature Eu Fig. 8 Optical response of the nanohybrids as determined by (a) photoluminescence spectra of all the nanohybrids and deconvoluted spectra of the nanohybrids (b) TiO2–aCNT, (c) TiO2–aNCNT and (d) NTiO2–aNCNT. © 2021 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2021, 11, 6748–6763 \| 6757 View Article Online Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. RSC Advances Paper is due to crystalline defects. These defects can be due to the transformations in the crystalline structure due to temperature changes, occurrence of dopants (which distort the crystalline structure) and amorphosity of the nanocrystals.44,45 In semiconductor photocatalysis, Eu enhances the absorption of light beyond the absorption edge. Eu is the reciprocal of the slope in a plot of the natural log of the absorption coeﬃcient [ln(R)] and photon energy (hn).45 The results show that the hybridizing TiO2 with aNCNTs is eﬀective in enhancing visible light absorption through synergistic eﬀect of doping TiO2 with N and hybridizing it with aNCNTs is eﬀective in enhancing visible light absorption through Eu. This is shown by the nanohybrid TiO2– aNCNT having the highest Eu of 1.07 eV (Fig. 7(d)). However, the synergy of doping TiO2 with N and hybridizing with aNCNT suppresses the eﬀect of aNCNTs in increasing the Eu as shown by the low Eu for the nanohybrid NTiO2–aNCNT (0.816 eV) compared to that of TiO2–aCNT (0.848 eV). 3.5.2 Photoluminescence spectrum for nanohybrids. The PL intensity indicates that hybridizing TiO2 and NTiO2 with aNCNT/aCNT suppresses charge recombination compared to commercial TiO2 (Fig. 8(a)). However, there is a signicant diﬀerence in the degree to which aCNT and aNCNT suppress charge recombination in that aNCNT-based nanohybrids (NTiO2–aNCNT and TiO2–aCNT) have a signicantly low PL intensity than the nanohybrid TiO2–aCNT whose band edge peak has an intensity of 2.71 106 CPS. Furthermore, a positive synergy exists between doping TiO2 with N and hybridizing with aNCNT in that the nanohybrid NTiO2–aNCNT has the lowest PL intensity of 6.24 105 CPS; a signicantly low intensity compared to that of TiO2–aNCT 6.59 105 CPS. The implications of the PL intensity on the photocatalytic activity of the nanohybrids is discussed in the sections for the photocatalytic activity of the nanohybrids (Section 3.6). The PL spectra of the nanohybrids is also used to infer the presence of crystal defects and uncapped bonds on the surface of crystalline materials such as TiO2. Charge recombination occurring at crystal defects is indicated by the peaks occurring at 460 nm while charge recombination due to dangling bonds is indicated by PL spectra not going back to 0 CPS at the longer wavelengths.46 Therefore, the PL spectra for the nanohybrids indicates that they have O vacancies but do not have any dangling bonds (Fig. 8(b)–(d)). A controlled occurrence of these O vacancies can enhance the photocatalytic activity of TiO2 in that, these sites introduce mid-band gap states closer to the VB hence providing an alternative conduction band in a lower energy state. The overall eﬀect is the reduction of the Eg.47 However, as it is in the case of most dopants, this reduction in the Eg is benecial if there is an eﬃcient means of sequestering photogenerated electrons away from the VB of the TiO2 since there charge recombination is reduced. It is, therefore, expected that the occurrence of these vacancies in the nanohybrids TiO2– aNCNT and NTiO2–aNCNT enhances the photocatalytic activity due to the low charge recombination. 3.6. Photocatalytic performance of the nanohybrids 3.6.1 Removal of the dye Reactive Red 120 (RR 120) from a single component system A. Adsorptive removal of the dye RR 120. The nanohybrid TiO2–aCNT had the highest adsorptive dye removal of 98.2% at 60 min of adsorption–desorption equilibrium (Table 5). While Table 5 A summary of the optical properties and average particle size compared to the adsorptive and photocatalytic performances of the nanohybrids Nanohybrid Eg (eV) PL intensity (106 CPS) kapp (102 min1) Particle size (nm) % dye adsorption % dye removed at 30 min under illumination TiO2–aCNT TiO2–aNCNT NTiO2–aNCNT 3.00 3.00 2.97 0.272 6.8 6.07 2.25 3.44 2.49 7.35 1.59 7.07 1.60 16.9 4.1 98.2 2.0 81.1 1.55 18.9 1.91 99 1.75 99 1.65 46 2.3 Fig. 9 Reactive Red 120 (CI) molecule. 6758 \| RSC Adv., 2021, 11, 6748–6763 © 2021 The Author(s). Published by the Royal Society of Chemistry View Article Online Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Paper the nanohybrid TiO2–aNCNT, showed the second highest overall adsorptive removal of the dye (81.1%), it showed the fastest adsorptive removal, reaching 82.9% within 30 min (Fig. 10(a)). The nanohybrid NTiO2–NCNT showed the lowest adsorptive removal and the highest leaching of the dye molecules reaching its maximum at 45 min and subsequently leaching oﬀ 6% of the already adsorbed dye. Given that adsorption is a factor of active surface area and charge diﬀerence between the catalyst and the pollutant, the high adsorptive capacity of the nanohybrids TiO2–aCNT and TiO2–aNCNT is typical of catalysts with a large active surface area.48 The surface is a factor of particle size, particle size distribution and dispersion. Small, monodisperse and well dispersed nanoparticles oﬀer high active surface area. The adsorptive behavior of the nanohybrids behave as predicted by the TEM micrographs of the nanohybrids in that the nanohybrids TiO2–aCNT and TiO2–aNCNT have the smallest particle size (7.35 1.5 and 7.07 1.6 nm, respectively) and are better dispersed (Table 5 and Fig. 3). NTiO2–aNCNT, on the other hand, has the highest particle size distribution (16.9 4.1 nm) and the lowest adsorption. It also suggests that at the operational pH for the adsorption, the TiO2–aNCT and TiO2–aCNT nanohybrid photocatalysts have an overall negative charge, hence they actively attract the dye molecules. Charge behavior of the dye RR 120 and its interaction with CNT-based photocatalysts has shown that the attractive forces are due to the positively charged sulte groups (R-SO3) aer the dissociation of the Na+ ion and the – interactions arising from p–p the cyclic rings in the RR 120 molecule (Fig. 9).49 Also, being an azo dye, the RR 120 molecule is rich in N and as such it is expected that in its interaction with the N-doped photocatalysts, the lone pairs of the N atoms will result in the repulsion between the dye molecules and the photocatalysts. The low adsorptive capacity of NTiO2–aNCNT and the highest adsorption of TiO2–aCNT could suggest that the RSC Advances repulsion due to these lone pairs is more pronounced than the possible attractive forces due to the – interactions between the cyclic carbon rings in the dye p–p molecules and the –C–C– making up of the aNCNTs and aNCNTs. B. Photocatalytic removal of the dye RR 120 under illumination. The TiO2–aNCNT nanohybrids demonstrates the highest photocatalytic degradation of the dye jumping from 81.1% to 99.1% aer 30 min under illumination while the removal by TiO2–aCNT only reaches 99% dye removal aer 120 min of illumination (Fig. 10(b), (c) and Table 5). The nanohybrid NTiO2–aNCNT shows improved photocatalytic removal by reaching 99.9 overall dye removal at contact time 300 min like all the nanohybrids at an overall apparent rst order constant of 2.49 102 min1, a rate constant that is higher than that for TiO2–aCNT (2.25 102). Kinetic studies of the photocatalytic removal of the dye demonstrate that the photocatalytic degradation of the dye follows pseudo rst order kinetics. A comparison of the photocatalytic performance of these nanohybrids with the physicochemical properties of the nanohybrids leads to the following observations: (i) The dye removal performance for the TiO2–aCNT nanohybrids is more dependent on the small particle size, monodispersity and good particle dispersion (properties which insinuate a large surface area) more than it does rely on the optical properties; (ii) The NTiO2–aNCNT nanohybrids, however, relies more on its good photocatalytic properties such as low Eg and high photosensitization in the visible to near infrared region. These properties make it more suited for visible light photocatalysis than all the nanohybrids. This means that the photocatalytic degradation by the TiO2–aNCNT photocatalytic nanohybrids proceeds through the production of the reactive oxidation species (O2c and cOH) more than the surface mediated redox degradation of the nanohybrids; Photocatalytic performance of the nanohybrids measured by (a) % dye removal during adsorption–desorption equilibration (in the dark) and photocatalytic degradation (under illumination), (b) photocatalytic degradation under illumination and (c) apparent ﬁrst order rate constants (kapp) for photocatalytic degradation under illumination. Fig. 10 © 2021 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2021, 11, 6748–6763 \| 6759 View Article Online Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. RSC Advances Paper Fig. 11 UV-vis spectra† of depicting degradation proﬁles for textile wastewater during photocatalytic treatment of the (a) TiO2–aNCNT (b) NTiO2–aCNT and (c) TiO2–aNCNT nanohybrids. Insets show the formation of peaks in the process of the oxidative treatment. (iii) The TiO2–aNCNT nanohybrids have high visible light photosensitization, high Eu but high Eg; characteristics which make it relatively better suited for visible light photocatalysis than TiO2–aCNT and NTiO2–aNCNT photocatalytic nanohybrids. The nanohybrid TiO2–aNCNT also has the smallest particle size and good particle size distribution. Therefore, it is the most suited nanohybrid for photocatalytic degradation because it possesses both the good characteristics and not the downside characteristics from the latter. 3.6.2 Photocatalytic treatment of industrial textile eﬄuent waste. The UV-vis spectra for the textile wastewater suggest that the water consists of mildly coloring dissolved organics. The color is assumed to be due to the presence of VAT dye nanoparticles. Typical textile wastewater also consists of organics such as aldehydes, alcohols, ketones and a wide range of organics from surfactants.50,51 Permissions to get the exact constituents of the dyeing, washing, rinsing and scouring chemicals could not be obtained from the source textile factory. The UV-vis spectra for the degradation of the organics in the wastewater suggest that it proceeds through the formation of intermediates that either absorb at a diﬀerent wavelength or react with each other to form a secondary intermediate that † The spectra of the samples are named according to the stage and time at which each sample was collected i.e. adsorptive (A) for adsorption–desorption equilibration and photocatalytic (P) for degradation under illumination. The times at which the samples are collected at each stage are indicated in Arabic numerals. 6760 \| RSC Adv., 2021, 11, 6748–6763 absorbs at a diﬀerent wavelength. This is indicated by the occurrence of isosbestic points between the spectra of aliquots collected at diﬀerent times for each of the nanohybrids. Isosbestic points are an indication of the changing composition of a sample.52 The quantity of some of these intermediates is prominent such that new peaks in the visible range appear for the TiO2–aCNT and NTiO2–aNCNT nanohybrids in the ranges 472–486 and 425–600 nm, respectively, aer 30 min during the adsorption–desorption equilibrium attainment stage (A30) (Fig. 11(a) and (b)). These peaks suggest that there some of the polluting components in the wastewater are broken down even in the dark by these nanohybrids. A classic example of a mass transfer observable in the UV-vis spectrum in the degradation of N-containing groups such as those typically contained by surfactants, to form nitrile and nitrate ions.53 Other Ccontaining moieties found in surfactants also form-visible light-absorbing groups.52 Another feature common to all the TiO2–CNT nanohybrids is the uctuation of the absorbance spectra for aliquots. For example, the aliquot collected at 30 min during sonication in the dark for the TiO2–aCNT nanohybrid has a high absorbance than the wastewater before degradation (Fig. 11). Similar observations are made on the nanohybrid NTiO2– aNCNT. The nanohybrid TiO2–aNCNT shows the highest adsorptive removal of the organics from the wastewater within the rst 30 min. This is evidenced by the decrease of the absorbance to almost zero across the whole spectrum Fig. 11(c). In addition to this, the aliquot collected aer © 2021 The Author(s). Published by the Royal Society of Chemistry View Article Online Paper Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Table 6 RSC Advances A comparison of the eﬃciency of the current work to previous literature Photocatalyst Application Result Ref. Magnetic MWCNT–TiO2 Photocatalytic degradation of malachite green (MG) 57 TiO2–Pt–MWCNT Photocatalytic hydrogen production under irradiation Photocatalytic degradation of malachite green Photocatalytic degradation of noroxacin, diclofenac and phenol 100% 20 ppm removal of MG at pH5 and catalyst loading of 200 ppm catalyst loading Catalyst reusable despite the bulk of TiO2–MWCNT and simple electrostatic attraction 2327 and 2091 mmol g L1 H2 produced in 2 h under irradiation of glycerol and methanol respectively 100% removal of MG in 6 min at 100 mg pollutant per 1 g of catalyst under solar irradiation k1app ¼ 1.248, 0.925, and 0.721 min1 for the degradation of, noroxacin, diclofenac and phenol All Ag3PO4@NC nanohybrids performed better than Ag3PO4 Energy band gap reduction from 2.7 to 2.3 eV and up to 280% increase in H2 evolution with incorporation of amorphous Carbon Specic capacity of 749 mA h g1 at current density of 0.2 A g1 7.046 101 ppm dye adsorption in 60 min and 90% dye removal in 180 min at k1app ¼ 17.2 103 min1 Ag3PO4@MWCNTs@Cr:SrTiO3 Ag3PO4@NC Amorphous C doped ZnxCd1xS H2 evolution under visible light Sn@aCNT Lithium-ion storage batteries Amorphous carbon doped ZnO/Zn Photocatalytic degradation of basic blue 41 (12.5 ppm) and 100 mg L1 catalyst loading under visible light TiO2–aCNT, TiO2–aNCNT, NTiO2– Photocatalytic degradation of Congo red Dye removal eﬃciency at 30 min adsorption and aNCNT (radiation microwave dye under LED white light 120 min photodegradation TiO2–aCNT – 69.4 and hydrothermal synthesis) 92.5%, TiO2–aNCNT – 89.2 and 99.2%, NTiO2–aNCNT – 42.9% and 82.6% TiO2–aCNT, TiO2–aNCNT, NTiO2– Photocatalytic degradation of RR 120 (20 Dye removal eﬃciency at 60 min adsorption and aNCNT ppm) at 100 ppm catalyst loading 300 min photodegradation TiO2–aCNT – 98.2 and 99.9%, TiO2–aNCNT – 81.1 and 99.9% NTiO2–aNCNT – 18.9% and 99.9% 120 min of degradation under illumination (P120) has the highest absorbance (Fig. 11(c)). These features also point to the possibility of the formation of UV-absorbing intermediates through a chemical reaction of intermediates formed from the degradation of non-UV absorbing organic moieties.54 VAT dyes, which are insoluble, can be broken down to water-soluble intermediates through the action of the radicals produced in AOPs. This would result in an increase in the concentration of dissolved organic moieties and hence an increase in the overall absorbance spectrum.55 However, in all the nanohybrids, the nal aliquots collected at 240 min of degradation under illumination (P240) has the lowest absorbance. This points to the fact that the concentration of the organic pollutants in the wastewater has decreased and hence in overall the catalysts are showing catalytic degradation for the organics in the textile wastewater. In essence, the nanohybrids are shown to eﬀectively break down textile dyes in industrial wastewater. High photocatalytic and adsorptive activity is desirable for photocatalysts to be incorporated into photocatalytic reactor membranes in order to accommodate the typical reduction in photocatalytic activity of the photocatalyst once in the PMR as it has been observed before.56 The proposed photocatalysts have the additional advantage of demonstrating the catalytic degradation of VAT dyes in industrial textile water as suggested by the formation of visible light absorbing intermediates. In as much diﬀerent photocatalysts are generally evaluated for eﬃcacy under © 2021 The Author(s). Published by the Royal Society of Chemistry 58 59 26 60 11 61 22 Current work diﬀerent environments, the photocatalytic activity of TiO2– aCNT and TiO2–aNCNT nanohybrids on single dye systems is comparable to the activity of photocatalytic nanohybrids that are made from crystalline CNTs (Table 6). We attribute the good activity to the high adsorption of the dye by these catalysts; a feature that is unique to these photocatalytic nanohybrids. This makes them oﬀer a niche for the use of photocatalytic membrane reactors that can be used to form potentially useful intermediates. 4. Conclusions This work has demonstrated that amorphous CNTs, which have been previously deemed to be of less value, can tailor the optical properties of TiO2 to enable visible light absorption through energy band gap reduction and visible light sensitization. In addition to this, these nanohybrids were synthesized through a simple and more reproducible microwave assisted hydrothermal method. The nanohybrids with the smallest particle size, narrowest particle size distribution, highest visible light sensitization, and lowest energy band gap were found to have the highest photocatalytic activity. The nanohybrids with a small particle size, good particle size dispersion but lower ability to absorb visible light have the highest adsorptive capacity and lowest degradation rate constant. Finally, these nanohybrids show a degree of photocatalytic activity for the degradation of organic pollutants in industrial wastewater. RSC Adv., 2021, 11, 6748–6763 \| 6761 View Article Online Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. RSC Advances Herein, we present another step in the direction towards energy eﬃciency, and economic industrial wastewater treatment through photocatalysis. The small particle size, narrow particle size distribution and the intimate contact between TiO2 and the graphitic material from aCNTs makes the subject nanohybrids better suited for the fabrication of photocatalytic reactor membranes with fewer micropores at the TiO2 polymer interface. The formation of visible light absorbing intermediates also opens a niche for the use of these nanohybrids in the fabrication of photocatalytic membrane reactors that can drive reactions for the generation of potentially useful intermediates from wastewater. Conﬂicts of interest There are no conicts to declare. Acknowledgements The authors would like to acknowledge the National Research Foundation for funding and the Institute for Nanotechnology and Water Sustainability of the University of South Africa for providing further funding and laboratory space for this work. Due appreciation goes to the Center for Sustainable Agriculture and Environmental Sciences of the University of South Africa for providing access to equipment for microwave synthesis. Lastly, our sincere gratitude to Ms Alice Magubane and Mr Victor Mashindi of the University of the Witwatersrand for assisting with CVD reactors for the synthesis of carbon nanotubes. References 1 W. Fang, M. Xing and J. Zhang, J. Photochem. Photobiol., C, 2017, 32, 21–39. 2 G. J. Kavarnos and N. J. Turro, Chem. Rev., 1986, 86, 401–449. 3 V. Etacheri, C. Di Valentin, J. Schneider, D. Bahnemann and S. C. Pillai, J. Photochem. Photobiol., C, 2015, 25, 1–29. 4 J. L. Won, J. M. Lee, S. T. Kochuveedu, T. H. Han, H. Y. Jeong, M. Park, J. M. Yun, J. Kwon, K. No, D. H. Kim and S. O. Kim, ACS Nano, 2011, 6, 935–943. 5 B. Makgabutlane, L. N. Nthunya, M. S. Maubane-Nkadimeng and S. D. Mhlanga, J. Environ. Chem. Eng., 2020, 104736. 6 Y. Lin, S. Wu, C. Yang, M. Chen and X. Li, Appl. Catal., B, 2019, 245, 71–86. 7 P. Ayala, R. Arenal, M. Rümmeli, A. Rubio and T. Pichler, Carbon, 2010, 48, 575–586. 8 L. Acauan, A. C. Dias, M. B. Pereira, F. Horowitz and C. P. Bergmann, ACS Appl. Mater. Interfaces, 2016, 8, 16444–16450. 9 M. Terrones, A. G. Souza and A. M. Rao, in Carbon Nanotubes, ed. A. J. D. S. Dresselhaus, Springer Berlin Heidelberg, Berlin, Topics in Applied Physics, 2008, vol. 111, pp. 531– 566. 10 S. D. Mhlanga, K. C. Mondal, R. Carter, M. J. Witcomb and N. J. Coville, S. Afr. J. Chem., 2009, 62, 67–76. 11 T. Zhao, Y. Liu and J. Zhu, Carbon, 2005, 43, 2907–2912. 12 B. Bera, NanoTrends, 2019, 21(1), 36–52. 6762 \| RSC Adv., 2021, 11, 6748–6763 Paper 13 D. Ganguly, D. Banerjee, B. Das, N. S. Das, U. K. Ghorai and K. K. Chattopadhyay, Mater. Res. Bull., 2019, 112, 307–313. 14 A. Rakitin, C. Papadopoulos and J. Xu, Phys. Rev. B: Condens. Matter Mater. Phys., 2000, 61, 5793–5796. 15 S. Sarkar, D. Banerjee, N. S. Das, U. K. Ghorai, D. Sen and K. K. Chattopadhyay, Phys. E, 2018, 97, 162–169. 16 L. Xu, Y. Zhang, X. Zhang, Y. Huang, X. Tan, C. Huang, X. Mei, F. Niu, C. Meng and G. Cheng, Bull. Mater. Sci., 2014, 37, 1397–1402. 17 L. Zhong, A. Tang, P. Yan, J. Wang, Q. Wang, X. Wen and Y. Cui, J. Colloid Interface Sci., 2019, 537, 450–457. 18 X. Zhou, L. Yu, X. Y. Yu and X. W. D. Lou, Adv. Energy Mater., 2016, 6, 1601177. 19 T. Zhao, C. Hou, H. Zhang, R. Zhu, S. She, J. Wang, T. Li, Z. Liu and B. Wei, Sci. Rep., 2014, 4, 1–7. 20 M. N. Ng and M. R. Johan, INEC 2010 - 2010 3rd Int. Nanoelectron. Conf. Proc., 2010, pp. 392–393. 21 J. Wang, C. Fan, Z. Ren, X. Fu, G. Qian and Z. Wang, Dalton Trans., 2014, 43, 13783–13791. 22 S. A. Zikalala, R. Selvaraj, F. Al Marzouqi, A. T. Kuvarega, B. B. Mamba, S. D. Mhlanga and E. N. Nxumalo, J. Environ. Chem. Eng., 2020, 8(5), 104082. 23 T. Xia, W. Zhang, Z. Wang, Y. Zhang, X. Song, J. Murowchick, V. Battaglia, G. Liu and X. Chen, Nano Energy, 2014, 6, 109– 118. 24 T. K. Han, L. B. Fen, N. M. Nee and M. R. Johan, Sci. World J., 2014, 2014, 847806. 25 S. Sarkar, D. Banerjee, N. S. Das and K. K. Chattopadhyay, Appl. Surf. Sci., 2015, 347, 824–831. 26 Y. Lin, C. Yang, S. Wu, X. Li, Y. Chen and W. L. Yang, Adv. Funct. Mater., 2020, 30, 1–13. 27 D. S. Dlamini, B. B. Mamba and J. Li, Sci. Total Environ., 2019, 656, 723–731. 28 Y. Zhao, Y. Tang and A. Star, J. Visualized Exp., 2013, 75, 1–7. 29 Z. N. Tetana, S. D. Mhlanga, G. Bepete and N. J. Coville, S. Afr. J. Chem., 2012, 65, 39–49. 30 K. Chizari, I. Janowska, M. Houllé, I. Florea, O. Ersen, T. Romero, P. Bernhardt, M. J. Ledoux and C. Pham-Huu, Appl. Catal., A, 2010, 380, 72–80. 31 NIST Photoelectron Spectroscopy Database 20 Version 4.1, https://srdata.nist.gov/xps/, accessed 9 June 2019. 32 L. I. Johansson, H. I. P. Johansson and K. L. Håkansson, Phys. Rev. B: Condens. Matter Mater. Phys., 1993, 48, 14520– 14523. 33 W. Li, R. Liang, A. Hu, Z. Huang and Y. N. Zhou, RSC Adv., 2014, 4, 36959. 34 M. C. Biesinger, B. P. Payne, A. P. Grosvenor, L. W. M. Lau, A. R. Gerson and R. S. C. Smart, Appl. Surf. Sci., 2011, 257, 2717–2730. 35 J. E. Krzanowski and R. E. Leuchtner, J. Am. Ceram. Soc., 1997, 80, 1277–1280. 36 D. Wu, M. Long, W. Cai, C. Chen and Y. Wu, J. Alloys Compd., 2010, 502, 289–294. 37 S. A. Zikalala, A. T. Kuvarega, B. B. Mamba, S. D. Mhlanga and E. N. Nxumalo, Mater. Today Chem., 2018, 10, 1–18. 38 M.-Q. Yang, N. Zhang and Y. Xu, ACS Appl. Mater. Interfaces, 2013, 5, 1156–1164. © 2021 The Author(s). Published by the Royal Society of Chemistry View Article Online Open Access Article. Published on 08 February 2021. Downloaded on 5/21/2024 5:49:10 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Paper 39 F. Tian, Y. Zhang, J. Zhang and C. Pan, J. Phys. Chem. C, 2012, 116, 7515–7519. 40 D. Ganguly, D. Pahari, N. S. Das, P. Howli, B. Das, D. Banerjee and K. K. Chattopadhyay, J. Electroanal. Chem., 2016, 778, 12–22. 41 F. Inoue, R. A. Ando and P. Corio, J. Raman Spectrosc., 2010, 42, 1379–1383. 42 P. Corio, K. C. Silva, N. A. Soares, F. Inoue and J. J. Santos, Chem. Phys. Lett., 2019, 723, 96–101. 43 B. Choudhury and A. Choudhury, J. Lumin., 2013, 136, 339– 346. 44 V. V. Kondalkar, S. S. Mali, N. B. Pawar, R. M. Mane, S. Choudhury, C. K. Hong, P. S. Patil, S. R. Patil, P. N. Bhosale and J. H. Kim, Electrochim. Acta, 2014, 143, 89–97. 45 M. V. Kurik, Phys. Status Solidi, 1971, 8, 9–45. 46 A. Kusior, M. Synowiec, K. Zakrzewska and M. Radecka, Crystals, 2019, 9, 163. 47 N. Kelaidis, A. Kordatos, S. R. G. Christopoulos and A. Chroneos, Sci. Rep., 2018, 8, 1–8. 48 W. S. Wang, D. H. Wang, W. G. Qu, L. Q. Lu and A. W. Xu, J. Phys. Chem. C, 2012, 116, 19893–19901. 49 E. Bazrafshan, F. K. Mostafapour, A. R. Hosseini, A. Raksh Khorshid and A. H. Mahvi, J. Chem., 2012, 2013, 1–8. 50 S. Ahirrao, in Industrial Wastewater Treatment, Recycling and Reuse, Elsevier Ltd., 2014, pp. 489–520. © 2021 The Author(s). Published by the Royal Society of Chemistry RSC Advances 51 K. Paździor, L. Bilińska and S. Ledakowicz, Chem. Eng. J., 2018, 120597. 52 C. Burgess, O. Thomas, V. Cerda, C. Gonzalez, E. Touraud, S. Spinelli, F. Theraulaz, B. Roig, M. Pouet, N. Azema, V. Mesnage, S. Vaillant, H. Descherf, E. Baures, J. Roussy, M. Domeizel and G. Junqua, UV-Visible spectrophotometry of water and wastewater, Elsevier, Amsterdam, 1st edn, 2007, vol. 27. 53 M. Joshi, R. Bansal and R. Purwar, Indian J. Fibre Text. Res., 2004, 29, 239–259. 54 D. D. Phong and J. Hur, Water Res., 2015, 87, 119–126. 55 X. R. Xu, H. Bin Li, W. H. Wang and J. D. Gu, Chemosphere, 2004, 57, 595–600. 56 A. T. Kuvarega, N. Khumalo, D. Dlamini and B. B. Mamba, Sep. Purif. Technol., 2018, 191, 122–133. 57 G. Daneshvar Tarigh, F. Shemirani and N. S. Maz’hari, RSC Adv., 2015, 5, 35070–35079. 58 N. Naﬀati, M. J. Sampaio, E. S. Da Silva, M. F. Nsib, Y. Arfaoui, A. Houas, J. L. Faria and C. G. Silva, Mater. Sci. Semicond. Process., 2020, 115, 105098. 59 Y. Lin, S. Wu, X. Li, X. Wu, C. Yang, G. Zeng, Y. Peng, Q. Zhou and L. Lu, Appl. Catal., B, 2018, 227, 557–570. 60 H. Li, G. Wang, X. Zhang and Z. Jin, Int. J. Hydrogen Energy, 2020, 45, 8405–8417. 61 S. Lanfredi, M. A. L. Nobre, P. S. Poon and J. Matos, Molecules, 2020, 25, 96–116. RSC Adv., 2021, 11, 6748–6763 \| 6763
W3100690219.txt	null	en		Evidence of urban heat island impacts on the vegetation growing season length in a tropical city	Landscape and urban planning	2,021	cc-by	6,948	Landscape and Urban Planning 206 (2021) 103989 Contents lists available at ScienceDirect Landscape and Urban Planning journal homepage: www.elsevier.com/locate/landurbplan Research Paper Evidence of urban heat island impacts on the vegetation growing season length in a tropical city Peter Kabano a, b, , Sarah Lindley a, Angela Harris a a Department of Geography, School of Environment, Education & Development, The University of Manchester, Oxford Road, UK Department of Urban and Regional Planning and Geo-information Management, Faculty of Geo-Information Science and Earth Observation (ITC), University of Twente, Enschede, the Netherlands b H I G H L I G H T S • Tropical urban phenology has key differences to temperate urban phenology. • In Kampala, growing season length increases along the urban–rural gradient. • Vegetation in the most built-up LCZ class had the shortest growing seasons. • Increases in surface temperatures resulted in shorter vegetation growing seasons. A R T I C L E I N F O A B S T R A C T Keywords: Tropics Local climate zones Landscape phenology Land surface temperature Urban heat island Knowledge about the impacts of urban heat islands (UHI) and associated thermal gradients on vegetation sea sonality (i.e. phenology) is vital for understanding spatial patterns in vegetation ecosystem functions. However, in contrast to temperate cites, there is little evidence to show how UHI influences landscape phenological pro cesses in the tropics. In this study, we examined vegetation phenological responses to urban form, distance from the city centre and surface temperatures, in the tropical city of Kampala, Uganda. Estimates of vegetation growing season length and land surface temperature were derived from MODIS satellite imagery for multiple years (2013–2015) and urban form was characterised using the Local Climate Zone (LCZ) classification. We showed that growing season length increased along the urban–rural gradient (p < 0.001) and was longest in the least built-up LCZ class (p < 0.001). Growing season length was significantly reduced as land surface temperature increased (p < 0.001). These findings contrast with results reported for temperate cities, where higher tem peratures are often associated with longer vegetation growing seasons. Our findings suggest that enhanced surface temperatures associated with UHI are a limiting factor to season length in the urban tropics. Urban planners in tropical cities should therefore account for vegetation sensitivity to UHI when developing targeted management strategies aiming to optimise the benefits accrued from vegetation. 1. Introduction By 2050, 68% of the world’s population will reside in urban areas (United Nations. (2019), 2019). Up to 90% of the projected growth of the global urban population will occur in Africa and Asia (United Na tions. (2019), 2019), where many cities are already vulnerable to climate change and hazards caused by urbanisation (du Toit et al., 2018). Vegetation is posited to significantly improve urban resilience through the provision of ecosystem functions, such as thermal regula tion, yet many cities in the global south are undergoing rapid vegetation loss (Lindley, Pauleit, Yeshitela, Cilliers, & Shackleton, 2018; Yao, Cao, Wang, Zhang, & Wu, 2019). It is therefore imperative to understand the impacts of urbanisation on vegetation to safeguard human and ecosystem health. One of the most obvious effects of urbanisation is the phenomenon known as the Urban Heat Island effect (UHI), i.e. where urban areas typically experience higher temperatures relative to their surrounding landscapes (Landsberg, 1981; Taha, 1997; Voogt & Oke, 2003). UHIs are caused by high proportions of impervious land cover types that promote heat storage and restrict natural radiative cooling through Corresponding author. E-mail addresses: ptkabano@gmail.com (P. Kabano), sarah.lindley@manchester.ac.uk (S. Lindley), angela.harris@manchester.ac.uk (A. Harris). https://doi.org/10.1016/j.landurbplan.2020.103989 Received 15 December 2019; Received in revised form 24 October 2020; Accepted 30 October 2020 Available online 13 November 2020 0169-2046/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). P. Kabano et al. Landscape and Urban Planning 206 (2021) 103989 evapotranspiration. UHI intensities are influenced by the size of cities and their characteristics in terms of urban morphology, biome, regional climate, seasonal changes in meteorological conditions and vegetation cover (Giridharan & Emmanuel, 2018; Imhoff, Zhang, Wolfe, & Bou noua, 2010; Roth, 2007). Urban vegetation mitigates UHI through increased thermal regulation via evapotranspiration (Norton et al., 2015) and the provision of shade (Li, Ratti, & Seiferling, 2018). In recent years, several studies have noted the importance of vegetation for UHI mitigation in tropical cities (e.g. Cavan et al., 2014; Feyisa, Dons, & Meilby, 2014; du Toit et al., 2018; Lindley et al., 2018). However, the specific impacts of urbanisation and UHI on vegetation phenology in the tropics remain less well studied. Vegetation phenology, defined as the seasonal timing of vegetation growth and reproduction (Fenner, 1998), is essential for primary pro duction and for sustaining many important ecosystem benefits for human populations (Denny et al., 2014). Vegetation responses to ur banisation have been studied extensively for temperate cities where UHIs result in earlier growing season start dates and longer growing season durations compared to surrounding rural areas (Jochner & Menzel, 2015). However, phenological patterns and processes within tropical cities are less well studied. The few urban tropical phenological studies that exist highlight differences in the timing of start of season tree budding compared to temperate cities (Gazal et al., 2008; Jochner, Alves-Eigenheer, Menzel, & Morellato, 2013). The evidence suggests that tree budding is more sensitive to UHI in temperate cities due to the sensitivity of temperate vegetation to springtime temperature increases (and photoperiod) after wintertime dormancy (Zhang, Friedl, & Schaaf, 2006). However, there remains a limited understanding of the effect of UHI on the timing of the end of the growing season and growing season duration in tropical cities. In tropical natural habitats leaf growth usually occurs in the wet season whereas leaf fall occurs mainly in the dry season (de Camargo, de Carvalho, Alberton, Reys, & Morellato, 2018; Williams, Myers, Muller, Duff, & Eamus, 1997). Given that UHI intensities are most significant during the dry season in tropical cities (Giridharan & Emmanuel, 2018; Roth, 2007), UHIs might have a greater influence on leaf fall than on leaf growth. Moreover, the formation of an Urban Dryness Island (UDI) effect and increased plant water requirements due to UHI-induced potential evapotranspiration (Hao, Huang, Qin, Liu, Li, & Sun, 2018; Luo & Lau, 2019; Wang, Hutyra, Li, & Friedl, 2017; Zipper, Schatz, Kucharik, & Loheide, 2017), may be more pronounced during the dry season. Consequently, the impact of UHI on leaf growth and development may vary depending on the season. To investigate these impacts there is a need for phenological observations that span the entire vegetation growing season. Satellite remote sensing can provide landscape scale phenological information on the start of the growing season (SOS), the end of the growing season (EOS) and the growing season length or duration (GSL) (Melaas, Wang, Miller, & Friedl, 2016; Zhang et al., 2003). Landscape phenology is inherently different from the phenology of individual species (Badeck et al., 2004), although information about both is useful for understanding phenology in urban environments (Jochner & Menzel, 2015). In temperate cities, vegetation growing in heavily built-up urban areas often has a long GSL due to elevated urban temperatures (Melaas et al., 2016; Zhang, Friedl, Schaaf, Strahler, & Schneider, 2004; Zhou, Zhao, Zhang, & Liu, 2016; Zipper et al., 2016). In contrast, the duration of the vegetation growing season declines along the urban–rural gradient as the degree of urbanisation diminishes (Zhang et al., 2004; Zhou et al., 2016). An equivalent understanding of landscape scale phenological processes in tropical cities is lacking. Whereas temperate regions experience extreme seasonal changes in temperature as the main trigger for vegetation phenology (along with photoperiod), changes in temperature are less drastic in the tropics. Tropical phenology is mainly controlled by rainfall (Clinton, Yu, Fu, He, & Gong, 2014; Zhang et al., 2006) but excess urban heat might act as a limiting (stress) factor in tropical urban contexts. In this study, we examined the impact of UHI intensities on vegeta tion phenology in the tropical city of Kampala, Uganda. Our objectives were to: (i) determine the spatial variability in landscape phenology in respect to degree of urbanisation (i.e. urban form and distance from the city centre); (ii) determine the combined effect of urban form and dis tance from the city centre on Land Surface Temperature (LST); (iii) establish how spatial patterns of LST vary across years; and (iv) assess the effect of variations in LST on phenology. 2. Materials and methods 2.1. Study area The study focussed on the equatorial city of Kampala in East Africa located at 00◦ 18′ 49′′ N 32◦ 34′ 52′′ E. Kampala has rapidly urbanised in recent years and the urban extent of the Kampala Greater Metropolitan Area (KGMA) now covers more than 800 km2 (Vermeiren, Van Rom paey, Loopmans, Serwajja, & Mukwaya, 2012). Our region of interest (ROI) covers an area extending 20 km from the city centre (approxi mately 1402 km2) and includes the KGMA. Kampala has a population of over 1.5 million inhabitants, and this is expected to reach 5.5 million by 2030 (United Nations. (2019), 2019). The city has a tropical rainforest equatorial climate (Af) according to the Köppen climate classification. There are two wet seasons (March-May and September-November) and the city has a mean annual precipitation of about 1200 mm. Torrential rains are often observed from March to May and July is normally the driest month. 2.2. Data Urban form, vegetation abundance, vegetation phenology and LST were characterised using remotely sensed satellite imagery for 2013–2015. We constrained our selection of satellite imagery to three years to minimise the effect of rapid changes in urban form across Kampala. The Local Climate Zone (LCZ) classification scheme (Stewart & Oke, 2012) was used to represent urban form, and LCZs were char acterised using imagery from the US Geological Survey Earth Explorer Landsat 8 Operational Land Imager (OLI). The Moderate Resolution Imaging Spectroradiometer (MODIS) sensor was used to estimate spatial and temporal patterns of LST, vegetation abundance and phenology. We obtained vegetation abun dance and phenology data using Vegetation Indices as proxy measures of canopy ‘greenness’ (Huete, Didan, van Leeuwen, Miura, & Glenn, 2011; Senanayake, Welivitiya, & Nadeeka, 2013; Yao et al., 2019; Yuan & Bauer, 2007). The subsections below provide further information on data sources and processing steps, and the overall methodology is summarised in Fig. 1. 2.2.1. Urban form and vegetation abundance The LCZ framework is a robust, objective and universal approach for characterising urban form for use in climatological research (Kotharkar & Bagade, 2018; Mushore et al., 2019; Stewart & Oke, 2012). There are seventeen classes contained in the LCZ framework, each representing distinct characteristics of surface cover and structure. We used the World Urban Database and Access Portal Tools (WUDAPT) LCZ classi fication method (Bechtel et al., 2015) for mapping Kampala’s LCZs. The WUDAPT method uses supervised machine learning to generate a citywide LCZ map from multispectral Landsat 8 OLI imagery in a 3-step process; namely: (i) acquisition and pre-processing of cloud-free im ages, (ii) creation of training areas in Google Earth and (iii) imple mentation of the classification in the open-source GIS software, System for Automated Geoscientific Analyses (SAGA-GIS). It is recommended that more than one satellite image is used in LCZ classification to minimise the impact of spectral changes in vegetation over time (Bechtel et al., 2015). We selected two Landsat 8 OLI scenes (LC81710602015074 and LC81710602015058), both with low cloud 2 P. Kabano et al. Landscape and Urban Planning 206 (2021) 103989 Fig. 1. Flow diagram of data processing steps. cover. The images were radiometrically calibrated to Top of the Atmo sphere reflectance and resampled from 30 m to 100 m. Resampling al lows the spectral signals of multiple urban features to be combined and thus facilitates attribution of local climate to zones (Bechtel et al., 2015; Stewart & Oke, 2012). Training areas with a minimum width of 200 m were digitised within Google Earth for the most common LCZ classes in Kampala: Compact midrise (LCZ2), Compact low-rise (LCZ3), Open midrise (LCZ5), Open low-rise (LCZ6), Large low-rise (LCZ8), Sparsely built (LCZ9), Dense trees (LCZA), Scattered trees (LCZB), Bare soil or sand (LCZF) and Water (LCZG). Subclass LCZ3_F was added to the selection to indicate compact low-rise neighbourhoods (LCZ3) with mostly bare soil surfaces (LCZF), as was done for the city of Nagpur in India (Kotharkar & Bagade, 2018). To minimise the effect of mixed pixels, we digitised polygons in loca tions with broadly homogenous land cover composition and avoided boundaries between any two given LCZ types. The number of training sample polygons per LCZ class ranged between 3 and 20, and depended on the area covered by each LCZ class and how well areas could be digitised. The LCZ classification was implemented in SAGA-GIS using its Random Forest classifier, which has high accuracy and computational performance for LCZ classification (Bechtel et al., 2015; Breiman, 2001). New training areas were iteratively selected, and LCZs reclassified to obtain an LCZ map that compared favourably with existing maps of Kampala’s urban morphology. Accuracy assessment of the final LCZ map was performed using the Semi-Automatic Classification QGIS plugin and a total of 764 test polygons (100 m cell size) selected through stratified random sampling (Congedo, 2016). The overall accuracy of the final LCZ map was 73.2% with a Kappa coefficient of 0.674. This is within the expected accuracy range, for instance, within the 60–89% range re ported for LCZ maps produced for 20 cities in China (Ren et al., 2019). Four of the eleven LCZ classes (i.e. Compact low-rise and bare soil (LCZ3_F), Open low-rise (LCZ6), Sparsely built (LCZ9) and Scattered trees (LCZB)) covered 87% of the ROI, and all LCZs had producer and user accuracies above 90% and 85% respectively. Given their high ac curacy statistics, distinctiveness and large spatial extent, these four LCZ classes (Fig. 2) were selected for further analysis. To validate our choice of LCZ classes, we ascertained that they were different in terms of sur face cover using a Kruskal-Wallis H-test performed on Enhanced Vege tation Index (EVI) data. We chose EVI for characterising surface cover (i. e. vegetation abundance) because it minimises canopy background variations and maintains sensitivity over dense vegetation in urban environments (Huete et al., 2011). EVI is an optimised combination of Blue, Red, and Near Infrared (NIR) bands and can be derived using the 3 P. Kabano et al. Landscape and Urban Planning 206 (2021) 103989 Fig. 2. A) Characteristics of candidate LCZs; B) High-resolution imagery for an example 1 km2 area of the candidate LCZ classes in Kampala. equation below: was used to smooth the EVI time series. This approach preserves important features of the dataset (i.e. maximum, minimum and width) whilst reducing the noise due to possible cloud cover (Yao et al., 2017). We set the adaptation strength to 3.0, seasonal parameter to 0.5 (2 seasons per year) and used a Savitzky-Golay window size of 3. We weighted the data for each pixel using the pixel reliability band (i.e. cloud-free assigned 1, marginal data assigned 0.5 and cloudy data assigned 0.1). We extracted the start (SOS), end (EOS) and length (GSL) of the vegetation growing season using the amplitude method. The amplitude method is better suited than the threshold method in urban settings because low vegetation cover in cities makes it hard to find a threshold for determining the start and end of the season (Zhou et al., 2016). The SOS and EOS were the dates that the fitted curve increased and declined to the proportion of the amplitude (set to 20%). EVI = 2.5(ρNIR − ρRED )/(L + ρNIR + C1 ρRED − C2 ρBLUE ) where ρ is reflectance; L is the canopy background adjustment factor; C1 and C2 are aerosol resistance weights. The coefficients are L = 1, C1 = 6 and C2 = 7.5 (Huete et al., 2011). We used the MOD13Q1 EVI product (16-day composites and a spatial resolution of 250 m) acquired from the Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC) of the National Aeronautics and Space Agency (NASA). To characterise vegetation abundance across Kampala whilst minimising the effects of seasonal changes in vegetation, we calculated the average of 23 MOD13Q1 EVI images for 2013 using the raster calculator function in ArcGIS 10.0. The vegetation abundance and LCZ maps were converted to polygon vector format and an intersect function of the overlay toolset in ArcGIS 10.0 used to acquire the LCZ classification at each EVI pixel location. 2.2.3. Land surface temperature We derived LST for 2013–2015 from the MODIS MOD11A2 LST product. The MOD11A2 LST product has a high temporal resolution (8day composite) and is a useful proxy for estimating surface UHI intensity (Yao et al., 2018). We again used the Savitzky-Golay smoothing algo rithm and TIMESAT 3.2 to identify the maximum LST for each of the three years as the basis for assessing impacts on vegetation development. As with the EVI data, the MODIS quality assessment images were used to 2.2.2. Vegetation phenology Time series analysis and extraction of vegetation phenology seasonal parameters was performed in the TIMESAT 3.2 program (Eklundh & Jönsson, 2015; Jonsson & Eklundh, 2004) using MOD13Q1 EVI images spanning three years (2013–2015) as shown in Fig. 3. The adaptive Savitsky-Golay algorithm which uses local polynomial fitting functions 4 P. Kabano et al. Landscape and Urban Planning 206 (2021) 103989 Fig. 3. Savitzky-Golay algorithm applied to the three-year MODIS EVI (Enhanced Vegetation Index) time series (MOD13Q1) for the candidate LCZ classes: (a) Compact low-rise (LCZ3_F); (b) Sparsely built (LCZ9); (c) open low-rise LCZ6; (d) Scattered trees (LCZB). Each data point (blue) represents the averaged EVI value of 10 randomly select pixels within each given LCZ category. Brown points represent the start and end of season. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) de-noise the data. model against a null model comprising the intercept only. The impor tance of each explanatory variable was determined using a likelihood ratio test (R function “drop1”) that compared the full model with reduced models. Variance inflation factors were computed from stan dard linear models to assess collinearity (Zuur, Ieno, & Elphick, 2010), and no issues were found. Normality and independence of the residuals were confirmed by inspecting QQ plots and plots of residuals against fitted values. Linear mixed models were also used to assess the influence of LST on SOS, EOS and GSL using LST and Year as fixed effects. This allowed us to examine the influence of LST patterns while controlling for meteoro logical differences across years. Location was included as a random ef fect and model diagnostics (significance of final full model and normality of residuals) performed as described in previous models. 2.3. Data analysis Vegetation phenology, vegetation abundance, LST and LCZ data were assigned to each MOD13Q1 EVI pixel as the unit of analysis. Firstly, all data was converted from raster to vector format (conversion toolbox in ArcGIS 10.0) due to differences in the spatial resolution of the input data (vegetation abundance & phenology, LCZ and LST; Fig. 4). The dissolve function (Manage data toolset in ArcGIS 10.0) was applied to the LST and LCZ vector data separately to merge polygons that had similar data and shared boundaries. The intersect function (overlay toolset in ArcGIS 10.0) was then used to acquire LCZ and LST data for each MOD13Q1 EVI pixel. To account for the diminishing effect of de gree of urbanisation along the urban–rural gradient, we obtained the distance between each pixel and the city centre using the Near Analysis tool in ArcGIS 10.0. Each data point therefore represented an EVI pixel location with its attributes (i.e. phenology, vegetation abundance, LCZ, distance from the city centre and LST) for each year. As with vegetation abundance, differences in LST and phenology among LCZs were computed using Kruskal–Wallis H-tests. A series of linear mixed models were used to analyse the effect of urban form (LCZ) and distance from the city centre on SOS, EOS, GSL and LST. We fitted a model for each response variable with LCZ and distance as explanatory variables. Year was used as a fixed effect to control for differences across years in the phenology models. In teractions between year and LCZ and between year and distance from the city centre were included in the LST model to account for varying effects of urban form and distance from the urban core across the three years. Location was used as a random effect to allow for correlated error terms caused by repeated observations (each year) at the same location. The modelling was done using the “lmer” function of the “lme4” package in R (Bates, Mächler, Bolker, & Walker, 2015; R Core Team, 2018). We used a likelihood ratio test to establish the significance of the final full 3. Results 3.1. Surface cover differences across LCZ types Vegetation abundance (i.e. mean EVI) differed significantly among the LCZs and between all LCZ pairs (Table 1). EVI decreased from the least to most built-up LCZ type. LCZ9 (Sparsely built) and LCZ6 (Open low-rise) had EVI values that were 17% and 34% lower respectively than those in LCZB. LCZ3_F (Compact low-rise and bare soil) recorded the lowest overall EVI at 47% lower than LCZB. 3.2. Phenology The combined effect of LCZ, distance from the city centre and year had a significant influence on SOS (likelihood ratio test: X2 = 1097, df = 6, p < 0.001), EOS (likelihood ratio test: X2 = 7963, df = 6, p < 0.001) and GSL (likelihood ratio test: X2 = 284.2, df = 11, p < 0.001). LCZ class had a significant influence on SOS, EOS, and GSL (Table 1; Table 2). LCZB (Scattered trees) experienced the earliest SOS and the latest EOS 5 P. Kabano et al. Landscape and Urban Planning 206 (2021) 103989 Fig. 4. Spatial distribution of input variables used in the analysis. These include: a) urban form represent by LCZ; b) vegetation abundance depicted by EVI; c) peak LST in the 2013 growing season; and (d) growing season length in 2013. The region of interest (area = 1402 km2) used for the analysis has been added for ease of visual comparison across images. Table 1 Kruskal-Wallis test results carried out on EVI (Enhanced Vegetation Index), LST (Land Surface Temperature) and GSL (Growing Season Length), and differences across LCZ classes in terms of mean (±99% confidence interval) EVI, LST and GSL. LCZ class Mean EVI Mean LST (◦ C) Mean GSL (days) LCZB. Scattered trees (area = 854 km2) LCZ9. Sparsely built (area = 277 km2) LCZ6. Open low-rise (area = 137 km2) LCZ3_F. Compact low-rise and bare soil (area = 134 km2) 0.435 ± 0.001 0.361 ± 0.001 0.285 ± 0.002 0.229 ± 0.002 32.57 ± 0.04 33.79 ± 0.06 35.37 ± 0.09 36.47 ± 0.08 110.0 ± 0.6 391,480 17 0.0001 15,451 3 0.0001 832.16 3 0.0001 Kruskal-Wallis test results 2 χ df p Table 2 Mixed Models testing whether phenology start of season (SOS), end of season (EOS) and growing season length (GSL) vary across years, Local Climate Zone (LCZ) class, distance from city centre and Land Surface Temperature (LST). DenDF and NumDF represent the Numerator and Denominator degrees of freedom. Pr(>F) is the significant coefficient (P) for the F statistic. 106.3 ± 1.1 100.5 ± 1.6 Phenology ~ degree of urbanisation Phenology ~ LST Year LCZ Distance Year LST SOS F value NumDF DenDF Pr(>F) 507.3 2 41,768 0.0001 17 3 21,487 0.0001 1.2 1 21,607 0.27 453.7 2 44,489 0.0001 1.7 1 35,017 0.1978 EOS F value NumDF DenDF Pr(>F) 4,128 2 41,883 0.0001 55.6 3 21,416 0.0001 1.2 1 21,539 0.28 3,351 2 44,458 0.0001 85.9 1 34,064 0.0001 GSL F value NumDF DenDF Pr(>F) 4,226 2 41,776 0.0001 119 3 21,400 0.0001 6.3 1 21,522 0.012 3,612 2 44,435 0.0001 71.7 1 34,793 0.0001 95.9 ± 1.6 dates resulting in longer growing seasons (Fig. 5). In comparison to LCZB, GSL was shorter in LCZ9 (Sparsely Built) (estimate = − 3.3 days, standard error = 0.5, p < 0.001), LCZ6 (estimate = − 8.6 days, standard error = 0.7, p < 0.001) and LCZ3_F (estimate = − 13.2 days, standard error = 0.7, p < 0.001). Distance from the city centre significantly (positively) influenced GSL but had negligible effects on SOS and EOS 6 P. Kabano et al. Landscape and Urban Planning 206 (2021) 103989 Fig. 5. Main effect plot for: (a) Start of Season; (b) End of Season; and (c) Growing Season in relation to LCZ class with error bars (95% confidence intervals). (Table 2). 4. Discussion Our results show that landscape phenology in the tropical city of Kampala is influenced by LST, distance from the city centre and degree of urbanisation (LCZ). During 2013-15, heavily built-up locations experienced high LST, early EOS and a short GSL in comparison to less built-up locations. LCZB (Scattered trees) had the highest vegetation cover and experienced the lowest LST, earliest SOS, latest EOS and longest GSL. LCZ9 (Sparsely built) and LCZ6 (Open low-rise) had GSLs that were 3 and 9 days shorter respectively than in LCZB. LCZ3_F (compact low-rise and bare soil) recorded the shortest GSL overall at 13 days shorter than in LCZB. The order of LCZs in respect to GSL was LCZB > LCZ9 > LCZ6 > LCZ3_F which mirrored the order of LCZs in respect to LST (LCZB < LCZ9 < LCZ6 < LCZ3_F). Furthermore, the decline in LST along the urban–rural gradient varied between years. Rural areas experienced temperatures that were 2 ◦ C cooler in 2013 in comparison to 2014 and 2015. The LCZs exhibited differences in surface cover characteristics. The relative order of vegetation abundance in each LCZ (i.e. EVI) was LCZB > LCZ9 > LCZ6 > LCZ3_F. This mirrored the order of LCZs in respect to LST (LCZB < LCZ9 < LCZ6 < LCZ3_F). Several studies have attributed lower temperatures within cities to higher vegetation abundance (e.g. Mushore et al., 2019; Senanayake et al., 2013; Yuan & Bauer, 2007) due to enhanced latent heat flux through evapotranspiration (Cavan et al., 2014; Feyisa et al., 2014). On the other hand, a high proportion of impervious land cover enhances thermal admittance and high heat storage resulting in higher temperatures (Landsberg, 1981). In the case of LST, a high amount of vegetation cover is particularly important in terms of its influence on surface albedo and shading (Taha, 1997). 3.3. Land surface temperature LST varied significantly across LCZs (Table 1) and was influenced by the combined effect of LCZ, distance from the city centre and year (likelihood ratio test: X2 = 37944, df = 14, p < 0.001). Despite some variation in LCZ LSTs between years (interaction between year and LCZ: F6, 43739 = 6.7, P < 0.001), the overall trend was similar, i.e. Scattered trees (LCZB) < Sparsely Built (LCZ9) < Open low-rise (LCZ6) < Compact low-rise (LCZ3_F). LST declined with increasing distance from the city centre across all years, but the magnitude of LST change from urban to rural areas varied across years (interaction between year and distance: F2, 43819 = 1247.7, P < 0.001). LST ranged between approximately 26–28 ◦ C (rural–urban) in 2014 and 2015 and around 24–28 ◦ C (rural–urban) in 2013 (Fig. 6). The Kruskal-Wallis H-test confirmed significant differences in LST across years (χ 2 = 8575.1, df = 2, P < 0.0001). 3.4. Relationship between LST and phenology The combined effect of spatial differences in LST and year had a significant effect on SOS (likelihood ratio test: X2 = 951.8, df = 3, p < 0.001), EOS (likelihood ratio test: X2 = 7610.7, df = 3, p < 0.001), and GSL (comparison of full with null model: X2 = 7717.5, df = 3, p < 0.001). LST had a significant negative effect on EOS (estimate = − 1 day, standard error = 0.1, p < 0.0001) and GSL (estimate = − 1 day, standard error = 0.1, p < 0.0001). However, LST had a negligible effect on SOS (Table 2). Fig. 6. Main effect plot for: (a) LST distribution with standard error bars across LCZ types in 2013, 2014 & 2015; and (b) LST distribution with confidence bands along an urban–rural gradient in 2013, 2014 & 2015. 7 P. Kabano et al. Landscape and Urban Planning 206 (2021) 103989 Our findings on the effect of urbanisation on landscape phenology in Kampala contrast with the established phenological patterns in temperate cities, where vegetation experiences earlier SOS, later EOS and longer GSL due to UHI (Melaas et al., 2016; Zhang et al., 2004; Zhou et al., 2016; Zipper et al., 2016). The long growing season observed in highly vegetated locations in Kampala agree with the phenological patterns observed by Guan et al. (2014) in tropical natural habitats. However, the cause and mechanisms determining longer growing sea sons were not well established in Guan et al. (2014). The Urban Dryness Island effect is a phenomenon that describes lower moisture availability in cities compared to the surrounding landscape as a consequence of high proportions of impervious land cover types (Hao et al., 2018; Luo & Lau, 2019; Wang et al., 2017). The spatial patterns of UDI are similar to the thermal gradients associated with UHI (Hao et al., 2018; Luo & Lau, 2019; Wang et al., 2017) and high plant water requirements have been attributed to UHI-induced potential evapotranspiration (Zipper et al., 2017). The UDI in Kampala is there fore also expected to have diminished from the most to least built-up LCZ category (LCZ3_F (Compact low-rise and bare soil) > LCZ6 (Open low-rise) > LCZ9 (Sparsely built) > LCZB (Scattered trees)). Observed phenology patterns in the city are likely to be driven by factors relating to both UHI and UDI. The spatial pattern of LST in 2013 differed from 2014 and 2015 due to the meteorological anomalies of the drought and El Niño effect in 2014 and 2015 respectively (Zhang, Dannenberg, Hwang, & Song, 2019). Regional temperature anomalies in 2014 and 2015 had strong effects on LST in rural areas, and the effect declined along the rural –urban gradient. These findings suggest that inter-annual differences in regional climate exacerbate the effect of UHI. References Badeck, F. W., Bondeau, A., Bottcher, K., Doktor, D., Lucht, W., Schaber, J., & Sitch, S. (2004). Responses of spring phenology to climate change. New Phytologist, 162(2), 295–309. https://doi.org/10.1111/j.1469-8137.2004.01059.x. Bates, D., Mächler, M., Bolker, B., & Walker, S. (2015). Fitting linear mixed-effects models using {lme4}. Journal of Statistical Software, 67(1), 1–48. https://doi.org/10 .18637/jss.v067.i01. Bechtel, B., Alexander, P. J., Bohner, J., Ching, J., Conrad, O., Feddema, J., … Stewart, I. (2015). Mapping local climate zones for a worldwide database of the form and function of cities. Isprs International Journal of Geo-Information, 4(1), 199–219. https://doi.org/10.3390/ijgi4010199. Breiman, L. (2001). Random forests. Machine Learning, 45(1), 5–32. Cavan, G., Lindley, S., Jalayer, F., Yeshitela, K., Pauleit, S., Renner, F., … Shemdoe, R. (2014). Urban morphological determinants of temperature regulating ecosystem services in two African cities. Ecological Indicators., 42, 43–57. https://doi.org/ 10.1016/j.ecolind.2014.01.025. Clinton, N., Yu, L., Fu, H. H., He, C. H., & Gong, P. (2014). Global-scale associations of vegetation phenology with rainfall and temperature at a high spatio-temporal resolution. Remote Sensing, 6(8), 7320–7338. https://doi.org/10.3390/rs6087320. Congedo, L. (2016). Semi-Automatic Classification Plugin Documentation. Release 6.0.1.1. https://doi.org/10.13140/RG.2.2.29474.02242/1. de Camargo, M. G. G., de Carvalho, G. H., Alberton, B. D., Reys, P., & Morellato, L. P. C. (2018). Leafing patterns and leaf exchange strategies of a cerrado woody community. Biotropica, 50(3), 442–454. https://doi.org/10.1111/btp.12552. Denny, E. G., Gerst, K. L., Miller-Rushing, A. J., Tierney, G. L., Crimmins, T. M., Enquist, C. A. F., … Weltzin, J. F. (2014). Standardized phenology monitoring methods to track plant and animal activity for science and resource management applications. International Journal of Biometeorology, 58(4), 591–601. https://doi. org/10.1007/s00484-014-0789-5. du Toit, M. J., Cilliers, S. S., Dallimer, M., Goddard, M., Guenat, S., & Cornelius, S. F. (2018). Urban green infrastructure and ecosystem services in sub-Saharan Africa. Landscape and Urban Planning, 180, 249–261. https://doi.org/10.1016/j. landurbplan.2018.06.001. Eklundh, L., & Jönsson, P. (2015). TIMESAT 3.2 with parallel processing Software Manual. ed. S. Lund and Malmö University. Fenner, M. (1998). The phenology of growth and reproduction in plants. Perspectives in Plant Ecology, Evolution and Systematics, 1(1), 78–91. Feyisa, G. L., Dons, K., & Meilby, H. (2014). Efficiency of parks in mitigating urban heat island effect: An example from Addis Ababa. Landscape and Urban Planning, 123, 87–95. https://doi.org/10.1016/j.landurbplan.2013.12.008. Gazal, R., White, M. A., Gillies, R., Rodemaker, E., Sparrow, E., & Gordon, L. (2008). GLOBE students, teachers, and scientists demonstrate variable differences between urban and rural leaf phenology. Global Change Biology, 14(7), 1568–1580. https:// doi.org/10.1111/j.1365-2486.2008.01602.x. Giridharan, R., & Emmanuel, R. (2018). The impact of urban compactness, comfort strategies and energy consumption on tropical urban heat island intensity: A review. Sustainable Cities and Society, 40, 677–687. https://doi.org/10.1016/j. scs.2018.01.024. Guan, K. Y., Wood, E. F., Medvigy, D., Kimball, J., Pan, M., Caylor, K. K., … Jones, M. O. (2014). Terrestrial hydrological controls on land surface phenology of African savannas and woodlands. Journal of Geophysical Research-Biogeosciences, 119(8), 1652–1669. https://doi.org/10.1002/2013jg002572. Hao, L., Huang, X., Qin, M., Liu, Y., Li, W., & Sun, G. (2018). Ecohydrological Processes Explain Urban Dry Island Effects in a Wet Region, Southern China. Water Resources Research. https://doi.org/10.1029/2018WR023002. Huete, A., Didan, K., van Leeuwen, W., Miura, T., & Glenn, E. (2011). MODIS Vegetation Indices. (B. Ramachandran, C. O. Justice, & M. J. Abrams, Eds.), Land Remote Sensing and Global Environmental Change: Nasa’s Earth Observing System and the Science of Aster and Modis (Vol. 11). https://doi.org/10.1007/978-1-4419-6749-7_ 26. Imhoff, M. L., Zhang, P., Wolfe, R. E., & Bounoua, L. (2010). Remote sensing of the urban heat island effect across biomes in the continental USA. Remote Sensing of Environment, 114(3), 504–513. https://doi.org/10.1016/j.rse.2009.10.008. Jochner, S., Alves-Eigenheer, M., Menzel, A., & Morellato, L. P. C. (2013). Using phenology to assess urban heat islands in tropical and temperate regions. International Journal of Climatology, 33(15), 3141–3151. https://doi.org/10.1002/ joc.3651. Jochner, S., & Menzel, A. (2015). Urban phenological studies – Past, present, future. Environmental Pollution, 203, 250–261. https://doi.org/10.1016/j. envpol.2015.01.003. Jonsson, P., & Eklundh, L. (2004). TIMESAT - a program for analyzing time-series of satellite sensor data. Computers & Geosciences, 30(8), 833–845. https://doi.org/ 10.1016/j.cageo.2004.05.006. Kotharkar, R., & Bagade, A. (2018). Evaluating urban heat island in the critical local climate zones of an Indian city. Landscape and Urban Planning, 169, 92–104. https:// doi.org/10.1016/j.landurbplan.2017.08.009. Landsberg, H. E. (1981). The Urban Climate. Retrieved from: Elsevier Science. https:// books.google.co.uk/books?id=zkKHiEXZGBIC. Li, X. J., Ratti, C., & Seiferling, I. (2018). Quantifying the shade provision of street trees in urban landscape: A case study in Boston, USA, using Google Street View. Landscape and Urban Planning, 169, 81–91. https://doi.org/10.1016/j. landurbplan.2017.08.011. Lindley, S., Pauleit, S., Yeshitela, K., Cilliers, S., & Shackleton, C. (2018). Rethinking urban green infrastructure and ecosystem services from the perspective of sub- 5. Conclusions In this paper, we provide substantial new evidence about the role of temperature as a limiting factor for GSL in tropical cities. Our study results for Kampala demonstrated that GSLs in LCZ6 (Open low-rise) and LCZ3_F (compact low-rise and bare soil) were 8.6 and 13.2 days shorter respectively compared to LCZB (Scattered trees). Shorter vegetation growing seasons, in turn, limit the provision of beneficial ecosystem functions. For instance, a shorter growing season will influence some aspects of urban agricultural productivity and regulating ecosystem roles such as those associated with human thermal comfort. Urban planning in tropical cities could focus on strategies that aim to mitigate UHI and extend the GSL, for example, through the enhancement of green spaces in highly built-up zones. Knowledge about the environmental processes and intrinsic attributes (surface cover, meteorology and phenology) of LCZ classes could provide useful information to support urban planning in Kampala. For example, city planners could aim to increase vegetation cover in LCZ3_F (compact low-rise and bare soil). Additionally, city planners could restrict further expansion of LCZ3_F in favour of LCZ classes that have higher vegetation cover, lower temper ature (and dryness) and therefore longer GSLs (e.g. LCZ6 and LCZ9). These strategies could also be applicable in other tropical cities that are faced with similar urbanisation challenges. The combined effect of UHI and elevated regional LST might have a more substantial impact on phenology than UHI alone. As the current study focussed solely on local-scale UHI effects on phenology, future work could explore the combined effect of UHI and LST anomalies at a regional scale. Acknowledgements We thank the manuscript reviewers for their helpful suggestions. This research was funded by the Commonwealth Scholarship Commis sion and Prince Albert II of Monaco through an Intergovernmental Panel for Climate Change Scholarship award. 8 P. Kabano et al. Landscape and Urban Planning 206 (2021) 103989 Williams, R. J., Myers, B. A., Muller, W. J., Duff, G. A., & Eamus, D. (1997). Leaf phenology of woody species in a North Australian tropical savanna. Ecology, 78(8), 2542–2558. Yao, R., Cao, J., Wang, L. C., Zhang, W. W., & Wu, X. J. (2019). Urbanization effects on vegetation cover in major African cities during 2001–2017. International Journal of Applied Earth Observation and Geoinformation, 75, 44–53. https://doi.org/10.1016/j. jag.2018.10.011. Yao, R., Wang, L., Huang, X., Guo, X., Niu, Z., & Liu, H. (2017). Investigation of urbanization effects on land surface phenology in Northeast China during 2001–2015. Remote Sensing, 9(1), 16. https://doi.org/10.3390/rs9010066. Yao, R., Wang, L., Huang, X., Niu, Y., Chen, Y., & Niu, Z. (2018). The influence of different data and method on estimating the surface urban heat island intensity. Ecological Indicators, 89, 45–55. https://doi.org/10.1016/J.ECOLIND.2018.01.044. Yuan, F., & Bauer, M. E. (2007). Comparison of impervious surface area and normalized difference vegetation index as indicators of surface urban heat island effects in Landsat imagery. Remote Sensing of Environment, 106(3), 375–386. https://doi. org/https://doi.org/10.1016/j.rse.2006.09.003. Zhang, Y., Dannenberg, M. P., Hwang, T., & Song, C. (2019). El Niño-southern oscillation-induced variability of terrestrial gross primary production during the satellite era. Journal of Geophysical Research Biogeosciences. https://doi.org/10.1029/ 2019jg005117. Zhang, X. Y., Friedl, M. A., & Schaaf, C. B. (2006). Global vegetation phenology from Moderate Resolution Imaging Spectroradiometer (MODIS): Evaluation of global patterns and comparison with in situ measurements. Journal of Geophysical ResearchBiogeosciences, 111(G4), 14. https://doi.org/10.1029/2006jg000217. Zhang, X. Y., Friedl, M. A., Schaaf, C. B., Strahler, A. H., Hodges, J. C. F., Gao, F., … Huete, A. (2003). Monitoring vegetation phenology using MODIS. Remote Sensing of Environment, 84(3), 471–475. https://doi.org/10.1016/s0034-4257(02)00135-9. Zhang, X. Y., Friedl, M. A., Schaaf, C. B., Strahler, A. H., & Schneider, A. (2004). The footprint of urban climates on vegetation phenology. Geophysical Research Letters, 31 (12). https://doi.org/10.1029/2004gl020137. Zhou, D. C., Zhao, S. Q., Zhang, L. X., & Liu, S. G. (2016). Remotely sensed assessment of urbanization effects on vegetation phenology in China’s 32 major cities. Remote Sensing of Environment, 176, 272–281. https://doi.org/10.1016/j.rse.2016.02.010. Zipper, S. C., Schatz, J., Kucharik, C. J., & Loheide, S. P. (2017). Urban heat islandinduced increases in evapotranspirative demand. Geophysical Research Letters, 44(2), 873–881. https://doi.org/10.1002/2016gl072190. Zipper, S. C., Schatz, J., Singh, A., Kucharik, C. J., Townsend, P. A., & Loheide, S. P. (2016). Urban heat island impacts on plant phenology: Intra-urban variability and response to land cover. Environmental Research Letters, 11(5). https://doi.org/ 10.1088/1748-9326/11/5/054023. Zuur, A. F., Ieno, E. N., & Elphick, C. S. (2010). A protocol for data exploration to avoid common statistical problems. Methods in Ecology and Evolution, 1(1), 3–14. https:// doi.org/10.1111/j.2041-210X.2009.00001.x. Saharan African cities. Landscape and Urban Planning, 180, 328–338. https://doi. org/10.1016/j.landurbplan.2018.08.016. Luo, M., & Lau, N. C. (2019). Urban expansion and drying climate in an urban agglomeration of east China. Geophysical Research Letters. https://doi.org/10.1029/ 2019GL082736. Melaas, E. K., Wang, J. A., Miller, D. L., & Friedl, M. A. (2016). Interactions between urban vegetation and surface urban heat islands: A case study in the Boston metropolitan region. Environmental Research Letters, 11(5), 11. https://doi.org/ 10.1088/1748-9326/11/5/054020. Mushore, T. D., Dube, T., Manjowe, M., Gumindoga, W., Chemura, A., Rousta, I., … Mutanga, O. (2019). Remotely sensed retrieval of local climate zones and their linkages to land surface temperature in harare metropolitan city, Zimbabwe. Urban Climate, 27, 259–271. https://doi.org/10.1016/j.uclim.2018.12.006. Norton, B. A., Coutts, A. M., Livesley, S. J., Harris, R. J., Hunter, A. M., & Williams, N. S. G. (2015). Planning for cooler cities: A framework to prioritise green infrastructure to mitigate high temperatures in urban landscapes. Landscape and Urban Planning, 134, 127–138. https://doi.org/10.1016/j.landurbplan.2014.10.018. R Core Team. (2018). R: A Language and Environment for Statistical Computing. Retrieved from. Vienna: Austria https://www.r-project.org/. Ren, C., Cai, M., Li, X., Zhang, L., Wang, R., Xu, Y., & Ng, E. (2019). Assessment of local climate zone classification maps of cities in china and feasible refinements. Scientific Reports. https://doi.org/10.1038/s41598-019-55444-9. Roth, M. (2007). Review of urban climate research in (sub)tropical regions. International Journal of Climatology, 27(14), 1859–1873. https://doi.org/10.1002/joc.1591. Senanayake, I. P., Welivitiya, W. D. D. P., & Nadeeka, P. M. (2013). Remote sensing based analysis of urban heat islands with vegetation cover in Colombo city, Sri Lanka using Landsat-7 ETM+ data. Urban Climate, 5, 19–35. https://doi.org/10.1016/J. UCLIM.2013.07.004. Stewart, I., & Oke, T. (2012). Local climate zones for urban temperature studies. Bulletin of the American Meteorological Society, 93(12), 1879–1900. Taha, H. (1997). Urban climates and heat islands: Albedo, evapotranspiration, and anthropogenic heat. Energy and Buildings, 25(2), 99–103. https://doi.org/10.1016/ S0378-7788(96)00999-1. United Nations. (2019). World Urbanization Prospects: The 2018 Revision. World Urbanization Prospects: The 2018 Revision. https://doi.org/10.18356/b9e995fe-en. Vermeiren, K., Van Rompaey, A., Loopmans, M., Serwajja, E., & Mukwaya, P. (2012). Urban growth of Kampala, Uganda: Pattern analysis and scenario development. Landscape and Urban Planning, 106(2), 199–206. https://doi.org/10.1016/J. LANDURBPLAN.2012.03.006. Voogt, J. A., & Oke, T. R. (2003). Thermal remote sensing of urban climates. Remote Sensing of Environment, 86(3), 370–384. https://doi.org/10.1016/s0034-4257(03) 00079-8. Wang, J. A., Hutyra, L. R., Li, D., & Friedl, M. A. (2017). Gradients of Atmospheric Temperature and Humidity Controlled by Local Urban Land-Use Intensity in Boston. Journal of Applied Meteorology and Climatology, 56(4), 817–831. https://doi.org/ 10.1175/JAMC-D-16-0325.1. 9
https://openalex.org/W4393356190	https://federalizm.rea.ru/jour/article/download/823/755	Russian	null	Systematic Approach to the Construction of a Law-Making Strategy (Using the Example of Strategic Planning of Spatial Development in Russia)	Federalizm	2,024	cc-by	6,746	5 5 DOI: http://dx.doi.org/10.21686/ 2073-1051-2024-1-5-20 Вопросы теории Е.В. КИСКИН СИСТЕМНЫЙ ПОДХОД К ПОСТРОЕНИЮ ПРАВОТВОРЧЕСКОЙ СТРАТЕГИИ (НА ПРИМЕРЕ СТРАТЕГИЧЕСКОГО ПЛАНИРОВАНИЯ ПРОСТРАНСТВЕННОГО РАЗВИТИЯ РОССИИ) В настоящее время в России правотворческая деятельность осущест вляется по модели тактического (ad hoc) реагирования на изменение ситуации, т.е. путем внесения множества локальных изменений в зако нодательство (так, в 2023 г. принято 685 федеральных и 9 федеральных конституционных законов). Вместе с тем быстрота и глубина проис ходящих в мире трансформаций, а также возрастающая сложность социальных взаимосвязей требуют перехода к стратегическому под ходу, т.е. к целенаправленному конструированию будущего правовыми средствами. Правотворческая стратегия позволяет снизить вариа тивность развития правового регулирования, поэтому ее исследование весьма актуально. В статье предпринимается попытка исследования правотворческой стратегии с позиций системной методологии. Такой подход позволяет рассмотреть стратегию комплексно, охарактеризо вать ее свойства, функции, состав и структуры, а также взаимосвязи с внешней средой. Теоретические выводы иллюстрируются на примере формирования стратегии пространственного развития России. Это направление стратегического планирования сейчас особенно актуально, поскольку необходимо реагировать на новые внешние (санкции, переори ентация экономических связей) и внутренние (увеличение числа субъектов Федерации, перераспределение логистических потоков, необходимость обеспечения технологического суверенитета) вызовы. В заключение автор делает выводы о необходимости институционализации правотворче ской стратегии и обеспечении системного похода при ее формировании. Ключевые слова: стратегия, правотворчество, системный подход, правотворческая стратегия, пространственное развитие, стратегия пространственного развития, регион. JEL: E27, Е61, К10, К19, O18 «Нет ничего практичнее хорошей теории». Эта максима, приписы ваемая психологу Курту Левину [1, с. 1], пожалуй, наиболее точно выра жает потребность научного осмысления правотворческой деятельности, особенно той ее части, которая связана с определением долгосрочных Federalism. 2024. Vol. 29. N 1 (113) 6 Е.В. КИСКИН перспектив развития правового регулирования. Быстрота и глубина происходящих в мире трансформаций, а также возрастающая сложность социальных взаимосвязей требуют перехода от тактического (ad hoc) реагирования на изменение ситуации к целенаправленному конструиро ванию будущего, в т.ч. правовыми средствами. Правотворческая стратегия выступает тем самым инструментарием, позволяющим снизить вариа тивность развития правового регулирования, придать этому процессу целенаправленность и рациональную обоснованность. В общественных науках стратегия изучается прежде всего экономи кой и в основном применительно к поведению частных коммерческих субъектов (фирм, корпораций) [2; 3]. Появляющиеся в последние годы ра боты, исследующие стратегический подход к государственному (шире – публичному) управлению, носят преимущественно прагматический и дидактический характер [4], а в юридической литературе рассматри ваются, как правило, отдельные аспекты правовых (в т.ч. правотворче ских) стратегий [5]. В то же время в современной постнеклассической науке системность сохраняет свое значение как важный методологический принцип [6]. Поэтому мы считаем необходимым предпринять попытку обосновать бо лее широкий (универсальный) подход к правотворческой стратегии. Несмотря на имеющееся мнение о невозможности построения об щей теории систем [7, с. 1 Стратегия пространственного развития Российской Федерации на период до 2025 года. Утверждена распоряжением Правительства Российской Федерации от 13 фев раля 2019 г. № 207-р. 2 Михаил Мишустин дал поручения по итогам стратегической сессии по развитию инфраструктуры. URL: http://government.ru/news/50202/ Е.В. КИСКИН СИСТЕМНЫЙ ПОДХОД К ПОСТРОЕНИЮ ПРАВОТВОРЧЕСКОЙ СТРАТЕГИИ (НА ПРИМЕРЕ СТРАТЕГИЧЕСКОГО ПЛАНИРОВАНИЯ ПРОСТРАНСТВЕННОГО РАЗВИТИЯ РОССИИ) 296–298], решение такой задачи применительно к конкретной области социальной (правовой) действительности мы по лагаем реальным и эвристически продуктивным. За основу (в качестве своего рода аксиоматической системы) примем вариант упорядочения общесистемных категорий, предложенный Валерием Николаевичем Сагатовским [8], что позволит обеспечить методологическое единство исследования. Теоретические выводы мы проиллюстрируем на примере форми рования правотворческой стратегии в сфере пространственного раз вития России. Выбор фактического материала не случаен. Страна ока залась в беспрецедентных для современной истории обстоятельствах. Внешнее санкционное давление, смена парадигмы внешнеполитических и внешнеэкономических отношений, территориальные изменения – все это повышает неопределенность дальнейшего развития государства, в т.ч. в части системы расселения, пространственной организации экономики, социальной и иных сфер общественной жизни. В то же время именно стратегия, как мы сказали, призвана опти мизировать процессы управления будущим [9, с. 9], повысить предска зуемость результата принимаемых правотворческих решений. Кроме того, в следующем году прекращает действие Стратегия про странственного развития Российской Федерации на период до 2025 года1. Правительство уже приступило к разработке концепции ее новой Федерализм. 2024. Т. 29. ¹ 1 (113) Системный подход к построению правотворческой стратегии 7 версии2. Поэтому теоретическое осмысление правотворческих аспек тов стратегического планирования именно в сфере пространственно го развития весьма актуально. Правотворческая стратегия как система Рассматривая правотворческую стратегию в качестве системы, мы характеризуем ее как целое, которое не сводимо к механической сум ме составляющих его частей, т.е. обладающее особыми интегративными характеристиками, придающими всей совокупности новые качества. Сложность исходного объекта ведет к тому, что в науке до настоящего времени нет единого понимания онтологического статуса стратегии. Она рассматривается как деятельность, процесс, набор правил, паттерн или план, а также как совокупность этих понятий [10]. Исходя из много образия стратегий, все эти подходы имеют эвристическую ценность. С точки зрения системного метода сущность правотворческой страте гии может быть познана в различии с другими объектами. Элементы си стемы, различные или тождественные по отношению к элементам других систем, характеризуются как свойства этой системы [8, с. 176]. Понятно, что критериев сходства и различия правотворческой стратегии с други ми системами может быть множество, остановимся лишь на наиболее существенных, с нашей точки зрения, свойствах стратегии. Правотворческая стратегия – гетерогенная система, состоит из эле ментов разной природы. Соответственно, она не может быть однознач но описана как совокупность одних лишь действий, или процессов, или правил, либо же как документ (группа документов). Учитывая, что правотворчество есть институционализированная деятельность по разработке, принятию, легитимации и трансляции общеобязательных нормативных текстов, то правотворческая стратегия должна рассматриваться как деятельностная система. Это означает, что ее элементами, т.е. первичными, далее не редуцируемыми (примени тельно к интегративным свойствам системы) составными частями будут субъекты, объекты и связи (отношения) между ними. Причем именно последние придают всей совокупности интегративные качества. Человеческая деятельность предполагает социальные взаимодей ствия и тем самым коммуникацию. Как мы неоднократно писали ранее, правотворчество имеет коммуникативную природу и мыслится как инте ракция субъектов, в ходе которой формируются социально признанные нормативные правовые тексты [11; 12]. Соответственно, с этой точки зрения правотворческая стратегия будет коммуникативной системой. Исходя из специфики правотворческой коммуникации, направлен ной на институционализацию общеобязательных велений, необходимо обратить внимание на прескриптивный (предписывающий) характер правотворческой стратегии. И в этом плане она выступает как норма Federalism. 2024. Vol. 29. N 1 (113) 8 Е.В. КИСКИН тивная система. При этом нормативность не означает, что правотворче ская стратегия состоит только из норм (правил), мы понимаем ее в более общем смысла как абстрактно-всеобщее выражение долженствования. Поэтому стратегия – это своего рода желаемый образ будущих норм. Из свойства гетерогенности следует также, что правотворче ская стратегия – это комплексная система, т.е. ее образуют элементы, одновременно функционирующие в других системах, причем в других по своей природе, с которыми правотворческая стратегия на этом осно вании связана сложными функциональными и иными зависимостями. Правотворческая стратегия как система Правотворческая стратегия, будучи самостоятельной системой, вместе с тем является компонентом системы более высокого порядка – публичной (государственной) управленческой стратегии. Применительно к сфере пространственного развития это означает, что правотворче ская стратегия в этой области должна рассматриваться как часть стра тегии пространственного развития, при этом часть неотъемлемая, поскольку в современном обществе правовое регулирование выступает основным инструментом социального управления. Сказанное также приводит нас к выводу что стратегия простран ственного развития как система – более широкое понятие, нежели до кумент с таким же названием, принятие которого предусмотрено ст. 20 Федерального закона от 28 июня 2014 г. № 172-ФЗ «О стратегическом планировании в Российской Федерации». В соответствии с п. 26 ст. 3 названного Федерального закона, стратегия пространственного раз вития Российской Федерации – документ стратегического планиро вания, определяющий приоритеты, цели и задачи регионального раз вития Российской Федерации и направленный на поддержание устой чивости системы расселения на территории Российской Федерации. В то же время в силу ч. 1 ст. 20 Федерального закона «О стратегическом планировании в Российской Федерации» стратегия пространственно го развития Российской Федерации разрабатывается в соответствии с ос новами государственной политики регионального развития Российской Федерации в целях реализации основных положений стратегии социаль но-экономического развития Российской Федерации и стратегии нацио нальной безопасности Российской Федерации, определяет приоритеты, цели и задачи регионального развития Российской Федерации и меры по их достижению и решению. А согласно ч. 6 этой же статьи по пору чению Президента Российской Федерации Правительство Российской Федерации разрабатывает основы государственной политики региональ ного развития Российской Федерации, являющиеся основой для определения приоритетов регионального развития Российской Федерации. Основы го сударственной политики регионального развития Российской Федерации утверждаются Президентом Российской Федерации. Обращает на себя внимание множественность документов – основы и стратегия – при едином предмете управления – региональном разви тии России. Причем первый из них логически предшествует второму: основы (и здесь, следуя тексту закона никак не избежать тавтологии) Федерализм. 2024. Т. 29. ¹ 1 (113) 9 Системный подход к построению правотворческой стратегии задают основы для определения приоритетов, которые в свою очередь формулируются стратегией. В юриспруденции сферу социальных взаимосвязей (общественных отношений), на которую направлено упорядочивающее воздействие права, называют предметом правового регулирования. Таким образом, с точки зрения правотворческого компонента, исходя из текста закона, оба документа – основы и стратегия – имеют общий предмет и логически связаны. Поэтому их можно рассматривать как составные части единой правотворческой стратегии в сфере регионального (пространственного) развития в широком системном понимании, изложенном выше. 3 Указ Президента Российской Федерации от 16 января 2017 г. № 13 «Об утвержде нии Основ государственной политики регионального развития Российской Федерации на период до 2025 года». Правотворческая стратегия как система Если сравнить содержание действующих Основ государственной политики регионального развития Российской Федерации на период до 2025 года3 (далее – Основы) и Стратегии пространственного развития на период до 2025 года (далее – Стратегия) в части определения ими пред мета своего регулирования, то можно заметить что Основы, как и пред усмотрено Федеральным законом «О стратегическом планировании в Российской Федерации», оперируют термином «региональное развитие», понимаемым как политическое и социально-экономическое развитие субъектов Российской Федерации и муниципальных образований. В то же время Стратегия, отходя от буквального смысла норм закона, заменяет понятие «региональное развитие» понятием «про странственное развитие», которое определяется как совершенствование системы расселения и территориальной организации экономики, в т.ч. за счет проведения эффективной государственной политики региональ ного развития. Как видим, в понимании Стратегии, пространствен ное развитие шире регионального, что представляется верным [13]. Поэтому подход законодателя, закрепленный в нормах Федерального закона «О стратегическом планировании в Российской Федерации», где, по сути, пространственное развитие сводится к региональному, видится сомнительным. С точки зрения системной методологии полагаем необхо димым проработать вопрос об унификации понятийного аппарата и устра нении множественности документов с пересекающимся содержанием. При этом определение пространственного развития, сформулированное в Стратегии, несмотря на дискуссионный характер [13], для утилитарных целей построения правотворческой стратегии в данной сфере мы полага ем вполне приемлемым, поскольку оно достаточно четко характеризует предмет регулирования (какие группы отношений надо регулировать). 4 Федеральный закон от 13 июля 2020 г. № 193-ФЗ «О государственной поддержке предпринимательской деятельности в Арктической зоне Российской Федерации». Функции правотворческой стратегии Приведенные признаки правотворческой стратегии могут быть обнаружены у множества иных систем. Чем же характеризуется ее уни кальность? 3 Указ Президента Российской Федерации от 16 января 2017 г. № 13 «Об утвержде нии Основ государственной политики регионального развития Российской Федерации на период до 2025 года». Federalism. 2024. Vol. 29. N 1 (113) 10 Е.В. КИСКИН Поскольку система как целое не сводима к сумме составляющих ее элементов, то именно особые взаимодействия, связи и отношения между этими элементами сообщают ей интегративные свойства, которые, про являясь в действии, рассматриваются как функции системы [8, с. 173]. Рассмотрим основные функции правотворческой стратегии. Главная ее функция состоит в оптимизации процессов управления бу дущим. При этом правотворческая коммуникация выстраивается таким образом, чтобы переход к желаемому правовому регулированию пред ставлял собой ряд последовательных шагов, в рамках которого посте пенно отдельные элементы настоящего заменялись на элементы констру ируемого будущего. Поэтому центральным звеном стратегии является образ желаемого будущего, который формируется через целеполагание. Подробнее о целях как компонентах правотворческой стратегии будет сказано ниже. Еще одна функция связана со снижением вариативности будущего правового регулирования. Стратегия предполагает рациональный отбор правотворческих решений для достижения заданных целей, решения поставленных задач и, соответственно, включает в себя критерии такого отбора. Например, Стратегией пространственного развития на период до 2025 года предусматривается реализация дополнительных направлений социально-экономического развития исторических поселений за счет сохранения и воссоздания памятников истории и культуры и истори чески ценных градоформирующих объектов. Таким образом, в качестве поддерживаемого государством, т.е. преимущественно обеспечиваемого правовыми средствами подхода, выбрана, по сути, консервация (сохра нение) наследия, а не ревитализация исторической среды или популя ризация туристского потенциала этих территорий. При этом какие-либо критерии (обоснования) выбора такого решения не приведены. Функция приоритизации реализуется путем ранжирования задач и определения последовательности их решения в условиях ограничен ности ресурсов. Так, Стратегией пространственного развития на период до 2025 года поставлена задача сокращения уровня межрегиональной дифференциации в социально-экономическом развитии субъек тов Российской Федерации. При этом Стратегией определен пере чень приоритетных геостратегических территорий (приложение № 4): эксклавные субъекты, регионы Северного Кавказа, Дальний Восток и Арктическая зона Российской Федерации. Соответственно, при вы равнивании регионов первостепенное внимание законодателя будет уделено созданию на этих территориях наиболее благоприятного ре жима развития экономики. Примерами такого первостепенного внима ния могут быть принятие закона о преференциальном осуществлении предпринимательской деятельности в Арктической зоне Российской Федерации4 или распространение режима особой экономической зоны Федерализм. 2024. Т. 29. ¹ 1 (113) 11 Системный подход к построению правотворческой стратегии на Курильские острова5. Функции правотворческой стратегии В то же время режим территорий опережаю щего развития в моногородах продолжает оставаться менее льготным, чем таковой на Дальнем Востоке6. Функция адаптации. Стратегия, формирующая образ будущего правового регулирования и пути его достижения, обеспечивает устой чивость правотворческой коммуникации в изменяющихся условиях. Недаром, в социальных науках данный термин появился в 50-е гг. XX в., когда существенное значение приобрела проблема реакции на неожидан ные изменения во внешней среде [2, с. 68]. Кроме того, стратегия суще ствует не просто в меняющейся, а в конфликтной (конкурентной) среде. Как пишет Лоуренс Фридман, «там, где цели и так легко достижимы, где задействованы неодушевленные предметы, а не другие люди, где ставки не велики, стратегия не так уж и необходима. В широком смысле, стратегия вступает в действие там, где имеется открытый или потенциальный кон фликт, столкновение интересов, требующее какого-то разрешения» [14, с. 9]. Право же всегда действует в остро конфликтной среде. Поэтому правотворческая стратегия выполняет также функцию преодоления со противления среды. Так, решение задачи по ускоренному социально- эконмическому развитию Дальнего Востока сдерживается продолжаю щимся миграционным оттоком населения7. Для того чтобы преодолеть этот негативный фактор, принимаются правотворческие решения, на правленные на закрепление постоянного населения и миграционный приток (например, льготное предоставление земельных участков (про грамма «Дальневосточный гектар»)8, субсидирование ставки по ипотеке (проект «Дальневосточная ипотека»)9 и др.). 8 Федеральный закон от 1 мая 2016 г. № 119-ФЗ «Об особенностях предоставления гражданам земельных участков, находящихся в государственной или муниципальной собственности и расположенных в Арктической зоне Российской Федерации и на других территориях Севера, Сибири и Дальнего Востока Российской Федерации, и о внесении изменений в отдельные законодательные акты Российской Федерации». 9 Постановление Правительства Российской Федерации от 7 декабря 2019 г. № 1609 «Об утверждении условий программы “Дальневосточная и арктическая ипотека”, Правил предоставления субсидий из федерального бюджета акционерному обществу “ДОМ.РФ” в виде вкладов в имущество акционерного общества «ДОМ.РФ», не увеличивающих его уставный капитал, для возмещения российским кредитным организациям и иным организациям недополученных доходов по жилищным (ипотечным) кредитам, предо ставленным гражданам Российской Федерации на приобретение или строительство жилых помещений на территориях субъектов Российской Федерации, входящих в состав Дальневосточного федерального округа, а также на сухопутных территориях Арктической зоны Российской Федерации, и внесении изменений в распоряжение Правительства Российской Федерации от 2 сентября 2015 г. № 1713-р». 9 Постановление Правительства Российской Федерации от 7 декабря 2019 г. 6 Федеральный закон от 29 декабря 2014 г. № 473-ФЗ «О территориях опережающего развития в Российской Федерации». 5 Федеральный закон от 18 марта 2023 г. № 84-ФЗ «О внесении изменений в от дельные законодательные акты Российской Федерации и о проведении на территории Калининградской области эксперимента по созданию условий для упорядоченной электронной торговли товарами». 5 Федеральный закон от 18 марта 2023 г. № 84-ФЗ «О внесении изменений в от дельные законодательные акты Российской Федерации и о проведении на территории Калининградской области эксперимента по созданию условий для упорядоченной электронной торговли товарами». 6 Федеральный закон от 29 декабря 2014 г. № 473-ФЗ «О территориях опережающего развития в Российской Федерации». 7 См.: Материалы парламентских слушаний на тему «О ходе реализации Основ государственной политики регионального развития Российской Федерации на период до 2025 года и Стратегии пространственного развития Российской Федерации на период до 2025 года». URL: http://region.council.gov.ru/activity/activities/parliamentary/150940/ 8 Федеральный закон от 1 мая 2016 г. № 119-ФЗ «Об особенностях предоставления гражданам земельных участков, находящихся в государственной или муниципальной собственности и расположенных в Арктической зоне Российской Федерации и на других территориях Севера, Сибири и Дальнего Востока Российской Федерации, и о внесении изменений в отдельные законодательные акты Российской Федерации». 9 Постановление Правительства Российской Федерации от 7 декабря 2019 г. № 1609 «Об утверждении условий программы “Дальневосточная и арктическая ипотека”, Правил предоставления субсидий из федерального бюджета акционерному обществу “ДОМ.РФ” в виде вкладов в имущество акционерного общества «ДОМ.РФ», не увеличивающих его уставный капитал, для возмещения российским кредитным организациям и иным организациям недополученных доходов по жилищным (ипотечным) кредитам, предо ставленным гражданам Российской Федерации на приобретение или строительство жилых помещений на территориях субъектов Российской Федерации, входящих в состав Дальневосточного федерального округа, а также на сухопутных территориях Арктической зоны Российской Федерации, и внесении изменений в распоряжение Правительства Российской Федерации от 2 сентября 2015 г. № 1713-р». 7 См.: Материалы парламентских слушаний на тему «О ходе реализации Основ государственной политики регионального развития Российской Федерации на период до 2025 года и Стратегии пространственного развития Российской Федерации на период до 2025 года». URL: http://region.council.gov.ru/activity/activities/parliamentary/150940/ 8 Федеральный закон от 1 мая 2016 г. № 119-ФЗ «Об особенностях предоставления гражданам земельных участков, находящихся в государственной или муниципальной собственности и расположенных в Арктической зоне Российской Федерации и на других территориях Севера, Сибири и Дальнего Востока Российской Федерации, и о внесении изменений в отдельные законодательные акты Российской Федерации». Функции правотворческой стратегии № 1609 «Об утверждении условий программы “Дальневосточная и арктическая ипотека”, Правил предоставления субсидий из федерального бюджета акционерному обществу “ДОМ.РФ” в виде вкладов в имущество акционерного общества «ДОМ.РФ», не увеличивающих его уставный капитал, для возмещения российским кредитным организациям и иным организациям недополученных доходов по жилищным (ипотечным) кредитам, предо ставленным гражданам Российской Федерации на приобретение или строительство жилых помещений на территориях субъектов Российской Федерации, входящих в состав Дальневосточного федерального округа, а также на сухопутных территориях Арктической зоны Российской Федерации, и внесении изменений в распоряжение Правительства Российской Федерации от 2 сентября 2015 г. № 1713-р». Federalism. 2024. Vol. 29. N 1 (113) Federalism. 2024. Vol. 29. N 1 (113) 12 Е.В. КИСКИН Предусматривая рациональный выбор и обоснование правотворче ских решений в процессе коммуникации, правотворческая стратегия выполняет функцию дополнительной легитимации правотворческих реше ний. Как мы отмечали, правотворческая стратегия нормативна, посколь ку предопределяет поведение как законодателя, так и участников регули руемых отношений, обусловливает принятие правотворческих решений, ограничивает усмотрение законодателя. Таким образом, стратегия обе спечивает обоснование и оправдание правотворческих решений. Ни действующая Стратегия пространственного развития на пери од до 2025 года, ни большинство других документов стратегического планирования не предусматривают мотивы принятия содержащихся в них решений. При этом, как мы писали выше, стратегия как си стема не сводима к одноименному документу. Поэтому мотивы при нятия решений могут быть опубличены отдельно. Важно отметить, что п. 7 Положения о содержании, составе, порядке разработки и утверждения стратегии пространственного развития Российской Федерации и подготавливаемых в ее составе предложений о совершен ствовании системы расселения на территории Российской Федерации и приоритетных направлениях размещения производительных сил на территории Российской Федерации (утвержденного Постановлением Правительства Российской Федерации от 20 августа 2015 г. № 870) предусмотрено, что при подготовке стратегии пространственного раз вития разрабатываются обосновывающие материалы, включающие в себя статистические, аналитические, картографические и иные мате риалы, определяющие и иллюстрирующие основные решения и поло жения стратегии. Указанные материалы не входят в состав документа, утверждаемого распоряжением Правительства Российской Федерации. При этом названным Положением обнародование данных мате риалов не предусмотрено, хотя это является весьма значимым с точки зрения легитимации (оправдания) принимаемых решений. Логическая структура правотворческой стратегии Теоретики системного подхода различают категории состава и струк туры системы. Если первая (состав) характеризует лишь набор (множе ство) элементов, входящих в систему, то вторая (структура) акцентирует внимание на отношениях между элементами, их упорядоченности. В за висимости от того, какой элемент будет выбран в качестве отправной точ ки, мы можем увидеть разные варианты построения отношений между элементами, т.е. разные структуры [8, с. 187–188]. Сказанное приводит к мысли о полиструктурности правотворческой стратегии как сиси темы. Применительно к разным правовым явления В.Н. Карташов рас сматривает логическую (логико-философскую), пространственную, генетическую, временню, стохастическую и иные структуры [15, с. 161–162]. В рамках настоящей работы мы обратим внимание на логи ческую структуру правотворческой стратегии. Теоретики системного подхода различают категории состава и струк туры системы. Если первая (состав) характеризует лишь набор (множе ство) элементов, входящих в систему, то вторая (структура) акцентирует внимание на отношениях между элементами, их упорядоченности. В за висимости от того, какой элемент будет выбран в качестве отправной точ ки, мы можем увидеть разные варианты построения отношений между элементами, т.е. разные структуры [8, с. 187–188]. Сказанное приводит к мысли о полиструктурности правотворческой стратегии как сиси темы. Применительно к разным правовым явления В.Н. Карташов рас сматривает логическую (логико-философскую), пространственную, генетическую, временню, стохастическую и иные структуры [15, с. 161–162]. В рамках настоящей работы мы обратим внимание на логи ческую структуру правотворческой стратегии. Федерализм. 2024. Т. 29. ¹ 1 (113) 13 Системный подход к построению правотворческой стратегии Внутренняя логика стратегии, как мы уже говорили (и здесь мы со гласны с Г.Г. Почепцовым), состоит в накоплении элементов будущего в настоящем, притом таких элементов, которые позволяют осуществить наиболее оптимальный переход к этому будущему. Специфика стратегии состоит в том, что она видит как будущее, так и настоящее одновременно. Поэтому, исходя из нее, можно понять потребности будущего в насто ящем, что именно будет нужно для того, чтобы сблизить эти две точки (будущего и настоящего) наименее болезненно [9, с. 40]. С этой точки зрения главным и неотъемлемым компонентом правотворческой стра тегии будут цели, поскольку, именно они характеризуют идеальный образ предполагаемого результата, т.е. желаемое состояние будущего правового регулирования. Цели правотворческой стратегии могут быть подразделены на вну тренние, обусловленные спецификой юридического типа социальной ре гуляции и более или менее стабильные в условиях данного общества, а также внешние, характеризующие желаемое состояние сферы соци альной действительности (общественных отношений), составляющих предмет моделируемого правового регулирования. К числу внутренних и постоянных целей правотворческой стра тегии относится прежде всего формирование эффективного и леги тимного нормативного правового текста. Federalism. 2024. Vol. 29. N 1 (113) Логическая структура правотворческой стратегии А внешние цели специфичны для каждой конкретной стратегии. Применительно к правотворче ской стратегии в исследуемой нами сфере внешние цели будут зависть от целей стратегии пространственного развития в целом, т.е. от мо делируемых результатов ее реализации – идеального состояния про странственной структуры экономики и системы расселения, которого планируется достичь. Следует отметить, что в действующей Стратегии простран ственного развития на период до 2025 года определена одна цель: обеспечение устойчивого и сбалансированного пространственно го развития Российской Федерации, направленного на сокраще ние межрегиональных различий в уровне и качестве жизни населения, ускорение темпов экономического роста и технологического развития, а также на обеспечение национальной безопасности страны. Как видим, такая цель не моделирует конечный результат, а характеризует процесс, т.е. ее достижение невозможно оценить. С точки зрения правотворче ства, исходя из такой цели, мы не сможем определить, что и как над лежит регулировать. Общепринято, что цели должны быть выполнимы и измеримы, т.е. снабжены индикаторами, позволяющими оценить степень их достижения. Следующий компонент логической структуры стратегии – это зада чи. Они чаще всего определяются как направления или пути достижения целей. Они описывают, что нужно сделать, чтобы обеспечить переход к конструируемому будущему. В Стратегии пространственного разви тия на период до 2025 года одна из задач сформулирована следующим образом: «сокращение уровня межрегиональной дифференциации в со Federalism. 2024. Vol. 29. N 1 (113) 14 14 Е.В. КИСКИН циально-экономическом развитии субъектов Российской Федерации, а также снижение внутрирегиональных социально-экономических раз личий: циально-экономическом развитии субъектов Российской Федерации, а также снижение внутрирегиональных социально-экономических раз личий: • за счет повышения устойчивости системы расселения путем со циально-экономического развития городов и сельских территорий; • за счет повышения конкурентоспособности экономик субъек тов Российской Федерации путем обеспечения условий для раз вития производства товаров и услуг в отраслях перспективных экономических специализаций субъектов Российской Федерации, приведенных по перечню согласно приложению № 1; • за счет улучшения транспортной доступности сельских террито рий; • за счет совершенствования территориальной системы органи зации оказания услуг социальной сферы, обеспечивающей ее оптимальную доступность с учетом современных технологий предоставления услуг; • за счет усиления межрегионального сотрудничества и координа ции социально-экономического развития субъектов Российской Федерации в рамках макрорегионов Российской Федерации, состав которых приведен согласно приложению № 2; • за счет формирования и развития минерально-сырьевых центров». Обращает на себя внимание, что первая часть («сокращение уров ня межрегиональной дифференциации…») этой формулировки больше подходит под понятие цели, т.к. характеризует конечный результат и может быть измерена. Применительно к правотворчеству соотношение цели и задачи может выглядеть, например, следующим образом (это лишь пример логического соотношения, абстрагируемся от целесообразности): • внешняя цель – повышение вклада малых и средних городов в экономический рост; • задача – установление в малых и средних городах более льготно го режима ведения предпринимательской деятельности по срав нению с крупными агломерациями. Приоритеты также выступают в качестве компонента логиче ской структуры правотворческой стратегии, поскольку они объективи руют интересы общества, позволяют ранжировать задачи и определить очередность их решения, т.е. в конечном счете – рационально распре делить ресурсы. В числе приоритетов пространственного развития до 2025 г. дей ствующая Стратегия называет развитие приграничных муниципальных образований с учетом их стратегического значения, существующего уровня и потенциала социально-экономического развития. Это озна чает, что применительно к сформулированной выше гипотетической задаче более льготный режим ведения предпринимательской деятель ности прежде всего должен быть обеспечен именно в приграничных му ниципалитетах. Федерализм. 2024. Т. 29. ¹ 1 (113) 15 Системный подход к построению правотворческой стратегии Формирование правотворческой стратегии невозможно без опреде ления вариантов достижения целей и решения задач, а также критериев отбора таких вариантов. Вариативность стратегии – важное свойство, обеспечивающее ее гибкость и в то же время устойчивость в сопро тивляющейся (конкурентной) среде. При этом выбор вариантов право творческих решений должен быть рационально обоснован, мотивирован, что обеспечит эффективность и легитимность будущего регулирования. Например, задача формирования и развития минерально-сырьевых центров в Арктической зоне может быть решена как за счет преимуще ственного использования вахтового метода освоения месторождений, так и за счет обустройства и развития постоянных поселений в Арктической зоне. циально-экономическом развитии субъектов Российской Федерации, а также снижение внутрирегиональных социально-экономических раз личий: Так, план реализации Стратегии пространственного раз вития Российской Федерации на период до 2025 года10 включает в себя, в частности, и меры правотворческого характера. 10 План реализации Стратегии пространственного развития Российской Федерации на период до 2025 года. Утвержден распоряжением Правительства Российской Федерации от 27 декабря 2019 г. № 3227-р. циально-экономическом развитии субъектов Российской Федерации, а также снижение внутрирегиональных социально-экономических раз личий: С точки зрения правового регулирования выбор такого метода мо жет потребовать, в частности, следующих мер: Формирование правотворческой стратегии невозможно без опреде ления вариантов достижения целей и решения задач, а также критериев отбора таких вариантов. Вариативность стратегии – важное свойство, обеспечивающее ее гибкость и в то же время устойчивость в сопро тивляющейся (конкурентной) среде. При этом выбор вариантов право творческих решений должен быть рационально обоснован, мотивирован, что обеспечит эффективность и легитимность будущего регулирования. фф удущ р у р Например, задача формирования и развития минерально-сырьевых центров в Арктической зоне может быть решена как за счет преимуще ственного использования вахтового метода освоения месторождений, так и за счет обустройства и развития постоянных поселений в Арктической зоне. С точки зрения правового регулирования выбор такого метода мо жет потребовать, в частности, следующих мер: • совершенствования положений гл. 47 «Особенности регулирова ния труда лиц, работающих вахтовым методом» Трудового кодек са Российской Федерации; • законодательного закрепления понятия «вахтовый поселок» и его статуса; • дополнения Градостроительного кодекса Российской Федерации нормами, устанавливающими требования к проектированию вахтовых поселков [16, с. 105]. Рациональные основания выбора такого решения понятны – повы шение рентабельности, снижение себестоимости и улучшение резуль татов финансово-хозяйственной деятельности предприятия, а также экономия средств государства на развитие социальной инфраструктуры. Выбор в сторону обустройства Арктики, постоянным населени ем может обеспечиваться теми же мерами привлечения населения, что и на Дальнем Востоке («арктический гектар» и «арктическая ипо тека»), а также совершенствованием пенсионного обеспечения лиц, работающих в Арктической зоне. Здесь основания выбора будут пре имущественно социального и геополитического характера. Прогнозы как компонент логической структуры правотворче ской стратегии позволяют оценить последствия проектируемых ре шений, возможные варианты развития моделируемых общественных отношений. Д.А. Керимов акцентировал внимание на соотношении прогнозов и целей. Прогнозирование, с его точки зрения, является не обходимым этапом становления правовой цели, которая всегда включает в себя те моменты предвидения, которые наиболее достоверны, строго, основаны на объективных закономерностях действительности, ее ре альных возможностях [17, с. 281]. Планы как компонент завершают логическую структуру правотвор ческой стратегии, поскольку обеспечивают последовательное, поэтапное достижение целей, т.е. постепенную замену элементов настоящего эле ментами конструируемого будущего. При этом с точки зрения вешней формы совершенно не принципиально, будет ли это отдельный до кумент, совокупность документов или отдельные положения в разных Federalism. 2024. Vol. 29. N 1 (113) Federalism. 2024. Vol. 29. N 1 (113) 16 Е.В. КИСКИН документах. Так, план реализации Стратегии пространственного раз вития Российской Федерации на период до 2025 года10 включает в себя, в частности, и меры правотворческого характера. документах. Некоторые направления формирования правотворческой стратегии в сфере пространственного развития Проведенное исследование, основанное на системной методологии, приводит как минимум, к двум важным выводам. Во-первых, правотворческая стратегия в сфере пространственного раз вития требует институционализации. Мы не настаиваем на принятии отдельного документа и даже на выделении в проектируемой стратегии пространственного развития отдельного раздела, но считаем нужным по думать о более четком описании предполагаемого правового регулирования, включая обоснование выбранного варианта такого регулирования. Последнее не вполне в духе российской традиции, которой свойственно оставлять за рамками правового акта мотивы его принятия, однако опубличивание оснований выбора правового регулирования способно повысить степень его легитимации. Эта задача может быть решена обнародованием части обосно вывающих материалов к стратегии, что, кстати, практиковалось в законо проектной работе в предреволюционный период. Так, проект Гражданского уложения Российской империи, подготовленный в 1892–1899 гг., был опу бликован в двенадцати томах, с подробными постатейными пояснениями, описывающими логику рассуждений авторов проекта [18]. Во-вторых, при разработке новой редакции стратегии простран ственного развития России важно обеспечить системность ее форми рования. Это требует решения трех задач. Первая – согласование положения Федерального закона «О страте гическом планировании в Российской Федерации» и подзаконных актов в части устранения двойственности понятий «региональное развитие» и «пространственное развитие», описывающих предмет стратегии. Вторая – оценка целесообразности формирования двух докумен тов стратегического планирования по одному (с точки зрения закона) предмету – основ государственной политики регионального развития и стратегии пространственного развития. Если принятие основ необ ходимо, например, с целью повышения статуса и юридической силы документа (утверждается Президентом Российской Федерации), то, учи тывая важность сферы пространственного развития, можно рассмотреть вопрос о повышении статуса объединенного документа. Третья – проектирование логически целостной структуры стратегии с учетом формулирования целей, задач, приоритетов, исходя из раз работанного наукой разграничения этих понятий, а также с учетом новых территориальных и геополитических реалий. 10 План реализации Стратегии пространственного развития Российской Федерации на период до 2025 года. Утвержден распоряжением Правительства Российской Федерации от 27 декабря 2019 г. № 3227-р. Федерализм. 2024. Т. 29. ¹ 1 (113) 17 Системный подход к построению правотворческой стратегии Список литературы 1. McCain K.W. “Nothing as Practical as a Good Theory”. Does Lewin’s Maxim Still Have Salience in the Applied Social Sciences? // Proceedings of the Association for Information Science and Technology. 2015. N 52 (1). P. 1–4. 1. McCain K.W. “Nothing as Practical as a Good Theory”. Does Lewin’s Maxim Still Have Salience in the Applied Social Sciences? // Proceedings of the Association for Information Science and Technology. 2015. N 52 (1). P. 1–4. 1. McCain K.W. “Nothing as Practical as a Good Theory”. Does Lewin’s Maxim Still Have Salience in the Applied Social Sciences? // Proceedings of the Association for Information Science and Technology. 2015. N 52 (1). P. 1–4. 2. Ансофф И. Стратегическое управление. М.: Экономика, 1989. 520 с. 2. Ансофф И. Стратегическое управление. М.: Экономика, 1989. 520 с. 3. Стратегия: пер. с англ. М.: Альпина Паблишер, 2022. 288 с. (Серия «Harvard Business Review: 10 лучших статей»). 3. Стратегия: пер. с англ. М.: Альпина Паблишер, 2022. 288 с. (Серия «Harvard Business Review: 10 лучших статей»). 4. Малган Дж. Искусство государственной стратегии: мобилизация власти и знания во имя всеобщего блага. М.: Изд-во Института Гайдара, 2020. 472 с. 4. Малган Дж. Искусство государственной стратегии: мобилизация власти и знания во имя всеобщего блага. М.: Изд-во Института Гайдара, 2020. 472 с. дарственной стратегии: мобилизация влас 5. Юридическая техника: Ежегодник. 2015. № 9: материалы XVI Меж- дународного научно-практического форума «Юртехнетика» на тему: «Стратегия, тактика, техника законотворчества (взаимосвязь, инновационные достиже ния, ошибки)» (Н. Новгород, 25–27 сентября 2014 г.) / гл. ред. В.М. Баранов. Н. Новгород: Кириллица, 2015. 851 с. 5. Юридическая техника: Ежегодник. 2015. № 9: материалы XVI Меж- дународного научно-практического форума «Юртехнетика» на тему: «Стратегия, тактика, техника законотворчества (взаимосвязь, инновационные достиже ния, ошибки)» (Н. Новгород, 25–27 сентября 2014 г.) / гл. ред. В.М. Баранов. Н. Новгород: Кириллица, 2015. 851 с. 6. Толпыкин В.Е. Системность как методологический принцип современ ной постнеклассической науки // Теория и практика общественного развития. 2011. № 7. С. 27–30. 6. Толпыкин В.Е. Системность как методологический принцип современ ной постнеклассической науки // Теория и практика общественного развития. 2011. № 7. С. 27–30. 7. Дубровский В.Я. Очерки по общей теории деятельности. М.: ННФ «Институт развития им. Г.П. Щедровицкого», 2011. 576 с. 8. Сагатовский В.Н. Основы систематизации всеобщих категорий. Томск: Издательство Томского университета, 1973. 432 с. 9. Почепцов Г.Г. Стратегия. Инструментарий по управлению будущим. М.: Рефл-бук, К.: Ваклер, 2005. 384 с. 10. Наумова Е.А. Список литературы Анализ определений стратегии и ее место в системе функ ционирования предприятия. Разнообразие взглядов и подходов к стратегии // Вестник ЮУрГУ. Серия: Экономика и менеджмент. 2011. № 8 (225). С. 111–114. Вестник ЮУрГУ. Серия: Экономика и менеджмент. 2011. № 8 (225). С. 111–114. 11. Кискин Е.В. Стратегия как компонент правотворческой технологии: коммуникативный подход // Юридическая техника. 2015. № 9. С. 292–298. 11. Кискин Е.В. Стратегия как компонент правотворческой технологии: коммуникативный подход // Юридическая техника. 2015. № 9. С. 292–298. 12. Кискин Е.В. Принципы как компонент правотворческой стратегии (на примере законодательства о выборах и референдумах) // Избирательное законодательство и практика. 2021. № 4. С. 11–14. 13. Жаворонкова Н.Г., Шпаковский Ю.Г. Правовые проблемы пространствен ного развития Российской Федерации: вопросы стратегического планирования // Юрист. 2020. № 5. С. 56–61. 14. Фридман Л. Стратегия: война, революция, бизнес. М.: Кучково поле, 2018. 767 с. 15. Карташов В.Н. Принципы права: понятие, структуры, функции // Юридическая техника. 2020. № 14. С. 158–166. 16. Попова О.В. Социальные аспекты нормативно-правового регулирования вахтового труда в северных регионах // Теория и практика общественного раз вития. 2021. № 12. С. 104–109. 17. Керимов Д.А. Методология права: Предмет, функции, проблемы фило софии права. М.: СГА, 2003. 521 с. 18. Проект книги первой Гражданского уложения с объяснительною за пискою. СПб.: Государственная типография, 1893. 263 с. Federalism. 2024. Vol. 29. N 1 (113) 18 Е.В. КИСКИН References 1. McCain K.W. “Nothing as Practical as a Good Theory”. Does Lewin’s Maxim Still Have Salience in the Applied Social Sciences? Proceedings of the Association for Information Science and Technology, 2015, No. 52 (1), pp. 1–4. f gy, , ( ), pp 2. Ansoff I. Strategicheskoe upravlenie [Strategic Management]. Moscow, Ekonomika, 1989, 520 p. (In Russ.). 3. Strategiia (Seriia “Harvard Business Review: 10 luchshikh statei”) [Strategy (Harvard Business Review Series: Top 10 Articles)]. Translated from English. Moscow, Al’pina Pablisher, 2022, 288 p. (In Russ.). 4. Malgan Dzh. Iskusstvo gosudarstvennoi strategii: mobilizatsiia vlasti i znaniia vo imia vseobshchego blaga [The Art of Public Strategy: Mobilizing Power and Knowledge for the Common Good]. Moscow, Izdatel’stvo Instituta Gaidara, 2020, 472 p. (In Russ.). 5. Yuridicheskaya tekhnika: Ezhegodnik. 2015. N 9: materialy XVI Mezhdunarodnogo nauchno-prakticheskogo foruma “Yurtekhnetika” na temu: “Strategiya, taktika, tekhnika zakonotvorchestva (vzaimosvyaz’, innovatsionnye dostizheniya, oshibki)” (Nizhnii Novgorod, 25–27 sentyabrya 2014 g.) [Legal Technique: Yearbook. 2015. No. 9: materials of the XVI International Scientific and Practical Forum “Yurtechnetics” on the topic: “Strategy, Tactics, Technique of Lawmaking (Interrelation, Innovative Achievements, Mistakes)” (Nizhniy Novgorod, September 25–27, 2014)], chief editor V.M. Baranov. Nizhniy Novgorod, Kirillitsa, 2015, 851 p. (In Russ.). 6. Tolpykin V.E. Sistemnost’ kak metodologicheskii printsip sovremennoi postneklassicheskoi nauki [Systematicity as a Methodological Principle of Modern Post-Non-Classical Science], Teoriia i praktika obshchestvennogo razvitiia [Theory and Practice of Social Development], 2011, No. 7, pp. 27–30. (In Russ.). ( ) 7. Dubrovskii V.Ia. Ocherki po obshchei teorii deiatel’nosti [Essays on the General Theory of Activity]. Moscow, NNF “Institut razvitiia im. G.P. Shchedrovitskogo”, 2011, 576 p. (In Russ.). 8. Sagatovskii V.N. Osnovy sistematizatsii vseobshchikh kategorii [Fundamentals of Systematization of Universal Categories]. Tomsk: Izdatel’stvo Tomskogo universiteta, 1973, 432 p. (In Russ.). 9. Pocheptsov G.G. Strategiia. Instrumentarii po upravleniiu budushchim [Toolkit for Managing the Future]. Moscow, Refl-buk, Kiev, Vakler, 2005, 384 p. (In Russ.). 10. Naumova E.A. Analiz opredelenii strategii i ee mesto v sisteme funktsionirovaniia predpriiatiia. Raznoobrazie vzgliadov i podkhodov k strategii [Analysis of Definitions of Strategy and Its Place in the System of Enterprise Functioning. Diversity of Views and Approaches to Strategy], Vestnik IuUrGU. Seriia: Ekonomika i menedzhment [Bulletin of SUSU. Series: Economics and Management], 2011, No. 8 (225), pp. 111–114. (In Russ.). 11. Kiskin E.V. Strategiia kak komponent pravotvorcheskoi tekhnologii: kommunikativnyi podkhod [Strategy as a Component of Law-Making Technology: a Communicative Approach], Iuridicheskaia tekhnika [Legal Technology], 2015, No. 9, pp. 292–298. (In Russ.). 12. Kiskin E.V. References Printsipy kak komponent pravotvorcheskoi strategii (na primere zakonodatel’stva o vyborakh i referendumakh) [Principles as a Component of Law- Making Strategy (Using the Example of Legislation on Elections and Referendums)], Izbiratel’noe zakonodatel’stvo i praktika [Electoral Legislation and Practice], 2021, No. 4, pp. 11–14. (In Russ.). 12. Kiskin E.V. Printsipy kak komponent pravotvorcheskoi strategii (na primere zakonodatel’stva o vyborakh i referendumakh) [Principles as a Component of Law- Making Strategy (Using the Example of Legislation on Elections and Referendums)], Izbiratel’noe zakonodatel’stvo i praktika [Electoral Legislation and Practice], 2021, No. 4, pp. 11–14. (In Russ.). 13. Zhavoronkova N.G., Shpakovskii Iu.G. Pravovye problemy prostranstvennogo razvitiia Rossiiskoi Federatsii: voprosy strategicheskogo planirovaniia [Legal Problems of Spatial Development of the Russian Federation: Issues of Strategic Planning], Iurist [Lawyer], 2020, No. 5, pp. 56–61. (In Russ.). 13. Zhavoronkova N.G., Shpakovskii Iu.G. Pravovye problemy prostranstvennogo razvitiia Rossiiskoi Federatsii: voprosy strategicheskogo planirovaniia [Legal Problems of Spatial Development of the Russian Federation: Issues of Strategic Planning], Iurist [Lawyer], 2020, No. 5, pp. 56–61. (In Russ.). Федерализм. 2024. Т. 29. ¹ 1 (113) 19 Системный подход к построению правотворческой стратегии 14. Fridman L. Strategiia: voina, revoliutsiia, biznes [Strategy: War, Revolution, Business]. Moscow, Kuchkovo pole, 2018, 767 p. (In Russ.). 15. Kartashov V.N. Printsipy prava: ponyatie, struktury, funktsii [Principles of Law: Concept, Structures, Functions], Yuridicheskaya tekhnika [Legal Technique], 2020, No. 14, pp. 158–166. (In Russ.). 16. Popova O.V. Sotsial’nye aspekty normativno-pravovogo regulirovaniia vakhtovogo truda v severnykh regionakh [Social Aspects of Legal Regulation of Rotational Work in the Northern Regions], Teoriia i praktika obshchestvennogo razvitiia [Theory and Practice of Social Development], 2021, No. 12, pp. 104–109. (In Russ.). 17. Kerimov D.A. Metodologiia prava: Predmet, funktsii, problemy filosofii prava [Methodology of Law: Subject, Functions, Problems of Philosophy of Law]. Moscow, SGA, 2003, 521 p. (In Russ.). , , p ( ) 18. Proekt knigi pervoi Grazhdanskogo ulozheniia s ob”iasnitel’noiu zapiskoiu [Draft Book of the First Civil Code with an Explanatory Note]. Saint Petersburg, Gosudarstvennaia tipografiia, 1893, 263 p. (In Russ.). Дата поступления – 05.02.2024 г. Федерализм. 2024. Т. 29. ¹ 1 (113) SYSTEMATIC APPROACH TO THE CONSTRUCTION OF A LAW-MAKING STRATEGY (USING THE EXAMPLE OF STRATEGIC PLANNING OF SPATIAL DEVELOPMENT IN RUSSIA) Currently in Russia, lawmaking is carried out according to the model of tactical (ad hoc) response to changing situations, i.e. by introducing many local changes to legislation (for example, in 2023, 685 federal and 9 federal constitutional laws were adopted). At the same time, the speed and depth of transformations taking place in the world, as well as the increasing complexity of social relationships, require a transition to a strategic approach, i.e. to the purposeful construction of the future by legal means. The law-making strategy allows us to reduce the variability in the development of legal regulation, so its study is a matter of current interest. The article attempts to study the law-making strategy from the perspective of systemic methodology. This approach allows us to consider the strategy comprehensively, characterize its properties, functions, composition and structure, as well as relationships with the external environment. The theoretical conclusions are illustrated using the example of the formation of a spatial development strategy for Russia. This area of strategic planning is now especially relevant, since it is necessary to respond to new external (sanctions, reorientation of economic relations) and internal (increasing the number of subjects of the Federation, redistribution of logistics flows, the need to ensure technological sovereignty) challenges. In conclusion, the author draws conclusions about the need to institutionalize the law-making strategy and to ensure a systematic approach in its formation. Keywords: strategy, lawmaking, systemic approach, lawmaking strategy, spatial development, spatial development strategy, region. JEL: К10, К19, Е61 Дата поступления – 05.02.2024 г. Federalism. 2024. Vol. 29. N 1 (113) 20 Е.В. КИСКИН КИСКИН Евгений Владимирович заместитель руководителя аппарата Комитета; Аппарат Совета Федерации Федерального Собрания Российской Федерации / ул. Большая Дмитровка, д. 26, г. Москва, 103426. e-mail: evkiskin@yandex.ru KISKIN Evgeny V. Deputy Head of a Committee Staff; Executive Office of the Federation Council of the Federal Assembly of the Russian Federation / 26, Bolshaya Dmitrovka Str., Moscow, 103426. e-mail: evkiskin@yandex.ru Для цитирования: Кискин Е.В. Системный подход к построению правотворческой страте гии (на примере стратегического планирования пространственного раз вития России) // Федерализм. 2024. Т. 29. № 1 (113). С. 5–20. DOI: http:// dx.doi.org/10.21686/2073-1051-2024-1-5-20 Федерализм. 2024. Т. 29. ¹ 1 (113)
https://openalex.org/W3129167872	https://www.frontiersin.org/articles/10.3389/fpls.2020.614837/pdf	English	null	Copper Ions Induce DNA Sequence Variation in Zygotic Embryo Culture-Derived Barley Regenerants	Frontiers in plant science	2,021	cc-by	11,039	Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; ATP, adenosine triphosphate; BAP, 6-benzylaminopurine; BER, base excision repair; DArTseqMet, Diversity Arrays Technology Sequencing Methylation Analysis; DH, doubled haploid; DMV, demethylation; DNMV, de novo methylation; metAFLP, methylation-sensitive Ampliﬁed Fragment Length Polymorphism; MethylRad, DNA methylation proﬁling method; MMR, mismatch repair; MSAP, methylation-sensitive ampliﬁcation polymorphism; NAA, 6-naphthalene acetic acid; NER, nucleotide excision repair; PacBio, Paciﬁc BioScience; ROS, reactive oxygen species; SAM, S-adenosyl-L-methionine; SV, sequence variation; TCIV, tissue culture-induced variation. ORIGINAL RESEARCH published: 04 February 2021 doi: 10.3389/fpls.2020.614837 ORIGINAL RESEARCH published: 04 February 2021 doi: 10.3389/fpls.2020.614837 ORIGINAL RESEARCH published: 04 February 2021 doi: 10.3389/fpls.2020.614837 Copper Ions Induce DNA Sequence Variation in Zygotic Embryo Culture-Derived Barley Regenerants In vitro tissue culture could be exploited to study cellular mechanisms that induce sequence variation. Altering the metal ion composition of tissue culture medium affects biochemical pathways involved in tissue culture-induced variation. Copper ions are involved in the mitochondrial respiratory chain and Yang cycle. Copper ions may participate in oxidative mutations, which may contribute to DNA sequence variation. Silver ions compete with copper ions to bind to the complex IV subunit of the respiratory chain, thus affecting the Yang cycle and DNA methylation. The mechanisms underlying somaclonal variation are unknown. In this study, we evaluated embryo- derived barley regenerants obtained from a single double-haploid plant via embryo culture under varying copper and silver ion concentrations and different durations of in vitro culture. Morphological variation among regenerants and the donor plant was not evaluated. Methylation-sensitive Ampliﬁed Fragment Length Polymorphism analysis of DNA samples showed DNA methylation pattern variation in CG and CHG (H = A, C, or T) sequence contexts. Furthermore, modiﬁcation of in vitro culture conditions explained DNA sequence variation, demethylation, and de novo methylation in the CHG context, as indicated by analysis of variance. Linear regression indicated that DNA sequence variation was related to de novo DNA methylation in the CHG context. Mediation analysis showed the role of copper ions as a mediator of sequence variation in the CHG context. No other contexts showed a signiﬁcant sequence variation in mediation analysis. Silver ions did not act as a mediator between any methylation contexts and sequence variation. Thus, incorporating copper ions in the induction medium should be treated with caution. Keywords: barley, copper, silver, metAFLP, somatic embryogenesis, mediation analysis Edited by: Ruslan Kalendar, University of Helsinki, Finland Edited by: Ruslan Kalendar, University of Helsinki, Finland Edited by: Ruslan Kalendar, University of Helsinki, Finland Reviewed by: Camilo López-Cristoffanini, University of Barcelona, Spain Elena Corredoira, Instituto de Investigaciones Agrobiológicas de Galicia (IIAG), Spain Caterina Catalano, alian National Research Council, Italy Reviewed by: Camilo López-Cristoffanini, University of Barcelona, Spain Elena Corredoira, Instituto de Investigaciones Agrobiológicas de Galicia (IIAG), Spain Caterina Catalano, Italian National Research Council, Italy Correspondence: Renata Orłowska r.orlowska@ihar.edu.pl Specialty section: This article was submitted to Technical Advances in Plant Science, a section of the journal Frontiers in Plant Science Received: 12 October 2020 Accepted: 22 December 2020 Published: 04 February 2021 Citation: Orłowska R, Zimny J and Bednarek PT (2021) Copper Ions Induce DNA Sequence Variation in Zygotic Embryo Culture-Derived Barley Regenerants. Front. Plant Sci. 11:614837. doi: 10.3389/fpls.2020.614837 Specialty section: This article was submitted to Technical Advances in Plant Science, a section of the journal Frontiers in Plant Science Specialty section: This article was submitted to Technical Advances in Plant Science, a section of the journal Frontiers in Plant Science Received: 12 October 2020 Accepted: 22 December 2020 Published: 04 February 2021 INTRODUCTION Costa et al., 2010) and to function as a cofactor of ethylene receptors for ethylene binding (Light et al., 2016). Moreover, silver ions induce sequence variation because of disturbances in DNA methylation processes during barley anther culture (Bednarek and Orłowska, 2020a). Barley (Hordeum vulgare L.) is a prominent grain used for human consumption, animal feeding, brewing, and renewable energy production (Tiidema and Truve, 2004; Schulze, 2007). Because of its importance as a food crop, barley receives much attention with regard to in vitro tissue culture and plant regeneration through somatic embryogenesis (Sharma et al., 2005). The improvement of agronomic traits, such as quality and disease resistance, and the development of gene modiﬁcation techniques require eﬃcient protocols for plant regeneration via in vitro tissue culture. However, the eﬃciency of plant regeneration through somatic embryogenesis varies with the plant genotype (Sharma et al., 2005). In barley, somatic embryogenesis has been performed using various explants, including mature and immature zygotic embryos (Lupotto, 1984; Bregitzer et al., 2000; Tiidema and Truve, 2004; Sharma et al., 2005; Ganeshan et al., 2006). The resultant regenerants showed phenotypic (Ruíz et al., 1992) and genotypic variation (Temel et al., 2014). Furthermore, barley regenerants derived through embryo culture also exhibited genetic and epigenetic variability (Bednarek et al., 2007). Although some pieces of evidence support the role of copper and silver ions in barley anther culture, limited information is available on the eﬀect of these ions on embryo-derived regenerants; an exception is our recent study on barley, where we optimized the culture conditions for maximizing the number of regenerants (Orłowska et al., 2020). It is not evident whether the L-methionine cycle is involved in zygotic embryo culture- induced variation. Since plant regeneration via zygotic embryo culture avoids chromosome doubling, the reestablishment of DNA methylation patterns via DNA replication might be facilitated (Piccolo and Fisher, 2014), limiting (epi)genetic pattern changes. However, the role of epigenetic processes, such as base excision repair (BER) (Drohat and Coey, 2016), nucleotide excision (NER) (Zhang et al., 2019), mismatch repair (MMR) (Marinus, 2012), 5-hydroxymethylcytosine (5- mC) oxidation (Globisch et al., 2010), and other modiﬁcations (Ito et al., 2011), in DNA methylation cannot be excluded (Liu and Lang, 2020). Active DNA demethylation can lead to C →T mutations, and 5-mC oxidation can also contribute to sequence variation (Nabel et al., 2012). INTRODUCTION Tissue culture-induced variation (TCIV) frequently arises during in vitro plant regeneration (Fiuk et al., 2010; Mikula et al., 2011a; Machczy´nska et al., 2014a). The process of plant regeneration requires cell reprogramming (Maki, 2002), which can result in genetic (sequence) and epigenetic (DNA demethylation and de novo methylation) changes (Miyao et al., 2011; Weckx et al., 2019; Orłowska and Bednarek, 2020). DNA methylation changes can further lead to sequence variation because of the activation of mobile elements (Orłowska et al., 2016) or perturbations in DNA repair systems (Wang et al., 2013) under in vitro tissue culture conditions. However, other reasons such as reactive oxygen species (ROS), plant growth regulators, number of subculture for TCIV cannot be excluded (Bairu et al., 2011; Krishna et al., 2016; Bednarek and Orłowska, 2020b). A key player in de novo DNA methylation processes, as well as in the methylation of other cellular compounds (Giovanelli et al., 1985), is S-adenosyl-L-methionine (SAM) (Cantoni, 1952), a product of the Yang cycle (Mäkinen and De, 2019). The Yang cycle is linked to the mitochondrial respiratory chain (Tripodi et al., 2018) via adenosine triphosphate (ATP), which is required for the synthesis of SAM (Markham and Pajares, 2009). Perturbations in the respiratory chain (Pu et al., 2015) aﬀect the L-methionine cycle (Kanyuka and Rudd, 2019). The lack or excess of SAM alkylating agent at the DNA methylation level leads to mobile element activation (Yilmaz et al., 2014) and/or mutations (Nabel et al., 2012). When copper and silver ions are included in tissue culture media, the mitochondrial respiratory chain enzyme, cytochrome c oxidase (Horn and Barrientos, 2008), may cause an imbalance, inﬂuencing the L-methionine cycle and altering relationships between DNA methylation and sequence variation. Additionally, copper and silver ions can induce abiotic stress (Speranza et al., 2013; Nawrot-Chorabik, 2017), leading to TCIV (Krishna et al., 2016). Plant regeneration via in vitro tissue culture involves the use of varying concentrations of diverse ingredients, such as copper (Cu2+) and silver (Ag+) ions (Orłowska and Bednarek, 2020). Copper ions participate in the electron transport chain (Pádua et al., 2010; Tan et al., 2010; Ravet and Pilon, 2013; Printz et al., 2016) and cell wall metabolism (Printz et al., 2016) and act as cofactors that facilitate ethylene receptors to bind to ethylene (Hirayama and Alonso, 2000; Light et al., 2016). Citation: Orłowska R, Zimny J and Bednarek PT (2021) Copper Ions Induce DNA Sequence Variation in Zygotic Embryo Culture-Derived Barley Regenerants. Front. Plant Sci. 11:614837. doi: 10.3389/fpls.2020.614837 February 2021 \| Volume 11 \| Article 614837 1 Frontiers in Plant Science \| www.frontiersin.org Copper-Induced Mutations During Somatic Embryogenesis Orłowska et al. Frontiers in Plant Science \| www.frontiersin.org INTRODUCTION Copper ions also protect cells against oxidative damage (Szöllösi, 2014) and contribute to the production of hydroxyl radicals via the Haber– Weiss cycle (Ravet and Pilon, 2013) and to a wide range of biochemical pathways aﬀecting DNA methylation (Cong et al., 2019; Puchkova et al., 2019). At sublethal concentrations, copper ions inﬂuence the Krebs cycle (Lauer et al., 2012). Changes in DNA methylation patterns have been evaluated using Diversity Arrays Technology Sequencing Methylation Analysis (DArTseqMet) (Pereira et al., 2020), semiquantitative methylation-sensitive ampliﬁcation polymorphism (MSAP) (Bednarek et al., 2017), methylRAD (Wang et al., 2015), methyl- seq (Brunner et al., 2009), PacBio (Rhoads and Au, 2015), and a variant of the MSAP approach (Baranek et al., 2016). Sequence variation and DNA methylation changes aﬀecting distinct DNA sequence contexts (symmetric and asymmetric) could be analyzed using the methylation-sensitive Ampliﬁed Fragment Length Polymorphism (metAFLP) method (Orłowska and Bednarek, 2020). This method takes advantage of the properties of Acc65I and KpnI isoschizomers, which recognize Silver ions aﬀect the number of green regenerants obtained via anther culture (Jha et al., 2007); induce embryogenic callus formation (Vain et al., 1989; Songstad et al., 1991); stimulate shoot induction, shoot formation (Purnhauser et al., 1987), and in vitro rooting (Wu et al., 2006; Jha et al., 2007; Würschum et al., 2015); inhibit callus necrosis (Wu et al., 2006); and aﬀect organogenesis (Paladi et al., 2017). Silver ions can also compete with copper ions to bind to the mitochondrial complex IV (Beyer, 1976; February 2021 \| Volume 11 \| Article 614837 Frontiers in Plant Science \| www.frontiersin.org 2 Copper-Induced Mutations During Somatic Embryogenesis Orłowska et al. the same restriction site but diﬀer in their sensitivity toward the DNA methylation pattern at the restriction site (Bednarek et al., 2007; Mikula et al., 2011b; Coronel et al., 2018). We recently used metAFLP to analyze DNA methylation patterns in barley regenerants derived via anther culture (Bednarek and Orłowska, 2020a,c; Bednarek et al., 2020). induction media throughout the experiment. Each variant of the experiment (M1–M9) had about 235 embryos. Primary explant- derived calli, somatic embryos, and embryo-like structures were observed several days after incubation on induction medium. Calli, embryo-like structures, and somatic embryos were transferred from the induction medium to the K4NB regeneration medium (Kumlehn et al., 2006) supplemented with 0.225 mg l−1 6-benzylaminopurine (BAP) at 21, 28, and 35 days, respectively. DNA Extraction Genomic DNA was extracted from the fresh leaves of 45 regenerants and a donor plant using the Plant DNeasy MiniPrep Kit (Qiagen, Hilden, Germany). DNA purity and concentration were tested using a spectrophotometer at wavelengths of 260 and 280 nm, whereas DNA integrity was veriﬁed by 1.2% agarose gel electrophoresis and ethidium bromide staining. DNA Analysis by Methylation-Sensitive Ampliﬁed Fragment Length Polymorphism The metAFLP analysis was performed as described previously (Bednarek et al., 2007), with minor modiﬁcations (Machczy´nska et al., 2014b). Brieﬂy, each DNA sample was separately digested with Acc65I/MseI and KpnI/MseI following adaptor ligation, pre-selective ampliﬁcation, and selective ampliﬁcation steps. Eight selective primer pairs were used for PCR ampliﬁcation (Supplementary Table S1). The P32-labeled PCR products were separated on polyacrylamide gels, and the gels were exposed to X-ray ﬁlms. The DNA banding patterns were converted into a binary contraposed matrix representing proﬁles of the Acc65I/MseI- and KpnI/MseI-derived platforms. The banding patterns were assigned to DNA sequence and DNA methylation change events and quantiﬁed, delivering information on sequence variation (SV), de novo DNA methylation (DNMV), and DNA demethylation (DMV). Furthermore, the metAFLP characteristics of varying DNA methylation contexts (CHH, CHG, and CG) were calculated (Orłowska and Bednarek, 2020). Twenty-four donor plants were cultivated in a growth chamber under controlled conditions (16-h light/8-h dark photoperiod, 16◦C day/12◦C night temperature, and 190 µE m−2 s−1 light intensity). Spikes with immature caryopses were collected 12–16 days after self-pollination. The caryopses were sterilized by soaking in 70% ethanol for 1 min, followed by stirring in 10% sodium hypochlorite for 20 min. The sterilized caryopses were rinsed several times with sterile water (Makowska et al., 2017) and used for immature zygotic embryo extraction. The extracted immature zygotic embryos were placed in Petri dishes (60 × 15 mm) containing solid N6L medium comprising macro- and micronutrients (Chu, 1978) and supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg l−1 6- naphthalene acetic acid (NAA), and 0.5 mg l−1 kinetin. These embryos were incubated at 26◦C under a 16 h light/8 h dark photoperiod to induce callus and somatic embryo formation. Nine diﬀerent types of induction media (based on N6L), each supplemented with varying concentrations of CuSO4 and AgNO3, were tested (trials M1–M9) (Table 1). Furthermore, diﬀerent durations of incubation on induction medium were tested (i.e., starting from planting the immature embryo on induction medium to when calli, ﬁrst somatic embryos, or embryo-like structures were placed on regeneration medium) (Table 1). In total, 2,150 immature embryos were placed on the INTRODUCTION Calli and embryo-like structures were incubated on regeneration medium at 26◦C, 16 h light/8 h dark photoperiod, and 50 µE m−2 s−1 light intensity. Green regenerated plantlets were transferred to 100 ml glass ﬂasks containing N6I rooting medium (Chu, 1978) supplemented with 2 mg l−1 indole-3- acetic acid (IAA). Subsequently, the regenerants were planted in a soil:sand mixture (3:1) and grown to maturity in a greenhouse under the same day/night regime as that described above for donor plants. Finally, 45 regenerants that originated from a single donor plant and equally representing each trial (ﬁve regenerants per trial) were used for further experiment. One donor plant and all regenerants derived from this donor constituted one set. the same restriction site but diﬀer in their sensitivity toward the DNA methylation pattern at the restriction site (Bednarek et al., 2007; Mikula et al., 2011b; Coronel et al., 2018). We recently used metAFLP to analyze DNA methylation patterns in barley regenerants derived via anther culture (Bednarek and Orłowska, 2020a,c; Bednarek et al., 2020). We assume that copper and possibly silver ions added to the in vitro tissue culture medium inﬂuence the mitochondrial respiratory chain, thus aﬀecting ATP production and inﬂuencing the Yang cycle. As the production of S-adenosyl L-methionine is concerned, the DNA methylation processes may be changed. Furthermore, copper ions participate in the oxidation of methylated cytosines, thus inducing mutations; although this has been veriﬁed in anther culture-derived barley regenerants, no information is available on embryo culture-derived regenerants. Understanding the processes that lead to TCIV in regenerants obtained via anther and embryo tissue culture may reveal putative diﬀerences and similarities between the two approaches. This study aims to assess the inﬂuence of copper and silver ions on the relationship between DNA methylation sequence contexts (CG and CHG, where H = A, C, or T) and sequence variation during the immature zygotic embryo culture of barley. Frontiers in Plant Science \| www.frontiersin.org MATERIALS AND METHODS Tissue Culture and Plant Regeneration Spring barley line NAD2 (Poznan Plant Breeders LTD., Nagradowice, Poland) was used to regenerate double-haploid (DH) plants (Orłowska and Bednarek, 2020) via spontaneous doubling (Segui-Simarro and Nuez, 2008), followed by the evaluation of generative progeny called donor plants (Supplementary Figure S1). Statistical Analysis Analysis of variance (ANOVA) was conducted in XlStat (Addinsoft, 2020). Excel add-in, whereas correlation and linear regression were performed in SPSS v 25 (IBM Corp, 2017). Mediation analysis was carried out in SPSS v 25 (IBM Corp, 2017) using PROCESS MACRO (Hayes, 2018). The Goodman test was Frontiers in Plant Science \| www.frontiersin.org February 2021 \| Volume 11 \| Article 614837 3 Copper-Induced Mutations During Somatic Embryogenesis Orłowska et al. TABLE 1 \| Characteristics of 45 barley regenerants derived via somatic embryogenesis under varying concentrations of copper and silver ions in the induction medium and different tissue culture durations (Time). TABLE 1 \| Characteristics of 45 barley regenerants derived via somatic embryogenesis under varying concentrations of copper and silver ions in the induction medium and different tissue culture durations (Time). Statistical Analysis Components added to the induction medium Length of induction Trial† Quantitative characteristics evaluated by metAFLP (%)§ Cu2+ (µM) Ag+ (µM) Time (days) CG_DMV CHG_DMV CG_DNMV CHG_DNMV SV 0.1 0 21 M1 0.495557 0.480178 0.495557 0.960355 1.919313 0.1 0 21 M1 0.495557 0.479323 0.495557 0.958647 1.918407 0.1 0 21 M1 0.487013 0.479323 0.974026 0.958647 1.917198 0.1 0 21 M1 0.487013 0.480178 0.974026 0.960355 1.918029 0.1 0 21 M1 0.487013 0.479217 0.487013 0.958433 2.395460 0.1 10 28 M2 0.491285 0.479133 0.000000 0.958267 3.354650 0.1 10 28 M2 0.487013 0.479964 0.487013 0.959928 2.396498 0.1 10 28 M2 0.484877 0.479133 0.969754 0.958267 3.353934 0.1 10 28 M2 0.512645 0.479217 0.000000 0.958433 2.398008 0.1 10 28 M2 0.489861 0.480178 0.489861 0.960355 2.398575 0.1 60 35 M3 0.489861 0.479323 0.979722 0.958647 1.918029 0.1 60 35 M3 0.489861 0.479217 0.489861 0.958433 2.876564 0.1 60 35 M3 0.495557 0.479323 0.495557 0.958647 1.918407 0.1 60 35 M3 0.491285 0.480178 0.491285 0.960355 1.918407 0.1 60 35 M3 0.491285 0.479217 0.491285 0.958433 2.396498 5 60 28 M4 0.487013 0.958267 0.487013 0.958267 2.875085 5 60 28 M4 0.504101 0.959928 0.000000 0.959928 1.918407 5 60 28 M4 0.491285 0.958433 0.491285 0.958433 1.917198 5 60 28 M4 0.491285 0.959928 0.491285 0.959928 1.918029 5 60 28 M4 0.487013 0.958433 0.487013 0.958433 1.916368 5 0 35 M5 0.504101 0.959928 0.000000 0.959928 1.918407 5 0 35 M5 0.487013 0.958433 0.974026 0.958433 1.916943 5 0 35 M5 0.491285 0.958267 0.000000 0.958267 2.875414 5 0 35 M5 0.487013 0.958433 0.487013 0.958433 1.916368 5 0 35 M5 0.487013 0.958433 0.487013 0.958433 1.916368 5 10 21 M6 0.484165 0.958433 0.968330 0.958433 1.916176 5 10 21 M6 0.484165 0.958267 0.968330 0.958267 2.395015 5 10 21 M6 0.487013 0.958134 0.487013 0.958134 2.874017 5 10 21 M6 0.487013 0.958134 0.487013 0.958134 2.874017 5 10 21 M6 0.487013 0.95933 0.487013 0.95933 2.875085 10 10 35 M7 0.484165 0.958025 0.968330 0.958025 3.352553 10 10 35 M7 0.484877 0.958025 0.969754 0.958025 3.832442 10 10 35 M7 0.491285 0.958025 0.491285 0.958025 4.790227 10 10 35 M7 0.487013 0.958025 0.487013 0.958025 3.832735 10 10 35 M7 0.504101 0.959113 0.000000 0.959113 3.355512 10 60 21 M8 0.489354 0.964543 0.978709 1.446815 3.371571 10 60 21 M8 0.493647 0.963542 0.493647 1.445313 3.370862 10 60 21 M8 0.489354 0.963542 0.978709 1.445313 3.370540 10 60 21 M8 0.492216 0.963388 0.984432 1.445081 3.852831 10 60 21 M8 0.493647 0.963542 0.493647 1.445313 3.370862 10 0 28 M9 0.492216 0.963542 0.984432 1.445313 3.371571 10 0 28 M9 0.497940 0.963542 0.49794 1.445313 3.371958 10 0 28 M9 0.492216 0.963542 0.984432 1.445313 3.371571 10 0 28 M9 0.489354 0.963542 0.978709 1.445313 3.370540 10 0 28 M9 0.489354 0.963542 0.978709 1.445313 3.370540 Mean 0.490954 0.800030 0.597614 1.066947 2.719493 SD 0.005934 0.22919 0.326757 0.204601 0.746831 †Trials (M1–M9) indicate conditions used to evaluate regenerants; SD, standard deviation. †Trials (M1–M9) indicate conditions used to evaluate regenerants; SD, standard deviation. § metAFLP, methylation-sensitive Ampliﬁed Fragment Length Polymorphism; CG_DMV and CHG_DMV, DNA demethylation of the CG sequence context; CG_DNMV and CHG_DNMV, de novo DNA methylation of the CHG sequence context; SV, sequence variation. †Trials (M1–M9) indicate conditions used to evaluate regenerants; SD, standard deviation. Frontiers in Plant Science \| www.frontiersin.org ( ) g § metAFLP, methylation-sensitive Ampliﬁed Fragment Length Polymorphism; CG_DMV and CHG_DMV, DNA demethylation of the CHG_DNMV, de novo DNA methylation of the CHG sequence context; SV, sequence variation. s used to evaluate regenerants; SD, standard deviation. RESULTS Although many embryos from 24 donor plants were plated, only in the case of one donor plant we succeeded in regeneration of plants that fulﬁlled requirements of our experimental design [a single donor plant and its regenerants representing all experimental conditions (trials M1–M9) and had at least ﬁve regenerants per trial]. All anther culture-derived and acclimatized donor plants were morphologically uniform in terms of leaf shape and color, plant height, spike number, and seed number (Supplementary Figure S2) and exhibited no diﬀerences compared with the source plant materials. In vitro immature zygotic embryo culture yielded as many as 45 regenerants from a single donor plant. Five regenerants were tested in each trail (M1–M9). All regenerated plants were morphologically identical to the donor plant. Regression analysis showed that the model encompassing SV, CHG_DMV, and CHG_DNMV was signiﬁcant [F(2) = 8.675, p = 0.001]. SV could be regressed on CHG_DMV and CHG_DNMV [Fchange(2, 42) = 8.675, p = 0.001] with R2adj = 0.259. Unstandardized and standardized coeﬃcients for CHG_DMV were 0.681 and 0.209, respectively, while those for CHG_DNMV were 1.55 and 0.425, respectively; however, only the last one (CHG_DNMV) was signiﬁcant (t = 3.02, p = 0.004). The Shapiro–Wilk test (0.86, p = 0.000) and Kolmogorov– Smirnov test with Lilliefors signiﬁcance correction (0.2, p < 0.01) violated the normality of the outcome (SV) variable. However, skewness (0.467, SE = 0.354) and kurtosis (−0.531, SE = 0.695) were not signiﬁcantly aﬀected. Visual inspection of the normal quantile–quantile (Q-Q) plot (data not shown) revealed almost normal distribution. Collinearity of the CHG_DNMV and CHG_DMV was not violated [Tolerance = 0.852, Variance inﬂation factor (VIF) = 1.174 for both variables]. Additionally, no outliers were detected based on Cook’s distance (0.2, SD = 0.03). In M1–M9 trials, a total of 335 Acc65I/MseI and 326 KpnI/MseI shared markers were ampliﬁed using eight selective primer pairs. The metAFLP proﬁles (Figure 1) allowed the identiﬁcation of four-digit banding patterns (events), with the prevailing “Z_1111” pattern reﬂecting a lack of change between the donor plant and its regenerants. The next most common events were “Z_1100” and “Z_0011,” which were assigned to the DNA methylation status of the donor and regenerant, respectively. No events corresponding to the “Z_0110” and “Z_1001” patterns (Supplementary Table S2) were detected. Statistical Analysis § metAFLP, methylation-sensitive Ampliﬁed Fragment Length Polymorphism; CG_DMV and CHG_DMV, DNA demethylation of the CG sequence context; CG_DNMV and CHG_DNMV, de novo DNA methylation of the CHG sequence context; SV, sequence variation. gth of induction Trial† Quantitative characteristics evaluated by metAFLP (%)§ Time (days) CG_DMV CHG_DMV CG_DNMV CHG_DNMV SV 21 M1 0.495557 0.480178 0.495557 0.960355 1.919313 21 M1 0.495557 0.479323 0.495557 0.958647 1.918407 21 M1 0.487013 0.479323 0.974026 0.958647 1.917198 21 M1 0.487013 0.480178 0.974026 0.960355 1.918029 21 M1 0.487013 0.479217 0.487013 0.958433 2.395460 28 M2 0.491285 0.479133 0.000000 0.958267 3.354650 28 M2 0.487013 0.479964 0.487013 0.959928 2.396498 28 M2 0.484877 0.479133 0.969754 0.958267 3.353934 28 M2 0.512645 0.479217 0.000000 0.958433 2.398008 28 M2 0.489861 0.480178 0.489861 0.960355 2.398575 35 M3 0.489861 0.479323 0.979722 0.958647 1.918029 35 M3 0.489861 0.479217 0.489861 0.958433 2.876564 35 M3 0.495557 0.479323 0.495557 0.958647 1.918407 35 M3 0.491285 0.480178 0.491285 0.960355 1.918407 35 M3 0.491285 0.479217 0.491285 0.958433 2.396498 28 M4 0.487013 0.958267 0.487013 0.958267 2.875085 28 M4 0.504101 0.959928 0.000000 0.959928 1.918407 28 M4 0.491285 0.958433 0.491285 0.958433 1.917198 28 M4 0.491285 0.959928 0.491285 0.959928 1.918029 28 M4 0.487013 0.958433 0.487013 0.958433 1.916368 35 M5 0.504101 0.959928 0.000000 0.959928 1.918407 35 M5 0.487013 0.958433 0.974026 0.958433 1.916943 35 M5 0.491285 0.958267 0.000000 0.958267 2.875414 35 M5 0.487013 0.958433 0.487013 0.958433 1.916368 35 M5 0.487013 0.958433 0.487013 0.958433 1.916368 21 M6 0.484165 0.958433 0.968330 0.958433 1.916176 21 M6 0.484165 0.958267 0.968330 0.958267 2.395015 21 M6 0.487013 0.958134 0.487013 0.958134 2.874017 21 M6 0.487013 0.958134 0.487013 0.958134 2.874017 21 M6 0.487013 0.95933 0.487013 0.95933 2.875085 35 M7 0.484165 0.958025 0.968330 0.958025 3.352553 35 M7 0.484877 0.958025 0.969754 0.958025 3.832442 35 M7 0.491285 0.958025 0.491285 0.958025 4.790227 35 M7 0.487013 0.958025 0.487013 0.958025 3.832735 35 M7 0.504101 0.959113 0.000000 0.959113 3.355512 21 M8 0.489354 0.964543 0.978709 1.446815 3.371571 21 M8 0.493647 0.963542 0.493647 1.445313 3.370862 21 M8 0.489354 0.963542 0.978709 1.445313 3.370540 21 M8 0.492216 0.963388 0.984432 1.445081 3.852831 21 M8 0.493647 0.963542 0.493647 1.445313 3.370862 28 M9 0.492216 0.963542 0.984432 1.445313 3.371571 28 M9 0.497940 0.963542 0.49794 1.445313 3.371958 28 M9 0.492216 0.963542 0.984432 1.445313 3.371571 28 M9 0.489354 0.963542 0.978709 1.445313 3.370540 28 M9 0.489354 0.963542 0.978709 1.445313 3.370540 Mean 0.490954 0.800030 0.597614 1.066947 2.719493 SD 0.005934 0.22919 0.326757 0.204601 0.746831 ants; SD, standard deviation. RESULTS A signiﬁcant mediation eﬀect of CHG_DMV and CHG_DNMV [independent variables (IDs)] on SV (outcome variable) through copper ions (mediator) (Table 3 and Figure 2) was evaluated based on signiﬁcant indirect eﬀects (IEs) of copper, as indicated by bootstrap values. The IEs were positive for the model with CHG_DMV and CHG_DNMV predictors. The signiﬁcance of mediation was assessed by the Goodman test (CHG_DMV: Z = 6.43, p < 0.001; CHG_DNMV: Z = 3.36, p < 0.001). The variance accounted for (VAF) value, represented as the beta coeﬃcient ratio of the IE to the total eﬀect, and the strength of the mediation were 331.4 (CHG_DMV) and 79.3%, respectively, indicating full and partial mediations (Hair et al., 2011), respectively. Conversion of events into the metAFLP quantitative characteristics related to DNMV, DMV, and SV of the CHH, CHG, and CG sequence contexts showed that all characteristics exhibited some level of variation (Table 1). CHG_DNMV and CHG_DMV prevailed over CG_DMV and CG_DNMV. Moreover, DNA demethylation was lower than de novo methylation in both methylation contexts, and SV was the most abundant type of variation identiﬁed among embryo- derived regenerants. Among all trials, M7–M9 trials with the highest concentration of copper ions in the induction medium showed the highest mean values of SV in regenerants, ranging from 3.37 to 3.83%, whereas in other trials, SV values mostly approximated to 2.0%. No mediation was evaluated for copper ion concentration when CG_DMV and CG_DNMV were used as predictors. Additionally, no mediation eﬀect of silver ions was detected in the relationship between any of the predictors (CG_DMV, CG_DNMV, CHG_DMV, and CHG_DNMV) (data not shown) (Table 3). The results of one-way ANOVA were only signiﬁcant for CHG_DMV [F(8) = 1009815.8, p < 0.0001, R2adj = 1], CHG_DNMV [F(8) = 428448.3, p < 0.0001, R2adj = 1], and SV [F(8) = 14.6, p < 0.0001, R2adj = 0.762] metAFLP characteristics, thus demonstrating mean diﬀerences between trials. Tukey’s test revealed signiﬁcant diﬀerences in CHG_DMV between three groups of trials: group A (M1, M2, M3), group B (M4, M5, M6, M7), and group C (M8, M9). Similarly, CHG_DNMV showed signiﬁcant diﬀerences between two groups of trials: group A (M1, M2, M3, M4, M5, M6, M7) and group B (M8, M9). SV showed Statistical Analysis February 2021 \| Volume 11 \| Article 614837 Frontiers in Plant Science \| www.frontiersin.org 4 Copper-Induced Mutations During Somatic Embryogenesis Orłowska et al. less variability among trials; diﬀerences in SV were observed among four partially overlapping groups of trials: group A(M7, M8, M9), B (M2, M8, M9), C (M2, M6, M9), and D (M1, M2, M3, M4, M5, M9). calculated based on t statistics evaluated for paths a and b of mediation analysis using a calculator for the Sobel test by K. J. Preacher1. Variance accounted for the mediation was expressed as the B coeﬃcient of indirect and total mediation eﬀects. Pearson correlation analysis showed that SV was signiﬁcantly correlated with Cu2+, CHG_DMV, and CHG_DNMV (Table 2). Furthermore, CHG_DMV and CHG_DNMV were correlated with Cu2+ but not with silver ion concentration. CHG_DNMV was correlated with the duration of incubation on the induction medium. Moreover, Cu2+ was correlated with SV. The correlation between CHG_DMV and CHG_DNMV was −0.62, while that between CG_DMV and CG_DNMV was 0.38. 1http://quantpsy.org/sobel/sobel.htm DISCUSSION To determine the quantitative SV and DNA methylation change characteristics of regenerants using the metAFLP approach, obtaining tissue culture explant from a DH genotype after the generative cycle is a prerequisite (Bednarek et al., 2007; February 2021 \| Volume 11 \| Article 614837 Frontiers in Plant Science \| www.frontiersin.org 5 Copper-Induced Mutations During Somatic Embryogenesis Orłowska et al. FIGURE 1 \| An example of the methylation-sensitive Ampliﬁed Fragment Length Polymorphism (metAFLP) proﬁle generated with CXG-AGC and MCAC selective primers. The Acc65I/MseI and KpnI/MseI reﬂected metAFLP platforms. Line 1 represents proﬁles of the donor, lines 2–7 and 8–13 proﬁles of regenerants obtained under M8 and M9 conditions, respectively. FIGURE 1 \| An example of the methylation-sensitive Ampliﬁed Fragment Length Polymorphism (metAFLP) proﬁle generated with CXG-AGC and MCAC selective primers. The Acc65I/MseI and KpnI/MseI reﬂected metAFLP platforms. Line 1 represents proﬁles of the donor, lines 2–7 and 8–13 proﬁles of regenerants obtained under M8 and M9 conditions, respectively. FIGURE 1 \| An example of the methylation-sensitive Ampliﬁed Fragment Length Polymorphism (metAFLP) proﬁle generated with CXG-AGC and MCAC selective primers. The Acc65I/MseI and KpnI/MseI reﬂected metAFLP platforms. Line 1 represents proﬁles of the donor, lines 2–7 and 8–13 proﬁles of regenerants obtained under M8 and M9 conditions, respectively. FIGURE 1 \| An example of the methylation-sensitive Ampliﬁed Fragment Length Polymorphism (metAFLP) proﬁle generated with CXG-AGC and MCAC selective primers. The Acc65I/MseI and KpnI/MseI reﬂected metAFLP platforms. Line 1 represents proﬁles of the donor, lines 2–7 and 8–13 proﬁles of regenerants obtained under M8 and M9 conditions, respectively. TABLE 2 \| Pearson correlation analysis of tissue culture conditions and metAFLP characteristics. Parameters† [Cu2+] [Ag+] Time CG_DMV CHG_DMV CG_DNMV CHG_DNMV SV [Cu2+] 1 0.000 0.000 −0.022 0.866* 0.250* 0.656 0.679 [Ag+] 0.000 1 0.000 0.082 0.002 −0.039 0.136 −0.070 Time 0.000 0.000 1 0.120 −0.003 −0.248 −0.328* 0.015 CG_DMV −0.022 0.082 0.120 1 −0.089 −0.622 0.090 −0.078 CHG_DMV 0.866 0.002 −0.003 −0.089 1 0.097 0.385 0.372 CG_DNMV 0.250* −0.039 −0.248 −0.622 0.097 1 0.393 0.164 CHG_DNMV 0.656** 0.136 −0.328* 0.090 0.385 0.393 1 0.505 SV 0.679 −0.070 0.015 −0.078 0.372 0.164 0.505 1 †[Cu2+] and [Ag+], concentrations of copper and silver ions, respectively, in the induction medium; Time, duration of incubation on the induction medium; CG_DMV and CHG_DMV, DNA demethylation of the CG and CHG sequence contexts; CG_DNMV and CHG_DNMV, de novo DNA methylation of the CG and CHG sequence contexts; SV, sequence variation. DISCUSSION § R2, amount of variance explained by the model; c’, direct effect of predictor (IDs) on the outcome (SV) while controlling for the mediator (Cu2+, Ag+); a, effect of the predictor (ID) on the mediator (Cu2+, Ag+); b, effect of the mediator (Cu2+, Ag+) on the outcome (SV); c, total effect focal predictor (CHG_DMV and CHG_DNMV, CG_DMV and CG_DNMV) on the outcome (SV); IE, indirect effect of the predictor (ID) on the outcome (SV) through the mediator. Asterisks indicate signiﬁcant values (p < 0.05, p < 0.01; *p < 0.001). ‡CI, conﬁdence interval; L and U, lower and upper limits, respectively, of 95% CI. TABLE 3 \| Outcomes of mediation analyses illustrating relationships between CHG_DMV/CHG_DNMV and SV assessing IEs. ( ); g , ( ); , q ( ) § R2, amount of variance explained by the model; c’, direct effect of predictor (IDs) on the outcome (SV) while controlling for the mediator (Cu2+, Ag+); a, effect of the predictor (ID) on the mediator (Cu2+, Ag+); b, effect of the mediator (Cu2+, Ag+) on the outcome (SV); c, total effect focal predictor (CHG_DMV and CHG_DNMV, CG_DMV and CG_DNMV) on the outcome (SV); IE, indirect effect of the predictor (ID) on the outcome (SV) through the mediator. Asterisks indicate signiﬁcant values (p < 0.05, p < 0.01; p < 0.001). ( p < 0.05, p < 0.01; p < 0.001). ‡CI, conﬁdence interval; L and U, lower and upper limits, respectively, of 95% CI. FIGURE 2 \| Simple mediation analysis illustrating the effect of copper ions on the relationship between sequence variation (SV) and DNA demethylation (DMV) and de novo DNA methylation (DNMV) in barley regenerants. (A,B) Relationship between CHG_DMV and SV (A) and between CHG_DNMV and SV (B) mediated by copper ions. p < 0.05, p < 0.01, p < 0.001. This was unexpected since copper nanoparticles (to a lesser extent than in the presence of zinc nanoparticles) have been shown to aﬀect chlorophyll-a in Pistia stratiotes L. (Araceae), leading to morphological variation (Olkhovych et al., 2016). It should be noted that copper and zinc function together to induce oxidative stress (Lee et al., 2002). Thus, the lack of morphological variation among embryo-derived regenerants was probably due to the constant concentration of zinc ions in the induction medium. DISCUSSION Data represent Pearson’s correlation coefﬁcients. Asterisks indicate signiﬁcant correlation (p < 0.05, *p < 0.01; one-tailed Student’s t-test). TABLE 2 \| Pearson correlation analysis of tissue culture conditions and metAFLP characteristics. †[Cu2+] and [Ag+], concentrations of copper and silver ions, respectively, in the induction medium; Time, duration of incubation on CHG_DMV, DNA demethylation of the CG and CHG sequence contexts; CG_DNMV and CHG_DNMV, de novo DNA methylation o SV, sequence variation. Data represent Pearson’s correlation coefﬁcients. Asterisks indicate signiﬁcant correlation (p < 0.05, *p < †[Cu2+] and [Ag+], concentrations of copper and silver ions, respectively, in the induction medium; Time, duration of incubation on the induction medium; CG_DMV and CHG_DMV, DNA demethylation of the CG and CHG sequence contexts; CG_DNMV and CHG_DNMV, de novo DNA methylation of the CG and CHG sequence contexts; SV, sequence variation. Data represent Pearson’s correlation coefﬁcients. Asterisks indicate signiﬁcant correlation (p < 0.05, p < 0.01; one-tailed Student’s t-test). Machczy´nska et al., 2014b; Orłowska and Bednarek, 2020; Orłowska et al., 2020). It is also crucial to have regenerants representing all trials from a single donor plant; otherwise, metAFLP quantitative characteristics might be disturbed by putative variation of donor plants [even if they originated from the same progenitor (Bednarek et al., 2007; Machczy´nska et al., February 2021 \| Volume 11 \| Article 614837 Frontiers in Plant Science \| www.frontiersin.org 6 Copper-Induced Mutations During Somatic Embryogenesis Orłowska et al. TABLE 3 \| Outcomes of mediation analyses illustrating relationships between CHG_DMV/CHG_DNMV and SV assessing IEs. Model† Effects§ 95% CI‡ R2 c’ a b c IE L U CHG_DMV→Cu2+→SV 0.647 −2.808* 15.442* 0.26* 1.214* 4.021 3.01 5.12 CHG_DNMV→Cu2+→SV 0.467 0.382 13.103* 0.112* 1.844* 1.461 0.41 2.11 CG_DMV→Cu2+→SV 0.465 −7.941 −15.221 0.124 −9.826 −1.885 −24.273 29.663 CG_DNMV→Cu2+→ 0.461 −0.013 3.125 0.124 0.376 0.389 −0.066 0.939 CHG_DMV→Ag+→SV 0.144 1.214 0.185 0.002 1.214 −0.0004 −1.723 0.179 CHG_DNMV→Ag+→SV 0.275 1.914** 17.663 −0.004 1.844 −0.07 −0.345 0.164 CG_DMV→Ag+→SV 0.01 −9.166 367.55 −0.002 −9.826 −0.066 −11.234 5.214 CG_DNMV→Ag+→SV 0.03 0.37 −3.167 −0.002 0.376 0.006 −0.084 0.163 †CHG_DMV and CHG_DNMV, DNA demethylation and de novo DNA methylation, respectively, of the CHG sequence context (predictors); Cu2+, copper ion concentration (mediator); Ag+, silver ion concentration (mediator); SV, sequence variation (outcome variable). DISCUSSION However, the absence of morphological variation does not necessarily imply that changes in DNA sequence and DNA methylation pattern related to tissue culture conditions were not induced. The metAFLP analysis revealed changes in SV as well as in the DNA demethylation and de novo methylation of distinct sequence contexts; this is consistent with the results obtained using anther culture-derived barley regenerants (Bednarek and Orłowska, 2020a). It should be stressed that CHH-related variation was not evaluated. This is because methylation of an asymmetric context is much less than that of symmetric contexts (Cokus et al., 2008). A higher level of diﬀerences between trials was caused by the comparison of CHG_DNMV and CHG_DMV with CG_DMV and CG_DNMV, which is surprising since CG methylation is presumably more abundant in plants than CHG methylation (Cokus et al., 2008). The identiﬁcation of increased variation within the CHG context might reﬂect the as-yet-unrecognized aspects of embryogenesis rather than metAFLP approach preferences. Finally, the amount of SV was either equal to or slightly greater than the total DMV and DNMV, which is not fully congruent with our previous studies on barley regenerants (Bednarek et al., 2007), where DNA methylation pattern changes exceeded those related to SV by at least two-fold. This diﬀerence in SV between the two studies could be attributed to varying copper ion concentrations used in the present study. Furthermore, increased SV in trials with the highest copper ion concentrations is consistent with studies on humans, thus documenting the role of copper ions in inducing mutations FIGURE 2 \| Simple mediation analysis illustrating the effect of copper ions on the relationship between sequence variation (SV) and DNA demethylation (DMV) and de novo DNA methylation (DNMV) in barley regenerants. (A,B) Relationship between CHG_DMV and SV (A) and between CHG_DNMV and SV (B) mediated by copper ions. p < 0.05, p < 0.01, **p < 0.001. 2015)] due to the so-called genotype eﬀect (Anu et al., 2004; Flinn et al., 2020). Having this in mind, only one experimental set included regenerants representing all trials with a minimum of ﬁve regenerants per trial. The other sets were underrepresented in diﬀerent trials and made repeated experiments impossible (Supplementary Figure S1). The immature zygotic embryo culture approach allowed the regeneration of a suﬃcient number of plants equally represented in all experimental trials diﬀering in induction medium composition and tissue culture duration. The regenerants were morphologically identical to the donor. DISCUSSION The reason why ANOVA results were insigniﬁcant for CG_DMV and CG_DNMV may be related to embryogenesis as CG methylation could be reestablished based on the DNA methylation pattern of chromosome homologs, whereas methylation of the CHG sequences may partly depend on epigenetic mechanisms (Saze et al., 2012; Niederhuth and Schmitz, 2014; Thiebaut et al., 2019; Liu and Lang, 2020), which are less precise than methylation mechanisms. This is supported by the fact that methylation changes aﬀecting CG contexts were lower than those related to the CHG context. In this study, SV showed a signiﬁcant correlation with CHG_DMV and CHG_DNMV, suggesting that DNA demethylation and de novo DNA methylation may explain DNA sequence variation. Linear regression of SV on CHG_DNMV (but not on CHG_DMV) indicates that de novo methylation of the symmetric CHG sequence context plays a vital role in the generation of SV. The lack of the identiﬁed CG sequence context may be attributed to the reestablishment of methylation patterns during cell division via replication mechanisms and the avoidance of epigenetic modiﬁcations during the process. By contrast, the contribution of CHG context in DNMV may indicate the participation of epigenetic pathways in the reestablishment of methylation patterns. This notion seems to be conﬁrmed by our studies of anther culture-derived barley regenerants (Orłowska and Bednarek, 2020), where the role of both CG and CHG contexts was demonstrated. The necessity of chromosome doubling in the case of anther culture-derived regenerants might explain the diﬀerence in DNA methylation context variation between the two types of plant regeneration methods. The results of this study are congruent with studies on Arabidopsis thaliana, indicating that methylated cytosines could be a source of mutations in the DNA sequence (Kiselev et al., 2019). It should be stressed, however, that linear regression analysis violated the assumption of normal distribution. Since normal distribution is assumed to be a prerequisite for linear regression, the results of this study should be treated with caution. To our surprise, silver ions failed to exhibit any signiﬁcant mediation between any DNA de novo methylation/demethylation and sequence contexts; this is not congruent with the results of anther culture (Orłowska and Bednarek, 2020) possibly because the cell wall of zygotic embryos limits the accessibility of ions in the cell, as suggested by Printz et al. (2016) for copper. This suggests that silver ions are less critical for zygotic embryo culture than for anther culture. DISCUSSION February 2021 \| Volume 11 \| Article 614837 Frontiers in Plant Science \| www.frontiersin.org 7 Copper-Induced Mutations During Somatic Embryogenesis Orłowska et al. aﬀecting ATP production. ATP is needed for the biosynthesis of SAM from methionine by SAM synthetase (Taylor et al., 2009). Furthermore, SAM, the product of the Yang cycle, is required for DNA methylation (Roje, 2006), which may lead to DNA SV in the presence of copper ions (Lee et al., 2002). The role of copper, as a mediator, in inducing SV from CG and CHG contexts has also been reported in barley anther culture (Bednarek and Orłowska, 2020a). Interestingly, however, in anther culture, SV was also related to CG_DMV, whereas in embryo-derived regenerants, only the role of the CHG context was revealed. The observed diﬀerence could be explained by the precise control over de novo methylation and demethylation of the CHG sequence context during embryogenesis compared with androgenesis and possibly the involvement of diﬀerent pathways aﬀecting DNA methylation changes during both processes. Our results are consistent with those of previous studies (Yruela, 2009; Printz et al., 2016), which showed that copper ions play a crucial role in plant cell function and participate in a complex network of interactions that inﬂuence the SV of CHG contexts in plants. Assuming that CHG_DNMV and CHG_DMV explain only a small fraction of SV shared among embryo-derived regenerants, as indicated by ANOVA, factors other than copper ions, such as mobile elements (Smith et al., 2012; Orłowska et al., 2016; Masuta et al., 2017), are involved in SV during plant tissue culture. Evidently, however, a higher concentration of copper ions in the induction medium led to a higher level of SV. This result is in agreement with data demonstrating copper toxicity in plants (Nawrot-Chorabik, 2017). that aﬀect the methylated cytosine residue within CG sequences (Lee et al., 2002). In the current study, the CHG contexts (speciﬁc to plants) were preferentially aﬀected by SV compared with the CG context. The results of ANOVA indicated that changes in CHG_DMV, CHG_DNMV, and SV could be explained by experimental trials. However, ANOVA failed to explain the variance between trials based on changes in CG_DMV and CG_DNMV. Thus, methylation changes aﬀecting CHG contexts might be typical for embryo-derived regenerants. Frontiers in Plant Science \| www.frontiersin.org SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2020. 614837/full#supplementary-material Supplementary Figure S1 \| Experimental design. Starting materials was represented by individual plants of the NAD2 barley line that were subjected to androgenesis (anther culture) to obtain DH regenerants. After a single round of generative cycle the progeny of a single regenerant encompassing twenty-four plants was used as a source of explants (immature zygotic embryos). The immature embryos of the given donor plant were subjected toward nine experimental conditions (M1-M9 trials) and formed a set. Only a single set resulted in regenerants that were regenerated under all trial conditions and was represented by at least ﬁve regenerants. In order to have equal representation of all regenerants in all trials ﬁve regenerants were used for further molecular (metAFLP) procedures. Supplementary Figure S2 \| Donor plants of barley. DISCUSSION Interestingly, the duration of barley zygotic embryo culture was not correlated with Cu2+ and SV. The result is in opposition to that of barley anther culture (Orłowska and Bednarek, 2020). The statistical insigniﬁcance of the duration of culture was probably caused by the presence of homologous chromosomes, which could facilitate the repair of putative SV. However, further investigation is needed to test this possibility. Nonetheless, SV induction during embryogenesis is certainly controlled diﬀerently than that during androgenesis. Alternatively, the diﬀerences in the mediations between anther culture and embryo culture, as shown previously, might be due to the way the two explant types have to switch their fate to an embryogenic one. Both CHG_DMV and CHG_DNMV were correlated with Cu2+, and Cu2+ was correlated with SV. Moreover, CHG_DMV and CHG_DNMV were also correlated with SV, indicating that mediation analysis between CHG_DMV and CHG_DNMV with SV, in the presence of Cu2+ (but not Ag+) as a mediator, is well grounded. Mediation analysis indicated that copper ions act as a mediator between CHG_DMV/_DNMV and SV during embryogenesis. The observed relationship is not surprising because copper inﬂuences the mitochondrial respiratory chain (Garcia et al., 2014), speciﬁcally complex IV (COX), as shown in humans (Borghouts et al., 2002), thus The putative limitation of this study is the sample size (number of regenerants). There is no simple formula that can be used to determine the minimum sample size for mediation analysis (Pan et al., 2018). Bootstrapping approach was suggested as a method of choice for sample sizes ranging from 20 to 80 (Shrout and Bolger, 2002; Preacher and Hayes, 2004). However, in some cases, mediation analysis with bootstrapping may overestimate eﬀects (Koopman et al., 2015). It was demonstrated February 2021 \| Volume 11 \| Article 614837 Frontiers in Plant Science \| www.frontiersin.org 8 Orłowska et al. Copper-Induced Mutations During Somatic Embryogenesis DATA AVAILABILITY STATEMENT however, that under some conditions, mediation analysis can be successfully performed on a sample size as small as 22 (Pan et al., 2018). Thus, our sample size of 45 regenerants fulﬁlls the requirement for mediation analysis. Furthermore, mediation analysis successfully identiﬁed signiﬁcant relationships between CHG_DMV/CHG_DNMV and SV mediated by copper ions. The signiﬁcance of the models was conﬁrmed by the Goodman test and bootstrapping of IEs. Assuming that our data are congruent with the biological role of copper ions in biochemical pathways, we believe that the mediation analysis results presented in this study reﬂect the real biological phenomenon. However, it would be of value to investigate a larger sample size to conﬁrm our results. The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author/s. FUNDING The present study demonstrates that diﬀerent sequence methylation contexts aﬀect SV in somatic embryo-derived regenerants because of the presence of copper ions in the induction medium. Copper ions may mediate SV, aﬀecting the mitochondrial respiratory chain, L-methionine cycle, and mutation-prone CHG DNA contexts. In embryo-derived regenerants, the CHG contexts are responsible for SV, when copper ions act as a mediator. The lack of a mediating eﬀect of silver ions during embryo culture was surprising. We speculate that the cell wall of zygotic embryos limits the accessibility of silver ions in the cell, and thus silver ions do not compete for cellular complexes involved in the respiratory chain. Zygotic embryo cultures may diﬀer in TCIV at the DNA sequence and methylation levels, suggesting that slightly diﬀerent mechanisms or pathways are involved during plant regeneration via anther and embryo culture. Studies dedicated to evaluating models that describe relationships between biochemical pathways and TCIV aim to develop practical tools for in vitro tissue culture manipulations, thus enhancing the understanding of and allowing control over parameters aﬀecting diﬀerent aspects of SV. Such models would be of value from both scientiﬁc and practical points of view. This research was funded by the Ministry of Agriculture and Rural Development, Poland (grant No. HORhn-801- PB-22/15-18). AUTHOR CONTRIBUTIONS RO and PB conceived the project, designed the research, analyzed the data, and wrote the ﬁrst draft of the manuscript. RO performed the experiments. RO and JZ prepared plant material. RO, PB, and JZ revised and ﬁnalized the manuscript. All authors contributed to the article and approved the submitted version. REFERENCES doi: 10.1007/ s00299-006-0252-0 Bregitzer, P., Campbell, R. D., Dahleen, L. S., Lemaux, P. G., and Cho, M.-J. (2000). Development of transformation systems for elite barley cultivars. Barley Genet. Newsl. 30, 10–12. Kanyuka, K., and Rudd, J. J. (2019). Cell surface immune receptors: the guardians of the plant’s extracellular spaces. Curr. Opin. Plant Biol. 50, 1–8. doi: 10.1016/ j.pbi.2019.02.005 Brunner, A. L., Johnson, D. S., Kim, S. W., Valouev, A., Reddy, T. E., Neﬀ, N. F., et al. (2009). Distinct DNA methylation patterns characterize diﬀerentiated human embryonic stem cells and developing human fetal liver. Genome Res. 19, 1044–1056. doi: 10.1101/gr.088773.108 Kiselev, K. V., Ogneva, Z. V., Dubrovina, A. S., Nityagovsky, N. N., and Suprun, A. R. (2019). Somatic mutations, DNA methylation, and expression of DNA repair genes in Arabidopsis thaliana treated with 5-azacytidine. Biol. Plant. 63, 398–404. Koopman, J., Howe, M., Hollenbeck, J. R., and Sin, H.-P. (2015). Small sample mediation testing: misplaced conﬁdence in bootstrapped conﬁdence intervals. J. Appl. Psychol. 100, 194–202. doi: 10.1037/a0036635 Cantoni, G. (1952). The nature of the active methyl donor formed enzymatically from l-methionine and adenosinetriphosphate1, 2. J. Am. Chem. Soc. 74, 2942– 2943. doi: 10.1021/ja01131a519 pp y Krishna, H., Alizadeh, M., Singh, D., Singh, U., Chauhan, N., Eftekhari, M., et al. Chu, C. C. (1978). “The N6 medium and its applications to anther culture of cereal crops,” in Proc. Symp. Plant Tissue Cul., ed H. Hu (Peking: Science Press), 45–50. Krishna, H., Alizadeh, M., Singh, D., Singh, U., Chauhan, N., Eftekhari, M., et al. (2016). Somaclonal variations and their applications in horticultural crops improvement. 3 Biotech 6:54. (2016). Somaclonal variations and their applications in horticultural crops improvement. 3 Biotech 6:54. Cokus, S. J., Feng, S., Zhang, X., Chen, Z., Merriman, B., Haudenschild, C. D., et al. (2008). Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219. doi: 10.1038/nature06745 Kumlehn, J., Serazetdinova, L., Hensel, G., Becker, D., and Loerz, H. (2006). Genetic transformation of barley (Hordeum vulgare L.). via infection of androgenetic pollen cultures with Agrobacterium tumefaciens. Plant Biotechnol. J. 4, 251–261. doi: 10.1111/j.1467-7652.2005.00178.x Cong, W., Miao, Y., Xu, L., Zhang, Y., Yuan, C., Wang, J., et al. (2019). Transgenerational memory of gene expression changes induced by heavy metal stress in rice (Oryza sativa L.). BMC Plant Biol. 19:282. Lauer, M. M., de Oliveira, C. B., Yano, N. L. I., and Bianchini, A. (2012). REFERENCES Bednarek, P. T., and Orłowska, R. (2020b). Plant tissue culture environment as a switch-key of (epi)genetic changes. Plant Cell Tissue Organ Cult. 140, 245–257. doi: 10.1007/s11240-019-01724-1 Addinsoft (2020). XLSTAT Statistical and Data Analysis Solution. Available online at: https://www.xlstat.com Bednarek, P. T., and Orłowska, R. (2020c). Time of in vitro anther culture may moderate action of copper and silver ions that aﬀect the relationship between DNA methylation change and the yield of barley green regenerants. Plants 9:1064. doi: 10.3390/plants9091064 Anu, A., Babu, K. N., and Peter, K. V. (2004). Variations among somaclones and its seedling progeny in Capsicum annuum. Plant Cell Tissue Organ Cult. 76, 261–267. doi: 10.1023/b:ticu.0000009246.24216.ea Bairu, M. W., Aremu, A. O., and Van Staden, J. (2011). Somaclonal variation in plants: causes and detection methods. Plant Growth Regul. 63, 147–173. doi: 10.1007/s10725-010-9554-x Bednarek, P. T., Orłowska, R., Koebner, R. M. D., and Zimny, J. (2007). Quantiﬁcation of the tissue-culture induced variation in barley (Hordeum vulgare L.). BMC Plant Biol. 7:10. Baranek, M., ˇCechová, J., Kovacs, T., Eichmeier, A., Wang, S., Raddová, J., et al. (2016). Use of combined MSAP and NGS techniques to identify diﬀerentially methylated regions in somaclones: a case study of two stable somatic wheat mutants. PLoS One 11:e0165749. doi: 10.1371/journal.pone.0165749 Bednarek, P. T., Orłowska, R., and Niedziela, A. (2017). A relative quantitative methylation-sensitive ampliﬁed polymorphism (MSAP). method for the analysis of abiotic stress. BMC Plant Biol. 17:79. Bednarek, P. T., Zebrowski, J., and Orłowska, R. (2020). Exploring the biochemical origin of DNA sequence variation in barley plants regenerated via in vitro anther culture. Int. J. Mol. Sci. 21:5770. doi: 10.3390/ijms21165770 Bednarek, P. T., and Orłowska, R. (2020a). CG demethylation leads to sequence mutations in an anther culture of barley due to the presence of Cu, Ag ions in the medium and culture time. Int. J. Mol. Sci. 21:4401. doi: 10.3390/ ijms21124401 Beyer, E. M. (1976). A potent inhibitor of ethylene action in plants. Plant Physiol. 58, 268–271. doi: 10.1104/pp.58.3.268 February 2021 \| Volume 11 \| Article 614837 Frontiers in Plant Science \| www.frontiersin.org 9 Copper-Induced Mutations During Somatic Embryogenesis Orłowska et al. Borghouts, C., Scheckhuber, C. Q., Werner, A., and Osiewacz, H. D. (2002). Respiration, copper availability and SOD activity in P. anserina strains with diﬀerent lifespan. Biogerontology 3, 143–153. Jha, A. K., Dahleen, L. S., and Suttle, J. C. (2007). Ethylene inﬂuences green plant regeneration from barley callus. Plant Cell Rep. 26, 285–290. REFERENCES Copper eﬀects on key metabolic enzymes and mitochondrial membrane potential in gills of the estuarine crab Neohelice granulata at diﬀerent salinities. Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 156, 140–147. doi: 10.1016/j.cbpc. 2012.08.001 Coronel, C. J., González, A. I., Ruiz, M. L., and Polanco, C. (2018). Analysis of somaclonal variation in transgenic and regenerated plants of Arabidopsis thaliana using methylation related metAFLP and TMD markers. Plant Cell Rep. 37, 137–152. doi: 10.1007/s00299-017-2217-x Lee, D. H., O’Connor, T. R., and Pfeifer, G. P. (2002). Oxidative DNA damage induced by copper and hydrogen peroxide promotes CG–>TT tandem mutations at methylated CpG dinucleotides in nucleotide excision repair- deﬁcient cells. Nucleic Acids Res. 30, 3566–3573. doi: 10.1093/nar/gkf478 Costa, C. S., Ronconi, J. V. V., Daufenbach, J. F., Gonçalves, C. L., Rezin, G. T., Streck, E. L., et al. (2010). In vitro eﬀects of silver nanoparticles on the mitochondrial respiratory chain. Mol. Cell. Biochem. 342, 51–56. doi: 10.1007/ s11010-010-0467-9 Drohat, A. C., and Coey, C. T. (2016). Role of base excision "repair" enzymes in erasing epigenetic marks from DNA. Chem. Rev. 116, 12711–12729. doi: 10.1021/acs.chemrev.6b00191 Light, K. M., Wisniewski, J. A., Vinyard, W. A., and Kieber-Emmons, M. T. (2016). Perception of the plant hormone ethylene: known-knowns and known- unknowns. J. Biol. Inorg. Chem. 21, 715–728. Fiuk, A., Bednarek, P. T., and Rybczy´nski, J. J. (2010). Flow cytometry, HPLC-RP, and metAFLP analyses to assess genetic variability in somatic embryo-derived plantlets of Gentiana pannonica scop. Plant Mol. Biol. Rep. 28, 413–420. doi: 10.1007/s11105-009-0167-3 Liu, R., and Lang, Z. (2020). The mechanism and function of active DNA demethylation in plants. J. Integr. Plant Biol. 62, 148–159. doi: 10.1111/jipb. 12879 Lupotto, E. (1984). Callus induction and plant regeneration from barley mature embryos. Ann. Bot. 54, 523–530. doi: 10.1093/oxfordjournals.aob.a086822 Flinn, B., Dale, S., Disharoon, A., and Kresovich, S. (2020). Comparative analysis of in vitro responses and regeneration between diverse bioenergy sorghum genotypes. Plants 9:248. doi: 10.3390/plants9020248 Machczy´nska, J., Orłowska, R., Ma´nkowski, D. R., Zimny, J., and Bednarek, P. T. (2014a). DNA methylation changes in triticale due to in vitro culture plant regeneration and consecutive reproduction. Plant Cell Tiss Organ Cult. 119, 289–299. doi: 10.1007/s11240-014-0533-1 Ganeshan, S., Chodaparambil, S. V., Båga, M., Fowler, D. B., Hucl, P., Rossnagel, B. G., et al. (2006). In vitro regeneration of cereals based on multiple shoot induction from mature embryos in response to thidiazuron. Plant Cell Tissue Organ Cult. 85, 63–73. REFERENCES doi: 10.1007/s11240-005-9049-z Machczy´nska, J., Orłowska, R., Zimny, J., and Bednarek, P. T. (2014b). Extended metAFLP approach in studies of the tissue culture induced variation (TCIV). triticale. Mol. Breed. 34, 845–854. doi: 10.1007/s11032-014-0079-2 Garcia, L., Welchen, E., and Gonzalez, D. H. (2014). Mitochondria and copper homeostasis in plants. Mitochondrion 19, 269–274. doi: 10.1016/j.mito.2014. 02.011 Machczy´nska, J., Zimny, J., and Bednarek, P. (2015). Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927). regenerants. Plant Mol. Biol. 89, 279–292. doi: 10.1007/s11103-015- 0368-0 Giovanelli, J., Mudd, S. H., and Datko, A. H. (1985). Quantitative analysis of pathways of methionine metabolism and their regulation in lemna. Plant Physiol. 78, 555–560. doi: 10.1104/pp.78.3.555 Maki, H. (2002). Origins of spontaneous mutations: speciﬁcity and directionality of base-substitution, frameshift, and sequence-substitution mutageneses. Ann. Rev. Genet. 36, 279–303. doi: 10.1146/annurev.genet.36.042602.094806 Globisch, D., Münzel, M., Müller, M., Michalakis, S., Wagner, M., Koch, S., et al. (2010). Tissue distribution of 5-Hydroxymethylcytosine and search for active demethylation intermediates. PLoS One 5:e15367. doi: 10.1371/journal.pone. 0015367 Mäkinen, K., and De, S. (2019). The signiﬁcance of methionine cycle enzymes in plant virus infections. Curr. Opin. Plant Biol. 50, 67–75. doi: 10.1016/j.pbi.2019. 03.002 Hair, J. F., Ringle, C. M., and Sarstedt, M. (2011). PLS-SEM: indeed a silver bullet. J. Mark. Theory Pract. 19, 139–152. doi: 10.2753/mtp1069-6679190202 Makowska, K., Oleszczuk, S., and Zimny, J. (2017). The eﬀect of copper on plant regeneration in barley microspore culture Czech. J. Genet. Plant Breed. 53, 17–22. doi: 10.17221/82/2016-cjgpb Hayes, A. F. (2018). Introduction to Mediation, Moderation, and Conditional Process Analysis. a Regression Bases Approach. New York, NY: Guilford Press. Marinus, M. G. (2012). DNA mismatch repair. EcoSal Plus 5:10.1128/ecosallus.7.2.5. Hirayama, T., and Alonso, J. M. (2000). Ethylene captures a metal! metal ions are involved in ethylene perception and signal transduction. Plant Cell Physiol. 41, 548–555. doi: 10.1093/pcp/41.5.548 Markham, G. D., and Pajares, M. A. (2009). Structure-function relationships in methionine adenosyltransferases. Cell. Mol. Life Sci. 66, 636–648. doi: 10.1007/ s00018-008-8516-1 Horn, D., and Barrientos, A. (2008). Mitochondrial copper metabolism and delivery to cytochrome c oxidase. IUBMB Life 60, 421–429. doi: 10.1002/iub.50 IBM Corp (2017). IBM SPSS Statistics for Windows 25. Armonk, NY: IBM Corp. Masuta, Y., Nozawa, K., Takagi, H., Yaegashi, H., Tanaka, K., Ito, T., et al. (2017). Inducible transposition of a heat-activated retrotransposon in tissue culture. Plant Cell Physiol. 58, 375–384. Ito, S., Shen, L., Dai, Q., Wu, S. C., Collins, L. REFERENCES Plant 7, 472–480. doi: 10.1093/mp/ sst165 Olkhovych, O., Volkogon, M., Taran, N., Batsmanova, L., and Kravchenko, I. (2016). The eﬀect of copper and zinc nanoparticles on the growth parameters, contents of ascorbic acid, and qualitative composition of amino acids and acylcarnitines in Pistia stratiotes L. (Araceae). Nanoscale Res. Lett. 11:218. Shrout, P. E., and Bolger, N. (2002). Mediation in experimental and nonexperimental studies: new procedures and recommendations. Psychol. Methods 7, 422–445. doi: 10.1037/1082-989x.7.4.422 Smith, A. M., Hansey, C. N., and Kaeppler, S. M. (2012). TCUP:a novel hAT transposon active in maize tissue culture. Front. Plant Sci. 3:6. Orłowska, R., and Bednarek, P. T. (2020). Precise evaluation of tissue culture- induced variation during optimisation of in vitro regeneration regime in barley. Plant Mol. Biol. 103, 33–50. doi: 10.1007/s11103-020-00973-5 Songstad, D. D., Armstrong, C. L., and Petersen, W. L. (1991). AgNO3 increases type II callus production from immature embryos of maize inbred B73 and its derivatives. Plant Cell Rep. 9, 699–702. doi: 10.1007/bf00235361 Orłowska, R., Machczy´nska, J., Oleszczuk, S., Zimny, J., and Bednarek, P. T. (2016). DNA methylation changes and TE activity induced in tissue cultures of barley (Hordeum vulgare L.). J. Biol. Res. 23:19. Speranza, A., Crinelli, R., Scoccianti, V., Taddei, A. R., Iacobucci, M., Bhattacharya, P., et al. (2013). In vitro toxicity of silver nanoparticles to kiwifruit pollen exhibits peculiar traits beyond the cause of silver ion release. Environ. Pollut. 179, 258–267. doi: 10.1016/j.envpol.2013.04.021 Orłowska, R., Pachota, K. A., Machczy´nska, J., Niedziela, A., Makowska, K., Zimny, J., et al. (2020). Improvement of anther cultures conditions using the taguchi method in three cereal crops. Electron. J. Biotechnol. 43, 8–15. doi: 10.1016/j. ejbt.2019.11.001 Szöllösi, R. (2014). “Chapter 3 - superoxide dismutase (SOD). and abiotic stress tolerance in plants: an overview,” in Oxidative Damage to Plants, ed. P. Ahmad (San Diego: Academic Press), 89–129. doi: 10.1016/b978-0-12-799963- 0.00003-4 Pádua, M., Cavaco, A. M., Aubert, S., Bligny, R., and Casimiro, A. (2010). Eﬀects of copper on the photosynthesis of intact chloroplasts: interaction with manganese. Physiol. Plant. 138, 301–311. doi: 10.1111/j.1399-3054.2009. 01335.x Tan, Y.-F., O’Toole, N., Taylor, N. L., and Millar, A. H. (2010). Divalent metal ions in plant mitochondria and their role in interactions with proteins and oxidative stress-induced damage to respiratory function. Plant Physiol. 152, 747–761. doi: 10.1104/pp.109.147942 Paladi, R. K., Rai, A. N., and Penna, S. (2017). Silver nitrate modulates organogenesis in Brassica juncea (L.). through diﬀerential antioxidant defense and hormonal gene expression. REFERENCES Sci. Horticult. 226, 261–267. doi: 10.1016/j. scienta.2017.08.038 Taylor, J. C., Bock, C. W., Takusagawa, F., and Markham, G. D. (2009). Discovery of novel types of inhibitors of S-adenosylmethionine synthesis by virtual screening. J. Med. Chem. 52, 5967–5973. doi: 10.1021/jm9006142 Pan, H., Liu, S., Miao, D., and Yuan, Y. (2018). Sample size determination for mediation analysis of longitudinal data. BMC Med. Res. Methodol. 18:32. Temel, A., Kartal, G., and Gozukirmizi, N. (2014). Genetic and Epigenetic Variations in Barley Calli Cultures. Biotechnol. Biotechnol. Equip. 22, 911–914. doi: 10.1080/13102818.2008.10817577 Pereira, W. J., Pappas, M. D. C. R., Grattapaglia, D., and Pappas, G. J. (2020). A cost-eﬀective approach to DNA methylation detection by methyl sensitive DArT sequencing. PLoS One 15:e0233800. doi: 10.1371/journal.pone.0233800 Piccolo, F. M., and Fisher, A. G. (2014). Getting rid of DNA methylation. Trends Cell Biol. 24, 136–143. doi: 10.1016/j.tcb.2013.09.001 Pereira, W. J., Pappas, M. D. C. R., Grattapaglia, D., and Pappas, G. J. (2020). A cost-eﬀective approach to DNA methylation detection by methyl sensitive DArT sequencing. PLoS One 15:e0233800. doi: 10.1371/journal.pone.0233800 Thiebaut, F., Hemerly, A. S., and Ferreira, P. C. G. (2019). A role for epigenetic regulation in the adaptation and stress responses of non-model plants. Front. Plant Sci. 10:246. q g j p Piccolo, F. M., and Fisher, A. G. (2014). Getting rid of DNA methylation. Trends Cell Biol. 24, 136–143. doi: 10.1016/j.tcb.2013.09.001 Tiidema, A., and Truve E. (2004). Eﬃcient regeneration of fertile barley plants from callus cultures of several Nordic cultivars. Hereditas 140, 171–176. doi: 10.1111/j.1601-5223.2004.01757.x Preacher, K. J., and Hayes, A. F. (2004). SPSS and SAS procedures for estimating indirect eﬀects in simple mediation models. Behav. Res. Methods Instrum. Comput. 36, 717–731. doi: 10.3758/bf03206553 Printz, B., Lutts, S., Hausman, J.-F., and Sergeant, K. (2016). Copper traﬃcking in plants and its implication on cell wall dynamics. Front. Plant Sci. 7:601. Tripodi, F., Castoldi, A., Nicastro, R., Reghellin, V., Lombardi, L., Airoldi, C., et al. (2018). Methionine supplementation stimulates mitochondrial respiration. Biochimica et Biophysica Acta (BBA) Mol. Cell Res. 1865, 1901–1913. doi: 10.1016/j.bbamcr.2018.09.007 Pu, X., Lv, X., Tan, T., Fu, F., Qin, G., and Lin, H. (2015). Roles of mitochondrial energy dissipation systems in plant development and acclimation to stress. Ann. Bot. 116, 583–600. doi: 10.1093/aob/mcv063 Vain, P., Yean, H., and Flament, P. (1989). Enhancement of production and regeneration of embryogenic type II callus in zea mays L. by AgNO3. Plant Cell Tissue Organ Cult. 18, 143–151. REFERENCES B., Swenberg, J. A., et al. (2011). Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303. doi: 10.1126/science.1210597 Mikula, A., Tomiczak, K., and Rybczynski, J. J. (2011a). Cryopreservation enhances embryogenic capacity of Gentiana cruciata (L.). suspension culture and February 2021 \| Volume 11 \| Article 614837 Frontiers in Plant Science \| www.frontiersin.org 10 Copper-Induced Mutations During Somatic Embryogenesis Orłowska et al. maintains (epi)genetic uniformity of regenerants. Plant Cell Rep. 30, 565–574. doi: 10.1007/s00299-010-0970-1 maintains (epi)genetic uniformity of regenerants. Plant Cell Rep. 30, 565–574. doi: 10.1007/s00299-010-0970-1 Roje, S. (2006). S-Adenosyl-L-methionine: beyond the universal methyl group donor. Phytochemistry 67, 1686–1698. doi: 10.1016/j.phytochem.2006.04.019 Mikula, A., Tomiczak, K., Wójcik, A., and Rybczynski, J. J. (2011b). ). Encapsulation-Dehydration Method Elevates Embryogenic Abilities of Gentiana Kurroo Cell Suspension and Carrying on Genetic stability of its Regenerants after Cryopreservation, Vol. 908. Leuven: International Society for Horticultural Science. Ruíz, M. L., Rueda, J., Peláez, M. I., Espino, F. J., Candela, M., Sendino, A. M. et al. (1992). Somatic embryogenesis, plant regeneration and somaclonal variation in barley. Plant Cell Tissue and Organ Cul. 28, 97–101. doi: 10.1007/ BF00039921 Saze, H., Tsugane, K., Kanno, T., and Nishimura, T. (2012). DNA methylation in plants: relationship to small RNAs and histone modiﬁcations, and functions in transposon inactivation. Plant Cell Physiol. 53, 766–784. doi: 10.1093/pcp/ pcs008 Miyao, A., Nakagome, M., Ohnuma, T., Yamagata, H., Kanamori, H., Katayose, Y., et al. (2011). Molecular spectrum of somaclonal variation in regenerated rice revealed by whole-genome sequencing. Plant Cell Physiol. 53, 256–264. doi: 10.1093/pcp/pcr172 Schulze, J. (2007). Improvements in cereal tissue culture by thidiazuron: a review fruit. Vegetable and Cereal Sci. Biotechnol. 1, 64–79. Nabel, C. S., Manning, S. A., and Kohli, R. M. (2012). The curious chemical biology of cytosine: deamination, methylation,and oxidation as modulators of genomic potential. ACS Chem. Biol. 7, 20–30. doi: 10.1021/cb2002895 Segui-Simarro, J. M., and Nuez, F. (2008). Pathways to doubled haploidy: chromosome doubling during androgenesis Cytogenet. Genome Res. 120, 358– 369. doi: 10.1159/000121085 Nawrot-Chorabik, K. (2017). Response of the callus cells of ﬁr (Abies nordmanniana). Vitro Heavy Metal Stress 59:25. doi: 10.1515/ﬀp-2017-0003 Sharma, V. K., Hansch, R., Mendel, R. R., and Schulze, J. (2005). Mature embryo axis-based high frequency somatic embryogenesis and plant regeneration from multiple cultivars of barley (Hordeum vulgare L.) J. Exp. Bot. 56, 1913–1922. doi: 10.1093/jxb/eri186 Niederhuth, C. E., and Schmitz, R. J. (2014). Covering your bases: inheritance of DNA methylation in plant genomes. Mol. REFERENCES doi: 10.1007/bf00047740 Puchkova, L. V., Broggini, M., Polishchuk, E. V., Ilyechova, E. Y., and Polishchuk, R. S. (2019). Silver ions as a tool for understanding diﬀerent aspects of copper metabolism. Nutrients 11:1364. doi: 10.3390/nu11061364 Wang, S., Lv, J., Zhang, L., Dou, J., Sun, Y., Li, X., et al. (2015). MethylRAD: a simple and scalable method for genome-wide DNA methylation proﬁling using methylation-dependent restriction enzymes. Open Biol. 5:150130. doi: 10.1098/rsob.150130 Purnhauser, L., Medgyesy, P., Czakó, M., Dix, P. J., and Márton, L. (1987). Stimulation of shoot regeneration in Triticum aestivum and Nicotiana plumbaginifolia Viv. tissue cultures using the ethylene inhibitor AgNO3. Plant Cell Rep. 6, 1–4. doi: 10.1007/bf00269725 Wang, X., Wu, R., and Liu, B. (2013). Tissue culture-induced genetic and epigenetic alterations in rice pure-lines, F1 hybrids and polyploids. BMC Plant Biol. 13:77. doi: 10.1186/1471-2229-13-77 Ravet, K., and Pilon, M. (2013). Copper and iron homeostasis in plants: the challenges of oxidative stress. Antioxid. Redox Signal. 19, 919–932. doi: 10.1089/ ars.2012.5084 Weckx, S., Inzé, D., and Maene, L. (2019). Tissue culture of oil palm: ﬁnding the balance between mass propagation and somaclonal variation. Front. Plant Sci. 10:722. Rhoads, A., and Au, K. F. (2015). PacBio sequencing and its applications. Genomics Proteomics Bioinform. 13, 278–289. doi: 10.1016/j.gpb.2015.08.002 February 2021 \| Volume 11 \| Article 614837 Frontiers in Plant Science \| www.frontiersin.org 11 Copper-Induced Mutations During Somatic Embryogenesis Orłowska et al. China: eﬀects of DNA repair genes polymorphisms on genetic damage. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 839, 13–20. doi: 10.1016/j.mrgentox. 2019.01.006 China: eﬀects of DNA repair genes polymorphisms on genetic damage. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 839, 13–20. doi: 10.1016/j.mrgentox. 2019.01.006 Wu, L. M., Wei, Y. M., and Zheng, Y. L. (2006). Eﬀects of silver nitrate on the tissue culture of immature wheat embryos. Russ. J. Plant Physiol. 53, 530–534. doi: 10.1134/s1021443706040157 Würschum, T., Tucker, M. R., Maurer, H. P., and Leiser, W. L. (2015). Ethylene inhibitors improve eﬃciency of microspore embryogenesis in hexaploid triticale. Plant Cell Tissue Organ Cult. 122, 751–757. doi: 10.1007/s11240-015- 0808-1 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Yilmaz, S., Marakli, S., and Gozukirmizi, N. (2014). BAGY2 retrotransposon analyses in barley calli cultures and regenerated plantlets. Biochem. Genet. 52, 233–244. doi: 10.1007/s10528-014-9643-z Copyright © 2021 Orłowska, Zimny and Bednarek. Frontiers in Plant Science \| www.frontiersin.org REFERENCES This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Yruela, I. (2009). Copper in plants: acquisition, transport and interactions. Funct. Plant Biol. 36, 409–430. doi: 10.1071/fp08288 Zhang, G.-H., Ren, J. C., Luo, M., Cui, J., Du, Y., and Yang, D. (2019). Association of BER and NER pathway polymorphism haplotypes and micronucleus frequencies with global DNA methylation in benzene-exposed workers of February 2021 \| Volume 11 \| Article 614837 Frontiers in Plant Science \| www.frontiersin.org 12
https://openalex.org/W2807097399	https://zenodo.org/records/3583593/files/235%20Analysis%20on%20The%20Impact%20of%20Reflectance%20in%20Optical%20Fiber%20Links.pdf	English	null	Analysis on The Impact of Reflectance in Optical Fiber Links	International journal of trend in scientific research and development	2,018	cc-by	4,518	ABSTRACT One of the major considerations with all types of fiber connection is the optical loss encountered The two main function of the transmitter are [2]: it serves as a light source launched into the optical fiber cable and to modulate the light by the binary data it receives from the source. The receiver has two functions: it detects the light coupled optical fiber cable and converts the light into electrical signal, and demodulates the light to determine the identity of the binary data that it represents. The connectors are used to attach the optical fiber and provide solid contact between receiver. The connector must align the optical fiber end precisely with the light source or receiver to prevent signal loss An optical fiber strand of pure glass used as the transmission medium for the signal [3]. It comprises of two concentric layers called the core and the cladding with different refractive indexes as shown in Figure 2 [4]. The index of refraction is a way of measuring the light in a material. Fiber could be multi short-haul, limited bandwidth and relatively low applications) or single-mode (suitable for high bandwidth and medium- and long One of the major considerati fiber–fiber connection is the optical loss encountered at the interface. The two main function of the transmitter are [2]: it serves as a light source launched into the optical fiber cable and to modulate the light by the binary data it receives from the source. The receiver has two functions: it detects the light coupled out of the optical fiber cable and converts the light into electrical signal, and demodulates the light to determine the identity of the binary data that it represents. The connectors are used to attach the optical fiber and provide solid contact between mated transmitter and receiver. The connector must align the optical fiber end precisely with the light source or receiver to An optical fiber is a long thin strand of pure glass used as the transmission medium for the signal [3]. It comprises of two concentric layers called the core and the cladding with different refractive indexes as shown in Figure 2 [4]. The index measuring the light in a material. Fiber could be multi-mode (suitable for haul, limited bandwidth and relatively low-cost mode (suitable for high- and long-haul applications). ABSTRACT One of the major considerations with all types of fiber connection is the optical loss encountered An optical fiber link is a part of an optic fiber communication system. Other components of the optic fiber link include the transmitter, connectors, and the receiver. The optical fiber could be single mode (for long distance transmission) or multi (for short distance transmission). This paper however, majors on the impact of reflectance in the single optical fiber. Reflectance is a hidden threat that increases Bit Error Rate, BER, (rate at which errors occur in transmission system) and reduces sy performance if not monitored or controlled. Optical Time Domain Reflectometer (OTDR) was used to measure the reflectance in single-mode fiber. Events measurements in OTDR heavily depend on good reflectance. The OTDR was able to establish the reflectance in every portion of the fiber under test. An average reflectance level of -14.9275 dB of 1550 nm signal over the span length of 20.422 km was achieved which is within the acceptable standard range. Hence, good quality performance transmissions can be achieved along these routes. An optical fiber link is a part of an optic fiber communication system. Other components of the optic fiber link include the transmitter, connectors, and the receiver. The optical fiber could be single- mode (for long distance transmission) or multi-mode or short distance transmission). This paper however, majors on the impact of reflectance in the single-mode optical fiber. Reflectance is a hidden threat that increases Bit Error Rate, BER, (rate at which errors occur in transmission system) and reduces system performance if not monitored or controlled. Optical Time Domain Reflectometer (OTDR) was used to mode fiber. Events measurements in OTDR heavily depend on good reflectance. The OTDR was able to establish the ce in every portion of the fiber under test. An 14.9275 dB of 1550 nm signal over the span length of 20.422 km was achieved which is within the acceptable standard range. Hence, good quality performance transmissions Keywords: Reflectance, OTDR, Fiber, Optical Fiber Link Reflectance, OTDR, Fiber, Optical Fiber Figure 2: Optical fiber structure Figure 2: Optical fiber structure ABSTRACT The two main function of the transmitter are [2]: it serves as a light source launched into the optical fiber cable and to modulate the light by the binary data it receives from the source. The receiver has two functions: it detects the light coupled optical fiber cable and converts the light into electrical signal, and demodulates the light to determine the identity of the binary data that it represents. The connectors are used to attach the optical fiber and provide solid contact between receiver. The connector must align the optical fiber end precisely with the light source or receiver to prevent signal loss An optical fiber strand of pure glass used as the transmission medium for the signal [3]. It comprises of two concentric layers called the core and the cladding with different refractive indexes as shown in Figure 2 [4]. The index of refraction is a way of measuring the light in a material. Fiber could be multi short-haul, limited bandwidth and relatively low applications) or single-mode (suitable for high bandwidth and medium- and long One of the major considerati fiber–fiber connection is the optical loss encountered at the interface. The two main function of the transmitter are [2]: it serves as a light source launched into the optical fiber cable and to modulate the light by the binary data it receives from the source. The receiver has two functions: it detects the light coupled out of the optical fiber cable and converts the light into electrical signal, and demodulates the light to determine the identity of the binary data that it represents. The connectors are used to attach the optical fiber and provide solid contact between mated transmitter and receiver. The connector must align the optical fiber end precisely with the light source or receiver to An optical fiber is a long thin strand of pure glass used as the transmission medium for the signal [3]. It comprises of two concentric layers called the core and the cladding with different refractive indexes as shown in Figure 2 [4]. The index measuring the light in a material. Fiber could be multi-mode (suitable for haul, limited bandwidth and relatively low-cost mode (suitable for high- and long-haul applications). Analysis on The Impact of Reflectance in Optical Fiber Links Analysis on The Impact of Reflectance in Optical Fiber Links Analysis on The Impact of Reflectance in Optical Fiber Links J. Ilouno 1Physics Department, University of Jos, Jos, Nigeria 2Physics Department, Kwararafa University, Wukari, Taraba State, Nigeria 3Backbone Connectivity Network, Abuja, Nigeria J. Ilouno1, M. Awoji2, J. Sani3 Physics Department, University of Jos, Jos, Nigeria Physics Department, Kwararafa University, Wukari, Taraba State, Nigeria Backbone Connectivity Network, Abuja, Nigeria Physics Department, Kwararafa University, Wukari, Taraba State, Nigeria J. Ilouno 1Physics Department, University of Jos, Jos, Nigeria 2Physics Department, Kwararafa University, Wukari, Taraba State, Nigeria 3Backbone Connectivity Network, Abuja, Nigeria J. Ilouno1, M. Awoji2, J. Sani3 Physics Department, University of Jos, Jos, Nigeria Physics Department, Kwararafa University, Wukari, Taraba State, Nigeria Backbone Connectivity Network, Abuja, Nigeria Physics Department, Kwararafa University, Wukari, Taraba State, Nigeria ISSN No: 2456 International Research ISSN No: 2456 - 6470 \| www.ijtsrd.com \| Volume International Journal of Trend in Scientific Research and Development (IJTSRD) International Open Access Journal www.ijtsrd.com \| Volume - 2 \| Issue – 4 Scientific (IJTSRD) International Open Access Journal ISSN No: 2456 International Research ISSN No: 2456 - 6470 \| www.ijtsrd.com \| Volume International Journal of Trend in Scientific Research and Development (IJTSRD) International Open Access Journal www.ijtsrd.com \| Volume - 2 \| Issue – 4 Scientific (IJTSRD) International Open Access Journal International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 Even when the two jointed fiber ends are smooth and perpendicular to the fiber axes, and the two fiber axes are perfectly aligned, a small proportion of the light may be reflected back into the transmitting fiber causing attenuation at the joint. This phenomenon is known as Fresnel reflection and is associated with the step changes in refractive index at the jointed interface (i.e. glass–air–glass) that leads to reflectance [5].Reflectance is the amount of light reflected from a single discontinuity in an optical fiber link such as a connector pair or the percentage of light reflected by a single component [1, 2]. Reflectance is expressed as the ratio of the intensity of light reflected to the incident light intensity in dB [6]. Light traveling down the fiber when it sees a change in refractive index, then reflection (reflectance) occurs. The most common causes of reflectance may arise from air gap between the connectors and dirt or residue left behind by the cleaning solution [7]. Connectors possess different ferrule end finishes to minimize reflectance as well as loss. Reflectance of low values is obtainable from fusion splicing and from careful designed mechanical joints. However, certain mechanisms can cause larger values of reflectance. These include optical interference produced in the cavity between two fiber end faces as well as reflection from a high-index layer formed on face of a highly polished fiber. Typical reflectance measurements require a large dynamic range. Accepted reflectance values in the optical telecommunication industry vary according to the connector type as seen in Table 1 [8]: Even when the two jointed fiber ends are smooth and perpendicular to the fiber axes, and the two fiber axes igned, a small proportion of the light may be reflected back into the transmitting fiber causing attenuation at the joint. Figure 3: Block diagra Figure 3: Block diagram of OTDR ii Figure 3 shows how light pulses are launched from the laser through the directional coupler into the optical fiber. The directional coupler channels light returned by the optical fiber to the avalanche photodiode. The avalanche photodiode then co the light energy into electrical energy. The electrical energy is sampled at a very high rate by the sample and-hold circuit. The sample maintains the instantaneous voltage level of each sample long enough for the analogy to digital converter to convert the electrical value to a numerical value. The numerical value from the analogy to digital converter processed by the DSP and the result is sent to the single-board computer to be stored in memory and displayed on the screen. The enti typically repeated many times during a single test of an optical fiber and coordinated by the timing. The OTDR will send the light constantly during certain period. The OTDR capture each sample in round time means the actually transmitti what the OTDR counts. The OTDR shows the time or distance on the horizontal axis and amplitude on the vertical axis. The horizontal axis’s unit is shown in meters or kilometers, and dB (decimal) in vertical axis. The trace generated by the OTDR can be used to identify the reflectance as seen in Figure 4. indicates high reflectance. Limits may occur to the dead zone (i.e. the distance or time where OTDR cannot detect or precisely localize event on the fiber of the OTDR. However, dead zones Figure 3 shows how light pulses are launched from the laser through the directional coupler into the optical fiber. The directional coupler channels light returned by the optical fiber to the avalanche photodiode. The avalanche photodiode then converts the light energy into electrical energy. The electrical energy is sampled at a very high rate by the sample- hold circuit. The sample-and-hold circuit maintains the instantaneous voltage level of each sample long enough for the analogy to digital converter to convert the electrical value to a numerical value. The numerical value from the analogy to digital converter processed by the DSP and the result is sent board computer to be stored in memory and displayed on the screen. The entire process is typically repeated many times during a single test of an optical fiber and coordinated by the timing. The OTDR will send the light constantly during certain period. I. INTRODUCTION Fiber optic or optical fiber link comprises of optical fiber, a transmitter, a receiver and connectors as seen in Figure 1[1]. Fiber optic or optical fiber link comprises of optical fiber, a transmitter, a receiver and connectors as seen @ IJTSRD \| Available Online @ www.ijtsrd.com fiber, a transmitter, a receiver and connectors as seen in Figure 1[1]. Figure 1: Optical fiber link @ IJTSRD \| Available Online @ www.ijtsrd.com \| Volume – 2 \| Issue – 4 \| May-Jun 2018 fiber, a transmitter, a receiver and connectors as seen Figure 2: Optical fiber structure Jun 2018 Page: 1372 Figure 2: Optical fiber structure Figure 1: Optical fiber link Figure 2: Optical fiber structure Figure 2: Optical fiber structure Figure 2: Optical fiber structure Figure 2: Optical fiber structure @ IJTSRD \| Available Online @ www.ijtsrd.com @ IJTSRD \| Available Online @ www.ijtsrd.com \| Volume – 2 \| Issue – 4 \| May-Jun 2018 Jun 2018 Page: 1372 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 This phenomenon is known as Fresnel reflection and is associated with the step changes in refractive index at the jointed glass) that leads to reflectance [5].Reflectance is the amount of light reflected from a single discontinuity in an optical fiber link such as a connector pair or the percentage of light reflected by a s expressed as the ratio of the intensity of light reflected to the incident light intensity in dB [6]. Light traveling down the fiber when it sees a change in refractive index, then reflection (reflectance) occurs. The most y arise from air gap between the connectors and dirt or residue left behind by the cleaning solution [7]. Connectors possess different ferrule end finishes to minimize reflectance as well as loss. Reflectance of low values is g and from careful designed mechanical joints. However, certain mechanisms can cause larger values of reflectance. These include optical interference produced in the cavity between two fiber end faces as well as index layer formed on the end face of a highly polished fiber. Typical reflectance measurements require a large dynamic range. Accepted reflectance values in the optical telecommunication industry vary according to the An OTDR can be used in the measurements of reflectance in optical fiber. A typical OTDR consists of eight basic components: the directional coupler, laser generator, time circuit, signal Digital Signal Processor (DSP), and analogy to digital converter, sample-and-hold circuit, and avalanche photodiode as shown in Figure 3 [9]. An OTDR can be used in the measurements of reflectance in optical fiber. A typical OTDR consists mponents: the directional coupler, laser generator, time circuit, signal-board computer, Digital Signal Processor (DSP), and analogy to digital hold circuit, and avalanche photodiode as shown in Figure 3 [9]. Figure 3: Block diagra Figure 3: Block diagram of OTDR Figure 3: Block diagra Figure 3: Block diagram of OTDR The OTDR capture each sample in round–trip time means the actually transmitting time is half of what the OTDR counts. The OTDR shows the time or distance on the horizontal axis and amplitude on the vertical axis. The horizontal axis’s unit is shown in meters or kilometers, and dB (decimal) in vertical he OTDR can be used to identify the reflectance as seen in Figure 4. High peak Limits may occur to the (i.e. the distance or time where OTDR cannot detect or precisely localize event on the fiber) dead zones boxes that value ustry v n Table ectors w value ustry v ectors w Table 1: Typical connectors with their reflectance onnectors with their reflectance Table 1: Typical connectors with their reflectance Connector Type Typical Reflectance Flat with air -20 dB Physical Contact (PC) -30 to 40 dB Ultra PC -40 to 50 dB APC (angled) -60 dB or higher onnectors with their reflectance Typical Reflectance 30 to 40 dB 40 to 50 dB 60 dB or higher For laser based systems, reflectance has the capacity to reduce system performance which affects the stability of the laser source. Also, in systems with more than a connector multiple reflections occur and this increases the level of signal noise at the o detector. The specific component at fault can be identified with optical equipment such as an Optical Time Domain Reflectometor, OTDR. For laser based systems, reflectance has the capacity to reduce system performance which affects the stability of the laser source. Also, in systems with more than a connector multiple reflections occur and this increases the level of signal noise at the optical detector. The specific component at fault can be identified with optical equipment such as an Optical @ IJTSRD \| Available Online @ www.ijtsrd.com @ IJTSRD \| Available Online @ www.ijtsrd.com \| Volume – 2 \| Issue – 4 \| May-Jun 2018 Jun 2018 Page: 1373 Page: 1373 Page: 1373 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 ournal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-647 International Journal of Trend in Scientific Research and Development (IJTS comply with TIA-455 (standard test procedures for fiber optics components) allow reflectance measurements to be made within the OTDR’s deadzone [7]. detected by the Avalanche photodiode receiver. Figure 3: Block diagra Figure 3: Block diagram of OTDR Flash drive OTDR test procedures Fiber Type: SM 96 CORE FIBER STERLITE Device: MTS 6000 Num.2487 Module: 8126LR Num.16131 The OTDR parameters were set as: integrator which improved the Signal to Noise Rati (SNR) by giving an arithmetic average over a numbe of measurements at one point. This signal was fed int a logarithmic amplifier and the average measuremen for successive points within the fiber were plotted an recorded with the chart recorder. The media converte was then used in converting the trace to readab format and retrieved with an external drive. The sam procedure was repeated for cores 02 to 24 of the fibe and results tabulated as seen in Table 2. Figure 5: OTDR connected to fiber under test III. RESULTS Table 2: Reflectance levels Fiber No. Reflectance (dB) 1 16 02 [ ] Figure 4: Reflectance Figure 4: OTDR Trace indicating Relectance Figure 4: Reflectance Figure 4: OTDR Trace indicating Relectance II. MATERIALS & METHODS Materials: 1. Single-Mode Patch cords 2. Power meter 3. Optical Time Domain Reflectometer (OTDR) 4. Media Converter/Transmission Equipment 5. Flash drive Figure 5: OTDR connected to fiber under test III RESULTS Figure 4: OTDR Trace indicating Relectance II. MATERIALS & METHODS II. MATERIALS & METHODS Figure 3: Block diagra Figure 3: Block diagram of OTDR The output of the photodiode receiver was driven by an integrator which improved the Signal to Noise Ratio (SNR) by giving an arithmetic average over a number of measurements at one point. This signal was fed into a logarithmic amplifier and the average measurements for successive points within the fiber were plotted and recorded with the chart recorder. The media converter was then used in converting the trace to readable format and retrieved with an external drive. The same procedure was repeated for cores 02 to 24 of the fiber and results tabulated as seen in Table 2. p y ( p fiber optics components) allow reflectance measurements to be made within the OTDR’s deadzone [7]. Figure 4: Reflectance Figure 4: OTDR Trace indicating Relectance II. MATERIALS & METHODS Materials: 1. Single-Mode Patch cords 2. Power meter 3. Optical Time Domain Reflectometer (OTDR) 4. Media Converter/Transmission Equipment 5. Flash drive OTDR test procedures Fiber Type: SM 96 CORE FIBER STERLITE Device: MTS 6000 Num.2487 Module: 8126LR Num.16131 The OTDR parameters were set as: Wavelength: 1550 nm Range (Km): 20.422 Acq. Time: 20s Resolution: 64cm Index: 1.46800 A power meter was used in testing for continuity along the cable before the measurements were taken. A single-mode patch cord was attached to the OTDR and to cable plant (core 01) under test via the patch y p output of the photodiode receiver was driven by an integrator which improved the Signal to Noise Ratio (SNR) by giving an arithmetic average over a number of measurements at one point. This signal was fed into a logarithmic amplifier and the average measurements for successive points within the fiber were plotted and recorded with the chart recorder. The media converter was then used in converting the trace to readable format and retrieved with an external drive. The same procedure was repeated for cores 02 to 24 of the fiber and results tabulated as seen in Table 2. Figure 5: OTDR connected to fiber under test III. RESULTS Table 2: Reflectance levels Fiber No. Reflectance (dB) 1 -16.02 2 -16.92 3 -14.85 4 -17.16 5 -16.83 6 -17.16 measurements to be made within the OTDR’s deadzone [7]. Figure 4: Reflectance Figure 4: OTDR Trace indicating Relectance II. MATERIALS & METHODS Materials: 1. Single-Mode Patch cords 2. Power meter 3. Optical Time Domain Reflectometer (OTDR) 4. Media Converter/Transmission Equipment 5. International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 10 17.09 11 -17.14 12 17.28 13 -16.20 14 -17.00 15 -16.89 16 -16.87 17 -16.88 18 -16.85 19 -17.16 20 -14.83 21 -14.74 22 -14.61 23 -15.15 24 -14.87 CUSSION reflectance level of -14.9275 dB was hich is within the standard specification. reflection events did not pose any threat he transmission capability or mechanical Therefore, it is not cost effective or s to remove the fibers with these small ents since they have little or no impact on tenuation. The small reflection recorded been caused by localized changed in the dex of the light carrying portion of the . However, measuring more than a 40 to poses a serious challenge or threat to the of an optical fiber link so must be voided. NCLUSION of reflectance on single-mode fiber optics zed using Optical Time Domain er (OTDR). Small reflection events were REFERENCES 1) G.P. Agrawal, Fiber-optic communication systems (New York: John Wiley & Sons ISBN 0- 47121571-6, 2002). 2) W. Etten & J. Plaats, Fundamentals of optical fiber communications (New York: Prentice Hall, 1991). 3) S. Dharamvir, Optical fiber based communication network, International Journal of Technical Research (IJTR), 1 (1), 2012, 43-46. 4) https://www.vialite.com/resourses/guides/single- mode-vs-mult-mode/ (2018) 5) M. Ilyas & H.T. Moftah, Handbook of optical communication networks (Florida, USA: CRC Press, 2003). 6) http://questtel.com/wiki/fiber-return-loss-and- reflectance (2018) 7) J.M. Senior, Optical Fiber Communications Principles and Practice, 3rd Edition (England: Pearson Practice Hall, 2009) 8) http://www.thefoa.org/tech/ref/testing/test/reflecta nce.html (2018) 9) S.K. Raghuwanshi, Experimental characterization of fiber optic communication link for digital transmission system. ICTACT Journal on Communication Technology, 5 (10), 2014, 868- 876. 1) G.P. Agrawal, Fiber-optic communication systems (New York: John Wiley & Sons ISBN 0- 47121571-6, 2002). 1) G.P. Agrawal, Fiber-optic communication systems (New York: John Wiley & Sons ISBN 0- 47121571-6, 2002). 2) W. Etten & J. Plaats, Fundamentals of optical fiber communications (New York: Prentice Hall, 1991). 2) W. Etten & J. Plaats, Fundamentals of optical fiber communications (New York: Prentice Hall, 1991). 3) S. Dharamvir, Optical fiber based communication network, International Journal of Technical Research (IJTR), 1 (1), 2012, 43-46. 4) https://www.vialite.com/resourses/guides/single- mode-vs-mult-mode/ (2018) 4) https://www.vialite.com/resourses/guides/single- mode-vs-mult-mode/ (2018) 5) M. Ilyas & H.T. Moftah, Handbook of optical communication networks (Florida, USA: CRC Press, 2003). II. MATERIALS & METHODS Materials: Figure 5: OTDR connected to fiber under test Figure 5: OTDR connected to fiber under test A power meter was used in testing for continuity along the cable before the measurements were taken. A single-mode patch cord was attached to the OTDR and to cable plant (core 01) under test via the patch panel at point A as shown in Figure 5. The OTDR was preset manually as stated above and it emitted light power pulses along the cable in a forward direction by the injection laser. The light pulses then bounced back and were measured by the factoring out of time and distances. The backscattered light was @ IJTSRD \| Available Online @ www.ijtsrd.com \| Volume – 2 \| Issue – 4 \| May-Jun 2018 Page: 1374 International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 10 -17.09 11 -17.14 12 17.28 13 -16.20 14 -17.00 15 -16.89 16 -16.87 17 -16.88 18 -16.85 19 -17.16 20 -14.83 21 -14.74 22 -14.61 23 -15.15 24 -14.87 IV. DISCUSSION An average reflectance level of -14.9275 dB was obtained which is within the standard specification. These small reflection events did not pose any threat or risk to the transmission capability or mechanical reliability. Therefore, it is not cost effective or advantageous to remove the fibers with these small reflection events since they have little or no impact on the fiber attenuation. The small reflection recorded might have been caused by localized changed in the refractive index of the light carrying portion of the optical fiber. However, measuring more than a 40 to 60 dB range poses a serious challenge or threat to the performance of an optical fiber link so must be technically avoided. V. CONCLUSION REFERENCES 1) G.P. Agrawal, Fiber-optic communication systems (New York: John Wiley & Sons ISBN 0- 47121571-6, 2002). 2) W. Etten & J. Plaats, Fundamentals of optical fiber communications (New York: Prentice Hall, 1991). 3) S. Dharamvir, Optical fiber based communication network, International Journal of Technical Research (IJTR), 1 (1), 2012, 43-46. 4) https://www.vialite.com/resourses/guides/single- mode-vs-mult-mode/ (2018) 5) M. Ilyas & H.T. Moftah, Handbook of optical communication networks (Florida, USA: CRC Press, 2003). 6) http://questtel.com/wiki/fiber-return-loss-and- reflectance (2018) 7) J.M. Senior, Optical Fiber Communications Principles and Practice, 3rd Edition (England: Pearson Practice Hall, 2009) 8) http://www.thefoa.org/tech/ref/testing/test/reflecta nce.html (2018) 9) S.K. Raghuwanshi, Experimental characterization of fiber optic communication link for digital transmission system. II. MATERIALS & METHODS ICTACT Journal on Communication Technology, 5 (10), 2014, 868- 876. International Journal of Trend in Scientific Research and Development (IJTSRD) IV. DISCUSSION s . t l r l n d e e transmission system. ICTA Communication Technology, 876. CONCLUSION V. CONCLUSION The impact of reflectance on single-mode fiber optics was analyzed using Optical Time Domain Reflectometer (OTDR). Small reflection events were measured and recorded as reflectance which poses no serious challenge to the system performance because their impact on the fiber attenuation was minimal or zero. @ IJTSRD \| Available Online @ www.ijtsrd.com \| Volume – 2 \| Issue – 4 \| May-Jun 2018 Page: 1375 @ IJTSRD \| Available Online @ www.ijtsrd.com \| Volume – 2 \| Issue – 4 \| Ma
https://openalex.org/W2170040488	https://europepmc.org/articles/pmc4060390?pdf=render	English	null	Serum C-X-C motif chemokine 13 is elevated in early and established rheumatoid arthritis and correlates with rheumatoid factor levels	Arthritis research & therapy	2,014	cc-by	9,074	Abstract Introduction: We hypothesized that serum levels of C-X-C motif chemokine 13 (CXCL13), a B-cell chemokine, would delineate a subset of rheumatoid arthritis (RA) patients characterized by increased humoral immunity. Introduction: We hypothesized that serum levels of C-X-C motif chemokine 13 (CXCL13), a B-cell chemokine, would delineate a subset of rheumatoid arthritis (RA) patients characterized by increased humoral immunity. Methods: Serum from patients with established RA (the Dartmouth RA Cohort) was analyzed for CXCL13, rheumatoid factor (RF) levels anticitrullinated peptide/protein antibody (ACPA) and total immunoglobulin G (IgG); Introduction: We hypothesized that serum levels of C-X-C motif chemokine 13 (CXCL13), a B-cell chemokine, would delineate a subset of rheumatoid arthritis (RA) patients characterized by increased humoral immunity. Methods: Serum from patients with established RA (the Dartmouth RA Cohort) was analyzed for CXCL13, rheumatoid factor (RF) levels, anticitrullinated peptide/protein antibody (ACPA) and total immunoglobulin G (IgG); other parameters were obtained by chart review. A confirmatory analysis was performed using samples from the Sherbrooke Early Undifferentiated PolyArthritis (EUPA) Cohort. The Wilcoxon rank-sum test, a t-test and Spearman’s correlation analysis were utilized to determine relationships between variables. um from patients with established RA (the Dartmouth RA Cohort) was analyzed for CXCL13, p ( ) y , rheumatoid factor (RF) levels, anticitrullinated peptide/protein antibody (ACPA) and total immunoglobulin G (IgG); other parameters were obtained by chart review. A confirmatory analysis was performed using samples from the Sherbrooke Early Undifferentiated PolyArthritis (EUPA) Cohort. The Wilcoxon rank-sum test, a t-test and Spearman’s correlation analysis were utilized to determine relationships between variables. Results: In both the Dartmouth and Sherbrooke cohorts, CXCL13 levels were selectively increased in seropositive relative to seronegative RA patients (P = 0.0002 and P < 0.0001 for the respective cohorts), with a strong correlation to both immunoglobulin M (IgM) and IgA RF levels (P < 0.0001). There was a weaker relationship to ACPA titers (P = 0.03 and P = 0.006, respectively) and total IgG (P = 0.02 and P = 0.14, respectively). No relationship was seen with regard to age, sex, shared epitope status or inclusion high-sensitivity C-reactive protein (hsCRP) in either cohort or regarding the presence of baseline erosions in the Sherbrooke Cohort, whereas a modest relationship with Disease Activity Score in 28 joints CRP (DAS28-CRP) was seen in the Dartmouth cohort but not the Sherbrooke cohort. * Correspondence: jonathan.d.jones@dartmouth.edu 1Division of Rheumatology, Geisel School of Medicine at Dartmouth College, One Medical Center Dr., Lebanon, NH 03756, USA Full list of author information is available at the end of the article © 2014 Jones et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Abstract Conclusion: Using both established and early RA cohorts, marked elevations of serum CXCL13 levels resided nearly completely within the seropositive population. CXCL13 levels exhibited a strong relationship with RF, whereas the association with clinical parameters (age, sex, DAS28-CRP and erosions) or other serologic markers (ACPA and IgG) was either much weaker or absent. Elevated serum CXCL13 levels may identify a subset of seropositive RA patients whose disease is shaped by or responsive to RF production. leukocyte antigen major histocompatibility complex class II DR β1 (HLA-DRB1) alleles containing the shared epi- tope [1]. The presence of IgG ACPAs and IgA-RF indicates that antibody heavy-chain class-switching has occurred, which is typically associated with T-cell–dependent B-cell maturation and differentiation [2,3]. Jones et al. Arthritis Research & Therapy 2014, 16:R103 http://arthritis-research.com/content/16/2/R103 Open Access Serum C-X-C motif chemokine 13 is elevated in early and established rheumatoid arthritis and correlates with rheumatoid factor levels Jonathan D Jones1, B JoNell Hamilton2, Gregory J Challener2, Artur J de Brum-Fernandes3, Pierre Cossette4, Patrick Liang3, Ariel Masetto3, Henri A Ménard5, Nathalie Carrier3, David L Boyle6, Sanna Rosengren7, Gilles Boire3 and William F C Rigby1,2 Introduction subsequently established the presence of a strong relationship between synovial CXCL13 mRNA expres- sion and serum CXCL13 level in a cohort of patients with long-standing RA [11]. Thus, synovial production appears to account for increased serum CXCL13 levels rather than serum CXCL13 elevation arising from a sys- temic reaction to joint inflammation. The confirmatory cohort consists of a subset of the pa- tients recruited from Sherbrooke, QC, Canada, as part of the longitudinal Early Undifferentiated Polyarthritis (EUPA) Cohort. This cohort varies from the Dartmouth RA Cohort in that it represents an early arthritis popula- tion, contains a greater proportion of seronegative patients and has predominantly patients who were DMARD- and corticosteroid-naïve at the time of inclusion. Cohort inclu- sion criteria included age ≥18 years, disease duration bet- ween 1 and 12 months and swollen joint count of three or more. In the subset reported herein, all RA patients ful- filled the 1987 ACR revised criteria. Seropositivity was de- fined as both an RF titer ≥40 IU/ml measured using Given those data, we hypothesized that serum CXCL13 levels would reflect the impact of CXCL13 on synovial inflammation and the shaping of the clinical and serolo- gic phenotype. We specifically wished to determine if CXCL13 levels identify a subset of RA patients, perhaps indicating a greater role of humoral immunity in disease pathogenesis. We undertook a cross-sectional analysis of circulating serum CXCL13 levels in RA patients followed at the Dartmouth-Hitchcock Medical Center. In this co- hort, we observed that CXCL13 expression was much higher in seropositive than seronegative RA patients. In addition, we observed that this relationship correlated most strongly with RF and not with ACPA. Subsequently, we saw identical relationships in an early RA cohort. We performed genetic, serologic and clinical analyses, which indicated that serum CXCL13 levels may identify a novel (and abundant) subpopulation of seropositive RA. Ad- ditional studies are required to assess the utility of this biomarker. Introduction Seropositive rheumatoid arthritis (RA) is an inflam- matory disease characterized by autoantibodies (im- munoglobulin G (IgG) anticitrullinated peptide/protein antibodies (ACPAs) and rheumatoid factor (RF)). These autoantibodies can appear years before the onset of clinical disease and are strongly linked to the human An important element of T-cell–dependent B cell mat- uration and differentiation is the formation of lymphoid follicles and germinal centers. Murine studies indicate the interaction of the C-X-C motif chemokine 13 (CXCL13) with C-X-C chemokine receptor type 5 (CXCR5) promotes Page 2 of 9 Jones et al. Arthritis Research & Therapy 2014, 16:R103 http://arthritis-research.com/content/16/2/R103 Jones et al. Arthritis Research & Therapy 2014, 16:R103 http://arthritis-research.com/content/16/2/R103 years (Table 1). Approval for this study was obtained from the Committee for the Protection of Human Subjects at Dartmouth College, and the patients provided their in- formed, signed consent to participate. Age and sex, disease duration, medication history, smoking status, seroposi- tivity (determined by clinical laboratory determination of RF >14 IU/ml using immunoturbidimetric measurement (Roche Diagnostics, Indianapolis, IN, USA) and/or anti– cyclic citrullinated peptide 2 (anti-CCP2) >5.0 U/ml by enzyme-linked immunosorbent assay (ELISA) (DiaSorin, Saluggia, Italy)) and high-sensitivity C-reactive protein (hsCRP) levels were recorded, and serum and DNA were collected. In some cases, Disease Activity Score in 28 joints CRP (DAS28-CRP) and Clinical Disease Activity Index (CDAI) scores were available from the clinical charts. this process through the recruitment of naïve B cells and follicular T cells to the lymphoid follicle [4-6]. Thus, it seems reasonable to posit that CXCL13 plays a role in the development of both IgG ACPAs and IgA-RF prior to the development of clinical signs and symptoms. In addition to the development of autoantibodies in the preclinical phase, CXCL13 has been associated with synovial inflammation in RA. A series of observations has established its production by multiple cell types in rheumatoid synovium, frequently in association with the formation of lymphoid follicular structures, including synovial T cells (but not T follicular cells) [7], monocytes/ macrophages [8] and follicular dendritic cells, endothelial cells and synovial fibroblasts [9]. In addition to its synovial production in RA, elevated serum levels of CXCL13 have been observed and were reported to be 1.7× higher in one small study of patients with active relative to quiescent disease [10]. Rosengren et al. Introduction Table 1 Patients in the Dartmouth RA Cohorta Patient demographics (N = 193) Seronegative Seropositive Number of patients (%) 30 (16%) 163 (84%) Average age, yr (range) 55.6 (29 to 72) 57.9 (19 to 92) Females, n (%) 24 (80%) 115 (71%) RA duration <2 yr 10 (33%) 41 (25%) SE status, n 14 116 SE alleles = 0 7 22 SE alleles = 1 7 56 SE alleles = 2 0 38 C4 status, n 14 111 C4 copies <4 9 30 C4 copies ≥4 5 81 Medication history, n No DMARDs 9 36 Nonbiologic DMARDs 10 54 TNF inhibitors 4 42 Other biologics 7b 31c aC4, Complement 4; DMARD, Disease-modifying antirheumatic drug; RA, Rheumatoid arthritis; SE, Shared epitope; TNF, Tumor necrosis factor. bAbatacept (n = 1) and rituximab (n = 6). cAbatacept (n = 2), rituximab (n = 27), tocilizumab (n = 1) and tofacitinib (n = 1). Table 1 Patients in the Dartmouth RA Cohorta Table 1 Patients in the Dartmouth RA Cohorta Patient samples The Dartmouth RA Cohort consists of patients recruited from the Dartmouth-Hitchcock Medical Center Rheuma- tology Clinic (Lebanon, NH, USA) who have established RA defined according to the American College of Rheu- matology (ACR) 1987 revised criteria [12]. This cohort represents a patient population with established RA whose disease duration extends, in some cases, longer than 20 Page 3 of 9 Jones et al. Arthritis Research & Therapy 2014, 16:R103 http://arthritis-research.com/content/16/2/R103 RapiTex RF (Dade Behring, Deerfield, IL, USA) and anti- CCP2 >20 U/ml using QUANTA Lite (Inova Diagnostics, San Diego, CA, USA), present concurrently at least once. Seronegativity was defined as negative RF and anti-CCP2 at all the visits. This subset was chosen randomly from among the Sherbrooke EUPA Cohort, with samples matched only for serostatus. The patients provided their signed, informed consent to participate, and study ap- proval was obtained from the Sherbrooke University Hospital Centre Institutional Review Board. Information gathered at enrollment included demographic details such as age, sex and time since onset of arthritis, as well as cli- nical details such as DAS28-CRP score, radiologic Sharp score and RF and ACPA status (Table 2). CXCL13 results due to the heterophilic activity of RF. We noticed that our range of CXCL13 levels was greater than levels reported elsewhere in the literature, so we con- firmed the results in the Dartmouth RA Cohort using a premade ELISA kit from the same company (R&D Systems), which showed minimal variation from the ori- ginal values we obtained (r = 0.95). Total levels of IgG were measured by ELISA (Immu- nology Consultants Laboratory, Portland, OR, USA), as were IgM RF levels (TheraTest Laboratories, Lombard, IL, USA) and IgA RF levels (Inova Diagnostics). In the Dartmouth RA Cohort, we additionally measured serum levels of IgG ACPA using a human QUANTA Lite CCP3 IgG ELISA kit (Inova Diagnostics). All analyses were done on the same sample or with samples from an aliquot iden- tical to the sample used to measure CXCL13. The one ex- ception was the measurement of anti-CCP2 levels (Inova Diagnostics/EUROIMMUN US, Morris Plains, NJ, USA) in the Sherbrooke EUPA Cohort, as it was measured either simultaneously with or within a few weeks after the serum collection used for the CXCL13 assay. Serum analysis Patient serum was stored at −80°C (Dartmouth RA Cohort) or −20°C (Sherbrooke EUPA Cohort) until analysis. CXCL13 levels were measured by ELISA according to the manufacturer’s instructions (Human CXCL13/BLC/ BCA-1 DuoSet; R&D Systems, Minneapolis, MN, USA). Several samples were kept and repetitively measured over the course of 3 weeks at 4°C, with no change in CXCL13 levels compared to freshly thawed serum. Serum samples were diluted 1:100, but the ELISA was repeated at a serum dilution of 1:10 in cases where low values were obtained. In six RF-positive patient samples, HeteroBlock reagent (Omega Biologicals, Bozeman, MT, USA) was titrated into the assay to eradicate the potential for false-positive DNA analysis I h D In the Dartmouth RA Cohort, HLA-DRB1 status (shared epitope) was obtained previously [13] through the American Red Cross Penn-Jersey Blood Services Region. Complement 4 (C4) allele copy number was also obtained as described previously [13] by Southern blot analysis, with confirmation by RT-PCR. HLA-DRB1 typing in the Sherbrooke EUPA Cohort was determined using sequence-specific primer PCR techniques as previously described [14]. Table 2 Recent-onset rheumatoid arthritis patients in the Sherbrooke Early Undifferentiated PolyArthritis Cohorta Table 2 Recent-onset rheumatoid arthritis patients in the Sherbrooke Early Undifferentiated PolyArthritis Cohorta Patient demographics (N = 339) Seronegative Seropositive Number of patients (%) 166 (49%) 173 (51%) Average age, yr (range) 54.2 (21 to 92) 65.6 (19 to 87) Females, n (%) 107 (64) 105 (61) Symptom duration, yr 0.44 0.45 SE status, n 120 132 SE alleles = 0 75 43 SE alleles = 1 44 59 SE alleles = 2 1 30 Mean DAS28-CRP (±SD) 5.10 (1.47) 5.04 (1.44) Mean CRP mg/L (±SD) 24.4 (33.9) 26.6 (35.6) Mean Sharp score erosions (±SD) 2.9 (6.1) 3.8 (7.0) Mean Sharp score narrowing (±SD) 3.4 (6.1) 2.2 (4.1) Tender joints 10.4 9.8 Swollen joints 10.5 10.2 Patient VAS score (100-mm scale) 54.2 53.9 aCRP, C-reactive protein; DAS28-CRP, Disease Activity Score in 28 joints C-reactive protein; SE, Shared epitope; VAS, Visual Analogue Scale. Seronegative vs seropositive analysis: Symptom duration, P = 0.85; DAS28-CRP, P = 0.72; CRP, P = 0.56; Sharp score erosions, P = 0.28; Sharp score narrowing, P = 0.09. CXCL13 is elevated in seropositive rheumatoid arthritis patients and correlates with immunoglobulin M rheumatoid factor The Dartmouth RA Cohort (N = 193) represents an established RA cohort with a variation in disease du- ration from <1 year to >20 years (Table 1). We first ana- lyzed serum CXCL13 levels in seronegative patients in relation to seropositive patients, as determined by the cli- nical laboratory data and chart history. Owing to the range of CXCL13 levels obtained (0 to >53,000 pg/ml) and the non-normal distributions, the data were log-transformed (log CXCL13). We identified a significant elevation in log CXCL13 levels in seropositive patients (by independent Student’s t-test), with a geometric mean values (95% CI) of 93 pg/ml in seronegatives (71.3 to 123.9) and 331 pg/ml in seropositives (250.0 to 430.5) (P = 0.0002) (Figure 1A). The addition of HeteroBlock did not alter the results, thus confirming that these findings were not due to the pre- sence of RF in the seropositive sera (data not shown). We examined the potential relationship of log CXCL13 levels to genetic markers (shared epitope status and com- plement C4B gene copy number deficiency) associated with autoantibody positivity in RA [13,15]. We saw no relation- ship between log CXCL13 levels in seropositive RA patients (n = 115 patients with HLA-DRB1 determination) in the presence or absence of the shared epitope (P = 0.91) (data not shown). Similarly, no relationship was seen (n = 111 patients with C4 (please italicize) gene copy number determination) with C4B gene copy number deficiency Serum IgM RF and IgG ACPA levels were measured in the seropositive patients and evaluated by Spearman correlation in relation to log CXCL13 levels. We found a highly significant relationship to IgM RF (r = 0.45, P < 0.0001) (Figure 1B), with a much weaker relationship Figure 1 Scatterplots illustrate strong correlation of serum CXCL13 with seropositivity and immunoglobulin M rheumatoid factor in the Dartmouth RA Cohort. (A) Log-transformed C-X-C motif chemokine 13 (CXCL13) values are higher in seropositive compared to seronegative rheumatoid arthritis (RA) patients by t-test (seropositive (n = 163), geometric mean (95% CI) = 331.1 pg/ml (250.0 to 430.5); seronegative (n = 30), geometric mean (95% CI) 93.3 pg/ml (71.3 to 123.9); P = 0.0002). (B) The log-transformed CXCL13 values of seropositive patients increase with higher rheumatoid factor (RF) levels when evaluated by Spearman correlation (P < 0.0001). CXCL13 is elevated in seropositive rheumatoid arthritis patients and correlates with immunoglobulin M rheumatoid factor (C) Tertile analysis of seropositive patients comparing the highest CXCL13 tertile (third tertile) to the first and second tertiles by Wilcoxon rank-sum test identifies a strong relationship with immunoglobulin M (IgM) RF (third-tertile IgM RF: mean = 182 ± 59 IU/ml; first- and second-tertile IgM RF: mean = 113 ± 74 IU/ml; P < 0.0001). (D) CXCL13 has a weaker relationship to IgG anticitrullinated peptide/protein antibody (ACPA) (P = 0.03). P < 0.05. Diagonal lines represent line of best fit. Figure 1 Scatterplots illustrate strong correlation of serum CXCL13 with seropositivity and immunoglobulin M rheumatoid factor in the Dartmouth RA Cohort. (A) Log-transformed C-X-C motif chemokine 13 (CXCL13) values are higher in seropositive compared to seronegative rheumatoid arthritis (RA) patients by t-test (seropositive (n = 163), geometric mean (95% CI) = 331.1 pg/ml (250.0 to 430.5); seronegative (n = 30), geometric mean (95% CI) 93.3 pg/ml (71.3 to 123.9); P = 0.0002). (B) The log-transformed CXCL13 values of seropositive patients increase with higher rheumatoid factor (RF) levels when evaluated by Spearman correlation (P < 0.0001). (C) Tertile analysis of seropositive patients comparing the highest CXCL13 tertile (third tertile) to the first and second tertiles by Wilcoxon rank-sum test identifies a strong relationship with immunoglobulin M (IgM) RF (third-tertile IgM RF: mean = 182 ± 59 IU/ml; first- and second-tertile IgM RF: mean = 113 ± 74 IU/ml; P < 0.0001). (D) CXCL13 has a weaker relationship to IgG anticitrullinated peptide/protein antibody (ACPA) (P = 0.03). P < 0.05. Diagonal lines represent line of best fit. Figure 1 Scatterplots illustrate strong correlation of serum CXCL13 with seropositivity and immunoglobulin M rheumatoid factor in the Dartmouth RA Cohort. (A) Log-transformed C-X-C motif chemokine 13 (CXCL13) values are higher in seropositive compared to seronegative rheumatoid arthritis (RA) patients by t-test (seropositive (n = 163), geometric mean (95% CI) = 331.1 pg/ml (250.0 to 430.5); seronegative (n = 30), geometric mean (95% CI) 93.3 pg/ml (71.3 to 123.9); P = 0.0002). (B) The log-transformed CXCL13 values of seropositive patients increase with higher rheumatoid factor (RF) levels when evaluated by Spearman correlation (P < 0.0001). Results to IgG ACPA (r = 0.17, P = 0.03) (Figure 1D). Of the 163 seropositive patients, 7 patients (4.3%) were positive for IgG ACPA but negative for IgM RF, and 8 patients (4.9%) were negative for IgG ACPA but positive for IgM RF. Evaluation of the CXCL13 values of these single-positive samples did not differ from the remaining double-positive samples (data not shown). Tertile analysis of CXCL13 values confirmed that IgM RF was higher in the third than in the first and second tertiles (mean = 182 ± 59 vs 113 ± 74 U/ml; P < 0.0001) (Figure 1C) but that IgG ACPA was not (mean = 511 ± 319 vs. 419 ± 339 U/ml; P = 0.09) (data not shown), as determined by Wilcoxon rank-sum test. Evaluation of log CXCL13 levels in relation to total IgG levels showed a weak but statistically significant relation- ship (r = 0.18, P = 0.02) (data not shown). Statistical analysis Statistical analysis was performed using STATA software version 12.1 (StataCorp, College Station, TX, USA). CXCL13, hsCRP and IgA RF levels were log-transformed because of the wide range and non-normal distribution of the data. Comparisons of two means were carried out by independent Student’s t-test or by Wilcoxon rank-sum test for non-normal distributions. Pearson correlation or Spearman correlation (in non-normal distributions) were used for analysis of log-transformed CXCL13 and other measures, such as IgM RF, ACPA, total IgG, age, hsCRP, DAS28-CRP, CDAI and erosions. For additional analysis of CXCL13 relationships to RF and ACPA, CXCL13 values from seropositive patients were divided into tertiles. The lower and upper cutoffs for the Dartmouth RA Cohort were 160 and 400 pg/ml, respectively. For the Sherbrooke EUPA Cohort, the lower and upper cutoffs were 150 and 1100 pg/ml, respectively. Because of clear overlap of RF values of the lower two CXCL13 tertiles, these values were combined for comparison to the highest tertile. Two- tailed P values <0.05 were considered significant. Page 4 of 9 Jones et al. Arthritis Research & Therapy 2014, 16:R103 http://arthritis-research.com/content/16/2/R103 Jones et al. Arthritis Research & Therapy 2014, 16:R103 http://arthritis-research.com/content/16/2/R103 CXCL13 is elevated in seropositive rheumatoid arthritis patients and correlates with immunoglobulin M rheumatoid factor (C) Tertile analysis of seropositive patients comparing the highest CXCL13 tertile (third tertile) to the first and second tertiles by Wilcoxon rank-sum test identifies a strong relationship with immunoglobulin M (IgM) RF (third-tertile IgM RF: mean = 182 ± 59 IU/ml; first- and second-tertile IgM RF: mean = 113 ± 74 IU/ml; P < 0.0001). (D) CXCL13 has a weaker relationship to IgG anticitrullinated peptide/protein antibody (ACPA) (P = 0.03). P < 0.05. Diagonal lines represent line of best fit. on of serum CXCL13 with seropositivity and immunoglobulin M rheumatoid factor in C-X-C motif chemokine 13 (CXCL13) values are higher in seropositive compared to seronegative Page 5 of 9 Jones et al. Arthritis Research & Therapy 2014, 16:R103 http://arthritis-research.com/content/16/2/R103 (P = 0.69), C4A deficiency (P = 0.56), or total C4 defi- ciency, defined as C4 gene copy number <4 (P = 0.35) (data not shown). (P = 0.69), C4A deficiency (P = 0.56), or total C4 defi- ciency, defined as C4 gene copy number <4 (P = 0.35) (data not shown). Relationships between CXCL13 and antibody levels in an early rheumatoid arthritis cohort We compared our findings in an established RA cohort to that of a well-characterized early RA cohort (the Sher- brooke EUPA Cohort) to address any potential confoun- ding by current or past therapy. This cohort consisted of a nearly equal number of seronegative and seropositive patients (n = 166 and 173, respectively) with an average disease duration of approximately 5 months (Table 2). As with established RA, a strong relationship with log CXCL13 levels and seropositivity had already been seen at the inclusion visit (P < 0.0001) (Figure 3A) with a geome- tric mean (95% CI) of 50.1 pg/ml (35.0 to 78.0) in seroneg- atives and 323.6 pg/ml (223.9 to 477.5) in seropositives. Similarly, we observed a strong relationship in the sero- positive patients when log CXCL13 levels were evaluated by Spearman correlation analysis against IgM RF levels (r = 0.54, P < 0.0001) (Figure 3B) as well as by tertile analysis (P < 0.0001) (Figure 3C). CXCL13 is elevated in seropositive rheumatoid arthritis patients and correlates with immunoglobulin M rheumatoid factor In comparison to the pa- tients with established RA in the Dartmouth RA Cohort, the recent-onset RA patients showed a stronger relation- ship between CXCL13 and IgG ACPA with P = 0.006 (r = 0.21) (Figure 3D) and P = 0.02 by tertile analysis (data not shown), respectively, but no relationship between serum IgG and CXCL13 level was observed (r = 0.11, CXCL13 relationships to other serologic and clinical features Within this cross-sectional analysis, we examined the rela- tionship of CXCL13 levels in relation to laboratory- reported hsCRP levels at the time of sample collection (n = 123 seropositive patients) and various clinical assess- ments of disease activity, namely, DAS28-CRP and CDAI. Log-transformed hsCRP (log CRP) and log CXCL13 showed only a trend toward significance (P = 0.07) (Figure 2A). Simultaneous measures of DAS28-CRP and CDAI on 23 and 22 seropositive patients, respectively, were available. The DAS28-CRP association was shown to be significant (r = 0.52, P = 0.01) (Figure 2B), whereas the CDAI showed only a trend toward a relationship to log CXCL13 (r = 0.38, P = 0.08) (data not shown). Further analyses included comparing log CXCL13 to age and sex, but the results were unremarkable (P = 0.28 and P = 0.34 respectively) (data not shown). Additionally, we found no relationship of log CXCL13 to smoking status as defined by never-smokers, past smokers and current smokers (P = 0.47). Figure 2 Scatterplots show minimal or no relationship of serum CXCL13 to high-sensitivity C-reactive protein or disease activity score in 28 joints C-reactive protein in the seropositive groups of the Dartmouth RA Cohort and the Sherbrooke Early Undifferentiated PolyArthritis Cohort. (A) In the Dartmouth RA Cohort, correlation analysis of log-transformed C-X-C motif chemokine 13 (LogCXCL13) and high-sensitivity C-reactive protein (LogCRP) does not show a relationship (n = 123, P = 0.07). (B) In the Dartmouth RA Cohort, correlation analysis of CXCL13 has a modest relationship with Disease Activity Score in 28 joints CRP (DAS28-CRP) (n = 23, P = 0.01). (C) In the recent-onset rheumatoid arthritis (RA) patients from the Sherbrooke Early Undifferentiated PolyArthritis (EUPA) Cohort, no relationship is identified between LogCXCL13 and LogCRP (n = 173, P = 0.08). (D) In the recent-onset RA patients from the Sherbrooke EUPA Cohort, no relationship is identified between LogCXCL13 and DAS28-CRP (n = 170, P = 0.28). Diagonal lines represent lines of best fit. Figure 2 Scatterplots show minimal or no relationship of serum CXCL13 to high-sensitivity C-reactive protein or disease activity score in 28 joints C-reactive protein in the seropositive groups of the Dartmouth RA Cohort and the Sherbrooke Early Undifferentiated PolyArthritis Cohort. (A) In the Dartmouth RA Cohort, correlation analysis of log-transformed C-X-C motif chemokine 13 (LogCXCL13) and high-sensitivity C-reactive protein (LogCRP) does not show a relationship (n = 123, P = 0.07). CXCL13 relationships to other serologic and clinical features (B) In the Dartmouth RA Cohort, correlation analysis of CXCL13 has a modest relationship with Disease Activity Score in 28 joints CRP (DAS28-CRP) (n = 23, P = 0.01). (C) In the recent-onset rheumatoid arthritis (RA) patients from the Sherbrooke Early Undifferentiated PolyArthritis (EUPA) Cohort, no relationship is identified between LogCXCL13 and LogCRP (n = 173, P = 0.08). (D) In the recent-onset RA patients from the Sherbrooke EUPA Cohort, no relationship is identified between LogCXCL13 and DAS28-CRP (n = 170, P = 0.28). Diagonal lines represent lines of best fit. s show minimal or no relationship of serum CXCL13 to high-sensitivity C-reactive protein or disease activity score ve protein in the seropositive groups of the Dartmouth RA Cohort and the Sherbrooke Early Undifferentiated Figure 2 Scatterplots show minimal or no relationship of serum CXCL13 to high-sensitivity C-reactive protein or disease activity score in 28 joints C-reactive protein in the seropositive groups of the Dartmouth RA Cohort and the Sherbrooke Early Undifferentiated PolyArthritis Cohort. (A) In the Dartmouth RA Cohort, correlation analysis of log-transformed C-X-C motif chemokine 13 (LogCXCL13) and high-sensitivity C-reactive protein (LogCRP) does not show a relationship (n = 123, P = 0.07). (B) In the Dartmouth RA Cohort, correlation analysis of CXCL13 has a modest relationship with Disease Activity Score in 28 joints CRP (DAS28-CRP) (n = 23, P = 0.01). (C) In the recent-onset rheumatoid arthritis (RA) patients from the Sherbrooke Early Undifferentiated PolyArthritis (EUPA) Cohort, no relationship is identified between LogCXCL13 and LogCRP (n = 173, P = 0.08). (D) In the recent-onset RA patients from the Sherbrooke EUPA Cohort, no relationship is identified between LogCXCL13 and DAS28-CRP (n = 170, P = 0.28). Diagonal lines represent lines of best fit. Jones et al. Arthritis Research & Therapy 2014, 16:R103 http://arthritis-research.com/content/16/2/R103 Page 6 of 9 Figure 3 Scatterplots demonstrate serum CXCL13 strongly correlates with seropositive rheumatoid arthritis and immunoglobulin M rheumatoid factor levels in the Sherbrooke Early Undifferentiated PolyArthritis Cohort, but has a weaker relationship to anticitrullinated peptide/protein antibody levels. (A) Log-transformed C-X-C motif chemokine 13 (LogCXCL13) is higher in seropositive than in seronegative RA patients as determined by t-test (seropositive: n = 173, geometric mean (95% CI) = 323.6 pg/ml (223.9 to 477.5); seronegative: n = 166, geometric mean (95% CI) = 50.1 pg/ml (35.0 to 78.0); P < 0.0001). CXCL13 relationships to other serologic and clinical features (B) As with the Dartmouth RA Cohort, a strong relationship is seen with LogCXCL13 and immunoglobulin M rheumatoid factor (IgM RF) in seropositive samples whether measured by Spearman correlation (P < 00001) (C) or by CXCL13 Figure 3 Scatterplots demonstrate serum CXCL13 strongly correlates with seropositive rheumatoid arthritis and immunoglobulin M rheumatoid factor levels in the Sherbrooke Early Undifferentiated PolyArthritis Cohort, but has a weaker relationship to anticitrullinated plots demonstrate serum CXCL13 strongly correlates with seropositive rheumatoid arthritis and immunoglobulin M or levels in the Sherbrooke Early Undifferentiated PolyArthritis Cohort, but has a weaker relationship to anticitrullinate Figure 3 Scatterplots demonstrate serum CXCL13 strongly correlates with seropositive rheumatoid arthritis and immunoglobulin M rheumatoid factor levels in the Sherbrooke Early Undifferentiated PolyArthritis Cohort, but has a weaker relationship to anticitrullinated peptide/protein antibody levels. (A) Log-transformed C-X-C motif chemokine 13 (LogCXCL13) is higher in seropositive than in seronegative RA patients as determined by t-test (seropositive: n = 173, geometric mean (95% CI) = 323.6 pg/ml (223.9 to 477.5); seronegative: n = 166, geometric mean (95% CI) = 50.1 pg/ml (35.0 to 78.0); P < 0.0001). (B) As with the Dartmouth RA Cohort, a strong relationship is seen with LogCXCL13 and immunoglobulin M rheumatoid factor (IgM RF) in seropositive samples, whether measured by Spearman correlation (P < 0.0001) (C) or by CXCL13 tertile analysis (third-tertile mean RF = 223 ± 57 IU/ml, first- and second-tertile mean RF = 141 ± 75 IU/ml; P < 0.0001). (D) A significant relationship is found between CXCL13 and anticitrullinated peptide/protein antibody (ACPA) by Spearman correlation (P = 0.006). P < 0.05. Diagonal lines represent lines of best fit. whether it was evaluated by Spearman correlation accor- ding to number of erosions (n = 123; r = −0.12, P = 0.17) (data not shown) or by t-test to compare the presence or absence of erosions (0 vs ≥1 erosions, P = 0.34; <5 vs ≥5 erosions; P = 0.95). P = 0.14) (data not shown). As we observed with the Dartmouth RA Cohort, we found no relationship with shared epitope status in the seropositives (n = 132; P = 0.38) (data not shown) or with smoking status (P = 0.62). Additionally, when we combined the data set from both cohorts, we continued to find no relationship with either shared epitope or smoking status (Additional file 1). CXCL13 relationships to other serologic and clinical features (B) As with the Dartmouth RA Cohort, a strong relationship is seen with LogCXCL13 and immunoglobulin M rheumatoid factor (IgM RF) in seropositive samples, whether measured by Spearman correlation (P < 0.0001) (C) or by CXCL13 tertile analysis (third-tertile mean RF = 223 ± 57 IU/ml, first- and second-tertile mean RF = 141 ± 75 IU/ml; P < 0.0001). (D) A significant relationship is found between CXCL13 and anticitrullinated peptide/protein antibody (ACPA) by Spearman correlation (P = 0.006). P < 0.05. Diagonal lines represent lines of best fit. Figure 3 Scatterplots demonstrate serum CXCL13 strongly correlates with seropositive rheumatoid arthritis and immunoglobulin M rheumatoid factor levels in the Sherbrooke Early Undifferentiated PolyArthritis Cohort, but has a weaker relationship to anticitrullinated peptide/protein antibody levels. (A) Log-transformed C-X-C motif chemokine 13 (LogCXCL13) is higher in seropositive than in seronegative RA patients as determined by t-test (seropositive: n = 173, geometric mean (95% CI) = 323.6 pg/ml (223.9 to 477.5); seronegative: n = 166, geometric mean (95% CI) = 50.1 pg/ml (35.0 to 78.0); P < 0.0001). (B) As with the Dartmouth RA Cohort, a strong relationship is seen with LogCXCL13 and immunoglobulin M rheumatoid factor (IgM RF) in seropositive samples, whether measured by Spearman correlation (P < 0.0001) (C) or by CXCL13 tertile analysis (third-tertile mean RF = 223 ± 57 IU/ml, first- and second-tertile mean RF = 141 ± 75 IU/ml; P < 0.0001). (D) A significant relationship is found between CXCL13 and anticitrullinated peptide/protein antibody (ACPA) by Spearman correlation (P = 0.006). P < 0.05. Diagonal lines represent lines of best fit. Figure 3 Scatterplots demonstrate serum CXCL13 strongly correlates with seropositive rheumatoid arthritis and immunoglobulin M rheumatoid factor levels in the Sherbrooke Early Undifferentiated PolyArthritis Cohort, but has a weaker relationship to anticitrullinated peptide/protein antibody levels. (A) Log-transformed C-X-C motif chemokine 13 (LogCXCL13) is higher in seropositive than in seronegative RA patients as determined by t-test (seropositive: n = 173, geometric mean (95% CI) = 323.6 pg/ml (223.9 to 477.5); seronegative: n = 166, geometric mean (95% CI) = 50.1 pg/ml (35.0 to 78.0); P < 0.0001). Relationships between CXCL13 and disease activity measures in an early rheumatoid arthritis cohort Despite a strong relationship of serum CXCL13 with IgM RF seropositivity, both established and recent-onset RA cohorts exhibited weaker correlations between CXCL13 and serum IgG and IgG ACPA levels. These data suggest that elevated serum CXCL13 levels might correspond to a process in which non-class-switched B cells producing IgM RF were promoted independently of follicle and ger- minal center formation that leads to immunoglobulin heavy-chain class switching and IgG ACPA. To begin to address this model, we analyzed the relationship between serum CXCL13 levels and IgA RF. Relationships between CXCL13 and disease activity measures in an early rheumatoid arthritis cohort The recent-onset RA patients drawn from the Sherbrooke EUPA Cohort showed no association between log CXCL13 serum levels and either age or sex (P = 0.77 and P = 0.43 respectively) (data not shown). A correlation between log CXCL13 and log CRP values were identified when sero- positive and seronegative patients were combined (n = 339; r = 0.14, P = 0.01) (data not shown). This association dis- sipated when evaluated only in the seropositives (n = 173; r = 0.13, P = 0.08) (Figure 2C). A similar relationship of serum CXCL13 to DAS28-CRP levels was shown in all pa- tients (n = 335; r = 0.12, P = 0.02), but this association also went away when the analysis was limited to seropositives (n = 170; r = 0.08, P = 0.28) (Figure 2D). There was no re- lationship between log CXCL13 levels and the presence of erosions at baseline in seropositive patients, regardless of Analysis of log-transformed CXCL13 and log-trans- formed IgA RF in the seropositive patients of both co- horts were strongly correlated (P < 0.0001) (Figure 4A and 4C). When evaluated by CXCL13 tertile analysis, the highest tertile had much higher IgA RF values than the first and second tertiles in the Dartmouth RA Cohort Jones et al. Arthritis Research & Therapy 2014, 16:R103 http://arthritis-research.com/content/16/2/R103 Page 7 of 9 Figure 4 Scatterplots illustrate a strong relationship of serum CXCL13 with immunoglobulin A rheumatoid factor in seropositive rheumatoid arthritis patients in both the Dartmouth RA Cohort (established RA) and recent-onset rheumatoid arthritis patients from the Sherbrooke Early Undifferentiated PolyArthritis Cohort. (A) Pearson correlation analysis of log-transformed C-X-C motif chemokine 13 (LogCXCL13) values and log-transformed immunoglobulin A rheumatoid factor (Log IgA RF) in the Dartmouth RA Cohort shows a strong association (P < 0.0001). Relationships between CXCL13 and disease activity measures in an early rheumatoid arthritis cohort (B) CXCL13 tertile analysis confirms this relationship (third-tertile IgA RF: geometric mean (95% CI) = 45.0 IU/ml (28.0 to 72.3), first- and second-tertile IgA RF: geometric mean (95% CI) = 11.2 IU/ml (7.6 to 16.3); P < 0.0001). (C) In the Sherbrooke Early Undifferentiated PolyArthritis Cohort, the relationship between LogCXCL13 and Log IgA RF persists, whether measured by correlation (P < 0.0001) (D) or by CXCL13 tertile analysis (third-tertile IgA RF: geometric mean (95% CI) = 74.1 IU/ml (51.4 to 106.7), first- and second-tertile IgA RF: geometric mean (95% CI) = 20.4 IU/ml (15.1 to 27.5); P < 0.0001). P < 0.05. Diagonal lines represent lines of best fit. Although an association between CXCL13 and ACPA was observed, it was not as strong a relationship as that seen with RF (IgM RF: r = 0.45 and r = 0.54 in the estab- lished and early RA cohorts, respectively; IgG ACPA: r = 0.17 and r = 0.21, respectively). These observations sug gest interesting and potentially specific associations of CXCL13 with both RF autoantibody formation and the pathogenesis of RA. (third-tertile geometric mean (95% CI) 45.0 IU/ml (28.0 to 72.3), first- and second-tertile geometric mean (95% CI) 11.2 IU/ml (7.6 to 16.3); P < 0.0001) (Figure 4B). A simi- larly strong correlation was seen in recent-onset, mostly untreated RA patients (third-tertile geometric mean (95% CI) 74.1 IU/ml (51.4 to 106.7), first- and second-tertile geometric mean (95% CI) 20.4 IU/ml (15.1 to 27.5); P < 0.0001) (Figure 4D). Thus, serum CXCL13 levels exhibited strong correlations with both IgM and IgA RF titers. We evaluated patients with very high CXCL13 values (top decile) and did not observe any significant variation in RF or ACPA values compared with the remaining pa- tients in the highest tertile (data not shown). Further, we did not identify any competing diagnosis (for example, lymphoma or cryoglobulinemia) or therapy to account for the very high levels of CXCL13. Relationships between CXCL13 and disease activity measures in an early rheumatoid arthritis cohort (B) CXCL13 tertile analysis confirms this relationship (third-tertile IgA RF: geometric mean (95% CI) = 45.0 IU/ml (28.0 to 72.3), first- and second-tertile IgA RF: geometric mean (95% CI) = 11.2 IU/ml (7.6 to 16.3); P < 0.0001). (C) In the Sherbrooke Early Undifferentiated PolyArthritis Cohort, the relationship between LogCXCL13 and Log IgA RF persists, whether measured by correlation (P < 0.0001) (D) or by CXCL13 tertile analysis (third-tertile IgA RF: geometric mean (95% CI) = 74.1 IU/ml (51.4 to 106.7), first- and second-tertile IgA RF: geometric mean (95% CI) = 20.4 IU/ml (15.1 to 27.5); P < 0.0001). P < 0.05. Diagonal lines represent lines of best fit. Figure 4 Scatterplots illustrate a strong relationship of serum CXCL13 with immunoglobulin A rheumatoid factor in seropositive rheumatoid arthritis patients in both the Dartmouth RA Cohort (established RA) and recent-onset rheumatoid arthritis patients from the Sherbrooke Early Undifferentiated PolyArthritis Cohort. (A) Pearson correlation analysis of log-transformed C-X-C motif chemokine 13 (LogCXCL13) values and log-transformed immunoglobulin A rheumatoid factor (Log IgA RF) in the Dartmouth RA Cohort shows a strong association (P < 0.0001). (B) CXCL13 tertile analysis confirms this relationship (third-tertile IgA RF: geometric mean (95% CI) = 45.0 IU/ml (28.0 to 72.3), first- and second-tertile IgA RF: geometric mean (95% CI) = 11.2 IU/ml (7.6 to 16.3); P < 0.0001). (C) In the Sherbrooke Early Undifferentiated PolyArthritis Cohort, the relationship between LogCXCL13 and Log IgA RF persists, whether measured by correlation (P < 0.0001) (D) or by CXCL13 tertile analysis (third-tertile IgA RF: geometric mean (95% CI) = 74.1 IU/ml (51.4 to 106.7), first- and second-tertile IgA RF: geometric mean (95% CI) = 20.4 IU/ml (15.1 to 27.5); P < 0.0001). P < 0.05. Diagonal lines represent lines of best fit. Figure 4 Scatterplots illustrate a strong relationship of serum CXCL13 with immunoglobulin A rheumatoid factor in seropositive rheumatoid arthritis patients in both the Dartmouth RA Cohort (established RA) and recent-onset rheumatoid arthritis patients from the Sherbrooke Early Undifferentiated PolyArthritis Cohort. (A) Pearson correlation analysis of log-transformed C-X-C motif chemokine 13 (LogCXCL13) values and log-transformed immunoglobulin A rheumatoid factor (Log IgA RF) in the Dartmouth RA Cohort shows a strong association (P < 0.0001). Conclusion In our present report, we demonstrate that serum levels of the B-cell chemokine CXCL13 exhibit a strong relation- ship with seropositive RA. The nature of this correlation appears to be particularly strong for both IgM and IgA RF, whereas there is a weaker relationship with IgG-ACPA. A particular strength of this finding is its presence to nearly identical degrees in both an early RA cohort and an estab- lished RA cohort. Elevations of serum CXCL13 did not consistently associate with disease duration, sex or mea- sures of disease activity in seropositive RA patients. More- over, CXCL13 levels did not appear to associate with other features of seropositivity, such as the shared epitope. These results suggest that elevated CXCL13 levels may possibly be used to identify a distinct subset of seroposi- tive RA patients that may either promote or result from the expansion of RF-producing B cells. This interpretation is further supported by the findings reported in a small study (n = 20) demonstrating that serum CXCL13 levels did not correlate with DAS28 measures [11]. Perhaps more important, the same study identified a strong relationship between serum CXCL13 protein and synovial CXCL13 mRNA expression [11]. Thus, serum CXCL13 levels appear to derive from a syn- ovial inflammation process characterized by the pro- duction of CXCL13. Although this interpretation differs somewhat from prior reports, these latter studies may have been confounded by inclusion of seronegative pa- tients or the inappropriate use of RF level as a criterion for disease activity [11,17,18]. Clearly, additional studies are needed to clarify this issue. Abbreviations ACPA: Anticitrullinated peptide/protein antibody; CDAI: Clinical Disease Activity Index; CXCL13: C-X-C motif chemokine 13; CXCR5: C-X-C chemokine receptor type 5; DAS28–CRP: Disease Activity Score in 28 joints–C-reactive protein; EUPA: Early Undifferentiated Polyarthritis Cohort; hsCRP: High-sensitivity C- reactive protein; Log CXCL13: Log-transformed C-X-C motif chemokine 13; RA: Rheumatoid arthritis; RF: Rheumatoid factor. The surprisingly strong correlation between serum CXCL13 levels and RF (IgM and IgA) titers, relative to that seen with either serum IgG or IgG-ACPA levels, in both an established RA cohort and a recent-onset, mostly untreated RA cohort may clarify the role of CXCL13 in autoantibody production. A similar but lesser relationship was observed in a recent cross-sectional analysis [16]. The most straightforward interpretation is that a greater pro- portion of circulating RF derives from synovial production relative to IgG-ACPA and IgG, which are presumably pro- duced at other sites, including the bone marrow. Despite its simplicity, this model must account for why both IgM RF and its class-switched counterpart, IgA RF, are affected. Alternatively, high levels of CXCL13 production may Additional file Additional file 1: Figure S1. Evaluation of combined data from seropositive patients from the Dartmouth and Sherbrooke cohorts. The evaluation did not identify any relationship with shared epitope status or smoking. (A) Log C-X-C motif chemokine 13 (CXCL13) levels do not vary based on the presence or absence of the shared epitope (n = 258, P = 0.73). (B) Log CXCL13 levels have no relationship with smoking whether comparing current smokers (n = 80) to past smokers (n = 129; P = 0.69), current smokers to never-smokers (n = 125; P = 0.28) or current smokers to both past and never-smokers (P = 0.42). Additional file 1: Figure S1. Evaluation of combined data from seropositive patients from the Dartmouth and Sherbrooke cohorts. The evaluation did not identify any relationship with shared epitope status or smoking. (A) Log C-X-C motif chemokine 13 (CXCL13) levels do not vary based on the presence or absence of the shared epitope (n = 258, P = 0.73). (B) Log CXCL13 levels have no relationship with smoking whether comparing current smokers (n = 80) to past smokers (n = 129; P = 0.69), current smokers to never-smokers (n = 125; P = 0.28) or current smokers to both past and never-smokers (P = 0.42). Murine models indicate that the function of CXCL13– CXCR5 interactions promotes recruitment of B cells and follicular T helper cells to the follicle and germinal cen- ters in secondary lymphoid organs [19]. In humans, the precise role of CXCL13, let alone rheumatoid synovium, is less clear. Synovial expression of CXCL13 has been as- sociated with diffuse lymphoid infiltration as well as the presence of lymphoid aggregates that resemble germinal centers [7,9,20]. However, the relationship of synovial histology to either inflammation or autoantibody pro- duction remains controversial [3,21,22]. Discussion We report a strong relationship between elevated serum CXCL13 levels and seropositive RA that was seen in an established disease cohort and confirmed in a mostly un- treated early RA cohort. Modest associations were seen with disease activity measures in established RA, but no associations were present in patients with early disease. We did not find any evidence to support a relationship between CXCL13 and HLA-DRB1 alleles containing the shared epitope or complement C4B deficiency. The most striking finding was the strength of the relationship bet- ween serum CXCL13 with IgM and IgA RF relative to that seen with ACPA titers (Figures 1, 3 and 4), an ob- servation not due to the heterophilic effects of RF. The presence of elevated CXCL13 levels in seropo- sitive patients from two distinct cohorts composed of patients with established and recent-onset RA suggests that this abnormality may be maintained over the course of the disease and may not be extensively influenced by disease duration or prior therapy. Supporting this notion is our observation that the mean CXCL13 levels in the two cohorts were nearly identical (330 vs 323 pg/ml). By extension, this result would predict the absence of a Page 8 of 9 Jones et al. Arthritis Research & Therapy 2014, 16:R103 http://arthritis-research.com/content/16/2/R103 correlation between serum CXCL13 and disease activity measures. Indeed, only a modest relationship with DAS28-CRP, and no relationship with hsCRP or CDAI, was seen in the Dartmouth RA Cohort. In addition, no re- lationship was observed with baseline hsCRP, DAS28-CRP or radiographic erosions in the recent-onset RA patients from the Sherbrooke EUPA Cohort (Figure 2A to 2D). Similar findings (modest correlation with DAS28-CRP and no correlation with erosive disease) were recently reported in a French cross-sectional cohort [16]. Thus, elevated serum CXCL13 levels did not simply reflect quantitative differences in synovial or systemic inflamma- tion between patients; rather, these data suggest the pre- sence of a qualitatively distinct subset of seropositive RA manifested by specific increases in both IgM and IgA RF. reflect a pathologic process in which synovial plasma cell production of RF is selectively enhanced relative to ACPA or other IgGs. Indeed, a strong CXCL13–RF relationship does not establish causality; therefore, another possibility is that elevated RF levels somehow drive increased pro- duction of CXCL13. Competing interests The authors declare that they have no competing interests. References 1. Arend WP, Firestein GS: Pre-rheumatoid arthritis: predisposition and transition to clinical synovitis. Nat Rev Rheumatol 2012, 8:573–586. 1. Arend WP, Firestein GS: Pre-rheumatoid arthritis: predisposition and transition to clinical synovitis. Nat Rev Rheumatol 2012, 8:573–586. 19. Finch DK, Ettinger R, Karnell JL, Herbst R, Sleeman MA: Effects of CXCL13 inhibition on lymphoid follicles in models of autoimmune disease. Eur J Clin Invest 2013, 43:501–509. 2. Williams DG, Moyes SP, Mageed RA: Rheumatoid factor isotype switch and somatic mutation variants within rheumatoid arthritis synovium. Immunology 1999, 98:123–136. 2. Williams DG, Moyes SP, Mageed RA: Rheumatoid factor isotype switch and somatic mutation variants within rheumatoid arthritis synovium. Immunology 1999, 98:123–136. 20. Shi K, Hayashida K, Kaneko M, Hashimoto J, Tomita T, Lipsky PE, Yoshikawa H, Ochi T: Lymphoid chemokine B cell-attracting chemokine-1 (CXCL13) is expressed in germinal center of ectopic lymphoid follicles within the synovium of chronic arthritis patients. J Immunol 2001, 166:650–655. 3. Humby F, Bombardieri M, Manzo A, Kelly S, Blades MC, Kirkham B, Spencer J, Pitzalis C: Ectopic lymphoid structures support ongoing production of class-switched autoantibodies in rheumatoid synovium. PLoS Med 2009, 6:e1. 3. Humby F, Bombardieri M, Manzo A, Kelly S, Blades MC, Kirkham B, Spencer J, Pitzalis C: Ectopic lymphoid structures support ongoing production of class-switched autoantibodies in rheumatoid synovium. PLoS Med 2009, 6:e1. 21. Cantaert T, Kolln J, Timmer T, van der Pouw Kraan TC, Vandooren B, Thurlings RM, Cañete JD, Catrina AI, Out T, Verweij CL, Zhang Y, Tak PP, Baeten D: B lymphocyte autoimmunity in rheumatoid synovitis is independent of ectopic lymphoid neogenesis. J Immunol 2008, 181:785–794. 4. Luther SA, Lopez T, Bai W, Hanahan D, Cyster JG: BLC expression in pancreatic islets causes B cell recruitment and lymphotoxin-dependent lymphoid neogenesis. Immunity 2000, 12:471–481. 4. Luther SA, Lopez T, Bai W, Hanahan D, Cyster JG: BLC expression in pancreatic islets causes B cell recruitment and lymphotoxin-dependent lymphoid neogenesis. Immunity 2000, 12:471–481. 5. Ansel KM, Ngo VN, Hyman PL, Luther SA, Forster R, Sedgwick JD, Browning JL, Lipp M, Cyster JG: A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature 2000, 406:309–314. 5. Ansel KM, Ngo VN, Hyman PL, Luther SA, Forster R, Sedgwick JD, Browning JL, Lipp M, Cyster JG: A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature 2000, 406:309–314. 22. Author details 1 1Division of Rheumatology, Geisel School of Medicine at Dartmouth College, One Medical Center Dr., Lebanon, NH 03756, USA. 2Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth College, One Medical Center Dr., Lebanon, NH 03756, USA. 3Division of Rheumatology, Sherbrooke University Hospital, 2500 University Boulevard, Sherbrooke, QC J1K 2R1, Canada. 4Department of Medicine, Sherbrooke University Hospital, 2500 University Boulevard, Sherbrooke, QC J1K 2R1, Canada. 5Research Institute of the McGill University Health Center, 2155 Guy St Montreal, QC H3H 2R9, Canada. 6Division of Rheumatology, University of California San Diego School of Medicine, 200 W Arbor Dr, La Jolla, CA 92103, USA 7H l Th S V ll R d S D CA 16. Sellam J, Rouanet S, Hendel-Chavez H, Miceli-Richard C, Combe B, Sibilia J, Loët X, Tebib J, Jourdan R, Dougados M, Taoufik Y, Mariette X: CCL19, a B cell chemokine, is related to the decrease of blood memory B cells and predicts the clinical response to rituximab in patients with rheumatoid arthritis. Arthritis Rheum 2013, 65:2253–2261. 17. Meeuwisse CM, van der Linden MP, Rullmann TA, Allaart CF, Nelissen R, Huizinga TW, Garritsen A, Toes RE, van Schaik R, van der Helm-van Mil AH: Identification of CXCL13 as a marker for rheumatoid arthritis outcome using an in silico model of the rheumatic joint. Arthritis Rheum 2011, 63:1265–1273. USA. 7Halozyme Therapeutics, 11388 Sorrento Valley Road, San Diego, CA 92121, USA. 18. Bugatti S, Manzo A, Benaglio F, Klersy C, Vitolo B, Todoerti M, Sakellariou G, Montecucco C, Caporali R: Serum levels of CXCL13 are associated with ultrasonographic synovitis and predict power Doppler persistence in early rheumatoid arthritis treated with non-biological disease-modifying anti-rheumatic drugs. Arthritis Res Ther 2012, 14:R34. doi:10.1186/ar4552 Cite this article as: Jones et al.: Serum C-X-C motif chemokine 13 is elevated in early and established rheumatoid arthritis and correlates with rheumatoid factor levels. Arthritis Research & Therapy 2014 16:R103. Jones et al. Arthritis Research & Therapy 2014, 16:R103 http://arthritis-research.com/content/16/2/R103 factor alpha, tumor necrosis factor receptor superfamily member 9, and macrophage colony-stimulating factor. Arthritis Rheum 2008, 58:2257–2267. analysis and drafted and critically revised the manuscript. All authors read and approved the final manuscript. 11. Rosengren S, Wei N, Kalunian KC, Kavanaugh A, Boyle DL: CXCL13: a novel biomarker of B-cell return following rituximab treatment and synovitis in patients with rheumatoid arthritis. Rheumatology (Oxford) 2011, 50:603–610. Authors’ contributions JDJ was responsible for the study design, data collection and analysis and drafting and critical revision of the manuscript. BJH performed data collection and analysis and critical revision of the manuscript. GJC carried out data analysis, specimen collection and critical revision of the manuscript. AJF, PC, PL, AM and HM were responsible for patient recruitment and study design and critical revision of the manuscript. NC performed data collection and analysis and critical revision of the manuscript. DLB was responsible for the study design and critical revision of the manuscript. SR was responsible for the study design and performed data analysis and critical revision of the manuscript. GB was responsible for the study design, performed data collection and analysis and drafted and critically revised the manuscript. WFCR was responsible for the study design, performed data collection and Page 9 of 9 Page 9 of 9 Jones et al. Arthritis Research & Therapy 2014, 16:R103 http://arthritis-research.com/content/16/2/R103 Acknowledgements Th k g This work was supported by National Institutes of Health grant R21 AR-061643 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (to WFCR), by Arthritis Foundation Clinical to Research Transition award 5918 (to JDJ) and by grants 00/201 and RG06/108 from The Arthritis Society and Canadian Institutes for Health Research grant MOP-110959 (to GB). AJF, PL, AM and GB are members of the Centre de Recherche Clinique Etienne-LeBel at the Centre Hospitalier Universitaire de Sherbrooke, which received a team grant from the Health Research Funds of Quebec. Since 2007, the Sherbrooke EUPA cohort has received financial support from the Canadian ArTHritis CoHort (CATCH) study, designed and implemented by investigators and financially supported initially by Amgen Canada and Pfizer Canada via an unrestricted research grant. As of 2011, further support was provided by Hoffmann-La Roche, Chemicals of Belgium (UCB) Canada, Bristol-Myers Squibb Canada, Abbott Laboratories and Janssen Biotech (a wholly owned subsidiary of Johnson & Johnson). 12. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS, Medsger TA Jr, Mitchell DM, Neustadt DH, Pinals RS, Schaller JG, Sharp JT, Wilder RL, Hunder GG: The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988, 31:315–324. 13. Rigby WF, Wu YL, Zan M, Zhou B, Rosengren S, Carlson C, Hilton W, Yu CY: Increased frequency of complement C4B deficiency in rheumatoid arthritis. Arthritis Rheum 2012, 64:1338–1344. 14. Carrier N, Cossette P, Daniel C, de Brum-Fernandes A, Liang P, Ménard HA, Boire G: The DERAA HLA-DR alleles in patients with early polyarthritis: protection against severe disease and lack of association with rheumatoid arthritis autoantibodies. Arthritis Rheum 2009, 60:698–707. 15. van der Helm-van Mil AH, Verpoort KN, Breedveld FC, Huizinga TW, Toes RE de Vries RR: The HLA-DRB1 shared epitope alleles are primarily a risk factor for anti-cyclic citrullinated peptide antibodies and are not an independent risk factor for development of rheumatoid arthritis. Arthritis Rheum 2006, 54:1117–1121. References Thurlings RM, Wijbrandts CA, Mebius RE, Cantaert T, Dinant HJ, van der Pouw-Kraan TC, Verweij CL, Baeten D, Tak PP: Synovial lymphoid neogenesis does not define a specific clinical rheumatoid arthritis phenotype. Arthritis Rheum 2008, 58:1582–1589. g y p 6. Gunn MD, Ngo VN, Ansel KM, Ekland EH, Cyster JG, Williams LT: A B-cell- homing chemokine made in lymphoid follicles activates Burkitt's lymphoma receptor-1. Nature 1998, 391:799–803. 6. Gunn MD, Ngo VN, Ansel KM, Ekland EH, Cyster JG, Williams LT: A B-cell- homing chemokine made in lymphoid follicles activates Burkitt's lymphoma receptor-1. Nature 1998, 391:799–803. 7. Manzo A, Vitolo B, Humby F, Caporali R, Jarrossay D, Dell’Accio F, Ciardelli L, Uguccioni M, Montecucco C, Pitzalis C: Mature antigen-experienced T helper cells synthesize and secrete the B cell chemoattractant CXCL13 in the inflammatory environment of the rheumatoid joint. Arthritis Rheum 2008, 58:3377–3387. 8. Carlsen HS, Baekkevold ES, Morton HC, Haraldsen G, Brandtzaeg P: Monocyte- like and mature macrophages produce CXCL13 (B cell-attracting chemokine 1) in inflammatory lesions with lymphoid neogenesis. Blood 2004, 104:3021–3027. 9. Takemura S, Braun A, Crowson C, Kurtin PJ, Cofield RH, O'Fallon WM, Goronzy JJ, Weyand CM: Lymphoid neogenesis in rheumatoid synovitis. J Immunol 2001, 167:1072–1080. 10. Rioja I, Hughes FJ, Sharp CH, Warnock LC, Montgomery DS, Akil M, Wilson AG, Binks MH, Dickson MC: Potential novel biomarkers of disease activity in rheumatoid arthritis patients: CXCL13, CCL23, transforming growth
https://openalex.org/W2750585446	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0210339&type=printable	English	null	<i>Sporosarcina pasteurii</i> can form nanoscale crystals on cell surface	bioRxiv (Cold Spring Harbor Laboratory)	2,017	cc-by	8,232	Tanushree Ghosh1, Swayamdipta BhaduriID1, Carlo Montemagno2†, Aloke KumarID3* 1 Department of Mechanical Engineering, University of Alberta, Edmonton, Alberta, Canada, 2 Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada, 3 Department of Mechanical Engineering, Indian Institute of Science, Bangalore, Karnataka, India † Deceased. * alokekumar@iisc.ac.in † Deceased. * alokekumar@iisc.ac.in a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. OPEN ACCESS Citation: Ghosh T, Bhaduri S, Montemagno C, Kumar A (2019) Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface. PLoS ONE 14(1): e0210339. https://doi. org/10.1371/journal.pone.0210339 Editor: Varenyam Achal, Guangdong Technion Israel Institute of Technology, CHINA Received: October 3, 2018 Accepted: December 20, 2018 Published: January 30, 2019 Copyright: © 2019 Ghosh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Tanushree Ghosh1, Swayamdipta BhaduriID1, Carlo Montemagno2†, Aloke KumarID3* Tanushree Ghosh1, Swayamdipta BhaduriID1, Carlo Montemagno2†, Aloke KumarID3* Abstract The bacterium Sporosarcina pasteurii (SP) is known for its ability to cause the phenomenon of microbially induced calcium carbonate precipitation (MICP). We explored bacterial partici- pation in the initial stages of the MICP process at the cellular length scale under two different growth environments (a) liquid culture (b) MICP in a soft agar (0.5%) column. In the liquid culture, ex-situ imaging of the cellular environment indicated that S. pasteurii was facilitating nucleation of nanoscale crystals of calcium carbonate on bacterial cell surface and its growth via ureolysis. During the same period, the meso-scale environment (bulk medium) was found to have overgrown calcium carbonate crystals. The effect of media components (urea, CaCl2), presence of live and dead in the growth medium were explored. The agar col- umn method allows for in-situ visualization of the phenomena, and using this platform, we found conclusive evidence of the bacterial cell surface facilitating formation of nanoscale crystals in the microenvironment. Here also the bulk environment or the meso-scale envi- ronment was found to possess overgrown calcium carbonate crystals. Extensive elemental analysis using Energy dispersive X-ray spectroscopy (EDS) and X-ray powder diffraction (XRD), confirmed that the crystals to be calcium carbonate, and two different polymorphs (calcite and vaterite) were identified. Active participation of S. pasteurii cell surface as the site of calcium carbonate precipitation has been shown using EDS elemental mapping with Scanning transmission electron microscopy (STEM) and scanning electron microscopy (SEM). Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface microorganisms that can produce urease (urea amidohydrolase) and hence are able to hydro- lyze urea to induce CaCO3 precipitation [1, 13–15]. Sporosarcina pasteurii (SP), formerly Bacil- lus pasteurii, is a non-pathogenic, endospore forming soil bacterium well known for its ability to precipitate CaCO3 through ureolysis [8]. S. pasteurii has attracted significant attention from researchers for its unique feature of CaCO3 precipitation [12, 16–20]. It is being investigated for possible usefulness in a multitude of applications including underground storage of carbon, healing masonry structures of archaeological importance, and long-term sealing of geologic cracks in large-scale structures [3, 15, 20–24].The MICP phenomenon has been exhaustively studied in bulk systems, sand columns [20, 25, 26], and bio-cementation processes [26, 27] to understand the strength provided by the CaCO3. The bacterial involvement has also been explored to understand its importance, control mineral deposition rate, urease activity, crystal size and participation of bacterial cells. Hammes and Verstraete [14] reported four very impor- tant parameters that affect MICP are pH, dissolved inorganic carbon (DIC), calcium concen- tration and available nucleation site. The saturation rate of carbonate ion concentration (CO3 2 −) is controlled by the first three parameters, and it is believed that bacterial cell surface at the nucleation site can facilitate stable and continuous CaCO3 deposition [15]. However, a bacte- ria-free solution loaded with urease enzyme can also induce CaCO3 precipitation. Mitchell and Ferris [17] studied the influence of bacteria on the nucleation of MICP in which a bacte- ria-free enzyme solution was compared to a bacteria induced environment. The authors reported significant positive effect of bacterial presence (referred as “bacteria-inclusive”) on the increase of size and growth rate of the precipitated crystal although the idea of bacterial control of MICP was rejected. The bacteria-free urease solution showed similarities with the bulk chemical precipitation, while differences were observed in the aqueous micro-environ- ment of bacteria [17]. Competing interests: The authors have declared that no competing interests exist. It has been suggested by other researchers that bacterial cell walls can serve as nucleation sites since bacterial cell surfaces carry negatively charged groups [3, 14, 23, 28–31]. These nega- tive charges can influence the binding of cations (e.g. Ca2+) on the cell surface and eventually act as nucleation sites when reacting with carbonate anions from the solution to form insoluble CaCO3 [1, 29, 32]. Introduction Funding: AK acknowledges partial support from NSERC, Canada. AK acknowledges partial support from the Startup Grant provided by the Indian Institute of Science. There were no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Biomineralization of calcium carbonate (CaCO3) or its precipitation due to chemical alteration of the environment induced by the microbial activity is known as microbiologically induced calcium carbonate precipitation (MICP) [1–4]. Bacteria can participate in the MICP phenome- non by means of various mechanisms such as urea hydrolysis [1, 2], photosynthesis [5, 6], sul- fate reduction [7, 8], anaerobic sulfide oxidation [9], biofilm formation [10] and mineral adsorption to extracellular polymeric substances [11, 12]. There has been significant interest in 1 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 Definitive proofs of involvement of bacterial cell surfaces in complex envi- ronments on the nucleation process are rare. Ghashghaei and Emtiazi [33] reported the pres- ence of nanocrystals of CaCO3 on cell walls of the bacteria Enterobacter ludwigii for experiments performed with a liquid culture. While the data is suggestive of the role of cell wall, a more definite proof is desirable. In a contrasting study, Bundeleva et al. [34] reported on MICP with the anoxygenic Rhodovulum sp., where they failed to show the existence of CaCO3 on or near live cells. The authors claim the existence of certain cell protection mecha- nisms against mineral encrustation at the vicinity of live bacteria and they offer the idea of mineral precipitation at a certain distance from the cell surface [34]. In another study, Harris et al. [10] investigated S. pasteurii biofilm and reported real time biochemical changes during CaCO3 precipitation occurred in the proximity of biofilms. They found rapid changes in the pH and Ca+2 concentrations near (within 600 μm) the biofilm during the MICP process. The metabolic activity (e.g. ureolysis) in the bacterial micro-environment (proximity of bacterial cells or biofilm) immediately changes the pH and induces CaCO3 precipitation. While this study explored the direct participation of live S. pasteurii biofilm in the MICP process, cellular level contribution by bacterium was not elucidated [10]. In a recent study, Zhang et al. [35] has denied the role of bacterial cell surface or any other negatively charged surface (such as poly- styrene microspheres with carboxyl or sulfonic group modified surfaces) in the CaCO3 precip- itation for their experimental conditions. In contradiction to Zhang’s report, the formation of CaCO3 crystals on the negatively charged surfaces were well studied by many researches [36– 38]. Negatively charged surfaces terminated with–COOH,–OH, and–NH2 functional groups 2 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface were reported in biomineralization processes of CaCO3 by Deng et al. [36]. Authors explored the effect of −OH and −NH2 terminated surfaces, where nucleation of amorphous calcium car- bonate (ACC) by adsorption of CaCO3 clusters from solution was obtained. However, direct formation of calcites was observed on −COOH surface which is more negatively charged. Cao et al. [38] showed similar bio-mineralization on the negatively charged hydrophilic polypep- tides at air-liquid interface. The experimental conditions in Zhang’s [35] study were not favor- able to facilitate CaCO3 precipitation on negatively charged surfaces. Also, the microscopic observations made by Zhang et al. [35] were not detailed enough to capture bacterial cell sur- faces at single-cell level. The participation of bacterial cell surface in the MICP process due to ureolytic activity and the proof of CaCO3 deposition on bacterial cell surfaces should be rein- vestigated. Thus, we see that the issue of the role of bacterial cells on mineral precipitation in MICP remains controversial and detailed studies delineating the exact mechanism leading to the bacterial cell surface deposition of CaCO3 in in-situ conditions are necessary. To the address the fundamental mechanism of MICP by S. pasteurii in complex environ- ments we designed several combinations of MICP compatible liquid medium to confirm direct evidence of CaCO3 precipitation on S. pasteurii cells. Single-cell level electron microscopy and detailed elemental analysis were used to explore initial stages of CaCO3 precipitation. We also designed a semisolid-agar column setup, which allows us to directly investigate in-situ mineral precipitation. Our setup consists of a 0.5% agar-column that is stab-inoculated and MICP pro- ceeds in the setup as downward traveling conspicuous mineral trails. We investigated the micro and macroenvironment of MICP and conclusively demonstrated the presence of nano- meter-sized crystals of CaCO3 on the bacterial cell surfaces. While this can be considered as definitive evidence that the cell wall does serve as a nucleation site, other mechanisms of nucle- ation are not ruled out. Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface Table 1. Experimental setups implemented for liquid culture studies. Experimental sample (n = 3) NB medium components and initial pH Urea Calcium chloride SP (Live/Dead) Initial pH NBUC-7 + + - 7 NBUC-9 + + - 9 NBUC-7SPdead + + + Dead 7 NBUC-7SPlive + + + Live 7 NBC-7SPlive - + + Live 7 NBU-7SPlive + - + Live 7 NBUC-9SPdead + + + Dead 9 NBUC-9SPlive + + + Live 9 NBUC-7 Urease + + - 7 ‘+’ or ‘-’ represents addition or deletion of components to the medium SP stands for Sporosarcina pasteurii SPdead or SPlive represents the addition of killed or live bacterial inoculums to NBUC medium NBC- only CaCl2 supplemented NB medium NBU-only urea supplemented NB medium Table 1. Experimental setups implemented for liquid culture studies. (ACS reagent 99%) and phenol red solution (#1072420100) were purchased from Sigma- Aldrich (Sigma-Aldrich, St. Louis, Missouri, USA). All the chemicals were used as purchased and solutions were prepared in Milli Q (18.2 MO) water. p p Different experimental combinations were prepared to observe the role of bacteria and the effect of composition of growth medium on the initiation of CaCO3 precipitation in liquid cul- ture. The effect of bacterial metabolic activity (in particular urease activity) and initial pH of growth medium were investigated for various combinations (see Table 1). Experiments were repeated in triplicate, unless otherwise mentioned. NBUC medium with initial pH of 7 and 9 were incubated at 30˚C without microbial inoculum and referred to as control set for each combination. The effect of proteins and salts present in the growth medium on the MICP pro- cess at two different pH was observed when no microbial inoculum were used. These served as media composition controls. Live S. pasteurii cells were immediately used as inoculum of NBUC, NBC and NBU media. The dead cells of S. pasteurii were prepared by heat killing of bacterial cells, autoclaving them at 121˚C for 20 min. Heat killed dead S. pasteurii cells were collected and washed with saline medium before addition to NBUC media. Table 1 shows all the setup where live and dead S. pasteurii are mentioned in subscript to the media composition. Microorganism and MICP conditions Sporosarcina pasteurii (Miquel) Yoon et al. ATCC 11859 used throughout the MICP study and was cultured in ATCC recommended Tris-YE medium for stock preparation and pilot culture [1]. Live S. pasteurii cells were collected by centrifugation and washed with saline water. The washing step was repeated until the pH of the supernantant became neutral (pH = 7) and then cells were resuspended in the culture medium. The cell count was maintained at 1×106 CFU/ ml of live bacteria approximately. For CaCO3 precipitation experiments, S. pasteurii cultures were prepared in nutrient medium with urea and CaCl2 supplements. The liquid CaCO3 pre- cipitation medium was prepared with nutrient broth (NB) containing urea (U) and CaCl2 (C) and together mentioned as NBUC throughout the study. The NBUC containing the following L-1 of double distilled water: 3g nutrient broth (NB); 20g Urea; 2.8g CaCl2. Nutrient Broth (NB) was prepared by mixing 5 g peptone; 3 g beef extract and 2 g sodium chloride per 1L of distilled water [39]. The pH of this medium was adjusted to 6.8 ± 0.2. Solution mixtures were sterilized by autoclave wet sterilization method (121˚C, 103421 Nm-2) for 15 min. Urea and CaCl2 solutions were filter-sterilized (PTFE syringe filters, pore size 0.2 μm, Sigma-Aldrich, St. Louis, Missouri, USA) and separately added to the NB medium before bacterial inoculation. All the liquid cultures were incubated in aerobic conditions at 30˚C with an orbital shaker operated at 120 rpm. A pH indicator dye (10 μL of phenol red) was added to all the control and test sets after 24 hours of incubation to visualize the pH change. All pH values were mea- sured using pH meter (Mettler Toledo S2 Handheld pH/mV Meter Field Kit) at 25 oC. Pep- tone, beef extract, urea, NaCl, HCl, NaOH and bacteriological agar were purchased from Fischer Scientific (Thermo-Fisher Scientific, Waltham, Massachusetts, USA). CaCl2. 2H2O 3 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 X-ray diffraction spectroscopy (XRD) and energy dispersive spectroscopy (EDS) Liquid medium and agar column samples were analysed for consolidated mineral precipita- tion. For XRD analysis, CaCO3 precipitate from agar column were collected, air dried and ground to powder. Ssamples were scanned by X-ray diffractometer (powder XRD, Rigaku Ultimate IV, Rigaku Corporation, Tokyo, Japan) and analysed for chemical components (Jade XRD pattern analysis software). EDS were performed alongwith SEM and TEM imaging on the bacterial cell surface and on to the surface of precipitated mineral. Chemical components were determined using EDS elemental mapping. MICP in semisolid-agar medium Visualization of MICP was finally aided by semisolid-agar columns, prepared by following Bang’s urea-CaCl2 liquid medium [1] with modification. The modified media contain (w/v) peptone 0.15%; beef extract 0.09%, sodium chloride 0.06% and CaCl2, 0.28%. The pH of the medium was adjusted to 7.0 and then 0.5% agar was added prior to autoclaving. Urea (2%) was added separately after autoclaving when media temperature cooled down to approxi- mately 50–60˚C. To create the agar columns, 10 ml of liquid agar was poured into upright test tubes and allowed to cool inside a biosafety cabinet, which finally resulted in columns of approximately 5 cm in length. Subsequently, these agar columns were inoculated by stabbing the free surface of the agar column with pre-cultured S. pasteurii using a stabbing needle. Fresh liquid media were poured on to the agar column to prevent drying of the agar surface. 4 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface The bacteria inoculated columns along with control sets were incubated at 30˚C for a maxi- mum duration of 7 days. Electron microscopy Bacterial broth cultures grown in different culture medium (experimental combinations from Table 1) were observed for CaCO3 deposition using field emmission scanning electron microscopy with EDS (Zeiss Sigma FESEM, Oberkochen Germany) and High resolution transmission elelctron microscopy (JEOL JEM-ARM200CF S/TEM with EDX, Massachusetts, US). For all the experimental setup with initial pH 7, culture aliquots were obtained after 24 hours of incubation at 30˚C. The setups with the initial pH 9, bacterial cells were collected within 1 hour of inoculation. Control sets were treated accordingly. The SEM and TEM sam- ples were prepared by fixing and dehydration in graded ethanol upto 100% ethanol[40, 41]. Agar columns were also investigated using both SEM and TEM. The cross-sections of the agar-column were fixed and dehydrated for SEM imaging. Every SEM sample was gold sput- tered (Denton Vacuum, Desk II, Moorestown, New Jersey) before imaging. y g g Prior to TEM, NBUC agar columns were cut at approximately mid-height (~ 2.5 cm) where bacterial activity were observed by optical microscopy. Agar slices were cut into small cubes and fixed in solution containing 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phos- phate buffer (pH = 7.4) for 30 min. The fixation process allows in-situ preservation of S. pas- teurii cells and its micro-environment. Post-fixation treatment of 1% Osmium tetroxide in 0.1 phosphate buffer was performed for 1 hour. Standard protocols for buffer washing and dehy- dration through graded alcohol were followed [42]. Samples were infiltrated with Spurr’s resin (1:1 of Ethanol: Spurr mixture) for 3 hours and then kept in 100% Spurr for 24 hours. Samples were then embedded in flat molds with fresh Spurr’s resin and cured overnight at 70˚C. Cured embedded resin capsules were sectioned in 70–90 nm thin sections using ultramicrotome (Reichert-Jung UltraCut E, Vienna, Austria) and mounted on copper grid for transmission electron microscopy (TEM, Philip-FEI, Morgangni 268, Oregon, USA) operated at 80kV. Results and discussion Fig 1 shows the parametric evaluation of the MICP phenomenon for nine different setups after 24 hours of culture. Phenol red (pH indicator dye) was added to all the nine cases to evaluate change in pH from neutral to alkaline (yellow/orange to pink). NBUC-7, NBUC-9 and NBUC- 7Urease denote the control sets, which were not inoculated with S. pasteurii (refer to Table 1). For sets NBUC-7SPlive, NBU-7SPlive and NBUC-7Urease the change of color indicates ureoly- tic activity. A comparison of NBUC-7SPdead and NBUC-7SPlive, suggests that bacterial activity led to change in pH; precipitation was also observed for NBUC-7SPlive. In the control set 5 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface Fig 1. Parametric evaluation of the MICP phenomenon by S. pasteurii in liquid media. Phenol red was used as pH indicator dye in culture tubes showing the final pH after 24h of culture. The text indicates the initial culture condition (see also Table 1). Fig 1. Parametric evaluation of the MICP phenomenon by S. pasteurii in liquid media. Phenol red was used as pH indicator dye in culture tubes showing the final pH after 24h of culture. The text indicates the initial culture condition (see also Table 1). https://doi.org/10.1371/journal.pone.0210339.g001 NBC-7SPlive (without urea), bacterial activity was unable to change medium pH and also no precipitation was observed even in the presence of same molar concentration of Ca+2 as NBUC-7SPlive. Hence NBC medium served as calcium positive control, where media conpo- nent unable to induce precipitation. On the other hand, NBU-7SPlive served as calcium nega- tive control and did not show any precipitation due to absence of Ca+2, despite a change in pH. The biophysical phenomenon of MICP has been investigated by several researchers and the following sequence of reactions (Eqs 1–6) has typically been suggested to lead to MICP [23, 26, 43]: COðNH2Þ2 þ H2O! ureolysis NH2COOH þ NH3 ð1Þ NH2COOH þ H2O ! NH3 þ H2CO3 ð2Þ 2NH3 þ 2H2O ! 2NHþ 4 þ 2OHð3Þ 2OH þ H2CO3 ! CO23 þ 2H2O ð4Þ Cell þ Ca2þ ! Cell Ca2þ ð5Þ Cell Ca2þ þ CO23 ! Cell CaCO3 ð6Þ COðNH2Þ2 þ H2O! ureolysis NH2COOH þ NH3 ð1Þ ð1Þ ð2Þ ð3Þ 2OH þ H2CO3 ! CO23 þ 2H2O Cell þ Ca2þ ! Cell Ca2þ ð5Þ Cell Ca2þ þ CO23 ! Results and discussion Cell CaCO3 ð6Þ ð6Þ The precipitates obtained from NBUC-9SPlive, NBUC-9SPdead and NBUC-7Urease was imaged using FESEM and the results are shown in Fig 2. Different morphologies of crystalline precipitates were observed in the different cases. For conditions of Fig 2A to 2D, precipitation within a very short time-scale (~ few minutes) was observed. For the NBUC-9SPdead set, rhom- bohedral crystalline precipiates were observed (Fig 2C and 2D). The morphology of precipi- tates for the NBUC-9SPlive set (Fig 2A and 2B), was distinctly different from the NBUC- 9SPdead set (Fig 2C and 2D). In Fig 2A and 2B, precipitation in the form of microspheres with a significant population of embedded bacterial cells was observed. These results suggest that prcipitation in the NBUC-9SPdead was chemical in nature, whereas NBUC-9SPlive set precipita- tion is likely both chemically and biochemically induced. The change in medium pH due to urease activity was supported by the urease enzyme supplemented (NBUC-7urease) test (Fig 2E and 2F). The MICP process is only observed for the NBUC-7SPlive and NBUC-9SPlive sets (see also Figure A in S1 File). 6 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface Fig 2. SEM images showing the effects of culture conditions on precipitate morphology. (a) Effect of initial pH of NBUC medium (NBUC-9SPlive) showing bacteria bound to crystalline microspheres. (b) Zoomed in section of (a). (c Typical calcite precipitation in NBUC-9SPdead medium with dead S. pasteurii cells. The zoomed in image (d) showing orthorhombic crystal structure of precipitates (e) precipitation induced by Urease (EC 3.5.1.5) supplemented condition (NBUC-7 Urease) where bacteria were absent. (f) zoomed in section of (e). https://doi org/10 1371/journal pone 0210339 g002 Fig 2. SEM images showing the effects of culture conditions on precipitate morphology. (a) Effect of initial pH of NBUC medium (NBUC-9SPlive) showing bacteria bound to crystalline microspheres. (b) Zoomed in section of (a). (c) Typical calcite precipitation in NBUC-9SPdead medium with dead S. pasteurii cells. The zoomed in image (d) showing orthorhombic crystal structure of precipitates (e) precipitation induced by Urease (EC 3.5.1.5) supplemented condition (NBUC-7 Urease) where bacteria were absent. (f) zoomed in section of (e). https://doi.org/10.1371/journal.pone.0210339.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 Nanoscale environment of S. pasteurii To investigate the nanoscale environment of the S. pasteurii cell surface, FESEM imaging along with EDS analysis were performed on the S. pasteurii cell surface at the single-cell level. Fig 3 depicts the data that was obtained for the NBC-7SPlive (Fig 3A to 3C) and NBUC-7SPlive (Fig 3D and 3F). Fig 3A and 3B shows no evidence of any precipitation on the cell surface, whereas for the NBUC-7SPlive set definitive precipitation can be observed on the cell surface. Further, EDS elemental map spectrum were obtained (at 20kV, dwelling time 50μs, process time 5s) for S. pasteurii cell surface from NBC-7SPlive (Fig 3C) and NBUC-7SPlive (Fig 3F) and they confirmed the presence of Ca+2 only on S. pasteurii cell from NBUC-7SPlive set. Note, that the NBC-7SPlive setup gives us an positive control where Ca+2 ions were present in same con- centration (25 mM CaCl2) as in NBUC-7SPlive set. For the NBUC-9SPdead set, no traces of cell surface deposition were found on the dead S. pasteurii cell. Fig 3E shows S. pasteurii cell sur- faces were observed to have islands of nano-sized deposition with strong intensities of Ca Kα. It provides strong support for the hypothesis that bacterial cell surfaces can serve as nucleation sites for CaCO3 precipitation or the MICP phenomenon. 7 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface Fig 3. SEM and EDS analysis of S. pasteurii. (a-b) SEM images of S. pasteurii control cells from NBC-7SPlive showing bacterial cells with no evidence of crystalline deposits on the cell wall. (c) EDS spectra collected on (b). (d-e) SEM images of S. pasteurii cells from NBUC-7SPlive clearly showing presence of crystalline deposits on the cell-wall. (f) EDS spectra collected on (e) and clearly showing presence of Ca. https://doi.org/10.1371/journal.pone.0210339.g003 Fig 3. SEM and EDS analysis of S. pasteurii. (a-b) SEM images of S. pasteurii control cells from NBC-7SPlive showing bacterial cells with no evidence of crystalline deposits on the cell wall. (c) EDS spectra collected on (b). (d-e) SEM images of S. pasteurii cells from NBUC-7SPlive clearly showing presence of crystalline deposits on the cell-wall. (f) EDS spectra collected on (e) and clearly showing presence of Ca. https://doi.org/10.1371/journal.pone.0210339.g003 The precipitation found in NBUC-7SPlive were mostly spherical, nano to micron sized and located on or adjacent to S. pasteurii cells. Nanoscale environment of S. pasteurii A plausible mechanism is schematically represented (Figure B in S1 File) and compared with FESEM images to relate all the observations. S. pas- teurii cells were observed to covered by CaCO3 either partially or fully. Spherical growth of CaCO3 was also observed in aggregation with S. pasteurii cells. The partially covered (partially encrusted) S. pasteurii cells were found to have islands of CaCO3 on cell surface. Whereas, fully encrusted cells found to have terminal spherical growth of CaCO3. The over-growth of nano/micron-sized CaCO3 may result in the death of fully encrusted bacteria leaving the impression of its existence. The structure of calcium carbonate precipitate may differ depend- ing on the media constituents (chemical content), pH, protein (enzyme content) and poly- meric substances (EPS content) and other medium additives. Thus, the morphology of the crystals can provide important clues about the precipitation pathway. Figs 2 and 3 also suggests that the MICP phenomenon can be segregated into two spatial domains (i) the nanoscale neighborhood of the cell surface (ii) the bulk medium, which can be called the meso-scale environment of the cell. Our study was aimed at understanding the relevance of the former. y g The S. pasteurii cell surface was further investigated by STEM associated with EDS (Fig 4). Fig 4A and 4B show STEM images of a single S. pasteurii cell obtained from NBC-7SPlive (Fig 4A ans 4B) and NBUC-7SPlive (Fig 4D and 4E). EDS elemental mapping was obtained for both samples. A cumulative map image was created for single-cell surface from NBC-7SPlive (Fig 4C) and NBUC-7SPlive (Fig 4F) to demonstrate co-localization of elements corresponding to EDS response. The distribution of individual elements of CaCO3 (C, O and Ca) were imaged separately for NBC-7SPlive (Fig 4Ci to 4Ciii) and NBUC-7SPlive (Fig 4Fi to 4Fiii). Fig 4 clearly shows the cell sample from NBUC-7SPlive encrused with calcium on the periphery of the cell surface indicating the nucleation of CaCO3. The map spectrum was quantified by using AZTech-EDS software and elemental signals were integrated along the S. pasteurii cell surface for NBC-7SPlive (Figs 5A and 4B) and NBUC-7SPlive (Figs 5C and 4D). The cross-sectional line profiles (Fig 5B and 5D) corresponding to image sections (Fig 5A-i and 5C-i) showed the intensities quantified for C, O and Ca. Nanoscale environment of S. pasteurii The line profile for Fig 5A-i is shown in Fig 5B and 5A very weak traces of Ca intensity on the cell boundary can be seen. On the other hand, image section of NBUC-7SPlive (Fig 5C-i) shows a strong signal from Ca along the cell boundary (Fig 5D) resulting in to two Ca-peaks. Remarkable increase in intensities for Ca at the fully encrusted cell towards both edges (Fig 5C-i, CaCO3 regions) was observed, which indicates a 8 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface Fig 4. STEM-BF and DF images and EDS elemental mapping on single S. pasteurii cells. (a) STEM-BF image of S. pasteurii cells from NBC-7SPlive. (b) zoomed in STEM-DF image at boxed site (c) A cumulative map created by overlaying EDS elemental mapping of (b). c(i-iii) show individual elemental distribution map for C, O and Ca along the S. pasteurii cell surface. (d) STEM-BF image of S. pasteurii cell from NBUC-7SPlive. (e) zoomed in STEM-DF image at boxed site (f) cumulative overlaid map f(i-iii) show individual elemental distribution map for C, O and Ca along the S. pasteurii cell surface. https://doi org/10 1371/journal pone 0210339 g004 Fig 4. STEM-BF and DF images and EDS elemental mapping on single S. pasteurii cells. (a) STEM-BF image of S. pasteurii cells from NBC-7SPlive. (b) zoomed in STEM-DF image at boxed site (c) A cumulative map created by overlaying EDS elemental mapping of (b). c(i-iii) show individual elemental distribution map for C, O and Ca along the S. pasteurii cell surface. (d) STEM-BF image of S. pasteurii cell from NBUC-7SPlive. (e) zoomed in STEM-DF image at boxed site (f) cumulative overlaid map f(i-iii) show individual elemental distribution map for C, O and Ca along the S. pasteurii cell surface. https://doi.org/10.1371/journal.pone.0210339.g004 https://doi.org/10.1371/journal.pone.0210339.g004 https://doi.org/10.1371/journal.pone.0210339.g004 cell surface coating of Ca. Similar increase in intensities along the S. pasteurii cell boundary were also found for C and O which confirms the formation of CaCO3 coating on S. pasteurii cell. The Ca content on S. pasteurii cells in NBUC medium was found to be ~600 fold higher along the bacterial cell than obtained in the NBC-SP cells. Nanoscale environment of S. pasteurii The Ca content on the NBC-SP cells were negligible compared to NBUC-SP cells, which rule out the adsorbtion of Ca+2 on the bac- terial extrapolymeric substances or to the medium components when ureolytic activity is absent.This gives us a direct evidance of S. pasteurii cell surface deposition The elemental con- tent were also quantified and compared for C, O and Ca from the intensities obtained from mapping EDS spectrum (Figure C in S1 File). Agar column study To understand growth of S. pasteurii in a less-mobilized and porous environment, an agar col- umn with stab culture was observed for a period of one week. The agar-cloumn samples were PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 9 / 15 Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface Fig 5. STEM and extracted line profile of CaCO3 deposited on S. pasteurii cell surface. The dark field TEM images representing the control S. pasteurii cell from NBC medium (a) and from NBUC medium (c). Scale bar is 500 nm. The area demarcated by the blue rectanges is the area where corresponding element intensities were integrated and presented along the middle blue line for a distance of 2 μm. The (b and d) EDS line scan showing the extracted intensity profile for K series of C, O and Ca. Solid lines (b) showing C, O and Ca intensities obtained from NBC-7SPlive cell surface where dashed line (d) intensiy profile for NBUC-7SPlive cell surface. https://doi.org/10.1371/journal.pone.0210339.g005 Fig 5. STEM and extracted line profile of CaCO3 deposited on S. pasteurii cell surface. The dark field TEM images representing the control S. pasteurii cell from NBC medium (a) and from NBUC medium (c). Scale bar is 500 nm. The area demarcated by the blue rectanges is the area where corresponding element intensities were integrated and presented along the middle blue line for a distance of 2 μm. The (b and d) EDS line scan showing the extracted intensity profile for K series of C, O and Ca. Solid lines (b) showing C, O and Ca intensities obtained from NBC-7SPlive cell surface where dashed line (d) intensiy profile for NBUC-7SPlive cell surface. https://doi.org/10.1371/journal.pone.0210339.g005 https://doi.org/10.1371/journal.pone.0210339.g005 fixed with glutaraldehyde allowing us to study the MICP phenomenon in-situ. Fig 6A shows the original culture tubes containg growth spans after day-1 and day-7. Extensive calcium car- bonate deposition can be prominently seen in Fig 6B after 7 days of incubation. The agar media acted as soft, porous and transperant nutrient riched environment to monitor the bacte- rial motility and calcite precipitation. Fig 6C depicts a scanning electron microscopy image of the agar column, where porous structures can clearly be seen. Fig 6D also shows the presence of deposits in the agar column after 7 days of bacterial activity. S. pasteurii bacterium posses flagellum, which allows it to navigate the porous structure of the agar column. PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 Agar column study The primary advantage of the agar-column experimental setup is in-situ visualization and easy access for supporting characterizations. The agar column was section at a depth of 2.5 cm after a period of 7 days and crystalline depositions were characterized by other forms of microscopy. Specifically, the agar colunm was characterized using optical microsopy, trans- mission electron microscopy (TEM), selected area diffraction pattern (SAED) and elemental characteization was determined by powder XRD and energy-dispersive X-ray spectroscopy (EDS). Optical microscopy of ultrathin (~80 nm) sections of the agar medium revealed Fig 6. MICP in agar column (a) Two samples containing the semi-solid 0.5% agar medium in two identical 20 mL test tubes. The one to the left is the control sample which is devoid of any bacterial cells. To the right is the S. pasteurii inoculated sample where the resulting mineral precipitation has left a conspicuous trail. Images were taken after 1 day and 7 days of the inoculation. (b) Blown out magnified image of the mineral deposition. (c-d) SEM image of agar showing pores in control set (c) and numerous mineral microspheres after 7 days (d) of incubation. https://doi org/10 1371/journal pone 0210339 g006 Fig 6. MICP in agar column (a) Two samples containing the semi-solid 0.5% agar medium in two identical 20 mL test tubes. The one to the left is the control sample which is devoid of any bacterial cells. To the right is the S. pasteurii inoculated sample where the resulting mineral precipitation has left a conspicuous trail. Images were taken after 1 day and 7 days of the inoculation. (b) Blown out magnified image of the mineral deposition. (c-d) SEM image of agar showing pores in control set (c) and numerous mineral microspheres after 7 days (d) of incubation. https://doi.org/10.1371/journal.pone.0210339.g006 https://doi.org/10.1371/journal.pone.0210339.g006 10 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface Fig 7. Optical and electron microscopy images of semi-solid agar grown CaCO3 microspheres and its XRD pattern. (a) TEM images of CaCO3 microspheres deposited far from SP cells (macro-environment) (b) An optical microscopy image and an ultrathin (80 nm) section of agar column with CaCO3 microspheres, stained with crystal violet. (c) TEM image of a portion of the ultrathin (80 nm) section. (inset) The SAED pattern of the crystalline structure. (d) XRD plot indicates the formation of calcite and vaterite polymorphs. Agar column study (inset) clearly demonstrates presence of vaterite. Fig 7. Optical and electron microscopy images of semi-solid agar grown CaCO3 microspheres and its XRD pattern. (a) TEM images of CaCO3 microspheres deposited far from SP cells (macro-environment) (b) An optical microscopy image and an ultrathin (80 nm) section of agar column with CaCO3 microspheres, stained with crystal violet. (c) TEM image of a portion of the ultrathin (80 nm) section. (inset) The SAED pattern of the crystalline structure. (d) XRD plot indicates the formation of calcite and vaterite polymorphs. (inset) clearly demonstrates presence of vaterite. https://doi.org/10.1371/journal.pone.0210339.g007 https://doi.org/10.1371/journal.pone.0210339.g007 depositions of crystalline mircrospheres profusely within the agar column. Optical and TEM imaging revealed that the visible white deposits within the column were mostly crystalline micropheres of about 10–50 μm in diameter (Fig 7A). TEM images of the microspheres (Fig 7B) shows a crystal violet stained corss-section of microsphere. Fig 7C shows the TEM image of the same with a SAED pattern (Inset of Fig 7C) which proves the crystalline nature of the precipitates and also consistent with the Miller indi- ces of calcium carbonate observed in XRD. Fig 7D shows the powder XRD result of the col- lected precipitate used to identify the crystalline phases of the inorganic compounds. The identified signature peaks of calcite (Fig 7D) at 2θ valus of 26.83o, 34.26o and 42.01o respec- tively corelated with lattice (hkl) indiced of (012), (104) and (110). Low intensity vaterite peaks (Fig 7D Inset) were also identified at 2θ values of 28.98o, 31.52o and 38.22o corelated with PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 11 / 15 Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface Fig 8. TEM images of ultrathin sections of the agar medium depicting the presence of S. pasteurii cells with cell surface depositions of nanometer sized CaCO3. https://doi org/10 1371/journal pone 0210339 g008 Fig 8. TEM images of ultrathin sections of the agar medium depicting the presence of S. pasteurii cells with cell surface depositions of nanometer sized CaCO3. https://doi org/10 1371/journal pone 0210339 g008 Fig 8. TEM images of ultrathin sections of the agar medium depicting the presence of S. pasteurii cells with cell surface depositions of nanometer sized CaCO3. https://doi.org/10.1371/journal.pone.0210339.g008 https://doi.org/10.1371/journal.pone.0210339.g008 lattice (hkl) indiced of (100), (101) and (102), respectively. TEM imaging of the micro-envi- ronment of bacterial cell surface shed light on the likely nucleation route for the calcite micro- spheres. Agar column study Additionally, urea hydrolysis by the bacterial cell may have resulted in a highly alkaline (rich in OH- ions) domain around the cell surface creating the “nanoscale neighborhood” which eventually served as sites of CaCO3 precipitation. Also, the possibilities of the presence of highly proteinaceous S-layer [29] or extra-polymeric substances (EPS)[10] in the nucleation process can not be discarded. Though the participation of bacterial cell surface in the CaCO3 precipitation is definite, the particular reason(s) behind still require more research. It is believed that the MICP process is driven primarly by urea hydrolysis [1, 10, 14, 17, 25, 35]. Caclium carbonate precipitation and the crystal morphology are likely influenced by two major factors: i) biochemical induction and/or ii) purely chemical induction. A process driven by biophysical/biochemical induction can be expected to be significantly slower than a purely chemical induction driven process. Culture media which were initially alkaline, resulted in a comparatively faster precipitation and hence were sampled after one hour; media with an ini- tially neutral pH resulted in a slower precipitation and hence were sampled after 24 hours. tially neutral pH resulted in a slower precipitation and hence were sampled after 24 hours. This difference in time-scales suggests that the former were chemically induced while the latter were biochemically induced. In case of biochemical induction, the process is likely to be extremely complex and we found that the nanoscale environment of the cell played a crucial role in the MICP process. Bacterial cell walls are expected to possess negatively charged units [3, 14, 23, 29], which likely resulted in a transport of Ca2+ ions towards the cell surface facilitat- ing nucleation at the cell surface. Additionally, urea hydrolysis by the bacterial cell may have Agar column study The 80 nm ultra-thin sections of agar column containing embedded bacteria shows in Fig 8 clearly depict the nanoscale environment of the bacterium cell surface. Magnified images indicate nanoscale spherical depositions on cell surface (black rectangle) and needle-like depo- sitions in the surrounding agar media. These results are definitive proof that SP can participate in the nucleation of crystals via its cell surface. However, we would like to reiterate that other nucleation mechanisms are not ruled out by our study. lattice (hkl) indiced of (100), (101) and (102), respectively. TEM imaging of the micro-envi- ronment of bacterial cell surface shed light on the likely nucleation route for the calcite micro- spheres. The 80 nm ultra-thin sections of agar column containing embedded bacteria shows in Fig 8 clearly depict the nanoscale environment of the bacterium cell surface. Magnified images indicate nanoscale spherical depositions on cell surface (black rectangle) and needle-like depo- sitions in the surrounding agar media. These results are definitive proof that SP can participate in the nucleation of crystals via its cell surface. However, we would like to reiterate that other nucleation mechanisms are not ruled out by our study. y y It is believed that the MICP process is driven primarly by urea hydrolysis [1, 10, 14, 17, 25, 35]. Caclium carbonate precipitation and the crystal morphology are likely influenced by two major factors: i) biochemical induction and/or ii) purely chemical induction. A process driven by biophysical/biochemical induction can be expected to be significantly slower than a purely chemical induction driven process. Culture media which were initially alkaline, resulted in a comparatively faster precipitation and hence were sampled after one hour; media with an ini- tially neutral pH resulted in a slower precipitation and hence were sampled after 24 hours. This difference in time-scales suggests that the former were chemically induced while the latter were biochemically induced. In case of biochemical induction, the process is likely to be extremely complex and we found that the nanoscale environment of the cell played a crucial role in the MICP process. Bacterial cell walls are expected to possess negatively charged units [3, 14, 23, 29], which likely resulted in a transport of Ca2+ ions towards the cell surface facilitat- ing nucleation at the cell surface. Author Contributions Conceptualization: Tanushree Ghosh, Aloke Kumar. Data curation: Tanushree Ghosh, Swayamdipta Bhaduri, Aloke Kumar. Data curation: Tanushree Ghosh, Swayamdipta Bhaduri, Aloke Kumar. Formal analysis: Tanushree Ghosh. Funding acquisition: Aloke Kumar. Investigation: Tanushree Ghosh, Swayamdipta Bhaduri, Aloke Kumar. Methodology: Tanushree Ghosh, Aloke Kumar. Resources: Carlo Montemagno. Supervision: Carlo Montemagno, Aloke Kumar. Validation: Tanushree Ghosh. Visualization: Tanushree Ghosh, Swayamdipta Bhaduri. Writing – original draft: Tanushree Ghosh, Swayamdipta Bhaduri, Carlo Montemagno, Aloke Kumar. Writing – review & editing: Tanushree Ghosh, Aloke Kumar. Supporting information Supporting information S1 File. (PDF) S2 File. (XLSX) S3 File. (XLSX) S4 File. (XLSX) Acknowledgments This study has drawn heavily on instrumentation like TEM, SAED, and XRD. Authors thank- fully acknowledged the technical contribution and instrument facility of NanoFAB, Faculty of Engineering, University of Alberta. Authors also thankfully acknowledged the TEM facility of the Department of Biological Science, University of Alberta. Conclusions In this work, we explored the MICP process by the bacterium Sporosarcina pasteurii. The par- ticipation and behavior of S. pasteurii cells at different pH of the medium as well as at different medium composition was investigated. Ureolytic S. pasteurii cells were found to possess cell surface CaCO3 and it was prominent deposited on the cell surface. To explore in situ, semi- solid agar (0.5%) was used to create a porous column, which was inoculated with a stab culture. As S. pasteurii cells migrated down the agar column they left conspicuous trail of crystals. Sam- ples of the agar column at different locations were taken and subjected to microscopy and we 12 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface found that the crystal train consisted of calcite microspheres, which on closer inspection were found to be an aggregate of needle-like nanoscale crystals. Moreover, cells whose surface con- tained calcite nanocrystals were also observed confirming the hypothesis that cell surface plays a role in nucleation. found that the crystal train consisted of calcite microspheres, which on closer inspection were found to be an aggregate of needle-like nanoscale crystals. Moreover, cells whose surface con- tained calcite nanocrystals were also observed confirming the hypothesis that cell surface plays a role in nucleation. PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 References 1. Stocks-Fischer S, Galinat JK, Bang SS. Microbiological precipitation of CaCO 3. Soil Biology and Bio- chemistry. 1999; 31(11):1563–1571. 2. Warren LA, Maurice PA, Parmar N, Ferris FG. Microbially mediated calcium carbonate precipitation: implications for interpreting calcite precipitation and for solid-phase capture of inorganic contaminants. Geomicrobiology Journal. 2001; 18(1):93–115. 13 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface 3. De Muynck W, De Belie N, Verstraete W. Microbial carbonate precipitation in construction materials: a review. Ecological Engineering. 2010; 36(2):118–136. 4. Sarikaya M. Biomimetics: materials fabrication through biology. Proceedings of the National Academy of Sciences. 1999; 96(25):14183–14185. 5. Thompson J, Ferris F. Cyanobacterial precipitation of gypsum, calcite, and magnesite from natural alka- line lake water. Geology. 1990; 18(10):995–998. 6. McConnaughey T, Whelan J. Calcification generates protons for nutrient and bicarbonate uptake. Earth-Science Reviews. 1997; 42(1–2):95–117. 7. Castanier S, Le Me´tayer-Levrel G, Perthuisot J-P. Ca-carbonates precipitation and limestone genesis —the microbiogeologist point of view. Sedimentary Geology. 1999; 126(1):9–23. 8. Hammes F, Boon N, de Villiers J, Verstraete W, Siciliano SD. Strain-specific ureolytic microbial calcium carbonate precipitation. Applied and environmental microbiology. 2003; 69(8):4901–4909. https://doi. org/10.1128/AEM.69.8.4901-4909.2003 PMID: 12902285 9. Warthmann R, Van Lith Y, Vasconcelos C, McKenzie JA, Karpoff AM. Bacterially induced dolomite pre- cipitation in anoxic culture experiments. Geology. 2000; 28(12):1091–1094. 10. Harris D, Ummadi JG, Thurber AR, Allau Y, Verba C, Colwell F, et al. Real-time monitoring of calcifica- tion process by Sporosarcina pasteurii biofilm. Analyst. 2016; 141(10):2887–2895. https://doi.org/10. 1039/c6an00007j PMID: 26939806 11. Kawaguchi T, Decho AW. A laboratory investigation of cyanobacterial extracellular polymeric secretions (EPS) in influencing CaCO 3 polymorphism. Journal of Crystal Growth. 2002; 240(1):230–235. 12. Arias JL, Ferna´ndez MaS. Polysaccharides and proteoglycans in calcium carbonate-based biominerali- zation. Chemical Reviews. 2008; 108(11):4475–4482. https://doi.org/10.1021/cr078269p PMID: 18652513 13. Bhaduri S, Debnath N, Mitra S, Liu Y, Kumar A. Microbiologically induced calcite precipitation mediated by Sporosarcina pasteurii. Journal of visualized experiments: JoVE. 2016(110). 14. Hammes F, Verstraete W. Key roles of pH and calcium metabolism in microbial carbonate precipitation. Reviews in environmental science and biotechnology. 2002; 1(1):3–7. 15. Phillips AJ, Gerlach R, Lauchnor E, Mitchell AC, Cunningham AB, Spangler L. Engineered applications of ureolytic biomineralization: a review. Biofouling. 2013; 29(6):715–733. https://doi.org/10.1080/ 08927014.2013.796550 PMID: 23802871 16. Bang SS, Galinat JK, Ramakrishnan V. Calcite precipitation induced by polyurethane-immobilized Bacillus pasteurii. Enzyme and microbial technology. 2001; 28(4):404–409. 17. Mitchell AC, Ferris FG. References The influence of Bacillus pasteurii on the nucleation and growth of calcium car- bonate. Geomicrobiology Journal. 2006; 23(3–4):213–226. 18. Achal V, Mukherjee A, Basu P, Reddy MS. Strain improvement of Sporosarcina pasteurii for enhanced urease and calcite production. Journal of industrial microbiology & biotechnology. 2009; 36(7):981–988. 19. Okwadha GD, Li J. Optimum conditions for microbial carbonate precipitation. Chemosphere. 2010; 81 (9):1143–1148. https://doi.org/10.1016/j.chemosphere.2010.09.066 PMID: 20947128 20. Dhami NK, Reddy MS, Mukherjee A. Biomineralization of calcium carbonates and their engineered applications: a review. Frontiers in Microbiology. 2013; 4(314):1–13. 21. Li M, Fu Q-L, Zhang Q, Achal V, Kawasaki S. Bio-grout based on microbially induced sand solidification by means of asparaginase activity. Scientific reports. 2015; 5(16128):1–9. 22. Phillips AJ, Cunningham AB, Gerlach R, Hiebert R, Hwang C, Lomans BP, et al. Fracture sealing with microbially-induced calcium carbonate precipitation: A field study. Environmental science & technology. 2016; 50(7):4111–4117. 23. Anbu P, Kang C-H, Shin Y-J, So J-S. Formations of calcium carbonate minerals by bacteria and its mul- tiple applications. SpringerPlus. 2016; 5(1):250. 24. Chunxiang Q, Jianyun W, Ruixing W, Liang C. Corrosion protection of cement-based building materials by surface deposition of CaCO 3 by Bacillus pasteurii. Materials Science and Engineering: C. 2009; 29 (4):1273–1280. 25. Tziviloglou E, Van Tittelboom K, Palin D, Wang J, Sierra-Beltra´n MG, Erşan YC¸ , et al. Bio-based self- healing concrete: from research to field application. Self-healing Materials. 2016:345–385. 26. Seifan M, Samani AK, Berenjian A. Bioconcrete: next generation of self-healing concrete. Applied microbiology and biotechnology. 2016; 100(6):2591–2602. https://doi.org/10.1007/s00253-016-7316-z PMID: 26825821 27. Ivanov V, Stabnikov V. Construction Biotechnology: Biogeochemistry, Microbiology and Biotechnology of Construction Materials and Processes: Springer; 2016. 14 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface 28. Morita RY. Calcite precipitation by marine bacteria*. Geomicrobiology Journal. 1980; 2(1):63–82. 29. Douglas S, Beveridge TJ. Mineral formation by bacteria in natural microbial communities. FEMS micro- biology ecology. 1998; 26(2):79–88. 30. Ehrlich HL. How microbes influence mineral growth and dissolution. Chemical geology. 1996; 132(1– 4):5–9. 31. Ehrlich HL. Geomicrobiology: its significance for geology. Earth-Science Reviews. 1998; 45(1):45–60. 32. Ferris F, Stehmeier L, Kantzas A, Mourits F. Bacteriogenic mineral plugging. Journal of Canadian Petro- leum Technology. 1997; 36(09). 33. Ghashghaei S, Emtiazi G. Production of calcite nanocrystal by a urease-positive strain of Enterobacter ludwigii and study of its structure by SEM. Current microbiology. 2013; 67(4):406–413. https://doi.org/ 10.1007/s00284-013-0379-5 PMID: 23677144 34. PLOS ONE \| https://doi.org/10.1371/journal.pone.0210339 January 30, 2019 References Bundeleva IA, Shirokova LS, Be´ne´zeth P, Pokrovsky OS, Kompantseva EI, Balor S. Calcium carbonate precipitation by anoxygenic phototrophic bacteria. Chemical Geology. 2012; 291:116–131. 35. Zhang W, Ju Y, Zong Y, Qi H, Zhao K. In situ real-time study on dynamics of microbially induced calcium carbonate precipitation at a single-cell level. Environmental science & technology. 2018. 36. Deng H, Wang S, Wang X, Du C, Shen X, Wang Y, et al. Two competitive nucleation mechanisms of calcium carbonate biomineralization in response to surface functionality in low calcium ion concentra- tion solution. Regenerative biomaterials. 2015; 2(3):187–195. https://doi.org/10.1093/rb/rbv010 PMID: 26814639 37. Rodriguez-Navarro C, Kudłacz K, Cizer O¨ , Ruiz-Agudo E. Formation of amorphous calcium carbonate and its transformation into mesostructured calcite. CrystEngComm. 2015; 17(1):58–72. 38. Cao H, Lin G, Yao J, Shao Z. Amphiphilic polypeptides as a bifunctional template in the mineralization of calcium carbonate at the air/water interface. Macromolecular bioscience. 2013; 13(5):650–659. https://doi.org/10.1002/mabi.201200334 PMID: 23610039 39. Ghosh T, Sarkar P. Isolation of a novel uric-acid-degrading microbe Comamonas sp. BT UA and rapid biosensing of uric acid from extracted uricase enzyme. Journal of biosciences. 2014; 39(5):805–819. PMID: 25431410 40. Nguyen JNT, Harbison AM. Scanning Electron Microscopy Sample Preparation and Imaging. Molecular Profiling: Springer; 2017. p. 71–84. 41. Fischer BTH E. R., Nair V., Hoyt F. H. and Dorward D. W. Scanning Electron Microscopy. Current Pro- tocols in Microbiology. 2012;CHAPTER: Unit2B. 2. 42. Glauert AM, Lewis PR. Biological specimen preparation for transmission electron microscopy: Prince- ton University Press; 2014. 43. Zhu T, Paulo C, Merroun ML, Dittrich M. Potential application of biomineralization by Synechococcus PCC8806 for concrete restoration. Ecological Engineering. 2015; 82:459–468. 15 / 15
https://openalex.org/W2487318092	https://europepmc.org/articles/pmc4967674?pdf=render	English	null	“Slalom”: Microsurgical Cross-Over Decompression for Multilevel Degenerative Lumbar Stenosis	BioMed research international	2,016	cc-by	3,694	Hindawi Publishing Corporation BioMed Research International Volume 2016, Article ID 9074257, 6 pages http://dx.doi.org/10.1155/2016/9074257 Hindawi Publishing Corporation BioMed Research International Volume 2016, Article ID 9074257, 6 pages http://dx.doi.org/10.1155/2016/9074257 Hindawi Publishing Corporation BioMed Research International Volume 2016, Article ID 9074257, 6 pages http://dx.doi.org/10.1155/2016/9074257 1. Introduction 10]. Longer skin incisions are necessary to reach 2 or more segments. The paravertebral muscles have to be retracted over a longer distance and the partial resection of the inferior and superior facets has to be performed at 2 or more segments on the same approach side. This produces higher unilateral collateral damage for muscles and joints which more or less counteracts the microsurgical philosophy of this approach.h Bilateral microsurgical so-called “cross-over decompression” through a unilateral approach has become a new minimally invasive surgical treatment option for degenerative lumbar spinal stenosis [1–7].h The main advantages of this technique are the diminished “access trauma” to the paravertebral muscles and to the facet joints. In particular the inferior facet contralateral to the approach side as well as its outer capsular surroundings can be preserved completely. The following paper describes a new surgical technique for selective multisegmental decompression through multiple microsurgical approaches with alternating approach sides (“Slalom” Technique). In cases of central spinal or foraminal stenosis associated with degenerative lumbar scoliosis decompression can be performed from the convex side, thus preserving the stability of the heavily loaded facet joint on the concave side [6].h Spine Center, Sch¨on Klinik M¨unchen Harlaching, Paracelsus Medical School Salzburg, Harlachinger Straße 51, 81547 M¨unchen, Germany Spine Center, Sch¨on Klinik M¨unchen Harlaching, Paracelsus Medical School Salzburg, Harlachinger Straße 51, 81547 M¨unchen, Germany Correspondence should be addressed to H. Michael Mayer; mmayer@schoen-kliniken.de Received 25 February 2016; Accepted 21 June 2016 Academic Editor: William B. Rodgers Copyright © 2016 H. M. Mayer and F. Heider. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. Selective, bilateral multisegmental microsurgical decompression of lumbar spinal canal stenosis through separate, alternating cross-over approaches. Indications. Two-segmental and multisegmental degenerative central and lateral lumbar spinal stenosis. Contraindications. None. Surgical Technique. Minimally invasive, muscle, and facet joint-sparing bilateral decompression of the lumbar spinal canal through 2 or more alternating microsurgical cross-over approaches from one side. Results. From December 2010 until December 2015 we operated on 202 patients with 2 or multisegmental stenosis (115 f; 87 m; average age 69.3 yrs, range 51–91 yrs). All patients were suffering from symptoms typical of a degenerative lumbar spinal stenosis. All patients complained about back pain; however the leg symptoms were dominant in all cases. Per decompressed segment, the average OR time was 36 min and the blood loss 45.7 cc. Patients were mobilized 6 hrs postop and hospitalization averaged 5.9 days. A total of 116/202 patients did not need submuscular drainage. 27/202 patients suffered from a complication (13.4%). Dural tears occurred in 3.5%, an epidural hematoma in 5.5%, a deep wound infection in 1.98%, and a temporary radiculopathy postop in 1.5%. Postop follow-up ranged from 12 to 24 months. There was a significant improvement of EQ 5 D, Oswestry Disability Index (ODI), VAS for Back and Leg Pain, and preoperative standing times and walking distances. 2. Methods 2.1. Surgical Goal. The goal of this technique is to achieve multisegmental bilateral decompression through separate unilateral microsurgical approaches. The spinal canal is reached through approaches with alternating the sides (e.g., These advantages usually get lost in cases with bi- or multisegmental pathologies which account for more than 50% of our own patient population in the last 17 years [3, 9, 2 BioMed Research International (a) (b) Figure 3: (a) From [8] with permission. Marking of the skin inci- sions to approach the levels L3-4-5-S1. (b) From [8] with permission. Graphic demonstration of a “Slalom” approach to stenotic levels L2- 3 from the right side, L3-4 from the left, and L4-5 again from the right side. Figure 1: From [8], with permission. Positioning of the patient in knee-thorax position with no pressure on abdomen. (a) Figure 1: From [8], with permission. Positioning of the patient in knee-thorax position with no pressure on abdomen. (a) (b) Cannulas (a) Cannulas (a) Cannulas (a) (b) Figure 2: (a) From [8] with permission. Localization of the level to be approached. Cannulas are inserted to mark the levels unde fluoroscopic control. (b) From [8] with permission. Lateral X-ray with graphic marking of the approach corridors to the levels L3-4 5-S1. Due to the lordotic angle of the lumbosacral junction, the level L4-5-1 can be approached through one small incision while the othe two levels are approached through separate skin incisions (“Gian Slalom”). Cannulas (b) Figure 3: (a) From [8] with permission. Marking of the skin inci- sions to approach the levels L3-4-5-S1. (b) From [8] with permission. Graphic demonstration of a “Slalom” approach to stenotic levels L2- 3 from the right side, L3-4 from the left, and L4-5 again from the right side. (a) (b) heaviness in the legs w/wo radicular symptoms, and w/wo associated deformity (e.g., degenerative lumbar scoliosis and degenerative spondylolisthesis) were included. There were no general contraindications to this approach. (b) 2.3. Surgical Technique. The patient is placed in a so-called knee-thorax position (Figure 1). He is kneeling on the surgical table with hips and knees flexed 90∘and shoulders in 90∘abduction and 90∘external rotation (attention: avoid overextension of the shoulders). Elbow (N. ulnaris) and wrist joint (N. medianus) and the shins are positioned on gel pads to avoid pressure sores. The abdomen should “hang” freely to maximally lower the pressure in the epidural veins. 2. Methods The cranial rim of the caudal lamina is undercut 2-3 mm. Thus the ipsilateral decompression is complete (Figure 5(a)). Now the OR table is tilted to the contralateral side and the assistant who fixes and guides the speculum tilts it to the contralateral side (Figure 5(b)). The surgical microscope is adjusted to give an oblique view to the contralateral part of the spinal canal. The yellow ligament is removed and the proximal lamina is undercut as is the superior facet on the contralateral side. Thus the central part as well as the lateral part (lateral recess) is completely decompressed as well (Figure 5(b)). Hemostasis is achieved with repeated irrigation with saline solution or the use of Floseal (Floseal Baxter Deutschland GmbH, Unterschleissheim, Germany). The table is tilted into the neutral position, the speculum is removed, and the fascia and skin are closed with resorbable intracutaneous sutures. Sub- muscular drainage without vacuum is inserted if necessary. A postop MRi shows the amount of decompression and the lack of “collateral damage” (Figure 6). This procedure is then repeated in the other segments with alternating skin incisions (see Figure 3). L1-2-3-4 (𝑛= 16), L2-3-4-5 (𝑛= 28), L3-4-5-1 (𝑛= 24), L1-2-3 + L5-1 (𝑛= 4), L2-3-4 + L5-1 (𝑛= 3), L1-2-3 + L5-1 (𝑛= 4), L2-3-4 + L5-1 (𝑛= 3), 4 segments (𝑛= 31): L1-2-3-4-5 (𝑛= 14), L2-3-4-5-1 (𝑛= 11), L1-2-3-4 + L5-1 (𝑛= 4), T12-L1-2-3-4 (𝑛= 2), 5 segments (𝑛= 12): L1-2-3-4-5-1 (𝑛= 11), T12-L1-2-3-4 + L5-1 (𝑛= 1). L1-2-3-4-5 (𝑛= 14), L2-3-4-5-1 (𝑛= 11), L1-2-3-4 + L5-1 (𝑛= 4), T12-L1-2-3-4 (𝑛= 2), 5 segments (𝑛= 12): L1-2-3-4-5-1 (𝑛= 11), T12-L1-2-3-4 + L5-1 (𝑛= 1). Per decompressed segment, the average OR time was 36 min, and the blood loss 45.7 cc. Patients were mobilized 6 hrs postoperatively and hospitalization averaged 5.9 days (which was mainly due to reimbursement regulations in Germany). A total of 116/202 patients did not need sub- muscular drainage. All patients received a soft lumbar brace (Lumbotrain, Fa Bauerfeind, Germany) for 4 weeks postop. Postop follow-up ranges from 12 to 24 months. There was a marked improvement of EQ 5D and Oswestry Disability Index (ODI) (Figure 7(a)). The same is true for the VAS for Back and Leg Pain (Figure 7(b)). All patients reported a significant improvement of their preop standing times and walking distances. A total of 27/202 patients suffered from a complication (13.4%). 2. Methods Patents with central, lateral, and foraminal stenosis of 2 and more lumbar levels with typical clini- cal symptoms (neurogenic claudication, buttock, leg pain, 3 BioMed Research International Figure 4: From [8] with permission. Graphic presentation of the interlaminar approach window with a minispeculum. f; 87 m; average age 69.3 yrs, range 51–91 yrs). In 202 patients a total of 577 segments were decompressed through separate approaches (see patient data listed below). All patients were suffering from symptoms typical for a degenerative lumbar spinal stenosis. All patients complained about back pain; however the leg symptoms were dominant in all cases. Patient data are as follows: 𝑁= 202, f : m (115 : 87), 𝑁= 202, f : m (115 : 87), age: average 69.3 yrs (range 51–91 yrs), age: average 69.3 yrs (range 51–91 yrs), follow-up: 12–24 mos, follow-up: 12–24 mos, Operated segments are as follows: Operated segments are as follows: 2 segments (𝑛= 84): L1-2-3 (𝑛= 8), L2-3-4 (𝑛= 14), L3-4-5 (𝑛= 32), L4-5-1 (𝑛= 22), T12-L1-2 (𝑛= 3), L2-3 + L4-5 (𝑛= 2), L1-2 + L3-4 (𝑛= 1), L3-4 + L5-1 (𝑛= 2), 2 segments (𝑛= 84): Figure 4: From [8] with permission. Graphic presentation of the interlaminar approach window with a minispeculum. L1-2-3 (𝑛= 8), L2-3-4 (𝑛= 14), L3-4-5 (𝑛= 32), L4-5-1 (𝑛= 22), T12-L1-2 (𝑛= 3), L2-3 + L4-5 (𝑛= 2 L1-2 + L3-4 (𝑛= 1) L3 4 + L5 1 (𝑛= 2) L1-2-3 (𝑛= 8), L2-3-4 (𝑛= 14), L3-4-5 (𝑛= 32), L4 5 1 (𝑛= 22) to the spinous process. Small attachments of the rotators are cut from the inferior lateral part of the superior lamina to expose the interlaminar window and the facet joint contour. A microspeculum (Piccolino, Medicon, Tuttlingen, Germany) is then inserted and the level of exposure is checked under fluoroscopic control (Figure 4).hi L2-3 + L4-5 (𝑛= 2), L1-2 + L3-4 (𝑛= 1), L3-4 + L5-1 (𝑛= 2), 3 segments (𝑛= 75): l The first step of decompression is to undercut the proxi- mal lamina with a high speed burr to expose the attachment of the yellow ligament medially and cranially. Then the spinal canal is opened and the yellow ligament is removed starting in the midline and then towards lateral cranial and finally along the lateral recess to expose the thecal sac and the root until it leaves the spinal canal around the caudal pedicle (Figure 5(a)). 2. Methods Lateral supports are important to secure the patient while the OR table is tilted during the procedure (Figure 1).h 2.3. Surgical Technique. The patient is placed in a so-called knee-thorax position (Figure 1). He is kneeling on the surgical table with hips and knees flexed 90∘and shoulders in 90∘abduction and 90∘external rotation (attention: avoid overextension of the shoulders). Elbow (N. ulnaris) and wrist joint (N. medianus) and the shins are positioned on gel pads to avoid pressure sores. The abdomen should “hang” freely to maximally lower the pressure in the epidural veins. Lateral supports are important to secure the patient while the OR table is tilted during the procedure (Figure 1).h Figure 2: (a) From [8] with permission. Localization of the levels to be approached. Cannulas are inserted to mark the levels under fluoroscopic control. (b) From [8] with permission. Lateral X-ray with graphic marking of the approach corridors to the levels L3-4- 5-S1. Due to the lordotic angle of the lumbosacral junction, the levels L4-5-1 can be approached through one small incision while the other two levels are approached through separate skin incisions (“Giant Slalom”). left-right-left). The rationale behind this is not only to decrease the amount of unilateral access damage but also to “balance” the trauma to the tissues on the way to the spinal canal (skin, muscles, facet joints, and lamina). Bilateral decompression of the central spinal canal and of the lateral recess is possible. If foraminal (“far lateral”) decompression has to be achieved as well, the approach has to be chosen from the contralateral side. The projection of the disc space on to the skin level is then marked under fluoroscopic control with cannulas (Figures 2(a)-2(b)). If there is a fixed lordosis sometimes 2 segments can be reached through one 20 mm skin incision. Figure 3 shows various skin incisions to 2-3-4-5 segments (Figures 3(a)-3(b)). Surgery is performed skin-to-skin with the help of a surgical microscope (Zeiss N 700, Zeiss, Oberkochen, Germany) with variable 400 mm focus length. About 5 mm paramedian, the dorsolumbar fascia is opened and the paravertebral muscles are bluntly and gently retracted from the lamina and the interlaminar window. Care has to be taken not to incise bigger attachments of the muscles 2.2. Indications. 2. Methods Dural tears occurred in 3.5%, epidural hematoma in 5.5%, a deep wound infection in 1.98%, and a temporary radiculopathy postop in 1.5%. 3. Results From December 2010 until December 2015 we operated on 202 patients with 2-segmental or multisegmental stenosis (115 4 BioMed Research International (a) (b) Figure 5: (a) From [8] with permission. Decompression of the ipsilateral side. (b) From [8] with permission. Decompression of the contra- lateral side. 4 BioMed Research International (b) (a) (b) (b) (a) Figure 5: (a) From [8] with permission. Decompression of the ipsilateral side. (b) From [8] with permission. Decompression of the contra- lateral side Figure 5: (a) From [8] with permission. Decompression of the ipsilateral side. (b) From [8] with permission. Decompression of the contra- lateral side. Figure 6: From [8] with permission. “Technical result” of decompression of one level (L3-4). Left: material that has been removed (yellow ligament, bone). Middle: MRI preoperatively; right: MRI postoperatively shows a complete decompression with preserved facet joint and minimum scar tissue formation in the muscles. Figure 6: From [8] with permission. “Technical result” of decompression of one level (L3-4). Left: material that has been removed (yellow ligament, bone). Middle: MRI preoperatively; right: MRI postoperatively shows a complete decompression with preserved facet joint and minimum scar tissue formation in the muscles. 4. Discussion 9, 10, 19]. The muscles are retracted only on one side and the area of the spinal canal which is exposed to the surrounding tissue remains small. This reduces the area of potential scar formation. Moreover, the integrity of the contralateral facet joint remains nearly completely intact. Degenerative lumbar spinal stenosis is gaining increasing importance. Growing life expectancy with higher demands towards quality of life and better diagnostic options have made spinal stenosis the most frequent pathology seen in spine centers around the Western World. In multisegmental stenosis however the sum of several unilateral interlaminar exposures leads to a more extensive unilateral muscle trauma. The removal of the medial part of the inferior facet in 2 or more levels on the same side may also lead to unilateral functional problems on the joint level. This gains even more importance in cases where spinal stenosis is associated with a degenerative deformity such as degenerative spondylolisthesis or de novo scoliosis. The “Slalom” Technique described in this paper leads to a more “balanced” collateral damage pattern thus keeping the full advantages of this minimal invasive approach. Conventional laminectomy with removal of posterior bony and ligamentous structures has been the gold standard of surgical treatment for decades. Although postoperative development of segmental instability is a multifactorial problem, unnecessary damage to anatomic structures which stabilize the functional spinal unit has always been a problem with this technique [11–14]. Moreover, the fact that the spinal canal is exposed more than what would be necessary just for a decompression increases the contact surface between paravertebral muscles and the dura is one of the reasons for extensive scar tissue formation and epidural fibrosis following conventional laminectomy which may lead to tethering of the cauda equina and radicular symptoms [12, 15–18]. Intraoperative blood loss was low, and submuscular drainage was necessary in only 42.5% of the cases. Minimal surgical trauma allows for early mobilization of the patient. Our success rates correspond well with those described for monosegmental approaches. 4. Discussion Microsurgical cross-over decompression through a uni- lateral approach significantly minimizes these problems [3, 4, 5 BioMed Research International Preoperative Preoperative 17 16 36 10 20 30 40 0.8 0.7 0.5 0 ODI 0.2 0.4 0.6 0.8 0 EQ 5D 12months 24months 12months 24months (a) Preoperative Preoperative VAS leg VAS back 22.3 19.2 47.8 0 20 40 60 0 20 40 60 32.3 26.1 45.7 12months 24months 12months 24months (b) igure 7: (a) EQ5 D and Oswestry Disability Index (ODI) preoperatively, as well as 12 and 24 mos postoperatively. (b) VAS for Leg and Back ain preoperatively as well as 12 and 24 mos postoperatively. Preoperative 0.8 0.7 0.5 0.2 0.4 0.6 0.8 0 EQ 5D 12months 24months Preoperative 17 16 36 10 20 30 40 0 ODI 12months 24months 24months 12months Preoperative (a) (a) Preoperative Preoperative VAS leg VAS back 22.3 19.2 47.8 0 20 40 60 0 20 40 60 32.3 26.1 45.7 12months 24months 12months 24months (b) 7 ( ) EQ5 D d O t Di bilit I d (ODI) ti l ll 12 d 24 t ti l (b) VAS f L d B 24months 12months Preoperative (b) Figure 7: (a) EQ5 D and Oswestry Disability Index (ODI) preoperatively, as well as 12 and 24 mos postoperatively. (b) VAS for Leg and Back Pain preoperatively as well as 12 and 24 mos postoperatively. The following limitations of the Slalom approach and of our short-term experience should not remain unmentioned. since this can be associated with the unilateral violation of the 2 inferior facets (L4 and L5) as mentioned above, we only recommend it in cases with wide isthmus interarticularis in order to prevent fatigue fractures postoperatively. In case a dural tear occurs intraoperatively on the ipsilat- eral approach side, repair is only possible with the use of dura clips and patches (e.g., TachoSil, Takeda Ltd., UK). In case of larger defects the approach has to be enlarged to perform a proper suture. This is also true for contralateral dural tears which then would require a contralateral approach.h p g p p y Our postop follow-up ranges between 1 and 2 years which may be too short for proof or disproof of a surgically induced progression of a preexisting deformity. References for degenerative lumbar stenosis,” The Journal of Bone & Joint Surgery—American Volume, vol. 73, no. 6, pp. 809–816, 1991. [1] F. Costa, M. Sassi, A. Cardia et al., “Degenerative lumbar spinal stenosis: analysis of results in a series of 374 patients treated with unilateral laminotomy for bilateral microdecompression,” Journal of Neurosurgery, vol. 7, no. 6, pp. 579–586, 2007. [17] T.-M. Markwalder, “Surgical management of neurogenic clau- dication in 100 patients with lumbar spinal stenosis due to degenerative spondylolisthesis,” Acta Neurochirurgica, vol. 120, no. 3-4, pp. 136–142, 1993. [2] M. O. Kelleher, M. Timlin, O. Persaud, and Y. R. Rampersaud, “Success and failure of minimally invasive decompression for focal lumbar spinal stenosis in patients with and without deformity,” Spine, vol. 35, no. 19, pp. E981–E987, 2010. [18] A. A. Ragab, M. A. Fye, and H. H. Bohlman, “Surgery of the lumbar spine for spinal stenosis in 118 patients 70 years of age or older,” Spine, vol. 28, no. 4, pp. 348–353, 2003. [19] C. E. Poletti, F. A. Simeone, and B. M. Onofrio, “Central lumbar stenosis caused by ligamentum flavum: unilateral laminotomy for bilateral ligamentectomy: preliminary report of two cases,” Neurosurgery, vol. 37, no. 2, pp. 343–347, 1995. [3] H. M. Mayer, “Micorsurgical decompression of acquired (degenerative) central and lateral spinal canal stenosis,” in Minimally Invasive Spine Surgery, H. M. Mayer, Ed., pp. 105–116, Springer, Berlin, Germany, 2000. [4] J. A. McCulloch, “Microsurgery for lateral zone stenosis,” in Essentials of Spinal Microsurgery, J. A. McCulloch and P. A. Young, Eds., pp. 453–486, Lippincott-Raven, Philadelphia, Pa, USA, 1998. [5] A. M. M¨usl¨uman, T. Cansever, A. Yilmaz, H. C¸avus¸o˘glu, ˙I. Y¨uce, and Y. Aydin, “Midterm outcome after a microsurgical unilateral approach for bilateral decompression of lumbar degenerative spondylolisthesis: clinical article,” Journal of Neu- rosurgery: Spine, vol. 16, no. 1, pp. 68–76, 2012. [6] L. Papavero, M. Thiel, E. Fritzsche, C. Kunze, M. Westphal, and R. Kothe, “Lumbar spinal stenosis: prognostic factors for bilateral microsurgical decompression using a unilateral approach,” Neurosurgery, vol. 65, no. 6, pp. ons182–ons187, 2009. [7] H. Toyoda, H. Nakamura, S. Konishi, S. Dohzono, M. Kato, and H. Matsuda, “Clinical outcome of microsurgical bilateral decompression via unilateral approach for lumbar canal steno- sis: minimum five-year follow-up,” Spine, vol. 36, no. 5, pp. 410– 415, 2011. [8] M. H. Mayer and F. Heider, “Selektive mikrochirurgische cross- over Dekompression mehrsegmentaler lumbaler Spinalkanal- stenosen,” Operative Orthop¨adie und Traumatologie, vol. 25, no. 1, pp. Competing Interests The lordotic curvature at the levels L4-5-S1 sometimes suggests a 2-segmental decompression through one small skin incision on the same side (“Giant Slalom”). However, The authors declare that they have no competing interests. BioMed Research International 6 References 47–62, 2013. [9] H. M. Mayer, J. List, A. Korge, and K. Wiechert, “Mikrochirurgie bei degenerativer lumbaler Spinalstenose,” Der Orthop¨ade, vol. 32, no. 10, pp. 889–895, 2003. [10] H. M. Mayer and A. Korge, “Microsurgical decompression of degenerative lumbar spinal stenosis,” European Spine Journal, vol. 18, no. 12, pp. 1989–1990, 2009. [11] K.-E. Johnsson, I. Redlund-Johnell, A. Ud´en, and S. Willner, “Preoperative and postoperative instability in lumbar spinal stenosis,” Spine, vol. 14, no. 6, pp. 591–593, 1989. [12] H. LaRocca and I. Macnab, “The laminectomy membrane: studies in its evolution, characteristics, effects and prophylaxis in dogs,” Journal of Bone and Joint Surgery—Series B, vol. 56, no. 3, pp. 545–550, 1974. [13] C. K. Lee, “Lumbar spinal instability (olisthesis) after extensive posterior spinal decompression,” Spine, vol. 8, no. 4, pp. 429– 433, 1983. [14] A. L. Nachemson, “Instability of the lumbar spine. Pathology, treatment, and clinical evaluation,” Neurosurgery Clinics of North America, vol. 2, no. 4, pp. 785–790, 1991. [15] S. Hall, J. D. Bartleson, B. M. Onofrio, H. L. Baker Jr., H. Okazaki, and J. D. O’Duffy, “Lumbar spinal stenosis: clinical fea- tures, diagnostic procedures, and results of surgical treatment in 68 patients,” Annals of Internal Medicine, vol. 103, no. 2, pp. 271–275, 1985. [16] J. N. Katz, S. J. Lipson, M. G. Larson, J. M. McInnes, A. H. Fossel, and M. H. Liang, “The outcome of decompressive laminectomy
https://openalex.org/W2898700983	http://revistaanestesiar.org/index.php/rear/article/download/610/1150	es		Consideraciones anestésicas con el uso del GLIOLAN®para la resección de gliomas malignos	Revista electrónica de AnestesiaR	1,970	cc-by-sa	1,078	ReaR REV ELECT ANESTESIAR- VOL 10 (7) :3 ISNN 1989 4090 Revista electrónica de AnestesiaR Julio 2018 FORMACION MÉDICA Consideraciones anestésicas con el uso del GLIOLAN® para la resección de gliomas malignos Benito Naverac H. Hospital Clínico Universitario Lozano Blesa de Zaragoza. Resumen Los gliomas cerebrales de alto grado son uno de los tumores infiltrativos más difíciles de tratar y su resección quirúrgica no suele ser completa. La utilización de marcadores fluorescentes (clorhidrato de 5 amino-levulínico (5 ALA) (Gliolan®) permite realizar una extirpación más completa de la lesión. Introducción Los gliomas cerebrales de alto grado son uno de los tumores infiltrativos más difíciles de tratar y su resección quirúrgica no suele ser completa. La utilización de marcadores fluorescentes (clorhidrato de 5 aminolevulínico (5 ALA) (Gliolan®) permite realizar una extirpación más completa de la lesión. El ácido 5 aminolevulínico (5-ALA) se trata de una molécula intracelular utilizada por Stummer en 1998. Es un precursor bioquímico natural del hemo que se metaboliza en la mitocondria a porfirinas fluorescentes como la protoporfirina IX. Tiene una absorción intestinal activa y atraviesa mal la barrera hematoencefálica sana, pero bien la alterada. La administración sistémica de 5-ALA provoca una sobrecarga del metabolismo celular de las porfirinas y la acumulación de PPIX en algunos epitelios y en las células de los gliomas de alto grado, siendo su concentración significativamente más alta en el tejido maligno que en el cerebro normal. Al utilizar una luz azul (longitud de onda de 400 nm) la PPIX es intensamente fluorescente. Bases de la técnica Se administra al paciente 5- ALA- vía oral (dosis 20mg/kg diluido en 50 ml de agua) unas dos horas antes de la inducción anestésica. Esta substancia es captada por las células tumorales. El efecto máximo de fluorescencia ocurre entre 6-9 horas después de su ingesta. ▪ ▪ Intraoperatoriamente, se enciende la luz ultravioleta del microscopio quirúrgico. En función de la fluorescencia, las zonas de más captación en rojo corresponden a tumor sólido, mientras que las zonas que se tiñen de rosa corresponden a células tumorales infiltradas o borde invasivo. El tejido sano se ve de color azul. Copyright ReAR. Rev Elect Anestesiar pertenece a la Asociación Anestesia Reanimación España. Entidad sin ánimo de lucro. ▪ Destacar también que el tejido necrótico no se tiñe y que algunas estructuras como los plexos coroides se tiñen de color rosáceo. Consideraciones anestésicas Requisitos para su utilización ▪ ▪ ▪ ▪ Los neurocirujanos deben ser experimentados en la cirugía de tumores malignos, con un conocimiento exhaustivo de la anatomía funcional del cerebro y deben haber completado un curso de formación sobre cirugía guiada por fluorescencia. El glioma debe ser de alto grado (Grado III y IV de la OMS). La principal desventaja es que el 5 ALA no proporciona información sobre la función que desempeña el tejido cerebral que rodea las células marcadas. Si se realiza una resección demasiado exhaustiva se corre el riesgo de aparición de déficit neurológico. El uso del Gliolan® es compatible con la utilización de técnicas de monitorización neurofisiológica intraoperatoria. Asimismo, se ha utilizado en paciente despierto. Contraindicaciones ▪ ▪ ▪ Hipersensibilidad al compuesto. Presencia de porfiria o hipersensibilidad a las porfirinas. Embarazo. Vía de la Síntesis del Grupo Hemo El 5 ALA mediante transformación enzimática se metaboliza a protoporfina IX (implicado por tanto, en el metabolismo de las porfirinas) de lo cual se desprenden las siguientes consideraciones anestésicas: Evitar fármacos desencadenantes de crisis porfíricas Se debe considerar al enfermo como susceptible de sufrir un ataque de porfiria, y evitar por tanto, el uso de fármacos desencadenantes de la crisis: tiopental, etomidato, pentazocina, fenitoina y diacepam… ▪ Se considera segura la utitilzación de propofol, fentanilo, remifentanilo, cloruro Copyright ReAR. Rev Elect Anestesiar pertenece a la Asociación Anestesia Reanimación España. Entidad sin ánimo de lucro. REV ELECT ANESTESIAR- VOL 10 (7) :3 ▪ ▪ ▪ mórfico y paracetamol entre otros. Pueden usarse con precaución Succinilcolina, rocuronio, cisatracurio, sevofluorano y desfluorano. Existe un caso de utilización de sugammadex en un caso de porfiria aguda sin complicaciones. Existen muchas listas de fármacos seguros e inseguros. En muchos casos los datos son conflictivos. Es recomendable consultar un listado actualizado y la evidencia científica más reciente al respecto. Evitar exposiciones directas de la piel y ojos a la luz ▪ ▪ Evitar fármacos fototóxicos y hepatotóxicos durante las primeras 24h No utilizar fármacos potencialmente fototóxicos como tetraciclinas, sulfonamidas, fluorquinolonas y extractos de hipericina. Efectos adversos descritos del uso del 5 ALA Se han descrito los siguientes efectos adversos: ▪ La exposición a la luz (solar, iluminación del quirófano o focos en las habitaciones) puede desencadenar reacciones fototóxicas tales como; edema e inflamación de la dermis, necrosis epidérmica, desestructuración de folículos pilosos y de glándulas sebáceas. (sondaje vesical, canalización de arteria y de vía central…). Utilización de gafas de sol para protección ocular. No explorar los ojos mediante el uso de linterna pupilar. ▪ ▪ ▪ Se debe de utilizar con precaución, en pacientes con enfermedad cardiovascular preexistente ya que se puede descender la presión arterial sistémica y pulmonar. Puede aparecer anemia, trombocitopenia y leucocitosis. Náuseas y alteración de enzimas hepáticas. Afortunadamente, la incidencia de estos efectos secundarios es extremadamente baja. Bibliografía 1. Es importante proteger al paciente de la exposición lumínica de piel y ojos durante al menos 24 horas con las siguientes medidas: 2. Mantener el quirófano en penumbra. Mantener la superficie cutánea cubierta el máximo tiempo posible. No iluminar directamente la piel a la hora de realizar técnicas 3. ▪ ▪ ▪ http://www.ema.europa.eu/docs/es_ES/ document_library/EPAR__Product_Information/human/000744/ WC500021790 Carretero, M; Negrete, N; Muñoz, G; Sánchez, J; Bernal, LM. Anesthetic treatment during resection of highgrade malignant gliomas after injection of 5-aminolevulinic acid to enhance intraoperative visualization. Rev Esp Anestesiol Reanim, 2010 vol. 57(6) pp. 393-5. (PubMed) Fernández-Candil, J; Pacreu, S; Villaba, G; Fernández, GS. Anesthetic approach and implications of the use of 5-aminolevulinic acid as a tumor marker in the resection of malignant Copyright ReAR. Rev Elect Anestesiar pertenece a la Asociación Anestesia Reanimación España. Entidad sin ánimo de lucro. 4. gliomas. Rev Esp Anestesiol Reanim, 2010 vol. 57(6) pp. 395-6. (PubMed) Buijs. EJ, Scholten JG, Ross JJ. Successful administration of sugammadex in a patient with acute porphyria: A case report. Eur J Anasthesiol. 2014 Aug (8) 439441. (HTML) Correspondencia al autor Helena Benito Naverac helenabenito@yahoo.es FEA de Anestesiología y Reanimación. Hospital Clínico Universitario Lozano Blesa de Zaragoza. Aceptado para blog en febrero de 2018. Copyright ReAR. Rev Elect Anestesiar pertenece a la Asociación Anestesia Reanimación España. Entidad sin ánimo de lucro.
W3113831354.txt	https://www.researchsquare.com/article/rs-6879/v1.pdf?c=1585613558000	en		Survey of human and bovine tuberculosis infection on dairy farms in southwestern China	Research Square (Research Square)	2,019	cc-by	6,108	Survey of human and bovine tuberculosis infection on dairy farms in southwestern China Shu Zhang (  dulianyouchao@qq.com ) Sichuan Center for Disease Control and Prevention https://orcid.org/0000-0001-6123-5418 Chuang Chen Sichuan Center for Disease Control and Prevention, Chengdu, Sichuan, China. Zhengyuan Rao Sichuan Center for Disease Control and Prevention, Chengdu, Sichuan, China. Aiguo Yang Sichuan Animal Epidemic Prevention and Control Center, Chengdu, Sichuan, China. Li Guo Sichuan Animal Epidemic Prevention and Control Center, Chengdu, Sichuan, China. Wei Hou Sichuan Animal Epidemic Prevention and Control Center, Chengdu, Sichuan, China. Liang Zhang Hongya Center for Disease Control and Prevention, Hongya County, Meishan, Sichuan, China. Xiulan Yang Hongya Center for Disease Control and Prevention, Hongya County, Meishan, Sichuan, China. Yan Liu Hongya Center for Animal Epidemic Prevention and Control, Hongya County, Meishan, Sichuan, China. Yumei Wu Hongya Center for Animal Epidemic Prevention and Control, Hongya County, Meishan, Sichuan, China. Research article Keywords: Zoonotic disease; Nontuberculous Mycobacteria; Susceptibility; Bovine Posted Date: October 18th, 2019 DOI: https://doi.org/10.21203/rs.2.16233/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/20 Abstract Background Tuberculosis is a zoonotic chronic infectious disease that can be transmitted through respiratory secretions, causing simultaneous infection in both people and cattle. The aim of our study was to identify the co-infections of Mycobacterium tuberculosis between human and cattle in dairy farms in Sichuan Province, southwest China.Results We selected 6 large-scale dairy farms and 5 small-scale dairy farms in Sichuan Province, southwest China, as research sites. A total of 378 dairy farm workers (except one pregnant woman)were screened for tuberculosis symptoms and examined by X-ray radiographs. One worker was diagnosed with tuberculosis, but the sputum culture result was negative, and no strain was obtained. The purified protein derivative (PPD) skin tests of 10224 dairy cows showed that 99 cows were positive, with a positive rate of 9.7‰. The Oesophageal-Pharyngeal (OP) secretions of PPD-positive cows were cultured, and 21 strains were obtained. After amplifying and sequencing the 16SrDNA, hsp65 and rpoB genes and the ITS region, sequence alignment in BLAST showed that these strains were nontuberculous mycobacteria (NTM)(18 Mycobacterium nonchromogenicum , 1 Mycobacterium hiberniae , 1 Mycobacterium arupense and 1 Mycobacterium chitae ).Conclusion This study indicated that PPD-positive cows on dairy farms were infected with NTM rather than Mycobacterium bovis . Cross-infection of tuberculosis between humans and cows on dairy farms has been controlled. Background Tuberculosisis a chronic zoonotic disease caused by the Mycobacterium tuberculosis complex(MTBC), which mainly includes M. tuberculosis(human-like), Mycobacterium bovis, Mycobacterium africanum and Mycobacterium microti. Humans and cattle share similar susceptibility to M. tuberculosis(human strain) and M.bovis[1-4]. As stated in previous reports, the infection status of bovine tuberculosis is grim. Livestock, cattle, especially dairy cows, are the most susceptible bovine to tuberculosis[5].Dairy cows can transmit tuberculosis to workers through oral secretions and feces. Milk secreted by cows with tuberculosis and dairy products that do not undergo pasteurization can also transmit tuberculosis to humans[6-7]. Approximately 3% of tuberculosis patients are infected with M.bovis by drinking milk or dairy products that are not sterilized[8-9]. In Asian and African countries and regions, 4.7% to 30.8% of tuberculosispositive cattle are infected with the human strain[10]. The more serious the prevalence of human tuberculosis on a farm is, the higher the proportion of cattle infected with human tuberculosis bacteria[11]. According to reports from 2010, the prevalence of bovine tuberculosis in China was 0-15% [12]. This indicates that there were simultaneous infections of tuberculosis in humans and cattle[13].Therefore, we conducted a tuberculosis survey of dairy cows and workers in large and small dairy farms in Sichuan Province, southwestern China, to identify simultaneous infections of tuberculosis in cows and humans. Full Text Page 2/20 Survey of human and bovine tuberculosis infection on dairy farms in southwestern China Chuang Chena, Zhengyuan Raoa, AiguoYang b, LiGuo b, WeiHou b, LiangZhang c, XiulanYang c, YanLiu d, YumeiWu d, Shu Zhang a* a Sichuan Center for Disease Control and Prevention, Chengdu, Sichuan, China. bSichuan Animal Epidemic Prevention and Control Center, Chengdu, Sichuan, China. c Hongya Center for Disease Control and Prevention, Hongya County, Meishan, Sichuan, China. d Hongya Center for Animal Epidemic Prevention and Control, Hongya County, Meishan, Sichuan, China. Corresponding author: Shu Zhang, dulianyouchao@qq.com Abstract Background: Tuberculosis is a zoonotic chronic infectious disease that can be transmitted through respiratory secretions, causing simultaneous infection in both people and cattle. The aim of our study was to identify the co-infections of Mycobacterium tuberculosis between human and cattle in dairy farms in Sichuan Province, southwest China. Results: We selected 6 large-scale dairy farms and 5 small-scale dairy farms in Sichuan Province, southwest China, as research sites. A total of 378 dairy farm workers (except one pregnant woman)were screened for tuberculosis symptoms and examined by X-ray radiographs. One worker was diagnosed with tuberculosis, but the sputum culture result was negative, and no strain was obtained. The purified protein derivative (PPD) skin tests of 10224 dairy cows showed that 99 cows were positive, with a positive rate of 9.7‰. The Oesophageal-Pharyngeal (OP) secretions of PPD-positive cows were cultured, and 21 strains were obtained. After amplifying and sequencing the 16SrDNA, hsp65 and rpoB genes and the ITS region, sequence alignment in BLAST showed that these strains were nontuberculous mycobacteria (NTM)(18 Mycobacterium nonchromogenicum, 1 Mycobacterium hiberniae, 1 Mycobacterium arupense and 1 Mycobacterium chitae).. Conclusion: This study indicated that PPD-positive cows on dairy farms were infected with NTM rather than Mycobacterium bovis. Cross-infection of tuberculosis between humans and cows on dairy farms has been controlled. Keywords: Zoonotic disease; Nontuberculous Mycobacteria; Susceptibility; Bovine Background Tuberculosisis a chronic zoonotic disease caused by the Mycobacterium tuberculosis complex(MTBC), which mainly includes M. tuberculosis(human-like), Mycobacterium bovis, Mycobacterium africanum and Page 3/20 Mycobacterium microti. Humans and cattle share similar susceptibility to M. tuberculosis(human strain) and M.bovis[1–4]. As stated in previous reports, the infection status of bovine tuberculosis is grim. Livestock, cattle, especially dairy cows, are the most susceptible bovine to tuberculosis[5].Dairy cows can transmit tuberculosis to workers through oral secretions and feces. Milk secreted by cows with tuberculosis and dairy products that do not undergo pasteurization can also transmit tuberculosis to humans[6–7]. Approximately 3% of tuberculosis patients are infected with M.bovis by drinking milk or dairy products that are not sterilized[8–9]. In Asian and African countries and regions, 4.7% to 30.8% of tuberculosispositive cattle are infected with the human strain[10]. The more serious the prevalence of human tuberculosis on a farm is, the higher the proportion of cattle infected with human tuberculosis bacteria[11]. According to reports from 2010, the prevalence of bovine tuberculosis in China was 0–15% [12]. This indicates that there were simultaneous infections of tuberculosis in humans and cattle[13].Therefore, we conducted a tuberculosis survey of dairy cows and workers in large and small dairy farms in Sichuan Province, southwestern China, to identify simultaneous infections of tuberculosis in cows and humans. Results Population survey A total of 378 workers in 5 large-scale dairy farms and 6 small-scale dairy farms were screened for symptoms; 377(a pregnant woman was not included)were examined by chest X-ray (DR).The symptom screening results showed that all the workers were asymptomatic; 1 person had an abnormal chest X-ray result, with suspected tuberculosis lesions; all 5 worker samples that were subjected to sputum smear and culture were negative. One patient with active pulmonary tuberculosis was clinically diagnosed and was referred for tuberculosis management and treatment. Investigation of dairy cows A total of 10224 dairy cows were examined by PPD skin tests. Ninety-nine cows were PPD positive, but none of them had suspicious symptoms. The OP secretions of 99 PPD-positive cows were cultured on acid LJ medium, and 21 samples were cultured for bacterial growth. The OP secretions from 88 cows that were adjacent to PPD-positive cows and 20 environmental samples were cultured, with negative results. Identification of species Page 4/20 The sequences of the 16SrDNA, hsp65, and rpoB genes and the ITS region were used to identify species by multilocus sequence analysis. The species of the 21 strains are presented in Table 1. All of the strains were nontuberculous mycobacteria (NTM). The MTBC was not detected. Drug sensitivity testing results The antimicrobial susceptibility testing results for the 20 isolates(Mycobacterium arupense did not grow)from the PPD-positive cows are shown in Table 2. Discussion The tuberculosis epidemic in China is serious; however, it is better than those in India and Indonesia. Sichuan Province is a populous province in southwestern China, and the epidemic of tuberculosis is very serious in this province. There are also many dairy cows raised in Sichuan Province. Tuberculosis is a zoonotic infectious disease caused by M. tuberculosis. Previous reports have shown evidence of tuberculosis cross-infection between humans and cattle[4,9]. We screened dairy farm workers for tuberculosis symptoms and performed X-ray chest radiographs, and all the cows were screened by PPD skin tests. OP secretions were obtained from PPD-positive cattle; the cows were fasted for 12 hours (with water accessible) to ensure that the OP secretions were not contaminated by ruminate. The results of the environmental sample cultures of the PPD-positive cattle were negative. Culture was also used to verify that the samples from OP secretions were not contaminated by environmental tuberculosis. According to previous reports in Chinese journals, the tuberculosis infection rate of cows on dairy farms in China is 10‰. In our study, the PPD-positive rate was approximately 10‰, which is consistent with previous reports. Through the culture of OP secretions from the PPD-positive cows, 21 isolates were obtained; however, they were recognized as NTM after sequencing and alignment the 16SrDNA, hsp65, rpoB gene and ITS region. This finding indicated that there may be an error in the 10‰ tuberculosis infection rate reported in the previous paper in dairy cows in China, as these cows are most likely infected with NTM. Currently, tuberculosis in dairy cows has been controlled, and NTM is the most likely infection source in PPD-positive cows. In this study, 21 NTM strains(18 Mycobacterium nonchromogenicum, 1 Mycobacterium hiberniae, 1 M.arupense, and 1 Mycobacterium chitae) were obtained. Research in Northern Ireland also cultured M. nonchromogenicum (a member of the Mycobacterium terrae complex, a nonpigmented, slow-growing species) from cattle, indicating that M. nonchromogenicum is a dominant Mycobacterium of cattle strains[17]. In previous reports, M. nonchromogenicum, M. hiberniae, and M. arupensewere also responsible for human infection and disease[18–22].The minimum inhibitory concentrations (MICs) of 21 kinds of drugs against M. nonchromogenicum, M. hiberniae and M. arupense were relatively high in the drug sensitivity test; in contrast, the MIC of M. chitae was low. Therefore, it is difficult to select appropriate drugs for the treatment of these NTMs. Thus, these MICs of NTMs will provid some Page 5/20 suggestions for the further research. We believe that our study makes a significant contribution to the literature because it is the first time to identify that PPD-positive dairy cows were infected with NTM but not Mycobacterium tuberculosis in southwestern China. Conclusions Our study indicated that the PPD-positive rate of cows on dairy farms in Sichuan Province which located in southwest China was approximately 10‰, but these cows were infected with NTM rather than Mycobacterium bovis and Mycobacterium tuberculosis. Cross-infection of tuberculosis between humans and cows on dairy farms has been controlled. Methods Research sites The study sites were five large-scale dairy farms and six small-scale dairy farms in Hongya County, Sichuan Province, southwest China. Investigation methods All the staff at the investigation sites were screened for clinical symptoms and examined by chest X-ray (DR). Then, sputum smears (night sputum, morning sputum and spot sputum)and sputum cultures were performed for those with suspected tuberculosis symptoms and suspected tuberculosis on chest X-ray. All dairy cows at the survey sites were screened by purified protein derivative (PPD) skin tests (purchased from Zhongmu CO., LTD., Chengdu, China). Then, Oesophageal-Pharyngeal (OP) secretions obtained from dairy cows (PPD-positive cows, cows with suspected tuberculosis symptoms, and adjacent cows of PPDpositive cows) that were fasted (water was provided) for 12 hours were subjected to culture; environmental samples of PPD-positive cows were collected for culture as well. Culture and DNA extraction After treatment with 4% NaOH, acid Löwenstein-Jensen (LJ) medium (purchased from Celnovte, CO., LTD., Zhengzhou, China) was used to culture the samples. Each sample was cultured in two tubes at 37°C for 1–2 months. DNA was extracted by the heating method using the culture strains. The culture strains were suspended in 100 µl of sterile distilled water and then heated at 99°C in a heating block for 20 min. The DNA was stored at –20°C. Page 6/20 Amplification and sequencing of the 16SrDNA, hsp65, and rpoB genes and the ITS region According to previous reports[14–16], the 16SrDNA, hsp65, and rpoB genes and the internal transcribed spacer (ITS)region were used to design the primers for DNA amplification, which were used to accurately identify the Mycobacterium strains. 16S rDNA PCR To amplify the 16S rDNA of Mycobacterium, the following primers were used: 16S-F(5’AGAGTTTGATCCTGGCTCAG–3’) and 16S-R(5’-AAGGAGGTGATCCAGCCGCA–3’). Two microliters of culture-suspended DNA was used as the DNA template in a 50 μl PCR mixture containing 25 μl of 2Taq Master Mix (purchased from Cwbiotech, CO., LTD., China), 1 μl of each forward and reverse primer (10 pmol/l) and 21 μl of double distilled water. The PCR cycling parameters were as follows: 95°C for 5 min for initial denaturation; followed by 35 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 58°C, and elongation for 1 min at 72°C; and final extension at 72°C for 10 min. hsp65 PCR The primers hsp65-F(5’-ATCGCCAAGGAGATCGAGCT–3’) and hsp65-R(5’-AAGGTGCCGCGGATCTTGTT– 3’) were used to amplify the hsp65 gene. The 50 μl PCR mixture contained 21 μl of double distilled water, 25 μl of 2Taq Master Mix (purchased from Cwbiotech, CO., LTD., China), 1 μl of each forward and reverse primer (10 pmol/l), and 2 μl of DNA template. The PCR cycling parameters were as follows: initial denaturation at 95°Cfor 5 min; followed by 35 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 63°C, and elongation for 1 min at 72°C; and a final extension at 72°C for 10 min. rpoB PCR rpob-F(5’-GGCAAGGTCACCCCGAAGGG–3’) and rpob-R(5’-AGCGGCTGCTGGGTGATCATC–3’) were used for the amplification of the rpoB gene. The 50 μl PCR mixture contained 21 μl of double distilled water, 25 μl of 2Taq Master Mix (purchased from Cwbiotech, CO., LTD., China), 1 μl of each forward and reverse primer (10 pmol/l), and 2 μlof DNA template of the culture suspensions. The PCR cycling parameters were as follows: initial denaturation at 95°Cfor 5 min; followed by 35 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 60°C, and elongation for 1 min at 72°C; and a final extension at 72°C for 10 min. 16SrDNA–23SrDNA internal transcribed spacer(ITS) PCR ITS -F(5’- AAGTCGTAACAAGGTARCCG –3’) and ITS -R(5’-TCGCCAAGGCATCCACC–3’) were used for the amplification of the ITS gene fragment. A 50 μl PCR mixture contained 21 μl of double distilled water, 25 μl of 2Taq Master Mix (purchased from Cwbiotech, CO., LTD., China), 1 μl of each forward and reverse primer (10 pmol/l), and 2 μl of DNA of the culture suspensions. The PCR cycling parameters were as Page 7/20 follows: initial denaturation at 95°Cfor 5 min; followed by 35 cycles of denaturation for 1 min at 95°C,annealing for 1 min at 60°C, and elongation for 1 min at 72°C; and a final extension at 72°Cfor 10 min. Sequencing of amplification products of the 16SrDNA, hsp65, and rpoB genes and the ITS region Gene amplification products of the 16SrDNA, hsp65, and rpoB genes and the ITS region were sequenced at Tsingke(Beijing，China). Alignment of sequences and identification of species The sequences of the amplified products(16SrDNA, hsp65, rpoB and ITS) were analyzed on the NCBI BLAST platform (https://blast.ncbi.nlm.nih.gov/Blast.cgi) by MegaBLAST for species identification. Drug sensitivity tests The in vitro drug susceptibility of these strains was evaluated by the broth dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). List of abbreviations MTBC: Mycobacterium tuberculosis complex; PPD: purified protein derivative; OP: OesophagealPharyngeal; NTM: nontuberculous mycobacteria; hsp65: heat shock protein 65; rpoB: β subunit of bacterial RNA polymerase; ITS: 16SrDNA–23SrDNA internal transcribed spacer Declarations Ethics approval and consent to participate This study was authorized by Sichuan Center for Disease Control and Prevention, and the Sichuan Medical Association. It’s ethical approval was obtained from the Research Ethics Committee of Sichuan Center for Disease Control and Prevention. These animals in this study were taken for routine diagnostics and not used for experimental studies, and the material consisted of Oesophageal-Pharyngeal Secretions(OP Secretions) used in this study were collected by local vets after verbal approvals from the Page 8/20 owners for further researching. All the staff involved in this study gave the informed consent in oral. According to the review of biomedical research ethics (SC/DL-GZ023) of Sichuan Centers for Disease Control and Prevention, collecting animal sputum culture isolates does not require ethical review. We drafted the written informed consents for the participants, but the owners of the dairy farms believed that oral consent was faster and more convenient, and supported oral consent. The ethical review committee agreed to this practice (Sichuan CDC ethical committee ethical review 4.7: Oral informed consent is allowed when signature would bring trouble to someone), so we used oral informed consent. Consent for publication Not applicable. Availability of data and materials All data support the results of this paper are included in this published article. Competing interests The authors declare that they have no competing interests. Funding This study was funded by the Sichuan Medical Association(Grant No. S16066). Authors’ contributions Chuang Chen supervised the project. Zhengyuan Rao, Aiguo Yang, Li Guo, Wei Hou, Liang Zhang, XiulanYang, YanLiu, YumeiWu and Shu Zhang were participated in the field investigation. Chuang Chen and Shu Zhang finished the lab work. Shu Zhang drafted and revised the manuscript. All authors read and approved the final manuscript. References 1. Michalak K, Austin C, Diesel S, et al. Mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants. Emerg Infect Dis. 1998;4:283–287. 2. Ocepek M, Pate M, Žolnir-Dovč M, et al. Transmission of Mycobacterium tuberculosis from human to cattle. J Clin Microbiol. 2005;43:3555–3557. Page 9/20 3. Suzuki Y, Matsuba T, Nakajima C. Zoonotic aspects of tuberculosis caused by Mycobacterium bovis. Kekkaku. 2010;85:79–86. 4. AmeniGK, Tadesse E, Hailu Y, et al. Transmission of Mycobacterium tuberculosis between farmers and cattle in Ethiopia. PLoS ONE. 2013;8(Suppl 10):e76891. 5. Rebuma F, Rea T, Alehegne W, et al. High Prevalence of Bovine Tuberculosis in Dairy Cattle in Central Ethiopia: Implications for the Dairy Industry and Public Health. PLoS One. 2012; 7(12): e52851. 6. Grange JM. Mycobacterium bovis infection in human beings. Tuberculosis (Edinb). 2001;81: 71–77. 7. de la Rua-Domenech R. Human Mycobacterium bovis infection in the United Kingdom: Incidence, risks, control measures and review of the zoonotic aspects of bovine tuberculosis. Tuberculosis (Edinb). 2001; 86: 77–109. 8. Grange JM, Daborn C, Cosivi O. HIV-related tuberculosis due to Mycobacterium bovis. Eur Respir J. 1994;7: 1564–1566. 9. Cosivi O, Grange JM, Daborn CJ, et al. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis. 1999;4: 59–70. 10. Thoen CO, LoBue PA, I de Kantor. Why has zoonotic tuberculosis not recerved much attention. Int J Tuberc Lung Dis. 2010;14(9):1073–1074. 11. Klein EJ, Boster DR, Stapp JR, et al. Humans as source of Mycobacterium tuberculosis infection in cattle, Spain. Emerg Infect Dis. 2011;17(12):2393–2395. 12. Thoen CO, Steele JH, Kaneene JB. Zoonotic tuberculosis Mycobacterium bovis and other pathogenic Mycobacteria. Mycobacterium bovis infection and control in China, Wiley Blackwell, 2014. 13. Hezekiah KA, Victor O A, Mutolib AS. Knowledge and practices about zoonotic tuberculosis prevention and associated determinants amongst livestock workers in Nigeria; 2015. PLoS One. 2018; 13(6): e0198810. 14. Springer B, Erik C. BÖttger, Philip Kirschner, et al. Phylogeny of the Mycobacterium chelonae-like organism based on partial sequencing of the 16S r RNA Gene and Proposal of Mycobacterium mucogenicum sp. nov. Int J Syst Bacteriol. 1995;45(2):262–7. 15. Kim H, Kim SH, Shim TS, et al. Differentiation of Mycobacterium species by analysis of the heatshock protein 65 gene(hsp65). Int J syst Evol Microbiol. 2005;55(Pt 4):1649–56. 16. Slany M, Svobodova J, Ettlova A, et al. Mycobacterium arupense among the isolates of nontuberculous mycobacteria from human, animal and environmental samples. VeterinarniMedicina. 2010;55(8):369–376. 17. Mc Corry TP, CM McCormick, MS Hughes, et al. Mycobacterium nonchromogenicum in nasal mucus from cattle in a herd infected with bovine tuberculosis. Vet Microb. 2004;99:281–285. 18. Sawai T, Inoue Y, Doi S, et al. A case of Mycobacterium nonchromogenicum pulmonary infection showing multiple nodular shadows in an immunocompetent patient. Diagn Microbiol Infect Dis. 2006;54(4):311–314. Page 10/20 19. Lai CC, Chen HW, Liu WL, et al. Fatal Meningitis Caused by Mycobacterium nonchromogenicum in a Patient with Nasopharyngeal Carcinoma. Clinical Infectious Diseases. 2008;46(2):325–326. 20. Kazda J, Cooney R, Monaghan M, et al. Mycobacterium hiberniae sp. nov. IntJ Syst EvolMicr. 1993; 43:352–357. 21. Cloud JL, Meyer JJ, Pounder JI, et al. Mycobacterium arupense sp. nov., a non-chromogenic bacterium isolated from clinical specimens. Int J Syst Evol Microbiol. 2006;56: 1413–1418. 22. Slany M, Pavlik I. Molecular detection of nontuberculous mycobacteria: advantages and limits of a broad-range sequencing approach. J Mol Microbiol Biotechnol. 2012;22(4):268–76. Results Population survey A total of 378 workers in 5 large-scale dairy farms and 6 small-scale dairy farms were screened for symptoms; 377(a pregnant woman was not included)were examined by chest X-ray (DR).The symptom screening results showed that all the workers were asymptomatic; 1 person had an abnormal chest X-ray result, with suspected tuberculosis lesions; all 5 worker samples that were subjected to sputum smear and culture were negative. One patient with active pulmonary tuberculosis was clinically diagnosed and was referred for tuberculosis management and treatment. Investigation of dairy cows A total of 10224 dairy cows were examined by PPD skin tests. Ninety-nine cows were PPD positive, but none of them had suspicious symptoms. The OP secretions of 99 PPD-positive cows were cultured on acid LJ medium, and 21 samples were cultured for bacterial growth. The OP secretions from 88 cows that were adjacent to PPD-positive cows and 20 environmental samples were cultured, with negative results. Identification of species The sequences of the 16SrDNA, hsp65, and rpoB genes and the ITS region were used to identify species by multilocus sequence analysis. The species of the 21 strains are presented in Table 1. All of the strains were nontuberculous mycobacteria (NTM). The MTBC was not detected. Drug sensitivity testing results Page 11/20 The antimicrobial susceptibility testing results for the 20 isolates(Mycobacterium arupense did not grow)from the PPD-positive cows are shown in Table 2. Discussion The tuberculosis epidemic in China is serious; however, it is better than those in India and Indonesia. Sichuan Province is a populous province in southwestern China, and the epidemic of tuberculosis is very serious in this province. There are also many dairy cows raised in Sichuan Province. Tuberculosis is a zoonotic infectious disease caused by M. tuberculosis. Previous reports have shown evidence of tuberculosis cross-infection between humans and cattle[4,9]. We screened dairy farm workers for tuberculosis symptoms and performed X-ray chest radiographs, and all the cows were screened by PPD skin tests. OP secretions were obtained from PPD-positive cattle; the cows were fasted for 12 hours (with water accessible) to ensure that the OP secretions were not contaminated by ruminate. The results of the environmental sample cultures of the PPD-positive cattle were negative. Culture was also used to verify that the samples from OP secretions were not contaminated by environmental tuberculosis. According to previous reports in Chinese journals, the tuberculosis infection rate of cows on dairy farms in China is 10‰. In our study, the PPD-positive rate was approximately 10‰, which is consistent with previous reports. Through the culture of OP secretions from the PPD-positive cows, 21 isolates were obtained; however, they were recognized as NTM after sequencing and alignment the 16SrDNA, hsp65, rpoB gene and ITS region. This finding indicated that there may be an error in the 10‰ tuberculosis infection rate reported in the previous paper in dairy cows in China, as these cows are most likely infected with NTM. Currently, tuberculosis in dairy cows has been controlled, and NTM is the most likely infection source in PPD-positive cows. In this study, 21 NTM strains(18 Mycobacterium nonchromogenicum, 1 Mycobacterium hiberniae, 1 M.arupense, and 1 Mycobacterium chitae) were obtained. Research in Northern Ireland also cultured M. nonchromogenicum (a member of the Mycobacterium terrae complex, a nonpigmented, slow-growing species) from cattle, indicating that M. nonchromogenicum is a dominant Mycobacterium of cattle strains[17]. In previous reports, M. nonchromogenicum, M. hiberniae, and M. arupense were also responsible for human infection and disease[18-22].The minimum inhibitory concentrations (MICs) of 21 kinds of drugs against M. nonchromogenicum, M. hiberniae and M. arupense were relatively high in the drug sensitivity test; in contrast, the MIC of M. chitae was low. Therefore, it is difficult to select appropriate drugs for the treatment of these NTMs. Thus, these MICs of NTMs will provid some suggestions for the further research. We believe that our study makes a significant contribution to the literature because it is the first time to identify that PPD-positive dairy cows were infected with NTM but not Mycobacterium tuberculosis in southwestern China. Conclusions Page 12/20 Our study indicated that the PPD-positive rate of cows on dairy farms in Sichuan Province which located in southwest China was approximately 10‰, but these cows were infected with NTM rather than Mycobacterium bovis and Mycobacterium tuberculosis. Cross-infection of tuberculosis between humans and cows on dairy farms has been controlled. Methods Research sites The study sites were five large-scale dairy farms and six small-scale dairy farms in Hongya County, Sichuan Province, southwest China. Investigation methods All the staff at the investigation sites were screened for clinical symptoms and examined by chest X-ray (DR). Then, sputum smears (night sputum, morning sputum and spot sputum)and sputum cultures were performed for those with suspected tuberculosis symptoms and suspected tuberculosis on chest X-ray. All dairy cows at the survey sites were screened by purified protein derivative (PPD) skin tests (purchased from Zhongmu CO., LTD., Chengdu, China). Then, Oesophageal-Pharyngeal (OP) secretions obtained from dairy cows (PPD-positive cows, cows with suspected tuberculosis symptoms, and adjacent cows of PPDpositive cows) that were fasted (water was provided) for 12 hours were subjected to culture; environmental samples of PPD-positive cows were collected for culture as well. Culture and DNA extraction After treatment with 4% NaOH, acid Löwenstein-Jensen (LJ) medium (purchased from Celnovte, CO., LTD., Zhengzhou, China) was used to culture the samples. Each sample was cultured in two tubes at 37°C for 1-2 months. DNA was extracted by the heating method using the culture strains. The culture strains were suspended in 100 µl of sterile distilled water and then heated at 99°C in a heating block for 20 min. The DNA was stored at -20°C. Amplification and sequencing of the 16SrDNA, hsp65, and rpoB genes and the ITS region According to previous reports[14-16], the 16SrDNA, hsp65, and rpoB genes and the internal transcribed spacer (ITS)region were used to design the primers for DNA amplification, which were used to accurately identify the Mycobacterium strains. Page 13/20 16S rDNA PCR To amplify the 16S rDNA of Mycobacterium, the following primers were used: 16S-F(5’AGAGTTTGATCCTGGCTCAG-3’) and 16S-R(5’-AAGGAGGTGATCCAGCCGCA-3’). Two microliters of culture-suspended DNA was used as the DNA template in a 50 μl PCR mixture containing 25 μl of 2Taq Master Mix (purchased from Cwbiotech, CO., LTD., China), 1 μl of each forward and reverse primer (10 pmol/l) and 21 μl of double distilled water. The PCR cycling parameters were as follows: 95°C for 5 min for initial denaturation; followed by 35 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 58°C, and elongation for 1 min at 72°C; and final extension at 72°C for 10 min. hsp65 PCR The primers hsp65-F(5’-ATCGCCAAGGAGATCGAGCT-3’) and hsp65-R(5’-AAGGTGCCGCGGATCTTGTT-3’) were used to amplify the hsp65 gene. The 50 μl PCR mixture contained 21 μl of double distilled water, 25 μl of 2Taq Master Mix (purchased from Cwbiotech, CO., LTD., China), 1 μl of each forward and reverse primer (10 pmol/l), and 2 μl of DNA template. The PCR cycling parameters were as follows: initial denaturation at 95°Cfor 5 min; followed by 35 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 63°C, and elongation for 1 min at 72°C; and a final extension at 72°C for 10 min. rpoB PCR rpob-F(5’-GGCAAGGTCACCCCGAAGGG-3’) and rpob-R(5’-AGCGGCTGCTGGGTGATCATC-3’) were used for the amplification of the rpoB gene. The 50 μl PCR mixture contained 21 μl of double distilled water, 25 μl of 2Taq Master Mix (purchased from Cwbiotech, CO., LTD., China), 1 μl of each forward and reverse primer (10 pmol/l), and 2 μlof DNA template of the culture suspensions. The PCR cycling parameters were as follows: initial denaturation at 95°Cfor 5 min; followed by 35 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 60°C, and elongation for 1 min at 72°C; and a final extension at 72°C for 10 min. 16SrDNA-23SrDNA internal transcribed spacer(ITS) PCR ITS -F(5’- AAGTCGTAACAAGGTARCCG -3’) and ITS -R(5’-TCGCCAAGGCATCCACC-3’) were used for the amplification of the ITS gene fragment. A 50 μl PCR mixture contained 21 μl of double distilled water, 25 μl of 2*Taq Master Mix (purchased from Cwbiotech, CO., LTD., China), 1 μl of each forward and reverse primer (10 pmol/l), and 2 μl of DNA of the culture suspensions. The PCR cycling parameters were as follows: initial denaturation at 95°Cfor 5 min; followed by 35 cycles of denaturation for 1 min at 95°C,annealing for 1 min at 60°C, and elongation for 1 min at 72°C; and a final extension at 72°Cfor 10 min. Sequencing of amplification products of the 16SrDNA, hsp65, and rpoB genes and the ITS region Page 14/20 Gene amplification products of the 16SrDNA, hsp65, and rpoB genes and the ITS region were sequenced at Tsingke(Beijing，China). Alignment of sequences and identification of species The sequences of the amplified products(16SrDNA, hsp65, rpoB and ITS) were analyzed on the NCBI BLAST platform (https://blast.ncbi.nlm.nih.gov/Blast.cgi) by MegaBLAST for species identification. Drug sensitivity tests The in vitro drug susceptibility of these strains was evaluated by the broth dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Abbreviations MTBC: Mycobacterium tuberculosis complex; PPD: purified protein derivative; OP: OesophagealPharyngeal; NTM: nontuberculous mycobacteria; hsp65: heat shock protein 65; rpoB: β subunit of bacterial RNA polymerase; ITS: 16SrDNA-23SrDNA internal transcribed spacer Declarations Ethics approval and consent to participate This study was authorized by Sichuan Center for Disease Control and Prevention, and the Sichuan Medical Association. It’s ethical approval was obtained from the Research Ethics Committee of Sichuan Center for Disease Control and Prevention. These animals in this study were taken for routine diagnostics and not used for experimental studies, and the material consisted of Oesophageal-Pharyngeal Secretions(OP Secretions) used in this study were collected by local vets after verbal approvals from the owners for further researching. All the staff involved in this study gave the informed consent in oral. According to the review of biomedical research ethics (SC/DL-GZ023) of Sichuan Centers for Disease Control and Prevention, collecting animal sputum culture isolates does not require ethical review. We drafted the written informed consents for the participants, but the owners of the dairy farms believed that oral consent was faster and more convenient, and supported oral consent. The ethical review committee agreed to this practice (Sichuan CDC ethical committee ethical review 4.7: Oral informed consent is allowed when signature would bring trouble to someone), so we used oral informed consent. Consent for publication Page 15/20 Not applicable. Availability of data and materials All data support the results of this paper are included in this published article. Competing interests The authors declare that they have no competing interests. Funding This study was funded by the Sichuan Medical Association(Grant No. S16066). Authors’ contributions Chuang Chen supervised the project. Zhengyuan Rao, Aiguo Yang, Li Guo, Wei Hou, Liang Zhang, Xiulan Yang, Yan Liu, Yumei Wu and Shu Zhang were participated in the field investigation. Chuang Chen and Shu Zhang finished the lab work. Shu Zhang drafted and revised the manuscript. All authors read and approved the final manuscript. References 1. Michalak K, Austin C, Diesel S, et al. Mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants. Emerg Infect Dis. 1998;4:283–287. 2. Ocepek M, Pate M, Žolnir-Dovč M, et al. Transmission of Mycobacterium tuberculosis from human to cattle. J Clin Microbiol . 2005;43:3555–3557. 3. Suzuki Y, Matsuba T, Nakajima C. Zoonotic aspects of tuberculosis caused by Mycobacterium bovis. Kekkaku. 2010;85:79–86. 4. AmeniGK, Tadesse E, Hailu Y, et al. Transmission of Mycobacterium tuberculosis between farmers and cattle in Ethiopia. PLoS ONE . 2013;8(Suppl 10):e76891. 5. Rebuma F, Rea T, Alehegne W, et al. High Prevalence of Bovine Tuberculosis in Dairy Cattle in Central Ethiopia: Implications for the Dairy Industry and Public Health. PLoS One. 2012; 7(12): e52851. 6. Grange JM. Mycobacterium bovis infection in human beings. Tuberculosis (Edinb). 2001;81: 71–77. Page 16/20 7. de la Rua-Domenech R. Human Mycobacterium bovis infection in the United Kingdom: Incidence, risks, control measures and review of the zoonotic aspects of bovine tuberculosis. Tuberculosis (Edinb). 2001; 86: 77–109. 8. Grange JM, Daborn C, Cosivi O. HIV-related tuberculosis due to Mycobacterium bovis. Eur Respir J. 1994;7: 1564–1566. 9. Cosivi O, Grange JM, Daborn CJ, et al. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis. 1999;4: 59–70. 10. Thoen CO, LoBue PA, I de Kantor. Why has zoonotic tuberculosis not recerved much attention. Int J Tuberc Lung Dis. 2010;14(9):1073-1074. 11. Klein EJ, Boster DR, Stapp JR, et al. Humans as source of Mycobacterium tuberculosis infection in cattle, Spain. Emerg Infect Dis. 2011;17(12):2393-2395. 12. Thoen CO, Steele JH, Kaneene JB. Zoonotic tuberculosis Mycobacterium bovis and other pathogenic Mycobacteria. Mycobacterium bovis infection and control in China, Wiley Blackwell, 2014. 13. Hezekiah KA, Victor O A, Mutolib AS. Knowledge and practices about zoonotic tuberculosis prevention and associated determinants amongst livestock workers in Nigeria; 2015. PLoS One . 2018; 13(6): e0198810. 14. Springer B, Erik C. BÖttger, Philip Kirschner, et al. Phylogeny of the Mycobacterium chelonae-like organism based on partial sequencing of the 16S r RNA Gene and Proposal of Mycobacterium mucogenicum sp. nov. Int J Syst Bacteriol. 1995;45(2):262-7. 15. Kim H, Kim SH, Shim TS, et al. Differentiation of Mycobacterium species by analysis of the heatshock protein 65 gene(hsp65). Int J syst Evol Microbiol. 2005;55(Pt 4):1649-56. 16. Slany M, Svobodova J, Ettlova A, et al. Mycobacterium arupense among the isolates of nontuberculous mycobacteria from human, animal and environmental samples. VeterinarniMedicina. 2010;55(8):369-376. 17. Mc Corry TP, CM McCormick, MS Hughes, et al. Mycobacterium nonchromogenicum in nasal mucus from cattle in a herd infected with bovine tuberculosis. Vet Microb. 2004;99:281-285. 18. Sawai T, Inoue Y, Doi S, et al. A case of Mycobacterium nonchromogenicum pulmonary infection showing multiple nodular shadows in an immunocompetent patient. Diagn Microbiol Infect Dis. 2006;54(4):311-314. 19. Lai CC, Chen HW, Liu WL, et al. Fatal Meningitis Caused by Mycobacterium nonchromogenicum in a Patient with Nasopharyngeal Carcinoma. Clinical Infectious Diseases. 2008;46(2):325-326. 20. Kazda J , Cooney R, Monaghan M, et al. Mycobacterium hiberniae sp. nov. IntJ Syst EvolMicr. 1993; 43:352-357. 21. Cloud JL, Meyer JJ, Pounder JI, et al. Mycobacterium arupense sp. nov., a non-chromogenic bacterium isolated from clinical specimens. Int J Syst Evol Microbiol. 2006;56: 1413-1418. 22. Slany M, Pavlik I. Molecular detection of nontuberculous mycobacteria: advantages and limits of a broad-range sequencing approach. J Mol Microbiol Biotechnol . 2012;22(4):268-76. Page 17/20 Tables Table 1.The species and strains obtained from the PPD-positive cows. NTM species identified Number of strains Mycobacterium nonchromogenicum 18 Mycobacterium hiberniae 1 Mycobacterium arupense 1 Mycobacterium chitae 1 Table 2. Distribution of antimicrobial susceptibility of the isolates. Page 18/20 Antimicrobial Drug The 18 isolates of M. MIC (µg/ml) of the MIC (µg/ml) of the agent concentration nonchromogenicum isolate of M. isolate of M. chitae range (µg/ml) MIC50(µg/ml) hiberniae MIC90 (µg/ml) moxifloxacin 0.0625-64 2 4 2 <0.0625 0.0625-64 4 4 4 <0.0625 tigecycline (TIG) 0.0625-64 >64 >64 32 <0.0625 clofazimine 0.03125-32 >32 >32 >32 <0.0625 cefoxitin (CFX) 0.25-256 64 >256 >256 <0.0625 vancomycin 0.25-256 >256 >256 >256 <0.0625 0.0625-64 8 >64 8 <0.0625 amikacin (AMK) 0.0625-64 16 64 16 <0.0625 azithromycin 0.0625-64 >64 >64 >64 0.125 0.25-256 256 >256 >256 <0.25 rifabutin (RFB) 0.0625-64 8 32 32 <0.0625 linezolid (LZD) 0.0625-64 64 >64 >64 <0.0625 minocycline 0.25-256 64 >256 64 <0.25 0.0625-64 16 >64 >64 <0.0625 0.25-256 32 64 64 <0.25 (MOX) gatifloxacin (GAT) (CFM) (VCM) streptomycin (SM) (AZM) sulfamethoxazole (SMZ) (MIN) clarithromycin (CLA) meropenem (MEM) Page 19/20 ethambutol 0.0625-64 1 1 1 <0.0625 0.0625-64 8 16 4 <0.0625 rifampicin (RIF) 0.0625-64 32 64 32 <0.0625 imipenem (IMP) 0.25-256 >256 >256 >256 <0.25 levofloxacin 0.0625-64 8 64 16 <0.0625 0.015625-16 >16 >16 >16 <0.015625 (EMB) capreomycin (CAP) (LFX) Bedaquiline Page 20/20
https://openalex.org/W3138093201	https://link.springer.com/content/pdf/10.17269/s41997-021-00501-y.pdf	English	null	Socio-demographic disparities in knowledge, practices, and ability to comply with COVID-19 public health measures in Canada	Canadian journal of public health	2,021	cc-by	9,762	ERROR: type should be string, got "https://doi.org/10.17269/s41997-021-00501-y\nCanadian Journal of Public Health (2021) 112:363–375 https://doi.org/10.17269/s41997-021-00501-y\nCanadian Journal of Public Health (2021) 112:363–375 SPECIAL SECTION ON COVID-19: QUANTITATIVE RESEARCH Abstract Objectives The effectiveness of public health interventions for mitigation of the COVID-19 pandemic depends on individual\nattitudes, compliance, and the level of support available to allow for compliance with these measures. The aim of this study was to\ndescribe attitudes and behaviours towards the Canadian COVID-19 public health response, and identify risk-modifying behav-\niours based on socio-demographic characteristics. g p\nMethods A cross-sectional online survey was administered in May 2020 to members of a paid panel representative of the\nCanadian population by age, gender, official language, and region of residence. A total of 4981 respondents provided responses\nfor indicators of self-reported risk perceptions, attitudes, and behaviours towards COVID-19 public health measures. Results More than 90% of respondents reported confidence in the ability to comply with a variety of public health measures. However, only 51% reported preparedness for illness in terms of expectation to work if sick or access to paid sick days. Risk\nperceptions, attitudes, and behaviours varied by demographic variables. Men, younger age groups, and those in the paid\nworkforce were less likely to consider public health measures to be effective, and had less confidence in their ability to comply. Approximately 80% of respondents reported that parents provided childcare and 52% reported that parents in the workforce\nprovided childcare while schools were closed. Methods A cross-sectional online survey was administered in May 2020 to members of a paid panel representative of the\nCanadian population by age, gender, official language, and region of residence. A total of 4981 respondents provided responses\nfor indicators of self-reported risk perceptions, attitudes, and behaviours towards COVID-19 public health measures. Results More than 90% of respondents reported confidence in the ability to comply with a variety of public health measures. However, only 51% reported preparedness for illness in terms of expectation to work if sick or access to paid sick days. Risk\ni\ni d\nd b h\ni\ni d b\nd\nhi\ni bl\nM\nd h\ni\nh\nid Methods A cross-sectional online survey was administered in May 2020 to members of a paid panel representative of the\nCanadian population by age, gender, official language, and region of residence. A total of 4981 respondents provided responses\nfor indicators of self-reported risk perceptions, attitudes, and behaviours towards COVID-19 public health measures. Abstract p\np\np\np\nResults More than 90% of respondents reported confidence in the ability to comply with a variety of public health measures. However, only 51% reported preparedness for illness in terms of expectation to work if sick or access to paid sick days. Risk\nperceptions, attitudes, and behaviours varied by demographic variables. Men, younger age groups, and those in the paid\nworkforce were less likely to consider public health measures to be effective, and had less confidence in their ability to comply. Approximately 80% of respondents reported that parents provided childcare and 52% reported that parents in the workforce\nprovided childcare while schools were closed. Conclusion Policies to help address issues of public adherence include targeted messaging for men and younger age groups,\nsocial supports for those who need to self-isolate, changes in workplace policies to discourage presenteeism, and provincially co-\nordinated masking and safe school policies. Socio-demographic disparities in knowledge, practices, and ability\nto comply with COVID-19 public health measures in Canada Amy L. Greer1,3 Received: 7 September 2020 /Accepted: 22 February 2021\n# The Author(s) 2021\n/ Published online: 24 March 2021 * Amy L. Greer\nagreer@uoguelph.ca; https://www.mathepilab.org * Amy L. Greer\nagreer@uoguelph.ca; https://www.mathepilab.org\n1\nDepartment of Population Medicine, University of Guelph,\nGuelph, Canada\n2\nMunk School of Global Affairs & Public Policy, University of\nToronto, Toronto, Canada\n3\nDalla Lana School of Public Health, University of Toronto,\nToronto, Canada 1\nDepartment of Population Medicine, University of Guelph,\nGuelph, Canada 2\nMunk School of Global Affairs & Public Policy, University of\nToronto, Toronto, Canada Résumé\nObj\nif 364 Can J Public Health (2021) 112:363–375 Résultats Plus de 90 % des répondants se sont dits certains de leur capacité de respecter de nombreuses mesures de santé\npublique. Par contre, 51 % seulement ont dit être préparés à respecter ces mesures s’ils attrapaient la maladie, c’est-à-dire pouvoir\ns’absenter du travail ou avoir droit à des congés de maladie payés. Les perceptions du risque, les attitudes et les comportements\nvariaient selon les caractéristiques démographiques. Les hommes, les jeunes et les personnes ayant un emploi rémunéré étaient\nmoins susceptibles de trouver les mesures de santé publique efficaces et moins sûrs de leur capacité de les respecter. Environ\n80 % des répondants ont indiqué que la garde des enfants était assurée par les parents, et 52 % ont indiqué que la garde des enfants\nquand les écoles étaient fermées était assurée par des parents ayant un emploi. Résultats Plus de 90 % des répondants se sont dits certains de leur capacité de respecter de nombreuses mesures de santé\npublique. Par contre, 51 % seulement ont dit être préparés à respecter ces mesures s’ils attrapaient la maladie, c’est-à-dire pouvoir\ns’absenter du travail ou avoir droit à des congés de maladie payés. Les perceptions du risque, les attitudes et les comportements\nvariaient selon les caractéristiques démographiques. Les hommes, les jeunes et les personnes ayant un emploi rémunéré étaient\nmoins susceptibles de trouver les mesures de santé publique efficaces et moins sûrs de leur capacité de les respecter. Environ\n80 % des répondants ont indiqué que la garde des enfants était assurée par les parents, et 52 % ont indiqué que la garde des enfants\nquand les écoles étaient fermées était assurée par des parents ayant un emploi. Conclusion Des messages ciblant les hommes et les jeunes, des soutiens sociaux aux personnes ayant besoin de\ns’isoler, des changements dans les politiques en milieu de travail pour dissuader le présentéisme, ainsi que des\npolitiques de port du masque et de sécurité à l’école coordonnées à l’échelle provinciale sont des mesures suscep-\ntibles d’atténuer les problèmes d’adhésion du public. Keywords COVID-19 . Attitude . Behaviour . Risk perception . Survey Keywords COVID-19 . Attitude . Behaviour . Risk perception . Survey Mots-clés COVID-19 . attitude . comportement . perception du risque . enquête Introduction public health measures. Thus, the objectives of this study are\nto (1) describe attitudes and behaviours towards the Canadian\nCOVID-19 public health response in May 2020, and\n(2) identify risk-modifying behaviours and resources to com-\nply with public health measures based on socio-demographic\nand household characteristics. This paper presents an initial\ndescriptive portrait of attitudes and perceptions among\nCanadians in the early stage of the pandemic to provide in-\nsights into improvements of programs and policies. The current coronavirus (COVID-19) pandemic represents a\nunique challenge for public health and health care systems. In\nthe absence of effective medical treatment options or a safe\nand effective vaccine, public health agencies have relied on\nnon-pharmaceutical interventions (NPIs) to mitigate transmis-\nsion of the virus. Physical distancing interventions act to re-\nduce the person-to-person contact rate in a population thereby\nreducing the likelihood of disease transmission. All Canadian\nprovinces and territories have instituted aggressive physical\ndistancing measures in response to the COVID-19 pandemic,\nincluding school closures, remote work, cancellation of mass\ngatherings, and the closure of all non-essential businesses. Résumé\nObj\nif Objectifs L’efficacité des mesures d’intervention en santé publique pour atténuer la pandémie de COVID-19 dépend des attitudes\nindividuelles, de la conformité, ainsi que du niveau d’aide disponible pour que les mesures soient respectées. Notre étude visait à\ndécrire les attitudes et les comportements à l’égard de la riposte de la santé publique canadienne à la COVID-19 et à cerner les\ncomportements modificateurs du risque d’après les caractéristiques sociodémographiques. Méthode Un sondage en ligne transversal a été administré en mai 2020 aux membres d’un comité rémunéré représentatif de\nl’âge, du sexe, des langues officielles et des régions de résidence de la population canadienne. En tout, 4 981 personnes ont fourni\ndes réponses à des questions indicatrices de leurs perceptions du risque, de leurs attitudes et de leurs comportements autodéclarés\nà l’égard des mesures de santé publique liées à la COVID-19. Objectifs L’efficacité des mesures d’intervention en santé publique pour atténuer la pandémie de COVID-19 dépend des attitudes\nindividuelles, de la conformité, ainsi que du niveau d’aide disponible pour que les mesures soient respectées. Notre étude visait à\ndécrire les attitudes et les comportements à l’égard de la riposte de la santé publique canadienne à la COVID-19 et à cerner les\ncomportements modificateurs du risque d’après les caractéristiques sociodémographiques. Objectifs L’efficacité des mesures d’intervention en santé publique pour atténuer la pandémie de COVID-19 dépend des attitudes\nindividuelles, de la conformité, ainsi que du niveau d’aide disponible pour que les mesures soient respectées. Notre étude visait à\ndécrire les attitudes et les comportements à l’égard de la riposte de la santé publique canadienne à la COVID-19 et à cerner les\ncomportements modificateurs du risque d’après les caractéristiques sociodémographiques. Méthode Un sondage en ligne transversal a été administré en mai 2020 aux membres d’un comité rémunéré représentatif de\nl’âge, du sexe, des langues officielles et des régions de résidence de la population canadienne. En tout, 4 981 personnes ont fourni Méthode Un sondage en ligne transversal a été administré en mai 2020 aux membres d’un comité rémunéré représentatif de\nl’âge, du sexe, des langues officielles et des régions de résidence de la population canadienne. En tout, 4 981 personnes ont fourni\ndes réponses à des questions indicatrices de leurs perceptions du risque, de leurs attitudes et de leurs comportements autodéclarés\nà l’égard des mesures de santé publique liées à la COVID-19. Data collection 2020) and posed questions about perceived risk Can J Public Health (2021) 112:363–375 365 of COVID-19 infection, as well as attitudes and behaviours\nregarding COVID-19 public health measures. Participants\nprovided information about their age, gender, province of res-\nidence, education level, employment status, household com-\nposition, household income, and the general size of their lo-\ncation of residence with options ranging from large city to\nrural. Participants were also asked whether they would be\nconsidered a priority risk group to receive the seasonal influ-\nenza vaccine as outlined by the Public Health Agency of\nCanada. The conditions meeting this criterion included chron-\nic respiratory disease, chronic heart disease, chronic kidney\ndisease, chronic liver disease, chronic neurological disease,\ndiabetes (all types), cancer, immunosuppression, dysfunction\nof the spleen, and/or BMI > 40 (National Advisory Committee\non Immunization (NACI) 2019). about attitudes toward COVID-19 public health measures. Chi-\nsquare analyses of individual contingency tables were conducted\nto further explore these data by respondent demographics and\nhousehold characteristics. The Bonferroni correction was applied\nfor each of the indicators of attitudes toward and ability to com-\nply with public health measures to account for multiple compar-\nisons within each measure. Therefore, a relationship was consid-\nered significant if the p-value was less than the corrected value\n(0.05/19 = 0.0026). Pairwise post hoc analyses with a Bonferroni\ncorrection were conducted for multi-level demographic and\nhousehold variables which showed significance in the chi-\nsquare analyses. q\ny\nLogistic regression models were developed to identify fac-\ntors associated with (1) mask use in the 24 hours prior to\nsurvey completion, (2) reporting direct contact with individ-\nuals outside of the respondent’s immediate household in the\n7 days prior to survey completion, and (3) reporting confi-\ndence in the ability to self-isolate with mild symptoms of\nCOVID-19. Univariable models were first assessed using a\nliberal p-value of less than 0.3 to determine eligibility for\ninclusion in the multivariable models. Variables included in\nthe initial full model for mask use included age, gender, risk\ngroup status, size of geographic region of residence, house-\nhold income, education level, employment status, household\ncomposition, and household size, as well as two indicators of\nperceived risk of COVID-19 to self and one indicator of\nperceived risk of transmission to others. Data collection Human behaviour is the main driver of respiratory disease\ntransmission and in the absence of a vaccine or other pharma-\nceutical interventions, mitigation requires large-scale behav-\niour change. As such, the effectiveness of public health inter-\nventions depends on the level of individual compliance and\nability to comply. Perceived risk due to COVID-19 and atti-\ntudes toward these measures have a large impact on the\nwillingness of people to make the behaviour changes\nnecessary for public health measures to be effective\n(Qazi et al. 2020). Perhaps more importantly, the ability\nto comply with public health measures such as self-\nisolation is dependent on an individual’s social and fi-\nnancial resources (Atchison et al. 2021). The study protocol was approved by the University of Guelph\nResearch Ethics Board (protocol #20-04-011) and the\nUniversity of Toronto Research Ethics Board (protocol\n#38251). The research company, Dynata, was contracted to\nconduct an electronic survey of Canadian adults between\nMay 7 and May 19, 2020. Participants were recruited from a\npanel of survey respondents and paid a nominal amount for\ncompleting the survey. Panelists who logged into their Dynata\naccount during the study period were directed to the survey if\nthey fit the quotas being targeted. Respondents provided in-\nformed consent after reading the study information by choos-\ning to continue to the survey questions. Representativeness of\nthe survey sample population was ensured by setting quotas\non age, gender, official language (English and French), and\nregion of residence (i.e., Atlantic, Quebec, Ontario, and West)\nbased on 2016 Canadian Census data (Statistics Canada\n2016a). Respondents could complete the survey in English\nor French. Enrollment into the survey within each stratum\nwas on a first-come, first-served basis. It is imperative that evidence be used to drive decision-\nmaking in order to provide the messaging and supports nec-\nessary to minimize transmission. It is important to identify\ngroups who are less likely to perceive COVID-19 as a risk,\nto perceive that public health measures are effective, and to\nhave the resources to comply, and more likely to engage in\nbehaviours associated with transmission of COVID-19. This\ninformation can be used to target messaging, develop policies,\nand provide supports to encourage uptake of the necessary The survey instrument was adapted from previous work\n(Jarvis et al. Data collection The initial full model\nassessing factors associated with engaging in an activity with\nnon-household contacts included respondents’ perceived ef-\nfectiveness of reducing contacts to mitigate transmission in\naddition to all variables included in the model described\nabove for mask use. The initial full model for confidence in\nthe ability to self-isolate included demographic characteristics\nand indicators of perceived risk as above in addition to work-\nplace indicators such as access to paid sick leave, expectation\nto work while sick, and the ability to work remotely. Household characteristics such as household size, whether\nthe household included children or a single parent, and\nwhether the household had access to a 14-day stockpile of\nsupplies were also included in the initial model. Further\ndetails of this regression model are located in the\nSupplementary Materials. COVID-19 risk perceptions were assessed by 3 statements\nand each response was recorded using a 6-level Likert scale\nranging from strongly agree to strongly disagree, and unsure. Perceived effectiveness of public health interventions to con-\ntrol COVID-19 was assessed by 8 questions, the responses to\nwhich were recorded using a 5-level Likert scale ranging from\nvery effective to not at all effective, and unsure. Respondents’\nconfidence that they could comply with various public health\nmeasures related to COVID-19 was assessed by 7 items and\nresponses were recorded using a 5-level Likert scale ranging\nfrom very confident to not at all confident, and unsure. Ability\nto comply with public health measures due to external influ-\nences was assessed using a 5-item Likert scale, ranging from\nstrongly agree to strongly disagree. Participants were also asked about their use of face masks. Adults living with children under the age of 15 years were\nasked to provide information about childcare provision during\nschool and daycare closures. The complete survey is provided\nin the Supplementary Materials. Results A total of 9120 survey responses were received between\nMay 7 and May 19, 2020. Survey responses were excluded\nfrom analysis if the survey was completed in less than\none third of the estimated completion time (n = 137), if the\nrespondent reported their age as less than 18 years (n = 23), or\nif the survey was discontinued prior to completion for any\nreason, including exceeding the age, gender, or province\nquotas (n = 3960). Responses with duplicated entries for gen-\nder, age, postal code, date, and contact names were considered\nduplicate responses and removed from the dataset (n = 19). Respondents who completed the entire survey and were not\nscreened out for any reason were included in the final sample,\nresulting in 4981 high-quality survey responses. Predictors of mask use The proportion of respondents who wore a mask in the 24\nhours prior to survey completion was 32.5% (95% CI, 31.2–\n33.8) for an average duration of 96.6 (SD 412.4) minutes. Respondents from Ontario (where physical distancing mea-\nsures were still in place at the time of the survey) reported the\nhighest level of mask use while those from Prince Edward\nIsland (where physical distancing recommendations were be-\nginning to relax at the time of the survey) reported the lowest\nmask use (Fig. 2a). The most common locations to wear a Data analysis Demographic characteristics of survey respondents were com-\npared with those from the 2016 Canadian Census in order to\nensure that the sample population was generally representative\nof the Canadian population. Attitudes towards the effectiveness of COVID-19 measures\nand confidence in individuals’ ability to comply with such mea-\nsures were aggregated to provide binary measures of agreement\n(strongly or somewhat); confidence (very or fairly); and per-\nceived effectiveness of measures (very or fairly); with the other\ncategory comprised of neutral responses, non-agreement, or un-\ncertainty. For a question regarding expectations of coworkers\nwith respect to working while ill, the responses “somewhat dis-\nagree” and “strongly disagree” were combined to form “dis-\nagree”, while all other responses were combined. The proportion\nof respondents who agreed, were confident, or thought each\nmeasure was effective was calculated for each of the questions pp\ny\nA backward elimination procedure was used to evaluate\nvariables for inclusion in the final multivariable regression\nmodels. Confounding was assessed by examining the vari-\nables in the model for changes once the potentially confound-\ning variable was excluded from the model. Once the final\nmodel was identified, all two-way multiplicative interaction\nterms involving age group with the other predictor variables\nwere assessed. Age group was of interest because it was sig-\nnificantly associated with most measures of perceived effec-\ntiveness and ability to comply with public health measures. Multicollinearity was assessed for each model using the Can J Public Health (2021) 112:363–375 366 variance inflation factor. All data were analyzed using\nRStudio Version 1.2.5033 (RStudio Team 2019). them to work if sick (Table 1). Fewer respondents in the youn-\ngest age group (18–29 years) reported having access to paid sick\nleave compared with those in the 30–39 years age group, and\nthose aged 30–59 years were more likely to have access to paid\nsick leave compared with respondents aged 60–69 years\n(Table 1). Demographics also predicted confidence in access to\na 14-day supply of food, and ability to find childcare (Table 1). Perceived effectiveness and confidence in the ability\nto comply with public health measures At least 87% of respondents considered each of the public\nhealth measures described to be effective in reducing the\ntransmission of COVID-19, with women and older indi-\nviduals expressing greater faith in public health mea-\nsures (Table 2). Those in the paid workforce were less\nlikely to agree that each of the public health measures\nis effective except school closures, where there was no\ndifference between groups. A detailed description of the respondent population is in-\ncluded in the Supplementary Materials (Table S1). For the\n4981 surveys, the proportion of respondents living in each\nprovince, the male to female ratio, and the proportion of re-\nspondents in each age category were comparable to the 2016\nCanadian Census of the population (data shown in Table S1,\nSupplementary Materials). More than 90% of respondents reported that they were\nconfident in their ability to comply with each of the five public\nhealth measures (Table 3), with greater confidence on most\nmeasures in women and older individuals. Lower-income in-\ndividuals were less confident in their ability to avoid public\ntransportation. Less confidence was seen in the paid work-\nforce, compared with those who were unemployed, retired,\nor working within the home. Childcare Table 1 describes respondents’ level of perceived risk in\nMay 2020 as well as indicators of preparedness in the event\nof illness. Overall, 61.5% of respondents agreed that COVID-\n19 would be a serious illness for them, 21.5% agreed that they\nare likely to catch the virus, and 71.5% agreed that they are\nlikely to transmit the virus if they do not follow public health\nadvice. Perceived risk of serious illness due to COVID-19\nincreased with increasing age beyond 50 years; however, per-\nceived risk of contracting the virus was highest in the 30–39\nyear age group and decreased with increasing age thereafter. Individuals who self-identified as being in a risk group were\nmore likely to agree that they are likely to catch the virus and\nexperience serious illness compared with other individuals,\nwhile those living with children under the age of 18 years or\nthose in the paid workforce were less likely to agree that\nCOVID-19 would be a serious illness for them compared with\nhouseholds containing no children or those not in the paid\nworkforce, respectively. Risk perception was also associated\nwith gender (Table 1). Respondents with household members who were 14 years of\nage or younger were asked about childcare provision when\nschools and daycares were closed due to the pandemic (n =\n930). More than 80% of respondents reported that a parent\nprovided childcare for their children during this time\n(Fig. 1a). Only 12.2% (95% CI, 10.1–14.3) of those requiring\nchildcare used providers who were not part of their household. Of the parents providing childcare, parents in the workforce\nprovided the greatest proportion of childcare duties (52%)\n(Fig. 1b). The wording of the questionnaire did not allow for\nan analysis of childcare by gender. Perceived preparedness A higher proportion of older individuals, those not in a risk\ngroup, and women reported that co-workers would not expect Can J Public Health (2021) 112:363–375 367 Table 1\nIndicators of perceived risk and preparedness in the event of illness stratified by socio-demographic characteristics. Values are reported as % (95% confidence interval), and those in bold font\nwere statistically significant between subgroups (p < 0.0026). Cells denoted by “-” signify that statistics were not run because the survey question was not relevant for one of the groups. Perceived preparedness Letters in superscript\nindicate levels at which pairwise comparisons were statistically significant with Bonferroni correction (p < 0.05)\nPerceived risk\nPreparedness in the event of illness\nLikely to contract\nCOVID-19 (n = 4981)\nLikely to be a serious\nillness for self (n = 4981)\nLikely to transmit to\nothers (n = 4981)\nMy co-workers would\nnot expect me to\nwork if ill (n = 2698)\nI would still get\npaid (n = 2698)\nI have enough food/supplies\nto last for 14 days (n = 4981)\nSomeone else would be\nable to look after my\nchildren (n = 930)\nOverall\n21.5% (20.4–22.7)\n61.5% (60.1–62.8)\n71.5% (70.3–72.8)\n50.9% (49.0–52.7)\n51.1% (49.2–53.0)\n70.8% (69.6–72.1)\n59.6% (56.4–62.7)\nGender\nWomen\n20.8% (19.3–22.4)\n61.0% (59.1–62.9)\n74.5% (72.8–76.2)\n55.7% (52.9–58.4)\n48.7% (45.9–51.5)\n72.9% (71.2–74.7)\n53.2% (48.7–57.6)\nMen\n22.1% (20.5–23.8)\n61.9% (60.0–63.8)\n68.4% (66.5–70.2)\n46.8% (44.2–49.3)\n53.3% (50.7–55.9)\n68.8% (66.9–70.6)\n66.2% (61.9–70.6)\nAge group\n18–29 yearsa\n25.7% d–f (22.7–28.8)\n45.1% c–f (41.6–48.6)\n70.9% (67.8–74.1)\n40.0% c, d (35.6–44.3)\n48.2% b (43.8–52.7)\n62.5% c–f (59.1–65.9)\n54.4% b, c, e (46.0–62.7)\n30–39 yearsb\n32.2% c–f (29.2–35.2)\n51.6% d–f (48.4–54.8)\n71.4% (68.6–74.3)\n43.3% c, d (39.8–46.8)\n55.3% a, e (51.7–58.8) 65.6% e, f (62.5–68.6)\n56.7% a, e (51.6–61.9)\n40–49 yearsc\n24.3% b, e, f (21.4–27.3) 54.9% a, d–f (51.4–58.3)\n70.3% (67.2–73.5)\n52.4% a, b (48.5–56.2)\n54.1% e (50.2–58.0)\n66.5% a, e, f (63.2–69.7)\n60.7% a, d, e (55.3–66.1)\n50–59 yearsd\n18.4% a, b (15.8–21.0)\n62.6% a-c, e, f (59.4–65.9)\n71.3% (68.3–74.4)\n63.4% a,b, e (59.4–67.4) 50.9% e (46.7–55.1)\n71.6% a, e, f (68.5–74.6)\n77.8% c, e (69.2–86.4)\n60–69 yearse\n13.4% a–c (11.3–15.6)\n72.9% a–c, d, f (70.1–75.7)\n70.6% (67.8–73.5)\n62.0% (55.9–68.2)\n41.4% b–d (35.1–47.6) 77.8% a–d (75.2–80.4)\n35.0% a, b, d (14.1–55.9)\n70+ yearsf\n13.8% a–c (11.1–16.5)\n85.3% a–d (82.5–88.0)\n75.6% (72.2–78.9)\n-\n-\n82.5% a–d (79.5–85.4)\n-\nRisk group\nYes\n29.8% (27.6–32.0)\n86.1% (84.4–87.7)\n74.0% (71.9–76.2)\n44.0% (40.3–47.6)\n52.7% (49.1–56.4)\n73.6% (71.5–75.8)\n63.4% (57.5–69.3)\nNo\n17.5% (16.2–18.8)\n49.6% (47.9–51.3)\n70.3% (68.8–71.9)\n53.4% (51.2–55.6)\n50.5% (48.3–52.7)\n69.4% (67.9–71.0)\n58.1% (54.4–61.9)\nHousehold children\nYes\n29.0% (26.4–31.7)\n55.8% (52.8–58.7)\n71.0% (68.3–73.6)\n48.3% (45.0–51.7)\n53.4% (50.1–56.8)\n66.1% (63.4–68.9)\n-\nNo\n19.3% (18.1–20.6)\n63.1% (61.6–64.7)\n71.7% (70.3–73.1)\n52.0% (50.0–54.3)\n50.0% (47.7–52.3)\n72.2% (70.8–73.6)\n-\nHousehold income\n$0–$60,000g\n21.8% (20.0–23.7)\n64.8% h (62.6–66.9)\n70.3% (68.3–72.4)\n51.0% (47.4–54.6)\n40.0% h, i (36.4–43.5)\n68.0% (65.9–70.1)\n49.3% h, i (42.8–55.8)\n$60,001–$110,000h\n22.9% (20.8–25.0)\n58.5% g (56.0–60.9)\n73.0% (70.8–75.2)\n51.6% (48.5–54.7)\n55.0% g, i (51.9–58.1)\n73.5% (71.3–75.6)\n62.2% g, i (57.2–67.2)\n> $110,000i\n22.4% (19.8–24.9)\n60.5% (57.5–63.4)\n73.1% (70.4–75.9)\n48.2% (44.7–51.7)\n57.8% g, h (54.3–61.3) 71.6% (68.8–74.3)\n65.1% g, h (59.6–70.7)\nDon’t know/prefer\nnot to answer\n14.5% (11.4–17.5)\n60.6% (56.3–64.8)\n68.4% (64.3–72.4)\n57.0% (50.1–63.9)\n47.0% (40.1–53.9)\n71.3% (67.4–75.2)\n56.5% (44.1–68.8)\nEmployment status\nPaid workforce\n26.2% (24.6–27.9)\n56.3% (54.4–58.1)\n70.6% (68.9–72.3)\n-\n-\n68.7% (66.9–70.4)\n61.5% (57.9–65.1)\nNot in paidworkforce 16.0% (14.5–17.5)\n67.6% (65.7–69.5)\n72.6% (70.8–74.5)\n-\n-\n73.3% (71.5–75.1)\n53.1% (46.4–59.8)\nLevel of education\nSecondary or lessj\n18.7% (16.5–20.9)\n61.7% (59.0–64.5)\n71.1% (68.5–73.6)\n53.6% (48.9–58.2)\n40.3% k, l (35.7–44.9)\n71.4% (68.8–74.0)\n50.3% (42.3–58.4)\nCollege/trades or\nother qualificationk\n21.7% (19.9–23.6)\n62.1% (59.9–64.3)\n70.0% (67.9–72.1)\n51.5% (48.4–54.6)\n50.7% j, l (47.6–53.8)\n71.4% (69.3–73.4)\n58.2% (53.0–63.4)\nUniversity degreel\n23.1% (21.2–25.0)\n60.6% (58.4–62.9)\n73.4% (71.4–75.4)\n49.5% (46.7–52.2)\n55.1% j (52.4–57.9)\n69.9% (67.8–72.0)\n63.8% (59.3–68.4)\nCan J Public Health (2021) 112:363 375 Can J Public Health (2021) 112:363–375 368 Table 2\nPerceived effectiveness of six different public health measures stratified by socio-demographic characteristics. Perceived preparedness Values are reported as % (95% confidence interval), and those in bold font were\nstatistically significant between subgroups (p < 0.0026) (n = 4981). Perceived preparedness However, there are\ndifferences in these indicators based on socio-demographic\nvariables and context. If our collective priority is to maintain\nan open economy, we need to ensure that individuals are able\nto comply with public health measures that prevent and con-\ntrol transmission of the virus. The results from this study have\nidentified a number of areas in which policies could help ad-\ndress issues of public adherence. mask were in supermarkets or other stores, anywhere\noutside the home, and walking on the street (Fig. 2b);\n42.7% (95% CI, 35.3–50.1) of mask-wearing transit-\nusers had worn a mask on transit in the past 24 hours. Factors associated with mask use are shown in Table 4;\nmask use was increased in households with more than one\nadult, with children, or with multiple generations; and in\nindividuals with university-level education, or reporting\nthat they would be at risk of serious illness with COVID-\n19 or at increased risk of developing COVID-19. Of the\nvariables assessed for interaction with age group, the\nonly interaction detected was between age and high-\nrisk conditions with younger (< 30 years), high-risk in-\ndividuals more likely to have reported mask use com-\npared with 40–49-year-old respondents who were not in\na risk group. p\nWhile confidence in the ability to comply with various\npublic health measures was high, younger age groups, those\nin the paid workforce, and, in some cases, those with lower\nincome were less likely to report confidence in the ability to\ncomply. Individuals need to feel supported in complying\nwith public health measures. Our findings of reduced confi-\ndence in ability to comply with public health measures are\nconsistent with other research demonstrating that those with\na low income (Wolf et al. 2020) and those in younger age\ngroups (Cvetković et al. 2020) are less prepared in the event\nof illness. Compliance in the event of self-isolation or quar-\nantine is at least partially dependent on preparedness and\nhaving the means to self-isolate; there is a need to develop\nsupports for those who need to self-isolate but may not have\nthe means to do so. Presenteeism risk (attending work while\nsick) was gendered, with more men than women anticipating\nthat co-workers would expect them to continue to work\nwhile sick; however, responses did not differentiate between\nemployer and co-workers. Perceived preparedness Letters in superscript indicate levels at which pairwise comparisons were statistically significant with Bonferroni correction (p < 0.05)\nPublic health measures\nReduce contacts\nSelf-isolate for\n14 days with severe\nrespiratory symptoms\nAvoid crowds\nStay home for 14 days when\nhousehold member has severe\nrespiratory symptoms\nSchool closures\nBusiness closures\nOverall\n93.7% (93.0–94.3)\n93.1% (92.4–93.8)\n94.0% (93.3–94.6)\n90.6% (89.8–91.4)\n87.2% (86.2–88.1)\n91.4% (90.6–92.2)\nGender\nWomen\n95.3% (94.4–96.1)\n94.9% (94.0–95.7)\n95.8% (95.0–96.6)\n92.5% (91.5–93.5)\n89.6% (88.5–90.8)\n93.4% (92.4–94.4)\nMen\n92.0% (91.0–93.1)\n91.3% (90.2–92.4)\n92.1% (91.1–93.2)\n88.8% (87.5–90.0)\n84.7% (83.2–86.1)\n89.4% (88.1–90.6)\nAge group\n18–29 yearsa\n90.4% c–f (88.3–92.5)\n89.9% e, f (87.8–92.0)\n89.8% d–f (87.7–91.9)\n86.2% d–f (83.8–88.6)\n85.8% (83.3–88.2)\n87.1% e, f (84.7–89.4)\n30–39 yearsb\n92.0% e, f (90.2–93.7)\n91.2% e, f (89.4–93.0)\n91.4% d–f (89.7–93.2)\n88.2% e, f (86.2–90.3)\n86.7% (84.6–88.9)\n88.9% e, f (86.9–90.9)\n40–49 yearsc\n91.3% a, e, f (89.3–93.2)\n92.5% (90.7–94.3)\n92.3% e, f (90.4–94.1)\n90.9% (88.9–92.9)\n85.8% (83.4–88.2)\n90.8% e, f (88.8–92.8)\n50–59 yearsd\n94.8% a, f (93.3–96.3)\n93.9% (92.3–95.5)\n95.4% a, b (94.0–96.8)\n92.2% a (90.4–94.0)\n88.1% (85.9–90.3)\n91.4% e, f (89.5–93.3)\n60–69 yearse\n95.9% a–c (94.7–97.2)\n95.4% a, b (94.1–96.7)\n97.1% a–c (96.1–98.2)\n92.7% a, b (91.1–94.4)\n88.2% (86.2–90.3)\n95.0% a–d (93.6–96.4)\n70+ yearsf\n98.1% a –d (97.1–99.2)\n95.8% a, b (94.2–97.3)\n98.1% a–c (97.1–99.2)\n93.9% a, b (92.0–95.8)\n88.4% (85.9–90.9)\n95.8% a–d (94.2–97.3)\nRisk group\nYes\n95.3% (94.2–96.3)\n93.4% (92.2–94.6)\n94.1% (92.9–95.2)\n90.3% (88.8–91.7)\n88.8% (87.2–90.3)\n92.8% (91.6–94.1)\nNo\n92.9% (92.0–93.8)\n92.9% (92.1–93.8)\n93.9% (93.1–94.7)\n90.8% (89.8–91.8)\n86.4% (85.2–87.6)\n90.7% (89.7–91.7)\nHousehold children\nYes\n92.6% (91.1–94.1)\n93.1% (91.6–94.5)\n93.1% (91.6–94.5)\n90.4% (88.7–92.1)\n87.6% (85.7–89.5)\n90.2% (88.4–91.9)\nNo\n94.0% (93.2–94.7)\n93.1% (92.3–93.9)\n94.2% (93.5–95.0)\n90.7% (89.8–91.6)\n87.0% (86.0–88.1)\n91.8% (90.9–92.7)\nHousehold income\n$0–$60,000\n93.2% (92.0–94.3)\n92.9% (91.7–94.1)\n94.3% (93.3–95.4)\n90.2% (88.9–91.6)\n87.8% (86.3–89.2)\n91.6% (90.3–92.8)\n$60,001–$110,000\n94.9% (93.8–96.0)\n94.8% (93.7–95.9)\n94.9% (93.8–96.0)\n92.4% (91.1–93.8)\n88.2% (86.6–89.8)\n93.1% (91.9–94.4)\n> $110,000\n92.5% (90.9–94.1)\n91.5% (89.8–93.2)\n92.0% (90.3–93.7)\n89.4% (87.5–91.2)\n85.1% (82.9–87.2)\n88.7% (86.7–90.6)\nDon’t know/prefer\nnot to answer\n94.0% (91.9–96.0)\n91.6% (89.2–94.0)\n93.8% (91.7–95.9)\n89.1% (86.4–91.8)\n86.1% (83.1–89.1)\n91.0% (88.5–93.5)\nEmployment status\nPaid workforce\n92.6% (91.6–93.5)\n92.2% (91.2–93.2)\n92.6% (91.6–93.6)\n89.3% (88.2–90.5)\n86.6% (85.3–87.9)\n89.4% (88.2–90.6)\nNot in paid workforce\n95.0% (94.1–95.9)\n94.1% (93.2–95.1)\n95.6% (94.7–96.4)\n92.2% (91.1–93.3)\n87.9% (86.5–89.2)\n93.8% (92.8–94.8)\nLevel of education\nSecondary or less\n93.0% (91.5–94.4)\n92.7% (91.2–94.2)\n93.8% (92.4–95.2)\n90.9% (89.2–92.5)\n88.1% (86.3–89.9)\n91.4% (89.8–93.0)\nCollege/trades\nor other qualification\n93.7% (92.6–94.8)\n93.5% (92.4–94.6)\n93.8% (92.8–94.9)\n90.2% (88.9–91.5)\n86.3% (84.7–87.8)\n90.5% (89.2–91.8)\nUniversity degree\n94.0% (93.0–95.1)\n92.9% (91.8–94.1)\n94.2% (93.1–95.2)\n90.9% (89.6–92.2)\n87.5% (86.0–89.0)\n92.3% (91.1–93.5)\n368\nCan J Public Health (2021) 112:363 Can J Public Health (2021) 112:363–375 369 to comply with such measures was high. Perceived preparedness More individuals with less edu-\ncation or income were at risk of not being paid if they took sick\nleave. Presenteeism has been shown to be prevalent among oc-\ncupations with high contact rates, including the care, welfare, and\neducation sectors (Aronsson et al. 2000). Determinants of\npresenteeism include job insecurity, workplace performance in-\ndicators that include attendance rates, and limited entitlement to\npaid sick leave (Kinman 2019). Investigators in Israel demon-\nstrated that paid sick time increases compliance with stay-home-\nwhen-sick policies from 57% with no compensation to almost\n100% when compensation was assumed (Bodas and Peleg\n2020). These findings highlight the need for a shift in workplace\nculture toward discouraging presenteeism and ensuring paid sick\ntime. Direct contact with non-household members The proportion of respondents who had engaged in an activity\nwith non-household contacts in the 7 days prior to survey\ncompletion was 24.4% (95% CI, 23.2–25.6) (Fig. 3a). More\nnon-household contact was reported for provinces which were\nmore advanced in the de-escalation of physical distancing\n(e.g., PEI) at the time of survey completion; however, in prov-\ninces where physical distancing was still in place during the\nsurvey period (e.g., ON), approximately 20% of respondents\nwere reporting non-household contacts in May 2020. Of the\nrespondents who reported non-household contacts, 62% re-\nported that this occurred once or twice in a 7-day period while\nalmost 23% reported having non-household contacts more\nthan 3 days out of the 7-day period prior to survey completion\n(Fig. 3b). Individuals in the youngest age group and those who\nreported an annual household income greater than $110,000\nwere more likely to have participated in an activity with\nsomeone outside their household (Table 5) compared\nwith older respondents and those earning less than\n$60,000, respectively. Perceived risk of serious illness\nwas associated with less interaction with individuals\noutside the household. Our finding that a majority of respondents with dependent\nchildren were responsible for childcare at the same time as\nmaintaining employment when schools and daycares were\nclosed due to the pandemic highlights the need for provincial-\nly co-ordinated plans with evidence-based infection preven-\ntion and control (IPAC) procedures for safe school function-\ning. There is mounting evidence that children are more sus-\nceptible and can transmit infection more readily than original-\nly thought, potentially contributing substantially to communi-\nty transmission (Hyde 2020). Recognition of transmission\namong children could lead to prolonged school closures in\nprovinces heavily impacted by the pandemic. Statistics\nCanada estimates that there are more than 10 million families Details of the regression model identifying factors associ-\nated with confidence in the ability to self-isolate with mild\nsymptoms of COVID-19 are located in Table S2 of the\nSupplementary Materials. Discussion At the time of data collection, Canadian provinces were in\nvarious stages of reopening the economy. Overall, the major-\nity of participants reported risk perceptions and attitudes about\nthe effectiveness of public health measures that are\nwell aligned with scientific evidence and the reported ability Table 3\nConfidence in the ability to comply with five different public health measures stratified by socio-demographic characteristics. Values are reported as % (95% confidence interval), and those in\nbold font were statistically significant between subgroups (p < 0.0026) (n = 4981). Discussion 1 a Respondents with children 14 years of age or younger (n = 930)\nreported on which individuals looked after the children in their household\nduring school and daycare closures due to the pandemic. b Respondents\nwho reported that parents provided childcare during school and daycare\nclosures (n = 769) also identified the employment circumstances of the\nparent who provided the childcare. The category “Parent in the\nWorkforce” includes those working remotely, those working part-time,\nthose who took leave from their job, and those who were unemployed due\nto COVID-19 but otherwise have been working parent who provided the childcare. The category “Parent in the\nWorkforce” includes those working remotely, those working part-time,\nthose who took leave from their job, and those who were unemployed due\nto COVID-19 but otherwise have been working Fig. 1 a Respondents with children 14 years of age or younger (n = 930)\nreported on which individuals looked after the children in their household\nduring school and daycare closures due to the pandemic. b Respondents\nwho reported that parents provided childcare during school and daycare\nclosures (n = 769) also identified the employment circumstances of the Fig. 1 a Respondents with children 14 years of age or younger (n = 930)\nreported on which individuals looked after the children in their household\nduring school and daycare closures due to the pandemic. b Respondents\nwho reported that parents provided childcare during school and daycare\nclosures (n = 769) also identified the employment circumstances of the parent who provided the childcare. The category “Parent in the\nWorkforce” includes those working remotely, those working part-time,\nthose who took leave from their job, and those who were unemployed due\nto COVID-19 but otherwise have been working parent who provided the childcare. The category “Parent in the\nWorkforce” includes those working remotely, those working part-time,\nthose who took leave from their job, and those who were unemployed due\nto COVID-19 but otherwise have been working parent who provided the childcare. The category “Parent in the\nWorkforce” includes those working remotely, those working part-time,\nthose who took leave from their job, and those who were unemployed due\nto COVID-19 but otherwise have been working An indication that public health messaging has been\nsuccessful in many cases, we found that both the per-\nceived ability to comply with public health measures\nand perceived effectiveness of such measures were very\nhigh. Discussion Letters in superscript indicate levels at which pairwise comparisons were statistically significant with Bonferroni\ncorrection (p < 0.05)\nPublic health measures\nReduce contacts\nSelf-isolate for 14 days\nwith severe respiratory\nsymptoms\nAvoid crowds\nStay home for 14 days\nwhen household member\nhas severe respiratory symptoms\nAvoid public transportation\nOverall\n93.7% (93.0–94.4)\n93.0% (92.3–93.7)\n93.3% (92.6–94.0)\n91.0% (90.2–91.7)\n90.9% (90.0–91.7)\nGender\nWomen\n94.6% (93.7–95.5)\n94.9% (94.1–95.8)\n94.7% (93.8–95.6)\n92.7% (91.6–93.7)\n92.9% (91.9–93.9)\nMen\n92.8% (91.8–93.9)\n91.1% (90.0–92.2)\n91.9% (90.8–92.9)\n89.3% (88.1–90.5)\n88.8% (87.6–90.1)\nAge group\n18–29 years a\n90.1% c–f (88.1–92.2)\n87.7% c–f (85.4–90.0)\n88.1.% c–f (85.8–90.4)\n85.6% c–f (83.2–88.1)\n85.3% c–f (82.8–87.8)\n30–39 years b\n92.6% e (90.9–94.3)\n90.6% e, f (88.7–92.5)\n89.2% c–f (87.2–91.2)\n87.5% c–f (85.4–89.6)\n86.8% c–f (84.7–89.0)\n40–49 years c\n92.0% a, e, f (90.1–93.9)\n92.1% a, e, f (90.3–94.0)\n91.8% a, c–f (89.9–93.7)\n89.7% a, c–f (87.5–91.8)\n89.4% a, b, e, f (87.3–91.5)\n50–59 years d\n94.3% a (92.8–95.9)\n94.2% a (92.7–95.8)\n95.6% a (94.3–97.0)\n93.9% a (92.3–95.5)\n92.1% a, b, f (90.3–93.9)\n60–69 years e\n96.7% a–c (95.6–97.8)\n96.4% a–c (95.3–97.6)\n97.2% a (96.2–98.3)\n94.2% a (92.7–95.6)\n95.0% a–c (93.6–96.4)\n70+ years f\n96.1% a, c (94.6–97.6)\n97.2% a–c (95.9–98.5)\n98.4% a (97.5–99.4)\n95.3% a (93.7–97.0)\n97.3% a–c (96.1–98.6)\nRisk group\nYes\n95.1% (94.0–96.1)\n92.8% (91.5–94.0)\n94.3% (93.1–95.4)\n91.0% (89.6–92.4)\n91.4% (90.1–92.8)\nNo\n93.0% (92.2–93.9)\n93.1% (92.2–94.0)\n92.8% (92.0–93.7)\n90.9% (90.0–91.9)\n90.6% (89.6–91.6)\nHousehold children\nYes\n92.6% (91.1–94.1)\n91.6% (90.0–93.3)\n91.7% (90.1–93.3)\n90.0% (88.2–91.7)\n89.2% (87.4–91.0)\nNo\n94.0% (93.2–94.7)\n93.4% (92.6–94.2)\n93.8% (93.0–94.5)\n91.2% (90.3–92.1)\n91.3% (90.5–92.2)\nHousehold income\n$0–$60,000 g\n93.2% (92.0–94.3)\n92.6% (91.4–93.8)\n93.3% (92.1–94.4)\n90.2% (88.8–91.5)\n89.3% h (87.9–90.7)\n$60,001–$110,000 h\n94.7% (93.6–95.8)\n94.5% (93.4–95.7)\n94.0% (92.8–95.1)\n92.0% (90.7–93.3)\n93.0% g (91.7–94.2)\n> $110,000\n93.8% (92.3–95.2)\n91.7% (90.0–93.4)\n92.7% (91.1–94.3)\n90.4% (88.6–92.2)\n90.8% (89.1–92.6)\nDon’t know/prefer\nnot to answer\n92.2% (89.9–94.5)\n92.4% (90.1–94.7)\n92.6% (90.3–94.9)\n91.6% (89.2–94.0)\n90.2% (87.7–92.8)\nEmployment status\nPaid workforce\n92.4% (91.4–93.4)\n91.4% (90.3–92.4)\n91.1% (90.1–92.2)\n88.9% (87.7–90.1)\n88.9% (87.7–90.1)\nNot in paid workforce\n95.2% (94.3–96.1)\n94.9% (94.0–95.8)\n95.8% (95.0–96.7)\n93.4% (92.4–94.4)\n93.2% (92.1–94.2)\nLevel of education\nSecondary or less\n92.7% (91.2–94.2)\n92.5% (91.0–94.0)\n93.1% (91.7–94.6)\n90.1% (88.4–91.8)\n90.4% (88.7–92.0)\nCollege/trades or\nother qualification\n93.6% (92.5–94.7)\n93.6% (92.5–94.7)\n93.7% (92.6–94.8)\n92.2% (91.0–93.4)\n91.5% (90.2–92.7)\nUniversity degree\n94.4% (93.4–95.4)\n92.8% (91.6–94.0)\n93.0% (91.8–94.1)\n90.2% (88.9–91.6)\n90.5% (89.2–91.9)\n370\nCan J Public Health (2021) 112:363–375 Can J Public Health (2021) 112:363–375 370 Can J Public Health (2021) 112:363–375 371 Fig. Discussion However, both indicators varied by age and gender, with children living in Canada (Statistics Canada 2020) and\nalmost 70% of families with dependent children have two\nemployed parents (Statistics Canada 2016b). Investing in ro-\nbust IPAC procedures for schools will allow schools to remain\nopen and parents to maintain employment. Fig. 2 Respondents were asked if they had worn a face mask in the 24 hours prior to survey completion. a represents reported mask use by province of\nresidence. b identifies the location(s) of mask use for respondents who reported wearing a mask in the previous 24 hours (n = 1617) Fig. 2 Respondents were asked if they had worn a face mask in the 24 hours prior to survey completion. a represents reported mask use by province of\nresidence. b identifies the location(s) of mask use for respondents who reported wearing a mask in the previous 24 hours (n = 1617) Fig. 2 Respondents were asked if they had worn a face mask in the 24 hours prior to survey completion. a represents reported mask use by province of\nresidence. b identifies the location(s) of mask use for respondents who reported wearing a mask in the previous 24 hours (n = 1617) 372 Can J Public Health (2021) 112:363–375 emphasizing the need for additional targeted messaging. in younger individuals was associated with poor compli-\nTable 4\nResults of a multivariable logistic regression analysis assessing factors associated with mask use in the 24 hours prior to survey completion. Discussion Values are reported as adjusted odds ratios (95% confidence interval) and those in bold font were statistically significant (p < 0.05) (n = 4981)\nVariable\nAdjusted OR (95% CI)\np Wald’s test\np (L-R test)\nHousehold composition\n0.005\nSingle person living alone (referent)\nAdults only living together\n1.32 (1.13–1.55)\n< 0.001\nFamily with children\n1.24 (1.01–1.52)\n0.04\n>2 generations living together\n1.71 (1.12–2.63)\n0.01\nGrandparents living with their grandchildren only\n1.16 (0.28–4.72)\n0.84\nAge category\n1\n18–29 years\n1.61 (1.23–2.10)\n< 0.001\n30–39 years\n1.21 (0.95–1.56)\n0.13\n40–49 years (referent)\n-\n-\n50–59 years\n1.22 (0.93–1.60)\n0.15\n60–69 years\n1.44 (1.08–1.92)\n0.01\nOver 70 years\n1.39 (0.98–1.99)\n0.07\nRespondent risk group\n1\nRespondent is in a high-risk group\n1.51 (1.06–2.15)\n0.02\nSize of the geographic region of residence\n< 0.001\nLarge city (referent)\nMedium-sized city\n0.74 (0.64–0.85)\n< 0.001\nLarge town\n0.61 (0.48–0.76)\n< 0.001\nSmall town\n0.53 (0.43–0.66)\n< 0.001\nRural place\n0.38 (0.29–0.50)\n< 0.001\nEducation level of respondent\n0.02\nSecondary or less (referent)\nCollege/trade/other qualification\n1.00 (0.85–1.18)\n1.0\nUniversity (bachelor degree or higher)\n1.21 (1.02–1.43)\n0.03\nEmployment status of respondent\n< 0.001\nUnemployed, student, retired, work within home (referent)\nEmployed FT, PT, self-employed\n1.32 (1.13–1.54)\n< 0.001\nPerceived risk of contracting the virus\n< 0.001\nLikely to contact the virus\n1.31 (1.12–1.52)\n< 0.001\nPerceived risk of serious illness due to COVID-19\n< 0.001\nCOVID-19 would be a serious illness for respondent\n1.61 (1.39–1.85)\n< 0.001\nInteraction between age category and respondent risk group\n< 0.001\n18–29 years in a risk group\n1.75 (1.06–2.90)\n0.03\n30–39 years in a risk group\n1.57 (0.98–2.53)\n0.06\n40–49 years, not high risk (referent)\n-\n-\n50–59 years in a risk group\n0.87 (0.54–1.39)\n0.56\n60–69 years in a risk group\n0.72 (0.46–1.13)\n0.15\nOver 70 years in a risk group\n0.89 (0.54–1.46)\n0.64 Table 4\nResults of a multivariable logistic regression analysis assessing factors associated with mask use in the 24 hours prior to survey completion. Values are reported as adjusted odds ratios (95% confidence interval) and those in bold font were statistically significant (p < 0.05) (n = 4981)\nVariable\nAdjusted OR (95% CI)\np Wald’s test\np (L-R test) Table 4\nResults of a multivariable logistic regression analysis assessing factors associated with mask use in the 24 hours prior to survey completion. Table 5 Results of a\nmultivariable logistic regression\nanalysis assessing factors\nassociated with engaging in an\nactivity with non-household con-\ntacts in the 7 days prior to survey\ncompletion. Values are reported\nas adjusted odds ratios (95%\nconfidence interval) and those in\nbold font are statistically signifi-\ncant (p < 0.05) (n = 4981) Discussion Values are reported as adjusted odds ratios (95% confidence interval) and those in bold font were statistically significant (p < 0.05) (n = 4981) in younger individuals was associated with poor compli-\nance with public health measures and is consistent with a\ngrowing body of evidence demonstrating that male gender\nand younger age groups engage in more COVID-19 risk\nbehaviours (Alsan et al. 2020; Seale et al. 2020). Younger\nadults tend to have larger contact networks than older in younger individuals was associated with poor compli-\nance with public health measures and is consistent with a\ngrowing body of evidence demonstrating that male gender\nand younger age groups engage in more COVID-19 risk\nbehaviours (Alsan et al. 2020; Seale et al. 2020). Younger\nadults tend to have larger contact networks than older emphasizing the need for additional targeted messaging. The finding that perceived risk of serious illness increased\nwith age group is consistent with past research (Bruine de\nBruin 2020; He et al. 2020) and is in line with empiric\nestimates of illness risk in older individuals (Public\nHealth Agency of Canada 2020). Perceived lack of risk Can J Public Health (2021) 112:363–375 373 Fig. 3 a Proportion of respondents reporting contact with non-household\nmembers in the 7 days prior to survey completion. More non-household\ncontacts were reported for provinces which were more advanced in the\nde-escalation of physical distancing (e.g., PEI) at the time of survey\ncompletion; however, in provinces where physical distancing was still\nin place during the survey period (e.g., ON), approximately 20% of re-\nspondents were reporting non-household contacts in May 2020. b The\nnumber of days in the past week respondents engaged in an activity with a\nnon-household contact, for those reporting such activity (n = 1216) in place during the survey period (e.g., ON), approximately 20% of re-\nspondents were reporting non-household contacts in May 2020. b The\nnumber of days in the past week respondents engaged in an activity with a\nnon-household contact, for those reporting such activity (n = 1216) Fig. 3 a Proportion of respondents reporting contact with non-household\nmembers in the 7 days prior to survey completion. More non-household\ncontacts were reported for provinces which were more advanced in the\nde-escalation of physical distancing (e.g., PEI) at the time of survey\ncompletion; however, in provinces where physical distancing was still Fig. Discussion 3 a Proportion of respondents reporting contact with non-household\nmembers in the 7 days prior to survey completion. More non-household\ncontacts were reported for provinces which were more advanced in the\nde-escalation of physical distancing (e.g., PEI) at the time of survey\ncompletion; however, in provinces where physical distancing was still Fig. 3 a Proportion of respondents reporting contact with non-household\nmembers in the 7 days prior to survey completion. More non-household\ncontacts were reported for provinces which were more advanced in the\nde-escalation of physical distancing (e.g., PEI) at the time of survey\ncompletion; however, in provinces where physical distancing was still Fig. 3 a Proportion of respondents reporting contact with non-household\nmembers in the 7 days prior to survey completion. More non-household\ncontacts were reported for provinces which were more advanced in the\nde-escalation of physical distancing (e.g., PEI) at the time of survey\ncompletion; however, in provinces where physical distancing was still in place during the survey period (e.g., ON), approximately 20% of re-\nspondents were reporting non-household contacts in May 2020. b The\nnumber of days in the past week respondents engaged in an activity with a\nnon-household contact, for those reporting such activity (n = 1216) The evidence for the efficacy of non-medical masks\nfor COVID-19 prevention continues to grow (Fisman\net al. 2020; Konda et al. 2020; Lyu and Wehby 2020;\nRodriguez-Palacios et al. 2020). While the survey ques-\ntion for mask use was not restricted to people who had\nleft their household in the previous 24 hours, fewer than adults (Mossong et al. 2008) which likely partially ex-\nplains these results. Recent increases in cases of\nCOVID-19 in adolescents and young adults have been\nattributed to greater mixing among this age group com-\nbined with lower adherence to physical distancing mea-\nsures (Goldstein and Lipsitch 2020). Supplementary Information The online version contains supplementary\nmaterial available at https://doi.org/10.17269/s41997-021-00501-y. Supplementary Information The online version contains supplementary\nmaterial available at https://doi.org/10.17269/s41997-021-00501-y. Acknowledgments We thank J. Lau for support in programming the\nsurvey. Author contributions All the authors contributed to the study conception\nand design. Survey design and data collection were performed by GB,\nAG, PL, and EM. Data analysis was conducted by GB. The first draft of\nthe manuscript was written by GB and all the authors commented on\nprevious versions of the manuscript. All the authors read and approved\nthe final manuscript. Data availability Aggregated data are available upon request from the\ncorresponding author. Data availability Aggregated data are available upon request from the\ncorresponding author. Data availability Aggregated data are available upon request from the\ncorresponding author. Code availability Code is available upon request from the corresponding\nauthor. Code availability Code is available upon request from the corresponding\nauthor. Declarations The funders had no role in study design, data collec-\ntion and analysis, decision to publish or preparation of the manuscript. Declarations The funders had no role in study design, data collec-\ntion and analysis, decision to publish or preparation of the manuscript. Ethics approval\nThe study protocol was approved by the University of\nGuelph Research Ethics Board (protocol #20-04-011) and the University\nof Toronto Research Ethics Board (protocol #38251). Ethics approval\nThe study protocol was approved by the University of\nGuelph Research Ethics Board (protocol #20-04-011) and the University\nof Toronto Research Ethics Board (protocol #38251). Consent to participate\nInformed consent was obtained from all individ-\nual participants included in the study. Consent for publication\nInformed consent was obtained from all indi-\nvidual participants included in the study. Discussion Variable\nAdjusted OR (95% CI)\np Wald’s test\np (L-R test)\nAge category\n< 0.001\n18–29 years\n1.74 (1.39–2.18)\n< 0.001\n30–39 years\n1.18 (0.95–1.48)\n0.14\n40–49 years (referent)\n-\n-\n50–59 years\n0.98 (0.78–1.24)\n0.88\n60–69 years\n1.22 (0.97–1.52)\n0.09\nOver 70 years\n0.90 (0.69–1.18)\n0.46\nHousehold income of respondent\n< 0.001\n$0–$60,000 (referent)\n-\n$60,001–$110,000\n1.09 (0.93–1.28)\n0.29\n> $110,000\n1.49 (1.25–1.78)\n< 0.001\nUnsure/prefer not to answer\n1.03 (0.82–1.30)\n0.80\nPerceived risk of COVID as a serious illness to self\n0.006\nNo (referent)\nYes\n0.83 (0.72–0.95)\n0.006 Adjusted OR (95% CI)\np Wald’s test\np (L-R test) Adjusted OR (95% CI)\np Wald’s test\np (L-R test) 374 Can J Public Health (2021) 112:363–375 for in-person learning, and to discourage presenteeism. Taken\ntogether, such measures are likely to mitigate the impact of the\nCOVID-19 pandemic in Canada. one third of respondents reported wearing a face mask\nin the 24 hours prior to survey completion. Mask use\nwas associated with household composition and the\nstrongest association was belonging to a household with\nmore than two generations living together, likely\nreflecting concern for the safety of older individuals in\nhouseholds. Increased mask use in the youngest age\ngroup may reflect younger individuals working in essen-\ntial service jobs at the time of the survey (e.g., grocery\nstores). As with other preventive measures, compliance\nwith masks was more likely in individuals with greater\nself-perceived risk. Limitations Funding GB and AG are supported by the Canada Research Chairs pro-\ngram. DF and AT are supported by the Canadian Institutes of Health\nResearch (CIHR). ZP is supported by the Natural Sciences and\nEngineering Research Council (NSERC). EM and PL are supported by\nHeritage Canada and the Social Sciences and Humanities Research\nCouncil (SSHRC). Funding to support data collection was provided by\nthe Public Health Agency of Canada (PHAC), The National\nCollaborating Centre for Infectious Diseases (NCCID), and the\nUniversity of Guelph. While every effort was made to ensure representativeness of\nthe study population, we note several potential biases, includ-\ning non-representativeness of the sample (a risk with any sur-\nvey), the online nature of the survey, which limits participa-\ntion to those who use the Internet, and self-report which intro-\nduces the potential for recall, response, and social desirability\nbiases. The large sample size means that statistical signifi-\ncance is seen with small absolute differences. The results are\nconsistent with the large body of research on risk attitudes and\nbehaviours and, on their own, would add little new knowledge\nto the literature. However, governments and public health of-\nficials have asked Canadians to comply with extraordinary\nmeasures. The value of this study lies in its assessment of\nthe ability to comply with these extraordinary measures during\na pandemic given a variety of socio-demographic characteris-\ntics. Finally, knowledge about COVID-19 and recommended\nbehaviours is changing rapidly. These data were collected in\nMay 2020 during a time in which provinces were in different\nphases of public health de-escalation and indoor masking or-\nders were not widespread, so these data are best interpreted as\na snapshot in time. References Mossong, J., Hens, N., Jit, M., Beutels, P., Auranen, K., Mikolajczyk, R.,\net al. (2008). Social contacts and mixing patterns relevant to the\nspread of infectious diseases. PLOS Medicine, 5(3), e74. https://\ndoi.org/10.1371/journal.pmed.0050074. Alsan, M., Stantcheva, S., Yang, D., & Cutler, D. (2020). Disparities in\ncoronavirus 2019 reported incidence, knowledge, and behavior\namong US adults. JAMA Network Open, 3(6), e2012403. https:// National Advisory Committee on Immunization (NACI). (2019). Canadian Immunization Guide Chapter on Influenza and\nStatement on Seasonal Influenza Vaccine for 2019–2020: An\nAdvisory Committee Statement (ACS). Ottawa. https://www. canada.ca/en/public-health/services/publications/vaccines-\nimmunization/canadian-immunization-guide-statement-seasonal-\ninfluenza-vaccine-2019-2020.html#List1. Accessed 24 Aug 2020. doi.org/10.1001/jamanetworkopen.2020.12403. Aronsson, G., Gustafsson, K., & Dallner, M. (2000). Sick but yet at work. An empirical study of sickness presenteeism. Journal of\nEpidemiology and Community Health, 54(7), 502–509. https://doi. org/10.1136/jech.54.7.502. Atchison, C., Bowman, L. R., Vrinten, C., Redd, R., Pristerà, P., Eaton, J.,\n& Ward, H. (2021). Early perceptions and behavioural responses\nduring the COVID-19 pandemic: a cross-sectional survey of UK\nadults. BMJ Open, 11(1), e043577. https://doi.org/10.1136/\nbmjopen-2020-043577. Public Health Agency of Canada. (2020). Coronavirus disease 2019\n(COVID-19): epidemiology update. https://health-infobase.canada. ca/covid-19/epidemiological-summary-covid-19-cases.html. Accessed 24 Aug 2020. j p\nBodas, M., & Peleg, K. (2020). Self-isolation compliance in the COVID-\n19 era influenced by compensation: findings from a recent survey in\nIsrael. Health Affairs (Project Hope), 39(6), 936–941. https://doi. org/10.1377/hlthaff.2020.00382. Qazi, A., Qazi, J., Naseer, K., Zeeshan, M., Hardaker, G., Zubairu, J., &\nKhalid, M. (2020). Analyzing situational awareness through public\nopinion to predict adoption of social distancing amid pandemic\nCOVID - 19. Journal of Medical Virology, 92(April), 849–855. https://doi.org/10.1002/jmv.25840. g\nBruine de Bruin, W. (2020). Age differences in COVID-19 risk percep-\ntions and mental health: evidence from a national US survey con-\nducted in March 2020. J Gerontol B Psychol Sci Soc Sci, 76, 1–6. https://doi.org/10.1093/geronb/gbaa074. Rodriguez-Palacios, A., Cominelli, F., Basson, A. R., Pizarro, T. T., &\nIlic, S. (2020). Textile masks and surface covers-a spray simulation\nmethod and a “universal droplet reduction model” against respirato-\nry pandemics. Frontiers in Medicine, 7, 260. https://doi.org/10. 3389/fmed.2020.00260. p\ng\ng\ng\nCvetković, V. M., Nikolić, N., Nenadić, U. R., Öcal, A., Noji, E. K., &\nZečević, M. (2020). Preparedness and preventive behaviors for a pan-\ndemic disaster caused by COVID-19 in Serbia. International Journal\nof Environmental Research and Public Health, 17(11), 1–23. https://\ndoi.org/10.3390/ijerph17114124. RStudio Team. (2019). RStudio: integrated development for R. Boston:\nRStudio, Inc. Seale, H., Heywood, A. E., Leask, J., Id, M. Conclusion Conflict of interest\nThe authors declare no competing interests. Conflict of interest\nThe authors declare no competing interests. The results of this study highlight the need for the develop-\nment of enhanced messaging and financial supports in order to\nfurther support improved compliance with public health mea-\nsures. Work is needed to identify strategies and develop tools\nfor targeted messaging to groups who are more likely to en-\ngage in risk behaviours, and social support is needed for lower\nincome individuals to enable periods of self-isolation and\nchildcare should they become ill, to permit schools to be open Open Access This article is licensed under a Creative Commons\nAttribution 4.0 International License, which permits use, sharing, adap-\ntation, distribution and reproduction in any medium or format, as long as\nyou give appropriate credit to the original author(s) and the source, pro-\nvide a link to the Creative Commons licence, and indicate if changes were\nmade. The images or other third party material in this article are included\nin the article's Creative Commons licence, unless indicated otherwise in a\ncredit line to the material. If material is not included in the article's 375 Can J Public Health (2021) 112:363–375 Creative Commons licence and your intended use is not permitted by\nstatutory regulation or exceeds the permitted use, you will need to obtain\npermission directly from the copyright holder. To view a copy of this\nlicence, visit http://creativecommons.org/licenses/by/4.0/. Konda, A., Prakash, A., Moss, G., Schmoldt, M., Grant, G., & Guha, S. (2020). Aerosol filtration efficiency of common fabrics used in re-\nspiratory cloth masks. ACS Nano, 14(5), 6339–6347. Lyu, W., & Wehby, G. L. (2020). Community use of face masks and\nCOVID-19: evidence from a natural experiment of state mandates in\nthe US. Health Affairs (Millwood), 39(8), 1419–1425. https://doi. org/10.1377/hlthaff.2020.00818. References S., Thomas, S., Durrheim,\nN., et al. (2020). COVID-19 is rapidly changing : examining public\nperceptions and behaviors in response to this evolving pandemic. PLoS ONE, 15(6), e0235112. https://doi.org/10.1371/journal.pone. 0235112. Fisman, D. N., Greer, A. L., & Tuite, A. R. (2020). Bidirectional impact\nof imperfect mask use on reproduction number of COVID-19: a next\ngeneration matrix approach. Infect Dis Model., 5, 405–408. https://\ndoi.org/10.1016/j.idm.2020.06.004. Goldstein, E., & Lipsitch, M. (2020). Temporal rise in the proportion of\nyounger adults and older adolescents among coronavirus disease\n(COVID-19) cases following the introduction of physical distancing\nmeasures, Germany, March to April 2020. Eurosurveillance,\n25(17), 22–25. https://doi.org/10.2807/1560-7917.ES.2020.25.17. 2000596. Statistics Canada. (2016a). 2016 Census. Catalogue Number 98-400-\nX2016003. Ottawa. https://www150.statcan.gc.ca/n1/en/catalogue/\n98-400-X2016003. Accessed 24 Aug 2020. Statistics Canada. (2016b). The rise of the dual-earner family with\nchildren. Ottawa. https://www150.statcan.gc.ca/n1/pub/11-630-x/\n11-630-x2016005-eng.htm. Accessed 24 Aug 2020. He, S., Chen, S., Kong, L., & Liu, W. (2020). Analysis of risk perceptions\nand related factors concerning COVID-19 epidemic in Chongqing,\nChina. J Community Health, 46, 278–285. https://doi.org/10.1007/\ns10900-020-00870-4. Statistics Canada. (2020). Table 17-10-0061-01 Estimates of the number\nof census families as of July 1st. Ottawa. https://doi.org/10.25318/\n1710006101-eng. Hyde, Z. (2020). COVID-19, children and schools: overlooked and at\nrisk. Med J Aust, 213(10), 444–446.e1. https://doi.org/10.5694/\nmja2.50823. Wolf, M. S., Serper, M., Opsasnick, L., O’Conor, R. M., Curtis, L. M.,\nBenavente, J. Y., et al. (2020). Awareness, attitudes, and actions\nrelated to COVID-19 among adults with chronic conditions at the\nonset of the U.S. outbreak. Annals of Internal Medicine, 173(2),\n100–109. https://doi.org/10.7326/m20-1239. Jarvis, C. I., Van Zandvoort, K., Gimma, A., Prem, K., Auzenbergs, M.,\nO’Reilly, K., et al. (2020). Quantifying the impact of physical dis-\ntance measures on the transmission of COVID-19 in the UK. BMC\nMedicine, 18(1), 124. https://doi.org/10.1186/s12916-020-01597-8. Publisher’s note Springer Nature remains neutral with regard to jurisdic-\ntional claims in published maps and institutional affiliations. Kinman, G. (2019). Sickness presenteeism at work: prevalence, costs and\nmanagement. British Medical Bulletin, 129(1), 69–78. https://doi. org/10.1093/bmb/ldy043."
https://openalex.org/W4312745225	https://journal.gumrf.ru/jour/article/download/209/209	Russian	null	RESEARCH OF NAVIGATIONAL AND HYDROGRAPHIC CHARACTERISTICS OF FLAW POLYNYAS OF THE EASTERN SECTOR OF THE NORTHERN SEA ROUTE	Vestnik Gosudarstvennogo universiteta morskogo i rečnogo flota imeni admirala S. O. Makarova/Vestnik Gosudarstvennogo universiteta morskogo i rečnogo flota imeni admirala S.O. Makarova	2,022	cc-by	5,123	For citation: Isaulova, Kristina Ya. “Research of navigational and hydrographic characteristics of flaw polynyas of the eastern sector of the Northern Sea Route.” Vestnik Gosudarstvennogo universiteta morskogo i rechnogo flota imeni admirala S. O. Makarova 14.3 (2022): 394–402. DOI: 10.21821/2309-5180-2022-14-3-394-402. DOI: 10.21821/2309-5180-2022-14-3-394-402 RESEARCH OF NAVIGATIONAL AND HYDROGRAPHIC CHARACTERISTICS OF FLAW POLYNYAS OF THE EASTERN SECTOR OF THE NORTHERN SEA ROUTE K. Ya. Isaulova Admiral Makarov State University of Maritime and Inland Shipping, St. Petersburg, Russian Federation Admiral Makarov State University of Maritime and Inland Shipping, St. Petersburg, Russian Federation The possibility of using the areas of flaw polynyas in the Eastern sector of the Northern Sea Route in planning and choosing the routes for navigation of transport ships is considered in the paper. It is noted that shipping in the Arctic seas is associated with an increased level of risk, shallow seas and difficult hydrometeorological conditions, and is also characterized by insufficient or completely absent hydrographic knowledge of the bottom topography. The main data characterizing the increase in the number of issued permits for ships navigation in the water area of the Northern Sea Route, and the percentage of ships depending on the ice class are given. It is noted that in the cold season, the main navigable straits of the Northern Sea Route are covered with fast ice. Due to circulation and under-ice currents, flaw polynyas, which can be used when planning navigation routes, are formed. The main flaw polynyas of the Eastern Sector of the Northern Sea Route are presented; data on their width, length and range of depths are provided. It is noted that the flaw polynyas of the East Siberian and Chukchi seas are deeper than the polynyas of the Laptev Sea. The ships classification depending on the draft is presented in the paper; an analysis of the characteristic routes for each category of ships is performed. An analysis of the data received from the automatic identification system of vessels has revealed that the choice of route is seasonal and directly depends on the ice conditions of the water area. The relevance of the topic is due to the need to increase the duration of navigation in the Eastern sector of the Northern Sea Route, in order to achieve targets for the volume of cargo transportation. This can be achieved by choosing the route of the transport vessel along the areas of ice polynyas, which are a strip of open water and young ice.l eywords: Northern Sea Route, coast ice, flaw polynya, easiest way, Eastern sector, year-round navigatio К. Я. Исаулова К. Я. Исаулова Выпуск 4 2022 год. Том 14. № 3 Выпуск 4 394 2022 год. Том 14. № 3 ФГБОУ ВО «ГУМРФ имени адмирала С. О. Макарова», Санкт-Петербург, Российская Федерация ФГБОУ ВО «ГУМРФ имени адмирала С. О. Макарова», Санкт-Петербург, Российская Федерация Для цитирования: Д р Исаулова К. Я. Исследование навигационно-гидрографических характеристик заприпайных полыней восточного сектора Северного морского пути / К. Я. Исаулова // Вестник Государственного универси- тета морского и речного флота имени адмирала С. О. Макарова. — 2022. — Т. 14. — № 3. — С. 394–402. DOI: 10.21821/2309-5180-2022-14-3-394-402. 3 Указ Президента Российской Федерации от 7 мая 2018 г. № 204 «О национальных целях и стратегических задачах раз- вития Российской Федерации на период до 2024 года». 2 Распоряжение Правительства Российской Федерации от 22 ноября 2008 г. № 1734-р «Транспортная стратегия Федерации на период до 2030 года». ФГБОУ ВО «ГУМРФ имени адмирала С. О. Макарова», Санкт-Петербург, Российская Федерация В работе исследована возможность использования районов заприпайных полыней Восточного сек‑ тора Северного морского пути при планировании и выборе маршрутов плавания транспортных судов. Подчеркивается, что судоходство в арктических морях связано с повышенным уровнем риска, мелковод- ностью морей и сложными гидрометеорологическими условиями, а также характеризуется недостаточной или полностью отсутствующей гидрографической изученностью рельефа дна. Приведены основные данные, характеризующие увеличение количества выдаваемых разрешений на плавание судов в акватории Север‑ ного морского пути, и процентное соотношение судов в зависимости от их ледового класса. Отмечается, что в холодное время года основные судоходное проливы Северного морского пути покрываются припаем. Ввиду циркуляции и подледных течений образуются заприпайные полыньи, которые могут использоваться при планировании маршрутов плавания. Перечислены заприпайные полыньи Восточного сектора Северного морского пути, содержатся данные по их ширине, длине и диапазону глубин. Отмечается, что заприпайные полыньи Восточно-Сибирского и Чукотского морей являются более глубоководными, чем полыньи моря Лап‑ тевых. В работе приведена классификация судов в зависимости от их осадки, выполнен анализ характерных маршрутов движения для каждой категории судов. Анализ данных, полученных от автоматической иден‑ тификационный системы судов, позволил выявить, что выбор пути следования носит сезонный характер и напрямую зависит от ледовых условий акватории. Актуальность темы обусловлена необходимостью увеличения сроков навигации в восточном секторе Северного морского пути для реализации целевых по‑ казателей по объемам грузоперевозок, что может быть достигнуто путем выбора маршрута следования транспортного судна по районам заприпайных полыней, представляющих собой полосу открытой воды и молодых льдов. Ключевые слова: Северный морской путь, припай, заприпайная полынья, выбор маршрута, восточный сектор, круглогодичная навигация. 1 Распоряжение Правительства Российской Федерации от 21 декабря 2019 года № 3120-р «План развития инфр Северного морского пути на период до 2035 года». Введение (Introduction) Развитие Северного морского пути (СМП) является одним из наиболее перспективных про- ектов в области транзитных грузоперевозок. В настоящее время действуют программы и указы, реализация которых обеспечивает стимулирование развития грузопотока и международного тран- зитного судоходства, строительство и модернизацию инфраструктуры морских портов, развитие навигационно-гидрографического обеспечения и строительство ледокольного флота. Реализуется ряд мероприятий, целью которых является превращение СМП в конкурентоспособную транспорт- ную магистраль [1]. Выполнение предусмотренных мероприятий должно осуществляться на базе следующих нормативных документов: «План развития инфраструктуры Северного морского пути на период до 2035 года» 1, «Транспортная стратегия Российской Федерации на период до 2030 года» 2, Указ Президента Российской Федерации от 7 мая 2018 года № 204 «О национальных целях и стра- тегических задачах развития Российской Федерации на период до 2024 года»3. В работах [2]–[4] отмечается, что судоходство в арктических морях характеризуется повы- шенным уровнем риска. К основным опасностям относятся низкие температуры, тяжелые ледовые условия, возможность обледенения верхних выступающих конструкций судна, а также быстро изменяющиеся гидрометеорологические условия. В работах [5]–[7] содержатся данные о недоста- точной или полностью отсутствующей гидрографической изученности рельефа дна и мелковод- ности арктических морей. На акватории СМП ежегодно отмечается увеличение объемов перевозимых грузов. Так, в 2021 г. объем грузоперевозок по трассам СМП составил 28,2 млн т, в 2020 г. целевой показатель федерального проекта «Северный морской путь» был превышен более чем на 13 %. К основным потребителям трасс СМП относятся компании, деятельность которых связана с переработкой и до- бычей природных ископаемых. Использование трасс СМП для транспортировки грузов из Европы в Азию в период благоприятных ледовых условий способно сократить время перевозки в два раза по сравнению с использованием Суэцкого канала. Выпуск 4 395 2022 год. Том 14. № 3 Выпуск 4 2022 год. Том 14. № 3 В период 2013–2021 гг. количество выданных разрешений на плавание судов в акватории СМП увеличилось в 2 раза. Соответствующие данные приведены на рис. 1. Более 45 % выданных разрешений относятся к судам, не имеющим ледового класса, 28 % — к судам ледового усиления 4 395 3 с возможностью плавания в арктических льдах (Arc 7 — Arc 4), 25 % — к судам ледового усиления с возможностью плавания в неарктических льдах (Ice1 – Ice3), более 1 % приходится на ледоколы. с возможностью плавания в арктических льдах (Arc 7 — Arc 4), 25 % — к судам ледового усиления с возможностью плавания в неарктических льдах (Ice1 – Ice3), более 1 % приходится на ледоколы. Рис. 1. Количество разрешений, выданных судам Администрацией СМП Рис. 1. Методы и материалы (Methods and Materials) При проведении исследования в качестве источников информации использовалис морские навигационные карты масштаба 1:500000 (Адмиралтейский номер: 11135, 1113 1142, 11143, 11144, 11146, 11147, 11149); – обзорные ледовые карты Северного Ледовитого океана, размещенные на сайте Арктического и Антарктического научно-исследовательского института; Выпуск 4 39 2022 год. Том 14. № 3 Выпуск 4 396 2022 год. Том 14. № 3 ф р р р р у – данные, полученные от судовых автоматизированных идентификационных систем, разме- щенные на морском портале Scanex Maritime. Информация включает данные о местонахождении судов, передаваемые с интервалом не более 10 мин, что позволяет выявить и сформировать основ- ные маршруты их движения. Введение (Introduction) Количество разрешений, выданных судам Администрацией СМП р р , выданных судам Администрацией СМП Для реализации целевых показателей по объемам грузоперевозок необходимо продлить пе- риод навигации в восточном секторе СМП, обеспечив в перспективе круглогодичную навигацию на всей акватории [8]. В работах [9], [10] отмечается, что наиболее сложным участком при обе- спечении круглогодичной навигации является Восточно-Сибирское море. Суровые гидрометео- рологические условия наблюдаются в районе архипелагов, разделяющих моря Арктики. В районе Новосибирских островов в период летней навигации чаще всего отмечаются неблагоприятные ледовые условия. С октября по май основные судоходные проливы СМП покрываются припаем, за исключением проливов Карские Ворота и Лонга. Припай представляет собой одну из форм не- подвижного ледяного покрова, расположенного у берега и распространяющегося в сторону моря на несколько десятков миль [11]. В арктических морях за счет циркуляции и подлёдных течений образуются заприпайные полыньи [12], которые могут использоваться для судоходства. Целью настоящей работы является исследование навигационно-гидрографических харак- теристик заприпайных полыней, определяемых благоприятными ледовыми условиями для судо- ходства. Результаты (Results) В 396 С октября акватория СМП покрывается ниласом и молодым льдом. К середине ноября, когда толщина молодого льда достигает 10–30 см, вдоль архипелага Северная Земля, Новосибирских остро- вов и побережья материка образуется припай. С ноября по май за припаем образуются заприпайные полыньи. Повторяемость полыней моря Лаптевых высока в течение всего периода осенне-зим- ней навигации (варьируется в интервале 57–100 %). Все полыньи в разные месяцы относятся к ста- ционарным или устойчивым. Наиболее устойчивые полыньи расположены в южной и восточной части моря с повторяемостью в пределах 65–70 %. Повторяемость заприпайных полыней Восточ- но-Сибирского моря ниже, чем моря Лаптевых, и составляет 41–89 %. На повторяемость и формиро- вание полыней в Чукотском море прямое воздействие оказывает циклон Алеутской депрессии [13]. В восточном секторе СМП формируется двенадцать заприпайных полыней, расположенных в море Лаптевых, Восточно-Сибирском и Чукотском морях. На рис. 2 цифрами обозначены названия за- припайных полыней. Рис. 2. Полыньи восточного сектора Северного морского пути: 1 — Восточная Североземельская; 2 — Северо-восточная Таймырская; 3 — Восточная Таймырская; 4 — Анабаро-Ленская; 5 — Западная Новосибирская; 6 — Северная Новосибирская; 7 — Восточная Новосибирская (запад); 8 — Восточная Новосибирская (восток); 9 — Айонская; 10 — Западная Чукотская; 11 — Восточная Чукотская; 12 — Северная Врангелевская Рис. 2. Полыньи восточного сектора Северного морского пути: Рис. 2. Полыньи восточного сектора Северного морского пути: 1 — Восточная Североземельская; 2 — Северо-восточная Таймырская; 3 — Восточная Таймырская; 4 — Анабаро-Ленская; 5 — Западная Новосибирская; 6 — Северная Новосибирская; 7 — Восточная Новосибирская (запад); 8 — Восточная Новосибирская (восток); 9 — Айонская; 10 — Западная Чукотская; 11 — Восточная Чукотская; 12 — Северная Врангелевская В табл. 1 приведен диапазон глубин в районе заприпайных полыней, полученный путем анализа картографических материалов, а также длина и ширина полыньи. Т б 1 В табл. 1 приведен диапазон глубин в районе заприпайных полыней, полученный путем анализа картографических материалов, а также длина и ширина полыньи. Таблица 1 Характеристики заприпайных полыней арктических морей восточного сектора Северного морского пути Полынья Диапазон глубин, м Ширина, км Длина, км Заприпайные полыньи моря Лаптевых Восточная Североземельская 34,0–2478,0 130 470 Северо-восточная Таймырская 12,6–294,0 100 210 Восточная Таймырская 8,4–49,5 55 185 Анабаро-Ленская 6,6–46,5 150 335 Западная Новосибирская 10,8–48,5 135 460 Заприпайные полыньи Восточно-Сибирского моря Северная Новосибирская 14,8–43,0 115 340 Восточная Новосибирская (запад) 14,6–37,5 75 250 Восточная Новосибирская (восток) 18,0–25,5 60 250 Айонская 10,0–37,5 35 130 Заприпайные полыньи Чукотского моря Западная Чукотская 14,2–44,5 25 200 Восточная Чукотская 21,4–45,0 35 520 Северная Врангелевская 15,0–36,5 45 200 В период с октября по май море Лаптевых покрыто льдами. Вдоль берега устанавливается припай, в западной, южной и восточной частях формируются заприпайные полыньи. Восточная Североземельская, Северо-восточная Таймырская и Восточная Таймырская полыньи расположены на востоке архипелага Северная Земля и вдоль восточной части п-ва Таймыр. Анабаро-Ленская и Западная Новосибирская полыньи простираются через море Лаптевых — от о-ва Большой Бегичев до северо-западной части о-ва Котельный. Глубины заприпайных полыней, расположенные с восточной стороны архипелага Северная Земля, уменьшаются с севера на юг, достигая минимального значения 10,0 м в районе Восточной Таймырской полыньи. Восточная Североземельская полынья является глубоководной, вдоль нее проходят изобаты 200–2200 м, минимальная глубина 34,0 м отмечается с северо-западной сторо- ны. Глубины в районе Северо-восточной Таймырской полыньи в среднем составляют 40–45 м, минимальные значения отмечаются в районе о-ва Малый Таймыр, центральная часть является глубоководной (проходят изобаты в 100 и 200 м). Полыньи моря Лаптевых, простирающиеся в южной части, характеризуются меньшими глу- бинами полыней западной части. В центральной части Восточной Таймырской полыньи проходит рекомендованный маршрут с курсом 183°–3°, на котором глубины превышают 35,0 м. Минимальные глубины расположены вдоль восточной стороны п-ва Таймыр. В районе Анабаро-Ленской заприпай- ной полыньи средняя глубина не превышает 23,0 м. Северо-западная часть полыньи и район дельты р. Лена мелководны, минимальная глубина составляет 6,6 м. В центральной части отмечается уве- личение глубин в направлении с запада на восток. В районе полыньи обнаружено значительное количество опасных глубин и банок. Среднее значение глубин в восточной и западной части Западной Новосибирской полыньи составляет 25 м, в центральной части — 39,0 м. Минимальные глубины расположены в юго-за- падной части полыньи в районе дельты р. Лена. Минимальная обнаруженная глубина равна 10,8 м. Заприпайные полыньи, расположенные вдоль архипелага Северная Земля, имеют практически неиз- менную ширину, равную 75 км. Максимальная ширина в районе пролива Шокальского равна 135 км. Средняя ширина заприпайных полыней, простирающихся от о-ва Большой Бегичев до о-ва Ко- тельный, не превышает 75 км. Наблюдается увеличение ширины полыньи севернее дельты р. Лена и ее сужение в восточной части моря Лаптевых. В Восточно-Сибирском море формируются четыре полыньи: Северная Новосибирская, Западная Новосибирская (на западе), Восточная Новосибирская (на востоке) и Айонская. Север- ная Новосибирская полынья расположена севернее о-вов Котельный и Новая Сибирь. Восточные Новосибирские полыньи простираются с северо-запада на юго-восток от 76 до 73 параллели с. ш. Айонская полынья расположена в восточной части Восточно-Сибирского моря, севернее о-ва Айон. Средняя глубина Северной Новосибирской полыньи составляет 26,0 м, максимальная — 40,0 м. Выпуск 4 397 2022 год. Том 14. № 3 4 397 Обсуждение результатов (Discussion) В зависимости от осадки все суда, осуществляющие судоходство на акватории СМП, условно можно разделить на три категории (табл. 2). Т б 2 Таблица 2 Категории судов в зависимости от осадки Категория Осадка, м Первая d > 9 Вторая 6 ≤ d ≤ 9 Третья d < 6 К первой категории относятся крупнотоннажные суда с осадкой более 9 м. Вторая ка- тегория характеризуется осадкой судов 6–9 м. К третьей категории относятся суда с осадкой менее 6 м. Транспортные суда при движении с запада на восток и обратно в районе архипелага Северная Земля и о-ва Врангеля имеют устойчивые маршруты движения, двигаясь по проливу Вилькицкого и проливу Лонга соответственно. В зависимости от периода навигации и осадки судов в районе Новосибирских островов существует три варианта движения: пролив Дмитрия Лаптева, пролив Санникова или альтернативная высокоширотная трасса севернее Новосибирских островов. В период с июля по октябрь большинство судов третьей категории (70 % от общего количе- ства судов с осадкой до 6 м) используют для прохода пролив Дмитрия Лаптева, остальные — про- лив Санникова. Такое распределение обусловлено обеспечением нужд северного завоза и доставкой грузов по рекам в арктические порты. В ноябре интенсивность судоходства снижается и полностью прекращается в декабре. В этот период суда с осадкой до 6 м используют высокоширотную аль- тернативную трассу, расположенную к северу от Новосибирских островов, частично проходящую по заприпайным полыньям. Суда второй категории в период с июля по октябрь используют исклю- чительно пролив Санникова. В октябре более 85 % судов проходят по проливу Санникова, осталь- ные — через пролив Дмитрия Лаптева и севернее Новосибирских остров. В ноябре все маршруты судов проходят исключительно севернее Новосибирских островов, в районе развития заприпайных полыней. Выпуск 4 99 2022 год. Том 14. № 3 Выпуск 4 9 2022 год. Том 14. № 3 Крупнотоннажные суда с осадкой более 9 м для прохода в районе Новосибирских островов используют два варианта маршрута: пролив Санникова и альтернативную высокоширотную трассу, часть которой проходит по заприпайным полыньям. В период с июля по октябрь более 70 % всех крупнотоннажных судов используют высокоширотную альтернативную трассу, в ноябре маршруты проходят исключительно по ней. 4 399 Анализ картографических материалов акватории СМП показал, что заприпайные полыньи моря Лаптевых имеют большую площадь по сравнению с полыньями Восточно-Сибирского и Чу- котского морей. Они превышают более чем в 2 и 6,5 раз площади заприпайных полыней Восточ- но-Сибирского и Чукотского морей соответственно, а также покрывают около 20 % от общей пло- щади моря Лаптевых. ского автономного округа, Северная Врангелевская полынья расположена с северо-западной сторо- ны о-ва Врангеля. Полыньи Чукотского моря характеризуются глубинами в диапазоне 27–30 м, ми- нимальная глубина, составляющая 14,2 м, расположена у берегов Чукотского автономного округа. Средние глубины Врангелевской полыньи составляют 30 м, минимальная — 15,0 м отмечается в южной ее части. Чукотская полынья простирается на сотни километров от Чаунской губы до Бе- рингова пролива. Западная часть Чукотской полыньи, ввиду своей малой ширины, называется прогалиной [15], ее восточная часть имеет ширину около 35 км, Северная Врангелевкая полынья достигает в ширину 45 км, в длину 200 км. ского автономного округа, Северная Врангелевская полынья расположена с северо-западной сторо- ны о-ва Врангеля. Полыньи Чукотского моря характеризуются глубинами в диапазоне 27–30 м, ми- нимальная глубина, составляющая 14,2 м, расположена у берегов Чукотского автономного округа. Средние глубины Врангелевской полыньи составляют 30 м, минимальная — 15,0 м отмечается в южной ее части. Чукотская полынья простирается на сотни километров от Чаунской губы до Бе- рингова пролива. Западная часть Чукотской полыньи, ввиду своей малой ширины, называется прогалиной [15], ее восточная часть имеет ширину около 35 км, Северная Врангелевкая полынья достигает в ширину 45 км, в длину 200 км. Восточные Новосибирские полыньи характеризуются малой гидрографической изученностью и практически полностью представляют собой «белые пятна» [14]. В западной части полыньи промер выполнен галсами через 1 км, средняя глубина составляет 24,0 м, минимальная — 14,8 м. В восточной части сведения о глубинах практически отсутствуют, минимальная обнаруженная глубина равна 18,0 м, средняя — 22,0 м. В южной части Айонской заприпайной полыньи про- ходит 20‑метровая изобата. Глубины изменяются в интервале 10–32 м, минимальная глубина отмечается на банке. Выпуск 4 2022 год. Том 14. № 3 Северная Новосибирская полынья имеет максимальную ширину развития севернее о-ва Новая Сибирь, составляющую 130 км, и среднюю ширину, равную 85 км. Максимальная ширина Восточ- ных Новосибирских полыней, которая отмечается в северо-западной части, составляет 75 км. Айон- ская полынья имеет наименьшую площадь среди полыней Восточно-Сибирского моря. Ее длина составляет 130 км, максимальная ширина в восточной части равна 40 км. В 398 2 В Чукотском море развиваются три полыньи: Западная и Восточная Чукотская, а также Север- ная Врангелевская. Западная и Восточная Чукотская полыньи простираются вдоль берегов Чукот- Выводы (Summary) На основе проведенного исследования можно сделать следующие выводы: На основе проведенного исследования можно сделать следующие выводы: 1. Глубины заприпайных полыней моря Лаптевых могут быть использованы для прохода судов второй и третьей категории с осадкой до 9 м. Заприпайные полыньи Восточно-Сибирского и Чукотского морей более глубоководны и в период осенне-зимней навигации могут использоваться судами с осадкой более 9 м. 1. Глубины заприпайных полыней моря Лаптевых могут быть использованы для прохода судов второй и третьей категории с осадкой до 9 м. Заприпайные полыньи Восточно-Сибирского и Чукотского морей более глубоководны и в период осенне-зимней навигации могут использоваться судами с осадкой более 9 м. 2. Зимняя навигация в Восточном секторе носит эпизодический характер, что обусловлено сложными гидрометеорологическими условиями и отсутствием достаточного количества мощных ледоколов. 3. Использование районов образования заприпайных полыней для прокладки маршрута дви- жения судна позволит продлить период навигации в холодное время года и сократить общее время перехода. Основная сложность использования заприпайных полыней заключается в возможности изменения их ширины в период развития. В отдельные годы с ноября по май может наблюдаться слабое развитие заприпайных полыней или их полное отсутствие. 4. Полученные данные могут быть использованы при планировании и выборе маршрутов движения судов в акватории СМП. и зависит от ледовых условий. В период летне-осенней навигации каждая категория судов имеет устойчивый маршрут движения. С октября по декабрь, когда акватория морей Восточного сектора СМП покрывается ниласом и молодым льдом, маршруты судов изменяются. СПИСОК ЛИТЕРАТУРЫ 1. Трегуб М. А. Перспективы развития Северного морского пути. Северный морской транзитный ко- ридор / М. А. Трегуб, К. А. Ярошенко-Соколовская // Системный анализ и логистика. — 2021. — № 1 (27). — С. 44–48. DOI: 10.31799/2077-5687-2021-1-44-48. 1. Трегуб М. А. Перспективы развития Северного морского пути. Северный морской транзитный ко- ридор / М. А. Трегуб, К. А. Ярошенко-Соколовская // Системный анализ и логистика. — 2021. — № 1 (27). — С. 44–48. DOI: 10.31799/2077-5687-2021-1-44-48. 2. Шаронов А. Ю. Задачи гидрометеорологического обеспечения круглогодичной навигации в Восточно-Сибирском море / А. Ю. Шаронов, В. А. Шматков // Вестник Государственного университета морского и речного флота имени адмирала С. О. Макарова. — 2018. — Т. 10. — № 1. — С. 170–182. DOI: 10.21821/2309-5180-2018-10-1-170-182. 3. Холопцев А. В. Анализ изменений ледовых условий на Северном морском пути в конце ХХ — начале ХXI века / А. В. Холопцев, С. А. Подпорин // Вестник Государственного университета морского и речного флота имени адмирала С. О. Макарова. — 2020. — Т. 12. — № 1. — С. 71–84. DOI: 10.21821/2309-5180-2020- 12-1-71-84. 4. Першин Н. В. Рациональное построение маршрутов плавания с учетом гидрометеорологических условий / Н. В. Першин // Ученые записки Российского государственного гидрометеорологического универ- ситета. — 2018. — № 52. — С. 61–66. 4. Першин Н. В. Рациональное построение маршрутов плавания с учетом гидрометеорологических условий / Н. В. Першин // Ученые записки Российского государственного гидрометеорологического универ- ситета. — 2018. — № 52. — С. 61–66. 5. Тезиков А. Л. Исследование факторов, влияющих на продолжительность навигации в акватории Северного морского пути / А. Л. Тезиков, Е. О. Ольховик // Вестник Государственного университета морского и речного флота имени адмирала С. О. Макарова. — 2020. — Т. 12. — № 4. — С. 734–744. DOI: 10.21821/2309- 5180-2020-12-4-734-744. 5. Тезиков А. Л. Исследование факторов, влияющих на продолжительность навигации в акватории Северного морского пути / А. Л. Тезиков, Е. О. Ольховик // Вестник Государственного университета морского и речного флота имени адмирала С. О. Макарова. — 2020. — Т. 12. — № 4. — С. 734–744. DOI: 10.21821/2309- 5180-2020-12-4-734-744. Выпуск 4 2022 год. Том 14. № 3 6. Решетняк С. В. История развития навигационно-гидрографического обеспечения в акватории Север- 6. Решетняк С. В. История развития навигационно-гидрографического обеспечения в акватории Север- 6. Решетняк С. В. История развития навигационно-гидрографического обеспечения в акватории Север- ного морского пути. Ч. 2: Комплексная система НГО акватории СМП в конце ХХ — начале ХХI в. (1990 г. — 6. Решетняк С. В. Обсуждение результатов (Discussion) Выбор маршрута движения судна в акватории СМП имеет сезонный характер REFERENCES 1. Tregub, Margarita A., and Kseniia A. Iaroshenko-Sokolovskaia. “Prospects for the development of the Northern Sea Route. Northern Sea Transit Corridor.” Sistemnyj analiz i logistika 1(27) (2021): 44–48. DOI: 10.31799/2077-5687-2021-1-44-48. 1. Tregub, Margarita A., and Kseniia A. Iaroshenko-Sokolovskaia. “Prospects for the development of the Northern Sea Route. Northern Sea Transit Corridor.” Sistemnyj analiz i logistika 1(27) (2021): 44–48. DOI: 10.31799/2077-5687-2021-1-44-48. 2. Sharonov, Andrei Yu., and Vladimir A. Shmatkov. “The problem of hydrometeorological maintenance of year-round navigation in the East Siberian Sea.” Vestnik Gosudarstvennogo universiteta morskogo i rechnogo flota imeni admirala S. O. Makarova 10.1 (2018): 170–182. DOI: 10.21821/2309-5180-2018-10-1-170-182. 2. Sharonov, Andrei Yu., and Vladimir A. Shmatkov. “The problem of hydrometeorological maintenance of year-round navigation in the East Siberian Sea.” Vestnik Gosudarstvennogo universiteta morskogo i rechnogo flota imeni admirala S. O. Makarova 10.1 (2018): 170–182. DOI: 10.21821/2309-5180-2018-10-1-170-182. 3. Kholoptsev, Aleksandr V., and Sergey A. Podporin. “Analysis of ice situation changes on the Northern Sea Route in the late XX – early XXI century.” Vestnik Gosudarstvennogo universiteta morskogo i rechnogo flota imeni admirala S. O. Makarova 12.1 (2020): 71–84. DOI: 10.21821/2309-5180-2020-12-1-71-84. 3. Kholoptsev, Aleksandr V., and Sergey A. Podporin. “Analysis of ice situation changes on the Northern Sea Route in the late XX – early XXI century.” Vestnik Gosudarstvennogo universiteta morskogo i rechnogo flota imeni admirala S. O. Makarova 12.1 (2020): 71–84. DOI: 10.21821/2309-5180-2020-12-1-71-84. 4. Pershin, N. V. “Rational creation of navigation routes taking into account hydrometeorological conditions.” Uchenye zapiski Rossiiskogo gosudarstvennogo gidrometeorologicheskogo universiteta 52 (2018): 61–66.f 4. Pershin, N. V. “Rational creation of navigation routes taking into account hydrometeorological conditi nye zapiski Rossiiskogo gosudarstvennogo gidrometeorologicheskogo universiteta 52 (2018): 61–66.f 4. Pershin, N. V. Rational creation of navigation routes taking into account hydrometeorological conditions. Uchenye zapiski Rossiiskogo gosudarstvennogo gidrometeorologicheskogo universiteta 52 (2018): 61–66.f 5. Tezikov, Aleksandr L., and Evgeniy O. Ol’khovik. “Studying the factors affecting the duration of naviga- tion in the Northern Sea Route water area.” Vestnik Gosudarstvennogo universiteta morskogo i rechnogo flota imeni admirala S. O. Makarova 12.4 (2020): 734–744. DOI: 10.21821/2309-5180-2020-12-4-734-744. 5. Tezikov, Aleksandr L., and Evgeniy O. Ol’khovik. “Studying the factors affecting the duration of naviga- tion in the Northern Sea Route water area.” Vestnik Gosudarstvennogo universiteta morskogo i rechnogo flota imeni admirala S. O. Makarova 12.4 (2020): 734–744. DOI: 10.21821/2309-5180-2020-12-4-734-744. 6. Reshetnyak, Sergei V. “The history of the development of navigation and hydrographic support in the waters of the Northern Sea Route. СПИСОК ЛИТЕРАТУРЫ Андрее ва // Сборник трудов IX Межвузовской научно-практической конференции аспирантов, студентов и курсан- тов «Современные тенденции и перспективы развития водного транспорта России». — СПб: ГУМР С. О. Макарова, 2018. — С. 443–447. р , 15. Захаров В. Ф. Роль заприпайных полыней в гидрохимическом и ледовом режиме моря Лаптевых / В. Ф. Захаров // Океанология. — 1966. — №24. — С. 168–179. 15. Захаров В. Ф. Роль заприпайных полыней в гидрохимическом и ледовом режиме моря Лаптевых / В. Ф. Захаров // Океанология. — 1966. — № 24. — С. 168–179. y g p , 15. Zakharov, V. F. “Rol’ zapripainykh polynei v gidrokhimicheskom i ledovom rezhime morya Laptevykh.” Okeanologiya 24 (1966): 168–179. 14. Andreeva, Ekaterina A. ““White spots” on the maps of the Northern Sea Route.” Sbornik trudov IX Mezh‑ vuzovskoj nauchno-prakticheskoj konferencii aspirantov, studentov i kursantov «Sovremennye tendencii i perspektivy razvitiya vodnogo transporta Rossii». SPb.: Izd-vo GUMRF im. adm. S. O. Makarova, 2018. 443–447. СПИСОК ЛИТЕРАТУРЫ История развития навигационно-гидрографического обеспечения в акватории Север- ного морского пути. Ч. 2: Комплексная система НГО акватории СМП в конце ХХ — начале ХХI в. (1990 г. — настоящее время) / С. В. Решетняк // Морской вестник. — 2019. — № 1 (69). — С. 117–123. 7. Решетняк С. В. Гидрографическая изученность подводного рельефа арктических морей России / С. В. Решетняк // Геодезия и картография. — 2006. — № 4. — С. 57–60. В 400 8. Гурлев И. В. Интеллектуализация транспортной системы Северного морского пути / И. В. Гурлев, А. А. Макоско, И. Г. Малыгин, В. Ю. Каминский // Морские интеллектуальные технологии. — 2021. — № 3–1 (53). — С. 228–235. DOI: 10.37220/MIT.2021.53.3.026. 9. Lee S. W. Economic Possibilities of Shipping through Northern Sea Route / S. W. Lee, J. M. Song // The Asian Journal of Shipping and Logistics. — 2014. — Vol. 30. — Is. 3. — Pp. 415–430. 9. Lee S. W. Economic Possibilities of Shipping through Northern Sea Route / S. W. Lee, J. M. Song // The Asian Journal of Shipping and Logistics. — 2014. — Vol. 30. — Is. 3. — Pp. 415–430. 10. Ольховик Е. О. Влияние льда на формирование судоходных маршрутов в акватории Северного морского пути / Е. О. Ольховик, Е. В. Андреева, А. Л. Тезиков // Вестник Астраханского государственно- го технического университета. Серия: Морская техника и технология. — 2019. — № 2. — С. 26–36. DOI: 10.24143/2073-1574-2019-2-26-36. 11. Marchenko N. Russian Arctic Seas: Navigational conditions and accidents / N. Marchenko. — Springer, 2012. — 296 p. 12. Номенклатура морских льдов. Условные обозначения для ледовых карт. — Гидрометеоиздат, 1974. — 80 с. 13. Атлас биологического разнообразия морей и побережий российской Арктики. — М.: WWF России, 2011. — 64 с. 14. Андреева Е. В. «Белые пятна» на картах акватории Северного морского пути / Е. В. Андрее- ва // Сборник трудов IX Межвузовской научно-практической конференции аспирантов, студентов и курсан- тов «Современные тенденции и перспективы развития водного транспорта России». — СПб: ГУМРФ им. адм. С. О. Макарова, 2018. — С. 443–447. 14. Андреева Е. В. «Белые пятна» на картах акватории Северного морского пути / Е. В. Андрее- ва // Сборник трудов IX Межвузовской научно-практической конференции аспирантов, студентов и курсан- тов «Современные тенденции и перспективы развития водного транспорта России». — СПб: ГУМРФ им. адм. 14. Андреева Е. В. «Белые пятна» на картах акватории Северного морского пути / Е. В. ИНФОРМАЦИЯ ОБ АВТОРЕ INFORMATION ABOUT THE AUTHOR Исаулова Кристина Яновна — аспирант Научный руководитель: Тезиков Александр Львович — доктор технических наук, профессор ФГБОУ ВО «ГУМРФ имени адмирала С. О. Макарова» 198035, Российская Федерация, Санкт-Петербург, ул. Двинская, 5/7 е-mail: isaulovakya@gumrf.ru Isaulova, Kristina Ya. — Postgraduate Supervisor: Tezikov, Aleksandr L. — Dr. of Technical Sciences, professor Admiral Makarov State University of Maritime and Inland Shipping 5/7 Dvinskaya Str., St. Petersburg, 198035, Russian Federation e-mail: isaulovakya@gumrf.ru Статья поступила в редакцию 2 февраля 2022 г. Received: February 2, 2022. REFERENCES Part 2: Integrated NGO system in the NSR water area at the end of the XX – beginning of the XXI century (1990 – present).” Morskoy Vestnik 1(69) (2019): 117–123. 6. Reshetnyak, Sergei V. “The history of the development of navigation and hydrographic support in the waters of the Northern Sea Route. Part 2: Integrated NGO system in the NSR water area at the end of the XX – beginning of the XXI century (1990 – present).” Morskoy Vestnik 1(69) (2019): 117–123. 7. Reshetnyak, S.V. “Hydrographic study of the arctic sees underwater relief of Russia.” Geodesy and Car‑ tography 4 (2006): 57–60. 8. Gurlev, Igor V., Alexander A. Makosko, Igor G. Malygin, and Valery Yu. Kaminsky. “Intellectualization of the transport system of the Northern Sea Route.” Marine intelligent technologies 3–1(53) (2021): 228–235. DOI: 10.37220/MIT.2021.53.3.026. Выпуск 4 1 2022 год. Том 14. № 3 9. Lee, Sung-Woo, and Ju-Mi. Song. “Economic Possibilities of Shipping through Northern Sea Route.” The Asian Journal of Shipping and Logistics 30.3 (2014): 415–430. 10. Olkhovik, Evgeniy Olegovich, Ekaterina Valerievna Andreeva, and Aleksandr Lvovich Tezikov. “Ice in- fluence on forming shipping routes in the water area of the northern sea route.” Vestnik of Astrakhan State Technical University. Series: Marine engineering and technologies 2 (2019): 26–36. DOI: 10.24143/2073-1574-2019-2-26-36. 11. Marchenko, N. Russian Arctic Seas: Navigational conditions and accidents. Springer, 2012 11. Marchenko, N. Russian Arctic Seas: Navigational conditions and accidents. Springer, 2012. 12 Nomenklatura morskikh l’dov Uslovnye oboznacheniya dlya ledovykh kart Gidrometeoizdat 4 401 3 y y y y , 13. Atlas biologicheskogo raznoobraziya morej i poberezhij rossijskoj Arktiki. M.: WWF Rossii, 2011. 14. Andreeva, Ekaterina A. ““White spots” on the maps of the Northern Sea Route.” Sbornik trudov IX Mezh‑ vuzovskoj nauchno-prakticheskoj konferencii aspirantov, studentov i kursantov «Sovremennye tendencii i perspektivy razvitiya vodnogo transporta Rossii». SPb.: Izd-vo GUMRF im. adm. S. O. Makarova, 2018. 443–447. y g p 15. Zakharov, V. F. “Rol’ zapripainykh polynei v gidrokhimicheskom i ledovom rezhime morya Laptevykh.” Okeanologiya 24 (1966): 168–179. INFORMATION ABOUT THE AUTHOR INFORMATION ABOUT THE AUTHOR Isaulova, Kristina Ya. — Postgraduate Supervisor: Tezikov, Aleksandr L. — Dr. of Technical Sciences, professor Admiral Makarov State University of Maritime and Inland Shipping 5/7 Dvinskaya Str., St. Petersburg, 198035, Russian Federation e-mail: isaulovakya@gumrf.ru Статья поступила в редакцию 2 февраля 2022 г. Received: February 2, 2022. Выпуск 4 40 2022 год. Том 14. № 3 Выпуск 4 402 2022 год. Том 14. № 3 В 402
https://openalex.org/W3189592846	https://accedacris.ulpgc.es/jspui/bitstream/10553/111920/1/localization_native_mms13.pdf	English	null	Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunofluorescence Microscopy and Biochemical Analysis	Crystals	2,021	cc-by	9,104	Citation: Oestreicher, Z.; Valverde-Tercedor, C.; Mumper, E.; Pérez-Guzmán, L.; Casillas-Ituarte, N.N.; Jimenez-Lopez, C.; Bazylinski, D.A.; Lower, S.K.; Lower, B.H. Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunoﬂuorescence Microscopy and Biochemical Analysis. Crystals 2021, 11, 874. https://doi.org/10.3390/ cryst11080874 Academic Editor: Abel Moreno Received: 6 July 2021 Accepted: 26 July 2021 Published: 28 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. p j g 5 School of Life Sciences, University of Nevada at Las Vegas, Las Vegas, NV 89154-4004, USA; dennis.bazylinski@unlv.edu * Correspondence: Lower.30@osu.edu; Tel.: +1-614-292-2265 p j g 5 School of Life Sciences, University of Nevada at Las Vegas, Las Vegas, NV 89154-4004, USA; dennis.bazylinski@unlv.edu Citation: Oestreicher, Z.; Valverde-Tercedor, C.; Mumper, E.; Pérez-Guzmán, L.; Casillas-Ituarte, N.N.; Jimenez-Lopez, C.; Bazylinski, D.A.; Lower, S.K.; Lower, B.H. Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunoﬂuorescence Microscopy and Biochemical Analysis. Crystals 2021, 11, 874. https://doi.org/10.3390/ cryst11080874 dennis.bazylinski@unlv.edu * Correspondence: Lower.30@osu.edu; Tel.: +1-614-292-2265 * Correspondence: Lower.30@osu.edu; Tel.: +1-614-292-2265 Abstract: Magnetotactic bacteria (MTB) biomineralize intracellular magnetite (Fe3O4) crystals sur- rounded by a magnetosome membrane (MM). The MM contains membrane-speciﬁc proteins that control Fe3O4 mineralization in MTB. Previous studies have demonstrated that Mms13 is a critical protein within the MM. Mms13 can be isolated from the MM fraction of Magnetospirillum magneticum AMB-1 and a Mms13 homolog, MamC, has been shown to control the size and shape of magnetite nanocrystals synthesized in-vitro. The objective of this study was to use several independent meth- ods to deﬁnitively determine the localization of native Mms13 in M. magneticum AMB-1. Using Mms13-immunogold labeling and transmission electron microscopy (TEM), we found that Mms13 is localized to the magnetosome chain of M. magneticum AMB-1 cells. Mms13 was detected in direct contact with magnetite crystals or within the MM. Immunoﬂuorescence detection of Mms13 in M. magneticum AMB-1 cells by confocal laser scanning microscopy (CLSM) showed Mms13 localization along the length of the magnetosome chain. Proteins contained within the MM were resolved by SDS-PAGE for Western blot analysis and LC-MS/MS (liquid chromatography with tandem mass spectrometry) protein sequencing. Using Anti-Mms13 antibody, a protein band with a molecular mass of ~14 kDa was detected in the MM fraction only. This polypeptide was digested with trypsin, sequenced by LC-MS/MS and identiﬁed as magnetosome protein Mms13. Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunoﬂuorescence Microscopy and Biochemical Analysis Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunoﬂuorescence Microscopy and Biochemical Analysis Zachery Oestreicher 1,2, Carmen Valverde-Tercedor 3, Eric Mumper 2, Lumarie Pérez-Guzmán 1, Nadia N. Casillas-Ituarte 1,2, Concepcion Jimenez-Lopez 4 , Dennis A. Bazylinski 5, Steven K. Lower 1,2 and Brian H. Lower 1,* 1 School of Environment & Natural Resources, The Ohio State University, Columbus, OH 43210, USA; zacheryoe@gmail.com (Z.O.); Lumieperez@gmail.com (L.P.-G.); casillas-ituarte.1@osu.edu (N.N.C.-I.); Lower.9@osu.edu (S.K.L.) 2 School of Earth Sciences, The Ohio State University, Columbus, OH 43210, USA; mumper.9@osu.edu 3 Instituto Universitario Investigaciones Biomédicas y Sanitarias (IUIBS), Universidad de Las Palmas de Gran Canaria (ULPGC), 35016 Las Palmas de Gran Canaria, Spain; carmen.valverde@ulpgc.es 4 Departmento de Microbiología, Facultad de Ciencias, Campus de Fuentenueva s/n, Universidad de Granada, 18071 Granada, Spain; cjl@ugr.es 2 School of Earth Sciences, The Ohio State University, Columbus, OH 43210, USA; mumper.9@osu.edu 3 Instituto Universitario Investigaciones Biomédicas y Sanitarias (IUIBS), Universidad de Las Palmas de Gran Canaria (ULPGC), 35016 Las Palmas de Gran Canaria, Spain; carmen.valverde@ulpgc.es 4 Departmento de Microbiología, Facultad de Ciencias, Campus de Fuentenueva s/n, Universidad de Granada, School of Earth Sciences, The Ohio State University, Columbus, OH 43210, USA; mumper.9@osu.edu 3 Instituto Universitario Investigaciones Biomédicas y Sanitarias (IUIBS), Universidad de Las Palmas de Gran Canaria (ULPGC), 35016 Las Palmas de Gran Canaria, Spain; carmen.valverde@ulpgc.es 4 Departmento de Microbiología, Facultad de Ciencias, Campus de Fuentenueva s/n, Universidad de Granada, 18071 Granada, Spain; cjl@ugr.es crystals crystals 1. Introduction Magnetotactic bacteria (MTB) are a group of prokaryotes, which biomineralize mag- netic crystals of magnetite (Fe3O4) and/or greigite (Fe3S4) surrounded by a lipid-bilayer, called the magnetosome membrane (MM) [1–4]. The magnetosome membrane (MM) originates as an invagination of the cytoplasmic membrane creating a vesicle in which the magnetite crystal nucleates and subsequently grows [5–9]. Proteins associated with the MM are believed to control the biomineralization of magnetosome crystals [10–21]. Individual magnetosomes are arranged in a chain(s) within a bacterium to maximize the magnetic dipole moment of the cell therefore allowing MTB to passively align and swim along Earth’s magnetic ﬁeld lines [1–4]. Presumably MTB use magnetotaxis in conjunction with chemotaxis to locate and maintain an optimum position in a water column for growth and survival [1–4]. Previous studies have shown that the MM contains proteins required for the biogenesis of intracellular magnetosomes and that the MM appears to be derived from the cytoplasmic membrane [9,12,13,15–19]. Some of these MM proteins are also found in the cytoplasmic membrane and fatty acid analysis suggests that the MM is derived from the cytoplasmic membrane [13,16,19]. Several MM proteins appear to control the formation of the magneto- some membrane and the biomineralization of the magnetosome crystals [2,6,7,10–17]. For example, in Magnetospirillum magneticum AMB-1, genomic and proteomic analysis have identiﬁed almost 100 magnetosome speciﬁc proteins, and at least a dozen of these proteins are believed to be involved in magnetosome biomineralization [3,4,16,22]. It has been demonstrated in Magnetospirillum species that several proteins, including Mms5, Mms6, Mms7 (MamD), Mms12 (MamF) and Mms13 (MamC) are tightly associated with the MM [3,4,6,9,12–16,18]. To date, inorganic synthesis of magnetite in the presence of some of these puriﬁed proteins has failed to yield crystals that are identical to those found within the magnetosomes of MTB [17,21]. The morphology of the magnetite crystals in MTB appears to be strain speciﬁc [2–4,23]. This suggests that the mineralization process and the function of individual proteins may vary by microbial strain. Therefore, it is vital to study individual proteins in all MTB species to determine similarities and differences between different bacterial species. There is great interest in understanding the protein-catalyzed mineralization process involved in magnetosome synthesis and the function of speciﬁc MM proteins in controlling the number, size, and morphology of nascent Fe3O4 nanocrystals. Peptides corresponding to the protein’s putative MM domain and catalytic domain were both identiﬁed by LC-MS/MS. Our results (Immunogold TEM, Immunoﬂuorescence CLSM, Western blot, LC-MS/MS), combined with results from previous studies, demonstrate that Mms13 and homolog proteins MamC and Mam12, are localized to the magnetosome chain in MTB belonging to the class Alphaproteobacteria. Because of their shared localization in the MM and highly conserved amino acid sequences, it is likely that MamC, Mam12, and Mms13 share similar roles in the biomineralization of Fe3O4 nanocrystals. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// Keywords: bacteria; biomineralization; magnetite; magnetotactic; magnetosome; nanocrystal; protein; TEM y protein; TEM creativecommons.org/licenses/by/ Crystals 2021, 11, 874. https://doi.org/10.3390/cryst11080874 https://www.mdpi.com/journal/crystals 2 of 13 Crystals 2021, 11, 874 2. Materials and Methods To purify magnetosomes and prepare cell soluble and membrane fractions of M. mag- neticum AMB-1 (ATCC 700264), the protocol described in [33] was followed. To clone mms13 (amb0951 in M. magneticum AMB-1; [22]), genomic DNA from M. magneticum AMB- 1 was obtained from the American Type Culture Collection (ATCC #700264). The gene amb0951 was ampliﬁed by the polymerase chain reaction (PCR) with the primers: fw1 (5′-CACCATGCGCTCCTGGCTG-3′) and rev2 (5′-TCAGGCCAGTTCGTCCCGC-3′). The PCR product was cloned into pET160/GW/D-TOPO according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). PCR products and the vector containing mms13 (pET160/GW/D-TOPO/mms13) were sequenced at the Plant-Microbe Genomics Facility at The Ohio State University. y y Escherichia coli TOP10 (Invitrogen, Carlsbad, CA, USA) and E. coli BL21 (Invitrogen, Carlsbad, CA, USA) cells were used for protein expression. Cells were cultured in 250 mL of Luria broth (LB) at 37 ◦C and induced with 1 mM isopropyl-1-thio-β-D galactopyranoside for 5.5 h. The recombinant Mms13 protein (rMms13) was insoluble and found in inclusion bodies. Therefore, 8 M urea was used to solubilize the inclusion bodies and rMms13 was puriﬁed under denaturing conditions using a nickel-nitrilotriacetic acid column (Qiagen, Hilden, Germany). rMms13 was renatured at 4 ◦C by dialysis against 1 L of buffer contain- ing 0.02 M Tris-HCl, 5 M NaCl, and 1 mM phenylmethanesulfonylﬂuoride (PMSF), pH 7.5. This buffer also contained 4 M, 2 M, 1 M, and 0.5 M urea for 2 h each. The ﬁnal dialysis was performed overnight at 4 ◦C in 1 L of buffer without urea. The protein was dialyzed several more times for 3 h each, at 4 ◦C against 1 L of fresh buffer (0.02 M Tris-HCl, 0.5 M NaCl, pH 7.5). This was the rMms13-protein fraction that was used for antibody production. p p y p To determine that the puriﬁed recombinant protein was indeed rMms13, the ﬁnal protein fraction was resolved by SDS-PAGE and stained with Coomassie R-250. The protein band corresponding to the molecular mass of rMms13 was excised and subjected to peptide mass ﬁngerprinting at the Mass Spectrometry & Proteomics Facility at The Ohio State University. Polyclonal antibodies against rMms13 were produced in a rabbit by ProSci Incorpo- rated (Poway, CA, USA). The ﬁnal concentration of the antibody (anti-Mms13) determined by direct ELISA was 1.4 mg/mL. These antibodies were used for Western blot and im- munogold labeling. 1. Introduction Among MM proteins, Mms13 and homologous proteins MamC and Mam12, are small (e.g., Mms13 is 145 amino acids) highly conserved protein within the magnetotactic Al- phaproteobacteria class and no obvious homologs have been identiﬁed in non-magnetotactic bacteria [3,4,9,12,13,16,18,22]. The secondary structure of MamC (a Mms13 homolog) re- veals two helicoidal transmembrane domains connected by a α-helix structured loop in the magnetosome lumen that contains several charged residues predicted to interact with the magnetite crystal [2–4,11,21,24]. Furthermore, it has been demonstrated that MamC plays a role in regulating the size and shape of magnetite crystals in MTB [2,4,14,21,25–27] and in in-vitro mineralization experiments [6,24–27]. Previous studies [12,13,15,16,18,28–32] have employed different methods and tech- niques to help deﬁne the localization of Mms13 and homologous proteins, MamC and Mam12, in MTB species belonging to the class Alphaproteobacteria. These studies used several techniques including plasmid-overexpressed fusion proteins, immunogold electron microscopy, immunoﬂuorescence microscopy, and biochemical and proteomic analysis of puriﬁed cell fractions. Indeed, the use of different methods and techniques to determine the localization of a protein within a cell is critical to validate results and avoid bias that can lead to false conclusions. The objective of this study was to use multiple independent methods on the same organism (M. magneticum AMB-1) to determine the subcellular location of native Mms13 with micron to nanometer scale resolution. We used (1) protein fractionation followed by Western blot analysis, (2) LC-MS/MS protein sequencing, (3) immunogold-Mms13 labeling followed by transmission electron microscopy (TEM) of cells, and (4) immunoﬂuorescence Crystals 2021, 11, 874 3 of 13 detection of Mms13 by confocal laser scanning microscopy (CLSM) on whole cells. To our knowledge, this is the ﬁrst time that immunogold TEM and immunoﬂuorescence microscopy have be used to examine the localization of native Mms13 in M. magneticum AMB-1 cells and puriﬁed magnetosomes. This work provides for a more complete un- derstanding of the subcellular localization of homologous proteins MamC, Mam12, and Mms13 in members of the magnetotactic Alphaproteobacteria group. This information is important to understanding protein function and identifying shared biomineralization pathways among MTB. 2. Materials and Methods Pre-immune serum was removed from the rabbit before injecting with antigen (rMms13) into the rabbit, which was later used as a control for immunogold labeling. For the Western blots, the protocol described in [33] was followed, using anti- Mms13 antibody (1:50,000) as primary antibody and goat anti-rabbit HRP (horseradish peroxidase) antibody (1:200) as secondary antibody and the Clean-Blot IP Detection Kit HRP (Thermo-Pierce, Rockford, IL, USA). PVDF membrane (Invitrogen) was blocked with 5% BSA, imaged using a Kodak Gel Logic 1500 Imager (Rochester, New York, NY, USA) and image processing (i.e., exposure, contrast) was applied to every pixel of the image using Adobe Photoshop. For protein identiﬁcation, protein bands were excised from polyacrylamide gel, di- gested with trypsin and sequenced by liquid chromatographic tandem mass spectrometry (LC-MS/MS). Protein identiﬁcation was performed by staff at The Ohio State University, Mass Spectrometry & Proteomics Facility. Proteins with a Mascot score of 50 or higher and Crystals 2021, 11, 874 4 of 13 a minimum of two unique peptides from one protein with a -b or -y ion sequence tag of ﬁve residues or better were accepted. a minimum of two unique peptides from one protein with a -b or -y ion sequence tag of ﬁve residues or better were accepted. p TEM grid preparation and immunogold labeling experiments of M. magneticum AMB- 1 cells and puriﬁed magnetosomes were done following the procedure described in [33,34]. Cells were placed on a TEM grid and incubated ﬁrst with anti-Mms13 (1:2000), and second with goat anti-rabbit IgG antibody conjugated with 10 nm gold (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:100 in 0.5% BSA in Tris-HCl. Puriﬁed magnetosomes (1:1250 diluted) were incubated with anti-Mms13 diluted 1:4000 and goat anti-rabbit IgG antibody (1:100) conjugated with 10 nm colloidal gold (Sigma-Aldrich, St. Louis, MO, USA). j g g ( g ) Fluorescence CLSM of whole, intact M. magneticum AMB-1 cells was performed as described by Oestreicher et al., 2016 [33,34]. Cells were harvested by centrifuging at 10,000× g for 10 min at 4 ◦C and ﬁxed in 4% paraformaldehyde for 5 min, placed on a glass slide and washed with PBS (phosphate buffered saline, pH 7.4). Cells were incubated and labeled with primary antibody (Anti-Mms13) at 1:400 dilution for 1 h at room temperature. 2. Materials and Methods Cells were washed 3X with PBS and labeled with secondary antibody, goat anti-rabbit IgG antibody conjugated to DyLight 488 (1:100; Thermo Fisher Scientiﬁc, Waltham, MA, USA). Cells were washed with PBS and analyzed using an Olympus FV 1000 CLSM (Olympus Corporation). 3. Results Cloning the PCR product into the pET160/GW/D-TOPO vector yielded a heterol- ogous expressed protein (rMms13), which preferentially accumulated in E. coli cells as inclusion bodies and had to be solubilized and puriﬁed using urea. The urea was removed and rMms13 renatured by extensive dialysis. The maximal yield in total protein, moni- tored by SDS-PAGE, was achieved using BL21 CodonPlus E. coli cells, under the following induction conditions: 1 mM IPTG, at 37 ◦C for 5.5 h. The rMms13 protein was puriﬁed and renatured by extensive dialysis in buffer contain- ing progressively less urea until the dialysis buffer contained no urea. The molecular mass of rMms13 was found to be approximately 18 kDa by SDS-PAGE (Figure 1) corresponding to the expected size of the Mms13 protein (~14.5 kDa) plus an additional ~4 kDa resulting from the pET160 vector. The ~18 kDa Coomassie-stained protein band (Figure 1, lane 3) was identiﬁed using LC-MS/MS. The polypeptide was found to correspond (p < 0.05) to the Mms13 protein from M. magneticum AMB-1. p g Figure 1. SDS-PAGE gel of puriﬁed recombinant Mms13 (rMms13) stained with Coomassie R-250. The lanes correspond to 10 µg of protein from non-induced Escherichia coli cells (lane 1), 10 µg of protein from induced E. coli cells (lane 2), and 5 µg of rMms13 protein puriﬁed using IMAC (lane 3). The black arrow shows the protein band that was excised from the gel, digested with trypsin, sequenced using LC-MS/MS to conﬁrm its identity as rMms13. The positions and molecular masses (in kilodaltons) of protein standards are indicated at the left (lane M). Figure 1. SDS-PAGE gel of puriﬁed recombinant Mms13 (rMms13) stained with Coomassie R-250. The lanes correspond to 10 µg of protein from non-induced Escherichia coli cells (lane 1), 10 µg of protein from induced E. coli cells (lane 2), and 5 µg of rMms13 protein puriﬁed using IMAC (lane 3). The black arrow shows the protein band that was excised from the gel, digested with trypsin, sequenced using LC-MS/MS to conﬁrm its identity as rMms13. The positions and molecular masses (in kilodaltons) of protein standards are indicated at the left (lane M). Crystals 2021, 11, 874 5 of 13 Western blot analysis was performed to determine whether Mms13 was present in the soluble fraction, cytoplasmic membrane fraction, or magnetosome membrane fraction of M. magneticum AMB-1. 3. Results A band corresponding to the expected molecular mass of Mms13 (~14.5 kDa) was only present in the magnetosome membrane fraction (Figure 2b, lane 3; Figure S1). No band was observed in the soluble fraction (Figure 2b, lane 1). No band was observed in the cytoplasmic membrane fraction (Figure 2b, lane 2). Figure 2. (a) Coomassie R-250 stained protein fraction of M. magneticum AMB-1 as resolved by SDS-PAGE. (b) Western blot of proteins solubilized from different fractions of M. magneticum AMB-1, resolved by SDS-PAGE and incubated with anti-Mms13 antibody. Lanes correspond to 10 µg soluble proteins (lane 1), 10 µg of cytoplasmic membrane proteins (lane 2), and 10 µg of magnetosome membrane proteins (lane 3). For comparison, molecular weight markers (lane M) were also resolved by SDS-PAGE. The positions and molecular masses (in kilodaltons) of protein standards are indicated using numbers down the left-side of the gel. The black arrow shows protein band that was excised, digested with trypsin and identiﬁed by LC-MS/MS peptide sequencing. (c) Mms13 amino acid sequence and locations of the four peptides that were identiﬁed by LC-MS/MS (numbered 1–4, red-colored, bold font that is highlighted and underlined). Mms13 was not detected by LC-MS/MS peptide sequencing in lanes 1 or 2. Figure 2. (a) Coomassie R-250 stained protein fraction of M. magneticum AMB-1 as resolved by SDS-PAGE. (b) Western blot of proteins solubilized from different fractions of M. magneticum AMB-1, resolved by SDS-PAGE and incubated with anti-Mms13 antibody. Lanes correspond to 10 µg soluble proteins (lane 1), 10 µg of cytoplasmic membrane proteins (lane 2), and 10 µg of magnetosome membrane proteins (lane 3). For comparison, molecular weight markers (lane M) were also resolved by SDS-PAGE. The positions and molecular masses (in kilodaltons) of protein standards are indicated using numbers down the left-side of the gel. The black arrow shows protein band that was excised, digested with trypsin and identiﬁed by LC-MS/MS peptide sequencing. (c) Mms13 amino acid sequence and locations of the four peptides that were identiﬁed by LC-MS/MS (numbered 1–4, red-colored, bold font that is highlighted and underlined). Mms13 was not detected by LC-MS/MS peptide sequencing in lanes 1 or 2. Figure 2. (a) Coomassie R-250 stained protein fraction of M. magneticum AMB-1 as resolved by SDS-PAGE. (b) Western blot of proteins solubilized from different fractions of M. magneticum AMB-1, resolved by SDS-PAGE and incubated with anti-Mms13 antibody. 3. Results Lanes correspond to 10 µg soluble proteins (lane 1), 10 µg of cytoplasmic membrane proteins (lane 2), and 10 µg of magnetosome membrane proteins (lane 3). For comparison, molecular weight markers (lane M) were also resolved by SDS-PAGE. The positions and molecular masses (in kilodaltons) of protein standards are indicated using numbers down the left-side of the gel. The black arrow shows protein band that was excised, digested with trypsin and identiﬁed by LC-MS/MS peptide sequencing. (c) Mms13 amino acid sequence and locations of the four peptides that were identiﬁed by LC-MS/MS (numbered 1–4, red-colored, bold font that is highlighted and underlined). Mms13 was not detected by LC-MS/MS peptide sequencing in lanes 1 or 2. This ~14 kDa band (Figure 2b, lane 3, black arrow) was excised from a polyacrylamide gel, digested with trypsin, and its amino acid sequence determined by LC-MS/MS. Four peptides were isolated and their amino acid sequences are shown in Figure 2c. LC-MS/MS analysis identiﬁed the ~14 kDa protein band contained within the magnetosome membrane fraction as the 145 amino acid magnetic particle protein Mms13 from Magnetospirillum magneticum AMB-1 (Figure 2c). Gel slices from lanes 1 and 2 (Figure 2), corresponding to ~14 kDa were excised and subjected to in-gel trypsin digestion followed by LC-MS/MS. Peptides corresponding to Mms13 were not detected by LC-MS/MS for the soluble fraction (Figure 2, lane 1) or for the cytoplasmic membrane fraction (Figure 2, lane 2). TEM analysis showed that the M. magneticum AMB-1 cells displayed Mms13 labeling along the magnetosome chain (Figure 3a–c). The nanogold particles were either detected within the matrix surrounding the magnetosomes or directly touching the Fe3O4 crystal (Figure 3d–f). It was also observed that some labeling appeared to occur distal to the Fe3O4 particles but typically nanogold particles were found to be in-line with the magnetosome chain (Figure 3a–c). In many instances, we observed that individual magnetosome particles were coated with numerous nanogold particles (Figure 3d–f). No nanogold labeling of the magnetosome chain was observed in either negative control when substituting 0.5% Crystals 2021, 11, 874 6 of 13 BSA for primary antibody (Figure 3g) or when 5% pre-immune serum was used in place anti-Mms13 (Figure 3h). Figure 3. Ultrathin sections of M. magneticum AMB-1. (a–c) A single cell showing magnetosomes labeled with Mms13 antibody and then labeled with goat anti-rabbit antibody conjugated with 10 nm colloidal gold. 3. Results (b) Three individual magnetosome particles showing multiple bound nanogold particles. (d–f) Magnetosome chain from M. magneticum AMB-1 cells showing nanogold particles bound directly to magnetosome crystal or observed within magnetosome membrane. (g) Negative control for immunolabeling showing an ultrathin section of M. magneticum AMB-1 treated exactly as in (a), but with 0.5% BSA substituted in place of the primary antibody. (h) Negative control treated exactly as in (a) but with 5% pre-immune serum substituted in place of the primary antibody. Dotted gray line (shown in a–c,g,h) is outline of bacterium. Scale bars provided for each image. Magnetosomes are large (i.e., ~70 nm) black particles arranged in a linear chain. Several individual magnetosomes are indicated with dotted black arrow and labeled Fe3O4. Colloidal gold particles are very small (i.e., ~10 nm) black spheres. Several individual nanogold particles are indicated with solid black arrow and labeled Au. BSA for primary antibody (Figure 3g) or when 5% pre-immune serum was used in place of anti-Mms13 (Figure 3h). BSA for primary antibody (Figure 3g) or when 5% pre-immune serum was used in place of anti-Mms13 (Figure 3h). Figure 3. Ultrathin sections of M. magneticum AMB-1. (a–c) A single cell showing magnetosomes labeled with Mms13 antibody and then labeled with goat anti-rabbit antibody conjugated with 10 nm colloidal gold. (b) Three individual magnetosome particles showing multiple bound nanogold particles. (d–f) Magnetosome chain from M. magneticum AMB-1 cells showing nanogold particles bound directly to magnetosome crystal or observed within magnetosome membrane. (g) Negative control for immunolabeling showing an ultrathin section of M. magneticum AMB-1 treated exactly as in (a), but with 0.5% BSA substituted in place of the primary antibody. (h) Negative control treated exactly as in (a) but with 5% pre-immune serum substituted in place of the primary antibody. Dotted gray line (shown in a–c,g,h) is outline of bacterium. Scale bars provided for each image. Magnetosomes are large (i.e., ~70 nm) black particles arranged in a linear chain. Several individual magnetosomes are indicated with dotted black arrow and labeled Fe3O4. Colloidal gold particles are very small (i.e., ~10 nm) black spheres. Several individual nanogold particles are indicated with solid black arrow and labeled Au. Immunoﬂuorescence of Dylight 488 tagged Anti-Mms13 allowed for the detection and localization of Mms13 within intact M. magneticum AMB-1 cells by CLSM coupled with Nomarski interference contrast technique. Mms13-labeling was observed only along the magnetosome chain of M. 3. Results magneticum AMB-1, occurring in the center and along the long axis of each cell (Figure 4d). No ﬂuorescence labeling occurred distal to the long axis of each cell (Figure 4). Negative controls were conducted on M. magneticum AMB-1 cells using buffer that lacked primary antibody (i.e., Anti-Mms13) or lacked secondary antibody (i.e., goat anti-rabbit IgG antibody conjugated to DyLight 488). No ﬂuorescence was observed in these two control experiments. 7 of 13 Crystals 2021, 11, 874 ure 4. Fluorescent labeled Magnetospirillum magneticum AMB-1 cells using Anti-Mms13 (1:400) as the primary antibody d goat anti-rabbit Dylight 488 (1:100) as the secondary antibody. (a) Nomarski image of M. magneticum AMB-1 cells The same view ﬁeld as (a) showing only ﬂuorescent image. (c) Merged Nomarski and ﬂuorescent images showing the tline of M. magneticum AMB-1 cells with green ﬂuorescent tag along the major axis of the cells co-localized with the gnetosome chain. (d) Enlargement of view ﬁeld shown in previous three panels. White arrow in each panel identiﬁes the me cell. Scale bars provided in each panel. Figure 4. Fluorescent labeled Magnetospirillum magneticum AMB-1 cells using Anti-Mms13 (1:400) as the primary antibody and goat anti-rabbit Dylight 488 (1:100) as the secondary antibody. (a) Nomarski image of M. magneticum AMB-1 cells. (b) The same view ﬁeld as (a) showing only ﬂuorescent image. (c) Merged Nomarski and ﬂuorescent images showing the outline of M. magneticum AMB-1 cells with green ﬂuorescent tag along the major axis of the cells co-localized with the magnetosome chain. (d) Enlargement of view ﬁeld shown in previous three panels. White arrow in each panel identiﬁes the same cell. Scale bars provided in each panel. 4. Discussion As shown in Table 1, previous reports have employed different methods to help deﬁne the localization of Mms13 and its homologs, MamC and Mam12, in four MTB species belonging to the class Alphaproteobacteria. In M. gryphiswaldense and M. magneticum AMB-1, the localization of MamC and Mms13, was examined using an inducible expression plasmid to overexpress recombinant GFP-MamC fusion protein in MTB and examine cells and puriﬁed magnetosomes using ﬂuorescence microscopy and spectroscopy [28,29]. Similar studies have not yet been employed on Mam12 in M. magnetotacticum MS-1 or on MamC in M. marinus MC-1. In other reports, immunogold labeling TEM of whole cells and puriﬁed magnetosomes was done with M. magnetotacticum MS-1 and M. marinus MC-1 [18,30]. Immunogold labeling TEM has not yet been conducted for MamC in M. gryphiswaldense or Mms13 in M. magneticum AMB-1. 8 of 13 Crystals 2021, 11, 874 Table 1. Studies conducted to examine subcellular localization of homologous proteins MamC, Mam12 and Mms13 in MTB belonging to Alphaproteobacteria [12,13,15,16,18,28–32,35–40]. Organism Gene Expression Protein Immunogold TEM Fluorescence Microscopy, Fluorescence Spectroscopy, or Enzyme Activity Biochemical and Proteomic Analysis of Puriﬁed MM Proteins Bacterial Cells Puriﬁed MM Bacterial Cells Puriﬁed MM Western Blot SDS-PAGE, 2D Gel, AA Sequence Magnetospirillum gryphiswaldense Plasmid Expression Recombinant MamC-GFP No No [28,35,37,39] [28] [28,38] [28] Genome Expression Native MamC No No No No No [12,13,35,40] Magnetospirillum magneticum AMB-1 Plasmid Expression Recombinant Mms13-GFP, Mms13-Luciferase, or Mms13-TSHR No [31] [29,32] [29,31,36] [29,31] [31] Genome Expression Native Mms13 ‡ ‡ ‡ ‡ ‡ ‡ [15,16] Magnetospirillum magnetotacticum MS-1 Plasmid Expression Recombinant Mam12-GFP No No No No No No Genome Expression Native Mam12 [18] [18] No No [18] [18] Magnetococcus marinus MC-1 Plasmid Expression Recombinant MamC-GFP No No No No No No Genome Expression Native MamC [30] [30] No No [30] [30] [Number] citation for journal article where experiment was published. The word “No” indicates that no studies have been published for this method of localization. ‡ Experiments that were completed and reported here in our study. AA, Amino Acid. GFP, Green Fluorescent Protein. MTB, Magnetotactic Bacteria. MM, Magnetosome Membrane. TEM, Transmission Electron Microscopy. SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, Two-Dimensional Gel Electrophoresis. TSHR, thyroid-stimulating hormone receptor. conducted to examine subcellular localization of homologous proteins MamC, Mam12 and Mms13 in MTB haproteobacteria [12,13,15,16,18,28–32,35–40]. [Number] citation for journal article where experiment was published. The word “No” indicates that no studies have been published for this method of localization. 4. Discussion Tanaka et al., 2006 conducted similar proteomic analysis of MM fractions isolated from M. magneticum AMB-1 [16]. They identiﬁed a total of 78 proteins associated with the MM fraction, including Mms13 [16]. p g Yoshino and Matsunaga, 2006 [31], Yoshino et al., 2010 [29], and Yoshino et al., 2021 [36] used a tetracycline-controlled inducible expression plasmid for M. magneticum AMB-1, using Mms13 as an anchor protein, to overexpress heterologous proteins, including green ﬂuorescent protein (GFP, molecular mass ~27 kDa), on the surface of magnetic particles. The plasmid containing mms13-GFP was overexpressed in M. magneticum AMB-1 and magnetic particles were extracted and collected from lysed M. magneticum AMB-1 cells using a neodymium-boron (Nd-B) magnet [29,31]. Yoshino et al., 2010 [29] used ﬂuorescence spectroscopy to demonstrate that magnetosomes extracted from M. magneticum AMB-1 were coated with the recombinant Mms13-GFP fusion protein [29]. M. magneticum AMB-1 cells transformed with a tetracycline-inducible Mms13-GFP plasmid were also observed by ﬂuorescence microscopy in the presence of anhydrotetracycline hydrochloride [29]. Yoshino and Matsunaga, 2006 [31] and Yoshino et al., 2010 [29] expressed two other recombinant Mms-13 fusion proteins (i.e., Mms13-Luciferase [31] and Mms13-FLAG-CD81 [29]) in M. magneticum AMB-1. They extracted the proteins from puriﬁed magnetic particles using 1% SDS and determined that both Mms13-Luciferase and Mms13-FLAG-CD81 fusion proteins were found attached to the magnetic particles using Western blotting and anti- luciferase antibody or anti-FLAG antibody [29,31]. Quinlan et al., 2011 [32] also developed an inducible plasmid expression system that used IPTG (isopropylthio-β-galactoside) to overexpress recombinant Mms13-GFP in M. magneticum AMB-1. When the mms13-GFP was overexpressed in M. magneticum AMB-1 and visualized by ﬂuorescence microscopy, green ﬂuorescence was observed along the length of the cell [32]. g g g The previous studies [15,16,29,31,32,36] on Mms13 expression and localization in M. magneticum AMB-1 (Table 1) focused on two types of experiments: (1) the use of ﬂuores- cence microscopy and spectroscopy or enzyme activity to examine plasmid-overexpressed recombinant fusion proteins (i.e., Mms13-GFP, Mms13-FLAG-CD81, Mms13-Luciferase, Mms13-TSHR) and (2) cellular fractionation, puriﬁcation of MM proteins, followed by amino acid sequencing or enzyme assays. While important, these experiments have limita- tions. For example, previous studies have demonstrated that fusion proteins can perturb a native protein’s activity, expression, function, and localization [41–44]. In Yoshino et al., 2010 [29], Yoshino and Matsunaga, 2006 [31], Quinlan et al., 2011 [32] and Yoshino et al., 2021 [36] an inducible plasmid expression system was used in M. 4. Discussion ‡ Experiments that were completed and reported here in our study. AA, Amino Acid. GFP, Green Fluorescent Protein. MTB, Magnetotactic Bacteria. MM, Magnetosome Membrane. TEM, Transmission Electron Microscopy. SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, Two-Dimensional Gel Electrophoresis. TSHR, thyroid-stimulating hormone receptor. In addition to microscopy, biochemical and proteomic analysis of the magnetosome membrane has been conducted on MamC, Mam12 and Mms13 in all four magnetotac- tic Alphaproteobacteria (Table 1) [12,13,15,16,18,28–32,35–40]. MamC is a highly conserved protein and one of the most abundant magnetosome-associated proteins [14,30]. When extracted from the cell, MamC, Mam12 and Mms13 have been found to associate with the MM fraction [12,13,15,16,18]. In-vivo genetic studies demonstrated that MamC regulates the size of magnetosome crystals in M. gryphiswaldense [14]. In-vitro biomineralization experiments using recombinant MamC demonstrated that MamC plays a role in controlling the size and shape of magnetite crystals in Magnetococcus marinus strain MC-1 [21,24,26]. p g y g By establishing the location of MamC, Mam12, and Mms13 within a bacterium and relative to the magnetosome chain, we gain signiﬁcant insight into if and how these pro- teins function to control the mineralization of Fe3O4 nanocrystals within the magnetosome. Consequently, this information is important for the in-vitro synthesis of Fe3O4 nanocrystals in the presence of puriﬁed proteins [15]. Moreover, establishing the subcellular localization of these three homologous proteins in members of the magnetotactic Alphaproteobacteria group allows for a better understanding of protein function and helps to identify shared biomineralization pathways among MTB (Table 1). Using immunogold stained ultrathin sections of bacterial cells and puriﬁed magnetosomes, native Mam12 was found to be local- ized to the magnetosome membrane of M. magnetotacticum [18]. Recombinant MamC-GFP was detected within the magnetosome chain of M. gryphiswaldense using immunoblotting and ﬂuorescence microscopy [28]. In addition, ﬁnally, immunogold labeling combined with TEM, showed that MamC was localized exclusively to the magnetosome chain of M. marinus strain MC-1 [30]. Previous studies, as summarized in Table 1, provide compelling, but not conclusive evidence, for the localization of Mms13 in M. magneticum AMB-1. Arakaki et al., 2003 conducted experiments with M. magneticum AMB-1, where they extracted proteins tightly bound to magnetic particles using 1% SDS (sodium dodecyl sulfate), separated them by two-dimensional gel electrophoresis and sequenced the stained proteins by Edman degradation [15]. They identiﬁed four proteins that were tightly bound to the puriﬁed Crystals 2021, 11, 874 9 of 13 magnetic particles including Mms13 [15]. 4. Discussion magneticum AMB-1 to overexpress comparatively large non-native proteins (i.e., GFP is ~27 kDa; CD81 is ~25 kDa; Luciferase is ~61kDa; TSHR, thyroid-stimulating hormone receptor is ~87 kDa) fused to the much small native Mms13 protein (i.e., Mms13 is ~12 kDa). The considerably larger size of the non-native protein (e.g., GFP is twice as large as Mms13, Luciferase is ﬁve-times larger than Mms13, TSHR is seven-times larger than Mms13) has steric consequences for protein folding, function and targeting that could alter expression and/or localization of the native protein [41–44]. In addition, the resolution limit of conventional ﬂuorescence microscopy is approximately 250 nm due to its dependance on the wavelength of the excitation light and microscope optics. This presents a challenge when imaging magnetosome particles, which are approximately 2–6 times smaller than the resolution offered by ﬂuorescence microscopy. The spatial resolution of the M. magneticum AMB-1 ﬂuorescence images pro- vided in Yoshino et al., 2010 [29] and Quinlan et al., 2011 [32] was not sufﬁcient to resolve magnetosome particles contained within the bacteria. TEM, on the other hand, which was conducted as part of our study on M. magneticum AMB-1, is ideally suited for obtaining the nm resolution required to image individual magnetosomes and Mms13 molecules within a bacterium. In the present study, we used Western blot analysis, LC-MS/MS protein sequencing, immunoﬂuorescence microscopy, and immunogold labeling TEM to establish the subcellu- lar location of native Mms13 in M. magneticum AMB-1. Western blot analysis showed that a polypeptide with a molecular mass similar to Mms13 (~14.5 kDa) was present in the MM Crystals 2021, 11, 874 10 of 13 10 of 13 fraction of M. magneticum AMB-1 (Figure 2b, lane 3). This ~14.5 kDa polypeptide was not detected in either the soluble fraction or cytoplasmic membrane fraction (Figure 2b, lanes 1 and 2). To determine the identity of this protein, LC-MS/MS peptide sequence analysis was performed on the protein band observed in the MM fraction (Figure 2b, lane 3, black arrow). LC-MS/MS analysis matched four peptides to Mms13 (Figure 2c). Peptide 1 was from the N-terminal end of Mms13 and peptides 2–4 were from the C-terminal end of Mms13 (Figure 2c). These results show that the mature Mms13 protein in M. magneticum AMB-1 contains at the very least amino acid residues 33–145 (Figure 2c). Thus, the mature Mms13 protein in M. 4. Discussion No ﬂuorescent labeling was observed outside the bacteria or distal to the mag- netosome chain (Figure 4). Thus, our ﬂuorescence measurements were consistent with TEM analysis that showed native Mms13 molecules localized to the magnetosome chain of M. magneticum AMB-1. These results were also consistent with our biochemical analyses that used LC-MS/MS and Western blotting to identify native Mms13 molecules within the MM fraction M. magneticum AMB-1. Fluorescence microscopy was used to conﬁrm the TEM analysis, which showed Mms13 localized to the MM and magnetite crystal surface. CLSM revealed ﬂuorescently labeled Mms13 molecules along the major axis of the bacterium (Figure 4). Green ﬂuores- cence DyLight 488 molecules were observed co-localized with the magnetosome chain (Figure 4). No ﬂuorescent labeling was observed outside the bacteria or distal to the mag- netosome chain (Figure 4). Thus, our ﬂuorescence measurements were consistent with TEM analysis that showed native Mms13 molecules localized to the magnetosome chain of M. magneticum AMB-1. These results were also consistent with our biochemical analyses that used LC-MS/MS and Western blotting to identify native Mms13 molecules within the MM fraction M. magneticum AMB-1. 4. Discussion magneticum AMB-1 contains both its hydrophobic, N-terminal putative mag- netosome membrane domain and its hydrophilic, C-terminal putative catalytic domain. These results are consistent with Arakaki et al., 2003 [15] and Tanaka et al., 2006 [16], which showed that Mms13 could be extracted from puriﬁed magnetosomes. Western blot analysis and LC-MS/MS protein identiﬁcation agreed with TEM analysis of M. magneticum AMB-1 (Figure 3). Immunogold electron microscopy showed Mms13 labeling along a bacterium’s magnetosome chain (Figure 3a–c). Nanogold particles were ob- served to be directly touching the Fe3O4 particles, within the magnetosome membrane sur- rounding the Fe3O4 particles or very close (<10 nm) to the magnetosome chain (Figure 3a–c). In many instances, multiple nanogold particles were found to be attached to a single mag- netosome crystal (Figure 3d–f). For example, in Figure 3f, we see a binding ratio of 3:1 (gold particle to magnetosome particle), with all nanogold particles touching or <10 nm from a magnetosome. These observations indicated that there was a strong afﬁnity of the Anti-Mms13-nanogold-label for the magnetosome chain. Control experiments were also performed using 5% pre-immune serum and no pri- mary antibody to conﬁrm the speciﬁcity of Anti-Mms13 antibody for Mms13 (Figure 3h). The pre-immune serum does not contain antibodies for Mms13 and thus nanogold par- ticles should not be detected in TEM images using pre-immune serum. Occasionally a small number of nanogold particles were observed outside the bacterial cell; however, no nanogold labeling occurred within or near the magnetosome chain (Figure 3g,h). This conﬁrmed that the results were not due to background labeling by the secondary antibody. Control experiments were also performed using 5% pre-immune serum and no pri- mary antibody to conﬁrm the speciﬁcity of Anti-Mms13 antibody for Mms13 (Figure 3h). The pre-immune serum does not contain antibodies for Mms13 and thus nanogold par- ticles should not be detected in TEM images using pre-immune serum. Occasionally a small number of nanogold particles were observed outside the bacterial cell; however, no nanogold labeling occurred within or near the magnetosome chain (Figure 3g,h). This conﬁrmed that the results were not due to background labeling by the secondary antibody. Fluorescence microscopy was used to conﬁrm the TEM analysis, which showed Mms13 localized to the MM and magnetite crystal surface. CLSM revealed ﬂuorescently labeled Mms13 molecules along the major axis of the bacterium (Figure 4). Green ﬂuores- cence DyLight 488 molecules were observed co-localized with the magnetosome chain (Figure 4). 5. Conclusions Deﬁning the localization of magnetosome proteins in MTB is a necessary step in determining their function(s) in magnetosome biomineralization. By understanding the mineralization process of Fe3O4 crystals in all MTB we can better understand the evolution of magnetotaxis and learn to reproduce the synthesis of Fe3O4 nanocrystals in-vitro. Such information could be explored in attempts to obtain tailored Fe3O4 magnets with speciﬁc morphology and size. By combining the results presented here with previous [12–16,18,21, 24,26–32,35–40], we can deﬁnitively conclude that homologous proteins MamC, Mam12, and Mms13 are localized to the magnetosome chain of all magnetotactic Alphaproteobacteria where the proteins likely function in the biomineralization of Fe3O4 nanocrystals. 11 of 13 11 of 13 Crystals 2021, 11, 874 Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/cryst11080874/s1, Figure S1. This Western blot is the same Western blot shown in Figure 2b, except that this image has not been modiﬁed (e.g., contrast, exposure, brightness) using image processing software (i.e., Adobe Photoshop). Lanes correspond to 10 µg soluble proteins (lane 1), 10 µg of cytoplasmic membrane proteins (lane 2), and 10 µg of magnetosome membrane proteins (lane 3). For comparison, molecular weight markers (lane M) are provided with the molecular masses (in kilodaltons) of protein standards indicated using numbers down the left-side of the gel. The black arrow shows protein band corresponding to Mms13. Author Contributions: Conceptualization, B.H.L., S.K.L., D.A.B. and C.J.-L.; methodology, Z.O., C.V.-T. and L.P.-G.; investigation, Z.O., C.V.-T., E.M., N.N.C.-I. and L.P.-G.; resources, B.H.L., S.K.L., D.A.B. and C.J.-L.; writing—original draft preparation, B.H.L., S.K.L., D.A.B., E.M. and C.J.-L.; writing—review and editing, B.H.L., N.N.C.-I. and S.K.L.; supervision, B.H.L., S.K.L., D.A.B. and C.J.-L.; project administration, B.H.L., S.K.L., D.A.B. and C.J.-L.; funding acquisition, B.H.L., S.K.L., D.A.B. and C.J.-L. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, B.H.L., S.K.L., D.A.B. and C.J.-L.; methodology, Z.O., C.V.-T. and L.P.-G.; investigation, Z.O., C.V.-T., E.M., N.N.C.-I. and L.P.-G.; resources, B.H.L., S.K.L., D.A.B. and C.J.-L.; writing—original draft preparation, B.H.L., S.K.L., D.A.B., E.M. and C.J.-L.; writing—review and editing, B.H.L., N.N.C.-I. and S.K.L.; supervision, B.H.L., S.K.L., D.A.B. and C.J.-L.; project administration, B.H.L., S.K.L., D.A.B. and C.J.-L.; funding acquisition, B.H.L., S.K.L., D.A.B. and C.J.-L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by U.S. 5. Conclusions National Science Foundation, grant number EAR-2038207 and EAR-1423939; and Ministerio de Economía y Competitividad, SPAIN and Fondo Europeo de Desarrollo Regional, FEDER grant numbers CGL2010-18274 and CGL2013-46612. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data sharing not applicable. Data Availability Statement: Data sharing not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Gorby, Y.A.; Beveridge, T.J.; Blakemore, R. Characterization of the bacterial magnetosome membrane. J. Bacteriol. 1988, 170, 834–841. [CrossRef] [ ] 2. Bazylinski, D.A.; Frankel, R.B. Magnetosome formation in prokaryotes. Nat. Rev. Microbiol. 2004, 2, 217–230. [CrossRef] [PubMed] 3. Lower, B.H.; Bazylinski, D.A. The bacterial magnetosome: A unique prokaryotic organelle. J. Mol. Microbiol. Biotechnol. 2013, 23, 63–80. [CrossRef] [PubMed] 2. Bazylinski, D.A.; Frankel, R.B. Magnetosome formation in prokaryotes. Nat. Rev. Microbiol. 2004, 2, 217–230. [CrossRef] [PubMed] 3. Lower, B.H.; Bazylinski, D.A. The bacterial magnetosome: A unique prokaryotic organelle. J. Mol. Microbiol. Biotechnol. 2013, 23, 63–80. [CrossRef] [PubMed] 4. Uebe, R.; Schüler, D. Magnetosome biogenesis in magnetotactic bacteria. Nat. Rev. Microbiol. 2016, 14, 621–637. [CrossRef] 63 80 [C oss e ] [ ubMed] 4. Uebe, R.; Schüler, D. Magnetosome biogenesis in magnetotactic bacteria. Nat. Rev. Microbiol. 2016, 14, 621–637. [CrossRef] 5 K ili A Li Z N D K J G I M ll b i i i i d b h i lik ] [ ] r, D. Magnetosome biogenesis in magnetotactic bacteria. Nat. Rev. Microbiol. 2016, 14, 621–637. [CrossRef] 4. Uebe, R.; Schüler, D. Magnetosome biogenesis in magnetotactic bacteria. Nat. Rev. Microbiol. 2016, 14, 5. Komeili, A.; Li, Z.; Newman, D.K.; Jensen, G.I. Magnetosomes are cell membrane invaginations organized by the actin-like protein MamK. Science 2006, 311, 242–245. [CrossRef] [PubMed] 6. Schüler, D. Genetics and cell biology of magnetosome formation in magnetotactic bacteria. FEMS Microbiol. Rev. 2008, 32, 654–672. [CrossRef] lar mechanisms of compartmentalization and biomineralization in magnetotactic bacteria. FEMS Microbiol 55. [CrossRef] [PubMed] 7. Komeili, A. Molecular mechanisms of compartmentalization and biomineralization in magnetotac Rev. 2012, 36, 232–255. [CrossRef] [PubMed] 8. Schüler, D.; Baeuerlein, E. Dynamics of iron uptake and Fe3O4 biomineralization during aerobic and microaerobic growth of Magnetospirillum gryphiswaldense. J. Bacteriol. 1998, 180, 159–162. [CrossRef] [PubMed] g p g yp 9. Schüler, D. Molecular analysis of a subcellular compartment: The magnetosome membrane in Magnetospirillum gryphiswaldense. Arch. Microbiol. 2004, 181, 1–7. [CrossRef] 10. Matsunaga, T.; Sakaguchi, T. Molecular mechanism of magnet formation in bacteria. J. Biosci. Bioeng. 2 10. Matsunaga, T.; Sakaguchi, T. Molecular mechanism of magnet formation in bacteria. J. Biosci. Bioeng. 2000, 90, 1–13. [CrossRef] 11. Arakaki, A.; Tanaka, M.; Matsunaga, T. Molecular mechanism of magnetic crystal formation in magnetotactic bacteria. In Biologic Magnetic Materials and Applications; Matsunaga, T., Tanaka, T., Kisailus, D., Eds.; Springer: Singapore, 2018; pp. 23–51. 11. Arakaki, A.; Tanaka, M.; Matsunaga, T. References Nudelman, H.; Perez-Gonzalez, T.; Kolushiva, S.; Widdrat, M.; Reichel, V.; Peigneux, A.; Davidov, G.; Bitton, R.; Faivre, D.; Ji L C t l Th i t f th h li l t t f M C d i d tit i t ti tid f it f ti 6. Jaba e a, .; Casa es t e a, S.; e á de V vas, .; e g eu , .; uaga o tes, . .; J e e ope , C. ote co se vat o method affects MamC-mediated biomineralization of magnetic nanoparticles. Cryst. Growth Des. 2019, 19, 1064–1071. [CrossRef] 27. Nudelman, H.; Perez-Gonzalez, T.; Kolushiva, S.; Widdrat, M.; Reichel, V.; Peigneux, A.; Davidov, G.; Bitton, R.; Faivre, D.; Jimenez-Lopez, C.; et al. The importance of the helical structure of a MamC-derived magnetite-interacting peptide for its function in magnetite formation. Acta Crystallogr. D Struct. Biol. 2018, 74, 10–20. [CrossRef] 27. Nudelman, H.; Perez-Gonzalez, T.; Kolushiva, S.; Widdrat, M.; Reichel, V.; Peigneux, A.; Davidov, G.; Bitton, R.; Faivre, D.; Jimenez-Lopez, C.; et al. The importance of the helical structure of a MamC-derived magnetite-interacting peptide for its function in magnetite formation. Acta Crystallogr. D Struct. Biol. 2018, 74, 10–20. [CrossRef] 28. Lang, C.; Schüler, D. Expression of green ﬂuorescent protein fused to magnetosome proteins in microaerophilic magnetotactic bacteria. Appl. Environ. Microbiol. 2008, 74, 4944–4953. [CrossRef] [PubMed] 29. Yoshino, T.; Shimojo, A.; Maeda, T.; Matsunaga, T. Inducible expression of transmembrane proteins on bacterial magnetic particles in Magnetospirillum magneticum AMB-1. Appl. Environ. Microbiol. 2010, 76, 1152–1157. [CrossRef] 30. Valverde-Tercedor, C.; Abadía-Molina, F.; Martinez-Bueno, M.; Pineda-Molina, E.; Chen, L.; Oestreicher, Z.; Lower, B.H.; Lower, S.K.; Bazylinski, D.A.; Jimenez-Lopez, C. Subcellular localization of the magnetosome protein MamC in the marine magnetotactic bacterium Magnetococcus marinus strain MC-1 using immunoelectron microscopy. Arch. Microbiol. 2014, 196, 481–488. [CrossRef] 31. Yoshino, T.; Matsunaga, T. Efﬁcient and stable display of functional proteins on bacterial magnetic particles using Mms13 as a novel anchor molecule. Appl. Environ. Microbiol. 2006, 72, 465–471. [CrossRef] 32. Quinlan, A.; Murat, D.; Vali, H.; Komeili, A. The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization. Mol. Microbiol. 2011, 80, 1075–1087. [CrossRef] 33. Oestreicher, Z.; Mumper, E.; Gassman, C.; Bazylinski, D.A.; Lower, S.K.; Lower, B.H. Spatial localization of Mms6 during biomineralization of Fe3O4 nanocrystals in Magnetospirillum magneticum AMB-1. J. Mater. Res. 2016, 31, 527–535. [CrossRef] 34. References Molecular mechanism of magnetic crystal formation in magnetotactic bacteria. In Biologic Magnetic Materials and Applications; Matsunaga, T., Tanaka, T., Kisailus, D., Eds.; Springer: Singapore, 2018; pp. 23–51. g pp g p g g p pp 12. Grünberg, K.; Wawer, C.; Tebo, B.M.; Schüler, D. A large cluster encoding several magnetosome proteins is conserved in different species of magnetotactic bacteria. Appl. Environ. Microbiol. 2001, 67, 4573–4582. [CrossRef] [PubMed] 12. Grünberg, K.; Wawer, C.; Tebo, B.M.; Schüler, D. A large cluster encoding several magnetosome proteins is conserved in different i f i b i A l E i Mi bi l 1 67 4573 4582 [C R f] [P bM d] 12. Grünberg, K.; Wawer, C.; Tebo, B.M.; Schüler, D. A large cluster encoding several magnetosome protein species of magnetotactic bacteria. Appl. Environ. Microbiol. 2001, 67, 4573–4582. [CrossRef] [PubMed] g g g g p es of magnetotactic bacteria. Appl. Environ. Microbiol. 2001, 67, 4573–4582. [CrossRef] [PubMed] 13. Grünberg, K.; Müller, E.-C.; Otto, A.; Reszka, R.; Linder, D.; Kube, M.; Reinhardt, R.; Schüler, D. Biochemical and proteomic analysis of the magnetosome membrane in Magnetospirillum gryphiswaldense. Appl. Environ. Microbiol. 2004, 70, 1040–1050. [CrossRef] 14. Scheffel, A.; Gärdes, A.; Grünberg, K.; Wanner, G.; Schüler, D. The major magnetosome proteins MamGFDC are not essential for magnetite biomineralization in Magnetospirillum gryphiswaldense, but regulate the size of magnetosome crystals. J. Bacteriol. 2008, 190, 377–386. [CrossRef] 15. Arakaki, A.; Webb, J.; Matsunaga, T. A novel protein tightly bound to bacterial magnetic particles in Ma strain AMB-1. J. Biol. Chem. 2003, 278, 8745–8750. [CrossRef] 16. Tanaka, M.; Okamura, Y.; Arakaki, A.; Tanaka, T.; Takeyama, H.; Matsunaga, T. Origin of magnetosome membrane: Proteomic analysis of magnetosome membrane and comparison with cytoplasmic membrane. Proteomics 2006, 6, 5234–5247. [CrossRef] [PubMed] 12 of 13 Crystals 2021, 11, 874 12 of 13 17. Kashyap, S.; Woehl, T.J.; Liu, X.; Mallapragada, S.K.; Prozorov, T.; Frankel, R.B. Nucleation of iron oxide nanoparticles mediated by Mms6 protein in situ. ACS Nano 2014, 8, 9097–9106. [CrossRef] [PubMed] y p 18. Taoka, A.; Asada, R.; Sasaki, H.; Anzawa, K.; Wu, L.F.; Fukumori, Y. Spatial localizations of Mam22 and Mam12 in the magnetosomes of Magnetospirillum magnetotacticum. J. Bacteriol. 2006, 188, 3805–3812. [CrossRef] [PubMed] g p g J pan, M.; Giacomelli, G.; Raschdorf, O.; Borg, S.; Plitzko, J.M.; Bramkamp, M.; Schuler, D.; Müller, F.-D. Ma 19. Toro-Nahuelpan, M.; Giacomelli, G.; Raschdorf, O.; Borg, S.; Plitzko, J.M.; Bramkamp, M.; Schuler, D.; Müller, F.-D. References MamY is a membrane-bound protein that aligns magnetosomes and the motility axis of helical magnetotactic bacteria. Nat. Microb. 2019, 4, 1978–1989. [CrossRef] [PubMed] 20. Ren, E.; Lei, Z.; Wang, J.; Zhang, Y.; Liu, G. Magnetosome modiﬁcation: From bio-nano engineering toward nanomedicine. Adv. Therap. 2018, 1, 1800080. [CrossRef] 21. Valverde-Tercedor, C.; Montalbán-Lopez, M.; Perez-Gonzalez, T.; Sanchez-Quesada, M.S.; Prozorov, T.; Pineda-Molina, E.; Fernandez-Vivas, M.A.; Rodriguez-Navarro, A.B.; Trubitsyn, D.; Bazylinski, D.A.; et al. Size control of in vitro synthesized magnetite crystals by the MamC protein of Magnetococcus marinus strain MC-1. Appl. Microbiol. Biotechnol. 2015, 99, 5109–5121. [CrossRef] [ ] 22. Matsunaga, T.; Okamura, Y.; Fukuda, Y.; Wahyudi, A.T.; Murase, Y.; Takeyama, H. Complete genome sequence of the facultative anaerobic magnetotactic bacterium Magnetospirillum sp. strain AMB-1. DNA Res. 2005, 12, 157–166. [CrossRef] [PubMed] 23. Pósfai, M.; Lefèvre, C.T.; Trubitsyn, D.; Bazylinski, D.A.; Frankel, R.B. Phylogenetic signiﬁcance of composition and crystal morphology of magnetosome minerals Front Microbiol 2013 4 1 15 [CrossRef] [PubMed] 22. Matsunaga, T.; Okamura, Y.; Fukuda, Y.; Wahyudi, A.T.; Murase, Y.; Takeyama, H. Complete genome sequence of the facultative anaerobic magnetotactic bacterium Magnetospirillum sp. strain AMB-1. DNA Res. 2005, 12, 157–166. [CrossRef] [PubMed] 23 Pósfai M Lefèvre C T Trubitsyn D Bazylinski D A Frankel R B Phylogenetic signiﬁcance of composition and crystal 23. Pósfai, M.; Lefèvre, C.T.; Trubitsyn, D.; Bazylinski, D.A.; Frankel, R.B. Phylogenetic signiﬁcance of morphology of magnetosome minerals. Front. Microbiol. 2013, 4, 1–15. [CrossRef] [PubMed] y y y g g p hology of magnetosome minerals. Front. Microbiol. 2013, 4, 1–15. [CrossRef] [PubMed] 24. Nudelman, H.; Valverde-Tercedor, C.; Kolusheva, S.; Perez-Gonzalez, T.; Widdrat, M.; Grimberg, N.; Levi, H.; Nelkenbaum, O.; Davidov, G.; Faivre, D.; et al. Structure-function studies of the magnetite-biomineralizing magnetosome-associated protein MamC. J. Struct. Biol. 2016, 194, 244–252. [CrossRef] 25. Rawlings, A.E.; Somner, L.A.; Fitzpatrick-Milton, M.; Roebuck, T.P.; Gwyn, C.; Liravi, P.; Seville, V.; Neal, T.J.; Mykhaylyk, O.O.; Baldwin, S.A.; et al. Artiﬁcial coiled coil biomineralization protein for the synthesis of magnetic nanoparticles. Nat. Commun. 2019, 10, 2873. [CrossRef] [PubMed] 26. Jabalera, Y.; Casares Atienza, S.; Fernández-Vivas, A.; Peigneux, A.; Azuaga Fortes, A.I.; Jimenez-Lopez, C. Protein conservation method affects MamC-mediated biomineralization of magnetic nanoparticles. Cryst. Growth Des. 2019, 19, 1064–1071. [CrossRef] 26. Jabalera, Y.; Casares Atienza, S.; Fernández-Vivas, A.; Peigneux, A.; Azuaga Fortes, A.I.; Jimenez-Lopez, C. Protein conservation method affects MamC-mediated biomineralization of magnetic nanoparticles. Cryst. Growth Des. 2019, 19, 1064–1071. [CrossRef] 27. 40. Lohße, A.; Uilrich, S.; Katzmann, E.; Borg, S.; Wanner, G.; Richter, M.; Boigt, B.; Schweder, T.; Schüler, D. Functional Analysis of the Magnetosome Island in Magnetospirillum gryphiswaldense: The mamAB Operon Is Sufﬁcient for Magnetite Biomineralization. PLoS ONE 2011, 6, e25561. [CrossRef] g p , , [ ] [ ] 42. Marshall, A.P.; Shirley, J.D.; Carlson, E.E. Enzyme-targeted ﬂuorescent small-molecule probes for bacterial imaging. Curr. Opin. Chem. Biol. 2020, 57, 155–165. [CrossRef] [PubMed] 43. Snapp, E. Design and use of ﬂuorescent fusion proteins in cell biology. Curr. Protoc. Cell Biol. 2005, 27, 21–24. [CrossRef] [PubMed] 44 Snapp E L Fluorescent proteins: A cell biologist’s user guide Trends Cell Biol 2009 19 649–655 [CrossRef] [PubMed] [ ] 41. Heo, M.; Nord, A.L.; Chamousset, D.; van Rijn, E.; Beaumont, H.J.E.; Pedaci, F. Impact of ﬂuoresce bacterial ﬂagellar motor. Sci. Rep. 2017, 7, 12583. [CrossRef] [PubMed] 43. Snapp, E. Design and use of ﬂuorescent fusion proteins in cell biology. Curr. Protoc. Cell Biol. 2005, 27, 21–24. [CrossRef] [PubMed] 44. Snapp, E.L. Fluorescent proteins: A cell biologist’s user guide. Trends Cell Biol. 2009, 19, 649–655. [CrossRef] [PubMed] , , [ ] [ ] 43. Snapp, E. Design and use of ﬂuorescent fusion proteins in cell biology. Curr. Protoc. Cell Biol. 2005, 27, 21–24. [CrossRef] [PubMed] eo, ; No , ; C o sse , ; j , ; e o , J ; e c , p c o o esce p o e s o o e bacterial ﬂagellar motor. Sci. Rep. 2017, 7, 12583. [CrossRef] [PubMed] 42. Marshall, A.P.; Shirley, J.D.; Carlson, E.E. Enzyme-targeted ﬂuorescent small-molecule probes for bacterial imaging. Curr. Opin. Chem. Biol. 2020, 57, 155–165. [CrossRef] [PubMed] 44. Snapp, E.L. Fluorescent proteins: A cell biologist’s user guide. Trends Cell Biol. 2009, 19, 649–655. [C [ ] [ ] 43. Snapp, E. Design and use of ﬂuorescent fusion proteins in cell biology. Curr. Protoc. Cell Biol. 2005, 27 44 Snapp E L Fluorescent proteins: A cell biologist’s user guide Trends Cell Biol 2009 19 649 655 [C [ ] rd, A.L.; Chamousset, D.; van Rijn, E.; Beaumont, H.J.E.; Pedaci, F. Impact of ﬂuorescent protein fusion ellar motor. Sci. Rep. 2017, 7, 12583. [CrossRef] [PubMed] References Oestreicher, Z.; Valverde-Tercedor, C.; Chen, L.; Jimenez-Lopez, C.; Bazylinski, D.A.; Casillas-Ituarte, N.N.; Lower, S.K.; Lower, B.H. Magnetosmoes and magnetite crystals produced by magnetotactic bacteria as resolved by atomic force microscopy and transmission electron microscopy. Micron 2012, 43, 1331–1335. [CrossRef] [PubMed] 35. Raschdorf, O.; Bonn, F.; Zeytuni, N.; Zarivach, R.; Becher, D.; Schüler, D. A quantitative assessment of the membrane-integral sub-proteome of a bacterial magnetic organelle. J. Proteom. 2018, 172, 89–99. [CrossRef] [PubMed] 36. Yoshino, T.; Tayama, S.; Maeda, Y.; Fujimoto, K.; Ota, S.; Wake, S.; Kisailus, D.; Tanaka, T. Magnetosome membrane engineering to improve G protein-coupled receptor activities in the magnetosome display system. Metabol. Eng. 2021, 67, 125–132. [CrossRef] [PubMed] 37. Mickoleit, F.; Lanzloth, C.; Schüler, D. A Versatile Toolkit for Controllable and Highly Selective Multifunctionalization of Bacterial Magnetic Nanoparticles. Small 2020, 16, 1906922. [CrossRef] 38. Ohuchi, S.; Schüler, D. In Vivo Display of a Multisubunit Enzyme Complex on Biogenic Magnetic Nanoparticles. Appl. Environ. Microbiol. 2009, 75, 7734–7738. [CrossRef] [PubMed] 39. Uebe, R.; Junge, K.; Henn, V.; Poxieitner, G.; Katzmann, E.; Plitzko, J.M.; Zarivach, R.; Kasama, T.; Wanner, G.; Pósfai, M.; et al. The cation diffusion facilitator proteins MamB and MamM of Magnetospirillum gryphiswaldense have distinct and complex functions, and are involved in magnetite biomineralization and magnetosome membrane assembly. Mol. Microbiol. 2011, 82, 818–835. [CrossRef] Crystals 2021, 11, 874 13 of 13 13 of 13 , , [ ] [ ] 43. Snapp, E. Design and use of ﬂuorescent fusion proteins in cell biology. Curr. Protoc. Cell Biol. 2005, 27, 21–24. [CrossRef] [PubMed] 44. Snapp, E.L. Fluorescent proteins: A cell biologist’s user guide. Trends Cell Biol. 2009, 19, 649–655. [CrossRef] [PubMed]
https://openalex.org/W2900028291	https://www.repository.cam.ac.uk/bitstream/1810/290595/1/Eustachian%20tube%20dysfunction%3a%20A%20diagnostic%20accuracy%20study%20and%20proposed%20diagnostic%20pathway.pdf	English	null	Eustachian tube dysfunction: A diagnostic accuracy study and proposed diagnostic pathway	PloS one	2,018	cc-by	15,371	Eustachian tube dysfunction: A diagnostic accuracy study and proposed diagnostic pathway Matthew E. SmithID1, Yemisi TakwoingiID2, Jon Deeks2, Cuneyt AlperID3, Manohar L. Bance1, Mahmood F. Bhutta4, Neil Donnelly1, Dennis Poe5, James R. Tysome1* 1 Cambridge Ear Institute, University of Cambridge, Cambridge, United Kingdom, 2 Institute of Applied Health Research, University of Birmingham, Edgbaston, Birmingham, United Kingdom, 3 Children’s Hospital of Pittsburgh, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States of America, 4 Brighton & Sussex University Hospitals NHS Trust, Brighton, United Kingdom, 5 Boston Children’s Hospital, Boston, Massachusetts, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * jrt20@cam.ac.uk Methods Index tests included two PROMs and 14 tests of ET opening (nine for obstructive, five for patulous ETD). In the absence of an accepted reference standard two methods were adopted to establish index test accuracy: expert panel diagnosis and latent class analysis. Index test results were assessed with Pearson correlation and principle component analy- sis, and test accuracy was determined. Logistic regression models assessed the predictive value of grouped test results. Data Availability Statement: All test data are available from the University of Cambridge data repository: https://doi.org/10.17863/CAM.26158. Funding: M.S. received funding from the Cambridge Hearing Trust. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Background and aims Citation: Smith ME, Takwoingi Y, Deeks J, Alper C, Bance ML, Bhutta MF, et al. (2018) Eustachian tube dysfunction: A diagnostic accuracy study and proposed diagnostic pathway. PLoS ONE 13(11): e0206946. https://doi.org/10.1371/journal. pone.0206946 Eustachian tube dysfunction (ETD) is a commonly diagnosed disorder of Eustachian tube opening and closure, which may be associated with severe symptoms and middle ear dis- ease. Currently the diagnosis of obstructive and patulous forms of ETD is primarily based on non-specific symptoms or examination findings, rather than measurement of the underlying function of the Eustachian tube. This has proved problematic when selecting patients for treatment, and when designing trial inclusion criteria and outcomes. This study aims to determine the correlation and diagnostic value of various tests of ET opening and patient reported outcome measures (PROMs), in order to generate a recommended diagnostic pathway for ETD. Editor: Kourosh Parham, University of Connecticut Health Center, UNITED STATES Received: August 14, 2018 Editor: Kourosh Parham, University of Connecticut Health Center, UNITED STATES Received: August 14, 2018 Accepted: October 21, 2018 Published: November 8, 2018 Copyright: © 2018 Smith et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Introduction The Eustachian tube (ET) is a dynamic tubular structure which normally only opens to facili- tate gaseous pressure regulation of the middle ear, but otherwise remains closed to prevent the transmission of sound, pressure, nasopharyngeal secretions and pathogens from the nose to the ear. Eustachian tube dysfunction (ETD) is a frequently applied diagnosis, used when abnormal ET function is believed to underlie any of a wide range of symptoms or middle ear (ME) abnormalities. Two distinct forms of the disorder are recognised, representing different ends of the spectrum of ET function: 1) obstructive ETD (OETD), in which tubal opening or patency is reduced, and 2) patulous ETD (PETD), in which the ET is too open[1]. Even within these two subtypes, the condition remains heterogeneous in terms of aetiology and presenta- tion, and it has been suggested that some patients with OETD have either predominantly active dysfunction (failure of muscle-controlled opening), or passive dysfunction (failure of pressure-related opening)[2]. Most ETD is diagnosed on the basis of the reported clinical history, examination of the ear and nasopharynx, and routine tests such as tympanometry. More objective tests of ET function have been proposed, with renewed efforts in validating and developing tests in recent years due to the need for outcome measures for emerging medical and surgical interventions. There is no reference (gold standard) method to diagnose OETD or PETD, and the clinical diagnosis of an experienced clinician has been the default reference standard for many years. In order to introduce objective outcome measures to trials of ETD interventions, tympanometric classifi- cation, Valsalva or breathing-related ME pressure change, patient reported outcome measures (PROMs) and many other tests have been proposed as potential diagnostic markers of ETD. Clinicians and researchers wishing to advance the diagnosis of ETD beyond clinical opin- ion currently have difficulty determining which tests should be used. Efforts have been made to describe the accuracy of tests that measure ET opening, and to develop PROMs[3], but diag- nostic accuracy research is complex in conditions such as ETD where the disorder is poorly defined and lacks a reference method for diagnosis. Test accuracy is measured by assessing the performance of the evaluated (index) test against the reference standard, within the same population of subjects with the suspected target disor- der[4]. To conduct test accuracy research in conditions such as ETD, a new reference standard can be synthesised. Conclusions ETD should be diagnosed on the basis of clinical assessment and tests of ET opening, as PROMs have no diagnostic value. Currently diagnostic uncertainty exists for some patients who appear to have intermittent ETD clinically, but have negative index test results. suggested tympanometry, sonotubometry and tubomanometry have the best diagnostic performance for obstructive ETD, and these are included in a proposed diagnostic pathway. suggested tympanometry, sonotubometry and tubomanometry have the best diagnostic performance for obstructive ETD, and these are included in a proposed diagnostic pathway. suggested tympanometry, sonotubometry and tubomanometry have the best diagnostic performance for obstructive ETD, and these are included in a proposed diagnostic pathway. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Results The expert panel diagnosis and PROMs results correlated with each other, but not with ET function measured by tests of ET opening. All index tests were found to be feasible in clinic, and acceptable to patients. PROMs had very poor specificity, and no diagnostic value. Com- bining the results of tests of ET function appeared beneficial. The latent class model Competing interests: The authors have declared that no competing interests exist. 1 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Diagnosis of Eustachian tube dysfunction Introduction This may be done through: 1) a predefined rule (composite reference stan- dard); 2) consensus among experts (panel diagnosis); 3) a statistical model based on the col- lected test results (latent class analysis)[5, 6]. A composite reference standard for ETD is not possible due to the lack of evidence relating existing tests to the condition. An expert panel can be used as a reference standard by establishing the presence or absence of a disorder in individual patients. The expert panel method is suited to conditions such as 2 / 28 Diagnosis of Eustachian tube dysfunction ETD where multiple sources of information such as patient characteristics, symptoms, exami- nation findings and other test results must be interpreted in a judicious way to reach a diagno- sis, particularly in the absence of an unequivocal definition of the disorder[5]. Latent class analysis uses a statistical model to combine different test results from each patient to construct a reference standard. The technique aims to determine the latent variable (diagnosis) on the basis of manifest variables (the test results). Latent class models perform well if there are large numbers of index tests, as they increase the possible test combinations and available degrees of freedom. However, these models require test results to be indepen- dent, and so not all possible ETD tests can be modelled together. Both diagnosis and test accuracy are heavily influenced by the situation and cohort in which they are assessed. Most tests for ETD have not been investigated in a representative clin- ical setting where patients have an uncertain diagnosis. In contrast, most tests have been assessed in a case-control design study, with cases selected to represent a single, often more severe form of ETD are matched with healthy controls. These two factors tend to overestimate test accuracy, as in practice the task is to differentiate subjects who are disease positive from subjects who have many of the same symptoms, but do not have the disorder[7, 8]. In addition, most tests have been assessed for diagnostic accuracy in isolation, missing the potential improvement that may be gained by combining test results, either informally as part of the diagnostic process, or mathematically in a risk calculator. Finally, the status of clinical assessment as the current default reference standard diagnosis for ETD has not been challenged. Instead, tests that do not support the clinical reference diag- nosis have been deemed inaccurate. Introduction The converse is of course possible. Without the assessment of multiple and varied tests in a single cohort, it has not been possible to establish the correla- tion between different diagnostic methods, and the accuracy of clinical diagnosis. This prospective diagnostic accuracy study aims to explore the relationship between results from a wide range of different methods for diagnosing ETD, and to establish their accuracy compared to a reference standard, generated using both latent class analysis and panel diagno- sis. We discuss the clinical implementation of these tests, and a recommended diagnostic pathway. Methods and materials Ethical approval was obtained from the UK Research Ethics Service. Written consent was obtained from all participants. Participants A single cohort of non-consecutive patients with symptoms or examination findings sugges- tive of ETD were selectively recruited from ENT outpatient clinics at Addenbrooke’s Hospital, Cambridge, between November 2016 and May 2017, according to the following criteria: Inclusion criteria. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Assessments Data were recorded on paper forms and in Microsoft Excel. Inclusion criteria. 18 years old One of the following findings (formalising current clinical practice): • at least 2 symptoms of ETD • at least 2 symptoms of ETD • AND/OR tympanic membrane retraction • AND/OR negative pressure tympanogram (-100daPa) list in S1 Table, and based upon a literature review. Exclusion criteria. 3 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Diagnosis of Eustachian tube dysfunction • Otoscopic findings that may prevent use of the full range of index tests (otitis media with effusion, tympanic membrane perforation$, cholesteatoma, discharging or infected ear) • Otoscopic findings that may prevent use of the full range of index tests (otitis media with effusion, tympanic membrane perforation$, cholesteatoma, discharging or infected ear) • Cardiac pacemaker (incompatible with the sonotubometry speaker) • Patients with cleft palate or craniofacial abnormality (due to concerns regarding their ability to perform the full range of tests, or to obtain interpretable results) • Inability to consent or poor understanding of written English Assessments Recruited patients initially underwent an assessment equivalent to the current standard of care in clinics without a specialist interest in ETD. Patients then underwent assessment with vari- ous tests for ETD (index tests). Standard-of-care assessment (4 components). Standard-of-care assessment (4 components). 1. A focused, standardised clinical history of the patient’s presenting complaint, screening for symptoms and medical history associated with ETD. 1. A focused, standardised clinical history of the patient’s presenting complaint, screening for symptoms and medical history associated with ETD. 2. Otoscopic examination: recording retraction, atelectasis, and other relevant features of the tympanic membrane. 2. Otoscopic examination: recording retraction, atelectasis, and other relevant features of the tympanic membrane. 3. Pure tone audiometry with air and bone conduction thresholds. 4. Middle ear pressure and compliance measured via tympanometry. Index tests. Two forms of ET function test were assessed as index tests: objective and semi-objective measures of ET opening, and symptom-based PROMS. Tests of ET opening for OETD (9 tests) and PETD (5 tests) were performed according to optimal methods previously identified[9, 10] or standard procedure (PETD tests). Details of the tests of ET opening, their various methods and the repeats performed can be found in Table 1. Two PROMs were com- pleted by patients: the CETDA[11] and ETDQ-7[12]. Composite measures of ET function were also assessed: the 5- and 7-item Eustachian Tube Scores (ETS and ETS-7) were calculated for each case, combining patient-reported symptoms with tubomanometry results in a simple numerical scale, with lower scores indicative of OETD[13]. All participants underwent all OETD and PETD tests in a single session, with a single asses- sor (MS). Although previous work suggested fatigue and test interactions were minimal[10], the index tests were applied in a partly-randomised manner; tympanometry and the nine step test were always applied first to prevent residual middle ear pressure influencing results, while the order of the other tests was randomised using an online random sequence generator[14]. PROMs were also completed in a randomised order. To assess feasibility in clinical practice, and the burden on the patient, for each test we also recorded: time taken to complete the test from start of patient instruction to end of data recording (min/sec); patient reported difficulty in completing the test (Likert scale 1–10, 1 = none, 10 = almost impossible); and discomfort during the test (Likert scale 1–10, 1 = none, 10 = almost unbearable). Diagnosis of Eustachian tube dysfunction Table 1. Tests of ET opening assessed, including manoeuvres used and number of repeats. ET Function Test Method (no. of measurements) Diagnostic outcome measured (unit) Other variables recorded (unit) ET function tested Obstructive ETD Tympanometry Patient at rest (1) ME pressure (daPa) Tympanic admittance (ml) - Active opening (ME pressure also influenced by mucosal gas exchange) Nine-step test 3 dry swallows, +400daPa in EAC (1) 3 dry swallows, - 400daPa in EAC (1) ME pressure change after ±400daPa (daPa) ME pressure change after equilibration (daPa) - Active opening Patient-reported assessment Valsalva (3) Dry Toynbee (3) Patient sensation (yes/no) - Passive opening (Valsalva) Passive and active opening (Toynbee) Observation of the tympanic membrane Valsalva (3) Dry Toynbee (3) Tympanic membrane movement (yes/no) Strength and speed of TM movement (weak/strong, fast/ slow) Passive opening (Valsalva) Passive and active opening (Toynbee) Tubo-tympano-aerodynamic- graphy (TTAG) Valsalva (3) Dry Toynbee (3) Peak positive and negative change in EAC pressure (daPa) NP pressure (daPa) Passive opening (Valsalva) Passive and active opening (Toynbee) Continuous impedance Valsalva (3) Dry Toynbee (3) Peak positive and negative change in tympanic impedance (ml) NP pressure (daPa) Passive opening (Valsalva) Passive and active opening (Toynbee) Sonotubometry Dry swallow (5) Peak EAC sound pressure level (dB) Duration of EAC recording (ms) - Active opening Tubomanometry Wet swallow, 300daPa (1) Wet swallow, 400daPa (1) Wet swallow, 500daPa (1) Peak EAC pressure change (daPa) R value (No unit) - Passive and active opening Tuboimpedance Wet swallow, 300daPa (1) Wet swallow, 400daPa (1) Wet swallow, 500daPa (1) Peak tympanic impedance change (ml) - Passive and active opening Patulous ETD Observation of the tympanic membrane 10 seconds heavy breathing (1) Breathing synchronous tympanic membrane movement (yes/no) - Passive closure TTAG 10 seconds heavy breathing (1) Breathing synchronous change in EAC pressure (daPa) - Passive closure Continuous impedance 10 seconds heavy breathing (1) Breathing synchronous change in tympanic impedance (ml) - Passive closure Sonotubometry 10 seconds heavy breathing (1) Breathing synchronous change in EAC sound pressure level (dB) - Passive closure Tubomanometry Wet swallow, 300daPa (1) Wet swallow, 400daPa (1) Wet swallow, 500daPa (1) R value (No unit) - Passive and active opening ME = middle ear, NP = nasopharyngeal, EAC = external auditory canal, TTAG = Tubo-tympano-aerodynamic-graphy. Expert panel diagnosis A six-person international expert panel was recruited from clinicians working and publishing in the field of ETD. The panel’s role was to provide a reference standard diagnosis of ETD, PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 4 / 28 https://doi.org/10.1371/journal.pone.0206946.t001 ME = middle ear, NP = nasopharyngeal, EAC = external auditory canal, TTAG = Tubo-tympano-aerodynamic-graphy. In addition for each test the following were recorded; time taken to complete the test (min/sec), patient reported difficulty to complete the test (Likert scale 1–10, 1 = none) and discomfort during the test (Likert scale1-10, 1 = none). Diagnosis of Eustachian tube dysfunction through the consensus opinion of experts. The six experts were divided into two groups, form- ing Panel A (MBa, MBh, ND) and Panel B (CA, DP, JT). To reduce assessor burden, study participants were randomly allocated into two groups for independent review by one of the two panels. To ensure that each panel had a similar case mix, allocation was stratified according to original clinic diagnosis (OETD / PETD / alternate diag- nosis). As part of the allocation process, a subgroup of 20 patients was allocated to both panels to assess inter-panel agreement. The random sampling was done by YT using PROC SUR- VEYSELECT in the SAS statistical package. Each patient’s history, examination and audiometry findings were presented to the panel on a separate case summary slide (Microsoft Powerpoint). Summary slides included informa- tion available in ENT outpatients (including the results of investigations (e.g. nasendoscopy or imaging), and the response to treatments initiated in the community (e.g. nasal steroid spray, or betahistine). Index test results were not presented to the panel to prevent incorporation bias falsely inflating test accuracy[8], with the exception of tympanometry, as this test is established and widely available in current ENT practice. Initially, each panel member assessed cases independently and without conferring. They were asked to diagnose ETD if they felt it was present, even if some symptoms, or the primary complaint, were thought to be due to another condition. Panel members were asked to con- sider three possible diagnoses: OETD, PETD, or an alternate diagnosis. Results from individual expert assessment were analysed, and where all three experts in a panel agreed on a diagnosis, this diagnosis was assigned. Disagreements were subsequently discussed at a panel teleconference (Fig 1) where panels A&B met separately. Summary data for each study participant were reviewed and panel members discussed the reasons for their individual diagnoses. The panels were directed to reach a consensus diagnosis if possible. Where consensus could not be reached, the diagnosis was made by a majority vote of 2–1. Rules were set in advance to assign a diagnosis in the event that there was disagreement between the panels A&B when diagnosing the subset of 20 shared cases: 1. Diagnoses made via individual diagnoses in agreement were assigned over those with initial disagreement 1. Diagnoses made via individual diagnoses in agreement were assigned over those with initial disagreement 2. Agreement between panels for the 20 shared cases was measured with Cohen’s Kappa. Agreement between panels for the 20 shared cases was measured with Cohen’s Kappa. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Diagnoses made at discussion via consensus were assigned over those made by a 2:1 vote 3. If rule 1 or 2 could not be applied, (i.e. there were conflicting diagnoses from panels A&B, made in an equivalent manner) the diagnosis was recorded as indeterminate. In addition for each test the following were recorded; time taken to complete the test (min/sec), patient reported difficulty to complete the test (Likert scale 1–10, 1 = none) and discomfort during the test (Likert scale1-10, 1 = none). https://doi org/10 1371/journal pone 0206946 t001 ME = middle ear, NP = nasopharyngeal, EAC = external auditory canal, TTAG = Tubo-tympano-aerodynamic-graphy. In addition for each test the following were recorded; time taken to complete the test (min/sec), patient reported difficulty to complete the test (Likert scale 1–10, 1 = none) and discomfort during the test (Likert scale1-10, 1 = none). PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 5 / 28 Statistical analysis and modelling Raw test data were initially analysed using descriptive statistics in accordance with diagnostic criteria described in the literature and previous work[9, 10]. Assessment of correlation and latent variables. Principle component analysis (PCA) was used to detect common latent variables within the test outcomes. PCA was performed using con- tinuous or ordinal variables from each of the tests and PROMs, and the panel diagnosis. To con- firm adequacy of sampling, the Kaiser-Meyer-Olkin (KMO) value was calculated (>0.6 adequate), and Bartlett’s Test of Sphericity performed. Components with an Eigenvalue <1 were disregarded. Pairwise correlations for the function tests, PROMs and panel diagnosis were per- formed by creating a correlation matrix (bivariate Pearson correlation). PCA and correlation were performed with SPSS 25 (IBM, Armonk, New York, USA). An explanatory description of PCA and latent class analysis (see below) is included in the Supporting Information (S1 File). PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 6 / 28 Diagnosis of Eustachian tube dysfunction Fig 1. Flow chart demonstrating the data collection process and generation of a reference standard, with subsequent analysis to assess test accuracy. https://doi org/10 1371/journal pone 0206946 g001 strating the data collection process and generation of a reference standard, with subsequent analysis to assess test accurac . Flow chart demonstrating the data collection process and generation of a reference standard, with subsequent analysis t Fig 1. Flow chart demonstrating the data collection process and generation of a reference standard, with subsequent analysis to assess test accuracy. https://doi.org/10.1371/journal.pone.0206946.g001 Test accuracy based on expert panel as reference standard. Estimation of the accuracy of individual tests: Using the panel diagnosis, 2x2 tables were generated for each index test. For tests measured on a continuous or ordinal scale, sensitivity and specificity were calculated at pre-specified and novel diagnostic thresholds (Table 2, column 3). As test repetitions were employed to improve repeatability of findings[10], novel thresholds using the repetitions were generated to allow interpretation. Receiver operating characteristic (ROC) curves were also plotted and the area under the curve (AUC) estimated. Thresholds at which sensitivity was maximised and the false positive rate was minimised were identified on ROC curves. Test per- formance was also summarised using Youden’s index (J, where J = sensitivity + specificity– 1) where 1 indicates a perfect test and 0 indicates no diagnostic value[15]. Accuracy was assessed for tests of both patulous and obstructive ETD. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Statistical analysis and modelling Development of a diagnostic model using multiple tests: The potential value of combining multiple tests was explored. Using penalised maximum likelihood estimation logistic regres- sion, multivariable models were developed for predicting the likelihood of OETD. The LASSO (least absolute shrinkage and selection operator) penalty term was used[16]. The strategy was to fit models to a fixed set of predefined tests, and the LASSO approach may shrink some PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 7 / 28 Diagnosis of Eustachian tube dysfunction Table 2. Summary of test results for ETD, with different interpretation methods. Diagnosis of Eustachian tube dysfunction Table 2. Test Parameter Interpretation for OETD Median % cases Expert Panel Diagnosis positive AUC (95% CI) Sensitivity (95% CI) Specificity (95% CI) Youden’s index J Obstructive ETD n = 57 OETD n = 57 non- OETD Tympanometry Patient at rest Raw data–ME pressure in daPa -11 - 0.66 (.56- .76) - - - <-50 daPa - 23.1 - 35.1 (22.9– 48.8) 91.2 (80.7– 97.0) 0.26 <-100 daPa - 15.4 - 26.3 (15.5– 39.6) 98.2 (90.6– 100.0) 0.25 Nine-step test +400daPa Raw data—daPa change -15 - 0.60 (.50- .71) - - - <5DaPa change - 25.0 - 31.6 (19.9– 45.2) 80.7 (68.0– 89.9) 0.12 <10DaPa change - 35.6 - 45.6 (32.3– 59.3) 73.7 (60.3– 84.4) 0.19 +ve equilibration Raw data—daPa change 6 - 0.62 (.52- .72) - - - <5DaPa change - 35.6 - 38.6 (25.9 52.4) 66.7 (52.9– 78.5) 0.05 <10DaPa change - 51.0 - 63.2 (49.3– 75.5) 61.4 (47.5– 74.0) 0.25 - 400daPa Raw data—daPa change 9 - 0.46 (.36- .55) - - - <5DaPa change - 47.1 - 59.6 (38.9– 66.0) 57.9 (44.0– 70.8) 0.11 <10DaPa change - 56.7 - 64.9 (51.1– 77.0) 49.1 (35.6– 62.7) 0.14 -ve equilibration Raw data—daPa change -3 - 0.58 (.48– 68) - - - <5DaPa change - 53.8 - 63.2 (49.3– 75.5) 50.9 (37.2– 64.3) 0.14 <10DaPa change - 66.3 - 75.4 (62.2– 85.8) 38.6 (25.9– 52.4) 0.14 ±400daPa Number results <5DaPa change /4 - - 0.60 (.49- .69) - - - together Number results <10DaPa change /4 - - 0.64 (.54- .74) - - - Any of 4 results <5DaPa change - 77.9 - 80.7 (68.0– 89.9) 38.6 (25.9– 52.4) 0.19 Any of 4 results <10DaPa change - 70.2 - 87.7 (76.3– 94.9) 29.8 (18.4– 43.4) 0.18 Patient-reported Valsalva Count no open out of 3 - - 0.61 (.51- .71) - - - 2 out of 3 +ve - 45.2 - 56.1 (42.3– 69.2) 61.4 (47.5– 74.0) 0.18 Dry Toynbee Count no open out of 3 - - 0.61 (.51- .71) - - - 2 out of 3 +ve - 66.3 - 77.2 (64.1– 87.2) 45.6 (32.3– 59.3) 0.23 Observed TM Valsalva Count no open out of 3 - - 0.68 (.58- .78) - - - 2 out of 3 +ve - 47.1 - 61.4 (42.3– 69.2) 61.4 (47.5– 74.0) 0.18 (Continued) PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 8 / 28 (Continued) Test Parameter Interpretation for OETD Median % cases Expert Panel Diagnosis positive AUC (95% CI) Sensitivity (95% CI) Specificity (95% CI) Youden’s index J Dry Toynbee Count no open out of 3 - - 0.67 (.58- .77) - - - 2 out of 3 +ve - 61.5 - 73.7 (60.3– 84.4) 54.4 (40.6– 67.6) 0.28 TTAG Valsalva Count no open out of 3 - - 0.59 (.49- .70) - - - 2 out of 3 +ve - 41.3 - 49.1 (35.6– 62.7) 70.2 (56.5– 81.5) 0.19 Dry Toynbee Count no open out of 3 - - 0.61 (.51- .71) - - - 2 out of 3 +ve - 52.9 - 59.6 (45.8– 72.4) 59.6 (45.8– 72.4) 0.19 Continuous impedance Valsalva Count no open out of 3 - - 0.61 (.51- .71) - - - 2 out of 3 +ve - 37.5 - 43.9 (30.7– 576) 71.9 (58.4– 83.0) 0.16 Dry Toynbee Count no open out of 3 - - 0.68 (.58- .77) - - - 2 out of 3 +ve - 52.9 - 66.7 (52.9– 785) 66.7 (52.9– 78.5) 0.33 Sonotubometry Dry swallow Raw data–mean dB 5 - 0.41 (.31- .51) - - <0dB count out of 5 - - 0.57 (.47- .68) - - - <5dB count out of 5 - - 0.58 (.47- .68) - - - <10dB count out of 5 - - 0.59 (.49- .70) - - - 3 out of 5 <0dB - 37.5 - 45.6 (32.3– 59.3) 70.2 (56.5– 81.5) 0.16 3 out of 5 <5dB - 47.1 - 56.1 (42.3– 69.2) 59.6 (45.8– 72.4) 0.16 3 out of 5 <10dB - 58.7 - 66.7 (52.9– 78.5) 47.4 (33.9– 61.0) 0.14 Tubomanometry 300daPa No open or R >1 - 56.7 - 68.4 (54.7– 80.0) 59.6 (45.8– 72.4) 0.28 400daPa No open or R >1 41.3 49.1 (35.6– 62.7) 70.2 (56.5– 81.5) 0.19 500daPa No open or R >1 39.4 47.4 (33.9– 61.0) 71.9 (58.4– 83.0) 0.19 All 3 pressures No open or R >1 out of 3 - - 0.65 (.55- .75) - - - Tuboimpedance 300daPa No open - 26.9 - 36.8 (24.4– 50.6) 84.2 (72.1– 92.5) 0.21 400daPa No open - 19.2 26.3 (15.5– 39.6) 86.0 (74.2– 93.7) 0.12 500daPa No open - 7.7 12.3 (5.0– 23.6) 96.5 (87.8– 99.5) 0.09 All 3 pressures No opens out of 3 - - 0.60 (.50- .70) - - - (Continued) PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 9 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Diagnosis of Eustachian tube dysfunction Table 2. (Continued) Test Parameter Interpretation for OETD Median % cases Expert Panel Diagnosis positive AUC (95% CI) Sensitivity (95% CI) Specificity (95% CI) Youden’s index J CETDA Total score Score 27 - 0.60 (.49- .69) - - - Score 17 - 81.8 - 87.7 (76.3– 94.9) 24.6 (14.1– 37.7) 0.12 ETDQ-7 Total score Score 25 - 0.59 (.48- .69) - - - Score 15 - 71.0 - 80.7 (68.0– 89.9) 24.6 (14.1– 37.7) 0.05 ETS Total score Score 4 - 0.60 (.50- .70) - - - Score 5 - 62.5 - 71.9 (58.4– 83.0) 43.9 (30.7– 57.6) 0.16 ETS7 Total score Score 6 0.67 (.57- .77) - - - Score 7 - 59.6 - 68.4 (54.7– 80.0) 47.4 (33.9– 61.0) 0.16 Patulous ETD n = 12 PETD n = 102 non- PETD Observed TM Heavy breathing +ve for change with breathing - 4.8 - 33.3 (9.9– 65.1) 99.0 (94.6– 100.0) 0.32 TTAG Heavy breathing +ve for change with breathing - 7.7 - 50.0 (21.0– 78.9) 98.1 (93.0– 99.8) 0.48 Impedance Heavy breathing +ve for change with breathing - 6.7 - 50.0 (21.0– 78.9) 99.0 (94.6– 100.0) 0.49 Sonotubometry Heavy breathing +ve for change with breathing or ET remains open after swallowing - 1.9 - 8.3 (0.2–38.4) 99.0 (94.6– 100.0) 0.07 Tubomanometry 300daPa R value < 0.2 - 5.8 - 33.3 (9.9– 65.1) 98.1 (93.0– 99.8) 0.31 400daPa R value < 0.2 - 4.8 - 33.3 (9.9– 65.1) 99.0 (94.6– 100.0) 0.32 500daPa R value < 0.2 - 5.8 - 25.0 (5.4– 57.1) 97.1 (91.6– 99.4) 0.22 Expert panel as the reference standard. CETDA = Cambridge Eustachian Tube Dysfunction Assessment, ETDQ-7 = Eustachian Tube Dysfunction Questionnaire 7,ETS (7) = Eustachian Tube Score (7), TTAG = tubo-tympanic aerodynamic graphy, TM = tympanic membrane htt //d i /10 1371/j l 0206946 t002 Expert panel as the reference standard. CETDA = Cambridge Eustachian Tube Dysfunction Assessment, ETDQ-7 = Eus (7) = Eustachian Tube Score (7), TTAG = tubo-tympanic aerodynamic graphy, TM = tympanic membrane Expert panel as the reference standard. CETDA = Cambridge Eustachian Tube Dysfunction Assessment, ETDQ-7 = Eustachian Tube Dysfunction Questionnaire 7,ETS (7) = Eustachian Tube Score (7), TTAG = tubo-tympanic aerodynamic graphy, TM = tympanic membrane Expert panel as the reference standard. CETDA = Cambridge Eustachian Tube Dysfunction Assessment, ETDQ-7 = Eustachian Tube Dysfunction Questionnaire 7,ETS (7) = Eustachian Tube Score (7), TTAG = tubo-tympanic aerodynamic graphy, TM = tympanic membrane coefficients to zero. Thus, the approach provided a form of variable selection in addition to correcting for optimism and preventing very extreme predictions[17]. Two models were designed containing tests grouped by current clinical application. In one model (Research model), both routine tests and function tests that are infrequently used were included. In a sec- ond, simpler model (Clinical model), only tests that are routinely available in clinical practice were included. Diagnostic thresholds were not applied to continuous variables. Tympanic pressure was considered by the clinical panel in making the final diagnosis of OETD, leading to potential for incorporation bias. Therefore, in sensitivity analyses this vari- able was excluded from some models, to explore whether the diagnostic value of the models was mainly driven by middle ear pressure. Assessment of diagnostic model performance and internal validation: Model performance was assessed in terms of discrimination and calibration. Discrimination is the ability of the 10 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Diagnosis of Eustachian tube dysfunction model to distinguish a patient with the endpoint OETD from a patient without OETD (with PETD or alternative diagnosis). The c-statistic for evaluating the discriminatory ability of a logistic regression model is equivalent to the ROC AUC. A c-statistic of 1.0 indicates perfect discrimination while a c-statistic of 0.5 indicates random discrimination[18]. For internal vali- dation of the model discrimination, bootstrapping with 200 samples was used to obtain opti- mism-adjusted estimates of model performance statistics[18–20]. Calibration, the agreement between observed endpoints and predictions[21] was also assessed, with methodology for this and internal validation in the Supporting Information (S1 File). Test accuracy based on latent class analysis. Latent class analysis (LCA) was used to combine multiple test results in order to estimate the proportion of patients with OETD, and the sensitivity and specificity of each of the index tests included in a 2-class latent class model. For LCA to be valid, the tests included in the analysis should be independent, measuring ETD in a distinct manner. Therefore, one test was selected for each aspect of ETD function (Table 1, column 5,), except where the test was considered sufficiently distinct (e.g. sonotubometry and impedance Toynbee for active opening). Results Data were obtained from 116 patients. Patient characteristics are outlined in Table 3. Six of the 116 patients were incorporated in analysis but had one missing test result due to: sonotubome- try contraindicated with pacemaker (n = 1); erratic impedance baseline (n = 2); and erratic sonotubometry baseline (n = 3). An additional five patients were recruited but were later excluded (one withdrew consent during testing, three were unable to perform tubomanome- try/tuboimpedance, and one could not complete the PROMs). One patient aspirated water during tubomanometry but quickly recovered, no other adverse events were recorded. When diagnosing ETD, there is no clear incentive to prioritise either sensitivity or specific- ity over the other. Therefore, for each test, the diagnostic threshold with the highest Youden’s index was selected for dichotomising ordinal and continuous test results, and LCA models were fitted. Four models were fitted: All-test model, with the panel diagnosis included as an index test alongside the tests of ET opening and PROMs; No-panel model, the all-test model without the panel diagnosis; No-PROM model, the All-test model without the PROM, and the Open-test model only including tests that measure ET opening. Each model containing a PROM included either the CETDA or ETDQ-7 (model variant A and B respectively). In addi- tional variations of the models, tympanic pressure was excluded, since results from this test were used by the panel in making the final diagnosis for each patient. LCA and regression analyses were performed using Stata 15 (StataCorp LP, College Station, Texas, USA). Assessment of fatigue during repeated testing. Patients were required to repeatedly per- form manoeuvres during assessment with TTAG, impedance and sonotubometry tests. To assess for evidence of fatigue and reducing ability, the nasopharyngeal pressures and results across the required 3–5 repetitions were compared using two-tailed AVOVA. Diagnosis of Eustachian tube dysfunction Table 3. Demographic and clinical characteristics of 116 participants. Characteristic No. (%) Mean ±SD Age, years 50 ±16.3 Male 50 (43) Test ear Contralateral ear Test ear Contralateral ear Right ear 59 (48) - Otoscopy normal 96 (83) 86 (76) Tympanogram type Type A 101 (87) 95 (82) Type B 0 15 (13) Type C 15 (13) 6 (5) Middle ear pressure, daPa -36.0 ±73.0 -46.9 ±50.2 PTA, dB 26.8 ±19.6 — PTA >20dB 61 (54) — Any symptoms present 113 (97) 69 (59) PTA = pure tone audiogram SD = standard deviation Table 3. Demographic and clinical characteristics of 116 participants. PTA = pure tone audiogram, SD = standard deviation the diagnoses agreed in 15 individuals (75%), with Cohen’s κ = 0.55 (95% CI 0.21–0.89), repre- senting a moderate strength of agreement[22]. the diagnoses agreed in 15 individuals (75%), with Cohen’s κ = 0.55 (95% CI 0.21–0.89), repre- senting a moderate strength of agreement[22]. Expert panel diagnosis Panel diagnoses were assigned as shown in Fig 2. Of the 116 patients, 57 (49.1%) had OETD, 12 (10.3%) had PETD and 45 (38.8%) had alternate diagnoses. For the remaining two (1.7%) patients, the panels could not agree and so the diagnosis for these patients was classified as indeterminate and they were excluded from further analysis. The proportion of cases assigned each primary diagnosis was similar between panels. In the 20 cases reviewed by both panels PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 11 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Patterns and correlations Principle component analysis suggested the presence of four significant latent components (Eigenvalue>1) underlying the results of the OETD tests of ET opening, PROMs and panel diagnosis. All tests of ET opening weighted positively on the first component (which Principle component analysis suggested the presence of four significant latent components (Eigenvalue>1) underlying the results of the OETD tests of ET opening, PROMs and panel diagnosis. All tests of ET opening weighted positively on the first component (which accounted for the greatest variance across all test results), while symptoms had a weak negative weighting on this component (Table 4). This pattern suggested that the first component repre- sented ET function in terms of tube opening, as expected with the dataset mainly formed from tests of ET opening. The tests loading strongly for component 2 were the two PROMs and the panel diagnosis, while the tests of ET opening loaded weakly or negatively for this component; this component therefore appeared to relate to symptoms. The similar loading for the PROMs and panel diagnosis is not surprising, as symptoms are likely to have been a strong driving fac- tor in the panel diagnosis. However, the loading patterns for components 1 and 2 suggest that symptoms and ET opening are distinct aspects of ETD which vary independently. For component 3, all tests measuring only passive ET opening had positive loading, while those measuring active and mixed opening all had negative loading. This suggests that passive and active ET dysfunction may be distinct, with some individuals expressing primarily passive or active dysfunction on tests of ET opening. Passive ET opening appeared more related to ETD symptoms than active opening on the basis of the loading for component 3. Loading for component 4 was overall weak with no clear pattern relating it to an attribute of ETD. PCA was not suitable to analyse PETD test results given the small PETD sample size. In the OETD correlation matrix incorporating OETD tests of ET opening, PROMs and the panel diagnosis (S2 Table), most tests of ET opening correlated with one another, with only 4 of 78 pairings demonstrating p0.05. All opening test pairs with differences in results that showed a p0.01 comprised tests that measured different aspects of ET function (e.g. Patterns and correlations active and passive opening), further suggesting that some patients may have predominantly active or PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 12 / 28 Diagnosis of Eustachian tube dysfunction Fig 2. Flow chart for the expert panel diagnostic process. Rules were set to assign a diagnosis in the event of disagreement between panels A&B: 1. Diagnoses made via individual agreement were assigned over those with initial disagreement; 2. Diagnoses made at discussion via consensus were assigned over those made by a 2:1 vote; 3. If rule 1 or 2 could not be applied, the diagnosis was recorded as indeterminate. https://doi.org/10.1371/journal.pone.0206946.g002 Fig 2. Flow chart for the expert panel diagnostic process. Rules were set to assign a diagnosis in the event of disagreement between panels A&B: 1. Diagnoses made via individual agreement were assigned over those with initial disagreement; 2. Diagnoses made at discussion via consensus were assigned over those made by a 2:1 vote; 3. If rule 1 or 2 could not be applied, the diagnosis was recorded as indeterminate. Fig 2. Flow chart for the expert panel diagnostic process. Rules were set to assign a diagnosis in the event of disagreement between panels A&B: 1. Diagnoses made via individual agreement were assigned over those with initial disagreement; 2. Diagnoses made at discussion via consensus were assigned over those made by a 2:1 vote; 3. If rule 1 or 2 could not be applied, the diagnosis was recorded as indeterminate. https://doi.org/10.1371/journal.pone.0206946.g002 https://doi.org/10.1371/journal.pone.0206946.g002 https://doi.org/10.1371/journal.pone.0206946.g002 passive ET dysfunction. The pairwise correlation between PROM scores and tests of ET open- ing was insignificant (p0.05) in almost all cases, demonstrating a limited relationship between a patient’s symptoms and ET opening, as seen in the PCA. Also as suggested by the PCA, in the matrix the panel diagnosis was found to be correlated with PROM scores and not significantly correlated with most tests of ET opening. The lack of correlation between the panel diagnosis and tympanometry suggested that panel knowledge of tympanometry results had not led to significant incorporation bias: an important finding when including the test in the statistical models. In contrast to OETD, the PETD correlation matrix (S3 Table) showed a different pattern, with the PETD panel diagnosis significantly correlated with the PETD tests of ET opening, and less strongly correlated with the PROMs. Again, the PROMs and tests of ET opening did not correlate. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Diagnosis of Eustachian tube dysfunction Table 4. Principle component analysis matrix. Aspect of OETD Test and parameter Component 1 2 3 4 Passive opening Observed Valsalva Count/3 0.79 -0.09 0.34 0.20 Reported Valsalva Count/3 0.65 -0.07 0.32 0.37 TTAG Valsalva Count/3 0.61 -0.31 0.60 0.01 Impedance Valsalva Count/3 0.64 -0.15 0.50 -0.23 Active opening Observed Toynbee Count/3 0.73 0.10 -0.15 0.32 Reported Toynbee Count/3 0.61 0.13 -0.32 0.53 TTAG Toynbee Count/3 0.66 -0.09 -0.23 0.01 Impedance Toynbee Count/3 0.77 0.12 -0.24 -0.24 Sonotubometry <5dB count/5 0.62 0.11 -0.32 -0.01 Both forms of opening Tubomanometry 300 & 400daPa R>1 or no open count/2 0.60 0.22 -0.11 -0.50 Tuboimpedance no-open count/3 0.61 0.17 -0.04 -0.52 Pressure equalisation Nine step ME change <10daPa count/4 0.50 0.23 -0.43 0.00 Tympanometry ME pressure daPa 0.44 -0.33 -0.04 0.27 Symptoms CETDA Total score -0.07 0.89 0.32 0.13 ETDQ-7 Total score -0.15 0.91 0.26 0.02 All Expert panel diagnosis OETD, PETD or alternate diagnosis 0.17 0.53 0.02 0.21 Table 4. Principle component analysis matrix. Patterns and correlations PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 13 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 https://doi.org/10.1371/journal.pone.0206946.t004 Test accuracy with panel diagnosis as reference standard Table 2 presents estimates of test accuracy from all obstructive and patulous ETD tests using expert panel diagnosis as the reference standard. Diagnostic performance measured by the AUC and Youden’s index was in many cases poor: no single test had sensitivity and specificity >65% or J>0.5. Some tests had high sensitivity or specificity, depending on the threshold applied. Tests for PETD performed better than those for OETD, with higher specificity. ETS and ETS-7 scores were not assessed beyond this stage as they are composite scores incorporat- ing the results of other index tests. The low number of PETD cases (12 panel-diagnosed cases) prevented further analysis of the diagnostic accuracy of tests for PETD using LCA or regression modelling. Selection of tests for inclusion in LCA and logistic regression models. For the regres- sion analyses, all nine OETD tests of ET opening were included with the CETDA or ETDQ-7 in the Research model (Table 5), following selection of the test variant and threshold with the PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 14 / 28 Diagnosis of Eustachian tube dysfunction highest Youden’s index (based on results in Table 2). For LCA the same process was adopted to identify suitable variables, although some tests were excluded to ensure that those incorpo- rated in the model were distinct, and not prone to similar test errors. To do this, a judgement was made on the basis of the underlying principle and methodology of each test. Selected tests were then grouped according to the four previously defined models (Table 5). For sonotubo- metry, a threshold of 5 or 10dB was not clearly superior, and so accuracy at each of the two thresholds was initially assessed in regression and LCA models. For tympanometry, thresholds of -50 and -100daPa were explored, with -50daPa found to give a higher accuracy, and corre- late more closely with other tests of ET opening. Multivariable diagnostic models. While using the expert panel as the reference standard, the benefit of combining test results was explored. The outputs from the Clinical and Research models are shown in Table 6. Patient-reported Valsalva, TTAG Valsalva, sonotubometry (both 5 and 10dB thresholds) and tuboimpedance were de-selected in one or both of the models due to shrinkage of their coefficients to zero. 1) Discriminatory ability of multivariable diagnostic models. Test accuracy with panel diagnosis as reference standard The c-statistic was superior for both the Clinical and Research models (Table 7, ROC curves in S1 Fig) when compared to equivalent AUC values for the individual index tests (Table 2). The choice of PROM included (CETDA or ETDQ-7) had little effect on the performance of the models. The addition of more complex tests mainly used in research did not markedly improve accuracy above that seen when only tests used in standard clinical practice were included (optimism corrected c-statistic was 0.74 for clinical models and 0.76–0.77 for research models). 1) Discriminatory ability of multivariable diagnostic models. The c-statistic was superior for both the Clinical and Research models (Table 7, ROC curves in S1 Fig) when compared to equivalent AUC values for the individual index tests (Table 2). The choice of PROM included (CETDA or ETDQ-7) had little effect on the performance of the models. The addition of more complex tests mainly used in research did not markedly improve accuracy above that seen when only tests used in standard clinical practice were included (optimism corrected c-statistic was 0.74 for clinical models and 0.76–0.77 for research models). 2) Calibration of multivariable diagnostic models. The calibration slope was between 0.79 and 0.83 for all regression models, as expected with relatively small datasets. This finding sug- gests that predictions made by the models regarding the probability of OETD are likely to be too extreme: low predictions too low, and high predictions too high[17] (calibration plots in S2 Fig). 2) Calibration of multivariable diagnostic models. The calibration slope was between 0.79 and 0.83 for all regression models, as expected with relatively small datasets. This finding sug- gests that predictions made by the models regarding the probability of OETD are likely to be too extreme: low predictions too low, and high predictions too high[17] (calibration plots in S2 Fig). PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Table 5. Table 5. Combined test models Latent class models Aspect of ETD evaluated Selected ET function test Regression variable Clinical model Research model Threshold for test positivity All-test Model No- panelModel No-PROM Model Open-test Model Symptoms CETDA score Total score ✓(a) ✓(a) Score 17 ✓(a) ✓(a) ETDQ7 score Total score ✓(b) ✓(b) Score 15 ✓(b) ✓(b) ME pressure equalisation Nine step test Tympanometry +400 change in daPa ME pressure in daPa ✓ ✓ ✓ +400 change <10daPa <-50daPa ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Passive opening Observed Valsalva Patient-reported Valsalva TTAG Valsalva No. opening out of 3 No. opening out of 3 No. opening out of 3 ✓ ✘ ✓ ✘ ✘ 2 out of 3 no open 2 out of 3 no open 2 out of 3 no open ✓ ✓ ✓ ✓ Active opening Sonotubometry Impedance Toynbee Observed Toynbee Patient-reported Toynbee No. opening out of 5 (>5dB) No. opening out of 3 No. opening out of 3 No. opening out of 3 ✓ ✓ ✘ ✓ 3 out of 5 <5dB 2 out of 3 no open 2 out of 3 no open 2 out of 3 no open ✓ ✘ ✓ ✘ ✓ ✘ ✓ ✘ Passive & active opening Tubomanometry Tuboimpedance No open or R>1 @30mbar No open @30mbar ✓ ✘ No open or R>1 @300daPa No open @300daPa ✓ ✓ ✓ ✓ All Expert panel NA NA NA Obstructive ETD diagnosis ✓ ✓ CETDA and ETDQ-7 included in separate models: model variant ‘a’ includes CETDA score and ‘b’ includes ETDQ7 score: the similarity of the PROMs precluded inclusion alongside one another in the models. ✓indicates tests selected for inclusion in the regression models and latent class analyses. Regression and LCA models evolved as tests were removed during analysis due to poor performance: ✘indicates that the index test was removed during analysis. ME = Middle Ear, TTAG = Tubo-tympano-aerodynamic-graphy https://doi org/10 1371/journal pone 0206946 t005 Fatigue. There was no evidence of a systematic change in patient manoeuvre ability (mea- sured by generated nasopharyngeal pressure) or test results across the immediate test repeats (ANOVA p value range 0.40–0.98) (S4 Table). Patient and assessor burden. All tests could be completed in a sufficiently short period of time by a single trained individual for them to be practical for use in routine clinics. Table 5. The mean time taken to perform all objective tests together was 40 min 21 sec (SD 283 sec), in addition to the time for PROM completion (not timed). The recorded time to complete each test included patient instruction, attempts to gain competence or useable data (if required), and data record- ing (Table 9). Tests were well tolerated overall, with difficulty and discomfort ratings highest for tubomanometry and tuboimpedance (Table 9), reported by patients to be due to the applied sudden-onset nasopharyngeal pressure during these tests. Latent class models LCA provided an opportunity to assess the expert panel diagnosis as an index test along with other index tests. Results from the four LCA models are shown in Table 8i-iii. The proportion of cases assigned to the OETD class was 31.6–32.5% in models without the panel diagnosis, and 38.5–40.2% in models with the panel diagnosis. Because the latent variable (the OETD diagnosis) is unobserved it was not possible to determine if it was the same cases classified as OETD in the models with similar results, but this is the assumption given the stability of the findings[23]. The addition of the panel diagnosis to the simpler models had a consistent effect: reducing the sensitivity and specificity of all other index tests by classifying a greater proportion of the cohort as OETD cases. In contrast, the addition of either the CETDA or ETDQ-7 to the models had little impact on class allocation, with the models suggesting the PROMs had no diagnostic value, J = -0.09 to 0.10. All models, with and without the panel diagnosis, demonstrated a consistent finding that tympanometry, sonotubometry and tubomanometry (using 300daPa) provided the greatest diagnostic value, being the only tests with J >0.5. Tests measuring ME pressure equalisation tended to have had higher specificity than tests measuring other aspects of ET opening. Tympanometry was excluded from early models (not shown) due to concern over incorpo- ration bias, but the effect on the model of including tympanometry seemed similar to the effect of other tests of ET opening, and unlike the effect of including the panel diagnosis. Sonotubo- metry interpreted with a 10dB threshold was included in some early LCA models, but was excluded due to model instability, unlike when sonotubometry was included at a 5dB threshold. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 15 / 28 Diagnosis of Eustachian tube dysfunction Diagnosis of Eustachian tube dysfunction Table 6. The coefficients (= log odds ratio) can be used to calculate a patient’s risk of having OETD. As continuous, raw data were used, tympanogram middle ear pressure and nine step pressure change have a negative predictive effect. A (CETDA) B (ETDQ-7) Clinical Model OR 95% CI Coefficient OR 95% CI Coefficient PROM (A or B) (per point) 1.05 0.99–1.10 0.05 1.03 0.98–1.07 0.03 Tympanometry (per dPa) 0.99 0.98–1.00 -0.01 0.99 0.98–1.00 -0.01 Observed Valsalva (per x/3) 2.61 1.00–6.82 0.96 2.64 1.02–6.82 0.97 Observed Toynbee (per x/3) 1.21 0.40–3.67 0.19 1.25 0.42–3.76 0.23 Reported Toynbee (per x/3) 1.68 0.60–4.71 0.52 1.67 0.60–4.63 0.51 Constant -2.40 -1.85 A (CETDA) B (ETDQ-7) Research Model OR 95% CI Coefficient OR 95% CI Coefficient PROM (A or B) (per point) 1.05 1.00–1.11 0.05 1.03 0.98–1.07 0.03 Nine step (per dPa change) 0.99 0.98–1.01 -0.01 0.99 0.98–1.01 -0.01 Tympanometry (per dPa) 0.99 0.98–1.00 -0.01 0.99 0.98–1.00 -0.01 Impedance Toynbee (per x/3) 2.30 0.89–5.96 0.83 2.25 0.88–5.75 0.81 Observed Valsalva (per x/3) 2.21 0.89–5.44 0.79 2.29 0.94–5.60 0.83 Tubomanometry (Y/N) 1.76 0.71–4.37 0.57 1.72 0.70–4.23 0.54 Constant -2.77 -2.14 OR = odds ratio. io) can be used to calculate a patient’s risk of having OETD. As continuous, raw data were used, tympanogram middle ear ave a negative predictive effect. audiometry. Although largely unmeasured, the assumption has been that the clinical diagnosis reliably translates to the ability of the ET to perform its physiological functions; opening to permit ME pressure equalisation and drainage, and protection of the middle ear. In recent years this link has been questioned, with reports that symptoms and the results of objective tests (tympanometry[24], tubomanometry[25, 26] and middle ear pressure equilibration tests [27]) were not closely matched. In this study PCA and correlation matrices demonstrated that patient symptoms measured by PROMs, and ET function measured by tests of ET opening for OETD and PETD, appear poorly correlated and vary independently. The panel diagnosis, as a close correlate of current clinical diagnosis, was found to be closely related to OETD symp- toms, but not the measured underlying ET function. Discussion Eustachian tube dysfunction is poorly defined and often presents a diagnostic challenge to cli- nicians. OETD or PETD diagnoses have often been applied to patients (or individual ears) based on clinical assessment relying on symptoms, otoscopy, tympanometry and pure tone PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 16 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Diagnosis of Eustachian tube dysfunction Table 8. All test latent class model: the panel diagnosis included as an index test alongside the tests of ET opening and PROMs. Selecting an ideal reference standard for OETD Two approaches for establishing test accuracy were explored. A consistent finding was that the tested PROMs had limited or no value as a diagnostic tool for OETD, highlighted by the very low Youden’s index and lack of influence on the latent class model output. While the PROMs consistently showed good sensitivity, the 71.0% to 81.8% overall PROM positive rate in the cohort resulted in very poor specificity. It is likely that the nature of the symptoms covered by the PROM items meant that patients with conditions such as hearing loss, Meniere’s Disease Table 7. Discriminatory ability of diagnostic models for OETD. Model c-statistic 95% CI Optimism corrected c-statistic Calibration slope Clinical A 0.76 0.67–0.84 0.74 (SD 0.02) 0.83 (SD 0.18) Clinical B 0.75 0.66–0.83 0.74 (SD 0.02) 0.79 (SD 0.19) Research A 0.79 0.70–0.86 0.77 (SD 0.02) 0.80 (SD 0.18) Research B 0.79 0.70–0.86 0.76 (SD 0.02) 0.79 (SD 0.19) https://doi.org/10.1371/journal.pone.0206946.t007 Table 7. Discriminatory ability of diagnostic models for OETD. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 17 / 28 Aspect of ET function evaluated Test All–Test Model a All test Model b Sens 95%CI Spec 95% CI J Sens 95% CI Spec 95% CI J Symptoms CETDA score 87.6 72.1–95.0 22.4 13.4–35.1 0.10 — — — — — ETDQ-7 score — — — — — 75.3 58.7–86.8 20.2 11.9–32.2 -0.05 ME pressure equalisation Tympanometry 52.4 27.4–76.2 100.0 0.0–100.0 0.52 54.6 30.7–76.6 100.0 0.0–100.0 0.55 Nine step test 62.4 43.3–78.2 81.8 65.2–91.5 0.44 61.7 42.6–77.8 80.2 66.1–89.4 0.42 Passive opening Observed Valsalva 74.4 56.5–86.7 73.8 54.8–86.7 0.48 75.6 57.9–87.5 73.2 55.9–85.5 0.49 Active opening Sonotubometry 77.6 55.1–90.7 71.4 55.8–83.2 0.49 79.5 58.0–91.6 71.4 56.5–82.7 0.51 Passive & active opening Tubomanometry 84.9 62.7–94.9 66.1 49.7–79.4 0.51 86.4 65.6–95.5 65.7 50.3–78.4 0.52 All Expert Panel 79.1 60.3–90.4 69.6 51.9–82.9 0.49 79.0 60.1–90.4 68.2 52.8–80.4 0.47 Percentage of cases in Class % 95% CI % 95% CI each class No OETD 59.8 39.9–76.9 61.5 44.1–76.3 OETD 40.2 23.1–60.1 38.5 23.7–55.9 Aspect of ET function evaluated Test No-Panel Model a No-Panel Model b Sens 95%CI Spec 95% CI J Sens 95% CI Spec 95% CI J Symptoms CETDA score 86.6 66.3–95.5 20.8 12.7–32.1 0.07 — — — — — ETDQ-7 score — — — — — 72.1 52.9–85.6 19.2 11.5–30.3 -0.09 ME pressure equalisation Tympanometry 61.2 30.4–85.1- 97.7 76.6–99.8 0.59 60.6 33.6–82.3 97.2 81.9–99.7 0.58 Nine step test 69.4 41.5–87.9 79.7 66.6–88.6 0.49 67.7 44.084.9- 78.7 65.9–87.7 0.46 Passive opening Observed Valsalva 76.1 54.5–89.5 68.7 53.3–80.8 0.45 76.9 55.2–90.0 68.8 54.8–80.1 0.46 Active opening Sonotubometry 87.0 58.2–97.0 69.9 53.5–82.4 0.57 89.2 59.6–97.9 70.7 55.7–82.3 0.60 Passive & active opening Tubomanometry 90.9 67.4–97.9 62.7 46.2–76.7 0.54 92.0 68.7–98.4 63.0 47.9–75.9 0.55 All Expert Panel — — — — — — — — — — Percentage of cases in Class % 95% CI % 95% CI each class No OETD 68.1 47.4–83.5 68.3 50.9–81.8 OETD 31.9 16.4–52.5 31.6 18.2–49.0 Aspect of ET function evaluated Test No-PROM Model Open-Test Model Sens 95%CI Spec 95% CI J Sens 95% CI Spec 95% CI J Symptoms CETDA score — — — — — — — — — — ETDQ-7 score — — — — — — — — — — ME pressure equalisation Tympanometry 54.1 29.8–76.5 100.0 0.0–100.0 0.54 60.0 32.5–82.3 97.6 79.5–99.8 0.58 Nine step test 62.0 42.9–78.0 80.6 66.2–89.9 0.43 68.0 43.5–85.4 79.4 66.5–88.2 0.47 Passive opening Observed Valsalva 75.2 57.5–87.2 73.3 55.6–85.7 0.49 76.2 55.0–89.3 69.1 54.4–80.7 0.45 Active opening Sonotubometry 78.9 57.3–91.3 71.3 56.3–82.8 0.50 87.7 59.5–97.2 70.7 55.2–82.5 0.58 Passive & active opening Tubomanometry 86.2 65.1–95.4 65.9 50.2–78.8 0.52 91.3 68.4–98.1 63.3 47.6–76.6 0.55 All Expert Panel 79.0 60.0–90.4 68.5 52.7–80.9 0.48 — — — — — Percentage of cases in Class % 95% CI % 95% CI each class No OETD 61.0 43.1–76.5 67.6 49.1–81.8 OETD 39.0 23.5–56.9 32.4 18.1–50.1 ‘a’ model includes the CETDA whereas ‘b’ model includes the ETDQ-7 No-panel latent class model: the All-test model without the panel diagnosis No-PROM latent https://doi.org/10.1371/journal.pone.0206946.t008 or temporomandibular joint (TMJ) dysfunction also scored highly, as has previously been demonstrated[11]. ‘a’ model includes the CETDA whereas ‘b’ model includes the ETDQ-7. No-panel latent class model: the All-test model without the panel diagnosis. No-PROM latent class model: the All-test model without the PROM). Open-test latent class model: only tests that measure ET opening included. Sens = sensitivity, Spec = specificity, J = Youden’s index (J) ( if >0.5),— = Not included, Impedance Toynbee (not shown) was excluded during analysis. Diagnosis of Eustachian tube dysfunction Table 9. Time to complete each test and patient-reported difficulty/discomfort. Reported by patient ET Opening Test Repetitions Time to complete Mean (SD) Difficulty /10 Median (range) Discomfort /10 Median (range) Tympanometry 1 1m 4s (34s) 1 (1–2) 1 (1–3) Nine-step test 1 6m 28s (82s) 1 (1–5) 1 (1–7) Patient-report & TM observation 6+1 3m 3s (51s) 1 (1–6) 2 (1–8) TTAG 6+1 5m 37s (78s) 1 (1–4) 2 (1–9) Impedance 6+1 6m 3s (85s) 2 (1–6) 1 (1–6) Sonotubometry 5+1 3m 53s (50s) 1 (1–6) 1 (1–6) Tubomanometry 3 7m 53s (138s) 3 (1–10) 3 (1–8) Tuboimpedance 3 6m 35s (143s) 3 (1–10) 2 (1–8) Values for patient-report and tympanic membrane (TM) observation were recorded together. TTAG = Tubo-tympano-aerodynamic-graphy. = additional PETD test for heavy breathing performed with OETD tests Table 9. Time to complete each test and patient-reported difficulty/discomfort. Values for patient-report and tympanic membrane (TM) observation were recorded together. TTAG = Tubo-tympano-aerodynamic-graphy. = additional PETD test for heavy breathing performed with OETD tests = additional PETD test for heavy breathing performed with OETD tests https://doi.org/10.1371/journal.pone.0206946.t009 according to LCA models without the panel (39% incorporating the panel). This difference had the effect of producing lower sensitivity values for the tests of ET opening when using the expert panel as the reference standard. There are two possible reasons for the differences between reference diagnoses 1. OETD was over-diagnosed clinically. Clinical diagnosis relies heavily on symptoms since examination and basic tests may both be normal. Symptoms are non-specific, as confirmed by the PROM performance, and so there may have been a high false positive rate in the panel diagnoses. Based on the PETD opening test results, clinical over-diagnosis of PETD may also have occurred. 2. Test of ET opening under-diagnose OETD. ET function may vary over time, and in some patients in whom OETD is intermittent the single testing episode may have occurred dur- ing a period of normal ET function, leading to a false negative. An example may be those patients who only suffer OETD during significant ambient pressure changes (baro-chal- lenge). By re-testing individuals after an interval[10], on a separate day, or even under dif- ferent conditions such as in a pressure chamber[28], it may be that intermittent OETD can be detected in more ears. The clearest difference between using the panel diagnosis and LCA was the percentage of individuals diagnosed as OETD: approximately 49% according to the panel, and 32% PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 18 / 28 https://doi.org/10.1371/journal.pone.0206946.t009 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Clinical and research implications: Diagnosing and measuring OETD This study suggests that current methods of clinical diagnosis alone (review of a patient’s history, examination, tympanometry and audiometry), even in the form of an expert pan- el’s consensus opinion, may be inadequate to diagnose OETD. The panel diagnosis ana- lysed as an index test appeared equivalent or inferior to some tests of ET opening. As indicated by the components of the PCA, if the panel diagnosis and tests of ET opening both measure ETD, they measure different aspects of the condition, and so are comple- mentary, rather than alternative diagnostic tools. While the PROMs were aligned with the panel diagnosis in the PCA, unlike the panel they were found not have diagnostic value due to poor specificity, and so analysis using the existing PROMs should be omitted from the diagnosis of ETD. It appears therefore that there are two aspects of OETD that should be incorporated into clinical and research practice when diagnosing ETD: the clinical assessment, representing patient history, examination and symptoms (rationalised in a manner that the tested PROMs cannot to ensure adequate specificity), and objective measures of ET function such as the tests of ET opening. Current methods of diagnosis based on clinical assessment[1] may not select the cohort of patients who would benefit most from interventions which are often designed to aid ET opening. They also appear inadequate in situations where a higher certainty of diagno- sis is required, such as when surgical interventions such as balloon Eustachian tuboplasty are being considered, or when conducting clinical research. This study builds on previous work to identify the most useful ET function tests in terms of reliability and accuracy[9, 10]. Given the potential issues with the flawed nature of the expert panel reference it appears that the results from the latent class models are more reliable, where tympanometry, sonotubometry and tubomanometry had the highest Youden’s index and therefore greatest potential diagnostic value for OETD. On the basis of our data using two current PROMs, PROMs should not be used in the diag- nosis of ETD, though they may have a role in quantifying and documenting symptoms, either to aid in the selection of confirmed ETD patients where surgical intervention may be justified, or if shown to be responsive, to monitor the course of disease over time or following treatment. Alternatively, the tests of ET opening may fail to diagnose those who only have a mild form of the disorder, perhaps because the obstructed ET still opens under the non-physiological conditions during many of the tests, or in the case of PETD, the ET opens and closes in a normal manner except during exercise or other provocation. The expert panel consensus process demonstrated that even experienced clinicians could not agree on the diagnosis for certain patients, and together with the poor correlation with tests of ET opening, this suggests that the panel diagnosis reference standard may be imperfect. However, completely removing the clinical acumen of an experienced clinician from the diag- nostic process for ETD risks the loss of a highly-sophisticated diagnostic ‘test’. Although the tests of ET opening were found to correlate with each other, and measure physiologically important aspects of ET function such as ME pressure equalisation, there are likely to be more aspects to normal ET function than can be objectively measured currently. The LCA No- PROM model (given the lack of diagnostic value from PROMs) therefore appears an attractive experimental means to combine clinical diagnosis with measured ET function. 19 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Diagnosis of Eustachian tube dysfunction Clinical and research implications: Diagnosing and measuring OETD Current PROMs are designed to be self-completed by the subjects, which introduces variability in the interpretation of the questions. Future research could be done into the development of an instrument that would act as a framework for taking the medical history, inquiring about a standard set of symptoms relevant to ETD. If validated, such an instrument might improve the initial selection of patients for testing. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 The role of test combinations Previous work has suggested that combining different ET function tests may improve diagnos- tic accuracy for ETD[2, 31, 32]. This was found to be the case in the regression models, where combining either basic, currently-used tests (Clinical model) or combining basic and advanced tests (Research model) improved the accuracy over the individual measures. Interestingly the additional improvement in accuracy using the Research model over the Clinical model was negligible, but this may be due to the potentially flawed expert panel reference standard, which was shown to relate poorly to the tests of ET opening included in the Research model. For the regression models to be clinically useful as a diagnostic tool, they must be inter- preted via a score chart, nomogram or calculator[17] using the coefficients identified for each test, and applying the patient’s test results to generate a probability of the patient having OETD. Given the reference standard on which the model was built, it was felt that a calculator based on our model would have limited clinical utility. However, the model- ling provides further evidence that the results of ET function tests should be combined, ideally mathematically. Clinical and research implications: Diagnosing and measuring PETD For PETD, only one reference standard was assessed as the sample size of affected cases (based on review of PETD tests of ET opening and the panel diagnosis) was deemed too small to allow reliable latent class analysis. Nonetheless the available data represent an advance in our understanding of tests for PETD, having investigated a number of tests in a single, clinically- relevant mixed cohort at the same point in time. The panel diagnosis and PETD tests of ET opening demonstrated significant correlation, suggesting that for PETD, the recognition of characteristic features in the history[29] may ensure that clinical diagnosis reflects the mea- sured ET function better than is the case for OETD. A diagnostic feature of PETD is reported to be movement of the tympanic membrane synchronised with heavy breathing, and the con- tinuous impedance and TTAG tests measuring this feature had the highest diagnostic accuracy in this study. Sonotubometry performed poorly using the tested protocol, however newer tech- niques may be superior[30]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 20 / 28 Diagnosis of Eustachian tube dysfunction Recommended ETD diagnostic pathway for clinical and research use On the basis of our findings, and a foundation of previously published work, we now have a much clearer understanding as to which tests of ET function should be employed, how these tests should be interpreted, and therefore, how ETD should be diagnosed. To guide clinicians and researchers, a diagnostic flow chart for ETD is proposed, incorporating the key assessment steps required to establish a diagnosis of OETD or PETD (Fig 3). The pathway incorporates tests selected on the basis of diagnostic performance, repeatability, feasibility and patient acceptability. Fig 3. Proposed diagnostic pathway for ETD. a, b While clinical assessment of a patient’s history and conventional examination may not be diagnostic of ETD, they are nonetheless an important means to identify suitable patients for investigation. c Effort should be made during assessment of the clinical history to identify habitual sniffing, as a negative middle ear pressure in these individuals may not indicate OETD, and further testing should be undertaken. d Described diagnostic thresholds are based on the equipment and protocols used in our study, but may require adjustment if alternative methods are used e TTAG is recommended if a tympanic membrane perforation is present. f A simple provocation test for use in clinic is asking the patient to exercise (jog on the spot or climb a flight of stairs) prior to testing. g Consider repeating tests on a separate occasion to improve sensitivity in patients with variable ET function. Patients with baro-challenge induced OETD may present in this group. https://doi.org/10.1371/journal.pone.0206946.g003 Fig 3. Proposed diagnostic pathway for ETD. a, b While clinical assessment of a patient’s history and conventional examination may not be diagnostic of ETD, they are nonetheless an important means to identify suitable patients for investigation. c Effort should be made during assessment of the clinical history to identify habitual sniffing, as a negative middle ear pressure in these individuals may not indicate OETD, and further testing should be undertaken. d Described diagnostic thresholds are based on the equipment and protocols used in our study, but may require adjustment if alternative methods are used e TTAG is recommended if a tympanic membrane perforation is present. f A simple provocation test for use in clinic is asking the patient to exercise (jog on the spot or climb a flight of stairs) prior to testing. Recommended ETD diagnostic pathway for clinical and research use g Consider repeating tests on a separate occasion to improve sensitivity in patients with variable ET function. Patients with baro-challenge induced OETD may present in this group. https://doi.org/10.1371/journal.pone.0206946.g003 https://doi.org/10.1371/journal.pone.0206946.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 21 / 28 Diagnosis of Eustachian tube dysfunction The pathway has been designed to enable the selection of patients for intervention, whether this be for clinical practice or research. For this reason, it has been designed to maximise speci- ficity when selecting individuals with OETD or PETD, as both clinicians and patients often want a high level of certainty in the diagnosis before initiating an intervention. Pathway stage 1: Clinical assessment. The pathway begins with clinical assessment, allowing the selection of patients suspected of having ETD, and initially targeting testing at the form of ETD thought to be most likely (OETD or PETD). Pre-selection of patients is standard practice when tests are applied in every field of medicine, ensuring that the tested population has a higher incidence of the target disorder, thereby reducing the cost and patient burden of unnecessary testing and improving the predictive value of the applied tests. Pathway stage 2: ET function tests. In patients suspected of having OETD with an intact TM without effusion, tympanometry should be applied as the first test of ET function. This is recommended because tympanometry has very high specificity, close to 100% in latent class models, although sensitivity was poor. Therefore, if ME pressure measured by tympanometry is lower than -50daPa patients can be diagnosed as having OETD without further testing. One exception to this results from the predisposition to inappropriate (often habitual) sniffing of some patients with PETD[33]. Clinicians should be aware that if patients admit to regular sniffing, this may cause a transient negative ME pressure in those with intermittent PETD, and locking of the ET at negative pressure may even create a temporary OETD. If tympanometry is normal, or the patient reports a history of inappropriate sniffing, fur- ther testing is warranted. Sonotubometry and tubomanometry demonstrated the best diagnos- tic performance for OETD, and while the two tests have good correlation, they do not diagnose exactly the same cohort. For these reasons, and to maximise diagnostic specificity, it is currently recommended that both tests should be positive if OETD is to be diagnosed with certainty. While the use of tubomanometry and sonotubometry is encouraged, they are currently not available in many centres. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Recommended ETD diagnostic pathway for clinical and research use The first reason for this is the intermittent nature of obstructive and patulous ETD in some individuals, as ET function may vary over even a relatively short period[10]. If clinical suspi- cion is high, repeat testing on another occasion is recommended in test-negative ears, to improve the likelihood that intermittent ETD is detected[34]. A second reason for an apparent absence of ETD during testing may be that the patient’s ETD only occurs under certain circumstances that cannot be replicated during testing, such as during baro-challenge. For PETD, it may be possible to replicate some circumstances that pro- voke symptoms at the time of testing, and the pathway includes a recommendation that exer- cise is used as a simple means of provocation[29]. Pathway stage 4: Current diagnostic uncertainty. The final stage involves an assessment as to why tests of ET opening may be negative. A diagnosis of Possible ETD is recommended for those patients who have symptom or findings suggestive of intermittent, or situation-spe- cific ETD (e.g. arising on baro-challenge), that we cannot detect with existing diagnostic tools if the patient is not affected by ETD at the time of testing. If a patient’s symptoms are continu- ous and present at, or around, the time of testing, it appears reasonable to label these individu- als as ETD Unlikely, particularly after testing on more than one occasion. The Possible ETD group clearly may contain individuals with an alternate diagnosis. In the absence of additional diagnostic methods for ETD, it may be difficult to finally exclude or detect ETD, and the clinician should consider investigating patients in this group for alternate conditions. Care should be taken when treating patients diagnosed as Possible ETD, and it is not recommended that this group is recruited to early-stage interventional trials. Active and passive ET function It is widely acknowledged that the ET may open actively (via muscular function) or passively (due to a pressure differential), and although a predominance of active or passive dysfunction is assumed in certain circumstances (e.g. cleft palate and rhinosinusitis respectively) the evi- dence of this distinction from objective testing has been limited[2, 35]. While the overall good correlation in this study between tests measuring only passive or active function suggests that in most cases the two vary together, a distinction between active and passive ET opening was supported by component 3 of the PCA. In future research, it would be valuable to measure active and passive dysfunction separately and determine if patients with a predominance of one or other dysfunction have a similar clinical course or response to interventions. Recommended ETD diagnostic pathway for clinical and research use If this is the case, observed Valsalva has the best diagnostic perfor- mance of the tests not requiring specialist equipment. This test does however have certain dis- advantages, the main one being a high degree of variation between patients regarding the nasopharyngeal pressure generated, even following training, that has been shown to affect the rate of ET opening and therefore test outcome[9, 10]. Observed Valsalva also only tests passive ET opening, therefore potentially missing individuals with OETD due to failure of active ET opening. If the tympanic membrane is not intact, basic methods for objectively measuring ET function are currently not available. For patients suspected of having PETD, the detection of ME pressure changes synchronised with nasopharyngeal pressure changes on breathing appears to be the best form of diagnostic test: although our own diagnostic study only incorporated a small sample size, the test is widely accepted, and the mechanism for positive test results with patency of the ET is clear. Imped- ance and TTAG methods of measurement are recommended for use in intact and perforated TMs respectively, as both tests were found to have comparable sensitivity, specificity and ease of use. If specialist equipment is not available, forced breathing with observation of the tym- panic membrane provides a diagnostic option for PETD if the tympanic membrane is intact. If patients have negative results from tests of ET opening on the side of the diagnostic path- way they started on (i.e. testing for obstructive or patulous forms of ETD), it is suggested that they cross over to undergo further testing for the other form of ETD. This step is suggested due to the possibility of initial incorrect targeting of testing, on the basis of non-specific symp- toms or examination findings. Pathway stage 3: Repeat testing. Patients with suspected ETD who have negative tests of ET opening for both OETD and PETD still cannot reliably be confirmed as non-ETD affected. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 22 / 28 Diagnosis of Eustachian tube dysfunction The first reason for this is the intermittent nature of obstructive and patulous ETD in some individuals, as ET function may vary over even a relatively short period[10]. If clinical suspi- cion is high, repeat testing on another occasion is recommended in test-negative ears, to improve the likelihood that intermittent ETD is detected[34]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Diagnosis of Eustachian tube dysfunction intermittent negative ME pressure, and the proposed diagnostic pathway has been designed to take account of this often-misdiagnosed group. The primary concern regarding the latent class analysis was that although efforts were made to select index tests for inclusion that were independent, this condition could have been violated if tests were prone to similar errors, or incorporation bias was an issue due to panel knowledge of patients’ symptoms and tympanometry results. The latter appears not to have been a problem as the PROMs were found to be non-diagnostic, and tympanometry results did not correlate with the panel diagnosis. Not all tests reported to measure or diagnose ETD could be included in this study. For more complex tests such as sonotubometry there are multiple methods described to per- form and interpret the tests that were not explored in this study[30, 36, 37]. Numerous tests have been described that require a perforated tympanic membrane, and these often allow greater analysis of tubal opening and closing[2]. Assigning equivalence between the results of different tests in ears with intact and perforated tympanic membranes will require further work, possibly utilising tests such as sonotubometry which do not require a specific tympanic membrane state. This study also did not examine the role of endo- scopic examination of the lumen of the cartilaginous ET. Although endoscopy during dynamic manoeuvres has not been reported to be a test of ET function, the findings may be of value in diagnosing the presence or cause of ETD, and differentiating between obstructive and patulous forms of the disease[38]. For those tests that were included, the findings should still be considered in the context of a relatively small sample size. The results of this study therefore provide direction for further research. This study was designed to overcome the limited applicability of existing case-control stud- ies by prospectively recruiting a clinically-relevant mixed cohort of individuals with highly var- ied forms and severity of presentation. The accuracy estimates from this study are therefore likely to be representative of patients at other institutions. However, to ensure viability of the study, in particular the ability to apply a wide range of tests to a single individual, certain exclu- sion criteria were applied, such as the need for an intact tympanic membrane and aerated ME. These requirements meant that diagnostic ability has not been assessed in all presentations of ETD, and ETD may be more severe or prevalent in ears with a perforated tympanic membrane or significant ME disease. Children also provide a separate population where the validity of extrapolating data from this study is unclear. The aetiology and natural course of ETD in chil- dren appears to differ from that in adults[39], and additional difficulty will arise in testing due to patient understanding and compliance. However, the recommended diagnostic process from this study provides a basis for further work in children. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Limitations of the study Both methods of classifying the disease status of individuals have flaws, though this study rep- resents an advance over previous diagnostic case-control studies that relied on a single imper- fect diagnostic method. While expert consensus may be expected to provide a more accurate diagnosis than a single clinician, the experts in this study were handicapped by not being able to question or examine the patients themselves, instead relying on a detailed case summary. In their normal practice the members of the expert panel may have had access to more test results than those provided here (audiometry and tympanometry), and so the expert diagnoses may not fully represent the diagnostic ability of the panel members. The case summaries presented to the panels reflected the information collected in standard practice in most ENT units, thus ensuring blinding to the index test results to prevent incorporation bias. Expert panel mem- bers drew attention to the importance of inappropriate (sometimes termed habitual) sniffing in patients with PETD[33]. The presence or absence of this symptom was not recorded in the case histories, potentially reducing the validity of tympanometry interpretation. Although PETD opening tests suggest this was not seen in this study, some patients may have PETD and PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 23 / 28 Acknowledgments The work of Lucy Truman, Jessica Bewick and Syed Hashmi is acknowledged in assisting with recruitment of patients. The work of Lucy Truman, Jessica Bewick and Syed Hashmi is acknowledged in assisting with recruitment of patients. Conclusions The findings in this study demonstrate the importance of measuring ET function in clinical practice. ETD should be diagnosed via clinical assessment combined with testing of ET open- ing specific to OETD and PETD, in contrast to previous recommendations that symptoms should be central to the diagnosis and definition[1]. The use of tests of ET opening may improve the selection of candidates for intervention, and allow the objective assessment of out- comes. Currently it is recommended that only patients with ETD diagnosed via positive tests of ET opening should be included in interventional trials. PROMs should not be used in the diagnostic process, but may have a role in characterising diagnosed cases and monitoring their response to treatment. Particular importance should now be given to reducing diagnostic uncertainty, and eliminating the need for a Possible ETD group, by developing practical ways to detect ears with intermittent or induced ETD. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 24 / 28 Diagnosis of Eustachian tube dysfunction Supporting information S1 Fig. Discriminatory ability of the logistic regression models (ROC curve). (TIFF) S2 Fig. Calibration plots for the logistic regression models (ROC curve). (TIFF) S1 Table. Symptoms associated with ETD (both obstructive and patulous) obtained via lit- erature review. (DOCX) S2 Table. OETD Test/PROM correlation matrix. Variables are the same as those recorded in Table 4. Bivarate Pearson correlation (r) shown, p value (two tailed) indicated by shading: dark grey shading p<0.01, and light grey shading p<0.05. (DOCX) S3 Table. PETD Test/PROM correlation matrix. Variables are the same as those recorded in Table 4. Bivarate Pearson correlation (r) shown, p value (two tailed) indicated by shading: dark grey shading p<0.01, and light grey shading p<0.05. (DOCX) S4 Table. Comparison of nasopharyngeal pressure and test outcomes across immediate repeats at a cohort level (x3 for all tests except sontotubometry which was repeated x5). If present, fatigue may be expected to affect either patient manoeuvre ability of test results. Con- tinuous variables only assessed. Tubomanometry and Tuboimpedance were not assessed as differences at 30/40/50mbar are expected. (DOCX) S1 File. Supporting information. (DOCX) S1 Table. Symptoms associated with ETD (both obstructive and patulous) obtained via lit- erature review. S2 Table. OETD Test/PROM correlation matrix. Variables are the same as those recorded in Table 4. Bivarate Pearson correlation (r) shown, p value (two tailed) indicated by shading: dark grey shading p<0.01, and light grey shading p<0.05. (DOCX) S3 Table. PETD Test/PROM correlation matrix. Variables are the same as those recorded in Table 4. Bivarate Pearson correlation (r) shown, p value (two tailed) indicated by shading: dark grey shading p<0.01, and light grey shading p<0.05. (DOCX) S4 Table. Comparison of nasopharyngeal pressure and test outcomes across immediate repeats at a cohort level (x3 for all tests except sontotubometry which was repeated x5). If present, fatigue may be expected to affect either patient manoeuvre ability of test results. Con- tinuous variables only assessed. Tubomanometry and Tuboimpedance were not assessed as differences at 30/40/50mbar are expected. (DOCX) S1 File. Supporting information. (DOCX) References 1. Schilder AGM, Bhutta MF, Butler CC, Holy CE, Levine LH, Kvaerner KJ, et al. Eustachian tube dysfunc- tion: consensus statement on definition, types, clinical presentation and diagnosis. Clin Otolaryngol. 2015; 40(5):407–11. https://doi.org/10.1111/coa.12475 PMID: 26347263 2. Doyle WJ, Swarts JD, Banks J, Casselbrant ML, Mandel EM, Alper CM. Sensitivity and specificity of eustachian tube function tests in adults. JAMA otolaryngology—head & neck surgery. 2013; 139 (7):719–27. https://doi.org/10.1001/jamaoto.2013.3559 PMID: 23868429; PubMed Central PMCID: PMC3792585. 3. Smith ME, Tysome JR. Tests of Eustachian tube function: a review. Clin Otolaryngol. 2015; 40(4):300– 11. https://doi.org/10.1111/coa.12428 PMID: 25851074. 4. Linnet K, Bossuyt PM, Moons KG, Reitsma JB. Quantifying the accuracy of a diagnostic test or marker. Clinical chemistry. 2012; 58(9):1292–301. Epub 2012/07/26. https://doi.org/10.1373/clinchem.2012. 182543 PMID: 22829313. 5. Reitsma JB, Rutjes AW, Khan KS, Coomarasamy A, Bossuyt PM. A review of solutions for diagnostic accuracy studies with an imperfect or missing reference standard. Journal of clinical epidemiology. 2009; 62(8):797–806. https://doi.org/10.1016/j.jclinepi.2009.02.005 PMID: 19447581. 6. Rutjes AW, Reitsma JB, Coomarasamy A, Khan KS, Bossuyt PM. Evaluation of diagnostic tests when there is no gold standard. A review of methods. Health technology assessment. 2007; 11(50):iii, ix-51. PMID: 18021577. 7. Rutjes AW, Reitsma JB, Di Nisio M, Smidt N, van Rijn JC, Bossuyt PM. Evidence of bias and variation in diagnostic accuracy studies. CMAJ. 2006; 174(4):469–76. https://doi.org/10.1503/cmaj.050090 PMID: 16477057; PubMed Central PMCID: PMC1373751. 8. Whiting P, Rutjes AW, Reitsma JB, Glas AS, Bossuyt PM, Kleijnen J. Sources of variation and bias in studies of diagnostic accuracy: a systematic review. Annals of internal medicine. 2004; 140(3):189– 202. PMID: 14757617. 9. Smith ME, Blythe AJC, Baker C, Zou CC, Hutchinson PJA, Tysome JR. Tests of Eustachian Tube Function: the Effect of Testing Technique on Tube Opening in Healthy Ears. Otol Neurotol. 2017; 38 (5):714–20. https://doi.org/10.1097/MAO.0000000000001375 PMID: 28306652. 10. Smith ME, Zou CC, Baker C, Blythe AJ, Hutchinson PJ, Tysome JR. The repeatability of tests of eusta- chian tube function in healthy ears. The Laryngoscope. 2017; 127(11):2619–26. Epub 2017/02/23. https://doi.org/10.1002/lary.26534 PMID: 28224679. 11. Smith ME, Cochrane IL, Donnelly N, Axon PR, Tysome JR. The Performance of Patient-reported Out- come Measures as Diagnostic Tools for Eustachian Tube Dysfunction. Otol Neurotol. 2018; 39 (9):1129–38. Epub 2018/08/15. https://doi.org/10.1097/MAO.0000000000001931 PMID: 30106847. 12. McCoul ED, Anand VK, Christos PJ. Validating the clinical assessment of eustachian tube dysfunction: The Eustachian Tube Dysfunction Questionnaire (ETDQ-7). The Laryngoscope. 2012; 122(5):1137– 41. https://doi.org/10.1002/lary.23223 PMID: 22374681 13. Author Contributions Conceptualization: Matthew E. Smith, James R. Tysome. Conceptualization: Matthew E. Smith, James R. Tysome. Data curation: Matthew E. Smith. Data curation: Matthew E. Smith. Formal analysis: Matthew E. Smith, Yemisi Takwoingi. Formal analysis: Matthew E. Smith, Yemisi Takwoingi. Formal analysis: Matthew E. Smith, Yemisi Takwoingi. Funding acquisition: Matthew E. Smith, James R. Tysome. Funding acquisition: Matthew E. Smith, James R. Tysome. Investigation: Matthew E. Smith, Cuneyt Alper, Manohar L. Bance, Mahmood F. Bhutta, Neil Donnelly, Dennis Poe, James R. Tysome. Methodology: Matthew E. Smith, Yemisi Takwoingi, Jon Deeks, James R. Tysome. Project administration: Matthew E. Smith, James R. Tysome. Resources: Matthew E. Smith. Supervision: Jon Deeks, James R. Tysome. Supervision: Jon Deeks, James R. Tysome. Writing – original draft: Matthew E. Smith. 25 / 28 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Diagnosis of Eustachian tube dysfunction Writing – review & editing: Matthew E. Smith, Yemisi Takwoingi, Jon Deeks, Cuneyt Alper, Manohar L. Bance, Mahmood F. Bhutta, Neil Donnelly, Dennis Poe, James R. Tysome. Writing – review & editing: Matthew E. Smith, Yemisi Takwoingi, Jon Deeks, Cuneyt Alper, Manohar L. Bance, Mahmood F. Bhutta, Neil Donnelly, Dennis Poe, James R. Tysome. References Schroder S, Lehmann M, Sauzet O, Ebmeyer J, Sudhoff H. A novel diagnostic tool for chronic obstruc- tive eustachian tube Dysfunction-The eustachian tube score. The Laryngoscope. 2014; 125(3):703–8. https://doi.org/10.1002/lary.24922 PMID: 25215457. 14. https://www.random.org/sequences. Accessed Nov 2017. 14. https://www.random.org/sequences. Accessed Nov 2017. 15. Youden WJ. Index for rating diagnostic tests. Cancer. 1950; 3(1):32–5. Epub 1950/01/01. PMID: 15405679. 16. Tibshirani R. Regression shrinkage and selection via the lasso. J R Statist Soc. 1996; 58:267–88. 17. Steyerberg EW, Vergouwe Y. Towards better clinical prediction models: seven steps for development and an ABCD for validation. Eur Heart J. 2014; 35(29):1925–31. Epub 2014/06/06. https://doi.org/10. 1093/eurheartj/ehu207 PMID: 24898551; PubMed Central PMCID: PMCPMC4155437. 18. Harrell FE Jr., Lee KL, Mark DB. Multivariable prognostic models: issues in developing models, evaluat- ing assumptions and adequacy, and measuring and reducing errors. Statistics in medicine. 1996; 15 (4):361–87. Epub 1996/02/28. https://doi.org/10.1002/(SICI)1097-0258(19960229)15:4<361::AID- SIM168>3.0.CO;2-4 PMID: 8668867. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 26 / 28 Diagnosis of Eustachian tube dysfunction 19. Smith GC, Seaman SR, Wood AM, Royston P, White IR. Correcting for optimistic prediction in small data sets. Am J Epidemiol. 2014; 180(3):318–24. Epub 2014/06/27. https://doi.org/10.1093/aje/kwu140 PMID: 24966219; PubMed Central PMCID: PMCPMC4108045. 20. Steyerberg EW, Eijkemans MJ, Harrell FE Jr., Habbema JD. Prognostic modelling with logistic regres- sion analysis: a comparison of selection and estimation methods in small data sets. Statistics in medi- cine. 2000; 19(8):1059–79. Epub 2000/05/03. PMID: 10790680. 21. Steyerberg EW, Vickers AJ, Cook NR, Gerds T, Gonen M, Obuchowski N, et al. Assessing the perfor- mance of prediction models: a framework for traditional and novel measures. Epidemiology. 2010; 21 (1):128–38. Epub 2009/12/17. https://doi.org/10.1097/EDE.0b013e3181c30fb2 PMID: 20010215; PubMed Central PMCID: PMCPMC3575184. 22. Altman DG. Practical statistics for medical research. New York, NY: Chapman & Hall/CRC Press.; 1999. 23. van Smeden M, Naaktgeboren CA, Reitsma JB, Moons KG, de Groot JA. Latent class models in diag- nostic studies when there is no reference standard—a systematic review. Am J Epidemiol. 2014; 179 (4):423–31. Epub 2013/11/26. https://doi.org/10.1093/aje/kwt286 PMID: 24272278. 24. Singh T, Taneja V, Kulendra K, Farr M, Robinson J, Rejali D. Balloon Eustachian tuboplasty treatment of longstanding Eustachian tube dysfunction. The Journal of laryngology and otology. 2017:1–6. https:// doi.org/10.1017/S0022215116009889 PMID: 28502258. 25. Liu P, Su K, Zhu B, Wu Y, Shi H, Yin S. Detection of eustachian tube openings by tubomanometry in adult otitis media with effusion. Eur Arch Otorhinolaryngol. 2016; 273(10):3109–15. https://doi.org/10. 1007/s00405-016-3938-0 PMID: 26894415. 26. References Jenckel F, Kappo N, Gliese A, Loewenthal M, Lorincz BB, Knecht R, et al. Endonasal dilatation of the Eustachian tube (EET) in children: feasibility and the role of tubomanometry (Esteve) in outcomes mea- surement. Eur Arch Otorhinolaryngol. 2014; 272(12):3677–83. Epub 2014/12/20. https://doi.org/10. 1007/s00405-014-3443-2 PMID: 25524643. 27. Teixeira MS, Swarts JD, Alper CM. Accuracy of the ETDQ-7 for Identifying Persons with Eustachian Tube Dysfunction. Otolaryngol Head Neck Surg. 2018; 158(1):83–9. Epub 2017/09/28. https://doi.org/ 10.1177/0194599817731729 PMID: 28949806; PubMed Central PMCID: PMCPMC5833308. 28. Mikolajczak S, Meyer MF, Felsch M, Jumah MD, Huttenbrink KB, Grosheva M, et al. Is there a differ- ence between active opening of the Eustachian tube in a hypobaric surrounding compared to a hyper- baric surrounding? Undersea Hyperb Med. 2015; 42(6):593–9. Epub 2016/01/09. PMID: 26742258. 29. Poe DS. Diagnosis and management of the patulous eustachian tube. Otol Neurotol. 2007; 28(5):668– 77. https://doi.org/10.1097/mao.0b013e31804d4998 PMID: 17534202. 30. Takata I, Ikeda R, Kawase T, Suzuki Y, Sato T, Katori Y, et al. Sonotubometric Assessment for Severity of Patulous Eustachian Tube. Otol Neurotol. 2017; 38(6):846–52. Epub 2017/04/25. https://doi.org/10. 1097/MAO.0000000000001413 PMID: 28437362. 31. Miyazawa T, Ueda H, Yanagita N. Eustachian tube function and middle ear barotrauma associated with extremes in atmospheric pressure. The Annals of otology, rhinology, and laryngology. 1996; 105 (11):887–92. https://doi.org/10.1177/000348949610501109 PMID: 8916865. 32. Kitajima N, Sugita-Kitajima A, Kitajima S. Altered eustachian tube function in SCUBA divers with alter- nobaric vertigo. Otol Neurotol. 2014; 35(5):850–6. Epub 2014/04/23. https://doi.org/10.1097/MAO. 0000000000000329 PMID: 24751737. 33. Ikeda R, Oshima T, Oshima H, Miyazaki M, Kikuchi T, Kawase T, et al. Management of patulous eusta- chian tube with habitual sniffing. Otol Neurotol. 2011; 32(5):790–3. Epub 2011/06/11. https://doi.org/10. 1097/MAO.0b013e3182184e23 PMID: 21659936. 34. Kobayashi T, Morita M, Yoshioka S, Mizuta K, Ohta S, Kikuchi T, et al. Diagnostic criteria for Patulous Eustachian Tube: A proposal by the Japan Otological Society. Auris, nasus, larynx. 2018; 45(1):1–5. Epub 2017/11/21. https://doi.org/10.1016/j.anl.2017.09.017 PMID: 29153260. 35. Casselbrant ML, Mandel EM, Seroky JT, Swarts JD, Doyle WJ. A pilot study of the ability of the forced response test to discriminate between 3-year-old children with chronic otitis media with effusion or with recurrent acute otitis media. Acta oto-laryngologica. 2011; 131(11):1150–4. Epub 2011/08/19. https:// doi.org/10.3109/00016489.2011.603137 PMID: 21846295; PubMed Central PMCID: PMCPMC3663051. 36. Asenov DR, Nath V, Telle A, Antweiler C, Walther LE, Vary P, et al. Sonotubometry with perfect sequences: First results in pathological ears. Acta oto-laryngologica. 2010; 130(11):1242–8. https://doi. org/10.3109/00016489.2010.492481 PMID: 20632904. 37. 38. Poe DS, Abou-Halawa A, Abdel-Razek O. Analysis of the dysfunctional eustachian tube by video endoscopy. Otol Neurotol. 2001; 22(5):590–5. PMID: 11568663. 39. Bluestone CD. Eustachian tube Structure, function, role in otitis media. Hamilton/London: BC Decker; 2005. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 Diagnosis of Eustachian tube dysfunction References Teixeira MS, Banks J, Swarts JD, Alper CM, Doyle WJ. Eustachian tube opening measured by sonotu- bometry is poorer in adults with a history of past middle ear disease. International journal of pediatric otorhinolaryngology. 2014; 78(4):593–8. https://doi.org/10.1016/j.ijporl.2014.01.005 PMID: 24491807; PubMed Central PMCID: PMC4017584. PLOS ONE \| https://doi.org/10.1371/journal.pone.0206946 November 8, 2018 27 / 28 Diagnosis of Eustachian tube dysfunction Diagnosis of Eustachian tube dysfunction 28 / 28
https://openalex.org/W1492327302	https://digital.library.unt.edu/ark:/67531/metadc1114205/m2/1/high_res_d/6165821.pdf	English	null	Factors affecting the behavior of bentonite fluids and their in-situ conversion into cement. Final report	null	1,984	cc-by	26,747	___- - -_ SAND84-7170 Unlimited Release UC-66~ SAND--84-7170 DE85 005141 Factors Affecting the Behavior of Bentonite Fluids and Their In-Situ Conversion Into Cement Final Report N. Guven, L. L. Carney, F. Malekahmadi, 1. J. Lee Department of Geosciences, Texas Tech University Lubbock, TX 79409 ‘Prepared by Sandia National Laboratories Albuquerque, New Mexico 87185 and tierrnore, California 94550 for the United States Department of Energy under Contract M-AW76DP00789 November 1984 SAND84-7170 Unlimited Release UC-66~ Factors Affecting the Behavior of Bentonite Fluids and Their In-Situ Conversion Into Cement Final Report N. Guven, L. L. Carney, F. Malekahmadi, 1. J. Lee Department of Geosciences, Texas Tech University Lubbock, TX 79409 N. Guven, L. L. Carney, F. Malekahmadi, 1. J. Lee Department of Geosciences, Texas Tech University Lubbock, TX 79409 ‘Prepared by Sandia National Laboratories Albuquerque, New Mexico 87185 and tierrnore, California 94550 for the United States Department of Energy under Contract M-AW76DP00789 DISCLAIMER This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency Thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof. SAND84-7170 Unlimited Release Unlimited Release DISCLAIMER This report was prepared as an account of work sponsored by an agency of the United Stab Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, exprcss or implied, or assumes any legal liability or responsi- bility for the accuracy, completencss, or uscfulncss of any information, apparatus, product, or proccss disclosed, or rcprtscnts that its usc would not infringe privately owned rights. Refer- ence herein to any specific commercial prcduct, proccss, or seMcc by trade name, trademark manufacturer, or otherwke docs not necessarily constitute or imply its endorsement, mom- mendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not ntctss8rily state or reflect thosc of the United States Government or any agency thereof. DISCLAIMER Printed in the united S t a b of America Availableliom N a t i d Techniad Information Servica UB. Department of Commerce 6zsSPortR7alRoad Springfield, A22161 Printed in the united S t a b of America Availableliom N a t i d Techniad Information Servica UB. Department of Commerce 6zsSPortR7alRoad Springfield, A22161 SAND84-7170 Unlimited Release SAND84-7170 bY N. Guven, L. L. Carney, F. Malekahmadi, and L. 3. Lee Department of Geosciences, Texas Tech University Lubbock, Texas 79409 Submitted to SANDIA NAnONAL -_ - - LABORATORIES . . -rad- I.---.Lv.. ,241 niwicinn 6' P Albuquerque, New Mexico 87185 ABSTRACT Bentonite i s the common viscosifier and f l u i d loss control additive i n water base d r i l l i n g fluids. lem due t o gelation a t high temperatures while d r i l l i n g , completing, o r cementing deep o i l and gas wells o r geothermal wells. tamination Prom the formation solids and formation f l u i d s may also a f f e c t Bentonite, however, creates i t s own prob- Furthermore mud con- the performance o f bentonite muds. develop a basic understanding o f the effects o f common salts, hydroxides, It i s therefore rather important t o and other mud additives on the rheology and other functions o f bentonite fluids. were autoclaved a t the temperature range 70 t o 600°F, and under a pressure o f 17,000 psi. was measured with a FANN 50C viscometer. plastic viscosity, gel strength, y i e l d point, pH, CEC, and f l u i d losses For t h i s purpose, bentonite f l u i d s containing these additives Subsequently, the high-temperature rheology o f the f l u i d s Other f l u i d properties such as l s o determined before and a f t e r autoclaving o f the fluids. The high- temperature reactions occurring i n these f l u i d s were studied with x-ray d i f f r a c t i o n and electron microscopy. A pure Na-bentonite f l u i d becomes highly viscous i n the temperature range 25O-45O0F, leading t o the gelation o f the f l u i d when bentonite con- centration reaches about 4% i n the fluid. The addition o f low molecular weight polymers seems t o control the viscosity only i n the l i m i t e d temper- ature range 300-400°F, but not a t higher temperatures. NaCl o r NaOH a t 1% concentration i n the f l u i d tremendously increases the "osmotic" swelling o f the bentonite and hence increases the fl d viscosity t o undesirably high values. bY N. Guven, L. L. Carney, F. Malekahmadi, and L. 3. Lee Department of Geosciences, Texas Tech University Lubbock, Texas 79409 Submitted to SANDIA NAnONAL -_ - - LABORATORIES . . -rad- I.---.Lv.. ,241 niwicinn 6' P Albuquerque, New Mexico 87185 Potassium salts and hydroxide f f e c t i v e l y control the f l u i d gelation a t high temperatures by promoting the conversion o f (swelling) smectite i n the f l u i d t o a (less-swelling) phase o f i l l i t e / s m e c t i t e mixed- i - layers. Salts and hydroxide o f calcium depress the f l u i d viscosity a t a l l temperatures. - layers. Salts and hydroxide o f calcium depress the f l u i d viscosity a t a l l temperatures. Another costly problem i n d r i l l i n g technology i s related t o the poor cement bonding o f the casing t o the formation. As a solution t o t h i s troublesome situation, the i n - s i t u conversion o f bentonite f l u i d s i n t o cement has been studied. by the addition o f lime t o bentonite f l u i d s a t and above 300°F. This conversion has been successfully achieved The ad temperature i t e , which o f free s i 1 tobermori t e i t i o n o f lime increases the f l u i d pH and promotes the high- reaction between 1 ime and smecti te. Consequently, tobermor- s a cement mineral, precipitates i n the fluid. The addition ca t o the bentonite/lime f l u i d does not increase the rate o f precipitation. The addition o f CaC12, however, t o the benton- ite/lime f l u i d enhances the i n - s i t u formation o f cement minerals i n ben- tonite fluid. The addition o f magnesia proves t o be detrimental both t o the cement conversion and t o the rheology o f the f l u i d but it provides a buffer f o r maintaining high pH i n the f l u i d . t o cause any noticeable retardation o f the cement reactions i n benton- i t e / l ime f l uids. Lignosulfonates do not seem ii TABLE OF CONTENTS ABSTRACT . . . . . bY N. Guven, L. L. Carney, F. Malekahmadi, and L. 3. Lee Department of Geosciences, Texas Tech University Lubbock, Texas 79409 Submitted to SANDIA NAnONAL -_ - - LABORATORIES . . -rad- I.---.Lv.. ,241 niwicinn 6' P Albuquerque, New Mexico 87185 . . . . . . . . . . . . . . . . . . EXECUTIVE SUMMARY. . . . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . METHODS OF INVESTIGATIONS. . . . . . I. BEHAVIOR OF THE BENTONITE-BASED 11. 111. . . . . . . . . . . . . . . . . FLU IDS AT HIGH TEMPERATURES . . . . . . . . . . . . . 1. Pure Bentonite Fluid . . . . . . . . . 2. Bentoni te/Polymer F1 u i ds . . . . . . . EFFECTS OF SODIUM CHLORIDE AND SODIUM HYDROXIDE ON BENTONITE FLUIDS. . . . . . . . . . . . . . 1. Bentoni te/NaCl F1 u i d . . . . . . . . . 2. Bentoni te/NaOH F1 u i d . . . . . . . . . 3. Bentonite/NaOH/Sil ica Fluid. . . . . . 4. Electron Microscopic Examination o f Mineral Reactions. . . . . . . . . . EFFECTS OF SALTS AND HYDROXIDE OF POTASSIUM ON BENTONITE FLUIDS. . . . . . . . . . . . . . 1. Bentoni te/KC1 F1 uid. . . . . . . . . . 2. Bentoni te/K2C03 Fl u i d. . . . . . . . . 3. Bentoni te/KOH F1 u i d. . . . . . . . . . 4. Mineral Reactions i n Bentonite Fluids Containing Salts and Hydroxide o f Potassium. . Page 1 V 1 4 7 7 2 1 29 29 29 32 40 47 47 47 54 54 iii Page IV. IN-SITU CONVERSION OF BENTONITE FLUIDS INTO CEMENT MINERALS . . . . . . . . . . . . . . . . . . . 75 1. Bentoni te/CaC12 F1 ui d. . . . . . . . . . . . . . . 75 2. Bentoni te/Lime F1 uid . . . . . . . . . . . . . . . 75 3. Bentonite/Lime/Sil ica F1 uid. . . . . . . . . . . . 82 4. bY N. Guven, L. L. Carney, F. Malekahmadi, and L. 3. Lee Department of Geosciences, Texas Tech University Lubbock, Texas 79409 Submitted to SANDIA NAnONAL -_ - - LABORATORIES . . -rad- I.---.Lv.. ,241 niwicinn 6' P Albuquerque, New Mexico 87185 Mineral Reactions in Bentonite Fluids Containing Silica, Lime, or CaC12 . . . . . . . 89 V. EFFECTS OF ADDITIVES ON THE CEMENT REACTIONS IN BENTONITIC DRILLING FLUIDS . . . . . . . . . . . . . . . . 105 1. Bentoni te/Lime/CaClZ Fluid . . . . . . . . . . . . 105 2. Bentoni te/Lime/Gypsum F1 uid. . . . . . . . . . . . 109 3. Bentoni te/Lime/Lignosul fonate F1 uid. . . . . . . . 109 4. Bentonite/Lime/MgO Fluid . . . . . . . . . . . . . 115 VI. SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . . . . . 124 i v EXECUTIVE SUMMARY These reactions involve: a) changes i n the morphology o f clay particles (smectite), b) changes i n the state o f hydration o f smectites, c) dissolution o f smectites, and e) precipitation o f new mineral phases t h a t are either detrimen or i n e r t t o the rheology o f the fluid. between smectite particles o f the bentonites and the additives i n the fluids. These reactions involve: a) changes i n the morphology o f clay particles (smectite), b) changes i n the state o f hydration o f smectites, Our investigations have shown that the above reactions involving smectite i n bentonite were the causes o f the problems t h a t have been experienced over the years while d r i l l i n g deep and hot wells with the bentonite fluids. A pure bentonite f l u i d with a Na-smectite as the main constituent becomes gelated (highly viscous) i n the temperature range 250-450°F. molecular polymer additive (a Cypan-type sodium polyacrylate with a molecu- l a r weight o f 940-1,400) i s found t o control the gelatin i n the temperature range o f 300-400°F but not a t higher temperatures. On the other hand, high The low molecular weight polymers (a sodium polyacrylate with a molecular weight i n the range o f 750,000 t o one m i l l i o n ) are found t o control the gelation o f the bentonite a t temperatures as high as 600°F. t i n g the r e l a t i v e amounts o f low molecular and high molecular polymers, a bentonite f l u i d can be designed t o give satisfactory performance a t low and high temperatures. Thus, by carefully selec- Interlayer cations o f the smectites can be easily exchanged by other cations which may predominate the f l u i d due t o mixing w i t h formation brines o r solids, o r due t o the mud additives. The rheological behavior o f the bentonite f l u i d s i s found t o be extremely sensitive t o the exchange- able cations i n the f l u i d . EXECUTIVE SUMMARY The behavior o f bentonite f l u i d s has been systematically studied under high temperatures and high pressures similar t o the down-hole condi- tions i n deep wells o r i n geothermal wells. The effects o f various salts, hydroxides and other mud additives have been examined on the properties o f the bentonite f l u i d s under the above conditions. The i n - s i t u conversion o f bentonite f l u i d s i n t o cement has been studied i n order t o improve the displacement o f d r i l l i n g f l u i d s by cement slurries. The purpose o f these investigations was t o ascertain a f u l l under- standing o f the viscosity changes (including gelation) i n bentonite based f l u i d s under down-hole conditions with temperature, p r The down-hole conditions were simulated by an autoclav Since the problems with d r i l l i n g f l u i d s u the borehole during s t a t i c conditions, t h one without the agitation. safety and economy. tures ranging from 300°F t o 600'F under the appropriate confining pressures up t o 16,000 psi. Subse o f salts and hydroxides , Cay and Mg, mud additives. The properties o f the strength, y i e l d PO l y arise near the bottom of toclaving process was a s t a t i c i c autoclave had also the advantages o f The bentonite f l u i d s have been autoclaved a t tempera- the f l u i d s were prepared i n the presence determined before and ti tes ( i t s hydration state, morphology, and mode o f aggregation) and other mineral reactions i n these fluids' have been died with x-r d electron micro- scopy. The results rev ed a close relationship between t h properties of the f l u i d s and the mineral reactions i n them. These reactions occur V V between smectite particles o f the bentonites and the additives i n the fluids. These reactions involve: between smectite particles o f the bentonites and the additives i n the fluids. EXECUTIVE SUMMARY Sodium hydroxides, especially NaC1, a t 1% con- centration i n the f l u i d causes the gelation o f the f l u i d up t o 500°F since v i the Na-ion increases the "osmotic" swelling o f the smectite particles. The presence o f free s i l i c a (1%) i n the f l u i d greatly depresses the viscos- i t y due t o the precipitation o f silicates such as analcime and albite. These s i l i c a t e precipitations may cause permeability damage i n the forma- tion. Salts and hydroxides o f potassium are found t o be very effective i n controlling the gelation o f bentonite a t high temperatures. This i s rela- ted t o the conversion o f ~ smecti t e t o ill i te/smecti t e mixed-1 ayers a t and above 400OF. The viscosity o f the bentonite f l u i d s i s greatly reduced by Ca-ions that enter i n t o the smecti t e interlayers by an ion-exchange reaction. Salts and hydroxides o f \calcium, therefore, depress the f l u i d visosity t o unacceptably low levels a t a l l temperatures. For instance, the high-tem- perature viscosity o f a 6% bentonite/l% lime f l u i d i s extremely low even before the autoclaving as shown i n Figure 1.1. The viscosity o f the f l u i d was raised t o an acceptable level only a f t e r increasing the bentonite con- centration t o 10%. Another costly problem i n d r i l l i n g geothermal and deep wells invol- ves the displacement o f the mud by a cement slurry a f t e r the casing i s placed. It i s again the characteristics o f bentonite that cause channel- ing o f cement slurries and the d i f f i c u l t y i n displacement o f the mud. To eliminate these problems, the- in-situ conversion o f bentonite fluids has pted by the addition o f lime. EXECUTIVE SUMMARY The presence o f l i m e i s found t o increase the f l u i d pH which dissolves the smectite and subsequently leads t o a reaction between l i m e ica. Tobermorite, a-common cement mineral, forms as the reac free s i l i c a t o the bentonite/lime f l u i d does not increase the rate o f t h i s cement reaction. However, the presence o f CaC12 i n the bentonite/lime and above 300OF. The addition o f v i i 30 I= z z! 10 a 3 P 5 BENTONITE/LIME FLUIDS AT VARIOUS BENTONITE CONCENTRATIONS (NOT AUTOCLAVED) 100 200 300 400 500 Figure 1.1 - The FANN 50C rheograms o f the bentonite/l% lime fluids a t various bentonite trati b f th t l i I= z z! 10 a 3 P 5 BENTONITE/LIME FLUIDS AT VARIOUS BENTONITE CONCENTRATIONS (NOT AUTOCLAVED) 100 200 300 400 500 Figure 1.1 - The FANN 50C rheograms o f the bentonite/l% lime fluids a t various bentonite concen t rati ons before the autocl avi ng . BENTONITE/LIME FLUIDS AT VARIOUS BENTONITE CONCENTRATIONS (NOT AUTOCLAVED) BENTONITE/LIME FLUIDS AT VARIOUS BENTONITE CONCENTRATIONS (NOT AUTOCLAVED) Figure 1.1 - The FANN 50C rheograms o f the bentonite/l% lime fluids a t various bentonite concen t rati ons before the autocl avi ng . fluid enhances the in-situ formation of cement minerals. Liqnosulfonates do not retard the cement reaction in the bentonitellime fluids. No cement minerals form when magnesia is added to the bentonite/lime fluid. fluid enhances the in-situ formation of cement minerals. Liqnosulfonates do not retard the cement reaction in the bentonitellime fluids. No cement minerals form when magnesia is added to the bentonite/lime fluid. i x INTRODUCTION While Wyoming bentonite has been used for many years t o obtain viscos- ity and thixotropy of drilling fluids, the intricate reaction mechanisms of bentonite have not been studied i n detail u n t i l now. Thousands of wells have been drilled using bentonite fluids and a number of bentonite- related problems has been experienced during the d r i l l i n g , cementing, com- pletion, or production of these wells. EXECUTIVE SUMMARY obstacle while d r i l l i n g geothermal wells or while d r i l l i n g i n deeper form- These problems may become a major ations w i t h high temperatures and under high pressures. Two of these prob- lems are found to be rather common and costly: a) high temperature gelation of bentonite-based muds, b) poor cement jobs behind casing after the borehole has been drilled and casing has been cemented i n the well- bore. The gelation of the mud creates problems with regard t o circulation, running logging tools tion fluids and cement slurries. the efficiency o f displacement of drilling f l u i d s by cement slurries. no success has been achieved i n increasing the efficiency or i n even obtaining a good bond between the casing and the formation. Various attempts were made t o improve B u t The investigations under the 'Sandia contracts #68-4752 and 32-2331 were therefore designed to obtain basic data on the bentonite fluids w i t h the above problems i n mind. Specifically, the -objectives of the projects were: _-- 1. To understand the conditions leading t o high-temperature gelation of bentonite muds. 1. To understand the conditions leading t o high-temperature gelation of bentonite muds. 2. To find remedies to control high-temperature gelation so t h a t a desirable drilling and cement job can be done. 2. To find remedies to control high-temperature gelation so t h a t a desirable drilling and cement job can be done. 1 1 3. To study the following factors which either promote o r retard the formations o f cement minerals i n bentonite/lime fluids: 3. To study the following factors which either promote o r retard the formations o f cement minerals i n bentonite/lime fluids: * lime/silica concentrations * Temperature * Lignosulfonate * Gypsum * MgO I n order t o develop a basic understanding o f the above troubles with bentonite f l u i d s and with related cement setting, the following f l u i d sys- tems were studied: A. Bentonite f l u i d s containing salts and hydroxides of sodium and potassium o r polymers. B. EXECUTIVE SUMMARY Bentonite f l u i d s containing lime and s i l i c a f o r the purpose o f understanding the i n - s i t u conversion o f the bentonitic f l u i d s i n t o cement. C. Effects o f additives on cement reaction i n bentonitic d r i l l i n g fl uids : a) Bentoni t e / l ime/CaC12 fl uid b) Bentonite/l ime/gypsum f l u i d c ) Bentonite/l ime/l ignosul fonate f l u i d Methods o f Investigations Bentonite f l u i d s were autoclaved a t the temperature range 300-600°F. The autoclave with a c e l l o f 600 m l capacity was manufactured by Autoclave Engineers, Erie, Pennsylvania. The c e l l was s t a t i c a l l y heated a t a rate o f 150"F/hour t o the desired temperature and the pressure was i n i t i a l l y produced by a Haskel air-driven l i q u i d pump. Temperature and pressure were monitored and automatically recorded. The autoclave was cooled i n a i r f o r 14 hours. c e l l i n t o a plastic b o t t l e f o r subsequent testing with various viscometers (FANN 50C, FANN 35A, and SANDIA) and a Baroid standard f i l t e r press. temperature was maintained f o r 6 hours by a temperature controller with the Barber-Colman analog and d i g i t a l set point controller. The fluid, a f t e r autoclaving, was transferred from the The The high-temperature viscosity o f the f l u i d s was usual l y measured with a FANN 50C viscometer and a few occasional tests were run with SANDIA viscometer. The FANN 50C viscometer i s equipped with a standard rotor cup with a sample capacity o f 50 m l and r o t o r cup speed o f 0-600 RPM. The sample can be heated up t o 500°F by an o i l bath which i s also used f o r cooling the sample. psi using nitrogen gas. the form o f rheograms f o r each f l u i d . plastic viscosity, gel strength, and y i e l d point were measured a t room temperature and atmospheric pressure with a FANN 35A viscometer. This instrument i s a direct reading viscometer that gives the plastic viscosity and y i e l d point d i r e c t l y from the two d i a l readings a t 300 RPM and a t 600 RPM. d) Bentonite/l ime/MgO f l u i d . d) Bentonite/l ime/MgO f l u i d . The above bentonite-based f l u i d systems have been autoclaved a t tempera- tures ranging from 300°F t o 600°F. been measured before and a f t e r autoclaving. The high temperature mineral reactions i n these f l u i d systems have been studied with x-ray d i f f r a c t i o n The properties o f these f l u i d s have 2 and analytical electron m i report. scopy. The results are described i n this and analytical electron m i report. scopy. The results are described i n this Methods o f Investigations Methods o f Investigations Similarly, gel strengths were determined by d i a l readings a t 3 RPM f i r s t a f t e r a run f o r 10 seconds then the second reading a f t e r 10 minutes. Pressures i n the sample cup were maintained a t 650 The viscosity/temperature variations are given i n Other f l u i d properties such as The gel strengths are reported i n pounds per 100 square feet. 4 4 The f l u i d loss properties o f the samples were measured by a Baroid Standard F i l t e r Press when the bentonite concentrations were high enough f o r a meaningfu'l measurement. The Baroid F i l t e r Press consists o f a f l u i d cup, a f i l t e r i n g medium, a pressurized nitrogen gas cylinder and regulator. The f i l t e r t e s t was run f o r 30 minutes under a pressure o f 100 psi o r u n t i l blsw-out occurred, a t which time the t e s t was stopped and t h e f i l - t r a t e was measured. .The mineral phases i n the f l u i d s were examined, before and a f t e r autoclaving, with x-ray d i f f r a c t i o n and analytical electron microscopy. For the x-ray diffraction, oriented clay films, were pyepared on glass slides from the fluids. The excess salts, hydroxides, and other soluble additives were washed o f f by centrifugation o f an aliquot o f each fluid. The oriented clay films were x-rayed upon air-drying and subsequently upon saturation with ethylene glycol. ped with a graphite monochromator and adjustable divergence s l i t was used f o r t h i s purpose. A P h i l l i p s x-ray diffractometer equip- After identification o f high-temperature mineral reactions i n the autoclaved f l u i d s with x-ray diffraction, the characteristic features of individual s o l i d particles were examined with a JEM-100 CX Analytical Electron Microscope. Methods o f Investigations This instrument allowed us t o use the following signals, generated by the interaction o f electrons with sol i d particles: - transmitted electrons which form transmission electron (TEM) images and selected area d i f f r a c t i o n (SAD) patterns, - secondary electrons which form SEM images o f the surface features o f the s o l i d particles, and - characteristic x-rays which give us the elemental spectral data related t o the chemistry o f the individual particles. Details o f 5 the energy-dispersive spectral analysis were given i n our previous Sandi a Report (SAND 82-7032). The intensities o f x-ray spectra were counted for 100 seconds i n the "area" mode i n which a selected area was scanned w i t h the electroh probe. collected and processed by an EG&G ORTEC EEDS I1 analyzer. silicon is a major element i n most o f the mineral particles, all The intensity data were Since the intensity data for the x-ray spectra were normalized w i t h refer- ence to the intensity o f Si-K line. graphs o f the x-ray spectra are related t o the sample holder and to The Cu- and Au-lines i n the the sample coating metal respectively. 6 1. Pure Bentonite Fluid The bentonite component of the water-base muds is the main viscosi- fer and f l u i d loss control additive i n these muds. In order t o understand the fundamental role of properties, we first ex B were usually prepared by using the raw bentonite, which con minerals, such as mica, quartz, feldspars, calcite and gyps t of a smectite. I t is, of course, t h s the desirable properties of the f l u i d regarding its nite on the fluid rheology and i puri f i ed bentoni t e f 1 ui ds . rheology and f l u i d loss control.’ In order to follow directly the contri- butions of the smecti te component, we prepared a pur{ fied Wyoming benton- , which is a Na-montmorillonite variety i n this ben- tonite. The purified Wyoming bentonite was obtained from the Baroid Division of NL Industries (Houston). I t was noted that the viscosi’ty of qs high w i t h increasing temperatures and turns ‘the mud into a g We, therefore, r i n a fluid state ows a significant fluid is found to be rather different under higher pressures. Figure l . l b 30 25 3 5 F 2 20 W 0 I P v 1: z 8 15 VI 5 -Z k! 10 a a P nm 5 6OOOF 50OOF \ 2% BENTONITE/H,O MINERAL COMPONENTS IN THE FLUIDS: 7OoF SMECTITE (11 i) 300'F SMECTITE (11 A) 4OOOF SMECTlTE (11 i) 50OOF SMECTITE (11 A) BOOOF SMECTITE (12.2i) 1 I I t I too 200 300 400 500 ( O F ) Figure 1.la - The FANN 50C rheograms .of the pure 2% bentonite f l u i d s after autoclavlng a t the temperatures marked on the curves. MINERAL COMPONENTS IN THE FLUIDS: 7OoF SMECTITE (11 i) 300'F SMECTITE (11 A) 4OOOF SMECTlTE (11 i) 50OOF SMECTITE (11 A) BOOOF SMECTITE (12.2i) 2% BENTONITE/H,O Figure 1.la - The FANN 50C rheograms .of the pure 2% bentonite f l u i d s after autoclavlng a t the temperatures marked on the curves. ’ TABLE: 1.la APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 5OC VISCOMETER FOR THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% PURIFIED BENTONITE Fann 50C Autoclaved at. The f i r s t number before the slash refers t o the gel strength a f t e r 10 seconds and the second number (following the slash) t o the gel strength a f t e r 10 minutes. Due t o the low clay content (2%) o f the fluid, the measurements o f A P I f i l t r a t e , cake thickness, and HP-HT f i l t r a t e were not feasible. These entries are l e f t blank i n t h i s and the other tables. Gel Strengths were determined as explained i n the t e x t . 1. Pure Bentonite Fluid temperature (OF) Temperature OF 70 300 400 500 600 75 6.2 7.7 11.8 21.8 28.9 100 3.9 , 6.4 8.7 16.4 25.6 150 3.1 4.1 5.8 12.5 19.3 200 3.0 2.7 4.5 9.5 15.4 4.3 1.9 3.7 7.7 14.95 250 300 6.9 2.0 4.2 7.7 18.7 , 350 9.8 3.1 4.5 7.4 20.4 400 10.4 3.0 2.9. 4.4 18.9 450 8.2 2.6 3.3 2.7 15.5 ‘500 . 7.7 10. 6.2 8 TABLE l . l b PROPERTIES.OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF TABLE l . l b PROPERTIES.OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF Autoclaved a t temperatures (OF) F1 u i d Property 70 300 400 500 600 Apparent Viscosity (centipoise) 6 10 20 38 49 Plastic Viscosity (centipoise) 4 8 13 20 19 Yield Point (lb/100 sq ft) 3 4 14 36 50 Gel Strength (lb/100 sq ft)** 11 7 212 3/3 9/10 14/15 API F i l t r a t e ‘(m1/30 min.)* Cake Thickness (inches) HP-HT F i l t r a t e (m1/30 min.) PH 8.2 7.6 7.4 7.2 6.8 CEC ( m i l l iequivalents/100 gms) 125 125 125 100 25 Due t o the low clay content (2%) o f the fluid, the measurements o f A P I f i l t r a t e , cake thickness, and HP-HT f i l t r a t e were not feasible. These entries are l e f t blank i n t h i s and the other tables. . Gel Strengths were determined as explained i n the t e x t . Due t o the low clay content (2%) o f the fluid, the measurements o f A P I f i l t r a t e , cake thickness, and HP-HT f i l t r a t e were not feasible. These entries are l e f t blank i n t h i s and the other tables. Gel Strengths were determined as explained i n the t e x t . The f i r s t number before the slash refers t o the gel strength a f t e r 10 seconds and the second number (following the slash) t o the gel strength a f t e r 10 minutes. 1. Pure Bentonite Fluid 10 shows the viscosity changes of the 2% purified bentonite fluids under the pressures up to 12,000 psi as obtained w i t h the SANDIA viscometer. The f l u i d viscosity is, i n general, increased by high pressure, whereas the high temperature causes a t h i n n i n g of the f l u i d . bentonite fluid shows steadily higher viscosity w i t h increasing tempera- tures and pressures i n Figure 1. l b as compared w i t h its viscosity curve i n Figure 1 .la under a constant pressure of 650 psi. drastically reduced w i t h the pressures above 4,000 psi. fluids which were autoclaved a t 300 and 400°F show a drastic t h i n n i n g up t o 375°F and 5000 psi, but they all show a similar increase i n viscosity w i t h increasing pressures and temperatures (Figure 1. lb). The untreated (70°F) The viscosi,ty hump is The bentonite The above results clearly indicat that the pure Na-bentonite swells viscosity hump i n the temperature tremendously above 200°F and develops range 250 to 450°F when the pressures are kept low. has been demonstrated w i t h the increase of clay concentration to a more realistic value such as 6%. 6% purified bentonite fluid whose initial pH was adjusted to 6.5 w i t h This viscosity hump Figure l.lc shows this viscosity hump for a d. A t this concentration the viscosity he f l u i d is about 90 a t 70°F and i t rises bout 300 cp a t i t becomes a thick gel for which we co In the range 250-400°F measure the vis- cosity. The visco drastically decre s from 300 cp a t 400°F t o about 00°F. For the were autoclaved a t 300, OO"F, this vis osity data i n claved a t 6 11 30 25 tn L z 5 F y 20 E - 8 15 v 3 I= z w K 2 10 n e 5 0 1000 1000 3000 4000 6000 8000 10,000 11,000 12,000 PRESSURE(PS 2% BENTONITE/H,O UNDER HIGH PRESSURE . (SANDIA VISCOMETER) 1 , I I I I I 3 ( . 100 200 300 400 500 600 ( O F ) Figure l . X-ray Diffraction Anal ysi s The purified NL bentonite consists of a Na-smectite (montmorillonite) w i t h a 11.3 A basal spacing i n air-dried state. upon glycol saturation. 0 0 I t fully expands to 17 A No changes i n the basal spacing of smectite were observed upon autoclaving a t the temperature range 300-600°F i n distil led water. 1. Pure Bentonite Fluid l b - The rheograms o f the pure 2% bentonite fluids after autoclaving a t the temperatures marked on them. Figure l . l b - The rheograms o f the pure 2% bentonite fluids after autoclaving a t the temperatures marked on them. 5. e J - 8 .J 0 0 8 0 0 0 0 P 0 0 .-- , \ ' It \ 7 \ I The other properties of the 2% purified bentonite fluids are given i n Table 1.lb. Due to the extreme low concentration, there is not enough clay to form a measurable f i l t e r cake. Therefore, no meaningful f l u i d loss values can be obtained. fluid show a large increase w i t h the high-temperature treatments (Table Plastic viscosity and yield point of the 1.lb). remains almost unchanged thereafter. The gel strength shows a major jump from 70 to 300°F; and i t The pH and CEC values show a signif- icant decrease w i t h the higher temperatures. Electron Microscopic Data The original smectite particles consist of well-dispersed t h i n films or aggregates of such folded films. Those morphological features remain unchanged after autoclaving the f l u i d a t 300 and 400°F as illustrated i n Figures 1.2 through 1.4. I, The t h i n smectite films seem to disintegrate into smaller pieces after autoclaving the fluid a t 500°F as displayed i n Figure 1.5, Lath-like particles are also found i n small quantities a t his temperature (Figure 1.6a-1.6b). The lath-shaped particles become the predominant components of the fluid after autoclaving a t 600°F. tion, there are small amount c t i t e films. Figures 1.7 through 1.10 display common forms of the lath-shaped smectite particles i n the fluid. The x-ray spectra obtained from the laths show very l i t t l e Mg and In addi- 14 15 15 Figure #: 1.4 F1 u i d : Ben toni te/H20/40O0 F Magnification: 17,000 X Film #: 3181 Description : Typical smectite particles showing f l u f f y morphology and t h i n films i n the f l u i d which was autoclaved a t 400°F. Figure #: 1.5 F1 uid: Bentonite/H20/50O0F Magnification: 14,000 X Film #: 3185 Des c r i p t i on : Commonly observed smectf t e particles consisting o f t h i n films. Some o f the films are broken i n t o smaller pieces a f t e r the reaction. Figure #: 1.4 F1 u i d : Ben toni te/H20/40O0 F Magnification: 17,000 X Film #: 3181 Figure #: 1.4 F1 u i d : Ben toni te/H20/40O0 F Magnification: 17,000 X Film #: 3181 Descri p t i on : Descri p t i on : The selected area electron diffraction pattern obtained the lath shown i n Figure 1.6b The following uni t-cell para are obtained m the pattern: 0 - a = 5.22 A 0 - a = 5.22 A 0 - a = 5.22 A Figure #: 1.7 Fluid: Bentonite/H20/60O0F Magnification: 27,000 X Film #: 3192 Figure #: 1.7 Fluid: Bentonite/H20/60O0F Magnification: 27,000 X Film #: 3192 Description : Typical smectite particles showing f l u f f y morphology and t h i n films i n the f l u i d which was autoclaved a t 400°F. Figure #: 1.5 F1 uid: Bentonite/H20/50O0F Magnification: 14,000 X Film #: 3185 Des c r i p t i on : Commonly observed smectf t e particles consisting o f t h i n films. Some o f the films are broken i n t o smaller pieces a f t e r the reaction. Figure #: 1.5 F1 uid: Bentonite/H20/50O0F Magnification: 14,000 X Film #: 3185 Figure #: 1.5 F1 uid: Bentonite/H20/50O0F Magnification: 14,000 X Film #: 3185 Commonly observed smectf t e particles consisting o f t h i n films. Some o f the films are broken i n t o smaller pieces a f t e r the reaction. Figure 1.6a F1 uid: Bentoni te/H20/50O0 F Magnification: 27,000 X Film #: 3187 Description: A fluffy smectite aggreg showing lath-like extensions devel oped in the autoclaved fluid. Figure #: 1.6b F1 ui d: Bentoni te/Hz0/50 Magnification: 27,000 X Film #: 3188 Description: Another smecti te aggregate showing three sets of laths 17 Figure 1.6a F1 uid: Bentoni te/H20/50O0 F Magnification: 27,000 X Film #: 3187 Description: A fluffy smectite aggreg showing lath-like extensions devel oped in the autoclaved fluid. A fluffy smectite aggreg showing lath-like extensions devel oped in the autoclaved fluid. Figure #: 1.6b F1 ui d: Bentoni te/Hz0/50 Magnification: 27,000 X Film #: 3188 Description: Another smecti te aggregate showing three sets of laths 17 Figure #: 1 . 6 ~ F1 uid: Bentonite/H20/50O0F Magnification: SAD Film #: 3189 Descri ption : Sets of laths which are developed i n the fluid auto- claved a t 600OF. The minute hexagonal crystals contain Fe, A1 , and Si i n their composition. 18 Figure #: 1.8 F1 ui d: Benton Magn i f i cat i on Film #: 3195 Figure #: 1.8 F1 ui d: Benton Magn i f i cat i on Film #: 3195 19 Figure #: 1.10a F1 uid: Bentonite/H20/60O0F Magnification: 45,000 X F i l m #: 3198 Description: Description: Image o f an elongated smec- t i t e p l a t e l e t which has formed i n the f l u j d during the autoclaving process. Image o f an elongated smec- t i t e p l a t e l e t which has formed i n the f l u j d during the autoclaving process. Figure #: 1.10b F l uid: Bentoni te/H20/60O0 F Magnification: SAD Film #: 3197 Description: The selected area electron d i f f r a c t i o n pattern from the smectite p a r t i c l e shown above. The following uni t-cel 1 par- ameters are obtained from the pattern: - a = 5.21 A - b = 8.98 A 0 0 Figure #: 1.10b F l uid: Bentoni te/H20/60O0 F Magnification: SAD Film #: 3197 High Molecular Gleight Polymer $ 1 The f l u i d consisting o f 2% bentonite and 0.25% high molecular weight I . 1 , polymer shows a considerably higher viscosity (Figure 1.11) below 300°F as - 3 'the.pure 2% bentonite fluid. o r abote 3OO0F, it becomes extremely low i n viscosity (Fig- However, when t h i s f l u i d i s - , . ure 1.11 and Tables 1.2a and 1.2b). Plastic viscosity, y i e l d point, and gel strength o f he fluid decrease dras c a l l y a t and above 300°F. Both . pH and CEC va?u steadily decrease w i t the increasing temperatures. Thus, the above data clearly indicate that:;he high molecular weight poly- mer seems tocontrol the f l u i d viscosity thinner .in the f l u i d a t 0" F- and above. 2. Bentonite/Polymer Fluids High and low molecular weight sodium polyacrylates (SPA) are comnon A CYPAN-.. . + u i d additives mainly f o r the purpose o f f l u i d loss control. type SPA with a molecular weight i n the range o f 750,000 t o used as the high molecular weight polymer. The low molecula had a molecular.weight o f 940-1,400. Description: The selected area electron d i f f r a c t i o n pattern from the smectite p a r t i c l e shown above. The following uni t-cel 1 par- ameters are obtained from the pattern: - a = 5.21 A - b = 8.98 A 0 0 The selected area electron d i f f r a c t i o n pattern from the smectite p a r t i c l e shown above. The following uni t-cel 1 par- ameters are obtained from the pattern: - a = 5.21 A - b = 8.98 A 0 0 20 K, indicating a b e i d e l l i t e - l i k e composition. area electron diffraction data i n Figures 1 . 6 ~ and 1.10b indicate a 1M polymorph f o r these b e i d e l l i t e laths. Furthermore, the selected c t o f t h i s polymer, we increased the I n 'Fact, the addition o f bentonite concentration o f the f l u i d t o 6%. 0.25% low molecular weight 'polyme the gelation o f the f l u i d and kept it i n a f l u i d state although with a o the6% bentonite fl 2 1 Iu N 15 5 2% BENTONITE 0.25% HIGH MOLECULAR WEIGHT POLYMER \ MINERAL COMPONENTS IN THE FLUIDS: 7OoF SMECTITE 3OOOF SMECTITE 4OOOF SMECTITE 500°F SMECTITE 6OOOF SMECTITE -75OF 600” F i I I I I I “-5OOOF 100 200 300 400 500 (” F) Figure 1.11 - The FANN 50C rheograms of the 2% bentonite/0.25% high molecular weight polymer after autoclaving at temperatures marked on the curves. MINERAL COMPONENTS IN THE FLUIDS: 7OoF SMECTITE 3OOOF SMECTITE 4OOOF SMECTITE 500°F SMECTITE 6OOOF SMECTITE Figure 1.11 - The FANN 50C rheograms of the 2% bentonite/0.25% high molecular weight polymer after autoclaving at temperatures marked on the curves. TABLE 1.2~1. ISCOSITIES (CP) MEASURED WITH E FANN 50C VISCOMETER FOR 2% BENTONITE / 0.25% HIGH MOL. WEIGHT POLYMER THE UNTREATED AND AUTOCLAVED FtUIOS CONSISTING OF TABLE 1.2~1. ISCOSITIES (CP) MEASURED WITH E FANN 50C VISCOMETER FOR 2% BENTONITE / 0.25% HIGH MOL. WEIGHT POLYMER THE UNTREATED AND AUTOCLAVED FtUIOS CONSISTING OF A u t o c l a v e d a t t e m p e r a t u r e ( O F ) 75 19.8 2.7 2.2 1.9 3.3 100- 15.5 2.1 1.5 1.3 2.8 Apparent Viscosity (centipoise) 20.0 3.5 3.0 2.5 4.0 Plastic Viscosity (centipoise) 16.0 3.0 3.0 2.0 4.0 Yield Point (lb/100 sq ft) 8.0 1.0 0.0 1.0 0.0 Gel Strength (lb/100 sq ft) 4/4 0.5/2 0.5/1 0.5/1 0.5/1 A P I F i l t r a t e (m1/30 min.) Cake Thickness (inches) HP-HT F i l t r a t e (m1/30 min.) PH 7.7 7.5 7.2 7.4 7.2 CEC (milliequivalents/lOO gms) 125 100 75 75 50 F1 u i d Property Autoclaved a t temperatures ( O F ) 70 300 400 500 600 , TABLE 1.2b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 0.25% HIGH MOLECULAR WEIGHT POLYMER TABLE 1.2b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 0.25% HIGH MOLECULAR WEIGHT POLYMER rather high viscosity. The viscosity o f the sample before autoclaving increases drastically above 250°F and reaches a maximum o f 166 cp a t about 375°F (Figure 1.12 and Tables 1.3a and 1.3b). The typical viscosity hump i s again developed since the viscosity drops steeply from 400 t o 500°F (Figure 1.12). When the 6% bentonite f l u i d with 0.25% low molecular weight polymer was autoclaved 300"F, the viscosity o f the f l u i d drops i n t o a range o f 10- a viscosity hump. The other properties o f the f l u i d such as gel strength and pH are also s i g n i f i c a n t l y lower a t 300°F than those o f the untreated fluids. t i e s including apparent and p l a s t i c viscosities increase greatly i n the f l u i d s autoclaved a t 400°F and above (Figure 1.12). autoclaved a t 500 and 600°F become a gel and lose t h e i r f l u i d properties. Thus, the low molecular weight polymer does not seem t o control the high vlscosity o f the bentonite f l u i d a t high temperatures. However, a l l these f l u i d proper- I n fact, the f l u i d s 25 180 - MINERAL COMPONENTS IN THE FLUIDS: 70" F SMECTITE 6% BENTONITE 300" F SMECTITE 400°F SMECTITE ~ 1 4 0 - POLYMER 50OoF SMECTITE v) 6OOOF SMECTITE 160 - 0.25% LOW MOLECULAR WEIGHT 5 (EXTREMELY HIGH VISCOSITY WHEN AUTOCLAVED AT 500 AND 600° F) '100 - ). k 400" F 75" F I I I I I 100 200 300 400 500 ~ ( O F ) Figure 1.12 - The FANN 50C rheograms of the 6% bentonite/0.25% low molecular weight polymer after autoclaving a t the temperatures marked on the curves. Figure 1.12 - The FANN 50C rheograms of the 6% bentonite/0.25% low molecular weight polymer after autoclaving a t the temperatures marked on the curves. TABLE 1.3a PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 6% BENTONITE / 0.25% LOW MOLECULAR WEIGHT POLYMER - Autoclaved a t temperatures (OF) F1 u i d Property 70 300 400 500 600 Apparent Viscosity (centipoise) 59.0 29.0 85.5 Plastic Viscosity (centipoise) 4.0 22.5 44.0 Yield Point (lb/100 sq ft) 37.5 12.5 83.0 >, c, >, cc 0 V 0 V tn ul > > c c I I r Gel Strength (lb/100 sq ft) 4/4 3/3 10/11 '5; u) APE F i l t r a t e (m1/30 min.) r .r Cake Thickness (inches) cn cn .r .r HP-HT F i l t r a t e (m1/30 min.) TABLE 1.3a PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 6% BENTONITE / 0.25% LOW MOLECULAR WEIGHT POLYMER TABLE 1.3b APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 6% BENTONITE / 0.25% LOW MOL. WEIGHT POLYMER TABLE 1.3b APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 6% BENTONITE / 0.25% LOW MOL. WEIGHT POLYMER - Fann 50C Autoclaved a t temperature (OF) Temperature OF 70 300 400 500 600 75 100 150 200 250 300 350 400 450 500 35 .O 25.1 21.1 19.3 30.5 127.9 162.8 161.8 103.8 23.9 27.8 . 80.0 19.5 56.9 14.3 42.6 * L) * L) 9- .r- 11.2 31.4 cn ln 0 0 ln 0 u ln F .F 10.0 24.9 > > r .c m m .C .- 12.5 23.7 r i 2' 2 15.8 22.8 aJ aJ 16.9 27.6 > > 22.8 36.1 23.3 50.9 28 1. Bentonite/NaCl Fluid A 4% bentonite and 1% NaCl f l u i d exhibits extremely high viscos with increasing temperatures as shown by the rheograms i n Figure 2.1 t i e s and by the data i n Tables 2,la and 2.lb. The untreated mud displays a viscos- i t y increase from 32.6 cp a t 70°F t o 181 cps a t 300°F and it shows a simi- l a r decrease from the maximum a t 300°F t o 11.3 cp a t 500°F. After auto- claving the f l u i d a t the temperatures 30O-50O0F, these viscosities are s i g n i f i c a n t l y lower although the viscosity hump i s s t i l l observed. When autoclaved a t 600°F the f l u i d exhibits again very high viscosities. The other properties o f the 4% bentonite/l% NaCl f l u i d s - a r e given i n Table 2.lb. The pH and CEC values o f the f l u i d s show a steady'decrease with higher temperatures. No new mineral phase was detected on the x-ray d i f f r a c t i o n analysis o f the fluids subject d t o high temperature treatments, The large reduc- t i o n of CEC a t h i temperature suggests a possible decomposition @is- o l ution) o f smec i n s a l t solution a t high temperatures. NaCl extremely increases the f l u i d viscosity, Thus NaCl a t 1% concentration level seems t o promote the 'lostnotic" swelling o f bentonite particles i .e. a maximum up-take o f water by them, 2 , Bentoni te/NaOH F1 u i d These reaction products are inert for the rheology of the f l u i d but they form a t the expense of smectite i n the fluid. Further- more, they may cause serious permeability damage i f they precipitate i n the reservoir. 2 , Bentoni te/NaOH F1 u i d The addition of be ing o f the bentonite i n bentonite concentration t o 8% i n order t o obtain any appreciable viscosity e needed t o inc a t room temperhture. However, as the temperature i s raised, the f l u i d 29 w 0 120 I- z 40 a P 2 20 MINERAL COMPONENTS IN THE FLUIDS 70* F SMECTITE 300° F SMECTITE 400° F SMECTITE 50OOF SMECTITE 600OF SMECTITE 400° F 4% BENTONITE 1% NaCl (HALITE) I n I 100 200 300 400 500 ( O F ) Figure 2.1 - The FANN 50C rheograms of the 4% bentonite/l% NaCl fluids after autoclaving a t the temperatures marked on the curves. Figure 2.1 - The FANN 50C rheograms of the 4% bentonite/l% NaCl fluids after autoclaving a t the temperatures marked on the curves. TABLE 2.la APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR 4% BENTONITE / 1% NaCl THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF Fann 50C Autoclaved a t temperature (OF) Temperature OF 70 300 400 500 600 32.6 42.4 92.0 83.1 158.3 100 35.6 35.9 68.7 57.7 114.2 150 58.2 4 65.8 56.7 109.7 200 103.8 49.2 75.2 68.5 120.9 250 159.5 55.4 79.1 76.8 115.5 300 181.3 49.8 76.8 73.7 103.8 350 154.3 24.8 47.8 56.1 83.4 56.1 n.d. 7.9 12.9 31.2 TABLE 2.la APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR 4% BENTONITE / 1% NaCl THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF becomes highly viscous and reaches a maximum of 267 cpsat 300°F as indi- cated by the viscosity data i n Table 2.2a. The rheograms i n Figure 2.2 and the data i n Tables 2.2a and 2.2b show that the fluid autoclaved a t 300°F displays the highest viscosity among the autoclaved bentoni te/NaOH fluids. and 5OOOF. The viscosity declines steadily for the fluids autoclaved a t 400 The fluid autoclaved a t 600°F becomes extremely t h i n . The reduction i n viscosity of the fluids autoclaved a t and above 400°F seems to be related to the dissolution of bentonite and subsequent precipitation of minerals such as analcime and albite. The amount of these new phases becomes larger as the temperature increases. In fact, the CEC values of \ the fluids similarly show a drastic decline w i t h the increasing tempera- tures (Table 2.2b). 3. BentonitejNaOHISil ica F1 uid Free silica was added t o the previous bentonite/NaOH fluid i n order t o study the effect of silica on the fluid. posed of a 2% bentonite/l% NaOH/l% silica has been autoclaved i n the tem- perature range 300-600°F. The properties of the untreated and autoclaved fluids are summarized by the rheograms i n Figure 2.3 and by the data i n Tables 2.3a and 2.3b. The viscosities of these fluids are extremely low and uniform for the untreated as well as for the autoclaved fluids. Simi- larly, the yield point and gel strengths of these fluids are low as shown For this purpose a f l u i d com- i n Table 2,3b, Thus, the addition of free silica seems to be a significant factor i n controlling the high temperature gelation of the smectite fluid. 32 Apparent Viscosity (centipoise) 35.0 51.0 77.0 Plastic viscosity (centipoise) 12.5 20.5 32.0 (lb/100 sq ft) 45.0 61.0 90.0 Gel Strength (lb/100 sq ft) ,34148 47/70 57/72 PI Filtrate (m1/30 min.) 28 24 ake Thickness (inches) ’ HP-HT Filtrate (m1/30 min.) PH CEC (milliequivalents/lOO gms) 100 63 50 7.7 6.8 6.7 TABLE 2.lb PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 4% BENTONITE / 1% NaCl Autoclaved at temperatures ( O F ) F1 uid Property 70 300 400 500 600 75.0 127.0 41.5 38.0 67.0 178.0 60/67 104/115 30 61 32 .6.8 6.8 50 50 TABLE 2.lb PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 4% BENTONITE / 1% NaCl 33 3! 0 P I- 2 W a 3 2 10 . ' I 4OOOF + I MINERAL COMPONENTS IN THE FLUIDS: 7OoF SMECTITE 3OOOF SMECTITE + ANALCIME* 4OOOF SMECTITE + ANALCIME* + ALBITE* 50OOF SMECTITE + ANALCIME* + ALBITE* 6OOOF SMECTITE + ANALCIME* + ALBITE* NEW PHASES FORMED IN THE FLUID 8% BENTONITE 1% NaOH Figure 2.2 - The FANN 50C rheograms o f the 8% bentonite/l% NaOH fluids after autoclaving a t the temperatures marked on the curves. 3! 0 P I- 2 W a 3 2 10 . 3. BentonitejNaOHISil ica F1 uid ' I 4OOOF + I MINERAL COMPONENTS IN THE FLUIDS: 7OoF SMECTITE 3OOOF SMECTITE + ANALCIME 4OOOF SMECTITE + ANALCIME* + ALBITE* 50OOF SMECTITE + ANALCIME* + ALBITE* 6OOOF SMECTITE + ANALCIME* + ALBITE* NEW PHASES FORMED IN THE FLUID 8% BENTONITE 1% NaOH Figure 2.2 - The FANN 50C rheograms o f the 8% bentonite/l% NaOH fluids after autoclaving a t the temperatures marked on the curves. MINERAL COMPONENTS IN THE FLUIDS: 7OoF SMECTITE 3OOOF SMECTITE + ANALCIME 4OOOF SMECTITE + ANALCIME* + ALBITE* 50OOF SMECTITE + ANALCIME* + ALBITE* 6OOOF SMECTITE + ANALCIME* + ALBITE* NEW PHASES FORMED IN THE FLUID MINERAL COMPONENTS IN THE FLUIDS: 7OoF SMECTITE 3OOOF SMECTITE + ANALCIME 4OOOF SMECTITE + ANALCIME* + ALBITE* 50OOF SMECTITE + ANALCIME* + ALBITE* 6OOOF SMECTITE + ANALCIME* + ALBITE* NEW PHASES FORMED IN THE FLUID I- 2 W a 3 2 10 I I 8% BENTONITE 1% NaOH Figure 2.2 - The FANN 50C rheograms o f the 8% bentonite/l% NaOH fluids after autoclaving a t the temperatures marked on the curves. Figure 2.2 - The FANN 50C rheograms o f the 8% bentonite/l% NaOH fluids after autoclaving a t the temperatures marked on the curves. TABLE 2.2a APPARENT VISCOSITIES (CP) MEASURED U I T H THE FANN 50C VISCOMETER FOR 8% BENTONITE / 1% NaO THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF Fann 50C Temperature O F A u t o c l a v e d a t t e m p e r a t u r e ( O F ) 75 13.7 55.0 30.3 17.9’ 3.9 100 22.0 9.1 15.6 9.1 2.5 150 51.9 18.7 11.0 6.2 1.9 200 110.9 24.7 9.1 4.7 1.7 250 . 184.8 43.6 11.0 4.4 1.3 300 267.1 252.5 13.7 5.6 1.2 350 267.1 252.5 16.6 7.3 1.2 35 TABLE 2.2b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% NaOH Autoclaved a t temperatures ( O F ) F1 u i d Property 70 300 400 500 600 Apparent Viscosity (centipoise) Plastic viscosity ’ (centipoise) Yield Point (lb/100 sq ft) Gel Strength (lb/100 sq ft) API F i l t r a t e (m1/30 min.) Cake Thickness (inches) HP-HT F i l t r a t e (m1/30 min.) PH CEC (milliequivalents/lOO gms) 4.5 2.5 3.0 3/4 12.3 100 69.5 37.5 63.5 47/64 16 4/32 12.2 88 41 - 53 32 34 18 38 4/ 5 6/ 7 40 50 4/32 5/32 10.2 7.5 56 25 4.5 3.0 2.0 1/ 2 7.3 12.5 TABLE 2.2b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% NaOH 36 2% BENTONITE 1% SILICA I- + 1% NaOH r. MINERAL COMPONENTS IN THE FLUIDS: . I 7OoF SMECTITE + QUARTZ. 300OF SMECTlTk + QUARTZ +.ANALCIME + ALBITE* 406OF SMECTITE + QUARTZ +-ANALCIME* + ALBITE* " ' 500OF SMECTlTE + QUARTZ + ANALCIME* + ALBITE* NEW PHASES FORMED IN THE FLUID 600°F - + QUARTZ + ANALCIME + ALBITE' _. I t . X-ray Diffraction Data The main reaction products i n this f l u i d are analcime and albite (Na- feldspar). Analcime (Na A1 Si2 0 6.H20) starts already to form a t 300°F i n small quantities (10%) but it becomes the major component of the f l u i d a t 400 and 500°F. About-80% of the f l u i d solids consists of analcime after autoclavi'ng the f l u i d a t 500°F. Albite (Na-feldspar) forms i n small quantities (10%) a t 500°F but it makes up about 80% o f the sample a t 600°F. No smectite remains i n the f l u i d after adtoclaving a t 600°F. tite readily dissolves a t -higher temperature i n high a1 kaline solution and i t reacts w i t h free silica w i t h the above reaction products. Thus, smec- MINERAL COMPONENTS IN THE FLUIDS: 7OoF SMECTITE 3OOOF SMECTITE + ANALCIME* 4OOOF SMECTITE + ANALCIME* + ALBITE* 50OOF SMECTITE + ANALCIME* + ALBITE* 6OOOF SMECTITE + ANALCIME* + ALBITE* NEW PHASES FORMED IN THE FLUID Figure 2.3 - The FANN 50C rheograms o f the 2% bentonite/l% NaOH/l% s i l i c a f l u i d s a f t e r autoclaving a t the temperatures marked on the curves. MINERAL COMPONENTS IN THE FLUIDS: Figure 2.3 - The FANN 50C rheograms o f the 2% bentonite/l% NaOH/l% s i l i c a f l u i d s a f t e r autoclaving a t the temperatures marked on the curves. TABLE 2.3a APPARENT VISCOSITIES (CP) MEASURED WITH THE FAN 50C VISCOMETER FOR THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% NaOH / 1% SILICA ~~~ ~~ Fann 50C Temperature OF Autoclaved at temperature ( O F ) 70 300 400 500 600 75 100 150 2.5 2.7 1.9 2.0 3.9 1.8 1.7 1.1 1.6 2.8 1.7 1.4 0.9 1.3 2.5 200 1.4 1.3 0.8 1.1 2.3 2 50 1.3 1.3 0.9 1.2 2.1 300 1.0 1 b . 2 1.3 1.2 2.0 350 0.9 1.3 1.5 1.2 2.4 400 0.8 1.2 2.2 1.1 2.7 450 0.5 1.3 3.1 0.9 3.1 500 1.2 4.8 0.4 1.0 8.1 TABLE 2.3a APPARENT VISCOSITIES (CP) MEASURED WITH THE FAN 50C VISCOMETER FOR THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% NaOH / 1% SILICA 38 TABLE 2.3b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% NaOH / 1% SILICA Autoclaved a t temperatures ( O F ) , Fluid Property 70 300 400 500 600 Apparent Viscosity (centipoise) 1.75 3.25 3.0 2.75 3.75 Plastic Viscosity (centipoise) 1.50 2.50 2.50 2.50 2.50 Yield Point (lb/100 sq ft) 0.50 1,50 . 1.0 0.50 2.50 Gel Strength (lb/lOO sq ft) 0.5/1 1/2 0.5/1 1/4 1/2 API F i l t r a t e (m1/30 min.) c Cake Thickness Cinch HP-HT F i l t r a t e (m1/30 min.) - 12.2 12.1 11.8 11.7 TABLE 2.3b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% NaOH / 1% SILICA 39 The pH of the fluid evolves from highly alkaline (11.8) a t 70°F to alka- line (9.Z) a t 600°F. This alkaline solution seems to dissolve most of the bentonite since the CEC values are highly reduced after autoclaving a t 500 and 600°F. 4. Electron Microscopic Examination of Mineral Reactions Smectite particles mainly occur as extremely t h i n films i n the pres- ence of NaOH and silica a t temperatures up to 300°F. 2.7 display the typical morphological features of smectite films after autoclaving a t various temperatures. A t 400°F and 500°F smectite films Figures 2.4 through exhibit minor changes which consist of folds and curled edges. Smectites occur very scarcely after autoclaving the fluid a t 600°F. As shown i n Figure 2.7, those that survived the treatment consist of thicker flakes which survive the extensive dissolution of smectite a t higher temperatures. Analcime forms as the first reaction product between Na-smectite and silica i n sodium-rich solution (NaOH). ite seems to be the main source of silica since analcime forms w i t h or The amorphous silica i n the benton- 40 Figure #: 2.4 Fluid: Bentonite/NaOH/SiO2/30OoF Magnification: 21,000 X Film #: 3152 Typical smectite thin films i n the fluid after autoclaving at 300" F. Figure #: 2.5 Fluid: Benton Magni f i cati on Film #: 3155 Des cr i p t i on : Typical smectite thin films i n the fluid after autoclaving at 300" F. Figure #: 2.5 Fluid: Benton Magni f i cati on Film #: 3155 Des cr i p t i on : Des cr i p t i on : Figure #: 2.6 F1 uid: Bentonite/NaOH/Si Q2/500 Magnification: 27,000 X , Film #: 3161 Description: Smectite f i l m s whose broken edges may be caused by the dissolution i n the f l u i d auto- claved a t 500°F. "F Figure #: 2.7 F1 uid: Bentoni te/NaOH/SiO2/60O0F Magnification: 35,000 X Film #: 3169 Description: Smectite flakes remaining una1 tered a f t e r the reaction. e. A 42 Figure #: 2.6 F1 uid: Bentonite/NaOH/Si Q2/500 Magnification: 27,000 X , Film #: 3161 Description: Smectite f i l m s whose broken edges may be caused by the dissolution i n the f l u i d auto- claved a t 500°F. "F Description: Figure #: 2.7 F1 uid: Bentoni te/NaOH/SiO2/60O0F Magnification: 35,000 X Film #: 3169 Description: Smectite flakes remaining una1 tered a f t e r the reaction. e. A 42 42 Figure #: 2.8 Fluid: Bentoni te/NaOH/SiO2/5OOoF Magnification: 500 X Film #: a 0000 Descri p t j on : A general view o f the f l u i d solids prepared d i r e c t l y on the Cu-boat. The spheres are analcime aggre- gates and the fibers are a l b i t e crystal s . Figure F1 uid: Magn i f Film # Descri #: 2.9 Bentoni te/NaOH/Si02/50O0 F cation: 3,000 X a 0000 tion: A magnified view o f the anal- cime crystal displaying well developed faces. The x-ray spectra obtained from the crystal are given below: Intensity I n tensity Element counts/100 sec. ratio/Si Na 711 .04 A1 34 75 .21 S i 16171 1.00 Fe 794 .05 Figure #: 2.8 Fluid: Bentoni te/NaOH/SiO2/5OOoF Magnification: 500 X Film #: a 0000 Figure #: 2.8 Fluid: Bentoni te/NaOH/SiO2/5OOoF Magnification: 500 X Film #: a 0000 Descri p t j on : A general view o f the f l u i d solids prepared d i r e c t l y on the Cu-boat. The spheres are analcime aggre- gates and the fibers are a l b i t e crystal s . Figure F1 uid: Magn i f Film # #: 2.9 Bentoni te/NaOH/Si02/50O0 F cation: 3,000 X a 0000 Descri tion: A magnified view o f the anal- cime crystal displaying well developed faces. The x-ray spectra obtained from the crystal are given below: A magnified view o f the anal- cime crystal displaying well developed faces. The x-ray spectra obtained from the crystal are given below: Intensity I n tensity Element counts/100 sec. ratio/Si Na 711 .04 A1 34 75 .21 S i 16171 1.00 Fe 794 .05 45 Figure #: 2.13 F1 uid: Bentoni te/NaOH/Si02/50O0F Magnification: 4,000 X Film #: a0004 Description : A magnified view o f t h crystals whic forms. Figure #: 2.13 F1 uid: Bentoni te/NaOH/Si02/50O0F Magnification: 4,000 X Film #: a0004 Description : A magnified view o f t h crystals whic forms. Description : A magnified view o f t h crystals whic forms. A magnified view o f t h crystals whic forms. 46 1. Bentonite/KCl Fluid& o f a 4% bentonite and 1% KC1 f l u i d shows a sharp increase from 18 cp a t 75°F t o a maximum o f 238 cp a t 360°F (Figure 3.1 and Tables 3.la and 3.lb). However the viscosities o f the autoclaved f l u i d s are drastically reduced. Thus, KC1 seems t o control the viscosity I o f the f l u i d a t ~ h i g h temp atures. This i s l s o important t o remember ' f when trying t o minimize formation damage effects o f the d r i l l i n g f l u i d . ' 2. Bentonite/5CO3 Fluid The untreated f l u i d containing 8% bentonite and 1% K2C03 becomes t i c a l l y a gel with ve high viscosities beyond our measurement capa- played by the data i n Figure 3.2 and Tables 3.2a and 3.2b. f l u i d autoclaved a t 300°F shows rather high viscosities with a maximum f 110 cp a t 300°F. It develops a typical viscosity "hump",within the temperature range 250-450" A t and above the temperature o f 400°F .viscosities o f the f l u i d s become rather acceptable (ranging from 10 cp). The pH ofathe f l u i d evolves gradually from 9.0 t o 7.0 during the temperature range 300 t o - 00°F. The CEC values, on the other hand, show /lo09 at- 400°F and t o s and also i n viscos- mineral reactions i - 1 .. I 600°F i n the bentonite/KCl fluid. Furthermore, a t and above 500"F, K-feld- spars also form i n the bentonite/K2C03 f l u i d . 47 15 Ei F 6 a 2 w 0 Y cn 0 0 2 > P I- 00 z w a 4 a 5 , \ 300° F - 4% BENTONITE 1% K CI (SYLVITE) MINERAL COMPONENTS IN THE FLUIDS 70° F SMECTITE 300° F SMECTITE 500OF SMECTITE 60OOF I/S MIXED-LAYER* NEW PHASE FORMED IN THE FLUID 4000~ SMECTITE (VISCOSITY IS VERY HIGH FOR THE UNTREATED FLUID) -- 500°F 100 200 300 400 500 Figure 3.1 - The FANN 50C rheograms of the 4% bentonite/l% KC1 fluids after autoclaving a t the temperatures marked on the curves. P 00 Figure 3.1 - The FANN 50C rheograms of the 4% bentonite/l% KC1 fluids after autoclaving a t the temperatures marked on the curves. TABLE 3.la Fann 50C .. Autoclaved Temperature OF 70 300 75 18.3 15.4 100 27,7 12.5 150 63.1 12.5 200 110.1 13.3 250 f 181.7 11.5 300 229.3 7.5 a t temperature (OF) 400 500 600 3.9 7.8 9.4 5.8 2.8 5.0 5.4 2.5 3.9 . 5.1 2.1 3.4 4.6 1.7 2.9 3.9 1.4 3.2 237.6 5.1 350 , 400 237. 4.1 , 2.3 1.2 3.3 450 - 229.3 -3.3 2.0 1.2 3.0 . 2. Bentonite/5CO3 Fluid 500 177.8 2.5 1.8 1.0 2.6 3.1 1.3 3.3 TABLE 3.la 49 TABLE 3:lb PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 4% BENTONITE / 1% KC1 Autoclaved at' temperatures ( O F ) F1 u i d Property 70 300 400 500 600 Apparent Viscosity (centipoise) 31.0 19.0 14.0 6.0 - - 8.0 Plastic Viscosity (centipoise) 6.0 9.5 10.0 4.0 7.0 Yield Point (lb/100 sq ft) 49.5 19.0 7.5 4.0 3.0 Gel Strength (lb/100 sq ft) 34/40 15/26 6/8 3/4 2/2 API F i l t r a t e (m1/30 min.) 38 40 92 Cake Thickness (inches) 5/32 4/32 7/32 HP-HT F i l t r a t e (m1/30 min.) PH 7.8 6.9 6.2 5.6 4.2 CEC ( m i l l iequivalents/100 gms) 100 75 63 63 50 TABLE 3:lb PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 4% BENTONITE / 1% KC1 50 90 80 0 n m F: 60 z W 0 >. 50 k v) 0 Y 40 6 I= U 4 n 30 2 20 10 300°F -+ 300" F - MINERAL COMPONENTS IN THE FLUIDS I 3000F4\ 4 7OoF SMECTITE '8% BENTONITE 1% K, CO, 500°F I/S MIXED-LAYER + K-FELDSPAR* 600°F I/S MIXED-LAYER + K-FELDSPAR* 'NEW PHASES FORMED IN THE FLUID I - 600" F 100 200 300 400 500 (" F) Figure 3.2 - The FANN 50C rheograms of the 8% bentonite/l% K2CO3 fluid after autoclaving a t the temperatures marked on the curves. Figure 3.2 - The FANN 50C rheograms of the 8% bentonite/l% K2CO3 fluid after autoclaving a t the temperatures marked on the curves. TABLE 3.2a APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% K2C03 Fann 50C A u t o c l a v e d a t t e m p e r a t u r e ( O F ) Temperature O F 70 300 400 500 600 - 75 100 150 200 2 50 300 350 400 450 5130 83.1 35.1 25.1 24.1 35.1 110.1 107.6 ? 2. Bentonite/5CO3 Fluid 29.1 20.8 40.4 18.3 12.2 10.2 9.8 11.0 14.5 20.8 30.3 13.7 20.0 15.4 11.3 8.6 8.7 6.6 9.3 6.6 11.0 7.5 12.8 7.0 12.5 6.5 11.6 6.0 10.4 6.7 11.8 5.0 TABLE 3.2a APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% K2C03 52 TABLE 3.2b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF II 8% BENTONITE / 1% $C03 Autoclaved a t temperatures ( O F ) F1 u i d Property 70 300 400 500 600 Apparent V i scos i ty (cen ti poi se ) 106.0 38.5 19.0 14.0 P1 as t i c V i scosi ty (centipoise ) 58.5 29.0 17.0 11.5 Yield Point (lb/100 sq ft) c, >r 95.5 19.0 4.0 5.5 r VI VI 0 u 19/89 2/4 2/4 2/2 Gel Strength (lb/100 sq ft) O F API F i l t r a t e (m1/30 min.) > Cake Thickness (inches) I r m r HP-HT F i l t r a t e (m1/30 min.) PH CEC ( m i l l iequivalents/lOO gms) 81 38 38 31 9.0' 7.8 7.4 7.0 TABLE 3.2b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% $C03 TABLE 3.2b 3. Bentonite/KOH Fluid The properties of an 8% bentonite and 1% KOH f l u i d proved t o be rather useful f o r the control of the high-temperature gelation o f benton- i t e . that we needed t o increase bentonite concentration t o 8%. The viscosity of the untreated 8% bentonite/l% KOH f l u i d was extremely low as compared The i n i t i a l viscosity o f the f l u i d a t room temperature was so low with the high viscosities o f the untreated bentonite/KCl and bentonite/ %C03 fluids. the viscosity enhancement i s rather small when the f l u i d was autoclaved a t 300°F and above. 600°F. peratures. These reductions i n CEC and pH are consistent with the mineral reactions a t higher temperatures. Smectite seems t o dissolve i n the alka- l i n e conditions a t lower temperature and illite/smectite mixed-layer and K-feldspar precipitate already a t 400°F. These reaction products become larger i n quantity a t temperatures 500 and 600°F. The decrease i n smec- t i t e content o f the f l u i d and the nature o f the reaction products cause, however, an increase i n f l u i d loss while they depress favorably the vis- cosity o f the fluids. As shown by the data i n Figure 3.3 and Tables 3.3a and 3.3b, The pH o f the f l u d evolves from 12.3 a t 70°F t o 7.2 a t The CEC values show a significant reduction with increasing tem- + Thus, the inclusion o f K -ion i n the mud system as KOH i s more favorable i n controlling the visocsity enhancement o f the bentonite fluids. A t the same time, the permeability damage from swelling smectites i n the formation can be also reduced by the K+-ion i n the d r i l l i n g f l u i d . 4. 3. Bentonite/KOH Fluid Mineralogical Reactions i n Bentonite Fluids Containing Salts and Hydroxides o f Potassium 4% Bentonite and 1% KC1 Fluid: The x-ray d i f f r a c t i o n data indicated a s l i g h t but steady decrease o f the basal reflection o f K-smectite from 54 i VI VI 15 a 5000F-~ 8% BENTONITE 1% KOH MINERAL COMPONENTS IN THE FLUIDS: 7OoF SMECTITE 3OOOF SMECTITE 4OOOF I/S MIXED-LAY ER + K-FELDSPAR 500°F I/S MIXED-LAYER + K-FELDSPAR 6OOOF I/S MIXED-LAYER + K-FELDSPAR I I I I I 100 200 300 400 500 ( O F ) Figure 3.3 - The FANN 50C rheograms o f the 8% bentonite/l% KOH f l u i d s a f t e r autoclav- i n g a t the temperatures marked on the curves. I i VI VI a 5000F-~ 8% BENTONITE 1% KOH I I I I I 100 200 300 400 500 ( O F ) Figure 3.3 - The FANN 50C rheograms o f the 8% bentonite/l% KOH f l u i d s a f t e r autoclav- i n g a t the temperatures marked on the curves. I Figure 3.3 - The FANN 50C rheograms o f the 8% bentonite/l% KOH f l u i d s a f t e r autoclav- i n g a t the temperatures marked on the curves. TABLE 3.3a APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE! 3. Bentonite/KOH Fluid UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% KOH Fann 50C Temperature O F Autoclaved at temperature (OF) 70 300 400 500 600 75 100 150 200 250 300 350 400 450 500 1.6 1.8 3.1 3.7 4.4 4.6 3.8 3.8 3.7 3.6 6.6 4.6 7.9 3.7 5.3 3.1 4.8 2.7 5.0 2.5 4.0 2.2 4.7 2.1 3.8 2.0 4.4 2.0 4.1 1.8 4.2 2.3 5.1 1.8 3.9 3.3 6.5 2.0 3.9 4.1 6.4 2.0 3.7 4.4 5.0 2.0 3.8 4.8 4.4 1.9 56 TABLE 3.3b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% KOH Autoclaved a t temperatures (OF) F1 u i d Property 70 300 .400 ~ 500 600 Apparent Viscosity (cen ti poi se ) 2.0 7.5 6.0 8.0 5.5 Plastic Viscosity (centipoise) 2.0 5.0 5.0 7.5 5.0 Yield Point (lb/100 sq f t ) 0.5 5.0 2.0 1.0 1.0 Gel Strength (lb/100 sq ft) 0,5/1 3.5 0.5/1 1/1 1/ 1 API F i l t r a t e (m1/30 min.) 60 48 50 Cake Thickness (inches) 6/32 . 4/32 3/32 HP-HT F i l t r a t e (m1/30 min.) PH 12.3 12.0 9.5 9.0 7.2 CEC ( m i l l iequivalents/lOO gms) 100 75 44 31 25 TABLE 3.3b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% KOH 57 0 0 12.0 A at room temperature to 11.3 A after the autoclaving at 500°F. This suggests the development of illite/smectite mixed-layers in small amounts. Large quantities of il lite/smectite mixed-layers with the predominance of non-swelling illite component form at 600°F and they display a basal spac- ing of 10.9 A. 0 The newly developed illite/smectite mixed-layers display morphologi- cal features which are very different from those o f original smectite part- icles. a mosaic of such thin platelets are observed during the electron micro- scopic examination of the fluid autoclaved at 500°F (Figures 3.4a and 3.5). Most of the smectite still maintains its typical fluffy morphology with thin folded films as in Figure 3.4a. Thin platelets of illite/smectite mixed-layers become the predominant particles in the fluid after autoclav- ing at 600°F. ite/smectite platelets. 3.6b givesthe unit-cell dimensions of - a = 5.17 A and - b = 9.06 A as expec- ted from a dioctahedral mica-type layers. 3. Bentonite/KOH Fluid The x-ray spectra in Figure 3.7b indicate significantly larger K than was found in Figure 3.4b for the original smectite. Thin platelets of illite/smectite mixed-layers or aggregates with Figures 3.6a and 3.7a give close views of these thin ill- The selected area diffraction pattern in Figure 0 0 8% bentonite + 1% KOH and 8% bentonite + 1% K2C03 Fluids: Illite/ smectite mixed-layers with predominantly non-swelling illite components develop in large quantities already at 400°F in these fluids. saturated smectite gives a basal spacing of 11.8 A at 70°F and it remains The K- O unchanged after autoclaving at 300°F in these fluids. tion shifts to 20.8 A in the presence of KOH at and above 400°F. It shows, however, a steady decrease from 10.9 A at 400°F to 10.5 A at 600°F. The original smectite particles consist of aggregates of thin and This basal reflec- 0 0 0 58 59 Figure #: 3.4a Fluid: Bentonite/KC1/50O0F Magnification: 13,000 X Film #: 3678 The comnonly observed smec- t i t e particles i n the sample. The smectite occurs as f l u f f y aggregate with folded edges, however, small and thin f i l m - l i k e platelets are also developed i n the system. The comnonly observed smec- t i t e particles i n the sample. The smectite occurs as f l u f f y aggregate with folded edges, however, small and thin f i l m - l i k e platelets are also developed i n the system. The comnonly observed smec- t i t e particles i n the sample. The smectite occurs as f l u f f y aggregate with folded edges, however, small and thin f i l m - l i k e platelets are also developed i n the system. 59 Intensity counts/100 sec. m 0 0 Energy i n keV 10.00 0 Energy i n keV 10.00 m 0 10.00 0 61 Figure #: 3.6a F1 uid: Bentoni te/KC1/60O0 F Magnification: 88,000 X Film #: 3697 Description : Image o f an isolated thin platelet o f I/S mixed-layer. Film #: 3696 Desc ri p t i on : Desc ri p t i on : A close view o f the t h i n platelets from the above micrograph. The edges o f the platelets are curled up because o f dehydration under vacuum. A close view o f the t h i n platelets from the above micrograph. The edges o f the platelets are curled up because o f dehydration under vacuum. Figure #: 3.8a F1 uid: Bentoni te/KOH/30O0F Magnification: 13,000 X Film #: 3711 61 Figure #: 3.7a Fluid: Bentonite/KC1/60O0F Magnification: 65,000 X Film #: 3695 Figure #: 3.7a Fluid: Bentonite/KC1/60O0F Magnification: 65,000 X Film #: 3695 62 Descri p t i on : A t 600°F there are also a few extremely well-crystallized mica (muscovite) platelets devel- a i l l u s t r a t e s such a muscovite along with However, a small number o f platelets uid autoclaved a t 5OO0F, the illite/smec- I t s SAD e l a t t e r shows again a 1M polymorph o f muscovite. The ure 3.13~ indicate that the K- and A l - content o f the smectite mixed-layers. solves i n increasing amounts i n 1 amounts o f i 1 1 i tates i n the f l u i d and above 400°F. This conversion of smectite t o a mixed-layered phase with predominantly (non-swelling) i l l i t e gnificantly larger than those i n i l l i t e / Thus, the smectite i n the original bentonite dis- solution a t and above 300°F. Large s with t h i n p l a t y morphology precipi- folded films which do not undergo any significant changes i n morphology after autoclaving a t 300'F. Common smectite particles i n the f l u i d are displayed i n Figures 3.8a and 3.9. with a submicron particle-size i s also developed i n the f l u i d a t 300'F. The typical thin platelets o f i l l i t e / s m e c t i t e mixed-layers develop i n the f l u i d a t and above 400OF. Such platelets have irregular outlines and a sub-micron size as shown i n Figure 3.10a. The selected area electron dif- fraction pattern i n Figure 3.10b indicates a domain micro-structure lead- ing t o the equally strong 6 inner spots i n the pattern. The x-ray spectra i n Figure 3.10~ show the K-content o f the p l a t e l e t i s larger than that i n the original smectite. In the t i t e platelets bec larger and better i n crystalline forms. Figure 3. l l a shows such thin platelets o f i l l i t e / s m e c t i t e mixed-layers. pattern i n Figure 3.11b indicates a 1M-stacking sequence. Descri p t i on : The commonly observed particles 'n the autoclaved fluid. Smec- i t e occurs as t h i n films and does not show s i g n i f i c a t n t changes i n morphology, though a small amount of t h i n plate- been observed. 62 m w m w m w 0 9 s > W Y r .I- B W E w 0 9 s > W Y r .I- B W E w 09 s > W Y r .I- B W E w > W Y r .I- B W E w folded films which do not undergo any significant changes i n morphology after autoclaving a t 300'F. Common smectite particles i n the f l u i d are displayed i n Figures 3.8a and 3.9. with a submicron particle-size i s also developed i n the f l u i d a t 300'F. The typical thin platelets o f i l l i t e / s m e c t i t e mixed-layers develop i n the f l u i d a t and above 400OF. Such platelets have irregular outlines and a sub-micron size as shown i n Figure 3.10a. The selected area electron dif- fraction pattern i n Figure 3.10b indicates a domain micro-structure lead- ing t o the equally strong 6 inner spots i n the pattern. The x-ray spectra i n Figure 3.10~ show the K-content o f the p l a t e l e t i s larger than that i n the original smectite. In the t i t e platelets bec larger and better i n crystalline forms. Figure 3. l l a shows such thin platelets o f i l l i t e / s m e c t i t e mixed-layers. pattern i n Figure 3.11b indicates a 1M-stacking sequence. Both the K-con- tent and the c r y s t a l l i n i t y o f these i l l i t e / s m e c t i t e platelets are enhanced a t 600°F as displayed i n Figure 3.12a. The SAD p a t t e r n ' i n Figure 3.12b indicates a stacking sequence o f 1M i l l i t e polymorph. Descri p t i on : Both the K-con- tent and the c r y s t a l l i n i t y o f these i l l i t e / s m e c t i t e platelets are enhanced a t 600°F as displayed i n Figure 3.12a. The SAD p a t t e r n ' i n Figure 3.12b indicates a stacking sequence o f 1M i l l i t e polymorph. A t 600°F there are also a few extremely well-crystallized mica (muscovite) platelets devel- a i l l u s t r a t e s such a muscovite along with However, a small number o f platelets uid autoclaved a t 5OO0F, the illite/smec- I t s SAD e l a t t e r shows again a 1M polymorph o f muscovite. The ure 3.13~ indicate that the K- and A l - content o f the smectite mixed-layers. solves i n increasing amounts i n 1 amounts o f i 1 1 i tates i n the f l u i d and above 400°F. This conversion of smectite t o a mixed-layered phase with predominantly (non-swelling) i l l i t e gnificantly larger than those i n i l l i t e / Thus, the smectite i n the original bentonite dis- solution a t and above 300°F. Large s with t h i n p l a t y morphology precipi- 65 Figure #: 3.9 Fluid: Bentonite/KOH/30O0F Magnification: 45,000 X Film #: 3712 Description: A close view o f a smectite aggregate showing typical thin film-1 i ke morphology. A close view o f a smectite aggregate showing typical thin film-1 i ke morphology. A close view o f a smectite aggregate showing typical thin film-1 i ke morphology. Figure #: 3.10a F1 uid: Bentonite/KOH/40O0F Magnification: 66,000 X Film #: 3710 Description: A close view o f an I/S mixed- layer occurring as an isolated thin platelet. Description: A close view o f an I/S mixed- layer occurring as an isolated thin platelet. 66 QI U 0 Energy In keV 10.00 QI U 0 Energy In keV 10.00 0 Energy In keV 10.00 QI U 0 Figure #: 3.10b F1 uid: Bentonite/KOH/40O0F Magnification: SAD Film #: 3709 Desc r i p t i on : Desc r i p t i on : The selected area electron diffraction pattern obtained from the I/S platelet i n the above micrograph. The calcu- lated cell parameters are: 0 a = 5.17 f .01 A - b = 8.88 -+ .01 A 0 0 a = 5.17 f .01 A - b = 8.88 -+ .01 A 0 0 a = 5.17 f .01 A - b = 8.88 -+ .01 A 0 Figure #: 3.11a F1 u i d : Bentoni te/KOH/50O0 F Magnification: 88,000 X Film #: 3454 Description: A magnified view o f an I/S mixed-layer occurring as t h i n platelet. 68 68 Figure #: 3.11b F1 uid: Bentoni te/KOH/500°F Magnification: SAD Film #: 3453 Description: The selected area electron diffraction pattern obtained from the above t h i n platelet. The following uni t-cell parameters are obtained: 0 - a = 5.19 2 0.01 A - b = 8.93 k 0.01 A 0 0 - a = 5.19 2 0.01 A - b = 8.93 k 0.01 A 0 Figure #: 3.12a Fluid: Bentoni te/KOH/60O0F Magnification: 66,000 X Film #: 3446 Description: An isolated illite platelet. The selected area electron ' diffract ion pattern i ndi cates the illite possesses a 1 - M type structure. \ tha Figure #: 3.12a Fluid: Bentoni te/KOH/60O0F Magnification: 66,000 X Film #: 3446 The selected area electron diffraction pattern obtained from the i l l i t e platelet shown i n the above micrograph. The calculated cell parameters are : The selected area electron diffraction pattern obtained from the i l l i t e platelet shown i n the above micrograph. The calculated cell parameters are : 0 a = 5.22 A - 0 b = 8.99 5 .03 A - Figure #: 3.13a F1 u i d: Bentoni te/KOH/60O0 F Magnification: 35,000 X Film #: 3702 Description: An isolated illite platelet. The selected area electron ' diffract ion pattern i ndi cates the illite possesses a 1 - M type structure. \ that 69 70 Figure #: 3.12b F1 uid: Bentoni te/KOH/600° F Magnification: SAD Film #: 3445 Figure #: 3.12b F1 uid: Bentoni te/KOH/600° F Magnification: SAD Film #: 3445 Des c ri p t i on : Des c ri p t i on : A close view of a well-crys- tal ized muscovite platelet. The x-ray spectral data i n Figure 3.13~ from the muscov- ite indicate a higher amount of K and A1 as compared w i t h the composition of I/S mixed- 1 ayers. 70 t l y related to the rheological behavior of the reaction product i n smaller quan- ove 400°F. Figure 3.14a displays f about 2 microns. The feldspar The x-ray spectra i n Figure 3.14a spar composition. ary, the mineral the fl uids a t and above 400°F can d-layers + K-feldspar. 72 Figure #: 3.13b F1 u i d: Bentoni te/KOH/60O0 F Magnification: SAD Film #: 3703 Description: Description: The selected area electron d i f - fraction pattern obtained from the mica platelet i n the above figure. The uni t-cel 1 parameters are calculated to be: - a = 5.17 i 0.01 A - b = 9.07 5 0.01 A 0 0 - a = 5.17 i 0.01 A - b = 9.07 5 0.01 A 0 0 - a = 5.17 i 0.01 A - b = 9.07 5 0.01 A 0 0 - b = 9.07 5 0.01 A 0 Figure #: 3.14a Fluid: Bentonite/KOH/60O0F Magnification: 44,000 X Film #: 3704 Description: A close view of a K-feldspar crystal w i t h a mosaic o f pseudo-hexagonal platelet. 72 counts/100 sec. U P Energy in keV 10.00 0 Energy in keV 10.00 0 U P U P 0 1. Bentonite/CaClp Fluid trong i n h i b i t i n g effect of calcium ion on the swell- e increased the bentonite concentration i n the fluid The viscosities of an to 8% i n order to have some measurable viscosities. bentonite/l% CaC12 f l u i d remain extremely low a t all temperatures as displayed i n Figure 4.1 and Table 4.la. The pH of the f l u i d shows a slight decli 7.7 a t 70°F t o 7.0 a t 600OF. However, the CEC values significantly decrease from 100 meq/100g a t 70°F t o 50 meq/lOOg a t 600°F (Table 4. lb). 2. Bentonite/Lime Fluid that "spud" muds are prepared from bentonite and me. The thixotropy obtained from such muds is related to the floccula- tion of the bentonite a t a high concentration. However, such a high viscous bentonite/lime fluid does not have any f l u i d loss control. major problem i n d r i l l i n g technology is related to the poor bonding between the cement wall. The additio tion of cement min A the casing and the filter cake on the borehole me to the bentonite fluid may lead t o the forma- t high temperatures. . We therefore examined the fects of lime addi t both low (2% bentonite/l% dand higher The rheological given by the rheogra 4.2b. The addition of lime decreases drastically the viscosity of the bentonite fluid a t all temperatures from 70 to 6OO0F, as compared w i t h rs of the 2% bentonite/l% lime f l u i d are data i n Tables 75 U m 5 8% BENTONITE 1% CaCI, MINERAL COMPONENTS IN THE FLUIDS: 7OoF SMECTITE 3OOOF SMECTITE 400" F SMECTITE 50OoF SMECITE 6OOOF SMECTITE 100 200 300 400 500 (" F) Figure 4.1 - The FANN 50C rheograms o f the 8% bentonite/l% CaC12 fluids which were autoclaved a t high temperatures up to 600°F. MINERAL COMPONENTS IN THE FLUIDS: 7OoF SMECTITE 3OOOF SMECTITE 400" F SMECTITE 50OoF SMECITE 6OOOF SMECTITE U m Figure 4.1 - The FANN 50C rheograms o f the 8% bentonite/l% CaC12 fluids which were autoclaved a t high temperatures up to 600°F. TABLE 4.la APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% CaC12 THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% CaC12 > fann 50C Autoclaved a t temperature ( O F ) Temperature OF 70 300 400 500 600 75 3.2 2.3 2.5 2.5 100 2.8 2.3 2.8 2.4 150 2.2 1.7 2.5 1.9 200 1.3 1.4 2.1 1.5 250 1.1 1.3 2.0 1.3 300 1.1 1.3 1.9 1.3 350 1.2 1.3 1.9 1.3 400 1.2 1.4 1.9 1.3 450 1.4 .1.5 2. 1.4 500 1.2 1.3 1.5 1.0 1.7 1.9 1.6 1.3 1.2 1.1 1.0 1.0 1.0 1.0 77 TABLE 4. lb PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% CaC12 . 2. Bentonite/Lime Fluid Autoclaved at -temperatures -(OF) Fluid Property 70 300 400 500 .- 600 Apparent Viscosity (centipoise) 2.0 2.25 Plastic Viscosity (centipoise) 1.5 2.0 Yield Point (lb/100 sq ft) 1.0 0.5 Gel Strength (lb/100 sq ft) API Filtrate (m1/30 min.) 0.5/2 1/2 2.75 2.5- 2.5 2.0 2.0 2.0 1.5 1.0 1.0 1/2 1/1.5 1/1.5 Cake Thickness (inches HP-HT Filtrate (m1/30 min. ) PH 7.7 6.8 6.9 7.1 7.0 CEC (milliequivalents/lOO gms) 100 69 62.5 56 50 TABLE 4. lb PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% CaC12 78 MINERAL COMPONENTS IN THE FLUIDS ~ O O F SMECTITE + CALCITE* U a 300°F SMECTITE + CALCITE* + TOBERMORITE* ~ 400°F SMECTITE + CALCITE* + TOBERMORITE* 500°F SMECTITE + CALCITE* + TOBERMORITE* + Ca-FELDSPAR* 600°F SMECTITE + CA MORlTE* + Ca-FELDSPAR* NEW PHASES FORME ) 2% BENTONITE 1% LIME '1 100 200 300 400 500 "F 4 Figure 4.2 - The FANN 50C rheograms of the 2% bentonite/l% lime fluids which were autoclaved a t the temperatures marked on the curves. MINERAL COMPONENTS IN THE FLUIDS ~ O O F SMECTITE + CALCITE 300°F SMECTITE + CALCITE* + TOBERMORITE* 400°F SMECTITE + CALCITE* + TOBERMORITE* 500°F SMECTITE + CALCITE* + TOBERMORITE* + Ca-FELDSPAR* 600°F SMECTITE + CA MORlTE* + Ca-FELDSPAR* NEW PHASES FORME ) MINERAL COMPONENTS IN THE FLUIDS ~ O O F SMECTITE + CALCITE 300°F SMECTITE + CALCITE* + TOBERMORITE* 400°F SMECTITE + CALCITE* + TOBERMORITE* 500°F SMECTITE + CALCITE* + TOBERMORITE* + Ca-FELDSPAR* 600°F SMECTITE + CA MORlTE* + Ca-FELDSPAR* NEW PHASES FORME ) Figure 4.2 - The FANN 50C rheograms of the 2% bentonite/l% lime fluids which were autoclaved a t the temperatures marked on the curves. TABLE 4.2a APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN %)C VISCOMETER FOR THE 2% BENTONITE / 1% LIME UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF Fann 50C A u t o c l a v e d a t t e m p e r a t u r e ( O F ) Temperature O F 70 300 400 500 600 75 100 150 200 250 300 350 400 450 500 3.2 2.7 2.3 1.4 1.8 3.0 2.3 1.9 1.2 1.6 2.7 2.1 1.8 0.9 1.3 2.2 1.9 1.7 0.2 1.0 2.3 1.7 1.4 0.3 0.9 2.1 1.5 1.0 0.4 1.0 1.9 1.3 0.9 0.8 1.0 1.8 1.2 0.9 0.9 0.9 1.9 1.5 1.0 0.4 0.8 2.0 1.7 1.1 1.0 0.8 TABLE 4.2a APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN %)C VISCOMETER FOR THE 2% BENTONITE / 1% LIME UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 80 TABLE 4.2b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% Ca(OH)2 Autoclaved a t temperatures ( O F ) F1 u i d Property 70 300 400 500 600 Apparent Viscosity (centipoise) 1.75 3.5 3.5 2.5 1.25 Plastic Viscosity (centipoise) 1.50 2.0 3.0 2.5 1.0 Yield Point (lb/100 sq ft) 0.50 3.0 1.0 0.0 0.5 Gel Strength (lb/100 sq ft) 0.5/4 2/2 2/2 0/5/0.5 0.5/0.5 API F i l t r a t e (m1/30 min.) Cake Thickness (inches) HP-HT F i l t r a t e (m1/30 min.) PH 12 11.6 11 9.2 9.2 CEC (milliequivalents/lOO gms) 125 50 50 25 25 TABLE 4.2b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% Ca(OH)2 81 the pure 2% bentonite f l u i d . evolving from 12 a t 70°F t o 9.2 a t 600"F, remains always highly alkaline. The CEC values drastically decrease from 125 meq/lOOg a t 70°F t o 25 meq/ The pH o f the 2% bentonite/l% lime fluid, lOOg a t 600°F. MINERAL COMPONENTS IN THE FLUIDS ~ O O F SMECTITE + CALCITE 300°F SMECTITE + CALCITE* + TOBERMORITE* 400°F SMECTITE + CALCITE* + TOBERMORITE* 500°F SMECTITE + CALCITE* + TOBERMORITE* + Ca-FELDSPAR* 600°F SMECTITE + CA MORlTE* + Ca-FELDSPAR* NEW PHASES FORME ) This indicates a substantial dissolution o f smectite i n alkaline conditions with the subsequent reaction products. The higher bentonite concentration i n 8% bentonite/l% lime f l u i d gives r i s e t o higher viscosity values f o r the untreated f l u i d and f o r those autoclaved a t 300 and 400°F as indicated by the rheograms i n Figure 4.3 and by the data i n Tables 4.3a and 4.3b. tures (500-600°F) the f l u i d viscosities drop drastically. However, a t higher tempera- The pH o f the f l u i d s reduces from 12 a t 70°F t o 7.4 a t 600OF. Similarly, the CEC values suffer a large decrease from 100 meq/100g a t 70°F t o 1.25 meq/100g a t 600" F. 3. Bentonite/Lime/Silica Fluid Free s i l i c a was added t o the bentonite/lime f l u i d with the expecta- t i o n t o enhance the cement formation since the cement reactions are gener- a l l y based on the reactions between lime and s i l i c a . f l u i d consisting o f 2% bentonite, 1% lime, and 1% s i l i c a was autoclaved a t temperatures from 300 t o 600OF. are represented by the data i n Figure 4.4 and i n Tables 4.4a and 4.4b. For t h i s purpose a The rheological properties o f t h i s f l u i d The viscosities o f t h i s f l u i d are extremely low a t a l l temperatures as it was the case for' the 2% bentonite/l% lime fluid. The pH range o f the f l u i d s i s also similar t o the previous bentonite/lime fluid, evolving from 12.8 a t 70°F t o 9.2 a t 600°F. One unusual parameter o'f the f l u i d i s i t s low CEC value (75 meq/100g) a t the ambient temperature. The CEC drops however t o 25 meq/100g a t 6OO0F, indicating a substantial decomposition o f smectite i n the a1 kal ine conditions a t high temperatures. co w 40 n g 35 5 p. z W 0 30 Y c E 15 U p. a 4 10 5 400" F It 750F\ \ "" : i MINERAL COMPONENTS IN THE FLUIDS: 70°F SMECTITE + CALCITE 300OF SMECTITE + CALCITE 4OOOF SMECTITE + CALCITE 5OOOF SMECTITE + CALCITE + Ca-FELDSPAR 600" F SMECTITE + Ca-FELDSPAR 8% BENTONITE 1% LIME Figure 4.3 - The FANN 50C rheograms o f the 8% bentonite/l% lime f l u i d s which were autoclaved a t the temperatures marked on the curves. MINERAL COMPONENTS IN THE FLUIDS: 70°F SMECTITE + CALCITE 300OF SMECTITE + CALCITE 4OOOF SMECTITE + CALCITE 5OOOF SMECTITE + CALCITE + Ca-FELDSPAR 600" F SMECTITE + Ca-FELDSPAR co w Figure 4.3 - The FANN 50C rheograms o f the 8% bentonite/l% lime f l u i d s which were autoclaved a t the temperatures marked on the curves. if the f i l t r a t i o n time was below 30 minutes, it i s indicated following the slash. 3. Bentonite/Lime/Silica Fluid TABLE 4.3a APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE 8% BENTONITE / 1% LIME UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF Fann 50C Temperature OF Autoclaved a t temperature (OF) 70 300 400 500 600 . 75 100 150 200 250 300 350 400 450 24.1 14.3 11.6 9.6 7.5 7.1 6.8 7.5 9.1 500 12.9 18.7 37.5 11.5 22.0 8.4 21.3 5.8 21.2 3.5 18.7 2.5 12.0 2.1 5.6 1.7 3.0 2.9 1.7 14.9 18.3 3.4 2.1 2.8 2.0 2.4 1.7 2.3 1.5 2.4 1.5 2.4 1.5 2.1 1.2 1.9 1.0 2.0 1.5 1.9 1.2 TABLE 4.3a APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE 8% BENTONITE / 1% LIME UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 84 TABLE 4.3b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% LIME Autoclaved a t temperatures (OF) I . 'Cake Thickness (inches) 'HP-HT F i l t r a t e ik1/30 min.) 45/1 60/2 . VH 12.0 10.4 10.0 7.8 7.4 CEC (milliequivalents/lOO gms) 100 50 56 50 12.5 HP-HT Cake Thickness (inches) 2 3/ 32 if the f i l t r a t i o n time was below 30 minutes, it i s indicated following the slash. TABLE 4.3b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 8% BENTONITE / 1% LIME 85 MINERAL COMPONENTS IN THE FLUIDS 70°F SMECTITE + QUARTZ + CALCITE 300°F SMECTITE + QUARTZ + CALCITE* + TOBERMORITE* 400°F SMECTITE + QUARTZ + CALCITE* + TOBERMORITE 500" F SMECTITE + QUARTZ + Ca-FELDSPAR* + TOBERMORITE* + CALCITE* 600°F SMECTITE + QUARTZ + Ca-FELDSPAR* + CALCITE* + TOBERMORITE* 5 NEW PHASES FORMED IN THE FLUID 2% BENTONITE 1% LIME 1% SILICA 30O"Fq. 0°F 100 200 300 400 500 O F Figure 4.4 - The FANN 50C rheograms o f the 2% bentonite/l% lime/l% silica fluids which were autoclaved a t the temperatures marked on the curves. Figure 4.4 - The FANN 50C rheograms o f the 2% bentonite/l% lime/l% silica fluids which were autoclaved a t the temperatures marked on the curves. 3. Bentonite/Lime/Silica Fluid TABLE 4.4b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / -1% LIME / 1% SILICA Autocal ved at temperatures ( O F ) F1 ui d Property 70 300 400 500 600 Apparent Viscosity (centipoise) 2.25 3.25 3.0 2.5 2.25 Plastic Viscosity (centipoise) 1.5 2.5 2.5 2.5 2.5 Yield Point (lb/100 sq ft) 1.5 1.5 1.0 1.0 0.5 Gel Strength (lb/100 sq ft) 1/2 1.512 114 111.5 0.513 API Filtrate (m1/30 min.) Cake Thickness (inches) HP-HT Filtrate (m1130 min.) PH 11.8 11.2 9.8 9.0 9.2 CEC (milliequivalents/lOO gms) 75 75 75 25 25 TABLE 4.4b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / -1% LIME / 1% SILICA 88 8% Bentonite/l% CaC12 Fluid The x-ray diffraction data show no new mineral phases formed i n the f l u i d except for halite. The latter is due to the exchange of Na i n smec- tite for calcium i n the solution, and halite forms when the chloride solu- tion w i t h Na has been dried a t room temperature. seems to be too low for the formation of tobermrite, a cement mineral. The pH of the f l u i d 2% Bentonite/l% Ca(OH)2 Fluid The main reaction products i n the 2% bentonite/l% lime fluids are calcite and tobermorite. to calcite even a t the ambient temperature when the fluid is exposed to C02 i n the air. Tobermrite is the cement mineral w i t h the formal composition 5 CaO. 6 Si02 . 5H20. i n large quantities a t ite forms i n small amounts a t 500°F but i n larger quantities a t 600°F. Calcite is generated by the conversion of lime T h i s readily happens during the air-drying of the f l u i d . Tobermorite forms only i n trace quantities a t 300°F b u t 0°F and 500°F. A Ca-feldspar similar to anorth- Consequently the tobermorite content is reduced t o about 20% a t 600OF. 8% Bentonite/l% Ca (0H)z Fluid X-ray d i f f r a c t i o n analysis shows that no tobermorite forms i n the system. This may be caused by the lower pH. values as well as lower dis- solved Ca/Si r a t i o s i n these fluids. product a t and below 500°F and- it.may form due t o the same reasons as dis; cussed above. A calcium-feldspar forms, however, i n small quantities a t 500°F and i n larger amounts a t 600°F. Smectites remain i n the system i n t h e i r Ca-forms with a basal spacing o f 15.2A. Calcite i s the prominent reaction 0 Electron Microscopic Observations ent o f the f i u i d a f t e r autoclaving a t 500°F. These tobermorites consist o f laths as shown i n Figures 4.9a and 4.9b. Some o f these laths display dissolution features. f l u i d a f t e r autoclaving a t 600OF. A typical tobemorite formed a t 600°F i s displayed i n Figure 4.10a with i t s SAD pattern i n Figure 4.10b. Tobermorite becomes the predominant compon- Tobermorite remains as a major component o f the found a t 400°F as shown i n Figure 4.8a along with i t s SAD pattern i n Fig- ure 4.8b. Calcite rhombs and irregular spherules occur also i n the f l u i d a f t e r autoclaving a t 400°F. Smectite particles decrease i n quantity i n the f l u i d a t and above 400°F. ent o f the f i u i d a f t e r autoclaving a t 500°F. These tobermorites consist o f laths as shown i n Figures 4.9a and 4.9b. Some o f these laths display dissolution features. f l u i d a f t e r autoclaving a t 600OF. A typical tobemorite formed a t 600°F i s displayed i n Figure 4.10a with i t s SAD pattern i n Figure 4.10b. Tobermorite becomes the predominant compon- Tobermorite remains as a major component o f the Electron Microscopic Observations Smectite particles are found t o have dissolved and disintegrated w i t h mperatures, Typical smectites are shown i n Figure 4.5a after autoclaving the f l u i d a t 300°F and i n Figure 4.5b after the treatment a t 600°F. gated w i t h smectite films. Tobermorite crystals occur a t 300°F as laths which are often aggre- Figures 4.6 and 4.7 display such tobermorite and smectite particles i n the fluid. Well-developed tobermorite laths are 89 90 Figure #: 4.5a F1 uid: Bentoni te/Ca( OH)2/3000F Magnification: 26,000 X Film #: 3129 Desc ri p t i on : Figure #: 4.5a F1 uid: Bentoni te/Ca( OH)2/3000F Magnification: 26,000 X Film #: 3129 Desc ri p t i on : Fluffy smectite t h i n films i n the autoclaved fluid. The x-ray spectra obtained from the smectite are given below: Intensity Intensity Element count/100 sec. ratio/Si Intensity Intensity Element count/100 sec. ratio/Si A1 859 .20 Si 30 32 1.00 Ca 594 .20 Fe 2 34 .08 Figure #: 4.5b F1 u i d: Bentoni te/Ca( OH)2/6000F Magnification: 20,000 X Film #: 3147 Description : Smectite flakes i n the auto- claved f l u i d . Figure #: 4.5b F1 u i d: Bentoni te/Ca( OH)2/6000F Magnification: 20,000 X Film #: 3147 Description : Smectite flakes i n the auto- claved f l u i d . 90 Figure #: 4.6 Film #: 3124 Description: Comonly observed aggregates of tobermori te and smecti te. The x-ray spectra obtained from the aggregate are given bel ow: Intensity Intensity Element counts/100 sec. ratio/Si A1 695 .20 Si 352 7 1.00 Ca 245 i .69 Fe 119 .03 91 Figure #: 4.6 Film #: 3124 Description: Comonly observed aggregates of tobermori te and smecti te. The x-ray spectra obtained from the aggregate are given bel ow: Intensity Intensity Element counts/100 sec. ratio/Si A1 695 .20 Si 352 7 1.00 Ca 245 i .69 Fe 119 .03 91 found a t 400°F as shown i n Figure 4.8a along with i t s SAD pattern i n Fig- ure 4.8b. Calcite rhombs and irregular spherules occur also i n the f l u i d a f t e r autoclaving a t 400°F. Smectite particles decrease i n quantity i n the f l u i d a t and above 400°F. 2% Bentonitell% Lime/l% Si02 Fluid The major reaction products i n t h i s f l u i d are tobermorite, calcite, Calcite and Ca-feldspar as i n the previous 2% bentonite/l% lime f l u i d . readily -forms even a t the ambient temperature due t o the reaction o f lime w i t h C02, which mostly takes place during the drying o f the mud i n the air. Tobermorite develops i n the f l u i d i n small amounts (5%) during autoclaving a t 300°F but i n larger amounts (1530%) a t higher temperatures (400-600°F). It i s interesting t o note that significantly less tobermorite i s formed i n t h i s f l u i d (with extra s i l i c a ) than i n the 2% bentonite/l% l i m e fluid. 92 Figure #: 4.8a F1 ui d: Bentoni te/Ca( OH)2/4000 Magnification: 9,000 X Film #: 3134 Description: #el 1 -devel oped tobermori t e plates i n the autoclaved fluid. 'F Figure #: 4.8b F1 uid: Bent0nite/Ca(OH)~/40O~F Film #: 3133 area electron diffraction pattern of the shown above. 93 / Figure #: 4.8a F1 ui d: Bentoni te/Ca( OH)2/4000 Magnification: 9,000 X Film #: 3134 Description: #el 1 -devel oped tobermori t e plates i n the autoclaved fluid. 'F #el 1 -devel oped tobermori t e plates i n the autoclaved fluid. Figure #: 4.8b F1 uid: Bent0nite/Ca(OH)~/40O~F Film #: 3133 area electron diffraction pattern of the shown above. area electron diffraction pattern of the shown above. 93 Figure #: 4.9a F1 uid: Bentoni te/Ca ( OH)2/5000 F Magnification: 16,000 X Film #: 3145 Description: Image o f the tobermorite fibers showing dissolution features. - Ca ( OH)2/5000 F ,000 X rmorite ssolution Ca(OH)2/5000F ,000 X o f the tober- m the above 94 Image o f the tobermorite fibers showing dissolution features. Figure #: 4.9b F1 uid: Bentoni te/Ca(OH)2/5000F Magnification: 50,000 X Film #: 3144 Description: A magnified view o f the tober- morite fibers from the above micrograph. Film #: 3144 Description: Description: A magnified view o f the tober- morite fibers from the above micrograph. 94 94 Figure #: 4.10a F1 ui d: Bentoni te/Ca( 0H)2/6000 F Magnification: 26,000 X Film #: 3149 Des c ri p t i on : Figure #: 4.10a F1 ui d: Bentoni te/Ca( 0H)2/6000 F Magnification: 26,000 X Film #: 3149 Description: Image o f a calcium s i l i c a t e hydrate (CSH) crystal. The x-ray spectra obtained from the crystal are given below: Intensity Intensity Element counts/100 sec. ratio/Si Intensity Intensity Element counts/100 sec. ratio/Si A1 - 536 .ll Si 4850 1.00 Ca 6211 1.28 Figure #: 4.10b Fluid: Bentonite/Ca(OH Magnification : SAD Film #: 3148 Description: The SAD pattern o f the crystal shown above. ),/6OOo F Figure #: 4.10b Fluid: Bentonite/Ca(OH Magnification : SAD Film #: 3148 Description: The SAD pattern o f the crystal shown above. ),/6OOo F 95 95 Thus the excess s i l i c a (fine quartz powder) does not promote the formation o f cement minerals i n the fluid. The tobermorite seems, therefore, t o be a product o f the reaction between lime and smectite (perhaps the amorphous s i l i c a i n the bentonite) but not between lime and s i l i c a from the f i n e quartz powder. Figure #: 4.11 F1 u i d: Bentoni te/Ca (OH)/Si 02/ Magnification: 5,000 X 300°F F i l m #: 3102 Figure #: 4.11 F1 u i d: Bentoni te/Ca (OH)/Si 02/ Magnification: 5,000 X 300°F F i l m #: 3102 Figure #: 4.11 F1 u i d: Bentoni te/Ca (OH)/Si 02/ Magnification: 5,000 X 300°F F i l m #: 3102 Description: Electron Microscopic Observations After autoclaving the f l u i d a t 300"F, smectite particles occur as thick aggregates as shown i n Figures 4.11 and 4.12. often contain dense and sometimes spherical particles o f c a l c i t e and tober- These aggregates morite that are entrapped i n them (Figure 4.13). Thus, there are no more well-dispersed t h i n films o f smectites .in the f l u i d but larger coagulated particles. o r ribbons as displayed i n Figures 4.14 and 4.15a. tron d i f f r a c t i o n pattern i n Figure 4.15b gives the unit-cell parameter o f a tobermorite, f o r the ribbon-like particle, i n Figure 4.15a. Sometimes, Tobermorite crystals appear i n the form o f t h i n laths, fibers, The selected area elec- these ribbons and laths seem t o j o i n each other and form platy particles o f tobermorite. Such a platy tobermorite with a mosaic o f laths i s i l l u s - trated i n Figure 4.16. strong Ca- and Si-lines as expected from a tobermorite. The x-ray spectra obtained from the p l a t e l e t give Tobermorite crystals that precipitate a f t e r autoclaving the f l u i d a t 400°F and a t 500°F seem t o improve i t s crystalline forms. They consist o f longer laths and fibers (Figure 4.17); and some o f them display a well- developed tabular morphology as shown i n Figure 4.18a. The SAD pattern i n Figure 4.18b and the x-ray spectra obtained from the p l a t e l e t i n Figure 4.18a confirm the i d e n t i t y o f tobermorite. cabbage-like aggregates after autoclaving the f l u i d a t 400 and 500OF. a tobermorite aggregate i s displayed i n Figure 4.19. Tobermorite laths often develop Such Calcite occurs as 96 Figure #: 4.11 F1 u i d: Bentoni te/Ca (OH)/Si 02/ Magnification: 5,000 X 300°F F i l m #: 3102 Description: A general view of aggregates after the reaction. Electron Microscopic Observations Smectite may retain its fluffy outlines, b u t most of them are partially fragmented, Calcite rhombs and Ca- and Si- containing spheres are corn- monly trapped i n the aggreg- ates. Figure #: 4.12 F1 uid: Bentoni te/Ca(OH)2/Si02/ Magnification: 13,000 X 300°F Film #: ‘3103 Descri p t i on : A magnified view of t tite aggregate from the above micrograph. A few tobermori t e laths are visible together with some cal ci um-ri ch spheres Description: A general view of aggregates after the reaction. Smectite may retain its fluffy outlines, b u t most of them are partially fragmented, Calcite rhombs and Ca- and Si- containing spheres are corn- monly trapped i n the aggreg- ates. Figure #: 4.12 F1 uid: Bentoni te/Ca(OH)2/Si02/ Magnification: 13,000 X 300°F Film #: ‘3103 Descri p t i on : A magnified view of t tite aggregate from the above micrograph. A few tobermori t e laths are visible together with some cal ci um-ri ch spheres Figure #: 4.13 F1 uid: Bentonite/Ca(OH)2/Si02/ Magnification: 33,000 X 300°F Film #: 3104 Description : Another magnified view of the smectite films from Figure 4.11. Figure #: 4.14 F1 uid: Bentoni te/Ca(OH)2/SiOp/ Magnification: 13,000 X 300°F Film #: 3105 Description: A general view of the tober- morite laths i n the sample, . some of them also occur i n plates. 98 Figure #: 4.14 F1 uid: Bentoni te/Ca(OH)2/SiOp/ Magnification: 13,000 X 300°F Film #: 3105 Figure #: 4.14 F1 uid: Bentoni te/Ca(OH)2/SiOp/ Magnification: 13,000 X 300°F Film #: 3105 Description: A general view of the tober- morite laths i n the sample, . some of them also occur i n plates. 98 98 99 99 99 Figure #: 4.16 F1 uid: Bentoni te/Ca(OH)2/Si02/ Magnification: 13,000 X 300°F Film #: 3107 Description: A p l a t y tobermorite which i s composed o f laths. The x-ray spectra o f t h i s plate are g i ven bel ow: I n tensity Intensity Element counts/100 sec. ratio/Si A1 167 .13 S i 1333 1.00 Ca 1438 1.08 Figure #: 4.17 F1 uid: Bentoni te/CA( OH)2/SiO/ Magnification: 20,000 X 400°F Film #: 3114 Desc r i p t i on : A closeup o f the tobermorite fibers and the smectite remains. 100 Figure #: 4.16 F1 uid: Bentoni te/Ca(OH)2/Si02/ Magnification: 13,000 X 300°F Film #: 3107 Description: A p l a t y tobermorite which i s composed o f laths. The x-ray spectra o f t h i s plate are g i ven bel ow: I n tensity Intensity Element counts/100 sec. ratio/Si A1 167 .13 S i 1333 1.00 Ca 1438 1.08 Figure #: 4.16 F1 uid: Bentoni te/Ca(OH)2/Si02/ Magnification: 13,000 X 300°F Film #: 3107 Description: Figure #: 4.17 F1 uid: Bentoni te/CA( OH)2/SiO/ Magnification: 20,000 X 400°F Film #: 3114 Desc r i p t i on : A closeup o f the tobermorite fibers and the smectite remains. 100 Figure #: 4.18 - F1 uid: Bentonite/Ca( Magnification: 33,000 X Film #: 3112 Deseription: Some o f the' tobermorite crystals, display tabular morphology with d i s t i n c t faces such as t h i s one, but they are not very comnon. The x-ray spectra obtained from the plate are given below: I n tens i ty Intensity Element counts/100 sec. ratio/Si A1 114 .08 S i 1513 1.00 Ca 1804 1.19 Figure #: 4.18b F1 uid: Bentoni te/CA( OH)2/Si02/400a Magni f i cation : SAD F i l m #: 3115 Descri p t i on: The SAD pattern o f the tobermoritc crystal shown above. < - b = 7.29w c = 21.8A 0 10 1 Deseription: Some o f the' tobermorite crystals, display tabular morphology with d i s t i n c t faces such as t h i s one, but they are not very comnon. The x-ray spectra obtained from the plate are given below: I n tens i ty Intensity Element counts/100 sec. ratio/Si A1 114 .08 S i 1513 1.00 Ca 1804 1.19 Figure #: 4.18b F1 uid: Bentoni te/CA( OH)2/Si02/400a Magni f i cation : SAD F i l m #: 3115 Descri p t i on: The SAD pattern o f the tobermoritc crystal shown above. < - b = 7.29w c = 21.8A 0 Figure #: 4.18b F1 uid: Bentoni te/CA( OH)2/Si02/400a Magni f i cation : SAD F i l m #: 3115 Descri p t i on: The SAD pattern o f the tobermoritc crystal shown above. < - b = 7.29w c = 21.8A 0 10 1 10 1 rhombs b u t mostly as spherules (Figure 4.20). 'Tobermorite laths become thicker and larger i n size after the f l u i d was autoclaved a t 6dO"F as typically shown i n Figures 4.21 and 4.22. A t this temperature a large number of a Ca-feldspar also precipitates i n the form of equant and thick crystals. Most of the smectite dissolves and dis clave temperature is increased from 400 t o 600°F. i ntegrates as the auto- 102 10 3 10 3 Figure #: 4.2 'Fluid: Benton Magni fi cat ion Film #: 3118 Description : A general vie te/Ca(OH)2/Si02/ 14, OOOX 600" F o f the commonly observed tobermorite i n t h i s fluid. / Figure #: 4.22 F1 uid: Bentonite/Ca(OH)2/Si02/ Magnification: 13,OOOX 600°F F i l m #: 3112 Descr i p t i on : Aggregates o f the tobermorite fibers. The x-ray spectra obtained from the aggregate are given below: I n tens i t y Element counts/100 sec. A1 10 7 S i 2063 Ca 1702 Fe 111 Intensity r a t i o/Si .05 1.00 .83 .05 Figure #: 4.2 'Fluid: Benton Magni fi cat ion Film #: 3118 Description : A general vie te/Ca(OH)2/Si02/ 14, OOOX 600" F o f the commonly observed tobermorite i n t h i s fluid. A general vie o f the commonly observed tobermorite i n t h i s fluid. Figure #: 4.22 F1 uid: Bentonite/Ca(OH)2/Si02/ Magnification: 13,OOOX 600°F F i l m #: 3112 Descr i p t i on : Aggregates o f the tobermorite fibers. The x-ray spectra obtained from the aggregate are given below: I n tens i t y Element counts/100 sec. A1 10 7 S i 2063 Ca 1702 Fe 111 Intensity r a t i o/Si .05 1.00 .83 .05 Aggregates o f the tobermorite fibers. The x-ray spectra obtained from the aggregate are given below: I n tens i t y Element counts/100 sec. A1 10 7 S i 2063 Ca 1702 Fe 111 Intensity r a t i o/Si .05 1.00 .83 .05 104 1. Bentoni te/Lime/CaC12 Fluid A f l u i d composed o f 2% bentonite/l% lime/l% CaC12 has been autoclaved a t high temperatures with the purpose o f increasing Ca2+-concentration i n the f l u i d with regard t o the formation o f tobermorite. o f the f l u i d are described by the rheograms i n Figure 5.1 and by the data i n Tables 5.la and 5.lb. These data show that the viscosities o f the f l u i d are extremely low a t a l l temperatures similar t o the other bentonite f l u i d s The characteristics with high Ca'+-concentrations. 70°F t o 8.9 a t 600"F, thus remaining always alkaline. drastically decrease from 75 meq/100g a t 70°F f o r the untreated f l u i d t o 12.5 meq/100g f o r the f l u i d autoclaved a t 600°F. This indicates most o f the smectite dissolv i n these a1 kal ine solutions a t high temperatures. The f l u i d pH steadily decreases from 12 d t The CEC values X-ray D i f f r a c t i on Anal ys i s Calcite, halite, tobermorite occur as the main reaction products i n t h i s f l u i d . Calcit readily forms by the carbonatization o f l i m e i m the air. Halite crysta izes while the chloride solution has dried. Sodium i s supplied from the Ca2+ i n the solution Na+ i n t h e o r i g i n a l clay. Tobermorite, the cement mineral, f i r s pears a t 300°F and it becomes a major component of the mud a t higher t e atures. autoclaving the f l u i d a t 600°F. c t i t e by the ion exchange reaction between the A calcium feldspar i s also formed while It i s very important-to note that smectite e n t i r e l y disappears from the f l u i d a t 400°F. Thus, the addition o f CaC12 t o the bentonite/lime f l u i d favors the t o t a l replacement o f smecti t e by tobermori te. 105 fi Y MINERAL COMPONENTS IN THE FLUIDS: 300" F SMECTITE + CALCITE + HALITE* + TOBERMORITE* 4OOOF CALCITE* + HALITE* + TOBERMORITE* 500°F HALITE* + TOMERMORITE* 600°F HALITE* + TOBERMORITE* + FELDSPAR* NEW PHASES FORMED IN THE FLUID 70" F SMECTITE + CALCITE + HALITE* - 2% BENTONITE 1% LIME 1% CaCI, 300" F 300" F, I I I I I 100 200 300 400 500 ( O F ) Figure 5.1 - The FANN 50C rheograms o f the 2% bentonite/l% lime/l% CaCl2 fluids which were autoclaved a t the temperatures marked on the curves. 3. Bentoni te/Lime/Lignosul fonate F1 uid A f l u i d with the c mposition o f 2% bentonite/l% lime/0.25% ligno- sul fonate exhibits e tremely low viscosities a t 11 temperatures/as shown by the data i n Figu 5.3 and i n Tables 5.3a an 5.3b. The f l u i d pH i evolves from 1210 a t 70°F and t o 8.5 a t 600"F, similar t o other bentonite/ l i m e fluids. Again the CEC values o f the f l u i d show'a reduction from 100 meq/100g a t 70°F 14 meq/100g a t 600°F. 2. Bentonite/Lime/Gypsum Fluid With the addition o f gypsum a new f l u i d was prepared with the com- position o f 2% bentonite/l% lime/l% gypsum. t h i s f l u i d are given i n Figure 5.2 and i n Tables 5.2a and 5.2b. f l u i d exhibits rather low viscosities as the other bentonite/lime fluids. The f l u i d pH and CEC values decrease significantly with the higher temper- atures. The measured properties o f This MINERAL COMPONENTS IN THE FLUIDS: 300" F SMECTITE + CALCITE* + HALITE* + TOBERMORITE* 4OOOF CALCITE* + HALITE* + TOBERMORITE* 500°F HALITE* + TOMERMORITE* 600°F HALITE* + TOBERMORITE* + FELDSPAR* NEW PHASES FORMED IN THE FLUID 70" F SMECTITE + CALCITE + HALITE* - Figure 5.1 - The FANN 50C rheograms o f the 2% bentonite/l% lime/l% CaCl2 fluids which were autoclaved a t the temperatures marked on the curves. TABLE 5. la APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE 2% BENTONITE / 1% Ca(OH)2 / 1% CaC12 UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF Temperature O F 70 300 400 500 600 75 1.1 2.2 1.5 1.7 1.4 100 0.7 2.1 1.4 1.8 1.2 TABLE 5. la APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE 2% BENTONITE / 1% Ca(OH)2 / 1% CaC12 UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 107 TABLE 5.lb PROPERTIES' OF THE UNTREATED AND' AUTOCLAVED FLUIDS CONSISTING OF - 2% BENTONITE / 1% LIME 1 1% CaC12 - -Autoclaved at temperatures - ( O F ) F1 uid Property 70 300 400 500 600 Apparent Viscosity (centipoise) 1.5 2.5 2.5 2.5 1.5 Plastic Viscosity (centipoise) 1.0 2.0 2.0 2.0 1.0 Yield Point (lb/100 sq ft) 1.0 1.0 1.0 1.0 1.0 Gel Stregth (lb/100 sq ft) 0.5/2 0.5/2 1/1 1/1.5 0.5/1 API Filtrate (m1/30 min.) Cake Thickness (inches) HP-HT Filtrate (m1/30 min.) PH 12 11.0 10.6 10.3 8.9 CEC (milliequivalents/lOO gms) 100 75 50 12.5 12.5 TABLE 5.lb PROPERTIES' OF THE UNTREATED AND' AUTOCLAVED FLUIDS CONSISTING OF - 2% BENTONITE / 1% LIME 1 1% CaC12 - 108 X-Ray Diffraction Analysis The main reaction products are calcite, tobermorite, and anhydrite i n t h i s fluid. Gyps during the autoclaving o f the f l u i d a t high temperatures o f 300-500°F. Tobermorite f i r s t forms i n small quantities a t 300°F but i n larger amounts a t higher temperatures. seems t o convert t o anhydrite by simple dehydration I n summary, gypsum does not seem t o give any special benefits i n bentoni t e / l ime fluids. X-Ray D i f f r a c t ion Analysis Tobermorite appears i n small quantities a t 300°F and i n larger amounts a t higher temperatures. Calcite forms again due t o the 109 r CI 0 2% BENTONITE 1% LIME 1% GYPSUM 50OoF MINERAL COMPONENTS IN THE FLUIDS: 7OoF SMECTITE + GYPSUM + CALCITE' 300OF SMECTITE + ANHYDRITE* + CALCITE* + TOBERMORITE* 4OOOF SMECTITE + ANHYDRITE* + CALCITE* + TOBERMORITE* 500°F SMECTITE + ANHYDRITE* + TOBERMORITE* + CALCITE* 6OOOF SMECTITE + TOBERMORITE* + CALCITE* NEW PHASES FORMED IN THE FLUID , 50OOF I / 600' F I I 1 I 100 200 300 400 500 (" F) Figure 5.2 - The FANN 50C rheograms of the 2% bentonite/l% lime/l% gypsum fluids after autoclaving a t temperatures marked on the curves. r CI 0 Figure 5.2 - The FANN 50C rheograms of the 2% bentonite/l% lime/l% gypsum fluids after autoclaving a t temperatures marked on the curves. X-Ray D i f f r a c t ion Analysis TABLE 5.2a APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% LIME / 1% GYPSUM Fann 50C A u t o c l a v e d a t t e m p e r a t u r e ( O F ) Temperature O F 70 300 400 500 600 75 1.4 3.9 2.2 4.2 1.2 100 1.2 3.3 1.9 3.8 1.0 150 200 0.8 2.8 0.7 2.7 1.8 3.7 1.0 1.8 3.5 1.0 0.8 2.6 1.7 3.3 1.0 250 300 0.8 2.5 1.7 3.1 1.0 TABLE 5.2a APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% LIME / 1% GYPSUM 11 1 TABLE 5.2b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% LIME / 1% GYPSUM Autoclaved a t temperatures ( O F ) Fluid Property 70 300 400 500 600 Apparent Vi scosi t y (cen ti poise) 2.0 6.0 4.0 7.0 1.5 Plastic Viscosity (centipoise) 1.5 3.0 2.5 3.5 1.5 Yield Point (lb/100 sq f t ) 1.5 6.0 2.5 7.5 0.0 Gel Strength (lb/100 sq f t ) l/2 31 4 3/ 5 4/4 0.5/1.5 API Filtrate (m1/30 min.) Cake Thickness (inches) HP-HT Giltrate (m1/30 min.) PH 12.6 11.5 11.6 11.4 9.4 CEC (mi 1 1 i equi Val ents/ 100 gms ) 100 BO 25 25 25 TABLE 5.2b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% LIME / 1% GYPSUM 112 15 6OOOF \ 75°F b MINERAL COMPONENTS IN THE FLUIDS 7 0 0 ~ SMECTITE + CALCITE* 3OOOF SMECTITE + CALCITE* + TOBERMORITE* 4OOOF SMECTITE + TOBERMORITE* + CALCITE* 600 OF SMECTITE + Ca-FELDSPAR* + TOBERMORITE* + CALCITE* SOOOF SMECTITE + TOBERMORlTE* + CALCITE' + Ca-FELDSPAR" NEW PHASES FORMED IN THE FLUID 2% BENTONITE 1% LIME 0.25% LIGNOSULFONATE 600' p ,/ 300°F 100 200 300 400 500 (" F) Figure 5.3 - The FANN 50C rheograms o f the 2% bentonite/l% lime/0.25% lignosulfonate f l u i d s a f t e r autoclaving a t the temperatures marked on the curves. 4. Bentonite/Lime/MgO Fluid A f l u i d w i t h the composition of 2% bentonite/l% lime/l% MgO showed rather interesting features. i n Figure 5.4 and Tables 5.4a and 5.4b. remain rather low a t all temperatures. all temperatures. The fluid pH remains highly a1 kaline without significant changes a t higher temperatures i n contrast t o the other f l u i d s described above. The CEC values, however, show an unusually low value of 75 meq/ The properties of t h i s f l u i d are sumnarized The viscosities of the f l u i d The f l u i d pH remains rather low a t lOOg a t 70°F and a decrease to 25 meq/100g a t 300°F. 15 MINERAL COMPONENTS IN THE FLUIDS 7 0 0 ~ SMECTITE + CALCITE 3OOOF SMECTITE + CALCITE* + TOBERMORITE* 4OOOF SMECTITE + TOBERMORITE* + CALCITE* 600 OF SMECTITE + Ca-FELDSPAR* + TOBERMORITE* + CALCITE* SOOOF SMECTITE + TOBERMORlTE* + CALCITE' + Ca-FELDSPAR" NEW PHASES FORMED IN THE FLUID 2% BENTONITE 1% LIME 0.25% LIGNOSULFONATE MINERAL COMPONENTS IN THE FLUIDS 7 0 0 ~ SMECTITE + CALCITE 3OOOF SMECTITE + CALCITE* + TOBERMORITE* 4OOOF SMECTITE + TOBERMORITE* + CALCITE* 600 OF SMECTITE + Ca-FELDSPAR* + TOBERMORITE* + CALCITE* SOOOF SMECTITE + TOBERMORlTE* + CALCITE' + Ca-FELDSPAR" NEW PHASES FORMED IN THE FLUID Figure 5.3 - The FANN 50C rheograms o f the 2% bentonite/l% lime/0.25% lignosulfonate f l u i d s a f t e r autoclaving a t the temperatures marked on the curves. TABLE 5.3a APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% LIME / 0.25% LIGNOSULFONATE Fann 50C Autoclaved .at temperature ( O F ) Temperature O F 70 300 400 500 600 75 100 1.1 0.8 0.9 0 .'8 1.3 150 200 250 1.1 0.7 0.8 ~ 0.7 1.1 1.1 0.7 0.5 0.6 1.1 1.1 0.6 0.5 0.6' r 1.0 300 1.1 0.6 0.4 0.6 1.0 350 400 1.0 0.6 0.4 0.6 1.0 1.0 0.7 0.4 0.5 1.0 450 0.9 0.9 0.3 0.8 1.1 500 0.8 1.0 0.2 0.8 1.3 TABLE 5.3a APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISCOMETER FOR THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% LIME / 0.25% LIGNOSULFONATE 114 TABLE 5.3b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% LIME / 0.25% LIGNOSULFONATE Autoclaved a t temperatures (OF) F1 uid Property 70 300 400 500 600 Apparent Viscosity (centipoise) 1.5 1.5 2.0 1.5 1.5 Plastic Viscosity (centipoise) 1.0 1.5 1.5 1.0 1.5 Yield Point (lb/l 0.5 0.5 1.0 0.0 .5/0,5 0.5/1 0.5/1 0.5/1 API F i l t r a t e (ml/ Cake Thickness HP-HT F i l t r a t e (m1/30 min.) PH 12-0 11.5 10.8 10.7 8.5 TABLE 5.3b 115 carbonatization of lime i n the air. A calcium-feldspar develops a t 500" and 600°F i n the fluid. Some of the smectite still remains i n the fluid even after autoclaving a t 6OO0F, whereas a large portion of smectite has been dissolved i n the alkaline solution fluids subjected to high tempera- tures. Although lignosulfonates are expected to give a temporary retarda- tion to the formation of cement minerals, we do not see such an effect i n the bentonite/lime f l u i d . - -- X-Ray Diffraction Analysis The addition of MgO to the bentonite/lime mud results i n unusual reaction products. MgO readily hydrates to brucite, Mg (OH)? i n the fluid a t 70°F, whereas lime similarly reacts w i t h C02 i n the a i r to calcite. Both calcite and brucite oecur as reaction products a t higher temperatures (300-6OO0F), indicating that the fluid is saturated w i t h respect to Ca2+ 2+ and Mg . A t higher temperatures some of the brucite seems to react w i t h C02 (in the f l u i d ) and form magnesite (MgC03). I t is interesting to note that magnesia readily reacts a t 300°F w i t h smectite and forms a serpentine 116 > L 10 MINERAL COMPONENTS IN THE FLUIDS 7OoF SMECTITE + CALCITE" +BRUCITE 300.F SMECTITE + CALCITE+ + BRUClTE++ SERPENTINE. + MAGNESITE+ , 4OOOF SERPENTINE'' + CALITE' + BRUCITE" + MAGNESITE+ 500OF SERPENTINE" + CALCITE* + BRUCITE' +MAGNESITE+ . 1 6 0 0 0 ~ SERPENTINE+'+ CALCITE+ + BRUCITE+ + MAGNESITE+ NEW PHASES FORMED IN THE FLUID - 50OOF 2% BENTONITE 1% LIME 1% Nlgo 500° F 400OF \ // ,-3OO"F 100 200 300 400 500 (" F) Figure 5.4 - The FANN 50C rheograms o f the 2% bentonite/l% lime/l% MgO fluids after autoclaving a t the temperatures marked on the curves. Figure 5.4 - The FANN 50C rheograms o f the 2% bentonite/l% lime/l% MgO fluids after autoclaving a t the temperatures marked on the curves. X-Ray Diffraction Analysis TABLE 5.4a APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISC THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% LIME / 1% MgO Fann 50C Autoclaved a t temperature (OF) Temperature F 70 a 300 400 500 600 75 1.7 2.7 2.5 2.7 1.5 100 1.7 2.6 2.3 2.4 1.2 150 1.5 2.6 2.2 2.3 1.1 200 2 50 1.3 2.7 2.3 2.3 1.0 1.2 2.8 2.3 2.4 1.0 300 1.3 2.8 2.5 2.5 1.0 350 400 1.2 2.7 2.6 2.6 1.0 1.4 2.7 2.6 2.6 1.0 450 1.0 3.5 3.7 4.4 1.0 500 1.2 4.5 11.5 20.6 1.3 TABLE 5.4a APPARENT VISCOSITIES (CP) MEASURED WITH THE FANN 50C VISC THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% LIME / 1% MgO 118 TABLE 5.4b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% LIME / 1% MgO Autoclaved a t temperatures ( O F ) F1 u i d Property 70 300 400 500 600 ~ Apparent Viscosi entipoise) 2.0 3.0 4.0 4.0 2.0 Plastic Viscosity (centipoise) 2.0 2.5 3.0 2.5 1.0 Gel Strength (lb/100 sq ft) 1/2 1/2 212 2/2 1/ 1 Yield Point (lb/100 sq ft) 0.5 1.5 1.5 2.5 1.5 Cake Thickness (inches) HP-HT F i l t r a t e (m1/30 min.) 11.6 11.8 11.8 11.6 12.2 PH TABLE 5.4b PROPERTIES OF THE UNTREATED AND AUTOCLAVED FLUIDS CONSISTING OF 2% BENTONITE / 1% LIME / 1% MgO 119 mineral (possibly amesite). by t h i s serpentine mineral when the f l u i d was autoclaved a t and above 400°F: Thus, no smectite remains i n the f l u i d a t and above 400°F. Fur- thermore, no cement mineral (tobermorite) crystallizes when MgO i s added t o the fluid, instead, a garnet-like phase has been found. I n fact, a l l the smectite has been replaced It i s also important t o remember the addition o f MgO maintains the f l u i d pH a t high alkaline values, thus providing a buffering agent f o r the water-base f 1 uids . Electron Microscopic Examination The comon reaction product i n the f l u i d a f t e r autoclaving a t and This phase i s not a swelling clay above 4000F i s a serpentine mineral. mineral l i k e smectite, and it, therefore, depresses the viscosity o f the fluid. A t higher temperatures, a f t e r autoclaving the f l u i d a t and above 500"F, a garnet-like mineral seems t o develop instead o f tobermorite. Typical serpentine and garnet particles are shown i n Figure 5.5a. Garnets occur as micron-sized spherical crystals whereas the serpentine develops hexagonal platelets with sub-micron dimensions. Close views o f these serpentine platelets are displayed i n Figures 5.6a and 5.7a. spectra i n Figure 5.6b give strong spectral lines o f Mg, Al, and S i . suggests an amesite o r aluminum l i z a r d i t e variety o f serpentine with the general chemical formula: The x-ray This 3+ EM93-x (AI, ~e ),I [siESx (AI, F ~ ~ + ) ~ I o5 ( 0 ~ The selected area electron d i f f r a c t i o n pattern i n Figure 5.7b gives - a and - b unit-cell parameters, which are compatible with the above serpentine species. 120 Figure #: 5.5a F1 ui d : Bentonite/ Lime/Mg0/600" F Magnification: 13,000 X Film #: 3687 Description : A general view of the commonly observed reaction products con- sisting o f serpentine and garnet. Figure #: 5.6a F1 uid: Bentonite/ Lime/Mg0/60OoF Magnification: 44,000 X Film #: 3683 Description : A close view o f the newly fomd serpentine pl a tel ets showing we1 1 -developed hexagonal out1 ines 12 I Figure #: 5.5a F1 ui d : Bentonite/ Lime/Mg0/600" F Magnification: 13,000 X Film #: 3687 Description : A general view of the commonly observed reaction products con- sisting o f serpentine and garnet. Figure #: 5.5a F1 ui d : Bentonite/ Lime/Mg0/600" F Magnification: 13,000 X Film #: 3687 Description : A general view of the commonly observed reaction products con- sisting o f serpentine and garnet. Figure #: 5.6a F1 uid: Bentonite/ Lime/Mg0/60OoF Magnification: 44,000 X Film #: 3683 Description : A close view o f the newly fomd serpentine pl a tel ets showing we1 1 -developed hexagonal out1 ines Description : A close view o f the newly fomd serpentine pl a tel ets showing we1 1 -developed hexagonal out1 ines A close view o f the newly fomd serpentine pl a tel ets showing we1 1 -developed hexagonal out1 ines 12 I 0 Energy i n keV 10.00 10.00 10.00 Figure #: 5.7a F1 uid: Bentonite/ Lime/MgQ/600 Magnification: 65,000 X F i l m #: 3686 Description: \-\ A close view o f a clay (serpentin p l a t e l e t with hexagonal outlines. e Figure #: 5.7a F1 uid: Bentonite/ Lime/MgQ/600 Magnification: 65,000 X F i l m #: 3686 Description: \-\ A close view o f a clay (serpentin p l a t e l e t with hexagonal outlines. Figure #: 5.7b F1 uid: Bentonite/ Lime/Mg0/600" Magnification: SAD F i l m #: 3685 Description: The selected area electron d i f t i o n pattern obtained from the p l a t e l e t shown i n Figure 5.7a. The c e l l parameters calculated from the pattern are: 0 4 b = 9.27 A e 'F 'f rac Figure #: 5.7a F1 uid: Bentonite/ Lime/MgQ/600 Magnification: 65,000 X F i l m #: 3686 Description: \-\ A close view o f a clay (serpentin p l a t e l e t with hexagonal outlines. e Figure #: 5.7b F1 uid: Bentonite/ Lime/Mg0/600" Magnification: SAD F i l m #: 3685 'F Description: The selected area electron d i f t i o n pattern obtained from the p l a t e l e t shown i n Figure 5.7a. The c e l l parameters calculated from the pattern are: 'f rac 0 4 b = 9.27 A 0 4 b = 9.27 A 0 4 b = 9.27 A 123 I n summary, the addition o f MgO i n the fluid prevents the formation o f cement minerals but the new phases (serpentine, garnet, magnesia etc.) do not lend any viscosity t o the fluid. 124 The Major Conclusions of the Investigation 1. Pure Na-bentonite f l u i d becomes highly viscous (gelation) i n the temperature range 250-450°F. 1. Pure Na-bentonite f l u i d becomes highly viscous (gelation) i n the temperature range 250-450°F. 1. Pure Na-bentonite f l u i d becomes highly viscous (gelation) i n the temperature range 250-450°F. 2. Low molecular weight polymer controls the viscosity only i n the limited temperature range 300-400°F. 2. Low molecular weight polymer controls the viscosity only i n the limited temperature range 300-400°F. 2. Low molecular weight polymer controls the viscosity only i n the limited temperature range 300-400°F. 3. High molecular weight polymer increases the f l u i d viscosity only for the untreated mud. claved a t and above 300OF. I t reverses this function when the f l u i d is auto- 3. High molecular weight polymer increases the f l u i d viscosity only for the untreated mud. claved a t and above 300OF. I t reverses this function when the f l u i d is auto- 3. High molecular weight polymer increases the f l u i d viscosity only for the untreated mud. claved a t and above 300OF. I t reverses this function when the f l u i d is auto- 4. Sodium hydroxide, especially NaCl, a t 1% concentration i n the f l u i d greatly increases the f l u i d viscosity (gelation) up t o 500°F. addition of 1% silica t o the fluid significantly reduces the viscosity but it leads to the precipitation of Na-silicates (analcime and The albite) which may cause permeability damage i n the formation. 4. Sodium hydroxide, especially NaCl, a t 1% concentration i n the f l u i d greatly increases the f l u i d viscosity (gelation) up t o 500°F. addition of 1% silica t o the fluid significantly reduces the viscosity but it leads to the precipitation of Na-silicates (analcime and The albite) which may cause permeability damage i n the formation. 5. Salts and hydroxides of potassium provide excellent controls on the f l u i d viscosity a t high temperatures. T h i s is related to the con- version of smectite to illite/smectite mixed-layers a t and above 400°F. 6. V I . SUMMARY AND CONCLUSIONS Factors control 1 ing the high-temperature behavior o ben toni t e f 1 u I ui and t h e i r i n - s i t u conversion i n t o cement have been studied. I n order t o understand the conditions leading t o high-temperature gelation, bentonite fluids have been autoclaved a t the temperature range 300-600°F f o r 16 hours under 16,000 psi with a systematic addition o f polymers (low and high molecular weight sodium polyacrylates), sodium chloride, sodium hydroxide, s i 1 ica , and salts and hydroxides o f potassi urn. Bentoni t e / l ime, bentonite/ CaC12, and bentonite/lime/silica f l u i d s have been examined f o r the purpose o f inducing the i n - s i t u conversion o f the mud i n t o cement. Effects o f the additives such as CaC12, gypsum, lignosulfonates, and MgO on the conversion - . rate o f mud t o cement were evaluated by studying the following f l u i d sys tems : bentonite/l ime/CaC12 bentoni t e / l ime/gypsum bentonite/l ime/l ignosul fonate bentoni t e / l ime/MgO bentonite/l ime/CaC12 bentoni t e / l ime/gypsum bentonite/l ime/l ignosul fonate bentoni t e / l ime/MgO bentonite/l ime/CaC12 bentoni t e / l ime/gypsum bentonite/l ime/l ignosul fonate bentoni t e / l ime/MgO bentonite/l ime/CaC12 bentoni t e / l ime/gypsum bentonite/l ime/l ignosul fonate bentoni t e / l ime/MgO The high-temperature rheologies o f the above f l u i d s were measured with a FANN 50C viscometer and with the SANDIA visc fore and after auto- rength , y i e l d point, pH, CEC, f l u i d loss, f i l t e r cake t were also meas- ured. The mineral reaction ave been studied with x-ray d i f f r a c t i o n and electron microscopy. Changes i n the properties o f the f l u i d s were correlated with the high temperature mineral reactions i n them. 12 5 The Major Conclusions of the Investigation Salts and hydroxides of calcium depress the fluid viscosity t o very low levels a t all temperatures. 6. Salts and hydroxides of calcium depress the fluid viscosity t o very low levels a t all temperatures. 7. The addition of lime increases the fluid pH and promotes the high- temperature reaction between bentonite and 1 ime w i t h the precipi ta- tion of tobermorite, the common cement mineral. 8. The addition of free silica t o the bentonite/lime fluid does not promote the formation of cement minerals i n the fluid. 9. The addition of 1% CaC12 t o the bentonite/lime f l u i d leads to the total conversion of bentonite (smecti te) into the cement mineral 126 tobermorite. Gypsum, on the other hand, does not give any benefits for the mud-to-cement conversion. tobermorite. Gypsum, on the other hand, does not give any benefits for the mud-to-cement conversion. tobermorite. Gypsum, on the other hand, does not give any benefits for the mud-to-cement conversion. 10. i Lignosulfonates do not seem to cause any appreciable retardation i n the conversion of the bentonitic mud into cement. 10. i Lignosulfonates do not seem to cause any appreciable retardation i n the conversion of the bentonitic mud into cement. 11. The addition of MgO to the bentonite/lime f l u i d prevents the precipi- tation of cement minerals b u t instead leads to the formation of a serpentine mineral. The latter phase i s a non-swelling layer sili- cate and i t does not lend any viscosity to the f l u i d . The addition o f magnesia becomes detrimental to the f l u i d viscosity. Magnesia provides, however, a reliable buffer for maintaining a high pH i n the f l u i d a t increasi temperatures. 11. The addition of MgO to the bentonite/lime f l u i d prevents the precipi- tation of cement minerals b u t instead leads to the formation of a serpentine mineral. The latter phase i s a non-swelling layer sili- cate and i t does not lend any viscosity to the f l u i d . The addition o f magnesia becomes detrimental to the f l u i d viscosity. The Major Conclusions of the Investigation Magnesia provides, however, a reliable buffer for maintaining a high pH i n the f l u i d a t increasi temperatures. 12 3 DISJ.RIBW?ION: TIP450(1-R-66-X46c (507) B. J. Livesay 129 Liverpool Cardiff, 92007 Harvey E. Mallory P.O. Box 54696 Tulsa, OK 74155 Ed Martin Superior oil Eastern Division P.O. Box 51108 OCS Lafayette, bisiana 70505 Gene Polk NL Baroid Albuquerque, El4 87110 6400 ~~ Blvd. N.E.8 365W D e l E. Pyle Union Geothermal Division Unim Oil Co. of California Union Oil Center Ixxs Angeles, a 90017 Attn: DeepakKenkeremath John C, -ley Ics Alamos National Labs Mail Stop 570 u -8 ~ l 4 a7545 might Smith Halliburton Drawer 1431 m-8 a 73533 Tan Turner Phillips Petroleum ccap?arry Geothermal operatiom 655 East 4500 South Salt fake City, UT 84107 Tan Warren Amom Prcduction canpany Research center P.O. Box 591 Tulsa, OK 74102 Tan Anderson Venture Innovations P.O. Ebx 35845 Houston, TX 77035 Ed aingman shell oil cblnpany Two shell Plaza P.O. Box 2099 Houston, TX 77001 Ben Bradford W e l l P.O. Box 2710 Tulsa, OK 74102 Jim Combs G e o t h d ksources Int'l 545 Middlefield m.8 mite 200 W O P X k , CA 94025 Larry Diamond Dyrn-Drill Wine, CA 92713 P.O. Box C-19576 Jcihn E. mtenot NL Petroleum Services P.O. Box 60087 Houston, TX 77205 Dr. Melvin Friedman Professor of Geology center for Tectcmocplysics and De. of Geolcgy Texas A&M University cbllqe Station, Tx 77843 Dr. Necip Guven (20) Dept. of Geosciences Texas Tech Vniv. Lubbodc, Texas 19409 James W. Langford Security Division ~resser Industries, Inc. P.O. Box 24647 Dallas, TX 75224 B. J. Livesay 129 Liverpool Cardiff, 92007 Harvey E. Mallory P.O. Box 54696 Tulsa, OK 74155 Ed Martin Superior oil Eastern Division P.O. Box 51108 OCS Lafayette, bisiana 70505 Gene Polk NL Baroid Albuquerque, El4 87110 6400 ~~ Blvd. N.E.8 365W D e l E. Pyle Union Geothermal Division Unim Oil Co. of California Union Oil Center Ixxs Angeles, a 90017 Attn: DeepakKenkeremath John C, -ley Ics Alamos National Labs Mail Stop 570 u -8 ~ l 4 a7545 might Smith Halliburton Drawer 1431 m-8 a 73533 Tan Turner Phillips Petroleum ccap?arry Geothermal operatiom 655 East 4500 South Salt fake City, UT 84107 Tan Warren Amom Prcduction canpany Research center P.O. Box 591 Tulsa, OK 74102 U.S. 9 U.S. GOVERNMENT PRINTING OFFICE: 1964-0-176-031/4196 U.S. GOVERNMENT PRINTING OFFICE: 1964-0-176-031/4196 The Major Conclusions of the Investigation Department of Energy (3) Geothennal Hydropower Tedmologies Divisiun Fbrrestal Bldg., CE 324 lo00 Independence Ave. S.W. Washington, D.C. 20585 D. Allen Attn: J. Bresee R e 'IbE W. P. Grace, WAL Nuclear & Geosciences Division 1512 3141 3151 6000 6200 6240 6241 6241 6300 6330 8214 L. A. L. J. Eridcscm (5) w. L. Garner (3) v. L. Dugan J. R. Kelsey (20) L. E. Duda (10) R. W. Lynch W. D. Weart M. A. M E. H. Beme R. K. Traeger 3141 L. J. Eridcscm (5) 129 *U.S. GOVERNMENT PRINTING OFFICE: 1964-0-176-031/4196
https://openalex.org/W1975595696	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0111343&type=printable	English	null	Podocalyxin Promotes Glioblastoma Multiforme Cell Invasion and Proliferation via β-Catenin Signaling	PloS one	2,014	cc-by	10,170	Introduction Glioblastoma multiforme (GBM) is by far the most common and most malignant primary adult brain tumor [1]. Despite great advances in surgery, chemotherapy and radiotherapy, the median survival is only 12 to 15 months for patients with GBM [2]. The poor prognosis of GBM is largely attributed to their rapid growth, invasiveness, and high rate of recurrence [3]. The highly invasive nature of GBM makes surgical resection non-curative, and it has also been proposed that invading cells may be more resistant to radiation and chemotherapy [3]. Therefore, it is important to identify and confirm potential therapeutic targets involved in the invasion and progression of GBM. b-Catenin (b-cat), originally identified as an essential regulator for E-cadherin-mediated cell-cell interaction, is a key component of the Wnt signaling pathway [10]. In most cells, b-cat is predominantly located at the plasma membrane in a complex with cadherins and a-catenin, which is resistant to mild detergent such as Triton X-100 and Nonidet P-40. This is the insoluble pool of b- catenin. Under normal conditions, small amount of soluble b-cat is present in the cytoplasm free from cadherin [11]. Wnt signals are transduced via specific cell surface receptors to activate a series of biochemical reactions involving a large protein complex consisting of b-catenin and glycogen synthase kinase-3b (GSK-3b), resulting in stabilization of soluble b-cat and therefore an increase in the soluble pool of b-cat [12]. The soluble b-cat interacts with the T cell factor (Tcf) family transcription factors to activate a number of downstream target genes such as c-Myc and c-Jun, which play important roles in the progression of cancers [11,13,14]. Increased b-cat signaling has been linked to progression of a variety of cancers, including prostate cancer, hepatocarcinoma and renal cell carcinoma [14–16]. Recent studies have suggested that b-cat signaling is a key contributor to the proliferation and invasiveness of GBM cells [17,18]. Podocalyxin (PODX) is a highly glycosylated and sialylated transmembrane protein, and a CD34 ortholog normally expressed on hematopoietc stem cells, hemangioblasts, vascular endothelial cells, podocytes, and a subset of neural progenitors [4]. The clinical significance of PODX in cancer progression has been investigated in many cancer types. PODXL expression is correlated with tumor grade in uterine endometrioid adenocarci- noma [5]. Its overexpression is an independent indicator of poor outcome in breast and colorectal carcinoma [6,7]. PODX also reportedly enhance in vitro invasion in breast cancer and prostate cancer cells [8]. Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution Lice use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant dat ta Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: This work was supported by Hunan Provincial Natural Science Foundation (grants #14A2256), Hunan, China. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: 13707315567@139 com Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: This work was supported by Hunan Provincial Natural Science Foundation (grants #14A2256), Hunan, China. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: This work was supported by Hunan Provincial Natural Science Foundation (grants #14A2256), Hunan, China. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: 13707315567@139.com * Email: 13707315567@139.com astrocytoma cell invasion and survival against apoptotic stress [9], suggesting that PODX also contributes to GBM progression. Podocalyxin Promotes Glioblastoma Multiforme Cell Invasion and Proliferation via b-Catenin Signaling Yu Liu, Yu-Gang Jiang* Department of Neurosurgery, The Second Xiangya Hospital, Central South University, Changsha, China Department of Neurosurgery, The Second Xiangya Hospital, Central South University, Changsha, China October 2014 \| Volume 9 \| Issue 10 \| e111343 Abstract Both podocalyxin (PODX) and b-catenin (b-cat) signaling reportedly play important roles in glioblastoma multiforme (GBM) progression. In this study, we for the first time explored crosstalk between PODX and b-cat signaling in GBM cells, and assessed its impact on GBM cell invasion and proliferation. Stable overexpression of PODX in LN-229 and U-118 MG human GBM cells increased the soluble/intracellular b-cat level, TOPflash luciferase reporter activity, the mRNA levels of b-cat signaling target genes, matrix metalloproteinase 9 (MMP9) expression/activity, and cell invasion and proliferation, which was abolished by selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 and selective b-cat signaling inhibitor CCT031374. On the other hand, stable knockdown of PODX in LN-229 and U-118 MG cells decreased the soluble b- cat level, TOPflash luciferase reporter activity, the mRNA levels of b-cat signaling target genes, MMP9 expression/activity, and cell invasion and proliferation, which was completely reversed by overexpression of a constitutively active b-cat mutant. In addition, overexpression of PODX induced p38 MAPK activity and inactivating phosphorylation of glycogen synthase kinase-3b (GSK-3b) at serine 389 in LN-229 and U-118 MG cells, which was abolished by PD169316, but not CCT031374; knockdown of PODX decreased p38 MAPK activity and inactivating phosphorylation of GSK-3b at serine 389 in both cell lines, which was not significantly affected by overexpression of constitutively active b-cat. In conclusion, this study indicates that PODX promotes GBM cell invasion and proliferation by elevating the soluble b-cat level/b-cat signaling through the p38 MAPK/GSK-3b pathway. Uncovering the PODX/b-cat signaling axis adds new insights not only into the biological functions of PODX and b-cat, but also into the molecular mechanisms underlying GBM progression. Citation: Liu Y, Jiang Y-G (2014) Podocalyxin Promotes Glioblastoma Multiforme Cell Invasion and Proliferation via b-Catenin Signaling. PLoS ONE 9(10): e111343. doi:10.1371/journal.pone.0111343 Editor: Rajesh Mohanraj, Faculty of Medicine & Health Sciences, United Arab Emirates Received August 13, 2014; Accepted October 1, 2014; Published October 28, 2014 iang. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted roduction in any medium, provided the original author and source are credited. Copyright: 2014 Liu, Jiang. This is an open-access article distributed under the terms of the Creative Commons Attribution License, use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2014 Liu, Jiang. Introduction A recent report has shown that PODX promotes October 2014 \| Volume 9 \| Issue 10 \| e111343 1 PLOS ONE \| www.plosone.org Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin Figure 1. Protein levels of podocalyxin (PODX) and b-catenin (b-cat) in glioblastoma multiforme (GBM) cells with overexpression o knockdown of PODX. In (A) LN-229 and (B) U-118 MG human GBM cells, protein levels of PODX and soluble and total b-cat were determined wi western blot analyses in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, lane 2), cells stably transfecte with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD16931 (25 mM) for 30 hours (PODX+PD, lane 4), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) f 30 hours (PODX+CCT, lane 5), cells stably transduced with scramble control shRNA (SC, lane 6), cells stably transduced with PODX-shRNA (lane 7), an cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b-cat (PODX-shRNA+active-cat, lane 8 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blotting was used as a loading control. The total b-cat protein level was not significant altered by PODX in both LN-229 and U-118 MG cells. Density of the PODX and the soluble b-cat blots was normalized against that of the GAPDH bl to obtain a relative blot density, which was expressed as fold changes to that of NC (designated as 1) to represent the relative protein content. Thre independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. ap,0.05 vs. controls (NC, VC an SC); bp,0.05 vs. PODX; cp,0.05 vs. PODX+PD; dp,0.05 vs. PODX+CCT. Figure 1. Protein levels of podocalyxin (PODX) and b-catenin (b-cat) in glioblastoma multiforme (GBM) cells with overexpression knockdown of PODX. In (A) LN-229 and (B) U-118 MG human GBM cells, protein levels of PODX and soluble and total b-cat were determined w Figure 1. Protein levels of podocalyxin (PODX) and b-catenin (b-cat) in glioblastoma multiforme (GBM) cells with overexpression o knockdown of PODX. Introduction In (A) LN-229 and (B) U-118 MG human GBM cells, protein levels of PODX and soluble and total b-cat were determined wit western blot analyses in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, lane 2), cells stably transfecte with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD16931 (25 mM) for 30 hours (PODX+PD lane 4) cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) fo Figure 1. Protein levels of podocalyxin (PODX) and b-catenin (b-cat) in glioblastoma multiforme (GBM) cells with overexpression or knockdown of PODX. In (A) LN-229 and (B) U-118 MG human GBM cells, protein levels of PODX and soluble and total b-cat were determined with western blot analyses in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, lane 2), cells stably transfected with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD, lane 4), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 30 hours (PODX+CCT, lane 5), cells stably transduced with scramble control shRNA (SC, lane 6), cells stably transduced with PODX-shRNA (lane 7), and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b-cat (PODX-shRNA+active-cat, lane 8). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blotting was used as a loading control. The total b-cat protein level was not significantly altered by PODX in both LN-229 and U-118 MG cells. Density of the PODX and the soluble b-cat blots was normalized against that of the GAPDH blot to obtain a relative blot density, which was expressed as fold changes to that of NC (designated as 1) to represent the relative protein content. Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. ap,0.05 vs. controls (NC, VC and SC); bp,0.05 vs. PODX; cp,0.05 vs. PODX+PD; dp,0.05 vs. PODX+CCT. doi:10.1371/journal.pone.0111343.g001 October 2014 \| Volume 9 \| Issue 10 \| e111343 PLOS ONE \| www.plosone.org 2 Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin Figure 2. Effects of podocalyxin (PODX) on b-catenin (b-cat) signaling luciferase reporter activities in glioblastoma multiforme (GBM) cells. Introduction (A) LN-229 and (B) U-118 MG GBM cells were transfected with TOPflash, a synthetic b-catenin luciferase reporter, or FOPflash, a negative control reporter for TOPflash. Thirty hours later, the luciferase activity was determined in normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 30 hours (PODX+CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b-cat (PODX-shRNA+ active-cat). The luciferase activity was expressed as fold changes to that of NC (designated as 1). ap,0.05 vs. controls (NC, VC and SC); bp,0.05 vs. PODX; cp,0.05 vs. PODX+PD; dp,0.05 vs. PODX+CCT; ep,0.05 vs. PODX-shRNA. doi:10.1371/journal.pone.0111343.g002 Figure 2. Effects of podocalyxin (PODX) on b-catenin (b-cat) signaling luciferase reporter activities in glioblastoma multiforme (GBM) cells. (A) LN-229 and (B) U-118 MG GBM cells were transfected with TOPflash, a synthetic b-catenin luciferase reporter, or FOPflash, a negative control reporter for TOPflash. Thirty hours later, the luciferase activity was determined in normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 30 hours (PODX+CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b-cat (PODX-shRNA+ active-cat). The luciferase activity was expressed as fold changes to that of NC (designated as 1). ap,0.05 vs. controls (NC, VC and SC); bp,0.05 vs. PODX; cp,0.05 vs. PODX+PD; dp,0.05 vs. PODX+CCT; ep,0.05 vs. PODX-shRNA. doi:10.1371/journal.pone.0111343.g002 kinase (MAPK) Assay kit (#9820) was purchased from Cell Signaling Technology. Selective b-cat signaling inhibitor CCT031374 was purchased from Tocris Bioscience (Bristol, UK). Dual-luciferase reporter assay system was purchased from Promega (Madison, WI, USA). Puromycin, G418, and selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Apparently, both PODX and b-cat signaling play important roles in GBM progression. Transfection and lentiviral transduction g LN-229 (CRL-2611) and U-118 MG (HTB-15) human GBM cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Human full length PODX cDNA was subcloned into pcDNA 3.1 expression vector. Human PODX shRNA plasmid (RHS3979-98487921) was purchased from Open Biosystems (Huntsville, AL, USA). Human b-cat cDNA clone (SC107921) was purchased from from Origene (Beijing, China) and the b-cat cDNA sequence lacking those encoding 151 amino-terminal residues was subcloned into pcDNA 3.1 to generate a constitutively active (DN151) b-cat expression vector. Anti-PODX (3D3) (39-3800) antibody and Lipofectamine 2000 transfection reagent were purchased from Life Technologies (Carlsbad, CA, USA). Anti-b-cat (C-18) (sc-1496) (epitope matched to the carboxyl terminal of human b-cat), anti-matrix metalloproteinase 9 (MMP9) (M-17) (sc-6841) and anti-glyceral- dehyde-3-phosphate dehydrogenase (GAPDH) (V-18) (sc-20357) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-GSK-3b antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). The anti- phospho-GSK-3b (serine 389) antibody was purchased from Millipore (Billerica, MA, USA). The SensoLyte 520 MMP-9 Assay Kit (#71155) was purchased from AnaSpec (Fremont, CA, USA). The Methylthiazoletetrazolium (MTT) Cell Proliferation Assay kit (ATCC #30-1010K) was purchased from American Type Culture Collection. The p38 mitogen-activated protein The PODX and the constitutively active (DN151) b-cat expression vectors were respectively transfected into LN-229 and U-118 MG cells using Lipofectamine 2000 transfection reagent (Life Technologies) according to the manufacturer’s instructions. Pools of stable transfectants were generated via selection with G418 (800 mg/mL) by the manufacturer’s protocol. Lentiviral transduction was performed in LN-229 and U-118 MG cells. Lentiviral particles were packaged with vector psPAX2 and vector pMD2.G according to the manufacturer’s instructions (Open Biosystems). A control virus containing a scrambled shRNA sequence that would not lead to specific degradation of any cellular mRNA was used as a negative control for PODX-shRNA lentiviral particles. Pools of stable transductants were generated via selection with puromycin (5 mg/mL). Introduction Our pilot study suggested that PODX could regulate b-cat signaling in GBM cells. In this study, we for the first time explored crosstalk between PODX and b-cat signaling in GBM cells, and assessed its impact on GBM cell invasion and proliferation. Western blot analysis In Western blot analyses, for whole cell lysates, cells were lysed in 250 mL of 26SDS loading buffer (62.5 mM TrisHCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromphenol blue, 5% 2- mercaptoethanol), and incubated at 95uC for 10 min. For soluble b-cat detection, cells were lysed in 0.1% Nonidet P-40 lysis buffer (0.1% Nonidet P-40, 10 mM HEPES, pH 7.5, 142.5 mM KCl, 5 mM MgCl2, and 1 mM EGTA). The lysates were centrifuged at 14,0006g for 10 min, and the supernatants were saved as soluble October 2014 \| Volume 9 \| Issue 10 \| e111343 PLOS ONE \| www.plosone.org 3 Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin Figure 3. Effect of podocalyxin (PODX) on mRNA levels of b-catenin (b-cat), C-Myc and C-Jun in glioblastoma multiforme (GBM) cells. The mRNA levels of b-cat and b-cat signaling target genes C-Myc and C-Jun were determined in (A) LN-229 and (B) U-118 MG GBM cells. Real- time RT-PCR was performed with normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 30 hours (PODX+ CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX- shRNA and stably transfected with constitutively active (DN151) b-cat (PODX-shRNA+active-cat). The mRNA level was expressed as fold changes to that of NC (designated as 1). p,0.05 vs. controls (NC, VC and SC). doi:10.1371/journal.pone.0111343.g003 Figure 3. Effect of podocalyxin (PODX) on mRNA levels of b-catenin (b-cat), C-Myc and C-Jun in glioblastoma multiforme (GBM) cells. The mRNA levels of b-cat and b-cat signaling target genes C-Myc and C-Jun were determined in (A) LN-229 and (B) U-118 MG GBM cells. Western blot analysis Real- time RT-PCR was performed with normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 30 hours (PODX+ CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX- shRNA and stably transfected with constitutively active (DN151) b-cat (PODX-shRNA+active-cat). The mRNA level was expressed as fold changes to that of NC (designated as 1). p,0.05 vs. controls (NC, VC and SC). doi:10.1371/journal.pone.0111343.g003 using SuperScript II reverse transcriptase (Life Technologies). Real-time quantitative PCR was performed on the LightCycler thermal cycler system (Roche Diagnostics, Indianapolis, IN, USA) using SYBR Green Mix (Life Technologies) as described by the manufacturer. The results were normalized against that of GAPDH in the same sample. The primers used are as follows: for b-cat (primers designed to measure both wild type b-cat and constitutively active DN151 b-cat mRNA levels), 59- GAT- CATGCTAGCATGGCAATTCCTGAG-39 (forward) and 59- AAGATCGGTACCTCAGTTATCTACAGG-39 (reverse); for c-Myc, 59-GGACGACGAGACCTTCATCAA-39 (forward) and 59-CCAGCTTCTCTGAGACGAGCTT-39 (reverse); for c-Jun, 59-CAAAGTTTGGATTGCATCAAGTG-39 (forward) and 59- cell lysate [14]. Equal amount of proteins for each sample were separated by 8–15% SDS-polyacrylamide gel and blotted onto a polyvinylidene difluoride microporous membrane (Millipore). Membranes were incubated for 1 hour with a 1:1000 dilution of primary antibody, and then washed and revealed using secondary antibodies with horseradish peroxidase conjugate (1:5000, 1 hour). Peroxidase was revealed with a GE Healthcare ECL kit (Shanghai, China). Proteins were quantified before being loaded onto the gel. Real-time quantitative reverse transcription PCR MMP9 activity assay MMP9 activity was measured with the SensoLyte 520 MMP-9 Assay Kit (AnaSpec) according to the manufacturer’s instructions [19,20]. The supernatants were collected and then incubated with 4-aminophenylmercuric acetate (AMPA) and MMP9 substrate. The fluorescence intensity at Ex/Em Wave lengths of 490 nm/ 520 nm were used as a measure of MMP9 activity. Each experiment was repeated for three times in duplicates. Luciferase Assay LN-229 and U-118 MG cells were transfected with TOPflash or FOPflash plasmids using Lipofectamine 2000 transfection reagent (Life Technologies). Plasmid PRL-CMV encoding Renilla reni- formis luciferase (at one fifth molar ratio to test plasmids) was co- transfected in each transfection as an internal control for data normalization. The luciferase assays were performed 30 hours after transfection with a dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Each experiment was repeated for three times in duplicates. MTT cell proliferation assay In vitro cell proliferation was determined with a MTT Cell Proliferation Assay kit as described by the manufacturer (ATCC). Briefly, cells were cultured at 156103 cells per well in 96-well tissue culture plates and incubated at 37uC for 15 or 30 hours. At the end of the culture period, cells were washed with phosphate- buffered saline, the MTT reagents were added according to the manufacturer’s recommendations, and the absorbance was mea- sured at 570 nm using an ELISA plate reader. Each experiment was repeated for three times in triplicates. Figure 4. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on glioblastoma multiforme (GBM) cell invasion. In vitro cell invasion assays were performed in (A) LN-229 and (B) U-118 MG GBM cells. Invading cells were fixed and counted in normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 30 hours (PODX+CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b-cat (PODX- shRNA+active-cat). Representative cell invasion images are shown. ap, 0.05 vs. controls (NC, VC and SC); bp,0.05 vs. PODX; cp,0.05 vs. PODX+ PD; dp,0.05 vs. PODX+CCT; ep,0.05 vs. PODX-shRNA. doi:10.1371/journal.pone.0111343.g004 Real-time quantitative reverse transcription PCR RNA was prepared from cells using TRIzol reagent (Life Technologies) followed by purification with TURBO DNA-free system (Ambion, Austin, TX, USA). The cDNAs were synthesized October 2014 \| Volume 9 \| Issue 10 \| e111343 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 4 Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin TAACATTATAAATGGTCACAGCACATG-39 (reverse); for Figure 4. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on glioblastoma multiforme (GBM) cell invasion. In vitro cell invasion assays were performed in (A) LN-229 and (B) U-118 MG GBM cells. Invading cells were fixed and counted in normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 30 hours (PODX+CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b-cat (PODX- shRNA+active-cat). Representative cell invasion images are shown. ap, 0.05 vs. controls (NC, VC and SC); bp,0.05 vs. PODX; cp,0.05 vs. PODX+ PD; dp,0.05 vs. PODX+CCT; ep,0.05 vs. PODX-shRNA. doi:10.1371/journal.pone.0111343.g004 GACTCATGACCACAGTCCATGC-39 (forward) and 59- AGAGGCAGGGATGATGTTCTG-39 (reverse). Each experi- ment was repeated for three times in duplicates. Cell invasion assay Transwell cell-culture chambers with 8-mm pore size (BD Biosciences, Bedford, MA, USA) for 24-well plates were coated with 50 mL Matrigel (10 mg/mL; BD Biosciences) (diluted 1:3). LN-229 and U-118 MG cells were seeded in the upper chamber at 36104 cells per well in RPMI 1640 serum-free medium. Complete medium (600 mL) with 10% fetal bovine serum was added to the lower chamber. After 30 hours of incubation, cells were fixed and stained with crystal violet. Invasion cells were counted in four random fields per chamber under microscope. Each experiment was repeated for three times in duplicates. p38 MAPK activity assay Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on expression of matrix metalloproteinase 9 (MMP9) i glioblastoma multiforme (GBM) cells The (A) mRNA and the (B) protein levels of MMP9 in LN-229 (left panel) and U-118 MG (right panel) GB Figure 5. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on expression of matrix metalloproteinase 9 (MMP9) in glioblastoma multiforme (GBM) cells. The (A) mRNA and the (B) protein levels of MMP9 in LN-229 (left panel) and U-118 MG (right panel) GBM cells were respectively determined by real-time RT-PCR and Western blot analyses in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, lane 2), cells stably transfected with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD, lane 4), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 30 hours (PODX+CCT, lane 5), cells stably transduced with scramble control shRNA (SC, lane 6), cells stably transduced with PODX-shRNA (lane 7), and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b-cat (PODX-shRNA+active-cat, lane 8). The mRNA and the protein levels of MMP9 were expressed as fold changes to those of NC (designated as 1), respectively. Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+ SD. ap,0.05 vs. controls (NC, VC and SC); bp,0.05 vs. PODX; cp,0.05 vs. PODX+PD; dp,0.05 vs. PODX+CCT; ep,0.05 vs. PODX-shRNA. doi:10.1371/journal.pone.0111343.g005 Statistical analysis constitutively active b-cat mutant, which lacks 151 amino-terminal residues (DN151), was stably overexpressed in cells expressing PODX-shRNA. In addition, as our pilot study had suggested that PODX could regulate the soluble b-cat level in GBM cells by a p38 MAPK-dependent mechanism (data not shown), we included a selective p38 MAPK inhibitor PD169316 in all experiments in this study [23]. As shown in Fig. 1, compared with the controls, PODX was overexpressed 5.4 and 5 folds in LN-229 and U-118 MG cells, respectively; on the other hand, the endogenous PODX level was knocked down approximately 80% and 75% in LN-229 and U-118 MG cells, respectively. While the total b-cat protein level was not significantly altered by overexpression and knock- down of PODX, overexpression of PODX increased the soluble b- cat level by 3.8 folds in LN-229 cells and by 3.2 folds in U-118 MG cells, which was abolished by PD169316 and CCT031374. On the other hand, knockdown of PODX decreased the soluble b-cat level by over 60% in both cell lines. Fig. 1 also shows that the constitutively active (DN151) form of b-cat was overexpressed in Statistical analyses were performed with SPSS for Windows 10.0 (SPSS Inc., Chicago, IL, USA). All data values were expressed as means6SD. Comparisons of means among multiple groups were performed with one-way ANOVA followed by post hoc pairwise comparisons using Tukey’s tests. A two-tailed p,0.05 was considered statistically significant in this study. p38 MAPK activity assay p y y p38 MAPK activity was measured with the p38 MAPK Assay kit (Cell Signaling Technology) according to the manufacturer’s instructions [21]. Briefly, cells were directly lysed in the culture dishes. Cell lysates were sonicated and centrifuged at 15,000 rpm for 10 minutes at 4uC. The supernatant containing equivalent amounts of protein (200 mg) was incubated by gentle rocking with 20 mL of immobilized phospho-p38-MAPK monoclonal antibody for 16 hours at 4uC. The immunoprecipitates were washed twice with the lysing buffer and pelleted by centrifugation. The p38 MAPK assay was carried out using ATF2 fusion protein (2 mg) as a substrate in the presence of 200 mM ATP and 16kinase buffer following the manufacturer’s recommendations. Samples were resolved on a 12% SDS-PAGE gel and visualized by autoradiog- raphy. TAACATTATAAATGGTCACAGCACATG-39 (reverse); for MMP9, 59-GTTCCCGGAGTGAGTTGA-39 (forward) and 59- TTTACATGGCACTGCAAAGC-39 (reverse); for GAPDH, 59- October 2014 \| Volume 9 \| Issue 10 \| e111343 5 PLOS ONE \| www.plosone.org Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin Figure 5. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on expression of matrix metalloproteinase 9 (MMP9) in glioblastoma multiforme (GBM) cells. The (A) mRNA and the (B) protein levels of MMP9 in LN-229 (left panel) and U-118 MG (right panel) GBM cells were respectively determined by real-time RT-PCR and Western blot analyses in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, lane 2), cells stably transfected with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD, lane 4), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 30 hours (PODX+CCT, lane 5), cells stably transduced with scramble control shRNA (SC, lane 6), cells stably transduced with PODX-shRNA (lane 7), and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b-cat (PODX-shRNA+active-cat, lane 8). The mRNA and the protein levels of MMP9 were expressed as fold changes to those of NC (designated as 1), respectively. Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+ SD. ap,0.05 vs. controls (NC, VC and SC); bp,0.05 vs. PODX; cp,0.05 vs. PODX+PD; dp,0.05 vs. PODX+CCT; ep,0.05 vs. PODX-shRNA. doi:10.1371/journal.pone.0111343.g005 Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin Figure 5. Effects of PODX on the protein levels of b-cat in GBM cells We stably overexpressed PODX in LN-229 and U-118 MG human GBM cells by stable transfection, and on the other hand stably transduced the cells with lentiviral shRNA to knock down PODX. CCT031374, a selective b-cat signaling inhibitor that decreases the intracellular/soluble b-cat level [22], was employed to inhibit b-cat signaling in cells overexpressing PODX. A October 2014 \| Volume 9 \| Issue 10 \| e111343 PLOS ONE \| www.plosone.org 6 ure 6. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on matrix metalloproteinase 9 (MMP9) activities in glioblast ultiforme (GBM) cells. In (A) LN-229 and (B) U-118 MG GBM cells, MMP9 activities were determined with a SensoLyte 520 MMP9 Assa aSpec) in normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells s nsfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+ s stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 30 hours (PODX+CCT), cells s nsduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX-shRNA and s nsfected with constitutively active (DN151) b-cat (PODX-shRNA+active-cat). The MMP9 activity was shown as fold changes to that o Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Ca Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin 6. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on matrix metalloproteinase 9 (MMP9) activities in glioblasto orme (GBM) cells. In (A) LN-229 and (B) U-118 MG GBM cells, MMP9 activities were determined with a SensoLyte 520 MMP9 Assay Figure 6. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on matrix metalloproteinase 9 (MMP9) activities in glioblastoma multiforme (GBM) cells. In (A) LN-229 and (B) U-118 MG GBM cells, MMP9 activities were determined with a SensoLyte 520 MMP9 Assay Kit (AnaSpec) in normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 30 hours (PODX+CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b-cat (PODX-shRNA+active-cat). Effects of PODX on transcriptional activities of b-cat in GBM cells As the above results suggested that PODX could significantly elevate the soluble b-cat level in GBM cells, we next examined whether PODX would activate b-cat signaling in GBM cells. As shown in Fig. 2, transcriptional activities of b-cat in LN-229 and U-118 MG cells were measured with TOPflash, a synthetic b-cat/ Tcf-dependent luciferase reporter [14]. Compared with controls, overexpression of PODX increased the luciferase activity of TOPflash by 10.6 folds in LN-229 cells and by 9.5 folds in U-118 MG cells, which was abolished by PD169316 and CCT031374. On the other hand, knockdown of PODX decreased the luciferase activity of TOPflash by approximately 70% in both cell lines, which was completely reversed by overexpression of DN151 b-cat. Little change was observed with FOPflash, a negative control reporter with mutated Tcf binding elements (Fig. 2) [14]. As shown in Fig. 3, real-time RT-PCR showed that overexpression or knockdown of PODX did not significantly affect b-cat mRNA levels in LN-229 and U-118 MG cells. However, the mRNA levels of b-cat signaling target genes (c-Myc and c-Jun) were increased over 3.3 folds by overexpression of PODX, which was abolished by PD169316 and CCT031374. Knockdown of PODX decreased the mRNA levels of c-Myc and c-Jun by over 60% in both cell lines, which was completely reversed by overexpression of DN151 b-cat (Fig. 3). Taken together, the results indicate that PODX can activate b-cat signaling in GBM cells by post-transcriptionally elevating the soluble b-cat level through a p38 MAPK-dependent mechanism. In addition, real-time RT-PCR with primers specif- ically designed to measure both wild type b-cat and DN151 b-cat mRNA levels showed that stable transfection of DN151 b-cat markedly increased the detected b-cat mRNA level, which was in agreement with the strong DN151 b-cat protein expression in Fig. 1. Effects of PODX on the protein levels of b-cat in GBM cells The MMP9 activity was shown as fold changes to that of NC (designated as 1). ap,0.05 vs. controls (NC, VC and SC); bp,0.05 vs. PODX; cp,0.05 vs. PODX+PD; dp,0.05 vs. PODX+CCT; ep,0.05 vs. PODX-shRNA. doi:10.1371/journal.pone.0111343.g006 October 2014 \| Volume 9 \| Issue 10 \| e111343 PLOS ONE \| www.plosone.org 7 Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin Figure 7. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on glioblastoma multiforme (GBM) cell proliferation. In (A) LN-229 and (B) U-118 MG GBM cells, methlythiazoletetrazolium (MTT) cell proliferation assays were performed for 15 or 30 hours in normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen- activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 15 or 30 hours (PODX+CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b cat (PODX shRNA+active cat) Cell proliferation at 15 and 30 the cells. It is apparent that CCT031374 and DN151 b-cat showed no significant effect on the protein level of PODX (Fig. 1), suggesting that inhibition or activation of b-cat signaling had no reciprocal effects on PODX expression. Effects of PODX/b-cat signaling on cell invasion and MMP9 expression/activity in GBM cells In (A) LN-229 and (B) U-118 MG GBM cells, the p38 MAPK activity was determined with a p38 MAPK Signaling Technology) by measuring phosphorylation of ATF2, a substrate of activated p38 MAPK. The levels of phosphorylated AT ere determined with western blot in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, la bly transfected with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein k inhibitor PD169316 (25 mM) for 30 hours (PODX+PD, lane 4), cells stably transfected with PODX and treated with selective b-cat sign Figure 8. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on p38 mitogen-activated protein kinase (MAPK) activities in glioblastoma multiforme (GBM) cells. In (A) LN-229 and (B) U-118 MG GBM cells, the p38 MAPK activity was determined with a p38 MAPK Assay kit (Cell Signaling Technology) by measuring phosphorylation of ATF2, a substrate of activated p38 MAPK. The levels of phosphorylated ATF2 (p- ATF2) were determined with western blot in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, lane 2), cells stably transfected with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD, lane 4), cells stably transfected with PODX and treated with selective b-cat signaling ects of podocalyxin (PODX)/b-catenin (b-cat) signaling on p38 mitogen-activated protein kinase (MAPK) activitie multiforme (GBM) cells. In (A) LN-229 and (B) U-118 MG GBM cells, the p38 MAPK activity was determined with a p38 MAPK A ng Technology) by measuring phosphorylation of ATF2, a substrate of activated p38 MAPK. The levels of phosphorylated ATF termined with western blot in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, lan nsfected with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein k or PD169316 (25 mM) for 30 hours (PODX+PD, lane 4), cells stably transfected with PODX and treated with selective b-cat sign Figure 8. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on p38 mitogen-activated protein kinase (MAPK) activities in glioblastoma multiforme (GBM) cells. In (A) LN-229 and (B) U-118 MG GBM cells, the p38 MAPK activity was determined with a p38 MAPK Assay kit (Cell Signaling Technology) by measuring phosphorylation of ATF2, a substrate of activated p38 MAPK. Effects of PODX/b-cat signaling on cell invasion and MMP9 expression/activity in GBM cells To examine effects of the PODX/b-cat signaling axis on GBM cell invasion, we performed in vitro cell invasion assays. Compared with the controls, overexpression of PODX increased cell invasion by approximately 2 folds in LN-229 and U-118 MG cells, which was abolished by PD169316 and CCT031374 (Fig. 4). On the other hand, knockdown of PODX decreased cell invasion by approximately 50% in both cell lines, which was completely reversed by overexpression of DN151 b-cat (Fig. 4). Figure 7. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on glioblastoma multiforme (GBM) cell proliferation. In (A) LN-229 and (B) U-118 MG GBM cells, methlythiazoletetrazolium (MTT) cell proliferation assays were performed for 15 or 30 hours in normal control cells (NC), cells stably transfected with the empty pcDNA3.1 vector (VC), cells stably transfected with PODX, cells stably transfected with PODX and treated with selective p38 mitogen- activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 15 or 30 hours (PODX+CCT), cells stably transduced with scramble control shRNA (SC), cells stably transduced with PODX-shRNA, and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b-cat (PODX-shRNA+active-cat). Cell proliferation at 15 and 30 hours was expressed as fold changes to that of NC (designated as 1). *p,0.05 vs. controls (NC, VC and SC) at 30 hours. doi:10.1371/journal.pone.0111343.g007 MMPs play a critical role in cancer cell invasion [24]. Among different MMPs tested, we found that the MMP9 expression was significantly altered by PODX/b-cat signaling in GBM cells. As shown in Fig. 5, compared with the controls, overexpression of PODX increased MMP9 expression at both the mRNA and the protein levels by over 4 folds in LN-229 and U-118 MG cells, which was abolished by PD169316 and CCT031374. Knockdown of PODX decreased MMP9 expression by approximately 60% in both cell lines, which was completely reversed by overexpression of DN151 b-cat (Fig. 5). Similar data trend was observed with the MMP9 activity (Fig. 6). October 2014 \| Volume 9 \| Issue 10 \| e111343 PLOS ONE \| www.plosone.org 8 Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin 8. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on p38 mitogen-activated protein kinase (MAPK) activiti stoma multiforme (GBM) cells. Effects of PODX/b-cat signaling on cell invasion and MMP9 expression/activity in GBM cells The levels of phosphorylated ATF2 (p- ATF2) were determined with western blot in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, lane 2), cells stably transfected with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD, lane 4), cells stably transfected with PODX and treated with selective b-cat signaling October 2014 \| Volume 9 \| Issue 10 \| e111343 PLOS ONE \| www.plosone.org 9 Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin inhibitor CCT031374 (50 mM) for 30 hours (PODX+CCT, lane 5), cells stably transduced with scramble control shRNA (SC, lane 6), cells stably transduced with PODX-shRNA (lane 7), and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b-cat (PODX-shRNA+active-cat, lane 8). The p-ATF2 content/p38 MAPK activity was shown as fold changes to that of NC (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. ap,0.05 vs. controls (NC, VC and SC); bp,0.05 vs. PODX; cp,0.05 vs. PODX+PD; dp,0.05 vs. PODX+CCT. doi:10.1371/journal.pone.0111343.g008 inhibitor CCT031374 (50 mM) for 30 hours (PODX+CCT, lane 5), cells stably transduced with scramble control shRNA (SC, lane 6), cells stably transduced with PODX-shRNA (lane 7), and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b-cat (PODX-shRNA+active-cat, lane 8). The p-ATF2 content/p38 MAPK activity was shown as fold changes to that of NC (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. ap,0.05 vs. controls (NC, VC and SC); bp,0.05 vs. PODX; cp,0.05 vs. PODX+PD; dp,0.05 vs. PODX+CCT. doi:10.1371/journal.pone.0111343.g008 models, we in the present study provide the first evidence supporting a crosstalk between PODX and b-cat signaling in cancer cells. We used two GBM cell lines as cell models in this study: (1) LN229, established from glioblastoma in the brain cortex of a 60-year-old female, showing epithelial morphology; (2) U-118 MG established from glioblastoma of a 50-year-old male, showing mixed morphology. Similar findings in the two cell models with relatively big background differences demonstrate a generalizable role of a PODX/b-cat signaling axis in GBM. Effects of PODX on p38 MAPK activities in GBM cells Effects of PODX on p38 MAPK activities in GBM cells The above results suggested that PODX could stimulate GBM cell invasion and proliferation by promoting b-cat signaling though a p38-dependent mechanism. Therefore, we next exam- ined effects of the PODX/b-cat signaling axis on p38 MAPK activity, which was measured by phosphorylation of ATF2, a substrate of activated p38 MAPK [21]. As evidenced by increased expression of phosphorylated ATF2, overexpression of PODX respectively induced p38 MAPK activity by 3.6 and 3.1 folds in LN-229 and U-118 MG cells, which was abolished by PD169316, but not CCT031374 (Fig. 8). On the other hand, knockdown of PODX decreased p38 MAPK activity by approximately 70% in both cell lines, which was not significantly affected by overexpres- sion of DN151 b-cat (Fig. 8). The results indicate that PODX and b-cat signaling are respectively upstream and downstream of p38 MAPK in the PODX/b-cat signaling axis. Effects of PODX on inactivating phosphorylation of GSK- 3b in GBM cells In line with the findings, our study showed that PODX increased the level of phosphorylated GSK-3b at serine 389 as well as the soluble b-cat level via p38 MAPK in GBM cells. GSK-3b reportedly is a downstream target of the p38 MAPK pathway [25,26]. Phosphorylation of GSK-3b at serine 389 by p38 enhances inactivation of GSK-3b and results in subsequent stabilization and accumulation of soluble b-cat [11,27]. Since our results had indicated that PODX could elevate the soluble b- cat level/b-cat signaling through p38 MAPK in GBM cells, we next examined effects of PODX on the level of inactivating phosphorylation of GSK-3b at serine 189 in GBM cells. As shown in Fig. 9, while the total GSK-3b was not significantly changed, overexpression of PODX respectively increased the level of phosphorylated GSK-3b (serine 389) by 3.2 and 3.0 folds in LN- 229 and U-118 MG cells, which was abolished by PD169316, but not CCT031374. On the other hand, knockdown of PODX decreased phosphorylation of GSK-3b at serine 389 by approx- imately 60% in both cell lines, which was not significantly affected by overexpression of DN151 b-cat (Fig. 9). Changes in the level of inactivating phosphorylation of GSK-3b at serine 189 (Fig. 9) showed similar data trend to that of the p38 MAPK activity (Fig. 8) and in agreement with changes in the soluble b-cat level (Fig. 1). Taken together, the results indicate that PODX elevate the soluble b-cat level/b-cat signaling in GBM cells via the p38 MAPK/GSK-3b pathway. Among different MMPs tested, we found that the MMP9 expression/activity was significantly altered by PODX/b-cat signaling, which is in agreement with a previous report that PODX induces MMP9 expression in GBM cells [9]. Our study has further revealed that b-cat signaling is a major mediator of Effects of PODX/b-cat signaling on cell invasion and MMP9 expression/activity in GBM cells Effects of PODX/b-cat signaling on GBM cell proliferation To examine effects of the PODX/b-cat signaling axis on GBM cell proliferation, we performed MTT cell invasion assays. Compared with the controls, overexpression of PODX signifi- cantly increased cell proliferation in LN-229 and U-118 MG cells after 30 hours of culture, which was abolished by PD169316 and CCT031374 (Fig. 7). On the other hand, knockdown of PODX significantly inhibited cell proliferation in both cell lines, which was completely reversed by overexpression of DN151 b-cat (Fig. 7). PODX is thought to regulate cell morphology and adhesion through its connections to intracellular proteins and to extracel- lular ligands [28]. b-cat is both a structural component of cell-cell contact sites and a signaling protein that activates the Wnt survival pathway [17,18]. In the absence of Wnt ligands, cytoplasmic/ soluble b-cat is constantly degraded by the axin complex. Wnt signals are transduced via specific cell surface receptors to deactivate the destruction complex, resulting in accumulation of cytoplasmic/soluble b-cat and transcriptional activation of b-cat/ Tcf-regulated genes [17]. In this study, overexpression and knockdown of PODX in GBM cells respectively increased and decreased the soluble b-cat level without significantly altering the expression of b-cat at the mRNA and the total protein levels, suggesting PODX elevates the soluble b-cat level/b-cat signaling in GBM cells by a post-translational modification mechanism. In addition, as a selective p38 MAPK inhibitor readily abolished the PODX-elevated soluble b-cat level without significantly altering the expression of PODX and b-cat, it indicates that PODX elevates the soluble b-cat level/b-cat signaling in a post- translational p38 MAPK-dependent manner in GBM cells. The underlying mechanisms will be uncovered in our future studies. PODX is thought to regulate cell morphology and adhesion through its connections to intracellular proteins and to extracel- lular ligands [28]. b-cat is both a structural component of cell-cell contact sites and a signaling protein that activates the Wnt survival pathway [17,18]. In the absence of Wnt ligands, cytoplasmic/ soluble b-cat is constantly degraded by the axin complex. Wnt signals are transduced via specific cell surface receptors to deactivate the destruction complex, resulting in accumulation of cytoplasmic/soluble b-cat and transcriptional activation of b-cat/ Tcf-regulated genes [17]. Effects of PODX on inactivating phosphorylation of GSK- 3b in GBM cells PODX reportedly enhances invasion in many cancers, includ- ing GBM [5–9]. Although there is evidence that PODX participate in epithelial-mesenchymal transition and interacts with different mediators of metastasis, the role of PODX is not yet fully understood [28]. Abnormal activation of b-cat signaling plays a pivotal role in the progression of a variety of cancers [17]. Recent studies have suggested that b-cat signaling is a key contributor to the proliferation and invasiveness of GBM cells. In our study, overexpression of PODX significantly increased GBM cell invasion, which was abolished by a selective b-cat signaling inhibitor that decreases the soluble b-cat level [22], suggesting that b-cat signaling is major downstream mediator of the promoting effect of PODX on GBM cell invasion. This was corroborated by the finding that knockdown of PODX significantly inhibited GBM dell invasion, which was completely reversed by overexpression of a constitutively active b-cat mutant. GSK-3b activity is a determinant of b-cat stabilization and accumulation of soluble b- cat (Sharma et al., 2002), and p38 MAPK has been shown to inhibit GSK-3b activity via phosphorylating serine 389 [25]. In line with the findings, our study showed that PODX increased the level of phosphorylated GSK-3b at serine 389 as well as the soluble b-cat level via p38 MAPK in GBM cells. PODX reportedly enhances invasion in many cancers, includ- ing GBM [5–9]. Although there is evidence that PODX participate in epithelial-mesenchymal transition and interacts with different mediators of metastasis, the role of PODX is not yet fully understood [28]. Abnormal activation of b-cat signaling plays a pivotal role in the progression of a variety of cancers [17]. Recent studies have suggested that b-cat signaling is a key contributor to the proliferation and invasiveness of GBM cells. In our study, overexpression of PODX significantly increased GBM cell invasion, which was abolished by a selective b-cat signaling inhibitor that decreases the soluble b-cat level [22], suggesting that b-cat signaling is major downstream mediator of the promoting effect of PODX on GBM cell invasion. This was corroborated by the finding that knockdown of PODX significantly inhibited GBM dell invasion, which was completely reversed by overexpression of a constitutively active b-cat mutant. GSK-3b activity is a determinant of b-cat stabilization and accumulation of soluble b- cat (Sharma et al., 2002), and p38 MAPK has been shown to inhibit GSK-3b activity via phosphorylating serine 389 [25]. Effects of PODX/b-cat signaling on cell invasion and MMP9 expression/activity in GBM cells In this study, overexpression and knockdown of PODX in GBM cells respectively increased and decreased the soluble b-cat level without significantly altering the expression of b-cat at the mRNA and the total protein levels, suggesting PODX elevates the soluble b-cat level/b-cat signaling in GBM cells by a post-translational modification mechanism. In addition, as a selective p38 MAPK inhibitor readily abolished the PODX-elevated soluble b-cat level without significantly altering the expression of PODX and b-cat, it indicates that PODX elevates the soluble b-cat level/b-cat signaling in a post- translational p38 MAPK-dependent manner in GBM cells. The underlying mechanisms will be uncovered in our future studies. Discussion Density of the p-GSK-3b (serine 389) blot was normalized against that of the total GSK-3b blot to obtain a relative blot density, which was expressed as fold changes to that of NC (designated as 1). Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. ap,0.05 vs. controls (NC, VC and SC); bp,0.05 vs. PODX; cp,0.05 vs. PODX+PD; dp,0.05 vs. PODX+CCT. doi:10.1371/journal.pone.0111343.g009 PODX-induced MMP9 expression in GBM cells, for a selective b- cat signaling inhibitor abolished the promoting effect of PODX on MMP9 expression. This was confirmed by the finding that direct activation of b-cat signaling by overexpressing a constitutively active b-cat mutant completely reversed the inhibitory effects of PODX-knockdown on MMP9 expression. The inducing effect of PODX/b-cat signaling on MMP9 expression/activity may at least partially explain for its promoting effect on GBM cell invasion. In addition, our results show that PODX/b-cat signaling activates MMP9 expression at the mRNA transcription level. Future studies are needed to examine whether this is a direct effect of b-cat/Tcf transcriptional activities, or further downstream of activated target genes of b-cat/Tcf signaling. Like several other solid tumors, GBM are considered to be driven by a small sub-population of cells known as glioma stem cells [29]. b-cat signaling reportedly is essential for cancer stem cell maintenance [30]. PODX is also a well-known stem cell marker, and is closely related to stem cell marker CD34 and to endoglycan [25]. Thus, based on our findings, it will also be interesting to examine in future studies the role of the PODX/b-cat signaling axis in glioma stem cells. In conclusion, our study indicates that PODX promotes GBM cell invasion and proliferation by elevating the soluble b-cat level/ b-cat signaling through the p38 MAPK/GSK-3b pathway. Uncovering the PODX/b-cat signaling axis adds new insights not only into the biological functions of PODX and b-cat, but also into the molecular mechanisms underlying GBM progression. Activation of b-cat signaling promote cell proliferation [17]. Indeed, our study has shown that PODX promotes GBM cell proliferation by elevating the soluble b-cat level/b-cat signaling. As the PODX/b-cat signaling axis enhances both invasion and proliferation of GBM cells, it may play an important role in GBM progression. Discussion Both PODX and b-cat has been found important for malignant progression in a variety of cancers [5–9,14–16]. Using GBM cell October 2014 \| Volume 9 \| Issue 10 \| e111343 PLOS ONE \| www.plosone.org 10 Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin 9. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on phosphorylated glycogen synthase kinase-3b (p- n glioblastoma multiforme (GBM) cells. In (A) LN-229 and (B) U-118 MG GBM cells, the levels of p-GSK-3b at serine 389 and tota termined with Western blot analyses in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC NE \| www.plosone.org 11 October 2014 \| Volume 9 \| Issue 10 \| s of podocalyxin (PODX)/b-catenin (b-cat) signaling on phosphorylated glycogen synthase kinase-3b stoma multiforme (GBM) cells. In (A) LN-229 and (B) U-118 MG GBM cells, the levels of p-GSK-3b at serine 389 and with Western blot analyses in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector Figure 9. Effects of podocalyxin (PODX)/b-catenin (b-cat) signaling on phosphorylated glycogen synthase kinase-3b (p-GSK-3b) levels in glioblastoma multiforme (GBM) cells. In (A) LN-229 and (B) U-118 MG GBM cells, the levels of p-GSK-3b at serine 389 and total GSK-3b were determined with Western blot analyses in normal control cells (NC, lane 1), cells stably transfected with the empty pcDNA3.1 vector (VC, lane 2), October 2014 \| Volume 9 \| Issue 10 \| e111343 PLOS ONE \| www.plosone.org 11 11 Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin cells stably transfected with PODX (lane 3), cells stably transfected with PODX and treated with selective p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 mM) for 30 hours (PODX+PD, lane 4), cells stably transfected with PODX and treated with selective b-cat signaling inhibitor CCT031374 (50 mM) for 30 hours (PODX+CCT, lane 5), cells stably transduced with scramble control shRNA (SC, lane 6), cells stably transduced with PODX-shRNA (lane 7), and cells stably transduced with PODX-shRNA and stably transfected with constitutively active (DN151) b-cat (PODX-shRNA+active-cat, lane 8). The total GSK-3b protein level was not significantly altered by PODX in both LN-229 and U-118 MG cells. References 15. Cheng BQ, Jiang Y, Zhu Q, Lin WG (2014) Wnt/b-catenin aids in regulating the proliferation of hepG2 cells mediated by thy-1. Genet Mol Res 13: 5115– 5127. 1. Zhang Z, Lin CC (2014) Taking advantage of neural development to treat glioblastoma. Eur J Neurosci doi: 10.1111/ejn.12655 1. Zhang Z, Lin CC (2014) Taking advantage of neural development to treat glioblastoma. Eur J Neurosci doi: 10.1111/ejn.12655 g j 2. Wen PY, Kesari S (2008) Malignant gliomas in adults. N Engl J Med 359: 492– 507. 16. VON Schulz-Hausmann SA, Schmeel LC, Schmeel FC, Schmidt-Wolf IG (2014) Targeting the wnt/beta-catenin pathway in renal cell carcinoma. Anticancer Res 34: 4101–4108. 3. Giese A, Bjerkvig R, Berens ME, Westphal M (2003) Cost of migration: invasion of malignant gliomas and implications for treatment. J Clin Oncol 21: 1624– 1636. 17. Kaur N, Chettiar S, Rathod S, Rath P, Muzumdar D, et al. (2013) Wnt3a mediated activation of Wnt/b-catenin signaling promotes tumor progression in glioblastoma. Mol Cell Neurosci 54: 44–57. 4. Nielsen JS, McNagny KM (2009) The role of podocalyxin in health and disease. J Am Soc Nephrol 20: 1669–1676. 18. Lee WS, Woo EY, Kwon J, Park MJ, Lee JS, et al. (2013) Bcl-w Enhances Mesenchymal Changes and Invasiveness of Glioblastoma Cells by Inducing Nuclear Accumulation of b-Catenin. PLoS One 8(6): e68030. 5. Yasuoka H, Tsujimoto M, Inagaki M, Kodama R, Tsuji H, et al. (2012) Clinicopathological significance of podocalyxin and phosphorylated ezrin in uterine endometrioid adenocarcinoma. J Clin Pathol 65: 399–402. 19. Pifer MA, Maerz T, Baker KC, Anderson K (2014) Matrix metalloproteinase content and activity in low-platelet, low-leukocyte and high-platelet, high- leukocyte platelet rich plasma (PRP) and the biologic response to PRP by human ligament fibroblasts. Am J Sports Med 42: 1211–1218. 6. Larsson A, Johansson ME, Wangefjord S, Gaber A, Nodin B, et al. (2011) Overexpression of podocalyxin-like protein is an independent factor of poor prognosis in colorectal cancer. Br J Cancer 105: 666–672. 7. Somasiri A, Nielsen JS, Makretsov N, McCoy ML, Prentice L, et al. (2004) Overexpression of the anti-adhesin podocalyxin is an independent predictor of breast cancer progression. Cancer Res 64: 5068–5073. 20. Spolidoro M, Putignano E, Munafo` C, Maffei L, Pizzorusso T (2013) Inhibition of matrix metalloproteinases prevents the potentiation of nondeprived-eye responses after monocular deprivation in juvenile rats. Cereb Cortex 22: 725– 734. 8. Discussion In addition, since both PODX and b-cat are essential drivers of malignant progression in many cancers [5–9,14–16], the PODX/b-cat signaling axis may also play important roles in the progression of other cancers besides GBM, which needs to be verified in future studies. Author Contributions Conceived and designed the experiments: YL Y-GJ. Performed the experiments: YL Y-GJ. Contributed reagents/materials/analysis tools: YL Conceived and designed the experiments: YL Y-GJ. Performed the experiments: YL Y-GJ. Contributed reagents/materials/analysis tools: YL Y-GJ. Contributed to the writing of the manuscript: YL Y-GJ. Proofreading: YL Y-GJ. Y-GJ. Contributed to the writing of the manuscript: YL Y-GJ. Proofreading: YL Y-GJ. 30. Wang Z, Wang N, Li W, Liu P, Chen Q, et al. (2014) Caveolin-1 mediates chemoresistance in breast cancer stem cells via b-catenin/ABCG2 signaling pathway. Carcinogenesis pii: bgu155. 29. Singh SK, Hawkins C, Clarke ID, Squire JA., Bayani J, et al. (2004) Identification of human brain tumour initiating cells. Nature 432: 396–401. mitogen-activated protein kinase (p38MAPK)/glycogen synthase kinase 3b (GSK-3b)/b-catenin signaling cascade. J Biol Chem 286: 44913–44920. 28. Kaprio T, Ferme´r C, Hagstro¨m J, Mustonen H, Bo¨ckelman C, et al. (2014) Podocalyxin is a marker of poor prognosis in colorectal cancer. BMC Cancer 14: 493. 27. Medina M, Wandosell F (2011) Deconstructing Its Activity. Int J Alzheimers Dis 2011: 479249. Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin ( b) b g g J 27. Medina M, Wandosell F (2011) Deconstructing GSK-3: The Fine Regul Its Activity. Int J Alzheimers Dis 2011: 479249. mitogen-activated protein kinase (p38MAPK)/glycogen synthase kinase 3b (GSK-3b)/b-catenin signaling cascade. J Biol Chem 286: 44913–44920. 27. Medina M, Wandosell F (2011) Deconstructing GSK-3: The Fine Regulation of Its Activity. Int J Alzheimers Dis 2011: 479249. 28. Kaprio T, Ferme´r C, Hagstro¨m J, Mustonen H, Bo¨ckelman C, et al. (2014) Podocalyxin is a marker of poor prognosis in colorectal cancer. BMC Cancer 14: 493. References Sizemore S, Cicek M, Sizemore N, Peng Ng K, Casey G, et al. (2007) Podocalyxin Increases the Aggressive Phenotype of Breast and Prostate Cancer Cells In vitro through Its Interaction with Ezrin. Cancer Res 67: 6183–6191. 21. Wang X, Wu H, Miller AH (2004) Interleukin 1alpha (IL-1alpha) induced activation of p38 mitogen-activated protein kinase inhibits glucocorticoid receptor function. Mol Psychiatry 9: 65–75. 9. Wu H, Yang L, Liao D, Chen Y, Wang W, et al. (2013) Podocalyxin regulates astrocytoma cell invasion and survival against temozolomide. Exp Ther Med 5: 1025–1029. 22. Ewan K, Pajak B, Stubbs M, Todd H, Barbeau O, et al. (2010) A useful approach to identify novel small-molecule inhibitors of Wnt-dependent transcription. Cancer Res 70: 5963–5973. 10. Chesire DR, Isaacs WB (2003) b-Catenin signaling in prostate cancer: an early perspective. Endocr Relat Cancer 10: 537–560. 23. Fu Y, O’Connor LM, Shepherd TG, Nachtigal MW (2003) The p38 MAPK inhibitor, PD169316, inhibits transforming growth factor beta-induced Smad signaling in human ovarian cancer cells. Biochem Biophys Res Commun 310: 391–397. 11. Cawthorn WP, Heyd F, Hegyi K, Sethi JK (2007) Tumour necrosis factor-alpha inhibits adipogenesis via a beta-catenin/TCF4 (TCF7L2)-dependent pathway. Cell Death Differ 14: 1361–1373. 12. Nusse R (1999) Repression and activation. Trends Genet 15: 1–3. 24. Li Y, Liao Q, Li K, Zhong D, Weng X, et al. (2012) Knockdown of endothelin A receptor expression inhibits osteosarcoma pulmonary metastasis in an orthotopic xenograft mouse model. Mol Med Rep 5: 1391–1395. 13. Gan XQ, Wang JY, Xi Y, Wu ZL, Li YP, et al. (2008) Nuclear Dvl, c-Jun, beta- catenin, and TCF form a complex leading to stabilization of beta-catenin-TCF interaction. J Cell Biol 180: 1087–1100. 25. Thornton TM, Pedraza-Alva G, Deng B, Wood CD, Aronshtam A, et al. (2008) Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation. Science 320: 667–670. 14. Sun P, Xiong H, Kim TH, Ren B, Zhang Z (2006) Positive inter-regulation between beta-catenin/T cell factor-4 signaling and endothelin-1 signaling potentiates proliferation and survival of prostate cancer cells. Mol Pharmacol 69: 520–531. 26. Zhang D, Guo M, Zhang W, Lu XY (2011) Adiponectin stimulates proliferation of adult hippocampal neural stem/progenitor cells through activation of p38 PLOS ONE \| www.plosone.org 12 October 2014 \| Volume 9 \| Issue 10 \| e111343 October 2014 \| Volume 9 \| Issue 10 \| e111343 Podocalyxin Promotes GBM Cell Invasion and Proliferation via b-Catenin Its Activity. References Int J Alzheimers Dis 2011: 479249. October 2014 \| Volume 9 \| Issue 10 \| e111343 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 13
https://openalex.org/W2009641748	https://www.beilstein-journals.org/bjoc/content/pdf/1860-5397-9-182.pdf	English	null	Bioinspired total synthesis of katsumadain A by organocatalytic enantioselective 1,4-conjugate addition	Beilstein journal of organic chemistry	2,013	cc-by	3,876	Abstract Katsumadain A, a naturally occurring inﬂuenza virus neuraminidase (NA) inhibitor, was synthesized by using a bioinspired, organocatalytic enantioselective 1,4-conjugate addition of styryl-2-pyranone with cinnamaldehyde, followed by a tandem Horner–Wadsworth–Emmons/oxa Michael addition. Bioinspired total synthesis of katsumadain A by organocatalytic enantioselective 1,4-conjugate addition Yongguang Wang, Ruiyang Bao, Shengdian Huang and Yefeng Tang* Letter Open Access Address: The Comprehensive AIDS Research Center, Department of Pharmacology & Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing, 100084, China Email: Yefeng Tang* - yefengtang@tsinghua.edu.cn * Corresponding author Keywords: bioinspired synthesis; catalysis; katsumadain A; natural product; organocatalytic 1,4-conjugate addition Beilstein J. Org. Chem. 2013, 9, 1601–1606. doi:10.3762/bjoc.9.182 Received: 30 May 2013 Accepted: 16 July 2013 Published: 06 August 2013 This article is part of the Thematic Series "Transition-metal and organocatalysis in natural product synthesis". Guest Editors: D. Y.-K. Chen and D. Ma © 2013 Wang et al; licensee Beilstein-Institut. License and terms: see end of document. Letter Open Access Address: The Comprehensive AIDS Research Center, Department of Pharmacology & Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing, 100084, China Email: Yefeng Tang* - yefengtang@tsinghua.edu.cn * Corresponding author Keywords: bioinspired synthesis; catalysis; katsumadain A; natural product; organocatalytic 1,4-conjugate addition Beilstein J. Org. Chem. 2013, 9, 1601–1606. doi:10.3762/bjoc.9.182 Received: 30 May 2013 Accepted: 16 July 2013 Published: 06 August 2013 This article is part of the Thematic Series "Transition-metal and organocatalysis in natural product synthesis". Guest Editors: D. Y.-K. Chen and D. Ma © 2013 Wang et al; licensee Beilstein-Institut. License and terms: see end of document. Introduction human inﬂuenza virus A/PR/8/34 of the subtype H1N1 (IC50 1.05–0.42 μM) by targeting the enzyme neuraminidase (NA) [4,5]. Moreover, it also inhibited the NA of four H1N1 swine inﬂuenza viruses with IC50 values between 0.59 and 1.64 μM. Therefore, katsumadain A represents an attractive lead struc- ture for the anti-flu drug discovery [6]. 2-Pyranone is a privilege structure that is often present in natural products and pharmaceuticals, many of which exhibit diverse molecular architectures and biological profiles [1,2]. For example, katsumadain A (1) and B (2), which were isolated from Alpinia katsumadai Hayata (Zingiberaceae), a chinese herbal drug used as an anti-emetic and stomachic agent, are two natural products bearing a diarylheptanoid scaffold that is incor- porated into the styryl-2-pyranone moiety [3]. Preliminary bio- logical evaluations showed that 1 and 2 feature anti-emetic activities on copper sulfate-induced emesis in young chicks. More recently, Rollinger et al. disclosed that katsumadain A (1) exhibited prominent in vitro inhibitory activity against the We recently reported the biomimetic total synthesis of katsumadain C [7], a natural product isolated from the same resource as katsumadain A and B [8]. As part of our continuous interest in the synthesis of bioactive 2-pyranone-derived natural products, we launched a project aiming to develop a highly effi- 1601 1601 Beilstein J. Org. Chem. 2013, 9, 1601–1606. designed as a fallback, in which katsumadain A could be accessed from the lactol 5a and phosphonate 6 via a tandem Horner–Wadsworth–Emmons (HWE)/oxa-Michael addition reaction [16]. In turn, 5a could be derived from 3 and cinnamaldehyde (7) by an organocatalytic enantioselective 1,4- conjugate addition followed by the hemiketal formation. cient route for the synthesis of katasumadain A as well as its analogues, which would pave the way for their application in further biomedical investigations. Biosynthetically, katsumadain A is assumed to be derived from styryl-2-pyranone 3 and alnustone (4) [9] through a 1,6-conju- gate addition/oxa-Michael addition cascade reaction (path a, Scheme 1). Indeed, both 3 and 4 are known natural substances. Apparently, the biosynthetic pathway represents the most straightforward and convergent approach to synthesize katsumadain A. However, its efficiency might be limited to some extent, given that α,β,γ,δ-unsaturated ketone 4 could undergo a competitive 1,4-conjugate addition to provide the other natural product katsumadain B (path b, Scheme 1). Introduction Actu- ally, the regioselectivity of a conjugate addition with α,β,γ,δ- unsaturated Michael acceptors remains a considerable chal- lenge, as it is heavily dependent on the steric and electronic nature of the substrates [10,11]. Moreover, the enantioselective 1,6-conjugate addition to acyclic dienones or dienoates mono- substituted at the β- and δ-position has rarely been investigated [12-15], thus leaving open the question of whether or not a biomimetic approach towards katsumadain A might succeed. Keeping these concerns in mind, an alternative strategy was Results and Discussion Our investigation was initiated by an investigation of the condi- tions that could effect the proposed biomimetic approach towards katsumadain A and katsumadain B. Both styryl-2-pyra- none 3 [17] and alnustone (4) [18] were synthesized according to the literature methods. First of all, the 1,6-conjugate addition of 3 towards 4 was attempted by employing various conditions, including different basic conditions (NaH, DBU, KHMDS) by activation of the nucleophile 3 or acidic conditions (AcOH, TMSOTf, Sc(OTf)3 and In(OTf)3) by activation of the elec- trophile 4. However, all of these reactions failed to provide satisfactory results and only lead to the recovery or the substan- tial decomposition of the starting material. We then turned our attention to the organocatalytic conjugated addition reaction. Among the various documented conditions [19-23], the 9-amino-9-deoxyepicinchona alkaloid-promoted Michael addi- Scheme 1: Proposed biosynthetic pathway and strategic analysis for synthesis of katsumadain A. Scheme 1: Proposed biosynthetic pathway and strategic analysis for synthesis of katsumadain A. Scheme 1: Proposed biosynthetic pathway and strategic analysis for synthesis of katsumadain A. 1602 Beilstein J. Org. Chem. 2013, 9, 1601–1606. Scheme 2: Preliminary results of the biomimetic synthesis of katsumadain A. tion is particularly attractive, mainly due to the availability of the catalyst and its superior reactivity towards the activation of the unsaturated ketone substrates through formation of the corresponding iminium intermediate [22]. To our delight, when we tried the standard conditions (30% catalyst A, 60% TFA, DCM, 96 h) in our case, we isolated a product in 25% isolated yield, which was proved to be the 1,4-adduct katsumadain B (2). Encouraged by this result, we further optimized the reac- tion by screening different solvents (CH3CN, THF, DMSO and MeOH) and additives (HCl, TFA and DMAP), aiming to improve the efficiency and selectivity (1,4- or 1,6-adduct) of the reaction. In most of the cases the 1,4-conjugate addition proceeded dominantly, while no or only trace amounts of the 1,6-adduct katsumadain A (1) was observed. The best result was obtained when the reaction was performed with a substoi- chiometric amount of catalyst A with MeOH as a solvent, in which katsumadain A and B were isolated in a 5:6 ratio with a combined yield of 33% (Scheme 2). Scheme 2: Preliminary results of the biomimetic synthesis of katsumadain A. selectivity issues, which we have struggled with in the afore- mentioned studies. Results and Discussion To validate this hypothesis, we performed a systematic investigation of the organocatalytic 1,4-conjugate addition by examining various reaction parameters, including organocatalyst, acid additive, solvent temperature, and reaction temperature (Table 1). The first reaction was performed by stir- ring a mixture of 3 and 7 in DCM at room temperature for 12 h With limited success regarding the the biomimetic synthesis of katsumadain A, we then moved towards the alternative ap- proach as described in Scheme 1. We envisioned that in this scenario an organocatalytic 1,4-conjugate addition [24-27] between 3 and 7 would circumvent both the reactivity and Table 1: Condition screening of organocatalytic 1,4-conjugate addition/hemiketalization of styryl-2-pyranone with α,β-unsaturated aldehydes. Entrya Catalyst Additive Solvent T (°C) Yield (%) ee (%)b 1 B none DCM 23 41 78 2 B BA DCM 23 78 91 3 C BA DCM 23 41 −81 4 D BA DCM 23 10 nd 5 B PNBA DCM 23 94 80 6 B BA MeOH 23 80 91 7 B BA CH3CN 23 78 75 8 B BA DMSO 23 79 93 9 B BA Toluene 23 66 91 10 B BA MeOH 0 78 92 11 B BA DCM 0 82 92 12 B BA DCM −20 45 93 aEach reaction was run with 3 (0.5 mmol) and 7 (0.6 mmol) in 2.0 mL solvent as shown above. bThe ee value was measured with the corresponding lactone product of 5a using chiral HPLC. Table 1: Condition screening of organocatalytic 1,4-conjugate addition/hemiketalization of styryl-2-pyranone with α,β-unsaturated aldehydes. Entrya Catalyst Additive Solvent T (°C) Yield (%) ee (%)b 1 B none DCM 23 41 78 2 B BA DCM 23 78 91 3 C BA DCM 23 41 −81 4 D BA DCM 23 10 nd 5 B PNBA DCM 23 94 80 6 B BA MeOH 23 80 91 7 B BA CH3CN 23 78 75 8 B BA DMSO 23 79 93 9 B BA Toluene 23 66 91 10 B BA MeOH 0 78 92 11 B BA DCM 0 82 92 12 B BA DCM −20 45 93 aEach reaction was run with 3 (0.5 mmol) and 7 (0.6 mmol) in 2.0 mL solvent as shown above. bThe ee value was measured with the corresponding lactone product of 5a using chiral HPLC. Table 1: Condition screening of organocatalytic 1,4-conjugate addition/hemiketalization of styryl-2-pyranone with α,β-unsaturated aldehydes. Results and Discussion It was found that the desired product 5a was obtained, albeit in moderate yield and enantioselectivity (Table 1, entry 1). To our delight, the usage of benzoic acid (BA) as an additive could dramatically improve the reaction by affording 5a in a good yield (78%) and a good ee value (91%, Table 1, entry 2). Besides the catalyst B, both Jørgensen catalyst C [29] and MacMillan catalyst D [30] were also tested in this reaction, but gave inferior results (Table 1, entries 3 and 4). As to the acid additive, p-nitrobenzoic acid (PNBA) was found to afford 5a in an excellent yield, but with a decreased ee value (80%). Furthermore, the solvent effect was also examined. Among the several solvents examined, both MeOH and DMSO proved to be suitable solvent systems (Table 1, entries 6 and 8) by furnishing comparable results with DCM, while MeCN and toluene led to modest results (Table 1, entry 7 and 9). Finally, we found that the reaction temperature has some influence on the outcomes, with a slightly improved enantioselectivity (92% ee) obtained at 0 °C (Table 1, entry 10 and 11). Although the best ee value (93%) was achieved at −20 °C, the reaction became sluggish and the yield dropped to 45% (Table 1, entry 12). It is noteworthy that 5a was isolated as a mixture of C-5 diastereoisomers (β-isomer/α-isomer = 5:1 to 7:1) in all of the above cases. For convenience, the ee value of 5a was determined with the corresponding lactone derivative. Furthermore, the absolute stereochemistry of 5a (β-isomer) was assigned as (7S,5R) by using the Mosher ester method (see Supporting Information File 1 for details). Having achieved the bicyclic core of katsumadain A in an efficient and enantioselective manner, we then moved towards its total synthesis through the proposed tandem Horner–Wadsworth–Emmons/oxa-Michael addition. As expected, deprotonation of 6 [31] with KHMDS at −40 °C for 0.5 h followed by the addition of the lactol 5a led to the forma- tion of katsumadain A as the only diastereoisomer in 52% yield, Table 2: Substrate scope of organocatalytic 1,4-conjugate addition/hemiketalization of styryl-2-pyranones with α,β-unsaturated aldehydes. Results and Discussion e 1: Condition screening of organocatalytic 1,4-conjugate addition/hemiketalization of styryl-2-pyranone with α,β-unsaturated aldehydes. aEach reaction was run with 3 (0.5 mmol) and 7 (0.6 mmol) in 2.0 mL solvent as shown above. bThe ee value was measured with the corresponding lactone product of 5a using chiral HPLC. 1603 Beilstein J. Org. Chem. 2013, 9, 1601–1606. To evaluate the substrate scope of the reaction, we then exam- ined different substituted styryl-2-pyranone and cinnamalde- hyde derivatives as Michael addtion donors and acceptors (Table 2). When styryl-2-pyranone 3a remained unchanged, a variety of cinnamaldehyde derivatives (7a–f) bearing either electron-withdrawing groups (4-Cl, 4-CF3 and 4-NO2, Table 2, entries 2-4) or electron-donating groups (4-MeO or 3,5-MeO, Table 2, entries 5 and 6) on the phenyl ring proved to be suit- able substrates, affording the corresponding products (5b–f) in good yields and enantioselectivities. Besides 3a, the Michael addition donors could also be extended to other substituted styryl-2-pyranone derivatives (e.g., 3b and 3c, Table 2, entries 7–11), all of which gave acceptable results. As proof-of-concept cases, the above outcomes indicate that the developed organocatalytic enantioselective 1,4-conjugate addition could be potentially applied to the synthesis of various bicyclic com- pounds bearing different aromatic moieties (Ar1 and Ar2), which paves the way to access katsumadain A and its analogues for further biomedical studies. To evaluate the substrate scope of the reaction, we then exam- ined different substituted styryl-2-pyranone and cinnamalde- hyde derivatives as Michael addtion donors and acceptors (Table 2). When styryl-2-pyranone 3a remained unchanged, a variety of cinnamaldehyde derivatives (7a–f) bearing either electron-withdrawing groups (4-Cl, 4-CF3 and 4-NO2, Table 2, entries 2-4) or electron-donating groups (4-MeO or 3,5-MeO, Table 2, entries 5 and 6) on the phenyl ring proved to be suit- able substrates, affording the corresponding products (5b–f) in good yields and enantioselectivities. Besides 3a, the Michael addition donors could also be extended to other substituted styryl-2-pyranone derivatives (e.g., 3b and 3c, Table 2, entries 7–11), all of which gave acceptable results. As proof-of-concept cases, the above outcomes indicate that the developed organocatalytic enantioselective 1,4-conjugate addition could be potentially applied to the synthesis of various bicyclic com- pounds bearing different aromatic moieties (Ar1 and Ar2), which paves the way to access katsumadain A and its analogues for further biomedical studies. in the presence of Hayashi catalyst B [28]. Supporting Information Supporting Information File 1 Experimental procedures and characterization data for synthetic 1, 3a–c, 5a–k and 9a–k. [http://www.beilstein-journals.org/bjoc/content/ supplementary/1860-5397-9-182-S1.pdf] Supporting Information File 1 Experimental procedures and characterization data for synthetic 1, 3a–c, 5a–k and 9a–k. [http://www.beilstein-journals.org/bjoc/content/ supplementary/1860-5397-9-182-S1.pdf] References 1. McGlacken, G. P.; Fairlamb, I. J. S. Nat. Prod. Rep. 2005, 22, 1. McGlacken, G. P.; Fairlamb, I. J. S. Nat. Prod. Rep. 2005, 22, 369–385. doi:10.1039/b416651p 369–385. doi:10.1039/b416651p 2. Goel, A.; Ram, V. J. Tetrahedron 2009, 65, 7865–7913. doi:10.1016/j.tet.2009.06.031 3. Yang, Y.; Kinoshita, K.; Koyama, K.; Takahashi, K.; Tai, T.; Nunoura, Y.; Watanabe, K. J. Nat. Prod. 1999, 62, 1672–1674. doi:10.1021/np990096e 4. Grienke, U.; Schmidtke, M.; Kirchmair, J.; Pfarr, K.; Wutzler, P.; Dürrwald, R.; Wolber, G.; Liedl, K. R.; Stuppner, H.; Rollinger, J. M. J. Med. Chem. 2010, 53, 778–786. doi:10.1021/jm901440f Scheme 3: Total synthesis of both enantiomers of katsumadain A. 5. Kirchmair, J.; Rollinger, J. M.; Liedl, K. R.; Seidel, N.; Krumbholz, A.; Schmidtke, M. Future Med. Chem. 2011, 3, 437–450. doi:10.4155/fmc.10.292 5. Kirchmair, J.; Rollinger, J. M.; Liedl, K. R.; Seidel, N.; Krumbholz, A.; Schmidtke, M. Future Med. Chem. 2011, 3, 437–450. doi:10.4155/fmc.10.292 Experimental apparently via the in situ generated intermediate 8. The spectro- scopic data of the synthetic katsumadain A were in accordance with those of the natural one [32]. However, we found that its optical rotation ([a]D25 = −75.4, c 0.40, EtOH) was quite different from the reported one ([a]D25 +3.7, c 0.40, EtOH), indicating that the naturally occurring 1 might exist as a racemic substance. Given that the two enantiomers of katsumadain A might show different behaviors in the biological studies from each other as well as from the racemic compounds, we also synthesized (+)-katsumadain A in a similar way by simply replacing the catatalyst B with its enantiomer in the organocat- alytic 1,4-conjugate addition (Scheme 3). Representative procedure for the organocatalytic 1,4-conju- gate addition: To a mixture of 3a (214 mg, 1.0 mmol) and 7a (163 mg, 1.2 mmol) in dry CH2Cl2 (5 mL) at 0 °C was added PhCOOH (24 mg, 0.2 mmol) and catatalyst B (50 mg, 0.2 equiv). The mixture was stirred at 0 °C for 10 h before being quenched by saturated aqueous NH4Cl. The mixture was extracted with DCM (3 × 10 mL), and the organic layers were washed with brine and dried over MgSO4. The organic solvent was removed under vacuum, and the residue was purified by column chromatography (CH2Cl2:ethyl acetate = 20:1) to give 5a (284 mg, 82% yield) as a light yellow solid. Supporting Information Supporting Information File 1 Experimental procedures and characterization data for synthetic 1, 3a–c, 5a–k and 9a–k. [http://www.beilstein-journals.org/bjoc/content/ supplementary/1860-5397-9-182-S1.pdf] Scheme 3: Total synthesis of both enantiomers of katsumadain A. Acknowledgements This work was financially supported by the National Science Foundation of China (21102081, 21272133), the Beijing Natural Science Foundation (2132037), the New Teachers' Fund for Doctor Stations (20110002120011), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, and the Ministry of Education. Results and Discussion Entrya Substrate (3 and 7) Yield of 5 (%)b ee value of 5 (%)c 1 3a: Ar1 = Ph; 7a: Ar2 = Ph 82 (5a) 92 2 3a: Ar1 = Ph; 7b: Ar2 = 4-Cl-Ph 59 (5b) 83 3 3a: Ar1 = Ph; 7c: Ar2 = 4-CF3-Ph 77 (5c) 82 4 3a: Ar1 = Ph; 7d: Ar2 = 4-NO2-Ph 79 (5d) 88 5 3a: Ar1 = Ph; 7e: Ar2 = 4-MeO-Ph 78 (5e) 87 6 3a: Ar1 = Ph; 7f: Ar2 = 3,5-MeO-Ph 70 (5f) 92 7 3b: Ar1 = 4-MeO-Ph; 7a: Ar2 = Ph 84 (5g) 82 8 3c: Ar1 = Furan; 7b: Ar2 = 4-Cl-Ph 88 (5h) 84 9 3c: Ar1 = Furan; 7d: Ar2 = 4-NO2-Ph 76 (5i) 91 10 3c: Ar1 = Furan; 7e: Ar2 = 4-MeO-Ph 75 (5j) 80 11 3c: Ar1 = Furan; 7g: Ar2 = Naphthyl 79 (5k) 90 aEach reaction was run with 3 (0.5 mmol) and 7 (0.6 mmol) in 2.0 mL solvent as shown above. bEach of 5a–k was obtained as a mixture of C-5 dia- stereoisomers (the ratio of α-isomer:β-isomer varied from 1:5 to 1:7. cThe ee value of 5a–k was measured with the corresponding lactone product using chiral HPLC. aEach reaction was run with 3 (0.5 mmol) and 7 (0.6 mmol) in 2.0 mL solvent as shown above. bEach of 5a–k was obtained as a mixture of C-5 dia- stereoisomers (the ratio of α-isomer:β-isomer varied from 1:5 to 1:7. cThe ee value of 5a–k was measured with the corresponding lactone product using chiral HPLC. 1604 Beilstein J. Org. Chem. 2013, 9, 1601–1606. Conclusion We accomplished the first enantioselective total synthesis of katsumadain A, a naturally occurring inﬂuenza virus neuraminidase (NA) inhibitor. The key elements of the syn- thesis featured a bioinspired, organocatalytic enantioselective 1,4-conjuate addition and a tandem HWE/oxa-Michael addition. Due to the high efficiency and flexibility of the synthetic route it is applicable to the syntheses of both enantiomers of katsumadain A as well as their analogues. Applications of these compounds in relevant biomedical studies are ongoing in this laboratory, and the progress will be reported in due course. 6. Grienke, U.; Schmidtke, M.; von Grafenstein, S.; Kirchmair, J.; Liedl, K. R.; Rollinger, J. M. Nat. Prod. Rep. 2012, 29, 11–36. doi:10.1039/c1np00053e 6. Grienke, U.; Schmidtke, M.; von Grafenstein, S.; Kirchmair, J.; Liedl, K. R.; Rollinger, J. M. Nat. Prod. Rep. 2012, 29, 11–36. doi:10.1039/c1np00053e 7. Zhang, P.; Wang, Y.; Bao, R.; Luo, T.; Yang, Z.; Tang 2012, 14, 162–165. doi:10.1021/ol2029433 7. Zhang, P.; Wang, Y.; Bao, R.; Luo, T.; Yang, Z.; Tang, Y. Org. L 2012, 14, 162–165. doi:10.1021/ol2029433 8. Yang, C.-S.; Wang, X.-B.; Wang, J.-S.; Luo, J.-G.; Luo, L.; Ko Org. Lett. 2011, 13, 3380–3383. doi:10.1021/ol201137v 9. Kuroyanagi, M.; Noro, T.; Fukushima, S.; Aiyama, R.; Ikuta, A.; Itokawa, H.; Morita, M. Chem. Pharm. Bull. 1983, 31, 1544–1550. doi:10.1248/cpb.31.1544 1605 Beilstein J. Org. Chem. 2013, 9, 1601–1606. 10.Csákÿ, A. G.; de la Herrán, G.; Murcia, M. C. Chem. Soc. Rev. 2010, 39, 4080–4102. doi:10.1039/b924486g License and Terms This is an Open Access article under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The license is subject to the Beilstein Journal of Organic Chemistry terms and conditions: (http://www.beilstein-journals.org/bjoc) The definitive version of this article is the electronic one which can be found at: doi:10.3762/bjoc.9.182 License and Terms 11.Silva, E. M. P.; Silva, A. M. S. Synthesis 2012, 44, 3109–3128. d i 10 1055/ 0032 1316778 11.Silva, E. M. P.; Silva, A. M. S. Synthesis 2012, 44, 3109–3128. doi:10.1055/s-0032-1316778 12.den Hartog, T.; Harutyunyan, S. R.; Font, D.; Minnaard, A. J.; Feringa, B. L. Angew. Chem., Int. Ed. 2008, 47, 398–401. doi:10.1002/anie.200703702 12.den Hartog, T.; Harutyunyan, S. R.; Font, D.; Minnaard, A. J.; Feringa, B. L. Angew. Chem., Int. Ed. 2008, 47, 398–401. 13.Bernardi, L.; López-Cantarero, J.; Niess, B.; Jørgensen, K. A. J. Am. Chem. Soc. 2007, 129, 5772–5778. doi:10.1021/ja0707097 13.Bernardi, L.; López-Cantarero, J.; Niess, B.; Jørgensen, K. A. J. Am. Chem. Soc. 2007, 129, 5772–5778. doi:10.1021/ja0707097 14.Murphy, J. J.; Quintard, A.; McArdle, P.; Alexakis, A.; Stephens, J. C. Angew. Chem., Int. Ed. 2011, 50, 5095–5098. doi:10.1002/anie.201100804 15.Tian, X.; Liu, Y.; Melchiorre, P. Angew. Chem., Int. Ed. 2012, 51, 6439–6442. doi:10.1002/anie.201202392 16.McLeod, M. C.; Wilson, Z. E.; Brimble, M. A. Org. Lett. 2011, 13, 5382–5385. doi:10.1021/ol202265g 17.Katritzky, A. R.; Wang, Z.; Wang, M.; Hall, C. D.; Suzuki, K. J. Org. Chem. 2005, 70, 4854–4856. doi:10.1021/jo050307m 18.Baranovsky, A.; Schmitt, B.; Fowler, D. J.; Schneider, B. Synth. Commun. 2003, 33, 1019–1045. doi:10.1081/SCC-120016367 19.Wang, J.-J.; Hu, Z.-P.; Lou, C.-L.; Liu, J.-L.; Li, X.-M.; Yan, M. 19.Wang, J.-J.; Hu, Z.-P.; Lou, C.-L.; Liu, J.-L.; Li, X.-M.; Yan, M. Tetrahedron 2011, 67, 4578–4583. doi:10.1016/j.tet.2011.04.086 Wang, J.-J.; Hu, Z.-P.; Lou, C.-L.; Liu, J.-L.; Li, X.-M.; Yan, M. Tetrahedron 2011, 67, 4578–4583. doi:10.1016/j.tet.2011.04.086 20.Zhu, X.; Lin, A.; Shi, Y.; Guo, J.; Zhu, C.; Cheng, Y. Org. Lett. 2011, 13, 4382–4385. doi:10.1021/ol201715h 21.Li, X.-M.; Wang, B.; Zhang, J.-M.; Yan, M. Org. Lett. 2011, 13, 21.Li, X.-M.; Wang, B.; Zhang, J.-M.; Yan, M. Org. Lett. 2011, 13, 374–377. doi:10.1021/ol102570b 22.Xie, J.-W.; Yue, L.; Chen, W.; Du, W.; Zhu, J.; Deng, J.-G.; Chen, Y.-C. Org. Lett. 2007, 9, 413–415. doi:10.1021/ol062718a 23.Liu, Y.; Liu, X.; Wang, M.; He, P.; Lin, L.; Feng, X. J. Org. Chem. 2012, 77, 4136–4142. doi:10.1021/jo3001047 77, 4136–4142. doi:10.1021/jo3001047 24.Erkkilä, A.; Majander, I.; Pihko, P. M. Chem. Rev. 2007, 107, 24.Erkkilä, A.; Majander, I.; Pihko, P. M. Chem. Rev. 2007, 107, 5416–5470. doi:10.1021/cr068388p 5416–5470. doi:10.1021/cr068388p 25.Almasi, D.; Alonso, D. A.; Nájera, C. Tetrahedron: Asymmetry 200 18, 299–365. doi:10.1016/j.tetasy.2007.01.023 25.Almasi, D.; Alonso, D. A.; Nájera, C. Tetrahedron: Asymmetry 200 18, 299–365. doi:10.1016/j.tetasy.2007.01.023 26.Tsogoeva, S. B. Eur. J. Org. Chem. 2007, 1701–1716. doi:10.1002/ejoc.200600653 26.Tsogoeva, S. B. Eur. J. Org. Chem. 2007, 1701–1716. doi:10.1002/ejoc.200600653 27.Rueping, M.; Merino, E.; Sugiono, E. Adv. Synth. Catal. 2008, 3 27.Rueping, M.; Merino, E.; Sugiono, E. Adv. S 2127–2131. doi:10.1002/adsc.200800340 2127–2131. doi:10.1002/adsc.200800340 28.Hayashi, Y.; Gotoh, H.; Hayashi, T.; Shoji, M. Angew. Chem., Int. Ed. 28.Hayashi, Y.; Gotoh, H.; Hayashi, T.; Shoji, M. Angew 2005, 44, 4212–4215. doi:10.1002/anie.200500599 29.Marigo, M.; Wabnitz, T. C.; Fielenbach, D.; Jørgensen, K. A. 29.Marigo, M.; Wabnitz, T. C.; Fielenbach, D.; Jørgensen, K. A. Angew. Chem., Int. Ed. 2005, 44, 794–797. doi:10.1002/anie.200462101 Angew. Chem., Int. Ed. 2005, 44, 794–797. 30.Austin, J. F.; MacMillan, D. W. C. J. Am. Chem. Soc. 2002, 124, 1172–1173. doi:10.1021/ja017255c 30.Austin, J. F.; MacMillan, D. W. C. J. Am. Chem. Soc. 2002, 124 1172–1173. doi:10.1021/ja017255c 31.Maloney, K. M.; Chung, J. Y. L. J. Org. Chem. 2009, 74, 7574–7576. doi:10.1021/jo901552k 32.The ee value of the synthetic (+)-1and (−)-1 was determined to be 80% and 81%, respectively, suggesting that partial racemization occurred in the HWE reaction, presumably via the intermediate 8. 32.The ee value of the synthetic (+)-1and (−)-1 was determined to be 80% and 81%, respectively, suggesting that partial racemization occurred in the HWE reaction, presumably via the intermediate 8. 1606
https://openalex.org/W3179897198	https://elib.dlr.de/143093/1/09477115.pdf	English	null	A Quantum Annealer for Subset Feature Selection and the Classification of Hyperspectral Images	IEEE journal of selected topics in applied earth observations and remote sensing	2,021	cc-by	10,663	A Quantum Annealer for Subset Feature Selection and the Classiﬁcation of Hyperspectral Images Soronzonbold Otgonbaatar, Mihai Datcu, Fellow, IEEE in which only certain edges are connected. In particular, the interaction among the particles is constrained [1], [2]. in which only certain edges are connected. In particular, the interaction among the particles is constrained [1], [2]. Abstract—Hyperspectral images showing objects belonging to several distinct target classes are characterized by dozens of spectral bands being available. However, some of these spectral bands are redundant and/or noisy, and hence, selecting highly- informative and trustworthy bands for each class is a vital step for classiﬁcation and for saving internal storage space; then the selected bands are termed a highly-informative spectral band subset. We use a Mutual Information (MI)-based method to select the spectral band subset of a given class and two additional binary quantum classiﬁers, namely a quantum boost (Qboost) and a quantum boost plus (Qboost-Plus) classiﬁer, to classify a two-label dataset characterized by the selected band subset. We pose both our MI-based band subset selection problem and the binary quantum classiﬁers as a quadratic unconstrained binary optimization (QUBO) problem. Such a quadratic problem is solvable with the help of conventional optimization techniques. However, the QUBO problem is an NP-hard global optimization problem, and hence, it is worthwhile for applying a quantum annealer. Thus, we adapted our MI-based optimization problem for selecting highly-informative bands for each class of a given hyperspectral image to be run on a D-Wave quantum annealer. After the selection of these highly-informative bands for each class, we employ our binary quantum classiﬁers to a two-label dataset on the D-Wave quantum annealer. In addition, we provide a novel multi-label classiﬁer exploiting an Error-Encoding Output Code (ECOC) when using our binary quantum classiﬁers. As a real-world dataset in Earth observation, we used the well- known AVIRIS hyperspectral image (HSI) of Indian Pine, north- western Indiana, USA. We can demonstrate that the MI-based band subset selection problem can be run on a D-Wave quantum annealer that selects the highly-informative spectral band subset for each target class in the Indian Pine HSI. We can also prove that our binary quantum classiﬁers and our novel multi- label classiﬁer generate a correct two- and multi-label dataset characterized by their selected bands and with high accuracy such as having been produced by conventional classiﬁers - and even better in some instances. A Quantum Annealer for Subset Feature Selection and the Classiﬁcation of Hyperspectral Images A D-Wave quantum annealer works as a metaheuristic pro- cess which is dedicated to tackle speciﬁc classes of optimiza- tion problems, e.g., QUBO problems. There are theoretical studies that a D-Wave quantum annealer can solve these QUBO problems faster than a conventional annealer (even for NP problems) [3], [4]. However, currently, there are no indications of computational advantages for real-world prob- lems. For practical applications, several studies are devoted to benchmark and assess a D-Wave quantum annealer for an operational planning and feature extraction from remotely- sensed images [5], [6]. For a real-world dataset in Earth observation, remotely sensed images differ in their image content representations due to the diverse satellite platforms with their different types of sensors. When we want to use a D-Wave quantum annealer with an Earth observation dataset, some of the challenges are the proper choice of appropriate remotely sensed images speciﬁed by their image content representations such as their spatial information, polarization states, spectral bands, and the embedding of a given dataset in the topology of a D- Wave quantum annealer. Here, we consider hyperspectral images (HSIs), and a selection of their highly-informative band subset is a very vital procedure in Earth observation. Hence, we use a Mutual Information (MI)-based optimization method to select the highly-informative band subset, and more importantly, we can easily embed and optimize the MI-based optimization method in the Pegasus topology of a D-Wave quantum annealer. Therefore, HSIs are one of the most proper datasets in Earth observation for a D-Wave quantum annealer than others. In particular, HSIs became an important ﬁeld of study to classify or identify objects in a ground scene such as roads, land cover, or agriculture since each object is characterized by a high-dimensional vector of the different spectral bands within the given full wavelength range. Due to the rich information content of the spectral bands, some of these bands carry more discriminatory information than others. Hence, some studies are focused on extracting highly- informative features or a dimensionality reduction of HSIs, for instance, by using deep learning networks or Principal Component Analysis (PCA) [7], [8]. On the other hand, some researchers focused on how to select a highly-informative band subset by using the concept of information theory; in particular, MI-based methods which provide a measure of independence between several spectral bands. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing en accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing 1 SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” A Quantum Annealer for Subset Feature Selection and the Classiﬁcation of Hyperspectral Images Moreover, these MI-based methods are based on prior or reference knowledge of the spectral signatures of objects; such knowledge can be Index Terms—Hyperspectral images, Mutual Information, Fea- ture selection, Quantum classiﬁer, Quantum Machine Learning, D-wave quantum annealer. Soronzonbold Otgonbaatar is with German Aerospace Center (DLR), Oberpfaffenhofen and the University of Siegen, Germany. e-mail: soronzon- bold.otgonbaatar@dlr.de. Mihai Datcu is with German Aerospace Center (DLR), Oberpfaffenhofen. e-mail: mihai.datcu@dlr.de. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ I. INTRODUCTION A A QUANTUM Annealer (QA) is a computing machine conﬁgured as a graph network G = (E, V ), at each vertex of which particles are residing, and its edges deﬁne the interaction strengths among these particles which are in quan- tum states ups or downs. For a D-Wave quantum annealer, the graph G has a speciﬁc network topology named Pegasus, This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citatio of Selected Topics in Applied Earth Observations and Remote Sensing SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” 2 obtained in speciﬁc spectral-signature databases of common ground targets [9], [10]. Fig. 1. An Indian Pine Hyperspectral Image: Ground truth. In this paper, we use an Indian Pine HSI of Indian Pine com- posed of l = 16 distinct classes. Each class is characterized by n = 200 bands (features); thus, the number of subsets of the features of a given class, e.g., yS = 1, is 2n combinations. A way to ﬁnd the best feature subset of this given class is to try all combinations. This is clearly computationally expensive for a large number of these features. Hence, in the ﬁrst part of this study, we introduce an MI- based subset feature selection problem as a global optimization problem for the Indian Pine HSI. Then we propose to optimize this MI-based band subset selection problem on a D-Wave quantum annealer. As a ﬁrst step, we map the MI-based band subset selection problem to a QUBO-based band subset selection problem. This is our ﬁrst problem mapping step. Secondly, we optimize this mapping problem on a D-Wave quantum annealer; quantum optimization [11], [12]; this part was strongly motivated by a feature selection tutorial offered by D-Wave [2]. Fig. 1. An Indian Pine Hyperspectral Image: Ground truth. We introduce the basics of a D-Wave quantum annealer and optimize the QUBO-based band subset selection problem for the Indian Pine HSI on a D-Wave quantum annealer (see Section V, quantum optimization). II. INTRODUCTION TO HYPERSCPECTRAL IMAGING A hyperspectral imaging sensor mounted on a satellite or aircraft measures the electromagnetic spectrum ranging from the visible to the near infrared wavelengths; for instance, the Imaging Spectroscopy and the Airborne Visible/Infrared Imag- ing Spectrometer (AVIRIS) sensor measures 224 continuous spectral bands ranging 400 nm to 2500 nm at 10 nm intervals [17]. Our contribution in this paper is then an attempt to bench- mark and assess a D-Wave quantum annealer for Earth obser- vation data and to recognize the challenges that are encoun- tered with real-world datasets and future quantum annealers or devices. Towards these goals, we are employing a D-Wave quantum annealer for feature selection and classiﬁcation of the Indian Pine HSI as a machine learning technique; our contribution consists of a three-step approach, As a real-world dataset of HSIs, we consider an Indian Pine HSI obtained by the AVIRIS sensor (see Fig. 1). This low-noise Indian Pine image having the spectral bands of X = {band1, . . . , band200} elements is a high-dimensional dataset. However, not all of these spectral bands are informa- tive for characterizing a speciﬁc class; in other words, some bands of X are redundant or noisy. It is advantageous to select a highly-informative band subset of the given spectral bands for a given class. Hence, we employ an MI-based band subset selection problem as a global optimization problem. 1) Feature selection on a D-Wave quantum annealer: the MI-based band subset selection, 2) Binary classiﬁcation on a D-Wave quantum annealer: the binary quantum classiﬁers to a two-label dataset characterized by those selected bands. I. INTRODUCTION Finally, we apply the binary quantum classiﬁers and the novel multi-label classiﬁer to the two- and multi-label dataset in Section VI, and in Section VII, respectively. We then draw a conclusion in Section VIII. In the second part of this study, we use binary quantum classiﬁers, namely a quantum boost (Qboost) and a quantum boost plus (Qboost-Plus) classiﬁer, in contrast to an adaptive boost (Adaboost) classiﬁer [13], [14]. We ﬁrst apply these quantum classiﬁers to a two-label dataset of the Indian Pine HSI, and secondly, we provide a novel multi-label classiﬁer via an ECOC when using our binary quantum classiﬁers [15], [16]; each resulting class is discriminated by the selected bands in the ﬁrst part of our study. We also benchmarked and assessed these binary quantum classiﬁers and the novel multi-label classiﬁer with respect to conventional classiﬁers, a Decision Tree Classiﬁer (DTC), a Support Vector Machine (SVM), and an Adaboost classiﬁer. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ III. INFORMATION THEORY AND MUTUAL INFORMATION-BASED BAND SUBSET SELECTION Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journa of Selected Topics in Applied Earth Observations and Remote Sensing CIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” 3 SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” 3 Class labels Y Selected Bands Xi Alfalfa band41 band47 band77 Corn-notill band13 band15 band17 Corn-mintill band3 band133 band190 Corn band49 band128 band175 Grass-Pasture band102 band143 band84 Grass-Trees band23 band40 band53 Grass-Pasture-mowed band61 band102 band109 Hay-windrowed band149 band150 band40 Oats band76 band85 band172 Soybean-notill band10 band145 band183 Soybean-mintill band12 band145 band180 Soybean-clean band4 band12 band136 Wheat band37 band82 band177 Wood band63 band102 band190 Building-Grass-Drives band18 band70 band109 Stone-Steel-Towers band79 band84 band104 TABLE I SELECTION OF THE BEST BAND SUBSET FOR EACH CLASS OF THE INDIAN PINE HSI BY USING THE QUBO-BASED BAND SUBSET SELECTION ON A D-WAVE QUANTUM ANNEALER. Class labels Y Selected Bands Xi Alfalfa band41 band47 band77 Corn-notill band13 band15 band17 Corn-mintill band3 band133 band190 Corn band49 band128 band175 Grass-Pasture band102 band143 band84 Grass-Trees band23 band40 band53 Grass-Pasture-mowed band61 band102 band109 Hay-windrowed band149 band150 band40 Oats band76 band85 band172 Soybean-notill band10 band145 band183 Soybean-mintill band12 band145 band180 Soybean-clean band4 band12 band136 Wheat band37 band82 band177 Wood band63 band102 band190 Building-Grass-Drives band18 band70 band109 Stone-Steel-Towers band79 band84 band104 P(Xn′) = Xn′ P10 n′=1 Xn′ , P(ym′) = ym′ P10 m′=1 ym′ , (1) (1) where Xn′ and ym′ represent the number of occurrences of band Xi and its class yS in the n′-th or m′-th bin, respectively. Their joint probability is deﬁned in the same way. For the selection of the band subset, we exploit Mutual Information (MI) which measures independence between band Xi and its class yS. It is deﬁned by I(Xi; yS) = X m′ X n′ P(Xn′, ym′) log P(Xn′, ym′) P(Xn′)P(ym′), (2) (2) and by Conditional Mutual Information (CMI) which is a measure of the dependence between band Xi and its class yS given another band Xj. The CMI can then be written as TABLE I SELECTION OF THE BEST BAND SUBSET FOR EACH CLASS OF THE INDIAN PINE HSI BY USING THE QUBO-BASED BAND SUBSET SELECTION ON A D-WAVE QUANTUM ANNEALER. I(Xi; yS\|Xj) = E(Xi\|Xj) −E(Xi\|yS, Xj), (3) (3) where E is the entropy that is a measure of the uncertainty of a random variable [18]. here ˜x1 = 1, ˜x2 = 1, and ˜x3 = · · · = ˜xn = 0. III. INFORMATION THEORY AND MUTUAL INFORMATION-BASED BAND SUBSET SELECTION On the other hand, we can interpret this matrix form that the ˜xn’s are for selecting a highly-informative band subset. Hence, we can express the MI-maximization problem expressed by Eq. (4) alternatively as These band subset selection techniques expressed by both Eq. (2) and Eq. (3) are named after a MI-based band subset selection problem which became popular in machine learning due to its strong mathematical foundation rooted in informa- tion theory. In the next sections, we pose the MI-based band subset selection problem as a global optimization problem. First, we map our MI-based band subset selection problem to a QUBO problem, and the QUBO problem to a QUBO-based band subset selection problem. Finally, we optimize the QUBO- based band subset selection problem on a D-Wave quantum annealer. max ⃗x [⃗xT Q⃗x], ⃗x = (˜x1, ˜x2, . . . , ˜xn)T , ⃗x ∈{0, +1}n, (6) (6) where T represents a transpose operation, and Q is repre- sented diagonal Qii = I(Xi; yS) and off-diagonal Qij = I(Xi; yS\|Xj) elements. We can even transform this maximiza- tion problem to a minimization problem by multiplying it by ”−1”. As a result, we have where T represents a transpose operation, and Q is repre- sented diagonal Qii = I(Xi; yS) and off-diagonal Qij = I(Xi; yS\|Xj) elements. We can even transform this maximiza- tion problem to a minimization problem by multiplying it by ”−1”. As a result, we have IV. PROBLEM MAPPING: THE QUBO-BASED BAND SUBSET SELECTION min ⃗x [⃗xT Q⃗x], ⃗x ∈{0, +1}n, (7) (7) A. Mapping of a Mutual Information-based problem to a QUBO problem where Qii = −I(Xi; yS) and Qij = −I(Xi; yS\|Xj) [11]. This form of the minimization problem over binary variables ⃗x is called a Quadratic Unconstrained Binary Optimization (QUBO) problem. In this part, we consider and pose the MI-based band subset selection problem as a global optimization problem [11], [12]. Moreover, the maximization over the subsets {Xi} can be written as The MI-based band subset selection problem is therefore equivalent to a QUBO problem when we write ”−I(Xi; yS)” and ”−I(Xi; yS\|Xj)” in the Q matrix, and minimize the Q matrix over the binary variables. max {Xi}  X Xi I(Xi; yS) + X Xi,Xj I(Xi; yS\|Xj)  , (4) (4) III. INFORMATION THEORY AND MUTUAL INFORMATION-BASED BAND SUBSET SELECTION 3) Multi-label classiﬁcation on a D-Wave quantum an- nealer: the ECOC generates a multi-label dataset when we are using our binary quantum classiﬁers. We select a highly-informative band subset for each class of the Indian Pine HSI; for instance, we consider the spectral bands X = {X1, . . . , X200} = {band1, . . . , band200} of a given class yS and ﬁnd its most informative band subset. To ﬁnd the highly-informative band subset for that speciﬁc class, we employ information theory; information is a function of probabilities. Hence, we represent band Xi and its correspond- ing class yS as probabilities. We derived the probabilities for the band Xi and its class yS by dividing them into ten bins in a histogram. The probability is then deﬁned as Moreover, the D-Wave quantum annealer may prove rele- vant even if we are not intending to demonstrate its advantage over a conventional annealer. The paper is structured as follows: We introduce the basics of hyperspectral imaging in Section II. We present the basics of information theory and MI-based band subset selection problem in Section III. In Section IV, we discuss the fun- damentals of a QUBO problem and demonstrate the problem mapping of an MI-based problem to a QUBO-based problem. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ TABLE II CLASSIFICATION ACCURACY OF THE DTC AND THE SVM. } 12: wS = wS/sum(wS). 13: end for 14: h = [h1, . . . , hN], hn ∈RS; {deﬁning an array to store the weak classiﬁer predictions.} 15: for i ←1, . . . , N do 16: h[i] = c[i](x); {storing the predicted classes.} 17: end for 18: h = h/N; {scaling h to the range of [−1/N, 1/N].} 19: return h. min ⃗x [⃗xT Q⃗x + γ n X i=1 (˜xi −k)2], ⃗x ∈{0, +1}n; (9) (9) where γ is a Lagrange multiplier. As an experiment for selecting the most informative band subset for the speciﬁc class of an Indian Pine HSI, we consider the band subsets with three elements (k = 3). 18: h = h/N; {scaling h to the range of [−1/N, 1/N].} 19: return h. variables are allowed to interact with others through the edges [19]. V. QUANTUM OPTIMIZATION: USING A D-WAVE QUANTUM ANNEALER In addition, the performance of a D-Wave quantum annealer strongly depends on mapping the binary variables of our QUBO problem expressed by Eq. (9) to the Pegasus topology. As it is possible to map (embed) our QUBO problem to the Pegasus topology as efﬁciently as possible, we employed a technique called minor-embedding which is offered by the company D-Wave systems [2], [19]. A. D-Wave quantum annealer We selected a highly-informative band subset characterizing the speciﬁc class of the Indian Pine HSI by optimizing the QUBO-based band subset selection problem in the form of Eq. (9). We optimized this optimization problem on a D- Wave quantum annealer, and we even benchmark the D-Wave quantum annealer with respect to its conventional version. A D-Wave Quantum Annealer is a quantum annealer for the special class of optimization problems, in particular, QUBO- like problems. Such a quantum annealer is a metaheuristic process evolving slowly enough from its initial energy Hi to its ﬁnal energy Hf in the form of a QUBO problem. The evolution process is expressed by This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ B. Mapping the QUBO problem to the QUBO-based subset band selection problem where Xi represents the bands of a given class yS of the Indian Pine HSI (see Fig. 1). To select a highly-informative band subset characterizing each class of the Indian Pine image (Fig. 1), we employ the QUBO problem described by Eq. (7) with an additional constraint Let us consider the band data X = {band1, . . . , band200} of a given class of Alfalfa or simply yS = 1 as an example case. We assume that Eq. (4) is maximized when we use the subset XS = {X1, X2} = {band1, band2}. We can express this result in a matrix form such that min ⃗x [⃗xT Q⃗x], s.t. n X i=1 ˜xi = k, ˜xi ∈{0, +1}, (8) (8) I(X1; yS) + I(X1; yS\|X2) + I(X2; yS) + I(X2; yS\|X1) ⇔ ⇔ ˜x1 ˜x2 I(X1; yS) I(X1; yS\|X2) I(X2; yS\|X1) I(X2; yS) ˜x1 ˜x2 , (5) where k is the number of bands (band subset) of interest, and n = 200 is the total number of given bands. Hence, we deﬁne the QUBO-based band subset selection problem as (5) This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing Algorithm 1 Fitting weak classiﬁers Fig. 2. Left: Ground truth, Right: Classiﬁcation of the l = 16 classes characterized by three highly-informative spectral bands shown in Table. I by using a Support Vector Machine (SVM). Algorithm 1 Fitting weak classiﬁers Algorithm 1 Fitting weak classiﬁers Algorithm 1 Fitting weak classiﬁers 1: INPUT: Training bands: (x, y) = (x1, y1), . . . , (xS, yS); {xS represents the three selected bands for a given class yS (Table I).} 2: y ∈{−1, +1}S; {S is the size of the input dataset, and y represents the two-label of the Indian Pine HSI (Table III).} 3: Initialize the weak classiﬁers: c = [c1, . . . B. Mapping the QUBO problem to the QUBO-based subset band selection problem ( ) {b i h i h f QUBO-based band selection PCA Classiﬁer DTC SVM DTC SVM Accuracy 0.74 0.81 0.72 0.81 TABLE II CLASSIFICATION ACCURACY OF THE DTC AND THE SVM. QUBO-based band selection PCA Classiﬁer DTC SVM DTC SVM Accuracy 0.74 0.81 0.72 0.81 yp [ ]( ), yp { , } 9: errm = wS · I(yp! = y)/sum(wS). 10: am = 0.5 · log 1 −errm errm . ( ) {b i h i h f m 11: wS = wS · exp(−am · yp · y); {boosting the weight of misclassiﬁed data.} B. Mapping the QUBO problem to the QUBO-based subset band selection problem , cN]; {DTCs.} 4: N; {the number of DTCs.} 5: wS = (1, . . . , 1)/S; {Assigning the same weight to each data element xS.} 6: for i ←1, . . . , N do 7: Fit a DTC, c[i], to the (x, y) with a weight wS. 8: yp = c[i](x), yp ∈{−1, +1}S. 9: errm = wS · I(yp! = y)/sum(wS). 10: am = 0.5 · log 1 −errm errm . 11: wS = wS · exp(−am · yp · y); {boosting the weight of misclassiﬁed data.} 12: wS = wS/sum(wS). 13: end for 14: h = [h1, . . . , hN], hn ∈RS; {deﬁning an array to store the weak classiﬁer predictions.} 15: for i ←1, . . . , N do 16: h[i] = c[i](x); {storing the predicted classes.} 17: end for 18: h = h/N; {scaling h to the range of [−1/N, 1/N].} 19: return h. 20: STOP ALGORITHM. 1: INPUT: Training bands: (x, y) = (x1, y1), . . . , (xS, yS); {xS represents the three selected bands for a given class yS (Table I).} S g } 2: y ∈{−1, +1}S; {S is the size of the input dataset, and y represents the two-label of the Indian Pine HSI (Table III).} } 2: y ∈{−1, +1}S; {S is the size of the input dataset, and y represents the two-label of the Indian Pine HSI (Table III).} ) } 3: Initialize the weak classiﬁers: c = [c1, . . . , cN]; {DTCs.} [ 1, , N]; { } 4: N; {the number of DTCs.} 5: wS = (1, . . . , 1)/S; {Assigning the same weight to each data element xS.} Fig. 2. Left: Ground truth, Right: Classiﬁcation of the l = 16 classes characterized by three highly-informative spectral bands shown in Table. I by using a Support Vector Machine (SVM). } 6: for i ←1, . . . , N do 7: Fit a DTC, c[i], to the (x, y) with a weight wS. S 7: Fit a DTC, c[i], to the (x, y) with a weight wS. S 8: yp = c[i](x), yp ∈{−1, +1}S. 9: errm = wS · I(yp! = y)/sum(wS). 10: am = 0.5 · log 1 −errm errm . B. Quantum optimization for the band subset selection Quantum optimization is an optimization of our QUBO- based band subset selection problem on a D-Wave quantum annealer. We performed our experiment in a classical annealer and a D-Wave quantum annealer. Both of these annealers selected the same band subset for each class of the Indian Pine HSI; we shown these selected band subsets in Table I while k = 3 in Eq. (9). H(t) = (1 −λ(t))Hi( ˆX) + λ(t)Hf( ˆZ), (10) 15: Optimize QUBO problem on a D-Wave quantum annealer 15: Optimize QUBO problem on a D-Wave quantum annealer α∗= min α " S X s=1 exp ( −ys N X i=1 αici(xs) ) /S # . (12) (12) 16: An optimal estimator weight vector: α∗. 16: An optimal estimator weight vector: α∗. 17: PREDICT: given the test band set (x1, . . . xt); 1 17: PREDICT: given the test band set (x1, . . . xt); 1 In contrast, a Qboost classiﬁer is an algorithm for ﬁnding an optimal estimator weight which takes only binary numbers αi ∈{0, +1}, and its loss is deﬁned by a squared loss denoted as L2. Hence, the Qboost classiﬁer is equivalent to a subset selection algorithm among many weak classiﬁers to approximately maximize the accuracy of the strong classiﬁer. In next section, we delve into the Qboost classiﬁer in more detail. 18: T = 1 t Pt t=1 PN i=1 α∗h[i](xt). 18: T = t P t=1 P i=1 α∗h[i](xt). C(xt) = sign PN i=1 α∗[i]h[i](xt) −T . t C(xt) = sign PN i=1 α∗[i]h[i](xt) −T . t C(xt) = sign PN i=1 α∗[i]h[i](xt) −T . ( ) P i=1 19: STOP ALGORITHM. ( ) P i 1 19: STOP ALGORITHM. and the SVM) for the multi-class classiﬁcation of the Indian Pine HSI (see Table. I) [8]. For this scenario, we present the classiﬁcation accuracy of the test dataset in Table. II. In general, these boosting algorithms start with assigning identical weights wS to our dataset. The weak classiﬁers classify these datasets, and if the data is misclassiﬁed then the weight of that data is increased (boosted). This procedure is repeated until no further improvement in the classiﬁcation accuracy can be seen. A DTC with a depth of one is considered as a weak classiﬁer; sometimes, it is called a decision stump classiﬁer. We already presented the steps for boosting a weight wS and the weak classiﬁer in Algorithm 1. These ﬁndings lead to the conclusion that our QUBO-based band subset selection method identiﬁed the highly-informative band subset, and it even helped to reduce a storage space and the computational load for training the given classiﬁers. (10) 13: end for 14: end for 15: Optimize QUBO problem on a D-Wave quantum annealer 16: An optimal estimator weight vector: α∗. 17: PREDICT: given the test band set (x1, . . . xt); 18: T = 1 t Pt t=1 PN i=1 α∗h[i](xt). C(xt) = sign PN i=1 α∗[i]h[i](xt) −T . 19: STOP ALGORITHM. Algorithm 2 Qboost classiﬁer 1: INPUT: h from (Algorithm 1) or given. 2: OUTPUT: The strong classiﬁer C. 3: Fit the weak classiﬁers to (x, y) (Algorithm 1): h (if h is not given). 4: The weak subset classiﬁer selection: α∗= minα[αT Qα], α ∈{0, +1}N; {QUBO problem} 5: Diagonal and off-diagonal elements of a matrix Q: 6: Q ∈RN×N. 7: for i ←1, . . . , N do 8: Qii = S/N 2 + λ −2(h[i])T · y. 9: end for 10: for i ←1, . . . , N do 11: for j ←i + 1, . . . , N do 12: Qij = (h[i])T · h[j]. 13: end for 14: end for 15: Optimize QUBO problem on a D-Wave quantum annealer 16: An optimal estimator weight vector: α∗. 17: PREDICT: given the test band set (x1, . . . xt); 18: T = 1 t Pt t=1 PN i=1 α∗h[i](xt). C(xt) = sign PN i=1 α∗[i]h[i](xt) −T . 19: STOP ALGORITHM. Algorithm 2 Qboost classiﬁer classiﬁcation accuracy; the weak classiﬁer is a classiﬁer that classiﬁes a given dataset better than random guessing [20]. 1: INPUT: h from (Algorithm 1) or given. g g 2: OUTPUT: The strong classiﬁer C. 3: Fit the weak classiﬁers to (x, y) (Algorithm 1): h (if h is not given). VI. A CASE STUDY OF A BINARY QUANTUM CLASSIFIER ON A D-WAVE QUANTUM ANNEALER FOR HYPERSPECTRAL IMAGE We have the Indian Pine HSI with 16 classes, where each class is characterized by three highly-informative bands selected by our QUBO-based band subset selection method shown in Table I. In this section, we analyse binary quantum classiﬁers, namely a quantum boost (Qboost) classiﬁer, and a quantum boost plus (Qboost-Plus) classiﬁer, for a two-label dataset of the Indian Pine HSI created as the binary output of a D-Wave quantum annealer; for instance, Alfalfa and Corn-notill, or Corn-mintill and Corn-notill, etc. Further, we benchmarked the classiﬁcation accuracy of our binary quantum classiﬁers with respect to conventional binary classiﬁers, such as a DTC, an SVM, and an Adaboost classiﬁer. B. A Qboost classiﬁer for a two-label dataset of the Indian Pine HSI This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ A. Basics of an Adaboost classiﬁer An Adaboost classiﬁer is an algorithm for ﬁnding an optimal estimator weight of many weak classiﬁers so that the classiﬁer C is maximized [21]; C(xS) = sign " N X i=1 αici(xS) # , ci(xS) ∈{−1, +1}, (11) where (xS, yS) represents a training dataset, and αi ∈[0, +1] is the estimator weight that is continuous-valued. Here, sign(f(xS)) = 1 if f(xS) > 0, sign(f(xS)) = −1 if f(xS) < 0, and sign(f(xS)) = 0 otherwise. The loss of the Adaboost classiﬁer is deﬁned as an exponential loss (10) (10) where ˆX, ˆZ are Pauli-x and -z matrices, Hi is the initial Hamiltonian of a system for a given time function of λ(t) = 0, and Hf is the QUBO problem with λ(t) = 1 [1]-[3]. To prove that we selected the highly-informative band subset for each class on a D-Wave quantum annealer, we performed the scene classiﬁcation for our Indian Pine HSI by using a Decision Tree Classiﬁer (DTC) and a Support Vector Machine (SVM) shown in Fig. (2) as a proof-of-concept. The hardware of the D-Wave quantum annealer has a speciﬁc graph topology G = (V, E) named Pegasus; its vertices represent binary variables ⃗x, and its edges deﬁne interaction strengths among the binary variables. However, the connectivity of these binary variables in the Pegasus topology is very constrained; in particular, only the certain binary In addition, we discovered that we needed at least a 10- dimensional parameter to reach the same accuracy as our proof-of-concept method when we apply the PCA for the dimensionality reduction and conventional classiﬁers (the DT This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” 5 This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing 5 SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” Algorithm 2 Qboost classiﬁer 1: INPUT: h from (Algorithm 1) or given. 2: OUTPUT: The strong classiﬁer C. 3: Fit the weak classiﬁers to (x, y) (Algorithm 1): h (if h is not given). 4: The weak subset classiﬁer selection: α∗= minα[αT Qα], α ∈{0, +1}N; {QUBO problem} 5: Diagonal and off-diagonal elements of a matrix Q: 6: Q ∈RN×N. 7: for i ←1, . . . , N do 8: Qii = S/N 2 + λ −2(h[i])T · y. 9: end for 10: for i ←1, . . . , N do 11: for j ←i + 1, . . . , N do 12: Qij = (h[i])T · h[j]. TABLE III  (14) ( ) where α∗represents the optimal estimator weight vector, S is the size of the training band dataset, and λ PN i=1 α0 i represents a 0-norm term. By expanding the squared loss function, we have α∗= min α,λ   N X i=1 N X j=1 αiαj S X s=1 ci(xs)cj(xs) ! + + N X i=1 αi λ −2 S X s=1 ci(xs)y(xs) !# , (15) (15) C(xS) = sign " 3 X i=1 αici(xS) # , ci(xS) ∈{−1, +1}, (19) where c1(xS), c2(xS), c3(xS) represent the DTC, SVM, and Qboost classiﬁers, respectively. In this scenario, we have h = [c1(xS), c2(xS), c3(xS)] in Algorithm 2, and this ensemble method is called a Qboost-Plus classiﬁer [2]. which is in the form of a QUBO problem while we deﬁne Qij = S X s=1 ci(xs)cj(xs), Qii = S/N 2 + λ −2 N X i=1 ci(xS)y(xS). (1 (16) B. A Qboost classiﬁer for a two-label dataset of the Indian Pine HSI Moving towards the Qboost classiﬁer, after having stopped boosting the weight of our dataset, the Qboost classiﬁer selects the weak subset classiﬁer so that the classiﬁcation accuracy of the strong classiﬁer is maximized. We executed the weak subset classiﬁer selection algorithm on a D-Wave quantum annealer as shown in Algorithm 2. Below, we explain the derivation of Algorithm 2 in detail. More importantly, the Qboost classiﬁer exploits the weight boosting by solving the weak subset classiﬁer selection problem on a D-Wave quantum annealer. We considered ﬁrst two types of boosting algorithms, a Qboost and an Adaboost algorithm (classiﬁer). The Qboost classiﬁer is a quantum version of an Adaboost classiﬁer. Here, we use two types of terminology for these classiﬁers, a strong classiﬁer C and a weak classiﬁer ci. The strong classiﬁer leverages many weak classiﬁers to achieve its high For the two-label dataset of the Indian Pine HSI, we deﬁne the training band dataset as (x1, y1), . . . , (xS, yS), the test band dataset as (x1, . . . , xt), and the strong classiﬁer, C(xS) ∈{−1, +1}, which is a binary classiﬁer in the form of [13], [20], This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” 6 Binary Classiﬁer Accuracy Classes DTC SVM Qboost Qboost-Plus Adaboost {1, 2} 0.99 0.99 0.99 0.99 0.99 {2, 3} 0.89 0.83 0.64 0.85 0.84 {3, 4} 0.88 0.92 0.83 0.92 0.90 {4, 5} 0.95 0.98 0.95 0.98 0.96 {5, 6} 0.99 0.99 0.98 0.99 0.98 {6, 7} 1.00 1.00 1.00 1.00 1.00 {7, 8} 0.95 0.99 0.99 0.99 0.99 {8, 9} 1.00 1.00 1.00 1.00 1.00 {9, 10} 1.00 0.99 0.99 0.99 0.99 {10, 11} 0.75 0.78 0.72 0.75 0.78 {11, 12} 0.83 0.86 0.83 0.86 0.84 {12, 13} 1.00 1.00 1.00 1.00 1.00 {13, 14} 0.99 0.99 0.99 0.99 0.99 {14, 15} 0.85 0.90 0.87 0.90 0.88 {15, 16} 0.99 0.99 0.99 0.99 0.99 TABLE III CLASSIFICATION ACCURACY OF THE DTC, SVM, Qboost, Qboost-Plus, AND Adaboost FOR THE TWO-LABEL OF THE INDIAN PINE HSI; {i, j} REPRESENTS THE TWO-LABELS, E.G., {1, 2} →Alfalfa and Corn-notill (FIG. 1). This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ B. A Qboost classiﬁer for a two-label dataset of the Indian Pine HSI BY A BOLD FONT WE NOTED THE HIGHEST ACCURACY VALUE OF THE QBOOST-PLUS CLASSIFIER WITH RESPECT TO THE ADABOOST CLASSIFIER. C(xS) = sign " N X i=1 αici(xS) # , ci(xS) ∈{−1, +1}, (13) where αi ∈{0, 1} is the estimator weight, and ci(xS) is the weak classiﬁer; we chose DTCs as our weak classiﬁers. Recent papers on theoretical studies [13], [14] and a prac- tical application for the remote sensing [22], are proposed to formulate the loss of the strong classiﬁer as a squared loss L2: α∗= min αi,λ   S X s=1 N X i=1 αici(xs) −y(xs) !2 + λ N X i=1 α0 i  , (14 Algorithm 3 A multi-label classiﬁer by using quantum binary classiﬁers via an ECOC • Training: We train each column of the coding matrix M ′ by quantum binary classiﬁers Cb = {C1, C2, . . . , C24}. dj = d(Cb(xt), M[j, :]), j = 1, 2, . . . , l. 15: return argminj dj; {codewords (a label) for the unlabeled data xt}. • Testing: For an unlabeled input xt, we evaluate Cb(xt) = {C1(xt), C2(xt), . . . , C24(xt)}, and then we assign Cb(xt) to the closest codewords in the coding matrix M by using an Euclidean/Hamming distance. 16: STOP ALGORITHM. selection method. Further, we leveraged these selected bands to benchmark our Qboost and Qboost-plus algorithms with respect to the classical classiﬁers. Our quantum classiﬁers clearly outperform the conventional classiﬁers for most of the binary instances of the Indian pine HSI. A. Benchmarking Qboost and Qboost-Plus for the multi-label classiﬁcation A. Benchmarking Qboost and Qboost-Plus for the multi-label classiﬁcation We run our experiment for the multi-label of the Indian Pine HSI via the ECOC by using the DTC, SVM, Qboost, Qboost- Plus, and Adaboost classiﬁer. Furthermore, we presented the classiﬁcation accuracy of our experiment in Table V. We compared also the classiﬁcation accuracy and the confusion matrix of the Qboost-Plus with one of the Adaboost classiﬁer (see Fig. 3). Their results again demonstrate that the Qboost- Plus classiﬁer beats the Adaboost classiﬁer when we leverage the ECOC technique for a multi-label classiﬁcation case. More importantly, we provided a novel multi-label classiﬁer via the ECOC technique when applying a quantum computing device yielding binary outputs. Algorithm 3 A multi-label classiﬁer by using quantum binary classiﬁers via an ECOC Algorithm 3 A multi-label classiﬁer by using quantum binary classiﬁers via an ECOC 1: INPUT: Training bands: (x, y) = (x1, y1), . . . , (xS, yS); {xS represents the three selected bands for a given class yS (see Table I).} y ∈{1, 2, . . . , 16}S; {y represents l = 16 distinct labels of the Indian Pine HSI, and S is a size of the training dataset.} } 2: OUTPUT: Quantum binary classiﬁers: Cb = {C1, C2, . . . , C24}. Cb = {C1, C2, . . . , C24}. { 3: CODING MATRIX: 3: CODING MATRIX: 4: Assign b = 24 codewords to each class (b > log2 l), and generate l by b coding matrix M for l = 16 distinct labels (see Table IV). 5: Construct S by b coding matrix M ′ for training classes y. 6: TRAINING: 7: for i ←1, . . . , b do 8: Construct two sets, Gi and Gi. Gi consists of all labels for which M ′[:, i] == 1, and Gi is the complement set. 9: Fit a quantum binary classiﬁer Ci to distinguish Gi from Gi by using Algorithm 2. • Coding matrix: We assign unique b-bits (codewords) to each class of the Indian Pine HSI such that b > log2 l where l = 16 is a number of classes; the classes are represented by a so-called coding matrix M ∈{0, 1}l×b (see Table IV), and M ′ ∈{0, 1}S×b for a training dataset with size S. In our case, each class is represented by b = 24 codewords generated randomly. 10: end for 11: TESTING: 11: TESTING: 12: Given an unlabeled data xt. 12: Given an unlabeled data xt. 13: Evaluate the trained quantum binary classiﬁers Cb(xt) = {C1(xt), C2(xt), . . . , C24(xt)} by employing the step 17 of Algorithm 2. 14: Compute an Euclidean/Hamming distance: dj = d(Cb(xt), M[j, :]), j = 1, 2, . . . , l. • Training: We train each column of the coding matrix M ′ by quantum binary classiﬁers Cb = {C1, C2, . . . , C24}. • Testing: For an unlabeled input xt, we evaluate Cb(xt) = {C1(xt), C2(xt), . . . , C24(xt)}, and then we assign Cb(xt) to the closest codewords in the coding matrix M by using an Euclidean/Hamming distance. VII. A NOVEL MULTI-LABEL CLASSIFIER FOR THE INDIAN PINE HSI ON A D-WAVE QUANTUM ANNEALER In the prior section, we exhibited that our quantum binary classiﬁers (Qboost and QboostPlus) classify the two-label of the Indian Pine HSI with high accuracy due to the binary output of a D-Wave quantum annealer. However, the Indian Pine HSI has 16 classes, and the quantum binary classiﬁers are needed to extend for the multi-label classiﬁcation. Hence, we propose a novel technique for the multi-label classiﬁcation via an Error-Correcting Output Code (ECOC), and namely, we leverage an ECOC technique to classify the multi-label of the Indian Pine HSI when using our binary quantum classiﬁers [15], [16], [23]. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ C. Benchmarking Qboost and Qboost-Plus for the two-class classiﬁcation Then we can write Then we can write We run our experiment in several scenarios for the two- label dataset of the Indian Pine HSI by using the DTC, SVM, Qboost, Qboost-Plus, and Adaboost classiﬁer. These scenarios are: α∗= min α [αT Qα], α ∈{0, +1}N. (17) (17) We optimized this problem on a D-Wave quantum annealer to select the weak subset classiﬁer in its quadratic form. 1) DTC for the two-label dataset of the Indian HSI. 2) SVM for the two-label dataset of the Indian HSI. Then we obtained the optimal estimator weight vector α∗, and the strong classiﬁer for the test band dataset becomes 3) Qboost with 30 weak classiﬁers for the two-label dataset of the Indian HSI; the weak classiﬁers are the DT classiﬁers with the depth three. C(xt) = sign " N X i=1 α∗ i ci(xt) −T # , T = 1 t t X t=1 N X i=1 α∗ci(xt), (18) 4) Qboost-Plus for the two-label dataset of the Indian HSI; the weak classiﬁers are a DTC, an SVM, and a Qboost classiﬁer. (18) 5) Adaboost with 30 weak classiﬁers for the two-label dataset of the Indian HSI; the weak classiﬁers are the decision stump classiﬁers. where (x1, . . . xt) are from the test band dataset of the Indian Pine HSI, and T is derived experimentally to increase the classiﬁcation accuracy of the strong classiﬁer C(xt) [13], [14]. We already the procedures of the Qboost classiﬁer in Algorithm 1 and Algorithm 2. All above scenarios used for benchmarking are the two- label classiﬁcation of the Indian Pine HSI, and we present the classiﬁcation accuracy of our experiment in Table III. We even compared the boosting algorithms, the Qboost-Plus and the Adaboost classiﬁer. Their results demonstrate that the Qboost- Plus classiﬁer performs the same as the Adaboost classiﬁer and even better in some instances. Secondly, we chose the DTC, SVM, and Qboost classiﬁers as weak classiﬁers instead of only a DTC. This method is sometimes called an ensemble method. By exploiting Eq. (13) and Eq. (15), we again formulated weak classiﬁers such that In this part, we selected the most highly-informative band of the Indian Pine HSI by using our QUBO-based band subset This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. C. Benchmarking Qboost and Qboost-Plus for the two-class classiﬁcation Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing his article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing 7 7 SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” Algorithm 3 A multi-label classiﬁer by using quantum binary classiﬁers via an ECOC VIII. DISCUSSION AND CONCLUSION a dataset play a vital role in quantum computers. In terms of a future work, we will design a hybrid quantum- classical network for Earth observation datasets which exploits both quantum computers (a quantum annealer and gate-based quantum computer) and a conventional computer. Such a hybrid network will be independent of the choice and size of datasets. In terms of a future work, we will design a hybrid quantum- classical network for Earth observation datasets which exploits both quantum computers (a quantum annealer and gate-based quantum computer) and a conventional computer. Such a hybrid network will be independent of the choice and size of datasets. ACKNOWLEDGMENT We would like to greatly acknowledge Gottfried Schwarz (DLR, Oberpfaffenhofen) for his valuable comments and contributions for enhancing a quality of our paper. Also, the authors gratefully acknowledge the Juelich Supercomput- ing Centre (https://www.fzjuelich.de/ias/jsc) for funding this project by providing computing time through the Juelich UNiﬁed Infrastructure for Quantum computing (JUNIQ) on a D-Wave quantum annealer. In the second part of our article, we ﬁrst tested a binary classiﬁcation for the Indian Pine HSI due to the binary output of our D-Wave quantum annealer. We proposed to employ two binary quantum classiﬁers, Qboost and Qboost-Plus, to our two-label dataset. Secondly, we provided an ECOC for the multi-label classiﬁcation of the Indian Pine HSI when applying our binary quantum classiﬁers. Here, the classes are characterized by the bands selected during the ﬁrst part of our study. We benchmarked these binary quantum classiﬁers and the novel multi-label classiﬁer in comparison to conventional classiﬁers that are a Decision Tree Classiﬁer, a Support Vector Machine classiﬁer, and an Adaboost classiﬁer. Our binary quantum classiﬁers and our novel multi-label classiﬁer even outperform these conventional classiﬁers for most instances of the two- and multi-label dataset. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VIII. DISCUSSION AND CONCLUSION In the ﬁrst part of this paper, we used an MI-based band subset selection technique as a global optimization approach for a real-world problem of the Indian Pine hyperspectral dataset on a D-Wave quantum annealer. We ﬁrst mapped An ECOC technique (see Algorithm 3 for a detailed procedure): An ECOC technique (see Algorithm 3 for a detailed procedure): rticle has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing cation in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Cit of Selected Topics in Applied Earth Observations and Remote Sensing SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” 8 (a) The Qboost-Plus classiﬁer via the ECOC technique Fig. 3. The confusion matrix for the l = 16 labels of the Indian Pine HSI. (b) The Adaboost classiﬁer via the ECOC technique (b) The Adaboost classiﬁer via the ECOC technique (a) The Qboost-Plus classiﬁer via the ECOC technique (b) The Adaboost classiﬁer via the ECOC technique Fig. 3. The confusion matrix for the l = 16 labels of the Indian Pine HSI. Fig. 3. The confusion matrix for the l = 16 labels of the Indian Pine HSI. a dataset play a vital role in quantum computers. this MI-based band subset selection problem to a QUBO- based band subset selection problem. Then we benchmarked and assessed the performance of a D-Wave quantum annealer compared to a conventional annealer. We demonstrated that the D-Wave quantum annealer correctly selects highly-informative bands competitive to a conventional annealer. To prove that our D-Wave quantum annealer selected the best bands for each class, we classiﬁed all 16 classes based on their three highly- informative bands by applying a Decision Tree Classiﬁer and a Support Vector Machine classiﬁer. Their classiﬁcation results exhibit that the selected bands are the highly-informative ones. Besides, the feature selection method saves storage space and reduces the computational load for the training process. SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” Datcu was the recipient of the National Order of Merit with the rank of Knight, for outstanding international research results, awarded by the President of Romania, in 2008, and the Romanian Academy Prize Traian Vuia for the development of the SAADI image analysis system and his activity in image processing in 1987. He was awarded the Chaire D’excellence Internationale Blaise Pascal 2017 for data science in earth observation and the 2018 Ad Astra Award for Excellence in Science. He has served as a Co-organizer for international conferences and workshops and as a Guest Editor for the IEEE and other journals. He is representative of Romanian in the Earth Observation Program Board (EO-PB). [14] V. S. Denchev, N. Ding, S. V. N. Vishwanathan, and H. Neven, “Robust classiﬁcation with adiabatic quantum optimization,” arXiv:1205.1148, May 2012. [Online]. Available: https://arxiv.org/abs/1205.1148 [15] T. G. Dietterich and G. Bakiri, “Solving multiclass learning problems via error-correcting output codes,” J. Artif. Int. Res., vol. 2, no. 1, p. 263–286, Jan. 1995. [16] A. Radoi and M. Datcu, “Multilabel annotation of multispectral remote sensing images using error-correcting output codes and most ambiguous examples,” IEEE Journal of Selected Topics in Applied Earth Observa- tions and Remote Sensing, vol. 12, no. 7, pp. 2121–2134, 2019. both in Strasbourg, France, University of Siegen, Germany, University of Innsbruck, Austria, University of Alcala, Spain, University Tor Vergata, Rome, Italy, University of Trento, Italy, Unicamp, Campinas, Brazil, China Academy of Science, Shenyang, China, Universidad Pontiﬁcia de Salamanca, Campus de Madrid, Spain, University of Camerino, Italy, and the Swiss Center for Scientiﬁc Computing, Manno, Switzerland. From 1992 to 2002, he had an Invited Professor Assignment with the Swiss Federal Institute of Technology (ETH Zurich), Switzerland. Since 2001, he had been initiating and leading the Competence Center on Information Extraction and Image Understanding for Earth Observation, Paris Institute of Technology, ParisTech, France, a collaboration of DLR with the French Space Agency (CNES). He has been a Professor holder of the DLR-CNES Chair at ParisTech. He has initiated the European frame of projects for image information mining and is involved in research programs for information extraction, data mining and knowledge discovery, and data science with the ESA, NASA, and in a variety of national and European projects. He is the Director of the Research Center for Spatial Information, UPB. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ REFERENCES Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing n accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” His research interests include explainable and physics-aware artiﬁcial intelligence, smart radar sensors design, and quantum machine learning with applications in Earth Observation. He has held Visiting Professor appointments with the University of Oviedo, Spain, the University Louis Pasteur and the International Space University, both in Strasbourg, France, University of Siegen, Germany, University of Innsbruck, Austria, University of Alcala, Spain, University Tor Vergata, Rome, Italy, University of Trento, Italy, Unicamp, Campinas, Brazil, China Academy of Science, Shenyang, China, Universidad Pontiﬁcia de Salamanca, Campus de Madrid, Spain, University of Camerino, Italy, and the Swiss Center for Scientiﬁc Computing, Manno, Switzerland. From 1992 to 2002, he had an Invited Professor Assignment with the Swiss Federal Institute of Technology (ETH Zurich), Switzerland. Since 2001, he had been initiating and leading the Competence Center on Information Extraction and Image Understanding for Earth Observation, Paris Institute of Technology, ParisTech, France, a collaboration of DLR with the French Space Agency (CNES). He has been a Professor holder of the DLR-CNES Chair at ParisTech. He has initiated the European frame of projects for image information mining and is involved in research programs for information extraction, data mining and knowledge discovery, and data science with the ESA, NASA, and in a variety of national and European projects. He is the Director of the Research Center for Spatial Information, UPB. He is a Senior Scientist and the Data Intelligence and Knowledge Discovery Research Group Leader with the Remote Sensing Technology Institute, DLR, and a delegate in the DLR-ONERA Joint Virtual Center for AI in Aerospace. He is a member of the ESA Working Group Big Data from Space and Visiting Professor with ESA’s Phi-Lab. Dr. Datcu was the recipient of the National Order of Merit with the rank of Knight, for outstanding international research results, awarded by the President of Romania, in 2008, and the Romanian Academy Prize Traian Vuia for the development of the SAADI image analysis system and his activity in image processing in 1987. He was awarded the Chaire D’excellence Internationale Blaise Pascal 2017 for data science in earth observation and the 2018 Ad Astra Award for Excellence in Science. He has served as a Co-organizer for international conferences and workshops and as a Guest Editor for the IEEE and other journals. He is representative of Romanian in the Earth Observation Program Board (EO-PB). Mihai Datcu (Fellow, IEEE) received the M.S. and Ph.D. SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” degrees in elec- tronics and telecommunications from the University Politehnica of Bucharest (UPB), Bucharest, Romania, in 1978 and 1986, respectively, and the ha- bilitation a Diriger Des Recherches degree in computer science from the University Louis Pasteur, Strasbourg, France, in 1999. Since 1981, he has been a Professor with the Department of Applied Electronics and Information Engineering, Faculty of Electronics, Telecommunications and Information Technology, UPB. Since 1993, he has been a Scientist with the German Aerospace Center (DLR), Wessling, Germany. His research interests include explainable and physics-aware artiﬁcial intelligence, smart radar sensors design, and quantum machine learning with applications in Earth Observation. He has held Visiting Professor appointments with the University of Oviedo, Spain, the University Louis Pasteur and the International Space University, both in Strasbourg, France, University of Siegen, Germany, University of Innsbruck, Austria, University of Alcala, Spain, University Tor Vergata, Rome, Italy, University of Trento, Italy, Unicamp, Campinas, Brazil, China Academy of Science, Shenyang, China, Universidad Pontiﬁcia de Salamanca, Campus de Madrid, Spain, University of Camerino, Italy, and the Swiss Center for Scientiﬁc Computing, Manno, Switzerland. From 1992 to 2002, he had an Invited Professor Assignment with the Swiss Federal Institute of Technology (ETH Zurich), Switzerland. Since 2001, he had been initiating and leading the Competence Center on Information Extraction and Image Understanding for Earth Observation, Paris Institute of Technology, ParisTech, France, a collaboration of DLR with the French Space Agency (CNES). He has been a Professor holder of the DLR-CNES Chair at ParisTech. He has initiated the European frame of projects for image information mining and is involved in research programs for information extraction, data mining and knowledge discovery, and data science with the ESA, NASA, and in a variety of national and European projects. He is the Director of the Research Center for Spatial Information, UPB. He is a Senior Scientist and the Data Intelligence and Knowledge Discovery Research Group Leader with the Remote Sensing Technology Institute, DLR, and a delegate in the DLR-ONERA Joint Virtual Center for AI in Aerospace. He is a member of the ESA Working Group Big Data from Space and Visiting Professor with ESA’s Phi-Lab. Dr. SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” 9 Soronzonbold Otgonbaatar received his B.Sc. and M.Sc. degrees in theo- retical physics from the National University of Mongolia, Mongolia, and the University of Siegen, Germany. Currently, he is pursuing his PhD degree in German Aerospace Center (DLR), Oberpfaffenhofen, Germany. His research interest spans over computational science, artiﬁcial intelligence (i.e. machine learning), quantum computing and algorithms, and quantum machine learning with practical applications in remote sensing data. [8] S. Prasad and L. M. Bruce, “Limitations of principal components analysis for hyperspectral target recognition,” IEEE Geoscience and Remote Sensing Letters, vol. 5, no. 4, pp. 625–629, 2008. [9] B. Guo, S. R. Gunn, R. I. Damper, and J. D. B. Nelson, “Band selection for hyperspectral image classiﬁcation using mutual information,” IEEE Geoscience and Remote Sensing Letters, vol. 3, no. 4, pp. 522–526, 2006. [10] S. Sarmah and S. K. Kalita, “Mutual information-based hierarchical band selection approach for hyperspectral images,” in Advances in Electronics, Communication and Computing, A. Kalam, S. Das, and K. Sharma, Eds. Singapore: Springer Singapore, 2018, pp. 755–763. [11] X. V. Nguyen, J. Chan, S. Romano, and J. Bailey, “Effective global approaches for mutual information based feature selection,” in Proceedings of the 20th ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, ser. KDD ’14. New York, NY, USA: Association for Computing Machinery, 2014, p. 512–521. [Online]. Available: https://doi.org/10.1145/2623330.2623611 [12] N. X. Vinh, S. Zhou, J. Chan, and J. Bailey, “Can high-order dependencies improve mutual information based feature selection?” Pattern Recognition, vol. 53, pp. 46–58, 2016. [Online]. Available: https://www.sciencedirect.com/science/article/pii/S0031320315004276 p p [13] H. Neven, V. S. Denchev, G. Rose, and W. G. Macready, “Qboost: Large scale classiﬁer training withadiabatic quantum optimization,” in Proceedings of the Asian Conference on Machine Learning, ser. Proceedings of Machine Learning Research, S. C. H. Hoi and W. Buntine, Eds., vol. 25. Singapore Management University, Singapore: PMLR, 04–06 Nov 2012, pp. 333–348. [Online]. Available: http://proceedings.mlr.press/v25/neven12.html Mihai Datcu (Fellow, IEEE) received the M.S. and Ph.D. degrees in elec- tronics and telecommunications from the University Politehnica of Bucharest (UPB), Bucharest, Romania, in 1978 and 1986, respectively, and the ha- bilitation a Diriger Des Recherches degree in computer science from the University Louis Pasteur, Strasbourg, France, in 1999. Since 1981, he has been a Professor with the Department of Applied Electronics and Information Engineering, Faculty of Electronics, Telecommunications and Information Technology, UPB. Since 1993, he has been a Scientist with the German Aerospace Center (DLR), Wessling, Germany. REFERENCES [1] E. Farhi, J. Goldstone, S. Gutmann, and M. Sipser, “Quantum computation by adiabatic evolution,” arXiv:0001106, Jan 2000. [Online]. Available: https://arxiv.org/abs/quant-ph/0001106 p g q p [2] (2021) A D-Wave quantum annealer. [Online]. Available: https: //cloud.dwavesys.com/leap [3] V. S. Denchev, S. Boixo, S. V. Isakov, N. Ding, R. Babbush, V. Smelyanskiy, J. Martinis, and H. Neven, “What is the computational value of ﬁnite-range tunneling?” Phys. Rev. X, vol. 6, p. 031015, Aug 2016. [Online]. Available: https://link.aps.org/doi/10.1103/PhysRevX.6. 031015 [4] E. Farhi, J. Goldstone, S. Gutmann, J. Lapan, A. Lundgren, and D. Preda, “A quantum adiabatic evolution algorithm applied to random instances of an np-complete problem,” Science, vol. 292, no. 5516, pp. 472–475, 2001. [Online]. Available: https: //science.sciencemag.org/content/292/5516/472 In the end, we realized how to leverage a quantum annealing device to extract knowledge and support real-world optimiza- tion problems in comparison to conventional machine learning techniques. In addition, we conceived strategies for formulat- ing and embedding real-world problems to the topology of a D-Wave machine. [5] T. Stollenwerk, B. O’Gorman, D. Venturelli, S. Mandr`a, O. Rodionova, H. Ng, B. Sridhar, E. G. Rieffel, and R. Biswas, “Quantum annealing applied to de-conﬂicting optimal trajectories for air trafﬁc management,” IEEE Transactions on Intelligent Transportation Systems, vol. 21, no. 1, pp. 285–297, 2020. pp [6] E. Rieffel, D. Venturelli, B. O’Gorman, M. Do, E. Prystay, and V. Smelyanskiy, “A case study in programming a quantum annealer for hard operational planning problems,” Quantum Information Processing, vol. 14, pp. 1–36, 2015. We must note however that our method is not intended to compete with a conventional method, but we intended to ﬁnd a proper dataset in Earth observation to evaluate an existing quantum algorithm on a D-Wave quantum annealer or the future quantum computers since the choice and the size of [7] F. Melgani and L. Bruzzone, “Classiﬁcation of hyperspectral remote sensing images with support vector machines,” IEEE Transactions on Geoscience and Remote Sensing, vol. 42, no. 8, pp. 1778–1790, 2004. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSTARS.2021.3095377, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing his article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. SPECIAL ISSUE ON “QUANTUM RESOURCES FOR EARTH OBSERVATION” He is a Senior Scientist and the Data Intelligence and Knowledge Discovery Research Group Leader with the Remote Sensing Technology Institute, DLR, and a delegate in the DLR-ONERA Joint Virtual Center for AI in Aerospace. He is a member of the ESA Working Group Big Data from Space and Visiting Professor with ESA’s Phi-Lab. Dr. Datcu was the recipient of the National Order of Merit with the rank of Knight, for outstanding international research results, awarded by the President of Romania, in 2008, and the Romanian Academy Prize Traian Vuia for the development of the SAADI image analysis system and his activity in image processing in 1987. He was awarded the Chaire D’excellence Internationale Blaise Pascal 2017 for data science in earth observation and the 2018 Ad Astra Award for Excellence in Science. He has served as a Co-organizer for international conferences and workshops and as a Guest Editor for the IEEE and other journals. He is representative of Romanian in the Earth Observation Program Board (EO-PB). [17] (2021) Airborne visible/infrared imaging spectrometer (aviris). [Online]. Available: https://aviris.jpl.nasa.gov/ [18] S. Barnett, Ed., Quantum Information. Oxford: Oxford university Press., 2009. [19] K. Boothby, P. Bunyk, J. Raymond, and A. Roy, “Next-generation topology of d-wave quantum processors,” arXiv:2003.00133, Feb 2020. [Online]. Available: https://arxiv.org/abs/2003.00133 [20] Y. Freund and R. E. Schapire, “A decision-theoretic generalization of on- line learning and an application to boosting,” Journal of Computer and System Sciences, vol. 55, no. 1, pp. 119–139, 1997. [Online]. Available: https://www.sciencedirect.com/science/article/pii/S002200009791504X [21] T. Hastie, S. Rosset, J. Zhu, and H. Zou, “Multi-class adaboost,” Statistics and Its Interface, vol. 2, no. 3, pp. 349–360, 2009. [22] E. Boyda, S. Basu, S. Ganguly, A. Michaelis, S. Mukhopadhyay, and R. R. Nemani, “Deploying a quantum annealing processor to detect tree cover in aerial imagery of california,” PLOS ONE, vol. 12, no. 2, pp. 1–22, 02 2017. [Online]. Available: https://doi.org/10.1371/journal.pone.0172505 [23] A. Berger, “Error-correcting output coding for text classiﬁcation,” In Proceedings of IJCAI-99 Workshop on Machine Learning for Informa- tion Filtering, 07 2001.
https://openalex.org/W2150034296	https://www.revistas.usp.br/posfau/article/download/48319/52177	Portuguese	null	Teses e dissertações	Pós. Revista do Programa de Pós-Graduação em Arquitetura e Urbanismo da FAUUSP	2,012	cc-by	515	MARCIA YOKO TERAZAKI Arquitetura e infraestrutura urbana: a Linha Norte-Sul e a Estação Ponte Pequena do metrô de São Paulo Data: 04.10.11 Banca: Profs. (as) Drs. (as): Luis Antonio Jorge, Regina Maria Prosperi Meyer e Renato Luiz Sobral Anelli IARA PIERRO DE CAMARGO Banca: Profs. (as) Drs. (as): Paulo Renato Mesquita Banca: Profs. (as) Drs. (as): Paulo Renato Mesquita Pellegrino, Marta Dora Grostein, Cataharina Pinheiro Pellegrino, Marta Dora Grostein, Cataharina Pinheiro O departamento de design gráfico da Cranbrook Academy of Art (1971-1995): novos caminhos para o design Data: 08.12.11 Banca: Profs. (as) Drs. (as): Carlos Roberto Zibel Costa, Maria Helena Werneck Bomeny e Priscila Lena Farias O departamento de design gráfico da Cranbrook Academy of Art (1971-1995): novos caminhos para o design Data: 08.12.11 Banca: Profs. (as) Drs. (as): Carlos Roberto Zibel Costa, Maria Helena Werneck Bomeny e Priscila Lena Farias Cordeiro dos Santos Lima, Yuri Tavares Rocha e Eduardo Antonio Gomes Marques BRAZ CASAGRANDE BRAZ CASAGRANDE Novo arrabalde: conservação e ocupação urbana na concepção do projeto de expansão da cidade de Vitória Data: 25.08.11 Banca: Profs. (as) Drs. (as): Maria Cristina da Silva Leme, Carlos Roberto Monteiro de Andrade e Eneida Maria Souza Mendonça BRAZ CASAGRANDE Novo arrabalde: conservação e ocupação urbana na concepção do projeto de expansão da cidade de Vitória Souza Mendonça Teses e dissertações 2o semestre 2011 2o semestre 2011 2o semestre 2011 ANDRÉ FONTAN KÖHLER Políticas públicas de regeneração urbana, preservação do patrimônio, lazer e turismo: padrões de intervenção pública e avaliação de resultados no Pátio de São Pedro Recife 1969 2008 Pedro, Recife, 1969-2008 Data: 15.12.11 VERÔNICA GARCIA DONOSO Banca: Profs. (as) Drs. (as): Maria Irene Queiroz A paisagem e os sistemas de espaços livres na urbanização contemporânea do interior paulista: estudo de caso da área entre São Carlos, Araraquara e Ribeirão Preto Data: 17.11.11 Banca: Profs. (as) Drs. (as): Eugênio Fernandes Queiroga, Ana Cecília Mattei de Arruda Campos e Silvia Selingardi Sampaio Ferreira Szmrecsanyi, Maria Lucia Bressan Pinheiro, José Carlos Garcia Durand, Madalena Pedroso Aulicino Ed d Alb t C N b e Eduardo Alberto Cusce Nobre e Eduardo Alberto Cusce Nobre LUZIA HELENA DOS SANTOS BARROS Requalificação dos aterros desativados (brownfields) no município de São Paulo: parques (greenfields) Raposo Tavares e Jardim Primavera Data: 16.12.11 DANIEL BONFIM DA SILVA Blocos de memórias: habitação social, arquitetura moderna e patrimônio cultural Data: 02.12.11 Banca: Profs. (as) Drs. (as): Nabil Georges Bonduki, Beatriz Mugayar Kühl, Paulo César Garcez Marins, Silvana Barbosa Rubino e Anna Beatriz Ayrosa Galvão Redes sociais virtuais: um estudo da formação, comunicação e ação social Data: 16.09.11 Banca: Profs. Drs. Carlos Roberto Zibel Costa, Daniela Kutschat Hanns e Caio Adorno Vassão Redes sociais virtuais: um estudo da formação comunicação e ação social Silvana Barbosa Rubino e Kutschat Hanns e Caio Adorno Vassão Anna Beatriz Ayrosa Galvão RENATO SEBASTIÁN RIOS MANTILLA Arquitetura – Jogo – Percepção: a casa como elemento lúdico Arquitetura – Jogo – Percepção: a casa como elemento lúdico Data: 13.12.11 Data: 13.12.11 Mazzilli, Maria Cecília Loschiavo dos Santos e Caio Adorno Vassão pós v.19 n.31 • são paulo • junho 2012 297 pós- 297 pós-
https://openalex.org/W4308265807	https://portal.research.lu.se/files/122805415/Fell_nder_Rebane_et_al_2022_Achieving_a_Data_Driven_Risk_Assessment_Methodology_for_Ethical_AI.pdf	English	null	Achieving a Data-driven Risk Assessment Methodology for Ethical AI	arXiv (Cornell University)	2,021	cc-by	12,661	Achieving a Data‐Driven Risk Assessment Methodology for Ethical AI DOI: 10.1007/s44206-022-00016-0 2022 Document Version: Publisher's PDF, also known as Version of record Link to publication Citation for published version (APA): Felländer, A., Rebane, J., Larsson, S., Wiggberg, M., & Heintz, F. (2022). Achieving a Data‐Driven Risk Assessment Methodology for Ethical AI. Digital Society, 1(2), 1-27. 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LUND UNIVERSITY PO Box 117 221 00 Lund +46 46-222 00 00 (2022) 1:13 Digital Society (2022) 1:13 https://doi.org/10.1007/s44206-022-00016-0 Digital Society (2022) 1:13 https://doi.org/10.1007/s44206-022-00016-0 Digital Society ORIGINAL PAPER * Jonathan Rebane jonathan@dsv.su.se 1 anch.AI (formerly AI Sustainability Center), Stockholm, Sweden 2 Department of Computer and Systems Sciences, Stockholm University, Stockholm, Sweden 3 Department of Technology and Society, Lund University, Lund, Sweden 4 Department of Computer and Information Science, Linköping University, Linköping, Sweden 5 Department of Industrial Engineering, KTH Royal Institute of Technology, Stockholm, Sweden Achieving a Data‑Driven Risk Assessment Methodology for Ethical AI Anna Felländer1 · Jonathan Rebane1,2 · Stefan Larsson1,3 · Mattias Wiggberg1,5 · Fredrik Heintz1,4 Received: 2 March 2022 / Accepted: 28 July 2022 © The Author(s) 2022 1 Introduction The evolving data-driven technology sector has resulted in AI solutions becoming pervasively implemented throughout much of society, such as for personalized rec- ommendations, health applications, and optimized processes (Jameson et al., 2002; Kalis et al., 2018; Tseng et al., 2021; Javaid et al., 2022; Groshev et al., 2021). These implementations demand a myriad of legal, ethical, and societal considera- tions which must be accounted for in order to develop ethical AI (eAI) solutions which sustain human values in an emerging data-driven era (Cath, 2018). The cost of ignoring eAI issues can be very high, with several high-profile AI systems ulti- mately needing to be shut down after risks inadvertently materialized and mas- sive reputational losses occurred (Wolf et al., 2017; Analytica, 2018; Lauer, 2021). These problems are not limited to isolated events, with over 1400 reports of AI caus- ing harm being reported in the Artificial Intelligence Incident Database (McGregor, 2020).f Effective and continuous risk management, utilizing risk assessment, is a vital component of ethics and compliance programs to anticipate and mitigate eAI risks before they occur. Risk management has backings in generalized ISO standards and should not be confused with ethical, financial, or operational auditing (Purdy, 2010). As of today, most means of risk management specific to the eAI landscape con- sists of a variety of guidelines, recommendations, and a handful of specified tools to account for and tackle individual challenges (Hagendorff, 2020; Jobin et al., 2019; Bellamy et al., 2019; Canca, 2020; Larsson, 2020). However, such resources have been criticized for being too abstract or technology-centered, lacking a direct focus on cross-functional organizational viewpoints and needs, such as the compatibility with standardized risk assessment models to bring principles to practice (Theodorou & Dignum, 2020). In addition, existing governance methodologies for eAI focus on research-focused processes rather than risk assessment, such as d’Aquin et al. (2018), or lack validation, emphasis on human rights, and cross-functional perspec- tives (Brendel et al., 2021). Proposals do exist for self-assessment, such as HLE- GAI (2020), and structured approaches on “ethics-based auditing” such as Brown et al. (2021); Floridi and Cowls (2019). However, requests have been made for the development and validation of methods that can be applied in reality to managing eAI organization risk, as a means to assure the legality, ethics, and robustness of AI systems Wright (2020); Theodorou and Dignum (2020); Brendel et al. (2021). Abstract The AI landscape demands a broad set of legal, ethical, and societal considera- tions to be accounted for in order to develop ethical AI (eAI) solutions which sus- tain human values and rights. Currently, a variety of guidelines and a handful of niche tools exist to account for and tackle individual challenges. However, it is also well established that many organizations face practical challenges in navigat- ing these considerations from a risk management perspective within AI governance. Therefore, new methodologies are needed to provide a well-vetted and real-world applicable structure and path through the checks and balances needed for ethically assessing and guiding the development of AI. In this paper, we show that a multidis- ciplinary research approach, spanning cross-sectional viewpoints, is the foundation of a pragmatic definition of ethical and societal risks faced by organizations using AI. Equally important are the findings of cross-structural governance for imple- menting eAI successfully. Based on evidence acquired from our multidisciplinary research investigation, we propose a novel data-driven risk assessment methodol- ogy, entitled DRESS-eAI. In addition, through the evaluation of our methodological implementation, we demonstrate its state-of-the-art relevance as a tool for sustaining human values in the data-driven AI era. Keywords Ethical AI · Sustainability · Risk assessment Keywords Ethical AI · Sustainability · Risk assessment 12341 456789) 3 Page 2 of 27 Digital Society (2022) 1:13 Digital Society (2022) 1:13 Digital Society 13 1 Introduction Furthermore, the proposal for an AI Act, published by the European Commission in April 2021 (Commission, 2021), puts much emphasis on assessments as a way to manage and mitigate high-risk use of AI systems. This proposal has faced critique in regard to its broad definition and potential for over-regulation of AI according to Glauner (2021), while others have characterized the proposal as an “auditing” regu- lation (Mökander et al., 2021). From an organizational viewpoint, risk assessment methodologies for technical systems exist to provide a linear structure for identifying and mitigating unregu- lated business risks with individualized risk assessment phases (Pandey, 2012). However, due to the multidisciplinary nature of AI solutions, what is needed 1 3 Digital Society (2022) 1:13 Page 3 of 27 13 are novel approaches, developed from a holistic multidisciplinary approach which incorporates technical, legal, and societal perspectives, with the objec- tive of detecting negative eAI externalities of organizations that would otherwise infringe on legal and human rights alongside organizational principles (Dignum, 2020). Research gaps needed to be filled are in relation to multi-stakeholder organizational perspectives for establishing core concepts with regard to the eAI risk landscape (Rodrigues, 2020). Such knowledge can then be leveraged as a basis for filling a gap in relation to the development of an eAI risk assessment methodology. Due to the rapidly progressing eAI landscape, such a methodology must be flexible in the sense that defined risks, concepts, and methods are flexible enough to accommodate an evidence-based evolution. It is well established that many organizations face practical challenges in navigating eAI considerations (Lauer, 2021; Rakova et al., 2021; Desouza et al., 2019). There is a general discussion regarding how ethical AI in organizations could be handled (Clarke, 2019). Yet the presence of proof-of-concepts where real organizations and real data have been tested is low. Therefore, new method- ologies are needed to provide a structure and path through the checks and bal- ances needed for ethically assessing an AI. The question we wish to answer in this paper is: How can a standardized approach to ethical AI risk assessment be constructed that is compatible with cross-functional organizational demands over a large variety of contexts? Our contribution addresses this question as follows: • Firstly, in Sect. 2, we report the findings of a multidisciplinary research investi- gation. 2 Research Approach To answer the question of how to provide an approach to eAI risk assessment that is compatible with different organization demands, we systematically approached the problem from a multi-disciplinary research perspective, which is further clarified below, to establish requirements needed for a well-vetted and real-world applicable risk assess- ment methodology in eAI. We specify the importance of a multi-disciplinary approach as vital in order to capture computer scientific notions of AI as well as humanistic and social-scientific notions of ethics-based governance. This process was based on steps which have been visualized in Fig. 1, including organizations specified in Table 1, and experts specified in Table 2, whom participated in cross-sector expert exercises. Details are further specified in subsequent sections. 1 Introduction This investigation was initiated as an eAI landscape review of risks, and then followed by cross-sector expert discussions which provided categorizations of unintended root causes of risks, which we call pitfalls. These discussions also identified fundamentals, which must be enacted by organizations within the eAI domain, in order to prevent pitfalls. These results, and subsequent content anal- ysis, helped to formulate the requirements needed for a standardized eAI risk assessment methodology that is compatible with regulatory demands across a large variety of contexts. • Next, in Sect. 3, we propose a novel data-driven methodology as a means to ensure that human values and rights are sustained for data-driven AI applications based on these results. We then leverage the discovered results to present a data- driven, cross-functional methodology implemented in the real world as a means to help ensure human values and rights are sustained in the data-driven AI era. • In Sect. 4, an initial evaluation of the methodology implementation is provided in the context of two case studies that demonstrate the effectiveness and feasibil- ity of the methodology including its data-driven development through iterative improvements. • Finally, in Sect. 5, we demonstrate how repeated applications of the implementa- tion on use cases have guided refinements. In addition, we outline the general data strategy of our implementation to provide a better understanding of how group-level data can and should be leveraged to refine implementations and pro- vide insights. 1 3 Page 4 of 27 Digital Society (2022) 1:13 Digital Society Digital Society 13 Fig. 1 Multidisciplinary research approach leading to the development, implementation, and evaluation of the proposed DRESS-eAI methodology Fig. 1 Multidisciplinary research approach leading to the development, implementation, and evaluation of the proposed DRESS-eAI methodology 2.1 Organizational and Societal eAI Risk Identification To establish the needs of a risk assessment methodology for organizational eAI risks which pose a threat to human values and rights, a systemic research process was conducted. As a first step, a literature review was performed by Larsson et al. (2019). This review included an assessment both quantitative and qualitative of the literature on fairness, transparency, and accountability in AI, in order to exhaustively examine the eAI landscape while identifying and discussing societal eAI risks which do not cause intentional harm. The review helped form a spring- board for future expert exercises on the topic. One outcome of this study was 1 3 Digital Society Page 5 of 27 13 Table 1 Participating organizations in the cross-sector expert exercises Organization Sector Boston Consulting Group Cross-sector Cirio (Law firm) Legal City of Malmö Public City of Stockholm Public Civil Rights Defenders Human rights Ericsson Private tech Google Sweden Private tech Human Rights Watch Human rights Karolinska Institute Public research KTH Royal Institute of Technology Public research Microsoft Sweden Private tech Sana Labs (AI for individualized learning) Tech start-up Stockholm School of Economics Public research Swedish institute for Standards Cross-sector Swedish Tax Agency Public Södertörn University Public research Telia Private tech The Institute for Futures Studies Public research Table 1 Participating organizations in the cross-sector expert exercises Organization Sector Boston Consulting Group Cross-sector Cirio (Law firm) Legal City of Malmö Public City of Stockholm Public Civil Rights Defenders Human rights Ericsson Private tech Google Sweden Private tech Human Rights Watch Human rights Karolinska Institute Public research KTH Royal Institute of Technology Public research Microsoft Sweden Private tech Sana Labs (AI for individualized learning) Tech start-up Stockholm School of Economics Public research Swedish institute for Standards Cross-sector Swedish Tax Agency Public Södertörn University Public research Telia Private tech The Institute for Futures Studies Public research a realization that much of the eAI landscape places a strong focus on technical rather than organizational risks. This realization motivated the need for highlight- ing organizational risks with society as the primary stakeholder. a realization that much of the eAI landscape places a strong focus on technical rather than organizational risks. This realization motivated the need for highlight- ing organizational risks with society as the primary stakeholder. Through an initial evaluation and iterative expert reviews of the topic, a list of the most common and distinctive risks related to eAI was composed. 2.1 Organizational and Societal eAI Risk Identification This con- sisted of eight organizational risks, whose definitions are backed by global human rights legislation and other external analysis by (General Assembly, 1948; Meek et al., 2016), including the commonality of issues addressed across ethics guide- lines (Jobin et al., 2019). The eight identified organizational risks are as follows: • Privacy intrusion—AI and data-driven solutions interfering with personal or sensitive data without regarding consent of the individual or groups whose data is collected, how data is shared or stored, agreement of the law, or other legitimate needs to protect the best interests of an individual or groups (right to privacy) • Amplified discrimination—AI and data-driven solutions which cause, facili- tate, maintain, or increase prejudicial decisions or treatment and/or biases towards race, sex, or any other protected groups obliged to equal treatment (right to fair treatment) • Violation of autonomy and independent decision making—AI and data- driven solutions which intentionally or unintentionally, and without consent, facilitate behavioral changes that manipulate independent decision making and social well-being (right to autonomy) 1 3 Page 6 of 27 Digital Society (2022) 1:13 13 Expert role No. • How should we define thematic categories as root causes of eAI risks in an organizational and societal context? • How should we define thematic categories as root causes of eAI risks in a How should we define thematic categories as root causes of eAI risks in an • How should we define thematic categories as root causes of eAI risks in an organizational and societal context? • How should we define thematic categories as root causes of eAI risks in an organizational and societal context? i organizational and societal context? • How can these risks be mitigated from broad society- and organizational-based perspectives? • How can these risks be mitigated from broad society- and organizational-based perspectives? Cross-sector exercises were conducted as part of an initiative together with the Swedish Innovation Agency (Vinnova), a government agency that administers state funding for research and development. Starting in 2018, participants were gathered to perform a series of exercises in Stockholm, Sweden which involved cross-sector experts in the domains such as civil society, public/private sector, tech start-ups, and policy. This included organizations spanning legal, technical, business, communica- tion, and sustainability/CSR from public and private domains, along with organiza- tions associated with the Stockholm, Sweden-based AI Sustainability Center startup. See Tables 1 and 2 for a full list of these organizations and the expert roles. Exer- cises were performed with the listed organizations, over the course of several years, consisting of round-table discussions, panel discussions, seminars, and joint analy- ses of ethical AI topics. Following these exercises, a content analysis of notes taken was performed by principle researchers to further refine and validate thematic con- tent which emerged. Such notes were subsequently shared with a total of 20 inde- pendent experts groups from Table 1 to provide independent perspectives and analy- sis on thematic content. This feedback was also integrated into the content analysis to validate and refine definitions surrounding the emergent themes. Evidence contin- ues to be collected through use cases to ensure identified categories are exhaustive of the eAI risk landscape from societal and organization perspectives. This selection was based on a need to form a multidisciplinary organizational perspective on societal, ethical, and legal considerations towards the eAI risk land- scape. The goal of exercises performed was to reflect on how the identified eAI risks would appear in each of these domains and identify which thematic categories form the root cause of each of these risks across all domains. 2.1 Organizational and Societal eAI Risk Identification of experts Policy area specialist 3 Regulator 2 Ethics philosopher 4 Standardization practitioner 4 Legal practitioner 10 Legal researcher 4 Policy think tank member 5 Human rights advocate 3 Human rights lawyer 2 Gender studies researcher 2 Government legal expert 3 Digital law expert 4 Tech company public affairs spokesperson 1 Data scientist 5 Economist 2 Global studies expert 1 Children’s rights advocate 1 AI startup founder 4 Municipal chief digital officer 3 EU law expert 2 Consultancy firm partner 4 Health technology researcher 2 Clinician 1 Machine learning researcher 10 Machine learning expert 5 Member of Swedish parliament 2 Journalist 2 Board representative from cross-industry multinational company 11 Data collection expert 1 Table 2 Description of experts from participating organizations in cross-sector exercises • Social exclusion and segregation—AI and data-driven solutions contributing to or maintaining an unfair denial of resources, rights, goods, and ability to participate in normal relationships and activities, whether in economic, social, cultural, organizational, or political arenas (right to inclusion) • Social exclusion and segregation—AI and data-driven solutions contributing to or maintaining an unfair denial of resources, rights, goods, and ability to participate in normal relationships and activities, whether in economic, social, cultural, organizational, or political arenas (right to inclusion) • Harm to safety—AI and data-driven solutions facilitating unwanted physi- cal harms to an individual or organization stemming from underdeveloped AI, and attributed to negligence from an organization (right to physical safety) • Harm to security of information—AI and data-driven solutions facilitating potential damage from unauthorized access of private data, due to faulty data protection and processing, or criminal activity (right to security of informa- tion) 1 3 3 Digital Society (2022) 1:13 Digital Society Page 7 of 27 13 • Misinformation and disinformation—AI and data-driven solutions which intentionally or unintentionally distribute information that is regarded as false and harmful to society (right to be informed) • Misinformation and disinformation—AI and data-driven solutions which intentionally or unintentionally distribute information that is regarded as false and harmful to society (right to be informed) • Misinformation and disinformation—AI and data-driven solutions which intentionally or unintentionally distribute information that is regarded as false and harmful to society (right to be informed) • Prevention of access to public service—AI and data-driven solutions contrib- uting to or maintaining a denial of public social assistance and service (right to public service access) • Prevention of access to public service—AI and data-driven solutions contrib- uting to or maintaining a denial of public social assistance and service (right to public service access) 2.2 Identification of Thematic Organizational Risk Source Categories (Pitfalls) The output of the initial eAI landscape review formed the basis for a series of cross- sector expert-based exercises in which thematic categories of root causes of eAI risks and in an organizational context were identified. The aims of the exercises were to answer the following questions which arose from the landscape review: • How should we define thematic categories as root causes of eAI risks in an organizational and societal context? Through this process, the experts reached a consensus on four common themes, which we refer to as pitfalls. 1 3 3 3 Digital Society (2022) 1:13 Page 8 of 27 13 Notes taken during these exercises were used in the previously specified content analysis approach to concretely narrow down and define the pitfall themes which emerged. These four pitfalls are: • Misuse/overuse of data—The AI application/solution could be overly intrusive, using private data, or it could be used for unintended purposes by others. This can include misinterpretations of primary users regarding implementation or deployment the AI application/solution.1 • Bias of the creator—Values and bias are intentionally or unintentionally pro- grammed by the creator who may also lack knowledge/skills of how the solution could scale in a broader context.2fi • Bias of the creator—Values and bias are intentionally or unintentionally pro- grammed by the creator who may also lack knowledge/skills of how the solution could scale in a broader context.2fi • Immature data and AI—Insufficient training of algorithms on datasets as well as lack of representative data could lead to incorrect and unethical recommenda- tions.3l • Immature data and AI—Insufficient training of algorithms on datasets as well as lack of representative data could lead to incorrect and unethical recommenda- tions.3l • Data bias—The data available is not an accurate reflection of reality or the pre- ferred reality and may lead to incorrect and unethical recommendations.4 • Data bias—The data available is not an accurate reflection of reality or the pre- ferred reality and may lead to incorrect and unethical recommendations.4 1 Further discussion on misuse/overuse of data: Brundage et al. (2018); Larsson (2021) 2 Further discussion on bias of the creator: Whittaker et al. (2019); Noble (2018) 3 Immature data and AI examples: Buolamwini and Gebru (2018); Shankar et al. (2017); Larsson (2019) 4 Data bias examples: Buolamwini and Gebru (2018); Shankar et al. (2017) 4 Data bias examples: Buolamwini and Gebru (2018); Shankar et al. (2017) 3 Immature data and AI examples: Buolamwini and Gebru (2018); Shankar et p ( ) ( ) ( 4 Data bias examples: Buolamwini and Gebru (2018); Shankar et al. (2017) 2.3 Identification of Thematic Organizational Ethical Requirements (Fundamentals) The next step of in the series of exercises was to discuss thematic categories for how to prevent and overcome such pitfalls in an organizational context. The results of this were the establishment of organization structural eAI foundations as thematic categories. Through this process, the experts identified four common themes, which we refer to as fundamentals, and are also echoed in much recent principled work on AI (Jobin et al., 2019). These 4 fundamentals consisted of: • Accountability—The need to stand accountable and justify one’s decisions an actions to its partners, users, and others with whom the system interacts. • Accountability—The need to stand accountable and justify one’s decisions and actions to its partners, users, and others with whom the system interacts. • Governance—Establishment of policies, principles, and/or protocols, and con- tinuous monitoring of their proper implementations. • Governance—Establishment of policies, principles, and/or protocols, and con- tinuous monitoring of their proper implementations. • Explainability—Ensure that algorithmic decisions, as well as any data driv- ing those decisions, can be explained to and understood by end users and other stakeholders using nontechnical terms. Explainability demands must meet a con- textually appropriate level to establish trust across stakeholders. • Explainability—Ensure that algorithmic decisions, as well as any data driv- ing those decisions, can be explained to and understood by end users and other stakeholders using nontechnical terms. Explainability demands must meet a con- textually appropriate level to establish trust across stakeholders. • Transparency—It must be possible to discover, trace, and detect how and why a system made a particular decision or acted in a certain way, and, if a system causes harm, to discover the root cause. Transparency demands must meet a con- textually appropriate level across entire systems to establish stakeholder trust. • Transparency—It must be possible to discover, trace, and detect how and why a system made a particular decision or acted in a certain way, and, if a system causes harm, to discover the root cause. Transparency demands must meet a con- textually appropriate level across entire systems to establish stakeholder trust. 1 3 1 3 Digital Society (2022) 1:13 Page 9 of 27 13 It was discovered through expert-based exercises and subsequent content analy- sis of notes and independent feedback that these categories have to be addressed as minimum requirements for any organization wishing to achieve eAI. 2.3 Identification of Thematic Organizational Ethical Requirements (Fundamentals) In addition, it was clear from within these discussions that meeting such requirements means that cross-functional considerations between roles must be taken into account from organizational levels to technical systems levels. Again, evidence to better define thematic content continues to be collected through use cases and further exercises to ensure these categories are exhaustive of the eAI risk landscape from societal and organization perspectives. 5 https://aisustainability.org/the-code/ 6 https://ec.europa.eu/info/sites/default/files/commission-white-paper-artificial-intelligence-feb2020_en. pdf 5 https://aisustainability.org/the-code/ 6i 3 Proposal of an eAI Risk Assessment Methodology As a realization of our results and to answer the question posed by this paper, we propose the following methodology, entitled the Data-driven Risk Assessment Meth- odology for Ethical AI (DRESS-eAI). DRESS-eAI is designed to focus on the detec- tion of pitfalls and enact the fundamentals relevant to most eAI use cases while being structured as a process that is familiar to organizations as it is comparable to the International Organization for Standardization (ISO) standard 31000:2009 for risk management (Purdy, 2010). This is an accepted standard for risk management developed by hundreds of risk management professionals over the course of four years, and has previously seen utilization within sustainability-focused methodology (Tiganoaia et al., 2019). The six process phases of the methodology are inspired directly by the ISO 31000:2009 risk management process with each phase being identified as a necessary step for systematically ensuring rigorous eAI practices of an organization. In addition, our aim has been to make the DRESS-eAI methodology compatible with any phase of an AI systems life cycle, while being fully compatible with a recent Declaration of eAI5. This declaration was issued as a response to aid organizations in preparing for the upcoming AI regulation recently proposed by the European Commission6. The declaration can be fulfilled directly through applying DRESS-eAI to achieve fundamentals as minimum requirements and overcome pit- falls which are root causes of eAI risks. We envision DRESS-eAI as a formative step towards establishing the requested common normative standards for high-risk AI solutions which may pose a risk to health, safety, and fundamental rights. Lack of cross-functional teams tackling eAI is a thematic issue that emerged within panel discussion exercises. To accommodate for this, we advocate that all these roles/functions spanning technical, legal, risk, compliance, commu- nications, CSR/sustainability, and HR are part of the process. Secondly, due to the specific individual risks of eAI projects, we acknowledge their relevancy in detection and mitigation as a core part of this framework. Due to eAI risk land- scape facing ongoing changes, we also acknowledge the need for DRESS-eAI and 1 3 Digital Society (2022) 1:13 Digital Society ( Page 10 of 27 Digital Society 13 Fig. 2 An overview of DRESS-eAI. The main use-case process phases are shown in the center. Linkages show the conceptual flow between core concepts and how collected data can be used to construct and refine implementations of the phases. 1 3 3 Proposal of an eAI Risk Assessment Methodology Also shown is how data outputs of use cases and external analyses are recorded in a database and used for generating insights and the iterative refining of existing catego- ries and internal processes Fig. 2 An overview of DRESS-eAI. The main use-case process phases are shown in the center. Linkages show the conceptual flow between core concepts and how collected data can be used to construct and refine implementations of the phases. Also shown is how data outputs of use cases and external analyses are recorded in a database and used for generating insights and the iterative refining of existing catego- ries and internal processes its implementations to be evolving through data-driven projects where generated structured data from use cases and external analyses are recorded and used for iterative refining of existing categories and internal processes. This will permit implementations of the methodology to remain up-to-date alongside performing as an analytical tool. Thus, when highlighting the methodology as being data- driven, this refers to the inclusion of a central database to store structured data generated from the different phases which can later be used to provide data-driven insights and refine implementations. Figure 2 provides a complete overview of the proposed methodology. We break the general process of the methodology into the following process phases for each eAI risk assessment use case. Such stages were based primarily on the ISO 31000:2009 risk management process: • Phase A: Problem definition/use case scoping—Establishing a use-case defi- nition including summary of challenges and identifying the project team. This should achieve a detailed description of the use case, including understanding of guiding policies/codes/values, key stakeholders, and technical specification. • Phase A: Problem definition/use case scoping—Establishing a use-case defi- nition including summary of challenges and identifying the project team. This should achieve a detailed description of the use case, including understanding of guiding policies/codes/values, key stakeholders, and technical specification. 1 3 1 3 Digital Society (2022) 1:13 Digital Society Page 11 of 27 13 • Phase B: Risk scanning/profiling—Capturing structured data related to the cur- rent state of achievement towards fundamentals and vulnerability towards pitfalls with data collection form multiple organization roles. 3 Proposal of an eAI Risk Assessment Methodology In addition, if a screening is performed from multiple perspectives, this phase can provide a gap analysis which is an indication how well a specific use case is conforming to the organi- zational standards.i • Phase B: Risk scanning/profiling—Capturing structured data related to the cur- rent state of achievement towards fundamentals and vulnerability towards pitfalls with data collection form multiple organization roles. In addition, if a screening is performed from multiple perspectives, this phase can provide a gap analysis which is an indication how well a specific use case is conforming to the organi- zational standards.i • Phase C: Risk assessment—Identification, evaluation, and prioritization of risk scenarios. For example, workshops should be used with a cross-functional team to identify ethical risk scenarios and what/which stakeholders could be impacted based on the risk exposure to pitfalls and fundamentals.i • Phase D: Risk mitigation measures—Identification of technical and non-technical mitigation measures and assigning ownership for actions. Identify risk mitigation measures that target the root cause of a risk scenario, or its effect. Plan for monitor- ing implementation of mitigating measures. • Phase E: Stakeholder engagement—Capturing stakeholder feedback. A neces- sary validation step for identified risk mitigation activities; focusing on those affected by the organization’s identified risk mitigation activities and what should be done to manage actual and potential impacts. • Phase F: Review and maintain—Conclusions from the completion of each phase and recommendations going forward. • Phase F: Review and maintain—Conclusions from the completion of each phase and recommendations going forward. 3.1 Implementation of DRESS‑eAI Finally, a use-case scoping survey is administered to capture a description of the AI solution that is to be assessed.i • In phase B: Risk scanning/profiling—We cover the eAI risk landscape with an exhaustive risk scan survey of over 150 questions tagged to and equally bal- anced according to relevant fundamental, pitfalls, and organizational role. These questions emerged as part of the same expert-based iterative process to exhaustively, and in a balanced manner, establish where an organization lies in the eAI risk landscape. This entails that each pitfall and fundamental is treated with equal priority in order to appropriately cover exposure to eAI pitfalls in the technically, legally, and societal defined risk landscape. Conceptually speaking, pitfalls may overlap with each other. However, for simplicity with our imple- mentation, each phase B question is tagged to a single pitfall. The decision of how to tag each question to a pitfall corresponded to which point in a AI’s life cycle the question was most associated to. Figure 3 provides an overview of how each pitfall was connected to the AI life cycle for the purposes of tagging questions. Tagging of fundamentals and roles were not associated to the AI life cycle; however, as stated, efforts were made to ensure that appropriate combi- nations of taggings were included to comprehensively cover the eAI risk land- scape. Questions can be answered by each role with four options: “yes,” “in- progress,” “not sure,” and “no.” We emphasize the role-based structuring of the questions to ensure the validity and comprehensiveness of answers, in addition to activating cross-functional cooperation across the organization. These roles include technical, legal, risk, compliance, communications, CSR/sustainability, business owner, and HR. See Table 3 for examples of these questions and their tagging structure, and Fig. 4 for an example summary report. Importantly, all structured data from this stage is captured in our database and used to produce group-level insights which can verify the ability of this phase to exhaustively cover the risk landscape, along with using outputs from this phase to provide insights within other phases. The output of this phase can be used to provide a gap analysis which is specified further in Sect. 3.2. 3.1 Implementation of DRESS‑eAI The above structure outlines and defines a generalized methodology for risk assess- ment within eAI. To explain how such a process can be enacted in the real world, we explain our implementation which has been applied and refined in relation to the case studies under examination. The chosen implementation relies on collecting structured data through cross- functional self-assessment surveys. It is important to note that the chosen imple- mentation may be prone to closed feedback loops, which can erroneously verify its own effectiveness and introduce data bias due to survey responses not reflect- ing true reality. As such, the implementation also collects and records qualitative feedback on the implementation directly through organizational stakeholders, per- mitting a deeper understanding of the implementation validity, rather than only rely- ing on quantitative evidence acquired through repeated surveys which may possess respondent errors. • In phase A: Problem definition/use case scoping—We perform workshops for the identification and detailing of an appropriate eAI use case. The outcome is a use- case definition including summary of challenges and detailed use-case descrip- tion based on a pre-defined template. To leverage the data-driven nature of the methodology, we administer three structured surveys to capture data which can later be leveraged for data-driven group-level insights between phases. Firstly, an organizational survey to capture general questions such as the organization’s size and domain. We also administer an organizational maturity survey to screen • In phase A: Problem definition/use case scoping—We perform workshops for the identification and detailing of an appropriate eAI use case. The outcome is a use- case definition including summary of challenges and detailed use-case descrip- tion based on a pre-defined template. To leverage the data-driven nature of the methodology, we administer three structured surveys to capture data which can later be leveraged for data-driven group-level insights between phases. Firstly, an organizational survey to capture general questions such as the organization’s size and domain. We also administer an organizational maturity survey to screen 1 3 3 Page 12 of 27 Digital Society (2022) 1:13 Digital Society 13 for the organizations preparedness for ethically high-risk AIs. Finally, a use-case scoping survey is administered to capture a description of the AI solution that is to be assessed.i for the organizations preparedness for ethically high-risk AIs. 3.1 Implementation of DRESS‑eAI i • In phase C: Risk assessment—We identify and characterize risk scenarios guided by information acquired in phase B, constructing a traditional heat map of risk scenarios to aid in prioritizing risk mitigation procedures on organiza- tional and use-case levels. Each risk scenario is tagged to a fundamental and a pitfall as well as one or more of the eight identified risks from Sect. 2. After a sufficient data collection period, we exploit our acquired database of risk scan surveys and risk scenarios to aid in the data-driven insight generation within and across phases. Risk scenarios are prioritized based on a qualitative analysis of likelihood and severity. Prioritized risk scenarios are characterized further, with input from additional interviews and focus meetings with the client if needed. • In phase D: Risk mitigation measures—Risk mitigation tools and recommenda- tions are determined which can be technical or non-technical. We identify risk owners for the prioritized risk scenarios, either taken in the project or identified improvements needed. We provide risk mitigation from both organization and use-case levels based on evidence acquired during evaluation. Each mitigation 1 3 1 3 Digital Society (2022) 1:13 Page 13 of 27 13 Digital Society 13 Table 3 Selected examples of eAI risk scanning questions demonstrating the tagging structure of fundamentals, pitfall, and cross-functional organizations roles Risk scanning question examples Question Fundamental Pitfall Organization role Have you tested model results for fairness with respect to different affected groups (e.g., tested for disparate error rates)? Governance Data bias Technical Have you defined what human bias means in the context of the solution and with regards to your organizations values or policies? Explainability Bias of the creator CR/CSR Are you confident in your organization’s ability to detect, then shut down a malfunctioning solution(s) in a timely manner, i.e., before any harm to people or society is caused? Governance Misuse/overuse Business owner Are the explanations that you provide about your solution easily accessible and in clear terms to external parties? Transparency Misuse/overuse Communications Do you have a person/function who is responsible for deciding when the algorithm(s) in the solution are mature enough/market ready? 3.1 Implementation of DRESS‑eAI Accountability Immature data/AI Technical Table 3 Selected examples of eAI risk scanning questions demonstrating the tagging structure of fundamentals, pitfall, and cross-functional organizations roles Risk scanning question examples Question Fundamental Pitfall Organization role Have you tested model results for fairness with respect to different affected groups (e.g., tested for disparate error rates)? Governance Data bias Technical Have you defined what human bias means in the context of the solution and with regards to your organizations values or policies? Explainability Bias of the creator CR/CSR Are you confident in your organization’s ability to detect, then shut down a malfunctioning solution(s) in a timely manner, i.e., before any harm to people or society is caused? Governance Misuse/overuse Business owner Are the explanations that you provide about your solution easily accessible and in clear terms to external parties? Transparency Misuse/overuse Communications Do you have a person/function who is responsible for deciding when the algorithm(s) in the solution are mature enough/market ready? Accountability Immature data/AI Technical 1 3 Digital Society (2022) 1 Page 14 of 27 Digital Society 13 Fig. 3 Demonstrating how the pitfalls were mapped along the AI life cycle to provide mutually exclusive categorization of phase B risk scan questions for the DRESS-eAI implementation. The stages shown rep- resent universally standard steps taken by organizational teams in developing and deploying AI solutions Fig. 3 Demonstrating how the pitfalls were mapped along the AI life cycle to provide mutually exclusive categorization of phase B risk scan questions for the DRESS-eAI implementation. The stages shown rep- resent universally standard steps taken by organizational teams in developing and deploying AI solutions measure is also tagged to a fundamental and pitfall as well as one or more of the eight identified risks from Sect. 2. After a sufficient data collection period, we leverage our database to provide data-driven recommendations and insights generation surrounding risk mitigation activities. Technical or non-technical risk-mitigating measures are identified and implemented in broader risk manage- ment/existing processes and assigned risk owners. Examples of risk mitigation measures: updated legal documents and processes, synthetic data for avoiding bias or to preserve privacy, tailored explainability models, training, establish- ing AI Ethical Principles or establishing an AI Ethical Board. Risk owners are identified within the organization and a plan is created for implementation and follow-up of actions. 3.1 Implementation of DRESS‑eAI • In phase E: Stakeholder engagement—we provide a summary of issues and rec- ommendations on the topic of risk mitigation, and how these can be addressed and enacted through stakeholder engagements. Steps include identifying and prioritizing stakeholders to engage with; deciding what type of input is needed • In phase E: Stakeholder engagement—we provide a summary of issues and rec- ommendations on the topic of risk mitigation, and how these can be addressed and enacted through stakeholder engagements. Steps include identifying and prioritizing stakeholders to engage with; deciding what type of input is needed Fig. 4 Example overview of use-case output for phase B categorized by fundamentals Fig. 4 Example overview of use-case output for phase B categorized by fundamentals Digital Society (2022) 1:13 Page 15 of 27 13 and whether to use existing stakeholder engagement forums /channels or tar- geted activities; collecting and analyzing information on key topics and problems addressed by stakeholders; stakeholder feedback captured in workshops and ana- lyzed in a report summarizing the activities and concerns raised; and potentially creating a modified risk mitigation plan to address feedback.i and whether to use existing stakeholder engagement forums /channels or tar- geted activities; collecting and analyzing information on key topics and problems addressed by stakeholders; stakeholder feedback captured in workshops and ana- lyzed in a report summarizing the activities and concerns raised; and potentially creating a modified risk mitigation plan to address feedback.i i • In phase F: Review and maintain—we report a summary of findings from apply- ing the DRESS-eAI implementation. An updated risk scan of the use case is conducted in order to track the effectiveness of risk mitigation activities taken over time. We also provide recommendations on how internal frameworks can be strengthened. Qualitative feedback on the implementation’s true impact is acquired. i • In phase F: Review and maintain—we report a summary of findings from apply- ing the DRESS-eAI implementation. An updated risk scan of the use case is conducted in order to track the effectiveness of risk mitigation activities taken over time. We also provide recommendations on how internal frameworks can be strengthened. Qualitative feedback on the implementation’s true impact is acquired. 3.2 Gap Analysis eAI principles and commitments made by organizations are often high level, and analyses are needed to ensure a minimization of gaps between higher aspirations and what is actually happening on product and developer levels (Mittelstadt, 2019). Such principles ultimately have little effect on practices if they are not directly tied to structures of accountability, incentives, and the ways of working in an organiza- tion. AI principles, codes, and guidelines also need to be combined with monitoring of their implementation, as well as consequences if they are not met. The phase B risk scanning survey output can be further used as a tool to identify possible gaps between stated ethical principles and higher aspiration and what might be happening on product or organizational level. For our DRESS-eAI implemen- tation, we also map organizational AI principles directly to risk scanning question results to facilitate the gap analysis described in Sect. 3.2. A general example of this output can be seen in Fig. 5. 4 Evaluation and Iterative Evolution of DRESS‑eAI Implementation As described, the DRESS-eAI risk assessment methodology has been structured to follow a data-driven iterative approach for refining implemented processes and concepts. We have implemented and tested this to our knowledge unique method- ology for assessing AI which is compatible to typical organizational structure and usable at any point in the life-cycle of an AI-system. We propose that any implemen- tation of our methodology should not remain a static snapshot, but a data-driven, iteratively evolving system, capturing information from each use-case for insights into the developing eAI landscape and for refining DRESS-eAI methodological implementations. In this section we outline the application of the DRESS-eAI methodology to two real organizational case studies, reporting the effectiveness of the current imple- mentation’s ability to detect and mitigate risk, along with reporting the data-driven 1 3 Page 16 of 27 Digital Society (2022) 1:13 Digital Society 13 Fig. 5 Example output of a gap analysis, showing how an organization’s AI principles are being achieved according to mappings to relevant DRESS-eAI’s use-case risk scan questions Fig. 5 Example output of a gap analysis, showing how an organization’s AI principles are being achieve according to mappings to relevant DRESS-eAI’s use-case risk scan questions evolution of our implementation dictated by quantitative and qualitative evidence acquired from each case study. 4.1.2 Outcomes After applying DRESS-eAI phases A, B, and C, it was identified that this use case was exposed to several ethical and societal risks. These mainly pertained to the pitfalls Bias of the creator and Data bias. Weaknesses in accountability and AI governance were identified. Risk scenarios were identified and nine of them were likely to occur and could result in severe impacts on people and society. These risk scenarios were prioritized for mitigation. Mitigating measures and risk own- ers were identified for each risk scenario identified. The mitigating actions taken in the project altered the solution to prevent misuse, such as updating UX interface to case workers to prevent misuse of model outputs; creating and communicating pur- pose statements to various stakeholders; and implementing methods for explaining model outputs and defining what needs to be explained and for whom (based on current and proposed future regulation). The effect is that the solution could then be scaled. Furthermore, applying DRESS-eAI highlighted the need for better AI governance broadly across the organization. One key finding is a lack of ownership of an ethical AI framework internally. Following this, a cross-functional group of internal stakeholders has now been initiated as a permanent ethical AI group, with the responsibility of sup- porting developers of AI solutions and advancing the organizations ethical AI maturity. In addition, it is highlighted that an approach for AI fairness and explainability was needed in order to serve future AI solutions. 4.1.1 Description This use case revolves around an AI system being used to classify job seekers based on personal data pertaining to job hiring, education, and language proficiency, as well as data about the condition and functioning of the labor market. This has, in other studies, been shown to be an area with inherent risks (Lopez, 2021). The data used for training input was generated from various sources. For new job seekers, a self-assessment survey was answered, and personal data was generated. For job seekers already known, data was gathered from a data lake where existing data about the job seeker was stored. Job seekers are then profiled using a deep learning model on 64 features. The output of the model was a prediction of how far from the labor market a job seeker is. Based on the outcome, job seekers are placed into three categories based on a rule-based selection. A human case worker would also be able to change the category 1 1 3 3 Digital Society (2022) 1:13 Page 17 of 27 Digital Society 13 a job seeker is placed in. Primary early concerns were raised regarding the risk of dis- criminating against sensitive groups, such as foreign-born women. a job seeker is placed in. Primary early concerns were raised regarding the risk of dis- criminating against sensitive groups, such as foreign-born women. 4.2.3 Input into Evolution of Implementation • Two risk scannings were required, with one on the organization level and one on the standard use-case level. This leads to the inclusion of the phase A organizational survey to aid in streamlining the implementation. g y g p • To foster better understanding around the terminology, a clearer distinction was made between transparency and explainability. • To foster better understanding around the terminology, a clearer distinction was made between transparency and explainability. • Validation that DRESS-eAI can be applied to a planning stage use case. • Validation that DRESS-eAI can be applied to a planning stage use case. 4.2.1 Description This use case began in an idea life cycle stage, where an AI system was used to monitor and select transactions on third-party marketplace platforms that should be reviewed for potential tax fraud. The AI system would be implemented in the own environment of third-party platforms. The AI system used information about the individual and the transaction as features for classification. 4.2.2 Outcomes Applying DRESS-eAI identified which types of eAI risks can occur when using AI to detect tax fraud, leading to an increased understanding and awareness of how prepared the organization was to handle such risks. Several eAI risk aspects were highlighted including a lack of clear organisational strategy for eAI; a lack of a sys- tematic approach to detect and handle ethical AI risks; lack of accountability for ethical AI risks; an inability to monitor AI systems; a large exposure to the pitfalls “Data bias” and “Bias of the creator”; a need to instate a central steering committee for overseeing eAI operations; and a need for competence development. Mitigating actions were then performed, resulting in the organization, a year later, having an established eAI policy and plan to establish an eAI steering committee. 4.1.3 Input into Evolution of Implementation • A need for separate use case and organizational risk mitigation was identified. i • Distinction added between risk mitigation measures that can be taken in the proje as opposed to what needs to change in the line organization. i • Distinction added between risk mitigation measures that can be taken in the project as opposed to what needs to change in the line organization. • When implementing DRESS-eAI, the need to involve representatives from busi- ness operations was identified. A role was then added to the survey, entitled business owner. It was found that when an AI system is part of a larger organi- zational process such as in this project, many risks are associated with lack of effective collaboration and/or instructions on how to use the AI system by the business unit. Specifying a business owner role enabled further cross-functional collaboration in identifying potential risk exposure and taking effective mitiga- tion actions. • Validation that DRESS-eAI can be applied to identify and mitigate eAI risk for a use case in the development life-cycle phase. • Validation that DRESS-eAI can be applied to identify and mitigate eAI risk for a use case in the development life-cycle phase. 1 3 Digital Society (2022) 1:13 Digital Society (2022) 1:13 Page 18 of 27 Digital Society (2022) 1:13 13 5.1 Refinement of Survey Tool Through Use‑Case Insights Any implementation of DRESS-eAI will demand the refinement of process tools to better accommodate for organizational needs and the developing eAI landscape. For our implementation, we recorded common feedback acquired during use cases in our database and made refinements based on group-level evidence. To help dem- onstrate this process, we present representative examples of how repeated use cases of our implementation resulted in the refinements of our data collection process. A complete overview of these examples can be viewed in Table 4.i In the first example, we examine a question pertaining to the data bias pitfall and governance fundamental. The question pertains to the needs of building a solution on the same data distributions in which it will be deployed. This is to help ensure it is not simply well fit to a training dataset and then underperforms on unfamiliar data examples in the real world. The original question was reported as incorrectly captur- ing the intention behind the question due to ambiguity. Since a technical role was intended to answer such a question, more detailed terminology about datasets and statistical distributions was included. In general, more exact terms for technical roles questions were added across questions. In the second example, an additional question was added to better capture the product owner’s input on whether or not they oversee the compatibility of the solu- tion to their organization’s values. This was part of the general refinement of the the risk scan to have the organizational product owner more involved in the risk scanning. This inclusion was noted as being crucial as product owners tended to understand the intended use and value of the solution more than other roles. To bet- ter identify potential vulnerabilities, more questions were added to have the product owner role as a larger part within the risk scan process, specifically asking them more organizationally related questions surrounding the governance and account- ability pitfalls. In the third example, it was reported that the original question could lead to incorrect responses due to ambiguities. The intention of the original question was to establish from the product owner whether or not they possess a general open data collection strategy; a lack of which could lead to insufficient data in terms of quality. However, the original wording of the question lead to misunderstandings that the question related to communicating human biases for selecting data. 5 Leveraging Group‑Level Results—Refinement of Data Collection Tool, Data Strategy, and Insights In this section, we examine the the data-driven aspect of the DRESS-eAI from two perspectives. Firstly, we demonstrate how repeated applications of use cases have guided the refinement of our implementation. Secondly, we outline the gen- eral data strategy of our implementation to provide a better understanding of how group-level data can and should be leveraged to refine implementations and pro- vide eAI insights both within and across DRESS-eAI phases. 1 3 Digital Society (2022) 1:13 Page 19 of 27 13 13 Table 4 Selected examples of eAI risk scanning questions showing the iterative evolution using case study evidence Risk Scanning Question Example 5 Version 1 Question Fundamental Pitfall Organization Role Do you have a diversity policy and procedures to ensure diversity in your organization? Governance Bias of the creator HR Do you have processes/approaches in place to ensure that there is diversity within your pool of designers and managers involved in the creation of the solution in terms of gender, culture, age, etc.? Governance Bias of the creator HR Updated Risk Scanning Question Example 5 Question Fundamental Pitfall Organization Role - (Question removed due to overlap with question below) Governance Bias of the creator HR Do you have processes/approaches in place to ensure that there is diversity within your pool of designers and managers involved in the creation of the solution in terms of gender, culture, age, etc.? Governance Bias of the creator HR 1 3 Page 20 of 27 Digital Society (2022) 1:13 Digital Society (2022) 1:13 Digital Society 13 5.1 Refinement of Survey Tool Through Use‑Case Insights This update rep- resents an example in which ambiguities in the questions were removed. In the fourth example, it was noted that the question was both ambiguous and interpreted incorrectly by respondents. The intention of the question was to high- light a data bias vulnerability due to having automated data bias processes. This could lead to data bias due to a lack of human oversight. The original wording of the question did not make this intention clear as thus lead to incorrect responses. Such a question is representative of similar questions that needed to be rephrased.i In the fifth example, we highlight a general case in which redundant questions needed to be removed. For the questions shown we noted from feedback that the general organisation question was sufficiently covered by a similar question and thus could be removed. 1 3 3 Page 21 of 27 13 Page 21 of 27 13 1 Risk Scanning Question Example 1 Version 1 Question Fundamental Pitfall Organization Role Is the distribution of demographic groups in your dataset representative of the reality you are trying to reflect? Governance Data bias TECH Updated Risk Scanning Question Example 1 Question Fundamental Pitfall Organization Role Is the distribution of demographic groups in your dataset representative of the distribution present in the population(s) where your solution is/are deployed Governance Data bias TECH Risk Scanning Question Example 2 Version 1 Question Fundamental Pitfall Organization Role - (Question needed to be added) - - - Updated Risk Scanning Question Example 2 Question Fundamental Pitfall Organization Role Are you as product owner involved in the design, development, auditing etc. of the solution to ensure that the solution conforms to your organizationial values Accountability Misuse/Overuse Product owner Risk Scanning Question Example 3 Version 1 Question Fundamental Pitfall Organization Role Do you communicate to relevant stakeholders about on what biases and values your data was selected and processed? Transparency Data bias Product owner Updated Risk Scanning Question Example 3 Question Fundamental Pitfall Organization Role Do you communicate to relevant stakeholders about on what grounds the data was selected and processed? Transparency Data bias Product owner Risk Scanning Question Example 4 Version 1 Question Fundamental Pitfall Organization Role Do you have an automated process for data validation? Governance Data bias TECH Updated Risk Scanning Question Example 4 Question Fundamental Pitfall Organization Role Do you have an automated process/approach with human oversight for data validation? 5.2 Data Strategy and Insights Importantly, we wish to highlight the relevancy for this methodology to be data- driven by having an underlying database capable of storing structured information from each use case to acquire group-level insights. Utilizing this data-driven back- bone of the methodology permits refinements in terms of efficiency, effectiveness, and deployability across a variety of contexts. We also model DRESS-eAI as a pro- cess that is continually refined though complementary external input such as new regulation, trends, and research on assessment methodology. Most importantly, the EU proposal for an AI Act is very likely to greatly impact European markets, stress- ing the need for these types of assessment. The data-driven backbone permits con- tinuous adaption of implementations to the changing eAI landscape. In practical terms, insights acquired from group-level data are exploited to improve each implementation in the following manner: • Providing summary reports on the general effectiveness of an implementation, and the state of the eAI landscape. • Permitting the benchmarking of eAI organizational status on per-sector and cross-sector levels. • Acquiring greater contextual information with less time burden on clients through personalized questionsi • Identifying deficiencies with existing surveys or tools. i • Developing internal and client dashboards and PR reports With the DRESS-eAI database, data insights can be acquired though the independ- ent analysis of each implementation phase. Of equal importance is the potential to understand how data from each phase is connected. Within the implementation applied for this study, we utilize a general approach for mapping phases together which can provide a structure for acquiring informative results though the means of statistical analysis and AI modeling. For our implementation, we build data rela- tions across various phases through common attributes for each output data table. More specifically, we achieve such relations by tagging all questions in phase B, risk scenarios in phase C, and mitigation measures in phase D with attributes of their respective pitfalls and fundamentals. Phase C risk scenarios and phase D risk miti- gation activities are also tagged to the eight risk categories identified and defined as part of this study. See Sect. 2 for an overview of the eight risks. 5.1 Refinement of Survey Tool Through Use‑Case Insights Governance Data bias TECH 3 Page 22 of 27 Digital Society (2022) 1:13 Digital Society (2022) 1:13 (2022) 1:13 Digital Society (2022) 1:13 13 5.3 Comparing DRESS‑eAI to Other Frameworks IEEE’s newly released IEEE Standard Model Process for Addressing Ethi- cal Concerns during System Design (IEEE 7000-2021) (IEEE, 2021) addresses a set of processes by which organizations can include consideration of ethical val- ues throughout the stages of concept exploration and development is established by this standard IEEE 7000-2021 supports organizations managers and engineers 1 3 Digital Society (2022) 1:13 Page 23 of 27 13 in transparent communication with selected stakeholders to look into ethical val- ues elicitation and prioritization. This involves traceability of ethical values through an operational concept, value propositions, and value dispositions in the system design. The standard is relevant to all sizes and types of organizations. While the IEEE 7000-2021 standard provides engineers and technologists with an implementa- ble process aligning innovation management processes, system design approaches, and software engineering methods to help address ethical concerns or risks during system design, the DRESS-eAI structure is more focused on using the organization’s own data as input for mitigating risk in their AI implementations. Hence, DRESS- eAI provides a value to the organization even though it is deployed after processes involving potential risks have been designed. In addition, through included case studies, DRESS-eAI has now proven to work in terms of real-world implementation the last three years.i in transparent communication with selected stakeholders to look into ethical val- ues elicitation and prioritization. This involves traceability of ethical values through an operational concept, value propositions, and value dispositions in the system design. The standard is relevant to all sizes and types of organizations. While the IEEE 7000-2021 standard provides engineers and technologists with an implementa- ble process aligning innovation management processes, system design approaches, and software engineering methods to help address ethical concerns or risks during system design, the DRESS-eAI structure is more focused on using the organization’s own data as input for mitigating risk in their AI implementations. Hence, DRESS- eAI provides a value to the organization even though it is deployed after processes involving potential risks have been designed. In addition, through included case studies, DRESS-eAI has now proven to work in terms of real-world implementation the last three years.i Ernst and Young recently surveyed and assessed the ecosystem of artificial intel- ligence risk assessment (AIRA) methodologies (Ezeani et al., 2021). 5.3 Comparing DRESS‑eAI to Other Frameworks Claiming to present a snapshot of the landscape at a certain point in time, the report aims to inform policymakers about the AI risk assessment landscape and provide emerging policy trends and leading practices. Based on the surveyed reports four leading prac- tices have been identified: categorization of risk, risk management, requirements for trustworthiness of AI, and relevant stakeholders for identifying and mitigating AI risk. Although formulated somewhat differently, all have their parallels with the DRESS-eAI methodology. 7 https://platform.anch.ai/auth/login 6 Limitations and Scope of DRESS‑eAI Limitations and scope of the DRESS-eAI framework are discussed in this section to help clarify the framework’s intended use and applicability. Firstly, we highlight that DRESS-eAI exists as an ethical AI risk assessment framework for organizations actively applying, or seeking to apply AI, and thus is not explicitly designed to support all functions of an ethics-based AI auditing frame- work. In this regard, DRESS-eAI does not explicitly support quantitative assess- ments of algorithmic bias and fairness which is achieved through direct analysis of training data and AI performance. DRESS-eAI also does not inherently provide full- compliance assessments tailored to regulations, such as those proposed in the EU AI Act (Commission, 2021). Furthermore, DRESS-eAI does not provide specific checklists nor guidelines to follow. It instead demands active participation of cross- functional engagement through an outlined playbook of steps to assess, report, and monitor comprehensive eAI risk exposure and mitigation plans on both organization and use-case levels. We would note that the demands of cross-functional work needed for DRESS-eAI are not always realistically achievable for logistical reasons. The economic, techni- cal, and expertise resources needed to complete an organizational-specific imple- mentation of the DRESS-eAI framework may not be available. Current demands for attaining AI expertise from IT, business, and legal perspectives suggest that the DRESS-eAI framework can most realistically be applied to organizations with 1 3 3 Page 24 of 27 Digital Society (2022) 1:13 13 at least a basic level of AI maturity. Furthermore, although DRESS-eAI explicitly integrates stakeholder engagement, the challenges of attaining meaningful stake- holder involvement within this field are reported by Costanza-Chock et al. (2022). To remedy several of these stated issues, we would defer organizations with limited resources to utilize, and gain inspiration, from the freely available digital implemen- tation of DRESS-eAI7. It should be noted this implementation does not explicitly support the entire stakeholder engagement stage.i Finally, we note that the specified implementation demands the integration of questionnaires for the risk scanning phase. This questionnaire-based approach to implementing the framework can inherently lead to self-reporting errors and bias, potentially hurting the validity of framework findings. For this reason, we would advocate having checks for data quality, such as an independent review of question responses, or multiple respondents answering and comparing their responses, to help ensure the reliability and validity of results. 7 Conclusion and Future Directions In this paper, we have outlined and motivated the problem of developing a vetted and real-world applicable approach to ethical AI risk assessment. We report the findings of our systematic multidisciplinary research approach to building defini- tions and establishing requirements needed for such a methodology. Importantly, our approach to involve cross-sector experts has highlighted a need for a methodology that incorporates cross-functional considerations that build on familiar organiza- tional processes. Leveraging this evidence, we then propose a novel methodology named DRESS-eAI. Furthermore, we fully describe our implementation and report the effectiveness and evolution of our implementation by describing several case studies and group-level insights. As ongoing work, we are actively employing the implementation of DRESS-eAI with organizations, continuously acquiring evidence to understand how our imple- mentation of the methodology can be further refined. Such evidence will permit additional group-level analyses, afforded by the data-driven backbone of DRESS- eAI, providing data-driven insights, while refining risk assessment tools and gap analyses. Funding Open access funding provided by Stockholm University. Partial financial support was received from Verket for innovationssystem (Vinnova). Funding Open access funding provided by Stockholm University. Partial financial support was received from Verket for innovationssystem (Vinnova). Data Availability The datasets generated during and/or analyzed during the current study are not publicly available due to privacy rights granted to participating organizations. References Analytica, O. (2018). Us fatality could slow down self-driving car testing. Emerald Expert Briefings Bellamy, R., Dey, K., Hind, M., Hoffman, S., Houde, S., Kannan, K., Lohia, P., Martino, J., Meht y yf S., Mojsilovic, A., Nagar, S., Ramamurthy, K., Richards, J., Saha, D., Sattigeri, P., Singh, M., Varshney, K., & Zhang, Y. (2019). Ai fairness 360: An extensible toolkit for detecting and miti- gating algorithmic bias. IBM Journal of Research and Development, 1. https://doi.org/10.1147/ JRD.2019.2942287i Brendel, A., Mirbabaie, M., Lembcke, T.-B., & Hofeditz, L. (2021). Ethical management of artifici intelligence. Sustainability, 13,. https://doi.org/10.3390/su13041974 Brown, S., Davidovic, J., & Hasan, A. (2021). The algorithm audit: Scoring the algorithms that sco us. Big Data & Society. https://doi.org/10.1177/2053951720983865if Brundage, M., Avin, S., Clark, J., Toner, H., Eckersley, P., Garfinkel, B., Dafoe, A., Scharre, P., Zeitzoff, T., Filar, B. et al. (2018). The malicious use of artificial intelligence: Forecasting, prevention, and mitigation arXiv preprint. https://doi.org/10.48550/arXiv.1802.07228 Buolamwini, J., & Gebru, T. (2018). Gender shades: Intersectional accuracy disparities in commercial gender classification. In Conference on fairness, accountability and transparency (pp. 77–91). PMLR. Canca, C. (2020). Operationalizing ai ethics principles. Communications of the ACM, 63, 18–21. https://doi.org/10.1007/s00146-021-01308-8i Cath, C. (2018). Governing artificial intelligence: ethical, legal and technical opportunities and chal- lenges. https://doi.org/10.1098/rsta.2018.0080i Clarke, R. (2019). Why the world wants controls over artificial intelligence. Computer Law and Secu- rity Review, 35, 423–433. https://doi.org/10.1016/j.clsr.2019.04.006 Commission, E. (2021). Proposal for a regulation of the european parliament and the council laying down harmonised rules on artificial intelligence (artificial intelligence act) and amending certain union legislative acts. EUR-Lex-52021PC0206. Costanza-Chock, S., Raji, I. D., & Buolamwini, J. (2022). Who audits the auditors? recommendations from a field scan of the algorithmic auditing ecosystem. In 2022 ACM Conference on Fairness, Accountability, and Transparency FAccT ’22 (p. 1571–1583). New York, NY, USA: Association for Computing Machinery. https://doi.org/10.1145/3531146.3533213 d’Aquin, M., Troullinou, P., O’Connor, N. E., Cullen, A., Faller, G., & Holden, L. (2018). Towards an “ethics by design” methodology for ai research projects. In Proceedings of the 2018 AAAI/ACM Conference on AI, Ethics, and Society (pp. 54–59). https://doi.org/10.1145/3278721.3278765i Desouza, K., Dawson, G., & Chenok, D. (2019). Designing, developing, and deploying artificial intel- ligence systems: Lessons from and for the public sector. Business Horizons, 63. https://doi.org/ 10.1016/j.bushor.2019.11.004 Dignum, V. (2020). Ai is multidisciplinary. AI Matters, 5, 18–21. https://doi.org/10.1145/337563 3375644 Ezeani, G., Koene, A., Kumar, R., Santiago, N., & Wright, D. (2021). Declarations Conflict of Interest The authors declare no competing interests. Digital Society (2022) 1:13 Page 25 of 27 Digital Society (2022) 1:13 13 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permis- sion directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. References A survey of artificial intelli- gence risk assessment methodologies - The global state of play and leading practices identified. Technical Report Trilateral Research.i Floridi, L., & Cowls, J. (2019). A Unified Framework of Five Principles for AI in Society. Harvard Data Science Review. https://doi.org/10.1162/99608f92.8cd550d1 1 3 Page 26 of 27 13 (2022) 1:13 Digital Society General Assembly, U. G. (1948). Universal declaration of human rights. UN General Assembly, 302, 14–25. Glauner, P. (2021). An assessment of the ai regulation proposed by the european commission. arXiv preprint. https://doi.org/10.48550/ARXIV.2105.15133 Groshev, M., Guimarães, C., Martín-Pérez, J., & de la Oliva, A. (2021). Toward intelligent cyber- physical systems: Digital twin meets artificial intelligence. IEEE Communications Magazine, 59, 14–20. https://doi.org/10.1109/MCOM.001.2001237f Hagendorff, T. (2020). The ethics of ai ethics: An evaluation of guidelines. Minds and Machines, 3 99–120. https://doi.org/10.1007/s11023-020-09517-8i p g HLEGAI (2020). Assessment list for trustworthy artificial intelligence. European Commission. HLEGAI (2020). Assessment list for trustworthy artificial intelligence. European Commission. IEEE (2021) Ieee standard model process for addressing ethical concerns during system design IEEE ( ) yi g p IEEE (2021). Ieee standard model process for addressing ethical concerns during system design. IEEE Std 7000-2021, (pp. 1–82). https://doi.org/10.1109/IEEESTD.2021.9536679 ( ) p g g y g Std 7000-2021, (pp. 1–82). https://doi.org/10.1109/IEEESTD.2021.9536679 Jameson, A., Konstan, J., & Riedl, J. (2002). Ai techniques for personalized re at 18th National Conference on Artificial Intelligence (AAAI).i meson, A., Konstan, J., & Riedl, J. (2002). Ai techniques for personalized recommendation. In Tutorial i Jameson, A., Konstan, J., & Riedl, J. (2002). Ai techniques for personalized recommendation. In Tutorial at 18th National Conference on Artificial Intelligence (AAAI).i Jameson, A., Konstan, J., & Riedl, J. (2002). Ai techniques for personali at 18th National Conference on Artificial Intelligence (AAAI).i at 18th National Conference on Artificial Intelligence (AAAI).i at 18th National Conference on Artificial Intelligence (AAAI).i i vaid, M., Haleem, A., Singh, R. P., & Suman, R. (2022). Artificial intelligence applications for industry Javaid, M., Haleem, A., Singh, R. P., & Suman, R. (2022). Artificial intelligence applications for industry 4.0: A literature-based study. Journal of Industrial Integration and Management, 7, 83–111. https:// doi.org/10.1142/S2424862221300040 i 4.0: A literature-based study. Journal of Industrial Integration and Management, 7, 83–111. https:// doi.org/10.1142/S2424862221300040 g Jobin, A., Ienca, M., & Vayena, E. (2019). The global landscape of ai ethics guidelines. Nature Machine Intelligence, 1, 389–399. https://doi.org/10.1038/s42256-019-0088-2 Kalis, B., Collier, M., & Fu, R. (2018). 10 promising ai applications in health care. References Harvard busine review.i Larsson, S. (2019). The socio-legal relevance of artificial intelligence. Droit et societe, 573–593. https doi.org/10.3917/drs1.103.0573i Larsson, S. (2020). On the governance of artificial intelligence through ethics guidelines. Asian Journal of Law and Society, 7, 1–23. https://doi.org/10.1017/als.2020.19 Larsson, S. (2021). Ai in the eu: Ethical guidelines as a governance tool. (pp. 85–111). https://doi.org/10. 1007/978-3-030-63672-2_4 Å Larsson, S., Anneroth, M., Felländer, A., Felländer-Tsai, L., Heintz, F., & Ångström, R. C. (2019). Sus- tainable ai: An inventory of the state of knowledge of ethical, social, and legal challenges related to artificial intelligence. i g Lauer, D. (2021). You cannot have ai ethics without ethics. AI and Ethics, 1, 21–25. https://doi.org/10. 1007/s43681-020-00013-4 Lopez, P. (2021). Bias does not equal bias: A socio-technical typology of bias in data-based algorithm systems. Internet Policy Review, 10, 1–29. https://doi.org/10.14763/2021.4.1598 McGregor, S. (2020). Preventing repeated real world ai failures by cataloging incidents: The ai incide database. arXiv preprint arXiv:2011.08512 Meek, T., Barham, H., Beltaif, N., Kaadoor, A., & Akhter, T. (2016). Managing the ethical and risk implications of rapid advances in artificial intelligence: a literature review. In 2016 Portland Inter- national Conference on Management of Engineering and Technology (PICMET) (pp. 682–693). https://doi.org/10.1109/PICMET.2016.7806752. IEEE. Mittelstadt, B. (2019). Principles alone cannot guarantee ethical ai. Nature Machine Intelligence, 1, 501 507. https://doi.org/10.1038/s42256-019-0114-4 p g Mökander, J., Axente, M., Casolari, F., & Floridi, L. (2021). Conformity assessments and post-market monitoring: a guide to the role of auditing in the proposed european ai regulation. Minds and Machines, (pp. 1–28). https://doi.org/10.1007/s11023-021-09577-4 Noble, S. U. (2018). Algorithms of oppression: How search engines reinforce racism. nyu Press. https:// doi.org/10.18574/9781479833641 Pandey, S. K. (2012). A comparative study of risk assessment methodologies for information system Bulletin of Electrical Engineering and Informatics, 1, 111–122. https://doi.org/10.12928/eei.v1i2.23 Purdy, G. (2010). Iso 31000: 2009 setting a new standard for risk management. Risk Analysis: An Inte national Journal, 30, 881–886. https://doi.org/10.1111/j.1539-6924.2010.01442.x Rakova, B., Yang, J., Cramer, H., & Chowdhury, R. (2021). Where responsible ai meets reality: Prac- titioner perspectives on enablers for shifting organizational practices. Proceedings of the ACM on Human-Computer Interaction, 5, 1–23. https://doi.org/10.1145/3449081 Rodrigues, R. (2020). Legal and human rights issues of ai: Gaps, challenges and vulnerabilities. Journal of Responsible Technology, 4. https://doi.org/10.1016/j.jrt.2020.100005 1 1 3 3 Digital Society (2022) 1:13 (2022) 1:13 Digital Society Page 27 of 27 13 Shankar, S., Halpern, Y., Breck, E., Atwood, J., Wilson, J., & Sculley, D. (2017). References No classification with- out representation: Assessing geodiversity issues in open data sets for the developing world. https:// doi.org/10.48550/arXiv.1711.08536 Theodorou, A., & Dignum, V. (2020). Towards ethical and socio-legal governance in ai. Nature Machine Intelligence, 2. https://doi.org/10.1038/s42256-019-0136-y Tiganoaia, B., Niculescu, A., Negoita, O., & Popescu, M. (2019). A new sustainable model for risk ma agementrimm. Sustainability, 11, 1178. https://doi.org/10.3390/su11041178 Tseng, M.-L., Tran, T. P. T., Ha, H. M., Bui, T.-D., & Lim, M. K. (2021). Sustainable industrial and opera- tion engineering trends and challenges toward industry 4.0: A data driven analysis. Journal of Indus- trial and Production Engineering, 38, 581–598. https://doi.org/10.1080/21681015.2021.1950227 g g p g Whittaker, M., Alper, M., Bennett, C. L., Hendren, S., Kaziunas, L., Mills, M., Morris, M. R., Rankin, J., Rogers, E., Salas, M. et al. (2019). Disability, bias, and ai. AI Now Institute, November. Whittaker, M., Alper, M., Bennett, C. L., Hendren, S., Kaziunas, L., Mills, M., Morris, M. R., Rankin, J., Rogers, E., Salas, M. et al. (2019). Disability, bias, and ai. AI Now Institute, November. Wolf, M., Miller, K., Grodzinsky, F. (2017). Why we should have seen that coming: Comments on micro- soft tay “experiment”, and wider implications. The ORBIT Journal, 1, 1–12. https://doi.org/10. 29297/orbit.v1i2.49 Wolf, M., Miller, K., Grodzinsky, F. (2017). Why we should have seen that coming: Comments on micro- soft tay “experiment”, and wider implications. The ORBIT Journal, 1, 1–12. https://doi.org/10. 29297/orbit.v1i2.49 Wright, S. A. (2020). Ai in the law: Towards assessing ethical risks. In 2020 IEEE International Confer- ence on Big Data (Big Data) (pp. 2160–2169). https://doi.org/10.1109/BigData50022.2020.9377950 right, S. A. (2020). Ai in the law: Towards assessing ethical risks. In 2020 IEEE International Confer- ence on Big Data (Big Data) (pp. 2160–2169). https://doi.org/10.1109/BigData50022.2020.9377950 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1 3
https://openalex.org/W2963976450	http://bit.fsv.cvut.cz/issues/01-19/full_01-19_04.pdf	English	null	FACTORS AFFECTING THE SUCCESS OF LOGISTICS ENTERPRISES IN VIETNAM	Business & IT	2,019	cc-by	6,487	FACTORS AFFECTING THE SUCCESS OF LOGISTICS ENTERPRISES IN VIETNAM Phung Thi Thanh1, Nguyen Thi Huyen Tran2, Tran Thi Phuong Thuy3 1Foreign Trade University Hochiminh city campus, 15 D5 Street 25 Ward Binh Thanh District, HochiminhCity, 084, Vietnam, thanhphung.ftu@gmail.com, (028.35127258) 2Foreign Trade University Hochiminh city campus, 15 D5 Street 25 Ward Binh Thanh District, HochiminhCity, 084, Vietnam, nguyenthihuyentran.cs2@ftu.edu.vn, (0909828275) 3Foreign Trade University Hochiminh city campus, 15 D5 Street 25 Ward Binh Thanh District, HochiminhCity, 084, Vietnam tranthiphuongthuy.cs2@ftu.edu.vn, (0908934282) Abstract Logistics has become quick developing and important industry which contributes large percentage in Vietnam’s GDP. However, with about 1,300 firms operating in this industry, only few firms have been successful in activity, the others have been facing mangy difficulties. The reason why this circumstance occurred was raised up encourage authors to conduct this research. Buy using FsQCA, some interesting results were found. Most important finding was that there consist different viewpoints between two groups of companies (leading companies and the others). As for all successful businesses in the industry, all factors affect the company's business results if they interact with each other and are combined to create a quite large level of influence for each logistics business. For other logistics businesses, their concern is not all of the seven elements. Two different perspectives are given when firms assess the factors that influence success. This implies somewhat conclude to success or unsuccess of logistics companies. 01/2019 01/2019 Business & IT Keywords Keywords Logistics, success, factors, management JEL Classification C52 DOI: https://doi.org/10.14311/bit.2019.01.04 Editorial information: journal Business & IT, ISSN 2570-7434, CreativeCommons license published by CTU in Prague, 2019, http://bit.fsv.cvut.cz/ Logistics, success, factors, management Introduction Globalization is a trend that has been strongly growing all over the world. This not only creates favorable conditions for countries to accelerate the import and export of goods and their trade with the rest of the world, but also smoothens the flow of goods, creating a broad market for fair competition on trade and investment. Along with this increasingly integrated process, logistics is increasingly focused by its importance. In recent years, especially after Vietnam's integration into the world, logistics has become an important factor contributing to the growth of the Vietnamese economy. Historically, the distribution of goods, exchanges of trade between regions in the country and with foreign countries have always been the essential activity of the national economy. If these activities are smooth and effective, it will greatly contribute to the development of the manufacturing industry. If these activities are stopped, it will negatively affect the whole production and life. In addition, for Vietnam, a country with strong and highly dependence on natural resources, logistics plays a more important role. Each geographic region has different terrain features, different mineral resources and different modes of work and practice, thus the distribution of industries, industrial parks, and economic centers must be in line with specific and general conditions in order to maximize the resources. In order to meet the increasing demand of the market, there are many positive changes of Vietnamese logistics service with the growing number of enterprises operating in this field. Some companies have found the right path to success and establish strong position in the logistics service market. In addition, there are small businesses, focusing on very small work areas, not creating more value so they cannot compete in domestic market. Therefore, the service of domestic logistics companies only meets about 25% of the actual demand. In the context of increasing import and export activities of Vietnam, exponentially expanding domestic trade and the increasing demand for logistics services, this issue needs to be paid special attention to restrict the loss of domestic enterprises on the domestic market. In order to help businesses grow and achieve success in the logistics industry, it is necessary first to identify the factors affecting the success of the logistics companies, thereby evaluating and promoting the strengths, improving the weaknesses in the operation of logistics enterprises. JEL Classification C52 orial information: journal Business & IT, ISSN 2570-7434, CreativeCommons license published by CTU in Prague, 2019, http://bit.fsv.cvut.cz/ Editorial information: journal Business & IT, ISSN 2570-7434, CreativeCommons license published by CTU in Prague, 2019, http://bit.fsv.cvut.cz/ 27 Business & IT 01/2019 Literature review In Vietnam, logistics is often understood as “logistics”, however, the word itself does not explain and embody the full meaning of the term logistics. Thus, the term was used in the original text of Article 233 of the Commercial Law 2005, which states: “Logistics is a commercial activity whereby a business entity conducts one or more works including delivery, transportation, warehousing, storage, customs clearance, other paperwork, customer consultation, packaging, marking, delivery or other related services under the agreement with customers for remuneration”. It can be seen that this concept is no longer appropriate to the reality because the role of logistics is increasingly expanding and not just the delivery of goods or customs clearance. Moreover, this service has developed to a new level, creating more value for the economy, integrating more and more deeply into the operation of enterprises, from the input of raw materials to the production and launching the goods into the market. With the logistics concepts mentioned above, it can be seen that logistics is a concept that can be understood in many angles. In broad terms, it is possible to understand that logistics begins with the optimization of the production cycle, not only with the flow of raw materials and fuels for the production, production of goods, but also with the act to bring the goods to the market, distribute to the end consumers. To do this, the logistics service provider must meet certain requirements in terms of expertise and resources, so that they can provide such a package of services. In the narrow sense, logistics is understood to be the same as freight forwarding, including many transport elements in the service delivery. Within the research scope of this study, logistics activity is mainly considered as the process of organizing and managing the flow of goods as well as other activities related to the import and export process of goods to optimize in terms of time and bring high economic efficiency. Introduction Logistics companies who want to succeed in this field should identify the key factors affecting their business results. In fact, there is very little empirical researches in Vietnam on this issue. Previous studies mainly looked at business success in qualitative aspects. Thus, the authors chooses to study the topic “Factors affecting the success of logistics enterprises in Vietnam” with the desire to point out the factors affecting the success of logistics enterprise, so that they have the basis to strongly develop their company. The authors focus on the factors affecting the success of logistics enterprises in Vietnam. These successful enterprises are among the 20 most successful logistics enterprises in 2015 according to the Vietnam Association of Logistics Services (VLA). In addition, the study also examines logistics enterprises in the industry to compare the impact of these factors. The study focuses on the period from 2007 to present for a number of reasons. Firstly, in 2007, Vietnam Freight Forwarders Association (VIFFAS) was renamed Vietnam Logistics Service Enterprise (VLA). This is a big turning point in the logistics industry in Vietnam, showing that Vietnam's logistics is gradually integrating with global trend. Secondly, the logistics industry has made great strides and there is no sign of slowing down to this point. Thus, it can be said that the period 2007 - now is a period of strong development of logistics services, so research in this period will bring practical value for research. 28 01/2019 Business & IT The study was conducted through 3 main steps: The first step: secondary and primary data collection of the 20 most successful logistics companies and other logistics enterprises in Vietnam; the second step: analysis of collected data; the last step: compare the results between two groups of enterprises. Regarding secondary data collection phase of the 20 most successful logistics enterprises in Vietnam, the authors collected data from the website of the Vietnam Association of Logistics Services (VLA). In addition, the authors also collected data from other businesses operating in this field. Regarding the analysis of primary data, the authors used fsQCA 2.0 to draw conclusions about the factors that really affect the success of the logistics businesses. 1. Concept of logistics service providers According to the definition of logistics business of the Business Dictionary, logistics service providers are understood as companies providing services that manage the flow of goods and materials from the beginning to the last point. These companies also provide transportation, packing and warehousing services. Brian Bass defines the logistics business based on the jobs, functions and tasks that the company will undertake. According to him, the logistics business is the organization that provides logistics solutions to other companies. These companies will specialize in the transportation, distribution, packaging, storage and storage of merchandise. Article 234 of the Commercial Law 2005 defines: “Traders engaging in the business of providing logistics services are qualified enterprises providing logistics services in accordance with the law”. These conditions are listed in the Decree No. 140/2007/ND-CP stipulating the Commercial Law on the conditions for trading in logistics services and the limitation of liability for traders providing logistic 29 01/2019 Business & IT services. Article 3 of this Decree states that “traders providing logistics services are traders providing logistics services to customers by themselves or by subcontractors performing one or more stages of that service”. Traders can be understood by the definition of Commercial Law 2005 as “legally established economic organizations that operate independently and regularly and have business registration”. services. Article 3 of this Decree states that “traders providing logistics services are traders providing logistics services to customers by themselves or by subcontractors performing one or more stages of that service”. Traders can be understood by the definition of Commercial Law 2005 as “legally established economic organizations that operate independently and regularly and have business registration”. Within the scope of this paper, logistics enterprises are understood as such enterprises that organize and provide transportation services for raw materials, materials and goods, strategies for control and cost optimization during transportation as well as other related processes. 2. The concept of success Success is a difficult concept to define because each person has a unique perspective on success. Some people define success based on financial criteria, some define success based on the material and spiritual results they achieve. So far, there is no definite definition of business success. The Merriam-Webster Dictionary defines success as “achieving wealth, respect or fame in reality. This is a defining paradigm of individual success rather than the success of the business”. Myers M. Dialectic (1994), in his research “A theoretical framework for the implementation of information systems”, states that “success can be achieved when the information system is perceived as success by the related people”. Success can be defined by the external category, from the perspective of people around, not from the perspective of each person. Wilson M, Howcroft D (2002) in his research “Re-conceptualizing failure: social shaping meets IS research”, defines “success is the survival of a company”. This is a fairly brief but comprehensive concept. In order to scale up its operations and be more successful, the first thing is that a company must survive in a competitive environment. This existence can be considered as the initial success of the business. DeLone W, McLean E (2004) in the study “Measuring e-commerce success: applying the DeLone and McLean information systems success model” states that “Net profit represents the most basic category of success”. In general, success is an intangible concept that is difficult to quantify. And there is no academic definition of business success. Understanding success depends on the views of individuals and groups. Within the scope of the research, the authors understand that success is the achievement of good business results, high performance and is evaluated based on financial manifestations such as turnover, profit and welfare, employees’ benefit and the position of the business in comparison with other businesses in the same field. Methodology The study was conducted in two phases, including preliminary research and formal research. The authors made preliminary research on the topic using qualitative methods to identify problem and research objectives. The authors have synthesized the basic theoretical foundations and available studies to underpin the subject. The formal quantitative study was conducted by direct interview and questionnaire with employees in logistics companies. In addition, online survey was also sent to employees in the subject group. The survey was designed on a scale from 1 to 7, with 1 meaning being completely disagreeable and 7 meaning being completely agreeable. The authors selected the survey of 20 most successful enterprises in 2015 according to the Vietnam Association of Logistics Services (VLA) to conduct the survey. In addition, the authors also conducted a survey of any business to compare the two groups. 30 01/2019 Business & IT For each business, the authors conducted a survey of one or two objects, from the results obtained, the authors used the average to obtain the final result from each company and use it for research. From the data collected, the authors used Microsoft Excel 2013 software to analyze descriptive statistics for some of the basic characteristics of the dataset. Data were then used for analysis through the Truth Table algorithm and fuzzy sets analysis using the fsQCA 2.0 tool to identify the factors affecting the success of the logistics enterprises in Vietnam. Fuzzy-set The Qualitative Comparative Analysis (fsQCA) is an analytical method of qualitative and quantitative analysis that is not based on statistical theory but on the basis of fuzzy sets. This method has many advantages over conventional regression analysis. FsQCA evaluates the degree of “need” and “sufficiency” of an influential factor based on hypothesis rather than simply analyzing the influence of independent and dependent variables (Fiss, 2007). While linear regression analysis requires large sample sizes and is often used for management studies (Rihoux, 2006; Woodside et al., 2012), fsQCA fits into smaller sample studies. The authors chose to use this analytical method to obtain the most accurate results on the impact of the influencing factors and the relationship between them. The analysis of the results will help to provide a general overview of the success of logistics enterprises and the factors that affect this success. For fsQCA, data collected from Likert scale 1 to 7 were converted to fsQCA data from 0 to 1 (Ragin, 2008). Methodology The authors used the Calibrate function to convert fsQCA's magnitude scale to scale in the data collection process and will use this algorithm to exploit and provide key data for the study. Truth Table is a data matrix consisting of 2k rows, where k is the number of factors of the model and each row represents a unique combination of research factors. After using this algorithm, the Truth Table will be filtered based on the frequency that shows the number of occurrences of the factor in the rows of the Truth Table and the consistency that indicates the relevance. The solutions that algorithms give to the results/phenomena needed to study (Ragin, 2008). According to Ragin, 2008; Leischnig, Henneberg and Thornton, 2014, when analyzing the condition of a phenomenon, the threshold of the consistency coefficient should be at least 0.8 and greater than 0.9. In this paper, the authors analyzed the results of the Truth Table algorithm based on the fsQCA study by Ragin (2008), Emmenegger (2011), Leischnig, Henneberg and Thornton (2014) The frequency is 1, the consistency factor is greater than 0.9. The solution to the problem of the research paper will be given after Truth Table filtering and in conjunction with the analytical algorithm. These solutions are produced with consistent coefficients and explanatory ratios, or raw coverage that draw conclusions about the impact of the factors and assess the level of their importance. Net coverage is an indicator of the level of explanation of each solution to the outcome/phenomenon and the importance of each solution (Ragin, 2008). The scaling factor should be at least 0.4, so the solutions offered are reliable and acceptable (Emmenegger, 2011). In the paper, the authors selected a net cover greater than 0.4. The survey to collect data is the official survey designed and adjusted by the authors after direct interview and consulted by some logistics experts and interviewees in some enterprises. The data were collected by direct survey through the paper survey of employees in the list of 20 most successful logistics enterprises in 2015 which the author selected and any logistics enterprises. In addition, the authors used email communications to send online surveys to the respondents. The data collection process was conducted by the authors in September and October 2017. The survey consisted of two parts. The first is the assessment, which is the most important part of the survey. Results The authors used Microsoft Excel 2013 software to process and analyze data. Each of the variables evaluated by the authors was based on two indices: mean value and dispersion or standard deviation. This standard deviation value indicates the degree of variability of the data. Regarding the quality of service, the average score is 5.71. Observed variables range from 4.97 to 6.3. The variable measuring “The large number of specialized containers and cargo handling equipment such as bar code scanners, weighing scales, forklifts, etc.” (DV3) has the lowest average value (4.97) and dispersion is fairly high, reaching 1.7. The variable measuring “The commitment of the company to ensure its customer service quality” (DV7) has the highest average value (6.3) and low dispersion (1.31). This shows that the business commitment of the service they provide affects the quality of the service but not so much. Regarding the customer relationship management, the average score for this criterion is 5.72. This is the highest score among the customer-related factors. Observed variables ranged from 5.27 to 6.17. The variable measuring “the company must use customer care management software” (QH5) has the lowest average value (5.27) and the dispersion of 1.21. The variable measuring “Staff must always listen to customer feedback and answer” (QH2) has the highest average value (6.17) and the dispersion is quite low (1.16). It can be concluded that the use of customer management software does not interfere with the management of the relationship between the company and the customer. The listening to new customers really affect this relationship. In terms of customer retention, the average score is 5.59 and the median values range from 5.13 to 6.1. The variable measuring “Companies have to invest heavily in communication activities to reach customers” (DT1) has the lowest value (5.13) and the dispersion reaches 1.31. The variable measuring “Companies must establish good relationships with customers from the first transaction” (DT3) has the highest average value (6.1) and the lowest dispersion (1.16). Communication is not the biggest factor affecting the relationship with customers of the enterprises. Regarding human resources, the average score for all sub-variables is 5.62. The mean value of the variables runs from 5.0 to 5.93. The variable measuring “Our staff's payroll ratio is higher than the rival firm for the same position” (NL5) has the lowest average value (5.0) and low dispersion (1.13). Methodology The 53 questionnaires were used for the respondents to evaluate the importance of variables in the research model based on a 7-point scale. The second part is the personal information of the respondents with two questions to collect personal information of the subject. 31 Business & IT 01/2019 After compiling the list of the 20 most successful logistics enterprises in Vietnam in 2015 and the list of other randomly surveyed logistics businesses, the authors sent the survey to 150 companies in two ways: go directly to the company to interview through questionnaire and receive survey results; send surveys via email to corporate email. The authors chose to survey two respondents in each company to make the most accurate assessment. After collecting data and summarizing the results, 82 questionnaires were completed in both online and paper forms. However, 13 questionnaires were excluded, of which 6 were rejected because the name of the company the respondents work was not identified, 7 questionnaires were rejected because the respondents does not work in logistics companies. The remaining sample size is 69, consisting of 30 samples from the successful business group and 39 samples from the other group. This sample size can be analyzed by fsQCA software (Leischnig, Henneberg and Thornton, 2014). Results The variable measuring “When choosing an employee for a new position, we choose based on the individual's learning and development potential when working in a company” (NL2) has highest average value of 5.93 and the lowest dispersion (1.09). It can be seen that the salary of the employees is one of interest of respondents but not the biggest concern. As for the corporate culture, the average value of this component is 5.93. The average value ranges from 5.80 to 6.03. The variable measuring “Company always allows employees to participate in public 32 01/2019 Business & IT discussions” (VH1) has the lowest average value (5.80) and the relatively low dispersion (1.05). The variable “Company always facilitates employees to connect and work together” (VH3) has the highest average value (6.03) and the dispersion of 1.11. This can be seen that the internal environment of a business is the most important variable in this component. discussions” (VH1) has the lowest average value (5.80) and the relatively low dispersion (1.05). The variable “Company always facilitates employees to connect and work together” (VH3) has the highest average value (6.03) and the dispersion of 1.11. This can be seen that the internal environment of a business is the most important variable in this component. Regarding the structure of the company, the average value is 5.9 and range from 5.63 to 6.33. The variable “Company must be flexible, adaptable to continuously changing environment” (CC1) has the lowest average value (5.63) and the dispersion is 1.25. The variable “Company should assign tasks that suit each employee to maximize their capacity” (CC2) has the highest average value (6.33) and the lowest dispersion (0.91). Regarding the leadership, the mean value is quite high and it is also the highest out of the four components measuring the influence of the internal factors of the enterprise (6.07). The variable “The leader must act as a facilitator, recognizing the individual's differences to maximize their potential” (LD1) has the highest mean value (6.13). The variable “The leader must set the right expectation for each employee, and reward incentives when the employee delivers the expected result” (LD2) has the lowest average value (6.0). The dispersion of variables in this component is not much different (from 1.13 to 1.18). Regarding components related to the business environment, the political component has an average value of 5.37 and a dispersion of 1.17. Results The average value of economics is 5.57 and the dispersion is 1.15. The technology component has an average value of 5.4 and a dispersion of 1.2. The labor component has an average value of 5.30 and a dispersion of 1.16. The competition component has an average value of 5.90 and a dispersion of 1.33. It can be seen that the factors of business environment are considerable interest as it is a condition for the business process to be better. In particular, for successful businesses, they focus more on the level of competition in the industry than are controlled and governed by the government such as politics, economics, policy relating to technology and workforce. In general, the components have relatively high average value, which can prove that the components have a certain degree of influence on the success of the logistics business. To determine and ensure that components that affect this success, fsQCA's fuzzy set analysis method will be used. For successful or unsuccessful businesses, the impact of the business environment on each business is the same because they work together and compete in the same market, with the same level of incentives for all businesses. Therefore, the authors only analyzed and evaluated the impact of the factors related to the customer and the internal factors of the business to determine the extent of their impacts on this success. The authors used the Truth Table algorithm for analysis as follows: Step 1: The three factors of customer and four factors of internal business are assigned to new variable, the value of which is calculated by the average math addition of the component variables in each factor; Step 2: The authors selected three benchmarks: a full membership point with a fuzzy score of 7, a cross point with a fuzzy point of 4, full nonmember ship with a fuzzy point of 1. The authors converted the magnitude scale 7 (from 1 to 7) into a scale of fsQCA (0.0 to 1.0) with the Calibrate function to run the Truth Table. Step 3: The Truth Table algorithm was used to analyze the effect on the variable “success”, seven variables are quality of service, customer relationship management, customer retention, human resources, corporate culture, company structure and leadership. Results 33 01/2019 Business & IT After filtering the Truth Table by frequency with 1 and consistency > 0.9, the results are shown in the Table 1 below: Table 1: Impact assessment of factors affecting the success of logistics enterprises (top 20) Factor Solution 1 Solution 2 Solution 3 Quality of service □ ■ ■ Customer relationship management ■ □ ■ Customer retention ■ □ ■ Human resources ■ ■ ■ Corporate culture ■ ■ ■ Company structure □ ■ ■ Leadership □ ■ ■ Raw Coverage 0.1353 0.1939 0.8512 Unique Coverage 0.0193 0.0081 0.659 Consistency 0.9691 1.00 0.936 Solution Coverage 0.8788 Solution Consistency 0.9336 Note: ■: the appearance of factor □: the absence of factor Table 1: Impact assessment of factors affecting the success of logistics enterprises (top 20) From the Table 1, with 7 variables included in the analysis, three solutions were proposed. However, only one solution affecting the success of the logistics business in Vietnam is the third option, satisfying the condition: “the net coverage of the solution is greater than 0.4”. With the appearance of all seven factors, the net coverage of the solution is very high, reaching 0.8512, which proves that the solution explains 85.12% of the components affecting the success of enterprises. Only 14.88% of other factors have impact but have not been included in the model. The consistency coefficient of the solution was also very high, reaching 0.936, indicating that this solution was correct in 93.6% of cases and had an effect on the success of the logistics enterprise (Ragin, 2008). Thus, looking at all three solutions, the two factors of human resources and corporate culture are present in all three solutions. It can be asserted that these two factors always have a moderate impact on the success of the business. Coverage or net coverage of all three solutions was 0.8788 and explained 87.88% whether internal and customer-related factors really affect the success of the business or not. The consistency of the solution was 0.9336, ranging from 0.8 to 0.9, which should be accepted and validated in practice. From the results of the analysis of Solution 1 and 2, it can be seen that the three elements of company structure, leadership and service quality are stronger than the two factors of customer relationship management and customer retention. Results The level of explanation for the solution increases with the presence of three factors in the second solution in comparison with Solution 1. However, the importance of these three factors is not too great and the business does not need to pay too much attention. Instead, they should concentrate on developing other factors. In comparison between Solution 3 and Solution 1, the presence of three factors: corporate structure, leadership and service quality in Solution 3 resulted in a net increase of over 71% over Solution 1 (from 0.1353 to 0.8512), the level of explanation of the solution to the success of the 34 01/2019 Business & IT business increased quite considerably. This proves that these three factors have great influence on the success of the business. If the enterprises do not have the resources to invest in all the elements, they should focus on these three factors first. business increased quite considerably. This proves that these three factors have great influence on the success of the business. If the enterprises do not have the resources to invest in all the elements, they should focus on these three factors first. In comparison between Solution 3 and 2, the presence of two factors: customer relationship management and customer retention in Solution 3 resulted in an explanation level of more than 63% over Solution 2 (from 0.1939 to 0.8512). This proves that these two factors also play big role in the success of the business. These are the aspects that businesses should invest heavily in developing larger companies in the market. It can be said that the impact level of the quality of service provided by enterprises, the structure of the organization as well as the leadership are greater than the rest. Businesses should focus on and prioritize these three factors first, then customer relationship management and customer retention. The impact of human resources and the cultural environment of the company do not account for large percentage so businesses do not need to focus too much on these factors. Results Like Solution 2, although the consistency of Solution 3 is less than 0.9, it is still acceptable because it stays between 0.8 and 0.9 (Ragin, 2008). This solution has an explanation level of 88.69% in practice. All three solutions have four common factors, including quality of service, corporate culture, corporate structure and leadership. This proves that these 4 factors have strong impact on the success of the enterprises but not high. The combination of these four factors is not so tight that it can have big impact on every company. The net coverage of the solution is 0.929, which could confirm that the combination of these three solutions account for 92.9% of the company's success. The solution consistency is 0.8687, indicating that this combination is appropriate in practice. By comparing Solution 1 and 2, it can be seen that the presence of two factors (customer retention factors and customer relationship management) in Solution 2 results in a significant increase in net coverage of the solution, at 58% (from 0.3012 up to 0.8825). This proves that these two factors play a very important role in the success of the business, higher than the impact of a combination of four factors (service quality, corporate culture, corporate structure and leadership). These two factors have very high levels of resonance and are interrelated, interacting and impacting the success of the enterprises. In comparison between Solution 1 and 3, in Solution 3 there are 6 factors affecting success, in addition to the four factors identified in Solution 1, there is also customer relationship management and human resources. With the emergence of these two factors, the net coverage of the solution increased (59.57%), more than the increase in solution 2 (from 0.3012 to 0.8969). Thus, it can be argued that the management of the customer relationship and the skilled workforce of each enterprise have great impact on the business performance and determine the level and speed the development of such company. Both Solution 2 and 3 are acceptable because they satisfy the condition of pure coverage. However, the variables that affect the success of the enterprises in the two solutions also change and that change leads to the increase of pure coverage. Results Table 2: Impact assessment of factors affecting the success of logistics enterprises (the other group) Factor Solution 1 Solution 2 Solution 3 Quality of service ■ ■ ■ Customer relationship management □ ■ ■ Customer retention □ ■ Human resources ■ Corporate culture ■ ■ ■ Company structure ■ ■ ■ Leadership ■ ■ ■ Raw Coverage 0.3012 0.8825 0.8969 Unique Coverage 0.0188 0.0056 0.0216 Consistency 0.9307 0.8939 0.8869 Solution Coverage 0.929 Solution Consistency 0.8687 sessment of factors affecting the success of logistics enterprises (the other group) Table 2: Impact assessment of factors affecting the success of logistics enterprises (the othe From the Table 2, there are 3 solutions, of which only Solution 2 and 3 are of high practical significance, satisfying the condition of pure coverage of 0.4. These two solutions are reliable in practice (Emmenegger, 2011). From the Table 2, there are 3 solutions, of which only Solution 2 and 3 are of high practical significance, satisfying the condition of pure coverage of 0.4. These two solutions are reliable in practice (Emmenegger, 2011). Solution 1 including service quality, corporate culture, corporate structure and leadership has low net coverage and low reliability because net coverage of this solution is 0.3012 <0.4. The four factors that appear only account for 30.12% of the success of the logistics enterprises. This is not a combination of reliable factors in practice. Solution 2 includes the following elements: service quality, customer relationship management, customer retention, corporate culture, corporate structure, leadership. This solution has a high net coverage and accounts for 88.25% of factors affecting the success of the enterprises. The consistency coefficient of this solution is 0.8939. The common consistency factor should reach a minimum of 0.8 and preferably greater than 0.9 when analyzing the condition and probability of a phenomenon or event (Ragin, 2008). Although the consistency coefficient is less than 0.9, this solution is still acceptable and still relevant in explaining the success of the enterprises. 35 Business & IT Business & IT 01/2019 Solution 3 includes the following elements: service quality, customer relationship management, human resources, corporate culture, corporate structure, leadership and customer retention. Net coverage of the solution is higher than any of the three solutions, reaching 0.8969, which accounted for 89.69% of the constituents of the enterprises’ success. 11.31% of other factors also have impact on this success but have not been included in the model for analysis. Conclusion and discussion This study is aimed at identifying factors affecting the success of logistics enterprises in Vietnam. The authors developed a scale of components that affect this success based on the studies by Alan C. Maltz (2000) and Dynamic Performance Multiplier Model (DMP) by Maltz et al. (2003), adapted to the logistics industry in Vietnam. The research model consists of 12 criteria: service quality, customer relationship management, customer retention, human resources, corporate culture, corporate structure, leadership, economics, technology, labor, competitive level are classified into 3 components that make up the success of logistics enterprises, including: factors related to customers, factors of business environment and factors related to the internal issues of enterprise. The research scale is designed and coded based on the Likert scale of 7. However, all businesses are influenced by factors of the business environment, so the factors related to the customer and the internal issues of business are prioritized to be included in the analysis. The data was analyzed and made official based on the results from fuzzy set analysis using the Truth Table algorithm on the fsQCA software. This software is suitable for medium-sized studies like the authors’ ongoing research. When analyzing in detail the influence of each factor of the two components relating to the customer and internal issues of business relating to the success of the logistics business, the authors obtained the results as Table 3: Table 3: A combination of factors that affect success of two business groups Successful groups of companies Factors Other group of companies ■ Service quality ■ ■ ■ Customer relations management ■ ■ ■ Customer retention ■ ■ Human resources ■ ■ Corporate culture ■ ■ ■ Corporate structure ■ ■ ■ Leaders’ ability ■ ■ Table 3: A combination of factors that affect success of two business groups Taking into account the impact of all seven variables on the success of the logistics business in Vietnam, for all successful businesses in the industry, all factors affect the company's business results if they interact with each other and are combined to create a quite large level of influence for each logistics business. For other logistics businesses, their concern is not all of the seven elements. Two different perspectives are given when firms assess the factors that influence success. Results Solution 3 has the appearance of the human resources factor and does not have the appearance of the customer retention factor, while Solution 2 has the appearance of this factor but does not have the human resources variable. The net coverage of Solution 3 is greater than Solution 2 but not significant (0.8969> 0.8825). It can be argued that the human factor has a stronger impact on customer retention and resonance with customer relationship management. The combination of customer relationship management and customer retention have impact more than 58% on success, while customer relationship management and human resources affect 59%. Thus, in addition to the four fundamental factors affecting the success of the enterprises, companies need to pay attention on the management of customer relationships, take appropriate measures to maintain relationships with customers as well as build high quality human resources. However, if the enterprises do not have sufficient resources, it is possible to temporarily abandon the relationship with the customer but still focus on the remaining factors. Enterprises should prioritize the development of the company's human resources rather than maintaining relationships with customers. 36 01/2019 01/2019 Business & IT Conclusion and discussion One view is that there are only six factors: quality of service, customer relationship management, customer retention, corporate culture, corporate structure and leadership are interlinked to influence the performance of a business. Human resources are not an important factor when considering the impact on the business. The other viewpoint is that customer retention has no impact on the business but instead is the human resources that the company has. It is human resources that join the other five factors to resonate with each other in relation to the success that the business wishes to achieve. 37 01/2019 Business & IT [1] Alan C.Maltz. (2000). Defining and Measuring Organizational Success: A Multi-dimensional Framework. Faculty of the Stevens Institute of Technology. [2] Emmenegger, Patrick. (2011). Job Security Regulations in Western Democracies: A Fuzzy Set Analysis. European Journal of Political Research 50. [3] Leischnig, A., Henneberg, S. C., & Thornton, S. C. ( 2014). Performing Configurational Analyses in Management Research: A Fuzzy Set Approach, (pp. 1-21). Bordeaux. References [1] Alan C.Maltz. (2000). Defining and Measuring Organizational Success: A Multi-dimensional Framework. Faculty of the Stevens Institute of Technology. [2] Emmenegger, Patrick. (2011). Job Security Regulations in Western Democracies: A Fuzzy Set Analysis. European Journal of Political Research 50. [3] Leischnig, A., Henneberg, S. C., & Thornton, S. C. ( 2014). Performing Configurational Analyses in Management Research: A Fuzzy Set Approach, (pp. 1-21). Bordeaux. [4] Ragin, Charles C. (2008). Redesigning social inquiry: Fuzzy sets and beyond. Chicago: University of Chicago Press. 38
https://openalex.org/W3111743606	http://repository.essex.ac.uk/31564/1/sim-4302-vyrgioti.pdf	English	null	On Queerness, Motherhood and the Gift of Belonging	Studies in the maternal	2,020	cc-by	2,727	Studies in the Maternal Studies in the Maternal Vyrgioti, M. 2020. On Queerness, Motherhood and the Gift of Belonging. Studies in the Maternal, 13(1): 20. DOI: https://doi.org/10.16995/sim.277 Vyrgioti, M. 2020. On Queerness, Motherhood and the Gift of Belonging. Studies in the Maternal, 13(1): 20. DOI: https://doi.org/10.16995/sim.277 ANNIVERSARY COLLECTION - POSITION PIECE On Queerness, Motherhood and the Gift of Belonging Marita Vyrgioti University of East London, GB mvyrgioti@uel.ac.uk This piece reflects on my experience as an intern for the journal Studies in the Maternal. I wish to suggest that this internship was something more than just an accumulation of professional skills. Starting from the discom fort of belonging as a queer woman, to a community structured around motherhood, I suggest that it is the disembodied image of the mother— psychoanalytically, and sociologically sustained—that did not allow me to explore links between queerness and the maternal in the first place. I then draw on a personal vignette about maternal friendships to consider homo sociality as an often-forgotten structure of support alongside heteronor mative family structures. Finally, I show that the capacity to psychically hold together queerness and the maternal (queer as maternal, and moth erhood as queer), can be engendered only after the question of how one belongs—peculiarly, queerly, uncomfortably, embarrassingly, socially—has been raised. Vyrgioti: On Queerness, Motherhood and the Gift of Belonging 2 2 Lee Edelman, No Future: Queer Theory and the Death Drive (Durham and London: Duke University Press, 2004). 1 Jacqueline Rose, Mothers: An Essay on Love and Cruelty (London: Faber and Faber Limited, 2018). Returning Sometime in autumn 2015, I started working as an Editorial Assistant for Studies in the Maternal. Through this editorial position, I was quickly exposed to the subject of motherhood and questions around maternal ethics. As a queer woman, I had not previously been preoccupied with the issue of motherhood for myself, but it was my encounter with MaMSIE, to some extent, which forced me to consider the mat ter. This piece reflects on my experience as an intern for the journal Studies in the Maternal. I wish to suggest that this internship was something more than just an accumulation of professional skills. This is because engaging with motherhood and the maternal, even through an editorial position, is in itself a disruptive experience. It invites one to consider what it means to be a subject that has been mothered, who can mother, who does or does not want to become a mother. It also invites one to ponder on the proliferation of contemporary representations of mothers—failing, excessive, frantic, omnipotent, hysteric, sexualised, misrepresented, boring, cruel, deserting, frail and devouring. As Jacqueline Rose points out, any intellectual task involving mothers sits in uncomfortable proximity with both love and cruelty.1 For these reasons, thinking about (our) mothers is inevitably and irreparably unsettling. Engaging with motherhood from a queer perspective creates an added disjunc ture. As Lee Edelman has argued, in being excluded from biological, heteronorma tive forms of reproduction, queer individuals pose a challenge to futurity.2 Instead of being guided by an ethical horizon generated by the capacity to reproduce (the exhausted motif of creating a better world for one’s children), queer sexualities embody the withdrawal from the possibility of a liveable future, occupying the place of negativity and death in contemporary culture. As a queer woman, my internship in MaMSIE was haunted by this particular deadly trajectory which, in the way I expe rienced it back then, stood in opposition to what I thought as maternal ethics. To illustrate this disjuncture more clearly, I wish to return to some of the online, remote Vyrgioti: On Queerness, Motherhood and the Gift of Belonging 3 Vyrgioti: On Queerness, Motherhood and the Gift of Belonging 3 and intellectual encounters that took place during my internship at MaMSIE. Returning By ask ing why I unwittingly ignored the physical, embodied dimension of motherhood back then, I hope to reflect on what being an intern in MaMSIE means to me now. Starting from the discomfort of belonging as a queer woman, to a community structured around motherhood, I suggest that it is the disembodied image of the mother—psychoanalytically, and sociologically sustained—that did not allow me to explore links between queerness and the maternal in the first place. I then draw on a personal vignette about maternal friendships to consider homosociality as an often- forgotten structure of support alongside heteronormative family structures. Finally, I show that the capacity to psychically hold together queerness and the maternal (queer as maternal, and motherhood as queer), can be engendered only after the question of how one belongs—peculiarly, queerly, uncomfortably, embarrassingly, socially—has been raised. 3 Maggie Nelson, The Argonauts (London: Melville House UK, 2015), 104. The Mother in the Workplace: the ‘No-body’ I wrote two short pieces for the MaMSIE blog: one on how the underfunding of the NHS has created the urge to centralise obstetric units in the UK, closing down local ones; and one about the missing refugee children in trafficking networks across Europe since the Syrian war. Both of those pieces helped me think about how large economic and political phenomena affect the lives of those most vulnerable. Return ing to these pieces, some three years and a PhD degree later (a form of birth in its own terms), I am struck by two things: first, by how pertinent these questions remain today; and second, and most crucially, by how my writing about motherhood pro foundly mismatched the proliferation of personal, reflective, physical, bodily writing that the maternal has come to occupy in queer scholarship. For example, in The Argonauts (2015), a show-stopping account of queer family-making, Maggie Nelson invites us to think of pregnancy as the work of constantly withdrawing from ‘fall ing forever, going to pieces.’3 There is an active effort of keeping it together, while the whole body stretched at its limits edges disintegration. Although Baraitser has Vyrgioti: On Queerness, Motherhood and the Gift of Belonging 4 argued that motherhood needs to be explored beyond the question of natality (chal lenging thus the assumption that the origins of the maternal subject lie in child birth), she suggests that motherhood feels like a similar rupture; an ‘interruption’ as she calls it which I think makes a pledge for thinking the maternal in relation to a grounded presence and physicality—4beyond melodrama, detachment, idealisation and sacredness. To further clarify the links between physicality and the maternal, I turn to another small piece I prepared for MaMSIE’s Facebook page, which commented on the witty banter that took place between James Cameron and Jeremy Corbyn during Prime Minister Questions in February 2016. The two men’s short exchange revolved around what their mothers would urge them to do in that current political juncture. In both Cameron’s and Corbyn’s fantasies, the ‘mother’ featured as a disembodied ethical figure: in the case of Cameron, inspiring patriotism by tidying up the messiness of masculinity (in his words ‘to straighten your tie and sing the national anthem’), and in the case of Corbyn, endorsing socialism by blasting austerity policies and con demning the underfunding of the NHS. 5 Herbert Marcuse, Eros and Civilization, 2nd ed. (Boston: Beacon Press, 1966[1955]); Norman O. Brown, Life Against Death: The Psychoanalytical Meaning of History, 2nd ed. (Connecticut: Wesleyan University Press, 1985[1959]). 4 Lisa Baraitser, Maternal Encounters: The Ethics of Interruption (London: Routledge, 2009). 6 Rose, Mothers: An Essay on Love and Cruelty. The Mother in the Workplace: the ‘No-body’ From their perspective, the figure of the mother is there to be narcissistically possessed as being on their side. While editing this short post, I remember reading through it, again and again, to find the words failing me that would express what I felt was going on in this exchange. There was a similar debate between Herbert Marcuse and his close friend Norman Brown, two post-Freudian philosophers who lived and worked in America in the 1950s. In their writings, they discuss how the new psychoanalytic, political subject can manage the life and death drives.5 Marcuse and Brown link the problem of the drives to the problem for the mother who is seen in an ambiguous place: while the undifferenti ated union with the mother promises relief from conflict and ambivalence, it is the forbidden desire for fusion that causes the subject’s incommensurate suffering. As Jacqueline Rose puts it, in the patriarchal imaginary, the mother marks both the Vyrgioti: On Queerness, Motherhood and the Gift of Belonging 5 desired object and the object wished ‘out of here’ or wished dead.6 Is it, then, that the ‘mother’ becomes sacred, idealised—taking the form of a law or an ethical guide—so that she, her body can be protected from our murderous attacks? Perhaps I was too lazy to do the work of thinking through Corbyn and Cameron’s witty exchange back when I was an Editorial Intern, thus failing to notice the disem bodied mother in their accounts: the mother is all law, no-body. Yet, if we accept that there is no such thing as ‘laziness’ from a psychoanalytic perspective, what was it that interrupted the process of imagining the mother as embodied in their accounts? Was it not the impossibility of thinking myself as a mother in an embodied way? Worse, was it not the impossibility of thinking myself as the daughter of a mother with a body (whatever this entails)? And even worse, was it not the impossibility of thinking of myself as the queer daughter of a non-queer mother? 6 Rose, Mothers: An Essay on Love and Cruelty. 7 Roberto Esposito, Communitas: The Origin and Destiny of Community, trans. Timothy Campbell (Stanford: Stanford University Press, 2010). Mothers Against the Future—or How I Realised my Mom’s Queerness When I received the invitation to contribute to the 10th anniversary issue of Studies in the Maternal, it was the same time as Penny, my mother’s best friend and long-term colleague passed away. Penny and my mom had been working together for more than a decade and had successfully implemented strategies to encourage recycling and to inspire environment-friendly behaviours among school students in Achaia—a province of Peloponnese in Greece. Besides their commitment to combating pollu tion and making the world a better place, Penny and my mother shared losses and separations in many different ways. Both suffered from chronic health conditions. Both had seen their children migrate abroad amidst the misery of the ten-year finan cial, social and political Greek crisis. Both saw their salaries and pension schemes axed at an age when they relied on them most. And both found mostly unhappiness in their declining marriages. Penny’s loss left both my mother and me bereft, not only because she was a wonderful person and I was used to her presence, especially during my long summer breaks in our summerhouse when they used to play cards and cook and chat after their morning swim. But with Penny, I felt that we were Vyrgioti: On Queerness, Motherhood and the Gift of Belonging 6 unwittingly sharing the task of maintaining a loving relationship with my ageing mother. My absence (since I was living abroad) and Penny’s presence gave me a sense of solace that my mother was being cared for. Their friendship also helped me recon figure my relationship with my mother from the perspective of homosociality. This is because their alliance of sustenance stretched the bonds of care outside the space of the heteronormative family, which in turns, problematised the fantasy of the ‘nucle arity’ of the family. As such, it allowed me to imagine a previously unimaginable connection between queerness and motherhood. Penny’s kind and caring presence aided the puncturing of an exclusively heterosexual system of support. Through her life and death, Penny embodied a maternal dependence on female friendships as homosocial forms of care, which eventually allowed me to envisage my mother as, in her own way, queer. The author has no competing interests to declare. The author has no competing interests to declare. Belonging Throughout this piece, I have suggested that the internship at MaMSIE was nothing but an ordinary professional experience. And it is the same extra-ordinariness that permeated the email invitation to write about it. Listing all the first names of every intern of MaMSIE during the last ten years in chronological order, as if Sigal and Lisa were speaking to all of us, to every one of us, made me think of this invitation as an invitation to reflect on how I belong to this community of maternal subjects. For Italian philosopher Roberto Esposito, community (or Communitas) is both a gift and a duty, an obligation.7 First, it is a gift because it engenders a question for retrospec tion (what did the internship on MaMSIE mean to you?) and offers the space for put ting it out there. Second, in Esposito’s understanding, community is a duty because it demands that we do the work of belonging by suspending our identifications with those social categorisations that pull us together or draw us apart. Esposito’s com munitas is not what is communally shared (‘we are all mothers and fathers’). Rather, it is about holding contradictory, murky, and embarrassing parts of ourselves together (being queer and maternal, being maternal without being a mother). Belonging, in Vyrgioti: On Queerness, Motherhood and the Gift of Belonging 7 this sense, is a duty because it creates the social, intellectual and psychic space where this work of ‘holding together’ our pieces needs to be done. Using the space offered by the community to repair the ruptures in ourselves is, after all, the greatest gift MaMSIE has given to me. Holding together the ‘queer’ and the ‘maternal,’ psychically, physically, socially is the gift, I hope, I have been able to give back. In loving memory of Penny. In loving memory of Penny. References Baraitser, L. 2009. Maternal Encounters: The Ethics of Interruption. London: Routledge. DOI: https://doi.org/10.4324/9780203030127 Baraitser, L. 2009. Maternal Encounters: The Ethics of Interruption. London: Routledge. DOI: https://doi.org/10.4324/9780203030127 Brown, NO. 1985. Life Against Death: The Psychoanalytical Meaning of History. 2nd ed. Connecticut: Wesleyan University Press. Brown, NO. 1985. Life Against Death: The Psychoanalytical Meaning of History. 2nd ed. Connecticut: Wesleyan University Press. Edelman, L. 2004. No Future: Queer Theory and the Death Drive. Durham and London: Duke University Press. DOI: https://doi.org/10.1215/9780822385981 Esposito, R. 2010. Communitas: The Origin and Destiny of Community. Translated by Timothy Campbell. Stanford: Stanford University Press. Edelman, L. 2004. No Future: Queer Theory and the Death Drive. Durham and London: Duke University Press. DOI: https://doi.org/10.1215/9780822385981 Edelman, L. 2004. No Future: Queer Theory and the Death Drive. Durham and London: Duke University Press. DOI: https://doi.org/10.1215/9780822385981 Esposito, R. 2010. Communitas: The Origin and Destiny of Community. Translated by Timothy Campbell. Stanford: Stanford University Press. London: Duke University Press. DOI: https://doi.org/10.1215/9780822385981 Esposito, R. 2010. Communitas: The Origin and Destiny of Community. Translated by Timothy Campbell. Stanford: Stanford University Press. Esposito, R. 2010. Communitas: The Origin and Destiny of Community. Translated by Timothy Campbell. Stanford: Stanford University Press. Marcuse, H. 1966. Eros and Civilization. 2nd ed. Boston: Beacon Press. Nelson, M. 2015. The Argonauts. London: Melville House UK. Rose, J. 2018. Mothers: An Essay on Love and Cruelty. London: Faber and Faber Limited. How to cite this article: Vyrgioti, M. 2020. On Queerness, Motherhood and the Gift of Belonging. Studies in the Maternal, 13(1): 20, pp. 1–7. DOI: https://doi.org/10.16995/ sim.277 Published: 08 December 2020 Copyright: © 2020 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. OPEN ACCESS Studies in the Maternal is a peer-reviewed open access journal published by Open Library of Humanities. How to cite this article: Vyrgioti, M. 2020. On Queerness, Motherhood and the Gift of Belonging. Studies in the Maternal, 13(1): 20, pp. 1–7. DOI: https://doi.org/10.16995/ sim.277 Published: 08 December 2020 Published: 08 December 2020 Copyright: © 2020 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. References See http://creativecommons.org/licenses/by/4.0/. OPEN ACCESS Studies in the Maternal is a peer-reviewed open access journal published by Open Library of Humanities. OPEN ACCESS Studies in the Maternal is a peer-reviewed open access journal published by Open Library of Humanities.
https://openalex.org/W4295927379	https://www.mdpi.com/2076-393X/10/9/1528/pdf?version=1663640725	English	null	FLU-v, a Broad-Spectrum Influenza Vaccine, Induces Cross-Reactive Cellular Immune Responses in Humans Measured by Dual IFN-γ and Granzyme B ELISpot Assay	Vaccines	2,022	cc-by	10,052	FLU-v, a Broad-Spectrum Inﬂuenza Vaccine, Induces Cross-Reactive Cellular Immune Responses in Humans Measured by Dual IFN-γ and Granzyme B ELISpot Assay Fredrik Oftung 1,, Lisbeth M. Næss 2, Ida Laake 1, Gregory Stoloff 3 and Olga Pleguezuelos 4 1 Department of Method Development and Analytics, Division of Infectious Disease Control, Norwegian Institute of Public Health, P.O. Box 222, N-0213 Oslo, Norway 2 Department of Infection Control and Vaccines, Division of Infectious Disease Control, Norwegian Institute of Public Health, P.O. Box 222, N-0213 Oslo, Norway 3 SEEK, London Bioscience Innovation Centre, 2 Royal College St, London NW1 0NH, UK 4 ConserV Bioscience, 77 Heyford Park, Bicester OX25 5HD, UK Correspondence: fredrik.oftung@fhi.no 1 Department of Method Development and Analytics, Division of Infectious Disease Control, Norwegian Institute of Public Health, P.O. Box 222, N-0213 Oslo, Norway 2 Department of Infection Control and Vaccines Division of Infectious Disease Control Norwegian Institute of 1 Department of Method Development and Analytics, Division of Infectious Disease Control, Norwegian Institute of Public Health, P.O. Box 222, N-0213 Oslo, Norway 2 Department of Infection Control and Vaccines, Division of Infectious Disease Control, Norwegian Institute of Public Health, P.O. Box 222, N-0213 Oslo, Norway 3 SEEK, London Bioscience Innovation Centre, 2 Royal College St, London NW1 0NH, UK 4 ConserV Bioscience, 77 Heyford Park, Bicester OX25 5HD, UK 2 Department of Infection Control and Vaccines, Division of Infectious Disease Control, Norwe Public Health, P.O. Box 222, N-0213 Oslo, Norway y 3 SEEK, London Bioscience Innovation Centre, 2 Royal College St, London NW1 0NH, UK 4 ConserV Bioscience 77 Heyford Park Bicester OX25 5HD UK * Correspondence: fredrik.oftung@fhi.no Abstract: Previous reports demonstrated that FLU-v, a peptide-based broad-spectrum inﬂuenza vaccine candidate, induced antibody and cellular immune responses in humans. Here, we evaluate cellular effector functions and cross-reactivity. PBMC sampled pre- (day 0) and post-vaccination (days 42 and 180) from vaccine (n = 58) and placebo (n = 27) recipients were tested in vitro for responses to FLU-v and inactivated inﬂuenza strains (A/H3N2, A/H1N1, A/H5N1, A/H7N9, B/Yamagata) using IFN-γ and granzyme B ELISpot. FLU-v induced a signiﬁcant increase in the number of IFN-γ- and granzyme-B-secreting cells responding to the vaccine antigens from pre-vaccination (medians: 5 SFU/106 cells for both markers) to day 42 (125 and 40 SFU/106 cells, p < 0.0001 for both) and day 180 (75 and 20 SFU/106 cells, p < 0.0001 and p = 0.0047). The fold increase from pre-vaccination to day 42 for IFN-γ-, granzyme-B-, and double-positive-secreting cells responding to FLU-v was signiﬁcantly elevated compared to placebo (medians: 16.3-fold vs. 1.0-fold, p < 0.0001; 3.5-fold vs. 1.0-fold, p < 0.0001; 3.0-fold vs. Citation: Oftung, F.; Næss, L.M.; Laake, I.; Stoloff, G.; Pleguezuelos, O. FLU-v, a Broad-Spectrum Inﬂuenza Vaccine, Induces Cross-Reactive Cellular Immune Responses in Humans Measured by Dual IFN-γ and Granzyme B ELISpot Assay. Vaccines 2022, 10, 1528. https:// doi.org/10.3390/vaccines10091528 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: broad spectrum inﬂuenza vaccine; FLU-v; clinical trial; dual ELISpot; cellular immune responses; cross-reactivity FLU-v, a Broad-Spectrum Inﬂuenza Vaccine, Induces Cross-Reactive Cellular Immune Responses in Humans Measured by Dual IFN-γ and Granzyme B ELISpot Assay Fredrik Oftung 1,*, Lisbeth M. Næss 2, Ida Laake 1, Gregory Stoloff 3 and Olga Pleguezuelos 4 1.0-fold, p = 0.0012, respectively). Stimulation of PBMC with inactivated inﬂuenza strains showed signiﬁcantly higher fold increases from pre-vaccination to day 42 in the vaccine group compared to placebo for IFN-γ-secreting cells reacting to H1N1 (medians: 2.3-fold vs. 0.8-fold, p = 0.0083), H3N2 (1.7-fold vs. 0.8-fold, p = 0.0178), and H5N1 (1.7-fold vs. 1.0-fold, p = 0.0441); for granzyme B secreting cells reacting to H1N1 (3.5-fold vs. 1.0-fold, p = 0.0075); and for double positive cells reacting to H1N1 (2.9-fold vs. 1.0-fold, p = 0.0219), H3N2 (1.7-fold vs. 0.9-fold, p = 0.0136), and the B strain (2.0-fold vs. 0.8-fold, p = 0.0227). The correlation observed between number of cells secreting IFN-γ or granzyme B in response to FLU-v and to the inﬂuenza strains supported vaccine-induced cross-reactivity. In conclusion, adjuvanted FLU-v vaccination induced cross-reactive cellular responses with cytotoxic capacity, further supporting the development of FLU-v as a broad-spectrum inﬂuenza vaccine. 1. Introduction Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Seasonal inﬂuenza virus continues to cause high morbidity and mortality every year, and sporadic epidemics and pandemics cause additional burdens to health care systems worldwide. For decades, seasonal ﬂu vaccines have included hemagglutinin (HA) isolated from the strains predicted to be in circulation in the coming season as the principal vaccine antigen. Due to the high mutation rate in the HA gene of inﬂuenza A and B viruses, updates to the vaccine are required to match this antigenic drift every year [1]. In addition, even more abrupt antigenic shifts, often involving re-assortment of the major surface antigens, https://www.mdpi.com/journal/vaccines Vaccines 2022, 10, 1528. https://doi.org/10.3390/vaccines10091528 Vaccines 2022, 10, 1528 2 of 14 can create new viruses with pandemic potential due to a lack of strain-speciﬁc protective immunity in the population [1]. Inﬂuenza vaccines that provide broad protection against diverse inﬂuenza strains are, therefore, highly needed to provide seasonal protection and pandemic preparedness against emergent inﬂuenza strains [1]. Several different approaches to develop broad spectrum inﬂuenza vaccines by targeting conserved regions of various inﬂuenza antigens to induce protective antibody and/or cell-mediated immune responses are being evaluated in clinical trials [2–5]. In this context, virus-like particles, protein or peptide-based subunit vaccines, virus-vector-based vaccines, and DNA/RNA constructs are among the most explored vaccine platform technologies for the efﬁcient induction of both antibody and cellular immunity [2]. FLU-v is an adjuvanted peptide vaccine consisting of four short peptides originat- ing from conserved regions of the M1, M2, NP-A, and NP-B proteins of the inﬂuenza virus that are manufactured using F-moc chemistry. Previous reports demonstrated that FLU-v induced cellular and humoral immune responses in both preclinical [4] and clini- cal studies [5–7], and reduced mild-to-moderate inﬂuenza disease in a human challenge study [8,9]. FLU-v immune responses were evaluated in a phase IIb clinical trial within the UNISEC Consortium framework [10]. The evaluation of cellular immune responses showed that a single dose of adjuvanted FLU-v was more effective than two doses of non-adjuvanted FLU-v in stimulating T cells positive for IFN-γ, TNFα, or IL-2 measured by multi-parametric ﬂow cytometry [5]. 2. Materials and Methods FLU-v is a 200 nM equimolar mixture of four lyophilized synthetic peptides (Table 1) ginated from conserved regions of the M1, M2, NP-A, and NP-B inﬂuenza virus pro- s [5]. The adjuvanted formulation is prepared by reconstituting lyophilized FLU-v in 5 mL of Montanide ISA-51 (Seppic) and 0.25 mL of water for injection, and mixing to ate a water-in-oil emulsion. The adjuvanted placebo formulation consisted of 0.5 mL water-in-oil emulsion without FLU-v. Montanide ISA-51 is made of a mineral oil and urfactant designed to increase antigen speciﬁc immune responses [11]. Healthy adult unteers in the vaccine (n = 58) or placebo (n = 27) groups received a subcutaneous injec- n of adjuvanted FLU-v or adjuvanted placebo on day 0, followed by a saline injection day 21, as determined by the randomization code. Study approval was obtained after cal review by the Dutch Central Committee on Research Involving Human Subjects erence NL55061.000.15), followed by approval from the competent authority (the Dutch nistry of Health, Welfare and Sport). Informed consent was obtained from each partici- t before proceeding with the trial interventions. The study was registered in EudraCT: 5-001932-38 [6,8]. le 1. Amino acid sequences of the peptides included in the FLU-v vaccine and the protein antigens y originate from. Peptide Name Protein Origin Amino Acid Sequence FLU-5 acetate M1 protein DLEALMEWLKTRPILSPLTKGILGFVFTLTVP FLU-7 acetate NP protein from A strains DLIFLARSALILRGSVAHKS FLU-8N acetate NP protein from B strains PGIADIEDLTLLARSMVVVR FLU-10 acetate M2 protein IIGILHLILWILDRLFFKCIYRLF FLU-v is a 200 nM equimolar mixture of four lyophilized synthetic peptides (Table 1) originated from conserved regions of the M1, M2, NP-A, and NP-B inﬂuenza virus pro- teins [5]. The adjuvanted formulation is prepared by reconstituting lyophilized FLU-v in 0.25 mL of Montanide ISA-51 (Seppic) and 0.25 mL of water for injection, and mixing to create a water-in-oil emulsion. The adjuvanted placebo formulation consisted of 0.5 mL of water-in-oil emulsion without FLU-v. Montanide ISA-51 is made of a mineral oil and a surfactant designed to increase antigen speciﬁc immune responses [11]. Healthy adult volunteers in the vaccine (n = 58) or placebo (n = 27) groups received a subcutaneous injec- tion of adjuvanted FLU-v or adjuvanted placebo on day 0, followed by a saline injection on day 21, as determined by the randomization code. 1. Introduction Moreover, adjuvanted vaccination also induced strong FLU-v-speciﬁc IgG responses that could play a role in antibody-dependent cellular cytotoxicity (ADCC) or complement activation against inﬂuenza-infected cells [5]. Utilizing leftover cryopreserved PBMC samples from the adjuvanted FLU-v group and adjuvanted placebo from the above-mentioned clinical phase IIb trial [5], we aimed to explore vaccine-induced effector functions and cross-reactive cellular immunity by means of measuring IFN-γ- and granzyme-B-producing cells by dual ELISpot assay in response to a panel of diverse inﬂuenza strains. 2.2. Dual ELISpot Assay 2.2. Dual ELISpot Assay PBMC was isolated from blood samples taken from all volunteers on day 0 (pre- vaccination), and days 42 and 180 post-vaccination. Cryo-preserved PBMC were thawed and washed in RPMI 1640 (15% FCS), and left to recover overnight in 5 mL RPMI 1640 (15% FCS and 100 U/mL penicillin/streptomycin) in a 37 ◦C humidiﬁed 5% CO2 incubator. Viable cells were counted in a MUSE automated cell counter (Millipore) using MUSE count and viability kit (Millipore). Viable cells were adjusted to 1 million cells/mL in RPMI 1640 (15% FCS) to reach a ﬁnal concentration of 100,000 cells/100 µL/well. All samples representing different time points from the same participant were tested on the same plate. p g p p p p FLU-v vaccine antigens were added to the cells as a mixture of the four synthetic peptides to reach a ﬁnal concentration of 4 µM/well (total peptide concentration). Phyto- hemagglutinin (PHA-P) was added to the assay as positive control at a ﬁnal concentration of 2 µg/mL, and complete cell culture medium (RPMI 1640 with 15% FCS) was used as neg- ative control. Based on previous in-house titration experiments, 100 hemagglutinin units (HAU)/mL of each inactivated inﬂuenza virus strain was used as the ﬁnal concentration. The inactivated strains used included A/California/7/2009 (H1N1), A/Shanghai/24/1990 (H3N2), B/Brisbane/9/2014 (Yamagata lineage), A/Vietnam/1194/2004 (NIBRG-14) (H5N1), and A/Anhui/1/2013 (NIBRG-268) (H7N9), all obtained from the National Institute for Biological Standards and Controls (NIBSC, United Kingdom). All antigens and controls were assayed in duplicates. Plates were incubated for 24 h in a 37 ◦C humidiﬁed 5% CO2 incubator. A human IFN-γ and granzyme B double color enzymatic ELISpot kit (Cellular Tech- nology Limited, Bonn, Germany) was used to enumerate the cells secreting IFN-γ and granzyme B after in vitro antigen stimulation. This assay allows for the simultaneous detection of two important markers related to protective immune responses against in- ﬂuenza. Dual detection of these parameters was performed by using a FITC-conjugated detection antibody for IFN-γ and a biotin-conjugated detection antibody for granzyme B, followed up with FITC-HRP and Strep-AP incubation for the visualization of red (INF-γ) and blue (granzyme B) spots, respectively. Double positive cells expressing both im- mune markers were detected as pink spots. The assay was performed following the manufacturer’s instructions. Spot forming units (SFU) were counted using the Immuno-Spot Series 6 Ultra-V plate analyzer (Cellular Technology Ltd.). 2. Materials and Methods Study approval was obtained after ethical review by the Dutch Central Committee on Research Involving Human Subjects (reference NL55061.000.15), followed by approval from the competent authority (the Dutch Ministry of Health, Welfare and Sport). Informed consent was obtained from each partici- pant before proceeding with the trial interventions. The study was registered in EudraCT: 2015-001932-38 [6,8]. Table 1. Amino acid sequences of the peptides included in the FLU-v vaccine and the protein antigens they originate from. Peptide Name Protein Origin Amino Acid Sequence FLU-5 acetate M1 protein DLEALMEWLKTRPILSPLTKGILGFVFTLTVP FLU-7 acetate NP protein from A strains DLIFLARSALILRGSVAHKS FLU-8N acetate NP protein from B strains PGIADIEDLTLLARSMVVVR FLU-10 acetate M2 protein IIGILHLILWILDRLFFKCIYRLF Table 1. Amino acid sequences of the peptides included in the FLU-v vaccine and the protein antigens they originate from. Vaccines 2022, 10, 1528 3 of 14 2.2. Dual ELISpot Assay This instrument was ISO certiﬁed (ISO 9001:2008), and counting options and procedures followed GCLP guidelines. The mean SFU for each duplicate sample was calculated and normalized by subtracting mean SFU counts for the negative control. Normalized SFU counts with values ≤0 were assigned a value of 0.5, and counts were ﬁnally multiplied by 10 to present the data as SFU/million cells. Samples not meeting the acceptance criteria for positive control (PHA stimulation > 20 SFU/105 cells) or negative control (medium only < 10 SFU/105 cells) were excluded from the statistical analysis. The discrepancies between the number of participants recruited to the study and the number of data points ﬁnally included in statistical analysis were due to missing donor visits and samples, an insufﬁcient number of viable cells in the sample to test all antigens, and an inability to meet the acceptance criteria for positive and negative controls in the assay. 3.1. ELISpot Responses to FLU-v In the FLU-v vaccinated group, the number of IFN-γ-producing cells detected after vaccine antigen stimulation was signiﬁcantly higher on day 42 (median: 125 SFU/million cells, p < 0.0001) and day 180 (median: 75 SFU/million cells, p < 0.0001) after vaccination compared to pre-vaccination (median: 5 SFU/million cells) (Table 2). Signiﬁcantly higher levels of FLU-v-speciﬁc granzyme-B-producing cells were also observed on day 42 (median: 40 SFU/million cells, p < 0.0001) and day 180 (median: 20 SFU/million cells, p < 0.0047) compared to pre-vaccination (median: 5 SFU/million cells) (Table 2). Finally, the number of vaccine-antigen-speciﬁc cells secreting both IFN-γ and granzyme B was signiﬁcantly higher on day 42 (median 20 SFU/million cells, p < 0.0001) and day 180 (5 SFU/million cells, p = 0.0059) compared to pre-vaccination. However, the median number of double positive cells was the same on day 180 as pre-vaccination, since the minimum possible number of cells was observed on both time points for about half of the participants. No signiﬁcant differences between pre- and post-vaccination were observed in the adjuvanted placebo group for any of the immune markers tested (Table 2). Signiﬁcant differences in the fold-increase between the FLU-v vaccinated group and the placebo group were seen for IFN-γ-producing cells from day 0 to day 42 (medians: 16.3-fold vs. 1.0-fold, p < 0.0001) and to day 180 (medians: 9.5-fold vs. 1.0-fold, p < 0.0001) (Table 3). The corresponding fold increases from pre- to post-vaccination for granzyme- B-secreting cells in the vaccine group were lower than those observed for the IFN-γ- secreting cells, but still signiﬁcantly higher than in the placebo group both on day 42 (medians: 3.5-fold vs. 1.0-fold, p < 0.0001) and on day 180 (medians: 2.0-fold vs. 1.0-fold, p = 0.0461) (Table 3). The fold increase for double positive cells in the FLU-v group was only signiﬁcantly different from placebo on day 42 (medians: 3.0-fold vs. 1.0-fold, p = 0.0012) (Table 3). ( ) In the FLU-v vaccinated group, the percentage of responders to the vaccine antigens was 75% (95% CI 61.2–85.1) on day 42 and 62.5% (95% CI 47–75.8) on day 180, based on IFN-γ-secreting cells; 54.3% (95% CI 40.2–67.8) on day 42 and 29.3% (95% CI 17.6–44.5) on day 180 for granzyme-B-secreting cells; and 32% (95% CI 17.2–51.6) on day 42 and 19% (95% CI 7.7–40.0) on day 180 for double positive cells. 3.1. ELISpot Responses to FLU-v Based on the responder deﬁnition used, no responders were detected in the placebo group for any of the markers used on day 42, whereas 6% were responders for IFN-γ and granzyme B on day 180. 3. Results Two different data analyses were used to facilitate the interpretation of the responses. Firstly, comparisons were made between the median number of positive cells at the different time points within the groups, allowing visualization of the pre-vaccination background levels. Secondly, the fold increase from pre-vaccination to post-vaccination was calculated and compared to placebo to show the vaccine effect more accurately. 2.3. Statistical Analyses The Wilcoxon signed rank sum test was used to compare the number of cells over time within each group, whereas the Mann–Whitney U test was used to compare the fold- increase between groups at the same time point. Non-parametric tests were chosen because the D’Agostino and Pearson Omnibus test and visual inspection of the histograms and QQ-plots demonstrated that the data were not normally distributed. Vaccine responders were deﬁned as participants with an SFU count of at least 40 SFU/million cells on days 42 or 180 post-vaccination, and showing a minimum of a two-fold increase in response from pre-vaccination to post-vaccination. p-values for comparison of percentage of respon- Vaccines 2022, 10, 1528 4 of 14 ders between groups were calculated with Fisher’s mid-P test. Spearman’s rank correlation coefﬁcients between the number of IFN-γ- or granzyme-B-secreting cells in response to the vaccine antigens and the different inactivated inﬂuenza strains were calculated. Statistical analyses were performed using Stata SE 16.0 (StataCorp LLC, College Station, TX, USA) and GraphPad 8.1.2 (Dotmatics, Boston, MA, USA) software. 3.2. ELISpot Responses to a Panel of Inﬂuenza Strains To evaluate the ability of the FLU-v vaccine to induce cross-reactive cellular immune responses, we also included a panel of relevant seasonal and pandemic inactivated inﬂuenza A and B strains when testing PBMC in ELISpot assays. Unlike the low baseline stimulation levels seen for the FLU-v antigens, higher levels of IFN-γ- and granzyme-B-producing cells responding to the inﬂuenza strains were already detected pre-vaccination in both groups (Table 2). Despite this, vaccination with adjuvanted FLU-v still induced a signiﬁcant increase in the number of IFN-γ-secreting cells (SFU/million) from day 0 to day 42 in Vaccines 2022, 10, 1528 5 of 14 response to all ﬁve virus strains tested (medians for H1N1: 128 vs. 310, p = 0.0001; H3N2: 268 vs. 555, p = 0.0001; H5N1: 460 vs. 668, p = 0.0001; H7N9: 130 vs. 268, p = 0.0026; and the B strain: 130 vs. 243, p = 0.0085) (Table 2). From day 0 to day 180, signiﬁcant differences in the number of IFN-γ-producing cells (SFU/million) were only observed for the H1N1 (medians: 128 vs. 240, p < 0.0163) and H5N1 (medians: 460 vs. 585, p = 0.0381) strains. In addition, signiﬁcant increases in the number of granzyme-B-secreting cells (SFU/million) from pre-vaccination to day 42 were also seen upon in vitro stimulation with the H1N1 (medians: 50 vs. 240, p < 0.0001), H3N2 (medians: 193 vs. 485, p = 0.0002), and H5N1 (medians: 305 vs. 650, p = 0.0041) strains. Although increases were also seen from day 0 to day 180, these were not statistically signiﬁcant (Table 2). Finally, an analysis of the double positive cell population, expressing both IFN-γ and granzyme B, showed signiﬁcantly higher cell numbers (SFU/million) on day 42 than on day 0 for H1N1 (medians: 25 vs. 125, p < 0.0011), H3N2 (medians: 135 vs. 220, p = 0.0064), H5N1 (medians: 245 vs. 305, p = 0.0220), and B inﬂuenza (medians: 10 vs. 30. p = 0.0046), as well as H1N1 (medians: 25 vs. 50, p = 0.0025) and H3N2 (medians: 135 vs. 205, p = 0.0449) on day 180 after vaccination. (Table 2). No differences in the placebo group were observed between pre- and post-vaccination in the number of cells secreting IFN-γ, granzyme B, or both factors with any of the inﬂuenza strains tested (Table 2). Table 2. 3.2. ELISpot Responses to a Panel of Inﬂuenza Strains The Mann–Whitney U test (MW) was used to compare fold increases between groups, and test the null hypothesis of equal distributions in the vaccine group and placebo group on day 42 and on day 180. Since we cannot reasonably assume that the distributions have the same shape, and only differ with respect to location, the Mann–Whitney U test cannot be interpreted as a comparison of medians. Signiﬁcant differences are indicated with p-values in bold. Antigen Median Fold Increase (n) (CI) Day 42 Median Fold Increase (n) (CI) Day 180 Adjuvanted FLU-v IFN-γ Granzyme B Double Positive Adjuvanted Placebo IFN-γ Granzyme B Double Positive p-Value (MW) Adjuvanted FLU-v IFN-γ Granzyme B Double Positive Adjuvanted Placebo IFN-γ Granzyme B Double Positive p-Value (MW) FLU-v 16.3 (48) (9.0–25.0) 1.0 (21) (1.0–1.0) <0.0001 9.5 (40) (4.0–19.0) 1.0 (17) (1.0–2.0) <0.0001 3.5 (46) (2.0–9.0) 1.0 (19) (0.5–1.0) <0.0001 2.0 (41) (1.0–5.0) 1.0 (16) (0.4–3.0) 0.0461 3.0 (25) (1.0–8.0) 1.0 (12) (1.0–1.0) 0.0012 1.0 (21) (1.0–4.0) 1.0 (7) (1.0–4.0) 0.30 H1N1 2.3 (47) (1.8–3.1) 0.8 (18) (0.5–2.0) 0.0083 1.9 (39) (1.4–3.0) 0.8 (15) (0.4–3.3) 0.23 3.5 (46) (2.1–4.4) 1.0 (16) (0.5–3.0) 0.0075 1.2 (40) (0.8–2.6) 0.9 (14) (0.1–5.5) 0.52 2.9 (25) (2.0–5.0) 1.0 (12) (0.6–1.3) 0.0219 1.8 (21) (1.0–6.6) 0.5 (7) (0.1–7.8) 0.11 Vaccines 2022, 10, 1528 6 of 14 Table 2. Cont. 3.2. ELISpot Responses to a Panel of Inﬂuenza Strains Antigen Treatment Group Median SFU (n) (CI) Day 0 IFN-γ Granzyme B Double Positive Median SFU (n) (CI) Day 42 IFN-γ Granzyme B Double Positive Median SFU (n) (CI) Day 180 IFN-γ Granzyme B Double Positive p-Value (Wilcoxon) Day 0–42 p-Value (Wilcoxon) Day 0–180 H5N1 Adjuvanted FLU-v 460 (45) (175–550) 668 (42) (530–810) 585 (37) (355–845) 0.0001 0.0381 305 (44) (160–495) 650 (41) (450–750) 325 (38) (115–570) 0.0041 0.69 245 (28) (110–335) 305 (24) (250–455) 260 (21) (175–465) 0.0220 0.18 Adjuvanted Placebo 493 (20) (250–890) 450 (17) (340–700) 470 (14) (170–1060) 0.73 0.80 490 (18) (120–800) 433 (16) (275–580) 360 (13) (105–650) 0.98 0.89 135 (11) (50–830) 230 (11) (80–725) 263 (6) (130–410) 0.76 0.56 H7N9 Adjuvanted FLU-v 130 (44) (75–210) 268 (40) (160–350) 215 (36) (70–280) 0.0026 0.27 50 (43) (15–100) 140 (39) (55–180) 50 (35) (15–105) 0.49 0.88 20 (28) (10–45) 60 (23) (20–115) 23 (20) (5–80) 0.06 0.47 Adjuvanted Placebo 150 (18) (40–430) 195 (16) (80–455) 140 (13) (30–265) 0.75 0.79 90 (18) (10–135) 83 (16) (10–260) 50 (12) (5–100) 0.99 0.58 20 (11) (5–170) 35 (11) (5–100) 30 (6) (5–45) 0.71 >0.99 B Adjuvanted FLU-v 130 (47) (75–200) 243 (46) (110–310) 135 (39) (60–230) 0.0085 0.63 25 (48) (10–75) 75 (45) (45–120) 40 (39) (15–75) 0.0601 0.27 10 (28) (5–20) 30 (25) (15–50) 20 (21) (10–50) 0.0046 0.21 Adjuvanted Placebo 133 (20) (40–270) 165 (18) (70–375) 100 (15) (40–265) 0.96 0.79 45 (19) (5–80) 45 (16) (15–110) 25 (14) (5–45) 0.99 0.49 10 (12) (5–35) 15 (12) (5–40) 10 (7) (5–50) 0.64 0.72 Table 2. Cont. Table 3. Median fold increase in number of IFN-γ-producing, granzyme-producing, and dou- ble positive cells (SFU/million cells) in the adjuvanted vaccine and placebo group from day 0 to days 42 and 180. Cells were stimulated with a mix of four synthetic peptides that composed the FLU-v vaccine or with whole inactivated inﬂuenza strains (A/California/7/2009 (H1N1), A/Shanghai/24/1990 (H3N2), A/Vietnam/1194/2004 (NIBRG-14) (H5N1), A/Anhui/1/2013 (NIBRG-268) (H7N9), and B/Brisbane/9/2014 (Yamagata lineage)). Fold increase on days 42 and 180 was deﬁned as the ratio between SFU counts on day 42 or 180, and SFU counts on day 0. n = number of samples to be included in the statistical analysis. CI = 95% conﬁdence interval. 3.2. ELISpot Responses to a Panel of Inﬂuenza Strains Median number of IFN-γ-secreting, granzyme-B-secreting, and double positive cells (SFU/million cells) in response to FLU-v and whole inactivated inﬂuenza strains (A/California/7/2009 (H1N1), A/Shanghai/24/1990 (H3N2), A/Vietnam/1194/2004 (NIBRG-14) (H5N1), A/Anhui/1/2013 (NIBRG-268) (H7N9), and B/Brisbane/9/2014 (Yamagata lineage)) in the group vaccinated with adjuvanted FLU-v and the placebo group on days 0, 42, and 180. n = number of samples to be included in statistical analysis. CI = 95% conﬁdence interval. Wilcoxon signed rank sum test was used to analyze differences between day 0 and 42 and between day 0 and 180 in each group. Signiﬁcant differences are indicated with p-values in bold. Antigen Treatment Group Median SFU (n) (CI) Day 0 IFN-γ Granzyme B Double Positive Median SFU (n) (CI) Day 42 IFN-γ Granzyme B Double Positive Median SFU (n) (CI) Day 180 IFN-γ Granzyme B Double Positive p-Value (Wilcoxon) Day 0–42 p-Value (Wilcoxon) Day 0–180 FLU-v Adjuvanted FLU-v 5 (49) (5–5) 125 (48) (50–200) 75 (40) (40–130) <0.0001 <0.0001 5 (48) (5–5) 40 (46) (10–60) 20 (41) (5–40) <0.0001 0.0047 5 (28) (5–5) 20 (25) (10–40) 5 (21) (5–20) <0.0001 0.0059 Adjuvanted Placebo 5 (22) (5–10) 5 (21) (5–10) 5 (17) (5–15) 0.83 0.14 5 (20) (5–10) 5 (19) (5–5) 5 (16) (5–15) 0.16 0.98 5 (12) (5–5) 5 (12) (5–5) 5 (7) (5–20) NA >0.99 H1N1 Adjuvanted FLU-v 128 (48) (50–260) 310 (47) (220–480) 240 (39) (95–400) <0.0001 0.0163 50 (47) (20–115) 240 (46) (95–380) 90 (40) (30–160) <0.0001 0.16 25 (28) (5–45) 125 (25) (30–170) 50 (21) (20–155) 0.0011 0.0025 Adjuvanted Placebo 265 (20) (75–410) 153 (18) (110–360) 160 (15) (70–390) 0.29 0.90 73 (18) (20–200) 65 (16) (20–125) 73 (14) (10–285) 0.76 0.62 23 (12) (10–70) 28 (12) (10–80) 30 (7) (5–155) 0.83 0.56 H3N2 Adjuvanted FLU-v 268 (48) (140–585) 555 (47) (355–695) 490 (39) (230–740) 0.0001 0.12 193 (48) (95–340) 485 (45) (360–630) 215 (40) (105–460) 0.0002 0.94 135 (29) (10–200) 220 (26) (155–385) 205 (22) (60–350) 0.0064 0.0449 Adjuvanted Placebo 510 (21) (205–800) 420 (19) (235–575) 460 (16) (230–865) 0.25 >0.99 235 (19) (70–530) 310 (17) (175–435) 310 (15) (95–480) 0.49 0.61 175 (12) (75–245) 138 (12) (75–230) 200 (7) (110–410) 0.42 0.78 Vaccines 2022, 10, 1528 6 of 14 Table 2. Cont. 3.2. ELISpot Responses to a Panel of Inﬂuenza Strains Antigen Treatment Group Median SFU (n) (CI) Day 0 IFN-γ Granzyme B Double Positive Median SFU (n) (CI) Day 42 IFN-γ Granzyme B Double Positive Median SFU (n) (CI) Day 180 IFN-γ Granzyme B Double Positive p-Value (Wilcoxon) Day 0–42 p-Value (Wilcoxon) Day 0–180 H5N1 Adjuvanted FLU-v 460 (45) (175–550) 668 (42) (530–810) 585 (37) (355–845) 0.0001 0.0381 305 (44) (160–495) 650 (41) (450–750) 325 (38) (115–570) 0.0041 0.69 245 (28) (110–335) 305 (24) (250–455) 260 (21) (175–465) 0.0220 0.18 Adjuvanted Placebo 493 (20) (250–890) 450 (17) (340–700) 470 (14) (170–1060) 0.73 0.80 490 (18) (120–800) 433 (16) (275–580) 360 (13) (105–650) 0.98 0.89 135 (11) (50–830) 230 (11) (80–725) 263 (6) (130–410) 0.76 0.56 H7N9 Adjuvanted FLU-v 130 (44) (75–210) 268 (40) (160–350) 215 (36) (70–280) 0.0026 0.27 50 (43) (15–100) 140 (39) (55–180) 50 (35) (15–105) 0.49 0.88 20 (28) (10–45) 60 (23) (20–115) 23 (20) (5–80) 0.06 0.47 Adjuvanted Placebo 150 (18) (40–430) 195 (16) (80–455) 140 (13) (30–265) 0.75 0.79 90 (18) (10–135) 83 (16) (10–260) 50 (12) (5–100) 0.99 0.58 20 (11) (5–170) 35 (11) (5–100) 30 (6) (5–45) 0.71 >0.99 B Adjuvanted FLU-v 130 (47) (75–200) 243 (46) (110–310) 135 (39) (60–230) 0.0085 0.63 25 (48) (10–75) 75 (45) (45–120) 40 (39) (15–75) 0.0601 0.27 10 (28) (5–20) 30 (25) (15–50) 20 (21) (10–50) 0.0046 0.21 Adjuvanted Placebo 133 (20) (40–270) 165 (18) (70–375) 100 (15) (40–265) 0.96 0.79 45 (19) (5–80) 45 (16) (15–110) 25 (14) (5–45) 0.99 0.49 10 (12) (5–35) 15 (12) (5–40) 10 (7) (5–50) 0.64 0.72 Table 3. Median fold increase in number of IFN-γ-producing, granzyme-producing, and dou- ble positive cells (SFU/million cells) in the adjuvanted vaccine and placebo group from day 0 to days 42 and 180. Cells were stimulated with a mix of four synthetic peptides that composed the FLU-v vaccine or with whole inactivated inﬂuenza strains (A/California/7/2009 (H1N1), A/Shanghai/24/1990 (H3N2), A/Vietnam/1194/2004 (NIBRG-14) (H5N1), A/Anhui/1/2013 (NIBRG-268) (H7N9), and B/Brisbane/9/2014 (Yamagata lineage)). Fold increase on days 42 and 180 was deﬁned as the ratio between SFU counts on day 42 or 180, and SFU counts on day 0. n = number of samples to be included in the statistical analysis. CI = 95% conﬁdence interval. 3.2. ELISpot Responses to a Panel of Inﬂuenza Strains The Mann–Whitney U test (MW) was used to compare fold increases between groups, and test the null hypothesis of equal distributions in the vaccine group and placebo group on day 42 and on day 180. Since we cannot reasonably assume that the distributions have the same shape, and only differ with respect to location, the Mann–Whitney U test cannot be interpreted as a comparison of medians. Signiﬁcant differences are indicated with p-values in bold. Antigen Median Fold Increase (n) (CI) Day 42 Median Fold Increase (n) (CI) Day 180 Adjuvanted FLU-v IFN-γ Granzyme B Double Positive Adjuvanted Placebo IFN-γ Granzyme B Double Positive p-Value (MW) Adjuvanted FLU-v IFN-γ Granzyme B Double Positive Adjuvanted Placebo IFN-γ Granzyme B Double Positive p-Value (MW) FLU-v 16.3 (48) (9.0–25.0) 1.0 (21) (1.0–1.0) <0.0001 9.5 (40) (4.0–19.0) 1.0 (17) (1.0–2.0) <0.0001 3.5 (46) (2.0–9.0) 1.0 (19) (0.5–1.0) <0.0001 2.0 (41) (1.0–5.0) 1.0 (16) (0.4–3.0) 0.0461 3.0 (25) (1.0–8.0) 1.0 (12) (1.0–1.0) 0.0012 1.0 (21) (1.0–4.0) 1.0 (7) (1.0–4.0) 0.30 H1N1 2.3 (47) (1.8–3.1) 0.8 (18) (0.5–2.0) 0.0083 1.9 (39) (1.4–3.0) 0.8 (15) (0.4–3.3) 0.23 3.5 (46) (2.1–4.4) 1.0 (16) (0.5–3.0) 0.0075 1.2 (40) (0.8–2.6) 0.9 (14) (0.1–5.5) 0.52 2.9 (25) (2.0–5.0) 1.0 (12) (0.6–1.3) 0.0219 1.8 (21) (1.0–6.6) 0.5 (7) (0.1–7.8) 0.11 Median Fold Increase (n) (CI) Day 42 Median Fold Median Fold Increase (n) (CI) Day 42 Vaccines 2022, 10, 1528 7 of 14 Table 3. Cont. Table 3. Cont. 3.2. ELISpot Responses to a Panel of Inﬂuenza Strains Antigen Median Fold Increase (n) (CI) Day 42 Median Fold Increase (n) (CI) Day 180 Adjuvanted FLU-v IFN-γ Granzyme B Double Positive Adjuvanted Placebo IFN-γ Granzyme B Double Positive p-Value (MW) Adjuvanted FLU-v IFN-γ Granzyme B Double Positive Adjuvanted Placebo IFN-γ Granzyme B Double Positive p-Value (MW) H3N2 1.7 (47) (1.3–2.0) 0.8 (19) (0.5–1.5) 0.0178 1.3 ((39) 0.8–2.5) 1.2 (16) (0.2–2.2) 0.43 1.8 (45) (1.1–2.6) 1.2 (17) (0.5–3.3) 0.33 0.9 (40) (0.7–1.6) 2.0 (15) (0.4–3.6) 0.39 1.7 (26) (1.0–2.6) 0.9 (12) (0.6–1.4) 0.0136 1.3 (22) (0.8–4.0) 1.5 (7) (0.2–4.0) 0.56 H5N1 1.7 (42) (1.2–2.4) 1.0 (17) (0.7–1.7) 0.0441 1.5 (37) (1.1–1.8) 1.2 (14) (0.4–3.0) 0.57 1.5 (41) (1.3–2.6) 0.9 (16) (0.7–2.6) 0.28 0.9 (38) (0.5–1.6) 1.6 (13) (0.6–3.2) 0.31 1.4 (24) (1.1–2.6) 0.9 (11) (0.6–2.1) 0.25 1.2 (21) (0.9–2.5) 2.2 (6) (0.5–12.7) 0.60 H7N9 2.2 (40) (1.2–3.3) 0.9 (16) (0.5–4.3) 0.25 1.2 (35) (0.9–3.0) 1.1 (13) (0.2–3.5) 0.42 1.7 (39) (0.6–4.0) 0.8 (16) (0.2–11.0) 0.62 1.0 (35) (0.3–3.0) 1.1 (12) (0.1–4.0) 0.75 2.1 (23) (0.5–3.3) 0.6 (11) (0.3–10.0) 0.59 1.0 (20) (1.0–2.0) 1.6 (6) (0.0–9.0) 0.68 B 1.5 (46) (1.0–2.5) 1.2 (18) (0.6–2.3) 0.51 1.3 (39) (0.6–2.3) 1.1 (15) (0.3–3.5) 0.97 2.0 (45) (1.0–3.3) 0.9 (16) (0.3–5.0) 0.24 0.8 (39) (0.4–1.6) 1.0 (14) (0.1–6.1) 0.88 2.0 (25) (1.0–6.0) 0.8 (12) (0.5–2.0) 0.0227 1.2 (21) (1.0–5.0) 1.5 (7) (0.1–5.0) 0.61 The overall IFN-γ fold increase levels from pre- to post-vaccination in the vaccinated group after stimulation with the inﬂuenza strains were lower than the response seen after stimulation with the vaccine antigens, but still signiﬁcantly higher than in the placebo group on day 42 for H1N1 (medians: 2.3-fold vs. 0.8-fold, p = 0.0083), H3N2 (medians: 1.7-fold vs. 0.8-fold, p = 0.0178), and H5N1 (medians: 1.7 vs. 1.0, p = 0.0441) (Table 3). Adju- vanted FLU-v vaccination also induced a signiﬁcantly higher fold increase for granzyme-B- producing cells in response to H1N1 (medians: 3.5-fold vs. 1.0-fold, p = 0.0075) on day 42 compared to placebo (Table 3). Moreover, analysis of the double positive cell population showed a signiﬁcant difference in fold increase between the vaccine and placebo group for H1N1 (medians: 2.9-fold vs. 1.0-fold, p = 0.0219), H3N2 (medians: 1.7-fold vs. 0.9-fold, p = 0.0136), and the B strain (medians: 2.0-fold vs. 0.8-fold, p = 0.0227) on day 42 after vaccination (Table 3). 3.2. ELISpot Responses to a Panel of Inﬂuenza Strains No signiﬁcant differences in fold increase from baseline to day 180 were observed between the vaccine and placebo groups for any of the strains tested. Median Fold Increase (n) (CI) Day 42 Median Fold Increase (n) (CI) Day 180 The overall IFN-γ fold increase levels from pre- to post-vaccination in the vaccinated group after stimulation with the inﬂuenza strains were lower than the response seen after stimulation with the vaccine antigens, but still signiﬁcantly higher than in the placebo group on day 42 for H1N1 (medians: 2.3-fold vs. 0.8-fold, p = 0.0083), H3N2 (medians: 1.7-fold vs. 0.8-fold, p = 0.0178), and H5N1 (medians: 1.7 vs. 1.0, p = 0.0441) (Table 3). Adju- vanted FLU-v vaccination also induced a signiﬁcantly higher fold increase for granzyme-B- producing cells in response to H1N1 (medians: 3.5-fold vs. 1.0-fold, p = 0.0075) on day 42 compared to placebo (Table 3). Moreover, analysis of the double positive cell population showed a signiﬁcant difference in fold increase between the vaccine and placebo group for H1N1 (medians: 2.9-fold vs. 1.0-fold, p = 0.0219), H3N2 (medians: 1.7-fold vs. 0.9-fold, p = 0.0136), and the B strain (medians: 2.0-fold vs. 0.8-fold, p = 0.0227) on day 42 after vaccination (Table 3). No signiﬁcant differences in fold increase from baseline to day 180 were observed between the vaccine and placebo groups for any of the strains tested. 3.3. Correlation Analysis Correlation analyses were performed between the number of cells secreting the dif- ferent markers detected on day 42 after stimulation with FLU-v antigens and stimulation with the individual strains in vaccinated individuals to determine whether the cellular responses to the FLU-v antigens were associated with responses to the inﬂuenza strains. Strong correlations were observed for the number of cells secreting INF-γ in response to FLU-v and in response to the A inﬂuenza strains, but to a lesser extent to the B strain (H1N1: r = 0.84, H3N2: r = 0.78, H5N1: r = 0.79, H7N9: r = 0.83, B: r = 0.60, p < 0.0001 for all correlations) (Figure 1). We also found similar correlations between the number of granzyme-B-producing cells after stimulation with FLU-v and the inﬂuenza strains (H1N1: r = 0.80, H3N2: r = 0.71, H5N1: r = 0.65, H7N9: r = 0.59, B: r = 0.61, p < 0.0001 for all correlations) (Figure 2), but not for double positive cells. Vaccines 2022, 10, 1528 8 of 14 1 for all H5N1 INF- SFU/million cells Figure 1. Correlation analysis between the number of IFN-γ-secreting cells (SFU/million ce response to the equimolar mix of the four synthetic peptides included in the FLU-v vaccine a response to whole inactivated influenza strains (A/California/7/2009 (H1N1), A/Shanghai/24 (H3N2), A/Vietnam/1194/2004 (NIBRG-14) (H5N1), A/Anhui/1/2013 (NIBRG-268) (H7N9) B/Brisbane/9/2014 (Yamagata lineage)) on day 42 in the FLU-v vaccinated group. Correlation ficients (r) and p-values according to the Spearman analysis are indicated. Figure 1. Correlation analysis between the number of IFN-γ-secreting cells (SFU/million cell response to the equimolar mix of the four synthetic peptides included in the FLU-v vaccine an response to whole inactivated inﬂuenza strains (A/California/7/2009 (H1N1), A/Shanghai/24/ (H3N2), A/Vietnam/1194/2004 (NIBRG-14) (H5N1), A/Anhui/1/2013 (NIBRG-268) (H7N9), B/Brisbane/9/2014 (Yamagata lineage)) on day 42 in the FLU-v vaccinated group. Correla coefﬁcients (r) and p-values according to the Spearman analysis are indicated. H5N1 INF- SFU/million cells H5N1 INF- SFU/million cells Figure 1. Correlation analysis between the number of IFN-γ-secreting cells (SFU/million cells) in response to the equimolar mix of the four synthetic peptides included in the FLU-v vaccine and in response to whole inactivated influenza strains (A/California/7/2009 (H1N1), A/Shanghai/24/1990 (H3N2), A/Vietnam/1194/2004 (NIBRG-14) (H5N1), A/Anhui/1/2013 (NIBRG-268) (H7N9), and B/Brisbane/9/2014 (Yamagata lineage)) on day 42 in the FLU-v vaccinated group. Correlation coef- ficients (r) and p-values according to the Spearman analysis are indicated. Figure 1. 3.3. Correlation Analysis Correlation analysis between the number of IFN-γ-secreting cells (SFU/million cells) in response to the equimolar mix of the four synthetic peptides included in the FLU-v vaccine and in response to whole inactivated inﬂuenza strains (A/California/7/2009 (H1N1), A/Shanghai/24/1990 (H3N2), A/Vietnam/1194/2004 (NIBRG-14) (H5N1), A/Anhui/1/2013 (NIBRG-268) (H7N9), and B/Brisbane/9/2014 (Yamagata lineage)) on day 42 in the FLU-v vaccinated group. Correlation coefﬁcients (r) and p-values according to the Spearman analysis are indicated. 9 of 14 9 of 14 Vaccines 2022, 10, 1528 Vaccines 2022, 10, 1528 H1N1 granz B SFU/million cells H3N2 granz B SFU/million cells H7N9 granz B SFU/million cells B granz B SFU/million cells Figure 2. Correlation analysis between the number of granzyme-B-secreting cells (SFU/milli in response to the equimolar mix of the four synthetic peptides included in the FLU-v vac in response to whole inactivated influenza strains (A/California/7/2009 (H1N1), A/Shanghai (H3N2), A/Vietnam/1194/2004 (NIBRG-14) (H5N1), A/Anhui/1/2013 (NIBRG-268) (H7N B/Brisbane/9/2014 (Yamagata lineage)) on day 42 in the FLU-v vaccinated group. Correlat ficients (r) and p-values according to the Spearman analysis are indicated. Figure 2. Correlation analysis between the number of granzyme-B-secreting cells (SFU/millio in response to the equimolar mix of the four synthetic peptides included in the FLU-v vaccine response to whole inactivated inﬂuenza strains (A/California/7/2009 (H1N1), A/Shanghai/24 (H3N2), A/Vietnam/1194/2004 (NIBRG-14) (H5N1), A/Anhui/1/2013 (NIBRG-268) (H7N9 B/Brisbane/9/2014 (Yamagata lineage)) on day 42 in the FLU-v vaccinated group. Corre coefﬁcients (r) and p-values according to the Spearman analysis are indicated. 4. Discussion Many efforts have been made over the years to make inﬂuenza vaccines more eff Numerous attempts have concentrated on improving the duration and titer of the ant H3N2 granz B SFU/million cells H1N1 granz B SFU/million cells H7N9 granz B SFU/million cells B granz B SFU/million cells B granz B SFU/million cells Figure 2. Correlation analysis between the number of granzyme-B-secreting cells (SFU/million cells) in response to the equimolar mix of the four synthetic peptides included in the FLU-v vaccine and in response to whole inactivated influenza strains (A/California/7/2009 (H1N1), A/Shanghai/24/1990 (H3N2), A/Vietnam/1194/2004 (NIBRG-14) (H5N1), A/Anhui/1/2013 (NIBRG-268) (H7N9), and B/Brisbane/9/2014 (Yamagata lineage)) on day 42 in the FLU-v vaccinated group. Correlation coef- ficients (r) and p-values according to the Spearman analysis are indicated. Figure 2. 3.3. Correlation Analysis Correlation analysis between the number of granzyme-B-secreting cells (SFU/million cells) in response to the equimolar mix of the four synthetic peptides included in the FLU-v vaccine and in response to whole inactivated inﬂuenza strains (A/California/7/2009 (H1N1), A/Shanghai/24/1990 (H3N2), A/Vietnam/1194/2004 (NIBRG-14) (H5N1), A/Anhui/1/2013 (NIBRG-268) (H7N9), and B/Brisbane/9/2014 (Yamagata lineage)) on day 42 in the FLU-v vaccinated group. Correlation coefﬁcients (r) and p-values according to the Spearman analysis are indicated. 4. Discussion Many efforts have been made over the years to make inﬂuenza vaccines more effective. Numerous attempts have concentrated on improving the duration and titer of the antibody Vaccines 2022, 10, 1528 10 of 14 10 of 14 response [12]. Although representing a valid short-term improvement, this is not a sustain- able long-term solution to the problem of antigenic drift and shift, and achieving broader protection continues to be a major goal [2,10]. The peptide-based FLU-v vaccine candidate aims to address this problem by combining short, conserved regions of the inﬂuenza pro- teins, M1, M2, and NP [4]. Previous studies have shown that FLU-v induces both antibodies and cellular immune responses in pre-clinical [4] and clinical settings [5–7,9], as well as protection against mild-to-moderate inﬂuenza disease in a human challenge study [9]. FLU-v-speciﬁc antibodies are not expected to be neutralizing, as they do not target epitopes on the viral surface, but they could bind to infected cells, triggering a cytotoxic response by means of activating complement or ADCC responses. The cellular immune responses triggered by FLU-v vaccination are biased towards Th1, as previously measured by multi- parametric ﬂow cytometry and cytokine ELISA [5], and the IgG antibodies generated include both IgG1 and IgG3 subclass components (unpublished data), which are relevant for ADCC responses. To further evaluate cellular effector functions and vaccine-induced cross-reactivity, PBMC samples from the UNISEC study [5,6] were assessed for in vitro responses to FLU-v antigens and a panel of inactivated heterosubtypic inﬂuenza strains by means of using dual ELISpot to enumerate IFN-γ- and granzyme-B-producing cells as markers for cytotoxic cellular responses. y p As demonstrated in this study, adjuvanted FLU-v vaccination induced a signiﬁcant increase in the number of vaccine-speciﬁc IFN-γ- and granzyme-B-secreting cells in re- sponse to the FLU-v antigens. Interestingly, the responses to FLU-v antigens were low pre-vaccination and in the placebo group, indicating that natural exposure to inﬂuenza does not generate signiﬁcant cellular responses to the viral protein regions covered by FLU-v, but they are induced through vaccination. A wide range of protein epitopes compete for binding to MHC class I and II molecules during an infection, leading to CD8+ and CD4+ T cell activation, respectively. Immunodominant epitopes are more successful in being pre- sented to the immune system than subdominant epitopes due to the intrinsic characteristics of the epitope sequences, genetic factors such as MHC alleles, and how the antigens are processed [13,14]. 4. Discussion However, epitope binding to MHC molecules does not always translate into activation of cells, leading to protective or cross-reactive responses. The FLU-v vaccine trains the immune system to respond to conserved epitopes that would normally not be presented due to competition with more immunodominant epitopes, which explains why a response is observed only after vaccination. Others have shown that vaccination with subdominant epitopes is a viable approach for inducing protection against respiratory viral infections [15,16]. Vaccination targeting non-dominant epitopes alone facilitated a broader and more potent response against murine lymphocytic choriomeningitis virus as compared to adding immunodominant epitopes [17]. As shown in this work, in vitro testing of PBMC for responses to heterosubtypic in- ﬂuenza strains conﬁrmed that FLU-v vaccination not only generates an immune response able to recognize naturally processed and presented inﬂuenza antigens, but also demon- strated that these responses are cross-reactive for diverse inﬂuenza A and B strains. A critical step in the development of broad-spectrum inﬂuenza vaccines is to demonstrate the induction of cross-reactive cellular responses contributing to protection from a variety of seasonal and pandemic inﬂuenza strains. However, demonstrating cross-protection in a clinical setting is challenging due to the fact that only a limited number of inﬂuenza subtypes circulate in the human population within a given season, and the emergence of those strains that pose a pandemic threat is difﬁcult to predict. It is possible to perform controlled human challenge studies, but only a handful of relevant strains have been approved for infection of volunteers. This situation calls for alternative approaches to demonstrate the broadness of cellular immunity. Employing in vitro immune assays to measure well established surrogate markers of protection may allow for the evaluation of predicted efﬁcacy against any inﬂuenza strains of interest, including those with pandemic potential isolated from animals. As a step in this direction, we have, in this study, demon- strated that cellular immune responses induced by FLU-v vaccination cross-recognized all Vaccines 2022, 10, 1528 11 of 14 11 of 14 inﬂuenza strains tested (H1N1, H3N2, H5N1, H7N9, and B inﬂuenza), as measured by an increase in IFN-γ- and granzyme-B-secreting cells detected in vitro. Additional support for vaccine-induced cross-reactive cellular immune responses was obtained by demonstrating a direct correlation between the responses to FLU-v and the responses to the individual viral strains. 4. Discussion Cross-recognition of the strains tested here is of importance because H1N1 and H3N2 have been the main circulating seasonal strains for many years [18], whereas H5N1 and H7N9 are strains of high concern due to their pandemic potential [19,20]. In contrast to the low background levels of responses to FLU-v antigens prior to vaccination, IFN-γ and granzyme B responses to the inﬂuenza strains were elevated before vaccination, thereby reducing the fold increase from pre- to post-vaccination. Although the innate responses to viral components may have contributed to higher pre-vaccination levels, it is also likely that adaptive cross-priming with naturally circulating seasonal inﬂuenza (H1N1 and H3N2) has induced pre-immunity to other strains not circulating among the study participants [21,22]. Naturally occurring cells cross-reacting with inﬂuenza strains not normally circulating in the tested population has also been observed by others [22–25]. The presence of pre-immunity at baseline may have reduced the ability to detect the true potential of FLU-v vaccination to induce cross-reactive cellular responses with the assay conditions used here. It is also likely that the use of live virus stimulation or transfection of target cells with inactivated virus would represent a more efﬁcient methodological approach for detecting CD8+ T cell responses, which are the main cell type with cytotoxic potential. This was not possible due to biosafety restrictions. p p y The mode of action for the current seasonal inﬂuenza vaccines is the generation of neutralizing antibodies against the major surface glycoprotein hemagglutinin (HA), com- monly quantiﬁed by the hemagglutinin inhibition (HI) assay and used as the traditional correlate of protection [3,26]. However, evaluation of universal inﬂuenza vaccines that work by activating T-cell responses requires alternative correlates of protection that should be standardized to be suitable for use in large multicenter clinical trials [3,26,27]. ELISpot is a functional, quantitative, and sensitive assay for the detection of cytokines and other immune markers at the single cell level, providing a suitable tool for assessing the im- munogenicity and biomarkers of vaccine efﬁcacy in clinical trials [28]. The dual ELISpot assay used here allows for the rapid measurement of IFN-γ-producing cells as a marker of Th1 responses, and granzyme-B-producing cells as a marker of cytotoxicity. Importantly, both of these immune parameters are associated with cell-mediated protection against inﬂuenza disease [3,29]. 4. Discussion It has been shown that the presence of both CD8+ and CD4+ T cells producing IFN-γ correlated with a low total symptom score after infection [30,31], and viral clearance and reduced shedding in the absence of speciﬁc antibodies [32]. Granzyme B contributes to protection by signaling the elimination of virus-infected host cells [33,34] using the apoptotic pathway, resulting in DNA fragmentation and a rapid loss of membrane integrity [35]. Although ELISpot does not discriminate between the different cell pheno- types, previous work indicated that CD8+ T cells with cytotoxic potential were induced by FLU-v vaccination, as detected by transfection of FLU-v into target cells exposed to splenocytes from vaccinated mice [4]. Moreover, depletion of CD8+ T cells resulted in reduced IFN-γ secretion from PBMC in FLU-v vaccinated human volunteers [8]. Low CD8+ T cell stimulation in vitro, as previously reported for this clinical study [5], may most likely be due to the assay conditions not being optimal for efﬁcient presentation of FLU-v antigens by MHC class I molecules rather than a lack of response. The efﬁcacy of FLU-v was tested in an H1N1 challenge study in human volunteers, and a single dose of adjuvanted FLU-v was more effective in reducing mild-to-moderate disease than two doses of adjuvanted FLU-v [9]; therefore, increasing the number of doses seems unlikely to provide additional beneﬁts, at least as measured with these parameters in a short-term perspective. p p p The reported IFN-γ response triggered by the FLU-v antigens in this study is consistent with previous results obtained with other immunological assays, such as IFN-γ ELISA and multi-parametric ﬂow-cytometry [5]. Detection of granzyme-B-secreting cells in vaccinated subjects provides additional evidence for the induction of cellular responses with cytotoxic Vaccines 2022, 10, 1528 12 of 14 12 of 14 capacity after FLU-v vaccination. In addition, detection of double positive cells, although at a low frequency, indicates that FLU-v activates a cell type that exhibits multifunctional properties and may play an important role in protection against inﬂuenza infection [36]. In summary, this study reports that adjuvanted FLU-v vaccination can induce cross- reactive cellular responses with potential cytotoxic capacity, as detected by dual IFN-γ and granzyme B ELISpot assays. References 1. Paules, C.I.; Sullivan, S.G.; Subbarao, K.; Fauci, A.S. Chasing Seasonal Inﬂuenza–The Need for a Universal Inﬂuenza Vaccine. N. Engl. J. Med. 2018, 378, 7–9. [CrossRef] [PubMed] 1. Paules, C.I.; Sullivan, S.G.; Subbarao, K.; Fauci, A.S. Chasing Seasonal Inﬂuenza–The Need for a Universal Inﬂuenza Vaccine. N. Engl. J. Med. 2018, 378, 7–9. [CrossRef] [PubMed] g J , , [ ] [ ] 2. Elbahesh, H.; Saletti, G.; Gerlach, T.; Rimmelzwaan, G.F. Broadly protective inﬂuenza vaccines: Desi Curr. Opin. Virol. 2019, 34, 1–9. [CrossRef] [PubMed] g 2. Elbahesh, H.; Saletti, G.; Gerlach, T.; Rimmelzwaan, G.F. Broadly protective inﬂuenza vaccines: Design and production platforms. Curr. Opin. Virol. 2019, 34, 1–9. [CrossRef] [PubMed] 2. Elbahesh, H.; Saletti, G.; Gerlach, T.; Rimmelzwaan, G.F. Broadly protective inﬂuenza vaccines: Design and production platforms. Curr. Opin. Virol. 2019, 34, 1–9. [CrossRef] [PubMed] 3. van Els, C.; Mjaaland, S.; Næss, L.; Sarkadi, J.; Gonczol, E.; Korsholm, K.S.; Hansen, J.; de Jonge, J.; Kersten, G.; Warner, J.; et al. Fast vaccine design and development based on correlates of protection (COPs). Hum. Vaccin. Immunother 2014, 10, 1935–1948. [CrossRef] [PubMed] 3. van Els, C.; Mjaaland, S.; Næss, L.; Sarkadi, J.; Gonczol, E.; Korsholm, K.S.; Hansen, J.; de Jonge, J.; Kersten, G.; Warner, J.; et al. Fast vaccine design and development based on correlates of protection (COPs). Hum. Vaccin. Immunother 2014, 10, 1935–1948. [CrossRef] [PubMed] 4. Stoloff, G.A.; Caparros-Wanderley, W. Synthetic multi-epitope peptides identiﬁed in silico induce protective immunity against multiple inﬂuenza serotypes. Eur. J. Immunol. 2007, 37, 2441–2449. [CrossRef] [PubMed] 4. Stoloff, G.A.; Caparros-Wanderley, W. Synthetic multi-epitope peptides identiﬁed in silico induce protective immunity against multiple inﬂuenza serotypes. Eur. J. Immunol. 2007, 37, 2441–2449. [CrossRef] [PubMed] p yp 5. Pleguezuelos, O.; Dille, J.; de Groen, S.; Oftung, F.; Niesters, H.G.M.; Islam, M.A.; Næss, L.M.; Hungnes, O.; Aldarij, N.; Idema, D.L.; et al. Immunogenicity, Safety, and Efﬁcacy of a Standalone Universal Inﬂuenza Vaccine, FLU-v, in Healthy Adults: A Randomized Clinical Trial. Ann. Intern. Med. 2020, 172, 453–462. [CrossRef] [PubMed] y 5. Pleguezuelos, O.; Dille, J.; de Groen, S.; Oftung, F.; Niesters, H.G.M.; Islam, M.A.; Næss, L.M.; Hungnes, O.; Aldarij, N.; Idema, D.L.; et al. Immunogenicity, Safety, and Efﬁcacy of a Standalone Universal Inﬂuenza Vaccine, FLU-v, in Healthy Adults: A Randomized Clinical Trial. Ann. Intern. Med. 2020, 172, 453–462. [CrossRef] [PubMed] 6. Data Availability Statement: Archives of the collected data are not publicly available. Acknowledgments: We thank the volunteers participating in the clinical trial (EudraCT: 2015-001932- 38); the WP leader for clinical trials in UNISEC, Eelko Hak; the clinical team conducting the study at the Isala Hospital in the Netherlands; and the laboratory team at the Norwegian Institute of Public Health (responsible person, Gro Ellen Korsvold). Conﬂicts of Interest: Fredrik Oftung, Lisbeth M. Næss, Ida Laake, and Olga Pleguezuelos declare no conﬂict of interest. Gregory Stoloff is a shareholder at SEEK. 4. Discussion Moreover, the results suggest that further optimization and usage of such in vitro assays may serve as a standardized approach to evaluate cross- reactive cell-mediated immune responses in clinical testing of universal vaccines, with options for assessing even potential pandemic strains not yet in human circulation. In conclusion, the data show that adjuvanted FLU-v is a promising broad-spectrum inﬂuenza vaccine candidate that warrants further testing for protective efﬁcacy against disease in clinical phase III trials. Author Contributions: Conception and design of the study, O.P., G.S. and F.O. Acquisition of data, F.O., L.M.N. and O.P. Analysis and interpretation of data, F.O., L.M.N., I.L. and O.P. Drafting or revising the article, F.O., L.M.N., I.L., G.S. and O.P. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by European Commission Directorate-General for Research and Innovation, European Member States within the UNISEC (Universal Inﬂuenza Vaccines Secured) project (FP7-Health No. 602012) and private funding (SEEK). Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and ethical approval was obtained from the Central Committee on Research Involving Human Subjects (CCMO) (reference NL55061.000.15). Informed Consent Statement: Prior to participation in the trial, written informed consent forms (ICF) were obtained from each subject. Data Availability Statement: Archives of the collected data are not publicly available. Data Availability Statement: Archives of the collected data are not publicly available. p f 7. Pleguezuelos, O.; Robinson, S.; Stoloff, G.A.; Caparrós-Wanderley, W. Synthetic Inﬂuenza vaccine (FLU-v) stimulates cell mediated immunity in a double-blind, randomised, placebo-controlled Phase I trial. Vaccine 2012, 30, 4655–4660. [CrossRef] 6. van Doorn, E.; Pleguezuelos, O.; Liu, H.; Fernandez, A.; Bannister, R.; Stoloff, G.; Oftung, F.; Norley, S.; Huckriede, A.; Frijlink, H.W.; et al. Evaluation of the immunogenicity and safety of different doses and formulations of a broad spectrum inﬂuenza vaccine (FLU-v) developed by SEEK: Study protocol for a single-center, randomized, double-blind and placebo-controlled clinical phase IIb trial. BMC Infect. Dis. 2017, 17, 241. [CrossRef] [PubMed] References Efﬁcient priming of CD8+ memory T cells speciﬁc for a subdominant epitope following Sendai virus infection. J. Immunol. 1997, 158, 4301–4309. p p g 16. Oukka, M.; Manuguerra, J.C.; Livaditis, N.; Tourdot, S.; Riche, N.; Vergnon, I.; Cordopatis, P.; Kosm against lethal viral infection by vaccination with nonimmunodominant peptides. J. Immunol. 1996, 157 17. Holst, P.J.; Jensen, B.A.; Ragonnaud, E.; Thomsen, A.R.; Christensen, J.P. Targeting of non-dominant antigens as a vaccine strategy to broaden T-cell responses during chronic viral infection. PLoS ONE 2015, 10, e0117242. [CrossRef] p g , , [ ] 18. Petrova, V.N.; Russell, C.A. The evolution of seasonal inﬂuenza viruses. Nat. Rev. Microbiol. 2018, 16, 47–60. [CrossRef] 19. Sutton, T.C. The Pandemic Threat of Emerging H5 and H7 Avian Inﬂuenza Viruses. Viruses 2018, 10, 4 The Pandemic Threat of Emerging H5 and H7 Avian Inﬂuenza Viruses. Viruses 2018, 10, 461. [CrossRef] 20. Uyeki, T.M.; Katz, J.M.; Jernigan, D.B. Novel inﬂuenza A viruses and pandemic threats. Lancet 2017 20. Uyeki, T.M.; Katz, J.M.; Jernigan, D.B. Novel inﬂuenza A viruses and pandemic threats. Lancet 2017, 389, 2172–2174. [CrossRef] 21. Greenbaum, J.A.; Kotturi, M.F.; Kim, Y.; Oseroff, C.; Vaughan, K.; Salimi, N.; Vita, R.; Ponomarenko, J.; Scheuermann, R.H.; Sette, A.; et al. Pre-existing immunity against swine-origin H1N1 inﬂuenza viruses in the general human population. Proc. Natl. Acad. Sci. USA 2009, 106, 20365–20370. [CrossRef] [PubMed] 22. Roti, M.; Yang, J.; Berger, D.; Huston, L.; James, E.A.; Kwok, W.W. Healthy human subjects have CD4+ T cells directed against H5N1 inﬂuenza virus. J. Immunol. 2008, 180, 1758–1768. [CrossRef] 23. van de Sandt, C.E.; Kreijtz, J.H.; de Mutsert, G.; Geelhoed-Mieras, M.M.; Hillaire, M.L.; Vogelzang-van Trierum, S.E.; Osterhaus, A.D.; Fouchier, R.A.; Rimmelzwaan, G.F. Human cytotoxic T lymphocytes directed to seasonal inﬂuenza A viruses cross-react with the newly emerging H7N9 virus. J. Virol. 2014, 88, 1684–1693. [CrossRef] [PubMed] y g g 24. Tu, W.; Mao, H.; Zheng, J.; Liu, Y.; Chiu, S.S.; Qin, G.; Chan, P.L.; Lam, K.T.; Guan, J.; Zhang, L.; et al. Cytotoxic T lymphocytes established by seasonal human inﬂuenza cross-react against 2009 pandemic H1N1 inﬂuenza virus. J. Virol. 2010, 84, 6527–6535. [CrossRef] [PubMed] 25. Lee, L.Y.; Ha do, L.A.; Simmons, C.; de Jong, M.D.; Chau, N.V.; Schumacher, R.; Peng, Y.C.; McMichael, A.J.; Farrar, J.J.; Smith, G.L.; et al. Memory T cells established by seasonal human inﬂuenza A infection cross-react with avian inﬂuenza A (H5N1) in healthy individuals. J. Clin. Invest. 2008, 118, 3478–3490. References van Doorn, E.; Pleguezuelos, O.; Liu, H.; Fernandez, A.; Bannister, R.; Stoloff, G.; Oftung, F.; Norley, S.; Huckriede, A.; Frijlink, H.W.; et al. Evaluation of the immunogenicity and safety of different doses and formulations of a broad spectrum inﬂuenza vaccine (FLU-v) developed by SEEK: Study protocol for a single-center, randomized, double-blind and placebo-controlled clinical phase IIb trial. BMC Infect. Dis. 2017, 17, 241. [CrossRef] [PubMed] p f [ ] [ ] 7. Pleguezuelos, O.; Robinson, S.; Stoloff, G.A.; Caparrós-Wanderley, W. Synthetic Inﬂuenza vaccine (FLU-v) stimulates cell mediated immunity in a double-blind, randomised, placebo-controlled Phase I trial. Vaccine 2012, 30, 4655–4660. [CrossRef] 13 of 14 13 of 14 Vaccines 2022, 10, 1528 8. Pleguezuelos, O.; Robinson, S.; Fernández, A.; Stoloff, G.A.; Mann, A.; Gilbert, A.; Balaratnam, G.; Wilkinson, T.; Lambkin- Williams, R.; Oxford, J.; et al. A Synthetic Inﬂuenza Virus Vaccine Induces a Cellular Immune Response That Correlates with Reduction in Symptomatology and Virus Shedding in a Randomized Phase Ib Live-Virus Challenge in Humans. Clin. Vaccine Immunol. 2015, 22, 828–835. [CrossRef] 9. Pleguezuelos, O.; James, E.; Fernandez, A.; Lopes, V.; Rosas, L.A.; Cervantes-Medina, A.; Cleath, J.; Edwards, K.; Neitzey, D.; Gu, W.; et al. Efﬁcacy of FLU-v, a broad-spectrum inﬂuenza vaccine, in a randomized phase IIb human inﬂuenza challenge study. NPJ Vaccines 2020, 5, 22. [CrossRef] [ ] 10. Liu, H.; Frijlink, H.W.; Huckriede, A.; van Doorn, E.; Schmidt, E.; Leroy, O.; Rimmelzwaan, G.; McCullough, K.; Whelan, M.; Hak, E. Inﬂuenza Vaccine Research funded by the European Commission FP7-Health-2013-Innovation-1 project. Vaccine 2016, 34, 5845–5854. [CrossRef] puis, L.; Deville, S.; Ascarateil, S.; Ganne, V. Montanide ISA 720 and 51: A new generation of water in oil nts for human vaccines. Expert. Rev. Vaccines 2002, 1, 111–118. [CrossRef] [PubMed] 11. Aucouturier, J.; Dupuis, L.; Deville, S.; Ascarateil, S.; Ganne, V. Montanide ISA 720 and 51: A new g emulsions as adjuvants for human vaccines. Expert. Rev. Vaccines 2002, 1, 111–118. [CrossRef] [PubMed 12. Podda, A. The adjuvanted inﬂuenza vaccines with novel adjuvants: Experience with the MF59-adjuvanted vaccine. Vaccine 2001, 19, 2673–2680. [CrossRef] 13. Sercarz, E.E.; Lehmann, P.V.; Ametani, A.; Benichou, G.; Miller, A.; Moudgil, K. Dominance and crypticity of T cell antigenic determinants. Annu. Rev. Immunol. 1993, 11, 729–766. [CrossRef] [PubMed] 14. Yewdell, J.W. Confronting complexity: Real-world immunodominance in antiviral CD8+ T cell responses. Immunity 2006, 25, 533–543. [CrossRef] 15. Cole, G.A.; Hogg, T.L.; Coppola, M.A.; Woodland, D.L. References [CrossRef] 26. Reber, A.; Katz, J. Immunological assessment of inﬂuenza vaccines and immune correlates of protection. Expert Rev. Vaccines 2013, 12, 519–536. [CrossRef] 27. Ward, B.J.; Pillet, S.; Charland, N.; Trepanier, S.; Couillard, J.; Landry, N. The establishment of surrogates and correlates of protection: Useful tools for the licensure of effective inﬂuenza vaccines? Hum. Vaccin. Immunother. 2018, 14, 647–656. [CrossRef] 28. Beebe, E.A.; Orr, M.T. Assessment of Antigen-Speciﬁc Cellular Immunogenicity Using Intracellular Cytokine Staining, ELISpot, and Culture Supernatants. Methods Mol. Biol. 2017, 1494, 313–320. [CrossRef] 29. Forrest, B.D.; Pride, M.W.; Dunning, A.J.; Capeding, M.R.; Chotpitayasunondh, T.; Tam, J.S.; Rappaport, R.; Eldridge, J.H.; Gruber, W.C. Correlation of cellular immune responses with protection against culture-conﬁrmed inﬂuenza virus in young children. Clin. Vaccine Immunol. 2008, 15, 1042–1053. [CrossRef] 30. Sridhar, S.; Begom, S.; Bermingham, A.; Hoschler, K.; Adamson, W.; Carman, W.; Bean, T.; Barclay, W.; Deeks, J.J.; Lalvani, A. Cellular immune correlates of protection against symptomatic pandemic inﬂuenza. Nat. Med. 2013, 19, 1305–1312. [CrossRef] 31 Wilkinson T M ; Li C K ; Chui C S ; Huang A K ; Perkins M ; Liebner J C ; Lambkin-Williams R ; Gilbert A ; Oxford J ; 30. Sridhar, S.; Begom, S.; Bermingham, A.; Hoschler, K.; Adamson, W.; Carman, W.; Bean, T.; Barcla Cellular immune correlates of protection against symptomatic pandemic inﬂuenza. Nat. Med. 2013 30. Sridhar, S.; Begom, S.; Bermingham, A.; Hoschler, K.; Adamson, W.; Carman, W.; Bean, T.; Barclay, W.; Deeks, J.J.; Lalvani, A. Cellular immune correlates of protection against symptomatic pandemic inﬂuenza. Nat. Med. 2013, 19, 1305–1312. [CrossRef] 31. Wilkinson, T.M.; Li, C.K.; Chui, C.S.; Huang, A.K.; Perkins, M.; Liebner, J.C.; Lambkin-Williams, R.; Gilbert, A.; Oxford, J.; Nicholas, B.; et al. Preexisting inﬂuenza-speciﬁc CD4+ T cells correlate with disease protection against inﬂuenza challenge in humans. Nat. Med. 2012, 18, 274–280. [CrossRef] [PubMed] p g y p p , , [ ] 31. Wilkinson, T.M.; Li, C.K.; Chui, C.S.; Huang, A.K.; Perkins, M.; Liebner, J.C.; Lambkin-Williams, R.; Gilbert, A.; Oxford, J.; Nicholas, B.; et al. Preexisting inﬂuenza-speciﬁc CD4+ T cells correlate with disease protection against inﬂuenza challenge in humans. Nat. Med. 2012, 18, 274–280. [CrossRef] [PubMed] 32. McMichael, A.J.; Gotch, F.M.; Noble, G.R.; Beare, P.A. Cytotoxic T-cell immunity to inﬂuenza. N. Engl. J. Med. 1983, 309, 13–17. [CrossRef] [PubMed] 33. McElhaney, J.E.; Gravenstein, S.; Upshaw, C.M.; Hooton, J.W.; Krause, P.; Drinka, P.; Bleackley, R.C. 33. McElhaney, J.E.; Gravenstein, S.; Upshaw, C.M.; Hooton, J.W.; Krause, P.; Drinka, P.; Bleackley, R.C. Granzyme B: A marker of risk for inﬂuenza in institutionalized older adults. Vaccine 2001, 19, 3744–3751. [CrossRef] 34. McElhaney, J.E.; Ewen, C.; Zhou, X.; Kane, K.P.; Xie, D.; Hager, W.D.; Barry, M.B.; Kleppinger, A.; Wang, Y.; Bleackley, R.C. Granzyme B: Correlates with protection and enhanced CTL response to inﬂuenza vaccination in older adults. Vaccine 2009, 27, 2418–2425. [CrossRef] , , [ ] [ ] 36. Brown, D.M.; Lee, S.; Garcia-Hernandez Mde, L.; Swain, S.L. Multifunctional CD4 cells expressing gamma interferon and perforin mediate protection against lethal inﬂuenza virus infection. J. Virol. 2012, 86, 6792–6803. [CrossRef] [ ] 35. Voskoboinik, I.; Whisstock, J.C.; Trapani, J.A. Perforin and granzymes: Function, dysfunction and Immunol. 2015, 15, 388–400. [CrossRef] [PubMed] ] isstock, J.C.; Trapani, J.A. Perforin and granzymes: Function, dysfunction and human pathology. Nat. Rev 88–400. [CrossRef] [PubMed] References Granzyme B: A marker of risk for inﬂuenza in institutionalized older adults. Vaccine 2001, 19, 3744–3751. [CrossRef] 14 of 14 14 of 14 Vaccines 2022, 10, 1528
https://openalex.org/W1120586323	https://scielo.conicyt.cl/pdf/estped/v40n2/art22.pdf	Spanish; Castilian	null	Discourse and Pedagogical Practice in High-performance School Frameworks	Estudios pedagógicos (Valdivia)	2,014	cc-by	9,236	* Estudio auspiciado por el Fondo Nacional de Ciencia y Tecnología (FONDECYT) del Gobierno de Chile, Proyecto N°1110478. RESUMEN El objetivo del artículo es analizar la relación entre el discurso sobre la práctica pedagógica y la práctica real en sala de clases de los profesores en establecimientos educativos de alto desempeño y bajo nivel socioeconómico de Santiago de Chile. Se implementa un estudio descriptivo de metodología mixta, entrevistas semiestructuradas a nueve docentes, y análisis de filmación de 34 horas de clases. Se encuentra coherencia entre el discurso referido al contexto institucional y el tipo de intervención del profesor, y desfase entre el discurso sobre la interacción profesor-estudiante y el tipo de intervención con ellos en la clase. Palabras clave: profesor, creencia, prácticas pedagógicas, relación profesor-alumno. Marco Antonio Villalta Páucar,a Diego Palacios Díaz.b Marco Antonio Villalta Páucar,a Diego Palacios Díaz.b a Escuela de Psicología, Facultad de Humanidades, Universidad de Santiago de Chile. Fono: 56-2-27184370. Correo electrónico: marco.villalta@usach.cl b Escuela de Psicología, Facultad de Humanidades, Universidad de Santiago de Chile. Fono: 56-2-27184370. Correo electrónico: diego.palacios@usach.cl ABSTRACT The aim of this paper is to analyze the relationship between the speech on teaching practice and the actual practice of teachers in educational institutions of high performance and low socioeconomic status in Santiago de Chile. A descriptive study of mixed methodology is implemented, with semistructured interviews of nine teachers, and film analysis of 34 hours of classes. There is coherence between the speech referred to the institutional context and the type of intervention by the teacher, yet there is a gap between the speech on teacher-student interaction and the type of intervention with their in class. Key words: teacher, beliefs, teaching practices, teacher-student relationship. Estudios Pedagógicos XL, N° 2: 373-389, 2014 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar* Discourse and Pedagogical Practice in High-performance School Frameworks Discurso e prática pedagógica em contextos de alto rendimento escolar Discurso y práctica pedagógica en contextos de alto rendimiento escolar* Discourse and Pedagogical Practice in High-performance School Frameworks Discurso e prática pedagógica em contextos de alto rendimento escolar 1. INTRODUCCIÓN Se encuentra un desfase entre lo que los profesores dicen y lo que efectivamente hacen como práctica pedagógica en sala de clases (Torrado y Pozo, 2006). Esta diferencia entre discurso y acción no es propia del campo educativo, es una realidad inherente a los estudios sociales, dado que el decir y el hacer respecto a algo está determinado por sus propias condiciones de realización (Taylor y Bogdan, 1998). La práctica cotidiana está influenciada por contingencias del contexto socio-espacial que no siempre pueden ser recogidas en el discurso que comúnmente se mueve en el campo de las explicaciones y las intenciones. El séptimo Informe sobre Desarrollo Humano en Chile (Programa de las Naciones Unidas para el Desarrollo [PNUD], 2009) analiza las prácticas cotidianas que obstaculizan y aquellas que potencian el desarrollo humano, definiendo las prácticas como “modos de actuar y relacionarse que despliegan las personas en espacios concretos de acción” (PNUD, 2009: 15), que articulan reglas institucionales, la subjetividad y el conocimiento práctico; el “saber hacer”. Estos tres elementos son las fuerzas que definen las posibilidades de desarrollo de las prácticas. Es el enfoque para el análisis de los discursos sobre la práctica que se considera en el presente estudio. En la educación, la reglas institucionales son constitutivas de la cultura institucional escolar, las cuales enmarcan los comportamientos, promueven valores, generan rituales y preservan tradiciones (Baeza, 2008). La cultura institucional es síntesis histórica de la acción coordinada de los actores educativos que se encuentran en el espacio escolar (Pérez Gómez, 1998). La subjetividad refiere al sentido personal que el sujeto da a su práctica, la cual es construida en la interacción con otros (PNUD, 2009). En tal sentido, el profesor construye su rol y tarea en el aprendizaje de los estudiantes, desde la experiencia personal relacionada al contexto en cual trabaja (Villalta, Martinic y Guzmán, 2011), y a sus teorías implícitas sobre el aprendizaje, adquiridas en la vida cotidiana de la escuela (Pecharromán y Pozo, 2006). El conocimiento práctico, fuertemente enmarcado por la cultura, reduce la incertidumbre y delimita las expectativas (PNUD, 2009); es el “saber hacer”–aunque no siempre eficaz– significativo para los actores educativos y, en consecuencia, resistente al cambio. Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Estudios Pedagógicos XL, N° 2: 373-389, 2014 á ó Resumo Analisa-se a relação entre o discurso sobre a prática pedagógica e a prática real dos professores em sala de aula, em estabelecimentos educativos com alto desempenho e baixo nível socioeconômico, em Santiago do Chile. Desenvolve-se um estudo descritivo com metodologia mista, com entrevistas semiestruturadas com nove docentes e análise da filmagem de 34 horas das aulas dos mesmos. Encontra-se coerência entre o discurso do contexto institucional e o tipo de intervenção do professor e uma defasagem entre o discurso sobre a interação professor- aluno e sobre o tipo de intervenção com os alunos em aula. Palavras chave: professor, crença, práticas pedagógicas, relação professor-aluno. 1. INTRODUCCIÓN Desde esta perspectiva se puede entender, por ejemplo, que las prácticas frontales de enseñanza sean hegemónicas en las aulas chilenas a pesar de que se modificaron las directrices nacionales de enseñanza e introdujeron nuevos recursos pedagógicos en el aula (Martinic y Vergara, 2007). El discurso del profesor sobre su práctica pedagógica integra una forma particular de interpretar la cultura institucional escolar, con sus creencias sobre identidad y rol en el proceso de enseñanza y aprendizaje, y con el oficio cotidiano de la enseñanza; lo cual confluye en una teoría sobre el “saber hacer” que, aunque posiblemente no tiene relación directa con la práctica pedagógica real en el aula, sí define las posibilidades de desarrollo de las mismas (Torrado y Pozo, 2006). El objetivo del artículo es analizar el discurso sobre la práctica pedagógica en relación a la práctica real en sala de clases que tienen los profesores de educación media en establecimientos educativos de alto desempeño y de bajo nivel socioeconómico de la Región Metropolitana. Es una investigación de tipo cualitativo, que toma como fuente de información 9 entrevistas semiestructuradas a profesores respecto a sus prácticas pedagógicas de aula en establecimientos educativos de alto desempeño, así como la filmación de las mismas. Son dos las preguntas que orientan el estudio: ¿qué elementos 374 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar del discurso constituyen la práctica pedagógica del docente en contextos educativos de alto rendimiento escolar?, y ¿cómo estos elementos discursivos se ponen de manifiesto en la práctica pedagógica en el aula? En el segundo título se exponen los referentes teóricos sobre cultura escolar y creencias del profesor sobre las prácticas y aprendizaje escolar; en el tercer título se presenta la metodología de estudio. A continuación, en el cuarto título se presentan los resultados del análisis de los discursos y del análisis de filmación de las clases observadas. Finalmente, en el quinto título se presenta la discusión y conclusiones del estudio. Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Estudios Pedagógicos XL, N° 2: 373-389, 2014 á ó Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Por otra parte, se encuentra que los cambios intencionados para el logro de aprendizaje en establecimientos educativos en contextos sociales vulnerables, en aspectos de la cultura escolar tales como el mejoramiento de los procesos organizacionales y las actividades de sala de clases, están asociados a logros de aprendizaje escolar (Volante et al., 2008). Esto pone en evidencia, por un lado, que la cultura escolar está constituida de patrones organizacionales aprendidos y, por lo tanto, éstos puede cambiar (Pirela de Faría y Sánchez de Gallardo, 2009); y, por otro lado, que el fracaso escolar en estos contextos es un tema de inequidad social y de calidad educativa, no de capacidades de los sujetos para aprender. Los estudios sobre la relación virtuosa entre cultura y aprendizaje escolar destacan aspectos como la relación emocional positiva entre profesores y estudiantes (Laboratorio Latinoamericano de Evaluación de la Calidad de la Educación [LLECE], 2010); el análisis colectivo para la toma de decisiones institucionales que contribuyen a una identidad institucional positiva (UNICEF y Ministerio de Educación de Chile [MINEDUC], 2004); prácticas de enseñanza sustentadas en el análisis de la experiencia profesional y que valoran la diversidad de los estudiantes (Murillo y Román, 2009); la satisfacción docente, elemento asociado a la promoción de climas positivos en el aula (Fernández y Cuadrado, 2008), entre otros. La relación entre cultura y aprendizaje escolar tiene como punto de encuentro vital los sucesos de sala de clases, lo cual ha sido foco de intervención para el mejoramiento educativo (Volante et al., 2008), espacio-tiempo donde se constituye la práctica pedagógica del docente (Villalta, Martinic y Guzmán, 2011). 2.1. CULTURA ESCOLAR Y APRENDIZAJE Según lo señala Baeza, la cultura escolar “es un sistema subyacente de normas, valores, rituales, tradiciones, ceremonias e historias que se acumulan a través del tiempo” (2008: 198), compartido por profesores, estudiantes y apoderados, y que configura determinadas expectativas, creencias y acciones. Es una construcción, síntesis histórica del “cruce de culturas” (Pérez Gómez, 1998) de quienes participan de ella e interactúan en el espacio y tiempo de la escuela. Desde esta perspectiva, existe una estrecha relación entre la cultura, en tanto ethos que impregna la vida en la escuela, y las prácticas sociales para convivir, enseñar y aprender en el espacio escolar. En este ethos coexisten diversas subculturas, como por ejemplo la cultura del docente, que define modos específicos de las prácticas educativas (Hargreaves, 2005), las que se encuentran asociadas a los aspectos estructurales de la organización de la escuela, y aportan a comprender el logro de los resultados educativos. La relación entre cultura y aprendizaje escolar ha sido documentada en diversos estudios para explicar el fracaso o el éxito educativo, destacando aspectos como el ambiente socioafectivo a nivel institucional, los niveles de autonomía que permite la estructura organizacional del establecimiento, y las posibilidades de profesionalizar el trabajo docente (Cornejo y Redondo, 2007); también los juicios positivos compartidos sobre la identidad institucional, tales como metas centradas en el aprendizaje, claridad en las normas de comportamiento al interior del establecimiento y las condiciones de infraestructura educativa (Villalta, 2014). La segmentación socioeconómica de la educación en Latinoamérica, especialmente en Chile, conlleva notorias diferencias en la calidad de la formación que reciben los estu- diantes, diferencias de capital cultural y, en consecuencia, la generación de expectativas y resultados de aprendizaje en relación directa con la inequidad estructural y simbólica del país (Baeza, 2008). La pobreza socioeconómica y de capital cultural –aspectos que confi- guran realidades sociales vulnerables a la exclusión social- está asociado a fracaso escolar (Ruíz y García, 2007). Por una parte, en los establecimientos que operan en sectores sociales vulnerables, el fracaso escolar suele ser atribuido además a las insuficientes capacidades cognitivas de los estudiantes, a su poca persistencia para enfrentar y superar dificultades, la baja autoestima, la carencia de redes sociales y de socialización primaria que les otorgue los códigos culturales para participar de modo efectivo en la escuela (Román, 2003). 375 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar En relación al aprendizaje escolar, se encuentra que las teorías implícitas que tienen los profesores acerca del aprendizaje ponen de manifiesto, entre otros aspectos, fuerte presencia de realismo ingenuo sobre el conocimiento, es decir, la idea del conocimiento como copia de la realidad (Martin et al., 2006); que las prácticas de enseñanza tienen distinto nivel de consistencia con la visión de mundo que manifiestan los profesores, y que se adaptan a las creencias sobre los dominios generales y específicos que es necesario desarrollar en el aprendizaje (Olafson & Schraw, 2006); que hay diferencias entre profesores con experiencia y estudiantes de pedagogía respecto al papel de los enfoques constructivistas en el proceso de enseñanza (Gómez y Guerra, 2012); que las creencias sobre el aprendizaje son diferenciadas desde la formación inicial para el profesor según el nivel de especialización –Educación Básica o Educación Media- (García y Sebastián, 2011); que las creencias cambian según el significado personal que tiene para el profesor su experiencia laboral en contextos sociales vulnerables (Barría et al., 2010), y; que las creencias tienen relación con procesos específicos de aprendizaje (Makuc, 2008). En síntesis, los estudios ponen de relieve, en primer lugar, que generalmente lo que los docentes piensan o creen no es siempre lo que realmente realizan en sus prácticas pedagógicas; aunque, no obstante ello, se debe reconocer y valorar que las creencias y teorías implícitas juegan un importante rol en términos de su función orientadora de la conducta de las personas. En segundo lugar, se pone en evidencia el valor altamente idiosincrático y contextual de las teorías implícitas, puesto que la evidencia empírica muestra de forma clara cómo estas concepciones varían y se adaptan según las particularidades contextuales y las demandas específicas a las que los docentes se ven enfrentados en su experiencia profesional cotidiana (Leal et al., 2009). Conocer las prácticas de enseñanza desde el discurso y la práctica real del profesor es una forma de abordar las disonancias encontradas entre discurso y práctica pedagógica partiendo de los aspectos donde ambas confluyen: se trata de acciones racionales que constituyen el ethos cultural de la institución escolar puesta de manifiesto en la sala de clases. Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Esto abre el desafío de desarrollar un abordaje metodológico que sitúe los procesos cognitivos del profesorado en una perspectiva ecológica para explicar las prácticas e interacciones que se dan en el contexto escolar, y que ofrece pautas para el comportamiento de los actores educativos. La comprensión de lo que los profesores creen y piensan sobre los fenómenos educativos, es un paso necesario para comprender cómo actúan en las aulas de clases. 3. MÉTODO Es un estudio de enfoque descriptivo interpretativo metodológicamente mixto, que combina técnicas de análisis cualitativas y herramientas cuantitativas en función de las preguntas y objetivo del estudio. 2.2. PENSAMIENTO DEL PROFESOR Y PRÁCTICAS DE ENSEÑANZA Las concepciones de los docentes acerca del aprendizaje y la enseñanza son elementos de central importancia al momento de comprender las conductas y prácticas que los profesores desarrollan en las aulas de clases. Diversos enfoques de investigación han estudiado cuáles son los procesos cognitivos que orientan las acciones del docente; diversidad de enfoques que han sido agrupados en lo que se ha denominado el paradigma del pensamiento del profesor (Marcelo, 1989). De acuerdo a Medina (2006), es posible reconocer dos tendencias en el estudio del paradigma del pensamiento del profesor: a) de los contenidos –diversos constructos que configuran el pensamiento educativo y la dimensión personal del profesorado: creencias, teorías implícitas, representaciones sociales, entre otros-, y b) de los procesos –es decir, la forma en que dichos constructos son modificados por las experiencias cotidianas del docente-. El tema del pensamiento del profesor se ha estudiado predominantemente desde la perspectiva de las creencias y las teorías implícitas que los docentes tienen acerca de distintos aspectos del proceso de enseñanza y aprendizaje. Se ha considerado que dichas creencias configuran teorías implícitas, esto es, un sistema o perspectiva conceptual personal e idiosincrático que permite a los docentes afrontar las diversas situaciones que se presentan en el contexto escolar (Medina, 2006), a partir de las cuales se ha buscado comprender por qué los docentes actúan de determinada manera en el ambiente de la sala de clases. Al respecto, se encuentra que las creencias tienen afinidad con la cultura social en tanto guía de la conducta de las personas (Chan & Elliott, 2002), y es posible encontrar disonancia entre lo que la persona cree y lo que efectivamente hace (Torrado y Pozo, 2006). 376 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar 1 En las identidades de los establecimientos se coloca un seudónimo para preservar el compromiso ético de proteger la confidencialidad de los informantes. 3.1. PARTICIPANTES Los casos de estudio se describen a dos niveles interdependientes, el contexto o esce- nario de las prácticas pedagógicas –los liceos–, y los casos de estudio propiamente tales –informantes–. El escenario de estudio está compuesto por liceos con las siguientes carac- terísticas: 377 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Estudios Pedagógicos XL, N° 2: 373-389, 2014 á ó Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar • Liceos de Alto Desempeño Académico. Se toma como criterio de inclusión aquellos que poseen subvención por Desempeño de Excelencia otorgado por el Sistema Nacional de Evaluación de Desempeño de Chile, cuyos criterios definen contextos favorables para lograr altos resultados de aprendizaje (MINEDUC, 2010). • Liceos de Alto Desempeño Académico. Se toma como criterio de inclusión aquellos que poseen subvención por Desempeño de Excelencia otorgado por el Sistema Nacional de Evaluación de Desempeño de Chile, cuyos criterios definen contextos favorables para lograr altos resultados de aprendizaje (MINEDUC, 2010). • Nivel Educativo. 2do Medio, de curriculum Científico Humanista y Técnico Profesional, pues es el nivel educativo de quienes rinden la prueba de evaluación del Sistema de Medición de Calidad de la Educación (SIMCE). Esto permite la comparación de logro educativo entre los establecimientos y estudiantes chilenos. • Nivel Educativo. 2do Medio, de curriculum Científico Humanista y Técnico Profesional, pues es el nivel educativo de quienes rinden la prueba de evaluación del Sistema de Medición de Calidad de la Educación (SIMCE). Esto permite la comparación de logro educativo entre los establecimientos y estudiantes chilenos. • Grupo Socioeconómico Medio Bajo. Determinado según la base de datos de la prueba nacional SIMCE 2010 en 2do Medio de acceso público en internet (http://www.simce.cl/). • Puntaje SIMCE 2010. Específicamente en 2do medio, por encima del Promedio Nacional y de sus pares institucionales y sociales en Matemáticas y Lenguaje. Eso asegura tomar establecimientos con altos logros de aprendizaje escolar (http://www.simce.cl/). • Puntaje SIMCE 2010. Específicamente en 2do medio, por encima del Promedio Nacional y de sus pares institucionales y sociales en Matemáticas y Lenguaje. Eso asegura tomar establecimientos con altos logros de aprendizaje escolar (http://www.simce.cl/). Los casos de estudio fueron profesores seleccionados con los siguientes criterios: a) Eficacia. Los profesores son nominados por sus pares o por el Equipo Directivo del establecimiento como “eficaces” en su labor profesional, en la generación de clima laboral positivo, en la relación con sus estudiantes y en el logro de resultados de aprendizajes. a) Eficacia. Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar 3.2. TÉCNICAS DE RECOLECCIÓN DE INFORMACIÓN Fueron consideradas para el estudio las siguientes técnicas de recolección de datos: Fueron consideradas para el estudio las siguientes técnicas de recolección de datos: • Entrevistas en profundidad con docentes. Se utilizaron a fin de conocer sus representaciones de la práctica pedagógica, los procesos cognitivos y conocimiento escolar que definen los objetivos pedagógicos con los cuales planifican su quehacer en el aula. Permitieron también conocer, desde la perspectiva de los docentes, las competencias profesionales vinculadas a sus prácticas pedagógicas. Se realizaron 9 entrevistas en profundidad. • Observación/filmación de sala de clases. Para analizar el tipo de intervención de docentes y estudiantes en el aula se observaron 3 sesiones por docente, las cuales fueron previamente acordadas con el/la profesor/a. Para el presente estudio se analizan en total 27 sesiones de registro de observación de las interacciones en la sala de clases. Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Los profesores son nominados por sus pares o por el Equipo Directivo del establecimiento como “eficaces” en su labor profesional, en la generación de clima laboral positivo, en la relación con sus estudiantes y en el logro de resultados de aprendizajes. b) Experiencia. Con cinco años o más de experiencia docente. Preferentemente en el mismo establecimiento para asegurar la relación cultura y práctica pedagógica en la explicación de la efectividad escolar. c) Voluntad. Para participar del estudio se requiere que el/la profesor/a permita el acceso a la filmación de sus clases. Se trabajó con 9 profesores de diversas disciplinas y se filmaron 3 clases consecutivas de cada uno de ellos (Tabla 1): Tabla 1. Liceo, Modalidad Curricular, Nivel Socioeconómico, N° de profesores, N° de clases filmadas y resultados SIMCE 2010 de establecimientos participantes N° Liceo1 Modalidad Nivel socioeconómico Profesores participantes N° total de clases filmadas SIMCE 2010 Lenguaje Matemáticas 1 SSB Técnico Profesional Medio bajo 2 6 264 278 2 EMP Científico Humanista Medio bajo 2 6 278 285 3 CJDC Científico Humanista Medio 3 9 287 296 4 SJPS Técnico Profesional Medio Bajo 2 6 293 303 Fuente: elaboración propia. Tabla 1. Liceo, Modalidad Curricular, Nivel Socioeconómico, N° de profesores, Tabla 1. Liceo, Modalidad Curricular, Nivel Socioeconómico, N° de profesores, N° de clases filmadas y resultados SIMCE 2010 de establecimientos participantes 1 En las identidades de los establecimientos se coloca un seudónimo para preservar el compromiso ético de proteger la confidencialidad de los informantes. 378 3.3. PROCEDIMIENTO DE ANÁLISIS DE DATOS 379 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Reelaboración Retoma la intervención de uno o más estudiantes para mostrar una visión más amplia del concepto que se trabaja (Cuadrado y Fernández, 2008). Síntesis Resumen de cuestiones más relevantes del contenido que se trabaja (Cuadrado y Fernández, 2008), conecta conceptos presentados. Pregunta de dominio de contenido Pide al estudiante que elija una respuesta entre un conjunto de alternativas, o que responda con el uso de información factual o conceptual (Nathan & Kim, 2009). Preguntas de elaboración de contenido Pide al estudiante que elabore una respuesta que conecte o aplique lo aprendido a nuevas situaciones (Nathan & Kim, 2009). Preguntas de metaproceso Pide al estudiante que justifique, explique, que desarrolle su intervención, o una intervención previa (Nathan & Kim, 2009). Evaluación cerrada Evaluación breve de aceptación o rechazo de respuesta del estudiante (Wells & Mejía, 2005). Evaluación elaborada El profesor confirma la respuesta (adecuada o no) del estudiante e incorpora nuevas elaboraciones a dicha respuesta (Wells & Mejía, 2005), o pregunta solicitando más información Fuente: elaboración propia. Tabla 3. Intervenciones del profesor vinculadas al proceso cognitivo y construcción del conocimiento escolar Tabla 3. Intervenciones del profesor vinculadas al proceso cognitivo y construcción del conocimiento escolar Intervención del estudiante Definición Validación de conocimientos escolares Expresiones verbales (“ya”, “aja”, repetición textual de lo que dice el profesor, o completamiento de frases, etc.) y no verbales (tomar nota, mirar al profesor, afirmar con la cabeza, etc.) que validan el contenido que entrega el profesor (Cobo, 1998). Reelaboración Retoma la intervención de uno o más estudiantes, o del profesor, para dar su visión del concepto que se trabaja. Propone Aporta nuevos elementos (habla de su experiencia, relaciona con aprendizajes previos) a la clase. Responde a dominio de contenido Elige una de las alternativas que presenta el profesor, produce una respuesta en los términos de conocimiento factual o conceptual que solicita el profesor. Responde con elaboración de contenido Elabora una respuesta para resolver una situación didáctica (la respuesta a preguntas abiertas, las respuestas a ejercicios de una guía, la interpretación de un texto, etc.). Metaproceso Habla sobre su propio razonamiento, vincula su intervención con intervenciones previas. 3.3. PROCEDIMIENTO DE ANÁLISIS DE DATOS Las entrevistas se analizaron mediante los principios de codificación Abierta y Axial de la Teoría Fundamentada (Strauss y Corbin, 2002) y el análisis Semántico de Contenido en perspectiva estructural (Navarro y Díaz, 1999): se realiza codificación abierta de los discursos, los cuales, en la codificación axial, son puestos en relación intra-textual con unidades de contexto extra-textual que estudios previos indican como estructurantes de la práctica cotidiana (PNUD, 2009; Villalta y Martinic, 2011): la subjetividad, el conocimiento práctico y la cultura institucional. La codificación axial permite establecer hipótesis teóricas sobre la relación entre las codificaciones abiertas y de éstas con los contextos de referencia, las cuales en su conjunto estructuran la práctica pedagógica.i Las filmaciones fueron analizadas según la intervención de profesor/a y estudiantes. La intervención es una categoría del Análisis de la Conversación (Villalta, 2009) para describir el proceso comunicativo de la clase. En la perspectiva conversacional, la intervención es la participación de cada interlocutor en relación de interdependencia ilocutoria para la construcción del proceso de enseñanza y aprendizaje. Para el análisis de las categorías de intervención de profesor (Tabla 2) y estudiantes (Tabla 3) se recogen los aportes de estudios previos (Villalta, Assael y Martinic, 2013). El análisis se hizo con apoyo computacional del software Videograph (Rimmele, 2009). Tabla 2. Intervenciones del profesor vinculadas al proceso cognitivo y construcción del conocimiento escolar Intervención del profesor Definición Presenta conocimientos escolares Explicita objetivos, reformula su propia intervención, uso de gestos para captar atención del estudiante y enfatizar aspectos de los contenidos (Cuadrado y Fernández, 2008), grafica contenidos en la pizarra o con uso de sistemas computacionales. Favorece participación del estudiante Expresiones afirmativas, repeticiones de la intervención del estudiante, gestos de apoyo como asentimiento con la cabeza, pasear por la sala atento a posibles consultas (Cuadrado y Fernández, 2008). Tabla 2. Intervenciones del profesor vinculadas al proceso cognitivo y construcción del conocimiento escolar Tabla 2. Intervenciones del profesor vinculadas al proceso cognitivo y construcción del conocimiento escolar Intervención del profesor Definición Presenta conocimientos escolares Explicita objetivos, reformula su propia intervención, uso de gestos para captar atención del estudiante y enfatizar aspectos de los contenidos (Cuadrado y Fernández, 2008), grafica contenidos en la pizarra o con uso de sistemas computacionales. Favorece participación del estudiante Expresiones afirmativas, repeticiones de la intervención del estudiante, gestos de apoyo como asentimiento con la cabeza, pasear por la sala atento a posibles consultas (Cuadrado y Fernández, 2008). 4. RESULTADOS Los resultados se presentan en dos dimensiones: 1) el análisis de contenido de las entrevistas, y 2) el análisis de las intervenciones en el aula. Ambas dimensiones se ponen en relación en el punto Conclusiones. 4.1. ANÁLISIS DE CONTENIDO DE ENTREVISTAS Las categorías abiertas se organizan en tres campos discursivos, los cuales se encuentran interrelacionados y estructuran la práctica pedagógica. El primero de ellos refiere al campo discursivo de la subjetividad, que agrupa un conjunto de textos que refieren a experiencias personales de los profesores, que se subdividen en dos categorías, una denominada formación continua, textos que indican el valor que los docentes dan a los cursos de postgrados o perfeccionamientos para sus prácticas profesionales de aula: “[…] a través de mis veinticinco años, yo creo que veintiuno o veintidós años siempre he estado estudiando algo, siempre he estado en algún curso de perfeccionamiento” (Profeso- ra, JDC). “[…] a través de mis veinticinco años, yo creo que veintiuno o veintidós años siempre he estado estudiando algo, siempre he estado en algún curso de perfeccionamiento” (Profeso- ra, JDC). La segunda categoría del campo discursivo subjetividad es denominada modelo docente, que contiene textos que refieren a la síntesis que hace el profesor de su experiencia como alumno, desde donde extrae una forma positiva de valorar la práctica en tanto intervención para abrir perspectivas de mundo y desafiar al estudiante: “… Tuve profesores […] que me decían, “mira, esto es así, pero si tú lo tomas de este otro punto de vista puede que no sea así”, o sea, ahí yo fui aprendiendo ciertas cosas, y esto, yo te estoy hablando de la época del colegio, que después se potenciaron mucho más en la Universidad” (Profesor, REMP). “… Tuve profesores […] que me decían, “mira, esto es así, pero si tú lo tomas de este otro punto de vista puede que no sea así”, o sea, ahí yo fui aprendiendo ciertas cosas, y esto, yo te estoy hablando de la época del colegio, que después se potenciaron mucho más en la Universidad” (Profesor, REMP). El segundo campo discursivo es el denominado del conocimiento práctico, que en el caso de los profesores se relaciona con dos categorías: (a) conocimiento de la disciplina que enseña, y (b) conocimiento de la interacción con los alumnos que le permite liderar el proceso comunicativo en la sala de clases. La categoría (a) está descrita por la sub-categoría especificidad del saber que refiere el valor que el docente otorga a los saberes de la disciplina que enseña, diferenciándolo de otros saberes escolares. 3.3. PROCEDIMIENTO DE ANÁLISIS DE DATOS Relación social Intervenciones que, en principio, no tienen ninguna relación con los contenidos escolares trabajados en clases. En colectivo No hay diálogo directo reconocible entre profesor y algún estudiante. Fuente: elaboración propia. 380 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar 4.1. ANÁLISIS DE CONTENIDO DE ENTREVISTAS La categoría (b) conocimiento de la interacción con los alumnos permite a los profesores discernir acciones que describen la sub-categoría promover la participación en el aula, la cual es una invitación abierta y constante a todos: “… debería haber alguna pregunta, debería haber algún… que pase una hora y media y no me pregunten nada… o sea no es una buena clase, no para mí […], entonces uno también tiene que tratar de soltar un poquito eso, preguntarle al niño que nunca te preguntó, hablar con la persona que no participa” (Profesor, SJ). “… debería haber alguna pregunta, debería haber algún… que pase una hora y media y no me pregunten nada… o sea no es una buena clase, no para mí […], entonces uno también tiene que tratar de soltar un poquito eso, preguntarle al niño que nunca te preguntó, hablar con la persona que no participa” (Profesor, SJ). Asimismo, la categoría (b) se estructura con la definición de determinadas prácticas que caracterizan al docente, las cuales se agrupan dentro de la sub-categoría cualidades del profesor, y refiere a los siguientes elementos: 1) establecer relación afectiva con los estudiantes, 2) comunicación efectiva, y 3) verificación constante de los aprendizajes: “Trato de establecer una relación más dialógica con los alumnos en la medida de lo posible” (Profesor, REMP). “Trato de establecer una relación más dialógica con los alumnos en la medida de lo posible” (Profesor, REMP). (Profesor, REMP). “Si el profesor se ve dubitativo los cabros no enganchan, no hay una empatía” (Profesor, JDC). “[…] Todavía me emociono cuando un alumno me dice “Me acuerdo cuando usted me dijo” […] asocian cosas de antes, en clases posteriores lo sacan otra vez, entonces igual, lo tienen, lo tienen, lo incorporaron”. (Profesora, SSB). “[…] Todavía me emociono cuando un alumno me dice “Me acuerdo cuando usted me dijo” […] asocian cosas de antes, en clases posteriores lo sacan otra vez, entonces igual, lo tienen, lo tienen, lo incorporaron”. (Profesora, SSB). El tercer campo discursivo es el contextual, que refiere al ámbito de la cultura escolar institucional que contiene y sostiene las prácticas pedagógicas de aula. 4.1. ANÁLISIS DE CONTENIDO DE ENTREVISTAS A continuación se presentan dos ejemplos de las disciplinas de Lenguaje e Historia: “De partida el lenguaje se usa en todas las asignaturas, un alumno, o una persona que no maneje las herramientas mínimas o las competencias mínimas del lenguaje le va a costar mucho entender historia, le va a costar entender ciencias […]” (Profesor Lenguaje, REMP). “De partida el lenguaje se usa en todas las asignaturas, un alumno, o una persona que no maneje las herramientas mínimas o las competencias mínimas del lenguaje le va a costar mucho entender historia, le va a costar entender ciencias […]” (Profesor Lenguaje, REMP). “… lo característico de la historia es que hay contenidos que tú puedes explicar en el presente […], el contenido histórico yo lo pongo en que los cabros desarrollen habilidades, por ejemplo un análisis comparativo, por ejemplo una síntesis, por ejemplo una evaluación, una interpretación…” (Profesor Historia, JDC). “… lo característico de la historia es que hay contenidos que tú puedes explicar en el presente […], el contenido histórico yo lo pongo en que los cabros desarrollen habilidades, por ejemplo un análisis comparativo, por ejemplo una síntesis, por ejemplo una evaluación, una interpretación…” (Profesor Historia, JDC). 381 Estudios Pedagógicos XL, N° 2: 373-389, 2014 á ó Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Estudios Pedagógicos XL, N 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar El conocimiento de la disciplina también está descrito por la sub-categoría estrategia de enseñanza, la cual, no obstante la especificidad disciplinar, comparte características de sistemático y desafiante para los estudiantes, “[…] yo presento mi clase sistematizada y ordenada […] entonces yo siempre presento una propiedad de algo, yo después lo demuestro, lo ejemplifico y digo: “ya, ahora te toca a ti”” (Profesor, JDC). “[…] yo presento mi clase sistematizada y ordenada […] entonces yo siempre presento una propiedad de algo, yo después lo demuestro, lo ejemplifico y digo: “ya, ahora te toca a ti”” (Profesor, JDC). 4.1. ANÁLISIS DE CONTENIDO DE ENTREVISTAS Dos sub-categorías referidas al aprendizaje escolar constituyen la descripción del contexto: a) la disciplina, entendida como el orden y cumplimiento de normativas: “(…) mantener ese orden, esta disciplina, esa constancia […] el respeto en la clase, sobre todas las cosas, y también es importante manejarse con los chiquillos y darles confianza, decirles que no existen obstáculos” (Profesor, JDC). Y b) la sistematicidad del proceso de enseñanza, que refiere a las acciones de planificación y evaluación constante de las acciones y resultados en el aula: “[…] La planificación de nuestras clases está hecha de manera centralizada, […] y ésa es como mi hoja de ruta para organizar mis tiempos, organizar mis materiales y todos los recursos que utilizo en clases” (Profesor, REMP). 382 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Estudios Pedagógicos XL, N° 2: 373-389, 2014 á ó Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Las categorías y subcategorías discursivas descritas son componentes de la práctica pedagógica que tiene como punto de mayor confluencia el conocimiento que el profesor tiene de la interacción con los estudiantes en el aula. Considerando el enfoque de estudio de las prácticas que propone el PNUD (2009), dichas categorías son organizadas en campos discursivos que refieren a la experiencia personal extra-aula –campo discursivo de subjetividad-, al conocimiento práctico de los sucesos de sala de clases –campo del conocimiento práctico-, y al contexto institucional extra-aula –campo discursivo contextual-, cuyas categorías operan como un todo sistémico (Figura 1). Figura 1. Elementos del discurso docente que estructuran la práctica pedagógica Fuente: elaboración propia. Figura 1. Elementos del discurso docente que estructuran la práctica pedagógica Fuente: elaboración propia. Fuente: elaboración propia. La puesta en relación de las categorías y sub-categorías definidas permite comprender que la formación continua que describen los profesores fortalece el conocimiento de la disciplina escolar que enseñan, permitiéndoles delimitar el valor y la especificidad de dicho saber, así como también las estrategias de la enseñanza. El conocimiento de la disciplina escolar no es suficiente para constituir el “saber hacer” de la práctica pedagógica en el aula. Se requiere del conocimiento de las interacciones con los estudiantes, esto es, la forma eficaz de relacionarse con ellos, para lo cual el profesor apela a su experiencia personal y profesional de donde construye su modelo docente, el cual le da sustentos prácticos para dos acciones: (a) promover la participación de los estudiantes en el aula, y (b) para discernir las cualidades del profesor en dicho contexto. No obstante, dichas acciones se sostienen o validan en dos elementos que caracterizan la cultura escolar de los establecimientos de alto rendimiento educativo: (1) el orden y la disciplina, lo cual enmarca los comportamientos posibles y deseables para ser miembro integrante de la comunidad escolar, y (2) la sistematicidad de la enseñanza, la cual se organiza y tiene soportes a nivel institucional. 383 4.2. ANÁLISIS DE LAS FILMACIONES DE CLASES Se filmaron tres sesiones consecutivas por cada docente, acumulándose un total de 27 clases. Cada clase duró en promedio una hora con 18 minutos. Las intervenciones del profesor y los estudiantes fueron categorizadas cada cinco segundos en todas las clases. Se observó en total 34 horas y 24 minutos de filmación. Tabla 4. Frecuencia porcentual de tiempo de intervenciones del profesor en las clases Categorías de Intervenciones del Profesor Frecuencia (unidades de 5 segundos) Porcentaje 1. Presenta conocimientos escolares. 13.461 54,3 2. Favorece participación de estudiantes. 8.025 32,4 3. Reelabora intervención de estudiantes. 184 0,7 4. Síntesis de los contenidos que presenta en clases. 20 0,1 5. Pregunta de dominio de contenidos factuales. 599 2,4 6. Pregunta que requiere elaboración de contenidos. 330 1,3 7. Preguntas de metaproceso. 123 0,5 8. Evaluación cerrada, breve de respuesta. 73 0,3 9. Evaluación elaborada, profundiza en la respuesta. 106 0,4 Total (unidades válidas) 22.921 92,4 Fuente: elaboración propia. Tabla 4. Frecuencia porcentual de tiempo de intervenciones del profesor en las clases Fuente: elaboración propia. En el 92% del tiempo total de clases observadas fue posible categorizar las intervenciones de los profesores en las clases (Tabla 4). El análisis de las intervenciones de los profesores indica que el 54,3% del tiempo de las clases los profesores presentaron conocimientos escolares (categoría 1), y el 32,4% del tiempo intervino para favorecer la participación del estudiante en la clase (categoría 2), con el empleo de gestos afirmativos a la intervención del estudiante, recorrer la sala para atender preguntas de estudiantes y repetir la intervención del estudiante. Se analiza la asociación entre la intervención del profesor con la intervención de los estudiantes en las clases observadas (Tabla 5), y se encuentra que hay asociación significativa [Chi-Cuadrado (64, N=22524) = 27476,903, p<.001; 38 casillas (46,9%) tienen una frecuencia esperada inferior a 5] entre las 9 categorías de intervenciones de los profesores con las 9 categorías de intervenciones de los estudiantes. La fuerza de la dependencia es moderada (V de Cramer=.39).i Se puede afirmar que intervenciones de los profesores e intervenciones de los estudiantes se encuentran en influencia recíproca. 4.2. ANÁLISIS DE LAS FILMACIONES DE CLASES Se observa que la mayor cantidad de unidades de tiempo de intervención de los alumnos es para validar conocimientos escolares que entrega el profesor (categoría 1 de estudiantes); más preciso aún, el 82,4% de esta intervención está asociada a la intervención de presentar conocimientos escolares del profesor (categoría 1 384 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Tabla 5. Asociación entre las intervenciones del profesor e intervenciones de los estudiantes en las clases observadas INTERVENCIONES DEL PROFESOR (Porcentaje) INTERVENCION DEL ESTUDIANTE (Porcentaje) 1.Validación conocimientos escolares 2.Reelaboración 3.Propone 4.Responde con dominio de contenidos 5.Responde con elaboración de contenidos 6.Metaproceso 7.Silencio 8.Relación social 9. En colectivo Total 1. Presenta conocimientos escolares 82,4 0,0 14,5 14,2 16,5 0,0 14,4 72,3 41,0 59,6 2. Favorece participación del estudiante 10,6 0,0 82,7 44,9 80,9 23,4 16,5 26,7 58,1 34,3 3. Reelabora 1,3 0,0 0,8 0,0 0,0 0,0 0,0 0,0 0,1 0,8 4. Síntesis 12,0 0,0 0,0 2,0 0,0 0,0 6,0 0,0 0,0 20,0 5. Pregunta de dominio de contenidos 2,7 100,0 1,2 30,2 0,8 0,0 57,7 1,0 0,3 2,6 6. Pregunta de elaboración de contenidos 2,0 0,0 0,0 2,7 1,6 0,0 3,1 0,0 0,3 1,5 7. Preguntas de metaproceso 0,0 0,0 0,2 0,0 0,0 76,6 0,0 0,0 0,0 0,3 8. Evaluación cerrada 0,2 0,0 0,4 7,1 0,0 0,0 2,1 0,0 0,1 0,3 9. Evaluación elaborada 0,7 0,0 0,4 0,5 0,2 0,0 0,0 0,0 0,1 0,5 Total % 100 100 100 100 100 100 100 100 100 100 Total 12.915 4 1.650 437 2.484 77 97 202 4.658 22.524 385 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar Estudios Pedagógicos XL, N° 2: 373-389, 2014 á ó del profesor). El cálculo porcentual por casilla indica que el 47,2% del tiempo que dura la clase se dedica a este tipo de intervenciones. También es interesante notar que la intervención de presentar conocimientos escolares concentra al 72,3% de intervenciones de la categoría relación social –que equivale a distracción- de los estudiantes, pero esto, en términos del tiempo total de la clase, equivale a menos del 1%. 4.2. ANÁLISIS DE LAS FILMACIONES DE CLASES La intervención de favorecer la participación del estudiante (categoría 2 del profesor) agrupa gran cantidad porcentual de las intervenciones de los estudiantes; el 82% de las intervenciones de proponer; el 44% de las intervenciones de responder con dominio de contenidos; el 80% de las intervenciones de responder con elaboración de contenido, y el 58% de las intervenciones denominadas en colectivo, que comúnmente refiere al trabajo en grupos de estudiantes en la sala. El análisis de las casillas indica que en el 12% del tiempo de las clases observadas, la intervención del profesor para favorecer la participación del estudiante (categoría 2 del profesor) coincide con situaciones donde los estudiantes no establecen una relación dialogal directa con el profesor (categoría 9 de estudiantes). De hecho, éste es el segundo tipo de intervención al cual los estudiantes dedican tiempo, y el 58% de los mismos están asociados a la categoría 2 del profesor. El tercer grupo de tiempo de las intervenciones de los estudiantes es para responder con elaboración de contenidos (categoría 5 de estudiantes), la cual está asociada a la categoría 2 del profesor: favorecer la participación del estudiante. El cálculo porcentual por casilla indica que el 8,9% del tiempo total de la clase se produce este encuentro de intervenciones entre profesor y estudiantes. En síntesis, los resultados presentados indican que cuando el profesor favorece la inter- vención de los estudiantes éstos diversifican el tipo de intervenciones que realizan en la clase. Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar La intervención del profesor del tipo favorece la participación del estudiante también tiene su correlato en la intervención de los estudiantes, que opera como condición y consecuencia para su realización. Dicho tipo de intervención está en consonancia con las subcategorías del campo discursivo de la subjetividad. En efecto, favorecer la participación del estudiante tiene relación con capacidades adquiridas en la formación continua para enseñar la disciplina y con el modelo docente que los profesores describen. Tal como se ha evidenciado en los estudios sobre creencias de los profesores en torno al aprendizaje, en esta investigación se constata que hay desfase entre lo señalado en el discurso y lo realizado en la práctica real de aula. En efecto, el discurso de los profesores pone el acento en la importancia de conocer y promover la interacción profesor-estudiantes en tanto meta y proceso para lograr el aprendizaje escolar, mientras que en las clases observadas la mayor parte del tiempo es de presentación de conocimientos escolares. No obstante, el discurso del campo de la subjetividad, sobre el modelo docente y las cualidades del profesor en la clase –comunicador efectivo, persona afectuosa y atento a verificar contenidos-, se pone también de manifiesto en el aula a través de la intervención denominada favorece la participación del estudiante, la cual, además, diversifica las formas de participación de los alumnos en la clase. Futuros estudios deben profundizar en el tipo de intervención del profesor relacionado al conocimiento de la disciplina escolar y la incidencia que esto puede tener en el tipo de intervención de los estudiantes. Del mismo modo, es importante indagar diferenciadamente en las concepciones de los docentes acerca del conocimiento propio de sus áreas disciplinares, a fin de comprender cómo dichas creencias orientarían las planificaciones y prácticas pedagógicas en el desarrollo de distintas asignaturas. 5. CONCLUSIONES En los liceos de altos resultados de aprendizaje la cultura escolar tiene normas y procedimientos que organizan la acción de los actores educativos en función de metas de aprendizaje. Dentro de estas condiciones institucionales se puede entender que la intervención del profesor en la clase sea, en más del 50% del tiempo que dura la clase, de presentar conocimientos escolares. Dicho tiempo es equivalente al tiempo que los alumnos mantienen el interés en la clase, validan, afirman y aceptan los contenidos entregados. Asimismo, cuando el profesor favorece la participación de los estudiantes, éstos diversifican los modos de intervenir en la clase. La intervención de presentar conocimientos escolares del profesor es hegemónica en las clases observadas, y esto es coherente con las categorías del campo discursivo contextual o de la cultura escolar analizadas en las entrevistas, en lo referente al valor que los profesores dan al orden y sistematicidad con que funciona el liceo. En liceos de alto rendimiento se prioriza la transmisión de contenidos escolares, y la institución se organiza para que eso sea posible. Por ello, el tipo de intervención hegemónica de los estudiantes en la clase está en consonancia con la intervención hegemónica de los profesores. Esto indica que dicha estructura comunicativa es un “saber hacer” que validan ambos interlocutores y tiene como condición la cultura institucional del orden. En dicho escenario, profesores y estudiantes comparten y sostienen recíprocamente las intervenciones que constituyen la interacción profesor-alumnos valorada en el liceo. 386 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar REFERENCIAS BIBLIOGRÁFICAS Baeza, J. (2008). El diálogo cultural de la escuela y en la escuela. Estudios Pedagógicos, vol. 34, n. 2, 193-206. Barría, P., Castro, A., Riveros, F. y Villalta, M. (2010). Experiencia y buenas prácticas pedagógicas en contextos sociales vulnerables de Santiago de Chile: Estudio sobre las creencias de los docentes. Ciencia y Aprendizaje, vol. 2, n. 1, 27-48. Chan, K. W. & Elliott, R. (2002). Exploratory Study of Hong Kong Teacher Education Students’ Epistemological Beliefs: Cultural Perspectives and Implications on Beliefs Research. Contemporary Educational Psychology, vol. 27, n. 3, 392-414. MINEDUC. (2010). Sistema Nacional de Evaluación del Desempeño de los Establecimientos Educacionales Subvencionados y de los Regidos por el Decreto Ley Nº 3166. Santiago: Ministerio de Educación. Recuperado de http://www.mineduc.cl/usuarios/sned/doc/201108031652460.FolletoDi- fusionSNED pdf consultado en mayo de 2011 MINEDUC. (2010). Sistema Nacional de Evaluación del Desempeño de los Establecimientos Educacionales Subvencionados y de los Regidos por el Decreto Ley Nº 3166. Santiago: Ministerio de Educación Recuperado de http://www mineduc cl/usuarios/sned/doc/201108031652460 FolletoDi y g p y g Educación. Recuperado de http://www.mineduc.cl/usuarios/sned/doc/201108031652460.FolletoDi- fusionSNED.pdf, consultado en mayo de 2011. Educación. Recuperado de http://www.mineduc.cl/usuarios/sned/doc/201108031652460.FolletoDi- fusionSNED.pdf, consultado en mayo de 2011. fusionSNED.pdf, consultado en mayo de 2011. Cobo, P. (1998). Análisis de los procesos cognitivos y de las interacciones sociales entre alumnos (16-17) en la resolución de problemas que comparan áreas de superficies planas. Un estudio de casos. Barcelona: Universitat Autònoma de Barcelona. Cornejo, R. y Redondo, J. (2007). Variables y factores asociados al aprendizaje escolar. Una discusión desde la investigación actual. Estudios Pedagógicos, vol. 3, n. 2, 155-175. Cuadrado, I. y Fernández, I. (2008). ¿Cómo intervienen maestros y profesores para favorecer el aprendizaje en secundaria? Un estudio comparativo desde el análisis del discurso. Infancia y Aprendizaje, vol. 31, n. 1, 3-23. Fernández, I. y Cuadrado, I. (2008). ¿Son conscientes los profesores de secundaria de los 387 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar recursos comunicativos verbales y no-verbales que emplean en el aula? Revista Iberoamericana de Educación, vol. 46, n. 6, 1-13. recursos comunicativos verbales y no-verbales que emplean en el aula? Revista Iberoamericana de Educación, vol. 46, n. 6, 1-13. García, M. y Sebastián, C. (2011). Creencias epistemológicas de estudiantes de pedagogía en educación parvularia, básica y media: ¿Diferencias en la formación inicial docente? Psykhe, vol. 20, n. 1, 10-23. Gómez, V. y Guerra, P. (2012). REFERENCIAS BIBLIOGRÁFICAS Teorías implícitas respecto a la enseñanza y el aprendizaje: ¿Existen diferencias entre profesores en ejercicio y estudiantes de pedagogía? Estudios Pedagógicos, vol. 38, n. 1, 25-43. Hargreaves, A. (2005). Profesorado, cultura y postmodernidad: Cambian los tiempos, cambia el profesorado. Madrid: Morata. Laboratorio Latinoamericano de Evaluación de la Calidad de la Educación (LLECE). (2010) Factores asociados al logro cognitivo de los estudiantes de América Latina y el Caribe. Santiago Laboratorio Latinoamericano de Evaluación de la Calidad de la Educación (LLECE). (2010). Factores asociados al logro cognitivo de los estudiantes de América Latina y el Caribe. Santiago: UNESCO-OREALC. Leal, F., Espinoza, C., Iraola, M. y Miranda, M. (2009). El contexto en la epistemología personal: Consideraciones teóricas y exploraciones empíricas. International Journal of Psychology, vol. 43, n. 1, 170-180. Makuc, M. (2008). Teorías implícitas de los docentes acerca de la comprensión de textos. Signos, vol. 41, n. 68, 403-422. Marcelo, C. (1989). El pensamiento del profesor. Barcelona: CEAC. Martin, E., Mateos, M., Martínez, P., Cervi, J., Pecharromán, A. y Villalón, R. (2006). Las concepciones de los profesores de educación primaria sobre la enseñanza y el aprendizaje. En J. Pozo, N. Scheuer, M. Pérez, M. Mateos, E. Martin y M. De la Curz, Nuevas formas de pensar la enseñanza y el aprendizaje. Las concepciones de profesores y alumnos (pp. 171-187). Barcelona: Graò. Martinic, S. y Vergara, C. (2007). Gestión del tiempo e interacción del profesor-alumno en la sala de clases de establecimientos de Jornada Escolar Completa en Chile. Revista Electrónica Iberoamericana sobre Calidad, Eficacia y Cambio en Educación, vol. 5, n. 5e, 3-20. Recuperado de http://www.rinace.net/arts/vol5num5e/art1.pdf, consultado en junio de 2011. Medina, J. (2006). La profesión docente y la construcción del conocimiento profesional. Buenos Aires: Editorial Lumen. Murillo, J. y Román, M. (2009). Mejorar el desempeño de los estudiantes de América Latina. Algunas reflexiones a partir de los resultados del SERCE. Revista Mexicana de Investigación Educativa, vol. 14, n. 41, 451-484. Nathan, M. & Kim, S. (2009). Regulation of Teacher Elicitations in the Mathematics Classroom. Cognition and Instruction, vol. 27, n. 2, 91-120. Navarro, P. y Díaz, C. (1999). Análisis de contenido. En J. Delgado y J. Gutiérrez (Coords.), Métodos y técnicas cualitativas de investigación en ciencias sociales (pp. 177-224). Madrid: Síntesis. Olafson, L. & Schraw, G. (2006). Teachers’ Beliefs and Practices within and across Domains. International Journal of Educational Research, vol. 45, n. 1-2, 71-84. Pecharromán, I. y Pozo, J. (2006). REFERENCIAS BIBLIOGRÁFICAS ¿Qué es el conocimiento y cómo se adquiere? Epistemología intuitiva en profesores y alumnos de secundaria. En J. Pozo, N. Scheuer, M. Puy, M. Mateos, E. Martin y M. De la Cruz, Nuevas formas de pensar la enseñanza y el aprendizaje. Las concepciones de profesores y alumnos (pp. 243-264). Barcelona: Graò. Pérez Gómez, Á. (1998). La cultura escolar en la sociedad neoliberal. Madrid: Morata. Pérez Gómez, Á. (1998). La cultura escolar en la sociedad neoliberal. Madrid: Morata. Pi l d F í L Sá h d G ll d M (2009) C lt di j i i Pirela de Faría, L. y Sánchez de Gallardo, M. (2009). Cultura y aprendizaje organizacional instituciones de Educación Básica. Revista de Ciencias Sociales, vol. 15, n. 1, 175-188. Programa de las Naciones Unidas para el Desarrollo (PNUD). (2009). Desarrollo humano en Chile. La manera de hacer las cosas. Santiago: PNUD. g Rimmele, R. (2009). What is Videograph? Videograph. The Program for Coding of Videos Rimmele, R. (2009). What is Videograph? Videograph. The Program for Coding of Videos. Recuperado de http://www.dervideograph.de/enhtmStart.html, consultado en mayo de 2011. , ( ) g p g p g f g f Recuperado de http://www.dervideograph.de/enhtmStart.html, consultado en mayo de 2011. Román, M. (2003). ¿Por qué los docentes no pueden desarrollar procesos de enseñanza cuperado de http://www.dervideograph.de/enhtmStart.html, consultado en mayo de 2011. Recuperado de http://www.dervideograph.de/enhtmStart.html, consultado en mayo de 2011. Román, M. (2003). ¿Por qué los docentes no pueden desarrollar procesos de enseñanza Román, M. (2003). ¿Por qué los docentes no pueden desarrollar procesos de enseñanza 388 Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar aprendizaje de calidad en contextos sociales vulnerables? Persona y Sociedad, vol. 17, n. 1, 113-128. Ruíz, G. y García, A. (2007). Las desigualdades de la escuela y del entorno. Revista Electrónica Iberoamericana sobre Calidad Eficacia y Cambio en Educación vol 5 n 5e 306-312 prendizaje de calidad en contextos sociales vulnerables? Persona y Sociedad, vol. 17, n. 1, 113-128. aprendizaje de calidad en contextos sociales vulnerables? Persona y Sociedad, vol. 17, n. 1, 113-128. Ruíz, G. y García, A. (2007). Las desigualdades de la escuela y del entorno. Revista Electrónica Iberoamericana sobre Calidad, Eficacia y Cambio en Educación, vol. 5, n. 5e, 306-312. p j y , , , Ruíz, G. y García, A. (2007). Las desigualdades de la escuela y del entorno. Estudios Pedagógicos XL, N° 2: 373-389, 2014 Discurso y práctica pedagógica en contextos de alto rendimiento escolar REFERENCIAS BIBLIOGRÁFICAS Revista Electrónica Iberoamericana sobre Calidad, Eficacia y Cambio en Educación, vol. 5, n. 5e, 306-312. Strauss, A. y Corbin, J. (2002). Bases de la investigación cualitativa. Técnicas y procedimientos para desarrollar la teoría fundamentada. Colombia: Universidad de Antioquia. Taylor, S. y Bogdan, R. (1998). Introducción a los métodos cualitativos de investigación. Barcelona: Paidós. Torrado, J. y Pozo, J. (2006). Del dicho al hecho: De las concepciones sobre el aprendizaje a la práctica de la enseñanza de la música. En J. Pozo, N. Scheuer, M. Pérez, M. Mateos, E. Martin y M. De la Cruz, Nuevas formas de pensar la enseñanza y el aprendizaje. Las concepciones de profesores y alumnos (pp. 205-228). Barcelona: Graò. UNICEF y Ministerio de Educación de Chile. (2004). ¿Quién dijo que no se puede? Escuelas efectivas en sectores de pobreza. Santiago: Ministerio de Educación-UNICEF. Recuperado de http:// www.unicef.cl/centrodoc/escuelas_efectivas/, consultado en junio de 2011. Villalta, M. (2014). Cultura escolar y prácticas de enseñanza en establecimientos de alto desempeño educativo. Aceptado en Universitas Psychologica, vol. 13, n. 1. _____., Assael, C. y Martinic, S. (2013). Conocimiento escolar y procesos cognitivos en la interacción didáctica de sala de clase. Aceptado en Perfiles Educativos, vol. 141. _____. y Martinic, S. (2011). Interacción didáctica y procesos cognitivos en sala de clase XXXIII Congreso Interamericano de Psicología, Medellín, Colombia. _____., Martinic, S. y Guzmán, M. (2011). Elementos de la interacción didáctica en la sala de clase que contribuyen al aprendizaje en contexto social vulnerable. Revista Mexicana de Investigación Educativa, vol. 16, n. 51, 1137-1158. _____. (2009). Una propuesta para el estudio de la interacción didáctica en la sala de clase. Estudios Pedagógicos, vol. 35, n. 1, 221-238. Volante, P., Cumsille, P., Denardin, F. y Müller, M. (2008). Análisis del cambio en los niveles de logro de escuelas de alta vulnerabilidad social. Estudios Pedagógicos, vol. 34, n. 2, 179-191. Wells, G. & Mejía, R. (2005). Toward Dialogue in the Classroom: Learning and Teaching through Inquiry. Working Papers on Culture, Education and Human Development, vol. 1, n. 4, 1-45. 389
https://openalex.org/W2141796410	https://journals.vgtu.lt/index.php/Transport/article/download/8645/7514	English	null	THE IMPROVEMENT OF EXTERNAL TRANSPORT COST EVALUATION IN THE CONTEXT OF LITHUANIA'S INTEGRATION INTO THE EUROPEAN UNION	Transport	2,003	cc-by	9,764	CONTEXT OF LITHUANIA S INTEGRATION INTO THE EUROPEAN UNION Margarita Uoraite Law University of Lithuania, Ateities g. 20, LT-2057 Vilnius, Lithuania. E-mail: misorait@ltu.lt Received 2003 06 02; accepted 2003 11 14 Margarita Uoraite Law University of Lithuania, Ateities g. 20, LT-2057 Vilnius, Lithuania. E-mail: misorait@ltu.lt Received 2003 06 02; accepted 2003 11 14 Abstract The accepted accession to the European Union in Lithuanian internal transportation is regulated by the co- ordination of national legal acts with the international conventions arid documents of various international organisations. This process was particularly accelerated when Lithuania started negotiations on the accession to the European Union. Future transport pricing policy of Lithuania has two objectives -to guarantee the covering of operational costs and to strive for economic efficiency. These objectives should not be coincident. Economic, institutional and legal refonns in Lithuania are implemented with the aim to co-ordinate the standards and regulations effective in Lithuania with the acts of the European Union. In the article on the basis of pricing of the peak, the social marginal, the environmental costs referring to the advanced research of Western and US scientists, were analysed and improved the evaluation of external Lithuanian transport costs and economic methods enabling the integration of external transport costs into the internal costs were offered. The improvement of external cost evaluation will enable the integration of external transport costs in line with the EU standards. One of the most actual problems of Lithuania is the growth of environmental impact and social subsequences caused by the increase of transport in Lithuania~ Increasing traffic jams, air pollution, growing rates of accidents and noise emission are the main problems of the Lithuanian transport system. Thus, in transport system, besides the internal costs, there exist the external costs that are not reflected in transport cost price. These costs are caused by air pollution, worsened health condition of people, decreased agrarian fertility, increased water pollution, etc. Therefore it is necessary to evaluate these external transport costs and to propose the ways of their reduction as well as their inclusion into the transportation price. The methodological basis of the article is the method of systematic and multicriteria! analysis aiming at the evaluation of the necessity of the inclusion of external transport costs into the transport price with the attitude towards the national and the carrier's positions. Research work of Western and US scientists was referred to the grounding of the necessity of external transport costs in Lithuania. CONTEXT OF LITHUANIA S INTEGRATION INTO THE EUROPEAN UNION Margarita Uoraite Law University of Lithuania, Ateities g. 20, LT-2057 Vilnius, Lithuania. E-mail: misorait@ltu.lt Received 2003 06 02; accepted 2003 11 14 Having analysed the experience of the scientists of US and Western countries, as well as the practice of Lithuanian scientists, a methodology for the evaluation of external transport costs in the country was offered. The application of the EU standards is tackled as well. Most ideas suggested in the article could be valuable for further formation and enhancement of external costs of economic technique in Lithuania thus approaching the national pricing to the EU standard system. The evaluation made in the article shall be applied for the implementation of external transport cost evaluation technique in Lithuanian transport. Keywords: external costs of transport, price theory. 229 229 ISSN 1648-4142 TRANSPORT http:lwww. vtu.lt/english/editions TRANSPORT- 2003, Vol XVIII, No 6, 229-240 2. Analysis of External Transport Costs, Prices and Pricing Theory Basing on this method the travel time spent in traffic jams is analysed. The "bottleneck" model was renewed and vastly investigated in the ar- ticles of Arnott [19], De Palma, A. and R. Lindsey (1998, 2000) [20, 21] and even nowadays traffic researchers often apply the model. Pricing of external environmental costs. Whereas the pricing of traffic jams reduces the number of kilometres covered and decreases the used fuel amounts thus the reduced air pollution is one of its positive re~ sults. Daniel and Behha (2000) [22] investigated this impact of traffic jams taxes according to a detailed model and settled that the taxes reduce the emission of vehicles from 10% to 30% with a social profit fluctuating from 15% to 30 %. Pany and Bento (2001) [22] have also investigated the profit of traffic jams pricing. They fo- cused an environmental impact and the impact of acci- dents. Daniel and Behha found that there is a great envi- ronmental benefit from the pricing of traffic jams (25 % of profit from a reduced traffic jam) provided that the fuel (petrol) tax by itself will not be able to internalise the external costs of air pollution. The principles of pric- ing of marginal costs maintain that instead of applying the traffic jams pricing for the improvement of air condi- tion it is necessary to carry out a special policy facilitat- ing the improvement of these particular external trans- pmt costs. The pollution caused by one vehicle does not depend on the traffic jams. Pricing in the case of traffic jams. Primarily the idea of using the pricing for the reduction of traffic jams was proposed more than eighty years ago by Pigou ( 1920) [3], then slightly later by Knight (1924) [4]. The theory demonstrated that traffic jams taxes lead to the internalisation of traffic jams costs by consumers. The theory of traffic jams pricing was developed by such sci- entists as Walters (1961) [5], Strotz (1964) [6], Mohring (1965, 1970, 1996) [7-9], Lvy-Lamber (1968) [10], Marchand (1968) [11], Vickrey (1968) [12], Keeler and Small (1989,1983,1992) [13-15] and DeVany (1976) [16], Jensen (2001) [17]. Vickrey's (1969) [18] "bottle- The importance of external transport costs (Table 1) is based on the fact that in the market economy the pub- lic resources decide upon the features of pricing. 2. Analysis of External Transport Costs, Prices and Pricing Theory Internal and external costs. Transportation costs are classified as internal and external costs. Internal costs of transportation emerge from the services of transporta- tion service providers and consumers. They emerge in the process of transportation. This can not be said of ex- ternal costs. The latter emerge when activities of one group make an impact on the other group without any remuneration of losses or compensation. If the loss is remunerated by compensation (as required by the Gov- ernment) and an external impact is evaluated, then the external costs become internal costs [2]. Optimal pollution tax Pollution - the ''polluter pays" principle. Figure demonstrates optimal pollution charges regarding the noise emission. General principles may be also success- fully applied to other fonns of pollution, such as air and water pollution. MPC means marginal private freight transportation costs (in tonnes). They comprise the work, fuel, maintenance costs. MEC means marginal environ- mental costs indicating marginal noise impact/disturbance expressed in monetary terms in each traffic level, and D is the curve of demand for transportation by road trans- port services. MSC is the curve of marginal social costs and it demonstrates the sum of MPC and MEC. On con- dition that road carriers regard their personal operating costs, then the production balance will be of the Q level A ' and the price or costs will be P A" However, if all the costs - personal and social are regarded, then Q* will be an optimal production level, and the price or costs will be P. The Q means a marginal profit equal to the mar- ginal social costs of transportation by road transport. The "polluter pays" principle states that carriers have to be aware of external costs, which they cause and pay pollu- tion, charge equivalent to ME C. It means that some goods will be shifted to other modes or that the industrial shift- ing will reduce general transport production. It is unprac- tical to directly charge consumers for pollution, there- fore for purchasing of a vehicle and for a yearly licence of the Road Fund several indirect methods were proposed, including the pollution tax. However, no method has of- fered an ideal "polluter pays" scheme, particularly the tax is not related to the actual use of a vehicle. neck" model is particularly differently viewed by vari- ous publication sources. 1. Introduction This process has particularly accelerated when Lithuania started negotiations on the accession to the European Union. Transport is one of the sectors of Lithuanian economy integrated into the European market. This ac- counts for the fact that transport presents a connecting- link facilitating the development of international trade and ensuring free movement of people and goods. The participation in the international trade decides the com- mon peculiarities of transport activity regulation. Even internal transportation is regulated by the co-ordination of national legal acts with the international conventions and documents of various international organisations. Future transport pricing policy of Lithuania has two objectives - to guarantee the covering of operational costs and to strive for economic efficiency. These ob- jectives should not be coincident. Economic, institu- tional and legal reforms in Lithuania are implemented with the aim to co-ordinate the standards and regula- tions effective in Lithuania with the acts of the Euro- pean Union [1]. M. lsoraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 230 p ME Optimal pollution tax p ME Optimal pollution tax 2. Analysis of External Transport Costs, Prices and Pricing Theory 2. Analysis of External Transport Costs, Prices and Pricing Theory If the prices reflect the shortage of goods, then principally the market will guarantee the effective distribution of re- sources. However, when the market prices, e.g. because of transport accidents, will reflect the existing shortcom- ings, such as weather purity, quality of environmental safety, infrastructure, etc., the transport demand will ex- ceed the socially optimal standard and the distribution of M. ISoraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 231 Table 1. Classification of transport costs Social costs Types of costs Internal/individual costs External costs Costs of transport services Fuel and vehicles costs, tickets/tariffs Other paid costs (for instance, vehicle storage costs) Infrastructure costs Consumers' fees, vehicles charges and fuel Uncovered infrastructure costs excise duty Costs of casualties Covered insurance and casualties costs Uncovered costs for casualties (i.e. pain and Costs of environmental safety Own losses Jam costs Costs of own time torments experienced by others) Uncovered environmental safety costs (i.e. noise paid by others) Delays/time consumption costs which are paid by others Table 1. Classification of transport costs Social costs Types of costs Internal/individual costs External costs Costs of transport services Fuel and vehicles costs, tickets/tariffs Other paid costs (for instance, vehicle storage costs) Infrastructure costs Consumers' fees, vehicles charges and fuel Uncovered infrastructure costs excise duty Costs of casualties Covered insurance and casualties costs Uncovered costs for casualties (i.e. pain and Costs of environmental safety Own losses Jam costs Costs of own time torments experienced by others) Uncovered environmental safety costs (i.e. noise paid by others) Delays/time consumption costs which are paid by others Table 1. Classification of transport costs resources will not be efficient. However the external costs are covered by others, not by transport users, i.e. by those affected by transportation: those falling ill because of air pollution, taxpayers, social insurance payers, infrastruc- ture costs payers, etc. Thus, the price system that does not reflect proper public costs is both ineffective and improper. External costs force transport users to react towards prices that are less than proper expenses, which increase the demand for transport, which make economic effect [23-27]. General input should be better reflected in the struc- ture of costs. Regarding the abundance oflegal acts con- cerning this issue, as well as the risk of competition varia- tion, it is necessary to implement a system defining tax payment for the infrastructure of all transport modes. 2. Analysis of External Transport Costs, Prices and Pricing Theory Table 2 demonstrates the indices of input of heavy vehicles moving in an open country with the speed of 100 km/h on a highway at non-peak hours. The presented numbers show the input related to the air pollution (costs related to health and harvest damages), climate changes (floods and harvest damages), infrastructure, noise emis- swn. Sustainable policy should be oriented towards com- plete internalisation of a social and environmental input. It is necessary to take decisive steps aiming at the disso- ciation of the growth of transport flows from the growth ofGDP, particularly in redistribution of transport modes, i.e. employing more railways, water and public trans- port. It is necessary to gradually change the transport charges by the more efficient means integrating expenses related to infrastructure, and external costs. These means firstly comprise the infrastructure users' tax, which is a particularly efficient measure for the management of traf- fic jams and for the reduction of environmental impact, and, secondly, - taxes for fuel, that enable the control of carbon dioxide emission. The foreseen measures should decrease the breach between costs and taxes. For instance, gradual tighten- ing of standards for vehicles gas emission should reduce air pollution. Price determination related to real costs arising from different types of engines, congestion and other external factors, shall not mean that the taxes will increase to all actors. The taxes will be higher in the places with more intensive traffic if compared with less devel- oped regions. It is most important to alter not a general level of taxes, but to change their structure that should be radi- cally transformed so that external and infrastructure costs were integrated into the transport price. The main principle of the collection of a tax for in- frastructure use is that the tax has to cover not only the costs related to the infrastructure, hut also to the external costs, related to roads accidents, air pollution, noise emis- sion and congestion. This concerns all transport modes and all consumers' categories, both private and commer- cial. M Isoraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 232 ment of an effective and sustainable transport system. Table 3 demonstrates constituent parts of costs of all transport modes. Regardless of different technologies it is possible to compare three categories by defining them as: infrastructure costs, users activity costs (users mon- etary and users travel time costs), carriers activity costs and social costs. higher traffic, the higher external costs. Five areas are de- fined where road traffic increases external costs: air pollu- tion, noise emission, accidents, use of land, road infra- structure. Air pollution and vehicles make an impact not only on the flora and fauna, people's health, but also on buildings, constructions, on water and soil, as well as on climate changes. Noise impacts the comfort of home as well as industry/production. For noise reduction additional funds are necessary for purchasing and installation of noise reducing insulation materials. Noise emission also makes a negative impact on recreation and tourism as well as on public health conditions. People do not wish to live in hotels or houses situated close to railway or bus stations and air- ports. Prices of flats and hotels situated near such objects are comparatively low. Accidents exercise not only a nega- tive impact on public health condition - the society suf- fers material losses caused by road accidents. For instance, the overturn of a vehicle with dangerous goods causes a spill ofhazardous materials. Use ofland also makes a nega- tive impact on soil as well as on general landscape. With the growth of road traffic the road infrastructure gets over- loaded. This fact is responsible for the costs of traffic jams. Increased traffic also increases the uncovered infrastruc- ture costs. p y Table 3 demonstrates constituent parts of costs of all transport modes. Regardless of different technologies it is possible to compare three categories by defining them as: infrastructure costs, users activity costs (users mon- etary and users travel time costs), carriers activity costs and social costs. The formula is applied with the aim to develop the Ferrari's transport costs definition model. It would be expedient to develop this model and to apply it for the detennination of transport prices in bigger Lithuanian towns, such as: Vilnius, Kaunas, Klaipeda. M Isoraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 According to the formula of costs determination, a transport enterprise has the costs of peak and non-peak services activity, the costs of changing a ce1tain route or route service activity and the indicated route costs (including the wear of ve- hicles and buildings). The costs of peak and non-peak time supply information for detern1ination of the peak and non-peak time tariffs. Information on the costs of activity is necessary for the definition of the activity defi- cit, the standards of tariffs and for the evaluation of changes proposed in a certain route service. While the distributed costs may be used for the evaluation of the capital used by a transport enterprise. If a transport user pays for the resources of use (e.g. use of energy and infrastructure), these costs may be con- sidered as internal costs. If a transport user makes an impact on the welfare of others (e.g. by polluting the air) without paying for that, then for others such costs be- come external costs. There is a clear relation to the prin- ciple "polluter pays", when the costs for pollution have to be covered. The fact that solutions in the market economy depend very much on market prices has par- ticular importance to external transpmt costs. Therefore pricing based on full social expenses is the principal ele- It is purposeful to calculate the costs of transport enterprise in bigger Lithuanian towns according to the following formula: (1) (1) (1) where: FC;1 -fully distributed costs for the i-th route at the t time period; C1m - average costs of the system- related peak time vehicle km (1 identifies a peak time period); M 1ir -services delivered by the vehicle km at Table 3. Constituents of costs of transport sectors Table 3. 3. Evaluation System of Social Subsequences and Environmental Impact of Transport Services Regarding the external costs it may be stated that external costs depend on the intensity of road traffic - the ructure costs (200 1) of a heavy vehicle moving on a motorway at l OOkm/h under the conditions Table 2. External and infrastructure costs (200 1) of a heavy vehicle moving on a motorway at l OOkm/h under the conditions of non-intensive traffic External and infrastructure costs Average (Euro) Air pollution 2,3-1,5 Climate changes 0,2-1,54 Infrastructure 2,1-3,3 Noise emission 0,7-4 Road accidents 0,2-2,6 Congestion 2,7-9,3 General 8-36 M Isoraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 M Isoraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 G 4(Lo -a) Lv +2a 2L0 +Lv = + p+t (3) v0 -v1 v1 v0 (3) L0 - distance (km) from the bridge to the sign indicating the need to slow down speed; Lv- the above and below length (km) of bridge (viaduct); v0 - speed of vehicle (km/h), when bridge is not closed; v1 - speed of vehicle (km/h), when traffic goes on the bridge; a- distance on both sides of bridge; p- coefficient; (when at the stretch (Lv + 2a) there is one traffic lane p = 2 , when two lanes - p = 1 ); t- interference with traffic (h), caused by the vehicle shifting traffic lanes. The average price of traffic flow is calculated as follows: (4) (4) ai -24 hours traffic intensity, vehicles per day; bi -time value of vehicles, LTL/h. The present system of charges of different transport modes in the EU countries does not induce the further efficiency and strengthening of the European transport system. Charges/taxes paid by road users significantly exceed the infrastructure costs. The average of road costs makes 1 % of GDP of the EU. Total revenues from road users (customs duties, vehicle tax and fuel dues) equal2 % of GDP of the EU. Average level of costs covered in European railways reaches 56 %. Coverage of railway costs is under research because the ratio of their correc- tion is not clear regarding the public services of railways. Transport charges/taxes should be precisely related to the coverage of infrastructure costs and to the external costs. To build or not to build an airport, seaport, road or rail- way infrastructure - this decision rests on the balance between all social costs and the increase of income. In- frastructure costs that are not covered have to be included into the transport price. M Isoraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 Constituents of costs of transport sectors Infrastructure costs - costs of land, capital, management, road signs maintenance Road Users costs- costs of vehicle ownership and activities, costs of time transport Carrier's costs External costs - costs of air pollution, noise, safety, traffic jams Costs of aviation system: capital and management costs Air Airport costs- costs of land, capital, maintenance, activities transport Carrier's costs User's costs - costs of time- time consumption External costs- costs of air pollution, noise emission, safety, traffic jams Infrastructure costs- costs of land, railway capital, management and maintenance costs Railway Rolling stock fleet costs- costs of capital, activities, maintenance User's costs- time costs- time consumption transport Carrier's costs External costs- costs of air pollution, noise emission, safety, traffic jams Infrastructure costs- capital, management costs Maritime Port costs- costs of land, capital, maintenance costs, activities costs transport Carrier's costs User's costs - costs of time - time consumption External costs - costs of air pollution, noise emission, safety M !Soraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 M !Soraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 233 233 the peak time for the i-th route at the time period of t ; where C2111 - average system-related non peak time vehicle km costs (2 identifies a non-peak time period); M 2it -non-peak time vehicles services delivered at the i-th route km at the time period of t; Clp- average system-related costs of peak time passengers; ~it -peak time passengers transported by the i-th route at the t period of time; C2p -average system-related costs of non-peak time passengers; P2it -passengers of non-peak time transported by the i-th routes at the t time period; fJic -ratio (or fraction) of additional non capital costs of enterprise suffered at the i-th route on the peak time of service; C NCt -general non capital costs of enterprise during the time period t; hie - ratio of enterprise's additional non-capital costs (fraction), obtained at the i- th route t non-peak time of service; f{j -ratio (or fraction) of enterprise's capital costs derived at the i-th route at the peak time of service; C Ct -general additional capital costs of enterprise during the period t ; j{; - ratio (fraction) of additional costs of enterprise capital which suffered at the i-th route at non-peak time of services. VK -average price of traffic flow (LTL/h): LS = E/xGxVK; M Isoraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 Road users' losses caused by traffic hindrances dur- ing the time of work are calculated by the following for- mula: Q = (Lxt P + 3 )x Elxqx Llilv; (5) (5) Q - road users' losses caused by the traffic hindrances during the surface handling installation work, LTL; L - length of road stretch, km; t P + 3 -average term of speed limitation on the object (basing on experience of regional enterprises), the tirrie duration of simple surface handling work in 1 km stretch of the road - 1 ,3 3 of 24 hours, that of handling with levelling layer- 2,33 of 24 hours, plus 3 more days for the speed limitation signs staying in place; EI - average yearly vehicles traffic intensity per 24 hours, vehicles per 24 hours; q -average price of 1 hour of vehicle delay time q = 14,52 LTL/h; ilv - average slow down of vehicles' flow caused by speed limitation applied during the repair time. Social costs of traffic jams. With the development of the sector of new transport modes and the reduction of congestion in towns, there is a system of identification of taxation for use of urban infrastructure (e.g. parking fee). The delay of standing and waiting may be calcu- lated by the method of modelling of traffic flows on road crossing. The method is based on the distribution model ofPuason, which is used for forecasting of random inci- dents at a certain time period. In this case an incident is the vehicles passing through the road crossing. Delays are caused when: Delays are caused when: a bridge is closed (its portentous condition) and the route has to be by-passed; Puason's distribution is expressed by the following function: a bridge is under repair and reconstruction, which causes traffic restriction. The economy of losses of time/time consumption value for the bridge is calculated as follows: p(mi'A)= P(X = m!A)= e-1A.Jm; (6) (6) LS = E/xGxVK; (2) m - the number of incidents at a certain period of time; 'A- average intensity of traffic of the investigated direction during a certain period of time. (2) LS = E/xGxVK; EI -traffic intensity (vehicle/hour); G- delay (vehicle); VK -average price of traffic flow (LTL/h): Modelling is applied in two cases: at peak hours (6 M ISoraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 234 conditions, weather conditions. conditions, weather conditions. hours per 24 hours) and at non-peak hours. During the modelling the moments of passing the road crossing in different directions are detennined in line with the re- quirements of road traffic rules and, after the evaluation of queuing, the delays are calculated. Time value should be recalculated every three years. Such costs of accidents are important for the policy of pricing, which are not covered by vehicle insurance premium, i.e. by a share of costs for hospital, production losses, loss of life. A great share of costs of accidents is already evaluated, however for defining external costs it would be purposeful to introduce measures promoting the decrease of accidents. Evaluation of noise emission costs. Traffic noise causes health problems. Noise interferes with mental activities. According to the World Health Organisation the noise emitted by transport is able to cause serious discomfort, as well as speech inhibition, high blood pres- sure, impact sleep, a stress. Road traffic is the greatest source of noise. Traffic noise causes problems at home, at work place, at school as well as in recreation areas. Therefore the improvement of transport means technol- ogy may be one of the ways towards the reduction of road traffic noise. The improvement of transport means technology and better planning of infrastructure plays a very important role in the reduction of noise emission. Producers of all transport means/vehicles make vast re- search into the reduction of noise (engine/tyre noise). However even such a measure as lowered speed limits will have small-scale int1uence if the road traffic and pmiicularly that of heavy transport vehicles is not con- stantly perfected. The main costs caused by accidents are the follow- ing: medical costs (e.g. hospital); costs of losses of pro- duction; material costs (e.g. vehicle repair costs); trans- portation costs (police, fire brigade, legal administration); insurance costs (vehicle, road, driver's education, etc.); non-material danger (personal grieffor relatives, friends); costs caused by casualties or injuries of persons. In many countries a significant share of these costs is precisely defined and, directly or non-directly, it is reimbursed by those related to the accidents. LS = E/xGxVK; Material costs are covered by vehicle insurance or directly by the owner. Insurance costs are reimbursed either by the owner of the vehicle or by the state (including infrastructure costs). Medical costs are covered by vehicle insurance. The remaining share of medical costs and components of other costs is considered as external costs. They are allocated/distrib- uted for traffic accidents. Following the present evalua- tion made by Dutchmen, a share of external costs in the context of common costs of accidents (including non- mateiial danger) ranges between 15 % and 25 %. Economic evaluation of the negative impact of noise. Negative impact of noise exercised on human be- ings is evaluated on the basis of provisional typical meth- odology. The evaluation is made of the noise emission sources, as well as of the spread of noise over a territory and of the level of noise isolated by the window. This may be differentiated according to habitable or cultural and domestic purpose premises. g g Costs of transport air pollution. Irrational use of natural resources, destruction of ecological balance may cause crucial changes in the habitation areas of human beings. There is no more activity of economy left that would not cause ecological subsequences. From the en- vironmental point of view the contradiction between human beings and the nature grows and causes more and more problems - local as well as global. Therefore in- dustrialists, politicians and citizens have consolidated their efforts seeking to retain our environment intact. These processes take place in the perspective of possible dramatical consequences and ce1iain negative changes may impact our economy, which would cause the lower- ing of social life standards. Many industrial countries managed to reach a number of significant achievements in the reduction of environmental pollution. It is gener- ally important to maintain the desirable environmental standards. One must admit that a great amount of efforts put for the reduction of environmental pollution is des- tined to the conection of former environmental mistakes. However, in the mass it is very important to retain the desirable quality ofthe environment [28-31]. Losses of people living in the acoustic discomfmi zone are identified according to the following fonnula: (7) (7) where B( I) -losses in LTL per capita per year regarding the noise level of premises in day time; N g (l) -number of inhabitants in the relevant zone at day. VBZPxYBZP)xAKZ(JPT)x/KPR; (9) In the course of the implementation of pollution re- duction policy and the implementation ofthe EU require- ments, since 1 January 1998 by the Decree of the Minis- try of Economy obligatory indices of fuel quality have been determined for the petrol and diesel fuel used in the country. The use of leaded gasoline was also prohibited. The rate of sulphur in diesel fuel is determined not to exceed 0,05%. where VCO -amount of exhausted pollution CO, kg/h; YCO- losses, LTL/t.; AKZ - multiplier for territory evaluation; JKPR -multiplier for evaluation of a street type. At present in Lithuania the only existing regulation means of mobile pollution consists of fuel quality stan- dards, vehicle pollution control programme (technical vehicles inspections and random road-side checks of gas emission), as well as customs duties for imported ve- hicles. In Lithuania the fuel standards in force are nearly adequate to the standards defined by the EU. The excise duty is not differentiated for fuels of different qualities. Most often in other countries the fuel tax is differenti- ated for leaded petrol and non-leaded petrol. Other regu- lation means existing in Lithuania are: pollution exhaust tests during the technical maintenance inspection of ve- hicles and fixed standards of exhaust. These fixed stan- dards of pollution in exhausted gas are checked every two years during the technical maintenance inspection of vehicles. Whereas vehicles are not produced in Lithuania, it is more important to control pollution dur- ing operation and to ensure the conformity of imported vehicles to the EU type approval of the date of their mak- mg. For ships carrying hazardous and dangerous goods (liquid or packed up) supplementary requirements are defined by the Klaipeda State Seaport shipping and Butinge Terminal shipping rules/regulations. All environmental safety methods may be divided into the methods of direct control and economical meth- ods. Methods of direct control are based on the applica- tion of administrative-legal measures (environmental quality standards, limitations, prohibitions, permissions). The advantages of administrative methods are in their transparency, clearness and easy application. How- ever they are not flexible and not economically effective from the public point of view. But the most important fact is that they do not meet the requirements of the prin- ciple of equivalence. LS = E/xGxVK; RUSH = (VCO x YCO + VCH x YCH + VNO x YNO + VSO x YSO + VTCxiTC+ VPBxYPB+ VTCxiTC+ VPBxYPB+ (9) LS = E/xGxVK; where B( I) -losses in LTL per capita per year regarding the noise level of premises in day time; N g (l) -number of inhabitants in the relevant zone at day. Losses in LTL per capita per year regarding the noise level of premises, Ld are found out by the following: B(l) =lOx (0,03xLd) -5,3. (8) (8) If the night transport t1ows were investigated, the losses caused by the noise at night-time would be identi- fied. Costs of transport accidents. There are great pub- lic regulation efforts aiming at the reduction of risks of accidents, irrespective of a model. Road transport acci- dent rates receive the most attention, if compared with other transport modes. Risk of accidents and their costs depend on many factors, such as: indices of transpmi means, road type, driver's characteristics, driver's behaviour, speed of vehicle, kilometres covered, traffic Transport impact on the environment is evaluated by 4 stages. In the first stage the transport emission is calculated and the driving conditions are evaluated, also the types of vehicles are determined by the fuel used (die- sel or petrol). In the second stage the concentration in M ISoraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 235 local or regional zones is identified. In the third stage the transport impact is evaluated, i.e. acute and chronic ill- nesses, attacks of asthma, taxes for hospitals and dam- age of production. In the fourth stage the costs of harm exercised by transport are evaluated. the guaranty of quality their samples are analysed, their specific, legally regulated parameters are checked (if an oil refinery is in a EU country, the product is tested at once, in other cases - while importing). These legal re- quirements are applied to the products reaching the mar- ket. Whereas the system of quality standards controls the production processes. The legal basis for these require- ments consists of the EU quality standards regulating the share of lead and sulphur combinations in fuel. Economic evaluation of the negative impact of gas emission. Losses of economy, which are caused by the environmental pollution by gas emission are expressed in monetary terms regarding urban zones and are defined by the formula: 'Quality standards of oil products' are very impor- tant for the preventive regulation of transport and energy emissions. The standards limit the quantities of sulphur, lead, aromatic hydrocarbon substances. The standards meet the EU requirements. M !Soraite I TRANSPORT- 2003, Vol XVIJL No 6, 229-240 Therefore, seeking to solve the problem of traffic jams in Vilnius, Kaunas and IUaipeda it is necessary to: 1. divide the town into zones; 1. divide the town into zones; 2. introduce in public transport the peak and non- peak time tariff. Tariffs of peak p1icing would enable: 1. the reduction of traffic jams; 2. saving of money for low-income people. The promotion of bicycles in Lithuania is in line with the European Union policy: It helps to reduce the emissions of C02 and other ham1ful mate1ials. It is in line with the requirements set in the Euro- pean Commission's 'Green Paper' dedicated to strengthening of citizens' role in the EU institutional structures. The requirements are the following: to guaranty free movement of European inhabitants regarding the economic, social and ecological fac- tors; For the evaluation of external transport costs in Lithuania a method of multicriteria] analysis was offered. One of the most important stages ofmulticriterial analy- sis consists of defining alternative qualifying meanings and significance of the criteria. After the calculation of meanings and significance of the criteria and by the ap- plication of methods ofmulticriterial analysis, the prior- ity of compared variants, their efficiency level and value are determined. Calculations are performed with the at- titude towards the positions of the state and the canier. The aim of calculations is to dete1mine the variant more properly in regard to the canier and the state - external costs covered by the state and external costs not covered by the state. The selection of the best variant was analysed from the point of view of state and a canier. Two vari- ants were taken: when the state covers the external trans- port expenses and, when the state does not cover them. The variants were evaluated according to the further pre- sented quantitative and qualitative c1iteria. Aiming at the identification of evaluations and the significance of the criteria a questionnaire of experts was employed. Basing on the summed up results of the experts' questionnaire the significance of every criterion was defined. The mechanism of multicriteria! complex proportional evalu- ation of integration of external costs into the transporta- to reach better macroeconomic results by saving funds in the field of public health; to reduce investments into transport infrastructure and its operation; to avoid additional costs caused by a long-term nega- tive environmental impact. M !Soraite I TRANSPORT- 2003, Vol XVIJL No 6, 229-240 M !Soraite I TRANSPORT- 2003, Vol XVIJL No 6, 229-240 236 ways; the development of biotransport in Lithuania; the establishment of a certification centre for certification of vehicles and their strategic parts as well as their opera- tion; the creation of the liquid fuels quality management system; the preparation of transport standards meeting the standard requirements of the European Union and ensuring of their implementation. Transport is responsible for the main share of urban noise. Transport noise causes discomfort not only for inhabitants, but for workers of various institutions and enterprises as well. Noise causes discomfort to small children, school children, especially in training and edu- cational areas and in recreation zones. Standards of noise rate in Lithuania slightly differ from those of the EU. Contrary to the EU, where the noise emission from envi- ronment is restricted, in Lithuania the rate of noise is determined by the highest permissible noise in living qua1ters or in working sunoundings. Noise makes a di- rect impact on human health and on the quality of habit- able and recreation areas. A particularly high negative impact on environment is exercised by transport as a dynamic source of noise. Physical noise parameters con- sist of the intensity of noise source (dB), frequency spectre of noise source (Hz), noise spread in the tenitory, noise reflections basing on the laws of construction, physics and acoustics. A negative impact of noise reveals itself by the decrease of hearing ability (in the case of long exposure to pem1anent noise source). A human ear re- acts differently towards noise sources of different fre- quencies. Principal noise sources are the flows of road transpmt in streets. In certain cases they generate up to 80-82 % of a general noise level in urban territories. Transport flows are dynamic noise sources. They practi- cally make a negative impact on all urban tenitories: dwelling-places, hospitals, sanatoriums, recreational zones, urban centres, as well as on communal and indus- trial tenitories. Traffic jams cause public problems. Growing amounts of vehicles increase this problem in Lithuania. In London, for the solution of the problem of traffic jams, the town is divided into several zones. According to this division it is most expensive to drive in the central zone of London. In the central zone the prices of public trans- port tickets are the highest. VBZPxYBZP)xAKZ(JPT)x/KPR; (9) Such regulation often locks fu1ther technical development towards the non-polluting tech- nologies, because companies do not have incentives for exceeding the defined control tasks based on existing standards and existing technologies. This is particularly characteristic of all countries with the authoritarian type of economy. 4. Application of the EU External Costs Evaluation Practice in Lithuania Economic measures may be divided into: ecologi- cal taxes, selling of permissions for pollution, ecological insurance, application of pricing mechanisms, subsidies, tax exemptions, policy of credits, compensation for suf- fered damages, systems of returning deposits, customs duties, fmes. Fuel quality is an important factor of pollution re- duction. In the European Union there are in force the legal acts comprising only those fuel components, which have an impact on air pollution emission (lead and sul- phur combinations). In some EU member countries the supplementary legal requirements are in force for differ- ent fuel characteristics, but in most countries other indi- ces are regulated by the market. Oil refining companies interested in forming a positive public image strive to ensure a good quality of fuel. It is important to define further trends of transport pollution reduction. The main trends of transport pollu- tion reduction are the following: the preparation of plans of measures for saving energy of all transport modes; the development of urban public transport and trolley-buses in particular; further electrification of Lithuanian rail- While products are submitted to the market, besides M !Soraite I TRANSPORT- 2003, Vol XVIJL No 6, 229-240 Covering of uncovered infrastructure costs. This criterion demonstrates that for covering of these costs it is necessary to relate them to the covering of in- frastructure costs. 3. Covering of uncovered infrastructure costs. This criterion demonstrates that for covering of these costs it is necessary to relate them to the covering of in- frastructure costs. 3. Covering of uncovered infrastructure costs. This criterion demonstrates that for covering of these costs it is necessary to relate them to the covering of in- frastructure costs. 4. Air pollution costs. External costs of transpmt pollution have to be based on the air pollution emission. It is done by defming charges which are directly based on em1ss1on. 4. Air pollution costs. External costs of transpmt pollution have to be based on the air pollution emission. It is done by defming charges which are directly based on em1ss1on. With a view of integration of external costs into the transport price, when the monetary value of such costs is not known, the crite1ia may be defined and the evalua- tion is made of the efficiency of this or that tax. 5. Costs of traffic jams. Aiming at their reduction it is necessary to introduce the tax for traffic jams. There are many measures restricting traffic jams, such as re- strictions of vehicles parking, public transport subsidies, decisions on land use aiming at fighting jams. Multicriteria! evaluation may be performed with the aim of evaluation whether it would be useful to intro- duce a tax for the use of one or other road. Multicriteria! analysis is an efficient method of presenting the data having qualitative or quantitative infonnation. Multi- criteria! analysis of external cost evaluation variants was performed in the following stages: 6. Transportation price per 1 passenger km. It shows the cost oftranspmtation of a passenger by 1 km. 6. Transportation price per 1 passenger km. It shows the cost oftranspmtation of a passenger by 1 km. 7. Traffic jams charges/taxes. Introduction of the traffic jams charges/taxes would increase the price of public transport ticket at peak hours, and would decrease it at non-peak time. M !Soraite I TRANSPORT- 2003, Vol XVIJL No 6, 229-240 In the Lithuanian strategy of environmental safety in the field of air safety the objective to 'reduce pollu- tion by transport emitted gases' is defined. The reduc- tion of pollution from mobile sources should lead to the use of vehicles meeting the EU standards. It should go together with the implementation of traffic flows regula- tion system and other measures of pollution reduction. The following principal priorities should be observed in a transport sector: Gradual progress towards the use of fuels meeting the EU requirements; Creation of independent national control system of fuel quality; Implementation of emitted gases neutralisation sys- tem; Promotion of the use of alternative fuels, etc. M. !Soraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 237 tion price in the road transport regarding the carrier and the state is presented. The results of the questionnaire poll of experts were processed and analysed. After the calculations of significance indices the level of priority and efficiency was identified. From the carrier's point of view it is more useful for the carrier when external costs of transport are covered by the state, i.e. in such case the efficiency level equals to 100%. Regarding the carrier the efficiency level is sufficiently low when external costs of transport are not covered by the state and it reaches 56,34 %. Comparing the variants from the state's point of view, when the state covers or does not cover external costs of transport, the priority is given to such variant when the state does not cover external expenses of trans- port. Comparing both views - that of the state and that of the carrier - it is possible to maintain that from the point of view of the state, when the state does not cover external costs of transport, it approaches to the pricing of social marginal transport costs. terion introduced for the reduction of traffic noise. Its purpose is to enlarge funds for purchasing noise control means, such as road isolation. Noise caused by transport may be reduced by the following means: traffic regula- tion, planning of road infrastructure, distance from in- habited areas, designing of roads in cuttings, bulwarks, noise isolation walls, windows insulation. 2. Costs of reduction of accidents. Costs of acci- dents include the whole number of costs, such as: medi- cal, loss of production, materials, transpmtation, insur- ance, non-material dangers, personal losses and injures. 3. M !Soraite I TRANSPORT- 2003, Vol XVIJL No 6, 229-240 Questionnaire poll and forming of data base; Questionnaire poll and forming of data base; Identification of the nomenclature, meanings and significance of qualitative characteristics; Making alternative variants of evaluation of exter- nal costs; Identification of the nomenclature, meanings and significance of qualitative characteristics; Tables 4-7 demonstrate the results obtained by the application of the method of multicriteria! analysis. The results obtained demonstrate that the integration of ex- ternal costs into the transportation price, when the costs are not covered, proves to be 10,89 %more efficient from the point of view of the state. The multicriteria! analysis enables the solution of numerous problems. This research deals with the calculations considering not only the po- sition of the state, but regarding the position of the car- rier as well. Making alternative variants of evaluation of exter- nal costs; Identification of significance of criteria; Multicriteria! analysis of variants of evaluation of external costs; Summary of the results. Summary of the results. Description of criteria 1. Introduction of noise reduction tax. It is a cri- Table 4. Increase of transportation price determined by the intemalisation of external costs in regard to the carrier - Title of criterion . Measure Significance of Not covered by state Covered by state criterion Noise reduction tax - Points 0,3 5 7 Reduction of accidents + Points 0,1 7 9 Not covered costs of infrastructure + Points 0,1 7 8 Costs of air pollution - Points 0,3 8 9 Costs of traffic jams - Points 0,2 6 7 Transportation costs for I km + LTUkm I 0,12 0,14 rtation price determined by the intemalisation of external costs in regard to the carrier Table 4. Increase of transportation price determined by the intemalisation of external costs in regard to t M Isoraite /TRANSPORT~ 2003, Vol XVIII, No 6, 229~240 238 'fable 5. Multicriteria! analysis of alternatives in regard to the carrier. Criterion of Compared variants Criteria analysed . Measure Not covered by the significance state Covered by the state Noise reduction tax ~ Points 0,3 0,1250 0,1750 Reduction of accidents + Points 0,1 0,0438 0,0563 Non covered infrastructure costs + Points 0,1 0,0467 0,0533 Air pollution costs ~ Points 0,3 0,1412 0,1588 Traffic jams costs ~ Points 0,2 0,0923 0,1077 Transportation costs for lkm + LTUkm 1 0,4615 0,5385 Sum of maximising normalised evaluated indices s+i 0,5520 0,6481 Sum of minimising normalised evaluated indices S _ 1 0,9105 0,4415 Significance Q; 0,8785 1,5594 Priority II I Efficiency degree N 1 56,34% 100% 'fable 5. Multicriteria! analysis of alternatives in regard to the carrier. Table 6. Growth of transportation costs determined by the intemalisation of external costs regarding the state Title of criterion . Measure Significance of the Covered by state Non-covered by state criterion Noise reduction tax + Points 0,3 7 9 Reduction of accidents ~ Points 0,2 8 9 Coverage of non-covered infrastructure costs + Points 0,3 7 8 Air pollution costs ~ Points 0,1 9 7 Traffic jams charges + Points 0,1 6 9 Transportation costs per I km + LTL/km I 0,13 0,14 Table 7. Multicriteria! Description of criteria analysis of alternatives in regard to the state Criterion of Compared variants Criteria analysed ' Measure Non-covered by significance Covered by state state Noise reduction tax + Points 0,3 0,1313 0,1688 Reduction of accidents - Points 0,2 0,0941 0,1059 Coverage of uncovered infrastructure costs + Points 0,3 0,14 0,16 Air pollution costs - Points 0,1 0,0563 0,0438 Traffic jams charge + Points 0,1 0,04 0,06 Transportation costs per I km + LTL/km 1 0,4815 0,5185 Sum of maximising normalised evaluated indices S+ 1 0,7928 0,9073 Sum of minimising normalised evaluated indices S_i 0,1504 0,1497 Significance Q 1 0,9424 1,0576 Priority II I Efficiency degree N i 89,11 % 100% * Growth of transportation costs determined by the intemalisation of external costs regarding the state Table 6. Growth of transportation costs determined by the intemalisation of external costs regarding the state Title of criterion . Measure Significance of the Covered by state Non-covered by state criterion Noise reduction tax + Points 0,3 7 9 Reduction of accidents ~ Points 0,2 8 9 Coverage of non-covered infrastructure costs + Points 0,3 7 8 Air pollution costs ~ Points 0,1 9 7 Traffic jams charges + Points 0,1 6 9 Transportation costs per I km + LTL/km I 0,13 0,14 Table 7. Multicriteria! analysis of alternatives in regard to the state Criterion of Compared variants Criteria analysed ' Measure Non-covered by significance Covered by state state Noise reduction tax + Points 0,3 0,1313 0,1688 Reduction of accidents - Points 0,2 0,0941 0,1059 Coverage of uncovered infrastructure costs + Points 0,3 0,14 0,16 Air pollution costs - Points 0,1 0,0563 0,0438 Traffic jams charge + Points 0,1 0,04 0,06 Transportation costs per I km + LTL/km 1 0,4815 0,5185 Sum of maximising normalised evaluated indices S+ 1 0,7928 0,9073 Sum of minimising normalised evaluated indices S_i 0,1504 0,1497 Significance Q 1 0,9424 1,0576 Priority II I Efficiency degree N i 89,11 % 100% * Mark + (-) shows that a relatively higher (lower) meaning of the criterion is more relevant to the Table 7. Multicriteria! analysis of alternatives in regard to the state Table 7. Multicriteria! * Mark + (-) shows that a relatively higher (lower) meaning of the criterion is more relevant to the carrier's requirements. 5. Conclusions 1. The inclusion of marginal external costs of trans- port into the prices of transport services will enable more precise assessment of national revenues generated in transport sector. 3. Pigou, Arthur C. The Economics of Welfare, first edition, Macmillan Company, London, 1920. 4. Knight, F. H. Some fallacies in the interpretation of social costs. Quarterly Journal of Economic, 1924, Vol38, p. 582- 606. 2. The application of the systematic method and multicriteria! complex evaluation method and the inclu- sion of external costs into the price of transport services allows: 5. Walters, Alan A. Theory & measurement of private & so- cial cost ofhigway congestion. Econometrica, Vol29, No 4, p. 676--699. Reprinted in Readings in Urban Econom- ics, eds., Matthew edel and Jerome Rothenberg, 1973, Macmillan, New York, p. 417--437. a) more objective setting of national budget; b) a carrier to look for more economic ways for the reduction of prices of transport services, which will im- prove not only carriers' financial indices, but will also allow him to last under heavy conditions of the market and competence. 6. Strotz, Robert H. Principles of urban transportation pricing. Highway Research Record, 1964, No 47. 7. Mohring, Herbert D. Urban Highway Investments. In: Pro- ceedings Measuring Benefits of Government Investments. Robert Dorfman, papers presented at a Conference of Ex- perts held November 7-9, 1963, The Brookings Institution, Washington, D. C., p. 231-291. 3. In the course of integration of external costs of transport services into the pricing of transport, the exter- nal costs become internal costs and in the carrier's ac- counts of costs all social marginal costs of transport are reflected. 8. Mohring, Herbet D. The peak load problem with increasing returns and pricing constraints. American Economic Re- view, September,1970, Vol60, No 4, p. 693-705. 4. It would be expedient to apply the principle of marginal costs for the improvement of the determination of transportation price, which would allow to reflect the costs of social subsequences in the price. 9. Mohring, H. and Anderson, D. Congestion costs and con- gestion pricing. University of California, Irvine (Working paper), March 1996. 10. Lvy-Lambert, H. Tarification des services qualit vmiable: application aux pages de circulation. Econometrica, 1968, 36(3--4), p. 564--574. 5. M. !Soraite I TRANSPORT- 2003, Vol XVIJL No 6, 229-240 239 Transport Research Conference' that took place in Riga on 13-14 April2000. Riga, 2000, p. 1-5. 5. Conclusions The inclusion of external costs into the pricing of transportation on the one hand would increase the price of such a service, however on the other hand it would induce the carrier to search for possibilities to reduce these costs: 11. Marchand, N. A note on optimal tolls in on imperfect envi- ronment. Econometrica, 1968, 36(3--4), p. 575-581. 12. Vickrey, WilliamS. Congestion Charges and Welfare: Some answers to Sharp's Doubts. Journal of Transport Econom- ics and Policy, January, 1968, Vol2, No I, p. 107-118. 5.1. To improve environmental quality (by the in- troduction of customs duties for imported old cars); 5.2. to reduce traffic jams (applying the Ferrari model of price definition, it is necessary to specify the price of transport services and to introduce appropriate peale time charge); 13. Small, Kenneth, A. A new highway pricing and investment policy. Washington: The Brooking Institution, 1989. 14. Small, Kenneth, A. The incidence of congestion tolls on urban highways. Journal of urban economics, 1983, Vol 13, p. 90--111. 5.3. after the introduction of noise charges the noise rate will decrease; 15. Small, K. A. Urban Transportation Economics. Fundamen- tals of Pure and Applied Economics. 1992 a. Harwood, Chur. 5.4. after the introduction of infrastructure charges for the use of railway infrastructure the funds will be found for the improvement and development of railway infrastructure; 16. DeVany, A. Uncertainty, waiting time, and capacity utili- zation: A stochastic theory of product quality. Journal of' Political Economy, 1976, 84, p. 523-540. 5.5. the losses caused by accidents being included into the road, railway, water, air transport infrastructure charges the uncovered costs of accidents will gradually decrease. 17. Jensen, M. W. and Ross. Hydrogen as a transportation fuel. Environmental, 2001, Vol43, No 3. 18. Vickrey, William S. Congestion theory and transport in- vestment. Am. Econ. Rev., 1969, Vol 59, p. 251-60. 19. Richard, Amott; Marvin, Kraus. When are anonymous con- gestion charges consistent with marginal cost pricing? Jour- nal of Public Economics, 1998, Vol67 (1 ), p. 45-64, Copy- right (c) Elsevier Science B. V. Description of criteria analysis of alternatives in regard to the state Criterion of Compared variants Criteria analysed ' Measure Non-covered by significance Covered by state state Noise reduction tax + Points 0,3 0,1313 0,1688 Reduction of accidents - Points 0,2 0,0941 0,1059 Coverage of uncovered infrastructure costs + Points 0,3 0,14 0,16 Air pollution costs - Points 0,1 0,0563 0,0438 Traffic jams charge + Points 0,1 0,04 0,06 Transportation costs per I km + LTL/km 1 0,4815 0,5185 Sum of maximising normalised evaluated indices S+ 1 0,7928 0,9073 Sum of minimising normalised evaluated indices S_i 0,1504 0,1497 Significance Q 1 0,9424 1,0576 Priority II I Efficiency degree N i 89,11 % 100% * Mark + (-) shows that a relatively higher (lower) meaning of the criterion is more relevant to the i ' i ents * Mark + (-) shows that a relatively higher (lower) meaning of the criterion is more relevant to the carrier's requirements. * Mark + (-) shows that a relatively higher (lower) meaning of the criterion is more relevant to the carrier's requirements. M. !Soraite I TRANSPORT- 2003, Vol XVIJL No 6, 229-240 2. ISoraite, M. From external costs towards fair and efficient pricing. In: Proceedings of the conference. 'Set of scien- tific reports of International Conference 'Nordic-Baltic References 1. ISoraite, M. Application of EU transport pricing policy in Lithuania. Transport (Transportas), Vilnius: Technika, Vol XN, No 4, 1999, p. 200-206. 20. De Palma, A.; Lindsey, R. Information and usage of congestible facilities under different pricing regimes. Ca- nadian Journal of Economics, 1998, 31(3), p. 666-692. 2. ISoraite, M. From external costs towards fair and efficient pricing. In: Proceedings of the conference. 'Set of scien- tific reports of International Conference 'Nordic-Baltic 21. De Palma, A. and Lindsey, R. Private toll roads: competi- tion under various ownership regimes. Annuals of Regional Science, 2000, 34(1), p. 13-35. M ISoraite I TRANSPORT- 2003, Vol XVIII, No 6, 229-240 240 27. Gudmundsson, H. and Hojer, M. Sustainable development principles and their implications for transport. Ecological economics, 1999, Vol 19 (3), p. 269-282. 22. Economic on Social Commission for Asia and Pacific. Asia Institute of Transport Development. Sustainable Transport Pricing and Charges. Principles and Issues. United Notion, 2000, p. 28-87. 28. Mozzarino, M. The economics of the greenhouse effect: evaluating the climate charge impact due to the transport sector in Italy. Energy policy, 2000, Vol 28, No 13, p. 957- 966. 23. Social costs. Draft report of the task force on the social costs of transport. European Conference of ministers of transport. Committee of deputies, 1997. Brussels, p. 37 - 56, p. 136-147, p. 94-120. 29. Nash, C. et al. ModifYing transport prices to internalise ex- ternalities: evidence from European case studies. Regional science and urban economics, 2001, Vol 31, No 4, p. 413- 431. 24. Towards fair and efficient pricing in transport- policy op- tions fort internalizing the external costs of transport in the European Union - green paper. European Commission, Belgium, 1996, p. 34. 30. Sperling, Daniel and Shaheen, Susan A. Transportation and energy: strategies for a sustainable transportation system. Berhley, CA: American Council for energy- efficient en- vironment, 1995. 25. External costs of transport. Accident, environmental and congestion costs in Western Europe. Zurich/Karlsruhe, March 2000, p. 2-3. 31. Feitelson, E. Introducing environmental equity dimensions into the sustainable transport discourse: issues and pitfalls. Transport and environment, 2002, Vol 7, No 2, p. 99- 118. 26. External costs of transport in externe. Edited by P. Eichel, S. Schmid, W. Krewitt, R. Freidrich, lER, Germany, 1997, p. 4.
https://openalex.org/W3018917159	https://europepmc.org/articles/pmc7176721?pdf=render	English	null	The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis	Nature communications	2,020	cc-by	18,805	1 Membrane Trafﬁc and Cell Division Lab, Institut Pasteur, UMR3691, CNRS, F-75015 Paris, France. 2 Sorbonne Université, Collège doctoral, F-75005 Paris, France. 3 Institut Pasteur, UTechS CB, F-75015 Paris, France. 4 Institut Pasteur, Mass Spectrometry for Biology Unit, C2RT, USR 2000, CNRS, F-75015 Paris, France. 5 Institut Pasteur, Proteomics Platform, Mass Spectrometry for Biology, C2RT, USR 2000, CNRS, F-75015 Paris, France. 6 Hub de Bioinformatique et Biostatistique – Département Biologie Computationnelle, Institut Pasteur, USR 3756 CNRS, F-75015 Paris, France. 7 UTechS Photonic BioImaging PBI (Imagopole), Centre de Recherche et de Ressources Technologiques C2RT, Institut Pasteur, Paris 75015, France. 8 Centre de Recherche en Cancérologie de Marseille (CRCM), Equipe labellisée Ligue 2018, Aix-Marseille Université, Inserm, CNRS, Institut Paoli Calmettes, 13009 Marseille, France. 9 KU Leuven, Department of Human Genetics, University of Leuven, B-3000 Leuven, Belgium. 10These authors jointly supervised: Neetu Gupta-Rossi, Arnaud Echard. ✉email: arnaud.echard@pasteur.fr ARTICLE Results Remarkably, cell membrane labeling with cell mask and scanning EM46 further demonstrated that the MBR+ fraction contained membrane- sealed, intact MBRs (Fig. 1d), with very similar shape and length, as observed in vivo14. Immunoﬂuorescence revealed that the 1–3 μm-sized objects sorted as MBR+ were indeed all GFP- positive MBRs and that the respective localization of key cyto- kinetic proteins [MKLP2, AuroraB, MKLP1, CEP55, RacGAP1, CHMP4B, ALIX, PRC1, and CRIK] was preserved (Fig. 1d). Thus, this original method to purify MBRs using ﬂow cytometry sorting allowed us to obtain intact and highly pure MBRs, which correspond to post-abscission midbodies. y The endosomal sorting complexes required for transport (ESCRT) machinery plays a critical and evolutionarily con- served role in cytokinetic abscission, both in Eukaryotes and in Archea20–31. This machinery is composed of several protein complexes (ESCRT-0 to III) and culminates with the poly- merization of ﬁlaments made of ESCRT-III components that contract in the presence of the ATPase VPS4 and ATP31–35. Remarkably, ESCRT-III-dependent helices of 17 nm ﬁlaments are observed at the abscission site by electron microscopy (EM), and ESCRT-III helical structures are often visible extending from the midbody to the abscission site10,36. Therefore, as in other topologically equivalent ESCRT-III-mediated events, including exosome biogenesis in multivesicular bodies (MVBs), retroviral budding or membrane repair, constriction of ESCRT- III ﬁlaments likely drives the ﬁnal membrane scission during cytokinetic abscission2–7,9. The midbody plays a fundamental role in cytokinesis, as it constitutes a protein-rich platform that recruits key compo- nents for abscission, including the ESCRT machinery2,3,6,9. It is well established that the MKLP1 kinesin targets CEP55 to the midbody, which in turn recruits, through both ESCRT-I TSG101 and ESCRT-associated protein ALIX, the entire ESCRT machinery29,37. After this initial recruitment to the midbody itself and prior to abscission, the ESCRT-III machinery is progressively enriched to the future abscission site on the midbody side12,13,20,21,25,30,31,36,38,39. y Since MKLP1 and CEP55 are only present at the midbody38, it remains elusive how, mechanistically, ESCRT-III components can localize to the abscission site. Another crucial related issue is to reveal how the ESCRT-III ﬁlaments could be coupled to the plasma membrane, as ﬁnal membrane constriction should require their tight association. We next performed proteomic and statistical analysis to (1) identify proteins detected in seven independent MBR+ prepara- tions and (2) identify proteins signiﬁcantly enriched in these preparations, as compared to MBR−, MBRE, and/or total cell fractions. Results C ytokinesis leads to the physical separation of daughter cells and concludes cell division. Final abscission occurs close to the midbody (or Flemming body), a prominent structure that matures at the center of the intercellular bridge connecting the two daughter cells and ﬁrst described by Walther Flemming in 18911–9. The scission occurs not at the midbody itself, but at the abscission site located at distance on one side of the midbody10–14. The ﬁrst scission is usually followed by a second cleavage on the other side of the midbody, leaving a free MidBody Remnant (MBR)8,12–15. Then, MBRs are either released or wander, tethered at the cell surface for several hours, before being engulfed and degraded by lysosomes14,16–19. The Flemmingsome reveals candidates for abscission. The proteome of intercellular bridges from CHO cells previously proved to be a particularly successful approach to identify pro- teins required for cytokinesis44. However, the use of detergents during the puriﬁcation steps precluded the recovery of crucial proteins for cytokinesis, for instance the ESCRT components44. In order to purify intact midbodies without detergent treatment and thus reveal the complete proteome of these abscission plat- forms, we took advantage of the fact that released MBRs can be easily detached from the cell surface by EDTA treatment, as we previously reported14. Differential centrifugations helped to enrich intact MBRs from EDTA-treated HeLa cells (“Midbody Remnant Enriched fraction” or “MBRE”, Fig. 1a) expressing the midbody-localized kinesin GFP-MKLP245 (which did not perturb the timing of abscission, Supplementary Fig. 1a). In order to improve the enrichment of MBRs, we developed an original protocol for isolating cell-free ﬂuorescent GFP-positive MBRs (“MBR+”) from EDTA-treated cells using ﬂow cytometry (Fig. 1a, b; Supplementary Fig. 1b). In parallel, and as a control, we isolated small particles of matched size (1–3 μm) and granu- larity (SSC) but negative for GFP-MKLP2 (“MBR−”) (Fig. 1a, b; Supplementary Fig. 1b). Western blot analysis demonstrated that the MBR+ population contained highly enriched known mid- body proteins [MKLP1, CRIK, PRC1, PLK1, and CEP55] and showed reduced contamination, as compared to MBR−, total cell lysate (Tot) and MBRE fractions, with intracellular compartments [calreticulin (endoplamic reticulum), GM130 (golgi), Tom22 (mitochondria), HistoneH3 (nucleus), EEA1 (endosomes)] (Fig. 1c; Supplementary Fig. 1c). As expected, proteins such as ALIX, which participates both in cytokinesis and endosomal sorting in interphase were less enriched (twofold). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z The Flemmingsome reveals an ESCRT-to- membrane coupling via ALIX/syntenin/ syndecan-4 required for completion of cytokinesis Cytokinesis requires the constriction of ESCRT-III ﬁlaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT- III machinery localizes to the abscission site. To reveal actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identiﬁed 489 proteins enriched in this organelle. Among these proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syn- decan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins are highly recruited ﬁrst at the midbody then at the abscission site, and their depletion delays abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 are essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efﬁcient scission, uncovering common requirements in cytokinesis, exosome formation and HIV budding. 1 Membrane Trafﬁc and Cell Division Lab, Institut Pasteur, UMR3691, CNRS, F-75015 Paris, France. 2 Sorbonne Université, Collège doctoral, F-75005 Paris, France. 3 Institut Pasteur, UTechS CB, F-75015 Paris, France. 4 Institut Pasteur, Mass Spectrometry for Biology Unit, C2RT, USR 2000, CNRS, F-75015 Paris, France. 5 Institut Pasteur, Proteomics Platform, Mass Spectrometry for Biology, C2RT, USR 2000, CNRS, F-75015 Paris, France. 6 Hub de Bioinformatique et Biostatistique – Département Biologie Computationnelle, Institut Pasteur, USR 3756 CNRS, F-75015 Paris, France. 7 UTechS Photonic BioImaging PBI (Imagopole), Centre de Recherche et de Ressources Technologiques C2RT, Institut Pasteur, Paris 75015, France. 8 Centre de Recherche en Cancérologie de Marseille (CRCM), Equipe labellisée Ligue 2018, Aix-Marseille Université, Inserm, CNRS, Institut Paoli Calmettes, 13009 Marseille, France. 9 KU Leuven, Department of Human Genetics, University of Leuven, B-3000 Leuven, Belgium. 10These authors jointly supervised: Neetu Gupta-Rossi, Arnaud Echard. ✉email: arnaud.echard@pasteur.fr 1 NATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications ARTICLE Results Interestingly, differential analyses indicated that the most abundant and most signiﬁcantly enriched proteins such as MKLP1 (KIF23) MKLP2 (KIF20A) Volcano plots demonstrated that, independently of the extracti method (eFASP or gel-based), these core cytokinetic proteins we more enriched in MBR+ as compared to MBR−, MBRE, or To (Supplementary Fig. 2), consistent with the results obtained western blots (Supplementary Fig 1c) GFP SSC-A 103 104 105 103 104 105 0 0 65 50 115 15 65 185 185 kDa Tot Total l SN Cell pellet Flow cytometry sorting GFPpos (MBR+) GFPneg (MBR–) MBR-Enriched (MBRE) Differential centrifugations MBR– MBR+ MBRE ALIX CEP55 CRIK CRIK (long exp.) PLK1 PRC1 TOM22 15 50 115 115 185 kDa Tot MBR– MBR+ MBRE EEA1 Histone H3 GAPDH MKLP1 Calreticulin GM130 PRC1 CHMP4B ALIX CRIK RacGAP CEP55 MKLP1 Aurora B MKLP2 Cell mask GFP- MKLP2 GFP- MKLP2 GFP- MKLP2 Cell mask 30 20 –log10(merged p-value) 10 0 0.0 2.5 5.0 7.5 10.0 Maximum of log2(MBR+/control) # of times found more ab. in MBR+ 1 2 3 Transport/Traffic/Vesicles Cell adhesion/Junction Actin-related Signal transduction/Signaling Microtubule-related Proteasome/Ubiquitin-related Mitosis/Cell division Cell cycle DNA-related RNA-related Translation/Protein biosynthesis Over-representation of GO term clusters in total flemmingsome Number of proteins 0 300 600 900 log10(p-value) > –1 –1 –25 –50 –75 –100 a f b c d e g Tot Tot Tot Tot Tot Tot MBR– MBR– MBR– MBR– MBR– MBR– MBRE MBRE MBRE MBRE MBRE MBRE MBR+ vs MBR+ vs MBR+ vs MBR+ vs MBR+ vs MBR+ vs Protein quantified in MBR+ and not in control MBR+ MBR– Traffic Actin Microtubule CPC ESCRT Translation Ubiquitin Proteasome Transport NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z ARTICL NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z in MBR+ 1 2 3 e GFP- MKLP2 Cell mask d d e PRC1 CHMP4B ALIX CRIK RacGAP CEP55 MKLP1 Aurora B MKLP2 Cell mask GFP- MKLP2 GFP- MKLP2 0.0 2.5 5.0 7.5 10.0 Maximum of log2(MBR+/control) Transport/Traffic/Vesicles Cell adhesion/Junction Actin-related Signal transduction/Signaling Microtubule-related Proteasome/Ubiquitin-related Mitosis/Cell division Cell cycle DNA-related RNA-related Translation/Protein biosynthesis Over-representation of GO term clusters in total flemmingsome Number of proteins 0 300 600 900 log10(p-value) > –1 –1 –25 –50 –75 –100 g Tot Tot Tot Tot Tot Tot MBR– MBR– MBR– MBR– MBR– MBR– MBRE MBRE MBRE MBRE MBRE MBRE MBR+ vs MBR+ vs MBR+ vs MBR+ vs MBR+ vs MBR+ vs Protein quantified in MBR+ and not in control Tot Tot Tot Tot Tot Tot MBR– MBR– MBR– MBR– MBR– MBR– MBRE MBRE MBRE MBRE MBRE MBRE MBR+ vs MBR+ vs MBR+ vs MBR+ vs MBR+ vs MBR+ vs Protein quantified in MBR+ and not in control Data 1 TAB2 TAB4 5 and Methods) For instance CRIK was f Traffic Actin Microtubule CPC ESCRT Translation Ubiquitin Proteasome Transport f g Transport/Traffic/Vesicles Cell adhesion/Junction Actin-related Signal transduction/Signaling Microtubule-related Proteasome/Ubiquitin-related Mitosis/Cell division Cell cycle DNA-related RNA-related Translation/Protein biosynthesis Over-representation of GO term clusters in total flemmingsome Number of proteins 0 300 600 900 log10(p-value) > –1 –1 –25 –50 –75 –100 g Microtubule CPC Translation Ubiquitin Proteasome Translation Data 1, TAB2, TAB4-5 and Methods). For instance, CRIK was found enriched >500-fold in MBR+ as compared to Total (Supplementary Data 1, TAB2, col I). Interestingly, differential analyses indicated that the most abundant and most signiﬁcantly enriched proteins, such as MKLP1 (KIF23), MKLP2 (KIF20A), RacGAP1, KIF4A, PRC1, KIF14, PLK1, CEP55, and CRIK (CIT) corresponded to well established proteins of cytokinesis (Fig. 1e). Data 1, TAB2, TAB4-5 and Methods). For instance, CRIK was found enriched >500-fold in MBR+ as compared to Total (Supplementary Data 1, TAB2, col I). Interestingly, differential analyses indicated that the most abundant and most signiﬁcantly enriched proteins, such as MKLP1 (KIF23), MKLP2 (KIF20A), RacGAP1, KIF4A, PRC1, KIF14, PLK1, CEP55, and CRIK (CIT) corresponded to well established proteins of cytokinesis (Fig. 1e). Volcano plots demonstrated that, independently of the extraction method (eFASP or gel-based), these core cytokinetic proteins were more enriched in MBR+ as compared to MBR−, MBRE, or Total (Supplementary Fig. 2), consistent with the results obtained by western blots (Supplementary Fig. 1c). Results Since it is notoriously difﬁcult to extract proteins from midbodies47, we used sodium dodecyl sulfate (SDS) to fully solubilize proteins from our different fractions after puriﬁcation. For mass spectrometry analysis, two methods for sample preparation were used and analyzed separately (SDS polyacryla- mide gel electrophoresis (PAGE) gel/in-gel digestion and enhanced ﬁlter-aided sample preparation (eFASP48)/in-solution digestion, and gave complementary results (Supplementary Fig. 2)). We detected a total of 1732 proteins with at least one unique identiﬁed peptide in the MBR+ preparations, constituting the Total Flemmingsome (Supplementary Data 1, TAB1), a name that we gave as a tribute to W. Flemming. g Here, we ﬁrst set up an original method, using ﬂow cyto- metry, for purifying intact post-cytokinetic MBRs, and identi- ﬁed 489 proteins enriched in this organelle by proteomics. Among them, we focused on the transmembrane protein syndecan-4 and associated proteins syntenin-1 (hereafter “syntenin”) and ALIX, all highly enriched. Indeed, ALIX directly interacts with ESCRT-III40 and we previously showed that syntenin can bind directly and simultaneously to the cytoplasmic tail of syndecans in vitro41,42. We thus hypothe- sized that ALIX-syntenin could mechanistically bridge the ESCRT machinery to the plasma membrane through the transmembrane proteoglycan syndecan-4. Interestingly, over- expression of syndecan-4 mutants that cannot be properly phosphorylated on the cytoplasmic tail was reported to perturb cytokinesis43. However, the underlying mechanism is unknown and whether syndecan-4 is actually required for cytokinesis has not been addressed. We here reveal that, together with ALIX, both syndecan-4 and syntenin are required for successful abscission in parallel to the TSG101 pathway, and promote the stable recruitment of the ESCRT-III machinery speciﬁcally at the abscission site. Among the 1732 proteins in MBR+, we deﬁned as the Enriched Flemmingsome (Supplementary Data 1, TAB2) a subset of 489 proteins signiﬁcantly enriched at least 1.3-fold with a false-discovery rate (FDR) < 5% as compared to MBRE, MBR−, or Tot (Fig. 1e, upper panel, Supplementary Fig. 1d and 2; Supplementary Data 1, TAB2-3) and/or quantitatively present in MBR+ but not detected in at least one other fraction (Fig. 1e, bottom panel; Supplementary NATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications 2 Data 1, TAB2, TAB4-5 and Methods). For instance, CRIK was found enriched >500-fold in MBR+ as compared to Total (Supplementary Data 1, TAB2, col I). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z GFP SSC-A 103 104 105 103 104 105 0 0 65 50 115 15 65 185 185 kDa Tot tal Cell pellet cytometry orting GFPpos (MBR ) MBR– MBR+ MBRE ALIX CEP55 CRIK CRIK (long exp.) PLK1 PRC1 TOM22 15 50 115 115 185 kDa Tot MBR– MBR+ MBRE b c MBR+ MBR– GFP SSC-A 103 104 105 103 104 105 0 0 b MBR+ MBR– GFP SSC-A 103 104 105 103 104 105 0 0 65 50 115 15 65 185 185 kDa Tot MBR– MBR+ MBRE ALIX CEP55 CRIK CRIK (long exp.) PLK1 PRC1 TOM22 b c MBR+ MBR– 65 50 115 15 65 185 185 kDa Tot MBR– MBR+ MBRE ALIX CEP55 CRIK CRIK (long exp. PLK1 PRC1 TOM22 c Total l SN Cell pellet Flow cytometry sorting GFPpos (MBR+) GFPneg (MBR–) MBR-Enriched (MBRE) Differential centrifugations a b c c a 15 50 115 115 185 kDa Tot MBR– MBR+ MBRE EEA1 Histone H3 GAPDH MKLP1 Calreticulin GM130 PRC1 CHMP4B ALIX CRIK RacGAP CEP55 MKLP1 Aurora B MKLP2 Cell mask GFP- MKLP2 GFP- MKLP2 GFP- MKLP2 Cell mask d 30 20 –log10(merged p-value) 10 0 0.0 2.5 5.0 7.5 10.0 Maximum of log2(MBR+/control) # of times found more ab. ARTICLE 2b), using antibodies recognizing endogenous proteins (staining speciﬁcity conﬁrmed in Supplementary Fig. 4c). Interestingly, super-resolution microscopy using structured illumination (SIM) showed that both syntenin and ALIX colocalized with CHMP4B at the outer rim of CHMP4B staining (Fig. 2c), a relative localization con- sistent with the molecular scheme presented in Fig. 2a and with the model presented in the ﬁnal ﬁgure. Time-lapse spinning-disk confocal microscopy further revealed a striking time-dependent and concomitant enrichment of these proteins, ﬁrst at the mid- body, then at the abscission site before the cut (Fig. 2d; Supple- mentary Movies 2–4). Thus syndecan-4, syntenin, ALIX, and CHMP4B extensively and dynamically colocalized during the terminal steps of cytokinesis, notably at the abscission site. bridge or the midbody and/or functionally involved in cytokin- esis, according to our literature search (Supplementary Data 1, TAB2 and dedicated website https://ﬂemmingsome.pasteur. cloud/). Proteins of the Enriched Flemmingsome were highly connected and based on the literature, many fell into known functional categories involved in cytokinesis, such as “actin- related”, “chromosomal passenger complex-related”, “microtu- bule-related”, “trafﬁc/transport-related”, or “ESCRT-related” (Fig. 1f; Supplementary Fig. 3). GO-term analysis revealed that 87.5% of proteins identiﬁed in MBR+ fell into 11 GO term clusters (deﬁned in Data 1, TAB7). The category “mitosis/cell division” was more signiﬁcantly overrepresented in MBR+ as compared to either total identiﬁed proteins (Fig. 1g) or MBRE (Supplementary Fig. 1e). This indicates that ﬂow cytometry-based MBR+ puriﬁcation is superior to MBRE puriﬁcation for obtaining proteins known to be implicated in cell division. Thus, our approach was highly successful at identifying 150 known cytokinetic proteins and, importantly, revealed 339 additional candidates potentially involved in cytokinesis/abscis- sion. In the rest of this study, we decided to focus on ALIX (PDCD6IP), syntenin (SDCBP) and syndecan-4 (SDC4) (Fig. 2a). Indeed, these three proteins were found among the most enriched in MBR+ compared to the other three fractions (Fig. 1e, highlighted in red) and are known to form a tripartite complex in interphase in the context of exosome formation41. Whether the same complex could be involved in cell division was unknown and could potentially reinforce the idea that exosome formation and cytokinetic abscission share common basic mechanisms. In addition, the implication of ALIX in abscission has been established (e.g., ref. 29) but its exact role remains elusive. Mechanism of syndecan-4 and syntenin recruitment by ALIX. We next investigated how syntenin and syndecan-4 are recruited to the intercellular bridge during cytokinesis. ARTICLE Fig. 1 Proteomics of highly pure and intact post-abscission midbodies revealed known and previously unknown proteins enriched in this organelle. a Midbody remnant puriﬁcation. HeLa cells (upper left picture) expressing GFP-MKLP2, a kinesin enriched in midbodies (MB) and midbody remnants (MBRs) were EDTA-treated (Total). After 70g centrifugation, the supernatant (SN) containing MBRs was processed either (1) by differential centrifugations leading to MBR-enriched fraction (MBRE) or (2) subjected to ﬂow cytometry sorting to purify GFP-positive MBRs (MBR+) and their GFP- negative counterpart (MBR−). b Representative pseudo-colored proﬁle of ﬂow cytometry sorting of MBRs. The MBR+ (14% total) and SSC-matched MBR−(44% total) were separated from remaining cells (1%). See Supplementary Fig. 1b. c Western blots of same amounts of protein extracts from Total (Tot), MBR-enriched (MBRE), ﬂow cytometry-sorted MBR−and MBR+ populations. Membranes were blotted repeatedly with indicated antibodies. See also Supplementary Figs. 1c and 6. d Upper left panel: MBR+ population analyzed with cell mask membrane marker. Each individual midbody is positive for GFP-MKLP2 (green) and cell mask (red) Scale bar: 6 μm. Upper right panel: scanning electron microscopy of an isolated MBR. Note the intact and sealed membrane. Lower panels: immunoﬂuorescence stainings of MBR+ for endogenous proteins or membrane marker (red), as indicated. Scale bars: 2 μm. e The Enriched Flemmingsome. Upper panel: merged volcano plot of the mass spectrometry analysis showing the maximum log2(fold change) in x-axis measured between MBR+ and the other fractions (MBRE, MBR−, or Total) and the corresponding –log10(merged p value) in y-axis. color code: proteins signiﬁcantly enriched in MBR+ when compared with 3 (red), 2 (blue), or 1 (green) of the other fractions. Bottom panel: proteins quantitatively present in MBR+ but not detected in at least two of the other fractions. ALIX (PDCD6IP), syntenin (SDCBP) and syndecan-4 (SDC4) circled in red. f STRING functional association network for the Enriched Flemmingsome. See Supplementary Fig. 3 for details. g GO-term over-representation clusters in the Total Flemmingsome. The size of each bar (x-axis) corresponds to the number of proteins in each cluster and the red gradient the enrichment p values coming from hypergeometric tests. Gray: p value > 0.1. site. Time-lapse spinning-disk confocal microscopy actually conﬁrmed that abscission occurred at the tip of the ALIX cone (Supplementary Fig. 4b, arrow and Supplementary Movie 1). Similarly, we observed colocalization at the confocal microscopy level between ALIX/syntenin and syntenin/syndecan-4, both at the midbody and at the abscission site (Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z Interestingly, 150 out of the 489 proteins (31%) of the Enriched Flemmingsome have been already localized to the furrow, the NATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications 3 3 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z ATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z Efﬁcient abscission requires Syndecan-4, syntenin, an F ti ll d l ti f ith ALIX t i 4 a b d c Tubulin CHMP4B ALIX Synt CHMP4B Tubulin 90 min 210 min 260 min 270 min Merge SDC4 Syntenin v v v GFP-SDC4 mScarlet-Syntenin 1 3 4 2 70 min 90 min 120 min 170 min Merge Syntenin ALIX v v v GFP-Syntenin ALIX-mScarlet 1 3 4 2 1 3 4 2 50 min 110 min 140 min 180 min Merge CHMP4B ALIX v v v CHMP4B-GFP ALIX-mScarlet SDC4 Syntenin Tubulin v v v v v ALIX Syntenin Tubulin v v v v v v v v v v ALIX CHMP4B Tubulin TM PDZ1 EFYA C-ter N-ter N-ter C-ter PDZ2 ECD (LYPXL)3 C-ter Syntenin Syndecan-4 ALlX N-ter Bro1 ESCRT-III TSG101 CEP55 100 % of ALlX-positive bridges 80 60 40 20 0 0 2 Distance (μm) 4 6 0 2 Distance (μm) 4 6 0 2 Distance (μm) 6 100 Normalized intensity (%) 80 60 40 20 0 100 Normalized intensity (%) 80 60 40 20 0 100 Normalized intensity (%) 80 60 40 20 0 100 % of Syntenin-positive bridges 80 60 40 20 0 100 % of SDC4-positive bridges 80 60 40 20 0 Without abscission site With abscission site Without abscission site With abscission site Without abscission site With abscission site F676 Synt ALIX ESCRT-III SDC4 4 a b SDC4 Syntenin Tubulin v v v v v ALIX Syntenin Tubulin v v v v v v v v v v ALIX CHMP4B Tubulin TM PDZ1 EFYA C-ter N-ter N-ter C-ter PDZ2 ECD (LYPXL)3 C-ter Syntenin Syn ALlX N-ter Bro1 ESCRT-III TSG101 CEP55 100 % of ALlX-positive bridges 80 60 40 20 0 0 2 Distance (μm) 4 6 0 2 Distance (μm) 4 6 0 2 Distance (μm) 6 100 Normalized intensity (%) 80 60 40 20 0 100 Normalized intensity (%) 80 60 40 20 0 100 Normalized intensity (%) 80 60 40 20 0 100 % of Syntenin-positive bridges 80 60 40 20 0 100 % of SDC4-positive bridges 80 60 40 20 0 Without abscission site With abscission site Without abscission site With abscission site Without abscission site With abscission site F676 Synt ALIX ESCRT-III SDC4 a b c Tubulin CHMP4B ALIX Synt CHMP4B Tubulin c d 90 min 210 min 260 min 270 min Merge SDC4 Syntenin v v v GFP-SDC4 mScarlet-Syntenin 1 3 4 2 70 min 90 min 120 min 170 min Merge Syntenin ALIX v v v GFP-Syntenin ALIX-mScarlet 1 3 4 2 1 3 4 2 50 min 110 min 140 min 180 min Merge CHMP4B ALIX v v v CHMP4B-GFP ALIX-mScarlet d transmembrane and cytosolic domains) was properly recruited, indicating that the extracellular domain is dispensable for syndecan-4 localization at the midbody or at the abscission site (Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z membrane and cytosolic domains) was properly recruited, ating that the extracellular domain is dispensable for can-4 localization at the midbody or at the abscission site Altogether, we conclude that ALIX recruits s turn, recruits syndecan-4 at the intercellular bridg interactions. 4 a b d c Tubulin CHMP4B ALIX Synt CHMP4B Tubulin 90 min 210 min 260 min 270 min Merge SDC4 Syntenin v v v GFP-SDC4 mScarlet-Syntenin 1 3 4 2 70 min 90 min 120 min 170 min Merge Syntenin ALIX v v v GFP-Syntenin ALIX-mScarlet 1 3 4 2 1 3 4 2 50 min 110 min 140 min 180 min Merge CHMP4B ALIX v v v CHMP4B-GFP ALIX-mScarlet SDC4 Syntenin Tubulin v v v v v ALIX Syntenin Tubulin v v v v v v v v v v ALIX CHMP4B Tubulin TM PDZ1 EFYA C-ter N-ter N-ter C-ter PDZ2 ECD (LYPXL)3 C-ter Syntenin Syndeca ALlX N-ter Bro1 ESCRT-III TSG101 CEP55 100 % of ALlX-positive bridges 80 60 40 20 0 0 2 Distance (μm) 4 6 0 2 Distance (μm) 4 6 0 2 Distance (μm) 6 100 Normalized intensity (%) 80 60 40 20 0 100 Normalized intensity (%) 80 60 40 20 0 100 Normalized intensity (%) 80 60 40 20 0 100 % of Syntenin-positive bridges 80 60 40 20 0 100 % of SDC4-positive bridges 80 60 40 20 0 Without abscission site With abscission site Without abscission site With abscission site Without abscission site With abscission site F676 Synt ALIX ESCRT-III SDC4 RE COMMUNICATIONS \| https://doi.org/10.1038/s41467 020 15205 z membrane and cytosolic domains) was properly recruited, ating that the extracellular domain is dispensable for ecan-4 localization at the midbody or at the abscission site 3f). These results indicate that syntenin and its interaction syndecan-4 are necessary for syndecan-4 localization at the e Altogether, we conclude that ALIX recruits syntenin turn, recruits syndecan-4 at the intercellular bridge throu interactions. ARTICLE First, siRNA- mediated depletion of ALIX strongly reduced the proportion of bridges positive for endogenous syntenin (Fig. 3a, b). In contrast, syntenin depletion had no effect on ALIX recruitment, and syndecan-4 depletion had basically no impact on syntenin or ALIX recruitment (Fig. 3b; Supplementary Fig. 4d). Importantly, upon reintroduction in ALIX-depleted cells, the ALIX F676D mutant that cannot bind to syntenin41,49 was unable to restore the recruitment of endogenous syntenin to the bridge, whereas wild-type ALIX could (Fig. 3c). Furthermore, a green ﬂuorescent protein (GFP)-tagged syntenin ΔALIX (a triple mutant LYP-LAA that cannot interact with ALIX41) was no longer recruited to the bridge, whereas GFP-syntenin wild-type and GFP-syntenin ΔSDC (harboring point mutations in PDZ1 and PDZ2 that dis- rupt the interaction with syndecans41,50) were recruited (Fig. 3d). Thus, ALIX recruits syntenin to the intercellular bridge and this requires a direct interaction between ALIX and syntenin. ALIX, syntenin, and syndecan-4 colocalize with ESCRT-III. In ﬁxed cells, endogenous ALIX and CHMP4B colocalized as two parallel stripes at the midbody (hereafter ﬁgured with white brackets) in bridges without observable abscission sites, as expected (Supplementary Fig. 4a). When bridges mature, the future abscission site also known as secondary ingression site (hereafter pointed with a white arrowhead) appears at the level of pinched and/or interrupted tubulin staining on one side of the midbody, typically 1–2 μm away, as previously characterized12,39. At this late stage, CHMP4B staining extends from the midbody to the abscission site, often appearing as a cone-shaped structure on one side of the midbody, as previously reported12,13,20,21,25,30,31,39 (Fig. 2b). We found that endogenous ALIX colocalized with CHMP4B both at the midbody and at the abscission site (Fig. 2b). To our knowledge, ALIX had not been described at the abscission q y We next observed that syndecan-4 failed to be correctly recruited at bridges upon ALIX depletion (consistent with the results above) or syntenin depletion (Fig. 3e). Furthermore, GFP- syndecan-4 Δsynt (a C-terminal YA motif deletion mutant that is essential for syntenin binding51) was no longer recruited to the bridge (Fig. 3f). Of note, the GFP-syndecan-4 ΔECD (a mutant that lacks the entire extracellular domain but retains its 4 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z Finally, we investigated why ESCRT-III did not accumulate normally at the abscission site when either ALIX, syntenin or syndecan-4 were depleted, using ﬂuorescent time-lapse micro- scopy in cells expressing a functional CHMP4B-GFP31. py p g In control cells, CHMP4B-GFP accumulated ﬁrst at the midbody then appeared on its side as a strong, large, cone-like signal pointing toward the abscission site (Fig. 5f; Supplementary Movie 5). Importantly, in ALIX-depleted cells, CHMP4B-GFP signal on the side of the midbody was less prominent and frequently disappeared as if it was unstable, something that we never observed in controls (Fig. 5g, yellow and cyan arrows highlight these transient pools of CHMP4B). As a consequence, the time between the ﬁrst occurrence of CHMP4B on the side of the midbody and actual abscission was delayed (Supplementary Fig. 5e, up), and abscission eventually occurred without large, cone-shaped concentration of CHMP4B at the abscission site (Fig. 5g; Supplementary Movie 6). In syntenin- or syndecan- depleted cells, we again observed a transient and unstable localization of CHMP4B-GFP on the side of the midbody (Fig. 5h, i, yellow and cyan arrows; Supplementary Movies 7 and 8). Abnormal behavior of CHMP4B at the future abscission site was thus observed upon either ALIX, syntenin or syndecan-4 depletion (Fig. 5j). This was accompanied by a delay between the accumulation of CHMP4B at the midbody and abscission (Supplementary Fig. 5e, bottom), and CHMP4B-GFP signal on the side of the midbody took longer to appear (Supplementary Fig. 5e, middle). As a consequence, the time between the ﬁrst occurrence of CHMP4B on the midbody side and actual abscission was delayed (Supplementary Fig. 5e, up). Although the phenotypes were similar, all the measured parameters were more impaired in ALIX-depleted cells as compared with ESCRT-III at the abscission site rely on syndecan-4/syntenin. We then investigated why abscission was delayed after ALIX, syntenin or syndecan-4 depletion. We ﬁrst quantiﬁed the pro- portion of intercellular bridges with no ESCRT-III at all (early bridges), with ESCRT-III localized only at the midbody (bridges without secondary ingression) and with ESCRT-III both at the midbody and at the abscission site (bridges with constricted/ interrupted tubulin staining, as in Fig. 2b). Depletion of either ALIX, syntenin or syndecan-4 considerably reduced the number of bridges with CHMP4B at the midbody + abscission site (Fig. 5a). This is consistent with the observed abscission delay in depleted cells (Fig. 4a–c). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z Nevertheless, time-lapse microscopy demonstrated that abscission was delayed in ALIX-depleted cells (Fig. 4a; Supplementary Fig. 5b), as previously reported, indi- cating that ALIX plays a speciﬁc role in abscission. Interestingly, abscission was also delayed after either syntenin or syndecan-4 depletions (Fig. 4b, c; Supplementary Fig. 5c, d). Abscission was fully restored by reintroducing either wild-type ALIX, syntenin or syndecan-4, respectively, ruling out off-target effects (Fig. 4a–c). Importantly, the ALIX F676D was unable to rescue the abscission defects due to ALIX depletion (Fig. 4a). Similarly, syntenin ΔALIX or syntenin ΔSDC failed to rescue the abscission defects observed after syntenin depletion (Fig. 4b). We conclude that ALIX, syntenin and syndecan-4 are important for normal abscission, and that the direct interactions between ALIX/synte- nin on the one hand and syntenin/syndecan on the other hand are critical. (Fig. 5b), syntenin ΔALIX (Fig. 5c), syntenin ΔSDC (Fig. 5c), or syndecan-4 Δsynt (Fig. 5d). In contrast, syndecan-4 ΔECD localized at the bridge and behaved similarly to the wild type for ESCRT-III recruitment (Fig. 5d). Conﬁrming previous reports29, we also observed that the presence of both TSG101 and ALIX is required for the recruitment of CHMP4B at the midbody itself (Fig. 5e). Strikingly, the depletion of both TSG101 and either syntenin or syndecan-4 essentially abolished the localization of CHMP4B at the abscission site (Fig. 5e). Altogether, we conclude that direct interactions between ALIX and syntenin on the one hand, and syntenin and syndecan-4 on the other hand are critical for the recruitment of ESCRT-III speciﬁcally at the abscission site but not at the midbody itself. The defects of CHMP4B localization at the abscission site, which were aggravated when TSG101 was depleted, are consistent with the delay in abscission observed after ALIX, syntenin and syndecan-4 depletion (Fig. 4). Syndecan-4/syntenin and TSG101 cooperate for abscission. TSG101 and ALIX were proposed to act in parallel for promoting abscission29, and we conﬁrmed that it is indeed the case, since co-depletion of TSG101 and ALIX led to a stronger delay in abscission (Fig. 4d, e, h). Interestingly, co-depleting either TSG101 and syntenin, or TSG101 and syndecan-4 also strongly increased the abscission delay, as compared to individual deple- tions (Fig. 4d, f–h). These results indicate that syndecan-4 and syntenin functionally cooperate with TSG101 for abscission. They also suggest that ALIX /syntenin/syndecan-4 and TSG101 act through distinct pathways to promote abscission. Syndecan-4/syntenin/ALIX stabilize ESCRTs at the abscission site. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z ARTICLE Fig. 2 Syndecan-4, syntenin, ALIX, and CHMP4B colocalize and are highly enriched ﬁrst at the midbody then at the abscission site. a The ESCRT-III- ALIX-syntenin-syndecan-4 complex. b Left panels: endogenous localization of ALIX, CHMP4B, syntenin, syndecan-4 (SDC4), and acetylated-tubulin in late bridges displaying abscission site in ﬁxed HeLa cells. The SDC4 antibody recognizes the ectodomain. Middle panels: intensity proﬁles along the bridge of the corresponding images with matched colors from left panels. Right panels: percentage (mean ± SD) of bridges without and with abscission sites (displaying a pinched tubulin staining on the midbody side) positive for either ALIX, syntenin and syndecan-4. n ≥20 cells, N = 3 independent experiments. One-sided Student’s t tests. c Structured illuminated microscopy images of endogenous ALIX/CHMP4B (left) and syntenin/CHMP4B (right), along with acetylated-tubulin staining in late bridges displaying abscission site. Arrows point to ALIX or syntenin localization at the outer rim of the CHMP4B staining. d Snapshots of time-lapse, spinning-disk confocal microscopy movies of cells co-expressing either CHMP4B-GFP/ALIX-mScarlet, GFP-Syntenin/ALIX- mScarlet, or GFP-SDC4/mScarlet-Syntenin. Selected time points show cells (1) before the recruitment of the ﬂuorescently-labeled proteins, (2) after their enrichment at the midbody, (3) after their appearance at the abscission site, and (4) after abscission. Time 0 corresponds to the time frame preceding furrow ingression. See also corresponding Supplementary Movies 2–4. b, d: Scale bars: 10 μm. c Scale bar: 1 μm. Brackets and arrowheads mark the midbody and the abscission site, respectively. led to a modest but reproducible increase of binucleated cells (Supplementary Fig. 5a). Nevertheless, time-lapse microscopy demonstrated that abscission was delayed in ALIX-depleted cells (Fig. 4a; Supplementary Fig. 5b), as previously reported, indi- cating that ALIX plays a speciﬁc role in abscission. Interestingly, abscission was also delayed after either syntenin or syndecan-4 depletions (Fig. 4b, c; Supplementary Fig. 5c, d). Abscission was fully restored by reintroducing either wild-type ALIX, syntenin or syndecan-4, respectively, ruling out off-target effects (Fig. 4a–c). Importantly, the ALIX F676D was unable to rescue the abscission defects due to ALIX depletion (Fig. 4a). Similarly, syntenin ΔALIX or syntenin ΔSDC failed to rescue the abscission defects observed after syntenin depletion (Fig. 4b). We conclude that ALIX, syntenin and syndecan-4 are important for normal abscission, and that the direct interactions between ALIX/synte- nin on the one hand and syntenin/syndecan on the other hand are critical. led to a modest but reproducible increase of binucleated cells (Supplementary Fig. 5a). NATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z 3f). These results indicate that syntenin and its interaction with syndecan-4 are necessary for syndecan-4 localization at the bridge. Altogether, we conclude that ALIX recruits syntenin that, in turn, recruits syndecan-4 at the intercellular bridge through direct interactions. Efﬁcient abscission requires Syndecan-4, syntenin, and ALIX. Functionally, depletion of either ALIX, syntenin or syndecan-4 NATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications 5 5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z ARTICLE siRNA: ALIX Synt siCtrl siALIX siSynt Tubulin SDC4 SDC4-positive bridges (%) siRNA: Synt + + + WT Synt Synt ΔALIX Synt ΔSDC +GFP-Synt WT +GFP-Synt ΔALIX +GFP-Synt ΔSDC siSynt Tubulin GFP-Syntenin- positive bridges (%) Synt ΔALIX Synt ΔSDC Synt ALIX SDC4 +Empty +Empty +WT ALIX +ALIX F676D siALIX siCtrl Tubulin synt P = 5 × 10–4 siRNA: 100 80 60 40 20 0 ALIX + + + + Empty WT ALIX ALIX F676D Syntenin-positive bridges (%) ALIX F676D Synt ALIX SDC4 P = 3 × 10–4 Syntenin-positive bridges (%) siRNA: SDC4 siCtrl siALIX siSDC4 Tubulin synt - ALIX 100 80 60 40 20 0 - Tubulin - Syntenin - SDC4 130 kDa - 100 kDa - 35 kDa - 35 kDa - 55 kDa - 55 kDa - siRNA: Synt P = 5 × 10–4 P = 0.005 P = 0.001 P = 0.001 P = 0.012 P = 0.002 NS P = 0.032 P = 0.003 NS P = 0.001 100 NS 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 Tubulin GFP-SDC4 WT GFP-SDC4 ΔECD GFP-SDC4: ΔECD GFP-SDC4 ΔSynt GFP-SDC4-positive bridges (%) SDC ΔSynt SDC ΔECD Synt ALIX SDC4 c d e f b a ΔSynt WT Ctrl Ctrl ALIX Ctrl SDC4 ALIX Ctrl GFP synt GFP SDC4 ALIX directly recruits syntenin and syntenin directly recruits syndecan-4 to the cytokinetic bridge. a Western blots of protein extracts from ce ed with control, ALIX, syntenin (Synt), or syndecan-4 (SDC4) siRNAs revealed with the indicated antibodies. Loading control: β-tubulin. b Left pane sentative intercellular bridges from control, ALIX or syndecan-4 siRNA-treated cells stained for acetylated-tubulin and endogenous syntenin, as ted. Right panel: percentage (mean ± SD) of bridges positive for syntenin after control, ALIX and syndecan-4 depletion. n = 31–53 cells, N = 3 endent experiments. (c) Left panels: representative intercellular bridges from control- or ALIX-depleted cells expressing either control (Empty), wi ALIX or ALIX F676D mutant (unable to interact with syntenin), and stained for acetylated-tubulin and endogenous syntenin, as indicated. Right pan ntage (mean ± SD) of bridges with syntenin recruitment in the corresponding conditions. n = 25–31 cells, N = 3 independent experiments. d Left s: representative intercellular bridges from syntenin-depleted cells expressing either GFP-syntenin wild-type, GFP-syntenin ΔALIX (unable to intera ALIX) or GFP-syntenin ΔSDC (unable to interact with syndecan-4). Acetylated-tubulin and GFP signals are shown. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z (c) Left panels: representative intercellular bridges from control- or ALIX-depleted cells expressing either control (Empty), wild- type ALIX or ALIX F676D mutant (unable to interact with syntenin), and stained for acetylated-tubulin and endogenous syntenin, as indicated. Right panel: percentage (mean ± SD) of bridges with syntenin recruitment in the corresponding conditions. n = 25–31 cells, N = 3 independent experiments. d Left panels: representative intercellular bridges from syntenin-depleted cells expressing either GFP-syntenin wild-type, GFP-syntenin ΔALIX (unable to interact with ALIX) or GFP-syntenin ΔSDC (unable to interact with syndecan-4). Acetylated-tubulin and GFP signals are shown. Right panel: percentage (mean ± SD) of bridges with GFP-tagged syntenin recruitment in the corresponding conditions. n = 14–33 cells, N = 4 independent experiments. e Left panels: representative intercellular bridges from control, ALIX or syntenin siRNA-treated cells and stained for acetylated-tubulin and endogenous syndecan-4, as indicated. Right panel: percentage (mean ± SD) of bridges positive for syndecan-4 after control, ALIX and syntenin depletion. n = 30–40 cells, N = 3 independent experiments. f Left panels: representative intercellular bridges from cells expressing either GFP-syndecan-4 wild-type, GFP-syndecan-4 ΔECD (deleted from its entire extracellular domain) or GFP-syndecan-4 ΔSynt (unable to interact with syntenin). Acetylated-tubulin and GFP signals are shown. Right panel: percentage (mean ± SD) of bridges with GFP-tagged syndecan-4 recruitment in the corresponding conditions. n = 23–41 cells, N = 3 independent experiments. b–f Scale bars: 2 μm. Brackets mark the midbody. Panels b–f: one-sided Student’s t tests. NS non signiﬁcant. Fig. 3 ALIX directly recruits syntenin and syntenin directly recruits syndecan-4 to the cytokinetic bridge. a Western blots of protein extracts from cells treated with control, ALIX, syntenin (Synt), or syndecan-4 (SDC4) siRNAs revealed with the indicated antibodies. Loading control: β-tubulin. b Left panels: representative intercellular bridges from control, ALIX or syndecan-4 siRNA-treated cells stained for acetylated-tubulin and endogenous syntenin, as indicated. Right panel: percentage (mean ± SD) of bridges positive for syntenin after control, ALIX and syndecan-4 depletion. n = 31–53 cells, N = 3 independent experiments. (c) Left panels: representative intercellular bridges from control- or ALIX-depleted cells expressing either control (Empty), wild- type ALIX or ALIX F676D mutant (unable to interact with syntenin), and stained for acetylated-tubulin and endogenous syntenin, as indicated. Right panel: percentage (mean ± SD) of bridges with syntenin recruitment in the corresponding conditions. n = 25–31 cells, N = 3 independent experiments. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z Right panel: percentage (mean f bridges with GFP-tagged syntenin recruitment in the corresponding conditions. n = 14–33 cells, N = 4 independent experiments. e Left panels URE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z ARTICL P = 3 × 10–4 Syntenin-positive bridges (%) siRNA: SDC4 siCtrl siALIX siSDC4 Tubulin synt 100 80 60 40 20 0 P = 0.012 b ALIX Ctrl - ALIX - Tubulin - Syntenin - SDC4 130 kDa - 100 kDa - 35 kDa - 35 kDa - 55 kDa - 55 kDa - siRNA: Synt a SDC4 ALIX Ctrl b a +Empty +Empty +WT ALIX +ALIX F676D siALIX siCtrl Tubulin synt P = 5 × 10–4 siRNA: 100 80 60 40 20 0 ALIX + + + + Empty WT ALIX ALIX F676D Syntenin-positive bridges (%) ALIX F676D Synt ALIX SDC4 P = 0.001 P = 0.002 NS P = 0.032 P = 0.003 c Ctrl siRNA: Synt + + + WT Synt Synt ΔALIX Synt ΔSDC +GFP-Synt WT +GFP-Synt ΔALIX +GFP-Synt ΔSDC siSynt Tubulin GFP-Syntenin- positive bridges (%) Synt ΔALIX Synt ΔSDC Synt ALIX SDC4 P = 0.001 NS 100 80 60 40 20 0 d GFP synt +Empty +Empty +WT ALIX +ALIX F676D siALIX siCtrl Tubulin synt c d c P = 5 × 10–4 100 NS 80 60 40 20 0 Tubulin GFP-SDC4 WT GFP-SDC4 ΔECD GFP-SDC4: ΔECD GFP-SDC4 ΔSynt GFP-SDC4-positive bridges (%) SDC ΔSynt SDC ΔECD Synt ALIX SDC4 f ΔSynt WT GFP SDC4 siRNA: ALIX Synt siCtrl siALIX siSynt Tubulin SDC4 SDC4-positive bridges (%) P = 0.005 P = 0.001 100 80 60 40 20 0 e Ctrl f siCtrl siALIX siSynt Tubulin SDC4 e e e nin and syntenin directly recruits syndecan-4 to the cytokinetic bridge. a Western blots of protein extracts from cells Fig. 3 ALIX directly recruits syntenin and syntenin directly recruits syndecan-4 to the cytokinetic bridge. a Western blots of protein extracts from cells treated with control, ALIX, syntenin (Synt), or syndecan-4 (SDC4) siRNAs revealed with the indicated antibodies. Loading control: β-tubulin. b Left panels: representative intercellular bridges from control, ALIX or syndecan-4 siRNA-treated cells stained for acetylated-tubulin and endogenous syntenin, as indicated. Right panel: percentage (mean ± SD) of bridges positive for syntenin after control, ALIX and syndecan-4 depletion. n = 31–53 cells, N = 3 independent experiments. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z Importantly, neither ALIX, syntenin nor syndecan-4 were individually required for correct ESCRT-III recruitment at the midbody itself (the proportion of bridges stuck at this earlier stage was actually increased) (Fig. 5a). As expected, the localization of ESCRT-III at the abscission site was restored in ALIX-, syntenin-, or syndecan-4-depleted cells upon expression of siRNA-resistant versions of the corresponding wild-type pro- teins (Fig. 5b–d). Importantly, the normal localization of ESCRT-III at the abscission site could not be restored by either ALIX F676D 6 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z syntenin- or syndecan-4-depleted cells (Fig. 5j; Supplementary Fig. 5e), perhaps because ALIX directly recruits ESCRT-III and/or possible alternative pathways (such as TSG101) that can partially compensate when syntenin or syndecan-4 are depleted. Alto- gether, we conclude that syndecan-4-syntenin-ALIX promotes stable recruitment of ESCRT-III at the abscission site and is thus key for efﬁcient cytokinetic abscission. enriched in puriﬁed MBR (Enriched Flemmingsome) (Fig. 1, Supplementary Data 1). To isolate MBRs, we developed an ori- ginal ﬂow cytometry-based protocol that yielded preparations that display three important features. First, the MBR+ fractions were highly pure. Second, the MBRs were obtained from unperturbed cells (no drugs for cell synchronization and no treatment for stabilizing actin or microtubules). Most importantly this puriﬁcation did not involve any detergents. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z This allowed us to identify both transmembrane (29 proteins, Supplementary f e g siRNA: Ctrl TSG101 + + + + + + + + siCtrl siALIX siSynt siSDC4 h d 130 kDa - 100 kDa - 55 kDa - 35 kDa - 70 kDa - 55 kDa - c P < 10–4 P = 7 × 10–4 P < 10–4 P < 10–4 P < 10–4 P < 10–4 P < 10–4 P < 10–4 P < 10–4 NS siRNA: Ctrl SDC4 + + + + Empty WT SDC4 b siRNA: Synt NS NS NS P = 0.007 P = 0.002 P = 0.038 P = 0.030 P = 0.016 P = 0.013 P = 0.020 P = 0.004 P = 0.001 P = 0.014 P = 0.036 P = 0.007 P = 0.004 P = 0.014 P = 0.008 NS NS NS P = 0.011 siCtrl + Empty siSDC4 + Empty siSDC4 + WT SDC4 1000 750 500 Time to abscission (min) 250 400 600 600 ALlX ALlX Ctrl Ctrl siRNA: siRNA: Ctrl TSG101 TSG101 SDC4 SDC4 SDC4 Syntenin Synt ALlX Synt Calreticulin 400 200 0 800 500 400 300 200 100 0 300 200 100 0 0 1000 750 500 Time to abscission (min) 250 0 1000 750 500 Time to abscission (min) 250 0 100 80 60 40 20 0 Cumulative fraction (%) Mean abscission time (min) Mean abscission time (min) Mean abscission time (min) 500 400 300 200 100 0 Mean abscission time (min) 100 80 60 40 20 0 Cumulative fraction (%) 100 80 60 40 20 0 Cumulative fraction (%) siCtrl + WT SDC4 siCtrl + GFP siSynt + GFP siCtrl + GFP siALIX + GFP siALIX + WT ALlX siALIX + ALlX F676D siSynt + WT Synt siSynt + Synt ΔALIX siSynt + Synt ΔSDC NS + + + + + GFP WT Synt Synt ΔALIX Synt ΔSDC a siRNA: ALIX + + + + GFP WT ALIX ALIX F676D NS NS Ctrl Ctrl 100 80 60 40 20 0 Cumulative fraction (%) 100 80 60 40 20 0 Cumulative fraction (%) 100 80 60 40 20 0 Cumulative fraction (%) 1000 750 siCtrl siTSG101 siALIX siTSG101 + siALIX P < 10–4 P < 10–4 P < 10–4 P < 10–4 P < 10–4 P < 10–4 siCtrl siTSG101 siCtrl siTSG101 siSDC4 siTSG101 + siSDC4 siSynt siTSG101 + siSynt 500 Time to abscission (min) 250 0 1000 750 500 Time to abscission (min) 250 0 1000 750 500 Time to abscission (min) 250 0 c P < 10–4 P < 10–4 P < 10–4 NS b NS NS siCtrl + Empty siSDC4 + Empty siSDC4 + WT SDC4 1000 750 500 Time to abscission (min) 250 0 1000 750 500 Time to abscission (min) 250 0 1000 750 500 Time to abscission (min) 250 0 100 80 60 40 20 0 Cumulative fraction (%) 100 80 60 40 20 0 Cumulative fraction (%) 100 80 60 40 20 0 Cumulative fraction (%) siCtrl + WT SDC4 siCtrl + GFP siSynt + GFP siCtrl + GFP siALIX + GFP siALIX + WT ALlX siALIX + ALlX F676D siSynt + WT Synt siSynt + Synt ΔALIX siSynt + Synt ΔSDC NS a NS NS c P < 10–4 P < 10–4 NS b NS NS siCtrl + Empty siSDC4 + Empty siSDC4 + WT SDC4 1000 750 500 Time to abscission (min) 250 0 1000 750 500 Time to abscission (min) 250 0 100 80 60 40 20 0 Cumulative fraction (%) 100 80 60 40 20 0 Cumulative fraction (%) siCtrl + WT SDC4 siCtrl + GFP siSynt + GFP 6D siSynt + WT Synt siSynt + Synt ΔALIX siSynt + Synt ΔSDC NS NS NS P < 10–4 1000 750 500 Time to abscission (min) 250 0 100 80 60 40 20 0 Cumulative fraction (%) siCtrl + GFP siALIX + GFP siALIX + WT ALlX siALIX + ALlX F676D a NS NS c P = 7 × 10–4 P < 10–4 P < 10–4 P < 10–4 siRNA: Ctrl + + Empty WT SDC4 b siRNA: Synt NS NS NS P = 0.002 P = 0.001 P = 0.014 P = 0.036 P = 0.007 P = 0.004 P = 0.014 P = 0.008 NS NS NS P = 0.011 siCtrl siSDC siSDC 500 Time to abscissi 250 400 600 500 400 300 200 100 0 300 200 100 0 0 1000 750 500 Time to abscission (min) 250 0 1000 750 500 Time to abscission (min) 250 0 100 80 60 40 20 0 Cumulative fraction (%) Mean abscission time (min) Mean abscission time (min) 500 400 300 200 100 0 Mean abscission time (min) 100 80 60 40 20 0 Cumulative fraction (%) 100 80 60 40 20 0 Cumulative fraction (%) siCtrl siCtrl + GFP siSynt + GFP siCtrl + GFP siALIX + GFP siALIX + WT ALlX siALIX + ALlX F676D siSynt + WT Synt siSynt + Synt ΔALIX siSynt + Synt ΔSDC NS + + + + + GFP WT Synt Synt ΔALIX Synt ΔSDC a siRNA: ALIX + + + + GFP WT ALIX ALIX F676D NS NS Ctrl Ctrl a Time to abscission (min) P = 0.014 P = 0.036 P = 0.007 P = 0.004 P = 0.014 NS 500 400 300 200 100 0 Mean abscission time (min) siRNA: ALIX + + + + GFP WT ALIX ALIX F676D Ctrl P = 7 × 10–4 P < 10–4 siRNA: Synt P = 0.001 P = 0.008 NS NS P = 0.011 600 500 400 300 200 100 0 Mean abscission time (min) + + + + + GFP WT Synt Synt ΔALIX Synt ΔSDC Ctrl siRNA: Ctrl SDC4 + + + + Empty WT SDC4 NS P = 0.007 P = 0.002 400 300 200 100 0 Mean abscission time (min) d 130 kDa - 100 kDa - 55 kDa - 35 kDa - 70 kDa - 55 kDa - ALlX ALlX Ctrl Ctrl siRNA: siRNA: Ctrl TSG101 TSG101 SDC4 SDC4 SDC4 Syntenin Synt ALlX Synt Calreticulin siRNA: Ctrl TSG101 + + + + + + + + siCtrl siALIX siSynt siSDC4 h P < 10–4 P = 0.038 P = 0.030 P = 0.016 P = 0.013 P = 0.020 P = 0.004 600 400 200 0 800 Mean abscission time (min) h h d d f e g + + + + + + siALIX siSynt siSDC4 P < 10–4 P < 10–4 P < 10–4 100 80 60 40 20 0 Cumulative fraction (%) 100 80 60 40 20 0 Cumulative fraction (%) 100 80 60 40 20 0 Cumulative fraction (%) 1000 750 siCtrl siTSG101 siALIX siTSG101 + siALIX P < 10–4 P < 10–4 P < 10–4 P < 10–4 P < 10–4 siCtrl siTSG101 siCtrl siTSG101 siSDC4 siTSG101 + siSDC4 siSynt siTSG101 + siSynt 500 Time to abscission (min) 250 0 1000 750 500 Time to abscission (min) 250 0 1000 750 500 Time to abscission (min) 250 0 e P < 10–4 P < 10–4 P < 10–4 100 80 60 40 20 0 Cumulative fraction (%) 1000 750 siCtrl siTSG101 siALIX siTSG101 + siALIX 500 Time to abscission (min) 250 0 f g 100 80 60 40 20 0 Cumulative fraction (%) C l i f i (%) P < 10–4 P < 10–4 P < 10–4 siCtrl siTSG101 siSynt siTSG101 + siSynt 1000 750 500 Time to abscission (min) 250 0 f Cumulative fraction (%) g 100 80 60 40 20 0 Cumulative fraction (%) P < 10–4 P < 10–4 P < 10–4 siCtrl siTSG101 siSDC4 siTSG101 + siSDC4 1000 750 500 Time to abscission (min) 250 0 g e enriched in puriﬁed MBR (Enriched Flemmingsome) (Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z 1, Supplementary Data 1). To isolate MBRs, we developed an ori- ginal ﬂow cytometry-based protocol that yielded preparations that display three important features. First, the MBR+ fractions were highly pure. Second, the MBRs were obtained from unperturbed cells (no drugs for cell synchronization and no treatment for stabilizing actin or microtubules). Most importantly this puriﬁcation did not involve any detergents. This allowed us to identify both transmembrane (29 proteins, Supplementary Data 1, TAB2, in blue) and membrane associated proteins in this organelle. This includes the 4 Rab proteins already involved in cytokinesis (Rab8, Rab11, Rab14, and Rab35)3, as well as syntenin- or syndecan-4-depleted cells (Fig. 5j; Supplementary Fig. 5e), perhaps because ALIX directly recruits ESCRT-III and/or possible alternative pathways (such as TSG101) that can partially compensate when syntenin or syndecan-4 are depleted. Alto- gether, we conclude that syndecan-4-syntenin-ALIX promotes stable recruitment of ESCRT-III at the abscission site and is thus key for efﬁcient cytokinetic abscission. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z d Left panels: representative intercellular bridges from syntenin-depleted cells expressing either GFP-syntenin wild-type, GFP-syntenin ΔALIX (unable to interact with ALIX) or GFP-syntenin ΔSDC (unable to interact with syndecan-4). Acetylated-tubulin and GFP signals are shown. Right panel: percentage (mean ± SD) of bridges with GFP-tagged syntenin recruitment in the corresponding conditions. n = 14–33 cells, N = 4 independent experiments. e Left panels: representative intercellular bridges from control, ALIX or syntenin siRNA-treated cells and stained for acetylated-tubulin and endogenous syndecan-4, as indicated. Right panel: percentage (mean ± SD) of bridges positive for syndecan-4 after control, ALIX and syntenin depletion. n = 30–40 cells, N = 3 independent experiments. f Left panels: representative intercellular bridges from cells expressing either GFP-syndecan-4 wild-type, GFP-syndecan-4 ΔECD (deleted from its entire extracellular domain) or GFP-syndecan-4 ΔSynt (unable to interact with syntenin). Acetylated-tubulin and GFP signals are shown. Right panel: percentage (mean ± SD) of bridges with GFP-tagged syndecan-4 recruitment in the corresponding conditions. n = 23–41 cells, N = 3 independent experiments. b–f Scale bars: 2 μm. Brackets mark the midbody. Panels b–f: one-sided Student’s t tests. NS non signiﬁcant. 7 NATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z human HeLa), (2) differences in actual organelles (intercellular bridges before abscission vs. post-abscission, free midbodies as generated at the time of abscission), (3) the membrane integ- rity of the organelles (use of detergents vs. detergent-free, thus preserving cytosolic components), (4) reduced contamina- tions and ﬁnally the relative quantiﬁcation of protein abundance (the Enriched Flemmingsome is based on signiﬁcant enrichment compared to control fractions, including total cell lysates). While our paper was in revision, a noncomparative proteomic analysis of a MBR preparation, based on isolation from the cul- ture media of HeLa cells by differential centrifugations associated with sucrose-gradient (no replica mentioned) has been pub- lished54. Interestingly, it showed 64% overlap with our Total Flemmingsome (558 out of 871 hits54 are found in the Total Flemmingsome). fold and a 5% FDR. This allows to include proteins that are signiﬁcantly enriched in MBRs and that are also present in the total cell lysate, as many proteins involved in cytokinesis (e.g. actin-related and trafﬁc-related) have also functions in interphase. The Flemmingsome thus represents a useful resource for the cytokinesis community and we created a website to share this data (https://ﬂemmingsome.pasteur.cloud/). Each hit in the Enriched Flemmingsome, in particular, was browsed in the lit- erature for an eventual function/localization linked to cytokinesis and this reference database will be regularly updated. Following up on the Enriched Flemmingsome, we found that ALIX, syntenin and syndecan-4 are indeed highly enriched at the midbody, then at the abscission site (Fig. 2). Functional analysis demonstrated that the three proteins are required for proper abscission (Fig. 4), and that they function together for ESCRT-III localization, speciﬁcally at the abscission site (Fig. 5). Of note, depletion of ALIX, syntenin or syndecan-4 revealed a speciﬁc role for these proteins in abscission rather than on intercellular bridge stability or integrity. Indeed, we observed only a modest, although reproducible, increase of binucleated cells (Supplementary Fig. 5a). For reasons that need further investigation but that we suspect to depend on differences in cell lines (e.g. HeLa ATCC vs. HeLa Kyoto), other studies reported a stronger increase in binucleated cells after ALIX depletion21,22. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z Nonetheless, our results indicate that ALIX, syntenin and syndecan-4 are impor- tant for promoting the coupling/interactions of the ESCRT-III machinery to the plasma membrane at the abscission site during the abscission step (see below) rather than for ﬁrmly attaching the plasma membrane to the midbody, the latter clearly depends on the physical interaction between MgcRacGAP and the membrane58. Our quantitative Flemmingsome was able to identify many bona ﬁde cytosolic (ESCRT-related, actin-related, microtubule- related) and membrane/vesicle associated proteins (e.g., Rab11, Rab35, Rab8, and Rab14) involved in cytokinesis3 but undetected in previous proteomic analysis (Fig. 1, Supplementary Data 1). Intriguingly, GO-term analysis indicated that ribosomal proteins were found enriched in the Flemmingsome, suggesting a func- tional interplay between cytokinesis and translation, as antici- pated by studies in Drosophila55,56. In addition, proteasome inhibition by MG132 has been previously found to delay abscission57 and here we revealed the presence of major protea- some components in the Enriched Flemmingsome (Fig. 1, GO- enrichment and Supplementary Data 1). Interestingly, we previously reported that syntenin can directly bridge ALIX to syndecan-1/4 in vitro, and that ALIX-syntenin- syndecan are key for budding of intraluminal vesicles in MVBs and exosome production41. This likely depends on the ability of ALIX to recruit the ESCRT-III machinery at the neck of intra- luminal vesicles in MVBs, but this could not be directly addressed given the small size of these necks. Here, we showed that the same module (ALIX-syntenin-syndecan-4) is used during cytokinesis at a much larger, micrometric scale, and found that it is actually critical for the stable association of ESCRT-III at the abscission site (Fig. 5). Of note, we did not detect any ALIX or syntenin at the plasma membrane in interphase cells, although syndecan-4 is clearly localized there (Supplementary Movie 4). This suggests that post-translational modiﬁcations of either ALIX, syntenin and/or syndecan-4, perhaps via phosphorylations43,59 regulate the formation of the tripartite complex at the midbody and at the abscission site during cytokinesis. Importantly, 31% of the proteins present in the Enriched Flemmingsome (150 proteins) were already demonstrated to be localized to the furrow, bridge or midbody during cytokinesis and/or to be involved in cytokinesis (Supplementary Data 1, TAB2). This asserts the strength of our proteomic study, as well as its potential for identifying additional candidates (339) important for cytokinesis. Discussion Most proteins involved in cytokinetic abscission strongly accu- mulate at the midbody3,9. Here, we identiﬁed 489 proteins ATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z ARTICLE Fig. 4 ALIX, syntenin, and syndecan-4 are required for successful abscission. a Transduced-HeLa cell lines expressing either GFP, wild-type ALIX-GFP or ALIX F676D-GFP were treated with either control or ALIX siRNAs. Abscission time (from furrow onset to abscission) was determined by phase-contrast time-lapse microscopy. Cumulative plot of the abscission times (upper panels) and mean abscission times (means ± SD) (lower panels) are shown. n = 111–156 cells, N = 3 independent experiments. b Abscission time was determined as above in transduced cell lines expressing either GFP, wild-type mCherry-syntenin, mCherry-syntenin ΔALIX, or mCherry-syntenin ΔSDC and treated with either control or syntenin siRNAs, as indicated. n = 105–147 cells, N = 3 independent experiments. c Abscission time was determined as above in HeLa cells treated with either control or syndecan-4 siRNAs, and transfected with either empty plasmid or plasmid expressing siRNA resistant transcript encoding syndecan-4. n = 130–179 cells, N = 3 independent experiments. Time axis were stopped at 1200 min. d Western blots of protein extracts from cells treated with control, ALIX, syntenin (Synt), syndecan-4 (SDC4), TSG101 siRNAs, or a combination of these siRNAs and revealed with the indicated antibodies. Loading control: calreticulin. e–g Abscission time was determined in cells treated with either control, ALIX, syntenin, syndecan-4, TSG101 siRNAs, or a combination of these siRNAs. Mean abscission times (means ± SD) are depicted in (h). n = 120–152 cells, N = 3 independent experiments. All measurements were carried out in parallel but have been displayed in three graphs for clarity. Upper panels a–c, e–g (time distribution): nonparametric and distribution-free Kolmogorov–Smirnov KS tests. NS nonsigniﬁcant. Lower panels a–c, h (mean abscission times): one-sided Student’s t tests. transmembrane proteins such as chloride channels (CLIC1 and CLIC4, previously localized at the midbody52), adhesion/tether- ing/signaling molecules (ITGA3, PlexinB2, ICAM1, BST2, and CD44) and tetraspanins (CD9, CD9-P1, and TM4SF1) whose potential functions during cytokinesis will be studied in the future. These three points are key improvements, when com- paring with previous proteomes of intercellular bridges, which already proved to be seminal in identifying essential proteins in cytokinesis44,53. Of note, 68 and 29% of the ﬁnal list of Skop et al. (160 proteins from CHO cells) were respectively present in our Total Flemmingsome (1732 proteins) and Enriched Flemmingsome (489 proteins) (Supplementary Data 1, TAB6). The difference in extent of protein recovery between both studies could be explained by (1) differences in cell origins (hamster CHO vs. NATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z To deﬁne the Enriched Flemmingsome, we decided to arbitrarily put the threshold of enrichment to 1.3- Our results therefore reveal that ESCRT-III recruitment during cytokinesis relies on two successive, separable phases (Fig. 6). 9 TURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z CEP55 directly interacts with and recruits both TSG101 and at the midbody37 (Fig. 6a). At the midbody, ESCRT-III onents are recruited in parallel, directly by ALIX and ectly by ESCRT-I/II29. Accordingly, both TSG101 and must be simultaneously depleted to prevent ESCRT-III recruitment at the intercellular bridge29. However, CEP55 ca account for ESCRT-III localization at the abscission site since absent from this location (Supplementary Fig. 5f). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z We now fo that ALIX plays a key additional role in cytokinesis: it rec syntenin, which in turn interacts with the transmembrane pro - GAPDH 55 kDa - 35 kDa - 100 kDa - 130 kDa - - ALIX ALIX siRNA ALIX + + + + Empty WT ALIX ALIX F676D - Tubulin Unspecific SDC4 siRNA - SDC4 FL - SDC4 ΔECD 35 kDa - 15 kDa - 55 kDa - 60 Bridges with CHMP4B at the abscission site (%) 40 20 0 60 Bridges with CHMP4B at the abscission site (%) 40 20 0 60 Bridges with CHMP4B at the abscission site (%) 40 20 0 25 kDa - siRNA: SDC4 + + + + + Empty WT SDC4 SDC4 ΔECD SDC4 ΔSynt ALIX: – WT F676D SDC4: – WT ΔECD ΔYA Synt: – WT ΔALIX ΔSDC 35 kDa - 55 kDa - - Tubulin Synt siRNA - Syntenin Synt + + + + + Empty WT Synt Synt ΔALIX Synt ΔSDC 10 0 70 80 90 100 110 120 130 140 150 180 170 160 siSDC4 Abscission CHMP4B-GFP 10 0 70 80 90 100 110 120 130 140 150 180 170 160 siSynt Abscission CHMP4B-GFP 0 10 60 70 80 90 100 110 160 170 180 210 200 190 siALIX Abscission CHMP4B-GFP 90 80 70 60 50 40 30 20 10 0 siCtrl Abscission CHMP4B-GFP Tubulin CHMP4B no CHMP4B CHMP4B at midbody + abscission site siCtrl siALlX siSynt siSDC4 siCtrl siALlX siSynt siSDC4 siTSG101 siTSG101 + siALlX siTSG101 + siSynt siTSG101 + siSDC4 CHMP4B at midbody 100 Category of bridges (%) 80 60 40 NS NS P = 0.044 P = 0.037 P = 0.026 P = 0.021 P = 0.028 P = 0.041 P = 0.003 P = 0.009 P = 0.009 P = 0.003 P = 0.005 P = 0.003 P = 0.009 P = 0.007 P = 0.006 NS NS NS NS NS NS P = 0.015 P = 0.014 P = 0.039 P = 0.019 P = 0.007 P = 0.004 P = 0.010 NS NS NS P = 0.001 P = 0.001 P = 0.006 P = 0.026 P = 0.012 P = 0.008 P = 0.011 NS NS NS NS NS P = 0.028 20 0 100 Category of bridges (%) 80 60 40 20 0 P = 5 × 10–4 P < 10–4 P = 8 × 10–4 P = 6 × 10–4 no CHMP4B CHMP4B at midbody + abscission site CHMP4B at midbody V a e g h j i b c d f Ctrl siRNA: Ctrl siRNA: Ctrl 100 Cytokinesis with abnormal CHMP4B-GFP behavior (%) 80 60 40 20 0 siCtrl siALlX siSynt siSDC4 P < 10–4 P < 10–4 P = 8 × 10–4 \| p // g/ / siCtrl siALlX siSynt siSDC4 siTSG101 siTSG101 + siALlX siTSG101 + siSynt siTSG101 + siSDC4 P = 0.021 P = 0.028 P = 0.041 P = 0.003 P = 0.009 P = 0.009 P = 0.003 P = 0.005 P = 0.003 P = 0.009 P = 0.007 P = 0.006 NS NS NS NS NS NS 100 Category of bridges (%) 80 60 40 20 0 P = 5 × 10–4 P < 10–4 P = 8 × 10–4 no CHMP4B CHMP4B at midbody + abscission site CHMP4B at midbody e e a 60 Bridges with CHMP4B at the abscission site (%) 40 20 0 Synt: – WT ΔALIX ΔSDC 35 kDa - 55 kDa - - Tubulin Synt siRNA - Syntenin Synt + + + + + Empty WT Synt Synt ΔALIX Synt ΔSDC P = 0.001 P = 0.001 P = 0.006 P = 0.026 NS NS NS c siRNA: Ctrl - Tubulin Unspecific SDC4 siRNA - SDC4 FL - SDC4 ΔECD 35 kDa - 15 kDa - 55 kDa - 60 Bridges with CHMP4B at the abscission site (%) 40 20 0 25 kDa - siRNA: SDC4 + + + + Empty WT SDC4 SDC4 ΔECD SDC4 S SDC4: – WT ΔECD ΔYA P = 0.019 P = 0.007 P = 0.004 P = 0.010 NS NS NS d Ctrl d b - GAPDH 55 kDa - 35 kDa - 100 kDa - 130 kDa - - ALIX ALIX siRNA ALIX: – WT F676D b c ALIX + + + + Empty WT ALIX ALIX F676D 60 Bridges with CHMP4B at the abscission site (%) 40 20 0 P = 0.012 P = 0.008 P = 0.011 NS NS P = 6 × 10–4 siRNA: Ctrl 90 80 70 60 50 40 30 20 10 0 siCtrl Abscission CHMP4B-GFP f f 10 0 70 80 90 100 110 120 130 140 150 180 170 160 siSDC4 Abscission CHMP4B-GFP 10 0 70 80 90 100 110 120 130 140 150 180 170 160 siSynt Abscission CHMP4B-GFP 0 10 60 70 80 90 100 110 160 170 180 210 200 190 siALIX Abscission CHMP4B-GFP siCtrl Abscission CHMP4B-GFP g h i Cytokinesis with abn CHMP4B-GFP behav 80 60 40 20 0 siCtrl siALlX siSynt siSDC4 P < 10–4 P = 8 × 10–4 h i First, CEP55 directly interacts with and recruits both TSG101 and ALIX at the midbody37 (Fig. ARTICLE 6 Working model: ALIX-syntenin-syndecan-4 couples the ESCRT-III machinery to the plasma membrane at the abscission site for efﬁcient scission. a ESCRT-III localization at the midbody depends on its recruitment by ALIX (blue) and ESCRT-I/-II (violet), which are targeted to the midbody by MKLP1-associated CEP55 (gray). This ﬁrst step does not require syntenin or syndecan-4. b ESCRT-III localization at the abscission site, located on one side of the midbody, depends on the tripartite ALIX- syntenin-syndecan-4 module. ALIX-syntenin (red), by directly coupling ESCRT-III (yellow) on the one hand and the transmembrane protein syndecan-4 (green) on the other hand is proposed to help maintain ESCRT- III polymers at the abscission site until the ﬁnal cut. expression of syndecan-4 cytoplasmic tail alone was unable to rescue ESCRT-III localization at the abscission site (Supplemen- tary Fig. 5h), indicating that both the transmembrane and the cytoplasmic tail of syndecan-4 are critical. In summary, the chain of interactions between ALIX/syntenin/syndecan-4 appears cri- tical for proper ESCRT-III localization at this site and thus abscission (Figs. 4 and 5). Furthermore, this model explains previous observations showing that depleting ALIX alone, which has little effect on ESCRT-III recruitment at the midbody itself, indeed has an impact on abscission29. Interestingly, co-depletion of TSG101 and either syntenin or syndecan-4 did not abolish ESCRT-III recruitment at the midbody (ALIX is still present), but strongly impacted on ESCRT-III localization at the abscission site (Fig. 5e) and consequently on abscission (Fig. 4d–h). This sug- gests that ALIX and TSG101 actually cooperate for localizing ESCRT-III at the abscission site (where they both localize), and not only for recruiting ESCRT-III at the midbody. Future studies will be required to understand how ALIX (via syntenin/syndecan- 4) and TSG101 (via an unknown mechanism) promote abscission through distinct pathways. Syndecan-4 Syntenin ALIX ESCRT-I/II ESCRT-III Microtubules Midbody Midbody Abscission site CEP55 CEP55 CEP55 ESCRT-l/ll ALlX ESCRT-lll ESCRT-lll at midbody a Midbody CEP55 a CEP55 ESCRT-l/ll ESCRT-lll ALlX ESCRT-lll at abscission site b ESCRT-III Syntenin Syndecan-4 Abscission b Syndecan-4 Syntenin ALIX ESCRT-I/II ESCRT-III Microtubules Midbody Abscission site CEP55 Syndecan-4 Syntenin Abscission Abscission g p y In other related ESCRT-dependent membrane scission events, such as exosome formation in MVBs and HIV budding, ALIX also plays a key role4,7. As mentioned above, we previously found that the ALIX-syntenin-syndecan module is essential for proper exosome scission41. ARTICLE ARTICLE Fig. 5 Persistent recruitment of ESCRT-III to the abscission site depends on the syndecan4-syntenin-ALIX module. a Cells treated with the indicated siRNAs were stained for endogenous CHMP4B and acetylated-tubulin. CHMP4B localization in late cytokinetic bridges was classiﬁed into three categories: (1) no staining, (2) CHMP4B localized only at the midbody, or (3) CHMP4B localized both at the midbody and at the abscission site (see representative images). The proportion of each category was quantiﬁed in control and depleted cells. n = 49–83 cells, N = 3 independent experiments. b–d Cells were depleted for either ALIX (b), syntenin (c), or syndecan-4 (d) and transfected with control plasmid (−) or with plasmids encoding either wild type or mutant versions of ALIX, syntenin, or syndecan-4. Upper panels: western blots were revealed with the indicated antibodies. Loading controls: GAPDH or β-tubulin. Lower panels: percentage (mean ± SD) of bridges with CHMP4B at the abscission site in each condition. b n = 28–31 cells; c n = 32–103 cells, d n = 33–56 cells. N = 3 independent experiments. e Cells were treated with control, ALIX, syntenin, syndecan-4, TSG101 siRNAs, alone or in combination. CHMP4B localization was quantiﬁed as in (a). n = 26–66 cells, N = 3 independent experiments. f–i HeLa cells stably expressing CHMP4B–GFP were treated with either control (f), ALIX (g), syntenin (h), or syndecan-4 (i) siRNAs and recorded by spinning-disk confocal time-lapse microscopy every 10 min. Zooms of the intercellular bridges are displayed. Time 0 corresponds to the frame preceding the arrival of CHMP4B at the midbody. Brackets mark the midbody. Arrows point toward pools of CHMP4B on the midbody side. Red arrows correspond to the CHMP4B leading to abscission (last time frame). Yellow and cyan arrows point to transient and unstable CHMP4B pools observed in depleted cells. See corresponding Supplementary Movies 5–8. j Quantiﬁcation of cytokinesis with abnormal CHMP4B-GFP behavior (disappearance of signal on the side of the midbody and fragmented cones) for each condition reported in (f–i). n = 24–38 cells from 5 independent experiments. One-sided Fisher’s exact tests. a, f–i Scale bars: 2 μm. Brackets mark midbodies in (a, f–j). Panels a–e: means ± SD and one-sided Student’s t tests. NS nonsigniﬁcant. Syndecan-4 Syntenin ALIX ESCRT-I/II ESCRT-III Microtubules Midbody CEP55 CEP55 ESCRT-l/ll ALlX ESCRT-l/ll ESCRT-lll ALlX ESCRT-lll ESCRT-lll at midbody ESCRT-lll at abscission site Midbody Abscission site a b CEP55 CEP55 ESCRT-III Syntenin Syndecan-4 Abscission Fig. ARTICLE TSG101 and ALIX cooperate and are both important in HIV budding, but TSG101 plays a more prominent role, in contrast to abscission (refs. 29 and Fig. 4). Remarkably, HIV appears to have hijacked and simpliﬁed this ALIX-syntenin- syndecan module, since the GAG protein (which is tightly asso- ciated to the plasma membrane through myristoylation) contains the ALIX-interacting LYPxL motif (that is found three times in syntenin), bypassing the need for syndecan-syntenin in the scis- sion step49,60–62. Thus, our study suggests that the coupling of the ESCRT-III machinery to a membrane protein via ALIX-syntenin or equivalent modules represents a critical requirement for efﬁ- cient scission during cytokinesis, exosome formation, and retro- viral budding. Fig. 6 Working model: ALIX-syntenin-syndecan-4 couples the ESCRT-III machinery to the plasma membrane at the abscission site for efﬁcient Fig. 6 Working model: ALIX-syntenin-syndecan-4 couples the ESCRT-III machinery to the plasma membrane at the abscission site for efﬁcient scission. a ESCRT-III localization at the midbody depends on its recruitment by ALIX (blue) and ESCRT-I/-II (violet), which are targeted to the midbody by MKLP1-associated CEP55 (gray). This ﬁrst step does not require syntenin or syndecan-4. b ESCRT-III localization at the abscission site, located on one side of the midbody, depends on the tripartite ALIX- syntenin-syndecan-4 module. ALIX-syntenin (red), by directly coupling ESCRT-III (yellow) on the one hand and the transmembrane protein syndecan-4 (green) on the other hand is proposed to help maintain ESCRT- III polymers at the abscission site until the ﬁnal cut. scission. a ESCRT-III localization at the midbody depends on its recruitment by ALIX (blue) and ESCRT-I/-II (violet), which are targeted to the midbody by MKLP1-associated CEP55 (gray). This ﬁrst step does not require syntenin or syndecan-4. b ESCRT-III localization at the abscission site, located on one side of the midbody, depends on the tripartite ALIX- syntenin-syndecan-4 module. ALIX-syntenin (red), by directly coupling ESCRT-III (yellow) on the one hand and the transmembrane protein syndecan-4 (green) on the other hand is proposed to help maintain ESCRT- III polymers at the abscission site until the ﬁnal cut. syndecan-4 (Fig. 6b). Importantly, the ALIX-syntenin-syndecan-4 module is required to stably maintain ESCRT-III components at the abscission site. In the absence of either ALIX, syntenin or syndecan-4, ESCRT-III components can polymerize and extend from the midbody toward the abscission site. However, ESCRT- III recruitment at the abscission site takes longer and is unstable, resulting in delayed abscission (Fig. 5). Cell cultures. HeLa cells CCL-2 (ATCC) and HeLa GFP-MKLP245 were grown in Dulbecco’s Modiﬁed Eagle Medium (DMEM) GlutaMax (31966; Gibco, Invitrogen Life Technologies) supplemented with 10% fetal bovine serum and 1× penicillin–streptomycin (Gibco) in 5% CO2 at 37 °C. The HeLa GFP-MKLP2 cells were cultured in G418 (Gibco) after FACS selection. CHMP4B-GFP and GFP- syndecan-4 stable cell lines were generated by electroporating HeLa ATCC cells with the corresponding plasmids, followed by G418 selection (Gibco) and selected by FACS sorting. GFP, ALIX-LAP-GFP WT, ALIX-LAP-GFP F676D, mCherry- syntenin WT, mCherry-syntenin ΔALIX and mCherry-syntenin ΔSDC stable cell ARTICLE We thus propose that ALIX-syntenin, by physically coupling ESCRT-III on the one hand and the transmembrane protein syndecan-4 on the other hand, help to maintain ESCRT-III polymers at the abscission site until the ﬁnal cut (Fig. 6b). Consistently, CHMP4B and syndecan-4 displayed correlated patterns at the abscission site in cells that were ﬁxed shortly before the actual cut (Supple- mentary Fig. 5g). Furthermore, in syndecan-4-depleted cells, the NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z 6a). At the midbody, ESCRT-III components are recruited in parallel, directly by ALIX and indirectly by ESCRT-I/II29. Accordingly, both TSG101 and ALIX must be simultaneously depleted to prevent ESCRT-III recruitment at the intercellular bridge29. However, CEP55 cannot account for ESCRT-III localization at the abscission site since it is absent from this location (Supplementary Fig. 5f). We now found that ALIX plays a key additional role in cytokinesis: it recruits syntenin, which in turn interacts with the transmembrane protein 10 NATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications First, CEP55 directly interacts with and recruits both TSG101 and ALIX at the midbody37 (Fig. 6a). At the midbody, ESCRT-III components are recruited in parallel, directly by ALIX and indirectly by ESCRT-I/II29. Accordingly, both TSG101 and ALIX must be simultaneously depleted to prevent ESCRT-III recruitment at the intercellular bridge29. However, CEP55 cannot account for ESCRT-III localization at the abscission site since it is absent from this location (Supplementary Fig. 5f). We now found that ALIX plays a key additional role in cytokinesis: it recruits syntenin, which in turn interacts with the transmembrane protein 10 ARTICLE pp y g pp p y pp y g Western blot experiments after siRNA treatment were carried out as follows: cells treated with siRNAs were lysed in NP-40 extract buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% NP-40) containing protease inhibitors. Totally, 20 µg of lysate were migrated in 10% or 4–15% gradient SDS-PAGE gels (Bio-Rad Laboratories), transferred onto polyvinylidene ﬂuoride membranes (Millipore) and incubated with corresponding antibodies in 5% milk in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween20, followed by horseradish peroxidase-coupled secondary antibodies (1:20,000, Jackson ImmunoResearch) and revealed by Flow cytometry sorting. MBRs were detached from HeLa GFP-MKLP245 cells with EDTA-treatment as described above. The SN from the ﬁrst 70 g centrifugation was collected. Sorting of MBR+ and MBR−particles was performed on a BD Biosciences FACS ARIA III. Neutral Density ﬁlter 1.0 has been used to detect small particles. Totally, 65,000 particles were gated on a pseudo-color plot looking at GFP vs. SSC-A parameters, both in log scales, as indicated. Cells have been excluded from the sorting gates after analysis of an unstained cell suspension as control (Fig. 1b; Supplementary Fig. 1b). The MBR+ (GFP-positive population) and MBR−(GFP-negative counterpart) populations sorted by ﬂow cytometry were concentrated by 60 min centrifugation at 1200g at 4 °C. The proteins from all the samples were solubilized in 2% SDS and further prepared for in-gel or in-solution digest. y chemiluminescence (GE Healthcare). For western blots against syndecan-4, cell extracts were treated with heparinase (AMS.HEP-ENZ III) and chondroitinase (AMS.E1028-02) for 3 h at 37 °C before migration. Uncropped western blots are displayed in Supplementary Fig. 7. Immunoﬂuorescence and image acquisition. HeLa cells were grown on cover- slips and then ﬁxed either with paraformaldehyde (PFA) 4% for 20 min at room temperature, with methanol for 3 min at −20 °C, or with trichloroacetic acid 10% for 20 min at room temperature. Cells were then permeabilized with 0.1% Triton- X100, blocked with PBS containing 0.2% bovine serum albumin (BSA) and suc- cessively incubated for 1 h at room temperature with primary (Supplementary Table 1) and secondary antibodies diluted in PBS containing 0.2% BSA with DAPI staining (0.5 mg/ml, Serva). Cells were mounted in Mowiol (Calbiochem). ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z lines were generated by lentiviral transduction of HeLa ATCC cl-2 cells and selected by FACS sorting. lines were generated by lentiviral transduction of HeLa ATCC cl-2 cells and selected by FACS sorting. for each channel with ZEN software and then corrected for chromatic aberration using 100-nm TetraSpeck microspheres (ThermoFisher Scientiﬁc) embedded in the same mounting media as the sample. The SIMcheck plugin in imageJ was used to analyze the quality of the acquisition and the processing in order to optimize parameters for resolution, signal-to-noise ratio, and reconstruction pattern63. Transfections and siRNAs. Plasmids were transfected in HeLa cells for 24 or 48 h using X-tremeGENE 9 DNA reagent (Roche). For silencing experiments, HeLa cells were transfected with 25 nM siRNAs for 48 h using HiPerFect (Qiagen) or Lipo- fectamine RNAiMAX (Invitrogen), following the manufacturer’s instructions. siRNAs against Luciferase (used as control, 5′CGUACGCGGAAUACUUCGA3′), ALIX (5′CCUGGAUAAUGAUGAAGGA3′), Syntenin (5′GAAGGACUCUCAAA UUGCA3′), Syndecan-4 (5′GUGAGGAUGUGUCCAACAA3′) and TSG101 (5′CC UCCAGUCUUCUCUCGUC3′) have been synthetized by Sigma. In rescue experiments, cells were ﬁrst transfected for 72 h with siRNAs using HiPerFect, then cotransfected by plasmids encoding untagged proteins and either GFP or H2B-GFP to detect transfected cells, using X-tremeGENE 9 DNA reagent for an additional 24 h. SiRNA-resistant versions of ALIX, syntenin and syndecan-4 have been obtained by mutating 6 bp of the siRNA-targeting sequence using NEBaseChanger (NEB). Time-lapse microscopy. For time-lapse phase-contrast imaging, HeLa cells were plated on glass bottom 12-well plates (MatTek) and put in an open chamber (Life Imaging) equilibrated in 5% CO2 and maintained at 37 °C. Time-lapse sequences were recorded every 10 min for 48 h using an inverted NikonEclipse TiE micro- scope with a ×20 0.45 NA Plan Fluor ELWD controlled by Metamorph software (Universal Imaging). For time-lapse ﬂuorescent microscopy, images were acquired using an inverted Eclipse TiE Nikon microscope equipped with a CSU-X1 spinning disk confocal scanning unit (Yokogawa) and with a EMCCD Camera (Evolve 512 Delta, Photometrics). Images were acquired with a ×60 1.4 NA PL-APO VC and MetaMorph software (MDS). Statistics and reproducibility data. All values are displayed as mean ± SD (standard deviation) for at least three independent experiments (as indicated in the ﬁgure legends). Signiﬁcance was calculated using unpaired, one-sided t tests or one- sided exact Fisher’s tests, as indicated. For comparing distribution of abscission times, a nonparametric Kolmogorov–Smirnov test was used. In all statistical tests p > 0.05 was considered as non signiﬁcant. ARTICLE Images were acquired with an inverted TiE Nikon microscope, using a ×100 1.4 NA PL- APO objective lens or a ×60 1.4 NA PL-APO VC objective lens and MetaMorph software (MDS) 7.8.0.0 driving a CCD camera (Photometrics Coolsnap HQ). Images were then converted into 8-bit images using ImageJ software (NIH). Pur- iﬁed MBR+ from ﬂow cytometry were concentrated at 1200g and a 5 μl-drop was incubated overnight on a glass coverslip. The MBR+ particles were processed for immunoﬂuorescence as described above for cells. Cell Mask (C10045, Thermo- Fisher) staining was performed on the GFP-MKLP2 adherent cells as indicated by the manufacturer, and then the MBR+ particles puriﬁed by ﬂow cytometry as described above. Preparation of samples for mass spectrometry. In gel digestion: In-gel digestion was performed by standard procedures64. Proteins (10 μg) were loaded on a SDS- PAGE gel (4–20% gradient, Expedeon). The electrophoretic migration of the gel was stopped after the stacking and the gel was stained with Coomassie Blue (InstantBlue™, Expedeon) and each lane was cut into three gel bands. Gel slices were washed several times in 50 mM ammonium bicarbonate, acetonitrile (1:1) for 15 min at 37 °C. Disulﬁde bonds were reduced with 10 mM DTT and cysteine alkylated with 55 mM IAA. Trypsin (Promega) digestion was performed overnight at 37 °C in 50 mM ammonium bicarbonate. Peptides were extracted from the gel by two incubations in 10% formic acid, acetonitrile (1:1) for 15 min at 37 °C. Extracts were dried in a Speed-Vac, and resuspended in 2% acetonitrile, 0.1% formic acid prior to LC–mass spectrometry (MS)MS analysis. For each sample (Total, MBRE, MBR+, and MBR−) ﬁve independent preparations were run on SDS-PAGE; an experimental replicate was made as an internal control for the MBR+/MBR− samples (numbered 3 and 4, Supplementary Data 1, TAB3). In-solution digestion (eFASP): Protein samples extracted in SDS were digested using eFASP protocol48. Filter units and collection tubes were incubated overnight in passivation solution: 5% (v/v) TWEEN®-20. All buffer exchanges were carried out by centrifugation at 14,000 g for 10 min. Brieﬂy, 10 µg of proteins from each sample were transferred into 30,000 MWCO centrifugal unit (Microcon® Centrifugal Filters, Merck) completed to 200 μL with exchange buffer (8 M urea, 0.2% DCA, 100 mM ammonium bicarbonate pH 8). Disulﬁde bonds were reduced with 5 mM TCEP (Sigma) for 1 h. Proteins were buffer-exchanged with three Structured illumination microscopy (SIM). ARTICLE p values are indicated in the Figures. Provided representative images have been observed reproducibly as indicated below: Fig. 1a (n > 20 cells), Fig. 1b (n > 30 FACS sortings), Fig. 1c (n = 2 blots), Fig. 1d (n > 20 midbodies), Fig. 2b (n > 30 ICBs), Fig. 2c (n > 3 ICBs), Fig. 2d (n > 10 movies), Fig. 3a (n = 2 blots), Fig. 4d (n = 2 blots), Supplementary Fig. 4a (n > 30 ICBs), Supplementary Fig. 4b (n = 3 movies), Supplementary Fig. 5b (n > 400 movies), Supplementary Fig. 5f (n > 20 cells), and Supplementary Fig. 5g (n > 20 cells). Plasmid constructs. Human ALIX, syntenin, and syndecan-4 cDNAs were sub- cloned into Gateway pENTR plasmids and eGFP, mScarlet or untagged transient expression vectors were generated by LR recombination (Thermo Fisher). All point mutations have been generated using NEBaseChanger (NEB), including ALIX F676D, syntenin ΔALIX (Y4A, P5A, Y46A, P47A, Y50A, and P51A)41, syntenin ΔSDC (K119A, S171H, D172E, K173Q, K203A, K250S, D251H, and S252E)50 syndecan-4 ΔECD (deleted from E19 to E145 included), syndecan-4 Δsynt (deleted for the last two amino acids, Y197 and A198)51. GFP-syndecan-4 was constructed by fusing the eGFP sequence between the syndecan-4 M1–E145 and E142–A198 sequences. Western blots. Western blot experiments comparing samples used in proteomic studies: Protein extracts from Total, MBRE, MBR+ and MBR−fractions were obtained directly after addition of 2% SDS to the samples. Proteins from approximately 2.5 × 106 MBR+ particles from ﬂow cytometry were loaded after lyophilization and resuspended in Laemmli 1× loading buffer on a 4–12% gradient SDS-PAGE gel (Bio-Rad). Serial dilutions (2-fold) were carried on from 5 to 10 μg extracts from Tot and MBRE preparations for comparisons between the samples and SyproRuby protein blot stain (Bio-Rad) used to determine the protein con- centrations in the different samples. Lanes with same levels of protein are shown in Fig. 1c and indicated as lane 1 and serial dilutions mentioned from this lane in Supplementary Fig. 1b. Uncropped blots are displayed in Supplementary Fig. 6. Western blot experiments after siRNA treatment were carried out as follows: cells treated with siRNAs were lysed in NP-40 extract buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% NP-40) containing protease inhibitors. ATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications Methods Cell culture Cell cultures. HeLa cells CCL-2 (ATCC) and HeLa GFP-MKLP245 were grown in Dulbecco’s Modiﬁed Eagle Medium (DMEM) GlutaMax (31966; Gibco, Invitrogen Life Technologies) supplemented with 10% fetal bovine serum and 1× penicillin–streptomycin (Gibco) in 5% CO2 at 37 °C. The HeLa GFP-MKLP2 cells were cultured in G418 (Gibco) after FACS selection. CHMP4B-GFP and GFP- syndecan-4 stable cell lines were generated by electroporating HeLa ATCC cells with the corresponding plasmids, followed by G418 selection (Gibco) and selected by FACS sorting. GFP, ALIX-LAP-GFP WT, ALIX-LAP-GFP F676D, mCherry- syntenin WT, mCherry-syntenin ΔALIX and mCherry-syntenin ΔSDC stable cell 11 URE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications ARTICLE Totally, 20 µg of lysate were migrated in 10% or 4–15% gradient SDS-PAGE gels (Bio-Rad Laboratories), transferred onto polyvinylidene ﬂuoride membranes (Millipore) and incubated with corresponding antibodies in 5% milk in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween20, followed by horseradish peroxidase-coupled secondary antibodies (1:20,000, Jackson ImmunoResearch) and revealed by chemiluminescence (GE Healthcare). For western blots against syndecan-4, cell extracts were treated with heparinase (AMS.HEP-ENZ III) and chondroitinase (AMS.E1028-02) for 3 h at 37 °C before migration. Uncropped western blots are displayed in Supplementary Fig. 7. Western blots. Western blot experiments comparing samples used in proteomic studies: Protein extracts from Total, MBRE, MBR+ and MBR−fractions were obtained directly after addition of 2% SDS to the samples. Proteins from approximately 2.5 × 106 MBR+ particles from ﬂow cytometry were loaded after lyophilization and resuspended in Laemmli 1× loading buffer on a 4–12% gradient SDS-PAGE gel (Bio-Rad). Serial dilutions (2-fold) were carried on from 5 to 10 μg extracts from Tot and MBRE preparations for comparisons between the samples and SyproRuby protein blot stain (Bio-Rad) used to determine the protein con- centrations in the different samples. Lanes with same levels of protein are shown in Fig. 1c and indicated as lane 1 and serial dilutions mentioned from this lane in Supplementary Fig. 1b. Uncropped blots are displayed in Supplementary Fig. 6. bl f d f ll Sample preparation for mass spectrometry. HeLa GFP-MKLP245 were detached from ﬂasks with 0.05% trypsin diluted in 0.02% EDTA (25300; Gibco, Invitrogen Life Technologies) and plated at 8 × 105 cells/well on 10-cm dishes for 3 days. Cells were rinsed 3-times with HBSS and then incubated in 2 mM EDTA/HBSS for 10 min at 37 °C to detach MBRs from the cell surface. The “Total” fraction represented the whole fraction of detached cells including the EDTA-supernatant (SN). The cells were pelleted by centrifugation (5 min at 70g). The “MBRE” was adapted from ref. 14: the SN from the ﬁrst centrifugation was centrifuged again (5 min at 70g) and SN from this step was aliquoted to 300 μl for another cen- trifugation (10 min at 70g). The MBRs were concentrated from the last SN by 60 min centrifugation at 1200 g. ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z The proteins associated to an adjusted p value inferior to a FDR of 5% have been considered as signiﬁcantly differentially abundant proteins. Finally, the proteins of interest are therefore those which emerge from this statistical analysis supple- mented by those which are considered to be absent from one condition and present in another. Results of these differential analyses are summarized in Supplementary Fig. 2 and Supplementary Data 1. The merged volcano plot (Fig. 1e, upper panel) is a summary of the six comparisons MBR+ vs. control (MBR−, MBRE, or Total; using either eFASP or Gel). The x-axis represents the maximum log2 fold-change between MBR+ and the different controls. The “merged p value” (y-axis) has been obtained using the Fisher’s method from the different p values that have been measured in the comparisons. Note that these two quantities have been computed only when data were available. rounds of 200 μL of exchange buffer. Buffer was then exchanged for an alkylation buffer (50 mM iodoacetamide, Urea 8 M, 100 mM ammonium bicarbonate pH 8) in the dark for 1 h. One wash with 200 μL of exchange buffer was done to remove the alkylating agent, followed by three buffer exchanges with 200 μL of digestion buffer (0.2% DCA/50 mM ammonium bicarbonate buffer pH 8). Totally, 100 μL of digestion buffer containing 1:50 ratio of sequencing-grade modiﬁed trypsin (Promega) per amount of protein was added to the retentate. Proteolysis was carried out at 37 °C overnight. Three rounds of 50 μL of recovery buffer (50 mM ammonium bicarbonate pH 8) were used to elute the peptide-rich solution. Then peptides were processed as described in ref. 48 and resuspended in 2% acetonitrile, 0.1% formic acid prior to LC–MS/MS analysis. For eFASP, three independent replicates were made for Tot and MBRE and two independent replicates for MBR+ and matched MBR−ﬂow cytometry-sorted samples (Supplementary Data 1, TAB3). y replicates were made for Tot and MBRE and two independent replicates for MBR+ and matched MBR−ﬂow cytometry-sorted samples (Supplementary Data 1, TAB3). UpsetR graph and STRING functional association network. The Upset graph (Supplementary Fig. 1d) represents the distribution of the signiﬁcant proteins coming from the different statistical analyzes. The Venn diagram represents the numbers of differentially abundant proteins in function of the kind of protein sample preparation (In-Gel or eFASP). Functional association network was determined by STRING73 and displayed using Cytoscape74. MS analysis. ARTICLE Tryptic peptides from in-gel digestion were analyzed on a Q Exactive HF instrument (Thermo Fisher Scientiﬁc, Bremen) coupled with an EASY nLC 1000 chromatography system (Thermo Fisher Scientiﬁc). Sample was loaded on an in-house packed 50 cm nano-HPLC column (75 μm inner diameter) with C18 resin (1.9 μm particles, 100 Å pore size, Reprosil-Pur Basic C18-HD resin, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) after an equilibration step in 100% solvent A (H2O, 0.1% FA). Peptides were ﬁrst eluted using a 2–7% gradient of solvent B (ACN, 0.1% FA) during 5 min, then a 7–23% gradient of solvent B during 80 min, a 23–45% gradient of solvent B during 40 min and ﬁnally a 45–80% gra- dient of solvent B during 5 min all at 250 nL min−1 ﬂow rate. The instrument method for the Q Exactive HF was set up in the data dependent acquisition mode. After a survey scan in the Orbitrap (resolution 60,000), the 10 most intense pre- cursor ions were selected for HCD fragmentation with a normalized collision energy set up to 28. Charge state screening was enabled, and precursors with unknown charge state or a charge state of 1, 7, 8, and >8 were excluded. Dynamic exclusion was enabled for 45 s. GO cluster diagrams. The over-representation analyses of GO-term clusters in Fig. 1g and Supplementary Fig. 1e have been performed using hypergeometric tests. GO-terms have been grouped following 11 different functional categories (details in Supplementary Table 1, TAB7). Hypergeometric tests were performed to test the overrepresentation hypothesis for each cluster using R software. All the proteins identiﬁed by MS have been used as background for the hypergeometric tests. A signiﬁcantly low p value means the proportion of proteins related to a GO-term cluster is signiﬁcantly superior in the considered list [Total Flemmingsome (Fig. 1g) or proteins more abundant in MBR+ than MBRE (Supplementary Fig. 1e)] than in this background. Tryptic peptides from eFASP digestion were analyzed on a Q Exactive plus instrument (Thermo Fisher Scientiﬁc, Bremen) coupled with an EASY nLC 1000 chromatography system (Thermo Fisher Scientiﬁc) and processed as described above. References l 1. Paweletz, N. Walther Flemming: pioneer of mitosis research. Nat. Rev. Mol. Cell Biol. 2, 72–75 (2001). 2. Addi, C., Bai, J. & Echard, A. Actin, microtubule, septin and ESCRT ﬁlament remodeling during late steps of cytokinesis. Curr. Opin. Cell Biol. 50, 27–34 (2018). 3. Fremont, S. & Echard, A. Membrane trafﬁc in the late steps of cytokinesis. Curr. Biol. 28, R458–R470 (2018). 4. Scourﬁeld, E. J. & Martin-Serrano, J. Growing functions of the ESCRT machinery in cell biology and viral replication. Biochem. Soc. Trans. 45, 613–634 (2017). 5. Stoten, C. L. & Carlton, J. G. ESCRT-dependent control of membrane remodelling during cell division. Semin. Cell Dev. Biol. 74, 50–65 (2017). 6. D’Avino, P. P. & Capalbo, L. Regulation of midbody formation and function by mitotic kinases. Semin. Cell Dev. Biol. 53, 57–63 (2016). 5. Stoten, C. L. & Carlton, J. G. ESCRT-dependent control of membrane remodelling during cell division. Semin. Cell Dev. Biol. 74, 50–65 (2017). 6. D’Avino, P. P. & Capalbo, L. Regulation of midbody formation and function by mitotic kinases. Semin. Cell Dev. Biol. 53, 57–63 (2016). Statistical analysis for proteomic studies. For the differential analyses, proteins identiﬁed in the reverse and contaminant databases and proteins “only identiﬁed by site” were ﬁrst discarded from the list of identiﬁed proteins. Then, proteins exhibiting fewer than two quantiﬁed values in at least one condition were discarded from the list. After log2 transformation of the leftover proteins, LFQ values were normalized by median centering within conditions (normalized function of the R package DAPAR67). Remaining proteins without any LFQ value in one of both conditions have been considered as proteins quantitatively present in a condition and absent in another (Supplementary Data 1, TAB4). They have therefore been set aside and considered as differentially abundant proteins. Next, missing values were imputed using the imp.norm function of the R package norm68. Proteins with a fold-change under 1.3 have been considered not signiﬁcantly differentially abun- dant. Statistical testing of the remaining proteins (having a fold-change over 1.3) was conducted using a limma t test69 thanks to the R package limma70. An adaptive Benjamini–Hochberg procedure was applied on the resulting p values thanks to the function adjust.p of R package cp4p71 using the robust method described in ref. 72 to estimate the proportion of true null hypotheses among the set of statistical tests. 7. Hurley, J. H. ESCRTs are everywhere. EMBO J. 34, 2398–2407 (2015). Data availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE75 partner repository with the dataset identiﬁer PXD013219. The Flemmingsome Website: https://ﬂemmingsome.pasteur.cloud/ All material described in this paper will be made available to readers and be sent upon request. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE75 partner repository with the dataset identiﬁer PXD013219. The Flemmingsome Website: https://ﬂemmingsome.pasteur.cloud/ All t i l d ib d i thi ill b d il bl t d d b t All material described in this paper will be made available to readers and be sent upon request. Received: 24 April 2019; Accepted: 21 February 2020; Published online: 22 April 2020 Protein identiﬁcation and quantiﬁcation. All data were searched using Andro- meda65 against a Human Uniprot database (downloaded in 2015 08 18, 20,204 entries), usual known MS contaminants and reversed sequences of all entries. Andromeda searches were performed choosing trypsin as speciﬁc enzyme with a maximum number of two missed cleavages. Possible modiﬁcations included car- bamidomethylation (Cys, ﬁxed), oxidation (Met, variable), and Nter acetylation (variable). The mass tolerance in MS was set to 20 ppm for the ﬁrst search then 4.5 ppm for the main search and 20 ppm for the MS/MS. Maximum peptide charge was set to seven and ﬁve amino acids were required as minimum peptide length. The “match between runs” feature was applied for samples having the same experimental condition with a maximal retention time window of 1 min. One unique peptide to the protein group was required for the protein identiﬁcation. A FDR cutoff of 1% was applied at the peptide and protein levels. Quantiﬁcation was performed using the XIC-based LFQ algorithm with the Fast LFQ mode as described in ref. 66. Unique and razor peptides, included modiﬁed peptides, with at least two ratio count were used for quantiﬁcation. ARTICLE Peptides were ﬁrst eluted using a 2–5 % gradient of solvent B (ACN, 0.1% FA) during 5 min, then a 5–22% gradient of solvent B during 150 min, a 22–45% gradient of solvent B during 60 min and ﬁnally a 45–80% gradient of solvent B during 10 min all at 250 nL min−1 ﬂow rate. The instrument method for the Q Exactive Plus was set up in the data dependent acquisition mode. After a survey scan in the Orbitrap (resolution 70,000), the 10 most intense precursor ions were selected for HCD fragmentation with a normalized collision energy set up to 28. Charge state screening was enabled, and precursors with unknown charge state or a charge state of 1, 7, 8, and >8 were excluded. Dynamic exclusion was enabled for 45 s. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. NATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications ARTICLE SIM was performed on a Zeiss LSM 780 Elyra PS1 microscope (Carl Zeiss, Germany) using C Plan-Apochromat 63×/ 1.4 oil objective with a 1.518 refractive index oil (Carl Zeiss). The ﬂuorescence signal is detected on an EMCCD Andor Ixon 887 1 K. Raw images are composed of ﬁfteen images per plane per channel (ﬁve phases, three angles), and acquired with a Z-distance of 0.11 μm. Acquisition parameters were adapted from one image to one other to optimize the signal to noise ratio. SIM images were processed separately 12 ARTICLE Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006). 33. Schoneberg, J., Lee, I. H., Iwasa, J. H. & Hurley, J. H. Reverse-topology membrane scission by the ESCRT proteins. Nat. Rev. Mol. Cell Biol. 18, 5–17 (2017). 65. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011). 66. Cox, J. et al. Accurate proteome-wide label-free quantiﬁcation by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteom. 13, 2513–2526 (2014). 34. Henne, W. M., Stenmark, H. & Emr, S. D. Molecular mechanisms of the membrane sculpting ESCRT pathway. Cold Spring Harbor Perspect. Biol. 5, (2013) https://doi.org/10.1101/cshperspect.a016766. 67. Wieczorek, S. et al. DAPAR & ProStaR: software to perform statistical analyses in quantitative discovery proteomics. Bioinformatics 33, 135–136 (2017). p g p p 35. Schoneberg, J. et al. ATP-dependent force generation and membrane scission by ESCRT-III and Vps4. Science 362, 1423–1428 (2018). g 35. Schoneberg, J. et al. ATP-dependent force generation 68. A. N. A. Norm: analysis of multivariate normal datasets with missing values. (R Package Version, 2013). 36. Goliand, I. et al. Resolving ESCRT-III spirals at the intercellular bridge of dividing cells using 3D STORM. Cell Rep. 24, 1756–1764 (2018). 69. G.K S. limma: Linear Models for Microarray Data. in Bioinformatics and Computational Biology Solutions Using R and Bioconductor. Statistics for Biology and Health (eds Gentleman R., Carey V. J., Huber W., Irizarry R. A., Dudoit S.). (Springer, New York, NY, 2005). 37. Lee, H. H., Elia, N., Ghirlando, R., Lippincott-Schwartz, J. & Hurley, J. H. Midbody targeting of the ESCRT machinery by a noncanonical coiled coil in CEP55. Science 322, 576–580 (2008). 38. Hu, C. K., Coughlin, M. & Mitchison, T. J. Midbody assembly and its regulation during cytokinesis. Mol. Biol. Cell 23, 1024–1034 (2012). 70. Ritchie, M. E. et al. limma powers differential expression analyses for RNA- sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015). 39. Renshaw, M. J., Liu, J., Lavoie, B. D. & Wilde, A. Anillin-dependent organization of septin ﬁlaments promotes intercellular bridge elongation and Chmp4B targeting to the abscission site. Open Biol. 4, 130190 (2014). 71. Giai Gianetto, Q. et al. Calibration plot for proteomics: a graphical tool to visually check the assumptions underlying FDR control in quantitative experiments. Proteomics 16, 29–32 (2016). ARTICLE 199, 1047–1055 (2012). g 19. Fazeli, G., Trinkwalder, M., Irmisch, L. & Wehman, A. M. C. elegans midbodies are released, phagocytosed and undergo LC3-dependent degradation independent of macroautophagy. J. Cell Sci. 129, 3721–3731 (2016). 50. Grootjans, J. J., Reekmans, G., Ceulemans, H. & David, G. Syntenin-syndecan binding requires syndecan-synteny and the co-operation of both PDZ domains of syntenin. J. Biol. Chem. 275, 19933–19941 (2000). y 51. Grootjans, J. J. et al. Syntenin, a PDZ protein that binds syndecan cytoplasmic domains. Proc. Natl Acad. Sci. USA 94, 13683–13688 (1997). 20. Carlton, J. G. & Martin-Serrano, J. Parallels between cytokinesis and retroviral budding: a role for the ESCRT machinery. Science 316, 1908–1912 (2007). 1. Grootjans, J. J. et al. Syntenin, a PDZ protein that binds synde g y 21. Morita, E. et al. Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis. EMBO J. 26, 4215–4227 (2007). 52. Berryman, M. A. & Goldenring, J. R. CLIC4 is enriched at cell-cell junctions and colocalizes with AKAP350 at the centrosome and midbody of cultured mammalian cells. Cell Motil. Cytoskeleton 56, 159–172 (2003). 22. Carlton, J. G., Agromayor, M. & Martin-Serrano, J. Differential requirements for Alix and ESCRT-III in cytokinesis and HIV-1 release. Proc. Natl Acad. Sci. USA 105, 10541–10546 (2008). y 53. Capalbo, L. et al. The midbody interactome reveals unexpected roles for PP1 phosphatases in cytokinesis. Nat. Commun. 10, 4513 (2019). 23. Samson, R. Y., Obita, T., Freund, S. M., Williams, R. L. & Bell, S. D. A role for the ESCRT system in cell division in archaea. Science 322, 1710–1713 (2008). l l l f h k l l ll 54. Peterman, E. et al. The post-abscission midbody is an intracellular signaling organelle that regulates cell proliferation. Nat. Commun. 10, 3181 (2019). 23. Samson, R. Y., Obita, T., Freund, S. M., Williams, R. L. & Bell, S. D. A role for the ESCRT system in cell division in archaea. Science 322, 1710–1713 (2008). 24. Agromayor, M. et al. Essential role of hIST1 in cytokinesis. Mol. Biol. cell 20, 1374–1387 (2009). 55. Eggert, U. S. et al. Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets. PLoS Biol. 2, e379 (2004). 24. Agromayor, M. et al. Essential role of hIST1 in cytokinesis. Mol. Biol. cell 20, 1374–1387 (2009). 25. Schiel, J. A. et al. ARTICLE FIP3-endosome-dependent formation of the secondary ingression mediates ESCRT-III recruitment during cytokinesis. Nat. Cell Biol. 14, 1068–1078 (2012). 56. Sanchez, C. G. et al. Regulation of ribosome biogenesis and protein synthesis controls germline stem cell differentiation. Cell Stem Cell 18, 276–290 (2016). 57. Straight, A. F. et al. Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 299, 1743–1747 (2003). 26. El Amine, N., Kechad, A., Jananji, S. & Hickson, G. R. Opposing actions of septins and Sticky on Anillin promote the transition from contractile to midbody ring. J. Cell Biol. 203, 487–504 (2013). 58. Lekomtsev, S. et al. Centralspindlin links the mitotic spindle to the plasma membrane during cytokinesis. Nature 492, 276–279 (2012). y g 27. Matias, N. R., Mathieu, J. & Huynh, J. R. Abscission is regulated by the ESCRT-III protein shrub in Drosophila germline stem cells. PLoS Genet. 11, e1004653 (2015). g y 59. Sun, S. et al. Phosphorylation-dependent activation of the ESCRT function of ALIX in cytokinetic abscission and retroviral budding. Dev. Cell 37, 581 (2016). 28. Eikenes, A. H. et al. ALIX and ESCRT-III coordinately control cytokinetic abscission during germline stem cell division in vivo. PLoS Genet. 11, e1004904 (2015). 60. Strack, B., Calistri, A., Craig, S., Popova, E. & Gottlinger, H. G. AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 114, 689–699 (2003). 29. Christ, L. et al. ALIX and ESCRT-I/II function as parallel ESCRT-III recruiters in cytokinetic abscission. J. Cell Biol. 212, 499–513 (2016). 61. Fisher, R. D. et al. Structural and biochemical studies of ALIX/AIP1 and its role in retrovirus budding. Cell 128, 841–852 (2007). 30. Fremont, S. et al. Oxidation of F-actin controls the terminal steps of cytokinesis. Nat. Commun. 8, 14528 (2017). 62. Sette, P. et al. HIV-1 Nucleocapsid Mimics the Membrane Adaptor Syntenin PDZ to Gain Access to ESCRTs and promote virus budding. Cell Host Microbe 19, 336–348 (2016). 31. Mierzwa, B. E. et al. Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis. Nat. Cell Biol. 19, 787–798 (2017). 63. Ball, G. et al. SIMcheck: a toolbox for successful super-resolution structured illumination microscopy. Sci. Rep. 5, 15915 (2015). 32. Christ, L., Raiborg, C., Wenzel, E. M., Campsteijn, C. & Stenmark, H. Cellular functions and molecular mechanisms of the ESCRT membrane-scission machinery. Trends Biochem. Sci. 42, 42–56 (2017). 64. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z 13. Lafaurie-Janvore, J. et al. ESCRT-III assembly and cytokinetic abscission are induced by tension release in the intercellular bridge. Science 339, 1625–1629 (2013). 42. Friand, V., David, G. & Zimmermann, P. Syntenin and syndecan in the biogenesis of exosomes. Biol. Cell 107, 331–341 (2015). biogenesis of exosomes. Biol. Cell 107, 331–341 (2015). 43. Keller-Pinter, A. et al. Syndecan-4 promotes cytokinesis in 43. Keller-Pinter, A. et al. Syndecan-4 promotes cytokinesis in a phosphorylation dependent manner. Cell. Mol. Life Sci. 67, 1881–1894 (2010). 14. Crowell, E. F., Gaffuri, A. L., Gayraud-Morel, B., Tajbakhsh, S. & Echard, A. Midbody remnant engulfment after cytokinesis abscission in mammalian cells. J. Cell Sci. 127, 3840–3851 (2014). dependent manner. Cell. Mol. Life Sci. 67, 1881–1894 (20 44. Skop, A. R., Liu, H., Yates, J. 3rd, Meyer, B. J. & Heald, R. Dissection of the mammalian midbody proteome reveals conserved cytokinesis mechanisms. Science 305, 61–66 (2004). 15. Dubreuil, V., Marzesco, A. M., Corbeil, D., Huttner, W. B. & Wilsch- Brauninger, M. Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin-1. J. cell Biol. 176, 483–495 (2007). 45. Maliga, Z. et al. A genomic toolkit to investigate kinesin and myosin motor function in cells. Nat. Cell Biol. 15, 325–334 (2013). , ( ) 46. Fremont, S. & Echard, A. Studying cytokinesis and midbody remnants using correlative light/scanning EM. Methods Cell Biol. 137, 239–251 (2017). 16. Salzmann, V. et al. Centrosome-dependent asymmetric inheritance of the midbody ring in Drosophila germline stem cell division. Mol. Biol. Cell 25, 267–275 (2014). correlative light/scanning EM. Methods Cell Biol. 137, 239–2 47. Mullins, J. M. & McIntosh, J. R. Isolation and initial characterization of the mammalian midbody. J. Cell Biol. 94, 654–661 (1982). mammalian midbody. J. Cell Biol. 94, 654–661 (1982). 17. Kuo, T. C. et al. Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity. Nat. Cell Biol. 13, 1214–1223 (2011). 48. Erde, J., Loo, R. R. & Loo, J. A. Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments. J. Proteome Res. 13, 1885–1895 (2014). 49. Lee, S., Joshi, A., Nagashima, K., Freed, E. O. & Hurley, J. H. Structural basis for viral late-domain binding to Alix. Nat. Struct. Mol. Biol. 14, 194–199 (2007). 18. Chai, Y. et al. Apoptotic regulators promote cytokinetic midbody degradation in C. elegans. J. Cell Biol. References l 8. Dionne, L. K., Wang, X. J. & Prekeris, R. Midbody: from cellular junk to regulator of cell polarity and cell fate. Curr. Opin. Cell Biol. 35, 51–58 (2015). 9. Mierzwa, B. & Gerlich, D. W. Cytokinetic abscission: molecular mechanisms and temporal control. Dev. Cell 31, 525–538 (2014). 10. Guizetti, J. et al. Cortical constriction during abscission involves helices of ESCRT-III-dependent ﬁlaments. Science 331, 1616–1620 (2011). 11. Schiel, J. A. et al. Endocytic membrane fusion and buckling-induced microtubule severing mediate cell abscission. J. Cell Sci. 124, 1411–1424 (2011). 12. Elia, N., Sougrat, R., Spurlin, T. A., Hurley, J. H. & Lippincott-Schwartz, J. Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission. Proc. Natl Acad. Sci. USA 108, 4846–4851 (2011). 13 TURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications ARTICLE 40. Katoh, K. et al. The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting. J. Biol. Chem. 278, 39104–39113 (2003). 72. Pounds, S. & Cheng, C. Robust estimation of the false discovery rate. Bioinformatics 22, 1979–1987 (2006). 73. Szklarczyk, D. et al. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 47, D607–D613 (2019). 41. Baietti, M. F. et al. Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat. Cell Biol. 14, 677–685 (2012). 14 NATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15205-z A.E. wrote the paper with the help of C.A., N.G.R., M.D., T.D., Q.G.G., J.C.R., M.M., and P.Z. 74. Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003). biomolecular interaction networks. Genome Res. 13, 2498–2504 75. Perez-Riverol, Y. et al. The PRIDE database and related tools and resources in 2019: improving support for quantiﬁcation data. Nucleic Acids Res. 47, D442–D450 (2019). Correspondence and requests for materials should be addressed to A.E. Peer review information Nature Communications thanks Jeremy Carlton, Ulrike Eggert, Harald Stenmark and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Complexité du Vivant ED515, contrat n° 2412/2016 and AMX. A.P. received a fellowship from the Doctoral School Complexité du Vivant ED515, contrat n°2611 bis/2016 and Fondation ARC pour la recherche sur le cancer (DOC20190508876). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Competing interests p g The authors declare no competing interests. Acknowledgements Supplementary information is available for this paper at https://doi.org/10.1038/s41467- 020-15205-z. Supplementary information is available for this paper at https://doi.org/10.1038/s41467- 020-15205-z. We thank R. Basto, G. Hickson, J. Mathieu, J.-R. Huyhn, R. Shaughnessy, M. Serres, and T. Wai for critical reading of the paper; the Echard Lab members for helpful discussions; the Recombinant antibodies platform (TAb-IP, Institut Curie, Paris) and the DSHB (University of Iowa) for antibodies. GFP-MKLP2 cells were from the Hyman Lab MPI- MCBG Dresden45. UTechS PBI is part of the France–BioImaging infrastructure network (FBI) supported by the French National Research Agency (ANR-10-INSB-04; Invest- ments for the Future), and acknowledges support from ANR/FBI and the Région Ile-de- France (program “Domaine d’Intérêt Majeur-Malinf”) for the use of the Zeiss LSM 780 Elyra PS1 microscope. We thank P.H. Commere from the Utechs CB, Institut Pasteur for FACS sorting. This work has been supported by Institut Pasteur, CNRS, and ANR (AbCyStem, Cytosign) to A.E. C.A. received a fellowship from the Doctoral School Complexité du Vivant ED515, contrat n° 2412/2016 and AMX. A.P. received a fellowship from the Doctoral School Complexité du Vivant ED515, contrat n°2611 bis/2016 and Fondation ARC pour la recherche sur le cancer (DOC20190508876). Correspondence and requests for materials should be addressed to A.E. © The Author(s) 2020 Author contributions C.A. carried out and analyzed the experiments presented in Figs. 2–5 and Supplementary Fig. 4 and 5; N.G.R. and A.P. in Fig. 1a–d and Supplementary Fig. 1b, c; S.F. in Fig. 1d and Supplementary Fig. 1a; F.M. in Fig. 3c; N.GR. and S.S. setup the ﬂow cytometry- puriﬁcation protocol for Fig. 1b, Supplementary Fig. 1b; N.G.R. and A.P. setup MBRE protocols. F.C. and M.R. assisted with technical help. T.D, M.D., J.C.R. and M.M. designed and carried out the mass spectrometry studies; Q.G.G. did statistical analyses and Fig. 1e–g, Supplementary Fig. 1d, 1e, 2, and 3; N.G.R., Q.G.G., M.M., M.D., J.C.R. and T.D. contributed to the Supplementary Data 1; H.M. created the website. We acknowledge the help of Thomas Menard from the IT Department at the Institut Pasteur for this work. A.S. carried out and analyzed the experiments presented in Fig. 2c. P.Z. provided reagents and helpful discussions. A.E. conceived the project and secured funding. N.G.R. and A.E. supervised the proteomic data; A.E. supervised the other data. © The Author(s) 2020 15 NATURE COMMUNICATIONS \| (2020)11:1941 \| https://doi.org/10.1038/s41467-020-15205-z \| www.nature.com/naturecommunicatio
https://openalex.org/W2753283927	https://www.nature.com/articles/cddis2017432.pdf	English	null	p73 is required for appropriate BMP-induced mesenchymal-to-epithelial transition during somatic cell reprogramming	Cell death and disease	2,017	cc-by	13,141	p73 is required for appropriate BMP-induced mesenchymal-to-epithelial transition during somatic Marta Martin-Lopez1, Laura Maeso-Alonso1, Sandra Fuertes-Alvarez1, Diego Balboa2, Virginia Rodríguez-Cortez3, Jere Weltner2, Inmaculada Diez-Prieto1,4, Andrew Davis5, Yaning Wu5, Timo Otonkoski2, Elsa R Flores5, Pablo Menéndez3,6,7, Margarita M Marques8 and Maria C Marin,1 The generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming holds great potential for modeling human diseases. However, the reprogramming process remains very inefficient and a better understanding of its basic biology is required. The mesenchymal-to-epithelial transition (MET) has been recognized as a crucial step for the successful reprogramming of fibroblasts into iPSCs. It has been reported that the p53 tumor suppressor gene acts as a barrier of this process, while its homolog p63 acts as an enabling factor. In this regard, the information concerning the role of the third homolog, p73, during cell reprogramming is limited. Here, we derive total Trp73 knockout mouse embryonic fibroblasts, with or without Trp53, and examine their reprogramming capacity. We show that p73 is required for effective reprogramming by the Yamanaka factors, even in the absence of p53. Lack of p73 affects the early stages of reprogramming, impairing the MET and resulting in altered maturation and stabilization phases. Accordingly, the obtained p73-deficient iPSCs have a defective epithelial phenotype and alterations in the expression of pluripotency markers. We demonstrate that p73 deficiency impairs the MET, at least in part, by hindering BMP pathway activation. We report that p73 is a positive modulator of the BMP circuit, enhancing its activation by DNp73 repression of the Smad6 promoter. Collectively, these findings provide mechanistic insight into the MET process, proposing p73 as an enhancer of MET during cellular reprogramming. Cell Death and Disease (2017) 8, e3034; doi:10.1038/cddis.2017.432; published online 7 September 2017 The MET process, a rate-limiting step during reprogram- ming, is tightly linked with the epithelial phenotype and the pluripotent state of iPSCs.6,9 MET, as well as its reversal epithelial-to-mesenchymal transition (EMT), has roles in developmental biology and metastasis, highlighting the fact that reprogramming and tumor progression share some similarities.10 Consistently, reprogramming requires, like tumor progression, that successive barriers must be over- come to reach stemness.11 The nature of these barriers are not completely understood, but the involvement of tumor suppressors like p5312–15 or Rb116 as reprogramming hurdles has been reported, launching the hypothesis that tumor suppression mechanisms must be acting as blockades of cellular reprogramming, maintaining the differentiated state and genomic stability. 1Instituto de Biomedicina (IBIOMED) and Departamento de Biología Molecular, University of León, University of Leon, Campus de Vegazana, Leon, Spain; 2Research Programs Unit, Molecular Neurology, Biomedicum Stem Cell Center, University of Helsinki, Haartmaninkatu 8, Helsinki, Finland; 3Josep Carreras Leukemia Research Institute, Department of Biomedicine. School of Medicine, University of Barcelona, Casanova 143, Barcelona, Spain; 4Departamento de Medicina, Cirugía y Anatomía Veterinaria, University of León, Campus de Vegazana, León, Spain; 5Department of Molecular Oncology, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL, USA; 6Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain; 7Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), ISCIII, Madrid, Spain and 8Instituto de Desarrollo Ganadero and Departamento de Producción Animal, University of León, Campus de Vegazana, León, Spain Corresponding author: MC Marin, Departamento de Biologia Molecular, IBIOMED, Universidad de Leon, Campus de Vegazana, Leon 24071, Spain. Tel: +34 987 291793; Fax: +34 987 291 998; E-mail: carmen.marin@unileon.es Received 01.3.17; revised 20.7.17; accepted 25.7.17; Edited by G Raschellà Citation: Cell Death and Disease (2017) 8, e3034; doi:10.1038/cddis.2017.432 Ofﬁcial journal of the Cell Death Differentiation Association Citation: Cell Death and Disease (2017) 8, e3034; doi:10.1038/cddis.2017.432 Ofﬁcial journal of the Cell Death Differentiation Association OPEN www.nature.com/cddis Results p73 is required for effective reprogramming by OSKM and OSK, independent of p53. To analyze the effect of total p73 deficiency on reprogramming, particularly in the context of p53-loss, we sought to compare the reprogramming efficiencies of WT, p73KO, p53KO and double mutants DKO-MEFs. We transfected early passage MEFs (P4) with piggyBac transposon vectors encoding OSKM regulated by a doxycycline (Dox)-inducible system.23 Reprogramming was monitored according to previously defined morphological criteria (emergence of small cells forming compact round colonies with well-defined borders), as well as alkaline phosphatase (AP) activity.24,25 After two weeks, small colonies began to appear in WT and p73KO cultures, and colonies with ESC-like morphology were collected at day 22. While WT cultures displayed typical ESC-colonies at this point, p73KO cultures exhibited a significantly lower number of irregular AP+ colonies (Figure 1a), indicating that lack of p73 blunted the reprogramming efficiency. Next, we addressed whether p53-induced reprogramming barriers could be accountable for the observed effect. Thus, we analyzed the expression level of p21CIP1 and miR-34a.12,20 Lack of p73 neither augmented the basal expression levels of these two p53-targets in MEFs, nor its induction after 7 days of Dox-treatment (Supplementary Figure 1a). To identify the phase of reprogramming affected by p73 deficiency, we characterized the expression kinetics of initiation, maturation and stabilization phase markers,6 collect- ing RNA samples at 7, 12 and 17 days after Dox-treatment (Figure 2a). The initiation phase (Figure 2b) is defined by the induction of epithelial-associated genes with a concomitant decrease of mesenchymal genes.9 Therefore, we examined the expression of epithelial markers like E-cadherin (which is upregulated during METand crucial for reprogramming21) and Epcam. We found that in p73KO cultures upregulation of Cdh1 or Epcam was significantly reduced, while the mesenchymal associated gene Snail showed a significant increase in the absence of p73 (Figure 2c), suggesting p73 requirement for appropriate MET establishment. Dysregulation of MET genes impairs MEF reprogramming,7 thus, we speculated that in p73-deficient cells the maturation and stabilization stages would be altered. Nanog expression begins to increase during maturation phase30,31 (Figure 2c). In agreement with an altered maturation, p73KO-iPSCs dis- played attenuated Nanog expression and delayed kinetics profile (Figure 2d), as well as highly reduced levels of Lin28, a gene required for iPSCs maturation32 (Figure 2c). p73 is required for appropriate BMP-induced mesenchymal-to-epithelial transition during somatic to act as an enabling factor.19 A variety of p53-induced protective pathways impair reprogramming including, but not limited to, those involved in the regulation of cell growth, mainly by p53-downstream targets p21Cip1 or miR-34.12,20 In addition, the p53–p21 axis can also restrain reprogramming by the inhibition of the MET.21 However, reports regarding p73 role in cellular reprogramming are inconclusive.19,22 DNp73 over- expression was reported to increase human iPSCs generation efficiency,22 while other report concluded that p73 had no role in reprogramming, proposing that the DNp73 effect was due to its p53-dominant-negative effect.19 Here, we demonstrate that p73 deficiency impairs MEFs reprogramming efficiency by abating BMP-induced MET, even in the absence of p53. In agreement, p73-deficient iPSCs have an attenuated epithelial phenotype and alterations in pluripotency markers expres- sion. We report, for the first time, that p73 is part of the BMP circuit, enhancing BMP signaling, at least in part, by DNp73 transcriptional repression of the Smad6 promoter. p73 deficiency impairs MET resulting in an altered maturation and stabilization phases. Both Trp73 isoforms, TA- and DNp73, were upregulated during reprogramming, being DNp73 significantly induced during the early stages of the process (Figure 2b). We used an alternative model to confirm p73 isoforms upregulation: reprogrammable-MEFs (Rep-MEFs)28 displayed primary mouse-ES colony-like structures 5 days after Dox-treatment and, by day 9, colonies were AP+ (Supplementary Figures 2a,b). qRT-PCR analysis confirmed DNp73 as the predominant isoform induced during Rep-MEFs reprogramming (Supplementary Figure 2c). p p g g ( pp y g ) To substantiate specific p73-isoform requirement for reprogramming, we attempt to reprogram TAp73 and DNp73-specific knockout MEFs.29 We carry out the repro- gramming experiments (Supplementary Figure 3a) and 5 days upon Sendai Virus transfection, the first mouse-ES colony-like structures began to be detected in WTand, to a lesser extent, in the TAp73-deficient MEFs (Supplementary Figures 3b,c). After 21 days, WT colonies progressed and were efficiently reprogrammed, becoming AP+ and expressing the pluripo- tency marker SSEA-1 (Supplementary Figures 3d,e). How- ever, neither TAp73 −/ −, nor DNp73 −/ −, colonies progressed to a reprogrammed state by the end of the experiment (Supplementary Figure 3d), indicating that while DNp73 appears to be the predominantly expressed isoform, both isoforms appear to be required for the complete reprogramming. p73 is required for appropriate BMP-induced mesenchymal-to-epithelial transition during somatic Embryonic stem cells (ESCs) are defined by their ability to proliferate by symmetrical cell divisions and to give raise to all specialized cell types (pluripotency).1 The possibility of generating induced pluripotent stem cells (iPSCs), with similar ESC-stemness, by the overexpression of the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM),2 has created new opportunities for developmental biology, disease modeling and regenerative medicine.3–5 g iPSCs generation from mouse embryonic fibroblasts (MEFs) is a slow and inefficient process in which fibroblasts gradually lose their mesenchymal identity and assume an embryonic gene expression pattern. Functional genomics studies have defined three phases during fibroblast OSKM-induced reprogramming (termed initiation, maturation and stabilization), and uncovered an early mesenchymal-to- epithelial transition (MET) that marks the initiation phase,6,7 which is dependent upon intrinsic BMP signaling. Indeed, BMP-SMAD signaling activation promotes iPSCs generation in the early reprogramming phase, confirming its role in the induction and maintenance of pluripotency.8 Members of the p53 family, comprised by the transcription factors p53, p63 and p73, share many functional properties, but also perform unique, and some time antagonist, biological functions.17 Inhibition of the p53 pathway increases iPSCs generation efficiency,12–15,18 whereas p63 has been reported p73 modulates BMP signaling during reprogramming M Martin-Lopez et al 2 to act as an enabling factor.19 A variety of p53-induced protective pathways impair reprogramming including, but not limited to, those involved in the regulation of cell growth, mainly by p53-downstream targets p21Cip1 or miR-34.12,20 In addition, the p53–p21 axis can also restrain reprogramming by the inhibition of the MET.21 However, reports regarding p73 role in cellular reprogramming are inconclusive.19,22 DNp73 over- expression was reported to increase human iPSCs generation efficiency,22 while other report concluded that p73 had no role in reprogramming, proposing that the DNp73 effect was due to its p53-dominant-negative effect.19 Here, we demonstrate that p73 deficiency impairs MEFs reprogramming efficiency by abating BMP-induced MET, even in the absence of p53. In agreement, p73-deficient iPSCs have an attenuated epithelial phenotype and alterations in pluripotency markers expres- sion. We report, for the first time, that p73 is part of the BMP circuit, enhancing BMP signaling, at least in part, by DNp73 transcriptional repression of the Smad6 promoter. Results Moreover, expression of Pecam1 was significantly decreased at the end of the stabilization phase in p73KO cells (Figure 2c), altogether demonstrating that lack of p73 impairs MET and alters the subsequent phases of the process resulting in a defective and inefficient reprogramming process. ( pp y g ) Trp53 −/ −significantly accelerated MEFs reprogramming kinetics; however, Trp73 −/ − attenuated this p53KO- enhancing effect (Figure 1b). Lack of c-MYC delayed and attenuated WT-MEFs reprogramming26 and in this setting, p73KO cultures were severely affected (Figure 1c). p53 deficiency boosted OSK-reprogramming efficiency (Figure 1d), but lack of p73 also decreased p53KO- enhancing effect in these conditions (Figure 1d). To rule out the possibility that the observed effect was due to different MEFs proliferative indexes,27 we analyzed growth curves from early passage MEFs littermates and found, at this early passages, no significant differences between either WT and p73KO, nor p53KO and DKO growth kinetics (Supplementary Figure 1b). Trp53 −/ −significantly accelerated MEFs reprogramming kinetics; however, Trp73 −/ − attenuated this p53KO- enhancing effect (Figure 1b). Lack of c-MYC delayed and attenuated WT-MEFs reprogramming26 and in this setting, p73KO cultures were severely affected (Figure 1c). p53 deficiency boosted OSK-reprogramming efficiency (Figure 1d), but lack of p73 also decreased p53KO- enhancing effect in these conditions (Figure 1d). To rule out the possibility that the observed effect was due to different MEFs proliferative indexes,27 we analyzed growth curves from early passage MEFs littermates and found, at this early passages, no significant differences between either WT and p73KO, nor p53KO and DKO growth kinetics (Supplementary Figure 1b). Trp53 also blocks somatic cell reprogramming by inhibiting MET through mechanisms independent of the proliferation or apoptosis pathways;21 thus, we sought to investigate whether p53 deficiency could counteract p73 requirement for MET induction. p53 deficiency resulted in a strong Cdh1 and Epcam induction21 (Figure 3). Lack of p73 significantly reduced this Cell Death and Disease p73 modulates BMP signaling during reprogramming M Martin-Lopez et al 3 WT p73KO p53KO DKO WT p73KO p53KO DKO Figure 1 Trp73 deficiency impairs reprogramming efficiency, even in the absence of p53. Results MEFs of the indicated genotypes, cultured and treated identically, were transfected with OSKM (a and b) or OSK factors (c and d) and the reprogramming efficiency was monitored by quantification of alkaline phosphatase positive colonies (AP+) after either 22 days for WTand p73KO (a and b) or 17 days for p53KO and DKO (c and d) of doxycycline treatment. Representative scanned plates and photomicrographs (10 × ) of the colonies are shown for each condition. Two independent reprogramming experiments were performed, including at least three biological replicates from the indicated genotypes (with the exception of p53KO-MEFs, n = 2), with two independent transfections per replicate. Mean ± S.E.M. are represented, equal variance. Student's t-test was performed to evaluate statistical differences; Po0.05, Po0.01, *Po0.001 p53KO DKO WT p73KO p53KO DKO p53KO DKO WT p73KO Figure 1 Trp73 deficiency impairs reprogramming efficiency, even in the absence of p53. MEFs of the indicated genotypes, cultured and treated identically, were transfected with OSKM (a and b) or OSK factors (c and d) and the reprogramming efficiency was monitored by quantification of alkaline phosphatase positive colonies (AP+) after either 22 days for WTand p73KO (a and b) or 17 days for p53KO and DKO (c and d) of doxycycline treatment. Representative scanned plates and photomicrographs (10 × ) of the colonies are shown for each condition. Two independent reprogramming experiments were performed, including at least three biological replicates from the indicated genotypes (with the exception of p53KO-MEFs, n = 2), with two independent transfections per replicate. Mean ± S.E.M. are represented, equal variance. Student's t-test was performed to evaluate statistical differences; Po0.05, Po0.01, Po0.001 induction despite p53 absence, highlighting p73 importance for the epithelial phenotype. However, Trp73 loss did not affect the suppression of mesenchymal markers in this context (Figure 3), confirming that mesenchymal and epithelial transcriptional regulators are controlled by independent pathways,9 and that p73 regulates primarily the epithelial profile. It is noteworthy that lack of p73 had a stronger repressive effect in the induction of Nanog, Lin28 and Pecam1 expression, which levels are close to WT values in the DKO cells (Figure 3), altogether suggesting that these cells might not be attaining a fully reprogrammed state. that p73 was not necessary to maintain self-renewal once the iPSC line was established (Figure 4a). WT- and p53KO-iPSCs lines expressed key molecular markers for pluripotency, like Nanog, SSEA-1 (Figure 4b) or Lin28 (Figure 4c). Results However, p73KO-iPSCs showed slightly lower levels of Nanog and SSEA-1 (Figure 4b). Lin28 analysis revealed that p73-deficient cells did not achieve appropriate expression levels (Figure 4c), supporting the idea that these cells might not have attained full pluripotency. Nevertheless, when cultured under differentiating conditions,33 all derived lines showed efficient embryoid body formation and positive staining for lineage markers of the three germ layers (Figure 4c). p73 is dispensable for iPSC self-renewal but required for a complete reprogrammed state. We obtained iPSCs lines by selecting primary-colonies with ESC-like morphology and expanding them by successive passages in the absence of Dox. We had severe difficulties establishing p73-deficient colonies, since many of them detached from the culture surface and did not survive after the second passage. We had only a 60% ± 7.14 (p73KO) and 50% ± 8.47 (DKO) success rate of colony establishment, compared with 90% ± 3.47 and 80%± 3.41 for the WT- and p53KO-colonies, respectively. Nevertheless, the established colonies were grown and maintained in culture for more than 20 passages, indicating p73 is dispensable for iPSC self-renewal but required for a complete reprogrammed state. We obtained iPSCs lines by selecting primary-colonies with ESC-like morphology and expanding them by successive passages in the absence of Dox. We had severe difficulties establishing p73-deficient colonies, since many of them detached from the culture surface and did not survive after the second passage. We had only a 60% ± 7.14 (p73KO) and 50% ± 8.47 (DKO) success rate of colony establishment, compared with 90% ± 3.47 and 80%± 3.41 for the WT- and p53KO-colonies, respectively. Nevertheless, the established colonies were grown and maintained in culture for more than 20 passages, indicating Colony morphology is one of the criteria to identify bona-fide ESC. In this regard, WT- and p53-iPSC colonies had the expected refractive appearance with tight and well-defined borders. However, p73KO-iPSC colonies, and DKO, were less compact, and had polygonal morphology and diffuse borders (Figure 5a). Moreover, at late passage, some of the DKO-iPSC lines lost mESC-colony morphology (Figure 5b), suggesting that they were not stable. Results several layers of cells (Figure 5d, dotted arrows) in which E-Cadherin was sharply localized at the plasma membrane (white arrows). To the contrary, p73KO-colonies were almost flat (1-2 cell layers) and had lower and diffuse expression of E-Cadherin. Reports have shown that β-catenin localization is a determinant factor of the pluripotent capacity.35 In accor- dance, β-catenin colocalized with E-Cadherin at the plasma membrane in the WT cells from the colony center that were establishing tight cell–cell interactions (Figure 5d, white arrows), but not in cells at the edge of the colony (arrowheads) which are prone to differentiate.35 p73KO cells had a diffuse distribution of E-Cadherin through the plasma membrane and displayed abundant cytoplasmic β-catenin (short arrows), indicating that E-Cadherin/β-catenin interactions were lost in many p73KO cells (yellow arrows). mRNA expression analysis of Cdh1 and Epcam confirmed significantly reduced levels of these epithelial genes in p73-deficient cells (Figure 5e), altogether indicating that lack of p73 resulted in an impaired epithelial phenotype. Thus, our data suggest that p73 deficiency leads to defects on MET establishment during the initiation phase of OSKM-induced reprogramming, which not only result in decreased reprogramming efficiency, but also several layers of cells (Figure 5d, dotted arrows) in which E-Cadherin was sharply localized at the plasma membrane (white arrows). To the contrary, p73KO-colonies were almost flat (1-2 cell layers) and had lower and diffuse expression of E-Cadherin. Reports have shown that β-catenin localization is a determinant factor of the pluripotent capacity.35 In accor- dance, β-catenin colocalized with E-Cadherin at the plasma membrane in the WT cells from the colony center that were establishing tight cell–cell interactions (Figure 5d, white arrows), but not in cells at the edge of the colony (arrowheads) which are prone to differentiate.35 p73KO cells had a diffuse distribution of E-Cadherin through the plasma membrane and displayed abundant cytoplasmic β-catenin (short arrows), indicating that E-Cadherin/β-catenin interactions were lost in many p73KO cells (yellow arrows). mRNA expression analysis of Cdh1 and Epcam confirmed significantly reduced levels of these epithelial genes in p73-deficient cells (Figure 5e), altogether indicating that lack of p73 resulted in an impaired epithelial phenotype. Results In agreement, p73-deficient iPSCs had reduced levels of E-Cadherin (Figure 5c), crucial for colony compaction and full pluripotency maintenance.34 Orthogonal projections of confocal microscopy images con- firmed that WT-iPSC colonies were dome-shaped, with Cell Death and Disease p73 modulates BMP signaling during reprogramming M Martin-Lopez et al 4 t=0 t=7 t=12 t=7 t=7 t=17 t=17 t=17 t=0 t=17 t=12 Figure 2 Lack of p73 impairs METresulting in altered maturation and stabilization phases. (a) Overview of the experimental design of MEFs reprogramming by doxycycline inducible OSKM transfection and sample collection during the initiation, maturation and stabilization phases of the process. (b and c) Analysis of the expression kinetics profile of markers during the reprogramming process of WT (b and c) and p73KO (c) MEFs. RNA samples were collected at the indicated days and expression analysis was performed by qRT-PCR: (b) TA and DNp73; (c) Cdh1, Snail, Nanog, Epcam, Lin28 and Pecam1. Expression of the indicated genes was normalized to 18S and set to 1 for non-transfected MEFs in each graph (t = 0). Analysis was performed with data from two independent experiments, with at least three biological replicates from the indicated genotypes, with two replicates per sample. Mean ± S.E.M. are represented, equal variance. Student's t-test was performed to evaluate statistical differences. Po0.05, Po0.01, Po0.001 t=0 t=7 t=12 t=7 t=7 t=0 t=17 t=12 t=17 t=17 t=17 Figure 2 Lack of p73 impairs METresulting in altered maturation and stabilization phases. (a) Overview of the experimental design of MEFs reprogramming by doxycycline inducible OSKM transfection and sample collection during the initiation, maturation and stabilization phases of the process. (b and c) Analysis of the expression kinetics profile of markers during the reprogramming process of WT (b and c) and p73KO (c) MEFs. RNA samples were collected at the indicated days and expression analysis was performed by qRT-PCR: (b) TA and DNp73; (c) Cdh1, Snail, Nanog, Epcam, Lin28 and Pecam1. Expression of the indicated genes was normalized to 18S and set to 1 for non-transfected MEFs in each graph (t = 0). Analysis was performed with data from two independent experiments, with at least three biological replicates from the indicated genotypes, with two replicates per sample. Mean ± S.E.M. are represented, equal variance. Student's t-test was performed to evaluate statistical differences. Po0.05, Po0.01, Po0.001 lead to alterations in the establishment of cell junctions involving E-cadherin. Cell Death and Disease Results RNA samples were collected at the indicated days and expression analysis was performed by qRT-PCR: Cdh1, Snail, Nanog, Epcam, Lin28 and Pecam1. Expression of the indicated genes was normalized to 18S and set to 1 for non-transfected MEFs in each graph (t = 0). Analysis was performed with data from two independent experiments, with at least three biological replicates from the indicated genotypes (two biological replicates in the case of p53KO cells), with two replicates per sample. Mean ± S.E.M. are represented, equal variance. Student's t-test was performed to evaluate statistical differences; Po0.05, Po0.01, Po0.001 TAp73, significantly reduced BMP4-induced Smad6 mRNA levels (Figure 7a), suggesting that Smad6 could be a DNp73 transcriptional target. levels of phosphorylated-Smad1/5/8, was remarkably reduced in the obtained p73KO-iPSC clones (Figure 6c). reduced in the obtained p73KO iPSC clones (Figure 6c). We evaluated p73 expression after BMP4 treatment of serum-deprived embryonal carcinoma P19 cells, known to upregulate Smad6 in response to BMPs.37 As depicted in Figure 6d, BMP-induced DNp73 levels, but not TAp73, correlated with increased levels of other BMP targets like Id1 or Smad6. Moreover, ectopic expression of DNp73 in serum- deprived P19 cells, enhanced BMP4-induced Id1 expression (Figure 6e, left panel) and prolonged Smad1/5/8 phosphoryla- tion (Figure 6e, right panel). DNp73 role as an enhancer of BMP signaling was also investigated in the Tet-OFF inducible cell line H1299-DNp73.38 In this system, reported to secrete low levels of endogenous BMP4,39 cells cultured in the presence of serum, but without BMP4 treatment, displayed detectable levels of p-Smad-1/5/8 that were enhanced upon induction of DNp73 expression (Figure 6f). Moreover, in serum-deprived conditions, DNp73 expression enhanced BMP4-induced Smad1/5/8 activation (Figure 6g), demonstrat- ing that DNp73 expression potentiated BMP signaling cascade. p g Thus, to address whether SMAD6 was transcriptionally regulated by DNp73, we performed an in silico prediction of p53-responsive elements within the human SMAD6 gene using p53Family-Target-Genes data base.40 This analysis unveiled a p53-binding site located between nt −2769 to −2737 from the transcription start site (position +1), that was partially conserved in the murine Smad6 promoter (Supplementary Figure 4a). We analyzed DNp73 ability to antagonize BMP4-induced activation of the Smad6 promoter using a Smad6-Luc reporter37 in P19 cells. DNp73, but not TAp73, significantly repressed BMP4 activation of the reporter gene (Figure 7b). As TA- and DNp73 can antagonistically regulate certain genes, we investigated whether DNp73 could repress BMP4-induced Smad6 promoter activation, in the presence of TAp73. Results Thus, our data suggest that p73 deficiency leads to defects on MET establishment during the initiation phase of OSKM-induced reprogramming, which not only result in decreased reprogramming efficiency, but also p73 is a positive modulator of the BMP circuit required for the OSKM-induced BMP signaling during the initia- tion phase of reprogramming. Next, we sought to identify the mechanism underlying p73 requirement for MET estab- lishment. MET induction is dependent upon the intrinsic activation of BMP cascade.6 We investigated if p73 deficiency affected OSKM-activation of BMP signaling by measuring Id1 expression, the immediate early BMP response gene.36 As expected, WT cells underwent a 2.4-fold increase in Id1 expression after 7 days of Dox-treatment, but no Id1 induction was detected in p73KO cells (Figure 6a), suggesting a role of p73 as a BMP signaling modulator. BMP signal is fine-tuned by feedback mechanisms orchestrated, among others, by the intracellular Smad inhibitor, Smad6.37 We observed a significant induction of Smad6 expression in p73KO cells when compared with WT cells (Figure 6b, left panel). Accordingly, higher levels of Smad6 were detected in p73KO-MEFs (Figure 6b, right panel). Moreover, BMP signaling cascade activation, which was measured by the Cell Death and Disease p73 modulates BMP signaling during reprogramming M Martin-Lopez et al 5 t=17 WT t=17 t=7 WT t=7 t=17 t=12 WT t=17 WT t=12 WT t=17 t=17 t=17 WT t=17 t=7 WT t=7 Figure 3 p53 deficiency could not fully counteract p73 requirement during reprogramming. Expression kinetics analysis of METand pluripotency markers during the p53KO and DKO-MEFs reprogramming process. RNA samples were collected at the indicated days and expression analysis was performed by qRT-PCR: Cdh1, Snail, Nanog, Epcam, Lin28 and Pecam1. Expression of the indicated genes was normalized to 18S and set to 1 for non-transfected MEFs in each graph (t = 0). Analysis was performed with data from two independent experiments, with at least three biological replicates from the indicated genotypes (two biological replicates in the case of p53KO cells), with two replicates per sample. Mean ± S.E.M. are represented, equal variance. Student's t-test was performed to evaluate statistical differences; Po0.05, Po0.01, Po0.001 t=7 WT t=7 t=7 WT t=7 Figure 3 p53 deficiency could not fully counteract p73 requirement during reprogramming. Expression kinetics analysis of METand pluripotency markers during the p53KO and DKO-MEFs reprogramming process. Results DNp73 was capable of significantly block BMP4-induced transcriptional activation independently of TAp73 expression (Figure 7c). Thus, physiological upregula- tion of TAp73 during the process was not opposing DNp73 modulation of BMP cascade. Next, we performed chromatin immunoprecipitation (ChIP) assays in H1299 cells, in 10% serum conditions to demonstrate that endogenous DNp73 could directly bind to the SMAD6 promoter p53-RE. Cross-linked cellular extracts were immunoprecipitated and the interacting DNA was quantified by qPCR using primers specific for the p53-REs of the SMAD6 and p21CIP1 promoters,41 and compared to DNp73 directly binds the SMAD6 promoter and represses its BMP-induced activation. DNp73 capacity to lengthen BMP cascade activation, together with the higher levels of Smad6 detected in p73KO-MEFs, lead us to hypothesize that DNp73 might be a direct repressor of this BMP inhibitor, modulating in this way, the BMP-negative feedback loop. Supporting this idea, overexpression of DNp73, but not Cell Death and Disease p73 modulates BMP signaling during reprogramming M Martin-Lopez et al 6 lldown as background. As expected, p21CIP1 showed ± 5 38-fold enrichment compared to IgG (P = 0 024; promoter p53-RE (6.80 ± 1.73; P = 0.004; Figure 7d, panel) demonstrating that this gene is a direct DN Tuj-1 CD31 AFP Ectoderm Mesoderm Endoderm WT p73KO p53KO DKO iPSCs lines Nanog SSEA-1 Merge + DAPI WT p73KO p53KO DKO WT p73KO p53KO DKO 4 p73 is dispensable for iPSCs self-renewal and in vitro pluripotency, but p73-deficient cells have decreased pluripotency marker expression and might no ull stemness. Three clones for each genotype were analyzed. All the p73-defective clones displayed the altered phenotype (a–c) iPSCs of the indicated genotyp under proliferating and non-differentiating conditions and analyzed. (a) Representative phase contrast images (Objective 5 × ; Scale: 250 μm) of iPSCs c ding to the 20th passage. (b) Confocal microscopy analysis of pluripotency markers Nanog (red) and SSEA-1 (green). DAPI was used to visualize nuclei. Ob ale: 80 μm. (c) Quantification of Lin28 expression by qRT-PCR. Mean values ± S.E.M. from duplicates of at least three clones per genotype from two indep nts are shown, equal variance. Expression was analyzed by qRT-PCR, normalized to 18S. Student's t-test was performed to evaluate statistical differences P 1, **Po0.001. (d) iPSCs lines of the indicated genotype were differentiated by embryoid body (EB) formation under differentiating conditions and lineage mar m layers were analyzed: Tuj-1 (ectoderm, green), CD31 (mesoderm, red) and AFP (endoderm, red). Results At least three clones per genotype were analyzed. DAPI was nuclei. Objective 20 × . Scale: 80 μm WT p73KO p53KO DKO iPSCs lines Nanog SSEA-1 Merge + DAPI WT p73KO p53KO DKO Tuj-1 CD31 AFP Ectoderm Mesoderm Endoderm WT p73KO p53KO DKO Endoderm Figure 4 p73 is dispensable for iPSCs self-renewal and in vitro pluripotency, but p73-deficient cells have decreased pluripotency marker expression and might not have attained full stemness. Three clones for each genotype were analyzed. All the p73-defective clones displayed the altered phenotype (a–c) iPSCs of the indicated genotype were cultured under proliferating and non-differentiating conditions and analyzed. (a) Representative phase contrast images (Objective 5 × ; Scale: 250 μm) of iPSCs cultures corresponding to the 20th passage. (b) Confocal microscopy analysis of pluripotency markers Nanog (red) and SSEA-1 (green). DAPI was used to visualize nuclei. Objective 20 × . Scale: 80 μm. (c) Quantification of Lin28 expression by qRT-PCR. Mean values ± S.E.M. from duplicates of at least three clones per genotype from two independent experiments are shown, equal variance. Expression was analyzed by qRT-PCR, normalized to 18S. Student's t-test was performed to evaluate statistical differences Po0.05, Po0.01, Po0.001. (d) iPSCs lines of the indicated genotype were differentiated by embryoid body (EB) formation under differentiating conditions and lineage markers of three germ layers were analyzed: Tuj-1 (ectoderm, green), CD31 (mesoderm, red) and AFP (endoderm, red). At least three clones per genotype were analyzed. DAPI was used to visualize nuclei. Objective 20 × . Scale: 80 μm promoter p53-RE (6.80 ± 1.73; P = 0.004; Figure 7d, right panel), demonstrating that this gene is a direct DNp73 transcriptional target. This enrichment was not detected when ChIP was performed in a region at the vicinity of the IgG-pulldown as background. As expected, p21CIP1 showed a 13.7 ± 5.38-fold enrichment compared to IgG (P = 0.024; Figure 7d, left panel). More importantly, we observed a significant in vitro direct interaction of DNp73 with the SMAD6 Cell Death and Disease p73 modulates BMP signaling during reprogramming M Martin-Lopez et al β-catenin E-cadherin WT p73KO p53KO DKO DKO WT p73KO p53KO DKO E-cadherin E-cadherin WT p73KO p73KO Merge re 5 Lack of p73 results in altered iPSC colony morphology and attenuated epithelial phenotype. (a–e) iPSCs of the indicated genotype were cultured under proliferatin ferentiating conditions and analyzed. Results Scale: 20 μm Quantification of Cdh1 and Epcam expression by qRT-PCR. Mean values ±S.E.M. from duplicates of at least three clones per genotype (two biological replicates in the case of p53 rom two independent experiments are shown, equal variance. Student's t-test was performed to evaluate statistical differences Po0.05, Po0.01, Po0.001 β-catenin E-cadherin WT p73KO p73KO Merge gure 5 Lack of p73 results in altered iPSC colony morphology and attenuated epithelial phenotype. (a–e) iPSCs of the indicated genotype were cultured under prolifera -differentiating conditions and analyzed. (a and b) Representative phase contrast images of iPSCs culturescorresponding to late passages(Objective 20 × in a and 10× in b μm). Three clones for each genotype were analyzed. All the p73-defective clones displayed the altered phenotype. (c) Confocal microscopy analysis of E-cadherin immuno ) (Objective 20× ; Scale: 80 μm). (d) Orthogonal projections of three-dimensional reconstruction images of iPSCs immunostained with β-catenin (red) and E-cadherin bodies. The lateral views are indicated by the dotted arrows and indicate the layers of cells that compose the colony. Co-localization of β-catenin and E-Cadherin at the mbrane in tight cell–cell interactions is indicated by white arrows, and cell with no co-localization at the plasma membrane are marked with arrowheads. In p73KO cells wit ibution of E-Cadherin and cytoplasmic β-catenin are marked by arrowheads and cell without E-Cadherin/β-catenin interactions by yellow arrows. Objective 63× . Scale: 20 β-catenin E-cadherin WT p73KO p73KO Figure 5 Lack of p73 results in altered iPSC colony morphology and attenuated epithelial phenotype. (a–e) iPSCs of the indicated genotype were cultured under proliferating and non-differentiating conditions and analyzed. (a and b) Representative phase contrast images of iPSCs culturescorresponding to late passages(Objective 20 × in a and 10× in b; Scale: 250 μm). Three clones for each genotype were analyzed. All the p73-defective clones displayed the altered phenotype. (c) Confocal microscopy analysis of E-cadherin immunostaining (red) (Objective 20× ; Scale: 80 μm). (d) Orthogonal projections of three-dimensional reconstruction images of iPSCs immunostained with β-catenin (red) and E-cadherin (green) antibodies. The lateral views are indicated by the dotted arrows and indicate the layers of cells that compose the colony. Co-localization of β-catenin and E-Cadherin at the plasma membrane in tight cell–cell interactions is indicated by white arrows, and cell with no co-localization at the plasma membrane are marked with arrowheads. Results (a and b) Representative phase contrast images of iPSCs culturescorresponding to late passages(Objective 20 × in a and 10× in b; S m). Three clones for each genotype were analyzed. All the p73-defective clones displayed the altered phenotype. (c) Confocal microscopy analysis of E-cadherin immunost Objective 20× ; Scale: 80 μm). (d) Orthogonal projections of three-dimensional reconstruction images of iPSCs immunostained with β-catenin (red) and E-cadherin (g dies. The lateral views are indicated by the dotted arrows and indicate the layers of cells that compose the colony. Co-localization of β-catenin and E-Cadherin at the p ane in tight cell–cell interactions is indicated by white arrows, and cell with no co-localization at the plasma membrane are marked with arrowheads. In p73KO cells with d tion of E-Cadherin and cytoplasmic β-catenin are marked by arrowheads and cell without E-Cadherin/β-catenin interactions by yellow arrows. Objective 63× . Scale: 20 μ fication of Cdh1 and Epcam expression by qRT-PCR. Mean values ±S.E.M. from duplicates of at least three clones per genotype (two biological replicates in the case of p5 i d d i h l i S d ' f d l i i l diff P 0 0 P 0 01 P 0 001 WT p73KO p53KO DKO DKO WT p73KO p53KO DKO E-cadherin E-cadherin DKO β-catenin E-cadherin WT p73KO p53KO DKO E-cadherin E-cadherin WT p73KO p73KO Merge Figure 5 Lack of p73 results in altered iPSC colony morphology and attenuated epithelial phenotype. (a–e) iPSCs of the indicated genotype were cultured under proliferating non-differentiating conditions and analyzed. (a and b) Representative phase contrast images of iPSCs culturescorresponding to late passages(Objective 20 × in a and 10× in b; Sc 250 μm). Three clones for each genotype were analyzed. All the p73-defective clones displayed the altered phenotype. (c) Confocal microscopy analysis of E-cadherin immunostain red) (Objective 20× ; Scale: 80 μm). (d) Orthogonal projections of three-dimensional reconstruction images of iPSCs immunostained with β-catenin (red) and E-cadherin (gre antibodies. The lateral views are indicated by the dotted arrows and indicate the layers of cells that compose the colony. Co-localization of β-catenin and E-Cadherin at the plas membrane in tight cell–cell interactions is indicated by white arrows, and cell with no co-localization at the plasma membrane are marked with arrowheads. In p73KO cells with diff distribution of E-Cadherin and cytoplasmic β-catenin are marked by arrowheads and cell without E-Cadherin/β-catenin interactions by yellow arrows. Objective 63× . Results In p73KO cells with diffuse distribution of E-Cadherin and cytoplasmic β-catenin are marked by arrowheads and cell without E-Cadherin/β-catenin interactions by yellow arrows. Objective 63× . Scale: 20 μm. (e) Quantification of Cdh1 and Epcam expression by qRT-PCR. Mean values ±S.E.M. from duplicates of at least three clones per genotype (two biological replicates in the case of p53KO) from two independent experiments are shown, equal variance. Student's t-test was performed to evaluate statistical differences Po0.05, Po0.01, Po0.001 Cell Death and Disease p73 modulates BMP signaling during reprogramming M Martin-Lopez et al 8 t=7 t=12 75 kDa 50 kDa 75 kDa 50 kDa HA- DNp73 p-Smad 1/5/8 β-actin H1299-DNp73 cells 75 kDa 50 kDa 75 kDa 50 kDa HA- DNp73 p-Smad 1/5/8 β-actin Non treated cells +BMP4 (12 h) Vector HA-DNp73 P19 cells p-Smad 1/5/8 β-actin 50 kDa 75 kDa 50 kDa 0 12 18 0 12 24 48 72 h 0 12 18 h 48 24 12 0 12 24 48 0 12 24 48 h +DNp73 +BMP4 H1299-DNp73 cells -DNp73 +BMP4 +DNp73 -BMP4 t=7 t=12 MEFs p-Smad 1/5/8 β-actin P19 cells P19 cells 50 kDa 75 kDa 50 kDa 1 2 1 2 3 p73KO WT iPSCs Figure 6 p73 is a positive modulator of the BMP circuit required for the OSKM-induced BMP signaling during the initiation phase of reprogramming. (a and b) Analysis of th expression kinetics profile of markers during reprogramming process of WTand p73KO-MEFs. RNA samples were collected at the indicated days and expression analysis wa performed by qRT-PCR: (a) Id1 (b) Smad6 (left panel). Expression of the indicated genes was normalized to 18S and set to 1 for non-transfected MEFs in each graph (t = 0). ( Quantification of Smad6 expression by qRT-PCR in MEFs (right panel). Expression was normalized to 18S. Analysis was performed with data from two independent experiment with at least three biological replicates from the indicated genotypes, with two replicates per sample. Mean ± S.E.M. are represented, equal variance. Student's t-test wa performed to evaluate statistical differences. Po0.05, Po0.01, Po0.001. (c) Western blot analysis of BMP signaling cascade activation in WTand p73KO-iPSC clone (d) Quantification of Id1, Smad6, TAp73 and DNp73 expression by qRT-PCR after BMP4 treatment (0–50 ng/ml BMP4) in serum-deprived P19 cells. (e–g) P19 cells we transfected with DNp73 expression plasmid and after 18 h, cells were serum-deprived for 24 h and then, treated with BMP4. Results (d) Quantification of Id1, Smad6, TAp73 and DNp73 expression by qRT-PCR after BMP4 treatment (0–50 ng/ml BMP4) in serum-deprived P19 cells. (e–g) P19 cells were transfected with DNp73 expression plasmid and after 18 h, cells were serum-deprived for 24 h and then, treated with BMP4. At the indicated time points BMP cascade activation was analyzed by quantification of Id1 by qRT-PCR (e, left panel) or phospho-Smad1/5/8 expression by western blot assay (e, right panel). (f and g) The Tet-OFF inducible cell line H1299-DNp73 was cultured on serum conditions (e) or serum-deprived (g) before inducing DNp73 expression in the presence or absence of BMP4 (g). BMP cascade activation was analyzed by phospho-Smad1/5/8 expression by western blot assay. Equal amounts of total protein were loaded and β-actin serves as loading control. Note that HA (Y11) antibody detects the exogenous DNp73 protein expression. At least two independent experiments were performed with similar results such as p53, p16INK or Rb1, that control cell proliferation, differentiation and cell death, serve as key regulators limiting cell reprogramming and maintaining cell fate and genomic stability.43 The members of the p53 family are transcription factors known to regulate such processes in somatic and stem cells.17 The emerging picture portraits an interconnected network in which p63 and p73 share many p53-functional properties, but also perform unique, and some time antago- nist, biological functions.44,45 Along the same line, p53 and p63 seem to play opposites roles in MEFs reprogramming. Trp53 abatement enhances and accelerates reprogramming yielding defective iPSCs with genomic instability and in vivo tumorigenic potential,21 while Trp63, DNp63 in particular, has been reported to act as an enabling factor.19 In this regard, the identified SMAD6 p53-RE but with no p53-RE homology (1700 bp downstream of the p53-RE, Supplementary Figure 4a). ChIP analysis with anti-DNp73 antibody in WT- and p73-deficient iPSCs revealed a 16.28 ± 4.14-fold enrich- ment compared to IgG (P = 0.00244; Figure 7d, lower panel) in WT-iPSCs but not in p73KO-iPSCs, further demonstrating the specificity of DNp73 direct interaction with the Smad6 promoter. Results At the indicated time points BMP cascade activatio was analyzed by quantification of Id1 by qRT-PCR (e, left panel) or phospho-Smad1/5/8 expression by western blot assay (e, right panel). (f and g) The Tet-OFF inducible cell lin H1299-DNp73 was cultured on serum conditions (e) or serum-deprived (g) before inducing DNp73 expression in the presence or absence of BMP4 (g). BMP cascade activatio was analyzed by phospho-Smad1/5/8 expression by western blot assay. Equal amounts of total protein were loaded and β-actin serves as loading control. Note that HA (Y1 antibody detects the exogenous DNp73 protein expression. At least two independent experiments were performed with similar results t=7 t=12 MEFs t=7 t=12 p-Smad 1/5/8 β-actin 50 kDa 75 kDa 50 kDa 1 2 1 2 3 p73KO WT iPSCs 75 kDa 50 kDa 75 kDa 50 kDa HA- DNp73 p-Smad 1/5/8 β-actin Non treated cells +BMP4 (12 h) Vector HA-DNp73 P19 cells 0 12 18 0 12 18 h P19 cells P19 cells 75 kDa 50 kDa 75 kDa 50 kDa HA- DNp73 p-Smad 1/5/8 β-actin Non treated cells +BMP4 (12 h) Vector HA-DNp73 P19 cells 0 12 18 0 12 18 h P19 cells P19 cells 75 kDa 50 kDa 75 kDa 50 kDa HA- DNp73 p-Smad 1/5/8 β-actin H1299-DNp73 cells 0 12 24 48 72 h p-Smad 1/5/8 β-actin 50 kDa 75 kDa 50 kDa 48 24 12 0 12 24 48 0 12 24 48 h +DNp73 +BMP4 H1299-DNp73 cells -DNp73 +BMP4 +DNp73 -BMP4 Figure 6 p73 is a positive modulator of the BMP circuit required for the OSKM-induced BMP signaling during the initiation phase of reprogramming. (a and b) Analysis of the expression kinetics profile of markers during reprogramming process of WTand p73KO-MEFs. RNA samples were collected at the indicated days and expression analysis was performed by qRT-PCR: (a) Id1 (b) Smad6 (left panel). Expression of the indicated genes was normalized to 18S and set to 1 for non-transfected MEFs in each graph (t = 0). (b) Quantification of Smad6 expression by qRT-PCR in MEFs (right panel). Expression was normalized to 18S. Analysis was performed with data from two independent experiments, with at least three biological replicates from the indicated genotypes, with two replicates per sample. Mean ± S.E.M. are represented, equal variance. Student's t-test was performed to evaluate statistical differences. Po0.05, Po0.01, Po0.001. (c) Western blot analysis of BMP signaling cascade activation in WTand p73KO-iPSC clones. Cell Death and Disease Discussion Real-time PCR using specific primers to amplify p53-RE of the human or mouse Smad6 promoter, or p53-RE in the p21Cip1 promoter as control, were performed and the data were normalized to input chromatin samples of each case and to IgG values = 1. Additional control was performed immunoprecipitating with either rabbit IgG or anti-TAp73 and anti-DNp73 specific antibodies, and PCR amplifying the ChIP product with primers specific to a region at the vicinity of the identified p53-RE (1700 bp downstream of the p53-RE), but without homology to this site (H1299 control). Experiments were repeated four times per duplicate. Bars represent mean values ± S.E. M; equal variance. Student's t-test was performed to evaluate statistical differences; Po0.05, Po0.01, Po0.001 demonstrate that lack of p73 decreased the reprogramming efficiency; however, we obtained p73KO-iPSC clones and showed that p73 is dispensable for iPSC self-renewal. Moreover, p73KO-iPSCs could be differentiated into cells of the three germ layers, including neural progenitor cells, but a close analysis of p73KO-iPSCs revealed that p73 deficiency resulted in decreased pluripotency markers, altered morphol- ogy and attenuated epithelial phenotype. information concerning p73 role in the reprogramming process is contradictory. DNp73 was reported to increase human iPSCs generation efficiency,22 while other study concluded that p73 has no role in MEFs reprogramming and proposed that DNp73 effect was mediated by its p53-dominant negative effect.19 To shed light on this issue, we reprogrammed total Trp73 knockout MEFs, with or without Trp53, with the Yamanaka factors. Our data demonstrate that the reduced reprogramming efficiency of p73-deficient MEFs cannot be explained by enhanced p53 activity, as previously proposed,19 supporting a specific p73 function during reprogramming, independent of DNp73-dominant-negative effect over p53. Thus, while the data presented in this work is in accordance with DNp73 positive role during reprogramming, it is in contradiction with the second report.19 This one indicated that p73 deficiency had no effect on iPSC generation, self- maintenance or pluripotency. Nevertheless, some of our results are in partial consonance with their observations. We information concerning p73 role in the reprogramming process is contradictory. DNp73 was reported to increase human iPSCs generation efficiency,22 while other study concluded that p73 has no role in MEFs reprogramming and proposed that DNp73 effect was mediated by its p53-dominant negative effect.19 To shed light on this issue, we reprogrammed total Trp73 knockout MEFs, with or without Trp53, with the Yamanaka factors. Discussion (b and c) Transcriptional analysis was performed with the reporter vector pGL2-mSmad6-promoter (-3123)-luc, a BMP-responsive reporter construct that includes the p53-RE, together with the indicated expression vectors in the presence or absence of BMP4. (c) TAp73 (0.2 ug) was co-transfected with increasing amounts of DNp73 (0.2–0.6 μg) before BMP4 treatment. Luciferase activity was normalized by the Renilla activity of the same lysate. Bars represent mean values ± S. E.M of at least four experiments; ns: not significant. (d) ChIP analysis of H1299 cells cultured in 10% serum conditions were performed using isotypic-antibody (rabbit IgG), anti- HA or anti-DNp73 specific antibodies. Real-time PCR using specific primers to amplify p53-RE of the human or mouse Smad6 promoter, or p53-RE in the p21Cip1 promoter as control, were performed and the data were normalized to input chromatin samples of each case and to IgG values = 1. Additional control was performed immunoprecipitating with either rabbit IgG or anti-TAp73 and anti-DNp73 specific antibodies, and PCR amplifying the ChIP product with primers specific to a region at the vicinity of the identified p53-RE (1700 bp downstream of the p53-RE), but without homology to this site (H1299 control). Experiments were repeated four times per duplicate. Bars represent mean values ± S.E. M; equal variance. Student's t-test was performed to evaluate statistical differences; Po0.05, Po0.01, Po0.001 Figure 7 Smad6 is a direct DNp73 transcriptional target. (a–c) P19 cells were transfected with either (a) TA-, DNp73 or vector control, or (b,c) Smad6-promoter reporter system. After 18 h cells were serum-deprived for 24 h and then treated with BMP4. At the indicated time points samples were collected and analyzed. (a) Quantification of Smad6 expression level was analyzed by qRT-PCR. (b and c) Transcriptional analysis was performed with the reporter vector pGL2-mSmad6-promoter (-3123)-luc, a BMP-responsive reporter construct that includes the p53-RE, together with the indicated expression vectors in the presence or absence of BMP4. (c) TAp73 (0.2 ug) was co-transfected with increasing amounts of DNp73 (0.2–0.6 μg) before BMP4 treatment. Luciferase activity was normalized by the Renilla activity of the same lysate. Bars represent mean values ± S. E.M of at least four experiments; ns: not significant. (d) ChIP analysis of H1299 cells cultured in 10% serum conditions were performed using isotypic-antibody (rabbit IgG), anti- HA or anti-DNp73 specific antibodies. Discussion Somatic cell reprogramming is an inefficient process in which successive barriers must be overcome to reach the pluripotent state.27,42 It is well established that tumor suppressor genes Cell Death and Disease p73 modulates BMP signaling during reprogramming M Martin-Lopez et al Figure 7 Smad6 is a direct DNp73 transcriptional target. (a–c) P19 cells were transfected with either (a) TA-, DNp73 or vector control, or (b,c) Smad6-promoter reporter system. After 18 h cells were serum-deprived for 24 h and then treated with BMP4. At the indicated time points samples were collected and analyzed. (a) Quantification of Smad6 expression level was analyzed by qRT-PCR. (b and c) Transcriptional analysis was performed with the reporter vector pGL2-mSmad6-promoter (-3123)-luc, a BMP-responsive reporter construct that includes the p53-RE, together with the indicated expression vectors in the presence or absence of BMP4. (c) TAp73 (0.2 ug) was co-transfected with increasing amounts of DNp73 (0.2–0.6 μg) before BMP4 treatment. Luciferase activity was normalized by the Renilla activity of the same lysate. Bars represent mean values ± S. E.M of at least four experiments; ns: not significant. (d) ChIP analysis of H1299 cells cultured in 10% serum conditions were performed using isotypic-antibody (rabbit IgG), anti- HA or anti-DNp73 specific antibodies. Real-time PCR using specific primers to amplify p53-RE of the human or mouse Smad6 promoter, or p53-RE in the p21Cip1 promoter as control, were performed and the data were normalized to input chromatin samples of each case and to IgG values = 1. Additional control was performed immunoprecipitating with either rabbit IgG or anti-TAp73 and anti-DNp73 specific antibodies, and PCR amplifying the ChIP product with primers specific to a region at the vicinity of the identified p53-RE (1700 bp downstream of the p53-RE), but without homology to this site (H1299 control). Experiments were repeated four times per duplicate. Bars represent mean values ± S.E. M; equal variance. Student's t-test was performed to evaluate statistical differences; Po0.05, Po0.01, Po0.001 9 Figure 7 Smad6 is a direct DNp73 transcriptional target. (a–c) P19 cells were transfected with either (a) TA-, DNp73 or vector control, or (b,c) Smad6-promoter reporter system. After 18 h cells were serum-deprived for 24 h and then treated with BMP4. At the indicated time points samples were collected and analyzed. (a) Quantification of Smad6 expression level was analyzed by qRT-PCR. Discussion Thus, lack of DNp73 expression during the initial steps of reprogramming would lead to increased levels of Smad6, which in turn, will blunt OSKM-induced BMP signaling. Consequently, MET induction would be impaired, resulting in an attenuated epithelial phenotype (with reduced Cdh1 and Epcam levels) and subsequent altered maturation and stabilization phases. Cell culture. Mouse Embryonic Fibroblasts (MEFs), P19 cells and H1299- DNp73β cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-Glutamine. MEFs were maintained on 0.1% gelatin-coated plates. The Rep-MEFs, which contain a unique copy of the doxycycline inducible polycistronic cassette encoding the OSKM factors, were derived from the in vivo reprogrammable mice i4F28 and were kindly provided by Dr. Manuel Serrano (Institute for Research in Biomedicine, Barcelona, Spain). The DNp73 null MEFs29 and non-published TAp73Δtd/Δtd were provided by Dr. Elsa Flores (Moffitt Cancer Center, Florida, USA) and were reprogrammed using a replication-defective and persistent Sendai virus (SeV) policistronic vector encoding the OKSM genes, reported to efficiently reprogrammed MEFs.50 p BMP signaling fails to induce an epithelial phenotype in the absence of OSKM,6 suggesting that BMP regulation of MET is dependent on cell-intrinsic factors. Thus, it is tempting to speculate that p73, in addition to its BMP-enhancing function, could act as a pro-epithelial factor. This idea is supported by the fact that p73-deficient iPSC colonies showed altered morphology and attenuated epithelial phenotype. p73KO- iPSCs displayed defective intercellular interactions with low and diffuse expression of E-Cadherin as well as cytoplasmic localization of β-catenin. These intercellular interactions are crucial for colony compaction and full pluripotency maintenance.34 Therefore, the altered morphology of the p73-deficient colonies could reflect the link between pluripo- tency and the requirement for certain intercellular interactions that might be defective in the absence of p73. Supporting the idea that p73-deficient cells might not be attaining full stemness, it is noteworthy their low levels of Lin28, considered necessary to obtain fully reprogrammed and stable iPSCs.32 P19 mouse embryonal carcinoma cells were kindly provided by Dr. Han Li (Spanish National Cancer Research Centre, Madrid, Spain). H1299-DNp73β cells were a gift of by Dr Xinbin Chen (University of California, Davis, USA). These cells stably express the DNp73β isoform under control of a tetracycline-inducible system ‘Tet-off”.38 Cells were cultured in the presence of tetracycline (2 μg/ml). When it was required, to induce DNp73 expression, cells were cultured without tetracycline. Discussion All the protocols used within this study had the appropriate institutional committee approval. Breeding and genotyping of wild-type mice (WT), Trp73 knockouts (p73KO), Trp53 knockouts (p53KO) and the double mutants Trp73 −/ −; Trp53 −/ −(DKO) were performed as described before.47 Generating the TAp73Δtd/Δtd mouse. The cre-loxP strategy was used to generate the TAp73 conditional knockout reporter allele (TAp73fltd). Genomic p73 DNA from intron 1 to intron 3 was amplified from mouse genomic DNA (C57BL/6). A neomycin resistance gene (neo) flanked by frt sites was inserted in intron 3. LoxP sites were cloned into the endogenous locus 5′ to exon 2 and 3′ of the frt-flanked neo cassette. tdTomato was cloned upstream of the 5′ loxP site and the synthetic CAG promoter was cloned downstream of the 3′ loxP site. The modified p73 locus was cloned into pL253.48 Mouse embryonic stem cells (G4) electroporated with the targeting vector were analyzed by Southern blot analysis for proper targeting of the TAp73 conditional knockout reporter allele. Resulting chimaeras were mated with C57BL/6 albino females and genotyped as described below. Mice with germline transmission of the targeted allele (TAp73fltd) were intercrossed to generate homozygous mice (TAp73fltd/fltd). TAp73fltd/fltd mice were intercrossed with Zp3-Cre (C57BL/6) transgenic mice.49 TAp73fltd/+; Zp3-Cre mice were intercrossed to generate TAp73fltd/Δtd; Zp3-Cre mice, which were subsequently intercrossed to generate TAp73Δtd /Δtd mice. All procedures were approved by the IACUC at University of Texas M.D. Anderson Cancer Center. Trp53 role in reprogramming is not limited to the regulation of cell growth rate,13 but can also restrain the process by the inhibition of the MET.21 The BMP-induced mesenchymal-to- epithelial transition is required during the early steps of reprogramming.6 Here, we demonstrated that lack of p73 results in an attenuated MET transition that could account for the reduced reprogramming efficiency detected in p73- deficient cultures. We propose, for the first time, a model in which p73 is part of the BMP circuit, acting as a positive modulator of the signaling cascade, required for the OSKM- induced BMP signaling during the initiation phase of reprogramming. DNp73 was the predominant isoform induced during the initiation stage of MEFs reprogramming. In agreement, we demonstrate that DNp73 is an enhancer of BMP cascade through direct transcriptional repression of the BMP signaling inhibitor, Smad6. Discussion Our data demonstrate that the reduced reprogramming efficiency of p73-deficient MEFs cannot be explained by enhanced p53 activity, as previously proposed,19 supporting a specific p73 function during reprogramming, independent of DNp73-dominant-negative effect over p53. Thus, while the data presented in this work is in accordance with DNp73 positive role during reprogramming, it is in contradiction with the second report.19 This one indicated that p73 deficiency had no effect on iPSC generation, self- maintenance or pluripotency. Nevertheless, some of our results are in partial consonance with their observations. We During OSKM-mediated reprogramming, the majority of cells never complete the process, and only a small number become iPSCs.11 We provide evidence that the lack of p73 function reduces even further this number. In DKO-MEFs, despite the absence of p53, lack of p73 still hinders reprogramming efficiency, suggesting that p73 performs a required function that cannot be by-passed by elimination of p53 stress–response barriers. Cell growth rate is a key parameter controlling reprogramming.27 In this regard, reports indicated that p73-deficient MEFs had decreased S phase and Cell Death and Disease p73 modulates BMP signaling during reprogramming M Martin-Lopez et al 10 their long-term cell growth rate became different from WT- cultures after 6 passages in culture,46 opening the possibility that the observed effect was due to differences in the MEFs proliferative index. However, in our hands, daily growth curves from early passage MEFs littermates revealed no significant differences between WT and p73KO growth kinetics. This indicates that p73 deficiency did not affect proliferating kinetics at these early passages, when the reprogramming was performed, therefore allowing the comparison of the repro- gramming kinetics between WT vs. p73KO or p53KO vs. DKO. However, it is possible that, at latter passages, proliferating rates differences will begin to arise in p73-deficient MEFs.46 Nevertheless, in agreement with previous reports,46 p53 elimination accelerates proliferation dynamics (Supplementary Figure 1b, compare right column panels with left), and p53-deficient MEFs had an accelerated reprogram- ming kinetic respect to WT- and p73KO-MEFs. during somatic cell reprogramming. Our findings provide mechanistic insight into the MET regulation, supporting a specific p73 function, independent of DNp73-dominant- negative effect over p53, and highlighting the yin-yang role of the p53 family members as regulators of the reprogramming process. Materials and Methods Mice husbandry and animal breeding. Animal experiments were conducted in agreement with European (Council Directive 2010/63/UE) and Spanish regulations (RD 53/2013) on the protection of experimental animals. Discussion Eighteen hours after transfection, cells were serum-deprived for 24 h, followed by BMP4 treatment (5 ng/ ml) as described before. Luciferase activity was assayed using the Dual-Luciferase Reporter System (Promega) in a Berthold's luminometer. Firefly luciferase values were normalized to the corresponding Renilla luciferase levels. The Rep-MEFs can be reprogrammed in vitro upon addition of doxycycline.28 Early passage Rep-MEFs-WT were seeded at 200 000 cells/well of 6-well plate. Next day, Rep-MEFs were treated with Dox at 1μg/ml (in iPSC media) to activate the cassette during the whole process. Colonies appear around day 5 after treatment and were stained for AP activity at day-1628 TAp73 and DNp73-specific knockout MEFs29 were infected with the tetracistronic SeV vector encoding OKSM factors at MOI of 3. Tetracistronic SeV was developed, generated, concentrated and tittered as previously described.50,51 Briefly, two days before SeV transduction, 50 000 cells were plated in 6-well plates. Then, cells were transduced with SeV vectors during 2 h at RT. Fresh media was added and cells were incubated at 37 °C o/n. Next day, 25 000 cells were harvested and plated onto MEF irradiated feeder cells with human embryonic stem cell (ESC) medium/MEF conditioned media supplemented with 8 ng/ml basic fibroblast growth factor (Miltenyi, Bergisch Gladbach, Germany). After 3 days of transduction, the culture medium was changed every other day until the analysis of reprogramming efficiency. Chromatin immunoprecipitation. ChIP analysis was carried out as previously described.55 Briefly, 20x106 H1299 cells, WT-iPSC or p73KO-iPSC were fixed with 1% formaldehyde for 10 min at room temperature. The reaction was stopped by addition of 0.125 mM glycine for 10 min at room temperature and cells were washed with PBS and lysed in 0.7% SDS lysis buffer. Cross-linked chromatin was fragmented by sonication to an average size of 400 bp using a Bioruptor® sonicator (Diagenode, Liege, Belgium). Chromatin was immunoprecipitated with the following antibodies: anti-p73 N terminal (Abcam, Cambridge, UK) and anti-p73 Delta N (38c674) (Abcam). Antibodies and cell lysates were incubated overnight at 4 °C before the addition of protein G-coupled magnetic beads (Dynabeads, Invitrogen) for 4 h at 4 °C. Negative controls were prepared by incubating parallel samples with non- immune rabbit IgG and anti-HA (Santa Cruz Biotechnology) antibodies. In vitro differentiation of iPSCs. Discussion kindly provided by Dr Hämäläinen (Biomedicum Stem Cell Centre, Helsinki, Finland) including: (a) PB-TET-OSKM-IRES-βgeo, for the expression of mouse Oct4, Sox2, Klf4 and c-Myc (OSKM) from a doxycycline (Dox)-inducible polycistronic construct, (b) PB-CAG-rtTA, encoding the reverse tetracycline transactivator (rtTA) and (c) pCAG-PBase, the expression vector for the transposase. Two independent reprogramming experiments were performed, each including at least three biological replicates from the indicated genotypes (with the exception of p53KO- MEFs, n = 2), with two independent transfections per replicate. Early passage MEFs from the indicated genotypes (WT, p73KO, p53KO and DKO) were transfected with the Neon electroporation device (ThermoFisher, MA, USA), according to the manufacturers instruction. Briefly, 200 000 cells (up to passage 4) were electroporated with 1 μg PB-TET-mOSKM or PB-TET-mOSK, 0.5 μg PB-rtTA and 0.5 μg pCAG-PBase plasmid. Expression of the transgenes was induced the following day by treatment with 1.5 μg/ml Doxycycline (Dox) in iPSC media (DMEM supplemented with 15% FBS, 2 mM L-Glutamine, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol and 1000 U/ml leukemia inhibitory factor, LIF). Colonies were manually picked and cultured on mouse fibroblast-inactivated feeder cells in iPSC media without Dox. Alkaline phosphatase (AP) staining was performed with Alkaline Phosphatase Detection Kit (Merck, Darmstadt, Germany) according to the manufacturer’s instructions. Transgene expression was confirmed in MEFS from the four genotypes by β-galactosidase activity (genes encoding OSKM were linked to a lacZ reporter). Western blot analysis. Immunoblot was performed as previously described54 with the following primary antibodies: rabbit anti-pSmad1/5 (Ser463/465) 1:1000 (Cell Signaling), rabbit anti-HA (Y11) 1:1000 (Santa Cruz Biotechnology, TX, USA), rabbit anti-actin (20–33) 1:10 000 (Sigma, MO, USA), followed by the appropriate HRP-conjugated secondary antibodies (Pierce). The enhanced chemiluminiscence was detected with Super Signal West-Pico Chemiluminiscent Substrate (Pierce). Luciferase assay. A plasmid containing a fragment of the mouse Smad6 promoter, inserted into pGL2-Basic vector (-3231-Luc),37 was kindly provided by Dr. Kato and Dr. Miyazono (University of Tsukuba, Japan). This reporter construct contains the putative p53-RE identified in the mouse Smad6 promoter region. P19 cells were transfected with 0.125 μg of the -3123-luc-pGL2-basic, 0.0625 μg of pRLNull renilla and 0.6 μg of either pcDNA3-HA-TAp73α, pcDNA3-HA-ΔNp73α or pcDNA3 expression vectors, using Lipofectamine™2000 Transfection Reagent (Invitrogen) following the manufacturer's protocol. For the co-transfection experiments, P19 cells were co-transfected with a fixed amount of TAp73 (0.2 μg) and different amounts of DNp73 from 0.2 to 0.6 μg. Discussion Cells were harvested and embryoid bodies (EBs) were prepared by the hanging drop procedure as previously described.52 Briefly, 20 μl-cell suspension drops (30 000 cell/ml in iPSC medium without LIF) were cultured for 4 days hanging from the lid of a Petri dish. EBs were then flushed down with EB medium and kept in suspension in bacteriological dishes for 3 additional days. Then, EBs were transferred to 0.1% gelatin-coated tissue culture plates and media was changed every 2 days. After 15 days, cells were fixed with 4% para-formaldehyde and used for further analysis. The protein-DNA cross-links were reversed in elution buffer (1% SDS and 50 mM Tris-HCl), followed by RNase treatment overnight at 65 °C. The eluted material was incubated with proteinase K for 3 h at 45 °C, and the DNA was purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany). Real-time qPCR was performed using SYBR Green PCR kit (Bio-Rad, CA, USA) in a StepOnePlus Real- Time PCR System (Applied Biosystems). The signals were normalized to the input (non-immune rabbit IgG immunoprecipitation). Primers encompassing the p53-RE in the human SMAD6 promoter were as follows: 5′-CACTTTGGGAGGCTAGGG and 5′-CCGCCAAGTAGCTGGAAC (amplicon, 150 bp). Primers encompassing the non- specific binding site (control) in the human SMAD6 promoter were as follows: 5′-GGACCAATCCCGACTTTACA and 5′-TAGGTGAGGGATCACGCTTT (amplicon, 218 bp). Primers encompassing the p53-RE in the mouse SMAD6 promoter were as follows: 5′-CAGGCAGGGAACTCTTTCAG and 5′-GTAGCTGGCAACCACCATTA (amplicon, 152 bp). Immunostaining. Immunofluorescence was performed as described.33 The following primary antibodies and dilutions were used: rat anti-CD31 1 : 1000 (BD, Franklin Lakes, NJ, USA), mouse anti-Tuj-1 1 : 1000 (Covance, Princeton, NJ, USA), mouse anti-AFP 1 : 1000 (Inmunostep, Salamanca, Spain), rabbit anti-Nanog 1:1000 (Chemicon, Billerica, MA, USA), mouse anti-SSEA-1 (MC-480) 1 : 100 (Pierce, Waltham, MA, USA), rabbit anti-E-cadherin 1 : 60 (Cell Signaling, MA, USA), mouse anti-E-cadherin 1:200 (Cell Signaling, Danvers, MA, USA), rabbit anti- β-catenin 1 : 200 (BD). Secondary antibodies were: Alexa 647 goat anti-IgG rabbit (Molecular Probes, Eugene, OR, USA), Alexa 488 goat anti-IgG mouse (Molecular Probes), Alexa 568 donkey anti-IgG rat (Molecular Probes), Cy3 donkey anti-IgG rabbit (Jackson Immunoresearch, West Grove, PA, USA) and FITC donkey anti-IgG mouse (Jackson Immunoresearch). Images were obtained with NIKON Eclip- seTE2000 and ZEISS LSM 800 confocal microscope. Sequence analysis and statistical analyses. Discussion Mouse Embryonic Fibroblasts were isolated from 13.5 days postcoitum embryos of the above-mentioned genotypes using standard procedures. For the analysis of cell growth rates, early passage cells derived from littermates were seeded at a concentration of 10 000 cells per cm2 (for daily counts) or 20 000 cells per cm2 (for serial passages following a 3T3 protocol). For experiments with P19 and H1299 cells involving BMP4 treatment, cells were serum-deprived (0.2% FBS) for 24 h, before the treatment with 0.5 to 50 ng/ml human BMP4 (Peprotech, Rocky Hill, NJ, USA) as indicated. Transfection of P19 cells was performed using Lipofectamine™2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's instructions. Eighteen hours after transfection, cells were serum-deprived as described and cells were treated with 5 ng/ml BMP4. Reprogramming of mouse embryonic fibroblast cells and culture of iPSCs. Mouse iPSCs were generated using the piggyBac (PB) transposition system, as previously described.23 The PB transposon vectors were y y p g Taken together, our data reveal that p73 is a positive modulator of the BMP circuit, required for BMP-induced MET Cell Death and Disease p73 modulates BMP signaling during reprogramming M Martin-Lopez et al p73 modulates BMP signaling during reprogramming M Martin-Lopez et al p73 modulates BMP signaling during reprogramming M M ti L t l 11 PCR in a StepOnePlus Real-Time PCR System (Applied Biosystems) using FastStart Universal SYBR Green Master (ROX) (Roche). All protocols were performed according to the manufacturer´s instructions. Primers sequences and conditions were described before53 and are indicated in the Supplementary Table 1. mRNA expression levels were calculated according to the formula: relative expression of gene = 2^(Ct internal reference-Ct gene), using 18S mRNA expression as internal reference. kindly provided by Dr Hämäläinen (Biomedicum Stem Cell Centre, Helsinki, Finland) including: (a) PB-TET-OSKM-IRES-βgeo, for the expression of mouse Oct4, Sox2, Klf4 and c-Myc (OSKM) from a doxycycline (Dox)-inducible polycistronic construct, (b) PB-CAG-rtTA, encoding the reverse tetracycline transactivator (rtTA) and (c) pCAG-PBase, the expression vector for the transposase. Two independent reprogramming experiments were performed, each including at least three biological replicates from the indicated genotypes (with the exception of p53KO- MEFs, n = 2), with two independent transfections per replicate. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 31. David L, Polo JM. Phases of reprogramming. Stem Cell Res 2014; 12: 754–761. 32. Tanabe K, Nakamura M, Narita M, Takahashi K, Yamanaka S. Maturation, not initiation, is the major roadblock during reprogramming toward pluripotency from human fibroblasts. Proc Natl Acad Sci USA 2013; 110: 12172–12179. 1. Chambers I, Smith A. Self-renewal of teratocarcinoma and embryonic stem cells. Oncogene 2004; 23: 7150–7160. 33. Fernandez-Alonso R, Martin-Lopez M, Gonzalez-Cano L, Garcia S, Castrillo F, Diez-Prieto I et al. p73 is required for endothelial cell differentiation, migration and the formation of vascular networks regulating VEGF and TGFbeta signaling. Cell Death Differ 2015; 22: 1287–1299. 2. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126: 663–676. 3. Avior Y, Sagi I, Benvenisty N. Pluripotent stem cells in disease modelling and drug discovery. Nat Rev Mol Cell Biol 2016; 17: 170–182. 34. Redmer T, Diecke S, Grigoryan T, Quiroga-Negreira A, Birchmeier W, Besser D. E-cadherin is crucial for embryonic stem cell pluripotency and can replace OCT4 during somatic cell reprogramming. EMBO Rep 2011; 12: 720–726. 4. Velasco I, Salazar P, Giorgetti A, Ramos-Mejia V, Castano J, Romero-Moya D et al. Concise review: Generation of neurons from somatic cells of healthy individuals and neurological patients through induced pluripotency or direct conversion. Stem Cells 2014; 32: 2811–2817. 35. Rosowski KA, Mertz AF, Norcross S, Dufresne ER, Horsley V. Edges of human embryonic stem cell colonies display distinct mechanical properties and differentiation potential. Sci Rep 2015; 5: 14218. 5. Ramos-Mejia V, Fraga MF, Menendez P. iPSCs from cancer cells: challenges and opportunities. Trends Mol Med 2012; 18: 245–247. 36. Korchynskyi O, ten Dijke P. Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. J Biol Chem 2002; 277: 4883–4891. 6. Samavarchi-Tehrani P, Golipour A, David L, Sung HK, Beyer TA, Datti A et al. Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 2010; 7: 64–77. 37. Ishida W, Hamamoto T, Kusanagi K, Yagi K, Kawabata M, Takehara K et al. Smad6 is a Smad1/5-induced smad inhibitor. Conflict of Interest 22. Lin Y, Cheng Z, Yang Z, Zheng J, Lin T. DNp73 improves generation efficiency of human induced pluripotent stem cells. BMC Cell Biol 2012; 13: 9–9. The authors declare no conflict of interest. p p 23. Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hamalainen R et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 2009; 458: 766–770. Acknowledgements. We thank Dr. Manuel Serrano (Institute for Research in Biomedicine, Barcelona, Spain) for kindly providing the Rep-MEFs. This work was supported by Grants SAF2012-36143 and SAF2015-71381-R from Spanish Ministerio de Economía y Competitividad and cofinanced by FEDER funds, and Grant LE310U14 from Junta de Castilla y León (to MCM.), and by Grant 257157 from the Academy of Finland (to TO). Work in P.M lab is supported by the European Research Council (CoG-2014-646903) and the Spanish Ministry of Economy- Competitiveness (SAF-SAF2013-43065) to PM. PM acknowledges the financial support from the Obra Social La Caixa-Fundaciò Josep Carreras, and Generalitat de Catalunya. PM is investigator of the Spanish Cell Therapy cooperative network (TERCEL). MML and SFA were holders of predoctoral fellowships from the Junta de Castilla y León. LMA is supported by a predoctoral scholarship from the Asociación Española contra el Cáncer (AECC). 24. Meissner A, Wernig M, Jaenisch R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol 2007; 25: 1177–1181. 25. Maherali N, Hochedlinger K. Guidelines and techniques for the generation of induced pluripotent stem cells. Cell Stem Cell 2008; 3: 595–605. 26. Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 2008; 26: 101–106. 27. Hanna J, Saha K, Pando B, van Zon J, Lengner CJ, Creyghton MP et al. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 2009; 462: 595–601. 28. Abad M, Mosteiro L, Pantoja C, Canamero M, Rayon T, Ors I et al. Reprogramming in vivo produces teratomas and iPS cells with totipotency features. Nature 2013; 502: 340–345. 29. Venkatanarayan A, Raulji P, Norton W, Chakravarti D, Coarfa C, Su X et al. IAPP-driven metabolic reprogramming induces regression of p53-deficient tumours in vivo. Nature 2015; 517: 626–630. 30. Buganim Y, Faddah DA, Cheng AW, Itskovich E, Markoulaki S, Ganz K et al. Publisher’s Note Cheung AKL, Phoon YP, Lung HL, Ko JMY, Cheng Y, Lung ML Roles of T 14. Marion RM, Strati K, Li H, Murga M, Blanco R, Ortega S et al. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature 2009; 460: 1149–1153. 44. Meletis K, Wirta V, Hede SM, Nister M, Lundeberg J, Frisen J. p53 suppresses the self- renewal of adult neural stem cells. Development 2006; 133: 363–369. 44. Meletis K, Wirta V, Hede SM, Nister M, Lundeberg J, Frisen J. p53 su 15. Utikal J, Polo JM, Stadtfeld M, Maherali N, Kulalert W, Walsh RM et al. Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature 2009; 460: 1145–1148. 45. Gonzalez-Cano L, Herreros-Villanueva M, Fernandez-Alonso R, Ayuso-Sacido A, Meyer G, Garcia-Verdugo JM et al. p73 deficiency results in impaired self renewal and premature neuronal differentiation of mouse neural progenitors independently of p53. Cell Death Dis 2010; 1: e109. 16. Kareta MS, Gorges LL, Hafeez S, Benayoun BA, Marro S, Zmoos AF et al. Inhibition of pluripotency networks by the Rb tumor suppressor restricts reprogramming and tumorigenesis. Cell Stem Cell 2015; 16: 39–50. 46. Talos F, Nemajerova A, Flores ER, Petrenko O, Moll UM. p73 suppresses polyploidy and aneuploidy in the absence of functional p53. Mol Cell 2007; 27: 647–659. g 17. Collavin L, Lunardi A, Del Sal G. p53-family proteins and their regulators: hubs and spokes in tumor suppression. Cell Death Differ 2010; 17: 901–911. 47. Gonzalez-Cano L, Fuertes-Alvarez S, Robledinos-Anton N, Bizy A, Villena-Cortes A, Farinas I et al. p73 is required for ependymal cell maturation and neurogenic SVZ cytoarchitecture. Dev Neurobiol 2016; 76: 730–747. pp 18. Li H, Collado M, Villasante A, Strati K, Ortega S, Canamero M et al. The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 2009; 460: 1136–1139. barrier for iPS cell reprogramming. Nature 2009; 460: 1136–1139. 48. Liu P, Jenkins NA, Copeland NG. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res 2003; 13: 476–484. 19. Alexandrova EM, Talos F, Moll UM. p73 is dispensable for commitment to neural stem cell fate, but is essential for neural stem cell maintenance and for blocking premature differentiation. Cell Death Differ 2013; 20: 368–368. 49. Lewandoski M, Wassarman KM, Martin GR. Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line. Conflict of Interest Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell 2012; 150: 1209–1222. Discussion In silico prediction of putative p53-response elements (p53-RE) within the human SMAD6 promoter was performed with the p53FamTaG data base.40 Sequence alignments, either between the identified p53-RE and the human SMAD6 gene (ENSG00000137834) or the mouse Smad6 (ENSMUSG00000036867) gene, were carried out using the ‘Pairwise sequence alignment’ (EMBOSS Needle) software, available from the European Bioinformatics Institute. RNA isolation and real-time qRT-PCR analysis. Total RNA from cultured cells was isolated using TRI reagent solution (Ambion, TX, USA) and purified with a Nucleospin RNA cleanup kit (Macherey-Nagel, Düren, Germany), according to the manufacturer’s instructions. DNase treatment was performed separately using RQ1 DNase (Promega, WI, USA) in the presence of Protector RNase inhibitor (Roche, Basel, Switzerland). First strand cDNA was synthesized using up to 2 μg of total RNA and the High Capacity RNA-to-cDNA kit (Applied Biosystems, Carlsbad, CA, USA). Gene expression was analyzed by real-time qRT- Statistical analyses were performed using the Student’s two-tailed t-test. Values with Po0.05 were considered statistically significant (Po0.05, Po0.01, *Po0.005). The mean ± S.E.M. of each value is represented. Figure legends include, when necessary, specific details regarding the number of replicates and independent replicates. Cell Death and Disease p73 modulates BMP signaling during reprogramming M Martin-Lopez et al p73 modulates BMP signaling during reprogramming M Martin-Lopez et al p73 modulates BMP signaling during reprogramming M Martin-Lopez et al 12 Conflict of Interest The authors declare no conflict of interest. Conflict of Interest The authors declare no conflict of interest. Conflict of Interest The authors declare no conflict of interest. Publisher’s Note Characterization of bone morphogenetic protein- responsive element in the mouse Smad6 promoter. J Biol Chem 2000; 275: 6075–6079. 7. Polo JM, Hochedlinger K. When fibroblasts MET iPSCs. Cell Stem Cell 2010; 7: 5–6. 7. Polo JM, Hochedlinger K. When fibroblasts MET iPSCs. Cell 8. Hayashi Y, Hsiao EC, Sami S, Lancero M, Schlieve CR, Nguyen T et al. BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence. Proc Natl Acad Sci USA 2016; 113: 13057–13062. 38. Liu G, Nozell S, Xiao H, Chen X. ΔNp73β is active in transactivation and growth suppression. Mol Cell Biol 2004; 24: 487–501. 9. Li R, Liang J, Ni S, Zhou T, Qing X, Li H et al. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 2010; 7: 51–63. 39. Langenfeld E, Hong CC, Lanke G, Langenfeld J. Bone morphogenetic protein type I receptor antagonists decrease growth and induce cell death of lung cancer cell lines. PloS One 2013; 8: e61256. 10. Goding CR, Pei D, Lu X. Cancer: pathological nuclear reprogramming? Nat Rev Cancer 2014; 14: 568–573. 40. Sbisà E, Catalano D, Grillo G, Licciulli F, Turi A, Liuni S et al. p53FamTaG: a database resource of human p53, p63 and p73 direct target genes combining in silico prediction and microarray data. BMC Bioinformatics 2007; 8: S20–S20. 11. Takahashi K, Yamanaka S. A decade of transcription factor-mediated reprogramming to pluripotency. Nat Rev Mol Cell Biol 2016; 17: 183–193. 41. Marques-Garcia F, Ferrandiz N, Fernandez-Alonso R, Gonzalez-Cano L, Herreros- Villanueva M, Rosa-Garrido M et al. p73 plays a role in erythroid differentiation through GATA1 induction. J Biol Chem 2009; 284: 21139–21156. 12. Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, Nakagawa M et al. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 2009; 460: 1132–1135. 42. Qin H, Diaz A, Blouin L, Lebbink Robert J, Patena W, Tanbun P et al. Systematic identification of barriers to human iPSC generation. Cell 158: 449–461. 13. Kawamura T, Suzuki J, Wang YV, Menendez S, Morera LB, Raya A et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 2009; 460: 1140–1144. 43. Cheung AKL, Phoon YP, Lung HL, Ko JMY, Cheng Y, Lung ML Roles of Tumor Suppressor Signaling on Reprogramming and Stemness Transition in Somatic Cells 2013. 43. Publisher’s Note Curr Biol 1997; 7: 148–151. 20. Choi YJ, Lin CP, Ho JJ, He X, Okada N, Bu P et al. miR-34 miRNAs provide a barrier for somatic cell reprogramming. Nat Cell Biol 2011; 13: 1353–1360. 21. Brosh R, Assia-Alroy Y, Molchadsky A, Bornstein C, Dekel E, Madar S et al. p53 counteracts reprogramming by inhibiting mesenchymal-to-epithelial transition. Cell Death Differ 2013; 20: 312–320. 50. Nishimura K, Sano M, Ohtaka M, Furuta B, Umemura Y, Nakajima Y et al. Development of defective and persistent Sendai virus vector: a unique gene delivery/expression system ideal for cell reprogramming. J Biol Chem 2011; 286: 4760–4771. Cell Death and Disease p73 modulates BMP signaling during reprogramming M Martin-Lopez et al 13 Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 51. Okano S, Yonemitsu Y, Nagata S, Sata S, Onimaru M, Nakagawa K et al. Recombinant Sendai virus vectors for activated T lymphocytes. Gene Therapy 2003; 10: 1381–1391. 52. Li X, Claesson-Welsh L, Shibuya M. VEGF receptor signal transduction. Methods Enzymol 2008; 443: 261–284. 53. Tomasini R, Tsuchihara K, Wilhelm M, Fujitani M, Rufini A, Cheung CC et al. TAp73 knockout shows genomic instability with infertility and tumor suppressor functions. Genes Dev 2008; 22: 2677–2691. 54. Fernandez-Garcia B, Vaque JP, Herreros-Villanueva M, Marques-Garcia F, Castrillo F, Fernandez-Medarde A et al. p73 cooperates with Ras in the activation of MAP kinase signaling cascade. Cell Death Differ 2007; 14: 254–265. 55. Vaque JP, Fernandez-Garcia B, Garcia-Sanz P, Ferrandiz N, Bretones G, Calvo F et al. c-Myc inhibits Ras-mediated differentiation of pheochromocytoma cells by blocking c-Jun up- regulation. Mol Cancer Res 2008; 6: 325–339. r The Author(s) 2017 Supplementary Information accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis) Supplementary Information accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis) plementary Information accompanies this paper on Cell Death and Disease website (http://www.natur ion accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis) Cell Death and Disease Cell Death and Disease
https://openalex.org/W4385331881	https://www.jomswsge.com/pdf-168382-92622?filename=A good restorative.pdf	English	null	A good restorative justice practice in an italian school	Journal of Modern Science	2,023	cc-by-sa	7,174	GIUSEPPE LIVERANO JOURNAL OF MODERN SCIENCE TOM 2/51/2023 www.jomswsge.com DOI: https://doi.org/10.13166/jms/168382 Giuseppe Liverano University of Bari Aldo Moro, Italy giuseppe.liverano@uniba.it ORCID: 0000-0003-1011-0054 Keywords: Restorative justice, Education, Training, Skills, Behavioural transformation, Inclusion Abstract School is the formal context deputed to the education and learning of children, but it is also the first and most important place for socialisation with non-chosen peers and non-neighbouring adults. From these premises one can only conclude that pupils and students who display behaviour that transgresses the rules of civil and democratic coexistence manifest an educational need. Just as error is an opportunity for learning, a school incapable of inclusion would manifest a lack of planning and an inability to value the uniqueness of each one. In the direction of promoting transformative educational actions from a constructive point of view, of the errors committed, an italian school, has developed a project aimed at transforming the transgression of a school rule into an opportunity for individual and systemic-relational growth. The contribution highlights briefly, the salient aspects of the project and the results in terms of learning some competences for life through the approach of committed pedagogy oriented towards inclusion. Wyższa Szkoła Gospodarki Euroregionalnej im. Alcide De Gasperi w Józefowie 288 A GOOD RESTORATIVE JUSTICE PRACTICE IN AN ITALIAN SCHOOL 1. Introduction Daily national and international news reports show, with much greater frequency, episodes of violence at school, aggressive and antisocial behaviour, bullying, attitudes that systematically violate school rules (tardiness, vandalism, unjustified absences), by pupils of all ages, often caused by toxic relationships between pupils and between pupils and teachers or by excessive aggression and marginality (De Laet et al. 2015; Rudasill et al., 2010 ). The world of education, with respect to this problem, reacts by implementing, almost invariably, zero-tolerance policies that by using punitive and exclusionary practices aim to control and manage pupils’ behaviour (Lodi et al., 2021, p.1). These policies exacerbate disciplinary problems and exacerbate racial, gen der and socioeconomic status disparities (Gwathney, 2021; Ingraham et al., 2016), emphasising the need for alternative approaches to school discipline management, as well as approaches that aim to promote wellbeing through out the school community. Thus, where education was supposed to explode as a liberating force, it turns out, instead, to be a mechanism with high po tential for domination and power (Hooks, 2020a, pp. 33-34). Many schools, reflecting on the failure of such punitive practices from the point of view of educational outcomes, are increasingly trying to use restorative practices to implement an educational project that, through a multi-level approach, can produce a significant impact in terms of sustainability in the relationships within a complex community such as the school and the whole environment (McCold, 2008) and a more qualitative presence of each individual subject. The aim is to transform conflicts, misbehaviour that have occurred within a school context into opportunities for transversal growth through the recovery and development of strong bonds, a unique and unrepeatable learning experience that each subject has the opportunity to have (Hooks, 2020a), a sense of com munity, built on values such as trust, responsibility, and the well-being of the subjects involved. Removing weak individuals who repeatedly commit school offences from school would be tantamount to admitting the school’s total lack of planning and the failure of the educational alliance with families and the local area, which, together, are responsible for educating and supporting the educational success of each pupil and student amidst difficulties that are Journal of Modern Science 2/51/2023 289 GIUSEPPE LIVERANO increasingly complex to manage. 1. Introduction One must therefore ask oneself whether a system of sanctions aimed at removing children from school, forcing them to stay at home where, due to particular family situations, it is difficult for them to improve their behaviour and education, is legitimate. In the same way, it is legitimate to ask oneself whether it is sufficient to remove children from school to make amends for bad behaviour, or whether, on the other hand, the school, as an educating community, should re-establish a positive relationship between the one who caused an injury or offence and the one who received it. Implementing a restorative justice educational project, in a system still based on punitive reintegration logics can represent the real educational transgression (Hooks, 2020a) able to produce a transformation of behaviour, to foster the understanding of certain educational values and the development of life skills capable of promoting effective and sustainable relationships within the school community. The restorative justice paradigm, in fact, does not simply aim to help those who have received an offence but is concerned with elaborating a path of reintegration for those who have committed wrongdoing (Wright, 2002), therefore it pursues educational goals. This paradigm, com bining the fundamental elements of ecological systems theory, which argues that individual behaviour is determined by multiple causes and is sensitive to multiple influences from the microsystems in which each subject moves and operates (Broffenbrenner, 2005) and the psychology of affect (Higgins, 1987) proposes a model in which restorative practices help to develop strong bonds and offer the opportunity for the student to value his/her presence, express his/her emotions, to live the school experience with pure joy (hooks, 2020a), to take public responsibility for his/her behaviour and to reintegrate into the school community (Acosta et al., 2019). An error-repair project, therefore, if it is to be educational, must promote an opportunity for individual and sys temic growth, which aims to enhance the social-relational dimension “with the broader goal of building safe school communities that promote well-being” (Lodi et al., 2021, p. 1). On the basis of these concepts, the following pages will describe the salient features of a project in which restorative practices provided the possibility for different forms of marginality to be transformed into radical creative possibilities and space par excellence (Hooks, 2020a). Wyższa Szkoła Gospodarki Euroregionalnej im. Alcide De Gasperi w Józefowie 290 A GOOD RESTORATIVE JUSTICE PRACTICE IN AN ITALIAN SCHOOL 2. Educational restorative justice for a more make sustainable school The case that we want to highlight through this contribution describes an educational project started in 2019, which the headmaster of the Istituto com prensivo Lentini-Einstein in Mottola, in the province of Taranto, strongly wanted to implement in his school, as an alternative proposal to the classic punishment, due to problematic behaviour repeatedly manifested by the students. For some time there had been episodes in which pupils had been involved in acts of bullying, offensive phrases even towards teachers, defacement of the school gardens, homophobic and xenophobic writing on the walls inside and out side the school. But what had strongly motivated the school’s management to adopt a different approach from that of suspension with stay-at-home were the phrases that the punished pupils punctually expressed immediately after the announcement of the sanction for the weekend: ‘Thank you, headmaster, you really needed it’! The school, therefore, had to find the courage to go against the tide, capable of intercepting students’ needs but also of being attentive to their subjective dimension without jeopardising its social mission, its ethical, inclusive and sustainability value (Riva, 2020). As a choice to be made to try to transform a complex situation into a constructive, but equally joyful and exciting practice, the school decided to naturally converge towards a comprehensive and complete pedagogical approach (Acosta et al., 2019) that was not only to address the single verified problem, but through an experience in which the subject could talk about his or her own history and make use of this experience in a systemic-relational perspective on several levels, was to:f 1. Improving the relationship between school staff and students, paying atten tion to the language used so as not to use offensive words and expressions; 2. Contribute to generating a cultural change aimed at student under standing and awareness of error, so as to foster the construction of a model of a peaceful and exciting school; 3. Improving the relationship with civil society, which must take charge of permanent and constant education; 3. Improving the relationship with civil society, which must take charge of permanent and constant education; 4. Improving the quality of school life through a more meaningful and productive presence both individually and socially; Journal of Modern Science 2/51/2023 291 GIUSEPPE LIVERANO 5. 2. Educational restorative justice for a more make sustainable school Increasing the value of the educational success of so-called ‘fragile’ pu pils in terms of performance in the study subjects and transversal skills; 6. Developing the willingness and ability to want to correct a behaviour, in a free and spontaneous way in order to think critically about oneself, as an act of resistance and opposition to the habit of letting oneself go (Hooks, 2020a, p. 32-33), through the involvement of all school staff called upon to support and encourage students since enthusiasm is generated by a collective effort (Hooks, 2020a); 7. Respect clear and consistent rules, and above all aimed at not privi leging anyone but ensuring fairness of treatment, which is known to produce greater effectiveness (Thapa et al., 2013; Thornberg, et al., 2018), to assume responsible and above all conscious behaviour; 8. Making the community more sustainable in terms of relationships also includes the elimination of all forms of inequality, as a supportive and healthy climate supports students to develop higher levels of assertive ness, empathy and other social skills (Were, 2017). Indeed, it is well known that restorative practices with a comprehensive approach and with the involvement of all actors in a school context produce significant improvements in both attitudes (social skills and self-esteem of participants) and behavioural measures (delinquency, disciplinary referrals, school performance, graduation rates) also in relation to the duration of in terventions (McCold, & Wachtel, 2002). An intervention that is restorative of the individual student, but transformative for the entire school community, represents a democratic practice that everyone converges towards because it represents a model of educational practice that can highlight how, more partic ipative and more serene students are more likely to change their behaviour in a positive direction also as a result of better collaboration with those in charge of the school (Wachtel, 2016 ). In this specific case, the aim of the Einstein- Lentini Comprehensive school was to implement an intervention that would help its students develop social and emotional skills to facilitate the re-es tablishment of relationships, to improve collaboration, and to create a sense of belonging to a community, in which every existence is intimately linked to that of others. These elements are fundamental for improving behaviour Wyższa Szkoła Gospodarki Euroregionalnej im. 2. Educational restorative justice for a more make sustainable school Alcide De Gasperi w Józefowie 292 A GOOD RESTORATIVE JUSTICE PRACTICE IN AN ITALIAN SCHOOL in the classroom and in the community as a whole, school performance, and for fostering quality attendance, peaceful coexistence and well-being (Drewery, 2014; Morrison, 2015). The proposal was also promoted so that the entire school community, through a collective effort, could draw out from each pupil the skills to improve the liveability and well-being within the school commu nity and the ability to collaborate in educational activities within the school and in civil society, so as to make each pupil capable of interacting and being productive in a perspective of sustainability. Activating the partnership with local stakeholders meant for the school to seek new proximity collaborations for the children’s education and to build a single body with it, understood as a space for educational reflection. Relating with external agencies became an opportunity to come out of an isolation that prevented it from performing its educational task in the best possible way, to improve its social function and to give the school assessment process the chance to fulfil a global task, not to limit it to being a thermometer that only measures the level of learning of contents, but to authentically assess the maturity of the children, their sense of citizenship, their ability to be responsible in building better environments in which they live in a shared and democratic way. The proposal was brought to the attention of the collegiate bodies of the comprehensive school and then to civil society to promote short and long-term actions that included the dissemination and acceptance of the project proposal, training meetings with teachers, students and students’ families, training meetings with the students’, teachers’ and students’ parents’ representatives, the drawing up of the table of ‘critical actions’ to which corresponded in a restorative perspective the relevant educational actions to be freely chosen, the certification attesting to the social commitment of solidarity, care, sustainability by a mixed evaluation group made up of teach ers, students and students’ parents. In essence, the educational intervention comprised five moments considered essential: 1. Boys who had committed ‘critical’ actions of any kind were given the opportunity to speak so that they could hear what they had to say;h 2. 2. Educational restorative justice for a more make sustainable school The moment of awareness of the critical action committed through an educational action that was not to turn out to be a humiliating and frustrating moment for the pupil but an emancipating practice, Journal of Modern Science 2/51/2023 293 GIUSEPPE LIVERANO an educational act that liberates and heals, a work of self-definition in which the generative identity is in continuous becoming never sepa rated from that of others [...] so that the sharing of the experience could create awareness that translates into a clear conception of committed pedagogy [...] in an individual and collective movement (Bocci et al., 2022, p. 77-78), a privileged time and space in which to demonstrate an understanding of who has been harmed and how (Thorsborne & Vinegrad, 2008, p. 29 );h an educational act that liberates and heals, a work of self-definition in which the generative identity is in continuous becoming never sepa rated from that of others [...] so that the sharing of the experience could create awareness that translates into a clear conception of committed pedagogy [...] in an individual and collective movement (Bocci et al., 2022, p. 77-78), a privileged time and space in which to demonstrate an understanding of who has been harmed and how (Thorsborne & Vinegrad, 2008, p. 29 );h 3. The school had to adopt an attitude not of exclusion and punishment but of understanding, affection, closeness and inclusion, and remedial action had to be based on these principles;h 4. The sustainability assessment of the remedial action performed by the pupil; 5. The young people were given the opportunity to narrate the learning experience, as through the telling of their version of the story they used what S. Kalogeras calls an ancient human technology (Kalogeras, 2013), the therapeutic, formative, orientative and transformative power of which is also known from a systemic-relational perspective (Batini 2010; Cavell, 2010; Batini 2015, 2019). The first moment was considered fundamental to improving the quality of relations between the different actors in the school community and, in general, the quality of the experience, as it was crucial that the pupils under stood the usefulness and educational function of what they had to do and not interpret it as an exercise of power by the management and an attempt to publicly humiliate them. Supporting the Student voice paradigm (Flutter & Ruddock, 2004, p. 2. Educational restorative justice for a more make sustainable school 135) was the best and most democratic way for the school to facilitate dialogue and rapprochement between the parties as well as the best way to handle conflicts between students where there had been verbal and non-verbal offence. In such cases, it sometimes becomes difficult to establish who is right and who is not. The possibility of a debate between those who allegedly suffered the offence, those who committed it, the teachers and the management can facilitate the interpretation and understanding of how the events occurred. Moreover, recounting what had happened became Wyższa Szkoła Gospodarki Euroregionalnej im. Alcide De Gasperi w Józefowie 294 A GOOD RESTORATIVE JUSTICE PRACTICE IN AN ITALIAN SCHOOL an opportunity for teachers to help their pupils also to develop skills in argu mentation, managing emotions, listening to the thoughts of others, expres siveness, time management, welcoming diversity, and understanding the reasons and feelings of others. This procedure, therefore, was considered the most suitable to restore harmony and balance in relations between students and between students and teachers. In order to support a systemic-relational change perspective, the involvement of all the school staff and actors outside the school context, appropriately and previously trained, to educate pupils to have proactive, pro-social attitudes, and to manage the possible embar rassment arising from the pupil’s exposure to an unforeseen educational experience, was fundamental in order not to disregard the expectations and objectives of the action (Acosta et al., 2019). Moreover, having attributed some of the pupils’ misbehaviour to a complex problem that also included a lack of affection in the pupils and of extracurricular educational support, the school had decided to adopt, for these particular cases, an approach geared towards affectivity and the promotion of the pupils’ sense of self-efficacy and esteem, the management of their emotions, through continuous expressions of en couragement to transform attitudes, language and behaviour, some of them being already very much affected by feelings of shame and frustration. Table 1 describes the main elements of the remedial actions carried out by the pupils over three years of the project and reported in the teachers’ evaluation rubric. Table 1. Skills assessment reporting Pupil type Critical action committed Selected restorative action Type of educational intervention Mode of activity Learner-reported learning outcomes and competences No. 2. Educational restorative justice for a more make sustainable school 3 pupils 1st and 2nd year secondary school Verbal and physical bullying against fellow students Care and support activities in a family home for minors Care education Pupils engaged in childcare activities Reduced misconduct, better academic performance, caring skills and hosting skills No. 2 pupils 1st and 3rd year secondary school Verbal and physical bullying against fellow students Care and support activities in a children’s home Education for reading, reception and care The pupil engaged in book reading activities with minors Improved inclusion and welcoming practices; improved social skills (e.g. empathy, awareness and responsibility); pro-social behaviour; Table 1. Skills assessment reporting Journal of Modern Science 2/51/2023 295 GIUSEPPE LIVERANO GIUSEPPE LIVERANO No. 2 pupils 3rd and 4th year sup. sec. Classroom vandalism with destruction of desks Restoration and carpentry activities in a family home and school Manual skills education Pupils repaired desks they had completely destroyed with the help of school staff Reduction of misconduct (e.g. bullying); less use of exclusionary disciplinary measures (e.g. notes and suspensions); improvement of social and emotional skills (e.g. empathy, awareness and responsibility); promotion of fairness; pro-social and positive behaviour; No. 1 pupil 4th year Upper Sec. Act of vandalism to theatre workshop equipment in particular to the curtains in the theatre workshop room Accompaniment in the perfor mance activities of a community for minors at social risk Emotional education The pupil together with other classmates and some school staff created new tents Reducing deviant behaviour, developing responsibility and caring attitudes No. 1 pupil 3rd year Upper Sec. Act of vandalism to library books. The pupil scribbled several books Reading, archiving and book lending activities in the municipal library Social and emotional learning through book illustrations The pupil was accompanied in reading and reflection activities and emotional management Improved social skills (e.g. empathy, awareness and responsibility); improved emotional skills, attitudes, academic performance; development of responsibility and caring No. 4 pupils 1st, 2nd and 4th year sup. sec. Act of littering and defacing public green spaces by pupils caught littering and kicking up flower beds School gardening and landscaping activities Environmental and Citizenship Education Pupils restored defaced greenery and engaged in landscaping activities accompanied by school staff Reflection, understanding and implementation of the concept of sustainability, cultural transformation in schools, development of caring skills, citizenship skills No. A GOOD RESTORATIVE JUSTICE PRACTICE IN AN ITALIAN SCHOOL No. 3 1st and 2nd year pupils Religious verbal offence by pupils towards other pupils Care and support activities in a community for foreigners Intercultural Education Pupils were accompanied in care activities by children of other nationalities and without parents Inclusive culture and reducing inequalities; self-awareness; capacity building for peaceful conflict resolution; improving positive peer relations; promoting equity; valuing differences No. 6 pupils 2nd and 3rd year sup. sec. Public lewdness of 4 pupils caught in effusion in the gymnasium and 2 pupils in the bathroom Training activities at a social cooperative Sexuality education No. 4 pupils (2M and 2F) were caught lewdly in the gymnasium and no. 2 pupils in the bathroom Better management of emotions and feelings, greater sense of responsibility and awareness, greater sense of trust in the school community No. 4 pupils 4th year sup. sec. Defacing and defacing walls in a public place. The pupils were caught on school cameras defacing the walls of the building Restoration of school walls Citizenship Education The pupils were helped by school staff to restore the walls Development of responsibility and awareness, sense of belonging to a community, social and citizenship skills No. 4 pupils 3rd and 4th year secondary school Verbal offence of a sexual nature by some pupils towards fellow pupils Education activities on respect for diversity at a training institution Education on sexuality and diversity Pupils were trained with sex education activities Safer schools; inclusive culture and reducing inequalities; promoting equity; No. 4 pupils 3rd and 4th year sup. sec. Verbal insults of a physical nature by two pupils towards the Italian teacher and by two pupils towards a mathematics teacher Training activities at a training organisation Education in corporeity Pupils were trained through activities to educate them about their bodies and respect for diversity more positive school climate and better social relations between teachers and students conflict management; emotional and social skills Table 1. In skills assessment reporting, offenses committed, educational actions and skills explored are highlighted Table 1. In skills assessment reporting, offenses committed, educational actions and skills explored are highlighted The table shows that the pupils caught engaging in critical behaviour are predominantly male (35 out of 40). An interesting datum is that relating to the type of deviance into which the critical behaviour was falling. 2. Educational restorative justice for a more make sustainable school 6 4th and 5th grade pupils Drug abuse by pupils caught abusing soft drugs Care and support activities in a community for recovering drug addicts Education for care and solidarity Pupils were accompanied in drug abuse prevention activities and training on the effects on health, they also took care of young drug addicts Decreasing health risk behaviour, developing greater responsibility and awareness, building safe and engaging school communities Wyższa Szkoła Gospodarki Euroregionalnej im. Alcide De Gasperi w Józefowie 296 A GOOD RESTORATIVE JUSTICE PRACTICE IN AN ITALIAN SCHOOL In fact, out of 40 critical behaviours, 29 (72% in light gray) fall in the area of so cial deviance, while 6 (15% in dark gray) fall in the area of moral deviance, Journal of Modern Science 2/51/2023 297 GIUSEPPE LIVERANO 3 (about 7% in white) fall in the area of religious deviance and finally 2 (about 5% again in light gray) in the area of legal/social deviance (defacement of walls). With regard to the results that the remedial educational activities have produced, a more positive school climate, a greater sense of commu nity, better social relations and greater cooperation between the subjects are evident. Nevertheless, there is evidence of better individual scholastic performance of the pupils involved in the remedial actions, but also of the other pupils in the classes to which they belong. In other words, it seems that the action generated a widespread positive influence to the point of activating processes of cultural modification not only of the classes involved but of the entire school. With regard to competences, the pupils involved in the reparative actions, but also the others who indirectly played a role in the reintegration process, seem to have been able to develop social and emotional competences, of expressiveness, of awareness and responsibility, but above all of citizenship and care. Thus two boys, respectively from the 3rd D (high school of science with language specialisation) and 4th C (high school of science with applied science specialisation):h “The restorative practice experience made me realise that one can make mistakes, but that an approach aimed at understanding the fragility of children is useful in understanding the effects that wrong behaviour can have on others. Doing something for others with the help of the school and others in the area helped me to understand how important certain values are and to develop a greater capacity to live with others, to accept myself for who I am, not to be afraid to show my emotions and feelings. I felt welcomed, understood and accompanied throughout and today I feel an active part of this school community” (pupil 3 D). This repair experience helped me to transform my idea about my school. Today I realised that my school wants to value me and take care of my growth. I had the opportunity to develop a path that helped me bring out parts of me that I was repressing. A GOOD RESTORATIVE JUSTICE PRACTICE IN AN ITALIAN SCHOOL I will continue after school to engage in collaborative activities to help those in need. Through this experience, I have improved my ability to be with others, to accept those Wyższa Szkoła Gospodarki Euroregionalnej im. Alcide De Gasperi w Józefowie 298 A GOOD RESTORATIVE JUSTICE PRACTICE IN AN ITALIAN SCHOOL who are different from me. My school and the co-workers were like parents who took care of me (Pupil 4C). With regard to the critical issues and limitations encountered by the school, two in particular stand out: the non-total involvement of all the teachers, since a small number of them did not believe too much in the effects that an educational project of error repair can have, and the risk of transforming a practice into a self-referential propaganda tool for the school, which may be distracted from understanding what the real perception of the problem is and the importance of having to invest all its resources, in a collective sense, to achieve results. Poor management of the practice, moreover, could easily turn into an instrument of power that, instead of guaranteeing every pupil the possibility of an edifying educational experience, could expose critical pupils to frustrating and humiliating situations and, consequently, accentuate inequalities. 3. Discussion The results of the project proposal show how, in about three years of activ ity, about 40 critical behaviours were matched by just as many spontaneous initiatives of educational reflection that were able to act to develop serene relationships and strong bonds between children and between children and adults, wellbeing at school, transversal skills and the strengthening of so cial-relational skills, as well as greater autonomy, the development of identity, awareness and responsibility (Lerner et. al., 2011). In order to establish the type of reparation, with respect to the misbehaviour assumed, the pupil was invited to reflect on the act performed, a reflection that almost automatically turned into a request to repair the damage done. Thus to an offence of a xen ophobic nature the student’s choice almost always fell to doing a month of care and assistance at an association that hosted people from other nations, to a behaviour of taking soft drugs the choice was oriented towards volun teering at a rehabilitation community for drug addicts, a writing on the walls of the school corresponded to some weekends repainting the entire façade, Journal of Modern Science 2/51/2023 299 GIUSEPPE LIVERANO a defacement of the school gardens corresponded to a few weekends dedicated to gardening and cleaning the school grounds from all forms of organic and inorganic waste, the breaking of some school desks corresponded to a few weekends of DIY with wood, of course, with the complicity and support of the school staff. Finally, the moment when each student was given the opportunity to recount his or her reparative experience became a formative space in which individual and collective reading and reflection alternated fluidly and educated the subjects to acquire a narrative, observational, reflective, hermeneutic, transformative forma mentis (Liverano, 2021). The story told by a student who has just embarked on a remedial action represents the link between what he has committed and what he has learnt, between his past and his present, between unexpressed potential and expressed potential, what holds together two different moments of the same life, its transformation, change, tracing its horizon of meaning. 3. Discussion Stories help the subject to create a bridge between memory and the present and to grasp the general and no longer fragmented vision of his or her existence (Liverano, 2021), they are a tool for expanding the boundaries of one’s world and offer the subject the possibility of placing his or her experiences against a broader and more meaningful background (Batini & Giusti, 2010). And in this case, they have served to bring home the possibility that each student has to expand his or her range of action within and beyond a school community. Storytelling is an effective antidote to the loss of meaning in educational and work contexts, to media bombardment, and to the disintegration of the self (Liverano, 2021) and, in this case, proved functional in understanding the value of a meaningful and productive pres ence at school. The stories help the subject enter into the historical flow, be come aware of what has been accomplished and transform behaviour, and, in a shared group analysis, clarify objectives and help understand that within a community one shares a common origin and perspective. The children were able to understand the meaning of their mistakes and trace different learning trajectories and behaviours. Sharing opinions, learning, and the effects of a narrative carried out in a group, moreover, turns out to be a powerful tool for creativity and innovation, as well as for enhancing skills and meta-com petences (Batini & Giusti, 2009, 2010). Ultimately, the project carried out by the Lentini-Einstein Comprehensive school reinforced what other studies Wyższa Szkoła Gospodarki Euroregionalnej im. Alcide De Gasperi w Józefowie 300 A GOOD RESTORATIVE JUSTICE PRACTICE IN AN ITALIAN SCHOOL have already been able to highlight (Kehoe et al., 2018; Gregory et al., 2015; Farr et al., 2020; Norris et al., 2019), namely that interventions involving re storative practices not only represent alternative forms of error management and reparation, but meaningful moments for pupils “to tell their opinions and emotions, lower the level of disciplinary disparity between students of different races, cultures and genders, participate proactively in school decision-making processes” (Lodi et al., 2021), develop life skills, and reduce school dropout and dropout (Rideout, et al., 2010; Darling-Hammond et al., 2020). In this specific case, it fostered the development of social and emotional skills that enabled the promotion of pro-social behaviour. 3. Discussion In addition, the individuals involved in the activities demonstrated improved relationships, willingness to collaborate better with the school community, and the ability to understand and develop an awareness of mistakes, including through a greater sense of responsibility and better listening skills. The children were also able to develop a more solid and defined identity through a greater sense of trust in the com munity in which they felt accepted, valued and more secure, which fostered a stronger sense of belonging. Experience has shown that collaboration and a collective effort oriented towards a common goal and towards the enhance ment of each pupil’s subjective experience, can be effective in improving the sense of community and trust in it, which is in turn able to reduce school inequalities, promote empowerment, processes of inclusion, equity, valorisa tion and well-being for all (Lustick et al., 2020; Oxley & Holden, 2021). This is one of the many reasons that lead one to think that from now on educational interventions of error repair can be used not only as compensatory practices but in a preventive key of welcoming and caring for people, relationships, and communities (Gregory, et al. 2016; Lodi et al., 2021), taking care, however, to verify that each practice is really feasible and is shaped to the characteristics of each school. 4. Conclusions Restorative justice is an alternative educational and disciplinary approach to the traditional approach of assigning punishments as a result of school-based Journal of Modern Science 2/51/2023 301 GIUSEPPE LIVERANO offenses. This case study focused on exploring a restorative justice practice within an Italian comprehensive school. In Italy, the restorative justice ap proach is beginning to be used as an alternative method to punishment and as an educational method for enhancing interpersonal skills or attempting to transform attitudes and behaviors at school. in this case study, it was high lighted how a restorative justice practice acts at the individual, community and organizational levels. The key stakeholders in this educational intervention stated that the practice of justice restorative generated personal and commu nity reflection geared toward creating a school context that is more equitable, more respectful of spaces and rules, and more relational. The effectiveness of a restorative justice intervention is revealed through its ability to promote equality and equity, to become a moment of individual and collective reflection that leads to a growth of the sense of responsibility and awareness and its ability to be an instrument of inclusion. For this reason to happen it is essential that the intervention is educational and excludes in its original design idea any form of discrimination and humiliation for those who are called to carry out a reparative task as an alternative to the classic punishment. In the concept of restorative justice, therefore, the ethical dimension occupies a fundamental role, which must be the background to every intervention in such a way that every reparative action and practice can be, for the subject, a strategy to grow and not an instrument of mortification or capable of generating discomfort and offense in turn. The effectiveness of a practice of restorative justice lies in its ability to rehabilitate the subject who has caused damage or offense, in the sense of making him aware of the effects of his gesture and helping him in a process of cultural transformation, towards a more peaceful, supportive, reflective, sustainable model, in which the community no longer plays the role of judging subject, but of organ that supports, supports and helps in difficulty. 4. Conclusions This is why the effectiveness of a restorative justice practice also depends on its ability to enable a subject of social and emotional skills that help him to manage his social dimension with more balance and harmony and to weave meaningful relationships. In this sense, the school community has the fun damental role of designing the most effective and efficient solutions to be educational and help those in difficulty to develop knowledge and skills that act on character and personality, to promote new lifestyles, more peaceful and Wyższa Szkoła Gospodarki Euroregionalnej im. Alcide De Gasperi w Józefowie 302 A GOOD RESTORATIVE JUSTICE PRACTICE IN AN ITALIAN SCHOOL supportive. If the school community, as a system of people, fails to support this idea of education, that is, aimed at supporting and helping its members in a fair and inclusive way to favor the process of emancipation and freedom, it will have failed in its task and in its educational mission. For this reason it is essential that in the choice to promote new educational paradigms within school contexts there is the participation of all and, the commitment in their respective roles, is systemic and organic and not improvised and uneven. The punitive system within schools now seems to be no longer able to satisfy the desired effects, because it indisposes even more students who do not feel understood, acting negatively on their motivation and involvement in educa tional actions, but above all because, as a model it does not allow to support the needs of students of freedom, expressiveness, to have a voice and a central role in schools. The choice of adherence to a model of restorative justice must therefore be able to represent a cultural object and be promoted and shared by all. Differently where there will be teachers who will promote different educational models (punitive and restorative), there will be students whose growth process will be characterized by a style of behavior full of doubts and insecurities. Journal of Modern Science 2/51/2023 303 GIUSEPPE LIVERANO References Acosta, J., Chinman, M., Ebener, P., Malone P., Phillips A., Wilks A. (2019). Evaluation of a Whole-School Change Intervention: Findings from a Two-Year Cluster- Randomized Trial of the Restorative Practices Intervention. J Youth Adolescence, 48, 876-890. https://doi.org/10.1007/s10964-019-01013-2f Batini, F. (2019). Gli effetti dell’orientamento narrativo: 20 anni di ricerca. In Soresi, S., Nota, L., Santilli, S. (a cura di), Il contributo dell’orientamento e del counseling all’Agenda 2030, Padova: Cluep. Batini, F. (2015). Scegliere o progettare? L’orientamento narrativo come risposta al cambiamento del bisogno di orientamento. Pedagogia oggi, Vol. n. 1.̀ Batini, F. (2012). L’orientamento alla prova della contemporaneità. Metis. Mondi educativi. Temi, indagini, suggestioni, 2, (1). Batini, F. (2012). Orientamento narrativo. Voci della Scuola, IX. Batini, F., Giusti, S. (2010). Imparare dalle narrazioni, Milano: Unicopli. Bronfenbrenner, U. (2005). Ecological systems theory. In U. Bronfenbrenner (Ed.), Making human beings human: Bioecological perspectives on human development, 106-173. Thousand Oaks, CA: Sage Publications Ltd. h Darling-Hammond, S., Fronius, T.A., Sutherland, H., Guckenburg, S., Petrosino, A., Hurley, N. (2020). Effectiveness of restorative justice in US primary and secondary schools: a review of quantitative research. Contemp. Sch. Psych. 24, 295-308. https:// doi.org/10.1007/s40688-020-00290-0 g Cavell, S. (2010). Little did I know: Excerpts from memory. Stanford: Stanford Univers De Laet, S., Colpin, H., Vervoort, E., Doumen, S., Van Leeuwen, K., Goossens, L., Verschueren, K. (2015). Developmental trajectories of children’s behavioural en gagement in late primary school: both teachers and peers matter. Developmental Psychology, 51 (9), p. 1292. https://doi.org/10.1037/a0039478 Drewery, W. (2014). Restorative practice in New Zealand schools: Social development through relational justice. Educational Philosophy and Theory, 48 (2),191-203. https:// doi.org/10.1080/00131857.2014.989951 Farr, B., Gandomi, M., De Matthews, D.E. (2020). Implementing Restorative Justice in an Urban Elementary School: A Principal’s Commitment and Experiences Eliminating Exclusionary Discipline. J. Cases Educ. Leadersh. 2020, 23, 48-62. https://doi.org/10.1177/1555458920922888 Flutter J., Ruddock J. (2004). Consulting pupils: what’s in it for schools. London: Routledge Falmer.f Gwathney, A.N. Offsetting Racial Divides: Adolescent African American Males & Restorative Justice Practices. Clin. Soc. Work J. 2021, 49, 346-355. https://doi-org. proxy.libraries.rutgers.edu/10.1007/s10615-021-00794-z Wyższa Szkoła Gospodarki Euroregionalnej im. Alcide De Gasperi w Józefowie 304 A GOOD RESTORATIVE JUSTICE PRACTICE IN AN ITALIAN SCHOOL Gregory, A., Clawson, K., Davis, A., Gerewitz, J. (2015). The Promise of Restorative Practices to Transform Teacher-Student Relationships and Achieve Equity in School Discipline. J. Educ. Psychol. Consult. 25, 325-353. doi: #10.1080/10474412.2014.929950 p y Higgins, E. T. (1987). Self-discrepancy: a theory relating self and affect. Psychological Review, 94(3), 319-340. References https://doi.org/10.1037/0033-295X.94.3.319 gg y y gf Review, 94(3), 319-340. https://doi.org/10.1037/0033-295X.94.3.319 p g Hooks, B. (1994b), Teaching to transgress education as the practice of freedom, New York, Routledge. Translated by It., Teaching to transgress. L’educazione come pratica della libertà, Milan: Meltemi, 2020. Hooks, B. (2020a). Teaching to transgress. Education as the practice of freedom. Milan: Meltemi. Kehoe, M., Bourke Taylor, H., Broderick, D. (2018). Developing students’ social skills using restorative practices: a new framework called HEART. Soc. psychicolo. educ. 21, 189-207. https://doi.org/10.1007/s11218-017-9402-1 Kalogeras, S. (2013). Storytelling: An ancient human technology and critical-creative pedagogy for transformative learning. International Journal of Information and Communication Technology Education, 9(4), 113-122. Doi: 10.4018/ijicte.2013100108i Ingraham, C.L., Hokoda, A., Moehlenbruck, D., Karafin, M., Manzo, C., Ramirez, D. (2016). Consultation and Collaboration to Develop and Implement Restorative Practices in a Culturally and Linguistically Diverse Elementary School. J. Educ. Psychol. Consult. 26, 354-384. https://doi.org/10.1080/10474412 .2015.1124782 Lerner, M., Lerner, J. V., Benson, J. B. (Eds.) (2011). Advances in child development and behaviour. 195-228. London: Elsevier. Vol. 41. Liverano, G. (2021). Online narrative guidance at the time of Covid-19: analysis of best practices. “Q-Times”.Journal of Education, Technology and Social Studies - QTimes - webmagazine Year XIII - No. 4, 2021 doi: 10.14668/QTimes_13416 Lodi, E., Perrella, L., Lepri, G.L., Scarpa, M.L., Patrizi, P. (2022). Use of Restorative Justice and Restorative Practices at School: A Systematic Literature Review. Int. J. Environ. Res. Public Health, 19, p. 96. https://doi.org/10.3390/ ijerph19010096 Lustick, H., Norton, C., Lopez, S.R., Greene-Rooks, J.H. (2020). Restorative Practices for Empowerment: A Social Work Lens. Child. Sch. 42, 89-97. doi: 10.1093/cs/cdaa006 McCold, P. (2008). Evaluation of a restorative milieu: Restorative practices in con Lustick, H., Norton, C., Lopez, S.R., Greene-Rooks, J.H. (2020). Restorative Practices for Empowerment: A Social Work Lens. Child. Sch. 42, 89-97. doi: 10.1093/cs/cdaa006 McCold, P. (2008). Evaluation of a restorative milieu: Restorative practices in con text. In H. Ventura Miller (Ed.) Restorative justice: From theory to practice. 99-137. Bingley, UK: Emerald Group Publishing Limited. McCold, P., & Wachtel, T. (2002). Restorative justice theory validation. In: E. Weitekamp, H.-J. Kerner (Eds.) Restorative justice: Theoretical foundations. 110-142. Devon, UK: Willan Publishing. Journal of Modern Science 2/51/2023 305 GIUSEPPE LIVERANO Morrison, B. (2015). Restorative justice in education: Changing lenses on educa tion threer’s. Restorative Justice: An International Journal, 3 (3), 445-452. doi: 10.1080/20504721.2015.1109367h Norris, H. (2019). The impact of restorative approaches on well-being: An evaluation of happiness and engagement in schools. Confl. Resolut. Wyższa Szkoła Gospodarki Euroregionalnej im. Alcide De Gasperi w Józefowie References Q., 36, 221-234. https:// doi.org/10.1002/crq.21242 g q Oxley, L., Holden, G.W. (2021). Three positive approaches to school discipline: are they compatible with principles of social justice? Educ. Child Psychologist. 38, 71-81. Rideout, G. , Karen, R. , Salinitri, G. , & Marc, F. ( 2010 ). Measuring the impact of restorative justice practices: outcomes and contexts . Journal of Educational Administration and Foundations , 21, 35-60. Riva, M. G. (2020). Orientamento. Una pratica educativa tra soggettività, conoscenza, orientamenti e condizionamenti. Pedagogia più didattica, Vol. 6, No. 2, Trento: Erickson. g g Rudasill, K. M., Reio, T. G., Stipanovic, N., Taylor, J. E. (2010). A longitudinal study of student-teacher relationship quality, difficult temperament, and risky behaviour from childhood to early adolescence. Journal of School Psychology, 48(5), 389-412. doi: 10.1016/j.jsp.2010.05.001hf Thapa, A., Cohen, J., Guffey, S., Higgins-D’Alessandro, A. (2013). A review of school climate research. Review of Educational Research, 83 (3), 357-385. https://doi. org/10.3102/0034654313483907 Thorsborne, M., Vinegrad D. (2008). Rethinking behaviour management: Restorative practice in schools. Milton Keynes: Speech Mark Publishing.h p y g Thornberg, R., Wänström, L., Pozzoli, T. and Gini, G. (2018). Victim prevalence in bullying and its association with teacher-student and student-student relationships and classroom moral disengagement: a path analysis at the classroom level. Education research papers, 33 (3), 320-335.https://doi.org/10.1080/02671522.2017.1302499i p p g Wachtel, T. (2016). Defining restoration: building a global learning network of restoration practices.The 15th IIRP World Conference. h Were, K. (2017). Promoting Social and Emotional Wellbeing and Responding to Mental Health Problems in Schools. In: Bährer-Kohler, S., Carod-Artal, F. (eds) Global Mental Health Springer, Cham. https://doi.org/10.1007/978-3-319-59123-0_11 Wright, M. (2002). The restorative justice paradigm. VOMA Connect. res. Practice. Suppl. II. Wyższa Szkoła Gospodarki Euroregionalnej im. Alcide De Gasperi w Józefowie 306
https://openalex.org/W3020534866	https://europepmc.org/articles/pmc7235503?pdf=render	English	null	Employing drug delivery strategies to create safe and effective pharmaceuticals for <scp>COVID</scp>‐19	Bioengineering & translational medicine	2,020	cc-by	6,432	Employing drug delivery strategies to create safe and effective pharmaceuticals for COVID-19 While the FDA can and seems willing to reduce the reg- ulatory burdens that can otherwise slow down progress, there is an immunological limit to the speed at which clinical trials can be ethically performed.10 This point has been underscored by previous reports from similar coronaviruses showing that anti-spike IgG antibodies induced by an experimental vaccine was complicit in promoting a pro- inflammatory reaction in the lung, exacerbating acute respiratory dis- tress syndrome (ARDS), and potentially leading to death.11,12 Once approved, it must then be manufactured at scale, though the parallel creation of vaccine production facilities customized for each of the top candidate vaccines currently underway could speed this pro- cess.13 Therefore, while a vaccine may ultimately be our savior, cur- rent best-case scenario estimates put the availability of a clinically viable vaccine at 12–18 months.14,15 Even that would be a two- or threefold improvement compared to the original mumps vaccine, which holds the record for the shortest time between virus isolation and vaccine development (1945–1948). Unfortunately, that vaccine yielded only short-term protection and was replaced several decades later by a more potent, long-lasting vaccine.16 The paradigm that prevention is more effective than treatment holds true across much of medicine. Vaccination against infectious disease, which is responsible for some of the greatest and most cost- effective improvements in public health, is perhaps the best example of this principle in action.1 Despite confidence expressed by the United States and other countries prior to SARS-CoV-2, it has become clear that we were ill-prepared to rapidly respond and mitigate a viral outbreak with a comprehensive response plan. Put simply, we have failed to provide our citizens with the tools necessary to stop the spread of coronavirus disease 2019 (COVID-19). This failure is most glaring in the lack of protection for healthcare workers, who have lacked adequate access to the personal protective equipment (PPE) they rely on to avoid contracting the disease themselves.2 We have, in short order, met “Disease X,” the unexpected and severe infectious disease that the World Health Organization and others such as Bill Gates had feared could quickly escalate and become an worldwide pandemic.3,4 Thus far, a majority of COVID-19's impact has been felt in countries that are most integrated into the global economy and are fairly well equipped from a healthcare perspective. Employing drug delivery strategies to create safe and effective pharmaceuticals for COVID-19 current estimate of ~1%5,6 due to the lack of adequate medical facili- ties and equipment as well as quarantining procedures that are more difficult to implement in those settings.7 current estimate of ~1%5,6 due to the lack of adequate medical facili- ties and equipment as well as quarantining procedures that are more difficult to implement in those settings.7 The outbreak of the novel SARS-CoV-2 pathogen and corresponding coronavirus disease 2019 (COVID-19) have had an enormous impact on both global health and the daily lives of billions of people world- wide. With a proven vaccine at least a year from being fully tested for safety and efficacy, there may be an opportunity to rapidly repurpose existing drugs in order to prevent SARS-CoV-2 infections and improve outcomes for patients already infected with COVID-19. At present, more than 40 different drugs are being explored for efficacy against COVID-19, including antivirals and immune modulating compounds. Unfortunately, many of these drugs are associated with side effects that limit their use to the most severe cases and thereby prevent their use as prophylactics. This commentary describes drug formulation strategies that can be used to maintain the efficacy of these drugs through controlled release, targeted delivery, and nonviral nucleic acid delivery. If successful, these approaches could enable the expanded use of drugs to reduce the mortality of this devastating disease and equip healthcare providers with the tools to accelerate our recovery from this pandemic and improve our response to the next outbreak of a novel pathogenic virus. In response to the global COVID-19 pandemic, there has been a major emphasis on developing a vaccine for SARS-CoV-2—and right- fully so. Herd immunity for COVID-19 does not appear likely to come to our rescue,8 so developing a vaccine that confers long-lived protec- tion is, and should be, our primary goal. However, our ability to develop a vaccine suitable for clinical use in a timely manner remains to be seen. Even with new strategies in vaccine development, which enabled Moderna (Cambridge, MA) and the National Institute of Allergy and Infectious Disease (NIAID) to design, produce, and admin- ister their mRNA-1,273 vaccine to humans in Phase I clinical trials just 63 days after the viral genome sequence was first reported,9 there is still a long way to go before it, or another vaccine, is proven to be safe and effective. Revised: 22 April 2020 Accepted: 22 April 2020 Revised: 22 April 2020 Accepted: 22 April 2020 Received: 14 April 2020 DOI: 10.1002/btm2.10163 DOI: 10.1002/btm2.10163 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Author. Bioengineering & Translational Medicine published by Wiley Periodicals, Inc. on behalf of The American Institute of Chemical Engineers. p p y eering & Translational Medicine published by Wiley Periodicals, Inc. on behalf of The American Institute of Chemical Engineers. wileyonlinelibrary.com/journal/btm2 1 of 7 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, properly cited. Employing drug delivery strategies to create safe and effective pharmaceuticals for COVID-19 If (or more likely when) the disease reaches critical levels in low- and middle-income countries, we expect to see an increase in the death rate from our In the meantime, social distancing has been implemented in many locales and by most accounts has been at least moderately successful in reducing the spread of COVID-19.17 New and more high- throughput tests have also been developed, both for determining the presence of an active infection via viral RNA and previous infection via antibody titer analysis.18,19 Convalescent plasma therapy may also 1 of 7 Bioeng Transl Med. 2020;5:e10163. https://doi.org/10.1002/btm2.10163 2 of 7 EDITORIAL EDITORIAL current formulation is suitable for use.31,32 Hydroxychloroquine showed a similar lack of efficacy in a U.S. trial.33 help improve outcomes in patients with severe COVID-19, though availability may be limited due to the (albeit decreasing) scarcity of donor plasma and difficulty obtaining it.20,21 There are also a number of postinfection therapeutics being evaluated in the clinic for their potential ability to reduce the severity of COVID-19.22,23 Most of these drugs are repurposed small molecule antivirals and immune- modulating antibodies either already approved for other indications (e.g., chloroquine, hydroxychloroquine, ribavirin, favipiravir) while others have progressed through early stage clinical trials, but have not yet received FDA approval (e.g., remdesivir, galidesivir, leronlimab). While there are numerous reports of their in vitro efficacy, their ther- apeutic value for humans remains unclear at present. With COVID-19 spreading at an alarming rate and the FDA helping to facilitate safety and efficacy testing, some of these drug trials should achieve suffi- cient enrollment to draw conclusions about their efficacy with appro- priate statistical power. Fortunately, there is a strong precedence for the value of pre- exposure prophylaxis (PrEP) when the side effects of antivirals are low in the form of HIV drugs, such as Truvada (emtricitabine/ tenofovir disoproxil). Employing drug delivery strategies to create safe and effective pharmaceuticals for COVID-19 Truvada inhibits reverse transcriptase to prevent HIV from creating DNA from its RNA, thereby preventing it from inte- grating into the host cell genome and replicating.34 Because this enzyme is not native or necessary for human cell function, inhibition with Truvada is not associated with highly pervasive or severe side effects, enabling its widespread used as both a prophylactic and a postexposure therapy.35 However, Truvada itself is unlikely to have efficacy against SARS-CoV-2 because it does not encode or use reverse transcriptase in its replication process.23 If an effective drug for treating COVID-19 with infrequent and/or mild side effects is identified, we may be able to rapidly transition to evaluating its use for PrEP. However, if that drug does have side effects, how can we reduce its toxicity while maintaining efficacy against COVID-19 to create a favorable value proposition for prophylactic use? We may be able to reduce the undesirable side effects of a drug through medici- nal chemistry, controlled release, or targeted delivery. Using medicinal chemistry to alter a drug's therapeutic window or prolong its biologi- cal half-life is a tried and true approach with many examples of suc- cess. However, this direct chemical modification would be limited to altering small molecule drugs and often involves a slow and empirical development process to develop a single drug substance, which likely cannot be completed and fully tested within the duration of an out- break.36 Alternatively, drug delivery systems are unique in their ability to provide solutions for drugs that have promise, but are not suffi- ciently safe in a traditional formulation to administer to patients. This can be achieved by improving absorption, increasing intracellular delivery, maintaining drug concentrations within a small therapeutic window, or providing a high drug gradient between the organ of inter- est (e.g., lungs) and systemic circulation. Though the potential impact of these strategies on the more than 100 drugs being evaluated for COVID-19 is difficult to summarize concisely, Table 1 provides a gen- eral perspective on the properties of drugs that may benefit the most from targeted delivery or controlled release formulations. If proven effective, these drugs offer a couple of key advantages from a rapid response perspective. First, there is vastly more safety data available for these drugs than for novel vaccines. Employing drug delivery strategies to create safe and effective pharmaceuticals for COVID-19 These drugs have been used in hundreds to thousands of people for those that have entered Phase III trials to billions of people for marketed drugs with a long history of use.24 The number of patients enrolled in the Phase I Moderna/NIAID vaccine trial (45)25 pales in comparison, as would be expected at this stage. Second, the ability to be effective after exposure to SARS-CoV-2 enables clinical trials to dramatically narrow down the patient population to be treated and allows out- comes to be measured on the order of weeks. Contrast this approach with standard Phase III vaccination testing which requires a large cohort and long-term follow-up studies to confirm safety and effi- cacy.26 Lastly, they often have more broad spectrum activity, which makes it more likely that they will remain functional even if SARS- CoV-2 mutates rapidly, though that does not appear to be the case at present.27 Depending on their activity, these could even serve as tools to combat the next viral Disease X that arrives sometime in the future.23 Thus far, there has been little discussion about using these drugs as prophylactics rather than postexposure treatments, which is pre- sumably due to their potential side effects. For example, chloroquine, a drug approved to treat a variety of ailments including malaria, has a small therapeutics index (only two- or threefold higher the daily dose) resulting in potentially fatal acute cardiovascular toxicity.28 Even with as-directed use, it is associated with high frequencies of nausea, diar- rhea, vomiting, muscle weakness, vision loss after prolonged use, and a bevy of other symptoms. The antiviral mechanism of chloroquine is unclear and potentially multifactorial, though some evidence suggests that prophylactic use prevents some viruses from infecting cells by disruption endosomal function.29,30 Whereas there is little motivation for taking chloroquine preemptively in its current state due to severe side effects and uncertain benefits for COVID-19, its use could poten- tially provide a net benefit when there is an active infection. However, based on recent studies using chloroquine in patients with COVID-19, including a double-blind Phase 2 clinical study in Brazil which had to be halted due to safety issues, it does not appear promising that the The development of sustained release platforms could enable the use of an array of drugs that otherwise exhibit harmful side effects. Employing drug delivery strategies to create safe and effective pharmaceuticals for COVID-19 • Controlled release devices would be easy to formulate because of the inherently stability of small molecules and may be especially useful for drugs with short half-lives, small therapeutic indices, or expensive/ complicated production processes. • Often used for immune regulation. • Good candidates for repurposing against novel pathogenic viruses if they target conserved proteins (e.g., the coronavirus spike protein), but likely difficult to isolate, validate, and produce on the timeline of a viral outbreak. • May be possible to determine safety prior to the outbreak of a novel pathogenic virus and thereby accelerate the timeline to implementation, though virus-specific efficacy would of course need to be evaluated. • Potentially more specific than small molecule drugs, leading to reduced off-target effects. • Antibodies that competitively bind with proteins on the patient's cells to prevent viral entry may disrupt their normal physiological function and therefore have undesirable effects. • Highly specific viral-targeted proteins are unlikely to benefit a great deal from targeted or controlled release systems owing to potentially large therapeutic indices; however, less specific proteins may benefit from targeted delivery to avoid high concentrations in off-target tissues • Local delivery could help to limit abnormal physiological function to only the target tissue where it is having a beneficial antiviral effect. • Controlled release devices may be difficult to develop because of the generally poor stability of proteins at 37C for extended periods of time and may not be necessary for antibodies with long half-lives, like endogenous IgG. • Controlled release devices may be difficult to develop because of the generally poor stability of proteins at 37C for extended periods of time and may not be necessary for antibodies with long half-lives, like endogenous IgG. • siRNA against cell surface proteins known to facilitate viral entry can be evaluated ahead of time to determine safety and suggest efficacy against related viruses to speed implementation against novel pathogenic viruses. • Can be rapidly customized for novel viral pathogens once the sequence is known and achieve somewhat predictable efficacy, though safety requires evaluation on a case-by-case basis. • Would benefit greatly from improved non-viral delivery formulations since poor delivery efficacy would allow viruses to enter or replicate in cells that have not received RNA. • siRNA can be rapidly customized in response to identification of the host protein being used for cell entry or viral replication. Employing drug delivery strategies to create safe and effective pharmaceuticals for COVID-19 For example, lopinavir and ritonavir, an HIV drug combination which is currently under evaluation as a COVID-19 treatment, has common side effects that include diarrhea, nausea, and liver damage.37 These drugs have a half-life of ~4–6 hr,38 meaning that systemic concentra- tions can vary by a factor of eight between peak and trough. Develop- ing a controlled-release formulation that exhibits zero-order release kinetics to maintain the minimum effective drug concentration could mitigate these side effects by reducing the steady-state drug concen- tration by as much as eight-fold and reducing the hepatic processing burden by 81%. Although the ability to achieve zero-order in vivo release kinetics with an oral or injectable delivery system largely remains elusive, even formulations that exhibit readily achievable first-order release kinetics could assist in reducing toxicity. Not all 3 of 7 3 of 7 3 of 7 EDITORIAL TABLE 1 COVID-19 drug categories and their potential for synergy with drug delivery systems Indirect (host target) • These drugs may have varying level of specificity and activity for viruses depending on their mechanism of action and how conserved the drug target is between viruses. • Often u • Potentia prevent commo • Often used for immune regulation. • Often used for immune regulation. • Often used for immune regulation. • Potentially broad activity for use in response to or to prevent many viral infections because they act on common host machinery. • Highly suitable for rapid repurposing against novel viral pathogens, but new drug development unlikely on a timeline relevant for outbreak response. • Because they act on host cellular machinery, they often interfere with normal physiological function, sometimes resulting in undesirable off-target effects. • Targeted delivery would enhance the local drug concentration at the site of infection (e.g., lungs) while maintaining a low systemic concentration, thus limiting side effects. • Targeted delivery may not be useful for drugs with activity against a target that is unique to viral entry or replication; however, drugs with less specific activity could benefit from targeted delivery to limit side effects. • Controlled release devices would be easy to formulate because of the inherently stability of small molecules and may be especially useful for drugs with short half-lives, small therapeutic indices, or expensive/ complicated production processes. 4 of 7 EDITORIAL intended.52,53 Another series of papers showed the ability to limit the effects of Ebola virus after exposure using lipid nanoparticles to deliver siRNA targeting an Ebola virus protein.54-56 In the last of these papers, nonhuman primates still exhibited signs of advanced Ebola virus disease, but 100% survived whereas no animals in the control group survived. This work was published in April 2015, 14 months before that outbreak had ended, though the Ebola virus was well- known before that 2-year outbreak.56 factor of 50 or more compared with traditional oral administration once first-pass metabolism is accounted for. One particularly promis- ing approach is the hitchhiking of drug-loaded nanocarriers on red blood cells.40 Intravenous administration of these constructs improved delivery to the lungs by ~40-fold and therefore could be used to achieve an effective local concentration without requiring a high systemic drug concentration. The preparation of inhalable parti- cles for local delivery is perhaps an even simpler approach, so long as the safety and utility concerns can be addressed.41 These strategies could provide safe and effective dosing even when there would other- wise be no therapeutic index (i.e., adverse events begin to occur before the drug is effective).42 However, this historical precedence for advanced formulations lagging behind the outbreak that stimulates their development may not hold true for COVID-19 since there is no precedence for the mag- nitude of COVID-19 in recent times or the resources being made available for its elimination.57,58 On the spectrum of rapid response readiness, the repurposing of existing drugs with broad-spectrum activity and known side effects that can be mitigated with advanced drug delivery techniques should be a top priority. Virus-targeting small molecule antivirals may be easy enough to formulate and can be tested for efficacy against SARS-CoV-2 in parallel. However, inter- feron therapy, which targets the host immune system to reduce dis- ease severity and has shown efficacy against SARS-CoV-2 in vitro,59 may pose a greater formulation challenge. In addition to protein stabil- ity concerns, the short biological half-life and off-target effects of interferons can yield severe and undesirable side effects when admin- istered via traditional formulations.60-62 To overcome these obstacles, there has been a concerted effort to develop advanced interferon for- mulations ranging from sequestration in nanogels for oral delivery63 to implantable devices releasing interferons with zero order.64 Inhala- tion of atomized interferon alpha has been recommended by Chinese guidelines in some patients with COVID-19 with uncertain results.23,65 An ideal drug formulation would exhibit high potency against SARS-CoV-2, have an excellent safety profile, and be produced via an inexpensive and scalable process. In addition, it would be very helpful if delivery systems were modular to enable their facile customization with new drugs. This could also enable a multidrug treatment to pre- vent the induction of resistance, which has been observed for some antivirals.43,44 The co-delivery of multiple drugs with different mecha- nisms of action simultaneously using either a combined (e.g., in the same particle) or preferably modular approach (e.g., blending particles containing different drugs) to enable novel virus flexibility could pre- vent viruses from developing resistance, including cross-resistance.45- Controlled-release systems may be employed to ensure a consistent, effective level of drug is present to avoid applying a selective pressure for drug resistance without concerns over poor patient compliance.46 Similarly, targeted drug delivery systems could avoid dose-limiting toxicity to ensure the effect of antivirals is sufficiently high to prevent the replication of all viral mutants present.47 Traditional controlled release and targeted delivery approaches may not be well-suited for the delivery of biomacromolecular therapeutics due to their potential loss of higher order structure and thus bioactivity during formulation and release.48,49 Fortunately, as of 2018, 77 of the 88 FDA-approved antivirals were small molecules,50 which historically have been easier to formulate.51 In the best-case scenario, we would have a formula- tion that acts on both SARS-CoV-2 as part of a broad spectrum of activity to have a therapy at the ready (i.e., tested for safety) for future outbreaks of novel viral pathogens, so that their efficacy against these pathogens could be rapidly evaluated and implemented to prevent or treat the disease. Beyond these “off-the-shelf” approaches, the next tier of priori- ties would be to employ platforms that can be easily customized to SARS-CoV-2, such as molecular imprinted polymers (MIPs) and nucleic acid therapeutics. Employing drug delivery strategies to create safe and effective pharmaceuticals for COVID-19 • Given the similar nature of most siRNAs, and to a lesser extent mRNAs, formulations would likely be broadly applicable to future customized therapies. • mRNA may be used to increase the expression of protective proteins. • In either case, efficient local delivery would be desired to avoid substantial modification of the patient physiology (e.g., systemic side effects) while maintaining efficacy at the site of viral replication and delivery. • Controlled release formulations may be challenging to develop due to the lack of RNA stability; however, if stability concerns can be overcome, prolonged release could help to maintain optimally altered expression. • Controlled release would be especially beneficial for prophylactic use if RNA stability concerns can be overcome through modification or other means. • The pulmonary delivery of mRNA encoding antibodies against a virus is being evaluated, though it is not clear that this would be meaningfully more effective than untargeted delivery since antibodies are secreted and circulate systemically. Targeted drug delivery may offer a similar or even superior ability to reduce toxicity in some cases, particularly for respiratory infections. Because the lungs comprise only about 2% of total body weight, targeted delivery could decrease the amount of drug required by a drugs under evaluation for COVID-19 are likely to benefit from this approach, however. Chloroquine, for example, has a biological half-life of up to 50 days and thus peak-to-trough systemic drug concentra- tions are unlikely to vary dramatically between daily doses.39 1. Plotkin S. History of vaccination. Proc Natl Acad Sci U S A. 2014;111: 12283-12287. 1. Plotkin S. History of vaccination. Proc Natl Acad Sci U S A. 2014;111: 12283-12287. 2. Cleveland Clinic Lerner Research Institute Theory Division. UV sterili- zation of personal protective equipment with idle laboratory biosafety cabinets during the COVID-19 pandemic. medRvix. 2020. https://doi. org/10.1101/2020.03.25.20043489. 3. World Health Organization. List of Blueprint priority diseases. 2018. http://www.who.int/blueprint/priority-diseases/en/ April 1, 2020. 3. World Health Organization. List of Blueprint priority diseases. 2018. http://www.who.int/blueprint/priority-diseases/en/ April 1, 2020. 4. Gates B. The next outbreak? We're not ready. TED Talks. 2015. https://www.ted.com/talks/bill_gates_the_next_outbreak_we_re_not_ ready?language=en 2020. 4. Gates B. The next outbreak? We're not ready. TED Talks. 2015. https://www.ted.com/talks/bill_gates_the_next_outbreak_we_re_not_ ready?language=en 2020. 5. Rajgor DD, Lee MH, Archuleta S, Bagdasarian N, Quek SC. The many estimates of the COVID-19 case fatality rate. Lancet Infect Dis. 2020. https://doi.org/10.1016/S1473-3099(20)30244-9. Even though deaths and infections appear to be approaching their apex in some areas thanks to increased awareness and social distancing, we are likely still in the early stages of life with COVID-19. The worst wave of infections has still yet to hit many cities and countries, so it is too soon to estimate when we can resume normal societal operations, though some studies have painted a bleak outlook.69 With the work of tens of thousands of dedicated scientists, healthcare providers, and front line workers and some luck, our vaccine development efforts will pay dividends in short order and render the production of safer COVID- 19 treatments and prophylactics temporarily obsolete. However, if first- generation vaccines prove ineffective or the SARS-CoV-2 virus mutates at a rate that prevents long-lived immunity, drug formulations could help sooner than later. Regardless of the readiness of these formulations for the current COVID-19 pandemic, we have now seen the havoc that a Disease X can wreak on our society and would be wise to develop both technology and social measures to mitigate the impact of the next Dis- ease X. In some ways, we are fortunate that this virus is related to previ- ous viral pathogens (MERS-CoV and SARS-CoV), which enabled us to have some basic understanding of this new virus as well as some tools ready in advance of its arrival.23,70,71 In other ways, such as SARS-CoV- 2's propensity to remain asymptomatic, yet transmissible early in an infection,72 we were not. Kevin J. McHugh https://orcid.org/0000-0001-6801-4431 Kevin J. McHugh https://orcid.org/0000-0001-6801-4431 CONFLICT OF INTEREST The author has no conflicts of interest to declare. If we are able to develop these high-efficacy, low-toxicity formula- tions, the next question is, of course, who should be taking these drugs prophylactically and when should they take them? The answer likely depends on the residual side effects and severity of the disease they are preventing, though from an ethical standpoint it is pretty clear that any use should be voluntary. If they have an exceptional safety profile and it is cost-effective to produce them, their use could be very widespread during periods of viral outbreak. If they are expensive, but effective or have a less clear net benefit to the average person, their distribution could be more targeted to high-risk populations. Providing effective, low-toxicity prophylactics to healthcare workers might be the most direct benefit to society. The value of healthcare workers in the face of a pandemic is well-appreciated by most, but we must do a better job of providing them with safe working conditions than we have during the current COVID-19 pandemic. These workers disproportionately interact with infected individuals, which increases their chance of contracting the disease. They also interact closely (and physically) with many people, which both increases their risk of contracting the disease and spreading it to others. Further, their frequent interaction with other healthcare workers creates the potential for a transmission nexus. Lastly, they also disproportionately interact with individuals likely to experience the worst COVID-19 outcomes, such as immunocompromised patients and patients with other comorbidities.68 If we can augment the protection provided by PPE using pharmaceutical interventions, we may be able to stymie the spread of the disease and maintain a healthcare workforce operating at full capacity when they are most needed. Kevin J. McHugh Kevin J. McHugh Department of Bioengineering, Rice University, Houston, TX Correspondence Kevin J. McHugh, Department of Bioengineering, Rice University, 6100 Main Street, MS-142, Houston, TX 77005. Email: kevin.mchugh@rice.edu MIPs, also referred to as synthetic antibodies, could be a direct substitute for convalescent plasma therapy.66 However, unlike convalescent plasma therapy, which is limited by the need for healthy, willing donors who have previously contracted the disease,67 MIP only requires a viral template, which can be generated created in a laboratory setting. This could be an especially important treatment in the early weeks of an outbreak when there is yet to be a sizable population of recovered patients. Nucleic acid therapies are particu- larly intriguing because of our ability to sequence a pathogenic viral genome soon after the outbreak has started and rapidly and inexpen- sively synthesize short RNA sequences as well as their potential to exhibit high specificity and be used after exposure. We have seen the inherent speed advantages of working with nucleic acids instead of proteins in the rapid production of a vaccine by Moderna and the NIAID, yet there is a long tail to those studies before efficacy can be determined. As a postexposure drug treatment, the efficacy of siRNA therapy could be evaluated in weeks rather than years. Whereas developing potent small molecule and protein therapeutics de novo in response to a viral epidemic (with or without advanced delivery plat- forms) does not appear possible on a relevant timeline, this After recent outbreaks including Ebola virus, Zika virus, severe acute respiratory syndrome-related coronavirus (SARS-CoV), middle east respiratory syndrome-related coronavirus (MERS-CoV), norovirus, N1N1pdm09 virus (swine flu), and a variety of avian flu viruses there was a flurry of activity to not only develop a vaccine, but also pre- and postexposure therapeutics. Unfortunately, or perhaps fortunately because outbreaks were mostly limited in duration and spread, these development efforts were largely unable to help with the outbreak that prompted their development. A nanoparticle formu- lation of ivermectin (a drug currently being explored for SARS-CoV-2 activity) that enhances intestinal absorption and exhibits controlled release to extend the duration of therapeutic drug levels was publi- shed 3 years after the end of the Zika virus outbreak for which it was 5 of 7 EDITORIAL generalizable approach seems much more well-suited for rapid thera- peutic development. Infect Dis Clin N Am. 2010;24:809-833. 46. Edagwa B, McMillan J, Sillman B, Gendelman HE. Long-acting slow effective release antiretroviral therapy. Expert Opin Drug Deliv. 2017; 14:1281-1291. 25. Moderna. Moderna's work on a potential vaccine against COVID-19. 2020. https://www.modernatx.com/modernas-work- potential-vaccine-against-covid-19. 47. Cao S, Woodrow KA. Nanotechnology approaches to eradicating HIV reservoirs. Eur J Pharm Biopharm. 2019;138:48-63. 26. Singh K, Mehta S. The clinical development process for a novel pre- ventive vaccine: an overview. J Postgrad Med. 2016;62:4-11. 48. van der Walle CF, Sharma G, Kumar MR. Current approaches to stabilising and analysing proteins during microencapsulation in PLGA. Expert Opin Drug Deliv. 2009;6:177-186. 27. Achenbach J. ‘The coronavirus isn't mutating quickly, suggesting a vaccine would offer lasting protection. Washington Post2020. https://www.washingtonpost.com/health/the-coronavirus-isnt-mutating- quickly-suggesting-a-vaccine-would-offer-lasting-protection/2020/03/ 24/406522d6-6dfd-11ea-b148-e4ce3fbd85b5_story.html. 49. McHugh KJ, Guarecuco R, Langer R, et al. Single-injection vaccines: Progress, challenges, and opportunities. J Control Release. 2015;219: 596-609. 50. Chaudhuri SS, Symons JA, Deval J. Innovation and trends in the development and approval of antiviral medicines: 1987-2017 and beyond. Antivir Res. 2018;155:76-88. 28. Weniger H. Review of side effects and toxicity of chloroquine. Bul World Health. 1979;79:A906. 29. Hong W. Combating COVID-19 with chloroquine. J Cell Biol. 2020. https://doi.org/10.1093/jmcb/mjaa015. 51. Frokjaer S, Otzen DE. Protein drug stability: a formulation challenge. Nat Rev Drug Discov. 2005;4:298-306. 30. Keyaerts E, Li S, Vijgen L, et al. Antiviral activity of chloroquine against human coronavirus OC43 infection in newborn mice. Anti- microb Agents Chemother. 2009;53:3416-3421.31. 52. González Canga A, Sahagún Prieto AM, José Diez Liébana M, et al. The pharmacokinetics and interactions of Ivermectin in humans—a- mini-review. AAPS J. 2008;10:42-46. 53. Surnar B, Kamran MZ, Shah AS, et al. Orally administrable therapeutic synthetic nanoparticle for Zika virus. ACS Nano. 2019;13:11034- 11048. 31. Borba MGS, FdA V, Sampaio VS, et al. Chloroquine diphosphate in two different dosages as adjunctive therapy of hospitalized patients with severe respiratory syndrome in the con- text of coronavirus (SARS-CoV-2) infection: preliminary safety results of a randomized, double-blinded, phase IIb clinical trial (CloroCovid-19 study). medRxiv. 2020. https://doi.org/10.1101/ 2020.04.07.20056424. 54. Geisbert TW, Hensley LE, Kagan E, et al. Postexposure protection of Guinea pigs against a lethal ebola virus challenge is conferred by RNA interference. J Infect Dis. 2006;193:1650-1657. 55. Geisbert TW, Lee AC, Robbins M, et al. Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study. Lancet. 2010;375:1896- 1905. 32. Juurlink DN. 6 of 7 6 of 7 6 of 7 EDITORIAL EDITORIAL 37. Cao B, Wang Y, Wen D. A trial of Lopinavir–ritonavir in adults hospi- talized with severe Covid-19. N Engl J Med. 2020;382:1787–1799. https://doi.org/10.1056/NEJMoa2001282. 17. Lewnard JA, Lo NC. Scientific and ethical basis for social-distancing interventions against COVID-19. Lancet Infect Dis. 2020. https://doi. org/10.1016/S1473-3099(20)30190-0. 38. Chandwani A, Shuter J. Lopinavir/ritonavir in the treatment of HIV-1 infection: a review. Ther Clin Risk Manag. 2008;4:1023-1033. 18. Yelin I, Aharony N, Shaer-Tamar E, et al. Evaluation of COVID-19RT- qPCR test in multi-sample pools. medRxiv. 2020. https://doi.org/10. 1101/2020.03.26.20039438v1. 39. Schrezenmeier E, Dörner T. Mechanisms of action of hydro- xychloroquine and chloroquine: implications for rheumatology. Nat Rev Rheumatol. 2020;16:155-166. 19. Amanat F, Nguyen T, Chromikova V, et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. medRxiv. 2020. https://doi.org/10.1101/2020.03.17.20037713v1. 40. Brenner JS, Pan DC, Myerson JW, et al. Red blood cell-hitchhiking boosts delivery of nanocarriers to chosen organs by orders of magni- tude. Nat Commun. 2018;9:1-14. 20. Shen C, Wang Z, Zhao F, et al. Treatment of 5 critically ill patients with COVID-19 with convalescent plasma. JAMA. 2020;323:1582– 1589. https://doi.org/10.1001/jama.2020.4783. 41. Anselmo AC, Gokarn Y, Mitragotri S. Non-invasive delivery strategies for biologics. Nat Rev Drug Discov. 2019;18:19-40. 21. Duan K, Liu B, Li C, et al. Effectiveness of convalescent plasma therapy in severe COVID-19 patients. Proc Natl Acad Sci U S A. 2020;117:9490–9496. https://doi.org/10.1073/pnas. 2004168117. 42. Aliot E, De Roy L, Capuccci A, et al. Safety of a controlled-release flecainide acetate formulation in the prevention of paroxysmal atrial fibrillation in outpatients. Ann Cardiol Angeiol. 2003;52:34-40. 43. Imai M, Yamashita M, Sakai-Tagawa Y, et al. Influenza a variants with reduced susceptibility to baloxavir isolated from Japanese patients are fit and transmit through respiratory droplets. Nat Microbiol. 2020; 5:27-33. 22. Li G, Clercq ED. Therapeutic options for the 2019 novel coronavirus (2019-nCoV). Nat Rev Drug Discov. 2020;19:149-150. 23. Harrison C. Coronavirus puts drug repurposing on the fast track. Nat Biotechnol. 2020;38:379-381. https://doi.org/10.1038/d41587-020- 00003-1. 44. Kormuth KA, Lakdawala SS. Emerging antiviral resistance. Nat Microbiol. 2020;5:4-5. 24. Institute of Medicine (US) Committee on the Economics of Antimalar- ial Drugs. The cost and cost-effectiveness of antimalarial drugs. In: Arrow KJ, Panosian C, Gelband H, eds. Saving lives, buying time: eco- nomics of malaria drugs in an age of resistance. Washington D.C: The National Academies Press; 2004:61-78. 45. Strasfeld L, Chou S. Antiviral drug resistance: mechanisms and clinical implications. 1. Plotkin S. History of vaccination. Proc Natl Acad Sci U S A. 2014;111: 12283-12287. There are few certainties about what the next Disease X will look like; therefore, establishing broad-spectrum pharma- ceutical formulations to treat, or better yet prevent, infections may offer a key tool in future fights against novel viral pathogens. 6. Lipsitch M. Estimating case fatality rates of COVID-19. Lancet Infect Dis. 2020. https://doi.org/10.1016/S1473-3099(20)30246-2. 6. Lipsitch M. Estimating case fatality rates of COVID-19. Lancet Infect Dis. 2020. https://doi.org/10.1016/S1473-3099(20)30246-2. 7. COVID-19 Clinical Research Coalition. Global coalition to accelerate COVID-19 clinical research in resource-limited settings. Lancet. 2020; 395:1322–1325. https://doi.org/10.1016/S0140-6736(20)30798-4. 8. Bock W, Adamik B, Bawiec M, et al. Mitigation and herd immunity strategy for COVID-19 is likely to fail. medRxiv. 2020. https://doi.org/ 10.1101/2020.03.25.20043109v1. 9. Cohen J. With record-setting speed, vaccine makers take their first shots at the new coronavirus. Science. 2020. https://doi.org/10. 1126/science.abb9996. 10. Callaway E. Coronavirus vaccines: five key questions as trials begin. Nature. 2020;579:481. 11. Liu L, Wei Q, Lin Q, et al. Anti–spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection. JCI Insight. 2019;4:123158. 12. Li X, Geng M, Peng Y, Meng L, Lu S. Molecular immune pathogenesis and diagnosis of COVID-19. J Pharm Anal. 2020;10:102-108. https:// doi.org/10.1016/j.jpha.2020.03.001. 13. Noah T, Gates B. Bill Gates on fighting coronavirus. The Daily Social Distancing Show. 2020. https://www.youtube.com/watch?v= iyFT8qXcOrM 14. The Lancet Infectious Diseases. Challenges of coronavirus disease 2019. Lancet Infect Dis. 2020;20:261. 15. Hodgson J. The pandemic pipeline. Nat Biotechnol. 2020. https://doi. org/10.1038/d41587-020-00005-z. 16. Centers for Disease Control and Prevention. Epidemiology and pre- vention of vaccine-preventable diseases. In: Hamborsky J, Kroger A, Wolfe S, eds. The Pink Book. 13th ed. Washington D.C: Public Health Foundation; 2015:247-260. Safety considerations with chloroquine, hydro- xychloroquine and azithromycin in the management of SARS-CoV-2 infection. CMAJ. 2020;192:E450-E453. https://doi.org/10.1503/ cmaj.200528. 56. Thi EP, Mire CE, Lee AC, et al. Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primates. Nature. 2015;521: 362-365. 33. Magagnoli J, Narendran S, Pereira F, et al. Outcomes of hydro- xychloroquine usage in United States veterans hospitalized with Covid-19. medRxiv. 2020. https://doi.org/10.1101/2020.04.16. 20065920.34. 57. Callaway E, Cyranoski D, Mallapaty S, Stoye E, Tollefson J. The coro- navirus pandemic in five powerful charts. Nature. 2020;579:482-483. 34. Masho SW, Wang CL, Nixon DE. Review of tenofovir-emtricitabine. Ther Clin Risk Manag. 2007;3:1097-1104. 58. Reperant LA, Osterhaus ADME. AIDS, Avian flu, SARS, MERS, Ebola, Zika… what next? Vaccine. 2017;35:4470-4474. 35. Riddell J, Amico KR, Mayer KH. HIV preexposure prophylaxis: a review. JAMA. 2018;319:1261-1268. 59. Lokugamage KG, Schindewolf C, Menachery VD. SARS-CoV-2 sensi- tive to type I interferon pretreatment. bioRxiv. 2020. https://doi.org/ 10.1101/2020.03.07.982264v1. 36. Leeson PD, Springthorpe B. The influence of drug-like concepts on decision-making in medicinal chemistry. Nat Rev Drug Discov. 2007;6: 881-890. 60. Thitinan S, McConville JT. Interferon alpha delivery systems for the treatment of hepatitis C. Int J Pharm. 2009;369:121-135. EDITORIAL 7 of 7 61. Brassard DL, Grace MJ, Bordens RW. Interferon-alpha as an immuno- therapeutic protein. J Leukoc Biol. 2002;71:565-581. 68. Guan WJ, Liang WH, Zhao Y, et al. Comorbidity and its impact on 1590 patients with COVID-19 in China: a nationwide analysis. Eur Respir J. 2020;2000547. https://doi.org/10.1183/13993003.00547- 2020. 62. Sleijfer S, Bannink M, Van Gool AR, et al. Side effects of interferon- alpha therapy. Pharm World Sci. 2005;27:423-431. 63. Kim Y, Thapa M, Hua DH, Chang KO. Biodegradable nanogels for oral delivery of interferon for norovirus infection. Antivir Res. 2011;89:165-173. 69. Ferguson NM, Laydon D, Nedjati-Gilani G, et al. Impact of non- pharmaceutical interventions (NPIs) to reduce COVID19 mortality and healthcare demand. Imperial College. 2020. doi: https://doi.org/ 10.25561/77482. 64. Lesinski GB, Sharma S, Varkner KA, et al. Release of biologically func- tional interferon-alpha from a nanochannel delivery system. Biomed Microdevices. 2005;7:71-79. 70. Prompetchara E, Ketloy C, Palaga T. Immune responses in COVID-19 and potential vaccines: lessons learned from SARS and MERS epi- demic. Asian Pac J Allergy Immunol. 2020;38:1-9. 65. Jin YH, Cheng ZS, Cheng H, et al. A rapid advice guideline for the diagnosis and treatment of 2019-nCov infected pneumonia. Mil Med Res. 2020;7:4. 71. Kim E, Erdos G, Huang S, et al. Microneedle array delivered recombi- nant coronavirus vaccines: immunogenicity and rapid translational development. EBioMedicine. 2020;102743. https://doi.org/10.1016/j. ebiom.2020.102743. 66. Graham SP, El-Sharif HF, Hussain S, et al. Evaluation of molecularly imprinted polymers as synthetic virus neutralizing antibody mimics. Front Bioeng Biotechnol. 2019;7:115. 72. Wölfel R, Corman VM, Guggemos W, et al. Virological assessment of hospitalized patients with COVID-2019. Nature. 2020. https://doi. org/10.1038/s41586-020-2196-x. 67. Roback JD, Guarner J. Convalescent plasma to treat COVID-19: pos- sibilities and challenges. JAMA. 2020;323:1561–1562. https://doi. org/10.1001/jama.2020.4940.
https://openalex.org/W3136445611	https://ieeexplore.ieee.org/ielx7/6287639/9312710/09385116.pdf	English	null	Design and Analysis of Simultaneous Wideband Input/Output Matching Technique for Ultra-Wideband Amplifier	IEEE access	2,021	cc-by	7,183	eceived February 26, 2021, accepted March 13, 2021, date of pu Received February 26, 2021, accepted March 13, 2021, date of publication March 24, 2021, date of current version March 30, 2021. Digital Object Identifier 10.1109/ACCESS.2021.3068394 I. INTRODUCTION bandwidth enhancement technique are presented. Section III provides experimental results and the characterization of the LNA. Finally, conclusions are provided in Section IV. The demand for radio frequency (RF) and high data rate com- munication systems has led to the use of higher frequencies and larger bandwidths [1]–[11]. While the size of transistors continues to shrink, the supply voltage must be scaled down proportionally, due to the reli- ability of the gate oxide [5]. However, the low transconduc- tance of the MOSFET at high frequencies affects signiﬁcantly the design of low voltage and low power RF front-ends. To improve circuit performance, a network for simultaneous wideband input and output matching is proposed in this work. Wideband input matching is achieved by a feedback resistor at the input inductor of the cascode input stage. Moreover, an inductive divider is used at the output of the LNA for a better reﬂection coefﬁcient S22. Using these approaches, the small signal gain and the NF of the UWB LNA covering all UWB frequencies, are improved. under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VOLUME 9, 202 TO-PO WANG , (Senior Member, IEEE) TO-PO WANG , (Senior Member, IEEE) Department of Electronic Engineering, National Taipei University of Technology, Taipei 10608, Taiwan e-mail: tpwang@ntut.edu.tw , ( , ) Department of Electronic Engineering, National Taipei University of Technology, Taipei 10608, Taiwan ( ) Department of Electronic Engineering, National Taipei University of Technology, Taipei 10608, Taiwan e-mail: tpwang@ntut.edu.tw ABSTRACT A simultaneous wideband input/output matching technique for ultra-wideband (UWB) low-noise ampliﬁer (LNA) is proposed in this paper. Feedback resistors leading the gate inductors combined with inductive dividers at output ports achieve an extended bandwidth and good input/output return loss. Moreover, Q-factor improved vertical solenoid inductors are used in the matching networks for high gain and low noise ﬁgure (NF). The proposed matching technique, not only enhances the bandwidth, but also achieves a high gain and a low NF for the fabricated 3.1-10.6-GHz monolithic 180-nm CMOS UWB ampliﬁer. Operating at low supply voltage, the measured power consumption is 18.9 mW, the measured gain of the UWB LNA is 15.02 dB, and the NF is 3.1 dB. Moreover, the measured input/output reﬂection coefﬁcients S11 and S22 are lower than −9.4 dB and -15.8 dB, respectively, covering the full-band UWB frequencies. Compared to previously published full-band 3.1-10.6-GHz 180-nm CMOS UWB LNAs, the proposed LNA measurements demonstrate high gain, low NF, low supply voltage, low power dissipation, and good input/output reﬂection coefﬁcients. INDEX TERMS Low-noise ampliﬁer (LNA), noise ﬁgure (NF), ultra-wideband (UWB). II. PROPOSED MATCHING TECHNIQUE FOR UWB LNA The operating principles and the performance limitations of several circuit topologies for wideband LNAs are presented. The ﬁrst topology, shown in Fig. 1(a), uses a resistive ter- mination of the input port to provide an input impedance of 50 . Although this topology attains a good reﬂection coefﬁcient (S11) over the band of interest, the noise perfor- mance that is achieved is low. Fig. 1(b) illustrates the second architecture using a self- biased inverter ampliﬁer with resistive feedback as the ﬁrst stage of the UWB LNA design [7]. With dual feedback and LC-ladder matching network, the architecture provides broadband matching for a common-source ampliﬁer. How- ever, the noise performance is limited because of the input matching network where resistive losses increase the mini- mum possible noise ﬁgure (NFmin) of the circuit. This paper is organized as follows. In Section II, the pro- posed UWB LNA with wideband input and output match- ing networks is introduced and design considerations of the The third circuit topology employing a common-gate MOSFET device is shown in Fig. 1(c). The advantage of The associate editor coordinating the review of this manuscript and approving it for publication was Yuh-Shyan Hwang. 46800 VOLUME 9, 2021 T.-P. Wang: Design and Analysis of Simultaneous Wideband Input/Output Matching Technique for UWB Amplifier FIGURE 1. Circuits with widely used wideband input matching topologies: (a) resistive termination, (b)self-biased inverter amplifier with a feedback resistor, (c)common-gate MOSFET device, (d)resistive shunt-shunt feedback. FIGURE 2. Circuit topology of the proposed wideband LNA employing the simultaneous wideband input/output matching technique (feedback resistors RF 1, RF 2 leading gate inductors Lg2, Lg3 and combining with inductive dividers at output ports). FIGURE 2. Circuit topology of the proposed wideband LNA employing the simultaneous wideband input/output matching technique (feedback resistors RF 1, RF 2 leading gate inductors Lg2, Lg3 and combining with inductive dividers at output ports). design considerations of the proposed matching technique and application for an UWB ampliﬁer are presented in details as follows. A. SIMULTANEOUS WIDEBAND INPUT/OUTPUT MATCHING CONSIDERATIONS To achieve a high gain, the matching circuit at the input of the LNA is essential. Fig. 3(a) shows a widely used narrowband input matching technique; the input impedance of the circuit can be derived as [12, p. 173] Zg = s(Lg1 + LS1) + 1 sCgs1 + gm1 LS1 Cgs1 (1) (1) where gm1 is the device transconductance and Cgs1 is the parasitic capacitance between the gate and the source ter- minals of the active device M1. Moreover, the inductors LS1 and Lg1 are selected to resonate with Cgs1 and to cancel the imaginary part of the input impedance. The value of the Cgs1 is 150 fF. The values of the LS1 and Lg1 are 0.16 nH and 1.4 nH, respectively. Setting the imaginary part of (1) to zero, the resonance frequency can be expressed as where gm1 is the device transconductance and Cgs1 is the parasitic capacitance between the gate and the source ter- minals of the active device M1. Moreover, the inductors LS1 and Lg1 are selected to resonate with Cgs1 and to cancel the imaginary part of the input impedance. The value of the Cgs1 is 150 fF. The values of the LS1 and Lg1 are 0.16 nH and 1.4 nH, respectively. Setting the imaginary part of (1) to zero, the resonance frequency can be expressed as FIGURE 1. Circuits with widely used wideband input matching topologies: (a) resistive termination, (b)self-biased inverter amplifier with a feedback resistor, (c)common-gate MOSFET device, (d)resistive shunt-shunt feedback. fo1 = 1 2π q Cgs1 Lg1 + LS1 (2) common-gate (CG) LNA is its low input impedance, which can easily be adjusted and matched to 50 . Note that the dominant noise source in CMOS devices is channel thermal noise [6], [10]. This source of noise is typically modeled as a shunt current source at the output circuit of the MOS- FET. Fig. 1(d) depicts the widely used architecture for UWB LNAs. Based on the cascode conﬁguration and on a resistive shunt-shunt feedback, the architecture allows extending the bandwidth of the LNA [10] and improving the gain, the sta- bility and the isolation between the output and the input. (2) (2) At matching, the input impedance of the LNA is equal to Zo, the resistance of the signal source. A. SIMULTANEOUS WIDEBAND INPUT/OUTPUT MATCHING CONSIDERATIONS Given that the real part of (1) is equal Zo, it follows that LS1 = ZoCgs1 gm1 (3) (3) Therefore, the inductance of LS1 can be determined by the parameters Zo, Cgs1, and gm1. Although the input matching network consisting of LS1, Lg1, and Cgs1 shown in Fig. 3 (a) can be perfectly achieved, the matching is suitable only for a narrowband LNA design. For wideband input matching, resistive shunt-shunt feedback is adopted in regular LNAs, as shown in Fig. 3(b). However, the noise performance and To enhance the circuit performance of an UWB ampliﬁer in terms of bandwidth, gain, NF, supply voltage, and DC power dissipation, a novel bandwidth enhancement technique (feed- back resistors RF1, RF2, leading gate inductors Lg2, Lg3 and inductive dividers LD3 and LD4 combined with CF2 and RF2 at output ports) is proposed in this work, as shown in Fig. 2. The VOLUME 9, 2021 46801 T.-P. Wang: Design and Analysis of Simultaneous Wideband Input/Output Matching Technique for UWB Amplifier FIGURE 3. Circuit topology with (a) narrowband matching, (b) regular matching with RF lagging Lg1, without inductive divider at output, and (c) proposed simultaneous wideband input/output matching technique (feedback resistor RF leading gate inductor Lg1 and combining with inductive divider at output port). where Cgs1 and Cd2 are the parasitic capacitors. In a typical design,s2Cgs1 Lg1 + LS1 + sgm1LS1 ≪1, s2Cd2(LD1 + LD2) ≪1, and gm1 h sLD2\|\| sLD1 + 1 sCd2 i ≫1 are sat- isﬁed. Consequently, the expression of Zf _in in (4) can be approximated by (5) Zf _in = RF gm1 h sLD2\|\| sLD1 + 1 sCd2 i + 1 gm1 (5) (5) Setting the imaginary part of (5) to nil allows determining the resonance frequency fo2 of the circuit in Fig. 3(c) fo2 = 1 2π√Cd2 (LD1 + LD2) (6) (6) where Cd2 is the parasitic capacitance between the drain of M2 and the inductor LD1. The value of the Cd2 is 250 fF. Moreover, the inductors LD1 and LD2 are selected to pro- vide the required output impedance. The values of the LD1 and LD2 in this work are 0.4 nH and 8.9 nH, respectively. Considering (1) and (5), the overall input impedance (Zin) of the proposed circuit shown in Fig. 3(c) can be written as (7), as indicated earlier. A. SIMULTANEOUS WIDEBAND INPUT/OUTPUT MATCHING CONSIDERATIONS 3(c) is vo2 vin1 = vo1 vin1 · vo2 vin2 vin1 vin1 vin2 = −gm1 sLd2R2 F1 Cgs1(Lg2+LS1)(RF1+sLd2) s2 + s ωo,in1 Qin1 + ω2 o,in1 × −gm3 sLd4R2 F2 Cgs3(Lg3+LS3)(RF2+sLd4) s2 + s ωo,in2 Qin2 + ω2 o,in2 (13) where ωo,in1 = 1 q Cgs1 Lg2 + LS1 (14) Qin1= (RF1 + sLd2) q Cgs1 Lg2 + LS1 (RF1+sLd2) gm1LS1+Cgs1 sLg2 QLg2 +RF1 +gm1RF1Ld2 (15) ωo,in2 = 1 q Cgs3 Lg3 + LS3 (16) Qin2 = (RF2 + sLd4) q Cgs3 Lg3 + LS3 (RF2 + sLd4) gm3LS3 + RF2Cgs3 + gm3RF2Ld4 (17) = −gm1 sLd2R2 F1 Cgs1(Lg2+LS1)(RF1+sLd2) s2 + s ωo,in1 Qin1 + ω2 o,in1 × −gm3 sLd4R2 F2 Cgs3(Lg3+LS3)(RF2+sLd4) s2 + s ωo,in2 Qin2 + ω2 o,in2 (13) Zout = Zout_wo_fb\|\|Zf_out (11) (11) Fig. 5 shows the calculated output return loss S22 with respect to the frequency for Zout_wo_fb and Zout of the pro- posed LNA. It is indicated that not only the output reﬂection coefﬁcient S22 is signiﬁcantly improved, but also the band- width (S22 <−10 dB) is effectively extended. (13) ωo,in1 = 1 q Cgs1 Lg2 + LS1 (14) (14) Consider the regular output matching circuit topology shown in Fig. 3(b), the overall output impedance can be driven as ωo,in1 = q Cgs1 Lg2 + LS1 (14) Qin1= (RF1 + sLd2) q Cgs1 Lg2 + LS1 (RF1+sLd2) gm1LS1+Cgs1 sLg2 QLg2 +RF1 +gm1RF1Ld2 (15) ωo,in2 = 1 q Cgs3 Lg3 + LS3 (16) Qin2 = (RF2 + sLd4) q Cgs3 Lg3 + LS3 (RF2 + sLd4) gm3LS3 + RF2Cgs3 + gm3RF2Ld4 (17) Z′ out = Z′ f_out\|\| sLD2\|\| Zo2\|\| 1 sCd2 (12) (12) which is also plotted in Fig. 5. Compared to the conventional design, the presented inductive divider circuitry at output not only achieves much better output return loss (S22), but also extends the bandwidth. A. SIMULTANEOUS WIDEBAND INPUT/OUTPUT MATCHING CONSIDERATIONS Wang: Design and Analysis of Simultaneous Wideband Input/Output Matching Technique for UWB Amplifier FIGURE 5. Calculated output return loss S22 with respect to the frequency for Zout_wo_fb, Zout of the proposed topology in Fig. 3(c) and Z′ out (RF lagging Lg1) of the conventional topology in Fig. 3(b). the overall input impedance of the circuit can be written as Z′ in = sLg1 + Z′ f _in\|\| sLS1 + 1 sCgs1 + gm1 LS1 Cgs1 . (8) (8) Z′ in is plotted in Fig. 4 and it is found that the proposed input matching circuitry (feedback resistor RF leading gate inductor Lg1 and combining with inductive divider at output) achieves a better input return loss from 3.1 to 10.6 GHz than the regular circuit in Fig. 3 (b) (feedback resistor RF lagging gate inductor Lg1). g For UWB ampliﬁers, the design of output impedance matching is also critical. However, it is seldom considered for UWB LNAs in the literature. Considering the proposed cir- cuit topology in Fig. 3(c), the output impedance Zout without feedback resistor RF can be expressed as Zout_wo_fb = sLD2\|\| Zo2\|\| 1 sCd2 + sLD1 = sLD2 s2Cd2LD1Zo2 + sLD1 + Zo2 s2Cd2Zo2 (LD1+LD2) + s (LD1 + LD2) + Zo2 (9) FIGURE 5. Calculated output return loss S22 with respect to the frequency for Zout_wo_fb, Zout of the proposed topology in Fig. 3(c) and Z′ out (RF lagging Lg1) of the conventional topology in Fig. 3(b). (9) Ld2, and Ld4, respectively. For simpliﬁcation of the analy- sis procedure, the parasitic capacitance Cgd1 and Cgd3 are neglected. Moreover, it is assumed that the currents ﬂowing through the intrinsic resistors (ro1, ro3) and the forward-body biased currents (gmb1vbs1, gmb3vbs3) are much smaller than the current sources (gm1vgs1 and gm3vgs3). Therefore, the small- signal gain of the proposed UWB LNA can be expressed as where Zo2 is the output impedance of the cascode stage. The output impedance Zf_out looking into the feedback resistor RF can be written as Zf_out = RF + 1 sCin \|\|Zg (10) (10) vo2 vin1 = vo1 vin1 · vo2 vin2 where Cin is a DC block, and its value is 4.11 pF. The combination of an inductor LD2 and a capacitor Cbyss is the RF choke. Considering (9) and (10), the overall output impedance of the proposed circuit in Fig. A. SIMULTANEOUS WIDEBAND INPUT/OUTPUT MATCHING CONSIDERATIONS Zin = Zg\|\|Zf _in (7) (7) In this UWB LNA, the circuit parameters are Cgs1 =150 fF, Cd2 =250 fF, LD1 =0.4 nH, LD2 =8.9 nH, LS1 =0.16 nH, Lg1 =1.4 nH, gm1 =27 mS, and RF =190 . Fig. 4 plots the calculated input return loss S11 with respect to frequency for Zg, Zf_in, and Zin (= Zg//Zf_in) of the proposed matching circuit in Fig. 3(c). As seen in Fig. 4, the wideband input matching can be effectively achieved by introducing a pole of frequency fo2. FIGURE 3. Circuit topology with (a) narrowband matching, (b) regular matching with RF lagging Lg1, without inductive divider at output, and (c) proposed simultaneous wideband input/output matching technique (feedback resistor RF leading gate inductor Lg1 and combining with inductive divider at output port). FIGURE 4. Calculated input return loss S11 with respect to the frequency for Zg, Zf_in, Zin (= Zg//Zf_in; RF leading Lg1) of the proposed topology in Fig. 3(c) and Z′ in (RF lagging Lg1) of the conventional topology in Fig. 3(b). the gain of the circuit in Fig. 3(b) are still limited, compared to Fig. 3(a). To further improve the input/output reﬂection coefﬁcients and small-signal gain, a cascode resistive shunt-shunt feed- back with a bandwidth enhancement technique (feedback resistors RF leading gate inductor Lg1 and combining with inductive dividers at output port) is proposed in this work, as shown in Fig. 3(c). The input impedance of the proposed circuit can be written as Zin = Zg//Zf_in, where Zg is the input impedance of the ampliﬁer stage without the feedback circuit, and Zf_in is the impedance looking into the feedback resistor RF. The impedance Zg can be written as (1), and the passives Lg1, LS1, Cgs1 are determined by (2)-(3). The impedance Zf_in can be formulated as The impedance Zf_in can be formulated as Zf _in = RF + h sLD2\|\| sLD1 + 1 sCd2 i 1 + gm1 h sLD2\|\| sLD1+ 1 sCd2 i [s2Cgs1(Lg1+LS1)+sgm1LS1+1][s2Cd2(LD1+LD2)+1] (4) FIGURE 4. Calculated input return loss S11 with respect to the frequency for Zg, Zf_in, Zin (= Zg//Zf_in; RF leading Lg1) of the proposed topology in Fig. 3(c) and Z′ in (RF lagging Lg1) of the conventional topology in Fig. 3(b). Consider the circuit topology in Fig. 3(b) of regular match- ing with feedback resistor RF lagging gate inductor Lg1, (4) VOLUME 9, 2021 T.-P. B. SMALL-SIGNAL GAIN AND NOISE FIGURE CONSIDERATIONS (17) Fig. 6 shows the small-signal equivalent circuit of the pro- posed UWB LNA plotted in Fig. 2, where Cgs1, Cgd1, Cgs3, and Cgd3 are the parasitic capacitances, and RLg2, RLd1, RLd2, and RLd4 are losses from the on-chip inductors Lg2, Ld1, The ωo,in1 and ωo,in2 are the series resonance frequen- cies, and Qin1 and Qin2 represent the Q-factors of input net- works at the frequencies (ωo,in1 and ωo,in2) for LNA’s ﬁrst 46803 VOLUME 9, 2021 T.-P. Wang: Design and Analysis of Simultaneous Wideband Input/Output Matching Technique for UWB Amplifier FIGURE 6. Small-signal equivalent circuit for the proposed full-band 3.1- to 10.6-GHz UWB LNA employing the simultaneous wideband input/output matching technique (feedback resistors RF 1, RF 2 leading gate inductors Lg2, Lg3 and combining with inductive dividers at output ports). FIGURE 6. Small-signal equivalent circuit for the proposed full-band 3.1- to 10.6-GHz UWB LNA employing the simultaneous wideband input/output matching technique (feedback resistors RF 1, RF 2 leading gate inductors Lg2, Lg3 and combining with inductive dividers at output ports). FIGURE 7. Microphotograph of the fabricated 3.1-10.6-GHz monolithic UWB amplifier. and second stages, respectively. According to Section II(A) and Fig. 4, it is known that the created series resonance frequencies (ωo,in1 and ωo,in2) indeed improves the input reﬂection coefﬁcient (S11) and effectively extended the bandwidth. Moreover, the Q-factor of the input matching network (Qin1) can be improved by using fully-integrated on-chip vertical solenoid inductors due to improved Q-factor (QLg2) than that of conventional planar inductors, leading to high gain. The noise factor of the proposed circuit topology with simultaneous wideband input/output matching technique (feedback resistor RF leading gate inductor Lg1 and combin- ing with inductive divider at output port) shown in Fig. 3(c), is expressed in (34). It is known that the coefﬁcients (κ and ξ) in (31) and (33) have inﬂuences on overall NF in (35). It is also observed that the coefﬁcients (κ and ξ) are func- tion of ωo,in1. By utilizing the proposed matching technique, the bandwidth is extended due to the series resonance fre- quency (ωo,in1). Moreover, it minimizes the coefﬁcients (κ and ξ) in (31) and (33), leading to a reduced noise factor F and noise ﬁgure NF. From (34) and (35), it is found that using Q- factor improved vertical solenoid inductors in this work can lead to reduced NF. FIGURE 7. Microphotograph of the fabricated 3.1-10.6-GHz monolithic UWB amplifier. 0.75-V supply voltage can be generated from a bandgap circuit [16]. Fig. 8(a) shows the measured and simulated S-parameters of the UWB LNA with simultaneous wideband input/output matching technique (feedback resistors RF1, RF2 leading gate inductors Lg2, Lg3 and combining with inductive dividers at output ports). It is observed that the measured maximum gain (S21) of the UWB LNA is 15.02 dB. VOLUME 9, 2021 In addition, the measured 3-dB bandwidth of the UWB LNA is 2.4 GHz to 13 GHz, while the gain (S21) varies from 12.02 dB to 15.02 dB. This results in 138% fractional bandwidth. Fig. 8(b) illustrates the measured, simulated, and calculated input return loss (S11) of the LNA. As shown from this ﬁgure, the measured input return loss (S11) of the UWB LNA is below −9.4 dB. Fig. 8(c) illustrates the measured, simulated, and calculated output return loss (S22) of the UWB LNA. It shows that from 3.1 to 10.6 GHz, the measured S22 of the LNA is below −15.8 dB. According to Fig. 8(b) and Fig. 8(c), the derived formulas (7) and (11) in Section II can be used to evaluate the input and output impedances. Moreover, to minimize the body leakage current, the 5-k resistors are inserted between the bodies and supply voltages (Vb1, Vb2, and Vb3), as shown in Fig. 2. This can lead to the minimized body leakage currents of 59.7 pA, 35.8 pA, and 2.05 pA, respectively. III. EXPERIMENTAL RESULTS Moreover, the IIP3 greater than −18.1 dBm is acceptable for a UWB system [23]. Fig. 12 depicts the measured and simulated group delay of the fabricated UWB LNA. It observed that a minimum group delay of 71.3 ps is achieved. The measured and simulated stability factor (K-factor) is shown in Fig. 13, indicating that the UWB LNA is stable (K > 1) in the entire frequency band. Table 1 summarizes the measured performance of the proposed UWB LNA shown in Fig. 2, and compares them to the performances of previously published full-band 3.1-10.6-GHz 180-nm CMOS UWB LNAs. It is conﬁrmed from table 1 that the proposed UWB LNA achieves low sup- ply voltage with low DC power dissipation of 18.9 mW, high gain of 15.02 dB, low NF of 3.1 dB, and good input/output return loss −9 4 dB/−15 8 dB It is observed that the output FIGURE 8. Measured and simulated (a) S21, (b) S11, and (c) S22 of the fabricated 3.1-10.6-GHz UWB LNA employing the proposed simultaneous wideband input/output matching technique (feedback resistors RF1, RF2leading gate inductors Lg2, Lg3 and combining with inductive dividers at output ports). This justiﬁes the transistor noise model used in this work for predicting NF. FIGURE 9. Measured and simulated noise figure of the fabricated 3.1-10.6-GHz UWB LNA. FIGURE 10. Measured input-referred third-order intercept point (IIP3) of the fabricated 3.1-10.6-GHz UWB LNA. FIGURE 9. Measured and simulated noise figure of the fabricated FIGURE 9. Measured and simulated noise figure of the fabricated 3.1-10.6-GHz UWB LNA. FIGURE 9. Measured and simulated noise figure of the fabricated 3.1-10.6-GHz UWB LNA. FIGURE 10. Measured input-referred third-order intercept point (IIP3) of the fabricated 3.1-10.6-GHz UWB LNA. FIGURE 10. Measured input-referred third-order intercept point (IIP3) of the fabricated 3.1-10.6-GHz UWB LNA. By inspecting Fig. 10, the value of IIP3 for the UWB LNA is −6 dBm. Fig. 11 shows the simulated IIP3 of the fabricated 3.1-10.6-GHz wideband LNA, and the average IIP3 over the entire frequency band is −6.1 dBm. It is known that the linearity (IIP3) of a receiver is typically dominated by the following stages (e.g., mixer or IF ampliﬁer) [22]. Moreover, the IIP3 greater than −18.1 dBm is acceptable for a UWB system [23]. Fig. 12 depicts the measured and simulated group delay of the fabricated UWB LNA. It observed that a minimum group delay of 71.3 ps is achieved. III. EXPERIMENTAL RESULTS The LNA was fabricated in a 180-nm CMOS process and the size of the fabricated chip is 0.945 × 0.82 mm2, exclud- ing the testing pads. The microphotograph of the chip is represented in Fig. 7. On-wafer probing was used to char- acterize the UWB LNA. The losses of the measurement setups were de-embedded and calibrated in the experimen- tal results. In this work, the MOSFETs (M1-M3) operate in saturation region with supply voltages of VDD1 =1.5 V and VDD2 =0.75 V, leading to the measured overall DC power dissipation of 18.9 mW. In [13]–[15], the adopted 1.5-V supply voltage is also widely used for low-voltage low-power 180-nm CMOS circuits. For applications, the used The measured and simulated NF of the proposed UWB LNA is depicted in Fig. 9. It is observed that the measured minimum NF of the LNA is 3.1 dB. Moreover, the mea- sured NF of the LNA covering the full-band UWB frequency (3.1-10.6-GHz) varies between 3.1 dB to 4.4 dB. Further- more, the differences between the calculated noise ﬁgure (35) and the measured result are within 1 dB in entire UWB. 46804 VOLUME 9, 2021 VOLUME 9, 2021 T.-P. Wang: Design and Analysis of Simultaneous Wideband Input/Output Matching Technique for UWB Amplifier T.-P. Wang: Design and Analysis of Simultaneous Wideband Input/Output Matching Technique for UWB Amplifier FIGURE 8. Measured and simulated (a) S21, (b) S11, and (c) S22 of the fabricated 3.1-10.6-GHz UWB LNA employing the proposed simultaneous wideband input/output matching technique (feedback resistors RF1, RF2leading gate inductors Lg2, Lg3 and combining with inductive dividers at output ports). This justiﬁes the transistor noise model used in this work for predicting NF. Fig. 10 represents the measured input-referred third-order intercept point (IIP3) of the UWB LNA. The characterization is carried out by using two-tone tests and the frequencies FIGURE 9. Measured and simulated noise figure of the fabricated 3.1-10.6-GHz UWB LNA. FIGURE 10. Measured input-referred third-order intercept point (IIP3) of the fabricated 3.1-10.6-GHz UWB LNA. By inspecting Fig. 10, the value of IIP3 for the UWB LNA is −6 dBm. Fig. 11 shows the simulated IIP3 of the fabricated 3.1-10.6-GHz wideband LNA, and the average IIP3 over the entire frequency band is −6.1 dBm. It is known that the linearity (IIP3) of a receiver is typically dominated by the following stages (e.g., mixer or IF ampliﬁer) [22]. III. EXPERIMENTAL RESULTS The measured and simulated stability factor (K-factor) is shown in Fig. 13, indicating that the UWB LNA is stable (K > 1) in the entire frequency band. By inspecting Fig. 10, the value of IIP3 for the UWB LNA is −6 dBm. Fig. 11 shows the simulated IIP3 of the fabricated 3.1-10.6-GHz wideband LNA, and the average IIP3 over the entire frequency band is −6.1 dBm. It is known that the linearity (IIP3) of a receiver is typically dominated by the following stages (e.g., mixer or IF ampliﬁer) [22]. Moreover, the IIP3 greater than −18.1 dBm is acceptable for a UWB system [23]. Fig. 12 depicts the measured and simulated group delay of the fabricated UWB LNA. It observed that a minimum group delay of 71.3 ps is achieved. The measured and simulated stability factor (K-factor) is shown in Fig. 13, indicating that the UWB LNA is stable (K > 1) in the entire frequency band. FIGURE 8. Measured and simulated (a) S21, (b) S11, and (c) S22 of the fabricated 3.1-10.6-GHz UWB LNA employing the proposed simultaneous wideband input/output matching technique (feedback resistors RF1, RF2leading gate inductors Lg2, Lg3 and combining with inductive dividers at output ports). Table 1 summarizes the measured performance of the proposed UWB LNA shown in Fig. 2, and compares them to the performances of previously published full-band 3.1-10.6-GHz 180-nm CMOS UWB LNAs. It is conﬁrmed from table 1 that the proposed UWB LNA achieves low sup- ply voltage with low DC power dissipation of 18.9 mW, high gain of 15.02 dB, low NF of 3.1 dB, and good input/output return loss −9.4 dB/−15.8 dB. It is observed that the output return loss (S22) is signiﬁcantly improved by using the pro- posed technique. In addition, ﬁgures of merit (FOM) for LNA This justiﬁes the transistor noise model used in this work for predicting NF. Fig. 10 represents the measured input-referred third-order intercept point (IIP3) of the UWB LNA. The characterization is carried out by using two-tone tests, and the frequencies of these two-tones for the UWB LNA are at the max- imum gain frequency of 4.9 GHz with 1-MHz spacing. 46805 VOLUME 9, 2021 T.-P. Wang: Design and Analysis of Simultaneous Wideband Input/Output Matching Technique for UWB Amplifier TABLE 1. Performance summarized and compared with previously published full-band 3.1-10.6-GHz 180-nm CMOS UWB LNAs. FIGURE 11. Simulated IIP3 of the fabricated 3.1-10.6-GHz UWB LNA. FIGURE 12. III. EXPERIMENTAL RESULTS Measured and simulated group delay of the fabricated 3.1-10.6-GHz UWB LNA. presented in [19] can be written as FOM1 GHz mW FIGURE 13. Measured and simulated stability factor (K-factor) of the fabricated 3.1-10.6-GHz UWB LNA. FOM2 = 20 · log10 Gain[lin] × BW[GHz] NF[lin] −1 × PDC[mW] × A[mm2] ! . (19) FOM3 = 20 · log10 Gain[lin] × BW[GHz] × IIP3[mW] NF[lin] −1 × PDC[mW] × A[mm2] ! . (20) Table 1 shows that the proposed LNA achieves superiors FOM1 and FOM2 of 2.15 and 6.65, respectively, and a com- parable FOM3 of −5.35. IV. CONCLUSION This work has proposed an UWB LNA using a simultaneous wideband input/output matching technique (feedback resistors RF1, RF2 leading gate inductors Lg2, Lg3 and combined with inductive dividers at output ports). Wideband input matching is achieved by the addition of a TABLE 1. Performance summarized and compared with previously published full-band 3.1-10.6-GHz 180-nm CMOS UWB LNAs. FIGURE 13. Measured and simulated stability factor (K-factor) of the fabricated 3.1-10.6-GHz UWB LNA. FIGURE 11. Simulated IIP3 of the fabricated 3.1-10.6-GHz UWB LNA. FIGURE 12. Measured and simulated group delay of the fabricated 3.1-10.6-GHz UWB LNA. presented in [19] can be written as FOM1 GHz W FOM2 = 20 · log10 Gain[lin] × BW[GHz] NF[lin] −1 × PDC[mW] × A[mm2] ! . (19) FOM3 = 20 · log10 Gain[lin] × BW[GHz] × IIP3[mW] NF[lin] −1 × PDC[mW] × A[mm2] ! . (20) (19) (20) Table 1 shows that the proposed LNA achieves superiors FOM1 and FOM2 of 2.15 and 6.65, respectively, and a com- parable FOM3 of −5.35. Table 1 shows that the proposed LNA achieves superiors FOM1 and FOM2 of 2.15 and 6.65, respectively, and a com- parable FOM3 of −5.35. FIGURE 12. Measured and simulated group delay of the fabricated 3.1-10.6-GHz UWB LNA. NOISE FIGURE FORMULATION In this appendix, the NF of the proposed circuit shown in Fig. 3(c) is analyzed. It noted that the dominant noise source for MOSFET devices is the channel thermal noise [6], [10]. Moreover, this type of noise source is typically modeled as a shunt current source at the output circuit of a MOSFET, and its value can be formulated as Sa,in,d ω0,in1 Sa,in,d ω0,in1 = i2 n,d 1f ω2 0,in1R2 S n gm1LS1 RS + Cgs1 1 + Rgate1 RS + RF1 RS + sLg2 RSQLg2 o2 (25) i2 n,d = 4kTγ gd0 · 1f (21) (21) (25) where k is the Boltzmann constant, T is the absolute temper- ature, γ is a bias-dependent factor, gd0 is the zero-bias drain conductance of the device, and f is the frequency. Operating in saturation, the value of γ is 2/3 for long-channel devices. As for a deep-submicron MOSFET, (21) has to be modiﬁed as Substituting (21) into (25), the formula (25) can then be rewritten as Sa,in,d ω0,in1 Sa,in,d ω0,in1 Sa,in,d ω0,in1 = 4kTγ gd0 ω2 0,in1R2 S n gm1LS1 RS + Cgs1 1 + Rgate1 RS + RF1 RS + sLg2 RSQLg2 o2 (26) as i2 n,d = 4kTγ gm1f α (22) (22) (26) where gm is the transconductance of MOSFET, and α is the ratio of gm to the zero-bias drain conductance gd0. The values of α and γ of deep-submicron MOSFETs are typically less than 1 and greater than 1, respectively [20]. Thus, this will result in a raised channel thermal noise i2 n,d for the deep- submicron MOSFET. where gm is the transconductance of MOSFET, and α is the ratio of gm to the zero-bias drain conductance gd0. The values of α and γ of deep-submicron MOSFETs are typically less than 1 and greater than 1, respectively [20]. Thus, this will result in a raised channel thermal noise i2 n,d for the deep- submicron MOSFET. In addition, a gate noise current i2n,g will be induced due to the capacitive coupling from a channel noise current i2 n,d. From [21], the induced noise current at gate terminal is presented by i2n,g = 4kTδgg · 1f (27) (27) From Fig. IV. CONCLUSION This work has proposed an UWB LNA using a simultaneous wideband input/output matching technique (feedback resistors RF1, RF2 leading gate inductors Lg2, Lg3 and combined with inductive dividers at output ports). Wideband input matching is achieved by the addition of a pole resulting from an inductive divider at the output port and whose frequency fo2 is given by (6). Moreover, the wideband presented in [19] can be written as (18) 46806 VOLUME 9, 2021 T.-P. Wang: Design and Analysis of Simultaneous Wideband Input/Output Matching Technique for UWB Amplifier FIGURE 14. Equivalent circuit of the proposed circuit in Fig. 3(c) including the noise sources. FIGURE 14. Equivalent circuit of the proposed circuit in Fig. 3(c) including the noise sources. Moreover, the output noise power density due to the series resistors RLg2 and Rgate1 can be written as output matching is signiﬁcantly improved owing to the RF leading Lg at the input port and the inductive divider at the output port, as illustrated in Fig. 5. Furthermore, the NF of the LNA is improved owing to the series resonance frequency (ωo,in1) in the wideband input matching network, leading to a low noise ﬁgure covering the full UWB. Measurement results show that the fabricated UWB LNA, using simulta- neous wideband input/output matching technique, compares favorably with similar designs. Sa,RLg2,Rgate1 ω0,in1 Sa,RLg2,Rgate1 ω0,in1 = 4kT RLg2 + Rgate1 g2 m1 ω2 0,in1R2 S n gm1LS1 RS + Cgs1 1 + Rgate1 RS + RF1 RS + sLg2 RSQLg2 o2 (24) Furthermore, the dominant noise contributor internal to a UWB LNA is the channel current of MOSFET for the ampli- ﬁer’s ﬁrst stage. Its output noise power density arising from this channel thermal source around the frequency ωo,in1 is Furthermore, the dominant noise contributor internal to a UWB LNA is the channel current of MOSFET for the ampli- ﬁer’s ﬁrst stage. Its output noise power density arising from this channel thermal source around the frequency ωo,in1 is REFERENCES Sa,in,g,u ω0,in1 Sa,in,g,u ω0,in1 = ξ · Sa,in,d ω0,in1 ξ a,in,d 0,in1 = 4kTγ ξgd0 ω2 0,in1R2 S n gm1LS1 RS + Cgs1 1 + Rgate1 RS + RF1 RS + sLg2 RSQLg2 o2 (32) where ξ ω0,in = δα2 5γ 1 −\|c\|2 1 + 1 ω2 0,inR2 SC2 gs1 ! (33) ξ a,in,d 0,in1 = 4kTγ ξgd0 ω2 0,in1R2 S n gm1LS1 RS + Cgs1 1 + Rgate1 RS + RF1 RS + sLg2 RSQLg2 o2 (32) [9] A. Ismail and A. A. Abidi, ‘‘A 3-10-GHz low-noise ampliﬁer with wide- band LC-ladder matching network,’’ IEEE J. Solid-State Circuits, vol. 39, no. 12, pp. 2269–2277, Dec. 2004. [10] C.-F. Liao and S.-I. Liu, ‘‘A broadband noise-canceling CMOS LNA for 3.1–10.6-GHz UWB receivers,’’ IEEE J. Solid-State Circuits, vol. 42, no. 2, pp. 329–339, Feb. 2007. where where [11] A. I. A. Galal, R. K. Pokharel, H. Kanay, and K. Yoshida, ‘‘Ultra-wideband low noise ampliﬁer with shunt resistive feedback in 0.18-m CMOS pro- cess,’’ in Proc. Top. Meeting Silicon Monolithic Integr. Circuits RF Syst. (SiRF), 2010, pp. 33–36. ξ ω0,in = δα2 5γ 1 −\|c\|2 1 + 1 ω2 0,inR2 SC2 gs1 ! (33) (33) [12] B. Razavi, RF Microelectronics. Upper Saddle River, NJ, USA: Prentice-Hall, 1998. To evaluate the NF reduction of the UWB ampliﬁer, the calculated total output noise including (23), (24), (30), and (32) is divided by the total output noise due to the source resistance (23). Assuming the bandwidth is 1 Hz, it yields the noise factor F, which can be derived as [13] J. Y. Lee, H. K. Park, H. J. Chang, and T. Y. Yun, ‘‘Low-power UWB LNA with common-gate and current-reuse techniques,’’ IET Microw., Antennas Propag., vol. 6, no. 7, pp. 793–799, May 2012. [14] U. Yodprasit and C. C. Enz, ‘‘A 1.5-V 75-dB dynamic range third- order Gm-C ﬁlter integrated in a 0.18-/spl mu/m standard digital CMOS process,’’ IEEE J. Solid-State Circuits, vol. 38, no. 7, pp. 1189–1197, Jul. 2003. F = 1 + sLg2 RsQLg2 + Rgate1 Rs + γ (κ + ξ)gd0 RSg2 m1 (34) [15] B. Vaz, J. Goes, and N. Paulino, ‘‘A 1.5-V 10-b 50 MS/s time-interleaved switched-opamp pipeline CMOS ADC with high energy efﬁciency,’’ in Symp. VLSI Circuits. Dig. Tech. Papers, 2004, pp. 432–435. (34) Consequently, the noise ﬁgure can be given as [16] V. Ivanov, R. ACKNOWLEDGMENT The author would like to thank the Taiwan Semiconduc- tor Manufacturing Company, Limited (TSMC) for chip fabrication, and the Taiwan Semiconductor Research Insti- tute (TSRI) for supporting. He also thank Shih-Hua Chiang for his efforts. c ≡ in,gi∗ n,d q i2n,gi2 n,d (29) (29) For this work, the values of δ and c are 3.4 and 1.0j for the 180-nm MOS, respectively [24]. As a result, the output noise power density from the internal noise current at the frequency of interest ωo,in1 can be divided into two categories. The ﬁrst term represents the combined effect of the drain noise current and the correlated portion of the gate noise current, and it is given by REFERENCES [1] S. Schmickl, T. Faseth, and H. Pretl, ‘‘An RF-energy harvester and IR-UWB transmitter for ultra-low-power battery-less biosensors,’’ IEEE Trans. Circuits Syst. I, Reg. Papers, vol. 67, no. 5, pp. 1459–1468, May 2020. [2] A. R. A. Kumar, A. Dutta, and B. D. Sahoo, ‘‘A low-power reconﬁg- urable narrowband/wideband LNA for cognitive radio-wireless sensor net- work,’’ IEEE Trans. Very Large Scale Integr. (VLSI) Syst., vol. 28, no. 1, pp. 212–223, Jan. 2020. Sa,in,d,in,g,c ω0,in1 = κ · Sa,in,d ω0,in1 = 4kTγ κgd0 ω2 0,in1R2 S n gm1LS1 RS + Cgs1 1 + Rgate1 RS + RF1 RS + sLg2 RSQLg2 o2 (30) where κ ω0,in = δα2 5γ \|c\|2 +  1 + \|c\| ω0,inRSCgs1 s δα2 5γ   2 (31) Sa,in,d,in,g,c ω0,in1 [3] S. Sattar and T. Z. A. Zulkiﬂi, ‘‘A 2.4/5.2-GHz concurrent dual-band CMOS low noise ampliﬁer,’’ IEEE Access, vol. 5, pp. 21148–21156, 2017. [4] S. A. S. Mohamed and Y. Manoli, ‘‘Design of low-power direct-conversion RF front-end with a double balanced current-driven subharmonic mixer in 0.13 µm CMOS,’’ IEEE Trans. Circuits Syst. I, Reg. Papers, vol. 60, no. 5, pp. 1322–1330, May 2013. [5] J. Sharma and H. Krishnaswamy, ‘‘216- and 316-GHz 45-nm SOI CMOS signal sources based on a maximum-gain ring oscillator topol- ogy,’’ IEEE Trans. Microw. Theory Techn., vol. 61, no. 1, pp. 492–504, Jan. 2013. where where κ ω0,in = δα2 5γ \|c\|2 +  1 + \|c\| ω0,inRSCgs1 s δα2 5γ   2 (31) [6] A. A. Abidi, ‘‘High-frequency noise measurements on FET’s with small dimensions,’’ IEEE Trans. Electron Devices, vol. ED-33, no. 11, pp. 1801–1805, Nov. 1986. (31) [7] C.-T. Fu, C.-N. Kuo, and S. S. Taylor, ‘‘Low-noise ampliﬁer design with dual reactive feedback for broadband simultaneous noise and impedance matching,’’ IEEE Trans. Microw. Theory Techn., vol. 58, no. 4, pp. 795–806, Apr. 2010. The second term is with the uncorrelated gate current noise, which can be written as [8] E. A. Keehr and A. Hajimiri, ‘‘A wide-swing low-noise transconduc- tance ampliﬁer and the enabling of large-signal handling direct-conversion receivers,’’ IEEE Trans. Circuits Syst. I, Reg. Papers, vol. 59, no. 1, pp. 30–43, Jan. 2012. NOISE FIGURE FORMULATION 14, the output noise power density due to the source resistance RS at the frequency of interest ωo,in1 can be formulated as where gg = ω2C2 gs 5gd0 · (28) (28) Sa,source ω0,in1 = 4kTRSg2 m1 ω2 0,in1R2 S n gm1LS1 RS + Cgs1 1 + Rgate1 RS + RF1 RS + sLg2 RSQLg2 o2 (23) The δ is the coefﬁcient with a value of 4/3 for long-channel devices. The channel noise current i2 n,d and the induced gate noise current i2n,g are closely related with a coefﬁcient c, The δ is the coefﬁcient with a value of 4/3 for long-channel devices. The channel noise current i2 n,d and the induced gate noise current i2n,g are closely related with a coefﬁcient c, (23) VOLUME 9, 2021 46807 T.-P. Wang: Design and Analysis of Simultaneous Wideband Input/Output Matching Technique for UWB Amplifier which is deﬁned as [20], [21] which is deﬁned as [20], [21] which is deﬁned as [20], [21] REFERENCES Brederlow, and J. Gerber, ‘‘An ultra low power bandgap operational at supply from 0.75 v,’’ IEEE J. Solid-State Circuits, vol. 47, no. 7, pp. 1515–1523, Jul. 2012. Consequently, the noise ﬁgure can be given as NF = 10 log10 F (35) (35) [17] B. Park, S. Choi, and S. Hong, ‘‘A low-noise ampliﬁer with tunable interference rejection for 3.1- to 10.6-GHz UWB system,’’ IEEE Microw. Wireless Compon. Lett., vol. 20, no. 1, pp. 40–42, Jan. 2010. For this work, the values of α, γ , and gd0 for the 180-nm MOSFETs are 0.6, 1.8, and 2.5 mS respectively [24], [25]. Moreover, the value of Rgate1 for the W/L =152µm/180nm NMOS in this work is 42 . [18] J. F. Chang and Y. S. Lin, ‘‘DC~ 10.5 GHz complimentary metal oxide semiconductor distributed ampliﬁer with RC gate terminal network for ultra-wideband pulse radio systems,’’ IET Microw., Antennas Propag., vol. 6, no. 2, pp. 127–134, Jan. 2012. 46808 VOLUME 9, 2021 T.-P. Wang: Design and Analysis of Simultaneous Wideband Input/Output Matching Technique for UWB Amplifier [19] J. Borremans, P. Wambacq, C. Soens, Y. Rolain, and M. Kuijk, ‘‘Low-area active-feedback low-noise ampliﬁer design in scaled digital CMOS,’’ IEEE J. Solid-State Circuits, vol. 43, no. 11, pp. 2422–2433, Nov. 2008. TO-PO WANG (Senior Member, IEEE) received the Ph.D. degree from National Taiwan University, Taipei, Taiwan. In 2009, he joined the Faculty of the Department of Electronic Engineering, National Taipei Univer- sity of Technology, Taipei, where he is currently a Professor. [20] D. K. Shaeffer and T. H. Lee, ‘‘A 1.5-V, 1.5-GHz CMOS low noise ampli- ﬁer,’’ IEEE J. Solid-State Circuits, vol. 32, no. 5, pp. 745–759, May 1997. [20] D. K. Shaeffer and T. H. Lee, ‘‘A 1.5-V, 1.5-GHz CMOS low noise ampli- ﬁer,’’ IEEE J. Solid-State Circuits, vol. 32, no. 5, pp. 745–759, May 1997. [21] A. van der Ziel, Noise in Solid State Device and Circuits. New York, NY, USA: Wiley, 1986. [21] A. van der Ziel, Noise in Solid State Device and Circuits. New York, NY, USA: Wiley, 1986. Dr. Wang was a recipient of the Best Paper Award of the Symposium on Nano Device Tech- nology (SNDT), in 2012, and the IEEE Ser- vice Award, in 2017. REFERENCES He was the General Secretary of the 2017 IEEE International Conference on Consumer Electronics-Taiwan (ICCE-Taiwan), the Session Chair of the 2017 IEEE International Conference on Consumer Electronics-Taiwan (ICCE-Taiwan), the 2012 IEEE Topic Meeting on Sili- con Monolithic Integrated Circuits in RF System (2012 SiRF), the 2012 IEEE International Conference on Wireless Information Technology and Systems (ICWITS), and the 2012 IEEE International Conference on Electron Devices and Solid-State Circuits (IEEE EDSSC). He also serves as an Associate Editor for IEEE MICROWAVE AND WIRELESS COMPONENTS LETTERS (MWCL). [22] B. Razavi, RF Microelectronics, 2nd ed. London, U.K.: Pearson, 2012. [23] H. Zheng, S. Lou, D. Lu, C. Shen, T. Chan, and H. C. Luong, ‘‘A 3.1 GHz–8.0 GHz single-chip transceiver for MB-OFDM UWB in 0.18-µm CMOS process,’’ IEEE J. Solid-State Circuits, vol. 44, no. 2, pp. 414–426, Feb. 2009. [24] A. J. Scholten, L. F. Tiemeijer, R. van Langevelde, R. J. Havens, A. T. A. Zegers-van Duijnhoven, and V. C. Venezia, ‘‘Noise modeling for RF CMOS circuit simulation,’’ IEEE Trans. Electron Devices, vol. 50, no. 3, pp. 618–632, Mar. 2003. [25] A. J. Scholten, H. J. Tromp, L. F. Tiemeijer, R. Van Langevelde, R. J. Havens, P. W. H. De Vreede, R. F. M. Roes, P. H. Woerlee, A. H. Montree, and D. B. M. Klaassen, ‘‘Accurate thermal noise model for deep-submicron CMOS,’’ in IEDM Tech. Dig., Dec. 1999, pp. 155–158. 46809 VOLUME 9, 2021
https://openalex.org/W4248359127	https://ccsenet.org/journal/index.php/jas/article/download/45851/34584	English	null	Reviewer Acknowledgements for Journal of Agricultural Science, Vol. 8, No. 12	Journal of agricultural science	2,016	cc-by	816	Journal of Agricultural Science; Vol. 8, No. 12; 2016 ISSN 1916-9752 E-ISSN 1916-9760 Published by Canadian Center of Science and Education Journal of Agricultural Science; Vol. 8, No. 12; 2016 ISSN 1916-9752 E-ISSN 1916-9760 Published by Canadian Center of Science and Education Journal of Agricultural Science; Vol. 8, No. 12; 2016 ISSN 1916-9752 E-ISSN 1916-9760 Journal of Agricultural Science; Vol. 8, No. 12; 2016 ISSN 1916-9752 E-ISSN 1916-9760 Published by Canadian Center of Science and Education Journal of Agricultural Science; Vol. 8, No. 12; 2016 ISSN 1916-9752 E-ISSN 1916-9760 214 ournal of Agricultural Science is recruiting reviewers for the journal. If you are interested in becoming a eviewer, we welcome you to join us. Please find the application form and details at ttp://recruitment.ccsenet.org and e-mail the completed application form to jas@ccsenet.org Reviewers for Volume 8, Number 12 Aman Chandi, DuPont Crop Protection, USA Amir Raza, Nuclear Institute for Food and Agriculture, Pakistan Ana Sanches Silva, National Institute of Health Dr Ricardo Jorge, Portugal Aureliano Albuquerque Ribeiro, Federal University of Ceara, Brazil Beatriz Sevilla-Moran, National Institute for Agricultural and Food Research and Technology, Spain C. Nasopoulou, University of the Aegean, Greece César A. Rosales Nieto, Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, Mexico Chaminda Shaman Herath, Coconut Research Institute of Sri Lanka, Sri Lanka Chao Di, University of Pennsylvania Medical Center, USA Chee Kong Yap, Universiti Putra Malaysia, Malaysia Eleonora Nistor, Banat University, Romania F. Bimbo, University of Foggia, Foggia, Italy Fernando Goulart, Universidade Federal de Minas Gerais, Brazil Francesco Vizzarri, University of Molise, Italy Graciele Araújo de Oliveira Caetano, UFVJM/UNIFAJ, Brazil Gustavo Perdoncini, Naturovos Company, Brazil Hoang Van Long, Da Nang Institute for Socio-Economic Development, Viet Nam Iuliana Vintila, Dunarea de Jos University, Romania Jose Manuel Brotons, Miguel Hernandez University, Spain N. Kishore, Palamuru University, India Paulo Gustavo Martins, Federal University of Jequitinhonha and Mucuri Valleys, Brazil Phokele Maponya, Agricultural Research Council-VOPI, South Africa R. Karina Gallardo, Washington State University, USA Rene Murrieta-Galindo, El Colegio de Veracruz (COLVER), Mexico Richard N. Onwonga, University of Nairobi, Kenya Sharifah Rahmah, Universiti Malaysia Terengganu, Malaysia Shivanand Hegde, University of Texas Medical Branch, USA Vipan Kumar, Montana State University, USA Willian Rodrigues Macedo, UFV Campus Rio Paranaíba, Brazil Wu-yi Liu, Fuyang Normal University, China Journal of Agricultural Science is recruiting reviewers for the journal. If you are interested in becoming a reviewer, we welcome you to join us. Please find the application form and details at http://recruitment.ccsenet.org and e-mail the completed application form to jas@ccsenet.org Journal of Agricultural Science is recruiting reviewers for the journal. If you are interested in becoming a reviewer, we welcome you to join us. Please find the application form and details at http://recruitment.ccsenet.org and e-mail the completed application form to jas@ccsenet.org p g p pp j @ g Reviewers for Volume 8, Number 12 Aman Chandi, DuPont Crop Protection, USA Amir Raza, Nuclear Institute for Food and Agriculture, Pakistan Ana Sanches Silva, National Institute of Health Dr Ricardo Jorge, Portugal Aureliano Albuquerque Ribeiro, Federal University of Ceara, Brazil Beatriz Sevilla-Moran, National Institute for Agricultural and Food Research and Technology, Spain C. Nasopoulou, University of the Aegean, Greece César A. Rosales Nieto, Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, Mexico Chaminda Shaman Herath, Coconut Research Institute of Sri Lanka, Sri Lanka Chao Di, University of Pennsylvania Medical Center, USA Chee Kong Yap, Universiti Putra Malaysia, Malaysia Eleonora Nistor, Banat University, Romania F. Bimbo, University of Foggia, Foggia, Italy Fernando Goulart, Universidade Federal de Minas Gerais, Brazil Francesco Vizzarri, University of Molise, Italy Graciele Araújo de Oliveira Caetano, UFVJM/UNIFAJ, Brazil Gustavo Perdoncini, Naturovos Company, Brazil Hoang Van Long, Da Nang Institute for Socio-Economic Development, Viet Nam Iuliana Vintila, Dunarea de Jos University, Romania Jose Manuel Brotons, Miguel Hernandez University, Spain N. Kishore, Palamuru University, India Paulo Gustavo Martins, Federal University of Jequitinhonha and Mucuri Valleys, Brazil Phokele Maponya, Agricultural Research Council-VOPI, South Africa R. Karina Gallardo, Washington State University, USA Rene Murrieta-Galindo, El Colegio de Veracruz (COLVER), Mexico Richard N. Onwonga, University of Nairobi, Kenya Sharifah Rahmah, Universiti Malaysia Terengganu, Malaysia Shivanand Hegde, University of Texas Medical Branch, USA Vipan Kumar, Montana State University, USA Willian Rodrigues Macedo, UFV Campus Rio Paranaíba, Brazil Wu-yi Liu, Fuyang Normal University, China Reviewers for Volume 8, Number 12 Reviewers for Volume 8, Number 12 Reviewer Acknowledgements Journal of Agricultural Science wishes to acknowledge the following individuals for their assistance with peer review of manuscripts for this issue. Their help and contributions in maintaining the quality of the journal are greatly appreciated. Aman Chandi, DuPont Crop Protection, USA Amir Raza, Nuclear Institute for Food and Agriculture, Pakistan Amir Raza, Nuclear Institute for Food and Agriculture, Pakistan Ana Sanches Silva, National Institute of Health Dr Ricardo Jorge, Portugal Aureliano Albuquerque Ribeiro, Federal University of Ceara, Brazil 214 Vol. 8, No. 12; 2016 Journal of Agricultural Science jas.ccsenet.org Xianlei Ma, Nanjing Agricultural University, China Xianlei Ma, Nanjing Agricultural University, China 215
https://openalex.org/W4224293574	https://www.nature.com/articles/s41467-022-29701-x.pdf	English	null	Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures	Nature communications	2,022	cc-by	12,980	Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures Susanne Fleig 1,2,13,14, Tamar Kapanadze1,2,14, Jeremiah Bernier-Latmani3, Julia K. Lill4, Tania Wyss 3,5, Jaba Gamrekelashvili 1,2, Dustin Kijas1,2, Bin Liu 6, Anne M. Hüsing 2, Esther Bovay7, Adan Chari Jirmo 6,8, Stephan Halle 9, Melanie Ricke-Hoch 10, Ralf H. Adams 7, Daniel R. Engel 4, Sibylle von Vietinghoff2,11, Reinhold Förster 9, Denise Hilﬁker-Kleiner10,12, Hermann Haller2, Tatiana V. Petrova 3 & Florian P. Limbourg 1,2✉ Susanne Fleig 1,2,13,14, Tamar Kapanadze1,2,14, Jeremiah Bernier-Latmani3, Julia K. Lill4, Tania Wyss 3,5, Jaba Gamrekelashvili 1,2, Dustin Kijas1,2, Bin Liu 6, Anne M. Hüsing 2, Esther Bovay7, Adan Chari Jirmo 6,8, Stephan Halle 9, Melanie Ricke-Hoch 10, Ralf H. Adams 7, Daniel R. Engel 4, Sibylle von Vietinghoff2,11, Reinhold Förster 9, Denise Hilﬁker-Kleiner10,12, Hermann Haller2, Tatiana V. Petrova 3 & Florian P. Limbourg 1,2✉ Susanne Fleig 1,2,13,14, Tamar Kapanadze1,2,14, Jeremiah Bernier-Latmani3, Julia K. Lill4, Tania Wyss 3,5, Jaba Gamrekelashvili 1,2, Dustin Kijas1,2, Bin Liu 6, Anne M. Hüsing 2, Esther Bovay7, Adan Chari Jirmo 6,8, Stephan Halle 9, Melanie Ricke-Hoch 10, Ralf H. Adams 7, Daniel R. Engel 4, Sibylle von Vietinghoff2,11, Reinhold Förster 9, Denise Hilﬁker-Kleiner10,12, Hermann Haller2, Tatiana V. Petrova 3 & Florian P. Limbourg 1,2✉ Tertiary lymphoid structures (TLS) are lymph node-like immune cell clusters that emerge during chronic inﬂammation in non-lymphoid organs like the kidney, but their origin remains not well understood. Here we show, using conditional deletion strategies of the canonical Notch signaling mediator Rbpj, that loss of endothelial Notch signaling in adult mice induces the spontaneous formation of bona ﬁde TLS in the kidney, liver and lung, based on molecular, cellular and structural criteria. These TLS form in a stereotypical manner around parenchymal arteries, while secondary lymphoid structures remained largely unchanged. This effect is mediated by endothelium of blood vessels, but not lymphatics, since a lymphatic endothelial- speciﬁc targeting strategy did not result in TLS formation, and involves loss of arterial spe- ciﬁcation and concomitant acquisition of a high endothelial cell phenotype, as shown by transcriptional analysis of kidney endothelial cells. This indicates a so far unrecognized role for vascular endothelial cells and Notch signaling in TLS initiation. 1 Vascular Medicine Research, Hannover Medical School, 30625 Hannover, Germany. 2 Department of Nephrology and Hypertension, Hannover Medical School, 30625 Hannover, Germany. 3 Vascular and Tumor Biology Laboratory, Department of Oncology UNIL CHUV and Ludwig Institute for Cancer Research, Lausanne, Switzerland. 1 Vascular Medicine Research, Hannover Medical School, 30625 Hannover, Germany. 2 Department of Nephrology and Hypertension, Hannover Medical School, 30625 Hannover, Germany. 3 Vascular and Tumor Biology Laboratory, Department of Oncology UNIL CHUV and Ludwig Institute for Cancer Research, Lausanne, Switzerland. 4 Department of Immunodynamics, Institute for Experimental Immunology and Imaging, Medical Research Centre, University Hospital Essen, 45147 Essen, Germany. 5 SIB Swiss Institute of Bioinformatics, Lausanne 1015, Switzerland. 6 Hannover Medical School, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany. 7 Max-Planck- Institute for Molecular Biomedicine, 48149 Muenster, Germany. 8 Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany. 9 Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany. 10 Department of Cardiology and Angiology, Hannover Medical School, 30625 Hannover, Germany. 11 Division of Medicine I, Nephrology section, UKB Bonn University Hospital, Bonn, Germany. 12 Department of Cardiovascular Complications of Oncologic Therapies, Medical Faculty of the Philipps University Marburg, 35037 Marburg, Germany. 13Present address: Department of Geriatric Medicine (Medical Clinic VI), RWTH Aachen University Hospital, 52074 Aachen, Germany. 14These auhtors contributed equally: Susanne Fleig, Tamar Kapanadze. ✉email: limbourg.ﬂorian@mh-hannover.de ARTICLE 22-29701-x NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications ARTICLE ARTICLE Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures TLSs develop in a variety of chronic inﬂammatory lesions3 and occur in auto- immune kidney diseases like Lupus nephritis4, ANCA-associated glomerulonephritis5, membranous glomerulonephritis6 or IgA- Nephritis7, but also in kidney transplants8. During infection, TLSs are beneﬁcial and are associated with pathogen clearance and increased survival. However, TLS can destroy normal kidney tissue and exacerbate autoimmune diseases and chronic rejection, suggesting that TLS are therapeutic targets in these conditions1. TLS resemble lymph nodes in cell composition and structure and form functional germinal centers9. Lymphocytes are attracted by chemokines like CXCL13 (B cells) and CCL19 (T cells) secreted by local stroma, and they in turn secrete lymphotoxins, which promote stroma differentiation towards lymphoid tissue ﬁbro- blastic reticular cells (FRC) and follicular dendritic cells (FDC)10. T While many studies have focused on immune cell contribution, little is known about the vascular regulation of TLS generation. TLSs contain specialized high endothelial cells (HECs), often organized in high endothelial venules (HEV), that recruit passing myeloid cells and lymphocytes via peripheral lymph node addressin PNAd, a glycoprotein ligand for L-selectin (CD62L) expressed by high endothelial venules in lymph nodes required for lymphocyte egress9. HECs strongly differ from lymph node capillary endothelial cells (ECs) in transcriptional signature11–13. HECs demonstrate enrichment in transcripts involved in the regulation of inﬂammatory response, leukocyte migration, and lymph node development. Interestingly, Notch signaling com- ponents and its downstream targets, as well as endothelial cell differentiation markers, are strongly downregulated in HEC11. Histologically, we observed signiﬁcant, but localized lympho- cytic inﬁltrations, consistent with TLSs23, clustered around segmental and interlobar arteries in kidneys of RbpjΔEC mice, but not in littermate controls (Fig. 1D, E). Aside from these inﬁltrations, the overall kidney architecture was preserved, although TLS displaced normal renal structures (Fig. 2A, Supplementary Fig. 2A). In areas of lymphocyte inﬁltration, Masson Trichrome and Sirius Red staining demonstrated focal interstitial matrix deposition and areas of ﬁbrosis (Supplementary Fig. 2A). Overall kidney function as measured by serum creatinine and proteinuria was not altered in RbpjΔEC mice; however, kidney Havcr1 gene expression (kidney injury molecule 1) was signiﬁcantly increased, suggesting subclinical renal injury (Supplementary Fig. 2B–D). Interestingly, 3D reconstruction of light sheet microscopy images of CD31- and B220-stained whole kidneys conﬁrmed extensive but stereotyped periarterial expan- sion of TLS along the segmental and interlobar arteries, forming central conglomerates in the renal medulla (Fig. 1E, Supplemen- tary Movie 1). Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures Thus, loss of endothelial Notch signaling induced spontaneous formation of renal TLS around second and third- order arteries without overt systemic or chronic inﬂammation. g y g Canonical Notch signaling is an evolutionary conserved, cell- contact dependent signaling pathway14. Activation of one of four membrane-bound Notch receptors by Notch ligands leads to Notch receptor intracellular domain (NICD) cleavage and translocation to the nucleus, where it associates with DNA-bound Rbpj and initiates transcription of target genes. Notch is a key player in vasculo- and angiogenesis during development14–16, and regulates arterial phenotype of endothelial cells and arterial EC identity in the adult17,18. Inversely, suppression of Notch by COUP-TF2 permits venous endothelial phenotype19. y Since RbpjΔEC mice have a pan-endothelial deletion of Notch signaling, we next analyzed liver and lung, parenchymatous organs prone to develop TLS24–28, and also the heart29. Consistent with a general role of Notch signaling in the regulation of parenchymatous TLS, we found regular TLS in all livers and lungs analyzed, but not the heart (Fig. 1G, Supplementary Fig. 2E–G). Here we show that conditional loss of Notch signaling by deletion of the canonical mediator Rbpj in blood vascular- endothelial cells modiﬁes arterial endothelial identity and shifts it to an HEV-like phenotype, which in turn is associated with the spontaneous formation of TLS in mouse kidney, liver, and lung. Our results highlight an essential and unexpected role of arterial endothelium in formation of TLS and suggest targeting the endothelial Notch pathway as a novel approach for modulating organ-speciﬁc immunity. TLSs show regular lymphoid and stromal tissue architecture and germinal center formation. To characterize in more detail the TLS that formed spontaneously in RbpjΔEC mice, we stained kidney cross-sections with markers of proto-typical lymphoid tissue cell components. As a general principle, TLSs were orga- nized around a central artery and were structured by a network of stromal cells expressing the FRC marker podoplanin30. This network was connected to the central artery and showed occa- sional enlarged and ﬁlled lymph vessels (Fig. 2A). Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures HECs demonstrate enrichment in transcripts involved in the regulation of inﬂammatory response, leukocyte migration, and lymph node development. Interestingly, Notch signaling com- ponents and its downstream targets, as well as endothelial cell differentiation markers, are strongly downregulated in HEC11. Canonical Notch signaling is an evolutionary conserved, cell- contact dependent signaling pathway14. Activation of one of four membrane-bound Notch receptors by Notch ligands leads to Notch receptor intracellular domain (NICD) cleavage and translocation to the nucleus, where it associates with DNA-bound Rbpj and initiates transcription of target genes. Notch is a key player in vasculo- and angiogenesis during development14–16, and regulates arterial phenotype of endothelial cells and arterial EC identity in the adult17,18. Inversely, suppression of Notch by COUP-TF2 permits venous endothelial phenotype19. Here we show that conditional loss of Notch signaling by deletion of the canonical mediator Rbpj in blood vascular- endothelial cells modiﬁes arterial endothelial identity and shifts it to an HEV-like phenotype, which in turn is associated with the spontaneous formation of TLS in mouse kidney, liver, and lung. Twelve weeks after induction, RbpjΔEC showed a higher frequency of mature B-lymphocytes (CD45+, CD19+, B220+) and T-lymphocytes (CD45+, CD3+) in the kidney by ﬂow cytometry, while B cell frequencies were reduced in peripheral blood and bone marrow (Fig. 1B, Supplementary Fig. 2A, B; 1L). The increase in renal B cells was driven by follicular B lymphocytes, not progenitors or other subtypes (Fig. 1C; Supplementary Fig. 1C, D). No changes were detected in B cell bone marrow niche cytokine expression of Il7 and Cxcl12 (Supplementary Fig. 1F). Furthermore, cell numbers and frequencies of B and T cells in secondary lymphoid organs such as the spleen or lymph nodes were comparable between RbpjΔEC and control mice (Fig. 1B), and spleen size was not changed (Supplementary Fig. 1G). RbpjΔEC mice also showed an increased frequency of dendritic cells in the kidney, bone marrow and spleen (Fig. 1B, lower panel; Supplementary ﬁg. 1H). In contrast, we observed no difference in neutrophilic granulocytes, monocyte subsets or macrophages between RbpjΔEC and control mice, and no signs of overt systemic inﬂammation (Fig. 1B, lower panel, supplementary Fig. 1H). Together, these ﬁndings suggest active lymphocyte recruitment to the kidney. T ertiary lymphoid structures (TLSs) are de novo generated lymphoid structures in non-lymphoid organs like the kidney that develop in response to chronic inﬂammation and sustain chronic immune responses1,2. Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures 4 Department of Immunodynamics, Institute for Experimental Immunology and Imaging, Medical Research Centre, University Hospital Essen, 45147 Essen, Germany. 5 SIB Swiss Institute of Bioinformatics, Lausanne 1015, Switzerland. 6 Hannover Medical School, Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany. 7 Max-Planck- Institute for Molecular Biomedicine, 48149 Muenster, Germany. 8 Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany. 9 Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany. 10 Department of Cardiology and Angiology, Hannover Medical School, 30625 Hannover, Germany. 11 Division of Medicine I, Nephrology section, UKB Bonn University Hospital, Bonn, Germany. 12 Department of Cardiovascular Complications of Oncologic Therapies, Medical Faculty of the Philipps University Marburg, 35037 Marburg, Germany. 13Present address: Department of Geriatric Medicine (Medical Clinic VI), RWTH Aachen University Hospital, 52074 Aachen, Germany. 14These auhtors contributed equally: Susanne Fleig, Tamar Kapanadze. ✉email: limbourg.ﬂorian@mh-hannover.de 1 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x T ertiary lymphoid structures (TLSs) are de novo generated lymphoid structures in non-lymphoid organs like the kidney that develop in response to chronic inﬂammation and sustain chronic immune responses1,2. TLSs develop in a variety of chronic inﬂammatory lesions3 and occur in auto- immune kidney diseases like Lupus nephritis4, ANCA-associated glomerulonephritis5, membranous glomerulonephritis6 or IgA- Nephritis7, but also in kidney transplants8. During infection, TLSs are beneﬁcial and are associated with pathogen clearance and increased survival. However, TLS can destroy normal kidney tissue and exacerbate autoimmune diseases and chronic rejection, suggesting that TLS are therapeutic targets in these conditions1. TLS resemble lymph nodes in cell composition and structure and form functional germinal centers9. Lymphocytes are attracted by chemokines like CXCL13 (B cells) and CCL19 (T cells) secreted by local stroma, and they in turn secrete lymphotoxins, which promote stroma differentiation towards lymphoid tissue ﬁbro- blastic reticular cells (FRC) and follicular dendritic cells (FDC)10. While many studies have focused on immune cell contribution, little is known about the vascular regulation of TLS generation. TLSs contain specialized high endothelial cells (HECs), often organized in high endothelial venules (HEV), that recruit passing myeloid cells and lymphocytes via peripheral lymph node addressin PNAd, a glycoprotein ligand for L-selectin (CD62L) expressed by high endothelial venules in lymph nodes required for lymphocyte egress9. HECs strongly differ from lymph node capillary endothelial cells (ECs) in transcriptional signature11–13. Results and discussion Loss of endothelial Notch signaling induces spontaneous for- mation of TLSs. To study the role of Notch signaling in vascular and immune homeostasis in the kidney, we generated Cdh5CreERT2;Rbpjﬂ/ﬂ(RbpjΔEC) transgenic mice by crossing conditional alleles of the canonical Notch effector Rbpj and an endothelial-speciﬁc and inducible Cre-recombinase20,21. We then induced Cre-recombinase activity by Tamoxifen injections at 7–9 weeks of age22, after completion of developmental angio- genesis and vascular remodeling (Fig. 1A), and conﬁrmed recombination of the Rbpj locus and downregulation of the Notch target gene Hey1 (Supplementary Fig. 1A, B). g y p g TLS contained B and T cells organized into distinct T and B cell-zones (Fig. 2B), and staining for GL7- and IgD-positive B cells showed regular germinal center formation (Fig. 2C)31, which was in line with a follicular B cell phenotype by ﬂow cytometry (Fig. 1C). Furthermore, a high percentage of B cells were KI67+, NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x Fig. 1 Spontaneous periarterial formation of TLS in conditional endothelial Rbpj mutant mice. A Induction protocol for Cdh5CreERT2; Rbpjﬂ/ﬂmice and Cre-negative littermates. B Flow cytometry, % of live cells, box plots with mean, 25–75th percentile (Inter-Quartile-Range, IQR, bounds of box) and total range (min-max, whiskers); Mann–Whitney test, two-tailed. CTRL n = 9, KO n = 6 mice; 3 independent experiments. C Flow cytometry representative plots for % follicular B lymphocytes in Ctrl and RbpjΔEC kidneys. Numbers indicate % of live CD45 + (upper panel)/% of CD45+/CD19+ cells (lower panel). D PAS staining of representative parafﬁn-embedded kidney sections in different arterial segments (sketch on left created with biorender.com). Upper row, interlobar arteries, lower row segmental arteries, magniﬁcation ×50 (overview, bar = 1000 µm) and boxed details magniﬁcation ×200 (bar = 50 µm). Inset: arterial lumen (arrowhead) within TLS structure. Experiment independently repeated with similar results >×3. E Quantiﬁcation of inﬁltrated area [in mm2] per transversal kidney cross-section (sum of all inﬁltrated areas per section). N = 10 mice per group, Mann–Whitney test, two-tailed, exact p-value 0.0021; Graph: Scatter dot blot, mean, standard deviation (SD). **p < 0.01. F Whole-mount kidney staining and light sheet imaging of CD31 (red) and B220 (green) of RbpjΔEC kidney; 3D reconstruction with IMARIS software; ventral view left with a ﬁlter for larger vascular structures; sagittal view middle and right, with magniﬁcation of inset. Representative image, kidneys of n = 3 mice were stained. G Representative images of IF stained liver (upper) and lung (lower image) TLS in RbpjΔEC mice, B220 positive cells in green. Organs of n = 3 mice were stained with similar results. Scale bars as marked. Source data are provided as a Source Data ﬁle. To test whether TLS formation was secondary to lymphatic-EC targeting of Notch signaling, we conditionally deleted Rbpj with the lymphatic-EC speciﬁc43, inducible Prox1CreERT2;Rbpjﬂ/ﬂ mouse model (RbpjΔLEC, Fig. 3F; recombination control in Supplementary Fig. 3B, C). In contrast to RbpjΔEC mice, which showed lower body weight at the end of the observation period (Supplementary Fig. 2H), age-matched RbpjΔLEC mice had normal weight compared to littermate controls and also showed no cardiac phenotype (Fig. 3G). Importantly, there was no evidence of TLS development in RbpjΔLEC mice, neither by histologic examination of the periarterial area covered by lymphatic/mononuclear cells (Fig. 3H), nor by immunoﬂuores- cent staining and quantiﬁcation of B-/T-lymphocytes (Fig. 3I). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x Thus, induced Notch loss of function in lymphatic EC is not sufﬁcient to induce spontaneous TLS formation. Together, these data demonstrate that loss of function of vascular-endothelial Notch signaling promotes TLS formation. honeycomb pattern into the periphery (Fig. 2G). Furthermore, immunostaining also revealed expression of the TNF-superfamily member RANKL in the periphery of TLS, which is expressed by lymphoid tissue inducer cells and mesenchymal lymphoid tissue organizer (LTO) cells37 and induces B cell chemokines in FRC38 (Fig. 2H). To corroborate a TLS molecular signature, we analyzed gene expression of whole kidney samples from control or RbpjΔEC mutant mice, which revealed signiﬁcant upregulation of Cxcl13, Cxcr5, Cxcr4, Ccl19, and Baff, prototypical genes involved in attraction and accumulation of B cells, and to some extent T cells, in TLS (Fig. 2J). This extended analysis demonstrates on a molecular, cellular, and structural level the formation of TLS after induced loss of function of endothelial Notch signaling in adult mice. TLS formation occurs independent of cardiac or lymphatic disease phenotypes but involves arteries as general guiding structures. To address the role of potential confounding expla- nations for the development of TLS we performed several control experiments. RbpjΔEC mice develop cardiac failure around 13–16 weeks of induction (Supplementary Fig. 2I)29. In order to investigate whether renal TLS formation in RbpjΔEC mice was secondary to cardiac failure we examined a genetically different mouse model of heart failure induced by myocardial-restricted deletion of Stat3 (αMHCCre;Stat3ﬂ/ﬂ= Stat3ΔMyoc)39. Male Stat3ΔMyoc mice develop age-related heart failure with dilatative cardiomyopathy (DCM) associated with lower myocardial capil- lary density starting at 6 months of age (Fig. 3A)39. However, although mice developed the full clinical picture of heart failure, indicated by an increased heart weight and expression of Nppa (ANP) (Fig. 3B), there was no evidence for TLS formation in the kidney of mutant mice, neither by lymphocyte quantiﬁcation by ﬂow cytometry (Fig. 3C) nor histologic examination (Fig. 3D). Furthermore, gene expression proﬁling also did not show TLS- associated changes observed in RbpjΔEC mutant mice (no differ- ence in kidney mRNA for Cxcl13, Cxcl12, Cxcr5, Cxcr4, and Ccl19, supplementary ﬁg 3A. Thus, heart failure per se does not lead to spontaneous development of TLS. To examine whether arteries are common guiding structures for TLS formation in kidney we analyzed an unrelated, inﬂammatory mouse model of unilateral ischemia-reperfusion injury (Supplementary Fig. 4A–C). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x indicating active proliferation (Fig. 2F, I). Thus, these data demonstrate a B cell phenotype consistent with mature TLS. We next studied the stromal components in TLS. CXCL13 is the major chemokine expressed by FDC attracting B-lymphocytes during the formation of lymphoid structures30,32. B cells in RbpjΔEC kidneys clustered around CXCL13-expressing cells (Fig. 2D), which co-expressed CD21/35, consistent with FDC phenotype28,33 (Fig. 2E). In spleen or lymph nodes, stromal ﬁbroblastic reticul cells (FRC) form conduits that guide B and T cells30. Stroma ce in RbpjΔEC TLS stained positive for FRC-markers podoplan (PDPN, Fig. 2A) and ER-TR7 (Fig. 2G)1,10,34–36, forming condu distinct from PDPN+/Lyve1+ lymphatic vessels (Fig. 2A, inset Notably, the conduit network formed by FRC in TLS was center around and closely attached to the central artery, extending in \| ( ) \| h d \| (Fig. 2E). In spleen or lymph nodes, stromal ﬁbroblastic reticular cells (FRC) form conduits that guide B and T cells30. Stroma cells in RbpjΔEC TLS stained positive for FRC-markers podoplanin (PDPN, Fig. 2A) and ER-TR7 (Fig. 2G)1,10,34–36, forming conduits distinct from PDPN+/Lyve1+ lymphatic vessels (Fig. 2A, insets). Notably, the conduit network formed by FRC in TLS was centered around and closely attached to the central artery, extending in a indicating active proliferation (Fig. 2F, I). Thus, these data demonstrate a B cell phenotype consistent with mature TLS. 3 ATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x Eleven weeks after injury, TLSs were found around segmental arteries in a stereotyped manner (Fig. 3K, Suppl. Figure 4D), which corroborates previous observations in several unrelated injury models, but also human kidneys10. This suggests periarterial organization as a general principle for TLS formation. Loss of endothelial Notch signaling induces an EC phenotype shift from arterial to high endothelial signature and is asso- ciated with chronic inﬂammatory kidney disease. Notch is a key regulator of endothelial identity. Active Notch signaling induces and maintains an arterial phenotype15,44, while low Notch sig- naling is associated with a venous phenotype, including the HEC phenotype found in lymph nodes11. We therefore hypothesized that induced endothelial Notch loss-of-function would lead to arterial dedifferentiation and an HEC phenotype shift, thereby promoting TLS formation. Compared to kidneys of littermate controls, kidneys of RbpjΔEC mice showed signiﬁcantly reduced expression of the arterial marker Efnb2, involved in the arterial speciﬁcation, but upregulated expression of Aplnr, identiﬁed as a venous marker which is downregulated at the onset of arterial speciﬁcation45 (Fig. 4A). Our genetic targeting strategy also affects lymphatic endothelial cells40. Since TLS in RbpjΔEC mice showed lymph vessels ﬁlled with mononuclear cells (see Fig. 2A, purple arrowheads), which might be caused by obliteration of efferent lymph vessels as described in the setting of CLEC2-deﬁcient lungs41, we ﬁrst stained whole kidneys with antibodies to B220 and LYVE1/Prox1 and imaged TLS/lymphatic architecture by light sheet micro- scopy. After 3D reconstruction, efferent lymphatic vessels appeared open with a continuous lumen within and outside the TLS structure (Fig. 3E, Supplementary movie 2), resembling normal renal lymphatics42. g Furthermore, in addition to reduced expression of the Notch target gene Hey1 (Supplementary Fig. 1A), the Notch signaling components Notch1, Dll4 and Jag1, which are enriched in arterial endothelium45,46, were also downregulated in RbpjΔEC kidneys, consistent with loss of arterial EC identity (Fig. 4A). y g At the same time, expression of peripheral lymph node addressin PNAd, glycoprotein ligands expressed by high NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunicati 4 4 ARTICLE NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x Fig. 2 Molecular, cellular and structural composition of periarterial TLS. A–I Immunoﬂuorescence staining and confocal laser scanning microscopy of representative RbpjΔEC kidney samples, merged and single channels as indicated. “A” indicates artery. Optical Magniﬁcation 200x; different scan areas (see scale bars). Scale: solid bar 50 µm, dotted bar 10 µm (2E). Each micrograph is representative of at least 4 biological replicates. J Whole kidney mRNA expression, relative fold change to control gene Rps9, n = 10/group. Graphs: Scatter dot blot, mean, SD (whiskers). Mann–Whitney test, two-tailed, Exact p-values: Cxcl13, p = 0.0003; Cxcl12, p = 0.393; Cxcr5, p = 0.0433; Cxcr4, p = 0.0288; Ccl19, p = 0.0052; Baff, p = 0.0007; Rankl, p = 0.0005. Source data are provided as a Source Data ﬁle. Fig. 2 Molecular, cellular and structural composition of periarterial TLS. A–I Immunoﬂuorescence staining and confocal laser scanning microscopy of representative RbpjΔEC kidney samples, merged and single channels as indicated. “A” indicates artery. Optical Magniﬁcation 200x; different scan areas (see scale bars). Scale: solid bar 50 µm, dotted bar 10 µm (2E). Each micrograph is representative of at least 4 biological replicates. J Whole kidney mRNA expression, relative fold change to control gene Rps9, n = 10/group. Graphs: Scatter dot blot, mean, SD (whiskers). Mann–Whitney test, two-tailed, Exact p-values: Cxcl13, p = 0.0003; Cxcl12, p = 0.393; Cxcr5, p = 0.0433; Cxcr4, p = 0.0288; Ccl19, p = 0.0052; Baff, p = 0.0007; Rankl, p = 0.0005. Source data are provided as a Source Data ﬁle. showed no evidence of PNAd expression (Fig. 4B; Supplementary Fig. 4E). Furthermore, PNAd decorated the apical aspects of arterial endothelium, as observed in differentiated HEC in lymph nodes. In addition, peritubular capillaries and vasa recta in the papilla also expressed PNAd. endothelial venules in lymph nodes required for lymphocyte egress via L-selectin (CD62L)47, was markedly enhanced in selected vascular beds in mutant kidneys. Remarkably, PNAd staining revealed strong expression in arterial endothelium in central TLS arteries, while endothelium in control kidneys 5 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x Functionally HEC create “pockets” for mononuclear cells on per sample Principal component analysis revealed predominant Functionally, HEC create “pockets” for mononuclear cells on their basal side before transmigration47,48. Consistent with transformation to HEC functionality, we observed B-lymphocyte clusters underneath the endothelium and within the vascular wall of central arteries of TLS in RbpjΔEC mice (Fig. 4C). ARTICLE Fig. 3 TLS formation in models of heart failure, conditional lymphatic-EC deletion of Rbpj or kidney ischemia reperfusion. A Experimental set up for analysis of cardiomyocyte restricted deletion of Stat3 (Stat3ΔMyoc). B Heart weight to femur length ratio (HW/FL, p = 0.0159) and cardiac ANP (Nppa) mRNA expression, p = 0.0079; CTRL n = 5, KO n = 5. Mann–Whitney test, 2-tailed, Graphs: Scatter dot blot, Mean, SD (whiskers). C Quantiﬁcation by lymphocytes by ﬂow cytometry of kidney homogenates; % of live cells, box plots with mean, IQR (25–75%, bounds of box) and total range (min-max: whiskers); CTRL n = 5, KO n = 5. D PAS staining of representative, parafﬁn-embedded kidney sections. Quantiﬁcation of inﬁltrated area [in mm2] per transversal kidney cross-section (sum of all inﬁltrated areas per section), N = 8 biological replicates per group, Mann–Whitney test, two-tailed, p = 0.51. Graph: Scatter dot blot, Mean, SD (whiskers). E RbpjΔEC Whole kidney staining for B220 (TLS) and Prox1/Lyve1 for lymphatic collecting vessels, light sheet microscopy, ventral view, 3D reconstruction via IMARIS software; scale bar: left image 1000 µm; insets are magniﬁcations of boxed detail, scale bar 150 µm. Exemplary image, kidneys from N = 3 mice stained. F Induction protocol for lymphatic endothelial-restricted deletion of Rbpj (RbpjΔLEC). G Body weight (n = 8/group, p = 0.3409) and heart weight to femur length ratio (n = 7/group, p = 0.9272) in 20–22-week-old mice (12 week after KO induction, from two independent experiments); Graphs: Scatter dot blot, mean (box), SD (whiskers). Mann–Whitney test, two-tailed. H Parafﬁn-embedded kidney sections, PAS staining, optical magniﬁcation: ×50 left, ×200 detail, scale bar: 1000 µm and 50 µm, as indicated. Quantiﬁcation of inﬁltrated area [in mm2] per transversal kidney cross-section (sum of all inﬁltrated areas per section), N = 11 CTRL, N = 8 RbpjΔLEC mice per group, Mann–Whitney test, two-tailed, p = 0.7168. Graph: Scatter dot blot, mean (box), SD (whiskers). I Upper panels: immunoﬂuorescence staining, optical magniﬁcation: 200x, scale bar: 50 µm. Lower panels: quantiﬁcation of periarterial B and T cells per microscopic image (each value = mean #cells per periarterial area of all such areas per one cross-section). CTRL n = 8, KO n = 7 mice analyzed from 2 independent experiments, Mann–Whitney test, 2-tailed. Graphs: Scatter dot blot, mean (box), SD (whiskers). ARTICLE J Immunoﬂuorescence staining as indicated and confocal laser scanning microscopy of kidney 11 weeks after ischemia reperfusion (I/ R) injury, optical magniﬁcation ×200, scale bar: 50 µm; see also Supplementary Fig. 4; representative picture; n = 3 animals in I/R-injury group. Source data are provided as a Source Data ﬁle. kidney and liver in RbpjΔaEC mice (Supplementary Fig. 6B), nor an increase in dendritic cells, as would be expected in TLS (Supplementary Fig. 6C). Histologically, there were no observable perivascular B cell inﬁltrates in RbpjΔaEC kidney, liver or heart (Supplementary Fig. 6D). To evaluate Cre-dependent targeting in this adult mouse model we analyzed Cre-dependent reporter gene expression in the kidney of BmxCreERT2; Gt(ROSA)26Sortm4(ACTB- tdTomato,-EGFP)Luo/J mice (BmxCreERT2;mTmG). While EC recom- bination in proximal segmental arteries was generally high, recombination in second and third-order renal arteries was incomplete (Supplementary Fig. 6E). Thus, our data suggest that loss of proximal arterial EC Notch signaling is not sufﬁcient to induce TLS formation. The role of lower segmental arterial EC Notch signaling in TLS formation deserves further study. Supplementary Data ﬁle 2). On whole gene expression level, endothelial cell differentiation gene signatures, matrix organiza- tion and EC barrier function along with Notch signaling signatures were signiﬁcantly downregulated in mutant EC. g g y g To compare the observed gene expression pattern changes of renal EC to previously described gene signatures derived from single-cell RNAseq analysis of various kidney EC populations, we ﬁrst performed GSEA with deﬁned renal arterial cell transcrip- tomic signatures49,50. In each arterial transcriptomic signature - i. e. large artery, cortical artery, cortical arteriole, medullary arteriole, arteriole efferent—signiﬁcant and consistent downregulation was registered in RbpjΔEC EC (Fig. 5C). In contrast, GSEA using as gene signature the transcriptomic proﬁles of homeostatic HEC12 revealed signiﬁcant upregulation in mutant EC (NES = 2.3, Fig. 5D). Together, these ﬁndings demonstrate that loss of renal endothelial Notch signaling induces an EC phenotype shift characterized by renal arterial dedifferentiation and HEC gene upregulation. We here show that loss of endothelial Notch signaling in adult mice induces the spontaneous formation of bona ﬁde TLS, based on molecular, cellular, and structural criteria, in several parenchymatous organs. This effect is mediated by the endothe- lium of blood vessels, but not lymphatics, since a lymphatic-EC- speciﬁc targeting strategy did not result in TLS formation; nor was this secondary to heart failure alone. ARTICLE While our screening analysis of mutant mice did not ﬁnd evidence for TLS formation in the heart, this certainly does not rule out the possibility of TLS formation in the heart, which might occur in structures not included in our analysis or in a different time frame. Since our data suggested a close link between loss of endothelial Notch signaling and TLS formation we next analyzed the expression of Notch target genes in various kidney diseases associated with TLS formation in the European Renal cDNA Bank (ERCB) cohort repository51. To this end, we compared human kidney biopsy RNAseq datasets from healthy living kidney donors with various forms of inﬂammatory glomerulone- phritis causing either nephrotic syndrome (focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), membranous glomerulonephritis) or nephritic syndrome (IgA- nephritis, systemic lupus erythematodes (SLE), rapid-progressive glomerulonephritis (RPGN)). Expression of the Notch target gene HES1 was signiﬁcantly and uniformly decreased in all disease entities compared to living donor (Fig. 5E). Furthermore, while expression of the Notch target HEY1 was signiﬁcantly down- regulated only in FSGS, the Notch target and related gene product HEYL was signiﬁcantly decreased in all inﬂammatory kidney diseases except membranous GN. This demonstrates that Notch downregulation is associated with TLS-forming chronic inﬂam- matory kidney diseases, which may indicate involvement in human TLS formation. y TLS formation in mutant mice occurred in a stereotypical manner around second and third-degree renal arteries, which was also observed in several unrelated conditions of kidney injury and during physiological aging in both human and mouse kidneys10. This is a so far underappreciated pattern of renal TLS organization. Interestingly, TLS generation has also been described around major non-renal arteries during athero- sclerosis development23,53, which suggests a general mechanism of TLS formation guided by arterial structures. Systematic studies are required to investigate this hypothesis further. A key role for arteries in the generation of local TLS is also suggested by the fact that arteries in the center of TLS displayed key features of a HEC phenotype: EC decorated with PNAd at the luminal side, leukocyte pockets, integration into FRC conduit networks expressing CXCL13 and, by experimental design, low Notch signature9,11. Why cuboidal EC shape was not observed is unclear, but EC height is sensitive to vasoconstriction, pressure, and most importantly, tissue ﬁxation, which could account for the ﬂat appearance of EC54. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x Furthermore, whole kidney gene expression analysis revealed robust upregula- tion of cell-adhesion molecule Madcam-1, another HEV marker, and borderline upregulation of P-selectin (Selp), L-selectin (Sell), and Vcam1 in RbpjΔEC kidneys (Fig. 4D). per sample. Principal component analysis revealed predominant clustering by genotypes, corroborating genetic interference in mutant kidney ECs (Fig. 5A). Hey1 and Jag1 were downregulated in RbpjΔEC endothelial cells, corresponding to whole kidney QRT results (DeSeq2 results are provided in Supplementary Data ﬁle 1, a heatmap of differentially regulated genes in Suppl. data ﬁle 4; a Volcano plot with differentially regulated genes in Supplementary Fig. 5D). Using gene set enrichment analysis (GSEA), we found that loss of Rbpj in kidney ECs resulted in 1837 gene sets being signiﬁcantly downregulated, while 123 gene sets were signiﬁcantly upregulated. A selection of up- and downregulated Gene Ontology (GO) terms with their respective normalized enrich- ment score (NES) is shown in Fig. 5B (full results in To characterize the global gene expression changes of endothelial Notch loss of function we isolated kidney ECs from 3 biological replicates per group (Supplementary Fig. 5A–C) and performed bulk-EC-RNA deep sequencing with >60 million reads NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x ARTICLE ARTICLE However, apical PNAd expression, Finally, to test whether loss of vascular-arterial EC Notch signaling is sufﬁcient to induce TLS formation, we generated arterial EC-speciﬁc BMXCreERT2;Rbpjﬂ/ﬂmice (RbpjΔaEC)52 and repeated the experiment (Supplementary Fig. 6A). By ﬂow cytometry we found no increase in CD19+B220+ B cells in 7 7 NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications ARTICLE ARTICLE Fig. 4 Endothelial signatures in TLS development. A Whole kidney mRNA expression, n = 10 mice per group from 2 independent experiments, Mann–Whitney test, two-tailed. Exact p-values: Aplnr, p = 0.003; Efnb2, p = 0.0068; Notch1, p = 0.0789; Dll4, p = 0.0101; Jag1, p = 0.0033; Graphs: scatter dot blot, mean, SD (whiskers). B Immunoﬂuorescence staining and confocal microscopy of RbpjΔEC kidney sections. Segmental artery (upper panel), peritubular capillaries (middle panel) and papillary region (lower panel), optical magniﬁcation: ×200, scale bar: 50 µm. Representative image, n = 3/group stained. C Immunoﬂuorescence staining and confocal microscopy of central TLS artery (segmental), inset with higher magniﬁcation. B lymphocytes in subendothelial pockets (white arrowheads). Optical magniﬁcation: ×200, all scale bars 50 µm. Representative image, n = 5/group stained. D Whole kidney mRNA expression, relative fold change, n = 10/group. Mann–Whitney test, two-tailed. Exact p-values: Selp p = 0.089; Sell p = 0.052; Madcam p = 0.0052; Vcam p = 0.071; Graphs: scatter dot blot, mean, SD (whiskers). Source data are provided as a Source Data ﬁle. HEC in secondary lymphatic structures develop after birth and express Nr2f2, which encodes the master venous regulator NR2F2 (COUP-TFII), a repressor of Notch signaling19. At the same time, genes associated with arterial speciﬁcation, e. g. Efnb2, Notch1, Dll4, and several Notch target genes, are uniformly down- regulated in HEC, when compared to capillary EC11. Conversely, active Notch signaling induces and maintains, in a dose- dependent manner, an arterial EC phenotype15,44,45,57,58. In fact, Notch signaling activity deﬁnes a developmental trajectory from venous to capillary to early/late arterial EC, in which HECs align to the low Notch signaling spectrum11,45. adaptive immune response arising de novo in affected organs67–69. The functional dependence on arterial signature changes, which involves downmodulation of Notch signaling by inﬂammatory conditions and regional conversion to HEC phenotype, could represent an evolutionary conserved mechan- ism to assemble lymphoid structures in affected organs, since Notch signaling is an ancient and evolutionary conserved signaling pathway. Our observation also provides a rationale for the described association of old age and development of TLS10, since Notch signaling components and Notch-dependent arteries and vascular networks in the bone decrease with age. This decrease can be rescued by endothelial overexpression of Notch, suggesting impairment of endothelial Notch signaling with age70. Methods Mi dh Mice. Cdh5CreERT2; Rbpjf/f (B6-Tg(Cdh5(PAC)-cre/ERT2)1Rha Rbpsuh tm3Hon/ Rbrc) were generated by crossing Cdh5(PAC)-CreERT2 mice71 with Rbpjf/f mice20; BmxCreERT2;Rbpjf/f mice (B6-Tg(Bmx(PAC)-cre/ERT2)1Rha Rbpsuh tm3Hon/Rbrc) were generated by crossing Bmx(PAC)-CreERT21Rha,52 mice with Rbpjf/f mice20. Cdh5CreERT2;TdTomato+/+ mice were generated by crossing Cdh5(PAC)-CreERT2 mice71 with B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Jackson Labora- tories Strain #:007914). αMHCCre;Stat3f/f mice were described before39. LDLr−/−mice were purchased from Jackson Laboratories (strain 002207,72). All mice were housed under speciﬁc pathogen-free conditions (Type 22 polysulfon IVC systems with positive pressure) in the animal facility of Hannover Medical School with a 14/10 h light/dark cycle (generated by a 400 lx light source), 21 ± 2 °C ambient temperature and 50 ± 5% relative humidity, and were supplied with autoclaved water and food (Altromin TPF-1324) ad libitum. All experiments were performed with male mice and age- matched littermate controls with the approval of the local animal welfare board (LAVES, Lower Saxony, Animal Studies Committee, protocol numbers 15/1944, 18/2931, and 18/2973; breeding and echocardiography on αMHCCre;Stat3f/f on protocols 33.12-42502-04-18/2807 and 33.19-42502-05-18A271). BMXCreERT2;mTmG mice were generated by crossing BMX(PAC)-Cre/ ERT21Rha,52 mice with Gt(ROSA)26SORtm4(ACTB-tdTomato-EGFP)Luo/J mice73. A recombination control experiment was performed with approval of LANUV, Northrhine-Westphalia, protocol number Az 81-02.04.2019.A114. p , p Prox1CreERT2; Rbpjf/f mice were generated by crossing Prox1-ERT2 mice43 with Rbpjf/f mice20; and Prox1CreERT2;mTmG mice were generated by crossing Prox1CreERT2 mice43 with Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J mice73. Mice were provided water and food (Scientiﬁc Animal Food & Engineering, R150) ad libitum. Mice were on a 12 h light/dark cycle and kept at 22 °C ± 2 °C with a relative humidity of 55%±10%. Experiments with these mice were performed with the approval of the Animal Ethics Committee of Vaud, Switzerland, protocol number VD2914. Our analysis also revealed that secondary lymphoid tissues, such as lymph nodes and spleen, were largely unaffected in mutant mice, judged by size and cell numbers. A small but signiﬁcant increase in dendritic cell numbers was noted, which is consistent with TLS development, due to their role in TLS formation66. Disruption of endothelial Notch signaling is expected to have the greatest impact in vascular beds with high Notch signaling activity, e. g. arteries and certain capillary beds, but little effects in veins or HEV with low Notch signaling activity11,45. Our data therefore are in line with the hypothesis that change in the arterial signature is involved in TLS formation. The capacity to form TLS represents a sustained form of an Experimental procedures. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701- together with the absence of MAdCAM-1, is a key feature of mature HEC in peripheral LN HEV mediating lymphocyte recruitment in a CD62L-dependent manner55, which, in combination with leukocytes in subendothelial pockets, suggests acquisition of HEC function after Notch loss-of-function. In addition, upregulation of several cell-adhesion molecules, such as Sell, Selp, Madcam1, or Vcam1, was observed in whole kidney RNA, which was not reﬂected in EC transcriptomic analysis (Supplementary Table 1), suggesting that upregulation of these cell-adhesion molecules occurs in recruited leukocytes or a distinct population of reticular stromal cells of lymphoid tissues, which support lymphocyte recruitment56. 8 CO C O \| ( ) \| h d \| together with the absence of MAdCAM-1, is a key feature of mature HEC in peripheral LN HEV mediating lymphocyte recruitment in a CD62L-dependent manner55, which, in combination with leukocytes in subendothelial pockets, suggests acquisition of HEC function after Notch loss-of-function. In addition, upregulation of several cell-adhesion molecules, such as Sell, Selp, Madcam1, or Vcam1, was observed in whole kidney RNA, which was not reﬂected in EC transcriptomic analysis (Supplementary Table 1), suggesting that upregulation of these cell-adhesion molecules occurs in recruited leukocytes or a distinct population of reticular stromal cells of lymphoid tissues, which support lymphocyte recruitment56. Sell, Selp, Madcam1, or Vcam1, was observed in whole kidney RNA, which was not reﬂected in EC transcriptomic analysis (Supplementary Table 1), suggesting that upregulation of these cell-adhesion molecules occurs in recruited leukocytes or a distinct population of reticular stromal cells of lymphoid tissues, which support lymphocyte recruitment56. NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications 8 ARTICLE Lastly, the fact that TLSs develop without alteration of secondary lymphoid structures (in terms of size and cell composition, aside from dendritic cell content) even suggest a potential therapeutic angle speciﬁc for TLS without affecting secondary lymphoid structures, and thus general immunosuppression. Maintenance of arterial phenotype requires continuous Notch signaling activated by arterial shear stress, which mediates anti- inﬂammatory effects of Notch signaling, while deletion of Notch1 induces loss of arterial speciﬁcation and disruption of anti- inﬂammatory gene networks, which promotes pro-inﬂammatory responses of EC and atherosclerosis development44,57,59,60. Our ﬁnding of a loss of an arterial gene signature in the transcriptome of RbpjΔEC mice—speciﬁcally shown in kidney endothelium via RNAseq and GSEA—corroborates the notion of active main- tenance of arterial phenotype by Notch signaling, but also demonstrates an EC phenotype shift towards HEC with development of TLS in the kidney. This also provides another basis for the pro-inﬂammatory effects and atherosclerosis development observed in mice with disrupted endothelial Notch signaling, since atherosclerosis is accompanied by arterial TLS formation23,57,60. Furthermore, the reciprocal link between Notch signaling and vascular inﬂammation61,62 might provide a conceptual framework to understand TLS formation via an EC phenotype shift. Pro-inﬂammatory cytokines and lipids down- regulate Notch signaling in arterial EC in vitro and in vivo60, which in responsive EC might induce a HEC phenotype shift and local development of TLS. Our data therefore support the hypothesis of an antigen-independent mechanisms of TLS generation mediated by prolonged inﬂammation2, which, via downmodulation of Notch signaling and HEC induction, promotes TLS formation. At the same time, this also implies a novel aspect of endothelial dysfunction, a key driver of vascular disease63. A molecular link between loss of Notch signaling, inﬂammation, and upregulation of PNAd expression is provided by the interaction with NF-kB, which is suppressed by Notch signaling and required for proper PNAd expression and HEC phenotype64,65. NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x ARTICLE Methods Mi dh At 7–9 weeks of age, Cdh5CreERT2; Rbpjf/f mice or Cre- negative littermate controls were injected 1500 µg Tamoxifen i.p. (dissolved in medium chain fatty acids at a dose of 10 mg/ml, volume 150 µl/dose) on ﬁve consecutive days and euthanized at the indicated time points. Prox1CreERT2; Rbpjf/f or Cre-negative littermate controls were injected 1500 µg Tamoxifen i.p. (dissolved in medium chain fatty acids at a dose of 10 mg/ml, volume 150 µl/dose) on ﬁve consecutive days. Kidneys were collected and ﬁxed in 4% PFA for histological analysis and immunostaining. f/f αMHCCre;Stat3f/f mice or Cre-negative littermate controls were euthanized at 7–8 months of age after veriﬁcation of heart failure phenotype via echocardiography in sedated mice (2% isoﬂurane inhalation, connected to a rodent ventilator) using a TURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications 9 ARTICLE ARTICLE The DEG list was used for generation of PCA plot using built in functionality in Qlucore omics explorer with default settings. Output counts were used for further analysis with Qlucore Omics explorer (Sweden). Data were log2 transformed, 1.1 was used as a threshold and low expression genes (<30 reads in all samples) were removed from the analysis. Principal component analysis (PCA) was performed on 370 differentially expressed genes (DEGs) after variance ﬁltering (ﬁltering threshold 0.05) selected by two-tailed Student’s t test with the Benjamini-Hochberg (B-H) correction (p < 0.02, FDR < 0.0199731). The DEG list was used for generation of PCA plot using built in functionality in Qlucore omics explorer with default settings. Propidium Iodide (Fluka) was used to exclude dead cells. Cells were analyzed on LSR II ﬂow cytometer (BD Biosciences, acquisition software BD FACSDiva Software v8.0.1) and data were analyzed using FlowJo software v8.0.1 (TreeStar). List of antibodies and dilutions used for ﬂow cytometry in Supplementary Table 1. Kidney endothelial cell isolation. For puriﬁcation of EC from kidneys, we combined MACS-based pre-enrichment and FACS strategies. Single-cell suspen- sions were prepared from kidneys as described above. After Fc block and sub- sequent staining with anti-CD31-PE and anti-CD45-FITC, cells were washed and incubated with 1:5 diluted anti-PE microbeads (Miltenyi Biotech, 130-048-801) for 15 min. Positive selection of magnetically labeled cells was performed using LS columns and MidiMax Separator in combination with MACS Multistand (all from Miltenyi Biotec) according to manufacturer’s instructions. After elution from columns, cells were pelleted, ﬁltered through 40 µm mesh and proceeded for sorting on a FACSAria IIu cell sorter (Becton-Dickinson). Total yield of EC (CD31+CD45−cells) was about 5 × 105 to 1 × 106 live cells per kidney. RNA was isolated directly after sorting using a Qiagen RNEasy micro kit according to the manufacturer’s protocol. Gene set enrichment analysis (GSEA). GSEA was conducted according to the method described in 200577 using the gene sets of the Gene Ontology (GO) Bio- logical Processes resource78,79. First, genes were ﬁltered to only retain the ones that had a mean expression level above log2(normalized counts+1) = 3.23, which yielded 18,862 genes. Next, the ordered Wald statistics calculated by DESeq2 were provided to the “gseGO” function of the clusterProﬁler package (v. 4.0.580) in R (v. 4.0.1), using parameters eps = 1e-60, minGSSize = 30, and maxGSSize = 2000. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x 770 (Visual Sonics) as described74 and tissues were collected for further is. Male LDLr−/−mice underwent unilateral ischemia reperfusion injury in ischemia) of the kidney at 8-10 weeks of age75 and were fed a high fat diet 0-70) for 10 weeks starting 1 week after surgery. animal studies were undertaken in accordance with German Animal re legislation and with the European Communities Council Directive 2010/ U for the protection of animals used for experimental purposes. All experiments were approved by the Local Institutional Animal Care an Advisory Committee and permitted by the relevant local authority fo protection. Tissue and cell preparation for ﬂow cytometry. Mice were sacriﬁce was collected from the Vena cava in Na2EDTA containing tubes; splee NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications experiments were approved by the Local Institutional Animal Care and Research Advisory Committee and permitted by the relevant local authority for animal protection. Vevo 770 (Visual Sonics) as described74 and tissues were collected for further analysis. Male LDLr−/−mice underwent unilateral ischemia reperfusion injury (27 min ischemia) of the kidney at 8-10 weeks of age75 and were fed a high fat diet (C1090-70) for 10 weeks starting 1 week after surgery. All animal studies were undertaken in accordance with German Animal Welfare legislation and with the European Communities Council Directive 2010/ 63/EU for the protection of animals used for experimental purposes. All Vevo 770 (Visual Sonics) as described74 and tissues were collected for further analysis. Male LDLr−/−mice underwent unilateral ischemia reperfusion injury (27 min ischemia) of the kidney at 8-10 weeks of age75 and were fed a high fat diet (C1090-70) for 10 weeks starting 1 week after surgery. All animal studies were undertaken in accordance with German Animal Welfare legislation and with the European Communities Council Directive 2010/ 63/EU for the protection of animals used for experimental purposes. All All animal studies were undertaken in accordance with German Animal Welfare legislation and with the European Communities Council Directive 2010/ 63/EU for the protection of animals used for experimental purposes. All Tissue and cell preparation for ﬂow cytometry. Mice were sacriﬁced and blood was collected from the Vena cava in Na2EDTA containing tubes; spleens, kidneys, Tissue and cell preparation for ﬂow cytometry. Mice were sacriﬁced and blood was collected from the Vena cava in Na2EDTA containing tubes; spleens, kidneys, NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications 10 ARTICLE With this method, an enrichment score (ES) was calculated for each gene set by decreasing or increasing a Kolmogorov-Smirnov statistic according to the mag- nitude of the Wald statistic of each gene (using p = 1 as in Equation 1 of Sub- ramanian et al, 2005). One thousand permutations were performed to obtain randomized ES and calculate the normalized enrichment score (NES) by dividing the real ES by the mean of the randomized ES values. The p-value associated with each gene set was adjusted by using the Benjamini-Hochberg procedure81. The list of signiﬁcant gene sets with adjusted p-value < 0.05 was manually parsed and representative GO terms were selected to create a barplot of normalized enrichment scores (full GSEA results in Supplementary Table 1). Finally, we used the same methods and functions to calculate enrichment scores, normalized enrichment scores and p-values for a custom list of gene signatures that we compiled from kidney endothelial single-cell RNAseq publications, deﬁning arterial EC or HEC12,49,50. ARTICLE Fig. 5 Kidney endothelial cell RNAseq and gene set enrichment analysis (GSEA). A Principal Component Analysis of RbpjΔEC vs. Control kidney EC transcription analysis, n = 3/group, (Variance ﬁltering 0.05, Student’s T test followed by the B-H correction (p < 0.02, FDR < 0.0199731) showing biological replicates of each group clustering together (370 genes) (more information in “Methods”). B GO-term GSEA—selected signiﬁcantly up- (red) and downregulated (blue) gene sets sorted by normalized enrichment score (NES). C Volcano plots of individual genes with expression changed in RbpjΔEC compared to control, with log2 (fold change) on x-axis and –log10 (adjusted p-value) on the y-axis; in red, genes belonging to selected marker gene sets for different kidney arterial segments (from refs. 49,50). NES and p value upper right corners; see “Methods” (GSEA) for statistics. D Homeostatic HEC marker gene set from12 in red in same volcano plot, NES and p value upper right corner. See Methods (GSEA) for statistics. E Human kidney biopsy RNAseq (GSE: EC Notch target genes in different glomerular diseases downregulated as compared to living kidney donor biopsies as control. Abbreviations and sample number: LD, living kidney donor (n = 21); MemGN, membranous glomerulonephritis (n = 18); FSGS, focal segmental glomerulosclerosis (n = 17); MCD, minimal change disease(n = 13); RPGN, rapid-progressive glomerulonephritis (n = 21); IgA, Iga-Nephritis (n = 25); SLE, systemic lupus erythematodes (n = 32). Single samples (dot) plus mean, IQR (box) and total range (min-max: whiskers). Statistic: Brown-Forsythe and Welch (1 way) ANOVA with Dunnet’s multiple comparisons; exact adjusted p-values: HES1, compared to LD: MemGN <0.0001; FSGS, 0.068; MCD, <0.0001; RPGN, 0.0003; IgA, 0.0035; SLE, 0.0347. HEY1, compared to LD: MemGN, 0.3236; FSGS, 0.0076; MCD; 0.5432; RPGN, 0.1433; IgA, 0.5891; SLE, 0.9987. HEYL, compared to LD: MemGN, 0.119; FSGS, 0.002; MCD, 0.0050; RPGN, < 0.0001; IgA, 0.0085; SLE, 0.0021. Source data are provided as supplementary ﬁles. BCL to FASTQ conversion. BCL ﬁles were converted to FASTQ ﬁles using bcl2fastq Conversion Software version v2.20.0.422 (Illumina). bones and para-aortic lymph nodes were excised and kept on ice during pre- paration. Spleens and lymph nodes were pressed, resuspended in PBS (Millipore) and ﬁltered through a 70 µm mesh; blood and bone marrow from tibia and femur were ﬁltered as above. Erythrocytes were removed from splenic and blood cell suspensions by red blood cell lysis buffer (Biolegend). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x ARTICLE ARTICLE bones and para-aortic lymph nodes were excised and kept on ice during pre- paration. Spleens and lymph nodes were pressed, resuspended in PBS (Millipore) and ﬁltered through a 70 µm mesh; blood and bone marrow from tibia and femur were ﬁltered as above. Erythrocytes were removed from splenic and blood cell suspensions by red blood cell lysis buffer (Biolegend). Raw data processing and quality control. Raw data processing was conducted by use of nfcore/rnaseq (version 1.4.2) which is a bioinformatics best-practice analysis pipeline used for RNA sequencing data at the National Genomics Infrastructure at SciLifeLab Stockholm, Sweden. The pipeline uses Nextﬂow, a bioinformatics workﬂow tool. It pre-processes raw data from FastQ inputs, aligns the reads and performs extensive quality control on the results. The genome reference and annotation data were taken from GENCODE.org (Mus musculus; GRCm38.p6; release M25). Kidneys were minced to small pieces (<1 mm), then digested with Collagenase II (Worthington) 500U/ml for 2× 21 min at 37 °C, interrupted by 1–2 courses of tissue dissociation (GentleMACS, program B). Cells were washed with PBS and ﬁltered through a 70μm mesh several times before proceeding with staining. For CD23 staining experiments, kidneys were pressed and ﬁltered as indicated above and lymphocytes were isolated using double (70–40%) percoll (GE Healthcare) density gradient centrifugation. After extensive washing cells from all organs were resuspended into PBS, counted using a Countess II automatic cell counter (Thermoﬁsher Scientiﬁcs) and used for ﬂow cytometry. Normalization and differential expression analysis. Normalization and differential expression analysis were performed with DESeq276 (Galaxy Tool Version 2.11.40.2) with default settings except for “Output normalized counts table”, “Turn off outliers ﬁltering”, and “Turn off independent ﬁltering”, all of which were set to “True”. Flow cytometry. Cells were resuspended in buffer containing 2% fetal calf serum (Biochrom), 2 mM Na2EDTA (Roth) and 0.05%NaN3 (AppliChem) and stained using antibodies listed in the table after Fc receptor blocking with TrueStain fcX anti-CD32/16 (Clone 93, Biolegend). Biotinylated antibodies were bound by Streptavidin PE-Dazzle 594. Output counts were used for further analysis with Qlucore Omics explorer (Sweden). Data were log2 transformed, 1.1 was used as a threshold and low expression genes (<30 reads in all samples) were removed from the analysis. Principal component analysis (PCA) was performed on 370 differentially expressed genes (DEGs) after variance ﬁltering (ﬁltering threshold 0.05) selected by two-tailed Student’s t test with the Benjamini-Hochberg (B-H) correction (p < 0.02, FDR < 0.0199731). References Nat. Commun. 10, 1157 (2019). following kidney injury and transplantation. Nat. Commun. 10, 1157 (2019). 9. Ager, A. High endothelial venules and other blood vessels: critical regulators of lymphoid organ development and function. Front. Immunol. 8, 45 (2017). 10. Sato, Y. et al. Heterogeneous ﬁbroblasts underlie age-dependent tertiary lymphoid tissues in the kidney. JCI Insight 1, e87680 (2016). g y j y p 9. Ager, A. High endothelial venules and other blood vessels: critical regulators of lymphoid organ development and function. Front. Immunol. 8, 45 (2017). For whole-mount staining, we followed an adapted iDisco-Protocol83 with an added bleaching step and cleared the organs with ethyl cinnamate (ECI84). In short, kidneys were harvested and postﬁxed in 4%PFA for 2 h; dehydrated in ethanol (50%, 70%, 100%) bleached with ETOH with 5% DMSO/ 5%H2O2; rehydrated stepwise; washed; incubated with permeabilization solution83 at 37 °C for 2 days; blocked with blocking solution83 at 37 °C for 2 days; Incubated with 5 µg/ml anti- B220-AF647 (Clone RA3-6B2, Biolegend #103226) in PTwH/ 5%DMSO/ 3% Donkey Serum, 37 °C, 3 days. Washed 4–5× until the next day; dehydrated in ethanol/H2O series and then cleared in ECI at room temperature. For the B220 and Prox1/LYVE1 costaining, we incubated with anti-Prox1 (R&D AF2727, 1:100) and Anti-LYVE1 (R&D BAF2125, 1:100) for 2 days, washed for 24 h, and incubated with anti-goat-Cy3 (1:500) and anti-B220-AF647 (1:100) for another 2 days at 37 °C. Imaging using light sheet microscopy; for CD31/B220, we used a LaVision BioTec Ultramicroscope (Imaging Center Essen), for B220/Prox1, Lyve1 a LaVision BioTec UltraMicroscope II at Hannover Medical School (different wavelength laser equipment). For 3D Image reconstruction and movie animation, Imaris Software 7.6.5 Version I (Bitplane/Oxford Instruments) was used. 9. Ager, A. High endothelial venules and other blood vessels: critical regulators of lymphoid organ development and function. Front. Immunol. 8, 45 (2017). 10. Sato, Y. et al. Heterogeneous ﬁbroblasts underlie age-dependent tertiary lymphoid tissues in the kidney. JCI Insight 1, e87680 (2016). 10. Sato, Y. et al. Heterogeneous ﬁbroblasts underlie age-dependent tertiary lymphoid tissues in the kidney. JCI Insight 1, e87680 (2016). 11. Lee, M. et al. Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing. Nat. Immunol. 15, 982–995 (2014). 12. Veerman, K., Tardiveau, C., Martins, F., Coudert, J. & Girard, J. P. Data availability The RNA sequencing data generated in this study, including raw sequencing ﬁles and a table of preprocessed counts per gene per sample, are publicly available in the NCBI’s Gene Expression Omnibus under accession number GSE193544. The mouse reference genome and annotation (GRCm38) used to align sequencing reads are available from GENCODE (https://www.gencodegenes.org/mouse/release_M25.html), and the Gene Ontology gene sets are available here: http://geneontology.org/. Tubulointerstitial Hey1, Hes1, and HeyL expression were analyzed in public datasets from the European cDNA bank cohort, the Nephrotic Syndrome Study Network, and the Vasculitis Clinical Research Consortium51, obtained at NCBI: GSE104948, and GSE104954. ) O.C.T.-embedded kidneys were cryosectioned into 6-μm sections and mounted on Superfrost slides (Fisher Scientiﬁc). For immunoﬂuorescence and laser scanning microscopy, sections were washed in 1X PBS, blocked in 10% normal donkey serum (Vector Laboratories), and incubated with primary Abs (against PDPN, Clone RTD4E10, abcam ab11936, dilution 1:400; Lyve1, R&D, BAF2125, dilution 1:100; B220, Clone RA3-6B2, Life Technologies 14-0452-82, dilution 1:100; CD3, DAKO A0452, dilution 1:100; CXCL13, R&D, AF470, dilution 1:1000; KI67, Thermo Fisher 14-5698-82, dilution 1:100; NG2, Millipore #AB5320, dilution 1:100; ER-TR7, BMA, dilution 1:100; Rankl, Clone IK22/5, eBioscience 14-5952-81, dilution 1:100; PNAd, Clone MECA79, Biolegend, dilution 1:100; Erg, Clone EPR3864, Abcam ab92513, dilution 1:400, Prox1, R&D, AF2727, dilution 1:100; CD31, Clone 390, Biolegend 122407, dilution 1:100; anti-GFP, AvesGFP-1010, dilution 1:500) and appropriate ﬂuorescence labeled secondary antibodies (Anti- host, AF488, AF555 or AF647, Life Technologies, dilution 1:500) or with directly labeled antibodies (Anti-asma-AF488, Clone 1A4, BD biosciences, dilution 1:300; asma-Cy3, Clone 1A4, Sigma C6198, dilution 1:300; GL7-FITC, Biolegend 144603, dilution 1:100; IgD-AF647, Clone 11-26c.2a, Biolegend #405707, dilution 1:100; CD21/35-FITC, Clone 7G6, BD Pharmingen 561769, dilution 1:100). DAPI (4,6- Diamidino-2-phenylindole, Invitrogen, Germany; dilution 1:2000) was used for counterstaining of nuclei and slides were mounted in Immunoselect Antifading Mounting Medium (Dianova, Germany). For CXCL13 staining, a TSA-Cy3 signal ampliﬁcation kit was used (SAT704A001KT, Perkin Elmer, USA). Images were acquired using Leica TCS SP8 AOBS (Leica Microsystems, Germany) confocal microscope with a ×20 objective or Zeiss LSM980 confocal microscope (Zeiss, Germany) with a ×25 objective. All other data are provided in the Supplemental Information, Supplemental Data ﬁles and the Source Data ﬁle accompanying this article. Source data are provided with this paper. Received: 14 May 2020; Accepted: 21 March 2022; Received: 14 May 2020; Accepted: 21 March 2022; ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x Essential DNA Green Master Mix on a LightCycler 96 system from Roche (acquisition software: LightCycler96 Version 1.1.0.1320 (© 2011 Roche Diagnostics International)) according to the manufacturer’s instructions. The expression of each speciﬁc gene was normalized to the expression of Rps9 and calculated by the comparative CT (2-ΔΔCT) method82. Primer sequences in Supplementary Table 2. unless otherwise indicated. The signiﬁcance of differences was calculated using the tests indicated in the respective ﬁgure legend (two-tailed Mann–Whitney test unless otherwise stated) with conﬁdence interval of 95%. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Tissue ﬁxation, embedding, and stainings. Immunohistochemistry and immu- noﬂuorescence staining in mice were performed as previously described18 with modiﬁcations. Mice were euthanized; kidney, liver, lung, spleen, heart and mesenterial lymph nodes were isolated and ﬁxed in 4% buffered paraformaldehyde (PFA). Organs were either embedded in parafﬁn or cryopreserved in sucrose and embedded in Tissue-tek O.C.T. compound (Sakura, Germany). Parafﬁn blocks were cut with a rotation microtome (Leica) at 1–2 µm thickness and stained according to routine histological protocols (hematoxylin eosin, periodic acid Schiff, Masson trichrome, Sirius red). References 1. Ruddle, N. H. Lymphatic vessels and tertiary lymphoid organs. J. Clin. Invest. 124, 953–959 (2014). 2. Yin, C., Mohanta, S., Mafﬁa, P. & Habenicht, A. J. R. Editorial: tertiary lymphoid organs (TLOs): powerhouses of disease immunity. Front. Immunol 8, 21–23 (2017). Quantiﬁcation of TLS area. Kidneys were cut in half along the axial plane and mounted face down, resulting in sections representing the middle of the kidney with hilar, papillar, medullary, and cortical fractions. In PAS stained sections, area covered by mononuclear cells was measured in the periarterial loose interstitial tissue area (vessel nerve sheath) along all (typically longitudinally cut) segmental and all (typically cross sectioned) interlobar arteries per section. As the kidneys/ sections were of similar size, we did not normalize the measured area to the section size. 3. Pitzalis, C., Jones, G. W., Bombardieri, M. & Jones, S. A. Ectopic lymphoid- like structures in infection, cancer and autoimmunity. Nat. Rev. Immunol. 14, 447–462 (2014). 4. Dorraji, S. E. et al. Mesenchymal stem cells and T cells in the formation of tertiary lymphoid structures in lupus nephritis. Sci. Rep. 8, 7861 (2018). 5. Steinmetz, O. M. et al. Analysis and classiﬁcation of B-cell inﬁltrates in lupus and ANCA-associated nephritis. Kidney Int. 74, 448–457 (2008). 5. Steinmetz, O. M. et al. Analysis and classiﬁcation of B-cell inﬁltrates in lupus and ANCA-associated nephritis. Kidney Int. 74, 448–457 (2008). 6. Segerer, S. & Schlondorff, D. B cells and tertiary lymphoid organs in renal inﬂammation. Kidney Int. 73, 533–537 (2008). p y , ( ) 6. Segerer, S. & Schlondorff, D. B cells and tertiary lymphoid organs in renal inﬂammation. Kidney Int. 73, 533–537 (2008). 6. Segerer, S. & Schlondorff, D. B cells and tertiary lymphoid organs in renal inﬂammation. Kidney Int. 73, 533–537 (2008). Whole-mount staining and optical clearing. For CD31-staining, we directly labeled anti-CD31-antibody (clone 390, Biolegend) with ﬂuorochrome AF790 using a direct labeling kit from Thermo Fisher Scientiﬁc (Cat: A20189). Five micrograms of antibody in PBS were injected via the tail vein. After 10–30 min, mice were euthanized, perfused through the left ventricle with PBS + 2 mM EDTA and then with buffered 4% PFA. 7. Pei, G. et al. Renal interstitial inﬁltration and tertiary lymphoid organ neogenesis in IgA nephropathy. Clin. J. Am. Soc. Nephrol. 9, 255–264 (2014). 8. Cippa, P. E. et al. A late B lymphocyte action in dysfunctional tissue repair following kidney injury and transplantation. RNA sequencing b Library generation. Two nanograms of total RNA were used for library preparation with the ‘SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian’ (#634413; Takara/Clontech) according to conditions recommended in user manual #063017. Generated libraries were barcoded by dual indexing approach and were ﬁnally ampliﬁed with 12 cycles of PCR. Fragment length distribution of generated libraries was monitored using the ‘Bioanalyzer High Sensitivity DNA Assay’ (5067- 4626; Agilent Technologies). Quantiﬁcation of libraries was performed by use of the ‘Qubit® dsDNA HS Assay Kit’ (Q32854; ThermoFisher Scientiﬁc). Gene array data analysis. Tubulointerstitial Hey1, Hes1, and HeyL expression were analyzed in public datasets from the European cDNA bank cohort, the Nephrotic Syndrome Study Network, and the Vasculitis Clinical Research Consortium51 obtained at NCBI GSE104948, and GSE104954. Sequencing run. Equal molar amounts of six libraries in total were pooled for a common sequencing run. Accordingly, each analyzed library constitutes 16.6% of overall ﬂowcell capacity. The library pool was denatured with NaOH and was ﬁnally diluted to 2 pM according to the Denature and Dilute Libraries Guide (Document # 15048776 v02; Illumina). A volume of 1.3 ml of denatured pool was loaded on an Illumina NextSeq 550 sequencer using a High Output Flowcell for single reads (20024906; Illumina). Sequencing was performed with the following settings: Sequence reads 1 and 2 with 38 bases each; Index reads 1 and 2 with 8 bases each. Quantitative real-time PCR analysis. Total RNA was puriﬁed from cell lysates using Nucleospin RNA II kit (Macherey Nagel). After purity and quality check, RNA was transcribed into cDNA employing a cDNA synthesis kit (Invitrogen) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using speciﬁc primers (see Supplementary Table 5) with FastStart 11 TURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications MMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications Code availability All bioinformatic tools and methods used in this manuscript have been published previously; we therefore did not deposit any code in a public repository. For further information please contact the corresponding author. Source data are provided with this paper. ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x 47. Girard, J. P., Moussion, C. & Forster, R. HEVs, lymphatics and homeostatic immune cell trafﬁcking in lymph nodes. Nat. Rev. Immunol. 12, 762–773 (2012). 18. Limbourg, A. et al. Notch ligand Delta-like 1 is essential for postnatal arteriogenesis. Circ. Res. 100, 363–371 (2007). g You, L. R. et al. Suppression of Notch signalling by the COUP g 19. You, L. R. et al. Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity. Nature 435, 98–104 (2005 48. Mionnet, C. et al. High endothelial venules as trafﬁc control points maintaining lymphocyte population homeostasis in lymph nodes. Blood 118, 6115–6122 (2011). 20. Han, H. et al. Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision. Int Immunol. 14, 637–645 (2002). 49. Kalucka, J. et al. Single-cell transcriptome atlas of murine endothelial cells. Cell 180, 764–779 e720 (2020). 21. Benedito, R. et al. The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis. Cell 137, 1124–1135 (2009). 50. Dumas, S. J. et al. Single-cell RNA sequencing reveals renal endothelium heterogeneity and metabolic adaptation to water deprivation. J. Am. Soc. Nephrol. 31, 118–138 (2020). g g 22. Gamrekelashvili, J. et al. Regulation of monocyte cell fate by blood vessels mediated by Notch signalling. Nat. Commun. 7, 12597 (2016). mediated by Notch signalling. Nat. Commun. 7, 12597 (2016 51. Grayson, P. C. et al. Metabolic pathways and immunometabolism in rare kidney diseases. Ann. Rheum. Dis. 77, 1226–1233 (2018). 23. Hu, D. et al. Artery tertiary lymphoid organs control aorta immunity and protect against atherosclerosis via vascular smooth muscle cell lymphotoxin β receptors. Immunity 42, 1100–1115 (2015). y 52. Ehling, M., Adams, S., Benedito, R. & Adams, R. H. Notch controls retinal blood vessel maturation and quiescence. Development 140, 3051–3061 (2013). p y 24. Ding, G. Y. et al. Distribution and density of tertiary lymphoid structures predict clinical outcome in intrahepatic cholangiocarcinoma. J. Hepatol. 76, 608–618 (2021). q p 53. Haverich, A. A surgeon’s view on the pathogenesis of atherosclerosis. Circulation 135, 205–207 (2017). 54. Belisle, C. & Sainte-Marie, G. The narrowing of high endothelial venules of the rat lymph node. Anat. Rec. 211, 184–191 (1985). 25. Li, H. et al. Peritumoral tertiary lymphoid structures correlate with protective immunity and improved prognosis in patients with hepatocellular carcinoma. Front Immunol. 12, 648812 (2021). 55. ARTICLE Mebius, R. E., Streeter, P. R., Michie, S., Butcher, E. C. & Weissman, I. L. A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+ CD3- cells to colonize lymph nodes. Proc. Natl Acad. Sci. USA 93, 11019–11024 (1996). 26. Fleige, H. et al. Manifold roles of CCR7 and its ligands in the induction and maintenance of bronchus-associated lymphoid tissue. Cell Rep. 23, 783–795 (2018). 27. Jia, J. et al. Cholesterol metabolism promotes B-cell positioning during immune pathogenesis of chronic obstructive pulmonary disease. EMBO Mol. Med. 10, e8349 (2018). 56. Katakai, T. et al. Organizer-like reticular stromal cell layer common to adult secondary lymphoid organs. J. Immunol. 181, 6189–6200 (2008). 28. Fleige, H. et al. IL-17-induced CXCL12 recruits B cells and induces follicle formation in BALT in the absence of differentiated FDCs. J. Exp. Med. 211, 643–651 (2014). 57. Mack, J. J. et al. NOTCH1 is a mechanosensor in adult arteries. Nat. Commun. 8, 1620 (2017). 58. Duarte, A. et al. Dosage-sensitive requirement for mouse Dll4 in artery development. Genes Dev. 18, 2474–2478 (2004). 29. Jabs, M. et al. Inhibition of endothelial notch signaling impairs fatty acid transport and leads to metabolic and vascular remodeling of the adult heart. Circulation 137, 2592–2608 (2018). 59. Theodoris, C. V. et al. Human disease modeling reveals integrated transcriptional and epigenetic mechanisms of NOTCH1 haploinsufﬁciency. Cell 160, 1072–1086 (2015). 30. Fletcher, A. L., Acton, S. E. & Knoblich, K. Lymph node ﬁbroblastic reticular cells in health and disease. Nat. Rev. Immunol. 15, 350–361 (2015). 60. Briot, A. et al. Endothelial NOTCH1 is suppressed by circulating lipids and antagonizes inﬂammation during atherosclerosis. J. Exp. Med. 212, 2147–2163 (2015). 31. Alsughayyir, J., Pettigrew, G. J. & Motallebzadeh, R. Spoiling for a ﬁght: B lymphocytes as initiator and effector populations within tertiary lymphoid organs in autoimmunity and transplantation. Front. Immunol. 8, 1639 (2017). 61. Briot, A., Bouloumie, A. & Iruela-Arispe, M. L. Notch, lipids, and endothelial cells. Curr. Opin. Lipidol. 27, 513–520 (2016). 32. Legler, D. F. et al. B cell-attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/ CXCR5. J. Exp. Med. 187, 655–660 (1998). 62. Krishnasamy, K. et al. Blood vessel control of macrophage maturation promotes arteriogenesis in ischemia. Nat. Commun. 8, 952 (2017). 3. Vita, J. A. Endothelial function. Circulation 124, e906–e912 ( 33. Krautler, N. J. ARTICLE et al. Follicular dendritic cells emerge from ubiquitous perivascular precursors. Cell 150, 194–206 (2012). 64. Drayton, D. L. et al. I kappa B kinase complex alpha kinase activity controls chemokine and high endothelial venule gene expression in lymph nodes and nasal-associated lymphoid tissue. J. Immunol. 173, 6161–6168 (2004). 34. Yi, T. et al. Oxysterol gradient generation by lymphoid stromal cells guides activated B cell movement during humoral responses. Immunity 37, 535–548 (2012). 65. Gamrekelashvili, J. & Limbourg, F. P. Rules of attraction: endothelial Notch signalling controls leucocyte homing in atherosclerosis via VCAM1. Cardiovasc. Res. 112, 527–529 (2016). 35. Sixt, M. et al. The conduit system transports soluble antigens from the afferent lymph to resident dendritic cells in the T cell area of the lymph node. Immunity 22, 19–29 (2005). 66. Halle, S. et al. Induced bronchus-associated lymphoid tissue serves as a general priming site for T cells and is maintained by dendritic cells. J. Exp. Med. 206, 2593–2601 (2009). 36. Grabner, R. et al. Lymphotoxin beta receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE-/- mice. J. Exp. Med. 206, 233–248 (2009). 67. Barone, F. et al. Stromal ﬁbroblasts in tertiary lymphoid structures: a novel target in chronic inﬂammation. Front. Immunol. 7, 477 (2016). 37. Mueller, C. G. & Hess, E. Emerging functions of RANKL in lymphoid tissues. Front. Immunol. 3, 261 (2012). 68. Boehm, T., Hess, I. & Swann, J. B. Evolution of lymphoid tissues. Trends Immunol. 33, 315–321 (2012). 38. Hess, E. et al. RANKL induces organized lymph node growth by stromal cell proliferation. J. Immunol. 188, 1245–1254 (2012). 69. Nishikawa, S. I., Hashi, H., Honda, K., Fraser, S. & Yoshida, H. Inﬂammation, a prototype for organogenesis of the lymphopoietic/hematopoietic system. Curr. Opin. Immunol. 12, 342–345 (2000). 39. Hilﬁker-Kleiner, D. et al. Signal transducer and activator of transcription 3 is required for myocardial capillary growth, control of interstitial matrix deposition, and heart protection from ischemic injury. Circ. Res. 95, 187–195 (2004). p 70. Kusumbe, A. P. et al. Age-dependent modulation of vascular niches for haematopoietic stem cells. Nature 532, 380–384 (2016). deposition, and heart protection from ischemic injury. Circ. Res. 95, 187–195 (2004). haematopoietic stem cells. Nature 532, 380–384 (2016). 71. Kusumbe, A. P., Ramasamy, S. K. & Adams, R. H. Coupling of angiogenesis and osteogenesis by a speciﬁc vessel subtype in bone. Nature 507, 323–328 (2014). 40. Zhang, F. et al. References Single-cell analysis reveals heterogeneity of high endothelial venules and different regulation of genes controlling lymphocyte entry to lymph nodes. Cell Rep. 26, 3116–3131 e3115 (2019). 13. Brulois, K. et al. A molecular map of murine lymph node blood vascular endothelium at single cell resolution. Nat. Commun. 11, 3798 (2020). 13. Brulois, K. et al. A molecular map of murine lymph node blood vascular endothelium at single cell resolution. Nat. Commun. 11, 3798 (2020). 14. Adams, R. H. & Alitalo, K. Molecular regulation of angiogenesis and lymphangiogenesis. Nat. Rev. Mol. Cell Biol. 8, 464–478 (2007). g 14. Adams, R. H. & Alitalo, K. Molecular regulation of angiogenesis and lymphangiogenesis. Nat. Rev. Mol. Cell Biol. 8, 464–478 (2007). 15. Lawson, N. D. et al. Notch signaling is required for arterial-venous differentiation during embryonic vascular development. Development 128, 3675–3683 (2001). Serum and urine measurements were performed with an automated Olympus AU400. Serum and urine measurements were performed with an automated Olympus AU400. 16. Nielsen, C. M. et al. Deletion of Rbpj from postnatal endothelium leads to abnormal arteriovenous shunting in mice. Development 141, 3782–3792 (2014). Statistics. Results are expressed as box plots with min/max whiskers, mean and 25–75%-range (box) or as scatter dot plots with mean ± standard deviation. N numbers are biological replicates of experiments performed at least three times 17. Villa, N. et al. Vascular expression of Notch pathway receptors and ligands is restricted to arterial vessels. Mech. Dev. 108, 161–164 (2001). 12 NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecom Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Correspondence and requests for materials should be addressed to Florian P. Limbourg. Peer review information Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Peer review information Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. The authors declare no competing interests. 82. Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101–1108 (2008). Funding 81. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300 (1995). ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-022-29701-x 78. Ashburner, M. et al. Gene ontology: tool for the uniﬁcation of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25–29 (2000). H.H. provided necessary materials or animals; T.W., J.G., B.L., and A.C.J. provided bioinformatic analyses; S.F. and F.P.L. wrote and edited the manuscript; All authors read, commented and corrected the manuscript; F.P.L. conceived and directed the study. 79. Gene Ontology C. The Gene Ontology resource: enriching a GOld mine. Nucleic Acids Res. 49, D325–D334 (2021). 80. Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProﬁler: an R package for comparing biological themes among gene clusters. OMICS 16, 284–287 (2012). ARTICLE Lacteal junction zippering protects against diet-induced obesity. Science 361, 599–603 (2018). 72. Ishibashi, S. et al. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J. Clin. Invest. 92, 883–893 (1993). 41. Reed, H. O. et al. Lymphatic impairment leads to pulmonary tertiary lymphoid organ formation and alveolar damage. J. Clin. Invest. 129, 2514–2526 (2019). 73. Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double- ﬂuorescent Cre reporter mouse. Genesis 45, 593–605 (2007). 42. Jafree, D. J. et al. Spatiotemporal dynamics and heterogeneity of renal lymphatics in mammalian development and cystic kidney disease. Elife 8, e48183 (2019). 74. Hoch, M. et al. Erythropoietin preserves the endothelial differentiation capacity of cardiac progenitor cells and reduces heart failure during anticancer therapies. Cell Stem Cell 9, 131–143 (2011). 43. Bazigou, E. et al. Genes regulating lymphangiogenesis control venous valve formation and maintenance in mice. J. Clin. Invest. 121, 2984–2992 (2011). 75. Wang, L. et al. Labile heme aggravates renal inﬂammation and complement activation after ischemia reperfusion injury. Front. Immunol. 10, 2975 (2019). 44. Fang, J. S. et al. Shear-induced Notch-Cx37-p27 axis arrests endothelial cell cycle to enable arterial speciﬁcation. Nat. Commun. 8, 1–14 (2017). 45. Su, T. et al. Single-cell analysis of early progenitor cells that build coronary arteries. Nature 559, 356–362 (2018). 76. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). 46. le Noble, F., Klein, C., Tintu, A., Pries, A. & Buschmann, I. Neural guidance molecules, tip cells, and mechanical factors in vascular development. Cardiovasc. Res. 78, 232–241 (2008). 77. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression proﬁles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005). 13 NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications Additional information 83. Renier, N. et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910 (2014). Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-022-29701-x. 84. Klingberg, A. et al. Fully automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using lightsheet microscopy. J. Am. Soc. Nephrol. 28, 452–459 (2017). Correspondence and requests for materials should be addressed to Florian P. Limbourg. © The Author(s) 2022 Acknowledgements Schematics were created with BioRender.com. We thank the Central Animal Facility, Research Core Facility Cell Sorting and Research Core Unit Laser Microscopy of Hannover Medical School for support. We thank Stefan Sablotny, Michaela Beese, Anja Standke, Herle Chlebusch, Birgit Brandt, Anja Bubke, and Petra Berkefeld for excellent technical support. We thank Zulrahman Erlangga for his help optimizing our kidney EC isolation protocol, and Heiko Schenk for his help in obtaining a chicken-anti-GFP anti- body. We thank Taija Makinen for providing Prox1-CreERT2, Freddy Radtke for Rbpjf/f mice and Ingmar Mederacke for providing B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato) Hze/J (Jackson Laboratories Strain #:007914) mice for breeding. Funded by intramural grants from Hannover Medical School (Hochschulinterne Leistungsförderung II, Clinical Scientist Program “Junge Akademie”, and Ellen-Schmidt-Habilitationsförderung ESP) to S.F., and by grants from Deutsche Forschungsgemeinschaft to T.K. (Ka5549/2-1), to S.v.V. (VI508/7-1), to J.G. (Ga2443/3-1), and to F.P.L. (Li948-7/1), as well as DFG KFO311 (HI 842/10-2 to D.H.-K. and RI 2531/2-2 to M.R.-H.) for the Stat3ΔMyoc-experiment. DFG- funded CRC 1348 (Dynamic Cellular Interfaces) to R.H.A. Grants by the Swiss League for Cancer Research (KFS-4895-08-2019, to T.V.P.) and the Swiss National Science Foun- dation (CRSK-3_190435 to J.B.-L.). Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints Author contributions © The Author(s) 2022 S.F., T.K., J.B.-L., J.G., J.L., D.K., E.B., A.H., S.H. did the experiments; S.F., T.K., F.P.L. designed experiments and analyzed data; D.H.K., M.R.H., T.P., D.R.E., S.v.V., R.H.A., NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunications 14 NATURE COMMUNICATIONS \| (2022) 13:2022 \| https://doi.org/10.1038/s41467-022-29701-x \| www.nature.com/naturecommunicati
https://openalex.org/W3025106235	http://eprints.whiterose.ac.uk/162005/14/1-s2.0-S2666123320300192-main.pdf	English	null	Decomposing the drivers of residential space cooling energy consumption in EU-28 countries using a panel data approach	Energy and built environment	2,020	cc-by	13,845	Article: t c e Andreou, A orcid.org/0000-0003-0892-6759, Barrett, J orcid.org/0000-0002-4285-6849, Taylor, PG orcid.org/0000-0001-7456-3744 et al. (2 more authors) (2020) Decomposing the drivers of residential space cooling energy consumption in EU-28 countries using a panel data approach. Energy and Built Environment, 1 (4). pp. 432-442. ISSN 2666-1233 https://doi.org/10.1016/j.enbenv.2020.03.005 https://doi.org/10.1016/j.enbenv.2020.03.005 This is a repository copy of Decomposing the drivers of residential space cooling energy consumption in EU-28 countries using a panel data approach. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/162005/ White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/162005/ Version: Published Version Version: Published Version Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Decomposing the drivers of residential space cooling energy consumption in EU-28 countries using a panel data approach dreas Andreou a , ∗ , John Barrett a , Peter G. Taylor a , b , Paul E. Brockway a , Zia Wadud b , c a Sustainability Research Institute, School of Earth and Environment, University of Leeds, Leeds, LS2 9JT, UK b Low Carbon Energy Research Group, School of Chemical and Process Engineering, University of Leeds, Leeds, LS2 9JT, UK c Spatial Modelling and Dynamics Research Group, Institute for Transport Studies, University of Leeds, Leeds, LS2 9JT, UK a b s t r a c t These mod- els provide limited value to the policy making process as they do not facilitate a broader discussion about the relative importance of the var- ious factors driving air-conditioning use in diﬀerent EU Member States. These historical estimates are subsequently mixed with crude assump- tions about the future development of modelled parameters, such as a 100% AC technology saturation rate [10 , 11] or using current diﬀusion data from United States as a proxy [12 , 13] , to deﬁne ceiling values for EU-28 space cooling energy consumption. The adoption of such simpli- ﬁed methodologies limits understanding about the potential trajectories residential AC markets could follow in the near-future and how diﬀerent capacity and more eﬀective management of summer time peak loads; issues which are already evident in Mediterranean EU-28 countries [8] . ∗ Corrresponding author. E-mail address: eeaan@leeds.ac.uk (A. Andreou). Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ Energy and Built Environment 1 (2020) 432–442 Contents lists available at ScienceDirect ∗ Corrresponding author. E-mail address: eeaan@leeds.ac.uk (A. Andreou). https://doi.org/10.1016/j.enbenv.2020.03.005 Received 16 November 2019; Received in revised form 20 March 2020; Accepted 20 March 2020 Available online 11 May 2020 2666-1233/© 2020 The Author(s). Published by Elsevier B.V. on behalf of Southwest Jiaotong University. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) a b s t r a c t Keywords: Space cooling AC diﬀusion Decomposition analysis Panel data model Peak demand While space cooling currently represents less than 1% of ﬁnal energy use in the residential sector of the Euro- pean Union (EU-28), it was the fastest growing end-use during the 2000-15 period with a mean annual growth rate of 6% per year. Currently, little is known about factors which have driven regional air-conditioning (AC) energy consumption over time, since the literature is limited to cross-sectional studies that lack diﬀerentiation between climatic and non-climatic inﬂuences. Future projections for the EU’s electricity sector may therefore neglect the potential implications of rapidly growing AC demand. We develop a novel decomposition framework, which breaks down residential space cooling energy consumption in EU-28 countries into the eﬀect of diﬀerent components from 2000 to 2015. Decomposition is extended to panel data models identifying speciﬁc drivers of space cooling’s climate-sensitive components. Finally, we explore scenarios of residential AC energy consumption up to 2050 and evaluate their impact on summer time peak loads. AC diﬀusion was found to be the key driver of space cooling energy consumption, but this eﬀect was partly counterbalanced by eﬃciency gains. While weather inﬂuences AC equipment ownership rate in EU-28 households, personal income has a larger marginal eﬀect. In baseline scenarios, AC diﬀusion saturates by 2050, while modestly increasing sectoral ﬁnal energy use. Still, our range of scenario values for space cooling energy consumption in 2050 exceed the majority of those originat- ing from recently published projections. In a future renewables-driven electricity system, energy security risks may emerge from a scenario of fast AC up-take in new and renovated buildings, especially for colder European countries where modelled peak cooling electricity demand is shown to outgrow the projected expansion of solar capacity. These ﬁndings have important implications for the EU’s strategy to decarbonise energy supply. capacity and more eﬀective management of summer time peak loads; issues which are already evident in Mediterranean EU-28 countries [8] . In the absence of granular household end-use consumption data, studies analysing the EU’s current space cooling energy use involve es- timates obtained mostly through bottom-up technology-based energy models. These in turn depend on technical parameters gathered over tiny time frames, overlooking past variation of AC use [5 , 9] . 1. Introduction More speciﬁcally, this paper tackles the following research questions: Nomenclature FEU Final energy use (TWh/y) A, S, I Activity, Structural, Intensity parameters Hou Number of households (hh) Diﬀ Air-conditioning diﬀusion (%) Qspec Useful speciﬁc cooling demand (useful kWh/hh •y) Eﬀ Eﬃciency indicator of air-conditioning systems Sat Air-conditioning saturation (%) X Predictor variables of air-conditioning diﬀusion Pop Population INC Personal income (2011$/pop PPP) TMP Mean outdoor temperature ( ◦C) t Time trend �, �, �, �Estimated model parameter coeﬃcients � Residual error AREA Useful household area (m 2 /hh) CDD Cooling degree days ( ◦C •d) SSP Shared socio-economic pathway RCP Representation concentration pathway Peak Potential peak electricity demand (GW) Cap Rated capacity (kW) SEER Seasonal energy eﬃciency ratio w Share of air-conditioning technologies (%) NrAC Number of air-conditioning units new New and renovated households old Old households AC Air-conditioning y Year c Country NUTS Nomenclature of Territorial Units for Statistics n Number of NUTS regions JJA June-July-August d Day tech Type of air-conditioning technology warm Warm group of EU-28 countries cold Cold group of EU-28 countries Nomenclature FEU Final energy use (TWh/y) A, S, I Activity, Structural, Intensity parameters Hou Number of households (hh) Diﬀ Air-conditioning diﬀusion (%) Qspec Useful speciﬁc cooling demand (useful kWh/hh •y) Eﬀ Eﬃciency indicator of air-conditioning systems Sat Air-conditioning saturation (%) X Predictor variables of air-conditioning diﬀusion Pop Population INC Personal income (2011$/pop PPP) TMP Mean outdoor temperature ( ◦C) t Time trend �, �, �, �Estimated model parameter coeﬃcients � Residual error AREA Useful household area (m 2 /hh) CDD Cooling degree days ( ◦C •d) SSP Shared socio-economic pathway RCP Representation concentration pathway Peak Potential peak electricity demand (GW) Cap Rated capacity (kW) SEER Seasonal energy eﬃciency ratio w Share of air-conditioning technologies (%) NrAC Number of air-conditioning units new New and renovated households old Old households AC Air-conditioning y Year c Country NUTS Nomenclature of Territorial Units for Statistics n Number of NUTS regions JJA June-July-August d Day tech Type of air-conditioning technology warm Warm group of EU-28 countries cold Cold group of EU-28 countries Nomenclature FEU Final energy use (TWh/y) A, S, I Activity, Structural, Intensity parameters Hou Number of households (hh) Diﬀ Air-conditioning diﬀusion (%) Qspec Useful speciﬁc cooling demand (useful kWh/hh •y) Eﬀ Eﬃciency indicator of air-conditioning systems Sat Air-conditioning saturation (%) X Predictor variables of air-conditioning diﬀusion Pop Population INC Personal income (2011$/pop PPP) TMP Mean outdoor temperature ( ◦C) t Time trend �, �, �, �Estimated model parameter coeﬃcients � Residual error AREA Useful household area (m 2 /hh) CDD Cooling degree days ( ◦C •d) SSP Shared socio-economic pathway RCP Representation concentration pathway Peak Potential peak electricity demand (GW) Cap Rated capacity (kW) SEER Seasonal energy eﬃciency ratio w Share of air-conditioning technologies (%) NrAC Number of air-conditioning units new New and renovated households old Old households AC Air-conditioning y Year c Country NUTS Nomenclature of Territorial Units for Statistics n Number of NUTS regions JJA June-July-August d Day tech Type of air-conditioning technology warm Warm group of EU-28 countries cold Cold group of EU-28 countries i. 1. Introduction What was the main driving force of EU’s residential space cooling energy consumption in the time period 2000-15? This question is tackled with index decomposition analysis (IDA), which helps link the variation of household air-conditioning energy consumption to relevant activity, structural and intensity components. ii. Which are the speciﬁc drivers of the climate-sensitive components of space cooling energy consumption? This objective is achieved by extending decomposition analysis to a set of panel data econometric models aiming to explain the inﬂuence of climatic and non-climatic factors on national AC penetration rates and households’ useful space cooling demand. iii. What are the impacts of future AC diﬀusion trajectories on electricity-based ﬁnal energy use for space cooling in the EU-28 res- idential sectors and potential peak cooling electricity demand, as projected up to 2050? This paper develops baseline AC diﬀusion scenarios incorporating projections of socio-economic and climatic data, while alternative policy cases consider unit eﬃciency targets and AC installation rates in new and renovated buildings. The rest of the paper is organised as follows: Section 2 describes the novel modelling framework adopted in this study and summarises the data requirements. Section 3 presents the results of the histori- cal analysis (2000-15) and the future scenario modelling (2016-50). Section 4 then discusses potential areas for EU policy intervention, while section 5 concludes. 2. Methodology factors, including climate change and economic growth, could aﬀect its evolution. Even if the impact of these two factors is taken into consid- eration, projections of AC diﬀusion are based on functions which were not calibrated using historical data for the EU-28 region only [14–16] . factors, including climate change and economic growth, could aﬀect its evolution. Even if the impact of these two factors is taken into consid- eration, projections of AC diﬀusion are based on functions which were not calibrated using historical data for the EU-28 region only [14–16] . 1. Introduction Global energy consumption for space cooling has increased three- fold between 1990 and 2016 and has been accompanied by a tremen- dous growth in air-conditioning (AC) sales [1 , 2] . The diﬀusion and use of air-conditioning across the globe has been strongly linked to chang- ing climatic and economic conditions [3] . In the European Union (EU- 28), while residential space cooling currently forms a minor share of sectoral ﬁnal energy use (0.6% in 2015) it was the fastest growing household end-use during the time period 2000-15, recording an aver- age consumption growth rate of 6.3% per year ( Fig. 1 ) [4] . Residential air-conditioning also has an enormous future growth potential in the EU-28 as less than 10% of household ﬂoor area is currently cooled [5] . Since space cooling in EU-28 households is usually supplied through electric room air-conditioners (RACs) [6] , the expected growth of resi- dential AC markets across Europe [7] will intensify pressure on national electricity sectors. This translates into a need for additional generating In the absence of granular household end-use consumption data, studies analysing the EU’s current space cooling energy use involve es- timates obtained mostly through bottom-up technology-based energy models. These in turn depend on technical parameters gathered over tiny time frames, overlooking past variation of AC use [5 , 9] . These mod- els provide limited value to the policy making process as they do not facilitate a broader discussion about the relative importance of the var- ious factors driving air-conditioning use in diﬀerent EU Member States. These historical estimates are subsequently mixed with crude assump- tions about the future development of modelled parameters, such as a 100% AC technology saturation rate [10 , 11] or using current diﬀusion data from United States as a proxy [12 , 13] , to deﬁne ceiling values for EU-28 space cooling energy consumption. The adoption of such simpli- ﬁed methodologies limits understanding about the potential trajectories residential AC markets could follow in the near-future and how diﬀerent ∗ Corrresponding author. E-mail address: eeaan@leeds.ac.uk (A. Andreou). https://doi.org/10.1016/j.enbenv.2020.03.005 Received 16 November 2019; Received in revised form 20 March 2020; Accepted 20 March 2020 Available online 11 May 2020 2666-1233/© 2020 The Author(s). Published by Elsevier B.V. on behalf of Southwest Jiaotong University. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) A. Andreou, J. Barrett and P.G. 1. Introduction Taylor et al. Energy and Built Environment 1 (2020) 432–442 Energy and Built Environment 1 (2020) 432–442 This paper uses the novel Integrated Database of the European En- ergy Sector (IDEES), published by the European Commission’s Joint Re- search Centre (JRC) in 2018, which provides consistent and detailed data about the residential space cooling sector of EU-28 countries over an extended time period (2000-15) [4] . This permits the development of a multi-method modelling framework for studying historical trends of space cooling energy consumption, which is inclusive of the broader non-technology factors. We use the term ‘space cooling (or AC) energy consumption’ to equate to the ‘electricity-based ﬁnal energy use for space cooling’ from this point onwards in the paper. Moreover, it adopts a more scenario-based approach to evaluate potential future pathways of residential AC energy consumption for diﬀerent EU-28 countries. Fig. 1. Indexed evolution of EU-28 residential ﬁnal energy use by end-use (2000-15) [4] 2.3. Panel data econometric modelling (2000-15) Diﬀusion ( Diﬀ) of residential air-conditioners in EU-28 countries dur- ing the baseline period is studied in a panel data setting through an “s-shaped ” logistic curve; a common functional form ﬁrst adopted by McNeil and Letschert [23 , 24] to construct a global AC diﬀusion curve. Similar to [25] , we modify the logistic function via Eqn. (5) to account for intra-country data variation: � �� = � × � × � (1) (1) � �� = � × � × � where FEU , ﬁnal energy use of a sector or for a speciﬁc end-use, is ex- pressed as the product of Activity ( A ), Structure ( S ) and Energy Inten- sity ( I ). The ﬁrst component, namely A , denotes the primary driver of ﬁnal energy use, while S captures additional parameters having an im- pact on its size. On the other hand, factor I represents energy consumed per unit of activity, which is inﬂuenced by weather, building and AC technology characteristics, as well as lifestyle patterns [21] . In the case of residential space cooling energy consumption, the number of house- holds and diﬀusion rate of AC equipment is respectively ascribed to the activity and structural parameter [12] . The intensity indicator is then de- ﬁned as space cooling energy consumed per air-conditioned household. Space cooling energy consumption ( FEU AC ) in EU-28 countries is there- fore expressed in annual steps (TWh/y) between 2000 and 2015, using Eqn. (2) : Dif � �,� = Sa � � 1 + �� exp (�X �,� ) (5) (5) Saturation ( Sat ) represents the maximum attainable up-take level of air-conditioning in residential buildings which is invariant with time, measured in years ( y ), and can be unique for each EU-28 country ( c ). Without imposing any ad-hoc restrictions, Sat can theoretically vary be- tween 0 and 100%. The horizontal position of the logistic curve is ad- justed by the constant �, while its slope is controlled by X , an array of variables expected to have a positive or negative temporal impact on AC diﬀusion rates. Besides local climate, a number of studies have concluded that grow- ing personal income has been a strong determinant of worldwide AC up-take ( [25] for China; [26] for United States; [27] for Mexico). 2.1. General modelling framework The analysis is performed across two overlapping layers, as shown in Fig. 2: (A) Traditional decomposition analysis [17–19] is ﬁrst Fig. 1. Indexed evolution of EU-28 residential ﬁnal energy use by end-use (2000-15) [4] 433 433 Energy and Built Environment 1 (2020) 432–442 A. Andreou, J. Barrett and P.G. Taylor et al. Fig. 2. General modelling framework Fig. 2. General modelling framework Fig. 2. General modelling framework space cooling energy consumption (kWh/hh) via the cooling system’s eﬃciency indicator, Eﬀ. employed to quantify the eﬀect of changes in diﬀerent components (i.e. household numbers, unit AC eﬃciency, useful speciﬁc cooling demand and AC diﬀusion) on historical variation (2000-15) of EU-28 residential AC energy consumption. Panel data models are then utilised to study the sensitivity of AC penetration rates and useful speciﬁc cooling de- mand to climatic and non-climatic inﬂuences. (B) Finally, scenarios are developed to analyse the impact of distinct AC diﬀusion trajectories on EU-28 sectoral space cooling energy consumption in the time period 2016-50, as well as on potential peak cooling electricity demand. Fu- ture baseline AC diﬀusion estimates are derived from the econometric model developed in (A), when enacted with country-level projections of climatic and socio-economic data. These are benchmarked against two policy cases concerning unit eﬃciency improvements and diversi- ﬁed installation rates, for which the assumptions are presented in the following sections. Log Mean Divisia Index – method I (LMDI-I) is the preferred tool to explain the year-to-year variation of residential AC energy consumption via the contribution of the 4 pre-selected components, due to its theo- retical and methodological advantages [22] . These advantages include leaving no residual term since absolute annual FEU AC changes over time can be completely decomposed to individual components, in an additive fashion through Eqn. (3) : Δ� � � �� = Δ� � � �� + Δ� � � �� + Δ� � � �� − Δ� � � �� (3) Δ� � � �� = Δ� � � �� + Δ� � � �� + Δ� � � �� − Δ� � � �� (3) where diﬀerences in FEU AC between a speciﬁc year, y , and base year, 0, equates to the sum of partial temporal eﬀects arising from changes in household numbers, AC appliance ownership, useful speciﬁc cooling demand and eﬃciency improvements. 2.1. General modelling framework Given the logarithmic form of the decomposition, the impact of individual components on FEU AC , such as that of the housing stock is calculated via Eqn. (4) : 2.2. Decomposition analysis (2000-15) Sectoral-level decomposition analysis is a very useful tool for energy policy-making, especially in the context of residential sector mitigation strategies where it has been applied to understand the temporal dynam- ics of energy consumption and the corresponding carbon emissions [20] . This approach oﬀers the advantage of attributing changes of ﬁnal energy use to a set of pre-speciﬁed factors which can be unique for each end- use service, thus facilitating the design of more tailored energy reduc- tion policies. In its simplest form, index decomposition analysis (IDA) is performed via Eqn. (1) : Δ� � � �� = � � � �� − � � � � � 0 �� − �� 0 × (� �� − � �� 0 ) (4) (4) 2.3. Panel data econometric modelling (2000-15) Evi- dence has also shown that energy eﬃciency improvements and reduc- tion in equipment prices has a reinforcing eﬀect on consumer purchasing decisions with respect to air-conditioners [28] . Bringing these elements � � � �� = �� × �� × �� ÷ �� (2) (2) � � � �� = �� × �� × �� ÷ �� The response of AC diﬀusion to weather variation is captured through TMP JJA which accounts for mean outdoor temperature in the summer months June-July-August (JJA), while a TMP JJA lag (K = 1) is subsequently added to control for the delayed impact of extreme heat events on ownership rates [25] . Since the impact of a unit change of temperature is expected to be stronger in areas with larger population [3] , we adjust TMP JJA , through Eqn. (7) , to account for the heteroge- neous distribution of residents across each EU-28 country, by applying weights corresponding to 2014’s population count of “Nomenclature of territorial units for statistics – level 3 ” (NUTS-3) sub-regions: Cooling degree days ( CDDs ) characterize the climate-sensitive part of useful speciﬁc cooling demand. They quantify the annual sum of daily deviations of mean outdoor air temperature from a pre-speciﬁed ﬁxed threshold [35] , in line with Eurostat’s deﬁnition 1 , below which no me- chanical space cooling is needed to restore thermal comfort in house- holds. Using a high temperature threshold (here set at 24 ◦C) ensures that days with low average temperature, when building energy demand is essentially climate-insensitive, are excluded from CDD calculations. CDDs in essence capture the cumulative eﬀect of warm temperatures on speciﬁc AC demand more eﬀectively than using an absolute measure of temperature, which was the approach used in the AC diﬀusion model. As in the standard FE model speciﬁcation, �s refer to country-level factors and � to the residual error term. TM � JJA �,� = ∑� ��=1 TM � JJA NUTS ,� Po � NUTS ,� ∑� ��=1 Po � NUTS ,� (7) (7) where n is the number of NUTS-3 sub-regions in each EU-28 country. Instead of estimating Eqn. (6) through Ordinary Least Square (OLS) regression, which would yield 28 unique sets of model coeﬃcients cor- responding to each EU-28 country, the eﬀect of weather and income on residential AC diﬀusion is more accurately identiﬁed via ﬁxed-eﬀects (FE) panel data estimation. Unlike OLS, panel data models can han- dle a data structure involving both a temporal and spatial dimension, thereby recognising the heterogeneity of behaviours observed in in- dividual countries. Moreover, panel data estimators control for time- invariant, country-speciﬁc characteristics ( �s) which can be correlated with explanatory parameters, thereby reducing omitted variable bias [29] . 2.4. Scenario analysis (2016-50) Future pathways of space cooling energy consumption ( FEU AC ) and potential peak cooling electricity demand ( Peak AC ) in the EU-28 re- gion are evaluated though a scenario modelling process which extends the analysis to the period 2016-50. The scenarios focus on incorporat- ing anticipated changes in the stock and eﬃciency of residential air- conditioners in accordance with the speciﬁcations of a baseline and two policy cases. In the baseline case, country-level AC penetration rates are projected in annual steps up to 2050 via the diﬀusion model in Eqn. (6) , for combinations of personal income ( INC ) and mean summer tempera- ture ( TMP JJA ) trajectories. Future personal income is derived from the Intergovernmental Panel on Climate Change’s Shared Socio-economic Pathways (SSPs), which provide plausible narratives for the long-term evolution of various socio- economic drivers [36] . Baseline AC diﬀusion scenarios adhere to 3 SSPs which cover the full spectrum of projection uncertainty, including a “middle-of-the-road ” trajectory (SSP2) and a fast vs. slow economic growth case (SSP5/SSP3). With respect to increasing summer temper- atures across Europe, Representative Concentration Pathway (RCP) 8.5 was selected since it describes the high-end of projected climate change, whereby radiative forcing is boosted up to 8.5 Wm -2 in 2100 due to un- mitigated greenhouse gas emissions [37] . Matching future Sat levels in EU-28 countries with present-day AC diﬀusion rates observed in regions of United States with similar climatic conditions – the so called “Climate Maximum ” [12 , 21 , 23] – would cloud analysis with strong assumptions about the pace of evolution of regional AC markets. Instead, a diﬀerent approach is followed to determine these ceiling values empirically. EU-28 countries are ﬁrst split in two groups according to long-term (1995-2015) cooling degree days ( CDDs ), mea- sured in ◦C •d, each of them assumed to reach a unique saturation point: countries with higher than average CDDs are labelled as warm, while the rest of them are named as cold. The performance of the Diﬀmodel is iteratively evaluated (at steps of 10%) for various group-level Sat values through the adjusted-R 2 criterion. Sat cold is constrained to be always smaller or equal to Sat warm , while both are set to vary above the highest current AC diﬀusion level in each region. A combination of saturation points that maximise the model’s goodness-of-ﬁt are ﬁnally adopted. � � � �� = �� × �� × �� ÷ �� where Hou is the number of households (hh) and Diﬀthe share of resi- dential buildings equipped with air-conditioning (%), conforming to the adopted framework. Moreover, useful speciﬁc cooling demand (useful kWh/hh), captured through Qspec , is converted into units of speciﬁc 434 Energy and Built Environment 1 (2020) 432–442 A. Andreou, J. Barrett and P.G. Taylor et al. Energy and Built Environment 1 (2020) 432–442 has been chosen to study the within-country variation of Qspec : together, our model explicitly accounts for weather and income changes and implicitly controls for evolving energy eﬃciency standards and AC prices through a time trend, t . Rearranging Eqn. (5) and taking log- arithms on both sides produces the following linear model given in Eqn. (6) : spe � �,� = �� + �1 ARE � �,� + �2 AREAS � �,� + �3 CD � �,� + � �,� (8) (8) spe � �,� = �� + �1 ARE � �,� + �2 AREAS � �,� + �3 CD � �,� + � �,� Useful ﬂoor area ( AREA ) is selected as a more straightforward ex- planatory parameter of Qspec ; in larger households, containing more rooms and communal areas, maintaining desired indoor temperature level demands either more intense use of existing space cooling equip- ment or acquisition of additional AC units, both having a signiﬁcant eﬀect on Qspec [34] . Moreover, past research has shown that household ﬂoor area is strongly connected with personal income [3] and so the in- clusion of both variables in the FE panel data model has been avoided. A quadratic ﬂoor area term ( AREASQ ) is also included to capture any additional non-linear eﬀects. ln ( Sa � � Dif � �,� − 1 ) = ln (�� ) + �1 � + �2 IN � �,� + � ∑ � =0 �3 � − � TM � JJA �,� − � + � �,� (6) where INC denotes annual personal income in individual EU-28 coun- tries, as approximated by per capita GDP which is adjusted to represent between-country price-level diﬀerences based on purchasing power par- ity (PPP). � � � �� = �� × �� × �� ÷ �� In the FE case, generated model coeﬃcients resemble the eﬀect on the dependent variable – on average across EU-28 countries – from a unit change in the independent variable along the temporal dimension. A Hausman test is also performed to conﬁrm the superiority of the FE estimator to the random-eﬀects (RE) one [30] . 1 �� = ∑� � =1 { ( � � � � − 21 ◦C ) , � � � � > 24 ◦C 0 , � � � � ≤ 24 ◦C , where d is the number of 2.4. Scenario analysis (2016-50) �� ′ �,� = �� ,� + ( � �� ′ �� − � �� ) + ( � �� ′ �� − � �� ) + ( � �� ′ � −1 − � �� −1 ) �� , 2015 (12) �� ′ �,� = �� ,� + ( � �� ′ �� − � �� ) + ( � �� ′ �� − � �� ) + ( � �� ′ � −1 − � �� −1 ) �� , 2015 (12) Our assumptions for this high penetration scenario is that diﬀusion rates for air-conditioning in new and renovated buildings, �� ′ �� , in- crease from around 17% in 2015, in 10-year time steps, from 80% in 2021-2030, to 90% in 2031-40, and eventually reach full saturation (100%) in 2041-50. These values are benchmarked against baseline Diﬀnew projections constructed to 2050 by means of linear extrapolation based on historical diﬀusion data (2000-15). The fraction of new and renovated buildings in the total housing stock per year in EU-28 coun- tries is assumed to remain constant at 3% in the future; which equals the ambitious building stock renovation target set in the EU’s Directive on Energy Performance of Buildings [41] and lies within the range of reno- vation rates selected in [42] for Germany’s household sector. Since this set of rules does not aﬀect AC purchasing decisions in old households, the NrAC old terms can be removed from Eqn. (12) as they cancel each other. Based on these assumptions, the modiﬁed AC diﬀusion parameter can be calculated under this scenario using Eqn. (13) : We subsequently devise two policy case scenarios, whose aim is to assess the sensitivity of future AC energy consumption trajectories to varying assumptions for factors which can be tackled by policy makers, namely the level of unit eﬃciency improvements and diﬀusion rates in new and renovated buildings. In this way, we demonstrate the wide range of possible outcomes in 2050 under various policy regimes, in- stead of attempting to quantify the possibility of each scenario being realised in the future. Case 1 - Unit Eﬃciency Improvement : We explore the size of energy savings which could be achieved due to signiﬁcant increases in AC equipment eﬃciency. 2.4. Scenario analysis (2016-50) The sensitivity of country-level FEU AC to growing AC up-take is as- sessed in 2016-50 by adjusting the Diﬀfactor in Eqn. (2) to match the respective scenario’s value, while holding household count ( Hou ), AC eﬃciency ( Eﬀ) and useful speciﬁc cooling demand ( Qspec ) constant at the level in 2015. Future residential AC energy consumption is therefore estimated using Eqn. (9) : � � � �� ,� = � � � �� , 2015 × �� ,� �� , 2015 (9) Useful demand for space cooling per unit of activity has been pre- viously econometrically estimated based on cross-sectional models us- ing climatic variables, in the form of CDDs, and economic development indicators, such as personal income [21] and household expenditures [31 , 32] . In a cross-sectional setting, the positive eﬀect of income on spe- ciﬁc cooling demand was demonstrated to diminish in wealthier coun- tries, as occupants choose to use their AC equipment irrespective of their ﬁnancial status [33] . Since the vast majority EU-28 nations are high- income economies a diﬀerent model speciﬁcation, described in Eqn. (8) , (9) We deﬁne potential peak cooling demand as the maximum theoret- ical load which a national electricity system would have to sustain if 1 �� = ∑� � =1 { ( � � � � − 21 ◦C ) , � � � � > 24 ◦C 0 , � � � � ≤ 24 ◦C , where d is the number of days in year, y , and TMP d is daily mean outdoor temperature [59] . 435 Energy and Built Environment 1 (2020) 432–442 A. Andreou, J. Barrett and P.G. Taylor et al. Energy and Built Environment 1 (2020) 432–442 Table 1 Composition and technical parameters of buildings RAC stock at the EU-15 level [6] AC technology w (%) Cap (kW) SEER Split systems 61.8 3.50 3.22 Multi-split systems 5.3 16.0 2.12 Single-duct systems 15.7 10.5 4.75 Packed units 17.2 4.75 2.12 Table 1 Composition and technical parameters of buildings RAC stock at the EU-15 level [6] expanding Eqn. (9) to incorporate changes in space cooling eﬃciency: expanding Eqn. 2.4. Scenario analysis (2016-50) Tighter minimum energy performance standards (MEPS) and stricter labelling schemes are the main mecha- nisms which currently enforce improvements in the performance of air- conditioners in EU-28 markets [1 , 3] . These measures along with techno- logical change are expected to have a major contribution in alleviating the foreseen pressure on electricity systems exerted by peak AC demand [38 , 39] . �� ′ �,� = �� ,� + 3% × ( �� ′ �� − �� ) + ( � �� ′ � −1 − � �� −1 ) �� , 2015 (13) �� ′ �,� = �� ,� + 3% × ( �� ′ �� − �� ) + ( � �� ′ � −1 − � �� −1 ) �� , 2015 This scenario adopts the 20% and 30% general eﬃciency goals en- dorsed through the EU’s mid-term energy strategy for 2020 and 2030, while it assumes that super-eﬃcient units dominate the market by the end of mid-21 st century, increasing average AC eﬃciency by 40% in 2050, relative to 2015. Based on JRC-IDEES data, the eﬃciency param- eter ( Eﬀ) of residential AC systems at the EU-28 level is equal to 3.0 in 2015. Incremental technical improvements, imposed in the future through this policy scenario, result in assumed EU-level Eﬀfactors of 3.6, 3.9 and 4.2 in 2021-30, 2031-40 and 2041-50, respectively for our study. While our assumed average AC eﬃciency of 4.2 in 2050 is slightly higher than the one adopted by JRC in [40] (~4.0), this is a feasible tar- get since it is still lower than the Eﬀvalue of the best available technol- ogy currently marketed in the EU-28 region [1] Electricity-based ﬁnal energy use for space cooling in 2016-50 is calculated via Eqn. (11) , after (13) 2.4. Scenario analysis (2016-50) (9) to incorporate changes in space cooling eﬃciency: � � � �� ,� = � � � �� , 2015 × �� ,� �� , 2015 × �� ,� �� , 2015 (11) (11) A similar approach is followed in obtaining modiﬁed potential peak cooling electricity demand ( Peak AC ) estimates through applying eﬃ- ciency improvements on the current SEER values of individual RAC tech- nologies in Table 1 . Case 2 - New Buildings AC Rates : About 3% of housing stock across the EU-28 region every year (2000-15) consists of new and renovated buildings, which generally display higher AC diﬀusion rates than ex- isting, non-renovated, ones [4] . In 2015, 17.1% of new and renovated EU-28 households were equipped with an air-conditioner, while only 8.9% of old residential buildings had one installed. Future develop- ments in the construction industry could facilitate easier installation of air-conditioning in the former group of buildings, as shown by the experience of the USA [26] . A drop in installation costs could work as an incentive for investing in room AC units; an economic behaviour which if mimicked by more and more newly-constructed households could abruptly transform the EU’s residential space cooling market. the full residential AC stock in a given year was operating at nameplate capacity; a condition which is more likely be fulﬁlled during periods of high extreme temperature. Maximum peak cooling demand ( Peak AC ) is then calculated as the product of total annual AC stock in a coun- try and full space cooling (electric) load. Stock size is ﬁrst obtained as a percentage from the projections performed through the AC diﬀusion model ( Eqn. (6) ) in the 2016-50 period and then converted into number of units ( NrAC ) using the number of houses in 2015. The latter param- eter is estimated with the help of EU-15 inventory data obtained from Pezzutto et al . [6] , including diﬀerent RAC systems’ rated capacity ( Cap ) and seasonal energy eﬃciency ratio ( SEER ), as well as their respective share ( w ) in total buildings’ sector stock. Estimation of potential peak cooling demand is given in Eqn. (10) : the full residential AC stock in a given year was operating at nameplate capacity; a condition which is more likely be fulﬁlled during periods of high extreme temperature. 2.4. Scenario analysis (2016-50) Maximum peak cooling demand ( Peak AC ) is then calculated as the product of total annual AC stock in a coun- try and full space cooling (electric) load. Stock size is ﬁrst obtained as a percentage from the projections performed through the AC diﬀusion model ( Eqn. (6) ) in the 2016-50 period and then converted into number of units ( NrAC ) using the number of houses in 2015. The latter param- eter is estimated with the help of EU-15 inventory data obtained from Pezzutto et al . [6] , including diﬀerent RAC systems’ rated capacity ( Cap ) and seasonal energy eﬃciency ratio ( SEER ), as well as their respective share ( w ) in total buildings’ sector stock. Estimation of potential peak cooling demand is given in Eqn. (10) : This “bad case ” scenario is assumed to have an additive eﬀect on baseline residential AC diﬀusion trajectories. The diﬀusion parameter is modiﬁed each year ( Diﬀ’ ) to account for the growing number of new and renovated units, �� ′ �� , in total air-conditioning stock, added to the previous year’s stock, �� ′ � −1 , as well as to old households’ AC equipment replacements and additions , �� ′ �� , as demonstrated by Eqn. (12) : � �� ,� = �� ,� × ∑4 ��ℎ =1 � ��ℎ × �� ℎ ÷ �� ℎ ∑4 ��ℎ =1 � ��ℎ (10) (10) where tech = split, multi-split, single-duct and packed systems; which are currently the technologies with highest diﬀusion in the household sec- tor. Amongst available room systems ( Table 1 ), split AC units form the largest portion of the installed RAC stock (~60%), while they have the smallest average capacity size and highest conversion eﬃciency factor. Given the absence of data about the future composition of residential AC stock, peak cooling electricity demand scenarios in 2050 are built based on the assumption that the relative share of individual technolo- gies remains unchanged. 3.1. Decomposition analysis of FEU AC (2000-2015) During the time period 2000-15, EU-28 residential space cooling en- ergy consumption increased from 6.4 to 15.8 TWh/y. Fig. 3 displays the results of the additive decomposition for all 1-year time bands, whereby the annual variation in EU-aggregate FEU AC levels, represented by dia- mond markers, is broken down into the contribution of single compo- nents ( Hou, Diﬀ, Qspec, Eﬀ), illustrated by the uniquely coloured column bars. As an example of this method, the + 0.88 TWh/y change in FEU AC observed between 2007 and 2008 comprises the 0.12, 1.23 and 0.08 TWh/y positive impact attributed respectively to housing stock, AC dif- fusion and speciﬁc cooling demand, and to the 0.55 TWh/y negative eﬀect from eﬃciency improvements. Descriptive statistics for all variables in the 2000-15 period are presented in Table 2 . In absolute terms, Italy is the country with by far the largest residential AC energy consumption having mean an- nual FEU AC levels of 5.4 TWh/y, which is 4 times larger than the quantity consumed by either Spain or Greece. Italy, Spain and Greece were collectively responsible for 70% of EU-aggregate residential space cooling energy consumption in 2015. Small northern EU-28 coun- tries such as Estonia and Latvia had negligible FEU AC levels in 2015 ( < 0.001 TWh/y). Fig. 3 shows that the diﬀusion of AC units in residential buildings had the strongest increasing impact on EU-28 space cooling energy con- sumption across all 15 time bands, having a mean eﬀect (2000-15) on FEU AC of + 1.02 TWh/y. The second largest contribution to FEU AC vari- ation is attributed to AC system eﬃciency, with an average decreasing eﬀect being half the size of diﬀusion-related one (-0.51 TWh/y). Useful speciﬁc cooling demand and housing stock size had smaller inﬂuences on annual AC energy consumption levels in the EU-28 region, with an average impact amounting to + 0.04 and + 0.08 TWh/y, respectively. This highlights the signiﬁcance of studying "extensive margins" in more detail [40] , relating partly to understanding the role climatic and non- climatic factors play in residential AC adoption. In per household terms, Cyprus and Malta, which are the two hottest EU-28 countries according to the long-term CDD criterion (1995-2015), recorded the highest levels of useful speciﬁc cooling demand, with Qspec values of 4974 useful kWh/hh •y and 3977 useful kWh/hh •y, respec- tively, when averaged over the 2000-15 period. 2.5. Data requirements were obtained from the World Bank [43] , expressed in constant inter- national dollars for the year 2011. National and NUTS-3 EU-28 popula- tion statistics, as well as annual (1995-2015) cooling degree days ( CDDs ) were sourced from Eurostat [44 , 45] . Monthly mean, near-surface, temperatures during the historical pe- riod (2000-15) were retrieved for every EU-28 country from Climatic Re- search Unit’s (CRU’s) time-series dataset, which is available in 0.5°x0.5° resolution [46] . For this task, the geographic centroid of each NUTS-3 sub-region was previously matched to the 4 nearest grid points and grid temperatures were aggregated to the NUTS-3 level via inverse distance weighting (IDW) interpolation. The coordinates of these geographic cen- tres were extracted from shapeﬁles depicting the geometries of NUTS-3 regions in 2013 [47] . 3. Results 3.1. Decomposition analysis of FEU AC (2000-2015) 3.1. Decomposition analysis of FEU AC (2000-2015) On the other hand, EU- 28 countries found on the low-end of the annual Qspec distribution - Latvia (364 useful kWh/hh •y) and Lithuania (392 useful kWh/hh •y) - do not coincide with those having the lowest number of long-term CDDs , namely Ireland and Sweden. Finally, the highest residential AC diﬀusion rates in 2015 were recorded in Croatia (48%), Greece (33%) and Italy (30%). Croatia’s exceptionally high penetration rates is unex- pected, given its milder summer seasons and lower consumer aﬄuence levels. Moreover, the largest diﬀusion eﬀect on FEU AC ( + 1.5 TWh/y) is found for the 2011-12 time band, which co-occurs with a 49.3% rel- ative increase in EU-wide cooling degree days, whose annual change is also plotted in Fig. 3 . One would expect that the Diﬀeﬀect would have peaked in 2002-3, as a result of the severe heatwave which struck the Eu- ropean continent; however, this is not evident from EU-aggregate data. On the other hand, useful speciﬁc cooling demand inﬂuences FEU AC the most in the 2010-11 time band, during which mean CDDs across the EU dropped by 19.2%. While this ﬁnding is counterintuitive, one should also note that this eﬀect peaks due to a sharp increase in Qspec lev- els across Italy, which indeed faced increasing CDDs in 2010-11. It is therefore vital to evaluate the speciﬁc drivers of Diﬀand Qspec , while accounting for the heterogeneous behaviour of EU-28 countries; an ob- jective which is tackled next. In order to devise baseline scenarios of AC penetration rates in EU-28 households, long-term projections regarding the annual growth of GDP per capita (PPP-adjusted) were collected for 3 SSP storylines from the database of International Institute for Applied Systems Anal- ysis [36] and then averaged over the time period 2015-50. Daily mean, near-surface, temperatures were extracted for the same time- frame from 19 regional climate projections, which have been performed under the EURO-CORDEX downscaling experiment and simulate the eﬀects of an extreme climate change scenario (RCP8.5) [48] . These Table 3 Long-term mean (2015-50) annual growth rates of baseline variables in the EU-28 region Annual growth rate (%) Category Variable Low-Range Mid-Range High-range Socio-economic INC 1.1 1.2 2.2 Climatic TMP JJA n/a (RCP8.5) 2.5. Data requirements For the purposes of index decomposition analysis, the JRC-IDEES database was accessed to obtain information regarding space cooling en- ergy consumption ( FEU AC ), household numbers ( Hou ), air-conditioning stock and eﬃciency status of AC systems ( Eﬀ) [4] . Based on these data, response variables of panel data models (i.e., useful speciﬁc cooling de- mand ( Qspec ) and AC diﬀusion ( Diﬀ)) were derived. Moreover, econo- metric modelling required the input of PPP-adjusted GDP data which 436 Energy and Built Environment 1 (2020) 432–442 A. Andreou, J. Barrett and P.G. Taylor et al. Table 2 Descriptive statistics of country-level variables during the historical period 2000-15 Table 2 Descriptive statistics of country-level variables during the historical period 2000-15 Variable Symbol Mean Std. Dev. Max Min AC energy consumption (TWh/y) FEU AC 0.40 1.07 7.84 0 Useful speciﬁc cooling demand (useful kWh/hh •y) Qspec 1,656 1,070 5,241 333 AC diffusion (%) Diff 6.4 9.3 48.6 0 Number of households (hh) Hou 7,459,383 9,998,632 40,558,210 134,669 AC system eﬃciency Eff 2.18 0.48 3.62 1.40 Personal income (2011$/pop PPP) INC 32,684 14,914 98,646 8,811 Household ﬂoor area (m 2 /hh) AREA 89.9 21.9 142.6 41.8 Mean JJA temperature (°C) TMP JJA 19.35 3.35 29.19 13.19 Cooling Degree Days ( ◦C •d) CDD 108 174 781 0 Table 2 Descriptive statistics of country-level variables during the historical period 2000-15 were obtained from the World Bank [43] , expressed in constant inter- national dollars for the year 2011. National and NUTS-3 EU-28 popula- tion statistics, as well as annual (1995-2015) cooling degree days ( CDDs ) were sourced from Eurostat [44 , 45] . projections were then translated into monthly, country-level, temper- ature statistics following the same aggregation procedure as in sub- section 2.2 . Table 3 provides a summary of climatic and non-climatic assumptions governing AC diﬀusion projections under the baseline case. projections were then translated into monthly, country-level, temper- ature statistics following the same aggregation procedure as in sub- section 2.2 . Table 3 provides a summary of climatic and non-climatic assumptions governing AC diﬀusion projections under the baseline case. projections were then translated into monthly, country-level, temper- ature statistics following the same aggregation procedure as in sub- section 2.2 . Table 3 provides a summary of climatic and non-climatic assumptions governing AC diﬀusion projections under the baseline case. Table 5 (6) is run initially without a lagged temperature variable, while the ob- tained R 2 (adj.) statistic is compared for all potential combinations of Sat warm (50-100%) and Sat cold . (20-100%). Model diagnostics which in- volved application of an F-test for country-level eﬀects and a Hausman test demonstrated the suitability of ﬁxed-eﬀects over the pooling and random-eﬀects panel data estimator, accordingly. The optimal model ﬁt is achieved when the saturation parameter for warm countries is set at 60%, while that for cold ones at 30%. niﬁcant, albeit at lower conﬁdence level (p < 0.1). This ﬁnding suggests that EU-28 households respond to warmer weather through purchasing AC units at two time steps, one occurring the same year during which a heat event took place and another the year after. The marginal impact of lagged temperature on the response variable is however smaller than the one attributed to contemporaneous TMP JJA . One should also note that inclusion of a lagged TMP JJA term in the model does not come without a cost, as model parameter estimation is less accurate and resulting R 2 (adj.) statistic is smaller compared to the basic speciﬁcation. Comparing the size of temperature and income marginal eﬀects on AC diﬀusion requires a standardization procedure which puts both pre- dictors on the same measurement scale. Re-scaling variables eﬀectively harmonizes FE regression coeﬃcients as they now represent the re- sponse of dependent variable due to a standard deviation of either TMP JJA or INC (Column (3)). The new income estimate is about 5 times larger than the one for temperature, implying that growing household aﬄuence levels have larger inﬂuence on AC purchasing decisions in the EU-28 region, compared to increasing outdoor temperatures. The greater importance of income versus temperature on AC diﬀusion is re- ﬂected in the ratio of INC to TMP JJA eﬀect on penetration, which we obtain as around ~5 in this paper, being roughly similar to a study re- ported for Chinese provinces (~6) [25] . Table 4 reports FE estimator’s output following implementation of the empirically-derived saturation levels. Column (1) includes generated coeﬃcients, along with their robust standard errors. 2 Due to the transformation of the logistic model, a negative change in one of the variables on the right-hand side of the equation results in an increase of Diﬀ variable, when other terms are kept constant. Table 5 FE estimation results of AC diﬀusion model with Sat warm = 60% and Sat cold = 30% Variables (1) (2) (3) INC (000$/pop) -0.086 ∗ ∗ ∗ (0.033) -0.078 ∗ ∗ (0.031) -0.150 ∗ ∗ ∗ (0.046) � �� (°C) -0.043 ∗ ∗ ∗ (0.014) -0.034 ∗ (0.019) -0.030 ∗ ∗ ∗ (0.009) � �� −1 (°C) -0.025 ∗ (0.014) Trend -0.137 ∗ ∗ ∗ (0.011) -0.142 ∗ ∗ ∗ (0.012) -0.152 ∗ ∗ ∗ (0.013) ln ( �� ) 7.503 ∗ ∗ ∗ (1.117) 7.570 ∗ ∗ ∗ (1.095) 3.964 ∗ ∗ ∗ (0.110) Observations 448 420 448 F-test 128.5 ∗ ∗ ∗ 130.8 ∗ ∗ ∗ Hausman test 18.0 ∗ ∗ ∗ 11.5 ∗ ∗ R 2 (adj.) 0.753 0.745 0.727 Statistically signiﬁcant ∗ at the 10%, ∗ ∗ at the 5% and ∗ ∗ ∗ at the 1% conﬁdence level. Note: Standard errors in the parenthesis are clustered by country (a la Arellano covariance matrix). FE estimation results of AC diﬀusion model with Sat warm = 60% and Sat cold = 30% FE estimation results of speciﬁc cooling demand model FE estimation results of speciﬁc cooling demand model Variables AREA (m 2 /hh) -36.595# (23.465) AREASQ 0.339 ∗ ∗ (0.145) CDD ( ◦C •d) 0.103 ∗ (0.062) ( �� ) 2004.117 ∗ ∗ (907.279) Observations 432 F-test 440.2 ∗ ∗ ∗ Hausman test 8.3 ∗ ∗ R 2 (adj.) 0.394 Statistically signiﬁcant at # at the 12%, ∗ at the 10%, ∗ ∗ at the 5% and ∗ ∗ ∗ at the 1% conﬁdence level. Statistically signiﬁcant ∗ at the 10%, ∗ ∗ at the 5% and ∗ ∗ ∗ at the 1% conﬁdence level. Note: Standard errors in the parenthesis are clustered by country (a la Arellano covariance matrix). garia (145 ◦C •d), Portugal (170 ◦C •d), Italy (201 ◦C •d), Spain (205 ◦C •d), Greece (300 ◦C •d), Malta (606 ◦C •d) and Cyprus (678 ◦C •d), which together represented 26.2% of total EU-28 housing stock in 2015 (57.7 million units). The rest 20 EU-28 countries (i.e., Austria, Belgium, Czech Republic, Denmark, Estonia, Finland, France, Germany, Hun- gary, Ireland, Latvia, Lithouania, Luxembourg, Netherlands, Poland, Romania, Slovakia, Slovenia, Sweden and United Kingdom) were as- signed to the cold group. The residential AC diﬀusion model in Eqn. 3.2. Panel data modelling of Diﬀand Qspec (2000-2015) 3.2. Panel data modelling of Diﬀand Qspec (2000-2015) EU-28 countries qualifying in the warm group, exceeding the region’s average long-term annual CDDs (102 ◦C •d), are Croatia (121 ◦C •d), Bul- 437 Energy and Built Environment 1 (2020) 432–442 A. Andreou, J. Barrett and P.G. Taylor et al. Fig. 3. Decomposition of residential space cool- ing energy consumption at the EU28-level with comparison to change in cooling degree days [LHS: Decomposition of 4 drivers of annual res- idential AC energy consumption (FEU AC ): Use- ful speciﬁc cooling demand (Qspec); Number of households (Hou); AC diﬀusion (Diﬀ); AC system eﬃciency (Eﬀ) Fig. 3. Decomposition of residential space cool- ing energy consumption at the EU28-level with comparison to change in cooling degree days [LHS: Decomposition of 4 drivers of annual res- idential AC energy consumption (FEU AC ): Use- ful speciﬁc cooling demand (Qspec); Number of households (Hou); AC diﬀusion (Diﬀ); AC system eﬃciency (Eﬀ) RHS: Annual change in cooling degree days (CDD)] Fig. 3. Decomposition of residential space cool- ing energy consumption at the EU28-level with comparison to change in cooling degree days [LHS: Decomposition of 4 drivers of annual res- idential AC energy consumption (FEU AC ): Use- ful speciﬁc cooling demand (Qspec); Number of households (Hou); AC diﬀusion (Diﬀ); AC system eﬃciency (Eﬀ) 3.3. Future scenarios of residential AC energy consumption (2016-2050) This section explores the range of potential FEU AC and Peak AC out- comes under diﬀerent trajectories of air-conditioning market develop- ment in the 2016-50 period. AC diﬀusion levels are projected in the future using the basic Diﬀmodel speciﬁcation (without a TMP JJA lag). In the baseline case, residential air-conditioning markets across warm and cold countries reach almost full saturation by the end of the pro- jection period, increasing EU-aggregate, mid-range, AC diﬀusion from 9.2% in 2015 to 37.6% in 2050. Saturation is virtually reached under all personal income and summer temperature projections in 2050, albeit at varying paces. A steeper AC diﬀusion curve at intermediate market de- velopment stages, arising from a high-income (SSP5) and extreme tem- perature trajectory (maximum of multi-model ensemble), would result in a higher amount of cumulative energy consumption over the period (2016-50). With respect to capacity-related impacts, potential peak cooling elec- tricity demand in the EU-28 household sector increases from 43.3 GW in 2015 (26.7/16.6 GW in warm/cold countries) to 177.7 GW in 2050, under the mid-range baseline projection, with 103.7 GW attributed to cold countries. Moreover, in agreement with FEU AC ﬁndings , Peak AC is aﬀected the most under the extreme AC diﬀusion scenario, recording a 9-fold increase by 2050 (401.9 GW, of which 304.9 GW is in cold countries). As anticipated, the smallest change in peak cooling electric- ity demand is projected for the strong unit eﬃciency case (127 GW in 2050, of which 74.1 GW is in cold countries). While knowledge about the future size of potential peak cooling de- mand is essential for electricity capacity upgrades, what is also of prin- cipal value for electricity network operators is the timing of integration of new plants to the grid. Given the fast-growing residential AC mar- kets and ambitious EU plans to decarbonise electricity grids by 2050, adequate provision of renewable capacity will be required to manage peak loads emerging during summer, mostly in the form of solar which has high seasonal output potential. Fig. 5 benchmarks the growth of Peak AC under the 3 scenarios against the projected expansion of solar- based generating capacity [49] , separately for warm and cold EU-28 countries. Potential peak residential AC demand across cold countries is shown to outgrow forecasted expansion of solar capacity during most of the projections period. Table 5 Results show that both personal income ( INC ) and contemporaneous mean summer tem- perature ( TMP JJA ) exhibited a highly statistically-signiﬁcant (p < 0.01) positive eﬀect on AC up-take in households during the time period 2000- 15, while a strong trend is also present 2 . Overall model performance is deemed very good, as independent variables collectively explain 75% of observed variation in data. The model of useful speciﬁc cooling demand in Eqn. (8) is run after ensuring the suitability of the FE estimator via the same conﬁrmatory tests. Ireland is the only EU-28 country with zero cooling degree days for all years in the sample (2000-15), thus it is excluded from the analysis. The estimated FE model generally has less explanatory power relative to the AC diﬀusion one, with an R 2 statistic close to 0.4 ( Table 5 ). Param- eter estimation shows the presence of a non-linear relationship between useful speciﬁc cooling demand and household ﬂoor area, since the AR- EASQ term has a statistically-signiﬁcant coeﬃcient. The marginal eﬀect Delayed temperature eﬀects are assessed by re-running the AC diﬀu- sion model with a summer temperature variable lag (Column (2)). Inter- estingly, the coeﬃcient of � �� −1 also turns out to be statistically sig- 438 A. Andreou, J. Barrett and P.G. Taylor et al. Energy and Built Environment 1 (2020) 432–442 Fig. 4. Past and future residential AC energy consumption at the EU-28 level under diﬀerent scenarios. Note: Error bars represent the range of uncertainty in RCP and SSP projections Energy and Built Environment 1 (2020) 432–442 A. Andreou, J. Barrett and P.G. Taylor et al. Fig. 4. Past and future residential AC energy consumption at the EU-28 level under diﬀerent scenarios. Note: Error bars represent the range of uncertainty in RCP and SSP projections of AREA on Qspec ( �1 + 2 �2 �� ) becomes positive when household ﬂoor area exceeds 54 m 2 /hh. This is much lower than the EU’s average household area recorded in the 2000-15 period (89.5 m 2 /hh). in 2015). Despite its growth, AC energy consumption in the baseline case still accounts for a modest share of EU’s residential total (1.9%) and electricity-based (6 %) ﬁnal energy use in 2050, as projected by the International Energy Agency (IEA) in their Reference Technology Scenario (RTS) [2] . Table 5 On the other hand, the temporal eﬀect of weather on useful spe- ciﬁc cooling demand is less evident. Although the estimated CDD co- eﬃcient exhibits the correct ( + ) sign, it is only marginally signiﬁcant (p ≈0.097), while its inclusion has a minor impact on model perfor- mance. Nevertheless, the soft link between Qspec and CDD demonstrated through our results supports the argument brought forward later in peak cooling electricity demand calculations that simultaneous AC use across multiple households would more likely occur during extreme heat conditions. As expected, the sharpest increase of EU-28 residential AC energy consumption in the future is estimated under the “New Buildings AC rates ” scenario (104.1 TWh/y in 2050 for the mid-range trajectory). Due to the radical transformation of national AC markets, the cold group of countries account for the largest share of household space cooling en- ergy consumption in 2050 (60.1%), with Germany and France having a combined contribution of 54.7% to total FEU AC , as opposed to 11.5% in 2015. On the other hand, the future share of Spain, Italy and Greece in FEU AC drops to a third. Under this scenario, space cooling in 2050 rep- resents about 3.7% of ﬁnal residential energy and 11.7% of electricity use at the EU level. Fig. 4. Past and future residential AC energy consumption at the EU-28 level under diﬀerent scenarios. Note: Error bars represent the range of uncertainty in RCP and SSP projections Energy and Built Environment 1 (2020) 432–442 4.2. Policy recommendations Passive cooling needs to receive further support in the Energy Performance of Buildings Directive [41] , as a tool which can not only enhance energy conservation eﬀorts in residential buildings, but more importantly can help minimise the chance of mass penetration of mechanical space cool- ing technologies in the future. Our paper generally ﬁnds important diﬀerences from other studies which also accounted for the eﬀects of temperature and income on AC diﬀusion and useful speciﬁc cooling demand. The JRC projected EU-28 residential AC energy consumption to reach 33 TWh/y by 2050, with- out the inﬂuence of climate change, and to increase up to 78 TWh/y under an RCP8.5-like set of climate simulations [14] . While the JRC’s highest FEU AC estimate lies within the range of values obtained in this paper (38-104 TWh/y), they predict a much stronger impact on the growth of AC penetration rates for southern European countries in 2050 than our baseline case. These were shown to exceed the 80% diﬀu- sion level by 2050 and continue to ascend thereafter, due to higher as- sumed saturation rates and the determinant role of climate. On the other hand, mid-21 st century’s AC ownership rates across northern European countries in [14] were found to remain well below the 30% saturation point adopted in this paper. (ii) Encouraging further eﬃciency improvements : Our scenario analysis results show that increasing the eﬃciency of RAC systems by 40% in 2050 signiﬁcantly reduces EU-28 space cooling energy con- sumption and potential peak cooling electricity demand. In the base- line case, faster diﬀusion of residential air-conditioners in the medium term results in higher annual growth rates of space cooling energy consumption relative to those observed in the 2041-50 period, when AC markets have approached saturation ( Fig. 4 ). Earlier action to enforce stricter minimum AC performance standards could therefore reduce the excess space cooling loads which electricity systems will have to sustain over successive years in the medium term. Achieving faster penetra- tion of highly-eﬃcient AC units in the EU-28 residential sector should not only rely on strengthening regulatory measures, such as mandatory MEPS. It will also require that national governments increase funding towards cooling-related research, prioritise consumer information pro- Dittmann et al . [9] presented similar trends to [14] about the spa- tial heterogeneity of future climatic impacts on the share of residential cooled areas in southern and northern EU-28 countries. 4.1. Comparison with previous studies 4.1. Comparison with previous studies 4.2. Policy recommendations Our ﬁndings agree with the general trend found in previous studies, which predict a signiﬁcant increase of electricity-based ﬁnal energy use for space cooling in the EU’s residential sector [3] . However, discrepan- cies arise between projections about the exact level of AC energy con- sumption in 2050. Our baseline FEU AC estimate for the EU-28 region in 2050 is roughly 2.5 times as large as the IEA’s RTS projection for space cooling energy consumption (21.8 TWh/y) [2] . The latest FEU AC esti- mate for 2050 for the EU-28 region is found in the JRC POTEnCIA cen- tral scenario (42.5 TWh/y) [40] , which compares well with our strong unit eﬃciency projection (38.4 TWh/y). Unlike this study’s modelling approach, these assessments did not examine potential implications of climate change on future residential AC energy consumption. In general, our ﬁndings highlight the expected important contribu- tion of future space cooling diﬀusion trajectories to the increasing stress felt by regional power systems, particularly through requiring additional capacity to meet summer time AC loads. A number of energy policies could therefore be promoted to limit the growth of EU-28 residential electricity use from AC growth to 2050: (i) Eﬀective promotion of passive cooling designs : Passive cool- ing comprises all those natural or passive techniques that can help main- tain indoor thermal comfort, while requiring minimal or zero energy in- put [3] . These can be split into processes preventing solar heat gains (e.g. better shading systems, roof and glazing properties), those modulating heat through utilisation of buildings’ thermal mass and those dissipat- ing heat (e.g., natural ventilation and evaporative cooling) [50] . Passive cooling needs to receive further support in the Energy Performance of Buildings Directive [41] , as a tool which can not only enhance energy conservation eﬀorts in residential buildings, but more importantly can help minimise the chance of mass penetration of mechanical space cool- ing technologies in the future. (i) Eﬀective promotion of passive cooling designs : Passive cool- ing comprises all those natural or passive techniques that can help main- tain indoor thermal comfort, while requiring minimal or zero energy in- put [3] . These can be split into processes preventing solar heat gains (e.g. better shading systems, roof and glazing properties), those modulating heat through utilisation of buildings’ thermal mass and those dissipat- ing heat (e.g., natural ventilation and evaporative cooling) [50] . 3.3. Future scenarios of residential AC energy consumption (2016-2050) This is especially the case for “New buildings AC rates ” scenario, whereby aggregate Peak AC increases by as much as twice (~1.9) the size of total solar capacity in cold countries, highlighting po- tential risks of electricity system failure if other sources of generating capacity are not added to meet peaking demand and alternative cooling options are not provided. In contrast to cold Member States, growth in solar capacity in warm countries catches up with that of potential peak Penetration levels of residential air-conditioning deviate signiﬁ- cantly from the Baseline scenario in the “New Buildings AC rates ” case, with the largest impact observed in cold countries where new and reno- vated buildings have low installation rates during the historical analysis period. Under mid-range trajectories, residential AC ownership rate in cold countries reaches 87.5% in 2050 without any signs of saturation, while diﬀusion in warm countries stagnates at 78.5%. Overall, aggre- gate space cooling diﬀusion in the EU-28 region reaches 85.1% in 2050 under this "bad case" scenario. Residential space cooling energy consumption is calculated at one- year time steps from 2016-50, under each of the 3 scenarios ( Fig. 4 ). In the baseline case, mid-range FEU AC across the EU increases by a factor of 3.4 in 2050 (53.7 TWh/y) relative to 2015. The contribution of cold countries to total FEU AC levels rises from 21.9% in 2015 to 39.5% in 2050, whereas that of warm states declines from 78.1% to 60.5% in the same time period. Italy and Spain together represent the largest portion of EU-28 space cooling energy consumption (46.4% compared to 59.8% 439 A. Andreou, J. Barrett and P.G. Taylor et al. Energy and Built Environment 1 (2020) 432–442 Fig. 5. Ratio of potential peak cooling electricity demand to solar-based capacity for cold (left) and warm (right) EU-28 countries Fig. 5. Ratio of potential peak cooling electricity demand to solar-based capacity for cold (left) and warm (right) EU-28 coun cooling demand much earlier in the 2016-50 period, with the latest turn- ing point occurring by 2025 under the extreme AC diﬀusion scenario. energy consumption. They estimated EU-27 residential ﬁnal electricity use for space cooling to reach 129-233 TWh/y [16] and 634-754 TWh/y [15] in 2050. Each range represents the diﬀerence in predicted AC en- ergy consumption levels for a constant climate case and a medium-high greenhouse emissions scenario in 2050. 4.2. Policy recommendations For a moderate climate change trajectory (RCP4.5), their EU-level estimate of FEU AC in 2050 amounts to 31 TWh/y, which is lower than the range of our estimates. Mima and Criqui [15 , 16] were the only studies which pro- jected a signiﬁcantly larger increase of future levels of space cooling 440 A. Andreou, J. Barrett and P.G. Taylor et al. Energy and Built Environment 1 (2020) 432–442 hold AC energy consumption in 2050 (54 TWh/y) is 32 TWh/y and 11 TWh/y higher than projections from IEA [2] and JRC [40] , respectively, which do not forecast potential temperature-driven increases of AC us- age in the future. When compared to other climate change impact as- sessments, our study generally ﬁnds important diﬀerences regarding the degree of north-south polarisation of AC diﬀusion in the EU-28 region [9 , 14] and the aggregate level of residential AC energy consumption in 2050 [15 , 16] . grammes about the beneﬁts of using energy eﬃcient AC units and pro- vide ﬁnancial incentives which increase the market availability of these products [1] . (iii) Amendment of renovation strategies : EU-28 Member States are obliged, under the Energy Eﬃciency Directive [51] , to publish long- term renovation strategies with a description of current housing stock and cost-eﬀective approaches to achieve deep renovation. While the ma- jority of countries were found to be compliant with this requirement [52] , there is still not a unifying approach towards curbing demand for space cooling. Moreover, the growing role of air-conditioning in achieving renovation goals has been overlooked by many of cold coun- tries. Updated strategies should parameterise anticipated changes in lo- cal climate characteristics, and also address other risk factors, such as increased consumers’ thermal comfort expectations [53] . Third, our study showed that electricity systems will have to sustain a higher level of peak cooling demand in the future if met by mechanical air-conditioners, which could challenge generation and network perfor- mance, subject to the size of electrical capacity installed [56] . [1] IEA, The Future of Cooling: Opportunities for energy-eﬃcient air conditioning, 2018 Paris, doi: 10.1787/9789264301993-en . [2] IEA, Energy Technology Perspectives 2017: Catalysing Energy Technology Transfor- mations, 2017 Paris, doi: 10.1787/energy_tech-2017-en . [3] M. Santamouris, Cooling the buildings – past, present and future, Energy Build 128 (2016) 617–638, doi: 10.1016/j.enbuild.2016.07.034 . [4] JRC, JRC-IDEES: Integrated Database of the European Energy Sector: Methodologi- cal note, 2017 Luxembourg, doi: 10.2760/182725 . 4.2. Policy recommendations This could be a particular issue in EU-28 countries whose power infrastructure is currently designed to face the highest loads during the winter season [57] ; a potential seasonal shift of peak electricity demand to summer months, as a result of increased AC usage, would have implications for improved inter-seasonal storage of renewable electricity, which could be then transmitted across the EU to places with high peak cooling de- mand. Future work could focus on devising future projections of actual peak cooling demand in EU-28 countries, which are based on functions that explain the variation in number of residential AC units being active in a region according to a set of climatic and non-climatic conditions, similar to [58] . (iv) Diversiﬁcation of cooling supply : While residential space cool- ing is usually supplied through electric room air-conditioners, decen- tralised, small-scale, production sites are emerging as alternative cold providers. Amongst available technologies, district cooling (DC) is con- sidered by the EU as an integral part of a future highly-eﬃcient space cooling sector [51] , as it oﬀers substantial environmental and primary energy savings beneﬁts [54] . Installing decentralised DC plants in urban areas with high cold demand density can more importantly increase ﬂex- ibility of cooling supply by reducing the stress on European electricity systems. 5. Conclusions This paper has developed and applied new approaches to decipher- ing drivers of past and future trends of electricity-based ﬁnal energy use for air-conditioning in EU’s residential sector; an end-use charac- terised by tremendous growth potential. A novel multi-method mod- elling framework was constructed, which used index decomposition analysis as a reference point for understanding the drivers of past space cooling energy consumption, then extended this to a set of panel data models estimating climatic and non-climatic eﬀects on AC components. Finally, a combination of the two methods led to the creation of sce- narios of residential AC energy consumption and potential peak cooling electricity demand in EU-28 countries up to 2050. CRediT authorship contribution statement Andreas Andreou: Conceptualization, Formal analysis, Methodol- ogy, Data curation, Investigation, Writing - original draft, Visualization, Software, Validation, Writing - review & editing. John Barrett: Con- ceptualization, Supervision, Visualization, Project administration, Re- sources. Peter G. Taylor: Conceptualization, Supervision, Visualization, Writing - review & editing, Funding acquisition. Paul E. Brockway: Conceptualization, Supervision, Visualization, Writing - review & edit- ing, Funding acquisition. Zia Wadud: Conceptualization, Supervision, Visualization, Methodology. Acknowledgments This PhD research is funded by the Priestley International Centre for Climate (PICC), which is based in University of Leeds, UK. Pe- ter Taylor gratefully acknowledges support from UK Research and In- novation through the Centre for Research into Energy Demand Solu- tions, grant reference number EP/R035288/1. Paul Brockway’s time was funded by the UK Research Councils, supported under EPSRC award EP/L024756/1 as part of the research programme of the UK Energy Research Centre (UKERC) and latterly under EPSRC Fellowship award EP/R024254/1. First, index decomposition analysis showed that penetration of air- conditioning and technical eﬃciency improvements, to a lesser extent, shaped past trends (2000-15) of space cooling energy consumption in EU-28 households. AC diﬀusion was by far the key driver in the past, contributing to the annual increase of EU-28 space cooling energy con- sumption on average by 1 TWh each year. This increasing eﬀect was only partly counterbalanced by AC unit eﬃciency gains over the same time period. Econometric analysis also suggested that both the diﬀusion of air-conditioning in households and useful speciﬁc energy demand de- pend on temperature variation. However, personal income was found to be the most important determinant of past AC diﬀusion, having a ﬁve times larger marginal eﬀect compared to mean summer temperature. The authors declare no conﬂict of interest. The authors declare no conﬂict of interest. Declaration of competing interest However, the size of DC systems in terms of peak demand capacity is presently limited to 1.7 GW in cold EU-28 countries and 0.5 GW in warm ones [55] , which we estimate represents only 10% and 2% of the cold and warm sub-regions’ potential peak cooling electricity demand in 2015 respectively. Furthermore, local authorities need to design a com- bination of ﬁscal incentives and bonus mechanisms for DC suppliers in order to overcome market obstacles and increase this technology’s share in EU-28 space cooling supply. In addition to minimising market risks, innovation in building engineering could facilitate easier connection of buildings to nearby decentralised systems. Data Repository A complete set of input and results datasets for this paper have been deposited at the University of Leeds Data Repository at doi: 10.5518/821 . [4] JRC, JRC-IDEES: Integrated Database of the European Energy Sector: Methodologi- cal note, 2017 Luxembourg, doi: 10.2760/182725 . References Second, through scenario analysis we devised three potential trajec- tories of AC diﬀusion and unit eﬃciency, based on which EU-28 ag- gregate residential space cooling energy consumption grows from 16 TWh/y in 2015 to 38-104 TWh/y in 2050. This represents an increase in the share of space cooling in EU-level residential ﬁnal electricity use from 2% in 2015 to 4-12% in 2050. Our baseline estimate of house- 441 A. Andreou, J. Barrett and P.G. Taylor et al. Energy and Built Environment 1 (2020) 432–442 [5] RESCUE, EU District cooling market and trends WP2.3, 2014 Stockholm https://www.euroheat.org/wp-content/uploads/2016/04/RESCUE_EU_Cooling_ Market.pdf . household cooling energy consumption, Appl. Energy 88 (2011) 2191–2200, doi: 10.1016/j.apenergy.2011.01.010 . household cooling energy consumption, Appl. Energy 88 (2011) 2191–2200, doi: 10.1016/j.apenergy.2011.01.010 . [35] R. Fazeli, M. Ruth, B. Davidsdottir, Temperature response functions for resi- dential energy demand - A review of models, Urban Clim 15 (2016) 45–59, doi: 10.1016/j.uclim.2016.01.001 . [6] S. Pezzutto, M. De Felice, R. Fazeli, L. Kranzl, S. Zambotti, Status quo of the air- conditioning market in europe: Assessment of the building stock, Energies 10 (2017) 1253, doi: 10.3390/en10091253 . /j [36] K. Riahi, D.P. van Vuuren, E. Kriegler, J. Edmonds, B.C. O’Neill, S. Fujimori, N. Bauer, K. Calvin, R. Dellink, O. Fricko, W. Lutz, A. Popp, J.C. Cuaresma, S. KC, M. Leimbach, L. Jiang, T. Kram, S. Rao, J. Emmerling, K. Ebi, T. Hasegawa, P. Havlik, F. Humpenöder, L.A. Da Silva, S. Smith, E. Stehfest, V. Bosetti, J. Eom, D. Gernaat, T. Masui, J. Rogelj, J. Streﬂer, L. Drouet, V. Krey, G. Luderer, M. Harmsen, K. Takahashi, L. Baumstark, J.C. Doelman, M. Kainuma, Z. Klimont, G. Marangoni, H. Lotze-Campen, M. Obersteiner, A. Tabeau, M. Tavoni, The Shared Socioeconomic Pathways and their energy, land use, and greenhouse gas emissions implications: An overview, Glob. Environ. Chang. 42 (2017) 153–168, doi: 10.1016/j.gloenvcha.2016.05.009 . [7] S. Pezzutto, R. Fazeli, M. De Felice, W. Sparber, Future development of the air- conditioning market in Europe: an outlook until 2020, Wiley Interdiscip. Rev. Energy Environ. 5 (2016) 649–669, doi: 10.1002/wene.210 . t, T. Masui, J. Rogelj, J. Streﬂer, L. Drouet, V. Krey, G. Ludere [8] M. Izquierdo, A. Moreno-Rodríguez, A. González-Gil, N. García-Hernando, Air conditioning in the region of Madrid, Spain: An approach to electricity consumption, economics and CO2 emissions, Energy 36 (2011) 1630–1639, doi: 10.1016/j.energy.2010.12.068 . [9] F. Dittmann, P. Rivière, P. References Kolokotsa, Passive cooling dissipation techniques for build- ings and other structures: The state of the art, Energy Build 57 (2013) 74–94, doi: 10.1016/j.enbuild.2012.11.002 . j [51] European Parliament, Directive 2012/27/EU on Energy Eﬃciency, Oﬀ. J. Eur. Union L315 (2012) 1–56, doi: 10.3000/19770677.L_2012.315.eng . [24] M.A. McNeil, V.E. Letschert, Modeling diﬀusion of electrical appli- ances in the residential sector, Energy Build 42 (2010) 783–790, doi: 10.1016/j.enbuild.2009.11.015 . [52] JRC, Assessment of second long-term renovation strategies under the Energy Eﬃ- ciency Directive, 2019 Luxembourg, doi: 10.2760/973672 . j [25] M. Auﬀhammer, Cooling China : The Weather Dependence of Air Conditioner Adop- tion, Front. Econ. China. 9 (2014) 70–84, doi: 10.3868/s060-003-014-0005-5 . [53] B. Aebischer , G. Henderson , G. Catenazzi , M. Jakob , Impact of climate change on thermal comfort, heating and cooling energy demand in Europe, ECEEE 2007 Sum- mer Study, 2007, pp. 859–870 . [26] J. Biddle, Explaining the spread of residential air conditioning, 1955-1980, Explor. Econ. Hist. 45 (2008) 402–423, doi: 10.1016/j.eeh.2008.02.004 . [54] ECOHEATCOOL, Possibilities with more district cooling in Europe WP5, 2006 Brus- sels https://www.euroheat.org/wp-content/uploads/2016/02/Ecoheatcool_WP5_ Web.pdf . [27] L.W. Davis, P.J. Gertler, Contribution of air conditioning adoption to future en- ergy use under global warming, Proc. Natl. Acad. Sci. 112 (2015) 5962–5967, doi: 10.1073/pnas.1423558112 . [28] D. Rapson, Durable goods and long-run electricity demand: Evidence from air conditioner purchase behavior, J. Environ. Econ. Manage. 68 (2014) 141–160, doi: 10.1016/j.jeem.2014.04.003 . [55] DG ENER, Mapping and analyses of the current and future (2020 - 2030) heating/ cooling fuel deployment (fossil/renewables) WP2, 2016 Brussels https://ec.europa.eu/energy/sites/ener/ﬁles/documents/mapping-hc-ﬁnal_ report-wp2.pdf . [29] M. Auﬀhammer, E.T. Mansur, Measuring climatic impacts on energy consump- tion: A review of the empirical literature, Energy Econ 46 (2014) 522–530, doi: 10.1016/j.eneco.2014.04.017 . [56] D. Connolly, Heat Roadmap Europe: Quantitative comparison between the electric- ity, heating, and cooling sectors for diﬀerent European countries, Energy 139 (2017) 580–593, doi: 10.1016/j.energy.2017.07.037 . [30] B. Baltagi , Econometric Analysis of Panel Data, 3rd ed., John Wiley & Sons, Chich- ester (UK), 2008 . [57] L. Wenz, A. Levermann, M. Auﬀhammer, North–south polarization of European elec- tricity consumption under future warming, Proc. Natl. Acad. Sci. 114 (2017) E7910– E7918, doi: 10.1073/pnas.1704339114 . [31] V. Daioglou, B.J. van Ruijven, D.P. van Vuuren, Model projections for household energy use in developing countries, Energy 37 (2012) 601–615, doi: 10.1016/j.energy.2011.10.044 . [58] D. Burillo, M.V. Chester, B. Ruddell, N. References http://appsso.eurostat.ec.europa.eu/nui/show.do?dataset = demo_r_pjangrp3& lang = en . [17] M. Reuter, M.K. Patel, W. Eichhammer, Applying ex post index decomposition anal- ysis to ﬁnal energy consumption for evaluating European energy eﬃciency policies and targets, Energy Eﬃc 12 (2019) 1329–1357, doi: 10.1007/s12053-018-09772-w . [45] ESTAT, Environment and energy, Energy Stat. (2015) http://appsso.eurostat.ec.europa.eu/nui/show.do?dataset = nrg_chdd_a&lang = en . [18] C. Achão, R. Schaeﬀer, Decomposition analysis of the variations in residen- tial electricity consumption in Brazil for the 1980-2007 period: Measuring the activity, intensity and structure eﬀects, Energy Policy 37 (2009) 5208–5220, doi: 10.1016/j.enpol.2009.07.043 . [46] I. Harris, P.D. Jones, T.J. Osborn, D.H. Lister, Updated high-resolution grids of monthly climatic observations - the CRU TS3.10 Dataset, Int. J. Climatol. 34 (2014) 623–642, doi: 10.1002/joc.3711 . [47] ESTAT, GISCO: Geographical Information and Maps, Adm. Units / Stat. Units. (2013) https://ec.europa.eu/eurostat/web/gisco/geodata/reference-data/administrative- units-statistical-units . [19] H. Nie, R. Kemp, Index decomposition analysis of residential energy consumption in China: 2002-2010, Appl. Energy. 121 (2014) 10–19, doi: 10.1016/j.apenergy.2014.01.070 . [48] D. Jacob, J. Petersen, B. Eggert, A. Alias, O.B. Christensen, L.M. Bouwer, A. Braun, A. Colette, M. Déqué, G. Georgievski, E. Georgopoulou, A. Gobiet, L. Menut, G. Nikulin, A. Haensler, N. Hempelmann, C. Jones, K. Keuler, S. Kovats, N. Kröner, S. Kotlarski, A. Kriegsmann, E. Martin, E. van Meijgaard, C. Moseley, S. Pfeifer, S. Preuschmann, C. Radermacher, K. Radtke, D. Rechid, M. Rounsev- ell, P. Samuelsson, S. Somot, J.F. Soussana, C. Teichmann, R. Valentini, R. Vau- tard, B. Weber, P. Yiou, EURO-CORDEX: new high-resolution climate change pro- jections for European impact research, Reg. Environ. Chang. 14 (2014) 563–578, doi: 10.1007/s10113-013-0499-2 . j p gy [20] X.Y. Xu, B.W. Ang, Analysing residential energy consumption us- ing index decomposition analysis, Appl. Energy. 113 (2014) 342–351, doi: 10.1016/j.apenergy.2013.07.052 . [21] M. Isaac, D.P. van Vuuren, Modeling global residential sector energy demand for heating and air conditioning in the context of climate change, Energy Policy 37 (2009) 507–521, doi: 10.1016/j.enpol.2008.09.051 . [22] B.W. Ang, Decomposition analysis for policymaking in energy: Which is the preferred method? Energy Policy 32 (2004) 1131–1139, doi: 10.1016/S0301-4215(03)00076-4 . [49] European Commission, EU Reference Scenario 2016 - Energy, transport and GHG emissions Trends to 2050, 2016 Luxembourg, doi: 10.2833/9127 . [23] M.A. McNeil, V.E. Letschert, Future Air Conditioning Energy Consumption in De- veloping Countries and what can be done about it: The Potential of Eﬃciency in the Residential Sector, 2008 Berkeley (CA) https://escholarship.org/uc/item/ 64f9r6wr . [50] M. Santamouris, D. References Stabat, Space Cooling Technology in Europe: Tech- nology Data and Demand Modelling, Heat Roadmap Europe WP3.2, 2017 Paris https://heatroadmap.eu/wp-content/uploads/2018/11/HRE4_D3.2.pdf . [37] K. Riahi, S. Rao, V. Krey, C. Cho, V. Chirkov, G. Fischer, G. Kindermann, N. Nakicen- ovic, P. Rafaj, RCP 8.5-A scenario of comparatively high greenhouse gas emissions, Clim. Change 109 (2011) 33–57, doi: 10.1007/s10584-011-0149-y . [10] S. Werner, European space cooling demands, Energy 110 (2016) 148–156, doi: 10.1016/j.energy.2015.11.028 . [11] W. Sparber, S. Pezzutto, Potential and actual space heating and cooling con- sumption in Europe (EURAC Research), RHC Annu. Event, 2014 Brussels, doi: 10.13140/RG.2.1.1744.6249 . [38] N. Shah, M. Wei, V. Letschert, A. Phadke, Beneﬁts of Leapfrogging to Supereﬃciency and Low Global Warming Potential Refrigerants in Room Air Conditioning, 2015 Berkeley (CA), doi: 10.2172/1397235 . [12] M. Jakubcionis, J. Carlsson, Estimation of European Union residen- tial sector space cooling potential, Energy Policy 101 (2017) 225–235, doi: 10.1016/j.enpol.2016.11.047 . [39] A. Phadke, N. Abhyankar, N. Shah, Avoiding 100 New Power Plants by Increasing Eﬃciency of Room Air Conditioners in India : Opportunities and Challenges, 2014 Berkeley (CA) https://ies.lbl.gov/publications/avoiding-100-new-power-plants . y p g p g p p [40] JRC, The POTEnCIA Central scenario: An EU energy outlook to 2050, 2019 Luxem- bourg, doi: 10.2760/32835 . [13] G. Henderson , Home air conditioning in Europe – how much energy would we use if we became more like American households ? in: ECEEE 2005 Summer Study, 2005, pp. 541–550 . [41] European Parliament, Directive 2018/844/EU on Energy Performance of Buildings, Oﬀ. J. Eur. Union. L 156 (2018) 75–91 . pp [14] JRC, Assessment of the impact of climate change on residential energy demand for heating and cooling, 2018 Luxembourg, doi: 10.2760/96778 . [42] M. Olonscheck, A. Holsten, J.P. Kropp, Heating and cooling energy demand and related emissions of the German residential building stock under climate change, Energy Policy 39 (2011) 4795–4806, doi: 10.1016/j.enpol.2011.06.041 . [15] S. Mima, P. Criqui, Assessment of the impacts under future climate change on the energy systems with the POLES model, Int. Energy Work., 2009 Venice, Italy https://halshs.archives-ouvertes.fr/halshs-00452948 . [43] World Bank, Economy and Growth, World Dev. Indic, 2018 https://data.worldbank.org/indicator/NY.GDP.MKTP.PP.KD . p [16] S. Mima, P. Criqui, The Costs of Climate Change for the European Energy System, an Assessment with the POLES Model, Environ. Model. Assess 20 (2015) 303–319, doi: 10.1007/s10666-015-9449-3 . [44] ESTAT, Population (Demograhy, Migration and Projections), Reg. Data. (2014). References Johnson, Electricity demand plan- ning forecasts should consider climate non-stationarity to maintain re- serve margins during heat waves, Appl. Energy. 206 (2017) 267–277, doi: 10.1016/j.apenergy.2017.08.141 . [32] B.J. van Ruijven, D.P. van Vuuren, B.J.M. de Vries, M. Isaac, J.P. van der Sluijs, P.L. Lucas, P. Balachandra, Model projections for household energy use in India, Energy Policy 39 (2011) 7747–7761, doi: 10.1016/j.enpol.2011.09.021 . [59] ESTAT, Cooling and heating degree days, Energy Stat. (2019) https://ec.europa.eu/eurostat/cache/metadata/en/nrg_chdd_esms.htm . [33] A. Levesque, R.C. Pietzcker, L. Baumstark, S. De Stercke, A. Grübler, G. Luderer, How much energy will buildings consume in 2100? A global perspective within a scenario framework, Energy 148 (2018) 514–527, doi: 10.1016/j.energy.2018.01.139 . [34] G.Y. Yun, K. Steemers, Behavioural, physical and socio-economic factors in 442
https://openalex.org/W2148034839	https://hal.archives-ouvertes.fr/hal-00804579/document	English	null	Mapping Cortico-Striatal Connectivity onto the Cortical Surface: A New Tractography-Based Approach to Study Huntington Disease	PloS one	2,013	cc-by	8,278	To cite this version: Linda Marrakchi-Kacem, Christine Delmaire, Pamela Guevara, Fabrice Poupon, Sophie Lecomte, et al.. Mapping Cortico-Striatal Connectivity onto the Cortical Surface: A New Tractography- Based Approach to Study Huntington Disease.. PLoS ONE, 2013, 8 (2), pp.e53135. 10.1371/jour- nal.pone.0053135. hal-00804579 Abstract Huntington disease (HD) is associated with early and severe damage to the basal ganglia and particularly the striatum. We investigated cortico-striatal connectivity modifications occurring in HD patients using a novel approach which focuses on the projection of the connectivity profile of the basal ganglia onto the cortex. This approach consists in computing, for each subcortical structure, surface connectivity measures representing its strength of connections to the cortex and comparing these measures across groups. In this study, we focused on Huntington disease as an application of this new approach. First, surface cortico-striatal connectivity measures of a group of healthy subjects were averaged in order to infer the ‘‘normal’’ connectivity profile of the striatum to the cortex. Second, a statistical analysis was performed from the surface connectivity measures of healthy subjects and HD patients in order to detect the cortical gyri presenting altered cortico-striatal connectivity in HD. Lastly, percentage differences of connectivity between healthy subjects and patients were inferred, for each nucleus of the striatum, from the connectivity measures of the cortical gyri presenting a significant connectivity difference between the two groups. These percentage differences characterize the axonal disruptions between the striatum and the cortex occurring in HD. We found selective region-specific degeneration of cortical connections predominating for associative and primary sensorimotor connections and with relative preservation of limbic connections. Our method can be used to infer novel connectivity-based markers of HD pathological process. Citation: Marrakchi-Kacem L, Delmaire C, Guevara P, Poupon F, Lecomte S, et al. (2013) Mapping Cortico-Striatal Connectivity onto the Cortical Surface: A New Tractography-Based Approach to Study Huntington Disease. PLoS ONE 8(2): e53135. doi:10.1371/journal.pone.0053135 Editor: Bogdan Draganski, Centre Hospitalier Universitaire Vaudois Lausanne – CHUV, UNIL, Switzerland Editor: Bogdan Draganski, Centre Hospitalier Universitaire Vaudois Lausanne – CHUV, UNIL, Switzerland Received March 5, 2012; Accepted November 28, 2012; Published February 6, 2013 eived March 5, 2012; Accepted November 28, 2012; Published Copyright: 2013 Marrakchi-Kacem et al. This is an open-access article distributed under the terms of the Creative Commons Attribu unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. akchi-Kacem et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. HAL Id: hal-00804579 https://hal.science/hal-00804579v1 Submitted on 26 Mar 2013 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Abstract Funding: TRACK-HD is supported by the Child Health and Development Institute CHDI/High Q Foundation, a not for profit organization dedicated to finding treatments for Huntington disease. This work was also supported by the Ecole des Neurosciences Paris-Ile-de-France (NucleiPark project), by the ANR/MNP/2009 (NucleiPark project) and by the Association France Parkinson (NucleiPark project). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: linda.marrakchi@gmail.com Mapping Cortico-Striatal Connectivity onto the Cortical Surface: A New Tractography-Based Approach to Study Huntington Disease Linda Marrakchi-Kacem1,2, Christine Delmaire3,4, Pamela Guevara1,2,5, Fabrice Poupon1,2, Sophie Lecomte1,2, Alan Tucholka6, Pauline Roca1,2, Je´roˆ me Yelnik7,8,9,10,11, Alexandra Durr7,8,9,10,12, Jean-Franc¸ois Mangin1,2, Ste´phane Lehe´ricy4,7,8,9,10, Cyril Poupon1,2 1 NeuroSpin, Commissariat a` l9Energie Atomique (CEA), Gif-Sur-Yvette, France, 2 Institut Fe´de´ratif de Recherche IFR49, Gif-Sur-Yvette, France, 3 De´partement de Neuroradiologie, Centre Hospitalier Re´gional Universitaire de Lille, Lille, France, 4 Centre de Neuro-Imagerie de Recherche CENIR, AP-HP, Hoˆpital de la Salpeˆtrie`re, Paris, France, 5 University of Concepcion, Concepcion, Chile, 6 Department of Radiology, CHUM, Notre Dame Hospital, Montreal, Canada, 7 Universite´ Pierre et Marie Curie-Paris 6, Centre de Recherche de l9Institut du Cerveau et de la Moelle e´pinie`re, UMR-S975, Paris, France, 8 Inserm, U975, Paris, France, 9 CNRS, UMR 7225, Paris, France, 10 ICM – Institut du Cerveau et de la Moe¨lle e´pinie`re, Paris, France, 11 De´partement de Neurologie, Centre d’Investigation Clinique, AP-HP, Hoˆpital de la Salpeˆtrie`re, Paris, France, 12 De´partement de Ge´ne´tique et Cytoge´ne´tique, AP-HP, Hoˆpital de la Salpeˆtrie`re, Paris, France February 2013 \| Volume 8 \| Issue 2 \| e53135 Mapping Cortico-Striatal Connections on the Cortex parameters: sagittal 3D MPRAGE T1-weighted: FOV = 2566256 mm2, matrix 256256, Flip angle = 10u TE/ TR = 2.98 ms/2.3 s, slice thickness TH = 1.1 mm, inversion time TI = 900 ms, 160 slices per slab, read bandwidth RBW = 240Hz/ pixel; Single-shot twice refocused spin-echo diffusion-weighted (DW)-EPI: FOV = 2566256 mm2, TH = 2 mm, matrix 128128, TE/TR = 86ms/12 s, GRAPPA 2, partial Fourier factor 6/8, 80 slices, RBW = 1630 Hz/pixel, b-value = 1000 s/mm2, 50 non collinear directions uniformly distributed; Dual-echo 2D gradient field map: FOV = 176 224mm2, matrix 6464, TE1/TE2 = 4.92/ 7.3, TH = 3.5 mm, TR = 0.5 s, 37 slices, RBW = 200 Hz/pixel, flip angle = 60u. parameters: sagittal 3D MPRAGE T1-weighted: FOV = 2566256 mm2, matrix 256256, Flip angle = 10u TE/ TR = 2.98 ms/2.3 s, slice thickness TH = 1.1 mm, inversion time TI = 900 ms, 160 slices per slab, read bandwidth RBW = 240Hz/ pixel; Single-shot twice refocused spin-echo diffusion-weighted (DW)-EPI: FOV = 2566256 mm2, TH = 2 mm, matrix 128128, TE/TR = 86ms/12 s, GRAPPA 2, partial Fourier factor 6/8, 80 slices, RBW = 1630 Hz/pixel, b-value = 1000 s/mm2, 50 non collinear directions uniformly distributed; Dual-echo 2D gradient field map: FOV = 176 224mm2, matrix 64*64, TE1/TE2 = 4.92/ 7.3, TH = 3.5 mm, TR = 0.5 s, 37 slices, RBW = 200 Hz/pixel, flip angle = 60u. tion between the basal ganglia and the cortex or analysed connectivity measures in the cortical space. Only one study measured changes of connectivity between the striatum and the cortex in HD using tractography [13] but focusing only on the frontal cortex. All the previous studies relying on tractography techniques (deterministic, probabilistic or bayesian) were based on tracking connections between regions of interest (ROI) which may present several limitations when studying the connections of the basal ganglia. First, they did not take into account the loops in which these structures are involved and can therefore create false direct connections between deep structures or between deep structures and the cortex. Second, they were restricted to the ROIs that were selected. Other approaches have been proposed recently, such as Tract-Based Spatial Statistics TBSS 14] which provide scalar measures including FA, MD or parallel (lI) and transverse (lH) diffusivity on a FA-based skeleton of tracts and allows the statistical comparison of these values between groups. T1-weighted data analysis Deep nuclei segmentation. In order to investigate cortico- striatal connectivity profiles, we segmented not only the striatum but also other deep nuclei that are involved in cortico-subcortical loops. These nuclei were used in the cortico-striatal tracts selection step. Segmentation of the deep nuclei was performed from T1- weighted data using a deformable model with regions evolving in competition with topology constraints described in [15]. The automatic segmentation included three basal ganglia nuclei in each hemisphere (the left and right caudate nucleus (LCd and RCd, respectively), the left and right putamen (LPu and RPu), the left and right globus pallidus (LGP and RGP), and the thalamus, the main output structure of the basal ganglia (left and right thalamus: LTh and RTh, respectively). All the automatic segmentations were checked by an expert (CD) and corrected manually accordingly. To address these limitations, we have developed a novel tractography-based approach to study the connections of the basal ganglia and the thalamus, which takes into account the known anatomy of cortico-basal ganglia loops. This approach projects the subcortico-cortical connectivity directly on the cortical surface, allowing whole brain analysis. It provides surface connectivity measures that can be used in group comparison studies to detect putative pathology-related modifications in the connections of the basal ganglia to the cortex. To illustrate this method we used it in the case of Huntington disease to investigate cortico-striatal connections modifications in HD. Cortex segmentation and parcellation. FreeSurfer v5.0 was used to extract the surface of the cortex for all the subjects [16]. A spherical resampling was performed as proposed by [17] in order to have the same number of vertices for all the subjects and a direct correspondence between these vertices across subjects. This property allowed accurate and direct matching of the surfaces of different subjects. In addition, to represent the information stemming from different subjects, an average surface was computed for each population from the individual surfaces extracted for all the subjects. The cortex of each subject was then subdivided into regions of interest using the FreeSurfer gyrus segmentation [18]. These regions were used for the computation of local connectivity statistics across subjects. Cortex segmentation and parcellation. FreeSurfer v5.0 was used to extract the surface of the cortex for all the subjects [16]. Mapping Cortico-Striatal Connections on the Cortex This method only highlights diffusion changes that are restricted to the skeleton of the main brain fibre tracts and does not provide information on the target cortical areas which connectivity is affected by the disease. T1-weighted data analysis A spherical resampling was performed as proposed by [17] in order to have the same number of vertices for all the subjects and a direct correspondence between these vertices across subjects. This property allowed accurate and direct matching of the surfaces of different subjects. In addition, to represent the information stemming from different subjects, an average surface was computed for each population from the individual surfaces extracted for all the subjects. The cortex of each subject was then subdivided into regions of interest using the FreeSurfer gyrus segmentation [18]. These regions were used for the computation of local connectivity statistics across subjects. The paper is organized as follows: 1) Introduction of the processing pipeline used to project the connectivity information onto the cortical surface, 2) description of the methods developed to infer the connectivity profile of each nucleus to the cortex, 3) use of the surface connectivity measures for the detection of cortical areas presenting abnormal connectivity with the striatum in HD. Materials and Methods Our approach was based on the computation of surface connectivity measures characterizing the density of the connec- tions between the striatum and the cortex. This approach used both T1-weighted data to extract the deep structures and the cortex, and high angular resolution diffusion-weighted data (HARDI) to recover the anatomical connectivity. The following sections describe the different steps of image processing required to compute the surface connectivity measures and to analyze them using adequate statistics. Tissues and CSF extraction. The gray matter GM, white matter WM and cerebrospinal fluid CSF were extracted using SPM5 software. We computed the intracranial volume of each subject by summing the volumes of GM, WM and CSF. This volume was used to normalize connectivity measures in the prospect of performing group comparison. Diffusion data analysis Diffusion data analysis Artifact removal and registration. Diffusion-weighted data were corrected from artefacts as follows. Slice outliers due to spikes Introduction the Brownian motion of water molecules within the tissue like the mean/ transverse/ and parallel diffusivities (MD, lH, lI) and the fractional anisotropy (FA) [4,5,6]. In addition, dMRI allows inferring their anatomical connectivity using tractography tech- niques [7]. Huntington disease (HD) is a neurodegenerative disorder, caused by CAG repeat expansion in the HTT gene, which is located on chromosome 4 and encodes huntingtin [1]. A hallmark of HD is the progressive degeneration of the striatal medium-size spiny neurons, which represent the greatest neuronal populations of the striatum [2]. Therefore, the striatum is a part of the basal ganglia that is early and severely affected by the disease [2,3]. In healthy volunteers, several studies relying on dMRI and tractography have analysed the connectivity of the basal ganglia [7,8,9,10] as well as their main output structure, the thalamus [11]. Diffusion MRI was also used to delineate the associative, sensorimotor and limbic functional territories of the basal ganglia in healthy volunteers based on their cortical connectivity information [7,8,10]. Recently, this method of parcellation of the basal ganglia into functional territories was used to investigate dMRI-based measures in each territory in patients with HD [12]. However, this study did not investigate the connectivity informa- Neuroimaging is increasingly used to investigate basal ganglia disorders as it can provide precise description and characterization of basal ganglia damage. While structural MRI provides some macroscopic information to characterize the atrophy of these structures, such as their volume, diffusion-weighted MRI (dMRI) can be used to characterize changes of their structure at the microscopic scale by means of rotationally invariant measures of February 2013 \| Volume 8 \| Issue 2 \| e53135 1 PLOS ONE \| www.plosone.org Mapping Cortico-Striatal Connections on the Cortex Mapping Cortico-Striatal Connections on the Cortex or motion of the subject during the acquisition of the k-space were detected and corrected using the technique described in [19] and implemented in the BrainVISA/Connectomist-2.0 diffusion tool- box. Distortions stemming from susceptibility effects were estimated using the field map acquired together with the DW data and were also corrected using the BrainVISA/Connectomist- 2.0 diffusion toolbox. Motions occurring during the acquisition were corrected using an affine registration, matching any DW data to the reference T2-weighted data acquired at b = 0s/mm2. The diffusion-weighted directions were modified accordingly. Last, the corrected DW data were matched to the T1-weighted data using a rigid 3D transform estimated by an automatic registration algorithm based on mutual information. surface if its distance to the surface is less than a given distance (2.0 mm in our case), which corresponds to the resolution of the DW data (Figure 1.C). This distance is considered to take into account the misregistration due to putative imperfect artefact removal. The tracts were first resampled with a 0.5 mm step which corresponds to half the resolution of T1-weighted images. This selection process allowed discarding the tracts that do not intersect both the striatum and the cortical surface. As for the tracts intersecting both the striatum and the cortical surface, two cases were obtained: either the tract links the striatum nucleus directly to the cortex (see blue segment in Figure 1.A) in which case the tract is kept, or the tract links the striatum nucleus to the cortex through an intermediate nucleus (see red segment in Figure 1.A) in which case the tract is discarded. Tractography. To recover the anatomical connectivity of each subject, a tractography technique was employed. First, a robust mask of the brain was built from the T1-weighted data as described in [20]. The advantage of such a mask compared to the classical FA-thresholding based masks, is that it is based on the anatomy of each subject and is not altered by FA modifications due to a given pathology. In fact if a classical FA-thresholding mask was used, some fiber pathways could be missed by the tractography algorithm because they are not covered by the mask due to Huntington’s disease related FA modifications in the voxels belonging to these pathways. Through this careful selection process, all false positive tracts entailing connectivity measures are removed leading to clean subcortico-cortical tractograms. Structural database Images were obtained in 15 symptomatic HD patients (8 women, 7 men, aged 46.466.54) and 15 age and gender matched healthy volunteers (8 women, 7 men, aged 46.4611.76) using a Tim Trio 3T MRI system (Siemens, Erlangen). All subjects were prospectively included in the frame of a clinical project dedicated to the study of HD (Track-HD project) [3]. All patients had a genetically proven HD with an abnormal number of CAG repeats ranging from 39 to 47. Clinical characteristics of HD patients are shown in Table 1. All subjects signed an informed consent and the study was approved by the Local Ethical Committee (CPP Ile de France 6). Data were acquired using the following sequence Table 1. Clinical characteristics of the subjects. Table 1. Clinical characteristics of the subjects. Table 1. Clinical characteristics of the subjects. HD patients Healthy volunteers Gender (M/F) 7/8 7/8 Age (years) 46.4+26.54 46.4+211.76 CAG 43.47+21.64 - Burden 357.9+251.38 - doi:10.1371/journal.pone.0053135.t001 doi:10.1371/journal.pone.0053135.t001 PLOS ONE \| www.plosone.org 2 Surface cortico-striatal connectivity measures The ultimate goal of this work was to compare the connectivity profiles of the striatum to the cortex between different groups and to detect a pathology related connectivity change. To this aim, we computed connectivity matrices to evaluate for each subject the number of tracts linking each vertex of the cortical surface to each nucleus of the striatum. From such connectivity matrices, we inferred for a given population P, the connectivity profile of each nucleus to each region of the cortical surface. Cortico-striatal connectivity matrix. For each subject s, the number of tracts connecting each nucleus n to each region of the cortex was obtained by computing the intersection between the cortical surface and the fibre tracts linking n to it and obtained following the procedure described in 2.3.3. The values related to each nucleus n were stored in a line of a sparse connectivity matrix [25]. For each tract linking a nucleus n to a triangle of the cortex surface, the intersection point of the triangle and the tract (or the projection of the closest point belonging to the tract on the triangle) divides the triangle into 3 small triangles corresponding to the areas a1, a2 and a3 (Figure 2). For each vertex vi, i[{1, 2, 3} of the triangle, the weight ai a1za2za3 was added to the vertex position corresponding to vi in the connectivity matrix in order to provide a higher weighting to the closest vertex to the projection point. When all the tracts are processed, a connectivity matrix containing the values of connectivity Cs(n,v) of each nucleus n to each vertex position v of the cortex is obtained. For each vertex v and each nucleus n the connectivity value Cs(n,v) provides an estimation of the number of tracts linking n to v. Selection of the cortico-striatal tracts. Connections be- tween the cortex and the basal ganglia plus the thalamus were organized into specific circuits, according to [23]. According to this model, the striatum receives afferents from the cerebral cortex. Most areas of the neocortex except the primary visual and auditory areas have projections onto the striatum. The striatum projects to the external and internal segments of the globus pallidus and the internal globus pallidus projects in turn to the thalamus. The thalamus sends efferents to the cerebral cortex. Recent anatomical investigations have revealed a more complex organization [24]. Surface cortico-striatal connectivity measures If the fibre tracts linking the striatum to the cortical surface are selected from the whole brain tractogram without taking into account the segmentation of other structures like the globus pallidus or the thalamus, indirect connections between the striatum and the cortex would be included (see blue segments in Figure 1.A), leading to false positives. Cortico-striatal connectivity profile. Before comparing connectivity measures across groups it is important to define some cortical regions of interest. We focused on the gyri obtained from the FreeSurfer parcellation described in 2.4. This parcella- tion consisted of 34 cortical regions in each hemisphere.. To restrict for each nucleus the number of cortical regions to be analyzed, we extracted for each nucleus its connectivity profile to the cortex which means the gyri to which it is the most connected. This connectivity profile was inferred from the connectivity measures contained in the connectivity matrix of each healthy subject. For each subject s, we computed the surface connectivity measure Cs(n,r) representing the number of tracts linking a nucleus n to a gyrus r. We developed a selection process that took into account the segmentation of the cortical surface and of several subcortical nuclei (caudate nucleus, putamen, globus pallidus and thalamus). For each tract belonging to the whole brain tractogram obtained after the tractography step, the intersection with each subcortical nucleus and with the cortical surface was computed. The tract was considered as intersecting the nucleus if at least a segment of minimum length of the tract intersects the nucleus (to avoid tangent fibres, see Figure 1.B) and as intersecting the cortical February 2013 \| Volume 8 \| Issue 2 \| e53135 PLOS ONE \| www.plosone.org 3 Mapping Cortico-Striatal Connections on the Cortex Figure 1. Cortico-striatal tracts selection. (A) If all basal ganglia were not taken into account in the selection process, direct connections (blue segment) but also indirect connections (red segment) of the striatum to the cortex would be studied. (B) A tract was considered as intersecting a nucleus if at least 2 points of the tract intersected the nucleus. (C) A tract was considered as intersecting the cortical surface if its distance to the surface was less than 2 mm. doi:10.1371/journal.pone.0053135.g001 Figure 1. Cortico-striatal tracts selection. Surface cortico-striatal connectivity measures (A) If all basal ganglia were not taken into account in the selection process, direct connections (blue segment) but also indirect connections (red segment) of the striatum to the cortex would be studied. (B) A tract was considered as intersecting a nucleus if at least 2 points of the tract intersected the nucleus. (C) A tract was considered as intersecting the cortical surface if its distance to the surface was less than 2 mm. doi:10.1371/journal.pone.0053135.g001 obtain average connectivity measures per surface unit: Results We compared the cortico-striatal connectivity of the two populations of healthy subjects and patients by detecting differences in surface connectivity measures. These differences were first detected using a statistical comparison and then quantified by computing a percentage difference of connectivity between the two populations. In order to take into account the intra-subject variability of surface connectivity measures we normalized the connectivity measures by the intracranial volume. Other normalization criterions were also investigated (brain or deep nuclei volumes, whole brain fibre tract number, fibre tracts crossing deep nuclei number) but were discarded because they were pathological and thus can hide pathological connectivity information if used for the normalization. Mapping Cortico-Striatal Connections on the Cortex surface unit. We defined this set of gyri as the connectivity profile of the nucleus n. where ANCHealthy(n,r) and ANCHuntington(n,r) represent the average normalized number of fibre tracts connecting n to r for healthy subjects and HD patients respectively. Deep nuclei volumes The volumes of the basal ganglia were measured from the manually corrected automatic segmentations. The volumes of all nuclei were systematically decreased in patients compared to controls (caudate nuclei: 43.2% and putamen: 43.6%, Figure 3). Surface cortico-striatal connectivity measures Surface connectivity measures were inferred between each nucleus of the striatum (caudate nucleus and putamen) and all the cortical gyri. For each nucleus and for each gyrus, an average connectivity measure per surface unit was computed for the group of healthy subjects as described in equation (2). The values obtained for each nucleus were mapped onto an average cortical surface computed from healthy subjects, and represented using a red color gradient palette showing the strength of the connectivity to each gyrus (Figure 6(A)). The connectivity profiles of each nucleus (which means the cortical gyri having an average connectivity value greater than 1 tract per surface unit) were represented in Figure 6(B). Percentage difference. For a given population P, we computed average normalized connectivity measures ANCP(n,r) between each nucleus n and each cortical region of interest r that belongs to its connectivity profile and that presents a significant connectivity difference between the group of healthy subject and HD patients. These measures were obtained by summing up the normalized connectivity measures of all the subjects s belonging to the population P and then by dividing the sum by the number of subjects of the population (card(P)): Streamline probabilistic tractography The tracts linking the striatum to the cortex were extracted from the whole set of tracts using the new selection procedure that we described in 2.3.3. An example of result of the selection of the fibre tracts linking the striatum to the cortex is provided in Figure 4 for one healthy subject and one HD patient, clearly depicting a reduced number of tracts obtained for HD patients compared to controls. In order to highlight the importance of incorporating anatomical prior knowledge in the tract selection process, we represented in Figure 5 the results of the selection of tracts passing through the striatum on a healthy subject using 2 different procedures: (A) by selecting all the tracts that intersect the striatum (a tract was considered as intersecting the striatum if at least 3 points of the tract are inside the striatum). (B) by selecting the tracts linking each nucleus of the striatum to the cortex as described in 2.3.3 and as was done in this paper. The Figure 5 clearly shows that the new selection procedure removes all the indirect tracts that connect each nucleus to the cortex and keeps only direct ones. Statistical test. We assessed the significance of the connec- tivity differences between the two groups. To this aim, we computed for each subject s the normalized surface connectivity measures NCs(n,r) between each nucleus n and each gyrus r belonging to the connectivity profile of n: NCs(n,r)~ Cs(n,r) IVs ð3Þ ð3Þ Were IVs represents the intracranial volume of subject s computed as described in 2.3.3. These normalized values were compared for the subjects of each population using a Mann-Whitney test. The choice for this statistical test was motivated by the lack of assumption about the Gaussianity of the data. For a given population P, the connectivity NCs(n,r) was considered as a random variable X P n,r taking different values for the different subjects s. Using the Mann-Whitney test on the two random variables X PH n,r and X PHD n,r corresponding to the two populations of controls and patients respectively, we isolated for each nucleus n the set of gyri presenting a significant difference of connectivity, among the gyri which are part of its connectivity profile. A significance level a = 0.05 was used and a False Discovery Rate correction for multiple comparison was applied. obtain average connectivity measures per surface unit: Cs(n,r)~ X v[r Cs(n,v) ð1Þ ð1Þ AH(n,r)~ P s[H Cs(n,r) Ss(r) card(H) ð2Þ ð2Þ We also computed for each subject the surface Ss(r) of each gyrus. The value Ss(r) was obtained by summing the areas of the triangles of the cortical mesh belonging to the gyrus r. For each nucleus n, we isolated the set of gyri r for which the value AH(n,r) was greater than a threshold set to 1 tract per For each subject, we normalized each connectivity measure Cs(n,r) by the surfaceSs(r). We then averaged these values across the subjects belonging to the population H of healthy subjects to Figure 2. Connectivity matrix of a nucleus n to the cortex surface. v1, v2 and v3 represent the vertices of a triangle which is intersected by a fibre tract coming from n. doi:10.1371/journal.pone.0053135.g002 Figure 2. Connectivity matrix of a nucleus n to the cortex surface. v1, v2 and v3 represent the vertices of a triangle which is intersected by a fibre tract coming from n. doi:10.1371/journal.pone.0053135.g002 February 2013 \| Volume 8 \| Issue 2 \| e53135 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 4 Mapping Cortico-Striatal Connections on the Cortex Group comparison Among the cortical regions constituting the cortical connectivity profile of each nucleus, the gyri presenting a significant difference of connectivity between the two populations were detected using a Mann-Whitney test (Table 2). Cortical regions associated with non significant p-values were not considered for further analysis. The percentage differences of connectivity PDC(n, r) obtained for each nucleus on the selected cortical regions are shown in Figure 7. ANCP(n,r)~ P s[P NCs(n,r) card(P) ð4Þ ANCP(n,r)~ P s[P NCs(n,r) card(P) ð4Þ ð4Þ For the caudate nucleus, cortical regions depicting significantly reduced connections predominated in the parietal lobes (posterior parietal regions and primary sensory area), followed by the frontal lobes (dorsal and ventral lateral, frontopolar, lateral orbito-frontal and primary motor). For the left caudate nucleus, the reduction ranged from 33.8% for the left frontal superior gyrus to 76.1% for the left posterior parietal area. For the right caudate nucleus, the PDC values ranged from 31.5% for the right lateral orbito-frontal gyrus to 62.8% for the right posterior parietal area. We measured the percentage difference of connectivity (PDC) between two populations, using the average normalized connec- tivity of healthy subjects as a reference. For a given cortical region r and a given nucleus n, the percentage difference of connectivity between the two groups of subjects was defined as follows: PDC(n,r)~ ANCHealthy(n,r){ANCHuntington(n,r) ANCHealthy(n,r) ð5Þ ð5Þ February 2013 \| Volume 8 \| Issue 2 \| e53135 PLOS ONE \| www.plosone.org February 2013 \| Volume 8 \| Issue 2 \| e53135 PLOS ONE \| www.plosone.org 5 Mapping Cortico-Striatal Connections on the Cortex Mapping Cortico-Striatal Connections on the Cortex Figure 3. Volumes of the deep nuclei for healthy subjects and HD patients. Points represent mean values and bars standard deviations. doi:10.1371/journal.pone.0053135.g003 or healthy subjects and HD patients. Points represent mean values and bars standard deviations. Figure 3. Volumes of the deep nuclei for healthy subjects and HD patients. Points represent mean values and bars standard deviations. doi:10.1371/journal.pone.0053135.g003 For the putamen, cortical regions with reduced connections predominated in associative temporal, the dorsal and ventral frontal areas, and parietal regions (posterior parietal and angular cortices), followed by the primary sensorimotor cortex. The PDC values for the left putamen varied from 18.0% for the left precentral gyrus to 48.0% for the left middle temporal gyrus. The PDC for the right putamen ranged from 28.9% for the right dorso lateral prefrontal gyrus to 58.5% for the right middle temporal gyrus. Mapping Cortico-Striatal Connections on the Cortex approach to investigate cortico-striatal connectivity in HD patients and showed a selective region-specific degeneration of cortical connections of the striatum by providing quantitative measures of the percentage difference of connectivity between each nucleus and cortical area. Our method for studying connectivity between the cortex and deep brain structures differs from previous ones in healthy volunteers, which relied on tractography-based subdivision of the basal ganglia [7,8,9,10] and thalamus [10,11] based on their connectivity profile with predefined regions of the cortex. In contrast, we focused on the circuit linking the deep nuclei to the cortex and we mapped the connectivity information onto the cortical surface. In order to quantify territory-specific disconnec- tions between the striatum and the cortex, we developed a processing pipeline working on the cortical surface. It consisted in computing surface connectivity measures of the cortico-striatal connectivity which gave access to the strength of connection between each nucleus and any cortical region, and enabled the inference of a percentage difference of connectivity between each of these nuclei and any cortical region between healthy subjects and patients. This tool efficiently provided the functional areas of the cortex presenting significant modifications of connectivity in HD patients compared with controls. belonging to its connectivity profile. Mapping Cortico-Striatal Connections on the Cortex Cortical region of interestLCd RCd LPu RPu Frontal (32) Frontal Pole 0.009552 0.233960 0.287755 0.393731 (27) Rostral Middle Frontal 0.000103 0.000653 0.000031 0.002905 (28) Superior Frontal 0.004221 0.005372 0.475195 0.042595 (3) Caudal Middle Frontal 0.018101 0.016334 0.042595 0.186256 (12) Lateral Orbito Frontal 0.000087 0.000488 0.005372 0.006046 (14) Medial Orbito Frontal 0.491727 0.246865 0.135847 0.046492 (17) Para Central 0.001000 0.007623 0.154764 0.070447 (18) Pars Opercularis 0.001511 0.000754 0.009552 0.006795 (19) Pars Orbitalis 0.007542 0.000103 0.001727 0.009552 (20) Pars Triangularis 0.000195 0.000653 0.000653 0.006795 (24) Precentral 0.001149 0.003733 0.003733 0.002905 Parietal (22) Post Central 0.000228 0.000869 0.001319 0.000087 (29) Superior Parietal 0.000053 0.000420 0.002905 0.001000 (8) Inferior Parietal ------------- ------------- 0.003733 0.000653 (25) Precuneus ------------- ------------- 0.145098 0.022127 (31) Supra Marginal ------------- ------------- 0.164846 0.004766 Temporal (30) Superior Temporal ------------- ------------- 0.003733 0.000311 (15) Middle Temporal ------------- ------------- 0.000267 0.000031 (9) Inferior Temporal ------------- ------------- 0.000142 0.000142 (33) Temporal Pole ------------- ------------- 0.127008 0.409773 (34) Transverse Temporal ------------- ------------- 0.409772 0.001727 (13) Lingual ------------- ------------- ------------- ------------- (7) Fusiform ------------- ------------- ------------- ------------- (16) Para Hippocampal ------------- ------------- ------------- ------------- (6) Entorhinal ------------- ------------- ------------- ------------- Occipital (21) Peri Calcarine ------------- ------------- ------------- ------------- (11) Lateral Occipital ------------- ------------- ------------- ------------- (5) Cuneus ------------- ------------- ------------- ------------- Cingulate (26) Rostral Anterior Cingulate 0.175343 0.287755 0.102921 0.393731 (2) Caudal Anterior Cingulate0.018101 0.186256 0.102921 0.118578 (23) Posterior Cingulate 0.032462 0.377868 0.246865 0.442287 (10) Isthmus Cingulate ------------- ------------- 0.475195 ------------- The regions that did not belong to the connectivity profile of the nucleus are represented by dashes (-----). The rows correspond to the cortical regions of interest and the columns correspond to the p values of the Mann-Whitney test for the left caudate, left putamen, right caudate and right putamen respectively. The significance levels obtained with the FDR multi-comparison correction were equal to: 0.010168, 0.008319, 0.003991, and 0.004193 for the LCd, RCd, LPu and RPu respectively.The p-values that were lower than the significance level were represented in bold. doi:10.1371/journal.pone.0053135.t002 Our results confirm the selective region-specific degeneration of cortico-striatal connections in HD. First, we found a greater degeneration of associative temporal, parietal and frontal cortico- striatal connections and a relative preservation of limbic connec- tions. There was also relatively larger reductions in primary sensory than motor connections. Mapping Cortico-Striatal Connections on the Cortex These results are in line with histological data in HD patient brains which showed prominent degeneration of both sensorimotor and associative parts of the striatum in late stages of HD as well as comparably less involvement of the ventral limbic striatum [2]. Using diffusion imaging, several studies have shown that the basal ganglia and cortico-striato-pallidal networks are affected by the pathological process [12,13,26,27]. Using ROI approaches, higher FA and MD values have been reported in the putamen, the globus pallidus and the caudate nucleus in symptomatic HD patients [26] and in presymptomatic gene carriers [13,27]. In the white matter, studies using voxel-based or ROI analysis have found lower FA values in frontal white matter, the corpus callosum, the internal capsule and the white matter underlying the central areas in premanifest gene carriers [27,28,29] and HD patients [27,29]. Other studies investigated diffusion measures along the skeleton of tracts using TBSS [14,30,31]. They reported decreased FA and increased lI and lH in several associative white matter fasciculi in HD patients [31] and in the corticospinal tract in premanifest gene carriers [30]. Only two studies investigated cortico-striatal connectivity using probabilistic tractography [12,13]. Kloppel et al. (2008) have demonstrated reduced connectivity between the frontal cortex and the body of the caudate nucleus in presymptomatic gene carriers [13]. One study parcellated the striatum into subregions based on connectivity with the cerebral cortex [12]. These authors found larger diffusion changes in sensorimotor striatal subregions of the caudate nucleus and putamen, which correlated with motor symptoms. They suggested that the motor cortico-striatal circuit was selectively vulnerable in HD. The regions that did not belong to the connectivity profile of the nucleus are represented by dashes (-----). The rows correspond to the cortical regions of interest and the columns correspond to the p values of the Mann-Whitney test for the left caudate, left putamen, right caudate and right putamen respectively. The significance levels obtained with the FDR multi-comparison correction were equal to: 0.010168, 0.008319, 0.003991, and 0.004193 for the LCd, RCd, LPu and RPu respectively.The p-values that were lower than the significance level were represented in bold. We also provided quantitative measures of the percentage difference of the connectivity between each nucleus and cortical area. For each nucleus, the percentage difference of connectivity was greater than 18% in several cortical regions including the frontal cortex. Discussion doi:10.1371/journal.pone.0053135.g006 February 2013 \| Volume 8 \| Issue 2 \| e53135 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 7 7 Mapping Cortico-Striatal Connections on the Cortex Discussion In this work, we have developed a novel approach to infer and map the connectivity of deep brain nuclei (basal ganglia and thalamus) directly onto the cortical surface. We applied this Figure 4. Cortico-striatal fibre tracts in one healthy subject and one HD patient. Only 1% of the actual fibre tracts were randomly selected and represented for a better rendering. In the patient, the volume of the striatum was reduced and the number of tracts obtained for each nucleus was decreased. doi:10.1371/journal.pone.0053135.g004 Figure 4. Cortico-striatal fibre tracts in one healthy subject and one HD patient. Only 1% of the actual fibre tracts were randomly selected and represented for a better rendering. In the patient, the volume of the striatum was reduced and the number of tracts obtained for each nucleus was decreased. doi:10.1371/journal.pone.0053135.g004 February 2013 \| Volume 8 \| Issue 2 \| e53135 PLOS ONE \| www.plosone.org 6 Mapping Cortico-Striatal Connections on the Cortex Figure 5. Selection of the connections of the striatum for a healthy subject. (A) selection of the tracts that intersect the striatum, (B) selection of only the direct tracts that link the striatum to the cortex. Only 1% of the actual fibre tracts were randomly selected and represented for a better rendering. doi:10.1371/journal.pone.0053135.g005 Figure 5. Selection of the connections of the striatum for a healthy subject. (A) selection of the tracts that intersect the striatum, (B) selection of only the direct tracts that link the striatum to the cortex. Only 1% of the actual fibre tracts were randomly selected and represented for a better rendering. Figure 5. Selection of the connections of the striatum for a healthy subject. (A) selection of the tracts that intersect the striatum, (B) selection of only the direct tracts that link the striatum to the cortex. Only 1% of the actual fibre tracts were randomly selected and represented for a better rendering. doi:10.1371/journal.pone.0053135.g005 Figure 6. Connectivity profile of the striatum to the cortex. (A) Average connectivity measures per surface unit for each nucleus and each gyrus. (B) Cortical gyri constituting the connectivity profile of each nucleus. doi:10.1371/journal.pone.0053135.g006 Figure 6. Connectivity profile of the striatum to the cortex. (A) Average connectivity measures per surface unit for each nucleus and each gyrus. (B) Cortical gyri constituting the connectivity profile of each nucleus. February 2013 \| Volume 8 \| Issue 2 \| e53135 Figure 7. Percentage difference of connectivity for the striatum nuclei. The figure shows the percentage difference of connectivity between healthy subjects and HD patients, obtained for each nucleus of the striatum and cortical gyri belonging to the connectivity profile of the nucleus and presenting a significant connectivity difference between healthy subjects and HD patients. doi:10.1371/journal.pone.0053135.g007 References 18. Desikan RS, Segonne F, Fischl B, Quinn BT, Dickerson BC, et al. (2006) An automated labeling system for subdividing the human cerebral cortex on MRI scans into gyral based regions of interest. Neuroimage 31: 968–980. 1. The Huntington’s Disease Collaborative Research Group (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 72: 971–983. 19. Dubois J, Dehaene-Lambertz G, Hertz-Pannier L, Santoro G, Mangin JF, et al. (2010) Correction strategy for infants diffusion-weighted images corrupted with motion. Stockholm, Sweden. In Proceedings of the ISMRM 17th Annual Meeting. 2. Vonsattel JPG (2008) Huntington disease models and human neuropathology: similarities and differences. Acta Neuropathol 115: 55–69. 2. Vonsattel JPG (2008) Huntington disease models and human neuropathology: similarities and differences. Acta Neuropathol 115: 55–69. 3. Tabrizi SJ, Langbehn DR, Leavitt BR, Roos RA, Durr A, et al. (2009) Biological and clinical manifestations of Huntington’s disease in the longitudinal TRACK- HD study: cross-sectional analysis of baseline data. Lancet Neurol 8: 791–801. 20. Guevara P, Duclap D, Marrakchi-Kacem L, Riviere D, Cointepas Y, et al. (2011) Accurate tractography propagation mask using T1-weighted data rather than FA. Montreal, Canada. In Proceedings of the ISMRM 18th Annual Meeting. 4. Le Bihan D (2001) Diffusion Tensor Imaging: Concepts and Applications. Journal of Magnetic Resonance 13: 534–546. 5. Pierpaoli C, Basser PJ (1996) Toward a quantitative assessment of diffusion anisotropy. Magn Reson Med 36: 893–906. 21. Descoteaux M, Angelino E, Fitzgibbons S, Deriche R (2007) Regularized, fast, and robust analytical Q-ball imaging. Magnetic Resonance in Medicine 58: 497–510. 6. Basser PJ, Jones DK (2002) Diffusion-tensor MRI: theory, experimental design and data analysis – a technical review. NMR Biomed 15: 456–467. 22. Perrin M, Poupon C, Cointepas Y, Rieul B, Golestani N, et al. (2005) Fiber tracking in q-ball fields using regularized particle trajectories. Inf Process Med Imaging 19: 52–63. 7. Lehericy S, Ducros M, Krainik A, Francois C, Van de Moortele PF, et al. (2004) 3-D Diffusion Tensor Axonal Tracking shows Distinct SMA and Pre-SMA Projections to the Human Striatum. Cereb Cortex 14: 1302–1309. g g 23. Alexander GE, DeLong MR, Strick PL (1986) Parallel organization of functionally segregated circuits linking basal ganglia and cortex. Annu Rev Neurosci 9: 357–381. 8. Lehericy S, Ducros M, Van De Moortele PF, Francois C, Thivard L, et al. (2004) Diffusion tensor fiber tracking shows distinct corticostriatal circuits in humans. References Annals of Neurology 55: 522–529. 24. Redgrave P, Rodriguez M, Smith Y, Rodriguez-Oroz MC, Lehericy S, et al. (2010) Goal-directed and habitual control in the basal ganglia: implications for Parkinson’s disease. Nat Rev Neurosci 11: 760–772. gy 9. Leh SE, Ptito A, Chakravarty MM, Strafella AP (2007) Fronto-striatal connections in the human brain: A probabilistic diffusion tractography study. Neuroscience Letters 419: 113–118. 25. Roca P, Riviere D, Guevara P, Poupon C, Mangin JF (2009) Tractography- based parcellation of the cortex using a spatially-informed dimension reduction of the connectivity matrix. Med Image Comput Comput Assist Interv 12: 935– 942. 10. Draganski B, Kherif G, Kloppel S, Cook PA, Alexander DC, et al. (2008) Evidence for Segregated and Integrative Connectivity Patterns in the Human Basal Ganglia. The Journal of Neuroscience 28: 7143–7152. Basal Ganglia. The Journal of Neuroscience 28: 7143–7152. 11. Behrens T, Johansen-Berg H, Woolrich MW, Smith SM, Wheeler-Kingshott CAM, et al. (2003) Non-invasive mapping of connections between human thalamus and cortex using diffusion imaging. Neuroscience 6: 750–757. 26. Douaud G, Behrens TE, Poupon C, Cointepas Y, Jbabdi S, et al. (2009) In vivo evidence for the selective subcortical degeneration in Huntington’s disease. Neuroimage 46: 958–966. 12. Bohanna I, Georgiou-Karistianis N, Egan GF (2011) Connectivity-based segmentation of the striatum in Huntington’s disease: Vulnerability of motor pathways. Neurobiol Dis. 27. Rosas HD, Tuch DS, Hevelone ND, Zaleta AK, Vangel M, et al. (2006) Diffusion tensor imaging in presymptomatic and early Huntington’s disease: Selective white matter pathology and its relationship to clinical measures. Mov Disord 21: 1317–1325. 13. Kloppel S, Draganski B, Golding CV, Chu C, Nagy Z, et al. (2008) White matter connections reflect changes in voluntary-guided saccades in pre- symptomatic Huntington’s disease. Brain 131: 196–204. 28. Reading SAJ, Yassa MA, Bakker A, Dziorny AC, Gourley LM, et al. (2005) Regional white matter change in pre-symptomatic Huntington’s disease: A diffusion tensor imaging study. Psychiatry Research: Neuroimaging 140: 55–62. 14. Smith SM, Jenkinson M, Johansen-Berg H, Rueckert D, Nichols TE, et al. (2006) Tract-based spatial statistics: voxelwise analysis of multi-subject diffusion data. Neuroimage 31: 1487–1505. 29. Rosas HD, Lee SY, Bender AC, Zaleta AK, Vangel M, et al. (2010) Altered white matter microstructure in the corpus callosum in Huntington’s disease: Implications for cortical disconnection. NeuroImage 49: 2995–3004. 15. Marrakchi-Kacem L, Poupon C, Mangin JF, Poupon F (2010) Multi-contrast deep nuclei segmentation using a probabilistic atlas. ISBI’10; Piscataway, NJ, USA. IEEE Press. 61–64. 30. Acknowledgments The authors would like to thank Marie Chupin and Olivier Colliot for useful discussions about inter-subjects normalization aspects. Mapping Cortico-Striatal Connections on the Cortex Mapping Cortico-Striatal Connections on the Cortex frame of functional areas. We applied this novel approach in the frame of a clinical study of HD and we showed that it adequately and efficiently detected differences in cortico-striatal connectivity between healthy subjects and HD patients. Reduction in connectivity predominated in associative and sensorimotor regions in good agreement with the known pathophysiology of HD. In the future, this approach will be used to investigate other neurode- generative pathologies involving the basal ganglia, and improve- ments will be done to provide accurate information about the affected areas, including longitudinal changes and correlation between the cortico-basal ganglia connectivity modifications and the clinical stage of patients. cortex in accordance with several morphological studies, which reported cortical atrophy in these regions [3,33,34]. Regional variations in cortico-striatal changes were also observed in accordance with the heterogeneity of the cortical atrophy reported in HD [33]. In line with a previous study, we found greater involvement of the primary sensory and posterior parietal cortex [12]. However, in contrast to this study, reduction in connections did not predominate in the primary motor network, which was as affected as other frontal connections, and we observed reduced temporal connections. Differences between studies may reflect differences in the methodology or in the selection of subjects. Overall our data suggests involvement of associative and sensorimotor connections of the striatum, with associative connections more affected than sensorimotor ones, and relative preservation of limbic connections. Reduced connections may represent the basis of altered cognitive functions particularly in domains that engage fronto-striatal circuitry (e.g., executive functions, motor and psychomotor speed) 35,36]. Conclusions Collected the subjects and participated to the acquisition of the database: AD. Conceived and designed the experiments: LM-K CP CD S. Lehe´ricy. Performed the experiments: LM-K CD S. Lecomte. Analyzed the data: LM-K CD CP S. Lehe´ricy JY. Contributed reagents/materials/analysis tools: LM-K CP FP AT PG PR JFM. Wrote the paper: LM-K CP CD S. Lehe´ricy. In this paper, we introduced a novel approach for the study of the cortico-subcortical connectivity which consists in computing surface connectivity measures. Projecting the connectivity profiles onto the cortical mantel was relevant as it enabled to detect the atrophy of the cortical connections of the striatum directly in the Mapping Cortico-Striatal Connections on the Cortex The important involvement of frontal connection was in good agreement with previous reports [13,32]. Cortico- striatal connectivity differences also predominated in associative areas (parietal, frontal, and temporal) and in the sensorimotor PLOS ONE \| www.plosone.org February 2013 \| Volume 8 \| Issue 2 \| e53135 February 2013 \| Volume 8 \| Issue 2 \| e53135 8 Mapping Cortico-Striatal Connections on the Cortex igure 7. Percentage difference of connectivity for the striatum nuclei. The figure shows the percentage difference of connectiv ealthy subjects and HD patients, obtained for each nucleus of the striatum and cortical gyri belonging to the connectivity profile of the resenting a significant connectivity difference between healthy subjects and HD patients. oi:10.1371/journal.pone.0053135.g007 Figure 7. Percentage difference of connectivity for the striatum nuclei. The figure shows the percentage difference of connectivity between healthy subjects and HD patients, obtained for each nucleus of the striatum and cortical gyri belonging to the connectivity profile of the nucleus and presenting a significant connectivity difference between healthy subjects and HD patients. doi:10.1371/journal.pone.0053135.g007 February 2013 \| Volume 8 \| Issue 2 \| e53135 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 9 Mapping Cortico-Striatal Connections on the Cortex g 36. Rosas HD, Hevelone ND, Zaleta AK, Greve DN, Salat DH, et al. (2005) Regional cortical thinning in preclinical Huntington disease and its relationship to cognition. Neurology 65: 745–747. 35. Solomon AC, Stout JC, Weaver M, Queller S, Tomusk A, et al. (2008) Ten-year rate of longitudinal change in neurocognitive and motor function in prediagnosis Huntington disease. Mov Disord 23: 1830–1836. 34. Nopoulos PC, Aylward EH, Ross CA, Johnson HJ, Magnotta VA, et al. (2010) Cerebral cortex structure in prodromal Huntington disease. Neurobiol Dis 40: 544–554. 33. Rosas HD, Salat DH, Lee SY, Zaleta AK, Pappu V, et al. (2008) Cerebral cortex and the clinical expression of Huntington’s disease: complexity and heteroge- neity. Brain 131: 1057–1068. Mapping Cortico-Striatal Connections on the Cortex 35. Solomon AC, Stout JC, Weaver M, Queller S, Tomusk A, et al. (2008) Ten-year rate of longitudinal change in neurocognitive and motor function in prediagnosis Huntington disease. Mov Disord 23: 1830–1836. 36. Rosas HD, Hevelone ND, Zaleta AK, Greve DN, Salat DH, et al. (2005) Regional cortical thinning in preclinical Huntington disease and its relationship to cognition. Neurology 65: 745–747. References Stoffers D, Sheldon S, Kuperman JM, Goldstein J, Corey-Bloom J, et al. (2010) Contrasting gray and white matter changes in preclinical Huntington disease: an MRI study. Neurology 74: 1208–1216. 16. Fischl B, Sereno MI, Tootell RBH, Dale AM (1999) High-Resolution Intersubject Averaging and a Coordinate System for the Cortical Surface. Human Brain Mapping 8: 272–284. 31. Nave RD, Ginestroni A, Tessa C, Giannelli M, Piacentini S, et al. (2010) Regional distribution and clinical correlates of white matter structural damage in Huntington disease: a tract-based spatial statistics study. AJNR Am J Neuroradiol 31: 1675–1681. 17. Argall BD, Saad ZS, Beauchamp MS (2006) Simplified Intersubject Averaging on the Cortical Surface Using SUMA. Human Brain Mapping 27: 14–27. February 2013 \| Volume 8 \| Issue 2 \| e53135 February 2013 \| Volume 8 \| Issue 2 \| e53135 PLOS ONE \| www.plosone.org 10 Mapping Cortico-Striatal Connections on the Cortex Mapping Cortico-Striatal Connections on the Cortex 32. Backman L, Robins-Wahlin TB, Lundin A, Ginovart N, Farde L (1997) Cognitive deficits in Huntington’s disease are predicted by dopaminergic PET markers and brain volumes. Brain 120 (Pt 12): 2207–2217. 33. Rosas HD, Salat DH, Lee SY, Zaleta AK, Pappu V, et al. (2008) Cerebral cortex and the clinical expression of Huntington’s disease: complexity and heteroge- neity. Brain 131: 1057–1068. y 34. Nopoulos PC, Aylward EH, Ross CA, Johnson HJ, Magnotta VA, et al. (2010) Cerebral cortex structure in prodromal Huntington disease. Neurobiol Dis 40: 544–554. February 2013 \| Volume 8 \| Issue 2 \| e53135 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 11
https://openalex.org/W2090102613	https://www.ams.org/proc/2008-136-02/S0002-9939-07-09118-6/S0002-9939-07-09118-6.pdf	English	null	The order of a group of even order	Proceedings of the American Mathematical Society	2,007	public-domain	4,162	PROCEEDINGS OF THE AMERICAN MATHEMATICAL SOCIETY Volume 136, Number 2, February 2008, Pages 397–402 S 0002-9939(07)09118-6 Article electronically published on October 25, 2007 PROCEEDINGS OF THE AMERICAN MATHEMATICAL SOCIETY Volume 136, Number 2, February 2008, Pages 397–402 S 0002-9939(07)09118-6 Article electronically published on October 25, 2007 PROCEEDINGS OF THE AMERICAN MATHEMATICAL SOCIETY Volume 136, Number 2, February 2008, Pages 397–402 S 0002-9939(07)09118-6 Article electronically published on October 25, 2007 c ⃝2007 American Mathematical Society Reverts to public domain 28 years from publication (Communicated by Jonathan I. Hall) Abstract. We will give an estimation of the order of a group of even order by the order of the centralizer of an involution using the classiﬁcation of ﬁnite simple groups. Received by the editors August 15, 2006. 2000 Mathematics Subject Classiﬁcation. Primary 20D05, 20D06. Key words and phrases. Finite simple groups, centralizers of involutions. The author was supported in part by Grant-in-Aid for Scientiﬁc Research (No. 16540030), Japan Society for the Promotion of Science. 2000 Mathematics Subject Classiﬁcation. Primary 20D05, 20D06. The author was supported in part by Grant-in-Aid for Scientiﬁc Research (No. 16540030 Japan Society for the Promotion of Science. Received by the editors August 15, 2006. 1. Introduction YAMAKI 398 class of involutions and the Sylow 2-subgroup of G is not cyclic or a general- ized quaternion, then \|G\| < \|CG(t)\|3 + mπ\|CG(t)\|2 for an involution t and for mπ =max{\|CG(t)\|, \|Inv(C−1 G (v))\|; v ∈Ω} without using the classiﬁcation of ﬁnite simple groups. Furthermore they showed that the orders of the sporadic simple groups Th and Ly can easily be determined by those of the centralizers of involu- tions using their result. class of involutions and the Sylow 2-subgroup of G is not cyclic or a general- ized quaternion, then \|G\| < \|CG(t)\|3 + mπ\|CG(t)\|2 for an involution t and for mπ =max{\|CG(t)\|, \|Inv(C−1 G (v))\|; v ∈Ω} without using the classiﬁcation of ﬁnite simple groups. Furthermore they showed that the orders of the sporadic simple groups Th and Ly can easily be determined by those of the centralizers of involu- tions using their result. Our notation is standard (see Atlas [5], Suzuki [13]). 1. Introduction Let G be a group of even order and t an involution in G. Brauer and Fowler [2] proved that if G is a ﬁnite simple group of even order, then \|G\| < (\|CG(t)\|2)!. It is well known that \|G\| < \|CG(u)\|3 for some involution u in G if G has more than one conjugacy class of involutions (see Suzuki [13, p. 127]). It has been conjectured by K. Harada that if G has precisely one conjugacy class of involutions and G is not 2-rank 1, then \|G\| < \|CG(u)\|3 for an involution u in G. The purpose of this note is to aﬃrmatively prove Harada’s conjecture using the classiﬁcation of ﬁnite simple groups which is now complete. Namely we will prove: Theorem 1. Let G be a group of even order. Suppose that G has precisely one conjugacy class of involutions and G is not 2-rank 1. Then \|G\| < \|CG(t)\|3 for an involution t in G. Combining a well known fact mentioned above we have: Corollary 1. Let G be a group of even order. Suppose that G is not 2-rank 1. Then \|G\| < \|CG(t)\|3 for some involution t in G. Corollary 1. Let G be a group of even order. Suppose that G is not 2-rank 1 Then \|G\| < \|CG(t)\|3 for some involution t in G. Remark 1. Let G be a ﬁnite group with 2-rank 1. Then the Sylow 2-subgroup of G is cyclic or a generalized quaternion. Let t be an involution in G. Then G = CG(t)O(G) by Burnside’s or Brauer-Suzuki’s theorem (see Suzuki [13, p. 144, p. 306]). If CG(t) ∩O(G) = 1 and \|O(G)\| > \|CG(t)\|2, then \|G\| > \|CG(t)\|3. Thus the conclusion of Theorem 1 does not hold true in general if G is 2-rank 1. Remark 2. Let π be the connected component of the prime graph of G containing 2 (see Williams [14], Kondrat’ev [12], Iiyori-Yamaki [11]). Let Ωbe the set of π- elements of G. For v ∈Ωdeﬁne Inv(C−1 G (v)) = {x ∈G x−1vx = v−1, x2 = 1}. Recently Harada and Miyamoto [9] proved that if G has precisely one conjugacy 397 H. 2. Finite simple groups The purpose of this section is to prove two lemmas on ﬁnite simple groups using the classiﬁcation. Lemma 1. Let G be a ﬁnite simple group with precisely one conjugacy class of involutions. Then \|G\| < \|CG(t)\|3 for an involution t in G. Proof. Let G be a non-abelian simple group with precisely one conjugacy class of involutions and t an involution in G. It follows that G is isomorphic to one of the groups for suitable q: A5, A6, A7, L2(q), L3(q), L4(q), U3(q), U4(q), G2(q),2G2(q),3D4(q3), Sz(q), M11, M22, M23, J1, J3, McL, ON, Ly, Th. A5, A6, A7, L2(q), L3(q), L4(q), U3(q), U4(q), G2(q),2G2(q),3D4(q3), Sz(q), M11, M22, M23, J1, J3, McL, ON, Ly, Th. Then we have the following tables which are self-explanatory. We refer to Atlas [5] for the sporadic simple groups, Burgoyne-Williamson [3], Suzuki [13], Williams [14] for the simple groups of Lie type over the ﬁeld of odd characteristic, and Aschbacher- Seitz [1], Suzuki [13], Dye [6, 7, 8] for those over the ﬁeld of even characteristic. It follows from Tables 1–4 below that \|G\| < \|CG(t)\|3, which completes the proof. □ Corollary 2. Let G be a ﬁnite simple group. Then \|G\| < \|CG(t)\|3 for some involution t in G. Lemma 2. There exists no ﬁnite simple group G such that G has k conjugacy classes of involutions for k ≥2 and all involutions in G are conjugate under Aut(G). Table 1. Alternating Groups G \|CG(t)\| \|G\| A5 = L2(4) 22 60 = 22.3.5 A6 = L2(32) 23 360 = 23.32.5 A7 23.3 2520 = 23.32.5.7 Table 2. Groups of Lie type over the ﬁeld of even characteristic G \|CG(t)\| \|G\| L2(q) q (q + 1)q(q −1) L3(q) q3(q −1)(3, q −1)−1 (q3 −1)(q2 −1)q3(3, q −1)−1 U3(q) q3(q + 1)(3, q + 1)−1 (q3 + 1)q3(q2 −1)(3, q + 1)−1 Sz(q), q = 22k+1, k ≥1 q2 (q2 + 1)q2(q −1) Table 1. Alternating Groups G \|CG(t)\| \|G\| A5 = L2(4) 22 60 = 22.3.5 A6 = L2(32) 23 360 = 23.32.5 A7 23.3 2520 = 23.32.5.7 Table 2. Groups of Lie type over the ﬁeld of even characteristic Table 2. 2. Finite simple groups Groups of Lie type over the ﬁeld of odd characteristic G \|CG(t)\| \|G\| L2(q), q ≡1(4) q −1 q(q2 −1)/2 L2(q), q ≡−1(4) q + 1 q(q2 −1)/2 L3(q) (q −1)2q(q + 1)(3, q −1)−1 q3(q2 −1)(q3 −1)(3, q −1)−1 L4(q), q ≡5(8) (q −1)3q2(q + 1)2/2 q6 3 i=1(qi+1 −1)/4 G2(q) q2(q2 −1)2 q6(q6 −1)(q2 −1) 2G2(q), q=32k+1, k≥2 q(q2 −1) q3(q3 + 1)(q −1) U3(q) q(q+1)(q2−1)(3, q+1)−1 (q3+1)q3(q2−1)(3, q+1)−1 U4(q), q ≡3(8) (q −1)2q2(q + 1)3/2 q6 3 i=1(qi+1 −(−1)i+1)/4 3D4(q3) q4(q2 −1)(q6 −1) q12(q8 + q4 + 1)(q6 −1)(q2 −1) Table 4. Sporadic simple groups G \|CG(t)\| \|G\| M11 24.3 24.32.5.11 J1 23.3.5 23.3.5.7.11.19 M22 27.3 27.32.5.7.11 M23 27.3.7 27.32.5.7.11.23 J3 27.3.5 27.35.5.17.19 McL 27.32.5.7 27.36.53.7.11 ON 29.32.5.7 29.34.5.73.11.19.31 Ly 28.34.52.7.11 28.37.56.7.11.31.37.67 Th 215.34.5.7 215.310.53.72.13.19.31 Proof. By our assumption all centralizers of involutions in G are isomorphic, and in particular any involution is contained in the center of a Sylow 2-subgroup of G. It is almost trivial that this does not happen when G is isomorphic to An (n ≥8) or one of the sporadic simple groups (see Atlas [5]). We assume that G is a group of Lie type. Proof. By our assumption all centralizers of involutions in G are isomorphic, and in particular any involution is contained in the center of a Sylow 2-subgroup of G. It is almost trivial that this does not happen when G is isomorphic to An (n ≥8) or one of the sporadic simple groups (see Atlas [5]). We assume that G is a group of Lie type. Case 1. Let G be a group of Lie type over the ﬁeld of odd characteristic. Then G is a group of component type. We look at the components of G to know whether the centralizers of involutions are isomorphic or not (see Burgoyne-Williamson [3] or Williams [14]). It will be easily seen that the centralizers of involutions in G are not isomorphic. Let G = Ln(q), n −2 = r + s, r > 0, s > 0 and d = (n, q −1). If n is odd, G contains centralizers of type Ar × As. It follows that n = 3 and k = 1, a contradiction. Suppose that n is even. If s is even or q ≡1(2d), then there exist centralizers of type Ar × As. 2. Finite simple groups Groups of Lie type over the ﬁeld of even characteristic G \|CG(t)\| \|G\| L2(q) q (q + 1)q(q −1) L3(q) q3(q −1)(3, q −1)−1 (q3 −1)(q2 −1)q3(3, q −1)−1 U3(q) q3(q + 1)(3, q + 1)−1 (q3 + 1)q3(q2 −1)(3, q + 1)−1 Sz(q), q = 22k+1, k ≥1 q2 (q2 + 1)q2(q −1) Table 2. Groups of Lie type over the ﬁeld of even characteristic G \|CG(t)\| \|G\| L2(q) q (q + 1)q(q −1) L3(q) q3(q −1)(3, q −1)−1 (q3 −1)(q2 −1)q3(3, q −1)−1 U3(q) q3(q + 1)(3, q + 1)−1 (q3 + 1)q3(q2 −1)(3, q + 1)−1 Sz(q), q = 22k+1, k ≥1 q2 (q2 + 1)q2(q −1) Table 2. Groups of Lie type over the ﬁeld of even characteristic G \|CG(t)\| \|G\| L2(q) q (q + 1)q(q −1) L3(q) q3(q −1)(3, q −1)−1 (q3 −1)(q2 −1)q3(3, q −1)−1 U3(q) q3(q + 1)(3, q + 1)−1 (q3 + 1)q3(q2 −1)(3, q + 1)−1 Sz(q), q = 22k+1, k ≥1 q2 (q2 + 1)q2(q −1) THE ORDER OF A GROUP OF EVEN ORDER 399 Table 3. Groups of Lie type over the ﬁeld of odd characteristic G \|CG(t)\| \|G\| L2(q), q ≡1(4) q −1 q(q2 −1)/2 L2(q), q ≡−1(4) q + 1 q(q2 −1)/2 L3(q) (q −1)2q(q + 1)(3, q −1)−1 q3(q2 −1)(q3 −1)(3, q −1)−1 L4(q), q ≡5(8) (q −1)3q2(q + 1)2/2 q6 3 i=1(qi+1 −1)/4 G2(q) q2(q2 −1)2 q6(q6 −1)(q2 −1) 2G2(q), q=32k+1, k≥2 q(q2 −1) q3(q3 + 1)(q −1) U3(q) q(q+1)(q2−1)(3, q+1)−1 (q3+1)q3(q2−1)(3, q+1)−1 U4(q), q ≡3(8) (q −1)2q2(q + 1)3/2 q6 3 i=1(qi+1 −(−1)i+1)/4 3D4(q3) q4(q2 −1)(q6 −1) q12(q8 + q4 + 1)(q6 −1)(q2 −1) Table 4. Sporadic simple groups G \|CG(t)\| \|G\| M11 24.3 24.32.5.11 J1 23.3.5 23.3.5.7.11.19 M22 27.3 27.32.5.7.11 M23 27.3.7 27.32.5.7.11.23 J3 27.3.5 27.35.5.17.19 McL 27.32.5.7 27.36.53.7.11 ON 29.32.5.7 29.34.5.73.11.19.31 Ly 28.34.52.7.11 28.37.56.7.11.31.37.67 Th 215.34.5.7 215.310.53.72.13.19.31 Proof. By our assumption all centralizers of involutions in G are isomorphic, and in particular any involution is contained in the center of a Sylow 2-subgroup of G. Table 3. 2. Finite simple groups If r = s and q ̸≡1(2d), then there is a centralizer of type Ar(q2). These centralizers of involutions are not isomorphic. This proves our lemma for G = Ln(q). Let G = Un(q), n −2 = r + s and t a 2-part of n. If n is odd, then t = 1 and there are centralizers of type 2Ar × 2As for 0 ≤r ≤(n −1)/2. It follows that n = 3 and k = 1. This is not our case. Suppose that n is even. If q ≡−1(2t), there are centralizers of type 2Ar × 2As for 0 ≤r ≤(n −2)/2. If q ≡1(2t), there are 400 H YAMAKI H. YAMAKI 400 centralizers of type 2Ar × 2As for r odd, 0 ≤r ≤(n −2)/2 and of type 2A(n−2)/2. They are not isomorphic. centralizers of type 2Ar × 2As for r odd, 0 ≤r ≤(n −2)/2 and of type 2A(n−2)/2. They are not isomorphic. Let G = S2m(q) and m = r +s, r > 0, s > 0. If r ̸= s or r = s is even, then there exist centralizers of type Cr × Cs. Assume r = s. Then G contains a centralizer of type Am−1 if q ≡1(4) or of type 2Am−1 if q ≡−1(4). This proves our lemma for S2m(q). Let G = O2n+1(q) and r + s = n, 0 ≤r ≤n −1, 2 ≤s ≤n. G contains a centralizer of type Bn−1. If q ≡1(4), there exist centralizers of type Br × Ds. If q ≡−1(4), then there exist centralizers of type Br × Ds when s is even or of type Br × 2Ds when s is odd. This proves our lemma for G when n ≥3. Let G = O+ 8 (q). Then G contains a centralizer of type D2 × D2. There exists a centralizer of type A3 if q ≡1(4) and type 2A3 if q ≡−1(4). They are not isomorphic. Let G = O− 8 (q). Then G contains a centralizer of type D2 × 2D2. There exists a centralizer of type 2A3 if q ≡1(4) and of type A3 if q ≡−1(4). They are not isomorphic. Let G = O+ 2n(q). Suppose that n is even and n ≥6. There exist centralizers of type Dr × Ds where r is even and r + s = n, 2 ≤r ≤(n −2)/2. 2. Finite simple groups If q ≡1(4) or n is divisible by 4, then there exists a centralizer of type Dn/2 × Dn/2. If q ≡−1(4), then G contains a centralizer of type 2Dn/2 × 2Dn/2. They are not isomorphic. Suppose that n is odd and n ≥5. There exist centralizers of type Dn−1 and of type Dr × Ds, r + s = n, 2 ≤r ≤(n + 1)/2 for r even. They are not isomorphic. Let G = O+ 2n(q). Suppose that n is even and n ≥6. There exist centralizers of type Dr × Ds where r is even and r + s = n, 2 ≤r ≤(n −2)/2. If q ≡1(4) or n is divisible by 4, then there exists a centralizer of type Dn/2 × Dn/2. If q ≡−1(4), then G contains a centralizer of type 2Dn/2 × 2Dn/2. They are not isomorphic. Suppose that n is odd and n ≥5. There exist centralizers of type Dn−1 and of type Dr × Ds, r + s = n, 2 ≤r ≤(n + 1)/2 for r even. They are not isomorphic. Let G = O− 2n(q). Suppose that n is even and n ≥6. There exist centralizers of type Dn−1 and of type 2Dn−1. They are not isomorphic. Suppose that n is odd and n ≥5. There exist centralizers of type Dn−1 and of type Dr × 2Ds where r is even and r + s = n, 2 ≤r ≤(n + 1)/2. They are not isomorphic. Let G = E6(q). There exist centralizers of type D5 and of type A1 × A5. 2 2 2 Let G = 2E6(q). There exist centralizers of type 2D5 and of type A1 × 2A5. Let G = 2E6(q). There exist centralizers of type 2D5 and of type A1 × 2A5 Let G = E7(q). Then G contains a centralizer of type A1 ×D6. If q ≡1(4), then there exist centralizers of type E6 and of type A7. If q ≡−1(4), then there exist centralizers of type 2E6 and of type 2A7. Let G = E7(q). Then G contains a centralizer of type A1 ×D6. If q ≡1(4), then there exist centralizers of type E6 and of type A7. If q ≡−1(4), then there exist centralizers of type 2E6 and of type 2A7. Let G = E8(q). There exist centralizers of type D8 and A1 × E7. 2 Let G = F4(q). 2. Finite simple groups There exist centralizers of type B4 and of type 2A7. 3 2 We note that the groups 3D4(q), G2(q), 2G2(q) have a unique conjugacy class of involutions when q is odd. We note that the groups 3D4(q), G2(q), 2G2(q) have a unique conjugacy class of involutions when q is odd. Case 2. Let G be a group of Lie type over the ﬁeld of even characteristic. If G is a classical simple group and G is not S4(q), then G contains a non-central involution, which is not our case (see Dye [6, 7, 8]). If G = S4(q), q even, then G has three conjugacy classes of involutions and all of them are central. Two of them have the same cardinalities but the third one is diﬀerent from the other two. If G is an exceptional group of rank 2, then G has two conjugacy classes of involutions and one of them is non-central. If G is an exceptional group of rank greater than 2, then it follows from Aschbacher-Seitz [1] that we have Table 5 of CG(t)/O2(CG(t)) for an involution t ∈G (see also Chigira-Iiyori-Yamaki [4], Kondrat’ev [12]) which eventually yields our lemma for G. We note that F4(q) has four conjugacy classes of involutions, and two of them are fused by the graph automorphism. □ □ 401 THE ORDER OF A GROUP OF EVEN ORDER Table 5 G CG(t)/O2(CG(t)) S6(q) F4(q) S6(q) S4(q) SL(2, q) × SL(2, q) SU(6, q) 2E6(q) SO(7, q) × Zq+1 (SL(2, q) × SL(2, q)) : Zq+1 SL(6, q) E6(q) S6(q) × Zq−1/(3.q−1) SL(2, q) × SL(3, q) SO+(12, q) S8(q) × SL(2, q) E7(q) SL(2, q) × S6(q) F4(q) S6(q) E7(q) E8(q) S12(q) F4(q) × SL(2, q) S8(q) 3 The proof of Theorem 1 Table 5 G CG(t)/O2(CG(t)) S6(q) F4(q) S6(q) S4(q) SL(2, q) × SL(2, q) SU(6, q) 2E6(q) SO(7, q) × Zq+1 (SL(2, q) × SL(2, q)) : Zq+1 SL(6, q) E6(q) S6(q) × Zq−1/(3.q−1) SL(2, q) × SL(3, q) SO+(12, q) S8(q) × SL(2, q) E7(q) SL(2, q) × S6(q) F4(q) S6(q) E7(q) E8(q) S12(q) F4(q) × SL(2, q) S8(q) 3 The proof of Theorem 1 3. The proof of Theorem 1 Proof. Since G has one conjugacy class of involutions it is easy to see that N = S. Let t ∈S be an involution. If S has one conjugacy class of involutions, then Lemma 5. Suppose that N = S × S × S × · · · × S where S is a non-abelian simple group. Then N = S and Theorem 1 holds true for G. Acknowledgments The author would like to thank the anonymous referee for informing him of a slightly diﬀerent proof of Theorem 1 which avoids the use of Lemma 2 and a proof of Lemma 2 without detailed case by case analysis. Actually the referee proved that if G has m conjugacy classes of involutions for m ≥2, then \|G\| < \|CG(x)\|3/m for some involution x in G with the use of the Thompson order formula and a counting argument (see Suzuki [13, p. 125–127]). Then he or she applied similar arguments in Lemmas 3, 4 and 5 to prove Theorem 1 by induction. Thus the proof relies less on the classiﬁcation of ﬁnite simple groups. 3. The proof of Theorem 1 Since t is a central involution in a Sylow 2-subgroup of G, it follows that (G : CG(t)) = k(S : CS(t)) and k is odd. This does not happen in ﬁnite simple groups by Lemma 2. □ (S : CS(t)) = (G : CG(t)). Since \|CS(t)\|3 > \|S\| by Lemma 1, it follows that \|CG(t)\|3 > \|G\|. Suppose that S has k (≥2) conjugacy classes of involutions. Since G has precisely one conjugacy class of involutions, all of the centralizers of involutions in S are isomorphic to CS(t). Since t is a central involution in a Sylow 2-subgroup of G, it follows that (G : CG(t)) = k(S : CS(t)) and k is odd. This does not happen in ﬁnite simple groups by Lemma 2. □ By Lemmas 3, 4 and 5 we may assume that G is a non-abelian simple group. It follows from Lemma 1 that Theorem 1 holds true for G. The proof of Theorem 1 is complete. References [1] M. Aschbacher and G. Seitz, Involutions in Chevalley groups over ﬁelds of even order, Nagoya Math. J. 63 (1976), 1–91. MR0422401 (54:10391) [1] M. Aschbacher and G. Seitz, Involutions in Chevalley groups over ﬁelds of even order, Nagoya Math. J. 63 (1976), 1–91. MR0422401 (54:10391) [2] R. Brauer and K. A. Fowler, On groups of even order, Ann. Math. 62 (1955), 565–583. MR0074414 (17:580e) [2] R. Brauer and K. A. Fowler, On groups of even order, Ann. Math. 62 (1955), 565–583. MR0074414 (17:580e) [3] N. Burgoyne and C. Williamson, Centralizers of involutions in Chevalley groups of odd char- acteristic, Mimeographed notes (1972). [4] N. Chigira, N. Iiyori and H. Yamaki, Non-abelian Sylow subgroups of ﬁnite groups of even order, Invent. Math. 139 (2000), 525–539. MR1738059 (2001c:20042) [5] J. Conway, R. T. Curtis, S. Norton, R. Parker, and R. Wilson, Atlas of ﬁnite groups, Claren- don Press, Oxford, 1985. MR827219 (88g:20025) [6] R. H. Dye, On the conjugacy classes of involutions of the simple orthogonal groups ove perfect ﬁelds of characteristic two, J. Algebra 18 (1971), 414–425. MR0276366 (43:2113) [7] R. H. Dye, On the involution classes of the linear groups GLn(K), SLn(K), PGLn(K), PSLn(K) over ﬁelds of characteristic two, Math. Proc. Cambridge Phil. Soc. 72 (1972), 1–6. MR0294519 (45:3589) [8] R. H. Dye, On the conjugacy classes of involutions of the unitary groups Um(K), SUm(K), PUm(K), PSUm(K) over perfect ﬁelds of characteristic 2, J. Algebra 24 (1973), 453–459. MR0308287 (46:7401) [9] K. Harada and M. Miyamoto, On the order of a group of even order, To appear in J. Algebra [9] K. Harada and M. Miyamoto, On the order of a group of even order, To appear in J. Algebra. [10] B. Huppert and N. Blackburn, Finite Groups III, Springer-Verlag, Berlin, 1982. MR662826 (84i:20001b) [10] B. Huppert and N. Blackburn, Finite Groups III, Springer-Verlag, Berlin, 1982. MR662826 (84i:20001b) [11] N. Iiyori and H. Yamaki, Prime graph components of the simple groups of Lie type over the ﬁelds of even characteristic, J. Algebra 155 (1993), 335–343. Corrigenda 181 (1996) 659. MR1212233 (94e:05268) [12] A. S. Kondrat’ev, Prime graph components of ﬁnite simple groups, Math. USSR Sbornik 67 (1990), 235–247. MR1015040 (90h:20018) ( ), ( ) 13] M. Suzuki, Group theory II, Springer-Verlag, Berlin, 1986. MR815926 (87e:20001) [13] M. Suzuki, Group theory II, Springer-Verlag [14] J. S. Williams, Prime graph components of ﬁnite groups, J. Algebra 69 (1981), 487–513. 3. The proof of Theorem 1 Let G be a minimal counterexample to Theorem 1. Suppose that G is not simple. Let N be a minimal normal subgroup of G. Then N is an elementary abelian p-group or a direct product of isomorphic non-abelian simple groups. Lemma 3. Suppose that N is an elementary abelian 2-group. Then Theorem 1 holds true for G. Proof. Let t ∈N be an involution. Since G has one conjugacy class of involutions, \|N\| −1 = (G : CG(t)) and CG(t) ⊇N. It follows that \|G\| < (\|CG(t)\| −1)\|CG(t)\| < \|CG(t)\|3. □ Proof. Let t ∈N be an involution. Since G has one conjugacy class of involutions, \|N\| −1 = (G : CG(t)) and CG(t) ⊇N. It follows that \|G\| < (\|CG(t)\| −1)\|CG(t)\| < \|CG(t)\|3. □ Lemma 4. Suppose that N is an elementary abelian p-group for odd prime p. Then Theorem 1 holds true for G. Proof. The group G/N satisﬁes the condition of Theorem 1. Let t be an invo- lution in G. Then by induction \|CG/N(tN)\|3 > \|G/N\|. Note that CG(t)N/N = CG/N(tN). Let ⟨t, u⟩be a four group in G. Since t ∼u ∼tu in G, Brauer- Wielandt’s formula (see Huppert-Blackburn [10, p. 290]) implies that \|N\| = \|CN(t)\|\|CN(u)\|\|CN(tu)\|/\|CN(⟨t, u⟩)\|2 = \|CN(t)\|3/\|CN(⟨t, u⟩)\|2. Since \|CG(t)N\|3/\|N\|3 > \|G\|/\|N\| it follows that \|CG(t)N\|3 > \|G\|\|N\|2. This yields \|CG(t)\|3 ≥\|CG(t)\|3/\|CN(⟨t, u⟩)\|2 > \|G\| □ which completes the proof. □ which completes the proof. □ which completes the proof. Lemma 5. Suppose that N = S × S × S × · · · × S where S is a non-abelian simple group. Then N = S and Theorem 1 holds true for G. Lemma 5. Suppose that N = S × S × S × · · · × S where S is a non-abelian simple group. Then N = S and Theorem 1 holds true for G. Proof. Since G has one conjugacy class of involutions it is easy to see that N = S. Let t ∈S be an involution. If S has one conjugacy class of involutions, then 402 H. YAMAKI (S : CS(t)) = (G : CG(t)). Since \|CS(t)\|3 > \|S\| by Lemma 1, it follows that \|CG(t)\|3 > \|G\|. Suppose that S has k (≥2) conjugacy classes of involutions. Since G has precisely one conjugacy class of involutions, all of the centralizers of involutions in S are isomorphic to CS(t). References MR617092 (82j:20054) Department of Mathematics, Kumamoto University, Kumamoto 860-8555 Japan E-mail address: yamaki@gpo.kumamoto-u.ac.jp -mail address: yamaki@gpo.kumamoto-u.ac.jp Current address: JICA, Maipu 1300, Piso 21, C1006ACT Buenos Aires, Argentina E-mail address: yamaki.hiroyoshi@gmail.com
https://openalex.org/W2902787537	https://peerj.com/articles/5943v0.3/submission	English	null	Peer Review #2 of "A needle in a haystack: Mesozoic origin of parasitism in Strepsiptera revealed by first definite Cretaceous primary larva (Insecta) (v0.1)"	null	2,018	cc-by	8,961	A needle in a haystack: Mesozoic origin of parasitism in Strepsiptera revealed by first definite Cretaceous primary larva (Insecta) 1 Institut für Zoologie und Evolutionsforschung, Spezielle Zoologie und Entomologie, Friedrich-Schiller Universität Jena, Jena, Germany 2 Department of Zoology, Faculty of Science, Charles University Prague, Praha, Czech Republic 3 Käshofen, Germany Corresponding Author: Hans Pohl Email address: hans.pohl@uni-jena.de Twisted winged insects (Strepsiptera) are a highly specialized small order of parasitic insects. Whether parasitism developed at an early or late stage in the evolution of the group was unknown. Here we record and describe the first definite Mesozoic strepsipteran primary larva embedded in Burmese amber (~99 million years ago). This extends the origin of the parasitism back by at least ~50 million years, and reveals that this specialized life style has evolved in the Mesozoic or even earlier in the group. The extremely small first instar displays all diagnostic characters of strepsipteran immatures of this stage and is nearly identical with those of Mengenillidae, one of the most “ancestral” extant strepsipteran taxa. This demonstrates a remarkable evolutionary stasis over ~100 million years. The new finding strongly weakens the case of small larvae embedded in Cretaceous amber interpreted as strepsipteran immatures. They differ in many structural features from extant strepsipteran primary larvae and are very likely parasitic beetle larvae. Manuscript to be reviewed A needle in a haystack: Mesozoic origin of parasitism in Strepsiptera revealed by first definite Cretaceous primary larva (Insecta) Hans Pohl Corresp., 1 , Jan Batelka 2 , Jakub Prokop 2 , Patrick Müller 3 , Margarita I Yavorskaya 1 , Rolf G Beutel 1 1 Institut für Zoologie und Evolutionsforschung, Spezielle Zoologie und Entomologie, Friedrich-Schiller Universität Jena, Jena, Germany 2 Department of Zoology, Faculty of Science, Charles University Prague, Praha, Czech Republic 3 Käshofen, Germany Manuscript to be reviewed 1 A needle in a haystack: Mesozoic origin of parasitism in Strepsiptera revealed by first 2 definite Cretaceous primary larva (Insecta) 3 Hans Pohl1, Jan Batelka2, Jakub Prokop2, Patrick Müller3, Margarita I. Yavorskaya1, Rolf G. 4 Beutel1 5 6 1 Institut für Zoologie und Evolutionsforschung, Friedrich-Schiller-Universität Jena, Germany 7 2 Department of Zoology, Faculty of Science, Charles University, Praha, Czech Republic 8 3 Käshofen, Germany 9 10 Corresponding author 11 Hans Pohl 12 Email address: hans.pohl@uni-jena.de 13 1 A needle in a haystack: Mesozoic origin of parasitism in Strepsiptera revealed by first 2 definite Cretaceous primary larva (Insecta) PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) 14 Abstract 15 Twisted winged insects (Strepsiptera) are a highly specialized small order of parasitic insects. 16 Whether parasitism developed at an early or late stage in the evolution of the group was 17 unknown. Here we record and describe the first definite Mesozoic strepsipteran primary larva 18 embedded in Burmese amber (~99 million years ago). This extends the origin of the parasitism 19 back by at least ~50 million years, and reveals that this specialized life style has evolved in the 20 Mesozoic or even earlier in the group. The extremely small first instar displays all diagnostic 21 characters of strepsipteran immatures of this stage and is nearly identical with those of 22 Mengenillidae, one of the most “ancestral” extant strepsipteran taxa. This demonstrates a 23 remarkable evolutionary stasis over ~100 million years. The new finding strongly weakens the 24 case of small larvae embedded in Cretaceous amber interpreted as strepsipteran immatures. They 25 differ in many structural features from extant strepsipteran primary larvae and are very likely 26 parasitic beetle larvae. 27 Manuscript to be reviewed 45 intersegmental membranes with the sclerotized anterior part of their body (Pohl & Beutel, 46 2008). The primary larvae of Strepsiptera are among the smallest known metazoans with an 47 average size of ca. 230 µm, comparable to unicellular ciliates of the genus Paramecium (Pohl, 48 2002). This size reduction enables the female to produce a huge number of offspring and the 49 minute primary larvae are able to penetrate relatively small insect hosts such as Delphacidae 50 (Auchenorrhyncha) with an adult size of 1.5–6 mm (Pohl & Beutel, 2008). The body of the 51 primary larvae is elongated oval to nearly drop shaped. The head is semicircular with well- 52 developed stemmata with cornea lenses. Antennae are missing and the labrum is not present as a 53 separate element. Sternal plates inserted between the coxae with spinulae and fringes of 54 microtrichia on the posterior border of the abdominal sternites produce capillary forces enabling 55 the larvae to stick to and crawl along wet surfaces (Pohl & Beutel, 2008). A unique apomorphic 56 feature within Holometabola is the presence of one pair of long and strongly developed bristle- 57 like cerci. They are inserted on the terminal abdominal segment XI, with strong muscles attached 58 to their base. They enable the minute larvae to jump very efficiently, an ability secondarily lost 59 in the primary larvae of the Stylopidae (Pohl & Beutel, 2005). 60 The relatively sparse Cretaceous fossil record of adult strepsipteran males suggests that 61 they were already a specialized group of insects in the late Mesozoic. It is very likely that the 62 highly modified antennae with flabellate appendages and numerous specialised dome-shaped 63 chemoreceptors were used for finding females over a relatively large distance as in extant species 64 of the group. Likewise, the uptake of food was apparently very limited at least, as suggested by 65 the strongly simplified mouthparts. Strepsipteran females from the Mesozoic have not yet been 66 discovered yet. As with most cases of parasitism documented in the fossil record (e.g. Nagler & 67 Haug, 2015), Mesozoic Strepsiptera provide only indirect evidence of this specialization. Based 68 on the small size of adult males (ca. 1.5–3 mm), it was assumed, that the species were already 69 endoparasites of other insects (Pohl & Beutel, 2008). 28 Introduction 29 Strepsiptera is a highly specialized small order of holometabolous insects (Pohl & Beutel, 2008; 30 2013). The phylogenetic placement of the group, one of the longest controversies in systematic 31 entomology, was only recently clarified (Niehuis et al., 2012; Peters et al., 2014; Misof et al., 32 2014; Kjer et al., 2016). The oldest recorded fossils are known from Cretaceous Burmese amber 33 (Grimaldi, Kathirithamby & Schawaroch, 2005; Pohl & Beutel, 2016; Engel et al., 2016), 34 indicating a minimum age of ca. 100 mya. An age of origin of ca. 120 mya was estimated based 35 on molecular data (McMahon, Hayward & Kathirithamby, 2011). However, the confirmed sister 36 group relationship with Coleoptera (Misof et al., 2014) implies that the group originated already 37 in the early Permian or late Carboniferous (McKenna et al., 2015; Toussaint et al., 2017). 29 Strepsiptera is a highly specialized small order of holometabolous insects (Pohl & Beutel, 2008; 30 2013). The phylogenetic placement of the group, one of the longest controversies in systematic 31 entomology, was only recently clarified (Niehuis et al., 2012; Peters et al., 2014; Misof et al., 32 2014; Kjer et al., 2016). The oldest recorded fossils are known from Cretaceous Burmese amber 33 (Grimaldi, Kathirithamby & Schawaroch, 2005; Pohl & Beutel, 2016; Engel et al., 2016), 34 indicating a minimum age of ca. 100 mya. An age of origin of ca. 120 mya was estimated based 35 on molecular data (McMahon, Hayward & Kathirithamby, 2011). However, the confirmed sister 36 group relationship with Coleoptera (Misof et al., 2014) implies that the group originated already 37 in the early Permian or late Carboniferous (McKenna et al., 2015; Toussaint et al., 2017). 38 Extant strepsipterans are characterized by a conspicuous sexual dimorphism. The winged 39 males are free living but extremely short-lived. With well-developed sensory organs and a highly 40 efficient flight apparatus they are able to find the females within their very short life span of only 41 few hours. Females are wingless and morphologically strongly simplified. They develop as 42 endoparasites of other insects. Only the females of the basal extant Mengenillidae (and probably 43 Bahiaxenidae) leave their host and are able to move actively. The females of the majority of the 44 species (Stylopidia) are permanently endoparasitic and only penetrate the host’s abdominal PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed 75 Confirmed fossil records of strepsipteran primary larvae are from the Eocene and Miocene, 76 respectively. The oldest fossil from Eocene brown coal (Geisel Valley, Germany) was initially 77 described as a first instar of a scale insect (Coccoidea) (Haupt, 1950). It was later assigned to the 78 extant strepsipteran genus Stichotrema (Myrmecolacidae) (Kinzelbach & Lutz, 1985). Finally, it 79 was placed as Stylopidia incertae sedis based on a re-examination and a cladistic analysis of 80 characters of primary larvae of all extant families of Strepsiptera (Pohl, 2009). Strepsipteran 81 primary larvae associated with its parent female and its host (Auchenorrhyncha: Delphacidae) 82 are reported from Dominican amber (Poinar, 2004), which is currently attributed to the Miocene: 83 15–20 mya (Iturralde-Vinent & MacPhee, 1996; Ituralde-Vinent, 2001). Other very small 84 “triungulin” larvae assigned to Strepsiptera were described from the Late Cretaceous 85 (Campanian) amber of Manitoba, Canada (Grimaldi, Kathirithamby & Schawaroch, 2005), and a 86 “planidium” from the Upper Cretaceous (Santonian) amber of the Taimyr Peninsula, Siberia 87 (Kathirithamby et al., 2017). The interpretation of the immatures treated in the earlier study was 88 discussed critically, pointing out an entire series of features in conflict with an assignment to 89 Strepsiptera (Beutel et al., 2016). The “planidium” from the Upper Cretaceous amber 90 (Kathirithamby et al., 2017) is most likely a parasitic beetle larva based on the following 91 features: small size (ca. 0.53 mm), conical head, absence of distinctly developed stemmata, 92 coarse ctenidia, large pretarsal adhesive pad, absence of abdominal segment XI and terminal 93 bristles (Batelka et al., accepted) (see below). 94 Kathirithamby et al. (2017) used the term “planidium” for primary larvae of Strepsiptera. 95 However, this is only appropriate for legless larvae of parasitic Diptera or Hymenoptera (Askew, 96 1971; Stehr, 1991). Triungulinid was introduced by Pierce (1909) for first instars of Strepsiptera 97 based on their similarity with primary larvae of Meloidae or Ripiphoridae (Coleoptera), which 98 were addressed as triungulins. However, as the structural affinities are only superficial and 99 obviously non-homologous, and the first instars differ in important features (e.g. nine abdominal 100 segments in Meloidae and Ripiphoridae versus 11 in primary larvae of Strepsiptera), we prefer 101 the neutral term primary larva for first instars of Strepsiptera. Manuscript to be reviewed However, conclusive evidence by direct 70 detection of definite strepsipteran larvae was lacking, and therefore direct evidence for an 71 endoparasitic life style of the immature stages of Mesozoic strepsipterans. Previously, 72 endoparasitism was only documented by fossil representatives of Stylopidia from Eocene 73 deposits, the strepsipteran subgroup with permanently endoparasitic females (Kinzelbach & 74 Pohl, 1994; Henderickx et al., 2013). PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed 106 The ordinal assignment among Strepsiptera is discussed. Based on the described features the 107 position of other putative strepsipteran larvae (Grimaldi, Kathirithamby & Schawaroch, 2005; 108 Kathirithamby et al., 2017) is critically re-evaluated. The new fossil larva clearly confirms that 109 Mesozoic Strepsiptera were already endoparasites of other insects. The origin of endoparasitism 110 is extended back by ca. 50 million years to a minimum of ca. 100 Ma. 111 113 Material 114 The piece of Burmese amber with the strepsipteran larva came from deposits in the Hukawng 115 Valley of Myanmar. The age is estimated as ca. 99 Ma (earliest Cenomanian (Shi et al., 2012)). 114 The piece of Burmese amber with the strepsipteran larva came from deposits in the Hukawng 115 Valley of Myanmar. The age is estimated as ca. 99 Ma (earliest Cenomanian (Shi et al., 2012)). 116 The amber with the strepsipteran first instar (accession number BU-002386) is integrated 117 into the collection of the Institute of Zoology, Chinese Academy of Sciences (Box 92, Beichen 118 West Road, Chaoyang District, Beijing, 100101, P. R. China) and will be deposited in the Three 119 Gorges Entomological Museum, Chongqing, China after 2027. It is from a mining locality at 120 Noije Bum (near Tanai Village, 26°21’33.41’’N, 96°43’11.88’’E) (Cruickshank & Ko, 2003; 121 Grimaldi, Engel & Nascimbene, 2009). The size of the piece of amber is 26 x 22 x 10 mm. 122 Syninclusions are listed in the following: Acari (9 specimens), orthopteran nymph (1), Psocodea 123 (1), Sternorrhyncha (1), apocritan Hymenoptera (2), Berothidae (Neuroptera) (1), Elateridae (1), 124 primary longipedes larvae of Ripiphoridae (46), Polyphaga with unclear affinity (2), 125 “nematoceran” species of Diptera with unclear affinity (2), brachyceran species of Diptera with 126 unclear affinity (2). 127 As important syninclusions are embedded very close to the strepsipteran larva, it was not 128 possible to isolate the larva and trim the amber piece into a thin plate and mount it on a glass 129 microscope slide. This precludes examining the first instar with oil-immersion lenses and phase 130 or differential interference contrast at a magnification of 1000 x. Therefore, some structures of 131 the minute larva could not be evaluated. 132 133 Specimen imaging p g g 134 The piece of amber was temporarily mounted on coverslips using glycerine. The specimen was 135 observed under two different microscopes: An Axio Zoom.V16 with a PlanNeoFluar Z 1.0x 136 (Carl Zeiss Microscopy GmbH) was used for the overview images and the images were saved as PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed Claws forming a trident with 102 spatulate setae occur in phoretic primary larvae of some genera of Meloidae (Bologna, Turco & 103 Pinto, 2010), but are completely lacking in strepsipteran larvae. 104 In the present study we describe a minute larva from a piece of Burmese amber, with a 105 habitus and a set of observable features unambiguously confirming a placement in Strepsiptera. 104 In the present study we describe a minute larva from a piece of Burmese amber, with a 105 habitus and a set of observable features unambiguously confirming a placement in Strepsiptera. 104 In the present study we describe a minute larva from a piece of Burmese amber, with a 105 habitus and a set of observable features unambiguously confirming a placement in Strepsiptera. PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed 137 CZI files. For observations and for measurements ZEN 2.3 lite (blue edition) (Carl Ze 138 Microscopy GmbH) was used. For higher magnifications, an Olympus IX81 invert 139 fluorescence microscope with UIS2 objectives, equipped with an ORCA-AG monochromatic 1 140 bit CCD camera (Hammatsu) was used. The mirror images were superimposed with Cell^ 141 software (Olympus Soft Imaging Solutions). Sets of photographs were analyzed with F 142 (Schindelin et al., 2012). 143 Single images were exported with ZEN 2.3 lite or Fiji respectively. Some images we 144 combined with Zerene Stacker (Zerene Systems LLC, Richland, USA). The photographs we 145 processed using Adobe Photoshop® CS6 (Adobe System Incorporated, San Jose, USA) a 146 arranged as plates. Adobe Illustrator® CS6 (Adobe Systems Incorporated, San Jose, USA) w 147 used for the lettering of the plates. Image stacks of the Olympus IX81 microscope were used f 148 the drawings and description. 149 150 Comparative taxonomy and terminology 151 Data on the morphology of Strepsiptera larvae are taken from (Pohl, 2000; 2002) and t 152 morphological nomenclature used in these studies is applied. Additionally, primary larvae 153 Eoxenos laboulbenei Peyerimhoff, 1919 and Mengenilla chobauti Hofeneder, 1910 (bo 154 Mengenillidae) were examined (research collection of H.P. at Phyletisches Museum). F 155 comparison primary larvae of E. laboulbenei embedded in Canada balsam on glass microsco 156 slides were examined with the same microscope (Olympus IX81) as the fossil. 157 158 Results 159 Preservation 160 Strongly depressed dorsoventrally, dorsal and ventral side thus difficult to distinguish, especia 161 in abdominal region. Part of left side of head and thorax covered by debris (Fig. 1A). 162 163 Morphology 164 First instar extremely small, total length excluding terminal bristles 197 µm. Head semicircul 165 with recognizable stemmata but lacking antennae. Sides of body subparallel, slightly conve 166 Dorsum smooth, without recognizable surface structures and largely devoid of setae. Termin 137 CZI files. For observations and for measurements ZEN 2.3 lite (blue edition) (Carl Zeiss 138 Microscopy GmbH) was used. For higher magnifications, an Olympus IX81 inverted 139 fluorescence microscope with UIS2 objectives, equipped with an ORCA-AG monochromatic 12- 140 bit CCD camera (Hammatsu) was used. The mirror images were superimposed with Cell^R 141 software (Olympus Soft Imaging Solutions). Sets of photographs were analyzed with Fiji 142 (Schindelin et al., 2012). 143 Single images were exported with ZEN 2.3 lite or Fiji respectively. Manuscript to be reviewed Some images were 144 combined with Zerene Stacker (Zerene Systems LLC, Richland, USA). The photographs were 145 processed using Adobe Photoshop® CS6 (Adobe System Incorporated, San Jose, USA) and 146 arranged as plates. Adobe Illustrator® CS6 (Adobe Systems Incorporated, San Jose, USA) was 147 used for the lettering of the plates. Image stacks of the Olympus IX81 microscope were used for 148 the drawings and description. Manuscript to be reviewed 167 abdominal segment XI with two pairs of strongly developed bristles. Specimen preserved in 168 ventral position 167 abdominal segment XI with two pairs of strongly developed bristles. Specimen preserved in 168 ventral position 168 ventral position 169 Exposed part of head capsule semicircular in ventral view, with evenly rounded anterior 170 margin and greatest width at hind margin. Visible part distinctly shorter than maximum width. 171 Six large individual stemmata with cornea lenses recognizable posterolaterally on left side in 172 strongly pigmented area, only three visible on right side (st in Figs. 1A, B, 2A). Labrum not 173 present as separate element. Evenly rounded anterior margin of head capsule apparently forming 174 sharp edge, lacking median emargination (Figs. 1A, B, 2A, B). Antennae not recognizable as 175 prominent structures, largely reduced. Antennal field likely represented by small circular 176 structure adjacent to anteriormost stemma, visible through translucent cuticle (af in Figs. 1B, 177 2A). Mandibles not visible. Ventral side of head medially covered by strongly modified maxillae 178 and labium. Maxillae medially fused, forming slightly curved, transverse plate-like structure 179 (Figs. 1B, 2B). Maxillary palps recognizable as circular spots on posterior maxillary margin (mp 180 in Figs. 1B, 2B). Details of labium not visible. Paired anterior tentorial arms visible in 181 posterolateral cephalic region (te in Figs. 1B, 2B). Ecdysial sutures not visible. One seta 182 recognizable on dorsal surface (fs in Fig. 1 B). Cuticle without recognizable surface 183 modifications. 184 Pro-, meso- and metathorax subequal in length on ventral side. Only inflicted lateral margin 185 of nota visible (Fig. 1). Thoracic segments continuously widening from anterior to posterior. 186 Prothorax with one seta on lateral pronotal margin, visible on right side. Conspicuous sternal 187 plate inserted between coxae (prsp in Figs. 1B, 2A). Anterior part of sternal plate broad, 188 posterior part with triangular apex. Spinulae on posterior margin not visible. Meso- and 189 metathorax very similar to prothorax, but sternal plate only visible on the former. Legs only 190 partly visible, short, composed of large, transverse coxa, trochanterofemur, tibia, and one- 191 segmented pad-like tarsus (Fig. 1B). Tarsus without claws. Hind leg with long seta on mesal 192 margin of coxa. 193 Abdomen composed of eleven segments. Segment II broadest, following segments slightly 194 tapering posteriorly (Fig. 1). Only lateral margins of tergites visible, with two lateral setae on 195 most of them (Figs. 150 Comparative taxonomy and terminology 151 Data on the morphology of Strepsiptera larvae are taken from (Pohl, 2000; 2002) and the 152 morphological nomenclature used in these studies is applied. Additionally, primary larvae of 153 Eoxenos laboulbenei Peyerimhoff, 1919 and Mengenilla chobauti Hofeneder, 1910 (both 154 Mengenillidae) were examined (research collection of H.P. at Phyletisches Museum). For 155 comparison primary larvae of E. laboulbenei embedded in Canada balsam on glass microscope 156 slides were examined with the same microscope (Olympus IX81) as the fossil. 157 158 Results 159 Preservation 160 Strongly depressed dorsoventrally, dorsal and ventral side thus difficult to distinguish, especially 161 in abdominal region. Part of left side of head and thorax covered by debris (Fig. 1A). 162 163 Morphology 164 First instar extremely small, total length excluding terminal bristles 197 µm. Head semicircular, 165 with recognizable stemmata but lacking antennae. Sides of body subparallel, slightly convex. 166 Dorsum smooth, without recognizable surface structures and largely devoid of setae. Terminal 151 Data on the morphology of Strepsiptera larvae are taken from (Pohl, 2000; 2002) and the 152 morphological nomenclature used in these studies is applied. Additionally, primary larvae of 153 Eoxenos laboulbenei Peyerimhoff, 1919 and Mengenilla chobauti Hofeneder, 1910 (both 154 Mengenillidae) were examined (research collection of H.P. at Phyletisches Museum). For 155 comparison primary larvae of E. laboulbenei embedded in Canada balsam on glass microscope 156 slides were examined with the same microscope (Olympus IX81) as the fossil. 160 Strongly depressed dorsoventrally, dorsal and ventral side thus difficult to distinguish, especially 161 in abdominal region. Part of left side of head and thorax covered by debris (Fig. 1A). y depressed dorsoventrally, dorsal and ventral side thus difficult to distinguish, especially minal region. Part of left side of head and thorax covered by debris (Fig. 1A). 164 First instar extremely small, total length excluding terminal bristles 197 µm. Head semicircular, 165 with recognizable stemmata but lacking antennae. Sides of body subparallel, slightly convex. 166 Dorsum smooth, without recognizable surface structures and largely devoid of setae. Terminal PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed 1B, 3A, B). Sternites I–IX half as long as thoracic segments. One short seta 196 visible on right side of sternite III, inserted close to hind margin. Sternites IV–VI with two setae 197 inserted laterally close to hind margin (Fig. 1B). Very fine parallel longitudinal lines on margins PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed 203 203 204 Diagnosis 205 Differs from primary larvae of Eoxenos laboulbenei by the presence of six stemmata and from 206 primary larvae of Mengenilla chobauti (both Mengenillidae) and all other known primary larvae 207 of Strepsiptera by the strongly developed second pair of bristles on hind margin of segment XI. 208 209 Discussion 210 The larva can be easily and unambiguously assigned to Strepsiptera, based on several diagnostic 211 features and also apomorphic character states shared with other first instars of the order. Like 212 extant strepsipteran primary larvae (Pohl, 2000) the fossil has an elongated oval to drop shaped 213 body, with a semicircular head with large stemmata with cornea lenses, but lacking a separate Manuscript to be reviewed 198 of sternites I–VIII may represent spinulae, or alternatively tergal furrows visible through cuticle 199 (Fig. 3A). Tergites IX and X fused, about as long as segments VI–VIII combined, forming large 200 plate-like structure covering terminal segment XI (Fig. 4A). Hind margin of segment XI 201 truncated, with pair of very strongly developed bristles inserted close to midline, and second 202 similar but shorter pair more laterally (Figs. 1B, 3B, 4B). 203 204 Diagnosis 205 Differs from primary larvae of Eoxenos laboulbenei by the presence of six stemmata and from 206 primary larvae of Mengenilla chobauti (both Mengenillidae) and all other known primary larvae 207 of Strepsiptera by the strongly developed second pair of bristles on hind margin of segment XI. 208 209 Discussion 210 The larva can be easily and unambiguously assigned to Strepsiptera, based on several diagnostic 211 features and also apomorphic character states shared with other first instars of the order. Like 212 extant strepsipteran primary larvae (Pohl, 2000) the fossil has an elongated oval to drop shaped 213 body, with a semicircular head with large stemmata with cornea lenses, but lacking a separate 214 labrum and also antennae as visible prominent structures. One pair of long and strongly 215 developed bristles inserted on the terminal abdominal segment XI is present (Figs. 1, 5A, B). 216 Within Strepsiptera, the larva shows close structural affinities with first instars of Eoxenos 217 laboulbenei, especially due to the identical equipment with bristles on the last abdominal 218 segments (Figs. 4, 5). A placement close to the root of Strepsiptera s.l. or s.str. is likely. 219 However, as only the male adults of extant Bahiaxenos and the extinct †Mengea, †Protoxenos 220 (both Eocene Baltic amber), †Cretostylops, †Kinzelbachilla, and †Phthanoxenos, (Cretaceous 221 Burmese amber) are known (Grimaldi, Kathirithamby & Schawaroch, 2005; Pohl, Beutel & 222 Kinzelbach, 2005; Bravo et al., 2009; Pohl & Beutel, 2016; Engel et al., 2016), a precise 223 phylogenetic assessment is not possible. It is conceivable that the larva belongs to one of the 224 three strepsipteran species known from the same fossil site, but a verification is not possible with 225 the information at hand. 226 A major point demonstrated here is that this Cretaceous primary larva of Strepsiptera differ 198 of sternites I–VIII may represent spinulae, or alternatively tergal f 199 (Fig. 3A). Manuscript to be reviewed Tergites IX and X fused, about as long as segments VI– 200 plate-like structure covering terminal segment XI (Fig. 4A). 201 truncated, with pair of very strongly developed bristles inserted 202 similar but shorter pair more laterally (Figs. 1B, 3B, 4B). 203 204 Diagnosis 205 Differs from primary larvae of Eoxenos laboulbenei by the prese 206 primary larvae of Mengenilla chobauti (both Mengenillidae) and a 207 of Strepsiptera by the strongly developed second pair of bristles on 208 209 Discussion 210 The larva can be easily and unambiguously assigned to Strepsipter 211 features and also apomorphic character states shared with other 212 extant strepsipteran primary larvae (Pohl, 2000) the fossil has an 213 body, with a semicircular head with large stemmata with cornea 214 labrum and also antennae as visible prominent structures. On 215 developed bristles inserted on the terminal abdominal segment X 216 Within Strepsiptera, the larva shows close structural affinities 217 laboulbenei, especially due to the identical equipment with b 218 segments (Figs. 4, 5). A placement close to the root of Strep 219 However, as only the male adults of extant Bahiaxenos and the e 220 (both Eocene Baltic amber), †Cretostylops, †Kinzelbachilla, and 221 Burmese amber) are known (Grimaldi, Kathirithamby & Schaw 222 Kinzelbach, 2005; Bravo et al., 2009; Pohl & Beutel, 2016; E 223 phylogenetic assessment is not possible. It is conceivable that th 224 three strepsipteran species known from the same fossil site, but a v 225 the information at hand. 226 A major point demonstrated here is that this Cretaceous prim 198 of sternites I–VIII may represent spinulae, or alternatively tergal furrows visible through cuticle 199 (Fig. 3A). Tergites IX and X fused, about as long as segments VI–VIII combined, forming large 200 plate-like structure covering terminal segment XI (Fig. 4A). Hind margin of segment XI 201 truncated, with pair of very strongly developed bristles inserted close to midline, and second 202 similar but shorter pair more laterally (Figs. 1B, 3B, 4B). 203 198 of sternites I–VIII may represent spinulae, or alternatively tergal furrows visible through cuticle 199 (Fig. 3A). Tergites IX and X fused, about as long as segments VI–VIII combined, forming large 200 plate-like structure covering terminal segment XI (Fig. 4A). Hind margin of segment XI 201 truncated, with pair of very strongly developed bristles inserted close to midline, and second 202 similar but shorter pair more laterally (Figs. 1B, 3B, 4B). 204 Diagnosis 210 The larva can be easily and unambiguously assigned to Strepsiptera, based on several diagnostic 211 features and also apomorphic character states shared with other first instars of the order. Like 212 extant strepsipteran primary larvae (Pohl, 2000) the fossil has an elongated oval to drop shaped 213 body, with a semicircular head with large stemmata with cornea lenses, but lacking a separate 214 labrum and also antennae as visible prominent structures. One pair of long and strongly 215 developed bristles inserted on the terminal abdominal segment XI is present (Figs. 1, 5A, B). 216 Within Strepsiptera, the larva shows close structural affinities with first instars of Eoxenos 217 laboulbenei, especially due to the identical equipment with bristles on the last abdominal 218 segments (Figs. 4, 5). A placement close to the root of Strepsiptera s.l. or s.str. is likely. 219 However, as only the male adults of extant Bahiaxenos and the extinct †Mengea, †Protoxenos 220 (both Eocene Baltic amber), †Cretostylops, †Kinzelbachilla, and †Phthanoxenos, (Cretaceous 221 Burmese amber) are known (Grimaldi, Kathirithamby & Schawaroch, 2005; Pohl, Beutel & 222 Kinzelbach, 2005; Bravo et al., 2009; Pohl & Beutel, 2016; Engel et al., 2016), a precise 223 phylogenetic assessment is not possible. It is conceivable that the larva belongs to one of the 224 three strepsipteran species known from the same fossil site, but a verification is not possible with 225 the information at hand. 226 A major point demonstrated here is that this Cretaceous primary larva of Strepsiptera differ 227 only in minimal details from extant immatures of basal genera of the order (Mengenillidae). This PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) 260 Conclusions 260 Conclusions 261 Detailed investigations of Burmese amber revealed the first definitive strepsipteran primary larva 262 from the Cretaceous. Diagnostic features are the size of less than 200 μm, an elongated oval 263 body, a semicircular head, and stemmata with cornea lenses in a strongly pigmented area. 264 Apomorphic character states shared with other primary larvae of Strepsiptera are the lack of a 265 separate labrum and prominent antennae, the medially fused maxillae, sternal plates, and the 266 presence of a pair of long and strongly developed bristles inserted on the terminal abdominal 267 segment XI. An evolutionary stability over ca. 100 million years is revealed as the Cretaceous 268 primary larva of Strepsiptera differs only in minimal details from extant Mengenillidae, the 269 sistergroup of the vast majority of the Strepsiptera (ca. 97 % of the species). The recently 270 described “planidium” assigned to Strepsiptera by Kathirithamby et al. (2017) differs in many 271 characters from fossil and extant primary larvae of Strepsiptera and is very likely a parasitic 272 beetle larva. Manuscript to be reviewed 228 documents a high evolutionary stability over ca. 100 million years and clearly suggests that these 229 extremely miniaturized larvae were already parasitic and produced in high numbers. 228 documents a high evolutionary stability over ca. 100 million years and clearly suggests that these 229 extremely miniaturized larvae were already parasitic and produced in high numbers. 230 Large stemmata for identification of a host and the abdominal jumping apparatus are 231 features clearly linked with parasitism, and the extremely small size suggests a very high number 232 of offspring like in extant groups (Pohl & Beutel, 2008). 233 The finding of a Cretaceous primary larva nearly identical with those of the extant genus 234 Eoxenos sheds new light on recently described “planidia” assigned to Strepsiptera 235 (Kathirithamby et al., 2017). Considering the clearly demonstrated evolutionary stability of the 236 tiny first instars, it appears highly unlikely that at the same time aberrant and unusually large 237 strepsipteran primary larvae occurred. The “planidia” described by Kathirithamby et al. (2017) 238 differ in many features from strepsipteran larvae, such as for instance much larger size (ca. 0.5 239 mm), anteriorly conical head, absence of large stemmata (“presumably two stemmata”), presence 240 of a membranous cervix, presence of ctenidia, posteriorly widening meso- and metanota, absence 241 of sternal plates, strongly widened femora, lack of setae and spines on the abdominal sternites, 242 only ten abdominal segments, lacking terminal bristles (on segment XI in Strepsiptera), and 243 consequently the lack of a jumping apparatus. It is much more likely that these were simply 244 miniaturized parasitic beetle larvae (Beutel et al., 2016), although their family placement is still 245 open to debate (Batelka et al., accepted). All observed features are compatible with a placement 246 in polyphagan beetles. The presence of coarse ctenidia and a pad-like pretarsal adhesive device 247 are apomorphic features linking it clearly with beetle larvae very likely belonging to the 248 cucujiform family Ripiphoridae (Beutel et al., 2016; Batelka et al., accepted). PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 289 Beutel RG, Zhang WW, Pohl H, Wappler T, Bai M 2016. A miniaturized beetle larva in 290 Cretaceous Burmese amber: reinterpretation of a fossil “strepsipteran triungulin.” Insect 291 Systematics & Evolution 47:83–91. 292 Bologna MA, Turco F, Pinto JD 2010. 11.19. Meloidae Gyllenhal, 1810. In: Leschen RAB, 293 Beutel RG, Lawrence JF eds. Handbook of Zoology. New York, 681–693. 93 Beutel RG, Lawrence JF eds. Handbook of Zoology. New York, 681–693 294 Bravo F, Pohl H, Silva-Neto A, Beutel R 2009. Bahiaxenidae, a “living fossil” and a new family 295 of Strepsiptera (Hexapoda) discovered in Brazil. Cladistics 25:614–623. 94 Bravo F, Pohl H, Silva-Neto A, Beutel R 2009. Bahiaxenidae, a “living fossil 295 of Strepsiptera (Hexapoda) discovered in Brazil. Cladistics 25:614–623. 296 Cruickshank RD, Ko K 2003. Geology of an amber locality in the Hukawng Valley, Northern 297 Myanmar. Journal of Asian Earth Sciences 21:441–455. 298 Engel MS, Huang D, Breitkreuz LCV, Azar D, Cai C, Alvarado M 2016. A new twisted-wing 299 parasitoid from mid-Cretaceous amber of Myanmar (Strepsiptera). Cretaceous Research 300 58:160–167. 301 Grimaldi DA, Engel MS, Nascimbene PC 2009. Fossiliferous Cretaceous amber from Myanmar 301 Grimaldi DA, Engel MS, Nascimbene PC 2009. Fossiliferous Cretaceous amber from Myanmar 302 (Burma): Its rediscovery, biotic diversity, and paleontological significance. American 01 Grimaldi DA, Engel MS, Nascimbene PC 2009. Fossiliferous Cretaceous am 301 Grimaldi DA, Engel MS, Nascimbene PC 2009. Fossiliferous Cretaceous amber from Myanmar 302 (Burma): Its rediscovery, biotic diversity, and paleontological significance. American 303 Museum Novitates 3361:1–71. 302 (Burma): Its rediscovery, biotic diversity, and paleontological significance. American 303 Museum Novitates 3361:1–71. 304 Grimaldi D, Kathirithamby J, Schawaroch V 2005. Strepsiptera and triungula in Cretaceous 305 amber. Insect Systematics & Evolution 36:1–20. 306 Haupt H 1950. Die Käfer (Coleoptera) aus der eozänen Braunkohle des Geiseltals. Geologica. 307 Schriftenreihe der geologischen Institute der Universitäten Berlin, Greifswald, Halle, 308 Rostock 6:VII–168. 309 Henderickx H, Bosselaers J, Pauwels E, van Hoorebeke L, Boone M 2013. X-ray micro-CT 310 reconstruction reveals eight antennomeres in a new fossil taxon that constitutes a sister clade 311 to Dundoxenos and Triozocera (Strepsiptera: Corioxenidae). Palaeontologia Electronica 312 16:1–16. 313 Iturralde-Vinent, MA 2001. Geology of the amber-bearing deposits of the greater Antilles. 314 Carribean Journal of Science 37:141–166. 315 Iturralde-Vinent MA, MacPhee RDE 1996. Age and paleogeographical origin of Dominican 316 amber. Science 273:1850–1852. 17 Kathirithamby J, Perkovsky EE, Falin ZH, Engel MS 2017. 274 Acknowledgments 275 We are grateful to Mr. Weiwei Zhang for providing pieces of amber used in the context of 276 another publication, and to Assoc. Prof. Ming Bai for arranging this loan. The authors thank 277 Ondřej Šebesta (Charles University) for his help with examination setup on the Olympus IX81 278 and Birgit Perner (Leibniz-Institut für Alternsforschung – Fritz-Lippmann-Institut, Jena) for 279 providing us access to the Axio Zoom.V16. We also thank Benjamin Naumann (Friedrich- 280 Schiller-Universität Jena) for his help with the Axio Zoom.V16. Finally, we would like to thank 281 Kenneth De Baets, Kateřina Votýpková and one anonymous reviewer for their helpful 282 suggestions, which improved the manuscript. 283 275 We are grateful to Mr. Weiwei Zhang for providing pieces of amber used in the context of 276 another publication, and to Assoc. Prof. Ming Bai for arranging this loan. The authors thank 277 Ondřej Šebesta (Charles University) for his help with examination setup on the Olympus IX81 278 and Birgit Perner (Leibniz-Institut für Alternsforschung – Fritz-Lippmann-Institut, Jena) for 279 providing us access to the Axio Zoom.V16. We also thank Benjamin Naumann (Friedrich- 280 Schiller-Universität Jena) for his help with the Axio Zoom.V16. Finally, we would like to thank 281 Kenneth De Baets, Kateřina Votýpková and one anonymous reviewer for their helpful 282 suggestions, which improved the manuscript. 283 284 References 285 Askew RR 1971. Parasitic insects. London: Heinemann Educational Books Ltd. 286 Batelka J, Prokop J, Pohl H, Bai M, Zhang W, Beutel RG accepted. Highly specialized 287 Cretaceous beetle parasitoids (Ripiphoridae) identified with optimized visualization of 288 microstructures. Systematic Entomology. 285 Askew RR 1971. Parasitic insects. London: Heinemann Educational Books Ltd. 286 Batelka J, Prokop J, Pohl H, Bai M, Zhang W, Beutel RG accepted. Highly specialized 287 Cretaceous beetle parasitoids (Ripiphoridae) identified with optimized visualization of 288 microstructures. Systematic Entomology. 286 Batelka J, Prokop J, Pohl H, Bai M, Zhang W, Beutel RG accepted. Highly specialized 287 Cretaceous beetle parasitoids (Ripiphoridae) identified with optimized visualization of 288 microstructures. Systematic Entomology. PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed A putative twis 317 Kathirithamby J, Perkovsky EE, Falin ZH, Engel MS 2017. A putative twisted-wing parasitoid 318 planidium (Insecta: Strepsiptera) in Taimyr Upper Cretaceous amber. Cretaceous Research 319 69:106–112. 318 planidium (Insecta: Strepsiptera) in Taimyr Upper Cretaceous amber. Cretaceous Research 319 69:106–112. 318 planidium (Insecta: Strepsiptera) in Taimyr Upper Cretaceous amber. Cretaceous Research 319 69:106–112. PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed 320 Kinzelbach R, Lutz H 1985. Stylopid larva from the Eocene – a spotlight on the phylogeny of the 321 stylopids (Strepsiptera). Annals of the Entomological Society of America 78:600–602. 322 Kinzelbach R, Pohl H 1994. The fossil Strepsiptera (Insecta: Strepsiptera). Annals of the 323 Entomological Society of America 87:59–70. 324 Kjer KM, Simon C, Yavorskaya M, Beutel RG 2016. Progress, pitfalls and parallel universes: a 325 history of insect phylogenetics. Journal of the Royal Society Interface 13:20160363. 326 McKenna DD, Wild AL, Kanda K, Bellamy CL, Beutel RG, Caterino MS, Farnum CW, Hawks 326 McKenna DD, Wild AL, Kanda K, Bellamy CL, Beutel RG, Caterino MS, Farnum CW, Hawks 327 DC, Ivie MA, Jameson ML, Leschen RAB, Marvaldi AE, McHugh JV, Newton AF, 328 Robertson JA, Thayer MK, Whiting MF, Lawrence JF, Ślipiński A, Maddison DR, Farrell 329 BD 2015. The beetle tree of life reveals that Coleoptera survived end‐Permian mass 330 extinction to diversify during the Cretaceous terrestrial revolution. Systematic Entomology 331 40:835–880. 332 McMahon DP, Hayward A, Kathirithamby J 2011. The first molecular phylogeny of Strepsiptera 333 (Insecta) reveals an early burst of molecular evolution correlated with the transition to 334 endoparasitism. PLoS ONE 6:e21206. Manuscript to be reviewed 335 Misof B, Liu S, Meusemann K, Peters RS, Donath A, Mayer C, Frandsen PB, Ware J, Flouri T, 336 Beutel RG, Niehuis O, Petersen M, Izquierdo-Carrasco F, Wappler T, Rust J, Aberer AJ, 337 Aspöck U, Aspöck H, Bartel D, Blanke A, Berger S, Böhm A, Buckley TR, Calcott B, Chen 338 J, Friedrich F, Fukui M, Fujita M, Greve C, Grobe P, Gu S, Huang Y, Jermiin LS, Kawahara 339 AY, Krogmann L, Kubiak M, Lanfear R, Letsch H, Li Y, Li Z, Li J, Lu H, Machida R, 340 Mashimo Y, Kapli P, McKenna DD, Meng G, Nakagaki Y, Navarrete-Heredia JL, Ott M, 341 Ou Y, Pass G, Podsiadlowski L, Pohl H, Reumont von BM, Schütte K, Sekiya K, Shimizu S, 342 Ślipiński A, Stamatakis A, Song W, Su X, Szucsich NU, Tan M, Tan X, Tang M, Tang J, 343 Timelthaler G, Tomizuka S, Trautwein M, Tong X, Uchifune T, Walzl MG, Wiegmann BM, 344 Wilbrandt J, Wipfler B, Wong TKF, Wu Q, Wu G, Xie Y, Yang S, Yang Q, Yeates DK, 345 Yoshizawa K, Zhang Q, Zhang R, Zhang W, Zhang Y, Zhao J, Zhou C, Zhou L, Ziesmann 346 T, Zou S, Li Y, Xu X, Zhang Y, Yang H, Wang J, Wang J, Kjer KM, Zhou X 2014. 347 Phylogenomics resolves the timing and pattern of insect evolution. Science 346:763–767. 348 Nagler C, Haug JT 2015. From fossil parasitoids to vectors: insects as parasites and hosts. Manuscript to be reviewed 350 Niehuis O, Hartig G, Grath S, Pohl H, Lehmann J, Tafer H, Donath A, Krauss V, Eisenhardt C, 351 Hertel J, Petersen M, Mayer C, Meusemann K, Peters RS, Stadler PF, Beutel RG, Bornberg- 352 Bauer E, McKenna DD, Misof B 2012. Genomic and morphological evidence converge to 353 resolve the enigma of Strepsiptera. Current Biology 22:1309–1313. 350 Niehuis O, Hartig G, Grath S, Pohl H, Lehmann J, Tafer H, Donath A, Krauss V, Eisenhardt C, 351 Hertel J, Petersen M, Mayer C, Meusemann K, Peters RS, Stadler PF, Beutel RG, Bornberg- 352 Bauer E, McKenna DD, Misof B 2012. Genomic and morphological evidence converge to 353 resolve the enigma of Strepsiptera. Current Biology 22:1309–1313. 354 Peters RS, Meusemann K, Petersen M, Mayer C, Wilbrandt J, Ziesmann T, Donath A, Kjer KM, 355 Aspöck U, Aspöck H, Aberer A, Stamatakis A, Friedrich F, Hünefeld F, Niehuis O, Beutel 356 RG, Misof B 2014. The evolutionary history of holometabolous insects inferred from 357 transcriptome-based phylogeny and comprehensive morphological data. BMC Evolutionary 358 Biology 14:52. 359 Pierce W 1909. A monographic revision of the twisted winged insects comprising the order 360 Strepsiptera Kirby. Bulletin of the United States National Museum 66:1–232. 361 Pohl H 2000. Die Primärlarven der Fächerflügler – evolutive Trends (Insecta, Strepsiptera). 362 Kaupia, Darmstädter Beiträge zur Naturgeschichte 10:1–144. 363 Pohl H 2002. Phylogeny of the Strepsiptera based on morphological data of the first instar 364 larvae. Zoologica Scripta 31:123–134. 365 Pohl H 2009. The oldest fossil strepsipteran larva (Insecta: Strepsiptera) from the Geisel Valley, 366 Germany (Eocene). Insect Systematics & Evolution 40:333–347. 366 Germany (Eocene). Insect Systematics & Evolution 40:333–347. 367 Pohl H, Beutel RG 2005. The phylogeny of Strepsiptera (Hexapoda). Cladistics 21:328–374. 368 Pohl H, Beutel RG 2008. The evolution of Strepsiptera (Hexapoda). Zoology 111:318–338. 369 Pohl H, Beutel RG 2013. The Strepsiptera-Odyssey: the history of the systematic placement of 370 an enigmatic parasitic insect order. Entomologia 1:e4. 369 Pohl H, Beutel RG 2013. The Strepsiptera Odyssey: the history of the systematic placement of 370 an enigmatic parasitic insect order. Entomologia 1:e4. 371 Pohl H, Beutel RG 2016. †Kinzelbachilla ellenbergeri – a new ancestral species, genus and 372 family of Strepsiptera (Insecta). Systematic Entomology 41:287–297. 373 Pohl H, Beutel RG, Kinzelbach R 2005. Protoxenidae fam. nov (Insecta, Strepsiptera) from 374 Baltic amber – a “missing link” in strepsipteran phylogeny. Zoologica Scripta 34:57–69. 375 Poinar G 2004. Manuscript to be reviewed d l 335 Misof B, Liu S, Meusemann K, Peters RS, Donath A, Mayer C, Frandsen PB, Ware J, Flouri T, 336 Beutel RG, Niehuis O, Petersen M, Izquierdo-Carrasco F, Wappler T, Rust J, Aberer AJ, 337 Aspöck U, Aspöck H, Bartel D, Blanke A, Berger S, Böhm A, Buckley TR, Calcott B, Chen 338 J, Friedrich F, Fukui M, Fujita M, Greve C, Grobe P, Gu S, Huang Y, Jermiin LS, Kawahara 339 AY, Krogmann L, Kubiak M, Lanfear R, Letsch H, Li Y, Li Z, Li J, Lu H, Machida R, 340 Mashimo Y, Kapli P, McKenna DD, Meng G, Nakagaki Y, Navarrete-Heredia JL, Ott M, 341 Ou Y, Pass G, Podsiadlowski L, Pohl H, Reumont von BM, Schütte K, Sekiya K, Shimizu S, 342 Ślipiński A, Stamatakis A, Song W, Su X, Szucsich NU, Tan M, Tan X, Tang M, Tang J, 343 Timelthaler G, Tomizuka S, Trautwein M, Tong X, Uchifune T, Walzl MG, Wiegmann BM, 344 Wilbrandt J, Wipfler B, Wong TKF, Wu Q, Wu G, Xie Y, Yang S, Yang Q, Yeates DK, 345 Yoshizawa K, Zhang Q, Zhang R, Zhang W, Zhang Y, Zhao J, Zhou C, Zhou L, Ziesmann 346 T, Zou S, Li Y, Xu X, Zhang Y, Yang H, Wang J, Wang J, Kjer KM, Zhou X 2014. 347 Phylogenomics resolves the timing and pattern of insect evolution. Science 346:763–767. 348 N l C H JT 2015 F f il i id i i d h 335 Misof B, Liu S, Meusemann K, Peters RS, Donath A, Mayer C, Frandsen PB, Ware J, Flouri T, 36 Beutel RG, Niehuis O, Petersen M, Izquierdo-Carrasco F, Wappler T, PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed Evidence of parasitism by Strepsiptera in Dominican amber. Biocontrol 49:239– 376 244. 377 Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, 378 Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, 379 Tomancak P, Cardona A 2012. Fiji: an open-source platform for biological-image analysis. 380 Nature Methods 9:676–682. PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed 381 Shi G, Grimaldi DA, Harlow GE, Wang J, Wang J, Yang M, Lei W, Li Q, Li X 2012. Age 382 constraint on Burmese amber based on U–Pb dating of zircons. Cretaceous Research 383 37:155–163. 384 Silvestri F 1941. Studi sugli “Strepsiptera” (lnsecta). I. Ridescrizione e ciclo dell’Eoxenos 385 Laboulbenei Peyerimhoff. Bolletino del Laboratorio di Zoologia generale e agraria della 386 Facoltà Agraria in Portici 31:311–341. 387 Stehr F 1991. Immature insects. Michigan: Kendall/Hunt. 387 Stehr F 1991. Immature insects. Michigan: Kendall/Hunt. 88 Toussaint EFA, Seidel M, Arriaga-Varela E, Hájek J, Král D, Sekerka L, Sho 388 Toussaint EFA, Seidel M, Arriaga-Varela E, Hájek J, Král D, Sekerka L, Short AEZ, Fikáček M 389 2017. The peril of dating beetles. Systematic Entomology 42:1–10. 390 Voigt E 1938. Weichteile an fossilen Insekten aus der eozänen Braunkohle des Geiseltales bei 391 Halle (Saale). Nova Acta Leopoldina, Neue Folge 6:1–38. g 391 Halle (Saale). Nova Acta Leopoldina, Neue Folge 6:1–38. 393 PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed 394 Figure 1 Strepsiptera primary larva in Burmese amber, ventral view. A. Photomicrograph with 395 an Axio Zoom.V16 with a PlanNeoFluar Z 1.0x. B. Drawing based on photomicrographs with an 396 Olympus IX81 inverted fluorescence microscope with UIS2 objective. Abbreviations: af = 397 antennal field, cb = caudal seta, cx = coxa, fe = femur, fs = frontal seta, lcb = lateral caudal seta, 398 mp = maxillary palp, mssp = mesosternal plate, mt = metanotum, mx = maxilla, prsp = prosternal 399 plate, sbsIX/X = segmental border between abdominal sternites IX/X, sbtVIII/IX = segmental 400 border between abdominal tergites VIII/IX, sI–sIX = abdominal sternites I–XI, st = stemmata, ta 401 = tarsus, te = tentorium, ti = tibia, X = abdominal segment X, XI = abdominal segment XI. 402 403 Figure 2 A, B. Strepsiptera primary larva in Burmese amber, head, pro-, and mesothorax, ventral 404 view. C, D. Eoxenos laboulbenei, head, pro-, and mesothorax, ventral view. Photomicrographs 405 with an Olympus IX81 inverted fluorescence microscope with UIS2 objective. Abbreviations: af 406 = antennal field, cos = coxal seta, lb = labium, mp = maxillary palp, mssp = mesosternal plate, 407 mx = maxilla, prsp = prosternal plate, st = stemmata, te = tentorium, ti = tibia. 408 408 409 Figure 3 A, B. Strepsiptera primary larva in Burmese amber. A. Meso-, metathorax, and anterior 410 abdominal segments, ventral view. B. Terminal abdominal segments. C, D. Eoxenos laboulbenei. 411 C. Abdominal segments III–IX, ventral view. D. Terminal abdominal segments. 412 Photomicrographs with an Olympus IX81 inverted fluorescence microscope with UIS2 413 objective. Abbreviations: cb = caudal bristle, lcb = lateral caudal bristle, sbsIX/X = segmental 414 border between sternite IX and X, sbsX/XI = segmental border between sternite X and XI, 415 sbtVIII/IX = segmental border between tergite VIII and IX, ti = tibia, X = abdominal segment X, 416 XI = abdominal segment XI. 417 418 Figure 4 Terminal segments of Strepsiptera primary larva in Burmese amber, drawings based on 419 photomicrographs with an Olympus IX81 inverted fluorescence microscope with UIS2 objective. 420 A. Dorsal view. B. Ventral view. Abbreviations: sbsIX/X = segmental border between abdominal 421 sternites IX/X, sbtVIII/IX = segmental border between abdominal tergites VIII/IX, X = 422 abdominal segment X, XI = abdominal segment XI. 423 409 Figure 3 A, B. Strepsiptera primary larva in Burmese amber. A. Manuscript to be reviewed Meso-, metathorax, and anterior 410 abdominal segments, ventral view. B. Terminal abdominal segments. C, D. Eoxenos laboulbenei. 411 C. Abdominal segments III–IX, ventral view. D. Terminal abdominal segments. 412 Photomicrographs with an Olympus IX81 inverted fluorescence microscope with UIS2 413 objective. Abbreviations: cb = caudal bristle, lcb = lateral caudal bristle, sbsIX/X = segmental 414 border between sternite IX and X, sbsX/XI = segmental border between sternite X and XI, 415 sbtVIII/IX = segmental border between tergite VIII and IX, ti = tibia, X = abdominal segment X, 416 XI = abdominal segment XI. 409 Figure 3 A, B. Strepsiptera primary larva in Burmese amber. A. Meso-, metathorax, and anterior 410 abdominal segments, ventral view. B. Terminal abdominal segments. C, D. Eoxenos laboulbenei. 411 C. Abdominal segments III–IX, ventral view. D. Terminal abdominal segments. 412 Photomicrographs with an Olympus IX81 inverted fluorescence microscope with UIS2 413 objective. Abbreviations: cb = caudal bristle, lcb = lateral caudal bristle, sbsIX/X = segmental 414 border between sternite IX and X, sbsX/XI = segmental border between sternite X and XI, 415 sbtVIII/IX = segmental border between tergite VIII and IX, ti = tibia, X = abdominal segment X, 416 XI = abdominal segment XI. PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed 424 Figure 5 Primary larva of Eoxenos laboulbenei, drawings based on scanning electron 425 micrographs (modified from Pohl, 2000). A. Ventral view. B. Dorsal view. Abbreviations: af = 426 antennal field, cb = caudal bristle, cx = coxa, fe = femur, fs = frontal seta, lb = labium, lcb = 427 lateral caudal bristle, mp = maxillary palp, mx = maxilla, prsp = prosternal plate, sI–sXI = 428 abdominal sternite I–XI, st = stemma, ta = tarsus, ti = tibia, tI–tX = abdominal tergite I–X. PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed Figure 1 Strepsiptera primary larva in Burmese amber, ventral view. Strepsiptera primary larva in Burmese amber, ventral view. A. Photomicrograph with an Axio Zoom.V16 with a PlanNeoFluar Z 1.0x. B. Drawing based on photomicrographs with an Olympus IX81 inverted fluorescence microscope with UIS2 objective. Abbreviations: af = antennal field, cb = caudal seta, cx = coxa, fe = femur, fs = frontal seta, lcb = lateral caudal seta, mp = maxillary palp, mssp = mesosternal plate, mt = metanotum, mx = maxilla, prsp = prosternal plate, sbsIX/X = segmental border between abdominal sternites IX/X, sbtVIII/IX = segmental border between abdominal tergites VIII/IX, sI–sIX = abdominal sternites I–XI, st = stemmata, ta = tarsus, te = tentorium, ti = tibia, X = abdominal segment X, XI = abdominal segment XI. PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed Strepsiptera primary larva in Burmese amber in comparison with an extant primary larva Strepsiptera primary larva in Burmese amber in comparison with an extant primary larva. A, B. Strepsiptera primary larva in Burmese amber, head, pro-, and mesothorax, ventral view. C, D. Eoxenos laboulbenei, head, pro-, and mesothorax, ventral view. Photomicrographs with an Olympus IX81 inverted fluorescence microscope with UIS2 objective. Abbreviations: af = antennal field, cos = coxal seta, lb = labium, mp = maxillary palp, mssp = mesosternal plate, mx = maxilla, prsp = prosternal plate, st = stemmata, te = tentorium, ti = tibia. Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed Figure 3 Strepsiptera primary larva in Burmese amber in comparison with an extant primary larva. A, B. Strepsiptera primary larva in Burmese amber. A. Meso-, metathorax, and anterior abdominal segments, ventral view. B. Terminal abdominal segments. C, D. Eoxenos laboulbenei. C. Abdominal segments III–IX, ventral view. D. Terminal abdominal segments. Photomicrographs with an Olympus IX81 inverted fluorescence microscope with UIS2 objective. Abbreviations: cb = caudal bristle, lcb = lateral caudal bristle, sbsIX/X = segmental border between sternite IX and X, sbsX/XI = segmental border between sternite X and XI, sbtVIII/IX = segmental border between tergite VIII and IX, ti = tibia, X = abdominal segment X, XI = abdominal segment XI. PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed Figure 4 Terminal segments of Strepsiptera primary larva in Burmese amber, drawings based on photomicrographs with an Olympus IX81 inverted fluorescence microscope with UIS2 objective. A. Dorsal view. B. Ventral view. Abbreviations: sbsIX/X = segmental border between abdominal sternites IX/X, sbtVIII/IX = segmental border between abdominal tergites VIII/IX, X = abdominal segment X, XI = abdominal segment XI. PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed Manuscript to be reviewed Figure 5 Primary larva of Eoxenos laboulbenei, drawings based on scanning electron micrographs (modified from Pohl, 2000). A. Ventral view. B. Dorsal view. Abbreviations: af = antennal field, cb = caudal bristle, cx = coxa, fe = femur, fs = frontal seta, lb = labium, lcb = lateral caudal bristle, mp = maxillary palp, mx = maxilla, prsp = prosternal plate, sI–sXI = abdominal sternite I–XI, st = stemma, ta = tarsus, ti = tibia, tI–tX = abdominal tergite I–X. PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018) Manuscript to be reviewed PeerJ reviewing PDF \| (2018:07:30075:2:0:NEW 6 Oct 2018)
W1968520702.txt	null	fr		Polyarthrite chronique destructrice révélant une polychondrite atrophiante	The Pan African medical journal	2,014	cc-by	425	Open Access Images in medicine Polyarthrite chronique destructrice révélant une polychondrite atrophiante Faten Frikha 1,&, Zouhir Bahloul1 1 Service de Médecine Interne, CHU Hédi Chaker, 3029 Sfax, Tunisie & Corresponding author: Faten Frikha, Service de Médecine Interne, CHU Hédi Chaker, 3029 Sfax, Key words: Polyarthrite chronique, polychondrite atrophiante Received: 09/11/2013 - Accepted: 12/11/2013 - Published: 26/01/2014 Pan African Medical Journal. 2014; 17:59 doi:10.11604/pamj.2014.17.59.3574 This article is available online at: http://www.panafrican-med-journal.com/content/article/17/59/full © Faten Frikha et al. The Pan African Medical Journal - ISSN 1937-8688. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Image en medicine La polychondrite atrophiante (PCA) est une affection rare caractérisée par une atteinte inflammatoire des cartilages avec une prédilection pour ceux du nez, du pavillon des oreilles et de l'arbre laryngotrachéobronchique. Des manifestations extracartilagineuses variées (rhumatologiques, oculaires, dermatologiques cardiovasculaires, audiovestibulaires ou rénales) sont possibles et parfois révélatrices. Patiente de 53 ans hospitalisée pour une polyarthrite déformante et destructrice et des épisodes d'ischémie digitale. A l'interrogatoire, on trouvait la notion de bourdonnement des oreilles avec diminution de l'acuité auditive. L'examen ostéoarticulaire révélait une atteinte déformante des mains et des pieds (A). L'examen clinique objectivait un effondrement de la pyramide nasale avec une destruction complète de la cloison cartilagineuse (B). La voix était dysphonique et il existait une surdité de perception bilatérale. A la biologie, il y avait un syndrome inflammatoire biologique avec une VS: 125 mm 1ère heure et une CRP à 49 mg/l. Le facteur rhumatoïde, les anticorps anti-CCP ainsi que les ANCA étaient négatifs. La radiographie des mains montrait une atteinte destructrice des MCP, des IPP et des IPD bilatérale et les images d'acro-ostéolyse de certaines phalanges (C). Le scanner du massif facial objectivait une Ostéolyse de la cloison nasale, un Comblement bilatéral du sinus maxillaire et des cellules ethmoïdales antérieures (D). Le diagnostic de PCA avec arthropathie destructrice était retenu. Un traitement par corticothérapie était instauré. L'évolution était partiellement favorable. Figure 1 : A)atteinte déformante des mains et présence de lésions ulcéro-nécrotiques de la main gauche; B)Effondrement de la pyramide nasale; C)radiographie des mains : une atteinte destructrice et des images d?acro-ostéolyse de certaines phalanges des doigts; D)Scanner du massif facial : Ostéolyse de la cloison nasale. Comblement bilatéral du sinus maxillaire et des cellules ethmoïdales antérieures Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Page number not for citation purposes 1
https://openalex.org/W3132748960	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0246511&type=printable	English	null	Searching for biological feedstock material: 3D printing of wood particles from house borer and drywood termite frass	PloS one	2,021	cc-by	5,931	PLOS ONE RESEARCH ARTICLE Searching for biological feedstock material: 3D printing of wood particles from house borer and drywood termite frass Rudy Plarre1, Andrea Zocca1, Andrea Spitzer1, Sigrid Benemann1, Anna A. Gorbushina1,2, Yuexuan Li1,3, Anja Waske1, Alexander Funk1, Janka Wilbig1, Jens Gu¨nsterID1,4* Rudy Plarre1, Andrea Zocca1, Andrea Spitzer1, Sigrid Benemann1, Anna A. Gorbushina1,2, Yuexuan Li1,3, Anja Waske1, Alexander Funk1, Janka Wilbig1, Jens Gu¨nsterID1,4* 1 Bundesanstalt fu¨r Materialforschung und -pru¨fung BAM, Berlin, Germany, 2 Institute of Geological Sciences, Freie Universita¨t Berlin, Berlin Germany, 3 State Key Laboratory for Manufacturing Systems Engineering, School of Mechanical Engineering, Xi’an Jiaotong University, Xi’an, Shaanxi, China, 4 Institute of Non-Metallic Materials, TU Clausthal, Clausthal-Zellerfeld, Germany a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * jens.guenster@bam.de * jens.guenster@bam.de OPEN ACCESS Citation: Plarre R, Zocca A, Spitzer A, Benemann S, Gorbushina AA, Li Y, et al. (2021) Searching for biological feedstock material: 3D printing of wood particles from house borer and drywood termite frass. PLoS ONE 16(2): e0246511. https://doi.org/ 10.1371/journal.pone.0246511 Editor: Amitava Mukherjee, VIT University, INDIA Received: October 9, 2020 Accepted: January 20, 2021 Published: February 19, 2021 Editor: Amitava Mukherjee, VIT University, INDIA Received: October 9, 2020 Accepted: January 20, 2021 Published: February 19, 2021 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0246511 Abstract Frass (fine powdery refuse or fragile perforated wood produced by the activity of boring insects) of larvae of the European house borer (EHB) and of drywood termites was tested as a natural and novel feedstock for 3D-printing of wood-based materials. Small particles produced by the drywood termite Incisitermes marginipennis and the EHB Hylotrupes baju- lus during feeding in construction timber, were used. Frass is a powdery material of particu- larly consistent quality that is essentially biologically processed wood mixed with debris of wood and faeces. The filigree-like particles flow easily permitting the build-up of wood- based structures in a layer wise fashion using the Binder Jetting printing process. The qual- ity of powders produced by different insect species was compared along with the processing steps and properties of the printed parts. Drywood termite frass with a Hausner Ratio HR = 1.1 with ρBulk = 0.67 g/cm3 and ρTap = 0.74 g/cm3 was perfectly suited to deposition of uni- formly packed layers in 3D printing. We suggest that a variety of naturally available feed- stocks could be used in environmentally responsible approaches to scientific material sciences/additive manufacturing. PLOS ONE PLOS ONE Additive manufacturing Adding material to form an object instead of subtracting material from an excessively large block is a new trend in manufacturing technologies which is currently stimulating an entire industry [2]. In the majority of additive manufacturing processes, the material is added layer by layer. The raw material (feedstock) is fed into the process as a powder/granulate, paste or suspension, as it is in a state optimized for the layer deposition process. In the manufacturing process itself, the feedstock is used to build up the desired object and it is simultaneously trans- ferred into a state possessing its final physical properties, or at least providing enough mechan- ical strength to transfer the configured object to further processing steps. Adding instead of subtracting material implies more than just flexibility in design. Multi material processing, the generation of unique properties and functionalities and functionally graded materials are just some facets intrinsic to AM. One of the most popular and widespread additive manufacturing technologies, the “Binder Jetting” (BJ), is making use of a powdery material as feedstock [3, 4], see also Fig 1. A layer of powdered material is first spread as a layer and subsequently the corre- sponding layer information of the object manufactured is selectively inscribed by a printing head, spraying individual droplets of a binding liquid onto the powder layer, thus selectively consolidating the powder and defining the cross-section of the object in a respective layer. The popularity of BJ is based on the fact, that it virtually can accept all powdery materials which provide sufficient flowability to be spread as a homogeneous thin layer. This flexibility has stimulated the creativity of many research groups envisioning the use of abundant materi- als, such as sand [5–7], or recyclable material in powdery form for upgrading waste for the manufacture of new products [8]. Costs and resources for the initial material synthesis can be saved. However, the refinement of powdery raw materials to powders well suited for BJ remains a mandatory processing step if the recycled material is not directly obtained in the appropriate powdery state [9]. Here a strategy of coupling the development of the most promising AM approaches to con- siderations of the use of natural or Nature-recycled materials as feedstocks was applied. Introduction Copyright: © 2021 Plarre et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Anthropogenic perturbations of natural ecosystems are omnipresent: materials and products of human activity are superimposed on natural cycles everywhere. According to Schellnhuber 1999 [1] there are two main components: the ecosphere N and the human factor H. N consists of intricate linkages between the atmosphere, hydrosphere, cryosphere, lithosphere, biosphere, etc, while the human factor H aggregates all actions and products along with a metaphysical component of human activity. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Sustainable coevolution of the ecosphere and the anthroposphere requires fresh scientific attitudes and approaches, including completely new ways of manufacturing. The ever-increas- ing human impact on the planet requires the deliberate coupling of natural feedstocks to novel Funding: The author(s) received no specific funding for this work. PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 1 / 12 PLOS ONE 3D printing of wood particles from house borer and drywood termite frass Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. manufacturing process. This way the life cycle of the products and materials can be determined early in production. Substituting dedicated feedstocks for additive manufacturing (AM) with surplus natural materials is one way to substantially increase AM sustainability while concomi- tantly providing high-value outputs for “pre-owned” materials/products. As a general rule for all production processes, natural and recycled feedstocks should take preference to dedicated ones–especially in the context of a circular economy. Deliberately developing naturally available feedstock constitutes an environmentally responsible scientific approach in material sciences. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 Additive manufacturing In the present work the powdery material which remains when house borer larvae or drywood ter- mites feed on wood, the so called frass, is used as a novel feedstock. 3D printing of wood chops [10–14] or plastic-wood composites has already been proven to be a feasible way for obtaining objects with wooden haptics. Dedicated feedstocks have been processed from wood and have been adapted to the respective printing process by refining it with polymeric additives [15–18]. In terms of sustainability, 3D printing of wood-based materials from house borer and termite frass is going one step further as it is not only using naturally occurring but also naturally pro- cessed materials directly as feedstocks. In the BJ process, the binder system, which is used to consolidate the powdery material to form an object, has to fulfill multiple requirements. As a liquid, it has to be of an appropriate viscosity and surface tension to be dosed by a commercial printer head [4, 19, 20]. In order to penetrate the deposited layer, it must also moisten the powdery feedstock. Moreover, it ideally PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 2 / 12 PLOS ONE 3D printing of wood particles from house borer and drywood termite frass Fig 1. Schematic of the Binder Jetting (BJ) process. https://doi org/10 1371/journal pone 0246511 g001 Fig 1. Schematic of the Binder Jetting (BJ) process. https://doi.org/10.1371/journal.pone.0246511.g001 https://doi.org/10.1371/journal.pone.0246511.g001 interacts with the powdery material to form a strong interparticle adhesion. In most cases, addition of a binder does not result in a significant densification of the powder. Hence, BJ is typically providing porous parts requiring a post treatment for densification [21, 22]. interacts with the powdery material to form a strong interparticle adhesion. In most cases, addition of a binder does not result in a significant densification of the powder. Hence, BJ is typically providing porous parts requiring a post treatment for densification [21, 22]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 Feedstocks from timber A major interest exists in 3D printing for ecofriendly and recycled materials, particularly in Binder Jetting [11–16, 23, 24]. BJ can utilize basically anything that can be powdered to an appropriate particle size. The particle size of the powders is essential for obtaining sufficient flowability for the deposition of defect-free layers: In case of too fine powders, the flowability will be poor, in case of too coarse-grained powders the definition of the part will be imprecise [25]. Wood particles may be obtained as byproducts from wood machining, such as saw dust, or are deliberately processed from wood. For obtaining suitable feedstocks for 3D printing, in most published works the wood particles are mixed into a polymer or mixed with other bind- ing phases [15, 17, 26–29]. On the other hand, timber can also be naturally processed into printable powders by e. g. insects feeding on wood. We have used small particles from feeding byproducts of the European house borer Hylotrupes bajulus and drywood termite Incisitermes marginipennis as raw material for 3D-printing. Insects in construction timber share several anatomic and physiological features which make them to appear perfectly adapted to this environment. Wood is an inhomogeneous pori- ferous matrix containing mainly cellulose and lignin. Their relative amounts vary between 3 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 PLOS ONE 3D printing of wood particles from house borer and drywood termite frass heartwood and sapwood as well as in the early and late wood of the annual rings. This results in local strength differences and an uneven distribution of essential nutrients for the insects. The larvae of wood boring beetles or drywood termites have strong mandibles which allow abrasion of all parts. The cellulose is the main hydrocarbon source usually digested with the aid of cellulase-producing microorganisms. However, the rare nitrogen containing elements of wood are the limiting factors. In order to access as much nitrogen as possible much more wood is consumed by wood feeding insects than actually needed for development. Larvae of H. bajulus, e. g. excavate throughout the sapwood leaving extensive tunnels filled with frass. The frass contains either loosely chopped off wood particle (debris) or dense-packed faeces. The latter is of cylindrical shape made up out of himidigested cellulose/lignin conglomerates. Destructions of insects feeding on build-in timber can cause severe danger and precautions are needed. Feedstocks from timber Laboratories like BAM therefore rear large pest populations to test different con- trol strategies for efficacy evaluation in pest control. The rearing byproducts like the frass were of no further use and discarded. However, after being modified by the insect digestive system the former non-uniform wooden material is changed to a homogeneous compact cellulose-lig- nin mixture and becomes suitable for further technical applications, such as 3D printing, with- out any further processing. In the present study we have evaluated, the so called frass, as feedstocks for 3D printing. The morphology of the drywood termite frass is quite different to house borer frass. While drywood termite frass appears as six-sided pellets almost uniform in size the frass from house borer is sawdust-like and more irregularly shaped. In contrary to dry- wood termites, the house borer larvae digest only part of the abraded wood with their frass containing loosely chopped off debris as well as more dense-packed faeces. Materials and methods Larvae of European house borer (EHB) were reared at constant conditions of 28 ± 2˚C and 75 ± 5% r.h. During the first days after hatching from the eggs, larvae were manually inserted into pine, Pinus sylvestris, sapwood blocks (1.5 x 2.5 x 5 cm3) enriched with peptone and yeast. This enrichment with nutrients was carried out by impregnating the sapwood with an aqueous solution of 1% peptone and 0.3% yeast at low pressure of 100 to 200 mbar for 30 minutes to speed up development. Two larvae per wood block were allowed to feed for approximately six months before being individually transferred into pure pine sapwood blocks (3 x 4 x 5.5 cm3), not enriched with any nutrients. While feeding in the wood, the larvae produce debris and fae- ces which are left behind in the frass tunnels. Debris is undigested wooden material derived from abrasion processes when the larvae’s mandibles carve on the wood. It usually bypasses the larvae during movement through the wood. While debris are of undefined structure, the faeces are densely packed into cylindrical pellets when leaving the larva’s hindgut. As the wood is increasingly consumed by the larvae, frass (debris and faeces) eventually trickle out and can be collected in larger amounts. Using a vibratory sieve shaker (Analysettre 3 spartan, Fritsch, 55743 Idar-Oberstein, Ger- many) EHB frass was sieved for 30 min at 1 mm amplitude. A fraction with particle size distri- bution of 45 μm– 100 μm, which amounts to 17% of the total frass, 57% above 100 μm and 26% below 45 μm, was then used for 3D-printing, see also Fig 2. The frass particles show a con- siderable flowability although the particle shape is rather flake-like than spherical. The Haus- ner ratio (HR), as a measure of flowability [29], was determined according to the relation shown in Eq 1: rTap=rBulk ¼ HR ð1Þ ð1Þ where HR is the Hausner ratio, ρBulk displays the freely settled bulk density of the powder and here HR is the Hausner ratio, ρBulk displays the freely settled bulk density of the powder and where HR is the Hausner ratio, ρBulk displays the freely settled bulk density of the powder and PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 4 / 12 PLOS ONE 3D printing of wood particles from house borer and drywood termite frass Fig 2. Materials and methods a) European house borer (Hylotrupes bajulus) full grown larva (top) and adult beetle (bottom); b) the sieved frass (in a particle size fraction of 45 to 100 μm) produced by larvae and used for 3D-printing. https://doi org/10 1371/journal pone 0246511 g002 rupes bajulus) full grown larva (top) and adult beetle (bottom); b) the sieved frass (in a particle size fraction of 45 to 100 μm) inting Fig 2. a) European house borer (Hylotrupes bajulus) full grown larva (top) and adult beetle (bottom); b) the sieved frass (in a particle size fraction of 45 to 100 μm) produced by larvae and used for 3D-printing. https://doi.org/10.1371/journal.pone.0246511.g002 https://doi.org/10.1371/journal.pone.0246511.g002 ρTap the bulk density after a given number of tapping cycles, at which the bulk density is in a plateau, in g/cm3. The tapped density was determined according to ISO 787–11 with a STAV 2003 type equipment from J. Engelsmann AG, Germany. With ρBulk = 0,14 g/cm3 and ρTap = 0,18 g/cm3, the EHB frass particles have a Hausner ratio of 1.25 which displays a fair flowability according to the classification [30]. Within this study, frass from drywood termites has been considered as feedstock for 3D printing, as well, see also Fig 3. Termites rely on fungi, protists and bacteria that live in their Fig 3. a) Drywood termites (Incisitermes marginipennis), soldier and worker with frass; b) SEM micrograph of single pellets of drywood termite frass. https://doi org/10 1371/journal pone 0246511 g003 Fig 3. a) Drywood termites (Incisitermes marginipennis), soldier and worker with frass; b) SEM micrograph of single pellets of drywood termite frass. Fig 3. a) Drywood termites (Incisitermes marginipennis), soldier and worker with frass; b) SEM micrograph of single pellets of drywood termite frass. https://doi org/10 1371/journal pone 0246511 g003 Fig 3. a) Drywood termites (Incisitermes marginipennis), soldier and worker with frass; b) SEM micrograph of single pellets of drywood termite frass. https://doi.org/10.1371/journal.pone.0246511.g003 https://doi.org/10.1371/journal.pone.0246511.g003 5 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 PLOS ONE 3D printing of wood particles from house borer and drywood termite frass gut to break down the wood and digest lignin and cellulose. In comparison to the feeding byproducts of EHB the drywood frass contains six-sided pellets almost uniform in size and reveal an excellent flowability required for the layer wise buildup of wooden structures, see also Fig 3. The pellets are very compact and composed of fine fibers and particles. Materials and methods Their good flowability, HR = 1.1 with ρBulk = 0,67 g/cm3 and ρTap = 0.74 g/cm3, making them perfectly suited for the deposition of uniformly packed layers in 3D printing. The termite pellets have been imaged by 3D X-ray computed tomography using a commer- cial ZEISS Xradia 620 Versa X-ray microscope. An acceleration voltage of 80 kV and power of 10 W was used. The X-ray spectrum was filtered on the source side by using a device specific LE1 filter. A geometrical magnification of around 6.8x (with a source to object distance of 25 mm and object to 145 mm) and optical magnification of 0.4x results in an effective pixel size of 10 μm. Since the pellets are light and experience some degree of electrostatic repulsion, they have been fixed on adhesive tape that has been rolled up for the measurement, see also Fig 4. The 3D tomographic dataset with 801 angular object projections was reconstructed using ZEISS reconstructor software, the processing of the reconstructed data was carried out using the software package Avizo (Thermo Fisher Scientific, USA). Length, Breadth and Width are ferret diameters of a measured 3D object. The length and width are the longest and shortest ferret diameters of an object, respectively. The breadth of the object was measured to be the longest ferret diameter orthogonal to the object’s length. Fig 4 shows the 3D representation of the tomographic dataset of the termite pellets. The rendered image of the pallets demonstrates that the pellets are of uniform, approximately ellip- soidal shape and have a narrow size distribution. The facetted 6-fold symmetry of the pellets, Fig 4. Rendered X-ray tomographic dataset of the termite pellets studied, 180 pellets (left). The length, breadth and width of each particle as determined from the 3D data (right). The average length of the pellets is around 1050 μm, its breath (or width) around 580 μm, yielding an aspect ratio of 1.8. Fig 4. Rendered X-ray tomographic dataset of the termite pellets studied, 180 pellets (left). The length, breadth and width of each particle as determined from the 3D data (right). The average length of the pellets is around 1050 μm, its breath (or width) around 580 μm, yielding an aspect ratio of 1.8. Materials and methods https://doi.org/10.1371/journal.pone.0246511.g004 https://doi.org/10.1371/journal.pone.0246511.g004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 6 / 12 PLOS ONE 3D printing of wood particles from house borer and drywood termite frass seen in the SEM image of Fig 3, is less obvious to observe from the 3D rendered image. The measurements of the geometrical dimensions of each of the 180 pellets are plotted in the graph of Fig 4, also. Length and breadth/width differ significantly and are around 1050 μm and 580 μm, respectively. The similarity between width and breadth indicates a well-rounded shape. The size of the individual pellets is large for BJ. In order to adapt the printing process to the size of the pellets, layer thicknesses of minimum 600 μm have been evaluated and 800 μm was found optimal for the deposition of smooth layers. Using a commercial 3D printing machine (RX-1, Prometal RCT GmbH, Augsburg, Ger- many) specimens from European house borer and drywood termites were printed. The binder used was a water based commercial system provided by ExOne GmbH, Augsburg Germany (PM-B-SR2-02, viscosity of 10.7 mPas @ 1000s-1). Cross-linking of the binder was carried out after each layer printing in the thermal curing station of the printer. The precision of the printed geometries depend on several processing parameters (e. g. powder particle size, flow- ability, and layer thickness) as well as binder saturation. With fine standard powders, with a typical particle size < 60 μm, the printer can achieve a volumetric resolution of better than 100 μm in all three dimensions. For adapting the printer to the new powders, printing parame- ters such as binder saturation, layer thickness and curing time were varied. The binder satura- tion is a parameter useful to evaluate the amount of binder used to glue a certain quantity of powder, because it gives the ratio between the volume of binder spread out in a volumetric unit (voxel) and the free volume, not filled with powder, in the same voxel. Consequently, a saturation S of 100% corresponds to all porosity of the powder bed is filled by binder. A satura- tion of 100% was chosen for printing EHB frass and 166% for termite frass, respectively. The layer thickness which turned out to be most appropriate for a reproducible deposition of uni- form layers was 100 μm for EHB and 800 μm for termite frass. Fig 5 is showing the models of the structures printed. PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 Materials and methods The cube in Fig 5A, with dimensions of 9 mm3 and rectangular struts, was chosen to evaluate the printing accuracy especially for Fig 5. Schematic drawing, including dimensions, of the specimens printed. https://doi.org/10.1371/journal.pone.0246511.g005 Fig 5. Schematic drawing, including dimensions, of the specimens printed. https://doi.org/10.1371/journal.pone.0246511.g005 Fig 5. Schematic drawing, including dimensions, of the specimens printed. https://doi.org/10.1371/journal.pone.0246511.g005 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 7 / 12 PLOS ONE 3D printing of wood particles from house borer and drywood termite frass the fine powders from ESB. The model in Fig 5B is designed for getting an impression about the possibility to reproduce fine structural features with particles as large as 1050 μm. It con- sists of an inner frame with 2 mm open space and an outer frame with 4 mm open space. Powder-based 3D-printing by Binder Jetting (BJ) of EHB frass A cubic structure was printed according to the model from Fig 5A, see Fig 6A. The model structure is reproduced very well during the printing process except for the bottom plane. Clearly a convex instead of planar shape can be recognized. This distortion arises from an oversaturation with binder. Also, indicative for an oversaturation is the reduction in the diam- eter of the printed capillaries. Instead of the designed square shaped cross section of 1mm edge length, the edge length is reduced to approximately 500 μm. The dimensional tolerance of printed parts associated to the printing parameters chosen is in the range of 200 μm. Due to the low packing density of the ESB frass, parts obtained from 3D printing have shown low mechanical strength and were considered as not useful in the as-printed state. On the other hand, they show considerably well detail quality and could serve as preforms for fur- ther processing steps, such as infiltration, giving them more mechanical strength. The frass particle were hardly compacted during layer deposition. Addition of binder did not result in a significant densification. Fig 6B shows details of the fluffy structure of the as-printed part. PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 Powder-based 3D-printing by Binder Jetting (BJ) of drywood termite frass From Fig 4 an average 580 μm breadth/width and 1050 μm length of the particles is deduced. This means that the approximately spheroidal particles have an aspect ratio of around 1.8. Overall, the 3D analysis reveals that termite pellets have a narrow size distribution and ellipsoi- dal shape. Accordingly, frass from dry wood termites reveals an excellent flowability and, thus, relatively high packing densities could be obtained during layer deposition. The approximately four times higher packing density, as compared to the EHB frass, appears promising for even load bearing applications of printed parts, without further processing. One general problem with compacted porous particles, i.e., the frass pellets, however, exists in the BJ process: Driven by capillary forces, the binder is drawn into the fine network of pores within the individual pellets. Primarily, all capillaries within the pellets must be filled before a sufficient amount of binder remains available for gluing the pellets to each other effectively. In the competition among capillaries with smaller diameters within individual pellets and Fig 6. Images of the manufactured specimens from European house borer (EHB) frass. a) Photograph of a printed cubic sample. b) Light microscope image of the sample from a), showing the wooden chops and macroscopic channels. https://doi.org/10.1371/journal.pone.0246511.g006 Fig 6. Images of the manufactured specimens from European house borer (EHB) frass. a) Photograph of a printed cubic sample. b) Light microscope image of the sample from a), showing the wooden chops and macroscopic channels. https://doi.org/10.1371/journal.pone.0246511.g006 8 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 PLOS ONE 3D printing of wood particles from house borer and drywood termite frass capillaries with larger diameters between individual pellets, the smaller diameters are provid- ing a stronger drag force for the binder uptake. Hence, a lot of binder is consumed before excessive binder is available for interparticle gluing. Therefore, in the printing process a high binder saturation of the powder bed is required for consolidating a structure while an excessive amount of binder stays in the pellets. Upon binder uptake of the frass particles, no swelling and loss of structural integrity could be observed. In order to compensate the binder uptake of the single pellets a binder saturation of 166% was chosen. It was found difficult to deposit lay- ers on top of the first printed cross sections, due to adhesion of the binder saturated pellets to the recoater. Powder-based 3D-printing by Binder Jetting (BJ) of drywood termite frass This problem could be solved by applying a gas flow throughout the powder bed, providing an additional force stabilizing already deposited material. This technology has been introduced recently for the deposition of powders with poor flowability [31]. Fig 7 shows parts printed from termite frass according to the models shown in Fig 5. In order to get an impression about the possibility to reproduce fine structural features in BJ with particles as large as 1050 μm, the model structure from Fig 5B has been printed, see Fig 7C. It can be seen from Fig 7C that the inner square frame of the model with ca. 2 mm open space is not well reproduced, while the outer, with ca. 4 mm open space, is reasonably well resolved. Accordingly, the dimensional tolerance of printed parts is in the range of 1000 μm. Fig 7. a) Binder jetting powder bed filled with drywood termite frass, b), c) part printed according to Fig 5B, d) printed part similar to the one shown in Fig 5A. ) d d b d f ll d h d d f b) ng powder bed filled with drywood termite frass, b), c) part printed according to Fig 5B, d) printed part similar to the one show Fig 7. a) Binder jetting powder bed filled with drywood termite frass, b), c) part printed according to Fig 5B, d) printed pa in Fig 5A Fig 7. a) Binder jetting powder bed filled with drywood termite frass, b), c) part printed according to Fig 5B, d) printed part similar to the one shown in Fig 5A. Fig 7. a) Binder jetting powder bed filled with drywood termite frass, b), c) part printed according to Fig 5B, d) printed part similar to the one shown in Fig 5A. https://doi.org/10.1371/journal.pone.0246511.g007 https://doi.org/10.1371/journal.pone.0246511.g007 9 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 PLOS ONE 3D printing of wood particles from house borer and drywood termite frass The frass pellets are approximately one order of magnitude larger than powders typically used in 3D printing. However, for printing wooden objects like furniture [16], coarse grained pow- ders are beneficial for the deposition of thicker layers and higher building rates could be achieved. Structural details are reproduced on a scale of several millimeters but definitely not below one millimeter. Conclusions Here we considered wood processed by the drywood termites Incisitermes marginipennis and the European house borer (EHB) Hylotrupes bajulus as feedstocks for 3D printing. This approach follows the general strategy of developing naturally available feedstocks as environ- mentally responsible substrates in material sciences. The quality of the powdery feedstocks, the so-called frass, provided by these insects during feeding in construction timber was very differ- ent in terms of processability in 3D printing. EHB frass reveals a flaky structure with poor packing density, whereas termite frass is consisting of pellets of almost uniform size and is packing very well. Despite of the different packing densities, both feedstocks could be spread out into thin homogeneous layers for the build-up of structures in the Binder jetting 3D print- ing process. At a size of 580 μm along the short- and 1050 μm at the long-axis, pellets of termite frass are approximately one order of magnitude larger than powders typically used in 3D printing. In printing wooden objects like furniture, coarse-grained powders allow the deposi- tion of thicker layers and higher construction rates. The fine, sawdust-like frass particles of EHB, with their smaller particle size, are well suited to the printing of filigree structures. Both feedstocks do not qualify for printing of “ready to use” structures, as their mechanical strength is low, but printed structures are useful as preforms for further processing, such as infiltration, for improving mechanical strength. With their better flowability and packing density, this especially applies for structures printed from termite frass. Author Contributions Conceptualization: Rudy Plarre, Andrea Zocca, Sigrid Benemann, Anna A. Gorbushina, Yuexuan Li, Anja Waske, Janka Wilbig, Jens Gu¨nster. Conceptualization: Rudy Plarre, Andrea Zocca, Sigrid Benemann, Anna A. Gorbushina, Yuexuan Li, Anja Waske, Janka Wilbig, Jens Gu¨nster. Data curation: Andrea Spitzer, Alexander Funk. Supporting information Supporting information S1 Fig. (JPG) S2 Fig. (JPG) S3 Fig. (JPG) S4 Fig. (JPG) S5 Fig. (PNG) S6 Fig. (JPG) S7 Fig. (TIF) S8 Fig. (JPG) PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 10 / 12 PLOS ONE 3D printing of wood particles from house borer and drywood termite frass S9 Fig. (JPG) S1 File. (XLSX) S2 File. (XLS) S3 File. (XLSX) References 11 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 PLOS ONE 3D printing of wood particles from house borer and drywood termite frass 17. Petchwattana N, Channuan W, Naknaen P, Narupai B. 3D printing filaments prepared from modified poly (lactic acid)/teak wood flour composites: an investigation on the particle size effects and silane cou- pling agent compatibilization. Journal of Physical Science. 2019; 30: 169–188. 18. Xu WY, Zhang X, Yang P, Långvik O, Wang XJ, Zhang YC, et al. Surface engineered biomimetic inks based on UV cross-linkable wood biopolymers for 3D printing. ACS Appl. Mater. Interfaces. 2019; 11: 12389–12400. https://doi.org/10.1021/acsami.9b03442 PMID: 30844234 19. Kang HR. Water-Base Ink-Jet Ink. I. Formulation. J. Imaging Sci. 1991; 351: 79–88 20. Bai Y, Wall C, Pham H, Esker A, Williams CB. Characterizing Binder–Powder Interaction in Binder Jet- ting Additive Manufacturing Via Sessile Drop Goniometry. J. Manuf. Sci. Eng. 2019; 141(1): 011005 21. Fleisher A, Zolotaryov D, Kovalevsky A, Muller-Kamskii G, Eshed E, Kazakin M, et al. Reaction bonding of silicon carbides by binder jet 3D-printing, phenolic resin binder impregnation and capillary liquid sili- con infiltration. Ceram. Int. 2019; 45: 18023–18029. 22. Zocca A, Colombo P, Gomes CM, Gu¨nster J. Additive Manufacturing of Ceramics: issues, potentialities and opportunities J. Am. Ceram. Soc. 2015; 98(7): 1983–2001. 23. Park SJ, Lee JE, Lee HB, Park J, Lee NK, Son Y, et al. 3D printing of bio-based polycarbonate and its potential applications in ecofriendly indoor manufacturing. Addit. Manuf. 2020; 31: 100974. 24. Henke K, Treml S. Wood based bulk material in 3D printing processes for applications in construction. Eur. J. Wood Prod. (2013); 71: 139–141. 25. Bai Y, Wagner G, Williams CB. Effect of Particle Size Distribution on Powder Packing and Sintering in Binder Jetting Additive Manufacturing of Metals. J. Manuf. Sci. Eng. 2017; 139: 081019. 26. Markstedt K, Håkansson K, Toriz G, Gatenholm P. Materials from trees assembled by 3D printing- Wood tissue beyond nature limits. Appl. Mater. Today. 2019; 15: 280–285. 27. Kariz M, Sernek M, Kuzman MK. Effect of humidity on 3D-printed specimens from wood-PLA filaments. Wood Res. 2018; 63: 917–922. 28. Ayrilmis N. Effect of layer thickness on surface properties of 3D printed materials produced from wood flour/PLA filament. Polym. Test. 2018; 71: 163–166. 29. Guessasma S, Belhabib S, Nouri H. Microstructure and mechanical performance of 3D printed wood- PLA/PHA using fused deposition modelling: effect of printing temperature. Polymers. 2019; 11: 1778. PLOS ONE \| https://doi.org/10.1371/journal.pone.0246511 February 19, 2021 References 1. Schellnhuber H.-J., Wenzel V. Earth System Analysis: Integrating Science for Sustainability. Berlin, Springer, 1998; IWK. 2. Gibson I, Rosen D, Stucker B. Binder Jetting. Additive manufacturing technologies Springer, New York, NY, 2015; 205–218. 3. Chavez LA, Ibave P, Wilburn B, Alexander IV D, Stewart C, Wicker R, et al. The influence of printing parameters, post-processing, and testing conditions on the properties of binder jetting additive manu- factured functional ceramics. Ceramics. 2020; 3: 65–77. 4. Ziaee M, Crane NB. Binder jetting: A review of process, materials, and methods. Addit. Manuf. 2019; 28: 781–801. 5. Upadhyay M, Sivarupan T, Mansori ME. 3D printing for rapid sand casting-A review. J. Manuf. Process. 2017; 29: 211–220. 6. Walker J, Harris E, Lynagh C, Beck A, Lonardo R, Vuksanovich B, et al. 3D printed smart molds for sand casting. Int. J. Met. 2018; 12: 785–796. 7. Almaghariz ES, Conner BP, Lenner L, Gullapalli R, Manogharan GP, Lamoncha B, et al. Int. J. Met. 2016; 10: 240–252. 8. Gu H, Bashir Z, Yang LT. The re-usability of heat-exposed poly (ethylene terephthalate) powder for laser sintering. Addit. Manuf. 2019; 28: 194–204. 9. Meteyer S, Xu X, Perry N, Zhao YF. Energy and material flow analysis of binder-jetting additive manufacturing processes. Procedia CIRP. 2014; 15: 19–25. 10. Kam D, Layani M, BarkaiMinerbi S, Orbaum D, BenHarush SA, Shoseyov O, et al. Additive Manufactur- ing of 3D Structures Composed of Wood Materials. Adv. Mater. Technol. 2019; 4: 1900158. 11. Kariz M, Sernek M, Kuzman MK. Use of wood powder and adhesive as a mixture for 3D printing. Eur. J. Wood Prod. 2016; 74: 123–126. 12. Rosenthal M, Henneberger C, Gutkes A, Bues CT. Liquid Deposition Modeling: a promising approach for 3D printing of wood. Eur. J. Wood Prod. 2018; 76: 797–799. 13. Wahab MS, Wagiman A, Ibrahim M. Development of Wood-Based Composites Material for 3D Printing. Applied Mechanics and Materials. 2013; 315: 987–991. 14. Wimmer R, Steyrer B, Woess J, Koddenberg T, Mundigler N. 3D printing and wood. Pro Ligno. 2015; 11: 144–149. 15. Ayrilmis N, Kariz M, Kwon JH, Kuzman MK. Effect of printing layer thickness on water absorption and mechanical properties of 3D-printed wood/PLA composite materials. The International Journal of Advanced Manufacturing Technology. 2019; 102: 2195–2200. 16. Pringle AM, Rudnicki M, Pearce JM. Wood Furniture Waste–Based Recycled 3-D Printing Filament. For. Prod. J. 2018; 68: 86–95. References https://doi.org/10.3390/polym11111778 PMID: 31671901 30. Hausner H. H. (1981). "Powder Characteristics and their effect on powder processing." Powder Tech- nology 30 [1] 3–8. 31. J. Gu¨nster (BAM), A. Zocca (BAM), C. Gomes (BAM), T. Mu¨hler (TU Clausthal) "Apparatus and method for stabilizing a powder bed by means of vacuum for additive manufacturing and corresponding pro- cess” EP 2900455 B1 12 / 12
https://openalex.org/W2810863695	https://internationalbreastfeedingjournal.biomedcentral.com/track/pdf/10.1186/s13006-018-0171-z	English	null	Exploring parental perceptions and knowledge regarding breastfeeding practices in Rajanpur, Punjab Province, Pakistan	International breastfeeding journal - Electronic Edition -	2,018	cc-by	10,072	Exploring parental perceptions and knowledge regarding breastfeeding practices in Rajanpur, Punjab Province, Pakistan Rubeena Zakar1, Muhammad Zakria Zakar1, Lubna Zaheer2 and Florian Fischer3* Rubeena Zakar1, Muhammad Zakria Zakar1, Lubna Zaheer2 and Florian Fischer3* Zakar et al. International Breastfeeding Journal (2018) 13:24 https://doi.org/10.1186/s13006-018-0171-z Zakar et al. International Breastfeeding Journal (2018) 13:24 https://doi.org/10.1186/s13006-018-0171-z Open Access Abstract Background: Exclusive breastfeeding is significantly associated with strong infant immunity and optimal development. The importance of breastfeeding is underestimated. Parental lack of knowledge and unhealthy practices regarding breastfeeding deprive infants of their basic right to mother’s milk. In developing countries, including Pakistan, with high child mortality and malnutrition, healthy breastfeeding practices can bring positive changes in child health status. From this perspective, the present study aims to understand parents’ knowledge, attitudes and practical encounters with breastfeeding practices and the factors that prevent them from adopting such practices. Methods: A qualitative study was carried out in both rural and urban settings in Rajanpur District of Punjab Province, Pakistan. We conducted 12 focus-group discussions (FGDs) that involved 38 mothers and 40 fathers with children aged under two years who were being breastfed. A thematic content analysis of data collected through FGDs was performed manually. The themes were both inductive and deductive in nature. Results: The study found that a majority of participants believed that the first thing given to an infant after birth should not be breast milk but honey, rose flower, or goat’s milk from the hands of an elder in the family or a religious person. No cleanliness measures were practised in this regard. The participants had misconceptions about the benefits of colostrum, which frequently prevented it being given to newborns. Participants reported many factors, such as: insufficient milk syndrome (slow growth of infants due to insufficient daily breast milk intake), a mother’s high workload, lack of social support, the influence of culturally designated advisors, and the promotion and marketing strategies of infant formula companies, that undermined exclusive breastfeeding efforts and encouraged mothers to switch to infant formula. Conclusions: Culturally acceptable and integrated public health interventions are needed to improve the breastfeeding- related health literacy and practices of parents, grandparents and communities. This will ultimately reduce the high infant mortality and malnutrition rates in Pakistan. Keywords: Exclusive breastfeeding, Parental perceptions, Breastfeeding knowledge Keywords: Exclusive breastfeeding, Parental perceptions, Breastfeeding knowledge * Correspondence: f.fischer@uni-bielefeld.de 3School of Public Health, Department of Public Health Medicine, Bielefeld University, Bielefeld, Germany Full list of author information is available at the end of the article * Correspondence: f.fischer@uni-bielefeld.de 3School of Public Health, Department of Public Health Medicine, Bielefeld University, Bielefeld, Germany Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background health, these studies have not explored the phenomenon in detail and merely provide numerical associations of one variable with another [11, 20–22]. In contrast, very few qualitative studies have been conducted to analyse the knowledge and behaviour of parents around breastfeeding practices and its benefits for the mother and child, particu- larly within the context of Pakistan [23, 24]. This study aims to explore the knowledge, attitudes and practical en- counters of parents (both mothers and fathers) concerning exclusive breastfeeding and colostrum and their overall breastfeeding practices, and the factors that prevent them from adopting such practices. We attempted to under- stand the influence of cultural and spiritual beliefs on these practices, and the benefits of breastfeeding for both infant and mother. Thus, through understanding the perspective of parents and healthcare professionals – specifically pub- lic health experts and healthcare managers – we can create awareness about optimal breastfeeding practices through campaigns and other health-promotion avenues, in a man- ner geared specifically towards parents; possibly saving lives and raising health standards. g Breastfeeding behaviours depend upon the cultural prac- tices and perceptions that guide the actions of mothers in making decisions about the duration and frequency of breastfeeding [1, 2]. Empirical evidence demonstrates that exclusive breastfeeding for the first six months after birth contributes significantly to strong infant immunity and op- timal development [1, 2]. Timely initiation of breastfeeding plays an important role in preventing infections and infant mortality [3, 4]. Breast milk is not only a primary source of nutrition for newborns but also functions as an anti-infective agent and protects breastfed infants from acute respiratory infections and diarrhoea [5]. Additionally, breastfeeding is beneficial for the mother. It is associated with elevated emotional attachment of the mother to the infant [6], reduced risk of breast and endometrial cancer [7] and increased duration of postpartum amenorrhea and consequent birth spacing [8], as well as several other health benefits such as lower risk of osteoporosis [9]. The World Health Organization (WHO) and the United Nations Children’s Fund (UNICEF) recommend the initi- ation of breastfeeding within the first hour after birth and exclusive breastfeeding for the first six months, especially in resource-deprived countries [10]. The WHO also advo- cates that breastfeeding should be continued along with complementary foods up to two years of age or beyond [10]. © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 12 Page 2 of 12 Zakar et al. International Breastfeeding Journal (2018) 13:24 Background However, unhealthy breastfeeding practices are com- mon in developing countries like Pakistan, where child mortality and malnutrition rates are high, and infections are widespread due to unhygienic conditions [11, 12]. The World Breastfeeding Trends Initiatives report highlights that, in Pakistan, 22% of neonatal deaths could be pre- vented if newborns were breastfed within an hour of birth [13]. Nevertheless, due to cultural stereotypes and false beliefs, the importance of breastfeeding is underestimated, and infants remain deprived of their basic right to breast milk, leaving them vulnerable to infections and, thus, increasing the burden of disease [3–5]. One can gauge the situation from the fact that, in the Punjab in 2013, only 10.6% of mothers breastfed their newborns within one hour of birth and only 17% of children were exclusively breastfed [14]. Study design The study was based on a qualitative research design. Data were collected through 12 focus-group discussions (FGDs) with mothers and fathers of children aged under two years in both rural and urban settings in Rajanpur District of Punjab Province, Pakistan. Rajanpur is situated in southern Punjab and has three Tehsils (administrative divisions) and 44 union councils (local government bodies). It has one of the poorest socio-economic indicators related to maternal and child health among all the districts of Punjab Province [14]. Rajanpur was selected as our study setting due to our interest in understanding the contextual factors behind child mortality and poor maternal and child health. Despite the traditional socio-economic environment, people are not practising exclusive and optimal breastfeeding, which can overcome deficiencies in child nutrition. Although about 98% of mothers breastfeed their children, very few of them (14%) initiate timely breastfeeding or practise exclu- sive breastfeeding [14]. Figure 1 presents the summary of union council and participant selection for the FGDs. There are a number of cultural practices that hinder optimal breastfeeding practices, such as offering a pre-lacteal feed. A significant percentage (74%) of mothers offer a pre-lacteal feed to their newborn [14]. Because of pre-lacteal feeding, an infant is deprived of the protective benefits of colostrum. Extensive research has proved that alternative feeding increases the risk of diarrhoea and acute respiratory-tract infections [15–17], as well as poor breastfeeding outcomes, such as delayed initiation and early cessation of breastfeeding [18, 19]. A purposive sampling technique was used to select mothers who had children aged under two years and were currently breastfeeding or trying to breastfeed their child. The sample was restricted in terms of the child’s age in order to minimise recall bias. Mothers who had a child with a severe illness or feeding problems (such as cleft lip or palate) were not included in the study. The data were collected from six union councils (two union councils from each Tehsil). In total, 12 FGDs were conducted with 38 mothers and 40 fathers (six in each category, out of which three were in urban and three in rural areas). The Although scholars have conducted extensive quantitative studies on exclusive breastfeeding and child and maternal Zakar et al. International Breastfeeding Journal (2018) 13:24 Page 3 of 12 Fig. Study design 1 Sample selection in research setting for FGDs six FGDs with mothers were arranged for 10:00 am because the mothers were relatively free at that time while their older children were at school. All of the six FGDs with fathers were organised on Sundays (as this is their day off and fathers could spare enough time for the meeting). The participants were served tea and refreshments before the interview to create a friendly environment. The FGDs with mothers and fathers were conducted by female and male interviewers respectively, usually in the participants’ national language (Urdu). The interviewers were experi- enced in qualitative interviewing and were trained in the FGD guide before going into the field. In some cases, when participants were not comfortable with Urdu, interviews were conducted in their local language (i.e. Siraiki or Punjabi). The local LHWs were also present during the FGDs to facilitate the sessions as they had good rapport with all the participants, being members of the same com- munity. Moreover, the presence of LHWs did not influence the content discussed in the sessions because they acted as moderators and translated certain parts of messages for researchers and participants if required. Each FGD lasted for between 90 and 120 min. participants did not belong to the same families and were not couples. There were six or seven participants in each FGD. Table 1 presents an overview of the profile charac- teristics of participants recruited for FGDs. All the participants were recruited with the help of lady health workers (LHWs). LHWs are female community health workers who live in the same community where they work. Each LHW is responsible for an average of 1000 people. LHWs register the population of their service area and act as liaison between the community and health facilities. They provide antenatal care, vaccinations for children under two and health-education information on hygiene and sanitation. For the recruitment of study par- ticipants, we contacted LHWs because they knew all the families in their localities. They went door to door in their localities and explained the study objectives to mothers and fathers and invited them to participate in the FGDs. LHWs provided a list of people who had agreed to partici- pate to the researchers. Data analysis y A thematic content analysis was performed manually to analyse the data. All audio-recorded FGDs were transcribed verbatim in Urdu. The transcripts were then translated into English. Both deductive and inductive reasoning were ap- plied in the data analysis [25]. The transcripts were read several times by the authors. A cross-check of the transla- tions was performed to address the possibility of detracting from the contextual meaning of response statements. The field notes taken by the researchers during FGDs were also considered in order to aid the data analysis. Firstly, the ini- tial codes were highlighted, first independently and then in joint sessions involving all the authors. A listing of all the codes was prepared. Response statements referring to the same theme were extracted and written out separately. In the second stage, the groupings of common themes/codes referring to broader categories were identified. Afterwards, thematic categories were coded to reveal the patterns and interplay of categories. The researchers moved back and forth in the transcribed data and searched for similar oc- currences or repeating ideas about the same phenomenon. The refining process continued until all the instances of contradictions and similarities had been explained. Finally, the findings were presented as theoretical constructs to explain the phenomenon being studied [26]. To maintain the credibility of the responses, all the authors discussed the findings. Multiple researchers performed thematic content analysis and the initial findings were shared with the LHWs and participants to obtain their comments on whether the research findings and interpretations reflected the meanings intended and related to their personal experi- ences. This allowed the participants to provide corrections for errors as well as clarifications. participants who could not read and write, the interviewer read the consent form aloud and took their thumb impres- sion instead of a signature. All the FGDs were audio re- corded with the permission of the participants; notes were also taken during the discussions. After each discussion, the researchers discussed the topics with each other and with the LHWs to clarify the concepts and their meanings. y p g The study used an FGD guide to collect data from mothers and fathers. The guide was developed on the basis of an extensive review of the literature on the topic and consisted mainly of deductive themes. Data analysis Also, we held infor- mal discussions with lactating mothers and with fathers, as well as other stakeholders such as mothers-in-law, doctors, midwives, LHWs and elders in the community to incorpor- ate contextual elements into the FGD guide. The guide was pilot tested with a group of five breastfeeding mothers be- fore the commencement of the FGDs. The guide consisted of topics such as: the first feed given to the child after birth, the use of and misconceptions about colostrum, the timing and frequency of breastfeeding, the benefits of breastfeed- ing for both child and mother, perceptions about partner support, and problems related to breastfeeding. Ethical clearance for the study protocols was obtained from the In- stitutional Review Board, University of the Punjab, Pakistan. Throughout the research process, we assured the Study design The researchers contacted the fathers and mothers who had expressed an interest in participating in the study and arranged a time and venue for the meeting according to their convenience. Before the start of the FGDs, all participants were informed about their voluntary participation, the confiden- tiality of recorded data and anonymity. Written informed consent was provided by all participants. For those The study was conducted in 2013 over a four-month period in a community setting. Eleven FGDs were conducted at LHWs’ offices, and one FGD with fathers was arranged at the house of one of the participants. The Zakar et al. International Breastfeeding Journal (2018) 13:24 Page 4 of 12 Page 4 of 12 Table 1 Profile characteristics of participants Variables Mothers (n = 38) Fathers (n = 40) Age (in years) Less than 25 17 9 25 and above 21 31 Number of children Less than 5 23 19 5 and above 15 21 Age of child (in years) Less than 1 year 24 27 1–2 years 14 13 Educational attainment No formal schooling 11 5 1–5 years of schooling 13 9 6–10 years of schooling 9 17 Above 10 years of schooling 4 9 Family monthly income (in PKRa) < 20,000 12 13 20,000–40,000 19 22 > 40,000 7 5 Family system Joint family 33 32 Nuclear family 5 8 a1 PKR = 107 USD confidentiality and anonymity of data. The data transcripts were shredded after use in data analysis. Results The areas which were discussed with mothers and fathers were related to parents’ breastfeeding knowledge and their practices. Particularly, information was sought on the use of colostrum, the exclusivity breastfeeding and the benefits of breastfeeding for both mother and infant. During the course of interviews, we tried to understand how different cultural practices can influence the breastfeeding practices of parents. Thirteen main themes were identified during the data analysis, which are presented in detail in the following section. Misconceptions about colostrum and alternatives to breastfeeding About half of the mothers (16 of 38) and fathers (20 of 40) believed that breast milk produced during the last month of pregnancy and soon after delivery (colostrum) is not suitable for a small baby. They perceived colostrum as “too heavy” [in direct translation from local language] for the newborn and believed that it cannot be digested by the infant’s fragile digestive system. Some mothers viewed colostrum as “decayed material” containing germs and other impurities. They believed that it became decayed due to its long stay in the breast during pregnancy. Therefore, discarding colostrum to clean the breast was considered a necessary and wise step. Since the colostrum was perceived as not good for the baby, goat’s or sheep’s milk was given for the first two or three days. Mothers considered goat’s milk to be very light and easily digestible as it is relatively “thin and light” compared to the milk of a buffalo or cow. Some- times Sonf [a locally produced herb which is perceived to have a good effect on digestion] was also boiled with goat’s milk and the extract was given to the baby. There was a belief among mothers that during the process of birthing, babies can ingest various smelly and dirty fluids. Thus, it is considered necessary to give goat’s milk to clean and drain that fluid out of the stomach. In order to clean the abdomen of the baby, Arqs [rose water] is exclusively given to newborns for one or two days for “cleaning purposes” and then after two days breast milk is started. Mothers reported that this behaviour is usu- ally followed by most mothers as it has been practised for generations. “Labouring-class mothers are too busy in their household and farm work and they don’t have time to properly sterilise the bottle: they don’t have fridges or other facilities. Formula milk is, therefore, not feasible for them… We earn our living with great difficulty and can only feed our child with breast milk.” (G3, P4, M28). There was a general perception among the participants that, after discarding colostrum for a few days, the breast milk becomes healthy and easily digestible for the new- born. Mothers agreed that breastfeeding increases the intimacy between a mother and her baby and that it has a positive health impact for both the baby and the mother. Benefits of breastfeeding for the mother Some male participants said that they were not aware of any specific benefits of breastfeeding for a mother, but they were sure that it is a healthy activity. Almost all the mothers perceived breastfeeding as a natural activity having many benefits without any harm. A farmer with no formal schooling stated: “It is the command of Allah; so why think about any benefit? Order is order.” (G1, P6, F32). Overall, the discussion demonstrated several benefits, as realised by both mothers and fathers. These included the fact that breast milk is readily available round the clock, and it was considered cost-effective and to have the required nutrients to strengthen an infant. However, not a single participant reported anything about improv- ing mothers’ dietary nutrients during lactation. There was much concern about making an infant fat, which implied that the fatness was considered to be healthy and powerful. The benefits of breastfeeding were seen by participants as being more important and advanta- geous for labouring-class families, who did not own a re- frigerator or sophisticated heating appliances in the kitchen. A female farm worker reported that: Infant’s first given feed The data analysis revealed that most of the participating mothers and fathers practised feeding a child with ghur- ati. This refers to the first food in the form of sweet things given to the newborn from the hands of some Zakar et al. International Breastfeeding Journal (2018) 13:24 Page 5 of 12 healthy for the newborn. .. So it is advisable to wait for some time before feeding.” (G2, P4, M22). healthy for the newborn. .. So it is advisable to wait for some time before feeding.” (G2, P4, M22). healthy for the newborn. .. So it is advisable to wait for some time before feeding.” (G2, P4, M22). elderly and pious person in the family immediately after birth. This is done with the understanding that the child will grow up with the qualities of the person who gave the first food. These things might be honey, sugar or Arq [extract of rose flower], which are called ghurati in their native language. Almost all the respondents strongly believed in ghurati as it has a cultural and ritual significance. Some of the respondents considered it a religious obligation, so the act was religiously sanctified, and the validity of the action was rarely questioned. The cleanliness of the hands giving this food was not consid- ered at all in this context. Benefits of breastfeeding for the infant Invariably, all the participants considered breastfeeding extremely beneficial for the health and growth of a new- born. Some of the respondents believed that “it is a sin if the baby is deprived of breast milk”. The respondents at- tached some cultural notions of power/strength to breast milk. Most of the participants mentioned that breast milk provides “preventive energy” for babies; if the baby has proper breastfeeding, “hundreds of diseases and ail- ments can be warded off”. One father mentioned that breast milk contains some “special and lasting power… the impact and power of breast milk lasts for 40 years.” (G6, P3, F34) Another one stated: “Breastfeeding is good for an infant. Medical knowledge has not yet discovered and counted the benefits of this natural gift [referring to breast milk] to the infant. Our forefathers used to be healthy and powerful because they got proper breastfeeding. There was no concept of diabetes, blood pressure or ulcers because the children were exclusively breastfed then…” (G4, P1, F41). “After a caesarean operation, the mother cannot feed, as the baby is kept in a nursery. How can the mother feed the baby? The lady doctor prescribes some imported formula milk. So the baby is started feeding with bottle milk and then it gradually gets used to bottle feeding. Almost all the babies born through caesarean are bottle-fed.” (G2, P2, M32). Negative connotations were implied for formula feed- ing. Mothers as well as fathers believed that rich people and fashionable women choose to formula feed their babies. Socio-cultural belief system concerning breastfeeding Some of the mothers and fathers believed that breast milk transmitted behavioural traits from mother to baby. However, linking breast milk with a mother’s religiosity and piety is a tricky proposition. The misbehaviour of a child in later stages of life are blamed on the breast milk and poor attitudes of the mother. These arguments were supported by religion: “Formula milk is dekhawa [snobbery] and a show of wealth and modernity: wealthy people buy expensive milk to show their wealth.” (G5, P4, F43). We found contradictory opinions about the effects of breastfeeding on mothers’ bodies. Some women believed that breastfeeding practices could improve the feminine shape of the female body. However, others perceived that the practice de-shapes the female body. These mothers avoided breastfeeding because they believed that it would make their bodies lose shape. Misconceptions about colostrum and alternatives to breastfeeding Furthermore, they reported that the risk of cys- tic formations in the breast and many other ailments had been reduced. Some male participants were of the view that poor families could not afford to buy infant formula and other alternatives so they relied on breast- feeding for their infants. They believed that if a mother was not producing milk due to weakness or health com- plications, a child might grow up with deficiencies and weak immunity. In addition, breastfeeding was perceived to be a natural birth prevention mechanism. Despite enumerating many benefits of breastfeeding for mothers, some of the participants believed that these benefits g In addition, there is a cultural belief among mothers that milk comes three days after birth. One mother who recently gave birth to her fourth child and lives in a rural area, stated: “When a woman delivers a child, she is in great pain; she cannot eat properly, and her body is dirty. In such a state, how could she feed her child? There may be no milk and even if there is some, it may not be Zakar et al. International Breastfeeding Journal (2018) 13:24 Page 6 of 12 Zakar et al. International Breastfeeding Journal (2018) 13:24 could only be “materialised” if the mother got proper food and was healthy. “Breastfeeding is a command of Allah. His commands are always beneficial and good for the health and well- being of mankind… The training of a child begins with breastfeeding.” (G2, P1, M29). could only be “materialised” if the mother got proper food and was healthy. “If the mother is weak [referring to anaemia], living in a poor and unhealthy environment and has no access to nutritious food, she will get less benefit from breastfeeding. Rather, breastfeeding will drain her energy and make her dhaancha [referring to her body structure turning into skin and bones].” (G1, P2, M36). Causes of bottle-feeding and non-production of breast milk Participants were asked: why do some mothers prefer bottle-feeding instead of breast milk? The main reason stated was that mother’s milk was not available in suffi- cient quantity or that some mothers did not have any milk at all. It was rarely reported that a healthy mother opted to bottle-feed her child. The mothers who had undergone caesarean section also reported not breast- feeding. Nevertheless, they could not explain any rela- tionship between caesarean section and non-production of breast milk. One mother, who had herself undergone the experience of caesarean section, said: “After the op- eration, the doctor prescribed strong medicine [referring to antibiotics], which dried up the milk.” Another woman believed that the food produced by excessive use of fertilisers and pesticides may cause an insufficiency or total absence of breast milk. Some mothers reported that, in the case of delivery in a private hospital, doctors prescribed and encouraged the use of expensive infant formula after a caesarean operation. Desired duration and frequency of breastfeeding Desired duration and frequency of breastfeeding We found a common perception about the duration and frequency of breastfeeding. It was believed that the mother should breastfeed the baby as long as possible. Nevertheless, they had no clear idea about the desirable duration of breastfeeding. The women reported having breastfed for between seven months and four years. It was also reported that the duration of breastfeeding depended on factors such as: 1) the occurrence of the next pregnancy; 2) the quantity of milk produced; and 3) the quality of milk. While discussing the duration of breastfeeding, some of the male participants made refer- ence to the Holy Quran, which stipulates a duration of two and a half years. In the light of this Quranic refer- ence, some male participants considered it sinful not to breastfeed a baby. They considered breast milk an “inali- enable right of the baby” and very important for his/her proper growth and development. y Some people were concerned about the poor quality of milk, which made the baby ill. Upon such suspicions about the quality of their milk, breastfeeding was stopped by mothers. Some people believed that in cer- tain circumstances, due to the mother’s ailments or un- known reasons, the mother’s milk becomes poisonous; it makes the baby ill and pale [referring to anaemia], and breastfeeding should be stopped immediately. The local culture had a technique to “empirically verify” the qual- ity of milk. One mother stated: “Sometimes the mother’s milk is poisonous. In such cases, the mother’s milk is ex- tracted and is given to small insects. If the insect dies, it is construed that the milk is poisonous.” (G2, P7, M37) There were also other reasons that discouraged exclusive breastfeeding. Some participants stated that some mothers start infant formula as supplementary food and others use it to increase the weight of the baby. One mother said: “Mother’s milk is thin, so the baby remains thin and weak. If you want to increase the weight of the baby, you may give bottle milk.” (G6, P1, M35). “Breastfeeding duration is not in the hands of mothers; it depends on the next pregnancy. So, it should be given until the next pregnancy or as long as milk comes.” (G3, P5, F29). Some participants underlined certain medical condi- tions when breastfeeding is to be avoided. Mothers’ diet during lactation Most of the male participants considered that no special diet is needed during lactation under normal circum- stances. They further reported that a mother only needs special food in cases when she is too weak or the pro- duction of milk is not sufficient. Female participants also believed that no special diet is required; what is normally available at home is sufficient. Nevertheless, they under- lined the importance of ghee [clarified butter made from the milk of a buffalo or cow], and believed that a lactat- ing mother should eat it regularly. One mother of four children opined: “The mother should eat at least 5 kg of ghee during Chila [the post-partum period lasting for 40 days].” (G6, P5, M39). However, some respondents thought that fruits and vegetables were also desirable food, but not essential. One father stated: “The mother should drink plenty of cow’s milk. If she cannot arrange milk due to poverty, then lassi [a sweet or savoury drink made from a yogurt or buttermilk base with water] may be the best option.” (G3, P4, F42). One mother of four children opined: “The mother should eat at least 5 kg of ghee during Chila [the post-partum period lasting for 40 days].” (G6, P5, M39). Desired duration and frequency of breastfeeding One of the male participants reported: “When the mother is suffer- ing from tuberculosis or any other malignant disease, she should avoid breastfeeding. Because, the local cul- ture believes, the disease could be transmitted to the baby through the breast milk.” (G3, P4, F36) A female participant shared: “If the mother is ‘too weak’ she ought to stop breastfeeding. It could be harmful to the health of the mother as well as the baby. Milk from a mother who is too weak may not be beneficial for her baby.” (G2, P5, M23) Nevertheless, other participants disagreed with this idea. Benefits of breastfeeding for the infant They further re- ported that some “modern women” who took more care of their health perceived that breastfeeding would “If a noble and God-fearing mother breastfeeds, it will make the baby noble and God fearing as well.” (G4, P2, F48). “Breast milk has an impact on the behaviour of an infant. If the mother is deviant, sinful or of ‘loose character’ she may not be allowed to breastfeed. In such cases, cow’s milk is better.” (G3, P5, M36). Zakar et al. International Breastfeeding Journal (2018) 13:24 Page 7 of 12 Page 7 of 12 weaken their bodies and would have a negative impact on their fitness. The respondents believed that mostly educated, middle-class families think in this way. Desired duration and frequency of breastfeeding Knowledge about exclusive breastfeeding The data analysis revealed that mothers do not practise exclusive breastfeeding, which has positive health impli- cations for both child and maternal health. Cultural practices were preventing mothers from doing so. The lack of health literacy and guidance about healthy breastfeeding were contributing to the poor health of children. The majority of mothers and fathers were un- able to comprehend what exclusive breastfeeding was about. Irrespective of the fact that an infant needs only the mother’s milk during the first six months, people preferred alternative ways to feed their baby, which were mainly backed by non-scientific sources. Both mothers and fathers believed that keeping a child on mother’s milk for six months would not make the baby fat. The strength needed to crawl was associated with the semi-liquid feed given from the fourth month. However, some respondents thought that fruits and vegetables were also desirable food, but not essential. One father stated: “The mother should drink plenty of cow’s milk. If she cannot arrange milk due to poverty, then lassi [a sweet or savoury drink made from a yogurt or buttermilk base with water] may be the best option.” (G3, P4, F42). It may be interesting to note that there was more stress on “what not to eat” instead of “what to eat” dur- ing the lactation period. The participants largely adhered to the local belief system of a “hot” and “cold” classifica- tion of things. Invariably, all the participants were of the view that “hot things” (e.g. eggs, chicken, beef, fish etc.) should be avoided. They stated some reasons behind this belief. One mother of four young children reported: Page 8 of 12 Zakar et al. International Breastfeeding Journal (2018) 13:24 Zakar et al. International Breastfeeding Journal (2018) 13:24 Page 8 of 12 Zakar et al. International Breastfeeding Journal (2018) 13:24 next pregnancy and, second, it prevents the wastage of blood.” (G1, P3, M25). next pregnancy and, second, it prevents the wastage of blood.” (G1, P3, M25). “The intake of excessive hot food turns the breast milk hot and therefore it is difficult for the small baby to di- gest it.” Secondly, sour things (like all citrus fruits, such as lemons etc.) were also considered harmful for the breastfeeding mother and her baby. Hard-to-digest things (like lentils and pulses, especially Dal channa) were also considered harmful. They also said that mothers should not take excessive tea or spicy food. Lactation-related problems experienced by mothers Another female participant explained: “The responsi- bility of the husband is to earn and provide necessary household things for the wife; this is enough, it is better to mind his own business.” (G6, P2, M32). Lactation-related problems experienced by mothers Various problems related to breastfeeding were dis- cussed. The most commonly reported problem was tears on the nipples, which caused a lot of pain to the mother. One mother shared: “Due to tears on my nipple, it was virtually impossible for me to breastfeed my baby.” (G4, P2, M23) Some of the women reported the problem of breast abscesses and said that severe pain in the breast was an obstacle to breastfeeding. Most of these problems were treated with home remedies and spiritual treat- ments. About half of the female participants reported the problem of producing insufficient breast milk for the child. A majority of these women said that this was due to the lack of a proper diet eaten by the mother. Some women stated that, due to their extensive engagement in household and farm activities, they could not feed their babies frequently enough, and this infrequent suckling led to a reduced production of milk. Mostly the female participants believed that a woman should first consult her mother-in-law and other elderly wise women and she may also inform her husband if there is something serious. One woman in her mid-thirties reported: “Informing the husband about women-related problems may be good but it is useless. Men in villages have no information or training about women’s health problems.” (G5, P2, M36) Some of the female participants highlighted the issue of discrimination against female babies. One mother of three girls and one son shared: “My husband never took any interest in breastfeeding-related issues when I had female babies. He never cared about my diet during lactation. But recently, I gave birth to a baby boy. Now he takes a lot of interest and asks me about my diet.” (G5, P4, M38). Women’s perceptions about lactational amenorrhea Some of the mothers considered lactational amenorrhea harmful to the health of the mother. While explaining the ill effects, one mother explained: Inter-spousal communication There was a complex normative system regulating inter-spousal communication in the local culture. Cul- tural norms usually encouraged the involvement of hus- bands in issues related to reproductive health. When male participants were asked about their communication with their wives in matters relating to reproductive health, at the outset, the response was negative. A ma- jority of the female participants reported that their hus- bands were not at all concerned with lactation and pregnancy-related things and “they ought not to be.” One male participant, who taught at a local high school, stated: “A lactating mother must keep a balance in her food intake to provide healthy milk for her child. She must not eat ‘too hot’ or ‘too cold’ food.” (G3, P2, F45) In the words of one mother-in-law stated by a mother [said in informal conversation]: “A mother must not keep eating ‘like a goat’; rather she should be careful and responsible.” (G1, P5, M39). One mother said: “No one would appreciate it if a man takes an interest in women-related issues, they [men] may not have proper knowledge and may offer bad advice.” (G4, P1, M29). Information system and dissemination “If mahwari [referring to the menstrual cycle] is stopped, that means decayed and “bad” blood is being accumulated within the body. Definitely this would have dangerous consequences for the health of women. Sometimes it causes a constant headache or weakening eyesight, or it may cause general decay of the whole body.” (G3, P1, M34). Participants employed different information-seeking mechanisms in rural and urban areas. The rural respon- dents were mostly illiterate or semi-literate and their main source of information was their elders and rela- tively more informed neighbours. “People discuss things with trusted people and then make decisions accord- ingly”, observed an illiterate female respondent. How- ever, in urban areas the literacy rates were high and people received information from written material, the mass media – especially TV debates – and advertise- ments, healthcare staff and educated friends. However, a majority of the mothers disagreed with this comment and thought that missing cycles has no ill ef- fects on the body. One mother shared: “Missing men- strual cycles is good in two ways: First, it prevents the Page 9 of 12 Page 9 of 12 Zakar et al. International Breastfeeding Journal (2018) 13:24 Participants were asked about their information-seeking and dissemination mechanisms. A majority of female (30 of 38) and male (32 of 40) participants reported interper- sonal communication as the most effective way to seek and share information. It was also pointed out that group discussions were important for providing information. Participants stated that advertisements on television and the radio also provided information, but that such one-way information is usually ignored by end-users. Some of the female participants suggested that illustration-based, easy-to-understand leaflets and pamphlets could be useful to improve and update their level of information. culture attached some sort of “honour to the first feed” and there were a number of symbolic activities connected with this honour [26]. Usually, the mother was not the one to whom this honour accrued be- cause she was considered impure due to the very process of birthing. There was a deeply held belief that the baby may acquire behavioural traits from the person who first feeds him/her. Hence, some family patriarch (usually the grandfather or a notable religious figure) whose behaviour deserved to be emulated did the job. Discussion This study explored the knowledge, attitudes and practices of mothers and fathers with children under two years of age regarding their breastfeeding practices. Breastfeeding is not a matter of the awareness of mothers; rather, the be- haviour is governed by a host of stakeholders, such as the mother-in-law, husband, neighbours and relatives of the in-laws, local healers and religious leaders. All act as advi- sors to the lactating mother and define the appropriate “quality” and “suitability” of her breast milk. The mother’s sexuality and a further pregnancy are also considered to “spoil” her breast milk, and these ideas create misconcep- tions that such spoiled milk can cause diarrhoea, vomiting and weakness in the child [27]. y Our study found many misconceptions about the introduction of colostrum. Such misconceptions are not limited to mothers in Pakistan; mothers in India [33] and Bangladesh [34] also believe that colostrum may harm the newborn because it is stored in the breast for the whole of the pregnancy. For that reason, adequate initiatives for improving knowledge about breastfeeding practices are needed. The participants had diverse ideas about the benefits of breastfeeding for both mother and newborn. Overall, the local culture considered breast- feeding a beneficial and natural activity and emphasised its continuation. However, these benefits and desirability were conditional, and strictly regulated by various cul- tural beliefs and local techniques to check the quality of the breast milk [31]. How these cultural beliefs intersect with medical technology, information, the market and globalisation did not fall within the scope of this re- search. Nevertheless, it was noted that these beliefs vary between social classes. Such beliefs also differ with re- spect to the level of education of the parents, their in- come and their level of health literacy [35]. We suggest that breastfeeding needs to be viewed, not in narrow terms as a mother-child dyad, but as taking place within a wider household and community environ- ment in which other actors and informal communication networks operate and influence such practices. Particu- larly, the influence of the grandmother and culturally des- ignated advisors and caregivers play a crucial role in mothers’ decision-making about the dis/continuation of breastfeeding [28]. The data also reveals that, sometimes, discontinuing or terminating breastfeeding may not be the decision of the mother. Information system and dissemination Although the health and immunological benefits of colostrum are well documented, “the local culture attached different meaning and importance to this body fluid as the cultural understanding is different than the universal physiological facts” [30]. In Pakistan, as in many developing countries, there is a negative cultural construction about the nature and effects of colostrum; it is largely considered unclean and unhealthy for the newborn. This belief results in the discarding of the colostrum and a delay in breastfeeding while the newborn is given something else as a pre-lacteal feed [31]. Statistical evidence demonstrates that, in the Pun- jab, the prevalence of pre-lacteal feeding is 75% [32]. Due to lack of knowledge, cultural myths and traditional practices, only 17% of children under six months of age are exclusively breastfed in Pakistan [32]. Zakar et al. International Breastfeeding Journal (2018) 13:24 Discussion It may be erroneously assumed to be due to her lack of awareness, but is rather precipitated by her feelings of disempowerment in the face of powerful advisors who may readily blame her for negligence or in- appropriate behaviour. One needs to understand the inter- connections between social relations, resources, sexuality, embodiment, power, and nurturance, which are all impli- cated in the challenge of feeding a newborn infant [29]. Cultural beliefs were found to be a major obstacle standing in the way of exclusive breastfeeding. We found that there was little accurate knowledge or awareness among people about exclusive breastfeed- ing practices. The socio-cultural patterns of breast- feeding practice are considered to be part of their religious traditions and a centuries-old, time-tested exercise [22]. After birth, breastfeeding was delayed due to various ritual and symbolic reasons. The local The present study found that insufficient milk syn- drome was another factor undermining exclusive breast- feeding and encouraging the use of infant formula. On the pretext of the mother having insufficient breastmilk production, a decision regarding supplementing or ter- minating breastfeeding was made [36]. However, Page 10 of 12 Zakar et al. International Breastfeeding Journal (2018) 13:24 Zakar et al. International Breastfeeding Journal (2018) 13:24 Zakar et al. International Breastfeeding Journal (2018) 13:24 In Pakistan, about 50% of the rural population receives treatment from quacks and traditional healers [44]. As in other developing countries, these unsup- portive and untrained individuals may undermine breastfeeding efforts [37]. “insufficient” is a subjective quantity depending on the perception of the mother or her advisors, and the nor- mal quantity was never operationalised. Other studies have reported that the claim of having insufficient milk may be attributable to women using it as a culturally ac- ceptable explanation for using infant formula [37]. Many studies have provided evidence that women continue to produce breast milk of adequate quality and quantity even when they themselves eat a minimal diet [38]. Many problems were linked with breastfeeding; among others, problems of suckling, nipple pain and breast ab- scesses were discussed. It was noted that there are many culturally designated advisors, caregivers, community workers, and family elders who continue to provide guid- ance to the mother. Sometimes, this advice is contradict- ory and creates confusion and anxiety for mothers [45]. As in other developing countries, this research docu- mented that mothers experience various dietary and be- havioural restrictions (e.g. Discussion not to eat “hot food” or have sex during breastfeeding, etc.). Such restrictions are sometimes too stringent and harsh, so that women feel “unqualified” to produce “good milk”; and instead of giving “bad milk” to the baby, the very process of breastfeeding is termi- nated. Sometimes these regimented requirements and restrictions are imposed by partisan family members to ad- vance their vested interests; for example, a mother-in-law may want to make breastfeeding difficult for the mother and then press her to engage in household work; or a hus- band may put restrictions on breastfeeding to enable the resumption of sexual activity, which is culturally barred during the breastfeeding period [39]. Previous studies have reported that, despite knowing the importance and benefits of breast milk, people think that infant formula makes the baby healthier [46]. This belief, though bio-medically not proven, may be due partly to the watery nature of breast milk and partly to the marketing strategies of infant formula companies, which present their product as being of superb quality [47]. This research has identified that various physical, so- cial and ideological ecologies create a given cultural con- text, which supports or constrains decisions about breastfeeding, especially its duration and exclusivity [42]. It was noted that the mother’s physical environment and familial relations influence feeding patterns. More specif- ically, the mother’s household workload, living patterns, and reproductive schedule modify maternal decisions re- garding the continuity of breastfeeding. In short, the overall social and institutional environment within which the mother is situated is considered an important pre- dictor of breastfeeding behaviour. Elaborate cultural techniques to declare milk “poison- ous and dirty” may be another example of the active role taken by advisors and stakeholders in the process of breastfeeding [40]. For example, it has been observed that women who question the quality of their breast milk have a strong incentive to stop breastfeeding in order to avoid their baby becoming sick. Women’s anx- ieties about the quality of their milk may be increased both by folk healers (who float insects in the milk) and by hospital pathology laboratories (which perform pseudo-scientific analysis for “bacteria” and “pus cells”). For these reasons, women are special targets of commodification, when their bodies are valued for their reproductive potential [41]. Limitations A major strength of this study was the comparatively large design with 12 FGDs. In addition, the combination of a deductive and inductive approach in data analysis has led to a comprehensive assessment of topics related to parental perceptions and knowledge regarding breast- feeding practices. As for any qualitative study design, the findings may not be generalisable. However, we expect that the results provide a good picture for similar popu- lations. Interviews were conducted by trained inter- viewers. Nevertheless, the use of different languages may also influence the results. In addition, it has to be noted that the focus-group discussants may have provided so- cially desirable answers. The results also showed that there was a lack of social support. Notwithstanding the verbal claims about the virtues of breastfeeding ranging from the commandment of Allah to the high status of breast- feeding mothers, the reality is that mothers often find little social support to continue breastfeeding. For example, their workload is not decreased, and appro- priate food is not provided. Rather, sometimes, a breastfeeding mother is blamed for the poor or poisonous quality of her milk. In this situation, the mother discontinues breastfeeding for fear of being blamed for making the baby sick by providing unhealthy breast milk. The data showed that various local conceptions and beliefs about the attributes of breast milk and its alternatives, could have consider- able impact on the pattern of breastfeeding [42, 43]. Conclusions The breastfeeding-related health literacy of parents, grand- parents and community needs to be improved to reduce the high infant mortality rates in Pakistan. This can be achieved through targeted, culturally acceptable, and inte- grated public health interventions at individual, commu- nity, and national levels. Such interventions include: Zakar et al. International Breastfeeding Journal (2018) 13:24 Page 11 of 12 Page 11 of 12 Page 11 of 12 engaging social and family decision-makers, involving men to motivate and support mothers, establishing peer-support social networks, and creating awareness through mass media about the benefits of the early and timely initiation of breastfeeding. These are imperative for promoting healthy breastfeeding practices in Pakistan. A more prac- tical approach would be to train LHWs and midwives about the importance of child and maternal nutrition, which can be achieved through encouraging exclusive breastfeeding. Additionally, media campaigns and talk shows should be used to disseminate information about the dietary intake of mothers who are breastfeeding. Efforts should be directed towards changing the main sources of information regarding the postpartum period and breast- feeding in order to eradicate false perceptions. 4. Mullany LC, Katz J, Li YM, Khatry SK, LeClerq SC, Darmstadt GL, Tielsch JM. Breastfeeding patterns, time to initiation, and mortality risk among newborns in southern Nepal. J Nutr. 2008;138:599–603. 5. Morrow AL, Ruiz-Palacios GM, Jiang X, Newburg DS. Human-milk glycans that inhibit pathogen binding protect breast-feeding infants against infectious diarrhoea. J Nutr. 2005;135(5):1304–7. 6. Renfrew MJ, Lang S, Woolridge MW. Early versus delayed initiation of breastfeeding. Cochrane Database Syst Rev. 2000;2:CD000043. 7. Tryggvadóttir L, Tulinius H, Jórunn E, Eyfjord JE, Sigurvinsson T. Breastfeeding and reduced risk of breast cancer in an Icelandic cohort study. Am J Epidemiol. 2001;154(1):37–42. 8. Jeroen K, Ginneken V. Prolonged breastfeeding as a birth spacing method. Stud Fam Plan. 1974;5:201–6. 8. Jeroen K, Ginneken V. Prolonged breastfeeding as a birth spacing method. Stud Fam Plan. 1974;5:201–6. 9. Stuebe A. The risks of not breastfeeding for mothers and infants. Rev Obstet Gynecol. 2009;2(4):222–31. 9. Stuebe A. The risks of not breastfeeding for mothers and infants. Rev Obstet Gynecol. 2009;2(4):222–31. 10. WHO & UNICEF. Global strategy for infant and young child feeding. Geneva: World Health Organization; New York: United Nation Children’s Fund; 2003. 10. WHO & UNICEF. Global strategy for infant and young child feeding. Geneva: World Health Organization; New York: United Nation Children’s Fund; 2003. 11. Acknowledgements g We acknowledge support of the publication fee by Deutsche Forschungsgemeinschaft and the Open Access Publication Funds of Bielefeld University. We thank Liz Sourbut for proof-reading this manuscript. g We acknowledge support of the publication fee by Deutsche Forschungsgemeinschaft and the Open Access Publication Funds of Bielefeld University. We thank Liz Sourbut for proof-reading this manuscript. 14. Multiple Cluster Indicator Survey 2014. Retrieved from http://bos.punjab.gov. pk/system/files/4.Nutrition.pdf 14. Multiple Cluster Indicator Survey 2014. Retrieved from http://bos.punjab.gov. pk/system/files/4.Nutrition.pdf 15. Tariku A, Biks GA, Wassie MM, Gebeyehu A, Getie AA. Factors associated with prelacteal feeding in the rural population of Northwest Ethiopia: a community cross-sectional study. Int Breastfeed J. 2016;11:14. 15. Tariku A, Biks GA, Wassie MM, Gebeyehu A, Getie AA. Factors associated with prelacteal feeding in the rural population of Northwest Ethiopia: a community cross-sectional study. Int Breastfeed J. 2016;11:14. Abbreviations 12. Baker D, Taylor H, Henderson J. Inequality in infant morbidity: causes and consequences in England in the 1990s. ALSPAC study team. Avon longitudinal study of pregnancy and childhood. J Epidemiol Community Health. 1998;52:451–8. 12. Baker D, Taylor H, Henderson J. Inequality in infant morbidity: causes and consequences in England in the 1990s. ALSPAC study team. Avon longitudinal study of pregnancy and childhood. J Epidemiol Community Health. 1998;52:451–8. Abbreviations FGD: Focus-group discussion; LHW: Lady Health Worker; UNICEF: United Nations Children’s Fund; USD: United States Dollar; WHO: World Health Organization 13. Gupta A, Arora V, Bhatt B. The state of the world’s breastfeeding Pakistan report card. Delhi: World Breastfeeding Trends Initative; 2006. 13. Gupta A, Arora V, Bhatt B. The state of the world’s breastfeeding Pakistan report card. Delhi: World Breastfeeding Trends Initative; 2006. Availability of data and materials 16. Gedefaw M, Berhe R. Determinates of childhood pneumonia and diarrhea with special emphasis to exclusive breastfeeding in north Achefer District, Northwest Ethiopia: a case control study. Open J Epidemiol. 2015;5(2):107. Authors’ contributions d d h d RZ designed the study. RZ, MZZ, LZ and FF analysed and interpreted the data. RZ drafted the manuscript. MZZ, LZ and FF revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript. 17. Lamberti LM, Walker CLF, Noiman A, Victora C, Black RE. Breastfeeding and the risk for diarrhea morbidity and mortality. BMC Public Health. 2011;11(S3):S15–27. 17. Lamberti LM, Walker CLF, Noiman A, Victora C, Black RE. Breastfeeding and the risk for diarrhea morbidity and mortality. BMC Public Health. 2011;11(S3):S15–27. 18. Raheem RA, Binns CW, Chih HJ, Sauer K. Determinants of the introduction of prelacteal feeds in the Maldives. Breastfeed Med. 2014;9(9):473–8. 19. Ludvigsson JF. Breastfeeding intentions, patterns, and determinants in infants visiting hospitals in La Paz, Bolivia. BMC Pediatric. 2003;3:5. Conclusions Amini M, Salarkia N, Eshrati B, Djazayery A. Poor breastfeeding as a probable cause of childhood malnutrition: exploring mothers’ and caregivers’ views on breastfeeding via a qualitative study in Damavand, Iran. Razavi Int J Med. 2013;1(1):30–4. 11. Amini M, Salarkia N, Eshrati B, Djazayery A. Poor breastfeeding as a probable cause of childhood malnutrition: exploring mothers’ and caregivers’ views on breastfeeding via a qualitative study in Damavand, Iran. Razavi Int J Med. 2013;1(1):30–4. Author details 1 25. Burnard P, Gill P, Stewart K, Treasure E, Chadwick B. Analysing and presenting qualitative data. Br Dent J. 2008;204(8):429–32. 25. Burnard P, Gill P, Stewart K, Treasure E, Chadwick B. Analysing Author details 1Institute of Social and Cultural Studies, University of the Punjab, Lahore, Pakistan. 2Institute of Communication Studies, University of the Punjab, Lahore, Pakistan. 3School of Public Health, Department of Public Health Medicine, Bielefeld University, Bielefeld, Germany. 26. Auerbach CF, Silverstein LB. Qualitative data: An introduction to coding and analysis. New York: New York University Press; 2003. 27. Gunnlaugsson G, Einarsdóttir J. Colostrum and ideas about bad milk: a case study from Guinea-Bissau. Soc Sci Med. 1993;3:283–8. Received: 20 July 2017 Accepted: 12 June 2018 Received: 20 July 2017 Accepted: 12 June 2018 28. Esterik PV. Contemporary trends in infant feeding research. Annu Rev Anthropol. 2002;31:257–78. 29. Sonko A, Worku A. Prevalence and predictors of exclusive breastfeeding for the first six months of life among women in Halaba special woreda, southern nations, nationalities and peoples’ region/SNNPR/, Ethiopia: a community based cross-sectional study. Arch Public Health. 2015;73:53. Consent for publication Not applicable. Consent for publication Not applicable. 23. Nazlee N, Bilal R, Latif Z, Bluck L. Maternal body composition and its relationship to infant breast milk in rural Pakistan. Food NutSci. 2011;2(9):932–7 Competing interests 24. Premani ZS, Kurji Z, Mithani Y. To explore the experiences of women on reasons in initiating and maintaining breastfeeding in urban area of Karachi, Pakistan: an exploratory study. ISRN Pediatr. 2011; https://doi.org/10.5402/2011/514323. Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate Ethical approval was obtained by the Institutional Review Board, University of the Punjab, and also from the hospitals and nursing institutes where data collection took place. Written informed consent was provided by study participants. 20. Khalil H, Khalil A. Professional mothers and breastfeeding practices. Annals of King Edward Medical University. 2000;6:60–6. 21. Zafar SN, Bustamante GM. Breastfeeding and working full time: experiences of nurse mothers in Karachi. Int J Car Sci. 2008;1(3):132–3. 22. Hussain M. Decline in breastfeeding: what are the factors responsible? Gomal J Medical Sci. 2003;1:7–10. References References 1. Kumar L, Shahnawaz K, Varma G, Choudhary SK, Gupta A, Singh JB. Knowledge, attitude and practices of nourishing mothers about breast feeding, attending urban health center: a cross-sectional study from Kishanganj, Bihar. JEMDS. 2014;3:1681–90. 2. WHO. Global strategy for Infant and Young Child Feeding. A joint WHO/ UNICEF statement. Geneva: World Health Organization; 2003. 3. Edmond KM, Zandoh C, Quigley MA, Amenga-Etego S, Owusu-Agyei S, Kirkwood BR. Delayed breastfeeding initiation increases risk of neonatal mortality. Pediatr. 2006;117:380–6. 1. Kumar L, Shahnawaz K, Varma G, Choudhary SK, Gupta A, Singh JB. Knowledge, attitude and practices of nourishing mothers about breast feeding, attending urban health center: a cross-sectional study from Kishanganj, Bihar. JEMDS. 2014;3:1681–90. 1. Kumar L, Shahnawaz K, Varma G, Choudhary SK, Gupta A, Singh JB. Knowledge, attitude and practices of nourishing mothers about breast feeding, attending urban health center: a cross-sectional study from Kishanganj, Bihar. JEMDS. 2014;3:1681–90. 30. Raghu L, Pallavi G, Shamsa Z, Zelee H, Aisha Y, Sharad I, Siham S, et al. Understanding care and feeding practices: building blocks for a sustainable intervention in India and Pakistan. Ann N Y Acad Sci. 2014;1308:204–17. 2. WHO. Global strategy for Infant and Young Child Feeding. A joint WHO/ UNICEF statement. Geneva: World Health Organization; 2003. 31. Wright AL. Cultural interpretation and intercultural variability in Navago beliefs about breastfeeding. Am Ethnol. 1993;4:781–96. 3. Edmond KM, Zandoh C, Quigley MA, Amenga-Etego S, Owusu-Agyei S, Kirkwood BR. Delayed breastfeeding initiation increases risk of neonatal mortality. Pediatr. 2006;117:380–6. 32. Indu K, Sharma AB. Early initiation of breastfeeding: a systematic literature review of factors and barriers in South Asia. Int Breastfeed J. 2016;11:17. Page 12 of 12 Zakar et al. International Breastfeeding Journal (2018) 13:24 33. National Institute of Population Studies, Islamabad, Pakistan. Pakistan Demographic and Health Surveys. Calverton: MEASURE DHS. 2013. Retrieved from http://pdf.usaid.gov/pdf_docs/pnaeb738.pdf 34. Parveen S, Sareen IB, Dahiya BR. Breast feeding practices in post IMNCI era in rural community of Haryana. Indian J Pub Health Res Develop. 2012;4:205–9. 35. Haider R, Rasheed S, ST G, Hassan N, Pachon H, Islam S, et al. Breastfeeding in infancy: identifying the program-relevant issues in Bangladesh. Int Breastfeed J. 2010;5:21. 36. Islam MA, Rahman MM, Mahalanabis D. Clinical sciences division, International Centre for Diarrhoeal Disease Research, Bangladesh. Dhaka Eur J Clin Nutr. 1994;6:416–24. 37. Ahluwalia IB, Morrow B, Hsia J. Why do women stop breastfeeding? Findings from the pregnancy risk assessment and monitoring system. Zakar et al. International Breastfeeding Journal (2018) 13:24 References Pediatr. 2005;6:1408–12. 38. Dettwyler KA, Fishman C. Infant feeding practices and growth. Annu Rev Anthropol. 1992;1:171–204. 39. Jelliffe DB, Jelliffe EFP. Human milk in the modern world. Oxford: Oxford University Press; 1978. 39. Jelliffe DB, Jelliffe EFP. Human milk in the modern world. Oxford: Oxford University Press; 1978. 40. Buskens I, Jaffe A, Mikhatshwa H. Infant feeding practices: realities and mindsets of mothers in southern Africa. AIDS Care. 2007;19(9):1101–9. 40. Buskens I, Jaffe A, Mikhatshwa H. Infant feeding practices: realities and mindsets of mothers in southern Africa. AIDS Care. 2007;19(9):1101–9. 41. Mull D. Mother’s milk and pseudoscientific breast milk testing in Pakistan. Soc Sci Med. 1992;11:1277–90. 41. Mull D. Mother’s milk and pseudoscientific breast milk testing in Pakistan. Soc Sci Med. 1992;11:1277–90. 42. Sharp LA. The commodification of body and its parts. Annu Rev Anthropol 2000;29:287–328. 42. Sharp LA. The commodification of body and its parts. Annu Rev Anthropol. 2000;29:287–328. 43. McDade TW. Parent-offspring conflict and the cultural ecology o breastfeeding. Hum Nat. 2001;1:9–25. 43. McDade TW. Parent-offspring conflict an breastfeeding. Hum Nat. 2001;1:9–25. breastfeeding. Hum Nat. 2001;1:9–25. 44. Zakar MZ. AIDS, culture, and body politics in Pakistan. Lage: Verlag Hans Jacobs; 2004. 45. Sharma IK, Byrne A. Early initiation of breastfeeding: a systematic literature review of factors and barriers in South Asia. Int Breastfeed J. 2016;11:17. 46. Rosenberg KD, Eastham CA, Kasehagen LJ, Sandoval AP. Marketing infant formula through hospitals: the impact of commercial hospital discharge packs on breastfeeding. Am J Public Health. 2008;98:290–5. 47. Aubel J. The role and influence of grandmother on child nutrition: culturally designated advisors and caregivers. Matern Child Nutr. 2012;8(1):19–35.
https://openalex.org/W1990092996	https://europepmc.org/articles/pmc3717009?pdf=render	English	null	The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus (Litopenaeus) vannamei	BMC veterinary research	2,013	cc-by	7,884	* Correspondence: Kallaya@biotec.or.th †Equal contributors 1Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI rd, Bangkok 10400, Thailand 2Shrimp-Virus Interaction Laboratory, National Center for Genetic Engineering and Biotechnology, Soi Yothi, Rama VI rd, Bangkok 10400, Thailand Full list of author information is available at the end of the article The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus (Litopenaeus) vannamei The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus (Litopenaeus) vannamei Amornrat Tangprasittipap1,2†, Jiraporn Srisala2†, Saisunee Chouwdee1,2, Montagan Somboon4, Niti Chuchird4, Chalor Limsuwan4, Thinnarat Srisuvan6, Timothy W Flegel1,3,5 and Kallaya Sritunyalucksana1,2,5* © 2013 Tangprasittipap et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Abstract Background: The microsporidian Enterocytozoon hepatopenaei was first described from Thailand in 2009 in farmed, indigenous giant tiger shrimp Penaeus (Penaeus) monodon. The natural reservoir for the parasite is still unknown. More recently, a microsporidian closely resembling it in morphology and tissue preference was found in Thai-farmed, exotic, whiteleg shrimp Penaeus (Litopenaeus) vannamei exhibiting white feces syndrome (WFS). Our objective was to compare the newly found pathogen with E. hepatopenaei and to determine its causal relationship with WFS. Results: Generic primers used to amplify a fragment of the small subunit ribosomal RNA (ssu rRNA) gene for cloning and sequencing revealed that the new parasite from WFS ponds had 99% sequence identity to that of E. hepatopenaei, suggesting it was conspecific. Normal histological analysis using tissue sections stained with hematoxylin and eosin (H&E) revealed that relatively few tubule epithelial cells exhibited spores, suggesting that the infections were light. However, the H&E results were deceptive since nested PCR and in situ hybridization analysis based on the cloned ssu rRNA gene fragment revealed very heavy infections in tubule epithelial cells in the central region of the hepatopancreas in the absence of spores. Despite these results, high prevalence of E. hepatopenaei in shrimp from ponds not exhibiting WFS and a pond that had recovered from WFS indicated no direct causal association between these infections and WFS. This was supported by laboratory oral challenge trials that revealed direct horizontal transmission to uninfected shrimp but no signs of WFS. Conclusions: The microsporidian newly found in P. vannamei is conspecific with previously described E. hepatopenaei and it is not causally associated with WFS. However, the deceptive severity of infections (much greater than previously reported in P. monodon) would undoubtedly have a negative effect on whiteleg shrimp growth and production efficiency and this could be exacerbated by the possibility of horizontal transmission revealed by laboratory challenge tests. Thus, it is recommended that the PCR and in situ hybridization methods developed herein be used to identify the natural reservoir species so they can be eliminated from the shrimp rearing system. RESEARCH ARTICLE Open Access Results Small subunit ribosomal RNA gene fragment analysis When the MF primers designed from the ssu rRNA sequences of Enterocytozoon species [12] were used with template hepatopancreatic DNA extracts from P. vannamei infected with the microsporidian, a 951 bp amplicon was obtained (Additional file 1). This was within the range of the expected size of approximately 900–1000 bp, based on conserved regions of Enterocytozoon ssu rRNA sequences listed at GenBank (FJ496356) and the previous amplicon of 886 bp obtained from P. monodon infected with Enterocytozoon hepatopenaei [12]. Cloning and sequencing of 3 clones revealed 100% identical sequences for 2 clones and only 2 variable nucleotides for a third clone. A consensus se- quence was concluded from the two identical clones and a 913 bp portion of the amplicon sequence (excluding the primer sequences, Genbank accession no. KF362130) was subjected to a general BLASTN search that yielded hits only for microsporidian sequence records. The top hits from the BLASTN search included Enterocytozoon isolated from P. monodon (GenBank FJ496356) at 96% identity, Nucleospora salmonis (GenBank U10883) at 89% identity and E. bieneusi (GenBank : AY257180) at 89% identity. The MF1 and MR1 primers were also used to amplify the ssu rRNA gene target from archived material of infected P. monodon used in previously published work [12] that gave rise to the GenBank record FJ496356. A consensus se- quence was established from 3 clones also of 913 bp each and named Pm-Entero (Genbank accession no. KF362129). Clustal alignment of our 913 bp sequence from P. vannamei with our 913 bp sequence from P. monodon revealed 99% identity, indicating that infections arose from the same microsporidian species (Figure 1). Histopathology and in situ hybridization of infections in P. vannamei As previously reported for infections of E. hepatopenaei in P. monodon, the number of hepatopancreatic cells showing spore formation in P. vannamei (Figure 2) was small, giving a superficial impression that the extent of the infections was very limited. The size of the spores (approximately 1 μm in length and less than 1 μm in width) and cytoplasmic location were also similar to those previously described for E. hepatopenaei in P. monodon. However, differences in P. vannamei included spore formation exclusively in B cells (Figure 2) and extensive infection of the medial and prox- imal tubule epithelial cells of the hepatopancreas in the ab- sence of spores, as revealed by in situ hybridization (Figure 3). This was not the situation for previous reports on E. hepatopenaei in P. monodon, where relatively few cells produced spores or showed recognizable plasmodia, and only those cells were positive by in situ hybridization [12,13]. The cells giving positive in situ hybridization re- actions in P. vannamei were restricted to the central re- gion of the HP and did not extend to the distal region composed of E-cells. In the transitional zone between the medial and distal cells, pinpoint positive in situ hybridization reactions suggested that early infection stages occurred as the HP cells differentiated from E cells into B, F and R cells. Negative control slides using the GFP-DIG-labled probe gave no positive in situ hybridization reactions (not shown). At low and medium magnification by H&E staining (Figure 3a,c,e), Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Page 2 of 10 (Table 2). However, a later set of samples (Table 3) gave less clear-cut results, in that some ponds without signs of WFS gave a high prevalence (9/10 shrimp) posi- tive for E. hepatopenaei by PCR (albeit mostly for the nested step), while other ponds with gross signs of WFS gave a low prevalence of PCR positive shrimp (4/10). In addition, one recovered pond with no signs of WFS (Table 4) had a high prevalence of shrimp (8/9) with ex- tensive infections as determined by in situ hybridization. This constituted a poor correlation between gross signs of WFS and severity of E. hepatopenaei infection. of P. monodon. In 2010, E. hepatopenaei was also reported from P. monodon exhibiting white feces syndrome (WFS) in Vietnam [11]. Here we report widespread infections of a microsporidian conspecific with E. hepatopenaei that was found in Thai-cultivated, exotic whiteleg shrimp P. vannamei exhibiting WFS. In addition, a nested PCR de- tection protocol is described together with its use in examining whiteleg shrimp from culture ponds and from oral challenge tests using hepatopancreatic tissue from shrimp with microsporidian infections. of P. monodon. In 2010, E. hepatopenaei was also reported from P. monodon exhibiting white feces syndrome (WFS) in Vietnam [11]. Here we report widespread infections of a microsporidian conspecific with E. hepatopenaei that was found in Thai-cultivated, exotic whiteleg shrimp P. vannamei exhibiting WFS. In addition, a nested PCR de- tection protocol is described together with its use in examining whiteleg shrimp from culture ponds and from oral challenge tests using hepatopancreatic tissue from shrimp with microsporidian infections. Using the digoxygenin (DIG) labeled probe to con- firm the validity of the PCR test, most (4/6) of the ex- tensive in situ positive samples were 1st-step PCR positive (2 exceptions Table 3, Pond7 YOT4 & Pond6 YOT). Specimens that showed light positive in situ hybridization reactions (n = 7) were either 2nd-step (nested PCR) positive only (n = 4) or gave negative PCR reactions (n = 2), while one gave a 1st-step positive re- sult (Pond 13 BAP-1). Of the 11 specimens that gave negative in situ hybridization test results, 6 gave nega- tive PCR test results and 5 gave 2nd-step positive PCR results. Background in the giant tiger shrimp Penaeus (Penaeus) monodon and the banana prawn Penaeus (Feneropenaeus) merguiensis [2-4]. It resembles morphologically the microsporidian Agmasoma penaei reported to infect Penaeus (Litopenaeus) setiferus and Penaeus (Farfantepenaeus) duorarum [1,5] in the Americas. More recently in Thailand it has been reported to also infect the same tissues in Penaeus (Litopenaeus) vannamei [6-8]. Spores from an unidentified microspridian have also been reported in muscles of P. monodon from Madagascar [9]. Several microsporidians have been reported as pathogens of penaeid shrimp [1]. Of these, two species have been reported to infect cultivated shrimp in Thailand. One of these is a species of Agmasoma previously called Thelohania that infects muscle tissue and connective tissue The other microsporidian reported from Thailand was a newly described species Enterocytozoon hepatopenaei [10] restricted to tubule epithelial cells of the hepatopancreas Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 PCR and in situ hybridization detection of the microsporidian in P. vannamei With a preliminary field sample of 11 shrimp taken from one WFS pond and 10 from a nearby normal pond, PCR tests using primers specific for E. hepatopenaei (Table 1) revealed that 10/11 shrimp from the WFS pond were positive while all 10 from the normal pond were negative Page 3 of 10 Page 3 of 10 Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Pv-Entero GATGGCTCCCACGTCCAAGGATGGCAGCAGGCGCGAAAATTGTCCACTCTTTTGAGAGGA 60 Pm-Entero GATGGCTCCCACGTCCAAGGGATGCAGCAGGCGCGAAAATTGTCCACTCTTTTGAGAGGA 60 AY257180 GATGGCTCCCACGTCCAAGGACGGCAGCAGGCGCGAAACTTGTCCACTCCTTACTGGGGA 60 ******************. ***********.****** : :. Pv-Entero GACAGTTATGAAACGTGAGTAGAAGGGTCGAGTGTAAAAACCTTGACGTGAAGCAATTGG 120 Pm-Entero GACAGTTATGAAACGTGAGTAGAAGGGTCGAGTGTAAAAACCTTGACGTGAAGCAATTGG 120 AY257180 GACAGTCATGAGACGTGAGTATAAGACCTGAGTGTAAAGACCTTAGGGTGAAGCAATTGG 120 ** .***** . ******.*.. ********* Pv-Entero AGGGCAAGTTTTGGTGCCAGCAGCCGCGGTAATTCCAACTCCAAGAGTGTCTATGGTGGA 180 Pm-Entero AGGGCAAGTTTTGGTGCCAGCAGCCGCGGTAATTCCAACTCCAAGAGTGTCTATGGTGGA 180 AY257180 AGGGCAAGCTTTGGTGCCAGCAGCCGCGGTAACTCCAACTCCAAGAGTGTCTATGGTGGA 180 **** ******************* *********************** Pv-Entero TGCTGCAGTTAAAGGGTCCGTAGTCGTAGATGCAATTAAAAGGTGGTGTTAAAAGCCATT 240 Pm-Entero TGCTGCAGTTAAAGGGTCCGTAGTCGTAGATGCAATTAAAAGGTGGTGTTAAAAGCCATT 240 AY257180 TGCTGCAGTTAAAGGGTCCGTAGTCGTGAATGCAATTAAATGTCGTTGTTCAATAGCGAT 240 ***********************..********: * **.:. .: Pv-Entero GAGTTTGTTGAGAGTAGCGGAACGGATAGGGAGCATGGTATAGGTGGGCAAAGAATGAAA 300 Pm-Entero GAGTTTGTTGAGAGTAGCGGAACGGATAGGGAGCATGGTATAGGTGGGCAAAGAATGAAA 300 AY257180 GAGTTTGCTGATGTTTGCGGAACGGATAGGGAGTGTAGTATAGACTGGCGAAGAATGAAA 300 ***** * . :**************** ..***. .******* Pv-Entero TCTCAAGACCCCACCTGGACCAACGGAGGCGAAAGCGATGCTCTTAGACGTATCTGGGGA 360 Pm-Entero TCTCAAGACCCCACCTGGACCAACGGAGGCGAAAGCGATGCTCTTAGACGTATCTGGGGA 360 AY257180 TCTCAAGACCCAGTTTGGACTAACGGAGGCGAAGGCGACACTCTTAGACGTATCTTAGGA 360 *******.. * ********. .*********** .* Pv-Entero TCAAGGACGAAGGCTAGAGTATCGAAAGTGATTAGACACCGCTGTAGTTCTAGCAGTAAA 420 Pm-Entero TCAAGGACGAAGGCTAGAGTATCGAAAGTGATTAGACACCGCTGTAGTTCTAGCAGTAAA 420 AY257180 TCAAGGACGAAGGCAGGAGTATCGAAAGTGATTAGACACCGCTGTAGTTCCTGCAGTAAA 420 ************:.****************************** :**** Pv-Entero CTATGCCGACAATGCTGGGTGTTGCGAGAGCGATGCTTGGTGTGGGAGAAATCTTAGTTT 480 Pm-Entero CTATGCCGACAATGCTGGGTGTTGCGAGAGCGATGCTTGGTGTGGGAGAAATCTTAGTTT 480 AY257180 CTATGCCGACAG--------CCTGTGTGTGAGAATACGTGGGCGGGAGAAATCTTAGTGT 472 *******. ::.: . * *************** * Pv-Entero TCGGGCTCTGGGGATAGTACGCTCGCAAGGGTGAAACTTAAAGCGAAATTGACGGAAGGA 540 Pm-Entero TCGGGCTCTGGGGATAGTACGCTCGCAAGGGTGAAACTTAAAGCGAAATTGACGGAAGGA 540 AY257180 TCGGGCTCTGGGGATAGTACGCTCGCAAGGGTGAAACTTAAAGCGAAATTGACGGAAGGA 532 ********************************************************** Pv-Entero CACTACCAGGAGTGGATTGTGCTGCTTAATTTAACTCAACGCGGGAAAACTTACCAGGGT 600 Pm-Entero CACTACCAGGAGTGGATTGTGCTGCTTAATTTAACTCAACGCGGGAAAACTTACCAGGGT 600 AY257180 CACTACCAGGAGTGGATTGTGCTGCTTAATTTAACTCAACGCGGGAAAACTTACCAGGGT 592 ******************************************************** Pv-Entero CAAGTCTATCGTAGATTGGAGACATGAGGTAGACAAGAGTGGTGCATGGCCGTTGGAAAT 660 Pm-Entero CAAGTCTATCGTAGATTGGAGACATGAGGTAGACAAGAGTGGTGCATGGCCGTTGGAAAT 660 AY257180 CAAGTCATTCGTTGATCGAATACGTGAGAATGGCAGGAGTGGTGCATGGCCGTTGGAAAT 652 **:::* . .*.::..******************** Pv-Entero TGATGGGGCGACTTTTAGCTTAAGTGCTGGAACCAGTGAGATCTTCTAGACAGGTGTTAT 720 Pm-Entero TGATGGGGCGACTTTTAGCTTAAGTGCTGGAACCAGTGAGATCTTCTAGACAGGTGTTAT 720 AY257180 TGATGGGGCGACCTTTAGCTTAAATGCTTAAACCAGTGAGACCTCCTTGACAGGTGTTCT 712 ******** ******. .******* :*******. Pv-Entero TTAGGCACAGGAGGGAGAAGGCAATAACAGGTCCGTGATGCCCTTAGATATCCTGGGCAG 780 Pm-Entero TTAGGCACAGGAGGGAGAAGGCAATAACAGGTCCGTGATGCCCTTAGATATCCTGGGCAG 780 AY257180 GTAA-CACAGGAGGGTGGAGGCTATAACAGGTCCGTGATGCCCTTAGATATCCTGGGCAG 771 . *******:.**:********************************* Pv-Entero CAAGCGCAATACAATATCTCTTGAGAAGACAAAGCAATTTGAGATGAGTAGGATTAGCTT 840 Pm-Entero CAAGCGCAATACAATATCTCTTGAGAAGACAAAGCAATTTGAGATGAGTAGGATTAGCTT 840 AY257180 CAAGCGCAATACAATATCTCTTCAGTAGACAAAGTGATTTGAGATGAGTAGGATCTACGT 831 ****************** :****** .**************** :.* * Pv-Entero TTGTAAATAAGCTATGAATGAGGAATTCCTAGTAACAGTGTCTCATCAAGGCATTGTGAA 900 Pm-Entero TTGTAAATAAGCTATGAATGAGGAATTCCTAGTAACAGTGTCTCATCAAGGCATTGTGAA 900 AY257180 TTGTAAATACGTAGTGAATAAGGAATTCCTAGTAACGGTGCCTCATCAAGGCATGGTGAA 891 ********. :.***.************.* *********** * % Identity Pv-Entero TGTGTCCCTGTTC 913 Pm-Entero TGTGTCCCTGTTC 913 99% AY257180 TGTGTCCCTGTTC 904 88% ********* Figure 1 (See legend on next page.) TGCTGCAGTTAAAGGGTCCGTAGTCGTAGATGCAATTAAAAGGTGGTGTTAAAAGCCATT 240 TGCTGCAGTTAAAGGGTCCGTAGTCGTGAATGCAATTAAATGTCGTTGTTCAATAGCGAT 240 ***********************..********: * **.:. .: TCGGGCTCTGGGGATAGTACGCTCGCAAGGGTGAAACTTAAAGCGAAATTGACGGAAGGA 540 Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Page 4 of 10 (See figure on previous page.) Figure 1 Clustal-W alignment of microsporidian small subunit rRNA sequences. The ssu rRNA sequence of Enterocytozoon hepatopenaei from P. monodon (Pm-Entero)(Genbank accession no. KF362129) and the microsporidian from P. vannamei (Pv-Entero) (Genbank accession no. KF362130) are compared with the matching region of the ssu rRNA gene of E. bieneusi (GenBank AY257180). Regions of 100% identity between Pv-Entero and Pm-Entero are outlined in grey background while 100% identity among all three species is indicated by asterisks. Discussion Because of identical tissue specificity, identical spore size and a sequence identity of 99% obtained in comparison of ssu rRNA gene fragments of 913 bp, we concluded that the microsporidian newly found in P. vannamei was identical to E. hepatopenaei previously reported from P. monodon. However, we sequenced only 3 clones (98-99% identity) from each shrimp species to obtain the consen- sus sequences we used for phylogenetic comparison. Thus, according to veterinary sampling tables [14], could and a sequence identity of 99% obtained in comparison of ssu rRNA gene fragments of 913 bp, we concluded that the microsporidian newly found in P. vannamei was identical to E. hepatopenaei previously reported from P. monodon. However, we sequenced only 3 clones (98-99% identity) from each shrimp species to obtain the consen- sus sequences we used for phylogenetic comparison. PCR and in situ hybridization detection of the microsporidian in P. vannamei that originated from a shrimp pond experiencing a WFS outbreak. Control shrimp were fed hepatopancreatic tissue from normal shrimp. Upon sampling the control shrimp on days 2, 4 and 7 after challenge, it was found that 1 in 3 sampled shrimp on each day gave weak microsporidian amplicons for the 2nd-step PCR (Figure 4), indicating that approximately 1/3 of the stock shrimp had already ac- quired light microsporidian infections before the experi- ment was initiated. Similarly, in the test group, one of the 3 sampled shrimp gave a weak 2nd-step microsporidian amplicon on day 2 after challenge, while stronger reactions were obtained on days 4 and 7, with two of these giving 1st-step positive results (Figure 4). None of the shrimp in either the test or control group showed gross signs of WFS or any mortality over the 7-day test period. These results indicated that E. hepatopenaei could be transmitted hori- zontally in P. vannamei by cannibalism, and this contrasted with Agmasoma penaei where transmission to shrimp is indirect from an alternate host [2-4]. there were no distinctive cytoplasmic features that re- vealed the microsporidian elements giving rise to positive in situ hybridization reactions in adjacent tissue sections. At the highest magnification by H&E staining (oil emer- sion lens), basophilic, cytoplasmic inclusions of highly variable, shape, size and number (Figure 3g) were present in the H&E stained cells and some of these may have been of microsporidian origin but they could not be unequivo- cally distinguished from other normal, basophilic cytoplas- mic structures of the host. Because of this phenomenon, histopathological evaluation of the severity of these infec- tions by H&E staining might be misleading, if the criterion used was the number of cells showing spores or other eas- ily recognizable microsporidian structures. Laboratory challenge tests Because of the association of Enterocytozoon infections with WFS in P. vannamei (this study) and P. monodon [11], a preliminary laboratory challenge test was carried out to de- termine the possibility of direct transmission using normal P. vannamei fed with hepatopancreatic tissue of shrimp Discussion BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 have missed sequences of other copies of the ssu rRNA gene that might have been present below the level of 64% Table 3 PCR and in situ hybridization results for microsporidia in a second set of shrimp ponds Normal shrimp Pond 3 CV5 (n=10) In situ Pond 7 YOT4 (n =5) In situ sample number 1st PCR 2nd PCR Hyb 1st PCR 2nd PCR Hyb 1 - + - - - - 2 - - + - - - 3 - + + - - ND 4 + + ++ - - ND 5 - - - - - ND 6 - + - 7 - + + 8 - - + 9 - + + 10 - + + Summary Infected 9/10 Infected 0/5 WFS shrimp Pond 13 BAP (n=10) In situ Pond 6 YOT (n=10) In situ sample number 1st PCR 2nd PCR Hyb 1st PCR 2nd PCR Hyb 1* + + + + + ++ 2 - + - + + ++ 3* - - - + + ND 4 - - - - + ++ 5 - - - + + ++ 6 - - ND + + ND 7* - - ND - + ++ 8* - - ND + + ND 9 - + - - - ND 10 - + - - - ND Summary Infected 4/10 Infected 8/10 * Histology of shrimp in these ponds showed no signs of microsporidian infection but 4 showed signs of severe bacterial infections of the HP that may have masked any microsporidians present. The remaining 6 samples looked normal with no signs of microsporidian infection. Detection of microsporidia by PCR and in situ hybridization in 2 normal and 2 WFS ponds. Hyb ++ indicates extensive positive in situ hybridization reactions in HP tissue while Hyb + indicates light focal positive reactions and Hyb – indicates no reaction. ND Not done. Table 3 PCR and in situ hybridization results for microsporidia in a second set of shrimp ponds of the specimens from Pond 13 BAP. Of particular import- ance was a third set of samples from a recovered WFS pond where most of the shrimp (8/9) showed heavy E. hepatopenaei infections by in situ hybridization (Table 4) despite the absence of white feces. Unfortunately none of the HP samples of these latter shrimp were prepared for PCR. Discussion Table 4 In situ hybridization results of shrimp samples for a pond recovered from WFS Recovered WFS shrimp pond Sample number In situ Hybridization 1 ++ 2 ++ 3 - 4 ++ 5 ++ 6 ++ 7 ++ 8 ++ 9 ++ Summary Infected 8/9 Table 4 In situ hybridization results of shrimp samples for a pond recovered from WFS Table 4 In situ hybridization results of shrimp samples for a pond recovered from WFS Discussion Thus, according to veterinary sampling tables [14], could Table 1 Primer sequences used Primer name Sequence (5′→3′) Size of amplicon (bp) MF1 CCG GAG AGG GAG CCT GAGA 951 MR1 GAC GGG CGG TGT GTA CAA A 1st PCR step 779 ENF779 CAG CAG GCG CGA AAA TTG TCC A ENR779 AAG AGA TAT TGT ATT GCG CTT GCT G Nested PCR step 176 ENF176 CAA CGC GGG AAA ACT TAC CA ENR176 ACC TGT TAT TGC CTT CTC CCT CC DIG microsporidian probe 411 ENF411 AGG TGG TGT TAA AAG CCA TTG AG ENR176 TAC CTC ATG TCT CCA ATC TAC GAT A DIG GFP probe 513 GFPF513 TTC ATC TGC ACC ACC GGC AAC CTG GFPR513 CTG GTA GTG GTC GGC GAG CTG CAC Table 2 PCR results from a preliminary sample of shrimp from a WFS pond and a normal pond nearby WFS pond Normal pond Sample # 1st PCR Nested PCR 1st PCR Nested PCR 1 + + - - 2 + + - - 3 - + - - 4 + + - - 5 + + - - 6 - - - - 7 + + - - 8 + + - - 9 + + - - 10 - + - - 11 + + Table 1 Primer sequences used Primer name Sequence (5′→3′) Size of amplicon (bp) MF1 CCG GAG AGG GAG CCT GAGA 951 MR1 GAC GGG CGG TGT GTA CAA A 1st PCR step 779 ENF779 CAG CAG GCG CGA AAA TTG TCC A ENR779 AAG AGA TAT TGT ATT GCG CTT GCT G Nested PCR step 176 ENF176 CAA CGC GGG AAA ACT TAC CA ENR176 ACC TGT TAT TGC CTT CTC CCT CC DIG microsporidian probe 411 ENF411 AGG TGG TGT TAA AAG CCA TTG AG ENR176 TAC CTC ATG TCT CCA ATC TAC GAT A DIG GFP probe 513 GFPF513 TTC ATC TGC ACC ACC GGC AAC CTG GFPR513 CTG GTA GTG GTC GGC GAG CTG CAC Table 2 PCR results from a preliminary sample of shrimp from a WFS pond and a normal pond nearby WFS pond Normal pond Sample # 1st PCR Nested PCR 1st PCR Nested PCR 1 + + - - 2 + + - - 3 - + - - 4 + + - - 5 + + - - 6 - - - - 7 + + - - 8 + + - - 9 + + - - 10 - + - - 11 + + Table 2 PCR results from a preliminary sample of shrimp from a WFS pond and a normal pond nearby Page 5 of 10 Tangprasittipap et al. Discussion Overall, these results and those of the oral challenge test indicated an unlikely causal association between WSF and infection with E. hepatopenaei. On the other hand, it is possible that the severity of E. hepatopenaei infections could be increased by the unknown underlying causes of WFS, giving a superficial impression of causation. The extensive nature of the E. hepatopenaei infections in many of the specimens examined suggests that there would be a high energy demand for the developing para- site and that this would have a negative effect on host growth. Given the difficulty in assessing the severity of infection by normal H&E staining, it would seem pru- dent to monitor ponds for E. hepatopenaei by PCR, especially if growth rates are lower than predicted in the absence of other more obvious causes. We have anec- dotal information that one Thai farming operation has now adopted a policy of terminating and restocking cul- ture ponds that show a high prevalence and severity of E. hepatopenaei infection indicated by one-step PCR positive reactions within the first month of cultivation, since their records show that these ponds exhibit uneco- nomic shrimp growth. Our tests (not shown) have revealed that E. hepatopenaei is not present in the post larvae that originate from SPF stocks of P. vannamei and are used to stock cultivation ponds in Thailand. This indicates that infections found in the cultivation ponds occurred after the ponds were stocked, and it suggests that they resulted by transmission from a natural pond source such as an unknown local res- ervoir species. This contention is supported by the fact that E. hepatopenaei was discovered in Thailand in indigenous * Histology of shrimp in these ponds showed no signs of microsporidian infection but 4 showed signs of severe bacterial infections of the HP that may have masked any microsporidians present. The remaining 6 samples looked normal with no signs of microsporidian infection. Detection of microsporidia by PCR and in situ hybridization in 2 normal and 2 WFS ponds. Hyb ++ indicates extensive positive in situ hybridization reactions in HP tissue while Hyb + indicates light focal positive reactions and Hyb – indicates no reaction. ND Not done. Table 4 In situ hybridization results of shrimp samples for a pond recovered from WFS normal with no signs of microsporidian infection. Detection of microsporidia by PCR and in situ hybridization in 2 normal and 2 WFS ponds. Hyb ++ indicates extensive positive in situ hybridization reactions in HP tissue while Hyb + indicates light focal positive reactions and Hyb – indicates no reaction. ND Not done. Table 4 In situ hybridization results of shrimp samples for a pond recovered from WFS Recovered WFS shrimp pond Sample number In situ Hybridization 1 ++ 2 ++ 3 - 4 ++ 5 ++ 6 ++ 7 ++ 8 ++ 9 ++ Summary Infected 8/9 Recovered WFS shrimp pond have missed sequences of other copies of the ssu rRNA gene that might have been present below the level of 64% prevalence in the template DNA and that might have dif- fered from our consensus sequences by more than 1 or 2%. Thus, a more detailed genetic analysis of the whole, small and large subunit RNA genes, ITS regions and per- haps other genes would be needed to fully confirm our conclusion that E. hepatopenaei infects both of these shrimp species. have missed sequences of other copies of the ssu rRNA gene that might have been present below the level of 64% prevalence in the template DNA and that might have dif- fered from our consensus sequences by more than 1 or 2%. Thus, a more detailed genetic analysis of the whole, small and large subunit RNA genes, ITS regions and per- haps other genes would be needed to fully confirm our conclusion that E. hepatopenaei infects both of these shrimp species. We have no explanation regarding the discrepancy be- tween a strong 1st-step positive PCR test result despite the appearance of a light in situ hybridization reaction for one Summary Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Page 6 of 10 Figure 2 Agarose gels showing nested PCR microsporidian-specific amplicons using 100 ng of total DNA template from hepatopancreatic tissue obtained from P. vannamei not challenged (A) or orally challenged (B) with the microsporidian. Lane 1-3:Three specimens 2 days post-challenge; Lane 4-6: Three specimens 4 days post-challenge, Lane 7-9: Three specimens 7 days post-challenge; Lane 10: plasmid positive control. The amplicon sizes for the 1st and 2nd-step PCR are 779 and 176 bp, respectively. Table 4 In situ hybridization results of shrimp samples for a pond recovered from WFS Figure 2 Agarose gels showing nested PCR microsporidian-specific amplicons using 100 ng of total DNA template from hepatopancreatic tissue obtained from P. vannamei not challenged (A) or orally challenged (B) with the microsporidian. Lane 1-3:Three specimens 2 days post-challenge; Lane 4-6: Three specimens 4 days post-challenge, Lane 7-9: Three specimens 7 days post-challenge; Lane 10: plasmid positive control. The amplicon sizes for the 1st and 2nd-step PCR are 779 and 176 bp, respectively. Conclusions P. monodon long before it was found in exotic P. vannamei [10,13]. Thus, our results indicate that the best strategy for controlling its infections in cultivation ponds would be to focus on identification of the reservoir species and on elim- inating them from the shrimp cultivation system. This ap- proach has been successful for the control of Agmasoma penaei in cultivated shrimp in Thailand. It would also be worthwhile (because of the capability of horizontal trans- mission of E. hepatopanaei) to determine whether or not its residual spores in a disease outbreak pond are elimi- nated by current procedures for preparation of ponds be- tween cultivation cycles. Although we have shown that the microsproidian E. hepatopenaei is often found in cultivated P. vannemei exhibiting WFS, it is unlikely that the parasite is causally as- sociated with WFS, although the severity of E. hepatopenaei infections may be exacerbated by the underlying causes of WFS. Since this microsporidian is not present in Thai SPF stocks, pond infections are probably initiated by transmis- sion from one or more local reservoir species. Thus, the most effective control strategy would be to identify the res- ervoir species and exclude it (them) from the shrimp pro- duction system. The PCR method and in situ hybridization Figure 3 Photomicrographs of microsporidian spores in hepatopancreatic cells. Microsporidian spores (arrows) inside B cells of the hepatopancreatic tubule epithelium (left) and free in the tubule lumen (right). Such cells were present in the section in low numbers, giving a misleading impression of the extent of the severe microsporidian infection evident by in situ hybridization as seen in Figure 2. The magnification bar applies to both images. Figure 3 Photomicrographs of microsporidian spores in hepatopancreatic cells. Microsporidian spores (arrows) inside B cells of the hepatopancreatic tubule epithelium (left) and free in the tubule lumen (right). Such cells were present in the section in low numbers, giving a misleading impression of the extent of the severe microsporidian infection evident by in situ hybridization as seen in Figure 2. The magnification bar applies to both images. Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Page 7 of 10 Page 7 of 10 Figure 4 Photomicrographs of infected hepatopancreatic tissue of P. vannamei. The adjacent sections of shrimp tissue stained with H&E (column 1) and with the in situ hybridization probe (column 2) showing that hepatopancreatic cells of P. Conclusions vannamei infected with the microsporidian cannot be easily detected by H&E staining even though extensive infection is revealed by in situ hybridization (dark brown to black staining). (a/b) Low magnification showing that positive reactions are restricted to the medial and proximal tubule epithelial cells of the shrimp hepatopancreas (HP) while the distal E cells are negative. Note that B cells dominate in the infected region. (c/d) Medium high magnification showing pinpoint positive, in situ hybridization reactions in the region of the HP adjacent to the distal E cells region. (e/f) High magnification clearly showing the difficulty in identifying infected cells by H&E staining but their clear revelation by in situ hybridization. (g/h) Very high magnification, emphasizing the features described in (e/f). Figure 4 Photomicrographs of infected hepatopancreatic tissue of P. vannamei. The adjacent sections of shrimp tissue stained with H&E (column 1) and with the in situ hybridization probe (column 2) showing that hepatopancreatic cells of P. vannamei infected with the microsporidian cannot be easily detected by H&E staining even though extensive infection is revealed by in situ hybridization (dark brown to black staining). (a/b) Low magnification showing that positive reactions are restricted to the medial and proximal tubule epithelial cells of the shrimp hepatopancreas (HP) while the distal E cells are negative. Note that B cells dominate in the infected region. (c/d) Medium high magnification showing pinpoint positive, in situ hybridization reactions in the region of the HP adjacent to the distal E cells region. (e/f) High magnification clearly showing the difficulty in identifying infected cells by H&E staining but their clear revelation by in situ hybridization. (g/h) Very high magnification, emphasizing the features described in (e/f). method developed here will be useful in identifying reser- voir species. harvesting of fish and crustaceans (http://www.dpi. nsw.gov.au/agriculture/livestock/animal-welfare/gene- ral/fish/shellfish; 30 March 2013) with respect to details regarding the transport of the shrimp and their laboratory maintenance. With respect to processing the shrimp for histological analysis or for killing at the end of an experi- ment, the salt water/ice slurry method was used as recommended in the Australian guidelines. Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 the shrimp were swabbed with 70% ethanol and the cara- pace was removed so that approximately 100 mg of outer hepatopancreatic tissue (excluding contamination from the internal portion of the stomach and midgut) could be trans- ferred aseptically to DNA extraction buffer (see below). The remainder of the whole hepatopancreas (including the an- terior midgut caecum, the posterior chamber of the stom- ach and a portion of the midgut) was injected with Davidson’s fixative and processed for histological analysis as previously described [15]. A second set of juvenile P. vannamei specimens exhibiting white feces syndrome were collected from an intensive shrimp farm in Chanthaburi province, Thailand during August –September 2011 to- gether with grossly normal shrimp from a nearby pond. Some of these shrimp were processed for DNA extraction from hepatopancreatic tissue as described above while the remaining were used in tests for transmission of the microsporidian by feeding of infected hepatopancreatic tis- sue to normal shrimp. the shrimp were swabbed with 70% ethanol and the cara- pace was removed so that approximately 100 mg of outer hepatopancreatic tissue (excluding contamination from the internal portion of the stomach and midgut) could be trans- ferred aseptically to DNA extraction buffer (see below). The remainder of the whole hepatopancreas (including the an- terior midgut caecum, the posterior chamber of the stom- ach and a portion of the midgut) was injected with Davidson’s fixative and processed for histological analysis as previously described [15]. A second set of juvenile P. vannamei specimens exhibiting white feces syndrome were collected from an intensive shrimp farm in Chanthaburi province, Thailand during August –September 2011 to- gether with grossly normal shrimp from a nearby pond. Some of these shrimp were processed for DNA extraction from hepatopancreatic tissue as described above while the remaining were used in tests for transmission of the microsporidian by feeding of infected hepatopancreatic tis- sue to normal shrimp. region of the ssu rRNA sequence of E. hepatopenaei (Genbank : FJ496356) using Clustal-W2 (http://www.ebi.ac. uk/Tools/msa/clustalw2/). Among three clone sequences, two sequences were 100% identical, whereas one was differ- ent at 2 /951 bases analyzed. The clone named MF12 was used as the positive control template for PCR reactions. To verify the GenBank record FJ496356 for the ssu rRNA sequence of the microsporidian E. hepatopenaei from P. Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 monodon [12], we used the same PCR protocol described above to re-amplify and re-clone the ssu rRNA gene fragment from archived DNA used as the template that gave rise to the FJ496356 record. The amplicon was cloned and 3 clones were sequenced to obtain a consen- sus sequence. Preparation of DNA templates Hepatopancreatic tissue was homogenized in lysis buffer (50 mM Tris–HCl, pH 8.0, 50 mM EDTA, 1% SDS, 10mM NaCl) containing 5 μg/ml proteinase K. Genomic DNA was isolated and purified by the phenol-chloroform method [16] and concentrations were determined by measuring UV absorption at 260 nm. All DNA templates were adjusted to a concentration of 50 ng/μl with distilled water for PCR tests. PCR detection of the microsporidian infection in P. vannamei Two pairs of specific primers, ENF779/ ENR779 and ENF176/ENR176 were designed from the amplicon de- scribed above for a nested PCR protocol to enhance specifi- city and sensitivity for detection of the new microsporidian (Table 1). The PCR amplification was carried out in 25 μl reaction mixture containing, 200 mM dNTP, 1.5 mM MgCl2, 0.1 mM primers and 0.625 unit Taq polymerase (Invitrogen). For the first step PCR reaction, the protocol consisted of initial denaturation at 94°C for 3 min followed by 35 cycles of denaturation at 94°C for 20 sec, annealing at 58°C for 20 sec and extension at 72°C for 45 sec with a final extension at 72°C for 5 min. The second, nested PCR reac- tion was carried out using 1 μl of the first PCR product as template. The PCR reaction conditions consisted of initial denaturation at 94°C for 3 min followed by 35 cycles of de- naturation at 94°C for 20 sec, annealing at 64°C for 20 sec and extension at 72°C for 20 sec with an additional exten- sion at 72°C for 5 min. The PCR products were visualized by 1.5% of agarose gel electrophoresis, ethidium bromide staining and gel placement on a UV transillumator. To de- termine the sensitivity of detection, 10-fold serial dilutions (106 – 1 copies of plasmid) were prepared and subjected to the nested PCR protocol. Sources of WFS and grossly normal farmed shrimp specimens Since the Ethical Principles and Guidelines for the Use of Animals of the National Research Council of Thailand (1999) apply to vertebrates only and there is no official standard for invertebrates, we adapted its principles to shrimp. We also followed the guidelines of the Austra- lian, New South Wales state government for the humane One set of shrimp samples was collected from Surathani province in southern Thailand on 28 October 2010 and consisted of shrimp from 2 ponds exhibiting WFS, 1 pond recovered from WFS and 2 normal ponds. After stunning, Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Page 8 of 10 Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Cloning of a microsporidian small subunit ribosomal RNA gene fragment A microsporidian ssu rRNA gene fragment was amplified from WFS P. vannamei by PCR as previously described [12]. Briefly, the primers MF1 and MR1 (Table 1) were designed from an ssu rRNA fragment of Enterocytozoon hepatopenaei isolated from P. monodon and relative to po- sitions 242–260 and 1165–1183, respectively, of Genbank record FJ496356. The PCR process was carried out in a 25 μl reaction mixture containing PCR buffer, 0.2 mM dNTP, 1.5 mM MgCl2, 0.1 μM primers, 0.625 units Taq DNA polymerase (Invitrogen), and 1 μl template. The PCR proto- col consisted of 35 cycles of denaturation at 94°C for 20 sec, annealing at 64°C for 20 sec, and extension at 72°C for 45 sec, followed by a 5 min final extension at 72°C. An srRNA gene fragment of 900–1,000 bp was amplified and cloned using a pGEMT-easy cloning Kit (Promega). Plas- mids were extracted from three clones, purified using a plasmid extract kit (Geneaid) and sequenced in both direc- tions by two universal vector primers, SP6 and T7, by Macrogen, Korea. The consensus sequence obtained (minus the primers) was subjected to a BLASTN (http:// www.ncbi.nlm.nih.gov/BLAST) search against the GenBank database and then it was aligned with the corresponding References h 1. Lightner DV: A handbook of pathology and diagnostic procedures for diseases of penaeid shrimp. Baton Rouge, LA: World Aquaculture Society; 1996. 1. Lightner DV: A handbook of pathology and diagnostic procedures for diseases of penaeid shrimp. Baton Rouge, LA: World Aquaculture Society; 1996. 2. Flegel TW, Boonyaratpalin S, Fegan DF, Guerin M, Sriurairatana S: High mortality of black tiger prawns from cotton shrimp disease in Thailand. In Diseases in Asian Aquaculture I. Edited by Shariff M, Subasinghe RP, Arthur JR. Manila: Fish Health Section, Asian Fisheries Society; 1992:181–197. y 3. Pasharawipas T, Flegel TW, Chaiyaroj S, Mongkolsuk S, Sirisinha S: Comparison of amplified RNA gene sequences from microsporidian parasites (Agmasoma or Thelohania) in Penaeus merguiensis and P. monodon. Asian Fisheries Science 1994, 7:169–178. 4. Pasharawipas T, Flegel TW: A specific DNA probe to identify the intermediate host of a common microsporidian parasite of Penaeus merguiensis and P. monodon. Asian Fish Sci 1994, 7:157–167. 5. Kelly JF: Tissue specificities of Thelohania duorara, Agmasoma penaei, and Pleistophora sp., microsporidian parasites of pink shrimp, Penaeus duorarum. J Invertebr Pathol 1979, 33:331–339. 3. Pasharawipas T, Flegel TW, Chaiyaroj S, Mongkolsuk S, Sirisinha S: Comparison of amplified RNA gene sequences from microsporidian parasites (Agmasoma or Thelohania) in Penaeus merguiensis and P. monodon. Asian Fisheries Science 1994, 7:169–178. 4. Pasharawipas T, Flegel TW: A specific DNA probe to identify the intermediate host of a common microsporidian parasite of Penaeus merguiensis and P. monodon. Asian Fish Sci 1994, 7:157–167. 5. Kelly JF: Tissue specificities of Thelohania duorara, Agmasoma penaei, and Pleistophora sp., microsporidian parasites of pink shrimp, Penaeus duorarum. J Invertebr Pathol 1979, 33:331–339. 6. Limsuwan C, Chuchird N, Laisutisan K: Efficacy of calcium hypochlorite on the prevalence of microsporidiosis (Thelohania) in pond-reared Litopenaeus vannamei. Kasetsart J Nat Sci 2008, 42:282–288. 7. Laisutisan K, Prasertsri S, Chuchird N, Limsuwan C: Ultrastructure of the microsporidian Thelohania (Agmasoma) penaei in the Pacific white shrimp Litopenaeus vannamei). Kasetsart Univ Fish Res Bull (Thailand) 2009, 33:41–48. 8. Prasertsri S, Limsuwan C, Chuchird N: The effects of microsporidian (Thelohania) infection on the growth and histological changes in pond- reared Pacific white shrimp (Litopenaeus vannamei). Kasetsart J Nat Sci 2009, 43:680–688. Laboratory challenge tests Received: 1 November 2012 Accepted: 24 June 2013 Published: 15 July 2013 Pacific white shrimp weighing 6–8 grams were obtained from a farm in Chanthaburi province, Thailand, and accli- mated at the Aquaculture Business Research Center Labora- tory, Faculty of Fisheries, Kasetsart University for 1 week. During the acclimatization period, shrimp were fed with commercial pellet feed. After that, 60 shrimp were randomly stocked in 6 aquaria (80 l each) with 10 shrimp per aquar- ium. The shrimp were divided into 2 groups of 30 animals (i.e., 3 aquaria) per group. Shrimp from the treatment group were fed with hepatopancreatic tissue of E. hepatopenaei- infected shrimp every day for 7 days (once a day and an- other meal fed with commercial pellet feed) while shrimp from the control group were fed with a commercial pellet feed twice a day. Dissolved oxygen (DO), Salinity, pH, and temperature during the acclimation period and the experi- ment were maintained at 4 ppm, 25 ppt, 7.8–8.0, and 28°C, respectively. The test was terminated after 7 days. The shrimp were sampled (one from each aquarium) on days 2, 4 and 7 after the feeding for PCR testing using the E. hepatopenaei primers. They were also monitored for mor- tality and signs of WFS. Authors’ contributions AT carried out the in situ hybridization studies, histological examination and drafted the manuscript. SC carried out the histological examination. JS carried out the molecular cloning and sequence analysis. MS, NC and CL participated in the experimental designs, performed bioassays and specimen collection. TS participated in design of the study and preparation of the specimens. TF and KS participated in the design of the study and coordination and completed the manuscript. All authors read and approved the final manuscript. Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Page 9 of 10 10 min at 37°C. Each section was overlaid with 200 μl of pre-hybridization solution [4 × SSC and 50% (v/v) deionized formamide] and incubated at 37°C for 30 min before the so- lution was replaced with 200 μl of hybridization mix containing the DIG-labeled probe (approximately 20 ng/ slide) and covered with a coverslip. The hybridization reaction was carried out at 42°C for 20 h in a humid cham- ber to avoid evaporation. After the sections were washed with high stringency, they were incubated with 0.5% blocking solution (Roche, Germany) for 30 min at room termperature. The sections were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (1:500 dilution). Unbound antibody was washed off twice and equilibrated in detection buffer (100 mM Tris–HCl, 100 mM NaCl and 50 mM MgCl2, pH 9.5). The signal was de- veloped by addition of NBT-BCIP substrate (Roche, Germany) and counterstaining was accomplished with Bis- marck brown Y (Sigma, USA). The slides were observed and photographed using an Olympus microscope with a digital camera. 10 min at 37°C. Each section was overlaid with 200 μl of pre-hybridization solution [4 × SSC and 50% (v/v) deionized formamide] and incubated at 37°C for 30 min before the so- lution was replaced with 200 μl of hybridization mix containing the DIG-labeled probe (approximately 20 ng/ slide) and covered with a coverslip. The hybridization reaction was carried out at 42°C for 20 h in a humid cham- ber to avoid evaporation. After the sections were washed with high stringency, they were incubated with 0.5% blocking solution (Roche, Germany) for 30 min at room termperature. The sections were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (1:500 dilution). Unbound antibody was washed off twice and equilibrated in detection buffer (100 mM Tris–HCl, 100 mM NaCl and 50 mM MgCl2, pH 9.5). The signal was de- veloped by addition of NBT-BCIP substrate (Roche, Germany) and counterstaining was accomplished with Bis- marck brown Y (Sigma, USA). The slides were observed and photographed using an Olympus microscope with a digital camera. Acknowledgements This work was supported by grants from National Research Council of Thailand. A Post doctoral fellowship granted to Dr. A. Tangprasittipap was from the Office of the Higher Education Commission (CHE) and Mahidol University under the National Research University Initiatives. Author details 1Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI rd, Bangkok 10400, Thailand. 2Shrimp-Virus Interaction Laboratory, National Center for Genetic Engineering and Biotechnology, Soi Yothi, Rama VI rd, Bangkok 10400, Thailand. 3National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency (NSTDA), Thailand Science Park, Phaholyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand. 4Aquaculture Business Research Center, Kasetsart University, Bangkok 10900, Thailand. 5Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI rd, Bangkok 10400, Thailand. 6Department of Livestock Development, 69/1 Phayathai Road, Bangkok 10400, Thailand. Additional file 9. Toubiana M, Guelorget O, Bouchereau JL, Lucien-Brun H, Marques A: Microsporidians in penaeid shrimp along the west coast of Madagascar. Dis Aquat Org 2004, 58:79–82. Additional file 1: Agarose gel analysis of PCR amplicons obtained using MF primers from [12]. Lane 1: MF12 plasmid positive control template; Lane 2: Total DNA extract template from a heavily infected shrimp; Lane 3: Same as lane 2 but lightly infected; Lane 4: Negative control DNA template from an uninfected shrimp; Lane 5: Distilled water template negative control. 10. Tourtip S: Histology, ultrastructure and molecular biology of a new microsporidium infecting the black tiger shrimp Penaeus monodon. PhD Thesis. Bangkok: Mahidol University; 2005. 11. Ha NTH, Ha DT, Thuy NT, Lien VTK: Enterocytozoon hepatopenaei Parasitizing on tiger shrimp (Penaeus monodon) infected by white feces culture in Vietnam, has been detected (In Vietnamese with English abstract). Agriculture and rural development: science and technology (translation from Vietnamese) 2010, 12:45–50. 11. Ha NTH, Ha DT, Thuy NT, Lien VTK: Enterocytozoon hepatopenaei Parasitizing on tiger shrimp (Penaeus monodon) infected by white feces culture in Vietnam, has been detected (In Vietnamese with English abstract). Agriculture and rural development: science and technology (translation from Vietnamese) 2010, 12:45–50. In situ hybridization A Di PCR l b li In situ hybridization A Dig-PCR labeling Kit (Roche, Germany) was used to prepare a probe for in situ hybridization using the primers shown in Table 1. A similarly labeled GFP-Dig probe was used as a negative control. Plasmid clones MF12 and pEGFP–N1 (Clontech) containing relevant inserts were used as templates for Enterocytozoon sp. and the negative control, respectively. Dig-labeled probes were purified using a PCR purification kit (Geneaid) and labeling efficiency was deter- mined by dot blot hybridization. Shrimp were fixed in Davidson's fixative overnight before processing for routine paraffin embedding [15]. Tissue sections were digested with 10 μg/ml Proteinase K (Invitrogen, USA) in TNE buffer for Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Page 9 of 10 Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 The authors declare that they have no competing interests. Competing interests h h d l h The authors declare that they have no competing interests. Page 10 of 10 Page 10 of 10 Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 Tangprasittipap et al. BMC Veterinary Research 2013, 9:139 http://www.biomedcentral.com/1746-6148/9/139 12. Tourtip S, Wongtripop S, Stentiford GD, Bateman KS, Sriurairatana S, Chavadej J, Sritunyalucksana K, Withyachumnarnkul B: Enterocytozoon hepatopenaei sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships. J Invertebr Pathol 2009, 102(1):21–29. 13. Chayaburakul K, Nash G, Pratanpipat P, Sriurairatana S, Withyachumnarnkul B: Multiple pathogens found in growth-retarded black tiger shrimp Penaeus monodon cultivated in Thailand. Dis Aquat Org 2004, 60:89–96. g 14. Cameron A: Survey toolbox for aquatic animal diseases; a practical manual and software package. Canberra: Australian Centre for International Agricultural Research; 2002. 15. Bell TA, Lightner DV: A handbook of normal shrimp histology. Baton Rouge, LA: World Aquaculture Society; 1988. 16. Sambrook J, Russell DW: Molecular cloning:a laboratory manual 3rd Edition. Cold Spring Harbor: Cold Spring Harbor Press; 2001. doi:10.1186/1746-6148-9-139 Cite this article as: Tangprasittipap et al.: The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus (Litopenaeus) vannamei. BMC Veterinary Research 2013 9:139. 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https://openalex.org/W4367052092	https://figshare.com/articles/journal_contribution/Economic_Consequences_of_Greylisting_by_the_Financial_Action_Task_Force/23274677/1/files/41022329.pdf	English	null	Economic Consequences of Greylisting by the Financial Action Task Force	Risks	2,023	cc-by	20,369	Article Economic Consequences of Greylisting by the Financial Action Task Force † Louis de Koker 1,2,* , John Howell 3 and Nicholas Morris 1 1 La Trobe Law School, La Trobe University, Bundoora, VIC 3086, Australia; n.morris@latrobe.edu.au 2 Department of Mercantile and Labour Law, University of the Western Cape, Cape Town 7535, South Africa 1 La Trobe Law School, La Trobe University, Bundoora, VIC 3086, Australia; n.morris@latrobe.edu.au 2 Department of Mercantile and Labour Law, University of the Western Cape, Cape Town 7535, South Africa 3 JH Dragon Network, Guildford GU5 0UU, UK; john.howell@jh-co.com * Correspondence: l.dekoker@latrobe.edu.au † This paper is an extended and revised version of our paper presented at the Third Bahamas Empirical Anti-Money Laundering Conference, hosted in January 2022 by the Central Bank of The Bahamas. 1 La Trobe Law School, La Trobe University, Bundoora, VIC 3086, Australia; n.morris@latrobe.edu.au 2 Department of Mercantile and Labour Law, University of the Western Cape, Cape Town 7535, South Africa 3 JH Dragon Network, Guildford GU5 0UU, UK; john.howell@jh-co.com † This paper is an extended and revised version of our paper presented at the Third Bahamas Empirical Anti-Money Laundering Conference, hosted in January 2022 by the Central Bank of The Bahamas. † This paper is an extended and revised version of our paper presented at the Third Bahamas Empirical Anti-Money Laundering Conference, hosted in January 2022 by the Central Bank of The Bahamas. Abstract: This study considers the impact of the FATF’s greylisting process from a market perspective. The results are intended to inform the development of public policy and improvement of market signalling. The study develops a theoretical market impact model and identiﬁes the indicators which may impact banking operations and institutional decisions. It is explicitly market-oriented in that the model seeks to reﬂect how stakeholders in ﬁnancial and non-ﬁnancial markets typically respond to signals sent out by the FATF. The authors ﬁnd that the FATF’s greylisting signalling has changed over time and distinguish four broad periods reﬂecting different messaging. The study uses ﬁnancial, trade, and other variables derived from the World Bank’s ‘World Development Indicators’ databank to explore evidence of impact in each of the different phases of the FATF’s approach to greylisting. The study uses a pooled cross-section and time series approach with ﬁxed effects, based on a sample of 177 countries and 3540 country-years of data from 2000 to 2020. Citation: de Koker, Louis, John Howell, and Nicholas Morris. 2023. Economic Consequences of Greylisting by the Financial Action Task Force. Risks 11: 81. https:// doi.org/10.3390/risks11050081 Keywords: FATF; greylisting; blacklisting; remediation; anti-money laundering Article Economic Consequences of Greylisting by the Financial Action Task Force † The study examines impacts on net ofﬁcial development assistance, the banking environment (non-performing loans, risk premiums), net foreign assets, indebtedness, and market capitalisation. It ﬁnds signiﬁcant correlations between many ﬁnancial variables and FATF listing events, including an apparent reduction in development assistance during greylisting periods which endured after the country was delisted. This is of signiﬁcant concern, as such reductions may impact disproportionately on developing economies. risks risks risks Citation: de Koker, Louis, John Howell, and Nicholas Morris. 2023. Economic Consequences of Greylisting by the Financial Action Task Force. Risks 11: 81. https:// doi.org/10.3390/risks11050081 1. Introduction This study investigates economic risks and impacts for countries included in the list of ‘jurisdictions under increased monitoring’, also known as the ‘grey list’, by the Financial Action Task Force (FATF), the global standard-setter for Anti-Money Laundering and Counter Financing of Terrorism and Proliferation (AML/CFT/CPF). Countries on the grey list have failed to adhere appropriately to the FATF standards but committed themselves to resolving the identiﬁed strategic deﬁciencies within agreed timeframes, during which they are subject to increased monitoring by the FATF. In the ﬁrst quarter of 2023, 24 jurisdictions were listed on the FATF grey list, including Nigeria, South Africa, and Türkiye (FATF 2023). Academic Editor: Mogens Steffensen Received: 29 January 2023 Revised: 11 March 2023 Accepted: 17 April 2023 Published: 25 April 2023 Received: 29 January 2023 Revised: 11 March 2023 Accepted: 17 April 2023 Published: 25 April 2023 g y g g y Studies have found that the FATF typically moved listed countries, predominantly developing economies, to higher levels of compliance with FATF standards within a rea- sonable period of time because governments responded with concern about the negative economic impact that listing might impose (Sharman 2009; Morse 2022; FATF 2015). The studies are, however, divided on whether greylisting actually created negative economic consequences. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). This study considers FATF’s greylisting impact from a market perspective, i.e., as it affects both private and public sector stakeholders. The study identiﬁes different phases in market signalling engendered by the FATF’s greylisting process, develops a theoretical https://www.mdpi.com/journal/risks Risks 2023, 11, 81. https://doi.org/10.3390/risks11050081 Risks 2023, 11, 81 2 of 32 market impact model, and identiﬁes indicators to be studied which may impact both banking operations and institutional decisions. It explores evidence of impact in each of the phases using indicators derived from the World Bank’s ‘World Development Indicators’ databank. The study is novel in that it considers the whole time period over which the FATF has been operating; divides this period into phases during which the FATF’s signalling and processes varied; explores empirical evidence on the impact of listings across all countries for which usable data is available, and not just a sample of countries; seeks to identify the different impacts of listing, remediation, and delisting; and examines whether the impact occurs immediately or after a time lag. The approach is explicitly market-oriented, in that the theoretical model used for identifying variables which may be impacted seeks to reﬂect how stakeholders in ﬁnancial and non-ﬁnancial markets may typically respond to the signals sent out by the FATF. Using a pooled cross-section and time series approach with ﬁxed effects, based on a sample of 177 countries and 3540 country-years of data from 2000 to 2020, the study ﬁnds signiﬁcant correlations between many ﬁnancial variables and FATF listing events. This does not of itself prove causation, as it may be that the FATF listing and these variables are simply co-determined. p y A major ﬁnding is that net ofﬁcial development assistance (ODA), International Bank for Reconstruction and Development (IBRD) loans, and International Development Associ- ation (IDA) credits seem to reduce during greylisting periods and this reduction endures after the country becomes delisted. This is of signiﬁcant concern as such reduction may impact disproportionately on developing economies and their populations. The study ﬁnds evidence of improvements in the banking environment (non-performing loans, risk premiums) as well as some evidence of reductions in net foreign assets and increased indebtedness in the earlier listing phases. In Period III (2010–2015), which saw the most intense FATF activity, listing is correlated with a decline in market capitalisation, although remediation seems to reverse this, encouraging lenders and improving net ﬁnancial ﬂows and exchange rates. Negative correlation between net ODA, IBRD and IDA loans and credits and listings are evident in Periods III and IV (2016–2021), with the exception that net ODA is positively correlated in the remediation period in Period IV. There is also signiﬁcant evidence of correlation between the remediation and delisting process and reduced GDP growth rates. These ﬁndings are of concern from a development policy perspective and should inform further investigation of the use of greylisting along with a FATF review of its market signalling impact as well as the regulatory and market signalling responses of its members and other regulators. Measures resulting in economic hardship for countries when they are working with the FATF to improve their compliance levels require closer policy scrutiny. The remainder of this paper is organised as follows. It contains a section that describes the background to the study, a literature review, a section that describes the materials and methods used, followed by the results, a discussion of the results, and the conclusions. 2. Background The Financial Action Task Force (FATF), housed at the OECD in Paris, is the inter- governmental global standard-setter for anti-money laundering, and counter-terrorist and proliferation ﬁnancing (AML/CTF/CPF). More than 200 jurisdictions are committed to ensuring technical compliance with the FATF standards, known as the ‘Forty Recommen- dations’, as well as effective implementation of compliant AML/CTF/CPF measures. The FATF monitors progress on adoption and implementation of its standards. With FATF-style regional bodies (FSRBs), it assesses whether countries have the necessary legal and institutional frameworks in place and how effectively they are implementing them. This peer review process, known as Mutual Evaluation, applies an agreed methodology (Kyriakos-Saad 2005; FATF 2004b, 2013–2021). Risks 2023, 11, 81 3 of 32 3 of 32 The FATF has no formal powers of compulsion but its standards, especially Recom- mendations 10 (Customer due diligence) and 19 (Higher-risk countries), require countries to ensure that their ﬁnancial institutions apply appropriate countermeasures where countries fail to meet the standards appropriately (FATF 2012–2023). These measures include apply- ing enhanced due diligence measures to business relationships and transactions relating to countries where this is called for by the FATF, and also where enhanced risk requires them to adopt such risk mitigation measures. Within this context, the FATF has been advising speciﬁc action against countries that fail to show adequate commitment to compliance (Drezner 2008; Nance 2015; Morse 2017). In 2000 and 2001 the FATF listed 23 countries and territories publicly in its Non-Cooperative Countries or Territories (NCCT) program. According to the FATF, this process was highly successful as “all of the jurisdictions listed in 2000 and 2001 made signiﬁcant progress and the last country was removed from the list in October 2006” (FATF 2022a). Since 2007, and with improved processes adopted in 2009, the FATF’s International Co- operation Review Group (ICRG) has identiﬁed, examined and worked with jurisdictions that were failing to adequately implement AML/CFT/CPF measures. A jurisdiction that enters the ICRG review process as a result of a disappointing mutual evaluation has 12 months to collaborate with the FATF or its FSRB to address the identiﬁed deﬁciencies and avoid possible public identiﬁcation and formal review by the FATF. The FATF then prioritises the review of those countries with more signiﬁcant ﬁnancial sectors, e.g., USD 5 billion or more in ﬁnancial sector assets. 2. Background At this point, when the country has committed to addressing the identiﬁed deﬁciencies, the FATF may move to have it placed on its so-called greylist (FATF 2022a). 2.1. Black and Greylisting The FATF currently publishes two listing statements at the end of each of its three plenary meetings, in February, June, and October. The two statements reﬂect the different levels of risk posed at any given time by the deﬁciencies in the listed jurisdictions: • High-Risk Jurisdictions subject to a Call for Action (previously called “Public State- ment”, more generally known as its blacklist): these high-risk jurisdictions have sig- niﬁcant strategic deﬁciencies in their AML/CFT/CPF regimes and FATF calls on all jurisdictions to implement “counter-measures” against these countries to miti- gate the risks they pose. These measures slow down and even prevent commercial engagements, reinforcing economic sanctions against the countries currently listed. • High-Risk Jurisdictions subject to a Call for Action (previously called “Public State- ment”, more generally known as its blacklist): these high-risk jurisdictions have sig- niﬁcant strategic deﬁciencies in their AML/CFT/CPF regimes and FATF calls on all jurisdictions to implement “counter-measures” against these countries to miti- gate the risks they pose. These measures slow down and even prevent commercial engagements, reinforcing economic sanctions against the countries currently listed. • High-Risk Jurisdictions subject to a Call for Action (previously called “Public State- ment”, more generally known as its blacklist): these high-risk jurisdictions have sig- niﬁcant strategic deﬁciencies in their AML/CFT/CPF regimes and FATF calls on all jurisdictions to implement “counter-measures” against these countries to miti- gate the risks they pose. These measures slow down and even prevent commercial engagements, reinforcing economic sanctions against the countries currently listed. For a number of years only two countries, Iran and North Korea, were ‘blacklisted’. Myanmar was added in October 2022 but in a special category where FATF calls for enhanced due diligence measures proportionate to the risks to be applied, rather than more drastic countermeasures. Myanmar was added due to unsatisfactory progress with its action plan to address its strategic deﬁciencies and this listing reﬂects elements of the dark-grey list that the FATF published in Period III, discussed below. For a number of years only two countries, Iran and North Korea, were ‘blacklisted’. Myanmar was added in October 2022 but in a special category where FATF calls for enhanced due diligence measures proportionate to the risks to be applied, rather than more drastic countermeasures. 2.2. Phases in the FATF’s Greylisting Process As explained above, the FATF currently publishes two lists, a so-called blacklist and a greylist (Riccardi 2022; Morse 2022). This study focuses on the greylist. Some authors refer to both grey- and blacklisting as “blacklisting” but, given the difference between the impact on blacklisted countries (e.g., on Iran and North Korea) and the impact on the large number of countries that are greylisted, the term “greylisting” is used here to distinguish the two lists and listing processes. g p Since 2000 FATF’s communications and signals regarding greylisting have changed on several occasions. These communications are made in formal, public statements to inform risk mitigation measures to be taken by countries and ﬁnancial institutions and changes in messaging could impact on market responses (Ederington and Lee 1993; Farka and Fleissig 2012). Previous impact studies differentiated mainly between the NCCT period and the post-NCCT period based on changes in the listing processes and have not speciﬁcally considered different messaging periods in the listing processes. The authors distinguish four main periods. The ﬁrst period commenced with the NCCT listings, which is classiﬁed as a greylisting process as it is more closely aligned in nature with the latter than with blacklisting and the accompanying countermeasures. With this as the starting point, and considering the FATF’s labelling, messaging, and market signalling in its statements, the following four periods are identiﬁed: • Period I—2000–2006: following discussions that began in 1998 (FATF 2007a), the FATF in February 2000 agreed on 25 criteria to be applied to identify countries with “detri- mental rules and practices which impair the effectiveness of their money-laundering prevention and detection systems, as well as the results of their judicial enquiries in this area” (FATF 2000). The FATF published its ﬁrst list of 15 “non-compliant countries or territories” (NCCTs) in June 2000 (FATF 2000) and added another 8 jurisdictions in 2001 (FATF 2001). The public naming and shaming of these countries put pressure on them to improve their compliance levels (FATF 2003a, 2015; Sharman 2009; Morse 2022). No further countries were added but listed countries were delisted when they reached the desired compliance level. The last country on the list, Myanmar, was delisted in October 2006. 2.1. Black and Greylisting Myanmar was added due to unsatisfactory progress with its action plan to address its strategic deﬁciencies and this listing reﬂects elements of the dark-grey list that the FATF published in Period III, discussed below. For a number of years only two countries, Iran and North Korea, were ‘blacklisted’. Myanmar was added in October 2022 but in a special category where FATF calls for enhanced due diligence measures proportionate to the risks to be applied, rather than more drastic countermeasures. Myanmar was added due to unsatisfactory progress with its action plan to address its strategic deﬁciencies and this listing reﬂects elements of the dark-grey list that the FATF published in Period III, discussed below. • Jurisdictions under Increased Monitoring (previously called “Improving Global AML/ CFT Compliance: On-going process”, more generally known as the greylist): these jurisdictions are actively working with the FATF to address the identiﬁed strategic deﬁciencies in their AML/CFT/CPF regimes. These countries have committed to swiftly resolving these within agreed timeframes. Other countries are advised to consider this information and, though the FATF explicitly states that it does not call for enhanced due diligence in relation to transactions and business linked to these countries, such risk mitigation measures are often triggered by a listing. The neat division between these two lists was blurred for some time in the period 2011–2015 by the publication of a list of jurisdictions that had strategic deﬁciencies and that had not committed to an action plan to address them or had not made sufﬁcient progress implementing that plan, the so-called dark-grey list. In these cases, FATF signalled that enhanced due diligence measures may be appropriate as it explicitly called on its members to consider the risks arising from the deﬁciencies associated with each jurisdiction. Risks 2023, 11, 81 4 of 32 To be removed from FATF monitoring, a jurisdiction must address all (or nearly all) the components of its action plan as conﬁrmed by an on-site visit. 2.2. Phases in the FATF’s Greylisting Process 2.2. Phases in the FATF’s Greylisting Process In October 2009 Iran, Pakistan, São Tomé and Príncipe and Turkmenistan still appeared on the same series of public statements (FATF 2009). The remaining countries were added to the new Period III statements that commenced in February 2010. • Period III—2010–2015: in June 2009 FATF adopted new review procedures providing a more important role for its ICRG regional review groups. Each reviewed jurisdiction also had an opportunity to engage the regional review group to discuss the results of the mutual evaluation report and to develop an action plan with the FATF to address the deﬁciencies of concern. The FATF now speciﬁcally requested high-level political commitment to implement these action plans (FATF 2010a; Morse 2017). The new procedures led to the publication of three lists, from June 2010 (FATF 2010b, 2010c), following a February 2010 iteration that listed the remaining Period II countries separately (FATF 2010a; FATF Watch 2013): # Firstly, a list identifying “[j]urisdictions subject to a FATF call on its members and other jurisdictions to apply countermeasures to protect the international ﬁnancial system from the ongoing and substantial money laundering and ter- rorist ﬁnancing (ML/TF) risks emanating from the jurisdiction” (the so-called “blacklist”), where FATF called for jurisdictions to apply counter-measures; # Secondly, a list identifying “[j]urisdictions with strategic AML/CFT deﬁcien- cies that have not committed to an action plan developed with the FATF to address key deﬁciencies where the FATF called on its members to consider the risks arising from the deﬁciencies associated with each jurisdiction . . . ”, as described in each statement (the so-called “dark-grey list”), where FATF called for countries to consider the risks arising from the identiﬁed deﬁciencies; g # Thirdly, a list of jurisdictions with strategic AML/CFT deﬁciencies for which they have developed an action plan with the FATF (the so-called “light-grey list”). The FATF did not explicitly call for any measures to be taken in relation to these countries, though the listing would enliven normal FATF-related due diligence measures in the market to mitigate any jurisdictional risks. While the situations differ among jurisdictions, each jurisdiction has provided a written high-level political commitment to address the identiﬁed deﬁciencies. From February 2011 the latter list was augmented by a list of “jurisdictions not making sufﬁcient progress”, which identiﬁed those countries on the light-grey list that could be moved to the dark-grey list if they failed to take action expeditiously. 2.2. Phases in the FATF’s Greylisting Process A ﬁnal formal NCCT report relating to the monitoring of Myanmar’s compliance was adopted in October 2007 (FATF 2007a) but for practical impact purposes and to distinguish clearly between Periods I and II for this study, the year in which the last country was delisted is taken as the last year of Period I. During this phase the FATF labelled the listed countries as “non-compliant”. Its communication of listing and delisting dates during this period had a measure of clarity that is not as evident in later periods. The NCCT listing process did not follow a comprehensive compliance assessment of each country. The listings gave rise to political and economic development concerns. It was furthermore clear that the process could be strengthened technically. As a result, the IMF, World Bank and the FATF jointly adopted an agreed mutual evaluation methodology in October 2002. (Kyriakos-Saad 2005). The methodology was revised in 2004 (FATF 2004b) after FATF standards themselves were revised and expanded in 2003 (FATF 2003b). This period also saw the adoption of ﬁnancing of terrorism standards by the FATF (FATF 2004a). • Period II—2007–2009: this was a brief, transitional period between the conclusion of the NCCT period and the commencement of a more standardised process. • Period II—2007–2009: this was a brief, transitional period between the conclusion of the NCCT period and the commencement of a more standardised process. In October 2007 the FATF issued a public statement on Iran, expressing its concern that Iran’s deﬁcient AML/CFT regime posed a signiﬁcant vulnerability to the international ﬁnancial system and announcing that FATF members were advising their ﬁnancial institu- tions to consider the related risks for purposes of enhanced due diligence (FATF 2007b). The FATF did not have a formal black- or greylist at this stage but the language used in the Risks 2023, 11, 81 5 of 32 statement resembles those in greylisting statements, and was clearly intended to trigger risk management responses by the market. This was followed, from February 2008, by broader public statements on Iran, the northern part of Cyprus, Pakistan, São Tomé and Príncipe, Turkmenistan, and Uzbekistan, pointing to risks relating to the deﬁciencies in their AML/CFT systems (FATF 2008a, 2008c). Later statements in this series recognised improvements by the northern part of Cyprus (FATF 2008b) and Uzbekistan (FATF 2009), although they were not formally or clearly “delisted”. 2.2. Phases in the FATF’s Greylisting Process p y The FATF’s messaging around the listed jurisdictions changed further in 2020 when the list heading changed to “Jurisdictions under Increased Monitoring” with the following new introductory text (FATF 2020): “Jurisdictions under increased monitoring are actively working with the FATF to address strategic deﬁciencies in their regimes to counter money laundering, terrorist ﬁnancing, and proliferation ﬁnancing. When the FATF places a jurisdiction under increased mon- itoring, it means the country has committed to resolve swiftly the identiﬁed strategic deﬁciencies within agreed timeframes and is subject to increased monitoring”. In October 2022 it strengthened its statement that it is not calling for enhanced due diligence measures by adding that the FATF Standards “do not envisage de-risking, or cutting-off entire classes of customers, but call for the application of a risk-based approach (FATF 2022b). The second paragraph of the statement now reads (FATF 2023): “The FATF and FATF-style regional bodies (FSRBs) continue to work with the juris- dictions below as they report on the progress achieved in addressing their strategic deﬁciencies. The FATF calls on these jurisdictions to complete their action plans expedi- tiously and within the agreed timeframes. The FATF welcomes their commitment and will closely monitor their progress. The FATF does not call for the application of enhanced due diligence measures to be applied to these jurisdictions. The FATF Standards do not envisage de-risking, or cutting-off entire classes of customers, but call for the application of a risk-based approach. Therefore, the FATF encourages its members and all jurisdictions to take into account the information presented below in their risk analysis.” “The FATF and FATF-style regional bodies (FSRBs) continue to work with the juris- dictions below as they report on the progress achieved in addressing their strategic deﬁciencies. The FATF calls on these jurisdictions to complete their action plans expedi- tiously and within the agreed timeframes. The FATF welcomes their commitment and will closely monitor their progress. The FATF does not call for the application of enhanced due diligence measures to be applied to these jurisdictions. The FATF Standards do not envisage de-risking, or cutting-off entire classes of customers, but call for the application of a risk-based approach. 2.2. Phases in the FATF’s Greylisting Process From June 2011, the dark-grey list itself had added to it “countries with strategic AML/CFT deﬁciencies that have not made sufﬁcient progress in addressing the deﬁciencies” (FATF 2011). The complexity of the signalling using black, dark-grey, light-grey and warning lists was enhanced when FSRBs began to issue their own lists and statements and some of these were published by the FATF on its website (Nechaev 2014). The Caribbean Financial Action Task Force (CFATF), the FSRB for the Caribbean, for example, greylisted Belize and Guyana, but then blacklisted them in November 2013, with the CFATF calling on its members to consider implementing countermeasures to protect their ﬁnancial systems (CFATF 2013). In May 2014 Belize was removed from the CFATF blacklist, but Guyana was greylisted by the FATF in October 2014, delisted in October 2016 (FATF 2016), and exited the CFATF processes in November 2016 (CFATF 2016). During this period the FATF published its current set of standards (FATF 2012–2023) as well as its current mutual evaluation methodology (FATF 2013–2021). Risks 2023, 11, 81 6 of 32 • Period IV—2016–2021: from February 2016, the FATF’s greylist (now called “Im- proving Global AML/CFT Compliance: On-going Process”) was restricted to the jurisdictions previously listed on the light-grey list, i.e., “jurisdictions that have strate- gic AML/CFT deﬁciencies for which they have developed an action plan with the FATF.” Listing therefore held a more positive message than in the previous period where the dark-grey listing coloured the tone of greylisting. • Period IV—2016–2021: from February 2016, the FATF’s greylist (now called “Im- proving Global AML/CFT Compliance: On-going Process”) was restricted to the jurisdictions previously listed on the light-grey list, i.e., “jurisdictions that have strate- gic AML/CFT deﬁciencies for which they have developed an action plan with the FATF.” Listing therefore held a more positive message than in the previous period where the dark-grey listing coloured the tone of greylisting. Changes were made to the language of the statements too. In October 2019, for example, the FATF added explicitly that it “does not call for the application of enhanced due diligence measures to be applied to these jurisdictions”, but that it encourages its members “to take into account the information presented below in their risk analysis”. 2.2. Phases in the FATF’s Greylisting Process Therefore, the FATF encourages its members and all jurisdictions to take into account the information presented below in their risk analysis.” These language changes communicated a more positive message about the greylisted countries: they have strategic deﬁciencies but they are committed to ﬁxing the problems and have implemented an action plan to do so reasonably expeditiously and subject to external monitoring. Furthermore, the FATF made it clear that, while the information in the statement may be relevant to a risk assessment, the mere listing of a country on this list does not amount to a call for the application of enhanced due diligence measures. The likelihood of a severe market reaction to such a listing would therefore tend to decline. 2.3. Other Lists This study focuses on the FATF’s greylist and its economic impact on listed countries. It is however necessary to note that, while important, this is only one of a number of country risk lists published by nations or civil society that are likely to be taken into regard in a risk-based AML/CFT country risk assessment, for example the Financial Secrecy Index (Tax Justice Network 2022); the US International Narcotics Control Strategy Report (US Department of Justice 2022); the Corruption Perceptions Index (Transparency International 2021) and the Basel AML Index (Basel Institute on Governance 2022). In addition, lists of country risk indicators and country risk proﬁles are also available commercially to subscribers. These lists themselves are often informed, in part, by FATF country listings, and their weighting in the methodologies of those lists may not reﬂect the more constructive market signalling by the FATF in the current listing phase. The impacts of those lists are out of the scope of this study, but in principle they may modify or intensify the impacts of Risks 2023, 11, 81 7 of 32 FATF listings. Clear messaging by the FATF can prevent or at least counteract such changes and hence the study focuses on the FATF and its messaging. FATF listings. Clear messaging by the FATF can prevent or at least counteract such changes and hence the study focuses on the FATF and its messaging. A particularly important development was the adoption of a European Union (EU) AML/CFT-related list of high-risk third countries in 2016, with listing rules revised in 2020 (European Commission 2020, 2022). Countries that are greylisted by the FATF are usually added to the EU list, unless they are exempted. The EU can add any country to their list, but EU and European Economic Area members are exempted. This exemption owes more to realpolitik than to principle. Least-developed countries, as identiﬁed by the UN, are also exempted unless such a country is identiﬁed as presenting a threat to the EU ﬁnancial system or is designated as an offshore ﬁnancial centre (European Commission 2020). While the EU professes to support the FATF’s processes, there are various points of tension between the two listing processes, e.g., the EU may add its own requirements to any FATF country action plan and a FATF delisting does not necessarily lead to an EU delisting of that country (Dalip 2020). 2.3. Other Lists Importantly, the listing triggers an obligation of EU institutions to adopt enhanced due diligence measures in relation to a greylisted country, even though the FATF does not call for such measures (Fourth EU AML Directive 2015): Member States should at least provide for enhanced customer due diligence measures to be applied by the obliged entities when dealing with natural persons or legal entities established in high-risk third countries identiﬁed by the Commission. Reliance on third parties established in such high-risk third countries should also be prohibited. Countries not included in the list should not be automatically considered to have effective AML/CFT systems and natural persons or legal entities established in such countries should be assessed on a risk-sensitive basis. Member States should at least provide for enhanced customer due diligence measures to be applied by the obliged entities when dealing with natural persons or legal entities established in high-risk third countries identiﬁed by the Commission. Reliance on third parties established in such high-risk third countries should also be prohibited. Countries not included in the list should not be automatically considered to have effective AML/CFT systems and natural persons or legal entities established in such countries should be assessed on a risk-sensitive basis. Other countries, including the UK, also require enhanced due diligence measures in relation to greylisted countries. Their regulatory expectations are therefore not aligned with the FATF’s market signalling in the current period, although the FATF’s messaging may inﬂuence some of the enhanced measures that regulated institutions may choose to adopt. The growing number of relevant country lists, and especially the operation of the EU high-risk third countries list, may blur the clear proportional attribution of any economic impact that the FATF greylist may have. Where a negative impact is identiﬁed, the market prominence and political weight of the FATF lists would support a strong argument for a signiﬁcant share in such impact. 3. Literature Review A number of studies have analysed economic data to determine whether greylisting impacted negatively on a jurisdiction, especially from an international political perspective. Given the questions pursued in this paper, the literature review focused on the key studies where an extensive analysis of economic data was undertaken. Kudrle (2008) studied changes in Bank for International Settlements data on ﬁnancial ﬂows to and from tax havens to determine whether there was evidence of a negative impact. The study focused on 46 jurisdictions and data in the quarters before and after FATF and OECD tax haven listing and delisting. The study considered total assets and liabilities, and the associated component non-bank assets and liabilities, as well as their counterparts for the loan and deposit data over the period for which the latter were available. The study produced mixed results and concluded that “no substantial and consistent impact of blacklisting on banking investment in and out of the tax havens was found across 38 jurisdictions.” j Balakina et al. (2017) explored whether “blacklisting” had a measurable negative economic effect on a country, called the “stigma effect”. Drawing on the Global Financial Development Database they studied the relationship between international capital move- ments and FATF listing and delisting events in 126 countries from 1996 to 2014. Variables used for the analysis included total Foreign Claims (Bank Flow), size of ﬁnancial insti- tutions and markets, the degree to which individuals can and do use ﬁnancial services, the efﬁciency of ﬁnancial intermediaries and markets in intermediating resources and Risks 2023, 11, 81 8 of 32 8 of 32 facilitating ﬁnancial transactions, the stability of ﬁnancial institutions and markets, and the level of macroeconomic stability. The authors concluded that the stigma effect did not exist. facilitating ﬁnancial transactions, the stability of ﬁnancial institutions and markets, and the level of macroeconomic stability. The authors concluded that the stigma effect did not exist. y g A number of studies found more conclusive evidence of listing and economic impact correlations. Farías and Almeida (2014) analysed AML ratings, greylisting and corruption indicators in 36 Latin American and Caribbean countries between 1960 and 2010, and noted that poor FATF ratings and high levels of corruption can both have a downward impact on domestic and international investment. 3. Literature Review They also found evidence that illicit ﬂows can increase the depth of ﬁnancial markets, but that the impact varies depending on, for example, the strength of ties with main ﬁnancial centres. In general, the authors are conﬁdent that their work demonstrates that FATF listings “have indeed penalized countries and territories that were target of the body’s critical reviews”. g y Collin et al. (2016) used SWIFT data to explore the impact of AML regulation on payment ﬂows. They found that greylisted countries faced “up to a 10 percent decline in cross border payments received from other jurisdictions, but no change in the number sent”. Greylisted countries are also more likely to see a decline in payments from other countries with weak AML/CFT institutions. Their conclusions were based on monthly counts of cross-border payment messages recorded in the SWIFT system between January 2004 and August 2014 and listings by the FATF over the same period. In the period examined, the number of such messages rose from around 20 million/month to around 40 million/month, with substantial seasonal variation. Correlations were observed between such messages and exports, with an overall correlation of 0.33. They noted that the poorest countries were most likely to be greylisted: “countries with a GDP per capita under $20,000 face greylisting rates as high as 40%”. To identify the covariance of FATF greylisting against other ‘treatment’ measures, the authors also explored the impact of the US International Narcotics Control Strategy Report ratings and US, EU or UN sanctions. Control variables included GDP per capita (World Bank), a country’s democracy score (POLITY IV), the World Bank World Governance Indicators, a dummy variable indicating whether or not there is an ongoing conﬂict, and the country’s current exchange rate against the dollar. Tests were carried out for ‘leads’ and ‘lags’ in the responses. g p The authors found that the impact is “more complex than recent narratives have suggested. There was no evidence that countries that have frequently been mentioned as sources of de-risking or sources of pressure to de-risk are the ones where there are substantial declines in payment ﬂows to and from grey-listed countries”. Morse (2017) analysed the interaction between “reputation, market enforcement, and international cooperation”, focusing on the global campaign to combat terrorist ﬁnancing. 3. Literature Review Her PhD thesis also provides some detail of speciﬁc countries and their experience with being put on non-complier lists, including Thailand, Turkey, Philippines, Guyana, and Panama. Morse examined the effect of the FATF greylisting on cross-border bank-to-bank liabilities between February 2010 and December 2015 and concluded that cross-border bank ﬂows decline and investors demand higher relative yields for sovereign debt when a jurisdiction is listed. The analysis focused on non-FATF member countries with comparable levels of compliance. A variety of methodologies were applied, including interviews with 15 ﬁnance industry professionals, fuzzy regression discontinuity, ‘non-instrumented analysis of sovereign spreads’, a Cox Proportional Hazards model, and a case study of Thailand. The latter provided a graphical presentation of trends in cross-border liabilities and in relation to listing and delisting. No particular pattern was found. Correlational evidence was cited that the non-complier list affected the risk premium for long-term debt. p p g Later, Morse (2019) analysed growth in cross-border liabilities for 39 countries, 10 of which were grey-listed, ﬁnding a large signiﬁcant impact of c. 15–16%. Morse reﬁned her analysis and found strong and statistically signiﬁcant results ranging from an approximately Risks 2023, 11, 81 9 of 32 10.6 percent decrease to a 12.6% and 12.8% decrease in liabilities in certain speciﬁcations (Morse 2022). Case-Ruchala and Nance (2020) replicated Morse’s 2019 model for a wider range of cases and found that the inclusion of more comprehensive data erased the effect. They argue that Morse’s ﬁndings were largely based on her (imbalanced) selection of cases, and that “[their] ﬁndings strongly suggest that being listed by FATF does not lead to a signiﬁcant change in investment patterns”. They also examined the impact of listing on cross-border portfolio asset investments using the IMF Coordinated Portfolio Investment Survey 9 data in a similar series of regressions in relation to the wider range of cases and again found no effect of listing. Jayasekara (2021) analysed the short-term economic implications of AML/CFT policies. The study focused on seven economies selected from the FATF’s 2019 listings—Ethiopia, Sri Lanka, Tunisia, Serbia, Afghanistan, Lao PDR, and Guyana. It found that the domestic currency of monitored jurisdictions depreciated during the review period. The study also found evidence that limitations on fund transfers and correspondence banking relationships may have negative impacts on the capital markets of listed countries. 3. Literature Review Kida and Paetzold (2021), published as an IMF working paper, found that “gr[e]y- listing results in a large and statistically signiﬁcant reduction in capital inﬂows”. The study used data from 89 emerging and developing countries in 2000–2017, and identiﬁed greylisting using FATF public statements. During the period, 78 of the countries were greylisted at least once, and the response to this event was explored using a machine- learning process known as lasso. They found statistically signiﬁcant declines in capital inﬂows of 7.6%, in FDI inﬂows of 3.0%, in portfolio inﬂows of 2.9% and in other investment inﬂows of 3.6%. The studies to date have therefore provided mixed results about the economic im- pact of greylisting by the FATF. The studies have however not identiﬁed the changes in the FATF’s listing-related market signalling over time or studied these to determine whether they may provide a clearer answer. Did the changes in language impact on market responses? As discussed in Section 2.2 above, the authors identify different signalling periods in the FATF’s greylisting processes. This study therefore set out to determine: • whether listing and delisting had signiﬁcant correlations with economic indicators, either immediately or potentially over one and two years after listing and delisting; and • whether listing and delisting had signiﬁcant correlations with economic indicators, either immediately or potentially over one and two years after listing and delisting; and • if any correlations were identiﬁed, whether there was evidence of any differences across the key periods of greylisting. across the key periods of greylisting. 4.1. A FATF Greylisting Economic Impact Model 4.1. A FATF Greylisting Economic Impact Model Previous studies selected relevant economic indicators without comprehensively explaining how they relate to greylisting, to the operation of the market, and to each other. It was therefore important to develop a theoretical impact model to inform the selection of indicators for the study and to assist in explaining any ﬁndings. There is a well-developed literature on how signalling affects competitive behaviour dating back at least to the 1980s (Heil and Robertson 1991). This literature provides some guidance on how ﬁrms, markets, and industries may react to particular signals, and in particular the importance of consistency, clarity, and aggressiveness of the signal (Fiske and Taylor 1984). It also provides some evidence that (suitably designed) public policy signals can have a beneﬁcial effect in integrated markets (Avdjiev et al. 2012). Drawing on this literature, we have developed an impact model, summarized in Figure 1. Risks 2023, 11, 81 10 of 32 ndard- 10 of 32 ndard- Figure 1. FATF Greylisting Impact Model. This model summarises the authors’ understanding of how FATF listing signals could aﬀect the economy of a jurisdiction. FATF standards and statements are supposed to be reﬂected in the national regulations and policies of regulated businesses and profes- sions. Non-regulated businesses and clients are indirectly aﬀected if they depend on these sectors to function. Donor/lender governments and near-market international lending in- Figure 1. FATF Greylisting Impact Model. The model tracks the broad high-level assumptions that underpin the FATF standards. These derive from a ﬁrst-world and historical and generalised view of national ﬁnancial systems and economies, albeit a view that is changing. However, the high-level basic market functions assumed by the model still exist. Importantly, there are standardised data on them to enable this analysis. Thi d l i th th ’ d t di f h FATF li ti i l ld Model Model. Figure 1. FATF Greylisting Impact Model. Figure 1. FATF Greylisting Impact Model. Figure 1. FATF Greylisting Impact Model. Figure 1. FATF Greylisting Impact Model. This model summarises the authors’ understanding of how FATF listing signals could aﬀect the economy of a jurisdiction. FATF standards and statements are supposed to be reﬂected in the national regulations and policies of regulated businesses and profes- sions. 4.1. A FATF Greylisting Economic Impact Model Non-regulated businesses and clients are indirectly aﬀected if they depend on these sectors to function Donor/lender governments and near market international lending in The model tracks the broad high-level assumptions that underpin the FATF standards. These derive from a ﬁrst-world and historical and generalised view of national ﬁnancial systems and economies, albeit a view that is changing. However, the high-level basic market functions assumed by the model still exist. Importantly, there are standardised data on them to enable this analysis. sectors to function. Donor/lender governments and near-market international lending in- stitutions also act in this space, as do market-based national and international investors. Greylisting may decrease opportunities for, and increase the cost of, doing business: • Governments may tighten the regulation of trade with the listed country, or with connected entities, or even prohibit business • Firms in, or linked to, the listed country (e.g., by ownership or trade) may need to This model summarises the authors’ understanding of how FATF listing signals could affect the economy of a jurisdiction. FATF standards and statements are supposed to be reﬂected in the national regulations and policies of regulated businesses and professions. Non-regulated businesses and clients are indirectly affected if they depend on these sectors to function. Donor/lender governments and near-market international lending institutions also act in this space, as do market-based national and international investors. undertake more checks to comply, and be seen to be complying, with regulations R l i h i l i li d f d d p Greylisting may decrease opportunities for, and increase the cost of, doing business: • Resulting changes in aggregate volume, variety, quality and cost of goods and ser- vices may impact national proﬁtability • Governments may tighten the regulation of trade with the listed country, or with connected entities, or even prohibit business • Firms in, or linked to, the listed country (e.g., by ownership or trade) may need to undertake more checks to comply, and be seen to be complying, with regulations • Resulting changes in aggregate volume, variety, quality and cost of goods and services may impact national proﬁtability Once trade is affected by listing signals there are likely to be ﬁrst- and second-order effects. These are brieﬂy outlined in Section 5, together with indicators that could be considered to reﬂect and measure these effects. 4.2. First-Order Effects 4.2.1. Increased Supplier Costs 4.3.1. Downward Proﬁtability Spiral 4.3.1. Downward Proﬁtability Spiral Higher reference rates and higher corporate spreads (linked to perceptions of lowered proﬁtability and debt service capability) are a double shock to the debt markets, with knock-on effects in equity markets. This may produce further reductions in proﬁtability, tax payment and debt service/equity raising capacity, plus an increase in non-performing loans. # Possible indicators: bank nonperforming loans to total gross loans (%)—important for showing incipient failures of companies, though also an indicator of reckless lending. Market capitalisation of listed domestic companies (current US$) would be expected to fall with MER and rise on delisting. 4.2.3. Reduced National Income and Expenditure 4.2.3. Reduced National Income and Expenditure Direct aid to a listed country may fall as donors negotiate measures to address the new risk proﬁle. The effect would add to national budgetary pressures. One way for government to relieve this might be to reduce ESI payments. This, however, would further lower ﬁrm proﬁtability. # Possible indicators: net ofﬁcial development assistance and ofﬁcial aid received [current US$], IFC, private nonguaranteed [NFL, current US$] IBRD loans and IDA credits [DOD, current US$]. The focus here is on changes in access to capital given degrees of politicisation. 4.3. Second-Order Effects 4.3. Second-Order Effects 4.2.4. Reduced Access to Capital International bond and loan markets are likely to see listing negatively, putting up- wards pressure on both government borrowing levels and corporate spreads. Some credi- tors may no longer lend or may reduce investment levels. Governments and international lending organisations may react in a similar fashion, including by increasing the condi- tionality (and thus expense) of lending. There would be less liquidity. Of the liquidity that exists some would be needed to cover higher costs of indebtedness. # Possible indicators: risk premium on lending (lending rate minus treasury bill rate, %), Lending interest rate (%), Interest rate spread (lending rate minus deposit rate, %). Testing should show ballooning/tightening of both indicators on listing/delisting # Possible indicators: risk premium on lending (lending rate minus treasury bill rate, # Possible indicators: risk premium on lending (lending rate minus treasury bill rate, %), Lending interest rate (%), Interest rate spread (lending rate minus deposit rate, %). Testing should show ballooning/tightening of both indicators on listing/delisting # p g ( g y , %), Lending interest rate (%), Interest rate spread (lending rate minus deposit rate, %). Testing should show ballooning/tightening of both indicators on listing/delisting ) g ( ) p ( g p ) Testing should show ballooning/tightening of both indicators on listing/delisting 4.2.1. Increased Supplier Costs Firms may amend internal policies and operating practices to respond to greylisting risk signals. Firms may amend internal policies and operating practices to respond to greylisting risk signals. Risks 2023, 11, 81 11 of 32 11 of 32 At the margin, higher resulting regulatory risks may be unmanageable or inadequately compensated. Firms made inefﬁcient and unproﬁtable (through inability to manage risk or pass on cost increases) may reduce the range of goods on offer or exit the market entirely. 4.2.2. Reduced Firm Proﬁtability, Tax Payments and Debt Service Capacity Reduced supply and increased cost of factor goods and services may reduce gross proﬁt margins. This in turn would reduce ﬁrm headline tax liability and debt/equity service capacity. # Possible indicators: tax revenue totals, dividend levels # Possible indicators: tax revenue totals, dividend levels # Possible indicators: private non-guaranteed loans, commercial, banks and other creditors (NFL, current US$). 4.4.1. Time Lags The effects described may not all impact at the same time. Market impacts may be immediate and slight but become signiﬁcant as iterations of the negative cycle take cumulative effect. Impacts linked to structural factors (such as multilateral lending and aid) may be slow to show but signiﬁcant. # Possible leading indicators: Exchange rates and monthly GDP (current LCU)/GDP (US$)) with both falling with listing and rising with delisting; Net ﬁnancial ﬂows, bilateral (NFL, current US$) with ﬂows falling as commercial activity and FX values diminish (except for inﬂows from release of foreign holdings); foreign direct invest- ment, net inﬂows (BoP, current US$) again, falling with listing, rising with delisting; foreign direct investment, net outﬂows (BoP, current US$)—rising with listing, falling with delisting. g # Possible lagging indicators: net foreign assets (current LCU)—falling as assets are liquidated, possibly at a discount, to cover local trading losses and as a result of LC depreciation; external debt stocks, total (DOD, current US$) rising and falling as trade drops off; net acquisition of ﬁnancial assets (% of GDP) (though it is important to exclude external buying on weakness); claims on central government, etc. (% GDP); claims on other sectors of the domestic economy (% of GDP). 4.4.2. Persistence Negative impacts may endure and ﬂuctuate whilst the causes of listing are uncorrected. They may return to normal when the FATF signals that its concerns have been addressed. However, they may also persist after successful correction and delisting, for example if private sector conﬁdence and public sector surpluses are slow to return. Where ﬁrms have exited the market permanently, reduced supply of their own offerings and loss of demand for their factor goods may maintain pressure on the economy. The model is therefore intended to help examine if removal of FATF grey listing acts in the same way as raising a blind—immediately readmitting sunlight—or whether it resembles removing a clothes iron—where outcomes depend on the fabric, the level of retained heat, and whether permanent damage has (already) occurred. 4.4. Time Lags and Endurance 4.4. Time Lags and Endurance 4.5. Empirical Testing Using the model as a theoretical basis, a global FATF greylisting database was con- structed for the period from 2001 to 2020. This brought together data on the ﬁnancial and economic outcomes for all countries with data on when FATF and related events occurred, including publication dates for relevant listing statements. The focus was on NCCT list- ings, light- and dark-grey listings prior to 2016, and greylisting from 2016 onwards. For consistency, the handful of FSRB listings was added on the assumption that international market responses to FATF and FSRB greylisting would tend to be similar. The principal source of economic and ﬁnancial data was the World Bank’s ‘World Development Indicators’ databank, updated in 2022. Countries where the data were too sparse to be used were excluded from the analysis, so that the ﬁnal analysis was based on 177 countries and 3540 country-years of data. For these remaining countries, some interpolation of missing data was also undertaken. Yti = αi + δt + Σk βk Xkti + εti (1) (1) Yti = αi + δt + Σk βk Xkti + εti 4.3.2. Reduced Government Income The continuing drop in proﬁtability may reduce government income, adding to pres- sure in sovereign debt and corporate capital markets. Knock-on effects on ESI payments, again impacting ﬁrm proﬁtability, may cause the cycle to repeat in a negative feedback loop. # Possible indicators: private non-guaranteed loans, commercial, banks and other creditors (NFL, current US$). Risks 2023, 11, 81 12 of 32 12 of 32 where: Yti is the economic or ﬁnancial variable which may be correlated with or affected by listing. This may either be the level of a variable or the year-on-year change in a variable. Ai is the cross-section effects, a vector of dummy variables indicating cross-section I (ﬁxed effects). Yti is the economic or ﬁnancial variable which may be correlated with or affected by listing. This may either be the level of a variable or the year-on-year change in a variable. y y g This may either be the level of a variable or the year-on-year change in a variable. Ai is the cross-section effects, a vector of dummy variables indicating cross-section I (ﬁxed effects). y y y g Ai is the cross-section effects, a vector of dummy variables indicating cross-section I (ﬁxed effects). ) δt is a vector of dummy variables indicating time t (ﬁxed effects). Risks 2023, 11, 81 13 of 32 13 of 32 Xkti are the k independent variables that vary over cross-section and time. Xkti are the k independent variables that vary over cross-section and time. βk are the respective coefﬁcients indicating the effect of Xk on Y. εti are the stochastic errors that vary over both cross-section and time. p y βk are the respective coefﬁcients indicating the effect of Xk on Y. p g εti are the stochastic errors that vary over both cross-section and time. Yti = αi + Σk βk Xkti + εti (2) (2) Yti = αi + Σk βk Xkti + εti Many of the regulatory actions being explored coincide with the chaos caused by the Global Financial Crisis (GFC), so dummy variables for the two years of 2007 and 2008 have been included. Thus, the study uses a hybrid system of ﬁxed effects with country variation and some limited time variation. For each of the four FATF listing periods described above, dummy variables were established for the listings, remediation period, i.e., the period spent on the list, and delisting of listed countries, giving a total of twelve possible variables for each country. The magnitude and signiﬁcance of the coefﬁcients on these variables then provide an indication of the effect of FATF regulatory action in the period with respect to the ﬁnancial or economic indicator chosen as the dependent variable. where: p The detailed econometric results are presented in Annex A, grouped into ﬁrst-order and second-order effects reﬂecting the indicators selected for testing in the theoretical model. The tables presented there are colour-coded to denote signiﬁcance at the 95% (dark blue), 90% (blue), and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Those tables present results for all the possible indicators identiﬁed for testing above. The discussion below focuses on those economic and ﬁnancial indicators that were found to be correlated in a signiﬁcant way. 5. Results 5.1. Period I: 2000–2006 5.1. Period I: 2000–2006 In the ﬁrst FATF regulatory period, the econometric results, which are presented in more detail in Appendix A, reﬂect correlations with risk-oriented indicators and external assets and debts. These are broadly consistent with the FATF greylisting economic impact model outlined in Section 5. The more signiﬁcant ﬁndings are provided in Table 1 below. Table 1. Correlations with indicator variables in 2000–2006 period. Table 1. Correlations with indicator variables in 2000–2006 period. Table 1. Correlations with indicator variables in 2000–2006 period. 2000–2006———————————————> Dependent Variable Initial Listing I t-Stat Remediation I t-Stat Delisting I t-Stat Risk Premium y-on-y change 0.13279 0.270 −0.4811 −1.355 −0.3618 −0.979 Risk Premium t + 1 y-on-y change 0.20510 0.417 −0.5222 −1.470 −0.2848 −0.771 Risk Premium t + 2 y-on-y change 0.16417 0.334 −0.4923 −1.390 −0.3713 −1.008 Bank nonperforming loans to total gross loans (%) y-on-y change 0.01477 0.714 −0.0628 −4.207 −0.0443 −2.856 Bank nonperforming loans to total gross loans (%) t + 1 y-on-y change 0.01204 0.571 −0.0605 −3.981 −0.0058 −0.365 Bank nonperforming loans to total gross loans (%) t + 2 y-on-y change −0.02732 −1.280 −0.0198 −1.286 −0.0056 −0.347 Net foreign assets (current LCU)/GDP y-on-y change 1.30816 0.908 −2.3546 −2.264 −0.1584 −0.146 Net foreign assets (current LCU)/GDP t + 1 y-on-y change 2.08712 0.874 −2.2532 −1.307 −0.7302 −0.407 Risks 2023, 11, 81 14 of 32 Table 1. Cont. 2000–2006———————————————> Dependent Variable Initial Listing I t-Stat Remediation I t-Stat Delisting I t-Stat Net foreign assets (current LCU)/GDP t + 2 y-on-y change 1.70945 1.184 −2.3595 −2.265 −0.9868 −0.911 External debt stocks, total (DOD, current US$)/GDP y-on-y change 0.01426 0.237 −0.0227 −0.523 −0.0362 −0.801 External debt stocks, total (DOD, current US$)/GDP t + 1 y-on-y change 0.08197 1.361 −0.0534 −1.229 −0.0211 −0.467 External debt stocks, total (DOD, current US$)/GDP t + 2 y-on-y change 0.09511 1.583 −0.0987 −2.277 −0.0622 −1.380 Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Table 1. Cont. 5.1. Period I: 2000–2006 Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Among the leading indicators identiﬁed in the theoretical model, there is strongly signiﬁcant evidence that bank non-performing loans fell during the remediation periods and on delisting, and also some weakly signiﬁcant evidence that risk premiums were reduced. Net foreign assets declined during the period in which countries were listed, and debts rose on listing, which provides some support for the ﬁndings of Collin et al. (2016), but there is some evidence of reduction in debt stocks after a time lag. 5.2. Period II: 2007–2009 As discussed above, the three-year period 2007–2009 has been identiﬁed as a transition period from the NCCT listing to the introduction of a more formal black- and greylist. Empirical ﬁndings from this period need to be treated with caution as they are based on a limited number of country-years of data and would be skewed by the nature of the handful of countries that were singled out. g Table 2 shows the beginnings of a more institutional response in this period, although weaker market-based effects, for example on exchange rates and growth, persist. Table 2. Correlations with indicator variables in 2007–2009 period. 2007–2009———————————————————–> Dependent Variable Initial Listing II t-Stat Remediation II t-Stat Delisting II t-Stat Bank nonperforming loans to total gross loans (%) y-on-y change −0.1900 −5.012 0.35048 12.134 −0.0452 −1.005 Bank nonperforming loans to total gross loans (%) t + 1 y-on-y change 0.3060 7.919 −0.04547 −1.544 −0.0365 −0.794 Bank nonperforming loans to total gross loans (%) t + 2 y-on-y change −0.0146 −0.372 −0.06165 −2.068 −0.0364 −0.784 Exchange Rate (GDP(LC)/GDP(US$)) y-on-y change −0.0229 −0.304 0.07801 1.359 0.0316 0.353 Exchange Rate (GDP(LC)/GDP(US$)) t + 1 y-on-y change 0.0684 0.782 −0.02396 −0.359 0.0332 0.320 Table 2. Correlations with indicator variables in 2007–2009 period. Table 2. Correlations with indicator variables in 2007–2009 period. 15 of 32 Risks 2023, 11, 81 Table 2. Cont. 5.2. Period II: 2007–2009 2007–2009———————————————————–> Dependent Variable Initial Listing II t-Stat Remediation II t-Stat Delisting II t-Stat Exchange Rate (GDP(LC)/GDP(US$)) t + 2 y-on-y change 0.0381 0.306 −0.06340 −0.670 −0.0210 −0.143 Year-on-year change in GDP (current US$) 0.1271 1.519 −0.0884 −1.386 −0.0484 −0.487 Year-on-year change in GDP (current US$) t + 1 −0.0657 −0.779 −0.0170 −0.265 −0.0499 −0.498 Year-on-year change in GDP (current US$) t + 2 1.9502 0.247 0.5420 0.090 0.6712 0.072 Foreign direct investment, net inﬂows (BoP, current US$)/GDP −0.0726 −0.843 0.05570 0.849 0.8954 8.760 Foreign direct investment, net inﬂows (BoP, current US$)/GDP t + 1 0.2355 2.715 0.06811 1.031 0.1598 1.551 Foreign direct investment, net inﬂows (BoP, current US$)/GDP t + 2 0.0658 0.757 0.00441 0.067 0.2296 2.225 Foreign direct investment, net outﬂows (BoP, current US$)/GDP −0.0653 −0.868 −0.005 −0.085 0.7838 8.767 Foreign direct investment, net outﬂows (BoP, current US$)/GDP t + 1 0.2354 3.084 0.012 0.199 0.1466 1.616 Foreign direct investment, net outﬂows (BoP, current US$)/GDP t + 2 0.0345 0.440 −0.002 −0.032 0.34 3.666 External debt stocks, total (DOD, current US$)/GDP y-on-y change −0.2850 −2.586 0.16403 1.953 −0.0199 −0.152 External debt stocks, total (DOD, current US$)/GDP t + 1 y-on-y change −0.2067 −1.871 0.29364 3.490 −0.0051 −0.039 External debt stocks, total (DOD, current US$)/GDP t + 2 y-on-y change −0.0134 −0.122 0.16421 1.957 −0.0038 −0.029 Net ofﬁcial development assistance and ofﬁcial aid received (current US$)/GDP 0.0191 0.896 −0.01339 −0.825 0.0072 0.286 Net ofﬁcial development assistance and ofﬁcial aid received (current US$)/GDP t + 1 0.0144 0.677 −0.00945 −0.582 0.0031 0.122 Net ofﬁcial development assistance and ofﬁcial aid received (current US$)/GDP t + 2 −0.0343 −1.606 0.02859 1.756 −0.0006 −0.025 2007–2009— Risks 2023, 11, 81 16 of 32 16 of 32 Table 2. Cont. 2007–2009———————————————————–> Dependent Variable Initial Listing II t-Stat Remediation II t-Stat Delisting II t-Stat IBRD loans and IDA credits (DOD, current US$)/GDP 0.0548 1.367 −0.09248 −3.026 −0.0104 −0.217 IBRD loans and IDA credits (DOD, current US$)/GDP t + 1 0.0506 1.354 −0.07549 −2.649 −0.0082 −0.184 IBRD loans and IDA credits (DOD, current US$)/GDP t + 2 0.0427 1.250 −0.06014 −2.312 −0.0060 −0.149 Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. 5.2. Period II: 2007–2009 Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Table 2. Cont. Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). The impact on bank non-performing loans is more nuanced, with evidence of both positive and negative effects. Unlike other periods, this period does show some strongly signiﬁcant effects on foreign direct investment, both inﬂows and outﬂows. There are also strongly signiﬁcant negative impacts on IBRD loans and IDA credits, perhaps indicating that the same things that concern FATF also concern the ofﬁcials who are deciding on the allocation of such loans and credits. 5.3. Period III: 2010–2015 The third period of FATF listings—a period characterised by higher levels of compli- ance with FATF listing signals by compliant countries and institutions—seems to have had more wide-ranging correlations, including with investment ﬂows and with bank behaviour, as predicted by the theoretical model by Collin et al. (2016). This is also a period of more complex signalling, including the use of the dark-grey list that may have increased market responses to greylisting in general. Table 3 summarises the more signiﬁcant ﬁndings. Table 3. Correlations with indicator variables in 2010–2015 period. Table 3. Correlations with indicator variables in 2010–2015 period. 2010–2015———————————————————> Dependent Variable Initial Listing III t-Stat Remediation III t-Stat Delisting III t-Stat Market capitalisation of listed domestic companies (current US$) y-on-y change −2.8970 −2.548 2.9748 4.406 −0.2035 −0.182 Market capitalisation of listed domestic companies (current US$) t + 1 y-on-y change 7.5329 6.666 0.0019 0.003 −0.1970 −0.178 Market capitalisation of listed domestic companies (current US$) t + 2 y-on-y change 0.1351 0.119 −0.7050 −1.042 −0.6876 −0.615 Private non-guaranteed loans, commercial banks and other creditors 0.0046 1.263 0.0076 3.549 0.0091 2.569 Private non-guaranteed loans, commercial banks and other creditors t + 1 −0.0033 −0.900 0.0075 3.425 0.0040 1.102 Table 3. Correlations with indicator variables in 2010–2015 period. 17 of 32 Risks 2023, 11, 81 Table 3. Cont. 5.3. Period III: 2010–2015 2010–2015———————————————————> Dependent Variable Initial Listing III t-Stat Remediation III t-Stat Delisting III t-Stat Private non-guaranteed loans, commercial banks and other creditors t + 2 0.0049 1.320 0.0021 0.954 −0.0027 −0.755 Exchange Rate (GDP(LC)/GDP(US$)) y-on-y change −0.0668 −3.294 0.0426 3.536 0.0117 0.591 Exchange Rate (GDP(LC)/GDP(US$)) t + 1 y-on-y change 0.0489 2.076 0.0315 2.252 0.0292 1.265 Exchange Rate (GDP(LC)/GDP(US$)) t + 2 y-on-y change −0.0088 −0.264 0.0140 0.704 0.0114 0.347 Year-on-year change in GDP (current US$) 0.0909 4.039 −0.0372 −2.782 −0.0146 −0.662 Year-on-year change in GDP (current US$) t + 1 0.0410 1.805 −0.0850 −6.311 −0.0860 −3.860 Year-on-year change in GDP (current US$) t + 2 −1.9131 −0.901 −1.1836 −0.939 −0.8024 −0.385 Net ﬁnancial ﬂows, bilateral [NFL, current US$] −0.0024 −1.327 0.0058 5.404 0.0057 3.214 Net ﬁnancial ﬂows, bilateral [NFL, current US$] t + 1 −0.0010 −0.570 0.0052 5.014 0.0055 3.191 Net ﬁnancial ﬂows, bilateral [NFL, current US$] t + 2 −0.0014 −0.759 0.0056 5.305 0.0022 1.237 Net foreign assets (current LCU)/GDP y-on-y change −1.3917 −1.958 1.0722 2.541 −0.5792 −0.830 Net foreign assets (current LCU)/GDP t + 1 y-on-y change −1.1231 −0.953 0.9118 1.304 −0.0233 −0.020 Net foreign assets (current LCU)/GDP t + 2 y-on-y change 4.1504 5.832 −0.5239 −1.240 0.1003 0.144 Claims on other sectors of the domestic economy (% of GDP t + 1)/GDP y-on-y change −0.0087 −1.158 0.0128 2.867 0.0090 1.217 Claims on other sectors of the domestic economy (% of GDP t + 1)/GDP t+1 y-on-y change 0.0026 0.350 0.0098 2.194 0.0118 1.587 Claims on other sectors of the domestic economy (% of GDP t + 1)/GDP t+2 y-on-y change 0.0132 1.649 0.0026 0.539 0.0103 1.319 Net ofﬁcial development assistance and ofﬁcial aid received (current US$)/GDP −0.0009 −0.153 −0.0066 −1.936 −0.0098 −1.749 Risks 2023, 11, 81 18 of 32 Table 3. Cont. 5.3. Period III: 2010–2015 2010–2015———————————————————> Dependent Variable Initial Listing III t-Stat Remediation III t-Stat Delisting III t-Stat Net ofﬁcial development assistance and ofﬁcial aid received (current US$)/GDP t + 1 −0.0040 −0.702 −0.0047 −1.380 −0.0083 −1.479 Net ofﬁcial development assistance and ofﬁcial aid received (current US$)/GDP t + 2 −0.0074 −1.287 −0.0021 −0.608 −0.0070 −1.234 IBRD loans and IDA credits (DOD, current US$)/GDP 0.0045 0.413 −0.0352 −5.486 −0.0352 −3.328 IBRD loans and IDA credits (DOD, current US$)/GDP t + 1 0.0028 0.276 −0.0307 −5.145 −0.0302 −3.062 IBRD loans and IDA credits (DOD, current US$)/GDP t + 2 0.0025 0.274 −0.0253 −4.648 −0.0235 −2.604 Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Table 3. Cont. 2010–2015— Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). In this period FATF listing activity correlates with a decline in the market capitalisa- tion of companies in the countries selected for listing, but this rebounds quite quickly, a trend reinforced during the remediation process. The remediation process also seems to encourage lenders, as does delisting. After an initial decline, remediation is also positive for exchange rates, although negative for GDP growth. The same pattern of improvement in ﬁnancial situation is evident for net ﬁnancial ﬂows, and for net foreign assets, reinforcing the ﬁndings of Balakina et al. (2017). These ﬁndings also reinforce those of Morse (2017). 5.3. Period III: 2010–2015 g g This period sees the emergence of a correlation between the remediation process plus delisting and a decline in net ODA, as well as, with a lag, the listing process itself. It is possible that this reﬂects FATF attention exacerbating institutional disquiet about a country (causation?), or that the same issues that FATF is concerned about also concern aid ofﬁcials (correlation?). There is also more strongly signiﬁcant evidence of a negative impact on IBRD loans and IDA credits throughout the remediation and delisting periods which persists after one- and two-year lags. Again, this may reﬂect greater caution in institutional policies and procedures following the listing. 5.4. Period IV: 2016 Onwards From 2016, greylisting was restricted to those countries with an action plan, i.e., those countries that were actively working with the FATF to address strategic deﬁciencies in their AML/CFT/CPF regimes. This meant that the greylisting public statements may have been viewed as less alarming by the market, ﬂagging these countries as jurisdictions with problems but also with political commitment and action plans to address the problems. At the same time that the FATF was stepping away from calling for enhanced due diligence measures in relation to greylisted countries, the EU and other countries were requiring their institutions to do precisely that. Table 4 summarises the more signiﬁcant results. Risks 2023, 11, 81 19 of 32 Table 4. Correlations with indicator variables in 2016–2020 period. Table 4. Correlations with indicator variables in 2016–2020 period. Table 4. Correlations with indicator variables in 2016–2020 period. 5.4. Period IV: 2016 Onwards 2016 onwards———————————————————-→ Dependent Variable Initial Listing IV t-Stat Remediation IV t-Stat Delisting IV t-Stat Bank nonperforming loans to total gross loans (%) y-on-y change −0.0522 −2.163 0.0329 2.459 0.0198 1.083 Bank nonperforming loans to total gross loans (%) t + 1 y-on-y change −0.0453 −1.843 0.0276 2.024 0.0251 1.346 Bank nonperforming loans to total gross loans (%) t + 2 y-on-y change −0.0460 −1.847 0.0240 1.738 0.0239 1.271 Private non-guaranteed loans, commercial banks and other creditors 0.0120 1.399 −0.0070 −1.479 0.0031 0.474 Private non-guaranteed loans, commercial banks and other creditors t + 1 0.0035 0.407 −0.0015 −0.304 −0.0130 −1.985 Private non-guaranteed loans, commercial banks and other creditors t + 2 0.0073 0.832 −0.0070 −1.455 −0.0084 −1.276 Exchange Rate (GDP(LC)/GDP(US$)) y-on-y change −0.0532 −1.109 0.0792 2.982 0.0654 1.802 Exchange Rate (GDP(LC)/GDP(US$)) t + 1 y-on-y change 0.0631 1.133 −0.0170 −0.552 0.0166 0.393 Exchange Rate (GDP(LC)/GDP(US$)) t + 2 y-on-y change 0.0497 0.629 −0.0233 −0.532 0.0943 1.576 Year-on-year change in GDP (current US$) 0.0272 0.511 −0.0713 −2.417 −0.0778 −1.930 Year-on-year change in GDP (current US$) t + 1 −0.0314 −0.584 −0.0366 −1.231 −0.0597 −1.47 Year-on-year change in GDP (current US$) t + 2 −1.0759 −0.214 −0.2282 −0.082 −2.7520 −0.72 Net ofﬁcial development assistance and ofﬁcial aid received (current US$)/GDP −0.0346 −2.555 0.0474 6.319 −0.0376 −3.663 Net ofﬁcial development assistance and ofﬁcial aid received (current US$)/GDP t + 1 −0.0556 −4.095 0.0580 7.718 −0.0408 −3.973 Net ofﬁcial development assistance and ofﬁcial aid received (current US$)/GDP t + 2 −0.0584 −4.297 0.0604 8.027 −0.0385 −3.739 IBRD loans and IDA credits (DOD, current US$)/GDP 0.0042 0.163 −0.0250 −1.765 −0.0249 −1.29 IBRD loans and IDA credits (DOD, current US$)/GDP t + 1 0.0060 0.254 −0.0193 −1.467 −0.0193 −1.07 IBRD loans and IDA credits (DOD, current US$)/GDP t + 2 0.0113 0.521 −0.0125 −1.041 −0.0117 −0.71 Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. 5.4. Period IV: 2016 Onwards Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Risks 2023, 11, 81 20 of 32 There is strongly signiﬁcant evidence that listing coincided with a reduction in non- performing loans and (less signiﬁcantly) an improvement in the willingness of lenders to grant loans, but that this reversed during the remediation period. Remediation seems to have coincided with an improvement in exchange rates, but also with a reduction in growth rates. The negative correlation between net ODA and listings and delistings persists into this period, with more strongly signiﬁcant coefﬁcients. However, there are now important differences in that the impact during the remediation process seems to be positive and the negative impact on delisting lasts for longer. This may be because some ODA is in fact allocated to selected countries to help with the remediation process. This raises possible concern that the ODA contributed to assist with institutional reform may come at the expense of funding non-AML/CFT/CPF development activities (“crowding out”?) and may reduce future ODA. There is also signiﬁcant evidence of correlation between the remediation and delisting process and reduced GDP growth rates, which may be related. An empirical ﬁnding which stands out in all the periods is the correlation between FATF listings and reductions in ODA loans, IBRD loans and IDA credits. There are several possible explanations for this. One is that FATF listing creates a period of uncertainty, during which ofﬁcials are hesitant to commit to loans in the relevant country. A second is that the same issues which cause FATF to list a country also cause ofﬁcials to downgrade that country’s credit-worthiness. A third is that FATF scrutiny distracts government ofﬁcials, and so delays other processes, including agreements on bilateral and multilateral loans. In Period IV, we also noted a positive correlation during the remediation and delisting phases, which may indicate a diversion of funds from other activities to those related to AML/CFT remediation. This is an area where more detailed investigation on a country-by-country basis, beyond the scope of this article, could well yield valuable insights. 6.1. Linking the Findings and the Model Over-compliant stakeholders may be less responsive to positive news emanating from listed countries (De Koker and Symington 2014). This would be expressed in the model as a delisted country still experiencing prohibitions and adverse terms of trade caused by enduring market sentiment or regulatory expectations. Disruption of the FATF’s general signalling could also be caused by interference from other macro signals such as market crises, political bargains and other competing review processes. g g Over time, however, FATF stakeholders would be expected to become more familiar with the FATF process and to observe that unless greylistings become blacklistings (till now an event of global signiﬁcance, and very rare) they apparently inevitably resolve over time. Stakeholders would also develop the ability to balance speciﬁc and general signals and note their individual relevance to greylisting outcomes. g y g In Period I the FATF’s general listing signal was new and, for the reasons described above, very strong. The speciﬁc signals were more focused. In terms of their effects, the correlation of foreign exchange rate falls on listing and delisting in this period are consistent with signals that combined to create/conﬁrm high-level negative country opinions and thus hardened terms of credit and trade in markets inﬂuenced by FATF norms. At the same time, there may be disinvestment by criminal and marginal players put off by the increase in scrutiny and cooperation associated with listing and the improved AML/CFT measures signalled by delisting. The decrease in foreign holdings seen could also follow from lower ﬁrm/government purchasing power, due to currency depreciation, and sales of foreign assets to cover cash shortfalls or in response to regulatory pressure (especially host regulatory pressure on listed-country-owned ﬁrms). The increase in ODA after delisting would correspond to a country having met delisting conditionality criteria. The lags support the notion of a learning process on all sides. In contrast, the signalling during Periods II and III was not as well-deﬁned as that during Period I. The public statements during Period II were not particularly informative and the Period III messaging was complex with dark- and light-grey lists, warning lists and public statements, as well as lists issued by FSRBs and published by the FATF. Period III also coincided with IMF and G7/G20 attempts to limit the negative impacts of the Global Financial Crisis on the many smaller countries, now members of FSRBs. 6.1. Linking the Findings and the Model 6.1. Linking the Findings and the Model The key FATF compliance audiences are countries (government ofﬁcials), markets (regulated institutions and investors), and society (civil society, commentators, etc). To secure the desired responses from these audiences, the FATF has to craft its standards, guidance, statements, and conduct to communicate information that would elicit the desired responses. p The model reﬂects how listing signals could reasonably affect trade and public ﬁnance, and thus the relevant economy. With respect to greylisting, these are: A speciﬁc signal to the listed country expressing concern about the seriousness of the identiﬁed AML/CFT deﬁciencies, the risk they pose to the global AML/CFT, and the urgency with which the deﬁciencies have to be remedied. It is conveyed as a formal and objective ﬁnding based on agreed standards and made by trained evaluators and peers after a carefully managed process that includes dialogue and review. A speciﬁc signal to the listed country expressing concern about the seriousness of the identiﬁed AML/CFT deﬁciencies, the risk they pose to the global AML/CFT, and the urgency with which the deﬁciencies have to be remedied. It is conveyed as a formal and objective ﬁnding based on agreed standards and made by trained evaluators and peers after a carefully managed process that includes dialogue and review. A general signal to the rest of the AML/CFT/CPF audiences, drawing attention to the ﬁndings and how stakeholders may react to the risks posed by the iden- tiﬁed deﬁciencies. This signal is less deﬁned and more open to interpretation, depending on speciﬁc country knowledge and dealings as well as own risk expo- sure. Responses are also informed by expectations of appropriate behaviour by counterparts and national regulators. It is important to note that the market response to the FATF signal has often reﬂected lags and delays. A negative impact may only eventuate a year or even two years after listing. As greylisting conveys not one signal but two, maintaining the correct balance or blend is essential. Otherwise, action taken by target groups may lead to outcomes the FATF did not intend. Firms, the media, and even national regulators may, for example, overact Risks 2023, 11, 81 21 of 32 21 of 32 or over-comply with the general signal. This is a common precautionary reaction to new information being received or new and old information being combined. 6.2. Limitations The study and the research questions focus on correlations rather than causation. There is a likelihood that any improvements in a country’s AML/CFT/CPF system are the consequence of its listing (Morse 2022) but this has to be determined at a country level as other drivers may also be relevant. Similarly, a negative economic impact may be discernible after a country was listed, and a positive impact after its delisting but, given the complexity of global, regional and national socio-political and economic factors, it cannot be stated with conﬁdence that those impacts were indeed caused, or even signiﬁcantly affected by, the listing and delisting. Simply demonstrating a correlation between an improved AML/CFT regime and FATF actions does not prove that FATF actions cause such improvements. Collinearity between explanatory variables in a regression equation can cause bias to estimated coefﬁcients, and also generate spurious measures of a particular variable’s signiﬁcance. In addition, explanatory factors used in regression equations may themselves be endogenous, meaning that they may be co-determined with the variable they are supposed to be explaining. For example, reductions in overseas investment and lending may be associated with FATF listing but in principle it could be that the same underlying concerns that drive the listing of a country also drive private sector concerns about inward investment and lending. Thus, a reduction in inward investment following a listing may not be caused by the listing. It is hard to tell without some model of causation. Listing considerations include information that is publicly available to the market, e.g., gaps in law and law enforcement. A plausible hypothesis is, however, that vulnerabilities that were suspected but could not be fully observed by honest private agents are veriﬁed or falsiﬁed by ofﬁcial action by an independent expert authority with some private information, such as the FATF. The unambiguous signal to the market from the FATF reduces information asymmetry from the perspective of the private agents and underpins a plausible hypothesis that listing itself makes a signiﬁcant contribution to the relevant impact. g p The existing empirical literature on the impact of FATF actions often does not address these questions adequately, despite there being a well-developed literature on causality and endogeneity as applied to other topics. It is for this reason that the empirical ﬁndings are described as correlations between listings and the economic and ﬁnancial variables, rather than the effects of listings. 6.2. Limitations This study is intended to inform further work that may help to shed more light on the issue by undertaking a more detailed micro- and meso-level investigation, taking into consideration the different periods in the FATF listing process. Given the dataset used in the study, the econometric analysis focused on the relevant calendar years of listing, remediation, and delisting. This would obscure short-term impacts, e.g., short-lived spikes in foreign exchange rates, that do not impact on annual rates. The full set of econometric results is attached as Attachment A. 6.1. Linking the Findings and the Model There were also internal adjustments between key FATF stakeholders, with rebalancing between especially IMF (Article IV) and FATF processes helping to rebalance FATF signalling towards the technical assistance and speciﬁcs (carrot) whilst maintaining the opportunity to name and shame generalities (stick). The increase in IBRD and IDA loans after delisting is again consistent with delisting signals being more readily discerned by markets and near-market multilateral actors with a corresponding positive impact on credit, goods and services. p g p p g Period IV saw a return to clearer signalling with the publication of only one FATF greylist, the former light-grey list, with countries that had deﬁciencies but were working on an agreed action plan to address them expeditiously. The language of the statements was also adjusted during this period to prevent unnecessarily negative market responses to these countries. In addition, the market would anticipate a fast resolution of the concerns followed by a delisting. As a result of all of these changes, negative market responses would tend to be limited. The more constructive approach by the FATF correlates with more positive outcomes in relation to positive aid and concessional lending correlations, possibly reﬂecting positive signals into credit markets, with knock-on effects in equity market conﬁdence. Positive or neutral responses would however be limited as the EU and other national regulators continue to call for enhanced due diligence measures in relation to greylisted countries, even when the FATF explicitly stated the contrary. g y p y y Period IV therefore still reﬂects some concerns: net ODA, IBRD loans and IDA credits seem to be reduced on listing and delisting, GDP growth rates are impacted, and bank non-performing loans may be adversely affected. These ﬁndings are of concern from a development policy perspective. It is possible that risk managers and risk management Risks 2023, 11, 81 22 of 32 22 of 32 processes in the ODA, IBRD loan, and IDA credit space are less responsive to the signalling changes in this phase than their corporate and commercial counterparts, or are less free to respond appropriately. These ﬁndings should be further investigated but they should prompt the FATF to review its listings market signalling and the regulatory requirements of its members to limit unintended negative economic consequences. 7. Conclusions This study identiﬁed four distinct periods in the FATF’s greylisting of countries that were not appropriately compliant with FATF standards, based on changes in its messag- ing and signalling in its processes and statements. The econometric analysis identiﬁed correlations between listing and delisting and a range of negative and positive economic indicators. The latter were identiﬁed using a FATF greylisting impact model that was developed to support the study. The study also provides an explanation of the potential causal drivers that may explain the correlations. Risks 2023, 11, 81 23 of 32 The study ﬁnds signiﬁcant correlations with several of the economic and ﬁnancial variables which are suggested as possible indicators by the economic and ﬁnancial model, but not with all. One- or two-year lags are evident in a number of cases. Several of the ﬁndings reinforce those previously identiﬁed in the literature, but there are also some differences. Importantly, negative indicators are less visible in the dataset in Period IV (2016). This may be due to changes in the list itself as the FATF, from February 2016, only added countries with strategic deﬁciencies that committed themselves to a plan of action and a timeline to address the concerns. The signalling by the FATF was also more positive in relation to the listed countries. The market, on the other hand, had also come to expect a delisting to follow a listing. This combination of factors may result in more muted negative market responses. Net ODA, IBRD loans and IDA credits seem, however, to be reduced on listing and delisting, while GDP growth rates are impacted, and bank non-performing loans may be adversely affected. These ﬁndings are of concern from a development policy perspective. Punitive economic consequences, especially in relation to development aid and fund- ing, would seem to run counter to the spirit and objective of the Period IV listings. The listing informs the global community that a country with strategic deﬁciencies has given its commitment to the FATF to actively and speedily address them. While punitive action may be required to move a country towards a commitment to address such deﬁciencies, there seems to be little need for punitive measures once that commitment has been extracted and it is clear that the country has implemented an agreed plan to address remaining problems. 7. Conclusions Should a country fail to act on its commitment, the FATF still has the option to list the country on its blacklist. In October 2022 the FATF blacklisted Myanmar for its failure to implement its action plan as agreed. The FATF is therefore alive to this option. FATF listing processes that trigger negative economic consequences for countries that are committed to resolving deﬁciencies and are swiftly acting in terms of an agreed action plan to do so, should be subjected to close policy scrutiny by all FATF stakeholders. Improvements may avoid unintended and unwarranted negative consequences. Despite the change in the FATF’s listing processes and language, the EU and other countries are requiring their institutions to adopt risk-based enhanced due diligence mea- sures in relation to greylisted countries. Many of these countries are leading members of the FATF but adopt such measures even though the FATF is explicitly stating that it does not call for enhanced due diligence in relation to greylisted countries. The FATF operates by consensus and members who require their own institutions to implement enhanced due diligence measures therefore agree with the position taken by the FATF. The market signalling in this regard is confusing and should be clariﬁed. The push for enhanced due diligence measures in these cases is said to be based on the AML/CFT-related risks posed by these jurisdictions, including the need for a consistent response to those risks. Such risks however are generally at their highest when the country is assessed for FATF compliance. In many cases, countries then improve their assessed compliance levels in an unsuccessful attempt to avoid greylisting. The risks they pose, as assessed by FATF processes, therefore tend to be lower by the time they are listed, compared to the risk levels during the initial assessment period. If the EU and other countries were concerned about risk, calls for a risk-based response to the mutual evaluation report rather than to the later listing would be more appropriate. The indications of a potential negative impact on development aid and funding in the current period are particularly concerning. Greylisting may cause economic and social harms that may not be fully appreciated by the FATF and its development stakeholders. 7. Conclusions The identiﬁed correlations should be further investigated but, even in the absence of further clarity, the FATF should consider its market signalling impact as well as the regulatory and market signalling responses of its members and other regulators to limit any unintended negative economic consequences for listed countries. It should of course be noted that these ﬁndings are general and should not be inter- preted as an absence or presence of negative responses or indicators for any particular Risks 2023, 11, 81 24 of 32 24 of 32 country. Country proﬁles differ widely, and some may have been negatively affected, although on balance most other peers were not affected. country. Country proﬁles differ widely, and some may have been negatively affected, although on balance most other peers were not affected. The ﬁndings and the model suggest least greylisting harm is likely when the FATF uses strong speciﬁc FATF signals that are to the point, combined with clear general signals which reinforce positive impacts and reassure unsettled markets. Positively framed and communicated greylisting need not have unintended and/or over-enduring effects on a national economy and may correct unfounded perceptions of risk that accompanied earlier periods in the FATF’s greylisting. These signals should, however, also be reﬂected by the FATF members’ own national regulations and regulators, and those of non-FATF members, to have their appropriate effect on the market. Future decisions regarding greylisting should ideally be informed by data and research. In particular, it would be helpful to strengthen the understanding of causation. This study identiﬁed correlations and more work is required to deepen insights into causation. With the data available and the model that was developed it is possible to prospectively assess the likely economic impact of a listing on a country. An assessment of the likely impact (intended and unintended effects) would be a useful inclusion in future listing discussions of the FATF, especially coupled with planning for development assistance to address such impact and safeguards/mechanisms to check and correct unintended consequences. Author Contributions: Conceptualisation, L.d.K., N.M., J.H.; methodology, L.d.K., N.M., J.H.; vali- dation, N.M.; formal analysis, N.M.; investigation, L.d.K., N.M., J.H.; resources, L.d.K.; data curation, N.M., L.d.K.; writing—original draft preparation, L.d.K., N.M., J.H.; writing—review and editing, L.d.K., N.M., J.H.; visualisation, N.M., J.H. All authors have read and agreed to the published version of the manuscript. 7. Conclusions Funding: This paper reﬂects work undertaken during a commission by the Deutsche Gesellschaft für Internationale Zusammenarbeit GmbH (GiZ). Contract number: 81263810; Project processing number: 18.2126.3-009.00. Data Availability Statement: Publicly available datasets were analysed in this study. This data can be found here: https://databank.worldbank.org/source/world-development-indicators which was accessed between 16 April 2021 and 9 May 2022 Additional data used in this analysis is available on request from the corresponding author. Acknowledgments: This study was supported by an advisory panel consisting of Peter Andrews (regulatory economist), Sue Jaffer (regulatory economist) and Mike Levi (criminologist). The authors acknowledge with appreciation the valuable and constructive discussions with the advisory panel members, and the research assistance and support provided by Alette de Koker and Sue Jaffer. The paper however reﬂects the views of the authors and not necessarily those of individual advisory panel members. The authors further acknowledge support provided by the FATF secretariat who shared some data on listing dates, and constructive comments from Dr Julia Morse and other participants on an early draft presented at the Third Bahamas Empirical Anti-Money Laundering Conference, hosted in January 2022 by the Central Bank of The Bahamas. Louis de Koker acknowledges the support received from La Trobe University and from Wolfson College, Oxford, and CeFIM at SOAS who accommodated him as a visiting scholar and enabled the completion of this work. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funder framed the question but had no involvement in the design of the study or in the collection, analysis and interpretation of data, apart from making constructive comments on an early draft of the original report that included country studies. The funder had no role in the decision to publish the results. Appendix A. Econometric Results Risks 2023, 11, 81 25 of 32 Table A1. Summary of Pooled Cross-section Time Series regressions with Fixed Effects, First Order Correlations, Immediate, one- and two-year lags. T-stat 80% 1.282 T-stat 90% 1.645 T-stat 95% 1.96 https://www.stat.colostate.edu/inmem/gumina/st201/pdf/Utts-Heckard_t-Table.pdf (accessed on 5 February 2021) Signiﬁcant? Appendix A. Econometric Results http://uregina.ca/~gingrich/tt.pdf (accessed on 5 February 2021) Negative impact Positive impact 2000–2006—————————————————— ——> 2007–2009—————————————————— ——> 2010–2015—————————————————— ——> 2016 onwards———————————————— ————> Diagnostics Dependent Variable Initial List- ing I t- Stat Reme- diation I t- Stat De- listing I t- Stat Initial List- ing II t- Stat Reme- diation II t- Stat De- listing II t- Stat Initial List- ing III t- Stat Reme- diation III t- Stat De- listing III t- Stat Initial List- ing IV t- Stat Reme- diation IV t- Stat De- listing IV t- Stat GFC Du- mmy t- Value R- Square F- Stat Outliers Net ofﬁcial development assistance and ofﬁcial aid eceived (current US$)/GDP 0.0050 0.434 −0.0005 −0.04 0.0103 1.176 0.0191 0.896 −0.01339 −0.83 0.0072 0.286 −0.001 −0.15 −0.0066 −1.94 −0.0098 −1.75 −0.035 −2.56 0.0474 6.319 −0.0376 −3.66 0.0004 0.221 0.8235 74.0 51 Net ofﬁcial development assistance and ofﬁcial aid eceived (current US$)/GDP t + 1 0.0116 1.001 0.0008 0.094 0.0133 1.517 0.0144 0.677 −0.00945 −0.58 0.0031 0.122 −0.004 −0.70 −0.0047 −1.38 −0.0083 −1.48 −0.056 −4.10 0.0580 7.718 −0.0408 −3.97 0.0041 2.057 0.8202 72.3 63 Net ofﬁcial development assistance and ofﬁcial aid eceived (current US$)/GDP t + 2 0.0104 0.893 0.0018 0.215 0.0025 0.291 −0.034 −1.61 0.02859 1.756 −0.0006 −0.03 −0.007 −1.29 −0.0021 −0.61 −0.0070 −1.23 −0.058 −4.30 0.0604 8.027 −0.0385 −3.74 0.0034 1.713 0.8166 70.6 69 FC, private nonguaranteed NFL, current US$)/GDP 0.0000 0.136 −0.0001 −0.23 −0.0002 −0.48 0.0002 0.179 0.00002 0.024 0.0002 0.188 0.0001 0.515 0.0000 0.299 0.0001 0.476 0.0004 0.564 −0.0001 −0.41 0.0002 0.367 −0.0002 −1.701 0.0451 0.75 33 FC, private nonguaranteed NFL, current US$)/GDP t + 1 −0.0003 −0.66 0.0001 0.128 −0.0001 −0.18 −0.0001 −0.07 0.00007 0.089 0.0002 0.208 −0.0003 −0.97 0.0002 0.966 0.0002 0.761 0.0004 0.639 −0.0005 −1.48 −0.0006 −1.28 0.0001 1.543 0.0478 0.80 34 FC, private nonguaranteed NFL, current US$)/GDP t + 2 −0.0004 −0.75 0.0002 0.518 −0.0002 −0.46 −0.0003 −0.31 0.00009 0.114 0.0001 0.075 0.0001 0.308 0.0001 0.698 −0.0002 −0.59 −0.0001 −0.21 −0.0001 −0.41 −0.0001 −0.29 0.0004 3.845 0.0498 0.83 33 BRD loans and DA credits DOD, current US$)/GDP −0.0033 −0.15 0.0084 0.534 −0.0013 −0.08 0.0548 1.367 −0.09248 −3.03 −0.0104 −0.22 0.0045 0.413 −0.0352 −5.49 −0.0352 −3.33 0.0042 0.163 −0.0250 −1.77 −0.0249 −1.29 −0.0129 −3.440 0.6058 24.3 83 BRD loans and DA credits DOD, current US$)/GDP t + 1 0.0012 0.063 0.0045 0.304 −0.0031 −0.20 0.0506 1.354 −0.07549 −2.65 −0.0082 −0.18 0.0028 0.276 −0.0307 −5.15 −0.0302 −3.06 0.0060 0.254 −0.0193 −1.47 −0.0193 −1.07 −0.0134 −3.844 0.5967 23.4 93 BRD loans and DA credits DOD, current US$)/GDP t + 2 0.0032 0.174 0.0006 0.048 −0.0047 −0.34 0.0427 1.250 −0.06014 −2.31 −0.0060 −0.15 0.0025 0.274 −0.0253 −4.65 −0.0235 −2.60 0.0113 0.521 −0.0125 −1.04 −0.0117 −0.71 −0.0118 −3.697 0.5972 23.5 77 Table A1. Appendix A. Econometric Results Summary of Pooled Cross-section Time Series regressions with Fixed Effects, First Order Correlations, Immediate, one- and two-year lags. Risks 2023, 11, 81 26 of 32 T-stat 80% 1.282 T-stat 90% 1.645 T-stat 95% 1.96 https://www.stat.colostate.edu/inmem/gumina/st201/pdf/Utts-Heckard_t-Table.pdf (accessed on 5 February 2021) Signiﬁcant? http://uregina.ca/~gingrich/tt.pdf (accessed on 5 February 2021) Negative impact Positive impact 2000–2006—————————————————— ——> 2007–2009—————————————————— ——> 2010–2015—————————————————— ——> 2016 onwards———————————————— ————> Diagnostics Dependent Variable Initial List- ing I t- Stat Reme- diation I t- Stat De- listing I t- Stat Initial List- ing II t- Stat Reme- diation II t- Stat De- listing II t- Stat Initial List- ing III t- Stat Reme- diation III t- Stat De- listing III t- Stat Initial List- ing IV t- Stat Reme- diation IV t- Stat De- listing IV t- Stat GFC Du- mmy t- Value R- Square F- Stat Outliers Risk Premium y-on-y change 0.13279 0.270 −0.4811 −1.355 −0.3618 −0.979 −0.1212 −0.134 0.0459 0.067 0.0133 0.012 −0.0108 −0.045 −0.0429 −0.298 0.0390 0.164 0.0480 0.084 0.0083 0.026 −0.0757 −0.174 −0.0130 −0.154 0.0484 0.81 10 Risk Premium t + y-on-y change 0.20510 0.417 −0.5222 −1.470 −0.2848 −0.771 0.1350 0.150 −0.0466 −0.068 −0.0008 −0.001 −0.0306 −0.126 −0.0165 −0.114 −0.0534 −0.224 −0.1740 −0.303 0.0706 0.222 −0.2060 −0.474 −0.0340 −0.404 0.0484 0.81 10 Risk Premium t + y-on-y change 0.16417 0.334 −0.4923 −1.390 −0.3713 −1.008 0.0013 0.001 −0.0249 −0.036 −0.0163 −0.015 −0.0531 −0.219 −0.0152 −0.106 −0.0629 −0.265 0.0980 0.171 −0.0532 −0.168 −0.0494 −0.114 −0.0217 −0.258 0.0480 0.80 10 Spread y-on-y hange −0.05828 −0.206 0.0594 0.291 −0.0098 −0.046 −0.7295 −1.406 0.0664 0.168 −0.0362 −0.059 0.0514 0.368 −0.0273 −0.329 0.0180 0.131 −0.0497 −0.151 0.0224 0.122 −0.0205 −0.082 0.0077 0.159 0.0136 0.22 13 Spread t + 1 y-on-y change 0.00711 0.025 0.0445 0.218 −0.0211 −0.099 −0.0966 −0.186 0.1055 0.267 0.0082 0.013 −0.0168 −0.120 0.0084 0.102 0.0179 0.131 0.0780 0.236 0.0133 0.073 −0.0101 −0.040 0.1319 2.728 0.0150 0.24 13 Spread t + 2 y-on-y change 0.07750 0.271 −0.0231 −0.112 −0.0150 −0.070 0.6489 1.239 −0.6832 −1.712 0.0084 0.013 −0.0300 −0.213 0.0294 0.351 0.1138 0.823 −0.0769 −0.231 0.0610 0.330 0.0429 0.170 0.1368 2.803 0.0161 0.26 12 Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Appendix A. Econometric Results Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Results based on WDI 27 April 2022 update. Changes in variable after FATF intervention. Country ﬁxed effects relative to USA. Constant set to zero. Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Results based on WDI 27 April 2022 update. Changes in variable after FATF intervention. Country ﬁxed effects relative to USA. Constant set to zero. Risks 2023, 11, 81 27 of 32 Table A2. Second-Order Correlations, Immediate, one- and two-year lags. Appendix A. Econometric Results 2000–2006—————————————————— ——> 2007–2009—————————————————— ——> 2010–2015—————————————————— ——> 2016 onwards———————————————— ————> Diagnostics Dependent Variable Initial List- ing I t- Stat Remed- iation I t- Stat De- listing I t- Stat Initial List- ing II t- Stat Remed- iation II t- Stat De- listing II t- Stat Initial List- ing III t- Stat Remed- iation III t- Stat De- listing III t- Stat Initial List- ing IV t- Stat Remed- iation IV t- Stat De- listing IV t- Stat GFC Dummy t- Value R- Square F- Stat Outliers Bank nonperforming oans to total gross loans (%) y-on-y change 0.01477 0.714 −0.0628 −4.207 −0.0443 −2.856 −0.1900 −5.012 0.35048 12.134 −0.0452 −1.005 0.0078 0.767 −0.0045 −0.748 −0.0039 −0.387 −0.0522 −2.163 0.0329 2.459 0.0198 1.083 −0.0147 −4.149 0.4683 13.96 51 Bank nonperforming oans to total gross loans (%) t + y-on-y change 0.01204 0.571 −0.0605 −3.981 −0.0058 −0.365 0.3060 7.919 −0.0455 −1.544 −0.0365 −0.794 0.0017 0.166 −0.0057 −0.917 −0.0021 −0.210 −0.0453 −1.843 0.0276 2.024 0.0251 1.346 −0.0065 −1.795 0.4799 14.63 52 Bank nonperforming oans to total gross loans (%) t + 2 y-on-y change −0.0273 −1.280 −0.0198 −1.286 −0.0056 −0.347 −0.0146 −0.372 −0.0617 −2.068 −0.0364 −0.784 0.0058 0.552 −0.0038 −0.614 0.0057 0.549 −0.0460 −1.847 0.0240 1.738 0.0239 1.271 0.0061 1.669 0.4924 15.38 42 Market capitalisation of isted domestic companies current US$) y-on-y change −0.2683 −0.116 0.4054 0.244 0.1524 0.088 −0.4235 −0.100 0.70354 0.218 0.2270 0.045 −2.8970 −2.548 2.9748 4.406 −0.2035 −0.182 −0.1188 −0.044 0.3837 0.258 0.3571 0.175 −0.0870 −0.221 0.0618 1.04 17 Market capitalisation of isted domestic companies current US$) t + 1 y-on-y change −0.2737 −0.119 0.4150 0.251 0.3050 0.177 0.1466 0.035 0.02755 0.009 −0.1598 −0.032 7.5329 6.666 0.0019 0.003 −0.1970 −0.178 −0.2016 −0.075 0.3610 0.244 0.2331 0.115 −0.0625 −0.160 0.0736 1.26 17 Market capitalisation of isted domestic companies current US$) t + 2 y-on-y change −0.0321 −0.014 −0.0341 −0.020 −0.0105 −0.006 0.2318 0.055 −0.3837 −0.119 −0.3391 −0.067 0.1351 0.119 −0.7050 −1.042 −0.6876 −0.615 −0.1936 −0.072 0.1397 0.094 −0.1728 −0.085 −0.1173 −0.297 0.0561 0.94 15 Private non-guaranteed oans, commercial banks and other creditors −0.003 −0.400 −0.0006 −0.106 −0.0034 −0.619 −0.0074 −0.550 0.00228 0.222 −0.0020 −0.123 0.0046 1.263 0.0076 3.549 0.0091 2.569 0.0120 1.399 −0.0070 −1.479 0.0031 0.474 0.0027 2.132 0.2638 5.68 58 Private non-guaranteed oans, commercial banks and other creditors t + 1 0.00031 0.042 −0.0026 −0.478 −0.0037 −0.671 −0.0053 −0.390 0.00205 0.197 −0.0001 −0.008 −0.0033 −0.900 0.0075 3.425 0.0040 1.102 0.0035 0.407 −0.0015 −0.304 −0.0130 −1.985 0.0055 4.309 0.2739 5.98 60 Private non-guaranteed oans, commercial banks and other creditors t + 2 −0.003 −0.398 −0.0024 −0.437 −0.0010 −0.171 −0.0039 −0.283 0.00171 0.164 −0.0010 −0.064 0.0049 1.320 0.0021 0.954 −0.0027 −0.755 0.0073 0.832 −0.0070 −1.455 −0.0084 −1.276 0.0027 2.080 0.2863 6.36 58 Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Appendix A. Econometric Results Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Results based on WDI 27 April 2022 update. Changes in variable after FATF intervention. Country ﬁxed effects relative to USA. Constant set to zero. Table A2. Second-Order Correlations, Immediate, one- and two-year lags. Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Results based on WDI 27 April 2022 update. Changes in variable after FATF intervention. Country ﬁxed effects relative to USA. Constant set to zero. Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Results based on WDI 27 April 2022 update. Changes in variable after FATF intervention. Country ﬁxed effects relative to USA. Constant set to zero. Risks 2023, 11, 81 28 of 32 Table A3. Leading Indicators. Appendix A. Econometric Results Leading Indicators 2000–2006—————————————————— ——> 2007–2009—————————————————— ——> 2010–2015—————————————————— ——> 2016 onwards————————————————————> Diagnostics Dependent Variable Initial List- ing I t- Stat Reme- diation I t- Stat De- listing I t- Stat Initial List- ing II t- Stat Reme- diation II t- Stat De- listing II t- Stat Initial List- ing III t- Stat Reme- diation III t- Stat De- listing III t- Stat Initial List- ing IV t- Stat Reme- diation IV t- Stat De- listing IV t- Stat GFC Du- mmy t- Value R- Square F- Stat Outliers Exchange Rate GDP(LC)/GDP(US$)) y-on-y change −0.04769 −1.160 0.0137 0.462 −0.0054 −0.176 −0.0229 −0.304 0.07801 1.359 0.0316 0.353 −0.0668 −3.294 0.0426 3.536 0.0117 0.591 −0.0532 −1.109 0.0792 2.982 0.0654 1.802 −0.0561 −7.990 0.2190 4.45 40 Exchange Rate GDP(LC)/GDP(US$)) + 1 y-on-y hange 0.00851 0.178 0.0099 0.288 −0.0498 −1.389 0.0684 0.782 −0.02396 −0.359 0.0332 0.320 0.0489 2.076 0.0315 2.252 0.0292 1.265 0.0631 1.133 −0.0170 −0.552 0.0166 0.393 0.0060 0.735 0.1894 3.70 33 Exchange Rate GDP(LC)/GDP(US$)) + 2 y-on-y hange −0.00269 −0.040 −0.0229 −0.469 −0.0649 −1.276 0.0381 0.306 −0.06340 −0.670 −0.0210 −0.143 −0.0088 −0.264 0.0140 0.704 0.0114 0.347 0.0497 0.629 −0.0233 −0.532 0.0943 1.576 0.0172 1.485 0.1537 2.88 29 Year-on-year hange in GDP current US$) −0.0714 −1.565 0.0358 1.088 0.0433 1.265 0.1271 1.519 −0.0884 −1.386 −0.0484 −0.487 0.0909 4.039 −0.0372 −2.782 −0.0146 −0.662 0.0272 0.511 −0.0713 −2.417 −0.0778 −1.930 0.1054 13.517 0.3553 8.74 39 Year-on-year hange in GDP current US$) t + 1 −0.0296 −0.644 0.0268 0.807 0.0486 1.407 −0.0657 −0.779 −0.0170 −0.265 −0.0499 −0.498 0.0410 1.805 −0.0850 −6.311 −0.0860 −3.860 −0.0314 −0.584 −0.0366 −1.231 −0.0597 −1.469 −0.0232 −2.948 0.3229 7.56 37 Year-on-year hange in GDP current US$) t + 2 −0.2503 −0.058 0.2917 0.094 1.8897 0.585 1.9502 0.247 0.5420 0.090 0.6712 0.072 −1.9131 −0.901 −1.1836 −0.939 −0.8024 −0.385 −1.0759 −0.214 −0.2282 −0.082 −2.7520 −0.724 −0.0619 −0.084 0.0583 0.98 11 Net ﬁnancial ﬂows, bilateral NFL, current US$]/GDP 0.00224 0.611 −0.0026 −0.979 −0.0017 −0.603 −0.0198 −2.952 0.02172 4.246 0.0086 1.074 −0.0024 −1.327 0.0058 5.404 0.0057 3.214 0.0053 1.250 0.0010 0.420 0.0062 1.915 −0.0020 −3.183 0.4097 11.01 55 Net ﬁnancial ﬂows, bilateral NFL, current US$]/GDP t + 1 0.00292 0.825 −0.0034 −1.331 0.0011 0.398 −0.0401 −6.176 0.05370 10.848 0.0068 0.877 −0.0010 −0.570 0.0052 5.014 0.0055 3.191 0.0027 0.664 0.0014 0.594 0.0013 0.413 −0.0003 −0.545 0.4215 11.55 58 Net ﬁnancial ﬂows, bilateral NFL, current US$]/GDP t + 2 0.00314 0.864 −0.0027 −1.030 0.0001 0.023 −0.0218 −3.276 0.04168 8.205 0.0001 0.016 −0.0014 −0.759 0.0056 5.305 0.0022 1.237 −0.0022 −0.511 0.0029 1.246 −0.0034 −1.067 0.0018 2.825 0.3921 10.23 59 Foreign direct nvestment, net nﬂows (BoP, urrent US$)/GDP −0.00824 −0.175 −0.0115 −0.338 −0.0148 −0.421 −0.0726 −0.843 0.05570 0.849 0.8954 8.760 0.0018 0.078 0.0070 0.511 −0.0044 −0.195 −0.0030 −0.054 −0.0053 −0.175 0.0082 0.198 0.0290 3.610 0.4418 12.55 45 Foreign direct nvestment, net nﬂows (BoP, urrent US$)/GDP t + 1 −0.02129 −0.450 −0.0083 −0.242 −0.0049 −0.138 0.2355 2.715 0.06811 1.031 0.1598 1.551 0.0027 0.116 0.0005 0.033 −0.0028 −0.123 −0.0084 −0.152 −0.0030 −0.097 −0.0032 −0.076 0.0048 0.597 0.4467 12.80 44 Table A3. Appendix A. Econometric Results Leading Indicators. Risks 2023, 11, 81 29 of 32 Table A3. Cont. Leading Indicators 2000–2006—————————————————— ——> 2007–2009—————————————————— ——> 2010–2015—————————————————— ——> 2016 onwards————————————————————> Diagnostics Dependent Variable Initial List- ing I t- Stat Reme- diation I t- Stat De- listing I t- Stat Initial List- ing II t- Stat Reme- diation II t- Stat De- listing II t- Stat Initial List- ing III t- Stat Reme- diation III t- Stat De- listing III t- Stat Initial List- ing IV t- Stat Reme- diation IV t- Stat De- listing IV t- Stat GFC Du- mmy t- Value R- Square F- Stat Outliers Foreign direct nvestment, net nﬂows (BoP, current US$)/GDP t + 2 −0.01821 −0.384 −0.0023 −0.068 −0.0177 −0.498 0.0658 0.757 0.00441 0.067 0.2296 2.225 0.0021 0.089 −0.0036 −0.257 −0.0176 −0.768 −0.0118 −0.213 −0.0010 −0.031 0.0059 0.142 0.0031 0.382 0.4684 13.97 40 Foreign direct nvestment, net outﬂows (BoP, current US$)/GDP −0.00881 −0.215 −0.0027 −0.091 −0.0058 −0.188 −0.0653 −0.868 −0.005 −0.085 0.7838 8.767 −0.0028 −0.140 0.0033 0.277 0.0032 0.162 −0.0030 −0.062 0.0016 0.060 0.0005 0.013 0.0230 3.276 0.3438 8.31 44 Foreign direct nvestment, net outﬂows (BoP, current US$)/GDP t + 1 −0.01271 −0.305 −0.0023 −0.078 −0.0053 −0.169 0.2354 3.084 0.012 0.199 0.1466 1.616 −0.0031 −0.150 0.0031 0.253 0.0007 0.036 −0.0033 −0.067 0.0016 0.060 0.0011 0.029 0.0015 0.211 0.3561 8.77 44 Foreign direct nvestment, net outﬂows (BoP, current US$)/GDP t + 2 −0.01180 −0.276 −0.0024 −0.077 −0.0111 −0.345 0.0345 0.440 −0.002 −0.032 0.34 3.666 −0.0008 −0.040 0.0007 0.053 −0.0021 −0.104 −0.0046 −0.092 0.0015 0.053 −0.0006 −0.016 −0.0005 −0.068 0.3825 9.82 46 Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Results based on WDI 27 April 2022 update. Changes in variable after FATF intervention. Country ﬁxed effects relative to USA. Constant set to zero. Table A3. Cont. Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Appendix A. Econometric Results Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Results based on WDI 27 April 2022 update. Changes in variable after FATF intervention. Country ﬁxed effects relative to USA. Constant set to zero. Notes: The period ‘Initial listing’ refers to the calendar year in which the listing occurs, ‘Remediation’ refers to the calendar year(s) including the year in which listing occurs, and ‘Delisting’ to the calendar year in which delisting occurred. Colour-coding denotes signiﬁcance at the 95% (dark blue), 90% (blue) and 80% (light blue) conﬁdence levels and whether the effect is positive (green) or negative (pink). Results based on WDI 27 April 2022 update. Changes in variable after FATF intervention. Country ﬁxed effects relative to USA. Constant set to zero. 30 of 32 Risks 2023, 11, 81 References All Politics is Global: Explaining International Regulatory Regimes. Princeton: Prin Drezner, Daniel W. 2008. All Politics is Global: Explaining International Regulatory Regimes. Princeton: Princeton University Press. Ederington, Louis H., and Jae Ha Lee. 1993. How markets process information: News releases and volatility. The Journal of Finance 48: 1161–91. [CrossRef] European Commission. 2020. Methodology for Identifying High-Risk Third Countries under Directive (EU) 2015/849. Commission Staff Working Document SWD(2020) 99. Available online: https://ec.europa.eu/info/sites/default/ﬁles/business_economy_eu ro/banking_and_finance/documents/200507-anti-money-laundering-terrorism-ﬁnancing-action-plan-methodology_en.pdf (ac- cessed on 10 August 2022). g European Commission. 2022. EU Policy on High-Risk Third Countries. Available online: https://ec.europa.eu/info/business-econo my-euro/banking-and-ﬁnance/financial-supervision-and-risk-management/anti-money-laundering-and-countering-ﬁnanci ng-terrorism/eu-policy-high-risk-third-countries_en#objectives (accessed on 10 August 2022). Farías, María Elisa, and Monica Arruda de Almeida. 2014. Does saying ‘yes’ to capital inﬂows necessarily mean good business? The effect of antimoney laundering regulations in the Latin American and the Caribbean economies. Economics and Politics 26: 96–127. Available online: https://ideas.repec.org/a/bla/ecopol/v26y2014i1p96-127.html (accessed on 10 August 2022). Farka, Mira, and Adrian R. Fleissig. 2012. The effect of FOMC statements on asset prices. International Review of Applied Economics 26: 387–416. [CrossRef] FATF. 2000. Annual Report 1999–2000. Available online: https://www.fatf-gafi.org/en/publications/Fatfgeneral/Fatfannualreport19 99-2000.html (accessed on 10 August 2022). FATF. 2001. Review to Identify Non-Cooperative Countries or Territories: Increasing the World-Wide Effectiveness of Anti-money Laundering Measures (June 2001). Available online: https://www.fatf-gafi.org/media/fatf/documents/reports/2000%202001 %20NCCT%20ENG.pdf (accessed on 10 August 2022). %20NCCT%20ENG.pdf (accessed on 10 August 2022). FATF. 2003a. Annual Report 2002–2003. Available online: https://www.oecd.org/newsroom/2789358.pdf (accessed on 10 August 2022). FATF. 2003b. The Forty Recommendations. Available online: https://www.fatf-gafi.org/media/fatf/documents/recommendations/p dfs/FATF%20Recommendations%202003.pdf (accessed on 10 August 2022). FATF. 2004a. FATF IX Special Recommendations. Available online: https://www.fatf-gafi.org/content/dam/fatf/documents/reports /methodology.pdf (accessed on 10 August 2022). FATF 2004b Methodology for Assessing Compliance with the FATF 40 Recommendations and FATF 9 Special Recommendations FATF. 2004a. FATF IX Special Recommendations. Available online: https://www.fatf-gafi.org/content/dam/fatf/documents/reports /methodology.pdf (accessed on 10 August 2022). FATF. 2004b. Methodology for Assessing Compliance with the FATF 40 Recommendations and FATF 9 Special Recommendations. Available online: https://www.fatf-gafi.org/publications/fatfrecommendations/documents/methodologyforassessingcompli ancewiththefatf40recommendationsandfatf9specialrecommendations.html (accessed on 10 August 2022). FATF. 2004b. Methodology for Assessing Compliance with the FATF 40 Recommendations and FATF 9 Special Recommendations. Available online: https://www.fatf-gafi.org/publications/fatfrecommendations/documents/methodologyforassessingcompli ancewiththefatf40recommendationsandfatf9specialrecommendations.html (accessed on 10 August 2022). p g FATF. 2007a. Annual Review of Non-Cooperative Countries and Territories 2006–2007: Eighth NCCT Review. Available online: https://www.fatf-gafi.org/media/fatf/documents/reports/2006%202007%20NCCT%20ENG.pdf (accessed on 10 August 2022). FATF. 2007a. Annual Review of Non-Cooperative Countries and Territories 2006–2007: Eighth NCCT Review. Available online: https://www.fatf-gafi.org/media/fatf/documents/reports/2006%202007%20NCCT%20ENG.pdf (accessed on 10 August 2022). FATF. 2007b. Statement on Iran. Available online: https://www.ﬁncen.gov/sites/default/ﬁles/shared/FATFOct2007.pdf (accessed on FATF. 2007a. References Avdjiev, Stefan, Patrick McGuire, and Nikola A. Tarashev. 2012. The Social Value of Policy Signals. Bank for International Settlements Working Paper No. 386. Basel: Bank for International Settlements. Available online: https://www.bis.org/publ/work386.pdf (accessed on 10 August 2022). ( g ) Balakina, Olga, Angelo D’Andrea, and Donato Masciandaro. 2017. Bank secrecy in offshore centres and capital ﬂows: Does blacklisting matter? Review of Financial Economics 32: 30–57. Available online: https://EconPapers.repec.org/RePEc:eee:revfin:v:32:y:2017:i:c: p:30 57 (accessed on 10 August 2022). [CrossRef] Basel Institute on Governance. 2022. Basel AML Index. Available online: https://index.baselgovernance.org/ (accessed on 10 August 2022) Basel Institute on Governance. 2022. Basel AML Index. Available online: https://index.baselgovernance.org/ (accessed on 10 August 2022). ) Case-Ruchala, Devin, and Mark Nance. 2020. FATF blacklists don’t work the way you think they do. Paper presented at the Second International Research Conference on Empirical Approaches to AML and Financial Crime Suppression, Nassau, Bahamas, 27–29 January; Available online: https://bahamasamlconference.centralbankbahamas.com/assets/images/pdf/2021/Evaluating% 20 ff t %20 f%20FATF%20li ti %20 %20C R h l %20 d%20N df ( d 10 A t 2022) International Research Conference on Empirical Approaches to AML and Financial Crime Suppression, Nassau, Bahamas, 27 29 January; Available online: https://bahamasamlconference.centralbankbahamas.com/assets/images/pdf/2021/Evaluating% 20effects%20of%20FATF%20listing%20-%20Case-Ruchala%20and%20Nance.pdf (accessed on 10 August 2022). January; Available online: https://bahamasamlconference.centralbankbahamas.com/assets/images/pdf/2021/Evaluating% 20effects%20of%20FATF%20listing%20-%20Case-Ruchala%20and%20Nance.pdf (accessed on 10 August 2022). 20effects%20of%20FATF%20listing%20-%20Case-Ruchala%20and%20Nance.pdf (accessed on 10 August 2022). g p g CFATF. 2013. CFATF Decides to Call for Counter Measures against Belize and Guyana. Available online: htt publications/high-risk-and-other-monitored-jurisdictions/documents/cfatf-ps-nov2013.html (access g p g 2013. CFATF Decides to Call for Counter Measures against Belize and Guyana. Available online: https://w des to Call for Counter Measures against Belize and Guyana. Available online: https://www.fatf-gafi.org/ g y p ublications/high-risk-and-other-monitored-jurisdictions/documents/cfatf-ps-nov2013.html (accessed on 1 p g j p ( g ) CFATF. 2016. CFATF Public Statement on Haiti, Suriname and Guyana. Available online: https://www.fatf-gafi.org/content/fatf-gafi/ en/publications/High-risk-and-other-monitored-jurisdictions/Cfatf-public-statement-haiti-suriname-guyana-nov2016.html (ac- cessed on 10 August 2022). g Collin, Matthew, Samantha Cook, and Kimmo Soramaki. 2016. The Impact of Anti-Money Laundering Regulation on Payment Flows: Evidence from SWIFT Data. Center for Global Development Working Paper No. 445. Washington, DC: Centre for Global Development. [CrossRef] p Dalip, Andrew. 2020. The EU’s 2020 Methodology vs. the FATF Greylist. ACAMS Today. Available online: https://www.acamstoday.o rg/the-eus-2020-methodology-vs-the-fatf-greylist/ (accessed on 10 August 2022). De Koker, Louis, and John Symington. 2014. Conservative corporate compliance: Reﬂections on a study of compliance responses by South African banks. Law in Context 30: 228–56. Available online: https://search.informit.org/doi/epdf/10.3316/informit.2519 08955045471 (accessed on 10 August 2022). ( g ) Drezner, Daniel W. 2008. References Annual Review of Non-Cooperative Countries and Territories 2006–2007: Eighth NCCT Review. Available online: https://www.fatf-gafi.org/media/fatf/documents/reports/2006%202007%20NCCT%20ENG.pdf (accessed on 10 August 2022). FATF. 2007b. Statement on Iran. Available online: https://www.ﬁncen.gov/sites/default/ﬁles/shared/FATFOct2007.pdf (accessed on 10 August 2022). FATF. 2007b. Statement on Iran. Available online: https://www.ﬁncen.gov/sites/default/ﬁles/shared/FATFOct2007.pdf (accessed on 10 August 2022). 31 of 32 Risks 2023, 11, 81 FATF. 2008a. FATF Statement—20 June 2008. Available online: https://www.fatf-gafi.org/countries/n-r/pakistan/documents/fatfsta tement-20june2008.html (accessed on 10 August 2022). FATF. 2008b. FATF Statement Concerning Iran, Uzbekistan, Turkmenistan, Pakistan, São Tomé and Príncipe and the Northern Part of Cyprus—16 October 2008. Available online: https://www.fatf-gafi.org/countries/a-c/cyprus/documents/fatfstatementconcer ningiranuzbekistanturkmenistanpakistansaotomeandprincipeandthenorthernpartofcyprus-16october2008.html (accessed on 10 August 2022). g FATF. 2008c. Statement—28 February 2008. Available online: http://www.fatf-gafi.org/countries/n-r/pakistan/documents/fatfstate ment-28february2008.html (accessed on 10 August 2022). y g FATF. 2009. FATF Statement Concerning Iran, Uzbekistan, Turkmenistan, Pakistan and São Tomé and Príncipe—October 2009. Available online: https://www.fatf-gafi.org/countries/n-r/pakistan/documents/fatfstatementconcerningiranuzbekistanturkmenistan pakistanandsaotomeandprincipe-october2009.html (accessed on 10 August 2022). pakistanandsaotomeandprincipe-october2009.html (accessed on 10 August 2022). FATF. 2010a. Annual Report 2009–2010. Available online: https://www.fatf-gafi.org/publications/fatfgeneral/documents/fatfannualr eport2009-2010.html (accessed on 10 August 2022). FATF. 2010a. Annual Report 2009 2010. Available online: https://www.fatf gafi.org/publications/fatfgeneral/documents/fatfannualr eport2009-2010.html (accessed on 10 August 2022). p g FATF. 2010b. Improving Global AML/CFT Compliance: Update On-Going Process—February 2010. Available online: https://www.fatf-gafi.org/publications/high-risk-and-other-monitored-jurisdictions/documents/improvingglobalamlc ftcomplianceupdateon-goingprocess-february2010.html (accessed on 10 August 2022). FATF. 2010c. Public Statement—February 2010. Available online: https://www.fatf-gafi.org/publications/high-risk-and-other-monit ored-jurisdictions/documents/fatfpublicstatement-february2010.html#:~:text=The%20FATF%20reafﬁrms%20its%20call,Irania n%20companies%20and%20ﬁnancial%20institutions (accessed on 10 August 2022). 011. FATF Public Statement—24 June 2011. Available online: https://www.fatf-gafi.org/publications/high- FATF. 2011. FATF Public Statement—24 June 2011. Available online: https://www.fatf-gafi.org/publication it d j i di ti /d t /f tf bli t t t 24j 2011 ht l ( d 10 A t 2022) FATF. 2011. FATF Public Statement—24 June 2011. Available online: https://www.fatf-gafi.org/publications/high-risk-and-other-m onitored-jurisdictions/documents/fatfpublicstatement-24june2011.html (accessed on 10 August 2022). FATF. 2011. FATF Public Statement—24 June 2011. Available online: https://www.fatf-gafi.org/publication onitored-jurisdictions/documents/fatfpublicstatement-24june2011.html (accessed on 10 August 2022) nitored-jurisdictions/documents/fatfpublicstatement-24june2011.html (accessed on 10 August 2022). FATF. 2012–2023. International Standards on Combating Money Laundering and the Financing of Terrorism & Proliferation: The FATF Recommendations. Available online: https://www.fatf-gafi.org/en/topics/fatf-recommendations.html (accessed on 21 April 2023). FATF. 2013–2021. Methodology for Assessing Technical Compliance with the FATF Recommendations and the Effectiveness of AML/CFT Systems. Available online: https://www.fatf-gafi.org/en/publications/Mutualevaluations/Fatf-methodology.html (accessed on 21 April 2023). p FATF. 2015. Annual Report 2014–2015. Available online: https://www.fatf-gafi.org/media/fatf/documents/reports/Annual-report-2 014-2015.pdf (accessed on 10 August 2022). FATF 2016 I Gl b l AML/CFT C l O G P 21 O b 2016 A l bl l h // f f f FATF. 2016. Improving Global AML/CFT Compliance: On-Going Process—21 October 2016. References The methodology for assessing compliance with anti-money laundering and combating the ﬁnancing of terrorism standards. In Current Developments in Monetary and Financial Law, Vol. 3. Washington, DC: International Monetary Fund, chp. 16. Available online: https://www.imf.org/external/np/leg/amlcft/eng/pdf/cdmﬂ_v3.pdf (accessed on 10 August 2022). Morse, Julia C. 2017. From Blacklists to Bankers: Reputation, Market Enforcement, and International Cooperation. Ph.D. dissertation, Princeton University, Princeton, NJ, USA. Available online: https://dataspace.princeton.edu/handle/88435/dsp01h702q9046 (accessed on 10 August 2022). ( g ) Morse, Julia C. 2019. Blacklists, market enforcement, and the global regime to combat terrorist ﬁnancing. International Organization 73: 511–45. Available online: https://www.cambridge.org/core/journals/international-organization/article/abs/blacklists-mark et-enforcement-and-the-global-regime-to-combat-terrorist-financing/C40086326EAD3EB5857AAEDD52FB98C3 (accessed on 10 August 2022). [CrossRef] g ) Morse, Julia C. 2022. The Bankers’ Blacklist: Unofﬁcial Market Enforcement and the Global Fight against Illicit Financing. Ithaca: Cornell University Press. y Nance, Mark T. 2015. Naming and shaming in ﬁnancial regulation: Explaining variation in the Financial Action Task Force on Money Laundering. In The Politics of Leverage in International Relations. Edited by H. Richard Friman. Palgrave Studies in International Relations Series; London: Palgrave Macmillan, pp. 123–42. y Nance, Mark T. 2015. Naming and shaming in ﬁnancial regulation: Explaining variation in the Financial Action Task Force on Money Laundering. In The Politics of Leverage in International Relations. Edited by H. Richard Friman. Palgrave Studies in International Relations Series; London: Palgrave Macmillan, pp. 123–42. g pp Nechaev, Vladimir. 2014. Speech by FATF President Vladimir Nechaev at the 3rd Caribbean Conference on the International Financial Services Sector. Available online: https://www.fatf-gafi.org/en/publications/Fatfgeneral/Speech-nechaev-ifss.html (accessed on 10 August 2022). g pp Nechaev, Vladimir. 2014. Speech by FATF President Vladimir Nechaev at the 3rd Caribbean Conference on the International Financial Services Sector. Available online: https://www.fatf-gafi.org/en/publications/Fatfgeneral/Speech-nechaev-ifss.html (accessed on 10 August 2022). g ) Riccardi, Michele. 2022. Money Laundering Blacklists. New York: Routledge. g ) Riccardi, Michele. 2022. Money Laundering Blacklists. New York: Routledge. Riccardi, Michele. 2022. Money Laundering Blacklists. New York: Routledge. Sharman Jason C 2009 The bark is the bite: International organizations and blacklisting Review of International Political Economy 16: Sharman, Jason C. 2009. The bark is the bite: International organizations and blacklisting. Review of Internat 573–96. [CrossRef] Tax Justice Network. 2022. Financial Secrecy Index. Available online: https://fsi.taxjustice.net/ (accessed on 10 August 2022). Transparency International. 2021. Corruption Perceptions Index. Available online: https://www.transparency.org/en/cpi/2021 (accessed on 10 August 2022). g US Department of Justice. 2022. 2022 International Narcotics Control Strategy Report. Available online: https://www.state.gov/2022-i nternational-narcotics-control-strategy-report-2/#:~:text=The%202022%20International%20Narcotics%20Control,trade%20in %20Calendar%20Year%202021 (accessed on 10 August 2022). g US Department of Justice. References Available online: https://www.fatf-gafi.or g/publications/high-risk-and-other-monitored-jurisdictions/documents/fatf-compliance-october-2016.html#Guyana (accessed on 10 August 2022). on 10 August 2022). FATF. 2020. Jurisdictions under Increased Monitoring—21 February 2020. Available online: https://www.fatf-gafi.org/publications/h igh-risk-and-other-monitored-jurisdictions/documents/increased-monitoring-february-2020.html (accessed on 10 August 2022). FATF. 2020. Jurisdictions under Increased Monitoring 21 February 2020. Available online: https://www.fatf gafi.org/publications/h igh-risk-and-other-monitored-jurisdictions/documents/increased-monitoring-february-2020.html (accessed on 10 August 2022). FATF. 2022a. More about the International Co-operation Review Group (ICRG). Available online: http://www.fatf-gafi.org/publicatio ns/high-risk-and-other-monitored-jurisdictions/more/more-on-high-risk-and-non-cooperative-jurisdictions.html?hf=10&b =0&s=desc(fatf_releasedate) (accessed on 10 August 2022). FATF. 2022a. More about the International Co-operation Review Group (ICRG). Available online: http://www.fatf-gafi.org/publicatio ns/high-risk-and-other-monitored-jurisdictions/more/more-on-high-risk-and-non-cooperative-jurisdictions.html?hf=10&b =0&s=desc(fatf_releasedate) (accessed on 10 August 2022). FATF. 2022b. Jurisdictions under Increased Monitoring—21 October 2022. Available online: https://www.fatf-gafi.org/content/fatf-g afi/en/publications/High-risk-and-other-monitored-jurisdictions/Increased-monitoring-october-2022.html (accessed on 10 January 2023). y FATF. 2023. Jurisdictions under Increased Monitoring—24 February 2023. Available online: https://www.fatf-gafi.org/en/publication s/High-risk-and-other-monitored-jurisdictions/Increased-monitoring-february-2023.html#:~:text=Paris%2C%2024%20Febru ary%202023%20%2D%20Jurisdictions,terrorist%20financing%2C%20and%20proliferation%20ﬁnancing (accessed on 21 April 2023). ) FATF Watch. 2013. A Proliferation of Lists? 2013. Available online: https://fatfwatch.com/category/fatf/ (accessed on 10 August 2022). FATF Watch. 2013. A Proliferation of Lists? 2013. Available online: https://fatfwatch.com/category/fatf/ (accessed on 10 August 2022). ) Fiske, Susan T., and Shelley E. Taylor. 1984. Social Cognition. Chicago: Addison-Wesley. Fiske, Susan T., and Shelley E. Taylor. 1984. Social Cognition. Chicago: Addison-Wesley. Fourth EU AML Directive. 2015. Directive (EU) 2015/849 of the European Parliament and of the Council of 20 May 2015 on the Prevention of the Use of the Financial System for the Purposes of Money Laundering or Terrorist Financing, Amending Regulation (EU) No 648/2012 of the European Parliament and of the Council, and Repealing Directive 2005/60/EC of the European Parliament and of the Council and Commission Directive 2006/70/EC. Ofﬁcial Journal of the European Union L 141: 73–117. Heil, Oliver, and Thomas S. Robertson. 1991. Toward a Theory of Competitive Market Signalling: A Res Management Journal 12: 403–18. [CrossRef] Jayasekara, Sisira Dharmasri. 2021. Deﬁcient regimes of anti-money laundering and countering the ﬁnan digital banking and ﬁnancial inclusion. Journal of Money Laundering Control 24: 150–62. [CrossRef] Kida, Mizuho, and Simon Paetzold. 2021. The Impact of Gray-Listing on Capital Flows: An Analysis Using Machine Learning. IMF Working Paper. Available online: https://www.imf.org/-/media/Files/Publications/WP/2021/English/wpiea2021153-print- pdf.ashx (accessed on 10 August 2022). Kida, Mizuho, and Simon Paetzold. 2021. The Impact of Gray-Listing on Capital Flows: An Analysis Using Machine Learning. IMF Working Paper. Available online: https://www.imf.org/-/media/Files/Publications/WP/2021/English/wpiea2021153-print- pdf.ashx (accessed on 10 August 2022). 32 of 32 Risks 2023, 11, 81 Kudrle, Robert Thomas. 2008. Did blacklisting hurt the tax havens? Journal of Money Laundering Control 12: 33–49. [CrossRef] Kyriakos-Saad, Nadim. 2005. References 2022. 2022 International Narcotics Control Strategy Report. Available online: https://www.state.gov/2022-i nternational-narcotics-control-strategy-report-2/#:~:text=The%202022%20International%20Narcotics%20Control,trade%20in %20Calendar%20Year%202021 (accessed on 10 August 2022). 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https://openalex.org/W423480287	https://scholarlypublications.universiteitleiden.nl/access/item%3A3075315/view	English	null	The potential of bioorthogonal chemistry for correlative light and electron microscopy: a call to arms	Journal of chemical biology	2,015	cc-by	3,664	The potential of bioorthogonal chemistry for correlative light and electron microscopy: a call to arms Daphne M. van Elsland1,2 & Erik Bos3 & Herman S. Overkleeft1,2 & Abraham J. Koster3 & Sander I. van Kasteren1,2 Received: 2 December 2014 /Accepted: 27 April 2015 /Published online: 26 May 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com about its subcellular location and context can be obtained. CLEM studies that involve fluorescence microscopy may benefit from fluorescent markers that can be attached to mol- ecules of interest to allow their identification and localization. To date, most readily this has been done by fluorescent fusion proteins, by fluorescent antibody labelling or by the chemical modification of a protein with a fluorescent detection group [1–3]. As well as these fluorescent detection moieties, struc- tures must be present in the CLEM sample that are both EM and LM detectable in order to correlate (overlay) the LM image with the EM image. Examples of such EM/LM detect- able structures are fluorescently labelled cellular structures that are suitable to be identified by EM through their distinct morphology (e.g. stained nuclei) or fluorescently labelled electron-dense particles (e.g. fluorescent microspheres) [1–4]. Abstract With correlative light and electron microscopy (CLEM), the ultrastructural cellular location of a biomolecule of interest can be determined using a combination of light microscopy (LM) and electron microscopy (EM). In many cases, the application of CLEM requires the use of markers that need to be attached to a biomolecule of interest to allow its identification and localization. Here, we review the potential of bioorthogonal chemistry to introduce such markers for CLEM. Keywords Bioorthogonal chemistry . Click chemistry . Electron microscopy . CLEM Keywords Bioorthogonal chemistry . Click chemistry . Electron microscopy . CLEM J Chem Biol (2015) 8:153–157 DOI 10.1007/s12154-015-0134-4 J Chem Biol (2015) 8:153–157 DOI 10.1007/s12154-015-0134-4 REVIEW REVIEW * Sander I. van Kasteren s.i.van.kasteren@chem.leidenuniv.nl * Abraham J. Koster a.j.koster@lumc.nl 1 Division of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands 2 Institute for Chemical Immunology, Leiden, The Netherlands 3 Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands Introduction The above-mentioned labelling approaches have been very successfully applied to CLEM imaging of specific proteins in their cellular context. However, they carry some limitations. First, the use of fluorescent fusion proteins requires genetic manipulation of the cell, which can be difficult and can affect the function of the protein of interest [5]. An alternative to genetic manipulation is antibody labelling. However, for spec- imens prepared for CLEM, antibody labelling is an elaborate process of which the success rate is notoriously low due to lack of availability of functional antibodies [6]. Finally, all these labelling approaches do not readily allow imaging of non-templated biomolecules, such as glycans and lipids. Correlative light and electron microscopy (CLEM) is an im- aging technique that combines the virtues of light microscopy (LM) with those of electron microscopy (EM). With this tech- nique, specific molecular and cellular structures in a cell can be identified with LM after which ultrastructural information Abraham J. Koster and Sander I. van Kasteren share the corresponding authorship. * Sander I. van Kasteren s.i.van.kasteren@chem.leidenuniv.nl Bioorthogonal chemistry is a powerful new labelling tool that circumvents the disadvantages mentioned above and al- lows for the imaging of a wide range of biomolecules. Its mechanism [7] relies on the introduction of a small abiotic chemical group (one that is non-reactive with other chemical functionality found in the cell) into a biomolecule of interest which can be specifically reacted with a detection moiety using a so-called bioorthogonal chemical reaction, a reaction of the 1 Division of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands 2 Institute for Chemical Immunology, Leiden, The Netherlands 3 Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands 1 Division of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands J Chem Biol (2015) 8:153–157 154 Fig. 1 Bioorthogonal chemistry for imaging. a General approach: a biologically inert group is incorporated into a biomolecule class in a living cell and selectively visualized using chemistry specific for this bioorthogonal group. b An azide-labelled N-acetylmannosamine analogue is converted to CMP-sialic acid in vivo. Azido-sialic is then incorporated in bioorthogonal r Huisgen cycload [26], the inverse the photoclick re Fig. 1 Bioorthogonal chemistry for imaging. a General approach: a biologically inert group is incorporated into a biomolecule class in a living cell and selectively visualized using chemistry specific for this bioorthogonal group. Introduction b An azide-labelled N-acetylmannosamine analogue is converted to CMP-sialic acid in vivo. Azido-sialic is then incorporated into the nascent glycoproteins. c–f Recently applied bioorthogonal reactions for imaging, such as the copper-catalysed Huisgen cycloaddition (c) [25], the strain-promoted cycloaddition (d) [26], the inverse electron-demand Diels-Alder cycloaddition (e) [27], or the photoclick reaction (f) [28] incorporated into the nascent glycoproteins. c–f Recently applied bioorthogonal reactions for imaging, such as the copper-catalysed Huisgen cycloaddition (c) [25], the strain-promoted cycloaddition (d) [26], the inverse electron-demand Diels-Alder cycloaddition (e) [27], or the photoclick reaction (f) [28] incorporated into the nascent glycoproteins. c–f Recently applied bioorthogonal reactions for imaging, such as the copper-catalysed Huisgen cycloaddition (c) [25], the strain-promoted cycloaddition (d) [26], the inverse electron-demand Diels-Alder cycloaddition (e) [27], or the photoclick reaction (f) [28] Fig. 1 Bioorthogonal chemistry for imaging. a General approach: a biologically inert group is incorporated into a biomolecule class in a living cell and selectively visualized using chemistry specific for this bioorthogonal group. b An azide-labelled N-acetylmannosamine analogue is converted to CMP-sialic acid in vivo. Azido-sialic is then tag with a detectable group that is essentially background-free in biological systems (Fig. 1a) [8]. As this labelling strategy makes use of a small chemical group to tag a biomolecule of interest, it minimally interferes with the structure of the labelled makes use of a small chemical group to tag a biomolecule of interest, it minimally interferes with the structure of the labelled 155 J Chem Biol (2015) 8:153–157 There are now also numerous bioorthogonal reactions available for labelling these tags (thoroughly reviewed in [24] and [10]). Examples of the most often used labelling strategies are illustrated in Fig. 1c–f. The copper-catalysed Huisgen cycloaddition (ccHc) is well known for its high reac- tion rate and selectivity and is often used on fixed sample material, as copper is toxic to cells (Fig. 1c) [25]. The strain- promoted cycloaddition reaction is a faster alternative of the Staudinger ligation and allows in vivo labelling as there is no need of copper catalysis during this reaction (Fig. 1d) [26]. The inverse electron-demand Diels-Alder cycloaddition is a fast bioorthogonal reaction that does not require catalysis. An example of such a reaction is the cycloaddition of s-tetrazine and trans-cyclooctene derivatives (Fig. 1e) [27]. It is also possible to use photoactivatable chemical groups for so- called photoclick-reactions. Introduction Nitrile imine mediated [1,3]-dipo- lar cycloaddition reaction and has been employed to selective- ly functionalize an alkene genetically encoded in a protein inside E. coli cells [28]. The reaction procedure was reported to be simple, straightforward and non-toxic to E. coli cells (Fig. 1f). This variety in labelling strategies and chemical re- actions highlights the versatility of the approach as it can be altered upon experimental settings. It is even possible to ‘mul- tiplex’ different bioorthogonal reactions [29], i.e. to first label one class of biomolecule (in this case a proteasome subunit) using one bioorthogonal reaction and then perform an addi- tional bioorthogonal reaction on a second class and a third reaction on a third class of bioorthogonal groups. This ap- proach shows that the reactions are not only just bioorthogonal but also mutually orthogonal to one another. biomolecule and as such minimally affects cellular biochemis- try [9]. Since the initial development of the Staudinger-Bertozzi ligation, bioorthogonal labelling chemistry has evolved rapidly. Currently, a wide-ranging chemical toolkit is available of both tags for incorporation into biomolecules and reactions for sub- sequent labelling of these tags [10]. The choice of tag and modification chemistry can therefore be optimized and tailored for the specific biological hypothesis [7]. The introduction of a bioorthogonal tag into a biomolecule of interest occurs most readily by the metabolic incorporation of a tagged biomolecule building block. One of the approaches that exemplify the metabolic incorporation strategy was report- ed by Saxon et al. [11]. They synthesized a cell-permeable azide-tagged N-acetylmannosamine analogue (Ac4ManNAz) which was administered to mammalian cells during cell culture [12]. Inside these cells, the acetyl groups were removed from Ac4ManNAz after which it was passed on to the steps of the sialic acid biosynthetic pathway where it was converted to SiaNAz. After conversion to the nucleotide sugar CMP- SiaNAz, SiaNAz is incorporated into various glycoconjugates by sialyltransferase enzymes. With this approach, Saxon et al. produced cells containing azide-tagged sialoglycans and visu- alized these using the Staudinger-Bertozzi reaction (Fig. 1b). Since this first inception, the applications and classes of biomolecules that can be labelled with this approach have expanded rapidly. For instance, Salic and Mitchinson nicely demonstrated that bioorthogonal tags can be incorporated into newly synthesized DNA of both cultured cells and mouse tissues after metabolic incorporation of the tagged nucleic acid 5-ethynyl-2′-deoxyuridine [13]. Introduction An additional example is the metabolic incorporation of bioorthogonal tags into lipids in order to study protein lipidation and lipid trafficking (thor- oughly reviewed in [14]). Likewise, metabolic incorporation of bioorthogonal chemical tags has been reported for proteins [15]. Kiick et al. showed the incorporation of bioorthogonal tags in the proteome of Escherichia coli (E. coli) cells upon addition of the tagged amino acid azidohomoalanine. They showed that azidohomoalanine is incorporated at sites where the amino acid methionine naturally resides. Hatzenpichler et al. showed that tagging of proteins using this abiotic amino acid is a successful approach to study newly synthesized pro- teins in individual microorganisms within environmental sam- ples [9]. In addition to the proteome-wide metabolic incorpo- ration of bioorthogonal tags, single proteins can also be mod- ified by using amber codon suppression (reviewed in [16]). Although genetic modification is needed for this approach— with the same downsides as other genetic techniques—it is a great addition to the bioorthogonal toolkit. Attachment of abi- otic tags to covalent enzyme inhibitors allows even to selec- tive visualize active populations of enzymes in a complex mixture [17–19]. Moreover, it can be applied to the tagging of biomolecules in living multicellular organisms, such as Caenorhabditis elegans [20], zebrafish [21, 22] and mice [23]. We anticipate that bioorthogonal chemistry will be a pow- erful and useful addition to the CLEM-labelling toolkit. It would allow the imaging of non-protein biomolecules, and it precludes the need for genetic tagging and antibody labelling. Furthermore, the fate of biomolecules labelled by these ap- proaches can even be monitored during the degradation pro- cess. For example, a protein labelled with bioorthogonal ami- no acids can be imaged, even when it is proteolytically de- graded, as—unlike reporter proteins—the tags survive this catabolic pathway [30]. To the best of our knowledge, bioorthogonal reactions have not been combined with CLEM imaging up to now. This is surprising, since fluorescent imag- ing of bioorthogonal tags has become a commonplace over the last decade and a half [7]. In this review, we will highlight some of the inroads that have been made towards the CLEM imaging of bioorthogonal reactions. Bioorthogonal labelling for CLEM imaging To allow EM imaging of bioorthogonal tags, an electron- dense group is required that can be introduced using a bioorthogonal reaction. The most commonly used EM- 156 J Chem Biol (2015) 8:153–157 detectable groups are gold nanoparticles (GNPs) [31]. How- ever, GNPs in combination with bioorthogonal labelling have not been explored for this purpose. This is remarkable, since the inverse use of bioorthogonal chemistry—to synthesize protein/DNA-modified gold particles—has been reported [32, 33]. In these studies, no change in GNP size was observed and hydrophobic, organometallic and hydrophilic moieties could be introduced onto the particles. Brennan et al. [34] used a similar biochemical approach to produce biomolecule- modified gold particles. They first produced azide-modified gold particles and 4-pentynoic acid-modified lipase and reacted the two using a copper(II) catalyst with ascorbate- reducing agent to generate the active Cu(I) species in situ. Under reducing conditions, the thiol-gold linkage appeared stable, and gold particles modified with lipase were observed. These examples indicate that GNPs have the potential to be used in combination with bioorthogonal chemistry as a label- ling strategy for EM samples. of virus infection using a near-infrared Qdot variant [39], highlighting the power of this approach. A second alternative approach by which the subcellular location of fluorophores can be made EM-visible is by photoconversion of diaminobenzidine (DAB) [40]. This ap- proach uses fluorophores to photooxidize DAB, which results in precipitates after reaction with osmium. These precipitates are electron-dense and therefore EM-detectable. Such fluorophores are readily available as detecting agents for bioorthogonal reactions, and numerous examples exist of the use of these to label biomolecules. However, again, no exam- ples have been reported of the approach where first the fluorophore is used to image a bioorthogonal label followed by photoconversion of DAB to allow for EM imaging. A final example of an approach that has great potential for CLEM imaging of enzyme activities is the use of an aggre- gating probe. Ye et al. [41] reported the use of a probe that upon cleavage by the apoptosis-related caspases 3 or 7 cy- clizes and precipitates to form insoluble fluorescent nanoaggregates. Rather than imaging these aggregates by CLEM, the authors imaged them by super-resolution micros- copy and conventional confocal microscopy. They also showed these probes could be applied to the in vivo imaging of tumour apoptosis after treatment with doxorubicin. 1. van Rijnsoever C, Oorschot V, Klumperman J (2008) Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections. Nat Methods 5(11): 973–980 Conclusion The examples and strategies discussed here highlight the pow- er of bioorthogonal chemistry for the labelling of biomole- cules in a cellular context. To the best of our knowledge, bioorthogonal chemistry has not been explored for CLEM imaging, although many inroads have been made. We antici- pate that bioorthogonal chemistry will enable CLEM imaging of molecules for which the current toolkit is not amenable, such as non-genetically template biomolecules, temporal sub- populations of proteins (those expressed in a given time win- dow), or the imaging of enzymatically active subpopulation of a protein species. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, dis- tribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Bioorthogonal labelling for CLEM imaging Besides these electron-dense EM-detectable particles, bioorthogonal fluorophore introduction could also be used for EM imaging; namely, in combination with CLEM. Especially considering that fluorescent bioorthogonal labelling strategies are very well established in the field. However, as stated above, to perform CLEM with this particular strategy, detectable moi- eties are required that are visible in both imaging modalities (e.g. fluorescent electron-dense nano or microparticles). One approach to circumvent this would be to directly react the bioorthogonal tags with such electron-dense fluorophores. Quantum dots (Qdots) are suitable candidates for this purpose [35]. As with GNP-modification, significant development has gone into the modification of Qdots with biomolecules using bioorthogonal strategies. Again, they have not yet been used for introducing these fluorophores for CLEM. In the case of CLEM labelling, one difficulty with the modification of Qdots using ‘classic’ copper-catalysed Huisgen reaction (Fig. 1c) is fluorescence quenching [36]. Fluorescence quenching was circumvented by using either the strain-promoted [3+2]-cy- cloaddition reaction (Fig. 1d) [36] or the tetrazine-norbornene inverse electron-demand Diels-Alder reaction (Fig. 1e) [37]. In the former reaction, cyclooctyne-modified cadmium selenide/zinc sulphide (CdSe/ZnS) core-shell Qdots were modified with cyclooctyne groups and as such used to image the presence of azide-containing sugars on the surface of cul- tured CHO cells, analogous to the work performed by the Bertozzi group. However, in these experiments, no CLEM was performed. Zhang et al. [38] recently used a similar ap- proach to image the intracellular presence of viruses. CdSe/ZnS Qdots were modified with an azide-containing out- er coating. These particles were then reacted with dibenzocyclooctyne-modified viruses that had been used to infect GFP-expressing A549 cells. This approach allowed the imaging of viral infection in these cells with good selec- tively. Recently, the same group published the in vivo imaging References Roth J (1996) The silver anniversary of gold: 25 years of the col- loidal gold marker system for immunocytochemistry and histo- chemistry. Histochem Cell Biol 106(1):1–8 10. Lang K, Chin JW (2014) Bioorthogonal reactions for labeling pro- teins. ACS Chem Biol 9(1):16–20 11. Saxon E, Bertozzi CR (2000) Cell surface engineering by a modi- fied Staudinger reaction. Science 287(5460):2007–2010 32. Rahim MK et al (2013) Bioorthogonal chemistries for nanomaterial conjugation and targeting. Nanotechnol Rev 2(2):215–227 12. Keppler OTet al (2001) Biochemical engineering of the N-acyl side chain of sialic acid: biological implications. Glycobiology 11(2): 11R–18R 33. Algar WR et al (2011) The controlled display of biomolecules on nanoparticles: a challenge suited to bioorthogonal chemistry. Bioconjug Chem 22(5):825–858 13. Salic A, Mitchison TJ (2008) A chemical method for fast and sen- sitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105(7):2415–2420 34. Brennan JL et al (2006) Bionanoconjugation via click chemistry: the creation of functional hybrids of lipases and gold nanoparticles. Bioconjug Chem 17(6):1373–1375 14. Hang HC, Wilson JP, Charron G (2011) Bioorthogonal chemical reporters for analyzing protein lipidation and lipid trafficking. Acc Chem Res 44(9):699–708 35. Giepmans BN et al (2005) Correlated light and electron microscop- ic imaging of multiple endogenous proteins using Quantum dots. Nat Methods 2(10):743–749 15. Kiick KL et al (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger liga- tion. Proc Natl Acad Sci U S A 99(1):19–24 36. Bernardin A et al (2010) Copper-free click chemistry for highly luminescent quantum dot conjugates: application to in vivo meta- bolic imaging. Bioconjug Chem 21(4):583–588 ( ) 16. Xie J, Schultz PG (2006) A chemical toolkit for proteins—an ex- panded genetic code. Nat Rev Mol Cell Biol 7(10):775–782 37. Han H-S et al (2010) Development of a bioorthogonal and highly efficient conjugation method for quantum dots using tetrazine- norbornene cycloaddition. J Am Chem Soc 132(23):7838–7839 17. Speers AE, Adam GC, Cravatt BF (2003) Activity-based protein profiling in vivo using a copper(I)-catalyzed azide-alkyne [3+2] cycloaddition. J Am Chem Soc 125(16):4686–4687 38. Zhang P et al (2012) Click-functionalized compact quantum dots protected by multidentate-imidazole ligands: conjugation-ready nanotags for living-virus labeling and imaging. J Am Chem Soc 134(20):8388–8391 18. Ovaa H et al (2003) Chemistry in living cells: detection of active proteasomes by a two-step labeling strategy. Angew Chem Int Ed 42(31):3626–3629 39. References 157 J Chem Biol (2015) 8:153–157 23. Chang PV et al (2010) Copper-free click chemistry in living ani- mals. Proc Natl Acad Sci U S A 107(5):1821–1826 2. Ellisman MH et al (2012) Picking faces out of a crowd: genetic labels for identification of proteins in correlated light and electron microscopy imaging. Methods Cell Biol 111:139–155 24. Ramil CP, Lin Q (2013) Bioorthogonal chemistry: strategies and recent developments. Chem Commun 49(94):11007–11022 3. Bos E et al (2014) Vitrification of Tokuyasu-style immuno-labelled sections for correlative cryo light microscopy and cryo electron tomography. J Struct Biol 186(2):273–282 25. Tornøe CW, Christensen C, Meldal M (2002) Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1, 3-dipolar cycloadditions of terminal alkynes to azides. J Org Chem 67(9):3057–3064 4. Brown E, Verkade P (2010) The use of markers for correlative light electron microscopy. Protoplasma 244(1–4):91–97 5. Thomas CL, Maule AJ (2000) Limitations on the use of fused green fluorescent protein to investigate structure-function relationships for the cauliflower mosaic virus movement protein. J Gen Virol 81(Pt 7):1851–1855 26. Agard NJ, Prescher JA, Bertozzi CR (2004) A strain-promoted [3+ 2] azide-alkyne cycloaddition for covalent modification of biomol- ecules in living systems. J Am Chem Soc 126(46):15046–15047 27. Blackman ML, Royzen M, Fox JM (2008) Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels-Alder re- activity. J Am Chem Soc 130(41):13518–13519 6. Griffiths G, Lucocq JM (2014) Antibodies for immunolabeling by light and electron microscopy: not for the faint hearted. Histochem Cell Biol 142(4):347–360 28. Song W et al (2008) Selective functionalization of a genetically encoded alkene-containing protein via Bphotoclick chemistry^ in bacterial cells. J Am Chem Soc 130(30):9654–9655 7. Patterson DM, Nazarova LA, Prescher JA (2014) Finding the right (bioorthogonal) chemistry. ACS Chem Biol 9(3):592–605 8. Kolb HC, Finn MG, Sharpless KB (2001) Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Ed 40(11):2005–2021 29. Willems LI et al (2012) Triple bioorthogonal ligation strategy for simultaneous labeling of multiple enzymatic activities. Angew Chem Int Ed 51(18):4431–4434 9. Hatzenpichler R et al (2014) In situ visualization of newly synthe- sized proteins in environmental microbes using amino acid tagging and click chemistry. Environ Microbiol 16(8):2568–2590 30. Ouellette SP et al (2011) Chlamydia species-dependent differences in the growth requirement for lysosomes. PLoS One 6(3), e16783 31. References Pan H et al (2014) Noninvasive visualization of respiratory viral infection using bioorthogonal conjugated near-infrared-emitting quantum dots. ACS Nano 8(6):5468–5477 ( ) 19. Willems LI, Overkleeft HS, van Kasteren SI (2014) Current devel- opments in activity-based protein profiling. Bioconjug Chem 25(7): 1181–1191 40. Cortese K, Diaspro A, Tacchetti C (2009) Advanced correlative light/electron microscopy: current methods and new developments using Tokuyasu cryosections. J Histochem Cytochem 57(12): 1103–1112 20. Ullrich M et al (2014) Bio-orthogonal labeling as a tool to visualize and identify newly synthesized proteins in Caenorhabditis elegans. Nat Protoc 9(9):2237–2255 ( ) 21. Laughlin ST et al (2008) In vivo imaging of membrane-associated glycans in developing zebrafish. Science 320(5876):664–667 41. Ye D et al (2014) Bioorthogonal cyclization-mediated in situ self- assembly of small-molecule probes for imaging caspase activity in vivo. Nat Chem 6(6):519–526 22. Prescher JA, Dube DH, Bertozzi CR (2004) Chemical remodelling of cell surfaces in living animals. Nature 430(7002):873–877
W2504784152.txt	null	de		Lehrbuch der physiologischen Chemie, mit Einschluss der physikalischen Chemie der Zellen und Gewebe und des Stoff- und Kraftwechsels des tierischen Organismus. In Vorlesungen	null	1,923	public-domain	296,428	^ 7 LEHRBUCH DER PHYSIOLOGISCHEN CHEMIE mit Eiiisclilulj der physikalischen Cliemie der Zellen und Gewebe und des Stoff- und Kraftwechsels des tierischen Organismus IN VORLESUNGEN Von EMIL ABDERHALDEN PROFESSOR DR. MED. trr PHIL. H. C. DIREKTOR DES PHYSIOLOGISCHEN INSTITUTES D. UNIVERSITÄT HALLE A.S. Fünfte, neu bearbeitete Auflage. I. TEIL. \m ORGANISCHEN NAHRUNGS8T0KFE UND Hill VKHHAIJEN IM ZELLSTOFFWECHSEL. MIT > FIGUREN. URBAN & SCHWARZENBERG N., WIEN BERLIN FRIEDRICHSTRASSE 105b I., 1923. M A H L E RST R ASSE 4 QP 5/V r.i Alle Rechte, gleichfallH das der Übersetzung in die russische Sprache vorbehalten. Englische und russische Übersetzung erschienen. I'rinted in Austria. — Copyright, 1922, by Urban & Schwarzenberg, Berlin. üeriuaiii Vorwort Auf allen Teilgebieten der physiologisch^chemischen Forschung sind Fortschritte erzielt worden. Sie spiegeln sich in der Dar' neuen Auflage des Lehrbuches wider. Es ist immer eine große Freude, an Stelle von bloßen Vermutungen und von Stellung der festgefügte Tatsachen mitteilen zu können. Es sei in dieser Hinsicht hervorgehoben, daß unsere Kenntnisse über den stufenweisen Abbau der Kohlehydrate wesent^ lieh gefördert und ferner ganz enge Beziehungen zwischen Chole^ Sterin und der Cholsäure und ihren Verwandten aufgefunden Wahrscheinlichkeitsschlüssen worden sind. Die Art der Darstellung ist sich gleich geblieben. Es ist ab'- sichtlich nicht auf die Eigenschaften, die Darstellungsmethoden der einzelnen Verbindungen usw., eingegangen worden. Es Hinsicht auf das Handbuch sei in dieser biochemischen Arbeitsmethoden, Verlag Schwarzenberg, Berlin— Wien, das Bio^ Bd. I IX, Urban & chemische Handlexikon, Bd. I X, Verlag Julius Springer, Berlin, und das Handbuch der biologischen Arbeitsmethoden, Bd. I X, Verlag Urban & Schwarzenberg, Berlin Wien, hingewiesen. Möge die neue Auflage wieder Freunde finden Sie soll Kunde davon geben, daß trotz aller Nöte der Zeit der Forschergeist un^ gebrochen und der Fortschritt der Wissenschaft unaufhaltbar ist. — der — — — ! Halle a. S., im Oktober 1922. Emil Abderhalden. InhaltsYerzeiclinis. Seite Vorlesung I. Einleitung 1 Vorlesung IL Kohlehydrate. Allgemeines. haltige Kohlehydrate. — Monosaccharide. — Glukuronsäure. — Stickstoft- — Zyklosen 12 Vorlesung III. Kohlehydrate. II. 46 Polysaccharide. Glukoside Vorlesung IV. Bildung der Kohlehydrate im Pflanzenorganismus. Die Rolle der Biattfarbstoffe bei der Synthese von organischer Substanz aus Kolilensäure und Wasser. Die Herkunft der Asymmetrie der Bausteine der Lebewesen Kohlehydrate. III. 75 Vorlesung V. Kohlehydrate. IV. Verhalten der Kohlehydrate im tierischen Organismus. Abbau der zusammengesetzten Kohlehydrate im Darmkanal. Wirkung der Darmflora 9ö auf die Kohlehydrate Vorlesung VI. Kohlehydrate. V. Verhalten der Kohlehydrate im Zellstofifwechsel. Ihr Auf- und .111 Abbau. Die Stoffwechselzwischen- und -endprodukte der Kohlehydrate . . Vorlesung VII. Kohlehydrate. des Kohlehydratstoftwechsels. — Alimentäre — Zuckerzentrum. — Bedeutung der Nebennieren für den Zncker- VI. Die Glukosurie. stoff'wechsel der Regulation 142 Leber Vorlesung VIII. Kohlehydrate. VII. Die Beziehungen der Pankreasdrüse zum Kohlehydratstoft- 108 wechsel Vorlesung IX. Kohlehydrate. VIII. Diabetes melitus 17.S Vorlesung X. Bildung der Azetonkörper. Phlorhizinglukosnrie. Herkunft der Kohlehydrate im tierischen Organismus Kohlehydrate. IX. 1!^9 Vorlesung XL Fettstoffe und ihre Bausteine. Fettsäuren und Glyzerin 210 Inhaltsverzeichnis. VI Seite Vorlesung XII. Fette mit hochmolekularem einwertigem Alkohol als Baustein. Sterine. Gallensäuren Sterinester. 224 . Vorlesung XIII. Phosphatide und ihre Bausteine 238 Vorlesung XIV. Fette. Phosphatide. Sterine. Bildung der Fette, Phosphatide und ihrer Bausteine, sowie der Sterine im Pflanzenreich. Verhalten der Fette und Phosphatide im tierischen Organismus. Ihr Abbau im Darmkanal 253 Vorlesung XV. Fette. Phosphatide. Sterine. im 2. Das Verhalten der Fette, Phosphatide und Sterine tierischen Organismus. wechselzwischenBausteine Ihre Beteiligung am Zellstoffwechsel. Die^Stotf- und -endprodukte der Fette und Phosphatide und ihrer 271 Vorlesung XVI. Fette. Phosphatide. Sterine. 3. Die Wechselbeziehungen der Bausteine der Fette zu denen der Eiweißstoife und zum Traubenzucker. Das Verhalten der Phosphatide und der Sterine im Zellstoffwechsel 29U Vorlesung XVII. Eiweißstoffe und ihre Bausteine. Aminosäuren 310 Vorlesung XVIII. Eiweißstoffe und ihre Bausteine. 2. Aminosäuren. im Eiweißmolekül. Peptone. Polypeptide Die Art ihrer Verknüpfung 340 Vorlesung XIX. Eiweißstoffe und ihre Bausteine. als Bestandteile 3. Die Struktur der Eiweißstoffe. Polypeptide der Peptone 3ßl Vorlesung XX. Eiweißstoffe und ihre Bausteine. teilung, 4. Eigenschaften der Eiweißstoffe. Ihre Ein- Zusammensetzung und ihr Vorkommen 383 Vorlesung XXL Eiweißstoffe und ihre Bausteine. 5. Bildung der Aminosäuren und der Eiweißim Pflanzenorganismus. Assimilation des Stickstoffs. Sein Kreislauf stoff'e in der Natur. Herkunft der übrigen am Aufbau der Proteine beteiligten Elemente 40.5 Vorlesung XXII. Eiweißstoffe und ihre Bausteine. 6. Der Eiweißstoffwechsel der Pflanze. Die Beziehungen der Aminosäuren zu den Betainen und Alkaloiden. Abbau der Aminosäuren in den höheren Pflanzen, ferner durch Bakterien und Hefe- 432 zellen Vorlesung XXIII. Eiweißstoffe und ihre Bausteine. 7. Verliaiten der Eiweißstoffe Organismus. Ihr Abbau im Magendarmkanal im tierischen 4(11 Inlialtsverzeichnis. VII Seite Vorlesung XXIV. Eiweißstoffe und ihre Bausteine. 8. Verhalten Organismus. Ihr Abbau im Darmkanal der Eiweißstoöe im tierischen 485 Vorlesung XXV. Eiweißstoffe und ihre Bausteine. Die Wirkung der Darmflora U. Verhalten der Aminosäuren im Darmkanat. ... 504 Vorlesung XXVI. ihre Bausteine. 10. Verhalten der von der Darmwand aufgenommenen Abbaustufen der Proteine jenseits des Darmkanals Eiweißstoffe und 5'<i8 Vorlesung XXVII. Eiweißstoffe und ihre Bausteine. 11. Die Bildung von Aminosäuren im Zellstoff- wechsel 545 Vorlesung XXVIII. Eiweißstoffe und^ihre^^Bausteine. 12. Der Abbau der Aminosäuren im ZeUstoff- wechsel. Die Endprodukte des Eiweiß- und Aminosäurestoffwechsels .... 5(57 Vorlesung XXIX. Eiweißstoffe und ihre Bausteine. 13. Der Abbau der Aminosäuren im Zellstoff- wechsel. Neubildung von Aminosäuren 593 Vorlesung XXX. Eiweißstoffe und ihre Bausteine. thesen im 14. Verwendung von Aminosäuren zu Syn- Ihre Beziehungen zu den Kohlehydraten und Fette und der Phosphatide. Stoffwechselprodukte, die Zellstoffwechsel. den Bausteinen der EiweißabkömmAufbau mit keiner der be- in direkten Beziehungen _zu bestimmten Aminosäuren stehen. zusammengesetzter Natur, die in ihrem kannten Eiweißabbaustufen übereinstimmen linge 617 Vorlesung XXXI. Nukleoproteide. Nukleinsäuren und ihre Bausteine 643 Vorlesung XXXII. Nukleoproteide. Nukleinsäuren und ihre Bausteine. 2. Entstehung der Nukleoproteide und der Nukleinsäuren nebst ihren Bausteinen in der Pflanzenund Tierwelt. Ihr Verhalten im tierischen Organismus. Die Stoffwechselendprodukte der Bausteine der Nukleinsäuren. Störungen des Purinstoffwechsels 661 Vorlesung XXXIII. Blatt- und Blutfarbstoff. Chlorophyll und Hämoglobin. Ihre Herkunft und ihr Verhalten im tierischen Organismus. Die Beziehungen des Häma687 tins zum Gallenfarbstoff und zum Urobilin. Sonstige Farbstoffe . Sachverzeichnis . . 719 Vorlesung I. Einleitung. Die Physiologie wird allgemein als die Lehre vom Leben be- zeichnet. Die Lebenserscheiniingen der einzelnen Zellen, bestimmter Organe und aus verschiedenen Zellstaaten aufgebauter Organismen sind es. die der Physiologe zu erforschen strebt. Er will wissen, weshalb die einzebe Zelle ,.lebt", wodurch sie sich von einer toten unterscheidet, welche Funktionen sie erfüllt, und wie diese in allen einzelnen Teilvorgängen sich ergeben und untereinander zusammenhängen usw. Durchblättern wir die vorhandenen Hand- und Lehrbücher der Physiologie, dann finden wir. je mehr die Verfasser bestrebt sind, Vorgänge in Lebewesen zu schildern, die mit exakten Methoden analysierbar sind, um so weniger vom Leben selbst. Auf Schritt und Tritt begegnen wir Forschungsmethoden, Arbeitshypothesen und Vorstellungen, die ohne weiteres die nahen Beziehungen der Physiologie zu den exakten Naturwissenschaften klarlegen. Besonders eng verknüpft ist die Physiologie in allen ihren Teilen mit den beiden Disziplinen Physik und Chemie. Jeder Fortschritt auf diesen Gebieten, sei es in der Methodik, in den Fragestellungen oder in den Forschungsresultaten, spiegelt sich unmittelbar in der physiologischen Forschung wieder. Den tiefgehenden Einfluß der erwähnten Nachbargebiete auf die Forschung auf dem Gebiete der Physiologie zeigt ohne weiteres die Entwicklungsgeschichte der Physiologie. Es sei unter anderem daran erinnert, welch große Bedeutung für die physiologische Forschung die Beobachtung Galvanis (1790), daß ein mit zwei verschiedenen Metallen in Berührung kommender Froschschenkel zuckt, hatte. Die Elektrophysiologie entwickelte sich von dieser Feststellung und den an sie anschließenden Forschungen Voltas aus. Den fundamentalen Forschungen des genialen Physikers und Physiologen HfJmhoUz verdankt die Optik und Akustik ihre genauesten Grundlagen. Ein mächtiges Fundament der gesamten Physiologie bildet der durch Robert Mayer (1842) geführte Beweis, daß die Summe der Energieformen im Weltall unveränderlich ist. Rubner verdanken wir den ersten exakten Beweis dafür, daß das Gesetz der Erhaltung der Energie auch für den tierischen Organismus gilt. Damit ist eine Grundlage geschaffen worden, die uns gestattet, die Zellvorgänge vom energetischen Standpunkte aus in exaktester Weise zu verfolgen. Eine Fülle von Fragestellungen aller Art war die Folge dieser Erkenntnis. Abderhalden. Physiologische Chemie. I. Teil. 5. Anfl. J 2 I. Vorlesuug. Gewaltig war von jeher auch der Einfluß der führenden Chemiker Entwicklung der Physiologie. \'on ihnen stammen zum allergrößten Teil die Grundlagen unserer Kenntnisse des chemischen Aufbaues der Nahrungsstoi'fe und der Zellbestandteile. Jnsfiis Liehic/, Wähler, Lavoisicr, Forscher, die an der Wiege des so gewaltig aufgeblühten Forstanden, wandten ihr Interesse bereits in schungsgebietes ..Chemie" hervorragendem Maße chemischen Vorgängen in lebenden Zellen zu. Die Beziehungen der chemischen Forschung zu Vorgängen in der lebenden Natur haben sich im Laufe der Zeit mehr und mehr gefestigt: sei es, daß der Chemiker der Pflanzen- und Tierzelle ihre Geheimnisse bei der Synthese bestimmter Stoffe ablauscht, sei es, daß er möglichst lückenlos die Zellbestandteile in reinem Zustand abtrennt und durch Ab- und Umbau ihre Natur, Struktur und Konfiguration aufzuklären sucht. Welch gewaltigen Einfluß die Forschungen der Chemiker auf diesen Gebieten gehabt haben, zeigt am besten ein Blick auf die Entwicklung unserer Kenntnisse des Stoffwechsels einzelner Zellen, ganzer Organe und aus mannigfaltigen Zellstaaten zusammengesetzter Organismen. So sind unsere Kenntnisse des Fettstoffwechsels unmittelbar mit der Aufklärung der Struktur der Fette durch Chevreul und Berthelot verknüpft. Das Schicksal der Kohlehydrate, der Eiweißstoffe, der Nukleinsäuren und ihrer Abkömmlinge im Tier- und Pflanzenorganismus wäre heute noch in tiefes Dunkel gehüllt, wenn nicht vor allem Emil Fischer, der geniale Förderer chemischer und physiologisch-chemischer Forschungen, die Struktur dieser Stoffgruppen und ihre Beziehungen zueinander klargelegt hätte. W'ie reizvoll gestalten sich ferner die Forschungen von WiUstätter über die Blatt- und Blütenfarhstoffe. W'ie fesselt uns das erfolgreiche Willicmi Küster, Hans Fischer, R. WiUstätter Vordringen jener Forscher die den Aufbau des Blutfarbstoffes und insbesondere seines eisenhaltigen Anteiles, des Hämatins. zu enträtseln suchen! Die festgestellten engen Beziehungen zwischen Blatt- und Blutfarbstoff rufen nach einer durch lückenlose Tatsachen belegten Feststellung der Ursachen der biologisch ganz verschiedenen Funktionen der genannten kompliziert gebauten Verbindungen. Gleichzeitig möchten wir erfahren, ob sie direkte Beziehungen zueinander unterhalten, und ob sie in Pflanze und Tier von gemeinsamen Bausteinen aus ihren Ursprung nehmen. Eine Fülle ungelöster Probleme harrt des Augenblicks, in dem die letzte flülle fällt, die uns den Einblick in die Gesamtstruktur der genannten Verbindungen noch behindert. Vorläufig überspinnen zahlreiche Hypothesen die von der exakten, experimentellen Forschung offen gelassenen Lücken. p]in weiteres wichtiges Beispiel, wie die Aufklärung der Struktur von Verbindungen Zusammenhänge klar legt, zeigt uns die Chemie des Cholesterins und der Gallensäuren bzw. eines ihrer Bausteine, der Cholsäure. Die Forschungen von Windaus und Borsrhe haben bei der Zerlegung dieser Verbindungen zu einer eng verw^andten Abbaustufe geführt, die eindeutig ergibt, daß die erwähnten Stoffe in enger Beziehung zueinander stehen. W^eitere mächtige Impulse sind seit den grundlegenden Arbeiten can't Hojß, Arrhenius und Osf/rald?^ von dem (Gebiete der physikalischen Chemie ausgegangen. Die neueren Anschauungen über das Verhalten gelöster Stoffe in Flüssigkeiten ergaben ganz neue Forschungsrichtungen, insbesondere bei Problemen der Ptesorption, des Stofftransportes und der Ausauf die — — Einleitung. ;-5 Scheidung. Hatte man begonüen. die Wirkung bestimmter \'erbindungen im Organismus in erster Linie mit ihrem chemi.^chen Bau in Beziehung zu bringen, so sah man bald ein. dal;, ihre physikalischen Eigenschaften nicht vernachlässigt werden dürfen. \"on zahlreichen Gesichtspunkten aus hat das Studium des kolloiden Zustandes eine Summe von Anregungen für die Erforschung vieler Zelivorgänge erbracht. Immer mehr erkennen \N-ir, wie bedeutungsvoll der physikalische Zustand der Zellbestandteile für Wir stehen im Beginn einer reiche Frucht verihre Funktionen ist. sprechenden Forschung. So sehen wir denn von allen Seiten die physiologische Forschung, von den Fortschritten der Xachbargebiete Physik und Chemie mächtig beeinflußt und gefördert, und es \\-ill fast .scheinen, als ob das P^orschungsgebiet des Physiologen zur Zeit da seine Grenzen findet, wo das objektive Experiment, das Messen und Wägen aufhört, über die Berechtigung einer solchen Begrenzung kann mau verschiedener Meinung sein. Man wird im Prinzip dem Physiologen Bunge recht geben müssen, wenn er zum Ausdruck bringt, daß die Grundlage der physiologischen Forschung in erster Linie eine genaue Kenntnis der Funktionen derjenigen (Jrgane sein müsse, mit denen wir all das in unser Bewußtsein aufnehmen, was in der Natur vorgeht. Es sind dies unsere Sinnesorgane. Eine genaue Kenntnis der Art und Weise, wie die einzelnen Wahrnehmungen uns zum Bewußtsein kommen, in welcher Art wir die einzelnen Eindrücke festlegen, wie wir sie mit dem übrigen Inhalt unseres Bewußtseins in Zusammenhang bringen und im einzelnen Fall willkürlich oder unwillkürlich reproduzieren, würde ohne Zweifel die Art unserer Naturbeobachtung tief beeinflussen und in Fällen uns vor übereilten Analogieschlüssen und dem überi'agenEinfluß von Hypothesen aller Art bewahren. Die Erforschung der manchen den erwähnten Probleme ist mehr und mehr von den Physiologen vernachlässigt und den Psychologen überlassen worden. Wie mir scheint mit Unrecht, denn eine ersprießliche Erforschung der psychischen Eigenschaften der Zellen erfordert mehr als jedes andere Sondergebiet der Physiologie eine gründliche Kenntnis der Methoden und Ergebnisse der experimentell-physiologischen Forschung, sollen nicht Psychologen und Physiologen aneinander vorbei arbeiten. Die moderne Psychologie gestaltet sich immer mehr zu einer experimentellen Wissenschaft aus. Methoden, die in der übrigen Physiologie Verwendung finden, werden direkt oder verändert angewandt. So besteht denn die Hoffnung, daß wohl der interessanteste und zugleich wichtigste Teil der Physiologie sich wieder enger an die übrigen Teile dieses Forschungsgebietes anschließen wird und dereinst, wenn die richtigen Methoden und vor allem neue Wege gefunden sind, führend für manches Gebiet der Physiologie werden wird. Man wird nun die Frage aufwerfen, ob denn die Physiologie keine eigenen Methoden besitzt. Wir haben bis jetzt den tiefen Einfluß, den die Forschungsgebiete Chemie und Physik von jeher auf die physiologische Forschung ausgeübt haben, besprochen und dadurch vielleicht den Eindruck erweckt, als wäre die Physiologie ein unselbständiges Forschungsgebiet. Dem ist nun durchaus nicht so. Die Physiologen haben zahlreiche eigene Methoden geschaffen. Das direkte f^xperiment an Pflanze und Tier führte zu eigenartigen, dem Versuchsobjekt und den besonderen Fragestellungen angepaßten Versuchsanordnungen. Manche Beobachtungen wirkten 1* 4 I. Vorlesung. befruchtend auf andere Forschungsgebiete, und vor allem hat die Ihysiologie der Physik und Chemie manche neue Methode zugeführt und auf mancherlei Probleme jener Gebiete befruchtend gewirkt. Überall, wo wir hinblicken, finden wir in der physiologischen Forschung das Bestreben, Brücken zu den exakten Wissenschaften hinüber zu schlagen. Sind auch die Methoden, mit denen ein bestimmter Befund erhoben wird, eigener Art. und steht er zunächst scheinbar ohne jede Analogie da, so ruht der Forschergeist nicht eher, bis die Beziehungen zu bekannten Vorgängen in der unbelebten Natur aufgefunden sind. Der Forscher braucht Erklärungen für die von ihm gemachten Beobachtungen, soll seine Forschung nicht auf die Dauer eines bestimmten Planes entbehren. Der Lehrer ist ebenfalls darauf angewiesen, die durch das Experiment der Natur entlockten Geheimnisse in gegenseitige Beziehungen zu bringen. So füllen denn neben Tatsachen Theorien und Hypothesen oder, wie Semper sie sehr bezeichnend nennt, „llastvorstellungen" einen wesentlichen Teil des Forschungsgebietes „PhyManche davon sind mit den Tatsachen so eng verwoben, siologie" aus. daß ihr Vorhandensein fast ganz vergessen ist und sie als festgewonnenes Gut behandelt werden. Der gleichen Erscheinung begegnen wir bei den sogenannten exakten Naturwissenschaften, der Physik und Chemie. Bei beiden Gebieten bilden Theorien und Hypothesen mächtige Stützpfeiler. Es sei nur an die hypothetischen Vorstellungen der verschiedenartigen Ätherschwingungen erinnert, an die Annahme bestimmter Strukturen und Konfigurationen bestimmter Verbindungen usw. Alle diese Vorstellungen, die eine große Summe von Einzelbeobachtungen zu einem einheitlichen Bild zusammenfassen und der Forschung ganz bestimmte Wege weisen, sind uns so geläufig geworden, daß wir ihre wahre Natur oft ganz übersehen und geneigt sind, mit den herrschenden Anschauungen in Widerspruch stehende Beobachtungen als fehlerhafte anzusehen, oder aber auch durch eine besondere, sogenannte Hilfshypothese mit der Haupthypothese zu vereinigen. Auf dem Gebiete der Physiologie spielen die Theorien und Hypothesen besonders große Rolle. Ihre Anzahl ist eine sehr große. Man kann über die Bedeutung von Erklärungsversuchen gemachter Feststellungen verschiedener Ansicht sein. Ganz entbehrlich werden die erwähnten Hilfsmittel unserer Forschung nie werden. Sie beleben das nackte Tatsachenmaterial der experimentellen Forschung. In einem Punkte sind wohl alle Forscher einig, nähmlich darin, daß Theorie und Hypothese der tatsächlichen Beobachtung nicht gleichwertig sind. Niemals dürfen Tatsachen einer herrschenden Theorie angepaßt werden. Stets hat sich die Erklärung nach den Versuchsresultaten zu richten. Das Umgekehrte darf eine ganz nie der Fall sein. Den Theorien und Hypothesen ist im allgemeinen kein langes Leben Sie werden meist an Hand einiger weniger Beobachtungen beschieden. und suchen diesen gerecht zu werden, und gleichzeitig eröffnen Ausblicke für weitere Forschungen. Bald werden Tatsachen bekannt, die der herrschenden Anschauung widersprechen. In diesem Augenblick muß die alte Vorstellung fallen und neuen Anschauungen Platz machen. Manche Hypothese erweist sich auf Jahre hinaus als fruchtbare Führerin auf dunklen Pfaden. Sie führt auf schwierigem Gelände über mancherlei aufgestellt sie Einleitung. 5 Hindernisse zu einer kleinen Anhöhe, von der aus der Forscher erkennt, ist, das Licht der Wissenschaft eine kleine Strecke vorwärts zu tragen, gleichzeitig bemerkt er aber, daß ein viel einfacherer Weg ihn weiter geführt haben würde, und verfolgt er diesen zurück, dann wird ihm klar, daß dieser Pfad mit längst begangenen Wegen in direktem Zusammenhang steht, ja vielleicht entdeckt er sogar, daß der von ihm gewählte Pfad bereits Spuren, die andere Forscher zurückgelassen haben, aufweist. Sehr häufig erweist sich ein solcher Weg auch als Irrweg. Der Forscher darf dann nicht stehen bleiben, sondern er muß den Weg schleunigst verlassen und einen Pfad einschlagen, der die vorliegenden Tatsachen als Marksteine hat. Schon diese wenigen Bemerkungen zeigen, welch hohe Bedeutung Theorien und Hypothesen für die gesamte Naturforschung haben. Sie erfüllen ihre Aufgabe, wenn sie locker aufgebaut und leicht entfernbar angebracht sind. Sie können aber dann zu einer großen Gefahr für den Fortschritt jeder Forschung werden, wenn sie als feste Barrikaden erst nach jahrelangem Ansturm fallen. Muß schon der Forscher den Wert von Hypothesen und Theorien genau abschätzen können und in der Lage sein, fern von jeder Schulmeinung zu forschen, so ist es noch in viel höherem Maße notwendig, daß der Studierende die Grenzen zwischen Tatsachen und bloßen Anschauungen und Erklärungsversuchen genau überblicken kann. Die Tatsachen werden dauernd ihren Wert behalten. Die Theorien und Hypothesen dagegen sind vergänglich. Derjenige, der jetzt den Stand unserer Kenntnisse auf dem Gebiet der Physiologie in sich aufnimmt, wird daß es ihm geglückt den raschen Fortschritten der gesamten Forschung leicht in wenigen Jahren neuen Ergebnissen der experimentellen Physiologie ratlos gegenüberstehen, wenn ihm nicht das die einzelnen Tatsachen zusammenfassende Geflecht von Hypothesen kenntlich gemacht wird. Aus diesem Grunde werden wir Tatsachen und Anschauungen stets scharf gegeneinander abheben. Es wird dann demjenigen, der der Forschung weiter folgen will, nicht schwer fallen, alte Vorstellungen durch neue zu ersetzen, wenn er die Tatsachen als gesichertes Gut immer wieder unverändert und nur durch neue Befunde ergänzt, wiederfindet. Es unterliegt keinem Zweifel, daß durch diese Art der Darstellung der Eindruck einer gewissen Unsicherheit in der ganzen Forschung hervorgerufen wird. Eine elegante Überkleidung der zahlreichen noch vorhandenen Lücken unserer Kenntnisse auf dem Gebiete der Physiologie durch weit ausblickende Hypothesen mag für den Augenblick mehr Befriedigung geben. Sie nimmt jedoch dem Studierenden die Möglichkeit, mit den weiteren Fortschritten der Wissenbei schaft Schritt zu halten. Aus den gegebenen Darlegungen ergibt sich ohne weiteres, daß für fruchtbringendes Studium der Physiologie eine gründliche Kenntnis der Grundtatsachen der Physik und Chemie unerläßlich ist. Vor allem kann nicht genug auf den hohen Wert praktischer Übungen auf beiden (iebieten hingewiesen werden. E.xperimentelle Wissenschaften können vom Lernenden nie und nimmer nur aus Vorlesungen und Büchern in vollster Tiefe ausgeschöpft werden. Der eigene, wenn zunächst auch noch ungeschickte Versuch lehrt mehr als die eingehendste Schilderung eines solchen! Die Physiologie unterhält jedoch nicht nur enge Beziehungen zu den genannten beiden Disziplinen, sondern auch zur Anatomie. Eine genaue ein ' 6 I. Vorlesung. Kenntnis des makroskopischen und mikroskopischen Baues der einzelnen Organe der Pflanzen- und Tierorganismen bildet die Grundlage, von der aus erst ein Verständnis der Funktionen der einzelnen Gewebs- und Zellarten möglich ist. Die Funktionen der einzelnen Organe sind meistens an ganz verschiedenen Pflanzen- und Tierarten studiert worden. Bald wurde durch vergleichende Untersuchungen an verschiedenen Arten eine breitere Basis zum Verständnis der Bedeutung eines bestimmten Organes geschaffen, bald bedingten besondere Verhältnisse, dali das Studium der Funktionen bestimmter Gewebe nur bei einer ganz bestimmten Tierklasse erfolgreich durchgeführt werden konnte. Diese Hinweise mögen genügen, um zu zeigen, daß vergleichende Studien auf dem Gebiete der Zoologie und Botanik den Einblick in die Funktionen bestimmter Organe ganz wesentlich erweitern. Auch die Berücksichtigung entwicklungsgeschichtlicher Ergebnisse Ontogenese und Phylogenese liefert manche neue Gesichtspunkte. Von ganz besonders hoher Bedeutung ist für das Studium der Physiologie die jetzt so hoch entwickelte Vererbungslehre. Von diesem Forschungsgebiete aus erhalten wir fortlaufend neue Anregungen — — der mannigfaltigsten Art. Nachdem wir festgestellt haben, welche Kenntnisse erforderlich sind, um das Studium der Physiologie erfolgreich in Angriff nehmen zu können, wollen wir uns noch die Frage vorlegen, welche Bedeutung die Physiologie für die übrigen medizinischen Disziplinen hat. Sie bildet kurz gesagt die Grund- für die gesamte Pathologie. Die Beziehungen zwischen den Forschungsgebieten Pathologie und Physiologie waren noch nie so innige, wie es zurzeit der Fall ist. Der Pathologe betrachtet die Funktionen der nach irgend einer Richtung veränderten Organe nicht mehr als etwas vollständig Neuartiges, sondern er vergleicht sie mit den Funktionen des gesunden Organes und sieht in ihren Abänderungen den Ausdruck einer Anpassung an veränderte Bedingungen. Fände diese nicht statt, dann läge etwas ..Pathologisches'' vor! Aus dieser engen Zusammenarbeit von Physiologie und Pathologie hat nicht nur die letztere Disziplin großen Nutzen gezogen, sondern auch die erstere. Die Beobachtungen der Kliniker an erkrankten Individuen haben nicht nur manche Befunde, die durch Tierversuche erhoben worden sind, ergänzt und erweitert, sondern in vielen Fällen hat die Pathologie die Physiologie bei der Erforschung der Funktionen bestimmter Organe erst auf die rechte Spur gebracht. Eine überaus wertvolle Ergänzung der Wechselbeziehungen zwischen Physiologie und Pathologie liefert die pathologische Anatomie. Die genaue Lokalisation bestimmter Veränderungen einzelner Gewebsarten gibt dem vom Pathologen erhobenen Befund in vielen Fällen erst eine sichere Deutung. Von besonderer Wichtigkeit ist es. daß die bestimmten Erkrankungen zugrunde liegenden Organveränderungen experimentell durch ganz bestimmte Eingriffe hervorgerufen werden können. Die dann auftretenden Folgeerscheinungen lassen sich durch derartige \ersuche eindeutiger studieren, weil sich Komplikationen aller Art ausschließen lassen. Es besteht kein Zweifel, daß dieses junge P'orschungsgebiet unsere Kenntnisse der Funktionen der einzelnen Organe bedeutend vertiefen wird. Einen großen Ansporn wird die erwähnte Forschung erhalten, wenn unsere Kenntnisse und vor allen Dingen auch die Forschungsmethoden auf dem Gebiete des Zellstoffwechsels reichhaltigere geworden sind. Dei" einfache lage Eiuloituug. 7 Befund einer morphologischen Veränderung- bestimmter Zellarten kann in keinem Falle ein abschließendes urteil über ihren Funktionszustand abgeben, es sei denn, daß die Zellen ganz zerstört seien. Eine Zelle kann schwere, sofort ins Auge fallende Veränderungen aufweisen und trotzdem ihre verschiedenartigen Funktionen oder doch einzelne davon uneingeschränkt oder doch in genügender Weise durchführen. Es kann aber auch der Fall eintreten, daß ein Organ dem pathologischen Anatomen als ganz normal erscheint, und trotzdem können bestimmte Zellarten in manchen ihrer Funktionen vollständig versagen. Die Ergänzung rein morphologischer Betrachtung der Zellen durch eine Prüfung ihrer einzelnen Funktionen wird die Bedeutung der Ergebnisse dieses Forschungsgebietes ganz wesentlich erhöhen. Ein besonders reizvolles Oebiet das immer engere Beziehungen zur Physiologie gewinnt, stellen all die mannigfaltigen Abwehrmaßregeln gegen dar. HierAmöben. Bakterien usw. fremdartige Stoffe und Zellarten her gehören die den Infektionen mit bestimmten Zellarten folgenden — — Reaktionen des befallenen Organismus. Das Studium dieser Erscheinungen hat zu Ergebnissen geführt, die für die Physiologie von grundlegender Bedeutung geworden sind. Im Laufe der Zeit haben sich auf dem Gebiete der Immunitätsforschung Anschauungen entwickelt, die in engster Beziehung zu Vorstellungen getreten sind, die der Physiologe sich über den Zellstoffwechsel gebildet hat. daß die Pharmakologie mit der in gewissem Sinne angewandte Physiologie darstellt. Ein fruchtbringendes Studium der Pharmakologie und der Toxikologie ist ohne gründliche Kenntnisse der normalen Funktionen der einzelnen Organe undenkbar. Wir wollen wissen, wo die einzelnen Verbindungen angreifen, welche Zellen sie beeinflussen, welche Funktionen sie (\|uantitativ oder qualitativ verändern. Das therapeutische Handeln des Arztes wird dann ein zielbewußtes sein, wenn er im einzelnen Falle die Wirkung des von ihm gewählten Nicht unerwähnt darf experiment eilen Therapie bleiben, ebenfalls Mittels genau überblicken kann. Selbst Gebiete, die scheinbar mehr als andere klinische Fächer ein zeigen, wie Chirurgie. Geburtshilfe, Dermatologie usw.. stehen heute viel mehr als je in engstem Zusammenhang mit der physiologischen Forschung. Vor allem sind es die Wechselbeziehungen der einzelnen Organe zueinander, die zu gemeinsamen F'or- rein praktisches Gepräge schungen auf all diesen Gebieten rufen. Der Chirurg muß z. B. wissen, ob bestimmte Organe lebenswichtig sind. Um Muskeln. Sehnen usw. zu verpflanzen, muß er die Funktionen der einzelnen in Frage kommenden Gelenke ganz genau kennen. Ein erfolgreicher Orthopäde ohne gründliche Kenntnisse aller Einzelheiten der normalen Muskel- und Skelettfunktionen ist undenkbar. Den Geburtshelfer interessieren alle Teile der Physiologie, insbesondere die Fragen des Stoffaustausches zwischen Muttermund Kind, Fragen des Wachstums usw. Daß endlich ein fruchtbringendes Studium auf den Gebieten der Pathologie der Sinnesorgane und des gesamten Nervensystems ohne genaue Kenntnis der Funktionen der entsprechenden normalen Organe unmöglich ist, braucht kaum besonders hervorgehoben zu werden. g I. V'orlesuug. Nachdem wir nun die Stellung der Physiologie zu den mit ihr in Beziehung stehenden Forschungsgebieten erörtert haben, wollen wir zu der eingangs erörterten Frage nach der Berechtigung der Definition der Physiologie als Lehre vom Leben zurückkehren. Wir haben bereits betont, daß im wesentlichen der Physiologe in erster Linie jenen Vorgängen sein Interesse zuwendet, die er mit exakten Methoden erforschen kann. Sein Bestreben ist. all die komplizierten Zellvorgänge auf (jesetzmäliigkeiten zurückzuführen, die wir auch in der unbelebten Natur antreffen. Der Forscher ist dann befriedigt, wenn es ihm geglückt ist, einen Vorgang, der scheinbar das Vorhandensein eines Lebewesens zur Voraussetzung hat, ohne dieses und in gleicher Art durchzuführen. Unaufhaltsam dringt die Forschung immer weiter vor. Es wird angestrebt, bestimmte Funktionen auf besondere Zellarten zurückzuführen. Sind solche Bestrebungen erfolgreich, dann wird versucht, die Zellen als solche auszuschalten und die gleichen Vorgänge mit aus ihnen bereiteten Auszügen durchzuführen. Ein besonders lebhaft bearbeitetes Gebiet ist zurzeit die lückenlose Erforschung der Umwandlung bestimmter Stoffe im Zellstoffwechsel. Es genügt uns nicht mehr, nur mit den Anfangs- und Endprodukten des Stoffwechsels vertraut zu sein. Wir wollen alle Zwi.schenstufen kennen lernen, weil bekannt geworden ist, daß von solchen aus sich oft interessante Beziehungen zu Verbindungen ergeben, die ganz anderen Gruppen von Stoffen als die Ausgangsmaterialien angehören. Ferner besitzen solche Zwischenprodukte oft wichtige Wirkungen. Man stellt nun in gewissem Sinne der einzelnen Zellart Fragen. Der Physiologe zieht alle Möglichkeiten, die beim Abbau eines bestimmten Stoffes sich ergeben können, in Betracht. Es werden alle nur denkbaren Abbaustufen synthetisch dargestellt und der Zelle vorgelegt. Bleiben sie unberührt, dann wird geschlossen, daß die betreffende Verbindung der Zelle fremd ist. Findet dagegen ein weiterer Abbau durch sie statt oder verwertet sie das dargebotene Material sonstwie, dann nimmt man an, daß die betreffende Verbindung der Zelle vertraut ist. Auf diesem Wege sind schon eine große Zahl interessanter Ptesultate erhalten worden. Man wird freilich nicht allen gezogenen Schlußfolgerungen zustimmen können. Die Erfahrung hat uns gelehrt, daß kein Organ für sich besteht und somit auch keine Zellart. Alle stehen unter sich in Wechselbeziehungen. Sie ergänzen sich, helfen sich aus, senden sich Werkzeuge zur Bearbeitung bestimmter Materialien, geben vorbereitete Stoffe zur weiteren Verwandlung weiter usw. Arbeiten wir mit einem isolierten Organe allein, dann begeben wir uns der Hilfeleistungen durch andere Organe. Ferner hat es sich gezeigt, daß jede Zellart zur Durchführung ihrer mannigfaltigen Aufgaben ganz bestimmter Bedingungen bedarf. Diese einzuhalten oder gar nachzuahmen, ist im p]inzelfalle infolge unserer mangelhaften Kenntnisse auf diesem Gebiete vielfach noch unmöglich. Wir können uns nur bestreben, möglichst „physiologische" Bedingungen herzustellen. Die Zelle arbeitet immer mit Spuren. Eine Anhäufung von Abbauprodukten kann an sich schon zu den schwersten Störungen führen. Wir greifen mit unseren Methoden gar oft recht roh in das feine (letriebe der Zellen ein und glauben dann, die auftretenden Reaktionen der Zellen als normale auffassen zu dürfen. Vor Fehlschlüssen auf diesem Gebiet der Forschung schützt uns nur die Anwendung möglichst vieler verschiedenartiger Forschungsmethoden. Zunächst wird der ganze Organismus studiert, dann das überlebende Organ, EiuleitnuLT. 9 folgt das Studium besonderer Zellarten, endlich schaltet man. wie schon oben erwähnt, jedes organisierte Wesen aus und arbeitet mit Auszügen aus Zellen. Auch der umgekehrte Weg ist schon erfolgreich begangen worden. Erst dann, wenn alle angewandten Methoden zu dem gleichen Befunde führen, darf das erhaltene Resultat als ein gesichertes betrachtet werden. Genau in der gleichen Weise arbeitet der Physiologe, der mit phy- dann und physikalisch-chemischen Methoden arbeitet. Auch den einzelnen Vorgang auf bestimmte Zellen oder Zellanteile zu begrenzen. Die erhaltenen Befunde sucht er mit Gesetzmaliigkeiten und Erscheinungen in Einklang zu bringen, die sich in der unbelebten Natur finden, oder er sucht sogar Modelle herzustellen, die entsprechende Erscheinungen zeigen, wie das untersuchte lebende Material. So sehen wir denn die Physiologen auf verschiedenen Wegen jene Vorgänge in der belebten Natur studieren, die sich mit Methoden angreifen lassen, die wir verändert und angepaßt oder auch in ganz der gleichen Weise bei der Erforschung von Erscheinungen in der unbelebten Natur verwenden. Wir greifen jene ^'orgänge im Geschehen der Zellen heraus, die sich auf Gesetzmäßigkeiten, die sich in der unbelebten Natur finden, zurückführen lassen. Alle jene \'orgänge, die dem gegenwärtigen Stande der exakten Naturwissenschaften entsprechend einer Erklärung noch unzugänglich sind bzw. nur durch rein metaphysische Spekulationen ohne sicheren Untergrund dem Verständnis in mehr oder weniger befriedigender Weise zugänglich gemacht werden können, werden zurzeit vom Physiologen nur wenig oder garnicht berücksichtigt. Die Physiologie, wie sie heute gelehrt wird, umfaßt nicht die ganze Lehre Tom Leben, sondern sie bringt in der Hauptsache nur jene Teiltunktionen der Zellen zur Darstellung, die zurzeit mit genauen Methoden erforschbar sind. Im wesentlichen wird das chemische und physikalische Geschehen sikalischen er ist bestrebt, studiert. Wir heben diesen Umstand deshalb besonders hervor, w^eil er uns jeder Diskussion über die Frage nach einer sogenannten Lebenskraft enthebt. Man hat früher vor mancher Fragestellung halt gemacht, weil man sich vorstellte, daß die lebenden Zellen mit Kräften arbeiten, die der unbelebten Natur fehlen. Es Avürde. wenn man sich einer solchen Vorstellung unbedingt beugen würde, mancher Fragestellung eine enge Grenze gezogen. Die Wissen.schaft verträgt solche Einengungen nicht. Sie anerkennt keine Grenzen. So ist denn auch die Annahme einer besonderen Lebenskraft heftig bekämpft worden. Weder ist es bis jetzt geglückt, eine besondere, nur der belebten Natur eigene Energieform eindeutig nachzuweisen, noch ist es gelungen, zu beweisen, daß tatsächlich in der belebten Natur keine Energieform wirksam ist, die nur dieser eigen ist. Unsere Kenntnisse sind gegenwärtig auf allen Gebieten der Physiologie noch so lückenhaft, daß keine Zeit übrig bleibt, um das Problem der sogenannten Lebenskraft in Angriff zu nehmen. Jeder auch noch so winzig kleine Fortschritt auf dem (iebiet der experimentellen Forschung ist zurzeit unendlich viel bedeutungsvoller als eine noch so lange Abhandlung über Sein oder Nichtsein einer Lebenskraft! Es gibt keine gegebene Form der Darstellung der Funktionen der Lebewesen und Zellen. Man kann die Funktionen der Organismen von den JQ I. Yorlesimg. mannigfaltigsten Gesichtspunkten aus darstellen. Es ist gewiß nicht das Richtige, wenn die Schilderung der einzelnen Zellfunktionen ausschließlich vom Experiment beherrscht wird. Wir müßten eigentlich hinausgehen und die Lebewesen in ihrem ganzen Werden, Sein und Vergehen an Ort und Stelle verfolgen. Jedes, auch das kleinste Lebewesen bietet in der Natur draußen eine Fülle der interessantesten Erscheinungen. Anatomisch ganz rätselhafte Organe werden uns ihrem Wesen nach sofort klar, wenn wir ihre Funktionen beobachten. Die Biologie vermag uns erst in ihrer Gesamtheit ein umfassendes Bild der Vorgänge in den einzelnen Lebewesen zu geben. Wir werden im folgenden uns in der Hauptsache an die höher und jene Funktionen betrachten, die mit physikalisch-chemischen Forschuniu: und chemischen der Methoden verfolgt worden sind. Wir werden mit der Frage beginnen, welche Stoffe der pflanzliche und vor allem der tierische Organismus braucht, um seinen Bestand zu erhalten, zu wachsen, sich zu vermehren und seine mannigfaltigen Bedürfnisse zu bestreiten. Wir werden jeden einzelnen dieser Stoffe, Nahrungsstoffe genannt, zunächst einer chemischen Natur nach betrachten und ihn dann auf seinem Weg im Verdauungsapparat verfolgen. Wir werden uns die Frage vorlegen, was mit ihm im gesamten Verdauungskanal geschieht, in welcher Form er zur Resorption gelangt, und wie er jenseits der Darmw^and verarbeitet wird. Wir werden festwelchen besonderen Bedürfnissen der einzelne Nahrungsstoffe stellen, dient, welche Beziehungen er zu besonderen Funktionen und Organen hat, und in welcher Form er schließlich den Körper wieder verläßt. Wir werden so die Verdauung, die Resorption, die Assimilation und den Abbau zu den Stoffwechselendprodukten und damit den Zellstoffwechsel kennen lernen. Der gesamte Stoffwechsel wird vom qualitativen Standpunkt aus betrachtet an uns vorüberziehen. Wir werden dann nach der Quantität der zur Erhaltung des Lebens unter bestimmten Bedingungen notwendigen Nahrungsstoffe fragen. Dieses Problem läßt sich. wie wir sehen werden, von zwei Gesichtspunkten aus betrachten. Wir werden die Nahrungsstoffe einmal als Körper mit bestimmtem chemischem Aufbau betrachten und dann deren Energieinhalt bei der Besprechung der Frage nach dem Nahrungsbedarf als Grundlage wählen. Diese Art der Darstellung wird am besten die inneren Zusammenhänge der einzelnen Umwandlungen der verschiedenen Nahrungsstoffe und die Beziehungen bestimmter Stoffe zu besonderen Zellfunktionen klarlegen. Möge sie dazu anregen, keinen Vorgang für sich allein zu betrachten, sondern überall nach Zusammenhängen zu suchen Die gewählte Art der Schilderung der einzelnen Stoffwechselvorgänge soll in erster Linie zum Nachdenken anregen und gleichzeitig auch ein klares Bild der Art und Weise geben, wie der Physiologe bestimmten organisierten Lebewesen halten Fragestellungen nachgeht. Die Physiologie ist ein Forschungsgebiet, das, wie kaum ein anderes, nur mit Gedankenarbeit erschlossen werden kann. Niemand wird mit Erfolg Physiologie studieren, wenn er nicht den Ergebnissen dieses Forschungsgebietes denkend folgt. In diesem Sinne bedeutet die Physiologie für das Studium der Medizin das. was die {Philosophie für zahlreiche andere Disziplinen. Der Arzt steht am Krankenbett vor einer Summe von Erscheinungen mannigfaltigster Art. Als echter Forscher wird er nicht ruhen, bis er sie Eiuleitüug. U so weit es möglich ist, auf ihre Grundursachen zurückgeführt hat. Seine Kenntnisse der normalen Funktionen der einzelnen Organe werden für sein ganzes Handeln maßgebend sein. Er wird nicht da und dort planlos ein Symptom nach dem anderen bekämpfen, sondern versuchen, durch Behebung der gemeinsamen Ursache eine Besserung der verschiedensten krankhaften Erscheinungen herbeizuführen. Das physiologische Denken wird dem Arzte bald eine große Überlegenheit über alle jene verschaffen, bei alle, denen mit jedem Symptom, mag es noch so mannigfachen Ursachen entspringen, sich fast automatisch eine bestimmte Behandlungsart verknüpft. Nur der denkende Arzt wird auf die Dauer mit Erfolg und Befriedigung wirken. Er wird die medizinische Wissenschaft führen und ihr ein bestimmtes Gepräge und vor allem auch eine bestimmte Stetigkeit geben. Die Ergebnisse der Physiologie lassen sich nicht auswendig lernen. Das Erworbene würde toter Ballast bleiben. Nur das wirklich denkend ..Assimilierte" wird uns vertraut und läßt sich mit Erfolg zu neuer Gedankenarbeit verwenden. Es kann das demjenigen, der Physiologie zu studieren beginnt, nicht eindringlich genug eingeprägt werden. Das Erlernte bleibt gar häufig ..gehirnfremd". Nur das Gedachte und mit Gedankenarbeit Errungene wird ..gehirneigen" und bleibt uns vertraut. Die gewählte Form der Darstellung führt zu Wiederholungen. Wir halten das für keinen Fehler. Es wird ein und derselbe Befund von ganz verschiedenen Gesichtspunkten aus betrachtet. Dadurch wird erreicht, daß jede einzelne Tatsache Beziehungen mannigfaltigster Art zu anderen Ergebnissen erhält. Kein Geschehnis innerhalb des Organismus besteht für sich! Es hat erst dann allgemeineres Interesse, wenn wir seine mannigfaltigen Folgeerscheinungen möglichst lückenlos übersehen. Die Physiologie muß eine Schule der Logik sein! Es muß die Zeit kommen, in der kein Natur- forscher ohne gründliche Kenntnis der Physiologie und kein Arzt ohne tiefes Eindringen in die Grundtatsachen der Naturwissenschaften und der gesamten Biologie denkbar ist. Ärzte waren früher unter den Führern der naturwissenschaftlichen Forschung mit an erster Stelle! Jeder Versuch, die naturwissenschaftliche Vorbildung des Mediziners zu verkürzen, muß sich bitter rächen. Eine festgefügte, allen Ansprüchen entsprechende Grundlage ist die erste Bedingung bei der Aufführung eines Gebäudes Wer möchte die \'erantwortung tragen, wenn das immer mannigfaltiger sich entwickelnde Gebäude der medizinischen Wissenschaften auf ein immer mehr eingeengtes Studium der Grundlagen der ganzen Forschung aufgebaut würde Wer kann heute sagen, was für den Mediziner entbehrlich, und was unbedingt notwendig isty Ein gründliches Studium der Naturwissenschaften und der Physiologie wird dem Arzt eine stetige Quelle von Werten sein, die sein ganzes inneres Leben mitbestimmen wird. Befriedigung kann nur der Arzt haben, der mit seinem Handeln auf dem sicheren Boden der Wissenschaft steht. Er wird frei von jeder Schulmeinuug bleiben und sich keinen Dogmen unterwerfen. Sein Wissen gibt ihm Mittel an die Hand, um jede neue Errungenschaft sofort auf ihren inneren Wert zu prüfen. Kurz, je gründlicher die naturwissenschaftliche Ausbildung des Arztes ist, um so besser wird er bei entsprechendem praktischen Können und bei wirklicher Eignung zum Beruf den Kampf ums Dasein bestehen. ! ".•' Vorlesung IL Kohlehydrate.^) Allgemeines. — Monosaccharide. ~ Kohlehydrate. Glukuronsäure. - — Stickstoffhaltige Cyclosen. Die Kohlehydrate sind in der Natur weit verbreitet. Wir treffen sie vor allen Dingen in der Pflanzenwelt in einer kaum zu übersehenden Fülle mannigfaltigster Formen. Diesen entsprechen auch die verschiedenartigsten Funktionen, die sie in der Pflanzenzelle erfüllen. In der Tierwelt treten die Kohlehydrate gegenüber anderen Stoffen, insbesondere den Eiweißstoffen, zurück. Sie finden sich nicht nur in geringerer Menge, sondern es ist auch ihr Formenreichtum ein geringerer als in der Pflanzen- Nahrungsstoffe Kohlehydrate für den tierischen Die wichtigsten Repräsentanten der Kohlehydrate sind schon lange bekannt, am längsten wohl der Rohrzucker, der schon vor dem Beginne unserer Zeitrechnung in Indien aus dem Safte des Zuckerrohres durch Einkochen in fester Form gewonnen worden ist. Jetzt bildet neben dem Zuckerrohr die Zuckerrübe'-) ein weiteres Ausgangsmaterial. Frühzeitig w^urde auch der Traubenzucker erkannt. Rein dargestellt wurde er jedoch erst von Marggraf in der Mitte des 18. Jahrhunderts. Im Jahre 1615 hat ferner BartoUeti'^) ein drittes Glied dieser Gruppe aus der Milch isoliert, nämlich den Milchzucker. Nennen wir noch die Zellulose und die Stärke, dann sind die Glieder der Kohlehydratgruppe erschöpft, die zu jener Zeit bekannt waren, als durch die Entdeckungen von Lavois/er und Scheel e die organische Chemie begründet wurde. Sehen wir von einzelnen, allerdings sehr bedeutungsvollen Beobachtungen ab z. B. von Kirchhofe Befund, daß Stärke durch Kochen welt. Als sind die Organismus von größter Bedeutung. — ) Für eingehendere Studien dieser Gruppe vnu Verbindungen sind zu empfohlen: Kmil Fischer: Untersuchungen über Kohlehydrate und Fermente (1884 1908). J. Springer. Berlin 1909; ferner Edmund <). v. Lipjjmann : Die Chemie der Zuckerarten. Friedrich Vieweg & Sohn. 1904. 2 Bde 3. Auh. Brauuschweig. B. Tollens: Kurzes IIandl)uch der Kohlehydrate. 2 Bde. 2. Aufl. (1898). Biochemisebes Handlexikon, herausgegeben von Emil Abderhalden. 2. 1911 mit Ergänzungsbänden 8, 9 (^K Gräfe, Zeinple'n, Nevherf/, Bewalde, Euler, Lundberg). Handlnich der biologischen Arbeitsmethoden. Herausgegeben von Emil Abderhalden, Aht. I. Teil .ö. bearbeitet von ZejHjj/e« und Nord. — — — — '-) 79. P^ntdecker ist Marggraf (1747): Ber. d. Berliner Akademie der Wissensch. 1749 (1747). 'j Fabricio Bartolleti: Encyclop;if'dia dogmatica. 1615. Kohlehydrate. 13 mit verdünnten Säuren in Traubenzucker zerfällt i), und daß derselbe Vojgang' durch einen im Getreide oder Malz'-) vorhandenen Stoff hervorgerufen so blieb bis in die neueste Zeit hinein die Chemie und damit unwird mittelbar die Physiologie der Kohlehydrate in ein großes Dunkel gehüllt, das erst den wichtigen Forschungen von Kiliani und vor allem von Emil. Fischer wich. Wir werden im Verlauf der folgenden Betrachtungen sehen, wie eng die Entwicklung der Chemie der Kohlehydrate mit derjenigen der Chemie im allgemeinen und insbesondere der Strukturlehre verknüpft ist, und welch bedeutungsvolle Ausblicke sich mit der fortschreitenden Erkenntnis des Aufbaues der Kohlehydrate für große Gebiete dei- Biologie ergeben haben. Die Kohlehydrate bestehen ganz allgemein aus den Elementen C. H und 0. Sauerstoff und Wasserstoff finden sich im allgemeinen im Verhältnis OH,. Dies ist auch der Grund, von 1:2, also gerade so, wie beim Wasser weshalb man dieser Gruppe von Verbindungen den Namen Kohlehydrate gegeben hat, eine Bezeichnung, die übrigens nicht charakteristisch für diese Gruppe allein ist, denn die Chemie kennt andere Verbindungen (Essigsäure, Milchsäure), welche ebenfalls dieses Verhältnis von Sauerstoff und Wasserstoff aufweisen und nicht zur Zuckergruppe gehören. Früher definierte man die Kohlehydrate auch als Verbindungen mit 6 oder einem Vielfachen von 6 Kohlenstoffatomen. Auch dieses Merkmal ist durch die Auffindung von Zuckern mit weniger und mehr als 6 und vor allem durch die Synthese von Zuckern mit 7, 8 und 9 Kohlenstoffatomen durch Emil Fischer hinfällig geworden. Eine scharf abgegrenzte Definition für alle Kohlehydrate läßt sich überhaupt nicht g:eben, weil die einzelnen (irlieder dieser Gruppe zum Teil recht verschiedene Eigenschaften zeigen. Man kann die Kohlehydrate ganz allgemein als Aldehyde oder Ketone mehr- — , = wertiger Alkohole auffassen. In die Struktur Verhältnisse der Zuckerarten erhalten wir am besten einen Einblick, wenn wir die Oxydationsstufen verschieden wertiger Alkohole verfolgen, und zwar wollen wir zunächst die Oxydation an einer primären Alkoholgruppe eintreten lassen. Der einfachste ein- der Methylalkohol. Er geht bei gelinder Oxydation in Formaldehyd und bei stärkerer in Ameisensäure über. Umgekehrt kann man sich den Formaldehyd durch Reduktion aus Ameisensäure und den Methylalkohol durch Reduktion aus ersterem entstanden denken. Gehen wir von einem mehrwertigen Alkohol aus, dann stehen zwei primäre Alkoholgruppen zur Oxydation zur Verfügung. W^ir erhalten mit zunehmender wertige Alkohol ist Oxydation den Aldehyd, eine einbasische, und schließlich durch Umwandlung der letzten primären Alkoholgruppe in die Karboxylgruppe eine zwei basische Säure. Die folgende Übersicht soll diese Beziehungen klarlegen. Säuren: zweibasische einbasische Alkohol: Aldehvd: H)c.oH H.(<;j H. (<;;„ Methylalkohol Formaldehyd Ameisensäuio einwertiger Alkohol Monose ') -) Kirchhoff: Journ de pharmacie. 74. 199 (1811). Kirchoff: Schweigger?, .fournal. 14. 389 (1814). 14 II. Vorlosiiiiir. Alkohol: CH, . CHo . Aldehyd: OH OH Gly kol zweiwertiger Alkohol CHo . OH Glvkolose (Glykolaldehyd) Säuren: einbasische zweibasische y\0H CH2 OH . Glykolsäure OH ^OH CH .OH CH .OH y CH2 . OH Glyzerin OH Oxalsäure Biose CH, .OH CH .OH ,^0 CH .OH CH, . OH (ilvzerose CH, . OH Glvzerinsäure OH Tartronsäure dreiwertiger Alkohol CH.. 1 . OH Triose (Aldose] Kohlehydrate. ]ö Alkohol: Aldehvd: Säuren: einbasische zweibasische CH .OH CH .OH CH .OH . CH .OH ' I I CH .<:)H CH .OH CH CH CH OH CH . ( )H . i . . oH CH )H CH ( : . . oH (JH i I oH CH .OH CH .OH CH CH,.OH CH., .OH CH..OH sechswertiger Alkohol Hexose . CH .OH C<qjj (Aldose) primären Alkoholgriippe eine sekundäre oxyKeton. das bei weiterer Oxydation unter Aufspaltung des Moleküls in Säuren zerfällt, von denen, wie aus der unten mitgeteilten Formel leicht ersichtlich ist, jede weniger Kohlenstoffatome besitzt, als das Keton selbst. Wird an diert, Stelle einer dann erhalten wir ein Alkohol II. Iß Vorlesung. Reihenfolge durch Reduktion schließlich von Säuren ausgehend zu Alkoholen gelangt, vermag auch die Zelle die Glieder dieser Körperklassen durch Oxydation bzw. durch Reduktion je nach Bedarf ineinander umzuwandeln. Die gegebene Übersicht enthüllt uns gleichzeitig die Einteilung der Kohlehydrate. Wir haben Verbindungen kennen gelernt, die wir von verschiedenwertigen Alkoholen ableiten können. Die Namen der Zuckerarten sind durch die Endsilbe -ose und die Anzahl der Kohlenstoffatome gekennzeichnet. Im strengen Sinne der Definition der Kohlehydrate können wir als einfachsten Zucker den Formaldehyd bezeichnen. Er ist als onose aufzufassen. Es folgt dann die Gruppe der Di- bzw. Biosen, die sich von einem zweiwertigen Alkohol ableitet. Die Triosen zeigen Beziehungen zu dreiwertigen, die Tetrosen zu vier- und die Pentosen zu fünf wertigen Alkoholen. Endlich folgen die für uns wichtigsten einfachen Zuckerarten, die Hexosen. Sie sind Aldehyde oder Ketone sechswertiger Alkohole. Es würden dann die Heptosen. Getösen, Nonosen usw. folgen. Von manchen dieser letzteren Zuckerarten sind in der Natur nur die entsprechenden Alkohole oder die zugehörigen Säuren aufgefunden worden. So wurde z. B. schon im Jahre l^'M in den Früchten von Laurus persea^), später in den unreifen Samen, den Blättern und dem Perikarp von Persea gratissima (Avocado- oder Aligatorbirne) der siebenwertige Alkohol Persei t, auch d-Mannoheptid genannt, nachgewiesen. Es ist nun geglückt, daneben eine Heptose zu isolieren. Sie hat die Struktur der d-Mannoketo- M heptose^): OH OH H H CH, (OH) CO C . . . C . C . C . CH2 (OH). H H OH OH Sie läßt sich durch Reduktion in den erwähnten Alkohol d-Perseit über- führen. Wahrscheinlich ist die Beobachtung, wonach Sorbosebakterien diesen Alkohol in eine Ketoheptose verwandeln =^), so zu deuten, daß die erwähnte d-Mannoketoheptose entsteht. Ferner ist der Alkohol Volemit aus Lac- tarium volemus^) und den Rhizomen mancher Primulaarten dargestellt worden. 5) Auch er ist ein siebenwertiger Alkohol. Aus Sedum spectabile ist ferner eine Heptose, Sedoheptose genannt, gewonnen worden. ß) Schon bei den Triosen ergeben sich zwei Möglichkeiten. Einmal kann ein Aldehyd vorliegen oder aber ein Keton. Wir bezeichnen Zuckerarten, die durch eine Aldehydgrupi)e ausgezeichnet sind, als Aldosen und solche, aufweisen, als Ke tosen. Die ein Karbonyl die eine Ketogruppe Nomenklatur sei an einem Beispiel erörtert. Der Traubenzucker, dem wir fortwährend begegnen werden, gehört zu den Hexosen. Er ist ferner eine Aldose. Wir können ihn somit als Aldohexose bezeichnen. Damit — — Avequin: Annales Chim. m6cl., Phvs. et Toxicol, 7. 467 (1831). F. B. La Forqe: Journ. of biol. Chem. 28. 511 (1917). Bull, de la ) Brrtrand: Compt. rend. de l'Acad. des Sciences. 147. 201 (1908). Soc. chim. de France [4]. 5. (529 (1909). ) Bourquelot: Bull, de la Soc. mycol. de France. 5. 122 (1891). 796 (1902). ^) Bougault und Allard: Compt. rend. de l'Acad. des Sciences. 135. F. B. La Forge: «) La Forge und Hudson: J. of biol. ehem., 30. 61 (1917). Ebenda 42. 367 (1920V ) ^) — — Kohlehydrate. \'J sind die Strukturverhältnisse dieses Zuckers charakterisiert. Wir leiten ihn von einem sechswertigen Alkohol ab und lassen durch Oxydation Wegnahme von zwei Wasserstoffatomen mittels Sauerstoffs — — eine primäre Alkoholgruppe in eine Aldehydgruppe übergehen. Wir kennen auch eine Hexose, die gleichzeitig Ketose ist. Es ist dies der Fruchtzucker. Er ist, wie oben hervorgehoben wurde, durch Oxydation einer sekundären Alkoholgruppe eines sechswertigen Alkohols entstanden. Der Fruchtzucker ist somit eine Ketohexose. Wir haben noch eine gemeinsame Bezeichnung für alle die angeführten Wir nennen sie einfache Zucker oder auch Saccharide. Diese Bezeichnung soll zum Ausdruck bringen, daß diese Zuckerarten sich in keine Zucker. einfacheren Spaltstücke zerlegen lassen, die auch noch den Charakter der Kohlehydrate besitzen, wenigstens gelingt dies nicht auf einlachem Wege. Im allgemeinen erhalten wir bei Abbau- und Spaltungsversuchen Verbindungen, die den Charakter der Zuckerarten verloren haben. Von diesen einfachen Zuckern können zwei, drei, vier und mehr zusammentreten und eine neue Verbindung bilden. Wir sprechen dann von Di-, Tri-, Tetra-, Pentaund Polysacchariden oder ganz allgemein von zusammengesetzten Zuckern. Wir geben im Namen kurz gesagt die Anzahl der untereinander verknüpften einfachen Zucker an. Diese Bezeichnung gilt selbstverständlich für die Aldosen und Ketosen. ferner für die Pentosen, Hexosen usw. Charakterisiert wird ein zusammengesetzter Zucker einmal durch die Anzahl der an seinem Aufbau beteiligten einfachen Zucker. Wir wollen diese als Bausteine bezeichnen. Ferner ist maßgebend die Art dieser Bausteine. Es kann der gleiche Baustein ausschließlich mehrfach vertreten sein. Es können sich aber auch alle möglichen einfachen Zuckerarten zusammenfinden. Vor allem ist auch die Art der Bindung zwischen den einzelnen Bau- steinen von größter Bedeutung für die Eigenschaften des einzelnen zusammengesetzten Zuckers. Gleichartige Bausteine können unter sich ganz verschieden verknüpft sein, so daß selbst bei der gleichen Anzahl gleichartiger Bausteine ganz verschiedenartige Verbindungen entstehen können. Unden Aufbau eines Polysaccharides beteiligt, dann ist die Zahl der Kombinationsmöglichkeiten je nach der Anzahl der Bausteine eine sehr mannigfaltige. Schon durch die verschiedene Reihenfolge der einzelnen Zucker können isomere Polysaccharide mit vollständig verschiedenen Eigenschaften entstehen. Wir wollen, um diese Verhältnisse klarzulegen, annehmen, daß eine Tetrose A mit einer Pentose B und einer Hexose C zu einem Trisaccharid vereinigt sei. Es sinci folgende Konibinationen allein durch die verschiedene Reihenfolge dieser drei Zuckerarten denkbar: A, B, C; A, C, B: B, A, C; B, C. A: C, A, B: C, B, A. Würden wir vier oder fünf und mehr verschiedene Bausteine vereinigen, dann würden wir zu noch weit mehr, allein durch die Art der Reihenfolge der einzelnen einfachen Zucker bedingten Isonierion kommen. Selbstverständlich wird die Zahl der möglichen Verbindungen noch dadurch außerordentlich gesteigert, daß die Art. wie die Bausteine unter sich veiknüpft sind, wechsich verschiedene einfache Zucker am seln kann. Diese Ausführungen sollen das Verständnis für die Art und Weise wecken, wie die Tier- und vor allem die Pflanzenzelle aus verhältnismäßig Abderhal de u . Physiologische Chemie. I. Teil, '). Aurt. 2 II. 2g Vorlesung. wenigen Bausteinen eine gewaltige Fülle der mannigfaltigsten Produkte hervorgehen lassen kann. Jede einzelne V^erbindung hat besondere Eigenschaften und erfüllt ganz besondere Aufgaben. Wir werden dieser Möglichwenigen Verbindungen eine große Zahl isomerer ^'erkeit, aus einigen hindungen aufzubauen, immer \vieder begegnen.^ Dieser kurze Überblick über die Gruppe der Kohlehydrate hat uns schon sehr viele Kenntnisse über diese Verbindungen vermittelt. Wir erkennen aber sofort, daß sie sehr lückenhaft sind, wenn wir erwähnen, daß wir z. B. mehrere Hexosen kennen, die gleichzeitig Aldosen sind. Jede dieser Aldosen mit 6 Kohlenstoffatoraen liefert einen sechswertigen Alkohol. Ferner kennen wir die zugehörigen ein- und zweibasischen Säuren. Sie zeigen je nach ihrer Abkunft ganz verschiedene Eigenschaften, und wir können aus der Art eines Alkohols bzw. der ein- und zweibasischen Säuren schließen, von welcher Aldohexose sie herstammen. Es ist klar, daß die gegebenen Strukturformeln nicht ausreichen, um das Vorkommen verschiedener Aldohexosen verständlich zu machen. W'ir empfinden auch noch eine andere Lücke. Wir haben die Dar- stellung der Struktur der Kohlehydrate mit einer Definition der Zuckerarten begonnen. Wir können uns damit nicht zufrieden geben. Wir möchten gerne erfahren, auf welchem Wege die Struktur der Zucker aufgeklärt worden ist. Zwar gibt die Umwandlung der Aldosen in Alkohole und in Säuren uns manchen Anhaltspunkt. Der Chemiker gibt sich jedoch nie mit derartigen Überführungen allein zufrieden. Sie könnten ja unter mehr oder weniger tiefgi'eifenden Umsetzungen erfolgen. Erst dann, wenn es dem Chemiker gelungen ist die fragliche Verbindung synthetisch aufzubauen, ist für ihn und die Wissenschaft die Frage der Struktur endgültig gelöst. Wir wollen uns im folgenden der Frage zuwenden, auf welchem Wege die Struktur der Kohlehydrate erwiesen worden ist. Ferner wird zu entscheiden sein auf welche Art das Vorhandensein mehrerer ganz verschiedener Aldohexosen, Aldopentosen usw. zu , erklären ist. Wie auf fast allen Gebieten der physiologischen Chemie, so ist auch hier, wie bereits angedeutet wurde, erst durch die synthetische Bereitung der kommenden Verbindungen ein klares Bild der Entstehung und der Umwandlungen der Kohlehydrate im pflanzlichen und tierischen Organismus in Frage — erhalten worden. Es ist zunächst Emil Fischer gelungen, aus Glyzerin demselben Glyzerin, dem wir noch bei der Besprechung der Fette begegnen werden durch gelinde Oxydation eine Substanz herzustellen, welche die charakteristischen Eigenschaften der Zucker besitzt. Diese Verbin- — Glyzerose (C3 H« O3) genannt, enthält nun allerdings nur die Hälfte des Kohlenstoffs. Wasserstoffs und Sauerstoffs des Traubenzuckers (Cg H12 Oß). Als es jedoch gelang, durch Einwirkung von verdünnter Lauge zwei Moleküle der Glyzerose zusammenzufügen und so einen wahren dung, Zucker mit 6 Kohlenstoffatomen darzustellen, zweifelte niemand mehr daran, daß die Glyzerose als Glied der Kohlehydratreihe aufzufassen sei. Diese Synthese ist deshalb noch von ganz besonderem Werte, weil durch lich sie sich glückte sogar stanz, indem Beziehungen zu den Fettsubstanze ergeben. SchließSynthese aus einer noch einfacheren Grundsub- die Emil Fischer, von Formaldehyd, H. C\ij ausgehend, durch Kohlehydrate. Polymerisation (6xCH, O^Cg HiaO«) zu demselben Zucker gelangte, wie er aus Glyzerin, bzw. der Glyzerose erhalten Diese 19 worden war. ^) vom Formaldehyd ausgehende Synthese einer Hexose fesselt unser Interesse noch deshalb in ganz besonderem Maße, weil, wie wir noch sehen werden, nach der Auffassung von Adolf v. Baeyer-) die chlorophyllhaltigen Blätter die Kohlensäure der Luft zusammen mit aufgenommenem Wasser zu Formaldehyd reduzieren und aus diesem dann durch Kondensation Zucker aufbauen. Mit dieser Hypothese können wir uns leicht die Bildung verschiedener Zuckerarten mit verschiedenem Kohlenstoffgehalt erklären, ist es doch denkbar, daß beim Aufbau der höheren Zucker in der Natur dieselben Zwischenstufen auftreten, wie sie bei der künstlichen Synthese beobachtet worden sind. Bei genauerer Untersuchung hat sich nun gezeigt, daß der aus GlyFormaldehyd durch Polymerisation entstandene Zucker mit 6 Kohlenstoff atomen dem Traubenzucker in seinen Eigenschaften nicht ganz entspricht. Er wurde deshalb auch mit einem besonderen Namen, nämlich Acrose, belegt. Diese Verschiedenheit gibt sich vor allem darin kund, daß die Lösung der synthetisch gewonnenen Aldohexose nicht imstande ist, die Polarisationsebene zu drehen. Wir bezeichnen solche Verbindungen als optisch inaktive im Gegensatz zu den optisch aktiven. Zu den letzteren gehören nun die in der Natur vorkommenden Zuckerarten 3) und insbesondere auch die Hexosen. Schon Biot) hat die wichtige Beobachtung gemacht, daß Ptohrzucker die Ebene des polarisierten Lichtes dreht. Diese Eigenschaft wurde bald technisch verwertet»), um den Gehalt von zuckerhaltigen Säften an Rohrzucker festzustellen. Da die verschiedenartigen Zucker ein ganz verschiedenes Drehungsvermögen besitzen, haben wir in dieser Eigenschaft ein wertvolles Hilfsmittel, um die einzelnen Ziickerarteu zu charakterisieren und zu unterscheiden. zerose, bzw. aus Der Acrose nun, wie schon erwähnt, die optische Aktivität zunächst auffallenden Erscheinung ergab sich, daß die Acrose aus optisch entgegengesetzt wirkenden Stücken zusammenDie Acrose gesetzt ist. Es ist gelungen, sie in diese zu zerlegen. kann je nach den gewählten Bedingungen in P'ruchtzucker oder Mannose oder Traubenzucker umgewandelt werden. Damit war der Schlul.W ganz. Als Ursache fehlt dieser stein der Synthese von in der Natur vorkommenden Zuckerarten gelegt. Die optische Aktivität fast aller in der Natur vorkommenden Kohlenstoffverbindungen hat erst durch die bekannte fruchtbare Hypothese Le Bth und van't Ho/f's''') (1874) eine befriedigende Erklärung gefunden. ) Vgl. Emil Fischer: Die Chemie der Kohlehydrate imd ihre Bcdeutuuic für die Physiologie. August Hirschwald. Berlin 1894; Synthesen in der Purin- und Zuckergruppe. Vicweg & Sohn. Braunschweig 190.3. Untersuchungen über Kohlehydrale und Fermente (1884—1908). J. Springer. Berlin 1909. -) A. V. Baeyer: Ber. d. Deutschen Chera. Ges. 3. H3 (1870). ') Über das Vorkommen einer optisch inaktiven Pentose im Harn siehe weiter unten. ) Biot: Comptes rendus de l'Acad. d. Sciences. 10. 264: 16. r)19 (1848). ^) C'lerget: Compt. rend. de PAcad. d. Sciences. 16. 1000(1843); 22. 1138 (184G); 23. 256 (1846); 26. 240 (1848). ^) Vgl. .7. H. van't Iloß': Die Lagerung der Atome im Räume. 2. Auflage. Vieweg & Sohn. Braiiuschweig 1894. - V(m't Hoff: Dix annees daus Phistoire d'une theorie. Abdruck der denkwürdigen Abhandlung ran'/ llojh: P. M. Bazendijk. Rotterdam 1887. — II. Diese beiden Forscher Vorlesuui^. haben unabhängig von einander die Asym- metrie des Moleküls, die bereits Pasteur^) in geistvoller Weise am Beider Rechts- und Linksweinsäure zur Erklärung dieser optischen Antipoden herangezogen hatte, auf diejenige des einzelnen Kohlenstoffatoms zurückgeführt. Die Asymmetrie eines Kohlenstoffatoms ist durch dessen Bindung mit vier verschiedenen Massen bedingt. Jedes einzelne asymmetrische Kohlenstoffatom einer Verbindung bedingt zwei verschiedene Formen, was sich leicht veranschaulichen läßt, wenn man mit van't Hof sich die Affinitäten eines Kohlenstoffatoms nach den Ecken eines Tetraeders gerichtet denkt, in dessen Mitte das Kohlenstoffatom sich findet. Fig. 1 gibt uns ein Bild dieser Art von Isomerie. Beide Formen verhalten sich zueinander wie Bild und Spiegel oder wie der linke Handschuh spiel Fig. 1. R^. Hj, Rj. Rf sind die verschiedenen Massen, mit denen das Kohlenstoffatora in Bindung steht. zur Deckung zu bringen. Durch Vereinigung zweier solcher Moleküle entsteht ein Molekül einer optisch inaktiven polyraeren Verbindung. Somit sind von jeder Kohlenstoffverbindung mit einem asymmetrischen Kohlenstoff atom drei Modifikationen bekannt, nämlich zwei optisch aktive Formen, eine Links- und eine Rechtsform, und eine aus der Vereinigung dieser entstehende inaktive Verbindung. Diese ist, obgleich sie zwei asymmetrische Kohlenstoffatome enthält, dennoch optisch inaktiv, weil die beiden Molekülhälften gleich stark, aber in entgegengesetztem Sinne auf den polarisierten Lichtstrahl wirken. Ein Beispiel möge die erwähnten Beziehungen zwischen optischer Aktivität und Asymmetrie eines oder mehrerer Kohlenstoffatome näher erläutern. Bei der Hydrolyse mancher Eiweißkörper erhält man als einfachste Spaltprodukte Säuren, die durch den Besitz einer XH., Aminogruppe ausgezeichnet sind. Solche, „Aminosäuren" genannte Spaltprodukte sind das Glykokoll und das Alanin. Glykokoll ist seiner Struktur nach Aminoessigsäure. während dem Alanin die Konstitution einer z-Amino- zum rechten, d. h. sie sind nicht = propionsäure zukommt. La chimie daiis Tespace. 1876. (Deutsch von Herrmann Räume. 1877.) : Die Lagerung der Atome im — K. Auivers: Die Entwicklung der Stereochemie. Karl Winters Uuiv.- Buchhaudlung, Heidelberg 1890. ') Pasteur: Recherches sur la dissymötrie moleculaire des produits orgauiques tfber die Asymmetrie naturels. Legons de chimie professöes en 1860. Paris 1861. bei natürlich vorkommenden organischen Verbindungen. tJbersetzt und herausgegeben von M. nnA H. Ladenburg Ostwald^ Klassiker der exakten Wissenschaften. Nr. 28. Vgl. ferner: II. Landolf : Das optische Drehungsvermögen organischer Substanzen und Brauuschweig 1898. dessen praktische Anwendungen. 2. Auflage. Vieweg & Soh Eine ganz besonders klare und übersichtliche Darstellung gibt A. Werner: Lehrbuch der .Stereochemio. Gustav Fischer. Jena 1904. — — : i. — Kohlehydrate. 21 H V/CO OH H3 C\r«/CO OH Essigsäure. Propionsäure. H\p/CO OH H3 C \p/CO OH H^^NH^ H^V^NHa Aminoessigsäure = z-Aminopropionsäure = Glykokoll. Alanin. Aus diesen Strukturformeln ergibt sich ohne weiteres, daß weder die Essigsäure noch die Propionsäure, noch die Aminoessigsäure ein asymmetrisches Kohlenstoffatom besitzt, denn mit keinem der Kohlenstoffatome sind vier verschiedene Massen verknüpft.^) Die Karboxvlgruppe, ^ — H und die Methylgruppe, — C;-H, die in den obigen Formeln nicht t;\.:^„ ^^H ausge- H schrieben sind, enthalten auch kein solches. Alle die genannten Verbindungen sind nur in einer Form bekannt. Sie sind alle an und für sich optisch inaktiv. Ganz anders liegen die Verhältnisse beim Alanin. Bei diesem ist das mit einem Sternchen bezeichnete Kohlenstoffatom mit vier verschiedenen Massen beladen. Es ist daher asymmetrisch. Das Alanin mu(5 somit in verschiedenen For- men vorkommen. In der Tat dreht die aus Eiweiß erhaltene a-Aminopropionsäure das polarisierte Licht, und zwar nach rechts. Man bezeichnet sie daher als d-a-Aminopropionsäure d-Alanin (d dextrogyr, rechisdrehend). Wir können durch die Synthese ein inaktives Alanin darstellen. Dieses muß nach der oben erwähnten Hypothese aus zwei Hälften bestehen, von denen die eine ebensoviel nach rechts dreht, wie die andere nach links. Das ist in der Tat der Fall, denn man kann die inaktive, auch als d, 1-Alanin bezeichnete a-Aminopropionsäure in ihre beiden Komponenten zerlegen. Man erhält d-Alanin und 1-Alanin. Beide kann man durch geeignete Mittel wieder in d, 1-Alanin überführen. 1-Alanin und d-Alanin sind die beiden optischen Antipoden des inaktiven Alanins. Glykokoll können wir nicht spalten. Es ist symmetrisch gebaut. P]s drängt sich ganz von selbst die Frage auf, in welcher Weise wir die Besonderheit im Aufbau optisch isomerer Verbindungen zum Ausdruck bringen können. Bleiben wir bei unserem Beispiel d- und 1-Alanin. Es ist nicht denkbar, daß beiden die oben angeführte Strukturformel zukommt. Nun läßt sich die \'erschiedenheit solcher Verbindungen nur zur Darstellung bringen, wenn wir, der Wirklichkeit entsprechend, uns die Atomgruppen nicht in einer Ebene, sondern im Pvaume um das asymmetrisch"e Kohlenstoffatom gelagert denken. Wir denken uns dann diese räumliche Anordnung der Atomgruppen in die Ebene projiziert und erhalten so die sogenannten Konfigurationsformeln. Für d- und 1-Alanin bringen wir den Unterschied, wie folgt, zum Ausdruck: = = H. I. CH3 CH3 I I NH2 C H . . I COOK ) H C NH, . . 1 CO OH In den Formehi sind die gleichen Massen durch Fettdruck lu'rvor?ehnlien. li- 22 Vorlesiiug. Zurzeit können wir nicht aussagen, ob Formel I bzw. II die d-, bzw. darstellt. Es ist, wie wir später erfahren werden, bis heute nicht geglückt, eine der beiden Formen mit voller Sicherheit in direkte Beziehung zu einer optisch aktiven Verbindung mit bekannter Konfiguration zu bringen. Wir haben die Möglichkeit, die eben mitgeteilten ^'orstellungeu auf I-Form ihre Richtigkeit zu prüfen. Wird einer in ^'erbindung, die ein asym- Kohlenstoffatom besitzt und infolgedessen optisch aktiv ist. die Asymmetrie aufgehoben, dann muß auch die optische Aktivität verschwinden. Es sei dies an einem Beispiel erörtert. Ersetzen wir im optisch aktiven Amylchlorid das Chlor durch Wasserstoff, so entsteht das optisch inaktive Pentan: Dieses besitzt zweimal die Gruppe CH3: metrisches H3 C\p/CH9 . Cl H3 C\,^/CH., . Pentan Amylchlorid Durch Vertauschung zweier Substituenten am asymmetrischen Kohlenmuß eine Umkehrung des optischen Drehungsvermögens eintreten, wenn die geschilderte Vorstellung der Konfiguration optisch isomerer Verbindungen richtig ist. Es ist nun in der Tat der Beweis geführt worden, daß mit der Umstellung zweier Substituenten am asymmetrischen Kohlenstoffatom die optische Drehung sich umkehrt.^) Das folgende einfache stoffatom Beispiel gibt einen klaren Einblick in das ganze Problem, vgl. S. 23.-) \Yir haben bisher nur von Verbindungen mit einem asymmetrischen Kohlenstoffatom gesprochen. Mit dem Auftreten von zwei und mehr asymmetrischen Kohlenstoff atomen muß die Zahl der isomeren Verbindungen gesetzmäßig steigen und 2" betragen, wenn n die Zahl der asymmetrischen Kohlenstoffatome bedeutet. Die praktische Erfahrung hat diese Voraussetzung in glänzendster Weise bestätigt und wohl kein Gebiet der Chemie hat der Lehre Le Beh und varit Hojfs eine so mächtige Stütze gebracht, wie gerade der Ausbau der Kohlehydratchemie nach diesen Gesichtspunkten durch Emil Fischer. Wir haben bereits hervorgehoben, daß z. B. mehrere, in ihren Eigenschaften ganz verschiedene Zuckerarten der empirischen Formel C^ H^o Og bekannt sind. Es seien nur erwähnt der Traubenzucker, die Mannose und die Galaktose. Nun enthalten diese Zuckerarten der Formel CeHi-^Uß. wie jede der unten mitgeteilten Konfigurationsformeln der Aldohexosen zeigt, nicht weniger als vier asymmetrische Kohlenstoffatome. Sie sind durch ein kenntlich gemacht. Nach der obigen Berechnung müssen somit 2* 16 möglich sein verschiedene Verbindungen mit der eben angeführten = Es unterliegt keinem Zweifel, daß dem auch so ist. denn es sind bereits nicht weniger als 14 dieser Formen dargestellt. Für jede einzelne dieser Verbindungen ist der geometrische Aufbau an Hand der Theorie aufgeklärt und die Konfiguration des einzelnen Moleküls durch bestimmte Formeln dargestellt worden. Die folgende Übersicht ^) Struktur existieren. ) Emil Fischer und Fritz Brauns: Sitzungsber. der Preuß. Akad. der Wiss. 714 (1914). Ber. d. Deutschen. Chem Ges. 47. 3181 (1914). Emil Abderhalden >mA %onAVcA/ra7d.- Ber. d. Deutschen Chem. Ges. 48. 1847(1915). Eine klare Vorstellung über die Struktur und Konfiguration asymmetrisch gebauter Verbindungen kann man aus derartigen, gewissermaßen in eine Ebene projizierten Formeln nie erhalten. Sie ist nur zu erreichen, wenn man au Hand eines Modells eine räumliche Betrachtung der Lagerung der einzelnen Atomgruppen vornimmt. ") •') 23 Kohlehydrate. E — — ~r >. c c >^. II. '24 die von Hexosen.'-) zeigt Emil Fischer Vorlesuncf. aufgestellten Konfigurationsformeln ^) P^O der P^O 0\jj I i H— C— OH HO— C— H HO— C— H— C— OH H— C— OH HO— C— H H— ->^C— OH HO— C— H-C— OH HO— C— H— C— OH HO— C— 1 I HO- -C— I I I H— C— OH 1 HO -C— CH. OH 1-Glukose CH2 OH CH2 OH l-Mannose C I d-Maiinose H— C— OH CH2 OH d-Glukose P^O //O 0\H \H 1 H— C— OH HO— C— H— C-OH HO— C— H— C— OH HO HO— C— H— C— OH HO— C— H C— OH H_C-OH HO— C— H— C— OH HO— C— i HO-C— H ' H— C-OH I I I C— i I I I CH. OH CH2OH CH2OH 1-Gulose d-Idose 1-Idose I I CH2 OH d-Gulose ') Emil Fischer hat [Ber. d. Deutschen Chem. Ges. 45. 461 (1912)] die Frage nach der Struktur der Glukose wieder aufgerollt und gibt ans mehrfachen Gründen die hier wiedergegebenen Aldehydformeln zugunsten der von Tollens [Ber. d. Deutschen ehem. Ges. 26'. 2406 (1893)1 aufgestellten ..Oxydo-Formeln" auf: CHüH oder ()bwohl diese Formeln vielen Beobachtungen au Zuckern, wie dem Auftreten isomerer Glukoside, ferner der Multirotatiou, besser Rechnung tragen als die ,,Garl)onylFormeln", wollen wir die letzteren beibehalten, weil sie in übersichtlicherer Weise die Beziehungen der Zuckerarten zu dem entsprechenden Alkohol und den zugehörigen Säuren wiedergeben. Von den Oxydoformeln werden wir bei der Darstellung der (ilukoside Gebrauch machen. ) Auch aufgestellt. für die übrigen kohlenstoft'ärmereu Zucker sind Konfigurationsformeln — Vgl. zu dem Problem der Konfiguration und insbesondere der Zusammen- II. 26 Voiiesunff. mit Cyanwasserstoff (Blausäure) zu binden. Durch Verseifung des geCyanhydrins und nachfolgende Reduktion erhält man, wie E. Fischer und später Kiliani i) gezeigt haben, einen neuen Zucker, der sich von dem Ausgangsmaterial durch den Mehrgehalt an einem Kohlenstoffatom unterscheidet. Es ist auf diesem Wege nicht nur gelungen, von den einfachsten Gliedern der Kohlehydratgruppe zu den Hexosen zu gelangen, sondern darüber hinaus zu Zuckern mit 7, 8 und 9 Kohlenstoffbildeten atomen. 2) Die folgenden Formeln zeigen diese Synthese am Beispiel einer Triose CN CH.OH C<^ CH. OH + HCN = CH.OH 4- 2 H.^ 0-NH I- I CH.,.OH [ Kohlehydrate. Dihydrofurans gegeben worden der Glukose zur Mannose-): «ines H< ) . HC- H— C— OH ist.») 27 Sie vermittelt die Beziehung HC- HO CH HO— C— . HC- I I (JH— C— — HO— C H— H— C— OH H— H— C— OH I I . H()_C— >- I GH., 1 H OH H— C— OH 1 CH, OH Olukal . d-Glukose H— CH, . OH d -Mannose Umgekehrt kann aus einem Zucker bestimmter Zusammensetzung ein solcher mit niederer Kohlenstoffanzahl hervorgehen. So beobachteten Salkoicski und NeKheir/ ^j, daß aus der zum Traubenzucker in engster Beziehung stehenden Ghikuronsäure bei intensiver f'äulnis Kohlensäure abgespalten wird. Es entsteht eine Pentose, nämlich die Xylose: c4 1 28 Kohlehydrate. 29 Salko/rski i) oelang es, die Ursache des eigenartigen Verhaltens des Harnes aufzuklären. Er entdeckte nämlich, daß eine Pen tose in ihm enthalten war. Dieser Befund einer Pentose im Urin blieb nicht vereinzelt. -j Man erkannte bald, daß eine eigenartige Abweichung des Stoffwechsels vorlag. Sie erhielt die Bezeichnung Pentosurie. Der zunächst liegende Gedanke war. daß die im Harn ausgeschiedene Pentose direkt der Nahrung entstammte, das heißt, daß nach erfolgter Aufnahme einer solchen diese unverändert durch die Nieren zur Ausscheidung kam. Diese Vermutung hat sich bei den meisten Fällen von Pentosurie nicht bestätigt. ^) Es sind keine direkten Beziehungen zu den Pentosen der Nahrung gefunden worden.) Kurze Zeit nach der Entdeckung der ersten Pentosurie wurde festgestellt, daß Pentosen am Aufbau mancher Kernsubstanzen beteiligt sind. In diesen finden sich eigenartige Säuren. Nukleinsäuren genannt, gebunden. Sie liefern bei ihrer Spaltung unter anderen Bausteinen Pentosen. A.Kossel^) vermutete schon das Vorkommen von solchen in aus Hefezellen ge- machte ihr Vorhandensein Nukleinsäure, und H((iumarsfe)i aus der Pankreasdrüse isolierten Kernsubstanz wahrscheinlich. Es sind dann zahlreiche Untersuchungen ausgeführt worden, die alle den Beweis erbrachten, daß in der Tat am Aufbau zahlreicher Nukleinsäuren Pentosen beteiligt sind.') Der Gehalt der einzelnen Organe an Pentosen schwankt mit der Menge der vorhandenen Kernsubstanzen. ^) Wir kennen mehrere Pentosen. Bis jetzt sind die folgenden in der Natur aufgefunden worden: Arabinose, Xylose und Ptibose. Sie gehören ohne Ausnahme zu den Aldosen. Ihre Konfigurationsformeln sind die folgenden: wonnener in ''') der r>A^ P^^^ nA^ Y^H H— C— OH H— C— OH H— C— OH H— C— OH H— C— OH ^\H yXH 1 I I HO— C— H HO— C— H HO— C— H H— C— OH I CH, OH 1-Arabinose I I CH2 OH OH GH., d-Uibose. ») 1-Xylose E. Salkowski uud Jastrowitz: Zeutralbl. f. med. Wisseuschafteu, Xr. 19 iiud 35. 593 (1892). Die chronische Pentosurie, -) M. Bial: Berliner kliu. vVocheuschr, Xr. 21 (1904). Berliner Klinik. 19. Heft 226 (1907). ') M. Bial und F. Bhimenfhal : Deutsche med. Wocheuschr. 22 (1901). — F. Blum: Zeitschr. f. klin. Med. 59. 244 (1906). ) Es gibt allerdings eine Ausscheidung von Pentoseu, die abhiingig ist von deren ') 337 u. — Zufuhr. Sie wird am besten als alimentäre Pentosurie bezeichnet 5) A. Kossei: Verhandl.der phys.Ges., Berlin, im Arch.f.Pbys. (u. Anat.) 157(1893). - Vgl. auch Ferd. «) 0. Hammarsten: Zeitschr. f. physiol. Chemi(>. 19. 19 (1.S94). J. Wohlfimiuth : Zeitschr. für Blumenthal: Zeitschr. f. klin. Med. 34. 160 (1898). — physiol. Chemie. 37. 375 (1903). ') E. Salkowski: Zeitschr. f. physiol. Chemie. 27. 507 (1899). ") Georo Grund: Zeitschrift für physiol. Chemie. 35. 111 (1902). Ernst Bendix und Erich Ebstein: Zeitschr. f. allgeni. Physiologie. 2. Heft — Vgl. auch — 1 (1902) E. Ebstein: Zentralbl, f. Stoffwechsel- und \'erdauungskrankheiten. 3. 503 (1902). Vereins d. Deutsch. Zuckerindustrie. 59. 642. ®) B. Tollens u. F. Rorive: Ztschr. d 1. 30 II. Vorlesung. Bis vor kurzem galt die 1-Xylose als die einzige sogenannte OrganBang ') hatte sie zum erstenmal aus einem zusammengesetzten Eiweißkörper, Proteid genannt, der Pankreasdrüse abgeschieden. Xeuberg-} bezeichnete sie ai?f Grund seiner Beobachtungen als 1-Xylose. Es ist jedoch zweifelhaft, ob diese Pentose am Aufbau von Zellbestandteilen Anteil pentose. Jedenfalls ist bis jetzt als Pentose in bestimmten Nukleinsäuren ausschheßlich die d-Ribose nachgewiesen. 3) Die im Harn vorkommende Pentose wird von Neuherg für inaktive Arabinose gehalten.) Es scheint jedoch nach L«/^2a^oä) auch die 1-Arabinose auftreten zu können. Es ist nicht ausgeschlossen, daß die sogenannte Harnpentose nicht Arabinose, sondern eine andere Pentose ist, wenigstens deuten eigene Beobachtungen an einem Fall von Pentosurie darauf hin. Sie ist auch als Ketopentose (d-Lyxose bzw. Xyloketose)") angesprochen worden.') Da somit die Natur der Harnpentose noch nicht klargestellt ist, so können wir auch über ihre Herkunft und Entstehung^ wie nichts aussagen. Dieses Beispiel zeigt uns besonders eindringhch sehr die eindeutige Beantwortung von Stoffwechselfragen von einer klaren Erkenntnis des Aufbaues der an ihm beteiligten Produkte abhängig ist. Zu jeder der Pentosen gehören je ein Alkohol und zwei Säuren, wie die folgende Übersicht zeigt: hat. , Aldose Arabinose Xylose Ribose Alkohol Arabit Xylit iVdonit (Ribit) Einbasische Säure Zweibasische Säure Arabonsäure Xylonsäure Ribonsäure Trioxyglutarsäure Xylo-Trioxyglutarsäure Ribo-Trioxyglutarsäure. Im Pflanzenreich sind verschiedentlich Pentosen aufgefunden worden, Methylgruppe besitzen, sogenannte Methylpentosen. So ist die Methylpentose Fukose') im Seetang, in der Alge Porphyria lacinata und ferner in vielen Blättern und Blüten enthalten. Weitere Methylpentosen sind die Rhamnose, die Chinovose, die Rhodeose und die Isorhodeose Die Konfiguration der 1-Rhamnose, die wir als Beispiel der Stuktur einer Methylpentose hier anführen, ist von EniU Fischer und Karl Zach^) durch Überführung von Traubenzucker in diese Verbindung ganz aufgeklärt worden: die eine Ivar Bami: Zeitschr. f. physiol. Chcmio. 31. 411 (1900/01). Carl Neiiberg : Berichte d. Deutschen Chem. Gesellsch. 35. 1467 (1902j. Berichte d. Deutscheu Chem. Gesellsch. 42. 1198,. ') P. A. Levene und Jacobs: HaisermuX Wenzel: Monatshefte f. Chemie. 31. 357(1910). '2102, 2469. 2474(1909). ) ('arl Neuherg : Berichte der Deutschen Chem. Gesellsch. 33. 2243 (1900). Archiv f. exper. Path. ^) Biccardo Luzzato: Hofmeistern Beiträge. 6. 87 (1904). Suppl.-Bd. 366 (1908). II. Pharmak. •<) AlmaHiller: .1. of biul. Chem. 30. 129 (1917). - Vgl. ') F. A. Levene und F.B. La Forge: J. of biol. Chem. 18. :-^19 (1914). anch Ernsf Zerner wüd liudolßne WalUich: Monatshefte f. Chemie. 35. 1025 (1914). ) Bieler und Tollens: Berichte der Deutschen Chem. Gesellsch. 22. 3062 (1889). Ma</uenne, Compt. rend. de l'Acad. Günther und l'ollens: Ebenda. 23. 2585 (1890). ') ') — — — — des Sciences. 106. 603 (1892). ") Emil Fischer und Karl /.ach: 376] (1912). Berichte der Deutschen Chem. Gesellsch. 45. Kohlehydrate. ;\\ OHOHH H i CH, . i I C C . . C .0 I C . . Cf ^\H H H OHOH 1-Rhamnose. Die Methvlpentosen kommen fast gar nicht in freiem Zustande in der Natur vor. Sie sind fast durchweg Bausteine komplizierter und sehr mannigfaltig zusammengesetzter Zuckerarten. Auch nehmen sie am Aufbau von Verbindungen teil, die außer Zuckerarten noch andere Bausteine aufweisen. Ob ihnen im tierischen Organismus eine liolle zukommt, und ob sie am Aufbau von Zellbestandteilen Anteil haben, ist noch unbekannt. Die Pentosen haben manche Eigenschaften mit den Aldohexo.sen gemein. Es ist dies auch leicht verständlich, weil sowohl die Pentosen als auch die Hexosen als Aldehyde mehrwertiger Alkohole gemeinsame Züge in chemischer Beziehung tragen. Wir werden einige Reaktionen der einfachen Kohlehydrate bei den, Hexosen im Zusammenhang besprechen und wollen hier nur hervorheben, daß zum Xachweis der Pentosen Farbreaktionen M ausgearbeitet worden sind. Erhitzt man z.B. eine pentosehaltige Flüssigkeit mit Orzin l-Methyl-3, 5-dioxybenzol und rauchender Salzsäure, dann tritt eine blauviolette bis grünliche Färbung auf. Mit = = 1,3,5-Trioxybenzol und starker Salzsäure erhält man eine kirschrote Färbung. Die Lösung zeigt einen charakteristischen Absorptionsstreifen im Grün zwischen D und E. Werden Pentosen mit starken Phloroglucin man Furfurol = Furanaldehvd: Mineralsäuren erhitzt, so erhält CH — CH CH C.Cf \H Methvlpentosen liefern y.-Methylfurfurol: CH— CH ,0 C C/ \H II CH, . II C . O Cber die Entstehung der Pentosen im pflanzlichen Organismus haben wir auf Grund von Versuchen keine Erfahrung. Chemisch kann man .sich ihre Bildung, wie bereits geschildert, wohl vorstellen, und zwar kommen einfachsten läßt alle früher besprochenen Bildungsarten in Betracht. sich ihre Entstehung vom Formaldehyd, dem hypothetischen ersten Am Assimilationsprodukt der Kohlensäure der Luft durch die grünen Blätter, unter Zusammentritt von ö Molekülen dieser Verbindung (öxCH., C5H10O5) ableiten. Es wäre auch denkbar, daß die. wie schon ange- = •) Vgl. hierzu: B. Tollem im Handbuch der biochemischen Arbeitsmethoden. 2C Senberfi: Der Harn. 1. 3.%64 (1910). Urban & Schwarzenlierg. Berlin-Wien. 1910. — (1911). J. Springer. Berlin. II- 32 Vorlesung. führt, durch Oxydation des Glyzerins erhaltene Glyzerose das Ausgangsprodukt wäre, von der aus durch Anlagerung von zwei Kohlenstoffatomen die Pentosen sich bilden könnten. Endlich ist es möglich, daß die Pentosen durch Abbau aus kohlenstoffreicheren Zuckern z. B. aus Glukose, entstehen. Über die Bildung der Zucker der Fünfkohlenstoffreihe im tierischen Organismus wissen wir gar nichts. Es ist naheliegend, ihre Herkunft aus dem Gehalt der Nahrung an Pentosen abzuleiten. Bestimmte Beziehungen sind jedoch noch nicht verfolgt worden. AVir werden im Laufe dieser Vorlesungen bald erfahren, daß die tierische Zelle auch umfassende Synthesen ausführen kann. Sie kann ferner spalten, oxydieren und reduzieren. Nichts spricht dagegen, daß im tierischen Organismus die Pentosen in entsprechender Weise gebildet werden, wie es oben für die Pflanzenzelle geschildert worden ist. Wir kommen nun zu der für den tierischen Organismus wichtigsten Gruppe der Monosaccharide, zu den Hexosen der allgemeinen Formel Cg Hjo Oß. In einer vorläufigen Betrachtung haben wir bereits gesehen, daß nach der Le Bel-van't Hoß'schen Regel 16 isomere Verbindungen denkbar sind. Von diesen kommen für uns nur die d -Glukose, die d -Galaktose und die d- Fruktose in Betracht. Bevor wir auf diese Zuckerarten näher eingehen, sollen hier einige der wichtigsten Reaktionen der einfacheren Kohlehydrate im allgemeinen erörtert werden, soweit sie für das Verständnis des physiologischen Verhaltens und des Nachweises der verschiedenen Zuckerarten bedeutungsvoll sind. Die einfachen Zucker sind entsprechend ihrer Aldehydbzw. Ketonnatur sehr leicht oxydierbar. Sie reduzieren deshalb beim Erwärmen in alkalischer Lösung Metalloxyde. Auf dieser Eigenschaft beruht ein Teil der qualitativen und auch der quantitativen Methoden des Zuckernachweises, so die Trommersohe, die Fehlingsche, die Böffchersche, die Almen- Xi/lamlersche Probe. Kocht man z. B. eine alkalisch gemachte Zuckerlösung mit einem Kupferoxydsalz, Cu 0. so erhält man i\.bscheidung von ziegelrotem Kupferoxydul, Cu, 0. Beim Erhitzen einer Lösung von Zucker mit Natronlauge tritt Zer- setzung ein {Mooresche Probe). Die Flüssigkeit färbt sich braun, es entstehen Milchsäure, Brenzkatechin, Ameisensäure und andere Stoffe. Wird zu V2 cni^ einer verdünnten wässerigen Traubenzuckerlösung ein Tropfen einer lOVoig^i Lösung von a-Naphtol in azetonfreiem Alkohol und nun 1 cm^ konzentrierter Schwefelsäure vorsichtig und langsam zugesetzt, so entsteht an der Berührungsstelle beider Schichten ein violettroter Ring. Beim Umschütteln nimmt das ganze Gemisch diese Farbe an. Diese Reaktion (Molisch- Udrdnszki/), die zum Zuckernachweis in Proteinen etc. oft verwendet wird, beruht auf der Bildung von Furfurol aus Zucker unter der Einwirkung der Schwefelsäure. 1) Der Fruchtzucker ist dadurch ausgezeichnet, daß beim Erwärmen seiner Lösung mit verdünnter Salzsäure und etwas Resorcin 1, 3-Dioxybenzol eine Rotfärbung eintritt.^) = ) f. — L. H. Molisch: Wiener Monatshefte. 7. 198 (1886). B. Reinbold: Archiv 12. 358 (1888). phvsiol. Chemie. — (1904). d. f. — j IM und I'echmann: Chem. Zentralbl. 781 (1885). Deutschen Chem. Ges. 20. 181 (1887). Diese Reaktion — v. Udrdnszki/: Zeitschr. Physiol. 103. 581 d. ges. Seliivanoff : Berichte übrigens nicht nur auf 2'h. ist Kohlehydrate. ;-};-} Wird eine Zuckerlösung eingedampft und weiter erwärmt oder Zucker direkt erhitzt, so tritt vor der Verkohlung ein charakteristischer Geruch auf. Die gebildete Masse nennt man Karamel.'j Im Gegensatz zu den Pentosen liefern die Hexosen beim Erhitzen mit rauchender Salzsäure nicht Furfurol, sondern Lävulinsäure^)^ Azetylpropionsäure: CH3 CO CH.^ CH., CO OH und Ameisensäure. Intermediär entsteht besonders leicht aus Ketohexosen Oxymethylfurfurol') — — — — HC— CH II II ^O C.C^ (OHjHaC.C H O Dieses bedingt die charakteristischen Reaktionen mit Resorzin und Salzsäure usw. Bei längerer Einwirkung von Säuren entstehen schwarze Massen, die Hu min Substanzen genannt worden sind. Sehr empfindhch sind die Monosaccharide (mit Einschluß des Milchzuckers und der Maltose) gegen Alkali. Schwache Alkalien bewirken Umlagerungen, die weiter unten erwähnt sind. Bei längerer Einwirkung erfolgen tiefgreifende Veränderungen.) Es bilden sich z. B. Methylglyoxa) und Milchsäure. Eine wichtige und für die Erforschung der Zuckerarten so bedeutungsvoll gewordene Reaktion ist die Vereinigung vieler Zuckerarten mit Hydrazinen in essigsaurer Lösung unter Wasseraustritt zu Hydrazonen. Eine solche Verbindung ist die mit Phenylhydrazin.^) Wird eine ungefähr lOVoige wässerige Lösung z. B. von Traubenzucker mit einer Auflösung von Phenylhydrazin in verdünnter Essigsäuse versetzt und dann die Mischung 10 15 Minuten auf dem Wasserbade gekocht, dann scheiden sich bald feine gelbe Nadeln ab. Diese Verbindung heißt Glukosazon.*) Sie ist entstanden durch Zusammentritt von einem Molekül Zucker und zwei Molekülen Phenylhydrazin, und zwar vollzieht sich diese Reaktion in zwei Abschnitten. Zunächst verbindet sich der Zucker mit einem Molekül der Base zu einem Hydrazon der Formel: — CH,(0H).[CH(0H)l3.CH(0H).C0H + NHo.NH.CeH^ = CH,(0H) [CH(0H)l3 CH(OH) CH = N— NH C,H, + H^O. . . . . die Fruktose beschränkt, doch tritt sie hei dieser hesouders leicht eiu. Vgl. über diese Reaktion: Älb. v. Ekenstein und .7. J. Blanksma: Berichte d. Deutschen Chem. Gesellsch. 43. 2355 (1910). ') Vgl. über ihre Zusammensetznug: Mary Cimningham und Charles Doree: .1. Chem. Soc. 111. 589 (1917). ) A. V. Grote, E. Kehrer und H. Tollens: Anualcn der Chemie und Pharmacie. 206. 207 (1881). C. Wehner und B. Tollens: Ebeiula. 243. 314 (1888). - Berichte d. Deutschen Chem. Gesellsch. 33. 1286 (1900). ') A. V. Ekenstein und J. ./. Blanksma: Berichte d. Deutschen Chem. Gesellsch. Vgl. auch: J. A. Middendorp: Rec. trav. chim. Pavs-Bas. 98. 1 (1919). 43. 2355 (1910). — — ) Vgl. hierzu J. N. Xef: Liebi;/^ Annaleu. 376. ^) Emil Fischer: Berichte d. 1 (1910). Deutscheu Chem. Gesellsch. 17. .'i79 (1884): 20. 821 (1887). ) Entsprechend spricht man von Galaktosazou, Arabiuosazon. Xylosazon, Maltosazon etc. etc. AbderhaldeD, l'hysioloijisclie Choraie. I.Teil, 5. AuH. 3 34 Vorlesung. II- Da diese Verbindung in Wasser sehr leicht löslich ist, kommt diese* Phase der Reaktion beim Traubenzucker nicht zur Beobachtung. i) Beim Erwärmen mit überschüssigem Phenylhydrazin tritt nun an der mit einem * bezeichneten Alkoholgruppe eine eigenartige Oxydation ein. Es wird diese Gruppe vorübergehend in Carbonyl verwandelt, und dieses bindet dann ein zweites Molekül Phenylhydrazin: CHaCOH) [CH(0H)]3 . . C— CH CH^.HN.N N.NH.CeH,. Wie wir bald sehen werden, besitzt der Fruchtzucker, die Fruktose, keine Aldehyd-, sondern eine Ketogruppe. Er hat folgende Struktur: CH.OH i CO I OH— C— H— C— OH I H^C— OH CHoOH. Hier erfolgt nun die Reaktion mit Phenylhydrazin in umgekehrter Reihenfolge, wie die folgenden Formeln zeigen: CH2(0H) [CH(0H)]3 . . C— CUMm ) QH^.HN.N CH2(0H).\|CH(0H)], .C— CH II CfiH, II .HN.N N.NH.C.H,. Schließlich entsteht somit dasselbe (Jsazoii, wie das beim Traubenzucker erhaltene. Alle natürlichen und künstlichen einfachen Zucker, welche die Fehlingsche Lösung reduzieren (mit Einschluß der Disacch aride Milchzuckei- und Maltose), geben die genannte Reaktion. Die erhaltenen Produkte sind sehr charakteristisch und zur Erkennung und Abscheidung von Zuckern das wertvollste Hiltsmittel, das wir besitzen. Eine sehr wichtige Eigenschaft der natürlichen Zucker ist bereits erwähnt, nämlich ihre optische Aktivität. Auf ihr beruhen quantitative Bestimmungsmethoden. Sie gab ursprünglich auch die Grundlage zur Einteilung der Zuckerarten ab. So wurde der rechtsdrehende Traubenzucker als d-Glukose und der linksdrehende Fruchtzucker, der dementsprechend auch Lävulose genannt wird, als 1-Fruktose aufgeführt. A'm?7 Fischer schlug dann vor, diese Bezeichnungen in anderem Sinne zu verwenden und durch die Vorzeichen die Zusammengehörigkeit bestimmter Gruppen hervorzuheben. Aus diesem Grunde ist die} Fruktose als d-Fruktose be') Sie läßt sich hingegen bei der Mannoso Wasser sehr schwer lösliches Phenvlhvdraznn leicht isolieren, hihlet. weil diese ein in Kohlehydrate. ;,ö zeichnet und so ihre nahe Verwandtschaft zur d-Glukose, dem Traubenzucker, zum Ausdruck gebracht worden. Ohne Zweifel kann die Verwendung des gleichen Vorzeichens für den Hinweis auf das optische Verhalten und auf genetische Beziehungen leicht Anlaß zu Mißverstandnissen geben, besonders, wenn in keiner Weise kenntlich gemacht wird, was mit ihm zum Ausdruck gebracht werden soll. Wir möchten deshalb vorschlagen, das Vorzeichen dem Substrat voranzustellen, wenn es sich darum handelt, das optische Verhalten zu kennzeichnen und es hinter diesen in Klammern anzubringen, wenn genetische Beziehungen vermerkt werden sollen. Von diesen Gesichtspunkten aus bezeichnen wir die in der Natur vorkom- mende Form des Fruchtzuckers als l-Fruktose (d). ^) Die biologisch interessanteste Reaktion vieler Zucker ist ihre Spaltbarkeit durch bestimmte Organismen. So zerlegen verschiedene Hefearten Traubenzucker in Alkohol und Kohlensäure (alkoholische Gärung): C, H, 2 0« Das Bacterium = 2 Cs U, OH -f 2 COo. lactis zerlegt die Glukose dagegen in zwei Moleküle Gärungsmilchsäure (Milchsäuregärung): Cg Hl 2 Oß — 2 C3 Hg O3. Schließlich zerfällt der Traubenzucker unter der Einwirkung von bein Buttersäure. Kohlensäure und Wasserstoff (Buttersäuregärung): C,H,.,Og = (',HgOo + 2H2 4- 2C0o. Es soll an dieser Stelle auf die Vorgänge nicht näher eingegangen werden. Wir werden noch (lelegenheit haben, in ausführlicher Weise zu stimmten Mikroben zeigen, wie sehr die sterische Konfiguration der einzelnen Zuckerarten von Einfluß auf ihre Gärfähigkeit ist. Wir werden ferner erfahren, wie Ennl Fischer -) mit Hilfe dieser Reaktionen und der sich ergebenden Resultate zu Fragestellungen und Schlußfolgerungen gekommen ist, die für die Biologie von weittragendster Bedeutung geworden sind. Sie bilden das Fundament unserer ganzen Kenntnisse der Fermentreaktionen. Wir werden ferncierfahren, daß den einzelnen Vorgängen, die hier in einfachen Formeln zum Ausdruck gebracht sind, in Wirkhchkeit eine ganze Reihe von Um- setzungen entsprechen. Die Hexosen können wir zunächst in zwei Gruppen teilen, nämlich in Aldosen und Ke tosen. Von den ersteren interessieren uns, wie schon erwähnt, der Traubenzucker =:d-Glukose und die d-Galaktose. Die Konfiguration dieser beiden Zucker ist auf S. 24, 25 wiedergegeben. Eine weitere Aldohexose ist die Mannose. Sie ist im Pflanzenreich verbreitet, findet sich jedoch im tierischen Organismus nicht. Von Ketosen kommt nur der Fruchtzucker, auch Fruktose oder Lävulose genannt, in Betracht. Die folgende Übersicht gibt die Beziehungen dei- drei Aldosen zugehörigen Alkohol und zu den entsprechenden Säuren wieder =^): ) 55. Vgl. hierzu zum auch Karl Freudenberq und Fritz Brauns: Bor. d. D. Cheui. Ges. 1339 (1922). '•') Emil Fischer: Bedeutung der Stereochemie pbysiol. Chemie. 26. 60 (1898/99). ^) Vgl. über die Oxydation von Zuckern: Ges. 54. 456 (1921); 55. 7.'i (1922). A". für die Physiologie. Zeitschr. Kiliani. Ber. d. t. Deutschen ehem. 3* 36 LI. Vorlesung. Einbasische Säure Zweibasische Säure Dulcit 1) Glukonsäure Galaktonsäure Zuckersäure Schleim säure Mannit Mannonsäure Mannozuckersäure. Aldose Alkohol Glukose Galaktose Mannose Sorbit 1) Ebenso, wie die einzelnen Aldohexosen sich durch die Struktur und Konfiguration ihres Moleküls unterscheiden, sind in dieser Beziehung auch die entsprechenden Säuren in ihrem inneren Aufbau verschieden. Es seien an zwei Beispielen die Beziehungen von Aldose, Alkohol und den zugehörigen Säuren dargestellt: P^O Kohlehydrate. 37 der Reduktion, wie die Aldosen, auch Alkohol, und zwar ein Gemisch von Mannif und Sorbit. Zwischen der Glukose, der Fruktose und der Mannose bestehen enge Beziehungen. Sie gehen unter der Einwirkung ganz verdünnter Alkalien ineinander über. ^) Aus jeder einzelnen Zuckerart bilden sich die beiden anderen. Es bleibt jedoch ein Teil der ursprünglichen Ver- bindung bestehen. Es kommt zu einem Gleichgewicht: Glukose ^ tose ^ ^ Fruk- y Mannose. L.Michaelis und P. Bona^), welche die Umwandlung der (Glukose durch Alkali genauer verfolgten, kommen zum Schlüsse, daß ihre Geschwindigkeit der Hydroxylionenkonzentration proportional und der Wasserstoffionenkonzentration umgekehrt proportional ist. Gleichzeitig haben die genannten Autoren festgestellt, daß der Traubenzucker seiner Natur nach eine Säure ist, d. h. Wasserstoffionen abdissoziieren kann. Wir erhalten dann H- (Wasserstoffion) und den Rest des Glukosemoleküls als Zuckerion. Dieses kann an der Stelle, 'an der das H- abgespalten wurde, dieses wieder anlagern. Das H' kann aber auch an anderer Stelle eintreten und so den Umlagerungsvorgang bewirken. Bei dieser Gelegenheit sei auch darauf hingewiesen, daß die Glukose in zwei stereoisomeren Formen vorkommt, die leicht ineinander übergehen und sich in wässeriger Lösung das Gleichgewicht halten. Wir werden auf diese beiden Formen später bei der Besprechung der Glukoside und der durch Fermente herbeigeführten Synthesen zurückkommen. Die Fruktose ist im Pflanzenreich sehr verbreitet. Sie findet sich in freiem und gebundenem Zustande. In ersterer Form ist sie. meist mit anderen Zuckern gemischt, Bestandteil vieler Früchte. Fruktose entsteht ferner bei der Hydrolyse bestimmter Reservekohlehydrate der Pflanzen, so des Inulins, dem Reservestoff der Wurzelknollen von Dahlien, der Helianwichthus-, Topinambur-, Alant- und Atractylisarten etc. Am tigsten ist zucker. lekül ihr Bei Vorkommen mit dem Traubenzucker zusammen im Rohrseiner Hydrolyse entsteht 1 Molekül Glukose und I Mo- Fruktose. Unter den Produkten des Tierreiches ist der Fruchtzucker nicht gefunden worden. Im Honig kommt er stets neben Glukose vor Nach reichlichem (^enuß von Früchten kann im Harn zuweilen Fruktose auftreten. Ob sich auch normalerweise Fruktose in den tierischen Geweben findet»), ist recht zweifelhaft. Interessant ist das Vorkommen einer Fruktosurie. ) Ihr Wesen ist noch nicht erkannt. häufig des Sorbits iu Sorbose durch ein Bakterium, B. Xyliuum, bewirkt wird. Vgl. G. Bertrand: ittude biochimiqne de la Bacterie du Sorbose. Paris. Gauthiers- Villars 1905. ') C. Ä. Lobry de ßrvgn und W. Alberta van Ekenstein: Ber. d. Deutschen Cheni. Gesellsch. 28. 3078 (1895). L. Michaelis und P. Kona: Biochem. Zeitschr. 47. 447 (1912). ('. Neuberff Vgl. 0. Adler und R. Adler: Fßügers Archiv. 110. 99 (1905). Rudolf 0/ner: Monatsund //. Sfraus.'i: Zeitschr. f. physiol. Chemie. 36. 233. (1902). hefte f. Chemie. 25. 1153 (1904) und 26. 1165 (1905). Zeitschr. f. physiol. Chemie. 45. ») «) 359 (1905). — — ) L. Borchardt: Zeitschr. f. physiol. Chemie. 55. 241 (1908); 60. 411 (1909). W. Voit: Zeitschr. f. physiol. Chemie. 68. 122 (1908). — H. K. Barrmscheen: Biochem. Zeitschr. 127. 222 (1922). '^^ II. \'orlesiuig. Die hervorragendste Rolle von allen Monosacchariden spielt unzweifelim tierischen Organismus die Glukose, der Traubenzucker. Er bildet die Form, in der die Kohlehydrate ganz allgemein zur Resorption und zur Überführung von Organ zu Organ, von den Reservestätten zu den Verbrauchsstätten kommen. ') Die Glukose findet sich stets im Blute, und zwar in ganz bestimmter Menge. Der Gehalt des Blutes an Traubenzucker schwankt unter normalen Verhältnissen nur innerhalb enger Grenzen. Er beträgt durchschnittlich 0"05 0"l"/o h^i verschiedenen Tierarten.-) Glukose ist auch in einzelnen Organen (Muskeln) aufgefunden worden. Der normale menschliche Harn enthält keinen Traubenhaft — -^j zucker.) Im Pflanzenreich ist der Traubenzucker sehr verbreitet, teils als solcher, teils in mächtigen Ablagerungen als Reservestoff, z. B. als Stärke, ferner Form der verschiedenartigsten Zellulosearten. Als letztes Glied dieser Reihe haben wir noch die Galaktose zu betrachten. Im Pflanzenreich findet sie sich als Bestandteil mancher Verbindungen, so z. B. des D ig ton ins, des Sapotoxins etc. Man kennt als Gerüstsubstanz in i auch zahlreiche polymere Verbindungen der Galaktose, sogenannte Galaktane, die zum Teil bei der Hydrolyse ausschließhch Galaktose Hefern. zum Teil daneben auch andere Zuckerarten. Lippmann hat d-Galaktose in freiem Zustand an Früchten von Epheu beobachtet.^) Die gleiche Feststellung konnte ich bei der Untersuchung eines weißen Belages der gleichen Früchte machen. Im Organismus ist die Galaktose hauptsächlich in VerGlukose als Milchzucker und ferner als Baustein des Cerebrons nachgewiesen worden. Über die Bildung der Galaktose im tierischen Organismus wissen wir nichts Genaues. Wir können nur \q\mutungen äußern und annehmen, daß sie durch Umlagerung aus Glukose hervorgeht. Galaktose ist in einzelnen Fällen im Säuglingsharn angetroffen worden.6) Die Galaktosurie ist in diesem Falle ohne Zweifel auf den aufgenommenen Milchzucker zurückzuführen. bindung tierischen mit Wir kennen außer der Glukonsäure und der Zuckersäure noch eine einbasische Säure, die sich direkt auf den Traubenzucker zurückführen läßt. Es ist dies die Glukuronsäure. Sie ist dadurch ausgezeichnet daß sie die Aldehydgruppe noch besitzt. Schon (). Schmiedebercj und Hans Met/er'') f. ') PJs gilt dies gewiß auch für das wissenschaftl. Botanik. 50. 200 (1911), ') PHaiizeiiroicli. Vgl. IC. Huhlund: .lalirbüclier Daß im Bluto Traubeuzuckcr vorkommt, wurde von Dobson 181;") entdeckt. Bang: Der Blutzucker. A. F. Bergmann, Wiesbaden 191H. Vgl. auch Jvar — ') Berthold Öpp/er: Zeitschr. f. phvsiol. ('hemie. 75. 71 (1911). B. Bernier: de Pharm, et Chim. (7) 9. 493 (1914)."— (Mto Folin (.1. of i)iol. Chem. 22. 327 (UtK)) ist der Ansicht, daß auch normaler Harn Zucker enthält. Vgl. auch Stanley h'. Benedict, Emil Osterberg und Isaac Neuwirfh: .1. of l)iol. Chem. 34. 217 (1918). ) Sehr wichtige und eingehende IJutersuchungen über alimentäre Glukosurie verdanken wir Bernhard Schöndorff: Fßiiger^ Archiv. 121. 572 (1908). '') Edmund r. Lippmann: Berichte der Deutscheu Chem. Gesellschaft. 43. 3611 J. (1910). Leo Langfitein und Franz Steinitz: llofineister^ Beiträge. 7. 075 (190(5). 0. Schmiedeberg und Hans Meyer: Zeitschr. f. physiol. Chemie. 3. 422 (1879). Vgl. auch die Beol)achtungen von Ma:r Jatf'e: Berichte d. Deutsch. Chem. (iesellsch. 7. ) ') 16(;9 (1874). — Kohlehydrate. 39 vermuteten, dali die (ilukuronsäure ein Derivat des Traubenzuckers sei. Bewiesen wurde diese Annahme durch die Cberführung der Glukuronsäure in d-Zuckersäure durch T/iierfeider ^) und vor allem durch ihre Synthese von der d-Zuckersäure aus (E. Fischer und Pilot^'). Damit war auch ihre Konfiguration festgestellt.^) Sie ergibt sich aus der folgenden Zusammenstellung: 40 'I- Vorlpsuug. aus bestimmten Aminosäuren durch BakIndol im Darmkanal gebildet werden. Die (ilukuronsäure paart sich außerdem mit zahlreichen \erbindungen bestimmter Konstitution, die dem Organismus neben den Nahrungsstoffen zum Teil zu therapeutischen Zwecken, zum Teil, um bestimmte Fragestellungen zu studieren, absichtlich zugeführt worden sind. Sie paart sich mit (Jliedern der aliphatischen (mit Alkoholen, Aldebekanntesten sind hyden, Ketonen etc.) und der aromatischen Reihe. die Verbindungen mit Kampfer und Chloral. Die Zahl der beobachteten gepaarten Glukuronsäuren ist eine sehr große.') Ihre Konstitution ist von C. Xeuberg und E. Xeimaiin'^) durch die Synthese der Phenolglukuronlind terien Am C. NeuA'. Salkowski und geführten Nachweis, daß das künstlich erhaltene Produkt mit dem natürlich gewonnenen in seinen Eigenschaften übereinstimmt. Die Phenolglukuronsäure besitzt nach diesen Autoren die folgende Konstitution säure aufgeklärt worden und durch den von herg'^) CH . (J_CW1. Phenolrest HC OH CO OH. von Art von (Jlukuronsäureverbindungen sind Außer dieser Jqf^) und später von Maynm-Lcvy'^) (Jlukuronsäurepaarlinge beobachtet worden, die als Ester aufzufassen sind. Sie sind unbeständiger als die oben erwähnten Glukuronsäureverbindungen vom Glukosidtypus. Hierher gehört z. B. die nach Benzoesäureeingabe auftretende Benzoyl-glukuronsäure: C^H . OC . C, Hf. HC OH CO OH. ') Namentlich llildehmndt hat eine sehr ^rnße Zahl dieser Verbindungen studiert. Vergleiche Carl Xculierg und Rewahl : Biochem. Handlexikon. 2. 521 (1911), ferner Yuho Hämäläinen: Skand. Archiv f. Physiol. 23. H() (1909). ') Carl Neuberi/ und Wilheh» Xeimatw: Zeitschr. f. pbysiol. ('hemie. 44. 114(190.o). K Salkowski und C. Neuhen/: Biochem. Zeitsclir. 2. 307 (1907). M. Jaffe: Zeitschr. f. physiol. Chemie. 43. 374 (19U4). - Vgl. hier/u auch E. Sal) A. Magnus-Levy : Biochem. Zeitschr. 6. ."i02 (1907). kowski: Zeitschr. f. physiol. Chemie. 1. 2ö. (1877) und 4. 135 (1880). V) ) Kohlehydrate. 41 Die Kenntnis der Glukiirons<äure und ihrer Eigenschaften ist prakvon allergrößter Bedeutung, weil eine große Anzahl von Medikamenten von ihr gebunden werden., Die Glukuronsäurepaarlinge gelangen dann im Harn zur Ausscheidung. Die Glukuronsäure selbst reduziert Metalloxyde bei alkalischer Reaktion. Viele Paarlinge, die an und für sich nicht reduzieren, werden durch das Alkali gespalten. Es tritt dann beim Erwärmen der Lösung Abscheidung von reduziertem Metalloxyd ein. Dieses Verhalten der Glukuronsäure kann leicht zu Täuschungen Veranlassung geben. Gewiß sind eine große Anzahl von sogenannten vorübergehenden Zuckerausscheidungen im Harn auf eine vermehrte Glukuronsäureausscheidung in Form leicht hydrolysierbarer Paarlinge zurückzuführen. So beobachtete ich selbst einen Fall von angeblicher Zuckerausscheidung im Gefolge von Scharlach bei einem Kinde. Die Untersuchung des Harnes ergab starke Reduktion von Kupferoxyd in alkalischer Lösung. Der Harn zeigte jedoch kein Gärungsvermögen. Ferner drehte er nach links. Das Vorhandensein von Glukuronsäure bzw. von (ilukuronsäurepaarlingen kann mittelst Farbreaktionen festgestellt werden. Mit a-Naphtol, Phlorogluzin und Orzin erhält man die gleichen Färbungen, wie mit den Pentosen. Einen sehr schönen Farbstoff erhält man, wenn man eine Glukuronsäure enthaltende Flüssigkeit mit alkoholischer Naphtoresorzinlösung und rauchender Salzsäure versetzt. i) Der sich bildende Farbstoff löst sich in Äther mit blau- oder rotvioletter Farbe. tisch Wir werden auf die Glukuronsäure und ihre Bedeutung noch zurückkommen. Es sei schon hier bemerkt daß wir allen Grund haben anzu, nehmen, daß sie auch in der tierischen Zelle aus (Tlukose durch Oxydation der primären Alkoholgruppe hervorgeht. Kurz erwähnt sei noch, daß auch Zucker mit verzweigter Kohlenstoff kette bekannt geworden sind. Eine solche besitzt die aus dem Apiin, einer in der Petersilie vorkommenden Verbindung, gewonnene Apiose. 2) Sie eine ;i-Oxymethylerythrose,i;Jj-[^J[j>C(OH). ist liegt keinem Zweifel, . C<;J^. Es unter- daß die Apiose nicht der einzige Vertreter dieser Klasse von Verbindungen Im Anschluß an CH OH ist. Zuckerarten seien noch zwei Klassen \'erbindungen erwähnt, die ohne Zweifel Beziehungen zu diesen haben. Es sind dies die stickstoffhaltigen Kolileliydrate und gewisse aromatische Verbindungen. Diese letzteren hat man, um ihre nahe Zugehörigkeit zu den Kohlehydraten hervorzuheben, mit der Silbe -ose ausgezeichnet. Die andere Silbe des Namens der Vertreter dieser Gruppe Cyclosen besagt uns, daß zyklische, d. h. aromatische Verbindungen die einfachen von — — vorliegen. Von den Stickstoff enthaltenden, zu den Kohlehydraten in Beziehung stehenden Verbindungen sei in erster Linie (ilukosamin (Chitosamin) genannt. Es ist bei der Spaltung von Chitin aufgefunden worden. — ('. ) B. Tollens: Berichte der DeutKchen Ghem. Gesellschaft. 41. 1788 (1908). Vgl. hierzu auch Mandel Tollens: Münchener med. Wochenschr. 56. 652 (1909). und C. Neuberg: Biochem. Zeitschr. 13. 148 (1908). — ) E. Vorgerichten: Liehigs Annalen. 321. 71 — C Neuberg Ebenda. 24. 436 (1910). : (1902). 42 II- Vorlesimg. ist eine zusammengesetzte Verbindung, die insbesondere bei den Arthropoden sehr verbreitet ist. ^) Große Mengen Chitin finden wir ferner bei den Crustaceen.-) Der Ilummerpanzer ist das beste und ergiebigste Ausgangsmaterial für Cliitin und das daraus zu gewinnende Glukosamin. Chitinmembranen sind auch bei manchen Pilzen ^) und Flechten aufgefunden worden. Die Struktur des Chitins ist noch nicht aufgeklärt. Nach den einen) ist es ein polymeres Monoazetyldiglukosamin, nach anderen^) ist je ein Molekül Glukosamin mit drei Molekülen Azetylglukosamin unter Austritt von yier Molekülen Wasser verbunden. Chitin Das Glukosamin findet sich außer in Chitin interessanterweise als Baustein mancher Eiweißstoffe, so des Muzins. Ferner ist es Baustein der aus verschiedenen Geweben gewonnenen Mukoitinschwefelsäure. Die Konstitution des Glukosamins ist von Emil Fischer und Hermann Es ist als ein Derivat des Traubenzuckers oder der d-Mannose zu betrachten, in dem das in a-Stellung befindliche Hydroxyl durch die Aminogruppe NH., ersetzt ist. Seine Konfigurationsformel ist folgende. Wir stellen sie, um die nahen Beziehungen zur Glukose zum Ausdruck zu bringen, neben diese'): Leuchs'^) aufgeklärt worden. — C — NH^ H— C — OH H HO— C— HO — (^ H— C — OH H — C— OH H — C — OH H — C — OH I H I I I CH, OH CHo OH d-Glukosamin. d-Glukose Das Glukosamin ist eine sehr interessante Verbindung. Sie vermittelt den Übergang zu den Oxy-a-aminosäuren, denen wir bald als Spaltprodukte der Proteine begegnen werden, und so schlägt das Glukosamin gewissermaßen eine Brücke von den Kohlehydraten zu den Bausteinen der EiweißVgl. D. II. Westc.r: Zoolog. .Talirbuch. Abt. f. System. 2«. 531 (1910). Ledderhose hat das ülukosamiu entdeckt; vgl. Zeitscbr. f. physiol. Chemie. 2. 213 (1878/1879) und 4. 139 (1880). Vgl. auch H. SteiideJ : Ebenda. 34. 353 (1902). ') E. WinUrstein : Berichte der Deutschen Chem. (iesellsch. 27.3113(1894) und 28. 168 (1895). Emil Seholl: Monatshefte f. Chemie. 29. 1023 (1908). ^'. ) Th. R. q/fer: Biochem. Zeitscbr. 7. 120 (1908). Fränkel und A. Kelhi Monatshefte f. Chemie. 29. 1023 (1908). - Vgl. auch 0. r. Fürth und M. Russo: Hofmeistern Beiträge. 8. 163 (190(5). Emil Loewtj: Biochem. Zeitscbr. 23. 47 (1909). — Hugo Brach: Biochem. Zeitscbr. 38. 4(i8 (1912). 5) .7. C. Irvine: .iourn. Cbeni. Soc. 95/96. 546 (1909). ) Emil Fischer und tiermann Leuchs : Berichte der Deutschen. Chem. Gesellscb. 35. 3787 (1902). Ebenda. 36. 24 (1903). ') James J r rine \.]oura. of chem. Soc. 101. 1128 (1912)] hat d-ülukosamin in d-Glukose übergefiibrt. Vgl. aucli P. A. Lerene: .Iourn. of biol. Chem. 36. 73, 89 ») -) — ~ — — - — (1918). Kohlehydrate. 43 körper. Über die physiologische Bedeutung des Glukosaniins wissen wir vorläufig noch nichts. Bei Verfütterung von Glukosaniin konnte bis jetzt keine Beziehung zum Kohlehvdratstoffwechsel aufgefunden werden^), womit natürlich nicht bewiesen ist, daß das im Eiweiß vorhandene, in bestimmter Form gebundene Glukosamin beim Abbau dasselbe Verhalten zeigt, wie die künstlich in freiem Zustande zugeführte Verbindung. Hervorgehoben sei noch, daß aus Lycoperdon eine interessante, aus zwei Molekülen Glukosamin und Ameisensäure bestehende Verbindung, das Lycoperdin, gewonnen worden ist.^) Bei der Zerlegung der zuerst aus Knorpel und später auch aus anderen Geweben erhaltenen Chondroitinschwefelsäure ist ferner eine dem Glukosamin isomere Verbindung, das Chondrosamin, erhalten worden. Es hat nach Levene^) die Konstitution eines von der Pentose d-Lyxose sich ableitenden Hexosamines. Von den Cjcloseii ist nur eine \'erbindung bekannt, die, soweit unsere Kenntnisse reichen, sich regelmäßig im Organismus der höher organisierten Tiere vorfindet nämlich der i-Inosit. Er ist von Scherer) im Muskel entdeckt worden. Später wurde nachgewiesen, daß der Inosit in den verschiedensten Organen, kurz in allen Zellen des tierischen Organismus anzutreffen ist. Er tritt auch immer im Harn auf.^) Es ist die Frage aufgeworfen worden, ob der Inosit vorgebildet ist oder aber erst sekundär aus einer kompliziert gebauten Verbindung abgespalten wird.') Soviel ist sicher bewiesen, daß freier Inosit in tierischen Geweben vorkommt.-) Ob er daneben auch als Baustein am Aufbau zusammengesetzter Verbindungen teilnimmt, ist unbekannt. Wegen seines Vorkommens im Muskelgewebe, seines süßen Geschmackes und seiner mit den Hexosen gemeinsamen empirischen Zusammensetzung wurde der Inosit früher unter die Zucker eingereiht und kurz als Muskelzucker bezeichnet. Nachdem festgestellt worden war. daß eine aromatische Verbindung vorliegt, und es ferner nicht glücken wollte, direkte chemische und biologische Beziehungen zwischen den Zuckern und dem Inosit herzustellen war man eher geneigt, die Cyclosen und insbesondere den Inosit als eine für sich abgeschlossene Gruppe zu betrachten. Erst Xenherg^) schlug wieder eine Brücke vom Inosit zu den Kohlehydraten, indem es ihm glückte, aus dieser Verbindung Furfurol zu bilden. Da der Inosit auch in der Pflanzenwelt sehr verbreitet ist. so versuchte man, zuerst bei dieser Übergänge zu den Kohlehydraten zu finden. — Vgl. auch Kurt Mei/er: ') Manfred Bial : Berliuer kliu. Wocheuschr. 44 (1905). Hofmeisters, Beiträge. 9. 134 (1907). ") Y.Kotahe und Y. Sera: Zeitschrift für physiol. Chemie. 88.43 (1913); 89.482 a914). ») P. A. Levene uud F. B. La Forge; The J. of biol. Chern. 15. 1.^6 (1913): 18. 127.240 (1914); 20. 434 (1915). - P. A. Lecene; Ebenda. 31. 608 (1917». ) Scherer: Liehig?, Aunal. 73. 322 (1850). ') Vgl. hierzu Emil Sfarkenstein:' Zeitschrift für exper. Fath. u. Ther. 5. 378 (1908). «) F. Rosenberger: Zeitschr. f. physiol. Chemie. 56. 373. (1908): 57. 4ii4 (1908); 58. 3fi9 (1909). '") Emil Starkenstein: Zeitschr. f. physiol. Chemie, ) C. Neuberg: Biochem. Zeitschr. 9." 5.^1 (1908). 58. 162 (1908). 44 II- Vorlesuug. Es ist nicht gelungen, die Entstehung der Cyclosen irgendwie aufzuklären. Fütterungsversuche an Tieren führten zur Feststellung, daß ein Teil des Inosits unverändert im Harn wieder erscheint, i) Ein Teil scheint als Zellbaustein verwendet zu werden. Sehr wahrscheinlich tritt beim Abbau des Inosits Milchsäure 2) auf. Wenn wir alles, was wir über den Inosit wissen, zusammenfassen, dann kommen wir zum Resultate, daß es weder für den Pflanzenorganismus noch den Tierorganismus erwiesen ist, daß seine Bildung über die Kohlehydrate geht oder bei seinem Abbau solche als Zwischenstufen auftreten. Wir können somit die Beziehungen zu den Kohlehydraten zurzeit nur als sehr wahrscheinliche hinstellen. Der Inosit hat die Formel eines Hexaoxy-hexahydrobenzols^): CHOH Hü HC GH OH HO HC CHOH CHOH Der Inosit findet sich im Pflanzenreich zum Teil als solcher, zum Teil in Form von Methylestern. So ist der im Kautschuk von Borneo enthaltene Bornesit ein Monomethylester des i-Inosits. Im Milchsaft von Castilloa elastica und dem Kautschuk von Gabon findet sich der Dam- = Inositdimethylester. Von besonderem Interesse ist eine zunächst den Samen von Brassica nigra), bald aber auch in anderen Samen &) entdeckte, Inosit und Phosphorsäure enthaltende Verbindung. Sie ist Phytin genannt worden. Ihre Konstitution ist noch nicht ganz aufgeklärt. WahrscheinHch handelt es sich um einen Inosithexa-^) oder einen Inositpentaphosphorsäureester.') Außerdem ist im Weizenkorn ein Inositmonophosphat aufgefunden worden.^) Derartige Verbindungen von organischen mit anorganischen Bestandteilen«) erwecken unser Interesse in ganz besonders hohem Maße, bonit in Vgl. Emil Starkenstein: Biochera. Zeitschr. 30. 56 (1910). Mayer: Biochem. Zeitschr. 2. 398 (1907) und E. Starkenstein: Biochem. Zeitschr. 30. 56 (1910). Vgl. auch R. J. Anderson: Jourii. of biol. Chem. 25. 391 (1916); R. J. Anderson und A. W. Bosworth: Ebenda. 25. 399 (1916). •) Vgl. seine Synthese: Heinrich Wieland und Robert S. Wishart: Berichte der Deutschen Chem. Gesellsch. 47. 2082 (1914). ) Palladin: Zeitschr. f. Biol. N. F. 13. 191 (1895). ») E. Schulze und E. Winterstein: Zeitschr. f. physiol. Chemie. 22. 91 (1896): 40. 120 (1904). E. Winterstein: Berichte der Deutschen Chem. Gesellsch. 30. 2299 (1897). Pasternak: Revue gön^rale de Botan 12. 5 (1900); Compt. rend, de l'Acad. des Sciences 137. 202, 337, 439 (1903); Compt. rend. de la Soc. de biol. 55. 1190 (1903). •) C. Neuberg: Biochem. Zeitschr. 9. 558 (1908). Vgl. hierzu auch E. Slarkenstein: Ebenda. 30. 56 (1910). Wlad. Vorbrodt: Anzeigen Akad. Wiss. Krakau. Serie A. 414 (1910). Angelo Contardi: Atti Royal. Accad. dei Lincei [5]. 19. I. 23 (1910). /'. W. BoutVgl. ferner Robert Henry Aders Plimmer: The Biochem. .1. 7. 157. (1913). uell: J. Americ. Chem. Soc. 39. 491 fl917). S. I'osternak: C. r. 169. 138 (1919). ') Vgl. ./. ß. Rather: J. Americ. Chem. Soc. 40. 523 (1918). ») A. J. Anderson : .). of Biol. Chem. 18. 441 (1914); 20. 463, 475, 483, 493 (1915). ') ) — P — — — — — — •) Vgl. auch die Bildung von Vorlesung VT und Bd. II ^. XIX — — Hexo se ph ospha t bei der alkoholischen Gärung: 45 Kohlehydrate. weil unsere Kenntnisse über die Art und Weise, wie die einzelnen Zellbausteine unter sich verknüpft sind, noch überaus dürftige sind. daß in Organen von Rochen und Haifischen Erwähnt sei noch eine Cyclose aufgefunden worden ist^j, die dem Inosit isomer, aber nicht mit ihm identisch ist. Sie hat den Namen Scvilit erhalten. Ferner enthält die Miesmuschel, Mytilus edulis, eine Cyclose, Mytilit genannt, der folgenden , Struktur«) CH OH CH OH HO HC . . . HO. HC CH OH . C /\. OH ') Chemie. ) CH3 Staedeler nad i^rericÄs.- Jahresbericht der Chemie. .550(1858); .lourn. f. prakt. 48 (1858). D. Ackermann: Berichte der Deutschen Chem. Gesellsch. 54. 1938 (1921). [1.] 73. Vorlesung III. Kohlehydrate. Polysaccharide. Glukoside. Wir haben eine ganze Anzahl verschiedener einfacher Zuckerarien kennen gelernt, die sich alle als Aldehyde oder Ketone mehrwertiger Alkohole auffassen lassen. Sie unterscheiden sich teils durch die verschiedene Anzahl der Kohlenstoffatome, teils durch das Vorhandensein einer Aldehyd- oder einer Ketongruppe oder aber einzig und allein durch die Konfiguration des Moleküls. Diese einfachen Zucker finden sich meist nur in kleinen Mengen als solche in der Natur. Sie stellen eine Stufe im Aufoder Abbau zusammengesetzter Zucker dar. Die Monosaccharide werden von der Tier- und Pflanzenzelle und vor allem von der letzteren in der mannigfaltigsten Weise miteinander verknüpft. Wir erhalten, wie schon erwähnt, Verbindungen, die zwei, drei und mehr einfache Zucker als Bausteine besitzen. Wir unterscheiden, je nach der Anzahl der am Aufbau der zusammengesetzten Zuckerarten beteiligten einfachen Zucker, die den allgemeinen Namen Saccharide tragen, Di-, Tri-. Tetra- usw. -sacch aride. Ist uns die Anzahl der vorhandenen Bausteine nicht bekannt, dann sprechen wir ganz allgemein von Polysacchariden. Man kann aber auch alle Zucker, die mehrere Saccharide gebunden enthalten, als Polysaccharide bezeichnen und dann diejenigen besonders abtrennen, bei denen die Anzahl der Bausteine genau bekannt ist. Wir beginnen mit der Besprechung der Disaccharide. Sie sind nach der eben besprochenen Einteilung als Zucker zu bezeichnen, an deien Aufbau zwei Saccharide beliebiger Art beteiligt sind. Es können zwei Pentosen oder zwei Hexosen zu einer einheitlichen Verbindung verknüpft sein, oder aber es ist eine Hexose mit einer Pentose verbunden. So hat man in der sogenannten Vicianose"), einem Zucker, der in Vicia angustifolia von Bertrand entdeckt worden ist, ein Molekül d-Glukose mit einem Molekül 1-Arabinose vereinigt gefunden. Die Zahl der Disaccharide, die für uns hier in Betracht kommen, ist nicht sehr groß. Es spielen als Nahrungsstoffe für den tierischen (7. — ') Gabriel Bertrand: Compt. reud. de lAcad. des Sciences. 143. 832 (1907). Bertrand und G. Weismüller: Ebenda. 147. 252 (1908); 1.50. ISO (1910): 151. 884 (1910). Kohlehydrate. 47 Organismus eigentlich nur der Rohrzucker und der Milchzucker eine Der erstere wird, da er ausschließlich im Pflanzenreich vorkommt, nur von manchen Herbivoren und Omnivoren aufgenommen. Auch dem Milchzucker kommt als Nahrungsstoff keine allgemeine Bedeutung zu, denn er wird seinem Vorkommen in der Milch entsprechend nur während der Säuglingsperiode zugeführt. Einzig beim Menschen spielt die Milch als Nahrungsmittel das ganze Leben hindurch eine mehr oder weniger große Rolle. Ein drittes in der Pflanzen- und Tierwelt verbreitetes Disaccharid ist der Malzzucker. Er findet sich nie in größeren Mengen, weil er nur als Zwischenstufe auftritt, sei es, daß ein Polysaccharid zerfällt oder aber ein solches aus einfachen Bausteinen gebildet wird. Auf das Pflanzenreich beschränkt ist die aus zwei Monosacchariden bestehende Zellobiose. Rolle. Sie ist eine wichtige Abbaustufe der bald zu besprechenden Zellulose. Viel- fach beschrieben worden ist eine der Maltose sehr nahe stehende Zuckerart, die Isomaltose. Sie ist angeblich aus verschiedenen Organen gewonnen Da sie jedoch in keinem Falle mit Sicherheit festgestellt worden können wir sie nicht als Bestandteil des tierischen oder pflanzlichen Organismus aufführen. Wir erwähnen sie nur deshalb hier, weil wir bei der Besprechung der Synthese von Disacchariden durch Fermente auf die Isomaltose stoßen werden. Sie ist von Emil Fischer^) bei der Einwirkung von kalter rauchender Salzsäure auf Traubenzucker erhalten worden. Bei Gelegenheit der Besprechung von durch Fermente bewirkten Synthesen werden wir auch eine in der Natur noch nicht aufgefundene Isolaktose ') kennen lernen. Sämtliche Disaccharide kann man sich aus dem Zusammentritt von zwei Molekülen einfacher Zucker unter Austritt von einem Molekül Wasser entstanden denken. Das folgende Beispiel bringt zum Ausdruck, wie zwei Hexosen sich zu einem Disaccharid vereinigen: worden. ist, Ce H,2 O, -h C„ H,., 0«-H2 = C,o_ H,., 0„. Umgekehrt zerfällt jedes Disaccharid der erwähnten Art unter der Einwirkung von Säuren oder bestimmten Fermenten unter Wasseraufnahme in zwei Moleküle Hexosen; C,2 n,, 0„ -h H., = C„ H,, 0, + C« H,., (),. Bevor wir auf die einzelnen Disaccharide eingehen, wollen wir uns mit der Frage ihrer Konstitution befassen. Die Tatsache allein, daß die Disaccharide unter Wasseraustritt aus zwei Molekülen von Monosacchariden hervorgehen, genügt uns nicht. Wir werden z. B. bald vernehmen, daß es verschiedene Disaccharide gibt, die aus zwei Molekülen Traubenzucker aufgebaut sind. Ohne Zweifel muß in diesen die Bindungsart der Traubenzuckermoleküle eine verschiedene sein, sonst könnten wir nicht verstehen, weshalb die aus den gleichen Bausteinen bestehenden Verbindungen so verschiedene Eigenschaften haben können. Es ist bis jetzt nicht geglückt, eines der bekannten Disaccharide synthetisch zu bereiten und so dessen Struktur aufzuklären. Wir sind noch auf Vermutungen angewiesen. Doch Emil Fwc/ier; Berichte der Deutsclien Chem. Gesellsch. 28. 3024 (1895). ^ Emil Fischer: Berichte der Deutschen Chem. Gesellsch. 23. oöS? (1890). ') III- N nrlesung. 48 hat Emil Fischer'^) eiuen Weg ausfindig gemacht, der vorläufig zu einem Disaccharid vom Typus der Treh alose, einem Zucker, der zuerst im Mutterkorn aufgefunden worden ist-), geführt hat. Der Rohrzucker, auch Saccharose oder Saccharobiose genannt, ist, wie schon erwähnt, ein Disaccharid der Pflanzenwelt^). Erspielt als Reservewohl allen Phanerogamen eine wichtige Rolle und findet sich in seiner Hauptmenge vor allem, ganz entsprechend seiner Funktion, in nicht chlorophylhaltigen Geweben abgelagert. In kleineren Mengen ist er jedoch in allen Pflanzeuteilen gefunden worden. In größeren Massen begegnet man dem Rohrzucker in den Stengeln der Zucke rhirse und des Zuckerrohres, ferner im Saft einiger Palmenarten und demjenigen des Zuckerahorns, der Birke, des Johannisbrotbaumes etc. Erhebliche Mengen von Rohrzucker finden sich auch in den reifen Früchten und Blättern zahlreicher Gewächse. Von ganz hervorragender Bedeutung für die Gewinnung des Rohrzuckers ist, wie bekannt, die Zuckerrübe geworden, die neben dem Zuckerrohr die Hauptquelle des Handelsproduktes Rohrzucker darstellt. stoff bei In den Geweben des tierischen Organismus ist der als Nahrungsund Genußmittel so wichtige Rohrzucker bis jetzt nie nachgewiesen worden. Er muß somit, bevor er in das Blut und die Zellen übergeht, eine Umwandlung erleiden. Der Rohrzucker entspricht, wie bereits Liebig im Jahre 1834 nachFormel C12H23O11. Er zerfällt unter der Einwirkung von hydrolysierenden Mitteln (Chemikalien, Fermenten) in je ein Molekül d-Fruktose und ein Molekül d-Glukose. Da in diesem Spaltungsgemisch die d-Fruktose stärker nach links dreht, als die d-Glukose nach rechts, so dreht es nach links, d. h. in entgegengesetztem Sinne, wie der nach rechts drehende Rohrzucker. Man nennt aus diesem Grunde das durch Spaltung des Rohrzuckers entstehende Gemisch der beiden Hexosen Invertzucker und den ganzen Vorgang der Hydrolyse Inversion.) Die Bildung des Invertzuckers ist zuerst 1830 von Dubrun/aut^) beobachtet worden. Gemische von Frucht- und Traubenzucker sind übrigens in der Natur sehr verbreitet (Honig, Früchte etc.). wies, in seiner Zusammensetzung der Der Rohrzucker reduziert in alkalischer Lösung Metalloxyde nicht. Er unterscheidet sich dadurch scharf von den übrigen, oben angeführten Disacchariden. Wir können aus diesem Verhalten des Rohrzuckers schließen, daß er keine freie Aldehyd- oder Karbonylgruppe besitzt. Dieser Annahme trägt die folgende Struktur- und Konfigurationsformel Rechnung«): ') Emil Fischer und Konrad Delhriick: Berichte der Deutschen Chem. Gesellsch. 2776 (1909). ^) Mitscherlich: Wigqers: Aunalen der Chemie und Pharmazie. 1. 173 (1832). .1. f. prakt. Chemie. [1.] 73. 68 (1858). ') Über seine Verbreitung vgl. z. B. E. Schulze und ^'. Frankfurt : Zeitschr. für physiol. Chemie. 20. öll (1895). ) Dieser Ausdruck wird ganz unzutrett'end auch ganz allgemein für die Hydrolyse der zusammengesetzten Kohlehydrate in einfache Zucker gebraucht. ) Dubrunfaut: Comptes rend. de FAcad. des Sciences. 25. 308 (1847); 29. 51 (1849); 42. 901 (1856). ) Emil Fischer: Berichte der Deutsclien Chem. Gesellsch. 26. 2400 (1893). Vgl. auch Max hcrgmanfi und W. N. Hmvorth: J. of chem soc. 1J7. 199 (1920). Arthur Miekeln) Ber. d. D. Chem. (ies. 55. 1392 (1922). 42. — — — : Kohlehydrate. 49 CHo.OH CH2.OH Fruktoserest. Spaltet man den Rohrzucker z. B. durch Kochen mit Säuren, dann tritt alsbald Reduktionsvermögen auf, weil ein Zerfall des Üisaccharids in seine reduzierenden Bausteine einsetzt. Der Rohrzucker gärt auch nicht direkt. Die Gärung setzt ein. sobald das Disaccharid in seine Bausteine zerlegt ist. Ebenso, wie das eben besprochene Disaccharid, kommt der Milchzucker, auch Laktose oder Laktobiose genannt, als solcher in der Natur vor und ist schon 1615 von Fabricio Bartoletti in der ..Encyclo])aedia dogmatica" beschrieben und 1700 von Tesfi und ferner 1715 von Val/isnrri Glukoserest in der Schrift ..De praestantia lactis" als ein neu entdecktes Arzneimittel verkündet worden. Der Milchzucker findet sich in verschiedenen Mengen in der Milch aller Säugetiere. Bei Wöchnerinnen tritt er oft in geringen Mengen im HarnM auf. Bei Kühen ist er gleichfalls einige Tage vor und nach der Geburt im Urin gefunden worden. Wird das Säugen abgebrochen, so wird Milchzucker durch die Nieren ausgescliieden. Eingehende Studien über die Herkunft des Milchzuckers der Milch hat CIi. Porcher-) ausgeführt. Er fand, daß nach Exstirpation der Brustdrüsen bei säugenden Ziegen und Kühen bald eine starke Vermehrung des Zuckergehaltes des Blutes auftritt. Es kommt zur Ausscheidung von Zucker durch die Nieren. Der im Harn vorkommende Zucker erwies sich als Trauben- und nicht als Milchzucker. Diese Versuche machen es sehr wahrscheinlich, daß die Laktose erst in der Brustdrüse, und zwar offenbar nur aus Glukose gebildet wird und nicht aus Traubenzucker und Galaktose der Nahrung. Im Pflanzenreiche ist der Milchzucker bis jetzt nicht gefunden worden, Bei der Hydrolyse zerfällt der Milchzucker unter Wasseraufnahme in je ein Molekül Glukose und Galaktose. In die Art der Vereinigung dieser beiden Bausteine gibt die folgende Formel einen Einblick): ^'j ) Vgl. Leblanc und Guillot: Compt. reud. de lAcad des Sciences. 34. 580 (1852). — Franz Hofmeister: Zeitschr. ph\siol. Chemie. Kjl (1877'78). — P. Kaltenbach 360 '(1878 79), — F. A. Lamaire: Zeitsch. physiol. Zeitschr. physiol Chemie. — Chemie. 21 442 (1895 96j. — Ludwig: Wiener klin. Wncheuschr 305 (1899). : 1. f. f. 2. f. M. Kaufmann imd //. Magne: Compt. roud, de l'Acad. des Sciences. 143. 779 (1906). ") Vgl. Ch. Porcher: Compt. rend. de TAcad. des Sciences. 141. 73, 4()7 (1905). D. Nol-l Pafon und E. /'. Carthcart auch Carlo Foä Archiv, fisiol. 5. Heft 6 (1909). Journ. of Physiol. 42. 179 (1911). ') Die Angabe Bouchardats über den Befund von Milchzucker in derjoifon Frucht von Achras sapota ist noch unbestätigt. Compt. rend. de l'Acad. des Sciences. 73 462(1871). W. y. Ilaworth und ) Denhani und Woodhouse J. ehem. Soc. 111. 244 (191S). A. II. r„n der Haar: Roc. Trav chim 37. G. C.Leitch: Ebenda. 113 188 (1918). — — : : — — 251 (1918). AbderbaldKO. Physiologische Chemie. I.Teil, 5 Aiitl. 4 111. 50 Vorlesung. CH2.OH CH.OH CH2 . OH !— O.H.C Galaktoserest (ilukoserest Bei der Oxydation mit Salpetersäure erhält man aus ihm SchleimCO OH /(CH OH J CO OH. Ihre Bildung ist auf die Galaktose zurückzuführen (vgl. S. 36). Der Milchzucker reduziert Metalloxyde in alkalischer Lösung. Er wird von gewöhnlicher Hefe nicht vergoren, dagegen gibt es mancherlei Pilze und Pflanzen, welche die Laktose in Alkohol und Kohlensäure spalten können. Der Milchzucker unterliegt leicht der früher erwähnten Milchsäure- und Buttersäuregärung (vgl. diese S. 35). Bei allen diesen Umsetzungen geht ohne Zweifel eine Spaltung in die Bausteine voran. Im Gegensatz zu den bisher besprochenen beiden Disacchariden tritt der Malzzucker, auch Maltose, Maltobiose, Ptyalose, Cerealose genannt, nie in größeren Mengen auf. Er ist, wie schon erwähnt, ein Durchgangsprodukt, das meist sofort weiter gespalten oder zum Aufbau noch komplizierter gebauter Verbindungen verwendet wird. Die wichtigste Bildung der Maltose ist die durch fermentative Spaltung von Stärke im Pflanzenreich und von Glykogen im tierischen Organismus. Schon im Jahre 1785 beobachtete Irvine und 1815 Kirchhofe), daß Malzauszug Stärke abbaut. Der hierbei entstehende Zucker ist zuerst von de Saussure (1819) isoliert worden. Duhrunfaut^) (1847) nannte ihn Maltose und untersuchte ihn eingehender. Das im Malz enthaltene wirksame Prinzip, die sogenannte Diastase, wurde von Fa//en und Fersoz^) isoliert. Die Stärke zerfällt nicht einfach in Maltosemoleküle, sondern es entstehen zunächst komplizierter gebaute Abbaustufen, in deren Zusammensetzung wir zurzeit keinen genauen Einblick haben. Das erste wohl definierbare Abbauprodukt der Stärke und des Glykogens nach erfolgter Hydrolyse durch Diastase ist, neben einigen schon recht gut charakterisierten Dextrinen, das Disaccharid Maltose. Die Maltose gehört zu den reduzierenden Zuckern. Hefe vergärt sie. Sie ist auch der Milchsäure- und Buttersäuregärung fähig. Allen diesen Umwandlungen geht die Hydrolyse des Disaccharids in seine Bausteine voraus. Erst diese werden in Alkohol und Kohlensäure, bzw. Milchsäure, bzw. Buttersäure, Wasserstoff und Kohlensäure verwandelt. Der Maltose kommt folgende Struktur und Konfiguration zu): säure . ') Kirchhoff: Schweiffers Jourii. 15. 389. Dubrunfauf: Annales de chimie et de physique. \|3.\| 21. 178 (1848). ") Payen und Persoz: Ebenda. 2. ö.'j, 56. 73 und 337. Berichte d. Deutschen Chem. Gesellsch. 27. 2988 (1894). ) Emil Fischer: W. Lee Lewis und Siegel A. Buckborough: Nef: Liebigs. Annalen. 403. 299 (1914). Journ. Americ. Chem. Soc. 36. 2385 (1914). ^) — — Kohlehydrate. H— C— OH CH2 . Öl HO— C— OH Olukoserest Glukoserest Die gleiche Stellung, welche die Maltose im Abbau der Stärke und des Glykogens und den nächsten Abbaustufen dieser Polysaccharide einnimmt, besitzt die Zellobiose gegenüber der Zellulose. Es ist das Verdienst von Hans Pringsheim^), diesen Nachweis geführt zu haben. Die Zellobiose ist zuerst von Skraiq) und König beim künstlichen Abbau der Zellulose erhalten worden. Bei der Hydrolyse liefert die Zellobiose zwei Moleküle Traubenzucker. Sie reduziert gärt aber nicht. Eine Spaltung der Zellobiose in ihre Bausteine erfolgt, wenn man auf sie einen wässerigen Auszug aus Aspergillus niger oder einen Kefirauszug einwirken läßt. 3) Dieses Spaltvermögen kommt auch den sogenannten Zellulosebakterien zu, die im Darminhalt anzutreffen sind.^) Der Zellobiose kommt die folgende Formel zu :^) '^) , HO.HC- GH, H— C— OH HO-C— I Die HO— C— — I -C— HO— C— . OH Glukoserest seien OH H— C— OH H— C H— C— CH., . :CH Glukoserest am Aufbau der einzelnen Disaccharide beteiligten Monosaccharide im folgenden nochmals übersichtlich zusammengestellt. Durch Hydro') Hans Frinc/sheim -) Zd. U. Skraup : Zeitschr. f. phjsiol. Chemie. 78. 266 (1912). und J. König: Ber. d. Deutschen Chem. Gesellsch. 34. 1115 (19U1)-, Monatsh. f. Chemie. 22. 1011 (1901). Sciences. 149. 1385 ^) G. Bertrand und Holderer: Compt. reud. de lAcad. des Emil Fischer und G. Zemplen: Liehiiß Annalen. 365. 1 (1909); 150. 180(1910). (1909); 372. 254 (1910). H. Pringsheim ) Vgl. H. Prine/sheim: Z. f. physiol. Chemie. 78. 2^6 (1912). und Magnus von Merkatz: Eheuda. 105. 173 (1919). =) Vgl. M. Bergmann und H. Schotte: Ber. d. Deutschen Chem. Ges. 54. 440 (1921). — — 4* III- 52 Vorlesung. — — Aufnahme von einem Molekül Wasser entstehen die Bausteine der Disaccharide. und umgekehrt erhalten wir diese selbst, wenn die entsprechenden Monosaccharide unter Wasserabspaltung in bestimmter Weise lyse zusammentreten. Saccharose Laktose (Rohrzucker) (Milchzucker) -fHsOX Fruktose Glukose (FruehtzuckerJ (Traubenzucker) , '+H2 0\ Galaktose Glukose (Traubenzucker) Saccharose Laktose Maltose Zellobiose (Malzzucker) X+H-^Ox (jlukose (Traubenzucker) Glukose (Traubenzuckerj \ — R^0/ Maltose Z+H^OX Glukose (Traubenzucker) Glukose (Traubenzucker) \— H^O/ Zellobiose Es sind uns auch Zuckerarten bekannt, die bei der Hydrolyse Moleküle einfacher Zucker liefern. Es sind dies die Trisaccharide. An ihrem Aufbau können ausschließlich Pentosen beteiligt sein, oder es sind solche mit Hexosen zusammengefügt. So liefert z. B. die Rhamninose. die als Xanthorhamnin in den Früchten von Rhamnus infectoria aufgefunden wurde, bei der vollständigen Spaltung unter Aufnahme von zwei Molekülen Wasser ein Molekül d-Glukose und zwei Moleküle Rhamnose (Methylpentose vgl. S. 31). Schließlich gibt es Trisaccharide, die nur aus Hexosen aufgebaut sind. Recht verbreitet ist die Raffinose, auch Melitriose genannt. Sie besteht aus je einem Molekül d-Fruktose, d-Glukose und d-Galaktose. Im Eschenmanna ist ferner ein Trisaccharid beobachtet worden, das aus zwei Molekülen d-Galaktose und einem Molekül d-Glukose besteht. Ein weiteres Trisaccharid, die Gentianose, hefert bei der vollständigen Hydrolyse ein Molekül. Fruktose und zwei Moleküle Glukose. Es sind noch weitere Trisaccharide bekannt. Sie sind bis jetzt nur im Pflanzenreich mit Sicherheit aufgefunden worden. Sie müssen jedoch ohne Zweifel als Abbaustufen und Zwischenstufen beim Aufbau höher molekularer Polysaccharide auftreten. Hans Pringsheini i) hat bei der Hydrolyse von Steinnußspänen, die beim Abbau die früher erwähnte Mannose (vgl. S. 24, 27,34) liefern, eine aus drei Molekülen dieses Monosaccharids bestehende Verbindung beobachtet. Im Pflanzenreich sind auch Tetrasaccharide aufgefunden worden. Ein solches ist die Stachyose, auch Lupeose genannt. Sie findet sich im Eschenmanna, in den Wurzeln von Stachys tuberifera und auch in den unterirdischen Teilen von Lamium album.'^j Dieser Zucker liefert bei der drei ) Hans Pringsheim: Zeitsclir. f. physiol. Chemie. 80. 376 (1912). — Planta: Landwirtsch. V'ersuchsstationen. 25. 473 (1877). E. Schulze und Planta: Ber. d. Deutschen Chem. Gesellsch. 23. 1692 (1890); 24. 2705 (1891). Tanret: (Jompt. rend. de l'Acad des Sciences. 134. 1586 (1902); 136. 1569 (1903); Bull, de la Soc. de Science. \|3.1 27. 947 (1902): 29. 888 (1903). -) — Kohlehydrate. Hydrolyse zwei Moleküle d-Galaktose und je ein 53 Molekül d-Glukose und d-Fruktose.i) Pentasaccharide und Zuckerarten, die eine noch größere, genau bekannte Anzahl von Monosacchariden enthalten, sind bis jetzt weder auf dem ^Vege der Synthese dargestellt, noch sind sie in der Natur nachgewiesen worden. Daß sie vorhanden sein müssen, darüber besteht kein Zweifel. Derartige Zucker bilden sich, wie der Befund hochmolekularer, kristallisierter Dextrine bewiesen hat -), beim Abbau hochmolekularer Polysaccharide. Daß wir erst einzelne davon kennen, ist darauf zurückzuführen, daß die Zuckerarten an und für sich sehr schwer zur Kristallisation zu bringen sind. Sobald wir aber amorphe Körper vor uns haben, fällt die Entscheidung, ob ein Produkt einheitlich ist, oder aber ein Gemisch darschwer. In den meisten Fällen sind unter diesen stellt, außerordentlich Umständen erst dann bestimmte Schlüsse möglich, wenn die vermutete Substanz synthetisch gewonnen worden ist. Man kann die Eigenschaften der als identisch angesehenen Verbindungen dann genau vergleichen. Die Synthese muß somit bei der Auffindung komplizierter gebauter Veibindungen die Führung tibernehmen. Es ist dies eine auf dem Gebiete der Kohlehydrate sehr schwierige Aufgabe. Wir haben in der letzten Vorlesung schon hervorgehoben, daß drei verschiedene Bausteine allein schon durch die Art der Reihenfolge zu sechs isomeren Trisacchariden führen. Sind noch mehr Bausteine vorhanden, dann steigt die Zahl dieser Art von isomeren Verbindungen sehr rasch an. Dazu kommt noch, daß wir bereits bei den Disacchariden Verbindungen kennen gelernt haben, die dieselben Monosaccharide gebunden enthalten. Sowohl die Maltose als die Zellobiose liefern bei der Hydrolyse zwei Moleküle Traubenzucker. Da diese beiden Disaccharide in chemischer, physikalischer und biologischer Hinsicht sich ganz verschieden verhalten, so müssen wir annehmen, daß die beiden Bausteine in den genannten Verbindungen in verschiedener Weise verknüpft sind. Neben den Isomerien, die allein schon durch eine verschiedene Aufeinanderfolge verschiedenartiger Bausteine bedingt sind, kommen also noch weitere Möglichkeiten der Strukturverschiedenheiten durch die Art der Bindung hinzu. Diese Überlegungen machen es verständlich, weshalb die Aufklärung der Struktur der Polysaccharide nur langsame Fortschritte macht. Noch ein anderer Umstand bereitet der Forschung fast unüberwindbare Hindernisse. Diejenigen Zuckerarten, die mehr als zwei Monosaccharide gebunden enthalten, kommen nur äußerst selten für sich allein in größeren Mengen vor. Sie sind vielmehr meistens mit ungezählten anderen Polysacchariden gemischt. Da uns jede Kenntnis der Eigenschaften der einzelnen höher molekularen Polysaccharide fehlt, sind wir auf einen glücklichen Zufall angewiesen, wenn es uns glückt, aus einem solchen heterogenen Gemisch eine einheitliche Verbindung abzutrennen. Wir verlassen somit bei der Besprechung der weiteren Polysaccharide den sicheren Boden, auf dem wir bis jetzt wandelten, und begeben uns zur Besprechung von Zuckerarten, von denen wir nur aussagen können, welche ') C. E. Schulze: Ber. Neuberg und ") S. d. Deutschen Chem. Gesellsch. 43. 2230 (191U). Lachmann: Biochem. Zeitschr. 24. 171 (1910). Vgl. weiter unten. — Vgl. IH- Vorlesung. 54 Bausteine übrig bleiben, wenn der Abbau unter Wasseraufnahme vollständigdurchgeführt ist. Wir können jedoch bis auf wenige Ausnahmen (vgl. S. f)2) nicht angeben, wie oft der einzelne Baustein wiederkehrt. Wir bezeichnen aus diesem Grunde die Anzahl der am Aufbau eines bestimmten Polysaccharids beteiligten Monosaccharide nicht mit einer Zahl, sondern mit n. Für die ganze Auffassung des Abbaus der Polysaccharide zu einfacheren Produkten ist die Frage ihrer Struktur von grundlegender Bedeutung. Auf das gleiche Problem stoßen wir, wenn wir uns die Frage nach dem Aufbau von aus einer ganzen Reihe von einfachen Zuckern zusammengesetzten Kohlehydraten vorlegen. Während man sich bis vor kurzem vorstellte, daß in den hochmolekularen Polysacchariden die einzelnen Bausteine in entsprechender Weise untereinander verbunden sind, wie wir es bei den Disacchariden dargestellt haben, neigt man jetzt mehr der Auffassung zu. daß dem Aufbau der Polysaccharide Ringkomplexe zugrunde liegen, die unter sich anhydridartig verknüpft sind.i) In den wiederholt erwähnten Polysacchariden Stärke und Glykogen wären in diesem Falle Anhydromaltose- und vielleicht auch Anhydroisomaltosekomplexe mit einander verknüpft, während bei dem Polysaccharid Zellulose Anhydrozellobiosegruppen vereinigt wären. Nach dieser Vorstellung der Struktur der Polysaccharide würden bei ihrem Abbau zwei Vorgänge zu unterscheiden sein. Einmal die Loslösung von Anhydridkomplexen voneinander unter Bildung kleinerer Moleküle. Dazu käme dann noch die Aufspaltung der Anhydridbindung. -j Da wir von einem großen Teil der zwischen den hochmolekularen und den nach der Anzahl der Bausteine wohl charakterisierten Polysacchariden stehenden Zuckerarten ganz sicher wissen, daß sie ein äußerst mannigfaltiges Gemisch und nicht chemische Individuen darstellen, so verlassen wir zunächst den bisherigen Gang der Darstellung und sehen uns nach Polysacchariden um, die wir einigermaßen charakterisieren können. Von diesen aus kehren wir dann zu Verbindungen zurück, an deren Aufbau eine geringere Anzahl von Monosacchariden beteiligt ist. Wir werden von selbst auf diese stoßen, wenn wir den Abbau der hochmolekularen Zuckerarten verfolgen. Die Zahl der Polysaccharide, die gewissermaßen am anderen Ende der Reihe der Kohlehydrate stehen, wenn wir von den Monosacchariden ausgehen, ist eine sehr große. Es unterliegt keinem Zweifel, daß viele hierher gehörende Glieder der Kohlehydratreihe, die bei verschiedenen Individuen des Tier- und Pflanzenreiches auftreten und die gleiche Funktionen erfüllen, zwar unter gleichen Namen aufgeführt sind, jedoch sicher in Einzelheiten einen ganz verschiedenen Aufbau zeigen, l'berall macht sich eben bemerkbar, daß wir uns auf unsicherem Boden befinden, weil die Frage des Aufbaues und vor allem auch der Einheitlichkeit der Polysaccharide zurzeit nur ungenügend oder auch gar nicht beantwortet werden Vgl. hierzu auch /'. Karrer und A. P. Smirnoff: Helv. chim. Acta. 5. 124 (1921 Vgl. zu diesen Fragen Hans Prinqsheim und F. Eissler: Berichte der Deutschen -Chem. Ges. 46. 2959 (1913); Berichte der Deutschen Pharmaz. Ges. 27. 4 (1917). Hans Pringsheim und A. Aronowsky Berichte der Deutschen Chem. Ges. 54. 1281 (1921). Hans Prinffsheim und Walter Pcrsch: El)enda. 54. .3161 (1921). Aime' Pictef und Jean Sarasin: Arch. sciences phys. et nat. Gcneve. [4]. 46. ö (191S); Helvet chim. acta. P. Karrer: 1. 87 (1918). A'. Hess und Ernst Messmer: Ebenda. 54. 499 (1921). /'. Karrer und Helv. chim. acta. 3. 866(1920); 4. 169, 263(1921) Widmer: Ebenda. ') ). ") — : — — 4. 174(1921). — — Kohlehydrate. kann. So sprechen wir. 55 um nur ein Beispiel zu erwähnen, von Zellulose. Wir treffen diese überall im Pflanzenreich an. Manche dieser Zellulosearten zeigen Verschiedenheiten, manche können wir jedoch zur Zeit mit unseren chemischen Hilfsmitteln nicht unterscheiden. Dali trotzdem auch Zellulosearten, die uns zunächst als identisch erscheinen, ganz verschieden sein müssen, lehrt uns ihr \'erhalten gegenüber gewissen von den Zellen der Pflanzen- und Tierwelt hervorgebrachten Stoffen den Fermenten. Wir werden später vernehmen daß manche von diesen unter bestimmten Bedingungen einen Abbau -unter Wasseraiifnahme durchführen. Sie zerleg-en jedoch nicht jedes Substrat sondern immer nur ganz bestimmte Arten von solchen. Dieser Umstand ermöglicht eine äußerst feine Unterscheidung von scheinbar gleichartigen Verbindungen auf biologischem Wege. . , . Wir kennen Polysaccharide, die vollständig aus Pentosen aufgebaut Andere enthalten neben diesen auch Hexosen.') Am wichtigsten sind für unseren Organismus diejenigen Polysaccharide, die ganz aus Hexosen bestehen. Unter diesen kommt wieder denjenigen die größte Bedeutung zu. die AI dosen enthalten. Wir kennen auch ein in gewissen Pflanzen vorkommendes Polysaccharid, das ganz aus Molekülen der Ketohexose Fruchtzucker besteht.-) Es ist dies das Inulin. Es ist in Algen und vor allem sind. — vor allem bei KomReservekohiehydrat bei vielen Phanerogamen aufgefunden worden. Seine Konstitution ist in weitgehender Weise vor allem durch Ha)is Prinrisheun aufgeklärt worden. Es sind im Inulin neun Fruktosemoleküle gebunden, und zwar in Form von drei Anhydrotrifruktosekomplexen.-i Im tierischen Organismus kommen keine Polysaccharide vor. an deren Aufbau Fruktose beteiligt ist. als positen — Die Polysaccharide der Pentosen sind als Pento sane bezeichnet worden. Ein chemisch einheitliches Pentosan dürfte zurzeit wohl kaum bekannt sein. Sehr verbreitet sind in der P'f lanzenweit auch Methylpentosane.) Sie haben diesen Namen erhalten, weil sie bei der Hydrolyse Methylpentosen liefern. Der Gehalt des Holzes an Metbylpentosanen beträgt etwa 50/0 daneben findet man etwa 10— 2üo/o Pentosane. Die jungen Pflanzenteile weisen weniger Pentosane auf als die älteren. In Gräsern und Futterkräutern sind je nach dem Reifezustand verschiedene Mengen von Pentosanen enthalten. Bei der Ernährung der Pflanzenfresser spielen sie 5 sicher eine bedeutsame Rolle. '1 Vgl. über die \eiliieituiig ..gemischter" Polysaccharide 11. a. EL Xchulzi und Ch. Godet: Zeitschr. f. physiol. Chemie. 61. 279 (1909). Vintilesco: Recherches ./. biochim. sur quelques sucres et glucosides. Paris 1910. Mary Daries Suartz: Nutrition investigations ou the carhohydrates of Lichens, Algae aud related suhstances. New Haven. Conn. 1911. Hier findet sich viel Literatur. -) Über die Konstitution vgl. ./. C. Iruine uud K. St. Sterlr : .lourn. of ehem. soc. London. 117. 1474 (1920). 7/ Priiiguheim uud A. Aronowski/ : Berichte der Deutschten Chem. Ges. 54. 1281 (1921). Hans Pringsheim und Max Lassmann: Ebenda. 55. — — — — — 1409 (1922). Hans Pringsheim und Älcjc.' Aronoirsky Ebenda. 55. 1414 (1922). Die Fruktose scheint in der v-Forni (vgl. hierzu Anmerkung 4, S. 67), der wahrscheinlich ein Äthylenoxydrinir zukommt, in der Auhydrotrifruktose vorhanden zu sein. Vgl. hierzu auch .!/«./• Bpiqmann und Arthur Mickelei/: Ber. d. I). ("hem. Ges. 55. 1392 (1922). ) Über ihre Bedeutung siehe: A'. Miuake .lourn. of the College of Agriculture Tokio. 4. 327 (1912). : •') : III. \ Öllösung. 5() in der Pflanzenwelt die sogenannten Manna ne. vollständigen Hydrolyse Mannose. Sie sind ein sehr wichtiges Reservekohlehydrat der Palmensamen. Sie sind aber auch in vielen anderen Pflanzenarten aufgefunden worden. Es scheint auch bei dieser (Jruppe nicht geglückt zu sein, einheitliche Individuen zu isolieren. Zu erwähnen wären noch die Galaktane, die aus (jalaktose aufgebaut bei der Hydrolyse Galaktose und .sind und die Manno-galaktane, die Mannose liefern. Da es sich bei allen diesen Polysacchariden sicher um Gemische aller möglichen Verbindungen handelt, seien sie hier nur angeführt, um ein Bild von der großen Mannigfaltigkeit der Polysaccharide in der Pflanzenwelt zu geben. Von Polysacchariden, die im wesentlichen nur in der Pflanzenwelt vorkommen und die, soweit unsere Kenntnisse reichen, auch nur für diese von Bedeutung sind, seien noch die folgenden erwähnt. Von den verschiedensten Pflanzen werden, besonders, wenn Zellen erkranken oder verletzt werden, eigenartige Produkte, Gummisubstanzen genannt, abgehat auch sondert. Sie sind nicht einheitlich zusammengesetzt. Man Sehr verbreitet sind Sie liefern bei der gummiartige Substanzen im Harn gefunden und von tierischem Gummi gesprochen. Diese Bezeichnung ist jedoch eine ganz willkürliche, denn niemand hat diese Polysaccharide so charakterisiert, daß ihre Zugehörigkeit zu Gummiarten feststeht. Auch die pflanzlichen Gummisubstanzen sind mehr nach ihrem Vorkommen und ihren Eigenschaften als nach ihrer Zusammensetzung zusammengefaßt worden. Es handelt sich wohl immer um ein (remisch der mannig-faltigsten Polysaccharide. Eingehend untersucht ist eine Gummiart aus Hefe. ») Sie liefert bei der Hydrolyse Mannose und (tlukose. In Samen, Holzkörpern, Rinden von Bäumen usw. sind Polysaccharide aufgefunden worden, die sich in mancher Beziehung scharf von der Zellulose unterscheiden. Die ganze Gruppe hat von K. Schulze-) den Namen Hemizellulosen erhalten. Auch sogenannte Reservezellulosen hat man unterschieden. Erwähnt seien ferner die in Getreidearten so häufigen Amylane. Weit verbreitet sind auch die Pflanzenschleime. Sie finden sich meist in besonderen sogenannten Schleimzellen. Bei der Hydrolyse liefern sie Pentosen und Hexosen meistens Arabinose und Galaktose. Der Agar-Agar, der zu mannigfaltigen Zwecken Verwendung findet, — z. B. als Nährboden für Bakterien, ist ein solcher Schleimstoff. Er findet den Florideen. Zu erwähnen sind ferner die Pektinstoffe. Sie finden sich in fleischigen Früchten, in Wurzeln usw. und enthalten unter anderem Arabinose, Galaktose, Methylpentosen und Methylalkohol. Es gelang v. FelUnherg^ den Nachweis zu führen, daß das Pektin der Methylester der Pektinsäure ist. Die Pektinsäure selbst liefert bei der Hydrolyse d-(ialaktose und d-Galakturonsäure.^) Die letztere hat entsprechend dei- d-Galaktose die Konfiguration: sich in Meeresalgen, — E. Salkoirski: Berichte der Deutscbeu Chem. Gesellsch. 27. 4',)7, 925 (1894). und A. Fodor: Zeitschr. f. plivsiol. Chemie. 72. 339 (1911). -) E. Schulze, E. Steiyer u. W. Maxirell : Zeitschr. f. physiol. Chemie, 14. 227 (1890). ') Vgl. Th. r. Fellenherq : Mitt. a. d. Gebiete d. Lebensmitteluntersucbungen (veröffentl. vom Schweizer (iesnudheitsamt). 5. 172. 22.5 (1914): 7. 42 (1916); 8. ') //. Etiler 1 (1916). — Biochem. Z. 85. 118 (1918). ^) Berichte — Felix Ehrlich: Chem. Ztg. 41. 197 (1917). Vgl. Svntheso Emil d. Detitscheu Chem. Tiesellsch. 23. 937 (1890); 24. 2142(1891). Fischer: 57 Kohlehydrate. ^0 H . C HO C . HO C . . UH . . H C OH . . CO OH. EhrlirJi ist geneigt, Zellmembranen als das die Konstitution des Pektins Ca-Mg-Salz einer komplexen galaktose-methoxy-tetragalakturonsäiire von pflanzlichen Anhydro-arabino- aufzufassen. Wenn man bedenkt, daß jeder dieser hier kurz aufgezählten druppen eine Unzahl einzelner Individuen entsprechen dürfte, dann erkennt man. wie große, ja gewaltige Lücken die Chemie der Kohlehydrate noch aufweist, (ileichzeitig gewinnt man auch den Eindruck, daß nicht allein, wie man lange Zeit betonte, die Eiweißstoffe in unübersehbarer Mannigfaltigkeit auftreten und geradezu den Charakter der einzelnen Zellarten bedingen. Die Kohlehydrate stellen besonders in der Pflanzenwelt Verbindungen dar. die mindestens so mannigfaltiger Aufbaumöglichkeiten fähig sind, wie das bei den Eiweißstoffen der Fall ist. Wir gehen nun zur Besprechung derjenigen Polysaccharide des Pflanzenreiches über, die vom Pflanzen- und Allesfresser stets in mehr oder weniger großer Menge aufgenommen werden und als Nahrungsylum und stoffe eine große Rolle spielen. Es sind dies die Stärke Reservesogenanntes typisches ein ist Die erstere die Zellulose. kohlehydrat der Pflanzenwelt. Wir verstehen darunter ein Kohleum im geeigneten wird, hvdrat. das für spätere Zeiten abgelagert Augenblick durch Abbau allen möglichen Zwecken wieder zugänglich gemacht zQ werden. Ihrer Funktion entsprechend finden wir Anhäufungen = Am ' von Stärke in Form von charakteristisch gestalteten Körnern in Samen. Knollen usw. Wir begegnen der Stärke auch, und das ist besonders interessant, in den Chlorophyllkörnern der Blätter. Es läßt sich durch einfache Versuche direkt beweisen, daß an diesen Stellen .Stärke aus Kohlensäure und Wasser gebildet worden ist. und zwar unter dem Einflüsse der Sonnenenergie. ^ Die Stärkekörner i der Pflanzenart in ganz verschieMan glaubte als Sphärokristalle. eine Zeitlang, die Stärke als chemisches Individuum an-prechen zu dürfen. Die genauere Analyse hat jedoch ergeben, daß sie ein r;emi.-ch darstellt, «j Ma'juenne^-)' hat zwei Bestandteile der Stärke scharf unterschieden. denen Formen ') auf. treten je nach Man betrachtet sie Vgl. Vorlesan? IV. Man wird bei' genauerer Kenntnis der Bestandteile der Stärke diesen Namen nur noch als morphologischen Begriff anwenden und die chemischen Individuen als solche anführen. Mai 1904. Bull, de 'i Vgl. L. Maquenne: Annales de thim. et de Phvsique (8). 2. Ann. de Chim. et de Phy(1906): 1 la soc. chim. de Paris. 3^ serie. Nr. 18—19. -) 58 lil- Vorlesung. nämlich die Amylose und das Amylopektin. Das letztere enthält Phosphorsäure gebunden. \) Die Amylose löst sich in Alkalien, liefert keinen Kleister und bedingt die indigoblaue Färbung des Stärkekornes, wenn man dieses mit einer Lösung von Jod in Jodkali in Gegenwart von Jodwasserstoff zusammenbringt, ^j Diese Farbe ist nur in der Kälte beständig. Beim Erwärmen verschwindet sie. um beim Abkühlen wieder zu erscheinen. Eine Lösung von Amylopektin wird mit Jod violett bis rot gefärbt, es bildet Kleister und ist in Alkali unlöslich. Die Kleisterbildung der Stärke ist somit auf diesen Bestandteil zurückzuführen. Bei der Entstehung des Kleisters aus Stärkekörnern beobachtet man, daß diese in warmem Wasser zunächst «unter bedeutender Wasseraufnahme quellen. Sie platzen schließlich. Weder die Stärke noch der Kleister reduzieren Metalloxyde. Amylose und Amylopektin unterscheiden sich auch dadurch schart, erstere von einem Ferment, Diastase genannt, erst dann angewährend die Diastase griffen wird, wenn sie sich in Lösung befindet, Amylopektin sehr leicht direkt spaltet. Nach Gruzewska ^) entspricht die Amylose dem Innern des Stärkekornes, während das Amylopektin seine daß die Hülle darstellt. Sehr wichtig ist die Beobachtung, daß die Stärke kein einheitlich zusammengesetztes Gemisch der genannten Körper darstellt. Es enthalten nämlich die verschiedenen Stärkesorten die Anteile Amylose und Amylopektin in verschiedenen Mengen.) Vielleicht steht damit in Zusammenhang, daß die verschiedenen Stärkearten von Diastase aus Milz, PankreasSo enthält z.B.: saft und Speichel verschieden rasch abgebaut werden. •") > Haferstärke Kastanienstärke Bohnenstärke Reisstärke . . . . . . . . 71,5" ,, 67,OVo 75,50 .... 68,5% Kartoffelstärke- Weizenstärke Roggenstärke Amylopektin und 28,50/0 Amylose " r ^- '^ ^. •• 73,Oo/o '^'^fi^U ]• 24,5Vo r ,- 31,5''/o ,. „ 27,OVo ,• o2,5",o ,,• . . . . . 67,5''/o ,. ,. . . . 78,5Vo r r 21.5« Lassen wir auf die Stärke Säuren oder bestimmte Fermente einwirken, dann erfolgt ein x\bbau, der über zahlreiche Zwischenstufen führt, bis wir schließlich auf die einfachsten Bausteine stoßen. Diese sind Traubenzuckermoleküle. Die Stärkekomponenten sind somit als Polysaccharide der d- Glukose aufzufassen. Verfolgen wir den Abbau der Stärke genauer, dann beobachten wir, daß zunächst Produkte auftreten, die auch noch viele — siqup (8j. 9. 179 (190(5). Vgl. auch H. van Laer : Bull, de la Soc. Chiin. Belgique. 18. (1906). Z. Grtizewf^ka Eugene Fouard: C. r. de l'Acad. d. Sc. 148. .o02 (1909). Journ. de physiol. et de pathol. gener. 14. 7 (1912). Vgl. auch Ch. Tanrel : Cr. de lAcad. 159. .^30 (1914); Bull. Soc. Chim. de France. [4]. 17. 83 (1915). ') Vgl. A. W. Thomas: Biochem. Bull. 3. 402 (1914). Max Samec: Internat. Zeitschr. f. physikal -ehem. Biol. 1'. 173(1914). Kolloid-chem. Beihefte. 6. 23 (1914). John C. Northrop und ,/. M. Nelson: Journ. Americ. Chem. 38. 472 (1916). '^) Vgl. H. von Euler und Karl Mi/rhäck: Arkiv för Kemi, Mineral., Geol. 8. 1 (1921). Ä. Lottermoser: Zeitschr. f. Elektrochemie. 27. 496 (1921). ) Z. Gruzeivska: J. de physiol. et de pathol. gen. 14. 7 (19121. ) Charles Tanret: C. r. de l'Acad. 158. 1353 (1914). ^) Vgl. Marius Pauletig: Zeitschr. f. physiol. Chemie. 100. 74 (1917). — — — — — — — Knlileliyilrate. f)9 (ilukosemoleküle gebunden enthalten, jedoch ganz andere Eigenschaften Sie lösen sich z. B. leicht in Wasser auf. als die Stärkeanteile zeigen. Bald kann man auch reduzierende Produkte beobachten. Die Jodreaktion fällt auch anders aus als mit Stärke selbst. Zunächst beobachtet man andere Farben: violette, braune, rötliche bis burgunderrote Farbentöne usw. Schließlich bleibt bei weiterem Abbau jede Färbung aus. Ein genaues Bild des Verlaufs des Abbaus der Bestandteile der wenn deren Struktur aufgeklärt ist. Stärke wird sich erst geben lassen , Soviel ist sicher, daß die Zerlegung der großen Moleküle in kleinere über mancherlei Zwischenstufen erfolgt, bis schließlich der Abbau stein Glukose durchgeführt bi.> zum Bau- ist. Man hat versucht, die den Stärkeanteilen noch nahestehenden Abbaustufen zu charakterisieren. \ or allem wählte man hierzu die Färbung mit Jod. Man hat auch bestimmte Verbindungen unterschieden und sie durch Namen ausgezeichnet. Es unterliegt keinem Zweifel, daß für den Forscher all die gemachten Beobachtungen sehr wertvoll sind, für uns sind die einzelnen Namen einstweilen noch entbehrlich weil mit wenigen Ausnahmen mit keinem ein erwiesen einheitliches Individuum verknüpft ist. ^j Wir ziehen es deshall) vor, das ganze große (Temisch der auf die Stärkekomponenten folgenden Abbaustufen mit einem einzigen Namen. nämUch Dextrine, zu bezeichnen. Dieser Name umfaßt somit, das sei ganz besonders betont, kein chemisches Individuum sondern ein großes Gemisch aller möglichen Zwischenstufen im Abbau der Stärkeanteile bis zur Glukose herab. Der Begriff Dextrine ist somit ein mehr biologischer als chemischer. W^ir wollen uns nun der Frage zuwenden, ob es nicht geglückt ist, unter all diesen Abbaustufen die eine oder andere zu isolieren und al.^ cht^misches Individuum zu charakterisieren. Trotz aller aufgewandten Mühe konnte bis vor kurzem nur ein Disaccharid, nämlich die Maltose, als wohlcharakterisierte Abbaustufe nachgewiesen werden. Sie zerfällt unter der Einwirkung eines besonderen Fermentes in zwei Moleküle 1-Glukose. Es scheint aber, daß neuere Versuche erfolgreicher sind und bald weitere gut charakterisierte, einfachere Polysaccharide zur Kenntnis bringen werden. 2j Es sind nämlich kristallisierte Dextrine isoliert worden, und . , zwar eine a-Hexaamylose, (Cß Hio 05)6 ",) und eine -/.-Tetraamylose Ferner ist eine ß-Hexaamylose gewonnen worden ) In diesen Polyamylosen sind Di- bzw. Triamylosekomplexe vereinigt. Die Diamylose ist als eine Anhydromaltose erkannt worden. Sobald die Struktur und Konfiguration einzelner Bruchstücke des Ausgangsprodukte? bekannt sein werden, wird man sich eine klarere \'orstellung von den einzelnen Vorgängen beim Abbau eines Polysaccharidmoleküls machen können. Wie S. 54 ausgeführt, folgen .'^ich Loslösung von (Cf, Hio05)4. Vgl. /. r. Blake: Journ. of Americ. Chem. Soc. 40. G23 (1918). F. Schardhif/er: Zeitschr. f. Uutcrs. d. Nahrungs- u. Genußmittel. 6. 874 (1903). //. I'ringsheim und A. LantZentralbl. f. Bakt. u. Parasitenkunde. 22. 98 (19ü8). H. Fringsheim und hans: Berichte d. Deutschen Chem. Gesellsch. 45. 2533 (1912). Franz Eissler : Berichte d. Deutscheu Chem. Gesellsch. 47. 2565 (1914). M W. Biltz und IC. Trnfhe: Berichte der Deutschen Chem. Gesellsch. 46. 1377 M — 1913). -) — — — Vgl. auch Wilhelm Biltz: Ebenda. 46. 1532 (1913). Vgl. hierzu Hans Pringsheim und Walter Persch; Ber. d. Deutschen Chem. Gesellsch. 55. 1425 (1922). Hans Pringsheim und Diamaudi l>ernikos: Ebenda. 55. 1433 (1922). Hans Pringsheim und Kurt r^oldsfein : Ebenda. 55. 1446 (1922). ) — — TU. Vorlesung. 60 Anhydridkomplexen und Aufsprengung von Anhydridbindungen. Es werden offenbar Komplexe mit kleinerem Molekulargewicht, als es dem Ausgangsprodukt zukommt, frei, die in manchen und vielleicht den wesentlichsten Zügen noch dessen Charakter besitzen. Mit der Aufsprengung der Anhydridbindungen zu den Bausteinen selbst werden Aldehyd- und Ketogruppen frei und damit treten Eigenschaften auf, die dem ursprünglichen PolysacDie Aufspaltung der Anhydridringe erfolgt charid nicht eigen sind. nicht erst in dem Augenblicke, in dem der Baustein Glukose vielmehr ohne Zweifel schon innerhalb zusammengesetzter Verbindungen. Vielleicht darf man der Art des weiteren Abbaues solcher Komplexe die Erfahrungen über den stufenweisen Abbau von ihrer Struktur nach einigermaßen bekannter einfacherer Polysaccharide zugrunde legen. So hat z. B. das Studium des Abbaus von Trisacchariden durch Fermente ergeben, daß diese stets einen Baustein nach dem anderen loslösen. Das gleiche gilt für die Einwirkung von Chemikalien. So spaltet z. B. Säure bei geringer Erwärmung aus Raffinose zunächst d-Fruktose ab. Es bleibt ein aus Glukose und Galaktose bestehendes Disaccharid übrig, die Melibiose. Bei weiterer Erwärmung wird diese dann in d-Galaktose und d-Glukose zerDurch Fermente läßt sich der Abbau der Raffinose in zwei Richtungen legt. vollziehen. Fermente aus Oberhefezellen zerlegen dieses Trisaccharid so, daß Melibiose und Fruktose entstehen. Die Fermentgruppe Emulsin bildet Rohrzucker und Galaktose 2): sicherlich frei wird, ^ ) Hydrolyse mit Oberhefenfermenten oder verdünnten Säuren Melibiose H OH CH, . OH 0— C I HO . C . H.C.OH H.C- CH5OH Galaktoserest Fruktoserest Glukoserest Kaffinosc H OH Rohrzucker Hydrolyse mit Emulsin — ) Scheibler: Berichte der Deutsch. Chem. Gesellsch. 18. 1799 {1885). B. Tollens und Gans: Ebenda. 21. 21.oü (1888). Scheibler und Mittel itieier: Ebenda. 22. 1B78. (1899); 26. 2930 (1893). ») C. Neuberg: Biochem. Zeitschr. 3. 519. 535 (1907). Vgl. auch Zeitschrift de Vereines der Deutschen Zuckerindustrie. 67. 463 (1917J. — Kohlehydrate. 61 Das Studium des stufenweisen Abbaus solcher Polysaccharide gibt uns eine weitere Möglichkeit zusammengesetzte Zuckerarten zu charakterisieren. Da das Ausgangsmaterial uud die sich bildenden Spaltstücke ein verschiedenes optisches Verhalten zeigen, läßt sich die Art dos Abbaus durch Verfolgung des Drehungsvermögens genau feststellen, vorausgesetzt, daß das optische Verhalten der in Betracht kommenden Abbaustufen bekannt und die gegenseitige Beeinflussung der einzelnen Komponenten auf das optische Verhalten festgestellt ist. Das andere Polysaccharid der Pflanzenwelt, das uns hier interessiert, im allgemeinen für die Pflanze eine andere Bedeutung als die Stärke. Sie hat im großen und ganzen mechanische Funktionen zu erfüllen. Sie ist die Stütz- und Abgrenzungssubstanz der Pflanzenwelt. Die Zellulose findet sich in der Tierwelt nur ganz vereinzelt. Eine Zellulosedie Zellulose, hat art ist z.B. bei Tunikaten nachgewiesen worden. ') Sie hat den Namen Daß es sich bei der aus dem Mantel von Phallusia Tunicin erhalten. mammillaris gewonnenen Substanz um ein Polysaccharid handelt, das der sehr nahe steht und sogar wahrscheinlich mit ihr identisch ist, beweist der Umstand, daß es geglückt ist, aus Tunicin Zellobiose^), ein charakteristisches Abbauprodukt der Zellulose, zu gewinnen. Zellulose Die Zellulose ist in erster Linie durch ihre außerordentliche Widerstandsfähigkeit gegen alle möglichen EinwiFkungen ausgezeichnet. Sie ist nicht nur sehr resistent gegen Chemikalien, sie wird auch von Fermenten nur schwer angegriffen. Gewisse Lebewesen vermögen sie abzubauen. Vor allem sind es Mikroorganismen, denen diese Fähigkeit zukommt. Die Zellulose ist in den gewöhnlichen Lösungsmitteln, in verdünnten Säuren und Alkalien ganz unlöslich. Sie löst sich nur in Kupferoxydammoniak (Schiveizersches Reagens s). Bringt man Zellulose, z. B. Filtrierpapier, in heiße, konzentrierte Schwelelsäure, dann erhält man Abscheidung von Kohlenstoff. Läßt man sie bei gewöhnlicher Temperatur mit konzentrierter Schwefelsäure stehen, dann bilden sich zunächst Schwefelsäureester. Läßt lO» man auf Zellulose Essigsäureanhydrid und Schwefelsäure bei 105 einwirken, dann gelangt man zur Oktaacety Izellobiose.) Es sei auch noch kurz auf die Zellulosenitrate hingewiesen, die wegen ihrer besonderen Eigenschaften mannigfaltige Anwendungen, z. B. als Sprengmittel, gefunden haben. Die Zellulose färbt sich mit Jod und Schwefelsäure blau. Es sind noch sehr viele Farbreaktionen beschrieben worden. Sehr schön ist die Violettfärbung mit Chlorzinkjodid. — 1 Wird die Zellulose durch Kochen mit verdünnten Säuren hydrolysiert, Die einzelnen Zwischenprodukte dann liefert sie schließlich Traubenzucker. sind auch hier nicht bekannt. Nur das Disaccharid Zellobiose ist näher =^) — ') Berthelot: WiiUerstein: Aniiales de Cbim. et de l'hys. 56. 149 (1859). ZeitBcbr. f. physiol. Chemie. 18. 46 (1893). •) luid G. Zemplen: Zeitschr. f. physiologische Chemie. 72. .^8 Fj. Abderhalden /•-'. (1911). prakt. Chemie. 76. 109. 344 (1857). König: Monatshefte f. Chemie. 22. 1031 (1901). Charles F. (iross ') Nach M. Cunningham (J. Chem. Soc. 113. 173 11918]. und K.J. Heran: (Ebenda. 113. 182 \|1918\|) gibt es Zellulosearteu. bei deren Abbau nicht Glukose entsteht. ») E. Schweizer: .Inurn. ) Zd. H. Skraup und f. ./. — III. H2 Vorlesuug, charakterisiert worden. Sie liefert zwei Moleküle Traubenzucker. Die Konstitution der Zellulose ist ebenso wie die der bereits besprochenen Polysac- charide mit noch in wesentlichen Funkten strittig, i) Eine für die Pflanzenwelt bedeutungsvolle Kombination von Zellulose liegt im Holze vor. Holzsubstanz Lignin — — Im tierischen Organismus ist bis jetzt nur ein Polysaccharid dieser Reihe aufgefunden worden, das ausschließlich aus Traubenzucker besteht. Es dies das Glykogen. Diesem kommt biologisch die gleiche Bedeutung zu, wie z. B. der Stärke, dem Reservekohlehydrat der Pflanze. Der tierische Organismus speichert überschüssige Kohlehydrate in Form von Glykogen in den verschiedensten Organen auf. Dieses Polysaccharid wurde fast zur gleichen Zeit von CUindo ßemard -) und V. Beulen ^) entdeckt. ist Claude Bernard beobachtete schon im Jahre 1848 den hohen Gehalt der Leber an Traubenzucker) und fand, daß sie erst nach langem Hungern sehr zuckerarm wird. Wenige Jahre später glückte ihm auch der Nachweis, daß die Glukose in der Leber nicht unmittelbar als solche vorhanden erst allmählich aus einer Vorstufe entsteht. Er stellte fest, ist, sondern daß die einem eben getöteten Hunde entnommene Leber nach der Ausspülung des Blutes und längerer Durchleitung (40 Minuten) von Wasser keinen Zucker an die Spülflüssigkeit mehr abgab. Auch konnte durch Auskochen eines Leberstückchens kein Zucker erhalten werden. Wohl aber ließ sich solcher in reichlicher Menge nachweisen, wenn die frische Leber z. B. 24 Stunden gelegen hatte. Dies brachte Claude Bernard auf den Gedanken, daß in der Leber eine Substanz vorhanden sei, die in W^asser sich schwer Das löst und unter der Mitwirkung der Lebersubstauz Zucker liefert. Lebergewebe muß „lebend" sein, wie der folgende Versuch zeigt. Wird nach dem vollständigen Auswaschen der Leber die eine Hälfte gekocht, so zeigt es sich, daß dieses Leberstück keinen Zucker mehr bildet, wohl aber der andere nicht gekochte Teil. Claude Bernard hat aber nicht nur die Entstehung des Zuckers aus einer offenbar kompliziert gebauten Vorstufe verfolgt, sondern es ist ihm auch gelungen, diese darzustellen.'') Die von ihm angewandte Methode zur Darstellung des (ilykogens ist in ihren Grundzügen auch heute noch dieselbe. Sie beruht auf der Beobachtung, daß Alkohol ') Vgl. hierzu besdiidert; Kurt Hess und IV. Wittclsbach: Zeitschr. f. EleklroK. Hess und Eriisf Messmer: Berichte d. Deutsch. Chem. chemie. 26. 232 (1920). Kurt JJcss: Berichte d. Deutsch. Chem. Gesellsch. 54. (iosellsch. 54. 834 (1921). — — — — P. Karrer Knrf Hess: Zeitschr. f. augevvaiidte Chemie. 34. 49 (1921). 2S68 (1921). /'. Karrer und Fr. Widmer: und ('. Nägeli: Helv. ehem. acta. 4. 169 (1921). J. C. Ircine und Ch. W. Snn/ar: ^. Chem. Soc. London. Ebenda. 4. 174 (1921). Karl Frendenhery : Berichte d. Deutsch. Chem. Gesellsch. 54. 117. 1489 (1920). R. (). Herzoy und W. Jancke: Zeitschr. f. Physik. 3. 196 (1920). 767 (1921). -) Claude i?ip/-narrf.Compt. rend. de l'Acad. des Sciences. 41. 461 (1855); 44. 1325 (1857); 48. 77, 673, 884 (1859). ') V. Hensen: Virchoin^ Archiv. 11. 395 (1857). ) Vgl. Claude Bernard und Barreswil: Compt. rend. de l'Acad. des Sciences. 27. Eine ausgedehnte Studie über das Glykogen verdanken wir E. F. W. 514 (1848). Bonn. l'lläyer: Das Glykogen und seine Beziehungen zur Zuckerkrankheit. 2. Aufl. Eine vollständige Zusammenstellung der Arbeiten von Claude Martin Hager. 1905. Bernard findet sich in: L'oeuvre de Claude Bernard. l'aris. .1. B. Bailliere et Fils. ^) Claude Bernard: Lc^ons sur la l'hysiologie et la Pathologie du Systeme uerveux. Vgl. auch (iazette medicale. 2i<. III (1857). 1. 467 (1857). — — — — * — — — Kohlehydrate, 6H aus einer alkalischen Lösung der Organe das Glykogen fällt. Durch Wiederholung der Auflösung in Kalilauge und Fällung mit Alkohol kann das Iiohglykogen gereinigt werden. August KeknU ») hat das Glykogen nach der oben angegebenen Methode zuerst Stickstoff- und aschefrei dargestellt. Das Glykogen stellt ein feines, weißes, amorphes Pulver dar. Es ist geruch- und geschmacklos. Cber sein Molekulargewicht liegen sehr widersprechende Angaben vor. Die einen Autoren schreiben ihm ein sehr hohes zu. andere halten eine einfachere Zusammensetzung für wahrscheinlicher. Letztere dürften nach neueren Forschungen recht behalten. 2) Das (Glykogen quillt in kaltem Wasser und löst sich nur scheinbar auf. Die Lösung zeigt hierbei deutliche Opaleszenz. Daß eine wirkliche Lösung nicht eingetreten daß das Glykogen nicht durch tierische Memist, beweist der Umstand, branen diffundiert. Außerdem hat Gatin-Gruznrska ^) gezeigt, daß in Wasser gelöstes Glykogen gegenüber dem elektrischen Strome sich ganz wie ein Kolloid verhält. Ks wandert, und zwar zur Anode.) Das Glykogen dreht nach rechts. Seine Lösung färbt sich mit Jod je nach der Konzentration gelbbraun, rotbraun bis tiefrot. Kupferoxydhydrat wird von Glykogen in alkalischer Lösung in Lösung gehalten, jedoch nicht reduziert."^) (ianz entsprechend, wie die übrigen Polysaccharide, zerfällt auch (ilykogen beim Kochen mit verdünnten Mineralsäuren in seine einfachsten Bausteine, und zwar entsteht ausschließlich T r a u b e n z u c k e r. Glykogen wird auch durch diastasische Fermente abgebaut. Von Abbauprodukten sind Dextrine und Maltose mit Sicherheit nachgewiesen. Im übrigen liegen die Verhältnisse genau so, wie bei der Stärke, indem wir vorläufig auch hier bei den höher molekularen Verbindungen (Dextrinen) keine Gewähr für deren Einheitlichkeit haben, ebensowenig, wie wir wissen, ob das Glykogen selbst ein einheitHches chemisches Individuum darstellt. Interessant ist, daß beim stufenweisen Abbau des Glykogens entsprechende Produkte isoliert werden konnten, wie bei der Zerlegung der Stärke, indem auch hier Hexa- und Tetraamylose zur Beobachtung kamen. ^) Unentschieden ist noch die Frage, ob das in den Geweben abgelagerte Glykogen im freien Zustande vorhanden ist, oder ob es nicht vielmehr wenigstens zum Teil gebunden vor- kommt. Das Glykogen ist im gesamten Tierreich sehr verbreitet und findet sich in ist die den verschiedenartigsten Geweben. ^) Eine Hauptablagerungsstätte es in der Zellsubstanz eingelagert. Der Kern Leber. In dieser ist M August Keknlr: Pharmaz. Zeutrall)!. 300 (1H58). Z. Gatin-Gruzewska: Pfiüger^ Archiv. 103. 282 (1<)04). 2) Zeitschr. f. physiol. Chemie. 46. 293 (1905). ^) Z. Gatin-Gruzeuska: F/lüf/ers Archiv. G.d/Errico: Pflügers Archix. 115.359(1906).— 103. 287 (1904). - K. r. Knaßl-Lenz: — Fil. Bofiazzi iiud Leonhard Wacker: Zeitschr. f. physiol. Chemie. 71. 143 (1911). ) Vgl. hierzu auch F. Botazzi : Atti R. Acc. dei Lincei Roma. [5. \| 18. II. S7 (1909). ^) Bezüglich ") der außerordeutlich wichtigen quantitativen Bestimmung des (ily- (Zitat 4, S. 62), 61 u. 67 u. tt'. Uans Pringsheim und S/ephanie Lichtentitcin : Berichte der kogeijs vgl. Pflüger: 1. c. Deutschen Hieni. Gesellsch. 49. 364 (1916). ') Vgl. bezüglich des mikmcliemischen Nachweises des Glykogens Dietrich Barj'urth: Archiv für mikroskopische Anatomie. 25. 259 (1885) und Edgar Gierke : Das Glvkogen in der Morphologie dos Zellstoftwechsels. Habilit.-Schrift. (i. Fischer. Jena 1905. Vorlesung. (34 III- bleibt stets frei vou Glykogen. Der Glykogengehalt der Leber ist abhängig vom Ernährungszustand des Tieres. Die Leber enthält dieses Polysaccharid wenn auch in geringen Mengen. den der Leber der Wirbeltiere nach Funktion und Bau ent.sprechendeu (Jrganen vieler "Wirbellosen aufgefunden worden, so bei Krebsen, Mollusken usw. bereits in frühen Entwicklungsstadien'), Es ist auch in Eingehende Studien sind namentlich über die Verteilung des Glykogens in der Leber von Gastropoden gemacht worden. Es zeigte sich, daß der Glykogengehalt dieses Organs ganz von denselben Bedingungen adhängig ist, wie bei den Wirbeltieren. Bei Limax und Helix-) konnte durch Hunger nach 20 21 Tagen das gesamte (Glykogen zum Verschwinden gebracht werden. Nach der Fütterung trat im Verlauf von 9 lOStunben wieder Glykogen auf. Es wird zunächst in der Bindesubstanz abgelagert und dann erst in dem Epithel der Leber. Beim Hungern verschwindet letzteres zuerst. Bei den Gastropoden ist die Leber der Hauptstapelplatz des Glykogens. Alle übrigen Organe kommen als Ablagerungsstätten kaum — — in Betracht. Auch bei anderen niederen Organismen außer den Mollusken und Gastropoden ist Glykogen recht verbreitet. So wies schon Chuale Bemard Glykogen in Fliegenlarven, den Raupen mancher Insekten, in Regenwürmern, Bandwürmern usw. nach. Andere Autoren fanden es bei Echiuodermen, Holothurien, Polypen, Schwämmen usw. Auch bei Protozoen (Vorticellen, Opalinen, Chilodon, Amöben, Rhizopoden). ferner bei Pilzen ^j ist Glykogen nachgewiesen worden. Eingehend untersucht ist von Clautnan^) und Arthur Harden und WiU'mtH John Young"-') der Gehalt der Hefezellen an Glykogen. Der Nachweis des Glykogens ist nicht in allen Fällen einwandfrei geführt und vor allem ist noch ganz unentschieden, ob nicht trotz großer Ähnlichkeit zum Teil von Glykogen ganz verschiedene Substanzen vorlagen. Jedenfalls gehören alle diese Stoffe nach ihrer biologischen Bedeutung zur Gruppe der Reservekohlehydrate. ") Bei den Wirbeltieren kommt auch den Muskeln eine bedeutende Die Verteilung des Glykogens in den einverschiedenen Organen ^) ist eine sehr ver- Rolle als Glykogenspeicher zu. zelnen Muskeln schiedene, ^l auch und den Glykogen findet sich nicht nur in den quergestreiften, sondern den glatten Muskeln und ist auch in den Muskel fibrillen ent- in — J) E. Piiiiger: Pflügers Archiv. 95. 19 (1901). Ebenda. 102. 305 (1904). Vgl. z. B. //. Erhard: Verhaudl. der Deutscheu Zool. Gesellsch. 22. Jahresver. H. Erhard und F. Zieglwallner ^ Zeitschr. f. Biol. 58. ö41 Sammlung in Halle 1912. ') — (1912). ^) Errera: Das Epiplasma der Ascomvceten und das Glykogen der Pflanzen, Brüssel 1882 und Compt. rend. de l'Acad. des" Sciences. 101. 253 (1885). ) Clautrian: Mem. couronn. Acad. Roy. Belg. 53 (1895). ^) Arthur Harden und William John Younq: Transactions of the Chemical Society. 81 (1902). ) Bezüglich des Vorkommens von Glykogen unter pathologischen Verhältnissen, namentlich in Neubildungen vgl. 0. Lubarsch: Glykogendegeneration in 0. Lubarsch und R. Ostertag: Ergebnisse. 1. Jahrg. 2. 166 (1895). ') Bernhard Schöndorff: Pflüger?, Archiv. 90. 191 (1903). ) August Cramer: Zeitschr. f. Biologie. 24. 78 (1888). Kohlehydrate. 65 Der Gehalt der Muskeln an Glykogen ist abhängig vom allgemeinen Ernährungszustand. Wir werden bald sehen, daß dem Glykogen der Muskeln eine ganz besondere Bedeutung zukommt, und daß es in Beziehung zu ihrer Arbeitsleistung steht. Auch dem Muskelapparate der Wirbellosen fehlt das Glykogen nicht und hat hier dieselbe Bedeutung, wie bei den halten. \\irbeltieren. ist ferner in der Pankreasdrüse, in den kleineren Verdauungsapparates, den Lungen, den Nieren, den Geschlechtsdrüsen, im Nervensystem i), den Epithelien, der Bindesubstanz, im Knorpel 2). den Blut- 3) und Lymphgefäßen Glykogen Drüsen des nachgewiesen worden. Außer den bis jetzt aufgeführten Kohlehydraten sind verschiedene zu den Polysacchariden gehörende Verbindungen beschrieben worden, die teils im Blute, in der Milch und namentlich im Harn beobachtet worden sind. Sie führen zum Teil, wie wir schon S. 56 erwähnt haben, den Namen tierisches Gummi), zum Teil werden sie als dextrinartige Substanzen ö) usw. bezeichnet. Letztere sind namentlich im Harn von Diabeteskranken in größerer Menge aufgefunden worden. Es sollen jedoch auch in jedem normalen Harn solche Produkte vorhanden sein, wenigstens werden die beim Kochen von Harn mit Mineralsäuren sich bildenden Huminsubstanzen. allerdings ohne sichere Grundlagen, als Beweis für die Anwesenheit von Kohlehydraten aufgefaßt. ^) Sicheres wissen wir, wie schon betont, einstweilen über diese Produkte nicht und ebenso unbekannt ist vorläufig ihre biologische Bedeutung. Am nächsten liegend ist namentlich bei den im Harn auftretenden komplizierter gebauten Kohlehydraten der Gedanke, daß wir es mit Produkten zu tun haben, die dem vollständigen Abbau in den Geweben entgangen sind. Im Anschluß an die besprochenen Polysaccharide sei noch auf diejenigen Verbindungen hingewiesen, an deren Aufbau stickstoffhaltige Kohlehydrate beteiligt sind. Ein Dipentosamin, d. h. eine — — Biose eines Pentosamins eine Verbindung, die entsprechend aufzufassen ist, wie das Glukosamin ist z. B. aus Pferdeleber isoliert worden. ^) In diesem Zusammenhange sei auch auf die S. 42 ff., 173 erwähnten stickstoffhaltigen, den Kohlehydraten nahestehenden Verbindungen hingewiesen. Über ihre biologische Bedeutung wissen wir zur Zeit leider so gut wie garnichts. ») H. Erhard: Biolog. Zentralbl. 31. 472 (1911) und Archiv 442 (1912). f. , Zellforschung. 8. Pietro Guizzetto: Zeutralbl. f. Pathol. 21. Juni (1910). Eine viel umstrittene Fiage ist. ob das Blutplasma selbst Glykogen enthält oder aber, ob der Gehalt des Blutes an Glykogen nur auf denjenigen der weißen Blutkörperchen zurückzuführen ist. Es scheint, daß ab und zu Glykogen im Plasma sich vorfindet, gewöhnlich dürfte jedoch sein Vorkommen auf die Leukozyten beschränkt sein. ) IL A. Landwehr: Zentralbl. f. d. med. Wisseusch. Nr. 21. 369 (1885). Vgl. auch K. Baisch: Zeitschr. f. phvsiol. Chemie. 18. 193 (1894) und 19. 339 i:i895). «) ') — — Ebenda. 20. 249 (1895). ') Vgl. z. B. K. r. Alffhan: Helsingfors. üsakevhtiö Weilin und Göös Aktie- iiolag (1904). ") Vgl. die Beobachtung von Emil Abderhalden und physiol. Chemie. 46. 19 (1905j. ') Th. R. Offner: Hofmeistern Beiträge. 8. 399 (1906). Abderhalden, I. Teil, Physiologische Chemie. 5. Aufl. Fritz Prcgl: Zeitschr. 5 f. m. Vorlesung. 66 Wir haben bis jetzt ausschließlich Verbindungen betrachtet, an deren Aufbau nur Kohlehydrate teilnehmen. Wir würden ein sehr unvollständiges Bild der Bedeutung dieser Körperklasse entwerten, wenn wir nicht noch hervorheben würden, daß die Saccharide in mannigfaltiger Art am Aufbau aller möglichen Substanzen sich beteiligen, die auch aus anderen Bausteinen als aus Kohlehydraten bestehen. Man hat alle derartigen Verbindungen Glukoside genannt. Man kann diesen Begriff auch auf die Polysaccharide ausdehnen und diese dann als solche Glukoside auffassen, die nur Kohlehydrate enthalten. Es ist jedoch zweckmäßiger, den Namen Glukoside ganz auf jene Verbindungen zu übertragen, die neben Kohlehydraten auch andere Arten von Bausteinen besitzen. Die Zahl der Glukoside ist eine ganz gewaltige.^) Sie sind vor allen Dingen im Pflanzenreich weit verbreitet. Man kann die große Klasse der Glukoside einmal ganz allgemein nach ihrer empirischen Zusammensetzung einteilen und die Anwesenheit oder das Fehlen von Stickstoff als Abgrenzung benutzen. Man kann ferner die Art der vorhandenen Kohlehydrate der Einteilung zugrunde legen und z. B. stickstoffhaltige Glukoside, die bei der Hydrolyse Glukose liefern, für sich betrachten. Alle diese Einteilungs- formen haben deshalb etwas Gezwungenes an sich, weil in den meisten Fällen nicht die Zuckerkomponenten, sondern die anderen Bausteine der Glukoside unser besonderes Interesse fesseln und durch die erwähnte Gruppierung Verbindungen getrennt werden, die wiegen sonst gleichen oder nahe verwandten, nicht zuckerartigen Bausteinen zusammengehören und auch nach ihren biologischen Eigenschaften enge Beziehungen zueinander haben. Emil Fischer 2) hat die Glukoside auf Grund der Beobachtung, daß die verschiedenartigen Vertreter dieser Körperklasse sich gegenüber Fer- menten ganz charakteristisch verhalten, in zwei große Klassen eingeteilt. 3) Es gibt Glukoside, die von Bierhefe und den aus dieser darstellbaren, ferm enthaltigen Auszügen gespalten werden. Diese Glukoside hat Emil Fischer a-Glukoside genannt. Andere Glukoside widerstehen den Fermenten der Bierhefe vollständig, sie lassen sich aber durch Fermente, Emulsin genannt, die in süßen und bitteren Mandeln enthalten sind, zerDie Glukoside, die dieser Pteihe angehören, sind als ^-Glukoside worden.) Die Glukoside, die in der Pflanzenwelt vorkommen, gehören fast ausnahmslos der letzteren Reihe an. Boiirqiiolot hat auf Grund dieser Beobachtungen eine biochemische Methode zum Nachweis bestimmter Glukoside im Pflanzenreich ausgearbeitet. legen. bezeichnet •'>) Die beiden genannten Pieihen von Glukosiden werden durch die folgenden einfachsten Glukoside vertreten: 'J Vgl. u. a. ./. ./. ( . ran Rijn: Die Glukoside. Gebr. Bornträger. Berlin 1900. Emil Fischer: Berichte der Deutschen Chcm. Gesellsch. 27. 29G5 (1865); 28. 1145 (1895); Zeitschr. f. physiol. Chemie. 26. 60 (1898). ') Außerdem hat Emil Fischer [Berichte der Deutscheu Chcm. Gesellsch. 47. 1980 Vgl. auch J. ('. Iriine, Ahr. W. Fijfe, (1914)] ein y-Methylglukosid aufgefunden. Th. Ferciral: Journ. Cham. Soc. 107. 524 (1915). ) Ganz eindeutig ist übrigens die erwähnte Trennung mittels Hefe und Emulsin nicht, wie wir iu Band II, Vorlesung XV'III noch erfahren werden. ^) Em. Bourquelot : Arch. de l'harmacie. 245. Vgl. weitere Literatur 172 (1907). im Kapitel Synthesen durch Fermente. -) — — : Kohlehydrate, 67 CH, . (J . C . H H— C— OH HO-C—H H— H— C-OH H— H— C— OH CH2 OH a-Methyl-d-glukosid. CH, OH ß-Methyl-d-glukosid. Diese Glukoside, die durch Erhitzen von Glukose mit methylalkoliefern bei der Spaltung nicht Salzsäure gewonnen werden Wird a-Methylglukosid hydrolysiert dann erhält die gleiche Glukose.^) man eine Glukose, die sehr stark dreht. Man hat sie als a- Glukose bezeichnet. 3) Aus dem ß-Glukosid erhält man eine weniger stark drehende, ß-Glukose genannte Form. Die beiden Glukosearten sind durch die folgenden, die nahen Beziehungen zu den a- und fi-Glukosiden ohne weiteres zum Ausdruck bringenden, die Butylenoxydbindung enthaltenden Formeln charakterisiert worden): holischer ' ) , , OH— C-H H-C— oH HO— C— CH.2OH a-d-Glukose. ') GH., OH ä-d-Glukose. Emil Fischer: Berichte der Deutschen Chem. Gesellsch. 26. 2400 (1893); 28. 1151 (1895). Armstrong: Jourü. Chem. Soc. 83. 1305 (1904). Über eine weitere Glukoseform vgl. Emil Fischer : Berichte der Deutscheu Ä. \V. Fijfe und James Colquhoun Irvine Chem. Gesellsch. 47. 1980 (1914). 7A. F. Hogg: Journ. Cbem. Soc. 107. 524 (1915). ) Äußer der a- und ji- d- Glukose ist noch eine andere, mit 7 bezeichnete Form zur Beobachtung gelangt, für die eine Äthylenoxydbindung im Molekül angenommen wird: -) ') — , HO-C-H H-C-OH l>0 H— C/ I HO— C-H HO-C—H und I H-C— OH H-C— OH I I I 1 CH., I H-C— OH H_C-OH OH CHjOH Vorlesung, III. 68 An Stelle der Methylgruppe kann man synthetisch alle möglichen Verbindungen mit Glukose verknüpfen und z. B. phenolartige Produkte darstellen. Selbstverständlich kann man auch andere Zuckerarten wählen. Man kann die Art des beteiligten Zuckers im Namen des Glukosids zum Ausdruck bringen, indem man z. B. von Pentosiden, Hexosiden usw. und noch spezieller von Arabinosiden, Xylosiden, Galaktosiden usw. spricht. Der Name Glukosid ist nicht ganz glücklich gewählt, weil er an und für sich auf die Glukose hinweist. Man könnte von dem allgemeinen Namen der Bausteine der Kohlehydratreihe Saccharid ausgehen und von Saccharosiden sprechen, allein dann kommt man wieder mit der Saccharose, dem Eohrzucker, in Konflikt.^) Zu den Glukosiden des Pflanzenreiches gehört eine große Reihe von Verbindungen, die ganz besondere Wirkungen auf den tierischen Organismus Es seien entfalten. Sie sind zum Teil wichtige Arzneimittel geworden. In den Blättern von einige der bekanntesten Glukoside hier angeführt. Arctostaphylos uva ursi und auch in anderen Pflanzen ist das Arbutin aufgefunden worden. Es hat die folgende Konstitution: O.CeHiiOs j HC CH 1 I HC CH Xc/^ I ÖH Bei der Hydrolyse erhält man d- Glukose und Hydrochinon. Neben dem Arbutin kommt meist auch ein methyliertes Arbutin vor. Von besonderem Interesse ist das Glukosid Phlorhizin. Es findet sich in der Rinde des Apfel-, Bim-, Kirsch- und Pflaumenbaumes. Besonders reich an Phlorhizin ist die Wurzelrinde. 2) Bei der Spaltung mit Säure liefert dieses Glukosid unter Wasseraufnahme d-Glukose und Phloretin.^) Dieses letzläßt sich weiter in Phlorogluzin und Phloretinsäure = p-Oxyphenyl-propionsäure zerlegen. Unter geeigneten Bedingungen erhält man aus dem Phlorhizin nach Cremer und Seuffert^) unter Abspaltung von Phloretinsäure Phlorogluzinglukosid = Phlorin. Die Synthese dieses tere Leider ist die Nomenklatur und Auffassung dieser Formen, in denen die Glukose in Lösung auftritt, wobei sich zwischen den verschiedenen Formen Gleichgewichte ausbilden, noch nicht einheitlich. ) In vieler Beziehung nicht unzweckmäßig wäre die Bezeichnung der gesamten Kohlehydrate als Karbohydrosen. Die Bezeichnung Karbohydroside würde dann die „Glukoside" umfassen. ) de Koninck: Anualen d. Chemie. 15. 75. 258 (1835). ») J. Liebig: Annalen d. Chemie. 30. 217 (1839). Strecker: Ebenda. 74. 184 (1850). Synthese: Emil Fischer und Ostnan Nouri: Berichte d. Deutschen Chem. Gcsellscb. 50. 611 (1917). ) Joseph Schulter: Zeitschr. f. Biol. 56. 274 (1911). M. Cremer: Münchener med. Wochenschr. Nr. 32 (1911). M. Cremer und R. W. Seuffert: Berichte der Deutschen Chem. Gesellsch. 45. 2565 (1912). — — — — 69 Kohlehydrate, Glukosides ist von Emil Fischer und H. Strauss ^) ausgeführt worden. Die folgenden Formeln geben einen Einblick in den Abbau des Phlorhizins: CHo . GH., . C(J . C HO/^\o C CH (OH) CH (OH) . . I H \ OH P-Oxy- ÖH . . C CH (OH) CH^ OH. . I H . . 70 III. Vorlesung. In Gaultheria procumbens ist ein Glukosid, Gaultherin genannt, aufgefunden worden, das bei der Hydrolyse Glukose und Salizylsäuremethylester liefert. Unbekannt in ihrer Konstitution sind die Glukside Adonin und Adonitin. Beide finden sich in der Pflanze Adonis. Sie wirken beide auf das Herz. Ein stark wirkendes Herzgift ist auch das Cheiranthin, das in den Blättern und Samen des Goldlacks sich findet. Von hervorragender Bedeutung sind die verschiedenen Digitalisglukoside i) aus DigitaUs. Sie wirken alle mehr oder weniger ausgesprochen auf das Herz. Auch Strophantin aus den Samen von Strophantus Combe ist ein ausgesprochenes Herzmittel. Am Aufbau dieses letzteren Glukosids sind Mannose und Rhamnose beteihgt. Ein interessantes stickstoffhaltiges Glukosid Es hat ist das Amygdalin.^) die folgende Konfiguration s): Glukoserest Glukoserest D OH H OH H H OH H H HC-C — C — C — C — CH, — — C — C — C — C — C~CH..OH. H OH H H OH H OH I NC.CH.aH, O Blau- Benzaldesäure- hydrest rest Bei der Einwirkung von Emulsin zerfällt Amygdalin dehyd, Blausäure und zwei Moleküle d-Glukose): • in Benzal- = 2 Ce H,.. 0, -\-G,R,. CHO + HCN. C,o Ha, NOu + 2 Hg Durch Fermente der Hefe erfolgt die Spaltung in anderer Art. Es entstehen d-Glukose und Mandelnitrilglukosid^), d.h. es wird nur ein Glukoserest entfernt. ") Man kann das Amygdalin als ein Glukosid eines Disaccharids auffassen. Die Natur des letzteren ist noch nicht ganz aufgeklärt. Maltose, die nach ihrem Verhalten gegenüber Emulsin und Hefefermenten und insbesondere gegenüber der sogenannten Maltase als ein Vgl hierzu: 0. Schmiedeberg : Archiv f. exper. Path. u. Phanuak. 3. 1(5 (1874). Kiliani: Archiv f. Pharmacie. 3. 284 (1874). 230. 250 (1892); 233. 319 (1895); 234. 273 (1896). Berichte der Deutschen Chem. Gesellsch. 31. 24.54 (1898): 34. 3591 (1901); 40. 2996 (1907): 49. 701 (1916); 52. 200 ') — 0. Schmiedeher q und Koppe: Ebenda. — — (1918). ) Entdeckt von liobiquet und Boufron-Chalard : Ann. de Chini. et de Phys. 44 352 (1829). ") Auld: .Tourn Chem. Soc. 93. 1276 (1908). ) J. Liehig und F. Wähler: Ann. de Chim. et de Physique. 64. 185 (1837). Vgl. auch ebenda. 22. 1 (1837). ^) Vgl. seine Synthese: Emil Fischer und Max Bergmann: Berichte der Deutschen Chem. Gesellsch. 50. 1047 (1917). «) E. Fischer: Berichte der Deutscheu Chem. Gesellsch. 27. 2989 (1894); 28. 1508 (1895). [21. — Kohlehydrate. a-Glukosid aufzufassen mehr um eine Kombination ist, 7 liegt sicher nicht von a- und vor. fi-Olukose. Es handelt sich vielDas aus Amygdalin darstellbare Mandelsäurenitrilglukosid ist als das l-Mandelsäurenitril-liglukosid erkannt worden, i) Interessanter Weise kommt in den BKättern von Sambucus niger ein d-Mandelsäurenitril-ß-glukosid, Sambu- nigrin genannt, vor.-) Endlich ist das in frischen Blättern von Prunus aufgefundene Prunolaurasin als Gemisch der beiden erwähnten Glukoside festgestellt worden. Schließlich sei noch erwähnt, daß das Alizarin, der bekannte Krappfarbstoff, in der Mutterpflanze Rubia tinctoria, auch als Glukosid (Rub- laurocerasus =') ervthrinsäure) vorkommt. Auch der Indigo findet sich im Pflanzenreich nicht als solcher, sondern in Form des Glukosids Indikan. Bei der Hydrolyse erhält man aus diesem Glukose und Indoxyl. Dieses letztere ist farblos. Erst das bei der Oxydation sich bildende Indigotin zeigt die schön blaue Farbe: Ci,H,,N06 + H,0 = CjU^ + iVllj^ Glukose Indoxyl Indikan ^CgH.NO + ()., = CisHioN-^O -, -f 2H.,(). Indigotin. Eine außerordentlich interessante Gruppe von Glukosiden stellen auch die Anthocyane dar.) Sie sind als Farbstoffe der Blüten und Früchte weit verbreitet. So zerfällt das Anthocyan der Kornblume, das Cyanin, bei der Hydrolyse in zwei Moleküle Glukose und ein Molekül Cyanidin (eigentliche Farbstoffkomponente). Das gleiche Diglukosid des Cyanidins findet sich in dem Anthocyan von Rosa gallica. In den findet sich der Farbstoff Idäin. Er enthält ein Molekül Cyanidin uud ein Molekül Galaktose. Der Farbstoff der Weintraube, das Önin, liefert bei der Spaltung je ein Molekül Glukose und Ö nid in. Das Anthocyan der Heidelbeere, Myrtillin genannt, besteht aus Glukose und Myrtillidin. Die Stockrose enthält die gleiche Farbstoffkomponente Myrtillidin plus Glukose. Bei der Spaltung des Anthocyans des Rittersporns, Delphinin genannt, entstehen zwei Moleküle Glukose, zwei Moleküle p-Oxybenzoäsäure und ein Molekül Delphinidin. Aus dem letzteren sind durch Spaltung mit Alkali Phloroglucin und Gallussäure gewonnen worden. Mit den Anthocyanen sind die Flavone, z. B. das Quercetin. das sich in der Eichenrinde, in den Blüten der Kastanie usw. in Form des Glukosids Quercitrin findet, nahe verwandt. Preißelbeeren ') Vgl. R. J. Caldwell und S. L. Coiirfauld: .1. of ehem. Soc. 91. 666. 671 (1907); Bourquelot und Herissey : J. de Pharmacic et de Chimie. (6.) 26. 5 (1907). ^) Bourquelot und Jle'rissei/: J. de Pharmacie et de ('himie. ((>.) 26. ö (1907). ) Herissey: J. de Pharmacic et de Chimie. (6.) 23. 5 (190(5). ) Vgl. die Arbeiten von Richard Willsfätfer: Sitzuntrsbcricht der Preußischen Akad. d. Wiss. 402 (1914); Richard Willsfätfer und Heinrich Mallison: Ebenda. 769 (1914); Richard Wilhfätter und ImzIo Zechmeisfer: Kbenda. S86 (1914); Richard Richard WillWillstätter: Berichte d. Deutschen Chem. Gesellsch. 47. 2S31 (1914). sf (Älter, Thomas J. Nolan, Heinrich Mallison, Eimer K. lioUon, Walter Mieg. Ernst H. Zollinger, Karl Martin: Liebiys Annaion. 408. 1 (1914/l."i). — III- 72 Vorlesung. Die Farbstoffkomponenten der Anthocyane sind nach den tiefschürfenden Untersuchungen von Eichard WiUstätfcr als Derivate eines Phonylbenzopyrylium zu betrachten. Diese Beispiele mögen genügen, um zu zeigen, daß die Pflanzenwelt zur Herstellung der mannigfaltigen Farbstoffe als Bausteine Kohlehydrate gemeinsam mit anderen Verbindungen verwendet. Nur erwähnt sei noch, daß die Anthocyane mit Säure rote, mit Alkali blaue Farbstoffe bilden. Endlich kommt noch eine neutrale Form vor, die violett ist. Viele Verschiedenheiten der unser Phenolbetain Auge entzückenden Blütenfarben sind durch das \^orkommen dieser drei — — VerbindungGformen bedingt. Interessant ist. daß auch die (jruppe der Tanuine zu den Glukosiden gehört, und zwar sind die Alkoholgruppen der (ilukose mit Gallus- säure esterartig verbunden. \) Auch die in der Natur so verbreiteten siden zuzurechnen. 2) Saponine sind den Gluko- Die Aufzählung dieser wenigen Ghikoside der Pflanzenwelt mag genügen, um einen Einblik in die Verbreitung dieser Körperklasse in der Pflanzenwelt und ihre Bedeutung zu geben. Wir wollen nur noch hervorheben, daß wir ihre Anwesenheit oft auch am Vorhandensein derjenigen Fermente erkennen können, die imstande sind, bestimmte Glukoside in ihre Bausteine zu zerlegen. .^ueh im tierischen Organismus hat man Glukoside aufgefunden oder mit anderen Worten Verbindungen, die bei der Hydroly.se neben anderen Bausteinen Zucker liefern. Zwei solchen Klassen von Verbindungen sind wir bereits begegnet, nämlich den Nukleinsäuren und Produkten der Gehirnsubstanz, die Galaktose enthalten. Als Baustein vieler Nukleinsäuren ist die d-Ptibose, eine Pentose, festgestellt worden. Außerdem gibt es Nukleinsäuren, an deren Aufbau Hexosen beteiligt sind. Wir kommen auf diese Verbindungen noch zurück.''} An dieser Stelle sei auch der von F. A. Levcne ) bei der Darstellung von Nukleinsäuren aus der Milz beobachteten Säure gedacht, die an und für sich nicht reduziert, wohl aber nach dem Kochen mit Säuren. Levptie nennt sie Gl ukoth ionsäure. Er hält sie für eine Ätherschwefelsäure. Von welcher Art die Kohlehydratkomponente ist, ist bis jetzt nicht festgestellt. John A.Mandel und F. A. Lerenc'') konnten diese Säure auch ') Emil Fischer und Karl Freudenberq : Berichte d. Deutsch, ('hem. GesellschFmil Vgl. auch ebenda. 46. 1116 (1913); 47. 2485 (1914). 915 (1912). Karl Freudenberq Berichte der Fischer und R.Strauß: Ebenda. 45. 3773 (1912). Emil Fischer: Ebenda.' 52. 809 (1919). Deutschen Chem. Gesellsch. 52. 177 (1919). Emil Fischer und M. Bergmann: Ebenda. 52. 809 (1919). ) A. W. van der Haar: Archiv f. Pharmazie. 250. 424 (1912); 251. 217 (1913): E. Winferstein und Ber. d. D. Chem. Ges. 54. 3142, 3148 (1921); 55. 1054 (1922). M.Maxim: Helv. chim. Acta. 2. 195 (1919). ) Vgl. über die Synthese von Glukosiden der Purine: Emil Fischer und BurckEmil Fischer: hardt Ilelferich: Berichte d. Deutsch. Ghem. Gesellsch. 47. 210 (1914). Ebenda. 47. 3193 (1914). Emil Fischer und A'. r. Fodor: Ebenda. 47. 1058 (1914). ) F. A. Levene: Zeitschr. f. physiol. Chemie. 37. 400 (1903). ^) John A. Mandel und F. A. Levene: Zeitschr. f. physiol. Chemie. 45. 386 (1905); ferner Biochem. Zeitschr. 4. 78 (1907). J. A. Mandel und Carl Neuberg: Biochem. W. A. Jacobs: Journ. of experim. Med. 4. Zeitschr. 13. 142 (1908). J'. A. Lerene und 557 (1908). 45. — — — — — : — — — — — Kohlehydrate. 73 aus der Niere, der Leber, dem Pankreas und der Milchdrüse in allerdings recht geringen Mengen gewinnen. Es scheinen übrigens gepaarte Schwefel- säureverbindungen in Verbindung mit Kohlehydraten im Organismus ziemzu sein. Eine sehr interessante Verbindung ist aus Knorpel und Amyloid dargestellt worden. Man hat sie Chondroitinschwefelsäure') genannt. Sie ergibt bei der Hydrolyse Schwefelsäure, Essigsäure, eine dem Glukosamin isomere Verbindung, Chondros am in genannt-) (vgl. auch S. 43), und Glukuronsäure. 3) Hier ist auch die aus dem schleimigen Inhalt des Schweinemagens und anderen Proteinen der gleichen Art gewonnene, gepaarte Schwefelsäure des Mucins zu nennen. Sie ist Mucoit in Schwefel säure genannt worden und liefert bei der Spaltung d-Glukosamin. Nach Abspaltung der Schwefelsäure bleibt Mucoitin übrig. Dieses ist ein Disaccharid, bestehend aus Glukuronsäure lich verbreitet und d-Glukosamin. Es enthält ferner eine Azetylgruppe.) Über die Bedeutung dieser glukosidartigen Verbindung ist zurzeit nichts bekannt. Sehr unsicher sind auch unsere Kenntnisse über das von Dredisel >) beschriebene Je kor in. Es ist zuerst in der Pferdeleber, später auch in der Leber eines Delphins und dann von Bald} •5) in demselben Organ und der Milz anderer Tiere, in den Muskeln und dem Blut des Pferdes und im Menschengehirn aufgefunden worden. Das Jekorin enthält Schwefel und Phosphor und einen Kohlehydratkomplex, der von Mrmasse'') als Glukose angegeben wird. ^) Es ist vorläufig ganz unmöglich, etwas über die Zusammensetzung des Jekorins auszusagen. Höchstwahrscheinlich stellt es überhaupt keine einheitliche Substanz, sondern ein Gemisch ganz verschiedenartiger Produkte dar. Über seine Bedeutung läßt sich nach dieser Sachlage vorläufig gar nichts aussagen, ^j Von weittragender Bedeutung ist ohne Zweifel die Beobachtung, daß die Glukose bei der Gärung mit Hefe sich mit Phosphorsäure verbindet M Carl Th. Mörncr: Zeitschr. f. physiol. Chemie. 20. Mitteilung über die Chondroitinschwefelsäure findet sich in: — Die erste 357 (1895). Skand. Archiv f. Physiol. N. P. KrawVgl. ferner: Zeitschr. f. physiol. Chemie. 23. 311 (1897). 1. 210 (1889). B. Ocldi: Archiv f. exper. kow: Archiv f. exper. Path. u. Pharmak. 40. 195 (1898). 0. Schmiedeberg : Archiv f. exper. Path. u. Pharmak. Path. u. Pharmak. 33. 370 (1894). A. Orgierund ('. Nenberg : Zeitschr. f. physiol. Chemie. 28.355(1891); 87. 47 (1920). Vgl. auch Agnes Kelh/: Beiträge zur ehem. Physiol. u. Pathol. 5. 377 37. 407 (1903). KuraKondo: Biochem. Zeitschr. 26. \\Q> {\%\0). Josef Hebting: Biochem. (1904). Zeitschrift. 63. 353 (1914). «) P. A. Levene und F. B. La Forge: .Journ. of. Biol. Chem. 18. 123 (1914); 20. Vgl. auch P. A. Levene: Journ. F. A. Levene: Ebenda. 31. 609 (1917). 433 (1915). of Biol. Chem. 26. 143. 155 (1916). ^) F. A. Levene und F. B. La Forge: Journ. of Biol. Chem. 15. 69. 155 — — — — — — — — — - (1913). ) F. A. Levene und ./. Löpez-Siidrez: Journ. of Biol. Chem. 36. 105 (1918); 467 (1921). ^) E.Drechsel: Berichte der Sachs. Gesellsch. der Wisseusch. 1886. 44 und Zeitschrift f. Biolog. 33. 85 (1896). «) Baldi: Archiv f. (Anat. u.) Physiol. 1887. Suppl. 100. ') Faul Manasse: Zeitschr. f. physiol. Chemie. 20. 478 (1895). 8) B. Bing: Skand. Archiv f. Physiol. 9. 166 (1900). Vgl. auch .7. Meinertz: Zeitschr. f. physiol. Chemie. 46. 376 (1905) und M. Siegfried und H.Mark: Ebenda. 46. 492 (1905). 45. '') III. V'orlesung. 74- und so ein Glukosid liefert. ') Kohlehydrate. Auch Mannose und Fruktose bilden eine Hexosediphosphorsäure, CoHio04(P04H2)2. Diese Beobachtung hat an Interesse noch dadurch bedeutend gewonnen, als vieles dafür spricht, daß auch in unseren Geweben und insbesondere in den Muskelzellen eine offenbar identische Glukosidbildung stattfindet. Wir kommen^ auf diesen Vorgang noch eingehend zurück. 2) — 1) A. Barden und W. J. Younq: Biochem. Zeitschr. 32. 173 (1911). W.J. Young L.r.Lebedef: Biochem. Zeitschr. 28. 213 (1911); Proceed. Rov. Soc. 81. 528(1909). 36. 248 (1912). Hans Etiler: Zeitschr. f. physiol. Chemie. 74. 15 (1911); 76. 281 (1911). H. Euler und A. Fodor: Biochem. Zeitschr. 36. 401 (1911). K. Langheld: Berichte d. Deutsch. Chem. Gesellsch. 45. 1125 (1912). H. Euler und David Johannsson: Zeitschr. f. phvsiol. Chem. 80. 205 1912. Hans Euler, E. Thorin und D. Johannsson: Ebenda. 79. 375 (1912). C. Neuherg und H. Pollak: Biochem. Zeitschr. 26. 514 (1910). C. Neuberg und E. Kreisch mer : Ebenda. 36. 5 (1911). Carl Neuberg, Eduard Färber, Adam Lerite und Eru^in Schwenk: Ebenda. 83. 244 (1917). C. Neuberg: — — — — — — — — — — Ebenda. 88. 432 (1918). -) Nach Beobachtungen von Carl Neuberg (Biochem. Zeitschr. 103. 320 [1920]) ist die Bildung der Hexosediphosphorsäure nicht Bedingung für den Eintritt der alkoholischen Gärung. Vorlesung IV. Kohlehydrate. III. Bildung der Kohlehydrate im Pflanzenorganismus. Die Rolle der Blattfarbstoffe bei der Synthese von organischer Substanz aus Kohlensäure und Wasser. Die Herkunft der Asymmetrie der Bausteine der Lebewesen. Wir haben die wesentlichsten Tatsachen über den chemischen Aufbau der verschiedenartigen Kohlehydi-ate und ihr Vorkommen im Pflanzenund Tierreich kennen gelernt und wollen nun an Hand dieser Kenntnisse uns der Frage zuwenden, welche Bedeutung die Kohlehydrate für den pflanzlichen und tierischen Organismus besitzen, und welchen Umwandlungen sie unterworfen werden. Wir müssen, bevor wir an diese Aufgabe herantreten, einige Bemerkungen allgemeiner Art vorausschicken. Wir werden im Laufe der Vorlesungen erkennen, daß der tierische Organismus und ebenso der Pflanzenorganismus bestimmter Stoffe bedarf, um leben zu können. Alle Vorgänge im Tier- und Pflanzenreich werden von den folgenden grundlegenden Gesetzen beherrscht. Einmal gilt für die belebte Natur genau gleich, wie für die unbelebte, das Gesetz der Erhaltung des Stoffes. Es kann weder in der Pflanze noch im Tier irgend ein Stoff oder ein Element verloren gehen oder aus nichts entstehen. Diese Tatsache erleichtert die Verfolgung des V^erhaltens bestimmter Verbindungen oder auch einzelner Elemente im Pflanzen- und Tierorganismus ganz außerordentlich. Wir wissen, daß ein aufgenommener Stoff nicht verschwinden kann und ebenso gewiß ist es, daß ein ausgeschiedenes Produkt nicht aus nichts hervorgegangen ist, sondern Beziehungen zu Verbindungen oder Elementen haben muß, die dem Organismus einmal zugeführt worden sind. ^) Die zweite grundlegende Tatsache ist, daß auch für die belebte Natur ganz allgemein das Gesetz von der Erhaltung der Energie volle (Gültigkeit hat. Die Energie kann die mannigialtigsten Formen annehmen, niemalg kann sie jedoch verschwinden oder aus nichts entstehen. Die Summe der Energie, in w^elcher Form sie auch auftreten mag, bleibt sich gleich. Wir werden bald hören, daß auf dieser zuerst von Bubner für den ') An diesen Grundtatsachen wird nichts geändert, auch wenn einwandfrei bewiesen würde, daß ein Element aus einem anderen entstehen kann. Die Summe einfachster Teilchen, die in diesem Falle als Bausteine der Elemente in Betracht kämen, würde stets gleich hleihen. 76 I^ Vorlesung. tierischen Urganismus einwandfrei bewiesenen Grundlage die Lehre des ganzen Energiewechsels aufgebaut ist. Die Pflanzen- und Tierorganismen brauchen beständig Stoffe aller Art. Die einfache Beobachtung zeigt uns das. Eine Pflanze, der Avir nichts zuführen, verdorrt bald und ebenso geht das Tier zugrunde, wenn wir es hungern lassen. Wir bezeichnen jene Stoffe, die zur Erhaltung des Lebens von Pflanze und Tier notwendig sind, als Nahrungsstoffe. Diese haben je nach ihrer Art verschiedene Funktionen im Zelleben zu erfüllen. Jede Zelle besteht aus bestimmten Bestandteilen. Eine Klasse von solchen haben wir bereits mit der Gruppe der Kohlehydrate kennen gelernt. Eine Zelle kann sich vermehren, z. B. durch Teilung, oder sie kann an und für sich zunehmen und z. B. wachsen. Aber auch dann, wenn sie scheinbar unver- ändert bleibt, d. h. wenn sie ..erwachsen" ist, ist ihr Inhalt nicht ein für allemal unveränderlich. Die Zelle ist weder einer Maschine, noch einem Laboratorium mit feststehender Einrichtung, noch einem Gebäude mit mannigfaltigen Abteilungen vergleichbar. Kein einziger dieser oft gebrauchten Vergleiche deckt sich mit dem Wesen der Zelle auch nur entfernt. Sie sind im Gegenteil zu vermeiden, weil sie ganz unrichtige Vorstellungen erwecken. Die einzelne Zelle verbraucht beständig Zellinhaltsstoffe zu ganz bestimmten Zwecken. Die entstehenden Lücken müssen ergänzt werden. Dazu braucht die Zelle Nahrungsstoffe. Fortwährend vollführt die Zelle auch mit Energieumsatz verknüpfte Leistungen, sei es, daß sie sich z. B. bewegt, oder daß sie eine bestimmte Temperatur innehält usw. Nun haben wir bereits festgestellt, daß keine Zellart Energie aus nichts bilden kann. Zu jeder Art von Leistung ist Energie in irgend einer Form notwendig. Diese muß irgend woher genommen ^verden. Wir werden bald sehen, daß die Pflanzen, soweit sie Chlorophyll führen, Sonnenlicht als Energiequelle verwenden können. Die Pflanze kann aber auch chemische Energie in andere Energieformen umwandeln, genau so, wie das Tier seine Energiearten aus solcher gewinnt. Durch Spaltung und vor allen Dingen durch Oxydation von organischen Verbindungen wird Energie frei und der Zelle zu ihren mannigfaltigen Vorgängen zur Verfügung gestellt. Beim Abbau durch Spaltung oder Oxydation werden bestimmte Verbindungen in einfache Produkte zerlegt, die für die Zelle in der schließlich erreichten Form keine Bedeutung mehr haben. Wir sprechen dann von Endprodukten des Stoffwechsels. Die Zelle scheidet derartige Stoffe aus. Es ist klar, daß die Zelle bald unfähig zu weiteren Leistungen wäre, würde nicht neues Material zugeführt. Es muß die Zelle Nahrungsstoffe in sich aufnehmen, um eine Quelle für den Energieverbrauch zu besitzen. Mit der Feststellung, daß jede Zelle der Zufuhr bestimmter Stoffe zur Aufrechterhaltung ihres Baues und zur Vollbringung von mit Energieumsatz verknüpfter Leistungen bedarf, haben wir die Bedeutung der Nahrungsstoffe keineswegs erschöpft. Wir werden noch erfahren, daß sie mannigfaltigen anderen Zwecken dienen, z. B. zur Bereitung von ^kretund Inkretstoffen. Nachdem wir erkannt haben, daß jede Zelle bestimmte Stoffe zur Verfügung haben muß, um ihren Bau aufrecht erhalten und alle ihre verschiedenartigen Leistungen befriedigen zu können, wollen wir uns der wichtigen Frage zuwenden, welcher Art die Nahrungsstoffe sind. Wir unterscheiden anorganische und organische Nahrungsstoffe. Zu den Kohlehydrate. 77 Kohlehydrate, die Fette und die Eiweißstoffe mit ihren Bausteinen. Ferner dürften für manche Tierarten auch die Phosphatide und die Nukleoproteide mit ihren Bausteinen oder letzteren gehören die doch einzelner derselben zu den organischen Nahrungsstoffen zu rechnen sein, während andere tierische Organismen diese zuletzt genannten Gruppen von Verbindungen synthetisch aus den Bausteinen der Kohlehydrate, Fette und namentlich der Eiweißstoffe zu bereiten in der Lage sind. Es kommen noch eine Reihe von organischen Nahrungsstoffen hinzu, über deren Bau sich zur Zeit nichts aussagen läßt. Sie wirken in Spuren und sind Vitamine, Nutramine, akzessorische Nahrungsstoffe usw. genannt worden. Sie nehmen ohne Zweifel keine Sonderstellung für sich ein. Wir wissen z. B., daß manche Bausteine von Eiweißstoffen und manche Mineralstoffe unentbehrlich sind. Auch der Sauerstoff und das Wasser gehören zu den unersetzbaren Nahrungsstoffen. Das Besondere an der erwähnten Gruppe von Nahrungsstoffen ist, daß wir über ihre Natur nicht unterrichtet sind, und daß ganz geringe Mengen davon genügen, um bestimmte Zellfunktionen in normalen Bahnen verlaufen zu lassen. Sobald die Konstitution der erwähnten Stoffe geklärt sein wird, werden wir ihnen im Zusammenhang mit den übrigen organischen Nahrungsstoffen eine Vorlesung widmen. Vorläufig können wir nur Beweise füi' ihre Uneutbehrlichkeit für bestimmte Leistungen der Zellen beibringen.^) Die anorganischen Nahrungsstoffe umfassen Wasser, ferner das Gas Sauerstoff. Die Pflanze Mineral Stoffe und nimmt außerdem als gasförmigen Nahrungsstoff noch Kohlensäure auf. Für manche Mikroorganismen und niederen Pflanzenarten ist ferner freier Stickstoff ein Nahrungsstoff. Die Einteilung der Nahrungsstoffe in die genannten zwei großen (iruppen entspricht den Gepflogenheiten der Chemie. Wir bringen mit der erwähnten Abgrenzung der Nahrungsstoffe die Zugehörigkeit ihrer einzelnen Vertreter zu bestimmten Gruppen von chemischen Verbindungen zum Ausdruck. Die beiden Klassen von Nahrungsstoffen unterscheiden sich vom physiologischen Standpunkt aus betrachtet ganz wesentlich. Die organischen Nahrungsstoffe haben mit den anorganischen das gemeinsam, daß sie am Aufbau der Zellen beteiligt sind, ihnen eigentümlich ist. daß sie bei der Spaltung und vor allem bei der Oxydation Energie liefern. In den organischen Nahrungsstoffen ist chemische Energie aufgespeichert. Die Erfahrung hat gezeigt, daß die Pflanzen beim Aufbau ihrer Zellsubstanzeu von anderen Grundstoffen ausgehen, als die tierischen Organismen. Es gilt dies in erster Linie von der Bereitung der organischen Substanzen. Streng genommen dürfen wir allerdings Tier und Pflanze nicht ohne weiteres einander gegenüber stellen. Wir kennen nämlich Pflanzen, die sich, was die Bildung der organischen Stoffe anlietrifft, genau so verhalten, wie tierische Organismen. Es sind dies jene Pflanzen, die des Blattfarbstoffes und insbesondere des Chlorophylls entbehren. Wir kennen andererseits Tiere, die Chlorophyll besitzen und mittelst (Vortizellen, Flagellaten [Dimystax Perrieri], Planarien, Hydra etc.) dieses, ihnen im (Gründe genommen allerdings fremden Materiales gleiche Stoffwechselvorgänge vollziehen, wie sie den Pflanzen eigen sind. Es handelt sich in diesen Fällen um ein Zusammenleben, eine Symbiose, z. B. mit ') Vgl. Bd. II, Vorlesung .\X1I1. IV. Vorlesung. 78 der interessantesten Beispiele einer Symbiose dieser Art die bekanntlich aus Pilzen und Algen bestehen. Wenn \nr im folgenden von den Leistungen der Pflanzen sprechen, sind immer diejenigen pflanzlichen Organismen gemeint, die Chlorophyll besitzen. Wir wissen schon seit längerer Zeit, daß die Pflanze imstande Eines Algen. bilden die Flechten i), Blattfarbstoffes und des Sonnenlichtes aus und Wasser organische Substanz aufzubauen. Schon frühzeitig erkannte man im Pflanzenorganismus ein Wesen, das ist, mittelst des Kohlensäure der kompliziertesten Synthesen auszuführen imstande nur darauf hingewiesen, daß all die ungezählten, nur zum Teil schon bekannten Verbindungen organischer Natur, die in den verschiedenartigsten Pflanzen sich finden, aus einfachsten Stoffen durch Synthesen zum Teil sicher kompliziertester Natur entstanden sind. Man glaubte zunächst, daß nur Lebewesen und von diesen wiederum nur die Pflanzen Synthesen ausführen könnten, bis es Wähler^) im Jahre 1828 glückte, isozyansaures Ammonium in Harnstoff überzuführen. Bald folgten ungezählte Synthesen. Jeder Tag bringt neue, synthetisch im Laboratorium gewonnene Verbindungen. Der Chemiker schreckt vor keinen Schmerigkeiten zurück und tritt in mancher Beziehung mit der Pflanze in Wettbewerb. Mancher Farbstoff und auch schon einige Alkaloide werden im Laboratorium rationeller gewonnen, als es durch Anbau jener Pflanzen möglich ist, die die gleichen Produkte bilden. Auf diese Weise sind schon die dem Anbau bestimmter Pflanzen wiederholt große Länd.erstrecken dienten, zur Gewinnung von Nahrungsmitteln, wie Kartoffeln, Getreide usw., frei geworden. Es sei z. B. an die Gewinnung des Krappfarbstoffes, des Alizarins (1, 2-Dioxyathrachinon) durch Graebe und Liehermann (1868) erinnert. Dieser schöne Farbstoff wurde früher ausschließlich durch Anbau von Ptubia tinctoria gewonnen. Diese Pflanze bildet ein Glukosid, die Ruberythrinsäure, das bei der Spaltung neben Glukose Alizarin eine große ist. Es Fülle sei , liefert. ^) Der tierische Organismus vermag, soweit unsere Kenntnisse reichen, aus Kohlensäure und Wasser keine organische Substanz zu bilden. Dadurch unterscheidet er sich scharf vom Pflanzenorganismus. Zum Aufbau organischer Substanz braucht er organische Grundstoffe, wenigstens gilt dies für die höher organisierten Tiere. Diese Feststellung führt zu dem Schlüsse, daß der tierische Organismus unmittelbar auf das Pflanzenreich angewiesen ist. Ohne die grundlegende synthetische Arbeit der Pflanzenwelt ist ein Leben tierischer Zellen, von wenigen Ausnahmen abgesehen, undenkbar. Das Tier übernimmt aus der Pflanzenwelt bestimmte Grundstoffe. Der Pflanzenfresser, auch Horbivore genannt, stellt die Beziehungen zur Pflanzenwelt gemeinsam mit dem Omnivoren, dem Allesfresser, direkt her, indem von diesen Tieren Pflanzen mit ihrem Inhalt als Nahrung aufgenommen werden. Der Fleischfresser, der Carnivore, unterhält nur indirekte Beziehungen zum Pflanzenreich. Er verspeist Tiere, die ihrerseits direkt oder indirekt — ) Schwendener: Nägelis Beiträge z. wiss. Bot. H. 2, 3 u. 4. Leipzig 1860—68. Vgl. auch de Barij: Die Erscheinungen der Symbiose. Straßburg. Karl J. Fischer (1879). 0. Her tu: ig : Die Symbiose oder das Genossenschaftsleben im Tierreich. Jena (1883). — ==) Wähler: Poggendarf?, Arinalen. =») Vgl. hierzu S. 71. 3. 177 (1825); 12. 253 (1828). Kohlehydrate. ihre Körpersubstanz aus Materialien 79 aufgebaut haben, die Pflanzen ent- nommen sind. Lange Zeit war man geneigt, dem keit zu Synthesen abzusprechen. tierischen Organismus jede FähigPflanzen- und Tierreich sollten sich in arbeiten. Es sollte die Pflanze aus einorganische Kohlensäure und "Wasser gewissem Sinne in die Hiinde wie fachsten Verbindungen Substanzen aufbauen, die der tierische Organismus dann übernehmen und abbauen sollte. Dieser Abbau führt zum großen Teil zu Verbindungen, von denen die Pflanze bei ihren Synthesen ausgegangen ist. So würde sich ein einfacher Kreislauf der einzelnen Verbindungen und Elemente ergeben. Gleichzeitig umfaßt dieser Kreislauf auch den der Energie. 1 g Zucker aus Kohlensäure und "Wasser zu bilden, sind, worauf wir noch eingehend zurückkommen, rund 4 große Kalorien notwendig. Genau die gleiche Energiemenge wird frei, wenn 1 g Kohlehydrat bis zu Kohlensäure und "Wasser abgebaut wird. In diese Vorstellung, wonach dem tierischen Organismus Synthesen versagt sein sollten, wurde die erste Bresche gelegt, als TJrt ij, Keller und Wühler der Frage nachgingen, ob der tierische Organismus aromatische Verbindungen abzubauen vermag, und was aus ihnen wird. Es wurde zu diesem Zwecke Benzoesäure verfüttert. Im Harn des Versuchstieres (Säugetier) fanden sich nur geringe Mengen freier Benzoesäure, dagegen ließ sich in größerer Menge eine Verbindung, die bei der Spaltung Benzoesäure und GlykokoU lieferte, gewinnen. Es hatte somit eine Synthese statt- — — Um gefunden, und zwar hatten sich unter Abspaltung eines Moleküles "Wasser, Benzoesäure und GlykokoU zu Hippursäure vereinigt. Die gleiche Synthese kann unter bestimmten Bedingungen im Pieagenzglas vollzogen werden. Es war somit zum ersten Male geglückt, im tierischen Organismus eine Synthese nachzuweisen. Dieser Beobachtung folgten bald zahlreiche andere. Jetzt wissen wir, daß die tierische Zelle viele Synthesen ausführen kann, doch finden ihre Fähigkeiten in dieser Richtung insofern eine Grenze, als es ihr versagt ist, bei der Synthese von organischen Substanzen direkt von anorganischen Produkten, wie "Wasser und Kohlensäure, auszugehen. Die Pflanze muß hier Vorarbeit leisten und ein organisches Gerüst bereitstellen, von dem aus die tierische Zelle dann weiter baut. Die fi'üher gezogene scharfe Grenze zwischen Pflanzen- und Tierwelt hat sich noch nach einer anderen Richtung beträchtlich verwischt. Es hat sich nämlich bald herausgestellt, daß die Pflanze nicht nur Synthesen ausführt, sondern genau so, wie das Tier, organische Verbindungen abbaut. Die Pflanzenzelle spaltet genau so, wie die Tierzelle, ja sie oxydiert auch und bildet manche Stoffwechselendprodukte der gleichen Art. wie sie vom tierischen Organismus hervorgebracht werden. Diese kurzen Bemerkungen, kommen, rechtfertigen einzelnen Falle auf die wir noch ausführlich zurück- wenn wir bei der Besprechung der einzelnen Nahrungsstoffe stets auf das Pflanzenreich zurückgreifen und uns die Frage vorlegen, wie die Pflanze diese aufbaut. Wir werden dann in jedem Organismus es. festzustellen einsetzt, versuchen, bei welcher Stufe der tierische um seine Synthesen durchzuführen. — Wilhelm Keller (uud Wühler): ) Ure: Prov. medic. and surg. Journ. (1841). Lje6i>s Annalen. 43. 108 (1842).— F. Wühler \\m\ F. Frerichs: Ebenda. 65. 33.0(18-18). IV. ^'orlesung. gQ Die Bildung von Kohlenwasserstoffverbindungen im Pfianzenorganis- mus aus Kohlensäure und Wasser ist von grundlegender Bedeutung. Diese Synthese beherrscht das ganze Leben von Pflanze und Tier. Wir können von diesen Gesichtspunkten aus vermuten, daß die ersten Lebewesen auf der Erde nicht Tiere, sondern Pflanzen waren, vorausgedaß immer die gleichen Lebensbedingungen maßgebend waren. setzt, Jedenfalls müssen die ersten Pioniere Lebewesen gewesen sein, die imstande waren, aus einfachsten Stoffen, wie Kohlensäure, Wasser, Stickstoff usw., organische Substanzen aufzubauen. Da der tierische Organismus selbst nicht in der Lage ist, bei seinen Synthesen von diesen Baustoffen auszugehen, so Avar er auf das Vorhandensein von Vertretern der Pflanzenwelt angewiesen. Dieses Abhängigkeitsverhältnis Pflanzenreich ist geblieben. des Tierreiches vom Man hat zunächst vermutet, daß die Pflanze außer Kohlensäure noch andere Kohlenstoffverbindungen verwenden kann. Man dachte z. B. an die Aufnahme von Karbonaten durch die Wurzeln oder gar von organischen Kohlenstoffverbindungen. Die Möglichkeit, daß manche dieser Verbindungen von der Pflanze verwertet werden können, ist durch viele Versuche erwiesen worden. Unter natürlichen Bedingungen kommt jedoch wohl nur die Kohlensäure der Luft als Quelle für Kohlenstoff in Betracht. Schon Ingeuhousz'^) (1119) und Theodor de SaHssure^-) (1804) hatten dies klar erkannt. Die Kohlensäure wird durch die Spaltöffnungen der Blätter aufgenommen.^) Sie verschwindet und an ihrer Stelle erscheint ein anderes das die Blätter auf dem gleichen Wege verläßt. Dieses Gas ist SauerDie Pflanze nimmt Kohlensäure auf, verwertet diese und scheidet Sauerstoff aus. Dieser Vorgang vollzieht sich nur bei Gegenwart von (ias, stoff. Chlorophyll und von Sonnenenergie. Man hat sich selbstverständlich mit der Feststellung dieser Grundtatsachen der Kohlensäureassimilation nicht begnügt, sondern den ganzen Vorgang nach den verschiedensten Richtungen aufzuklären versucht. W^ir stehen an der Wiege der gesamten organischen Substanzen der Pflanzen- und damit auch der Tierwelt. Es ist daher nicht verwunderlich, wenn Forscher aus allen möglichen Gebieten der gesamten Naturwissenschaften sich zusammengefunden haben, um den fundamental wichtigen Vorgang der Kohlensäureassimilation durch Chlorophyll enthaltendes Pflanzengewebe möglichst in Vor allem allen Einzelheiten klarzustellen. was aus der Kohlensäure wird. daß Verbindungen entstehen, die außer Kohlenstoff und Sauerstoff noch zum mindesten Wasserstoff enthalten. Es muß somit mit der Kohlensäure noch eine zweite Verbindung in Reaktion treten. interessiert uns die Frage, Man erkannte bald, ) Ingenhousz: Experimeuts iipou Vofietables. Loudou 1879 und Essais oii tlie foods of plants aud the renovation of soils. 1796. ^) Theodor de Saiisstire: Recherches chimiqucs sur la v(^getatiou. Paris 1804. Vgl. auch bezüglich dieser Fragen W. Pfeffer: rfhiiizenbiologie. 1. W. Eugelmanu. Leipzig 1907. Friedrich Czapek: Biochemie der Pllauzen. 1. 409. Gustav Fischer. Jeua 1905. — Eduard O.v. Lipj)mann: Die Chemie der Zuckerartcu. 2. Halbbaud. 1747. Vieweg & Sohn. Braunschweig 1904. J. Sachs: Geschichte der Botanik. 494 ff. A. Hansen: Geschichte der Assimilation. Arb. d. l)ot. lust. zu Wüizburg. 2. (1875). — — — : — 537 (1882). ^) Vgl. über den Einfluß des Kohlensäuregehaltes der Luft auf die Vegetation E. Reinau: Kohlensäure und Pflanzen. Verlag Wilhelm Knapp. Halle a. S. 1920. Kohlehydrate. 81 Es ist dies das Wasser. Als. Produkt der Assimilation von Kohlensäure und Wasser fiel bald die Stärke, also ein Kohlehydrat, auf. Dieses Polysaccharid läßt sich sehr leicht nachweisen, z. B. mittelst der Jodreaktion (vgl. S. 58). Setzen wir z. B. ein chlorophyllhaltiges Blatt, bei dem wir durch Untersuchung eines Stückes davon festgestellt haben, daß es frei von Stärke ist, dem Sonnenlichte aus, so färbt sich das Blatt nunmehr, wenn wir es, am besten nach Entfernung des Blattfarbstoffes, mit Jodlösung zusammenbringen, infolge seines Stärkegehaltes intensiv blau. Hatten wir einen Teil des Blattes vor der Belichtung mit einer für Licht undurchlässigen Masse, z. B. Stanniol, bedeckt, dann bleibt dieser bei der Jodeinwirkung ungefärbt. Diese Beobachtungen hatten bis in die neueste Zeit hinein die ganze Frage nach der Assimilation der Kohlensäure und des W^assers beherrscht. Man fragte ganz allgemein nicht mehr nach der Synthese organischer Verbindungen, sondern nach der Bildung von Kohlehydraten und suchte, die übrigen zahllosen organischen Verbindungen der Pflanzenzellen direkt oder indirekt in Beziehung zu bestimmten Kohlehydraten zu bringen. Es ist sehr fraglich, ob diese Auffassung die richtige ist. Es hat ohne Zweifel mehr Wahrscheinlichkeit für sich, daß zunächst eine oder auch mehrere Verbindungen aus Kohlensäure und Wasser gebildet werden, die zunächst noch keiner bestimmten Klasse von Verbindungen der Zellen angehören, sondern vielmehr ein Baumaterial darstellen, aus dem alle möglichen Verbindungen hervorgehen können. Es wäre somit die erste Aufgabe die, nach solchen Stoffen zu suchen und dann deren Beziehungen zu den Zellbestandteilen des Pflanzenorganismus festzulegen. Bevor wir auf die Resultate dieser Forschungen eingehen, wollen wir uns mit der Frage beschäftigen, welche Rolle das Sonnenlicht und das Chlorophyll bei der Bildung organischer Substanzen aus Kohlensäure und Wasser spielen. Wir müssen zunächst daran erinnern, daß Kohlensäure und Wasser stabile Verbindungen darstellen. Wir können sie nur unter Anwendung von Energie in ihre Bestandteile zerlegen. Beide Verbindungen reagieren nicht von sich aus zusammen. Das vorhandene Gleichgewicht in diesen Verbindungen muß gestört werden. Die dazu notwendige Energie wird nun durch das Sonnenlicht geliefert. Die Lichtenergie nach den neuesten Forschungen sind die Lichtstrahlen als transversale elektromagnetische Schwingungen des Äthers aufzufassen allein genügt auch nicht, um Kohlensäure und Wasser in irgend einer Form zur Reaktion zu bringen, es muß noch ein weiterer Faktor eingreifen. Es ist dies der Blatt färb stoff und insbesondere das Chlorophyll. Streng genommen genügen Kohlensäure, Wasser, Chlorophyll und Lichtenergie auch noch nicht, um eine Bildung von organischen Verbindungen in die W^ege zu leiten und zu ermögUchen, denn alle Versuche, das Pflanzen entzogene Chlorophyll an Stelle von chlorophyllhaltigem Gewebe anzuwenden, veiliefen bis jetzt rcsultatlos. Es müssen ohne Zweifel noch andere Bedingungen, die wir zurzeit noch nicht kennen, für die Kohlensäure- 1) und — — ) Der Vorgang der Kohlensäureassimilatiou ist von dem der Verwertimg des Wassers nicht zu trennen. Gewöhnlich spricht man nur von Kohlensäureassimilatiou und nennt als Resultat die Bildung organischer Substanz. Es ist gewiß richtiger, stets die Assimilation des Wassers mit zu nennen, denn ohne Zufuhr von Wasserstoff ist eine Bildung von Kohlenwasserstoffverbindungen unmöglich. Abderhalden, Physiologische Chemie. I.Teil, 5. Aufl. ß I^ 82 • Vorlesung. Wasserassimilation maßgebend sein. Wichtig ist, daß das Chlorophyll in den Pflanzenzellen in kolloider Form enthalten ist.i) Nach neueren Versuchen sind assimilatorisch wirksam der ganze sichtbare Bezirk des Spektrums und der Hauptteil des Ultravioletts. Nur das Infrarot ist ganz unwirksam. 2) Vergleicht man die Absorption des Lichtes durch grüne Blätter mit seiner assimilatorischen Wirkung, so findet man, da& beide nicht immer parallel verlaufen. Die Assimilation nimmt vom äußersten Rot bis zu den Fraunhofer sehen Linien B C stark zu und sinkt dann langsam zum violetten Ende des Spektrums ab. Die Abnahme erfolgt nicht gleichmäßig. Es finden sich neben dem erwähnten Hauptmaximum der AssimiBis zur Fraunhofersehen Linie E laufen lation mehrere Nebenmaxima. Absorption und Assimilation ziemlich parallel. Hinter E steigt die Absorption an, während die Assimilation fällt. Der Blattfarbstoff ist chemisch und physikalisch eingehend untersucht worden. Wir werden später erfahren, daß er manche Ähnlichkeit in seinem Bau mit dem eisenhaltigen Anteil des Blutfarbstoffes, dem Hämatin, hat, nur finden wir beim Chlorophyll an Stelle des Eisens Magnesium,^ wie Willst äff er bewiesen hat. Es mag vorläufig genügen, wenn wir mitteilen, daß das Chlorophyll eine organische Verbindung darstellt, an deren Aufbau neben Kohlen-, Wasser- und Sauerstoff auch Stickstoff beteiligt ist. Das gewöhnliche grüne" Chlorophyll absorbiert Lichtstrahlen von ganz bestimmter Wellenlänge. Umfassende Studien über die chemische Wirkung von Lichtstrahlen haben zu der Erfahrung geführt, daß nur solche Strahlenarten wirksam sind, die absorbiert werden.^) Es braucht jedoch nicht umgekehrt jede Strahlenart, die zur Absorption kommt, eine Wirkung zu entfalten. Der Blattfarbstoff als solcher besteht aus zwei Anteilen, nämlich dem eigentlichen Chlorophyll und dem Carotin, bzw. dessen Oxydationsprodukt, dem Xanthophyll.) Die erstere Komponente ist grün, die letztere gelb. Durch diese werden blaue und blauviolette Strahlen (Fraunhofersehe Linie F) absorbiert, durch dasChlorophyll rote und gelbe Strahlen {Fraunhofersehe Linien B und C). Die größte Bedeutung für die Kohlensäure- und Wasserassimilation wird den roten und gelben Strahlen zugeschrieben. Jedoch ist nicht ausgeschlossen, daß auch die blauen und violetten Strahlen in besonderen Fällen eine entscheidende Rolle spielen.^») Für die blaugrünen Süßwasseralgen und die roten Meeresalgen liegt die maximale Assimilation in anderen Teilen des Spektrums. Encjelmanfi^} wies nach, daß Lichtstrahlen verschiedener Wellenlängen in jedem Falle ceteris paribus um so stärker assimilierend wirken, je mehr sie von dem : ,. Vgl. hierzu D. Iwanoicski: Ber. der Deutschen Botan. Gesellsch. 31.600(1914.) f. Botanik. 13. 195 (1921). -) Vgl. A. Ursprung: Ber. der Deutscheu Botan. Gesellsch. 35. 44 (1917): 36 73, 86 (1918). ») J. W. Ih. V. Grotthuss (1818) vgl. in IMivald^ Klassikern. :Nr. 152. 101. Draper: Phil. Magaz. (3). 19. Vgl. auch A. Crsprung: Ber. der Deutschen Botan. Gesellsch. 36. 73 (1918). ) Der gelbe Farbstoff soll das Chlorophyll vor der zerstörenden Einwirkung desy Lichtes schützen. Vgl. D. hvanoivski: Ber. der Deutschen Botan. (iesellsch. 31. 613 (1914). ) Vgl. hierzu C. Timiriazeff: Bull, de Congn's iuternat. de botan. et d'horticult. Th. W. Engelmann: ä, St. P^ters'bourg. 1884. Proceed. Roy. Soc. 72. 424. (1903). Botan. Ztg. 42. 81 (1884). '') Th. W. Engelmann: Botan. Ztg. Nr. 1 und 2 (1883). ) — Vgl. auch K. Stern: /. — — - — Kohlehydrate. 83 betreffenden Farbstoff absorbiert werden. Das zur Eigenfarbe komplementäre farbige Licht ist deshalb im allgemeinen das assimilatorisch wirksamste. So ist die bestimmte Farbe der assimilatorisch wirksamen Teile der in verschiedenen Wassertiefen lebenden Pflanzen als Anpassung aufzufassen. Die spektroskopische Analyse des durch verschieden dicke Wasserschichten hindurchgegangenen Lichtes zeigt, daß die roten Strahlen vom Wasser sehr stark, die grünen und blaugrünen dagegen viel weniger absorbiert werden. Mit zunehmender Tiefe werden sich somit blaugrüne und grüne Formen in bezug auf Assimilation mehr und mehr im Nachteil gegenüber solchen befinden, die rote oder gelbe Farbstoffe enthalten. Aus diesen Umständen heraus ist es verständlich, weshalb in größeren Tiefen die roten und gelben Formen im Kampfe ums Dasein den Sieg davontragen. Engehnann^) und Gaidiikow-) haben die an der Flora des Meeres und der Seen beobachtete Anpassung des Chlorophylls an die in bestimmten Tiefen vorkommenden Strahlen bestimmter Wellenlänge auch experimentell nachzuahmen versucht. Die Alge Oscillaria sancta ist bei gewöhnWurde diese Algenart auslichem Tageslicht violett bis braunviolett. schließlich mit Licht bestimmter Wellenlänge bestrahlt, dann nahm sie im \'erlauf von zwei Monaten die der Lichtart komplementäre Farbe an. Kulturen von Oscillaria sancta. die rotem Lichte ausgesetzt waren, wurden grün und umgekehrt rot, wenn grünes Licht auf sie einwirkte. =') Wir haben den Vorgang der Kohlensäureassimilation bereits als eine Reduktion charakterisiert, indem wir angaben, daß dabei Sauerstoff in Freiheit gesetzt wird. Eufielmann -) hat diesen Umstand dazu verwendet, um in außerordentlich geistvoller Weise die Bedeutung der Lichtstrahlen und insbesondere von Strahlen bestimmter Wellenlänge für die Verwertung der Kohlensäure durch einen Chlorophyll enthaltenden Organismus zu beweisen. Engelmann benutzte nämlich Bakterien, die auf Sauerstoff angewiesen sind, als Reagens auf dieses Gas. Er brachte auf einen Objektträger einen Algenfaden und gab bestimmte, aerobe, d. h. auf Sauerstoff angewiesene Bakterien (Bacterium therm o) zu ihrer Nährflüssigkeit hinzu. Nun bedeckte er das Präparat mit einem Deckglas und dichtete es ab. Die Bakterien bewegten sich zunächst lebhaft. Bald stellten sie jedoch, als das Präparat unbelichtet blieb, ihre Bewegung ein. Sobald jedoch Lichtstrahlen Zutritt zu dem Algenfaden hatten, fingen die Bakterien wieder an. sich lebhaft zu bewegen. Die Erklärung dieser Erscheinung ist die folgende. Das Präparat enthielt nach der Anfertigung Sauerstoff. Dieser diente den Bakterien als Nahrungsstoff. Sie verwendeten ihn, um aus organischen, von ihnen aufgenommenen Substraten Energie frei zu machen. Sie brauchten solche zur Bewegung. vollständig luftdicht — ^'ach >) Th. W. EngeJmann: Archiv f. (Anat. u.) Physiol. Suppl. 333 (1902). Versuchen von jV. Gaidukow: Ebenda. 214 (1903). ) X. Gaidukoir: Anhang zu den Abhandlungen der Kgl. Preuß. Akademie der Wissensch. (1902). Berichte der Deutschen Bot. Gesellsch. 21. 484 (1903). ) Vgl. hierzu auch W. Magnus und B. Schindler: Berichte der Deutschen Bot. Gesellsch. 30. 314 (1912). Diese Autoren konnten diesogenanntechromatische AdaptaVgl. auch B. Boresch: Jahrb. f. wissenschaftl. Botanik. 52. tion nicht bestätigen. Alfred Heilbronn: Annales de l'Institut oc^anogr. 5. 1 (1911). 145 (1912). ) Th. W. 'Engehnann: Botan. Ztg. 419(1882); 1 (1883); 80 (1884); 64(1886); rjUigers Archiv. 25. 285 (1881): Verhandl. d. Amsterdamer Akad. 1894. 292 (1887). 26. 537 (1881); 27. 485 (1882); 30. 95 (1883). — — — — — 1^- Vorlesung. g4 Sobald der Sauerstoff aufgebraucht war, stellten Wurde nun der Algenfaden sie dann nahm ihre Bewegung ein. Assimilation von Kohlensäure auf. Dabei wird, wie schon erwähnt, freier Sauerstoff abgeschieden. Dieser diente den Bakterien wieder als Nahrungsstoff, und so konnten sie belichtet, sich wieder bewegen. er die Gleichzeitig lieferten die Bakterien dem Algenfaden Kohlensäure, denn diese bauen organische Substanzen bis zu Kohlensäure und Wasser ab. Wir sehen somit unter dem Mikroskop unmittelbar einen äußerst interessanten Kreislauf sich vollziehen. Man die Bakterien in diesem Versuche als feinstes Reagens auf miniBillionstel Milligramm! von Sauerstoff verwenden. malste Spuren Sehr schöne Resultate ergeben auch, wie Beijerinck'^) gezeigt hat, Leuchtbakterien. Diese brauchen zum Leuchten zur Oxydation Sauerstoff. wenn Sauerstoff fehlt und leuchten Sie stellen das Leuchten sofort ein wieder auf, wenn der Algenfaden im genannten Versuche belichtet wird. Man hat sich mit der Erkenntnis, daß bei der Assimilation der Kohlensäure der Luft und des Wassers Strahlenarten bestimmter Wellenlänge und ferner der Blattfarbstoff eine ausschlaggebende Rolle spielen, selbstverständlich nicht zufrieden gegeben. Wir wollen wissen, in welcher Beziehung der Blattfarbstoff zum ganzen Vorgang steht. Nimmt er aktiv kann — — — — , an der Entstehung der organischen Verbindungen aus Kohlensäure und Wasser teil, oder wirkt er nur als Vermittler? Eingehende Studien von Richard Wülstätter und Arthur Stoll 2) haben ergeben, daß neben dem Chlorophyll noch eine fermentartig wirkende Substanz bei der Assimilation der Kohlensäure beteiligt ist. Schon die Beobachtung, daß Chlorophyllgehalt und Assimilationsgröße stark voneinander abweichen können, zeigt, daß nicht nur der erstere in Betracht kommt. Die erwähnten Forscher stellen sich vor, daß Kohlensäure und Chlorophyll eine Verbindung eingehen, die dann durch das erwähnte Ferment unter Sauerstoffabspaltung zerlegt wird. Der Zutritt der Kohlensäure zu den das Chlorphyll führenden Chloroplasten wird offenbar durch eine diese adsorbierende Substanz vermittelt. Sie kann als Kohlensäureakkumulator aufgefaßt werden. Möglicherweise sind Eiweißstoffe mit freien Aminogruppen die Aufnahmestelle für Kohlensäuremoleküle. Unzweifelhaft sichergestellt ist, daß der abgespaltene Sauerstoff nicht aus Wasser, sondern aus der Kohlensäure stammt. Bestimmungen des assimilatorischen Koeffizienten CO -j-^ ergaben, daß er bei gesteigerter und lang dauernder Wo — 35" konstant und genau gleich 1 Assimilation bei einer Temperatur von 10 ist, d. h. es wird der gesamte Sauerstoff der Kohlensäure bei der Assimilation entbunden. Die von Wülstätter und Stall s) nach dieser Richtung ausgeführten Versuche waren nicht einfach. Es mußte der Einfluß des Sauerstoffverbrauches und der Kohlensäurebildung ausgeschaltet werden. Dazu waren Kontrollversuche in strömendem Gas im Dunkeln notwendig. Der Unterschied im Gehalt der Luft an Kohlensäure vor und nach dem — ) Beijerinck: Botan. Ztg. 744(1890). Vgl. aMch Hans Molisch: Naturvvisseuschaftliche Rundschau. 20. 505 (1905). ^) Richard Willstätter und Arthur Stoll: Berichte der Deutschen Chem. Gesellsch. 48. 1540 (1915). ') Richard Willstätter und Arthur Stoll: Berichte der Deutschen Chem. Gesellsch. 50. 1777 (1917). Kohlehydrate. 85 Strömen über die Blätter im Dunkeln zeigte den Betrag der Atmung an. Derjenige zwischen dem im Dunkeln und bei Belichtung über die verwendeten Blätter geleiteten Gase ergab die assimilatorische Leistung. Der CO,- und Oa-Unterschied zwischen dem Versuchsgas und dem im Dunkeln über die Blätter geleiteten Gas ließ den respiratorischen Quotienten erkennen. Endlich gab die CO2- und Og-Differenz zwischen dem Gase im Dunkelversuch und dem Versuch bei Belichtung den Assimilationsquotienten ohne Einfluß der Kohlensäurebildung und des Sauerstoff Verbrauches. Die erwähnte wichtige Beobachtung über die Größe des assimilatorischen Koeffizienten führte die genannten Forscher 1) zu einem Versuche der Erklärung des ganzen Assimilatiönsvorganges. Zunächst ist bemerkenswert, daß Lösungen von Chlorophyll in organischen Lösungsmitteln nicht mit Kohlensäure reagieren. Dagegen bedingt das Einleiten auch von stark verdünnter Kohlensäure in kolloide, wässerige Chlorophyllösungen eine Spaltung des Chlorophylls in Magnesiumkarbonat und Phäophytin. 3) Das letztere wird ausgeflockt. Die Reaktion des Chlorophylls mit Kohlensäure findet ihr Ende nach seiner vollständigen Zerlegung. Man kann nach den ganzen Ergebnissen das Chlorphyll als die sekundäre Magnesiumverbindung des Phäophytins auffassen. Die beiden Valenzen, mit denen Magnesium an Stickstoffatome gebunden ist, werden bei seiner Abspaltung durch Kohlensäure gelöst. Bevor die Spaltung herbeigeführt wird, tritt eine leicht dissoziierbare Zwischenverbindung auf, die als primäre Magnesiumverbindung des Phäophytins aufzufassen ist. Die folgende Gleichung gibt den Vorgang wieder 2): li :: ; / \cX • ;; + co^ + h, o Mg ^ ' : ^ \c^ ^c/ e/ \c_ ' II j II II II : ug-o-cC^^ ^^cII II II Kohlensäureverbindung des Chlorophyll Chlorophylls. Auf Grund dieser Reagenzglasversuche kann man schließen, daß das auch in der Pflanze mit Kohlensäure eine Verbindung eingeht. 3) Ein Vergleich des Verhaltens der Kohlensäure gegenüber einer kolloiden Chlorophyllösung und dem in Blättern enthaltenen Farbstoff ergab bedeutsame Unterschiede. Im Blatte ist das Chlorophyll gegen die spaltende Wirkung der Kohlensäure viel widerstandsfähiger. Ferner ist Chlorophyll Bichard Willstütter und Arthur Stoll : Berichte der Deutschen Chem. Gesellsch. Vgl. auch: Untersuchungen über die Assimilation der Kohlensäure. auch William Küster: Zeitschr. f. physiol. Vsrl. hierzu .Julius Springer, Berlin 1918. Chemie. 110. 93 (1920). -) Vgl. hierzu die Chemie des Chlorophylls und seiner Abbaustufen in Vorlesung 1) 50. 1791 (1917). XXXII. 3) Vgl. 188 (1920). hierzu auch O. Warburq: Biochem. Zeitschr. 100. 230 (1919); 103. IV. Vorlesung. gg die Geschwindigkeit der Aufnahme dieses Gases durch das im Blatt befindliche Blattgrün viel größer als bei der Adsorption durch eine kolloide Lösung. Nachdem festgestellt war, daß Kohlensäure selbst oder ein Kohlensäurederivat an Chlorophyll gebunden wird, entsteht nunmehr die grundlegende Frage, wie es zur Sauerstoffabspaltung und zum ersten umgewandelten Assimilationsprodukt kommt. Wühtätter und Htoll sind der Ansicht, daß das absorbierte Licht im Chlorophyllmolekül selbst chemische Arbeit leistet. Die mit ihm zugeführte Energie soll eine Verschiebung der Valenzen im Kohlensäuremolekül bewirken und damit eine Umgruppierung der Atome. Es sind folgende Möglichkeiten gegeben: 1. Bildung H C=(J von Formvlhvdroperoxvd oder Perameisensäure: 2. Entstehung von Formaldehydperoxvd: /" \ ()— C^OH. Im ersteren oder Fall I () stellt sich die Abspaltung von Sauerstoff unter Bildung von Formaldehyd. wie folgt dar: \n '>N Kohlensäureverbindung Chlorophyllformaldehydperoxyd. des Chlorophylls Die Abspaltung des gesamten Sauerstoffs könnte auf einmal erfolgen. Manches spricht jedoch dafür, daß zunächst, wie es die folgenden Formeln ausdrücken, ein Atom Sauerstoff abgegeben wird, und daß dann der ganze Vorgang sich wiederholt: /^^ I I \NH H •>Mg + H., c<"^ix ^NH —^ H 11. cf1; + ' .. ( ).,- > Die gegebenen Formeln stehen in vollem Einklang mit allen Beobachtungen der genannten Forscher.^; Die erwähnte Konstanz des assimila) Vgl. hierzu auch die Ausführungen von Frl. Gertrud Woker: Pfliitjera Archiv. 176. H (1919). Kohlehydrate. 87 torischen Koeffizienten zeigt, daß das Reduktionsprodukt der Kohlensäure mit dem Chlorophyll gebunden bleibt, bis die ganze molekulare Sauerstoffmenge abgespalten ist. Erst dann kann ein neues Kohlensäuremolekül eintreten, wenn das zuvor aufgenommene Molekül zur Formaldehydstufe desoxydiert worden ist.\) Im Zusammenhang mit der Feststellung, daß Chlorophyll bestimmte Lichtstrahlen festhält und chemische Wirkungen ausgelöst werden, haben die folgenden Beobachtungen ein erhöhtes Interesse. Schon seit langer Zeit ist bekannt, daß das Licht chemische Wirkungen entfalten kann. Es sei z. B. an die Vorgänge bei der Photographie erinnert, ferner an das Abblassen von belichteten, nicht „lichtechten" Farben (Tapeten, Stoffen etc.). Man hat die diesen direkt verfolgbaren Vorgängen zugrunde liegenden Reaktionen genauer studiert und damit begonnen, bestimmte chemische Verbindungen der Lichtvvirkung im allgemeinen oder auch Strahlen bestimmter Wellenlänge auszusetzen. 2) Es wurde z. B. festgestellt, daß belichtetes Azeton in Methan und Essigsäure zerfällt: CHj.CO.CHg-h r= CH, + CH3 COOK. Auch Synthesen, Oxydationen und Reduk-h H., . tionen sind beobachtet worden. Handelt es sich bei diesen Vorgängen um unmittelbare Wirkungen bestimmter Strahlenarten, so ist in anderen Fällen noch die Anwesenheit eines weiteren Stoffes nötig, um die Reaktion zu vermitteln. Setzen wir z. B. Benzoesäure Lichtstrahlen aus, so können wir mit den uns zur Verfügung stehenden Methoden keine Veränderungen an ihr nachweisen. Geben wir jedoch zu der Lösung etwas Ferrisulfat, dann erhalten wir bei Belichtung Salizylsäure. An Stelle von Eisensalzen kann man auch andere, z. B. solche des Mangans und Urans, verwenden. Xeuher(f) hat gezeigt, daß eine ganze Anzahl biologisch wichtiger \'erbindungen, wie Zuckerarten, Alkohole, Aminosäuren die Bausteine der Eiweißstoffe Fette usw. unter dem Einfluß von Lichtstrahlen bei Anwesenheit eines sogenannten Katalysators eben eines der erwähnten Metallsalze in charakteristischer W^eise verändert und zum großen Teil abgebaut werden.) So wird z. B. Milchsäure in Gegenwart von Uranylsulfat bei Belichtung und Sauerstoffzufuhr, wie folgt, zerlegt^): — — — — CH3.CH(OH).CO()+H+UO: + 2H = CH3.C<2-f CO, + 2HoO-fU •". Wir kennen außer den erwähnten Lichtwirkungen noch andere Arten. Die eine steht ') wahrscheinlich in mancher Beziehung der zuletzt Vgl. über weitere Theorien über die Kohlensäureassimilatiou: bespro- Schröder: Die Hypothesen über die chemischen Vorgänge bei der Kohleusäureassimilation. G. Fischer, Jena 1917. ^) Es sei auf die zahlreichen Versuche von G. Ciamician und P. Silber verwiesen. Vgl. Berichte der Deutschen Chem. Gesellsch. Jg. 33—48 (1900—1915). — E. l'aterno: <iazz. chim. ital. 44. I. 237. II. 99 (1914). R. Ciusa und A. Piergallini: Atti R. Accad. — <lei Lincei [5.] 28. I. 821 (1914). 3) Carl Neuberq: Biochen» Zeftschr. 13. 305 (1908); 17. 270 (1909): 27. 210 <1910); 29. 279 (1910); 39. 158 (1912); 67. 59 (1914). ) Vgl. die Literatur über dieses Gebiet bei A. Jesionek: Lichtbiologie und Lichtpathologie. Praktische p]rgebnisse auf dem Gebiete der Haut- und Geschlechtskrankheiten. Jg. 2. J. F. Bergmann. Wiesbaden. 1912. Carl Neiiber;/: Beziehungen des Lebens zum Licht. Allg. med. Verlagsanstalt. Berlin. 1913. 5) Iwan Bolin: Z. f. physikalische Chemie. 87, 490 (1914). — IV. Vorlesung. 8g ebenen für recht zeigen nahe. Manche Verbindungen, ja ganze Zellen, die an und sichtbares Licht nicht wahrnehmbar beeinflußt werden, von eingreifende Erscheinungen, sobald geringe Mengen durch sieb tief fluoreszierenden Stoffen zugegen sind. Verschiedene Protozoen, manche Tappeiner i) festwie Fermente und sogenannte Toxine werden , unter den erwähnten Bedingungen schwer geschädigt. Man hat von einer Sensibilisierung gesprochen und sich z. B. vorgestellt, daß die fluoreszierenden Stoffe die Fähigkeit besitzen, Strahlenarten von bestimmter Wellenlänge in solche von anderer umzuwandeln. Unwirksame Strahlenarten könnten so zu chemisch wirksamen werden. Ja die Möglichkeit ist durchaus gegeben, daß eine ganz andere Energieform gebildet wird. Man hat die Wirkung der fluoreszierenden Stoffe als photogestellt hat, dynamische bezeichnet. Es ist von größtem Interesse, daß Chlorophyllösungen photodynamisch wirksam sind, wie vor allem Hausmann^) gezeigt hat. Das Chlorophyll ist von diesen Gesichtspunkten aus als Überträger und wahrscheinlich gleichzeitig auch als ein Umwandler von Lichtenergie zu betrachten. An und für sich lichtunempfindliche Stoffe werden auf diesem Wege der Einwirkung der Lichtenergie zugänggemacht. Wir haben schon hervorgehoben, daß als erstes, ohne weiteres erkennbares Produkt der Kohlensäure- und Wasseiassimilation die Stärke erkannt worden ist. Es ist sehr unwahrscheinlich, daß dieses kompliziert gebaute Polysaccharid direkt aus der Reduktion von Kohlensäure und Wasser hervorgeht. Wir müßten denn schon annehmen, daß die Chlorophyll führenden Pflanzenzellen ihre Synthesen in jeder W^eise anders durchführen, als Avir es uns in Analogie mit den Beobachtungen im chemischen Laboratorium vorstellen. Eine solche Möglichkeit darf nicht ohne weiteres von der Hand gewiesen werden. Ihre Besprechung ist jedoch unfruchtbar, weil wir zurzeit keinen einzigen Beweis für eine solche Annahme besitzen. Wenn wir die Synthese eines kompliziert gebauten, zusammengesetzten Stoffes erforschen wollen, dann fragen wir zunächst nach den Komponenlich am Aufbau der betreffenden Verbindung beteiligt sind. Es redudann zunächst das Problem der Synthese der Verbindung selbst auf das der Gewinnung ihrer Bausteine. Bei der Synthese der Stärke ist zuerst die Frage nach dem Aufbau der Bausteine, nämlich der Traubenzuckermoleküle, zu beantworten. Wie entsteht Glukose beim Assimilationsvorgang aus Kohlensäure und Wasser? Es sind vom rein chemischen Standpunkt aus viele Möglichkeiten gegeben. Einmal kann die Glukose direkt gebildet werden, sie kann aber auch auf Umwegen über die verschiedenartigsten Zwischenstufen entstehen. Niemand wird wohl die Glukose als primäres Assimilationsprodukt der Kohlensäure und des Wassers ansehen. Es müssen als erste synthetische Produkte noch einfachere Verbindungen ten, die ziert sich entstehen. ') //.;;. rappeiner und A. Jodlbauer: Die sensibilisierende Wirkung fluoreszierender Substanzen. Leipzig. ¥. C. W. Vogel. 1907. V^gl. auch ('. Amsler und E. F. Pick: A. f. exporim. Path. n. Pharm. 82. 86 (1917). Fort2) Walther Nansinann: Biochem. Zeitschr. 16. 294 (1909); 30. 276 (1910). schritte der Naturwissenschaften. 6. 243 (1912). Vgl. ferner Biochem. Zeitschr. 77. Ergebnisse der Physiologie. XVI. 288 (1917). 268 (1916). — — — — Kohlehydrate. 89 A.v. Baeyer'^) hat die Hypothese aufgestellt, daß als erstes Assimider Formaldehyd in Betracht kommt. CO., + HgO HCOH + Oa- Nach dieser Formel würden Kohlensäure und Wasser unter Freiwerden von Sauerstoff vereinigt. Der gebildete Formaldehyd könnte nun, da er an und für sich sehr reaktionsfähig ist, im Mittelpunkt aller möglichen Synthesen stehen. Wir haben bereits-) hervorgehoben, daß es geglückt ist, im Laboratorium aus Formaldehyd durch Polymerisation eine Hexose zu bereiten: 6 X HCOH^Cg Hj.^Og. Der in der Pflanze gebildete Traubenzucker könnte dann unter Wasseraustritt mit anderen Glukosemolekülen zusammen zu Stärke vereinigt werden. Würden sich nur fünf Formaldehydmoleküle vereinigen, dann kämen wir zu Pentosen. Man könnte auch an die Entstehung einer Triose, z. B. der früher erwähnten Glyzerose'), denken und diese in den Mittelpunkt ungezählter Syn- = lationsprodukt thesen stellen. Während noch vor kurzem zahlreiche andere Möglichkeiten von ersten Assimilationsprodukten in Frage kamen, entheben uns die Seite 86 ff. mitgeteilten Ergebnisse von Wilhtätter und Stoll weiterer Erörterungen. Sie decken sich in allen Einzelheiten mit der Annahme des Formaldehyds als erstem Assimilationsprodukt. ) Mit der Assimilation von Kohlensäure und W^asser unter Vermittlung des Chlorophylls und der Wirkung der Sonnenstrahlen als Energiequelle ist nicht nur die erste Synthese von organischer Substanz im Reiche der Organismen verknüpft, sondern gleichzeitig auch die Asymmetrie der Zellbau- steine und der meisten organischen Verbindungen im Pflanzenund Tierreich überhaupt. Wir haben bereits darauf hingewiesen &), daß die in der Natur vorkommenden Kohlehydrate asymmetrische Kohlenstoffatome besitzen. Auf ihrem Vorkommen beruht die optische Aktivität dieser Verbindungen. Außer den Kohlehydraten sind noch zahlreiche andere Verbindungen durch den Besitz asymmetrischer Kohlenstoffatome ausgezeichnet. Mit ganz verschwindend geringen Ausnahmen findet sich von den optisch-aktiven Formen immer nur eine bestimmte Komponente in der Natur vor. So treffen wir immer d-Glukose an, ferner d-Galaktose usw. Piazemkörper d. h. Verbindungen, die durch das Vorkommen von gleichen Mengen links- und rechtsdrehender Komponenten optisch-inaktiv sind die beiden gleich großen, jedoch entgegengesetzt gerichteten Drehungen heben sich auf kommen in der Natur fast gar nicht vor. Die Pflanze nimmt mit der Kohlensäure eine Kohlenstoffverbindung auf, die kein asymmetrisches Kohlenstoffatom besitzt. Mit der Assimilation, d. h. mit der ersten Synthese setzt die Asymmetrie ein. Die Pflanzenzelle vollführt die erste asymmetrische Synthese. Die einmal erworbene Asymmetrie der organischen Verbindungen überträgt sich mit , — — dem Futter auf den tierischen Organismus. in Man hat viel darüber nachgedacht, wie die asymmetrische Synthese der Organismenwelt bei der Assimilation der Kohlensäure zustande') ') =•) A. i\ Baeyer: Berichte der Deutschen Chem. Gescllsch. ) Vgl. die gesamte einschlägige Julius Springer. Berlin. 1918. 5) 3. 63 (1870). Vgl. S. 18. Vgl. S. 18. S. 19. Literatur liei E. Willstätter und A. Stoll: I.e. I^- Vorlesung. 90 kommt. Es liegen verschiedene Möglichkeiten vor. Es wäre denkbar, daß zuerst razemische Verbindungen entstehen würden, die sekundär zur Spaltung kämen, wobei die eine optisch-aktive Form dem Abbau unterliegen würde, während die andere erhalten bliebe. Diese Annahme hat sehr wenig Wahrscheinlichkeit für sich, weil die direkte Beobachtung gezeigt hat, daß die Zellen in erster Linie jene optisch-aktiven Verbindungen abzubauen und die in der Natur vorkommen, während sie gerade die optischen Antipoden schwer angreifen können, ja oft sogar ganz unverändert lassen. Mehr Wahrscheinlichkeit hat die folgende Annahme, die allerdings das Auftreten der ersten asymmetrisch gebauten Verbindung in der Organismenwelt nicht erklärt, für sich. Sie hat nämlich zur Voraussetzung, daß bereits eine optisch-aktive Verbindung vorhanden ist. Diese könnte bewirken, daß die eine der beiden optischen Antipoden viel rascher gebildet wird, als die andere. umzuwandeln vermögen, Es ist versucht worden, im Reagenzglas asymmetrische Synthesen auszuführen. Dabei sind Beobachtungen gemacht worden, die unmittelbar zeigen, wie eine bereits vorhandene asymmetrische Verbindung für die Entstehung einer neuen optisch-aktiven Form bestimmend sein kann. Bosenthaler^) zeigte zunächst, daß man bei der Einwirkung eines bestimmten Fermentgemisches, Emulsin genannt, auf Blausäure und Benzaldehyd rechtsdrehendes Mandelsäurenitril linksdrehende Mandelsäure erhält. CH (OH) CN . . Benzaldehyd enthält kein und durch Verseifung ('HO Cg Hg. asymmetrisches Kohlenstoffatom, daraus HCN M- Cg H5 . = wohl aber das durch die Synthese gewonnene Mandelsäurenitril. Das asymmetrische Kohlenstoffatom ist in vorstehender Formel mit einem bezeichnet. Das wirksame Prinzip bei dieser Synthese, das Emulsin, ist ohne Zweifel selbst asymmetrisch gebaut und bedingt wahrscheinlich durch direkte Bindung mit den zu vereinigenden Komponenten den nun in einer Richtung verlaufenden Aufbau des Mandelsäurenitrils. Würde man einfach Blausäure und Benzaldehyd ohne Anwesenheit des Emulsins vereinigen, dann käme man zu einem optisch inaktiven Mandelsäurenitril, das man jedoch dadurch als Razemkörper charakterisieren könnte, daß man es sekundär in seine beiden optisch aktiven Anteile zerlegen würde. Derartige Spaltungen sind schon in großer Zahl ausgeführt worden, und man ist auf diesem, vor allem von Emil Fischer beschrittenen Wege zu den in der Natur vorkommenden optisch aktiven Komponenten gelangt. In diesem Falle wird ein Umweg eingeschlagen, indem zunächst der Razemkörper dargestellt wird. Streng genommen ist eine derartige Synthese, wenn sie von Verbindungen ausgeht, die kein asymmetrisches Kohlenstoffatom enthalten, auch eine asymmetrische, denn sie erzeugt dieses. Es kommt optisch nur nicht zur Geltung, weil die beiden optischen Antipoden, die genau gleich stark, jedoch in entgegengesetzter Richtung drehen, in gleichen Mengen zugegen sind. Sie müssen durch besondere Maßnahmen von einander getrennt werden. Wie wir schon betont haben, schlägt die Pflanzenzelle diesen inWeg kaum ein. Sie bildet offenbar primär optisch aktive Substanzen, d. h. sie läßt von den möglichen optisch aktiven Formen fast direkten ') L. Rosent haier: Biochem. Zeitschr. 14. 238 (1908): 26. 1 (1910). Kohlehydrate, 91 ausnahmslos immer nur die eine hervorgehen. Emil Fischer hat den Begriff der asymmetrischen Synthese für die primäre Bildung optisch aktiver Verbindungen vorbehalten.^) Die erwähnte Bildung von optisch aktivem Mandelsäurenitril unter Verwendung von Emulsin ist eine solche asymmetrische Synthese. G. Bredig und P. S. Fiske-) ist es geglückt, eine asymmetrische Synthese durchzuführen, zu der an Stelle des seiner Natur nach unbekannten Emülsins Alkaloide verwendet wurden. Sie wählten als Ausgangsmaterialien auch Blausäure und Benzaldehyd und erhielten, wenn sie das Alkaloid Chinin verwendeten, rechtsdrehendes und bei Verwendung von Chinidin liuksdrehendes Cyanhydrin. Durch Verseif ung entstanden die entsprechenden optisch aktiven Mandelsäuren. Von besonderem Interesse ist die Beobachtung, daß das zugesetzte Alkaloid mit dem Cyanhydrin eine Verbindung eingeht. Es ist sehr wahrscheinlich, daß dieser Vorgang bestimmend für den Verlauf der optisch asymmetrischen Synthese ist. Die interessanten Feststellungen von Posenthaler und von Bredig und Fiske stützen die eben geäußerte Ansicht über die Entstehung der optisch asymmetrischen Synthesen im Pflanzenreich beim Assimilationsvorgang. Die Pflanzenzelle enthält optisch aktive Substanzen genug, welche die gleiche Ptolle spielen können, wie das Emulsin bzw. die Alkaloide Es wäre wohl denkbar, daß den Alkain den erwähnten Versuchen. loiden bei bestimmten asymmetrischen Synthesen eine ausschlaggebende Rolle zufällt. Es ist auch die Ansicht ausgesprochen worden ), daß die optisch asymmetrische Synthese dadurch bewirkt wer4e, daß infolge unregelmäßiger Reflexion des linear polarisierten Anteils des Himmelshchtes an den Wasserflächen des Meeres zirkulär polarisiertes Licht entstehen soll. Die Drehung der Polarisationsebene des Lichtes durch den Erdmagnetismus bewirkt, daß hierbei weder an einem Punkte der Erde, noch auf der ganzen Erdoberfläche gleiche Mengen beider Lichtformen entstehen. Nun ist durch Cofton^) festgestellt worden, daß optisch aktive, gefärbte Flüssigdie Komponenten des zirkulär polarisierten Lichtes verschieden stark absorbieren. Die eine optisch aktive Form bevorzugt rechts zirkulär polarisiertes Licht und die andere das links zirkulär polarisierte. Nun daß nur solches Licht chemische bereits hervorgehoben worden, ist Wirkungen entfalten kann, das absorbiert wird. Würde nun die eine Art des zirkulär polarisierten Lichtes allein vorhanden sein oder doch stark keiten überwiegen, dann könnte man sich vorstellen, daß die Synthese einer be- ) Vgl. über asymmetrische Synthesen. Emil Fischer: Berichte der Deutschen Chem. Gesellsch. 27.^3230 (1894). Zeitschr. f. physiol. Chemie. 26. 87 (1898). W. Marckual<l : Berichte der Ueutschen Chem. Gesellsch. 37. 349 und 1368 (1904). Alexander Mc Kenzie: Journ. Chem. Soc. 85. 378 (1904); 85. 1249 (1904); 87. 1373 (1905); 89. 365 (1906); - Alexander Mc Kenzie und Henni Wren: .lonrn. Chem. Soc. 91. Vgl. auch K. Fajans: Zeitschrift, f. physikal. Chemie. 73. 1215 (.1907); 97. 473 (1910). — — • — — 25 (1910). — Vgl. ferner 2) G. Bredig und /'. S. Fiske: Biochem. Zeitschr. 46. 7 (1912). auch G. Brediq und K. Fajans: Berichte der Deutschen Chem. (iesellsch. 41. 752 (1908). K. H. Daicin: Journ. of Physiol. 30. 253 (1904); 32. 199 (1905). ^) A. Byk: Zeitschr. f. physikal. Chemie. 49. 641 (1904). - Vgl. auch über dieses Problem Emil Erlenmeyer : Biochem. Zeitschr. 52. 439 (1913). ) A. Cotton: Annal. de Chim. et Physique [VII]. 8. 347 (1896). — 92 i^'- Vorlesung. stimmten optisch aktiven Form begünstigt wird. Diese Hypothese würde primäre Auftreten optisch aktiver Formen ohne bereits bestehende optisch asymmetrische Verbindungen erklären. Sie ist jedoch zurzeit experimentell noch zu wenig gestützt. das Die Bildung organischer Substanz aus Kohlensäure unter Einfügung von Wasserstoff unter Sauerstoffabspaltung ist noch von anderen Gesichtspunkten aus von allergrößtem Interesse. Wir werden bald vernehmen, daß der tierische Organismus die aus Kohlensäure und Wasser von der Pflanze aufgebauten Substanzen übernimmt und sie wieder mit Sauerstoff vereinigt. Über viele Abbaustufen herüber gelangen wir wieder zu den Ausgangsmaterialien der ursprünglichen Synthese, nämlich zu Kohlensäure und Wasser. Diese werden vom Tiere ausgeschieden und in gewissem Sinne der Pflanze wieder zur Verfügung gestellt. Die Synthese kann von neuem beginnen. Jedesmal wird Sonnenenergie in Form von chemischer Energie gespeichert. Das Tier übernimmt mit den organischen Verbindungen eine ganz bestimmte Menge Energie, die wieder vollständig frei wird, sobald die organische Verbindung restlos zu Kohlensäure und Wasser abgebaut ist. Es ist somit in letzter Linie Sonnenenergie, mit der unsere Auch die Pflanze kann Zellen wirtschaften! bar machen, wenn spaltet sie die sich diese Energiequelle nutz- von ihr aufgebauten organischen Verbindungen und oxydiert. Wir sehen einen interessanten Kreislauf von Stoff und Energie uns. Er ist kein im ganzen Umfang in kurzen Zeiträumen ab- vor laufender, indem stets große Massen von Kohlen-, Wasser- und Sauerstoff für mehr oder weniger lange Zeit dem Kreislauf entzogen bleiben. Jede Pflanze und jedes Tier hält in seiner Zellsubstanz die genannten Elemente und in den genannten Verbindungen Energie zurück. Erst wenn das einzelne Individuum zugrunde geht, dann bietet sich Gelegenheit, das organische Gerüst des ganzen Zellstaates niederzureißen und auf Umwegen den Kohlenstoff, den Wasser- und Sauerstoff dem Kreislauf zurückzugeben. Die Pflanze vermag die vom Tier gebildeten Substanzen nicht direkt zu übernehmen. Ebensowenig kann ein Individuum der Pflanzenwelt organische Substanzen, die beim Tode eines anderen Individuums zur Verfügung gestellt werden, direkt verwerten. Pflanzen- und Tierreich sind hier durch das gewaltige Heer der Mikroorganismen verknüpft. Diese verhindern, daß große Massen organischer Substanz verloren gehen. Diese kleinsten Lebewesen ermöglichen erst die innigen Beziehungen zwischen Tier- und Pflanzenwelt. Sie zerlegen die Bausteine der toten Zellen und führen sie schließlich zu ihren Grundsubstanzen zurück, unter anderem zu Kohlensäure und Wasser. Die Arbeit der Mikroorganismen reicht nicht aus, um die gewaltige Masse von organischer Substanz, die die Pflanze ununterbrochen aufbaut, schließlich wieder zu zerlegen. Gewaltige Kohlenlager i) erzählen uns von Leichen ungezählter Pflanzenarten. Der Kohlenstoff dieser Kohlenlager entstammt auch der Kohlensäure der Luft. Sie wird dieser wieder zugeführt, wenn die Kohle verbrannt wird. Bis das geschieht, bleiben gewaltige Mengen von Kohlenstoff dem Kreislauf entzogen. Auch ungezählte Tier') Vgl. hierzu Jkinnenhcrf/: Gebr. Bornträger, Berlin. Geologie der Steinkohlenlager. I. 1908; II. l'.ll'2. Kohlehydrate. 93 leichen sind unter bestimmten Bedingungen dem Abbau durch Bakterien zum Teil wenigstens entzogen worden. Davon zeugen die großen Massen von Petroleum, die an bestimmten Stellen der Erde sich angesammelt dann bilden sich Kohlensäure und Wasser. Das Licht, das uns die Flamme des ist Sonnenhcht, das vor Tausenden von Jahren haben.i) Verbrennen wir dieses, wird Energie Petroleums entgegenstrahlt die Erde beschienen hat! Gleichzeitig frei. Große Mengen von Kohlensäure sind der Atmosphäre im Laufe der dem Umwege der Synthese durch die Pflanzenwelt entzogen worden. Es ist dies jene Kohlensäure, die an Basen, wie Kalk und Magnesia, gebunden, am Aufbau gewaltiger Schichten der Erdrinde beteiUgt ist. Wird sie durch eine stärkere Säure, z. B. durch Kieselsäure, aus ihrer Verbindung mit den erwähnten Basen verdrängt, dann kehrt sie in die Atmosphäre zurück. Sie ist damit dem Kreislauf des Kohlenstoffs und Sauerstoffs wieder eingereiht. Zeit direkt, d. h. nicht auf kurze Überblick über die Entstehung organischer Substanz Organismenwelt enthüllt uns interessante Wechselbeziehungen zwischen Pflanzen- und Tierreich. Wir erkennen, daß die Chlorophyll enthaltende Zelle von entscheidender Bedeutung für Sein und Nichtsein aller Organismenarten ist. Die Betrachtung des Kreislaufs der Stoffe und der Energie ergibt ebenfalls interessante Einblicke in das Werden und Vergehen in der Natur. Wir sehen, daß ein bestimmter Stoff, wie z. B. der Kohlenstoff, in ewig wechselndem Reigen bald der nichtorganisierten, bald der organisierten Welt angehört. War der Kohlenstoff eben noch Bestandteil einer organischen Verbindung, ja vielleicht am Aufbau „lebender" Zellsubstanz beteiligt, so ist er im nächsten Augenblick schon wieder in Form der Kohlensäure der unbelebten Natur übergeben, um wieder an anderer Stelle des Kreislaufs am Aufbau von Zellen sich zu Dieser in der beteiligen. Die einzelne Zelle ruht nie, solange sie Lebensfunktionen zeigt. Der ununterbrochene Wechsel in ihrem Haushalt ist charakteristisch für das Zelleben. Auch der kompliziert gebaute Organismus, den wir als einen Staat von Zellen auffassen können, befindet sich nie im Gleichgewicht. Lassen wir unseren Blick weiter schweifen auf die gesamte große Natur, dann erkennen wir unschwer, daß auch sie in ständigem Wechsel begriffen ist. Auch die unbelebte Natur ruht nicht. Überall bahnen sich Umwandlungen an. Die meisten verlaufen außerordentlich langsam. Die Zelle dagegen läßt in rascher Reihenfolge Vorgang auf Vorgang und Umwandlung auf Umwandlung folgen. Eine auch nur kurze Pause in ihrem gesamten Getriebe bedeutet meistens den Tod des Indi\1duums. Es entstehen wieder neue Zellen, wenn seine Bestandteile, wie oben erwähnt, zerlegt sind und So geht ein neues Lebewesen die gebildeten Bausteine übernommen hat. Bald geein Baustein und ein Element von Individuum zu Individuum. hört ein Stoff der Pflanzenwelt, Natur bald der Tierwelt, bald der unbelebten an. Verfolgen wir einen bestimmten Stoff in seinem Kreislauf von der unbelebten Natur zur belebten, so drängt sich uns unmittelbar die Frage ) 1. Vgl. C. Engler: Entstehung des Erdöls. 269 (1910). Fortschritte der Naturwissenschaften. 94 I^ • ^ orlesung. — Kohlehydrate. auf. wie es kommt, daß nun dieser Stoff in einer Zelle an Vorgängen teilnehmen kann, die wir als Lebenserscheinungen deuten. Wir stoßen bei dieser Fragestellung immer wieder auf die Tatsache, daß nur eine bereits vorhandene Zelle imstande ist, den zugeführten Stoff den ..Lebensvorgängen" einzufügen. Mit der ersten Zelle beginnen die Rätsel. Solange es uns nicht möghch ist, im Reagenzglas aus einem Gemisch von organischen Stoffen und anorganischen Verbindungen eine lebende Zelle zusammenzufügen, so lange wird man nicht imstande sein, physikalisch und chemisch zu erklären, was eigentlich Leben ist. Wir müssen uns daher, wie schon früher betont, damit bescheiden, möglichst viele der Lebensvorgänge auf Vorgänge und Gesetzmäßigkeiten zurückzuführen, die sich der unbelebten Natur ablauschen lassen. Vorlesung V. Kohlehydrate. IV. Verhalten der Kohlehydrate im tierischen Organismus. Abbau der zusammengesetzten Kohlehydrate im Darmkanal. Wirkung der Darmflora auf die Kohlehydrate. Die Pflanzen enthalten, wie wir wiederholt hervorgehoben haben, eine gewaltige Fülle der verschiedenartigsten Kohlehydrate. Diese übernimmt der Pflanzenfresser mit seiner Nahrung. Das Gleiche gilt für den Omnivoren, wenn er Pflanzenkost als Nahrung wählt. Nur der reine Fleischfresser nimmt wenig Arten von Kohlehydraten auf, indem, wie wir schon betont haben, im tierischen (Jrganismus diese Verbindungen an Menge und Mannigfaltigkeit zurücktreten. Die Tatsache, daß auch der Pflanzenfresser in seinen Geweben nur über relativ wenige Kohlehydratarten verfügt, trotzdem er fortwährend die verschiedenartigsten Vertreter dieser Klasse von Nahrungsstoffen, insbesondere der Gruppe der Pentosen. Methylpentosen und der Hexosen aufnimmt, zeigt ohne weiteres, dali umfassende Umwandlungen stattfinden müssen, ehe im allgemeinen die Kohlehydrate der Nahrung unseren Geweben zugeführt werden. Sie beginnen bereits im Verdauungskanal. Während wir über die Beziehungen der Stärke, des Glykogens, des liohr-, Malz- und Milchzuckers und der einfachen Zucker, wie der Glukose, Fruktose und Galaktose zu den Kohlehydraten der tierischen Zellen recht gut unterrichtet sind, fehlt uns zurzeit noch ein vollkommen klarer Einblick in das Verhalten der großen Anzahl der übrigen Vertreter der Kohlehydrate. Wir müssen uns vorläufig damit begnügen, zum Ausdruck zu bringen, daß diese alle ohne Ausnahme einem weitgehenden Abbau im Verdauungskanal unterliegen, sofern es sich um Polysaccharide oder Glukoside handelt. Die sich bildenden Bausteine werden dann weiter verwandelt, bis sie den Körperzellen angepaßt sind. Wohl die meisten erlangen Beziehungen zum Traubenzucker. Es bestehen hier noch sehr große Lücken in unseren Kenntnissen, und es wird noch sehr vieler Arbeit bedürfen, bis wir über das Schicksal jeder einzelnen Art von Kohlehydraten lückenlos unterrichtet sind. Wir werden uns hier mit jenen Kohlehydraten befassen, über deren Verhalten im tierischen Organismus wir genauere Kenntnisse haben. Es sind dies die Polysaccharide: Glykogen, Stärke, Zellulose und die Disaccharide Milchzucker, Maltose und Rohrzucker. Von den Monosacchariden Glukose. Fruktose und Galaktose wissen wir, daß sie normaler- V. Vorlesung. 96 weise im Verdauungskanal, sofern sie nicht von den im Darmkanal lebenden Mikroorganismen angegriffen und verwendet werden, keinem weiteren Abbau unterliegen. Diese einfachsten Bausteine sind bereits zur Aufnahme in die Gewebe vorbereitet. Galaktose und Fruktose scheinen zuvor in Glukose umgewandelt zu werden. Diese Umlagerung vollzieht sich auch im Reagenzglase sehr leicht.^) Sämtliche Polysaccharide dagegen können nicht ohne weiteres zur Auf- nahme in die Gewebe gelangen. Es zeigt dies ohne weiteres die folgende Wenn wir irgend ein Tier selbst w^ochenlang mit Stärke füttern, begegnen wir trotzdem diesem Kohlehydrat nicht jenseits des Darmkanales in den Gew^eben. Der tierische Organismus besitzt kein der aufgenommenen Stärke identisches Polysaccharid. Das Gleiche gilt für den Rohrzucker. Selbst bei ausschließlicher Zufuhr dieses Disaccharids begegnen wir unter normalen Verhältnissen der Saccharose nicht im Blute und den Zellen des tierischen Organismus. Diese direkten Beobachtungen zeigen schon, daß offenbar die genannten Kohlehydrate vor ihrer Aufnahme durch die Darmwand einer weitgehenden Umwandlung unterliegen. Wir kennen noch eine Methode, um diese Annahme experimentell zu prüfen. Normalerweise führen wir dem tierischen Organismus alle festen und flüssigen Nahrungsstoffe durch den Verdauungskanal zu. Wir sprechen von einer Aufnahme der Nahrung per os. Nur der Sauerstoff hat eine bei manchen Tierklassen andere Eingangspforte, nämlich die Lungen die Kiemen und die Haut. Wir können nur durch Überschwemmung des Magendarmkanals mit einem bestimmten Nahrungsstoff unter Umständen erzwingen, daß auch noch nicht umgewandelte Stoffe zur Aufnahme gelangen. Wir überrumpeln gewissermaßen die Zellen des Verdauungskanales und verhindern, daß der Abbau vollständig durchgeführt wird. Auf noch einfachere Weise erreichen wir den Eintritt von nicht umgewandelten Nahrungsstoffen, indem wir diese mit Umgehung des Darmkanals direkt in die Blutbahn einspritzen intravasculäre Zufuhr oder aber sie unter die Haut oder in die subkutane Zufuhr Bauchhöhle intraperitoneale Zufuhr bringen. Wir sprechen ganz allgemein von einer parenteralen Einverleibung 2) der Nahrungsstoffe. Wählen wir Rohrzucker zu diesem Versuche, dann beobachten wir, daß nach ganz kurzer Zeit der Harn Rohrzucker enthält, und zwar wird fast das gesamte parenteral zugeführte Disaccharid durch die Nieren die Bereitungsstätten des Harnes ausgeschieden. Offenbar liegt dieser Entfernung des Rohrzuckers aus dem Organismus und insbesondere aus dem Blut die Tatsache zugrunde, daß die Körperzellen mit dem Rohrzucker als solchem nichts anzufangen wissen. Tatsache. so — — — — ') ) träge. 4. Vgl. hierzu S. ,37. ,,parenteral" Der Ausdruck — — — — — =') stammt von Carl Oppenheimer: Hofmeister?, Bei- 267 (1903). — Vgl. hierzu E. Weinland: Zeitschr. f. Biol. 47. 279 (1907). E. Abderhalden (Carl Brahm, G. Kapjberaer, E. Ba/hstnann, F. Wildermuth, L. Griqorescu) : Zeitschr. f. physiol. Chemie. 64. 529 (1910); C9. 23 (1910); 71. 367 (1911); 90. "388, 419 (1914). Vgl. auch Fritz Voit: Deutsches Archiv f. klin. Med. 58. 523 (1897). Lafayette ß. Mendel and Philip U. Mitchell: Amer. Journ. of Physiol. 14. 239 (1905). Walter Brasch: Hahilit.-Scbrift. München. R. Oldenhourg. 1907. Lafayette B. Mendel and Israel S. Kleiner: Amer. Journ. of Physiol. 26. 3'.)6 (1910). Ernst Ileilner: Zeitschr. f. Biol. 56. 75 (1911). Ferner Emil Abderhalden: Die Ahderhaldensche Reaktion. '•') — — 5. Auflage, J. Springer, 1922. — — — — Kohlehydrate. Wir wollen nun. nachdem wir festgestellt haben, qy poi- os aufge- nommene Polysaccharide nicht als solche jenseits der Darmwand auftreten, dal» uns die Frage vorlegen, was mit diesen im ^erdauungskanal geschieht. Zunächst kommen die aufgenommenen Xahrungsstoffe in der Mundhöhle mit dem Speichel, dem Sekret der verschiedenen Speicheldrüsen in Berührung. Die Nahrung wird, wie man sich ausdrückt, eingespeichelt. Die innige Vermengung mit dem Speichel wird durch den Kauakt herbeigeführt. Die Ziihne zerkleinern die Nahrung. Dabei werden Zellen eröffnet und ihr Inhalt der Einwirkung des Speichels ausgesetzt. Wenn wir in der Tierreihe die Gestalt der Zähne und ferner die Ausbildung des Kiefergelenkes genau verfolgen, dann erkennen wir ohne weiteres die innige Anpassung an die Art der aufgenommenen Nahrung. Die Herbivoren haben Zähne mit mächtigen Mahlflächen. Die Pflanzennahrung muß zerrieben werden, sollen die von Zellulose umschlossenen Zellen ausgiebig eröffnet werden. Der Karnivore zerschneidet und zerreißt seine an und für sich zum großen Teil weiche Nahrung. Beim Omnivoren finden wir Merkmale des reinen Herbivoren- und Karnivorengebisses vereinigt. Beim Herbivoren gestattet das Kiefergelenk ausgiebige seitliche Bewegungen. Sie sind beim Omnivoren beschränkt und beim Karnivoren so die gut, wie ausgeschlossen. Der Speichel hat verschiedene Funktionen. Eine davon ist das Schlüpfrigmachen des Bissens, damit er ohne Widerstand durch die Speiseröhre in den Magen gleiten kann. Der Speichel ist ferner imstande, manche Polysaccharide zu spalten. Zur Feststellung der Wirkung irgend eines in den Darmkanal sich ergießenden Sekretes stehen uns verschiedene ^Methoden zur Verfügung. Einmal können wir einzelne Nahrungsstoffe verfüttern und dann nach gewisser Zeit die Tiere toten und den Inhalt bestimmter Teile des Darmkanales untersuchen und seine Bestandteile mit dem verfütterten Materiale vergleichen. Oder wir legen an bestimmten Abschnitten des Darmkanales Öffnungen an, so daß das Darmrohr in Verbindung mit der Außenwelt steht. Wir sprechen von der Bildung einer Fistel. Wir halten diese gewöhnlich verschlossen und öffnen sie nur, wenn wir den Inhalt des betreffenden Darmabschnittes herauslassen wollen. Eine solche Fistel würde rasch zuwachsen, wenn man nicht eine sogenannte Kanüle einheilen lassen würde. Diese stellt ein Ptohr dar, das auf beiden Seiten umgebogene Ränder Der eine dieser Ränder ruht auf der Schleimhaut des Darmrohres, der andere auf der äußeren Haut. Die Fisteln können wir auch dazu benützen, um in das Darrarohr sich ergießende Sekrekte aufzufangen. Diese lassen wir dann außerhalb des tierischen Organismus im Reagenzglas auf beliebige Nahrungsstoffe einwirken. Wir stellen fest, ob eine Änderung eintritt und. falls eine solche erfolgt, welcher Art sie ist. Die gemachten Beobachtungen vergleichen wir dann mit den bei der Untersuchung des Darminhaltes gemachten Befunden, wobei wir natürlich Darmabschnitte wählen, in denen das gleiche Sekret seine Wirkung entfaltet, dessen Einfluß auf bestimmte Nahrungsstoffe wir untersucht haben. P^s ist dies eine weitere .Methode, um die Wirkung eines bestimmten Sekretes auf einzelne Verbindungen zu verfolgen. Beim Speichel liegen die Verhältnisse sehr einfach weil er sich in den Anfangsteil des Darmrohres ergießt. Wir können ihn leicht gewinnen besitzt. , Abderhalden. Physiologische Chemie. .^. Aufl. 7 9^ ^ • Vorlesuug, und ferner das zerkaute, eingespeichelte Produkt direkt aus der Mundhöhle entfernen. Wenn wir Kleister i), d. h. Stärke, die wir mit Wasser erwärmt haben, in den Mund nehmen, ihn kauen und dann nach etwa 5 Minuten wieder ausspucken, dann beobachten wir, daß das Gemisch nach Zusatz von Alkali und Kupfersulfat beim Erhitzen deutliches Reduktionsvermögen zeigt. Vor dem Einführen des Kleisters in die Mundhölile ist er auf die Fähigkeit zu reduzieren untersucht worden. Ein Reduktionsvermögen war nicht nachweisbar. Der Kleister färbte sich mit Jodjodkaliumlösung intensiv blau. Nach längerem Verw-eilen in der Mundhöhle finden wir überhaupt keine Blaufärbung mehr, es zeigen sich vielmehr rotviolette bis rotbraune Farbtöne. Wir schließen aus diesen Beobachtungen, daß der Kleister verändert eine Spaltung erfolgt ist. Das komist. Alles spricht dafür, daß pliziert gebaute Molekül ist in einfachere Bruchstücke zerlegt worden. Es kommt das ganze Gemisch der Abbaustufen zur Beobachtung. Gegen diese Deutung des erhobenen Befundes läßt sich ein gewichtiger Einwand erheben. In der Mundhöhle ist Speichel zum Kleister getreten. Dieser könnte selbst worden reduzierende Stoffe enthalten und auch bewirken, daß die Jodreaktion der diesen Einwand auf seine Berechtigung zu Stärke verändert wird. prüfen, sind Kontrollversuche notAvendig. Wir sammeln eine größere Menge Speichel. Die gesamte Menge teilen wir, nachdem wir ihn gut vermischt haben, in vier gleiche Teile. Einen Teil prüfen wir auf reduzierende Stoffe. Es tritt keine Reduktion ein. Einen anderen Teil der Speichelflüssigkeit lassen wir auf Kleister einwirken. Wir stellen sofort nach erfolgter Vermischung mit einer kleinen Probe des Gemisches die Jodreaktion an und überzeugen uns, daß eine schöne Blaufärbung erfolgt. Nun stellen wir Speichel und Kleister in den Brutschrank. Es ist;, dies ein abgeschlossener, gegen Wärmeverluste geschützter Raum, der beständig auf 37" erwärmt ist. Von Zeit zu Zeit entnehmen wir Proben und prüfen auf Reduktionsvermögen und stellen die Jodreaktion an. Wir beobachten, daß nach einiger Zeit reduzierende Substanzen auftreten. Das Reduktionsvermögen nimmt mit der Dauer der Einwirkung des Speichels zu. Ferner, beobachten wir, daß mit der Zunahme des Reduktionsvermögens die Jodreaktion Farben zeigt, die darauf hinw^eisen, daß Dextrine enstanden sind. Die vierte Speichelportion endlich erhitzen wir für kurze Zeit auf 100''. Dann setzen wir sie zu Kleister und bringen das Gemisch in den Brutschrank. Es tritt kein Reduktionsvermögen auf. Die gleich stark bleibende Jodreaktion läßt darauf schließen, daß die Stärke nicht verändert worden ist. Um Zu einem w^eiteren Versuche verwenden wir nichtgekochten Speichel und unveränderte Stärkekörner. Wir stellen fest, daß der Speichel nur sehr langsam einwirkt. Wir können diesen Versuch auch unter dem Mikroskop vornehmen. Wir sehen, wie das geschichtete Stärkekorn allmählich ..angefressen" wird. Es tritt Verflüssigung bestimmter Teile des Kornes auf. Diese Beobachtungen zeigen uns, daß das gequollene Stärkekorn viel leichter vom Speichel zerlegt wird, als das genuine Korn. Ganz die gleiche Feststellung können wir machen, wenn wir eine Scheibe einer rohen ') Vgl. hierzu S. ÖH. Kohlehydrate, 99 Kartoffel mit Speichel zusammeiibrins4en und gleichzeitig eine gekochte Kartoffelscheibe genau gleich behandeln. Während die erstere noch fast unverändert ist, hat die gekochte Kartoffelscheibe nach kurzer Zeit ihre Stärkekörner ganz eingebüßt.^) Sie sind zu Dextrinen und noch tieferen Abbaustufen zerlegt worden. Die erwähnten Beobachtungen führen zum Schlüsse, daß im Speichel ein Stoff vorhanden sein muß, der imstande ist, Stärke zu spalten. Er muß durch hohe Temperatur so verändert werden, daß er unwirksam wird. Es geht dies daraus hervor, daß der gekochte Speichel Stärke nicht mehr angreift. Dieser eigenartige Stoff gehört seinem 'ganzen Wesen nach zu den Fermenten. Er hat den Namen Diastase erhalten.-) Da dieses Ferment StärkemAmylum hydrolytisch zerlegen kann, bezeichnet man die Diastase auch als amylo-lytisches Ferment. Es sei gleich hier bemerkt, daß dieses Ferment auch im Dünndarm vorkommt und ferner offenbar in allen Zellen des tierischen und auch des pflanzlichen Organismus enthalten ist. Weitere Studien haben ergeben, daß die Diastase nicht nur durch hohe Temperaturen unwirksam gemacht wird, sondern daß ihre Wirksamkeit in hohem Grade von der Reaktion der Umgebung, in der sie wirken soll, abhängig ist. Sie wirkt am besten bei schwach alkalischer bis neutraler Reaktion. Sehr empfindlich ist sie gegen Säuren oder, besser ausgedrückt, gegen H-Ionen. Nachdem man erkannt hatte, daß der Speichel ein amylolytisches Ferment enthält, hat man festzustellen versucht, auf welche Art der ilbbau der Stärke zustande kommt. Man fand, daß er ein hydrolytischer ist und über Dextrine führt. Frühzeitig werden auch schon einfachere Spaltstücke und vor allem auch Traubenzucker moleküle frei. Von gut definierten Abbaustufen ist neben der Glukose die Maltose 3) zu nennen. Sie wird durch ein besonderes Ferment, die Malta se), in zwei Moleküle Traubenzucker zerlegt. Ferner ist es geglückt, kristallisierte Hexa- und Tetraamylose unter den Abbaustufen nachzuweisen. &) Die gleiche oder doch sehr ähnliche Art des Abbaues kann man herbeiführen, wenn man verdünnte Säure auf Stärke einwirken läßt und dafür sorgt, daß die einzelnen Zwischenstufen langsam zum weiteren Abbau gelangen. Mit der Feststellung, daß Speichel Stärke zerlegt, ist bereits gezeigt worden, daß in der Mundhöhle die Verdauung einsetzt. Es fragt der rohen Stärke eine sehr gute. ist die Ausnutzung zu 94"/^ ausnutzen (gekochte Stärke wird zu 997o verwertet), ist beim Wiederkäuer die Ausnutzung beider Arten von Stärke gleich gut. Vgl. u. a. W. Völtz: Landwirtsch. .Tahrb. 50. 455 (1917); Zeitschr. f. Spiritusindustrie. 40. 167 (1917). '') Die Diastase des Speichels ist auch Ptyalin genannt worden. Es ist besser, diese Bezeichnung nicht zu verwenden, weil bis jetzt keine Beobachtung vorliegt, die beweist, daß die Diastase des Speichels eine andere ist, als die des Pankreassaftes und der Tier- und Pflanzenzellon überhaupt. ') Vgl. J. Seec/en: Zentrall)l. f. med. VVissensch. 14. 849 (187G) und lyiügers ArMuschiv. 19. 106 (1879)! Ferner Offo yasse: Pßüffers Archiv. 14. 473 (1877). culus und v.Mering: Zeitschr. f. physiol. Chemie. 1. 395 (1877/78); 2. 403 (1878/79); Horace T. Brown und John 4. 93 (1880). Ebenda. 5. 185 (1881). V. Merim/: E. Külz und J. Voi/el Jleron: Liehiffs Annalen. 199. 165 (1879); 204. 228 (1880). Zeitschr. f. Biol. 31. 108 (1895). llamhurcjer: Fßügers Archiv. ) M. C. Tebh: Journ. of Physiol. 15. 421 (1894). 60. 543 (1885). ') Vgl. S. 59 und 63. ') Beim PÜauzenfresser Während Schweine diese nur — — — ~ — — V. Vorlesung. [QO sich nun, ob auch andere Kohlehydrate durch Speichel gespalten werden. Rohrzucker und Zellulose werden nicht angegriffen, wohl aber Glykogen. Dieses Polysaccharid des tierischen Organismus wird auch durch ein zur Gruppe der Diastase gehörendes Ferment stufenweise zerlegt. Es entstehen Dextrine, Maltose und Glukose. Wie es scheint, kommt nur der Omnivore dazu, Stärke und Glykogen zu zerlegen, indem den reinen Karnivoren eine Diastase des Speii) In der Tat spielt beim Karnivoren die chels ganz zu fehlen scheint, Kohlehydratverdauung durch den Speichel lange keine so bedeutungsvolle Rolle, wie beim Omnivoren und vor allem beim Herbivoren. Das Glykogen ist viel leichter angreifbar, als das genuine Stärkekorn. Außerdem nimmt der Karnivore weniger Kohlehydrate auf, als der Herbivore. Dazu kommt eine große Erschwerung der Ausnutzung der Kohlehydrate der Pflanze durch die Zellulose, die den Zellinhalt einhüllt. In der Zellulose haben wir früher schon ein Polysaccharid kennen gelernt, das außerordentlich widerWir können hier schon erwähnen, daß der tierische standsfähig ist.2) Organismus von sich aus über keine Stoffe verfügt, um die Zellulose aufzuschließen. Dem Pflanzenfresser würden nicht nur die Kohlehydrate des von Zellulose umgebenen Zellinhaltes verschlossen bleiben, sondern auch seine übrigen Bestandteile, wenn er nicht über besondere Einrichtungen und Hilfsmittel verfügen würde, um die Zellulosewand zu durchbrechen. Eine Maßnahme zur Eröffnung von Pflanzenzellen haben wir bereits kennen gelernt, nämlich die gründliche Zermahlung der Nahrung mittelst besonders eingerichteter Mahlzähne. Es sind noch andere Einrichtungen getroffen, um die Ausnutzung der Pflanzennahrung zu einer möglichst günstigen zu gestalten. So besitzen manche Vogelarten einen Kropf, in dem die aufgenommenen Körner bei Körpertemperatur aufgeweicht werden. Es kommt hierbei ohne Zweifel noch ein anderes Moment zum Ausdruck. Die aufgenommenen Nahrungsmittel bestehen aus Zellen. Diese enthalten Fermente aller Art, die im Kropf zum Teil wenigstens ihre Wirkung entfalten und den Abbau in die Wege leiten können. ^) Die feuchte Wärme des Kropfes begünstigt ohne Zweifel die Wirkung dieser Zellfermente. Bei manchen Vögeln sehen wir ferner, daß ein Magenteil, der sog. Muskelmagen, so eingerichtet ist, daß in ihm Körner usw. zermahlen werden können. Besonders großartige Anpassungen an die Pflanzennahrung finden wir beim Wiederkäuer. Er besitzt vier Magenabteilungen. Davon ist nur eine eigentlicher Magen. Die drei übrigen Abschnitte gehören ihrer Funktion nach noch zur Mundhöhle. Die Bissen w^erden bei den Wiederkäuern gut zermahlen und eingespeichelt verschluckt und gelangen in den sogenannten Vormagen, auch Pansen genannt. Die ganz flüssigen Anteile sammeln sich hauptsächlich im Netzmagens Retikulum. Im Pansen wirkt nun feuchte Wärme auf die verschluckten Bissen ein. Das ganze Material unterliegt einem Mazerationsvorgang. Ungezählte Mikroorganismen wirken mit, um die Zellulose einzuschmelzen. Auch können die Fermente der Nahrung von innen heraus den — ^) Bidder und Schmidt: Die Verdauungssäfte und der Stoffwechsel. 14 (1852). Claude Bernard: Legons sur les propriötfe physiologiques et les altöratious patholoLafayetti B.Mendel and Frank giques des liquides des Torganisme. II. 249 (1859). F. Underhill: The Journ. of Biological Chemistry. 3. 135 (1907). — =>) Vgl. S. 61. •) Ellenberger: Ebenda. 18. 119(1906). Skandin. Archiv f. l'hvsiol. 18. 306 (1906). — F. Berf/mann: Kohlehydrate. 101 Abbau der Zellbestandteile fördern. Ferner wirkt der verschluckte Speichel Die schon etwas erweichte Masse wird nun nochmals in die Mundhöhle befördert. Es beginnt das Wiederkauen. Wieder wird alles zermahlen und nochmals innig mit Speichel vermischt. Ist auch jetzt der Bissen noch nicht genügend zerkleinert, dann wird er wieder dem Vormagen übergeben. Er steigt dagan später nochmals auf. Bildet jedoch der Bissen nunmehr eine halbweiche Masse, dann wird er durch eine außerordentlich interPsalterium (Omasus) essante Einrichtung direkt dem Blatte rmagen und von da dem eigentlichen Magen, dem Labmagen, Abomasus, zuweiter. = geführt. Die Speiseröhre setzt sich nämlich, in einen Kanal, genannt Schlundrinne, fort. Dieser leitet die Bissen bis zum Blättermagen. Das Rohr ist in Wirklichkeit kein vollkommen geschlossenes. Es bildet sich nämlich dadurch, daß die Ränder einer flachen Rinne sich erheben und Erschlaffen diese, dann haben wir wieder die so ein Rohr herstellen. flache Rinne und der Ösophagus hört mit der Einmündung in den Pansen Es kann somit die Speiseröhre ihren Inhalt bald nur in den Vorauf. magen und den Netzmagen ergießen, bald reicht sie bis zum Blättermagen. Man kann sich auch vorstellen, daß die Speiseröhre einst bis zum eigentlichen Magen ein geschlossenes Rohr bildete, und daß dann um ihr Ende Aussackungen sich gebildet haben, in die die W^and der Speiseröhre mit einbezogen wurde. Auch bei anderen Herbivoren als den Wiederkäuern finden wir Vorrichtungen, die ohne Zweifel bezwecken, daß einerseits die Zellulose möglichst weitgehend aufgeweicht wird und gleichzeitig der Speichel möglichst lange seine Wirkung entfalten kann. So ist z. B. beim Pferde der Magen ganz scharf in zwei Abschnitte geteilt. Nur diejenige Hälfte des Magens besitzt eine richtige Schleimhaut mit Drüsen, die mit dem Duodenum in Verbindung steht. Der Teil des Magens, in den die Speiseröhre mündet, ist seinen ganzen Einrichtungen und Funktionen nach als eine Mazerationskammer aufzufassen. Er stellt eine primitivere Einrichtung mit gleichem Zweck, wie die drei ersten Abteilungen des Wiederkäuermagens, dar. Selbst bei denjenigen Tieren, bei denen der Magen anatomisch nicht in verschiedene Teile zerlegt ist, findet sich eine wichtige Einrichtung, um dem Speichel noch für längere Zeit seine Wirkung zu belassen. Um diese zu verstehen, müssen wir hervorheben, daß die Magenschleimhaut Drüsen besitzt, die den sogenannten Magensaft absondern. Dieser ist vor allem dadurch ausgezeichnet, daß er stark sauer reagiert. Er enthält freie Salzsäure. Nun ist, wie schon hervorgehoben wurde, die saure Reaktion unverträglich mit der Wirksamkeit der Diastase. Würden nun die verschluckten Bissen sofort mit Magensalt durchtränkt werden, dann wäre die Wirkung des Speichels auf die oben erwähnten Polysaccharide und ihre Abbaustufen mit einem Schlage aufgehoben. Die genauere Verfolgung des Verhaltens der durch den Ösophagus dem Magen zugeführten Bissen hat nun gezeigt, daß die zu verschiedenen Zeiten verschluckten Bissen sich in der Weise schichten, daß die zuletzt in den Magen gelangten zentral von den vorher aufgenommenen zu liegen kommen.') Nicht alle Teile des — ) Ellenherger : Fßügers Archiv. 114. 93 (1906i. F. GriUzner: Fßiif/ers Archiv. A. Scheunen Arthur Scheuner f : /^/rt^ers Archiv. 169. 201 (1917). 106.463(1905). nud Fritz Kiok : Ebeuda. 193. 16(1922). Einil Abderhalden unä Ernst Wertheimer : Ebenda. 194. 168 (1922). — — — ^ • Vorlesung. 102 Mageninhaltes werden gleichzeitig von Magensaft durchtränkt, vielmehr dauert es einige Zeit, bis auch die von der Magenwand abgelegeneren Mageninhaltsanteile mit ihm in Berührung kommen. Die der Magenwand zuucächst gelegenen Nahrungsschichten werden vom Magensaft zuerst verdaut und dabei verflüssigt. Es entsteht sogenannter Chymus. Dieser wird durch den Pförtner, den Pylorus, ins Duodenum entlassen. Dadurch rücken neue Schichten des Mageninhaltes in den Bereich der Magensaftwirkung, bis schließlich der Speicheldiastase die Bedingungen zur Wirkung ganz entzogen werden. Überlegt man sich diese Verhältnisse genau, dann ergibt sich von selbst, daß man über den Umfang der Verdauung der Stärke und des Glykogens und ihrer Abbaustufen durch die Speicheldiastase keine genaueren Angaben machen kann. Er ist gewiß außerordentlich großen Schwankungen unterworfen. Es kommt in Betracht, wie intensiv die Einspeichelung war, wie lange der Bissen in der Mundhöhle verweilte, wie weit bei Aufnahme von Pflanzennahrung die Zellulosewände zermalmt und aufgeweicht waren und endlich, wie lange es dauert, bis der Mageninhalt durchsäuert ist. Alles in allem ist die Kohlehydratverdauung durch den Speichel nur Je umfassender erleichtert sie ist, als eine vorbereitende aufzufassen. um so mehr wird die Verdauung im Dünndarm und abgekürzt. Sie arbeitet dieser vor. Wir haben bereits festgestellt daß die jNIundverdauung mittelst des Speichels teils in besonders organisierten Magenabschnitten, teils ohne solche durch die Art der Ablagerung der dem Magen zugeführten Bissen im Magen noch längere Zeit fortdauern kann. Es fragt sich nun, ob der Magen nicht auch eine eigene, d. h. durch von seiner Schleimhaut mit Drüsen hervorgebrachte Stoffe bewirkte Verdauung von Kohlehygraten hat. Es ist dies nicht der Fall.^) Der Magen gibt keine auf Kohlehydrate eingestellten Fermente ab. Dagegen kommt der schon er- ihren wähnten Salzsäure eine gewisse Bedeutung zu, indem diese stark genug ist, um z. B. Stärke, Dextrine, Rohrzucker usw. zu spalten. Doch spielt diese Hydrolyse quantitativ kaum eine Rolle. Aus dem Magen gelangt der Speisebrei, der Chymus, zunächst in das Duodenum, um sich dann von hier aus in ganz dünner Schicht über den ganzen Dünndarm mit seiner durch Zotten stark vergrößerten Oberfläche auszubreiten. Hier im gesamten Dünndarm setzt die Verdauung der Kohlehydrate ganz umfassend ein. Der Speisebrei enthält je nach der Art der aufgenommenen Nahrung noch verschiedene zusammengesetzte Kohlehydrate nebst ihren Abbaustufen. Wir treffen auf noch unangegriffene Stärke, wenn solche zur Aufnahme gelangte. Das Glykogen ist beim Karnivoren, da er keine Speicheldiastase zu besitzen scheint, ebenfalls noch unabgebaut. Wir müssen ferner beim Herbivoren und Omnivoren noch große Mengen von Zellulose erwarten, da, wie schon betont worden ist, der tierische Organismus von sich aus dieses Polysaccharid nicht zerlegen kann. Ferner müssen wir noch eines Disaccharids gedenken, das ') Nach F. Beugen und G. llaane: l'ßii(/ers Arcliiv. 106. 267 (1904) liefert der Magen des Schweines ein diastatisches Ferment, ferner soll nach //. Friedenthal : Ar- Suppl. 383 (1899) im Magensaft des Hundes ein bei saurer f. (Anat. u.) Physiol. Reaktion wirksames diastatisches Ferment enthalten sein. Diese Angaben bedürfen dringend der Kachprüfung chiv ! Kohlehydrate. \()}\ zwar /Normalerweise bei den Tiereu nur während eines kurzen Lebensabschnittes aufgenommen wird, nämlich des Milchzuckers. Er wird mit der Milch wahrend der Säuglingsperiode zugeführt. Nur der Mensch und manche Haustiere nehmen auch später noch Milch auf. Der Milchzucker wird erst im Dünndarm gespalten. Zunächst unterliegen die Nahrungsstoffe dem von den Drüsen des Duo- denums hervorgebrachten Safte. Dazu kommt dann das von der Pankreasdrüse abgegebene Sekret. Endlich wirken jene Abscheidungen, die von den ungezählten Drüschen der gesamten Dünndarmschleimhaut geliefert werden, auf den Chymus ein. Man hat die Wirkung des Pankreassaftes und diejenige der Sekrete all der genannten Drüschen für sich studiert. Man nennt ihre Absonderung kurz Darmsaft. Der Pankreassaft enthält eine oder mehrere Diastasen, d. h. amylolytische Fermente. Stärke und Glykogen werden in der gleichen Weise gespalten, wie von der Diastase des Speichels. Niemand weiß, ob nur eine bestimmte Diastase wirksam ist oder, ob nicht für bestimmte Stärkearten und ihre Komponenten und insbesondere auch für das Glykogen besondere Diastasen in Wirkung treten. Manches spricht dafür, dalj es auf bestimmte Dextrine eingestellte Fermente gibt. Bekannt ist die Maltase, die Maltose hydrolysiert. Sie findet sich im Pankreasund Darmsaft. Im Pankreassaft ist auch ein Ferment gefunden worden, das den Milchzucker in Glukose und Galaktose zerlegt. Man hat es Laktase genannt. Es verschwindet einige Zeit nach Aufhören der Milch- bzw. Milchzuckerzufuhr. Interessanterweise tritt die Laktase wieder im Darmkanal auf. wenn man wieder Milch zuführt, i) Dem Pankreassaft scheint ein auf Ptohrzucker eingestelltes Ferment zu fehlen. Dafür findet sich ein solches im Darmsaft. Man hat es Saccharase genannt oder auch Invertin. Der letztere Name erinnert an die Bezeichnung Invertzucker."^) Der Name Saccharase steht in Beziehung zum Namen Saccharose, Rohrzucker. Diese Art der Benennung der Fermente hat den großen Vorzug, daß sie sofort an das Substrat erinnert, das vom betreffenden Fermente verändert wird. Der Darmsaft enthält ferner amylolytisch wirkende Fermente und auch Laktase. Er kann somit den Pankreassaft ergänzen, ja sogar ersetzen. Wir erkennen aus der gegebenen Darstellung, daß im Darmkanal auf die verschiedenen Kohlehydrate unserer Nahrung eingestellte Fermente vorhanden sind. Sie vermögen, die aus mehreren Bausteinen bestehenden Kohlehydrate vollständig in ihre einfachsten Bausteine zu zerlegen. Nur die Zellulose widersteht den Fermenten des Verdauungskanals. =^) Nun — ') E. Weinland: Zeitschr. f. Biol. 38. 607 (1899); 40. 386 (1900). F. A. Bainbridge: Joura. of Phvsiol. 31. 98 (1905). Vgl. auch R. H. Aders FUmnier: Jouru. of Physiol. 34. 93 (1906); 35. 20 (1906/07). ./. Ibrahim und L. Kaimiheimer: Zeitschr. f. physiol. Chemie. 62. 287 (1909). li. Botnpiani: Arch. de Farmacol. sperim. 19. 423 (1915). — -) — — Vgl. hierzu S. 48. ^) Interessanterweise verfügt die Weinbergschnecke im Magensaft über Fermente, die Zellulose lösen. Auch mit Leberpaukreasextrakt dieses Tieres wurde Abbau von Zellulose beobachtet. Vgl. W. Biedermann und F. Moritz: P/iiigers. Archiv. 73. 219 (1898); E. Müller: Ebenda. 83. 619 ( 1901 ). D. H. Bierrij und ./. Giaja : Biochem. Zeitschr. 40. 370 (1912) haben Leberpankreassaft von Helix pomatia auf Mannane und (ialaktaue einwirken lassen. Es erfolgte ein Abbau. Auch der Magendarmsaft des Flußkrebses greift diese Polysaccharide an. Im Pflanzenreich sind Fermento, die die verschiedensten Polysaccharide zerlegen können, sehr verbreitet. — 104 \. Vorlesung. hat aber die Erfahruog gezeigt, daß die Herbivoren die Zeliulosö ganz gut verwerten können '), ja im Verdauungskanal der Wiederkäuer werden sogar schwach oder mäßig verholzte Zellwände in weitgehendem Maße abgebaute) Auch für die Omnivoren ist die Zellulose kein wertloser Ballast. Der Karnivore dagegen vermag die Zellulose gar nicht auszunutzen. 3) Sie ist ihm ganz ungewohnt. Er erhält ja normalerweise dieses Polysaccharid nie und ist Der deshalb in keiner Weise auf sie eingerichtet. Organismus zeigt die Art seiner Nahrung nicht nur und ferner in der Ausbildung seines Magens, sondern auch durch den Bau seines Darmkanales. Vor allem fällt auf. daß der reine Herbivore den längsten Dünndarm aufweist. Den kürzesten finden wir beim Karnivoren. Der Omnivore nimmt eine Mittelstellung ein. Der lange Darm ist der Auf.Schließung der Zellulose angepaßt.) Eine besondere Bedeutung kommt bei manchen tierische in der Organisation seines (lebisses an Tieren (z. B. Pferd, Kaninchen) dem Coecum für die Zellulosezerlegung Diese erfolgt interessanterweise unter Vermittlung kleinster Lebewesen. Es sind dies Bakterien eigener Art, die den Dünndarm bevölkern. Die Zahl ihrer Arten ist erheblich. Man hat die ganze Mikroorganismenwelt auch kurz als Darmflora bezeichnet. Sie lebt auf Kosten der zugeführten Nahrungsstoffe. Die kleinsten Lebewesen vermehren sich, führen ihren eigenartigen, unseren Körperzellen ganz fremden Stoffwechsel durch, scheiden fremdartige Stoff Wechselprodukte aus und gehen schließlich auch zugrunde. Die Mikroorganismen, die unseren Darm bewohnen, schädigen uns durch Wegnahme von Nahrungsstoffen und liefern ohne Zweifel manches Stoffwechselprodukt, das für unsere Körperzellen nicht gleichgültig ist. Dafür nützen sie uns dadurch, daß sie den Abbau der Zellulose in die Wege leiten. Setzt man Darmsaft ohne weiteres zu Zellulose, so beobachtet man nach einiger Zeit, daß sie verändert wird. Sie geht schließlich in Lösung.^) Es entstehen allerlei Abbauprodukte, u. a. niedrige Fettsäuren (Essigsäure, Buttersäure, Valeriansäure), Kohlensäure und ferner Methan. 7) AVird jedoch der Darmsaft vor der Einwirkung auf die Zellulose sorgfältig von Bakterien befreit durch Filtration durch ein Bprkefeld-Filter dann vermag er zu. 5) — — , ^) Vgl. u. a. W. Jlennebcrfj uud F. Stohmann: Beiträge zu einer rationellen Fütterung der Wiederkäuer. Brauuschweig 1860 und 1864; Zeitschr. f. Biol. 21. 613 (1885). r. Knieriem: Ebenda. 21. 67 (1885). U. Weiske, B. Schulze und E. Flechsig: Ebenda. 22. 373 (1886). F. Wolf: Landwirtsch. Jahrb. 49. Supp. III (1887). N. Zun/z: jytügers Archiv. 49. 477 (1891). -) Vgl. G. Haberlandt und .Y. Zun/z: Sitzungsber. d. Akad. d. Wissensch. 41. 686 (1915). — Vgl. auch G. Haberlandt: Mikroskopische Untersuchungen über Zellwandver- — — — dauung. Gebr. Bornträger, Berlin 1918. ») Vgl. u. a. //. r. Hösslin: Zeitschr. f. Biol. 54. 395 (1910). A. Scheunert und E. Loetsch: Biochem. Zeitschr. 20. 10 (1909); Zeitschr. f. physiol. Chemie. 65. 219 (1910). H. Lohrisch: Ebenda. 69. 143 (1910). ) Füttert man aus einem Wurf Kaninchen einen Teil ausschließlich mit Milch und einen anderen mit Heu, dann zeigt es sich, daß die auf die letztere Art ernährten Tiere einen bedeutend längeren Dünndarm besitzen als die mit Milch gefütterten. ^) A'. Zuniz: Zentralbl. f. Thvsiol. 19. 581 (1905). W. rstjanzew: Biochem. Zeitschrift. 4. 154 (1907). ") Vgl. z. B. Viktor Hofmeister: Archiv f. wissenschaftl. u. prakt. Heilkunde. 11. Heft 1/2 (1885). 'j Vgl. u. a. \i'. Ellenberffer, A. Scheunert, W. Griimner uud A.Hopff'e: Zeitschr. f. physiol. Chemie. 96. 236 n915). — — — Kohleliyilratp. 105 diese nicht anzugreifeu. i; Zellulose abbauende Bakterienarten sind aus dem Verdauungsschlauch von Herbivoren isoliert und für sich gezüchtet worden.-) Die Mikroorganismen spielen in der Natur eine sehr wichtige Rolle. Sie vermitteln die Beziehungen zwischen vorhandenen Individuen und der Nachwelt. Stirbt ein Individuum, dann stellen sie sich in großer Zahl ein. Eine Art arbeitet der anderen vor, bis schließlich der ganze kunstvolle Bau all der ungezählten Zellarten der Leiche zerstört ist. Aus den verbleibenden Trümmern blüht neues Leben empor, indem zunächst Pflanzen sich ihrer bedienen, um von neuem Zellen aufzubauen. Dann folgt das Tier, das die eben erblühte Pflanze verzehrt und so den Kreislauf des Lebens fortsetzt. Nun dringen diese Pioniere des Lebens in unseren Verdauungskanal ein und vollziehen auch da ihre Abbautätigkeit. Manche der hier vorhandenen Zelltrümmer werden von ihnen weitgehend zerlegt. Mancher wertvolle Baustein wird unseren Zellen vorenthalten, weil Bakterien ihn so verändern, daß er für unsere (Gewebszellen nicht mehr verwertbar ist. In vielen Fällen geht der Abbau so weit, daß die synthetischen Fähigkeiten der tierischen Zelle nicht ausreichen, um die Trümmer wieder zusammenzuzimmern. Für den Karnivoren ist die Darmflora wohl kaum von Nutzen, dagegen sind sie dem reinen Herbivoren ganz unentbehrlich. Nicht nur würde er große Mengen kostbaren Materiales mit der Zellulose verlieren, sondern außerdem noch viele von dieser eingeschlossene Substanzen. Die gleichen Gesichtspunkte nur kann sich dieser im Notfall seinen gelten auch für die Omnivoren Nahrungsbedarf zum größeren Teil aus animalischer Nahi'ung decken. Es ist noch nicht klargelegt, wie sich das Wechselspiel zwischen der Darmflora und den Köi-perzellen vollzieht. AVir können uns etwa folgendes Bild davon machen. Zunächst wird die Zellulose durch von bestimmten Bakterien hervorgebrachte Fermente gespalten. Es entstehen lösliche Abbaustufen, die man etwa den De.xtrinen vergleichen kann. Nun greifen wahrscheinlich Fermente in den weiteren Abbau ein, die im Darmsaft und vielleicht auch im Pankreassaft enthalten sind. Wir erhalten Traubenzucker,, ferner auch Zellobiose.ä) Diese wird dann durch die Wirkung eines besonderen, von be.stimmten Bakterienarten gebildeten Fermentes. Zellobiase genannt), in zwei Moleküle Traubenzucker zerlegt. Diese Bruchstücke werden resorManche Dextrine und tieferen Abbaustufen werden jedoch von biert. bestimmten Bakterienarten in anderer Richtung abgebaut. Es entstehen die genannten Fettsäuren und ferner Kohlensäure und Methan. Es gehen durch die Bildung der letzteren beiden Produkte immer gewisse Mengen der Zellulosebausteine für den Wirt verloren. Leider kann man den Abbau der Zellulose durch die Darmflora . außerhalb des Darmes nicht gut in allen Teilen nachahmen. Es fehlt nämlich ein wichtiger Vorgang vollkommen. Es ist dies die Wegnahme — V) H. Tappeiner: Zeitschr. f. Biol. 19. 228 (1883); 20. 52 (1884); 24. 105 (1888). Biedermann iiud Moritz: Hoppe-Sei/ler: Zeitschr. f. phvsiol. Chemie. 10. 401 (1886). Erich Midier: Ebenda. 83. 619 (1901). Fflügers Archiv. 73. 291 (1898). Vgl. auch Arthur -) \r. EUenberger: Archiv f. (Auat. u.) Physiol. 139 (1906). H. v. Hoesslin und E.J.Lesser: Scheunert: Zeitschr. f. phvsiol. Chemie. 38. 9 (1906). — — — — Zeitschr. f. Biol. 54. 47 (1912). ') Vgl. S. 51. ) Zellen des tierischen Organismus bringen nach allen vorliegcudcn Erfahrungen keine Zellobiase hervor. Vgl. hierzu: H. Pringsheivi und Magnus v. Merkatz: Zeitschr. f. physiol. Chemie 105. 178 (1919). ^ X06 • Vorlesung. der gebildeten Abbaustufen. Im Darinkanal wird fortwährend resorbiert. Ferner ergießen sich immer wieder neue Sekretraengen. Endlich sind die Lebensbedingungen im Darrakanal für die Mikroorganismen ganz andere Wir sehen an diesem Beispiel, wie außerordentlich als im Reagenzglas. schwer es ist, eine relativ einfache Fragestellung klar zu beantworten. Bei jedem Vorgange spielen eine Summe von einzelnen Momenten mit, Ein Prozeß ist qualitativ und die zusammen ein Endresultat bedingen. quantitativ von anderen \'orgängen abhängig. Die geringste Störung in den notwendigen Bedingungen verschiebt sofort das ganze Bild. Kennen wir sie nicht bis in alle Einzelheiten, dann müssen wir in der Beurteilung der Resultate von Laboratoriumsversuchen äußerst vorsichtig sein. Als sicher festgestellte Tatsache können wir betrachten, daß durch Vermittlung einer geeigneten Darmflora Zellulose aufgeschlossen und damit, soweit die Abbaustufen von den Mikroorganismen nicht zu weit abgebaut sind, für unsere Körperzellen verwertbar gemacht werden kann. Die Karnivoren scheiden der Nahrung zugesetzte Zellulose mit den Fäzes wieder vollständig aus. Die Omnivoren verw^erten einen erheblichen Teil der zugeführten Zellulose. Der JVIensch verhält sich der Zellulose gegenüber je nach der Art und vor allem der Zubereitung der Zellulose sehr verschieden.!) Der reine Herbivore mit seinen besonderen Einrichtungen und vor allem mit seinem sehr langen Dünndarm ist am besten auf die Verarbeitung der Zellulose eingerichtet. Bei der Beurteilung der Bedeutung einer mehr oder weniger ergiebigen Zelluloseverdauung muß in Betracht gezogen werden, daß, solange aus diesem Polysaccharid bestehende Zellwände unverletzt sind der gesamte Zellinhalt der Wirkung der Verdauungsfermente entzogen bleibt. Es geht also nicht nur Zellulose verloren, wenn diese unangegriffen bleibt, sondern zugleich das gesamte wertvolle Material an anderen Kohlehydraten, an Fett, Eiweiß usw'., das von ihr umschlossen bleibt, soweit nicht die Zellfermente in der Lage sind, von innen heraus durch Abbau diffundierbare Produkte hervorzubringen. Aus diesem Grunde ist eine mechanische Zerkleinerung und damit Eröffnung der Pflanzenzellen eine so bedeutsame Hilfe-) für eine gute Ausnutzung.^) Die Frage nach der Bedeutung der Darmflora für die Ausnutzung der Nahrungsbestandteile ist durch die folgenden Versuche einer direkten l'rüfung unterzogen worden. Xiiftall und Thierfeider ^) entnahmen schwangeren Meerschweinchen kurz vor Eintritt der Geburt durch den sog. Kaiserschnitt die Jungen und brachten sie unter peinlichster Einhaltung der Asepsis in einen sorgfältig sterilisierten Käfig. Bekanntlich werden , — ) Vgl. u. a. JI. Lohri.sch: Zeitsclir. f. experim. Path. u. Ther. 5. 478 (1909). ü. V. Hoesslin: Zeitschr. f. Kinderheilkunde. 1. 81 (1911). 2) Vgl. auch: Hans Fried cnthal: Fßüf/ers Archiv. 144. 152 (1912). G. v. Bergmann und Fr. W. Sirauch: Therapeut. Monatshefte. 27. .lauuar (1918). Aher nicht nur von diesem Gesichtspunkte aus ist eine mechanische Zertümmerung der Pflanzenzellen für die Verdauung von größter Bedeutung, es hat sich herausgestellt, daß Trypsin unveränderte Pflanzenzellen nicht angreift, daß jedoch die V'erdauung sofort in Gang kommt, wenn man aus ihnen die in Alkohol, Äther und Chloroform löslichen Bestandteile entfernt hat. Es scheint, daß diese Stofl'e das Kindringen der Fermente hindern. Vgl. IT. Biedermann: Fßüf/em Archiv. 174. 358 (1919). Georg JI. F. Nnttall und IJ. Thierfelder : Zeitschr. f. physiol. Chemie. 21. 109 (1895/9G); 22.' 62 (189G/97). — •') ') Kohlehydrate. die Meerschweinchen 107 im Gegensatz zu den ihnen verwandten Tieren voll- kommen entwickelt zur Welt gebracht. Sie können sofort nach der Geburt dieselbe Nahrung, wie die erwachsenen Tiere, aufnehmen. Es gelang nun, die auf die genannte Art aus dem Mutterleibe entfernten Meerschweinchen einige Zeit in steriler (8 Tage) mit sterilisierter Nahrung (Milch und Cakes) und Umgebung am Leben zu erhalten. Die Versuchstiere nahmen normaler Weise an Gewicht zu. Daß die Meerschweinchen in der Tat gebUeben waren, bewies die Untersuchung der Tiere nach dem Abschlüsse des Versuches. Damit wäre bewiesen, daß der tierische Organismus auch ohne Bakterien auskommt. Leider ist der angeführte \'ersuch nicht ganz vollwertig. Er beweist nur, daß Meerschweinchen Milch und Cakes bei Ausschluß von Bakterien gut verwerten können, worüber an und für sich ein Zweifel nicht bestehen konnte. Er besagt jedoch nichts darüber, wie die Ausnutzung sich verhalten hätte, wenn den Meerschweinchen ihre normale, an Zellulose reiche Nahrung verabreicht worden wäre. Es ist wohl ganz zweifellos, daß bei Verabreichung von Zellulose das Fehlen von Bakterien sich sehr deutUch bemerkbar gemacht hätte. Das zeigen denn auch deutlich die Versuche von ScJwttelim. i) Er wählte als Versuchstiere Hühnchen, die steril ausgebrütet wurden und in bakterienfrei in steril gehaltenen Räumen sterile Nahrung erhielten. Die Versuchstiere zeigten trotz reichlicher Nahrungsaufnahme fortwährend Hunger und gingen in derselben Zeit zugrunde, wie ohne Nahrung belassene Hühnchen. Sobald dem Futter Bakterien aus Hühnerfäzes zugesetzt wurden, erholten sich die Tiere und nahmen an Gewicht zu. Ferner hat Moro^) ganz ähnliche Ver- suche mit den Larven der Knoblauchkröte ausgeführt. Es gelang ihm, diese 35 Tage lang steril aufzuziehen. Es ergab sich, daß die sterilen Larven gegenüber den Kontrolltieren, die in Wasser, das Fäzes des Muttertieres enthielt, aufgewachsen waren, ganz erheblich im Gewicht und in ihrer gesamten Entwicklung zurückblieben. Wir wollen nicht unerwähnt lassen, daß die erwähnten Beobachtungen möglicher Weise eine ganz andere Ur- sache haben, als bis jetzt vermutet worden ist. Es hat sich nämlich immer mehr gezeigt, daß bei der Ernährung geringe Mengen von eigenartigen, vorläufig Nutram ine genannten, zum Teil außerordentlich leicht veränderlichen Stoffen eine bedeutsame Rolle spielen. Es ist nicht ausgeschlossen, daß diese Produkte bei der Sterilisierung der Nahrung zerstört worden sind und dadurch der ungünstige Ausfall der Versuche bedingt war. Die Versuche müssen unter Zusatz der lebenswichtigen Nutramine wiederholt werden. 3) W^ie schon erwähnt, greift die Darmflora nicht nur die Zellulose an, sondern auch andere Nahrungsstoffe und vor allem auch andere Kohlehydrate. Es sind uns zurzeit noch lange nicht alle derartigen Vorgänge bekannt. Sie spielen sicher beim Abbau und der Verwertung der Pentosane eine Rolle. Pflanzenfresser nutzen z. B. die Pentosane der Vegetabilien bis zu ßO^/o aus. ) Sicher leiten auch bei dieser Gruppe von PolyM. Schottelius: Archiv für Hygiene. 34. 210 (1899) uud 42. 48 (1902). Moro: Jahrbuch der Kinderheilkunde. 62. H. 4 (1905). ^) Vgl. S. 77 und Band II die Vorlesung über Nutramine. ) W. E. Stone: American Chem. J. 14. 9 (1892). Weiske: Z. f. physiol. Chemie. Albin r. Rudno RudzinsU: 2». 489 (1895). B. Slorotzow: Ebenda 34. 181 (1901). Ebenda. 40. 317 (1904). ») 2) — — - "^ X08 • Vorlesung. sacchariden Bakterien den Abbau ein. Eingehend studiert ist der Abbau der Glukose durch bestimmte Bakterienarten. So wissen wir, daß Traubenzucker in Buttersäure, Kohlensäure und Wasserstoff zerlegt werden kann. ^) Wir sprechen von einer Buttersäuregärung. Ferner kann (llukose in Milch säuregär ung. Es sei ferner zwei Moleküle Milchsäure zerfallen daran erinnert, daß Hefezellen aus Glukose Alkohol und Kohlensäure bilden alkoholische Gärung. Die beiden ersten Gärungen treten können ohne Zweifel oft in mehr oder weniger großem Umfange ein, während über ein Vorkommen der alkoholischen Gärung im Darmkanal sicheres nicht bekannt ist. Erwähnt sei, daß im Magen derartige Vorgänge unter — — normalen Umständen nicht auftreten, weil die im Magensaft vorhandene Salzsäure die Mikroorganismen am Wachstum verhindert und viele direkt vernichtet. Sie stellen sich ein, sobald die Bildung des sauren Magensaftes aus irgend einer Ursache vermindert oder ganz eingeschränkt ist. Wir werden später auf die erwähnten Gärungen noch zurückkommen. Die Bildung der genannten Abbaustufen erfolgt nicht in so einfacher und direkter Weise, wie es nach der Formulierung der einzelnen Spaltungen erscheinen mag. Fassen wir nun zusammen, was wir über die Verdauung der Kohlehydrate wissen, dann ergibt sich das folgende Bild. Der tierische Organismus verfügt über eine ganze Anzahl von Stoffen, die er in bestimmten Zellen hervorbringt und an bestimmten Stellen in den Darmkanal abgibt. Sie sind Fermente genannt worden. Manche davon sind ausschließlich auf ganz bestimmte Kohlehydrate eingestellt, von anderen ist uns zurzeit eine streng spezifische Wirkung nur insoweit bekannt, als die betreffenden Fermente nur Vertreter der Kohlehydratreihe abbauen. So baut die Laktase nur Laktose, die Maltase nur Maltose und die Saccharase nur Saccharose ab. Von der Diastase dagegen können wir zurzeit nur aussagen, daß sie viele Polysaccharide zerlegt. Vielleicht liegen die Verhältnisse in Wirklichkeit so, daß die sogenannte Diastase ein Gemisch von Fermenten darstellt. Da wir jedoch diejenigen Polysaccharide, die von der Diastase abgebaut werden, ihrer Struktur nach noch nicht genau kennen und vor allen Dingen nicht in der Lage sind, die Polysaccharide alle einzeln in reinem Zustande zu gewinnen, so vermögen wir selbstverständlich auch nicht die Frage zu entscheiden, ob nicht unter dem Sammelnamen Diastase Fermente verborgen sind, die nur auf ganz bestimmte Bindungsarten eingestellt sind. Wesentlich ist, daß im Darmkanal alle Kohlehydrate zusammengesetzter Natur zerlegt werden können. Eine Ausnahme bildet nur die Zellulose, deren Abbau durch die Darmfloia vermittelt wird. Hier kommt der tierische Organismus mit seinen Hilfsmitteln nicht aus. Welche Bedeutung hat nun dieser Abbau? Wir werden später erfahren, daß tierische Membranen sogenannte kolloide Stoffe nicht durchlassen. Zu diesen gehören die Stärke und das Glykogen. Mit der Umwandlung in Dextrine vollzieht sich eine Überführung in nicht kolloide, lösliche Produkte. Diese durchdringen tierische Membranen. Falls die Verdauung nur den Zweck haben sollte, mit der Nahrung aufgenommene kolloide und daher nicht aufnahmefähige Substanzen in lösliche Spaltstücke überzuführen, dann würde der Abbau zu Dextrinen vollständig genügen. ') Vgl. S. 35. Kohlehydrate. 109 Nun wissen wir jedoch, daß auch Fermente im Darmliaual vorhanden sind, die ganz einfache, leicht lösliche Polysaccharide, wie die Disaccharide Rohrzucker, Milch- und Malzzucker unter Wasseraufnahme zerlegen können. Es sind somit Einrichtungen getroffen, um alle Polysaccharide bis zu den IJausteinen abzubauen. Es ist schwer, ja zurzeit ganz unmöglich, zu entscheiden, wie weit in Wirklichkeit im Darmkanal die Polysaccharide abgebaut werden. Es ist möglich, daß nur die einfachsten Bausteine zur Resorption gelangen, es ist jedoch auch denkbar, daß Bruchstücke von Polysacchariden resorbiert w^erden, die noch mehr als einen Baustein gebunden enthalten. So viel ist sicher, daß der Abbau soweit durchgeführt wird, bis nichts mehr an den charakteristischen Bau des aufgenommenen Polysaccharids erinnert. Es sei in dieser Beziehung noch besonders auf den Rohrund Milchzucker hingewiesen, die beide nie ungespalten zur Aufnahme gelangen. Es wird durch Fermentwirkung Baustein von Baustein gelöst, bis Abbaustufen entstanden sind, die der tierische Organismus als Ausgangsmaterialien zu neuen Synthesen oder zu anderen Zwecken benutzen kann. Die Verdauung wandelt charakteristisch gebaute Verbindungen zu indifferenten, „neutralen" Stoffen ab. Gelangen komplizierter gebaute Kohlehydrate zur Resorption, dann kann noch in der Darm^vand eine Spaltung zu den einfachsten Bausteinen erfolgen. Endlich können noch die Leberzellen eingreifen, an denen das vom Darm kommende Blut vorübergeführt wird, ehe die Übergabe an den großen Kreislauf erfolgt. Wir werden immer wieder die Frage nach dem Abbau der verschiedenartigsten Nahrungsstoffe im Magendarmkanal nur in nicht völlig befriedigender Weise beantw^orten können. Dieser Umstand findet seine Begründung darin, daß wir immer nur eine bestimmte Phase der Verdauung untersuchen können. Ob wir nun ein Tier mitten in der Verdauung töten und bestimmten Teilen des Darmes den Inhalt rasch entnehmen, oder ob wir Chymus aus Fisteln gewinnen, immer unterbrechen wir die Verdauung in einem bestimmten Augenblicke. Wir müssen daher immer alle möglichen Abbaustufen antreffen. Das Vorhandensein einer großen Menge von noch kompliziert gebauten Abbaustufen und das Zurücktreten der einfachsten Bausteine kann man ganz gut so deuten, daß eben die einfachsten Bausteine fortw'ährend von der Darmwand aufgenommen werden, während die zur Resorption noch nicht reifen Dextrine usw. weiter zerlegt worden wären, wenn wir den Abbau nicht künstlich unterbrochen hätten. Wir können leider die Verdauung außerhalb des Darmes noch nicht vollständig nachahmen. Im Darmkanale selbst finden sich Verhältnisse, die wir zurzeit nicht übersehen können. Einerseits vermögen wir die Resorption nicht zu ersetzen. Im Reagenzglas bleiben die Abbaustufen liegen. Ferner ergießen sich in den Darmkanal lortw'ährend neue Sekrete aus den verschiedenen Drüsen. Die Reaktion des Chymus wird in feinster Weise geregelt und eingestellt. Immer wieder greifen neue Momente ein. Solange wir nicht in der Lage sind, den ganzen Verdauungsvorgang künstlich bis in alle Einzelheiten nachzuahmen, wird die Frage nach dem Abbau der einzelnen Nahrungsstoffe nicht genau beantwortet werden können. Es liegt hier ein Forschunsgebiet vor, das noch kaum bearbeitet ist. Es gibt noch eine andere Möglichkeit, um zu prüfen, wie weit der Abbau der Nahrungsstoffe in Wirklichkeit geht, bevor eine Aufnahme in den V. Vorlesung. Kohlehydrate. 110 großen Kreislauf erfolgt. Es ist dies die Untersuchung des Blutes auf die Abbaustufen der Polysaccharide nach A'erfütterung von solchen. Es ist bis jetzt im Blute mit absoluter Sicherheit nur Traubenzucker nachgewiesen worden. Dextrine, Maltose usw. scheinen normalerweise nicht im Blute vorzukommen. Es sei in diesem Zusammenhange auch an die eingangs erwähnten Versuche mit parenteraler Zufuhr von Nahrungsstoffen erinnert.^) Die erhaltenen Resultate stützen die Ansicht, daß bei der Verdauung tiefe Abbaustufen und wahrscheinlich die einzelnen einfachsten Bausteine entstehen. Wichtig ist auch die Tatsache, daß man die gesamten Kohlehydrate der Nahrung durch Traubenzucker ersetzen kann. Wir haben bereits erwähnt, daß die Kohlehydrate von den Zellen der Darmwand aufgenommen werden. Es scheint, daß auch von der Magenwand einfache Kohlehydrate findet jedoch in der ') Vgl. S.96. resorbiert werden können. Hauptsache im Dünndarm statt. Die Resorption Vorlesung VI. Kohlehydrate. V. Verhalten der Kohlehydrate im Zellstoffwechsel. Ihr Auf- und Abbau. Die Stoffwechselzwischen- und -endprodukte der Kohlehydrate. Wir wollen nun versuchen, den resorbierten Kohlehydraten von der weg- auf allen ihren Wegen, bis sie in die Stoffwechselendprodukte Kohlensäure und Wasser verwandelt sind, zu folgen. Wir stoßen dabei sofort auf große Schwierigkeiten. Normalerweise erfolgt nämUch der Abbau der Polysaccharide stufenweise. Diese Einrichtung bewirkt, daß von JNIoment zu Moment immer nur Spuren resorptionsfähiger Produkte Darmwand Diese bilden sich an den verschiedensten Stellen der großen Oberfläche des Dünndarms. Sie werden von den Darmzellen aufgenommen und offenbar rasch weitergegeben. Es sind zwei Wege möglich, um die resorbierten Stoffe den Körperzellen zuzuführen. Einmal können sie direkt dem Blute Pfortader tibergeben werden, oder aber der Transport erfolgt zunächst auf dem Lymphweg. Im letzteren Falle können wir die gesamten aufgenommenen Stoffe dadurch einer Kontrolle unterwerfen, daß wir da, wo der Ductus thoracicus sich in die Vena anonym a ergießt, eine Fistel anlegen und den durch sie ausfließenden Chylus sammeln. Es hat sich gezeigt, daß die resorbierten Kohlehydrate in der entstehen. — — Hauptsache direkt der Blutbahn übergeben werden. Wir könnten nun das Pfort ad erblut auffangen und feststellen, was für Kohlehydrate zur Aufnahme gelangen. Derartige Versuche sind auch durchgeführt worden. Man konnte zeigen, daß der Gehalt des Pfortaderblutes an Traubenzucker anstieg. Manche Autoren wollen auch wie Dextrine, beobachtet haben, doch sind die Versuche nicht mit genügender Genauigkeit durchgeführt worden, um Beweiskraft zu haben. Ferner müßte man a priori erwarten, daß das Blut des großen Kreislaufes anzeigt, was für Arten von Kohlehydraten zur Piesorption gelangen, wenn man solche verfüttert. Die nach dieser Pachtung durch- Polysaccharide, geführten Untersuchungen ergaben keine sicheren Befunde. Es zeigte sich zunächst überraschende Tatsache, daß auch nach Aufnahme großer Mengen von Kohlehydraten der Gehalt des Gesamtblutes an diesen in recht engen Grenzen unverändert bleibt. Wir stehen folgenden Tatsachen gegenüber. Wir beobachten, daß ein Tier in wenigen Stunden eine größere Menge von Kohlehydraten aufnimmt, verdaut und resorbiert. Wir können diese Menge recht genau die 1 1 2 VI. Yorlesuug. bestimmen, indem wir das Gewicht der verfütterten Kohlehydrate festDie Menge davon, die im Darmkanal ein Raub der Darmflora wird, wenn wir von den reinen Pflanzenfressern ausgehen, so gering, daß ist. wir sie vernachlässigen dürfen. Bestimmen wir den Gehalt des Blutes an Traubenzucker vor und nach der Verfütterung der Kohlehydrate, dann finden wir einen nur unbedeutenden Unterschied. Wo sind die Kohlehydrate geblieben V stellen. Es sind folgende Möglichkeiten gegeben. Einmal könnte der Abbau der Kohlehydrate im Darmkanal oder in der Darmwand oder endlich in der Leber über die Monosaccharide hinausgehen. Es könnten also Abbaustufen entstehen, die nichts mehr mit den Kohlehydraten gemein haben. Es sind nach dieser Richtung mehrere Hypothesen aufgestellt worden. So vermutete z. B. Pavy ^) eine Umwandlung der resorbierten Kohlehydrate in Fett in der Darmwand. Andere Forscher dachten an die Bildung einfacherer Abbaustufen ans dem Traubenzucker. Diese sollten dann das Baumaterial für die verschiedenartigsten Synthesen abgeben oder aber von den Zellen weiter gespalten werden. Keine dieser Ansichten konnte experimentell bestätigt werden. Gegen die letztere Anschauung spricht folgender Umstand: Wir haben früher schon hervorgehoben, daß die Zellen Energie zu ihren verschiedenartigen Verrichtungen brauchen. Diese erhalten sie, indem sie organische Stoffe spalten und oxydieren. Interessanterweise geht nun die Loslösung der einzelnen Bausteine aus zusammengesetzten Verbindungen unter W^asseraufnahme fast ohne Wärmetönung vor sich, d. h. es wird sozusagen keine Energie frei, ferner verursacht der umgekehrte Vorgang, nämlich die Wiederaneinanderfügung der Bausteine unter Wasserabspaltung fast keinen Energieverbrauch. Sobald aber ein Baustein weiter gespalten wird, wird meistens Energie frei und umgekehrt verläuft im allgemeinen die Synthese von einfacheren Spaltstücken aus mit Energieverbrauch. Würden die Kohlehydrate im Darmkanal über die Bausteine hinaus gespalten, dann müßte im Darmrohr Energiq frei werden, von der den Körperzellen nur in beschränktem Maße etwas zugute käme nämhch in der Hauptsache nur den benachbarten Darmzellen. Der tierische Organismus müßte dann jenseits der Darmwand oder in dieser selbst Energie aufwenden, wenn er die aufgenommenen Spaltstücke wieder zu bestimmten Verbindungen vereinigen wollte. — Die meisten Forscher stehen wohl auf dem Standpunkte, daß in der Hauptsache Traubenzucker in das Blut der Pfortader übergeht. Da nun der Zuckergehalt des Blutes in engen Grenzen unverändert bleibt, so könnte man daran denken, daß die Spuren von Glukose, die von Augenblick zu Augenblick zur Piesorption kommen, sofort von den Körperzellen aufgenommen werden. Nun treffen wir in den Körperzellen den resorbierten und der Blutbahn übergebenen Traubenzucker auch nicht oder nur zum geringsten Teil unverändert an. Das wäre verständlich, wenn die Körperzellen den Zucker sofort weiter verarbeiten würden. Sie könnten ihn z. B. abbauen. Ein derartiges Verschwinden großer Mengen von Traubenzucker müßte sich nachweisen lassen. Die (ilukose liefert nämlich beim Abbau schließlich als letzte Stoffwechselprodukte Kohlensäure und W^asser. Wenn ) F. W. Pari/: Über den Kohlehydratstoffwechsel. Deutsche Ausgabe von Ktirt Moeckel. Wilh. Engelmann. Leipzig 1901. Kohlehydrate. 113 wir nun ein Tier in einen Apparat einsperren, der uns gestattet, genau zu bestimmen, wieviel Sauerstoff es verbraucht und wieviel Kohlen- säure es ausscheidet, dann können wir leicht verfolgen, ob die Verfütterung von Kohlehydraten bei sonst ganz gleich bleibenden Bedingungen einen Einfluß auf den Sauerstoffverbrauch und die Kohlensäureausscheiduug hat. Es ist dies nicht der Fall. Noch auf einem anderen Wege muß sich entscheiden lassen, ob die aufgenommeneu Kohlehydrate direkt abgebaut werden. Wir haben bereits erwähnt i), daß beim Abbau der Kohlehydrate zu den Endprodukten Kohlensäure und Wasser die gleiche Energiemenge frei wird, die verwandt worden ist, um sie aus Kohlensäure und Wasser aufzubauen. Bestimmen wir bei einem ruhenden Tier den Energie Wechsel vor und nach Verfütterung von Kohlehydraten, dann finden wir keine Anhaltspunkte dafür, daß solche zu den erwähnten Stoffwechselendprodukten abgebaut worden sind. Der Stoffumsatz bleibt unbeeinflußt Schon diese Beobachtungen ergeben das wichtige Gesetz, daß der Stoffwechsel der Zellen des tierischen Organismus von diesen selbst reguliert wird und unter normalen Verhältnissen nicht durch die aufgenommenen Kohlehydrate direkt beeinflußt werden kann. Da worden ist, daß die resorbierte Glukose den Geweben auch nur annähernd in ihrer Gesamtheit anzutreffen ist und andrerseits feststeht, daß sie nicht völlig abgebaut wird, bleibt nun nur noch die eine Möglichkeit übrig, daß sie in anderer Weise als durch Abbau verändert worden ist. Entweder ist sie in den Körperzellen so umgewandelt worden, daß sie keine direkten Beziehungen zu den Kohlehydraten mehr hat oder aber, es ist z. B. ein anderes Kohlehydrat aus der Glukose gebildet worden. einerseits festgestellt weder im Blute noch in Am schnellsten mußte man ohne Zweifel dem Verbleib des resorbierten Traubenzuckers auf die Spur kommen, indem man systematisch allen Möglichkeiten nachging. Da die Glukose den Blutweg einschlägt, so war es am zweckmäßigsten, dieser Bahn zu folgen. Das gesamte vom Darm kommende Blut durchfließt bei den Säugetieren 2) zunächst die Leber. Es treten hier ganz eigenartige Kreislauf Verhältnisse auf. Das Blut wird durch die Bildung von ungezählten ^'erzweigungen und Verästelungen auf eine sehr große Oberfläche ausgebreitet. Die feinsten Zweige, die in die Venae centrales der Leberläppchen übergehen, umschließen Leberzellen. Es wird auf diese Weise das mit resorbierten Stoffen aUer Art beladene Blut an zahlreichen Leberzellen vorbeigeführt. Sollte nicht schon hier die aufgenommene Glukose verändert werden? Diese Vermutung ist schon deshalb angebracht, weil Beobachtungen vorhegen, wonach der Zuckergehalt des Pfortaderblutes von der Resorption von Kohlehydraten direkt abhängig ist, während wir im Blut, das sich aus der Lebervene in die Vena cava inferior ergießt, bereits einen solchen Einfluß nicht immer sicher feststellen können. Offenbar ist dem Blute Glukose entzogen worden. Nun haben wir früher schon darauf hingewiesen, daß im tierischen Organismus ein Polysaccharid vorkommt, das Glykogen, das bei der M Vgl. S. 79. ^) Andere Tierklassen haben zum Teil einen „Is'ierenpfortadcrkreislauf". A b eih al d e n il , Vh.vPiol(igi8che Cherair. T. Teil, 5. Aufl. g ^'^i- J[14 Vorlesung. Spaltung unter VVasseraufnahme Glukose liefert. Dieses Kohlehydrat ist, wie schon früher erwähnt, in der Leber entdeckt worden. i) Bald wurde man darauf aufmerksam, daß die Leberzellen nicht immer gleich viel Glykogen enthalten, sondern daß ihr Gehalt an diesem Polysaccharid von der Nahrungsaufnahme und insbesondere von der Zufuhr von Kohlehydraten abhängig ist. Man machte sich nun bierten Traubenzuckers im folgendes Bild über das Verhalten des resortierischen Organismus. Die Glukose wird den Leberzellen zugefühi't. Diese belassen dem Blut eine bestimmte Menge davon. Das Zuviel wird von ihnen aufgenommen und sofort zu dem Polysaccharid Glykogen aufgebaut. Ein Molekül Glukose wird an das andere angelagert. Jedesmal wird ein Molekül Wasser frei. Wahrscheinlich geht die Synthese über die Maltose. Man nimmt allgemein an, daß Fermente an dieser Synthese beteiligt sind. Das Glykogen ist ein Kolloid. Es lagert in den Leberzellen, ohne direkte Beziehungen zu den Bausteinen der Zelle zu haben. Es ist als ein Reservestoff aufzufassen, der dem Stoffwechsel so lange entzogen bleibt, bis Bedarf an Glukose auftritt. Das Glykogen wird dann nicht als solches von der Leberzelle abgegeben, sondern es erfolgt zunächst ein Abbau, und zwar tritt eine Fermentgruppe in Wirksamkeit, die vollständig entsprechend arbeitet, wie die Diastase der Säfte des Darrakanals. Es entstehen Dextrine. Auch Maltose tritt auf. Diese wird durch die Maltase in zwei Moleküle Traubenzucker zerlegt. Die Glukose geht ins Blut über und wird jener Stelle zugeführt, an der sie gebraucht wird, bzw. sie ersetzt einen Mindergehalt des Blutes an Glukose, weil Organzellen bereits dem Blute Traubenzucker entzogen und so den Gehalt des Blutes an diesem Kohlehydrat herabgedrückt haben. Das Glykogen spielt im tierischen Organismus eine ganz entsprechende Rolle wie in der Pflanzenwelt die Stärke. Diese Anschauungen sind durch die folgenden Beobachtungen sicherworden. Wir wollen annehmen, daß wir zehn Kaninchen, die dem gleichen Wurf entstammen und ganz gleichmäßig ernährt worden sind, zehn Tage ohne jede Nahrungszufuhr lassen. Nun töten wir fünf von diesen Tieren, entnehmen sofort jedem die Leber und stellen fest, wieviel Glykogen sie enthält. Dem Reste der Versuchstiere geben wir Stärke oder Rohrzucker oder Traubenzucker zu fressen. besten verfüttern wir den einen Tieren das eine Kohlehydrat und den anderen ein anderes. Etwa sechs bis acht Stunden nach der Fütterung töten wir auch diese Tiere und stellen sofort den Glykogengehalt der Leber fest. Bei den Hungertieren finden wir entweder nur Spuren von Glykogen oder eine ganz geringe Menge, wiihrend alle jene Tiere, die kurz vor der Tötung Kohlehydrate erhalten hatten, viel von diesem Polysaccharid in der Leber aufweisen. Diese Versuchsanordnung können wir dazu benutzen, um festzustellen, ob eine bestimmte verfütterte Substanz Glykogenbildner ist, d. h. ob nach ihrer Aufnahme in den Organismus in der Leber Glykogen entstanden ist. 2) gestellt Am 1) Vgl. hierzu S. 62 ff. Beobachtungen bei E. W. Fßiiger: Das Glykogen und seine Beziehungen zur Zuckerkrankheit. 2. Aufl. Maitin Hager. Bonn. 1905; Max Cremer: Ergeb. d. Physiol. 1. 803 (1902). -) Vgl. die Literatur über derartige Kohlehydrate. 115 Es sei schon hier erwähnt, daß wir die Leber noch auf andere Arten olykogenarm machen können. Eine dieser Methoden ist die Leistung von Arbeit. Lassen wir zum Beispiel einen Hund bis zur Erschöpfung laufen, dann erweist sich seine Leber als glykogenarm. Die gleiche Wirkung erhält man nach Vergiftung von Tieren mit Strychnin. Dieses Gift bewirkt erhöhte Reflexerregbarkeit. Bei der geringsten Erschütterung treten Muskelkrämpfe ein. Auch dadurch werden s^orhandene Glykogenvorräte schließlich erschöpft. •- Es fragt sich nun, ob der erwähnte Befund, daß nach Verfütterung von Kohlehydraten die Leber einen hohen Glykogengehalt aufweist, eindeutig beweist, daß die resobierte Glukose in Form von Glykogen abgelagert worden Man hat selbstverständlich diesen ^'ersuch oft wiederholt und immer als Kontrolltiere Hungertiere oder aber durch Arbeit erschöpfte Tiere benutzt. Es wäre gesucht, wollte man annehmen, daß zufälligerweise die getöteten ist. Kontrolltiere weniger Glykogen in der Leber besessen hätten als die übrigen Dagegen läßt sich ein anderer, sehr gewichtiger Einwand erheben. Er ist zwar sehr unbequem und wird von vielen Forschern als zu weit- Tiere. gehend betrachtet und doch sind im Laufe der Zeit manche Beobachtungen bekannt geworden, die zeigen, daß man die Beweise für bestimmte Befunde nicht eindeutig genug fordern kann. Man könnte sich nämlich ganz gut vorstellen, daß die Verfütterung der Kohlehydrate eine indirekte Wirkung entfaltet. Es könnten Stoffe in Glykogen übergehen, die bereits im Hungertier vorhanden sind.. Die verfütterten Kohlehydrate könnten beispielsweise zu bestimmten Zellfunktionen herangezogen werden, die beim Hungertier bisher auf Kosten von Stoffen bestritten wurden, die jetzt zur Glykogenbildung frei werden. Wir verfügen über zahlreiche Beobachtungen, die eindeutig erweisen, daß die Bausteine des Glykogens nicht von Kohlehydraten herzustammen brauchen. Gewisse Bausteine der Eiweißstoffe können z. B. Kohlenstoff ketten zur Glykogenbildung liefern. Wahrscheinlich führt der Weg immer über die Glukose. Warum sollte nicht auch nach der Kohlehydratfütterung eine solche Art der Glykogenbildung einsetzen? Der direkte Versuch mußte entscheiden. sich studieren, d. h. losgelöst W'ir können die Lel)er für vom übrigen Organismus. Am besten eignet die Schildkrötenleber, i) Sie läßt sich gut durchgelungen, auch die Säugetierleber zu derartigen Versuchen zu verwenden. -) Ein derartiger Versuch wird in seinen wesentlichsten Punkten, wie folgt, durchgeführt. W-ir wählen eine bestimmte Flüssigkeit, um die Leber zu durchströmen. Entweder nehmen wir Blut oder eine sich zu solchen Studien bluten. Es ist sogenannte Nährlösung. Diese besteht aus Wasser, in dem bestimmte Salze in bestimmter Konzentration vorhanden sind. Der Durchspülungsflüssigkeit können wir nun bestimmte Substanzen zusetzen. In unserem Falle wählen wir zunächst Glukose. W'ir setzen z. B. Blut eine ganz bestimmte Menge davon zu. Nun lassen wir es durch die Pfortader ein- und durch die Lebervene abfließen. W^ir wiederholen diesen Kreislauf längere Zeit. Nun analysieren wir erstens die gesammelte Durchströmungsflüssigkeit auf Zucker und finden, daß fast der ganze zugesetzte Zucker verschwunden wir rasch den Glykogengehalt der Leber. Wir ist. Zwei ens bestimmen 1) ^) Karl Grube: I'Jlüqers Arr-hiv. 107. 490 (1905). J. de Met/er: J. internat. de Piiysiol. 8. 204 (1909). VI. Vorlesung. IIQ möchten gerne wissen, ob und um wieviel die Leber an diesem Polysaccharid zugenommen hat. Es wird deshalb beim Beginn des Versuches ein Stück der Leber fortgenommen und sein Gehalt an Glykogen bestimmt. AVir nehmen an, daß die Leber das Glykogen annähernd gleichmäßig verteilt enthält. Es hat sich gezeigt, daß diese Annahme nur bedingt richtig ist. Immerhin begeht man keinen großen Fehler, wenn man die gesamte Leber wiegt, dann in einem gewogenen Teil das Glykogen bestimmt und berechnet, wie viel Glykogen das zur Durchblutung verbleibende Leberstück enthält. Die Zunahme an Glykogen am Schlüsse der Durchblutung entsprach ungefähr der aus dem Blute verschwundenen Glukose. wohl kaum mit der anders gedeutet werden, als Durchspülungsflüssigkeit zugeführten Glukose Glykogen gebildet haben. Man hat die geschilderte Methodik dazu benutzt, um verschiedene Verbindungen als Bildungsmaterial für Glykogen zu prüfen. Fruchtzucker und Galaktose liefern Glykogen, die Disaccharide Rohr- und Milchzucker i) nicht, obwohl sie jene Komponenten enthalten, aus denen die Leberzellen das Polysaccharid bereiten. Es muß der Synthese eine Spaltung der genannten Disaccharide in ihre Bausteine vorausgehen. Die Leberzellen können diese Hydrolyse nicht durchführen, deshalb sind die genannten Disaccharide für Pentosen vermochten die Leberzellen auch nicht sie nicht verwertbar. zur Glykogenbildung zu verwerten, dagegen erwiesen sich z. B. Glyzerin und andere Verbindungen, auf die wir noch zurückkommen, als Glykogen- daß Dieser Befund Leberzellen die kann aus der bildner. -) Die positiven Befunde einer Glykogenbildung aus einem bestimmten Materiale sind wohl eindeutig, nicht aber die negativen. Es darf nicht daraus, daß bei einem solchen Durchblutungsversuch eine bestimmte Verbindung nicht das erwartete Produkt liefert, geschlossen werden, daß nunmehr der betreffende Organismus, dessen Organ man untersucht hat, die betreffende Umwandlung überhaupt nicht zu vollziehen vermag. Einmal kann ein anderes Organ den Vorgang durchführen, und ferner ist es denkbar, daß mehrere Zellarten zusammenwirken müssen, um ein bestimmtes Produkt in ein anderes umzuwandeln. Das eine Organ bereitet den Vorgang vor, ein anderes vollendet ihn. So wissen wir mit Sicherheit, daß bestimmte Aminosäuren Glukose und damit den Baustein des Glykogens liefern. Ist jedoch die Zufuhr der Aminosäuren auf die Leberzellen beschränkt, dann scheint, wie schon bemerkt, keine Verwertung zur Glykogenbildung einzutreten. ») • allein Wir müssen uns nun die Frage vorlegen, ob \nr mit der Tatsache auskommen, daß die Leberzellen Traubenzucker in Form von Gly- kogen speichern können. Es ist klar, daß diese Ablagerung eine Grenze haben muß. Jede einzelne Zelle kann nur eine bestimmte Menge von Glykogen aufnehmen. Wir können mit dem Mikroskop die Schollen von Glykogen sehen und feststellen, daß sich mit ihrem Verschwinden die Zellstruktur nicht ändert, weil das Polysaccharid eben nichts mit dem Bau 1) ) ') Vgl. Ernst Weinland: Zeitschr. f. Bk>logie. 40. 374 (1900). Vgl. avich Vorlesung X. Karl Grube: Fßüfjers Archiv. 122. 451 (1908). — Karl Grube: 1. c. Kohlehydrate. 117 der Zelle zu tun hat. Wir erblicken Zellen, die mit Glykogen vollgepfropft andere enthalten weniger davon. sind, Direkte Versuche haben ergeben, dalj die Leber des Menschen etwa IbOg Glykogen aufnehmen kann. Was geschieht, wenn mehr Glukose als zur Bildung dieser 150 ^^ Glykogen notwendig ist, zugeführt wird? Die Möglichkeit einer Mehrzufuhr ist immer gegeben. Vor allen Dingen wird normalerweise die Leber ihre Speicher nie ganz geleert haben. Es wird sich somit meist schon nach einer relativ geringen Traubenzuckerzufuhr die Frage ergeben, wo die Glukose bleibt, nachdem die Leber mit Gly- kogen beladen ist. Man fand bald, daß wohl alle Körperzellen Glykogen speichern können. Vor allen Dingen stellen die Muskelzellen wichtige Speicher für Glykogen dar. Auch hier hat man direkte Versuche durchgeführt und Muskelgewebe mit Blut durchströmt, dem man Glukose zugesetzt hatte. Sie verschwand zum größten Teil. Gleichzeitig trat in den Muskelzellen Glykogen auf. Schließlich könnten aber auch diese Speicher alle gefüllt sein und weitere Zuckermassen zur Resorption gelangen. Was geschieht dann mit der Glukose V Der Zuckergehalt des Blutes und der Gewebe steigt auch in diesem Falle unter normalen Verhältnissen nicht an. Eingehende Unter- imergeben, daß der tierische Organismus in Fett umzuwandeln und in dieser Form Glukose zu speichern. Die Überführung soll zum Teil schon in der Leber suchungen stande haben Zucker ist, einsetzen, i) Der Beweis, daß Kohlehydrate in Fett übergeführt werden können, kann auf verschiedene Arten geführt werden. Einmal kann man einem möglichst fettarmen Tiere eine Nahrung zuführen, die neben \iel Kohlehydraten Eiweiß und möglichst wenig Fett enthält. Das Tier wird dann, nachdem es an Körpergewicht zugenommen hat, geschlachtet und sein Gesamtgehalt an Fett festgestellt. Gewöhnlich ist diese Versuchsanordnung in der Art durchgeführt worden, daß man Tiere des gleichen Wurfes hungern ließ. Ein Teil der Tiere wurde dann getötet und der Fettgehalt des gesamten Organismus bestimmt. Dieser Wert gab dann die Grundlage für die Berechnung der Fettzunahme nach Verabreichung einer fettarmen und kohlehydratreichen Nahrung. Es ist klar, daß bei derartigen \'ersuchen festgestellt werden muß, w'as von den verabreichten Nahrungsstoffen zur Resorption gelangt, d. h. man darf nicht einfach den Gehalt der Nahrung an Fett, Kohlehydraten und Eiweiß in Rechnung setzen, sondern man muß den Kot auf Stickstoffund Fettgehalt untersuchen. Leider sind nicht alle in dieser Richtung ausgeführten Versuche einwandfrei, weil nicht immer der Fettgehalt der Nahrung richtig bestimmt worden ist. Es liegen jedoch genügend einwandfreie Versuche vor, die für die Annahme der Überführung von Kohlehydraten in Fett beweisend sind. -) - So gibt ') ») ') z. B. X. Tscherwinsky ^) die folgenden Zahlenwerte an: P. lunkersdorf: Pftüger^ Archiv. 192. 305 (1921). Vgl. Charniewsky': Zeitschr. f. Biol. 20. 179 (1884). N. Tscherwinsky : Landwirtsch. Versuchsstationen. 29. 317 (1883). j^ Tl. Vorlesung. J[g Eiweiß Fett Das vier Monate mit Gerste gefütterte Tier ^ enthielt Das Kontrolltier enthielt der Nahrung waren 0,69 l,o6 Ar/ H,56 /rg 7.49 0,66 ,. Somit hatte das erstere Tier angesetzt. In 2,52 kg 0,96 ihm . 9,25 hg ,. zugeführt worden Unterschied . . . ,. ,. — 5,93 kg + 7,90 kg Das verwendete Versuchstier, ein junges Schwein, hatte somit 7,9 kg die nicht aus Nahrungsfett stammen konnten und für die das Eiweiß der Nahrung gar nicht in Betracht kommt, angesetzt. Fett, Die gleiche Feststellung, nämUch die Fähigkeit des tierischen OrgaKohlehydrate in Fett umzuwandeln, ist auch durch eine andere Versuchsanordnung bewiesen worden. Es wurde der Gaswechsel i) eines mit viel Kohlehydraten ernährten Tieres verfolgt. Kennt man den Gehalt des möglichst fettarmen und kohlehydratreichen Futters an Kohlenstoff, Eiweiß und Kohlehydraten, so kann man einmal durch die Bestimmung des Stickstoffgehaltes des Harnes den im Körper zurückgebliebenen Anteil an Eiweiß ermitteln und .aus der ausgeatmeten Kohlensäure plus dem mit dem Harnstoff den Körper verlassenden Kohlenstoff den im Körper verbleibenden berechnen. Man fand dabei für den im Körper verbliebenen Kohlenstoff derart hohe Werte, daß nur die Annahme übrig bleibt, daß aus den zugeführten Kohlehydraten Fett gebildet worden ist. Wir wollen nicht verschweigen, daß nicht bewiesen ist, daß die Fähigkeit der Fettbildung aus Kohlehydraten allen tierischen Organismen zukommt. Man hat zu den Versuchen Tiere gewählt, die zur Fettmast geeignet sind. Es ist wohl möglich, daß nicht jede Tierart in gleichem Maße Kohlehydrate in Fett umwandeln kann. Es sind sicherlich auch individuelle Unterschiede vorhanden. Vielleicht spielt auch das Alter eine gewisse Rolle. Jedenfalls muß das ganze Forschungsgebiet noch nach allen Seiten erweitert und vertieft werden. Es ist uns nicht bekannt, ob der tierische Organismus in erster Linie die Lagerstellen der Leberzellen für Glykogen füllt und dann die übrigen Speicher und endlich bei zu großem ,.Andrang" an Glukose zur Umwandlung in Fett greift. Es ist eine solche Annahme nicht sehr wahrscheinlich. Der Organismus wird je nach Bedarf das Glykogen an verschiedenen Stellen entstehen lassen und ebenso zur Fettbildung aus Kohlehydraten nicht nur dann greifen, wenn für die überschüssige Glukose kein Unterkommen miehr vorhanden ist. Es fragt sich nun, was aus den Glykogenvorräten wird. Wie wir schon gesehen haben, verschwindet das Glykogen allmählich aus den Speichern, sobald keine Nahrung zugeführt und sobald Arbeit geleistet wird. Der erste, der die Beziehungen des Glykogens zur Muskelarbeit klar erkannte, war Claude Benmrd:^) Er fand, daß im Winterschlaf befindliche Tiere große Mengen von Glykogen in der Leber besitzen, und zwar in den nismus, — dieser 1) Es wäre erwünscht, wenn mit Vgl. Bubner: Zeitscbr. f. Biol. 22. 272(1886). Methode noch mehr und länger dauernde Versuche ausgeführt würden. ) Claude Bernard: Compt. reud. de lAcad. des Sciences. 48. 673 [vgl. Anmer- kung pag. 683 (1859)]. Kohlehyilratc. 119 in dem Gewebe der Muskeln und den Lungen. Sobald die Tiere erwachten und sich bewegten, sah Claude Bernard das Glykogen schwinden. Ferner machte er die Beobachtung, daß bei gut genährten Säugetieren und \'ögeln das Muskelgewebe in Ruhe sei es spontan, sei es künstlich nach Durchtrennung des dieses versorgenden Nerven Glykogen anhäuft, das wieder verschwindet, sobald der Muskel Arbeit Leberzellen, — — leistet. Direkte ^'ersuche sind von S. Weiß'^) ausgeführt worden. Er verden Glykogengehalt der hinteren Extremitäten des Frosches. Die Muskeln des einen Beines wurden durch elektrische Reizung bis zur Erschöpfung zur Kontraktion gebracht, während die andere Extremität, die Das Glykogen der tätigen Muskeln zeigte zur Kontrolle diente, ruhte. im ^'ergleich zu dem der ruhenden eine Abnahme um 2427 oO^So/o. Endlich hat Th. C/iandelon^-) folgenden Versuch angestellt. Er durchschnitt einem Kaninchen die Nn. ischiadici und crurales und fand in den gelähmten Muskeln nach 2 ^5 Tagen eine Zunahme an Glykogen von glich — — 5-51— 172-4VoAuf andere Weise ist endlich Eduard Küh^) zu demselben Resultate Er ließ einen gut genährten Hund einen schweren Wagen ziehen. Das Versuchstier wog 45ö0% und lief im ganzen 1) Stunden 40 Minuten. Es wurde durch Verbluten getötet. Die Glykogenbestimmung ergab einen Gesamtbestand von 5205 </, d. h. pro Kilogramm Tier M4<7 Glykogen. Der Glykogengehalt eines gut genährten, nicht ermüdeten Hundes beträgt pro Kilogramm bis 38 g. KiUz hat an weiteren drei Hunden denselben Versuch mit demselben Erfolge wiederholt. gelangt. Es sind im Laufe der Zeit eine große Anzahl von Untersuchungen immer eindeutiger erbrachten, daß die Muskelzellen in erster Linie auf Kosten von Kohlehydraten ihre Leistungen vollziehen. Diese Anschauung stieß zunächst auf großen Widerstand, weil die Lehre von Justus Liebig^), wonach der Muskel in erster Linie Eiweißkörper als Energiequelle benutzen sollte, großen Anklang gefunden hatte. Sie geriet ins Wanken, als die Forscher Fick und WisUcenus^) bei der Leistung einer bedeutenden Steigarbeit Besteigung des 1956 wt mitgeteilt worden, die alle den Beweis — dem — gelegenen Faulhorns in exakter Weise feststellen Umsatz der Eiweißstoffe in keiner Weise den Aufwand an Energie decken konnte, der notwendig war. um ihr Körpergewicht auf die genannte Höhe zu bringen. Wir kommen später noch auf diesen Versuch zurück. Es folgten dann sogenannte Stoffwechselversuche. Man Keß z. B. Tiere eine bestimmte Arbeit leisten und stellte den Verbrauch an Eiweißstoffen fest. Alle diese Versuche führten zum Resultate, daß die Lehre Liebigs unhaltbar ist. Die Muskeln verwenden bei ihrer Tätigkeit in erster Linie Kohlehydrate. über Spiegel des Brienzersees konnten, daß der Weiß: Sitzuugsber. der Wiener Akad. der Wiss. 04. Abt. 1. Vgl. auch If. Marcuse Chandelon: I'fiiiffers Archiv. 13. 62(5 (1876). rjlüc/erB Arch. 39. 425 (1886)."— Eduard Manche: Zeitschr. f. Biologie. 25. 163 (1889). ^) Eduard Külz: Beiträge zur Keuntnis des Glykogens. 41 (1891). ) Justus Liehig: Chemische Briefe. 1857. ^) A. Fick und J. Wislicenus : Vierteljahresschrift der Züricher naturf. Gesellsch. 10. 317 (1865). Vgl. auch Vgl. dazu ./. Liebiq: Liebig?, Auualeu. 153. 1 (1870). C. TojV; Zeitschr. f. Biol. 6. 305 (187(1). ') S. -) 'Jh. — — — 120 ^ ''• Vorlesung. Von ganz besonderer Bedeutung wurde die folgende Feststellung. Die Kohlehydrate werden schließlich von den tierischen Zellen zu Kohlensäure und AVasser abgebaut. Zum Abbau einer bestimmten Menge von Kohlehydraten zu Kohlensäure und Wasser ist eine ganz bestimmte Quantität von Sauerstoff notwendig. Ferner bildet sich eine bestimmte Menge von Kohlensäure. Stellt man den verbrauchten Sauerstoff der gebildeten Kohlensäure gegenüber, dann ergibt sich, daß die Volumina beider Gase gleich sind: CO, -7—=- = 1. Man nennt das Verhältnis von CO2 den respiratorischen Quotienten. Kein anderer Nahrungsstoff den gleichen Wert. Der respiratorische Quotient ist für die Fette und Eiweißstoffe kleiner als 1. Diese Tatsache ermöglicht es, festzustellen, ob bei einer bestimmten Leistung tierischer Zellen hauptsächlich oder gar ausschließlich Kohlehydrate verbraucht werden, oder ob sie sich auf Kosten anderer Nahrungsstoffe vollzieht. Es zeigte sich, daß bei großen Arbeitsleistungen normal ernährte Tiere einen respiratorischen Quotienten aufweisen, der 1 sehr nahe kommt. zu 0-2 liefert Nachdem wir spalten festgestellt haben, daß die Muskelzellen Kohlehydrate und oxydieren, um direkt oder indirekt Energie zur Leistung von Arbeit zu erhalten, ergibt sich ganz von selbst die Fragestellung, in welcher Form die Kohlehydrate zur Oxydation gelangen. Wird Glykogen als solches zu Kohlensäure und Wasser abgebaut? Es ist dies nach allen Beobachtungen ohne Zweifel nicht der Fall Die Zelle arbeitet möglichst ökonomisch. Jede einzelne Zellart stellt ein Glied in einem großen, in mannigfaltiger Weise zusammenarbeitenden Zellstaate dar. Zwar hat jede Zellart eigene Aufgaben zu erfüllen und geht daher eigene Wege, sie muß aber in mancher Beziehung sich doch dem gemeinsamen Haushalte aller anderen Zellen anschließen. Eine der für das Leben der Zellen wichtigsten gemeinsamen Arbeiten aller Zellarten ist die Einhaltung einer bestimmten Körpertemperatur. Der Organismus verfügt über mancherlei Einrichtungen, um diese in engen Grenzen gleich zu erhalten. Es sei hier nur kurz erwähnt, daß der Umfang der Oxydationsvorgänge die Wärmebildung steigern oder einschränken kann, und daß ferner Vorrichtungen vorhanden sind, um die Wärmeabgabe je nach Bedarf zu regeln. Würde die Zelle ein Polysaccharid ohne weiteres in Kohlensäure und Wasser zerlegen, dann würde mit einem Schlage jene Wärmemenge frei werden, die beim Aufbau des betreffenden Kohlehydrates aus Kohlensäure und Wasser aufgewendet worden ist. Der Zelle wäre es sehr erschwert, wenn nicht unmöglich gemacht, den Energieverbrauch der augenblicklichen Leistung anzupassen. Eine iVbstufung der Infreiheitsetzung der Energie wäre nicht oder doch nur recht unvoll- kommen möglich. Alle Körperzellen verfügen nun über Einrichtungen, u m die zusammengsetzten Nahrungstoffe in einfachere Bruchstücke zu zerlegen. Der Abbau ist ein hydrolytischer. Er wird durch Fermente herbeigeführt, die die Zelle selbst bereitet. Sie sind ihre Werkzeuge, mit denen sie sich das zu oxydierende Material vorbereitet. Aus Glykogen werden durch Diastase Dextrine gebildet. Ferner entstehen Maltose und Glukose. Es vollziehen sich in den Körperzellen ganz entsprechende Vorgänge, wie im Darmkanal. Während jedoch die Fermente im Verdauungskanal be- Kohlehydrate. 121 ganz verschiedenartige Materalien abzubauen haben, steht die immer vor gleichen Aufgaben. Dank der Tätigkeit der Verdauungsdriisen fließt den Körperzellen unentwegt ein gleichartiges Nährmaterial zu. Es entstehen Abbaustufen, die der Zelle vertraut sind. Es unterliegt keinem Zweifel mehr, daß die einzelne Zelle Traubenzucker abbaut. Dieser ist ihr entweder direkt zugeführt worden, oder sie hat ihn selbst durch Spaltung aus einem Polysaccharid, z. B. aus Glykogen, bereitet. Es fragt sich nun, welche Abbaustufen durchlaufen werden, bis Kohlensäure und Wasser als letzte Stoffwechselprodukte entstanden sind. Alles spricht dafür, daß eine direkte Oxydation von Glukose zu den genannten Produkten wohl kaum stattfindet. Der Abbau ist vielmehr ein stufenweiser. Sein Studium fesselt uns aus vielen Gründen in besonders hohem Maße. Die genaue Kenntnis aller Stufen, die beim Abbau einer Verbindung durchlaufen werden, gibt uns einen tiefen Einblick in die Vorgänge des Zellstoffwechsels und die Leistungen der einzelnen Zellarten. Wir wollen nicht nur wissen, welche Wege eingeschlagen werden, um zu den einzelnen Stoffwechselprodukten zu gelangen, Aveil uns die einzelnen Phasen wichtige Einblickein die Art und Weise geben, wie die Zelle arbeitet, sondern vor allem auch deshalb, weil die neueste Forschung mit Sicherheit ergeben hat. daß die tierische Zelle imstande ist, Verbindungen bestimmter Art in solche ganz anderer Struktur zu verwandeln. So können die Kohlehydrate, wie schon betont, in Fett übergehen. Welche Zwischenstufe beim Abbau der Glukose bildet den Ausgangspunkt zu dieser Synthese? Welches ist das einfachst gebaute Material, das noch zu Synthesen Verwendung finden kann? Gibt es nur eine Art des Abbaues oder paßt die einzelne Zelle die Zerlegung der Glukose bestimmten Zwecken an? Das sind alles Fragen von grundlegender Bedeutung für die ganze Auffassung des Stoffwechsels. ständig einzelne Zelle Die Beantwortung der gestellten Fragen ist außerordentlich schwierig. kann auf folgenden Wegen versucht werden. Wir verfüttern bestimmten Tieren alle jene Verbindungen, die wir von der Glukose ableiten können. Wir stellen uns z. B. die Frage, ob Glukonsäurei) vom tierischen Organismus verwertet wird. Dann gehen wir zur Zuckersäure^) über. Oder wir geben Milchsäure usw. Führen wir diese Substanzen per os zu, dann drohen uns Störungen von selten der Darmflora. Die Darmbakterien können die verfütterten Produkte weiter umwandeln. Umgehen wir den Darmkanal und spritzen wir die Stoffe in die Blutbahn, dann schaffen wir zum vorneherein ganz unnatürliche Bedingungen. Der tierische Organismus arbeitet immer in sehr großen Verdünnungen. Wollen wir diesem Umstände Rechnung tragen, dann müssen wir entweder viel Flüssigkeit zuführen, oder aber die Substanz muß sehr langsam in Spuren dem Blute übergeben werden. Es ist klar, daß unter diesen Umständen die erhaltenen Resultate nicht Sie eindeutig sein können. In vieler Beziehung erhalten wir, wie es schon i)ei der Frage nach der Herkunft des Glykogens der Leberzellen erörtert worden ist-), bestimmtere Ergebnisse, wenn wir ein einzelnes Organ mit einer Flüssigkeit durchströmen, der wir die zu prüfende Substanz zusetzen. Wir sprechen von ') '-) Vgl. S. 36. Vgl. hierzu S. 116. ^ I- J22 Vorlesung. Versuchen am ..überlebenden"' Organ. Verschwindet von dem zugeführten Produkte etwas und können wir gleichzeitig beweisen, daß dem verschwundenen Teil bestimmte, neu auftretende Verbindungen entsprechen, dann dürfen wir aus dem Befunde schließen, daß bestimmte Zellarten die zugeführte Verbindung verwerten können. Der negative Befund besagt nichts, w^eil der Einwand durchaus zu recht besteht, daß entweder die richtigen Bedingungen nicht getroffen worden sind, oder aber die Zusammenarbeit mehrerer Zellarten notwendig ist, um die betreffende Verbindung zu verarbeiten. Manche Studien sind mit einzelligen Lebewesen gemacht worden. Wir können diese auf bestimmten Nährmaterialien züchten. Die Erfahrung hat gezeigt, daß verschiedene Zellarten eine bestimmte Verbindung in ganz verschiedener Weise abbauen und verwerten. Ja, man kann oft geradezu aus der Art der Verwendung einer bestimmten Substanz auf die Art der Zellen schließen. Unter Berücksichtigung der vorhandenen, fast unüberwindlichen Schwierigkeiten, die sich dem Bestreben entgegenstellen, den stufenweisen Zuckerabbau durch direkte Versuche zu erschließen, darf es nicht überraschen, wenn wir zurzeit die Frage nach den einzelnen Abbaustufen der Glukose nur zum Teil genau beantworten können. Wir müssen uns in der Hauptsache damit begnügen, zu erörtern, welche Abbaustufen nach dem jetzigen Stand unseres Wissens in Betracht kommen. Erwähnt sei noch, daß die Zerlegung der Glukose Glukolyse genannt worden ist. Die Fermente, die diese herbeiführen, heißen glukolytische. Sie sind sicherlich nicht einheitlich in ihrer V^'irkung. Man wird sie späterhin nach dieser zu gruppieren haben. zu erwähnen, daß im Harn in verschiedener Menge eine zur Glukose in direkter Beziehung steht. Es ist dies die Glukuronsäure.i) Sie findet sich nicht in freiem Zustande, sondern Wir sprechen von ist immer mit bestimmten Verbindungen gekuppelt. Zunächst Säure ist auftritt, die Sie drehen nach links, während die Glukuronsäure selbst nach rechts dreht. Wir haben schon erwähnt, daß es zwei Formen von Glukuronsäurepaarlingen gibt.-) Alkohole können sich direkt mit Glukuronsäure paaren. Es sind eine große Anzahl derartiger Verbindungen verfüttert und der Harn dann auf den entsprechenden Paarung untersucht worden. 3) Diese Untersuchungen führten zu außerordentlich wichtigen Resultaten. Man fand nämlich, daß der tierische Organismus manche Verbindungen, die an und für sich zur Kuppelung mit Glukuronsäure ungeeignet sind, soweit verändert, bis eine Paarung eintreten kann. Auf diese Weise konnte man die Leistungen der tierischen Zellen nach mancher Richtung eindeutig klarstellen. Nach Verfütterung von Aldehyden fand man im Harn Glukurondurch Reduktion aus diesen säure mit der dem Aldehyd entsprechenden gepaaarten Gl ukuron säuren. , •) '') Vgl. S, 39-41. Vgl. hiezu S. 40. ^) Besonders Jajf'e. llUdcbrand, Kcuhcrf/, llümäläintn und Ihonias haben sich mit derartigen Untersuchungen beschäftigt. Vgl. die Literatur in Emil Fromm: Die chemischen Schutzmittel des Tierkörpers liei Vergiftungen, S. 22—24. Karl J. Trüluier, Straßl)urg 1903. Ferner Biochem. Handlexikon. Bd. VII. Kapitel Terpene und ätherische Öle, bearbeitet von Bartelt (1911) und Bd. II. .ö21 (1911), Glukuronsäurepaarlinge, bearbeitet von Neuberg und Retvald. — Kohlehydiate. 123 gebildeten Alkoholgruppe gepaart. Ein Beispiel möge diesen Vorgang be- Nach Verabreichung von Chloralhydrat findet man im Harn die Urochloralsäure. Diese besteht aus Glukuronsäure und Trichloräthylalkohol. Dieser ist durch Reduktion aus dem Chlorallegen. sogenannte hydrat entstanden: CI3 C . C\^ H2 . + 2H = Clg C Chloralhydrat CI3C . . CH.3.OH + H2 0. Trichloräthylalkohol CH2.OH + C^HjoO, = CI3C CH2.O CHgOg + H2O. . Glukuronsäure . Urochloralsäure. Auch Ketone werden reduziert. In anderen Fällen führt die Oxydation zum gewünschten Ziele. So wird z. B. Nitrotoluol zunächst in Nitrobenzylalkohol übergeführt. Dieser verbindet sich dann mit Glukuronsäure: NO-, . CgH, CH3 + . Nitrotoluol NO., . CßH, CH2.OH + CßHioO, . = NO2 C« H, CH2.OH. . . Nitrobenzylalkohol. = NO2 CßH, CH.2.0 CßHgOe + U,0. . . . Man bat auch Wasseranlagerung beobachtet. Thujon=Tanazeton Thujonhydrat über: CiqH^sO 4- H.O rzz Cq H,, OH. geht in ' . Thujonhydrat Thujon . CioH,, . . OH + CßH.oO, = O.C,oH^,O.C6H,Oe + H.,0 . Thujonhydrat glukuron säure. Nicht jede Tierart vermag in allen Fällen derartige Umwandlungen zu vollziehen. Es ergeben sich interessante Unterschiede. Dieses Forschungsgebiet ist trotz sehr vieler Einzelbeobachtungen nur zum kleinsten Teil systematisch bearbeitet. Ein weiterer Ausbau dieser Untersuchungen wird ohne Zweifel der vergleichenden Physiologie manche wichtige Befunde liefern. Den ganzen Vorgang hat man als Schutzwirkung aufgefaßt. Durch die Kuppelung werden der Zelle schädliche oder doch die Zellfunktionen störende Substanzen unschädlich gemacht und gleichzeitig in eine Form übergeführt, in der sie leicht aus dem Körper durch die Nieren entfernt werden können. 1) Die wichtigste, jedoch sicher nicht die einzige Bildungsstätte der gepaarten Glukuronsäuren ist die Leber. Es fragt sich nun, ob wir in der Glukuronsäure ein normales Abbauprodukt des Traubenzuckers vor uns haben. Man könnte sich die folgende Vorstellung zu eigen machen. Der Abbau des Trauben- ') Durch die ümwaudlung und Kuppelung werden gleichzeitig stark oberfläclieuaktive Substanzen in weniger aktive und daher auch weniger adsorbierbare verwandelt. Vgl. L. lierczeUer: Bfochem. Zeitschr. 84. 75 (1917). 124 ^'I- Vorlesung. Zuckers führt über die Glukuronsäure. Diese ist die erste oxydative Abbaustufe. Finden sich Substanzen vor, die zur Kuppelung mit ihr geeignet sind, so wird sie mit diesen gepaart und dadurch vor dem weiteren Abbau geschützt. AMr erhalten so Kenntnis von einer Abbaustufe der Glukose, die sonst unserer Beobachtung entgehen würde. Gegen diese Annahme sind verschiedene Einwände möglich. Vor allem hervorgehoben worden, daß es nicht recht verständlich sei, weshalb bei der Oxydation der Glukose die Aldehydgruppe, die doch so leicht Sauerstoff aufnimmt, verschont bleibe und eine primäre Alkoholgruppe zuerst der Oxydation unterliege. Um dieser Schwierigkeit in der Vorstellung der Entstehung der Glukuronsäure aus der Glukose zu begegnen, hat man die Annahme gemacht i), daß diese sich zuerst mit der zu paarenden Verbindung kupple und erst dann die Oxydation einsetze. In diesem Falle wäre die Glukuronsäure kein normales Abbauprodukt der Glukose, sondern ihre Entstehung wäre ausschließlich an die vorausgehende Kuppelung der Glukose gebunden. Nun können wir im Reagenzglas Glukose mit \Vasserstoffsuperoxyd in Glukuronsäure überführen.^) f^erner ist die Möglichkeit durchaus gegeben daß die Zelle den Sauerstoff an beliebige zur Aufnahme von solchem befähigte Stellen des Moleküls anlagern kann. Es werden die Oxydationen höchstwahrscheinUch durch Fermente vermittelt. Für diese sind bei ihrer Wirkung bestimmte Strukturverhältnisse maßgebend. ist , Es spricht somit nichts gegen , die Annahme, daß die Glukuronsäure beim Abbau der Glukose ist. Leider verfügen wir über keine eindeutigen Beobachtungen, die beweisen, daß die Glukuronsäure im Zellstoffwechsel regelmäßig entsteht. 3) Es ist zwar behauptet worden, daß im Harn freie Glukuronsäure auftreten könne, allein ein ganz normales Zwischenprodukt keine dieser Angaben hält einer kritischen Würdigung stand. Die verschiedenen Glukuronsäurepaarlinge sind verschieden leicht spaltbar. Vor allem zerfallen die ätherartig gebundenen Paarlinge leicht. So kann es .sich leicht ereignen, daß der Harn z. B. nach einigem Stehen freie Glu, kuronsäure aufweist. In diesem Zusammenhang sei darauf hingewiesen, daß die Glukuronsäure Beziehungen zu den Pentosen besitzt. Durch Kohlensäureabspaltung gelangt man zur Xylose.) Es ist möglich, daß die Zellen des tierischen Organismus diesen Weg einschlagen, wenn sie aus Hexosen zu Pentosen gelangen wollen. Sicher festgestellt ist, daß in den Geweben aus Traubenzucker Milchsäure entstehen kann. Unentschieden ist zurzeit noch, ') Vgl. hierzu Emil Fischer uud 0. Pilotif: Ber. d. Deutscheu Chem. Gesellsch. 24. 521 (1801). ) Ä. Jolles: Biochein. Zeitschr. 34. 242 (1911). ') Johannes Biberfeld [Biochem. Zeitschr. 65. 479 (1914)] macht gegen die Annahme, daß die Glukuronsäure das erste Oxydationsprodukt beim Abbau der Glukose sein könnte, geltend, daß Hunde und Kaninchen die subkutan und intravenös einge- führte Säure sehr bald quantitativ im Harn ausscheiden. Diese Beobachtung läßt sich nicht ohne weiteres auf die normalen Verhältnisse üliertragcn. Es könnte sehr wohl einen Unterschied ausmachen, je nachdem die Glukuronsäure in Zellen entsteht oder aber außerhalb solcher zugeführt wird. ) Vgl. S. 27. Kohlehydrate. 125 ob der Abbau immer über diese Zwischenstufe führt oder nur in bestimmten Fällen. Es sei gleich an dieser Stelle hervorgehoben, daß wir mehr als eine Quelle für die Milchsäure kennen. Gewisse Aminosäuren z. B. liefern beim Abbau auch diese Säure. Auch beim Durchleiten von Glyzerin durch die Leber konnte Milchsäure erhalten werden. Die entstehende Ver- bindung ist die d-Milchsäure. Man ist unausgesetzt bemüht gewesen, die Einzelheiten des Kohlehydratstoffwechsels von den verschiedenartigsten Gesichtspunkten aus aufzuklären. Eine unübersehbare Anzahl von P'ragestelluugen drängt sich hier zusammen. Sie sind vom brennendstem Interesse. Wir wissen, daß mit der Aufklärung des Abbaues einer bestimmten Art von Verbindungen gleichzeitig tiefe Einblicke in die Umsetzung auch andersartiger Verbindungen gewonnen werden. Es kommt noch hinzu, daß Kohlehydratstoffwechsel und Muskeltätigkeit unzweifelhaft in engster Beziehung zu einander stehen. Mehr und mehr festigt sich die Ansicht daß die Muskelzelle sich ihren Energiebedarf aus Traubenzucker oder einem Abkömmling davon , erschließt. Nun vollziehen sich alle Umsetzungen in Zellen an kleinsten Mengen. werden die verschiedensten Umwandlungsstufen durchDas ist der Grund, weshalb es nur so schwier gehngen will, in eindeutiger und direkter Weise den Abbau bestimmter Verbindungen in bestimmten Geweben klarzulegen. Glücklicherweise wurde man immer mehr darauf aufmerksam, daß die Hefezelle den Zucker in gleicher oder doch sehr ähnlicher Weise abbaut, wie die Muskelzelle. Es seien die wichtigsten Punkte zusammengestellt. Bei der alkoholischen Gärung spielt ohne Zweifel die Phosphorsäure eine bedeutsame Holle. Sie ist zwar offenbar nicht ganz unentbehrlich ist doch Zuckervergärung auch ohne sie beobachtet worden i), doch gewännt man mehr und mehr den Eindruck, als ob ihr im allgemeinen eine große Wichtigkeit zukomme, und zwar vermutet man, daß sie in ihrer Verbindung mit Zucker" die Geschwindigkeit des Gärvorganges regelt. 2) Es bildet sich eine esterartige Verbindung zwischen Zucker und In kurzer Zeit laufen. , Phosphorsäure.) Sie ist als Fruktosediphosphorsäure erkannt worden. Nun hat vor allem Emlxlen mit seinen Schülern gezeigt, daß die Phosphorsäure in der Muskelzelle eine sehr wichtige Rolle spielt. Zunächst gelang ihm die Isolierung einer Substanz, die beim Abbau Phosphorsäure und Milchsäure liefert.) Er nannte sie Laktazidogen und ist der An- — ») Vgl. auch //. v. Ihder: CarlNeuherg: Biochem. Zeitschr. 103. 320 (1920). Ebenda. 86. 191 (1918). ^) 0. Meyerhof: Zeitschr. f. phvsiol. Chemie. 102. 191 (1918). 3) L. Iiranoff: Zeitschr. f. physiol. Chem. 50. 281 (1907). A. Barden uud ^f^ I. Young: Proceed. of the Royal Soc. (B). 80. 299 (1908): 81. 528 (1909): 82. 321 (1909). Biochem. Zeitschr. 32. 173. 177 (1911). A.v. Lehedew: Biochem. Zeitschr. 20. 114 (1909); 28. 213 (1909); 36. 248 (1911); 39. 155 (1912). Biochem. J. 12. 87 (1918). H. Euler H. Euler uud G. Lundeqiisf : Zeitschr. f. physiol. Chemie. 72. 97 (1911). und A. Fodor: Biochem. Zeitschr. 36. 401 (1911). H. Etiler, Ohlsen, Büchström und Berggren: Zeitschr. f. pliysiol. Chemie. 76. 4f)8 (1912); 77. 394 (1912). Zeitschr. f. Gärungsphysiol. 1. 203 (1912). C. Neuberg: Biochem. Zeitschr. 88. 432 (1918). ) G. Emhden, F. Kalberlah und H. Engel: Biochem. Zeitschr. 45. 5 (1912). G. Emhden, W. Griesbach uud Ernst Schmitz: Zeitschr. f. physiol. Chemie. 93. 1 (1914); 98. 181 (1917). G. Emhden und Frifz Laqncr: Ebenda. 98. 181 (1917); 113. 1 G. Emhden, Ernst Schmitz und Peter Meincke: Ebenda. 113. 10 (1921). (1921). — — — — — — — — — 126 ^^I- Vorlesung. sieht, daß sie mit der Fruktosediphosphorsiiure identisch sei. Embden konnte ferner feststellen, daß während der Muskeltätigkeit gebundene Phosphorsäure frei wird.i) Auch die Hefezelle vermag Kohlehydratester zu zerlegen. Die Spaltung des Zuckers durch Hefe wird einem Komplex von P'ermenten zugeschrieben, der einstweilen den Namen Zymase^) führt. Später wird man die einzelnen Fermente im Zusammenhang mit ihrer besonderen Wirkung bezeichnen, sobald das Wesen des stufenweisen Abbaues des Zuckermoleküls noch genauer erkannt ist. Die Zymase wird inaktiv, wenn man aus Hefe bereiteten Saft 3) der Dialyse unterwirft. Dialysat und nichtdialysierender Teil sind für sich unfähig, Zucker zu zerlegen. Gibt man jedoch beide Lösungen zusammen, dann tritt wieder Gärung ein. An Stelle des Dialysates kann man auch gekochten, filtrierten Preßsaft aus Hefe verwenden. Es nimmt somit bei der Gärung ein Stoff in irgend einer Weise teil, der kochbeständig und dialysierbar ist. Seine Natur ist noch nicht aufgeklärt.) Man hat einstweilen diesen unbekannten Stoff K oferment genannt.^) Es ist von größter Bedeutung, daß sich bei der Verfolgung der Atmungsvorgänge ganz entsprechende Beobachtungen machen ließen, wie bei der alkoholischen Gärung. Meyerhof ^) verfolgte den Atmungsvorgang abgetöteter Hefezellen, d. h. er bestimmte den Sauerstoffverbrauch und fand, daß er aufhörte, wenn mit Azeton abgetötete Zellen abzentrifugiert und mehrfach mit Wasser gewaschen wurden. Wurde jedoch der wässerige Auszug aus Hefe, der für sich auch keine Atmung zeigte, zu den Zellen hinzugefügt, dann stellte sich wieder Sauerstoffverbrauch ein. Es ist somit in das Wasser ein Stoff übergegangen, der beim Atmungsvorgang eine bedeutsame, unentbehrliche Rolle spielt. Er ist ziemlich kochbeständig und dialysierbar. Bereitet man sich aus getrockneter Hefe einen Mazerationssaft, indem man auf sie Wasser einwirken läßt und dann filtriert oder zentrifugiert, dann zeigt der ungelöste Ilückstand keinen Sauerstoffverbrauch, wohl aber der AusWird dieser der Dialyse unterworfen, dann verliert der nichtdialysierende Teil das Vermögen, Sauerstoff zu verbrauchen, aber auch das Dialysat atmet nicht. Gibt man beide Lösungen zusammen, dann stellt sich sofort wieder Atmung ein. Man erkennt ohne weiteres die große Ähnlichkeit, die im ^ergleich zu den bei der alkoholischen Gärung gemachten Beobachtungen vorliegt. Man darf vermuten, daß Atmung und Beginn des Gärvorganges innig miteinander verknüpft sind. Wir werden später bei der Besprechung der Zellatmung auf diese Befunde zurückzug. ') G. Embden und II. Lawaczeck : Zeitschr. f. physiol. Chemie. 127. 181 (1922)Vgl. auch G. Emhden, Eduard Gräfe und Ernst Schmitz: Ehenda. 113. 67 (11121). G. Embden und Erich Adler: G. Emhden und Ed. Gräfe: Ebenda. 113. 108 (1921). Ebenda. 113. 201 (1921). Vgl. a. u. Emil Abderhalden m^d A. Eodor: Fermentforschung. 5. 138 (1921). — — '•') ^) Durch Auspressen oder Mazerieren von Hefe gewonnen. ) Man vermutet, daß Ketosäuren wirksam sind. Vgl. S. 127 ff. Jlardcn und W. I. Young: Proceed. of the Royal Soc. (B). 77. 405 (190ß); E. Büchner und Anfoni: Zeitschr. f. physiol. (B). 80. 299 (1908). E. Büchner und Klatte: Biochem. Zeitschr. 8. 520 (1908). Chemie. 41. 131 (1900). E. Buchner und //. Hahn: Ebenda. 19. 191 (1909); 27. 418 (1910). •) Otto Mei/erhof: Zeitschr. f. physiol. Chemie. 101. 165 (1918): 102. 1, 185 Pjlüger^ Archiv, 170. 367, 42«, 1918; 175. 20 (1919). (1918). 5) (B). 78. //. 369 (1906): — — — — Kohleliydrate. kommen. 127 Im Zusammenhang- mit den erwähnten Feststellungen an HefeBeobachtung von größter Tragweite. Wird Muskel- zellen ist die folgende substanz zerkleinert und dann mit Wasser gründlich ausgezogen, dann verliert sie die Fähigkeit, Sauerstoff zu verbrauchen. Wird jedoch der wässerige Auszug wieder zugefügt, dann setzt die Atmung wieder ein Man kann ihn vorher kochen, ohne daß der Erfolg des Zusatzes beeinträchtigt wird. Besonders wichtig ist nun die Feststellung, daß die durch Ausziehen mit Wasser inaktivierte Muskelsubstanz auch durch Zusatz von gekochtem und filtriertem Hefemazerationssaft wieder atmungsfähig \N'ird, und umgekehrt kann man der Fähigkeit, Sauerstoff zu verbrauchen, beraubten Hefezellen durch Muskelkochsaft diese wiedergeben. i) ! Es gelang dann weiterhin aus allen tierischen Organen durch Kochen jenen Stoff oder Stoff komplex zu gewinnen, der notwendig ist, damit die Atmung vor sich gehen kann. Gleichzeitig enthält das Kochwasser auch das für die alkoholische Gärung unentbehrliche Koferment. Es liegt sehr nahe, den sogenannten Atmungskörper, eben jenen Stoff, der für die Atmung unentbehrlich ist, und das Koferment der alkoholischen Gärung für identisch zuhalten. Wir wollen jedoch dem für eine solche Auffassung noch nicht restlos genügenden Tatsachenmaterial nicht vorgreifen. W^eitere Forschungen werden die Zusammenhänge erst vollkommen kliiren. Erst wenn die Natur des Kofermentes bekannt sein wird, wird man klar sehen und auch feststellen können, in w'elcher Art und Weise es in den ganzen Vorgang der Zuckerspaltung eingreift.2) In den tierischen Geweben findet sich ferner ein Hemmungskörper, der die Zymase in ihrer Wirkung beeinflußt. Aus diesem Grunde sind kalte Gewebsauszüge unwirksam. Erst durch Auskochen erhält man ein wirksames Koferment. ») Interessant ist auch die Beobachtung, daß die Atemtätigkeit durch Hexosediphosphorsäureester gesteigert wird. Auch hierin ist eine Analogie zum Gärungsvorgang vorhanden. Diese Feststellungen mögen genügen, um zu zeigen, mit welcher Berechtigung wir Forschungsergebnisse, die an einzelligen Lebewesen und insbesondere an Hefezellen erhalten worden sind, auf unseren Organismus übertragen. Den größten Einfluß auf die ganzen Vorstellungen über den Zuckerabbau durch Hefezellen hat die Entdeckung von Nei(berg^) ausgeübt, wonach diese und aus ihnen bereitete Preß- und Mazerationssäfte imstande sind, Brenztraubensäure, CH3.CO.COOH, unter Abspaltung von Kohlensäure zu zerlegen. Der Vorgang wird einem Ferment, Karboxylase genannt, zuj Von besonderem Interesse ist die Beobachtung, daß auch vollständig bis zu den Bausteinen abgebautes Fleiscli noch die Fähigkeit besitzt, atraungserregend zu wirken. Vgl. 0. Meijcrhof: Ffliiyer's, Archiv. 175. 20 (1919). ^) Carl Xeuherq nimmt Beziehungen der a-Ketosäuren zum Koferment an. Vgl. //. llarden: Biocheni. Jouru. 11. (54 (1917). Biochem. Zeitschrift. 88. 145 (1918). ) Vgl. zu diesen Problemen auch die Vorlesungen über Fermente im 2. Band. Dort wird auch auf das Wesen der ganzen Vorgänge eingegangen. ) Carl Neuberg und L. Karczuq: Ber. d. Deutschen Chcni. Gesellsch. 44. 2477 C. Neuberg und P. Rosenfhal: Ebenda. 51. 128 (1912); 61. 171 (1914). (1911). C. Nenberg und N. Iwanoff: Ebenda. 67. 1 (1914). Vgl. auch W. Palladin, N. Groniojf und iV. N. Moniererde: Ebenda. 62. 137 (1914). C. Neuberg und L. Czapski: Ebenda. 67. 9 (1914). Vgl. ferner C. Neuherg: Biochem. Zeitschr. 71. 1 (191;')). Vgl. ferner J. Bodndr Ebenda. 73. 193 (1910)."— Ä. Bau: Ebenda. 73. 340 (1916). — — — — : — — — Vi. Vorlesung. 12^ geschrieben. Seine Wirkung ist vom oben erwähnten Koferment unabhängig. Beim Abbau der Brenztraubensäure entsteht neben Kohlensäure Azetaldehyd: CH3 . CO COOK = CO2 + CH3 C<H . . ist überall da aufgefunden worden, wo Zymase vorDer Abbau der Brenztraubensäure kann dabei in Einzelheiten ein verschiedener sein. So wurde bei Zucker spaltenden Bakterien kein Offenbar entstanden Ameisen- und Essigsäure Aldehyd beobachtet. 2) als Zwischenstufen. Als Endprodukt traten Kohlensäure und Wasserstoff auf. Es ist auch geglückt, den Nachweis zu führen, daß der Azetaldehyd Die Karboxylase handen ist.i) weiter in Äthylalkohol übergeführt wird.^) Es muß noch hervorgehoben werden, daß die Karboxylase nicht nur die Ketosäure Brenztraubensäure unter Kohlensäureabspaltung in den zügehörigen Aldehyd verwandelt, sie greift vielmehr ganz allgemein a-Keto- säuren an. Ebenso steht die Fähigkeit ,der Hefezellen, Azetaldehyd zu Äthylalkohol zu reduzieren, nicht allein da. Neuberg) hat in zahlreichen Versuchen gezeigt, daß Hefe viele andere Aldehyde in die entsprechenden Alkohole verwandelt. Es ist von größter Bedeutung, daß es gelungen ist, beider alkoholischen Gärung von Zucker Azetaldehyd nicht nur nachzuweisen, sondern durch Bindung mittelst eines Salzes der schwefeligen Säure in Form einer Komplexverbindung in großen Mengen festzulegen.^) Angewandt wurde unter anderem^) Dinatriumsulfit. 0=R Es reagiert, wie folgt, auf Aldehyde: R . C\jj -f Nag SO3 Bei Festlegung des AzetalCH(OH) + H2 dehyds entsteht in größerer Menge Glyzerin.') Man hat es in der Hand, seine Menge durch Steigerung oder Verminderung des Zusatzes von . . (0 .SO2 Na) -t- Na (OH). oder zu verkleinern. Von besonderem Ines auch gelingt, Azetaldehyd unter Glyzerinbildung durch Tierkohle abzufangen.«) Diese adsorbiert den Aldehyd lebhaft. Je mehr Azetaldehyd durch Festlegung der Umwandlung in Äthylalkohol entzogen wird, um so mehr Glyzerin entsteht, und zwar konnte in jedem Augenblick der Gärung auf ein Molekül Azetaldehyd ein solches Dinatriumsnlfit zu vergrößern teresse ) ist die Feststellung, C. Neuberq und Joh. daß Kerb : Bericht der Deutschen Chem. Gesellschaft 46. 2225 (1913). Vgl. L: Karezag und E. Schiff: Biochem. Zeitschr. 70. 325 (1915). C. Neuberff undKlsa Reinfurth: Biochem. Zeitschr. 89. 365 (1918); 92. 234 (1918). ) Vgl. Carl Neuberg und Mitarbeiter (Weide, Nord, Kerb): Biochem. Zeitschr. 60. 472 (1914); 62. 470, 477,' 482 (1914); 67.18,24,46,104,111 (1914); 92.96,111(1918). 5) Vgl. u. a. L. Karezar/ und L. Mikzdr : Biochem. Zeitschr. 55. 79 (1913); 70. «) ») — L. Karczafi und FAse Breuer: Ebenda. 70. 320 (1915). Vgl. hierzu Carl Neuberg, Julius Hirsch und Elsa Reinfurth: Biochem. Cari Neuberg und Elsa Reinfurth: Ebenda. 106. 281 Zeitschr. 105. .307 (1920). (1920) ; Berichte d. Deutschen Chem. GeseÜsch. 53. 1039 (1920). ') Vgl. hierzu auch W. Connstein nnd K. Liidecke: Ber. d. Deutschen Chem. Gesellsch. 52. 1385 (1919). ) Vgl. hierzu Emil Abderhalden: Fermentforschung. 5. 89, 110, 255 (1921). 317 (1915). ') — — Emil Abderhalden und Susi Glaubach: Ebenda. 6. 143 (1922). Kohlehydrate. 129 von Glyzerin. 1) Diese Feststellung zeigt, daß zwischen der Bildung dieses dreiwertigen Alkohols und derjenigen von Brenztraubensäure bzw. Azetaldehyd und Äthylalkohol wechselseitige Beziehungen bestehen. Carl Xeuberg -) hat neuerdings noch eine weitere Art des Abfangens der Abbaustufe Azetaldehyd aufgefunden, die weit über die Feststellung dieses Produktes als Zwischenstufe im Abbau von Zucker hinaus größte Bedeutung hat. Er setzte zur Gärflüssigkeit, wobei er entweder die Gärung beim Traubenzucker bzw. der Fruktose oder aber bei der Brenztraubensäure einsetzen ließ, aromatische Aldehyde hinzu und machte dabei die wichtige Beobachtung, daß diese mit Azetaldehyd zu einem Ketonalkohol vereinigt wurden. Bei Anwendung von Benzaldehyd entstand 1-Phenvlazetylkarbinol bzw. 1-Phenylbrenztraubenalkohol: CH3 CO.CH(OH).C6H5. Es hat somit eine Synthese stattgefunden, und zwar in einer bisher in dieser Form nicht bekannten Weise. Es findet Verkuppelung von Kohlenstoff aö Kohlenstoff statt. C. Neuberg schreibt diese Synthese einem besonderen Fermente, Karboligase genannt, zu. Da nun Benzaldehyd und Azetaldehyd an und für sich von Hefezellen nicht vereinigt werden, vielmehr die erwähnte Synthese nur eintritt, wenn Azetaldehyd im Augenblick der Entstehung beim Abbau von Zucker bzw. von Brenztraubensäure zugegen ist, so dürfte die folgende Formulierung des Vorganges der stattfindenden Umsetzung gerecht werden 3): . CH3 CO . + HOC CßH, . — CH3 CO CH(OH) >- . . /OH . c,n,'+ C^O \0H COOH + OHAH^ Brenztrauben- Benzaldehyd Phenylbrenztrauben- säure alkohol. Weitere Forschungen müssen lehren,' welche Bedeutung den gemachten Feststellungen im Zellstoffwechsel zukommen. Sie eröffnen weite Möglichkeiten der Synthese hochmolekularer Verbindungen aus einfacheren Bruchstücken unter Zusammenfügung von Kohlenstoff an Kohlenstoff. Wir werden auf diese Art von Synthesen von verschiedenen Gesichtspunkten aus noch zurückkommen. Kehren wir zur Frage der Zerlegung der Glukose bei der alkoholischen Gärung zurück, und betrachten wir zunächst die Bildung von Äthylalkohol unter der Annahme, daß Brenztraubensäure als Zwischenstufe des Abbaues von Glukose auftritt und diese durch die Karboxylase in Azetaldehyd übergeführt wird. Dieser kann dann, wie die folgenden Formeln zeigen, durch Wasserstoffanlagerung in Äthylalkohol übergehen : CH3.CO.COOH— C02 = CH3.C<,, ^H -1-112 Ho = CHo v^rij CH, I3 G<„ L.n2 OH. CH, o\„ + . . . C. Neuberg und J. Hirsch : Biochem. Zeitschr. 89. 954(1921). Carl Neuberg und Julius Hirsch: Biochem. Zeitschr. 115. 282 (1921). Carl Neuberg und Ludiriq Lieberinanii : Ebenda. 121. 311 (1921). Carl Neuberg und Heinz Ohle: Ebenda. 127. 327 (1922); 128. 610 (1922). ') Vgl. Carl Neuberg und Heinz Ohle: 1. c. ») ) Abderhalden, Physiologische Chemie. I.Teil. 5. Anfi. — — 9 YI. Vorlesung. 130 Es fragt sich nun, woher der Wasserstoff, der die Überführung des Eine Antwort auf diese Frage in Alkohol bewirkt, stammt. können wir erst geben, wenn wir noch tiefer in das Problem des Abbaues des Zuckermoleküls eingedrungen sind. Wie sein Abbau in allen Einzelnur soviel scheint wissen wir noch nicht genau heiten vor sich geht sicher zu sein, daß ein Produkt der Dreikohlenstoffreihe entsteht. Man hat an Glyzerinaldehyd, Milchsäure, Dioxyazeton und Methylglyoxal gedacht: Aldehyds , , CH2.OH CH3 CH2.OH CH3 CH.OH ^0 c<; H CH.OH CO CO COOH CH..OH Milchsäure Dioxyazeton Glyzerinaldehyd CH2 bzw. CH (OH) yyO Wi ^H H Methylglyoxal. Bei der alkoholischen Gärung hat man kleine Mengen von Azetaldehyd, Glyzerin und Milchsäure auch bei ungestörtem Ablauf gefunden, i) Nehmen wir an, daß beim Abbau des Zuckermoleküls Glyzerinaldehyd und Methylglyoxal durch Hefe entstehen, dann können wir uns folgendes Bild über die Entstehung aller bisher beobachteten Gärungsprodukte machen, wobei gleichzeitig ersichtlich ist, weshalb bei der Verhinderung der Überführung des Azetaldehyds in Äthylalkohol Glyzerin in entsprechender Weise entsteht: CHo.OH OH Brenztraubensäure Glyzerinaldehyd CH2.OH CH3 I CH.OH CH2.OH Aethylalkohol. CH2.OH Azetaldehyd Glyzerin (Wird Azetaldehyd abgefangen, dann reduziert der nicht zur Verwendung kommende Wasserstoff Glyzerinaldehyd zu Glyzerin!) ) Vgl. auch die Arbeiten von //. F.uTer, Lchcdiw u. a. in der Zeitschr. f. pbysiol. Chemie, 1911 und 1912 und in den Ber. d. Deutschen Cbem. Gesellsch. 1912. S. Kosly- — Kohlehydrate. Es ist nun 131 Neuherg'^) gelungen, noch eine dritte Form der Zucker- spaltung festzustellen. Läßt man die Gärung sich bei alkalischer Reaktion unter Anwendung von alkalisch reagierenden Salzen, wie Karbonaten, Bikarbonaten, Phosphaten usw., vollziehen, dann gehen zwei Moleküle Azetaldehyd unter Aufnahme von einem Molekül Wasser in Äthylalkohol und Essigsäure über. Auch in diesem Falle findet der sonst zur Hydrierung von Azetaldehyd zu Äthylalkohol dienende Wasserstoff zur Reduktion eines anderen Zuckerteils Verwendung. Es entsteht wiederum Glyzerin. Die Bildung der beiden ersten Produkte, der Essigsäure und des Äthylalkohols, kann man sich unter wechselseitiger Oxydation und Reduktion entsprechend der Cannizzaro?>Qh%\i Reaktion -) entstanden denken CH3.C<g\| CH,.<r CH3.CH0.OH H., ~^CH,.;COOH Da, wie erwähnt, zwxi Moleküle Azetaldehyd verwendet werden, um ein Molekül Essigsäure zu bilden, ist zu erwarten, daß auf ein Molekül dieser Säure zwei Moleküle Glyzerin kommen. Die Erfahrung bestätigt diese Annahme. Man kann die geschilderten drei, je nach den vorhandenen Bedin- gungen verlauf enden Arten des Zuckerabbaus 3), wie folgt, in empirischen Formeln zusammenstellen >'2C2H5 .OH-11. gewöhnliche alkoholische Gärung: CgHi^Og 2CO2. Vergärung unter Zusatz von Sulfit oder Tierkohle: 2. — CeHi,Oe 3. —> CHs.cCH-hCO^ + CsHgO^. Vergärung bei alkalischer Reaktion: 2C6H12O6 + H2O CH3 COOH -h C2 H5 OH -f- 2 C3 Hg O3 + 2 CO.,. Daß es sich bei diesen Reaktionen nicht um Vorgänge handelt, . — >- . die_ nur f'ir die Hefezelle Gültigkeit haben, zeigt die Beobachtung, daß auch" bei Gärungsvorgängen, an denen Bakterien beteiligt sind, Azetaldehyd eine ausschlaggebende Rolle spielt. Auch hier führte die Abfangemethode mittelst Sulfit (Kalziurasulfit) zur Festlegung dieses Aldehyds.; Wenn wir auch noch weit davon entfernt sind, alle Einzelvorgänge, die dem Zuckerabbau bis zu den jeweiligen Endprodukten zugrunde liegen, klar übersehen und an Hand eindeutiger Tatsachen verfolgen zu können, so haben doch die angeführten Beobachtungen unsere Kenntnisse über den stufenweise Abbau organischer Verbindungen und insbesondere des Zuckermoleküls oder, noch schärfer umschrieben, der a-Ketosäuren und ganz — S. Kosttjtschew und Hübbenet: tscheiv: Zeitschr. f. physiol. Chemie. 79. 130 (1912). Eduard Büchner und Ktirt Lanyheld: Berichte d. Deutschen Ebenda. 79. 359 (1912). Eduard Buchner, Kurt Langheld \mA Siegfried Skraup: Chem. Ges. 46.1972(1913). Carl Neuberg und Joh. Kerb :BGT.i. Deutsch. Chem. Gesellsch. Ebenda. 47. 2.550 (1914). 47. 2730 (1919). Vgl. ) Vgl. C. Neuberg und J. Hirsch: Biochem. Zeitschr. 100. 304 (1919). auch C. Neuberg, J. Hirsch und E. Reinfurth: Ebenda. 105. 307 (1920). ^) Vgl. Cannizzaro: Liebigs Annalen. 88. 129 (1853). ») Vgl. auch P. Thomas: Ann. Inst. Pasteur. 34. 162 (1920). ') Vgl. C. Neuberg und F. F. Nord: Biochem. Zeitschr. 96. 133, 158 (1919). — — — — 9 VI. Vorlesung. 132 besonders der Brenztraubensäure außerordentlich gefördert. Die bedeutsame Rolle, die der Azetaldehyd als Zwischenprodukt beim Abbau von Zucker bzw. von Brenztraubensäure spielt, hat noch durch die Feststellung, wonach Aldehyde die alkoholische Gärung anregen^), an Interesse gewonnen. Sie wirken vielleicht durch Aufnahme von Wasserstoff. Diese Annahme hat dadurch an Wahrscheinlichkeit gewonnen, daß der Nachweis geführt werden konnte, daß andere hydrierbare Verbindungen wie Ketone, Diketone und Disulfide, eine ähnliche Wirkung haben. 2) Wir kommen nun zu der wichtigen Frage zurück, in welchen Bahnen der Abbau des Traubenzuckers sich im tierischen Organismus vollzieht. Bietet schon die Hefezelle der restlosen Lösung der Frage nach allen Zwischenstufen des Zuckerabbaus, die bis zu den Stoff Wechselendprodukten führen, die größten Schwierigkeiten, so sind sie noch viel schwieriger zu beheben, wenn wir es mit einem ganzen Zellstaat zu tun haben. Die einzelnen Stoff wechselzwischenprodukte werden nur in Spuren gebildet und sofort nach ihrer Entstehung weiter verwandelt. Darüber besteht kein Zweifel, daß der Abbau auch in der tierischen Zelle stufenweise vor sich geht. Von allen zu erwartenden Zwischen- COOH I stufen ist bis jetzt nur die d-Milchsäure (l)») HO.C.H über jeden Zweifel I CH3 sichergestellt.) Die Konfiguration der d-Milchsäure ist durch Überführung COOK 1 von natürlicher 1-Äpfelsäuro HO.C.H in diese festgestellt worden.^) Es I CH, I CO OH 1-Äpfelsäure ') Vgl. C. Neuberg: Biochem. Zeitschr. 88. 145 (1918); C. Neuberg und M. Ehrlich: Ebenda. 101. 239, 276 (1919/20). Vgl. auch Emil Abderhalden: Fermentforschung. 5. 105 (1921). 2) Vgl. C. Neuberg nndi A.Levite: Biochem. Zeitschr. 91. 257 (1918); CNeuberq und F. F. Nord: Ber. d. D. Ch. Ges. 52. 2237 und 2248 (1919). C. Neuberg und M. Ehrlich: Biochem. Zeitschr. 101. 27(i (1920); hier findet sich weitere Literatur. ^) Nach der sterischen Beziehung der d-Milchsäure zur 1-Äpfelsäure und dieser zur 1- Weinsäure [vgl. Karl Freudenberg und Fritz Brauns: Ber. d. Deutschen Chem. Ges. 55. 1339 (1922). A. Wohl und B. 'Schellenberg : Ebenda. 55. 1404 (1922)] müßte die Milchsäure der angegebenen Konfiguration die Bezeichnung 1-Milchsäure tragen. Vgl. hierzu auch die Bezeichnung des Fruchtzuckers als d-Fruktose, obwohl sie nach links dreht. Es ist zu befürchten, daß bei Einführung der Bezeichnung 1-Milchsäure Verwirrung entstehen wird. Das Zweckmäßigste wäre wohl, die genetischen Beziehungen in einer Klammer hinter dem Namen der Verbindung anzugeben, also z. B. d-Milchsäure (1) zu schreiben. Deckt sich die Bezeichnung für den genetischen Zusammenhang mit derjenigen für das optische Verhalten, dann ist ein besonderer Hinweis auf erstere — — — nicht erforderlich. ) Vgl. auch D. L. Forster und Z>. M. Mogle: Biochem. J. 15. 672 (1921). ^) Karl Freudenberg : Berichte der Deutschen Chem. Gesellsch. 47. 2027 (1914). — Karl Freudenberg und Fritz Brauns: Ebenda. 55. 1339 (1922). R. Schellenberg: Ebenda. 55. 1404 (1922). — A. Wohl und Kohlehydrate. 135 war schon lange bekannt, daß Milchsäure besonders in den Muskeln auftritt^), jedoch wurde ihre Herkunft zweifelhaft, nachdem man erkannt hatte, daß auch die Aminosäure d-Alanin d-a-Aminopropionsäure Milchsäure liefern kann. Diese Verbindung steht außer zur d-Milchsäure = auch in sehr naher Beziehung zur Brenztraubensäure, wie die folgenden Formeln zeigen: COOH COOH I I NH.,.C.H + HO.C.H COOH ^ I CO +NH3 I CH3 CH3 d-Milchsäure (1) d-Alanin (1) CH3 Brenztraubensäure. Die letzten Zweifel darüber, ob auch aus Glukose Milchsäure entstehen kann, sind jetzt behoben. Es bleibt nun nur noch, festzustehen, wie die Bildung dieser Säure sich vollzieht. Es ist möglich, daß der Traubenzucker zunächst in zwei Moleküle d-Glyzerinaldehyd zerfällt und dieser dann in d-Milchsäure umgelagert wird 2): ^ H— C— OH I H— C— OH \, COOH I CH2 OH i HO— C-H I H— C— OH H— C— OH » I HO— C— ^//O C^' I H X' CHs* H— C— OH I I CH, OH d-Glukose CHo OH d-Milchsäure (Ij. 2 Moleküle d-Glyzerinaldehyd (d) Es gibt noch zahlreiche andere Möglichkeiten des Abbaus des TrauEs ist wohl denkbar, daß es vor der Spaltung in sich benzuckermoleküls. •) Vgl. von Noorden und G. Embden: Zentralbl. f. d. ges. Physiol. u. Pathol. d. Stoffw. N. F. 1. 1906. Die zahlreichen Arbeiten von G. Emhden und seinen Mitarbeitern vergleiche z. B. Biocbem. Zeitschr. 45. 1—206 (1912). Hier findet sich auch weitere Literatur. W. 31. Fletcher und F. Gowland Hopkins: Journ. of physiol. 25. 247 F. A. Levene und Johannes Müller: Zentralbl. f. Physiol. 21. 831 (1907). (1907). Rudolf G. M. Meyer: The Journ. of Biol. Chem. 11. 361 (1912); 12. 265 (1912). Türkei: Biochem. Zeitschr. 20. 431 (1909). 2) Emhden: Biochem. Zeitschr. 45. Adam Loeb: Ebenda. 50. 1—206 (1912). Walter Grieshach: Ebenda. 50. 457 (1913). Otto v. Fürth :B\oz\i&m. 451 (1913). Zeitschr. 64. 131, 155 (1914); 69. 199 (1915). — — — — — — — VI. Vorlesung. 134 Veränderungen erfährt, die die Spaltung vorbereiten. Auch als Abbaustufen kommen noch andere Verbindungen als die erwähnten in Frage. In erster Linie sei der Bildung von Methylglyoxal gedacht. i) Die Annahme dieser Abbaustufe hat dadurch an Wahrscheinlichkeit gewonnen, daß es geglückt ist, in den Geweben Fermente nachzuweisen, die Milchsäure verwandeln sie in '-) r^O y\H COOH /yO yxH —^ HO.C.H I f\(\av ' 0^^^ C(OH) I C=0 + H2 I CH. CH2 ^Hs , Methylglyoxal d-Milchsäure (1) Dieser Vorgang könnte auch reversibel sein. Das Ferment, das die Umwandlung vollzieht, ist Glyoxalase genannt worden. In diesem Zusammenhang sei auf die außerordentlich wichtige Tatsache hingewiesen, daß viele Beobachtungen dafür vorliegen, daß die einzelnen Umwandlungsvorgänge umkehrbarer Natur sind. Gleichzeitig erkennen wir beim tieferen Eindringen in die Einzelheiten des Abbaues der Kohlehydrate, daß es im allgemeinen keinen in sich abgeschlossenen Stoffwechsel der einzelnen organischen Nahrungsstoffe gibt. Der Abbau aller führt zu gemeinsamen Abbaustufen, und zwar finden sich diese in der Hauptsache im Gebiete der Drei- und Zweikohlenstoffreihe. Von ihnen aus kann der Abbau weiter gehen, oder aber es setzen Aufbauvorgänge ein. Von diesen Gesichtspunkten aus müssen wir jede einzelne Umwandlung der einzelnen Zwischenstufen betrachten und uns immer nach Übergangsverbindungen zu anderen Klassen von organischen Nahrungsstoffen umsehen. Einen weiteren Wendepunkt in der ganzen Auffassung des Abbaues des Traubenzuckers in unseren Geweben bedeutet der Befund von Steppt) wonach Azetaldehyd im Blute und im Harn feststellbar ist. Ferner konnte Hirsch^) zeigen, daß Froschmuskelbrei aus Kohlehydraten Azetaldehyd hervorgehen läßt. Damit ist der Azetaldehyd mediären Zuckerabbau festgestellt. als ein Abbaustoff im inter- Es ist naheliegend, die Entstehung des Azetaldehyds in entsprechender Weise anzunehmen, wie wir es S. 127 bei der alkoholischen Gärung ) Vgl. Dakin und Dudle;/: The Journ. of Biol. Chem. 14. 155 15. 127 15. 463 — F. A. Levene und G. M. Meyer: Ebenda. 14. 551 (1913).(1913); — Vgl. auch Carl Neuberg: Biochem. Zeitschr. 49. 502 (1913). — A. J. Ringer: Journ. of Biol. Chem. 15. 146 (1913). Carl Neuberg: Biochem. Zeitschr. 49. 502 (1913); 51. 484 (1913). — H. (1913). ) Dakin und H. W.Diidleg: I). Journ. of Biol. Chem. (1913). 14. 155, — 423 (1913); ) W. Stepp; Biochem. Zeitschr. 107. 60 (1920). W. Stepp und H. Lange: Deutsches Archiv f. klin. Med. 134. 47 (1920). W. Stepp und li. B'eulgen: Zeitschr. f. physiol. Chem. 114. 301 (1921); 119. 72 (1922). ) Julius Hirsch: Biochem. Zeitschr. 117. 113 (1921). — Kohlehydrate. geschildert haben, d. h. es 135 kommt als Vorstufe Brenztraubensäure in Frage, die unter Kohlensäureabspaltung den genannten Aldehyd liefert. In diesem Zusammenhang sei folgender Beobachtungen gedacht. Es konnte gezeigt werden 1), daß Brenztraubensäure beim Durchleiten durch die Leber zu d-Milchsäure reduziert wird. Es liegt auch hier ein umkehrbarer Vorgang vor: — CH3.CH(0H).C00H ^ H-, CH3.CO.COOH + n, Brenztraubensäure. Milchsäure Es wurde ferner festgestellt, daß aus Brenztraubensäure der Leber in Azetessig säure entsteht. Ein direkter Übergang ist kaum anzunehmen, vielmehr dürfte der Weg über Azetaldehyd führen. Es ist aber auch denkund diese Azet- bar, daß der gebildete Aldehyd in Essigsäure übergeht 2) essigsäure liefert: — H2O CH3 . C<H + 0—'h CH3 C<Qf\| 2 (CH3 COOK) :^ CH3 CO GH., COOH. . . . . . . + H2O Auch hier treffen wir wieder auf eine bedeutungsvolle Beziehung. Mit der Essigsäure und der Azetessigsäure stoßen wir auf Zwischenstufen, die zu der Fettsäurereihe hinüberführen! In diesem Zusammenhange sei noch des interessanten Befundes von Frkke 3) gedacht, wonach im Harn bei schwerem Diabetes melitus die Verbindung CH3 . CH (OH) CH, C\tt . . allerdings sehr kleinen Mengen aufgefunden werden konnte. Diese Verbindung ist in mehr als einer Hinsicht von Interesse. Sie zeigt Beziehungen zur ß-Oxv buttersäure (siehe hierzu Vorlesung X): CH3.CH(0H).CH,.C00H und zum Azetaldehyd. Schließlich sei noch erwähnt, daß man beim Durchleiten von Azetaldehyd durch die Leber und beim Vermischen dieser Verbindung mit Organbrei Äthylalkohol) erhält. Würde es geüngen, Brenztraubensäure im tierischen Organismus nachzuweisen und den Beweis eindeutig zu führen, daß diese Verbindung auch von Organmazerationssäften bzw. durch lebende Zellen unter Kohlensäureabspaltung in Azetaldehyd übergeht 5), dann ließe sich der in G. Embden und M. Oppenheimer: Biochem. Zeitschr. 45. 34 (1922). A. Loeb: Biochem. Zeitschr. 47. 118 (1912). G. Embden uud A. Loeb Zeitschr. f. physiol. Chemie. 88. 246 (1913). ^) Robert Fricke: Zeitschr. f. physiol. Chemie. 118. 218 (1922). ) Vgl. BatelliwnAL. Stern: Biochem. Zeitschr. 28. 147 (1910); 29. 130 (1910). ) — 2) — — J. Parnas: Biochem. Zeitschr. 28. 274 (1910). Gustav Embden und Karl Baldes: Ebenda. 45. 157 (1912). Vgl. hierzu auch Carl Neuberg : Biochem. Zeitschr. 51. 484 — (1913). 5) Vgl. M. Tschernorutzhi : Biochem. Zeitschr. 43. 486 (1912). und G. M. Meyer: Journ. of Biol. Chem. 17. 443 (1914). — P. A. Levene ^ I- Vorlesung. 136 Glukoseabbau nahe und vielleicht sogar vollBeziehung- zu demjenigen in der Hefezelie bringen. Es ist nicht ausgeschlossen, daß nur die richtigen Bedingungen noch nicht getroffen sind, und der Nachweis des \'orkommens und des Abbaues der Brenztraubensäure doch noch glückt. ^) in Zellen der Tiere in sehr kommen übereinstimmende Die Feststellung, wonach tierische Gewebe aus Azetaldehyd Äthylalkohol bilden können, hat durch die Beobachtung, daß in der Pflanzenwelt auch Alkohol gebildet wird, und die Mitteilung von StoMasa^), wonach Organe von Tieren normaler Weise in ihrem Stoffwechsel diese Verbindung bilden, ganz wesentlich an Interesse und Bedeutung gewonnen. Es ist jedoch bis heute nicht geglückt, die Frage klar zu entscheiden, ob tierische Gewebe unter normalen Verhältnissen Glukose über Alkohol abbauen. Man hat auch prinzipielle Bedenken gegen eine solche Annahme geltend gemacht. Wir wissen, daß der Alkohol ein ausgesprochenes Protoplasmagift Sollte nun die Zelle selbst eine Verbindung erzeugen, die ihr schädlich werden kann? Es ist dies durchaus möglich. Fast jedes Stoff wechselzwischenprodukt kann in genügender Konzentration Zellen schädigen. Die Zelle arbeitet unter normalen Verhältnissen immer nur in Spuren. Jede ist. Zwischenstufe Mird rasch durchlaufen. Kaum ist eine Stufe erreicht, so ist der Vorgang des Auf- oder Abbaues auch schon weiter geleitet. Das erschwert unsere Forschung auf diesem Gebiete so sehr. Wenn nicht' die Zelle eine Anomalie aufweist, die verhindert, daß gebildete Zwischenprodukte vollständig verwertet werden, oder wenn es uns nicht gelingt, durch experimentelle Eingriffe irgend ein Zwischenprodukt zur Anhäufung zu bringen, so entgehen uns die einzelnen Abbaustufen. Hier wird erst Klarheit eintreten, wenn die Mikrochemie uns M'eitere Hilfsmittel an die Hand gegeben hat. Überblicken wir alles, was wir bis jetzt über den (jlukoseabbau in unseren Geweben wissen, so können wir zur Zeit folgende Zusammenstellung geben, wobei noch zu bemerken ist, daß es fraglich ist, ob die Umwandlung direkt beim Traubenzucker einsetzt,, oder aber von einem noch unbekannten der Glukose nahestehenden Umwandlungsprodukt ausgeht. ") ) Eigene Beobachtungen machen es wahrscheinlich, daß dem Blut zugesetzte Brenztraubensäure unter Azetaldehydbildung zerlegt wird und ebenso scheinen Organe diesen Abbau vollziehen zu können, doch sind die erhaltenen Ergebnisse noch nicht eindeutig und sicher genug. ^) Vgl. hierzu u. a. Julius Stoklasa, John Jelinek und Eugen Vitek: Hofmeister?, Beitr. 3. 460 (1903) u. Zeitschr. f. Zuckerind. Böhmens. 27. 633 (1903). — Julius Stoklasa, Adolf Ernest und Karl Chocenski/ : Zeitschr. f. physiol. Chemie. 50. 303 (1907) u. 51. Julius Stoklasa und F. Czerny : Berichte d. Deutschen Chem. Ges. 36. 156 (1907). — - Jtilius Stoklasa: Pßüc/ers Archiv. 101. 311 (1904); Zentralbl. f. Phvsiol. 4058(1903). 17. 465 (1903); Berichte d. Deutschen Chem. Ges. 38. 664 (1905). Vgl. auch über die angebliche Auffindung von Äthylalkohol in normalen Organen und im Blut: JV. Ilufson Ford: .Journ. of Physiol. 34. 430 (1907). Felix Reach: Biochem. Zeitschr. 3. 326 — — (1907). ^) Vgl. hierzu Fritz Laqucr: Zeitschr. f. physiol. Chemie. 116. 169 (19:J1). Kohlehydrate. 137 Glykogen d-(Tlukose Veresterung mit Phosphorsäure (Laktazidogen) ^ I -r ). I Kohlehydrat der Dreikohlenstoffreihe ^_1 Beziehungen zum Glyzerin (Glyzerose, Methylglyoxal) it d-Milchsäure (1) Beziehung zur d-a-Aminopropion Brenztraubensäure ^ <1 säure -^ N = d-Alanin (1) I — Azetessigsäure -< -^^ ^ ~"^ Azetaldehvd Äthylalkohol > i Essigsäure \ Beziehungen zu höheren Fettsäuren. Wir kennen noch eine weitere Umwandlung des Traubenzuckers, die neue Beziehungen der Kohlehydrate zu anderen Gruppen von Verbindungen ermöglicht. A. Windaus und F. Knoop ^) beobachteten nämlich, daß bei der Einwirkung von Zinkhydroxyd-Ammoniak, Zn(OH).2.4NH3, auf Glukose im Sonnenlicht bei Zimmertemperatur Methylimidazol: CH. C NH >CH CH— sich bildet. Der Bildung dieser Verbindung aus Traubenzucker dürfte wohl eine Glukose in Methylglyoxal, CH3.CO.CHO, vorausgehen. mit Ammoniak und Formaldehyd leicht Imidazol. Diese Umwandlung erweckt deshalb unser besonderes Interesse, weil wir dem Imidazolring einmal bei einer Aminosäure, dem Histidin, begegnen und ferner bei den Purinbasen. Die Möglichkeit ist gegeben, daß der Traubenzucker auch im Zellstoffwechsel in Imidazol übergeführt werden kann und sich so Beziehungen zu stickstoffhaltigen Verbindungen ergeben, die sich nicht ohne weiteres aus der Struktur der einzelnen Produkte ab- Spaltung Dieses der liefert leiten lassen. Nach der jetzt herrschenden Annahme vollzieht sich der Abbau der Glukose in den Zellen teils ohne, teils mit Sauerstoff. Die ersten Eingriffe sind Spaltungen und molekulare Umlagerungen. Erst in späteren ») -) (1905). Vgl. S. 134. A. Windaus und F. Knoop: Berichte der Deutschen Beitr. z. chem. Physiol. u. Pathol. fi. 392 (1905). — Chem. Gesellsch. 38. 1166 VI- Vorlesung. 138 Phasen des Abbaus setzen Oxydationen ein. In diesem Zusammenhang sei ganz kurz ein Problem von allergrößtem Interesse gestreift, auf das wir später noch eingehend zurückkommen. i) Es handelt sich um die Lieferung der bei der Muskelarbeit notwendigen Energie. Wir wissen jetzt, daß die Muskelzelle als Maschine betrachtet, keine kalorische, sondern eine chemodynamische ist, d. h. es findet nicht eine Umwandlung von chemischer Energie in Wärme und eine weitere in Arbeitsenergie statt, vielmehr ist die Verwertung der chemischen Energie eine direkte. Ferner hat es sich herausgestellt, daß man bei der Muskeltätigkeit und der ihr zugrunde liegenden Vorgänge drei Phasen zu unterscheiden hat, nämlich: 1. die Kontraktion des Muskels. Sie vollzieht sich, ohne daß Sauerstoff verwendet wird. Somit stammt die zu dem erwähnten Vorgang notwendige Energie aus Spaltungsvorgängen. Sie wird bei der Zerlegung von Traubenzucker oder einer ihm verwandten Verbindung in Milchsäure in Freiheit gesetzt. Der Energiebedarf für den Kontraktionsvorgang ist gering. Es folgt dann 2. die Periode der Erschlaffung und an diese schließt sich 3. die Periode der Erholung und Wiederherstellung an. Es scheint, daß im ersten und zweiten Stadium, dem der Zusammenziehung und dem der Erschlaffung, in der Hauptsache physikalisch-chemische Kräfte eine Rolle spielen. Bei der ersteren wird offenbar Quellungsenergie verbraucht, die in der Zeit der Erschlaffung wiedergebildet wird. In der Periode der Erholung wird Sauerstoff zur Wiederherstellung jenes Zustandes verbraucht, der erforderlich ist, um auf einen Reiz hin. der in gewissem Sinne mit einem Anstoß vergleichbar ist, wieder eine Zusammenziehung auslösen zu können. Es ist selbstverständlich von allergrößtem Interesse alle diese Phasen in allen Einzelheiten mit den Teilvorgängen des Zuckerabbaus in Zusammenhang zu bringen. Gewiß hat jede Abbaustufe neben ihrer Bedeutung als Energiequelle noch eine ganz besondere Rolle bei bestimmten Phasen der Muskeltätigkeit zu spielen. Daß Arbeitsleistung ohne Sauerstoff möglich ist, beweisen u. a. die Beobachtungen von Hennami-), Pflüger^) und Bunge). Hermann wies nach, daß ein ausgeschnittener Muskel, aus dem kein Sauerstoff mehr auspumpbar ist, in einem sauerstofffreien Medium arbeiten und Kohlensäure erzeugen kann. Daneben beobachtete Hermann noch die Bildung einer Säure (Milchsäure). Pßüger gelang es, einen Frosch bei einer Temperatur von wenigen Graden über 0" in einer sauerstofffreien Atmosphäre 25 Stunden lang lebensfähig zu erhalten. Dabei schied das Versuchstier beträchtliche ') Vgl. hierzu: A. V. Hill: J. of Physiol. 46. 28, 435 (1913); 48. XI (1914); Physiol. Reviews. 2. 310 (1922). V. r. Weizsäcker: Sitzungsber. der Heidelberger Ak. d. Wissenschaften. Math.-naturw. Klasse. .Tg. 1917. 8. Abhaudl.; Münchner med. Wochenschr. Nr. 7, 247 und Nr. 8, 257 (1915). Vgl. auch .7. Parnas: Pflügers Arch. 134. 441 (1910). J. Parnas und E. Wagner: Biochem. Zeitächr. 61. 387 ./. Parnas: Zentralbl. f. Physiol. 30. 1 (1915). .7. Bernstein: Pflügers (1814). Arch. 159. 521 (1914). Olto Meyerhof: Pflügers Arch. 175. 88 (1919); 182. 232, 284 (1920); 185.11 (1920); 188. 114 "(1921); 195. 22 (1922). Vgl. auch J.v.Kries: Ebenda. 190. 66 (1921). W. Hartree und A. V. Hill: .Tourn. of physiol. 54. 84 Vgl. ferner Jakoh K. Parnas: Biochem. Zeitschr. 116. 71. (1920); 55. 133 (1921). 102 (1921). ^) Hermann: Untersuchungen über den Stoft'wechsel der Muskeln. Berlin 1867. ») E. Pßüger: Pßilgers Arciiiv. 10. 251 (1875). ) G. Bunge: Zeitschr. f. physiol. Chemie. 8. 48 (1883/84); 12. 565 (1888); 14. 318 (1889). — — — — — — — — — Kohlehydrate. Kohlensäuremengen aus. Schließlich wies wurm der Katze, Ascaris mystax, 4 139 G. v. Bunge nach — 5 Tage , daß der Spul- in vollkommen sauerstoff- Medien leben und sich gleichzeitig äußerst lebhaft bewegen kann. Weinland^) und später besser'^) haben weiterhin ausführliche Studien über den Stoffwechsel mancher Tiere in Abwesenheit von Sauerstoff gemacht freien und interessante Befunde erhalten. Um weitere Einbücke in mögliche Abbaustufen und Zwischenglieder zwischen verschiedenen Klassen von Verbindungen zu erhalten, hat man verschiedene Substanzen auf ihr Vermögen, in Glykogen überzugehen, geprüft. Die meisten dieser Versuche sind an der überlebenden Leber ausgeführt worden. Es zeigte sich, daß zu derartigen Versuchen sich nur das ganz lebensfrische Organ eignet. Es muß wenige Minuten nach dem Tode des Tieres zur Durchblutung kommen. 3) Aus Dextrose und Lävulose bilden die Leberzellen direkt Glykogen. Galaktose und Maltose werden nicht verwendet. Die erstere muß ohne Zweifel vorher in Glukose umgelagert und die letztere in ihre Komponenten gespalten sein. Diese Feststellungen, wonach die überlebende Leber imstande ist, Glykogen aus bestimmten Bausteinen aufzubauen, eröffneten die Möglichkeit, eine ganze Keihe von Verbindungen als Baumaterial für dieses Polysaccharid zu prüfen, die einerseits beim Abbau von Glukose sich bilden oder aber andrerseits als Stufen beim Aufbau von Traubenzucker aus nicht zuckerartigem Material in Frage kommen könnten. Dazu ist zu bemerken, das man wohl nicht fehl geht, wenn man annimmt, daß die Abbau- und Aufbaustufen zum Teil, wenn nicht sogar vollständig identisch sind, d. h. es sind wahrscheinlich die Auf- und Abbaureaktionen reversibel. Als Glykogenbildner erkannt worden: sind bis jetzt bei solchen Durchblutungsversuchen d-Milchsäure (1) CH3 CH(OH) COOK; Glyzerinsäure CH.,(OH) CH(OH). . . . .COOK; Glyzerinaldehyd CH^ (OH) CH (OH) . .CH.,(OH); . C\{^. Glykol CH.(OH). Glykolaldehyd CH2(0H).C\^. Glykolaldehyddikarbon- säure C00H.CH(0H).C0.C00H. Aus Glykolsäure, Glyoxylsäure, GH., (OH).COOH, C\{J-COOH. Brenztraubensäure), CH3.CO.COOH, vermochte die überlebende Leber kein Glykogen zu bilden. Auch Aminosäuren, wieAlauin und Serin, wurden nicht verwandelt. Nun darf man bei Versuchen an isolierten Organen die positiv ausgefallenen Versuche wohl ohne weiteres in dem Sinne verwerten, daß die betreffenden Substanzen den sie verwendenden Zellen, im vorliegenden Fall den Leberzellen, nicht fremd sind. Man darf vielmehr mit größter Wahrscheinlichkeit ) Von den wichtigen Arbeiten von E. Weinland sei genannt: Zeitschr. f. Bio- 55 (1901). Vgl. den zusammenfassenden Vortrag von E. J. Lesser: Das Leben ohne Zeitschr. f. Biologie. Sauerstoff. Verhandl. der physiol. Gesellschaft. Berlin. 37. 1 (1912). 51. 487 (19Ü7); 53. 533 (1909); 54. 1 (1910); 56. 467 (1911). ») Luchsinqer: In.-Diss. Zürich 1875. Grube: Pßüger^ kK)\\\AQil. 483(1905); 118. 1 (1907). J. Parnas und Julius Euer : Biochem. Zeitschr. 41. 386 (1912). //. K. Ban-enscheen : Biochem. Zeitschr. 58. 277 (1914). ») P. Mayer: (Biochem. Zeitschr. 40. 441 [1912]) hält die Glykogenbildung aus Brenztraubensäure für wahrscheinlich erwiesen. logie. 42. ^) — — ' — — ^ I- Vorlesung. X40 annehmen, daß die zur Glykogenbildung geeigneten Baumaterialien auch im normalen Stoffwechsel auftreten und in gleicher Weise Verwendung finden können. Dagegen darf man die negativ verlaufenen Versuche nicht als entscheidend dafür betrachten, daß der tierische Organismus nicht zu Glykogen führten, nicht in Kohlehydrate verwissen, daß zahlreiche Organe im Organismus zuarbeiten. Das eine fördert einen Vorgang bis zu einer bestimmten jene Stoffe, die wandeln kann. sammen Stufe, übergibt Wir diese dann einem anderen Gewebe, das nun seinerseits eine weitere Umwandlung vollzieht. Erst wenn der Versuch am überlebenden viele verschiedene Organe ausgedehnt worden ist und vor allem Versuche an kombinierten Organpräparaten, d. h. unter Verwendung Organ auf mehrerer gleichzeitig durchströmter Organe durchgeführt worden sind, kann man zu eindeutigen Ergebnissen gelangen. Endlich muß der Versuch sei es nun, daß man die Substanzen verfüttert oder unter Umgehung des Darmkanales in die Blutbahn bringt, herangezogen werden. am ganzen Tiere, aber Fassen wir nun zusammen, was wir über die Verarbeitung der Kohlehydrate in den Zellen der Gewebe wissen, dann ergibt sich folgendes die es ihnen ermöglichen, KohleBild. Die Zellen verfügen über Fermente hydrate, die aus mehreren einfachen Sacchariden aufgebaut sind, unter Wasseraufnahme in ihre Bausteine zu zerlegen. Soweit unsere jetzigen Kenntnisse reichen, kommen nur Polysaccharide der Hexosen für den Zellstoffwechsel in Betracht, wenn wir von den eigentlichen Glukosiden absehen. Die Zellen bilden über mehrere Zwischenstufen Dextrine genannt Glukose. In dieser Form kommt der Zucker im Organismus zum Transport. Die Synthese der Polysaccharide geht von der Glukose aus und ebenso beginnt der tiefere Abbau der Kohlehydrate wohl fast ausnahmslos bei ihr. Sie wird schließlich in Kohlensäure und Wasser übergeführt. Dabei wird ganz genau jene Energiemenge frei, die notwendig war. um sie aus Wasser und Kohlensäure aufzubauen. Es ist sehr unwahrscheinlich, daß jemals eine direkte Oxydation der Glukose zu den genannten Endprodukten stattfindet. Der Abbau der Glukose ist vielmehr ein stufenweiser. Es erfolgen zunächst Spaltungsvorgänge. Diese führen zu Abbaustufen, die uns den Weg zeigen, auf dem die Kohlehydrate mit Verbindungen anderer Körperklassen in Verbindung treten können. Ferner erkennen wir die Brücken, die umgekehrt von anderen Verbindungen zu den Kohlehydraten hinüberführen. Wir können zur Zeit nur einzelne dieser Abbaustufen mit einiger Wahrscheinlichkeit als im Zellstoffwechsel auftretend angeben. Es unterliegt keinem Zweifel, daß ihre Anzahl viel größer ist, als man im allgemeinen annimmt. , — — Dieser stufenweise Abbau ermöglicht es der Zelle, ohne Sauerstoffverbrauch das Glukosemolekül zu zerlegen. Die Zelle bereitet sich Verbindungen aller Art, die für sie oder andere Zellarten von größter Bedeutung sind. Bei jeder Zwischenstufe kann Halt gemacht und irgend eine Synthese in die Wege geleitet werden. Gleichzeitig setzt die Zelle auf diese Weise die in der Glukose enthaltene Energiemenge in Teilbeträgen in Freiheit. Die Zelle regelt beim stufenweisen Abbau ihren Energiewechsel in feinster Weise. Es eröffnen sich uns außerordentlich wichtige Einblicke in das feinere Getriebe der Zellen. Wir verstehen, weshalb trotz Anwesen- Kohlehydrate. 141 heit von Sauerstoff und Glukose und den die Oxydation vermittelnden Stoffen die Bildung von Kohlensäure und Wasser nicht direkt einsetzt. Es müssen Spaltungsvorgänge vorausgehen, die von der Zelle von Fall zu Fall bestimmter Weise in die Wege geleitet werden. Wenn wir von einer Leitung bestimmter Vorgänge durch Zellen sprechen, wollen wir damit nicht zum Ausdruck bringen, daß die Zelle durch ein eigenes Zentralorgan irgend welcher Art aktiv Vorgänge unterbricht, einleitet oder durchführt. Es ist vielmehr an eine Art von Selbstregulation zu denken, etwa in der Art. daß ein bestimmter Zustand nach physikalischen und chemischen Gesetzen bestimmte Vorgänge auslöst, sie zum Stillstand bringt oder in -rückläufigem Sinne beeinflußt. Diesen Gesetzmäßigkeiten müssen wir nachgehen. Wir kennen sie noch nicht und können sie auch nicht kennen, weil einmal immer noch der chemische und physikalische Bau der Zelle nur ungenügend erkannt ist, und ferner bisher nur der geringste Teil des Geschehens in den Zellen aufgeklärt ist. Sicher wird die Zukunft uns gestatten, die Zellvorgänge mit viel genaueren Ausdrücken zu beschreiben. Nicht unerwähnt wollen wir lassen, daß ohne Zweifel eine ganze Reihe von Organen und in erster Linie die Pankreasdrüse, die Nebenniere, die Schilddrüse und die Hypophyse in einzelne Stoff wechselvorgänge eingreifen und vor allem auch auf den Kohlehydratumsatz einen tiefgehenden Einfluß ausüben. in Vorlesung VII. Kohlehydrate. VI. — Alimentäre Glukosurie. — Zuckerzentrum. — Bedeutung der Nebennieren für den ZuckerstoifDie Regulation des Kohiehydratstoffwechsels. wechsel der Leber. Wir wollen zunächst zusammenfassen, was wir bis jetzt über das Verhalten der mit der Nahrung aufgenommenen Kohlehydrate im tierischen Organismus erfahren haben. Zunächst unterliegen die Kohlehydrate der Verdauung im Darmkanal, Die aus mehreren Bausteinen aufgebauten Verbindungen werden durch bestimmte Fermente zerlegt.« Schließlich bleiben im wesentlichen nur noch einfachste Bausteine übrig. Nichts erinnert mehr an den ursprünglichen, einer bestimmten Aufgabe angepaßten Bau. Wir sind nicht imstande, aus der Art der Bausteine Schlüsse auf die Struktur jenes Polysaccharids zu ziehen, dem sie entstammen. Die einfachen Abbaustufen werden resorbiert und dem Blute übergeben. Für den tierischen Organismus spielt als Transportzucker und als Material zu den verschiedensten Zwecken der Traubenzucker die Hauptrolle. Fruktose und Galaktose können leicht durch Umlagerung in Glukose übergeführt werden. Es dürfte im wesentlichen nur Traubenzucker dem Blute übergeben werden. Der Anteil des aufgenommenen Traubenzuckers, der augenblicklich keine Verwendung findet, wird in Form von Glykogen oder Fett abgelagert. Die Leber- und Muskelzellen sind die Hauptspeicher für das erwähnte Polysaccharid. Die einzelne Zelle beginnt ihre Umsetzungen beim Traubenzucker. Stehen ihr Polysaccharide, wie z. B. Glykogen, zur Verfügung, dann zerlegt sie diese zunächst durch Hydrolyse, bis der Abbau zu Glukose führt. Diese verwertet die einzelne Zelle je nach Bedarf. Bald wird sie neue Polysaccharide oder auch Glukoside bereiten, bald braucht sie Energie und spaltet, um solche zu erhalten, Glukose, bald werden Abbaustufen aus ihr als Ausgangsmaterial zur Synthese bestimmter Verbindungen verwendet. Eine solche Synthese ist die Bildung von Fett aus Kohlehydraten. Die Glukose kann nicht direkt in Fett übergehen. Sie muß in noch einfachere Bruchstücke zerlegt werden. Wir -begegnen, wenn wir den Abbau der Glukose durch die Zellen genau verfolgen, Abbaustufen, von denen aus eine Bildung der Komponenten der Fette verständlich wird. Es hat sich herausgestellt, daß die Glukose nicht direkt zu Kohlensäure und Wasser, den Stoff Wechselendprodukten der Kohlehydrate, abgebaut wird. Es erfolgt vielmehr ein stufenweiser Abbau, der in der Hauptsache mit einer Spaltung einsetzt. Erst bei tieferen Spaltstücken erfolgen im allgemeinen Oxydationen. Wahrscheinlich verläuft der Abbau des Traubenzuckers je nach Bedarf verschieden. Kohlehydrate. '^^^^ Wir haben nun bereits festgestellt, daß große Mengen von Kohlehydraten im tierischen Organismus in Form von Glykogen gespeichert werden können. Ferner können Kohlehydrate den Fettbestand vermehren. Weiterhin haben wir betont, daß z. B. die Muskelzellen Leistungen auf Kosten von Kohlehydraten vollführen. Ja, wir sahen, daß durch angestrengte Muskelarbeit die Glykogenspeicher ganz oder doch größtenteils geleert werden können. Wir beobachten, daß bestimmte Zellarten in Tätigkeit sind. Sie brauchen organische Verbindungen, in unserem Falle Kohlehydrate, um Energie zur Leistung von Arbeit zu gewinnen. Ist der Vorrat erschöpft, dann hört die Arbeit nicht auf. Der Muskel verkürzt sich weiter. Nach dem Gesetz der Erhaltung der Energie ist diese Tatsache nur so zu erklären, daß dem arbeitenden Muskel neues Nährmaterial zufließt. Das ist Er entnimmt dem Blute Kohlehydrate und andere in der Tat der Fall. Stoffe. Es wäre zu erwarten, daß nach kurzer Zeit der Zuckergehalt des Blutes sinken würde, da ihm, wie sicher festgestellt worden ist, Glukose durch die arbeitenden, über keine Vorräte mehr verfügenden Muskelzellen entzogen wird. Tatsächlich bleibt der Zuckergehalt des Blutes in engen Grenzen gleich.^} Also muß logischerweise, da das Gesetz der Erhaltung der Materie auch für die Lebewesen volle Gültigkeit hat, dem Blute von irgendwoher Glukose zufUeßen. Man kann an verschiedene Möglichkeiten eines Ersatzes denken. Wenn wir vom Gehalt des Blutes an Traubenzucker sprechen, meinen wir stets Glukose, die im Blutplasma gelöst, d. h. in freiem Zustand, vorhanden ist. Es wäre nun denkbar, daß im Blutplasma sich Polysaccharide finden, die dann, wenn der Glukosegehalt des Plasmas zu sinken beginnt, hydrolysiert werden. Ferner könnte Traubenzucker an andere Stoffe im Blute locker gebunden sein. Durch Spaltung solcher glukosidartigen Verbindungen würde dann der Gehalt des Blutes an freier Glukose wieder auf die Norm zurückgebracht. mehrfach behauptet worden, daß im Blute neben dem vorkomme. Es ist jedoch nicht geglückt, den Nachweis solcher Verbindungen eindeutig zu erbringen. Den Angaben, nach denen immer neben freiem Traubenzucker auch gebundener im Blutplasma sich befinden soll='), stehen andere gegenüber, die mit aller Entschiedenheit betonen, daß das Blutplasma ausschließlich freie Glukose enthalte. Es war bei dieser Sachlage die Aufgabe der weiteren Forschung, Methoden zu schaffen, die es ermöglichen, diese einfache Fragestellung nach Art des Vorkommens der Glukose im Blute einwandfrei zu entscheiden.) Zur Bestimmung des Blutzuckers ist es notwendig, die Eiweißkörper aus dem Plasma zu entfernen. Es ist nun möglich, daß Glukose In der Tat ist freien Traubenzucker 2) auch gebundener Der Blutzuckergehalt normaler Individuen schwankt zwischen 009— OlOVoIm Blutplasma sollen regelmäßig Pentosen in Mengen von 000(5— 0012 7o vorkommen. Vgl. J. W. Best: Arch. neerlaud. de physiol. de l'homme et des auimaux. 3. 222 (1919). ) -) ') R. Le'pine: Le diabete sucr6. Felix Alcan, Paris 1909. Hier finden sich zahlreiche Literaturangaben. ) Ivar Bang: Der Blutzucker. Bergmann, Wiesbaden 1913. In dieser ausgezeichneten kritischen Zusammenstellung finden sich alle wichtigen Arbeiten über dieses Gebiet. Vgl. ferner Richard Ege : Biochem. Zeitschr. 87. 92 (1918). Israel L. Kleiner: The Journ. of biol. Chemie. 34. 471 (1918). St. Bttszntjäh: Biochem. Zeitschr. 113. 52 (1921).— St. Rusznydk und G. Hetenyi : Ebenda. 121. 125 (1921). — — — ^^^' Vorlesung. 144 bei der Fällung der Proteine mitgerissen wird, und daß der Anschein entgebundene Glukose vor. Es ist aber auch denkbar, daß bei der Enteiweißung locker gebundener Traubenzucker aus seiner Bindung gelöst wird, und so nur freie Glukose zur Beobachtung kommt. Der nun Jahre umfassende Streit über die Art des Vorkommens des Traubenzuckers im Blute zeigt, wie kaum ein anderes Problem so deutlich, welch hohe und entscheidende Bedeutung für die biologische Forschung eine einwandSie führt zu Fortschritten, die dauernd ihren Wert fi'eie Methodik hat. behalten. Fehlen uns ausreichende Methoden, um eine bestimmte Fragestellung eindeutig zu beantworten, dann tasten wir uns mühsam durch unzuverlässige Beobachtungen hindurch und kommen nicht über Vermutungen steht, als läge hinaus. Die Erforschung der Art des Vorkommens des Zuckers im Blut ist durch die folgende Methode wesentlich geklärt worden. i) Sie beruht kurz auf folgender Überlegung. Bringen wir in einen Schlauch, der aus einer tierischen Membran besteht, ein Gemisch eines im kolloiden und eines nichtkoUoiden Zustand befindlichen Stoffes, dann wird der erstere nicht durch den Schlauch diffundieren können, wohl aber der letztere. Von diesem wird nach einiger Zeit soviel im Dialysat enthalten sein, Schlauches die daß die Flüssigkeit innerhalb und außerhalb des aufweist. Die gelösten Teilchen des Konzentration davon gleiche Nichtkolloids sind so lange durch die Membran hindurch gewandert Würden wir als Außen(diffundiert), bis Gleichgewicht erreicht ist. betreffende Nichtkolloid in flüssigkeit eine Lösung wählen, die das genau der gleichen Konzentration enthält, wie die Innenflüssigkeit, dann würde keine Diffusion eintreten, weil der Versuch bereits mit einem Gleichgewicht beginnt. Wenn wir nun in den Dialysierschlauch Blutplasma füllen, dessen gesamten Zuckergehalt wir ermittelt haben, so muß, falls ein Teil der analytisch festgestellten Glukose in gebundenem Zustande im Blutplasma verbanden ist, Traubenzucker von der Außenflüssigkeit in den Schlauchinhalt hineindiffundieren, falls wir dieser Glukose in der dem ermittelten Zuckergehalt entsprechenden Konzentration zufügen. Es ergab sich, daß Gleichgewicht besteht, d. h. es wanderte keine Glukose. Ein Gefälle war also nicht nachweisbar. Aus diesem Befunde ergibt sich, daß die analytisch festgestellte Zuckermenge in freiem Zustande im Blutplasma vorhanden ist. Unentschieden bleibt jedoch immer noch, ob außer dem mit der angewandten Methode aufgefundenen Zucker noch solcher vorhanden ist, der, weil in Bindung anwesend, der Bestimmung entgeht. Ferner ist noch der Einwand möglich, daß der locker gebundene Zucker schon kurze Zeit nach der im Blutentnahme in Freiheit gesetzt wird. 2) Wir dürfen aus den vorliegenden Ergebnissen mit größter Wahrscheinlichkeit schließen, daß die Glukose, die man im Blutplasma nach den gewöhnlichen Methoden bestimmt, in freiem Zustande in diesem enthalten ist. Offen bleibt dagegen immer noch die Frage, ob außerdem noch Verbindungen im Blute enthalten sind, die Zucker gebunden Soviel ist jedoch sicher, daß der sog. gebundene Zucker bei weitem nicht ausreichen würde, um den Zuckerbedarf des Blutes zu decken. enthalten. ) ») Leonor Michaelis und I'eier liona : Biochem. Zeitschr. 14. 476 (1908). Vgl. auch S. Gutmann und 0. Adler: Biochem. Zeitschr. 83. 11 (1917). Kohlehydrate. 145 wenn ihm Glukose durch arbeitende Zellen entführt wird. Xun enthält das Blut auch Zellen. Wir finden in ihm rote und weiße Blutkörperchen und Blutplättchen. Die weißen Blutkörperchen können Glykogen bilden. Man findet dieses Polysaccharid oft in ihrem Zelleib. Ohne Zweifel finden sich auch in den roten Blutkörperchen und den Blutplättchen Polysaccharide oder Glu- koside, kurz ^erbindungen, an deren Aufbau Glukose beteiligt ist. Diese Zellen haben alle auch ihren Kohlehydratstoffwechsel. Auch sie verwenden Glukose zu allen möglichen Vorgängen. Man darf nicht ohne weiteres daraus, daß diese Zellen Traubenzucker oder polymere Verbindungen davon enthalten, schheßen, daß sie etwa die im Blute entstehenden Lücken im Traubenzuckergehalt von sich aus ausgleichen. Vielmehr zehren all diese ihrerseit vom Traubenzuckergehalt des Plasmas. Während es Zellen früher nicht gelungen war, in den roten Blutkörperchen Glukose aufzufinden, ist es jetzt durch Verbesserung der Methodik möglich geworden, ihre Anwesenheit festzustellen. \) Ob den bisherigen Angaben, wonach die roten Blutkörperchen einiger Tierarten keine Glukose enthalten, eine besondere Bedeutung zukommt, erscheint zurzeit noch mehr als fraglich. Es ist unwahrscheinlich, daß die roten Blutkörperchen verschiedener Tierarten sich durch das Vorkommen oder das Fehlen von Glukose unterscheiden. Es wird gewiß darauf ankommen, in welcher Phase des Stoffwechsels man die roten Blutkörperchen untersucht. Wichtig ist, daß der Traubenzuckergehalt der roten Blutkörperchen nicht der gleich große ist, wie der des Plasmas. 2) Es handelt sich somit nicht um ein Diffusionsgleichgewicht. Die roten und sicherlich auch die übrigen Blutzellen sind in ihrem Kohlehydratgehalt selbständig.^) Scheiden somit die Zellen des Blutes als Quelle für den Traubenzucker des Plasmas so gut, wie vollständig, aus, so bleibt nichts anderes übrig, als der Frage nachzugehen, in w^elchen Beziehungen die Glyko- genspeicher zu dem Zuckergehalt des Blutes stehen. Wir haben ohne Zweifel zwei Arten von Ablagerungsstätten für Glykogen zu unterscheiden. Einmal die Muskelzellen, die gewissermaßen für sich selbst ein Depot errichten. Sie brauchen hauptsächlich Kohlehydrate zu ihren Leistungen. Die Leber dagegen lagert große Mengen von Glykogen ab. die ihre Zellen sicher nur zum kleinsten Teil verbrauchen. Die Leber stellt in gewissem Sinne einen Zentralspeicher dar, von dem aus die übrigen Speicher ) Vgl. die Literatur (L. Michaelis und Peter Bona, P. Bona und A. Döblin, D. Takahashi, Lyttkens und Sandgren, R. Lepine und Bouhid, A. Hollinger, E. Frank uud A. Brett Schneider, Moeckel, Bud. Hoeber u. a.) bei Ivar Bang, 1. c. ') Vgl. Bollg und Oppermann: R. Höher: ßiochem. Zeitschr. 45. 207 (1912). Ebenda. 48. 187 (1913); 49. 378(1913). Frank: Zeitschr. f. physiol. Chemie. 70. 129 W. Stepp: P. Bona und Döblin: Biochem. Zeitschr. 31. 215 (1911). (1910/11). Deutsches Archiv f. klin. Med. 124. 199 (1917). ^) Die Frage ob die roten Blutkörperchen für Glukose durchlässig sind, ist immer noch heiß umstritten. Vgl. z. B. B. Brinkman und F. van Dam: Arch. Internat, de Bich. Ege: Biochem. physiol. 15. 107 (1919); Biochem. Zeitschr. 108. 94 (1920). Zeitschr. 111. 189 (1920) 114. 88 (1921). - W. Falta und M. Bichter-Quittner: Ebenda. Ebenda. 107. 248 (1920). //. C. Hagedorn: ICO. 148 (1919); 129. 576 (1922). S. van Creveld und B. Brinkman: Ebenda. 119. M. Bönniger: Ebenda. 122. 258 (1921). Mir K. Onohara: The British Journ. of experim. Path. 2. 194 (1921). 65 (1921). scheint, daß jeder einzelne Forscher immer nur aussagen kann, ob die roten Blutkörperchen bei den von ihm gewählten Bedingungen für Traubenzucker durchlässig sind oder nicht. Auf keinen Fall dürfen die gemachten Beobachtungen ohne weiteres verallgemeinert werden. — — — — — — ; - — — Abderhalden, Physiologische Chemie. I. Teil, 5. Anfl. — 10 146 ^11- Vorlesung. und vor allem das Blut mit Traubenzucker versehen werden. Wir kommen zu diesem Schlüsse weil festgestellt worden ist, daß die Leber auch dann ihr Glykogen einbüßt, wenn ihre Zellen erwiesenermaßen keine vermehrten Leistungen aufweisen. Es braucht nur der Fall einzutreten, daß z. B. Muskeln dauernd Arbeit leisten und infolgedessen Energiequellen, vor allem Kohlehydrate, brauchen. Es fragt sich, ob nur die Leber ihr Glykogen hergibt, oder ob auch andere Organe und vor allem auch jene Muskeln dieses Polysaccharid zur Verfügung stellen, die ruhen. Es spricht sehr vieles für eine derartige Annahme. Es ist in gewissem Sinne im Bedarfsfall jede Zellart ein Spender von Zucker, die Glykogen enthält, wenn andere Gewebe und dadurch das Blut Bedarf an solchem haben. Der Umstand, daß, wie wir schon erwähnt haben, der ruhende Muskel mehr Glykogen aufweist, als der erschöpfte, beweist noch nicht, daß der erstere nicht auch Glykogen eingebüßt hat, weil der Glykogengehalt bei Beginn des Versuches nicht bekannt ist. Vielleicht hält der Muskel eine bestimmte „Notration" zäh zurück. Fußend auf den bisher mitgeteilten Tatsachen, können wir uns die Beziehungen zwischen den Verbrauchsstätten von Glukose, dem Blute und den Glykogenspeichern und insbesondere dem großen Lager in der Leber, wie folgt, vorstellen. Wir gehen am besten von einem Ruhestadium aus und nehmen an, daß wir ein gut ernährtes Tier vor uns haben, dessen Darmkanal jedoch leer ist, d. h. es ist seit einiger Zeit keine Nahrung aufgenommen worden. Die Glykogenspeicher sind gefüllt. Im Blute kreist eine bestimmte Menge von Traubenzucker. Nun beginnt eine Muskelgruppe zu arbeiten. Sie greift ihre Glykogen Vorräte an, baut das Polysaccharid hydrolytisch ab und verbraucht die gebildete Glukose in der früher geschilderten Weise. Nach einiger Zeit sind die Kohlehydratvorräte erschöpft. Die Muskelzellen beziehen Traubenzucker direkt oder durch Vermittlung der Lymphe aus dem Blute. Infolgedessen muß der Glukosegehalt des Blutes sinken. Da vom Darmkanal kein Zufluß an Kohlehydraten erfolgt, so wird der Mindergehalt an Traubenzucker von den Organen aus ausgeglichen, die noch Glykogenvorräte besitzen. In erster Linie wird die Leber zum Ersatz herangezogen. Ihre Zellen bauen in dem Maße Glykogen ab, als der Zuckerspiegel im Plasma sinkt. Immer neue Mengen von Traubenzucker werden nachgeschoben, so daß der Zuckergehalt des Blutes unter normalen Verhältnissen nie wesentlich unter die Norm sinkt. Dieser Umstand ist von größter Bedeutung. Ob das Tier bis zur Erschöpfung arbeitet oder hungert, immer findet sich im Blute annähernd der gleiche Traubenzuckergehalt. Viele Organe wachen darüber, daß er nicht sinkt. Die gegebene Darstellung der Wechselbeziehungen zwischen Organen, die Kohlehydratvorräte besitzen, und solchen, die Kohlehydrate brauchen, ergibt auf der einen Seite eine Stelle, an der dem Blute Traubenzucker entzogen wird und auf der anderen Seite einen Ort, von dem aus diesem wieder solcher zugeführt wird. Der Abfluß regelt gewissermaßen den Zufluß. So plausibel eine derartige Annahme auch ist, und so gut sie sich auch mit den bis jetzt erörterten Resultaten der Erforschung des Kohlehydratstoffwechsels deckt, so dürfen wir uns mit dieser Anschauung allein nicht zufrieden geben. Verschiedene Phasen in Kohlehydrate. [47 diesem Austauschvorgang bedürfen noch der Aufklärung und des experimentellen Beweises. Einmal müssen wir uns nach Versuchen umsehen, die eiiideutig belegen, daß tierische Zellen und insbesondere die Leberzellen Glykogen zu Traubenzucker abzubauen vermögen. P'erner wollen wir wissen, was die Leberzellen veranlaßt, das abgelagerte Polysaccharid im richtigen Augenblick in gerade ausreichender Menge zu spalten und den gebildeten Traubenzucker dem Blute zu übergeben. Wir wollen gleich erwähnen, daß man an eine vom Blute selbst herbeigeführte Regelung denken könnte. Es wäre möglich, daß die Leberzellen in feinster Weise auf einen bestimmten Gehalt des Blutes an Glukose eingestellt sind. Es könnte ein Gleichgewicht zwischen Blutzucker und dem Zucker-, bzw. Glykogengehalt der Leberzellen bestehen. Steigt der Gehalt des Blutes an Glukose, dann geht solcher in die Leberzellen über. Diese beantworten die erhöhte Traubenzuckerkonzentration mit einer Bildung von Glykogen. Fällt dagegen der Zuckergehalt des Blutes, dann setzt der umgekehrte Vorgang ein. Es ist durchaus möglich, daß diese Art der Regelung des Blutzuckergehaltes eine Rolle spielt, jedoch kommen sicher noch andere Momente in Betracht, wie wir gleich erfahren werden. Der Abbau des Glykogens in den Leberzellen ist gründlich studiert worden. Schon lange hatte man vermutet, daß er durch ein diastatisches Ferment eingeleitet wird und über Dextrine und Maltose schließlich ausschließlich zu Traubenzucker führt, i) Es war jedoch sehr schwer, einen eindeutigen Beweis für diese Annahme zu erbringen. Immer wieder tauchten Bedenken gegen die ausgeführten Versuche auf. Es wurde der Standpunkt verteidigt, daß nur die Leberzelle als Ganzes im „lebenden" Zustande den Abbau des Glykogens durchführen könne. Schon Wittich^) konnte im Jahre 1873 den Nachweis führen, daß es gelingt, aus der vollständig blutleeren, mit Alkohol gehärteten Leber mit Glyzerin einen Stoff auszuziehen, der Glykogen zu Traubenzucker abbaut. Pnvy^) zeigte ferner, daß man eine mit Alkohol behandelte Leber lange Zeit aufbewahren kann. Immer zeigen wässerige Auszüge der so aufbewahrten Leber diastatische Wirkung. Alle derartigen Versuche begegneten immer wieder dem Mißtrauen kritischer Forscher. Es wurde eingewendet, daß der Glykogenabbau durch Mikroorganismen herbeigeführt w^orden sei. Diese finden sich überall. Arbeitet man nicht mit allen Vorsichtsmaßregeln, um Infektionen zu vermeiden, dann kann man sicher damit rechnen, daß solche anwesend sind. Allerdings vermissen wir im einzelnen Falle den Beweis, daß Mikroorganismen Glykogen abbauen und vor allem in dem Maße zerlegen können, wie es bei der Verwendung der Leberauszüge beobachtet worden ist. Kontrollversuche mit gekochten Lebern ergaben keine wirksamen Extrakte. Man erhält mit diesen, wie man leicht feststellen kann, keinen Abbau von Glykogen, auch wenn man sonst unter den gleichen Bedingungen arbeitet, wie mit dem wirksamen Auszug. Schließlich hat E. Salkowski^) den Einwand, E. und — ') Muskulus und v. Merinq: Zeitschr. f. psysiol. Chemie. 2. 416 (1878/79). E. Kiltz W. Fan/: The Physiology of Carbohydrates. 125 u. 132. London 1894. — Vogl: Zeitschr. f. Biol. 31. 108 (1895). V. Wittich: Pßüf/ers Archiv. 7. 28 (1873). ^} E. b'alkowski: Deutsche med. Wochcnschr. Nr. 16(1888); Archiv f. (Anat. u.) Physiol. 554 (1890); Zentralbl. f. d. med. Wissensch. 27. Nr. 13. 227 (1889). S. 2) 10* ^'11- 148 Vorlesuug. daß der Glykogenabbau auf Bakterien zurückzuführen sei, dadurch widerlegt, daß er die Zuckerbildung auch in Chloroform wasser beobachtete. Da Chloroform in wässeriger Lösung jede Protoplasmawirkung und damit auch diejenige von Mikroorganismen aufhebt, so ist bewiesen, daß die Leberzelle als solche nicht notwendig ist, um die Diastase, die von ihr gebildet worden Salkowski hat z. B. den folgenden Versuch ist, zur Wirkung zu bringen. ausgeführt. Er entnahm einem Kaninchen, das 17 Stunden vor dem Tode 10 g in Wasser gelösten Rohrzucker per os erhalten hatte, die Leber. Sie wurde sofort zerhackt und zerrieben, nachdem die Gallenblase und die gröberen Gallengänge entfernt worden waren. Nun wurde der Leberbrei in zwei gleiche Hälften geteilt. Die eine wurde ohne weitere Maßnahmen mit Chloroformwasser zusammen in eine Flasche gefüllt. Die andere wurde vorher gekocht. Nach 68 Stunden wurde in beiden Versuchen der Gehalt an Glykogen und an Glukose- festgestellt. Bei dem Versuche, bei dem ungekochter Leberbrei angewandt worden war, fand Salkowski reichlich gekochter Zucker (4828^) und kein Glykogen. Der zweite Versuch ergab viel Glykogen und wenig Zucker (36ö(/). Durch das Leberbrei Kochen war eben die Diastase zerstört worden! Die erwähnten Versuche beAveisen, daß sich aus der Leber amylolytische Fermente isolieren lassen. Nun besteht jedes einzelne Organ nicht nur aus den ihm zugehörenden Zellen, sondern es enthält auch noch Lymphe und Blut mit ihren Formelementen. Da nun bereits durch Magendie^) im Jahre 1846 'festgestellt worden war, daß dem Blute eine diastatische Wirkung zukommt, war die MögUchkeit gegeben, daß beim Nachweis der Diastase in der Leber gar nicht ein Leberzellferment isoliert worden war, sondern ein dem Blute zugehörendes Ferment. Durch vollständiges Entbluten der Leber und gründliches Auswaschen des noch verbliebenen Blutes konnte die Diastase des Blutplasmas und diejenige der Lymphe ausgeschlossen werden. J. J. B. Madeod und B. G. Pearce und ferner E. J. Lesser-) konnten zeigen, daß auch eine blut- und lymphfreie Leber Diastase aufweist. Somit enthalten die Leberzellen eine eigene Diastase. Die Tatsache, daß das Blutplasma Diastase besitzt, führt bei der Frage nach dem Vorkommen dieses Fermentes in irgend welchen Zellarten zu der Forderung, daß nur mit einwandfrei entbluteten Organen gearbeitet werden darf. Durch die letzteren Feststellungen, die wir durch eigene an in ganz entsprechender Weise durchgeführten Versuchen gemachten Beobachtungen bestätigen können, ist bewiesen, daß die Leberzellen über einen Ferment- — komplex — — Diastase genannt — verfügen, der Glykogen unter Wasseraufnahme zerlegen kann. Er ist ausziehbar und wirkt auch fern von der Zelle. Wir wollen gleich bemerken, daß offenbar alle Körperzellen Diastase besitzen. Überall stoßen wir auf die Fähigkeit Glykogen abzubauen. ') — Magendie: Compt. rend. d. l'Acad. d. Sciences. 23. 189 (1846). Vgl. weiKurt Moeckel und Franz Eost : Zeitschr. f. physiol. Chem. 67. 433 tere Literatur bei (1910). ^) J. J. R. Madeod und li. G. Pearce: American Jouru. of riiysiol. 25. 25.") (1910). — Ernst J. Lesser: Biocliom. Zeitschr. 52. 471 (1913); vgl. aucli J. Grode undi'.,/. besser: Zeitschr. Biol. 60. 371 (1913); E.J. Lesser: Ebenda. 60. 388 (1913); Müncheuer med. Wochenschr. 60. 341 (1913). — Vgl. ferner Ivar Bang: Biochem. Zeitschr. 49. (1913). f. 1 Kohlehydrate. 149 Mit der Feststellung, daß Leberzellen einerseits über Glykogen verfügen und andrerseits Diastase besitzen, sehen wir noch nicht klarer in der Frage nach den Wechselbeziehungen zwischen Leberzellen und Blut. Im Gegenteil, es türmen sich neue Schwierigkeiten auf. Wir beobachten, daß die zerhackte Leber ihr Glykogen rasch verliert. Weshalb vermochte es neben der Diastase in der unversehrten, lebenden Zelle unverändert zu bleiben? Warum beginnt der Abbau, sobald wir die Leber aus dem Organismus entfernen, und warum erfolgt er besonders schnell, wenn wir die Leberzellen zerstören? Warum gibt die Leber unter normalen Bedingungen nur dann Glykogen ab, wenn ein Bedürfnis irgendwelcher Art vorliegt? Wie kommt es, daß die gleiche Zelle, die Glykogen mit Hilfe der Diastase abbaut, auch imstande ist, aus Glukose jenes Polysaccharid entstehen zu lassen? Das sind alles Fragen, die nach dem jetzigen Stand unseres Wissens nur zum Teil in exakter Weise beantwortet werden können. Wir werden später erfahren, daß die Möglichkeit besteht, daß die Fermentwirkung je den Kouzentrationsverhältnissen zwischen Ferment und Substrat bald zum Abbau, bald zum Aufbau führt. Es ist aber auch möglich, daß besondere Fermente die Synthese herbeiführen. Jedenfalls müssen wir annehmen, daß entweder die einzelnen Fermente in der Zelle auf irgend eine Art von den Substraten, auf die sie eingestellt sind, ferngehalten werden!) oder aber, es sind die Fermente in der Zelle in einem Zustand vorhanden, in dem sie unwirksam sind. Es ist aber auch möglich, daß das Substrat in einer P^rm in der Zelle zugegen ist, in der es von dem wirksamen Ferment nicht angegriffen werden kann. Entweder wird dann nach im geeigneten Augenblick das unwirksame Ferment in die wirksame Form übergeführt^), oder es wird das Substrat so hergerichtet, daß es angreifbar wird. Zerstören wir die Zelle, dann bringen wir alles durcheinander. Vor allem kann jetzt jener Stoff wirksam werden, der entweder das Ferment, wie man sich ausdrückt, aktiviert, d. h. in die wirksame Form überführt, Form gebracht. ») oder es wird das Substrat in die geeignete Die direkte Beobachtung am normalen Tier vermochte keinen weiteren Einblick in den Kohlehydratstoffwechsel zu geben. Es verläuft eben der Stoffwechsel der einzelnen Zelle in so fein geregelter Weise, daß es Vgl. hierzu F. Hofmeister: Die chemische Organisation der Zelle. Friedr. VieFerner: J. J. R. Macleod und G. R. Fearce: AmeriJ. Grode und E. J. Lesser: Zeitschr. f. Biol. can Journ. of Physiol. 25. 255 (1910). 60. 371 (1913). Die beiden Forscher machten die interessante Beobachtung, daß Leber, ') weg & Sohn. Braunschweig 1901. — — Muskeln uud unreife Eier vom Frosche in den Wintermonaten keinen oder doch fast keinen Glykogenschwund zeigen, wenn die Organe unverletzt sind. In den Sommermonaten zeigen d;e gleichen Orgaue postmortalen Glykogenschwund. Die zerkleinerten Organe verhalten sich bei Sommer- und Winterfröscheu gleich. Vgl. hierzu auch Schiff: Vgl. auch Untersuchungen über die Zuckerbestimmung in der Leber. Wiirzburg 1859. — E. J. Leiser: Biochem. Zeitschr. 119. 108 (1921). 2) Nach Irar Bang [Biochem. Zeitschr. 49. vator in Frage. ') 1 (1913)] kommt Kochsalz als Akti- In den Vorlesungen über Fermente kommen wir auf diese Möglichkeiten zurück. Wir werden erfahren, daß Zustandsänderungeu bewirken können, daß Formente wirksam oder auch unwirksam werden. Da die Absorption bei den Fermentvorgängen eine bedeutsame Rolle spielt, können physikalisch-chemische Einflüsse von entscheidender Bedeutung für sie sein. ^11- Vorlesung. 150 nie zu Anhäufungen von Zwischenprodukten kommt. Überall werden Spuren der einzelnen Substanzen gebildet. Ehe wir sie gefaßt haben, sind sie schon Wir stehen immer wieder vor der Tatsache, daß der Organismus so lange nichts über die Einzelheiten seines Zellstoffwechsels verrät, als alle Organe harmonisch zusammen arbeiten. Sehr oft enthüllen uns Störungen im Zellstoffwechsel mit einem Schlage wichtige Phasen in der Verwertung bestimmter Verbindungen. Die Pathologie hat von jeher der Physiologie große Dienste geleistet. Die Natur stellt dann, wenn bestimmte Organe versagen, einen Versuch an, der schon in sehr vielen Fällen der ganzen experimentellen Forschung bestimmte Wege gewiesen hat. Sehen wir im Gefolge der Erkrankung eines bestimmten Organes einen von der Norm abweichenden Verlauf im Abbau bestimmter Verbindungen auftreten, dann fragen wir zunächst, ob die beobachtete Abbaustufe deshalb entstanden ist, weil der Zellstoffwechsel der veränderten Zellen in andere Bahnen gedrängt ist, oder aber, ob eine sonst uns stets entgehende, normale Zwischenstufe im Ab- oder Aufbau bestimmter Substanzen zum Vorschein kommt, weil ihr weiterer Abbau aufgehoben oder doch verzögert ist. Beide Möglichkeiten fesseln unser Interesse im höchsten Maße. Wir müssen versuchen, die eine Möglichkeit auszuschließen. Der einzige W'eg ist bis jetzt der Versuch am Tiere. Wir können dasjenige Organ, das wir im Verdacht haben, daß es bestimmend in den Ab-, Auf- oder Umbau einer bestimmten Verbindung eingreift, ausschalten und w^eiter zerlegt oder fortgeführt. dann die Folgen studieren. Am besten wird ein solches Organ exstir- piert. Zeigen sich bestimmte Ausfallserscheinungen, dann können wir versuchen, diese durch Transplantation des fortgenommenen Organes zu beheben. Ja in manchen Fällen ist es sogar gelungen, Teilfunktionen eines Organes dadurch zu ersetzen, daß man das Organ oder aus ihm hergestellte Substanzen verfütterte. Bald ist die Pathologie auf diesem Gebiete führend gewesen, und es folgte der Tierversuch nach, bald ging die Beobachtung im Laboratorium voraus und warf Licht auf die Ätiologie mancher Symptome bestimmter Krankheiten. Wohl kaum ein anderes Gebiet gibt zur Zeit die engen Beziehungen zwischen Pathologie und Physiologie klarer wieder, als dasjenige der Störungen des Kohlehydratstoffwechsels. W^ir wenden uns nun zu den Erfahrungen, die die experimentelle Erforschung und die Pathologie über den Ablauf des Kohlehydratstoffwechsels ergeben haben. Wir haben wiederholt betont, daß wir normalerweise den größten Teil der Kohlehydrate in aus Bausteinen zusammengesetzter Form aufnehmen. Der Abbau erfolgt im Darmkanal stufenweise, so daß immer von Augenblick zu Augenblick nur Spuren von resorbierfähigen Abbauprodukten entstehen. Es liegt hier eine äußerst fein organisierte Regelung vor, die verhindert, daß der Leber und der großen Blutbahn auf einmal größere Mengen von Traubenzucker zugeführt werden. Was geschieht, jvenn wir diesen Schutz zur Verhinderung der Überschwemmung des Organismus mit Traubenzucker künstlich durchbrechen? Geben wir einem Tiere, z. B. einem Hunde, per os größere Mengen von Traubenzucker, dann beobachten wir, daß von einer geder Zufuhr an im Harn dieser Zucker anzutreffen ist.i) wissen Grenze M Vgl. hierzu u. a- Franz Ifofmeisier: Arch. f. experim. Path. u. Pharinak. Bernhard Schöndorf:' Pßügerii Arch. 121. 572 (1908). 240 (1889). — 25. Kohlehydrate. Wir sprechen von einer Glukosurie. ]^5]^ Wie kommt diese zustande? Eine Antwort gibt die Untersuchung des Zuckergehaltes des Blutes. Wir finden, daß dieses mehr Glukose enthält, als sonst. Man nennt diesen Zustand Hyperglukoplasmie. Es ist offenbar die Niere auf einen bestimmten Gehalt des Blutes an Traubenzucker eingestellt. Wird dieser überschritten, dann greift unter normalen Umstanden die Niere ein. Sie scheidet den Überschuß an Traubenzucker aus und drückt seine Menge im Blut auf die normale Grenze herab. WMe ist die Hyperglukoplasmie zustande gekommen? Durch die Zufuhr großer Mengen von Traubenzucker in den Darm ist auf einmal eine große Menge resorbierfähiges Kohlehydrat zur Stelle. Zunächst führt die Pfortader den Leberzellen mehr Zucker als sonst zu. Diese bauen Glykogen Sie können jedoch mit der Glukosezufuhr nicht Schritt halten. Es geht viel Traubenzucker in den großen Kreislauf über. Alle Körperzellen greifen ein, um das Zuviel an Glukose dem Blute wieder abzunehmen. Auch sie kommen nicht mit. Der Gehalt des Blutes an Glukose bleibt erhöht. Offenbar ist auch keine Zeit zur Umwandlung des Traubenzuckers in Fett. Nun greift die Niere ein und beseitigt die Hyperglukoplasmie durch Ausscheidung des Zuviel an Glukose. Die Glukosurie hört auf, sobald der Zuckergehalt des Blutes wieder auf die Norm herabgedrückt ist. auf. Es ist klar, daß man unmöglich eine bestimmte Menge von Traubenzacker angeben kann, die, wenn in kurzer Zeit zugeführt, zur Glukosurie führen muß.i) Es kommt selbstverständlich darauf an, in welchem Zustand der Organismus sich befindet. Ein Tier, das ruht und dessen Speicher gefüllt sind, wird viel bälder eine Glukosurie aufweisen als ein solches, das ausgehungert ist oder Arbeit leistet vorausgesetzt, daß die Leberzellen an und für sich in vollem Maße funktionstüchtig sind.-) Man hat die Menge Traubenzucker, die noch aufgenommen werden kann, ohne daß es zu einer Glukosurie kommt, als Assimilationsgrenze bezeichnet. Aus der ganzen Darlegung geht hervor, daß wir hier eine Hyperglukämie und infolgedessen eine Glukosurie vor uns haben, die beide einzig und allein durch die Durchbrechung der normalen Art der Ernährung bedingt sind. Hört die Zufuhr der einfachen Zucker auf, dann klingt die Hyperglukoplasmie allmählich wieder ab. Gleichzeitig wird die Glukosurie immer geringfügiger und schließlich tritt überhaupt kein Zucker mehr in den Harn über. Es handelt sich also um einen vorübergehenden Zustand. Man hat diese Art von Glukosurie die alimentäre genannt. Den gleichen Zustand können wir künstlich hervorbringen, indem wir Traubenzucker direkt in die Blutbahn einführen und dadurch eine Hyperglukoplasmie erzeugen. Die Nieren beantworten diese mit einer Ausscheidung von Glukose. Man könnte daran denken, den Einfluß der Leber auf den Kohlehydratstoffwechsel in der Art zu studieren, daß man sie als Speicher ausschaltet. Sie stellt nämlich, wie wir schon betont haben, in doppelter Weise einen Regulator des Kohlehydratumsatzes dar. Einmai verhütet sie — ') Vgl. hierzu u. a. lledirig Begemann : Arch. internat. de Pharmacodynamie et de Th^r. 22. 97 (1912). ^) Vgl. hierzu G. Comessati: Hofmeisters, BcUr. 9. 6()(1907). Grober: Deutsches Arch. f. klin. Med. 95. 137 (1909). H. Hohlweg und F. Veit: Zeitschr. f. Biol. 51, — 491 (1908). — ^^^- ]^52 ^ oilesuug. durch Glykogenspeicherung, daß in dem dem Körperkreislauf zuströmenden Blute zuviel Zucker verbleibt, und dann füllt sie wieder Lücken in seinem Zuckergehalt aus. Leider kann man beim Säugetier die Leber nicht vollständig aus dem Körper entfernen, ohne sein Leben zu gefährden, wohl aber läßt sich das Pfortaderblut unter Umgehung dieses Organes in den großen Kreislauf leiten. Man hat die Pfortader direkt mit der Vena cava inferior in Verbindung Nunmehr kommen die vom Darm resorbierten Stoffe direkt ins Blut des großen Kreislaufs. Direkte Schiidigungen des Kohlehydratstoffwechsels wurden nicht beobachtet. Freilich weisen die ausgeführten Untersuchungen noch große Lücken auf. Vor allem würde es uns interessieren, man nennt die Operation Bilzu erfahren wie ein so operierter Hund dung der Eckschen Anastomose bzw. Fistel zwischen Pfortader und Vena sich bei Arbeitsleistungen verhält, und welche Organe Cava inferior den arbeitenden Muskeln Kohlehydrate zur Verfügung stellen. i) Es scheint aus den verschiedenartigen, jedoch immer noch an Zahl der einzelnen Versuche und Dauer der Beobachtungszeit ungenügenden Untersuchungen hervorzugehen, daß die Leber für den normalen Ablauf des Kohlehydratstoffwechsels nicht ganz unentbehrlich ist.^) Der Zuckergehalt des Blutes soll nach den Angaben einiger Forscher abnehmen, sobald die Leber vollständig entfernt ist. Die Anlegung der £'cÄ:schen Fistel dagegen beeinflußt den Zuckergehalt des Blutes nicht. s) Die Leber steht in diesem Falle immer noch durch die Leberarterie und die ihr entsprechenden Lebervenen und ferner auch durch das Lymphsystem mit dem großen Kreislauf und damit den übrigen Zellen des Körpers in Beziehung. Es gibt auch noch andere Glykogenspeicher, .die helfend eingreifen können, wenn Bedarf an Glukose ist, doch dürfte sich das Fehlen der zentralen Vorratsstätte bei besonderen Ansprüchen an den Kohlehydratstoffwechsel geltend machen. Führt man den Tieren mit EcHcher Fistel andere Zuckerarten als Glukose zu, z. B. Galaktose, dann zeigt sich eine wesentliche Störung. Während z. B, ein Hund vor der Operation von per OS zugeführter Galaktose etwa 4 10% im Harn ausschied, verder Anlegung der Fistel 79% davon unverwertet den ließen nach Körper.) Es hängt dies offenbar damit zusammen, daß die Leber bei der Umwandlung von Nicht-Glukose in Glukose direkt oder indirekt eine hervorragende Rolle spielt, ja es scheint, daß sie in dieser Hinsicht unersetzbar ist. In ihr findet die Überführung von Milchsäure und anderen Produkten in Glukose und darüber hinaus in Glykogen statt. Da durch die überlebende Leber geleitete Galaktose von dieser nicht zu Glykogen aufgebaut^) wird, ist vielleicht der Einfluß dieses Organs bei der TTmwandlung in (Glukose kein direkter. Vielleicht gebracht. — , — — — ) Vgl. B. TuiHß und Vaufihan llailey: Pjliigera Arcliiv. 61. 551 (1895). W. Paii/ und li. L. Siau: Journ. of Pliysiöl. 29. 375(1903). Minkowski: Archiv, f. experim. Path. und Pharm. 21. 41 (1886). Fr. Schenk: T'fiüffcrs Archiv. 57. 553 (1894). Vgl. n. a. N. Biirdenko: Internat. I^citr. zur Path. und Ther. d. Ernährungs- F. — — ') störungen. ') 4. Vgl. 93 (1912). Michaud : Verhandlungen des Deutschen Kongresses für innere Medizin. — de Filippi: Zeitsclir. f. Binl 50. 38 (1908). F. Drauth : Arch. f. experim. Path. u. Pharm. 72. 457 (1913). Franke und Raabe: Sitzungsber. u. Abhandl. d. natui forsch. Gesellsch. Rostock 1912. ^) Vgl. K. Barrenschcm: Biochem. Zeitschr. 58. 277 (1914). 28. 561 (1911). ) — Kolileliychato. 153 hat es Einfluß auf die Größe der Resorption aus aber es wirkt mit anderen Organen zusammen. Es dem Darmkanal, oder ist aber auch möglich, daß beim Durchbiutungsversuch die Bedingungen für eine Umwandlung nicht genügen. Im gleichen Sinne, wie die erwähnten Beobachtungen bei Tieren mit Eckacher Fistel, sprechen auch Versuche am Menschen mit Lebererkrankungen, bei denen nach Zufuhr von großen Mengen (z. B. 40 f/ nüchtern) Galaktose oder Lävulose im Harn bedeutend mehr dieser Kohlehydrate im Harn erscheinen als beim normalen Individuum. i) Wir können von einer anderen Seite aus in sehr schöner Weise festin welch feiner W^eise die Zuckerabgabe von Seiten der Leber normalerweise geregelt wird. Claude Bernard, ein Physiologe, dem wir zum größten Teil die ganzen Grundlagen unserer Kenntnisse des Kohlehydratstoff Wechsels verdanken, hat nämlich entdeckt, daß man von einer bestimmten Stelle der Medulla oblongata aus bewirken kann, daß die Leberzellen mit einem Schlage ihr Glykogen abbauen. Das Blut wird plötzlich mit Traubenzucker überschwemmt. Der Hyperglukoplasmie folgt eine Glukosurie. Sie hält so lange an, als der Zuckergehalt des Blutes stellen, gesteigert ist. Der klassische Versuch von Claude Bernard^) ist der folgende. Es wird einem Kaninchen nach Festlegung des Kopfes die Spitze eines Troikarts in der Medianlinie auf das Os occipitale gleich an der Protuberantia occipitalis superior aufgesetzt und nun durchgestochen, bis man auf die Pars basilaris stößt. Das Instrument durchbohrt hierbei die Schädeldecke, das Kleinhirn und die hinteren und mittleren Stränge des verlängerten Markes. Die genaue Untersuchung der verletzten Stelle hat ergeben, daß sie nach oben durch den Ursprung der beiden Nn. acustici und nach unten durch eine Linie, die die Austrittsstellen der Nn. vagi verbindet, begrenzt wird. Es sei gleich erwähnt, daß man bei Verletzung des Zwischenhirns den gleichen Erfolg sieht. s) Offenbar liegt ein Sympathikuszentrum vor, dessen Bahnen Claude Bernard bei seiner Operation verletzte. Schon 1 2 Stunden nach der erwähnten Operation findet man Zucker im Harn. Die Zuckerausscheidung ist keine dauernde. Gewöhnlich verschwindet sie bei Kaninchen nach 5 6 Stunden. Bei Hunden dagegen dauert sie unter Umständen länger. Claude Bernard beobachtete bei letzteren während 7 Tagen Zuckerausscheidung. Der Gehalt des Harnes an Zucker ist im allgemeinen nicht sehr hoch. Seine Menge beträgt gewöhnlich 2 S^/o.) Claude Bernard gibt bereits als Ursache der Zuckerausscheidung eine Vermehrung des Blutzuckers an. Statt der üblichen 0"05 OlVo fand er über Oo"/o Zucker im Blute. Der Zuckerstich wie die geschilderte Operation gePiqüre nannt wird, gelingt auch bei Vögeln^) und Fröschen.'') — — — — ') Vgl. — — , H. Straitss: Deutsche med. Wochenschr. Nr. 37. 1780 (1913). — Ha/is Wörner und Kmil Reiss: Ebenda. Xr. 18 (1914). — Vgl. auch A. (rottschaJk: Zeitschr. f. die gesamte exper. Med. 26. 34 (1922). — G. Heti'nyi uud St. Liebmann: Klinische 1. 1204 (1922). Claude Bernard : Le(;ons (Cours du semestre d'hiver). 1854 55, pag. 289. ») Vgl. u. a. li. Aschner: Pjfüffera Archiv. 146. 114 (1912). -- Vgl. auch h\ Leschk'e: Zeitschr. f. exper. Path. u. Ther. 14. 167 (1913). Marita: The Tohoku .louru of exper. Med. 2. 403 (1921). Hedon: Diabete. Dictionuaire de Physiol. 4. 812. ^) M. Bernhardt: Virchoirs Archiv. 59' 407 (1874). ^) M. Schiff: Untersuchungen älter die Zuckerbiidung. Wiirzlturg 1859. AVochensclir. — ^) — ••) -!>'. ^^^- Vorlesung. 2^54 Von größter Bedeutung für die ganze Auffassung dieser iVrt von Er fand, daß der Glukosurie ist eine Beobachtung von F. W. Dock.^) Zuckerstich nur dann gelingt, wenn wohlgenährte Tiere verwendet werden, Die Oped. h. solche, die einen Glykogenvorrat in der Leber besitzen. ration versagt dagegen gänzlich, wenn Hungertiere zu diesen Versuchen benutzt werden. Naunyn-) kam zu demselben Resultate. Er zeigte ganz eindeutig, daß der Erfolg des Zuckerstiches ausschließlich vom Ernährungszustände der Versuchstiere abhängig ist. Immer findet man bei der Sektion der einige Zeit nach dem Zuckerstich getöteten Versuchstiere, daß die Leber glykogenfrei geworden ist. Daß die Leber die Eigenschaft, Zucker in Form von Glykogen aufzuspeichern, verloren hat, geht aus folgender Beobachtung hervor. Wird einem normalen, durch Hunger möglichst glykogenfrei gemachten Tiere eine Traubenzuckerlösung in die Mesenterialvene eingeführt, so erscheinen nur geringe Mengen Zucker im Harn. Wird derselbe Versuch mit einem Tiere, an dem der Zuckerstich ausgeführt worden ist, angestellt, so tritt nach der Injektion des Zuckers bald eine starke Glukosurie auf. 3) Wir müssen uns nun die Frage vorlegen, in welchem Zusammenhang der Zuckerstich und die Zucker Überschwemmung des Organismus stehen. Claude Beniard bewies durch den folgenden Versuch, daß die Nn. vagi Beziehungen zwischen der Leber und der erwähnten Stelle in der Medulla vermitteln.) oblongata sie ist Zuckerzentrum genannt worden Wird nämlich nach ihrer Durchschneidung am Halse der Zuckerstich ausgeführt, so ist er ebenso wirksam, wie wenn die Nn. vagi intakt sind. Reizt man den peripheren Stumpf des N. vagus, ohne dalj zuvor der Zuckerstich ausgeführt worden ist, so beobachtet man keine Glukosurie. Sie tritt jedoch alsbald auf, wenn das zentrale, d. h. das mit der Medulla oblongata zusammenhängende Ende gereizt wird. Bei einem solchen Versuche konnte Claude Bernard durch die Sektion nachweisen, daß der ganze Körper des Versuchstieres mit Zucker überschwemmt war. Am meisten Zucker fand er in den Lebervenen. Claude Beruard wies ferner nach, daß die Durchschneidung der Nn. vagi am Halse die Leber in der Folge zuckerfrei macht. Er schließt aus all diesen Versuchen, daß in der Medulla oblongata — — ein Zentrum zur Regelung des Zuckerumsatzes in der Leber vorhanden ist. Die Vermittlung, d. h. die Reizleitung, besorgt der N. vagus. Die Zuckerbildung nach Reizung des zentralen Vagussumpfes faßt Claude Bernard als einen reflektorischen Vorgang auf^), und zwar sollen die Lungenäste des Vagus die auf das Zuckerzentrum wirkenden Fasern führen, denn nach Durchschneidung der Nn. vagi über der Leber und unter der Lunge zeigte sich kein Einfluß auf die Zuckerbildung der Leber mehr. Es fragt sich nun, auf welchem Wege das Zuckerzentrum auf die Leber einen Einfluß ausübt. Claude Bernard durchschnitt das Rückenmark in verschiedener Höhe unter der Medulla oblongata und fand, daß die leitenden Bahnen in den oberen Teilen des Rückenmarkes liegen müssen," denn seine Durchschneidung unter dem ersten Dorsalwirbel hebt die Ein^) -) ») ') 5) F. W. Dock: Pflüger?, Archiv. 5. 571 (1872). B. Naunyn: Archiv f. experim. Path. niui Pharmak. 3. 85 (1875). Vgl. auch I). Eckhard: Beiträge zur Anat. u. Physiol. 8. 77 (187i)). Vgl. die Zusammenstellung von Leccnc : Zentralbl. f. Physiol. 8. 379 (1894). Vgl. /';. F. Pflüger: Das Glykogen. 1. c. 386. Kohlehydrate. 155 Wirkung des Zuckerzentrums auf die Zuckerbildung der Leber auf. Eine wichtige Bestätigung dieser Schlußfolgerung erbrachten C. Eckhardts'^) Versuche. Diese ergaben, daß nach der Durchschneidung beider Nn. vagi und der Nn. sympathici am Halse der Zuckerstich noch wirksam bleibt. Nach der Durchtrennung der beiden Nn. splanchnici hat er jedoch keinen Erfolg mehr. Dieses letztere Ergebnis weist darauf hin, daß die durch den Zuckerstich bewirkte Erregung der Zellen des Zuckerzentrums der Leber auf der Bahn der Nn. splanchnici in irgend einer Weise zugeleitet wird.^) Nach diesen Ergebnissen müssen wir annehmen, daß der Zuckerstoffwechsel der Leber direkt von einem Nervenzentrum aus geregelt wird. Die Nn. vagi leiten die zentripetalen Erregungen und die Nn. splanchnici ver- mittein die zentrifugalen. Unentschieden haben wir bis jetzt die Frage gelassen, ob nur die nach dem Zuckerstich Zucker abgibt, oder aber, ob die im Harn auftretende Glukose auch anderen Organen entstammt. Durch die Versuche von Moos^) und Moritz Schiff) ist bewiesen, daß nur der Zuckerstoffwechsel der Leber beeinflußt wird. Werden nämlich die Gefäße der Leber unterbunden, dann erscheint kein Zucker im Urin. Besonders schön Dieser Forscher erzeugte bei 8 zeigen dies die Versuche von Schiff'. gleich großen Fröschen durch den Zuckerstich Glukosurie. Nach 2 3 Vi. Stunden ließ sich Zucker im Harn feststellen. Nun wurde allen Versuchstieren die Leber bloßgelegt und aus der Bauchwunde herausgezogen. Dann wurden alle Gefäße dieses Organs und der Gallengang mit einer Fadenschlinge umfaßt. Bei vier Versuchen wurde die Fadenschlinge zugezogen, Während nun bei den letzteren die bei den übrigen dagegen nicht. Glukosurie fortdauerte, nahm sie bei den letzteren mehr und mehr ab und nach drei Stunden war der Harn zuckerfrei. Wir können die erste Phase der Wirkung des Zuckerstiches mit der alimentären Glukosurie in Parallele setzen. Bei dieser erfolgt die Überschwemmung des Blutes mit Zucker vom Darm, bei ersterem von der Leber aus. Sie hat ^aufgehört, den Glykogenabbau den vorliegenden Bedürfnissen anzupassen. Gleichzeitig vermögen die Leberzellen kein Glykogen mehr aufzubauen. Die Leber ist in gewissem Sinne aus dem Zuckerstoffwechsel ausgeschaltet. Wir können von diesen Gesichtspunkten aus die nach dem Zuckerstich auftretende Glukosurie als eine h'epatogene bezeichnen. Die Beobachtung Claude Bernards und derjenigen Forscher, die seine Feststellung ergänzt und erweitert haben, weist darauf hin, daß der Zuckerstoffwechsel und insbesondere der Glykogenauf- und -abbau sicher nicht ausschließlich direkt vom Blute aus geregelt wird. Die Leberzellen stehen vielmehr unter der Kontrolle eines nervösen Zentrums. Diesem werden offenbar durch die Nn. vagi beständig Nachrichten über den Stand des Zuckerumsatzes in der Leber übermittelt. Ferner empfängt das Zentrum auch Nachrichten über den Zuckerbedarf der Körperzellen und insbesondere des Blutes. Es ist möglich, daß das Zentrum direkt über den Zuckergehalt des Blutes unterrichtet wird, d. h. daß ein Ansteigen des Blutzuckergehaltes Leber — ) 2) ) Eckhard: Beitrage zur Anat. u/Phvsiol. 4. 138. Vgl. hiezu: J. J. R. Macleod: Americ. J. of Physiol. 19. Moes: Arch. f. wisseuschaftl. Heilkunde 4. 37. ('. 388 (1907). ) Moritz Schiff: Untersuchungen über die Zuckerbildung in der Leber. 76. Würzburg 1859. ^ü- Vorlesung. 156 das Zentrum erregt und dieses auf der Bahn der Nn. splanchnici die Leberzellen zur Glykogenbildung veranlaßt. Es wäre ja denkbar, daß im Anfang der Verdauung der resorbierte Zucker die Leber passiert und, ohne abgefangen zu werden, in den großen Kreislauf gelangt. Das wäre dann für das Zentrum das Zeichen zum Eingreifen. Umgekehrt könnte ein Sinken des Zuckergehaltes des Blutes bewirken, daß das am Zentrum vorüberfließende Blut dieses in dem Sinne beeinflußt, daß nunmehr die benachrichtigten Leberzellen (ilykogen abbauen. Normalerweise erfolgt das Wechselspiel zwischen Zuckerzentrum und Leberzellen ohne Zweifel in fein abgestufter Weise. Verletzen wir das Zuckerzentrum, oder reizen wir es auf irgend eine andere Art, dann zerstören wir durch brutalen Eingriff jede Regelung. Die Leberzellen w-erden in ihrer Gesamtheit beeinflußt. Sie schütten ihren Traubenzucker nach erfolgtem Abbau des Glykogens aus. Die Zellen sind aufs schwerste geschädigt. Sie sind auch nicht in der Lage, bei Reizung des Zuckerzentrums aus zugeführtem Traubenzucker Glykogen aufzubauen, wie aus den Beobachtungen von Freund und Popper^) hervorgeht. Sie konnten nur dann bei intravenöser Zufuhr von Glukose Glykogenbildung in der Leber nachweisen, wenn sie jede zerebrale Reizung fernhielten. Wir dürfen uns nun keineswegs mit der Feststellung zufrieden geben, daß unter bestimmten Bedingungen die Leberzellen aus zugeführtem Traubenzucker kein Glykogen bilden, vielmehr müssen wir der Frage nachgehen, warum das nicht der Fall ist. Es könnte z. B. sein, daß die Leberzellen für Glukose in bestimmten Fällen undurchlässig sind. Würde sie in die Zellen hineingelangen, so fände vielleicht der Glykogenaufbau statt. In einem solchen Falle wäre in Wirklichkeit gar keine direkte Störung der Glykogensynthese vorbanden, vielmehr würden Veränderungen der Zellgrenzschicht in der Durchlässigkeit für Traubenzucker vorliegen. Unklar bleibt die Beziehung des Zuckerzentrums bzw. der zur Leber verlaufenden motorischen Bahn zum Glykogen bzw. zur Dias tase. Bewirkt der nervöse Impuls, daß in der Lebervon ihm — — oder wird das Substrat das Glykogen das vielleicht irgendwie gebunden war, so verwandelt, daß der Abbau erfolgen kann? Oder liefert der N. splanchnicus gar den Aktivator etwa in Form eines InkretstoffesV Undenkbar wäre das nicht, denn wir kennen umgewandeltes Nervengewebe, das Inkretstoffe, die große Wirkungen entfalten, hervorbringt. So liefert der Sympathikusanteil der Nebenniere das Adrenalin. Warum sollten nicht die nervösen Zentren auch derartige Stoffe bilden und diese weiter geben? Es könnte aber auch sein, daß vom Nervensystem aus die physikalischen Bedingungen in den Zellen geändert werden und dadurch ein Zustand geschaffen wird, unter dem Diastase und Glykogen zusammenkommen und zusammenwirken können. SchließUch muß auch der folgenden Möglichkeit gedacht werden. Es ist wohl möglich, daß in der Leberzelle zwischen vorhandener Glukose und Glykogen vorrat eine Art von Gleichgewicht besteht. Verläßt Traubenzucker die Zelle, dann ist dieses gestört. P^s erfolgt ein Abbau von Glykogen, bis wieder ein Gleichgewichtszustand erreicht ist. Es wäre nun denkbar, daß vom Zuckerzentrum aus in irgend einer Weise die Durchlässigkeit der Grenzschichten der Leberzelle die Diastase aktiviert wird, ') Ernst Freund urjd Ilm/o I'oppcr : Biocliem. Zeitschr. 41. 5G. (1912). Kohlehydrate. 157 für Zucker verändert wird. Wir werden noch erfahren, daß geringfügige Veränderungen in der Zusammensetzung des Blutplasmas, z. B. eine Verschiebung im Gehalt an Kalziumion. von größter Bedeutung für den physikalischen Zustand von Zellgrenzschichten und damit unter anderem auch für ihre Durchlässigkeit für bestimmte Stoffe sein kann. Eine erhöhte Durchlässigkeit für Glukose könnte zur P'olge haben, daß der Abbau des vorhandenen Glykogens zwangsläufig erfolgt. Wir deuten diese Möglichkeit einer Erklärung der raschen Überführung des Glykogens der Leberzellen in Glukose nur an, weil zurzeit, wie wir noch sehen werden, eine befriedigende Erklärung dafür nicht vorliegt. Besondere Aufmerksamkeit hat man der Diastase der Leberzellen Zellen Nach den vorliegenden Beobachtungen derselben beim Zuckerstich nicht erkennbar, i) gewidmet. ist eine Vermehrung Mit der Feststellung, daß von einem Zentrum aus der Zuckerhausder Leber geregelt wird, ist die Mögüchkeit durchaus nicht ausgeschlossen, daß die Leberzellen auch vom Blute selbst aus zum Glykogenaufbzw, -abbau angeregt werden. Es sind eine ganze Anzahl von Stoffen bekannt geworden, nach deren Einführung in den Organismus Hyperglukoplasmie und darauf folgend Glukosurie eintritt. In den meisten Fällen ist nicht eindeutig genug festgestellt worden, an welcher Stelle sie eingreifen und eine Störung des Kohlehydratstoffwechsels bewirken. Für manche dieser Substanzen ist es sehr wahrscheinlich gemacht worden, daß sie auf das Zuckerzentrum wirken. Andere dürften dagegen die Leberzellen direkt beeinflussen und wieder andere greifen offenbar an verschiedenen Stellen zugleich schädigend in den normalen Ablauf des Zuckerstoffwechsels ein. Es darf nicht verschwiegen werden, daß manche Beobachtungen auf diesem Gebiete zu flüchtig gemacht sind, um bindende Schlüsse zuzulassen. Vor allem interessiert uns in jedem Falle von Glukosurie die Frage, ob Hyperglukoplasma besteht oder aber, ob die Zuckerausscheidung durch die Nieren eine andere Ursache hat. Sehr interessant ist die Beobachtung^), daß nach Einspritzung einer iVoigen Kochsalzlösung in die Blutbahn Glukosurie auftritt. Verschiedene Beoliachtungen machen es wahrscheinlich, daß das Kochsalz reizend auf das Zuckerzentrum einwirkt. So ist festgestellt worden, daß nach Dui'chschneidung der Nn. splanchnici die Kochsalzinjektion unwirksam war.=) Ferner trat die Glukosurie früher und viel stärker auf, wenn das Kochsalz durch Einspritzung in die Arteria vertebralis unmittelbar der Medulla oblongata zugeführt wurde, als wenn es einem peripheren Gefäße übergeben worden war. Martin H. Fischer) hat diese Beobachtungen erweitert und zunächst gezeigt, daß im Kochsalz dem Natriumion die Wirkung zukommt.") Ferner gelang es, die Glukosurie durch Einspritzung von Kalziumion aufzuheben. Natrium- und Kalziumion zeigen in diesem halt — ') E. Starkenstein: Biochem. Zeitschr. 24. J. J. R. Macleod uud 191 (1910). R. G. Pearce: Americau Journ. of Physiol. 25. 2.55 (1910); 27. 341 (1911); 28. 403 (1911). Vgl. auch St. Osato: The Tohoku .Joiiru. of exper. Med. 1. 1 (1920). -) C. Eckhard: Beitr. z. Auat. u. l'hvsiol. 8. 77 (1879). ) Külz: Eckhard?, Beiträge. 6. 177 (1872). ) Martin IL Fischer: Univ. of California Publications. Physiol. 1. 77 (1913); Vgl. auch Koichi Naito: The Tohoku. l'ftüqers, Archiv. 106. 80 (1904); 109 1 (1905) Journ. of exper. Med. 1. 131 (1920). ^) Auch Li, K, Sr erzeugen Glukosurie. NH^-lou ist dagegen ohne Eintluß. — — ^Ü- Vorlesung. ]58 Wir werden später noch mehr Beispiele Art kennen lernen. Die der Einbringung einer Kochsalzlösung bestimmter Konzentration (Ve molekulare Lösung) folgende Glukosurie hat nach neueren Beobachtungen sicher keine einheitliche Ursache. Es ist nämlich festgestellt worvor allem die Nierendeni), daß das Kochsalz verschiedene Körperzellen so beeinflußt, daß sie für Traubenzucker durchlässig und Darmzellen werden. Die Glukosurie könnte somit auch ohne Hyperglukoplasmie zuFalle eine antagonistische Wirkung. dieser — — stande kommen. 2) Auf Reizung des Zuckerzentrums ist die Glukosurie nach Eingabe von Morphium, Strychnin, Phosphor, Arsen, Uransalzen, Sublimat, Amylnitrit, Kurare, Chloral, Nitrobenzol, Chloroform, Äther, Azeton usw. zurückgeführt worden. Auch die nach Überladung des Blutes mit Kohlensäure bzw. bei Sauerstoff mangeP) eintretende Glukosurie Neuerdings wird vermutet, daß die Kohlensoll die gleiche Ursache haben. säure die Leberzellen direkt beeinflußt. Endlich wäre noch zu erwähnen, daß auch die nach Kohlenoxydver giftung (Leuchtgas!) auftretende Glukosurie mit dem Zuckerzentrum in Zusammenhang gebracht worden ist. Glukosurie beobachtet man zuweilen auch nach Gehirnerschütterungen. Man hat auch hier eine Reizung des Zuckerzentrums angenommen. Ferner fand man wiederholt Zucker im Harn, wenn Tiere, z. B. Katzen, lange Zeit gefesselt worden waren), und endlich ist Zuckerausscheidung im Harn insbesondere bei Fröschen nach starker Abkühlung festgestellt worden.^) Man vermutet, daß auch in diesen Fällen das Zuckerzentrum direkt oder indirekt erregt wird.") Zu ganz neuen Gesichtspunkten in der Frage nach dem Wesen der sogenannten Zuckerstichglukosurie führte die Entdeckung von Blvm'^), wonach das von der Nebenniere gebildete Adrenalin ebenfalls zu einer Hyperglukoplasmie und im Gefolge davon zu einer Glukosurie führt, vorausgesetzt, daß die Leber Glykogen enthält. s) Dem Adrenalin kommt die Formel eines 3,4-Dioxyphenyl-methylaminoäthanols zu: — — Frank John Bruce Macallnm: Univ. of California Public. 1. 125 (1904). Martin und Israel S. Kleiner: Journ. of Biol. Chemistry. 4. 395 (1907). H. Fischer und Gertrud Moore: American Journ. of Physiol. 19. 341 (1907). 2) Vgl. Louise Mc Danell und Frank P. Underhill .-Journ. of Biol. Cheni. 29. 273 (1 917). Ivar Bang und Ph. Senström: ') Vgl. Edie: Biochera. Journ. 1. 455 (1906). ) P. Vnderhill — Biochem. Zeitschr. 437 (1913). ) R. Böhm und F. A. Hoffmann: Arch. f. experim. Path. u. Pharm. 8. 271 und Vgl. auch W. B. Cannon (Ä. T. Shohl und W. S. Wright, D. de la Pas, 375 (1878). R. G. Hoskins): Americ. Journ. of Physiol. 28. 64 (1911); 29. 274, 280 (1911/12). G. N. Stewart und ./. M. Pogof: Journ. of exper. Med. 26. 637 (1917); Americ. Journ. of Physiol. 46. 90 (1918). ') Vgl. z. B. M. Loetoit: Zentralbl. f. Physiol. 21. 873 (1907); Arch. f. experim. Path. u. Pharm. 60. 1. 420 (1808/09). von Glukosurie auch mit dem Namen Diabetes be) Man hat alle Formen zeichnet. Man spricht; insl)esondere von einem Zuckerstichdiabetes, einem Fesseluugsund Kältediabetes. Wir ziehen es vor, den Namen Diabetes ganz für jene Krankheit vorzubehalten, die unter anderen Symptomen auch Glukosurie aufweist. ) E. Blum: Deutsches Archiv f. Idin. Medizin. 71. Okt (1901); P/%ers Archiv. Vgl. weitere Literatur bei A. Biedl: Innere Sekretion. Urbau & Schwar90. 617 (1902). 50. — — — zenberg, Berlin-Wien. 3. Aufl. 1916. 8) Fritz Hildebrandt: Arch. experim. Path. u. Pharm. 88, 80 (1920). Kohleliydrate. ] 59 CH 0H.Cf^\\|C.CH(0H).CH,.NH.CIl3. CH Das Adrenalin wird von der Marksubstanz der Nebenniere, die bekannt- vom N. syrapathicus abstammt, gebildet. Es entfaltet noch andere Wirkungen. Es wirkt ganz allgemein auf alle Organe ein, die vom N. sympathicus innerviert werden. Besonders wichtig und in die lich Augen fallend ist der Einfluß des Adrenalins auf die Blutgefäße. Es verengert diese und bewirkt dadurch ein Ansteigen des Blutdruckes. Was nun die Wirkung des Adrenalins auf den Kohlehydratstoffwechsel anbetrifft, so kommen vor allem zwei Möglichkeiten in Betracht. Es kann auf dem Wege des X. sympathicus eine Ptcizwirkung auf die Leberzellen ausüben oder aber einen direkten Einfluß auf diese entfalten. Für letztere Annahme sprechen Versuche von Ivar Bang.^) Er beobachtete nämlich, daß Adrenalin die überlebende Froschleber zum raschen Abbau ihres Glykogens veranlaßt. Gegen die i\.uslegung dieser Feststellung im Sinne einer normalen Funktion des Ardenalins ist einzuwenden, daß die Konzentration dieser Verbindung im Organismus wohl nie auch nur annähernd so groß ist, wie sie von Bang zu seinen Versuchen angewandt worden ist. Nun wissen wir aus zahlreichen Beobachtungen, daß für die Wirkung eines seine Konzentration von ausschlaggebender Bedeutung ist. Oft wirken kleine Dosen reizend und große lähmend. Wir dürfen in keinem Falle die physiologische Bedeutung eines Stoffes aus Versuchen ableiten, bei denen wir eine Verbindung unter Steigerung der Konzentration auf Stoffes ein Vielfaches des natürlichen Vorkommens zur Wirkung kommen lassen. Wir dürfen nur aussagen, daß der betreffende Körper bei einer bestimmten Konzentration bestimmte Wirkungen entfaltet. Darüber hinaus sind Schlußfolgerungen auf das Verhalten im Organismus ohne genügende Grundlage. Mehr und mehr fand die folgende Vorstellung von der Wirkung des Adrenalins auf den Zuckerstoffwechsel Anklang. Das Zuckerzentrum wirkt nicht direkt durch Vermittlung des N. splanchnicus auf die Leberzellen. Es sind vielmehr die Nebennieren zwischen dieses und die Leber eingeschaltet. Wird das Zuckerzentrum gereizt, dann werden die Nebennieren veranlaßt, Adrenalin abzugeben. Seine Menge richtet sich nach der Größe des Reizes. Es wird dann den Leberzellen zugeführt und veranlaßt einen den augenblicklichen Bedürfnissen entsprechenden Abbau von Glykogen und damit Zuckerabgabe an das Blut. Unter normalen Verhältnissen ist das Zusammenspiel von Zuckerzentrum und Nebennieren ein außerordentlich feines. Es wird nur soviel Adrenalin abgegeben, als notwendig ist, um ausreichende Mengen von Glykogen zum Abbau zu bringen. Beim Zuckerstich und sonstigen brutalen Eingriffen in das ganze zusammengekoppelte System wird viel Adrenalin deni Blute übergeben, und es erfolgt nun ein Massenabbau von Glykogen. ») Ivar Bang: Biochom. Zeitschr. 49. 81 (1913). 160 ^ il- Voiiesuug. Die geschilderte Auffassung der Stellung der Nebenniere zum Zuckerzentrum und zur Leber ist nicht ohne Widerspruch geblieben. i) Wir müssen deshalb sorgfältig prüfen, ob die vorliegenden Versuchsresultate mit ihr in Einklang stehen. Zunächst wies A. Mayer^) nach, daß der Zuckerstich nur dann gelingt, wenn die Nebennieren vorhanden sind. Kahn und Starkenstein^) konnten diese Beobachtung bestätigen. Nach doppelseitiger Nebennierenexstirpation der Zuckerstich ist erfolglos. Man zunächst glaubte diesen Umstand mit der Annahme erklären zu können, daß die Leber nach der Es würde dann der deshalb erfolglos sein, weil dieses Organ keinen Zucker mehr abzugeben hat. Es hat sich jedoch herausgestellt, daß der Verlust der Nebennieren das Glykogenspeicherungsvermögen der Leber nicht wesentlich beeinflußt.) Nur bei der Ratte wurde die Leber glykogenfrei gefunden.») Trotzdem die Leberzellen bei den übrigen Tieren viel Glykogen aufweisen, bleibt der Zuckerstich ohne Erfolg, wenn die Nebennieren fortEntfernung der Nebennieren ihr Glykogen einbüße. Zuckerstich genommen sind. Es wurde ferner beobachtet, daß die Nebenniere nach dem Zuckerstich an Adrenalin und an dem Substrat (sogenannte chromierbare Substanz^), die diese Verbindung hervorbringt, verarmt.") Durchschneidet so Seite, zeigt man beim Kaninchen den N. splanchnicus die Nebenniere der gleichen Seite nach auf der einen dem Zuckerstich keine Veränderungen, während sie bei der auf der anderen Seite befindlichen anzutreffen sind.^) Wichtig ist auch die Beobachtung, daß nach vollkommener Isolierung der Leber von nervösen Zentralorganen mittels Durchschneidung aller Nerven der Zuckerstich noch wirksam ist. Dagegen wurde er unwirksam, sobald die Nebennieren durch Durchschneidung aller Nervenbahnen isoliert wurden, während das übrige Splanchnikusgebiet mit dem Zentralnervensystem in Zusammenhang blieb. 9) Es ist nun bezweifelt worden, ob tatsächlich im Anschluß an den Zuckerstich eine ausreichende Zunahme^") an iVdrenalin im Blute stattfindet. Li dieser Richtung ausgeführte Untersuchungen!') haben ergeben, daß die ') Vgl. auch E. Wertheiltier uud G.Battez: — H. Freund und G. Marchand: Arch. (1910). f. Arcli. interuat. de Physiol. 9. 3ü3 exper. Path. und Pharm. 76. 324 fl914). A. Mayer: Compt. rend. de la Soc. de Biol. 58. 1123 (1906). R. H. Kahn uud- E. Starkenstein: Pßügers Archiv. 139. 181 (1911). ) R. H. Kahn und E. Starkenstein: J'ßügers Archiv. 139. ISl (1911). Sh. Kuriyama: Journ. of hiol. Chem. 34. 287, 29'.) '(1918). 5) 'O. Schwarz: Pßüffers Archiv. 134. 259 (1910). '^) Die Chromreaktion dieses Gewebes (auch chromaffines genannt) beruht auf seinem Gehalt an Adrenalin, vgl. W. Stoeltzner : Münchener med. Wochenschr. Nr. 22. = ) ») — 584 (1919). ') R. H. Kahn: Pßügers Archiv. 140. 209 (1911); 144. 2.51 396 (1912); 146. Jjuro Fnjii: Journ. of experim. Med. 1. 38 (1920): The Tohoku .Journ. 578 (1912). of exper. Med. i. 38 (1920). 8) A. Jarisch: Archiv f. experim. Path. u. Ther. 13. 520 (1913). R. 11. Kahn: J. Fujii: The Tohoku Journ. of exper. Med. 1. r/tüffers Archiv. 169. 347 (1917). 83 (1920). «) Adolf Jarisch: Pfliigers Archiv. 158. 478 (1914). >») Vgl", hierzu N. Watermann und H. J. Smit: PflWgcr^ Archiv. 124. 198 (1908). jY. Watermann: Ebenda. 141. 104 (1911). W. Falta und J. G. Priestleg: Berliner - J. Nigrin y Lopez: t^benda. 145. 311 (1912). klin. Wochenschr. Nr. 47 (1911). E. Starkenstein: Zeitschr. f. experim. Pathol. u. Ther. 10. 78 (1912). '•) Paul Trendelenburci und Kurt Fleischhauer : Zeitschr. f. d. ges. experim. Med. 1. 369 (1913). — — — — — — Kohlehydrate. [61 Verhältnisse nicht so einfach liegen können, wie angenommen worden ist. Die Vorstellung, daß der durch den Zuckerstich ausgeübte Reiz nur auf dem Wege über die Nebenniere wirken kann, ist ohne Zweifel zu eng gefaßt. 1) Vor allem ist zu prüfen, ob, wie schon oben erwähnt, das Adrenalin unter normalen Verhältnissen direkt auf die Leberzellen wirkt oder aber seinerseits nervöse Zentren oder Nervenbahnen beeinflußt und so indirekt auf den Glykogenabbau einwirkt. Ferner bleibt auch noch die M()glichkeit, daß durch die bewirkten nervösen Einflüsse die Leberzellen für die Einwirkung des Adrenalins vorbereitet werden. Wichtig sind nach dieser Richtung die Beobachtungen von Macleod und Pearce.-) Diese Forscher haben festgestellt, daß nach Entfernung des Plexus hepaticus die Reizung des N. splanchnicus unwirksam ist, obschon eine Hypersekretion der Nebennieren vorhanden ist. Wird jedoch der Plexus hepaticus gereizt, dann erhält man Hyperglukoplasmie, wenn die Nebennieren funktionstüchtig sind. Sind sie entfernt, dann bleibt die Überschwemmung des Blutes mit Zucker aus. Diese Resultate sprechen durchaus dafür, daß die Nebenniere und die Leber Beziehungen zueinander haben, die durch Nervenbahnen vermittelt werden, die im Plexus hepaticus verlaufen. Einer der größten Fehler in der Erforschung bestimmter Funktionen ohne Zweifel der, daß fast gewaltsam versucht wird, eine einzige Möglichkeit für ihre Erklärung festzulegen. Dabei deuten zahlreiche Beobachtungen darauf hin, daß der Organismus mehrere Sicherungen für besonders bedeutungsvolle Funktionen besitzt. Die Tätigkeit der Leber im Kohlehydratstoffwechsel ist eine mannigfaltige. Sie wandelt Verbindungen nicht kohlehydratartiger Natur in Glukose um. Sie speichert diese in Form von Glykogen und zerlegt dieses wieder je nach Bedarf. Die Leber bewirkt, daß jedes Minus an Blutzucker sofort ausgeglichen wird. Diese letztere Funktion können wir der Bildung und Abgabe von Sekretstoffen durch Driisenzellen vollständig an die Seite stellen. Wir kennen eine ganze Reihe von Organen, die an die Blutbahn Stoffe abgeben, die im gesamten Haushalt an irgend einer Stelle eine bedeutsame Rolle spielen. Stoffe, die nach außen, z. B. in den Darmkanal oder auf die äußere Haut abgegeben werden, bezeichnen wir als Sekrete. Stoffe, die innerhalb des Zellstaates bleiben und. dem Blute oder der Lymphe zur Weiterführung übergeben werden, nennen wir mit Boux am besten Inkrete. Ohne Zweifel ist von diesen Gesichtspunkten aus die Leber als eine Drüse aufzufassen, die Traubenzucker als Inkret dem Blute übergibt. Wir haben eine der interessantesten und am besten bekannten Wechselbeziehung zwischen verschiedenen Organen vor uns. Die Leber sendet z. B. Zucker zu den Muskelzellen, und diese übergeben dem Blute als ein Spaltstück von diesem Milchsäure. Aus dieser bauen die Leberzellen wieder Glukose auf. ist Wir zweifeln nicht daran, daß die Abgabe des erwähnten Inkretes, des Traubenzuckers, an das Blut von mehr als einer Stelle aus beeinflußt wird. Zunächst besteht die Möglichkeit, daß der Zuckergehalt des Blutes direkt als Reiz wirkt. Ferner ist es denkbar, daß jene Organe, die Zucker verbrauchen, in der Weise Einfluß auf die Leberzellen haben, daß vielleicht die dem Blute übergebenen Abbaustufen, z. B. die Milchsäure, An») Vgl. hierzu K. 2) J. J. R. Macleorl II. Kahn: Pßücjers Archiv. 169. 32H (1917). und li. G. Pearce: Americ. Journ. of Physiol. 29. 419 (1912). Abderhalden, Physiologische C'hcmii', I.Teil, 5. Anfl. \\ ^'11- 162 Vorlesung. stoß ZU Glykogenabbau geben. Ferner ist der Weg über Nervenbahnen und Zentren gegeben. Diese Art des Wechselspiels ist noch lange nicht aufgeklärt. Vielleicht spielt bei dieser Regelung des Zuckerstoffwechsels auch der Umstand eine Rolle, daß die Nervensubstanz selbst einen ganz regen Glukoseverbrauch aufweist.^) Es wäre denkbar, daß das Zuckerzentrum im besonderen für die ausreichende Versorgung des Nervengewebes mit Zucker sorgt. Die Nervenzellen des Zuckerzentrums stehen durch Nervenbahnen direkt mit den Leberzellen in Verbindung. Man darf ohne w^eiteres von inkretorischen Fasern sprechen. Außerdem unterhält das Zuckerzentrum Beziehungen zur Nebenniere. Auch zu ihr hin gehen inkretorische Bahnen. Es bleibt noch die Frage zu beantworten, ob nur das Adrenalin für die Beteiligung am Zuckerstoffwechsel in Betracht kommt. Mir scheint kein Anhaltspunkt dafür vorzuliegen, daß dieses den einzigen Inkretstoff der Nebenniere darstellt. Der Umstand, daß man durch Zufuhr immerhin beträchthcher Mengen von Adrenalin Hyperglukoplasmie hervorrufen kann, gibt uns noch nicht die Gewißheit, daß diese Verbindung nun auch normalerweise im Zuckerhaushalt Verwendung findet. Es könnten auch noch andere Stoffe mitwirken. Wir dürfen bei der Beurteilung der Beziehungen der Nebennieren zum Zuckerzentrum und zur Leber eines nicht außer acht lassen. Das Adrenalin wird, wie schon oben hervorgehoben, von der Marksubstanz der Nebenniere gebildet. Diese stammt vom N. sympathicus ab. Das Zuckerzentrum ist sicherlich auch ein Sympathicuszentrum. Vielleicht steht es noch mit anderen, übergeordneten, sympathischen Zentren im Zwischenhirn in Verbindung. Zur Leber ziehen zahlreiche sympathische Nervenbahnen. Aus diesen Feststellungen ergeben sich ohneweiteres enge Beziehungen aller dieser Organe zueinander. Fassen wir die wichtigsten Feststellungen zusammen, nämlich einmal die Tatsache, daß nach Entfernung der Nebennieren der Zuckerstich meistens vollständig unwirksam bleibt, obwohl die Leber Glykogen besitzt, und ferner die Beobachtung, daß er bei entnervter Leber wirksam ist, sofern wenigstens eine Nebenniere ihre Nervenbahnen noch besitzt, und endlich der Zuckerstich unwirksam ist, wenn die Nebenieren nervös übrige Splanchnicusgebiet mit dem Zenisoliert werden, dagegen das trum in leitender Verbindung bleibt, so ergibt sich mit größter WahrscheinUchkeit, daß die Nebennieren direkte Beziehungen zur Leber haben und in irgend einer Weise am Zuckerstoffw^echsel aktiv beteiligt sind. Vieles spricht dafür, daß ein von den Nebennieren abgegebenes Inkret, darunter das Adrenalin, 2) auf dem Wege der sympathischen Zentren und Nerven, die mit den Leberzellen in Verbindung stehen, auf diese einwirkt, s) Von größter Bedeutung wäre es, die Art der Beeinflussung der Leberzellen kennen zu lernen. Es bleibt das schon mehrfach berührte Problem zu ) (1917). Else Hirschberg und Hans Winterstein: Zeitscbr. — Else Hirschberg: Ebenda. 101. 248 (1918). f. physiol. Chemie. 100. 185 ') Adrenalin wird sehr leicht verändert. Es ist wohl möglich, daß es nicht selbst wirksam ist, sondern ein Umwandlungsprodukt. ^) Für die Frage der Bedeutung des Adrenalins für den Kohleliydratstoffwechscl kann die Mitteilung von G. N. Stewart und /. M. L'ogoß' [L of Pharm. 10. 1, 49 (1917)] von großer Bedeutung werden, wonach nach Exstirpation der rechten Nebenniere und der vom linken Semilunarganglion herkommenden Nervenfasern (Versuchstier Katze !) das Blut adrenalinfrei wird. Störungen irgend welcher Art waren keine zu beobachten ! Kohlehydrate. IQ-^ ergründen, weshalb in der Leberzelle Glykogen und Diastase sich zusammen finden, jedoch nicht aufeinander wirken, ehe nicht von außen ein Reiz eintritt. Man kann sich, wie schon einmal betont, vorstellen, daß in der Zelle Maßnahmen getroffen sind, die verhindern, daß die Diastase ihre Wirkung entfalten kann, sei es nun, daß sie inaktiviert ist, oder aber das Glykogen jene Gruppe besetzt enthält, die den ersten Angriff durch das Ferment vermittelt oder aber sich in einem physikalischen Zustand befindet, der die Fermentwirkung ausschließt. Der Möglichkeiten sind sehr viele. i) Es kommt auch in Frage, daß das Ferment in der Zelle vielleicht nicht zum Glykogen hin diffundieren kann, wenn nicht der ganze Zustand des Zellinhaltes sich ändert. Die Beobachtung, auf die wir noch eingehen, daß die Durchlässigkeit von Membranen durch an sich geringfügige Konzentrationsänderungen im Gehalt an bestimmten Ionen sich vollständig ändern kann, gibt vielleicht auch Hinweise auf die Möglichkeit der Reizwirkung. Einstweilen müssen wir uns damit begnügen, zum Ausdruck zu bringen, daß durch den Reiz in den Leberzellen Verhältnisse geschaffen werden, unter denen die Diastase das Glykogen zerlegen kann. Mir scheint die Annahme am wahrscheinlichsten, daß Zustandsänderuugen, d. h. physikalisch-chemische Veränderungen einsetzen, die es ermöglichen, daß die bis dahin getrennten Stoffe, Diastase und Glykogen, aufeinander einwirken. Nicht unerwähnt wollen wir lassen, daß beobachtet worden ist, daß nach dem Zuckerstich die Leberzellen das Glykogen in die Blut- und Lymphbahnen ausstoßen, und dann dort der Abbau einsetzt. Weitere Versuche müssen ergeben, ob dieser Vorgang die Regel ist und das gesamte Glykogen betrifft. 2) Auf die Möglichkeit einer Veränderung der Zellgrenzschichten im Sinne einer veränderten Durchlässigkeit für Zucker haben wir schon S. 156 hingewiesen. Man hat auch der Schilddrüse, den Nebenschilddrüsen, der Hypophyse, der Milz und anderen Organen direkte und indirekte Beziehungen zur Leber und zur Nebenniere zugeschrieben und daran gedacht, daß je nach Bedarf den Glykogenabbau fördernde und hemmende Substanzen von bestimmten Drüsen zur Verfügung gestellt werden. 3) Es liegen jedoch noch keine eindeutigen Resultate vor und vor allem ist noch ganz unbekannt, an welchen Stellen sie in den Kohlehydratstoffwechsel eingreifen.) Wie außerordentlich vorsichtig man bei allen Versuchen, auf experimentellem Wege an Tieren Störungen im Zuckerhaushalt festzustellen, sein muß, zeigen die Beobachtungen, wonach psychische Einflüsse den Zucker) 1877. Vgl. zu diesem Probleme auch 67. Bernard: Lecons sur le diabete. Paris — E. J.Lesser: Ergebnisse der inneren Medizin und Kinderheilkunde. XVI. 279 (1919). Vgl. F. Hofmeister: Kohlehydratstoffwechsel der Leber. Vortrag. NothnagelWien 1913. ^) Vgl. hierzu die interessanten Beobachtungen von H. Eppinqer, W. Falta und C. Rudinger: Wiener klin. Wochenschr. 21. Nr. 21. 752 (1908); Zeitschr. klin. Med. Heinrich Ritzmann : Arch. f. experim. 66. H. 1 u. 2 (1908) und #7. H. 5 u. 6 (1909). Path. u. Pharm. 61. 231 (1911). Frank P. Underhill und Warren W. Hilditch: Americ. Journ. of Physiol. 25. 66 (1909). Frank F. Underhill: Americ. Journ. of Physiol. 27. Weitere Literatur Soichiro Miura: Biochem. Zeitschr. 51. 423 (1913). 331 (1911). vgl. bei Artur Biedl: Innere Sekretion. Urban & Schwarzenberg. Berlin-Wien. 3. Aufl. T. Togawa: Biochem. Zeitschr. 109. 1 (1920). (1916). ) Vgl. auch Frank F. Underhill und Normann R. Blathertvick : Journ. of hiol. ^) Stiftung. — — — — — — Chem. 18. 87 (1914). 11 VII. Vorlesung. J^g4 gehalt im Blute wesentlich zu steigern vermögen. Es hat schon die Fesselung der Tiere 1), die Freilegung eines Gefäßes, die Narkose eine Erhöhung des Blutzuckergehaltes im Gefolge.-) Nicht unerwähnt wollen wir lassen, daß bei vielen Forschungen auf diesem Gebiete die angewandte Methodik der Zuckerbestimmung viel zu wünschen übrig läßt. Es wird vielfach von Hyperglukoplasmie und auch von Glukosurie gesprochen, obwohl meistens nur worden ist, daß ein bestimmtes Reduktionsvermögen vorliegt. Es ist durchaus möglich, daß neben Glukose, besonders in pathologischen Fällen noch andere reduzierende Stoffe anzutreffen sind. Alle diese Momente festgestellt , erschweren die Beurteilung der Ergebnisse vieler Versuche außerordentlich. Sie lassen auch verstehen, weshalb das ganze Forschungsgebiet nicht entsprechend der aufgewandten Mühe gefördert worden ist. Zum Schlüsse sei noch der Glukosurie bei hungernden Tieren») gedacht. Hofmeister^) beobachtete, daß bei jungen Hunden die Assimilationsgrenze für Kohlehydrate stark herabsinkt, wenn sie ungenügend ernährt werden. Die Ursache der mangelhaften Ausnutzung der zugeführten Kohlehydrate bei Unterernährung könnte darauf beruhen, daß diejenigen Organe, die den Zuckerstoffwechsel beherrschen, infolge des Hungerzustandes ihre Funktionen nicht in vollem Umfange durchzuführen vermögen. So kann vielleicht die Leber nicht rasch genug die ihr mit dem Blute zugeführte Glukose in Glykogen verwandeln. Es kommt infolgedessen zur Hyperglukoplasmie und im Anschluß daran zur Glukosurie. Nach neueren Erfahrungen scheint jedoch der Grund der Hyperglukoplasmie ein anderer zu sein. Es ist beobachtet worden &). daß geringe Säuremengen imstande sind, eine hepatogene Hyperglukoplasmie zu erzeugen. Es wird die Leber veranlaßt, ihr Glykogen abzubauen, bzw. verhindert, solches zu bilden. Auch sollen die Leberzellen Glykogen als solches abgeben. Eine Beteiligung der Nebenniere wurde ausgeschlossen. Nun ist bei der Glukosurie der Hungertiere auch eine Zunahme des Säuregehaltes des Blutes nachgewiesen worden. Sie dürfte durch eine Störung des Zellstoffwechsels infolge ungenügender Nahrungszufuhr bedingt sein. Durch Eingabe von Alkali konnte die Hyperglukoplasmie beseitigt werden.*) Ein ganz neues Licht auf manche Beobachtungen über das Auftreten von Glukosurie werfen hoch bedeutsame Versuche von H. J. Hcunhurger und li. BrmhnanJ) Sie stellten fest, daß das Vermögen der Frosch- niere, Glukose zurückzuhalten, in außerordentlich feiner Weise von der chemischen Zusammensetzung der Durchströmungsflüssigkeit abhängig ist. Wählt man. zum Durchspülen Bingersche Lösung, so wird von OP/o Glukose etwa ein Drittel zurückgehalten. Die verwendete Lösung enthielt: OTo/o NaCl, 2«/o Na HCO3, 001 «/« K Ol 1) '') Vgl. hierzu Zitat 4, S. 158. Vgl. E. Hirsch und //. Reinhach: Zeitschr. f. physiol. Chemie. 87. 122 (1913); 91. 292 (1914). ^) Hnngernde Menschen verhalten sich gleich. Es- gibt auch eine sogenannte Landstreicher-Gluko-surie. ) F. Hofmeister: Arch. f. cxperim. Path. und Pharmak. 26. 359 (1890). ) H. Elias: Biochem. Zeitschr. 48. 121 (1913). «) H. Elias und L. Kolb : Biochem. Zeitschr. 52. 331 (1913). ') H. J. Hamburger und R. Brinkman: Biochem. Zeitschr. 88. 97 (1918). Vgl. auch G. Barkan, Rh. Broemser und A. Hahn: Zeitschr. f. Biol. 74. 1 (1921). Ph. Broemser und Ä. Hahn: Ebenda. 74. 37 (1921). — — Kohlehydrate. 165 und 00075 »/o CaCl,. Wurde nun der CaCL-Gehalt auf 0005 oder auf dann erschien sofort in der aus dem Ureter fließenden Flüssigkeit, dem künstlichen Harn, der gleiche Zuckergehalt, wie er in der' Durchspülungsflüssigkeit enthalten war, d. h. es wurde kein Zucker zurückgehalten. Läßt man den CaCU-Gehalt gleich, und wird der K-Gehalt verändert, dann ist das Resultat ein gleiches. Für die Zurückhaltung der maximalen Zuckermenge ist ein ganz bestimmtes Verhältnis von Ca zu K notwendig. Besonders bedeutungsvoll ist die Feststellung, daß eine ganz bedeutende Vermehrung der Glukose-Zurückhaltung erhalten werden kann, wenn in der oben angeführten Durchströmungsflüssigkeit der Gehalt an NaHCOg auf 009 Vo gesteigert wird. Die Ursache dieser Erscheinung 0-01 5 Yo gebracht, auf die Reaktion der Durchströmungsflüssigkeit zurückzuführen. Ist sie wenig alkalisch, daß ihre Reaktion beim Durchströmen sauer wird, dann nimmt die Durchlässigkeit der Niere für Glukose zu. Bleibt der künstliche Harn infolge Steigerung des NaHCOs-Gehaltes der Durchspülungsflüssigkeit alkaUsch, dann ist die Zuckerretention vergrößert. Von den 0",lVo Zucker werden bei 0'09Vo NaHCOj doppelt so große Glukosemengen zurückgehalten als bei 0"02 "/o Na HCCJa. Gleichzeitig muß der Gehalt der Lösung an Ca Gl., auch etwas gesteigert werden, damit eine genügende Konzentration von Ca-Ionen zugegen ist. Wird der Gehalt an NaHCOg und entsprechend derjenige an CaClj noch mehr gesteigert, dann geht noch weniger Glukose durch die Nierenepithelien hindurch. Es gelang, allen Traubenzucker mit der folgenden Lösung zurückzuhalten: 0-5S NaCl, O-2850'o NaHCOj, 0-OP/o KCl, 0-02»/o Ca CL. gleichen Bei der Die Versuche fielen nicht immer gleich aus. Konzentration an den genannten Salzen kam es zuweilen auch zur Ausscheidung von Traubenzucker: immerhin war die Menge an zurückgehaltener Glukose größer als bei anderen Konzentrationen. Fügt man zur Durchspülungsflüssigkeit die Menge Traubenzucker, die dem normalen Gehalt ist so , des Froschblutes an diesem entspricht, nämlich O^Oö^/o, hinzu, dann wird bei der erwähnten Salzkonzentration das gesamte Kohlehydrat zurückgehalten. Interessant ist, daß bei der zuletzt erwähnten Zusammensetzung der Nährlösung das Kalium fehlen durfte. Ferner ist von großer Bedeutung, daß andere Zuckerarten, wie Rohrzucker, Milchzucker, Maltose, Raff inose, Fruchtzucker, 1-Arabinose ^und 1-Mannose, unter den gleichen Bedingungen, unter denen Glukose von den Glomerulusepithelien zurückgehalten wird, durchgelassen werden 1). Galaktose, 1-Xylose und d-Ribose werden teilweise zurückgehalten. Die einzelnen Kohlehydrate verhalten sich somit je nach ihrer Konfiguration verschieden. Die Feststellung, daß die Nierenepithelien durch Veränderung des Mengenverhältnisses der erwähnten Salze und insbesondere des Ca Clo und des NaHCOg bald mehr, bald weniger, bald gar nicht für Glukose durchlässig gemacht werden können, ist von allergrößtem Interesse. Sie zeigt uns, daß die genannten Zellen in ihrer Durchlässigkeit stark beeinflußbar sind. Es gibt Membranen, die für jeden Stoff durchlässig sind, andere lassen nur bestimmte Produkte durch. In den Zellgrenzschichten, die für die Ausscheidung von Substanzen und in ') H. J. Uaynburger und R. lirinkman: Proceed. of Koninkl. Akad. van Wetenschappen te Amsterdam'. 19. 98«), 997 (1917); 21. Nr. 4. 548 (1919); 22. 353, 360 (1919): Biochem. Zeitschr. 128. 185. 208 (1922). VII. Vorlesung. IQQ ' unserem Fall für Kohlehydrate aus dem Blute in die Nierenkanälchen hinein in Frage kommen, haben wir „Membrane" vor uns, deren Zustand und damit auch deren Durchlässigkeit für bestimmte Produkte nicht eint'ür allemal gegeben ist, vielmehr unterliegt er mit all den mit ihm verknüpften physikalisch-chemischen Eigenschaften, je nach den vorhandenen Bedingungen, Wandlungen. Von größtem Interesse ist, daß die Konfiguration der Substrate für das Durchdringen von Zellgrenzschichten von Bedeutung ist. Wir werden auf die erwähnten grundlegenden Versuche der Abhängigkeit der Eigenschaften der Zellgrenzschichten von den vorhandenen Bedingungen von verschiedenen Gesichtspunkten aus zurückkommen. Wir ersehen aus diesen Beobachtungen, wie sorgfältig alle Bedingungen festgeForschungen auf dem Gebiete des stellt werden müssen, unter denen Kohlehydratstoffwechsels unternommen werden. Eine Änderung in der Zusammensetzung des Blutes vermag schon eine Glukosurie zu erklären. Wir haben nicht nur den Zuckergehalt des Blutes in Rechnung zu setzen, sondern müssen auch feststellen, ob sich nicht die Nierenepithelien unter Bedingungen finden, unter denen sie an und für sich Zucker durchlassen. Eine Glukosurie kann somit mannigfache Ursachen haben. Fassen wir die uns bis jetzt bekannten zusammen. Einmal kann vom Darme aus soviel Zucker zuströmen, daß die Leber- und sonstigen Körperzellen mit ihm nicht fertig werden. Die Gründe dafür können mannigfaltige sein. Einmal kann der Zustrom so groß sein, daß die Glykogenbildung an sich nicht Schritt halten kann. Es kann aber auch sein, daß die Glykogen bildenden Zellen bereits mit Glykogen beladen sind. Die Nierenzellen greifen ein und entlassen den Überschuß an Zucker aus dem Plasma. Sie sind unter normalen Verhältnissen bis zu einer bestimmten Zuckermenge dicht für Glukose. Wird diese Grenze überschritten, dann beginnen sie durchlässig zu werden, und zwar kommen in erster Linie, wenn nicht ausschließlich die Glomerulusepithelien in Betracht, i) Sind die Leberzellen infolge irgend einer Störung in ihrer Fähigkeit, Glykogen zu bilden, eingeschränkt, dann muß es wiederum leicht zu einer Hyperglukoplasmie kommen, wenn vom Darm aus Glukose zur Aufnahme gelangt. Zwar können andere Körperzellen einspringen, jedoch hat das seine Grenzen. Behält dabei die Leber ihre Fähigkeit, aus anderen Materialien als aus Kohlehydraten Glukose aufzubauen, dann wird noch eine weitere Ursache der Überschwemmung des Plasmas mit Zucker hinzugefügt. Eine Hyperglukoplasmie tritt auch dann ein, wenn die Leber innerhalb kurzer Zeit ohne jede Regelung sich ihres Glykogenvorrates durch Abbau entledigt. Nun beherrschen auch die Nierenepithelien in gewissen Grenzen die Zuckerausscheidung. Sie können durch Änderung der Zusammensetzung des Blutes für Traubenzucker weniger oder mehr durchlässig werden. Von einer gewissen Grenze ab lassen die Nierenzellen auf alle Fälle solchen durch. Eine wirkliche Hyperglukoplasmie wird wohl im allgemeinen immer eine Glukosurie im (befolge haben. Wir haben aber auch erfahren, daß die Möglichkeit besteht, daß die Niere bei normalem Zuckergehalt, ja sogar bei vermindertem für Glukose durchlässig werden kann. ') H. J. Hamburger und R. lirinkinan: Biochem. Zeitschr. 94. 131 (1919). Kohlehydrate. 167 genau zu studieren haben, wo die immer nur eine Folgeerscheinung. Treffen wir auf eine Hyperglukoplasmie, dann interessiert es uns zu erfahren, Bei jeder Glukosurie werden wir Ursache zu suchen ist. Sie selbst ist ja diese zustande gekommen ist. Ist sie enteral (aUmentär) oder hepathogen bedingt? Im letzteren Falle ist zu prüfen, aus welchem Grunde die Leber versagt. Ist der Glykogenaufbau oder sein i\,bbau oder beides wie zugleich gestört? Hat sich die Durchlässigkeit der Zellgrenzschichten der Leberzellen für Glukose verändert? Liegt die Ursache primär in der Leber oder hat eine Beeinflussung von außen stattgefunden (Zuckerzentrum, Fortfall des Einflusses eines Organes mit Inkretbildung: Schilddrüse, Hypophyse, Nebenniere. Milz usw\)? Diese wenigen Hinweise mögen genügen, um zu zeigen, wie zahlreiche Fragen jeder Fall von Glukosurie uns aufgibt. Sie vermehren sich noch, wenn wir im folgenden weiter in das Problem des Kohlehydratstoffwechsels vordringen werden. Vorlesung VIII. Kohlehydrate. VII. Die Beziehungen der Panl<reasdrüse zum Kohiehydratstoffwechsel. Unsere Kenntnisse über den Kohiehydratstoffwechsel und insbesondere über die Regelung des Abbaues des Traubenzuckers im tierischen Organismus haben durch die von J. v. Mering und 0. Minkoivski^) im Jahre 1889 entdeckte Tatsache, daß Hunde, denen ihre Pankreasdrüse vollkommen fortgenommen ist, im Harn Zucker ausscheiden, eine wesentliche Erweiterung erfahren. Diese Beobachtung wurde bald bestätigt, bald wurde bestritten, daß die Entfernung der Pankreasdrüse eine Störung des Kohlehydratstoffwechsels im Gefolge habe. In vielen Fällen war auch die Glukosurie eine nur vorübergehende. Es bald geglückt, zu zeigen, worauf diese Unstimmigkeiten in den Resultaten der einzelnen Versuche zurückzuführen w-aren. Es kommt nämlich darauf an, daß die Pankreasdrüse vollständig entfernt wird.') Bleiben auch nur einzelne Zellnester bei der Operation zurück, dann vermögen diese allein den Kohiehydratstoffwechsel in normalen Bahnen zu halten, solange sie ihre in Frage kommenden Funktionen auszuüben imstande sind und keine besonderen Anforderungen an den Kohiehydratstoffwechsel gestellt werden! Es ist dies eine Entdeckung von größter Bedeutung. Bei weiteren Untersuchungen konnte gezeigt werden, daß auch Fische (SelaAmphibien (Frösche), Reptilien (Schildkröte) und Vögel^) die chier»), vollständige Pankreasexstirpation mit dem Auftreten einer Glukosurie beantworten. Es steht fest, daß in dem ganzen Tierreiche die Pan- ist kreasdrüse außer der Aufgabe, an den Darmkanal Verdauungsabzugeben, noch einen bedeutsamen, nicht ersetzbaren Einfluß auf den Kohiehydratstoffwechsel hat. saft ') (1890). J V. Mering und 0. Minkowski: Arch. f. experim. Path. u. Pharm. 26. 371 — O. Minkowski: Untersuchungen üher den I)iabetes. Leipzig. Vogel. 1893. Eduard Fflüqer Pflüger?, Archiv. 106. 181 (1905). (Anat. u.) Adehof: Zei'tschr. 28. 293 (1891). — Wilh. Marctise: Arch. =') : ») f. liiol. f. Pbysiol. 539 (1894). Diamare: Zentralhl. f. Physiol. 20. ()17 (1906); 21. 863 (1907). W. Rausch: Archiv, f. experim. Path. u. Pharmak. 37. 274 (1890); 39. 219 (1897). — 0. Minkoivski: Arch. f. experim. Path. u. Pharmak. 31. S,'i (189:5). — Max Cremer ) V. 5) und A. lütter: Zeitschr. f, Biol. 28. 459 (1891). Kohlehydrate. Zunächst dachte 1(39 man daran, daß das Fehlen der Pankreasdrüse des- halb eine Störung des Kohlehydratstoffwechsels zur Folge hat, weil ja mit dem Wegfall dieses Organes auch die Sekretion des Pankreassaftes den Darmkanal aufgehoben ist. Es wäre denkbar, daß nunmehr die Verdauung beeinflußt wird und sich indirekt Störungen aller Art anschließen, unter denen auch der Zuckerstoffwechsel leidet. Gegen diese Annahme spricht die Tatsache, daß die Glukosurie dann ausbleibt, wenn zwar jede Verbindung der Pankreasdrüse mit dem Darmkanal unterbunden ist, jedoch noch funktionstüchtige Organteile im Organismus zu- in rückgeblieben sind. Nunmehr suchte man die Glukosurie nach erfolgter vollständiger Entfernung der Pankreasdrüse in Zusammenhang mit jener Art der Zuckerausscheidung im Harn zu bringen, die nach Erregung des Zuckerzentrums eintritt. Es wäre denkbar, daß bei der Operation Nervenbahnen gereizt werden, die in Beziehung zum Zuckerzentrum stehen. Auch diese Annahme konnte als unhaltbar erwiesen werden. Einmal würde mit ihr sehr schwer die Tatsache in Einklang zu bringen sein, daß die Glukosurie ausbleibt oder höchstens eine vorübergehende ist, wenn von der Pankreasdrüse kleine Picste zurückbleiben. Es wäre sehr gesucht, wollte man annehmen, daß die Entfernung dieser letzten Pteste auf dem Wege von Nervenbahnen eine Reizung des Zuckerzentrums v^eranlaßt. Selbstverständlich bleibt bei nicht sorgfältiger Entfernung der Pankreasdrüse bald diese, bald jene Zellgruppe zurück und nicht etwa immer ein bestimmtes Organstück. Schon diese Beobachtungen beweisen die Unmöglichkeit, die nach Entfernung der Pankreasdrüse auftretende Glukosurie mit dem Zuckerzentrum und seinen Funktionen in direkten Zusammenhang zu bringen. Ganz besonders klar hat der folgende Versuch von Mi?ikowski am Hunde bewiesen, daß ohne jeden Zweifel die Pankreasdrüse als solche in den Kohlehydratstoffwechsel eingreift. Beim Hunde ist der unterste Teil des absteigenden Astes der Pankreasdrüse nicht mit dem Duodenum verwachsen. Er liegt vielmehr frei im Mensenterium. Dieses Stück trennte nun Minkowski^) so vom übrigen Pankreasgewebe, daß es im Zusammenhang mit dem Mensenterium blieb und seine Versorgung mit Blut- und Lymphgefäßen und Nerven nicht gestört wurde. Dieses Pankreasstück wurde nun aus der Bauchhöhle herausgezogen und unmittelbar unter der Har.t neben der Schnittwunde eingeheilt. Nachdem das Versuchstier diese Operation überstanden hatte, wurde die Bauchhöhle wiederum geöffnet und nun der gesamte Rest der zurückgebliebenen Drüse vollständig entfernt. Es blieb also nur das kleine, unter der Haut eingeheilte Stück der Pankreasdrüse übrig, trotzdem trat keine Glukosurie auf. Wurde nun auch dieser Teil des Pankreas exstirpiert, dann stellte sich sofort Zuckerausscheidung im Harn ein. Das gleiche Resultat erhält man, wenn man den größten Teil der Pankreasdrüse entfernt und nur ein Stück davon zurückläßt, das mit dem Darmkanal nicht mehr in Zusammenhang steht. Die so operierten Tiere erholen sich rasch. Die Wunden sind bald vernarbt. Man findet im Harn höchstens in den ersten Stunden nach der Operation Zucker. Das zurück) 0. Minkowski: Arch. f. experni. Path. u Pharm. 31 81 Hedon: Diahete pancröatiqne. Paris 1908. (1893). — Vgl. auch VIII. Vorlesung. 170 gelassene kleine Stück Pankreasdrüse genügt, um den Kohlehydratstoffwechsel aufrecht zu erhalten.') Xach einiger Zeit jedoch erfolgt Glukosurie. Tötet man das Versuchstier, dann findet man, daß das zurückgelassene Pankreasgewebe zugrunde gegangen ist. Die auftretende Glukosurie zeigt uns unmittelbar an^ daß nunmehr keine funktionstüchtigen Pankreasdrüsenzellen mehr vorhanden sind. Sie degenerieren offenbar deshalb, weil die Bildung des Pankreassekretes weiter geht. Es fehlt der Abfluß und infolgedessen kommt es zu Stauungen, die dann zum Untergang der Zellen führen. Nachdem einwandfrei festgestellt worden ist, daß der vollständigen Entfernung der Pankreasdrüse immer eine Glukosurie folgt, ergibt sich zunächst die Frage nach ihrer Ursache. Bis jetzt haben wir zwei Möglichkeiten für das Zustandekommen einer Zuckerausscheidung im Harn kennen gelernt. Einmal wird eine solche durch ein Zuviel an Zucker im Blutplasma verursacht. Ferner kann die Niere für Glukose durchlässig werden, d. h. es kommt zur Zuckerausscheidung, obwohl der Zuckergehalt des Blutes nicht gesteigert ist. Es sei an die Glukosurie nach Einführung von Kochsalzlösung in die Blutbahn erinnert 2) und an die Feststellungen über die Abhängigkeit der Durchlässigkeit der Glomerulusepithelien von der Zusammensetzung der Durchspülungsflüssigkeit. 3) Es hat sich erw^iesen, daß die Zuckerausscheidung, die sich bei pankreaslosen Tieren findet, durch eine Hyperglukoplasmie verursacht ist. Man trifft stets einen erhöhten Zuckerspiegel im Blutplasma an. Es hätte somit die Glukosurie nach Entfernung der Pankreasdrüse die gleiche Ursache, wie diejenige nach erfolgtem Zuckerstich. Schon die Tatsache jedoch, daß der letztere nur dann von Erfolg begleitet ist, wenn die Leber Glykogen aufweist, während die Pankreasexstirpation immer Hyperglukoplasmie im Gefolge hat, sofern die Entfernung der Drüse eine vollständige ist, beweist, daß beide Arten von Störungen des Zuckerhaushaltes nicht identisch sein können. Dazu kommt noch, daß die Zuckerausscheidung nach Pankreasexstirpation eine dauernde ist. Sie hält bis zum Tode des pankreaslosen Tieres an. Selbst dann, wenn das Tier hungert, erscheint beständig Glukose im Harn. Diese Feststellungen führen zu der weiteren Frage: Weshalb besteht nach erfolgter, vollständiger Pankreasexstirpation eine dauernde Hyperglukoplasmie? Es ist immer mehr erkannt worden, daß eine wesentliche Störung des Kohlehydratstoffwechsels nach Ausfall der Pankreasdrüse darin liegt, daß die Leber nicht mehr in der Lage ist, Glykogen in ausreichendem Maße aufzubauen. Man findet dieses Organ nach Pankreasexstirpation arm an Glykogen. Besonders wichtig ist die Beobachtung, daß nach Entfernung der Pankreasdrüse die Leber beim Durchblutungsversuch 32 Stunden bis 5 Tage nach der (Jperation weder Trauben- noch Fruchtzucker in Glykogen umzuwandeln vermochte.) Versuche, durch Zusatz von Pankreasextrakt die Glykogensynthese in Gang zu bringen, hatten ein negatives Ergebnis. Ferner ist festgestellt worden, dali die Froschleber nach Pankreasexstirpation ') W. Sandmejjer: Zeitschr. ') S. f. Biol. 31. 12 (1895). ") 157. Vgl. S. 164. ) H. K. liarrenscheen : Biochem. Zeitschr. 58. 277 (1914). Kohlehydrate. 171 einen vermehrten Abbau des Glykogens zeigt. ^) Es muß somit die Entfernung der genannten Drüse mit einer tiefgehenden Veränderung der Leberzellen verbunden sein. 2) Es ist verlockend, anzunehmen, daß die Pankreasdrüse einen oder mehrere Stoffe liefere, die den Glykogenaufbau in der Leber beherrschen und diese Vorstellung der Feststellung gegenüber zu stellen, wonach die Nebennieren Inkretstoffe abgeben, die umgekehrt den Glykogenabbau beherrschen. Man könnte dann im Wechselspiel dieser Wirkungen die ganze Regelung des Glykogenauf- und -abbaus in der Leber erblicken, wobei es, wie S. 162 ff. schon betont, zur Zeit nicht möglich ist, zu entscheiden, welcher Art die Störung im Grunde genom- meif ist. 3) Wir müßten nun der Frage nachgehen, ob wir die schweren Störungen des gesamten Zuckerstoffwechsels nach Ausfall der Funktionen der Pankreasdrüse damit erklären können, daß die Leberzellen aus irgend einem Grunde, w^obei auch eine veränderte Durchlässigkeit der Zellgrenzschichten der Leberzellen eine Kolle spielen könnte (vgl. S. In6). nicht mehr imstande sind, den ihnen zugeführten Traubenzucker zu Glykogen aufzubauen. Es drängen sich ohne weiteres Einwände auf. Zunächst denken wir an die übrigen Glykogenspeicher und ferner an die Umwandlung von Zucker in Fett! Sollten auch hier Störungen vorliegen, so daß der gesamte dem pankreaslosen Organismus zugeführte Traubenzucker und der in ihm entstehende in den Gew^eben und dem Blutplasma liegen bleiben. Es käme fortwährend zu einer Hyperglukoplasraie und infolge davon zu Glukosurie. Nun ist eine Störung des Glykogenaufbaues in den übrigen Körperzellen außer denen der Leber nicht festgestellt. Die Fähigkeit der Bildung von Fett aus Zucker bei Fehlen der Pankreasdrüse ist nicht eindeutig geprüft. Es ist wahrscheinhch unmöglich, sie zu verfolgen, weil der pankreaslose Organismus wohl überhaupt kaum zur Fettablagerung kommt. Ihm entgeht mit dem durch die Nieren ausgeschiedenen Zucker beständig wertvolles Energiematerial. Infolgedessen wird er alle organischen Nahrungsstoffe und Verbindungen, deren Energieinhalt er erschließen kann, zur Aufrechterhaltung des Stoffwechsels heranziehen. Auf können wir uns somit nicht ohne weiteres darauf stützen, alle Fälle daß der gesamte Organismus des pankreaslosen Organismus Glukose nicht mehr unterbringen kann. Daß jedoch in dieser Richtung eine schwere Störung vorliegt, daraufhin deutet auch die Beobachtung, daß pankreaslose Hunde von per os zugeführter und intravenös eingespritzter Glukose einen einer größeren Anteil durch die Nieren ausscheiden, als normale Tiere.) Einer der wichtigsten Befunde des gesamten Kohlehydratstoffwechsels schließt sich hier an. Man begann, die Menge der im Harn erscheinenden Glukose zu bestimmen und fand bald, daß mehr Traubenzucker im viel Harn erschien als Kohlehydrate mit der Nahrung aufgenommen 1) ) Fröhlich und Pollack: Arch. f. experim. Path. u. Pharm. 77. 265, 299 (1914). Vgl. Auch E. J. Lesser : Ergebnisse der inneren Medizin und Kinderheilkunde. XVI. 279 (1919). ') Zuelzer, — Vgl. hierzu z. B. Herter und Wakemann: Virchow?> krch\\ 169. 479 (1902). Dorn und Marxer: Deutsche med. Wochenschr. Nr. 32. 1908. — 0. Loewi: . — V. Falta und J. G. Priestley : Berliner Arch. f. experim. Path. u. Pharm. 58. 83 (1908). klin. Wochenschr. Nr. 47 (1911). ) F. M. Allen und J/ar?/ B. Wishart: The Journ. of biol. ehem. 13. 615 (1920). F. M. Allen: Americ. .Journ. o"f the med. Sciences. 160. 781 (1920). — VIII. Vorlesung. 1^2 waren. Zuerst dachte man daran, daß vorhandene Glykogenvorräte das Mehr an Zucker im Harn bedingten. Schließlich müßte jedoch dieser Zuschuß aufhören! Je genauer man die verabreichte Nahrung auf Kohlehydrate untersuchte, und je sorgfältiger die Zusammensetzung des Harnes studiert wurde, um so zwingender wurden die Beweise, daß der pankreaslose Hund mehr Traubenzucker im Harn ausscheidet als er Kohlehydrate einnimmt und besitzt, i) Somit war bewiesen, daß der tierische Organismus Traubenzucker aus Nichtkohlehydraten bildet. Die einen Forscher glaubten, daß eine Umwandlung von Fett in Kohlehydrate während andere die Eiweißkörper Ijzw. ihre Bausteine, die Aminosäuren, als Quelle für Glukose bezeichneten. Die letztere Annatime ist über jeden Zweifel sicher festgestellt, während eine Zuckerbildung aus Fett zwar durchaus nicht ausgeschlossen ist, jedoch bisher nicht eindeutig bewiesen werden konnte. 2) Somit hat die der Entfernung der Pankreasdrüse folgende Glukosurie zu der weittragenden Erkenntnis geführt, daß der tierische Organismus vorliege, Kohlehydrate aus bestimmten Aminosäuren bilden kann. Wir kommen auf diese Tatsache noch zurück. Handelt es sich bei dieser Bildung von Glukose um einen Vorgang, der etwas Abnormes, eben durch das Fehlen der Pankreasdrüse Bedingtes darstellt, oder kommt vielmehr eine Quelle für Zucker zum Vorschein, nur vielleicht in geringerem Umfange die auch sonst fließt, jedoch der direkten Beobachtung entgeht, weil der gebildete Traubenzucker normalerweise sofort weiter verwertet wird? Es wäre sehr gesucht, wollte man annehmen, daß die Zellen des — — tierischen Organismus, nachdem sie durch die Fortnahme der Pankreasdrüse schwer geschädigt sind, auf einmal zu dem unzweifelhaft sehr komplizierten Vorgang der UmAvandlung von Nichtzuckern in Kohlehydrate greifen, ohne auch sonst den gleichen Weg einzuschlagen. Eine ganze Kette von Vorgängen ist notwendig, bis Aminosäuren in Zucker verwandelt Wie sollte ein so komplizierter Vorgang plötzlich ohne jede Vorsind. bereitung von den Zellen bewältigt werden, wenn sie wirklich etwas ganz Neuartiges zu leisten hätten! Es ist vielmehr als sicher festgestellt anzunehmen, daß Ftets aus bestimmten Aminosäuren in einem dem Bedarf angepaßten Umfange Glukose entsteht. Nachdem wir wissen, daß beim Abbau von Eiweißbausteinen und von Glukose gemeinsame Abbaustufen entstehen (vergl. S. 137) und uns ferner bekannt ist, daß umkehrbare Vorgänge vorliegen, erscheint uns die Bildung von Zucker aus bestimmten Aminosäuren nicht mehr auffallend. Es fließen der Stoffwechsel mancher EiweiÜbausteine und derjenige der Glukose und sicherlich auch derjenige der Bausteine der Fette zusammen. Kehren wir nunmehr zu der grundlegenden Frage nach der Ursache der Hyperglukoplasmie- nach vollständiger Entfernung der Pankreasdrüse zurück. Wir haben festgestellt, daß nicht nur Kohlehydrate als Quelle für den Traubenzuckergehalt des Blutplasmas in Frage kommen, die mit der M Vgl. u. a. //. Lüthje: Deutsches Arch. f. kliu. Med. 79. 498 (1904); rfiiU/ers Archiv. 106. 160 (1904). /.. Mohr: Zeitschr. f. klin. Med. 52. 337 (1904). Eduard J'/lüf/er und Peter Junkersdorf : I'ßü(/erü Archiv. 131, 201 (1910). — — — ') Vgl. Vorlesung X, XI und XV. Vgl. auch August Krogh und Johannes Lindhard (Göran lAljestrand und Knud Gad): Biochem. .lourn. 14. 290 (1920). Kohlehydrate, 173 Nahrung aufgenommen werden oder bereits in den Geweben vorhanden viehnehr werden beständig Zuckermoleküle aus anderen Verbindungen und insbesondere aus bestimmten Aminosäuren gebildet. Von diesen Gesichtspunkten aus wird eine Hyperglukoplasmie verständlich, wenn man annimmt, daß die Leberzellen den Überschuß an Glukose nicht dem Blut- sind, plasma entziehen. Bevor man die Wechselbeziehung zwischen der Pankreasdrüse und der Leber im Hinblick auf den Glykogenaufbau erkannt hatte, suchte man die Störung des Zuckerstoffwechsels in der Hauptsache in demjenigen der Muskelzelle. Man vermutete, daß diese die Fähigkeit eingebüßt habe, das Zuckermolekül zu zerlegen. Es sollte die Pankreasdrüse einen Stoff liefern, der in irgend einer Weise den Abbau des Zuckers im Muskel leitet. In der Tat sind Versuche mitgeteilt worden, aus denen hervorzugehen schien, daß die Muskulatur von pankreaslosen Tieren nicht imstande ist, Glukose abzubauen^), jedoch ist diesen Ergebnissen widersprochen worden.2) Aus neueren Versuchen geht hervor, daß auch im pankreaslosen Organismus Glukose zum Abbau kommt und vor allem auch die Muskelzellen sich aus ihr Energie erschließen können. Es ist von größter Tragweite, eindeutig zu entscheiden, ob Muskelzellen pankreasloser Tiere Glukose abbauen und verwerten können oder nicht, denn im letzteren Falle wäre klar erwiesen, daß die erwähnten Zellen nicht ausschließlich auf Glukose als Energiematerial angewiesen sind, denn das pankreaslose Tier kann Muskelarbeit leisten. Wir möchten auf Grund der vorliegenden Arbeiten und an Hand eigener Erfahrungen der Ansicht zuneigen, daß die Annahme die zutreffende ist, wonach die Muskulatur des pankreaslosen Organismus Zucker zerlegen kann. Unentschieden bleibt, ob dei" ganze Vorgang glatt verläuft und qualitativ und quantitativ restlos ungestört ist. Hier müssen weitere Versuche Klarheit bringen. Es spricht manches dafür, daß die Störung des Kohlehydratstoffwechsels nach vollständiger Entfernung der Pankreasdrüse mit der Einschi'änkung bzw. Aufhebung der Glykogensynthese in der Leber nicht erschöpft ist. Wir müssen noch der Möglichkeit gedenken, daß die Pankreasdrüse nicht direkt auf die Leberzellen einwirkt und ihren Glykogenaufbau durch Abgabe bestimmter Stoffe regelt. Es könnte sein, daß sie in Beziehung zu anderen Organen, wie zur Schilddrüse, Hypophyse usw. steht und über diese Einfluß auf die erwähnten Zellen gewinnt. Von diesem Gesichtspunkte aus wäre es verständhch, weshalb es bis jetzt nicht in einwandfreier Weise gelungen ist, die nach erfolgter vollständiger Entfernung der Pankreasdrüse auftretende tiefgreifende Störung des Kohlehydratstoffwechsels durch Verfüttorung von Pankreasgewebe bzw. Einspritzung von Auszügen aus solchem wirksam zu beinflussen. Bevor in dieser liichtung nicht erfolgreiche und ('. Lovatt Evans: Jouru. of Physiol. 49. B7 (1914). — F. Verzdr: Ebenda. und A. v. Feje'r: Biochem. Zeitschr. 53. 140(1913). 66. 75 (1914).— <). Borges und IL Salomon: Ebenda. 27. 143 (1910). — Vgl. auch (). For') E. H. Starling — E. Verzdr ges: Ebenda. 27. Path. u. Parmak. — (1918). 171 (1915). H. K. — E. Eorschbach r.ud H. Schc'ifer: Archiv f. experim. (1910). 0. Loewi: Therapeutische Monatshefte. 32. 350 344 (1918). Moorhouse, S. W. Patterson und M. Stei^henson: Biochem. J. 9. 131 82. — -Jakob K. Parnas: ^) Vgl. Ernst J. Lesser: Biochem. Zeitschr. 103. 1 (1920). Ebenda. 116. 89 (1921). Fr. M. Allen und Mary B. Wishart: The Americ. J. of med. Sciences. 161. 165 (1921). — VIII. Vorlesung. 174 Versuche vorliegen, bleibt die Annahme, daß die Pankreasdrüse als solche Stoffe erzeugt, die irgendwie in den Kohlehydratstoffwechsel eingreifen, unbewiesen. Viel w^ahrscheinUcher erscheint einstweilen, daß mehrere Organe in der erwähnten Pachtung zusammenarbeiten. Eines davon ist die Pankreasdrüse. Es fragt sich nun, mit welchen w^eiteren Organen sie gemeinsam den Kohlehydratstoffwechsel beherrscht, und welcher Art diese Beziehungen sind. Schließhch könnte man auch daran denken, daß die Pankreasdrüse mit den Nebennieren zusammen arbeitet und diese in der Abgabe jener Stoffe beeinflußt, die den Glykogenabbau in Gang bringen, i) Vielleicht fällt mit dem Ausfall der Pankreasdrüse eine Hemmung fort, so daß die Leberzellen nur auf Glykogenabbau und nicht auch auf Glykogenaufbau eingestellt sind. Bei allen diesen Einflüssen kann es sich um solche physikalisch-chemischer Art handeln. Durch ganz geringfügige Verschiebungen in der Zusammensetzung von Zeilinhaltsstoffen oder auch nur solchen der Zellgrenzschichten können für die Funktionen der Zellen hochbedeutsame Zustandsänderungen bestimmter Zellbestandteile eintreten, die für den Verlauf bestimmter Zellfunktionen von ausschlaggebender Bedeutung sind. Erwähnt sei noch, daß man die bisher negativen Ergebnisse der Versuche, die Pankreasdrüse durch aus ihr gewonnene Stoffe zu ersetzen, auch auf eine hohe Empfindlichkeit der wirksamen Produkte zurückführen kann, doch fehlen bestimmte Anhaltspunkte für eine solche Annahme. Es ist auch denkbar, daß bessere Fiesultate zu gewinnen w^ären, wenn man unmittelbar nach der erfolgten Entfernung der Pankreasdrüse mit Ersatzversuchen einsetzen würde. Man muß damit rechnen, daß die einmal vorhandene Störung Zustände schafft, die nur schwer oder überhaupt nicht mehr überwindbar sind. Hatten auch, wie eben erwähnt, Versuche, die entfernte Pankreasdrüse durch Verfütterung von Gewebe dieses Organes oder auch von Auszügen aus ihm zu ersetzen, keinen eindeutigen Erfolg, so besteht, und das möchten wir besonders hervorheben, doch kaum ein Zweifel darüber, daß die Pankreasdrüse mittelst Inkretstoffen direkt oder indirekt auf den Kohlehydratstoffwechsel einwirkt. Es seien einige Belege für diese Annahme angeführt^): Zunächst sei an die auf S. 169 erwähnten Versuche erinnert, wonach ein kleines Stück der Pankreasdrüse genügt, um ihren Einfluß auf den Kohlehydratstoffwechsel aufrecht zu erhalten. Es lassen sich ferner zwei Tiere so vereinigen, daß das Blut des einen Organismus auch im Körper des anderen kreist. 3) Man nennt eine solche Vereinigung zweier Tiere Parabiose. Forschhach) vereinigte zwei junge Hunde. Dem einen Tiere wurde dann E. Heyon: Arch. internat. de physiol. 18. 213 (1921). Murlin und ./. E. Sweet (The J. of biol. Cheni. 28. 1 [1916]) sind der Ansicht, daß der Umstand, daß bei der Entfernung der Pankreasdrüse die in das Duodenum übertretende Salzsäure des Mageninhaltes infolge des Fehlens des Pankreassaftes nicht ausreichend durch Alkali neutralisiert werden kann, viel dazu beiträgt, daß der Kohleh} dratstoflWechsel gestört wird. Dieser Annahme widersprechen viele Beobachtungen, es sei z. B. an die Tatsache erinnert, daß ein kleines Stück der Pankreasdrüse genügt, nun eine Hyperglukoplasmie und damit eine Glukosurie zu verhindern. ') F. Sauerbrnch und 3f. Ileijde: Münchener med. Wochcnschr. Nr. 4. 908. E. Hedon: Arch. f. experim. Path. u. Pharm. 60. 131 (1909). ) J Forschbach : Deutsche med. Wochenschr. Nr. 21. 1908; Archiv, internat. de physiol. 13. 4 (1913). 1) 2) J. li. — Kohlehydrate. I75 Es trat bei diesem keine Glukosurie auf. war gleichzeitig die Pankreasdrüse weggenommen worden. Dieses litt an ausgesprochener Glukosurie. Der Ausfall dieses Versuches macht es im höchsten Grade wahrscheinlich, daß die die Pankreasdrüse entfernt. Einem einzelnen Kontrolltier Pankreasdrüse an das Blut Stoffe abgibt, die den Kohlehydratstoffwechsel direkt oder indirekt beeinflussen. Nur so kann man erklären, weshalb das Tier ohne Pankreas frei von Glukosurie blieb. Es wurden ihm unzweifelhaft vom gesunden und mit ihm vereinigten Tiere die von dessen Pankreasdrüse sezernierten Stoffe zugeführt. Zu dem gleichen Ptesultate führten So beobachtete Hedon^), daß die Glukosurie Bluttransfusionsversuche. rasch verloren ging, als er einem pankreaslosen Hunde Blutserum, das aus venösem Blut der Bauchspeicheldrüse eines gesunden Hundes stammte, in eine Vena mesaraica einführte. Einen der Parabiose ganz entsprechenden Zustand stellt jedes schwangere Tier dar. Carlson und Drennan-} legten sich die wichtige Frage vor, ob die Pankreasdrüsen der im Uterus befindlichen, beinahe ausgewachsenen Föten für die entsprechende Drüse des mütterkönnen. Sie exstirpierten hochträchtigen lichen Organismus eintreten Hündinnen die Pankreasdrüse. Es trat entweder keine oder doch nur eine geringfügige Glukosurie auf. In dem Augenblicke, in dem bei der Geburt das letzte Junge und damit die letzte Pankreasdrüse den mütterlichen Organismus verlassen hatte, stellten sich Hyperglukoplasmie und Glukosurie ein! Diese Beobachtungen beweisen, daß von den Pankreasdrüsen der Föten durch den Plazentarkreislauf Stoffe auf das Muttertier übergegangen waren, die den Kohlehydratstoffwechsel auch im mütterlichen Organismus trotz Fehlens seiner Pankreasdrüse in normalen Bahnen zum Ablauf brachten. Mit der Entfernung der Föten fiel dieses Inkret fort und die Störung des Kohlehydratstoffwechsels war da! Auch der folgende Versuch spricht im Sinne einer innersekretorischen Tätigkeit der Pankreasdrüse. BiedP) unterband bei einem Hunde den Ductus thoracicus, oder er leitete die in diesem be- findliche Lymphe nach außen ab und beobachtete, daß trotz Erhaltenseins einer funktionsfähigen Pankreasdrüse eine andauernde Glukosurie auftrat.^ Der Ausfall dieser Versuche weist darauf hin, daß die Pankreasdrüse jene Stoffe, die in den Kohlehydratstoffwechsel eingreifen, nicht direkt der Blutbahn übergibt. Sie werden vielmehr zunächst dem Ductus thoracicus zugeführt. Sie gelangen dann in diesem mit dem Chylus in den großen Kreislauf. Durch die Unterbindung des Ductus thoracicus bzw. durch die Ableitung der Ductuslymphe wird die Verbindung zwischen der Pankreasdrüse und den übrigen Organen des Hedon: Compt. rend. de la soc. biol. 71. 124 (1911). Ä. J. Carlson und F. M. Drennan: Americ. Journ. of Physiol. 28. 391 (1911). A. J. Carlson und //. Ginsburg: Vgl. auch F. M. Drennan: Ebenda. 28. 396 (1911). Vgl. hierzu Fr. M. Allen: The American J. of Physiol. 54. Ebenda. 36. 217 (1914). 451 (1921). Dieser Autor konnte die erwähnten Versuche nicht bestätigen. Es muß abgewartet werden, worauf der Widerspruch zurückzuführen ist. Vgl. ferner A. Biedl und ») Arfur Biedl: Zentralbl. f. Physiol. 12. (i24 (1899). Es wäre sehr erwünscht, Jh.. R. Offer: Wiener klin. Wochcuschr. Nr. 49. Iö3ü (1907). wenn dieser wiclitige Versuch wiederholt würde' ) Wie schon mehrfach betont, erhalten alle derartigen Versuche erst die volle Beweiskraft, wenn neben der Zuckerausscheidung im Harn auch der Zuckergehalt des 1) — ^) — — — — Blutplasmas ausreichend kontrolliert wird. '• \lll. Vorlesung. 1 7(5 Organismus unterbrochen. Infolgedessen bildet sich der gleiche Zustand wenn die Pankreasdrüse entfernt wäre. Ganz von selbst stoßen wir nun noch auf das folgende Problem. Wir haben bis jetzt eine Doppelfunktion der Pankreasdrüse kennen gelernt. Sie Seine hohe Bedeutung für die bildet und sezerniert den V'erdauungssaft. Kohlehvdratverdauung haben wir bereits besprochen. Ferner gibt sie an heraus, wie das Blut kretstoffe — direkt oder indirekt auf dem Wege der Lymphbahnen — In- Werden nun Sekret- und Inkretstoffe von den ab. gleichen Zellarten gebildet oder enthält die Pankreasdrüse zwei grundsätzlich verschiedene ZellartenV Die mikroskopische Betrachtung der Pankreasdrüse zeigt, daß in der Tat zwei verschiedene Die einen stehen in Zusammenhang Zellformen in ihr vorhanden sind.^) mit Ausführungsgängen und sind ohne Zweifel als die Bildungsstätten der Die anderen sind in Gruppen angeordnet und Sekretstoffe anzusprechen. entbehren dieser Beziehung zu Ausführungskanälen. Es ist auch heute noch nicht über jeden Zweifel festgestellt, ob man berechtigt ist, anzunehmen, daß die nach ihrem Entdecker 2) und ihrer Anordnung als Langerhanssche Inseln bezeichneten Zellarten allein für die Bildung von Inkretstoffen in Frage kommen. Nach der einen Ansicht stellen die in den Langerhans&chen Inseln vereinten Zellen Jugendformen von später Pankreassaft liefernden Drüsenzellen dar. Nach dieser Anschauung käme für die Beeinflussung des Kohlehydratstoffwechsels das gesamte Drüsenparenchym einheitlich in Frage^), d. h. eine Scheidung in Sekret- und Inkretzellen wäre nicht vorhanden. Nach der anderen Anschauung hätte man zwischen zwei Zellarten mit getrennten Funktionen zu unterscheiden. Es ist zurzeit nicht möglich, zu der einen oder anderen Ansicht endgültig Stellung zu nehmen. Mir scheint, daß diejenigen Forscher, die für die Sekret- und Inkretbildung gesonderte Zellarten fordern, zu sehr in der Meinung befangen sind, als würde die einzelne Zelle eine biologische Einheit darstellen. Je mehr wir in den physikalisch-chemischen Bau der Zellen Ein- blick erhalten, um so mehr erkennen wir, daß diese in vieler Beziehung beim Zellinhalt nicht um eine eine Vielheit darstellen. Es handelt sich Reaktionen sich einheitlich im ganzen können wir die Zellen als einen Komplex von zahlreichen Einzelteilchen im gewissem Sinne kann man an Einzelräume denken auffassen, die alle Orte besonderer Vorgänge und Funktionen darstellen. Die Zelle ist nicht die letzte biologische Einheit, vielmehr umf^aßt sie viele solche! Von diesen Gesichtspunkten aus bereitet es keine Schwierigkeiten, anzunehmen, daß ein und dieselbe Zeilart die Bestandteile des Pankreassaftes bereitet und zugleich Inkretstoffe homogene Lösung, in der bestimmte Zellinhalt vollziehen, vielmehr — Hunsemann — Veihandl. d. Deutschen Pathol. Gesellsch. 187 (1901); Berliner A. Weichselbaum : Sitzungsb. d. Akad. d. Wissensch. "NVien. 119. Abt. 111. 73. 1910. K. A. Heihery: Ergebnisse der Anatomie und Entwicklungsgeschichte. 19. 2. Mälfte. 1909 (1911). Beitr. z. pathol. Anat. u. allg. Path. Carl)/ Sei/farth: 'Seue 51. 178. (1911). Joh. Moldenhaiier : Inaug.-Diss. Bern 1909. Beiträt{e zur Kenntnis der Langerhünsscheu Inseln im menschlichen Pankreas und ihrer Beziehung zum Diabetes melitus. Gustav Fischer, Jena 1920. Fr. M. Allen: The J. of exper. Med. 31. 3(53 (1920); The J. of metabolic Research. 1. 5, 7ö. 16.'), 93, 221, 2Ö1 (1922). W. B. Martin: Ebenda. 1. 43 (1922). ^) Langerhans : Beiträge zur mikioskopischeu Anatomie der Bauchspeicheldrüsen. ) Idin. V. : Wochenschr. Nr. 20 (1912). — — — — — — — Berlin. Diss. 'l869. ^) Vgl. hierzu besonders Carl;/ Sei/j'arlli: Neue Beiträge 1. c. (Zitat 1. S. 176). Kohlehydrate. 177 die beim Kohlehydratstofiwechsel in irgend einer Weise mitwirken. Leider sind die Inselzellen so in das übrige Pankreasgewebe verilochten, daß es nicht möglich ist. nur sie zu entfernen.^) Wäre das durchführbar, dann wäre der Beweis leicht zu eibriugen, ob wirklich nur ihnen eine Bedeutung im Kohlehydratstoifwechsel zukommt. Überblicken wir alles, was wir über die Bedeutung der Pankreasdrüse für den Kohlehydratstoffwechsel kennen gelernt haben, dann ergeben sich eine ganze Anzahl von Kenntnissen von grundsätzlicher Bedeutung. Wir sind auf ein Organ gestoßen, das eine äußere und eine innere Sekretion hat. In der Leber und der Nebenniere hatten wir bereits Organe vor uns, die Inkrete liefern. Die erstere besitzt auch eine äußere Sekretion, nämlich in der Gallenbildung. Die Beschäftigung mit dem Pankreasdiabetes hat ferner zu der bedeutsamen Feststellung geführt, daß liefert, der tierische Organismus aus bestimmten Aminosäuren Glukose bilden kann. Im Mittelpunkt dieser ganzen Überführung stehen Verbindungen des Dreikohlenstoffsystems. Wir haben schon S. 137 darauf aufmerksam gemacht, daß die Brenztraubensäure ein solches Zwischenglied darstellt. Ferner haben wir erkannt, daß die Pankreas- drüse direkt oder indirekt Beziehungen zur Leber haben muß. Welcher Art diese sind, konnten wir nicht sicher feststellen, nur steht fest, daß der Glykogenaufbau in den Leberzellen gestört ist. Ferner haben wir hervorgehoben, daß die Synthese von Traubenzucker nicht eingeschränkt, ja sogar vielleicht gesteigert ist. Schließlich sei noch mit allem Nachdrucke betont, daß wir zwar beständig von einer Störung des Kohlehydratstoffwechsels nach vollständiger Entfernung der Pankreasdrüse gesprochen haben, jedoch davon überzeugt sind, daß darüber hinaus der gesamte Es läßt sich keine StoffStoffwechsel auf das tiefgehendste gestört ist. wechselart für sich allein betrachten. Wir sprechen zwar von Kohlehydrat-, Fett-, Eiweiß- bzw. Aminosäurestoffwechsel und halten diese Stoffwechselarten auseinander, wir sind uns jedoch dabei bewußt, daß sie alle innig zusammenhängen und ohne Zweifel voneinander und darüber hinaus auch vom Mineralstoffwechsel abhängig sind. Wir gehen zunächst den augenfälligsten Störungen nach, folgen allen Spuren, die sie hinterlassen und stoßen schließlich auf \'erbindungen, die keinen spezifischen Charakter mehr zeigen, denen wir nicht ansehen können, ob sie von Kohlehydraten oder Bausteinen der Fette oder der Proteine herstammen. In diesem Gebiete wurzelt vielleicht auch die wesentlichste Störung im Stoffwechsel nach f^ntfernung der Pankreasdrüse. Der Organismus erzeugt die Abbaustufen im allgemeinen in Spuren. Sie werden Schlag auf Schlag weiter verwandelt. Ergeben sich Störungen, häuft sich ein Produkt an. dann kann das dt r Anlaß sein, daß der ganze Stoffwechsel in eine bestimmte Richtung gedrängt wird. Diese (Jedanken sollen nur darauf hinweisen, daß man über einer Einzelerscheinung nie die Betrachtung des Gesamtstoffwechsels und der gesamten Vorgänge im Organismus aus den Augen verlieren soll. Gleichzeitig soll mit allem Nachdrucke betont werden, daß die ganze Frage der Bedeutung der Pankreasdrüse für den Zucker- und den Gesamtstofiwechsel noch im Flusse ist. Bei Selachiern ) Zentralbl. A bder f. li ist eiue solche Physiol. 18. 432 (1904). al den , TreDOung möglich. Physiologische Chemie. I.Teil. 5. Aufl. Vgl. Dietmare und Kuliabko 12 Vorlesung IX. Kohlehydrate. VIII. Diabetes melitus. erzeugte Hyperglunicht nur deshalb das größte Interesse geweckt, weil die Aussicht bestand, ein lückenloses l'.ild des Ablaufs des Kohlehydratstoffwechsels zu erhalten, sondern vor allem auch, weil wir eine Krankheit kennen, bei der eine Störung des genannten Es ist dies der Diabetes melitus, die ZuckerStoffwechsels vorliegt. Die auf verschiedene Arten experimentell koplasmie mit nachfolgender Glukosurie hat harnruhr. Bei dieser erscheint Traubenzucker im Harn, i) Ferner findet Schon im Mittelalter war sich wohl regelmäßig eine Hyperglukoplasmie. den indischen und arabischen Ärzten bekannt, daß bei der erwähnten Krankheit ein süß schmeckender Stoff im Harn erscheint. Es gelang jedoch erst Chevreul') im Jahre 1815, den Harnzucker in Kristallform zu gewinnen. Bouchardat^) und Pelifjot) haben ihn dann einwandfrei als Traubenzucker erkannt.-') Würden wir genau wissen, auf welche Art und Weise der tierische )rganismus seinen Kohlehydratstoff Wechsel regelt, würden wir alle Organe kennen, die ihn beherrschen und würde uns bekannt sein, wie jedes einzelne Organ wirkt, an welcher Stelle des gesamten Zuckerstoffwechsels die einzelnen Gewebe eingreifen, dann würden wir ohne Zweifel imstande sein, den Diabetes melitus therapeutisch wirksam anzugreifen, indem wir < oder, vielleicht besser ausgedrückt, die voneinander alihängigen Ursachen beseitigen könnten. Ja, es wäre denkbar, daß eine wirksame Prophylaxe eingeleitet werden könnte. Wartet aus diesen Gründen der Pathologe mit größter Ungeduld auf weitere Fortschritte auf dem Gebiete der experimentellen Erforschung des Kohlehydratstoffwechsels, so ist andererseits dem Physiologen ein von der Natur angestelltes Experi- die Ursache ') Vgl. hierzu J^Jduard von lyippmann : Zur (Joscliiclite (k's diabetischen Ziicker.s. 1 (1920). C'herrenl: Bull, de la Societc'^ philomati(iue. 148 (1815); Aunales de Chimie. (Iieraiker-Ztjr. 44. ^) 4.5 (1815). Bouchardat : Gompt. read, de TAcad. des Sciences. 6. 337 (1838). Peligot: Ebenda. 7. 106 (1838). ') Vgl. weitere historische Daten bei R. Lepine : Le diabete sucre. Felix ') ) Paris 1909. Alcaii. Kohlehydrate. 179 ment, wie es der Diabetes melitus darstellt, sehr willkommen. Winkt doch die Aussicht, dal» durch die Erforschung dieser Krankheit und insbesondere Ursachen Licht über den normalen x\blau\| des Kohlehydratstoffwechsels verbreitet wird. Man darf allerdings, das sei hier gleich mit voller Schärfe hervorgehoben, nicht ohne weiteres die an bestimmten Tieren gemachten Beobachtungen auf den Menschen übertragen und umgekehrt, die beim Diabetes melitus des Menschen erhaltenen Befunde ohne weiteres mit entsprechenden Zuständen bei Tieren in l'arallele stellen. Die Erfahi'ung hat zwar gezeigt, dali der Stoffwechsel bei allen Tieren von den gleichen (Grundgesetzen beherrscht wird, es finden sich jedoch in den p]inzelheiten, wie ein Stoff verwendet, umgewandelt und abgebaut wird, zum Teil recht erhebliche Unterschiede. Vor allem ist zu berücksichtigen, daß ein Karnivore, der auf große Eiweil')und geringe genuine \) Kohlehydratmengen eingestellt ist, sich anders veihalten kann, als ein Herbivore und auch als ein Omnivore. Nur unter exaktester Berücksichtigung aller einzelnen Momente dürfen Beobachtungen, die an verschiedenen Tierf^rten gewonnen sind, vergleichend verwertet werden. Nicht außeracht lassen darf man ferner bei allen e.xperimentell erzeugten Schädigungen des Stoft wechseis oder ganz allgemein von bestimmten Funkihrer daß die Natur im allgemeinen die Störungen bestimmter Art allmählich hervorgehen läßt. Es kann Jahre dauern, bis ein Versagen eines Organes voll in Erscheinung tritt. Unterdessen können alle möglichen Kompensationserscheinungen sich eingestellt haben oder aber, es sind mehr und mehr auch andere Organe in Mitleidenschaft gezogen worden. Bei den experimentell herbeigeführten Ausfallserscheinungen stellen wir den Organismus ganz plötzlich vor ganz neue Verhältnisse. Will man das Band zwischen Pathologie und experimenteller Forschung enger knüpfen, dann muß unser Bestreben darauf gerichtet sein, Methoden zu ersinnen, die es uns ermöglichen, bestimmte Organe allmählich auszuschalten oder noch besser nur bestimmte ihrer Funktionen zum Versagen zu bringen. tionen, Trotz gewaltiger Anstrengungen ist es bis heute noch nicht geglückt, Ursachen des Diabetes melitus restlos zu erkennen. Wir wissen in der Hauptsache nur, daß eine Hyperglukoplasmie vorliegt. Ein ganz wesentliches Hindernis in der Erforschung des Diabetes melitus bildet ohne Zweifel der Umstand, daß wir nicht in der Lage sind, einwandfrei zu entscheiden, ob dem Symptom der Hyperglukoplasmie und der Glukosurie eine einheitliche Ursache zugrunde liegt. Zwar wissen wir, daß es recht verschiedene Formen von Diabetes melitus gibt. Wir unterscheiden leichte und schwere Formen. Diese sind durch alle Übergänge untereinander die verknüpft. L'nter einer leichten Form verstehen wir eine (Glukosurie, die zurückgeht, sobald man die Kohlehydratzufuhr einschränkt. Ja, oft genügt Leistung körperhcher Arbeit, d. h. eine stärkere Inanspruchnahme der Kohlehydrate, um eine bestehende Glukosurie zum Verschwinden zu bringen.'-) r>ei den schweren Formen dauert die Zuckerausscheiduiif'' auch dann 't .jOnuin" soll hiev lieißeii. mit der Nahruiii;- ziigefühite Ktiiilehydiate. /iiui Unterschied vou dem Zucker, den sich der Orifanisnms selbst aus bcstimmteu Aniiiiosäureu und vielleicht auch aus Bausteinen der Fette bereitet. -') Es sei auch au dieser Stelle darauf hiiigewieseu, daß gewiß mancher Fall von „rasch vorübergehendem" Diabetes unrichtig diagnostiziert und durch (Uukuroii.'^.iiin' vorgetäuscht ist. Vgl. S. .37ff.. vgl. ferner/. 15. I'aid Mni/er : Berliner klin. Wochenschr. I^- Vorlosmiff 180 wenn mit der Nahruni^ keine Kohleliydrate verabieiclit werden sicher auch schon längst die Kohiehydratvorräte verbraucht sind. an. und Wir werden uns hier mit dieser wichtigen Stoffwechselkrankheit niiiinsoweit beschäftigen, als wir aus ihr Anhaltspunkte für die Physiologie des Kohlehydratstoffwechsels gewinnen können.') Zunächst wollen wir versuchen, an Hand der bisherigen Darlegungen ein Bild über die möghchen Arten der Störung des Kohlehydratstoffwechsels beim Diabetes zu geben. Es sei zunächst an die alimentäre (ilukosurie erinnert.-) Wir haben festgestellt, dal.) bei Zufuhr größerer Mengen von Kohlehydraten diejenigen Zellen, die Glykogen zu speichern vermögen und jene, die Kohlehydrate in Fett überführen, schließlich den Zufluß an (ilukose nicht mehr bewältigen können. Es bleibt eine größere Menge von Traubenzucker im Blute liegen. Die Nieren greifen ein und entfernen den Überschuß davon. Man kann sich nun wohl vorstellen, daß in manchen Fällen die Leberzellen auch bei einer Zufuhr von Kohlehydraten, die von gesunden Individuen spielend bewältigt wird, außerstande sind, zu verhindern, daß eine Hyperglukoplasmie entsteht. 3) Vermögen dann die anderen Zellen, die Glykogen zu bilden vermögen, nicht einzugreifen, dann folgt als Ausgleich der Hyperglukoplasmie eine Glukosurie. Selbstverständlich kann unter Umständen die Leber ganz normal funktionieren, es versagen jedoch die übrigen Zellen, die Glykogen bilden können. Auch bei diesem Zustande muß es zur Hyperglukoplasmie kommen, wenn die Zufuhr von Kohlehydraten gewisse Grenzen überschreitet. Man gebraucht in der Pathologie oft den Ausdruck „schwaches" oder ,,geschwächtes' Organ. Wir können mit dieser Bezeichnung ganz gut Unzulänglichkeiten in bestimmten Funktionen in Einklang bringen. Es ist denkbar, daß z. B. die Leberzellen nicht über genügend Fermente verfügen, um den Glykogenaufbau rasch durchzuführen, sei es, daß die Fermentvorstufe in zu geringer Menge zugegen ist, sei es, daß der Aktivator unzureichend wirkt. Es ist aber auch denkbar, daß die Leber- und vielleicht auch andere Zellen den Traubenzucker nicht rasch genug eintreten lassen. Vielleicht ist die Zellwand so beschaffen, daß dem Durchtritt des Traubenzuckers Schwierigkeiten entgegenstehen. Ist die Zufuhr von Traubenzucker eine geringe oder erfolgt sie zwar in größerem Maßstabe, jedoch über eine längere Zeit verteilt, dann können die Zellen mit derGlykogensynthese Schritt halten. Sie verhindern dadurch das Zustandekommen einer Hyperglukoplasmie. Nur dann, wenn den Zellen auf einmal viel Glukose zugeführt wird, vermögen sie nicht rasch genug mit ihm fertig zu werden. Würde — Nr. 13 fliKjS) Zeitschr. f. Idiu. Med. 47. H. 1/2 (1902). Nr. 27, 28 (18<n)) F. Blmne»thal und //. Wolf: Zeitschr. f. kiiu. Med. 52. H. 3/4 (1904). Emil Abderhalden: Zeitschrift f. physiol. Chem. 85. 9.') (1913). ) Es sei auf die Werke: Houchardnl : Du diabete sucre. Paris 1888. Claude Bernard: Lecons sur le diabete. Paris 1877. Deutsche Ausgabe (Posuer) Berlin 1878. r. Frerichs: Über den Diabetes. Berlin 1884. r. Noorden : Haudb. d. Pathol. d. Stoffwechsels. 2. Auti. 2. Ilirschwald. Berlin 1907; Zuckerkrankheit. 6. AuH. 1912. Naunyn: Diabetes melitus. 2. AuH. Wien 1906. R. Lepinc: Le diabete sucrd. Feli.\ Alcan. Paris 1909. Alfred GUion: Neuere l)ial)etesforschungen. Ergebn. d. inneren Med. u. Kinderheilkunde.' 9. 206' ü 91 2). Ludolf Knhl : Pathol. Physiol. 9. AuH. Carl mn Noorden und Hugo Salomon: Handbuch der F. C. W.Vogel. Leipzig 1918. Ernährungslehre. J. Springer, Berlin. 1920. hingewiesen. -) Vgl. S. 150. '( Vgl. liierzu //. Slanh : Zeitschr. f. klin. Med. 93. 89. 123 (1922). ; : — — —C — — — — • ~ — Knhlehyilratp. 1§1 der Traubenzucker nicht durch die Nieren ausgeschieden, dann würde in solchen Fällen schließlich die gesamte, den normalen Zuckerspiegel des Blutes überragende Glukosemenge ein Unterkommen finden. Die Xierenzellen greifen jedoch automatisch ein, sobald der Zuckergehalt des Blutes eine gewisse Grenze überschreitet. In solchen Fällen kann ein „geschwächtes", seine x\ufgaben nicht vollständig erfüllendes Organ sich wieder erholen, so daß diese Form des Diabetes melitus zur Ausheilung kommt. Eine besondere Ursache der Hyperglukoplasmie kijnnte unter Umständen dadurch entstehen, daß jene Zellarten, die (rlukose in Fett überführen, versagen, ."^ind die Glykogenspeicher gefüllt, dann wäre in diesem Falle kaum eine Möglichkeit gegeben, um eine weitere Zuckerzufuhr aus dem Blute abzulenken. Man hat gegen die Annahme, daß ein Versagen der Leberzellen bei der Glykogenspeicherung leichten Fällen von Diabetes melitus zugrunde liegen könne, eingewandt, daß die Leber die schwersten Veränderungen zeigen kann, ohne daß Zucker im Irin gefunden wird. Dieser Einwand ist nicht stichhaltig. ^Vir wissen, daß die Leber eine große Zahl von Funktionen ausführt, von denen jede bis zu einem gewissen Grade unabhängig von den übrigen ist und für sich allein gestört sein kann. So braucht nicht jede Erkrankung der Leber gerade die den Kohlehydratstoflwechsel regelnden Teile der Zellen zu treffen. Die Einschränkung der Assimilation.'^fähigkeit der Leber kann die mannigfachsten Gründe haben. Es kann sich um eine allgemeine Herabsetzung der Leistungsfähigkeit der Leberzellen handeln, oder aber es liegt eine besondere Störung vor. Sie kann z. B. einzig und allein dadurch bedingt sein, daß in den Leberzellen sich Bedingungen finden, die einem Glykogenaufl)au ungünstig sind. Ferner ist der Fall möglich, daß umgekehrt dem (ilykogenabbau Hindernisse entgegenstehen. Der Glykogengehalt eines Organes beweist noch lange nicht, daß die betreffenden Zellen im gegebenen Augenblick Glykogen zu bilden und auch abzubauen vermögen. Es kann sehr wohl der Fall eintreten, daß einmal gebildetes Glykogen liegen bleibt, und dadurch die Speicher für einen weiteren Zufluß versperrt sind, weil der Abbau des schon vorhandenen Polysaccharids gestört ist. Das im einzelnen Falle nachgewiesene Glykogen kann schon sehr lange an Ort und Stelle lagern! gewilj Ein weiterer Anlaß zur Entstehung einer Hyperglukoplasmie könnte dadurch gegeben sein, daß in den Beziehungen der Leber zum Zuckerzentrum an irgend einer Stelle eine Störung eingetreten ist. ^j Normalerweise ist der Zuckerstoffwechsel der Leber in feinster Weise geregelt. Wir haben früher schon betont, wie fein das Zusammenspiel der einzelnen Faktoren im Kohlehydratstoffwechsel sein muß, damit immer im richtigen Augenblick ein bestimmter \'organg einsetzt und ein anderer aufhört. Es ist denkbar, daß das Zuckerzentrum bei gewissen Zuständen leichter anspricht als sonst. Wir haben allen Grund anzunehmen, daß keine Zelle des tierischen Organismus mit Ausnahme derjenigen, die rein mechanische Funktionen erfüllen das Zusammenspiel mit anderen Zellen je ganz aufgibt. Die Zellen müs.sen stets bereit sein, um auf die ver.schiedensten Anforderungen entsprechend antworten zu können. Sie dürfen nicht auf bestimmte Nachrichten hin si(;h erst vorbereiten. So werden auch dem — ) Vgl. s. i.-i.sff. 1^- Vorlesuug. 1^2 Ziickcrzentrum mit seinen Zellen ununterbrochen Reize zui>eleitet. Sie sind bis zu einer gewissen Grenze zu schwach, um einen Erfolg zu haben. Erst wenn die „Reizschwelle'' erreicht oder überschritten ist. macht sich das Zuckerzentrum geltend. Bald wird ein Abbau, bald ein Aufbau von Glykogen eingeleitet. Das Zuckerzentrum kann nun unter Umständen sich in einem labileren Zustande befinden. Es genügen Reize, die weit unter der normalen Schwelle liegen, um die Leberzellen zu veranlassen, ihr Glykogen abzubauen, ja es ist denkbar, daß das Zuckerzentrum sich dauernd in Erregung befindet. Die Leberzellen sind dann als Glykogenspeicher mehr oder weniger ausgeschaltet. Diese Art der Hyperglukoplasmie mit nachfolgender Glukosurie würde etwa jenem Zustande an die Seite zu stellen sein, den wir nach erfolgreichem Zuckerstich eintreten sahen. Auch diese Art des Diabetes könnte in Heilung übergehen, sobald das Zuckerzentrum sich wieder auf die Norm einstellen würde. Selbstverständlich könnte die Ursache einer derartigen Hyperglukoplasmie auch an anderen Stellen der Wechselbeziehungen zwischen Zuckerzentrum und Leber sich finden. Man hat sogar an eine allgemeine erhöhte Erregbarkeit des N. sympathicus- gebietes gedacht i) und manche Fälle von Diabetes melitus ganz diesem Nervensystem zur Last gelegt. Die Möglichkeit, dal) tatsächlich vom X. sympathicus aus eine Abartung des Zuckerstoffwechsels bedingt werden könnte, würde um so verständlicher werden, wenn die vorliegenden Angaben, wonach im Zwischenhirn -) und in vielleicht noch höher organisierten Gehirnteilen sich Zentren finden, die den Kohlehydratstoffwechsel beherrschen, weitere Bestätigungen finden. Eigene Beobachtungen stützen die gemachten Befunde. Schließlich könnte man auch an ein eigentliches Sympathicuszentrum denken, das alle Bahnen des N. sympathicus direkt oder indirekt beherrscht. Das sogenannte Zuckerzentrum in der MeduUa oblongata wäre in diesem Falle entweder ein Zentrum zweiter Ordnung oder aber der Zuckerstich trifft Bahnen, die vom Zentrum erster Ordnung herabsteigen, um ins Rückenmark und von da durch die vorderen Wurzeln zu den sympathischen Ganglien bew. zum Grenzstrang zu gelangen. Die Störung einer einzigen Stelle, nämlich des Zentrums erster Ordnung, würde dann die mannigfaltigen Folgen erklären. Diese Möglichkeit fordert zu weiteren Studien über die zentralen Sympathicusbahnen auf. Da das Zuckerzentrum Beziehungen zur Nebenniere hat, so besteht auch die Möglichkeit, daß von ihr Störungen des Zuckerstoffwechsels ausgehen können, und man von einem adrenalen Typus des Diabetes melitus sprechen kann. Alle in der Vorlesung \'H erörterten Beziehungen zwischen Zuckerzentrum, Nebennieren und Leber kommen hier in 15etiacht. P)ei der Besprechung der Zuckerstichglukosurie gedachten wir auch des lünflusses von intravenös zugeführter Kochsalzlösung. AVir lernten eine Form von Glukosurie kennen, bei der die vermehrte Durchlässigkeit der Niereneitit hellen für Glukose eine Rolle spielte. I>esonders klar liegen die V'erhältnisse bei den Seite 164 angeführten Beobachtungen, wonach es möglich ist, die Dichtigkeit der Glomerulusepithelien für(ilukose innerhall) gewisser Grenzen nach Belieben zu erhöhen oder zu erniedrigen ) Claude Bernard schrieb dem Nervensystem schon eine große Rolle beim Diabetes melitus zu. Vgl. auch W. Falta : Zeitschr. f. Icliii, M(m1. m. Vi. h^^ (HM)8). -) H. Aschner: I'ßiuier^ Archiv. 146. 114 (1121 ^Kohlehydrate. lyß Es ist wohl möglich, daß es Fälle von Diabetes gibt, bei denen gar keine Hype iglukoplasmie vorliegt, sondern die Zuckerverluste nur dadurch bedingt daß die Durchlässigkeit der Nieren für Zucker verändert ist. In der Tat sind solche Fälle von Diabetes beobachtet worden. Man spricht von sind, einer renalen Form des Diabetes.') Wir kennen weiterhin Formen von Diabetes melitus, die so viele gemeinsame Züge mit jener Art von Hyperglukoplasmie mit nachfolgender Glukosurie aufweisen, die nach vollständiger Entfernung der Pan- kreasdrüse auftritt, daß man mit Recht vermutet, daß vor allem beiden schweren Arten von Zuckerharnruhr die genannte Drüse so verändert ist, daß sie ihre Rolle im Zuckerhaushalte des Organismus gar nicht oder doch nur unvollkommen erfüllen kann. In der Tat konnte man in manchen Fällen von Diabetes melitus anatomische Veränderungen in der Pankreasdrüse nachweisen -), aber selbst, wenn der pathologische Anatom keine Xeränderungen findet, ist es denkbar, daß trotzdem funktionelle Störungen vorhanden sind. Der Anatom sieht nur gröbere, morphologische Veränderungen. Er vermag zurzeit, Feinheiten in der Änderung der Struktur der Zellen nicht festzustellen. Das, was der pathologische Anatom beobachtet, ist sehr oft nur das Ende eines langwierigen Vorganges, der von feinsten Anfängen an fortschreitend, allmählich zu schweren, direkt sichtbaren Zellveränderungen geführt hat. Niemals darf man jedoch erwarten, daß bei jedem Diabetes melitus die Pankreasdrüse beteiligt sein muß. Gewiß ist eine Tatsache, der wesentlichsten Ursachen der daß trotz großer Anstrengungen die Erforschung des Diabetes melitus wenig tief in das "Wesen und die Ursachen dieser Stoffwechselstörung eingedrungen ist, darin begründet, daß man dieses ohne Zweifel recht mannigfaltige und nur durch das gleichartige Symptom der Glukosurie zusammengehaltene Krankheitsbild immer wieder von einem einzigen Gesichtspunkt aus zu betrachten bestrebt ist. Es gibt keinen einheitlichen Diabetes melitus! Aus diesem Grunde kann keine Therapie auf alle Fälle passen. Unser Bestreben muß darauf gerichtet sein, das. was unter dem Namen Diabetes melitus zusammengefaßt worden ist, in Gruppen mit gemeinsamer Ursache und entsprechendem Verlauf zu trennen. Leider verfügen wir immer noch nicht über genügend exakte Untersuchungen über den Zuckerhaushalt der Diabetiker, ja es fehlt sogar fast ganz an Feststellungen über das ^'erhalten des Blutzuckers, Zumeist ist nur das Reduktionsvermögen des enteiweißten Blutes bestimmt worden. Es wäre von größtem Interesse, zu erfahren, ob es wirkUch in allen Fällen Glukose entspricht. 3) Gründliche Forschungen auf diesem Gebiete würden sicherlich unsere Kenntnisse über das Wesen der verschiedenen Diabetesformen wesentlich föidtrn. Sie haben bereits zur Auffindung von Azetaldehyd im Blutplasma geführt.-') Vielleicht wird man auch auf Formen treffen, bei denen andere Organe, z. B. die Schilddrüse, die Nebennieren, die Hypophyse usw. primär beteiligt sind. Leo FoUak : Aich. f. cxperim. l'ath. ii. Phaimak. (i4. 415 John R. Mnriin uud f. kliu. Med. 102. I(i9 (1911). Walter L. Xiles: The Americau J. of the medical Sciences 153. 79 (1917). ^) (1911). Vgl. hierzu u. a. — W. Weiland: Arch. -) •') ) — Vgl. hierzu die Literatur S. 176. Vgl. u. a. W. Stepp: Z. f. phjsiol. Chemie. 97. 213 (191(i). Vgl. Literatur S. 135. ^^- Vorlesnnff. 134 Es ist verstandlich, dali die Erforsciiun<j- des Wesens des Diabetes unter dieser Vielheit der möglichen Ursachen leiden muß, und insbesondere auch deshalb, weil die verschiedenartigen F'ormen dieser Störungen klinisch noch gar nicht geschieden sind. Wir können Erfahrungen, die an einem Fall gewonnen sind, unter diesen Umständen gar nicht auf andere übertragen. Die erste Frage, die wir entschieden raelitus haben möchten, bauen ist die: oder nicht? Kann der Diabetiker Traubenzucker ab- beantwortet worden. Es jedoch nach den neuesten Feststellungen beim experimentellen Pankreasdiabetes i) und beim Diabetes melitus des Menschen mehr dafür-), dalJ der Diabetiker Glukose abbauen kann, als dagegen. Immerhin muli mit der Möglichkeit gerechnet werden, daß es Fälle von Diabetes gibt, bei denen der Glukoseabau wenigstens quantitativ gestört ist. ^) Sie ist sehr verschieden spricht Die Frage, ob zugeführter Traubenzucker verwertet wird oder nicht, läßt sich auf verschiedenen Wiegen prüfen. Einmal kann man die Menge ausgeschiedenen Zuckers vor und nach der Zufuhr von Kohlehydrat bei gleichbleibender Grundnahrung verfolgen. Diese Art der Feststellung des V^erhaltens von (rhikose im Organismus des Diabetikers enthält viele Möglichkeiten von Fehleniuellen. Einmal kann zugeführter Zucker im Darm durch Bakterien abgebaut werden. Ferner wissen wir, daß die Zuckerausscheidung bei vielen Diabetikern ganz außerordentlich stark durch psychische Momente beeinflußbar ist. Eine Erregung kann sofort ein Ansteigen der Glukosurie zur Folge haben. Wir haben S. 158 schon erfahren, daß auch bei Tieren Schreck und Angst den Zuckergehalt des Blutes des beeinflussen. Einwandfreier ist die (Verfolgung des Gaswechsels unter Be- stimmung des respiratorischen Quotienten. Unter diesem verstehen wir das Verhältnis der ausgeschiedenen Kohlensäure zum aufgenommenen Sauerstoff: CO,:!).,.) Er ist beim Abbau von Kohlehydraten zu Kohlensäure und Wasser genau = 1. Die Glukose ist sauerstoffreich. Die sauerstoffärmeren Fettsäuren und Aminosäuren Bausteine der Fette bzw. der — — Eiweißstoffe brauchen zur Umwandlung in die Stoffwechselendprodukte eine größere Sauerstoffzufuhr. Es ist daher der respiratorische Quotient beim Abbau dieser Verbindungen kleiner als 1. Führt man einem Diabetiker Kohlehydrate zu und nähert sich der respiratorische Quotient 1 oder würde er gar gleich 1. dann dürfte man auf eine Verwendbarkeit der Glukose schließen. Bleibt er jedoch unverändert unter 1, dann würde das dafür sprechen, daß das Vermögen. Zucker zu verwerten, verloren gegangen ist. Die in dieser Richtung ausgeführten Versuche ergaben, daß es Diabetesfälle gibt, bei denen unzweifelhaft Traubenzucker abgebaut worden kann. >} Es tauchen selbstverständlich auch hier wieder genau die gleichen Fragen auf. wie wir sie zur Erklärung der Ausfallserscheinungen beim experimentellen Panki-easdiabetes erörtert haben. Von welcher Stelle geht die Störung aus? Ist die Pankreasdrüse primär beteiligt, oder versagt sie, weil ein ') ) =) ) ) (1918). Vgl. S. 17.S. Vgl. u. a. Elliot P. Joslin: Arch. of iutprnal. Med. 16. ()0:3 (191."! ). Vgl. auch R. T. Woodyatf: J. of hiolog. ehem. 14. 441 (I91.S). Wir kommen auf diesen Quotienten noch eingehend zurück. Vgl. auch S. 120. Vgl. u. a. S. Bernsfnn und W. Falla: Dfnitsches; .\rchiv f. klin. Medizin. 127, 1 Hier findet sich weitere Literatur. — Kohlehydrate. \ ^5 anderes Organ ihr die für Itestimmte Funlctionen notwendigen Inkretstoffe nicht Uefert V Haben wir auch beim Diabetes meUtus eine Störung der dykogenbildung und -aut'bewahrung in der Leber vor uns? f'ür diese Annahme sprechen manche Beobachtungen. Eine Zeitlang huldigte man der Vorstellung, daß beim Diabetiker das Oxydationsvermögen ganz allgemein gestört sei. Seitdem wir wissen, daß der Zuckerabbau zunächst ohne jede Sauerstoffzufuhr durch Spaltungsvorgänge erfolgt, kommt einer solchen Annahme zum vornherein wenig Bedeutung zu. Es haben aber auch direkte Versuche ergeben, daß der Diabetiker kein allgemein herabgesetztes Oxydationsvermögen aufweist.^) Allerdings läßt sich zeigen, daß in schweren Fällen von Diabetis mehtus die Atmung der roten Blutkörperchen und vielleicht auch von anderen Zellarten stark herabgesetzt ist. 2) Sie läßt sich durch Zugabe von aus Hefe und Kleie gewonnenen Stoffen anfachen. Diese noch vereinzelte Beobachtung führt zu der \'ermutung, daß es Fälle von Diabetes melitus gibt, bei denen vielleicht infolge der zur Bekämpfung derHyperglukoplasniie und anderer Erscheinungen eingeleiteten einseitigen Ernährung sich ein Zustand hinzugesellt, wie wir ihn experimentell bei Tieren hervorrufen können, w^enn wir sie mit künstlich zusammengesetzten Nahrungsstoffgeinischen oder einseitig mit bestimmten Nahrungsmitteln, wie geschliffenem Keis, ernähren. Auch hierbei leiden die Oxydationen in den Zellen. Wir kommen später auf dieses Gebiet noch eingehend zurück. Es sei an dieser Stelle nur auf diesen Zusammenhang hingewiesen und darauf aufmerksam gemacht, daß manche therapeutische Maßnahme beim Diabetes melitus vielleicht hauptsächlich in dem Sinne wirkt, daß sie die einseitige Ernährung durchbricht und dem Organismus jene Stoffe zuführt, die zur Durchführung der Oxydationen in den Zellen notwendig sind. Wir haben schon auf S. 120 ff. darauf aufmerksam gemacht, wie verwickelt die einzelnen Fermentvorgänge in den Zellen verlaufen, und wie zahlreich die Stoffe sind, die zu ihrer Durchführung erforderlich sind. Es sei an die Fermentvorstufe, den Aktivator und das Koferment erinnert. Alle diese Produkte genügen für sich auch noch nicht. Es bedarf in feinster Weise eingestellter Bedingungen, damit der einzelne Fermentvorgang zustande kommt. Gewiß lohnt es sich, den Diabetes melitus und vor allem alte und schwere Fälle auch vom Gesichtspunkt der Zufuhr aller zur Durchführung des Stoffwechsels erforderlichen Nahrungsstoffe und vor allem auch der noch unbekannten (vgl. Band H, Vorlesung XXH) zu erforschen ( Das Studium der schweren Fälle von Diabetes hat zu einer weiteren wichtigen Feststellung geführt, die im Einklang mit Beobachtungen beim experimentellen Pankreasdiabetes steht. Es konnte nämlich in einwandWeise festge.stellt werden, daß die Zuckeraus Scheidung auch dann anhält, wenn in der Nahrung keine Kohlehydrate zugeführt werden.») Zunächst dachte man an die Zuckervorräte des Organismus, doch zeigten exakt durchgeführte Versuche bald, daß auch bei der Annahme freier ') Vgl. z. B. O. baunigarten : Archiv f. experiui. Path. 11. Parmak. 2. Ö3(1905). klin. Med. 19. — 0. Schultzen: Berliner klin. Wocheuschr. Nr. 85 (1875). — M. Nenki luul Sieher: Jouru. (1890). f. prakt. Chemie. N. F. 26. 35 (1882). - H. Leo: Zeitschr. — W. Weintraud nnd E. Laves: Zeitschr. f. physiol. Chem. f. 19. GOS. 629 (1894). — ) Emil Abderhalden und E. Werf heimer : Fßiit/eni Archiv. 194. ()47 (1922). Emil Abderhalden: Klinische Wochenschr. 1. Nr. 22 (1922). ') L. Mohr: Vgl. u. a. //. Lüthje: Deutsches Arch. f. klin. Med. 79.498 (1904). Zeitschr. f. klin. Med. 52. .337 (1904). A. (iiqoii und Massini: Deutsches Archiv, t. klin. Med. 96. 107 (1908). — — IX. Vorlesung. 186 solcher ein großer Teil des ausgeschiedenen Zuckers in seiner Herkunft unerklärt blieb. Man begann ferner die Menge des im Harn zur Ausscheidung gelangenden, gebundenen Stickstoffs (N) mit der des ausgeschiedenenTraubenZuckers (Dextrose D) zu vergleichen und fand bald Beziehungen zwischen den beiden Größen N und D. Alle Beobachtungen zwangen schließlich zu der Annahme, daß Zucker sich aus Eiweiß bzw. aus Aminosäuren bildet, d.h. man kam zu dem gleichen Schlüsse, den wir bei der Besprechung der Frage nach der Herkunft des Harnzuckers bei den der Pankreasdrüse beraubten Tieren gezogen haben. — wieviel Zucker einem Gramm Eiweißstickstoff und Landergyen'^ finden, daß auf 1 9 N etwa 6 q GluDiese Berechnung- ist nur für den Fall zutreffend, daß Man hat berechnet, entspricht. G'igon'^) kose kommen. ein Abbau von Glukose ausgeschlossen ist. Wird jedoch ein Teil des Traubenzuckers vom Organismus verwertet, dann ist sie nicht anwendbar. Die Widersprüche, die auf diesem Forschungsgebiete hervorgetreten sind, sind ohne Zweifel auf ein Übersehen dieses Umstandes zurückzuführen. Kann nämhch der Diabetiker Kohlehydrate verwerten, dann bringt das ^'erhältnis von Stickstoff zu Glukose im Harn gar nicht zum Ausdruck, wieviel Zucker aus Aminosäuren hervorgegangen ist, weilder Feststellung eine wechselnde Menge von (ilukose entgeht. Auch hier drängt sich uns die Frage auf, ob die Tatsache, daß beim Diabetes im Harn Zucker erscheint, der von bestimmten Aminosäuren herstammt, eine Eigenart dieser Stoff Wechselstörung darstellt, oder aber, ob infolge des wenigstens teilweisen Versagens des Ginkoseabbaus ein ganz normaler, sonst verborgener Vorgang erkennbar wird. Aus den gleichen schon S. 172 angeführten (Jründen sind wir der Ansicht, daß die Überführung von Aminosäuren in Glukose über stickstofffreie Verbindungen des Dreikohlenstoffsystems etwas ganz Normales darstellt. Denkbar ist es, daß der ganze Vorgang quantitativ verschoben ist und beim Diabetes mehr Zucker aus Nichtzuckern bereitet wird, als es im allgemeinen unter normalen A erhältnissen der Fall ist.'^) Für die Umwandlung von Abbaustufeu aus Aminosäuren in Glukose dürfte die folgende Überlegung einen Hinweis geben. Der tierische Organismus kann große Mengen von Kohlehydraten in Form von Glykogen und von Fett speichern. Ebenso kann er zugeführtes Fett, für das er gerade keine Verwendung hat, ablagern. Dagegen vermag er nicht Eiweili oder p]iweißabkömmlinge in größeren Mengen aufzubewahren. Jede Zelle hat eine bestimmte Menge ümsatzeiweiß zur Verfügung. Darüber hinaus kann sie neben den eigentlichen Zelleiweißstoffen, die am Bau der Zelle Anteil haben, keine Proteine unterbringen. Auch Aminosäuren können nicht in wesentlichen Mengen gespeichert werden. Die llierfiUirimg dieser Verbindungen in stickstofffreie Produkte unter Abspaltung der den Stickstoff tragenden Aminogruppe und die Bildung von Glukose aus diesen Umwandlungsprodukten ermöglicht es dem Organismus, indirekt EiweißA. Gigon: Deutsclies Arcli. f. kliu. Med. 97. 37(5 (1909). Landerffren: Nord. med. Arkiv. Abt. 2. Kr. 10 (1910). Es ist damit uiclit der erhöhte Aminosi'uireumsatz gemeint, der in den P'ällen notwendig wird, in denen für die Bestreitung des Energiebedarfes nicht genügend andere Quellen zur Verfügung stehen, vielmehr ist daran gedacht \vord(Mi. daß von den Zucker liefernden Aminosäuren ein höherer Prozentsatz als unter ^formalen Verhaltnissen diesem Umbau unterworfen wird. 1) ^) '•^) Kohlelivdrate. ] ^7 ahköinmlini^e im Körper zurückzuhalten. Über die gebildete Glukose kann natürlich auch die Fettbildung einsetzen, ^o wird verhindert, daß durch die Zufuhr von Eiweiß und seinen Abbaustufen der Zellstaat aus Mangel an Keservestätten für diese Produkte gezwungen wird, sie vollständig abzuhauen oder unausgenutzt aus dem Körper zu entlassen. Von diesen (Gesichtspunkten aus wird uns verständlich, daß die Bildung von (ilukose aus Nichtzuckern ein den Körperzellen ganz vertrauter A'organg ist und keineswegs einen für den Diabetiker und das pankreaslose Tier besonderen A'organg darstellen kann. Als Quelle für Zucker sind auch die Fette angesprochen worden. Es ist noch unentschieden, ob das der Fall ist.^) Es liegen noch eine große Reihe von Beobachtungen aller Art an Diabetesf allen vor. \on größter Bedeutung ist der schon S. 185 erwähnte Befund, wonach im Blut und im Harn des Diabetikers Azetaldehyd und ferner allerdings nur in schweren Fällen und auch dann nur in Spuren Aldol CH^ . CH(OH) . GH., . C\jj nachgewiesen werden konnte.-) Im Harne sind ab und zu neben Glukose auch andere Kohlehydrate aufgefunden worden, z. B. Maltose. Lävulose, Pentosen, doch ist der Beweis, daß jene Zuckerarten wirklich vorlagen, nicht immer ausreichend geführt. Mehr Sicherheit liegt in der Beobachtung, daß Diabetiker Lävulose verwerten können. 3) Bestätigt sich der Befund ganz allgemein, wonach der Fruchtzucker vom Diabetiker besser verwertet wird als Glukose, dann ergeben sich neue Fragestellungen. Im allgemeinen wird angenommen, daß die Lävulose bereits im Darmkanal oder aber auf dem Wege der Resorption in Dextrose umgelagert wird. Zur Glykogensynthese kann nur (jlukose als Ausgangsmaterial dienen. Der Umstand, daß nun die Fruktose beim Diabetiker eine Sonderstellung einnehmen soll, eröffnet neue Probleme. Sollte die Lävulose wir führen sie uns vornehmlich in Form von Rohrzucker zu auch unter normalen Verhältnissen einen Stoffwechsel für sich haben V Es ist dies wenig wahrscheinlich. Nun sind die Angaben über die Verwertung der Lävulose bei Diabetes durchaus nicht einheitlich. Bei einer eingehenden Verfolgung dieses scheinbar nicht so wichtigen und in Wirklichkeit doch sehr bedeutsamen Problems stößt man vielleicht auf eine Erklärung der noch unklaren Punkte. Es gelingt vielleicht zu zeigen, was fü)- Typen von Diabetikern Lävulose verwerten können, und welche nicht. Einstweilen können wir die gemachten Beobachtungen nur mitteilen. — — Wir bemerken auch hier wieder, daß beim Tieferschürfen in den un.s durch die im Kohlehydratstoffwechsel gestörten Organismen offenbar gewordenen Fi'agestellungen sich immer wieder Punkte ergeben, die uns unbefriedigt lassen. Es hat sich in der Forschung immer als sehr lohnend herausgestellt, solchen Unstimmigkeiten nachzugehen. Sie beweisen immer, daß irgend eine Beobachtung oder auch mehrere unrichtig gedeutet worden sind. Oder aber es liegen Fehler in den P'eststellungen vor. ^) Vgl. u. a. .7. K. Parnas uud //. Wagner: Biochem. Zeitschr. 127. 5.') (1922) Krof/h und Joh. Lindhard: The Bioclioin. J. 14. 290 (1920). ^) Vgl. die Literatur S. IBö. ') E. Külz: Beiträge zur Pathologie uud Therapie dos Diahetes melitus. Marburg 1874. S. 130. Worin Mii/Irr: rjlügerii Archiv. 34. Ö7(; (1884): 36.172 (18SÖ). Franz llofmeisf er: Arch. f. experim. I'ath. u. Pharm. 25. 240 (1889). —John Jfai/0. Minkoirski: Arch. f. exi>erim. crafl : Zeitschr. f. plivsiol. Chem. 19. 137 (1894). U. Sandnieyer: Zeitschr. t. Biol. 31. 12 (31) (1S94). Path. u. Pharm. 31. 108.(1898). ('. Fritz Voif: Zeitschr. f. Biol, 29. 147 (1892). A. Socin: Diss. Straßbnrg 1894. — — — .(. — — — — IX. Vorlesung. Igg Schließlich Kohlehydrate. müssen wir noch kurz \'orstellungeu über das Zustande- kommen der Hyperglukoplasmie und der Glukosurie streiten, die zwar immer noch da und dort auftauchen, in Wirklichkeit aber überholt sind. So dachte man daran, daß normalerweise im Blutplasma Glukose gespalten wird.') Diese (xlukolyse sollte beim Diabetes gehemmt sein. Nun ist erwiesen '-). daß wohl die Blutzellen Fermente besitzen, die Traubenzucker abbauen können, nicht aber das Plasma. Eine Zuckerzerstörung im Blute kommt sicherlich für die ganzen Probleme, die wir erörtert haben nicht in Frage. Die Blutzellen haben ihien eigenen Kohlehydratstoffwechsel, der sich in ihnen selbst vollzieht. Für die Erklärung der Glukosurie wurde hartnäckig die Hypothese verteidigt, daß im normalen Blute der Zucker nicht frei, sondern in Bindung zugegen sei. wenigstens sollte der größte Teil davon festgelegt sein.'M Diese Annahme wurde gemacht, um zu erklären, weshalb die Niere normaler W'eise keinen Zucker ausscheidet. Beim Diabetes sollte gebundener Zucker frei werden, und es deshalb zu seiner Entfernung aus dem Blute durch die Nierenepithelien kommen. Wir wissen jedoch jetzt genau, daß es im Blute keine in Betracht kommenden Mengen gebundenen Zuckers nur Glukosamin ist als Baustein von bestimmten Eiweißkörperu gibt ferner haben die auf S. 164 beschriebenen Versuche klar und festgelegt deutlich erwiesen, daß die Niere auch freien Zucker nicht durchläßt, sofern — — die richtigen , Bedingungen getroffen sind. Wir waren genötigt, diese unund immer wieder auf- richtigen Vorstellungen zu streifen, weil sie immer tauchen. Schließlich sei noch die wichtige Frage gestreift, ob beim Diabetiker Gewebe reicher an Zucker sind als beim normalen Individuum. Gibt neben der Hyperglukoplasmie eine Überschwemmung der Zellen mit Traubenzucker? Lassen sich von ihr aus manche Störungen, die beim Diabetes auftreten, erklären? Es Hegen in dieser ftichtung noch fast keine eindeutigen Versuche vor. Es scheint jedoch, als wären beim Diabetes die Gewebe nicht zuckerreicher als unter normalen Verhältnissen.) Es ist von größter Bedeutung, daß dieser Frage weiter nachgegangen wird. die es B. Lepine: Le diabete sucr^. Felix Alcau. Paris 1909. Bona uud A. Döblin : Biochem. Zeitschr. 32. 489 (1911). P. Bona und J. J. Macleod: Journ. of. biol. Chem. 15. 497 (1913). F. Arnheim: Ebenda. 48. .3.') (1913j. ') 2) r. ') Vgl. die Literatur S.143, 144. Vgl. Walter \V. l'almcr: .1. of biol. ) — — Chem. 30. 79 (1917). Vorlesung X. Kohlehydrate. IX. der Bildung Azetonkörper. Phlorhizinglukosurie. Herkunft hydrate im tierischen Organismus. der Kohle- In schweren Fällen von Diabetes melitus nimmt der Harn häufig einen eigentümlichen, obstartigen Geruch an, der schon den alten Ärzten aufgefallen ist. Die Ursache dieser Erscheinung ist. wie bereits C. Gerhardt ^) vermutete und später Tollens-) und v. Jaksch^) klar erkannten, die Azet- essigsäure (CH3 CO) CH2 COOH. Neben ihr findet man Azeton CH3 CO CH3 und. wie Stadelmann) und Minkowski'^) zeigten, in vielen CH (OH) CR, COOH. Alle drei VerFällen ß-Oxybuttersäure CR, bindungen stehen, wie ein Blick auf ihre Zusammensetzung lehrt, in direktem Zusammenhang. Die Azetessigsäure entsteht ohne Zweifel durch . . . . . . < . . )xydation aus der ß-Oxvbuttersäure CH, [i CH, CH OH + . = CO + H. 0. X CH, CH, C( )0H ß-Oxybuttersäure COOH Azetessigsäure. Gerhardt: Wiener med. Presse. Nr. 28. 673 (1895). Tollens: LieOigs Annaleii. 209. 30 (1881). 3) B. r. Jaksch: Ber. d. Deutscheu Cliem. Gesellsch. 15. 1496 (1882). Vgl. auch W. Petters: Prager Vierteljahrschr. 55. 81 il8ö7); Zeitsehr. f. phvsiol. Chemie. 7. 485 Ferner M. Ceresole : Ebenda. 15. 1326 (1882). (1883). ) E. Sfadelmann: Archiv f. experim. Pathol. u. Pharm. 17. 419 (1883). Vgl. auch E. Küh: Zeitsehr. f. Biol. 20. 165 (1884). ) 0. Minkowski : Zeutralbl. f. d. med. Wissensch. 242 (1884) und Archiv f. experim. Pathol. u. Pharm. 18. 35 u. 147 (1884). K. Külz: Ebenda. 18. 291 (1884) und Zeitschi, >) C. 2) — — — — — 0. Minkoirski : Archiv f. experim. 165(1884) und' Ebenda. 23.329(1887). Pathol. u. Pharm. 31. 183 (1893). T. Araki: Zeitschrift f. phvsiol. Chemie. 18. 1 f. Biol. 20. (1894). — X- Vorlesiiug. 190 Dieser Vorgang ist umkehrbar, denn es kann sich umgekehrt durcli Reduktion aus Azetessigsiiure (i-()\ybuttersäure bilden, ja es scheint, dali der letztere Vorgang sich leichter vollzieht als der erstere. ^l Aus der Azetessigsaure kann man sich leicht durch Abspaltung von Kohlensäure Azeton entstanden denken: CO) CUo COOR = CR, Azetessigsaure. (CH3 . . . . CH, + CO2. Azeton. ("() . Daß in vielen Fällen die ß-Oxybuttersäure im Harn fehlt, wäluiMhi beiden anderen Verbindungen vorhanden sind, ist nicht auffallend, denn wir können uns wohl vorstellen, daß erstere einmal der Oxydation entgeht, das andere Mal nicht. Die [i-Oxybuttersäure enthält, wie obige Formel zeigt, ein asymmetrisches () Kohlenstoffatom. d. h. sie ist optisch aktiv, und zwar kommt sie stets in der linksdrehenden Form zur Ausscheidung. Es ist vielfach versucht worden, die Herkunft dieser Verbindungen festzustellen und vor allem ihre Bedeutung für den Diabetes selbst zu ergründen. Vor allem ist bemerkenswert, daß das Vorkommen von Azeton im Harn nicht für Diabetes spezifisch ist. Es ist Azetonurie - richtiger ist die Bezeichnung Diazeturie, da meist nur Azetessigsaure vorhanden ist meist allerdings in beschränktem Maße, bei vielen fieberhaften Krankheiten beobachtet worden. Azeton und Azetessigsaure hat man auch bei langdauernder Inanition-) (Hunger, Kachexie) aufgefunden. In geringen Mengen wird Azeton besonders bei ausschließlicher Ernährung mit Eiw(Mß und Fett auch von Gesunden beständig ausgeschieden. Es verläßt übrit^cns nicht alles Azeton den Organismus durch die Nieren, ein Teil wird mir der Atemluft abgegeben. Man hat die ganze (iruppe der Verbindungen: Azeton, Azetessigsaure und ß-Oxybuttersäure auch Azetonkörper genannt. Es ist a priori nicht sehr wahrscheinlich, daß die Azetonkörper mir den Kohlehydraten in direktem Zusammenhang stehen und ihr Auftreten etwa anzeigt, daß ihr Abbau gestört ist. Viele Beobachtungen sprechen dafür, daß in schweren Fällen von Diabetes melitus der Abbau von Traubenzucker stark eingeschränkt ist. Trotzdem treffen wir unter Umständen So ist z. B. bei auf geradezu gewaltige Mengen von Azetonkörpern. einem Knaben die Ausscheidung von ;)4'2 // y-0.\ybuttersäure innerhalb von o Tagen beobachtet worden. Würde der Traubenzucker die Azetonkörper liefern, so wäre die erwälmte Beobachtung unerklärbar. Ferner ist festgestellt worden, daß man eine bestehende Diazeturie durch Verabreichung von Kohlehydraten vermindern, ja sogar unterdrücken kann. Verbindungen, die eine bestehende Diazeturie einschränken können, sind „antiketogene" die — , =^) — schrift. 27. 474 (1910). — L. Blum: Münchener med. Wochensohr. 57. 1451 (1910). — of Biol. Cheni. 97 D. Dabin: Journ. Americ. Med. Ass. 54. 1441 (1910): Ikikin: Ebenda. lOö (1910). (1910). — A. J. Wakemann und Jakscli: Über .\zctonuric und Diazeturie. Berlin lS9ö. — Friedrich Miilhr: experim. Pathol. ßriK/sc/i: Zeitschr. Berliner klin. Wochenschr. 428 (1887). — /•/'. ) 0. Neilbauer: V'erhandl. d. 27. Kongresses f. innere Medizin. 56() (llUi)). Friedinanii und Mause: Münchener med. Wochensclir. Nr. 34 (1910); Biocliem. Zeit- 8. .loiirn. //. //. -) 8. I). r. Tit. f. u. Iher. 1. 42f; (1905). ') Magnus- Lei: ji : Archiv 389 (1902). f. experim. l'athol. u. Pharm. ii-Oxyl)uttersäure -{-Azetessigsaure aus \Ludiriq Czapski: A. 77. 42. 143 (1899) und 45. — Bei einem anderen Fall schied ein 16.iähriger Diabetiker täglich ca. 100(/ 218 (1914)]. f. experim. Path. u. Plianii Kohlehydrate. 191 Körper gcuannt worden. Ferner hat die Erfahrung gelehrt, daß die völlige Weglassung der Kohlehydrate aus der Nahrung der Diabetiker oft direkt Diazeturie zur Folge hat, ja der Arzt ist oft genötigt, die an Kohlehydraten arme und an Eiweiß reiche Nahrung durch eine solche zu ersetzen, die Kohlehydrate in größeren Mengen enthält. Endlich wissen wir, daß jede Beschränkung der Kohlehydrate in der Nahrung zur vermehrten Ausscheidung von Azetessigsäure führen kann.i) Aus diesen Gründen hat man nach anderen Quellen für die Azetonkörper gesucht. In Betracht kommen die Fette und die Eiweißstoffe. Wir wollen das erhaltene Resultat gleich vorweg nehmen. Es ist festgestllt worden, daß Fettsäuren und ferner Aminosäuren Azetonkörper liefern können. Die Kenntnis der Konfiguration der 1-ß-Oxybuttersäure wäre für die Beurteilung ihrer Herkunft sicherlich von großer Wichtigkeit. Sie läßt sich in l-x-Aminopropionsäure überführen und ferner durch die Synthese mit d-Pr opyl engl ykol in Beziehung bringen.-) Was die Fettsäuren anbetrifft, so wissen wir seit den wichtigen Beobachtungen von Knoop'^) und ferner von Emhden^), daß der Abbau der normalen Fettsäuren in ganz charakteristischer Weise erfolgt. Es tritt näinlich in der Regel stets ein Verlust von zwei Kohlenstoffatomen in der Art ein, daß eine Spaltung zwischen dem y- und ß-Kohlenstoffatom erfolgt. Wir bezeichnen das dem Karboxyl benachbarte Kohlenstoffatom als das in z-Stellung befindliche. Das darauf folgende hat die ß-Stellung zum Karboxyl inne usw. Wir charakterisieren so jedes Kohlenstoffatom einer Kette durch Bezeichnung seiner Stellung zum Karboxyl. Durch einen Abbau der genannten Art gelangen wir, wenn wir von normalen Fettsäuren mit einer geraden Anzahl von Kohlenstoffatomeu ausgehen, schließlich zur Buttersäure. Diese kann leicht durch Oxydation am ß-Kohlenstoffatom in y-Oxybuttersäure übergehen und von ihr aus ist dann der Weg zur Bildung von Azetessigsäure und von Azeton offen. sCH^ Y CH3 I ßCH.OH 7. CH., X. Vorlesung. 192 Eine Fettsäure mit einer ungeraden Anzahl von Kohlenstoffatonien bei der gleichen Art des Abbaus natürlich niemals direkt Buttersäure liefern, weil auch die Abbauprodukte die ungerade Anzahl von Kohlenstoffatomen beibehalten. Die Annahme, dali die IJuttersäure als Abbaustufe höherer Fettsäuren eine (Quelle der [i-Oxybuttersäuie werden kann, hat durch mancherlei Beobachtungen eine große Sicherheit erlangt. Einmal konnte gezeigt werden, dali die Menge der Azetonkörper nach Eingabe von Buttersäure und von Kapronsäure ansteigt.') Ferner wurde beobachtet, daß nui' jene normalen Fettsäuren zur Vermehrung der Azetonkörper beitragen, die über eine gerade Anzahl von Kohlenstoffatomen verfügen.^) kann Immer wieder wurden auch die Eiweißkörper bzw. ihre Bausteine als Quelle der Azetonkörper bezeichnet. Es entwickelte sich bald ein lebhaftes Für und Wider diese Anschauung. Vor allem wurde gegen die Annahme einer Beteiligung der Proteine an der Bildung der Azetonkörper vorgebracht, daß ihre Menge in keinem direkten Verhältnisse zu der im Harn ausgeschiedenen Stickstoffmenge stehe. Wir werden später erfahren, daß die Eiweißstoffe im Gegensatze zu den Kohlehydraten Stickstoff enthalten. Dieser erscheint im Harn in Form bestimmter Verbindungen gebunden. Lange Zeit war man der Ansicht, daß man den Ablauf des Eiweißstoffwechsels ausschließlich durch Bestimmung des im Harne erscheinenden Stickstoffs verfolgen könne. Jetzt wissen wir jedoch, daß nur ein Teil des Kohlenstoffs des Eiweißmoleküls gleichzeitig mit dem Stickstoff im Stoffwechsel sein Ende findet. Nach Abspaltung der den Stickstoff enthaltenden Gruppe bleiben Kohlenstoffketten im Organismus zurück, die noch alle möglichen Funktionen übernehmen können. Wir haben ja bereits den Übergang dieser Verbindungen in Zucker kennen gelernt.^) Da diese Kohlenstoffketten in engster Beziehung zu Fettsäuren stehen, ist die Möglichkeit gegeben, daß von ihnen aus auch eine Bildung von Azeton körpern erfolgen kann. Das ganze Problem der Entstehung von Azetonkörpern aus Eiweiß wurde erst in dem Augenblicke einer eindeutigen Beantwortung zugänglich, als man es auf die Bausteine der Proteine, nämlich auf die Amino- säuren übertrug. Wir werden später erfahren, wie Aminosäuren in den Körperzellen abgebaut werden. Hier mag, da wir die Struktur der Bausteine der Eiweißstoffe noch nicht kennen gelernt haben, der Hinweis genügen, daß vor allem Emhdcti) den Nachweis führen konnte, daß in der Leber aus ganz bestimmten Aminosäuren Azetessigsäure bzw. Azeton gebildet wird, Avährend andere P)austeine des Eiweißmoleküls keine Azetonkörperbildner sind. So liefert z. B. die a-Amino-isobutylessigsäure Azeton, wenn man sie durch die überlebende Leber hindurch leitet, während die a-Amino-isovaleriansäure unter den gleichen Bedingungen kein Azeton gibt. Ferner geht a-Amino-valeriansäure ') ./. Vgl. z. B. L. Schwarz: Deutsches Archiv f. in [i-Oxybuttersäure s) kliu. Mediziu. 76. über; 233 (1903). — Bär und L. Blum: Archiv f. experim. Pathol. u. Phann. 53. 89 a90fi); 59. 321 (1908); «2. 129 (1910). -) \gl. weitere Literatur bei A. Magnus-Levy: Ergebnisse der inneren Medizin und Kinderheilkunde. 1. 352 (1908). ^) Vgl- Vorlesung VIII und IX. 11. Salomon und F. Schmidt: Hofmeistern Beiträge. 8. 129(1906). ) fjr Emhden, S. Kertess: Zeitschr. f. G. Emhden und /•'. Kalberluh: Kbenda. 8. 121 (1906). physiol. Chemie. 106. 258 (1919). G. Emhden und A. Marx: Hofmeistern, Beiträge. 11. 318 (1908). — — '") Kohlehydrate. 193 a-Amino-kapronsaiire und 7.-Ainino-l)uttersäure dagegen liefern nicht. Die folgenden Formeln mögen veranschaulichen, wes- diese Säure halb sich die erwähnten Unterschiede finden CH3 CH3 X- Vorlesung. 194 Diese Studien über die Herkunft der Azetonkörper haben einen außerordentlich interessanten und wichtigen Einblick in Stoffwechselvorgänge bestimmter Zellen ergeben. Überall stoßen wir auf den stufenweisen Abbau der einzelnen Verbindungen, und an allen Stellen treffen wir auf Abbaustufen, die Beziehungen zu den verschiedensten Gruppen von Stoffen vermitteln. Je weiter ein Produkt zerlegt wird, um so mehr verliert es seinen besonderen Charakter. Ist das komplizierte, zusammengesetzte Molekül in seine Bausteine zerlegt, dann geben diese noch xluskunft über die Art jener zusammengesetzten Verbindung, aus der sie hervorgegangen sind. Nichts verrät jedoch mehr den feineren Bau des Ausgangsmateriales. Werden nun auch noch die Bausteine stufenweise zerlegt, dann gelangen wir zu Abbaustufen, die schließlich vollständig indifferent sind. d. h. die aus den verschiedenartigsten Verbindungen entstanden sein können. Diese Abbaustufen bilden den Ausgangspunkt von Synthesen verschiedenster Art. Namentlich die Verbindungen des Dreikohlenstoff Systems bilden eine wichtige Durchgangsstufe im Ab- und Aufbau der verschiedenartigsten Produkte. Viele Beobachtungen sprechen dafür, daß die Bildung der Azetonkörper auch so erfolgen kann, daß ein weiter abgebautes Produkt durch Synthese z. B. in ß-Oxybuttersäure und über diese in Azetessigsäure übergehen kann. E. Friedmann^) hat die wichtige Beobachtung gemacht, daß Azetaldehyd beim Durchleiten durch die Leber in Azetessigsäure verwandelt wird. Friedmann stellt sich diese Synthese, wie folgt, vor: Zwei Moleküle Azetaldehyd werden zu Aldol kondensiert: CH3 CHO -f CH, CHO AzetaldeAzetalde. . — >-CH;, . CH (OH) . GH., . GHQ. Aldol hyd hyd Aldol würde dann zu Azetessigsäure oxydiert: CH3 . — CH3 CH (OH) CH (OH) CH, CHO -f . >^ . . . GH., . COOH + —y ß-Oxybuttersäure Aldol CH3 . CO . GH., . COOH + H., O. Azetessigsäure. Es sei im Zusammenhang mit diesen Feststellungen auch auf die 135 erwähnte Bildung von Azetessigsäure aus Essigsäure verwiesen. Es liegen noch mancherlei Beobachtungen über die Bildung von Azetonkörpernausverschiedenartigen Verbindungen vor. In manchen Fällen ist der Beweis, daß eine bestimmte Verbindung zur Entstehung von Azetonkörpern führt, nur indirekt erbracht worden. Derartig erhobene Befunde sind immer vieldeutig. Zusammenfassend können wir hervorheben, daß die Azetonkörper auf verschiedenen Wegen sich bilden können. Sie entstehen durch Abbau von aus Fetten stammenden FettS. '^j und ferner aus bestimmten Aminosäuren. Ferner ist worden, daß auch eine synthetische Bildung der Azetonkörper möglich ist. säuren festgestellt ) '') E. Friedmann: Hofmeisters, Beiträge. 11. 202 (19Ü8). Vgl. hierzu auch G. Katsch: Deutsches Archiv f. kliu. Med. 127. 210 (1918); 134. 59 (1920). Kohlehydrate. ]9;) Erwähnen wollen wir noch, daß mancherlei Beobachtungen vorliegen, Fette als die Hauptquelle der Azetonkörper hinweisen. So läßt .>;ich feststellen, daß während des Hungers zu einer Zeit, in der der Organismus seine Ausgaben zum größten Teil auf Kosten seines Fettbestandes die auf die bestreitet, die Diazeturie ansteigt. Die Tatsache, daß Kohlehydratzufuhr die Ausscheidung der Azetonkörper einschränkt, findet ungezwungen darin seine Erklärung, daß die Kohlehydrate Fett sparen. Sie kommen an seiner Stelle zum Abbau. Aus dem gleichen Grunde kann man in manchen Fällen durch vermehrte Eiweißzufuhr die Bildung der Azetonkörper herabsetzen. Interessant ist in diesem Zusammenhange auch die in neuerer Zeit oft bestätigte Beobachtung von Schwarz^), wonach das Blut von an Diabetes Leidenden oft auch dann einen erhöhten Fettgehalt aufweist, wenn kein Fett aufgenommen wird. Offenbar findet ein vermehrter Fetttransport nach verschiedenen Zellen hin statt. Wenn wir bei Störungen des Stoffwechsels auf bestimmte \'erbindungen stoßen, dann fragen wir uns zunächst, ob sie infolge des veränderten Stoffwechsels auftreten, d. h. der Störung ihre Entstehung verdanken, oder aber, ob uns Produkte entgegentreten, die zu den normalen Stoff wechselprodukten gehören. Wir nehmen sie vielleicht wahr, weil sie zu langsam oder gar nicht weiter abgebaut werden. Es spricht manches dafür, daß die Azetonkörper Verbindungen darstellen, die auch normalerweise im Zellstoffwechsel gebildet werden, wenigstens gilt dies für die ß-Oxy buttersäure. Vielleicht wird diese im Organismus des Diabetikers in einer sonst ungewohnten Richtung weiter zerlegt. Wahrscheinlich ist jedoch, daß auch die Azetessigsäure nebst dem Azeton zu den normalen Stoffwechselzwischenproduktengehören. Normalerweise tritt Abbau zu Kohlensäure und Wasser ein. Die Störung beim Diabetes melitus besteht vielleicht darin, daß der Abbau des Azetons nicht zu Ende geführt wird, ja er bleibt oft zum Teil sogar bei den Vorstufen, der Azetessigsäure und der ß-Oxybuttersäure. stehen. Manches spricht auch für eine weitere Störung der Art, daß der Abbau der ß-Oxyder normalerweise vielleicht über verschiedene Stufen führt. der Hauptsache in die eine Richtung, nämlich über Azetessigsäure und Azeton gedrängt ist. Nicht unerwähnt wollen wir lassen, daß man wiederholt daran gedacht hat^), die ß-Oxybuttersäure und die Azetessigsäure könnten irgendwie mit jenen Umwandlungsprodukten zusammenhängen, die beim Umbau von Aminosäuren in Glukose auftreten. Es ist jedoch nicht geglückt, einen eindeutigen Beweis für diese Ansicht zu erbringen. •) buttersäure, in Durch Emhden und Friedmann ist festgestellt worden, daß die Leberzellen die Bildung der Azetessigsäure aus mannigfaltigen Quellen und auf verschiedene Weisen durchzuführen vermögen. Wir kennen somit ein bestimmtes Organ, das die genannten Verbindungen hervorbringen kann. Unbekannt ist bis jetzt, ob nicht auch andere (iewebe die gleichen L'm- — ') Leo Schwarz: Deutsches Archiv f. klin. Medizin. 76. 233 (1903). Vgl. auch Ivar Bang: Biocheni. Zeitschr. 94. S.^'.l (1919). -) Vgl. 0. Minkotrshi: Archiv f. experim. Path. u. rharin.31. 189(1893): f'tiiigers Archiv. 111. 13 (190(5). v. Noorden: Handb. d. Path. d. Stoff'wechsels. 2. Aiitl. 2. (1907). ^) P]s ist auch vermutet worden daß die ß-Oxybuttorsäuro eiu nicht Jiorraales Stoffwechselprodukt darstellt. Vgl. hierzu u. a. 7^'. AVMfeojivr.- Verhandl. d. 27. Kongresses f. innere Medizin. Gitstar Kmbden und Louis Michaud: Ilofmeisfrr^ Bei566 (1910). — , — träge. 11. 332 (1908). 13 X. Voilesuug. \',]{^ Wandlungen vollziehen können. Interessant ist die Feststellung von Embden und Latfes^), dal» die Leber eines seiner Pankreasdrüse beraubten Hundes bedeutend mehr Azetessigsäure bildete, als das entsprechende Organ eines normalen Tieres. Nicht unerwähnt wollen wir bei dieser Gelegenheit lassen, daß man gegen die Verallgemeinerung der Ergebnisse von Durchblutungsversuchen an überlebenden (Jrganeu Einwände erheben kann. Wir wissen nicht, wie lange die einzelnen Zellen des betreffenden Organes in normalen Grenzen weiter tätig sind. Ferner sind ganz sicher die ^'ersuchsbedingungen den natürlichen Verhältnissen nur ganz roh angepaßt. Wir leiten eine bestimmte Verbindung längere Zeit an den gleichen Zellen vorbei. Normalerweise würde sie vielleicht unverändert mit dem Blute fortgeführt. Wir zwingen vielleicht das Organ zu Eingriffen, die es sonst in dem Umfang nicht ausFerner mrd unter normalen Verhältnissen das gebildete Produkt führt. sofort in irgend einer Weise verwertet. Es bleibt nicht liegen. Endlich muß immer wieder darauf hingewiesen werden, daß unter normalen Verhältnissen mehrere Organe zusammenarbeiten. Fehlen die Wechselbeziehungen zwischen den einzelnen Organen, dann ist es wohl denkbar, daß eine bestimmte Art des Abbaus undurchführbar ist und nun ein anderer Weg eingeschlagen wird. Derartige Bedenken sind durchaus berechtigt. Soll ein Ergebnis eindeutig sein, dann darf es sich nie auf nur eine N'ersuchsanordnung stützen. Wenn die Beobachtungen am gesamten Organismus und diejenigen am einzelnen überlebenden Organe sich decken, dann wäre es gesucht, wollte man nicht die einfachste Erklärung der gemachten Beobachtung in den Vordergrund stellen. Nun stehen die Feststellungen Emhdens und seiner Schüler in bestem Einklang mit Fütterungsversuchen. Es liegt somit in diesem Falle kein Grund vor, daran zu zweifeln, daß auch im gesamten Organismus der Leber eine wichtige Rolle in der Bereitung der Azetonkörper zukommt. Dem Vorkommen der Azetonkörper im Blute hat man lange Zeit eine wesentliche Bedeutung bei der Beurteilung des Verlaufes des Diabetes zugeschrieben. Man nahm an, daß die Anwesenheit der angeführten Säuren die Alkaleszenz des Blutes herabdrücke und so an und für sich schwere Störungen hervorrufen könne.'-) Man führte deshalb den Namen Azidosis für den ganzen Zustand ein. Diese Bezeichnung ist unrichtig und irrefühiend. Man sollte sie verlassen, nachdem festgestellt worden ist, daß die Reaktion des Blutes infolge besonderer Einrichtungen selbst bei Anwesenheit großer Säuremengen nicht verändei-t wird. Wir wollen den Ausdruck Azetonoplasmie anstelle der Bezeichnung Azidosis treten lassen. Er soll daran erinnern, daß Azetonkörper im Blute zugegen sind. Die Azetonoplasmie ist in mehr als einer Beziehung von größtem Interesse. Zunächst ist es auffallend, daß die wahre 'Azidität des Blutplasmas, d. h. die Wasserstoffionenkonzentration selbst bei einem hohen Gehalt des Plasmas an Säuren nicht verändert zu sein braucht.'^) Es rührt dies daher, daß in diesem Substanzen enthalten sind, die jeder Änderung der Wasserstoffionenkonzentration den größten Widerstand entgegensetzen. Vor allem sind es die Karbonate, Phosphate und manche Proteine, die in dieser Richtung wirksam sind. Wir werden später auf den Mechanismus der ) -} ») Gustav Embden und Leone Lottes: Jlofmeiyters Beiträge. 11. 327 (1908). Hermann Straub: Deutsches Arch. f. klin. Med. 109. 223 (1913). H. Benedict: Pßügers Archiv. 115. 106 (1906). Vgl. hierzu Kohlehydrate. 197 ganzen Regulation zurückkommen (vgl. Band II. Vorlesung IX), hier mag der Hinweis genügen, daß für die Neutralitätsregulation die sehwachen Säuren Kohlensäure und Phosphorsäure und unter Umständen noch Eiweiß in Frage kommen. \) Vergleicht man den Kohlensäuregehalt von normalem und von „azidotischem" Blut, dann findet man. daß das letztere weniger von dieser Säure enthält, und zwar ist unter Umständen nur noch physikalisch gelöstes (ias zugegen.-) Die Beobachtung, daß zwischen dem Grad der Azetonoplasmie und dem Kohlensäuregehalt des Blutes bestimmte Beziehungen bestehen, hat zu einer Methode geführt, die gestattet, aus dem letzteren den ersteren zu bestimmen. 3) Es treten die Kohlensäure und die Säuren Ji-Oxvbuttersäure und Azetessigsäure in Wettbewerb um das zur Verfügung stehende Alkali. Alle diese Säuren sind schwache Säuren. Für das Verteilungsgleichgewicht kommt das Massenwirkungsgesetz in Betracht. Wir haben in dem Mechanismus der Festhaltung eines bestimmten Reaktionszustandes im Blute keinen Sonderfall vor uns. Wir treffen überall im Organismus auf solche Einrichtungen. Für den Arzt ist die Azetonoplasmie von allergrößter Bedeutung. Dadurch, daß das Blut weniger Kohlensäure aufnehmen kann, als ohne die genannten Säuren, muß der Organismus Schaden erleiden. Es ist die ganze sogenannte innere Atmung, d. h. der (Tasaustausch zwischen Gewebe und Blut gestört. In der Tat beobachten wir bei einem hohen Gehalt des Blutes an [i-Oxybuttersäure und Azetessigsäure, daß schwere Erscheinungen auftreten. Man spricht von einem Koma diabeticum. Greift man nicht ein, so erfolgt der Tod. Voraus gehen zunehmende Dyspnoe, Somnolenz und Sinken der Körpertemperatur. Zur Bekämpfung des Komas führt man intravenös größere Mengen von Natriumbikarbonat ZU.+) Meist beobachtet man dann fast sofortige Erleichterung. Sie ist nicht etwa darauf zurückzuführen, daß durch das zugeführte Alkali die Säuren des Blutes neutralisiert werden. Die Reaktion des Blutes war ja nicht sauer. Vielmehr erleichtert das Alkali den Kohlensäureabtransport. Natürlich handelt es sich nur um eine vorübergehende Hilfe, weil das ^lehr an Alkali bald durch die Nieren entfernt ist. Eine verminderte Glukosurie nach der Alkalizufuhr darf man nicht ohne weiteres im Sinne einer Besserung des ganzen Zustandes deuten. Es ist durchaus möglich, daß im Sinne der S. 164 genannten Versuche die Niere für Zucker dichter wird. Daß das Koma diabeticum in der erwähnten Art auf den hohen an Säuren insbesondere tritt 3-Oxybuttersäure in den Vordergrund zurückzuführen ist, bezeugen die Ergebnisse jener Versuche, bei denen Tieren Säure zugeführt wurde. So zeigten Kaninchen, (iehalt des Blutes — — — M Vgl. hierzu L. J. Hendcrsohn Ergebnisse der Physiologie. 8. 254 (1909). : 1044 (1920t. -) W. Mc Kim Marriott: Journ. of biol. Chem. 18. 241 (1914). ') Vgl. Donald D. van Sli/ke (Glenn E. Cullen, Reqinald Fitz, Edgar Stillman): .lourn. of biol. Chem. 30. 289, .S47. 3()9. 389. 401, 40.ö (1917). //. Straub' nwA Klothilde Meier: Biochem. Zeitschr. 89. 156 (1918); Deutsches Archiv f. kliii. Med. 129. 54 (1919). Wüh. C. Studie und Donald D. ran Slyke : Journ. of biol. Chem. 41. 191 (1920). D. D. van Slyke und W. W. /)rt/mßr; Ebenda. 41. 567 (1920). G. E. CiiUn, Ebenda. 52. 501, 517 (1922). ( ) Vgl. hierzu auch J. R. Muri in und L. /'. 'rarer : The .1. of Biol. Chem. 28. Nr 1 Jouru. of biol. Chem. 1. — — — (1916). — X. Vorlesung. l^)^ denen Salzsäure verabreicht wurde, schwere Dyspnoe. i) Der Kohlensäure- gehalt des Blutes war stark herabgesetzt, die Ainmoniakmenge des Harnes erhöht. Der ganze Zustand besserte sich nach subkutaner Zufuhr großer Mengen von Natriumkarbonat. Erwähnt sei noch, daß man das Auftreten großer Mengen von [j-Oxybuttersäure im Blute beim Koma auf mangelhafte Oxydationsvorgänge im Körper zui-ückgeführt hat. In dieser Hinsicht ist es von Interesse, daß man im Koma oft im Harn Aminosäuren antrifft.-) Es sei ferner an die schon S. 185 mitgeteilte Beobachtung erinnert, wonach die roten Blutkörperchen und v.ahrscheinlich auch andere Zellarten bei schweren Fällen von Diabetes und insbesondere auch im Koma diabeticum eine stark herabgesetzte Atmung zeigen. Die Bildung der Azetonkörper ist übrigens nicht auf den Diabetes melitus allein beschränkt. Sie finden sich unter Umständen auch bei allen angeführten Arten von Glukosurie. Ein Zusammenhang mit dem gestörten Kohlehydratstoffwechsel ist auf alle Fälle vorhanden, nur braucht dieser nicht ein direkter zu sein. Man darf nicht außer acht lassen, daß in Wirklichkeit eine so tiefgreifende Stoff wechselstörung, wie sie durch die schweren und bleibenden Formen der Hyperglukoplasmie zum Ausdruck kommt, niemals für sich allein bestehen, d. h. auf eine bestimmte Klasse von Verbindungen beschränkt bleiben kann. Da, wie wir fortwährend betont haben, wederein Kohlehydi-at, noch ein Fett- oder Eiweißstoffwechsel für sich besteht, sondern vielmehr Wechselbeziehungen der mannigfaltigsten Art die erwähnten Stoffwechselarten auf das innigste miteinander verknüpfen, so läßt sich leicht ermessen, wie sehr der Gesamtstoffwechsel leiden muß, wenn eine Gruppe der wichtigsten Nähr- und Baumaterialien in Mitleidenschaft gezogen ist. Es ist deshalb wohl verständlich, daß über kurz oder lang die Stoffwechselstörung eine allgemeine wird und der Ab- und Aufbau der Eiweißkörper und Fette ebenfalls Not leidet. Von diesem Gesichtspunkte aus muß das schwere Krankheitsbild des Diabetes betrachtet werden, soll ein volles Verständnis des ganzen Umfanges der eingetretenen Störung erlangt werden. Nicht nur der Verlust an Spannkräften, der dem Körper durch die beständige Ausfuhr der großen Zuckermengen erwächst und der ja zum Teil außerdem durch andere Nahrungsstoffe gedeckt werden kann, beherrscht die ganze Krankheit und stempelt sie zu einer so schweren, sondern der Zusammenbruch des ganzen Zellstoffwechsels. Die abnorme Zusammensetzung des Blutes und der Gewebsflüssigkeit führt zu manchen sekundären Erscheinungen. Die Widerstandsfähigkeit der Gewebe und Zellen gegen Infektionen sinkt und so ruft eine Schädigung bald andere hervor. Ihre Summe verleiht dem Krankheitsbild Diabetes zu dem gemeinsamen Grundbild von I'all zu Fall seine eigenartigen Züge. Bei den l)is jetzt besprochenen Formen von (ilukosurie war eine Hyperglukoplasmie vorhanden. Nur bei der intravenösen Zufuhi- von Kochsalzlösung ließ sich feststellen,') daß neben der Überschwemmung des Blutes mit Glukose gleichzeitig als Ursache für die bestehende Glukosurie eine — ') Friedrich Walter: Arch. f. experini. l'ath. u. Pharmak. 7. 148 (1877). \"gl. auch Julius Pohl: Biochem. Zoitsclir. 18. 24 (1909). -) Enal Abderhalden : Zeitschr. f. physiol. Cbcm. 44. 17 (1905). Bei acht neueu Fällen von Koma diai)eticuin konnton 1-Tyrosin 1-Leucin, d-Alanin und auffallend große Mengen von GlykokoU isoliert werden. — , ') Vgl. S.' 157. Kohlehydrate. erhöhte tracht Durchlässigkeit der 199 Nierenepithehen für Traubenzucker kommt. Ferner haben wir S. 164 erfahren, dal3 man durch in Be- die Art der Zusammensetzung- der DurchspülungsfUissigkeit die Niere für Glukose durchlässig machen kann. Es gehngt, sie auch für diese innerhalb gewisser (Frenzen zu dichten. Wir kennen nun eine Form von (xlukosurie, bei der in der Kegel eine Hyperglukoplasmie vermißt wird, ja sehr oft findet man sogar einen geringeren Gehalt des lUutes an /ucker. Wir wollen gleich vorweg nehmen, daß festgestellt worden ist, daß bei dieser Art von (ilukosurie die Nieren beteiligt sind. Sie lassen Glukose aus dem Blute in den Harn übertreten. Diese Form von Zuckerausscheidung ist von V. Mering'^) entdeckt worden. Er spritzte Hunden Phlorhizin ein und beobachtete, daß Traubenzucker im Harn auftrat. Man spricht von einer Phlorhizin-Glukosurie. Dem Phlorhizin sind wir schon begegnet.^) Es gehört zur Gruppe der Glukoside und ist seiner Konstitution nach aufgeklärt. Es läßt sich einerseits in Phloretin und Glukose spalten und andrerseits in Phlorijn =: Plorogluzinglukosid und Phloretinsäure =p-Oxyphenylpropionsäure. Von den Spaltprodukten des Phlorhizins ergeben Phloretin und Phlorin beim Hunde eine Glukosurie, während Phlorogluzin und ferner die Phloretinsäure unwirksam sind. Beim Kaninchen trat auf Einspritzung von Phloretin keine Glukosurie auf.^) mehr oder weniger Nach parenteraler Zufuhr von Phlorhizin erscheint im Harne Phlorhizin glukuronsäure. Es hat somit eine Paarung mit Glukuronsäure stattgefunden. Es ist von großem Interesse daß dieser Paarling beim Kaninchen keine (Glukosurie verursacht. Beim Hunde ist seine W^irkung nicht ganz aufgehoben, jedoch stark eingeschränkt. Ein Teil des zugeführten Phlo rhizins wird in noch unbekannter Weise verändert. Die Phlorhizin-Glukosurie hat aus mehreren Gründen großes Interesse erlangt. Einmal ist es mit ihrer Hilfe durch systematische Studien gelungen, zu entscheiden, ob bestimmte Verbindungen Beziehungen zu den Kohlehydraten besitzen oder nicht. Ferner hat man das Phlorhizin besonders in letzter Zeit oft dazu verwendet, um zu prüfen, ob die Nieren normal funktionieren, d. h. durch Phlorhizin veranlaßt werden, Traubenzucker an den Harn abzugeben oder aber in anderer Weise zu reagieren.) Die Wirkung des Phlorhizins ist bei den verschiedensten Tieren bei Amphibien, Reptilien, Vögeln, Säugetieren und beim Menschen studiert worden.») Der Erfolg war qualitativ immer derselbe. Es erscheint nach Zufuhr einer bestimmten Dosis des Glukosides Traubenzucker im Harn, und zwar scheint so lange (ilukosurie zu bestehen, als sich Phlorhizin im Blute befindet. Wichtig ist die Beobachtung, daß Phlorhizin (ilukose in Form von Glukuronsäure bindet und auf diesem Wege dem Organismus ebenfalls Traubenzucker entzieht. Zunächst versuchte man die Glu, — ^) r. Zeitschr. f. Mering: Veihandluagen des Kmigiesses für innere Medizin 1886 und 1887kliu. 'Med. 14. 405 (1888) und 16. 431 (1889). Vgl. S. 68. Vgl. S. 68-69. Vgl. hierzu Jos. Schüller: /oitschr. f. Biol. 56. 274 (1911). Crenier: Müuchener med. NYochenschr. iS'r. 32(1911). M. ('retner und //. II'. Seußert: -) — "j M. — — Berichte der Deutschen Cheni. Gesellsch. 45. 2565 (1912). ) Vgl. z. B. \'icfor Blum: iS'ierenphysiologie und funi<tionelle Nierendiagnostik. Franz Deutike. Leipzig und Wien 1913. Vgl. die T-iteratur bei Crahaiu Lvfk: Krgchn. d. rhysiol. 12. 315 (1912). •') ^- Vorlesunsr. 200 kosurie nach Vergiftung mit Phlorhizin auf ähnliche Ursachen, wie sie den übrigen Formen der Zuckerausscheidung im Harn zugrunde liegen, zurückSo dachte man an eine Beeinflussung des Zuckerzen t rums. zuführen. Es zeigte sich jedoch, daß das Phlorhizin auch dann noch wirkte, als das untere Hals- oder das obere Brustraark durchschnitten worden war.^) Es wurde auch bald erkannt, daß die Leber kaum direkt beteiligt ist") Ferner wurde festgestellt, daß die Pankreasdrüse keine Veränderungen zeigt, und endhch die Zufuhr von Phlorhizin die nach vollständiger Entfernung der Pankreasdrüse bestehende Glukosurie noch zu steigern vermag. 3) Auch das Hungertier beantwortet die Eingabe von Phlorhizin mit Zucker- ausscheidung im Harn. Es sei gleich hier bemerkt, daßdasgenaueStudium der Herkunft des bei der Phlorhizinglukosurie ausgeschiedenen Zuckers ergeben hat. daß er sicher zum Teil von bestimmten Aminosäuren abstammt. Einen großen Fortschritt in der Erkenntnis der Stellung der Glukosnach Zufuhr von Phlorhizin zu den übrigen Formen von Zuckerausscheidung erbrachte das eingehende Studium des Verhaltens des Blutzuckers. Seine Menge ist, wie schon erwähnt, nicht regelmäßig erhöht. Es besteht im Gegenteil zuweilen auch eine Hypoglukoplasmie.) Nach eigenen Beobachtungen ist diese beim Frosche nach Phlorhizinzufuhr seltener anzutreffen als die Hyperglukoplasmie. urie Da trotz erheblicher Glukosurie der Zuckergehalt des Blutes nicht oder nur wenig unter die Form herabgesetzt ist, so geht schon aus dieser Beobachtung hervor, daß dem Blute andauernd Glukose zugeführt werden muß, denn sonst müßte bei der fortgesetzten Zuckerausscheidung durch die Nieren nach kurzer Zeit das Blut zuckerfrei werden. Der erste, der experimentelle Beweise für die Annahme erbrachte, daß der oder doch ein Angriffspunkt des Phlorhizins in der Niere zu suchen ist, war Zuntz.^) Er führte den folgenden wichtigen Versuch aus. Es wurden einem Hunde beide Ureteren freigelegt. Der aus ihnen hervorquellende Harn war frei von Zucker. Nun injizierte Z/m^^ in die Nieren- — arterie der einen Niere eine Lösung von Phlorhizin. Schon nach 1 2 Mider Ureter dieser Niere mehr Harn als das andere Grgan. Er reduzierte alkalische Kupferoxydlösung. Der von der anderen Niere sezemierte Harn war noch frei von Zucker. Erst einige Minuten später lieferte auch diese zuckerhaltigen Harn. Dieser Versuch zeigt, daß das Phlorhizin ohne Zweifel das Nierenepithel direkt an Ort und Stelle beeinflußt) Es gelangte im erwähnten Versuche zuerst zu jener Niere, in deren Arterie es eingespritzt worden war. Erst später wurde es der zweiten Niere zugeführt, um nun auch dort seine Wirkung zu entfalten. Man hat weiterhin den Versuch gemacht, die Wirkung des Phlorhizins auf bestimmte Nierenepithelien zu lokalisieren. Es konnten einmal die Epithelien der Glomeruli beeinflußt sein oder aber jene Zellen, die die nuten lieferte Le'pine: C. r. de l'Acad. des sc. 121. 450 (1896). J. E. Sweet und A. J. Ringer: The .Toiirn. of Bio). Chcra. 14. Vgl. auch J. H. Austin und A. J. Ringer: P^beiida. 14. 1.^9 (1913). ») ') •') ) 5) «) 13ö (1913). 0. Minkowski: Arch. f. experim. Patli. u. l'harmak. 31. 85 (1893). Vgl. z.B. Peter Junkersdorf: Pflügen^ Archiv. 131. 306 (1910). Zuntz: Arch. f. (Anat. u.) Physi'ol. 570 (1895). Vgl. auch A. Erlandsen: Biochem. Zoit^rhr. 23. 'V29 (1910): 24. iV. ]. (1910). — Kohlehydrate. l'( ) 1 Harnkanälchen bekleiden. Beim Frosche lielj sich die gestellte Frage leicht entscheiden, weil bei diesem die Glomeruli von der Nierenarterie versorgt werden, während die Harnkanälchen von Gefäßen aus der Renoportalvene umflochten sind. Nach Unterbindung der Nierenarterie, also nach Ausschaltung der Glomeruli, trat auf Zufuhr von Phlorhizin noch Glukosurie auf.^) Dieser Umstand beweist, daß das genannte Glukosid offenbar auch die Epithelien der Harnkanälchen zur Zuckerausscheidung veranlaßt. Ferner wurde beobachtet, daß nach erfolgter Phlorhizinvergiftung die Markmehr Zucker enthielt als die Rindenschicht.'-) Endlich konnte in den Harnkanälchen mehr Zucker festgestellt werden als in den schicht der Niere Glonieruluskapseln.3) Wir können uns somit folgendes Bild von der Wirkung des Phlomachen. Es hat eine spezifische Wirkung auf die Zellen der Harnkanälchen. Diese werden zur Abscheid ung von Traubenzucker angeregt.) Wir müssen kurz erwähnen, daß manche Forscher annehmen, daß der Harn durch rhizins Osmose, Diffusion und Filtration aus dem Blute sich bildet. Da jedoch der Harn eine andere Zusammensetzung als das Blut hat, so war man gezwungen, die erwähnte Vorstellung der Harnabsonderung durch eine Hilfshypothese zu ergänzen. Es soll der durch die Glomeruli abgeschiedene Harn in seiner Zusammensetzung durch eine Rückresorption von Wasser und ein- zelnen Bestandteilen des Harns in den Harnkanälchen verändert werden. Man könnte sich von der Grundlage dieser Hypothese aus vorstellen, daß normalerweise die Glomeruli auch Zucker abscheiden. Dieser würde jedoch unter normalen Verhältnissen durch Rückresorption wieder zurückgewonnen. Das Phlorhizin würde den Zellen der Harnkanälchen die Fähigkeit nehmen, den Traubenzucker wieder aufzunehmen. Nach einer zweiten, mit den vor- liegenden Tatsachen viel eher vereinbaren Theorie stellt die Harnbildung einen richtigen Sekretions Vorgang dar. Jede eineine Nierenzelle erfüllt Das Phlorhizin würde in bei der Harnbildung eine bestimmte Aufgabe. diesem Falle die Zellen der Harnkanälchen so beeinflussen, daß sie entgegen ihren sonstigen Leistungen die Sekretion von Glukose übernehmen. Man könnte auch an Folgendes denken. Die erwähnten Epithelien sind normalerweise auf einen bestimmten Gehalt des Blutes an Traubenzucker eingestellt. Bei Hyperglukoplasmie scheiden sie das Mehr an Glukose aus. Das Phlorhizin könnte nunmehr die Epithelien der Harnkanälchen so beeinflussen, daß sie auf einen anderen Zuckergehalt, und zwar auf einen niedrigeren eingestellt sind, und es deshalb zur Glukosurie bei normalem und sogar bei unternormalem Gehalt des Blutes an Zucker kommt. s) Es ist naheliegend, an eine Beeinflussung des lonengehaltes des Blutes im Sinne der S. 104 erwähnten Versuche zu denken. Eine Verschiebung im Gehalt des Koder Ca-Ions würde an und für sich schon genügen, um die Permeabilität der Nierenzellen zu beeinflussen. Es kann aber ebenso gut auch ein spezifischer Einfluß auf diese Zellen durch das Phlorhizin selbst stattfinden. ') B. Mosherg: Diss. Wiirzhurg 1898. M. Nishi:' Arch. f. experim. Path. u. Pharmak. 62. 3i>9 (1910). 3) A. Seeliq: Arch. f. experim. Path. u. Pharmak. 37. 106 (189(5). ) Vgl. hierzu auch 'Ih. P. Nash: The Journ. of biol. Ghem. 51. 171 (1922). Jsaac: Arch. f. experim. ^) Vgl. hierzu auch die Ansichten von K. Frank und Path. u. Pharmak. 64. 293 (1911). Erich Frank: Kheiida. 72. 387 (1913). 2) — .S'. X. Vorlesung. 202 Für diese letztere Ansicht spricht der folgende \'ersuch.i) Es wurde eine Froschniere von der Aorta aus durchspült, und zwar mit einer Nährlösung, die das Glomerulusepithel für den zugeführten Zuckergehalt undurchlässig machte. Wurde der Durchspülungsflüssigkeit 00004", o Phlorhizin zugefügt, so ließ dieses sofort Glukose durchtreten. Nun kann jedoch die Beeinflussung bestimmter Nierenepithelien durch das Phlorhizin unmöglich den Einfluß dieses Glukosids auf den Kohlehydratund den Gesamtstoffwechsel restlos erklären. Es müssen noch andere Wirkungen vorhanden sein. Die Sekretion des Traubenzuckers könnte allerdings an und für sich bewirken, daß sekundär Störungen aller Art sich einstellen. Wir haben schon betont, daß offenbar die Leberzellen und vielleicht auch andere Zellarten die Aufgabe haben, den Zuckergehalt des Blutes zu überwachen. Sinkt der Zuckerspiegel, dann wird Glykogen abgebaut und dem Blute Glukose übergeben. Da die Niere unter dem Einfluß von Phlorhizin immer wieder den Gehalt des Blutes an Zucker vermindert, würden auf diese Weise allmählich alle Vorräte aufgebraucht und, ohne Verwertung gefunden zu haben, ausgeschieden. Auch der eben synthetisch gebildete Traubenzucker^) würde dem Blute zur Ausfüllung der bestehenden Lücken übergeben, und so könnte man im Harn Traubenzucker in Erscheinung treten sehen der aus anderer Quelle als aus Kohlehydraten stammt. Viele Beobachtungen sprechen jedoch dafür, daß eine so einfache Erklärung der Phlorhizinglukosurie und ihrer Folgen nicht ausreicht, um alle Erscheinungen zu deuten. Das Phlorhizin hat offenbar mehrere Angriffspunkte. Wichtig ist in dieser Hinsicht die Beobachtung von I^iderJull^), wonach nach Ausschaltung der Nieren die Phlorhizinvergiftung stets zu einer Hyperglukoplasmie führt. Das Phlorhizin scheint auch den Eiweißstoffwechsel primär zu beeinflussen.) Daß er sekundär in Mitleidenschaft gezogen wird, ergibt sich aus dem Umstände, daß der mit Phlorhizin vergiftete Organismus kostbares Material in Form von Traubenzucker verliert. Es muß zur Bestreitung des Energiestoffwechsels zu einem vermehrten Eiweißabbau kommen. Es scheint jedoch, daß über den augenblicklichen Bedarf hinaus noch Eiweiß zerlegt wird. , — Durch fortgesetzte Zufuhr von Phlorhizin dreimal 2(/ Phlorhizin subkutan bei Hunden kann man eine maximale Menge von Glukose zur Ausscheidung bringen. Sie steht zum Harnstickstoff in einem bestimmten Verhältnis, wie zuerst Cremer und Ritter-') und später vor allem Graham Li(sk^) gezeigt haben. Der Quotient I):N') beträgt beim Hund ohne Pankreas etwa 2'J5, bei der Phlorhizinglukosurie kann er einen Wert von 8"65 erreichen.«) Er wird nur erhalten, wenn man das Glukosid ohne zu lange Unterbrechung Tag und Nacht zuführt. Interessanterweise erhält man bei der Phlorhizinglukosurie häufig schwere Formen von Azetono- — täglich — 1) B. Brinkman: Ned. Tijdschr. Geneesk. 2. 982 (1908). Vgl. auch Quaterhj: Journ. of experim. Med. XII. l->5 (1919). ^) Vgl. hierzu Vorlesung VI. 3) Frank J'. Underhül : The Journ. of Biol. Chemistry. 13. 15 (1912). ) Reilley, Nolan und G. Liisk: Americ. Journ. of Physiol. 1. 395 (1898). Junzi ('. Yoshikawa: Zeitschr. f. physiol. Chem. 75. 475 (1911). G. L. Wolf und Emil Osterberg: Americ. Journ. of Physiol. 28. 71 (1911). ')' Max Cremer und Riffer: Zeitschr. f. Biol. 29. 2ö() (1892). — «) Graham Lusk: Zeitschr. f. ') Vgl. S. 186. ") Graham Lusk: .Americ. Journ. of l'hvsiol. 22. 163 (1908). Biol. 30. 82 (1898). — Kohlehydrate. plasmie mit allen 208 ihren P'olgen. Der Kohlehydratmangel scheint auch hier ihr Auftreten zu begünstiuen. \Vir haben nunmehr mehrere Arten von Störungen des Kohlehydratstoffwechsels kennen gelernt, die alle zu dem eindeutigen Schlüsse führten, daß der tierische Organismus Glukose aus anderen Quellen als aus Kohlehydraten i)ilden kann, ^'on den zwei zur Verfügung stehenden, der Menge nach in Betracht kommenden Xahrungsstotfen. den Fetten und Eiweii)Stofl'en. ist nur für die letzteren eine direkte Beteiligung am Kohlehydratstoffwechsel erwiesen worden. Bestimmte Aminosäuren können direkt in Zucker umgewandelt werden. Diese Tatsache ist von grundlegender Bedeutung. Sie ist lange Zeit heiß umstritten worden. Zunächst ergab die Erfahrung bei schweren Fällen von Diabetes melitus, daß die zugeführten und im Organismus vorhandenen Kohlehydrate nicht ausreichen, um die große Menge der im Harn erscheinenden Glukose zu erklären, i) Dann folgten die wichtigen Beobachtungen am Hund ohne Pankreasdrüse.-) Auch hier findet man so große Mengen von Traubenzucker im Urin, daß keine andere Erklärung übrig bleibt, als daß Xichtzucker Kohlehydrate liefern können. Dazu kamen dann die Feststellungen bei der der Einspritzung von Phlorhizin folgenden Glukosurie. Die bei den erwähnten Formen der Störmig des Kohlehydratstoffwechsels gemachten Beobachtungen sind durch direkte Versuche erweitert Avorden. Man begnügte sich nicht mit der Feststellung, daß Aminosäuren Zucker liefern können. Man wollte wissen, welche von ihnen in Betracht kommen, und welche keine Zuckerbildner sind. Endlich versuchte man festzustellen, ob nicht auch andere Verbindungen im tierischen Organismus in Kohlehydrate überfiihrbar sind. Es stehen uns verschiedene Methoden zur ^'erfügung. um die Frage zu entscheiden, ob bestimmte Verbindungen Zuckerbildner sind oder nicht. Einmal kann man Tiere durch Hunger. Arbeit oder durch künsthche Maßnahmen, wie Strychninvergiftung. glykogenarm machen. Dann verfüttert man die zu prüfende Substanz und bestimmt darnach den Glykogengehalt des ganzen Organismus oder auch nur denjenigen der Leber. Man geht dabei von der Ansicht aus, daß die etwa gebildete Glukose sofort zu Glykogen aufgebaut wird. Diese Art der Prüfung der Beziehungen eines bestimmten Produkte.^ zu den Kohlehydraten hat viel an Beweiskraft verloren, weil Beobachtungen vorliegen, wonach eine praktisch glykogenfreie Leber ohne Nahrungszufuhr wieder glykogenhaltig werden kann. 3) Diese Feststellung deutet allein schon darauf hin, daß normalerweise Glykogen aus Produkten hervorgehen kann, die keine direkten Beziehungen zu den Kohlehydraten haben. Beim hungernden Tier findet ohne Zweifel, wie übrigens bei jedem unter normalen Verhältnissen lebenden Tiere auch, fortwährend eine Bildung von (ilukose aus Aminosäuren statt. Beweisender sind Versuche an überlebenden Organen. Der Durchspülungsflüssigkeit wird eine bestimmte Verbindung zugesetzt und verDiese Methode hat den großen folgt, ob sie zur Glykogenbildung führt. Lüthje: Deutsches Arch. f. klin. Med. 79. 498 (1904) und Zeitschr. Med. 43. 225 (1901). L. Mohr: Zeitschr. f. kliu. Med. b2. 337 (1904). -) /;. Pflnger uud /'. Junkersdorl rriiiffer? Archiv. 131. 201 (1910). ') \s\. K. PttiKier: l'ftägpr^ Archiv. 119. 117 (1907). — : f. kliu. ^- Vorlesung. 204 Vorteil, daß sie mehrfache Kontrollen zuläßt. Einmal kann man vor dem Versuche das betreffende Organ, z. B. die lieber, wiegen und dann in einem abgewogenen Teil davon das (ilykogen feststellen. Nach der Durchspülung wird wieder eine (jlykogenbestimmung vorgenommen. Die Durchspülungsflüssigkeit kann man endlich daraufhin prüfen, ob die zugesetzte Substanz verschwunden ist oder doch an Menge abgenommen hat. Der positive Befund einer Glykogenbildung beweist, daß die betreffende Substanz Zucker liefern kann, sofern nicht besondere Tnistände zur Annahme einer indirekten Wirkung zwingen. Der negative Befund beweist hingegen in keinem Falle, daß die betreffende Verbindung keine Beziehung zu den Kohlehydraten hat. Es können andere Organe als die Leber die Umwandlung in Zucker vollziehen, oder es ist die Zusammenarbeit mehrerer Organe notwendig. Hier müssen Fütterungsversuche den Beweis zu einem ein- deutigen gestalten. Eine weitere Möglichkeit, die Beziehungen bestimmter Verbindungen zu den Kohlehydraten zu studieren, ergeben die erwähnten Störungen des Kohle hydratstoff wechseis. Wir können z. B. einen Menschen mit Diabetes melitus oder einen pankreaslosen Hund in bestimmter Weise ernähren. Wir stellen den Quotienten D:N fest^) und fügen nun zu genau der gleichen Nahrung jene Verbindung hinzu, von der wir wissen wollen, ob sie Zucker liefern kann. Tritt eine entsprechende oder doch eine deutliche Erhöhung der Zuckerausscheidung ein, dann schließen wir, daß eine Zuckerbildung erfolgt ist. Genau gleich ist der Versuchsplan bei der (ilukosurie nach Vergiftung mit Phlorhizin, nur muß man, um zu brauchbaren Resultaten zu gelangen, mit dem genannten Glukosid durch andauernde Zufuhr die maximale Zuckerausscheidung innehalten. Gegen die Deutung dieser Versuche lassen sich Einwände erheben. Man kann z. B. an indirekte Wirkungen denken und annehmen, daß irgend eine andere Substanz gespart worden ist und so der Zuckerbildung zugänglich wurde. Eine solche Annahme hat etwas Gezwungenes an sich. Es nicht recht einzusehen, weshalb die eine Verbindung jene angesoll und eine andere vielleicht ganz ähnliche nicht. Dazu kommt, daß wir jeden Versuch dieser Art durch die Untersuchungen an der überlebenden l.eber stützen können. Ein anderer Einwand erscheint bedeutungsvoller, nämlich die Beeinflussung der Niere durch bestimmte Verbindungen. Sie kann so verändert werden, daß sie mehr Zucker durchläßt als zuvor. Es kann aber auch umgekehrt das Ausscheidungsvermögen der Niere für Glukose verringert werden. Infolgedessen müssen wir unbedingt verlangen, daß jeder Versuch der genannten Art durch Zuckerbestimmungen im Blute ergänzt wird. Es besteht die Möglichkeit, daß trotz erhöhtem Zuckergehalt des Blutes die Niere weniger Zucker ausscheidet als vorher. So ist z. B. die Beantwortung der Frage, ob Brenztraubensäure in (Jlukose übergehen kann, vorläufig daran gescheitert, daß diese ^'erbin(lung die Niere so stark schädigt, daß sie für Traubenzucker ..dicht" wird. 2) ist nommene sparende Wirkung haben M Vgl. S. 186. ') Paul Mayer: Biochcjii. Zoitsclir. 40. 441 (1912): 49. 480 (1913); 55. (1913). — Vgl. auch A. J. Ringer, K. M. Franke/ und L. Jonax: Tlic .lonin. of Biol. Chcm. 15. — 145 (1913). D. Dakin W. Jannei/: Klionda. 15. 177 (1918). — Ma.r Cremer: //. Bf'ilincr klin. iiuil A'. Wnclionschr Nr. 'M n91}). 1 Kohlehydrate. 205 Es ist schließlich noch ein weiterer wichtige!- Einwand gemacht worden. Aminosäuren. Fettsäuren und auch andere ^'erbindungen können auf die Glukosebildung in der Weise einwirken, daß sie den Glykogenabbau steigern! Ein solcher Einfluß wurde z. B. bei gesättigten Fettsäuren mit ungerader Kohlenstof fanzahl beobachtet, während er bei solchen mit gerader Anzahl vermißt wurde. i) Diese Beobachtungen zeigen, wie vorsichtig man in der Beurteilung einer Verbindung als Quelle für Wir wollen uns im Folgenden im Wesentlichen an Zucker sein muß Feststellungen halten, die vor allem vom Gesichtspunkte der chemischen Verwandtschaft bzw. gleicher oder doch ähnlicher Abbaustufen auf Beziehungen zur Glukose hinweisen. Schließlich möchten wir noch bemerken, daß der Umstand, daß eine Verbindung in bestimmter Konzentration einen Anreiz zum Abbau von Glykogen zu Glukose gibt, natürlich nicht ausschließt, daß sie zugleich in ihren Abbaustufen Quelle für Glukose ist. Es wird alles darauf ankommen, ob bei ihrem Abbau Verbindungen entstehen, von denen aus Traubenzucker entstehen kann. ! Wenn wir nunmehr zu der Besprechung der Frage übergehen, welche tierischen Organismus Kohlehydrate liefern Substanzen im können, werden wir unmittelbar auf die gleichen Fragestehungen wir bei der Frage nach der Art des Abbaues des Traubenzuckers erörtert haben.-) Wir kennen eine große Anzahl von Vorgängen im tierischen Organismus, die umkehrbar verlaufen. Es spricht sehr vieles dafür, daß auch im Zuckerauf- und -abbau Zwischenstufen auttreten, die je nach den Verhältnissen bald zum Glukosemolekül so treffen, die hinauf, bald zu einfacheren Abbaustufen hinunterführen. Wir haben schon wiederholt betont, daß in den Zellen des tierischen Organismus die Möglichkeit der Bildung von Verbindungen gegeben ist. von denen aus sich Brücken zu den verschiedensten Substanzen schlagen lassen. Eine solche Verbindung scheint die Brenztraubensäure zu sein. ^i Sie unterhält Beziehungen zu bestimmten Aminosäuren, vor allem zum Alanin. Ferner kann man sie sich aus Glukose entstanden denken. Endlich könnte sie auch Beziehungen zum Glyzerin, ja auch zu Fettsäuren haben. Einige dieser Beziehungen seien durch die folgenden Formeln veranschaulicht: CH3 — > CO OH CH, OH + 2 — CO CH." CH, . I i I CH NH, + . I I + XH3 . I COOH Alanin= a-AminoPropionsäure COOH Brenztraubensäure CH . I + 2 H., >^ 1 CH, OH Glyzerin . COOH Brenztraubensäure, Vgl. Leo Pollak: Biochem. Zeitscbr. 127. 120 (1922). Vgl. Vorlesung VI. ^) Vgl. hierzu C. Neuberg uud Joh. Kerb: Biochem. Zeitschr. 53. 406 Ferner H. D. Dakin und H. \V. Dudle;/: The Journ. of Biol. Chem. 14. öhb ') ") A. J. Ringer: Ebenda. 15. 145 (1913). (1913). (1913). — — X. Vorlesung. 206 COOH H C .OH HO C .H H C .OH CH . (3H + (^H3 H C.OH CH2 OH COOH I I CO + H., . CH3 BrenztraubenMilchsäure Glukose säure Als Abbaustule der Glukose kommt auch Methylglyoxal=Brenztraubensäurealdehyd in Betracht. 1) Auch diese Verbindung kann inBrenz- traubensäure übergehen: f^ Kohlehydrate. gewissermaßen 207 neutrale lrodukte aufzufassen sind. 8ie des Abbaus und Aufbaus ganz verschiedenartiger Verbindungen. Sie dürften in Verbindungen der Dreikohlenstoffund vielleicht auch der Zweikohlenstoffreihe zu suchen sein. Die verschiedenartigsten der oben erwähnten Versuchsanordnungen haben als Glykogenbildner die folgenden Substanzen ergeben. i) Der typische Baustein des Glykogens ist der Traubenzucker. Daß er direkt in das genannte Polysaccharid übergehen kann, ist durch Untersuchungen an der überlebenden Leber und an Muskeln eindeutig festgestellt worden, d -Fruktose und d-Galaktose, die beide, wie wir mehrfach betont haben leicht in Glukose umgelagert werden, sind selbstverständlich BaumateriaHen für das Glykogen. Ferner erwies sich als Ausgangsmaterial zur Glykogen bzw. Glukosebildung geeignet der Glykolaldehyd.-^) Ob auch Glyzerinaldehyd-^) in Frage kommt, ist noch unentschieden, jedoch recht wahr- decken, stellen die als am Kreuzungspunkte , Auch Glykol, Glyzerinsäure und d-Milchsäure scheinlich. liefern Glykogen. Baer und Parnas) stellen sich vor, daß die letztere Verbindung über Glyzerinsäure und Glykolaldehyd zu Glukose führt 3 [CH3 . GH (OH) COOK] 3 -}- . —> 3 ICH, —> (OH) . CH (OH) COOHj . Glyzerinsäure Milchsäure —y 0—3 COo-3 -f 3 [CH, . OH COHj . 3 H, Ge H,., O^. Glykolaldehyd Glukose. Emhden denkt dagegen an eine Umlagerung von Milchsäure in Glyzerinaldehyd. Zwei Moleküle dieser Verbindung verbinden 2 CH3 I . CHOH COOHJ . — Milchsäure >- würden sich dann zu Glukose CH (OH) COH] Glyzerinaldehyd 2 [CH, (OH) . . -^ G, H^.. (\. Glukose. Für das Glyzerin erbrachte zuerst Cremer^) den sicheren Beweis daß die mit Phlorhizin vergiftet waren es in Kohlehydrat übergehen kann. Lüthje^) hat den gleichen Befund bei Hunden ohne Pankreas erhoben. Ferner liefern Propylalkohol und Iso- — — er arbeitete mit Tieren, . butylalkohoH) Glukose. Ferner sollen Propionsäure, Valerian-, Heptyl-, Isobutyl- und Isokapronsäure in Traubenzucker übergehen können. Binger^), der diese Feststellungen gemacht hat, nimmt an, daß die Propionsäure im Mittelpunkt der Synthese steht und aus den genannten Fettsäuren zu) Vgl. hierzu S. 139. Es ist dort angeführt, welche Substauzen beim Durchleiten durch die überlebende Leber Glykogen lieferten. Manche Verbindung, die hierbei ein negatives Resultat ergab, zeigte sich als zur Kohlehydratbildung befähigt, als sie dem gesamten Organismus zur Verfügung gestellt wurde. Vgl. auch W. />. «) Baer und Parnas : Biochem. Zeitschr. 41. 386 (1912). Sanswyi und R. T. Woodyatt : Journ. of Biol. Chem. 24. 327, 343 (1916). =) 6r. Embden, K. Baldes und E. Schmitz: Biochem. Zeitschr. 45. 127 (1912). ./. l'arnas: Zentralbl. f. Physiol. 26. 671 (1912). J. SmedUy: Journ. of Physiol. 44. 203 (1912). ) Baer und Parnas: Biochem. Zeitschr. 41. 386 (1912). ) Max Cremer: Münchener med. Wochenschr. Nr. 49. 944 (1V)U2). Sitzungslier. d. Gesellscli. f. Morph, w. l'hvsiol. in München. 27. Mai 1902. «) H. Lüthje: Deutsches Archiv f. klin. Medizin. 79. 498 (1904) und 80. 101 (1905). ') A. J. Ringer, E. M. Franhel und L. Jonas: Journ. of. Biol. Chem. 14. 52ö (1913). «) A. J. Ringer: Journ. of Biol. Chem. 12. 511 (1912); 14. 43 (1913). A. J. Ringer, E. M. Franke! und L. Jonas: Ebenda. 14. .'i25 (1913). — — — ^- Vorlesung. 208 nächst gebildet wird. Auch Äpfelsäure, Bernsteinsäure und Fumarsäure erwiesen sich als Zuckerbildner.i) Die letztere Verbindung wird wahrscheinlich unter CÜ.j-Abspaltuug über Milchsäure iu Glukose umgewandelt 2): COO H CH r:: CH COOH -r H, O — CO, . . übergibt —> CH3 CH . . (OH). COOH. man der Leber Rohrzucker oder Milchzucker^), so ver- mag sie diese Disaccharide nicht zu verwerten, wenn sie nicht zuvor in ihre Bausteine zerlegt sind. Zahlreich sind die Versuche über das Verhalten von Eiweiß Stoffen zur Zuckerbildung. Die Untersuchungen sind erst in dem Augenblick eindeutig geworden, als man anfing, an Stellen von Proteinen ihre Bausteine, nämhch Aminosäuren, zu verwenden. Es zeigte sich bei Versuchen am gesamten Organismus, dali Glykokoll, Alanin, Cystin, Serin, Asparagin- und Glutaminsäure, Arginin und Ornithin Traubenzucker liefern), während Phenylalanin, Tyrosin, Tryptophan und auch Leuzin, Isoleuzin, Valin. Lysin, Histidin») keine direkten Beziehungen zu den Kohlehydraten haben sollen. Versuche an der überlebender Leber hatten, wie S. l;)9 erwähnt, für alle Aminosäuren das gleiche negative Ergebnis. Durch den Beweis, daß Glyzerin in Glukose übergehen kann, ist zwar eine Brücke von den Fetten zu den Kohlehydraten geschlagen, trotzdem ^\ird immer noch bezweifelt, daß die Fette normalerweise Zucker liefern. Das Glyzerin ist ein Baustein der Fette. Es tritt jedoch seiner den FettMenge nach gegenüber den übrigen Bestandteilen der Fette säuren stark zurück. Bis jetzt verfügen wir über keine überzeugenden Versuche, die eine irgendwie erhebliche Zuckerbildung aus Fett beweisen würden. ß) Auf der anderen Seite sind jedoch auch keine Beobachtungen bekannt geworden, die mit voller Schärfe Beziehungen zwischen den Fetten und den Kohlehydraten ausschließen würden. Die Frage, ob nicht doch in besonderen Fällen Fette als Quelle von Kohlehydraten in Betracht kommen, bleibt vorläufig noch eine offene. Der Umstand; daß Aminosäuren vollständig ausreichen, um den in Erscheinung tretenden Zucker, der von Xichtkohlehvdraten stammt, seiner Herkunft nach aufzuklären, genügt — — ) Max Cremer: Berl. klin. Wocheaschr. Nr. 31 (19131. — Vgl. auch Hans Schröder: 23 (1922). - W. Bürger: Ebenda 2. 19 (1922). Hans Müller: Helvet. chim. acta. 5. 163 (1922). — Vgl. auch Ä. Jung Beitr. z. Phvsiol. 2. -) Vgl. — — E. Abderhalden: Fermentforschung. 6. (1922). und H.Müller: Ebenda 5. 239 (1922). H.D. Dahin: .Journ. of biol. ehem. 52. 183 (1922). ') Vgl. u. a. Ernst Weinland: Zeitschr. f. Biol. 40. 374 (1902). ) Vgl. Graham Lusk: Amerc. Journ. of Physiol. 22. 174 (1908) und Ergebnisse Vgl. ferner: A. J. Ringer und Graham Lusk: Zeitschr. der Physiologie. 1. c. (1912). Max Cremer: Verhandl. d. physiol. Gesellsch. f. physiol. Chemie. 66. 10(i (1910). A. J. Ringer, E. M. Frankel und 29. November 1912. Med. Klinik. Nr. .oO (1912). L. Jonas: Journ. of Biol. Chem. 14. 525 und 539 (1913). ') H. D. Dakin: Jouru. of Biol. Chem. 14. 321 (1913). ) Vgl. zu diesem Probleme ./. Seegen: Die Zuckerbildung im Tierköi-per, ihr Umfang und ihre Bedeutung. Berlin 1890. J. Weiss: Zeitschr. f. physiol. Chemie. 24. 542 (1898). N. Zuntz: Verhandl. d. physiol. Gesellschaft. 14. Oktober 1898. E. Abderhalden und Feter Rona: Zeitschr. f. physiol. Chemie. 41. 303 (1904) — /•;. 0. Krummacher und E. WeinWeinland: Zeitschr. f. Biol. 49. 421 und 466(1908). A. Gigon: Deutsches Archiv f. klin. Medizin. land: Zeitschr. f. Biol. 52. 273 (1909). 97. 376 (1909). — Eduard Pßüger und Feter Junkersdorf Fßüger^ Archiv. 131. 201 Felix Lommel: Feter Junkersdorf: Fflügers Archiv. 137. 269 (1910). (1910). Archiv f. experim. Path. u. Pharm. 63. 1 (1910). — If. Erharf F. Zieglwallner Zeitschr. — — : — — — — — — : — — : f. Biol. 58. 541 (1912) Kohlehydrate. noch nicht, um eine Beteiligung der Fette 20 an der Zuckerbildung und gar auszuschließen. Man darf zur Zeit nicht mehr aussagen, ganz als dal) auch ohne eine Umwandlung von Fetten in Kohlehydrate der Zuckerbedarf des Organismus gedeckt werden kann. Die Aminosäuren reichen dazu aus. Die Beobachtungen über die Bildung von Kohlehydraten aus Verbindungen, die nicht zu dieser Gruppe von Stoffen gehören, zeigen uns, daß die tierische Zelle mannigfaltige Synthesen durchführen kann. Während man noch vor wenigen Jahren sich scheute, im tierischen Organismus umfassendere Synthesen anzunehmen, sind jetzt alle Schranken gefallen. Wir wissen, daß jede einzelne Körperzelle einer lieihe von Synthesen fähig ist. Es sei in diesem Zusammenhange nicht unerwähnt, daß die Frage erörtert worden [ist, ob nicht vielleicht der zur Resorption gelangende Traubenzucker bereits in der Darm wand umgewandelt wird. Pavij dachte an eine Umbildung in Fett.i) Diese Ansicht wurde durch direkte Versuche als unrichtig erwiesen.'^) Dagegen besteht die Möglichkeit, daß schon in den Zellen der Darmwand Aminosäuren zu Kohlehydraten in Beziehung treten, und vielleicht auch Glukose zum Teil wenigstens soweit zerlegt wird, bis Abbaustufen vorhanden sind, die zu allen möglichen anderen Verbindungen führen können. Eindeutige Beweise für solche Vermutungen sind jedoch bis jetzt nicht erbracht worden, Der Beziehungen der Kohlehydrate zu den Fetten haben wir wiederholt gedacht. Es ist ein tiefgehender Umbau nötig, ehe Kohlehydrate in die Bausteine der Fette und insbesondere in Fettsäuren umgewandelt sind. Am wahrscheinlichsten ist die Annahme, daß nicht Glukose als solche zur Umwandlung kommt, sondern eine ihrer tieferen Abbaustufen. Dem Umbau geht ein Abbau zu einem indifferenten Baumaterial voraus, dann setzt die Synthese ein. Es ist möglich, daß, wie schon wiederholt erwähnt, Brenztraubensäure^) bzw. Azetaldehyd und die aus ihm hervorgehende Essigsäure bzw\ Azetessigsäure das Ausgangsmaterial zur Bildung höhermolekularer Fettsäuren darstellt.) Die Bildung von Fettsäuren aus Kohlehydraten ist bei Ascaris, dem Spulwurm, direkt beobachtet worden, als dieser ohne Sauerstoff gehalten wurde. Er bewegte sich lebhaft und bildete Valeriansäure und ferner Kapronsäure aus Kohlehydraten.') Im übrigen ist der genauere Mechanismus der Überführung von Kohlehydraten in Fett im tierischen Organismus noch nicht aufgeklärt. Da auch Pflanzen diese Umwandlung zu vollziehen vermögen, und besonders in gewissen Samenarten einerseits Fettstoffe in Kohlehydrate und anderseits solche in erstere Verbindung übergehen, besteht die Hoffnung, daß durch eingehende Studien an diesem leicht zugänglichen Materiale die Zwischenstufen im Umbau der genannten Verbindungen bald auf- gefunden werden.'') ') F. W. Painj Über den Kohlehydratstoffwechsel. (Übersetzt von Kiot Mockel.) Wilh. Eugelmaiiu. Leipzig 1907. ) G. r. Bergmann und K. Reicher: Zeitschr. f. experim. Path. u. Ther. 5. 71)0(1909). ) Vgl. Ida\Smedli'ji uud Era Lubrzc/nska: The Biochemical Journ. 7. 3(54 (1913). — Carl Xeuberi/: Handbiicli der Biochemie. P>gäuziiugsl)and. 602 (1913). . Vgl. S. 137. G. r. Bunge: Zeitschr. f. phvsiol. Chemie. 8. 48 1 8,S3 14. 318 (1890). 1 884) E. Weinland: Zeitschr. f. Hiol. 42. .")o (1901); 43. 8(5 (1902). ) Vgl. Die möglichen Wege der Überführung von Kolilchydratcn in Fett in \"i>resuug Xlll. ) ) 1 Abderhalden, l'liy.siologisch« Chemie. I. Teil, 5. Aufl. ( - — : J4 Vorlesung XI. Fettstoffe und ihre Bausteine: Fettsäuren und Glyzerin. Wir sind den Fetten und ihren Bestandteilen schon wiederholt beEinmal wurde festgestellt, daß Kohlehydrate in Fette übergehen können 1), und ferner der eine Baustein der Fette, das Glyzerin, TraubenEndlich trafen wir auf die Fette, als die Herkunft zucker hefern kann. der Azetonkörper besprochen wurde. ~) Die Fette unterscheiden sich von den Kohlehydraten schon durch ihr ganz verschiedenes Aussehen und vor allem durch die eigenartigen Löslichkeitsverhältnisse. Sie sind mit wenigen Ausnahmen 3) unlöslich in Wasser, dagegen lösen sie sich in einer Reihe organischer Lösungsmittel, wie Äther, Chloroform, Benzol, Azeton, Schwefelkohlenstoff, Tetrachlorkohlenstoff. Manche Fette lösen sich auch ganz gut in Alkohol. Die Fette zeigen je nach ihrer Zusammensetzung einen ganz verschiedenen Schmelzpunkt. Manche sind bei Körpertemperatur flüssig, andere sind weich und wieder andere fest. Alle Fettarten sind in reinem Zustande farblos und ferner geruch- und geschmacklos. Schüttelt man ein flüssiges Fett energisch mit Wasser, dann erscheint dieses zuEs ist ganz undurchsichtig geworden. Bei genächst milchig getrübt. nauerem Zusehen erkennt man, daß die Trübung durch ungezählte, klein,ste Fetttröpfchen bedingt ist. Durch das Schütteln mit dem Wasser ist das Fett zerstäubt worden. Nach einiger Zeit hellt sich das Gemisch auf. Man beobachtet, daß größere Fetttropfen entstehen. Diese sammeln sich, da das spezifische Gewicht der Fette kleiner als 1 ist, an der Oberfläche des Wassers an. Schließlich schwimmt auf dem Wasser die ursprüngliche Fettschicht und das Wasser ist wieder ganz klar. Man nennt das erwähnte Zerstäuben des Fettes in Wasser Emulgieren des Fettes. Eine Emulsion, die nach einiger Zeit wieder verschwindet, wird als eine unbeständige bezeichnet. Eine beständige Emulsion wird erhalten, wenn man öl z. B. mit Gummi zusammen schüttelt. Das Gummi bindet dabei das ganze Wasser. Es bildet sich eine Emulsion von Öl in dem stark hydratisierten Kolloid Gummi.) gegnet. ') S. 117. Siehe Vorlesung X. ^) Diese betreffen Verbindungen zwischen Glyzerin und Fettsäuren mit niodorei Kohlenstoffatomzahl, die jedoch in der Natur keine Rolle spielen. ) Vgl. Martin H. Fischer und Mar Jan 0. Hooker: Kolloidz. 18. 129 (1916). ^) Fettstoffe und ihre Bausteine. 211 Durch die erwähnten physikalischen Eigenschaften sind die Fette scharf gegen die Kohlehydrate abgegrenzt, dagegen nicht gegen viele andere Verbindungen, die ganz ähnliche Löslichkeitsverhältnisse und auch sonst entsprechende Eigenschaften aufweisen. Charakteristisch für die Fette ist ihr Aufbau. Sie bestehen aus Kohlenstoff, Wasser- und Sauerstoff. Schon die Elementaranalyse, d. h. die Feststellung des Mengenverhältnisses dieser drei Elemente, zeigt einen sehr deutlichen Unterschied gegenüber den Kohlehydraten. Die Fette besitzen weniger Sauerstoff als diese. Wir haben auf diesen Umstand schon einmal hingewiesen, als festgestellt wurde 1), daß der respiratorische Quotient bei der Zerlegung von Zucker zu Kohlensäure und Wasser gleich 1 ist, während er bei den Fetten deshalb kleiner als 1 ausfällt, weil bei ihrem Abbau zu den Stoffwechselendprodukten mehr Sauerstoff zur Verfügung gestellt werden muß, als dies bei den Kohlehydraten der Fall ist. Die Fette gehören zu den zusammengesetzten Verbindungen. Sie lassen sich mit Alkalien, Säuren und auch mit Fermenten unter Wasseraufnahme spalten. Wir gelangen auf diese Weise zu den Bausteinen der Fette. Diese bestehen stets aus einem Alkohol und Fettsäuren. Der Alkohol der meisten Fettarten ist das Glyzerin. Dieses ist ein dreiwertiger Alkohol CHo . OH I CH CH, . . OH OH Seine Gegenwart läßt sich leicht erkennen, indem es bei Gegenwart energisch Wasser entziehender Mittel z. B. beim Erhitzen mit saurem schwefelsaurem KaUum in Akrolein übergeht. Dieses liefert stechende, die Schleimhäute stark reizende Dämpfe: — — GH., . OH CHo" r CH .OH — 2H2O = CH I! CH, . OH c<;h Glyzerin Akrolein. Die Fettsäurekomponenten sind mannigfaltiger Natur. Sie haben, soweit unsere bisherigen Kenntnisse reichen, nur das miteinander gemein, daß sie einbasisch sind. Sehr verbreitet sind die gesättigten Fettsäuren der Reihe C^ Hon O., oder Cn H2°+\ COOH, deren einfachstes Glied bekanntlich die Ameisensäure, COOH, ist. Jedes folgende Glied unterscheidet sich von dem vorhergehenden durch einen Mehrgehalt einer CHg-Gruppe: H ') . Vpl. S. 120. 14 XI. Vorlesung. '212 H COOH Ameisensäure. CH3 COOH Essigsäure. CH3 CHo COOH Propionsäure. CH3 CH, CH, COOH Buttersäure. CH3 CHo CH, CH.. COOH Valeri ansäure. CH3 CH., CH.. CH.. CH.. COOH Kapronsäure. CH3 CH2 CR, CHo CHo CH., COOH Heptylsäure. CH3 CH2 CH, CHo CH., CHo CHo COOH Octyl- = Capryl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . säure usw. Am verbreitetsten sind als Bausteine der Fette aus dieser Reihe von Fettsäuren die Palmitin- und die Stearinsäure: CH3 (CHsXi COOH und CH3 (CH2)i6 COOH. Beide sind bei gewöhnlicher Temperatur fest. Sie kristallisieren in Blättchen und haben ganz ähnliche Löslichkeitsverhältnisse, wie die Fette. Sie selbst sind unlöslich in Wasser, dagegen lösen sich ihre Alkalisalze leicht in Wasser. Die salzartigen Verbindungen mit den Erdalkalien und Schwermetallen sind dagegen in Wasser schwer löslich bis unlöslich. Von weiteren Fettsäuren dieser Reihe, die insbesondere im Pflanzenreich sehr verbreitet sind, wären noch zu erwähnen die Kaprinsäure (CH3 (CH.,)8 COOH), die Laurin- (CH3 (CHo),o COOH), die Myristin- (CH3 (CHj)i., COOH), die Arachin- (CH3 (CH^jis COOH) und' die . . . • . . . . . • Karnaubasäure (CH3 (CHo)oo COOH).i) . Sehr verbreitet sind als Bausteine der Fette auch ungesättigte Fettsäuren der Reihe Cn H.,n_o 0., oder Cn H.,n-i COOH. Der Haupt• Gruppe von Säuren die folgende Zusammensetzung: CH3 (CH,), CH vertreter dieser . ist die Ölsäure, CigHa^O.,. Sie hat = CH (CH.,), COOH. . . . Ferner gehört hierher die im Pflanzenreich und insbesondere im Rüböl und im Öl des Senfsamens enthaltene Erukasäure: CH3 Wie . (CH.,), . CH =r CH (CH-,)„ COOH. . . Formeln zeigen, enthalten die ungesättigten Fettsäuren eine Doppelbindung. An diese können sich Halogene und auch Sauerstoff anlagern. Die Ölsäure ist bei gewöhnlicher Temperatur flüssig. Namentlich in Fetten von Fischen, sogenannten Tranen, sind manche ungesättigte Fettsäuren dieser Reihe nachgewiesen worden, so im Dorschlebertran die Döglingsäure, C19H36O2, die Gadoleinsäure, C.^o H38 0,, und die diese Jekorinsäure, C19 H36 Oo. Eine weitere Klasse von ungesättigten Fettsäuren stellen diejenigen der Reihe CnH2n-4 02 dar. In verschiedenen Pflanzenteilen ist z. B. die L in Ölsäure, CigH,, Oo, als Baustein von Fetten aufgefunden worden. Besonders interessant sind von vielen Gesichtspunkten aus die sogenannten Oxyfettsäuren. Sie können gesättigt oder auch ungesättigt sein. Die Verbindungen dieser Reihe sind dadurch ausgezeichnet, daß sie eine oder ) Vgl. über diese VerbiiHluugen die Lelirljüclier der organischen Chemie. Von Werken, die weitere Einzelheiten über die Fettchemie enthalten, seien besonders hervorgehoben: F. Ulzer und ./. Klimont : Allgemeine und physi(dogiscbe Chemie der Fette. — Springer. Berlin (1906). 2. Autl. 1912. Biochemisches'liandlexikoii. 3. 1 (1911) [bearbeitet von Carl Brahm\\ 1. 912 (1911) [bearbeitet von Ernst Schmilz]. .T. Springer. Berlin 1911.— A. .Tolles: Chemie der Fette. 2. AuH. Karl J. Trühuer. Straüburg l'.»12. .T. — hmr Bang: Chemie und Biocliemie der Lipoide. J. F. Bergmann. Wiesbaden 1911. — W. Glikin: Chemie der Fette, lupoide und Wachsarten. Gebr. Bornträger. Leipzig 1913. Fottstott'o uml ihre Bausteine. 21o mehrere Oxygruppen besitzen. Wir kennen Mono- und Dioxyfettsäurcn bestimmter Fettarten. So hat Julinrd^) im Kizinusöl eine 1) i X y s t e a r i n s ä u r e auf jj;ef unden als Bestandteile CH3 (CHa), CH (OH) . . . CH (OH) . {CH.J, COOH. . Darmstädter und L/fschütz^) aus dem Wollfett die Lanozerinsäure: 0,7 Hb7 (0H)._, COOH. Auch ungesättigte Oxyfettsäuren sind beobachtet worden. Eine solche, nämlich die Rizinolsäure, CjyHa-, (OH) COOH, kommt im lUzinusöl vor. Schließlich sei noch erwähnt, daß die Möglichkeit besteht, daß auch aromatische Säuren am Aufbau von Fetten beteiligt sind. 2) Die Darstellung der Fette in reinem Zustande bereitet große Schwierigkeiten, Aveil sie mit wenig Ausnahmen nicht kristallisieren. In der Natur kommen die Fette wohl nie als chemisches Individuum für sich vor. Sie sind vielmehr mit anderen Fettarten gemischt. Da nun die Fette alle recht P'erner isolierten . . ähnliche Eigenschaften besitzen, ist eine Trennung eines Fettgemisches in Anteile sehr schwierig. Auch die Bausteine der Fette lassen sich oft nur schwer trennen. Der Alkohol kann immer leicht isoliert werden, seine dagegen hält es schwer, die verschiedenen Fettsäuren rein zu gewinnen und bei ungesättigten Fettsäuren sekundäre Veränderungen zu vermeiden. Es unterliegt keinem Zweifel, daß wir in der Kenntnis der einzelnen Bausteine der Fette erst in den Anfängen stehen. Je weiter die Methoden zur Isolierung der Fettsäuren ausgebildet werden, um so mannigfaltigeren Vertretern der Fettsäurereihe wird mau begegnen. In diesem Zusammenhange sei erwähnt, daß die Fettsäuren im Tier- und auch namentlich im Pflanzenreich als solche d. h. ohne mit einem Alkohol gebunden zu sein weit verbreitet sind. Meist begegnet man ihnen jedoch nur in geringen Mengen. In vielen Fällen dürften die in den Geweben auftretenden freien Fettsäuren Zwischenstationen im Aboder Aufbau von Fetten darstellen. Auch die Alkohole und insbesondere das Glyzerin kommen im Tier- und Pflanzenreich als solche vor. Wir haben bemerkt, daß die typischen Fette fast durchweg Glyzerin als Baustein besitzen. Es gibt jedoch auch Verbindungen, die andere Alkohole aufweisen. Von solchen seien erwähnt: Cetylalkohol, CigHgg OH, Oktadecylalkohol, CipHgy OH, Karnaubylalkohol, C24 H^g OH, Myricylalkohol, CsoHgi OH oder CajHßa OH. Auch Alkohole der aromatischen lleihe binden sich mit Fettsäuren zu Fetten. Die wichtigste Gruppe dieser Reihe stellen die Sterine dar. Von diesen ist im Tierreich das Cholesterin der wichtigste Vertreter. W^ir kommen auf diese zyklischen Alkohole noch zurück. Mit der Feststellung, daß die Fette bei der Hydrolyse einen Alkohol und Fettsäuren liefern, haben wir die Vertreter dieser Körperklasse noch nicht genügend charakterisiert. Wir wollen wissen, wie die einzelnen Bau- — — M JulUard: Bull, de la soc. chim. 3. ^) 2893 Ddrmsfädter und Lifschütz: Ber. 13, d. 238 (1895). Deutschen ehem. (lesellscli. 29. 1474, (189()). ') Von oranz besonderem Interesse ist die Beoltachtung, d;iß in den tialläpfelu zyklische Fettsiiure, die (' y kloga lli p h arsiiu re, vorkommt. \'gl. IlerniaiiH Kunz-Krausc Arch. f. Pharmazie. 242. 256 (1904). — Jlcrnianii Kidiz-Hrausr und Paul eine : Schelle: Ebenda. 242. 257 (1904). 248. 695 (1911). — //. Kitnz-h'ravf^e und l'diil Matücke: Ebenda. ^^ 214 \ orlesuug. steine miteinander vereinigt sind. Es ist das große Verdienst des Chemikers ChevreuP) (1811), die Konstitution der Fette in ihren Grundzügen aufgeklärt zu haben. Die Fette sind ganz allgemein esterartige Verbindungen eines Alkohols mit Fettsäuren. Ist der Alkohol mehrwertig, dann sind a priori mehreje Möglichkeiten gegeben. Einmal können alle Alkoholgruppen mit Fettsäuren verknüpft sein oder aber nur einzelne. Nach den bisherigen Erfahrungen scheinen immer alle verfügbaren Alkoholgruppen besetzt zu sein. Wir wollen die Konstitution eines am Beispiel Fettes, eines soge- nannten Glyzerides, d. h. eines Fettes, an dessen Aufbau Glyzerin beteiligt ist, erörtern. Die Zusammensetzung der Glyzeride ist vor allen Dingen durch die Arbeiten von Berthelot^) einwandfrei bewiesen worden. Er hat nämlich Fettsäureester des Glyzerins durch Synthese dargestellt (185-1). Er erhitzte Fettsäuren mit der entsprechenden, berechneten Menge Glyzerin im zugeschmolzenen Rohr auf etwa 200''C.äj Später hat man übersichtlichere Methoden aufgefunden. Man läßt z. B. auf das z-Monochlorhydrin des Glyzerins das Natriumsalz einer Fettsäure einwirken und erhält dann einen Monof ettsäuregiyzerinester a CH, . OH GH.,' . = CH I . OH I ß CH . OH I . + Na Gl. OH i a CH2 .Gl + Na OOC C^^H^, a-MonochlorStearinsanres hydrin. Natrium. . GH. OC C,, H3, -/-Monostearinsäure. . . glyzerinester. Zu gemischten Glyzeriden gelangt man), indem man auf a-Monochlorhydrin ein Molekül des Chlorids einer Fettsäure einwirken läßt>>), z.B. Myristinsäurechlorid. Es entsteht der Monomyristinsäureester.') Nunmehr kann man noch Stearinsäurechlorid auf den Ester einwirken lassen. Die Stearinsäure besetzt die noch freie Alkoholgruppe (ß-Stellung). Jetzt wird durch Einwirkung von Silbernitrit das Chlor durch die OH-Gruppe ersetzt und die M CherreuJ : Recherches chirniquesJ sur les corps gras d'origiue auimale. (1823). Paris — -) Berthelot: /ahlreiche Arbciteu in den Ann. chim. 5 und weitere. Vgl. aucli die Übersiclit bei J. Bellticci : (iazz. Chiui. ital. 42. II. 283 (1912). =) Berthelot: Aun. de chim. (3). 41. 216 (1854). ) Guth: Zeitschr. f. Biol. K. F. 26. 78 (1903). Krafft: Ber. d. Deutschen Cheui. Gesellsch. 36. 4339 (1903). •') Ad. Grün und li. Schreyer; Ber. d. Deutschen Cheni. Gesellsch. 45. 342U (1912). Vgl. auch Albrecht r. Skopnik: In.-Diss. Zürich 1909. ") Wir geben diese Synthese wieder, weil sie einen guten Einblick in den Auf- — — bau der Glyzeride geben, obwohl es namentlich nach den Arbeiten von Emil Fischer, ü. Bergmann und Bärwind : Ber. d. D. Cbem. Gesellsch. 53. 1589 (1920). 3Ia.t Bergmann, Erwin Brand und F. Dreyer: Ebenda 54. 936 (1920), nicht feststeht, ob die Fettsäuren bei der erwähnten Art des Aufbaus in a-Stellung eintritt. Vgl. über die Synthese von Glyzeriden mit sicher festgestellter Struktur die Arbeiten von Emil Abderhalden und Egon Eichwald (siehe S. 216, Zitat 3) und die erwähnte Arbeit von Bergmann, Brand und Drei/er: ferner <'. Amberger wwd K. Broinu/: Biochem. Zeitschr. 130. — 252 (1922). Verbindimg' Fettstoffe uml ihre Bausteine. a-Myristo-ß-stearin ist fertig.') 215 Schließlich und ein drittes Molekül Fettsäurechlorid einwirken lassen Alkoholgruppe kanu man noch die letzte freie Die folgenden Formeln geben die Synthese des besetzen. a-Myristo-^-stearins Avieder CH., 7. OH + ClOC . . CHa Ci3 H.,7 . OC . . C13 H^^ I I = CH .OH iCH .OH CH, y. CH2 Cl a-Myristinsäureester des z-Monochlorhydrins. Gl . + HCl . y.-Mono- Myristinsäure- chlor- Chlorid. hydrin. CH, . OC . CH, Ci3 H,7 " . CH = CH CH.2 . . . . OC . C13 H,, . C,, H35 l I . . OH + ClOC Ci7 H35 . Cl CH., OC + HCl Cl . a-Myristinsäure[i-stearinsäureester des Stearinsäurechlorid. X- Mono chlor hydrins. Geht unter Einwirkung von Silbernitrit über in CH2 . . OC Ci3 üo? . CH .O.OC.C17H35 CH2 . OH a-Myristin-ß-stearin. Man kann ferner auf Dichlorhydrin und Trichlorhydrin Salze von Fettsäuren einwirken lassen und erhält dann di- und trisubstituierte Produkte: CH., . Cl -f Na OOC . CH^ R^) .OCR . = CH .OH I I CH .OH + 2NaCl. 1 I CH2 Cl + Na OOC R a-Dichlorhvdrin. . . CH2 . . OC R . z, ') Als Zwischenprodukt entsteht der Salpetrigsäureester: CH,' . OOC R CH,' . I . OOC R . = CH., OOC R, I CH OOC . . I CH., . R, " + Ag NO3 Cl . . ! CHj . . NO -h Ag Cl. Dieser wird mit Wasser leicht verseift: CH.." . OOC R . CHjj I CH," OOC R, . . I OOC R . := CH . OOC . R, I CHj O NO -h H.,0 CH2 OH HNO,. R bedeutet den Rest der C- und H- Atome einer beliebigen Fettsäure. . -) . I . . -I- ^I- Vorlesung. 216 Ganz entsprechend lassen sichx, i-Esterimda, z, ß-Esteraus a, ß-Diehlorhydrin CH2 CH . . CH, Cl Cl Cl . und Trichlorhydrin CH : . gewinnen. Cl CH, OH CH, Cl Wir haben diese Synthesen deshalb so ausführlich wiedergegeben, weil . . um sich die Konstitution einer Verbindung einzuprägen, als die ^'ertiefung in ihren Auf- und Abbau. Von der eben gegebenen Grundlage aus können wir nunmehr mit Leichtigkeit die Struktur der verschiedenartigsten Eette verstehen. Früher war man der Ansicht, daß ein bestimmtes Fett nur eine bestimmte Fettsäureart enthalte. In der Tat gibt es derartige Fette. Es gibt Glyzeride, die drei Moleküle Stearinsäure an Glyzerin gebunden enthalten. Man hat solche Fette Tristearin genannt. Ferner können drei Moleküle Palmitinsäure vorkommen. Die Namen Tripalm itin, Triolein (drei Moleküle Ölsäure), ferner Triacetin, (drei Moleküle Essigsäure), Tributyrin, Trilaurin, Trimyristin usw. bedeuten immer, daß Verbindungen von Glyzerin mit drei Molekülen der entsprechenden Fettsäuren vorliegen. Als Beispiel wollen wir die Konstitutionsformel des Tripalmitins es keine bessere Methode gibt, wiedergeben GH., . OH HOOC . CH .OH + HOOC.CisHo^ CH., . OH Glyzerin. HOOC . CH, O OC C\, H3, . . . C^^ H3, = CH .O.OC.C15H3, + 3 Hj O. Ci5 H31 CH, . Drei Moleküle Palmitinsäure. O OC . . C,^ H31 Tripalmitin. Ein Molekül Glyzerin tritt mit drei Molekülen Fettsäure zusammen. Die Veresterung erfolgt unter Wasseraustritt. Es liegt genau der gleiche Vorgang vor, wie wenn z. B. ein einwertiger Alkohol mit einer Säure sich esterartig bindet. So entsteht z. B. der Propionsäureäthylesteri), wie folgt C, H^ + H., 0. CH, COOH + OH C, H, = CH3 CH., CO Umgekehrt erhält man aus dem Ester wieder seine Anteile, wenn man ihn unter AVasserauf nähme spaltet. Tripal- CH3 . . . . . . . mitin zerfällt unter Aufnahme 'von drei Molekülen Wasser in ein Molekül Glyzerin und drei Moleküle Palmitinsäure. Man nennt die Hydrolyse von Fetten auch Ve\|rseifung. Meistens führt man sie durch Kochen mit Alkali herbei. Man erhält dann neben dem Alkohol nicht direkt die Fettsäuren, sondern deren Salze. Die Salze der Fettsäuren sind Seifen genannt worden. Man spricht von Alkaliseifcn oder spezieller von Natrium') Nach der allgemeiuni Definition der F'ette, wonach sie esterartige Verbindungen von Fettsänren mit Alkoholen sind, ist auch diese Verbindung ein Fett, doch werden mit der Bezeichnung Fett meist gleichzeitig bestimmte Eigenschaften mit einge- schlossen. P^ine auf die eigcntliclien Fette begrenzte Definition dieser Körperklasse läßt sich kaum geben, es sei denn, man scliließe die ein- inul zweiwertigen, einfacher ge- bauten Alkohole ganz aus. Fettstoffe uuil ihre Bausteine. 217 Kalium- oder Lithiumseife, ferner von einer Kalkseife, wenn ein Kalksalz vorliegt usw. Wie wir schon angedeutet haben, kommen neben den eben besproFetten auch solche vor, bei denen die Alkoholgruppen des Glyzerins mit verschiedenen Fettsäuren verbunden sind. So gibt es Fette, die z. B. zwei verschiedene Fettsäuren enthalten. Die eine chenen davon muß dann zweimal vertreten sein. So kennen wir ein Fett, das aus einem Molekül Glyzerin, einem solchen von Stearinsäure und zwei Molekülen Palmitinsäure besteht. Es ist aus Talg^) und auch synthetisch erhalten worden. Ein solches Fett bezeichnet man als Stearo-dipalmitin. Dieser Xame gibt uns noch keine Auskunft über die Stellung der einzelnen Fettsäuren im Molekül des Glyzerins. Wie die folgenden Formeln zeigen, in denen die Stearinsäure mit S und die Palmitinsäure mit P bezeichnet ist, gibt es zwei isomere Verbindungen der gleichen Zusammensetzung: GH., . O .P GH., 1 CH . () . P ! . O P oder . GH . O . S I I GH., . () . GHo.O.P S II. I. Es genügt diesen Fällen die Hydrolyse und die genaue Bestimder Art und Menge der Spaltprodukte nicht, um die Konstitution mung in des Ausgangsmateriales festzustellen. Nur die Synthese der beiden stereoisomeren Formen und das Studium ihrer Eigenschaften kann eine Entscheidung herbeiführen. Form I muß übrigens optisch aktiv sein, denn sie enthält ein asymmetrisches Kohlenstoffatom (). -) Optisch-aktive Fette sind synthetisch dargestellt worden. 3) Sie drehen sehr schwach. Alle gemachten Beobachtungen lassen es wenig wahrscheinlich erscheinen, daß in der Natur optisch-aktive Glyzeride, die im Glyzerylrest asymmetrisch gebaut sind, eine Rolle spielen. Bei asymmetrischem Glyzerylrest ist außerdem zu erwarten, daß beide optisch-aktiven Formen ohne Bevorzugung der einen entstehen, und es so zur Bildung von inaktiven Razemkörpern kommt. Ein Fett kann bei symmetrisch gebautem Glyzerylrest optisch-aktiv sein, wenn es optischaktive Fettsäuren als Bausteine aufweist. Derart optisch-aktive (ilyzeride dürften schon eher in der Organismenwelt anzutreffen sein. Aus Hammel- und Rindertalg wurde z - Palmito- a, ß - distearin gewonnen.) Bömer-') entdeckte es auch im Schweinefett. Die aus Hammeltalg erhaltene Verbindung ist jedoch nicht identisch mit dem Palmito-distearin des Schweinefettes. Es muß die Stellung der Fettsäuremoleküle im Glyzerin eine verschiedene .sein. Im Schweinefett kommt auch ein Stearo-dipalmitin vor. Im Menschenfett soll Dioleo-stearin enthalten .sein. Auch einOleo') Willy Hansen: Arcli. f. Hvg. 42. 1 (1902). -) Vgl. "hierzu S. 19 ff. ') Vgl. Emil Abderhalden und E(/on Eichuald^Bor. tl. Deutscheu Cheni. (ies. 47. 1856, 2880 (1914); 48. 113. 1847 (1915). ) \'gl. liierzu Hans Kreis und Auf/K.st Hafner: Ber. d. Deutschen Chem. Gesellsch, Vgl. auch .7. Klimon't und E. Meisrls: Mouatsh. f. Chemie. 36. 1123. 276f) (1903). — ' 30. 341 (1909). ^) A. bünier: Zeitschr. f. Uutersuch. d. Xahiungsin. 25. 331 (1913). A. Bötnerxmd H. Merfen : Ebenda. 43. 101 (1922). — Vgl. auch XI- Vorlesung. 21 distearin ist bekannt. Im Gänsefett ist ferner ein a. - Stearo - x, ß - dipalmitin festgestellt.!) Es unterliegt keinem Zweifel, daß man noch auf manche derartige Fette treffen wird. Besonders interessant sind Fette, an deren Aufbau drei verschie- dene Fettsäuren beteiligt sind. So sind ein Palmito-oleo-stearin und ein Oleo-palmito-butyrin beschrieben worden. Es ist jedoch noch nicht eindeutig genug festgestellt, ob die betreffenden Verbindungen einheitlich waren. Bei drei verschiedenen Fettsäuren, die wir mit S, P und bezeichnen wollen, sind die folgenden stereoisomeren Formen möglich: M CH.2 CH . . () . . M P CH, . . P . CH, . . M Fettstofle und ibre Bausteine. 219 ist der Charakter der es aufbauenden Fettsäuren aufgeprägt. Fette, ganz aus Palmitin- oder Stearinsäure bestehen, haben einen relativ hohen Schmelzpunkt und sind bei gewöhnlicher Temperatur fest. Das Vorkommen von Ölsäure bedingt flüssige Fette. Man hat die flüssigen Fette auch Öle genannt Olivenöl. Rizinusöl usw. Sind verschiedene Fettsäuren mit Glyzerin verbunden, dann sind bei den aus ihnen bestehenden Giyzeriden möglichen Variationen in den physikalischen Eigenalle schaften, je nach ihrer Art und Menge, zu erwarten. Schon der Umstand, daß am Aufbau der Fette sich verschiedene Fettsäuren beteiligen können, und im einzelnen Molekül wiederum nicht die gleichen Komponenten sich zu wiederholen brauchen und in diesem Falle stereoisomere Formen möglich sind, zeigt uns, daß die Fette durchaus nicht, wie man früher anzunehmen geneigt war, eine wenig abwechslungsreiche Gruppe von Verbindungen darstellen. Sie können im Gegenteil außerordentlich mannigfaltig in ihrem Aufbau sein. Rechnet man hinzu, daß die Fette offenbar nie in reinem Zustande vorkommen, sondern unter sich in der mannigfaltigstens Weise und in ganz verschiedenen Mengenverhältnissen gemischt sind, dann versteht man, daß jede Tierart ihr eigenes Fett haben kann und innerhalb jedes einzelnen Organismus wiederum bestimmte Zellarten über eigenes Fett verfügen können. Jenes Fett, daß am Aufbau der Zellen teilnimmt, dürfte wohl einen besonders eigenartigen Aufbau aufweisen oder doch durch die Art seiner Mischung ein spezifisches Gepräge besitzen. Es sei hier schon kurz gestreift, daß manche Fettgemische in ganz geringen Mengen Stoffe noch unbekannter Natur gelöst enthalten, die einen bedeutsamen Einfluß auf das Wachstum und manche andere Zellfunktionen haben. 1) Wir kommen in Band II, in der Vorlesung XXIII auf diese Stoffe noch zurück. Ihre Kenntnis ist für die Abschätzung des Nährwertes von Fetten von großer Bedeutung. Es kann eine Fettart biologisch wertvoller erscheinen als eine andere, weil sie jene noch unbekannten Wachstumsstoffe" enthält und die andere nicht, obwohl vielleicht die Fette als solche, ohne jene Produkte gleichwertig sind. Fette die — ,, Die Fette sind im Pflanzen- und Tierreich ganz außerordentlich verbreitet. Es dürfte wohl keine Zelle geben, die nicht Fett besitzt. Es kommt zum Teil frei in der Zelle eingelagert vor und stellt in dieser Form offenbar einen Reservestoff, etwa entsprechend dem Glykogen, dar. Daneben findet sich immer Fett, das man nicht ohne weiteres nachweisen kann, weil es Beobachtungen nach wohl an andere Zellbestandteile gebunden ist. Tatsache ist von weittragender Bedeutung. Der Fettgehalt einer Zelle darf niemals nur nach dem direkt nachweisbaren Fette eingeschätzt werden. Es muß vielmehr neben dem direkt durch bestimmte Lösungsmittel abtrennbaren Fett auch jener Anteil festgestellt werden, der infolge seiner Bindung nicht in dieses übergeht. Um das gesamte Fett quantitativ bestimmen zu können, wird das zu untersuchende Gewebe nach erfolgter Trocknung mit Schwefelkohlenstoff, Tetrachlorkohlenstoff oder einem andern Lösungs- allen Diese mittel für Fette vollständig erschöpft. Dann spaltet man das vom „freien^' Fett befreite Gewebe mittelst Magensaft oder verdünnter Säure und zieht — 1) Vgl. u. a. F. G. Hopkins: J. of Physiol. 44. 425 (1912). Stepp: ErgebH. Aron: Biochem. nisse der inneren Medizin und Kinderheilkunde. 15. 257 (1917). Zeitschr. 92. 211, 103. 172 (1920). S. Rosenbuum: Ebenda. 109. 271 (1920). — H. Aron und B. Gralka: p:beuda. 115. 188 (1920). — — ^I- Vorlesung. 220 dann nochmals mit Fette lösenden Flüssigkeiten aus. Man erhält so die Menge des gebundenen Fettes. Die Summe des freien und des gebundenen Fettes ergibt dann die Gesamtmenge an Fett oder, genauer ausgedrückt, jener Substanzen, die in dem angewandten Lösungsmittel löslich sind. Wir kennen nämlich zur Zeit kein solches, das nur Fette aufnimmt. Infolgedessen darf man auch nicht einfach den Rückstand, der nach dem Verdunsten des Lösungsmittels verbleibt, als Fett in Rechnung setzen. Er enthält vielmehr neben diesem Cholesterin, Cholesterinester und Phosphatide. Eine genaue Fettbestimmung erfordert große Kenntnisse. Man hat vorgeschlagen, nicht die Fette als solche, sondern nach ihrer Spaltung die gebildeten Fettsäuren zu bestimmen, doch ist auch diese Methode nicht ganz genau, weil die l'hosphatide auch Fettsäuren liefern, ferner können Verbindungen zwischen solchen und Cholesterin vorhanden sein. Die Menge der Phosphatide kann man ziemlich genau bestimmen indem man als Maßstab ihren Baustein Phosphorsäure nimmt. Diese Bemerkungen sollen nur andeuten mit welchen Schwierigkeiten genaue Untersuchungen über den Fettgehalt von (Jeweben und Zellen ver, , knüpft sind. Sie lassen es verständlich erscheinen, weshalb auf dem Gebiete des Fettstoffwechsels so viele einander widersprechende Ergebnisse vorhanden sind. In den wenigsten Fällen sind ausreichende Methoden zur Anwendung gekommen. Vielfach hat man sich sogar mit Farbreaktionen begnügt und nach ihrem Ausfall den Fettgehalt geschätzt. P]s haben nämlich die Fette die Eigenschaft, mit manchen Farbstoffen charakteristische Färbungen anzunehmen. Selbstverständlich können derartige Methoden nie ([uantitative sein. Außer jenen Körperzellen, die neben anderen Stoffen auch in relativ geringen Mengen freies Fett aufweisen, gibt es noch solche, welche in der Hauptsache aus solchem bestehen. Man hat sie kurzweg als Fettzellen bezeichnet. Sie haben offenbar in besonders ausgesprochenem Maße die Fähigkeit, Fett zu speichern. Meistens finden sich viele solcher Zellen vereinigt. Sie bilden das sogenannte Fettgewebe, das im tierischen Organismus insbesondere im Unterhautbindegewebe und im Mesenterium oft in großer Masse auftritt. Auch in der Pflanzenwelt kommen solche Gewebe vor. Oft werden die Pflanzensamen mit großen Mengen von Fett ausgerüstet. Der tierische Organismus verwendet das Fettgewebe, wie wir noch erfahren werden, auch zu mechanischen Zwecken, z. B. als Schutz gegen Druck. Ferner spielt es vor allem als schlechter Wärmeleiter im Wärmehaushalt eine wichtige Rolle. Wenn man im allgemeinen vom Fette eines Tieres spricht, dann man jenes Fettgemisch, das die Fettdepots, das Fettgewebe, erfüllt. meint Man spricht von Menschenfett, von Hammeltalg, von Pferdefett usw. und charakterisiert es zunächst durch jene Temperatur, bei der es schmilzt, oder, wenn man es zum Schmelzen gebracht hat, wieder erstarrt. Jede Fettarfi) hat unter normalen \'erhältnissen einen ziemlich konstanten-) Schmelzbzw. Erstarrungspunkt. Die meisten derartigen Fette sind bei Körpertemperatur halbfest. Es kommen jedoch auch richtige Öle im Tierreich vor, es sei z. B. an den Lebertran erinnert. Er besteht aus den Fettsub') Nicht zu verwechselu mit dem chomischon Begriff eines liostinimtoii. eiiilieitJede ..Fettart" stellt ein lifstiminfes (iemisc.li von Fetten dar. Vgl. liifizu A'/. Grihi und ,1. Ciisladis: Hci'. d. Dciitsciicn Clicni. (icscliscli 45. lichen Fettes '') 3()t)l (lOl-i). ! Fettstotle uml 221 ihre liausteiue. stanzen der Leber von Fischen. Es seien die Schmelzpunkte^) einii^er der wichtigsten Fettarten angeführt"-): Schmelzpunkt Meuschenfett Gänsefett IT'ö" 26--;-i4'' Butterfett Pferdefett 28—330 Schweinefett Hühnerfett 3(j ca. 40« — 46" 33—40« 37— 40« Hundefett Hindertalg Hammeltalg 41—49« 44—51« Die angeführten Werte sind nicht so zu verstehen, als ob jede Tier- bestimmte Keservefettart verfügte. Es hat sich vielmehr gezeigt, daß das Fett ein und desselben Tieres je nach der Körperstelle, der es entnommen worden ist, sich verschieden verhält. Htnriques und Hansen'^) haben durch systematische \'ersuche festgestellt, daß das dicht unter der Haut liegende Fett den tiefsten und das im Inneren des Körpers sich befindende den höchsten Schmelzpunkt hat. Das Fett des Fettgewebes schwankt in seiner Zusammensetzung auch erheblich je nach der Art der Ernährung. Ferner scheint das Klima von Einfluß zu sein. art nur über eine Während die Kohlehydrate im allgemeinen sehr beständig sind, zeigen Liegen an der Luft Veränderungen. Zunächst ist zu bemerken, daß die Fette als solche in den Geweben sehr oft von Fettsäuren begleitet sind. Diese weisen ohne Zweifel auf Umsetzungen hin. Über sie führt der Auf- und Abbau von Fett. Man hat für die reinen Fette auch den Namen Neutralfett eingeführt. Auch solche zeigen sehr oft sekundäre Veränderungen. Man spricht von einem Ranzigwerden des Fettes.) Das Fett nimmt dabei sehr oft einen unangenehmen Geruch an. Auch sein Geschmack leidet stark. Das Ranzigwerden der Fette beruht auf komplizierten Umwandlungsvorgängen. Es sind noch nicht alle erkannt, nur soviel ist sicher, daß hydrolytische und oxydative Vorgänge zusammenwirken. Es scheint vor allem dem Lichte eine bedeutsame Rolle bei diesen Veränderungen des Fettes zuzukommen. Besonders leicht werden jene Fette umgewandelt, an deren Aufbau ungesättigte Fettsäuren beteiligt sind. Es entstehen 0.\vfettsäuren und auch Aldehyde aller Art. Ranzige Fette bilden beim Schütteln mit Wasser eine recht beständige Emulsion. viele Fette schon beim ) Die Fette sind uoch auf andere Weise charakterisiert worden. So h;it man B. festgestellt, wieviel Jod ein bestimmtes Fett aufzunehmen vermag. Mau nennt diesen Wert die Jodzahl. Alle diese indirekten Methoden, ja selbst die Schmelzpunktsbestimmung sind zurzeit nur Notbehelfe. Genaue Fettbestimniungsmethoden werden erst zur Verfügung stehen, wenn es gelungen ist, die Fette einzeln in chemisch reinem z. Zustande abzutrennen. Vgl. die Tabellen bei F. Ulzer und ./. Klimont: Allgemeine und physiologische J. Springer. Berlin. 190(5. ^) Henri</ties und Hansen: Chem.-Ztg. Kepetit. 20(1900). ) Vgl. hierzu u. a. Kitserl : Untersuchungen über das Kanzigwcrdeu der Fetto. Ferd. Diimmler. Berlin 1890. Lafar: Bakteriologische Studien über die Butter. über das Ranzigwerdeu r. Kleclci: Untersuchungen R. Oldenbourg. München 1895. der Butter. Th. Stauffer. Leipzig 1894. ^) Chemie der Fette. — — ^^- VorlesuiJtr. 222 Überblicken Nvir das, was wir über die verschiedenartigen Kette kennen gelernt haben, dann ergibt sich folgendes. Die Fette stellen ganz allgemein esterartige Verbindungen zwischen Alkoholen und Fettsäuren dar. Je nach der Art der Alkoholkomponeute lassen sich die folgenden Gruppenabgrenzen: l.Die eigentlichenFette. Sie sind Fettsäure-glyzerinester, und zwar bestehen die gewöhnlichen Fette aus Triglyzeriden. Diese lassen sich, je nachdem nur eine Art von Fettsäuren an ihrem Aufbau beteiligt sind oder verschiedene, in: a) gleichsäurige homoazide Fette und h) verschiedensäurige heteroazide Fette einteilen. Diese letzteren zerfallen wieder in zwei Untergruppen, je nachdem zwei oder aber drei verschiedene Fettsäuren am Bau des Fettes teilnehmen. Es seien diese für das Verständnis der Mannigfaltigkeit des Aufbaues der Fette grundlegenden Tatsachen durch die folgende Darstellung noch einmal in einfachster Weise verständlich gemacht. G bedeutet den dreiwertigen Alkohol Glyzerin. A, B und C sind verschiedene Fettsäuren. Es sind nun folgende Möglichkeiten gegeben: = G^A \A = Fettstofte und ihre Bausteine. 223 Wir unterscheiden ferner Fette, an deren Aufbau hochmolekulare, aliphatische, einwertige Alkohole und eine Fettsäure beteiligt sind. Diese Fette sind auch Wachse genannt worden. Wir kommen in der nächsten Vorlesung auf sie zurück. Endlich kennen wir, wie wir gleich erfahren werden, Fette, an deren Bau zyklische, einwertige Alkohole teilnehmen. Der typische Vertreter dieser Klasse von Fetten ist die Verbindung zwischen Cholesterin und einer Fettsäure. Es unterliegt keinem Zweifel, daß die weitere P'orschung diese Reihe erweitern wird. Es können sicher noch andere, z. B. zwei- und auch mehr als dreiwertige Alkohole Fette bilden. Gemeinsam ist allen diesen Fettarten ein sehr ähnliches physikalisches Verhalten und ferner der Umstand, daß sie Alkohol und Fettsäure sich unter Wasseraufnahme in ihre Anteile zerlegen lassen. Umgekehrt können sie aus den Bausteinen unter Wasserabspaltung gebüdet werden. Wir haben somit ganz entsprechende Beziehungen der Muttersubstanzen zu ihren Bausteinen, wie bei den Kohlehydraten. Ab- und Aufbau vollziehen sich hier, wie dort, unter Wasseraufnahme bzw. W^asserabspaltung. — — Vorlesung XII. Fette mit lioclimolekiilarem einwertigem Alkohol als Baustein: Wachse. Sterinester. Sterine. Gallensäuren. Wir haben Fettsäuren bereits auf Grund der Möglichkeit, daß verschiedenartige am Aufbau einer Fettart beteihgt sein können, darauf hingedaß die Zahl der verschiedenartigen Fette eine sehr große sein kann. Nun kann außerdem noch, wie schon erwähnt^), die Alkoholkomponente wechseln. In der Tat hat man in der Natur zahlreiche Verbindungen angetroffen, an deren Aufbau Fettsäuren und ferner ein anderer Alkohol als das Glyzerin beteiligt sind. Diese Fettarten zeichnen sich durch ihre besonderen Eigenschaften mehr oder weniger stark gegenüber den Glyzerideu aus. Man hat die meisten dieser Körper unter dem Namen Wachse zusammengefaßt. Sie kommen im Pflanzen- und Tierreich vor. Ea handelt sich, soweit unsere Kenntnisse reichen, stets um esterartige Verbindungen zwischen einem Molekül Fettsäure und einem einwertigen, hochmolekularen Alkohol. Es seien die wichtigsten dieser Verbindungen hier angeführt. Im Walrat, auch Cetin genannt, einer fettartigen Substanz, die sich im Schädel der Pottwale findet, ist Palmitinsäure-cetylester aufgefunden worden. 2) Er zerfällt bei der Hydrolyse in Palmitinsäure und Cetylalkohol, CH3 (CH.,),, CH, OH: wiesen, . . Cie H,3 . OC . . Ci5 H31 4- H, Palmitinsäure-cetyl- = C« H33 OH -h HOOC C,, H3, . . Cetylalkohol. Palmitinsäure. ester. Im Bienenwachs ist der Palmitinsäure-myricylester, C30 Hr, O.OC.CjsHsi, enthalten. . Eine Verbindung von Octadecetylalkohol, CH3 (CH2)i6 ('H.^ OH, mit Fettsäuren ist in dem Sekret der Bürzeldrüse der Vögel enthalten. 3) Aus chinesischem Wachs und aus Opiumwachs konnte der Cerotinsäure-cerylester, Cje H^ OC C25H5,, abgeschieden werden. Der gleiche Alkohol, mit Palmitinsäure verknüpft, findet sich im Mohnwachs. . . . 238. ') S. -) Heintz; Ber. ^) /'. d. Deutschen Chem. Gesellscli. 10. 3023 (1892). Röhmann: Hofmeisters, Beiträge 5. llü (1904). . . Fi'tte mit liocliiiiülekularcm oinwertigeni Von besonderem Interesse sind Alkohol esterartige als Baustein. 22^ \ erbindungen zwischtu der solche Ester nachwies, war E. SchulzeS) Er fand im Wollfett der Schafe Cholesterinester. Es wurden dann solche auch in verschiedenen epidermalen Gebilden (Haare, Nägel, Hufe, Hörner, Federn) aufgefunden.-^) Endlich hat Hürthlt^) im Blute den Palmitinsäure-cholesterinester festgestellt. Auch ein dem Alkohol Cholesterin und Fettsäuren. Der erste, Ölsäureester kommt in diesem vor. Im Wollfett ist ein Stearinsäureester beobachtet worden. Man hat diesen Fettarten erst in neuerer Zeit größere Aufmerksamkeit gewidmet und gefunden, daß sie sehr verbreitet sind und namentlich auch bei Verfettung von (ieweben eine große Rolle spielen. Pflw^er) erkannte als erster Cholesterinester in verfetteten Nieien. Diese Ester fallen dadurch auf, daß sie doppelbrechend sind. Sie scheinen regelmäßig eine Vermehrung zu erfahren, wenn Zellen verfetten. Die Cholesterinfrttsäureester rechnet man nicht zu den Wachsen. Sie bilden innerhalb der Fette eine Gruppe für sich und unterscheiden sich von den übrigen bis jetzt l)ekannten Fettarten in erster Linie dadurch, daß sie als Baustein einen zyklischen Alkohol besitzen. Das Cholesterin gehört zu der Klasse der Sterine. Unter diesem sind alle dem Cholesterin verwandten Verbindungen zusammengefaßt worden. Die Sterine sind im Tier- und Pflanzenreich sehr verbreitet. Sie kommen auch im freien Zustande vor. Es scheint, daß jede Zelle Sterine und wahrscheinlich auch Sterinester enthält. Die Sterine des Tier- und Pflanzenreiches sind verschieden. Man hat die dem letzteren zukommenden Vertreter dieser Körperklasse auch ganz allgemein als Sammelnamen Phytosterine bezeichnet. die Sterine des Tierreiches Dieser Einteilung entsprechend könnte mau Zoosterine nennen. Bei den Wirbeltieren im wesentlichen nur ein Sterin, nämlich das Cholesterin, vorzuDas IsoCholesterin ö), das im Wollfett, auch Lanolin genannt, gefunden worden ist und ferner das sogenannte Koprosterin des Kotes sind Umwandlungsprodukte des Cholesterins. Auch bei den Vögeln, Reptilien und Fischen konnte bis jetzt kein anderes Sterin als das Cholesterin isoliert werden. Besondere Sterine finden wir meist neben dem gewöhnlichen Cholesterin bei den Wirbellosen. "^j So ist das aus Puppen von Bombyx mori, dem Seidenspinner, gewonnene Sterin nicht identisch mit Cholesterin. Es ist Bombicesterin genannt worden. Im scheint kommen. E. Schulze: Ber. d. Deutscheu Chem. (jesellsch. 5. 1075 (1871); 6. 251 (1872). Oskar Liehreich: Jahresbericht über die Fortschritte der Chemie. 2164 (IH^O). ') Karl Hürthle: Zeitschr. f. physiol. Chem. 21. 331 (1895/96). Vgl. anch FJnriuird llepuer: Pßüqer^ Archiv. 73. 595 (1898). Vgl. auch Felix Kauders: Biochein. ') -) — — Zeitschr. 55. 96' (1913). ) Panzer: Zeitschr. f. physiol. Chem. 48. 519 (19(J()) und 54. 239 (1907/08). Vgl. feruer Aschoff; Münchener med. Wochcnschr. Nr. 7 (1910). A. Wiudaus: Zeitschrift f. physiol. (^Ihem. 68. 110 (1910). Leonhard Wacker: Zeitschr. f. physiol. Chem. — 80. — — 383 (1912). E. Schulze: Ber. d. Deutscheu Chem. Gcsellsch. 5. 1075 (1871); 6. 251 (1872). prakt. Chemie. (2). 7. 163 (1873). - A. Moreschi : Atti K. Acc. dei Lincei. Rom. (5). 19. II. 53 (1910). ^) Vgl. die Literatur (Doree, Leirkowitsch, Uetize, Hesse, Buriaii, Uitter, Cohen, Tanret, A. Menozzi und A. Moreschi u. A.) bei A. Wiudaus: Untersuchungen über Cholesterin. Arch. f. Pharm. 246. 117 (1908). JJauth: Inang.-Diss. Freiburg 1907. August Welsch: luaug.-Diss. Freiburg 1909. Über LujxMd vgl. yardus Henri Cohen: Inaug.-Diss. Utrecht 1906. 5) — E. Schulze: Journal f. — — Abele rh al (1h n . Thysiologischo Chemie. I. Teil, .'>. — .AuH. 15 XII. Vorlesung. 2i>6 Meersch wamm, Suberite.s domuncula. findet sich ein Spongosterin genanntes Sterin. Viel mannigfaltiger sind offenbar die Sterine der Pflanzenwelt. Wir kennen schon jetzt eine ganze Anzahl Vertreter dieser Körperklasse. Das scheint den Pflanzen vollständig zu bis jetzt in tierischen Geweben noch keine Phytosterine aufgefunden worden.^) Sehr verbreitet ist von den Phytosterinen das Sitosterin, 0,7 Utf, + H2 0. Es steht wahrscheinlich dem Cholesterin sehr nahe und scheint nur stereomer von ihm verschieden zu sein.'-) Aus den Calabarbohnen ist Stigm asterin, + H., 0, gewonnen worden. Von den eingehender untersuchten Cjn H4g Phytosterinen sind noch das Lupeol, das 7.- und ß-Amyrin und das Euphorbon zu nennen. Endlich ist zu erwähnen, daß im Mutterkorn eigentliche Cholesterin umgekehrt sind fehlen, Ergosterin vorkommt. Dieses Sterin in ist allen bis jetzt unter- aufgefunden worden. Neben dem Ergosterin enthält das Mutterkorn noch das Fungisterin. Es sind noch mancherlei Phytosterine beschrieben und mit besonderen Namen belegt worden, ohne daß jedoch stets der Nachweis erbracht worden wäre, daß die betreffenden Verbindungen völlig rein waren. Dazu kommt noch daß wir zurzeit die Konstitution der Sterine nicht vollständig kennen. Es ist wohl möglich, daß manche der zu den Sterinen gerechneten Verbindungen zu anderen Klassen von Substanzen gehören. suchten Pilzen , Am eingehendsten untersucht ist bis jetzt das Cholesterin. Es zyklischer, ungesättigter, einwertiger, sekundärer Alkohol. Es kristallisiert aus Chloroform oder wasserfreiem Äther in feinen, seidenglänzenden Nadeln. Aus wässerigem Alkohol bildet es charakteristische, große, rhombische Tafeln. Sie fühlen sich fettig an und glänzen perlmutterartig. Cholesterin ist in Wasser unlöslich, löslich in Äther, Chloroform und Benzol. Auch in Alkohol löst es sich besonders leicht in warmem. Cholesterin ist optisch aktiv. Am Lichte verändert es sich. 3) Besonders wichtig ist seine Eigenschaft, mit verschiedenen Verbindungen, so mit Saponin) Seifenstoffglukoside der Pflanzenwelt-^) eine Verbindung einzugehen. Für den quantitativen Nachweis des Cholesterins neben Cholesterinester ist besonders bedeutungsvoll die Beobachtung geworden, daß ein Molekül davon sich mit einem solchen von kristallisiertem Digitonin bindet. ist ein , — — Es sind zahlreiche Farbenreaktionen des Cholesterins bekannt. Löst man z. B. Cholesterin in Chloroform und wird mit konzentrierter Schwefelsäure unterschichtet, dann färbt sich die Chloroformlösung rot, während grün fluoresziert (Salkoivskii^ Probe). Fügt man zu einer Lösung von Cholesterin in Chloroform tropfenweise Essigsäureanhydrid und konzentrierte Schwefelsäure, dann färbt sich die Flüssigkeit zudie Schwefelsäure dann violett, blau und schließlich dunkelgrün {LiehermannBurchardü Reaktion). Übrigens sind diese Fai'breaktionen durchaus nicht erst rosenrot, ') ') ') Vgl. dazu aucli jMary Taylor Ellis: Biocliem. Jouru. 12. 154. 1(>0 u. 173 (liMS). A. Windaus iiiid K. RakUn: Z. f. physiol. Chem. 101. 22:5 (15118). E. Schulze und E. Wintersfein: Zeitschr. f. physiol. Chemie. 43. Sltj (1;K)4): 48. 546 (1906). ) ') Berlin Windaus: Ber. d. Deutschen Chem. Gesellsch. 42. 23S (190i)). Biochem. Haudlexikon. 7. 14.t (1912) (bearbeitet von Kohrrf). .1. A. 1912. Spriiiircr. Fette mit liochmolekularom ciDwertigom Alkohol als Baustein. für C'holesterin charakteristisch. Man rinen, sondern auch mit Harzsiiuren Ste- allen Das Cholesterin ist zuerst von Coiiradi^) und CrVew^) aus Gallensteinen worden. Ks macht oft bis zu 90% iind mehr dieser aus der (ialle nach ihrem Aussehen Steine genannt abscheidenden I'rodukte i,'-evvonnen sich erhält sie nicht nur mit und Terpenalkoholen. 227 — genauer untersucht. Er nannte es seinen ganzen p]igenschaf ten nach (lallenfett, Cholestearin. Erst später entdeckte man, daß das Cholestearin als solches seiner Struktur nach mit den Fetten nichts zu tun hat. Der Name Cholestearin ist zweckmäliig in Cholesterin verändert worden, um den Eindruck zu verwischen, als käme mit dem Namen ein Zusammenhang mit den Fetten zum Ausdruck. Bald fand man dann das Cholesterin in allen )rganen. Besonders reich an dieser Verbindung ist das Cehirn (ca. löVo fies trockenen Corpus callosum). In der Lebergalle findet man ca. e^/o Cholesterin. In den verschiedenen Organen kommen etwa 0'2 0"5Vo dieses Alhohols vor. Das Cholesterin hat die empirische Zusammensetzung C.27H46O. Von der Feststellung der einfachsten Formel einer Verbindung bis zur Aufklärung der Funktion und Stellung jeder einzelnen Atomgruppe ist meistens ein sehr weiter Weg zurückzulegen. Das kommt einem ganz besonders zum aus. Chevreul hat das Cholesterin < — Bewußtsein, wenn man die Chemie des Cholesterins verfolgt. Es bedurfte langer, unausgesetzter Bemühungen, um die Struktur des Cholesterins in d^n wesentlichsten Punkten aufzuklären.'^) Bis jetzt steht vor allem dank den Untersuchungen von A. Windaus folgendes fest. Der Sauerstoff ist in einer Hydroxylgruppe vorhanden. Schon frühzeitig wurde erkannt, daß Cholesterin sich verestern läßt.) Damit war das Vorkommen einer AlkoholCH (JH. gruppe bewiesen, und zwar handelt es sich um die (iruppe Durch die (Jxydation dieser sekundären Alkoholgruppe wurde das Keton Cholestenon erhalten.^») Cholesterin enthält eine Doppelbindung.6) Es geht unter Addition von einem Molekül Wasserstoff in den gesättigten sekundären Alkohol Dihydrocholesterin (auch ß-Cholestanol genannt), C.^y H48O, über.') Dieser kann leicht in den gesättigten Stammkohlenwasserstoff Cholestan, C^yH^g, verwandelt werden.^) Cholesterin enthält ein System von Ringen. Die 1 "Überlegung, daß Cholestan acht Wasserstoffatome weniger enthält, als das entsprechende gesättigte Paraffin, CsyHße.' führt zu der Annahme, daß im Cholesterin vier hydrierte — . Conradi: Inaug.-Diss. Jeoa 1775. Gren: Inang.-Diss. Halle 1788. Windaus, O. Diels und E. Ahdri^) Vgl. die Literatur (Maiithner und Stada, halden, L. Tsckugajew und Ä. Gasteß' und \V. Fomin, Richard Willstätter und Enrin W. Mayer u. A.) im Biochem .Handlexikon. 3. ^68 (1911) (bearbeitet von Ad. Windmis). Gustav Sfein: Inaug.-Diss. Freiburg Ad. Windaus: Hab.-Schrift. Freibnrg 19U2. Von neueren Arbeiten seien genannt: A. Windaus und C. Resan: Ber. d. Deut1905. ^. Windaus und C. (Hbrig: P'.beiida. 40. 2487 schen ehem. (;csell.s(:h. 46. 1240(1913). .1. Windaxx: P^bpuA. Windaus und <). Dalmer: P^benda. 5'2. ir)2(1919). (1913). <la. 52. 170 (1919). ••) Berlhelnt: Ann. chim. et phvs. 56. 51 (1850). ) 0. Diels und K. Abderhalden :liQY. d. Deutschen Clicm. (lesellsch. 37. H099 (1904). O. r. Fürth und Gustav Erlsenrcich : Biochem. Zeitsclir. 69. «) Vgl. hierzu auch 416 (1915). ') R. Willst üttcrnwA If J/r<//cr.- Ber. d. Deutschen Chem. (iesellsch. 41. 2199(1908). ») <>. Diels und Linn : Ber. d. Deutschen Che/n. (iesellsch. 41. 548 (1908). iiithiur: Momitvh. f. Chemie. 30. (;;i8 (T.K)'.)). ') ^) — — — — — — '. M 15-'^ XII. Vorlesuiiir. 228 Ringe vorhanden sind. Da nun die Oxydation von Cholesterin zu einer Dikarbousäure, CoTHiiO^, ohne Spaltung des Moleküls in kleinere Bruchstücke lisch führte), ist bewiesen, dal5 die gebunden sein sekundäre Alkoholgruppe zykmuß. Die folgenden Formeln geben den Verlauf der Oxydation wieder: ^ + '1 —y 4 CH, — CH OH ^^ ^ COOH COOH . + HA). Auch das Dihydrocholesterin, C27 H^g 0, läßt sich über das entsprechende Keton in die gleiche Dikarbousäure überführen."^) Nun mußte nachgeforscht werden, ob die ungesättigte, die Doppelbindung enthaltende Gruppe auch in einem Hinge oder in einer alipathischen Kette befindet. Eine ganze lieihe von Reaktionen ergaben einwandfrei, erstens, daß die Doppelbindung in einem Ring enthalten ist, und zweitens, daß sie einem anderen Ring angehört als dem, der die sekundäre Alkoholgruppe trägt.») Nun mußte durch weitere Studien die gegenseitige Stellung der (Jruppen CH.OH und CH:C bzw. CH:CH genauer festgestellt werden.^) Ferner war der Charakter sich der Seitenkette aufzuklären.^) Über die Stellung der Gruppen CH OH und gab die folgende Beobachtung Aufschluß. Das schon Cholesterins, das Cholestenon von der Formel . 1 \ \ CH C bzw. CH CH : : erwähnte Keton des r CH2— CO CH=:CH, vorsichtiger Oxydation eine Ketomonokarbonsäure und KohlenKetogruppe bleibt unverändert. Die ungesättigte Gruppe hinwird angegriffen. Es bildet sich ohne Zweifel zuerst die Keto- bei liefert säure. Die gegen dikarbonsäure C^tH^^O,^. Diese spaltet dann CO., ab und geht in die erwähnte Ketomonokarbonsäure CoHHiiO;^ über.'') Dieser ganze Vorgang ist nur erklärbar, wenn man annimmt, daß die Doppelbindung im Cholestenon y-Stellung zur Ketogruppe sich befindet in -i, ! I (JH2 CH —>. i CH \/V/ CO CH -t-40 1 1 1 . COOH —^^ CH^ CH^ COOH 1 CH2 CH \/\ CO COOH ! +<^'^.- [ \/ CO ) 0. Diels und E. Ahdirlialden : Bcr. d. Deutsclieu Clieni. (toscHscIi. 36. 3179 (1913); 37. 3092 (1904). ^) Windaus uud f'ibri(/: Bcr. d. Ueutschen Chem. Gesellsch. 47. 2387 (1914). ') Vgl. Windaus uud Stein: Ber. d. Deutscheu Chem. Gesellsch. 37. 3f)99 (1904). — Mauthner und Snida: Mouatsh. f. Chemie. 15. 86 (1894); 24. (552 (1904). — \'gl. auch Windaus: Ber. d. Deutschen Chem. Gesellsch. 50. 134 (1917). ) Vgl. Windaus: Ber. d. Deutschen Chem. Ges. 39. 2010 (190(5); 50. 133(1917). ^) Vgl. dazu: Windaus: Bcr. d. Deutschen Chem. Gesellsch. 42. 3772 (1909). Windaus und liesau: P^benda. 46. 1246 (1913). ^ Windaus: /oitschr. f. physiol. Chem. 102. 160 (1918). •) Vgl. A. Winduus: Ber. d. Deutsch. Chem. Ges. 50. 133 (1917). Vgl. weitere Beweise für diese Annahme: A. Windaus: Ebenda. 36. .3755 (1903): 39. 225(5 (1906). — Fette mit hochniolokulareni cinwertitfem Alkohol als Bausteiu. Nach diesen Feststellungen können wir sterins, wie folgt, wiedergeben die Konstitntion 229 des Chole- : CH CH CH., CH CH OH Zwei Ringe von den oben erwähnten vier hydrierten liingen sind somit in ihrem Bau im wesentlichen bekannt. Nun mußte noch festgestellt werden, in welchen Beziehungen die zwei noch unbekannten Ringsysteme zu den eben erwähnten stehen, welche Struktur sie haben, und endlich blieb noch die Natur der im Cholesterin vorhandenen Seitenkette aufzuklären. Es erwies sich, da(j sie aus acht Kohlenstoffatomen von folgender Anordnung besteht: (CH3)., . CH CH, . . CH., . CH, . . CH . CH^. t CH3 Die bis jetzt vorliegenden Feststellungen über die Struktur des Choführen nach Wmdaiis^) zu der folgenden Konstitutionsformel, wobei zu bemerken ist, daß noch nicht alle angenommenen Strukturverlesterins hältnisse eindeutig aufgeklärt sind:-) CH, H,C— CH CH, HC CH H,C CH HC CH, CH. CH, CH- CH,— CH,— CH, -CH CH. CH, C—CH, H,C2 ' n aCHvCH ' 1 H,C3 1 I i HCOH CH Cholesterin geht im Darmkanal unter Reduktion in Koprosterin Dieses enthält keine Itoppelbindung und besitzt zwei Atome über.^'j — \>1. auch A. W'indA. Windaus: ßer. d. Deutscher Chem. Ges. 53. 488 (1920). f. phvsiol. Chemie. 109. 18:3(1920); 115. 257 (1921); 117. 146(1921). -) Bezüglich der Numerierung der einzeluen Kohlenstoffatomc in den Ringen I und II sei auf die erwähnte Arbeit von Windaus verwiesen. ') St. Bondzyiiski und V. Ilumnirki: Zeitschr. f. phvsiol. Chem. 22. 390 (1896/97). Miiller: ZeitSt. Bondzi/nski : Ber. d. Deutscheu Chem. Gesellsch. 29." 476(1896). schrift f. phj'siol. Chem. 29. 129 (1900). Vgl. auch Charles Don'e und J. A. Gardner: Journ. Chem. Soc. London. 93. l(')2ö (1908). Vgl. insbesondere A. Windaus: Ber. d. aMsund H. Luders: Zeitschr. — — Deutschen Chem. Cesellsch. 49. 1724 (1917). — — ^11- Vorlestiug. 2.^0 Wasserstoff mehr als Cholesterin. Das Koprosterin ist niciit identisch mit dem normalen Dihydrocholesterin = [i-ChoIestanol, sondern ihm isomer und leitet sich von PseudoCholesterin, einer dem Stammkohlenwasserstoff des Cholesterins Cholestan isomeren Verbindung ab. Etwas von der letzteren Verbindung soll neben dem Ivoprosterin entstehen. i) Ferner ist aus Pferdefäzes noch ein sogenanntes Hippokoprosterin als Umwandlungsprodukt des Cholesterins beschrieben worden. Seine Natur ist noch nicht aufgeklärt, doch scheint es in der Nahrung vorgebildet, und zwar ein Phytosterin oder doch ein Abkömmling davon zu sein.-) Das aus Hauttalg gewonnene Isoc holest er in ist wahrscheinlich ein direktes Umwandlungsprodukt des Cholesterins. Erwähnt sei noch, dal.j auch Oxycholesterine beschrieben worden sind 3), so u. a. ein Dioxycholesterin. Ihre Natur und ihre Herkunft bleiben noch aufzuklären. Von größter P>edeutung ist die Feststellung, daü im tierischen Organismus noch ein Vertreter der Klasse der hydroaromatischen Verbindungen mit mehreren Ringsystemen vorkommt. Es hat sich nämlich herausgestellt, daß die Cholsäure, ein Baustein bestimmter Gallensäuren, in ihrer Struktur dem Cholesterin nahe steht. Diese Beobachtung führte zu der Fragestellung, ob die Cholsäure nicht ein Umwandlungsprodukt des Cholesterins sein könnte. Bire Bejahung würde uns gestatten, den zur Zeit noch vollkommen in Dunkel gehüllten Cholesterinstoff Wechsel zu erhellen. Der Umstand, daß Cholesterin und Cholsäure sich in ihrer Struktur sehr nahe stehen, rechtfertigt es, die Gallensäuren an dieser Stelle zu besprechen, obwohl wir auf Bausteine stoßen werden, für die uns manche Vorkenntnisse noch fehlen. Wir werden ihnen bei der Besprechung der Bausteine der Eiweißstoffe begegnen. Die Cholsäure kommt nämlich einerseits mit der Aminosäure Glykokoll gepaart vor, und andrerseits findet sie sich mit einer schwefelhaltigen Verbindung, dem T aurin, gekuppelt. Die erstere Verbindung hat die Struktur einer Aminoessigsäure, NHo.CHo. COOH. Taurin ist ein Umwandlungsprodukt der Aminosäure Cystin bzw. Cystein. Wir kommen auf seine Entstehung und seine Struktur noch zurück. Außer Cholsäure findet sich als Paarling noch die mit ihr eng verwandte Desoxycholsäure. Auch sie ist unter Austritt von einem Molekül Wasser entweder mit Glykokoll oder mit Taurin verbunden. Man spricht von einer Glykochol- und einer G ly kod e so xy cholsäure und ferner von einer Taurochol- und einer Taurodesoxycholsäure. In der liindergalle ist endlich noch eine Lithocholsäure genannte, der Cholsäure und Desoxycholsäuie nahe verwandte \'erbindung in geringeiMenge angetroffen worden.-) Wir wollen zunächst die Struktur der Cholsäure und der ihr verwandten \'erbindungen betrachten. Sie ist dank der unermüdlichen Forschungen vor allem von Wieland und Barsche in weitgehendem Malie und A. Vibrit/: ßcr. d. Deiitsclieii Chciii. Cicsellsch. 48. 8ö7 Fischar: Zcitsclir. f. physio'l. Chemie. 73. lia^ (1V)11). ') V'irl. Charles Dore'e und ./. A. Gardtier: rrocoed. of tlio loyal soc. 8(>. 212 (lilOB). ') J. Lißchiif:: Zcitsclir. f. physiol. Clioniie. 50. 487 (1907); 53. 140 (1907): 85. 17.') (1909); 63. 222 (1910); 106. 271 (1919); Hiocliem. Zoitschr. 48. 373 (19i:<): 52. 206 (1913). E. Sr.hreiher und IJndrd : El.(!nda. 49. 458 (1913). '') Nj^l. J/ans- Fischer: Zeitschr. f. phvsiol. Chemie. 73. 234 (1911). luich ^) (1915). Vgl. A. Wiitddu.s — //. — .V. /;. Schr;irn-: .]. — of Pliysiol. 44. 2(i5 (1912). Fette mit liochmolekulareni tMuwertigem Alkohol als Baustein. O'^l aufgeklärt worden.') Der Cholsiiure kommt die Formel C,^ H40 O5 zu. Sie ist eine gesättigte, einbasische Säure. Sie enthält drei Alkoholgruppen. Nimmt man diese bekannten (Jruppen aus der obigen Formel heraus, dann verbleibt noch der liest C^:^ II36 als in seinem Aufbau noch unvoll- ständig aufgeklärt'-) C,3H36(OH)3.C()OH. Der Cholsäure, zugrunde. C23 Hgr, (OH)^ .COOH, und der Desoxychoisiiure COOH, liegt der gesättigte Kohlenwasserstoff Cholan ll^oi Er ist um 4H2 ärmer als der entsprechende Paraffin kohlen- Cas H37 (0H)2 1'.,^ . Somit muß das Kohlenstoffgerüst der Cholsäure und der Desoxycholsäure ein alizyklisches System von vier gesättigten Kohlenstoffringen enthalten. Es galt nun, durch stufenweisen Abbau zu erforschen, in welcher Art die Ringsysteme autgebaut sind. Bei beiden Säuren hat bis jetzt der oxydative Abbau wasserstoff, C23H48. wichtigsten Resultate ergeben. Cholsäure, C24H4oOr, geht durch in eine um iMh ärmere Verbindung, C24H34O5, Dehydrocholsäure genannt, über.) Die Desoxycholsäure, C24H40O4, liefert eine die -^j , Oxydation um 2H2 ärmere Dehydro-desoxycholsäure, C^illsa ^^i-^) Das erstere Oxydationsprodukt enthält drei, das letztere zwei Sauerstoffatome in Ketonkarbonylform. Die Entstehung dieser Gruppen durch Oxydation i)eweist, daü in den genannten Ausgangsprodukten, der Chol- und Desoxycholsäure, sekundäre Alkoholgruppen enthalten sind. Cholsäure ist demnach C20 H33 (>CH 0H)3 COOH und Desoxycholsäure C.« U,, (>CH .OH). COOH. . . . Unter anderen Bedingungen liefert die Cholsäure bei der Oxydation neben noch unbekannten Oxydationsprodukten Biliansäure, C24H34O8"), und daneben in geringerer Ausbeute Isobiliansäure, C24H34O8.') Aus ) Vgl. auch .¥a;Y/// Scheiick: Zeitschr. 167 (1920); 112. HS (1921). f. plivsiol. Chemie. 107. 152 {1919j; 110. 2) Fritz Frec/l: Pflüger^ Archiv. 71. m'5 (1898); 72. 266 (1898). Monatsh. f. Chem. 32 (1903). 's. BÖndi und E. Mülhr: Zeitschr. f. physiol. Chemie. 47. 501 (1906). K. Lanpheld: Ber. d. Deutscheu Chem. Gesellsch. 41. 380 (1905). E. Letsclic: Zeitschr. f. physiol. Chemie. 61. 219 (1909). Fritz l'reql uud Havs Buchtala: PU)eiuia. S. B. Schri/rer: Jourii. of Physiol. 44'. 2()5 (1912). 74. 198 (1911). ^) Vgl. A. Panzer: Zeitschr. f. physiol. Chemie. 48. 192 (1906). Vgl. über die Koustitutioti der Cholsäure: Fritz l're(ß: Plheuda. 65. 163 (1910). Hugo Sclirötter, Richard Weizinbröck und Hi inhold Witt: Sitzuugsher. d. kaiserl. Akad. d. Wisseuscli. in Wien. 117. 1 (1908). K. Langheld: Ber. d. Deutschen Chem. Gesellsch. 41. 1023 Otto r. Fihth, E. Letsche: Zeitschr. f. physiol. Chemie. 61. 215 (1919). (1908). Ernst Jerusalem Emil Lenk uud H. ishihara : Biochem. Zeitschr. 20. 375 (1909): 26. 406 (1910); 43. 323 (1912). Heinrich Wieland und Friedrich Josef Weil: Zeitschr. f. physiol. Chemie. 80. 287 (1912). — 24. — — — — — ~ — , — — — — Lnt— Miilius: ) Olaf Hammarsten: Ber. d. Deutscheu Chem. (iesellsch. 14. 71 (1881). schinoff: p:bcuda. 18.3043(1885). Lassar-Colm: S^benda. 25.804(1892). Ber. d. Deutschen Chem. Gesellsch. 19. 2005 (1880); 20. 1980 (1887). — ^) Vgl. u. a. HVe/a«rfund E. liorrsch: Zeitschr. f. physiol. Chem. 106. 19(10919). //. Gotthard liulnheim: Zeitschr. f. physiol. Chemie. Cohn: Ber. d. Deutscheu Chem. Gesellsch. 32. (J84 (1899). Chemie. 24. 45 (1903). ) Fritz Preql Monatsh. f. Ciicmie. 24. 53 (19(«). «) : 25. 307 (1898). — Lassar- — /'V/V- T'renl: Monatsh.f. 232 Vorlosuug. -'^H- der Desoxycholsäure gehen hervor: CholansäiireM. CjiH;;,;!)^, und IsoVerbindunoen haben sich als Trikarbonsäuren erwiesen. Die beiden ersteren Säuren enthalten ferner zwei, die beiden letzteren je eine Ketonkarbonvloruppe. Die Säuregruppen, die bei der Oxydation gebildet werden, ohne dalj die Ausgangsmaterialien in Bruchstücke zerlegt werden, können nur so entstanden sein, daß Ringsprengung stattfindet: cholansäure-), CoiHä.iO^. Alle vier —> L-CO.CH2-' LcoOH COOH-'. Es ist geglückt, Biliansäure zu Cholansäure und Isobiliansäure zu Isocholansäure zu reduzieren.^) Die Biliansäure ist weiter zu Ciliansäure), C, HjoOjn oder C^iH^^Om, oxydiert worden.'') Acht dieser Sauerstoffatome geboren Karbonylgruppen an. Von weiteren erfolgreichen Versuchen sei erwähnt, dali es geglückt aus der Cholsäure alle drei Hydroxylgruppen als Wasser abzuspalten") und durch Wasserstoff zu ersetzen. Es bildete sich die dreifach unge- ist, Cholatrienkarbonsäure, C.,4H34 02. Die Desoxycholsäure liefert Bedingungen die zweifach ungesättigte Choladienkarbonsäure, C^iHsgO.,. Beide lassen sich zu der gleichen gesättigten Oholankarbonsäure, C^iHioOs, reduzieren. Diese Verbindung ist somit als sättigte den gleichen unter die gemeinsame Muttersubstanz der Cholsäure. der Deso.xycholsäure Lithocholsäure und der zu betrachten. Es ist von größter Bedeutung, daß bzw. aus Cholestan eine Verbindung, C.24H40O.., aus Cholesterin erhalten worden ist, die der erwähnten Cholankarbonsäure isomer ist. Geht man statt von Cholestan von einer aus diesem durch Umlagerung gebildeten Diastereomeren, dem Pseudoch ölest an, aus, dann erhält man bei der Oxydation mit Chrorasäureanhydrid eine Verbindung, die mit der aus Cholsäure bereiteten Cholankarbonsäure vollständig identisch ist.^) Mit dieser Feststellung ist die nahe Verwandtschaft zwischen Cholesterin und den erwähnten Gallensäurepaarlingen zur Gewißheit erhoben. Damit sind zahlreiche Fragestellungen über die Art der Beziehungen dieser \erbindungen im tierischen Organismus gegeben. Sie sind ohne Zweifel in der lüchtung der Abkunft der erwähnten Gallensäurepaarlinge vom Cholesterin und nicht umgekehrt zu suchen. Vom rein chemischen Standpunkt aus lassen sich die nahen Beziehungen zwischen Cholesterin und der Cholsäure durch die folgende Formel wiedergeben:' C27 H4« 4- 50 =CH3 CO CH3 + C,4 H,o 0,. . . Fritz Pregl: Monatsh. f. Chemie. 24. 49 (1903). Latschinof: Ber. d. Deutschen Chem. Gesellsch. 19. 1529 (188()). ') Vpl. ir. Borsche und Emmi/ Rosenkranz: Ber. d. Deutschen Chem. Oosellscli. 52. 342 (1919). W. Barsch : Kl)enda. 52. 1303 (19191. ) Lassar-Cohn: Ber. d, Deutschen Chem. Gesellsch. 32. 684 (1899). Frifz Frrdl: Monatsh. f. Chemie. 24. 57 (1903). Vgl. namentlich Marfin Schenck: Zeitschrift f. physiol. Chemie. 107. 152 (1919). ^) Marfin Schenck: Zeitschr. f. phvsi(d. Chemie, 87. 59 (1913); 89. 3(50 (1914); ») ') — — — 104. 2-i4 (1919). "> IJ. Wieland und IVril: Zeitschr. f. physiol. Chemie. 80. 287 (1912). A. Windaus: Nachrichten der Gesellschaft der Wissenschaften zu Göttingen. Math.-physik. Klasse 1919. Ber. d. Deutschen Chem. Ges. 52. HMf) (1919). Zeitschrift f. physiol. Chemie. 117. 146 (1921).' ') — — Fette mit hochmolekularein einwertigem Alkoliol als Baustein. 233 Nachdem es WielandX) gelungen ist. einen dritten Ring der Cholsäure aufzusprengen und seine Struktur aufzuklären, sind von den 24 Kohlenstoff atomen der Cholsäure und ihrer Verwandten 18 in ihrer Stellung im Molekül aufgeklärt. Wir sind somit nur noch über 6 Kohlenstoffatome im Unklaren. Auf Grund der Forschungen von Heinrich Wlcland'-) und W. Borsche'^) lassen sich für die drei Säuren Cholsäure, Desoxycholsäure und Lithocholsäure) die folgenden Strukturformeln aufstellen, wobei es noch fraglich ist, ob Ring II ein Fünf- oder Sechsring ist.'') CH.OH CH, CeH.o HCi £19 2 — CH CH., CH, COOH . . . ...iCH, p- 3^X12 HO HO CH.OH . CH, CH. Cholsäure C24H40O5. CH. CH, CeH,o H.Ci HO.HC< Q — CH CH.2 CH., COOH . . . — iCHa A >CH.OH CH., CH, Desoxycholsäure CoiH^,, O4. ) Vgl. insbesüudere H. Wielund iiutl A. Kulenkampd : Zeitschr f. phvsiol. Chemie. H.'Wieland uud //. Wieland: Ebenda. 108. 300 (1919) (1920). 108. 295 (1919/20). P. Wei/land: Ebenda. 110. 12.-5 (1920). II. Wieland und W. Schulenbnrq : Ebenda. 114. H. Wieland und Otto Schlichting : Ebenda. 119. 7(i (1922). 167 (1921). ') H. Wieland und W. Schnlenburg : Zeitschr. f. physiol. Chemie. 114. 1H7 (1921). Vgl. ferner H. Wieland und O. Schlichting: Ebenda. 119. 76(1922): 120. 227 (1922). H. Wieland und Franz Adickes: Ebenda. 120. 2.S2 (1922). ^) W. Barsche: Ber. d. Deutscheu Chem. (icsellscli. 52. 342 (1919). ) W. Barsche, 0. Weickert und Robert Meifcr : Ber. d. Deutschen Chem. Cresellsch. J. Hallwass: In. Diss. Göttingen 1922. 54. 8177 (1911). ^) Vgl. hierzu A. Windaus und W. Hü ekel : Nachr. d. Gesellsch. d. Wisseusch. zu Göttingen. Mathem.-phvsikal. Klasse. 1921. Wieland: Zeitschr. f. phvsinl. //. Chemie. 114. 174 (1921); flO. 90 (1922). — — — — — — — — 234 XII. Vorlesung, CH., CR — CH3 — CH CHa CHa COOK I CeHjo HC . . . CH, CH, HO HC< . . /\^ ^CH-, CH2 CHo Lithocholsäure C24H40O3. Ein Vergleich dieser Strukturformeln mit der S. 229 wiedergegebenen für Cholesterin zeigt ohne weiters die nahen Beziehungen. Wir wollen nun zur Besprechung der einzelnen gepaarten Gallensiiuren übergehen. Die GlykocholsäureM, besser Cholyl-glyzin genannt, besteht aus ('holsäure und (ilykokoll. Sie zerfällt in diese Bausteine durch Spaltung unter Wasseraufnahme und läßt sich aus ihnen unter Wasserabspaltung wieder aufbauen.-) Die Bindungsart zwischen der Cholsäure und dem (ilykokoll ist die gleiche, wie wir sie bei der Hippursäure und den Polypeptiden kennen lernen werden. Sie ergibt sich aus den folgenden Formeln: C.o H,3 (> CH ()H)3 CO NH GH, C0( )H + H, O . . Cholylrest C20 H3, (> . . . ^ Glyzinrest CH OH);, COOH -f NH2 CH2 COOH Cholsäure . . . . Glykokoll. Ganz entsprechend ist die Struktur der Glykodesoxycholsäure = Desoxycholyl-glyzin, nur ist an ihrem Aufbau an Stelle der Cholsäure Desoxycholsäure beteiligt. Das Cholyl-glyzin und Desoxycholyl-glyzin finden sich in der Galle des Menschen und der Pflanzenfresser. Derjenigen der Fleischfresser scheinen meistens zu fehlen. Gibt man zu einer Cholyl- bzw. Desoxycholyl-glyzin enthaltenden Flüssigkeit etw'as Rohrzucker und unterschichtet die Lösung mit konzentrierter Schwefelsäure, dann tritt an der Berührungsstelle beider Schichten eine Kotfärbung auf [Fettenkofers Reaktion). Man nimmt an, daß diese Farbreaktion durch Bildung von Furfurol zustande komme. Sie ist nicht für die genannten Gallensäuren charakteristisch. Sie wird vielmehr durch die Anwesenheit der C'holyl- bzw. Desoxycholylgruppe bedingt. Den Gallensäuren ist ferner eine erregende Wirkung auf das Herz gemein. P)ald folgt der vermehrten Herzfre(iuenz eine starke Verlangsamung. Bringt man gallensaure Salze auf das freigelegte Herz, dann läßt sich nach kurz vorübergehender Steigerung der Zahl der diese Gallensäuren eine bedeutende Verminderung derselben feststellen. in die Firscheinung tritt auch auf, wenn (iallensäuren gebracht werden. Die starke Herabsetzung der Pulszahl bei Herzkontraktionen Die gleiche P)lutbahn '> •-) Letschr: Zcitschr. t. i)h\siol. (jhemie. 60. Vgl. ihre Gewinnung: Vgl. S. Hondi und K. Müller: Zeitsclir. f. pli\si(.i. ( homio. 47. .')()! 4H3 (1909). (190f)). Pette mit hochmolekularem, einwertigem Alkohol als Baustein Ikterus auf ist den Übertritt von Gallensäuren das Blut in 2ii5 zurückzu- führen. Entsprechend den beiden gepaarten Gallensäuren, an deren Aufbau Glykokoll beteiligt ist, kennen wir auch zwei solche, die T au r in als BauIhre Entstehung aus Cystein ergibt sich aus folgender stein besitzen. Übersicht + CH,.SH — CH NH., . CH,.SO,.()H CH,.S(),.()H 3 CH >- . —y CH^ NH., . NH., i I COOH COOK —CO, Cystein Cysteinsäure Der Taurin. Taurocholsäure = Cholyl-taurin') kommt die folgende Struktur zu: C,„ H33 ( CH 0H)3 CO NH CH, CH, SO, OH + H, . . ( CH OH . Cholsäure . . . . — >- Taurinrest Cholylrest C,o H33 . )3 . COOH -f NH, CH, CH. S( . . . ), . OH Taurin. Ebenso wie es ein Desoxycholyl-glyzin gibt, kommt Taurin gekuppelt mit Desoxycholsäure in der Galle vor.-') Die Taurocholsäure bildet den Hauptbestandteil der (ialle des Hundes. Sie findet sich in geringen, wechselnden Mengen auch in der Galle des Menschen, der Rinder, der Schafe und Ziegen. Auch in der Fischgalle (Galle des Dorsches) hat man Cholyl-taurin gefunden. 1 )ie dritte, der oben erwähnten (iallensäurenkomponenten,dieLithoc holsäure, ist in sehr geringen Mengen in der Hindergalle enthalten. Über ihre Verbindungen ist noch nichts Genaues bekannt. Sie dürfte wohl auch mit Glykokoll bzw. Taurin gekuppelt vorkommen. Es sind außer den genannten Gallensäuren noch weitere beschrieben worden. So soll in der Menschengalle eine Fellin säure) vorkommen, ferner in der Galle des Schweines Hyocholsäure'^), doch sind die vorhandenen Angaben zu dürftig, um zu entscheiden, ob wirklich reine Verbindungen vorgelegen haben. Sicher festgestellt ist das Vorkommen einer in der Galle der (iänse aufgefundenen (iallensäure. Sie liefert bei der Spaltung Taurin und ) Vgl. hierzu llennaiiii Wieland und l'h. HildenbraiuJ : A. f. experini. Path. u. Fliarm. 85. 199 (1919). Vgl. auch //. Wieland: Khenda. 86. 79, 92 (1920). ^) Vffl. ihre Darstellung: Olaf I laminarsten : /eitschr. f. physiol. Chemie. 43. 127. (1904). ^) bei //. Taurodeso.xycholsäure Vgl. die Synthese der Glykodesoxycholsäure und der IIedici<i Stender: Zeitschr. f. phvsiol. Chemie. 106. 181 (1919). Wieland und ) (r. Schotten: Zeitschr. für physiol. Chemie. 10. 175 (188(5); U. 2(58 (1887). — Lassar-Cohn: Zeitschr. f. physiol. Chem. 19. .'i().S (1S94). — Vgl. auch //. /'. 7'. Önttn: Skand. Arch. f. Physiol. 16. 27:5 (1904). Vgl. aucii (Haf Hammarsien: ') Ad. Strecker: Liebigs Auualen. 70. 188 (18-19). Ebenda. 74. 123 (1911). Severin Jolin: Zeitschr. f. physiol. Chemie. 12. 512 (1888); 13. 205 (1889). — — ^'1 Vorlesung. 286 Chenocholsäure. Sie ist somit als ein Chenocholyl-tauriu Taurochenocholsäure') aufzufassen. bz\v. eine Besonders interessant ist die Feststellung einer neuen Klasse von gepaarten Gallensäuren, die nicht Glykokoll oder Taurin, sondern Schwefelsäure enthalten.'-) Ferner finden sich in diesen an Stelle von Chol- bzw. Deso.xycholsaure Alkohole, a- und ß-Scymnol genannt. Es liegt offenbar eine esterartige Verbindung zwischen der Schwefelsäure und dem Scymnol vor. Man hat die gepaarten Verbindungen Scymnolschwefelsäuren geSie sind von Olaf Hanuiiorsten in der Galle des Haifisches, nannt. Scymnus, entdeckt worden. 0. HarnnKu-den^ hat auch die Galle des Walrosses untersucht. Neben einer dem Cholyl-glyzin nahestehenden Verbindung wurde der Taurinpaarling der Cholsäure aufgefunden. Den Hauptbestandteil der Sie sind vorläufig Walrossgalle bilden Säuren noch unbekannter Natur. a-Phocaetaurocholsäure und a-Phocaecholsäure, [i-Phocaetaurocholsäure und ß-Phocaecholsäure genannt worden. Erwähnt sei ferner, daß aus den Bezoarsteinen, d. h. aus Steinen, die sich zuweilen im üarmkanal der Bezoarziogen bilden, eine sog. LithoEs f ellin säure) und eine Lithobilinsäure^) gewonnen worden sind. kommt der ersteren die Formel CooHaßO^ zu.") Wahrscheinlich steht sie einem Nahrungsbestandteil und hat keine Verwandtschaft mit Gallensäuren. Die Gallensäuren, die sehr wahrscheinlich aus Cholesterin hervorgehen, gelangen mit der Galle in den Darm und verlassen normalerweise den Organismus mit den Fäzes. Ein Teil der in den Darmkanal gelangten Gallensäuren wird wieder resorbiert und dann von neuem mit der Galle ausgeschieden.') Ob weitere Abbaustufen aus Gallensäuren in den Geweben oder unter der Einwirkung der Darmflora sich bilden können, wissen wir noch nicht mit genügender Sicherheit. Es sind allerdings Beobachtungen über die Umwandlung von Gallensäuren durch Bakterien gemacht worden, doch genügen sie nicht, um zu unterscheiden, ob im Darmkanal selbst in größerem Umfang Gallensäuren verändert werden. Es wäre ganz gut denkbar, daß bestimmte Umwandlungsprodukte zur Kesorption gelangen und vom Organismus verwertet werden. Von großem Interesse ist die Beobachtung, daß im Hautsekret der Kröten eine Verbindung, Bufotalin*) genannt, vorkommt, die zu den Gallensäuren in ganz naher Beziehung steht.') Sie hat die Zusammensetzung Coe Hje Or und enthält vier hydroaromatische Ringe. Zwei C- Atome gehören einer esterartig gebundenen Azetylgruppe an. Ikifotalin ist ein in Beziehung zu Heintz und W'isliccnus: Po(i(iendorlV Aiiu. il. lhysik u. Clioniio 108. .ö59 {18,")9). f. physiol. Chemie. 63iS (1868). •') Olaf Ifammar.^fen: Zeitschr. f. physiol. Chemie 84. 322 (1898). ^) Olaf I lammar stell : Zeitschr. f. physiol. Chemie 61. 4ö4 (1909). ) Jünger und Klares: Ber. d. Deutscheu Chcm. Gesellsch. 28 3045 (1895). ^) Giort/io /^o.süe»-; Gaz/. cliuica ital. 9. 462 (1879). ) H'öhler: Liebigs Ann. 41. 131. //««.« Fifichrr : Bor. d. Deutschen Chcm. ') — R. Otto: Zoitschr. — Gesellsch. 49. 2413 (1917). ') /;. Werthrimer: «) Vgl. 9) //. Wu'land und UViV.- Ber. d. Deutschen Chcm. Ges. 46. 3315 (1913). Wirland und JL Alles: Ber. d. Deutschen Chem. Ges. 55. 1789 (1922). Vgl. auch /-'. C. r, 174. .')()4 (1922). //. Flurif: Archiv — f. experim. I'uth. n. Pharniok. 81. 319 (1917). Fette mit hochmoleUiilaroiii, ouiwortigom Alkdhnl als Bausteiu. 237 Lakton und enthält zwei OH-Gruppen. Es findet sich im Ilautsekret und in dem Sekret der sogenannten (Jhrdrüsen der Kröten gebunden, und zwar liefert die Muttersubstanz, Bufotoxin genanntM, hei der Spaltung: Bufotalein, C.,^ H30 O3, Essigsäure, Wasser und Suberyl-arginiu. Die letztere Verbindung läßt sich durch Hydrolyse in die Diaminsäure Arginin (vgl. Vorlesung XVII) und Korksäure spalten. Dem Suberyl-arginiu kommt folgende Struktur zu: HOOC (CHo), CO NH C = NH . T. , . .. . , Korksaurerest (Suberylgruppe.) NH, . NH CH., GH., GH.. GH GOOH . . ' , . ' . . Argininrest. Die Karboxylgruppe der Suberylgruppe des Suberyl-arginins ist offenbar esterartig mit dem Bufotalin verknüpft. ^)H. Wieland ; Sitziingsber. d. bayr. Akad. d. Wisseusch. Math.-pliysikal. Kl. 329 (1920). Vorlesung XIII. Phosphatide und ihre Bausteine. Mit dem Namen Phosphatide hat Tlindidium^) eine große Klasse von Verbindungen bezeichnet, die zu den Fetten in mancher Hinsicht in nahen Beziehungen stehen. Sie sind gegenüber diesen durcli den Gehalt an Phosphorsäure und mindestens einer stickstoffhaltigen Base ausgeThudichum wies bereits darauf hin, daß diese Körperklasse zeichnet. mannigfaltig gebaute Vertreter umfaßt. In der Folgezeit war man jedoch geneigt, sie als einheitliche Verbindungen vom Typus des Lezithins aufzufassen. Die genauere Betrachtung der sogenannten Lezithinfraktionen ergab jedoch große Unstimmigkeiten im Verhältnis des Phosphorgehaltes zu dem des vorhandenen Stickstoffs. Schließlich kam man auf die lange verkannte Arbeit von Thudichum zurück und unterscheidet jetzt schon eine ganze Anzahl verschiedenartiger Phosphatide. Die Phosphatide sind im Pflanzen- und Tierreich stark verbreitet. 2) Sie fehlen keiner Zelle. In besonders großen Mengen finden sie sich in der Nervensubstanz (Gehirn ca. 20Vo), ini Eidotter, im Herzmuskel, in der Niere und der Leber (ca. S"/,, in diesen OrganenJ. Die meisten Phosphatide kristallisieren nicht. Sie stellen, getrocknet, wachsartige Massen dar. Sie sind farblos, riechen und schmecken nicht. In Wasser sind die Lipoide mit wenig Ausnahmen ganz unlöslich, dagegen bilden sie mit ihm eine Art von Emulsion. Die scheinbare Lösung sieht opaleszierend aus. Wie die Fette lösen sich auch die Phosphatide in Äther, Alkohol, Chloroform, Benzol usw. Die für die Beurteilung der Forschung auf dem Gebiete der Phosphatide wichtigste I^ügenschaft ist ihre leichte Zersetzlichkeit. Schon beim Liegen an der Luft und namentlich bei gleichzeitiger Belichtung tritt bei vielen Vertretern dieser Körperklasse Zersetzung ein. Manche Phosphatide sind gegen Alkali oder Säuren sehr empfindlich. Schon bei den Fetten konnten wir feststellen, daß mancb.e von ihnen im Laufe der Zeit Veränderungen erleiden. Bei den Phosphatiden treten diese sehr rasch ein, so daß man bei der Isolierung eines unbekannten Phosphatids zu') ./. Ludwig W. J liudichum: Die chemische Ivonstitutioii des (l(>liirns des Meuschen und der Tiere. Franz rictzcker. Tiibintren. 11)01. ) Vfrl. weitere Einzelheiten über Phosphatide hei hör /h/iu/: (,'lieniie und Biochemie der Lipoide. J. F. Bergmann. 1911. S. Fraotkcl: (iehirnclieniie. Ergebn. d. l'hysiol. 8. 212 (1909). F. A. Leiiene und Ida 1'. Rolf: l'liysi.d. IJiviews. 1. Nr. M. 1921. Hier findet sicli eine Übersicht über die Literatur. — — Phosphatide niul ihre Bausteine. nächst eigentlich nie weiß, ob der erhaltene Kiirper 2;)9 die nrspriingliche Verbindung darstellt oder aber schon weitgehend verändert und vielleicht schon zum Teil in seine Bausteine gespalten ist. Da man es in den einzelnen Fällen fast immer mit amorphen, schwer zu reinigenden Körpern zu tun hat, ist es oft ganz unmöglich, etwas über die Einheitlichkeit der Es ist aus den angeführten Gründen verisolierten Körper auszusagen. ständlich, daß viele sogenannte Phosphatide heiß umstritten sind. Dazu kommt noch, daß die Eigenschaften der Phosphatide durch Beimengungen stark beeinflußt werden. Auch dadurch können sich große Fehlerquellen ergeben. In vielen Fällen ist die Bestimmung des Phosphatidgehaltes von Organen ausschließlich auf den Gehalt eines Lösungsmittels an Phosphor basiert worden. In anderen Fällen hat man auch in bestimmten Extraktionsmitteln einfach das Verhältnis von Phosphor und Stickstoff' bestimmt. Alle derartigen Untersuchungen haben keine Beweiskraft. Die von Thudichum vorgeschlagene Einteilung der Phosphatide darf nur als Notbehelf betrachtet werden. Sie stützt sich auf das \'erhältnis von Phosphor und Stickgtoif in den einzelnen Phosphatiden. Sicher besagen die einzelnen Namen oft mehr, als wir wirklich wissen. Man wird erst dann klar sehen, wenn die Bausteine der Phosphatide genau bekannt sind, und man sich in jedem Falle auch ein Bild von der Struktur der einzelnen Verbindungen machen kann. Die Erforschung der Phosphatide steht in den allerersten Stadien. Sie wird ohne Zweifel neue Impulse erhalten, wenn die Synthese irgend eines Phosphatides geglückt ist. Hat man erst ein „Modell" für die einzelnen Gruppen von Phosphatiden, dann wird es nicht schwer sein, auf Grund von dessen Eigenschaften Methoden ausfindig zu machen, die jeder Zersetzung vorbeugen und Gewähr für einheitliche Verbindungen geben. Wir werden der gleichen Schwierigkeit in der Abgrenzung der einzelnen chemischen Individuen bei den Eiweißstoften begegnen. Auch bei diesen sind wir bei der Abtrennung einzelner Arten auf physikalische Methoden angewiesen. Möglicherweise sind die kristallisierenden Produkte einheitlich. Auch bei den hochmolekularen Polysacchariden konnten wir, soweit sie nicht in Kristallform zu bringen waren, nirgends den Nachweis erbringen, daß chemisch einheitliche Individuen vorlagen. Dieser Lücken in unseren Kenntnissen der einzelnen Gruppen von Verbindungen muß man sich immer klar bewußt bleiben, sonst wird man leicht ganz unrichtige Vorstellungen über die Bedeutung ganzer Forschungsgebiete erhalten. Unsere Anforderungen können, was die Beweisführung für das Vorkommen bestimmter chemischer Individuen anbetrifft, nicht scharf genug sein. Von den Phosphatiden ist bis jetzt am eingehendsten das von ]^auquelin (1811), von Coiierhe (1834) und vor allem von Gohlc//^) (1845) aufgefundene Lezithin untersucht. Wir wollen dieses zuerst besprechen und dann eine Übersicht über die Einteilung der Phosphatide und der bis jetzt dargestellten Vertreter dieser Körperklasse geben. Wenn wir von Lezithin sprechen, müssen wir gleich hervorheben, daß es mehr als fraglich ist, ob nur eine bestimmte Verbindung mit den Bausteinen des Lezithins im Pflanzen- und Tierreich vorkommt, oder ob nicht vielmehr eine ganze 1) M. Gobley: Conipt. reud. de l'Acad. des Scienc. 21. TBO. '.>88 (1845); 22. iWi (1846); 23. 654 (1847); Jnuni. de pharm, et de ohim. 17. 401; 18. 107 (1850) und 1<» 406 (1851). ^III- Vorlesung. 240 Anzahl verschiedener Lezithine existieren. Es ist uämHch bis heute noch nicht gelungen, die Art der einzelnen Bausteine des Lezithins bis in alle Einzelheiten festzustellen. Sicher nachgewiesen sind: Glyzerin, Phosphorsäure und Cholin, ferner enthält das Lezithin Fettsäuren. Ihre Natur immer noch ist ganz nicht Höchstwahrscheinlich enthalten klargestellt. kommen nicht alle Lezithine die gleichen Fettsäuren. Sehr wahrscheinlich Ihre Anwesenheit würde erklären, weshalb die Lezithine so leicht zersetzlich sind. Es finden sich auch Unterschiede in der Konstitution der aus den Lezithinen zu erhaltenden Glyzerylungesättigte Fettsäuren vor. phosphorsäure. Im Lezithin haben wir zunächst eine uns bereits von den Fetten her bekannte Verbindung von Fettsäuren mit dem dreiwertigen Alkohol Glyzerin. Zwei Alkoholgruppen sind esterartig mit ersteren verknüpft. Früher nahm man an, daß hauptsächlich Stearinsäure am Aufljau des Lezithins beteiligt sei. Jetzt herrscht die Ansicht vor, daß mindestens eine der beiden Fettsäuren ungesättigt ist.i) Mit der dritten Alkoholgruppe ist Phosphorsäure verbunden. An dieser sitzt endlich die stickstoffhaltige Base Cholin. Die folgende Formel gil)t die Struktur des Lezithins wieder, vorEs sei ausgesetzt, daß die vorliegenden Beobachtungen vollständig sind. bemerkt, daß manche Forscher das Vorkommen einer solchen Verbindung bestreiten, wieder andere halten die Formel für zu einfach. Es soll außer Cholin oder an Stelle des Cholins eine andere stickstoffhaltige Base vorhanden sein. Leider hat der Versuch einer Synthese von Lezithin der genannten Zusammensetzung bis jetzt keinen ?>folg g:ehabt."-j f GH., . OOC R ^) . Fettsäurerest Glvzerinrest CH . OOC . R, ^) I = a H, -f I l CHo— 0\ HO^P — :'H..CH3— 0/ Cholinrest \| I GH., . I Phosphorsäurerest I I N^fCHj), OH OH GH .OH i CH. 0\ /GH., HO/ \0H . . GH., . OH HO^P = 0-^2 Moleküle Fettsäuren + N^CCHs), Cholin. Glyzeryl-phosphorsäure. ) A. Erlandsen ist der Ausicht, daß am Aufl)au des Lezithins Säureu der LinolLinolenreihc beteiligt sind. Vgl. Zeitschr. f. physiol. Chem. 51. 71 (1907). Ad. 2) Fe.ter Berf/ell: Ber. d. Deutschen Chem. Gesellsch. 33. 2584 (1900). Vgl. auch K. Langheld, F. Oppmann fh-iin und F. Kade: Klieiida. 45. 33(57 (1912). und E. Mei/er: Ebenda. 45. 3753 (1912). ') R und R, bezeichnen die C- und H-Atome zweier verschiedener Fettsäuren. niid — — Phosphatide Baustoino. uiul ihre 241 + IL CH., CH OH . OH . + HOv HO^P = 0. HO- HO CH, OH Glyzerin. . Phosphorsäure. mau Lezithin mit Alkalien oder Barytwasser, dann erhält vorstehenden Formeln dargestellt ist, unter Aufnahme von drei Molekülen Wasser zwei Moleküle Fettsäuren, Cholin und ferner Gl yzeryl- phosphorsäure. Die letztere läßt sich weiter unter Wasseraufnahme in ein Molekül Glyzerin und ein solches von Phosphorsäure spalten. Glyzerin, die Fettsäuren, das Cholin und die PhosphorSpaltet man. wie in säure sind somit Bausteine der Lezithine. Durch Zusammenfügung müßte man unter Austritt von vier Molekülen Wasser zum Lezithin gelangen. Interessant ist die Beobachtung von Trier'^). daß einzelne Lezithine bei der Hydrolyse mit verdünnter Säure und auch mit Baryt Aminoäthylalkohol =: Oxäthylamin, von Tfitr auch Colamin dieser Bruchstücke genannt, liefern: CH-3 . CH., . OH NH., In der aufgestellten Formel ist angenommen worden, daß die beiden Fettsäuren in x- und ß-Stellung mit dem Glyzerin verbunden sind. Diese Annahme ist deshalb begründet, weil das Lezithin optisch-aktiv ist-). d. h. mindestens ein asymmetrisches Kohlenstoffatom enthält. Das Cholin enthält kein solches Kohlenstoffatom, folglich muß die optische Aktivität des Lezithins in der Art der Bindung der Fettsäuremoleküle mit dem Glyzerin begründet sein, vorausgesetzt, daß keine optisch-aktiven Fettsäuren an seinem Aufbau beteiligt sind. Es sind folgende beiden Möglichkeiten gegeben: CH, O — phosphorsaures Cholin CH — r . . < > . i ) 1 CH., F ettsäure A CIL und — Fettsäure A r CI1 . O .(> ! (IL . < ) II I Formel wenn II enthält nur dann ein asymmetrisches Kohlenstoffatom, die beiden Fettsäurereste verschieden 1) — Fettsäure A — phosphorsaures Cholin — Fettsäure B sind. Nun ist es UV Istäff er freorg Trier: Zeitschr. f. physiol. Chem. 76. 496(1912): vgl. aucli Julius Eppler Ebenda. 87. 233 (1913). Diakonow: Zentralbl. f. d. med. Wissensch. 438 (18G8l. — Franz Jliindeshatjen: prakt. Med. 28. 219 (1883). E. Gilson: Zeitschr. f. physiol. Chem. 12. 555 (1888). Adolf Strecker: Liebig?, Annalen. 148. 77 (1868). Abderhalden, Physiologische Chemie. I. Teil, 0. Anfl. 16 ^) Journ. f. — — XIU. Vorlesung. 242 und Liidecke'^) gelungen, durch Hydrolyse von Lezithin eine optisch-aktive Glvzeryi-phosphorsäure zu erhalten.'-) Esistdies nur bei der Gruppierung CH2 . OH — CH OH — CHo . . . P(0H2) Somit möglich. entspricht Formel I der Struktur dieser Glyzeryl-phosphorsäure. Neben dieser a-Gly- zeryl-phosphorsäure kommen olifenbar auch Derivate der [i-GlyzerylOP (OH). CH, (OH) in Phosphaphosphor säure, CH2 (OH) CH . tiden . . . . . vor.^') Stellt man sich auf den Standpunkt, daß die mitgeteilte J'ormel gewissermaßen das Modell für die Gruppe der Lezithine darstellt, dann kann man sich ohne weiteres vorstellen, auf welche Art ungezählte Verbindungen vom gleichen Typus zustande kommen können. Zunächst können die Fettsäuren verschieden sein. Es könnten Lezithine vorhanden sein die zwei gleiche , Fettsäuremoleküle enthalten, während vielleicht am Bau anderer zwei Im letzteren Fall sind je nach verschiedene Fettsäuren beteiligt sind. ihrer Anordnung isomere Verbindungen möglich. Schließhch ist zu berücksichtigen, daß, wie oben erwähnt, außer der optisch-aktiven Glyzerylphosphorsäure auch die symmetrische Form vorkommt. Li diesem Falle ist die optische Aktivität der Phosphatide dadurch bedingt, daß zwei verschiedene Fettsäuremoleküle vorhanden sind, oder aber es liegt die Asymmetrie in diesen selbst begründet. Auch hier sind isomere Formen möglich. Stets wiederkehrend sind das Glyzerin und die Phosphorsäure. Es scheint, daß auch das Cholin und vielleicht das Oxyäthylamin als ständige Bausteine der Lezithine aufzufassen sind. Manche Forscher sind allerdings der Ansicht, daß es lezithinartige Körper gibt, die noch andere stickstoffhaltige Basen besitzen. In diesem Falle müßte man entweder den Gruppennamen Lezithin für alle jene Verbindungen festhalten, die Cholin bzw. Oxyäthylamin als Baustein enthalten, oder aber man kann die Definition der Gruppe Aveiter fassen und jede Verbindung ihr zurechnen, die außer einem Molekül Glyzerin zwei Molekülen Fettsäuren und einem Molekül Phosphorsäure noch eine stickstoffhaltige Base enthält. In diesem Falle existieren außerordentlich viele Möglichkeiten neuer Verbindungen, indem dann nur noch das Glyzerin und die Phosphorsäure in jeder Lezithinart wiederkehren würden, während die Art der Fettsäuren und der stickstoffhaltigen Base in der verschiedensten Weise Avechseln könnte. Manche Beobachtungen deuten darauf hin, daß die einzelnen Phosphatide eine verschiedene Molekulargröße haben. Es ist möglich, daß es Verbindungen dieser Klasse gibt, in denen sich das Lezithin oder ein ähnlicher Rest wiederholt. Man muß dann die Zahl der wiederkehrenden Komplexe etwa in der Form bezeichnen, daß man von Monolezithiden und ferner von Polylezithiden spricht. Kennt man die Anzalil der zusammen vereinigten Moleküle Monolezithide, dann würde man von Di-, Tri- usw. Lezithiden sprechen. Als Verankerungsstcllen konnnen die freien )H-Gruppen der Phosphorsäure und die Glykolgruppe des (Jholins in Frage. , < ) Richard Willstätter und Karl Ludeckc: Bor. d. Deutsclieii Clicm. Gesellscli. 37. 3753 (1904). ^) Ihre Svutbcse vgl. Kinil Abderhalden uud Eqoii Eichtcald: Ber. d. Deutt^clion (Jheiii. Gcsellsch. 51. 1308 (lUlS). ") Vgl. hierzu (). Baillij: C. r. de Tac. des sc. 160. 395(li)15j; Ann. d. Cliiniie. Rev. genörale des sciences pures et appl. 29. 208 (1918). -- King [9]. 6. 96 (1917;; a,Dd I'ijmau: .Journ. Chem. Soc. 105. 1238 (1914). Phosphatide und ihre Bausteine. Diese Hinweise inögen geniigen, 04^^ um zu zeigen, daß schon innerhalb Gruppe der Lezithine die mannigfaltigsten Verbindungen möglich sind. Dazu kommt dann noch, daß die einzelnen Lezithine in vielfacher Weise gemischt in den Zellen vorkommen. Es sind, ganz entsprechend, der wie bei den Fetten, auch in dieser Richtung ungezählte Möglichkeiten Auf diese Weise können die Lezithine mit den übrigen gegeben. Bestandteilen der Zellen den Charakter einer Zellart mitbestimmen und ihr ein zelleigenes Gepräge geben. Von den Bausteinen der Lezithine sind uns das Glyzerin, die Fettsäuren und die Phosphorsäure schon vertraut. Noch nicht angetroffen haben wir die Kombination von Glyzerin und Phosphorsäure, die Glyzeryl-phosphorsäure, die stickstoffhaltige Base Cholin und das Oxyäthj'lamin. Die erstere Verbindung läßt sich aus ihren Bausteinen leicht synthetisch gewinnend) Sie ist in geringen Mengen in tierischen Geweben aufgefunden worden und ist wohl als Übergangsverbindung beim Ab- und auch Aufbau von Lezithinen aufzufassen. Cholin ist außer als Baustein des Lezithins auch frei in tierischen Geweben angetroffen worden. Es ist auch im Pflanzenreich außerSeine Konstitution ergibt sieh am besten aus seiner Wurtz^) gewann Cholin, indem er Trimethylamin auf eine konzentrierte wässerige Lösung von Äthylenoxyd einwirken ließ: ordentlich verbreitet. Synthese. ,CH,.CH,.OH //CH, /CH, CHov >o + N^cHs + \\.A)-yw^m., I m./ Äthylen- ^CHg \0H \CH3 Trimethylamin Cholin oxyd Cholin ist Wir können hydroxyd es somit ein Trimethyl-oxyäthyl-ammoniumhydroxyd. uns auch entstanden denken, indem wir in Ammonium- : n4-h ^H Atome Wasserstoff durch die Methylgruppe, CH3, ersetzen und das entstandene Trimethyl-amnioniumhydroxyd mit Glykol unter Wasseraustritt vereinigt denken: drei — — yeiiberg und J:'. Krctuchmer: Biochem. Zoitschr. 36. ö (1911). Lee J'yinan: .1. ehem. Suc. London. 105. 1238 (1914). o. Bailly: C'. r. de l'Acad. des sciences. 161. 077 (1915). -) A. Vgl. auch Wurtz: Liebig% Annalen. Suppl. 6. IIG (18B8); 6. 197 (18G8). ir. Giileicitsch: Zeitschr. f. physiol. Chem. 24. 513(1898). Martin Krüger nnd Peter Her gell: Ber. d. Deutschen Chem. Gesellsch. 36. 29Ü1 (1903). ) Vgl. Z.B. ('. Harald Kinq und F. — — 16* XIII. Vorlesung. 244 /CIL. (11.,. OH HO.ILC ,11 /CII3 N f- CH3 //CH3 I + H( ) . H., VCH3 C ->- N '{- CH3 + IL ( ^CH3 " \()J1 \()Il Trimethylammonium- Glykol Cholin hvdroxvd Dem (xlykol sind wir schon begegnet. 1) Alkohol in Beziehung zur Glykolose. Es steht Eine sehr wichtige Bildungsweise des Cholins ist zweiwertiger als aus die dem er- wähnten Oxyäthylamin. Laut man auf dieses .lodmethyl und methylalk«holische Kalilauge einwirken, dann entsteht Cholin. 2) Die nahen Beziehungen dieser \'erbindungen zueinander ergeben die folgenden beiden Formeln GH., GH, . . OII GH., NIL GH. OH . N<X[^}\|^ . ^011' Oxyäthylamin=: Aminoäthylalkohol Gholin. Es sei an dieser (Stelle darauf hingewiesen, daß dem Gholin verwandte Verbindungen sehr verbreitet sind. Allerdings haben sich manche, namentlich aus Pflanzen isolierte, scheinbar neue Produkte, nach sorgfälInteressant ist das tiger Reinigung als gewöhnliches Gholin erwiesen. Vorkommen von Azctylchol in, (GH3)3 .N.(OH).GH., GH., O. OC .GH.,, im Mutterkorn. Außer dem Gholin sind eine ganze Anzahl verwandter Xerbindungen im Tier- und Pflanzenreich angetroffen worden, die sich von ihm in ihrer Struktur unterscheiden. Sie lassen sich alle von Ammoniumliydroxyd ableiten. Sicher sind noch lange nicht alle derartigen Basen bekannt. Sie dürften vor allem auch als Bausteine der Phosphatide in Betracht ') . . kommen. Im Pflanzenreich sehr verbreitet ist das Betain.) Es ist auch im Organismus aufgefunden worden.^') Es ist durch Oxydation von Gholin und auch synthetisch gewonnen worden. Es tritt nicht als Baustein der Phosphatid!^ auf. Es hat folgende Struktur: tierischen Vgl. S. 14. Geor;/ Trier: Zeitsclir. f. pliysiol. Cheni. 80. 401) (191^2). ^) So sind das aus doni Fliegenpilz gewonnene Araanitiii und das aus Uirkensamen isolierte Fagin nichts weiter als gewöhnliches Cholin. ') 2) ist von IIiiscDiaiDi und Manne im Bocksdorn entdeckt worden. Liebi(/f. Sup. 2. 2S:} (lHf)3); 3. 24.i (1864). Vgl. auch Scheibler: Ber. d. Deutschen Cliem. (.esellsch. 2. 21)2 (lSf59): 3. lö.') (1870). K. Srhiihc und G. Trier: /eitschr. f. ) Es - .\iiiialen. physiol. '") — Chem. 80. Ö3 n'.)i2). Briecjer: Die l'toniiiine. Berlin 1885/8(). - Ackernicnin Kahnnigs- u. GenuLhnittel. 14. ()87 (H)07). 421 (1909). M. Ilenze: /eitsclu". f. physiol. Ciieniie. Wilson: .lourn. nf hi,d. Chein. 18. 17 (1914). Zeitsclir. f. — und /•'. Kitfscher: — Suira: Fffüffers Archiv. 78. 70. 2ö3 (19il).' — Dr. Wrir/hf und l'liospliatido ihre Bausteine. ('(M)ll 245 ^(11., oder als Anhydrid l\J)) ( CO N(— CH.j \\ch; Das Betain steht in naher Beziehung za einer Aminosäure, nämlich zum Glykokoll = Aminoessigsäure: CHo . C( )()H. NU, ein, Führen wir in die NiL-Gruppe, Aminogruppe genannt, Methylgruppen dann erhalten wir Betain: COOH. /CH3 CIL . I ^^CH.. M3H Wir werden, nachdem wir die Aminosäuren kennen gelernt haben, ganze Anzahl von betainartigen Verbindungen zu besprechen haben, die wir uns durch erschöpfende Methylierung der Aminogruppe der genannten Säuren entstanden denken können. Wir stoßen mit dieser Feststellung auf eine Wechselbeziehung zwischen Verbindungen eine vom Charakter des Betains und mancher Aminosäuren. Aus Gehirn und Blut isolierte Lichreirh^) eine Verbindung der Zusammensetzung eines Trimethyl-vinylammoniumhydroxydes. Sie wurde Neurin genannt: CH = GH., //CH3 Man kann es sich aus Cholin durch Dehydratation entstanden denken. Vorkommen dieser Verbindung im Gehirn. Da- Guleiritsch-) bestreitet das gegen soll sie in Nebennieren enthalten sein.") Neurin entsteht bei der Einwirkung von Bakterien auf Cholin.) Aus Hefezellen ist eine Verbindung der Struktur eines Dimethyl- propenylamins: ,CH =r CH . CH3 ^CH3 N^CH3 ( ') '-) •') ') 264. (J7() )H O. Liehreich: Liebi;/^ Aiiiialeti. 1,'U. 2!) (18(^0). IV. Gulewitsch: Zeitschr. f. phvsiol. Clioiii. 27. fiO (1W)9). A. Lohmann: Zeitschr. f. IJiol.'Sß. 1 (1<)11). A'. Schmidt: Arch. (1909). d. I'liannazic 229. 48t (1891). — A. Huckcrt: Kbenda. ^III- Vorlesuug. 246 worden. Sie hat den Namen Aschamini) erhalten. Ob sie ein Baustein von Phosphatiden ist oder aber für sich vorkommt, ist noch unaufgeklärt. isoliert Viel umstritten lierter Körper, ist ein aus dem Fliegenpilz, Amanita muscaria, iso- Muskarin genannt. 2) Es ist durch neuere Versuche immer geworden 3), ob eine Verbindung der beschriebenen Art und mit der ihr zugeschriebenen Wirkung auf das Herz sie bewirkt Stillstand in Diastole im Fliegenpilz vorgebildet vorkommt.) zweifelhafter — — Nach der Ansicht vieler Forscher kommt das Lezithin in den Zellen, wenigstens zum Teil, nicht frei vor, sondern es ist mit Vertretern anderer Körperklassen verknüpft. So sind Kombinationen mit Eiweiß und Kohlehydraten beschrieben worden. Eine solche Verbindung soll z. B. das Jekorin darstellen. Es ist dies ein Produkt, das von Drechsele) zunächst aus der Leber gewonnen und bald aus den verschiedensten Organen isoliert wurde. Da nun das Lezithin selbst noch nicht in kristallisiertem Zustande isoliert werden konnte, und ferner seine Konstitution noch nicht über jedem Zweifel erhaben feststeht, erübrigt es sich, die Frage zu besprechen, ob sogenannte Lezithide, d. h. Kombinationen von Lezithin mit anderen Verbindungen vorkommen. Sie läßt sich erst dann beantworten, \venn unsere Kenntnisse über die Lezithine vollständigere sind. Vorläutig kann man immer noch den Einwand erheben, daß die beobachteten Kombinationen auf Adsorptionserscheinungen beruhen^ d. h. keine chemischen Verbindungen darstellen. Außer dem eben besprochenen Lezithin ist eine weitere Verbindung in immer nähere Beziehungen zur Gruppe der Lezithine gebracht worden, nämlich das Kephalin. Es ist von T/iudichum'^) im Gehirn entdeckt w^orden. Später wurde es auch aus Eigelb dargestellt. Es scheint nach manchen Beobachtungen ziemlich verbreitet zu sein.^) Kephalin enthält, wie das eigentliche Lezithin, ein Molekül Glyzeryl-phosphorsäure und zwei Moleküle Fettsäuren, und zwar Stearinsäure und Kephalinsäure. Als stickstofi'h altige Base wurde Oxäthylamin=:Aminoäthylalkohol aufgefunden. 9) Wir hätten somit eine Verbindung vor uns, die nach der gegebenen Definition als Lezithin zu bezeichnen wäre. Levene und TFes^io) geben dem Kephalin folgende Formel: '^) Emil Abderhalden uud JI. Schaumann: Pßm/efS Archiv. 172. 1 (1918). Schmiedeherg uad Koppe: Das Muskarin, das gütige Alkaloid des Fliegenpilzes. Leipzig 1869. — E. Harnnck: Arch. f. cxperim. Path. u.Pharmak. 4. 168 (1875). Schmiedeherg uud Harnack: Ebenda. 6. 101 (1876). ^) Vgl. Arthur James Eirins: Biocheni. Jouru. 8. 209 (1914). //. H. Dale:^. ^) ^) — — Pharm, and cxperim. Ther. 6. 147 (1914). ) Albert B. Weinhagen: Z. f. physiol. Chemie. 105. 249 (1919): 112. 13 (1921). Drechsel: Jouru. f. prakt. Chem. 33. 425 (1886). Vgl. ferner S. 73. Thudichum: 1. c. •) M. Stern und N. Thierfeider : Zeitschr. f. physiol. Chem. 53. 371 (1907). ") Koch und Woods: .Tourn. of Biol. Chem. 1. 203 (1905/06). S. Fracnkel: Biochem. Zeitsclir. 16. 370 (1909). ) A. Jianmann: Biochem. Zeitschr. 54. 30 (1913). Montagne H. Benall: Ebenda. 55. 296 (1913). ") B. A. Lerene und S. Komatsii: .1. of biol. Chem. 39. 91 (1919). B. A. Levene und Jda B. Rolf: El)enda. 40. 1 (1919). Vgl. auch B. A. Lerene und d.J. West: Journ. of Biol. Chem. .35. 285 (1918). Vgl. hierzu auch Consin: .lourn. de Pliarm. et do Chim. 24. 101 (190(3); 25. 177 (1907). Jakob Bornas: Biochem. Zeit- — "•) '') — — — — — — Phosphatide imd ihre Bausteine. CH. . . OC . C\, . 247 H31 CH .U.()C.C„.H33 CH, .0.0— P = OH — CH, .CH. .NH,. Noch unsicherer sind unsere Kenntnisse über eine weitere Gruppe von Substanzen, die von manchen Forschern hier untergebracht worden Sie kommen im Nervengewebe und vielist. Es sind dies die MyeHne. leicht auch in anderen Geweben (Herzmuskel) vor. 1) Die bis jetzt besprochenen Phosphatide hatten das gemeinsam, daß in ihnen auf ein Phosphor ein Stickstoff kommt. Man hat, wie oben schon erwähnt, das Verhältnis von Phosphor zu Stickstoff" vorläufig als Grundlage einer Einteilung der Phosphatide gewählt und unterscheidet: 1. Monoamino-monophosphatide: 1N:1P. Hierher gehören die Lezithine-) und das Kephalin. Als ein Vertreter dieser 2. Monoamino-diphosphatide: 1N:2P. Klasse von Verbindungen ist das aus Herzmuskel isolierte Cuorin betrachtet worden.) Neuerdings wird seine Existenz bezweifelt.^) Weitere Monoamino-diphosphatide sind aus der Leber^) und aus Eigelb^) (Hydrolezithin und Hydrokephalin^) gewonnen worden. hört Dann folgen o. Diamino-monophosphatide: 2N: IP. Dahin gevon Thndichnm aus Gehirnsubstanz abgeschiedene Sphingo- das myelin. Dieses Phosphatid kristallisiert aus Alkohol in Nadeln. Bei seiner Spaltung wurden Phosphorsänre, Cholin, Sphingosin, Lignozerinsäure, CH3 .(CH2).22 COOH, und eine noch nicht erkannte Säure erhalten.') Ein ganz ähnliches Phosphatid hat Erlandsen^'^) aus Muskeln abgetrennt. Es enthält Glyzeryl-phosphorsäure, ferner soll eine Oxyfettsäure in ihm enthalten sein. Es fehlt noch die Aufklärung der stickstoffhaltigen Base. Endlich ist aus Eigelb'^) und ferner aus der Pankreasdrüse 1-) des Pferdes ein kristallisiertes Diaminophosphatid gewonnen worden. • Schrift 22. 411 (1910). 21. 41 — — Vgl. hierzu P. A. Levene und C. J. West: J. of biol. Chem. F. Fenger: J. Pharm, aud experim. Ther. 18. 51 (1921). (1916). ') Vgl. hierzu auch L. Äschoff: Beitr. z. pathol. Anat. u. z. allgem. Pathol. 47. 1 (1909). und Ida P. Rolf: The J. of biol. ehem. 46. 353 (1921 A. Erlandsen: Undersögelser voer Hjertets fosfatider. Kjöbeuhavii 1906. Zeitschrift, f. physiol. Chem. 51. 71 (1907). Hugh Mac Leaii [(Biocbem. .Jouni. 8. 453 (1914)] hält das Cuorin für unreines Lezithin. 5) F. A. Levene und S. Koniatsu: The J. of biol. chem. 39. 83, 91 (1919). Mac Lean, Buc/h and W. J. Grifßths: Biochemical J. 14. (515 (1920). «) A. Baskoff: Zeitschr. f. physiol. Chem. 57. 395 (1908); 60. 426 (1909). Vgl. auch F. A. Levene und Inqvaldsen: The J. of biol. chem. 43. 359 (1920). ") Mac Lean: Ebenda. 57. 304 (1908). F. A. Levene und Ida F. Rolf: The J. of biol. chem. 46. 193 (1921). F. A. 8) F. A. Levene und C. J. West: The J. of biol. chem. 35. 285 (1918). Levene und S. Komatsu: The J. of biol. ehem. 39. 83 (1919). 9) F. A. Levene: The Journ. of biol. chem. 15. 153 (1913): 18. 4.53 (1914); 24 69, 111 (1916). •') Vgl. auch F. Ä. Levene — ) 1. — — — — '0)yt. Erlandsen: Zeitschr. f. physiol. Chem. 51. 71 (1907). ") M. Stern und IL Thierfelder : Ebenda. 53. 370 (1907). 12) S. Fraenkel und Offer: Biochem. Zeitschr. 26. 53 (1910). XIII. Vorlesimg. 2 48 Wichtig: ist. daß auch in der Milch ein Monoamino- und ein Diaminophosphatid gewonnen worden sind. Schließlich scheint auch ein aus Nieren dargestelltes Phosphatid-) hierher zu o-ehören.3) Es soll an Stelle von Glyzerin Galaktose besitzen. Bei der Hydrolyse wurden gefunden: Phosphorsäure, Cholin, Sphingosin. Fettsäuren und Galaktose.) Schließlich ist ein Produkt der Klasse der Phosphatide beschrieben worden, an dessen Aufbau Lignocerinsäure und Glukosarnin neben Phosphorsäure beteiligt sein soll.'') Eine weitere, vierte Gruppe bilden die T r i a m i n o-m o n o p h o s p h a t i d e. In ihnen finden sich Stickstoff und Phosphor im Verhältnis von 3:1. Dahin wird das sogenannte Neottin^) gerechnet, das aus Eigelb erhalten wurde. Endlich wäre noch 5. der Triamino-diphosphatide zu gedenken. Sie weisen das Verhältnis o N 2 P auf. Ein derartiges Phosphatid ist als Bestandteil der Niere^) beschrieben. Ferner soll das Sahidin^), ein Phosphatid der Gehirnsubstanz, hierher gehören. Es ist sehr schwer zu sagen, ob dieser Art der Einteilung der Phosphatide zurzeit mehr Bedeutung als der einer vorläufigen Orientierung zukommt. Solange man nicht einmal die einzelnen Bausteine der verschiedenen Vertreter dieser Erlasse von Verbindungen kennt, wird man bei der ganzen Art der Isolierung mancher der als chemische Individuen beschriebenen Körper Zweifel darüber nicht ganz unterdrücken können, ob wirklich einheitliche Substanzen zur Beobachtung gelangt sind. Aus diesen Gründen kann es nicht aufiallen, wenn manche der angeführten Verbindungen angeihre Zahl ließe sich aus der Literatur noch leicht vermehren-') fochten ist. Wichtig ist jedenfalls als Wegweiser, daß man nach bestimmten Methoden aus bestimmten Organen Substanzen abscheiden kann, die das erwähnte Verhältnis von N zu P aufweisen, vorausgesetzt, daß nicht Gemenge vorliegen, ^^on dieser Grundlage aus werden sich sicher neue Wege finden, die uns einen klareren Einblick in die Gruppe der Phosphatide eröffnen, als es zurzeit der Fall ist. Nicht unerwähnt wollen wir lassen, daß eine Trennung von Phosphatiden nach den Löslichkeitsverhältnissen möglich ist. Man hat in Azeton lösliche, in diesem Lösungsmittel unlösliche, dagegen in Alkohol lösliche und endlich in Azeton und Alkohol : — — unlösliche Produkte unterschieden. ^o) Von großem Interesse ist die Feststellung, daß auch in der Pfianzen- welt sich zahlreiche, verschiedenartige Phosphatide finden. ^\) Manche scheinen sehr ähnlich gebaut zu sein, wie die entsprechenden Verbindungen M ^) Jouni. of Biol. Chem. 21. 539 (191;")). Ih. B. Osborne und A. J. Wakcman Es ist zu Unrecht Carnaubon genanut worden. Es enthält die angenommene : Carnaubasäure nicht. — ") H. Mac Lraii : Zeitschr. f. physiol. Chem. 64. 302 1 91U). Soc. 27. .Juni) (1912). Otto Rosenlieim und Uu()h Mac Lean: Biocheni. .lourn. 9. 103 (1916). S. Fraenkel und /'. Kafka: Biochem. Zoitschr. 101. 159 (1920). S. Fraenkel und Bti/allio: Biochem. Zeitschr. 9. 44 (1908). S. Fraenkel und Noqurira: Biochem. Zeitschr. 10. 3()() (1909). 6'. Fraenkel: Biochem. Zeitschr. 24. 268 (1910). ") Vgl. ) Dtmham und .Jacobson : ( •Journ. of Physiol. 45. (l'roceed. physiol. ) ') ") ') z. B. S. Fraenkel und /.. Dimitz: Biochem. Zeitsciir. 28. 295 (1910). — S. Fraenkel u. //. Elias: Ebenda. 28. 320 (]91(J) und weitere Arbeiten in dieser Zeitschritt. ") Vgl. P. A. Levene und 7'. Inr/raldscn: Tiic Journ. of biol. chem. 43. 359 (1920). — und F. Steiger: Zeitschr. f. physiol. Chem. 13. 365 (1889). O. llicstand: Inaug.-Diss. und E. Winterstein: El)enda. 40. 101 (1903). E. W'intersfein und O. Hiesland: Zeitschr. f. pliysiol. f'hemie. 47. 496. Zürich 1906. ") Vgl. E. Schulze F. Schtilze — — Phospliatiile mi<l ihre Bausteine. 249 des Tierreiches, ja in vielen Fällen ist die Übereinstinnnunji,- so groß, dati vielleicht identische Produkte vorliegen. Man muß jedoch bei derartigen Schlußfolgerungen sehr vorsichtig sein denn selbst wenn entsprechende Eigenschaften vorliegen und die Art und Mengen der einzelnen Bausteine die gleichen sind, ist doch noch die Möglichkeit von isomeren ^'erbindungen gegeben. Im Anschluß an die Phosphatide sei noch eines sehr wichtigen Produktes gedacht, das von Liebreich^) aus Gehirnsubstanz abgetrennt worden ist, nämlich des Protagons. Es findet sich in markhaltigen Nervenfasern. Es sollen jedoch auch ganz ähnliche Körper in 'den Blutkörperchen, den Leukozyten, Nieren, Nebennieren usw. vorkommen.-) Es ist noch unklar, welche Stellung diese Klasse von Substanzen zu den Phosphatiden hat, ja es ist noch umstritten, ob die als Protagone bezeichneten Substanzen einheitliche Verbindungen darstellen. Es fehlt nicht an Forschern, die der Meinung sind, daß das Protagon ein Gemisch von ganz verschiedenen Komplexen darstellt. ^'j Das Protagon aus Gehirnsubstanz Es enthält die Elemente C, II, (>, N, ist in Kristallform erhalten worden. P und S. Beim Kochen mit Barytwasser erhielt man charakteristische Bau, Fettsäuren, Gl yzeryl-phophor säure steine der Lezithine, nämlich und Cholin. Daneben treten als neuer Bestandteil die sogenannten Zerebroside in einer Menge von ca. SÖ^/o des Ausgangsmateriales auf. In welcher Form der Schwefel gebunden ist, ist noch unklar. Die Zerebroside enthalten keinen Phosphor und Schwefel. Sie sind stickstoffhaltig. Es sind zwei Vertreter dieser Klasse von Verbindungen bekannt, nämlich das Phrenosin bzw. Zerebron) unddasKerasin.") Beide sind sich sehr ähnlich. Sie unterscheiden sich hauptsächlich durch ihr optisches Verhalten. Sie linden sich nicht nur im Nervengewebe, man hat sie vielmehr auch in der Nebenniere, der Leber, im Eigelb usw. angetroffen.''') Bei der Spaltung des Phrenosins bzw. Zerebrons, CVsH.uNOy, erhielt Thierfeider Galaktose, Zerebrons äure und ferner die stickstoffhaltige Base Sphingosin. ') Die Zerebronsäure ist eine a-Oxysäure. Sie ist von Levene und Jacobs ^) durch Oxydation in Lignozerinsäure'-'), C24H48O2 =CH3. (€112)22 -COOH, — — E. Winterstein: Zeitschr. f. psychol. Chem. 58. 500 (1909). (1906); 54. 288 (1908). K. Smolcnski: Ebenda. 58. E. Winterstein uud K. i^moleiiski: Ebenda. 58. 506 (1909). E. Schulze und E. tichulzc uud G. Trier: Ebenda. 67. 4(5 (1910). 522 (1909). 17. Njcqovan: G. 7V-i>r; Ebenda. 73. 383(1911). Pfennin(/er: Ebenda. 71. 174 (1911). Georg Trier: Ebenda. 86. 407 (1913). Ebenda.' 76. 1 (1911). ) Liebreich: Liebig?, Aunalen. 134. 29 (1865). Vgl' weitere Literatur Thitdlchu>n, Kossei, Baumstark, W. Cramer, JV. J. Gies, 0. Roseiiheim und Chr. Tebb, Ganigct — — — — — — — und Blankenhorn und vor allem Thierfeldcr und Wörner, vgl. Biochem. Handlexikon. 250 (1911) (bearbeitet von W. Cramer). J. Springer. Berlin (1911). -) Vgl. Literatur: Biocliem. Handlexikon. 3. 250 (1911) 1. c. ') Vgl. hierzu Ale.r. L. l'curson: Biochem. Journ. 8. 616 (1914). Früher wurden Zerebron und Phrenosin als zwei verschiedene Verbindungen 3. •) Jetzt wissen wir, daß sie identisch sind. P. A. Leinene und C. J. West: The J. of Biol. Chem. 15. 193 (1913); 31. 635 (1917). ») Vgl. u. a. r. A. Levene uud C. J. West: J. of biol. Chem. 31. 649 (1917). ') Bei der Oxydation des Sphingosius wurde n-Trid'ecylsäure erhalten [/. .1. L'etührt. "•) ('. J. West: The Journ. of Biol. Chem. 18. 481 (1914)1. F. A. Levene und W. A. Jacobs: Journ. of Biol. Chem. 12. 839 (1912; 18. 477 Hermann Vgl. auch Otto Roseiiheim: Biochem. Journ. 10. 142 (1917). (1914). P. A. Levene Loeninq und //. 'Ihierfelcler: Zeitschr. f. physiol. Clieni. 74.282(1911). uud C'.'./. West: Journ. of Biol. Chem. 26. 115 (1916). P. Brigl: Zeitschr. f. physiol, Chemie. 95. 161 (1915). P. A. Levene und /''. A. Tai/lor: .). of l)iol. Chom. 52. 227(1922) ") Vgl. hierzu Meyer, Brod und Soyka : Monatsh. f. Chemie 34. 1113 (1913). Levene und ») — — — — — Xlll. Vorlesung. 250 Dieser Säure sind wir schon bei der Besprechung; des Sphingomyelins begegnet. Kerasin, C47 Hm NOg + H.^ 1). besteht aus 1 Molekül Sphingosin, Ololekül Galaktose und i Molekül Lignozerinsäure.-) Das andere Spaltstück des Zerebrons, das Sphingosin, ist als ein ungesättigter, zweiwertiger Aminoalkohol charakterisiert worden. 3) Es kommt ihm die Formel: ri7H3i(OH)2.NH.2 zu. Die Zerebroside können, je nach der Anordnung der einzelnen Bausteine, genau so, wie die Fette und Phosphatide, in mehreren stereoisomeren Formen vorkommen. Es können auch die einzelnen Bausteine ganz verschiedene sein. Die Galaktose ist vielleicht der einzige konstante Bestandteil der Zerebroside. Infolge ihres Gehaltes an einem Kohlehydrat können wir sie auch als Glukoside auffassen. Fettsäure und vielleicht auch die stickstoffhaltige Base können wechseln. Interessant ist, daß auch im Pflanzenreiche Substanzen angetroffen worden sind, die Beziehungen zu den Zerebrosiden zu haben scheinen.) H. Thierfeider ^) stellt sich die Konstitution des Kerasins und des Phrenosins bzw. Zerebrons, wie folgt, vor: tibergeführt worden. C23H47 — CO j Lignozerylgruppe NH I C12H..5 — CH = CH— GH— CH— CH2 Sphingosinrest. HO.CH— CHOH— CHOH — CH— CHOH— CHo.OH Galaktoserest } Kerasin. C23H^7 — CH — CO Zerebronsäurere st OH I NH C,2H.,5— CH = CH — CH— CH— CHJ^ Sphingosinrest 0^ OH CH — CHOH — CHOH — CH— CHOH— CHo OH . . } Galaktoserest Phrenosin bzw. Zerebron. 1) Rosenheim: Biochem. J. 10. 142 (leiC)). — Vgl. hierzu P. Briyl uud E. Fuchs: J. f. phvsiol. Chemie 119. 280(1922). n. Ihierfelder: Zeitschr. f. physiol. Chem. 85. 35 (1913); 89. 248 (1914); Ul. 1Ü7 (1914). P. A. Levene: J. of biol. Cheui. 15. 359 (1913). Posenheim: Biochem. J. 10. 142 (191(j). ^) — — — P. A. Levene und F. A. Taylor: 227 (1922). P. A. Lei'ene uud Jacobs: Jourii. Biul. Chem. 11. 547 (1912). Otto Piesser und //. Ihierfelder: Zeitschr. f. physiol. Chem. 77. 508 (1911). Karl Thomas und /'. A. Levene IL Thierfelder: Kbenda. 77. 511 (1912). und ('. J. West: Journ. ot Biol. Chem. 24. 63 (1916). ) Zellner: Monatshefte für Chemie. 32. 133. 1057 (1911). ) y/. Thierfelder: Zeitschr. f. physiol. Chemie. 89. 248 (1914). Vgl. auch PoJ. of biol. Chinii. 52. •') — — — — senheim: Biochem. Journ. 10. 142 (1916). Phosphatide und ihre Bausteine. 251 Schließlich sei noch erwähnt, daß Koch^) und Levene-) schwefelhaltige Verbindungen aus Gehirnsubstanz gewonnen haben, die in mancher Hinsicht den Phosphatiden nahe zu stehen scheinen. Sie sind Sulfatide genannt worden. Die von Levene erhaltene Verbindung ist frei von Phos- phorsäure. Wenn wir auf die besprochenen Gruppen von Verbindungen zurückblicken, dann erkennen wir aus der ganzen Darstellung, daß unsere Kennt- nisse noch sehr lückenhafte sind. Es unterliegt keinem Zweifei, daß vorläufig mancherlei \'erbindungen zu Gruppen vereinigt worden sind, die gar nichts miteinander zu tun haben. Ferner wird sich gewiß manche bereits mit besonderem Namen belegte Verbindung als eine verunreinigte, bereits bekannte Substanz herausstellen. Es liegt in der Natur der einzelnen Verbindungen, daß die Forschung so langsam fortschreitet. Vor allem führt die leichte Zersetzlichkeit einzelner Produkte zu großen Schwierigkeiten. Sehr unangenehm machen sich endlich die sehr ähnlichen Löslichkeits Verhältnisse geltend. Werden erst einmal die im Pflanzen- und Tierreich so außerordentlich verbreiteten Körper der besprochenen Gruppen genau bekannt sein, dann werden ohne Zweifel viele ungelöste Probleme über den Zellstoffwechsel wesentlich gefördert werden. ») ^) W. Koch: Zeitschr. f. phvsiol. P. A. Levene: Jouru. of Biol. Chem. 70. 94 (1910). Chem. 13. 463 (1913). Vorlesung XIV. Fette. Pliosplmtide. Sterine. Bildung der Fette, Phosphatide und ihrer Bausteine, sowie der Sterine im Pflanzenreich. Verhalten der Fette und Phosphatide im tierischen Organismus. Ihr Abbau im Darmkanal. Wir haben schon bei der Besprechung der Synthese der Kohlehydrate im Pflanzenorganisraus aus Kohlensäure und Wasser i) die Frage besprochen, ob die Bausteine der Fette als primäre Assimilationsprodukte zu betrachten sind oder aber sekundär aus bestimmten Kohlehydraten entstehen. Die Bildung des Glyzerins, sowie der mehrwertigen Alkohole überhaupt, ist leicht verständlich. Sie stehen in so nahen Beziehungen zu den Kohlehydraten, daß eine Umwandlung der einen Klasse von Stoffen in die andere und umgekehrt wohl auch der Zelle keine Schwierigkeiten bietet. Es sei in dieser Beziehung an die Glyzerose, eine Triose. erinnert. 2) Mit ihr steht das Glyzerin in nächster Beziehung: Fette, riiospliatide. Steiiue. 25?) Wahrscheinlicher ist die Annahme, daß zunächst ein Abbau zu einfacheren Produkten erfolgt bzw. die Bildung der Bausteine der Fette von einfacheren Assimilationsprodukten ausgeht. Man wird auch hier an Verbindungen der Dreikohlenstoftreihe und mit ihnen in Beziehung stehenden bildet.^) Substanzen denken. Es sei auf die S. 1H7 dargestellten Zusammenhänge zwischen Methylglyoxal, Brenztraubensäure, Azetaldehyd, Essig- säure und Azetessigsäure hingewiesen. Man kann sich auch vorstellen, daß zwei Moleküle Azetaldehyd zusammentreten-) und dann die Umwandlung in Butt er säure eintritt ='): CH, . r(^\\ + VE, . c<J^ Azet- Azet- aldehyd aldehyd = CH3 CH (OH) . . cn, C<H . Aldol CH3 CH, (JH., COOH Buttersäure. . . . Auch andere Aldehyde könnten in ganz entsprechender Weise verbunden werden und so zu mannigfaltigen Fettsäuren führen. \) Aldehyde und ferner Ketone spielen als Zwischenstufen zwischen verschiedenen Klassen von Verbindungen sicher eine große Rolle. Sie sind sehr reaktionsfähig. Vielleicht führen die Versuche von Neiiberg u. a.^) über die Oxydation verschiedener Verbindungen bei Einwirkung von Licht und ferner eines Ferriüder Uranylsalzes zu neuen Ergebnissen bei der Erforschung der Fettbildung aus Kohlehydraten in der Pflanze.") Es entstehen z. B. aus Alkoholen Aldehyde, aus Säuren Aldehyde oder Ketone, und zwar tindet oft gleichzeitig ein Abbau statt. Er kann jedoch auch ausbleiben. Auch Aminosäuren gehen in Aldehyde über. Sie verlieren bei dieser Umwandlung die Aminogruppe. Bei dieser Gelegenheit sei der schon S. 181 erwähnten Art der Umwandlung von Aldehyden gedacht. Wir haben dort festgestellt, daß zwei Moleküle eines bestimmten Aldehyds unter Wasseraufnahme in ein Molekül Säure und ein Molekül Alkohol übergehen können {Can7iizzaro^Q\i(^ Reaktion). Parnas'') war der erste, der auf diese Art der Umwandlung von Aldehyden in Geweben hinwies. So erhält man z. B. aus zwei Molekülen Azetaldehyd Essigsäure und Äthylalkohol: CH3 . (<[] Azetaldehyd ') + CH, C<2 + H., . = CH3 COOH + CH;, . Azetaldehyd Essigsäure . CH., . OH Äthylalkohol. Emil Fischer: Die Chemie der Kohlehydrate und ihre Augast Hirschwald. S. 28. 1894. Bedeutuiii,' fiir die i'hy- siolocie. Berliu. •-) ^) Vgl. hierzu S. 131. Vgl. hierzu A. Mcu/iiiis-Lery und L. F. Mei/er: Haudl». d. Biocheni. 4. 445 — A. Magnvs-Lery: Arch. (Anat. Physiol. 300 (1902). Vgl. hierzu auch Ida Sinedlei/ und Kra Lubrziinsla The Hiochem. Jouin, (1913). — Zentralhl. Physiol. 26. 91ö (1913). CarlNeMberg: Biochem. Zeitschr. 13. 3(J5 (1908); 29. 279(1910). — Vgl. auch 87. — Vgl. auch Alfred Bnirath: Jouru. prakt. Chem. 8«. 336 (1912). (1909). f.' u.) ") 3(')4 7. : f. '') 8. (2.) f. Vgl. auch 8. 87. •) ./. Parnafi: Biocliem. Zeitschr. 28. 275 (1910). Vyl. auch lidtfclli und /.. Stern: (Jonip. reud. Soc. de hi(d. 08. 742 (1910); 60. in2 (1910). Biocliem. Zeits.iirift. 29. 130 (1910). ') — — XIV. Vorlesung. 254 Ein Ferment, Aid eh yd mutase genannt, beschleunigt den Reaktionsverlauf so stark, daß die oben erwähnte Aldolbildung nicht eintritt. Ganz besonders bedeutungsvoll ist bei dieser Reaktion, die exotherm verläuft, der Umstand, daß durch eine Hydrolyse eine Oxydation herbeigeführt wird und zugleich eine Reduktion die Bildung der Säure entspricht der Oxydation, diejenige des Alkohols der Reduktion. Parnas weist auf den Umstand hin, daß im Tierreich Fette und Wachse vorkommen, in denen die Fettsäure und der Alkohol den gleichen Bau und die gleiche ^Anzahl Kohlenstotfatome aufweisen. Es liegt nahe, anzunehmen, daß beide Komponenten aus einer gemeinsamen Verbindung, — nämlich dem entsprechenden Aldehyd, hervorgegangen sind. Es sei an das Fett des ßürzeldrüsensekretes erinnert. Es enthält Oktadezylalkohol CigHj^gO, und Stearinsäure, CjgHogOa. Im Walrat finden sich Zetylalkohol, CiüHg^O, und Palmitinsäure, CieHssO.., esterartig verknüpft. Es ist immer von Interesse, derartigen Beziehungen nachzugehen und zu prüfen, inwieweit sich aus solchen Beobachtungen Aufschlüsse über die Entstehung bestimmter Verbindungen ergeben. Vorläufig sind wir immer noch auf Schlüsse angewiesen, die der Wahrscheinlichkeit entsprechen, jedoch nicht durch lückenlose Ergebnisse gesichert sind. Bei den Pflanzen dürften die Bausteine der Fette und damit diese selbst im Prinzip auf zwei Arten entstehen, nämlich einerseits durch sekundären Umbau von andersartigen Verbindungen und andrerseits als primäre Produkte der Kohlensäure- und Wasserassimilation. Im ersteren Falle kommt insbesondere die Umwandlung von Kohlehydraten, ferner von Alkoholen und auch von Säuren verschiedener Art in Fettsäuren in Betracht. Alkohole können zu dem entsprechenden Aldehyd oxydiert Averden und dieser würde dann entweder unter Aldolbildung in eine Säure verwandelt, oder aber es entstehen unter Wasseraufnahme aus zwei Molekülen des Aldehyds ein Molekül Alkohol und ein solches der entsprechenden Säure. Säuren können gleichfalls in Aldehyde übergehen oder anderweitig umgewandelt werden. Es sind jedenfalls in den meisten Fällen eingreifende Vorgänge notwendig, bis die Umwandlung in die entsprechenden Fettsäuren vollzogen ist. Manches spricht dafür, daß aus den verschiedenartigsten Verbindungen durch Abund Umbau zunächst eine Verbindung einfacher Konstitution gebildet wird, über die die Synthese zu Glyzerin und den Fettsäuren verläuft. Vielleicht treffen sich in diesen Verbindungen die Abbaustufen verschiedenartiger Produkte. Sie würden ein gemeinsames Durchgangsstadium für die Bildung der Bausteine der verschiedensten Körperklassen darstellen. Die gleichen Verbindungen könnten auch beim Assimilationsvorgang primär aus Kohlensäure und Wasser sich bilden. Carl Neiihcrq^) hat, wie schon S. 129 erwähnt Avorden ist, noch eine andere Möglichkeit der Bildung kohlenstoffreicherer Verbindungen aus kohlenstoftarmeren entdeckt. Hefezellen vermögen z. B. Azetaldehyd in statu nascendi mit Benzaldehyd zu Phenylazetylkarbinol bzw. Phenylbrenztraubensäure zu vereinigen: CH3 C (^ -f Ce U, C <g . . ') Vgl. die Literatur S. 129, Zitat —^ CH, CO CII (OH Ce h . -). . ). J Fette. Phosphatide. Sterine. 25f) Es ist wohl möglich, daß bei der Synthese von Fettsäuren diese unbekannte Art von fermentativer Verknüpfung von Kohlenstoß' an Kohlenstott" eine Rolle spielt. Das in Betracht kommende Ferment ist Karboligase genannt worden. Die Umwandlung von komplizierter gebauten Verbindungen und vor allem von Kohlehydraten in Fett wird man am besten an Samen verfolgen können. Wir kennen solche, die in großen Mengen Öl aus Zucker bereiten. Bis jetzt war es nicht möglich, Zwischenstufen in diesem Umwandlungsvorgang aufzufinden. Es scheint, daß der Traubenzucker zunächst gesättigte Fettsäuren liefert und diese dann zum Teil wenigstens in ungesättigte Verbindungen übergehen. i) Die Bildung der Phosphatide erfordert noch weitere Bausteine. Einmal muß Phosphorsäure zugegen sein. Diese nimmt die Pflanze in Form von Phosphaten durch die Wurzeln auf. Ferner muß die stickstoffhaltige Base gebildet werden. Wir werden bei der Besprechung der Bildung der Eiweilikörper bzw. der Aminosäuren in der Pflanze auf die Frage der Stickstortquelle des Pflanzenorganismus eingehen. Hier sei nur erwähnt daß der Befund von Aminoäthylalkohol bei der HydrolysemancherLezithinarten vielleicht den Weg anzeigt, auf dem Cholin und im Anschlüsse daran Betain entstanden sind. Man könnte sich z. B. vorstellen, daß zunächst Glykolaldebyd entsteht und dieser entspechend der Cannizza roschen Reakl)isher tion in Glykol säure und Glykol übergeht-j: ?\r COOK ?\h + H,0 = +1CH,.OH I CH,.OH Glykolaldehyd Glykolaldehyd CH.,.OH " + 1 I CH...OH CH„.OH Glykolsäure Glykol. Diese beiden Verbindungen könnten dann mit Ammoniak unter Ausvon einem Molekül Wasser auf direktem oder indirektem Wege die beiden folgenden Verbindungen ergeben: tritt CIL. OH COOH und j I CH2.NH2 Glykokoll = Aminoessigsäure CH2.NH2 Aminoäthylalkohol. Die erstere Verbindung ist eine Aminosäure und Baustein vieler EiDer Aminoäthylalkohol geht bei erschöpfender Methylierung in Cholin über 3): weißstoffe. /CH3 HO GH., CH, — N<[\| . . —^ HO CH, CH^ — N<^^^^ . - . \0H V^l. z. B. Scrgius Iranoir: Beiheft zum bot. Zentralbl. 28. 13!» (1911). Vgl. hierzu d.' Trier: Über einfache Ptianzenbasen und ihre Beziehungen zui -Vufbau der Eiweißstoil'e und Lezithine. Gebrüder Borutrüger. Berlin 1912. ') Vgl. auch S. 244. ») ') ^ orlesung. -^^^^ 95(3 Die geschilderte Art der Bildung des Aminoiithylalkohols aus Glykol bereitet insofern gewisse Schwierigkeiten, als nicht ohne weiteres verständlich durch die NH,ist, weshalb nur eine OH-Gruppe des zweiwertigen Alkohols Gruppe ersetzt wird. Trier nimmt an, daß nur eine solche zur Verfügung stehe. Die andere soll nämlich durch Phosphorsäure besetzt sein. Wir hätten Kohlensäure und Weg > Formaldehyd —> Glykolaldehyd —>-Glykol — — Aminoäthyl-phosphorsäureester — phorsäureglykolester Phosphor säürecholinester — Diglyzerid-phosphorsäure-cholinder Lezithinsynthese: dann etwa den folgenden Wasser >-Phos- ^ >^ >- >- ester. Diese Darstellung des möglichen, jedoch durchaus nicht bewiesenen N'erlaufes der Lezithmsynthese gibt nur einen Weg wieder. Selbstverständlich kann z. B. zunächst der Diglyzerid-phosphorsäure-glj^kolester entstehen und dann erst die Einfügung der Aminogruppe einsetzen, ja Trier hält es für wahrscheinlich, daß zunächst das ganze Molekül des Lezithins auf das Cholin aufgebaut und ganz zuletzt die Aminoäthylgruppe methyliert wird, falls diese nicht, wie z. B. im Kephalin erhalten bleibt. Schließlich sei erwähnt, daß man sich die Bildung des Aminoäthylalkohols auch, wie folgt, denken kann. Glykolaldehyd könnte direkt mit Ammoniak in Reaktion treten und Aminoazetaldehyd liefern. Dieser bis würde dann entsprechend der Cannizzaroschen Reaktion ein Molekül Aminoäthylalkohol und ein solches von Aminoessigsäure — Glykokoll liefern. , Fctto. Phosphatide. Steriue. 257 Selbstverständlich haben derartige Erörterungen über die mögliehen Wege einer bestimmten Synthese nur so lange einen Wert, als sie nicht in Widerspruch mit tatsächlichen Befunden treten. Sie gleichen dem Entwürfe eines Planes, dessen Einzelheiten durch das direkte Experiment festgelegt wer- den müssen. Neue Versuche bringen neue Beobachtungen. Der Plan wird ergänzt und verbessert. Oft muß er schließlich so umgeändert werden, daß ein ganz neues Bild entsteht. Mit der Besprechung der erwähnten Möglichkeiten der Synthese von Cholin in der Pflanze und wahrscheinlich in mancher Hinsicht auch im tierischen Organismus haben wir noch einen anderen Zweck verfolgt. Um in den Geist der physiologischen, mit chemischen Methoden angebahnten Forschung einzudringen, genügt es nicht, bestimmte Formeln zu kennen, man muß sich vielmehr in die ganze Denkungsweise des Chemikers vertiefen. Je mehr Wege man kennt, die zu einer bestimmten Verbindung hinführen, um so klarer wird der Einblick in den feineren Bau des einzelnen Moleküls. Über die Entstehung der übrigen Phosphatide lassen sich zurzeit schon deshalb keine Angaben machen, weil wir ihre Zusammensetzung nicht kennen. Auch über die Synthese der Sterine in der Pflanzenzelle vermögen wir nichts auszusagen. Angedeutet sei, daß vielleicht die sogenannten Zyklosen das Bindeglied zwischen den aliphatischen und zyklischen Verbindungen darstellen.') Doch sind noch viele andere Möglichkeiten gegeben. In der Pflanzenzelle kommt den Fetten, den Sterinen und Phosphatiden ohne Zweifel im Prinzip die gleiche Bedeutung zu. wie in der tierischen Zelle. Lager von Fett finden wir bei den mitten im Stoftwechsel stehenden Organen der Pflanzen selten, wohl aber in Samen. Knollen und Früchten. Die Zellen der Pflanzen verfügen genau, wie die Tierzellen, über alle Einrichtungen, um Fette und sicher auch Phosphatide aller Art in ihre Bausteine zu zerlegen. Die Pflanze kann so ein Fett in ein anderes überführen oder die Bausteine auch anderweitig versvertcn. Namentlich in fettreichen Pflanzensamen ist das Abbauvermögen von Fett genau studiert worden. Man beobachtete das Auftreten von Fettsäuren und Glyzerin. Der ümvvandlung von Fett in Kohlehydrate geht diese Spaltung ebenfalls voraus. Sie erfolgt, wie Müntz-) zuerst erkannte, durch ein Ferment, Lipase genannt. Es ist überall da auzutreften. wo Fettdepots sich finden, aber auch in jeder einzelnen Zelle. Ein sehr gut wirkendes lijjolytisches Ferment ist aus Rizinussamen isoliert worden.^) Es wirkt am intensivsten bei schwach saurer Reaktion. Es ist noch unentschieden, ob die Pflanzenzellen Fett vollständig bis zu den Bausteinen abbauen, wenn solches an andere Orte übergeführt werden soll. Es ist möglich, daß nur ein Teil des Fettes gespalten wird. Sobald freie Fettsäuren gebildet sind, tritt bei Anwesenheit von Alkali Emulsion des noch ungespaltenen Vgl. S. 48 ff. Müniz: Auu. — d. Chim. 22. 472 (1871). Vtil. ferner SrhiUzenberqer: (4). Internat, wissensch. Bild. 263 (1876). Green: Proc. of the Rovnl Soc. 48. 870 (1890). W. Sigmund: Sitzungsbcr. d. W. Akad. 99. 406 (18Ü0): 100. 328 (1891); 101. 540 (1892). ^) W. Connsfein, E. Hoi/er und i/. Warfenhcrg : Ber. d. Deutschen Chem. Gesellsch. 35. 3988 (1902). Connsfein: Arch. f. (Anat. u.l'Physiol. 1905. Verliandl. d. physiol. Gesellsch. Berlin. ) — — — Abderhalden, l'hysiologische Chemie. I. Teil, 5. Aufl. J7 XIY. Vorlesung. 258 Es ist denkbar, jedoch nicht scharf bewiesen, Fettes ein. dieser feinsten Verteilung durch die Zellwand hindurchtritt. daß Fett in Wir wollen nun zu der Frage übergehen, was mit den Fetten geschieht, ehe sie im tierischen Organismus den Körperzellen zugeführt werden. Das Verhalten der Phosphatide und Sterine im tierischen Organismus werden wir für sich besprechen. Die mit der Nahrung aufgenommenen Fette werden in der Mundhöhle chemisch nicht verändert. Der Speichel verfügt über keinen Stoff, der Fette zerlegen kann. Beim Kauakt wird manche Zelle zerstört und damit auch das in ihr enthaltene Fett wenigstens zum Teil freigelegt. Im übrigen kommen die Fette unverändert in den Magen. Während man enthalten in früherer Zeit annahm, daß im Magensaft ein Ferment sei, das Fette spalten kann, war man in neuerer Zeit geneigt, diesem jede Fähigkeit. Angehörige der Fettreihe zu zerlegen, abzusprechen. Diese Anschauung erhielt durch die folgende, interessante Beobachtung Er fand, daß bei fettreicher Xahrung eine Stütze. Duodenums also Chymus vermischt mit den Sekreten des in den Magen Darm- und Pankreassaft und Galle Duodenums zurücksteigt.-) Wir werden nun gleich erfahren, daß die beiden ersten von Boldyreff'^) Inhalt des — — — - der erwähnten Sekrete Lipase enthalten. Damit schienen nun die Befunde jener Forscher, die im Magensaft ein fettspaltendes Ferment gefunden hatten, erklärt. Sie hatten offenbar übersehen, daß sie Darmlipase in Händen hatten und nicht ein dem Magen selbst zukommendes Ferment. Andere Forscher, die jedes Fettspaltungsvermögen des Magensaftes vermißten, hatten dagegen das reine, nicht mit Duodenalinhalt vermischte Sekret des Magens untersucht. Mit dieser Schlußfolgerung schien das Problem des Vorkommens von Lipase im Magensaft endgültig gelöst zu sein. Bald tauchten jedoch große Bedenken auf. Laqueur^) und I)avi(hohn) haben nämlich nachgewiesen, daß die Lipase des Magens andere Eigenschaften zeigt, als das entsprechende Ferment des Darm- und Pankreassaftes. Die Lipase wird von den sie bildenden Zellen, wie otfenbar alle Fermente, nicht in aktiver Form abgegeben. Es muß erst noch ein weiterer Stoff auf die Ferment Vorstufe einwirken, bevor das Ferment seine Wirksamkeit erlangt. Wir werden gleich erfahren, daß das sogenannte Lipasezymogen, die Vorstufe der Lipase, der Pankreasdrüse und der Drüschen der Dünndarmschleimhaut durch Galle aktiviert wird. Die Lipase des Magens läßt sich jedoch durch sie nicht in die wirksame Stufe überführen. ^j Ferner gezeigt worden, daß Magenist besonders in neuerer Zeit einwandfrei saft, bei dem jede Beimischung von Duodenalinhalt ausgeschlossen war. 1) W. — Vgl. auch ./. Boa«: ZeiiBoldi/reff: Pßüqers Archiv. 121. 13 (1917). Ch. Con'pjmn.: Arch. de physiol Med. 10. 97 (1889); 17. 155 (1890). tr.ilblatt f. klin. — — UDrm. et pathol. 26. 125 (1898). Emil Abderhalden uml Florentin Mdigr^caanu: Emil Abderhalden iiud Alfred S^^hittenZeitschr. f. physiol. Chem. 57. 317 (1908). heim: Ebenda. 59. 230 (1909). '') Durch eine fettreiche Nahrung (Ölfrühstück) kann iniiu a\ich beim .Mc.uschon Duodenalinhalt in den Magen „locken'^ und ihn dann zu diagnostischen Zwecken aushebern. Ob nur fettreiche Nahrung die Antiperistaltik briwirkt. ist noch strittisr. ') E. Laqueur: Hofmeisters Beiträge. 8. 215 (1908). ) Heinrich Daoidsohn: Biochern. Zeitschr. 49. 249 (1913). =) Vgl. a'ich M. Takafa: The Tohoku J. of e.xperiin. Med. 2. 209 (1921). — Fette. Phosphatide. Sterine. 259 auch Fette spaltet, i) Allerdings erhält man nur dann eine umfassendere Verseifuno; von solchen, wenn sie emulgiert sind. Je feiner die Emulsion ist und eine je größere Oberfläche durch sie erzeugt wird, um so rascher erfolgt der Abbau des Fettes. Die Angabe, daß die Magenlipase Fett überhaupt nur dann spalten kann, wenn eine Emulsion zugegen ist, dürfte wohl kaum den Tatsachen entsprechen. Es ist nicht einzusehen, weshalb die aktive Lipase nicht auch nicht emulgiertes Fett angreifen kann. Sie vermag nur nicht in die groben Fettmassen einzudringen, und so bleibt ihre Wirkung eine sehr beschränkte. Ist jedoch eine Emulsion zugegen, dann bieten sich der Lipase ungezählte Angriffsstellen. Es scheint, daß während der Säuglingsperiode die Magenlipase eine bedeutsamere Rolle spielt, als später. In der Milch ist das Fett in feiner Emulsion vorhanden und damit ausgiebig angreifbar. In dieser Hinsicht ist ganz besonders bedeutungsvoll, daß die Emulsion des Milchfettes bei saurer Reaktion beständig ist, während diejenige jedes anderen Fettes bei dieser aufgehoben wird. Später, wenn die Milch als Nahrung ganz v\^egfällt, wie bei den Tieren, oder doch stark zurücktritt, wie beim Menschen, verliert offenbar die Fettverdauung im Magen viel an Bedeutung. < Jedenfalls dürfen wir es nach den erwähnten Ergebnissen sicher festgestellt betrachten, daß die Magenschleimhaut eine Lipase abgibt. Sie ist ohne Zweifel anderer Art als das im Darmkanal auftretende, fettspaltende Ferment. Dieser Punkt ist bei den Untersuchungen über die Magenlipase viel zu wenig berücksichtigt worden. Es wäre auch denkbar, daß die Magenlipase nur auf bestimmte Fettarten, z. B. die Milchfette, eingestellt ist, und durch diesen Umstand manche sieh widersprechende Angaben über das Vorhandensein einer Magenlipase beals sind, doch ist diese Annahme nicht sehr wahrscheinlich, weil bei den Fette spaltenden Fermente eine Einstellung auf feinere Straktnrver- dingt hältnisse zu fehlen scheint. -i Das Fett wird im Magen stets nur teilweise gespalten. Auch wenn der Duodenalinhalt in ihn zurüeksteigt und unter günstigen Bedingungen in ihm eine eigentlich dem Darme zugehörende Verdauung einsetzt, wird es wohl nie zu einer umfangreicheren Spaltung der Fette im Magen kommen. Unter normalen Verhältnissen spielt die Fettverdauung im Magen sicher eine nur unbedeutende Rolle. Sie setzt erst im Duodenum und dann im übrigen Darme in großem Umfange ein. Zunächst unterliegen die Fette einer physikalischen Umwandlung. Es tritt Zerstäubung in feinste Tröpfchen, Bildung einer Emulsion, ein. Die Bedingungen zu ihrer Entstehung sind im Darmkanal sehr günstige. Die Anwesenheit von Alkali im Darm- und Pankreassaft und das Vorhandensein von freien Fettsäuren bewirken die Entstehung von Seifen. Das Alkali ist zum großen Teil als Karbonat zugegen. Bei der Bindung des Alkalis an Fettsäuren — Cash: ) Vgl. zu dieser ganzen Frage: Marcet: The medical Times. 210(1858). F. Volhard: Arch. f. (Anat. u.) Phvsiol. 3-23 (1880). Of/ata: Ebenda. 515 (1881). Adolf Zinsser: Hofmeistern Beiträge. 7. 31 Zeitschr. f. klin. Med. 42. 414 (1900). Alberf Fromme: Ebenda. 7. 51 (1905). S. Lcvites: Zeitschr. f. physiol. nn05). — — — — — — E. S. London: Ebenda. 50. 125 (1906). — J. P. Sedgnük: Kopci Zeitschr. Ibrahim und -lahrb. Kinderheilk. N. F. 44. 194 (1906). — Biol. 53. 201 (1910). — Pesthy: Biochem. Zeitschr. 34. 147 (1911). — Jlanrir/i Chem. 49. 273 (1906). ./. f. 6". Davidnohn : Biochem. Zeitschr. 49. 249 (1913). = Vgl. z. B. Emil Abderhulden und A. Weil: ) /'. f. : v. Formentfoi-chung. 4. 76 (1921). 17 Xl\. Vorlesung. 9(50 wird die Kohlensäure frei. Sie unterstützt beim Entweichen das Zerstäuben der Fetttröpfchen, indem sie solche zerschlägt. Was die freien Fettsäuren anbetrifft, so finden sich solche wohl stets in den Fettgemischen der Nahrung. Ferner können sie auch bei der Spaltung von Fett im Magen entstanden sein. Die Fettverdauung im Darmkanal würde auf diese Weise durch die im Magen eingeleitete Fettspaltung wirksam unterstützt. Vielleicht liegt in diesem Punkte eine ganz wesentliche Bedeutung der Magenlipase. Durch die Emulgierung der Fette wird eine starke Oberflächen Vergrößerung bewirkt. Jeder größere Fetttropfen zerfallt in Tausende von kleinsten Tröpfchen. Auf diese Weise ist die Möglichkeit gegeben, daß die Lipase rasch die einzelnen Fetteilchen zerlegen kann. Es bietet sich ihr in jedem einzelnen Tröpfchen eine Angrift'smüglichkeit, Die Lipase entstammt, wie schon wiederholt erwähnt wurde, der Pankreasdrüse und fernerden Drüschen der Darmwand. Die Pankreaslipase ist von Claude Bernard ^) entdeckt worden, nachdem schon vor ihm Eherle ^) beobachtet hatte, daß sich Fette bei Zusatz von Pankreasgevvebe in eine Emulsion überführen lassen. Bernard bewies, daß die Bildung der Emulsion nichts der Pankreasdrüse Eigentümliches ist. Er beobachtete, daß die Anwesenheit einer Spur von Seifen genügt, um beim Schütteln von Fetten mit Wasser eine Flmulsion zu erzeugen. Das Wesentliche der Wirkung des Sekretes der Pankreasdrüse ist die Bildung von freien Fettsäuren und von Glyzerin aus Neutralfetten. Die Bildung dieser Spaltprodukte kommt der Lipase zu. Boldyreff'^) bewies das V^orkommen eines Fette spaltenden Fermentes im Darmsaft. Die Vorstufe der Lipase wird, wie schon erwähnt, durch Galle aktiviert. Fürth und Schütz) bewiesen, daß die Gallensäuren das hierbei wirksame Prinzip darstellen. Diese Beobachtungen machen es verständlich, weshalb die Fettverdauung so stark beeinträchtigt ist, wenn aus irgend einem Grunde der Zufluß der Galle zum Darme behindert wird. Die Lipase ist dann nur als Vorstufe zugegen und kann nicht in die aktive Form übergeführt werden. saft Wenn wir außerhalb des tierischen Organismus Pankreas- oder Darraauf fein emulgiertes Fett einwirken lassen, dann können wir nach einiger Zeit den Nachweis führen, daß es zum großen Teil in seine Bausteine zerlegt ist. Eine vollständige Spaltung tritt nicht ein. Als Grund dieser Erscheinung kimnte einmal eine hemmende Wirkung der sich bildenden Abbaustufen auf die Fermenthydrolyse in Betracht kommen. Sicher bewiesen ist, daß sich schließlich zwischen Ferment und den Spaltprodukten, wenn diese eine gewisse Konzentration erreicht haben, ein Gleichgewicht herstellt. Wird dieses durch Zusatz von Fettsäuren und Glyzerin so verschoben, daß die Konzentration an Spalt') l'aris Claude Bernard: Memoiro siir le pancröas et sur le röle ') 3) Eherle: Physiologie der Verdauung. Würzburg 1834. W. Boldyretf: Zentralbl. f. rhysiol. 18. 460 (1905); Chemie. 50. 394 (1907). ) ß. du suc paucreatique. 1856. O. 23. — f. pliysiol. Vgl. auch Fürth und ./. Schütz; Hofmeister?, Beiträge. 9. 28 (1906). Kmil F. 'rerroine: Biochem. 404 (1910). V. Magnus: /eitschr. f. physinl. (Ihemie. 48. 373 (1906). Zeitsciir. Zeitsciir. — Fette, l'hfvsphatitle. Steriue. 261 Produkten erhöht wird, dann bewirkt die Lipase eine Synvon Fett, d. h. die Lipase vollzieht nunmehr den umgekehrten Vorgang.!) Man hat aus den Beobachtungen über die Fett- these spaltung im Reagenzglas den Schluß ziehen wollen, daß auch im Darmkanal der Abbau der Fette kein vollständiger sein könne. Er ist jedoch keineswegs berechtigt, denn es kommt im Darmkanal nie zu einem solchen Gleichgewicht, weil einerseits die sich bildenden Spaltprodukte fortwährend durch Resorption entfernt Averden, und ferner immer wieder neue Lipase zugeführt werden kann. Theoretisch ist die Zerlegung der Fette in Glyzerin und Fettsäuren im Darmkanal sehr wohl möglich. Es kämen dann nur die Bausteine der Fette, die Fettsäuren bzw. Seifen und der Alkohol, meistens Glyzerin, zur Resorption. Wir hätten in diesem Falle ganz entsprechende Verhältnisse vor uns, wie bei den zusammengesetzten Kohlehydraten. Leider läßt sich die Frage nach dem Umfang des Abbaus der Fette im Darmkanal nicht exakt beantworten. Es liegen genau die gleichen Schwierigkeiten vor, wie wir sie bei der Besprechung der Verdauung der Polysaccharide im Darmkanal schon hervorgehoben haben. Die Untersuchung des Darrainhaltes ergibt stets neben den Bausteinen der Fette auch diese selbst. Nach der einen Fett vor der Aufnahme in seine Bausteine zerlegt. 2) Andere Forscher dagegen glauben, daß nur ein relativ kleiner Teil des Fettes gespalten wird. Der Rest soll in Form feinster Tröpfchen direkt zur Resorption gelangen.-^) Für beide Ansichten sind mit Hilfe verschiedener Methoden Unterlagen beigebracht worden. Es ist nicht geglückt, die eine oder die andere Meinung in exakter Weise ganz zu widerlegen, wohl aber sind so viele Ergebnisse bekannt geworden, die für eine jedenfalls sehr weitgehende bis vollständige Spaltung der Fette sprechen, daß wir eine solche als höchst wahrscheinlich vorhanden annehmen dürfen. Es stehen sich zurzeit zwei Ansichten gegenüber. Annahme wird das gesamte Die Vorstellung der Aufnahme von Fetttröpfchen erweckt zunächst aus folgendem Grunde Bedenken: Beim Abbau der Polysaccharide und der Eiweißstotte entstehen schon sehr frühzeitig wasserlösliche Produkte, die leicht durch tierische Membranen dilfundieren. Es wäre an und für sich wohl möglich, daß die Resorption w^enigstens zum Teil schon bei noch zusammengesetzten Abbaustufen einsetzen und der weitere Abbau je nach Bedarf in der Darmwand oder in den Geweben sich vollziehen würde. Die Erfahrung hat jedoch gezeigt, daß dies im allgemeinen nicht der Fall ist. Der Abbau geht im wesentlichen bis zu den Bausteinen. Es sei in dieser Hinsicht noch ganz besonders an das Verhalten des im Wasser relativ leicht löslichen Rohrzuckers erinnert. Er ist weder in der Darm wand noch jenseits derselben anzutreffen. Bei den Fetten liegen die Verhältnisse ganz anders. Sie sind in Wasser unlöslich. Wir kennen auch keine Zwischenstufen eines teilweisen Abbaus, die im Wasser löslich und daher diffun>) Vgl. ./. r. 533 (1901). de l'Acad. d. f. Biol. 36. 26. ^) 82. Kaxtle und A. S. Loeroiharf: Amer. Chem. Joiiru. 24. 391 (1900); Henri Pofterin: C. r. de la soc. biol. 53. 70 (1901). Sc. 136. 1152 (1903) und 138. 378 (104). //. — Hanriot: C. — z. B. /. Mtink: Virchowa Arcliiv. 95. 407 (1884). 0. Frank: Zeitschr. 568 (1898). E. Pflüyer: J'flih/ers Archiv. 80. 111 (1900); 81. 375 (1900); Vtrl. 303 (1900); 85. ') — Vgl. hierzu — 1 (1901); 86. 211( 1902). z. B. S. Exner: Pßiiffers Archiv. 84. ()28 (1901). XIV. Vorlesung. 262 dierbar sind. Die Frage spitzt sich somit hier, wie folgt, zu. Zellen der Darmwand in Wasser unlösliche Stoffe Können die aufnehmen, oder ist ihnen diese Möglichkeit versagt? Nun wandern Bakterien durch die Darmwand durch, jedoch nur dann, wenn eine anatomische Veränderung infolge einer »Schädigung der Epithelschicht der Darm- wand vorhanden ist. Ferner ist behauptet worden, daß Stärkekörner die Darmwand direkt passieren können, doch ist diesen Ergel)nissen immer wieder widersprochen worden. i) Immerhin bleibt zunächst die Möglichkeit bestehen, daß feinste Fetttröpfchen die Epithelzellen der Darmschleimhaut durchwandern können. Wäre dies der Fall, dann Aväre allerdings nicht ohne weiteres einzusehen, weshalb, wie die direkte Beobachtung ergibt, stets ein sehr beträchtlicher Teil des Fettes sich im Darminhalt in gespaltener Form nachweisen läßt. Unter der Voraussetzung, daß Fetttröpfchen die Darmzellen direkt durchdringen können, erscheint die Bedeutung der Lipase darauf beschränkt, etwas freie Fettsäuren zu erzeugen, um die Emulgierung des Fettes in Gang zu bringen. Je rascher ein Fett -aufgenommen würde, ein um so kleinerer Teil würde der Verseifung unterliegen Schließlich sei noch bemerkt, daß man auch daran gedacht hat, daß Leukozyten bei dem Fetttransport eine Rolle spielen. Sie sollen aus der Darmwand auswandern, sich mit Fett beladen und dann wieder in sie zurückkehren.-) Diese Vorstellung hat wenig Anklang gefunden. Sie bedarf dringend der Nachprüfung. Nun haben wir bei der Besprechung der Verdauung der Kohlehydrate und insbesondere derDisaccharide hervorgehoben, daß sie offenbar nicht nur den Zweck hat, Substanzen, die nicht durch tierische Membranen diffundieren können, in solche zu verwandeln, die diese Eigenschaft besitzen, sondern es wird durch den Abbau die spezifische Struktur der aus mehreren Bausteinen aufgebauten Verbindungen vernichtet und so den Körperzellen ein Baumaterial gel)oten, das keine besonderen, von Fall zu Fall wechselnden Eigentümlichkeiten mehr besitzt. Sollten vielleicht den Fetten entsprechende Verhältnisse vorliegen? In der Tat ist die Möglichkeit durchaus gegeben, wenn auch noch nicht streng genug bewiesen, daß die Fette der einzelnen Zellarten nicht allein durch das Mischungsverhältnis, in dem die einzelnen Fettarten sich vorfinden, sondern in weitgehender Weise auch durch die Art ihrer Zusammensetzung charakterisiert sind. In diesem Falle wäre es vei-ständlich, wenn die Fette vollständig in die indifferenten Bausteine zerlegt würden. Es bleibt jedoch die Möglichkeit, daß der Umbau sich erst in der Zelle vollzieht. Die einzelnen Fette und insbesondere die Glyzeride, die ja die Hauptrolle spielen, sind unter sich in ihren Eigenschaften recht ähnlich. Sie haben auch alle einen gleichen Bau. Sie stellen Ester dar. Die Art der Bindung zwischen den Fettsäuren und den Alkoholgruppen ist stets die gleiche. Es handelt sich somit bei den Fetten nicht um jene Mannigfaltigkeit der Bindungsarten und damit der Struktur, wie wir sie bei den Polysacchariden angetroffen haben. Es kommen diese einfacheren Verhältnisse ganz oftenbar auch in den Beziehungen zwischen den Fetten und dem auf sie eingcbei — Fritz Verzür : Biochem. Zcitsclir. 34. 8(5 1 91 1 ./. Voi(/t Kbciida. 36. H97 ( 1 91 1 ). Vgl. liiorzn A. Wiedersheim: Fcstschr. d. 56. Vers. deiitscluT Natiuf. uud Är/to zu Freiberg 1883. I'reusse: Archiv!, wisseusch. iitid prakt. Tierlioilkuiide. 11. 1 (188.')). ) ( ^) — ). : Fette. Phosphatide. Sterine. 26H Ferment zum Ausdruck. Es handelt sich stets um den gleichen Vorgang: Lösung der esterartigen Bindung zwischen je einer Alkoholgruppe und einer Fettsäure. Es genügt ölten bar eine Art von Fermenten, Stellten um in Betracht kommenden Glyzeride zu spalten. i) Sie auf bestimmte Strnkturverhältnisse eingestellt. 2) Es würde somit das Erscheinen von ganz verschiedenartigen Fetten in den Körperzellen an sie keine unlösbaren Aufgaben stellen. Sie können mit der Lipase, die sie besitzen, in allen Fällen auskommen. Bei den Kohlehydraten liegen in dieser Hinsicht die Verhältnisse ganz anders. Den Körperzellen fehlt die Möglichkeit, z. B. Rohrzucker oder Milchzucker zu spalten, weil ihnen die auf diese Produkte eingestellten Fermente fehlen. Die Zerlegung dieser Verbindungen in ihre Bausteine ist ein für alleraal in den Darmkanal verlegt. Aus all den angeführten Gründen ist es verständlich, weshalb die Frage der Resorption und des Transportes von ungespaltenem Fett trotz zahlreicher Untersuchungen immer noch eine unentschiedene ist. die Lipase, ist nicht die spezifisch Eine weitere große Schwierigkeit in der Beurteilung des Umfanges des Fettabbaues im Darmkanal bereitet die Beobachtung, daß bald nach Beginn der Fettverdauung in den Darmepithelien Neutralfett nachweisbar ist. 3) Diejenigen Forscher, die geneigt sind, eine direkte Aufnahme von Fetttröpfchen anzunehmen, halten das in den Darmepithelien feststellbare Fett für unverändertes Nahrungsfett. Jene Autoren jedoch, die eine Spaltung der Fette in Glyzerin und Fettsäuren als unerläßliche Vorbedingung der Resorption betrachten, nehmen an, daß diese Bausteine bereits in der Darmwand wieder zu Fett zusammengefügt werden. Nach einer fettreichen Mahlzeit können wir das Fett direkt im Blute beobachten. Das Blutplasma erscheint milchig getrübt. Beim Stehen setzt solches Blut 'oft treten von Fetten in der Blutbahn eine Fettschicht ab. Ferner in schönster läßt Weise das Auf- sich mittels des Ultra- mikroskops verfolgen. Dieses beruht auf dem gleichen Prinzip, mit Hilfe dessen wir feinste Stäubchen in der Luft durch Einfallen von Lichtstrahlen dunklen Raum sichtbar machen können. Man hat die kleinen in einen Fetteilchen Hämokonien genannt.) Es fragt sich nun, wie das synthetisch aufgebaute, bzw. nach der anderen Annahme direkt resorbierte Nahrungsfett aus den Zellen der Darmw^and herauskommt, und wie es schließlich in die Blutbahn gelangt. Findet auch wieder eine Spaltung in Fettsäuren und Glyzerin statt, oder wandern die feinen Fetttröpfchen durch die Zellwand? Es ist dies eine sehr schwierig zu beantwortende Frage. Anerkennt man, daß feinste Fetttröpfchen die Zellwand durchwandern können, dann ist nicht recht verständlich, weshalb a priori verneint wird, daß die Darmepithelzellen Nahrungsfett in feinster Emulsion aufnehmen können. ) Vgl. hierzu 19. Juli 1909. u. a. L. Morel uud E. Terroine: C. r. de TAcad. d. Sciences. — K. George Falk: Journ. of the Amer. Chem. Soc. 35. 601 (1903). — Emil Abderhalden und Egon Eichivald: Ber. d. Deutschen Chera. Ges. 47. 1856, 2880 (1914); 48. 113, 1847 (1915). 2) Yg\. Emil Abderhalden und Arthur Weil: Fermentforschung. 4. 76 (1921). 3) Noll: Pflüger?, Arch. 136. 208 (1910). A. Kreidl )Alfred Neumann: Zentralbl. f. Physiol. 21. (1907); 27. 214 (1913). und A. Neumann: Sitzungsber. d. Wiener Akad. Math.-naturw. Klasse. 120. (19111 — XIY. Vorlesung. 2(i4 Es ist ganz unmöglich, hier eine Entscheidung zu treffen. Lange man, daß die mikroskopische Verfolgung der Fettresorption zurzeit Zeit glaubte und des Fetttransportes volle Klarheit über die bestehenden Verhältnisse ergeben würde. Bald wurde Jedoch festgestellt, daß die Fette und Fettsäuren sich in mancher Hinsicht gegenüber vielen sogenannten Fettreagentien so ähnlich verhalten, daß es unmöglich ist, zu entscheiden, ob im einzelnen Falle Fette oder Fettsäuren zur Beobachtung kommen. Eine weitere Schwierigkeit bietet der Umstand, daß das Fett in der Zelle sich dem Nachweis ganz entziehen kann. Niemals darf aus dem negativen Ausfall von mikroskopischen Fettreaktionen der Schluß gezogen werden, daß kein Fett vorhanden ist! Ganze Lehren sind auf solchen Trugschlüssen errichtet worden! Steht man auf dem Standpunkte, daß die Fette vor ihrer Resorption in ihre Bausteine zerlegt werden, so liegt kein Grund vor, daran zu zweifeln, daß auch in den Darmepithelien das durch Synthese Allerentstandene Fett wieder vor dem Weitertransport gespalten wird. dings müßte sich dann sofort wieder eine Synthese anreihen, weil wir im Blute Fettsäuren beziehungsweise Seifen und Glyzerin höchstens in geringen Spuren auffinden, dagegen Fett in größerer Menge. Der Fettgehalt des Blutes zeigt direkte Beziehungen zur Fettaufnahme. Nun können wir jederzeit im Reagenzglas der Spaltung von Fett durch Herbeiführung der oben ') erwähnten Bedingungen die Synthese folgen lassen und umgekehrt nach BelieIten dieser Halt gebieten und den Vorgang wieder in die Richtung der Spaltung lenken. Weshalb sollte die Zelle diese Vorgänge nicht auch in jeder Richtung beherrschen! Die Darmepithelzelle nimmt Seifen, Glyzerin und wohl auch Fettsäuren auf Sie besitzt Lipase. Zunächst werden die Spaltprodukte angereichert. Ist eine gewisse Konzentration an ihnen erreicht, dann setzt die Synthese ein. Das gebildete Fett kann dann nach Bedarf wieder gespalten werden. Mit dieser Erklärung allein können wir uns nicht zufrieden geben. Wohl ist es denkbar, daß durch die immer wieder zufließenden Spaltstücke des Fettes und durch eventuelle Mehrproduktion von Lipase das Gleichgewicht zwischen Ferment und Substrat immer in der gleichen Richtung verschoben wird, wir kommen jedoch nicht über die Schwierigkeit der Erklärung des ganzen Vorganges hinaus, wenn wir erwähnen, daß kurze Zeit nach der Fettaufnahme auch schon solches im Blute erscheint. Wozu dient dann die vorübergehende Synthese in der Darm wand? Diese Fragestellung bietet nur dann fast unüberwindbare Schwierigkeiten, wenn man das ganze Problem der Fettverdauung und -resorption nur qualitativ und nicht auch quantitativ verfolgt. Niemand hat bis jetzt bewiesen, daß die gesamten resorbierten Bausteine der Fette in den Darmepithelien als Neutralfett auftreten. Nichts spricht dagegen, daß der bei weitem größte Teil der resorbierten Seifen, Fettsäuren und des Glyzerins direkt durch die Zellen der Darmwand in den Köri)er gelangt, und zwar wird, wie wir gleich erfahren werden, in der Hauptsache die Lymphbahn eingeschlagen. In der Lymphe vollzieht sich vielleicht erst die Synthese zu Fett. Es ist leicht möglich, daß hierbei die Leukozyten den eine wesentliche Rolle spielen, und insbesondere den Lymphknoten Mesenterialdrüsen - - in dieser Hinsicht eine ganz besondere Bedeutung zukommt. Vielleicht wird nur der allergeringste Teil der resorbierten vollständig — ') Vgl. S. 260. Fette. Phosphatide. Sterine. 26Ö Bausteine der Fette in den Darmepithelien zurückgehalten und in Neutral- fett verwandelt. Vielleicht liegt eine Regulation des Zustromes der Fettbestand- diesem Verhalten vor. Wir kommen somit zu dem Schlüsse, daß der Befund von Xeutralfett in den Darmepithelien während der Fettaufnahme der Anschauung, daß im Darmkanal die Fette vollständig gespalten werden, nicht widers])richt. Die Annahme daß schließlich alles aufgenommene Fett als solches in den Zellen der Darmwand wieder erscheint, ist unbewiesen. Es sprechen mehr Gründe dafür, daß Glyzerin, Fettsäuren und Seifen direkt den Lymphspalten zugeführt und hier Fett aus den Bausteinen gebildet wird. Ein wiederholter Ab- und Aufbau bereitet der Erklärung keine Schwierigkeiten, besonders, wenn man mit Bat/liss^) der Ansicht ist. daß ein und dassellte Ferment teile in nach den Konzentrations^'erhältnissen bald ab-, bald aufbauen kann. Verschiebungen in den Mengenverhältnissen zwischen Ferment und Substrat kann die Zelle in mannigfacher Weise vornehmen. Einmal kann sie für bestimmte Substanzen nur in der einen Richtung, z. B. von außen nach innen durchlässig sein. Es kommt so zur Anreicherung von solchen. Auch kann die Zelle gebildete Bausteine aus zusammengesetzten Verbindungen entlassen und so ihre Konzentration fortgesetzt vermehren. Dann kann sie Produkte durch Kuppelung mit anderen Zellbestandteilen festlegen und auf diesem Wege das Konzentrationsverhältnis verändern. Endlich vermag sie bald mehr, bald weniger aktives Ferment hervorzubringen. Schließlich besteht die Möglichkeit, daß die Zelle durch Änderung der Reaktion, der Innenkonzentration usw. Fermentvorgänge bald beschleunigt, bald hemmt. Alle diese verschiedenen Maßnahmen, welche die Zellen in jedem Augenblicke in der mannigfaltigsten Weise herbeiführen können, vermögen wir im einzelnen Reagenzglasversuch nicht nachzuahmen. Unsere Versuche sind viel zu grob und in ihren Bedingungen immer ganz einseitig eingestellt. Wir können nicht genug davor warnen, die Zellvorgänge zu schematisieren und ausschließlich vom Gesichtspunkte unserer Laboratoriumserfahrungen je zu beurteilen. Aus der großen Zahl der vorliegenden Beobachtungen über den Abbau der Fette im Darmkanal, ihre Aufnahme und ihr Verhalten in der Darmwand seien einige besonders überzeugende mitgeteilt. Wir müssen dabei vorausschicken, daß das resorbierte Fett in den Lymphbahnen des Mesenteriums dem Ductus thoracicus zugeführt wird. Sein Inhalt, auch Chylus genannt, ergießt sich dann in die Vena anonyma. Dieser Umstand erleichtert in mancher Beziehung das Studium des Überganges der Fette in den tierischen Organismus. Man kann nämlich den Ductus thoracicus ableiten, indem man ihn vor seiner Einmündung in die Vena anonyma durchschneidet und dann in die Haut einnäht, oder man bringt in seiner Wand eine seitliche ötfnung, eine Fistel, an. Zu (\|uantitativen nach außen Beobachtungen ist es unbedingt notwendig, den gesamten Inhalt des Ductus thoracicus nach außen abzuleiten. Verfüttert man nicht Neutralfette sondern ausschließlich Fettsäuren oder Seifen, dann erscheint im Chylus Neutralfett.-) Dieses Ergebnis beweist, daß der tierische Organismus imstande (1884). ') W. M. Bayliss: Journ. of Physiol. 46. 236 (1913). ") Radijeicski: — 1. Virchous, Archiv." 43. 26S (1868). — /. ist, Mnnk: Ebenda. 95. Munk und Rosenstein: Ebenda. 123. 230. 484 (1891). Fett 431 XIV. Vorlesung. 2(5(3 aufzubauen, und zwar mußte er in diesem B'alle außerdem noch das Glyzerin stellen, da dieser Alkohol nicht verfüttert wurde. Ferner wurden nüchterne Hunde teils mit Fett, teils mit einem Gemisch von Fettsäuren und Glyzerin gefüttert. Das Darmepithel zeigte das gleiche mikroskopische Bild.i) Ich selbst hatte oft Gelegenheit, bei Versuchen über den Ersatz der Nahrungsstofte durch ihre Bausteine nach Eingabe von Fettsäuren und Glyzerin in den Lymphbahnen des Mesenteriums beträchtliche Mengen von Fett zu sehen. 2) Argyrts und Frank") verfütterten Hunden Monoglyzeride. d. h Fette, in denen das Glyzerin nur eine Fettsäuregruppe trug. Im Chylus erschienen keine Monoglyzeride, sondern Triglyzeride, Die einfachste Erklärung dieses Befundes ist die Annahme einer vollständigen Hydrolyse des Monoglyzerids zu Glyzerin und Fettsäure. Daran schloß sich dann eine Svnthese von Triglyzeriden aus den gebildeten Fettsäure- und Glyzerinmolekülen. Dabei müssen von den letzteren welche unbenutzt übrig geblieben sein, weil die Fettsäuren von drei Molekülen der Monoglyzeride notwendig sind, um ein Molekül Triglyzerid zu bilden. Über den Ort der Verseifung des Monoglyzerids sagt allerdings dieser Versuch nichts aus. Sie kann sich im Darmkanal vollziehen, es ist jedoch nicht ausgeschlossen, daß die Hydrolyse erst in der Darmwand stattfindet. Interessant ist, daß Fettsäureäthylester) und -glykolester»), d. h. Fette, in denen an Stelle von Glyzerin Äthylalkohol bzw. der zweiwertige Alkohol Glykol zugegen ist, ganz gut ausgenutzt werden. Es wäre wichtig zu erfahren, ob nach erfolgter Aufnahme in der Darmwand auch hier Triglyzeride gebildet werden. Bei vielen Fetten muß ohne Zweifel unter allen Umständen der Resorption eine weitgehende Spaltung vorausgehen. Es sind dies jene Verbindungen, deren Schmelzpunkt höher als die Körpertemperatur liegt. Die Bildung einer Emulsion ist nicht möglich, so lange das Fett nicht in die flüssige Form übergeführt ist. Nun wird derartiges Fett ganz gut verwertet. I. Munk fand z. B., daß Hammeltalg, der bei ca. 50* schmilzt, vom Hunde bis zu 90"/o ausgenutzt wird, ^i Besonders interessant ist die Beobachtung, daß auch der bei öS" schmelzende Walrat zur Resorption gelangt. Daß ihr eine Zerlegung in die Bausteine vorausgehen muß, beweist die an einem Menschen mit einer Lymphfistel gemachte Beobachtung. Es trat nämlich im Chylus hauptsächlich Fett auf, an dessen Bau Palmitinsäure und Glyzerin beteiligt waren. Nun besteht der Walrat, wie wir schon früher mitgeteilt haben, aus Palmitinsäure und Zetylalkohol. Der letztere war somit durch Glyzerin ersetzt worden. Zur Entscheidung der Frage, ob wirklich die Bildung einer feinsten Emulsion genügt, um eine Aufnahme von Fett durch die Darmwand zu ») '') Perewoznikoff: Zbl. f. d. med. Wissensch. 851 (1874). Vgl. hierzu die Literatur Emil Abderhalden: Synthese der Zellbausteiue in Pflanze und Tier. J. Springer. Berlin 1912. ^) A. Argyris und O. Frank: Zeitschr. ) 0. Frank: Zeitschr. f. Biol. 36. f. Biol. 59. 5(i8 (1898). 143 (1912). — J. Mliller und Jlans Murscfi- hamer: Biochena. Zeitschr. 78. 63 (1916). ^) H. Heinrich Franck: Münchener med. Wochenschr. 65. 1216 (1918). 8) Immanuel Munk: Virchoir^ Archiv. 80. 10 (1880); ebenda. 95. 407 (1884).— 0. Frank: Archiv f. (Anat. u.) Vgl. auch Arnschink: Zeitschr. f. Biol. 26. 434 (1890). Phvsiol. 308 (1894). — Friedrich Müller: Zeitschr. — f. kliii. Med. 12. 45 (1887). Fette. Phosphatide. Sterine. 267 ermöglichen, sind die folgenden \'eisuclie ausgeführt worden.') Lanolin ein Fett, das sehr schwer verseif bar ist. Es bildet jedoch mit Wasser verrieben eine sehr feine Emulsion. Wurde eine solche einem Hunde verfüttert, dann fand man im Kot 97'5''/o des verabreichten Lanolins wieder, d. h. es war offenbar keine Spur von Lanolin zur Resorption gelangt. Die fehlenden ^2'b'>/o des verfütterten Fettes sind sicher auf die bei solchen Fütterungsversuchen nicht zu umgehenden Fehlerquellen zurückzuführen. Endlich hat man auch im Darmkanal hydrolysierbares Fett innig mit nicht verseif barem vermengt, eine Emulsion bereitet und dann das Gemisch verfüttert.'-) Es zeigte sich, daß das von Lipase spaltbare Fett resorbiert worden war, während das andere Fett z. B. weiches Paraffin vollständig unresorbiert blieb. Es wäre gesucht, wenn man annehmen wollte, daß die nicht durch Lipase verseifbaren Fette deshalb nicht zur Autnahme gelangt sind, weil sie in ihrer Zusammensetzung von der der gewöhnlichen Fette abweichen. Immerhin muß auch daran gedacht werden.-^) ist — — Im Anschluß an diese Versuche sei bemerkt, daß die Resorption der Fette um so leichter erfolgt, je niedriger sie schmelzen. Man darf jedoch diese Tatsache im Hinblick auf die mitgeteilten Beobachtungen nicht ohne weiteres im .Sinne einer direkten Aufnahme von ungespaltenem Fett verwerten. Daß hoch schmelzende Fette nur sehr langsam zur Resorption gelangen, kann sehr wohl seinen Grund darin haben, daß ihr Abbau stark verlangsamt ist. Das Ferment hat keine große Angriffsfläche, während die niedrig schmelzenden Fette leicht Emulsionen bilden und auf diese Weise der Lipase eine viel größere Angriffsmöglichkeit bieten. So wird Olivenöl zu 977''/o ausgenutzt. Von den zwischen 25 und 34" schmelzenden Fettarten (Gänse-, Schweinefett) werden bis zu 97"5''/o aufgenommen. Vom Hammeltalg dagegen, der bei 44 51" schmilzt, werden beim Menschen nur — 9r5"/o resorbiert, von dem bei 53" schmelzenden Walrat gar nur den Umfang der Fettresorption haben PcUenkofer und Voit^) Über 15"/o. und Bubrif^r'') Versuche angestellt. Erstere fanden, daß ein 35 kg schwerer Hund im Tage von 350 g Fett 98"/o resorbieren kann. Ebensoviel kann nach liiihner unter Umständen auch der menschliche Darm aufnehmen. Im allgemeinen werden jedoch nicht mehr als 100 120 g Fett vertragen. 90 bis — Für die Annahme einer Resorption von ungespaltenem Fett schienen Beobachtungen zu sprechen. Wie wir schon l)etont haben, liefert die Pankreasdrüse die Vorstufe der Lipase. Diese vermag, nachdem sie durch Gallensäuren aktiviert worden ist, Fette zu zerlegen. Dem Pankreassaft kommt noch eine weitere Bedeutung bei der Fettverdauung zu. Er führt Alkalikarbonat mit sich. Das Alkali dient zur Bildung von fettsauren Salzen (Seifen). Ihre Entstehung vermittelt das Eintreten der Emulsion. Unterbindet man nunmehr die Ausführungsgänge der Pankreasdrüse, oder entfernt man die Drüse, so findet doch die folgenden — Connstein: Archiv f. (Auat. u.) Pliysiol. 30 (18'.)9). Alexander r. Fekete: Archiv. 139. 211 (1911). W. R. Bloor: Jouru. of Biol. Chem. 15. lOö (1913). -) F. Henriqncs iiud C. Hansen: Zcntralbl. f. Physinl. 14. 313 (1900). ^) Es sei iu dieser Hinsicht z. B. darauf hingewiesen, daL'i wir Farbstoffe kennen, die von bestimmten Zellen aut'L,'enoninicn werden und von anderen nicht. ••) Fettenkofer und ('. Voit: Zeitschr. t. Biol. 9. 1 (1873). ') — Pjnif/cri> 5) M. Rnhner: Zeitschr. f. Biol. 15. ll.ö (1879). ^1^- Vorlesung. 268 noch eine Aufnahme von Fett statt. i) Sie ist zwar stark herabgesetzt, 5H" o jedoch sicher nachweisbar. Von Milchfett wurden z. B. noch 28 der v^erabreichten Menge aufgenommen.-) Dieser Befund ist nicht mehr auffallend und darf nicht mehr als Beweis für eine direkte Resorption — ungespaltenem Fett angeführt werden, seitdem wir wissen, daß die Darmes auch Lipase absondern. Auch sie wird durch Gallensäuren aktiviert. Fällt die Pankreaslipase auch fort, so kann somit der Darmsaft immer noch Fette spalten. Erschwerend für die Fettverdauung besonders bei Karnivoren dürfte bei Abwesenheit des Pankreassaftes die infolge der sauren Reaktion des Chymus unzureichende Bildung einer Emulsion sein. Aus dem Magen wird nämlich beständig saurer Chymus in den Darm befördert. Unter normalen Verhältnissen wird ein erheblicher Teil der zugeführten Säure im Darm durch das Alkali des Pankreassaftes Wird kein solcher sezerniert. so wird die Reaktion des neutralisiert. Chymus im Darme länger sauer bleiben, als unter normalen Verhältnissen. Daß die saure Reaktion der Bildung einer Emulsion hinderlich ist, beweist von kleinen Drüsen des — — der folgende Versuch. Übersättigt man eine Fettemulsion mit Säure, so Es treten größere Öltropfen sieht man sie allmählich verschwinden. ansammeln. Daß, die sich an der Oberfläche der Flüssigkeit auf. wie Teiclimann^) nachgewiesen hat, bei Kaninchen nach Unterbindung des Ductus pancreaticus die Fettresorption nicht wesentlich gestört ist, spricht nicht gegen die erwähnte Auffassung der Störung der Fettverdauung und -resorption nach Wegfall der Pankreassekretion. Die Sekrete des Dünndarmes der Herbivoren verfügen über größere Alkalivorräte als die der Karnivoren. Daß das Milchfett trotz des Fehlens des Pankreassaftes so ausgiebig zur Resorption gelangt, erklärt sich vielleicht aus folgendem Umstände. Wird Milch durch Lab zur Gerinnung gebracht, und dann das Gerinnsel durch Pepsin in Gegenwart von Salzsäure in Lösung gebracht, so erhält man eine sehr beständige saure Fettemulsion. Eine bedeutungsvolle Rolle spielt bei der Fettresorption vor allem auch die Galle. Ursprünglich hat man ihr einen direkten Einfluß auf die Darmepithelien zugeschrieben. Sie sollte diese zur Resorption anreizen. Die Bedeutung der Galle beruht jedoch, wie besonders Pflnger^) betont hat, im wesentlichen auf ihrem Lösungsvermögen für FettEine Mischung von Galle und Natriumkarbonat säuren und Seifen. vermag große Mengen von Stearin- und Palmitinsäure zu lösen. Die gallensauren Alkalisalze bringen viele wasserunlösliche Stofle in Lösung. ^) Besonders wichtig ist das Lösungsvermögen der Galle für die in Wasser so schwer löslichen Magnesia- und Kalkseifen. Bei dieser Gelegenheit wollen wir erwähnen, daß durchaus nicht alle bei der Spaltung von Fetten sich bildenden Fettsäuren als Salze im Darminhalt vorhanden zu sein ') Vgl. hierzu ./. /•. Mcring und (). Minkowski: Arch. 26. 371 (1890). ") f. Abelmami: luauir.-Diss. Dorpat. 1890. Bio]. 31. f. expor. l'atli. u. riiariuak. — Vgl. auch W. Sandmexjer: Zeitschr. 12 (1894). M. Tcichmanu: Inaug.-Diss. Breslau 1891. E. Fflüger: Pflilffers ArchW. 88. 299(1902); 88. 431 (1902); 90. 1 (1902). Vgl. auch G. RÖssi: Arch. di Fisiol. 4. 429 (1907). ^) Vgl. H. Wieland und //. Sorge: Zeitschr. f. pliysiol. Chemie. 97. 1 (191G). Vgl. auch (Jarl Nenbfrg: Biochem. Zeitschr. 76. 107 (1916). •'J ) — — Fette. brauchen. Einmal Bindung der ziierbare Verbindungen. ist I'hnspl'atiile. es fraglich, ob in Fettsäuren zugegen wohl die Löslichkeit der Seifen, als 269 jedem Falle genügend Basen zur sind. Man muß Stcriue. Ferner unter auch sind die Seifen dissodiesen Umständen stets sodiejenige der Fettsäuren be- rücksichtigen. Daß die Galle einen großen Einfluß auf die Resorption der Fette hat, geht aus den Versuchen von Düstre'^) hervor. Er unterband beim Hund den Ductus choledochus und stellte eine Fistel zwischen Gallenblase und Dünndarmmitte her. Bei fettreicher Nahrung erwiesen sich erst die unterhalb dieser Fistel befindlichen Chylusgefäße milchig getrübt. Die Galle allein scheint bei der Fettresorption nicht ausschlaggebend zu sein, vielmehr wirkt sie mit dem Pankreassaft zusammen. Es läßt sich dieser Umstand, wie Claude Bernard gezeigt hat, in sehr hübscher Weise beim Kaninchen zeigen. Bei diesem mündet der Ductus choledochus etwa 10 cm über dem Pankreasgang in den Dünndarm ein. Zwischen den beiden Einmündungssteilen bleiben die Chylusgefäße auch bei fettreicher Nahrung klar und durchsichtig. Erst unterhalb des Eintrittes des Pankreassaftes erblickt man milchig getrübte, fetthaltige Chylusbahnen. Diese Beobachtungen ergeben ein weiteres Moment für das \'erständnis der mangelhaften Fettausnützung nach Ausschaltung des Sekretes der Pankreasdrüse. Fehlen Galle und Pankreassaft, dann ist die Fettaufnahme fast völlig aufgehoben. Aus allen diesen Beobachtungen geht hervor, daß viele Momente zusammenwirken müssen, um die Verdauung und Resorption der Fette in richtigen Bahnen zu halten. Es sind uns noch lange nicht alle diese Bedingungen genau bekannt. Es ist deshalb leicht verständlich, daß der Versuch, die Fettverdauung und -resorption in isolierten Darmschlingen zu Wir haben beim Fettabbau etwas ganz Entsprechendes vor uns, wie bei dem der Polysaccharide. Die Spaltung erfolgt sicherlich nicht plötzlich in großem Umfange, sondern es dürften auch hier die fortwährend in kleinen Mengen entstehenden Abbaustufen immer bald zur Resorption kommen, so daß keine einseitige Anhäufung irgend eines Spaltproduktes stattfindet. Man könnte auch daran denken, daß der Abbau ein stufenweiser ist und zunächst aus dem studieren, widersprechende Resultate liefern mußte. -) ein Diglyzerid wird. Dieses könnte dann unter Verlust eines weiteren Fettsäuremoleküls in ein Monoglyzerid übergehen. Aus diesem erhielte man schließlich Glyzerin und das letzte Molekül Fettsäure. Für die Annahme eines solchen allmählichen Abbaues der Fette liegen jedoch keine Beweise vor. Sie läßt sich mittels Fetten prüfen, die optische Ak- Triglyzerid tivität besitzen. Ehe wir das Schicksal der Fette bzw. ihrer Spaltprodukte nach stattgehabter Resorption weiter verfolgen, müssen wir noch kurz die Frage erörtern, ob die Darmflora den Fetten bzw. ihren Bausteinen etwas anhaben kann. Es unterliegt keinem Zweifel, daß die im Darme vorhandenen Mikroorganismen Fett spalten können. Auch sie verfügen über Lipase. Ferner können sie die Bausteine der Fette weiter zerlegen und verändern. Was aus dem Glyzerin und den einzelnen Fettsäuren wird, ist noch nicht A. Das/re: Arch. de phvsiol. norm, et path. 22. 315 (1890). Vgl. hierzu z. B. H'. ('röner: Biochem. Zeitschr. 23. 97 (1909). - Otto v. Fürth und Julius Schütz: Hofmeister?: Beiträge. 10. 4()2 (1907). 1) 2) XIV. Vorlesung. 270 Fette. Phosphatide. Sterine. genauer festgestellt, .ledenfalls dürfen wir bei quantitativen Versuchen über die Fettresorption die Möglichkeit einer unter besonderen Verhältnissen vielleicht ganz beträchtlichen Wegnahme von Fett und seinen Bausteinen durch die Darmflora nicht vernachlässigen. Im Anschluß an die Fette sei kurz erwähnt, daß sehr vieles dafür spricht, daß die Phosphatide im Magendarmkanal in entsprechender Weise, wie die Fette, abgebaut werden, doch fehlt uns leider noch ein genauer Einblick in das Verhalten der verschiedenen Verbindungen dieser KörperEs ist dies nicht auffallend, wenn wir daran klasse im Darmkanal. erinnern, daß wir diese Gruppe von Stoffen erst in ihren Umrissen kennen. Überall, wo wir hinblicken, klaffen hier weite Lücken. Sobald wir über die Zusammensetzung und die Struktur von Verbindungen nicht ganz genau unterrichtet sind, verlieren wir jeden sicheren Grund bei biologischen Problemen. Etwas genauer untersucht ist das Verhalten des Lezithins gegenüber den einzelnen Verdauungssäften. Pankreassaft und Darmsaft zerlegen Lezithin!) in Fettsäuren und Glyzerylphosphorsäure. Auch Cholin wird frei, ob in seiner Gesamtheit, ist nicht festgestellt. Der Abbau des Lezithins braucht nicht einheitlich zu sein. Er könnte auch so verlaufen, daß Cholin Es läßt sich vorläufig über den z. B. an Phosphorsäure gebunden bleibt. Umfang der Lezithinspaltung im Darmkanal nichts aussagen. In Analogie zu den an anderen organischen Nahrungsstoffen gemachten Erfahrungen darf wohl angenommen werden, daß auch die Phosphatide bei der Verdauung in ihre Bausteine zerlegt werden. Ob dabei die Lipase bei diesem Abbau, wenigstens bei der Loslösung der Fettsäuremoleküle, mitwirkt, oder ob besondere Fermente in Frage kommen, wissen wir zurzeit nicht. Über das Verhalten der Sterine im Magendarmkanal wissen wir wenig. Die Sterinester dürften wohl gespalten werden. Im übrigen gelangen die Sterine wohl zum größten Teil unverändert zur Resorption. In den Fäzes findet man veränderte Sterine. Das Koprosterin stammt vom Cholesterin ab und geht durch Reduktion und Umlagerung aus ihm hervor. Wahrscheinlich liegt Bakterienwirkung vor. Das in den Pferdefäzes aufgefundene Hippokoprosterin wird auf Phytosterine der Nahrung zurückgeführt. '-) — •) Bokai/: Zeitschr. f. physiol. Chemie. Paul Mayer: Biochera. 1. 157 (1887). Zeitschr. 1. 39 (19üß). Ä. Clementi: Atti R. Accad. dei Lincei. Roma. (3.) 28. II. 465 (1919). -) Charle.'i Doree und J. A. Gardner: Proc. of tlie roval Soc. B. 80. 212 (1908). — Vorlesung XV. Fette. Phosphatide. Sterine. Fette, Phosphatide und Sterine im tierischen Organismus. Ihre Beteiligung am Zellstoffwechsei. Die Stoffwechselzwischen- und -endprodukte der Fette und Phosphatide und ihre Das Verhalten der Bausteine. Wir haben aus den vorliegenden Ergebnissen verschiedenartiger \'ersuche den Schluß gezogen, daß die Fette und Phosphatide im Darnikanal Diese höchstwahrscheinlich vollständig in ihre Bausteine zerlegt werden. gelangen zur Resorption. Ein Teil der aufgenommenen Bausteine der Fette wird in den Darmepithelien wieder zu Neutralfett aufgebaut. Es ist mehr als fraglich, ob die Abbauprodukte des gesamten Fettes zuerst in den Epithelzellen der Darmwand halt machen, um erst dann, vielleicht nach wieder Es ist näher liegend. erfolgter Spaltung, weiter befördert zu werden. anzunehmen, daß Seifen, Fettsäuren und Glyzerin direkt durch die Zellen hindurch in die Lymphspalten übergeführt werden und hier in NeutralÜber das Verhalten der Phosphatide nach erfolgter Refett übergehen. sorption der einzelnen Bausteine wissen wir nichts Sicheres. Es ist ml>glich, daß der Aufnahme in die Darmwand unmittelbar die Synthese folgt. Es ist jedoch sehr wohl denkbar, daß die einzelnen Bausteine den Körperzellen als solche zugeführt werden. Wir haben bereits erwähnt, daß die Fette zum grüßten Teil auf dem Lymphwege zum Transport gelangen. Schon die einfache Beobachtung zeigt uns an, daß die Lymphbahnen des Mesenteriums den Fetttransport in großem Umfange übernehmen. Wenn man z. B. bei einem hungernden oder nüchternen Tiere den Dünndarm hochhebt und damit das Mesenterium anspannt, dann kann man die Lymphbahnen, auch Chylusgefäße genannt. nur schwer erkennen. Führt man jedoch den gleichen Versuch bei einem Tiere aus, das etwa 2 6 Stunden vor der Tötung eine fettreiche Nahrung erhalten hat, dann erkennt man die Chylusbahnen ohne weiteres. Sie sind ganz prall mit Fett gefüllt und sehen infolgedessen mattweiß aus. Diese Beobachtung beweist nur, daß Fett auf dem Wege der Lymphbahnen zum Weitertransport gelangt. Sie schließt jedoch nicht aus. daß auch solches von den Pfortaderwurzeln aufgenommen wird. Es sind zwei Wege gegeben, um die Frage nach der direkten Aufnahme von Fett durch — '1 XV. Vorlesunff. -1 i zu entscheiden. Einmal kann man das Pfortaderblut während der Fettresorption auf Feit untersuchen, und ferner läßt sich der Chylus Bei des Ductus thoracicus aus einer Fistel sammeln und analysieren. gemischtem Futter steigt die Menge des in der Zeiteinheit ausfließenden die Blutbahn Er zeigt nur eine auffallende Änderung fast gar nicht an. Aussehens, wenn in der Nahrung Fett enthalten ist. Während wird er undurchsichtig. An ist, der Chylus sonst durchscheinend sind die Fette schuld. Der Chylus des Beschaffenheit der dieser Änderung Chylus enthält das Fett in feinster Emulsion. Führt man einen derartigen Versuch so aus. daß man zunächst den beim nüchternen Tiere ausfließenden Chylus auf Fett untersucht und dann nach Verabreichung einer bestimmten Fettmenge die Ductuslymphe während einer längeren Zeit sammelt und nunmehr den Mehrgehalt an Fett feststellt, dann findet man, daß die Es ist größte Menge des resorbierten Fettes den Lymphweg einschlägt. i) schwer, eindeutig festzustellen, ob stets auch Fett durch die Pfortaderwurzeln aufgenommen wird.'-) Eine exakte Entscheidung dieser Fragestellung ist deshall) so schwierig, weil vom resorbierten Fett sicher etwas in der Darmwand liegen bleibt und ohne Zweifel immer auch ein allerFerner dürften dings kleiner Teil davon von Bakterien verändert wird. auch Bestandteile der aufgenommenen Fette, ohne wieder zu Neutralfett umgewandelt zu werden, Verwendung finden. Schließlich kann auch Fett unresorbiert bleiben. Zu allen diesen Faktoren kommt noch der Umstand hinzu, daß es schwer hält, aus dem Chylus das Fett ohne Beimengungen abzuscheiden. Cbylus seines Es ist von großem Interesse, daß das Fett im Gegensatz zu den Kohlehydraten hauptsächlich den Lymphweg wählt. Es nimmt damit eine Sonderstellung ein. Das Fett umgeht zum großen Teil die Leber. Der Organismus hat nicht, wie bei den übrigen resorbierten Stoffen, die Möglichkeit, die aufgenommenen Fette vor dem Übergang in den großen Kreislauf einer Kontrolle durch die Leberzellen zu unterwerfen. Dieses eigentümliche Verhalten der Fette steht vielleicht mit einer Regulation der Bei den Kohlehydraten ist Fettzufuhr in das Blut im Zusammenhang. dem allgemeinen Kreislauf ein Speicher für sie vorgeschaltet, nämlich die Sie kann resorbierten Traubenzucker abfangen und so unter norLeber. malen Verhältnissen jeder größeren Überschwemmung des Blutes des großen Kreislaufes mit Zucker vorbeugen. Die Fettspeicher für die Fette liegen jedoch jenseits der Leber. Sie kann allerdings auch Fett aufnehmen, jedoch hat die Fettablagerung nach den Beobachtungen von liosenfeld') einen großen Einfluß auf die Glykogenspeicherung. Die Leberzellen können, wenn sie mit Fett gefüllt sind, weniger Glykogen ablagern, als sonst; ja man findet sogar oft, daß beide Produkte sich geradezu ausschließen. Die Umgehung Fettreiche Lebern enthalten häufig gar kein Glykogen. ») Arch. f. Vgl. 1. Munk und Höllenstein: Virchows Archiv. 123. 2,S0, 484 (1891) uud L Munk und Friedcntlial: Zentralbl. f. (Anat. u.) Physiol. 376 (1890). — Pliysiol. 15. •') (Anat. 297 (1901). — //../. Hamburger: Arch. f. Vgl. B. fr d'Errico: Arch. di Fisiol. 4 (1908). Georg Jeannovies uud Ernst P. Pick: Wiener kliu. Physiol. 554 (1900). u.) — Wochenschr. 23. Nr. IG (1910). G. Posenfeld: Bericht über den XIX. Kongreß für innere Medizin. (1901); Vgl. auch E. Pßüffer: Pßüger?, Archiv. Berliner kliii. Wochenschr. 47. 1268 (1910). =•) 119. 117 (1907j. — Fette, l'liospliatide. der Leber durch die Fette gibt crewissem Sinne freie Hand. dieser Sterine. bei der 278 Glykogenablagerung in Würde das Fett die Leber, ohne aufgehalten zu werden, passieren, dann könnte das Blut leicht in kürzester Zeit mit Fett überfüllt werden. Es scheint nämlich nicht rasch aus ihm abgegeben Averden zu können. Der Chylusstrom dagegen ist ein langsamer. Das aufgenommene Fett fließt infolgedessen mit dem Chylns dem Blute allmählich zu. ^'ielleicht werden mit dem Chylus dem Blute auch in geeignetem Verhältnis Substanzen zugeführt, welche die feine Zerstäubung des Fettes aucli in ihm aufrecht erhalten. Zudem kann in der Lymphe die Synthese des Fettes aus aufgenommenen Fettsäuren bzw. Seifen und Glyzerin zu Ende geführt werden. Die Fettsäuren und Seifen sind für das Blut nicht gleichgültig. Ihr Erscheinen in diesem würde zu St()rungen aller Art führen. Vielleicht hängt der Transport der Fette stellung der Fette durch die zusammen. Lymphe auch mit einer weiteren SondeiWir werden nämlich gleich erfahren, daß Nahrungsfette als solche im tierischen Organismus zur zu bringen. Bei den Kohlehydraten und Eiweißstotfen ist es nicht möglich, dem Körper nicht zukommende Verbindungen unter Benützung der natürlichen Zufuhrwege in den Geweben einzulagern. So können wir, wie früher schon erwähnt wurde, weder Rohrzucker, nocli Organismus auffinden, auch wenn wir diese SubStärke im tierischen stanzen lange Zeit hindurch in großen Mengen verfüttert haben. Führen wir derartige Verbindungen unter Umgehung des Darmkanals in den Körper ein, indem wir sie z. B. direkt in die Blutbahn einspritzen, dann werden sie entweder direkt durch die Niere ausgeschieden oder nachträglich in der Blutbahn noch gespalten. es leicht gelingt, Ablagerung ( Den Übertritt des Fettes in das Blut können wir bald nach der stattgehabten Resorption direkt beobachten. Wir haben schon daraufhingewiesen, daß das Blutplasma bei reichlicher Fettaufnahme milchig getrül)t erscheint. Oft kann man in solchen Fällen durch Zentrifugieren das Fett direkt als Rahm zur Abscheidung bringen. Ist die Fettaufnahme keine sehr große, dann können wir die feinsten Fetttröpfchen Hämokonien noch mittels des Ultramikroskops sehen ') Neben den Neutralfetten finden wir im Blute immer auch Glyzerin.'-) Es ist noch nicht sicher erwiesen, ol» sein Vorkommen mit der Aufnahme der Fette in direktem Zusammenhang steht. Es kann ebensogut dem Zellstoffwechsel entstammen oder sich beim — — Übergang der Fette des Blutes in die Gewebe bilden. Interessant ist der Befund, daß der Glyzeringehalt des Blutes in engen Grenzen konstant ist.') Ein Befund von Fettsäuren könnte die gleiche I^sache, wie das Vorkommen des Glyzerin haben. Das im Blute enthaltene Fett verschwindet nach einiger Zeit au.s diesem und wird dann in den Geweben angetroffen. Jede einzelne Körperzelle bedarf der Fette. Diese bilden unentbehrliche Bestandteile ihres Baues. Bald wird diese, bald jene Zelle Fett aufbauen. Andere Zellen verwerten das zugeführte Fett als Energiequelle, indem sie es, beziehungsweise seine Bausteine, abbauen und schließlich Kohlensäure und Wasser aus den letzten Vgl. z. B. Oskar Weltmann: Biochem. Zeitschr. 65. 440 (1914). P. Tangl und St. Weiser: Pfiliffers Archiv. 115. l.')2 (1906). ») Ernsf Schmitz: Biocliem. /eitschr. 45. 18 (19l2). >) ) Abdti rh al d e n . Physiologische Chemie. I.Teil, 5. Aufl. Jg XY. Vorlesung. 274 Bruchstücken bilden. Sicher werden die Fette und ihre Bausteine genau wie die Kohlehydrate, als Ausgangsmaterialien zu mannigfachen Synthesen Verwendung finden. Wir kommen auf diesen Punkt noch zurück. Dasjenige Fett, das nicht sofort verwendet wird, wird abgelagert. Der tierische Organismus verfügt über Zellen, die große Mengen von Fett aufnehmen können. Sie sind zu sogenannten Fettgeweben vereinigt. Mächtige Fettdepots finden sich unter normalen Ernährungsverhältnissen im Unterhautbindegewebe und zwischen den Blättern des Mesenteriums. Es können jedoch auch sonst an allen möglichen Stellen des Organismus sich Fettzellen bilden. So kann z. B. bei fetten Individuen die Niere von einer dicken Fettkapsel umschlossen sein. Auch am Herzen finden sich oft große Fettablagerungen. Es ist von Interesse, daß die Murmeltiere und, wie es scheint, die Winterschläfer überhaupt, noch besondere Fettdepots haben. so, Frage nach den Beziehungen der Fettdepots zum und dem gesamten Stoffwechsel erörtern, müssen wir der Frage nachgehen, auf welche Art und Weise das Fett die Blutbahn verläßt. Es ist trotz aller Bemühungen nicht geglückt, den Vorgang des Überganges des Fettes in die Gewebe klarzustellen. Das Fett kann als solches in Form feinster Tröpfchen die Endothelien der Blutgefäßkapillaren durchdringen- oder es werden die viel umstrittenen Storaata als Weg benützt. Es ist auch denkbar, daß die Fette wieder in ihre wasserli'tslichen Bausteine zerlegt werden, ehe die Gefäßwand passiert wird. Für diese Ansicht könnte ins Feld geführt werden, daß im Blutplasma lipoIvtische Fermente vorkommen. Ihre Menge läßt sich durch vermehrte Zufuhr von Fett steigern. ^j Auch im Hunger, wenn sich eine Lipoplasmie Ehe wir die Fettstofifwechsel vermehrten Fetttransportes findet, erfährt der Lipasegehalt des Blutplasmas eine Zunahme. 3) Auch Fermente, die Cholesterinfettsäureester) spalten, sind mit Sicherheit im Blute aufgefunden worden. Es ist trotz aller Bemühungen noch nicht gelungen, die Bedeutung der Blutlipase klarzustellen. Manche Autoren sprechen ihr die Fähigkeit, das im Blut vorhandene Fett zu spalten, ab. Jedenfalls ist es nicht geglückt, eine Bildung von Glyzerin und Fettsäuren im Blute zu beobachten. Allerdings dürfte es auch sehr schwer sein, die sich bildenden Spuren dieser Verbindungen festzustellen. Gegen die Annahme, daß eine Lipase das im Blut vorhandene Fett si)altet und auf diese Weise seinen Durchtritt durch die Blutgefäßwand ermöglicht, sprechen mancherlei Punkte. Es ist fraglich, ob das Blutplasma für Fettsäuren und Seifen ein ebenso gutes Lösungsmittel darstellt, wie die Galle. Vor allem erwecken jedoch die folgenden Beobachtungen Bedenken. Wir wissen, daß umgekehrt aus den Geweben bzw. der Lymphe Fett in die Blutbahn eintritt. Wir beobachten unter manchen Bedingungen eine starke Überschwemmung des Blutes mit Fett. Man spricht in solchen Fällen infolge ) Hanriot: Compt. rend. de la Soc. de Biol. 48. 925 (189()); Compt. reiid. de — Hanriot und Camus: Compt. rend. de l'acad. des sciences. 124. 235 (1897). — Arthus: Journ. de pliysiol. et path. 4. 455 (1902). — Michaelis und Peter Bona: Biochem. Zeitschr. 31. 345 (1911). — Pe/er Rona: Ebenda. 33. 412 (1911). — Peter Rona und J. Ibsen: Ebenda. 21. (1912). — G. Izar: Ebenda. l'acad. des sciences. 123. 7.ö3 (189R). Tj. 3!>. 40. 390. (1912). Emil Abderhalden und Peter Rona: Zeitschr. f. physiol. ("bem. 75.30(1911). Emil Abderhalden und Arno Ed. Lampe : Zeitschr. f. pliysiol. ('hem 78.396 (1912). ") .7. //. Schultz: Biochem. Zeitschr. 42. 255 (1912). -) ^) Fette. Phosphatide. Sterine. von 27Ö Lipoplasmie. Sie ist bei Diabetes ineiitus^j oft beobworden und auch gegen Ende der Hungerperiode."-i Offenbar einer achtet liegt ein vermehrter Fetttransport von den Fettgeweben zu den verschiedensten Kürperzellen vor. Aber nicht nur in diesen Fällen, sondern auch unter normalen Verhältnissen erfolgt der Fetttransport von den Reservestätten nach den Orten des Gebrauchs auf dem ßlutwege. Wie kommt nun dieses Fett in die Blutbahn V Wird es vorher gespalten? Ist dieses der Fall, dann ist es auffallend, dali das Blut Neutralfett führt. Wie und wo kommt die Bildung von Fett zustande? Nehmen die Blntzellen. die auch über Lipase verfügen, an der Fettsynthese teil, oder vollzieht sie sich im Blute selbst? Jedenfalls sind die Bedingungen zur Fettsynthese durch die Lipase des Blutplasmas keine besonders günstigen. Fettsäuren und Glyzerin können sich kaum im Blute anreichern, ohne Störungen zu verursachen, es sei denn, daß im Blutplasma die Fettsäuren bzw. Seifen in einer Form vorhanden sind, in der sie unschädlich sind. Jarisch ^) ist auf Grund der Beobachtung, daß dem Plasma bzw. Serum zugesetzte Seifen in Alkali und Fettsäuren zerfallen und die letzteren, wie vermutet wird, an Eiweil.1 gebunden werden, der Meinung, daß auf diese Weise an und für sich schwer lösliche Fettsäuren zur Lösung gelangen und sich dem direkten Nachweis entziehen. Wir sehen, daß sich uns überall große Schwierigkeiten entgegen.stellen. wenn wir den Fetttransport verfolgen. Sie verringern sich, wenn wir einen direkten Transport von Fett durch die Blutgefäßkapillaren nach beiden Richtungen annehmen. Es scheint uns nicht unwahrscheinlich, daß die Leukozyten am Fetttransport teilnehmen. So gut aber diese nach Belieben die ßlutbahu verlassen und wieder in sie eintreten, kimnten auch die feinen Fetttröpfchen als solche auf dem gleichen Wege in die Lymphe und von da in die Zellen der Gewebe übertreten. Erwähnt sei noch, daß man auch daran gedacht hat, daß das Neutralfett im Blute in anderer Weise als durch Hydrolyse in wasserlösliche, dialysierbare Produkte verwandelt wird, doch fehlen überzeugende Versuche.) Es folgt gleich noch eine weitere Schwierigkeit. Wie kommt das Fett aus der Lymphe in die einzelnen Zellen der Gewebe hinein? Müssen wir wieder eine Spaltung in wasserlösliche Produkte annehmen? Muß ferner umgekehrt das Fett wieder zerlegt werden, wenn es die Zelle verläßt, um an einem anderen Orte verwendet zu werden? Wir stehen hier vor ungelösten Problemen. Die Tatsache, daß wir überall in den Geweben Lipase antreifen, die, das sei hier gleich angefügt, auch durch Gallensäuren aktivierbar ist, macht es wahrscheinlich, daß ein direkter Übertritt der Fetttröpfchen durch die Zellwand Wir müssen jedoch ») die Möglichkeit eines kaum stattfindet.^) direkten Überganges zugeben. Vgl. S. 195. Vgl. hierzu Ivar Bang: Biochem. Zeitschr. 90. 383; 91. 104. 111. 224 fl918). A. Jarisch: Fflugen Archiv. 194. 873 (1922). ) W. Connstein und L. Michaelis: Fßiic/ers Archiv. 65. 473. (1897); 69. 76. (1897). Vgl. ferner Wilhelm Connstein : Ergebn. d. Physiol. 3. I. 194 (1904). G. MaiisJeld: Zentralbl. f. Physiol. 21. 666 (1907). Joh. Müller: Zeitschr. f. physiol. Chem. ) ') — 8(). — — 469 (1913). Vgl. hior/u die Beobachtung von E. Nierenstein: Zeitschr. f. allg. Physiol. 10. 137 (1909), der bei Paramüzien Fettspaltuug in den Vakuolen der Zellen beobachtet zu haben glaubt. Im Endoplasma erfolgt dann wieder Synthese zu Neutralfett. \<A. dazu auch ^^ Staniewicz: ßull. de l'Acad. de Cracov."l99 (1910). '") . 18 XV. Yorlesuug. 276 Der Lipase könnte ja in der Zelle ausschließlich die Bedeutung zukommen, Fett dann zu spalten, wenn sie selbst die Hausteine braucht. i) Wir wollen uns nun der Frage zuwenden, ob wirklich bewiesen ist. daß die Fettdepots durch das vom Darm aus resorbierte Fett gespeist werden. Zunächst erscheint diese Fragestellung sehr gesucht, denn weshalb sollten zwischen den aufgenommenen Fetten und den Fettdepots keine direkten Beziehungen bestehen? Um das aufgeworfene Problem zu verstehen, muß an die Tatsache erinnert werden, daß der tierische Organismus Kohlehydrate in Fett überführen kann. Daraus folgt, daß das Fett der Fettzelleii durchaus nicht von dem Fett der Nahrung abzustammen braucht. In der Tat ist lange Zeit die Lehre vertreten worden, daß nur aus Kohlehydraten gebildetes Fett im Kin-per abgelagert werde. Das mit der Nahrung aufgenommene Fett dagegen sollte direkt verbraucht Es waren umfassende Versuche notwendig, um die erwähnte Anschauung zu widerlegen. Heute wissen wir mit Bestimmtheit, daß das Fett im tierischen Organismus mindestens zwei Quellen entstammt. Einmal wird mit der Nahrung aufgenommenes Fett als solches abgelagert und den Zellen zur Verfügung gestellt, und ferner wird Fett aus Kohlehydraten gebildet. Die Beweise für die Fettbildung aus Kohlehydraten haben wir S. llTlf mitgeteilt. Sie hat a priori vieles für sich. Der tierische Organismus hat in seinen Geweben nur für eine bestinmite Menge von Kohlehydraten Raum. Die Glykogenmenge, die in der Leber, den Muskeln und den übrigen Organen abgelagert werden kann, ist eine begrenzte. Der tierische Organismus kommt jedoch oft in die Lage, größere Mengen von Kolilehydraten, als er augenblicklich als solche verwerten kann, aufzustapeln. Hier kommen nun die ausgedehnten Depots für Fett zu Hilfe. In ihnen können große Mengen von Kohlehydraten in Form von Fett untergebracht und Gänzlich unbis zum Augenblick ihres Gebrauches aufbewahrt werden. l)ekannt ist, an welcher Stelle des tierischen Organismus, in welchem Organ, diese Umwandlung sich vollzieht. Es ist möglich, daß die Leber diesen komplizierten Umbau durchführt, es ist aber auch denkbar, daß Averden. er bereits in der Darm wand erfolgt. Der Beweis, daß das resorbierte Fett als Quelle für dasjenige der Fettzellen in Betracht kommt, ist in folgender Weise geführt worden. Franz Ho/mami-) ließ einen Hund so lange hungern, bis er fettarm war. Der Eintritt dieses Zustandes kann erfahrungsgemäß durch die Kontrolle der Stickstoffausscheidung festgestellt werden. Das hungernde Tier braucht zunächst seine Glykogen- und schließlich auch seine Fettvorräte auf und spart auf diese Weise möglichst sein Eiweiß. Sind die Fettmassen aufgebraucht, dann tritt plötzlich ein großer Eiweißzerfall ein. .loiirii. of Biol. Cheiii. 2. 81)1, 415 (1907). Vgl. auch Peftenkofer Zoitschr. f. Biol. 8. 153 (1872) B. Schulze: Laiulwirtscli. .ialirh. 1. 57 (1SS2). und Voit: Zcitschr. f. Biol. 9. 1 (1873). F. Soxhlet: Zcitschr. d. laiulwirtschaftl. Vereines in Bayern. Aii,i,aistlieft 1881. //. IVefskr nnd F. llild: Ebenda. St. Chanieirski: Zcitschr. f. Biol, 20. 179 (1884). /';. Mfissl und F. Sfrohmai/rr /. Munk: Virchoirs Archiv. 101. 91 (1885). 10. 1 (1874). ') A. S. Loereharnf: 2) Franz Hofmann: — — — — — — — Sitznugsbcr. d. kais. Akad. d. Wissensch. 88. III. .hillheft. 1883 und Monatshefte f. Ciicni. E. Meissl. F. Strohmer und N. r. I.orenz: Zcitschr. f. Biol. 22. (\^ (188i;). 4. 801 (1883). ('. M. 1,'iilmer: Zcitschr. für Voit: Sitzungsber. d. Münchener Akad. 288 (1885). — - Biol. 22. 272 (188(5). Fette, rhosnluitide. Steriue. 277 Die Stickstoffausscheidung steigt unvermittelt an. Das ist nach 4—6 Wochen langem Hungern der Fall. Nun fütterte Hofmann ein Versuchstier mit viel Speck und wenig Fleisch. Das zugeführte Fett und Eiweiß waren genau bestimmt worden. Es ergab sich, daß der Hund in 5 Tagen 1854 g Fett und 254 // Eiweiß resorbiert und lüöo g Fett angesetzt hatte. Somit war der Beweis erbracht, daß Nahrungslett zum Aufbau des Körperfettes Verwendung gefunden hatte, da eine andere Quelle für diesen großen Fettansatz als das verabreichte Fett nicht vorhanden war. Auch andere konnten durch Fettfütterung und auch durch Verabreichung Forscher von Fettsäuren Fettansatz bei hungernden Hunden erzielen. Ein weiterer Beweis dafür, daß das Nahrungsfett und das abgelagerte Fett in direkten Beziehungen zueinander stehen, eine andere Weise erbracht worden, i) /. ist schließlich noch auf Munk fütterte einen 16 kcf schweren Hund nach 19tägigem Hungern 14 Tage lang mit aus Hammeltalg gewonnenen Fettsäuren. Das Körpergewicht des Versuchstieres, das während der vorhergehenden Hungerperiode um 32% gesunken war, stieg wieder um XI^Iq. Bei der Sektion des Hundes fand sich ein sehr reichlicher Fettansatz. Durch Auslassen dieses Fettes konnten etwa 1100// eines bei Zimmertemperatur festen und erst bei 40" schmelzenden Fettes isoliert werden. Nun schmilzt bekanntlich Hammeltalg bei dieser Temperatur, während das normale Hundefett einen niedrigeren Schmelzpunkt hat. 1. Mimk verwendete zu einem zweiten Versuche Kiiböl. Es konnte aus dem Fettpolster des Versuchstieres ein bei 23" schmelzendes Fett erhalten werden. Bei 14" schied sich eine körnig kristallinische Masse ab. Das gewonnene Fett enthielt 82"/o Ölsäure und 12"/n feste P^ettsäuren. Normales Hundefett enthält nur 66"/,, Ölsäure und 29"/o feste Säuren. Rüböl enthält ferner Erucasäure (Coo H40 O.J. Auch diese ließ sich aus dem Reservefett des Hundes isolieren. Wir können auf Grund umfassender Versuche die Angaben von /. MiDik bestätigen. Es gelingt in der Tat, große Mengen von Nahrungsfett in den Fettdepots anzuhäufen. Es ist nach mehreren Wochen, nachdem die Fütterung mit dem betreffenden Fett eingestellt worden ist, noch nachweisbar. Man kann einen „Rüböl"- und „Hammeltalg-Hund" ohne weiteres an der Beschaffenheit seines Fettes erkennen. Schließlich sei noch erwähnt, daß man den Übergang von unverändertem Nahrungsfett in die Milch-) und das Sekret der Talgdrüsen^') beobachtet hat. In der Folgezeit sind noch sehr viele Beobachtungen gemacht worden, aus denen sich die interessante Tatsache ergibt, daß die Zusammensetzung des Depotfettes durch die Art des Nahrungsfettes beeinflußt werden kann. So haben Pferde, die mit Hafer gefüttert werden, ein anderes Fett als Tiere, denen Heu verabreicht wird.) Liegt hier für die Fette ein allgemeines Gesetz vor? Unterliegt das Nahrungsfett nicht wie alle sonstigen Nahrungsstoffe einem TJm- — Radzijewski: Virchowi^ Archiv. 43. 268 (1871): 56. 211 (1884). Lebedew: l.Munk: Archiv f. (Anat. u.) 1882; PiUlger% Archiv. 31. 15 (1883). I'liysidl. 273 (1S83): Virchows Archiv. <>5. 41)7 (1884). V.?l. auch Gcorc/ Bosenfeld: Verhaudl. d. 17. Kongresses f. iuu. Med. .o03 (1899). ^) Vgl. /.. B. Engel: Verbandl. d. 22. Vers. d. Ges. f. Kinderheilkunde. Meran 19Ü5. Plafo: Verhäiidl. der Deutschen dermat. Ges. Breslau. 182 (1901). ) Vgl. hierzu (i. Rosenfeld: Ergebnisse der Pbysiol. 1. I. 651 (1902); 2. I. 80 (1903) mit viel Literatur. •) M(m1. Zentralbl. •') — — 278 -"^^^ • Vorlesung. bau, ehe es dem Körperbestand einverleibt wird? Wird das Fett in gewissem Sinne nicht körpereigen? Diese Fragen lassen sich nicht durch Prüfung des Depotfettes beantworten. Das Fett der Fettzellcn hat mit dem Bau der Zelle, der Struktur des Protoplasmas nichts zu tun. Wenn wir feststellen wollen, ob das Fett der Körperzellen selbst, d. h. dasjenige Fett, das einen nicht ersetzbaren Bestandteil der Zelle darstellt, durch die Art des aufgenommenen Fettes beeinflußbar ist, dann müssen w^ir das abgelagerte Fett ausschalten und nur das eigentliche Zellfett prüfen. Es ist nun nicht leicht, zu entscheiden, was eigentliches Zellfett Gehört das sichtbare Fett auch dazu, oder kommt nur jenes in Betracht, das erst in Freiheit gesetzt wird, wenn man die Zellstruktur zerstört? Diese Frage läßt sich zurzeit nicht entscheiden, dagegen dürfen w'ir mit Sicherheit annehmen, daß das in der Zelle gebundene Fett ihr auch als Bestandteil zugehört. Die Untersuchung dieses Fettes ergab'), daß es bei Hunden, die mit Rüböl gefüttert wurden und gewaltige Depots von dieser Fettart besaßen, die gleiche Zusammensetzung hatte, wie das entsprechende P^'ett von normalen Hunden und von Tieren, die ausschließlich Hammeltalg als Fett erhalten hatten. Da sich dieses Zellfett nicht im reinem Zustande abscheiden läßt, blieb nichts anderes übrig, als es zu verseifen und eine Bestimmung der Fettsäuren durchzuführen.-) ist! Durch die erwähnten Versuche ist festgestellt worden, daß das am Aufbau der Körperzellen beteiligte Fett nicht durch die Art des aufgenommenen Fettes beeinflußt wird. Jede Tierart bereitet ohne Zweifel eigenartige Fetlgemische, und wahrscheinlich sind die eigentlichen Zellfette bei der gleichen Tierart je nach den Organen wiederum verschiedene. Das Fett nimmt somit in dieser Beziehung keine Sonderstellung ein. Jede Tierart baut sieh eigenes Fett auf. Der Umstand, daß einseitige Ernährung mit einem bestimmten Fettgemisch dieses zu massenhafter Ablagerung in den Fettgeweben bringt, findet vielleicht im folgenden seine Erklärung. Das Fett wird, wie schon erwähnt, nach der Auffassung der meisten Forscher im Darmkanal in seine Bausteine zerlegt. ) Dabei entstehen Glyzerin und jene Fettsäuren, die am Aufbau des verfütterten Fettes beteiligt sind. Handelt es sich um Fette, die jene Fettsäuren enthalten, die der Organismus zum Aufbau seiner Fette braucht und sind sie in genügender Menge vorhanden, dann kann wohl bei dem Wiederaufbau der Fette in der Darmwand ein Fettgemiseh gebildet werden, das in gewissem Sinne „depoteigen" ist. Hat jedoch das l^'ctt andere Fettsäurekomponenten oder sind einzelne davon sehr stark vertreten und andere sehr wenig, dann vermag der Organismus in der Darmwand bzw. in der Lymphe offenbar das ihm sonst gewohnte Fett nicht zu bilden. Die Darmwandzellen bzw. die Leukozyten müßten schon die Eigenschaft haben, Fettsäuren umbauen zu können. Da sie diese Fähigkeit vielleichtnieht besitzen ') — jedenfalls nicht Emil Abderhalden und in ausreichenden» Maße C. liruluii: /oitsclir. f. pliysiol. C-lioniie. — so kommt , 65. .•}30(1909). dann Kigeue. nocli nicht veröfieiitliclitc Boohachtiiiigeii. ) Nimmt man an, daß feinste PVtttrupfcheu als solche die Davmwand passieren, ist das Eindringen von unverändertem is'ahrungsfett in die (jewebe besonders leicht verständlich '') Fette, l'liospliatide. Sterine. 27 9 es in gewissem ISinne automatisch wieder zur Synthese des Fettes, das zugeführt worden ist. Sind die Körperzellen genötigt, Zellfett zu bilden, dann können sie das körperfremde Depotfett spalten und die Fettsäuren umbauen. Es ist aber auch möglieh, daß das Zellfett hauptsächlich aus Kohlehydraten hervorgeht. Bei dieser KSynthese. die ol^enbar von einfachen Abbaustufen der Kohlehydrate ausgeht, können sicher alle Fettsäuren, die notwendig sind, um spezifisch gebaute Fettarten und Fettgemische herzustellen, gebildet werden. Das Depotfett ist sicherlich vornehmlich Energiequelle und in diesem Falle kann es in gewissen Grenzen gleichgültig sein, welchen Aufbau es hat, wenn es nur durch die Zelllipasen spaltbar ist und seine Bausteine weiter abgebaut werden können. Das Fett der Depots ist dem Glykogen vergleichbar. Dieses Reservekohlehydrat zeigt innerhalb der ganzen Tierreihediegleiche Zusammensetzung. Es hat keinen artspezifischen, sondern höchstens einen tierspezifischen Charakter. Das Reservefett liegt ebenso, wie das Glykogen außerhalb des Zellverbandes. Es bestimmt den Zellcharakter nicht mit. Das Reservefett braucht seiner ganzen Stellung und seiner Funktion nach keinen spezifischen Charakter zu haben. Es wäre nicht auffallend^ wenn es immer enge Beziehungen zur Art des Nahrungsfettes haben würde. Es brauchte nicht einmal tierspezifisch zusein. In Wirklichkeit sind jedoch je nach der Tierart erhebliche Unterschiede in der Zusammensetzung der Depotfette vorhanden. Es ist richtig, daß die Pflanzenfresser, die sich von Blättern und Stengeln von Gramineen ernähren, unter sich ein recht ähnlich aussehendes Fett von ähnlichem Schmelzpunkt aufweisen. Identisch sind die Fette jedoch nicht. Offenbar finden sich neben unveränderten Nahrungsfetten doch auch umgebaute Fette beigemischt. Auch die Fleischfresser haben bei Aufnahme der gleichen Fleischart durchaus nicht das gleiche Depotfett. Daß der tierische Organismus aufgenommenes Fett in weitgehender Weise umbauen kann, beweist die Beobachtung, daß Walrat in gewiihnliches Fett und Monoglyzerid in Triglyzerid umgewandelt werden. i) Wenn man übrigens besondere, leicht nachweisbare Fette in kleinen Mengen der Nahrung zusetzt, so kommt es nicht immer zur Ablagerung des betreffenden Fettes. So konnte Hunden wochenlang bis 20 (/ Rüböl pro Tag gegeben werden, ohne daß dieses in allen Fällen in den Fettdepots nachzuweisen war.-) Die Beobachtungen von Munk sind vor allem deshalb so wertvoll, weil sie uns in eindeutiger Weise beweisen, daß der tierische Organismus seine Depots direkt mit aufgenommenen Fetten füllt und nicht etwa nur mit Fett, das aus Kohlehydraten hervorgegangen ist. Es würde uns das Studium des Fett- und KohlehydratStoffwechsels sehr erleichtern, wenn wir das aus Kohlehydraten gebildete Fett von dem aus Nahrungsfett entstandenen unterscheiden könnten. Leider ist das nicht der Fall. Die einzige Beobachtung, die vorliegt, ist daß das aus Kohlehydraten gel)ildete Fett arm an (")lsäure sein soll.^) Dieser Befund genügt natürlich nicht zur Charakterisierung der Herkunft die. der Bestandteile der Fettlager. ») Vgl. S. 26H -) Eigene, nicht venitt'eutliclite Beobachtung. G. Rosenfeld: Ergebnisse der Thysiol. 1. I. ') Gf)]. (1902) ^^ 280 • Vorlesung. ist ohne Zweifel die Anpassung der bestimmte Funktionen. i) Das Fettgewebe ist nicht nur Vorratskammer, in der Überschüsse an Fett und Kohlehydraten abgelagert werden. Es hat auch rein mechanische Funktionen zu erfüllen. Die ganze Beschaffenheit der Haut ist wesentlich von der Zusammensetzung und der Menge des Fettgewebes im Unterhautbindegewebe aljhängig. An manchen .Stellen bildet das Fett einen direkten Schutz gegen mechanische Einwirkungen. Es wirkt als Polster. Eine besondere Rolle spielt das Fett der Augenhöhle. Es liefert für den Bulbus die Gelenkpfanne. Es ist von großem Interesse, daß das Fett, das mechanischen Funktionen dient, in Zeiten der Not Hunger am spätesten angegriifen wird. Sehr wichtig ist die Funktion des Fettes als schlechter Wärmeleiter. Das Fettgewebe ist ein guter Wärmeschutz. Beim Glykogen haben wir die Frage erörtert^), in welchen Beziehungen es zum Kohlehydratstotfwechsel der verschiedenen Organe steht. Wir kamen zum Schlüsse, daß die Miiskelzellen Glygogen brauchen, um Arbeit zu leisten. Sie zerlegen es stufenweise bis zu Traubenzucker. Dieser wird dann weiter abgebaut, bis schließlich nur noch Kohlensäure und Wasser als letzte Produkte übrig bleiben, oder es geht der Abbau je nach Bedarf nicht so tief. Die entstandenen Produkte können den Ausgangspunkt zu Synthesen aller Art bilden. Die Leberzellen bedürfen auch der Kohlehydrate. Sie verwenden jedoch sicherlich nur den allerkleinsten Teil des gespeicherten Glygogens selbst. Die Hauptmasse dieses Polysaccharids hat in der Leber nur seinen Stapelplatz. Wird irgendwo im Organismus Glukose gebraucht, dann springt die Leber mit ihren Vorräten ein, wenn nicht an >rt und Stelle sich Ablagerungen von Glykogen linden. Wir stellten fest, daß die Leber mithilft, den Zuckergehalt des Blutes möglichst konstant zu erhalten. Ferner lernten wir ein Zentrum kennen, das den Zuckerstotfwechsel beherrscht. Schließlich fanden wirmancherlei Anzeichen dafür, daß verschiedene Organe einen bestimmenden Einfluß auf den Glykogenauf- und -abbau haben und vielleicht auch auf die Zuckerbildung aus anderen Materialien als aus Kohlehydraten. Alle Fragestellungen, die sich beim Kohlehydratstoflweehsel ergaben, tauchen auch hier wieder auf. Wir wollen wissen, in welchen Beziehungen die in den Vorratskammern abgelagerten Fette zum Noch nicht j^enügend beachtet Fettgeraische der Depots an — — ( Fettstoffwechsel und damit auch zum Gesamt Stoffwechsel stehen. Es interessiert uns ferner zu erfahren, auf welche Art und Weise die Fettzellen veranlaßt werden, ihr Fett herzugeben, wenn Bedarf an solchen eintritt. Wir können leider kein so mannigfaltiges und in vielen Teilen genau bekanntes Bild des Fettstotitwechsels entwerfen, wie das bei den Kohlehydraten der Fall war. Die Fette und ihre Bausteine stellen, soweit unsere Kenntnisse bis jetzt reichen, kein für bestimmte Funktionen bevorzugtes Nährmaterial dar. Aus diesem Grunde verm()gen wir den Fetten im Organismus nicht so genau zu folgen. Ferner haben wir es nicht mit einem zentralen Depot, sondern mit sehr vielen .Ablagerungsstätten zu tun. Endlich beobachten wir, daß die Fettgewebe Vgl. hierzu 11'. h'tiäp/'cliiiaclicr und //. Lelr/KJor/l': Zeitsclir. f. e.xper. Patli //. lAndorir: .lalirl). f. Kiiitlerheilkuiidc. «6. 28(5 (1907). 133 (190ß). ^) Vgl. Vorlesung VI und folgende. ') 2. — F'ette. Phosphatide. Sterine. 281 das gespeicherte Fett nicht in kurzer Zeit völlig hergeben und sich dann wieder füllen, wie das unter bestimmten Verhältnissen bei der Leber mit dem Glykogen der Fall ist. Die Fettdepots halten im Gegenteil ihren Bestand beim erwachsenen Individuum in engen Grenzen aufrecht. Erst wenn Nahrungsmangel eintritt, wird den Depots in grülierem Umfange Material entnommen. Alle diese Umstände erschweren die Verfolgung des Fettstotlwechsels vom chemischen Standpunkte aus. Dazu kommt noch, daß in den Fettstottwechsel immer noch der Kohlehydratstoffwechsel hineinspielt. Fortwährend kann wieder aus Kohlehydraten Fett gebildet werden. Wir kennen auch nicht mit Sicherheit ein nervöses Zentrum, das den Fettstotfwechsel beherrscht. Damit soll nicht gesagt sein, daß er nicht von solchen aus geleitet sein kann.i) Unter Berücksichtigung des Verhaltens der Kohlehydrate im Zellstoffwechsel können wir uns etwa folgendes Bild vom Verlauf des Fettstoffwechsels in den einzelnen Zellen machen. Braucht eine Zelle Fett zu irgend einem Zwecke, dann kann sie solches durch Umwandlung von Kohlehydraten in dieses bilden, oder es werden, falls sie nicht selbst „Unisatzfett" besitzt, Fettzellen veranlaßt, ihr Fett zu liefern. Dieses wird zum Transport dem Blut übergeben, nachdem es vorher, wahrscheinlich Zum Transport die Fettzellen passiert hat. in seine Bausteine zerlegt, kommt es wahrscheinlich als Neutralfett. Soll Zellfett aus ihm werden, dann ist ein Umbau nötig. Er vollzieht sich entweder in der Zelle, die das neue Fett nötig hat, oder es wird nach der Ansicht mancher Forscher das Fett zunächst der Leber zugeführt. 2) Diese soll es spalten und aus den entstandenen Fettsäuren andere, z. B. ungesättigte, bilden. Wahrscheinlicher ist schon die Annahme, daß jede einzelne Körperzelle selbst ihr Fett bilden und aufbauen kann. Es kann jedoch auch sein, daß es der Zelle nur auf den EnergieIn diesem Fall zerlegt sie ohne Zweifel das inhalt des Fettes ankommt. Wir kommen ihr zugeführte Fett und verarbeitet die Bausteine weiter. ohne weiteres zu der Fragestellung, ob die Zellen Glyzerin und Fettsäuren direkt zu den Stoffwechselendprodukten, Kohlensäure und Wasser, abbauen, oder ob nicht vielmehr auch hier, ganz entsprechend, wie bei den Kohlehydraten, der Abbau ein stufenweiser ist. An diese Fragestellung schließt sich das Problem der W^echselbeziehungen der Fette und ihrer Wir können gleich vorausBausteine zu anderen Verbindungen an. schicken, daß die Beziehungen, die zu ganz andersartigen Verbindungen auch hier nicht von den Fettsäuren und dem Glyzerin, hinführen, sondern ohne Zweifel von einfacheren Abbaustufen der Bausteine der Fette ausgehen. Was das Glyzerin anbetritft, so sei daran erinnert, daß es Glukose liefern kann.^^ Es ist möglich, daß das Glyzerin über Glyzerose in Traubenzucker übergeht. Es ist jedoch auch denkbar, daß es zuvor tiefer abgebaut wird. Auch der Weg über Methylglyoxal, Brenztraubensäure, MilchNach L. R. Mülhir: VerhandJ. d. Deutschen Gesellsch. f. innere Medizin 428 am Boden des 3. Ventrikels ein .,Fettzeutrum" bctiuden. ^) Georg Joannovics und Eriist F. Pick: ^Viencr klin. Wochenschr. 23. Nr. 17 (1<»10). H. S. Raper: Journ. of Bio). Chem. 14. 117 (1Ü13). ') (191 Ij soll sich — ') Vgl. K. Schmitz: Biocliem. Zeitschr. 45. IK (1912). -^^ 282 • Vorlesung. säure ^) usw. ist g-eg;eben. Es koiumen alle ErörteruDgen, die bereits mehrfach angeführt worden sind, in Betracht. 2) Unsere Kenntnisse über den Abbau der Fettsäuren sind durch die Beobachtungen von F. Knoop °) außerordentlich erweitert worden. Wir wissen daß gesättigte, aliphatische und aromatische Fettsäuren durch Oxydation am fi-Kohlenstoffatom unter Abspaltung der a-Kohlenstoffgruppe nebst dem Karboxyl gekürzt werden. So schied ein Hund, dem Phenylpropionsäure, CßHß CH2 CHo jetzt, . . . f. COOH, eingegeben wurde. Benzoesäure, CgHö COOH, in Form von Hip pursäure aus.) Zimt säure, Cg H5 CH CH COOH, führte gleichCOOH, ergab falls zu Benzoesäure. Benzoylessigsäure, CßHg .CO CHo den gleichen Befund. Phenylbuttersäure, Cg Hß.CH., CHg .CH., COOH, CH., COOH, die dann als Phenazeturliefert Phenylessigsäure, Cß H5 . : . . . . . . . . säure zur Ausscheidung gelangt. &) Besonders interessant ist die Beobachtung, daß Phenylvaleriansäure, Benzoesäure, bzw. Hippursäure ergibt.^) Aliphatische, gesättigte Fettsäuren zeigen, wie schon betont, das gleiche Verhalten.") Die folgenden allgemeinen Formeln sollen zeigen, wie dieser Abbau unter „[i- Oxydation" und paarweiser Abspaltung von Kohlenstoff- atomen sich vollzieht. CH3 . CH.. CH3 CH, . . CH2 . CH, . CH, CH, . . CHo . CH, CHo CH2 . . . COOH COOH :« ß ' I CH, Wir haben . CH., . CH.3 . OOOH. schon auf diese Art des Abbaus der Fettsäuren bei der Besprechung der Herkunft der Azetonkörper hingewiesen. Die Ergebnisse der Fütterungsversuche 9) und die an durchbluteter Leber ausgeführten Untersuchungen ergaben in Übereinstimmung mit der geschilderten stufenweisen Zerlegung von Fettsäuren, daß nur solche Fettsäuren Azeton bzw. Azetessigsäure liefern, die eine gerade Anzahl von Kohlenstoffatomen besitzen. Es entsteht beim Abbau solcher Säuren, wie das obige Beispiel zeigt, Buttersäure. Diese wird durch [i-Oxydation in fi-Oxybutter säure übergeführt, aus der dann die erwähnten Verbindungen hervorgehen können. früher') Vgl. hierzu S. Oppenheimer: Biocheni. Zeitschr. 45. .SO (1912). uud XIII. Vgl. Vorlesung ^) Franz Knoop: Nofnicistcrs ßeitr. G. 150(1904). Vgl. ferner Max Koppel: Karl Thomas uud Herbert Schölte: Berichte d. Deutscheu Chem. Ges. 44. 3576 (1911). Zeitschr. f. physiol. Chemie 104. 141 (1919). ••) Vgl. hierzu S. 79. ^) Vgl. auch H. Thierfelder und Erich Schempp: J'Jliif/t'rs Archiv. 167. 280 (1917). - /A D. Dahin: The Journ, ^) Franz Knoop: Hofmeisters Bcitr. 6. 150(1904). ) X ^) — — Chem. 6. 221 (1909). Vgl. hierzu 6'. Kvibden, H. Salomon uud Fr. Schmidt: Hofmeisters Bcitr. 8. G. Kmbden und A. Marx: Ehenda 9. 318 (1908). 129 (1906). «) S. 192. ) Vgl. Jnlins Jiaer uud Lt'on Blum: Arch. f. expcrim. I'ath. u. Pharmak. J>5. L. Borchardt : Zentralbl. 89 (1906); 56. 92 (19061; 59. 321 (1908); 62. 129 (1910. Geelmutjden : Zeitf. d. gesamte Physiol. u. Path. d. Stoffwechsels. N. F. Nr. 5 (1906). of. l.iol. ') — — schrift f. physiol. Chem. 23. 431 (1897). — Fette. Pliospliatide. Steriue. 283 Die Tatsache, daß j^esättigte Fettsäuren eine Oxydation am ß-Kohlencrlahren, war überraschend. Wir sind es so gewohnt, Ergebnisse der reinen Chemie auf Vorgänge in Lebewesen zu übertragen, daß wir unwillkürlich stutzig werden, wenn uns ein Vorgang entgegentritt, der einen anderen, als den erwarteten Verlauf nimmt. Umgekehrt geben wir uns leicht zufrieden, wenn ein Geschehnis in Zellen entdeckt ist, das in gutem Einklang mit den Erfahrungen des chemischen Experimentes zu stehen scheint. Ein Tieferschürfen zur Aufklärung aller Einzelheiten des gesamten Vorganges unterbleibt dann oft. Ist eine Reaktion im Organismus entdeckt, die den auf Grund unserer Kenntnisse erwarteten Verlauf nicht nimmt, dann muß alles aufgeboten werden, um ihn auch außerhalb des Körpers im Reagenzglas nachzuahmen. Nur so gelingt es, mögliche Zwischenreaktionen aufzuklären. Es kann nicht genug betont werden, daß Vorgänge, die uns als sehr einfach erscheinen, und die wir ohne weiteres in eine einfache Formelgleichung bannen zu können glauben, in Wirklichkeit eine Kette von einander bedingenden Reaktionen erfordern. Vom erwähnten Gesichtspunkte aus hat Dahin ^) die Einwirkung von Wasserstoffsuperoxyd auf Fettsäuren studiert. Er fand, daß ihre Ammonsalze am \|i-Kohlenstofiatom oxydiert und zu Ketonen der nächst niederen Kohlenstoffreihe abgebaut werden. Ob diese Art des Abbaues auch im tierischen Organismus unter normalen Verhältnissen eintritt, ist nach den Versuchen von Herrmanns-) zweifelhaft. Immerhin ist der Beweis, daß man auch im Reagenzglas ß-Oxj^dationen vornehmen kann, bedeutungsvoll. Man hat versucht, den feineren Mechanismus der [i-Oxydation und der Abspaltung der zwei Kohlenstoffatome aufzuklären. Es unterliegt wohl keinem Zweifel, daß der ganze Vorgang stufenweise über Zwischenprodukte führt. Es sind verschiedene Möglichkeiten gegeben. Einmal kann zuerst die Oxydation am [i-KohlenstolTatom einsetzen und darauf die Kürzung der Kohlenstoffkette folgen. Die Bildung der ß-Oxybuttersäure aus Buttersäure und ferner die Entstehung von Phenyl-Ji-oxypropionsäure nach Verfütterung von Phenylpropionsäure an Katzen 3) geben vielleicht den Weg an, auf dem der Abbau der Fettsäuren einsetzt. Ferner wissen wir, daß ß-Oxybuttersäure Azetessigsäure und Azeton liefert, und aus Phenyl-[i-oxypropionsäure Benzoylessigsäure, bzw. Azetylbenzol Azetophenon, hervorgeht. Diese beiden letzteren Verbindungen gehen dann in Benzoesäure über. stotifatoni = CH3 1 284 Fette. Phosphatide. Steiiue. 285 Gewiß sind nicht alle Zwischenstufen, die in Wirklichkeit auftreten, wiedergegeben. Es sind nur diejenigen genannt, die durch Versuche sichergestellt sind. Ganz sicher sind zwischen jeder der genannten Stufen noch weitere Einzelreaktionen anzunehmen, t^s ist sehr unwahrscheinlich, daß der Abbau so sprungweise verläuft. Es sind auch ohne Zweifel noch andere Möglichkeiten des Abbaus vorhanden. Vor allem muß auch mit dem Falle gerechnet werden, daß nicht nur je zwei Kohlenstoflfatome zur Abspaltung kommen, es ist vielmehr durchaus möglich, daß z. B. eine Kohlenstotitkette unter Loslösung von vier Kohlenstoffatomen zum Abbau kommt. Jedenfalls zeigt das Studium jeder einzelnen organischen Verbindung immer wieder, wie wenig der so vielfach verwendete Ausdruck „eine Substanz verbrennt im tierischen Organismus zu Kohlensäure und Wasser" über den Vorgang der Entstehung der Stoftwechselendprodukte aussagt. Die Ergebnisse von Untersuchungen über den Abbau von [i-Ketonsäuren sprechen für die geschilderte Art der Zerlegung der Fettsäuren im Zellstoll'wechsel. Vielleicht entsteht die [i-Ketonsäure in manchen Fällen auch direkt aus der Fettsäure und nicht nur auf dem Umwege über die 'i-Hydroxy Verbindung. Die Bildung von Essigsäure beim Abbau von normalen, gesättigten Vielleicht steht die im Fettsäuren ist noch nicht eindeutig festgestellt. Harn-), wie es scheint, immer vorhandene Ameisensäure mit der 1511dung von Essigsäure im Zusammenhang. Sie könnte ein Abbauprodukt dieser letzteren und sein z. B. über Glyoxylsäure C^.v.COOH, in Ameisensäure übergehen, doch fehlt ein eindeutiger Beweis für diese Annahme. Es ist auch mitghch, daß normale, gesättigte Fettsäuren zunächst in 7-, fi-ungesättigte Verbindungen übergehen: R .^CH., GH., . . GOGH—>^R . GH GH GOGH. : . Diese könnten dann, wie die ungesättigten Säuren, weiter abgebaut werden. Das Studium der Abbaustufen ungesättigter Fettsäuren hat ergeben, daß diese offenbar in verschiedener Weise verändert werden. Einmal kann eine direkte Spaltung zwischen dem x- und \|i-Kohlen- stoffatom erfolgen: R GH GH . : [i tigte . GOGH— >-R . Gooll 7. Oder es wird Wasser aufgenommen und die x-. [i-ungesätVerbindung in eine gesättigte ß-Oxysäure übergeführt: R GH CH GOOH + H, >^R GH(OH) CH., GOOH. . : . O— . . . — M ^'gl. hierzu (i. Embdni und Michand: Hofmeistern Beitr. 11. 332 (1908). FricdA. J. Wakeinaii und //. J). JJakin: The .louru. of hiol. Chem. 6. 373 (1U09). ii/atvi Biochem. Zeitschr. 55. 436 (1913). -) SchdM 'l'hiidichu)n (F/Iiicfers Archiv. 15. 129 \|1S77]) war bel<aunt, daß der Ilani Ameisensäure enthält. Vgl. ferner //. f). Dakin und A. ./. Wakeinan: Journ. nf liiol. rheni. 9. 329 (1911). II. 1>. Dakin, Janiiey und Wakeniaini : .lourn. of l)i<d. Chem. 14. 341 (1913). Rudolf Strisoirer: Biochem. Zeitschr. 54. 189 (I913l. Vgl. auch K. Salkowski: Zeitschr. f. physiol. (Jhem. 104. IGl (1919). ^icht berücksichtigt ist viel- — /•-'. : — — — — — fach bei den Untersuchungen über die Ameisensäureltihluug im Organismus der Gehalt der Nahruugs- und Genuß mittel an dieser Säure! -^^'- 286 Vorlesung. So erhielt E. Friedniann'^) Azetessi^säure. als er Kroton säure durch die überlebende Leber leitete. Ihre Bildung erfolgt ohne Zweifel auf dem folgenden Wege: CH3.CH:CH.C00H + H,0 ->CH3.CH(0H).CHo.C00H + 0— HoO—> Krotonsäure [i-Oxybutt er säure CH3 CO CHo COOH. Azetessigsäure. . . . Es ist in jedem einzelnen Falle fraglich, ob man die an niedrigen Gliedern der Fettsäurereihe gemachten Beobachtungen unmittelbar auf das Verhalten der hochmolekularen Verbindungen übertragen darf. Ferner sind, um bestimmte Verbindungen vor dem vollständigen Abbau zu bevvabren, Fettsäuren mit aromatischen Resten verbunden und dann verfüttert worden. Daß hierbei der Abbau in bestimmter Weise beeinflußt werden kann, zeigen z. B. die Erfahrungen von Hermianns^) Er fand, daß Phenylazetessigester fast ausschließlich Benzylmethylketon ergab, während Benzylund Phenylpropylazetessigester nur ganz geringe Mengen des diesen Verbindungen entsprechenden Ketons lieferten. Offenbar hat der Phenylrest in der ersten Verbindung mit der kurzen aliphatischen Kohlenstolfkette seinen Einfluß mehr geltend gemacht als in der längeren aliphatischen Kette von Kohlenstoftatomen der letzteren Verbindungen. Es ist nun nicht gesagt, daß jede gesättigte, normale Fett- säure in der genannten Art unter [i-Oxydation und paariger Absprengung von Kohlenstoffatomen oder auf sonst einem der erwähnten Wege abgebaut werden muß. Man darf vorläufig nur von — ob einer sicher festgestellten Art des Abbaues direkt oder indirekt einsetzt, ist nicht wesentlich nun die ^-Oxydation — sprechen. Zahlreiche Versuche sind im Gange, um festzustellen, wann ein anderer Modus des Abbaues auftritt, und bei welcher Struktur des Moleküls der Abbau überPrüft man den Abbau aller nur denkbaren Zwischenstufen haupt ausbleibt. im Abbau einer bestimmten Verbindung, dann darf man hofifen, alle jene Stufen ausschalten zu können, die normalerweise nicht gebildet werden. In diesem Zusammenhang sei darauf hingewiesen, daß der Versuch unternommen worden ist, den Abbau der Glieder der normalen gesättigten :') — eine Sonderstellung nehmen die ersten ihrer Glieder, und zwar Ameisensäure und Essigsäure ein, und ferner auch die Propionsäure im Organismus in insofern, als bei ihr y- und ß-Oxydation beobachtet ist Fettsäurereihe — Zusammenhang mit dem physikalisch-chemischen Verhalten der einzelnen gesättigten Fettsäuren zu bringen.) Es ergaben sich in der Tat in mancher Hinsicht Anhaltspunkte. Es haben z. B. die Fettsäuren mit gerader Kohlenstoffatomzahl und diejenigen mit ungerader Schmelzpunkte, die deutlich das Bestehen von zwei zusammengehörenden Reihen zeigen, d. h. die einzelnen Glieder der Fettsäurereihe mit gerader Kohlenstoflfatomzahl und diejenigen — Junichi Mochizvki: ') E. Friedmann: Hofmeistern Beiträge 11. 371 (1908). E. Friedmann und C. Maasc: El)end:i. bh. 450 (191:5). Biochem. Zeitsclir. .55. 443 (1913). ^) Leo Herrmanns: \. c. Zitat. 2, S. 283. ») Vgl. E. Friedmann und C. Maase: Biochem. Zeitschr. 27. 97, 113 (1900). E. Friedmann und C. Maase: Ebenda. 27. E. Friedmann: Ebenda. 27. 119 (1910). H. D. Dahin: The Journ. of Biol. Chcm. .3. r)7 (1907); 5. 173 (19()S). 474 (1910). ) Vgl. hierzu K. Spiro: Ilelvet. chim. acta 4. 459 (1921): Biochem. Zeitschr. 127. 299 (1922). — — — — Fette. Phosphatide. Sterine. 287 der Reihe mit einer ungeraden Anzahl von Kohlenstoifatomeu bilden unter Es gehören z. B. zur .Säure sich eine eng zusammengehörende Gruppe. Cß nicht die Verbindungen C5 und C7, vielmehr C4 und C^ usw. Es kommt ohne Zweifel in dieser Zusammengehörigkeit die Eigenart des inneren Baues der Glieder einerseits der geraden und andrerseits der ungeraden Kohlenstoflatomzahl-Reihe zum Ausdruck. Entsprechend dem feineren Bau der Fettsäuren vollzieht sich ihr Abbau unter Abspaltung von Kohlenstotfatomen in gerader Anzahl. Es bleiben so die Abbaustufen wenigstens zunächst in der Reihe, zu der das Ansgangsprodukt gehört. Schließlich sei noch erwähnt, daß auch die Zerlegung von Fett- säuren mit verzweigter Kohlenstoffkette und von mehrbasischen Gliedern der Fettsäurereihe studiert worden ist.^) Es spricht alles dafür, daß der Abbau auch hier nicht nur auf eine bestimmte Art erfolgt.-) Es scheint, daß der erste Angritt" auf Fettsäuren mit verzweigter Kohle nstott- kette in der Weise erfolgt, daß eine Methylgruppe abgespalten bzw. durch Oxygruppe ersetzt wird. So beobachteten z. B. Baer und Bluin Übergang von Isobuttersäure in Milchsäure: die -> ^g^>CH COOH . oh'/^^ Isobuttersäure • '^) den ^^^^ Milchsäure. Von den mehrbasischen Säuren sind insbesondere die zweibasischen untersucht worden. Es ist jedoch bis jetzt noch nicht geglückt, eine bestimmte Art des Abbaues festzustellen. Interessanter weise wird Oxalsäure, COOH , im tierischen Organismus schwer und nach einigen Autoren über- I COOH haupt nicht angegriffen), während Malonsäure, Bernsteinsäure, Glutars äure usw. vollständig abgebaut werden. Die gemachten Beobachtungen ergeben das wichtige Resultat, daß auch die Fettsäuren nicht direkt in die Endprodukte Kohlensäure und Wasser zerfallen. Der Abbau ist vielmehr auch hier, wie bei den Kohlehydraten, ein stufenweiser. Die Spaltung kann stets Halt machen und bei der gebildeten Abbaustufe die Synthese einsetzen. Aus der vorliegenden Darstellung ergeben sich ohne weiteres die schon früher erörterten Beziehungen zu den Azetonkörpern. Von solchen Abbaustufen aus müssen wir auch nach Verbindungen suchen, die zu Angehörigen anderer Körperklassen führen. Bei jeder einzelnen im tierischen Organismus auftretenden Abbaustufe von organischen Verbindungen ergibt sich die Frage, ob sie von den Zellen des tierischen Organismus noch zu ) V'^gl. die Literatur (Embden und Mitarbeiter, Baer uud Blum, Friedman h und Mitarbeiter) bei H. D. Dahin: Oxydations etc. 1. c. Zitat 3, S. 306. ) Vgl. eine weitere Möglichkeit: Henry Stenley Kaper: The Biochemical. .1. <S. 320 (1916). ä) J. Baer und L. Blum: Archiv f. experim. Path. u. Pharm. 55. 89 (1906); 56. 92 (1906); 59. 321 (1908); 62. 129 (1910). ) Vgl. hierzu u. a. J. Pohl: Archiv, f. experim. Path. u. Pharm. 37. 413 (189.3); Zeitschr. f. experim. Path. u. Ther. 8. 308 (1910). //. Hildehrandt.: Zeitschr. für physiol. Chem. 35. 141 (1902). A. Brion: Zeitschr. f. physiol. Ghem. 25. 283 (189S). — — — Carl Neuherg und Ä Saneyoshi : Biochem. Zeitschr. 36. 32 (1911). ^^ 288 • Vorlesung. Synthesen Verwendung finden kann. Wann ist eine bestimmte Abbaustufe endgültig- als Baumaterial nicht mehr verwendbar? Ohne Zweifel vermag der tierische Organismus Wasser und Kohlensäure, die letzten Abbaustufen aller organischen Nahrungsstoffe, nicht mehr 7a\ organischer Substanz aufzubauen. Dennoch können auch diese letzten Glieder einer langen Keihe von Abbaustufen unter Umständen noch einmal Beziehungen zu organischen Verbindungen anknüpfen. Einmal können Teile von W^asser bei der Hydrolyse zusammengesetzter Verbindungen in die entstandenen Bruchstücke eintreten. Dann kann, wie wir eben bei der Besprechung des Abbaues der Krotonsäure zu [i-Oxybuttersäure gesehen haben, Wasser angelagert werden. Wir kennen sehr viele chemische Reaktionen, an denen Wasser direkt teilnimmt, ganz abgesehen von seinen übrigen wichtigen Funktionen im Zellhaushalt. Auch die Kohlensäure kann unter Umständen noch einmal in den Kreislauf der Stoffe im tierischen Organismus eintreten. Es sei auf die Beobachtung von Siegfried hingewiesen, daß Aminosäuren Kohlensäure unter Bildung von Karbaminosäuren binden können.^) Besonders interessant und wichtig ist die Feststellung derjenigen Abbaustufen aus jeder einzelnen organischen Verbindung, von der aus sich noch Produkte anderer Zusammensetzung und mit höherem Molekulargewicht bilden können. Die exakte Erforschung dieses Problems ist deshalb so wichtig, weil seine Lösung uns jene Substanzen bekannt gibt, die das einfachste Baumaterial des tierischen Organismus darstellen. Nun haben wir bereits früher erwähnt, daß für zahlreiche Vorgänge erwiesen ist, daß sie umkehrbar sind. Es sei z. B. daran erinnert, daß Azetessigsäure durch Reduktion in .S-Oxybuttersäure übergehen kann. 2) Gewiß sind viele der geschilderten ^'erbindungen, die wir beim Abbau der Fettsäuren kennen gelernt haben, auch in umgekehrter Reihenfolge untereinander verknüpft. Von besonderem Interesse ist die Beobachtung, daß Essigsäure in Azetessigsäure und damit auch in [i-Oxybuttersäure umgewandelt werden kann. Loeb'^) fand, daß Blut, dem Essigsäure zugesetzt war, beim Durchleiten durch die Leber Azetessigsäure aufwies. E. Friedmann) hält den folgenden Weg der Umwandlung von Essigsäure in Azetessigsäure für den wahrscheinlichsten: CH3 . COOH + CE, . C<[[ -H., —> CH, CH CH COOH + Essigsäure Azedaldehyd CH3 CH(OH) CH., COOH -fH., ß-Oxy buttersäure Es würde somit die Essigsäure, . . . : . — . H., —> Krotonsäure — CH3 CO >- . . CH., . COOH Azetessigsäure. falls die an überlebenden Organen gemachten Beobachtungen ohne weiteres auf Stoftvvechselvorgänge im tierischen Organismus übertragen werden dürfen, noch zu Synthesen verwendbar sein. Daß der angegebene Weg der Bildung der Azetessigsäure über die Krotonsäure und die ß-Oxybuttersäure möglich ist, beweist die Beobachtung, daß verfüttertes Furfurol in Furfurakrylsäure übergeht =^): ') -) •') Vgl. Vorlesung XVIII. Vgl. S. 190 und fernpr noch L. r. La(^rrniarh: Biochem. Zeitschr. 55. 458 (191.'5). Afhitn Loeb: Biochem. Zeitschr. 46. 118 (1912). } /•;. ^) M. Friedmann: Ehenda. 55. 43(5 (1913). Jaff'- lind Rudolf Cohn: Bor. d. Deutsch. Chem. Gesellsch. 20. 2311 (1887). 289 Fette. Phosphatide. Steriue. + C, H3 . Essigsäure Ein säure Zum C/^CH, COOH=:C, H. . Furfurol . () . CH CH COOH + H., 0. : . Furfurakrylsäure. obige Darstellung der Entstehung der KrotonÜbereinstimmung mit der eben geschilderten Synthese. Schlüsse sei noch erwähnt, daß man daran gedacht hat, daß Blick die zeigt . auf die der Abbau der Fettsäuren sich in der Hauptsache, ja vielleicht ausschließHat diese Annahme a priori wenig lich in der Leber vollziehen könnte. Wahrscheinlichkeit für sich, so haben auch direkte Versuche gezeigt, daß sie nicht zutrifft. 1) Damit soll nicht gesagt sein, daß den Leberzellen in der Umbildung von Fettsäuren nicht eine große Bedeutung zukommen kann. Im allgemeinen dürften jedoch auch andere Körperzellen befähigt sein, Fettsäuren zu zerlegen. 1) Vgl. z. B. Julius Bär: Biochem. Zeitschr. 127. 275 (1922). Abde rh al de u, Physiologische Chemie. I.Teil. 5. Aufl. 19 Vorlesung XVI. Fette. Phosphatide. Sterine. 3. Die Wechselbeziehungen der Bausteine der Fette zu denen der EiweißstofFe und zum Traubenzucker. Das Verhalten der Phosphatide und der Sterine im ZellstofFwechsel. Die Verfolgung der Wechselbeziehungen der Kohlehydrate zu Verbindungen, die nicht der Kohlehydratreihe angehören, hat nicht nur unsere Kenntnisse des Kohlehydratstofiwechsels außerordentlich gefördert, sondern uns gleichzeitig einen tiefen Einblick in den Zellstotfwechsel verschafft. Es ergab sich, daß die Zelle den Traubenzucker über mehrere Von derartigen Zwischenstufen zu Kohlensäure und Wasser abbaut. Zwischenprodukten zweigen jene Wege ab, die zu anderen Verbindungen hinführen, und umgekehrt ließ sich mit großer Wahrscheinlichkeit zeigen, daß von diesen aus sich wieder Beziehungen zum Zucker ergeben, wobei offenbar der gleiche Weg eingeschlagen wird. Diese reichen Ergebnisse lassen es verständlich erscheinen, daß man auch bei den Fetten Wechselbeziehungen zu anderen Verbindungen aufzufinden bestrebt war. Vor allem und ihre Abbaustufen interessiert uns die Frage, ob die Fette Beziehungen zu den Kohlehydraten und den Eiweißstoffen beziehungsweise zu ihren Bausteinen, den Aminosäuren, besitzen. Wir wollen zunächst ohne Berücksichtigung der vorliegenden Tatsachen das heiß umstrittene Gebiet der Wechselbeziehungen zwischen den Fetten und den genannten Verbindungen von den bisher erörterten Vorgängen im Zellstoffwechsel aus einer Betrachtung unterziehen. Wir beginnen mit der Frage der Umwandlung von Fett in Zucker. Es unterliegt nach den bisher gemachten Beobachtungen keinem Zweifel, daß Glyzerin in Glukose übergehen kann. Dürfen wir auch die Fettsäuren beziehungsweise ihre Abbaustufen als Quelle für Zucker im tierischen Organismus betrachten? Die Pflanzenwie sicher festgestellt ist, Fettsäuren in Zucker um. Überführung im einzelnen erfolgt, wissen wir nicht. Er ist möglich, daß sich durch [i-Oxydation höherer, gesättigter Fettsäuren schließlich z. B. Kapronsäurc bildet und diese dann in Griukose übergeht. Wahrscheinlicher ist jedoch die Annahme, daß die Kohlenstoffkette der Fettsäuren noch weiter verkürzt wird, und die Synthese von Zucker von einfacheren Abbauprodukten ausgeht. Wir können zurzeit die Zuckerbildung aus zelle wandelt, Wie die Fette. Phosphatide. Steriue. 291 Fettsäuren im tierischen Organismus nur als möglich hinstellen. Bewiesen Wir wollen gleich hier erwähnen, dal,! die meisten ist sie keinesfalls. Forscher der Ansicht sind, daß eine Zuckerbildung aus Fettsäuren im tierischen Stoft'wechsel nicht erfolgt. Es fehlt jedoch auch nicht an Stimmen, Umwandlung für wahrscheinlich halten. Die zweite P>age, nämlich die Beziehungen der Bausteine der Fette zu Aminosäuren, wollen wir erst erörtern^), wenn wir diese kennen gelernt haben und dafür hier auf die Frage eingehen, ob Aminosäuren Material zur Fettbildung liefern können. Vom theoretischen Standpunkte aus muß diese Frage unbedingt bejaht werden. Wir haben die eine solche nämlich festgestellt, daß Aminosäuren Traubenzucker liefern können. Ferner ist sicher bewiesen, daß Zucker im tierischen Organismus in Fett übergehen kann. Es können somit in der Tat im gebildeten Fett Kohlenstoftketten vorhanden sein, die einst Aminosäuren angehörten. Es wäre ein aussichtsloses Beginnen, wenn wir die Frage nach der Bildung von Fett aus Aminosäuren von diesen Gesichtspunkten aus beantworten wollten. Wir können den Aminosäuren nur bis zu den Kohlehydraten folgen. Die Umwandlung von solchen in Fette ist ein Problem für sich und hat nichts mehr mit den Beziehungen der Aminosäuren zu diesen zu tun. Wenn wir die Frage nach der Umwandlung von Aminosäuren in Fett stellen, so wollen wir erfahren, ob aus Bruchstücken von diesen irgend welcher Art direkt Fett oder ihre Bausteine hervorgehen können. Zunächst wäre zu prüfen, ob bestimmte Aminosäuren Glyzerin liefern können. Es sei daran erinnert, daß beim Abbau von Alanin Milchsäure und ferner auch Brenztraubensäure Wir haben bis jetzt keine Beweise dafür, daß aus diesen entstehen kann. Abbaustufen direkt Glyzerin gebildet werden kann. Dagegen ist es wohl möglich, daß die Glyzerose, die ja enge Beziehungen zur Milchsäure hat, den Übergang zu Glyzerin vermittelt. Schwieriger gestaltet sich die Beweisführung einer Überführung von Aminosäuren in Fettsäuren. Es fehlen uns noch die Kenntnisse über die Bildung höherer Fettsäuren aus einfacheren Verbindungen in der Zelle. Wir wissen nicht, ob eine Synthese aus niederen Fettsäuren eintritt. Möglich ist, wie wir S. 253fr. erörtert haben, eine derartige Synthese durchaus. Niedere Fettsäuren könnten sich aus Aminosäuren ganz gut bilden. Fassen wir alles zusammen, was wir vom rein theoretischen Standpunkte aus über die Bildung der Bausteine der Fette aus Aminosäuren wissen, so kommen wir zum Schlüsse, daß diese wohl möglich ist. doch fehlen uns noch zu viele Unterlagen, um eine solche Annahme in ihren Einzelphasen prüfen zu können. Nachdem wir festgestellt haben, daß vom theoretischen Standpunkte aus keine Tatsachen vorliegen, die eine Beteiligung der in den Aminosäuren enthaltenen Kohlenstoffketten am Aufbau der Bausteine der Fette ausschließen, wollen wir zu den Erfahrungen übergehen, die durch das direkte Experiment und mancherlei Beobachtungen an pathologisch veränderten Organen gemacht worden sind. Wir wollen gleich vorweg nehmen, daß zurzeit die Meinung den Vorrang hat, daß eine Fettbildung aus Eiweiß bzw. aus Aminosäuren nicht erwiesen ist. Es seien einige Ergebnisse der von verschiedenen Gesichtspunkten aus unternommenen Untersuchungen über die Fettbildung aus Aminosäuren ') Vgl. Vorlosung XXII. 19^ XVI. Vorlesuug. 292 Im Anschluß daran wollen wir dann die Frage besprechen, ob schon jetzt aus dem Umstände, daß eine Fettbildung aus Aminosäuren nicht eindeutig bewiesen werden konnte, der Schluß gezogen werden darf, daß der tierische Organismus diese Umwandlung überhaupt nicht vollzieht. Im Mittelpunkt der ganzen Besprechung über die Umwandlungen von Eiweiß in Fett standen früher die Versuche von Pettenkofer und Voii.^) Diese beiden Forscher hatten Hunde mit großen Mengen von mögliclist fettarmem Fleisch gefüttert. Sie prüften, ob im Harn der in Form von Eiweiß zugeführte Stickstoff wieder auftrat, und ob gleichzeitig auch die entsprechende Kohlenstoffmenge zur Ausscheidung kam. Für den Kohlendann stoff' kommen einmal die organischen Verbindungen des Harns und in Betracht. Exspirationsluft der Kohlensäure die vor allem Pettenkofer und Vo'd berechneten, daß zwar der gesamte eingeführte hier angeführt. Stickstoff zur Ausscheidung gelangte, dagegen nicht aller Kohlenstoff. Aus diesem Ergebnis zogen sie den Schluß, daß dem Eiweiß zugehörender Kohlenstoff zur Bildung von Fett verwendet worden war. Pß.üger^) griff die Beweiskraft der Ergebnisse dieser Versuche an. Er konnte zeigen, daß die den Berechnungen zugrunde gelegten Werte für Stickstoff und KohlenPettenkofer und Voit hatten stoff' im fettarmen Muskel unrichtig waren. jedoch ohne Zweifel dennoch eine sehr wichtige Beobachtung gemacht. Sie hatten nämlich durch ihre Versuche festgestellt, daß die alleinige Verfolgung der Stickstoff'zu- und -ausfuhr uns nicht über den Umfang des Eiweißstoffwechsels unterrichtet. Es kommt ohne Zweifel oft zur Zurückhaltung von Kohlenstoff im Körper in irgend einer Form. Nach dem damaligen Stand der Kenntnisse des Zellstoff wechseis war es sehr naheliegend, an eine Verwertung der im Körper zurückgebliebenen Kohlenstoftketten zum Aufbau von Fett zu denken. Jetzt wissen wir, daß bestimmte Aminosäuren Zucker liefern können. Diese Umwandlung könnte sehr gut das erwähnte Ergebnis der Versuche von Pettenkofer und Voit bedingt haben. Direkt beweisend für die Fettbildung aus Eiweiß schienen die Beobachtungen über die sogenannte Verfettung von Organen zu sein. Gewebe, die makroskopisch und auch mikroskopisch nur wenig Fett zeigen, können unter der Einwirkung bestimmter Stoffe nach kurzer Zeit sehr fettreich aussehen. So erhält man z. B. nach Vergiftung mit Phosphor fettige Degeneration der Leber. Gleichzeitig beobachtet man häufig Aminosäuren im Harn und auch im Lebergewebe. Es lag sehr nahe, diese Beobachtungen im Sinne einer Fettbildung aus Eiweiß zu deuten. Das Auftreten von Aminosäuren weist offenbar auf einen v^ermehrten Eiweißabbau hin. An seiner Stelle tritt Fett auf. Es schien somit das Problem der Fett- bildung aus Eiweiß eindeutig gelöst! Wie kaum ein anderes Beispiel, zeigt die Beantwortung der Frage nach der Umwandlung von Eiweiß in Fett, daß ausschließlich quanti- — Annalen. Suppl. 57. 361 (1862). M. Pettenkofer uud C. Voit: 371 (1870). Ebeuda. 7. 433 (1871). -- C. Voit: Handbuch der Physiologie des Gesamtstoffwechsels und der Fortpflanzung. Leipzig 1881. München. M. Kieger. 1883. ^) E. Pjfüger: P/ii((,ers Archiv. 50. 48 (330 und 396) (1891): 51. 229(1891): 52. Vgl. auch M. KumagaMa : Miiteü. d. mediz. 1 (1892): 68. 176 (1897); 77. 521 (1899). Fakultät der Universität Tokio. 3. 1 (1890). ') M. Pettenkofer C. Voit: Zeitschr. f. uud Biol. 5. ('. Voit: 106 (1869); Liebigs 6. — — Fette. Phosphatide. Steriue. 293 tative Versuche zu eindeutigen Schlußfolgerungen führen können. Das in der Leber zur Beobachtung kommende Fett braucht nicht an Ort und Stelle entstanden zu sein. Es kijnnte ja aus anderen Teilen des Organismus eingewandert sein. Ferner muß mit der Möglichkeit gerechnet werden, daß Zellfett, das sich unter normalen Verhältnissen der direkten Beobachtung entzieht, im Gefolge der Phosphorvergiftung freigeworden ist, und deshalb in Erscheinung tritt. i) Die ersten quantitativen Versuche rechneten nicht mit dem Vorkommen von in der Zelle gebundenem Fett. Sie mußten deshalb zu Irrtümern führen. Äthanasiii') berücksichtigte das gebundene, nicht direkt ausziehbare Zellfett. Er bestimmte den gesamten Fettgehalt von 124 Fröschen. Dann vergiftete er ebenso viele Tiere mit Phosphor und stellte wiederum den Fettgehalt des gesamten Körpers fest. Es hatte keine Zunahme des Gesamtfettgehaltes stattgefunden. Taylor^) fand sogar im Gegenteil eine Abnahme des Körperfettes nach Phosphorvergiftung. Versuche an Mäusen führten zum gleichen Resultate.) Während "iQ^oO/o Fett enthielten, gleich gefütterte Kontrolltiere im Körper l;3'8 wiesen die mit Phosphor vergifteten Tiere einen Fettbestand von nur 4' 13 TO^/o auf. Somit war also der gesamte Organismus an Fett verarmt. Die Leber der mit Phosphor vergifteten Mäuse dagegen besaß einen Gehalt an Fett von 7'4 374"/o, während normale Mäuse in diesem Organ nur 51 ll'BVo Fett enthalten. Es hatten somit alle anderen Gewebe auf Kosten der Leber an Fett eingebüßt. Der Gedanke ist naheliegend, daß die Vermehrung der Fettbestände der Leber in direkter Beziehung zu der Verminderung des Fettgehaltes der übrigen Gewebe steht. Bosenfeld°) hat diese Annahmt; direkt bewiesen, indem er aus dem Fettdepot eines mit Hammeltalg gefütterten Hundes ein Fett in die Leber einwandern sah, das seiner Zusammensetzung nach dem Fette des Depots entsprach. Ferner zeigte er, daß hungernde Hunde und Hühner nach Phosphorvergiftung keine Zunahme des Gesamtkörperfettes aufwiesen. Somit muß die alte Annahme, wonach bei der Phosphorvergiftung Eiweiß sich in Fett umwandelt, aufgegeben werden. Aus den vorliegenden Versuchen ergibt sich somit, daß nach der Phosphorvergiftung die Verteilung des Fettes in den einzelnen Geweben eine Veränderung erfährt. Gleichzeitig kommen jedoch auch infolge der schweren Schädigung der Zellen Fettsubstanzen zum Vorschein, die unter normalen Verhältnissen nicht in freiem Zustande im Gewebe enthalten sind.") Wenn diese Ansicht richtig ist, dann darf bei einem fettarmen Tiere die Verfettung einzelner Organe und insbesondere der Leber je nach dem Fettgehalt des Versuchstieres entweder gar nicht oder doch nur in ganz geringfügiger Weise — — — — Vgl. Georg Rosenfeld: Zeutralbl. f. inn. Med. Nr. 33 (190U). D. Athanasiii: Fflügers Archiv. 74. 511 (181)9). ä) Taylor: Journ. öf experim. Med. 4. 399 (1899). ) Fr. Kraus und A. Sommer: Hofmeister?, Beitr. 2. 86 (1902). °) Georij Rosenfeld: Verhaudl. d. Ivongr. f. inu. Med. 1894. Allg. med. Zeutralzeitung. Nr. 60 (1897); Nr. 89 (1900). Vgl. namentlich Fettbildinig. II. Teil. Ergebnisse der Physiologie. (Asher & Spiro.) Bergmann. Wiesbaden. 2. I. .ö9 (1903). Vgl. auch //. Gideon Wells: Zeitschr. f. physiol. Chem. 45. 412 (1905). ^) Vgl. hierzu auch L. Mohr: Verhaudl. d. Gesellscl). deutsch. Naturf. u. Ärzte Jounnovic und I'ick: Zeitschr. f. experim. Path. u. Therapie. 6. 184 (1909). (1910). Arno Kirsche: Biochem. Zeitschr, 55. 1(59 (1913). 1) -) — — — : — ~ XVL Vorlesung. 994 Es gibt Versuche Bosenfelds'^) bestätigen diese Annahme. außer Phosphor noch viele andere Gifte, die eine Verschiebung in der So beobachtet man Verfettungen nach Eingabe Fett Verteilung bewirken. von Arsen. Antimon. Alkohol. Chloroform. Ferner dürften manche von Bakterien abstammende Produkte die gleichen Folgen haben. Nach diesen Befunden können wir uns folgendes Bild von der PhosDer Phosphor schädigt die Leberzellen primär.-) phor\^ergiftung machen. auftreten. Die Ihr Bau wird" so verändert, kommen daß ihre Bestandteile zum Teil zum Abbau — Auftreten von Aminosäuren, Verschwinden des Glykogens — werden gebundene Anteile frei, es ist wohl möglich, daß das des gebundenen Fettes und der Eiweißabbau in direktem Zusammenhang stehen. Wir dürfen jedoch nicht übersehen, daß uns unbekannt ist, wieviel Eiweiß zum Abbau kommt. Außerdem könnten die in Erscheinung tretenden Aminosäuren auch mit dem gestörten ZellstoftIhr Abbau ist vielleicht an Ort und Stelle wechsel zusammenhängen. gehemmt. Als zweite Erscheinung ergibt sich dann die Fettwanderung. Ihre Ursache ist zurzeit noch in Dunkel gehüllt. Bei der Beurteilung der Ergebnisse der geschilderten Versuche dürfen wir einen sehr wichtigen Punkt nicht außer acht lassen. Es ist dies die Methodik des Fettnachweises. Sie hat in der letzten Zeit viele Verbesserungen erfahren. Immerhin wird noch manches Produkt als Fett So lange wir es mit mitbestimmt, das gar nicht zu diesem hinzugehört. Fettgemischen zu tun haben, und wir nicht wohl definierte Fette verfolgen können, dürfen wir die vorliegenden Versuche nicht als streng beweisend betrachten. Wir dürfen aus ihren Ergebnissen nur den Schluß ziehen, daß eine Fettbildung aus Eiweiß durch sie nicht bewiesen werden konnte. Die ganze Forschung über Verfettung. Fettwanderung und Fettinfiltration ist zurzeit im Fluß". Man hat erkannt, daß neben dem gewöhnlichen Fett die Cholesterinfettsäureverbindungen im Stoffwechsel eine bedeutsame Rolle spielen. Ein sogenanntes verfettetes Organ kann eine Abnahme an gewöhnlichem Fett und eine Zunahme an Cholesterinester zeigen und umgekehrt. ») Von großem Interesse ist ferner die Beobachtung, daß in fettfreie" Gewebsstücke Fett einwandert, wenn man sie Tieren in die Bauchhöhle bringt.) Gleichzeitig bemerkt man auch das Einwandern Die Gründe, weshalb toten Geweben Fett zugezahlreicher Leukozyten. Sollte es etwa als Lösungsmittel jener tragen wird, sind noch unklar. Abbaustufen dienen, die bei der durch die Leukozyten mit ihren Fermenten herbeigeführten Zertrümmerung des zerstörten Gewebes entstehen? Für die Annahme einer Fettbildung aus Eiweiß schienen ferner Versammelte suche von Hofmann an Fliegenlarven zu sprechen. Hofmann Fliegeneier und bestimmte in einem Teil davon den Fettgehalt. Den Rest der Eier züchtete er auf defibriniertem Blut. Dieses enthielt 032Vo Fett. gleichzeitig Freiwerden "") 1) G. Rosenfeld: Ergebu. 2) Auch die aus dem Körper eutferute Leber d. Pbysiol. 2. I. 59. (1903). zeigt Verfettung. ^Yeuu Phosphor — durch sie durcbgeleitet wird. Vgl. ('. Mnrrakis : Arch. f. (Anat. u.) Pbysiol. 94. (1904). L. Hess und /'. Saxl: Virchoiia Archiv. P. SaxI: Hofmeisters Beitr. 10. 447 (1907). Vgl. auch F. Fischler und K. Bardach: Zeitschr. f. physiol. 202. 148. (1910!. Chemie. 78. 435 (1912). ') Aschof: Beitr. z. pathol. Anat. u. z. allg. Pathol. 47. 1 (1909). ) Wilhelm Griesser: Ebenda. 51. 115 (1911). Hier tindet sich weitere Literatur. Franz Hofmann: Zeitschr. f. Biol. 8. 153 (1872). — '") — Fette. Phosphatide. Sterine. 295 Die Fliegeneier besaßen 4'9Vo Fettsubstanzen. Die auf dem Blut gewachsenen Maden zeigten schließlich einen Fettgehalt, der den der Eier und des Blutes um das Zehnfache übertraf. Nun waren jedoch einesteils die Fettbestimmungen nicht einwandfrei und andernteils muß der Einwand erhoben werden, daß die auf dem Blut sich bald entwickelnden Pilzmassen die Umwandlung von Eiweißbausteinen in Fett vollzogen haben könnten. 0. Frank^)^ der den Versuch mit möglichst entfettetem Fleisch wiederholte, kam zu keinem eindeutigen Resultate. Somit sind auch diese Versuche nicht geeignet, die Bildung von Fett aus Eiweiß zu beweisen. Schließlich sei noch der sogenannten Leichen wachs- oder Adipozirebildung^j gedacht. Unter diesem Vorgang versteht man die Umwandlung von Leichen und Leichenteilen in eine wachsähnliche Masse. Mehr und mehr sieht man aus den Muskeln das Eiweiß verschwinden und an seiner Stelle ein Gemisch hoher Fettsäuren und von Seifen auftreten. 3) Diese eigenartige Umwandlung beobachtet man besonders auf feuchten Begräbnisplätzen, wo eine langsame Zersetzung unter geringer Sauerstoffzufuhr vor sich geht. Durch genaue Verfolgung dieses Vorganges und vor allem durch direkte Versuche ist festgestellt worden, daß keine Umwandlung von Eiw^eiß in Fett vorliegt, sondern, daß das schon vorhandene Fett die Ursache der Leichenwachsbildung ist. Zum Teil kommt das an Ort und Stelle vorhandene Fett in Betracht, zum Teil handelt es sich um aus den übrigen Körperteilen durch Wasser eingespülte Abbauprodukte von Fettstoffen. Übrigens hätte auch der Beweis einer Abstammung des Fettes aus Eiweiß bei diesem Vorgang keine Rückschlüsse auf die Bildung von Fett aus solchem im lebenden tierischen Organismus gestattet, denn auch hier l)leibt der Einwand, daß Mikroorganismen aller Art den Umbau vollzogen haben könnten, offen. Daß bestimmte Bakterien aus Eiweiß bzw. aus Aminosäuren Fett bilden können, scheint sichergestellt zu sein.) Auf die Mitwirkung von Mikroorganismen wird auch die Fettbildung aus Eiweiß beim Reifen des Käses zurückgeführt. 5) Schließlich sei noch erwähnt, daß auch der Fettgehalt der Sekrete der Milch- und Talgdrüsen wiederholt auf Eiweiß zurückgeführt worden ist. Direkte Versuche brachten keine Bestätigung dieser Annahme. Eine eindeutige Entscheidung der Frage nach der Herkunft des Fettes der Sekrete ist übrigens schwer zu erbringen, weil der tierische Organismus über Fettvorräte verfügt und nicht auf das Fett der Nahrung angewiesen ist. Außerdem kommt stets die Umwandlung von Kohlehydraten in Fett in Betracht. — '^ Otto Frank: Zeitschr. f. Biol. 35. 549(1897). Vgl. ferner Emst Weinland: Ebenda. 51. 197 (1908); Biol. Zeutralbl. 29. 0(58 (1909). ^) Vgl. Julius Kratter: Zeitschr. f. Biol. 16. 455 (1880). Erman Vierteljahrosschrift f. gerichtl. Med. N. F. 37. 51 (1882). E. Safkowski: Festschr. für Virchoirs. Jubiläum. 23. (1891). K. B. Lehmann: Sitzungsber. d. physikal.-med. Gesellsch. zu Würzburg. 1888. Erwin Yoit: Sitzungsber. d. Gesellsch. f. Morphol. und rhvsiol. in München. 4. 50 (1888). Fr. Kraus: Arch. f. experim. Path. u. Pharmak. 22. 174(1887). ^) A'gl. über die Zusammensetzung des Adipozire Giovanni Issoglio: Giorn. Farm. Chim. 65. 361 (1916). B. F. L'iiffan' und M. J. Marshall: Journ.of. biol. Chem. 29. 319 (1917). Ä. Goy und E. Wende: Biochem. Zeitschr. 131. 8 (1922). •») -S; P. Beebe und B. Buxton: Americ. Journ. of Physiol. 12. 466 (1905). ^) Karl Windisch: Arbeiten a. d. Kaiserl. Gesundheitsamte. 17. (1600). //. Jacohsthal: PßU(/ers Archiv. 54. 484 (1893). — — — — — — — : — XVI. Vorlesung. 296 Nachdem wir die wichtig-sten Versuche, die von verschiedenen Gesichtspunkten aus unternommen worden sind, um das Problem der Bildung von Fett aus Aminosäuren im tierischen Organismus zu lösen, erwähnt haben, wollen wir uns die Frage vorlegen, ob wir aus den erhaltenen Ergebnissen den Schluß ableiten dürfen, daß die tierische Zelle die erwähnte Umwandlung nicht zu vollziehen vermag. Dieser Schluß erscheint uns nicht berechtigt. Die vorliegenden Versuche haben nur bewiesen, daß unter den gewählten Bedingungen eine Überführung von Aminosäuren in Fett nicht feststellbar ist. Es ist gewiß nicht auffallend, wenn ein durch Phosphor schwer geschädigtes Organ keine komplizierten Umwandlungen vollzieht, und wenn ferner in der Leiche ein so eingreifender Vorgang nicht mehr stattfindet. Um die Frage nach der Fettbildung aus Eiweiß beziehungsweise aus Aminosäuren entscheiden zu können, muß der Versuch auf den normalen Organismus und ferner auf das überlebende Organ übertragen werden. Man wird Beziehungen bestimmter Eiweißbausteine zu den Komponenten der Fette suchen müssen. Bei der Verfolgung des Fettstoffwechsels stoßen wir überall auf große Schwierigkeiten, sobald wir die Probleme schärfer fassen und eindeutige Antworten verlangen. Es ist keine dem Diabetes entsprechende Störung im \erlauf des Fettumsatzes und seiner Bausteine bekannt. Wir kennen keinen mangelhaften oder eigenartigen Abbau des Glyzerins und der Fettsäuren abgesehen von der Azetonkörperbildung. Würden wir Hemmungen des Abbaus der Bausteine der Fette kennen, dann würden sich unsere Kenntnisse über die W^echselbeziehungen der Fette zu anderen Verbindungen rasch erweitern. Wir könnten dann genau so, wie bei den Kohlehydraten, nachforschen, ob die Menge der gebildeten Fettsäuren, bzw. des Glyzerins der zur Verfügung gestellten Fettmenge und dem aus Kohlehydraten sich bildenden Fett entspricht oder, ob es notwendig ist. auch auf die Aminosäuren zurückzugreifen. Vielleicht gelingt es der Forschung, experimentell eine Störung im Abbau der Bausteine der Fette zu erzeugen! Eine Besonderheit des Fettstoffwechsels und im besonderen des Fettansatzes ist uns wohl bekannt, nämlich die sogenannte Fettsucht, Adipositas oder auch Obesitas genannt. Während beim normalen erwachsenen Individuum das Fettgewebe sich mit der übrigen Körpermasse in ein gewisses Gleichgewicht stellt das Körpergewicht ^vird in engen Grenzen auf gleicher Höhe erhalten sehen wir, daß manche Menschen immer mehr an Gewicht zunehmen. Dieses Anwachsen des Körpergewichtes ist, wie leicht festgestellt werden kann, auf eine oft ganz gewaltige Zunahme des Fettgewebes zurückzuführen. Derartige Individuen leiden schwer. Sie müssen beständig eine große Last in Gestalt von Fett mit sich umhertragen. Diese stetige „Belastung" bleibt nicht ohne tiefgehenden Einfluß auf das Herz. Seine Arbeitsleistung ist erhöht. Es kommt infolge davon oft zu Störungen. Wir können durch geeignete Maßnahmen starke Zufuhr von Fettbildnern: von Fett und vor allem von Kohlehydraten, ferner Einschränkung des Verbrauches: Mangel an Muskelarlieit schließlich jedes Individuum mehr oder weniger leicht zu einem Fettsüchtigen machen. Es sei an die Mästung der Straßburger Gänse und des Schlachtviehs erinnert. Gewiß kommen viele Fettsüchtige aus ganz denselben Gründen zu ihren gewaltigen Fettablagerungen. Es [»raucht die tägliche Zufuhr an Stoffen die Ausfuhr an Stoffwechselendprodukten gar nicht so sehi' zu übertreffen, um schließ- — — — , — — 297 Fette. Phosphatide. Sterine. im Laufe der Monate und Jaliie zu großen Ablaj>erungen zu führen. Es nun ohne Zweifel Personen, die außerordentlich zu Fettablagerungen neigen, während andere, wie man sich ausdrückt, weniger dazu disponiert sind. Man hat daran gedacht, daß bei zu Fettsucht neigenden Individuen der Stoffwechsel aus irgend welchen Ursachen herabgesetzt sei. Doch ist dieser Ansicht widersprochen worden. i) Es ist auch kaum zu erwarten, daß die Fettsucht etwas Einheitliches darstellt, vielmehr haben neuere Forschungen mehr und mehr ergeben, daß ihr keine einheitliche Ursache zugrunde liegt. Besonders bedeutungsvoll sind in dieser Hinsicht Beobachtungen an Tieren und Menschen mit ausgeschalteten oder gestörten Funktionen bestimmter Organe geworden. So fand man bei Hunden, denen die Thymusdrüse 2) fortgenommen war, ab und zu einen ganz außerordentlichen Fettansatz. Bekannt ist ferner die Fettsucht nach Kastration. Ihre Erklärung bereitet Schwierigkeiten, weil mit der Entfernung der Keimdrüsen ein tiefer Eingriff in eine ganze Reihe von Funktionen erfolgt. Das kastrierte Tier Schon dieser Umstand kann ist weniger lebhaft als das nicht kastrierte. von Einfluß auf den Stoffverbrauch im besonderen und auf den Fettumsatz sein. Besonders eingehend studiert ist die sogenannte hypophysäre Fettsucht. Sie wird in Zusammenhang mit einer Störung in der lieh gibt Inkretbildung des Mittellappens Schilddrüse ist der Hypophyse gebracht. Auch die Beziehung zum Fettstoffwechsel gebracht worden. Einstweilen ist es unmöglich, abzugrenzen, an w^ elcher Stelle das einzelne Organ in diesen eingreift. Es besteht die M()glichkeit, daß Fett gespart wird, weil andere Nahrungsstoffe in gesteigertem Maße verbraucht werden. Der vermehrte Fettansatz wäre in diesem Falle nur eine indirekte Es kann jedoch auch der Folge einer Störung irgend eines Organes. Es bleibt auch die Möglichkeit eines Fettumsatz direkt betroffen sein. vermehrten Fettansatzes infolge einer Störung im Abbau der Bausteine der Fette. Alle diese Möglichkeiten, die noch leicht vermehrt werden könnten, lassen es verständlich erscheinen, weshalb die bisherigen Stoffwecbseluntersuchungen noch wenig übereinstimmende Ergebnisse gezeitigt haben.-) Man wird versuchen müssen, diejenigen Fälle von Störungen im Fettstoffwechsel, die in ihren Erscheinungen gleichartig sind, auf ihre Ursache zu prüfen und umgekehrt, bei gleichen Organstörungen festzustellen, ob die Folgeerscheinungen sich entsprechen. Berücksichtigt man die Tatsache, daß die meisten Phosin phatide Glyzerin uiid Fettsäuren unter ihren Bausteinen beso drängt sich unmittelbar die Frage auf, ob die Fette keine Beziehungen zu Vertretern dieser Körperklasse haben. Wählen wir als Beispiel das Lezithin. Es enthält Glyzerin, Fettsäuren, Phosphorsäure und Cholin. Die Fettsäuren sind mit dem Glyzerin esterartig verbunden. Wir haben somit die gleiche Struktur, wie bei den Fetten, nur ist sitzen, das dritte Hydroxyl des Glyzerins esterartig mit Phosphorsäure verknüpft. An diese reiht sich noch das Cholin an. Phosphorsäurc steht dem Organismus zur Verfügung. Die Synthese von Glyzeryl-phosphorsäure dürfte der tierischen Zelle keine Schwierigkeiten bereiten. Es fragt sich nur, ob der ^) G. \g\. die Literatur bei A.Jaquef: Ergebnisse der Physiologie. 2. I. 553 (1903). c. Bergmann: Handb. -) Vgl. //. d. Biochemie. 4. II. 208 (15)10). Hausleiter: Zeitschr. f. experim. Path. 11. Ther. 17. 1 (1915). XVI. Vorlesung. 298 Organismus Fettsäuren umwandeln kann. Wir haben bereits erwähnt, daß beobachtet worden ist, daß in der Leber gesättigte Fettsäuren in ungesättigte übergeführt werden. ^j Es ist somit wohl möglich, daß die zur Synthese eines bestimmten Lezithins notwendigen Fettsäuren gebildet werden können. Es müßte der tierische Organismus nun nur noch die Fähigkeit besitzen, Cholin aufzubauen. Wir dürfen vielleicht die Frage schärfer fassen und fragen, ob die tierische Zelle Methylgruppen, z. B. tierische in Glykokoll = Aminoessigsäure einzuführen vermag: CHo COOH . ^\H GH.. . ^ I CH. COOH y'CHä Aminoessig- . CH., OH /CH3 j I CH3 ^ CH3 X ^OH ^OH Betain Cholin. säure=Gly kokoll Noch naheliegender ist die Annahme, daß das aus Phosphatiden gewonnene Oxyäthylamin zur Methylierung kommt. Dieses Amin könnte aus einer Aminosäure, und zwar aus Serin = y.-Amino-i-oxypropionsäure entstanden sein, und zwar durch Kohlensäureabspaltung-): GH., OH GH.. OH GH. OH /CH, . . . —> GH, N < ^g' I I GH . NH., —> I GH., . NH., \0H GOOH — GOo I Serin . Oxy-äthylamin Gholin. Wir werden bald erfahren, daß der tierische Organismus Methylgruppen -j, Azetyl-, Phenjiazetyl- und Benzoylgruppen an Aminogruppen anlagern kann. Es ist somit nicht ausgeschlossen, daß im tierischen Organismus Glykokoll in der eben dargestellten Weise durch Methylierung in Betain übergeführt wird. Dieses müßte dann allerdings noch zu Gholin reduziert werden): Seitdem wir wissen, daß der tierische Organismus aus ß-Oxybuttersäure durch Oxydation Azetessigsäure bildet, und diese durch Reduktion wieder in die erstere Verbindung verwandeln kann, erscheint ein solcher Reduktionsvorgang als nichts Ungewöhnliches. Da jedoch zurzeit gar keine Beweise für die Bildung von Gholin aus Glykokoll vorliegen, muß es selbstverständlich dahingestellt bleiben, ob der tierische Organismus sich Ghohn selbst darstellen kann. Wir heben das Problem der Bildung von Gholin im tierischen Organismus deshalb so sehr hervor, weil Stoflfwechselversuche vorliegen, die beweisen, daß manche Tiere Phosphatide auch dann in größerer Menge bilden können, wenn ihnen eine an solchen arme Nahrung zugeführt wird. So fütterte Fingerling »j ^) Vgl. auch //. S. liapcr: Journ. of Biol. Chem. 14. 117 (1913). Vgl. hierzu auch F. F. Nord: Biochem. Zeitschr. 95. 271 (1919). Vgl. hierzu F. Hofmeister : Archiv f. experimeutello Path. u. Tharm. 33. 198 ) Vgl. andere Möglichkeiten der Bildung von Cholin aus Aminosäuren. 1) ^) (1894). — W. Ilis: Ebenda. 22. 253 (1897). und Vorlesung XXII und XXX. ) Guatar Fingerlinfi : Biochem. Zeitschr. 38. 448 (1912). S. 255 Fette. Phosphatide. Sterine, 299 - Enten mit einer an organischen Phosphorverbindungen sehr armen Nahrung. legten ebenso viele Eier, wie Tiere, die normales Futter erhielten. Die Eier enthielten den normalen Gehalt an Phosphatiden bzw. organischen Phosphorverbindungen. Ale Callum^) ernährte ferner drei Hühner lange Zeit mit fast fettfreier Nahrung. In etwa SV-, Monaten legten die Tiere öl Eier. Diese enthielten etwas über O«';,, Phosphatide. Es mußte in diesen Versuchen nicht nur eine vollständige Synthese der Phosphatide aus Bestandteilen der Fette Fettsäuren und Glyzerin erfolgt sein, sondern es bildeten den eigentlichen Ausgangspunkt des Aufbaus unzweifelhaft die Kohlehydrate. Aus ihnen mußten, da der Fettgehalt der Nahrung bei weitem nicht ausreichte, zuerst Fette, bzw. ihre Bausteine gebildet werden. Schließlich konnte auch in zahlreichen Versuchen gezeigt werden, daß Hunde an Gewicht stark zunehmen, wenn ihnen in der Nahrung nur die Bausteine der Fette, der Kohlehydrate und Eiweißstoife verabreicht werden. -) Es wäre gesucht, wenn man annehmen wollte, daß in diesen Fällen neben den übrigen Stoffen nicht auch die Phosphatide vermehrt worden sind. Röhmann") fütterte Mäuse mit Kasein. Vitellin, Hühnereiweiß, Stärke, Fett. Salzen und Malz ohne Lezithin. Sie gediehen ganz gut. Auch junge Tiere ließen sich mit diesem Futter aufziehen. Wir kommen somit zum Schlüsse, daß Beobachtungen vorliegen, die beweisen, daß der tierische Organismus Phosphatide synthetisch bereiten kann. Er kann die einzelnen organischen Bausteine bilden und dann aus diesen und Phosphorsäure Phosphatide aufbauen. Es soll damit nicht gesagt sein, daß jeder tierische Organismus derartiger Synthesen fähig ist. ^j Mit der Möglichkeit, daß der eine oder andere Vorgang bei verschiedenen Tieren verschieden verläuft, müssen wir immer rechnen. Es unterliegt jedoch wohl kaum einem Zweifel, daß jeder tierische Organismus sich die verschiedenartigen Phosphatide seiner Zellen nach erfolgtem Abbau der in der Nahrung zugeführten Phosphatidarten aufbauen kann. Bereitet es schon sehr große Schwierigkeiten, den Fetten und ihren Bausteinen im Zellstoffwechsel zu folgen, so ist es zurzeit ganz unmöglich, etwas über das ^'erhalten der verschiedenen Phosphatide in den GeAveben auszusagen. Wir können nur in Analogie mit den Fetten annehmen, daß die vom Darm aufgenommenen Abbaustufen der Phosphatide entweder als solche zimi Transport kommen, oder aber es erfolgt schon in Darmwandzellen oder auch im Lymphgewebe eine Synthese. Auf alle Fälle wird die einzelne Körperzelle zum Aufbau ihrer eigenen Phosphatide von deren Bausteinen ausgehen müssen. Werden ihr diese nicht in freiem Zustand zugeführt, dann wird sie sich diese durch Abbau von Phosphatiden bereiten. Sicher festgestellt ist. daß jede einzelne Körperzelle lliosphatide als unentbehrliche Bestandteile enthält. Es gilt dies sowohl für die Pflanzen- als auch die Tierwelt. In besonders hervorragendem Maße Sie — — ^>) M E. y. Mc CaUiiiH, I. G. Halpin und A. H. Drescher: Journ. of. Bi(d. Chem. 13, 219 (1912). -) Emil Abderhalden : Zeitschr. f. physiol. Chemie. 77. 22 (1912). Synthese der Zellbausteine in Ptiauze und Tier. J. SpriuErer. Berlin 1912. ä) F. Röhmann: Biochem. Zeitschr. 64. 3u (1914). ) Vgl. dazu auch Tf. Dezani: Biocliimica a Terapia spuria. 11. 1 (1914). — — .S'. Stepp: Zeitschr. ^) Biol. 66. libO (1916). Vgl. hierzu z. B. T'. Jlenriqiies und 890 (1903). f. ('. Hansen: Skandin. Archiv f. l'hvsiol. 14. XVI. Yorlesuug. 300 die Phosphatide am Aufbau des gesamten Nervensystems beteiligt. So lange wir jedoch die einzelnen Vertreter dieser Körperklasse nicht genauer kennen, ist es unmöglich, ihre Beteiligung an bestimmten Zellvorgängen und vor allem die Art ihres Eingreifens in den Zell stotfWechsel durch genaue Versuche aul'zuklären. Hervorheben möchten wir noch die folgende wichtige Beobachtung. Das Gewebe des Zentralnervensystems besteht bekanntlich aus grauer und sind weißer Substanz. Beide enthalten Mineralstofie, Eiweißstoffe, Kohlehydrate, Phosphatide, Cholesterin und Cholesterinester. Es ist außerordentlich interessant, daß die Eiweißstoffe beider Substanzarten die gleichen Aminosäuren und diese in gleichen Mengenverhältnissen besitzen. ^) Trotzdem können in ihrer Struktur große L'nterschiede vorhanden sein. Immerhin weist die erwähnte Eeststellung auf eine große Ähnlichkeit der Proteine der grauen und weißen Substanz hin, und man gewinnt den Eindruck, als ob die mannigfaltigen Vertreter der Klasse der Phosphatide den morphologisch und funktionell verschiedenen Bestandteilen des Nervensystems ihre spezifischen Eigenschaften verliehen. Deir Umstand, daß der Infunktionsnahme mancher Nervenbahnen die Bildung der an Phosphatiden reichen Markscheide vorausgeht, weist auch auf ihre hohe Bedeutung hin. Die Phosphatide werden in den Zellen durch Fermente in ihre Bausteine zerlegt. Hierbei dürfte es zur Bildung von Glyzeryl-phosphorsäure, von Fettsäuren und Cholin kommen. Die erstere Verbindung wird dann weiter in Glyzerin und Phosphorsäure gespalten.-) Die stickstoffhaltige Base Cholin wird in allen Geweben ^\ in geringen und wechsehiden Mengen auch im Blutplasma) angetroffen. Sein Vorkommen ist zunächst mit dem Phosphatidstoffwechsel in mehr oder weniger direkten Zusammenhang zu bringen. Es ist beim Auf- und Abbau von Phosphatiden als Zwischenstation zu erwarten. Darüber hinaus kommt dem Cholin im Organismus noch eine besondere Bedeutung zu.^) Von besonders großem Interesse ist die Beobachtung von le Heux^)^ wonach Cholin die Darmbewegung anDas Cholin gehört regt. Es wirkt auf den Äuerhac]i?>Q\\Qn Plexus ein. offenbar in die Reihe jener Stoffe, die Inkretstoffe bzw. Endokretstoffe') genannt worden sind. Von besonderem Interesse ist die Beobachtung, daß wahrscheinlich nicht nur Cholin als solches, sondern esterartige Verbindungen mit Säuren als Erreger der Darmbewegung in Frage kommen. ') Emil Abderhalden iiud Arthur Weil: Zeitschr. f. pbysiol. Cheni. 81. 2U7 (1912): 83. 207 (1912). Paul Grosser und Jo.se/ Jlusler: Biochem. Zcitschr. 39. 1 (1912). W. Webster: Carl Schwarz und R. Lederer: Fflügers Archiv. 124. 353(1908). J. 'Gantrelet: C. r. de l'Ac. des Sciences. 148. The Biochem. .Toiirn. 4. 117 (1909j. Tosaku Kinoshita: G. Totani: Zeitschr. f. physiol. Chemie. 65. 86 (1910). 995 (1909). A. Lohniann: Zeitschr. f. Biologie. 56. 1 (1911). Pflü(fer% Archiv. 132. 606 (1910). M. Guggenheim und W. Löß'ler: Biochem. Zeitschr. 74. 208 (1917). -) ^) — — — — — — ' f. physiol. Chemie. 53. 31 (1907). B. (Jtto V. Eiirth und Carl Schivarz: l'/liif/er^ Archiv. 124. 427 (190(J). Carl Schnurz: Zcntralhl. f. Physiol. 23. Nr. 11 (1910)! «) J. W\ le Heux: Pfiiiger'i Archiv. 173. 8 (191H); 179. 177 (1920); 190. 280, Vgl. auch K. Arai: Ebenda. 193. 359 (1922): 195. 398 (1922). 301 (1921). Einil Abderhalden und E. Wertheimer: Ebenda. 194. 168 (1922j. ') Vgl. hiezu Emil Abderhalden: Pßügerü Archiv. 195. 482 (1922). — ) E. Letsche: Zeitschr. =) Vgl. z. — — B'ette. Phosphatide. Sterine. 3Q1^ Am wirksamsten ist der Essigsäurecliolinester.'l Als weniger wirksam erwies sich die Propionsäureverbindung-, und noch viel geringer ist die Wirkung des Es spricht vieles dafür, daß solche esterartige Verbindungen zwischen Cholin und Säuren durch Fermentvvirkung in der Darmwand gebildet werden.-) Erwähnt sei noch, daß Cholin bei intravenöser Zufuhr vorübergehende Blutdrucksenkung bewirkt.-^) Ob dieser Beobachtung eine Bedeutung für die Blutdruckregelung im Organisnms zukommt, steht dahin. Es ist auch beobachtet worden, daß bei Reizung von Muskeln ihr n-Buttersäureesters. Cholingehalt ansteigt.) Die Bausteine Glyzerin und Fettsäuren der Phosphatide werden in der gleichen Weise weiter verarbeitet, wie die entsprechenden Bestandteile der Fette. Die Phosphorsäure kann weiter Verwendung tinden, oder sie kommt durch die Nieren oder die Darm wand zur Ausscheidung. Über den weiteren Abbau des Cholins wissen wir noch nichts Genaues. Das im Harn in Spuren aufgefundene Trimethylamin») Cholin in direktem Zusammenhang: CH.> OH / CE, . steht vielleicht mit dem . ACH3 /CH3 N^CH3 NfCH3 VCH, \ OH \CH3 Cholin Trimethylamin. Es scheint übrigens im Harne nur zum geringsten Teil in freiem Zustande, sondern vielmehr in einer noch unbekannten Bindung vorzukommen.'') Ktitsriier') hat Basen aus dem Harn gewonnen, die Trimethylamin abspalten. Noch lückenhafter als unsere Kenntnisse über das Verhalten der Phosphatide im Zellstoffwechsel sind diejenigen über die Beteiligung der Sterine, und insbesondere des Cholesterins an bestimmten Zellvorgängen. Wir wissen nur so viel ganz sicher, daß bei Aufnahme von Pflanzennahrung Cholesterin in den tierischen Geweben erscheint, wenigstens konnte bis jetzt in keinem Falle ein Phytosterin jenseits des DarmDaraus geht herv^or, daß beim Pflanzenkanals aufgefunden werden. fresser das zugeführte Phytosterin in Cholesterin tibergeht. Reid Hunt: Jouru. Pharm. Therap. 7. 301 (1915). Vgl. hiezu J. W. Le Beu.r: Pflik/ers Archiv. 190. 260 (1921). ä) E. Geif/er und 0. Loeiri: Biochem. Zeitschr. 127. 174 (1922). ) Vgl. u. a. Sicah Vincent und W. Cramer: Journ. of Physiol. 30. 143 (1903). A. Lohmann: Pßiif/er^ Archiv. 118. 215 (1907): 128. 142 (1909). Sirale Vincent und Sheen: Journ. of Physiol. 29. 242 (1903). Emil Abderhalden und Franz Müller: Zeitschr. f. physiol. Chem'ie. 65. 420 (1910): 74. 253 (1910). Franz Müller: Pflügen Archiv. 134. 2H9 (1910). J. Pal: Zentralbl. f. Physiol. 24. (1910). L. B. Mendel und F. B. Underhill: Zentralbl. f. Physiol. 24. Nr. 7 (1910). Vgl. auch G. Modrakowski: Pflügers Archiv. 124. 601 (190«): 133. 291 (1910). L. Popielski: Ebenda. 128. 191, 222 (1909). Reid Hunt und R. de M. Tareau: Hygien. Laborat. Bulletin Nr. 73. 1 (1911). 5) Filippo de Filippi : Zeitschr. f. physiol. (heuiie. 49. 433. (1907). Vgl. auch Aldo Patta: Arch. d. Farmacol. sperim. 18' 284 (1914). 1) -) — — — — — — — — — — — «) Vgl. Takeda: Pfiügers Archiv. 129. 82 (1909). T. Kinoshita: Zentralbl. Physiol. 24. Nr. 17 (19iO). C. Doree und F. Golla: Biochem. Journ. 5. 306 (1911). ') Fr. Kutscher: Zeitschr. f. physiol. Chemie. 51. 457 (1907). Vgl. auch Kutscher und Lohmann: Ebenda. 48. 422 (1906): 49. 81 (1906). f. — — X\'I. Vorlesung. 3Q2 gleiche ist natürlich beim Omnivoren der Fall, wenn er Phytosterine aufnimmt. Das Cholesterin der Fleischnahrung braucht nicht umgewandelt zu werden, denn bis jetzt ist bei den höheren Tieren nur ein Zoosterin. Über das Verhalten des nämlich das Cholesterin, beobachtet worden. Cholesterins im Darmkanal, seine Resorption und seinen Transport nach den Gew^eben ist uns nichts Sicheres bekannt. Da nach dem jetzigen Stand unserer Kenntnisse kein Anhaltspunkt dafür vorliegt, daß der tierische Organismus Cholesterin aus einer anderen Gruppe von Verbindungen synthetisch bereiten kann, müssen wir annehmen; daß das mit der Nahrung aufgenommene Sterin zur Resorption kommt und die einzige Quelle für das Cholesterin des Organismus darstellt, i) Die Galle dürfte bei der Aufnahme der Sterine als Lösungsmittel eine bedeutsame Rolle spielen. Cholesterinfettsäureverbindungen werden wahrscheinlich im Darme in ihre Bausteine zerlegt. Bemerkt sei noch, daß manche Beobachtungen dafür sprechen, daß die Nebennieren direkte Beziehungen zum Cholesterinstoffwechsel haben, doch reichen die Befunde noch nicht aus, um bestimmte Schlüsse zu Sie sollen vor allem bei der Bildung von Cholesterinfettziehen.-) säureester eine Rolle spielen, doch darf als erwiesen angesehen werden, daß andere Gew-ebe diese Synthese auch vollziehen können, ja es fehlt nicht an B^orschern, die der Meinung sind, daß die Nebennieren die Cholesterinester nicht selbst bilden, sondern diese auf dem Blutwege zugeführt erhalten und speichern. 3) Auch die Corpora lutea sollen im Cholesterinstoifwechsel eine besondere Stellung einnehmen.) Jede einzelne Zelle besitzt Sterine. Es ist auffallend,^ daß in der Wirbeltierreihe nur ein Sterin anzutreffen ist, nämlich das Cholesterin. Es besitzt somit keinen arteigenen Charakter. Es steht damit mit dem Glykogen und auch mit manchem Reservefett in einer Linie. Durch die Das der Literatur über das Auftreten von großen Cholesterin') Manche Angaben mensren unter pathologischen Bedingungen eröttueu die Möglichkeit, daß der tierische Organismus Cholesterin aus irgend welchen Verbindungen bereiten kann. Dieser besonders von Ädrien Grigauf (Le cycle de la cholesterinemie, vgl. Zitat) gezogene Schluß ist jedoch so lange als verfrüht zu betrachten, als quantitative Bestimmungen über die mit der Nahrung zugeführten Sterine, den Gehalt des Organismus an Cholesterin und endlich die Ausscheidung dieser Verbindung nicht vorliegen. Zu bedenken ist vor allem, daß bis jetzt kein Beweis dafür vorliegt, daß der tierische Organismus der Ringbildung fähig ist. Es müßte also für die Cholesterinbildung schon ein Material in Frage kommen, daß zyklisch gebaut ist. Einstweilen ist ein solches unbekannt. Vgl. auch H. A. Klein: Ebenda. 29. 46ö Hugo Pribram: Biochem. Zeitschr. 1. 413 (19üG). (1910) und vor allem die zahlreichen Arbeiten von Gardner und seinen Mitarbeitern über die Bedeutung des Cholesterins für die tierische Zelle. Vgl. z. B. Charles Doree G. W. Ellis und und ./. A. Gardner: Procoed. of the Royal Soc. 80. 227 (1908). J. A. Gardner und P. E. Gardner: Ebenda. 84. 461 (1912); 85. 38ö (1912). ./. A. I'. E. Lander: Biochem. Lander: Proeeed. Roy. Soc. Lander. Soc. B. 87. 229 (1914). Dezani S. Lesani: Archiv, d. Farmacol. sperim. 17. 4 (1914). I. 9. 78 (1915). A. Chanffard, Gug Laroche und A. Grigauf: und F. Catoretti: Ebenda. 19. 1 (1914). J. Gardner und /'. W. Fox: Proeeed. of royal Soc. B. 92. Ann. de med. 8. 149 (1920). — — — — — — — & — — D. KUnkerf: Berliner klin. Wochenschr. 50. 820 (1913), hier tindet sich viel Literatur. Alhrecht und Weltmann: Wiener klin. Wochenschr. 483 (1911). — K. Katvainura: Die Cholesterinverfcttung. Gustav Fischer. Jena 1911. — L. Aschof: Wiener experiment. (1911). — L. Wacker und W. Uueck: Archiv klin. Wochenschr. Nr. 358 (1921). ') 1(5 f. Path. u. Pharm. 71. 373 (1913). ') Vgl. zu diesem Probleme M. Landau und J. W. Mc Nee: Beiträge zur pathol. Anat. und zur allg. Pathol. 58. 667 (1914). ) Adrien Grigaut: Lo cycle de la cholesteriuömie. G. Steinheil. Paris 1913. Fette. Phosphatide. Sterine. 305 Kuppelung- mit Fettsäuren können jedoch eigenartige \'erbin düngen hervorgehen, doch sind die Möglichkeiten verschiedener Verbindungen dadurch stark beschränkt, daß nur eine Hydroxylgruppe zur Bindung mit Fettsäuren zur Verfügung steht. Allerdings könnten wieder Verschiedenheiten dadurch bedingt sein, daii die Cholesterinester unter sich in ganz verschiedenem Mischungsverhältnis vorkommen und vielleicht auch mit den übrigen Fetten Einstv^eilen kennen wir in verschiedenartigstem Gemenge sich finden. allerdings nur wenige Cholesterinester als Bestandteile tierischer Zellen und des Blutplasmas. Bemerkenswert ist der schwankende Gehalt selbst paariger Organe an Cholesterin und besonders an Cholesterinester. Im Blutplasma soll das Verhältnis des freien Cholesterins zu Cholesterinester unter normalen Verhältnissen konstant sein.i) Auch die roten Blutkörperchen haben einen für jede Tierart charakteristischen, konstanten Gehalt an freiem Cholesterin.-) Manche Forscher sind der Ansicht, daß das Cholesterin als ein Stofiwechselendprodukt der Zellen aufzufassen ist. Es soll als solches hauptsächlich durch die Galle zur Ausscheidung gelangen. Diese Ansicht ist ohne Zweifel ganz unhaltbar und kann nur za einer Zeit entstanden sein, in der der Bau des Cholesterins unbekannt war. Jetzt wissen wir, daß es sehr wenig Wahrscheinlichkeit für sich hat, daß Cholesterin mit irgend einer Abbaustufe der Fette, Kohlehydrate und Eiweißstoffe in direktem steht. Das Cholesterin hat vielmehr höchst wahrscheinlich einen für sich abgeschlossenen Stoffwechsel. Sein Auftreten in jeder einzelnen Zelle, seine Beteiligung am Aufbau besonderer Fette, die Umwandlung der Phytosterine in das Zoosterin Cholesterin, das sind alles Momente, die es als ganz ausgeschlossen erscheinen lassen, daß dem Cholesterin im Zellstoffwechsel nicht eine hohe Bedeutung zukommt. Von größter Bedeutung ist der S. 230 erwähnte enge Zusammenhang des Cholesterins und gewiß der Sterine überhaupt mit den Paarungen der Gallensäuren: Cholsäure, Desoxycholsäure und Lithochol säure. Damit ist mit großer Wahrscheinlichkeit ein Weg des Abbaues von Cholesterin aufgewiesen. 3) Das Cholesterin ist in seiner Verbindung mit Fettsäuren ohne Zweifel als Zellbaustein zu betrachten. Das freie Cholesterin hingegen dürfte als ein wichtiges Agens in jeder einzelnen Zelle in irgend welche noch unbekannte Vorgänge eingreifen. An Vermutungen über die Zusammenhang Rolle des Cholesterins im Zellstoffwechsel fehlt es nicht, doch sind diese meistens wenig begründet. Meist wird das Cholesterin in seinen Funktionen mit den Phosphatiden in Zusammenhang gebracht. Vielleicht gibt die folgende Beobachtung Hinweise auf die Rolle, die das Cholesterin im tierischen Organismus spielt. Um die folgenden Versuche zu verstehen, müssen wir kurz vorausschicken, daß das Blut Plasma — eine eiweiß- und salzreiche Flüssigkeit Vgl. W. B. Bloor und A. Knudson: .T. of biol. ehem. 29. 7 (1917)Th. E. Iless-Thaijsen: Biochem. Zeitschr. 62. 115 (1914). ') Der Versach durch Verfütterung von Cholesterin den Gehalt der Galle an Gallensäuren zu steigern hatte keinen Erfolg. [\'gl. 3/. G. Foster C. W. llooper und G. H. Whipple: The J. of biol. Chem. 38. 421 (1919).] Das scldießt natürlich nicht aus, daß trotzdem die erwähnten Gallensäurepaarlinge von Cholesterin abstammen. 1) -) , 304 ^^ I- Vorlesimg. — — \ — Zellen besitzt. Zu den letzteren und außerdem Formelemente gehören die roten Blutkörperchen. Sie enthalten einen roten Farbstoff. Blutfarbstoff genannt. Wir können ihn in mannigfacher Weise von den Blutkörperchen abtrennen. Einmal können wir die Blutkörperchen durch verschiedene Maßnahmen zerstören. Wir kennen jedoch auch Mittel, die, ohne das Stroma der Zelle sichtbar zu verändern, schon in Spuren bewirken, daß die roten Blutkörperchen ihren Farbstoff abgeben. Man nennt diesen Vorgang Hämolyse und die Stoffe, die ihn bewirken, Hämol}"sine. Hämolysierende Wirkungen hat z.B. das Gift der Brillenschlange. Kobragift genannt. Hämolysine sind ferner aus Kreuz.^pinnen (Arachnolysin), aus Bakterienkulturen usw. gewonnen worden. Besonders wichtig ist, daß auch im Pflanzenreich hämolytisch wirkende Stoffe vorkommen. Dahin gehören die in einer Reihe von Pflanzen aufgefundenen Saponine. Sie gehören zu den Glukosiden.^) gift, Geben wir z. B. zu Rinderblut eine ganz geringe Menge von Kobradann erkennen wir bald das Eintreten der Hämolyse daran, daß das undurchsichtige Blut sich allmählich in eine durchsichtige, rote Entfernen Avir aus Blut das Plasma durch Zentri- vorher Lösung verwandelt. — fugieren die spezifisch schwereren Formelemente setzen sich am Boden des Zentrifugiergefäßes ab, das Plasma kann dann leicht abgehoben werden und waschen wir die roten Blutkörperchen mit isosmotischer Kochsalzlösung vollständig frei von anhaftendem Plasma, dann erhalten wir beim Zusatz von Kol)ragift zu den in isotonischer Kochsalzlösung suspendierten roten Blutzellen keine Hämolyse. Geben wir nunmehr Plasma zu der Suspension der roten Blutkörperchen hinzu, dann erhalten wir sofort Hämolyse.-) An Stelle von Plasma können wir nun auch, wie Ki/es"^) zeigte. Lezithin verwenden. Dieses wird unter der Wirkung des Kobragiftes in Die hämolysierende ein hämolytisch wirksames Produkt umgewandelt. Verbindung ist Lysozithin genannt^) und als Anhydrid des Monopalmityllezithins erkannt worden. s) Das Kobragift spaltet aus dem Lezithin Ölsäure ab. Es wirkt wie ein Ferment und enthält offenbar eine Phosphatidase, bzw. Lezithase. Der Abbau vollzieht sich in zwei Phasen. Zuerst entsteht das erwähnte stark hämolysierende Produkt und darauf eine hämolytisch unwirksame Substanz. Kalksalze begünstigen die fermentative Spaltung. 6) Interessanterweise kann man die Wirkung des Lezithins durch Zusatz einer Cholesterinemulsion aufheben. Das Cholesterin wirkt in gewissem Sinne als Antagonist des Lezithins. Wichtig ist, daß das — , Cholesterin seine Wirksamkeit einbüßt, wenn seine Hydroxylgruppe besetzt ist.') Ein Cholesterinester ist z. ß. unwirksam. •) ^) Vgl. S. 72. Die Sapouinhäiiiolyse wird durch Serum umgekehrt goliemmt. — Freslon S. FJexner und H. Noquchi: Jouru. of experim. Med. 6. Nr. 3 (1912). Berliner kliu. Wochenschr. Nr. 38 39 (1902); Zeitschr. f. physinl. Chem. 41. 273 ^) Kyes: — P. Kyes und Hans Sachs: Berliner (1904). Vgl. auch klin. 2—4 (1903). — Wochenschr. Nr. Ransom: Deutsche med. Wochenschr. 1901. ) C. Delezenne und Ledebt: Compt. rend. 155. 1101 (1903). ) C. Delezenne und E. Fourneau: Bull.Soc.Chim.de France. [4. \| B. Kndicke und H. Sachs: Biochem. Zeitschr. 76. 359 (1917). Vgl. iiierzu W. Hausmann: Ho/meisfers Beitr. 6. 517 (1905). halden und Le Counf: Z. f. exnerini. Path. u. Ther. 2. 199 (19fJ5). 8) '') 15.421 (1914). — Emil Abder- Fette. Phosphatide. Sterine. 3Q5 Die hemmende Wirkung des Cholesterins im erwähnten Versuche kann verschiedene Ursachen haben. Forschungen von Windaaa^) führten zu der Vermutung, daß bei der Beeinflussung der Kobragiftwirkung durch Sterine ähnliche Verhältnisse vorliegen, wie bei der Hemmung der Hämolyse mittelst Saponin durch die gleichen Verbindungen. Windaus konnte nämlich zeigen, daß beim Zusammenbringen von Cholesterin und Digieinem Saponin eine Verbindung auftritt. Es entsteht tonin Digitonin-cholesterid. Auch andere Saponine, wie Solanin und — — Zy kl am in, ergeben Cholesterinester mit Cholesterin wohlcharakterisierte dagegen binden sich Verbindungen. nicht. mit Saponinen Diese Beobachtungen erööhen die Möglichkeit, daß sich aus dem Kobragift, das ohne Zweifel ein Gemisch verschiedener Giftkomponenten darstellt, das bei der Erzeugung der Hämolyse wirksame Prinzip mittels Cholesterin als Cholesterid abtrennen läßt. Wir hätten uns nach diesen Ergebnissen die Wirkung des Cholesterins in der Weise vorzustellen, daß es das die Hämolyse bewirkende Produkt durch Bindung festlegt und sein Molekül dabei so verändert, daß es unwirksam wird. Bewiesen ist diese Annahme nicht. Es ist ganz gut möglich, daß das Cholesterin rein physikalisch wirkt. So ist z. B. seine hemmende Wirkung auf die Kobralezithidhämolyse auf eine stabile Emulgierung des Cholesterins zurückgeführt worden. Es ist von großem Interesse, daß nichtjede Blutkörperchenart und nicht alle Blutkörperchen der gleichen Art immer im gleichen Maße widerstandsfähig + Kobragift sind. Man könnte sich folgenoder empfindlich gegen Lezithin des Bild von dieser Erscheinung machen. Das rote Blutkörperchen enthält freies Cholesterin und Cholesterinester. Ferner dürfte neben freiem Phosphatid freiem Lezithin auch gebundenes zugegen sein. Verfügt die Zelle in einem gegebenen Moment über viel freies Cholesterin und wenig freies Lezithin, dann wird mehr Lezithin zugefügt w^erden müssen, um die Wirkung des Cholesterins zu überbieten. Umgekehrt wird beim Zusatz von ganz wenig Lezithin Hämolyse eintreten, wenn die Zelle wenig freies Cholesterin zur Verfügung hat. Es ist auch beobachtet worden, daß gewaschene rote Blutkörperchen ohne jeden Zusatz von Lezithin Hämolyse zeigten, wenn sie mit Kobragift in Berührung kamen. Man kann auf diesem Wege geradezu die Widerstandsfähigkeit von roten Blutkörperchen ..austitrieren". 2) Es ist möglich, daß innerhalb des Organismus Cholesterin und Phosphatide in ähnlicher Weise zusammenwirken, wie wir es bei dem besprochenen Reagenzglasversuch gesehen haben. Bald könnte die Zelle Cholesterinester verseifen und das freie Cholesterin zur Wirkung bringen, bald könnte solches gebunden und so seiner Wirkung beraubt werden. Auch di(^ Phosphatide werden vielleicht von der Zelle in gleicher Weise bald aktiviert, bald in den unwirksamen Zustand überführt. Die Zelle wird vielleiclit auf diese Art und Weise vor manchen Schädigungen, die ihr drohen. be\vahrt. — — Immer mehr bricht sich die Anschauung Bahn, daß Cholesterin. Cholesterinester, Phosphatide, Fette oder auch nur einzelne dieser Verbindungen, wie Cholesterin und Phosphatide, einen tiefgehenden Einfluß A. Windaus: Ber. d. Deutscheu Chera. Gesellsch. 42. 238 (1909). Vgl. hierzu u. a. Emil Abderhalden und We7-ney Bucital: Arch. f. wisseusch. prakt. Tierheilk. 37. H. 3 (1911). Emil Abderhalden und Ar f hur Weil: Khenila. ') ^) u. — 38. 1 (1912). Abderhalden, Physiologische Chemie. I.Teil, 5. Anfl. 20 X\I. Vorlesung. 306 auf den physikalisch-chemischen Zustand der Zellinhaltsstoft'e und vor allem auch auf die Blutbestandteile und insbesondere auf die Proteine besitzen. i) Eine Änderung in den normalen Wechselbeziehungen aller dieser Stoße zueinander, die durch ein bestimmtes Mengenverhältnis derselben bedingt sind macht sich in mannigfachen Störungen geltend. Wir werden später XII) auf diese wichtigen Funktionen bestimmter (vo-1. Band 2, Vorlesung V— Zell- und Blutinhaltsstofte noch zurückkommen. Wir kennen auch eine Überflutung des Blutplasmas mit Cholesterin und wissen, daß Cholesterinester eine Hypercholesterinoplasmie primär oder sekundär sich in größeren Mengen in veränderten Geweben ablagern können. So hat man bei Diabetes melitus ist noch fraglich wiederholt eine Hypercholesterinoplasmie beobachtet. Auch während der Schwangerschaft und bei bestimmten Formen der Erkrankung der Nieren-) ist ein Ansteigen des Gehaltes des Blutplasmas an Cholesterin beobachtet worden.") L. Wacker und JV. Hueck^) berichten, daß durch vermehrte Zufuhr von Cholesterin experimentell eine Atherosklerose o-enannte Veränderung der Arterienwand herbeigeführt werden kann. Ferner zeigte es sich, daß auch die sogenannten Xanthome (Xanthelasma) in großer Menge eigenartige, gelb gefärbte Geschwülste der Haut — — — — — Cholesterinester enthalten. &) Zahllos sind die Funktionen, die man den Phosphathiden im Zellund Gesamtstoffwechsel zugeschrieben hat.«) Der Befund von Eisen') in Phosphatiden hat zu der Annahme geführt, daß sie bei den Oxydationsvorgängen eine wichtige Rolle spielen. Man hat sie auch als Fermente und Aktivatoren von solchen angesprochen. Ferner hat man vermutet, daß sie imstande seien, Fermentwirkungen zu hemmen oder gar zu unterdrücken. ^) Eine große Rolle spielen die Phosphatide und die Sterine in der Immunität sforschung. Es ist hier nicht der Ort, auf diese BeSie werden ohne Zweifel erst dann eine sichere funde einzugehen. '•) Es sei z. B. veiwieseu auf B. Brmkman und E. ran Dam: Biochem. Zeitscbr. Fritz R. Brinkman und H. WastI Ebenda. 124. 25 (1921). Hier müßte auch die gesamte neuere Literatur Eichholz: Ebenda. 128. 310 (1922). über das Wesen des Lues-Nachweises nach Wassermann, Sachs - Georgi, Meinicke, Dold usw. angeschlossen werden, denn es stellt sicli immer mehr heraus, daß Zustaudsänderungeu von Plasmaproteinen lieim Zustandekommen der einzelnen Reak) 108. 35. 52, 61 (1920). — — — : tionen Vorbedingung sindl ) Vgl. u. a. Ä. Hahn u. E. Wolff: Z. f. kliu. Med. 92. 393 (1921). Adrian (rri(jaut: Le cycle de la cholesterincmie. G. Steinheil. Paris 1913. Vgl. auch W. li. nioor: .1. of biol. Chem. 49. 201 (1921). — P. Sisfo: Ann. di clin. — ) med. 11. 14 (1921). ) L. Wacker und W. Hueck: Münchener med. Wochenschr. 60. 2Ü97 (1913). — Vgl. auch Kaethe Detrey: Arch. of internal. Med. 17. 757 (1916). — ./. Prings') F. Pinkus und C. Pick: Deutsche med. Wochenschr. 1'426 (1908). Vgl. ferner: M. Verse: Zie(/lera Beitr. 52. Dezember Ebenda. 2445 (1908). F. Eosenthai und P. Braunisch: Z. f. klin. Med. 92. 429 (1921). (1911). P. Sisto: Gioru. di diu. med. 2. 210 (1921). der sog. Nut ramine ) Wir kommen auf die Frage noch bei^der Bespreciuing hciiii: — — — zurück. ') Vgl. W. Glikin: Ber. d. Deutschen Chem. (ies. 41. 910 (1908). — ilf. Gonner- mann: Biochem. Zeitschr. 95. 286 (1919).—- H. van den Brrf/h, /'. Müller und J. firoekZeit.schr. 108. 279 (1920). Vgl. hierzu .1/. Siegfried: Biochem. Zeitschr. 86. 98 (1918). Irar Bang: Chemie ) Vgl. hierzu die umfassende Übersicht bei der Lipoide. .1. F. Bergmann. Wiosb;ulen 1911. ntei/er: Biochem. ) und Biochemie Fette. Phosphatide. Sterine. 3QY Grundlage erhalten, wenn wir die Phosphatide besser kennen. Solange wir noch mit dem Sammelnamen „Lipoide" operieren müssen, und wir nicht genau wissen, was er eigentlich alles umfaßt, besteht die Gefahr, daß durchaus verschiedene Wirkungen auf eine bestimmte Gruppe von Verbindungen zurückgeführt werden. Beinahe alles, was man in der Zelle nicht genau definieren kann, wird in dem Sammelnamen „Lipoide" untergebracht, sofern das Produkt in den Löslichkeitsverhältnissen einigermaßen mit denen der Fette übereinstimmt. Wenn wir in der Literatur der Angabe begegnen, daß jemand mit Lezithinen gearbeitet und damit bestimmte Beobachtungen gemacht hat, dann können wir nie wissen, ob der Betreffende wirklich in Händen hatte, weil diese \'erbindung Dazu kommt, daß P^ette, Phosphatide und Lezithin so leicht veränderlich ist. Sterinester alle mög- Verbindungen mitlösen. Man findet z. B. die Phosphatide nie Es kann nicht genug betont werden, daß die Phosphatide aschefrei. ein ganz heterogenes Gemisch von Verbindungen verschiedener Art darstellen, und daß vor allem der Begriff „Lipoide" einen zurzeit chemisch gar nicht definierbaren Sammelbegriff für alle Verbindungen mit Löslichkeitsverhältnissen, die denen der Fette mehr oder weniger entsprechen, darstellt. Den Phosphatiden ist im \'erein mit den Fetten, Sterinen und Sterinestern eine besondere Bedeutung als Lösungsmittel für bestimmte Verbindungen zugeschrieben worden. i) Wir werden auf diesen Punkt später zurückkommen, wenn wir die Rolle der anorganischen Stoffe und insbesondere der Ionen und ferner der Kolloide im Zellstoft'vvechsel kennen gelernt lichen haben. Werfen wir zum Schluß noch einmal einen Blick auf das Verhalten der Fette, Phosphatide und Sterine im tierischen Organismus Wir sahen, daß er darauf eingerichtet ist. die zusammengesetzten Verbindungen im Darmkanal in ihre Bausteine zu zerlegen. Dieser Abbau ist notwendig, um zu Produkten zu gelangen, die in Wasser I löslich und diffundierbar sind. Gleichzeitig wird ferner der tierischen Zelle dem Gemisch der Bausteine Fette und Phosphatide zu bilden, die dem Bau der einzelnen Zellen angepaßt sind. Die die Möglichkeit gegeben, ans Fette gehen in Fettsäuren bzw. Seifen und Alkohol über, aus den Phosphatiden bilden sich ihre Bausteine, Fettsäuren bzw. Seifen, Glyzerin, Phosphorsäure und Cholin oder andere stickstoff'haltige Basen, sofern solche am Aufbau besonderer Vertreter dieser Körperklasse beteiligt sind. Dabei bleibt die Möglichkeit durchaus offen, daß die Hydrol3"se nicht in jedem Falle eine vollständige ist, vielmehr auch zusammengesetzte Bruchstücke, wie Glyzeryl-phosphorsäure und Cholin, in Verbindung mit Phosphorsäure zur Resorption gelangen. Die Sterinester werden höchstwahrscheinlich auch verseift. Die resorbierten Bausteine der Fette werden teilweise schon in der Darmwand zusammengefügt. Wie umfassend diese Synthese ist, und ob sie wirklich ausschließlich in den Darmepithelien erfolgt, ist noch nicht festgestellt. Jedenfalls entsteht nicht immer die dem aufgenommenen Es sind nämlich Beobachtungen bekannt. Fett entsprechende Verbindung. — ) Hans llorftt Meyer: Aicli. f. experim. t'ath. u. Thannak. 42. 10!) (lb'9'J). E. Orerfon: Vicrteljuhresschr. d. naturf. Gesellsch. in Züricli. 44. 88. (1894); Studium über die Xarkose. Zugleich eiu Beitrag zur allgemeiiieu Pharmakologie. Gustav Fischi r. Jena 1901. J'ßüfferH Archiv. 92. 115 (1902). — 20' XVI. Vorlesung. ;^()g die beweisen, daß schon kurz nach der stattgehabten Resorption Fettarten auftreten können, als die, die aufgenommen wurden. andere Ferner müssen wir annehmen, daß resorbiertes Phytosterin sehr bald in Cholesterin übergeführt wird, wenigstens konnte bis jetzt in tierischen Geweben kein solches aufgefunden werden. Es kann jenseits des Darmes auch dasjenige Fett mit der Nahrung zugeführt wurde, ja es kann auch als sich bilden, das solches zur Ab- lagerung kommen. Eine Beeinflussung des Charakters der tierischen Zellen tindet jedoch dadurch nicht statt, denn das an ihrem Bau beteiligte Fett Jede Zellart ist in seinem Aufbau vom aufgenommenen Fett unabhängig. baut sich eigenes Fett und bereitet eigene, charakteristische Gemische, wobei offenbar auch das Cholesterin, seine Ester und ferner Phosphatide mit einbezogen werden. Die Fette schlagen sicher zu einem großen Teil den Lymphweg ein. Nach neueren Beobachtungen wird auch Fett von den Pfortaderwurzeln aufgenommen. Schließlich gelangen die Fette, das Cholesterin und die Phosphatide oder ihre Bausteine ins Blut und werden dann auf noch nicht bekannte Weise schließlich den Körperzellen übermittelt. Diese verwenden die Bausteine der Fette in der mannigfaltigsten Weise. Sie werden entweder stufenweise abgebaut, oder sie dienen zur Synthese von Fett und Besondere Zellarten speichern Fett und bilden mächtige Phosphatiden. Depots, deren Inhalt zur Verfügung gestellt wird, wenn irgendwo Bedarf ist. Das Fett dieser Speicher rührt nicht nur von mit der Nahrung zugeführten Fetten her. sondern auch von umgewandelten Kohlehydraten. Wir haben immer wieder betont, daß unsere Kenntnisse des FettstofiVechsels noch recht dürftige sind. Einzig die Feststellung von Knoop, wonach die gesättigten Fettsäuren in charakteristischer Weise durch Oxydation am ß-kohlenstoffatom und Verlust der beiden endständigen Kohlenstoftatome lozw. allgemeiner ausgedrückt, durch Abspaltung einer geraden Anzahl von Kohlenstoffatomen verkürzt werden, ferner die sichergestellte Beziehung der Fettsäuren zu den Azetonkörpern und der Nachweis, daß Glyzerin in Glukose übergehen kann, haben das Dunkel, das noch über den einzelnen Vorgängen beim Ab-, Um- und Aufbau der Fette und ihrer Bausteine lagert, erhellt. Welch umfassende chemische Vorgänge die Körperzellen bei der Bildung von Fetten vollziehen ktinnen, zeigt am besten die Betrachtung der Zusammensetzung einiger fetthaltiger Sekrete. Wir haben bereits gesehen, daß die Bürzeldrüse der Vögel ein Sekret bildet, das ein Fett enthält, an dessen Aufbau Oktadezylalkohol als Alkoholkomponente beteiligt ist.i) Daneben finden sich auch Glyzerinfette. Das Bürzeldrüsensekret dient zur Einfettung des Gefieders, wodurch es vor der Benetzung mit Wasser geschützt wird. In der Haut finden sich verschiedenartige Drüschen, die besondere Sekrete ab.sondern. Sie enthalten alle Fett und scheinen die gemeinsame Funktion zu haben, die Haut durch Einfetten geschmeidig zu erhalten. Daneljen mögen auch noch andere Funktionen vorhanden sein. Ein solches Fettgemisch haben wir bereits wiederholt erwähnt, nämlich das Wollfett, auch Lanolin genannt.-; Man hielt es zunächst für eine ziemlich ein1) •') reich: Röhmann: Hofmeistern Beitr. 5. 110 (1904). — \gl auch S. 220. Hartmann: Über den Fettuachweis in der Schafwolle. Göttingeu. 1868. — TAehF. Berliner klin. Wochenschr. 7(il (1885); Arch. f. (Auat. u.) Physiol. 363 (1890). 309 Fette. Phosphatide. Sterine. heitliche Substanz. Jetzt wissen wir, daß der Name Lanolin nur ein Sanirael- name für sehr viele Komponenten ist. Dani/städfer und Li fschütz'^) geben als Bestandteile des Wollfettes an: Zerylalkohol, C.-, Hgs OH, Karnaubylalkohol, C..^ H^cj UH, daneben . . noch Laktone verschiedener Art zu finden sein. Ferner sind aus der Reihe der Sterine isoliert worden: Cholesterin, Isocholesterin"^) und ferner sog'. Oxycholesterine. Endlich sind zahlreiche Fettsäuren als Bestandteile des Lanolins bzw^ der in diesem vorkommenden Fette beschrieben worden. Von gesättigten Fettsäuren werden angeführt: sollen Lanozerinsäure, C30 Hr.o O4, Lanopalminsäure, Cig H32 O-j, MyristinCOOH, Karnaubasäure, C.23 Hi^ .COOH, Zerotinsäure, C.>6H53 COOH, Kapronsäure, C5 Hu COOH. Daneben sind noch ungesättigte Fettsäuren beobachtet worden. Schon diese Zusammenstellung säure, C13 H.^- . . . zeigt, welch kompliziertes Gemisch von Zellen der Hautdrüsen bereiten können. Angehitrigen der Fettreihe die Besondere fetthaltige Sekrete liefern ferner die Ohrschmalzdrüsen, Talgdrüsen und die Meihoni^ch^n Drüsen. Auch das Smegma ist ein fetthaltiges Sekret. Je mehr man die Funktion aller dieser Drüsen und Drüschen studiert hat, um so mehr hat man eingesehen, daß die alte Vorstellung, wonach ihre Sekrete dadurch zustande kommen sollten, die sich vielmehr, wie vor P^s handelt verfallen, unrichtig ist. allem Plafo für die Talgdrüsen feststellen konnte, um einen richtigen Sekretionsvorgang. :^) Das einzelne Sekret bereitet der Untersuchung große Schwierigkeiten, witW es schwer und zum Teil zurzeit überhaupt nicht in reinem Znstand erhalten werden kann. Schließlich wollen wir noch an jenes merkwürdige, fettreiche Sekret, Vernix caseosa genannt, erinnern, daß Zellen dem die Haut des Fötus überzogen ist. Ein sehr schönes Beispiel für die Tatsache, daß die Körperzellen eigene Fettarten bilden, liefern endlich die Zellen der Milchdrüse. Das Fett der Milch ist normalerweise ein ganz charakteristisches. Der Umstand, daß durch Zufuhr größerer Mengen von bestimmten Nahrungsfetten eine Ausscheidung solcher Fette durch die Milchdrüse erreicht werden kann, ist von keiner Bedeutung für die Auffassung der Entstehung der Bestandteile der Milch in den Milchdrüsenzellen. In der Butter aus Kuhmilch hat man hauptsächlich Triglyzeride beobachtet, an deren Aufbau Stea rin-, mit Palmitin- und Ölsäure, ferner Myristin-, Laurin- und Arachinsäure Man hat ferner noch Buttersäure, Kapron-. Kapryl-. Kaprinsäure, ferner Essigsäure und auch Ameisensäure beobachtet. Die weitere Forschung wird ohne Zweifel noch an zahlreichen Bei- beteiligt sind. zeigen können, daß jede einzelne Zellart sich eigene Fette und Phosphatide und vor allem auch charakteristische Gemische dieser Verbindungen bereitet. Gleichzeitig wird sicher auch festgestellt werden, in welcher Art die einzelnen Baumaterialien umgewandelt werden. spielen 1) L. Darmstädter und ./. Lifschüfz: Ber. d. Deutscheu Chem. Gesellsch. 28. 3133 Vgl. auch P. G. Unna: (1895); 29. 618. 1474 und 2890 (1896); 31. 97, 1112 (1898). P. G. Unna und ./. Lifschütz: Monatshefte d. prakt. Derniatol. Ebenda. 45. 1 (1907). Ä. Bitschke und P. G. Unna: Biochem. Zeitschr. 20. 469 (1909). 4.^. 334 (1907). Arthur Fraenkel: Berliner klin. Wochensclir. Xr. 12 (1905). -) Xach Röhmann [Biochem. Zeitschr. 77. 298 (1911)J fraglich. ') Pluto: Verband), d. Deutschen dermatol. (iesellscb. Breslau. 182 (1901). — — — — Vorlesung XVII. Eiweißstoffe und ihre Bausteine. Aminosäuren. Die Eiweißstoffel), auch Proteine genannt, stellen hochmolekulare, zusammengesetzte Verbindungen dar, an deren Aufbau stickstoffhaltige Bausteine beteiligt sind. Bis jetzt haben wir nur bei den Phosphatiden Stickstoff als regelmäßigen Bestandteil aller Angehörigen dieser Klasse von A'erl)indungen angetroffen. Er fand sich jedoch nicht in allen BauFerner haben steinen, sondern nur im Cholin bzw. einer anderen Base. interessierte Von diesen gelernt. kennen Kohlehydrate wir stickstoffhaltige uns ganz besonders das Glukosamin, weil es Beziehungen zu den Bausteinen der Eiweißkörper aufweist. Bei den Proteinen enthalten Scämtlidie Daneben gibt es auch solche, bis jetzt bekannten Bausteine Stickstoft". Vereinzelte Proteine enthalttni die außerdem noch Schwefel aufweisen. Phosphor. Es konnte bisher noch nicht mit Bestimmtheit festgestellt werden, in welcher Form der Phosphor in den betreffenden Proteinen gebunden ist. Es scheinen esterartige Verbindungen zwischen Phosphorsäure und den Bausteinen der Proteine vorzukommen, daneben dürfte Es ist ferner möglich, daß der sie auch salzartige Bindungen eingehen. Phosphor zum Teil auch organisch gebunden ist. Wir können die Eiweißstoffe als hochmolekulare, nur im kolloiden Zustand bekannte Verbindungen bezeichnen, die dadurch ausgezeichnet sind, daß an ihrem Aufbau die Elemente C, H, 0, N und S teilnehmen. Das letztere Element fehlt offenbar nur ganz vereinzelt. Ferner kommt, wie schon erwähnt, einzelnen Proteinen Phosphor zu. Die Elementaranalyse der Proteine ergibt mit wenis,- Ausnahmen annähernd gleiche MengenverVictor Griessmayer: Die Proteide ) Vgl. über diese Gruppe von Verbiuduiigeu der Getreidearten usw. Karl Winter. Heidelberg 1897. - Eduard Stranss: Studien über 0. Cohnheim: Chemie der Eiweißkörper. 3. Autl. Friedr. die Albuniinoide. Ebenda. 11)04. Vieweg A: Sohn. Braunschweig 1911. - Gustar Mann und 0. Cohnheim: Chemistry of Emil Fif^cher: Untersuchungen über the Proteids. Macniillan and Cic. London 1906. Emil AJ>derhaldcn: Aminosäuren. Polypeptide und l'roteine. J. Springer. Berlin 1906. Neuere Ergebnisse der Eiweißclieniie. Gustav Fischer. Jena 1909. -- B. H. A. Plimmer: The Chemical Constitution of tlie proteins. Monographs on Biochemistry. l.ongmann's Green and Cie. London 1911. -~ Handbuch der Biochemie. 1. 226— .'lOO (bearbeitet v.ui P. Bona, E. Abderhalden, Franz Samuely). G. Fischer. Jena 1909. - Biochem. Handlexikon. 4. 1—917 (bearbeitet von Th. ß. Osborne, Franz Samuely, Adolf Jiolhtt, Karl Rasl-e. Otto Neubauer, Helmufh Scheibler, Ge'za Zemple'n, Hans T'rinf/sheini, Ernst W'iiiferstein, Peter Bona). J. Spiinger. Berlin 1911. : — — Eiweißstotte und ihre Bausteiue. o].l — — 55" o C, 65 75% H. 15 18% N und OS 2"5% S. Der Sauerstottgehalt berechnet sich aus dem Unterschiede der Summe der erwähnten Elemente und 100" o- Erwähnt sei. daß die EiweißstoÖ'e sehr schwer ganz frei von Asche zu gewinnen sind. hältnisse der einzelnen Elemente. Man findet etwa 50 — — Selbstverständlich genügen die angegebenen Daten nicht, um die Gruppe der Proteine zu charakterisieren. Wir kennen eine ganze Reihe von Reaktionen, die von den Angehörigen der Klasse der Eiweißstotte werden. Sie beruhen zum Teil darauf, daß die Proteine ausim kolloiden Zustand vorkommen, zum Teil sind es Farbreaktionen, die auf das Vorhandensein bestimmter Bausteine und bestimmter Bindungsarteu im Molekül zurückzuführen sind. Wir werden auf diese Eigenschaften der Proteine noch zurückkommen. gegeben schließlich Eine Charakterisierung der verschiedenartigen Eiweißstoffe auf Grund ist außerordentlich schwierig, weil sie in ganz verschiedenen Zuständen im Pflanzen- und tierischen Organismus vorkommen. ihrer Eigenschaften VVir treffen in Flüssigkeiten an. z. B. in der Milch, im BlutWir können nicht ohne weiteres erkennen, ob diese wahre Proteine plasma usw. Li)sungen darstellen oder aber, ob sich in ihnen Verbindungen finden, die nur scheinbar gelöst sind. Füllen wir eine solche Flüssigkeit - z. B. Blut- — — ein solcher besteht aus einer einen Dialysierschlauch pflanzlichen Membran so beobachten wir. wenn wir ihn in destilliertes Wasser eintauchen, daß bald Salze und andere Verbindungen, wie Zucker, Harnstoff usw.. durch ihn hindurchtreten. Eiweiß- plasma in tierischen oder — . dagegen bleiben quantitativ im Schlauche zurück. Es auch nicht eine Spur von diesen Verbindungen durch den Dialysierschlauch hindurch. Mit Hilfe dieser einfachen Methode können wir leicht erkennen, ob eine Verbindung wirkliche Lösungen bildet oder nicht. Die ersteren Stoffe nennen wir Nichtkolloide ^j, die letzteren Kolloide. Auf die Eigenschaften und die Bedeutung der Abgrenzung dieser beiden durch viele Zwischengliedei- verknüpften Klassen von Verbindungen werden wir später eingehend zurückkommen. -i Erhitzen wir eint' eiweißhaltige Flüssigkeit, dann tritt bei einer bestimmten Temperatur Koagulation ein Die scheinbare Lösung trübt sich zunächst. Die Trübung nimmt mehr und mehr zu und schließlich geht das ganze Eiweiß in den festen Zustand über. Wir sprechen von Hitzekoagulation. Es zeigen jedoch nicht alle Eiweißstoffe dieses Verhalten. So ist z. B. der Leim bei gewöhnlicher Ten)peratui fest. Beim Erwärmen mit Wasser geht er in Lösung. Viele eiweißhaltige Flüssigkeiten geben auf Zusatz von Salzlösungen bei einem Ammonsulfat. Zinksulfat. Kochsalzlösungen usw. stoffe dring-t — — bestimmten Gehalte an dem betreffenden Salz Ausflockungen. Es hat das scheinbar gelüste Eiweiß seinen Zustand geändert und ist in Form grölterer Konglomerate ausgefallen, blanche Proteine lassen sich schon durch enero-isehcs Schütteln ausflocken. ') Sie sind trüiier^Kristalloide genannt worden. Dieser Name ist jedoch nicht sehr glücklich gewählt, weil er leicht mit der Fähigkeit, zu kristallisieren, in Zusammenhang gebracht werden kann. Nun iribt es „Kristalloide". die bis jetzt nicht in Kristallform zu bringen waren, und Kolloide, die Kristalle bilden. Die Einteilung in Kristalloide und Kolloide stammt von Th. Graham (Philosoph. Transactionsi. 151. Teil 1. 1S3(18<)1\|. ^) Vgl. Band 2 dieses Lehrbuches, Vorlesung V XII. — XVII. Vorlegung. 312 Neben diesen Eiweilistoffen, die scheinbare Lösung- bilden, kommen auch halbfeste, zähflüssige Proteine vor. Es sei z. B. an das Eiereiweiß erinnert. Schließlich gibt es ungezählte Formen von Eiweiß, die vollständig fest sind. Als Beispiel seien die sogenannten Koratinarten angeführt. Aus Keratin bestehen die Haare, die Nägel, die Hufe, die Hörner, die Geweihe, die Barten des Wales usw. (iroße Massen von Eiweiß sind in dieser Form im Tierreich abgelagert. in der Natur Schon diese kurze Übersicht läßt es verständlich erscheineo, daß sich ohne weiteres auf Grund ihrer Eigenschaften charakterisieren lassen. Dagegen umfaßt die folgende Definition alle Vertreter dieser Körperklasse. Sie stützt sich darauf, daß sämtliche Proteine die die Proteine nicht gleichen charakteristischen Bausteine besitzen, nämlich die Aminosäuren. Die Eiweißstoffe sind hochmolekulare, zusammengesetzte, im kolloiden Zustand — nur vom festen Zustand bis an die Grenze der wahren Lösung bekannte Verbindungen, an deren Aufbau in charakteristischer Weise untereinander verknüpfte Aminosäuren beteiligt sind. Diese organischen Säuren enthalten die für die Eiweißstoffe charakteristischen Elemente und Gruppen. Mit ihnen haben sie die Eigenschaft in allen Abstufungen seinen — auch als Basen wirksam sein zu können. Wir kommen auf dieses wichtige Verhalten der Proteine und ihrer Abkömmlinge noch zurück (vgl. Band 2, Vorlesung IX). Die Bausteine der Proteine werden aus diesen erhalten, indem man sie mit rauchender Salzsäure oder mit 25''/oiger Schwefelsäure mehrere Stunden kocht. ^) Das Eiweißmolekül wird dabei unter Wasseraufnahme in einfachere Bruchstücke zerlegt. Schließlich bleiben die Aminosäuren übrig. Genau so, wie bei den zusammengesetzten Kohlehydraten und den Fetten, läßt sich der Abbau unter Hydrolyse bei den meisten Proteinen mehr oder weniger Man nennt diese leicht auch mittels bestimmter Fermente herbeiführen. ganz allgemein Proteasen oder proteolytische Fermente. Wir beginnen die Besprechung der Chemie der Eiweißstoffe am lui besten mit der Erörterung der Struktur der einzelnen Annnosäuren. Anschlüsse daran werden wir die Frage zu beantworten haben, in welcher Art und Weise die einzelnen Eiweißbausteine unter sich im Eiweißraolekül verknüpft sind, und damit werden wir von selbst auf die Frage nach dem Aufbau des Eiweißes stoßen. gemein, sowohl als Säuren als Man bezeichnet solche Verbindungen als Ampholyte. Die Aminosäuren sind mit Ausnahme zweier Verbindungen — Prolin und Oxyprolin^) — dadurch charakterisiert, daß sie = zum Karboxyl eine AminoNH.^-Gruppe tragen. Eine ganze Reibe von Aminosäuren lassen sich direkt von Gliedern der gesättigten Fettsäurereihe der allgemeinen Formel C„ H.j„ O.^ oder in 7.-Stellung CnH.,n+i .COOH ableiten. Wir sind Besprechung der Bausteine der Fette H GOOH. Die die Aminosäure. . ') ilabei Mau kann auch durch Kochen diesen Fettsäuren bei der Ihr einfachstes Glied ist einfachste Aminosäure müßte die docli tritt mit Lauge Hydrolyse teilweise Razeniisierung der P'iiweiliabbanprodukte ein. -) Sollte Pyrrolidonkarboiisäure Kiweißbaustein sein, dann zu vermerken sein. •) Vgl. dazu S. 212. bereits begegnet.'') lici^licifiihrcu. winden drei Ansnalinicn Eiweißstoffe uud ihre Bausteine. olo ClK)H haben, d. h. sie würde aus der Ameisensäure durch Ersatz eines Wasserstoffatonis durch die NHo-Gruppe hervorgehen. Als Baustein der Proteine ist diese Verbindung bisher nicht beobachtet worden. Sie ist wegen ihrer leichten Zersetzlichkeit im freien Zustand überhaupt Wohl aber ist ihr Ammonsalz, NH., COO NH4, darnicht bekannt. .Struktur NH., . . . worden. Wir werden dieser Verbindung noch begegnen, wenn wir dem Abbau der Aminosäuren im tierischen Organismus bis zum stickstoffhaltigen Stolt'wechselendprodukt, nämlich dem Harnstoff, folgen werden. Diese einfachste Aminosäure wäre als Aminoameisensäure zu bezeichnen. Man hat sie auch Karbaminsäure genannt. Die einfachste Aminosäure, die als Bausteine verschiedener Proteine aufgefunden worden ist. leitet sich von der Essigsäure ab, wie die folgenden Formeln zeigen: gestellt CH3.COOH CH, . GOCH GH, .CoOi) X NH3/ oder NH., — COO'O GH, X b'^: H NH., Essigsäure Aminoessigsäure = Glykokoll. Die Aminoessigsäure, auch Glykokoll, Glyzin oder Leimsüß ist bereits im Jahre 1820 von Braconnot^) neben Leuzin aus Leim und aus Muskelfleisch erhalten worden. Interessanter weise kommt diese Aminosäure in erheblichen Mengen im Schließmuskel von Pecten irradians) in freiem Zustande vor. Sie ist auch im Harn verschiedener Tiere und ferner im Menschenharn gefunden worden.») Endlich ist Glykokoll auch aus Blutplasma in freiem Zustand gewonnen worden. ß) Glykokoll bildet monokline Kristalle. Sie sind in Wasser ziemlich leicht löslich (1:4). Glykokoll schmeckt süß. Wie die oben dargestellte genannt, Formel zeigt, enthält das Glykokoll kein asymmetrisches Kohlenstoffatom. Es ist daher optisch inaktiv. Wird eine wässerige Lösung von Glykokoll mit Kupferoxyd gekocht, dann nimmt diese eine blaue Farbe an. Es hat sich das Kupfersalz des Glykokolls gebildet. Es hat die folgende Struktur: NH., CHo COG\ . . \Cu. NHo . GH., . QOO/ Alle bekannten Aminosäuren geben solche Salze. Sie sind wegen ihrer verschiedenen Löslichkeit zur Trennung besonderem Werte. ') einzelner Aminosäuren von ganz You diesen beiden Formeln werden wir der Übersichtlichkeit wegen nur die Es sei jedoch ausdrücklich vermerkt, daß die Aminosäure und ins- erstere verwenden. N'gl. dazu K. G. Falk und besondere Glykokoll und Alanin in beiden bekannt sind. K. Sugiiira: Jouru. of biol. Chem. 34. 29. (1918). -) Nach der Formulierung Werner?,. Vgl. R. Willstätter: Ber. d. Deuschen Chem. Heinrich Ges. 35. 585 (1902). Spiro und Löffler: Helv. chim. acta. 2. 533 (1919). Biltz und Hans Paetzokl: Ber. d. Deutschen Chem. Ges. 55. 1066 (1922). ^) Braconnot: Ann. de Chim. et de Fhysique (2). 13. 114 (1820). ) Chiftenaen: Liebic/s Annalen. 178. 26() (1875). ') (t. Ernbäen und H. Reese: Hofmeister?, Beiträge. 7. 411 (1906). E. AbderF. Samuel)/: halden und A. Schiftenhelm: Zcitschr. f. phvsinl. Chomie. 47. 339. (1906). — — — f^benda. 47. 376 (1906). ') A. Bini/el: Zeitscbr. f. physiol. Chemie. 57. 382 (1908). — 314 XVII. Vorlesuug. Von großem Interesse ist die Beobachtung i), daß Aminosäuren mit Neutralsalzen gut kristallisierende Verbindungen von bestimmter Konstitution ergeben. So bildet Glykokoll mit Lithiurachlorid, Li Cl, zwei chemisch Die eine besteht ans einem Molekül Lithium einheitliche Verbindungen. Chlorid, einem Molekül Glykokoll und einem solchen von Wasser. An der zweiten Verbindung nehmen zwei Moleküle Glykokoll teil. Mit Kalziumchlorid sind die folgenden Verbindungen erhalten worden: 1. 2. 3. Ca GL. NHo CH, COOK. 3H.,0. CaClo CH2 COOK), 4H.,0. CaClo 3 (NH2 CH2 COOH). . . 2(m . . . . . . Die Aminogruppe kann mit den verschiedensten Säurechloriden in Reaktion treten. Wird z. B. Glykokoll in alkalischer Lösung mit Benzoyl- dann glyzin = Hippursäure: erhält chlorid geschüttelt-), C,H, CO . Cl man Benzoyl-glykokoll — Benzoyl- + H NH CH., COOH = CßH^ CO NH . . . . . GykokoU Benzoyl- CH., . . COOH + H Cl Hippursäure. chlorid Zahlreiche derartige Derivate dienen zur Charakterisierung einzelner Aminosäuren. Glykokoll, sowie alle bis jetzt bekannten y- Aminosäuren geben, wenn sie in wässeriger Lösung mit Triketohydrindeuhydrat (Xinhydrin) ^j, C6hXco>g(öh).„ gekocht werden, eine prachtvolle Blaufärbung.) Wird fein gepulvertes Glykokoll mit absolutem Alkohol, z. B. Äthylalkohol Übergossen und dann in das Gemisch sorgfältig getrocknetes Salzsäuregas eingeleitet, dann geht das Glykokoll in Li»sung. Kühlt sich die während der Reaktion siedend heiß gewordene alkoholische Lösung wieder ab, dann kristallisiert Glykokollcsterchlorhydrat aus: CH, . COOH 4- HO . C, H^ + H Cl NH., Äthylalkohol Glykokoll = CH., CO . . . C. H5 + H, NIL. HCl Glykokollcsterchlorhydrat Diese Verbindung ist zur Isolierung des Glykokolls sehr geeignet, da die Esterchlorhydrate der übrigen Aminosäuren fast alle in Alkohol leichter löslich sind. Wir werden gleich erfahren, daß Emil Fischer °) die Esterchlorhydrate und die aus ihnen leicht zu gewinnenden freien Aminosäureester benutzt hat, um die verschiedenen Aminosäuren zu trennen. 8ö. 1 Zeitsclir. f. physiol. Chemie. 81. ') P. J'fei/fer uud J. r. Modelski: (1918). Ber. d. Deutscheu Chem. Gesellsch. 45. 1289. 1938 (1915). Ber. d. Deutscheu Chom. Gesellsch. 19. 502 (1886): 2) J. Baum: 329 (1912); Zeitschr. f. physiol. Chemie. 9. 4G3 (1885). Siegfried litihemann : Trausactions of Chem. Soc. 97. 2025 (1910). Ygl.Emil Abderhalden uud Hubert Schmidt: Zeitschr. f. physiol. Cheuiie. 72. 37. Emil Abderhalden uud Arno Ed. I.ampr': Ebenda. 85. 1.S6 (1911): 85. 143 (1913). 3.57 (1913). (1913). — ir. Halle, E. Löwenstein und E. Pribram: Biochem. Zeitsclir. 55. c. S. 307. ^) Emil Eiftcher: Untersuchnuiron üIkm' Aiuinosäureu etc. 1. ») ) — Eiweißstofte iiud ihre Bausteiue. ;-il5 GlykokoU ist auch synthetisch dargestellt worden. Läßt man z. B. auf Monochloressigsäure Ammoniak einwirken, dann entsteht Aminoessigsäure ') Cl . CIL . COOH + XH, = XH, CHo . . COOIi + H CI. Die nächste homologe Verbindung der Essigsäure ist die Propionsäure: CH3 CH, COOH. In dieser können wir am z- und ß-Kohlenstott. . ß Wie schon erwähnt, sind alle bis jetzt atom Wasserstoftatome ersetzen bekannten, aus Eiweißstollten gewonnenen Aminosäuren '/-Aminosäuren, d. h. die Aminogruppe sitzt an dem der Karboxylgruppe l)enacbbarten Kohlenstoftatom. Die der Propionsäure entsprechende Aminosäure ist das Alanin = a -Am inopropionsäure: CHj . CH, . COOH Propionsäure CH3 . CH COOH . NH, x-Aminopropion säure. x\lanin enthält ein asymmetrisches Kohlenstoftatom. Es ist durch Fettdruck in der vorstehenden und den folgenden Formeln kenntlich gemacht. Alanin ist optisch aktiv, und zwar dreht das in den Proteinen enthaltene nach rechts. Man bezeichnet rechtsdrehende (dextrogyre) Verbindungen mit d, linksdrehende (lävogyrej mit 1 und mit dl razemische, optisch-inaktive Verbindungen. In letzteren sind gleiche Mengen der links- und rechtsdrehendeu Verbindungen vereinigt. Es heben sich daher die beiden gleich großen, jedoch entgegengesetzten Drehungen auf. Derartige Verbindungen lassen sich auf verschiedene Arten in ihre Komponenten zerlegen. Viele Zellarten verfügen über Fermente, die nur die in der X'atur vorkommende optisch-aktive Form zu spalten vermögen. So baut z. B. Hefe nur d-Alanin ab. ^j Läßt man sie auf d 1- Alanin einwirken, dann beobachtet man, daß nach einiger Zeit 1- Alanin entstanden ist. Die quantitative Verfolgung des ganzes Vorganges beweist, daß d- Alanin verschwunden ist. Die vorher optischinaktive Lösung ist optisch aktiv, und zwar linksdrehend geworden. Bei dieser Art von Spaltung von Razemkürpern. die man als biologische bezeichnet hat, verliert man stets die eine Hälfte des Razemkörpers. weil sie von den verwendeten Zellen zerlegt wird. Wir erhalten die in der X'atur nicht vorkommende optisch-aktive Form. Wir verfügen, namentlich dank den Untersuchungen Emil Fiscliera^). auch über chemische Methoden, die uns gestatten, die beiden in einem Razemkörper der erwähnten Art vereinigten optisch-aktiven Formen zu trennen. In diesem Falle erhalten wir beide Hälften des Razemkörpers in quantitativer Ausbeute. Emil Fischer) hat ferner die wichtige Beobachtung gemacht, daß bestimmte Aminosäuren, darunter auch das Alanin, durch bestimmte ReIf. Perkin und /Jujjj/ct: Liehiga Auualeu. 108. 11:^ (1858). F. Ehrlich: Biochem. Zeitschr. 1. 8 (1906). ') Emil Fischfr: Ber. d. Deutschen Cham. Gesellsch. 32. 245G (1899) und Untersuchungen über Aminosäuren usw. 1. c. (S. 307). ) Emil Fische}- und O. Emi? Warlturq : Liebir/S Annalen. 340. 1(50 (1905). Emil Fischer und K. Rash- : Ber. d. Deutschen Chem. Gesellsch. 39. 3995 (1906). Emil Fischer: Untersuchungen 1. c. (S. 310). Vgl. Fischer: Ebenda. 40. 491 (1907). besonders Liebi(/s Annalen. 381. 123 (1911). ) ^) — — XVII. Vorlesung. ;3L(3 agenzien daß so beeinflußt werden, sie ihre Läßt Konfiguration ändern. man z. B. auf d-Alanin in der Kälte Stickoxyd und Brom (Nitrosylbromid) einwirken, so erhält man unter Abspaltung der NH^-Gruppe und Ersatz Wird diese mit Ammoniak zudurch Brom l-a-Brompropion säure sammengebracht, dann entsteht wieder a-Aminopropionsäure. Sie dreht jedoch nicht nach rechts, wie das Ausgangsmaterial, sondern nach links. Umgekehrt liefert 1-Alanin über d-7.-Brompropionsäure d-Alanin: ^ d-Alanin — (NH3) + d--/-Brompropionsäure I t (NOBr) (NOBr) ' 1 —^ yr + 1-x-Brompropionsäure Bei der Einwirkung von d-Milchsäure (1). ^ (NH3) salpetriger ^ I 1-Alanin. Säure auf d-Alanin erhält man GOCH I HO G H . . CH3 Die Konfiguration des d-Alanins (1)-) ist die folgende: 3) COOH NH.3.G.H CH3 Alanin ist zuerst von Schilt zenherg er und Bourgeois^) als Baustein der Seide festgestellt worden. Es löst sich in Wasser ziemlich leicht. Es bildet, wie das Glykokoll, Salze und schmeckt süß.») Vom Alanin lassen sich eine ganze Anzahl von Aminosäuren ableiten, indem wir am ß-Kohlenstoffatom an Stelle eines Wasserstoftatoms bestimmte Gruppen eintreten lassen. So kennen wir eine Aminosäure, die ihrer Struktur nach als eine y.-Amino-ü-oxy-propionsäure zu bezeichnen ist. Sie hat den Namen Serin erhalten: ') Emil Fischer und — A'. Raske : Ber. d. Deutschen Chem. Gesellscb. 40. B718 — E. Fischer und W. Ä. Jacobs: Ebenda. 40. 1057 (1907). (1907); 41. 893 (1908). Carl Carl Neuberff und .1/. Silbermann: Zeitscbr. f. physiol. Chem. 44. 134 (1905). Neuberg: Biochem. /eitschr. 5. 451 (1907). h Vgl. hiezu S. 35. ) Vgl. A. Wohl und -ß. Schellenberg : Bei. d. Deutscheu Chem. (ies. 55. 1404 Vgl. auch G. W.Clough: J. Chem. Soc. 131. .526 (1918). (1922). ) P. Schützenherger und A. Bourgeois: Compt. reud. de TAcad. des Sciences. 81. Vgl. Jh. Weiß: Ber. d. Deutschen Chem. Gesellsch. 21. 1.529 (1888). 1191 (1875). die Synthese von Alanin A. Strecker: Liebif/s Anualen. 75. 29 (1850). — — — ^) neten F. gegen 297". — [a].Voo ~ + 2"7" in wässeriger Lösung, + lO^" in der berech- Menge Salzsaure gelöst. Eiweißstoft'e uuil ihre Bausteine. COOH COOH NHo C H NH. C H . . . ';^\1 . " I 1 CH3 CH, d-AIanin(l) . OH l-Serin(l). Serin ist von Cramer^) bei der Hydrolyse von Seidenleim entdeckt worden. 2) Es kommt auch im Schweiß vor, ^j Es ist optisch-aktiv. Die als Baustein der Proteine vorkommende Form dreht nach links.) Serin löst sich ziemlich leicht in Wasser und schmeckt süß. ") Eine weitere Aminosäure, die in naher Beziehung zum Alanin steht, das Zystein. Es ist eine a-Amino-fi-thiopropionsäure. Es unterscheidet sich vom Serin dadurch, daß es an Stelle der OH-Gruppe eine ist SH-Gruppe besitzt: GH., Diese Verbindung dation in ist .(SH).CH(NH).COOH. unbeständig. Sie geht sehr leicht unter Oxy- Zystin=üi- (x-amino-ß-thiopropionsäure) über. Diesem kommt die folgende Struktur zu: COOH GOOH I ! NHo.C.H NH.,.C.H I . I CH^.S S.CH., I-Zystin(l). Unter den Abbauprodukten der Eiweißstofife ist bis jetzt nur das 1-Zystin^) aufgefunden worden. Damit ist nicht ausgeschlossen, daß nicht auch das leicht in Zystin übergehende Zystein ganz allgemein als Baustein von Proteinen auftritt "). Im Linseneiweiß ist es ohne Zweifel enthalten.«) Von größerem Interesse ist der Nachweis, daß Zystein in Verbindung mit Glutaminsäure in verschiedenen Zellarten vorkommt.'-) Es handelt sich oöenbar um ein Produkt der Struktur: — Bhnil Fischer: Ber. (1. ') E. ('ramer: Journ. f. prakt. Chem. 96. 76 (1865). Deutschen Chem. Gesellsch. 40. 1.Ö01 (1907). 2) Emil Fischer und H. Leuchs: Ber. d. Deutschen Chem. Gesellsch. 35. 3787 (1902) haben Serin synthetisch aus Ammoniak, Blausäure und Glvkolaldehyd bereitet. 3) G. Emden und H. Tachau: Biochem. Zeitschr. 28. 330 (1910). 40. 1501 (1907). ) Vgl. Emil Fischer: Ber. d. Deutschen Chem. Gesellsch. Emil Fischer und W. A. Jacobs: Kbenda. 39. 2942 (1906). 6-83'' in Wasser gelöst, -\|- 14-45" in salzsaurer Lösung. 5) F. 228". [a]^^" — =— ) ') []d''' T'. = — 225° in salzsaurer Lösung. Arnold: [Zeitschr. f. physiol. Chemie. 70. 314 (1910)J beschreibt das Vor- kommen des Zystei'ns in Geweben und Zellen. — Mörner: Z. f. physiol. Ygl.Reiss: Graefes Arch. f. Ophthal. 80. 588 (1911). A. Jess: Z. f. physiol. Chemie 110. 266 (1920). Vgl. auca Emil Abderhalden ») F. G. Hopkins: The Biochem. J. 15. 286 1921). und E. Wertheimer: Pßügers Archiv. 197 (1922). 8) Chemie. 18. 61. — — XVII. Vorlesuug. 31J-; HS CH2 CH CO— NH CH ('H, CH, COOH . . . . . . . COOH \11, Glataminsäurerest. Zysteinrest. Dieser Verbindung kommt nach den vorliegenden Beobachtungen die Bedeutung eines Sauerstotit'iiberträgers in den Zellen zu. Das Zystin wurde zuerst als Bestandteil von Blasensteinen aufgeDerartige Konkremente finden sich im Harn bei einer befunden. 1' stimmten Stoftwechselanomalie der sogenannten Zystin urie. Später wurde das Zystin als Baustein verschiedener Proteine erkannt. 2) Seine KonstituFriedmann Neuherr/ und Erlenmeijer aufgeklärt worden. ist von Friedmann^) oxydierte Zystein zu ZysteYnsäure. Diese ergab nach Abspaltung von Kohlensäure Taurin: tion ^ CHo . SH CHo NH, .C.H— >^ NH, SOo . . OH CH. . SO., . OH — > CH, NH, CH + CO, . COOH COOH Zystein ZysteYnsäure Taurin. Dieser Verbindung sind wir schon bei der Besprechung der schwefelhaltigen Gallensäuren begegnet. In diesen findet sich Taurin mit Cholsäure beziehungsweise Desoxycholsäure gepaart. Emil Fischei-) ist es ferner gelungen, 1-Serin in 1-Zystin überzuführen und damit die nahe Verw^andtschaft beider Aminosäuren zu beweisen. Da ferner 1-Serin in d-Alanin verwandelt werden kann''), ist eine weitere Beziehung zwischen diesen drei Aminosäuren gegeben. Sie kommt in den angeführten Konfigurationsformeln ohne weiteres zum Ausdruck. 1-Zystin kristallisiert meistens in charakteristischen hexagonalen Tafeln, deren Seiten gleich lang sind. Es ist in Wasser sehr schwer löslich. Es ist leicht zu erkennen, indem man seine Lösung mit Alkali unter Zusatz eines löslichen Bleisalzes, z. B. Bleiazetat, kocht. Bald tritt eine graue, immer dunkler werdende Färbung und schließlich eine grauschwarze Fällung von Bleisulfid auf. Durch das Kochen mit Alkali wird das Zystin zerstört und Schwefel abgespalten. Diese Probe ist nur eine (lualifative, denn es wird nicht freigemacht. Man nennt diese Reaktion Schwefelbleiprobe. zur Besprechung der x-Aminobuttersäure. Wir können sie von dem vierten Gliede der normalen Fettsäurereihe der Buttersäure, CH3 CH, CHg COOH, ableiten. Sie kann jedoch auch als ein [i-substituiertes Alanin aufgefaßt werden, wie die folgenden Formeln zeigen: aller Schwefel Wir wenden uns nun . . . ') Wollaston: Philosophical Transact. 220 (1810). Verhandl. d. -)-K. Kiilz: Zeitschi'. f. Biol. 27. 415 (1890). 0. Kmmeriinn : Gesellsch. deutscher Naturf. u. Ärzte. 2.391 (1894). Vgl. auch K. A. Moerner: Zeitschrift f. physiol. Chem. 42. 349 (1904). ) K. Fried mann: Hofmeister?, Beiträge. 3. 1 (1902). Carl Neuberc/: Ber. d. Deutschen Chem. Gesellsch. 35. 31(51 (1902). Erlenmei/er jun.: Ebeuda. 36. 2720 — — — — (1903). ) Kmil Fischer ') Kmil Fischer uiifl Karl u. Karl lütske: Ber. d. Deutscheu (jhem. (1907). /,'askr : Khciida. 41. 893 (1908). (iesellsch. 40. 3717 Eiweißstofte nud ihre Bausteine. CH, CHo . (JH . COOH . 3jq CH3 äCH, r NH, '^ ' I I CH NH, NH2 xVE " I COOH 7.-AminobuttersRure ' i COOH a-Aminopropionsäure z-Amino-ßmethyl-propioiisäure. Diese Aminosäure ^ ist wiederholt als Baustein von Proteinen beschrieben worden. Die genauere Untersuchung hat jedoch stets ergeben, daß ihre Identifizierung eine ungenügende war. Sie ist jedoch neuerdings bei der fermentativen Spaltung von Lupinensameneiweili isoliert und genau identifiziert worden. -) Es ist möglich, daß die Aminobuttersäure in diesem Falle sekundär aus einer anderen Aminosäure hervorgegangen ist, doch ist diese Annahme nicht sehr wahrscheinlich. Ihr Vorkommen als Baustein von Proteinen ist durch diese Beobachtung sehr wahrscheinlich gemacht. Sie dreht nach rechts und kristallisiert in perlmutterglänzenden Blättchen. Sie schmeckt süß.'^) Wir kennen ferner eine Aminosäure, die fünf Kohlenstoftatome aufweist und der empirischen Zusammensetzung nach in Beziehung zur Valeriansäure steht. Es ist dies das sogenannte Valin. Es hat nicht die Struktur der normalen Valeriansäure, sondern die der Isovaleriansäure. Sie hat eine verzweigte Kohlenstoffkette: ^^'ym CR, COOH . >CH CH COOH nu (^113 . . l^xlj . I NHo Isoval er ansäure z-Amino-isovalerian säure. i somit eine a-Amino-isovaleriansäure. Betrachtet man des Alanins, dann kann man sie auch als eine x-AminoValin ist zuerst von E. Schuhe) in ;i-dimethyl-propionsäure auffassen. Keimlingen aufgefunden worden. Bald wurde dann erkannt, daß es am Aufbau fast aller Eiw-eißkürper teilnimmt. Das Valin der Proteine dreht Valin ist sie als ein Derivat ') Frederick William Foreman (Biochem. Zeitschr. ö6. (U)13)\| 1 gibt au. im NH., CH \ ' Kasein a- Amiuoisobuttersäure,;.,TTä^C für diese Annahme findet sich jedoch . COOH, gefunden in seiner Mitteihing nicht. zu haben. Ein Beweis Es wäre dies die erste Aminosäure mit tertiär gebundenem Kohlenstoft'. Sie müßte optisch inaktiv sein, da sie kein asymmetrisches Kohlenstoffatom enthält. Eigene Beobachtung. 8-12" in Wasser gelöst. F. 304". Vgl. Fmil Fischer und A. Mouuei/rat: ') [xf^" Emil Abderhalden, Hisitig Lang Ber. d. Deutschen Chem. Gesellsch. 33. 2383 (1900). Chang und Erich Wurm: Zeitschr. f. physiol. Chem. 72. 24 (1911). Synthese aus a-Brom/;. Schulze: Zeitschr. f. physiol. Chem. 17. 193 (1893). isovaleriansäure nud Ammoniak vgl. l)oi Fittig und Clark: Liebigs Annalen. 139. 200 '-') =+ •») aH66). — XML Vorlesung. 320 nach rechts. Das d-Valin bildet weiße, glänzende Blättchen. Es schmeckt schwach süß und gleichzeitig etwas bitter.^) Wir kennen auch eine Verbindung, die sich von der normalen Valeriansäure ableitet. Es ist dies das Arginin. »Seiner Konstitution nach Wir haben in ist es eine a-Amino-^-guanidino-n-valeriansäure. dieser Aminosäure neben der Aminogruppe noch mit dem ^-Kohlenstoffatom verknüpft das G u a n i d n i CH3 . CHo . CH„ y c^ . : OH.. . . COOK ß normale Valeriansäure. /NH., C=NH \NHo Guanidin.-) NH. /NH, C^NH I \NH . GH., GH, . . GH., . (_H . CGOH x-Amino-fVguanidino-n-valeri ansäure. Wird Arginin mit Baryt hydrolysiert. dann erhält man unter Aufnahme von einem Molekül Wasser Harnstoff und a-. S-Diaminovaleriansäure.3) Die letztere Verbindung ist auch Ornithin genannt worden. Der Verlauf dieser Spaltung ergibt sich aus der folgenden Formel: mh NH. NHr=G — NH j I . GH, GH., . . GH, (^H GO()HJ+ H, . = Arginin NH, /NH, C4=o NH., I I -f- GH., GH, . . GH., . CH GOOH . \NH., Harnstoff Ornithin. Interessanterweise erfolgt diese Art des Abbaus des Arginins auch Kossei und Dakin^), die es entdeckt haben, haben es Arginase genannt. Die Struktur des Arginins ist schließlich noch durch die folgende Synthese bewiesen worden^): durch ein Ferment. = + 6-42'' ia Wasser, + 28-7'' iu 20%iger Salzsäure gelöst. 1) F. 315». [«]^oo -) Nach Hans Krall [Jouru. Chem. Soc. KH3 die Formeln: NH, . C^ 1 ^N alkalisch 107. 1398 (1915)] kommeu dem Guanidin NH., und NH : C((^ zu. jo nachdem die Lösung sauer oder \\H ist. E. Schulze und E. Wintersfein: Zeitschr. f. physiol. Chem. 26. 1 (1898); 34. Ber. d. Deutschen Chem. üesellsch. 30. 2879 (1898). 128 (1901). ) Ä. Kossei u. ü. D. Z)aH«; Zeitschr. f. physiol. Chem. 41. 321 (1904); 42. 181 (1904). ^) E. Schulze und E. Winferstein: Ber. d. Deutscheu Chem. Geseilsch. 32. 3191 (1899); Zeitschr. f. physiol. Chom. 34. 128 (1901). ») — Eiweißstotle uud ihre Bausteine 321 NH. NH,^ ' I 1 C'H, . CH2 CH, Ornithin . . CH COOH + CN NH, = . - . Zyanamid. NH., NH., NH = C — NH 1 I . CH, Arginin. GH., . . GH., . CH COOH Die Konstitution des Ornithins ist einmal durch die »Synthese^j und ferner durch den folgenden, von Ellinger^) entdeckten biologischen Abbau sichergestellt worden. Ornithin geht unter der Wirkung von Bakterien in Kohlensäure und Tetramethylendiainin = Putre szin über: GH., . GH., . GH. . CH . GOO H - GH2 NH, NH., GH., . . GH, NH, Ornithin . GH., -1- CO, NH., Putreszin. Das in der Natur vorkommende Arginin ist die d-Form.) Es ist von E. Schulze und E. Steiger') in den Kotyledonen etiolierter Lupinenkeimlinge entdeckt worden. Arginin ist als Baustein der verschiedensten Proteine sehr verbreitet. Sehr interessant ist das Vorkommen von freiem d-Arginin bei Melolontha vulgaris^) und anderen Wirbellosen. Es ist Seine Lösung ist alkalisch, während die bis leicht löslich in Wasser. sauer reagieren. jetzt erwähnten Aminosäuren amphoter oder schwach Sein Geschmack ist schwach bitter.'') Den Bausteinen des Arginins, dem Harnstoff und dem Ornithin, werden wir noch mehrfach begegnen. Der erstere ist ein typisches Stoffwechselendprodukt des Aminosäurestoffwechsels bei manchen Tieren. Das Ornithin ist als solches bis jetzt nicht in der Natur aufgefunden worden. Als nächstes Glied der Fettsäurereihe folgen die Verbindungen der Wir kennen sieben isomere Verbindungen der genannten Zusammensetzung. Von drei derselben leiten sich Aminosäuren ab. Kapronsäurereihe, GuHp^O.,. Dem Valin am nächsten stehen ihrer Struktur nach das Leuzin und das Isoleuzin. Das letztere läßt sich in direkte Beziehun": zum Alanin — ^) Emil Fischer: Berichte der Deutschen Chem. Gesellsch. 34. 454 (1901). Zeitschr. f. physiol. Chein. 44. 448 (1905). L. Störensen : h'. Fischer uud G. Zemplm: Ber. d. Deutschen Chem. Gesellsch. 42. 4878 (1909). -) A. Ellinger: Zeitschr. f. physiol. Chem. 29. 334 (1910). ßer. d. Deutscheu Chem. Gesellsch. 'ai. 3184 (1899); 32. 3542 (1900). Vs^l. auch lM<l<»bi(rq : Ebenda. S. /'. — 19. 49. 780 (1886). ^) Vgl. über 210 (1905). die Spaltung — — von dl-Arginiu Otto Ricsser: Zeitschr. f. physiol. Chem. 5) E. Schulze und E. Steiger: Ber. d. Deutscheu Chem. Gesellscii, 19. 1177 (1886). D. Ackermann: Zeitschr. f. Biologie. 73. 319 (1921). «) F. 207«. •) Abderhalden, Physiologische Chemie. I. Teil, 5. AuU. 21 XVII. Vorlesung. 322 und Valin bringen. Es ß-äthylpropionsäure CH3 . CH COOH . hat die J1«^>CH a-Aminopropinnsäure Struktur einer a-Araino-ß-ra ethyl- : . CH COO i^JJ^^CH . . (^H . COOH NHo NH, a-Amino-Ji-dimethYlPropionsäuren Valin y.-Amino-ß-raethylß-äthyl-propionsäure Isoleuzin. = Isoleuzin ist von Felix Ehrlich^) in der Rübenmelasse aufgefunden worden. In der Natur kommt die rechtsdrehende Form vor. Isoleuzin besitzt zwei asymmetrische Kohlenstoffatome. Es existiert in zwei stereoiso- und dem d'-AUo-isoleuzin. nieren Formen, dem d-Isoleuzin these entstehen beide Formen. 2) Bei Syn- der d-Isoleuzin kristallisiert aus Wasser in feinen, glänzenden Blättchen Es schmeckt adstringierend und schwach bitter. Es ist ziemlich schwer li)8lich in kaltem Wasser. s) Das Leuzin ist ein Derivat der Isobutylessigsäure: pTT CH^CH^ :Ch'>CH ^3 . . COOH ChJ/^^ • ^^^ • ^j^ • COOH a-Amino-isobutylessigsäure =:Leuzin. ilsobutyl essigsaure ^ "^ z-Amino -isobutylessigsäure zu beist somit als eine Man könnte es auch, wenn man es von Alanin ableiten will, eine a-Amino-3-isopropyI-propionsäure nennen.) Es ist von Proust''} entdeckt worden und gehört zu den am meisten verbreiteten und oft in Leuzin zeichnen. großen Mengen auftretenden Aminosäuren. Seine Konstitution ergibt sieh aus seiner Synthese. Sie sei neben derjenigen des Isoleuzins angeführf') Ber. d. Deutschen Chem. üesellsch. 37. 1309 (1904); Zeitschr. Zuckeriiidustrie. 975 (1904). ) Vgl. die Synthese L. Bouveaiilt und Rane Locquin: Compt. read, de l'Acad. des Sciences. 141. 115 (1907). Rene Locquin: Bull, de la Soc. de chim (4). 1. 5'.»5. 601 (1907). Felix Ehrlich: Ber. d. Deutscheu Chetn. Üesellsdi. 41. 1453 (1908). W. Brasch \md E. Friedmann: Hofm''i'iterA Beitrii','e. 11. 37() {V-)0^). E. A'i'Urhalden, P. Hirsch und ./. Schuler: Ber. d. Deutschen Ghem. Gesellsch. 42. B394 (1909). Felix Ehrlich: ) d. Ver. f. — — ) F. 280«. — M^go = -f- 11-29« in wässeriger, -\|- 41-29« iu salzsaiirer — Losung (207oi?e Salzsäure). ) Wir weisen auf diese Zusammenhänge nur hiu, um das Einprägen der Konstitution der einzelnen Verbindungen zu erleichtern. ') Prousf: Ann. de Chera. et de Phys. (2). 10. 40(1819). Vgl. auch Braounof : Ebenda. (2). 13. 119 (1820). Mulder: Journ f. prakt. Chem. 16. 290 (1839); 17. 57 — — (1839). — — Limpricht: Liehiqs Annalen. 94. 243 (1S55). E. Schulde und A. Likiern ik Deutschen Chem. (Gesellsch. 24. Gß9 (1891). Zeitschr. f. physiol. Chem. 17. Emil Fischer: Ber. d. Deutschen Chem. Gesellsch. 33. 2372 (1900). 513 (1839). L. Bouveaiilt und Rend Locquin: Bull, de la soc. chim. (3). 31. 1180 (1904). ) B^.r. d. — — Kiweißstoftc nnd ihre Hausteine. Qy'>C}I a 1 s CH, . CH, . my a k oh o + 1 1 OH . d-Amylalkohol. 1 — H, ^jj'>CH CH2 . ^ CHO . + HCN + NH3 . CH. . + HCN + NH. — H. CH (NH.) ex ^^]\|'>CH. CIKNH.) . d-Va1er Isovaleroaminonitril + I CH . CH, . a 11 i n o n i t r 1 i 1 — NH3 ' CH, (. J5'>CH CH (NH,) COOH a-Amino-isobutylessigsänre = Leuzin links. CN . + 2 H2 2 H., i -NH3 Q^'yCE CH(J . d-Valeraldehyd. ~H,0 gjj'>Cn H, - P^{^!'>CH Isovaleraldehyd TH 32:') . CH (NH2) COOH y.-Amino-^i-methyl-ß-äthyl- propionsäure = Isoleiizin. Das bei der Spaltung von Proteinen gewonnene Leuzin dreht nach Es ist durch seine Schwerlüslichkeit in Wasser aus5i;ezeichnet. Cha- rakteristisch für das Leuzin ist sein blaßblaues Kupfersalz. Es ist in Wasser außerordentlich schwer löslich. 1-Leuzin schmeckt fade und etwas bitler. Es kristallisiert in farblosen, glänzenden Rlättchen.i) In neuester Zeit ist in Eiweißstoffen des Nervengewebes eine der normalen Kapronsäure entsprechende Aminosäure festgestellt worden. -j Sie hat die Konstitution einer a-Amino-n-kapronsäure. Sie ist Norh'uzin genannt worden: CH, . CH, . CH, . CH, . (;H . COOH NH, a-Amino-n-kapronsäure=Norleuzin (a-Amino-fi-propyl-propionsäure). Manche Beobachtungen machen es wahrscheinlich, daß das NorEs dreht nach rechts, kristallisiert aus Wasser Es schmeckt schwach in sechseckigen, zu Drusen vereinigten Blättchen. süß. In Wasser ist es schwer l(>slich.^) leuzin sehr verbreitet ') F. 297». [a]i^Q„ ist. = — 10-34" in Wasser, + löy in 20%'gei' Salzsiiure gelöst. Emil Abderhalden uud Arfhur Weil: Zeitschr. f. physiol. Chem. 81. 207 (1912); Vgl. über die Synthese dieser Verbindung Kinil Fi.fchn- : Unter84. 39 (1913). suchungen usw. 1. c. S. 310 und Emil Ahderhalden, ('. Fröhlich und Dionj/a Fuchs: Ebenda. -) — 454 (1913). In der ersten Arbeit ündet sicli die Literatur der früheren, allerdings unsicheren Beobachtungen über diese Verbindung. 8«>. ') F. 285«. \|ot]J\|,o = + 4-5" in Wasser, + 21« in 20'/oig<?r Salzsäure gelöst. 21 XVII. Vorlesimg. 324 \on der normalen Kapronsäure leitet sich noch eine weitere Aminosäure ab, nämlich das Lysin.i) Es besitzt zwei Aminogruppen. Die eine befindet sich am a- und die andere am e-Kohlenstoffatom. Das Lysin ist demnach eine z-, s-Diamino-n-kapronsäure: V ^ c CH2 . CHo . CH., . ß GH., a ClI . NH, COOn . NH, Die Entdeckung des Lysins verdanken wir E. Schulze^) Er fand es Keimpflanzen von Lupinus hiteus und bald darauf auch in anderen Keimlingen. Bald wurde es, nachdem Kossei und Kidsrlier'^) Methoden zur Isolierung dieser Aminosäure ausgearbeitet hatten, mit dem Arginin und dem noch zu besprechenden Histidin zusammen in fast allen Proteinen aufgefunden. Seine Konstitution wurde sowohl durch Abbau als auch durch die Synthese) erwiesen. Ellmgcr"") beobachtete, daß Lysin von Bakterien angegriffen wird. Es wird aus ihm Kohlensäure abgespalten. Es vernämlich das Pentamethylendiamin Kadaverin. bleibt ein Amin, Die Konstitution dieser Verbindung war bereits von Ladenhurg^') aufgeklärt worden. .Seine Bildung aus dem Lysin erfolgt nach der folgenden Gleichung: in = GH., GH., . . GH, . GH, . NH2 GH GOO H = GH., ; NHo . GH, . GH., . GH., . GH. + GO., NH, NH, Lysin Kadaverin. Lysin ist bis jetzt nicht in kristallisiertem Zustande erhalten worSeine Lösung reagiert alkalisch. Diese sowohl als auch das Lysin selbst ziehen lebhaft Kohlensäure an. Das salzsaure Salz dreht nach rechts. Man nennt das in der Natur vorkommende Lysin deshalb d-Lysin, obwohl noch nicht festgestellt ist, ob das freie Lysin auch rechtsdrehend ist. Lysin bildet mit Pikrinsäure ein sehr schim kristallisierendes Pikrat. In direkte Beziehung zum Alanin können wir drei Aminosäuren bringen, die im Gegensatz zu den bis jetzt besprochenen der aromatischen Reihe angehören. Ersetzt man in der a-Aminopropionsäure in [i-Stellung ein Wasserstotfatom durch den Phenylrest, dann gelangt man zu der z-Amino-ß-phenylpropionsäure Phenylalanin: den. = Wiederholt sind Beobachtuugeu mitgeteilt worden, die auf das Vorkommen Verliinduug hinweisen. Es ist vermutet worden, daß ein Methylo mithin vorliege, und zwar CH,' GH.,' CH, CH COOH ^) einer dem Lysin isomeren . . . . '1 I NH CH3 NH... Thomas: Berliner klin. Wochenschr. Nr. 18. 428 (1919). E. Schulze: Zeitschr. f. physiol. Ghem. 24. 18 (1898); 28. ^iSh (1899); 30. 27G . Vgl. hierzu =) (1900). — ») A. Kossei und F. Kutscher: Zeitschr. f. physiol. Ghem. 31. 165 (1900/01). E. Schulze und E. Winlcrslein: Krgebn. d. l'hysiol. 1. ;i2 (1902). ) Emil Fischer und Fritz Weigert: Ebenda. 35. :5772 Sörensen: ('. r. (1902). des travaux du Laborat. de Carlsberg. 6. 1 (1903). ) A. Ellinger: Zeitschr. f. physiol. Ghem. 29. 334 (1900). Vgl. Zitat 2, S. 321. — — «) Ladenhurri: Bor. d. Deutschen Ghem. (leselisch. 19. 780 (188«). Kiweiüstiiflo CH HC^\ und ihre Baiistoiiic. 825 XVII. Vorlesung. 326 COH HC^NCOH NHoC.H CH HC COOH CHo C- Dieser Vorgang vollzieht Geweben. 'j Dem Phenylalanin sehr nahe steht das Tyrosin. Es ist seiner Konnach eine a-Amino-ß-para-oxyphenyl-propionsäure == stitution sich Sie wird auch leicht in leicht zu Farbstoffen oxydiert. Para-oxyphenyl-alanin -j: C COOH CH HC I H NH. NH., .('.H HC CH. = Cr H, (OH) I . CH., . CH. COOH. Tyrosin. von .Goi-Hp-Btsdue:'') zum erstenmal in Vicia sativa nachgewiesen worden. Jetzt wissen wir, daß es zu den verbreitetsten Aminosäuren gehört. Sein Nachweis ist leicht. j Einmal ist es in Wasser außerordentlich schwer löslich. Dieser Umstand erleichtert seine Isolierung außerordentlich. Ferner gibt Tyrosin Farbreaktionen. Sie sind, das sei ausdrücklich hervorgehoben, nicht ihm eigen, sondern zum Teil von der aromatischen Natur, zum Teil von seinem Phenulcharakter abhängig. Tyrosin gibt mit konzentrierter Salpetersäure, wie das Phenylalanin, die Xanthoproteinreaktion. Sie tritt schon in der Kälte auf und ist sehr intensiv. Dabei entsteht 31ononitrotyrosin.^) Fügt man zu Tyrosin oder zu einer Liisung davon eine Lösung eines Merkurisalzes in starker Salpetersäure, die etwas salpetrige Säure enthält (MiUona Reagens j, dann erhält Ihr Auftreten läßt man nach einiger Zeit eine prachtvolle Rotfärbung. sich durch Erhitzten stark be^hleunigen. Die Lösung von Tyrosin gibt mit Milloii^ Reagens zuerst eine weiße Fällung. Es sind noch viele andere Farbreaktionen angegeben worden. Es darf bei ihrer Anwendung zum Nachweis des Tyrosins nie übersehen werden, daß die meisten dieser Reaktionen auch von anderen Phenolen gegeben werden. Man kann sich auf sie nur dann verlassen, wenn man das Tyrosin noch auf andere Tyrosin ist '^) ') Vtrl. Hr. Ji/ocli und /'. Ui/hi//er: Zeitschr. f. d. uosamte oxperiin. Med. 5. ITi) (1917). Vgl. seiuc .Syutlicsc KrleiDiK ijer und Lipp: Bor. d. Deutsclieu Chcni. (icscllscli 1544 (1882); Lieb^'f/s Aimalen. 219. IGl (189;}). ") Gorup-Besauez: Bit. d. Doutscli. CIutti. Ges. 10. 781 (1877). A. SchuJzr und J. Bnrbieri: Kl)ciida. 11. 710, 1233 (1878). ) Vgl. hierzu Otto Fürth uud W. Fleischwann: Biocheiii. .1. 127. 137 (1922). Vgl. A'. Inoni/e: Zeitschr. f. physiol. Chem. 81. 80 (1912). ) Lasmigne: Aun. de Chini. ot de Phys. (2). 45. 44ö (1830). Millon: C. r. de l'Acad. d. Sc. 28. 40 (1849). "') : 15. — '-) — Eiweißstoffe iiiui "hre Bausteine. 327 sichergestellt hat. Wir keiiucn auch einen biologit-chtn Nachweis des Tyrosins. Versetzt man die wässerige Lösung von Tyrosin mit einem wässerigen Auszug bestimmter Pilze z. B. von Russula delica dann tritt nach kurzer Zeit eine schöne rote Färbung aul. Die Bildung dieses FarbstoÖes ist auf die Wirkung einer Fermentgruppe, Tyrosinase genannt, zurückzuführen, ij Allmählich kommt es zur Abscheidung eines schwarzen Pigmentes. Tyrosinasen sind auch bei manchen Tieren aulgefunden worden.-) Das in der Natur vorkommende Tyrosin dreht nach links. 3) Es kiistallisiert in feinen Nadeln. Es schmeckt wohl infolge seiner Schwerlöslichkeit nicht. Erhitzt man Tyrosin auf 270", dann erhält man unter Kohlensäureabspaltung das dem Phenyläthylamin entsprechende p-Oxy- Weise — — — — phenyläthylamin): CßH, (OH) . GH., . C'H . 000 H = "I NH, Tyrosin C,H, (OH; . GH., . " GH., -f r GU " NH.3 p-0 xy-phenyläthylamin. In manchen Proteinen kommt ein Tyrosin vor, an dessen Aufbau Jod beteiligt ist. Diese Verbindung ist von Drecfiseh) unter den Spaltprodukten des Achsenskelettes der Koralle Gorgonia Gavolini aufgefunden worden. Er nannte sie Jodgorgosäure. Erst Hmze und Wheeler^) erkannten sie als r),ö-Dijodty rosin: OH J.G^^'^C.J HG , Xc/ 3, Dijodtyrosin ist m COOH I GH., 5-Dijodtyrosin. auch aus Spongin (Badeschwamm) gewonnen wor- den.^) Es dürfte insbesondere im Skelett der An thozoen weit verbreitet sein. ^) Es findet sich ferner als Baustein von spezifischen Proteinen der Schilddrüse und stellt sehr wahrscheinlich eines jener Produkte dar, die von Zellen 1) (1907). Ch. Berfrand: C. r. de l'Acad. d. Sc. 122. 1215 (ISUC); 123. 463 (1890); 145. 1352 — B. Chodat: Arch. des sciences pbys. et uat. 112. (4). 24 (1907). halden und M. Gi(fff/enheim: Zeitschr. f. physiol. Ciioniie. 54. 331 (1907). -) Vgl. die Vorlesungen über Fermente in Band II. = ca. 16" iu 47oiger Salzsäure gelöst. ^) F. 318. [a]^QO ) R. Schmitt und 0. Nasse: Liebi</s Ann. 133. 211 (1865). E. Drechsel: Zeitschr. f. Biol. 33. 90 (1896). M. Henze: Zeitschr. f. physiol. Chemie. 51. (54 (1907). Henry ^) — E. Abder- — ('. Wheeler und George S. Jamieson: Amer. Chem. .lourn. 33. 365 (1905). //. ('. Wheeler und C. O. Johns: Ebenda. 43. 11 (1910). ^) Harr;/ ('. Wheeler und L. ß. Mendel: Jourii. of biol. Chem. 7. 1 (1909). A. Oswald: Zeitschr. f. physiol. Chemie. 75. 353 (1911) 8) Carl Th. Monier: Zeitschr. f. physiol. Chemie. 51. 33 (1907); 55. 77. 223 (1908). •) — — \ VII. Xorlesuiiir. 328 dieses Organes bereitet und als Inkretstoffe der Blut- oder Lymphbahn übergeben werden, um in anderen Zellarten einen spezifischen Einfluß auf bestimmte Funktionen auszuüben. ^) Die dritte, bis jetzt bekannte aromatische Aminosäure ist das Tryptophan. Während Phenylalanin und Tyrosin der homozyklischen Reihe angehören, treffen wir bei dieser Aminosäure auf einen Vertreter der heterozyklischen Gruppe 2). Wir können das Tryptophan als eine z-Aminopropion säure auffassen, an deren ß-Kohlenstoft" ein Wasserstoffatom durch die Indolgruppe ersetzt ist. Das Tryptophan wäre demnach als eine a-Amino-ß-indol-propionsäure zu bezeichnen. Indol hat die folgende Konstitution: CH HC -CH = HC Je CH Folglich C« H, N. CH NH kommt dem Tryptophan die folgende Forniel zu: COOH NHo C CH HC . C- . H —C CHo . HC^^yC CH CH NH Indol- ('«H«N.CH, .CH.COOH. NH, Alan in. Man war dieser interessanten Aminosäure schon lange auf der Spur. leicht daran zu erkennen, daß ihre L()sung mit Chlor- oder Bromwasser eine rosarote bis leicht violette Färbung gibt. 3) Spaltet man EiweißstofTe mit dem proteolytischen Ferment Trypsin, dann beobachtet man Sie ist nach kurzer Zeit das Auftreten der erwähnten Farbreaktion. Beim Beginn der Verdauung ist die Reaktion nicht nachweisbar. Im Laufe der Hydrolyse verstärkt sie sich immer mehr, bis schließlich alles Tryptophan abgespalten ist. Nui' das freie Tryptophan gibt die genannte Reaktion. Dieser Umstand gestattet, die Abspaltung des Tryptophans aus Eiweiß zu verfolgen. Das im Eiweiß gebundene Tryptophan gibt auch Farbreaktionen. So erhält man beim Zusatz von Glyoxylsäure): — Ahelin: Biochem. Zeitschr. 102. 58 (1919): 116. 1;}S (1921). Ol(/a Schiff'niann: Pflüger^ Archiv. 195. 167 (1922). ) Vgl. über sein Vorkommen Otto Fürth und E. Nobel: Biochem. Zeitsclir. 109. 108 (1920); Otto Fürth und Fritz Lieben: Biocliem. Zeitschr. 109. 124 (1920); 122. .08 ("1921). Ma:v ron Grab: Ebenda. 121. 69 (1921). Otto Folin und ./. N. Looriei/: The J. of biol. Chem. 51. 421 (1922). ') TiedenKinn und Gmelin : Die Verdauung nach Versuchen. Heidelberg und Leipzig ('laude Bernard: C. r. de l'Acad. d. Sc. Suppl. 1 (185.'')). 1826. E. Stadelmann: Zeitschr. f. Biol. 26. 491 (1890). R. Neumeister: Ebenda. 26. 324 (1890). M.Nencki: Ber. d. Deutsch. (Jhera. (ies. 28. 560(1895). Vgl. auch ('. Neubcn/ und A^. Popowski: Biochem. Zeitschr. 2. .S57 (1907). ) Vgl. Sidncii W. Cole: Journ. of Physiol. HO Hll (1906). ') /j'mil Vgl. hiezu .7. Abderhalden und — — — — — — — Eiweißstofte und ihre Bausteine. c<o o29 XVII. Vorlesung. 330 Das Oxytryptophan kristallisiert schwerer Färbung. löslich als das Nadeln, i) Es ist in Wasser noch Mit Bromwasser gibt es keine in Tryptophan. Die Aminosäure Tryptophan scheint in naher Beziehung zu einer von der Schilddrüse gebildeten, Thyroxin-) genannten Verbindung zu stehen. Nach Kendüll und Osterherg^) kommt ihr die Formel einer 4, 5, 6-trihydro4. 5, 6-trijodo-2-oxy-[i-indolpropionsäure) zu: H H H =C-C C . . Cf.^TT bzw. \0H H H C=0 C' NH H H H j>C<^^C— H H H\ C c Y . OH N H Eine weitere heterozyklische Aminosäure ist das Histidin. Es ist a-Amino-ß-imidazolyl-propionsäure. Der Imidazolgruppe sind wir bereits begegnet s), als wir die Bildung von Methylimidazol aus Tranbenzucker bei der Einwirkung von Zinkhydroxyd-Ammoniak besprachen. eine Iraidazol hat die folgende Konstitution: CH— NH CH. CH— Imidazo ») -i200 F. 293. [a]^" ) E. C. ^— lr2»iiü u-Natronlauge gelöst. Kendall: Transact. Ass. Am. Phys. 30. 420 (1915); The J. of biol. ehem. 39. 125 (1919). '0 E. C. Kendall und A. E. Üsttrbery: The J. of biol. ehem. 40. 2(5f) (1919). Die A'erbinduug ist auffallend reich an Jod! Es müssen weitere Forschungen abgewartet werden, um ein endgültiges Urteil über die Konstitution dieser Verbindung und ihre Bedeutung abgeben zu können. ) ) S. 137. Eiweißstfifte und ihiP Bausteine 331 Dem a-Amino-,--iruidazolyl-alaiiin kommt folj^lieh die folji;ende Formel zu 1): COOK I NH., . (' . Alaninrest H CHo N Imidazolrest CH CH-NH Histidin ist von A. Kossei-) unter den »Spaltprodukten des Proteins 8turin entdeckt worden. Es ist sehr verbreitet. Das in der Natur vorkommende Histidin ist die d-Verbindung. Es bildet lange schmale Tafeln.^) Histidin löst sich leicht in Wasser. Sein Geschmack ist süß.) Seine Lösung reagiert alkalisch. Gibt man zu einer Lösung von Histidin Bromwasser hinzu, dann entfärbt sich dieses zunächst. Schließlich bleibt bei vorsichtigem Zusatz man nunmehr, dann entfärbt Lösung, um dann ganz plötzlich eine dunkel weinrote Färbung anzunehmen.^) Histidin gibt in großer Verdünnung in sodaalkalischer Lödie Farbe des Bromwassers bestehen. Erhitzt sich die N=N sung mit Diazobenzolsulfosäure. CgH/ des Histidins ist • eine Rotfärbung. Die Synthese 1 Pyman^) gelungen. Alle bis jetzt besprochenen Aminosäuren ließen sich direkt oder indirekt mit Gliedern der Reihe der gesättigten Fettsäuren in Verbindung bringen. Den größten Teil der Bausteine der Proteine konnten wir als in ß-Stellung substituierte y.-Aminopropionsäure betrachten. Es wird von den entwickelten Gesichtspunkten der Überblick aus über die einzelnen Aminosäuren wesentlich erleichtert. Wir kennen nun noch zwei Aminosäuren, die keine ohne weiteres erkennbaren Beziehungen zu den bisher besprochenen Eiweißbausteinen aufweisen, es sind dies die Iminosäuren Prolin und Oxyprolin. Die erstere ist die z-Pyrrolidin karbonsäure. Sie leitet sich vom Pyrrolidin ab. dem die folgende Konstitution zukommt: GH., GH., 1 i CHo CHo ^NH ^) Vgl. über die Konstitution fies Histidius: llerman» Puuhi : Zeitschr. f. physiol. /•'. Knoop und A. W'indaKs: Jfol?iich-tcrs Beiträge. 7. 144 Chemie. 42. 508 (1904.) (1905): 8. 40? (1906). -) A.Ko.ssel: ZaiHchr. f. pliysiol. Chemie. 22. 177 (1896/97). S. G. Hrdin: Ehendsi. — — 22. 191 (1896). ^) Emil Abderhalden und Arthtir Wnl: Zeitschr. f. pliysi(d. Chemie. 77. 435 (1912). ) F. 280". [x]^,, = 4- 40-15" in wässeriger Lösung. Franz Knoop: llofniriKtcr^ Beiträge. 11. 356 (1908). ®) Frank Leo l'yman .lonrn. nf the Chem. Soc. London. 99. '") : 1H8() (1911). XVII. Vorlesuug 3;>^> Folfilich hat die a-Py r roli d i n k a rbonsä ure. das Prolin, die lolj;ende Struktur •): OH2 C'H, CH2 eil. ('(Hjii. Das Prolin ist von Emil Fischer -) unter den Öpaltprodukten des Kaseins entdeckt worden. Es stellte sich bald heraus, daß es ganz allgemein verbreitet ist. Seine Gewinnung ist dadurch erleichtert, daß es im Gegensatz zu den übrigen Aminosäuren in absolutem Äthylalkohol löslich ist. Auch sein Kupfersalz löst sich in diesem. Es dreht nach links, schmeckt süß. ist in Wasser leicht löslich und bildet flache Nadeln. 3) die ist Die Konstitution des Oxyprolins, das von Emil Fischer) unter den Gelatine zusammensetzenden Aminosäuren aufgefunden worden ist, von Leuclts und Breicster-') festgestellt worden. Es ist eine y-Oxy- pyrrolidin-7 -karbonsäure: CU HO.CH I I CHo CH \ NH / . (H)OH. Das Oxyprolin der Proteine dreht nach links. Es schmeckt süß, ist Wasser und bildet farblose Tafeln."^) Es ist wiederholt bezweifelt worden, ob Prolin und Oxyprolin am Aufbau von Eiweißstoffen beteiligt sind. Namentlich Sörensen') wies darauf hin, daß a-Amino-S-oxyvaleriansänre leicht in Prolin übergeht, und somit diese Aminosäure die Vorstufe der y.-Pyrrolidinkarbonsäure sein könnte. Es ist jedoch in einwandfreier Weise gelungen, zu beweisen, daß Prolin ein primäres Spaltprodukt der Proteine ist.^) Die M()glichkeit, daß außerdem die genannte a-Amino-S-oxyvaleriansäure ein Baustein mancher Proteine ist, leicht löslich in bleibt oifen. Ihre leichte Überführbarkeit Prolin zeigen die folgenden in Formeln Vgl. seine Synthese: /i. Willsfütter: Ber. d. Deutsch. Cheni. Ges. 33. 1160(1900). -S'. 34. 454 (1901). P. L. Siirensen: C. r. des travaux du Lahorat. de Carlsberg. 6. 137 (1908). S. P. L. Söreusen und A. C. Andersen: Ebenda. 7. auch Emil Fischer und G. Zemplen: Ber. d. Deutschen Chem. Ges. 42. 72(190).— Vgl. ' 1) -- Emil Fischer: P^benda. — — 2989 (1909). (1913). 2) Emil Fischer: Zeitsclir. •') F. 220". [a] 2^,, ) Emil Fischer: Ber. ^) Hermann ) F. 270'. [a] 2o'o — //. f. physiol. Chemie. 33. l.')l (1901). = — Sl" in wässeriger Lösung. Lettchs: d. Deutsch. Chem. Ges. 35. 26()0 (1902). 7^. Brewster: Ber. d. Deutsch. Cliem. Ges. 46. 986 und ./. Leuchs und K. Bormann: p:i)enda. 52. 208(J (1919). = —76" in wässeriger Lösung. P. L. Sörensen und A. ('. Andersen : Zeitschr. f. physiol. Chemie. 56. 236 (1908)Emil AbderVgl. Emil Fischer und li. Böhnrr: Eb(Mid;i. 65. fl.') (1910). halden und Karl Kautzsch: Kbeiida. 78. 96 (1912). ') «) iS'. — iMweiüstortt; CH., und ihre Bausteine. 333 — CR, CH.,— CH-, CH.C'OOH OH.CH., ' — H.,0 i I i i CH COOH \NH/ OH^ H.NH/ a. - A 111 i n - S - ü X }' . Prolin. V a 1 e r i a 11 s ä u r e Alle bisher angeführten Aminosäuren sind einbasisch. Wir kennen nun zwei Dikarbonsäuren, die Asparaginsäure und die Glutaminsäure. Beide finden sich in fast allen Proteinen. Besonders die letztere kommt in großen Mengen vor. Einzelne der Eiweißstolfe der Pflanzenwelt bestehen zu einem Drittel aus Glutaminsäure. Die Asparaginsäure ist Amino-bernsteinsäure^): eine COOH COOH I I CH CH, . NH., " I I CHo . CHj i I COOH COOH Bern st ein säure Ami nobern st ein säure. Sie dreht nach links und kristallisiert in rhombischen Blättchen oder Säulen. Ihre Lösung reagiert und schmeckt sauer.-) Die l-Asparagiiisäure ist zum erstenmal von PUsson^) beobachtet worden. Als Baustein von luweißstoften wurde sie von Bitthausen und Kreussler und ferner von Hlasiioetz und Hahermann) erkannt. Sie kommt auch frei im Drüsensekret von Tritonium nodosum vor. 5) Die Glutaminsäure säure dar. Sie ist eine stellt die nächste Homologe der Asparagin- Amino-glutarsäure''): COOH CH., COOH XVII. Vorlesuug. 334 Glutaminsäure Die rhombisch-sphenoidiseh-hemiedrischen in kristallisiert Kristallen.^) Wird die Glutaminsäure auf 185 — 190^ erhitzt, dann geht sie unter Pyrrolidonkarbon säure über 2): Wasseraustritt in die COOH COOH CH NH CH NH + Hg . . — CHo^ CHo >- CHo CHs CO OH CO Glutaminsäure Py rro 1 i don k arb nsäure Es ist wohl möglich, daß diese als Baustein von Proteinen auftritt.-^) Sie wird leicht zu Glutaminsäure aufgespalten, weshalb ihr Nachweis sehr Die leichte Überführbarkeit der Glutaminsäure in erschwert ist. Pyrrolidonkarbonsäure und umgekehrt eröffnet Beziehungen zum Prolin und damit auch zum Oxyprolin. Durch Reduktion könnte die Pyrrolidonkarbonsäure auch in der Zelle in Pyrrolidinkarbon säure übergehen und umgekehrt die letztere durch Oxydation in erstere verwandelt werden. Die folgenden Formeln geben diese nahen Beziehungen wieder): CH2 . CH2 . CH COOH CHo . — CH., durch Reduktion —> " CH, " I — H2 I COOH NH, Glutaminsäure — CH, ' ^ I I CH COOH \NH/ CH^ . a-Pyrrolidonkarbonsäure -i \ \ -y CO CH COOH \NH/ . — a-Pyrrolidinkarbonsäure, durch Oxydation. Aus Kaseinogen ist eine Oxy-glutaminsäure COOH CH CH . . . CH2 . der Zusammensetzung COOH I I NH, OH gewonnen worden.^) Sie enthält zwei asymmetrische Kohlenstoffatome. Es Weitere Untersuchungen sind daher zwei stereoisomere Formen möglich. sind nötig, säure um ihre Verbreitung festzustellen. Sie scheint mit Glutamin- zusammen vielen Proteinen als Baustein anzugehören. + wässeriger Lösuug, in salzsaiirer -\|- 31"2°. ) F. 225°. [alooo= ») A. Menozzil und G.Appiani: Gazz. chim. ital. 22. (2). 105(1892); 24. (1). 370 Emil Abderhalden und Karl Kaiitzsch: Zoitschr. f. phvsiol. Chemie. 64. 447 '^4'' in — (1894). (1910): 68. 487 (1910); 78. 333 (1912). Emil Abderhalden und Karl Kautzsch: 1. c. (Fußnote '). ) Emil Fischer und Rctjinald Bochner: Ber. d. Deutschen Chem. Gesellsch. 44. Vgl. auch Emil Abderhalden und Karl Kautzsch: 1. c. (Fußnote 2). 1332 (1811). Emil Abderhalden und Rudolf Hanslian: Zeitschr. f. physiol. (Chemie. 81. 228 (1912). ) H. D. Dakin: Biochena. Jl. 12. 290 (1918). ) — — Eiweißstoff'e und ihre Bausteine. 335 Von der Asparaginsäure und der Glutaminsäure kommt im PflanzenDas Amid der ersteren Verbindung, das Asparagin, hat die folgende Konstitution: reich im freien Zustande das Säureami d vor. COOK CO NH2 . 1 I €H NH, . CH I . NH., I CH2 CH, " I i COOIi COÜH Asparaginsäure Asparagin. Asparagin ist zuerst in Spargelsprößlingen aufgefunden worden. 1) Es im Pflanzenreich ganz allgemein verbreitet. Es ist in Knospen, Laubtrieben, in Knollen, Zwiebeln usw. gefunden worden. In Keimen, die ohne Belichtung gelassen werden, findet starke Anhäufung von Asparagin statt. Es dreht nach links und kristallisiert in großen, rhombischen, linkshemieist drischen Kristallen. Das Glutamin wurde von Schuhe und Barbieri'-) in Kürbiskeim- lingen entdeckt. Es hat folgende Konstitution''): COOH COOH i:H CH NH., I NH., CH2 I CHg I . CH9" I CHo" I COOH CO NHo Glutaminsäure Glutamin. . Das aus Keimlingen isolierte Glutamin dreht nach rechts. Es kristallisiert in farblosen, rhombischen Tafeln. Es ist die MögHchkeit gegeben, daß Asparagin und Glutamin Bausteine der Proteine oder doch mancher Proteine sind. Für das Vorkommen von Glutamin als Eiweißbaustein kann vielleicht die Feststellung verwertet werden, daß der Mensch nach Zufuhr von Phenylessigsäure Phenylazetylglutamin im Harn ausscheidet. Sie erscheint zum Teil als solches, zum Teil mit Harnstoff gekuppelt.) Ferner spricht für ein Vorkommen von Amiden und insbesondere von Glutamin im Eiweiß die Beobachtung, daß zwischen dem bei der Hydrolyse von Proteinen frei werdenden Ammoniak 55. — ) Delaville: Ann. de chim. et de phys. 41. 298 (I8i)2). Robiquct juii. Ebenda 152 (1905). VaqueUn und Robiquet jun.: Ebenda 57. 88 (19Ü5). ') E. Schulze und J. Barbieri: Ber. d. Deutsch. Chem. Ges. 10. 199 (1877): 11. — • 712 (1878). Vgl. H. Thier fehler: Zeitschr. f. physiol. Chem. 114. 192 (1921). H. Thierfeider und C. P. Sherwin: Ber. d. Deutsch. Chem. Ges. 47. 2630 (1914) Vgl. auch C. P. Sherwin und W. Wolf: The J. of biol. chem. 37. 113 (19191. ») — ) 336 XVII. Vorlesung. und dem Gehalt an Dikarbonsäuren und insbesondere an Glutaminsäure enge Beziehungen bestehen, i) Außer den hier angeführten Aminosäuren sind noch mehrere als Bausteine von Proteinen besehrieben worden. Es ist jedoch bis jetzt keine davon eindeutig festgestellt worden. Eine Verbindung von der Zusammensetzung Cio H.,6 No Oy ist aus Kasein erhalten worden."-) Es ist fraglich, ob diese Aminosäure ein primäres Abbauprodukt darstellt. Wir werden auf die Frage, ob wir schon alle am Aufbau der Proteine beteiligten Aminosäuren kennen, noch zurückkommen. Hier sei nur erwähnt, daß Beobachtungen vorliegen, die darauf hindeuten, daß noch unbekannte Bausteine Es ist wohl vorhanden sind. Ihre Zahl dürfte jedoch nicht groß sein. möglich, daß manche Proteine durch besondere Bausteine ausgezeichnet sind. \oY allem bedürfen noch die schwefelhaltigen Abbauprodukte erhöhter Aufmerksamkeit. Manche Proteine enthalten außer den Aminosäuren als Baustein noch das bereits besprochene^) Olukosamin: Eiweißstorte und ihre Bausteiue. 337 nämlich Mukoitinschwefelsäure und Chondroitinschwefel säure. Für die erstere Verbindung ist die folgende, noch nicht gesetzter Natur, allen Teilen bewiesene Strukturformel aufgestellt in CH2.OH Schwefelsäiiregruppe OH . 80, . worden: CH, . OH 1 . CH OH Glukosainin- . SO2 . . CH gruppe I CH H . C . I CH OH Azetyl- H C OH . gruppe . I H.C.NH. CO . I CH3 H C NH CO CH, . CH ! . . . I OHH HC C C C C COOH . . I OHH . . — 0- — CH I HC C C C C COOH . . . . . . HÖH H H H OH H H Glukuronsäuregruppe Mukoitinschwefelsäure. Der Chondroitinschwefelsäure wird eine entsprechende Struktur zuerkannt, nur findet sich an Stelle des Glukosamins das d-Lyxohexosamin. Es sei von der Strukturformel nur der von der für die Mukoitinschwefelsäure aufgestellten abw^eichende Teil wiedergegeben: Schwefelsäuregruppe CH, HO . C . SO, . OH H . — I HC a . HC OH . TJl O I HC NH. CO.CH3 . <v o HC Azetylgruppe I hier schließt sich, wie oben, Glukuronsäure an. Die erwähnten stickstoffhaltigen Kohlehydrate stellen in gewissem Sinne einen Übergang von den Kohlehydraten zu den Abderhalden, Physiologische Chemie. I.Teil. 5. AuH. 22 XVII. Vorlesung. 338 Aminosäuren dar. Besonders interessant ist die Beziehung des GlukoEs besitzt die für diese charakteristischen Gruppen und zugleich eine Aminogruppe. Es sei gleich hier erwähnt, daß bis jetzt die Bemühungen, die Stellung des Glukosamins im Zellstoffwechsel aufzuklären, erfolglos waren. Vor allem ist es nicht geglückt, die Herkunft des Glukosamins im tierischen Organismus festzustellen. Selbst dann, wenn wir in der Nahrung kein Glukosamin zuführen, bildet der tierische Organismus fortwährend große Mengen Muzin in seinen Drüsen und damit auch Glukosamin. 1) Die wahrscheinlichste Annahme ist die, daß die Zellen des tierischen Organismus vielleicht auch nur diejenigen, die Muzin bilden Traubenzucker aminieren, d. h. eine Aminogruppe in sein Molekül einfügen. Es ist jedoch auch möglich, daß eine bestimmte Aminosäure als Ausgangsmaterial dient. So könnten das Norleuzin oder das Lysin in Glukosamin übergeführt werden. Die folgenden Formeln zeigen die nahen Beziehungen dieser Verbindungen zueinander: sarains zur Glukose. — — GH, CH3 . NH., 1 I CH, I- GH., GH, GH, I GH GH NH, . I . GOOH Lysin Norleuzin 1 GH I i GOOH GH OH . r NH, ' . NH., o G^„ Glukosamin. Der Versuch, durch Verfütterung von Glukosamin etwaige Beziehungen zu den Kohlehydraten aufzutinden, war bis jetzt auch erfolglos. 2) Endlich muß noch erwähnt werden, daß bei der Hydrolyse der meisten Proteine in oft ganz beträchtlichen Mengen Ammoniak in Erscheinung tritt. Es ist möglich, daß seine Entstehung zum Teil auf ZerDer größte Teil dürfte setzung von Eiweißbausteinen zurückzuführen ist. insbesondere Glutamin und Asparagin —, deren jedoch aas Säureamiden Vorkommen im Eiweiß immer mehr an Wahrscheinlichkeit gewonnen hat 3), herstammen. Bei der Hydrolyse der Proteine mit Säuren, insbesondere mit rauchender jNIan Salzsäure entstehen braun bis schwarz gefärbte, amorphe Massen. hat sie Huminsubstanzen und Melanoidine genannt.) Ihre Natur — Eiweißstoffe und ihre Bausteine. ;)H9 Diese Substanzen verdanken sicher sekunnoch nicht aufgeklärt. dären Zersetz una:en ihre Entstehung, blanche Aniinosänren, wie z. ß. das Tryptophan, werden ieiclit zersetzt. •) Tryptophan ist die Muttersubstanz der Melanoidine.-) lluminsubstanzen bilden sich dann, wenn Kohlehydrate zugegen sind. Auch Glukosamin liefert mit Aminosäuren zusammen erhitzt solche Produkte. Aus dem Befunde von lluminsubstanzen wollte man den Schluß ableiten, daß im Proteinniolekül Kohlehydrate enthalten seien. ^) Es ist jedoch bis heute nicht geglückt, außer dem Glukosamin Kohlehydrate mit Sicherheit als Baustein der Proteine festzustellen, wenn auch Befunde vorliegen, die im Sinne des Vorkommens von solchen im Eiweiß sprechen.^) Nicht selten sind Proteinen Kohlehydrate nur beigemengt. Namentlich die aus Pflanzen gewonnenen Eiweißstoife sind oft stark mit Kohlehydrate führenden Substanzen verunreinigt. Man kann diese leicht erkennen, indem man zu der Lösung des betreffenden Proteins einige Tropfen einer alkoholischen Lösung von a-Naphthol zufügt und dann vorsichtig mit konzentrierter Schwefelsäure unterschichtet. Es tritt an der Berührungsstelle beider Schichten eine violette Färbung auf. Diese Reaktion ist außerist 'j) ordentlich emplindlich.''') .V. ') F. G. Hopkins uud W. W. Cole: Ebenda. 30. 311 (1903). ('nie: Jnuru. of Physidl. 27. 418 (1901). — Sidneif — Emil Abderhalden und Martin Kenipe: Zeitschr. f. phvsiol. Chemie. 52. 207 (1907). 2) ^) O. Fürth und Fr. Lieben: Biochem. Zeitschr. 116. 224 (1921). Vgl. zu dieser Frage Leo Lanqstein: Ertrehnisse d. Physiol. 1. 63 (1902): 3 (1). 4ö3 (1904). Langstein: Biochem. Zeitschr. 127. 34 (1922). IL Molisch: Monatsheft f. Chem. 7. 198 (1888). ^) Vgl. über die dieser Reaktion zugrunde liegenden Vorgänge: Carl Nenberg Zeitschr. f. physiol. Chemie. 31. 564 (1990'01). ) Jjeo 5) Reaktion nach Molisch. 22 Vorlesung XVIII. Eiweißstoffe und ihre Bausteine. 2. Aminosäuren. Die Art ihrer Verknüpfung im Eiweißmolekül. Peptone. Polypeptide. Wir haben die Struktur der am Aufbau der Proteine beteiligten Aminosäuren im Zusammenhang mit den ersten sechs GHedern der Reihe der gesättigten Fettsäuren besprochen. Einige davon sind a-Aminoderivate der entsprechenden Fettsäuren, andere enthahen an anderen Kohlenstoüfatomen eine zweite Aminogruppe oder einen anderen Rest. Nur die beiden zweibasischen Verbindungen Asparaginsäure und Glutaminsäure und die heterozyklischen Iminosäuren Prolin und Oxyprolin gehören anderen Reihen an. Die Kenntnis der Konstitution der einzelnen Aminosäuren ist deshalb unumgänglich notwendig, weil nur sie uns gestattet, den Abbau jeder einzelnen im Zellstoflt'wechsel zu verfolgen. Eine ganze Reihe von Verbindungen, die teils im Darmkanal, teils in den Geweben und ferner im Harn auftreten, können wir mit ganz bestimmten Aminosäuren in direkte Beziehung bringen. Ist die Struktur der einzelnen Aminosäuren bekannt, dann bereitet es keine Schwierigkeiten, die Zusammenhänge zwischen den einzelnen Stoti'wechselz wischen produkten und ihnen klar zu übersehen. Es seien im folgenden die einzelnen Aminosäuren übersichtlich zusammengestellt, und zwar unter Berücksichtigung der Konfiguration, soweit diese bekannt ist (Alanin und seine substituierten Abkömmlinge): GH., .COOK Eiweißstoffc und iiire Bausteine 1341 GOCH COOH C()()H I i NH2 C H . NH2 C H . . . I NH., CH2 CH3 d-a-A rainopropionsäure— d-Alanin(l) . ( )H A m n 0- ß-o x ypropionsäure 1- 7.- . C I CH, I I CH NH2 . COOH d-a-Amino- butteisäure (d-5c-Amino-ß-methyl-propionsäure) CH, 1 . SH 1-a-Amino-ßthio-propion- säure = Zystein (1). C(X)H CH, H i =:1-Serin(l) CH3 . COOH XVIII. Vorlesuno'. a42 CH, CH3 . NH, I CH, CH., ! CH, CH, CH2 CH, I CH . CH NH, NH., . I I COOH d-a-A m n o-s- a m n o-n-k a p r n- COOH d - a - A mi n o - n - k II p r n säure i = d-Norleuzin i säure = d-Lysin. (a-Amino-ß-propyl-propionsäurc) CH3 CH3 CH3 C3H5 \/^ \/ CH CH I I CH.NH, CH, COOH CH NH, . I COOH d-a-A m n o-[i-in e t h y l-[i-ät h y 1propionsäure = d-Isoleuzin. l-a-Aniino-isobutylessigsäure 1-Leuzin i = (a-Amino-[i-isoproi)yI-pi"opiüiisäure) C.OH CH COOH HC'^Nc.OH HC I HC CH NH., . C H . HC NH., CH C . . H " I C— l-x-Ainino-y-pheDyl-propionsäure=:l-Plienylalanin C HC . C- (H.. 8, 4-Dioxyphenylalanini) OH (! COOH CH .1 CH. . OH C .( COOH I I 1 HC CH C Nil, .CH CH, l-a-Amino-ß-p-oxy-phenyl- propionsäure=:l-Ty rosin. CH HC NH, C C- . . H CHo a-Ainino-fi-H. ö-dijod-p-oxy- pheny l-propionsäure = 8, ö-Dijodtyiosin = Jodgorgosäure'-) Noch fraglich, oli Kiwciiibaustein. Carl Th. Mörner hat aus Priuinoa-Gorgoniu eiuc Biomgorgosäure = 3, 5Dibrom-tyrosi n dargestellt. [Zeitsclir. f. physiol. Chemie. 88. 124 (1U13)]. ^) ) Eiweißstoftc uud Bausteine. ilire 34H COOK C(K>H NHo.C.Il CH — C.CH, HC HC l-y--A CH m nö-\|i-i n d o 1-p r o p o n s ä u r e i NFI,.C.H HC^N: -C.CH, HC^^C\^jj/CH CH -Cs (..OH CH -Amin o-ß-o x y-i n d o 1-p r o p o n \-y. i = l-Tiyptophan. i säure = l-()xy-tryptoph an COOK NH— CH NH. (^ H . HC \ -C -CH., l-a-Amino-;i-imidazolyI-propionsäure = 1-Histidin. HO H, C; CIL Ho C CH.COOH . HC: H, C. NH 1 -a-lyr 10 Hd in kar honsäure 1-Prolin = ^CHs /CH.COOH NH 1-y-Oxy-a-pyrrolidin- karbonsäure = I-Oxyprolin. COOH I COOH CH., CH, CR, I I CH NH, CH NH, . . I COOH COOH l-Amino bernsteinsäure d-Aminoglutarsäure ::= d - G u t a m n s ä u r e = 1-Asparajj,insäure 1 i COOH H, C CH, CH., I I oc eil .COOH I CH.OH I \/ NH CH NH, . COOH ß - (J X y "S" u t a ni i n s ä u r e 1 PyrroHdonkarl)onsäure. XVllI. Vorlesung. 344 CO NH2 . CH, CHo CH.NH2 1 COOK Glutamin. halt Die Bausteine der Proteine können nach ihrer Art und ihrem C4ean einzelnen Gruppen in der folgenden Weise eingeteilt werden. I. Aminosäuren der aliphatischen Reihe. 1. Monoamino-monokarbonsäuren. Dahin gehören alle jene Aminosäuren, die eine Aminogruppe tragen und Monokarbonsäuren sind: Glykokoll, Alanin, Aminobuttersäure, Valin, Isoleuzin, Leuzin, Norleuzin. Das Serin, das auch hierhin gehört, ist weiterhin durch den Gehalt einer Oxygruppe ausgezeichnet. Es ist eine Monoamino-mono-oxy- monokarbonsäure. Das Zystin besteht aus zwei Molekülen Zystein, das als Monoamino-monothio-monokarbonsäure aufzufassen ist. 2. Diamino-monokarbonsäuren. Diese enthalten außer eine einer Karboxylgruppe zwei Aminogruppen. Hierher gehört das Lysin. Eine Gruppe für sich bildet das Arginin, das neben einer Aminogruppe noch die Guanidinogruppe enthält. Es wäre als eine Monoamino-monoguanidino-monokarbonsäure zu bezeichnen. Da sich Arginin und Lysin in vielen ihrer Eigenschaften verhältnisse aufweisen sehr ähnlich sind, z. B. ähnliche Fällungs- und beide alkalisch reagieren, hat man sie auch zusammen Diaminosäuren genannt. 1) Diese Bezeichnung ist insofern außer der in a-Stellung befindlichen auch gerechtfertigt, als das Arginin Aminogruppe auch in der Guanidinogruppe eine freie Aminogruppe trägt. Erwähnt sei, daß früher auch das Histidin zu den Diaminosäuren hinzugerechnet worden ist. Monoaniino-di-karbonsäuren: Hierher gehören die beiden zweibasischen Aminosäuren Asparagin säure und Glutaminsäure. Hier reiht sich als Oxysäure die Oxy-glutaminsäure an. 3. II. A. Diese das Aminosäuren der aromatischen Reihe. Angehörige der homozyklischen Reihe. Gruppe enthält nur Monoamino-monokarbonsäuren, nämlich Phenylalanin und das Tyrosin. Das letztere ist eine Monoamino') Vielfach ist auch der Name Hexoubasen gebräuchlich. Eiweißstnrto imd ihre Bausteine. 345 mono-oxy-monokarbonsäure. Ferner gehören hierher das o, ö-Dijodtyrosin und das B. Auch 3, ö-Dihrom-tyrosin. Angehörige der heterozyklischen Reihe. die Vertreter dieser Körperklasse sind Monokarbonsäuren. Sie Monoamino-monokarbonsäuren: Tryptophan und Hiund die Monoamino-mono-oxy-monokarbonsäure (Jxytryptophan. Ferner gehören hierher die Monoiniinomonokarbonsäure Prolin und die Monoimino-mono-oxy-nionokarbonsäure umfaßt stidin die Oxyprolin. III. Bausteine der Proteine, die keine Aminosänren Wir kennen Gruppe nur das Gluko- bis jetzt als Vertreter dieser sind. samin und das Ammoniak. Die einzelnen Aminosäuren haben viele Eigenschaften miteinander gemein. Nur die zweibasischen Verbindungen haben einen ausgesprochen sauren Charakter und die Diaminosäuren einen basischen. Die übrigen Aminosäuren haben einen doppelten Charakter, indem einerseits die Aminogruppe und andrerseits das Karboxyl sich geltend machen kann. Wir haben bereits erwähnt, daß die Aminosäuren sich verestern lassen. Wir gelangen dann zu Verbindungen, die 'ihrer Struktur nach den Fetten entsprechen, nur ist hier statt einer Fettsäure eine Aminofettsäure mit einem Alkohol esterartig verknüpft. Ferner bilden die Aminosäuren Salze. Das Kupfersalz der einzelnen Aminosäuren ist besonders oft zur Trennung von Aminosäuren verwendet worden. Wir haben ferner schon der interessanten Beobachtung gedacht, daß die Aminosäuren sich mit Neutralsalzen verbinden. \) Endlich sei nochmals bemerkt, daß die Aminogruppe der Aminosäuren sich leicht mit Säureradikalen verknüpfen läßt. Es sind zahlreiche derartige Verbindungen gewonnen worden. Manche davon sind besonders geeignet, um bestimmte Aminosäuren zu charakterisieren. Von besonderem Interesse ist die Beobachtung, daß die Aminosäuren Harnstoff sogenannte üraminosäuren bilden. Kocht man Aminosäuren und Harnstoff zusammen in wässeriger Lösung, dann entweicht Ammoniak, und es bildet sich die erw^ähnte Verbindung. Sie besitzt am x-Kohlenstoffatom an Stelle der NH-^-Gruppe die NH CO NHj- = Uraminogruppe. Die Bezeichnung dieser Verbindungen ergibt sich ganz mit . von selbst. Sie Alanin gewählt: CH3 sind 7.-Uraminoverbindungen.2) Es sei . als Beispiel das XVIII. Yorlesmig. 346 oder allgemeiner dargestellt, wobei R den Rest der Kohlenstottlvette be- deutet: R CüOH ^/COOH NHav.,^ + Nh'>^ '^ - ^\Nh" CO NHo + ^^' , ^NH, . Erwärmt man eine Mineralsäure, dann erhält solche man Uraminoverbindung das Anhydrid der mit verdünnter Verbindung, auch Hvdantoin ":enannt: P/COOH " ^\NH CO NH2 H,0 ^\NH CO NH Hvdantoin. . . . . a-Uraniinosäure Sehr interessant ist ferner die Beobachtung von Siegfried 1), daß Aminosäuren Kohlensäure unter Bildung von Karbaminosäuren binden krmnen. Sättigt man z. B. eine wässerige Lösung von Glykokoll unter Kühlung mit Kohlensäure, so entsteht Glykokollkarbonsäure. Sie läßt sich durch Zusatz von Kalkmilch zu dem Reaktionsgemisch als Kalksalz abscheiden CHo . /OH COOH +cfo I —> CHo COOH + Ca< .OH — H, —y . H2 2 i NH .COOH \0H NH^ Glykokoll \0H Glykokollkarbonsäure CH2 COO. . I >Ca NH .CHXK Glykokoll karbonsaures Kalzium. Aus der Monokarbonsäure Glykokoll ist eine Dikarbonsäure geDie Aminodikarbonsäuren liefern Aminotrikarbonsäuren: worden. COOH COOH I I CH NH., . ('H . NH COOH . I CH., CH., COOH COOH Asparaginsänre Karbamino-bern stein säure. Endlich sei noch erwähnt, daß die Aminosäuren sieh zu Aldehyden reduzieren lassen: ,^ R CH COOH -h 2 H = R CH Cf . . I NH., .)/. . . O , , + H.. O. I NH, ') M. Siegfried: Zeitschr. f. physiol. Chemie. 44. 85 (1905); 46. 401 (1905). Siegfried und H. Schmitz: Ebenda. 65. 'J95 (1910). EiweißstofiV und ihre Bausteine. 80 der ist z. B. aus Glykokoll Aininoazetaldeh yd. NU.j . diircli CHo . ;-J47 Reduktion mit Natiiumamalj^ani C^jj, erhalten worden. \) Wie wir schon erwähnt halten, zeij^en die verschiedenen Aminomit wenig Ausnahmen entsprechend ihrer gleichartigen Struktur recht ähnliche Eigenschaften. Dieser l'mstand erschwert ihre Gewinnung ganz außerordentlich. Dazu kommt noch, dalj ihre Eigenschaften nur dann scharf zum Ausdruck gelangen, wenn sie vollständig rein sind. Geringe säuren \'erunreinigungen beeinflussen z. ß. die Löslichkeitsverbältnisse sehr stark. Ferner haben die Aminosäuren die unerwünschte Eigenschaft, leicht Mischkristalle zu bilden, ja man beobachtet zuweilen Kombinationen salzartigeii Charakters zwischen Tyrosin und Lysin und vielleicht auch zwischen anderen basischen und sauren Aminosäuren. Dieses Verhalten der Aminosäuren läßt es leicht erklärlich erscheinen, weshalb die einzelnen Bausteine der Proteine erst allmählich erkannt wurden. Man entdeckte sie meistens in einer bestimmten Proteinart nur dann, wenn sie in besonders großer Menge unter seinen Abbauprodukten auftraten. Für einige Aminosäuren wurden auch bestimmte Derivate oder Salze entdeckt, die zu schwer löslichen, So läßt sich z. B. die Glutaminsäure als salzsaures Salz gut abscheiden -), weil dieses in Salzsäure schwer löslich ist. Auch Farbreaktionen waren manchmal leitend. Es sei z. B. an die Bromreaktion des Tryptophans erinnert. Endlich kennen wir Aminosäuren, die an und für sich so schwer löslich sind, daß sie sich relativ leicht von den übrigen Bausteinen der Proteine abtrennen lassen. So kann z. B. das sehr schwer lösliche Tyrosin leicht erkannt werden. Auch das Leuzin löst sich schwer in Wasser. Schließlich lassen sich einzelne Aminosäuren auch auslallen. So haben Hopkins und Coh'^) das Tryptophan durch Fällung mit Merkurisulfat in schwefelsaurer Lösung in reinem Zustand isolieren können. für diese charakteristischen Verl)indungen führten. Mit der Feststellung, daß eine größere Anzahl von verschiedenen Aminosäuren aus Efweißstoffen verschiedener Art erhalten werden können, war für die Kenntnis der Zusammensetzung der einzelnen Proteine noch nicht viel gewonnen. Es bestand die Möglichkeit, daß die einzelnen Eiweißstoffe sich durch die Art der an ihrem Aufbau beteiligten Aminosäuren unterscheiden. Es konnte aber auch sein, daß sie alle die gleichen Bau.steine aufweisen. Es ist klar, daß unter dieser Unsicherheit die ganze Erforschung der Chemie und der Physiologie der Proteine leiden mußte. Für bestimmte Aminosäuren wurden im Laufe der Zeit allgemein brauchbare Methoden zu ihrem Nachweis und auch zur annähernd (\|uantitativen Be«timmung ausgearbeitet. Tyrosin wird als schwer lösliche Aminosäure abgetrennt. Allerdings macht ihre quantitative Bestimmung oft große Schwierigkeiten, weil diese Aminosäure von anderen in Lösung gehalten wird. Die Glutaminsäure läßt sich, wie schon bemerkt, als salzsaures ') Carl Xeuhcr/) Bor. Ebenda. 41. 1019 (1908). : -) J. //. .1. Jiifthansen: .Iduni. Di-ntsrh. Clioiu. Ges. 41. f. prakt. Chemie. 99. 454 S)öt; (1908. (186(5). — — h'mil n.s-c/icr //. lUasunt: und Hahermann: Lichiq?, Aunalen. 169. 150 (1S73). •) /•'. (i. Hopkins und >'. W. Colc: ,lourn. of physiol. 27. : 41.S (19(H). XVIII. Vorlesung. 348 Salz abscheiden. Für die Verbindungen Lysin, Arginin und Histidin haben Kossei und Kutscher'^) ein Verfahren ausgearbeitet, um sie zunächst gemeinsam auszufällen und dann weiterhin jede einzelne Aminosäure für sich zu isolieren. Es beruht darauf, daß die erwähnten Aminosäuren mit Phosphorvvolframsäure fälll)ar sind. Es fehlte nur noch eine Methode, um die übrigen Aminosäuren, wie Glykokoll, Alanin, Serin usw. auch dann aufzufinden, wenn sie nicht in besonders großen Mengen vorhanden sind. Diese empfindliche Lücke füllte Emil Fischer'^) aus, indem er die sogenannte Estermethode zum Nachweis von Monoaminosäuren ausarbeitete. Dem Nachweis der einzelnen am Aulbau der Proteine beteiligten Aminosäuren muß zuerst die Hydrolyse des zusammengesetzten Produktes vorausgehen. 3) Die Spaltung muß eine vollständige sein. Gew(>hnlieh kocht man die Proteine mehrere Stunden mit rauchender Salzsäure oder mit 25o/oioer Schwefelsäure. Man kann auch Fermente zur Hydrolyse anwenden. Aus der stark verdünnten Lösung werden dann die drei Verbindungen Lysin, Arginin und Histidin mit einer Lösung von Phosphorwolframsäure gefällt. Aus dem Filtrat der Fällung entfernt man den Überschuß des Fälhmgsmittels mit Baryt. Vom phosphorwolframsauren Baryt wird abfiltriert, aus dem Filtrat der überschüssige Baryt mit Schwefelsäure quantitativ entfernt und nunmehr das Filtrat vom schwefelsauren Baryum vollständig eingedampft. Um Zersetzungen zu vermeiden, verdampft man bei niederer Temperatur unter gleichzeitiger starker Luftverdünnung innerhalb des Verdampfungsgefäßes. Jetzt führt man die im ^'erdampfungsrückstand befindhchen Aminosäuren in die salzsauren Ester über. Das Verfahren ist das schon bei der Gewinnung des Gykokolläthylesterchlorhydrates geschilderte. Der Rückstand wird mit absolutem Alkohol Übergossen und in diesen trockenes Salzsäuregas bis zur Sättigung eingeleitet. Hierbei gehen die Aminosäuren unter Entstehung ihrer Ester in Litsung. Die Bildung der Ester bzw. zunächst ihrer Chlorhydrate ist aus der folgenden allgemeinen Formel ersichtlich: R CH COOH + HO . . NH2 Aminosäure . C, H5 + H Gl = R CH CO . . . (> . C, H, ^ H, O. NH, HCl Aminosäure. Äthylalkohol esterchlorhydrat. Glykokollesterchlorhydrat kristallisiert bei genügender Konzender Lösung direkt aus. Zur Trennung der übrigen Aminosäuren Esterchlorhydraten zunächst die freien Ester darzustellen. Man kann die Ester auf verschiedene Weisen in Freiheit tration ist es notwendig, aus den setzen, z. B. mit Natronlauge: A. Kossei und 7«'. Ku/scJier: /eitschr. f. physiol. (Jhciiiio. 31. 165 (1900/01). Emil Fischer: Zeitschr. f. physiol. Chemie. 33. 151 (1901). •') Eviil AbderVgl. hierzu Emil Fischer: Untersuchuugeu usw. 1. c. S. 33.i. halden: Neuere Ergebnisse der Kiweißcheniie. Gustav Fischer. Jena 1909. ») ') — EiueiÜstoffc und ihre Bausteine. K CH CO . . . () . + Xa((>H) = K C.,H, . ClI CO O . . H49 . l'.Al, NH, HCl A iiiinosäureester- Aniinosäure- chlorliydrat ester. + NaCl + H, () NU, . Die meisten der Monoamino-nionokarbonsäureester lösen sich leicht Sie werden mit diesem aufgenommen und dann durch Verdampfen des Äthers in reinem Zustande erhalten. Die Ester stellen Flüssigkeiten mit bestimmtem Siedepunkt dar. Leider liegen diese nicht für alle Aminosäureester weit genug auseinander, um jeden einzelnen für sich durch Destillation isolieren zu können, wohl aber ist es möglich, bei der Destillation unter vermindertem Druck Fraktionen aufzufangen, die nur drei bis vier verschiedene Aminosäuren enthalten. Aus den Estern der Aminosäuren gewinnt man diese selbst durch Verseifen mit Wasser zurück: in Äther. R CH CO 0\ C, H5 + H, . . . " " = R CH COOH -f C, H^ OH . . . " I I Aminosäure- NH, Amine- Äthyl- äthylester säure alkohol. NH., Mittelst fraktionierter Kristallisation, der Darstellung von Derivaten und von Salzen wird nun das aus jeder einzelnen Destillationsfraktion gewonnene Aminosäuregeraisch in seine Anteile zerlegt, nachdem man vorher das in absolutem Äthylalkohol lösliche Prolin mit diesem Lösungsmittel abgetrennt hat. Diese kurze Schilderung der Methodik der Isolierung der einzelnen Aminosäuren soll zeigen, welche l'mv.ege notwendig sind, um sie alle zu erkennen. Es ist klar, daß eine derartige ^lethode nicht quantitativ sein kann. Einmal sind Verluste bei den zahlreichen Operationen unvermeidlich. Dazu kommt, daß bei der Esterbildung sich ein Vorgang vollzieht, der es ganz unmöglich macht, die Aminosäuren quantitativ in ihre Ester überzuführen, es ist dies das sich bildende Wasser. Endlich bewirkt die Art der Infreiheitsetzung der Ester aus ihren Chlorhydraten, daß stets ein Teil der Ester wieder verseift wird, ehe eine Aufnahme in den Äther erfolgt ist. Schließlich verläuft die Destillation der Ester nicht ohne Zersetzung. Diese Erfahrungen und Überlegungen mußten zu der Frage führen, inwieweit die Anwendung der Estermethode vergleichende l'ntersuchungen über die Beteiligung der einzelnen Aminosäuren am Aufbau verschiedener Proteine zuläßt. Die Erfahrung ergab, daß zwar den Ausbeuten an den verschiedenen Aminosäuren keine quantitative Bedeutung zukommt, daß man jedoch bei ein und demselben Protein bei gleichartiger Durchführung der Estermethode stets annähernd Werte erhält. Bei den meisten Proteinen erreichte die Menge die gleichen der identifizierten Aminosäuren nur etwa ') nO— GO" ') der zu erwartenden Mau darf hei der Berechuuug des I'rozentgelialtes der isolierteu Aminosäuren Mengen nicht einfach addieren, denn hei der Hydrolyse des Proteins ist Wasser aufgenommen worden. Dieses muß in Ahzug gchracht werden. ihre XVllI. Vdilesiing. 350 Sollte ein großer Teil der am Aufbau der Proteine beteiligten Bausteine noch unerkannt sein V Zur Entscheidung dieser wichtigen Frage ^vurden reine Aminosäuren einzeln und dann in Mischungen verestert, die Ester in Freiheit gesetzt und destilliert. Die Destillate wurden dann verseift und schließlich jede Aminosäure, die verwendet worden war, mögEs zeigte sich, daß l)is 400/0 Verluste lichst quantitativ zurückgewonnen. eingetreten waren. Daraus ergibt sich mit großer Wahrscheinlichkeit, daß der bei weitem gr()ßte Teil der Bausteine der Proteine Bausteine.') bekannt ist.^) Es sei gleich hier vorweg genommen, daß weit über hundert verschiedene Eiweißstoffe nach den Verfahren von Kosf^el und Knfsrher und Kmil Fischer untersucht worden sind. Es ergab sich die wichtige Tatsache, sie werden hauptsächlich durch daß mit ganz wenig Ausnahmen die verschiedenartigsten der Protamine bedingt die Gruppe — — Proteine die gleichen Aminosäuren enthalten. kSib unterscheiden sich zunächst dadurch, daß die einzelnen Aminosäuren in verschiedenem Mengenverhältnis vorhanden sind. Einzelnen Proteinen fehlt die eine oder die andere Aminosäure. Wir haben die Eiweißkörper als zusammengesetzte, im kolloiden Zustand vorkommende Verbindungen charakterisiert, an deren Aufbau Aminosäuren beteiligt sind. Wir können diese, genau so, wie die Monosaccharide bei den Kohlehydraten und die Fettsäuren und den Alkohol bei den Fetten, als Bausteine der Proteine bezeichnen. Mit der Erkenntnis, daß bei der vollständigen Spaltung der Proteine unter Wasseraufnahme Aminosäuren sich bilden, kimnen wir uns nicht begnügen. Wir möchten gerne wissen, wie die einzelnen Bausteine im Eiweißmolekül untereinander verknüpft sind. Verfolgt man den Abbau der Eiweißkörper durch Säuren, Alkalien und Fermente genauer, dann läßt sich leicht feststellen, daß die Hydrolyse stufenweise vor sich geht. Es bilden sich Produkte, die sich vom Eiweiß dadurch scharf unterscheiden, daß sie nicht mehr kolloiden Charakter haben. Sie enthalten jedoch noch mehrere Aminosäuren gebunden. Werden solche Substanzen abgetrennt und weiter gespalten, dann gelangt man schließlich auch zu Aminosäuren. Es ist versucht worden, derartige, noch zusammengesetzte Abbaustufen zu isolieren und zu reinigen. Eine ganze Reihe von Forschern haben Methoden erdacht, um möglichst einheitliche Produkte zu gewinnen. Man hat zunächst das große Gemisch der noch zusammengesetzten Abbaustufen der Proteine in zwei Gruppen eingeteilt, nämlich in Albumosen und Peptone. Jede dieser Gruppen läßt sich in weitere Bestandteile trennen. Als Trennungsmittel sind hauptsächlich gesättigte Xeutralsalzlösungen angewandt worden. Kühne war der erste, der begann, die Abbaustufen der Proteine systematisch durch Aussalzen zu trennen. Dieses Verfahren Ncnmcister, Siegfried, Pick, Haslwn, ist dann besonders von Hofmeister. Zum u. A.s) weiter ausgearbeitet worden. Unter dem Namen Albumosen ') Vgl. hierzu 'Ihomas B. Oshorne 305 (1910). und Breese Jones: Amer. Jouiii. of Pbysiol. 26. 455 f. phvsiol Chemie. 74. — Emil AhderJiaJden und Arthur Weil: Zeitschr. (1911); 77. 59 (1912). ^) Vgl. auch Fmü Abderhalden: Zeitschr. f. physiol. Chemie. 120. 207 (1922). •') Vgl. ir. Kühne: Verband], des naturhistor. med. Vereins zu Heidelberg. N. F. IC. Kühni' und R. II. Chittenden: Zeitschr. f. Biologie. 20. 11 (1884). 3. 2S5 (l«8o). — — KiiweilSstottc iiud ihre Bausteine. )Mi]. wurden alle jene Produkte zusamniengefaßt, die sich z. B. mit Aminonsulfatbzw. Zinksulfatlösung oder auch Kochsalzlösung aussalzen lassen. Fügt man zu einer Lösung von zusammengesetzten Eivveißabbaustufen eine Neutralsalz- dann beobachtet man einem bestimmten Grade der Sättigung zeigt das Filtrat bei weiterem Zusatz der Neutralsalzlösung wieder eine Fällung. So hat man z. B. bei Halbsättigung mit Amraonsulfatlösung eine Fällung beobachtet. Man nannte das gefallene Produkt primäre Albumosen. Ein Teil davon löst sich in Wasser, ein anderer bleibt ungelöst. Die ersteren sind Protalbumosen, die letzteren Heteroalbumosen genannt worden. Fügt man zum Filtrat der sogenannten primären Albumosen weiter Ammonsulfat hinzu, bis schließlich vollständige Sättigung erreicht ist, dann fallen die sogenannten Deuteroalbumosen. Sie lassen sich durch fraktionierte Sättigung mit Ammonsulfat auch wieder in Gruppen aufteilen. Die nicht aassalzbaren Produkte sind Peptone genannt worden. Auch diese wurden lösung, eine Fällung. man Filtriert bei diese ab, so fraktioniert. Die erwähnten Methoden haben zwar in keinem einzigen Falle zu einer erwiesenermaßen einheitlichen Substanz geführt, wohl aber ist es mit ihrer Hilfe geglückt, Produkte abzutrennen, die frei von bestimmten Bausteinen waren. Man kann Fällungen herbeiführen, die kein Tyrosin enthalten, während andere Fraktionen diese Aminosäure aufweisen. Ebenso kann man schwefelhaltige und schwefelfreie Präparate gewinnen. Eigene Beobachtungen haben ergeben, daß man jede mit einem besonderen Namen belegte Albumose und jedes Pepton unter geeigneten Bedingungen in weitere Anteile zerlegen kann. Die erwähnten Namen bezeichnen nicht chemische Verbindungen, sondern vielmehr Gemische von mehr oder weniger einfach zusammengesetzten Eiweißabbaustufen. Die Namen Albumosen und Peptone haben die gleiche Bedeutung, wie der Sammelname Dextrin. Dieser bezeichnet ein großes Gemisch von Abbaustufen von Polysacchariden. Ebenso umfassen die Namen Albumosen und Peptone eine große Reihe von verschiedenen Abbaustufen der Proteine. Wir haben bei den Dextrinen darauf hingewiesen, daß es gelungen ist, sie in einzelne Fraktionen zu trennen, die ein verschiedenes Verhalten zeigen. Da jedoch vorläufig mit den gewählten Bezeichnungen sich keine bestimmten Vorstellungen über die Natur der einzelnen Produkte verbinden lassen, haben wir auf eine Aufzählung der einzelnen Namen verzichtet. Aus dem gleichen Grunde wollen wir auch hier auf weitere Einzelheiten in der Erforschung der Albumosen und Peptone nicht eingehen. Ihre Kenntnis hat zurzeit nur für den Forscher selbst große Bedeutung. p]rwähnen wollen wir nur noch, daß es vorteilhafter wäre, den Namen Albumosen ganz fallen zu lassen und nur von Peptonen zu sprechen. Es hat sich nämlich herausgestellt'), daß synthetisch aus — — W. Kühne. Zcitschr. f. Biologie. 29. 1 (1892). R. Neumeister: Elieuda.2(>. 824 (189U). K. P. Pick: Zeitschr. f. physiol. Chemie. 24. 246 (1897); 28. 219 (1899); Hofmeislcr^ Beitrage. 2. (J81 (1902). E. Zum: Ebenda. 27. 219 (1899); Extrait du Bull, de M. Siegfried : Zeitsclir. TAcad. royale de Belgifjue. (Jlasso des Sciences. Nr. 8. 1911. f. physiol. Chemie. 38. 2.59 (1903). H. C. Ilaalam: Journal of Physiol". .•J2. 2(57 (1905): 36. 164 (1908). Vel. weitere Literatur hei O. Cohnheint: Chemie der Eiweißkorper. — — 1. c. S. (1907). — — 307. M Kmil Fischer und Kmil Abderhalden: Ber. d. Deutsch. (Jhem. Kmil Fischer und O. (iern(,roß: Ebenda. 42. 3544 (1909). — CJes. 40. 3544 XVIII. Vorlesuug. 352 einzelnen Aminosäuren aufgebaute Verbindungen sich gegenüber Neutralsalzlösungen ganz verschieden verhalten, je nach der Art der an ihrem Aufbau beteiligten Aminosäuren. Hochmolekulare Produkte können nicht aussalzbar sein, während z. B. Tyrosin oder Zystin enthaltende Verbindungen sich schon mit Ammonsulfat bzw. Kochsalz aussalzen lassen, wenn drei bis vier Aminosäuren vereinigt sind. Es kann somit die Aussalzbarkeit keinen Aufschluß über die Molekulargröße geben. Es gibt ohne Zweifel „Peptone", die ein höheres Mole- kulargewicht besitzen, als bestimmte „Albumosen". Unwillkürlich wird man geneigt sein, den Namen „Albumosen" mit den dem Eiweiß noch am nächsten stehenden Produkten in Zusammenhang zu bringen. In Wirklichkeit gibt es Peptone, die den Proteinen näher stehen als manche Albumosen. Spricht man nur von Peptonen, dann genügt die Einteilung in aussalzbare und nicht aussalzbare Produkte, wobei man sich sofort der Tatsache zu erinnern hat, daß die Abscheidbarkeit dui'ch Ammonsulfat usw^ nichts über die Molekulargröße der betreffenden Sul)stanz aussagt. Einen Schritt weiter in der Aufklärung der Zusammensetzung der Peptone bedeuten die Arbeiten von Siegfried.'^) Es ist ihm gelungen, aus Peptongemischen mit Phosphorwolframsäure Produkte abzutrennen, die neben einzelnen Monoaminosäuren hauptsächlich aus Lysin, Arginin und Histidin bestehen. Siegfried hat diese Produkte als Kyrine bezeichnet. Es liegen ohne Zweifel Produkte von einfacherer Zusammensetzung vor. Es ist wohl möglich, daß das eine oder andere Kyrin ein einheitliches Produkt darEs ist jedoch bis jetzt für keine dieser Verbindungen der Beweis stellt. der Einheitlichkeit geführt und noch weniger ist ihre Struktur ermittelt w^orden.2j Bevor wir auf weitere Bestrebungen, die Peptone in wohl charakeinheitliche chemische Verbindungen aufzuteilen, eingehen, wollen wir die Frage aufwerfen, in welcher Art und Weise wohl die einzelnen Aminosäuren untereinander vereinigt sind. Emü Fischer hat diese FrageSein Plan stellung zum ersten Male experimentell in Angriff genommen. war folgender. War es bislang trotz aller Anstrengungen nicht geglückt, (las Gemisch von allen möglichen, aus dem Eiweiß durch Abbau entstandenen, zusammengesetzten Abbaustufen in wohl charakterisierte, chemisch einheitliche Substanzen zu zerlegen, so mußte man versuchen, zunächst durch Synthese Verbindungen aus mehreren jener Aminosäuren aufzubauen, Das Studium der Eigenschaften die im P^iweißmolekül enthalten sind. derartiger Produkte ergab möglicherweise Mittel und Wege, um aus Peptonen entsprechende Verbindungen abzuscheiden und mit den synthetisch terisierte, =^) identifizieren. Dieser Weg zur Erforschung der Struktur der Natur vorkommenden Verbindungen war ein eigenartiger, denn bishei' hatte die Aufklärung einer von der Organismenwelt erzeugten Verl)indung die folgenden Stufen durchlaufen. Zuerst wurde durch Abbauvorsuche und durch Überführung der unbekannten Verbindung in bekannte dargestellten zu von t ;5 in ) M. Siegjrli'd: Bcr. (1. sächs. (ies. d. Wissciiscb. in Leipzig. Math.-physik. Klasse, Zoitschr. f. phj'siol. Cliemio. 43. 44 (1904); 97. 3^(191()). (1913). -) Vgl. auch I'.A.Lecene und ,/. ran der Schecr : Jouru. of A)iol. ehem. 22. 425 (1915). ') Kntil Fischer: Untersuchungen, 1. c. S. 335. / — uud ihre Bausteiae. Eiweißstoffe 353 die Struktur auf analytischem Wege erschlossen. Es wurde die Funktion jeder einzelnen Atomgruppe im Molekül festgestellt. Dann legte die Synthese den Schlußstein in der ganzen Aufklärung der Konstitution einer Verbindung. Hier ging nun die Synthese voraus. Sie konnte zu Verbindungen In diesem Falle führen, die gar nicht im Eiweißraolekül enthalten sind. hätte man andere Bindungsmöglichkeiten studieren müssen. Emil Fischer ging von der Bindungsart aus, die die wahrscheinlichste war.^) Er ver- kettete zwei und mehr Aminosäuren in der Weise, daß das Karboxyl der einen Aminosäure sich mit der Aminogruppe einer weiteren verband. Eine derartige Bindungsweise können wir als säureamidartige Verkuppelung bezeichnen. Wir sind dieser Art der Verknüpfung von zwei Molekülen bereits dreimal begegnet. Einmal besprachen wir Säureamide bei den Dikarbonsäuren: Asparagin- und Glutaminsäure. Es sind dies das Asparagin und das Glutamin. In beiden Verbindungen ist das eine Karbox yl mit einer NH, -Gruppe besetzt: Säureamid GH., Ferner haben wir die Bildung von Hippur säure aus Aminoessig- säure und Benzoylchlorid besprochen: säuieamidartige Verkettimg HOOC CE, NH IH + Gl OG V, B, = HOOG GH., NH OC C, E, + HCl Hippursäure. BenzoylcTilorid GlykokoU . . . . . . . . Wir erhalten Hippursäure auch dann, wenn Säugetiere Benzoesäure und GlykokoU zur Verfügung haben: HOOC CH2 NH H -t- GlykokoU Benzoesäure . . HO OC . C, E, r= HOOC CH., . . NH . 0(" . C« H^ + E, Hippursäure. den Gallensäuren (vgl. S. 230) zusammenProdukte kennen gelernt, in denen GlykokoU bzw. Taurin mit einer weiteren Verbindung säureamidartig verknüpft ist. Genau ebenso lassen sich nun zwei und mehr Aminosäuren vereinigen. Als Beispiel wollen wir von der Synthese einer Verbindung ausgehen, an deren Aufljau zwei Moleküle GlykokoU r=:Glyzin beteiligt sind. Wir erhalten unter Abspaltung von einem Molekül Wasser aus zwei Molekülen Glyzin die Verbindung Glyzyl-glyzin^): Endlich haben wir in gesetzte ) Yd. auch Franz Hofmeister: Über Bau uud Gruppierung der Eiweißkörper. Vgl. ferner auch die zahlreichen Ergebnisse der Physiologie. 1." 1. Abt. 7.39 (1902). Arbeiten von Ih. Curtius und seinen Schülern im Journ. f. prakt. Cheni. Aon 8818 an. -) Möglich sind auch die folgenden drei Formen: — NH, CH„ CO . . iJ . NH . CH, COO. . . : I NHj CH, C OH) N CH, COOH NH, Ch' C (OH): N CH, COO. . . . . 1^ ( : . . . . '. . Abderhalden, Physiologische Chemie. I. Teil, 5. Aufl. 1 23 XVIII. Vorlesung. 354 HOOC CH2 . NH H + HO OC CH^ — H, = NH, Glykokoll . . GlykokoU HOOC. CH^ NH. CO . CH^ Glyzyl-glyzin. . . . NH^.!) Das Glyzyl-glyzin läßt sich wieder unter Aufnahme von Molekül Wasser in zwei Moleküle Glykokoll spalten: HHO HOOC CH, XH OC CH2 . . . . . Glyzyl-glyzin einem NH2 = HOOC CH^ NH^ + HOOC CH^ NH^ Glykokoll Glykokoll. . . . . In ganz entsprechender Weise können wir aus zwei verschiedenen Aminosäuren derartige Verbindungen aufbauen. Als Beispiel seien Glyzylalanin und Alanyl-glyzin und ferner Alanyl-leuzin und Leuzylalanin angeführt: NH, .CH, .COiOH + HNH.CH.CHa— H,0 = .Vi/;..CÄ2C0.NH.CH.CH • I 1 COOK COOK Alanin Glykokoll Glyzyl-alanin. CH3 .CH .COiOH + HNH.CH« COOH — H^Or^ . I NH2 Glykokoll Alanin CH, .CH.CO.'Sn. CH.. COOH . 1 NHo Alanyl-glyzin. /CH3 CH3 . — H, CH COiOH + H NH CH CH, CH . . I . . \cH I COOH NH, Alanin Leuzin. /CH3 CHs . CH CO NH CH CH, CH . . . . . ^^^ COOH NH2 Alanyl-leuzin. Qjj'>CH . CH, . CH CO OH . NH, Leuzin ) Die- einzelnen Anteile, aus verschiedenen Druck gekennzeichnet. -\- H:NH CH CH3 . . — H3 = COOH Alanin. denen diese Verbindungen bestehen, sind durch Eiweißstoffe und ihre Bausteine. C^^yCH CH. . . 355 CH CO NH CH . . . CH^ . ' I I COOK XH, Leiizyl-alanin. Es lassen sich auch mehr als zwei Aminosäuren in der gleichen Weise vereinigen. Etnil Fischer hat eine Verbindung dieser Art aufgebaut, die achtzehn einzelne Aminosäuren in säureamidartiger Verkuppelung enthält, nnd Abderhalden und Fodor^) sind zu einer Kette von neunzehn Gliedern gelangt. Als Beispiel einer solchen Verbindung sei das aus den fünf Aminosäuren Glyzin, Alanin, Serin, Leuzin und Tyrosin aufgebaute Glyzylalanyl-seryl-leuzyl-tyrosin angeführt. Die folgende Formel soll zeigen, wie unter Abspaltung von vier Molekülen Wasser die neue Verbindung entsteht. Umgekehrt zerfällt sie wieder in ihre fünf Bausteine, sobald die gleiche Menge Wasser aufgenommen wird: OH\|H OH\|H OHjH OH) CO .^R HfOH HOOC (HO C, H, CH,) CH HN OC (C, Hg) CH Gehen wir von Monoamino-dikarbonsäuren aus, dann ergeben sich verschiedene Möglichkeiten. Asparaginsäure und Glutaminsäure NH. CHo . (7(9 . . NH . ("H(CH3 ) . CO . NH CH(CH . . . I . . . . . . . können die folgenden Verbindungen liefern. Als Beispiel dung von Asparaginsäure mit Glykokoll gewählt: . sei die Verbin- NH CH COOH COOH CO m..wL.co .CH. yn, ch nh, CHg CH2 . . . . 1 I COOH COOH Asparagyl-monoglyzin I. Glyzyl-aspara'ginsäure COOH I CH NH, . CH, CO. VF. CH^ COOH Asparagyl-monoglyzin IL . CO NH CH, COOH . . CO . . NH CH COOH . . CH NH CO CH NH^ . . . . CH NH . 1 I CH2 CH2 ' 1 I NH CH^ COOH Asparagyl-diglyzin CO . . CO . M E. Abderhalden und Ä. Fodor: . NH . CH, . COOH Glyzyl-asparagyl-diglyzin. Ber. d. Deutsch. Chcm. Ges. 49. 561 (1916). 23 XVIII. Vorlesung. 356 Auch die Diarainosäure Lysin läßt sich verschiedener Weise mit in Aminosäuren verbinden: CH., . CH2 CH., . GH.. . . CH CO .Y^ CH, . . . . CO OH " 1 I NH, NH-, CH, Lysyl-glyzin. CH., CH, CHo CH . . . . NH CO NH.> . CHo r . CH, . NH CO . CH., . CH., NH CO . . . . . ' . . NH,^ . '1 " . . CH, . CH., CH, CH., CH, XH, NH, Monoglyzyl-lysin H. CHo . . Monoglyzyl-lysin I. CH, CH, .^CH, CH COOH NH CO CH., COOH . . . . . CH2 NH, . . CH COOH . NH CO . biglyzyl-lysin. CH, CH CO . XH, . . CH, . NH . NH, CH, . . COOH NH CO CH, XH, . . . Diglyzyl-lysyl-glyzin. Hierzu sei erwähnt, daß Beobachtungen vorliegen, \'\"onach im Eiweiß Lysin eine NH., -Gruppe, und zwar die in s-Stellung befindliche frei hat.i) Von den angeführten Möglichkeiten kämen somit nur die beiden ersten für das Eiweiß in Frage. Emil Fischer hat derartige Verbindungen Polypeptide genannt. 2) Sind zwei Aminosäuren säureamidartig vereinigt, dann spricht man von einem Dipeptid, von einem Tripeptid, wenn sich drei Bausteine finden usw. Die oben (S. 355) mitgeteilte Verbindung wäre somit als ein Pentapeptid zu bezeichnen. Die Aminosäuren selbst sind in Analogie mit den Bezeichnungen Monosaccharid, Di-, Tri-, Tetra- und Polysaccharide als Monopeptide oder einfach als Peptide zu benennen. Gegenüber der Nomenklatur der Kohlehydrate besteht nur insofern ein scharfer L^nterschied, als der Name Polypeptid für Verbindungen mit bekannter Struktur festgelegt ist, während wir mit der Bezeichnung Polysaccharid keine bestimmte Struktur verbinden, sondern mit diesem Namen nur zum Ausdruck bringen wollen, daß mehrere Monosaccharide miteinander vereinigt sind. Über die Art ihrer Verkuppelung sagt der Name Polysaccharid gar nichts aus.-^j Die Struktur der Polypeptide ergibt sich ohne weiteres aus ihrer Synthese. F^s seien die einzelnen Arten der Synthese der Polypeptide kurz gescliildert. Das Studium der Synthese der einzelnen D. van Slyke und F. J. Birchard: J. of biol. Ohein. 16. 539 (1914). Emil Abderhalden : Neuere Vgl. Emil Fischer: Untersucbungeu, 1. c. S. 310. Ergebnisse der Eiweißchemie. 1. c. S. 310. \^gl. auch die Arbeiten von Theodor Curtius und seinen Schülern: z. B. Ber. d. Deutschen Obern. Gesellsch. 35. 3226 (1902); 37. 1284 1) ^) — — (1904). ^) Es muß dies hervorgehoben werden, weil leider vielfach definierbaren Produkte ganz einfach Polypeptide genannt werden. alle möglichen un- und ihre Bausteine. Eiweißstoft'e 357 Verbindungen gibt uns zusammen mit der Verfolgung der Abbauversuche am besten einen klaren Einblick in ihren Bau. Zunächst wurde beobachtet, daß Aminosäureester leicht unter Abspaltung von Alkohol in Anhydride, genannt 2, ö-Diketopiperazine übergehen. Es sei dies am Beispiel des GlykokoUesters dargestellt: /GH., .CO. O.CaHsi N— H\ ^H — 2C0H5.OH NH CA-OOC.CHy 2 Äthylalkohol Moleküle Glykokoll-äthylester ^CHo^ . CO \e = XH ^CO . GH., Glyzinanhydrid. Läßt man auf das entstandene Glyzinanhydrid Alkali einwirken, bildet sich, wenn die Hydrolyse vorsichtig ausgeführt wird, das Dipeptid Glyzyl-glyzin. Der Ring wird an einer Stelle zwischen einer NH GO-Biiidung unter Wasstreintritt aufgeschlossen: dann — .cn. . CO /C^ CO OH H • .- TiTTT J\ll ..• \V- oder \vrTT /^^ W.GH, \ In beiden Fällen NH.3 GH, CO glyzin : . . u" \C0 NH . GH., OH entsteht . -- / .. . die gleiche Verbindung, nämlich Glyzyl- CH2 COOH. Wird die Hydrolyse vollständig . durchgeführt, dann bilden sich zwei Moleküle Glykokoll. Wir können auch gemischte Anhydride darstellen, z. B. Glyzylalaninanhydrid oder Leuzyl-tyrosinanhydrid usw. Bei der Aufspaltung derartiger Diketopiperazine können zwei strukturisomere Dipeptide entstehen, wie das folgende Beispiel zeigt: Alaninrest CH, I CH C0\ . \NH + H, = XH. CH. CO NH GH (GH,) COOH . A'^ / \C0 . GH., Glyzinrest Glyzyl-alaninanhydrid bzw. Alanyl-glyzinanhydrid. . . . Glvzvl-alanin. . XVIII. Vorlesung. 358 Alaninrest NH = XH, CH{GH,) CO NH CH, COOK NH + H, ^ \ r \^0 jj . . . CH// . . . Alanvl-fflyzin. ^ Ji^ ^ ^ Glyzinrest Erfolgt die Aufspaltung des Alanyl-glyzinauhydrides bei I. dann erhalten wir Glyzyl- alanin. Tritt dagegen bei II ein Molekül Wasser ein, dann gelangen wir zu Alanyl-glyzin. Umgekehrt liefern auch beide Dipeptide das gleiche Anhydrid. Diese kurze Darlegung zeigt schon, daß diese Art der Synthese von Polypeptiden nicht weit führen kann. Einmal können wir nur Dipeptide gewinnen, und dann erhalten wir außerdem Gemische von zwei strukturisomeren Verbindungen, sobald wir von Diketo- piperazinen ausgehen, die Aminosäuren entstanden aus der Vereinigung von zwei verschiedenen sind. Die folgende Art der Synthese von Polypeptiden ermöglicht eine mannigfaltigere Art des Aufbaues. Sie ergibt zugleich ein sehr klares Bild der Struktur derartiger Verbindungen. Emil Fischer ging von Halogenazylchloriden aus. Als Beispiel wollen wir die Synthese von Glyzylglyzin wählen. Als die eine Komponente zum Aufbau des genannten Dipeptids nehmen wir Gly kokoll. Diese kuppeln wir in alkalischer Lösung mit Chlorazetylchlorid. Dieses Säurechlorid können wir leicht von der Essigsäure ableiten, wie die folgenden Formeln zeigen: CH, COOH Essigsäure GH., . . COOH GH., . CO . Gl i \| Gl Cl M onochlor essigsaure Chlorazetylchlorid. Die Bildung des Glyzyl-glyzins erfolgt in den folgenden Phasen: Cl.CHo. GOicr+HNH.CH2.COOH=Ö.Ci72.C'O.NH.GH,.COOH-l-HCl Chlorazetyl-gjlyzin. Chlorazetylchlorid GlykokoU aC^2.CO.NH.GH2.COOH + NH3=.Yi72.Ci72-CO.NH.CH,.COOH-\|-HCl.i> Glyzyl-glyzin. Chlor azetyl-glyzin Läßt man auf dieses Zunächst bildet sich Ammoniak einwirken, dann erhalten wir das gesuchte Dipeptid Glyzylglyzin. Dieses können wir nun z. B. mit dein dem Alanin entsprechenden a-Brompropionylbromid kuppeln. Wir erhalten y.-Brompropiony 1- Chlor azetyl-glyzin. glyzyl-glyzin. Durch Einwirkung von Ammoniak, d. h. durch Aminierung entsteht daraus das Tripeptid A 1 an yl- glyzyl-glyzin: Br . CH (GH3) GOBr . -f- a-Brompropionylbromid ^) Bzw. NH4 . Cl bei H NH A^ CH, COOH = CH, CO Glyzyl-glyzin . . . . . AnweucUiug eines Überschusses von Ammoniak. und ihre Bausteine. Eiweißstofi'e XH 359 CO NH CH, CO GH., CO OH + HBr y.-Brompropionyl-glyzyl-glyzin. Br. CH(CH,) . . . . . . . + NH3 = NH^ CH(CHJ CO NH CH, CO NH CH, COOH + HBr . . . . . . . Alanyl-glyzyl-glyzin. Wollen wir das Tetrapeptid Leuzyl-alanyl-glyzyl-glyzin darstellen, so verbinden wir das Tripeptid Alanyl-glyzyl-glyzin mit a-Bromiso- kapronylbromid und aminieren dann das entstandene a-Bromisokapronyl- alanyl-glyzyl-glyzin. BrXm(C,E,).COBr + HNH.CH(CH,).CO.l!sE.CE„CO.NH.CH,.COOH a-Bromisokapronylbromid Alanyl-glyzyl-glyzin = Br .CmC\B,) .CO XH CH(CH,) CO .l!^E.CE, .CO XH CH, COOH 7.- Bromisokapronyl- alanyl-glyzyl-glyzin -fNHa— HBr = NH,.CH(C,H9).C0..Y^. CH(CHJ CO .-^^iR .CR,. CO XH CH, COOH . . : . . . . . . . Leuzyl-alanyl-glyzyl-glyzin. Auf diese Weise können wir zu beliebig langen Ketten von Aminogelangen. Diese Methode der Darstellung von Polypeptiden gestattet immer nur die Verlängerung der vorhandenen Kette von säureresten Aminosäureresten von der freien Aminogruppe aus. Dort greift das einwirkende Öäurechlorid mit seiner Karboxylgruppe ein. In vielen Fällen möchte man gerne ein vorhandenes Polypeptid am Karboxylende verlängern. Emil Fischer ist es geglückt, auch diese Möglichkeit zu verEs gelang ihm, Aminosäuren und Polypeptide direkt entsprechenden Säureehloride zu verwandeln. So kann man aus Glykokoll Glyzylchlorid bereiten und damit direkt ein Polypeptid darstellen. So erhält rtian aus Glyzj^lchlorid und Alan in unmittelbar Glyzyl-alanin ohne den Umweg über die Halogenazylverbindung: wirklichen. in die NH2 CHo CO :C1 -h H NH CH, COOH = NH, Oö, 00 NH CH, C0( )H Glyzylchlorid Glyzin Glyzyl-glyzin. . . . . . . . . . . Nun kann man auch ein Polypeptid chlorieren und direkt mit einem zweiten Polypeptid kuppeln. Als Beispiel wollen wir annehmen, daß das Tetrapeptid Alanyl-glyzyl-leuzyl-valin chloriert worden sei und mit dem Hexapeptid Glyzyl-alanyl-glyzyl-leuzyl-glyzyl-tyrosin gewerde. Es entsteht das Dekapeptid Alanyl-glyzyl-leuzylvalyl-glyzyl-alanyl-glyzyl-leuzyl-glyzyl-ty rosin: kuppelt NH, CH(CH,) CO NH CH,. CO NH CH(C^ HJ 00 NH CH (C3 H^) CO Cl-hH AW CH, 00 NH CH (CH3) CO XH CH, CO NH CH (C.Hg) CO XH CH, CO NH CH (CH, CgH, OH) COOH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dekapeptid. Als Beispiel für die Struktur hochmolekularer Polypeptide sei diejenige des synthetisch aus 19 Aminosäuren dargestellten Polypeptids 1-Leuzyl- triglyzyl-l-leuzyl-triglyzyl-l-leuzyl-triglyzyl -1-leuzyl-pentaglyzyl-glyzin angeführt 1): ) Emil Abderhalden und A. Fodor: Ber. d. Deutsch. Chem. Ges. 49. 561 (191(3). XVIII. Vorlesung. Eiweißstoffe und ihre Bausteine. 3^30 CH3 CH3 CH CH3 CH3 \ / CH CH, CH CO . CH NH.. — (NH CHo C0)3 \|— NH CH CO — (NH CH^ . . . . . . CO)3\|2 — Leuzyl - triglyzyl - 1 - leuzyl - 1 - triglyzyl - 1 - leuzyl triglyzyl- 1 - - NH CH CO — (NH . . . CH2 . C0)5 — NH . CH., . COOH CH2 1 CH /X CH3 CH3 1 - leuzyl - pentaglyzyl - gly zin. von Verbindungen dieser Art darSchon manche Tripeptide zeigen eine Reaktion, der wir bei den Aminosäuren nicht begegnet sind, die jedoch von den Peptonen und auch den Proteinen gegeben wird. Es ist dies die Biuretreaktion. Gibt man zur Lösung eines solchen Polypeptids Natronlauge und dann vorsichtig stark verdünnte Kupfersulfatl()sung, dann erhält man eine rosarote bis violettblaue Färbung. 8ie ist verschieden je nach der Art des Polypeptids. Ferner geben die Polypeptide die Farbreaktionen der an ihrem Aufbau beteiligten Aminosäuren. Tyrosinhaltige Polypeptide geben die Xanthoproteinreaktion und eine Rotfärbung mit Millons Reagens. Findet sich nur Phenylalanin, dann ist die letztere Reaktion negativ, während die erste positiv ausfällt. Tryptophan verrät sich als Es sind gestellt bereits weit über hundert worden. Baustein eines Polypeptids durch den positiven Ausfall der Gly 0x3^1- säureprobe, dagegen fällt die Bromwasserprobe negativ aus.^) Erst, wenn das Tryptophan in Freiheit gesetzt wird, läßt es sich mit Bromwasser erkennen. Enthält ein Polypeptid Zystin, dann gibt dieses beim Kochen mit Alkali unter Zusatz von Bleiazetat Ausfällung von Bleisulfid (Schwefel bleiprobe). Die Polypeptide ergelien alle mit Triketohydriiidenhydrat (Ninhydrin) Blaufärbung. Einige davon lassen sich, wie schon erwähnt, aussalzen. Tyrosin und Zystin begünstigen als Bausteine in Polypeptiden die Aussalzbarkeit. doch scheint es auch auf die Anordnung der einzelnen Aminosäuren im Molekül anzukommen. Die hochmolekularen Polypeptide zeigen beim Kochen Erscheinungen, die an Koagulation erinnern. Während die einfacheren Polypeptide mehr oder weniger leicht zur Kristallisation zu bringen sind, sind die hochmolekularen Verbindungen dieser Art bis jetzt nur im amorphen Zustande bekannt. Interessant ist auch die Eigenschaft der wässerigen L(»snng der hochmolekularen Polypeptide, zu schäumen. Nach ihren allgemeinen Eigenschaften stehen die Polypeptide den Peptonen oder, besser ausgedrückt, deren Bestandteilen sehr nahe. ') E. Ahderhahlii) und Marliti h'cin/ji:: Ber. d. Deutschen Cliem. Ges. 40. 2737(1907). Vorlesung XIX. und ihre Bausteine. Eiweißstoffe 3. Die Struktur Polypeptide Peptone. der EiweißstofFe. als Bestandteile der Mit den Polypeptiden haben wir Verbindungen kennen gelernt, die mehrere Aminosäuren säureamidartig verknüpft enthalten. Sie entstehen unter Wasseraustritt aus Aminosäuren und zerfallen wieder in diese unter Wasseraufnahme. Wählt man zum Aufbau derartiger Verbindungen optisch-aktive Aminosäuren, dann erhält man auch optischaktive Polypeptide. Am wichtigsten sind zur Aufklärung der Beziehungen der Polypeptide zu Eiweiüabbaustufen und zum Eiweiß selbst jene Aminosäureketten, an deren Aufbau die Bausteine der Proteine teilnehmen. Verwendet man Aminosäurechloride, dann kann man die bei der Hydrolyse von Proteinen gewonnenen Aminosäuren direkt untereinander verbinden. Sind die Aminosäuren synthetisch bereitet worden, dann müssen diestj^ da die Synthese im allgemeinen zu inaktiven Verbindungen führt, in ihre optisch-aktiven Komponenten zerlegt werden. Nur diejenige Form, die der in der Natur vorkommenden entspricht, kann direkt zur Synthese Ver- wendung finden, wenn es sich darum handelt, Polypeptide zu bereiten, In manchen die die in der Natur vorkommenden Bausteine enthalten. Fällen ist jedoch auch die andere Komponente durch Überführung in die entsprechende x-Bromfettsäure zur Synthese von Polypeptiden verwertbar. Es ist dies dann der Fall, wenn jene Umw^andlung mit einem Wechsel der Konfiguration verknüpft ist. Durch Chlorierung der Bromfettsäure entsteht das zur Kuppelung mit einer beliebigen Aminosäure oder auch einem Polypeptid geeignete Halogenazylchlorid. Nach erfolgter Aminierung des entstandenen Produktes ist das gewünschte optisch -aktive Polypeptid fertiggestellt. Die folgenden Formeln mögen an Hand eines Beispiels Bildung von d- AI anyl-d- alanin die beiden Arten von Synthesen optisch-aktiver Polypeptide erläutern — — 1. Verwendung von d-Alanylchlor id und d-Alanin: NH2 CH (CH3J CO; Cl -f H NH CH(CH3) COOH = . . . . d-Alanylchlorid NH.3 . CH . (CH3) . d-Alanin . CO NH CH (CH3) COOH . . d-Alanyl-d- alanin. XIX. Vorlesung. 362 2. Ausgangsmaterial: dl-x-Brompropionsäure: CH3 CH COOH + NH3 = CH3 ( H COOK + HBr . . . . Br dl -x-Bromprop ionsäure dl-Alanin. Spaltung des Razemkörpers in d-Alaniu 1-Alanin + (NOBr) i d-x- Brom Propionsäure. (Chlorierung) d-x-Brompropionylchlorid CH3 . + H NH CH (CH3) COüH CH CO Cf . . . . Br CH3 . CH CO NH. CH(CH.) COOH , . . Br d-a-Brompro,'pionyl-d- alanin + NH3 CHg . CH CO XH. CH(CHJ COOH . . . NHo d-Alanyl-d- alanin. Wir haben bereits festgestellt, daß die Eigenschaften der Polymanchen Punkten große Ähnlichkeit mit denen der Peptone peptide in Es gilt dies vor allem für die höher molekularen Produkte. Selbstverständlich genügt dieser Umstand nicht, um den Schluß zu rechtfertigen, daß in den Proteinen und ihren Abbaustufen die Aminosäuren so untereinander verkettet sind, wie in den synthetisch gewonnenen Polypeptiden. zeigen. Die nächste Aufgabe war nun, zu versuchen, aus einem Gevon zusammengesetzten Abbaustufen Produkte abzutrennen, die in allen Eigenschaften und in ihrer Struktur vollständig mit bestimmten synthetisch gewonnenen Polypeptiden übereinstimmten. Leider zeigen die Polypeptide keine so charakteristi- misch schen Eigenschaften, daß es möglich wäre, das Vorhandensein einer bestimmten Verbindung dieser Art sofort zu erkennen. Manche ihrer Eigenschaften sind von ijestimmten Bausteinen abhängig. Die gleichen Eigenschaften, wie z.B. bestimmte Farbreaktionen, kehren immer wieder, so oft ein Polypeptid den diese Reaktion verursachenden Baustein enthält. Bedingt ein Baustein bestimmte Fällungsreaktionen, dann linden wir diese häufig bei allen Ver- Eiweißstoffe und ihre Bausteiue. 1-363 bindungen. die ihn enthalten. Hat sieh somit die Hoffnung, auf einem einfachen Wege aus Peptongemischen Polypeptide abzutrennen, auch nicht erfüllt, so ist es doch in einer ganzen Reihe von Fällen gelungen, aus Peptonen Produkte zu gewinnen und zu reinigen, die sich als chemisch die einheitlich erwiesen. Sie konnten ferner mit den entsprechenden, gleichen Aminosäuren und in gleicher Menge besitzenden, synthetisch bereiteten Polypeptiden identifiziert werden. Mit dieser Feststellung ist bewiesen worden, daß unter den Peptonen sich Verbindungen finden, die zu den Polypeptiden gehören und ferner, daß im Eiweißmolekül Aminosäuren säurearaidartig verkettet sind. Es ist wohl möglich, daß die Peptone überhaupt nur ein Gemisch der verschiedenartigsten Polypeptide darstellen, in denen stets die eine Aminosäure mit ihrer Aminogruppe in das Karboxyl der anderen eingreift. Es ist aber auch sehr gut denkbar, daß noch andere Bindungsarten von Aminosäuren vorhanden sind. Vor allem dürften die Oxy-aminosäuren Abwechslung in das sonst gleichförmige Bild der Struktur der Proteine und ihrer zusammengesetzten Abbaustufen bringen. Auch die Diamino- und Dikarbonsäuren lassen verschiedenartige Bindungen zu. Vor allem kann bald eine Karboxyl- bzw. eine Aminogruppe frei bleiben, oder es sind alle verfügbaren Bindungsmöglichkeiten ausgenützt. Das erste Polypeptid, das bei der Hydrolyse von Eiweiß erhalten worden ist, war ein aus Gly kokoll und Alanin bestehendes Dipeptid, das sich als Glyzyl-d-alanin erwies: NH., . CH.2 . CO . NH CH(CH^) CO OH. . . Es wurde erhalten ^ -), als Seidenfibroin mit TOVoiger Schwefelsäure drei Tage bei Zimmertemperatur hydrolysiert wurde. Der Abbau geht hierbei nicht vollständig bis zu Aminosäuren. Die Isolierung des Dipeptids erfolgte nich auf direktem Wege. Es wurden vielmehr die Abbaustufen zunächst in der schon geschilderten Weise in die Esterchlorhydrate übergeführt. Die aus diesen gewonnenen freien Ester zeigten nach einiger Zeit kristallinische Abscheidungen. Es hatten sich Anhydride gebildet. Diese können auf zwei Arten entstehen, einmal aus Estern der Aminosäuren und dann aus solchen von Dipeptidcn. Die erstere Möglichkeit mußte durch Entfernung der Aminosäureester durch Ausäthern ausgeschlossen werden. Das beobachtete Anhydrid konnte nunmehr nur noch aus einem Dipeptidester sich gebildet haben. Zu seiner Identifizierung dienten zunächt seine Eigenschaften, vor allem sein Drehungsvermögen. Ferner konnte es zum Dipeptid aufgespalten und schließlich auch in seine Bausteine zerlegt werden. Es zeigte sich, daß Gly kok oll und d-Alanin an seinem Aufbau beteiligt waren. Das beobachtete Anhydrid war somit Glyzyl-d-alaninanhydrid bzw. d-Alanyl-glyzin- anhydrid.^) Aus welchem Dipeptid es enstanden war, ob aus Glyzyl-d alanin oder aus d-Alanyl-glyzin, konnte nicht entschieden werden, weil beide Dipeptide das gleiche Anhydrid liefern. Schon diese Feststellung be») F.Diil 752 (190(5). Fischer uud Emil Abderhalden : Ber. d. Deutscheu Chem. Gesellsch. 39. — Enal -) Emil Fischer und Emil Abderhalden : Ebenda. 39. 231ö (IVIÜG). Abderhalden und Akikazu Suiva: Zeitschr. f. pbvsiol. Chem. 66. 13 (1910). ») Vgl. auch Emil Abderhalden: Zeitschr" f. physiol. (.'hemie. 120. 207 (1922). XIX. Vorlesuug. 3(34 weist, daß die Abtrennung von Polypeptiden auf dem erwähnten Wege nur be- weist, daß Dipeptide mit bestimmten Bausteinen in einem Gemisch von Eiweißabbaustufen anzutreiben sind, jedoch läßt sich nicht bestimmen, welches Dipeptid das Anhydrid geliefert hat. Ferner ist diese Methodik nur zur Isolierung von Dipeptiden geeignet. In ganz entsprechender Weise wurde unter den Abbaustufen der Seide Glyzyl-1-tyrosinanhydridi) gewonnen. Ferner glückte es, aus Elastin Glyzyl-l-leuzinanhydrid^), Glyzyl-d-alaninanhydrid^) und Glyzyl-d-valinanhydrid-) und aus Gelatine Glyzyl-I-prolinanhydrid^) zu isolieren. Die Gelatine war mit Trypsin verdaut worden. Glyzyl-1-phenylalanin) ist ferner aus dem Darminhalt gewonnen und außer als freies Dipeptid auch als Anhydrid identifiziert worden. Endlich aus Kaseinogen Isoleuzyl-valinanhydrid erhalten worden. •^) Ferner hat F. G. Hopkins^) aus Zellen des tierischen Organismus und auch aus Hefe eine aus zwei Aminosäuren bestehende Verbindung isoliert, der ohne Zweifel bei der Zellatmung eine bedeutungsvolle Rolle zukommt. Sie besteht aus den Bausteinen Zystein und Glutaminsäure und ist wahrscheinlich als Dipeptid zu betrachten.') Eine endgültige Entscheidung, ob sie wirklich die Struktur eines solchen hat, vermag erst eine Yergleichung mit den Eigenschaften der entsprechenden synthetisch dargestellten Verbindung zu geben. ist Aus diesen Befunden ergibt sich, daß unter den Abbaustufen der verschiedensten Proteine Dipeptide anzutreffen sind. Es ist schließlich auch gelungen, solche direkt abzutrennen und ihrer Struktur nach vollständig aufzuklären. So gelang es, aus Elastin d-Alanyll-leuzin^), 1-Leuzyl-d-alanin-'), 1-Leuzyl-glyzin und GlyzylI-leuzin^") darzustellen. Seidenfibroin ergab d-Alanyl-glyzin, Glyzyl1-tyrosin und Glyzyl-d-alanin.i') Aus Gliadin wurde 1-Leuzyld-glutaminsäurei"-) isoliert. Das gleiche Protein lieferte auch 1-Prolyl]-phenylalanin.^3) Unter den Verdauungsprodukten von Eiweiß, gewonnen aus dem Darminhalt, ließ sich endlich, wie schon erwähnt, Glyzyl1 "Phenylalanin nachweisen.") ') Emil Fischer und Emil Abderhalden : Ber. d. Deutscheu Chem. (liesellsch. 39. — Emil Abderhalden und Akikazu Siina: Zeitschr. f. physiol. Chemie. 231.T (1906). 66. 13 (1910). E7nil Fischer und Emil Abderhalden: Ebenda. 40. 3544 (1907). F. A. Letene und W. A. Bcatfy: Ebenda. 39. 2060 (1906). P. A. Lerene und G. B. Wallace: Zeitschr. f. phvsiol.' Chem. 47. 143 (1906). F. A. Levene: Ber. d. Deutschen Chem. Gesellsch. 43. 3168 (1910). > ) Emil Abderhalden : Zeitschr. f. physiol. Chem. 81. 315 (1912). '") JI. D. Dahin: Biochem. -louru. 12.' 290 (1918). «) F. 0. Hopkins: The l)iochemical .1. 15. 286 (1921). ') Vgl. auch S. 318. ) Emil Fischer und h'jiiil Abderhalden: Her. d. Deutschen Clicm. Gesellsch. 40. 3544 (1907). ») Emil Abderhalden: Zeitschr. f. plivsiol. Chem. 58. 373 (1908). '") Emil Abderhalden: Zeitschr. f. phvsiol. Chem. 62. 315 (1909). »') h'mil Abderhalden: Ebenda. 62. 315 (1909); 63. 401 (1909): 65. 417 (1910.) Emil Abderhalden und Ryngo Jnoui/: Ebenda. 80. 19S (1912). '-) PJmil Abderhalden und Emil Fischer: Bor. d. Deutschen Chem. Gesellsch. 40. 3544 (1907). '') Thomas B. Osborne und .S'. H. Clapp: Americ. Jouru.of Physiol. 18- 123 (1907). ') Emil Abderhalden: Zeitsch. f. physiol. Chemie. 81. 315 (1912). -) ^) — — - und ihre Bausteine. Eiweißstort'e 35Ö Aus der Seide ist noch ein Dipeptid in Form eines Derivates gewonnen worden. M Wir haben früher schon erwähnt, daß man in den Aminosäuren die freie Aminogruppe mit Säurechloriden verschiedener Art verbinden kann. So ergibt z. B. Glykokoli mit Ji-Naphtalinsuifochlorid, SO., Cl, die folgende Verbindung: C10H7 . . CH, . COOK C'Ho NH2 Glykokoli . COOH NH.SO., .CioH, Ji-Naphtalinsulfo-glyzin. Auch die Polypeptide verfügen, wie die wiederholt mitgeteilten Formeln beweisen, über mindestens eine freie Aminogruppe es können deren auch mehrere sein, wenn Diaminosäuren an ihrem Aufbau beteiligt und in diesen nicht beide Aminogruppen besetzt sind. Die freie Aminogruppe kann genau so wie in den Aminosäuren mit Säurechloriden in Reaktion treten. Diejenigen Aminosäuren, deren Aminogruppe mit dem Karboxyl der nächsten Aminosäure verbunden ist, vermögen keine solche Verbindung mehr einzugehen, denn ihre Aminogruppe ist ja bereits besetzt! Dieser Umstand ermöglicht es. zu entscheiden, welche am Aufbau eines Polypeptids beteiligte Aminosäure eine freie Aminogruppe trägt. Wird nämlich ein Polypeptid, in das man z. B. den Naphtalinsulforest verankert hat, vollständig hydrolysiert, dann erhält man alle jene Aminosäuren als solche, die nicht mit dem Säurechlorid in Reaktion treten konnten. Diejenige Aminosäure jedoch, die über die freie Aminogruppe verfügte, erscheint unter den Spaltprodukten als Naphtalinsulfoderivat. Das folgende Beispiel erläutert das eben Gesagte: — CK.. CO. -^HXE(CH,). CO. XH.CH(C^H^). CO. '^UX^.CH^.C^E.On " I I COOH JVi7.su, .CioH, ß-Naph ta 1 i ns u 1 fogly zy 1- alany 1-1 e uz yl-ty rosin + 3H.,0 CH, . COOH CH3 . + i NH.SOo.CjoH, + Cß H, OHj ( . CH^ . CH COOH . I NH, NH, Alanin (i-Naphtalinsulfoglyzin _ r-u'>CH ^^' . + ' i .,„ CH COOH Leuzin. . CH, CH COOH . . i NH2 Tvrosin. ) Emil Fischer und Emil Abderhalden : Ber. d. Deutschen C'hein. Gesellsch. 40. 3544 — Vgl. dazu auch Emil Fischer (und Peter Berr/ell) Bericht der NaturforscherIn dieser Mitteilung findet sich die erste Angabe über ein ß-Naphtalinsulfoderivat aus Abbauprodukten der Seide, das anscheinend einem Dipeptid entsprach. Vgl. auch Emil Abderhalden und Casimir Funk: Zeitschr. f. (1907). Versammlung. Karlsbad 1902. — physiol. Chem. 64. 436 (1910). : ^IX. Vorlesung. 366 Es ist mm bei der Hydrolyse von Seidenfibroin nach erfolgter Kuppelung der Spaltungsprodukte mit ß-Naphtalinsulfochlorid eine Verbindung erhalten worden, die bei der Hydrolyse mit verdünnter Salzsäure ß-Naphtalinsulfo-glyzin und d-Alanin ergab. Daraus folgt, daß die Verbindung die folgende Struktur gehabt hat: CH, CO NH CH(CH3) COOH. Nur diese Verbindung konnte die erwähnten . . . . " 1 NH. SO,. C^^n^ ß-Naphtalinsnlfoglyzyl-d-alanin Spaltprodukte liefern. Das strukturisomere ß-Naphtalinsulfo-d-alanyl-glyzin ergibt bei der Spaltung ß-Naphtalinsulfo-d- alanin und Glyzin: CH, . CH CO XH. CH. COOH+ H. . . I = CH, CH COOH + NH. CH, COOH . . . . I NH SO2 CioH, ß-Naphtalinsulfod-alanyl-glyzin . . NH . SO2 . CioH, ß-Naphtalin- Glykokoll. sulfo-d-alanin Mit dieser Feststellung war bewiesen, daß unter den Produkten der Hydrolyse des Seidenfibroins Glyzyl-d-alanin als Abbaustufe- partiellen auftritt. Aus Seidentibroin ist auch ein Tetra peptid gewonnen worden. 1) Es ergab bei der vollständigen Hydrolyse Glykokoll, d-Alanin und 1-Tyrosin. Die Bestimmung des Mengenverhältnisses, in dem die einzelnen Aminosäuren auftraten, zeigte, daß auf zwei Moleküle Glykokoll je ein Molekül d-Alanin und 1-Tyrosin kamen. Die Molekulargewichtsbestimmung ergab einen auf ein aus den genannten Bausteinen zusammengesetztes Tetrapeptid stimmenden Wert. Auch die Elementaranalyse lieferte Werte, die mit der Annahme eines solchen Polypeptides in Einklang standen. Mit dieser Feststellung war zunächst nur bewiesen, daß eine Verbindung vorlag, die Glykokoll, Alanin und Tyrosin in einem bestimmten Mengenverhältnis gebunden enthielt. Über die Struktur der Verbindung vermögen die erwähnten Befunde nichts auszusagen. Wir wollen an Hand dieses Tetrapeptides erläutern, welche Schwierigkeiten die Aufklärung der Struktur einer aus mehreren Aminosäuren zusammengesetzten Abbaustufe aus Proteinen aufweist. Gleichzeitig ergibt sich auch der Weg. den die Erforschung der Struktur des Eiweißmoleküls nehmen muß, wenn nicht ganz neue Gesichtspunkte in das ganze d^^orschungsgebiet hineingetragen und neue Methoden entdeckt werden. Setzen wir voraus, daß das erwähnte Produkt die Struktur eines Tetrapeptids hat, dann können wir aus den drei verschiedenen Aminosäuren, wobei die eine zweimal vertreten ist, zwölf strukturisomere Verbindungen aufbauen, Avenn wir die einzelnen Bausteine sich in verschiedener Reihenfolge Wir müßten somit ein Tetrapeptid nach dem andern synthetisch bereiten, an dessen Aufbau zwei Moleküle Glykokoll, ein Molekül folgen lassen. ') (1907). (1915)]. Emil Fischer und Emil Abderhalden: ßer. d. Deutschen Chem. Ges. 40. 3544 \'gl. auch P. A. Levene und J. ran der Scheer [Jouru. of biol. Chem. 22. 425 Diese Autoren beschreiben ein lysinhaltiges Tripeptid aus Kasein. — Eiweißstotie und ihre Bausteine. 367 d-Alanin und ein solches von 1-Tyrosin teilnehmen, und jedesmal das erhaltene Produkt in seinen Eigenschaften mit denjenigen der durch Abbau der Seide erhaltenen Verbindung vergleichen. Es kann sein, daß der Zufall bald zu der gleichen Verbindung führt, es ist jedoch auch möglich, daß erst die zwölfte Verbindung dem isolierten Produkte entspricht! Nehmen wir an, daß vier verschiedene Aminosäuren am Aufbau eines Polypeptides beteiligt sind, dann haben wir bereits 24 strukturisomere Verbindungen vor uns. Fünf verschiedene Aminosäuren führen zu 120 verschiedenen Pentapeptiden, wenn wir die Reihenfolge der Bausteine wechseln, und bei sechs verschiedenen Bausteinen kommen wir zu 720 strukturisomeren Hexapeptiden. Die folgende Übersicht i) gibt die Zahl der strukturisomeren Verbindungen wieder unter der Annahme, daß bis zu 20 verschiedene Aminosäuren an ihrem Aufbau beteiligt und nur ein Mal vertreten sind: XIX. Vorlesuug. 368 Wir werden auf diese große Zahl von isomeren Verbindungen, die und allein durch noch zurückkommen. Ein sich aus relativ wenigen, verschiedenen Bausteinen einzig die verschiedene Reihenfolge aufbauen lassen, Blick auf die gegebene Übersicht zeigt, daß es ganz unmöglich ist, die Struktur eines bestimmten Polypeptids durch die Synthese der entsprechenden Verbindung aufzuklären, sobald die Zahl der einzelnen Arten von Aminosäuren ansteigt. Wir müssen uns nach Methoden umsehen, die gestatten, einzelne dieser strukturisomeren Verbindungen auszuschalten. Zunächst können wir versuchen, ein solches Produkt durch vorsichtigen Abbau in einfachere Verbindungen zu zerlegen, die auch noch mehrere Aminosäuren gebunden enthalten. In jedem einzelnen Bruchstücke muß dann z. B. mit Hilfe von ß-Xaphtalinsulfochlorid festgestellt werden, welche Aminosäure die freie Arainogruppe trägt. Vorher überzeugt man sich auf die gleiche Weise, welche Aminosäure im Ausgangsprodukt, also in unserem Falle im Tetrapeptid, imstande ist, die Naphtalinsulfogruppe zu binden. Die Hydrolyse dieser Derivate ergibt dann einerseits Naphtalinsulfo-aminosäuren und freie Aminosäuren. Das erwähnte Tetrapeptid ließ sich direkt in zwei Dipeptide spalten. Sie wurden als Anhydride isoliert. Das eine erwies sich als Glyzyl-1-tyrosinanhydrid und das andere als Glyzyl-d-alaninanhydrid. Aus diesem Befunde können wir schließen, daß im isolierten Tetrapeptid einerseits Gly kokoll und Ty rosin und andererseits Gly kokoll und Alan in sich in irgend einer Reihenfolge folgen. Wäre es gelungen, die beiden Dipeptide als solche zu gewinnen, dann wäre diese Reihenfolge festgelegt gewesen, und es hätte nur noch entschieden werden müssen, wie die beiden Dipeptide im Tetrapeptid unter sich vereinigt waren. Es bleiben nach diesem Ergebnis der partiellen Hydroh^se des Tetrapeptids noch folgende Möglichkeiten. Wir bezeichnen der besseren Übersichtlichkeit wegen Glykokoll mit G, Alanin mit A und Tyrosin mit T: l. G-A-G-T. 2. 6. G-T-G-A. A-G-G-T. Verbindungen, wie kommen nicht in Frage, 3. 7. G-T-A-G. T-G-A-G. G-G-A-T, 4. 8. G-G-T-A, G-A-T-G. T-G-G-A. 5. A-G-T-G. A-T-G-G oder T-A-G-G, Hydrolyse nicht die erwähnten Spahprodukte liefern können. Die vier verbleibenden Tetrapeptide müßten alle dargestellt werden, wollte man nach den bisherigen Befunden die Struktur des auf analytischem Wege erhaltenen Tetrapeptids vollständig sichern. Es ist nämlich durchaus nicht ausgeschlossen, daß zwei oder mehrere strukturisomere Polypeptide in ihren Eigenschaften sich so außerordentlich nahe stehen, daß sie kaum zu unterscheiden sind. Wir besitzen noch eine sehr wichtige Methode, um die Struktur eines bestimmten Polypejjtids aufzuklären. Es ist dies die Verfolgung seines Abbaus mittels polarisierten Lichtes. Die Polypeptide sind, wie schon erwähnt, oi)tisch-aktiv. sofern optisch-aktive Aminosäuren an Die einzelnen Polypeptide zeigen, je nach ihrem Aufbau beteiligt sind. ihrer Struktur ein verschiedenes Drehungsvermögen. >) So dreht z. B. Glyzyl-d-alanin 50" nach links, während d-Alanyl-glyzin 50" nach ) weil sie bei der partiellen Emil Abderhalden uud Andor Fodor : Zeitsclir. f. physiol. Cliemie. 81. 1 (1912) — Vgl. weitere Literatur bei Emil Abderhalden: Die Abderhaldensche Reaktiou. lage. J. Springer. 1922. 5. Auf- PJiwcißstoftc ihre Baiistciiio. 1111(1 fi69 rechts dreht. Glyzyl-d-alanyl-l-leuzin zeigt [ajogo^:— 90", d-Alanyl11", l-Leuzyl-d-alanyl-glyzin glyzyl-1-leuzin 17", l-Leuzyl60". und das g'lyzyl-d-alanin + 20". Glyzyl-1-leuzyl-d-alaniii sechste strukturisomere Tripeptid dieser Reihe d-Alaii3^1-l-leuzyl-gly30". Wir kennen ferner das Drehungsvermögen aller beim Abl)aii zin Die Art der dieser Tripeptide in Frage kommenden Bruchstücke. — — — — Drehungsänderung beim Abbau von Polypeptiden kann uns bestimmte Anhaltspunkte über ihre Struktur geben. Eiin Beispiel m()ge diese Art der Aufklärung der Struktur von Polypeptiden veranschaulichen. Es sei die Struktur eines aus 1-Leuzin, d-Alanin und GlykokoU be- stehenden Tripeptides aufzuklären. Wir wollen voraussetzen, daß ihm die Struktur 1-Leuzyl-glyzyl-d-alanin zukomme. Dieses kann nun beim vorsichtigen Abbau entweder 1-Leuzin und Glyzyl-d-alanin liefern oder aber d-Alanin und 1-Leuzyl-glyzin. Das erstere Dipeptid dreht oO" nach links, das letztere 85" nach rechts. Das Tripeptid selbst zeigt, wie schon erwähnt, [ajj'j"' = + 20". Beobachtet man nun beim Abbau "eines solchen einer viel stärkeren Rechtsdrehung und nacli Zurückgehen des Drehungsvermögens, dann darf' man schließen, daß unter Abspaltung von d-Alanin zunächst 1-Leuzyl-glyziii entstanden ist, das dann weiter in seine Komponenten 1-Leuzin und Tripeptids das Auftreten einiger Zeit ein starkes GlykokoU zerlegt wird: -f 1 - 20" Leuzy - gly zy - d - al a n n — 1 1 1()-H" -t- i + ()" !>•(:)" H5" --Ö0" / \ 1-Leuzyl-glyzin d-Alanin + +2.6" 85" 1-Leuzin GlykokoU —^ lO-o" 0" Der Abbau kann jedoch auch in umgekehrter Reihenfolge vor sich gehen, indem zuerst 1-Leuzin frei wird. Hierbei muß die Drehung zunächst nach links abweichen. Schließlich erfolgt dann die Hydrolyse des gebildeten Glyzyl-d-alanins und damit geht die Linksdrehung wieder zurück. 20" 1-Leuzyl-glyzyl-d-alanin \ ^' 1-Leuzin Glvzvl-d-ahmin — lu-:-"." — 500 GlykokoU d-Alanin 0'> -\-'2-6" Abderhalden, Physiologische fhurain. 1. 'I'eil. 5. Aiit'l. 2>4 370 ^^''^- Vorlesung'. Stellt man für jedes einzelne in Betracht kommende, strukturisomere Polypeptid die bei seinem stufenvveisen Abbau m()glichen Verbindungen fest, und bestimmt man für jede mögliche Abbaustufe das Drehungsvermögen, dann lassen sich auf diesem Wege oft über die Struktur des fraglichen Produktes Anhaltspunkte gewinnen. Vor allem kann man mittels dieses Verfahrens, das man als optische Methode bezeichnen kann, erfahren, wann von einer bestimmten Abbaustufe im Hydrolysat die größte Menge gebildet worden ist. Es läßt sich dann die Hydrolyse unterbrechen und die durch die Feststellung des Drehungsvermögens erschlossene Verbindung isolieren. Wir werden gleich erfahren, daß der stufenweise Abbau besonders mit Fermenten erfolgreich durchgeführt werden kann. Kehren wir nun zu dem erwähnten Tetrapeptid zurück. Es hat sich mit großer Wahrscheinlichkeit herausgestellt, daß es der Verbindung GlyzyI-d-alanyl-glyzyl-1-ty rosin entspricht. Es ist nämlich gelungen, aus den Abbauprodukten der Seide das Tripeptid d-Alanyl-glyzyl1-tyrosin zif gewinnen. i) Ferner ist es geglückt, das Tetrapeptid unter Abspaltung von Glykokoll in dieses Tripeptid überzuführen, und dann d-Alanyl-glyzin ist es auch gelungen, aus ihm die beiden Dipeptide und Glyzyl-1-tyrosin zu erhalten.-) Zur Sicherung der Struktur des (Erwähnten Tripeptids wurde noch die Kuppelung mit [i-Naphtalin) Die nachträgliche Hydrolyse ergab freies sulfochlorid ausgeführt. ^'^ Glykokoll, ß-Naphtalinsulfo-d-alanin und ferner Mononaphtalinsulfo-1-tyrosin. Das freie Tyrosin reagiert mit zwei Molekülen Naphtalinsulfochlorid. Ein Naphtalinsulforest wird von der Amino- und der andere von der Hydroxylgruppe gebunden, wie die folgende Formel zeigt: . SO2 . C,o H7 NH . SO., . Cio H, C CHo CH C'OOH Di-ß-naphtalinsulfo-ty rosin. . . Das im obigen Versuch erhaltene Naphtalinsulfo-ty rosin trug nur an der Hydroxylgruppe den Naphtalinsulforest. Die Aminogruppe war frei, seiner Aminogruppe mit der Karboxyl l'olglich muß das Tyrosin mit gruppe eines der beiden anderen Bausteine des Tripeptids verankert ge-. wesen sein. Da das Alanin als Naphtalinsulfoverbindung unter den Spaltprodukten des Naphtalinsulfo-tripeptids erschien, kommt ihm in demTripeptid die freie Aminogruppe zu. Folglich bleiben für die aus den Aminosäuren Glykokoll, d- Alanin und 1-Tyrosin bestehende Verbindung nur noch die folgenden beiden Möglichkeiten der Struktur übrig. Die Stellen, an denen ^) Emil Abderhalden uud Andor Fodor : Zcitschr . f. physiol. Chemie. 81. 1 (1912). 5. Aufl. — Vgl. weitere Literatur bei Emil Abderhalden Die Abderhaldensche Reaktion. : J. Springer. 1922. Nach neueren Beobachtungen. Vgl. auch Emil Abderhalden und Casimir Funk: Ebenda. 64. 43B ) Emil Abderhalden: Zeitschr. f. physiol. Chemie. 72. 1 (1911). -) ^) (lUlÜ). EiweiBstofle und ihre Bausteine. 1 Naphtalinsiilforeste eintreten kimnen, sind durch ein * Hnj;edeutet Gruppen, an die sie sieh und die durch Fettdruck liervorgehoben. anhig-ern, OH* NH, CH, . CH . CO . \H . CH, . CO NH CH (CH.,) COOH . . . !i-Nap htalinsulfo-al an yl-g'lyzyl-ty rosin. OH* NH, CH3 . C NH CHo COOH [i-NaphtaHnsulfo-alanyl-tyrosyJ-^lyzin. CH CO AT/ CH{CH,) . . . . CO . . . Beide Verbindungen ergeben bei der Hydrolyse: !i-Naphtasulfoalanin, Mono-naphtaHnsulfo-tyrosin und GlykokoH. Das Tyrosin trägt in beiden Fällen den Naphtalinsulforest an der Hydroxylgruppe. Die übrigen vier strukturisomeren Verbindungen kommen nicht in Betracht, weil sie, wie die folgenden Formeln zeigen, die erwähnten Spaltprodukte nach vorausgehender Kuppelung mit ß-Naphtaiinsultbchlorid nicht liefern kimnen: OH* (] * NH; CH, .CO. NH CH (CHi) CO NH CH (CH.,) ß-Naphtalinsulf<t-glyzyl-alanyl-ty rosin. . . . . . COOH OH* C I NH CH, [i - . CO . NH CH (CH,) CO NH VW {CH,) COOH . . . N a p h t al n s u 1 fo-gl y z y 1 - 1 y r i . . sy - a a n 1 1 i n. 24* XI\. Vorlesung. OH^ NH, I C . CH, . CII . CO . yH . ('H.> . CO NH . . VH (CH,) . COÖH j-Naphtalinsulfo-tyr(»syl-fi;lyzyl-alanin. OH NH, I C . CH, CH CO XH CH [CH.,] CO. NH CH, ß-NaphtalinsuU'o-tyrosyl-alan yl-glyzin. . . . . . . . COOH Die beiden ersten Tripeptide liefern bei der Hydrolyse ß-Naphtalinsulfoglyzin, Mononaphtalinsulfotyrctsin und Alanin und die beiden letzteren Dinapbtalinsulfotyrosin, Alanin und Glykokoll. Die Schilderung;- der g-roßen .Schwierigkeiten, die der Identitizierung eines isolierten zusammengesetzten Abl>auproduktes aus Eiweißstoft'en entgegenstehen, soll einerseits einen Blick in das ganze große Arbeitsgebiet geben und gleichzeitig verständlich machen, weshalb die Fortschritte in der Auffindung von Polypeptiden unter den Abbaustufen der Proteine so langsame sind. Es sind eine ganze Anzahl von Produkten isoliert, die ohne jeden Zweifel die Struktur von Polypeptiden besitzen, es gelang jedoch ihre Synthese noch nicht. Solange ein Produkt nicht mit einem synthetisch dargestellten Polypeptid identifiziert worden ist, darf ihm auch keine bestimmte Bezeichnung gegeben werden. Es ist immer noch nir>glich, daß neben der säureamidartigen Verkettung auch andere Bindungsarten zwischen den einzelnen Aminosäuren vorhanden ist. Diese würden übersehen, würde man einfach jedes Produkt, das aus einer bestinnnten Anzahl von Aminosäuren besteht und dessen Analyse, Molekulargewichtsbestimmung nebst dem Resultat der vollständigen und teilweisen Hydrolyse auf ein Polypeptid hinweist, ohne weiteres als ein solches annelunen. Außer dem erwähnten Tetrapeptid aus Seide siud noch aus Edestin ganz gut charakterisierte Produkte abgetrennt worden.') So eine Verbindung, die bei der vollständigen Hydrolyse 1-Tryptophan. d-Glutaniinsäure und 1-Leuzin ergab. Eine andere wies 1-Tyrosin, Glykokoll und 1-Leuzin als Bausteine auf. Ferner wurde aus Edestin ein aus 1-Tryptophan und d-Glutaminsäure bestehendes Produkt gewonnen. Alle diese Verbindungen harren ihrer Identiiizierung. Sind derartige J'rodukte nicht zur Ki'istallisation zu bringen, dann ist es außerordentlich schwierig, den Nachweis zu führen, daß chemisch" einheitliche Snbstanzen vorliegen. Ja selbst kristallisierende Produkte erweisen sieh nur zu oft als Gemische! Mischt mau wohl charakterisierte, synthetisch bereitete Polypeptide, dann ist es fast unmöglich, das Gemisch wieder zu entwirren. Die Hauntschwicrißkeit auf dem iranzen Arbeitsgebiete liegt darin, daß jede Erfahrung über die beste .\rt des Abbaues der einzelnen Proteine ') J'Jiiiil AbilcrhaHen: /.oitsclir. f. pliysiol. (;iiciiiii\ 5S. ?ü\\ (l'.IOS). KiweiBstutt'e und ilire Bausteine. 37;> Es handelt sich uui ein vorsichtiges Vorwärtstasten. Bald ist die Hydrolyse zu weit i^-egangen, und es stört die große Menge von freien Aminosäuren die Isolierung der zusammengesetzten Abbaustufen. Oder aber es haben sich nur hochmolekulare Verbindungen gebildet. fehlt. Die beim stufenweisen Abbau aus Proteinen entstehenden Peptone ohne Zweifel ein recht mannigfaltiges Gemisch aller möglichen Abbaustufen dar. Tausende von einzelnen \'erbindungen mögen da gemischt sein. Ist schon die Trennung der einzelnen Aminosäuren ohne Anwendung der Estermethode fast unmöglich, so läßt sich ermessen, welche Schwierigkeiten noch zu überwinden sind, bis ein Peptongemisch auch nur einigermaßen entwirrt ist. Ja es könnte fraglich erscheinen, ob man in absehbarer Zeit überhaupt so weit kommen wird, ein Peptongemisch vollständig in Je mehr Aminosäuren eine Verbindung geseine Anteile zu zerlegen. bunden enthält, um so schwieriger wird ihre Identitizierung. Wahrscheinlich wird man vorläutig sich damit begnügen müssen, den Nachweis zu führen, was für Verbindungsarten ein Peptongemisch enthält. Es wäre schon von allergrößter Bedeutung, wenn man mit Sicherheit feststellen könnte, daß die säureamidartige Verknüpfung der Aminosäuren die ausschließliche oder doch bei weitem überwiegende ist. Wahrscheinlichkeitsgründe für eine solche Annahme liegen genug vor, ein eindeutiger Beweis steht jedoch noch aus. An Hand einiger aus Peptonen abgesonderter Polypeptide könnte man sich dann ein Bild von dem Verlauf des stufenweisen Abbaues machen. Ferner ließe sich dann auch ein bestimmter .Schluß auf die stellen Struktur der Proteine selbst ziehen. Es erscheint kaum möglich, daß jemals ein bestimmtes Protein mit all seinen Bausteinen synthetisch aufgebaut werden kann, wohl al)er besteht die Möglichkeit, daß die Synthese zu einem Produkte führt, das alle allgemeinen Eigenschaften eines Proteins in sich vereinigt. An einem solchen Modell" kihmte man dann Studien über seinen Abbau und seine ., und sein ^'erhalten gegenüber allen möglichen Agenzien machen und die gewonnenen Erfahrungen auf das Eiweiß übertragen. An Eigenschaften Identitizierung mit einem bestimmten Eiweißkörper ist zurzeit gar nicht zu denken, vor allem schon deshalb nicht, weil wir zurzeit für kein einziges Protein den eindeutigen Beweis besitzen, daß es im chemischen eine Sinne einheitlich ist. Melleicht sind alle Proteine Gemische verschiedener Eiweißkörper, vielleicht sind auch mehrere Eiweißmoleküle locker untereinander verknüpft. Die genaue Erforschung der Bestandteile der Peptone hat noch einen weiteren, sehr bedeutungsvollen Zweck. Es taucht immer wieder der Gedanke auf. daß die Aminosäuren gar nicht alle im Eiweiß vorgebildet Eine Anzahl davon soll sekundär entstanden sein. Je mehr Polymit verschiedenen Bausteinen mit synthetisch dargestellten Verbindungen identitiziert werden können, um so mehr wird derartigen Annahmen der Boden entzogen. Schon die Beobachtung, daß die Hydrolyse der Proteine mit Säuren, Alkalien und Fermenten zu den gleichen Aminosäuren führt'), macht es sehr unwahrscheinlich, daß irg(Mid eine der bis jetzt bekannten Aminosäuren im Eiweißmolekül nicht vorgebildet ist. seien. peptide f. ') Vgl. z. H. Emil Abderhalden, F. physiol. Chciiiic. 61. 20.Ö (190'.)). Mediqreceanu und L. Pincussohn : Zeitschr. XIX. Vorlesung. ;-^74 Erwähnt sei noch, daß bei der Hydrolyse von Proteinen aneh Anhydride, B. 1-Leuzininiid, l-l'henylalanyl-d-alaninanhydrid, 1-Leuzyl-d-valinan hydrid erhalten worden sind.') Ihre Menge ist gering und wie z. wechselnd. Wahrscheinlich handelt es sich um sekundär entstandene Produkte. Immerhin ist es auch nuiglich, daß solche Verbindungen im Eiweiß vorgebildet sind. Als sicher sekundär entstandene Produkte sind die Brenztraubensäure2). CR, .CO. COOK und die a-Thiomilchsäure, C;H,(()H) CH (SH). .COOH^) erwiesen. Die erstere ist auf Alanin, Serin oder Zystin zurückzuführen. Die letztere stammt von Zystin ab. Da die a-Thiomilchsäure aus der !i-Thioverbindung Zystein hervorgeht, muß bei ihrer Entstehung eine . Wanderung der Thiogruppe eintreten. Zur Aufklärung der Struktur zusammengesetzter Eiweißabkönnnlinge verfügen wir noch über mancherlei weitere Hilfsmittel. So besitzen wir mehrere Methoden, die uns gestatten, die Frage zu entscheiden, ob eine bestimmte, zusammengesetzte V erltindung der Eiweißreihe freie Aminogruppen besitzt oder nicht. Ferner können wir ihre Menge feststellen und auf (Irund der Kenntnis des Stickstoffgehaltes der Verbindung genau aussagen, ein wie großer Teil des Gesamtstickstoifes in Form von NH« vorhanden ist. 17.(1). Sli/ke benutzte die Beobachtung, daß aliphatische Aminogruppen mit salpetriger Säure in der folgenden Weise reagieren: R CH COOH . . -f- HNO2 = R CK COOH 4- H, . . + N2 OH NH., zu einer quantitativen Methode der Bestimmung freier Aminogruppen. Die CO NH-Bindung reagiert unter geeigneten Bedingungen nicht. Polypeptide geben somit nur den Stickstoif der freien Aminogruppe ab. Bei jeder Lösung einer CO NH-Bindung unter Aufnahme von Wasser bildet sich . . nunmehr auch in Reaktion tritt. Es läßt sich Methode der Abbau von Proteinen, Peptonen und Polypeptiden in den einzelnen Phasen verfolgen. Vor allem vermag man mit ihrer Hilfe zu entscheiden, ob ein bei der teilweisen Hydrolyse von Proteinen gewonnenes Produkt Aminosäuren beigemischt enthält und ferner dem ver- eine freie Aminogruppe, die mittelst dieser muteten Polypeptid entspricht. Wir haben sch.on bemerkt, daß man die freien Aminogruppen auch durch Besetzung mit Säureresten festlegen kann. Bis jetzt ist hauptsächlich [:i-Naphtalinsulfochlorid zur Kuppelung benützt worden.) Sobald höher molekulare Polypeptide vorliegen, ergibt diese Methode keine verläßlichen Resultate mehr. Das gleiche gilt von der schon erwähnten Me) (1907). Emil Ahcicrhahlcn niul Casimir Funk: Zcit><clir. f. physiol. ('lieniio. 53. 19 — Vgl. auch Ritthunsen: Die Kiwcißkörper iu (ietreidearten usw. Boiiu 1875. — — Rudolf Kohn: Zcitschr. physiol. Chemie. 22. l.")3 (1896/97); 29. 283 (1900). f. Sulaskin und Katharina KouaUirski: Elieuda. 38. 4(57 (1903). Vgl. 2) K. IL A. Mörner: Zeitsclir. f. physiol. Chemie 42. 121, 305 (1904). auch die Studien von Carl Th. Mörner: Ebenda. 101. lö (1917); 103. 80 (1918). E. Erirf/niann : Hof") Sufef: Zeitschr. physiol. ('heuiie. 20. 5()4 (189')). f. meister?. Beitr. 3. 184 (1902). ) Vgl. S. 'iVth. Emil Fischer und l'eter Heigell Bcr. d. Deutschen Clioin. (iesellscli. .S'. — — : 35. 3779 (1902). Kiweißstoffe und Baustciue.' ilire 375 Sie beruht auf der Eigenschaft thode Sü'fjr/rieds.^) Arninogriippen, sich mit Kohlensäure zu verbinden: R CH N<[\| + CO., rr: R CH . . . . der freien N<^^Qjj COOH (H)()H — — NH CO vermögen keine Kohlensäure aufmüßte man in einer zusammengesetzten Verbindung die freien Aminogruppen in der Weise bestimmen können, daß man Kohlensäure einwirken läßt, die entstandene Verbindung isoliert, dann die an den NHg-Gruppen gebundene Kohlensäure wieder abspaltet und ihre Menge genau bestimmt. Das Verhältnis von Gesamtstickstotf der Verbindung zur Menge der abgespaltenen Kohlensäure müßte dann ergeben, wieviel von der gesamten Stickstoffmenge in Form freier Aminogruppen zugegen ist. Leider ergab die Erfahrung, daß bei höher molekularen Produkten nicht alle freien NHo-Gruppen mit Kohlensäure in Reaktion treten. Auch durch Methylierung sind die freien Aminogruppen charakterisiert worden. 2) Endlich hat Kossei ^j Eiweißstoffe insbesondere Protamine nit riert und auf diesem Wege versucht, einen Einblick in ihre Struktur zu erhalten. Bei der Hydrolyse nitrierter Proteine erhält man Aminosäuren, die die Nitrogruppe tragen. Es reagieren auch in diesem Falle nur die freien Aminogruppen. Selbstverständlich muß bei allen diesen Methoden ausgeschlossen werden, daß durch sie Hydrolysen bewirkt werden, denn sonst würden natürlich mehr freie Aminogruppen in Erscheinung treten, als ursprünglich vorhanden waren. Schließlich haben Blum und Strauss in Proteine in verschiedener Weise Jod eingeführt und versucht, auf diesem Wege ihre Strukturverhältnisse und insbesondere das Verhalten der Tyrosin- und Histidingruppe aufzuklären. Je mehr Gruppen man im EiweißmolekUl kennzeichnen kann, Die Iminügrupi)en zunehmen, . folglich — — um so mehr wird es glücken, Einblick in den feineren Bau der Proteine und ihre Abkömmlinge zu gewinnen. Von den mit den erwähnten Methoden erhaltenen Ergebnissen sei z. B. hervorgehoben, daß allem Anschein nach alle a-Aminogruppen gebunden sind, dagegen ist z. B. im Lysin die s-NHa-Gruppe frei.) Eine weitere Methode zur Aufklärung der Struktur der Proteine und ihrer Abbaustufen und ferner zur quantitativen Feststellung freier — •) Vgl. S. 84Ü, ferner M. Siegfried: Zeitschr. f. physiol. Chem. 58. 226 (1908). M. Siegfried und H. Schmitz: Ebenda. 65. 307 (1910). Vgl. hierzu und zum Problem der freien Aminogruppen im Eiweiß überhaupt: J^'mil Fischer und Gluud: Liebigs Ann. 369. 247 (1909). E. Abderhalden und K. Kautzsch: Zeitschr. f. physiol. Chemie. 72. 44 (1911). Engeland: Bericht d. Deutschen chem. Gesellsch. 43. 2662 (1910). D. van Slyke und' Fred J Birchard: The J. of biol. Chem. 16. 539 (1914). — F. Blum und Th. Umbach: Zeitschr. f. physiol. Chem. 88. 285 (1913). F. Blum und E. Strauss: Ebenda. 112. 113 (1920). 6\ Edlbacher: "') — — — . — Zeitschr. — physiol. Chemie. 107. (1919); 108. 287 (1919/20); 110. 153 (1920); 112. 80(1921). S. Edlbacher und Berthold Fuchs: Ebcuda. 114. 133 (1921). A'. Felix: Ebenda. 110. 217 (1920); 116. 150 (1921). J. Herzig: Ebenda. 111. 223 (1920). ./. Herzig und Hans Lieb : Ebenda. 117. 1. (1921). ^) A. Kossei und E. L. Kennaway: Zeitschr. f. physiol. Chemie. 72. 486 (1911). ) Vgl. hierzu Skraup: Monatsschr. f. Chemie. 27. 631, 653 (1906). A. Kossei und Gawrilow: Zeitschr. f. physiol. Chemie. 81. 274 (1912). />. v. Slyke und Fred f. — .52 — - — J. Birchard: The Jourri. liidl. "chcni. 16. 539 (1914). — — XIX. ^'ürlosullg. '.'t'JQ NHo- bzw. Karbüxylj^ruppen beruht auf der Beobachtung von Schiff). daß es möglich wird, in Aminosäuren die Menge der Karboxylgruppen titrimetrisch zu bestimmen, wenn man die Funktion der Aminogruppe ausschaltet.'-) Dies wird erreicht, indem man zu einer Lösung von Aminosäuren unter geeigneten Bedingungen Formaehyd (Fladormol) hinzugibt: CH3 CH3 CH . + HCl )H = CH N NH., . COOK COüH Methylenverbindung des Alan ins. Formal- Alanin dehyd Die Formel vorliegende CH, + H., 0. : am' Beispiel zeigt des Alanins, daß die Aminogruppe mit einer Methylengruppe besetzt wird. Nunmehr reagiert die Verbindung als einbasische Säure. Wählen wir ein Polypeptid, z. B. das Tripeptid Diglyzyl-glyzin, dann ergibt die sogenannte Formoltitration sie ist von Sörensen^) zu einer quantitativen Methode ausgearbeitet eine freie Karl)oxylgruppe. Wird das Tripeptid in Glyzyl-glyziu worden und Glykokoll gespalten, dann werden zwei Karboxylgruppen und auch zwei Aminogruppen frei: — — CH2 CO . . \H CK, CO NH CH., COOH + H, O = . . . . . NH., Diglyzyl-glyzin. CH.> . COOH + CH., CO XH. CK-, . . I NH, . . COOH I NH, Glyzyl-glyzin. Glykokoll Bei der vollständigen Hydrolyse erhalten wir drei Moleküle Glykokoll. Jedes hat eine Araino- und eine Karboxylgruppen- ^): NH, CH., . CO . . XK CK, CO NH CH., COOH + 2 . . . . . H., = Diglyzyl-glyzin NH2 CH, ( OOH -f NH, CH, (OOH + NH, CH, ( (M)H Glykokdll. Glykokoll Glykokoll . . . . . ') H. Schiß: Liehicf^ AiinuhMi. 310. '.^5 (1899); 319. 59, 287 (19Ü1); 325. 848 (1902). ^) Wird durch Zusatz von Alkohol zu einer Lösung von Aminosäuren bzw. Poly- peptiden die Dissoziation dieser Verbindungen zurückgedrängt, so lassen sie sich durch Willstäiter und Krnst Vgl. h'ichard einfache alkalimetrische Titration bestimmen. Waldschmidt-Leitz: Berichte d. Deutschen chom. (Jes. 54. 2988 (19.^). ^) 8. /'. L. Sörensen: Compt. rend. des travaux de Laborat. de Carlsberg. 7. 1 Vgl. auch Handbuch der biochemischen Biochem. Zeitsciir. 7. 43 (1907). (1907). Arbeitsinethodeu. 6. 262(1912). (Be:irbeitet von //. .Jrsseu-Hansen.) Irban & Schwarzeiiherg Berlin-Wien 1912. ) Donald D. nan S/i/ke: Journ. of Biol. (_;hem. 9. 185 (1911). '') h'mil Abderhalden und />. J). ran Slifke: Zeitschr. f. physiol. Chem. 74. ö05 fünil Abderhalden und Hiololf llanslian: Ebenda. 77.^285 (1912). (1911). — — — Kiweilistoffe uud Der Verlauf einer solchen Hydrolyse läßt Je tiefer der Abbau und Karboxylgruppen treten in Erscheinung. titration direkt 377 ihre Bausteine. verfolg-en. sich mittelst der fi;eht, um so Formol- mehr NH.,- Fassen wir nun zusammen, was wir über die Struktur der Proteine und ihrer zusammengesetzten Abbaustufen wissen, dann ergibt sich das folgende Bild. Beim Abbau der Eiweißstoffe entstehen unter Wasseraufnahme einfacher zusammengesetzte Bruchstücke. Die lange Kette der Aminosäurereste löst sich zu kleineren Gliedern auf. Es werden teils einzelne Glieder abgesprengt, teils entstehen kleinere Ketten, die noch mehrere Aminosäuren gebunden enthalten. Der Abbau erfolgt nach allen unseren Kenntnissen in der Art, daß unter Lösung einer säureamidartigen Verkettung einerseits eine Aminogruppe. andrerseits eine Karboxylgruppe sich bildet. Überall schieben sich Wasserteile ein: OH CO : OH H CO.NH NH CO.NH NH2 H NH ^ K COOH ^4 COOH CO NH, Auf diese Weise wird eine Aminosäure von einer anderen losgelöst. es zur Sprengung einer Kette in Bruchstücke* die alle nocli Kommt mehrere Aminosäuren enthalten, so ist die Hydrolyse im Prinzip genau die gleiche: OH H CO.NH CO.NH CO NH CO NH CO NH CO.NH CO.NH COOH NH^ CO NH CO NH . . . . Hierzu ist allerdings zu bemerken, daß bis jetzt keine solche Aufspaltung mitten durch längere Ketten hindurch beobachtet werden konnte. Bis jetzt ließ sich nur mit Sicherheit eine Abspaltung einzelner Aminosäuren am Ende oder Anfang solcher Ketten feststellen. — PepDas sich bildende Gemisch von allen möglichen Abbaustufen besteht vielleicht ausschließlich aus Polypeptiden, d. h. tone genannt aus Ketten von Aminosäuren, die säureamidartig untereinander verbunden sind. Dafür spricht die Beobachtung, daß es gelungen ist, verschiedene Polypeptide unter den Abbaustufen von Eiweißstoffen zu gewinnen. Ferner haben alle Untersuchungen übereinstimmend ergeben, daß mit der fortschreitenden Hydrolyse immer mehr freie Amino- und Karboxylgruppen in Erscheinung treten. Ob wir nun die Hydrolyse mit SäunMi, Alkalien oder Fermenten durchführen, immer beobachten wir die gleiche Art des Abbaus. Die Beobachtung, daß es. gelingt, Proteine unter geeigneten Bedingungen mittelst Fermenten bis zu Aminosäure zu zerlegen, ergab die ersten Fingerzeige dafür, daß Ennl Fischer auf dem richtigen Wege war, als er — XIX. V^orlesuug. ;-^78 . daß die Aminosäuren im Eiweißmolekül säureamidartig verknüpft Wir wissen nämlich, daß bestimmte Fermente nur bestimmte Verbindungen anzugreifen vermögen. Es genügt die geringste Veränderung im Molekül, um dem Fermente die Möglichkeit, seine Wirksamkeit zu entfalten, zu nehmen. Wir haben bereits bei den Kohlehydraten Fermente kennen gelernt, die auf bestimmte Verbindungen eingestellt sind. Es sei nur an aiinaliiii. sind. die Diastase, die Maltase, Laktase usw. erinnert. Bei den Fetten lernten wir die Lipase kennen, die Fette unter Wasseraufnahme zerlegt. Beim Abbau der Proteine bis zu den Bausteinen sind sicherlich eine ganze Reihe von Fermenten beteiligt. Wir nennen diejenigen, die Proteine angreifen, Proteasen oder proteolytische Fermente. Die Bestandteile der l'eptone werden oöenbar durch andere Fermentgruppen zerlegt. Man hat vorläufig Peptasen oder peptoly tische Fermente genannt. Es sie die Peptone spalten, ist nun von allergrößtem Interesse, daß Fermente, auch viele der synthetisch dargestellten Polypeptide in ihre Bausteine zerlegen. Damit war ohne allen Zweifel der Beweis geführt, daß diese eine Struktur besitzen, die den betreffenden,-' auch Polypeptidasen genannten Fermenten vertraut ist. Die säureamidartige Verknüpfung der Aminosäuren in den Polypeptiden ist somit die in den Eiweißstoffen und ihren noch Aminosäuren in Bindung enthaltenden Abbaustufen auch vorhandene. Wir werden auf die interessanten Ergebnisse der Studien über das Verhalten der einzelnen Polypeptide gegenüber verschiedenartigen Fermenten noch eingehend zurückkommen, i) Die säureamidartige Verkettung von Aminosäuren ist die einzige Bindungsart, die bis jetzt im Eiweiß nachgewiesen ist. Wir haben schon betont, daß auch andere Möglichkeiten der Verknüpfung von Aminosäuren gegeben sind. So könnten die Oxysäuren ester- oder ätherartige Bindungen bilden. Ein abwechslungsreiches Moment bringen die Dikarbonsäuren und ferner Lysin und Arginin in die Struktur der Proteine und ihrer zusammengesetzten Abkömmlinge hinein. 2) A. Kassel^) hat für eine bestimmte Gruppe von Proteinen den Versuch unternommen, die Frage nach der Bindungsweise des Lysins und Arginins im Eiweißmolekül genauer aufzuklären. Wir kennen Eivveißarten, die fast ganz aus einer von diesen beiden Aminosäuren bestehen und das neben nur einzelne der bekannten Monoaminosäuren aufweisen. So enthält z. B. das aus reifen Hoden von Lachsen gewonnene Protein Salmin 89"/o Arginin, kein Lysin und Histidin. Ferner sind Serin, Valin und Prolin autgefunden worden. Da, Avie Kassel nachweisen konnte, im Arginin die freie sie erscheint nach der Nitrierung als Aminogruppc nicht besetzt ist Nitrogruppe und ferner auch die Bestimmung der Basizität verlangte, dat) mehr freie Aminogruppen vorhanden .sein müssen, als zugegen wären, wenn jedes Argininmolekül die verfügbaren NHa-Grruppen säureamidartig verkettet hätte, so ergibt sich die folgende Anordnung der Arginin- und Monoaminosäure-Moleküle im Salmin: — — 2) Vgl. Band 2, Vorlesung XVI. Vgl. hierzu S. 355, 350. ^) A. Kossei: Livie jubilairedu ') l'rot. Charles Richet. 211 (1912). — A. Kossei und F. Weiss: Sitzungsber. d. Heidelberger Akad. d. Wissensch. 2. Okt. 1912; 7. Abh. 1913. — 1{. Kherharcl Gross: Zeitschr. f. physiol. Chemie. 120. 107 (1922) und ihro Bausteine. Eiweißstofife ^NH., . CH CO- ?,T9 NH.('H.CO . -NH CH CO. . r I i CH Valin- CHa rest i res t CH, /\^ I CH3 CH3 CH, 111Arginin I CH, I I CH, CH, Arüiniii-i resi > I I NH NH I C=NH C=NH NH., NH, !-NH CH CO . Alaninrest NH CH CO -NH CH CO . . . . . Serin- \| \| CH3 1 rest CH., . OH CH, I I CH, Argini n- CH2 rest NH C=NH NH, Ein weiteres Protamin, das Sturin, aus Hoden vom Stör, besteht aus ca. 630/0 Arginin, S^/o Lysin und 12°/o Histidin. Ferner enthält es Alanin und Leuzin. Die Anordnung der Bausteine dürfte die folgende sein. NH CH CO NH CH CO - CR, CH, . . I I CH, CH, Argininrest . . Lysini CH, CH, rest NH CH, 1 I C=NH NH, NH, NH CH -NH CH CO. A a n n1 . . . NH CH CO CO- . . i ( rest \ 1 I CH, CH, CH, Leuzin- Histi- C (linrest N CH rest "^CH l CH—NH i CH, CH, XIX. A^oilesuug. jj^Q Man hat auch daran iicdacht. daß eine Art von Kern in j;('wisseni Sinne das Zentrum des Eiweißniolekiils bilde, um den herum sich dann die polypeptidartigen Ketten gruppieren. Für die Annahme, daß im Eiweiß nicht einfach eine lanji,e Kette von Aminosäuren enthalten ist. spricht Einmal deuten viele Beobaclitungen auf eine gr<)ßere Anzahl manches. von Karboxyl- und AminooTupj)en hin. Stellt nnin sich vor, daß das P^iweilimolekül eine fortlaufende Kette von säureamidartij;- verknüpften Bausteinen darstellt, dann muß man schon annehmen, daß die freien Amino- und Karboxylo;ruppen hauptsächlich durch die Diamino- bzw. Dikarbonsäuren bedingt sind. Wir können unter der gemachten Voraussetzung das Eiweißmolekül einfach als ein sehr hochmolekulares Polypeptid aufDas Studium eines synthetisch bereiteten, aus möglichst allen fassen. bekannten Aminosäuren aufgebauten Polypeptids wird zunächst am sichersten entscheiden können, ob seine Eigenschaften völlig mit denen der Proteine übereinstimmen. Vor allem wird es interessant sein, festzustellen, ob ein so hochmolekulares Polypeptid von Pepsinsalzsäure, dem proteolytischen Ferment des Magensaftes, gespalten wird. Dieses greift, wie wir gleich erfahren werden, Eiweiß an. Es entstehen zum Teil ganz einfache Abbaustufen vom Charakter der Polypeptide, die nur wenige Bausteine enthalten. Es werden jedoch keine Aminosäuren abgespalten. Trypsin dagegen, das proteolytische Ferment Das Freiheit. letztere des Pankreassaftes. setzt frühzeitig solche in Ferment zerlegt auch synthetisch bereitete PolyAlle bisherigen Erfahrungen peptide unter Abspaltung von Aminosäuren. zeigen, daß die in den Polypeptiden vereinigten Aminosäureketten, wie schon er-ivähnt, nicht an einer beliebigen Stelle durchbrochen werden, sondern es sich das am Anfang oder Ende der Kette stehende Glied ab. Pepsinsalzsäure hat keines der bis jetzt untersuchten Polypeptide gespalten. Diese Beobachtung läßt sich verschieden deuten. Es wäre denkbar, löst daß das Eiweißmolekül aus einer ganzen Anzahl von Polypeptidketten vielleicht zusammengesetzt ist, die unter sich in einer unbekannten Art zusammengefügt sind. unter Vermittlung von Oxy säuren esterartig — — CH2 OH -H H OOC . . GH., . NHo CIL . . OC . CH., . NH., I = I CH NH, . CH NH, . I I COOK COOH Serin Glykokoll Glyzyl-serinester. Das Pepsin vermag vielleicht diese Art von Verbindungen lösen, w^ährend Trypsin die säureamidartige Verkettung spaltet. Doch spricht gegen eine solche Annahme, daß das Pepsin zu ganz einfachen Abbaustufen führt. Es ist kaum anzunehmen, daß die esterartige Bindung von Aminosäuren sich im Eiweißmolekül oft wiederholt. Es müßten denn weitere, noch unl)ekannte Oxysäuren am Aufbau der Proteine beteiligt sein. Oder man müßte schon annehmen, daß außer dei- säureamid- und esterartigen Verkettung der Aminosäuren noch andere Art(;n von Bindungen zwischen den einzelnen Bausteinen des Eiweißmoleküls vorkommen. Es ist jedoch auch ganz gut möglich, daß das Pepsin auf zu Kiwoißstort'c uiul ilire Bausteine. ;-^gl hochmolekulare Polypeptide eingestellt ist und diese an anderen Stellen angreift, als das Trypsin und den Abbau in anderer Kichtung weiterführt. Das Studium hochmolekularer P()ly])eptide muß hier Klarheit bringen. Ferner wird man alle S(»nstigen J)indun<;smügliehkeiten von Aminosäuren und ferner von Polypei)tidketten untereinander bei der Synthese berücksichtigen müssen. Die bisher dargestellten hochmolekularen Polypeptide enthalten mehrfach die gleiche Aminosäure und insl)esondere Glykokoll in unmittelbarer Nachbarschaft. So hat das von Kind Fischer dargestellte, aus 18 Amino- säuren bestehende Polypeptid die Znsammensetzung: 1-Leuzy 1-triglyzyl1-leuzyl-triglyzyl-l-leuzyl-oktaglyzyl-glyzin. Das von Emil Abderhalden und A. Fodor gewonnene, aus 19 Bausteinen bestehende Polypeptid ist 1-Leuzy 1-triglyzyl-l-le uzyl-triglyzy 1-1-1 euzyl-triglyzy 1-1-leuzylpentaglyzyl-glyzin. Es ist wohl möglich, daß die Häufung der Glyzinreste im Molekül dem ganzen Bau des Polypeptides einen Charakter gibt, der von den in der Natur vorkommenden Verbindungen abweicht. Die l)isherige Beobachtung hat nändich ergeben, daß nmn beim Abbau vornehmlich auf Polypeptide trifft, an deren Aufbau verschiedene Aminosäuren beteiligt sind. Vielleicht ergibt ejn entsprechend aufgebautes hochmolekulares Polypeptid dem Pepsin gegenüber ein anderes Verhalten. Erst, w^enn alle diese Fragen entschieden sind, wird es möglich sein, etwas über die besondere Struktur der Proteine auszusagen. Vorläufig müssen wir uns mit dem allgemeinen Bauplane begnügen. Die Zahl der aus den bis jetzt bekannten Aminosäuren darstellbaien Polypeptide ist, wie auf S. 367 dargestellt wurde, eine außerordentlich große. Milliarden und aber Milliarden verschiedenartiger Kombinationen sind möglich. Dazu kommt dann noch, daß sich die Proteine wohl nie in den Zellen in reinem Zustande finden. Es handelt sich sicher immer um Gemische verschiedener Eiweißstoffe. Die Art des Gemisches und der Mischung selbst kann dem ganzen Produkte ein besonderes Gepräge geben. Wenn betont wird, daß jeder Art von Organismen besondere Eiweißstoffe zukommen, und behauptet wird, daß innerhalb eines bestimmten Organismus jede Zellenart ein eigen artigei» Proteingemisch besitzt und schließlich sogar von individuell verschiedenen Eiweißstoften gesprochen wird, so ist man geneigt, eine so weitgehende Spezialisierung der einzelnen Proteine für unmöglich zu halten, t'berlegt man sich jedoch, daß einmal durch die Art der Reihenfolge, durch dije Mengenverhältnisse, in denen die einzelnen Aminosäuren sich finden, und ferner vielleicht noch durch besondere Feinheiten der Struktur unbekannter .\rt die angegebene Anzahl von verschiedenen Polypeptiden noch in ganz unübersehbarer Weise gesteigert werden kann, dann rückt die erwähnte Vorstellung von arteigenen, organeigenen und gar individueneigenen Proteinen durchaus in den Bereich der Möglichkeit. Dazu konunt noch, wie schon erwähnt, der besondere Charakter, der durch die Art der Mischung einzelner, verschiedenartiger Proteine dem ganzen Gemenge gegeben werden kann. Wir besitzen in den Fermenten außeiordentlich feine Keagenzien auf Unterschiede in der Zusammensetzung und Struktur von bestinnnten \ erbindungen. Diese weisen uns schon darauf hin. daß die die verschiedenen Zellen eines bestimmten Organismus aufltaucnden Proteine nicht gleicluiitig XIX. Vorlesuug. Eiweißstoffe und p,^2 sein können. 1) Wir kennen Zellfermente, ihre Bausteine. die nur Eiweißstoffe der gleichen Es scheint nach neuen Erfahrungen, daß die gleichen Organe in der ganzen Tierreihe. Proteine aufweisen, die sich sehr ähnlich sind und bei weitem keine so großen Unterschiede unter sich aufweisen, wie z. B. die Organproteine ein und desselben Es scheint ein biologisches Gesetz zum Vorschein zu Tieres, kommen, das zeigt, daß alle Tiere zu gleichen Zwecken ähnliche ,,funktionsspezifische" Proteine bilden. 3) Diese Ähnlichkeit braucht nur darauf zu beruhen, daß in bestimmten Eiweißstoffen bestimmte Gruppierungen von Aminosäuren wiederkehren. Dieser Umstand genügt unter Umständen, Zellart abbauen, in denen sie enthalten sind.'-) um zu bewirken, daß ein bestimmtes Ferment den Abbau dieser Verbindungen Selbstverständlich kann trotzdem das Organeiweiß jeder eröffnen kann. Tierart einen artspezifischen Charakter haben. Die Art der Mischung der einzelnen Zellproteine kann dem Zelleiweiß ein so spezifisches Gepräge daß es sich scharf von jedem anderen Zellproteingemisch unterund doch kann eine Komponente in der ganzen Tierreihe wiederkehren. Das Ferment, das auf diese besondere Eiweißart einwirken kann, macht uns auf diese, sonst gar nicht in Erscheinung tretende Anordnung bestimmter Atomgruppen aufmerksam. geben, scheidet, ') ^) Vgl. Vorlesung XXV. Vgl. Emil Abderhalden: Die ÄbderhaldenHche Berlin 1922. ') Vgl. Emil Abderhalden: Reaktion. 5. Aufl. J. Springer. Münchener med. Wochenschr. 60. 238ö (1913). Vorlesung XX. Eiweißstoffe Eigenschaften der Eiweißstoffe. ihr und ihre Bausteine. Zusammensetzung und Ihre Einteilung, Vorkommen. Bei den Kohlehydraten stellten wir fest, daß wir die struktur auch die Konfiguration der Monosaccharide kennen. Ferner erkannten wir, daß in den Disacchariden zwei Moleküle von Monosacchariden unter Wasserabspaltung vereinigt sind. Aus dem Verhalten der einzelnen Disaccharide gegenüber Metalloxjden in alkalischer Lösung und aus anderen Reaktionen ließ sich erschließen, ob die Aldehyd- bzw. Ketongruppe in reaktionsfähigem Zustande im Molekül enthalten ist oder aber, ob diese Gruppen besetzt oder sonstwie „verdeckt" sind. In der Folge gelang es, für bestimmte Disaccharide Konstitutionsformeln zu entwerfen, die zwar noch nicht durch die Synthese bestätigt, jedoch mit den ganzen Eigenschaften der betreö'enden Verbindungen übereinstimmen. Die höheren Polysaccharide, die Tri- und Tetrasaccharide usw. vermochten wir nur noch durch die Zahl und Art der Bausteine zu charakterisieren. Dazu kommen dann noch besondere Eigenschaften. Dann verlor sich mit dem Aufsteigen in der Reihe der zusammengesetzten Kohlehydrate die auf Grund der Kenntnis der Zahl und Art der Bausteine gewählte Nomenklatur immer mehr. Wir standen schließlich einem Gemisch von allen möglichen Abbaustufen aus hochmolekularen Kohlehydraten gegenüber, das wir ganz allgemein als Dextrine bezeichneten. Wir nehmen an, daß es aus zahlreichen Polysacchariden besteht, die einzeln eine bestimmte Anzahl von Bausteinen aufweisen. Einzelne Dextrine sind als Polysaccharide von bestimmter Zusammensetzung erkannt, i) Sie scheiden aus der Gruppe der unbekannten Bestandteile der Dextrine aus. Auf diese Verbindungen folgen dann die hochmolekularen, kolloiden Polysaccharide, wie die Stärke, das Glykogen und ferner die Zellulose. Auch diese Verbindungen tragen den Namen Polysaccharide. Er sagt nur aus, daß viele", aber nicht, wie viele Monosaccharide am Aufbau eines bestimmten Kohlehydrates beteiligt sind. Ist die Anzahl der Bausteine bekannt, dann tritt an die Stelle der Silbe „Poly" die entsprechende Zahl. Über die Struktur der Polysaccharide sagt der Name gar nichts aus. und ,^ ») Vgl. S. 59. ^-'^- ;^^4 Vorlesung. Bei den Proteinen lernten wir zunächst die Bausteine, die Amino- säuren, kennen. Über die Struktur der Monopeptide sind wir vollständig Dagegen ist die Frage der Konfiguration der einzelnen unterrichtet. Der Umstand, daß bei der Verl)indungen eine zum Teil noch offene. \) linwandhing der einen Verbindung in eine andere sich Veränderungen in der Konfiguration vollziehen können, erschwert das Studium der Beziehungen der feineren Struktur der einzelnen Aminosäuren zueinander und vor allem die Feststellung des Zusammenhanges der Konfiguration einzelner Aminosäuren mit derjenigen anderer Verbindungen, wie insbesondere der Kohlehydrate. Es sei in dieser Beziehung daran erinnert, daß z. B. d-Alanin bei der Einwirkung von Nitrosylbromid nicht d-Brompropionsäure, sondern 1-Brompropionsäure liefert."-) Bekannt, wie schon S. 316 dargestellt, ist die Konfiguration der d-a-Aminopropionsäure und damit der in [i-Stellung substituierten, von ihr sich ableitenden Verbindungen. somit unsere Kenntnisse, was die Konfiguration den Aminosäuren augenblicklich noch hinter denen der Monosaccharide zurück, so sind wir auf der anderen Stehen anbetrifft, Seite bei bei jenen Verbindungen der Eiweißreihe viel besser über die Struktur unterrichtet, die aus mehreren Aminosäuren bestehen. Wir wissen, daß im Eiweißmolekül die Aminosäuren säureamidartig miteinander verknüpft sind. Es bleibt dabei offen, ob nicht daneben auch noch andere Bindungsarten sich finden. Mit dem Namen Polypeptid verbinden wir die Vorstellung einer ganz bestimmten Struktur. Auch bei den Proteinen kennen wir ein Gemisch von Abbaustufen, das Es sind dies die Peptone. noch nicht völlig entwirrt werden konnte. Es ließen sich aus manchen Peptongemisclien einzelne Polypeptide abscheiden. Wir haben die Peptone bereits als ein Gemisch von zahlreichen Produkten geschildert, die einerseits dem Eiweiß und andererseits den Aminosäuren nahestehen. Sie lösen sich in Wasser, diffundieren durch Dialysiermembrane, geben die Biuretreaktion und ferner jene Farbreaktionen, die an das ^'orhandensein bestimmter Bausteine geknüpft sind. Fehlen diese, dann ergeben die betreffenden Peptone auch die entsprechende Bausteinfarbreaktion nicht. "Mit Triketohydrindenhydrat geben die Peptone, Avie die Polypeptide und Aminosäuren Blaufärbung. Steigen wir noch weiter emjxtr. dann gelangen wir zu den Eiweißstoffen. Sie sind nur im kolloiden Zustand bekannt. Eine große Anzahl von ihi-en Eigenschaften hängt mit diesem zusammen. Wir wollen sie hier nur streifen. Ein volles Verständnis des Verhaltens der Proteine unter verschiedenen Bedingungen wird erst möglich sein, wenn wir einerseits das N'erhalten und andrerseits die Gnindeigenschaften der Kolloide ergel('>ster Stoffe örtert haben werden.'') Aus diesem Grunde wollen wir die Besprechung " der physikalischen Eigenschaften der Proteine einstweilen zurückstellen. Wir haben die lroteine als kolloide, aus Aminosäuren zusammengesetzte Verbindungen charakterisiert. Sie geben bestimmte Farbreaktionen. Keine davon ist für die Eiweißsubstanz als ') ^) ') solche charakteristisch. Vgl. hierzu S. ;n6tt. \gl. S. 3()2. Vgl. Band 'l. Vdrlcsuntr'Mi \' Die und folgende. Proteine geben, wie die Eiweißstoffe und ihre Bausteiae. 385 und verdünnter Kupfersulfatlösung- eine violettrote Färbung. Schon einzelne Tripeptide geben die Biuretreaktion. Offenbar ist sie von der Struktur der Verbindungen abhäno-iound ferner müssen mehrere Aminosäuren säureanüdartig miteinander verknüpft sein. Zwei Aminosäuren genügen noch nicht. Die Proteine o-eben mit Triketuhydrindenhydrat Blaufärbung. Diese Reaktion haben sie mit fast allen Abbaustufen der Eiweißstoffe bis herunter zu den Aminosäuren gemein. Nur bestimmte aussalzbare Peptone geben keine Farbreaktion. Wahrscheinlich liegt irgend eine anhydridartige Bindung innerhalb des Moleküls vor. Triketohydrindenhydrat reagiert ganz allgemein mit Verbindungen, die in z-Stellung zum Karboxyl eine Aminogruppe tragen, i) Peptone, mit Alkali bis violettblaue Die nun folgenden Farbreaktionen sind auch nicht typisch für Eiweiß, ja sie können teilweise oder auch vollständig fehlen. Sie sind alle dadurch charakterisiert, daß sie von der Anwesenheit bestimmter Aminosäuren abhängig sind. Fehlt einem Protein ein solcher Baustein, dann fällt die diesem zukommende Reaktion neoativ aus. nämlich Trotzdem kann die Verbindung ein echter Eiweißkörper Geben Proteine die sein. Xanthoproteinreaktion, dann deutet dies ganz allgemein an, daß sie aromatische Bausteine enthalten. Es können Phenylalanin, Tyrosin, Tryptophan und Oxytryptophan vorliegen. Erhält man mit Millons Reagens eine rote Färbung, dann beweist daß der betreffende Eiweißkörper Tyrosin besitzt. Wir können noch den weiteren Schluß ziehen, daß die Oxy-Gruppe des Tyrosins unbesetzt ist, denn es fällt die Reaktion mit Tyrosin, dessen Hydroxyl z. B. mit dem Naphtalinsulforest 2) verknüpft ist, negativ aus. Der Schluß, daß der positive Ausfall der Reaktion mit Millons Reagens die Anwesenheit von Tyrosin im Eiweißmolekül beweist, ist nur deshalb zulässig, weil wir keine andere Aminosäure kennen, die diese Art von Rotfärbung gibt. 3) dies, Unterschichten ^^ir eine Eiweißlösung, die wir mit einer Lösun»- von mit konzentrierter Schwefelsäure dann Glyoxylsäure vermischt haben, erhalten wir an der Berührungsstelle beider Schicliten einen violetten Rin»vorausgesetzt, daß das untersuchte Protein Tryptophan enthält. Die Bromreaktion auf diese Aminosäure fällt natürlich negativ aus, weil sie nur mit der freien Aminosäure eintritt. — Erhalten wir beim Kochen eines Eiweißes mit Alkali und Bleiazetat grauschwarzer Niederschlag Al)scheidung von Bleisulfid dann wissen — Avir. — , daß ein zystin haltiges Produkt vorliegt. Aus der Feststellung, daß diese Farbreaktionen charakteristisch für bestimmte Bausteine und nicht für Eiweiß selbst sind, ergibt sich "anz von selbst, daß wir die Eiweißkörper nicht durch sie kennzeichnen können. Es bleibt als Unterscheidungsmerkmal gegenüber den Peptonen in der Hauptsache nur ihr kolloider Charakter. Dieser dürfte einmal dadurch bedingt sein, daß die Proteine hochmolekulare Verbindungen darstellen. ') Auch andere Verbindungen geben mit Triketoliydrindeulijdrat unter (Geeigneten Bedingungen Blaufärbung. Ihr Auftreten genügt für j-ich allein nicht, um auf Eiweiß und seine Abbaustufeu zu schließen. -) Vgl. S. 370. ^j Oxytryptophan gibt auch eine Färbung mit Millons Reagens, doch zei^t ein anderes Rot. A b]de rhal den , Physiologische Chemie. I. I'oil, 5. Aufl. V>') sie XX. Vorlesung. 3g6 Es ist jedoch auch möglich, daß Besonderheiten in der Struktur mit maßgebend sind. Die Eiweißkörper lassen sich aus ihren scheinbaren Lösungen auf ganz verschiedene Arten abtrennen. Die Hitzekoagulation i) haben wir schon erwähnt. Die Proteine fallen auf Zusatz von Salzen der Sch>vermetalle. Besonders oft sind Eisenchlorid, Eisenazetat, Kupfersulfat und -azetat, Bleiazetat, Zink- und Uranylazetat zur Abscheidung von Proteinen verwendet w^orden. Ferner lassen sich die Eiweißstoffe mittels der sog. Alkaloidreagenzien ausfällen. Sie fallen mit PhosphorAuch mit Gerbsäure, Pikrinw^olframsäure und Phosphormolybdänsäure. säure, Trichloressigsäure, Ferrozyanwasserstoffsäure erhält man Fällungen. Unterschichtet man eine Eiweißlösung sehr vorsichtig mit konzentrierter Salpetersäure in der Kälte, dann tritt an der Beriihrungsstelle beider Schichten ein grauweißer Ring auf 2) Diese Eiweißprobe ist sehr empfindEndlich fallen die meisten Eiweißkörper auf Zusatz von Alkohol. lich. Einige davon sind allerdings in ihm löslich. Mit Hilfe dieser Fällungen kann man wohl Eiweiß als solches abscheiden und erkennen, dagegen sind die erwähnten Methoden in den meisten Fällen ungeeignet, um Gemische verschiedener Proteine zu trennen. Die einzige Methode^ die bis jetzt mit Erfolg zur Darstellungvon Proteinen Verw^endung gefunden .hat, ist die von Kilhne'^) eingeführte und dann namentlich von Hofnieister^) und seinen Schülern^) und später von Oshorne^) ausgearbeitete Methode des Aussalzens. Es sind verschiedene Salze zur Fällung angewandt worden. Bevorzugt sind Ammonsulfat und Zinksulfat. Auch Kochsalz, Natriumsulfat und Magnesiumsulfat linden Verwendung. Die Erfahrung hat gezeigt, daß bestimmte Eiweißsubstanzen für ein bestimmtes Salz eine bestimmte Fällungsgrenze besitzen. Es kommt nicht nur auf die Konzentration der Salzlösung an. Die Aussalzbarkeit eines Proteins ist vielmehr auch von der absoluten Menge des aussalzenden Salzes und derjenigen des auszusalzenden Eiweißes abhängig. Die Fällungsgrenze einer EiweißMan fügt zu der Lösung eines substanz läßt sich, wie folgt, bestimmen. bestimmten Proteins ganz allmählich eine konzentrierte Lösung eines bestimmten Salzes. Man beobachtet, daß nach Zusatz einer bestimmten Menge der Salzlösung eine Trübung erscheint. Es ist dies die untere Fällungsgrenze für das betreft'ende Protein und die angewandte Art Salzlösung. Nun fährt man mit dem Zusatz der Salzlösung fort, bis keine Fällung mehr erfolgt. Es ist dann die obere Fällungsgrenze erreicht. wiederum nur für das betreffende Protein und das verwendete Sie gilt Salz. Hat Vgl. hierzu S. P. L. Sörensen und E. Jürc/ensen: Biochom. Zeitschr. 31. 397 sicli weitere Literatur. -) IJellersche Probe. ") ir. Kühne: Verb, des Heidelberger naturhist.-med. Vereins. N. F. 3. 286 (188Ö). A. llcynsitis: Fftih/ers Arch. 34. b3U (1884): Zeitschr. f. Biol. 29. 1 (1892). ) F.Hofmeister: Arch. f. exper. Patb. u. Pharm. 24. 247 (1887); 25. 1 (1888). ') (1911). — Hier findet Pauli: Hofmeisters Beitr. 3. 225 (1902). Osborne und ./. F. Harris: Amer. Journ. of Physiol. 93. 436 (1905); V^gl. auch Ilandb. der Biochem. .lourn. of the Amer. C'bem. Soc. 25. 537 (1903). Arbeitsmethoden. 2. 270 420(1910). (Bearbeitet von Thomas B. Oshortie, Fr. N. Schviz Vgl. ferner IIikio und Franz Samuelij.) l'rban t^: Schwarzenborg. Berlin-Wien 1910. Wiener: Zeitschr. f. physiol. Cheni. 74. 29 (1911). ^) IV. «) T. B. — — — J^iweißstotfe und ihre Bausteine. uü^ man ein Gemisch verschiedener Proteine zu trennen, dann beobachtet man daß nach Erreichung der oberen Fällungsgrenze bei weiterem Zusatz der Salzlösung keine Ausflockung mehr erfolgt. Erst nach einer bedeutend höheren Konzentration der Salzlösung in der Eivveißlösung erscheint wieder Es ist die untere Fällungsgrenze eines anderen Proteins eine Trübung. sie erkennen zu können, entfernt man das zuerst ausgefallene erreicht. Protein durch Filtration und gibt dann zum Filtrat weiter Salzlösung hinzu. Ist für das zweite Protein die obere Fällungsgrenze erreicht, dann wird wieder filtriert und versucht, im Filtrat weitere Eiweißstofte auszusalzen, falls dieses noch solche enthält. Die Salzlösungen werden kalt gesätti^-t und ganz neutral verwendet. Mit Hilfe dieser Methodik ist es gelungen, aus Pflanzen und Tieren eine sehr große Anzahl von Proteinen abzutrennen. Die so gewonnenen Eiweißstofte bilden das Ausgangsmaterial der Studien über diese Klasse von Verbindungen. Außerdem stehen uns noch zahlreiche Eivveißsubstanzen zur Verfügung, die in der Natur in festem Zustande vorkommen. Es sind dies Proteine, die in der Tierwelt in gewissem Sinne die Zellulose und oft, Um die dieser verwandten Kohlehydrate in ihren Funktionen vertreten. Wie Polysaccharide der Pflanzenwelt als Abgrenzung von Zellen und Geweben und als Stütze dienen, so finden wir im tierischen Organismus zahlreiche Gebilde, die mechanischen Funktionen dienen und ganz aus Eiweiß aufgebaut sind. Diese Gruppe von Proteinen läßt sich im einzelnen Falle nur sehr schwer in einzelne Bestandteile auflösen, ohne daß sekundäre Veränderungen, wie Hydrolyse usw., eintreten. Infolgedessen hat man diese Produkte meist direkt als Ausgangsraaterial benutzt, ohne eine Reinigung zu versuchen. Hierher gehören die elastischen Fasern, die Keratinarten. die Seide, das Spongin usw. diese Wenn wir uns ein Urteil über die Zusammensetzung einer Verbindung und vor allem auch über ihre Struktur bilden wollen, dann müssen wir in erster Linie genau wissen, ob das Ausgangsmaterial einheitlich ist. Erst dann können wir den Resultaten der Abbauversuche eine bestimmte Bedeutung zuerteilen und auf ihnen weiterbauen. Sobald es uns gelingt, einen Körper in Kristallform zu bringen, ist die Möglichkeit gegeben, ihn zu reinigen. Bei amorphen Körpern ist es außerordentlich schwer, den Beweiß zu erbringen, daß sie chemisch einheitlich sind. Es ist in diesen Fällen die wendig, sich Darstellung zahlreicher Derivate, Salze usw. not- um zu erfahren, ob das Ausgangsmaterial um kolloide Verbindungen, dann stellen sich rein ist. Handelt es der Reinigung oft fast unüberwindliche Schwierigkeiten entgegen. Der kolloide Zustand ist nämlich durch die Eigenschaft, alle möglichen Stoffe aufzunehmen, zu adsorbieren, ausgezeichnet. Wie steht es nun mit den Eiweißstoffen? Dürfen wir die Proteine als chemisch einheitliche Verbindungen ansehen? Oder bedeutet der Name Protein Eiweiß nur einen Sammelbegriff für eine Klasse von einander nahestehenden Verbindungen, wie etwa der Name Stärke mehrere Verbindungen, wie Amylopektin und Amylose^), zusammenfaßt? Diese Fragen sind außerordentlich schwer zu beantworten. Es ist erst in neuester Zeit mit allen Methoden der modernen physikalisch- = Vgl. S. .58. 25* XX. Vorlesung, 3gg chemischen Methodik versucht worden, den Beweis zu führen, daß kristallisiertes Eieralbumin chemisch einheitlich ist.i) Bei so kompliziert gebauten Verbindungen läßt sich eine sichere Unterscheidung zwischen konstanten Gemischen und einheitlichen einfachen Produkten nach dem jetzigen Stand unserer Methoden wohl kaum durchführen. Die gebräuchlichen Methoden der Darstellung der Proteine ergeben keine Gewähr für ihre Reinheit. Wir können uns wohl vorstellen, daß eine ganze Anzahl von Proteinen die gleichen Fällungsgrenzen aufweisen. Ferner handelt es sich bei dem Aussalzen nicht um das Fällen eines bestimmten Proteins bei einer ganz bestimmten Salzkonzentration, sondern wir fällen von einem bestimmten Grad der Sättigung an, bis nichts mehr ausflockt, d. h. bis die obere Sättigungsgrenze erreicht ist. Es ist wohl möglich, daß mehrere Proteine innerhalb der unteren und oberen Sättigungsgrenze der beobachteten scheinbar einheitlichen Fällung ihre beiden Sättigungsgrenzen haben. Dazu kommt noch, daß die Erfahrung beim Ai-beiten von Gemischen zeigt, daß die einzelnen Komponenten sich in ihien Eigen- schaften oft stark beeinflussen. Selbst kristallisierende Verbindungen sind Sollte es oft erst auf vielen Umwegen von Beimengungen zu trennen. wirklich möglich sein, durch einfaches Aussalzen aus einem Gemisch von hochmolekularen Verbindungen chemisch einheitliche Körper abzuscheiden? Eine solche Annahme hat sehr wenig Wahrscheinlichkeit für sich. Sie läßt sich direkt prüfen, indem man Polypeptide, die mit Ammonsulfat aussalzbar sind, mischt und dann feststellt, ob noch für jedes Polypeptid die gleichen Sättigungsgrenzen, wie zuvor bei der einzelnen ^'erbindung vorhanden sind, und damit die Möglichkeit einer Trennung gegeben ist. Es entspricht auf alle Fälle eher den Tatsachen, Avenn wir den Namen Protein vorläufig als einen biologischen Sammelbegriff für hochmolekulare, kolloide, aus Aminosäuren zusammengesetzte Verbindungeu bezeichnen und hervorheben, daß die Methoden wohl gestatten, unter Innehaltung der empirisch gefundenen Bedingungen immer wieder die gleiche Gruppe von Eiweißstoften zu isolieren, daß hingegen zurzeit für keinen Vertreter der Proteine der Beweis geführt ist, daß er chemisch einheitlich ist. Es ist möglich, daß einheitliche Proteine isoliert sind, es ist auch denkbar, daß manche davon aus ganz wenigen Komponenten bestehen, es muß jedoch auch mit der Möglichkeit gerechnet werden, daß jedes einzelne der bis jetzt isolierten Proteine aus einer größeren Anzahl nahe verwandter Produkte besteht. bisherigen Wir sind nicht imstande, zur Isolierung der Proteine prinzipiell verschiedene Methoden sich folgen zu lassen. In den meisten Fällen wird ein und dasselbe Verfahren wiederholt angewendet. Wir salzen ein Protein aus, bringen es wieder in L()sung und fällen wieder. Erhalten wir nun bei mehrfacher Wiederholung der Aussalzung verschiedene Fraktionen, so könnte man vermuten, daß es geglückt sei, eine gewisse Reinigung zu erzielen. In manchen Fällen mag das auch richtig sein, doch dürfen wir nie außer acht lassen, daß die ?^iweißstotfe Kolloide mit allen ihren Eigenschaften darstellen. Vor allem stören die Zustandsänderungen der kolloiden Proteine die Beurteilung der sogenannten Reinigung eines Proteins außer•) Vgl. S. P. L. Sörensen und Marq. Höyrup: Zeitschr. f. physiol. Cheni. 103. 15 S. F. L. Sörensen: Ebenda. 103. 211 (1918); 106. 1 (1919). (1908); 103. 267 (1918). — Eiweißstotfe uud ihre Bausteine. 3y9 Schon beim Aussalzen zeigen manche Proteine Änderungen Wir bezeichnen bei Kolloiden den scheinbar gelösten Zustand als 8ol (Solutio, Lösung), den festen als Gel (abgeleitet von Gelatine). Der erstere Zustand kann in den letzteren durch alle möglichen Zwischenstufen übergehen und umgekehrt. In diesem Falle sprechen wir von einer reversiblen Zustandsänderung. Es tritt jedoch auch der Er ist Fall ein, daß der einmal erreichte Gelzustand beibehalten wird. irreversibel geworden. Die Eigenschaften eines Kolloids sind in hohem Maße von dem augenblicklichen Zustand und vor allem auch von den vorhandenen Bedingungen abhängig. Wir können uns wohl vorstellen, daß ein bestimmtes Protein sich bei wiederholtem Aussalzen einzig und allein deshalb fraktionieren läßt, weil das Fällen und Auflösen Zustandsänderungen ordentlich. ihres Zustandes. die die Eigenschaften einzelner Teile des Proteins stark beeinDiese Bemerkungen sollen nur andeuten, vor welchen Schwierigkeiten wir stehen, wenn wir die Frage beantworten und experimentell beweisen sollen, ob es unter den isolierten Proteinen chemisch einheitliche Individuen gibt. Wir haben schon früher erwähnt, daß in der Natur scheinbar gelöste, halbfeste und feste Proteine vorkommen. Die ersteren lassen sich nur so lange isolieren, als sie ihren genuinen Zustand beibehalten oder in diesen wieder zurückgebracht werden können. Ist der Zustand eines Proteins irreversibel verändert, dann ist jede Möglichkeit seiner Reinigung im Sinne einer Abtrennung etwa beigemengter Eiweißstofl'e ausgeschlossen. So ist ein Eiweißkörper, der durch die Hitze koaguliert worden ist, in seinen ganzen Eigenschaften so verändert, daß er in den meisten Fällen von anderen auf die gleiche Weise gewonnenen Proteinen nicht mehr durch seine Löslichkeit, seine Fällbarkeit usw. unterschieden werden kann, obwohl die einzelnen Proteine im genuinen Zustand ganz verschieden waren. Man hat diese Überführung genuiner Eiweißstoflfe in den irreversiblen, festen Zustand auch Denaturierung genannt, um anzudeuten, daß die Eigenschaften des Ausgangsmaterials verloren gegangen sind. Eine besondere Art der Gelbildung stellt die Gerinnung dar. Wir werden bei der Besprechung des Blutes erfahren, daß dieses unter bestimmten Bedingungen gerinnt. Es ist ein vorher gelöster Eiweißkörper, der ausfällt. Auch die Totenstarre ist auf die Gerinnung von Eiweiß zurückgeführt worden. Ferner läßt sich die Milchgerinnung auf die Umwandlung eines Proteins zurückführen. Wir werden auf diese biologisch wichtigen Gerinnungsvorgänge noch zurückkommen. Fassen wir das Ergebnis der bisherigen Bemühungen, Eiweißstoffe als chemische Individuen abzutrennen, zusammen, dann ergibt sich, daß dieses Ziel nicht erreicht worden ist^), oder besser ausgedrückt, wir besitzen zurzeit keine Kriterien dafür, ob die Reindarstellung von Eiweißstoffen gelungen ist. Dagegen haben Forscher, wie Kühne, Hofmeister, Osborne, Methoden ausgearbeitet, die es uns ermr)glichen, aus den einzelnen Ausgangsmaterialien immer wieder die gleiche Eiweißart zu erhalten. 2) Es ist gelungen, eine große Anzahl von Proteinen so zu charakterisieren, daß bewirkt, flussen. ') Vgl. auch hierzu Friedrich Obermayer \\nA\Bobert Willheim: Biochem. Zeitschr. 38. 311 (1912); 50. 369 (1913). -) A'gl. die Literatur zu diesen Prol)lenieu bei 0. Cohnheim: Chemie der Eiweiß- körper, 1. c. S. 307. XX. Vorlesung. 390 eine Einteilung- der verschiedenartigen Eiweißsubstanzen möglich geworden ist. Sie ist auch heute noch maßgebend. Ehe wir auf die Besprechung des Vorkommens und der Avichtigsten Eigenschaften der einzelnen Proteinarten eingehen, müssen wir noch der interessanten Beobachtung gedenken, daß es gelungen ist, eine ganze Anzahl von Proteinen in kristallisiertem Zustande abzuscheiden.^) Dieser Befund erweckte große Hoffnungen. Das Problem der Gewinnung einheitlicher Proteine erschien gelöst. Die genauere Betrachtung der Eigenschaften der Eiweißkristalle zeigte jedoch bald, daß das Kristallisationsvermögen an und für sich keine Gewähr für die Einheitlichkeit der gewonnenen Produkte ergibt. Es ist nämlich gelungen, den Beweis zu führen, daß Eiweißkristalle, ohne in ihrer Form Änderungen zu zeigen, andere Kolloide aufnehmen können. Eieralbumin kann z. B. muzinartige, an Glukosamin reiche Proteine enthalten und doch kristallisieren, ^j Unter geeigneten Bedingungen läßt sich jedoch ein solches Eiweiß reinigen 3), pnd man gewinnt dann wieder die gleichen Kristalle, die jedoch bei der vollständigen Hydrolyse weniger Glukosamin ergeben als die erste Kristallisation. Ferner läßt sich Hämoglobin, ein zusammengesetzter Eiweißkörper, mit anderen Proteinen zusammen zur Kristallisation^bringen.) Endlich hat Zsigfnondy^) auf ein eigentümliches Verhalten der kolloiden Goldlösung bei Anwesenheit von Eiweiß aufmerksam gemacht. Eine reine Goldlösung wird durch Zusatz von Elektrolyten, z. B. von Kochsalz, ausgefiockt. Ist dagegen Eiweiß zugegen, so bleibt die Fällung aus. Die Eiweißstotfe schützen gleichsam das kolloide Gold. Fr. X. Schulz und Zsigmondiß) haben nun nachgewiesen, daß die Fähigkeit der Eiweißstofife, das kolloide Gold vor dem Ausfallen zu schützen, sich für jeden Eiweißkörper zahlenmäßig ausdrücken läßt. Globulin vermag z. B. unter bestimmten Bedingungen ungefähr die 20 fache Gewichtsmenge Gold zu schützen. Wird eine Eiweißlösung, die mit Goldlösung gemischt ist, gefällt, so fällt das Gold mit. Man erhält einen homogenen, roten Niederschlag. Löst man das Globulin wieder auf, so geht auch das Gold wieder in Lösung. Es ist klar, daß in diesem Falle sehr leicht eine Verbindung zwischen Gold und Eiweiß vorgetäuscht werden könnte. Es ist von großem Interesse, daß auch kristallisiertes Eieralbumin Gold aufnimmt und mit diesem sich Umkristallisieren läßt. Auch Kupfer, Eisen, Kalziumoxyd etc. können von Eiweiß in kolloider Lösung gehalten werden. Sind wir zurzeit auch nicht imstande, das Kristallisationsvermögen einiger Eiweißarten allein als Beweis für ihre chemische Einheitlichkeit anzuführen, so ist doch diese Eigenschaft von allergrößter Bedeutung für die ßeinigung von Proteinen geworden. Die erhaltenen Kristalle ') Vgl. die Literatur bei Fr. N. Schulz: Die Kristallisation von Eiweißstoft'eu und Bedeutung für die Eiweißchemie. Gustav Fischer. Jena 1901; ferner in Handbuch der Biochem. Arbeitsmethoden. 2. 270—346 (1910). (Bearbeitet von Thomas B. Osborne und Fr. N. Schulz.) Urban & Schwarzenberg. Wien-Berlin 1910. ^) Eniil Abderhalden, Peter Berqell und Theodor Dörpinqhaus : Zeitschr. f. physiol. Chemie. 41. 530 (1904). Vgl. hierzu: S. P. L. Sörensen und Marq. Höt/rup : Zeitschr. f. phvsiol. Chem. ihre •') 103. 15 (1918). ) Emil Abderhalden: Zeitschr. ^) i?. ) f. physiol. (hemie.'ST. 484 (1903). Zsifimondy: Zeitsclir. f. aualyt. (Miemie. 40. 697 (1901). Fr. N. Schulz und R. Zsigmondy : Jlo/meisferü. Beiträge. 3. 137 (1902). Eiweißstoffe uucl ihre Baiisteiue. 39 [ lassen sich nämlich Umkristallisieren. Hierbei verbleibt immer eine Mutterlauge, die Verunreinigungen enthält. Durch wiederholtes Umkristallisieren läßt sich eine Reinigung erzielen. Allerdings sind wir bei der Umkristallisation mit Ausnahme des Hämoglobins bei ein und demselben Protein auf die gleiche oder doch ganz entsprechende Methode angewiesen. Es wäre vorteilhafter, wenn die gleichen Kristalle unter ganz verschiedenen Bedingungen zu erhalten wären. Bis jetzt waren vor allem drei Methoden Hofmeister'^) erfolgreich. Man entfernt lehrte zuerst, Eieralbuminkristalle darzustellen. sogenannten Globuline durch Halbsättigung der Eiweißlösung mit Ammonsulfatlösung. Das Filtrat der Globuline läßt man spontan eindunsten. Sobald die Konzentration der Lösung die untere Fällungsgrenze für das Eieralbumin erreicht hat, beginnt das Protein in Kristallform auszufallen.'-) S. F. L. Sörensen'i) hat es unternommen, auf breitester Grundlage an Hand des kristallisierten, gereinigten Eiereiweißes seine physikalischen, physikalisch-chemischen und chemischen Eigenschaften zu studieren. Es konnte entgegen den bis jetzt herrschenden Anschauungen gezeigt werden, daß die Kristallisation des Eieralbumins einer gewöhnlichen Kristallisation entspricht. Ferner ergab sich, daß das reine Eiereiweiß bis auf einen geringen Phosphorgehalt aschefrei ist. Auch andere Albumine lassen sich in der gleichen Weise zur Kristallisation bringen. Plasma- bzw. Serumalbumin, Oxjhämogiobin, sowie einige) ihm nahe verwandte Verbindungen, wie das Hämoglobin, das Methämoglobin und ferner das Hämozyanin^) kristallisieren besonders schön. Das Oxyhämoglobin läßt sich auf verschiedene Arten zur Kristallisation bringen.''') So erhält man z. B. prachtvolle Kristalle, wenn man zu einer Lösung von Oxyhämoglobin vorsichtig in der Kälte Alkohol zufügt. Man kann jedoch auch mit Ammonsulfat Kristalle abscheiden. Eine weitere Methode zur Darstellung von Eiweißkristallen beruht darauf, daß einige Proteine in lOo/oiger Kochsalzlösung löslich sind und beim Verdünnen der Lösung in Kristallform ausfallen. Es sind eine ganze Anzahl von Eiweißsubstanzen der Pflanzenwelt auf diesem Wege in Kristallform erhalten worden.') Vor allem sind die sogenannten Edestine zuerst die kristallisationsfähiii:. — ) Franz Hofmeister: Zeitschr. f. physiol. Chemie. 14. 165 (1889); 16. 187 (1891). \g\. ferner F. G. Hoplins und N. N. Pinkus: .Tour«, of Physiol. 23. 130 (1898). '-) Sehr begünstigt wird die Kristallisation durch Zusatz von Säuren. ä) S. P. L. Sörensen: Zeitschr. f. physiol. Chemie. 103. 211 (257) 1918): 106. S. P. L. Sörensen und Mar g. Höyrup: Ebenda. 103. 267 (1918). 1 (1919). ) A. Gürber: Sitzungsber. d. phvsik.-med. Gesellsch. zu Würzburg. 143 (1894). A. Michel: Ebenda. 29. 28 (1895). ^) Vgl. L. Frrdericq: Arch. de zool. expörim. 7. 535(1878). M. Henze: Zeitschr. f. physiol. Chemie. 33. 370(1901): 43. 290(1904). H'. I). Hallibiirfon: Journ. of. Phvsiol. 6. 300 (1885). Ernst Philippi: Zeitschr. f. physiol. Chemie. 104. 88 (1919). ^) Vgl. hierzu F. L. Hünefeld: Der Chemismus in der tierischen Oxydation. F. A. Brockhaus. Leipzig 1840. —' B. Reichert: Arch. f. (Anat. u.) Physiol. 197 (1849); 71 F. Hoppe-Seyler: Mediz. -ehem. Untersuchung. 2. 181 (1867). (1852). 0. Zinofski/: Zeitschr. f. physiol. Chemie. 10. 16 (1885;. G. Hüfner: Ebenda. 4. 382 (1880); 8. 366 G. Hüfner und ./. Otto: Ebenda. 7. 65 (1882). (1884). A.Jäderholm: Zeitschrift f. Biol. 20.419 (1848). Emil Abderhalden: Ebenda. 24. 545 (1898). '') O. Maschke: Zeitschr. f. prakt. Chemie. 74. 436 (1858). 0. Schmiedeberg: Zeitschr. f. physiol. Chemie. 1. 205 (1877). E. Drechsel: Zeitschr. f. prakt. Chemie. 19. 331 (1879). Georg Grübler: Ebenda. 23. 97 (1881). Vgl. weitere Literatur bei Osborne : Handbuch der Biochem. Arbeitsmethoden 1. c. S. 385, Zitat 3. — — — — — — — — — — — — — — — •^^' Vorlesung. 392 Eiweißkristalle sind auch wiederholt in der Natur vorgebildet beobAllerdings steht die Identitizierung der beobachteten Kri- achtet worden. stalle mit Eiweiß oft auf schwachen Füßen 1 So hat bereits im Jahre 1850 Th. Hartig^) im Klebermehl kristallinische Gebilde gesehen, die als Aleuronkristalle oder auch als Ptlanzenkristalloide bezeichnet worden sind. Die Eiweißnatur dieser Kristalle hat BrnUkofer-) festgestellt. Eiweißkristalle sind ferner namentlich in vielen Samen, wie z. B. in Kürbissamen. Hanfsamen, Rizinnssamen und vor allem in der Paranuß beobachtet worden. Ein sehr hübsches Demonstrationsobjekt für derartige Kristallbildungen gibt der zu den Orobancheen gehörende Schmarotzer, die Schuppen wurz'M, Lathraea squamaria. Sie enthält in den Zellkernen Proteinkristalle. Sehr Es läßt leicht sich mit kristallisiert ein aus Antiaris-Saft gewonnenes Protein. in Lösung bringen und scheidet OB" oiger Essigsäure sich bei ihrem Einengen in Kristallen ab.) Auch in tierischen GeAveben sind Kristalle oöenljar eiweißartiger Natur vereinzelt beobachtet worden. So hat man in den Darmepithelien des Mehlwurms, Tenebrio molitor, sechsseitige Tafeln festgestellt. s; R. List^) gibt an, in den Pigmentzellen der Radialnerven vom Sphaerechinus granularis Rhomboeder und Hexaeder beobachtet zu haben, die Eiweißreaktionen gaben. Auch die in den Eiern von Fischen und Amphibien beobachteten Dotterplättchen, rektanguläre und (\|aadratische Täfelchen, gehören hierher. Auch in den Eiern des Rehs sind derartige Produkte festgestellt worden") und ebenso in den Epithelien des Hodens des Menschen. 8) Großes Interesse ist von jeher der Frage nach dem Molekulargewicht der Eiweißkörper entgegen gebracht worden. ^i Man ist auf verschiedenen Wegen vorgegangen. Zunächst kann uns die elementare Zusammen set/.ung einen Anhaltspunkt geben. Vor allem ist der Schwefelgehalt berücksichtigt worden. Hat ein als ..rein" angesehener Eiweißkörper einen an Schwefel, dann muß sein Molekulargewicht 3200mal Gehalt von „ Wir erhalten nach dieser schwerer sein als dasjenige des Wasserstoffs. Berechnung nur Minimalzahlen, da wir nicht wissen, wieviel Atome Schwefel Der Schwefelgehalt der verschiedenen auf ein Molekül Eiweiß kommen. Es sind folgende Berechnungen Eiweißarten ist ein recht verschiedener. ausgeführt worden i"): P Th. Hartiq: Botan. Ztg. Nr. 50. 881 (1850). L. Radlkofer: t'ber Kristalle proteiiihaltiger Körper priauzlicheii und tierischen Ursprungs. W. Engelmaun. Leipzig 1859. ^) A. F. W. Schinipir: Zeitschr. f. Kristallographie. 1880. ) Y. Kotake und F. Knoop: Zeitscbr. f. physiol. Chemie. 75. 488 (1911). H. Kiliani: Ber. d. Deutsch. Chem. Ges. 46. (167 (19i;i). Berliner 5) Vgl. Jnh. Frevtzel: Arch. f. mikroskopische Anatomie. 26. 287. entomol. Zeitschr. 26 (1882) und W. BiedenncDin: PJiüf/ers Archiv. 72. 105 (1898). «) B. List: Anat. Anzeiger. 7. 185 (1897). ') V. r. Fbner: Sitzuogsher. d. kais. Akad. d. Wissensch. zu Wien. 110. Abt. 3 (1901). «) Lubarsch: l'irchown Archiv. 145. 317 u. 3B2 (]89()). ") Vgl. die übersichtliche Zusammenstellung und Besprechung der diese Frage berührenden Literaturhei/V. A'. .sWh//^.- Die Größe des Eiweißmoleküls. Gustav Fischer. Jena 1903. >"j Fr. N. Schulz: 1. c. S. 17. 1) -) — — Eiweißstofte und ihre Bausteine. 393 Molekulargewicht (unter der AnDahme, daß auf jedes Molekül Eiweiß "nr ein Atom Schwefel ,^ n Ol Schwetel m entfällt) »/o (»xyhämoglobin (Pferd) . . Edestin (kristallisiertj . . (kristallisiert) . Eieralbumin . Globulin Serumalbumin (krist.; Pferd) . 043 087 13 7440 3780 2460 2320 1800 138 189 Unter Berücksichtigung des Umstandes, daß diese Eiweißstoffe nicht nur ein Atom Schwefel pro Molekül enthalten, berechnet Fr. X. Schulz^) als Molekulargewicht für Seruraalbumin 5100, für Eieralbumin 4900, für Oxyhämoglobin 14 800 und für Edestin 7o00.-) Einen weiteren Anhaltspunkt für die Bestimmung des Molekulargewichtes geben uns die substituierten Eiweißkörper, vor allem das Oxyhämoglobin. Dieses enthält neben dem Eiweißkörper Globin einen eisenhaltigen Paarling. nämlich das Hämatin. Der Eisengehalt des Oxyhämoglobins aus Pferdeblut beträgt 0o35'/o, für andere Gxyhämoglobinarten haben die 04 O^ö^/o Bestimmungen zum Teil wechselnde Werte ergeben. Jedes Hämatinmolekül enthält ein Atom Eisen. Ein Eisengehalt von 04 0'b°/o verlangt ein Molekül von 14 000— 1 1 200, ein Schwefelgehalt von 0-43— O-ß?"/» erfordert ein solches von 14 800 9000 und ein Hämatingehalt von 4 5"/o ^) ein solches von 14 800 11800. Neuerdings haben Hüfner und Gansser^) das Molekulargewicht des Oxyhämoglobins direkt bestimmt und gefunden, daß es für Pferdeoxyhämoglobin 15115 und für Rinderhämoglobin 16 321 beträgt. Wir können als festgestellt betrachten, daß dem C)xyhämoglobin als solchem ein so hohes Molekulargewicht zukommt, dagegen ist es noch fraglich, ob der Eiweißpaarling des Oxyhämoglobins, das Globin, auch ein so gewaltiges Molekül darstellt. Die Annahme, daß auf ein Hämatinmolekül ein Globinmolekül kommt, ist nicht einwandfrei bewiesen. Es spricht manches dafür, daß mehrere Globinmoleküle mit einem Molekül Hämatin gebunden sind. Außerdem ist es sehr fraglich, ob das Globin selbst einheitlich ist. Jedenfalls liegt zurzeit kein zwingender Grund vor, aus dem Molekulargewicht des Oxyhämoglobins einen direkten Schluß auf die Mölekulargröße des Globins zu ziehen. Es ist auch versucht worden, den Gehalt von Eiweißfällungen mittelst Kupfer. Kalzium, Silber usw. an diesen Metallen als Maßstab für die Molekulargewichtsbestimmung zu verwenden.^) Es ist schwer zu sagen, ob man salzartige Verbindungen annehmen darf. Neuere Untersuchungen über Kolloide zeigen, wie außerordentlich vorsichtig man in der Beurteilung derartiger „Verbindungen" sein muß. Es können chemische Verbindungen vorliegen, es ist jedoch auch möglich, daß — Fr. N. Schulz: 1. c. S. — — — — ») — 392. Für sorgfältig gereiuigtes Eieralbumin wird neuerdings das Molekulargewicht auf 34 000 berechnet. Vgl. S. P. L. Sörensen, S. A. Christiansen, Marg. Hönrup, S. Goldschmidt und S. Palitzsch: C. r. du Lab. de Carlsberg. 12. 262 (1917). Vgl. auch R. Willstättcr und E. Waldschntidt-Leitz : Berichte d. Deutschen Chem. Ges. 54. 2988 ^) — (1921). ») ) 5) Fr. N. Schulz: Zeitschr. f. physiol. Chemie. 24. 449 (1898). G. Hüfner und E. Gansser: Arch. f. (Anat. u.) Phvsiol. 209 (1907). E. Harnack: Zeitschr. f. physiol. Chem. 5. 19S (1881). XX. Yorlesimg. 394 um Adsorptionserscheinungen handelt. Schon die Erfahrungen mit den Polypeptiden zeigen, wie vorsichtig man in der Beurteilung von Salzen sein muß. Es kommt ganz auf die Bedingungen an. unter denen man ein Kupfersalz darstellt. Man erhält bald „normale", bald ganz z. B. anormal zusammengesetzte Verbindungen.^) Es lassen sich somit über die Molekulargröße der Proteine vorläufig keine ganz sichergestellten Angaben machen. 2) Wahrscheinlich zeigen die einzelnen Eiw^eißstoffe untereinander auch in dieser Hinsicht große Unterschiede. Vielleicht wird es später möglich sein, das Molekulargewicht bestimmter Eiweißstotfe auf Grund der bei der vollständigen Hydrolyse aufgefundenen Aminosäuren festzustellen. Allerdings setzt auch diese Methode voraus, daß das Ausgangsmaterial ganz einheitlich ist. Wir haben wiederholt hervorgehoben, daß für die Eiweißstotfe das am meisten charakteristische Merkmal ihr Aufbau aus Aminosäuren und ihr kolloider Zustand ist. Die erstere Eigenschaft haben sie mit den Peptonen und Polypeptiden gemein. Der physikalische Zustand trennt sie von ihnen. Bevor wir die Frage der Beteiligung der einzelnen Aminosäure am Aufbau der verschiedenen Proteinarten erörtern, wollen wir die Einteilung der Eiweißstoffe und ihr Vorkommen kurz erörtern. Zunächst zerfallen die Eiweißstoffe in zwei große Gruppen, je nachdem sie als solche oder gepaart mit einer nicht eiweißartigen Verbindung vorkommen. Man nennt die ersteren einfache Eiweißstoffe oder Proteine und die letzteren zusammengesetzte Eiweißstoffe oder Proteide. Diese Art der Bezeichnung ist nicht ganz klar, denn es ist ganz gut möglich, daß es neben den einfachen Eiweißkörpern auch solche gibt, die aus mehreren Molekülen einfacher Proteine zusammengesetzt sind. Man könnte in diesem Falle von Polyproteinen sprechen. Weniger mißverständlich sind die Bezeichnungen Eiweiß Protein und Eiweißver-, bindungen Proteide. Die Proteide umfassen zwei Klassen von Verbindungen, die durch es sich = = Komponente scharf charakterisiert sind. Nukleoproteide und ferner die Farbstoffeiweißverbindungen mit den diesen nahestehenden Verbindungen. Die Nukleoproteide enthalten als nicht zum Eiweiß in Beziehung stehendem Paarung die Nukleinsäuren. Zu den Farbstoffeiweißverbindungen ge- die Art der nicht Es sind d^es eiweißartigen die Bei ihm findet sich eine eisenhaltige Verbindung mit einem Eiweiß besonderer Hämatin genannt Art, dem Globin, verknüpft. Zu dieser Gruppe gehören ferner das Phykoerythrin^) und das Phykozyan.) Auch das Hämozyanin ist ein Proteid. Seine Farbstofifkomponente ist noch nicht genau bekannt. Man hat ferner jene Eiweißstoffe, die Phosphor enthalten, zu den Proteiden hinzugerechnet und sie Phosphorproteide genannt. Es hat sich nämlich herausgestellt, daß der Phosphor, soweit die Art seines Vorkommens bekannt geworden ist, in Form von Phosphorsäure zugegen ist. Sie ist hört der Blutfarbstoff, das Hämoglobin. — — und K. Sugiura: Joiiru.'of Biol. Chcm. 10. 9 (1911); 13. 1 (1912). Unter anderem sei erwähnt, daß K. Ymnakami [The Biocliem. J. 14. 522 (1920)] für das Kaseinogen ein Molekulargewicht von nur 2000 annimmt. ) Vgl. U. Molisch: Botan. Zeitg. 177 (1894). ) H. Molisch: Ebenda. 131 (1895). ') ^) P. A. Kober EiweiOstotfe und ihre Bausteiue. 395 leicht als solche abspaltbar. ^j Man könnte vielleicht von Eiweißphosphaten sprechen. Ob der Phosphor ausschließlich in dieser Form in den Eiweißstoffen vorkommt, ist noch unentschieden. Endlich hat man Glukoproteide unterschieden.-) Sie sollen neben Eiweiß ein Kohlehydrat enthalten. Diese Gruppe ist zurzeit noch wenig klargestellt. Wenn wir von Proteiden sprechen, dann stellen wir uns Verbindungen vor, die ans Eiweiß und einem nicht eiweißartigen Paarung bestehen. Sie müssen aus diesen beiden Bruchstücken aufgebaut sein und auch in sie zerlegt werden können. Bei den sogenannten Glukoproteiden steht der Beweis, daß eine Spaltung in ein Kohlehydrat und in Eiweiß möglich ist. noch ganz aus. Vor allem ist es nie gelungen, das Glukosamin aus Müzinen abzuspalten, ohne daß gleichzeitig ein Abbau des Eiweißes eingetreten wäre. Vorläiitig spricht nichts gegen die Annahme, daß das Glukosamin am Aufbau des Eiweißmoleküls direkt teilnimmt. In diesem Falle gehören die Glukosamin führenden Eiweißstofte zu den Proteinen und nicht zu den Proteiden. Pie einfachen Ei weiß Stoffe, folgenden Gruppen einteilen: die Proteine, lassen sich in die Albumine. Sie sind sehr verbreitet. Man bezeichnet sie gewöhnlich nach dem Ort ihres Vorkommens. So spricht man von Plasma- bzw. Seruraalbumin. von Eieralbumin, von Milchalbumin usw. Die Albumine sind neutrale Eiweißkörper. Sie lösen sich in salzfreiem Wasser. Sie sind auch in verdünnten Salzlösungen, Säuren und Alkalien löslich. Den bisher bekannten Albuminen fehlt das Glykokoll. Die erwähnten drei Albumine sind in Kristallform erhalten worden. Auch im Pflanzenreich finden sich Albumine. Ein solches ist das Leukos in aus Gerste, Roggen und Weizen. Ferner gehören hierhin das Rizin aus der Rizinusbohne und das Legumelin aus Erbsen, Linsen und Wicken. Globuline. Sie finden sich überall mit den Albuminen zusammen. gehören zu den am meisten verbreiteten Proteinen, die wir kennen. Wir unterscheiden Plasma- bzw. Serumglobulin, Eierglobulin. Milchglobulin usw. Die Globuline sind in Wasser und in verdünnten Säuren unlöslich. Sie lösen sich in verdünnten Alkalien und verdünnten Neutralsalzlösungen. Verdünnt man eine Globulinlösung mit Wasser, dann beobachtet man das Auftreten einer Trübung. Sie deutet an, daß die Globuline ausgefallen sind. Das gleiche erreichen wir. wenn wir z. B. Blutplasma oder -serum der Dialyse gegen destilliertes Wasser unterwerfen. Es diffundieren Salze in die Außenflüssigkeit und damit werden den Globulinen die Bedingungen zur Lösung entzogen. Sie fallen aus, während die Albumine gelöst bleiben. Man kann die Globuline durch Ansäuern ihrer Lösungen fällen. Es genügt schon das Durchleiten von Kohlensäure durch eine Globulinlösung, um sie zur Abscheidung zu bringen. Die Globuline sind sehr empfindlich. Sie werden sehr bald denaturiert und damit unlöslich. Die Globuline sind ihrer ganzen Natur nach Säuren. Sie ') (1906). Vgl. R. H. Aders Flimmer uud W. M.'Bayliss: Jourii. of Phvsiol. — R. H. Aders Plimmcr und F. H. Scott: Ebenda. 38. 247 (1909j. 33. 439 -) Die erste Beobachtuug über den Gehalt der Müzine au reduzierenden Stoffen stammt von A. Eichwald: Liebigs Anualen. 134. 177 (1865). -^^^ Vorlesung. 396 Die Globuline enthalten im Gegensatz zu den Albuminen Gl y kokoll. Es sind zu dieser Gruppe noch einige Proteine hinzugerechnet worden, die in mancher Beziehung ein abweichendes Verhalten gegenüber den eigentlichen Globulinen zeigen. So sind unter pathologischen Verhältnissen im Harn Globulinarten beobachtet worden. Ferner hat man ein Globulin Myosin, Myogen der Muskelzellen mit einem besonderen Namen belegt. Endlich sind das Ovomuzin aus Eierklar, das Laktoglobulin — — aus Milch, das Lentoglobulin aus der Linse, Thyreoglobulin aus der Schilddrüse und der sogenannte Bence-Jonessche Eiweißkörper unter die Globuline eingereiht worden. Der letztere Eiweißkörper findet sich im Harn bei bestimmten Erkrankungen, vor allem bei Osteosarkomatose. Er kristallisiert. Dieses Protein ist leicht daran zu erkennen, daß es beim Erhitzen zunächst gerinnt, jedoch bei höherer Temperatur wieder in Lösung geht. Weitere Globuline sind das Perkaglobulin des Rogens von Fischen, das Fibrinogen des Blutes, das bei der Gerinnung das Fibrin liefert.^) Zahlreiche Globuline sind in der Pflanzenwelt, besonders in den Samen aufgefunden worden. Hierher gehören die Edestine (aus Hanf-, Sonnenblumensamen usw.), das Exzelsin aus der Paranuß, das Globulin aus Kürbissamen, aus Baumwollsamen, das Amandin aus Mandeln, das Juglansin aus der Walnuß, das Legumin aus Erbsen Linsen, Wicken, das Vizilin aus Erbsen, Linsen und der Saubohne, das Phaseolin aus Bohnen, das Glyzinin aus der Sojabohne, das Vi gn in aus der Kuherbse, das Konglutin aus Lupinensamen, das Korylin aus Haselnüssen usw. Manche von diesen Globulinen sind in kristallinischem Zustande erhalten worden. Eine Gruppe für sich bilden die Gluteline. Sie sind bisher nur im Pflanzenreich beobachtet worden und vorläufig noch unvollständig untersucht und abgegrenzt. Sie bilden mit Basen in Wasser lösliche Salze. Dahin gehört das Glutenin aus Weizensamen, das Maisglutenin und das Oryzenin aus Reissamen. Besser charakterisiert sind die Prolamine. Mit diesem Namen hat Oshorne jene Proteine belegt, die in Alkohol litslich sind. Bis jetzt kannten wir nur Vertreter dieser Klasse von Proteinen aus der Pflanzenwelt. Neuerdings ist mitgeteilt worden, daß auch in der Milch in Alkohol lösliche Proteine vorhanden sind. 2) Ihre Untersuchung ist noch nicht so weit durchgeführt, daß man die Frage entscheiden könnte, ob sie in die Gruppe der Prolamine hineingehören. Im Pflanzenreich sind mehrere Angehörige dieser Klasse von Proteinen aufgefunden worden. Es gehört hieher das Gliadin. Es findet sich im Weizenmehl. Es löst sich in 60 SOVoigem Alkohol. Das Gliadin bildet mit Säuren und Basen Salze, die zum Teil in Wasser löslich sind. Ferner ist aus Roggen- und Hafermehl ein Prolamin gewonnen worden. In den Samen der Gerste findet sich das Hör den in und im Mais das Zein. Den Prolaminen fehlt das Lysin. Im Gliadin sind geringe Mengen von Lysin gefunden worden. Es ist jedoch fraglich, ob ihm — ) Es scheint bei verschiedenen Tierarten die gleiche Zusammensetzung zu haben. Vgl. Boss Äiken Gortner und Alexander J. Würtz': Journ. Americ. Chem. Soc. 39. 2239 (1917). ^) Th. B. Oshorne, A. J Wakeman, Ch. S. Leavenuorth und i). L. Nolan: Journ. of biol. Chem. 33. 7, 243 (1918). . Eiweißstott'e diese Aminosäure selbst und ihre Bausteine. zukommt, oder oij anderen Proteins zurückzuführen sie 397 auf eine Beimengung- eines ist.^j Eine weitere Gruppe von Proteinen bilden die Müzine. Sie sind zunächst durch ihre physikalischen Eigenschaften ausgezeichnet. Die Müzine tinden sich in der Natur in zähflüssigem Zustand. Sie bedingen das Fadenziehen vieler Sekrete. Mau hat zahlreiche Muzinarten unterschieden und siß in Ermanglung einer Charakterisierung auf Grund besonderer Eigenschaften einfach nach ihrem Vorkommen benannt. So sprechen wir von einem Submaxillarismuzin, einem Gallenmuzin. einem Magen- und Darmmuzin, einem Serosamuzin usw. Auch in der M7/rtr^o>^schen Sülze der Nabelschnur finden sich Müzine. Besonders verbreitet sind sie ferner bei den Schnecken. Ferner treffen wir auf Angehörige der Reihe der Müzine bei den Fischeiern. Besonders große Mengen einer Muzinart lassen sich aus dem Inhalt von Ovarialkystomen gewinnen. Man hat dieses Muzin Pseudomuzin genannt und daneben noch ein Paramuzin unterschieden. Es unterliegt keinem Zweifel, daß unter den Müzinen manches Protein untergebracht worden ist. das gar nicht hierher gehört. Ferner sind wohl die meisten Müzine stark verunreinigt. Es ist fast unmöglich, diese zähflüssigen Proteine zu reinigen. Sie stellen Säuren dar. Den Müzinen stehen die Mukoide sehr nahe. Sie unterscheiden sich von den Müzinen dadurch, daß sie durch Säuren nicht fällbar sind. Zu dieser Gruppe gehören das Ovomukoid aus dem Eiereiweiß der Yogel- Serummukoid, das eier, das Harnmukoid. das Hyalomukoid aus dem Glaskörper, das Korneamukoid, ferner das Chondro-. Tendo- und Osseomukoid. Außerdem sind noch aus Ligamenten, aus der Haut, aus niederer Tierarten usav. Proteine gewonnen worden, die den Mukoiden nahe zu stehen scheinen. In den Müzinen und Mukoiden sind, wie S. 337 erwähnt, die Verbindungen Chondroitin- und Mukoitin- Eihüllen schwefelsäure gefunden Avorden.-j Chondroitinschwefelsäure ist aus der der Sklera, aus Sehnen und Knorpel erhalten worden. Mukoitinschwefelsäure aus der Hornhaut, dem Glaskörper, aus Serummukoid. Ovomukoid, Mukoid aus Ovarialzysten, aus Muzin der Magenschleimhaut. Aorta, Wir kommen nunmehr zu zwei Klassen von Proteinen, die ihren ganzen Eigenschaften und ihrer Zusammensetzung nach eine besondere Stellung einnehmen. Es sind dies die Histone und Protamine. Sie sind beide durch ihre basischen Eigenschaften ausgezeichnet. Sie enthalten viel Lysin, Arginin und Histidin oder auch nur eine dieser Aminosäuren. Gruppen von Proteinen tinden sich in der Natur nicht in Sie sind vielmehr mit sauren Verbindungen zu Proteiden verknüpft. Wir kennen Verbindungen von Histonen mit Nukleinsäuren. Ferner dürfte das mit dem Humatin verknüpfte Globin Beide freiem Zustande. auch zu den Histonen zu rechnen für das ') Vgl. 41H (1909). 14. — ^Manche Be(»bachtung spricht auch Emil Abderhalden und Casimir Funk: Zeitsclir. f. pbysiol. Chemie. 40. liomas B. Oshorne und Charles S. Leavenicorth: Jouru. of biol. Chem. '1 481 (1913y. -) sein. Vorkommen von Verbindunüen zwischen Histonen und Vgl. die Literatur S. 336. 398 •^-^- Vorlesung. Protaminen mit sauren Proteinen. Diese Kombinationen wären unter die Polyproteine einzureihen. Es sind Histone^) aus den roten Blutkörperchen der Vögel, aus der Thymusdrüse, aus dem Sperma von Fischen usw. isoliert worden. Sie enthalten etwa 20— SO^/o der oben erwähnten Aminosäuren. Die Protamine sind von Miescher^) entdeckt worden. Ihre genauere Kenntnis verdanken wir Kossel^) und seinen Schülern. Es sind die Protamine vor allem aus den Testikeln von Fischen gewonnen worden. Ihre Bezeichnung ergibt die Herkunft. Wir unterscheiden Salm in (Lachs), Klup ein (Hering), Skombr in (Makrele), Zyklopter in (Seehase, Cyclopterus lumpus), Zyprinine (Karpfen), Sturin (Stör), Accipenserin (Accipenser), Silurin (Wels) usw. Auch aus dem Sperma des Menschen ist ein protaminartiges Protein gewonnen worden. Erwähnt sei noch, daß Kosscl die zusammengesetzten, den Peptonen der übrigen Proteine entsprechenden Abbaustufen der Protamine Protone genannt hat.) Die Protamine bestehen zum allergrößten Teil aus den sog. Diaminosäuren. Die Monoaminosäuren treten an Menge ganz zurück. Es sind auch immer nur einzelne davon vorhanden Wir kommen nun zu einer Gruppe von Proteinen, die wir am besten nach ihrer Funktion charakterisieren können. Es handelt sich um Eiweißstoffe, die im tierischen Organismus mechanische Funktionen erfüllen. Man hat sie Gerüsteiweiße genannt, doch wird dieser Name nicht der Funktion aller Glieder dieser Reihe gerecht. Das Elastin z. B. kann wohl kaum oder doch nur zum Teil als Gerüstsubstanz bezeichnet werden. Früher hat man dieser Gruppe von Eiweißstolfen den Namen Albuminoide gegeben, besser paßt der Name Proteinoide. Man könnte sie auch, unter Berücksichtigung ihres reichen Gehaltes an Glykokoll, Glyzinamine nennen. Die Glieder dieser Reihe sind einmal dadurch charakterisiert, daß sie stets in festem Zustande auftreten und ferner fast ausschließlich aus Monoaminsäuren bestehen. Bei ihrer Einteilung hat man sich ganz einfach an ihr Vorkommen gehalten. Es seien zunächst die wichtigsten Proteine dieser Gruppe, die bei den Averteb raten sich finden, erwähnt. Die verschiedensten Schwämme bauen ihre Gerüstsubstanz aus dem Protein Spongin auf. Wir sind ihm schon einmal begegnet, als wir das Vorkommen von 3, o-Dijod-1-tyrosin besprachen. Spongin enthält diesen Baustein. Auch das Gorgonin, das Achsenskelett von Korallen, enthält Dijod- und Dibrom ^)-ty rosin. Kornein findet sich ebenfalls in Korallen und enthält Jod. Es bestehen auch die Hornröhren des Röhrenwurmes Onuphis und Spirographis aus Eiweiß (Onuphin und Spiro- — L. Lilienfeld: Vgl. 11. a. Ä. Kossei: Zeitschr. f. pliysiol. Chemie. 8. 511 (1883 '84). F. Mescher: Arch. f. exper. Path. u. Phannak. 37. 100(1896). Irar Baiu/: Zeitsclir. f. pliysiol. Chemie. 27. 463 (1897); 30. 508 (1900); Hofmeisters ßeitr. 4. 115, 331 u. 362 (1903).' F. Gonbmi: Arch. intcrnat. de Physiol. 8. 300 (1909). ) Friedrich Miesc/ier: Histochemische und pbvsiol. Arbeiten. 2. F. C. W. Vogel. ') Ebenda. 18. 476 (1894). — — — Leipzig 1897. ^) A. Kossei: Zeitschr. f. physiol. Chemie. 22. 176 (189()); 25. I(i5 (1898); 26. 588 (1899); Ber. d. Deutschen Chem. Ges. 34. 3214 (1901) und weitere Ar1)eiten von Mafhews, F. Kutscher, Dcikin, Goto u. A. in der Zeitschr. f. physiol. Chemie. A. Kossei und F. Weiss: ") M. Goto: Zeitschr. f. physiol. Chemie. 37. 94 (1903). — Ebenda. "•) 59. 281 (1909). Vgl. dazu Carl l'h. Monier: Zeitsciu-. f. phvsiol. Chemie. 88. 138 (1913). Eiweißstoffe und ihre Bausteine. 399 graphin). Das Konchiolin stellt die organische Grundsubstanz der Muschelschalen dar. Ein sehr interessantes Protein dieser Reihe ist der von manchen Muscheln abgeschiedene Byssus. Er besteht aus seideglänzenden Fäden, die der Seide sehr ähnlich sind. Auch in ihrem Gehalt an Aminosäuren finden sich Anklänge an diese. Die Seide gehört ebenfalls hierher. Die Raupen der verschiedensten Vertreter der Lepidopteren liefern Gespinste, die alle zur Eiweißgruppe gehören. Auch die Spinnen spinnen mit Eiweiß! Ferner sondern viele Käfer i) ein an Eiweiß reiches Sekret ab, das zu feinen Fäden ausgesponnen wird und bald erstarrt, nachdem es die Spinndrüse verlassen hat. Der Seidenfaden ist nicht einheitlich. Man kann aus ihm durch Auskochen mit Wasser den Seidenleim Serizin erhalten. Es bleibt dann das Seidenfibroin = zurück. Auch die Vertebraten verfügen über sehr verschiedenartige Proteine dieser Reihe. Es seien erwähnt das Elastin, das die Grundsubstanz des elastischen Gewebes darstellt, ferner die zahlreichen Keratinarten. Keratine finden sich in der Oberhaut, sie setzen die Haare, Federn, die Nägel, Hufe, Geweihe, Hörner, die Barten des Wales, den Panzer der Schildkröte, das Hörn des Nasenhorns usw. zusammen. Im Nervengewebe findet sich das Neurokeratin. Es sind ferner zu erwähnen: Proteine, die EihüUen aufbauen. Bis jetzt sind die EihüUen von Testudo graeca, von Scyllium stellare, die Vogeleihüllen (Ovokeratin) genauer untersucht w^orden. Mit Koilin ist der hornartige Überzug des Vogelmagens bezeichnet worden.-) Schließlich wären noch Vertreter dieser Gruppe von Proteinen zu erwähnen, die die Grundsubstanz des retikulären Bindegewebes bilden (Retikulin), und ferner in der Linse, in tierischen Membranen, im Knorpel und im Knochen anzutreften sind. auch das Kollagen und der Leim. Das erstere Hauptmenge der Grundsubstanz des lockeren Bindegewebes. Es findet sich ferner in Sehnen, Faszien, Bändern, im Knorpel und in Knochen. Auch das aus Fischschuppen gewonnene Kollagen gehört hierher. Der Leim, Gelatine oder Glutin genannt, entsteht aus dem Kollagen durch Lösen in kochendem Wasser. Der Leim erstarrt bei gewöhnlicher Temperatur und wird bei etwa '60" wieder verflüssigt. Von den Proteiden haben wir bereits den Eiweißanteil des HämoHierher gehören bildet die globins erwähnt, nämlich das Globin. Es ist durch einen auffallend hohen Gehalt an His tidin ausgezeichnet. Wir haben auch schon erwähnt, daß Histone nnd Protamine in den Nukleoproteiden enthalten sind. Diese letzteren sind am Aufbau der Kernsubstanzen beteiligt. Es sind eine ganze Anzahl verschiedener Nukleoproteide dargestellt und untersucht w^orden. Das Hauptinteresse wurde freilich nicht den Eiweißanteilen, sondern der an ihrem Aufbau beteiligten Nukleinsäure zugewandt. So sind Nukleoproteide aus den Blutkörperchen der Vögel, aus Eiern, aus Gehirn, aus Hefe, aus der Leber, aus den Nieren, Nebennieren, den Muskeln, der Milz, der Pankreasdrüse, der Schilddrüse, aus Fischsperraa usw. gewonnen worden. Es bleiben noch die Phosphorproteide zu besprechen. Es sind unter diesem Namen eine ganze Anzahl verschiedenartiger Eiweißsubstanzen Nach eigenen Feststellungen. Ö K. B. Uofmann und Fritz P/t^/.- Zeitschr. f. pli\>iol. Chemie. 52. 448(1907). ) X^' Vorlesung. 400 zusammengefaßt worden. Als gemeinsames Merkmal findet sich eigentlich nur der Gehalt an Phosphor bzw. an Phosphorsäure. Manche Forscher rechnen diese Eiweißstofte zu den Proteinen, andere zu den Proteiden. Bei manchen wissen wir nicht mit Sicherheit, ob ihnen der Phosphor wirklich zukommt. Wiederholt konnte festgestellt werden, daß er nur einer Verunreinigung zuzuschreiben war. Die Aufstellung der Gruppe der Phosphorproteide stellt nur einen Notbehelf dar, um einige Namen, die sich eingebürgert hatten und unrichtige Vorstellungen erwecken konnten, ganz auszuschalten. So nannte man früher manche der jetzt hier eingereihten Verbindungen Nukleoalbumine. Diese Bezeichnung führte zu Verwechslungen mit den Nukleoproteiden. Die zu den Phosphorproteinen gehörenden Eiweißsubstanzen besitzen saure Eigenschaften. Sie sind unlöslich in Wasser und lassen sich, wie die Globuline durch Säuren ausfällen. Hierher gehört das Kaseinogen, das in der Milch vorkommt.^) Es sind eine ganze Anzahl von Salzen des Kaseinogens dargestellt und untersucht worden. Auch Azidkaseine sind bekannt. Pepsinsalzsäure spaltet aus dem Kasein Phosphorsäure ab. Labferment 2), ein im Magensaft vorkommendes Ferment, führt das Kaseinogen in Kasein über. Bei der Milchgerinnung fällt das Kasein als Kalksalz aus. Es handelt sich nicht um einen eigentlichen Gerinnungsvorgang, sondern um eine richtige Fällung eines schwerlöslichen Salzes. Zu den Phosphorproteiden w^erden auch die Vi teil ine gerechnet. Sie finden sich im Dotter der Eier. Ferner wäre noch die Gruppe der Ichthuline zu erwähnen. Sie stehen den Vitellinen offenbar sehr nahe und finden sich in Fischeiern. Zahlreiche, phosphorhaltige Proteine bzw. Proteide sind in der Pflanzenwelt aufgefunden worden, doch erwiesen sie sich fast immer als unrein. oft vor. Namentlich Beimengungen von Phosphatiden kommen auch Phosphatido-proteide, doch ist ihr Vielleicht existieren Vorkommen nicht eindeutig bewiesen. auf die gegebene Einteilung der Proteine und Proteide daß eine sehr große Anzahl verschiedenartiger Eiweißstoffe im Tierund Pflanzenreich aufgefunden worden ist. Man erkennt aus der gegebenen Darstellung ohne weiteres, daß die uns zur Verfügung stehenden Methoden, um Eivveißstoffe zu charakterisieren, ganz unzulänglich sind. Fast jede einzelne Gruppe enthält sicher heterogene Produkte. Einzig die Albumine, die Globuline und die Prolamine nebst den Histonen und Protaminen sind gut charakterisiert. Immerhin umfaßt gewiß jede dieser Gruppen auch noch recht verschiedenartige Substanzen. So lange wir darauf angewiesen sind, die Eiweißkürper mittelst Fällungsreaktionen und zum großen Teil auch nur nach ihrem Vorkommen und ihren Funktionen gegeneinander abzuEin Blick zeigt, grenzen, die muß ihre Eintinlung unbefriedigend bleiben. Klassifizierung der IVoteine Vorläufig vermittelt allein eine Ver- und Proteide einzig und ständigung. Gewiß existieren Billionen verschiedener Eiweißarten und zahlreiche einzelne Gruppen werden sich bilden lassen, wenn erst einmal unsere Kenntnisse über vollkommenere die Zusammensetzung und die Struktur der Proteine sind. Wir wollen noch die Frage aufwerfen, ol) die bisher erhaltenen Resultate der Untersuchungen über den Gehalt verschiedener •) ^) Vgl. B. Blei/er und R. Seidl: Biochem. Zeitscli. 128. 48 (1922). Vgl. über dieses Ferment Band 2, Vorlesung XIX. EiweiüstortV imd ihre Bausteine. 4()\| Proteine an einzelnen Aminosäuren eine Abg-renzun^- der einzelnen EiweiUarten erinöj;lichen. Es sind über hundert verschiedene Eiweißstott'e vollständig- hydrolysiert und die entstandenen (ieniische an Aminosäuren nach den von Kosse/ und Kutscher und Emil Fischer geschaffenen Methoden auf die einzelnen Aminosäuren untersucht worden. Es wurden vor allem auch die Mengen, in denen die einzelnen Bausteine gewonnen werden konnten, berücksichtigt. Wie schon wiederholt betont worden ist. besitzen wir zurzeit keine Methoden, um die einzelnen Monoaminosäuren quantitativ zu bestimmen, dagegen ergibt die Estermethode, wenn sie stets unter den gleichen Bedingungen^angewandt wird, vergleichZahlen werte. Die Ergebnisse der vorliegenden Untersuchungen gestatten eine Einteilung der Proteine in vier große Gruppen. Einmal kennen wir Proteine, die fast nur aus Monoaminosäuren bestehen. Dahin gehören alle jene Eiweißstotte, die wir als Proteinoide oder Glyzinamine bezeichnet haben. Es ist von großem Interesse, daß, wie schon erwähnt, in den meisten dieser Proteine namentlich das GlykokoU stark vorwiegt. Daneben findet man häutig große Mengen von d-Alanin und 1-Tyrosin. liare Diese Gruppe v(m Eiweißstotfen ist durch alle möglichen Übergänge mit einer zweiten verknüpft, an deren Aufbau rund 10 lö^'/o Arginin. Lysin und Histidin beteiligt sind. Der Rest 90 85%, besteht aus Monoaminosäuren. Hierher gehören die Albumine, Globuline usw. d. h. alle jene Eiweißarten, die nicht zu den Proteinoiden und auch nicht zu den Histonen und Protaminen gehören. In dieser Reihe sind manche Vertreter ganz gut charakterisiert. iSo finden wir bei den Albuminen kein GlykokoU. während die Globuline diese Aminosäure in allerdings geringer Menge (höchstens 50/0) besitzen. Die in Alkohol löslichen Eiweißstoffe zeichnen sich durch das Fehlen von Lysin ^) aus, ferner besitzen sie einen auffallend hohen Gehalt an Glutaminsäure und Gxyglutaminsäure. Das Gliadin besitzt z. B. über 40Vo an diesen Aminosäuren. — — Als dritte Gruppe folgen die Histone. Sie besitzen etwa '30" Arginin. und Lysin. anderen Ende dieser Reihe stehen die Protamine, die fast nur aus den genannten Aminosäuren bestehen. Monoaminosäuren kommen nur in geringer Menge in ihnen vor. Am Histidin Wir können vorläufig auf Grund dieser Ergebnisse nur prüfen, ol» jene Einteilung, die sich auf die physikalischen Eigenschaften und zun« Teil direkt auf ihre morphologischen gründet, haltbar ist. Eine neue Klassifizierung lassen sie nicht zu. Dazu sind unsere Kenntnisse noch viel zu gering. Einen bleibenden Wert kann außerdem nur eine auf die Struktui Rücksicht nehmende Einteilung der Proteine beanspruchen. Man wird in Zukunft versuchen müssen. Eiweißstoffe, die sich nach ihren Eigenschaften und vor allem nach ihrem Gehalt an den einzelnen Aminosäuren sehr nahe stehen, dadurch voneinander abzugrenzen, daß man aus ihnen Bruchstücke isoliert, die noch mehrere Aminosäuren gebunden enthalten. Gelingt es auf diesem Wege, zu Verl)indungen zu gelangen, die nur dem einen der sonst sich ähnlichen Eiweißstoflfe zukommen, so wird dieses ^Merkmal genügen, um dieses Protein zu charakterisieren. Wir haben festgestellt, daß z. B. sechs strukturisomere Tripeptide mit drei verschiedenen Bausteinen bei ') Vgl. hierzu S. Alxlo rhalden , 39(5. Physiologische Cliemi.'. I TimI. r,. .\iiH 26 ^^- Vorlesung. 402 der vollständigen Hydrolyse die gleichen Mengen der gleichen Aminosäuren Das Ergebnis der teilweisen Hydrolyse müßte ergeben, welche Die folgenden sechs aus den drei Struktur das einzelne Tripeptid hat. Aminosäuren, A, B und C, aufgebauten, strukturisomeren Tripeptide liefern, wie das folgende Schema zeigt, ganz verschiedene Dipeptide, wenn z. B. in allen sechs Fällen das hinterste oder aber das vorderste Glied der Kette abgesprengt wird: liefern. A— b"^ A— t^^ B— A^ B-C^ C— A^ C— B^ . einander nahe stehende Proteine entweder zu gegen einander abzugrenzen, können zurzeit nur sehr unvollkommen durchgeführt werden, weil noch sehr viel Vorarbeit fehlt. Wir müssen jedoch unbedingt einem solchen Ziel zustreben, wenn es uns selino-en soll, unsere Kenntnisse der Natur der einzelnen Proteine zn Derartige Versuche, identifizieren oder vertiefen. Wir kennen andere Methoden, um bestimmte Proteine zu kennzeichnen. Es sind dies die sog. biologischen Methoden. Spritzen wir z. B. einem bestimmten Tiere ein Protein in die Blutbahn ein. dann erhalten wir nach einio-er Zeit mit dem Serum des gespritzten Tieres und dem gleichen Protein eine eigenartige Reaktion. Es entsteht nämlich eine Ausflockung, genannt Präzipitinbildung.^) Das Serum eines nicht vorbehandelten Tieres Erscheinung nicht. Oder wir warten nach der ersten Zufuhr des Eiweißstoffes einige Tage ab und spritzen das gleiche Protein wieder. Wir bemerken, daß das vorher ganz muntere Tier fast plötzlich die schwersten zeigt diese P>scheinungen zeigt. Es treten Krämpfe und auch Lähmungserscheinungen seine Körpertemperatur sinkt. SchließDas Tier fühlt sich kalt an lich stirbt es. Die geringste Spur eines Eiweißkörpers genügt, um diesen Zustand auszulösen. 2) Man spricht von einer Überempfindlichkeit und Den der nennt die erste Injektion' des Produktes Sensibilisierung. zweiten Injektion folgenden Zustand nennt man Schock. Wählen wir zur Sensibilisierung und zur zweiten Injektion zwei verschiedene Proteine, dann l)leibt der Schock aus. Endlich ist beobachtet worden, daß nach erfolgter parenteraler Zufuhr von Eiweiß im Blutplasma Fermente auftreten, die Eiweiß spalten können. Vielleicht führt die weitere Ausarbeitung dieser Forschung zum Nachweis von Fermenten, die streng spezifisch auf bestimmte, Es ist wohl als einheitlich charakterisierte Eiweißarten eingestellt sind.^) möglich, daß das Studium des fermentativen Abbaues der Proteine uns noch wichtige Fingerzeige über ihre Struktur ergeben wird. Jedenfalls sind die biologischen Methoden zur Unterscheidung der Eiweißstotfe den chemischen Einen klaren Einblick in zurzeit an Feinheit noch bei weitem überlegen. — auf. — L. MiVgl. z. B. L. Michaelis: Deutsche med. Wochenschr. Nr. 41 (1902). und Carl Oppenheimer: Archiv f. (Anat. u.) Physiol. Suppl. 336 (1902). F. Ohermayer und E. P. I'ick: Wiener klin. Wochenschr. 17. Nr. lU (1904); 19. Nr. 19 (1906). Ferner die Lehrbücher der Immunitätslehre. ) rhaeli/i — Die Anaphylaxie. Fortschr. d. Deutsclieii Kliuik. ") Vgl. hierzu u. a. E. Friedhcrger Hermann Pfeiffer: Das Prnl)lem der Kiweißanaphyla.xie. (lustav 619 (1911). Alfred Clemens Pirt/urt: Allergie. Julius Spriuger. Berlin 1910. Fischer. Jena 1910. Schittenh'elm: Über Anaphylaxie vom Staudpunkt der pathologischen Physiologie und Ferdinand Enke. der Klinik. Jahresber. über die Ergebn. der Immuuitätsforschung. : 2. — — — Stuttgart 1910. =') Vgl. die Literatur bei P^mil Abderhalden : Die Ahderli(iJden<-^c\\(i Reaktion..^. Antl. J. Springer 1922. EiwciBstoft'e und ihre Biiusteiuo. 403 Kiweißstntte des tierischen Organismus Eieralbu- min '( Glykokoll Alanin .... .... Seriü Zystin .... Valin .... 30 Norleuzin Leuziufraktion ') 2 2 Weiße Sub- Oxyhämo- stanz des Zentralner- globin des Pferdes 30 3H 0-y 0-8 0-23 4-2 0-6 03 0-3 12 10 OOß 10 20 10 1-0 vensystems 12 0-2 2-5 11 7 15 15 10-5 290 40 3-8 25 4-2 25 3 5 vor- 3-2 4-5 1-0 015 10 2 6 110 1-4 2-5 . . Phenylalauiü Tyrosin 45 10 . . . . . . . Histidin bulin') (ilobin aus . . Isoleuzin Kaseinogen glo- 3Ö . . Serum- handen .... Lysin Arginin Asparagiusäure Glutaminsäure . . . .... Prolin Oxyprolin . 2-0 40 3-0 5-8 4-8 4-3 5-4 4-5 20 44 015 1-5 2-5 20 12 90 8-5 10-4 3-6 11-0 vor- 1-5 9n 31 025 . Tryptopliau vor- vor- handen banden handen Ammoniak . 80 1-7 20 23 0-3 10 vor- vor- handen handen . Eiweißstoffe des Pflauzenorganismus A m n o i Glykokoll Alanin . 8 ä u Legumin Edestin aus aus Erbse Hanfsamen . .... Serin .... Zystin .... ValiQ .... 0-4 3-8 20 05 3fi Leukosin (TÜadin uns ülut>-in aus aus Weizen- Weizpnehi Weizenmehl 25 Ol 0-9 4-6 0-7 samen 111 0-9 4-5 03 0-45 0-3 002 02 11-5 60 6-0 25 vor- 0-25 handen Norleuzin Leuziufraktion") Isoleuzin . . . . . Phenylalanin Tyrosin 80 . 210 375 25 3-8 2-6 20 1-5 21 2-4 1-7 4-25 1-7 2 2 Lysin Arginin Asparagiusäure Glutaminsäure 5 11-7 5-3 165 140 3-3 2-8 2-75 6-0 34 4-7 4-5 3-4 1-2 .... 170 140 17 6-7 3-2 370 32 2-4 0-9 23-5 4-2 Histidin . . . . . . .... . Prolin Oxyprolin . . . Aniiuouiak ') . 20 . Tryptophan vor- vor- vor- vor- handen handen handen 10 handen 20 2-3 1-4 51 40 . Nach eigener, nocli nicht mitgeteilter Untersuchung. — bedeutet, daß die betreffende Aminosäure nicht bestimmt worden ' IS 19 Leuzinfraktion-' bedeutet, daß die Trennung in die ist. Leuziiiisomcreu durchgeführt wurde. 2G' iiiclit -^^^ Vorlesung. Eiweißstottc uud ihre Bausteine. 404 das Wesen der biologischen Reaktionen werden wir jedoch erst erhalten, wenn die chemische und vor allem auch die physikalisch-chemische Forschung weiter fortgeschritten ist. Die vorstehenden Tabellen geben einen Einblick in die Zusammensetzung einiger der wichtigsten Proteine. i) Die gefundenen Ausbeuten an den einMan muß außerdem bei Betrachzelnen Aminosäuren sind abgerundet. tung der einzelnen Werte sich daran erinnern, daß sie nur Minimalauswerte darstellen. So bleiben bei den meisten Monoaminosäuren genommen sind Ty rosin. Glutaminsäure und Zystin, die direkt bestimmt die Ausbeuten um 30— 40"/o hinter der Wirklichkeit worden sind zurück. Vgl. S. 349. In der Tier- und Pflanzenwelt linden sich Farbstoffe, Pigmente, die man mit mehr oder weniger Berechtigung mit dem Eiweiß und seinen Abbaustiifen in Zusammenhang gebracht hat. Namentlich die sog. Melanine hat man auf Eiweiß zurückgeführt. Es spricht sehr vieles dafür, daß die Proteine und vor allem gewisse Bausteine, wie das Tryptophan und das Tyrosin, das Ausgangsmaterial zur Bildung mancher Melaninarten darstellen, doch sind diese so sehr verändert, daß sich keine direkten Beziehungen zu bestimmten Verbindungen mehr erkennen lassen. — — ^) Die deu folgenden Tabellen zugrunde gelegten Resultate sind den Arbeiten von Emil Fischer, Emil Abderhalden und Thomas B. Osborne und ibren Scbülcru entnommen. Vgl. weitere Daten bei 0. Cohnheini : Chemie der p]iweißkörper. 1 c. S. 307: Emil Abderhalden: Neuere Ergebnisse der Eiwoißcbemie. 1. c. S. 307. Biocheni Hand- lexikon 2. 1. c. S. 307. Vorlesung XXI. Eiweiüstoffe und ihre Bausteine. Bildung der Aminosäuren und der Eiweißstoffe im Pflanzenorganismus. Assimilation des Stickstoffs. Sein Kreislauf in der Natur. Herkunft der übrigen am Aufbau der Proteine beteiligten Elemente. Die Eiweißstoffe enthalten mit ihren Bausteinen Kohlenstoff, Wasserstoff. Stickstoff und »Sauerstoff. Dazu kommt dann noch der Schwefel, der sich mit wenigen Ausnahmen in allen Proteinen tindet, jedoch von den bekannten Aminosäuren nur dem Zystin zukommt. Es unterliegt wohl kaum einem Zweifel, daß das Problem der Entstehung der Proteine in der Pflanzenwelt sich auf die Frage nach der Bildung der einzelnen Aminosäuren zurückführen läßt, denn es ist kaum anzunehmen, daß der Eiweißaufbau in der Pflanze in »anderer Weise als über diese erfolgt. Die erste Frage, die wir zu beantworten haben, ist die nach der Herkunft der einzelnen Elemente. Kohlenstoff, Wasserstoff" und Sauerstoff" entstammen primär sicher der Kohlensäure der Luft und dem Wasser. Den Stickstoff nimmt die Pflanze zum größten Teil in Form Aon Salpeter aus dem Boden auf. Der Schwefel wird Sulfaten entnonnnen. Der Phosphor der Phosphorproteide endlich ist auf aufgenommene Phosphate zurückzuführen. Über die Herkunft der einzelnen Elemente besteht kein Zweifel, dagegen stoßen wir sofort auf zurzeit noch unüberwindbare Schwierigkeiten, wenn wir die Frage zu beantworten suchen, wie die erwähnten Elemente im Pflanzenorganismus zu den einzelnen Aminosäuren zusammengefügt werden. Es ist bis jetzt nicht gelungen, diesen Vorgang aufzuklären. Weder kennen wir bestimmte Zwischenstufen, noch wissen wir, in welcher Phase der Kohlensäure- und Wasserassimilation der Stickstoff eingreift. Ja. es ist nicht einmal geglückt, das Auftreten der fertigen Aminosäuren eindeutig festzustellen. Wohl finden sich solche im Pflanzenorganismus. Wir wissen jedoch nicht, ol) sie eben synthetisch entstanden sind oder aber dem Abbau vorhandener Proteine entstammen. Auch der Ort der Aminosäurebildung ist noch viel umstritten. Würde man in den chlorophyllhaltigen Pflanzenteilen und insbesondere in den Blättern oder sonst einem Organ der Pflanzen Verbindungen in größerer Menge antreffen, die mit der Bildung von Aminosäuren oder von P^iweili in irgend welchen direkten Beziehungen ständen, dann müßte es gelingen, das Proldem der Eiweißbildung in der XXI. NOrlesuug. 406 Bei der Assimilation von Kohlensäure und Wasser wir bereits erwähnt haben, als erstes sicher erkennbares Assimilationsprodukt die Stärke nachweisen.') Es unterliegt keinem Zweifel, daß dieses große Molekül, das sich aus zahlreichen Traubenzuckermolekülen aufbaut, nicht direkt aus Wasser und Kohlensäure gebildet wird. Es müssen vielmehr zunächst einfachere Produkte entstehen. Als ein derartiges Assimilationsprodukt ist der Formaldehyd, HCOH, zu betrachten.-) Selbst hier, wo wir direkt die Entstehung eines Polysaccharides beobachten und seine Bildung beeinflussen können, ist es noch nicht gelungen, Einzelheiten im Werdegang dieser Substanz festzustellen. Den gleichen Schwierigkeiten der Aufklärung des Entstehungsmodus begegneten wir bei der Besprechung der Frage nach der Herkunft der Fette, Phosphatide und Überall stoßen wir auf Hypothesen, Sterine im Pflanzenorganismus. die ein Bild entwerfen, wie der Aufbau der einzelnen Verbindungen vor sich gehen könnte. Auch hier bei den Aminosäuren bzw. Proteinen bleibt uns nichts anderes übrig, als einige Wege zu erörtern, die von der Kohlensäure, dem Wasser und dem stickstoft'haltigen Ausgangsmaterial zu den einzelnen Aminosäuren hinführen können. Pflanze klarzustellen. läßt sich, wie •^) Es sind zunächst drei Möglichkeiten gegeben. Einmal kann die der Aminosäuren ein direkter Assimilationsvorgang sein, d.h. sie entstehen ohne Umwege aus den genannten Grundstoffen. Es ist jedoch auch möglich, daß die Bildung der Aminosäuren derjenigen anderer Stoffe folgt. So könnten z. B. zunächst Kohlehydrate sich bilden, und diese dann in Aminosäuren verwandelt Bildung werden. Endlich ist es denkbar, daß als erstes Assimilationsprodukt der Kohlensäure und des W^assers ein Produkt auftritt, von dem aus alle möglichen Verbindungen entstehen können. Solche indifterente Verbindungen, die als Ausgangsmaterial für die mannigfaltigsten Verbindungen in Betracht kommen können, sind die Milchsäure, die Brenztraubensäure und auch das Methylglyoxal. Alle drei Verbindungen stehen sich sehr nahe und können auch leicht in einander übergeführt werden^): -<— CH, -<— CH.OH CO —y 4-0 — H2O COOH Milch- Brenztrau- säure bensäure CH3 A— I I I COOH — H,0 +0 4- H.. CH3 I CH.OH CO — >- —0 CH3 C(\i ^ Methylglyoxal —y COOH 4-n2() Milchsäure. Diese Verbindungen könnten im Pflanzenorganismus auf zwei Arten entstehen, einmal als Zwischenstufe bei der Assimilation der Kohlensäure und des Wassers, und ferner auch als Abbaustufen der Kohlehydrate und ) •') ) Vgl. S. 81. Vgl. S. 86 und 89. Vgl. S. 252 ff. Vgl. S. 130 ff. EiweiIJstoffe iiud ihre Bausteine. 407 insbesondere des Traubenzuckers. Von der Brenz! raubensäure aus gelangen wir leicht zum AI an in. Wir werden später erfahren, daß festgestellt werden konnte, daß im tierischen Organismus Ketosäuren mit Ammoniak zusammen Aminosäuren liefern. Es liegt daher nahe, an den gleichen Vorgang im Pflanzenorganismus zu denken: v^H3 ^Hj + NH3 = CO + H., —> CH CH3 1 CO COOK I I Brenztrau- COO NH, Ammoniumsalz der bensäure Brenztraubensäure . NH., + H.3O. COOH . Alanin. Mit dem Alanin stehen das Serin als ein ,ä-Oxy-alanin und das Zystein als ein ß-Thio-alanin in nächster Beziehung. Im ersteren Falle brauchte nur eine Oxydation des Alanins zu erfolgen, im letzteren könnte man sich als Zwischenstufe ein Reaktionsprodukt zwischen Alanin und Schwefelsäure (aus aufgenommenen Sulfaten) denken, das dann durch Reduktion in Zj^stein übergehen würde. Dem umgekehrten Vorgange sind wir' schon begegnet, als wir die Bildung von Taurin aus Zystein bzw. Zystin besprachen. 1) Selbstverständlich könnte auch das Serin das Ausgangsmaterial darstellen, und dieses durch Reduktion Alanin liefern und ferner in entsprechender Weise, wie das Alanin, zum Zystein führen: CH, + CH2 . OH XXI. \orlesuiig. 408 Zurzeit vermag wie Phenylbrenztraubensäure usw., bereitstellen kann. uns die geschilderte Hypothese der Bildung von Aminosäuren in ihrer Wir können mit ihrer Hilfe zuallgemeinen Form nicht zu befriedigen. nächst nur die Entstehung der genannten drei Aminosäuren ohne weiteres ' ) ableiten. Für die Erklärung der Bildung der übrigen Aminosäuren müssen Das Problem ihrer Entstehung wir zu weiteren Hilfshypothesen greifen. vereinfacht sich dadurch, daß manche davon unter sich nahe Beziehungen aufweisen, so daß sich die Frage der Herkunft der einzelnen Bausteine Wir haben soeben auf auf diejenige einige)- weniger einschränken läßt. den engen Zusammenhang von Alanin, Serin und Zystein hingewiesen. Es sei ferner auf die Beziehungen von Norleuzin zu Lysin hingewiesen: CH3 . CH. GH., . . CH. . CH COOH CH. . . CH2 CHo . . CHo . (;H . COOH ' ". " ! I NH, Lysin -Amino-£-amino-n-kap ron- NH2 NHo a- Amin o-n-kapron säure 7- säure. Die gleichen Beziehungen, wie zwischen Alanin und Serin, haben wir bei anderen Aminosäuren zu den entsprechenden Oxysäuren: Phenylalanin und Tyrosin, Prolin und Oxyprolin, Tryptophan und Oxytryptophan, Glutaminsäure und Oxyglutaminsäure. auch Ferner lassen sich Prolin und Glutaminsäure über die Pyrrolidonkarbonsäure in Beziehung briniren: COOH COOH COOH CH NH CH NH 1 €H . NH., . . 1 H,0 CH. CH2 CH2 CH, CH, COOH CO CH, CH, i Glutamin- Pyrrolidon- Pyrrolidin- säure karbonsänre karbonsäure. l'rolin läßt sich ferner mit dem Arginin in Verbindung bringen. Durch Aufspaltung des l^rrolidinringes unter Wasseraufnahme entsteht a-.\mino-fVoxyvaleri ansäure. Diese könnte dann mit Guanidin unter Wasscraustritt Arginin liefern: ^) Vgl. Gesellscli. 35. liiorzii auch 2438 (1902). h'. Krhnmeyer und ./. Knnlin: Ber. d. Deutschen Cheiii Kiweißstortc uud ihre Bausteine COOK 4U9 COOH I CH NH . + H., CH, ( ) NH.. = GH., + I CH, — C = NH NHo CH, CHj OH a-Amino-^oxyvaleriansäure CH2 Pyrrolidinkarb 011 säure . Gnanidin COOH CH NH., . GH., I GHo GH^ . NH G<^h' + H^ . Argin in. Umwandlungen könnten Diese sein, d. h. es könnten die auch umkehrbar umgekehrter Folge selbstverständlich Beziehungen sich auch in aneinander reihen. Es ist wohl möglich, daß nicht nur ein einziger Weg bei der Bildung der Aminosäuren eingeschlagen wird, und z. B. nur für die genannten Verbindungen die Bildung in den Hauptphasen wirklich so verläuft, wie es geschildert worden ist. Erwähnt sei noch, daß auch die Glyzerose Glyzerinaldehyd als Ausgangsmaterial für die Bildung der genannten Aminosäuren in Betracht kommen kann. 1) Sie selbst könnte durch Kondensation von drei Molekülen Formaldehyd gebildet werden. Der Weg vom Glyzerinaldehyd zur Aminosäure würde über die Glyzerinsäure zu = Serin führen: GH, ,^0\ = _ 3(h.C<«) GH OH . GH, OH + . > OH . I GH . OH -H NH3 = 1 GOOH C\H Formaldehyd Glyzerose (Glyzerinaldehyd) CHo GH . . OH NH, -\- H., I GOOH Serin. •) S. 14 und S. 89. Glyzerinsäure XXI. Vorlesung. 410 Es wäre auch folgender CH, . I Weg möglich OH CH, . OH OH + NH3 = CH NH2 + H2 I CH . , . Lo Lo Glyzerose Aminoglyzerose. Nun könnten zwei Moleküle dieser Verbindung entsprechend der früher erwähnten V) Cannizzaroschen Reaktion ein Molekül Aminoglyzerin und ein Molekül Serin liefern: CHo CH OH . NH2 . CH, + Uo NH.2 . CH, + H, ^0 OH . -> CH CH, NH, . CH + ' Moleküle Aminoglyzerinaldehyd OH . NH^ . I CH, C<pj C<fj 2 CH I OH . . OH COOK Amino- a-Amino-[i- glyzerin oxyproprionsänre =: Serin. Diese Reaktion könnte sich wiederholen, indem das Aminoglyzerin erfolgter Oxydation zu Aldehyd von neuem in Reaktion tritt. Selbstverständlich kann auch der Glyzerinaldehyd selbst in der gleichen Weise Glyzerinsäure und Glyzerin liefern, wenn man zwei Moleküle davon zur Reaktion bringt. Es braucht die Bildung der Glyzerinsäure nicht unbedingt durch direkte Oxydation zu erfolgen. G. Trier-) vermutet, dali der von ihm unter den Spaltungsprodukten von Phosphatiden aufgefundene Aminoäthylalkohol ein Ausgangspunkt für die Synthese von Aminosäuren darstellt. Er könnte durch Einwirkung von Ammoniak auf Glykolaldehyd entstehen, der seinerseits wieder aus zwei Molekülen Formaldehyd hervorgegangen sein könnte: nach CH, OH CH, + NH3 = . NH, = Hc<;gi !)- i^^o Formaldehyd Glykol- Aminoazet- aldehyd aldehyd. + H2O. (!./o Zwei Moleküle des gebildeten Aminoazetaldehyds würden dann je Molekül Aminoäthylalkohol und Aminoessigsäure Glykokoll liefern = ein CH2 C<0 xl 2 . . NH, CH, + . C<0 H Moleküle Aminoazetaldehyd CH2 -h H, = \|.H, NH, . . OH Aminoäthyl- (^Ha + . NH.3 COOH Glykokoll. alkohol Vgl. S. 131. Vgl. hierzu: Georg Trier: Über einfache Pöanzeubaseu und ihre Beziehungen Aufbau der Eiweißstoffe und Lezithine. Gebr. Bornträger. Berlin 1912. ') ^) zum NH2 KiwL'ißstort'e und iiiro Baustoiiie. 411 Dem Azetaldehyd sind wir schon nielirfach begegnet, und zwar als Al)l)austufe des Traubenzuckers. Es sei an die folgenden Formeln erinnert M: cooH QO^n I CH . —^ OH - H, ^0 C\ >^ H CK, CH. Hrenztraubensäure CH, Milchsäure koll — CO2 — ! CO Azetaldehyd. Der Azetaldehyd könnte über die Aminoverbindung ebenfalls Glykound Aminoäthylalkohol liefern. Dieser Weg führt nicht weiter, als der zuerst geschilderte. Wir können uns die Bildung der einfachsten Glieder der Aminosäuren hypo- noch nicht die Bildung der höher molekularen Aminosäuren erklärt. Nun sind im Pflanzenreich wiederholt verschiedenartige Aldehyde nachgewiesen worden.'-) Ferner hat man die Entstehung von solchen reaktionsfähigen Verbindungen aus allen möglichen Verbindungen, wie Kohlehydraten, Fettsäuren usw. unter dem Einfluß von Lichtstrahlen beobachtet. Es wäre wohl möglich, daß ganz entsprechende Reaktionen, wie sie eben für die einfachen Aldehyde beschrieben worden sind, die höheren Glieder der Aminosäuren hervorgehen lassen. Ho gut die Glyzerose in Beziehung zum Serin und damit zum Alanin und Zystein bzw. Zystin treten kann, ist die Möglichkeit gegeben, daß von der Glukose Wege zum Norleuzin, der y.-Aminokapronsäure, fuhren. Als Zwischenglied könnte Glukosamin auftreten. Allerdings müßte eine umfassende Reduktion eintreten, um die CH(OH)-Gruppen in die CH.,-Gruppen überzuführen thetisch zurecht legen, jedoch ist damit C<n "^ C<i^ CH (h.c<h) > . ( CH )H -y 1 (CH.OH), . COOH <^"<>0H l"" I I I CH NH., ' \ (CH.OHj, . CH NH., -y -> . NH., I \ CH(OH), (CH.^), ^^'- ^'ol•lesllng. 412 Wir können die erwähnte Art der Bildung von Aminosäuren ganz allgemein als eine Aminierung von Säuren oder ihrer Derivate charakterisieren, und zwar wurde angenommen, daß Ammoniak an der Reaktion beteiligt ist. Es ist ferner daran gedacht worden, daß die Überführung von Alde- hyden in Aminosäuren im Pflanzenorganismus in der gleichen Art erfolgen könnte, wie diese Verbindungen im Laboratorium bereitet werden. Wir haben insbesondere bei der Besprechung der Synthese von Leuzin und Isoleuzin aus IsovaJeraldehyd bzw. d-Valeraldehyd diese Art der Bildung von Aminosäuren genauer geschildert, ^j Die folgende Formel gibt aus Azetaldehyd, Blausäure und Amdie Bildung von Alanin moniak wieder: CH3 . C<jj + HCN -h NH3 — H., = CH3 CH (NH/) CN. Durch ^'erseifung geht das gebildete Nitril säure = Alanin CH3 ist . '-; . . in x-Aminopropion- über: CH (NHaj CN + 2 H2 . — NH3 = CH3 CH (NHo . ) . COOH. Nach dieser Hypothese der Bildung der Aminosäuren in der Pflanze Blausäure an der Reaktion beteihgt. In der Tat hat man in vielen Pflanzen diese Verbindung aufgefunden ^j, doch ist es noch nicht ganz sicher erwiesen, ob sie primär in freiem Zustande vorkommt. Es ist nämlich eine ganze Reihe von Verbindungen, namentlich auch von Glukosiden bekannt geworden), die beim Abbau Blausäure abgeben und zum Teil leicht spaltbar sind. Diese könnte, solange sie in Bindung vorhandien ist, nicht in Reaktion treten. Es wäre ja denkbar, daß sie je nach Bedarf aus diesen Verbindungen in Freiheit gesetzt würd. Jedenfalls zeigen die zahlreichen Beobachtungen, daß viele Pflanzenarten Blausäure bilden können. Diese müßte aus dem aufgenommenen Salpeterstickstofl' in irgendeiner Weise hervorgehen. Nach Bach-'] kann man sich vorstellen, daß zunächst die Salpetersäure durch Forraaldehyd zu salpetriger Säure reduziert wird: 2 wie HNO3 -\- HC<[\| = 2 HNO, + H^O -f CO... Die salpetrige Säure könnte dann mit zwei Molekülen Formaldehyd, in Reaktion treten: folgt, .0 HNOo + 2 HC<u H = HCN -+ ') CO., + 2 H., O. Vgl. S. ;i23. Kiirf H. Mei/er Nach uutl IL Hopf [Ber. d. Deutscheu Chem. Ges. 54. 17(11) N. (1921)] entspricht die Blausäure in ihrer Koustitution dem Formonitril: ») Joriss-en: Bull de lacad. royale de Beige (3). 7. 256 (1884). (ircs-hqff: Ber. d. Deutscheu Chem. (ies. 23. 35, 48 (1890). Vgl. weitere Literatur im Biochem. Handlexikon. 1. 922 (1911) (bearbeitet von Ernst Schmilz). .1. Springer, Berlin 1911. Cito liarenna (Atti R. Accad. dei Lincei, Roma) (5). 23. 11. 222, 302 (1914) berichtet über das Auftreten von Blausäure bei der Keimung von Samen. M. Gard : ('. r. de l'ac. des sciences. 161. 10 (1915). Carl L. Aisberg und Otifi F. Black: .Tourn. of biol. Chem. 21. 601 (1915). ) Vgl. S. 70 die Spaltung von Amygdalin in Blausäure, Benzaldehyd und zwei ) — — — H.C : — — Moleküle Glukose. j Bach: Compt. rend. de Tacad. des sciences. 122. 1499 (1897). und l'liwoißstotte ilirc Bausteine. 41 o Nuch dieser Hypothese würde Glykokoll aus Forinaldeli yd, Blausäure und Ammoniak entsprechend dem Alanin entstehen: HC^pI + HCX —>^ H. ( \[!JJ + NH, ^ — H, O = H, C{f^' + 2 U, — NH3 ii2 W'OOH. Die Übertragung dieser Hypothese M auf die Bildung der übrigen Aminosäuren verlangt, wie die früher erwähnten Anschauungen, die entsi)rechenden Aldehyde. So müßten wir zur Bildung von Phenylalanin Phenylazetaldehyd, zur Bildung von Leuzin Isovaleraldehyd usw. zur Verfügung haben. -J Die bisher besprochenen Hypothesen gehen von der Annahme aus, dal') die Pflanzenzelle Ammoniak zur Verfügung hat. Nun nimmt jedoch die Phanze unter normalen Verhältnissen wohl immer den größten Teil des Stickstoffs in Form von Salpeter auf. Dieser entsteht im Boden aus Ammoniakverbindungen. Wir werden bald erfahren, daß der tierische Organismus beständig stickstoffhaltige Stoffwechselendprodukte nach außen abgibt. Zum Teil wird direkt Ammoniak abgegeben, zum großen Teil ist jedoch der Stickstoff in organischer Bindung vorhanden je nach der Tierart hauptsächlich in Form von Hai'nstoff oder Harnsäure. Diese Verbindungen werden im Boden zersetzt Es wird Ammoniak gebildet und dieser wird dann durch bestimmte Bakterien oxydiert.'^) Es entsteht zuerst salpetrige Säure bzw. Nitrit. Daraus geht dann Salpetersäure bzw. Nitrat, Sal- — peter, hervor: NH3 -h 3 = HNO2 + H2 0; HNO., -f = HNO3. Man nennt diese Überführung des Ammoniakstickstoffs in Salpeter- Nitrifikation und die Lebewesen, die diesen Vorgang vollnitrifizierende Bakterien. Es ist erst sehr spät gelungen), stickstoff ziehen, diese zu züchten. Sie zeigen nämlich ein sehr eigenartiges Verhalten in der Art ihrer Ernährung. ^) Sie gedeihen nur auf einem rein anorganischen Material. Aus Ammoniumkarbonat bzw. Ammoniumsulfat und Magnesiumkarbonat beziehen sie Stickstoff. Kohlenstoff' und Sauerstoff'. Außerdem steht ihnen der Sauerstoff der Luft zur Verfügung. Sie brauchen diesen zu ihren Oxydations Vorgängen. Die Oxydation des Ammoniaks zu salpetriger Säure, HNO.2, erfolgt durch Pseudomonas europaea und den Nitrosokokkus. Die weitere Oxydation zu HNO3 übernimmt das Bacterium nitrobacter. Vgl. liierzu ^J. Treiib: Anuales du jardiu botan. de Buiteiizorg. 8. (2). 85(li)12i. : d. Heidelberger Akad. d. Wiss. 9. Al)ii. .lahrg. 1910. ') Nicht unerwähnt sei gelassen, daß die S. 129 erörterten Möglichkeiten (UmBildung von Kohlcnstottketten mit höherer ('- Atomzahl auch hier in Frage kommen. ^) Vgl. H. Davy: Elemente der Agrikulturchemie. 408 (1814). ) S. Win()(irmL^k)i : Compt. rend. de Tacad. des sciences. 110. 1013 (1890). Vtrl. über den Atmtingsvorgang sell)st: Meyerhof: rjUiger.-^ Arch. 164. 353 (1916): KM». 240 (1917). - Vgl. auch daarder und Oscar IJaf/em: Bergens Museums Aarbok 1919 20. M'. Heraeus: ^) h\ Ihieppp : Tageblatt der Naturforschervers. \Vicsl)adcn 1887. Z.Mitralb. f. Bakt. 3. Nr. 13 (1887). ') — Hartwig Kränzen Sitzungsbcr. — — XXI. A'orlesung. 4J[4 Weder in der Pflanzen- noch in der Tierwelt treffen wir auf Verbindungen, an deren Aufbau oxydierter Stickstoff beteiligt ist. Stets begegnen wir der NH2- oder der NH-Gruppe oder aber Stickstoff, der mit Methylgruppen besetzt ist. Es ist von großem Interesse, daß im Kreislauf des Stickstoffs in der zwischen Tier und Pflanze liegenden Phase die Überführung in die höchst oxydierte Form stattfindet, die Pflanze muß den Nitratstickstoff wieder reduzieren. Man nimmt an, Daß diese Reduktion sich über das Nitrit vollzieht. Aus diesen Feststellungen darf nicht der Schluß gezogen werden, daß die Pflanze durchaus auf Nitratstickstoff angewiesen ist. Sie vermag auch Ammoniakstickstoff zu verwenden. Die Feststellung dieser Tatsache stieß auf Schwierigkeiten. Werden nämlich Ammon salze in den Boden, auf dem die Pflanze wächst, gebracht, so beginnt sofort die Überführung in Nitrat durch die vorhandenen Bakterien. Man mußte, um zu einer eindeutigen Lösung der Frage der direkten Verwertbarkeit des Ammoniakstickstofls durch die Pflanzen zu gelangen, diese auf sterilem Boden, d. h. bei völliger Abwesenheit von Mikroorganismen züchten. Es zeigte sich, daß die Pflanzen unter günstigen Bedingungen den Ammoniakstickstoff gerade so gut verwerten konnten, wie den Nitratstickstoff. Für die Annahme, daß die Pflanze den letzteren nach erfolgter Aufnahme zuerst oxydiert, bevor sie ihn benützt, liegt keine Beobachtung vor. Es ist dies auch nicht sehr wahrscheinlich. Unter normalen Verhältnissen wird die Pflanze immer in die Lage kommen, Nitratstickstoff reduzieren zu müssen. 1) Man hat nun daran gedacht, ob nicht schon an dieser Stelle Beziehungen zu Produkten angeknüpft werden, die bei weiteren Umwandlungen zu Aminosäuren führen. Es wäre denkbar, daß zugleich mit der Kohlensäure auch die Salpetersäure reduziert würde, und gleichzeitig die entstehenden Produkte in Reaktion treten. Es wäre in diesem Falle die Stickstoffassimilation eben so gut ein lichtchemischer Vorgang, wie die Assimilation von Kohlensäure und Wasser, ßaudisch-) suchte diese Ansicht durch Versuche zu stützen. Er zeigte, daß Nitrate und Nitrite bei Gegenwart von Methylalkohol oder von Formald^hyd durch Lichtenergie leicht bis zu Ammoniak oder Aminen reduziert Averden. Durch Zusatz von leicht oxydablen Substanzen ließ sich die Reduktion stark beschleunigen. Die Sauerstoffabspaltung konnte direkt verfolgt werden. BandtHch gründet auf seine interessanten Beobachtungen eine Hypothese der Bildung von Aminosäuren im Pflanzenorganismus. Er geht von dem Gedanken aus, daß die bei der Reduktion des Nitratstickstoffs auftretende reaktionsfähig Nitrosylgruppe y N eine ist, Aldehydgruppe, ähnliche — C/„i ^H Rolle . OH bzw. \N ^f!, die sehr im Organismus spiele, wie die die nach allen Beobachtungen ohne Zweifel bei Benjamine Moore: Proceed. Royal Soc. Serie B. 90. 158 (1918). Oskar Baudisch: Ber. d. Deutschen Chem^ (Jes. 44. 1009 (1911); 49. 1148, O. Baudisch und Zentralbl. f. Bakt. (2). 32. 520 (1917). 1159, 117G (1916). Erwin Mayer: Elicuda. 45. 1771 (1912); 46. 115 (1913). — Vgl. auch Oskar Baudisch uud ./. //. Krert: Ebenda. 45. 1775 (1912). 0. Baudisch uud G. Klin</er: Klienda. 49. 11C7 (191G). Vgl. dazu auch Oskar Locw: Biochem. Zeitschr. 41. 224 (1912). ') Vgl. hierzu '') — — — — Eiweißstofte und ihre Baiisteiue. 4I5 ungemein vielen Umsetzungen entsteht und zu den mannigfaltigsten Reaktionen führt. man Methylalkohol und Kaliumnitrit, so wird aus letzterem Belichtet abgespalten. Sauerstoff' Dieser reagiert Es entsteht in statu Er oxydiert den Methylalkohol zu Formaldehyd. nascendi mit dem gebildeten Nitrosylkalium. Formhydroxamsäure: CH3 (OH) + KNO, = KNO + + CH3 (OH) H C<H + H, 0. . Nitrosyl- Formal- Methylalkohol dehyd kalium n-^^ + KNO TT = H C^'^^ Formal- Nitrosyl- dehyd kalium Formhydroxam- saures Kalium. Als Zwischenprodukt IT tritt wahrscheinlich Xitrosomethylalkohol: /-VIT wT^C/j^" H/ \N0 auf. Dieser lagert sich in Azi-nitromethan. H,- C: NO . OH. um. Bei weiterer Belichtung verschwindet die Formhydroxamsäure. Es geht Reduktion weiter. Baudisch nimmt nun an, daß Azi-nitromethan die weiteren Synthesen in der Pflanze vermittelt. Es erscheint uns wenig wahrscheinlich, daß diese Art der Assimilation von Stick.stoff die gewöhnliche ist. Da wir jedoch keine einzige Tatsache kennen, die eine bestimmte Art der Einfügung des Stickstoffs in bestimmte Kohlenstoffketten sicherstellt, so ist jede Arbeitshypothese, die sich auf direkte Beobachtungen stützt, wertvoll. Es kann nicht genug betont werden, daß man einen großen Fehler begehen würde, wollte man den ganzen Assimilationsvorgang ausschließlich von einer bestimmten Vorstellung aus aufzuklären versuchen. Es ist sehr wohl möglich, daß die Pflanzenzelle nach mannigfachen Plänen arbeitet. Schließlich wollen wir noch einer interessanten Beobachtung von Walther Loeh'^) gedenken. Setzte er Dämpfe von Form am id der stillen elektrischen Entladung aus, so bildete sich Oxamid. Formamid ist als erstes Produkt der Einwirkung von Ammoniak auf Kohlenoxyd aufzufassen die CO + NH3 = H CO NHo Formamid . 2 H . . CO . NH., —2H — CO NH., \| CO Formamid. . >- NH. Oxamid. . Wird das Formamid in wässeriger Lösung angewandt, so bildet sich oxaminsaures Ammonium, das durch Reduktion das Ammoniumsal/. der Aminoessigsäure liefert, d. h. es hat sich Gly kokoll gebildet: ») Walther Loab: Ber. d. Deutschen Chem. Gesellsch. 46. 684 (1Ü13). 416 •'^•'^I \'()rlesuug. —> COO NU, +4H — COO . -iH.CO.NH.. + H, CO . H.,0 NH, . > — NH, \ NH, CH, NH., . i COOH CH., . NH, Glykokoll. — Es war somit geglückt, aus Kohlensäure, Wasser und Ammoniak das Formamid kann man sich, wie schon betont, aus Kohlensäure l»zw. Kohlenoxyd und Ammoniak entstanden denken eine Aminosäure zu gewinnen. Eine weitere Möglichkeit für die Bildung der Aminosäuren in der Pflanzenwelt ist damit gegeben. Für das Verständnis der Wege, die die Pflanze bei der Bildung der Aminosäuren einschlägt, ist es von der größten Bedeutung zu erfahren, ob sie nur liei Gegenwart von Licht energie eintritt. Es hat sich gezeigt, daß die Eiweißbildung auch im Dunkeln erfolgt. \'oraussctzung ist, daß der Pflanze Kohlehydrate zur Verfügung stehen. Es scheint somit für diesen Fall der ziemlich sichere Nachweis erbracht, daß eine Bildung von Aminosäuren auf Kosten von Kohlehydraten erfolgen kann. Die zur Reduktion der Salpetersäure notwendige Energie wird bei Abwesenheit von Liehtenergie durch Oxydationsvorgänge geliefert. Auch die Pflanzenzelle baut ihre organischen Stoße stufenweise ab und macht dabei Energie frei. Sie verbraucht Sauerstoff und atmet Kohlensäure aus. Am Tage wird dieser Vorgang durch die Kohlensäureassimilation und Sauerstoffabspaltung verdeckt. Es ist möglich, daß die Bildung der Aminosäuren überhaupt in keinem direkten Zusammenhang mit der Liehtenergie steht, sondern vielmehr immer in den gleichen Bahnen verläuft. Die Steigerung der Eiweißbildung bei Beleuchtung könnte vielleicht darauf beruhen, daß durch die Assimilation der Kohlensäure und des Wassers den Pflanzenzellen in vermehrter Menge Kohlehydrate und Materialien zur Bildung von Energie zur Verfügung gestellt werden. Es ist jedoch auch möglich, daß die Pflanze die Bildung der Aminosäuren auf verschiedene Arten bewirken kann. Bald benutzt sie vielleicht Lichtenergie, bald bereitet sie sich die nr)tige Energie durch Abbau organischer Verbindungen. Der Bildung von Nitrit- bzw. Nitratstickstoff aus Ammoniakstickstott' steht ein interessanter entsprechender A'organg zur Seite. Wir kennen nändich Lebewesen, die Schwefelwasserstoff zu Schwefel oxydieren können. Es sind dies die in der Natur sehr verbreiteten Schwefelbakterien.') Sie sind zuerst in Schwefel(\|uellen aufgefunden worden. Besonders eingehend untersucht worden ist die Gruppe Beggiatoa. Der Vorgang verläuft, wie folgt: H., S -f () =: II2 + S. Der gebildete Schwefel wird zunächst in der Zelle abgelagert oder auch sofort zu Schwefelsäure oxydiert. Beide Vorgänge, die Schwefelbildung und die Entstehung der Schwefelsäure, verlaufen exotherm. Die Sehwefolbakterien spielen im Kreislauf — ') (2). 121 769 IVinot/radski/ : Botan. Ztg. (1903). (1919). — ' M. /)i)</</eli: (1H77). Noujalirshl. 48.) — Ormelianshi/ Zentralbl. : der Natiirforsch. f. GesoUscli. Bakt. 14. Ziiricli. EiweiBstotie uud ihre Bausteine. 417 des Schwefels die gleiche Rolle, wie die nitritizierenden io dem des 8tickstotfes. Der tierische Organismus <i:ibt den "roßten Teil des Schwefels in oxj'dierter Form nach außen ab. Gehen Pflanzen und Tiere zugrunde, <lann entsteht bei der Zerstörung der organischen Substanz durch Mikroorganismen Schwefelwasserstoff. Er würde in die Luft entweichen, wenn nicht die genannten Bakterien eingreifen und durch Bildung von Schwefel diesen festlegen würden. Die gebildete Schwefelsäure bindet sich mit Basen zu Sulfaten und kann in dieser Form von den Pflanzen verwendet werden. Es muß dann wieder zu einer Reduktion kommen, denn in den Proteinen ist bis jetzt nur die ThioSH-Gruppe aufgefunden worden. Einzelheiten über den Vorgang der Reduktion sind nicht bekannt. = Interessant ist ferner die Beobachtung, daß es Bakterien gibt, die Wasserstoff oxydieren k(»nnen.i) Sie sind imstande, mit Kohlensäure und Wasserstoff auf einem mineralischen Nährboden zu leben und organische Substanz zu bilden. Nicht alle Pflanzen verfügen über Chlorophyll. Manche sind ebenso, wie der tierisclie Organismus, auf Kohlenstoflfketten angewiesen. Sie können diese nicht selbst von Grund aus, d. h. von Kohlensäure und Wasser ausgehend, bilden. Wir nennen derartige Pflanzen Parasiten. Außerdem kennen wir Pflanzen, die teils selbst Kohlensäure und Wasser assimilieren, teils bestimmten Organismen, auf deren Kosten sie leben, Nälirstofte ent- Es sind dies die Saprophyten. Es ist von großem Interesse, das Verhalten dieser Pflanzenarten und ferner von Gliedern der einfacheren Formen der Pflanzenwelt, wie der Pilze, Flechten und Bakterienarten, gegenüber bestimmten Arten von stickstoffhaltigen Nahrungsstoffen zu verfolgen. Man kann z. B. Pilze auf verschiedenen Aminosäuren züchten oder ihnen Polypeptide oder Peptone als ausschließliche Stickstofifquelle geben. Aspergillus niger z. B. gedeiht g:anz gut auf solchen Substraten.-) Er bereitet seine Proteine insofern unabhängig von der dargebotenen Stickstoftnahrung. als ihre Zusammensetzung z. B. von der Art der dargebotenen Aminosäuren nicht beeinflußt wird. 3) Offenbar wird aus den Aminosäuren der Stickstoff in Form von Ammoniak abgespalten und dann die Synthese der einzelnen Aminosäuren ziehen. in die Wege geleitet. Werden zusammengesetzte Verbindungen, wie Peptone oder Polypeptide oder Proteine gegeben, dann erfolgt wahrscheinlich eine Hydrolyse, der sich dann die Desaminierung der Bausteine (Aminosäuren) anschließt. Audi die chlorophyllfreien Parasiten dürften wohl kaum vom Wirte fertige Proteine bzw. hochmolekulare Peptone übernehmen, oline sie vorher umgebaut zu haben. Auch hier wird der Verwertung eine weitgehende Hydrolyse vorausgehen. 1) Kaserer: Zeutralbl. f. Bakt. 16. (2). 681 (190G). — Lebedeff: Ber. d. bot. sellschaft. 27. .598 (1909). Ge- — "-) Vgl. z. B. E. Marchai: Zeitsclir. f. Bakt. u. Parasitenkde. 1.(2). 1753 (1895). Beijerinch: Botan. Ztg. 45 (1890): Zentralbl. f. Bakt. 13. .S(i8 (1893). F. Czapeklieiu. GcsellHofmeister?. Beiträge. 1. 542 (1902). (). Emmerling: Ber. d. Deutsclieu Emil .scliaft. 35. 2289 (1902); Zeutralbl. f. Bakt. u. Parasitenkde. 10. (2). 273 (1903). Abderhalden und Y. Teruuchi: Zeitschr. f. physiol. Chemie. 47. 391 (1906). M. Raciborski: Bull, de l'Acad. des Sciences de l'racovie. (lasse des Sc. uiat. et nat. Oktober 1900. AI. Kossowic- : Zeitschr. f. Gäriiugsphysiol. 2. 51. 55. 59 (1912). — — ( — - — •') Emil Abderhalden und f'eter Rona: Zeitschr. t. physiol. Chemie. 64. 179 (1905). Abderhalden, Physiologische Chemie. I. Tei), 5. Aufl. 27 XXI. Vorlesung. 418 Besonders interessant ist die Beobachtung, daß es Pflanzen gibt, die mit mannigfaltigen Vorrichtungen zum Fangen und Verdauen von Tieren ausgestattet sind. Man hat sie fleischfressende Pflanzen genannt. Diese Bezeichnung ist nicht sehr glücklich gewählt, weil es sich nicht um ein Fressen, sondern um eine Verdauung handelt. Es darf jetzt wohl als sicher festgestellt betrachtet werden, daß diese eigenartigen Pflanzen, wie Drosera, Nepenthes, Dionaea, Utricularia usw. ein Sekret abgeben, das verdauen kann. Sicher werden nicht nur Eiweißstoffe zerlegt, sondern auch andere zusammengesetzte Nahrungsstoffe, wie Fette und Kohlehydrate. Das eiweißspaltende Ferment scheint in die Gruppe des Pepsins zu gehören. i) Es ist auffallend, wie wenig dieser in jeder HinNicht unerwähnt sicht interessante und wichtige Vorgang erforscht ist! wollen wir lassen, daß oft der Vermutung Ausdruck gegeben worden ist, daß die genannten Pflanzen mit Mikroorganismen zusammen die Verdauungsvorgänge vollziehen. Es soll sich eine Art von Symbiose finden. Manche Forscher sind sogar der Meinung, daß die Pflanze nur ein Sekret Insekten diene, während die abgebe, das zum Festkleben der Tiere Verdauung durch Bakterien herbeigeführt werden soll. — — Schließlich sei noch erwähnt, daß der Nachweis geglückt ist, daß auch chlorophyllhaltige Pflanzen organische Verbindungen verschiedener Art direkt aufnehmen können.-) Handelt es sich um zusammengesetzte Verbindungen oder um solche, die nicht ohne weiteres zur direkten Verwertung geeignet sind, so werden die Pflanzenzellen genau so, wie die Tierzellen, einen AI)- und Umbau eintreten lassen. Sehr mannigfaltigen Verhältnissen in bezug auf die Ernährung und den Stoffwechsel begegnen wir bei den Bakterien. Manche davon können wir direkt an der Eigenart, wie sie bestimmte Verbindungen abbauen, erkennen. Es sei an die Bildung von Essigsäure durch manche Bakterien 3). an die Buttersäurc liefernden Mikroorganismen usw. erinnert. Wir wissen aus Erfahrung, daß die Art der Zusammensetzung eines Nährbodens entscheidend für die Züchtung zahlreicher Mikroorganismen ist. Manche bevorzugen als Stickstoffquelle Nitrate oder Nitrite, andere verlangen Ammonsalze, wieder andere bestimmte Peptongemische und noch andere gedeihen nur auf eiweißhaltigen Flüssigkeiten. Für die künstliche Züchtung der einzelnen Bakterien ist die Auffindung des geeigneten Nährbodens das Entscheidende. Einmal können wir dann eine Reinkultur züchten und mit ihr Beobachtungen anstellen. Wir können ferner mittels eines bestimmten Nährbodens unter Umständen aus einem Gemisch von Mikroorganismen eine bestimmte Art herauszüchten. Es wird dies dann der Fall sein, wenn für die übrigen Lebewesen die Bedingungen ungünstige und für die eine Bakterienart günstige sind. Wir gelangen so zu einer Reinkultur, indem wir für zahlreiche Bakterien die Existenzbedingungen vernichten. Wird ) Emil Abderhalden und Yutaka"Teynuchi: Zcitschr. 21 (1906). f. phvsiol. Chemie. 49. — .7. Lanrent ^) Vgl. H. Acton: Proceed. of the Royal See. London. 47. 150 (1890). Mazc und I'errier: Ebenda. C. r. de l'Acad. des Sc. 125. 887 (1897); 135. 870 (1902). 10.35 (1905): ./. Lcßrre: C. r. de l'Acad. des Sc. 141. 211, ()()4, 834, 139. 470 (1904). V. drafe: Sitziiugsber. d. Kaiser]. Akademie d. 142. 287 (1906); 143. 322 (190(5). — — — Wissenscli. in Wien. Matli.-uaturw. Kl. 118. 1 (1909). ^) Vgl. hierzu /,. B. Franz Lafar: Die Essigsäuregärung. Gustav Fischer. .Jena 1913. Eiweißstoffe und ihre Bausteine. 419 Chemie der Proteine weiter fortgeschritten sein, dann wird man wahrscheinlich nicht mehr so sehr, wie bisher, auf tastende Versuche im Aufsuchen der geeigneten Art der Ernährung der Mikroorganismen angewiesen sein. Man wird durch das Studium des Abbaues bestimmter Abkömmlinge des Eiweißes feststellen, welche Abbaustufen gebildet werden, i) Die Eigenart des Abbaus bestimmter Verbindungen ist ohne Zweifel für manche Mikroorganismen ebenso charakteristisch, wie ihr morphologisches Verhalten und ihre übrigen biologischen Eigenschaften. Unter natürlichen Verhältnissen finden wir wohl nur selten Reinkulturen von Bakterien. Meistens treten Kolonien verschiedenartiger Vertreter dieser kleinsten Lebewesen auf. Sie bereiten oft zusammen den Nährboden für weitere Bakterienarten vor. Die Gewebe des Pflanzen- und Tierorganismus bilden häufig diesen Nährboden. GcAviß vermag sich mancher Bazillus nur deshalb anzusiedeln, weil schon ein anderer, wenig beachteter den Boden vorbereitet hat. Umgekehrt wird manches Lebewesen zugrunde gehen, weil es die vorhandenen Stoffe nicht angreifen kann. Auch die einzelne Zelle muß imstande sein, die ihr gebotenen organischen \'erbindungen in geeigneter Weise zu zerlegen, sei es nun, daß sie Baumaterial zum Ausbau des eigenen Zelleibes oder zum Aufbau einer neuen Zelle braucht, sei es, daß sie Energie zur Verfügung haben muß. Überall begegnen wir den gleichen Grundzügen im Stoffwechsel. Nirgends beobachten wir eine direkte Verbrennung von Substraten mittels des Sauerstoffs. Stets kehrt der stufenweise Abbau der einzelnen organischen Stoffe wieder. Überall finden sich Fermente, die in mehr oder weniger spezifischer Weise auf bestimmte Produkte eingestellt sind. Wird man die Bedingungen, unter denen ein bestimmtes Lebewesen den Abbau von Gewebe vollziehen kann, besser kennen, dann wird es auch möglich sein, bei bestimmten Infektionen diese so zu ändern, daß ein für das betreffende Lebewesen ungeeigneter Nährboden entsteht und damit seine erst die Daseinsbedingungen vernichtet sind. Von besonderem Interesse sind jene Bakterienarten, die aus Nitraten freien Stickstoff bilden. Sie sind außerordentlich verbreitet und bevölkern den Ackerboden in großer Zahl, Ihre Entdeckung verdanken wir Davy.^) Daß die Stickstoff bildung aus Nitraten erfolgt, ist jedoch erst von Gayon und ViipefU^) bewiesen worden. Die Abspaltung des Stickstoffs erfolgt wahrscheinlich in mehreren Teil Vorgängen. Es entsteht salpetrige Säure, und diese wird dann zu Stickstoff" reduziert. Es sind eine ganze Reihe von Mikroorganismen bekannt, die Nitrate in Nitrite verwandeln können. Hierher gehören z. B. das den Darm bevölkernde Bacterium coli commune und der Bacillus pyocyaneus. Die Abspaltung des Stickstoffs erfolgt durch das Bacterium denitrifieans. Der genauere Vorgang der Denitrifikation ist noch unbekannt. Bei dem Vorgang der Nitritbildungaus Nitraten und ferner der Stickstoffabspaltung aus den Nitriten wird Energie frei und ferner auch Sauerstoff", die beide von jenen Mikro') Vgl. z. B. Etnil Abderhalden, L. Fincussohn und Adolf Wal/er: /eitschr. für physiol. Chemie. 68. 471 (1910). Wolfgaruj Weichardt: Zeatralblatt f. d. gesamte. Physiol. u. Pathol. d. Stoffwechsels. 5. 131 (1910). Vgl. ferner die Lehrbücher und — — Sammelwerke über Mikroorganismen, z. B. Walther Kruse: Allgemeine Mikrobiologie. F. (J. W. Vogel. Leipzig 1910. -) ^) H. Danj: Elemente der Agrikultur-Chemie. 408 (1814). Gayon und Dupetit: Compt. rend. de l'Acad. des Scienr. 95. 1544 (1882j. 27* ^^I- Vorlesung. 420 Organismen, werden. die diese Umwandlungen vollziehen können, verwertet tierische Organismus übernimmt von der Pflanze gebundenen und zwar zum größten Teil in Form von Eiweiß. Er gibt ihn während des Lebens wieder in gebundener Form ab. Der Harn enthält stickstoffhaltige organische Verbindungen und ebenso der Kot. Diese Produkte werden zum größten Teil durch die nitritizierenden Bakterien des Bodens in Salpeter übergeführt. Die Pflanze nimmt den Stickstoff in dieser Form auf, reduziert ihn und verbindet ihn mit Kohlenstoff und WasserStirbt ein Tier, dann vollzieht sich der gleiche stoff zu Aminosäuren. Vorgang. Zunächst tritt ein umfassender Abbau der Zellbestandteile durch Die Reaktion der Zellen die in den Zellen enthaltenen Fermente ein. Der Stickstoff, ändert sich. Die Regulation des Stofl'wechsels hat aufgehört. Alles geht nun drunter und drüber. Proteine zerfallen in Peptone und Aminosäuren, Fette werden gespalten und Polysaccharide zerlegt. Bald dringen vom Darmkanal aus Bakterien in die Gewebe ein. Sie erhalten von außen Zuzug. Auch größere Tiere, wie Insekten mannigfaltiger Art und insbesondere Käfer, nehmen am Zerstörungsvorgang teil. Schließlich bleibt von der ganzen Struktur der Gewebe nichts mehr übrig. Es wird Baustein von Baustein gelöst. Nichts erinnert mehr an den feinen Bau der Bestandteile der Zellen, nichts mehr an die mannigfaltigen Vorgänge, die sich noch vor kurzem unter Vermittlung eben der Stoffe, die jetzt in Trümmern vor uns liegen, in ungezählten Zellen vollzogen haben. Aus den einzelnen \'erbindungen des Organismus sind durch alle möghchen Zwischenstufen hindurch Kohlensäure, Wasser, Ammoniak, Schwefelwasserstoff usw. entstanden. Mikroorganismen haben in letzter Linie diesen Aljbau vollendet. Sie vermitteln auch den Weg zu neuem Leben. Sie bilden aus Schwefelwasserstoff Schwefel und Schwefelsäure. Sie lassen aus dem Ammoniak Nitrate hervorgehen. Die organischen Bestandteile des zugrunde gegangenen Organismus sind auch zur Stelle, und so sind alle Bedingungen gegeben, um von neuem Leben erblühen zu lassen. Ein Samenkorn genügt ja eine einzige Zelle! Da. wo eben noch ungezählte Mikroorganismen ein hoch organisiertes Lebewesen in einen Trümmerhaufen verwandelt haben, sproßt ein Keimling empor. Er entfaltet sich mächtig. Neue Zellen und Gewebe werden gebildet. Das tote Material wird in Berührung mit den Zellen zu einem integrierenden Bestandteil von lebhaft funktionierenden Gebilden. Die Pflanze erblüht. Auch sie ist außerordentlich reichhaltig ausgestaltet und in jeder Beziehung ein Kunstwerk. Schon naht sich ein Tier und zerstört die ganze Pracht. Seine Zähne vernichten das feine (iefilge der Gewebe. Im Magendarmkanal werden die zusammengesetzten Zellbestandteile in ihre Bausteine zerlegt. Manche von ihnen werden Bestandteile von Zellen. Sie erfüllen neue Funktionen. Andere dienen nur als Energiequelle. Wieder andere liefern Fermente oder sonstige wichtige Sekret- und Inkretstoffe. Schließlich gelangen auch diese Stoffe fiüher oder später nach außen, um wieder von neuem in den ewig sich wiederholenden Reigen einzutreten. Bald ist ein Element Bestandteil des Bodens, von Gesteinen usw.. bald erfüllt es eine wichtige Funktion in einer Pflanzen- oder 'J'ierzelle. Ohne die Mikroorganismen wäre dieser Reigen unmöglich. Er würde durchbrochen. Die Tierwelt könnte zunächst auf Kosten der vorhandenen Pflanzen weiterleben. Es würde jedoch keine über Nitrite — Eiweißstotie und ihre Bausteine. 421 Brücke oder doch nur eine unzulängliche zu den Pflanzen hinüberführen. Zwar könnten viele Pflanzen mit Ammoniak auskommen. Die Umwandlung Es würden jedoch die Tierlcichen werden köniien. Zwar könnten die Zellfermente aus den zusammengesetzten Verbindungen bis zu einem gewissen Grade die Bausteine bereiten. Es würde jedoch die Ammoniaknicht unbedingt notwendig. in Nitrat ist nicht ihre in Bestandteile aufgelöst bildung sich gar nicht oder nur unvollständig vollziehen. Kurz, die Pflanzen würden die von ihnen dem Tier übergebenen .Stoffe nicht in einer für sie brauchbaren Form zurückerhalten. Zuerst würden die Pflanzenfresser notleiden. Das Pflanzenreich müßte zurückgehen, wenn sich nicht, was wohl möglich wäre. Pflanzenarten finden würden, die sieh den neuartigen Bedingungen anpassen könnten. Auch die Pflanzen werden, wenn sie zugrunde gehen, schließlieh von Mikroorganismen in ihre Bestandteile aufgelöst. Diese dienen dann anderen Pflanzen wieder als Nahrung. Es ist klar, daß der fortwährende Abbau aller möglichen organischen Materialien tierischer und pflanzlicher Herkunft dazu führen muß, daß der Boden die verschiedenartigsten Stufen der Zerlegung aufweist, d. h. auch immer neben Ammoniak und Salpeter organisch gebundenen Stickstoff enthält.^) Dazu kommen dann in großen Mengen zugrunde gehende Bakterien, die auch alle möglichen Zerfallsprodukte liefern. So hat man z. B. verschiedene Aminosäuren und Purinbasen-) aus dem Boden gewonnen. Manche Mikroorganismen bevorzugen diese Stickstoffquellen und gehen nicht direkt von Ammoniak oder gar Salpeter aus, wenn sie ihre Stoffwechselvorgänge durchführen. Die ungezählten Arten von kleinsten Organismen im Ackerboden zeigen, daß auch hier das Leben in vollster Blüte steht. Nicht immer vollzieht sich der Wechsel zwischen Pflanze und Tier für das einzelne Element so rasch. Oft wird das eine oder andere Element auf lange Zeit hinaus aus dem Reigen ausgeschaltet. Jahrtausende lang wird Kohlenstoff in den Kohlenlagern festgehalten. Ferner verkünden mächtige Salpeterlager in vielen Gegenden, daß auf lange Zeit hinaus Stickstoff festgelegt worden ist. Die gewaltigen Petroleumquellen enthalten ebenfalls Stoffe, die vor Tausenden A'on Jahren den Reigen zwischen Pflanze und Tier mitgemacht haben, nunmehr aber auf lange Zeit hinaus der unbelebten Natur übergeben sind. Der Mensch reiht diese Produkte wieder in den Kreislauf der Elemente ein. Kohle, die vor ungezählten Jahren der Atmosphäre in Form von Kohlensäure angehört hatte und in allen möglichen Pflanzen- und Tierarten Baustein von Zellen gewesen war, wird Sonnenenergie, durch Verbrennung wieder in Kohlensäure übergeführt. die vor Jahrtausenden den Erdball bestrahlt hatte, wird wieder freil Die Kohlensäure wird von Pflanzen assimiliert und damit gehört ihr Kohlenstoff wieder dem ewig wechselnden Reigen in der unbelebten und belebten Natur an. Bald baut er Zellen einer Pflanze auf, bald ist er Bestandteil z. B. einer hoch organisierten Zelle des Zentralnervensystems eines Tieres. Den Salpeter übergeben wir als Düngemittel einfach dem Hoden und bewirken dadurch allein, daß sein Stickstoff wieder in den Kreislauf eintritt. \g}. u. a. Osirald Schreimr iiml Edmund ('. S/iorei/: Jourii. of BioJ. Cliem. S. 8. 3.S1 (1910). -) Vgl. hierzu ('raiideau: ('ours (ragricultuie de Tecole forest. Cliimie et pliysiol. iippliipiee l'agriculture. Paris 1879. ) 385 (1910): ;'i XXI. Vorlesung. 422 So würde denn im großen und ganzen der Kreislauf des Stickstoffs wenn nicht die Stickstoff aus Salpeter abspaltenden Bakterien wären! Beständig wird in großer Menge Stickstoff aus seiner Bindung losgelöst und der Atmosphäre übergeben. Wir müssen zunächst feststellen, wie der Pflanzen- und Tierorganismus sich gegenüber dem freien Stickstoff verhält. Es wurde schon frühzeitig von Boussingault (1851) bewiesen, daß die chlorophyllhaltigen Pflanzen den Stickstoff' der Atmosphäre nicht verwenden können. Dagegen wird das Ammoniak der Luft verwertet. Es kommt jedoch unter normalen Verhältnissen nur in Spuren in der Atmosphäre vor. Auch spätere Versuche ergaben immer wieder, daß die höher organisierten Pflanzen mit dem freien Stickstoff nichts anzufangen wissen. Im tierischen Organismus nimmt er ebenfalls nicht an irgendwelchen Stoffwechselvorgängen teil und wird auch nicht aus seinen Verbindungen in Freiheit gesetzt. ^) als ein abgeschlossener erscheinen, Der Kreislauf des Stickstoffs zwischen Pflanze und Tier und umgekehrt vollzieht sich in gebundener Form. Es müßte somit jede Infreiheitsetzung von Stickstoff als eine Schädigung der Organismenwelt aufgefaßt werden, falls sich in der Natur keine Vorrichtungen fänden, um diese Verluste wieder auszugleichen. Allmählich müßte der Boden an gebundenem Stickstoff verarmen. Der Vegetation würden bald enge Grenzen gesteckt sein; ja schließlich müßten ihr die Existenzbedingungen entzogen werden. Es wird allerdings beständig ein Teil des atmosphärischen Stickstoffs bei elektrischen Entladungen der Luft oxydiert und dann mit den Niederschlägen (Regen, Schnee) dem Boden zugeführt, doch werden auf diesem Wege nur sehr geringe Mengen von Stickstoff' in die gebundene, der Pflanze zugängliche Form übergeführt. Auch bei der Verdunstung von Wasser werden geringe Mengen von Ammonium nitrit gebildet. genannten Arten der Bindung von Stickstoff Überführung von freiem Stickstoff in die gebundene Form durch Bakterien und Fadenpilze. Schon Bcrthelot-) hatte festgestellt, daß im Boden eine Anreicherung von Stickstoff stattfinden kann. Bald gelang es auch Bakterien nachzuweisen, die den freien Stickstofl" der Luft binden können. Zunächst wurde von Winogradshy ein streng anaerober Bazillus, Bacillus oder Clostridium Pasteurianum, -im Ackerboden aufgefunden. Er ist auch im Meeresboden vorhanden. Er kommt mit aeroben Bakterien zusammen vor. Diese beseitigen die letzten Spuren von Sauerstoff und vermitteln auf diesem Wege dem Bacillus Pasteurianum die Bedingungen zu seiner Tätigkeit. Die Bindung von Stickstoff" erfordert Energie. Sie wird durch Abbau von Kohlehydraten in den Zellen der Bakterien frei und zur Verfügung gestellt. Es ergab sich, daß nicht alle Kohlehydrate verwertet werden. Glukose, Lävulose und Rohrzucker werden vom Bacillus Pasteurianum leicht angegriffen, dagegen erwiesen sich Laktose, Viel wichtiger als die ist die =) — Au(/ust Kroqh: Skand. Arch. f. Physiol. 18. 364 (1906). Cai-l Ojopenhcimer: l'. 177 (1906); 4. 328 (1907). ^) Berthelof: C. r. de TAcad. d. Sc. 101. 775 (1885); 104. 205, 625 (1887); 106. 569, 1049, 1214 (1888); 107. 372 (1888); 108. 700 (1889); 109. 277, 417 (1889); 115. ) Biochem. Zeitschr. 569 (1892); 116. 842 (1893). Winogradski/: C. r. de l'Acad. — =) d. Sc. 116. 1385 (1893); 118. 353 (1894). Vgl. weitere Literatur bei Ilatis l'rinqshcim : Zeutralbl. f. Bakt., Parasitenkunde u. Infektionskrankheiten. 20. 248 (1908). Eiweißstort'e uud ihre Bausteine. 423 Stärke und Zellulose als nicht verwertbar. Bacillus Americanus zeigt ein ganz entsprechendes Verhalten, i) Wir kennen außerdem noch aerobe Bakterien, die Stickstoff binden können. Auch brauchen Kohlehydrate zur Assimilation des freien StickAckerboden und auch im Meere außerordentlich stark Humus und Kalk verbreitet. Sie gehören der Gattung Azotobakter an. begünstigen die Entwicklung dieser Bakterienarten außerordentlich. Es ist ferner festgestellt worden, daß einige Nostocarten Stickstoff binden können. Da diese Algenarten sich die Kohlehydrate zur Bestreitung des zur Stickstoffbindung erforderlichen Energieaufwandes selbst aufbauen können, so sind sie unter den Lebewesen ganz besonders günstig gestellt, denn sie können den Kohlenstoff, den Sauerstoff und den Stickstoff der Luft entnehmen und sind nur zur Gewinnung von Wasserstoff und vor allem der Salze auf den Boden angewiesen. Derartige Lebewesen müssen als Pioniere zur Schaffung von Lebensbedingungen für anspruchsvollere Lebewesen ganz besonders geeignet sein. Wir treffen sie denn auch z. B. Haben sie eine Zeitlang diese unwirtlichen auf verwitternden Gesteinen. Stellen bewohnt, dann werden durch sie allmählich Bedingungen geschaffen, unter denen andere Lebewesen, zunächst vor allem Bakterien, gedeihen können. Ganz allmählich folgen dann Algen, Pilze und Flechten, bis schließlich Kulturland gewonnen ist, auf dem Phanerogamen aller Art sich halten können. stoffs. sie Sie sind im Während die bisher genannten, freien Stickstoff" bindenden Lebe- wesen frei im Ackerboden oder im Schlick des Meeresgrundes und dem Plankton des Meer- und Süßwassers vorkommen, gibt es eine ganze Anzahl von Bakterien, die offenbar stets in Symbiose mit bestimmten Pflanzen Es ist eine alte Erfahrung, daß manche Pflanzen, wie z. B. die leben. Leguminosen, den Boden an Stickstoff anreichern, während andere, wie z. B. die Gramineen, diesem ausschließlich solchen entziehen. Aus diesem Grunde wird der praktische Landwirt im allgemeinen auf demselben Boden nie hintereinander Getreide bauen, sondern abwechselnd Leguminosen und ^'ollständig aufgeklärt worden ist der ganze Vorgang, der Gramineen, diesem eigentümlichen Verhalten zugrunde liegt, durch die Untersuchungen von Hellriegel-) und Wulfarthj) Sie stellten zunächst in einwandfreier Weise die Stickstoffbindung durch die Leguminosen fest und fanden, daß Zusammenhang mit der Ausbildung der sogenannten Wurzelknöllchen dieser Pflanzen steht. Es ließ sich auch zeigen, daß diese Fähigkeit in Boden kultivierte Leguminosen zur Knöllchenbildung gezwungen werden können, wenn ein Aufguß von gewöhnlichem Ackerbodep zugefügt wird. Offenbar müssen im Ackerboden Mikroorganismen vorhanden Durch Erhitzen verliert der sein, die die Knöllchenbildung veranlassen. Bodenaufguß seine Wirksamkeit. Ganz anders verhalten sich die Gramineen. Sie lassen sich in ihrer Stickstoffaufnahme durch Bodenaufguß auf sterilem nicht beeinflussen. Ihre Stickstoffassimilation ist nur abhängig vom Nitrat- Vgl. Hans Pringsheim: Zentralbl. f. Bakt. 16 (2). l\)h (li)OG); 20. 248 (1908). H. Hellrieqel: Tageblatt der Naturforscher- Vers. Berlin. 290 (188G). ') Willfarth': Wiesbaden. 302 (1887). Tageblatt der Naturforscher-Vers. H. Hellricyel und H. Willfarth : Zeitschr. d. Vereins f. Rübenzuckerindustrie. BeilageVgl. die weitere heft. 234 (1888) und Berichte d. Botau. Gesellsch. 7. 138 (1889). 1) «) — — Literatur bei J. Vogel: Zentralbl. f. Bakteriol. und Parasiteukunde. 15. 11. 83 (1905). XXI. 424 N'oilesviug. gehalt des Bodens. Der freie Stickstoff kommt für sie gar nicht in Betracht. Die Leguminosen dagegen sind im natürlichen Boden nicht unbedingt auf den vorhandenen Salpeterstickstoff angewiesen. Werden die Leguminosen auf sterilisiertem Boden gezüchtet, dann verhalten sie sich wie die Gramineen. Sie haben die Fähigkeit verloren, sich den freien Stickstoff' nutzbar zu machen, und sind nun gänzlich vom Nitratgehalt des Bodens abhängig. Die folgenden Versuche ergeben einen Einblick in die Bedeutung der Stickstoffassimilation durch knöllchenhaltige Leguminosen. Schlocsing und Laurent'^) kultivierten Leguminosen in sterilisiertem Sande und in sterilen Glaszylindern. Der Gehalt an Sauerstoff, Kohlensäure und StickBei den einen stoff der in diesen betindlichen Luft war genau bekannt. Versuchen wurde steriles Wasser zugegeben, bei den anderen solches, dem zerriebene Knöllchen zugefügt worden waren. Nach drei Monaten wurde die Luft aus den Zylindern entfernt und ihr Stickstoffgehalt bestimmt. Es ergab sich, daß er nur in den Versuchen abgenommen hatte, bei denen Wurzelknöllchen hinzugegeben w^ordeu waren. In zwei solchen Versuchen mit Beigabe von W^ui'zelknöUchen war das Ergebnis das folgende: Stickstoffgehalt der Luft bei Beginn des Versuches ., ., .. Abschluß .. ., Aufgenommener Stickstoi« 1 n 2'6'6\'2 cm'^ 2483'8c'///=ä 2652-1 2457-4 „ \—m-hmg .. =hi m<^ Noch deutlicher ergibt sich die Stickstoffaufnahme aus der folgenden Versuchsanordniing. In ^'ersuch III waren keine Wurzelknöllchen vorhanden, wohl aber in Versuch I und II, im Boden und in gesäten Leguminosensamen (Erbsen) bei Beginn des Stickstoff Versuches I II 326 mg o2-5 mg Stickstoff in der Erde am Schlüsse des Versuches 73-5 „ 40-6 „ Stickstoffgewinn durch die Pflanzen . , . HI Die Wurzelknöllchen enthalten Bakterien, wie Beijermck'^) bewiesen hat. Sie leben in Symbiose mit dem Träger der KniUlchen. Beijerinck nannte den Bazillus B. radicicola. Er ist aerob und in Land und Wasser weit verNeue Beobachtungen sprechen übrigens dafür, daß der den freien Es scheint, daß den Stickstoff assimilierende Pilz nicht einheitlich ist. verschiedenen Papilionaceenforraen verschiedene Bazillenarten zukommen. Es ist nur gelungen, nahe verwandte Vertreter dieser Pflanzenfamilie wechsc 1seitig erfolgreich mit ihren KniUlchenbakterien zu infizieren. Es gelang anzuz. B. nicht Robiniawurzeln mit Erbsenbakterien zur Knöllchenbildung regen. Sehr interessant ist es auch, daß Soja hispida in europäischem Gartenland oft keine Kn!tllchen erzengt, wohl aber, wenn es mit japanischer Erde geim))ft wird. Welche Bedeutung diese Entdeckungen erlangt haben, erhellt schon daraus, daß die Knöllchenbakterien Handelsprodukt gebreitet. worden sind. Eiwoißstoffe imd ihre Bausteiue. 425 Ks ist fraglich, ob diese Knüllchenbakterien nur auf die Klasse der Papilionaceen beschränkt sind. Es liegen Beobachtungen vor, wonach sie auch bei anderen Pflanzengattungen zu finden sind. So sollen sie den Ericaceen, Rhinantaceen, Elaeagnaceen, Cyeadeen, Koniferen usw. zukommen. Man hat auch vermutet, daß die oft mit den Wurzeln der höheren Pflanzen symbiotisch lebenden Fadenpilze eine ähnliche Rolle spielen, wie die KnöUchenbakterien. Einstweilen lassen die vorliegenden Versuchsergebnisse noch kein bestimmtes Urteil zu. \ie\e Beobachtungen an den wild wachsenden Pflanzen lassen auf eine weite Verbreitung derartiger Symbiosen schließen. Wir kennen viele Pflanzen, die Jahr für Jahr in derselben Fülle an demselben Standarte immer wieder anzutreffen sind, während manche Blüteperiode andere Arten plötzlich auftauchen, um nach einer kurzen mehr und mehr zurückzugehen. So kann der ganze Charakter einer Waldwiese und insbesondere die Flora eines Schuttplatzes in rascher Reihenfolge sich ändern, Aveil offenbar die nur kurze Zeit seßhaften Pflanzen ganz und gar abgesehen von anderen Lebensbedingungen auf den Xitratgehalt des .. Bodens angewiesen sind. Die KnöUchenbakterien sind von Beijerinck^) und Prazmoivski-) in Reinkultur erhalten worden. Sie brauchen Eiweißstoffe zu ihrer Entwicklung. Aus dieser Beobachtung ergeben sich vielleicht Aufschlüsse über die Art der Symbiose zwischen dem Wirte und den Bazillen. Zunächst dachte man daran, daß diese den freien Stickstoff aufnehmen, ihn in irgend einer Form, wahrscheinlich als Ammoniak, verwenden und dann entweder Ammonsalze oder auch Aminosäuren bzw. Peptone der sie beherbergenden Pflanze übergeben. Nach neueren Beobachtungen scheinen jedoch die Verhältnisse nicht so einfach zu liegen. Die Wirtpflanze liefert sehr wahrscheinlich den Wurzelbazillen in irgend einer Form Kohlehydrate und Eiweißstoffe. Auf diesem Nährboden gedeihen die Bazillen und binden nun Stickstoff. Entweder wandeln sie diesen in Ammoniak um, oder sie oxydieren ihn und übergeben dann das gebildete Assimilationsprodukt der Pflanze, mit der sie zusammenleben. Man konnte das Eindringen der Wurzelbakterien in die Wurzeln von Leguminosen direkt verfolgen. Man sieht zunächst Bakterienansammlungen an den Wurzelhaaren. Diese umgeben sich mit einer Hülle. Die Bakterienmasse dringt dann durch das Wurzelhaar in die Parenchymzellen der Wurzel ein. Diese Zellen beginnen sich lebhaft zu teilen. Die Wurzel schwillt an dieser Stelle an. Es ist ein Knöllchen entstanden. Die Hülle, die die Bakterien bis jetzt umgeben hat, wird aufgelöst. Die Bakterien sind nun im Zellsaft frei vorhanden und vermehren sich stark auf Kosten der vorhandenen und vom Wirte nachgelieferten Stoffe. Schließlich wird der — — Bakteroidgewebe genannt ganze Inhalt dieser infizierten Zellen von der Wirtpflanze mittelst besonders gebildeter Gefäßbündel aufgenommen. Es ist möglich, daß erst in diesem Momente der Wirt das von den Bakterien aufgesammelte Material übernimmt, es ist jedoch wahrscheinliclier, daß schon vorher ein lebhafter Austausch stattfindet. Bemerkt sei noch, daß die Leguminosen, wie sicher festgestellt worden ist, auch Nitrate aus dem Boden aufnehmen und verwerten können. Sie sind somit nicht unbedingt auf die Tätigkeit der Wurzelbakterien angewiesen. ') -J Beijrrinck: Bot. Zoitg. 725. (1888). Prazmouski: Landwiitsch. Versuchsstatioueu. 37 Ißl (1890). XXI. Vorlesung. 426 Außer den genannten Bakterienarten sind auch noch andere aufgefunden worden, die freien Stickstoff zu binden vermögen. So ist diese Eigenschaft bei thermophilen Bakterien^) entdeckt worden. Von weittragender Bedeutung ist die Feststellung, daß die auf Blättern von Rubiaceen und tropischen Myrsinaceen (Ardisia) vorkommenden Bakterien auch freien Stickstoff assimilieren können. 2) Ferner konnte festgestellt werden, daß eine ganze Reihe von Pilzen imstande ist, freien Stickstoff zu verwenden. 3) Es handelt sich vor allem um Pilzarten, die auf abgestorbenen Pflanzenteilen sich ansiedeln. Auch Aspergillus niger und Penicillium glaucum können freien Stickstoö' verwerten.) Die auf den verwesenden Pflanzenresten sich ansiedelnden Organismen verwenden als Energiequelle die Zellulose und zerstören diese zugleich. &) Schließlich sei noch darauf hingewiesen, daß der kleine Wasserfarn Azolla den Stickstoff der Luft assimilieren kann. Er lebt in Symbiose mit der Alge Anabaena.^) Leider wissen wir fast gar nichts über den eigentlichen Vorgang der Assimilation des freien Stick Stoff s.'^) Welches sind Diese Frage konnte bis jetzt noch nicht beantwortet werden. Die anaeroben Bakterien werden wohl Ammoniak bilden. Die aeroben oxydieren vielleicht den Stickstofl' zu Salpetersäure. Mit der Feststellung der Tatsache, daß auch freier Stickstoff zur Assimilation gelangen kann, scheint der Kreislauf des Stickstoffs, der durch die Auffindung der denitrifizierenden Organismen eine Lücke aufzuweisen schien, geschlossen. Wir haben jedoch noch einen Punkt außer acht gelassen. Der Boden hat nämlich die Eigenschaft, gewisse BestandEs ist dies z. B. für die zur Entwicklung der Pflanzen teile zu adsorbieren. so wichtigen Verbindungen Phosphorsäure, Kalisalze und Ammoniak der Fall. Sobald ihre Lösungen mit Bodenteilcheu in Berührung kommen, werden sie in schwer lösliche Verbindungen übergeführt und so vor der Auswaschung durch das Regenwasser bewahrt. Ganz anders verhalten sich nun die salpetersauren Salze, die Nitrate. Sie werden vom Boden nicht zurückgehalten. Sie sind leicht löslich in Wasser und werden beständig ausgewaschen, den Bächen und Flüssen zugeführt und erscheinen zuletzt im Meere. Die Menge des jährlich dem Festland auf diese Weise entzogenen Stickstoffs ist sehr groß. K. Brandt^) berechnet sie auf rund 40 000 Millionen kg pro Jahr. Wir stehen vor der Frage, auf welchem Wege diese großen Stickstoffmengen dem allgemeinen Kreislauf des Stickstoffs zurück- die ersten Assimilationsprodukte? ') Hans Pringsheim: Zentralbl. f. Bakt. 31. (2). 23 (1911). =) Vgl. M. Miehe: Biol. Zentralbl. 32. Nr. 1 (1912). — F. C. •wissensch. Botanik. 51 (1912). v. Faber: Jahrb. f. — ) Vgl. ferner Hermann Froehlich: Jahrb. d. wissensch. Bot. 45. H. 2 (1908). Charlotte Ternetz : Ber. d. Deutschen Bot. Gesellsch. 22. 267 (1904). ) Hermann Froehlich: Jahrb. d. wissensch. Bot. 45. H. 2 (1908). ^) Vgl. Vgl. z. B. Hans Fringsheim: Zentralbl. f. Bot. 26 (2). 221 (1910). — auch Hans und Ernst Frinqsheim: Ebenda. 26 (2). 227 (1910). «) A. Oes: Zeitschr. L Bot. 5. 145 (1913). Vgl. dazu Julius Stoklasa: Beitrag zur Kenntnis der chemischen Vorgänge bei der Assimilation des elementaren Stickstoffs durch Azotobakter und Radiobakter. Gustav Fischer. Jena 1909. Hier findet sich viel Literatur angegeben. ) Vgl. K. Brandt: Wissenschaftliche Untersuchungen. Herausgegeben von der Vgl. auch E. Schulze: Kommission zur Untersuchung der deutschen Meere. 1899 u. 1901. Der Kreislauf des Stickstoffs in der Natur und der Stoffwechsel im Meere. Schweizer, '') — landwirtsch. Zentralbl. 1902. Eiweißstoffe uuil ihre Bausteiue. 4^>7 gegeben werden. Daß dies der Fall sein muß, beweist der Umstand, daß trotz der nun seit Jahrtausenden erfolgenden Auslaugung des Festlandes die Vegetation nach wie vor sich weiterentwickelt. Zwischen dem Ptianzenund Tierleben auf dem Festland und dem Meere bestehen keine wesentUnterschiede. Auch die Meerespflanzen assimilieren Kohlensäure, benötigen zu diesem Vorgänge der Sonnenenergie, weshalb denn in den Tiefen, in die kein Licht dringt, das Pflanzenwachstum aufhört. Auch die Meerespflanzen brauchen Nitrate zum Aufbau ihrer Eiweißsubstanzen, und ebenso entnimmt die im Meer lebende Tierwelt ihr Eiweiß in letzter Linie ausschließlich der Pflanzenwelt. Die Vegetation im Meere kann die großen Stickstoffmengen, die ihr ständig zugeführt werden, nicht bewältigen. Dazu kommt dann noch, daß beständig bei der Fäulnis der abgestorbenen Pflanzen und Tiere des Meeres stickstoffhaltige Substanz in Ammoniak und dieses in Xitrit und in Nitrat übergeführt wird, genau so, wie dies auf dem Festlande der Fall ist. Wohl wirft ab und zu das Meer gew^altige Tangmassen ans Land. Die in ihnen enthaltene Stickstoftmenge ist jedoch viel zu gering, um einen Ausgleich zwischen dem dem Land entführten Stickstoff zu schaffen. Es ist nun von größtem Interesse, daß. wie schon erwähnt, auch im Meere sich denitrifizierende Bakterien vorflnden, die fortwährend Stickstoff in Freiheit setzen. Sie geben den dem Meere zugeführten Stickstoff" zum Teil dem Kreislauf zurück. Hier erhellt erst die große Bedeutung der Denitrifikation, die uns auf dem Festlande als eine wenigstens scheinbar unwillkommene Erscheinung entgegentrat. Zugleich wird nun die große Rolle, die den Stickstoff assimilierenden Bakterien zukommt, vollkommen klar. Mit der Auffindung der denitrifizierenden Bakterien hat sich auch ein scheinbarer Widerspruch gelöst. Auf dem Festland nimmt bekanntlich die Dichtigkeit der Pflanzen und Tierwelt vom Äquator nach den Polen hin und mehr und mehr ab. Im Meer ist dies nicht der Fall. Diese Erscheinung ist sehr auffallend, denn man müßte erwarten, daß in den tropischen Meeren mit ihrer Lichtfülle der Entwicklung viel bessere Bedingungen geschaffen wären, als in den dunklen arktischen Zonen. Es ist wohl denkbar, daß dieser Umstand mit der Tätigkeit der denitrifizierenden Bakterien zusammenhängt. Für sie und ihr Wirken sind die Bedingungen in den tropischen Meeren am günstigsten. Sie entwickeln sich bei einer Temperatur von 25 oO" am besten. Sie werden somit in den tropischen Meeren den Meerpflanzen viel mehr Stickstoff' entziehen als in den Meeren der arktischen Zone. Wir wollen gleich betonen, daß dies nur ein Erklärungsversuch ist. Wir wissen, daß das Wachstum aller Organismen an das Gesetz des Minimums gebunden ist, d. h. sämtliche lichen auch auch sie — die einem Organismus geboten werden, richten sich in ihrer Verwertung nach dem in der kleinsten Menge vorhandenen. AVenn auch der Meerespflanze massenhaft Stickstoft" in Form von Salpeter zur Verfügung steht, so könnte andrerseits der Phosphor z. B. in zu geringer Menge vorhanden sein. Die Pflanze könnte dann den gesamten Stickstoff nur in der dem vorhandenen Phosphor entsprechenden Menge verwerten. Es ist ferner denkbar, daß in den Meeren der verschiedenen Zonen die Ernährungsbedingungen auch nach anderen Richtungen verschieden sind. Zum Schlüsse wollen wir noch kurz die wichtige Frage streifen, ob das Wechselspiel zwischen denitrifizierenden Lebewesen und den freien Stickstoff assimilierenden so beschaffen ist. daß ein annäherndes Gleich- Stoffe, XXI. Vorlesung. 428 Diese Frage kann in dieser Form aus nahegewicht zustande kommt. liegenden Gründen wohl nie genau beantwortet werden. Einmal kennen wir sicher noch lange nicht alle denitrifizierenden Lebewesen. Ferner sind sicher auch noch mehr Organismen vorhanden, die freien Stickstolf binden können. Dazu kommt dann noch, daß die Tätigkeit dieser Lebewesen in hohem Grade von den vorhandenen Bedingungen abhängig ist. Wir haben gesehen, daß fast alle Stickstoff' bindenden Lebewesen einer Energiequelle bedürfen^ die sie nicht selbst beschatten können. Fehlt diese, dann ist ihrer Wirkung eine Schranke gesetzt. Die wesentlichste Störung in den Beziehungen zwischen den Stickstoff den genannten beiden Klassen von Lebewesen in Freiheit setzenden und solchen bindenden bewirkt der Mensch mit seiner Kultur. Zunächst entzieht er durch Bebauung mit Häusern usw. w-eite Länderstrecken dem Pflanzen Wachstum. Gleichzeitig — — stellt er an bestimmte Gebiete besonders hohe Anforderungen an die Ertrag- fähigkeit. Der Boden soll möglichst vorteilhaft ausgenützt werden. Das Gesetz der Erhaltung der Materie und der Energie gilt selbstverständlich auch für Dazu kommt dann noch das wichtige Gesetz, daß. sich die Pflanzenwelt! die Verwertung der einzelnen zur Verfügung stehenden Stotte nach dem im Minimum vorhandenen Produkte richtet. Es kann ein Ackerboden, der reich an Phosphorsäure, an Kalk usw. ist, wenig Pflanzen ernähren, wenn er z, B. nur über geringe Stickstottvorräte verfügt, es sei denn, daß Stick- Lebewesen einen Ausgleich schatten. Je weiter die Kultur fortschreitet, um so mehr wird die Wechselbeziehung zwischen Tier und Pflanze durchbrochen. Die Abfallstotte des tierischen Organismus werden zum Teil direkt Flüssen zugeleitet und zum Teil verbrannt. In diesem Falle wird gebundener Stickstoff in Freiheit gesetzt. Auch Leichen werden verbrannt. Ferner w-ird bei der ^'erbrennung von Kohle auch beständig der Vorrat der Erde an gebundenem Stickstott' vermindert. Dazu kommt noch die chemische Industrie, die große Vorräte an gebundenem Stickstott" zu allen möglichen Zwecken braucht. Es sei an die Herstellung von Schießpnlver, an die Gewinnung von Sprengstoffen, von Teerfarben, von stickstoffhaltigen Medikamenten usw. erinnert. Somit stehen gesteigerten Anforderungen an die Ertragfähigkeit des Bodens große Verluste an gebundenem Stickstott" gegenüber. Die Praxis hat schon lange ergeben, daß dieses Deflzit an gebundenem Stickstott" von der Xatur nicht eingeholt werden kann. Wohl kann der vorsichtige Landwirt bald Leguminosen, bald Getreide anbauen und dadurch dem Boden immer wieder Stickstott' aus der Luft durch Vermittlung der KnöUchenbakterien zuführen, es wird trotzdem immer Äcker geben, die an gebundenem Stickstoff' verarmen. Es kommt in dieser Beziehung auch viel auf die Bodenbeschaff'enheit an. Das rasche Auslaugen durch Wasser kann auch dazu beitragen, dem Boden vorhandene, kostbare Vorräte zu entreißen. Man war schon seit langem geni»tigt, dem Ackerboden gebundenen Stickstoff" zuzufüliren. Man spricht von Stickstoffdüngung. Es standen bis vor kurzem vornehmlich drei Quellen zur Gewinnung von gebundenem Stickstoff' zur Verfügung. Einmal tinden sich große Lager von Salpeter an verschiedenen Orten namentlich in Chile. Ferner enthält die Steinkohle in allerdings nur geringer Menge gebundenen Stickstoff", und endlich haben wir noch den Guano. Dieser besteht aus Exkrementen, verfaulten Eiern stoff assimilierende EiweiBstofte iiud ihre Bausteine. 429 und Kadavern von \ ögelo. Gewaltige Ansammlungen dieser Produkte ünden sich auf den fast regenfreien Küsteninseln Perus. Diese Quellen an gebundenem Stickstoff sind leicht erschöpfbar. Die Steinkohle ist bis jetzt noch in ganz ungenügender Weise als verwendet worden. Die größte Bedeutung hatte bisher der Salpeter für die Landwirtschaft. Er verdankt seine Entstehung nitriEs finden sich namentlich in Chile große Lager tizierenden Bakterien. von Natronsalpeter. Sie sind jedoch auch erschöpfbar Immer mehr Länderstrecken werden vor allem in Amerika einer stärkeren Ausnutzung zugeführt. Infolgedessen werden auch immer mehr Düngemittel verlangt. Man hat ausgerechnet, daß etwa in 25 Jähren die Salpeterlager erschöpft sein werden. Es ist dies unwahrscheinlich, denn einmal ist es schwer feststellbar, wie der Bedarf sich verhält, und wie groß die vorhandenen Lager in Wirklichkeit sind. Ferner findet immer wieder Neubildung von Salpeter statt. Immerhin wird der natürlich vorkommende Salpeter in absehbarer Zeit lange nicht mehr allen Anforderungen genügen können. Stickstotfquelle I Diese Tatsachen mußten eine große Beunruhigung hervorrufen. Man begann auszurechnen, wie lange die Lebewesen auf der Erde noch Daseinsbedingungen finden werden. Eine fortschreitende Abnahme des Vorrates an gebundenem Stickstoff müßte schließlich zu einer starken Beschränkung der Einwohnerzahl führen. Sie müßte so lange zurückgehen, bis die natürlichen (Quellen an gebundenem Stickstoff' wieder ausreichen und die Mitarbeit der freien Stickstoff' assimilierenden Lebewesen genügen würden. Von diesen Gesichtspunkten aus könnte man die Frage aufwerfen, ob die modernen Bestrebungen der Hygieniker für die weitere Zukunft zweckmäßig seien. Wir bekämpfen die Mikroorganismen und suchen ihnen die Existenzbedingungen zu nehmen. Manche dieser Lebewesen begnügen sich nicht damit, den toten Organismus zu zerstören und in seine Elemente zu zerlegen. Viele davon greifen vielmehr lebendes Gewebe an und geben ungezählte Oiganismen frühzeitig dem Kreislauf der Elemente zurück. Wir nehmen den Kampf gegen diese Lebewesen auf. Wir suchen die Lebensdauer des Menschen zu verlängern. Auch die Haustiere fügen wir nicht mehr dem Kreislauf der Elemente ein. Sie werden zunächst verzehrt und die aus ihnen hervorgehenden Stoff'wechselendprodukte verbrannt. Überall .schädigen wir im Interesse der Gegenwart und des einzelnen Individuums Vorgänge, die sich im Laufe der Jahrtausende ausgebildet und zu einem Zustand geführt haben, bei dem Mangel an gebundenem Stickstoff' kaum bestand. Es ist ganz verständlich, daß unter diesen Umständen immer mehr der Wunsch sich geltend machte, es möchte eine Zeit kommen, in der der tierische Organismus und insbesondere der Mensch nicht mehr in so hohem Maße von der Pflanzenwelt in Abliängigkeit stehen würde, wie es bis jetzt der Fall w ar. Immer wieder wurde der Hoffnung Ausdruck gegeben, es möchten Mittel und Wege aufgefunden werden, um die organischen Nahrungsstoffe künstlich darzustellen. Wie wir bald vernehmen werden, ist dieses Problem viel rascher, als man glaubte, im Prinzip gel()st worden. Seine Lösung hat aber eindringlich und klai' bewiesen, daß wir niemals der Pflanzen werden entraten können. Einmal liefern sie uns die Nahrungsstoffe viel billiger und dazu noch in viel zweckmäßigerer Form, als die Technik sie jemals hervorbringen kann. XXI. Vorlesung. 430 nser ganzes Bestreben muß daher darauf gerichtet sein, den Pflanzen und besonders den Kulturgewächsen möglichst günstige Bedingungen zu 1 ihrem Wachstum zu schaffen. Gelingt es uns, sie durch künstliche Maßnahmen besser zu ernähren, dann ist zugleich die Ernährungsfrage für den tierischen Organismus in der denkbar besten Weise gelöst. Nicht derjenige, der im Laboratorium künstliche Nährmittel für das Tier und den Menschen erzeugt, löst das Problem der Ernährung des Menschen- und Tiergeschlechtes für alle Zeiten, sondern derjenige, der der Pflanze hilft, die ihr zur Verfügung stehenden Energiequellen möglichst rationell auszunützen und ihr zugleich ausreichende Nahrungsstoffe zuführt. Es ist denn auch der Technik gelungen, der Gefahr der Verarmung des Bodens an gebundenem Stickstoff wirksam zu begegnen. Es waren verschiedene Wege erfolgreich. Einmal läßt sich der Stickstoff der Luft, der uns ja in unermeßlichen Mengen zur Verfügung steht, bei hohen Tem- peraturen mit Sauerstoff zu NO vereinigen. Daraus bildet sich dann das beim Einleiten in Wasser oder Alkalilaugen salpetrige Säure bzw. Nitrite und ferner Salpetersäure bzw. Salpeter liefert. Praktisch wird die Oxydation des Stickstoffs mittelst Elektrizität herbeigeführt.^) Es ist dies die gleiche Art der Stickstoffbindung, wie sie bei elektrischen Entladungen in der Atmosphäre (Blitz) stattfindet. Es ist ferner gelungen, NO2, Stickstoff und Wasserstoff unter hohem Druck und Anwendung von Katalysatoren direkt zu vereinigen.-) Dieses Verfahren ist das in Deutschland führende. Man hat ferner zur Bindung von Stickstoä von der Tatsache Gebrauch gemacht, daß er unter geeigneten Bedingungen sich mit Metallen zu Nitriden verbindet. 3) Aus diesen läßt sich der StickstoÖ' in Form von Ammoniak abspalten. Schließlich sei noch eines Verfahrens Erwähnung getan, das bis vor kurzem das am meisten angewandte war. Es beruht auf der Eigenschaft der Karbide, Stickstoff zu binden.) Kalzium karbid liefert z. B. mit freiem Stickstoff" unter Abscheidung von Kohlenstoff das Kalksalz des Zyanamids, ON.NHj: CaC, + N2 = CaCN2-f-C. Der Stickstoff des Kalziumzyanamids läßt sich mittelst gespannten Wasserdampfes leicht in Form von Ammoniak abspalten: Ca CNg -t3H20 CaCOa + 2Nfl3. Meistens wird das Gemisch Ca CN.^ -1- C direkt = . Düngemittel verwendet. Zahlreiche Studien sind über sein Verhalten im Boden und seine Verwertbarkeit angestellt worden. s) als ) Georg Erlwein: Elektrotechnische Zeitschrift. Heft 2 und 3. 1907. Weitere Literatur bei Eduard Donath und Karl Frentzd: Die technische Ausnutzuug des atmospärischen Stickstoffs. V. Deuticke. Leipzig- Wieu 1906. C. Frenzel: Fortschritte der — Naturwissenschaften 242 (1911). ') Haber und van Oordt : Zeitschr. f. anorgau. Chem. 43. 111 (1905); 44 341 (1905J; 47. 42 (190()). ^) Haber und van Oordt: Zeitschr. f. anorg:in. Chemie. 44. 341 (1905). Lipski: 2. — Elektrochemie. 15. 189. (1909). ) G. Erlwein: Zeitschr. f. angewandte Chemie. 16. 533(1903). Adolph Frank: Ebenda. 16. 536 (1903); 19. 835 (1906). Otto N. Witt: Chem. Industrie. 28. 689. (1905). ^) \'gl. z. B. F. Löhnis: Zentralbl. f. Bakt., Parasiteukunde u. lufektionskrankh. 2. 877 (1906). —'0. Schönherr: Chemiker-Ztg. 32. 578 (1908). Zeitschr. f. — — Eiweißstotfe und ihre Bausteiue. 481 Fassen wir die Bemühungen, den freien Stickstoff der Luft und auch der Industrie nutzbar zu machen, zusammen, dann ergibt sich, daß dieses Ziel erreicht ist. Die großen Lücken, die durch die künstliche Umwandlung von gebundenem Stickstoff in die freie Form in den Vorräten der Natur an solchem entstanden sind, der Pflanze sind durch den Umstand, daß wir jetzt soviel Stickstoff der Luft in eine für die Pflanze verwendbare Form überführen können, als notwendig ist, mehr als ausgeglichen worden. Die moderne Hygiene darf ihren Kampf gegen jene Lebewesen, die unser Dasein bedrohen, unbeschadet der durch ihre Methoden geschaffenen Durchbrechung des natürlichen Kreislaufes des Stickstoffs mit aller Energie weiterführen! Vorlesung XXIL Eiweißstoffe und ihre Bausteine. 6. Der Eiweißstoffwechsel der Pflanze. Die Beziehungen der Aminosäuren zu den Betainen und Ail<aioiden. Abbau der Aminosäuren in den höheren Pflanzen, ferner durch Bakterien und Hefezellen. Wir haben festgestellt, daß die Chlorophyll enthaltende Pflanze aus der Kohlensäure der Luft, aus Wasser, Salpetersäure bzw. Ammoniak und Schwefelsäure Eiweiß aufbauen kann. Es spricht alles dafür, daß zunächst die einzelnen Aminosäuren gebildet werden, und diese dann unter Wasseraustritt zu Eiweiß zusammentreten. Diese Synthese von Aminosäuren bzw. Eiweiß aus den Elementen ist auf die Pflanzenwelt beschränkt. Der tierische Organismus vermag mit den Elementen als solchen nichts anzufangen. Seine synthetischen Fähigkeiten sind nicht so umfassend. Es müssen ihm organische Verbindungen zur Verfügung stehen. Diese können allerdings ganz einfacher Natur sein. Der tierische Organismus ist somit ganz auf das Pflanzenreich angewiesen. Die Pflanze allein vermag Sonnenenergie direkt zu verwenden. Der tierische Organismus übernimmt solche in den organischen Nahrungsstoffen. Gleichzeitig erhält er zusammengesetzte Verbindungen mit bestimmter Struktur, die er weit abbauen und dann seinen eigenen Bauplänen anpassen kann. Das Tier ist mit allen seinen Einrichtungen, seinen Fermenten usw. auf die Art der Nahrung eingerichtet. Die erste Aufgabe, die zu erfüllen ist, ist in der ganzen Tierreihe in mehr oder weniger ausgedehnter Weise der Abbau der eine besondere Struktur aufweisenden, zusammengesetzten Verbindungen. Überall treffen wir auf Fermente, die Hydrolysen bewirken kiinnen. Die Zellen des tierischen Organismus enthalten die gleichen Bestandteile, wie diejenigen der Pflanzen. Ob wir nun Eiweißstoffe aus einer Pflanzenzellc oder solche aus einer Tierzelle zerlegen, immer treffen wir auf die gleichen Aminosäuren. Die Nukleinsäuren der Kerne der verschiedenartigsten Zellen liefern die gleichen Spaltprodukte. Überall finden wir Fettsäuren und Glyzerin als Bausteine der Fette. Einzig die Kohlehydrate zeigen im Pflanzenreich eine größere Mannigfaltigkeit als im Tierreich, linigekehrt verfügt das Tier offenbar über Eiweißstoffe, die der Pflanze fehlen. Diese besonderen Proteine spielen jedoch zum größten Teil eine mechanische Rolle und geh()ren den Zellen als solchen nicht un- Kiwcißstiirto und ihre Bausteiiit'. 41-i,'» mittelbar an. Im PHanzenreicli vertreten in gewissem Sinne die Zellulosearten und Fentosane die Gruppe der mechanische Funktionen erfüllenden Eiweißarten. Die Erkenntnis, daß die Pflanzen- und Tierzellen in den wesentlichsten Zügen einen gleichartigen Bau l)esitzen, führt von selbst zur Fragestellung, sich auch gleiche oder doch ähnliche Funktionen finden. Diese Frage wollen wir hier vom Standpunkt des Stoffwechsels und insbesondere desjenigen des Eiweißes bzw. der Aminosäuren beantworten. Es sei gleich hervorgehoben, daß je weiter wir in die einzelnen ob Stoffwechselvorgänge in der Pflanze vordringen, um so mehr Prozesse bekannt werden, die im Tierreich ihr Analogen haben. Wir müssen bei der Pflanze zwei Vorgänge trennen i), nämlich einerseits die Bildung von organischer Substanz aus den Elementen unter Mitwirkung von Sonnenenergie und andrerseits den Ab-. Auf- und Umbau jener \erbindungen. die den Pflanzenzellen als Resultat photochemischer Vorgänge zufließen. Bei der Synthese der organischen \'erl)indungen aus den Elementen entstehen Produkte, die nicht ohne weiteres für jede einzelne Pflanzenzelle mit ihren verschiedenartigen Funktionen geeignet sind. Es muß eine Anpassung an die besonderen Verhältnisse stattflnden. Auch die Pflanzenzelle hydrolysiert zusammengesetzte Verbindungen und baut aus den entstandenen Abbaustufen neue Produkte auf. Ferner kann sie gebildete Bausteine stufenweise weiter zerlegen und sich die in ihnen aufgespeicherte Energie genau so nutzbar machen, wie die Tierzelle. Endlich vermag die Pflanzenzelle aus den ihr zugeführten Stoffen Fermente und auch Sekretstofle aller Art zu bereiten. Wir kennen auch Stoffwechselendprodukte, doch werden wohl die meisten davon, da die Pflanze mit Ausnahme der Kohlensäure wohl kaum Auswurfstoffe abgibt, wieder von neuem zu Synthesen verwendet. Zahllos sind die Verbindungen, die die Pflanze synthetisch mit Hilfe ihrer Zellen aufbauen kann. Wir flnden im Pflanzenreich außer den oft genannten Nahrungsstoffen und ihren Bausteinen Angeh(»rige der Reihe der Kohlenw^asserstoffe, der Alkohole, der Phenole, der Aldehyde, Ketone und Säuren. Wir begegnen einer fast unerschöpflichen Mannigfaltigkeit in der Bildung von Riechstoffen, von Farbstoffen und ferner von Alkaloiden. In diesen Stoflen kennzeichnet sich die Eigenart des Pflanzenorganismus. Die meisten dieser Stoffe stellt die Tierzelle nicht dar. Typische Pflanzenprodukte sind ferner die Gerbstoffe, die Flechtenstoffe, die Saponine, die Bitterstoffe, die Harze und der Kautschuk. Die Zahl der von der Pflanzenwelt mit allen ihren Arten hervorgebrachten Verbindungen ist eine ganz gewaltige. Von den Grundstoffen Kohlensäure und Wasser nehmen alle diese Stoffe, die wir zum Teil meist auf großen Umwegen in mühsamer Arbeit im Laboratorium bereiten können, ihren Ausgang. Der tierische Organismus bereitet auch Verbindungen eigener Art. die der Pflanzenzelle vollständig fehlen. Es sei z. B. an den Blutfarbstoff, an die Gallensäuren, an das Adrenalin usw. erinnert, doch steht er in der Mannigfaltigkeit seiner Synthesen weit hinter der Pflanze zurück. Es kann das nicht daran liegen, daß das Tier die Sonnenenergie nicht direkt benutzen kann, denn die ') In Wirklichkeit dürften wohl beide X'orgängf die mannigfachsten Beziehungen zueinander unterhalten. Eine so scharfe Abgrenzung ist notwendig, um die iteiden Vorgänge zu verstehen. Abderhaldon, Physiologisphe Chemie. I. 'l'eil, 'i. Aufl. O^ XXII. Vorlesung. 434 meisten der genannten Verbindungen dürften auch in der Pflanze in keinen direkten Beziehungen zur Kohlensäure- und Wasserassimilation stehen. Die Pflanzenzelle wird vieiraehr diese Produkte aus den ihr zugeführten, aus der Assimilationstätigkeit hervorgegangenen organischen Verbindungen durch mehr oder weniger eingreifende Umwandlungen erst nachträglich Der tierische Organismus beschränkt sich im großen und ganzen Umbau der ihm mit der Nahrung zugeführten Verbindungen. Er vollzieht keine umfassenden Synthesen mehr. Auch dann, wenn (mneue Verbindungen formt, erkennen wir unschwer den Zusammenhang mit dem Ausgangsraaterial. Die Pflanzenzelle geht vielleicht zum Teil von den gleichen Materialien aus, wie die Tierzelle. Sie reduziert, oxydiert, bilden. auf einen aus bildet aliphatischen Kohlenstoff ketten aromatische, spaltet gebildete Ringe wieder auf usw. Nichts erinnert schließlich mehr an das Ausgangsprodukt als die elementare Zusammensetzung. Gewiß wird man in Zukunft die Entstehung der einzelnen Verbindungen genauer verfolgen können und all die Wege klarlegen, die die Pflanzenzelle einschlägt, wenn es gilt, bestimmte Verbindungen aufzubauen. In vielen Fällen sind wir leider bis jetzt nicht über die Isolierung einzelner Produkte hinaus gekommen. Es fehlt uns die Kenntnis ihrer Bedeutung für den Organismus, der sie gebildet hat. Ziemlich eingehend untersucht ist der Ei weiß st off Wechsel der Pflanze. Es unterliegt wohl keinem Zweifel mehr, daß er viele Züge mit dem des tierischen Organismus gemein hat. Die Pflanzenzellen verfügen über Fermente, die Eiweiß, Peptone^) und Polypeptide^) Aus diesen hydrolysieren können. Es entstehen Aminosäuren. kann die einzelne Zelle von neuem Eiweiß aufbauen, oder aber es knüpfen sich an diese Abbaustufen Umwandlungen anderer Art an. Besonders lebhaft ist der Abbau von Eiweißstoffen bei der Keimung von Samen. Das Reserveeiweiß wird stufenweise abgebaut. Es lassen sich alle möglichen Abbaustufen nachweisen. Man findet Peptone) und AminoEine Anhäufung dieser Produkte findet säuren") in buntem Gemisch. nicht statt. Die gebildeten Abbauprodukte werden fortgeführt und dienen =5) einerseits zur Synthese von Eiweiß in den neugebildeten Zellen, andrer- ») Gorup-Besanez Ber. d. Deutsch. Chem. Ges. 7. 569 (1874); 8. ].olO (187.M: A. Schemwrt Green: Philos. Trausact. Royal Soc. 178. 39 (1887). 673 (1876). W. Zaleski: Ber. d. Deutsch. und Grimmer: Zeitschr. f. physiol. Chem. 48. 27 (1906). Martin: Moncoroo : Jouru. d.' Th(5rap. 7. 6 (1880). Botan. Ges. 23. 133 (1905). O. Emmerlinq: Ber. d. Deutsch. Chem. Journ. of phys. 5. 313 (1884); 6. 336 (1885). : — 9. — — — — — — — Hahn und Geret : Zeitschr. f. Biol. 40. 117 (1900). 35. 195 (1902). Vgl. weitere Literatur hei Carl Fr. Kutscher: Zeitschr. f. physiol. Chem. 33. 59 (1901). Oppenheimer: Fermente und ihre Wirkungen. 4. Aufl. F. C. W. Vogel. Leipzig 1913. Ges. hahn, — ^) Emil Abderhalden, Y. Teruuchi, Alfred Schittenhelm, August Rillief, DammHans Pringsheim: Zeitschr. f. physiol. Chem. 49. 26 (1906): 55. 395 (1908); 57. 332 (1908); 59. 249 (1909). und .V. Castoro: Zeitschr. f. physiol. Chem. 41. 455 (1904); ) Vgl. E. Schulze 43. 170 (1904). J Reynolds Green und Henri/ Jackson : Proceed. of the Royal Soc. E. Schulze E. Schulze: Zeitschr. f. physiol. Chem. 47. 507 (1906). 77 (B). 69 (1905). E. Godlewski: Bull, de l'Acad. des Sc. und E. Winterstein: Ehenda. 65. 431 (1910). W. Zaleski: Beitr. de Cracovie. Classe des Sciences math. et nat. (B). 623 (1911). E. Schulze: Zeitschr. f. physiol. Chem. 71. 31 zum Bot. Zentralbl. (1) 27. 63 (1911). W. l'alladin: Biochem. Zeitschr. 39. 290 (1912); 42. 325 (1912); 44. 318 (1912). (1911). ) W. 1{. Mack: Zeitschr. f. physiol. Chemie. 42. 259 (1904). — — — — — — — Eiweißstotte und ihre Baii;;teinc. 435 seits sind sie auch Ausganj;sraaterial zur Bilduriii- anderer Verbindun>^en. Diese Übereinstimmung mit den Stoffwechselvorgängen im tierischen Orgaauch er baut ab, wenn er aus einem vorhandenen Eiweiß ein nismus wird nur durch die Beobachtung durchbrochen, anderes bereiten will daß die Ptlanzenzelle größere Mengen von Asparagin und ferner auch von Glutamin bildet. M Diesen beiden Verbindungen begegnet man besonders in keimenden Samen. Sie hängen in irgend einer Weise mit dem Eiweißstoffwechsel zusammen. Es sind verschiedene Hypothesen aufgestellt worden, um ihre Entstehung und ihre Bedeutung zu erklären. iMan könnte zunächst daran denken, daß diese Säureamide eine Zwischenstation in der Umwandlung bestimmter Aminosäuren in andere darstellen oder gar auf Beziehungen zu den Kohlehydraten hinweisen. Es wäre denkbar, daß aus solchen Bernsteinsäure bzw. Glutarsäure oder eine diesen nahestehende Verbindung entsteht, die dann mit Ammoniak zusammentritt. Wir wissen, daß die Pflanze desaminieren und ami- — nieren, d. h. — Aminogruppe aus Aminosäuren abspalten bzw. die in Verbindungen einfügen kann.'-) Das im ersteren Falle frei werdende Ammoniak würde dann zur Synthese zur Verfügung stehen, falls es nicht durch Reduktion aus aufgenommenen Nitraten gebildet wird. Man hat ferner die Bildung der Säureamide der Harnstoffbildung im tierischen Organismus an die Seite gestellt. In beiden Fällen wird gebildetes Ammoniak gebunden. Bei der Pflanze sind es die Asparagin- und die Glutaminsäure, die Ammoniak festlegen, beim Tier die Karbaminsäure: COOH + NH3 I CO . NH., . NH., + H2 I CH . CH NH., CH2 GH., j I COOH COOH Asparaginsäure Asparagin. NH2 COOH + NH3 = NE, CO NH., + H., O Karbaminsäure Harnstoff . . . Von diesem Gesichtspunkte aus ist die Bildung der Säureamide ein Schutz der Zellen gegen als freies Ammoniak aufgefaßt worden. Entstehen- Ammoniak wird festgelegt. Es liegt ein umkehrbarer Vorgang vor. Während der Harnstoff endgültiges Stoffwechselendprodukt darstellt, sind die Säureamide weiterer Umwandlungen fähig. Priunischnikoir^), der auf Grund zahlreicher Beobachtungen die erwähnte Auffassung der Bedeutung des der Säureamidbildung vertritt, hat die interessante Feststellung gemacht, daß eiweißreiche Pflanzen, wie Lupinen, gegen freies Ammoniak besonders empfindlich sind, während kohlehydratreiche sich als viel wider') E.Schulze: Landwirtsch. Versuchsstationen. 33. 118 (1886); Landwirtscli. Jalirl). Borodin: Bot. Ztg. 801 (1878). 35. 621 (1906). Ber. d. Deutsch, l.otaii. lies. 25. D. Pnamschnikoic und ./. Schuhur: Eltonda. 28. 2.53 (1910. 213 (1907). Vgl. auch Anton Stieffei-: Zeitschr. f. physiol. Chemie. 86. 24;") (1913). — — — — — H7. BHfkfwifudi ) N. Castora: Zeitschr. f. phvsiol. Chemie. 50. 525 (1907). •Biochem. Zeitschr. 41. 431 (1912). ^) Prianisc/inikoir: Die landwirtschaftlichen \ ersuchsstationen. d. 2(i7 (1922): Ber. Deutscheu Bot. Gesellsch. (1922). 28^^ XXII. NOrlesiing. ^'^^ Standsfähiger erweisen. Im HungerzAistand wird jede Pflanze gegen Ammoniak empfindlicher. Es ist die Möglichkeit seiner Festlegung eingeschränkt bis aufgehoben. Man hat noch an andere Möglichkeiten des Zusammenhangs der Säureamidbildung mit dem Stoffwechsel und insbesondere mit dem Eiweißstoffwechsel gedacht, ohne jedoch für sie eindeutige Beweise erbringen zu können. Soviel ist sicher, daß das Asparagin nicht unmittelbar mit der Eiweißsynthese verknüpft ist, denn es nimmt nicht in dem Maße, wie die übrigen Aminosäuren, mit der Eiweißbildung ab.') Ferner läßt sich zeigen, daß die Säureamide später, ohne daß eine entsprechende Bildung von Eiweiß sich nachweisen läßt, wieder ganz verschwinden, und zwar um so rascher und vollständiger, je mehr die Keime und Pflanzen dem Licht ausgesetzt sind.'-) Offenbar werden die aufgestapelten Amide zu verschiedenartigen Synthesen und sehr wahrscheinlich auch zur Bildung einzelner Aminosäuren verwendet. Wie der weitere Abbau der Aminosäuren sich vollzieht, welche Abbaustufen auftreten, und in welcher Weise der Kohlehydrat- und Fettstotiwechsel mit dem Eiweißstoffwechsel in Wechselbeziehung tritt, wissen wir nicht. Wir können nur vermuten, daß bei der Desaminierung der Aminosäuren die gleichen Produkte auftreten, wie wir sie im tierischen Organismus antreffen. Besser unterrichtet sind wir über den Abbau mancher Aminosäuren durch Bakterien und die Hefezellen. Wir kommen darauf gleich zurück. Von Interesse ist, daß in einigen Pilzen Harnstoff aufgefunden worden ist.^) Die große Verbreitung eines Fermentes, Urease genannt, in der Pflanzenwelt, das Harnstoff in Ammoniak und Kohlensäure spaltet, läßt darauf schließen, daß diese Verbindung ein häuflges Stoffwechselprodukt darstellt.) Es entgeht dem Nachweis, weil es sofort weiter gespalten wird. Die Spaltung des Harnstoffs erfolgt offenbar von seinem Hydrat aus^): /OH //OH —y C^^NH., 2NH, + CO... \NH., Ferner ist im Wickensumen^) und im Zuckerrübensaft ') Guanidin beobachtet worden. Es entstammt vielleicht dem Argin in, das diese Gruppe besitzt.^) ) -) agronom. ') E. Schuhe: Bor. il. Deutschen botau. Ges. 22. ;W1 (1904). Pfeffer: Jahrli. d. wissensehaftl. Botanik. 8. 538 (187'2). — Mcimier: Annales 6. 27;) (1880). Max limnberqcr und Anton Landsicdl: Monatsliot'to 26. 1109 (1905). — R. Gazr: Arch. d. riiannazic. 243. 78 (1905). ~ f. — Cliemio. 24. 218 (1903); .1. doris und M. Maxen': Conipt. reud. do l'acad. des sc. 147. 1488 (1908). V,irl. aucli den Befuiul von d-Butyltliioharnstoff im iitliorischen 01 von Caidaniino aniara. L. r. Mar Kunze: Archiv d. Pharm. 245. 657 (1908). ) Vgl. hierzu auch Kmil A. Werner: Dublin J. of med. scieucc. 4. 577 (1922). 5) n. S. Anistronr/ und Edir. Horton: riocped. of thc Royal Soc. 85. (B). 109 (19121. ") ?j. Schulze: Zcitschr. f. physiol. Chemie. 17. 193 (189."r): Her. d. Deutschen (^liom. Ges. 25. G58 (1892). hiMii. G(>s. 29. 2()45 (189(5). ') 0. r. Lippinann: Bor. d, Deutschen ») Vgl. S. 320. ( Kiwoifetortc und ihre Baiisteiiio. 437 Ferner bilden viele Pflanzen Indol.i) Dieses j^^eht ohne Zweifel aus dem Trytophan, der ß-lndol-a-aminopropionsäure hervor. Ferner ist auch Skatol=ß-MethyIindol in der Pflanzenwelt aufgefunden worden.') C HC CH C CH Tryp 1 CH CH HC^'^C phan — 7.- GH., . CH (OOIl \\if\ CH NH, HC CH . . C CH NH NH A m n 0- -i n d [i i p r o }) i o n s ä u r e 1 ( ) l n do1 (^11 HC (; c HC C CH . CH,, CH NH Skatol = ;i-Methylindol. Es ist sehr wahrscheinlich, daß auch der prachtvolle blaue Farbstott" vom Ti-yptophan aus gebildet wird. Er findet sich als Glukosid, Indikan genannt, in Indigoferaarten und in Isatis tinctoria. Indigo Glukoserest Indolrest CH HC/'^C — C-O— CH CH C CH . (CH . OH), . CH.. OH \/ II !l HC .CH NH Indikan. Indikan Glukose frei. läßt sich durch Fermente oder Säuren spalten. Es wird Gleichzeitig entsteht Jndigo: C'H eil HC^\c— CO OC c HC CU c NH C^^ CH c =C NH CH CH Indigo. Haiullexikon. 4. 844 (15)11) (lioarlicitet ') Vgl. die Literatur im Hioclieniisclieu von G. Zempl('n). .J. Springer. Berlin l'.lll. -- Ferner F. Weelntizi-n: Pharma/.. Weekblad. 45. 132ö (1908). CItristian A. Her/er: W. It. Dimsfan: l'harniaeol. .lourii. 19. lOlU (1888). H. Walbaum: Ber. d. Deutschen Cheni. (ies. 33. .lourn. of Biol. Chem. ö. 489 (li)()8). J. Sack: Pluirniaz. Weekblad. 48, HOT (1911). 19ü;{ (1900). '') — — XXII. Vorlcsiinsr. 438 Im Mutterkorn sind eine ganze Reihe von Verbindungen aufgefunden worden, die sich direkt von Aminosäuren ableiten lassen. Es sind dies Amine Para oxyphenyläthylamini) = Tyramin, Imidazolyläthylamin-) = Histamin. Pentamethylendiamin == Kadaverin^), und Agmatin.) Zum Teil verraten schon die Namen dieser Verbindungen Die erstere Verbindung ist auf Tyrosin, die zweite auf ihre Herkunft. Histidin, die dritte auf Lysin und die vierte endlich auf Arginin zurückzuführen. In allen Fällen liegt die gleiche Bildnngsart vor. Es ist Kohlensäure aus den Aminosäuren abgespalten worden. Dieser Art des Abbaues von Aminosäuren werden wir noch oft begegnen. Namentlich manche Bakterienarten, die auch unseren Darm bevölkern, vollziehen die Die folgenden Formeln geben der einzelnen Verbindun":en wieder: diese Umwandlung.-') OH HC/'^CH OH HC/'^CH C den Zusammenhang C . . NH, C . CH., . CH . COO H C Tyrosin = (i-Paraoxy])henyl7. - a m n pr j) i i . CH., . CH., + CO2 Paraoxyphenyläthylamin. nsäu re CH— NH CH— NH >CH C N N C CH-, CH-, —>^ I CH . NH., (^H., . NH., + CO., COOK Histidin = ß-lmidazolyly.-arainoprojjion säure Imidazolyl-äthylamin. — ') G. Barger: Trausact. of the Ghem. Soc. 95. G. Barqer und 1123 (1909). Vgl. seine Synthese: Ihde: Arch. f. experini. Tatli. u. Pharm. 61. 113 (1909). George Barqer und George Barqer: Transact. of the (_;hem. Soc. 95. 1123 (1909). G. s'. Walpöle: Ebenda. 95. 1720(1909): .Jouni. of Thysiol. 38. 343 (19091'— A. UUmann hat Tyramin in Btechdistelkörneru gefunden. Biochem. Zeitschr. 128. 402 (1922). II. — II. — G. Barqer Ackermann und /•'. Kutscher: Zeitschr. f. Biol. 54. 387 (1910). H. Dale:io\\vn. of Cheni. 97. 2592 (1910): Zentralbl. f. Thysiol. 24. Nr. 19 (1910); Transactions of the Chem. Soc. 97. 2592 (1910); .lourn. of riiysiol. 11. 1 (1910). Synthese: K. K. Korss-lcr und M. Th. Hanke: .lourn. of Anieric. Chem. Soc. 39. 497 (1919); 40. 1724 (1918). ^) Biedlümler: Sitzungsber. d. (Jes. z. Beförd. d. Natuiw. Marburg 1908. ) B. Kngeland und /'. Kutscher: Zentralbl. f. l'hysiol. 24. 479(1910). Vgl. über seine Entdeckung A. Kossei: Zeitschrift f. physiol. Chemie. 66. 257 (1910) und seine Synthese: A. Kossei: Sitzungsbericlite (ku- Heidelberger Akad. d. Wisseusch. 12. Abt. ^) und — //. — — (1910). '") 1. Vgl. 7.. 103 (1906). (191S): 3. 1 B. Takaokt Sasaki: Acta scholae mecbc. — M. Tsiidji: Ebenda. 2. (1919). 11.") (1918). Uiiiversitatis imp. in Kioto. — K. Jlirai: Ebenda. 2. 425 CH., r . GH., ' . GH., . GH., GH . . GOOjH -> GH.. GH., GH.. GH, GH, + GO^ . . . . ' ' ' 439 und ihre Bausteine. Eiweißstoft'e • I i NH, ' ^ I NHo NH, Pentamethy lendiamin = NH.2 Lysin Kadaverin. NH, /NH-, G=NH \nH GH, GH, GH, GH COO H Arginin = S-Guanidino-a-amino. . . . . valeriansäure NH, /NH, G^NH i \NH . GH2 „GHj . GH., . GH., + GO^ Agmatin = Aminobutylenguanidin. Aus Merkurialisarten und in der Kalamuswurzel hat man ferner Methylamin isoliert. Es entstammt ohne Zweifel dem GlykokoU: GH, GOOH —> NH., GlykokoU = Amino- GH3 + GO., NE, Methylamin, essigsaure Es hat Beziehungen zum Im Tabak ist Isoamylamin vorhanden. Leuzin GH3 CH3 CH3 GH3 \x GH \/ GH I I CH, CH3 I i GH NH, . GH, . NH, + GO, GOO H Leuzin = 7.-Aminüisol)utyl- Isoamylamin. essigsaure Es ist nicht leicht, festzustellen, welche Bedeutung diese Verbindungen im Pflanzenreich und z. B. im Mutterkorn zukommt. Vielleicht gibt die Beobachtung, daß solche Amine von Hefen und Schimmelpilzen leicht in die entsprechenden Alkohole übergeführt werden'), einen Anhaltspunkt über ihre weitere Verwendung: ) G. Ciamician und C. Ravenna: Atti K. Accad. dei Liucei. 20. 614 (1911). XXII. \orle8nii^'. 440 C« H, (OH) CIL, . . —> CH, + H, o NH, p-Oxyphenyläthylamin ( ', 11^ ( < >H) . CH^ . CH, Oll + NH^ i)-()x yphciiyläthylalkohol = Tyrosol. Es ist wohl möglich, daß aus Aminosäuren in Pflanzen häutig Amine hervorgehen. Der Umstand, daß man ihnen bis jetzt nur vereinzelt begegnet darauf zurückzuführen sein, daß sie nur in kleinen ist, dürfte vielleicht Mengen entstehen und immer gleich weiter verarbeitet werden. Im Pflanzenreich sind eine ganze Anzahl von Verbindungen aufgefunden worden, die zum Retain in naher Beziehung stehen. Wir haben früher darauf hingewiesen, daß das Betain selbst dem Glykokoll nahe steht. Man kann es sich aus diesem durch vollständige Methylierung des Aminostickstoffesi) hervorgegangen denken: bereits CH, . CHj COOH /CH3 C( )( )H CH-, . " I I N<CH3 —). NH, VCH3 ^OH Glykokoll — Aminoessigsäure . CO /CH3 -^ I NfCH3 + H, O VCH3 Betain \)— Anhydridform. Das Betain ist in zahlreichen Pflanzen aufgefunden worden. Seine zum Eiweißstoflwechsel ist noch nicht aufgeklärt. Nach den einen Stellung Autoren stellt das Betain ein Stott'wechselendprodukt dar. nach anderen nur ein Zwischenprodukt. Es könnte z. B. durch Reduktion in Cholin übergehen und damit Beziehungen zu den Phosphatiden, insbesondere zum Schließlich ist es nicht ausgeschlossen, daß eine Lezithin, anknüpfen. Rück Verwandlung in Glykokoll eintritt, ^i Es sind eine Reihe weiterer methylierter Verbindungen aus Pflanzen isoliert worden, die sich von bekannten Aminosäuren ableiten lassen. Wir wollen sie gleich der zugehörigen Aminosäure gegenüberstellen: CH., -CHo " I I CH, €H. (Ooll NH Prolin = z-Py rrolidinkarboiisäure. M li.Enqeland: Ber. d. Deutschen Clieiii. Gesellsch. 42. 2968 (1909). - E. Schulze Vgl. ferner über die BilG. Tr/e/-; Zeitschr. f. physiol. Chem. 67. 50 (1910). duiis dieser Körperkliisse Georg Trier: tiber einfache PHauzeiibasen uud ihre Beziehungen zum Aufbau der Kiweilistoffe und Le/ithine. Gel»r. Borntriiger, Berlin 1912. ^) Vielleicht entsteht aus Betain auch (llykolsiiure. Vgl. hierzu Felix Ehrlich uud Fritz Lange: Ber. d. Deutschen Chem. (iesellsch. 46. 274B (1911^). iiutl : Eiweißstoffe und ihre liausteiiie. CHo— CH. CR.,— GH., GH., " r " I I 441 CH.G(H)H l»zw. I ('H GH., ^NfcH, NfcH3 GH, ^GHs H)H Stachydrin = Dimethyl- + U. o GO . Anhydrid form betain der a-Pj'^rroHdin- karbonsäure i) GH HG^ ^G— G HC . GH.. GH GOOH . GH . NH., GH NH Tryptophan =: a-Amino-'i-indolpropionsäure. GH G— G.GH., .OH HG HG . GO OH GH XH GH OH Hypaphorin = TrimethyUjetain des Tryptophans.^) GH— NH GH— NH GH— NH iCH >GH -N -N >C.SH und GH., GH., N G GH.. /CH3 . 1 CH3 I CH . NH., Histidin= a-Amino- . . I GOGH GH Nf-GHg CH N(-GH3 GO- Vgh, O ß-imidazolyl- Trimethylbetain des propionsäure Histidins I \^GH3 GG Erji;othionein =: Tri- methylbetain des = Thiohistidins.) Herzyninä) E. Schulze und d. Trier: Ber. d. Deutscheu Cheiu. Gosellsch. 42. 46n4 (U)U9); R. Encielawl : Bor. d. Deutphysiol. Chem. 59. 233 (1909): 67. Sl (1910). f. scheu Chem. (iesellsch. 42. 29fi5 (1909); .Vnli. f. IMmmiMzio. 247. 4(53(1909). -- H. Willstätter: Ber. d. DiMitschcu (Ikmu. (icsollscli. 33. 1 1(5(') (190(1). - V?l. soini' Synthese: G. Trier: Diss. Zürich 1910. Wctensch. .\mstrni,ini. 19. 1250 (1911). ) van Rotnhur(/h: Koninkl. Al<. viiii ^) G. Barger und Arthur Jatnes Eirins: The Bioclicm. Journ. 7. 204 (1913). Vgl, die Synthese: F. Kutscher: Zcntnlbl. f. Pliysiol. 24. 775 (1910). •) G. Ii(tr<iir und .1../. h'irins: 'i'r;insact. "cheni. Soc 99. 233(') (1911). ») Zeitschr. — — XXII. Vorlesung. 442 Das St ach yd in ist in Stachysknolleni), in Chrysanthemum einerariifoliura -) in Galeopsis grandiflora und aufgefunden worden. Es ist ohne Zweifel noch bei manchen Labiaten und Kompositen anzutreffen. Das Hypaphorin findet sich in Erythrina Hypaphorus.^) Das ßetain des Histidins ist im Boletus edulis angetroffen worden.) Ferner ist es aus dem Champignon 5) gewonnen worden. Besonders interessant ist das von Tanret^) aus dem Mutterkorn isolierte, schwefelhaltige Ergothionein. Es ist ein Histidin, in dem in der Imidazolgruppe an Stelle eines Wasserstoffatoms eine Thiogruppe eingetreten ist. Ferner ist der Stickstoff der Aminogruppe stellt die Anhydridform dar. Es sind noch weitere Betaine aufgefunden worden, die in direkter Beziehung zu bestimmten Aminosäuren stehen. So enthalten Stachysarten die Betaine Betonizin und Turizin.^) Beide sind als Dimethylderivate des Oxyprolins erkannt worden. s) Sie sind isomer. Man kann sie als vollständig methyliert. Die gegebene Formel Oxy-stachydrine betrachten. HO CH HO (^H— CH2 CH, . . CH COOH CH, CH2 . CH CO . NH CHj CH3 Oxyprolin-betain Oxy-prolin (Betonicin und Turicin). In diesem Zusammenhang sei kurz erwähnt, daß im Pflanzenreich ein weiteres Betain sehr verbreitet ist, das zwar zu keiner bekannten Aminosäure direkte Beziehungen besitzt, jedoch großes Interesse verdient, weil es vielleicht das Ausgangsmaterial zur Synthese mancher kompliziert gebauter Alkaloide darstellt. Es von der Nikotinsäure ab: ist dies das Trigon ellin. Es leitet sich CH CH /\C— CO /\C—COOH HC HC li I. II I CH \^l CH HC \N^ HC CH3 Trigon ellin — Methyl- Nikotinsäure =:ß-Pyridinkarbonsäure betain der Nikotinsäure. A. V. Planta und E. Schulze: Ber. d. Deutschen Chem. Gesellsch. 26. 939 (1893). 2) K. Yoshimura und G. Trier: Zeitschr. f. physiol. Chem. 77. 290 (1912). ») Grcshqff-. Medcdeclingen uits Lands Plant. 7. 29 (1890); 25. 54 (1898). Vgl. auch G. Barger ) C. Benter: Zeitschr. f. physiol. Chemie. 78. 167 (1912). und A.J.Exins: Biochem. Jouru. 7. 204 (1913). 5) F. Kutscher: Zentralbl. f. Physiol. 24. 775 (1910); Zeitschr. f. Untersuch, der R. ICngeland und F. Kutscher: Zentralbl. ]S'ahrungs- und (ionußmittel. 21. 535 (1910). ') — — f. Physiol. 26. «) ') ^) .5()9 (1912). Tanret: Compt. reud. de l'acad. des scienccs. 149. 222 (1909). F. Schulze und G. Trier: Zeitschr. f. phys. Chem. 76. 258 (1911); 79. 235 (1912). A. Küng: A. Küng und G. Trier: Zeitschr. f. physiol. Chemie. 85. 209(1913). Ebenda. 85. 21 ?' (1913). — Eiweißstotfe und ihre Hausteine. 443 Das Trigonellin ist in d^n Samen des Bocksdorns, Trigonella foenuni graecum, entdeckt worden, i) Später wurde es in vielen anderen Pflanzen aufgefunden.-) Über das Vorkommen von Nikotinsäure in Pflanzen berichten IL Suzuki und S. Matsunaga. Es sind im Pflanzenreiche auch einfachere Methylderivate von Aminosäuren aufgefunden worden. So erwies sich das in der Rinde von Geoffroya surinamensis vorkommende Surinamin als ein Methyltyrosin. ) Ferner ist das in verschiedenen Pflanzen aufgefundene Ratanhin ebenfalls Methyltyrosin und mit dem Surinamin identisch '') -^'j OH . CßH, .CHo.CH.COOH I NH CH3 Surin am in = Ratanhin = p-Oxyphenyla-methylaminopropionsäure. . Schließlich sind noch Verbindungen bekannt geworden, die offenbar durch zwei Umwandlungen aus Aminosäuren entstanden sind. Ein solcher Körper ist das in Gerstenkeimen entdeckte Hordenin.'^) Es hat die Konstitution eines p-Oxyphenyl-dimethyl-äthylamins. ') Wie der Name besagt, handelt es sich um ein p-Oxyphenyl-äthylamin, dessen Entstehungs- weise aus Tyrosin wir schon kennen gelernt haben, der Aminogruppe zwei Methylgruppen trägt: yCH-CH^ OH C . C . CH., . CH, in dem der Stickstoff X.XII. Vorlesung. 444 Hyoscyamiis niuticns isolierte Base ab. ;^ie ist als ein Tetramethylputreszin erkannt worden M: (JH., . ni., . C'H. CH. . Kh CH, ^\H CH. . . CH., . ^\CH3 Tutreszin = Tetramethylen- CH, "^\CH., Tetraniethyl-piitreszin. diami n Diese Verbindungen werden zum Teil bereits zu den Alkaloiden gerechnet. Es lassen sich auch noch andere Angehörige dieser Körperklasse mit bestimmten Eiweißspaltprodukten in enge Beziehung bringen. 80 ist aus Kakteen neben einer Anhalin genannten, mit Hordenin (vgl. S. 44;^) identischen Verbindung ein Alkaloid, Mezcalin genannt, gewonnen worden, eines .H, 4, 5-Trimethoxyphenyl-äthylamins dem die Konstitution zukommt ^ ) C CHa . . CH., . NH, HC/^ CH CH, . C C CH, . C O CH3 . Vor allen Dingen sind Lysin und Argin in, bzw. das aus diesem letzteren gewinnbare Ornithin als Ausgangsmaterialien zurSynthese von be- stimmten Alkaloiden sehr geeignet. Das Ornithin liefert unter Kohlensäureabspaltung Putreszin. Dieses steht dem Pyrrolidin und durch dieses dem Pyrrol sehr nahe. Lysin zeigt über Kadaverin Beziehungen zu Piperidin und Pyridin. Vom Pyrrol und vom Piperidin gelangen wir zum Chinolin bzw. Isochinolin. Die folgenden Formeln zeigen die Möglichkeit derartiger Übergänge^): /NHo NH., C^NH \NH I . CH., . CR, (^H, . . CH COOH . + ILO —> Arginin /NH. C^O -f I \NH., Harnstoff NH., NH.. CH., ( ! . CR, CH, . CH COOH . - CO., —> = Diamino= valeriansäure >rnithin Willstätter und . 7., [i W. Ileulmer: Her. d. Deutschen (hcm. Gcsellsch. 40. 8869 ') B. ') Vgl. Ernst Späth: Monatsli. f. (^lieraio. 40. 129 (1919); 42. 97 (1921). Vgl. liierzu K. Dreclisel : Her. d. Deutschen Chem. (iesellsch. 23. 309() (1S9()). /'irfrt: Pharrnaz. /oitg. Nr. Hh und 8() (19()ö). (1907). ') — Aime KivvoiL'istort'e und ilire 445 Bausteiue CH., NHs und Ringschlnß CH, .CH, .CH, .CH, . I I CH., Putresziii CH, \/^ NH NH.. NH., CH., = Tctra- PyrroHdiii. methylendiamin Pyrrolidin geht durch ( Kydation über HC — CH HC H..C- nCH CH. Pyrrol Avird durch Reduktion übergeführt in H, C CH NH NH Pvrrolin. Pyrrol. CH, . CH., in: . CH, CH, AU. COOH . CO., CH, .CH, .CH, .CH, .CH, NH, NH, Lysin ==7.. 2-Diaminocapron- NH, NH, Kadaverin := Penta- säure niethvlendiamin CH.. /\ H, C (^H., H.,C CH. Piperidin geht durch Oxydation über in: NH, und Ringschluß XXII. Vorlesung. 446 — in das Molekül des Alkaloids. Noch verbreiteter ist die usw. Kupplung mit einem Alkoholrest, und zwar verbinden sich mit ihm OH- oder NH-Gruppen. Bis jetzt ist nur der Methylrest (CH3) beobachtet worden. An seine Stelle kann auch das Methylenradikal treten. Fictet^) ist der Ansicht, daß der Formaldehyd das methylierende Agens in der Pflanze darstellt. Er stellt sich den \'organg, wie folgt, vor: chicin) R — OH + H R — NH 4- H . . C<j^ — C<}:J —^ R - N CH3 + O. R — O CH3 + 0. >- . . Es seien einige der bekanntesten und ihrer Struktur nach aufgeklärter Alkaloide hier angeführt ^i, um zu zeigen, wie eng in der Tat die Beziehungen zu den erwähnten Abkömmlingen von Aminosäuren sind. Das a-Coniin, eines der Alkaloide des Schierlings, Conium maculatum, ist in der gleichen Pflanze finden sich auch d-, a-, n-Propyl-piperidin. ') N-Methylconiin) und Conhydrin: CH2 Ho C GH., Ho C CH2 "i I . . I CH CH (OH) \N/ H CH CHo CHo CH3 \N/ Ho C CHo" 'i Ho C . H , . . CH., . CH, Conhydrin = Hydroxyconiin a-Coniin CHo /\' Ho C CHo CH CH,H CHo »^iA2 CH, ^»^8 \N/ HoC . . . CH3 Methylconiin. Das gemeinsame Vorkommen der so nah verwandten Verbindungen läßt vermuten, daß diese auseinander hervorgehen und vielleicht den Weg der Synthese oder des Abbaus anzeigen. Es sind zahlreiche Alkaloide bekannt, die sich auf den Pyridinkern zurückführen lassen. So ist das Arekaidin, ein Alkaloid der Arekanuß, N-Methyl-tetrahydronikotinsäure.ö) Auch sein Methylester, das 40. — Ber. d. Deutsch. Chem. Ges. ») A. Pictet: Pharm. Ztg. Nr. 85 uud 86 (1905). Vgl. auch J. Gadamer: Chem. Ztg. 35. 183 (1911). 3771 (1907). Berlin ^) Ernst Winterstein und Georg Trier: Die Alkaloide. Gebr. Bornträger. — 1910. A. Ladenburg: Ber. d. Deutschen Chem. Gesellsch. 39. 2486 (1906). Fasson: Ebenda. 24. 1678 (1891). A. Wohl und .1. Johnson: ») Jahns: Arch. d. Pharmazie. 229. 669 (1891). Ber. d. Deutscheu Chem. Gesellsch. 41. 131 (1908). 3) ) — Eiweißstotfe uud ihre Bausteiue. 447 Arekolin, kommt in der Arekanuß vor. Seine nahen Beziehungen zum Trigonellin zeigt die Gegenüberstellung der beiden Formeln: CH CH /\ HC C - CO H^C/XC.COOH HjC HC CH. CH N .0 CH3 Arekaidin = N-Methyltetrahydro- nikotinsäure CH3 MethylTrigonellin betain der Nikotinsäure = CH C CO OCH3 H2 C . . I I H, C CH. N.CH3 Arekolin = N~Methyl-tetrahydronikotinsäure-raethylester. Das Pyrrolidin kommt auch als solches im Tabak vor.') Es ist auch aus Mohrrübenblättern gewonnen worden: [CH, r . CH." I GHo GHo \NH/: . Eine Kombination des Pyridin- und Pyrrolidinkernes findet sich im Nikotin. Es ist ein a-Pyridyl-ß-N-methyl-pyrrolidin^): Pyrrolidinkeru [ Pyridinkern CH3 CH N /\ HC CHHC CH \N£ -CH CH. Nikotin NH CH, — CH") CHo CH.COOH CH2-CH, x-Pyrrolidonkarbonsäure=:Prolin. Dem Pyrrolidinkeru sind wir schon begegnet, als wir das Prolin und Oxyprolin kennen lernten. Wir haben ferner auf die nahen Beziehungen der Pyrrolidonkarbonsäure hingewiesen, die leicht unter Wasserund Court: Ber. d. Deutscheu Chem. Gesellsch. 40. 8771 (1907). Pictet und Rotschy Pinner: Ber. d. Deutschen Chem. Ges. 26. 294 (1893). Ehenda. 37. 1225 (1904). ) ^) Pictet — XXll. Vorlesung. 448 Es ist wohl möglich, daß die abspaltung aus Glutaminsäure entsteht. Pflanze diesen Weg zur Bildung des Pyrrolidinringes einschlägt. Es sei in diesem Zusammenhange auch noch einmal auf das .Stachydrin verwiesen, welches das Dimethvl betain des Prolins ist.^) Zahlreiche Alkaloide bilden die Angehörigen der Solanaceen. Auch diese Verbindungen zeigen Beziehungen zum Pyrrolidin. So kommt z. dem Atropin die folgende Struktur zu'-): CH. — CH GH., GH., /GH = GH. G< >GH. i ! I N.GH;, GH Atropin (> . (»G .Vn . ^GH— GH^ I i GH.,— GH 'dessen . )H ( . GH., einen stellt Alkohol dessen Ester dar, Säurekomponente die Tropasäure^'j das I — Vll — CH- OH ^\GH\gooh I N. GH, GH. OH GH. . G— GH GH.. I Tropin und ist: GH.3 GH., B. HG HG -GH., GH \/ GH Tropasäure. Tropin Von großem Interesse ist der Befund, daß das Hyoscyamin, das Zuin Hyoscyamus niger aufgefunden worden ist^), in seiner sammensetzung mit dem Atropin vollständig übereinstimmt. Auch es ist zuerst ein Troposäure-tro))incster. Bei der Spaltung erhält man jedoch eine optisch-aktive Tropasäure, während das Atropin eine inaktive (razemische) Tropasäure steht liefert."') Auch das Kokain, ein Alkaloid der Blätter von Erythroxylon coca^), dem Atropin sehr nahe: GH., — GH GH GO O . . GH., . GH =r GH N.GH3 G/V).oG.(^ GH. \'H GH.,-GH^^^GM^ GH Kokain. ') Vgl. S. 441. Vgl. A. Ladenl»ir(r. Bcr. Ji d. Dcutschoii Ch(>ni. Gcsollscli. 35. 1162 (1902). A. Ladcnbvrg iiiul Riigheiimr: Bcr. d. Dcutsciion Choiii. (lescUscli. 13. 373 (1880). Willstätter: Auualeii der Chemie. 326. 12. (1903). ) Geiger und Ih-s-sr: Aimalen dci- Cheniie. 217. 82 (1883). ) ./. Gadamer: Arcliiv der Pli;irin;i/.io. 239. 294, 321 (1901). Vgl. aiicli Cushuii: Uli. of. •-) ') - — «) Phjsiol. 30. 17(; (1903). Mnuaini: Aniiülen der Clieiiiie. 114. 218 (18()0). Eiweißstorte und ihre Bausteine. 449 Kokain ist der Methylester des benzoylierten Ecgonins.i) Zahlreich sind die Alkaloide, die sich vom Chinolin ableiten. Es steht in Beziehungen zum Indolkern und damit ohne Zweifel zum Tryptophan. Hierher gehören die zahlreichen Chinaalkaloide. Sie finden sich in den echten Chinarinden. Der wichtigste Vertreter dieser Klasse ist das Chinin. Es ist das p-Methoxyderivat des Cinchonins^): CH. = CH (^H . N CH TT^XV/'\p, HC tCH — Vn — CH. CH., HC —N CH CH C CH.. I CH. CH OH CH . Chinolinrest Cinchonin. GH., = CH CH - CH - CH., . CH., CH ^^/XVX^^^ CH C CH., CH«— CO. CH. HC -CH — I CH OH . Chinin. Auch die St rychnos alkaloide (Strychnin) gehören zur Gruppe der Chinolinalkaloide. Schließlich wollen wir noch einen der zahlreichen Vertreter der Klasse der Isochinoline erwähnen. Es gehören die sogenannten Opiumalkaloide hierher. Das Papaverin») hat folgende Konstitution): Isochinolingruppe CH CH 4Ö0 XXII. \'orlesinig. Diese Beispiele mögen geniigen, um zu zeigen, welch kompliziert gebaute Verbindungen die Pflanzen darzustellen vermögen. Überall stoßen wir auf direkte und indirekte Beziehungen zu Bausteinen der Proteine. Zu den Alkaloiden gehören eine große Zahl von pharmakologisch wichtigen Verbindungen. Viele derselben verdanken ihre Entdeckung dem Umstände, daß bestimmte Drogen eigenartige Wirkungen auf den tierischen Organismus ausüben. Die genauere Untersuchung ergab dann, daß diese Eigenschaft auf bestimmte; Verbindungen zurückzuführen ist. Über die Bedeutung der Alkaloide für die Pflanze läßt sich nichts Bestimmtes aussagen. Während die einen Forscher ihnen eine große Rolle in ihrem Stoffwechsel zuschreiben und sie den inneren Sekreten des tierischen Organismus an die Seite stellen, vermuten andere Autoren, daß die Alkaloide Produkte des Zellstoffwechsels darstellen, die für die Die Pflanze soll durch die Bildung Pflanze ohne weiteren Nutzen sind. der Alkaloide unbrauchbar gewordene Produkte dem Stoffwechsel entziehen. Das Tier scheidet seine Stoffwechselendprodukte aus, die Pflanze dagegen genötigt, die Abfallprodukte des Stoffwechsels in eine Form überzuWir können diese Ansicht nicht für führen, in der sie unschädlich sind. die richtige halten. Es wäre eine immerhin recht auffällige Erscheinung, wenn die Pflanzenzelle zur Unschädlichmachung von Stoffwechselendproist Weshalb wählt dukten einen so komplizierten Weg einschlagen würde. dann innerhalb einer bestimmten Klasse von Pflanzen die eine Art diesen und eine andere einen ganz anderen Weg? In der Tierreihe flnden wir in den wesentlichsten Punkten Übereinstimmung in der Art der Stoffwechselendprodukte. Es wäre auffallend, wenn die Pflanze ein und denWir sind selben Zweck auf so zahlreiche Arten zu erreichen versuchte. vielmehr der Anschauung, daß die Alkaloide im Pflanzenreich eine bestimmte Rolle innerhalb des Stoffwechsels spielen. Man hat sie auch als In der Tat werden viele Pflanzen, die Alkaloide Schutzstoffe betrachtet. enthalten, von manchen Tieren gemieden. Man darf nicht aus dem Umstände, daß der Schutz kein vollständiger ist, den Schluß ableiten, daß den Alkaloiden in dieser Richtung keine Bedeutung zukommt. Hls könnte auch sein, daß die Alkaloide bei der Assimilation und der Synthese; neuer Zellbestandteile eine Rolle spielen. Würden die Alkaloide wirklich Stoff- wechselendprodukte darstellen, dann müßten wir erwarten, daß sie in um so größerer Menge anzutreflen wären, je älter die Pflanzen sind. Das ist nun sicher nicht der Fall. Ja, manche Forscher schildern eine Abnahme des Alkaloidgehaltes mit dem Alter. In diesem Falle wäre bewiesen, daß die Alkaloide nicht nur Stoffvvechselendprodukte sein können. Sie scheinen Es ist auch ganz gut vielmehr wieder umgewandelt werden zu können. denkbar, daß die Pflanze ein und dasselbe Produkt mannigfachen Zwecken Es sei in dieser Beziehung an die Kohlensäure im dienstbar macht. Sie ist ein Stoffvvechselendprodukt. hat tierischen Organismus erinnert. aber dabei noch wichtige Aufgaben im Zellhaushalte zu erfüllen. Sie wirkt auf das Atemzentrum und trägt zur Regulation der .Atmung bei. z. B. Leider fehlen noch systematische Untersuchungen ül»er die Bcdingimgen, unter denen die Alkaloide entstehen. Es ist bekannt, daß manche Pflanzen unter bestimmten Verhältnis.sen diejenigen Alkaloide, die sie sonst bilden, Die Aufklärung der Bedeutung der Alkaloide für die nicht herstellen. Pflanzenwelt wird sicher wichtige Einblick«' in das üanzc Stoffwechsel- Kiwoilistnttc Bausteine. ihrL- iiiul 451 betriebe der Pflanze erörtnen. Schon ans diesem Grunde sind systematische Forschnngen über ihre Bildung anzustreben, um alle Vor- und Zwischenstufen bei der Entstehung eines bestimmten Alkaloides kennen zu lernen und gleichzeitig die Bedingungen zu erfahren, unter denen sie gebil(l<'t Kiii'xnq^'xi werden. )fi>. idl-^-iP. / Nahe Beziehungen' zu den Bmndstotfen mancher Alkaloide hat Öer Blattfarbstoft', das Chlorophyll. Er enthält Pyrrolringe. Der Beziehungen der Farbstoffe der Indolgruppe zum Tryptophan haben wir schon gedacht. Erwähnt sei, daß das aus den \Yurzeln des Sauerdorns, Berberis vulgaris, gewonnene Berberin, ein gelber Farbstoff, der Isochinolinreihe angehört. Seinen ganzen Eigenschaften und seiner Konstitution nach ist es ein Alkaloid. Gewiß wird die weitere Forschung die Beziehungen zwischen den einzelnen Gruppen von Verbindungen es jetzt schon der Fall noch enger knüpfen, viel als ist. Wir wollen nun wieder zu der wichtigen Frage nach jenen Umwandlungen von Aminosäuren zurückkehren, die durch direkte Versuche Sie sind geeignet, uns Fingerzeige über zur Beobachtung gelangt sind. die Art des Abbaus der Eiweißbausteine in unseren Körperzellen zu geben, wissen w4r doch, daß in den Grundzügen bei allen Zellartcn die Stoffwechselvorgänge gleichartig sind, während in P^inzelheiten jede Zellait Hervorgehoben sei, daß auch hier die Besonderheiten aufweisen kann. Fragestellung nie lauten darf, welcher Weg wird beim Abbau bestimmter Aminosäuren eingeschlagen, vielmehr müssen wir von Wegen sprechen, denn sicherlich vollzieht sich der Abbau je nachBedarf und jenachden vorhandenen Bedingungen verschieden. Hinweisen wollen wir noch auf die hohe Bedeutung, die die Feststellung der Art des Abbaus von Aminosäuren durch bestimmte Zellarten für deren Charakterisierung hat. Durch das Studium des gesamten Stoffwechsels der einzelnen Zellformen bis in alle Einzelheiten kommen wir zu einer Festlegung der einzelnen Zelltypen, wie sie uns keine noch so genaue morphologische Forschung vermitteln kann. Fragen der Konstitution, Vererbung usw. können auf dieser Basis vi«l schärfer erfaßt werden. Es liegt hier ein Forschungsgebiet von ganz außerordentlicher Bedeutung vor uns. Bestimmte Bakterien spalten aus Aminosäuren Kohlensäure ab. Man erhält dann immer das um einen Kohlenstoff" ärmere Amin.. Wir sind diesen Verbindungen schon wiederholt begegnet. ^) Einmal sind Amine als solche in höheren Pflanzen aufgefunden worden, und ferner flnden sich Methylderivate von solchen. Die folgende allgemeine Formel unterrichtet über diese Art des Abbaus von Aminosäuren: CH., .NH... COO H Es ist dies nicht die einzige Art der Aminbildung. Es kann auch und die Amingruppe durch Abspaltung von Reduktion eintreten Ameisensäure entstehen: eine R CH . . NH., I COOH ') Vffl. S. :-521 ff.. .J24. + ..„ H := Rj'. OHi NHo i , -J . . 4 H CüOH. . XXII. Vorlesung. 452 Beide Arten der Bildung von Aminen aus Aminosäuren sind beobachtet worden. Wir haben bereits die Bildung von Methylamin aus Glykokoll, von Isoamylamin aus Leuzin, von p-Oxyphenyläthylamin aus Tyrosin, von Imidazolyläthylamin aus Histidin, von Agmatin aus Arginin und von Kadaverin aus Lysin besprochen. Das p-Oxyphenyläthylamin kommt stets im Emmentaler Käse vor und entsteht ohne Zweifel aus Tyrosin. 1) Aus Valin geht Isobutylamin hervor 2): CH3 0H3 \/ CH Eiweiüstoffe und ihre Bausteine. CH HC 453 CH C —> — C CHo CH COOH . . . HC — C CH. CH., + C CO.,. . . " ' " ' II 1 HC I II CH C I II HC NHo CH NH II CH C NH. CH NH Asparaginsäuro liefert ^Alanin M und Glutaminsäure v-Aminobuttersäure^): COOH I CH.NHo COOH CO, •+ —y CH NR, .NR. " . ' ' * I I ! aCR, CR, r CH,' I aCH., r r ß-Alauiu " I CH, COOH CO(.)H Asparaginsäure r fiCR I COOH COOH —h vCR, .NR, 1 [iCH.. " CO2 + Glutamin- y-Aminobutter- säure säure. Die y-Aminobuttersäure hat noch dadurch ein besonderes Interesse weil der Nachweis geglückt ist, daß eine bei der Fäulnis von ein y-Trimethyl-butyro-betain ist.) Pferdefleisch sich bildende Substanz erlangt, =^) /CH3 CH, . NH., yCHa . , , CR, ßCH., CH, aCR, yCR, ^OH iCR COOH COOH /CH3 N^CHg ^CH3 y-Amino- y-Triraethyl- buttersäure butyro-betain I . N^CH., \CH3 xCR, CO Anhydridform, Die gleiche Verbindung wurde im Harn nach Phosphorvergiftung beobachtet. 5) Es sei gleich hier angefügt, daß ein aus Fleisch und Fleischextrakt gewonnenes Produkt, das Karnitin^), wahrscheinlich einem y-Tri- methyl-a-oxy butyro-betain entspricht"): ~ Emil Abderhalden uud ') D. Achcrmann: Zeitschr. f. Biologie. 56. 87 (1911). Andor Fodor: Ebenda. 85. 112 (19i;J). Emil AbderZeitschr. f. physiol. Chemie. 69. 273 (191U). «) IJ. Ackermann — Emil Abderhalden, Geortf halden und Karl Katifzsch: Ebenda. 81. 294 (1912). : — Fromme uud Paul Hirsch: Ebenda. 85. =•) ) 5) ) 131 (1918). L. Brie(/er: Ptomaiue. 3. 28 (188G). R. Engeland und Fr. Kutscher: Zeitschr. f. plivsiol. Chemie. 69. 282 (1910). K. Takeda: Pßügern Archiv. 1.S3. 365 (1910). W\ Guleicitsrh und R. Krittihcrg: Zeitschr. f. phvsiol. Chemie. 45. 826 (1905). — — Krtmben/: Ebenda. 48. 412 (iy(J6): 53. 514 (1907). ') R. Engeland: Bericht, d. Deutsch. Chem. Ges. 42. 2457 (1909); 43. 2708 1910. Adolf Rollett: Zeitschr. f. Emil Fischer und Ch. Göddertz: Ebenda. 43. 3272 (1910). R. phvsiol. Chemie. 69. 60 (1910). 404 •HiCH-^iiäLXIl. V'oi-lesuug. CH3 \() CH., CH OH . C()- Diese Betaine entsprechen in ihrer Entstehung dem Hordeh'in.^J Wie dieses aus Tyrosin bzw. dem p-üxyphenyläthylarain hervorgeht, bilden sie dem dieser entsprechenden y-Amino- sich offenbar aus CTlutaminsäure bzw. buttersäure. Die Aminosäuren können auch so abgebaut werden, daß unter Al)spaltung der Aminogruppe Fettsäuren mit der dem Ausgangsmaterial gleichen Anzahl von Kohlenstoffatomen entstehen. Dieser Vorgang vollzieht sich unter Reduktion.- K CH CUCH + 2 H = K . . iriv.'V) NH., . CH., COUH + NH3. . ; -'mmmh;;-; ::•;•/. - < Wahrscheinlich verläuft dieser Abbau stufenweise, .1,^ .. ?H?:, ,- (J.'juii ij;iJ r>hf9l^'o v-}. ( (! fl ^ fr etwa, wie folgt: ninyr'.i'niiiooaiiak-y 'iO w.. .Mi'w-y -.r,i ff4/ .1-.,..., R.ÜHCOOH + H.,0 — NH3 —> ß^GHaCGOH + 2H — H^0f^>^ IJ CH, COOH. j[y Die auö^en einzelnen Aminosäuren sich bildenden Fettsäuren ergeben . . ohne weiteres aus ihrer Konstitution. Glykokoll liefert Essigsäure, Alanin Propionsäure, Valin J sovaleriansäure, Leuzin Isobutylessigsäure, Isoleuzin 3lethyl-äthyl-propionsäure-), Phenylalanin Phenylpropionsäure ). Tyrosin p-Oxyphcnyl-propionsäure), Tryptophan Indolpropionsäure.^i Histidin geht in Imidazolylpropionsäure'M über und aus Arginin bzw. Ornithin kann f^-Aminovaleriansänre^) erhalten werden. Asparaginsäuro liefert Hernsteinsäure.'') Aus Glutaminsäure dagegen entsteht nicht die zu erwartende Glutarsäure, sondern hauptsächlich Bernstein säure.'') Ihre Bildung erfordert ohne Zweifel mehrere Vorgänge. Man kann sich vorstellen, daij Kohlensäure und Ammoniak abgespalten werden und dann eine Oxydation ei'folgt: sich • 1) Vgl. ^) Carl NiHbcrq und ») SeNfrenni/: Mduatsli. f. Chemie'. 10. 908 (188i»)E. linnmtmn: lier. d. Deutsch. Chein. Ces. 12. 1401! (1879): /''. (x. Hopkins uud Cole: .louni. of Physiol. 29. 4öl (19üaj. ) 5) S. 443. F.. Hoseuberq: Bidclieiii. Zcitsclir. 7. ]'J9 (li)U7). 13. j;79 (ISSO) Ackermann Zeitschr. f. physiol. Chemie. 65. 508 (1910). : und //. Salkorski: Ber. d." Deutsch. Chem. (ies. 16. 1191 (1883): Hl. 77<i D. Ackfinnann: Zeitschr. f. physiol. Chemie. 60. 482 (1919); 69. 'J73 (1910). <1898). ) Hoppe-Sri/Ier: Zeitschr. f. physiol. Cheniie. 1. 213 (1877). Car/ Neiihrrf/ um! ('. Cappezznoli: Biocheni. Zeitschr. Eiml Abdiirhalde» und Andor 18. 424 (1909). ^) I). ') K. — - — Foilor: Zeitsciir. ") /y. 18 f. physiol. Chemie. 85. WnUher Hrd^ch und C. 112 (1913). Nri(/>('.r(/: :',sn (1909). — "'. — 13. 299 (1908). lirasch : Biuchem. Zeitschr. Biochom. Zeitschr. liorchurdt: Zeitschr, t. physiol. Ghemie. 59. 97 (1909). Eiweißstnftc und ihre Baiistpine. COO H CH 1 . 455 CHo . NH2 Eiweißstoffe und ihre Bausteine. C . CH. / \ . —> CH C'OOH . C I Ce H, CH . CH, /H,\ ' . CH,, 457 . COOH —y " CH ^NH^ NH, Ce ^IsH^ Try^jtophaD = lndol-a- Indolpropioii säure aminopropionsänre C CHo . x\CH . —^ COOH C CH, . /\CH —y CH /\ Cg H4 Cß H4 Cg H4 CH ^NH/' Indolessigsäure ^NH-^ Skatol Indol. ^NH^ Yjü unterliegt keinem Zweifel, daß diese Abbaustuien nicht direkt aus einander hervorgehen. Es sind vielmehr noch mehrere Zwischenstufen vorhanden. So ist z. B. als Übergangsstufe von p-Kresol zu Phenol die p-Oxybenzoesäure, Cg H4 (OH) COOH, angenommen worden. Wahrscheinlich kommen zum Teil auch Zwischenstufen ganz anderer Art vor. Sicher folgen sich die verschiedenartigsten Vorgänge in buntem Wechsel. Vor allem sind es Reduktions- und Oxydationsvorgänge, die den Abbau der einzelnen Verbindungen herbeiführen. Wir kennen noch nicht von allen Aminosäuren die Abbaustufen. So wissen wir z. ß. vom Zystin nur, daß Es erfolgt auch es u. a. schließlich Schwefelwasserstoff liefern kann. hier der Abbau über mehrere Stufen. Der Abbau der Aminosäuren durch Bakterien ist vielfach als ein Fäulnisvorgang bezeichnet worden. Die ihn bewirkenden Lebewesen werden Fäulnisbakterien und die entstehenden Verbindungen Fäulnisprodukte genannt. 1) Diese Bezeichnungen sind nicht mehr haltbar und werden besser ganz vermieden. Zahlreiche sogenannte Fäulnisprodukte entstehen ohne jede ,,Fäulnis", d. h. es brauchen nicht immer Produkte gebildet zu werden, die sich durch den Geruch als „faulig"' erweisen.-) Dazu konmit. daß viele von ihnen auch ohne Bakterien entstehen und z. B. Stoffwechselzwischenprodukte im Organismus höherer Pflanzen und Tiere sind. Es ist deshalb besser, ganz allgemein vom Abbau bestimmter Aminosäuren durch Bakterien zu sprechen. In Zukunft wird ohne Zweifel das Hauptgewicht auf das Studium des Abbaus einzelner Aminosäuren durch ganz bestimmte Bakterienarten gelegt werden. Die Art des Abbaus ist für manche Lebewesen ganz charakteristisch. 3 Ein sehr schönes Beispiel dafür, daß verschiedene Zellarten bestimmte Aminosäuren in besonderer Weise abbauen, liefern die wichtigen Beobachtungen von Felix Ehrlich) über die Spaltung solcher Verbindungen . ») E. und H. Salkowski: Ber. d. Deutschen Ghem. Ges. 13. 191, 2217 (1880); Zeitschr. M. Nencki: Ber. d. Deutscheu Chem. Ges. 8. 356 (187.T). physiol. Chem. 9. 8 (1884). ^) D. Ackermann und F. Kutscher haben den Namen Aporrhegme n eingeführt. Es scheint mir eine solche Bezeichnung nicht zweckmäßig, weil sie Produkte zusammen- — f. faßt, die eine ganz verschiedene Herkunft haben können. Vgl. weitere Literatur bei Marceli Nencki: Opera omnia. 1. 1)2, 113, 144.244, 24ß ff., 174, 537 usw. L. Brieger: Die Ptomaiue. Berlin 1886. ) F. Ehrlich: Ber. d. Deutschen Chem. Ges. 39. 4072 (lüOül; 40. 1U27. 2538 (1907); 44. 139 (1911); Zeitschr. d. Vereins f. Deutsche Zuckerindustrie. 55. 539 (1905); Biochem. Zeitschr. 1. 8 (1906): 2. .52(1908); 18.391 (1909); Breslauer Chem. Gesellsch. ^) 11. Februar 1910. — XXII. Vorlesung. 458 durch Hefezellen. Man hatte allgemein angenommen, daß die Fuselöle, die durch Hefe herbeigeführten Gärung auftreten, auf Kohlehydrate zurückzuführen seien. Ehrlich stellte fest, daß nicht diese sondern bestimmte Aminosäuren in Alkohol übergeführt werden. Man hat von einer ..alkoholischen Gärung" der Aminosäuren gesprochen. Die aus den einzelnen Aminosäuren entstehenden Alkohole ergeben sich aus dieser allgemeinen Formel von selbst: d-Valin liefert Isobutylbei der alkohol, 1-Leuzin Isoamylalkohol, Isoleuzin d-Amylalkohol, 1-Phenylalanin Phenyläthylalkohol Phenosol, Tyrosin p-Oxyphenyläthylalkohol. auch Tyrosol genannt, Histidin Imidazolyläthylalkohol Hystol und Tryptophan Indoläthylalkohol Tryptophol. Es sei die Bildung des zugehörigen Alkohols ohne Rücksicht auf die Zwischenstufen am Beispiel des Lcuzins und Tyrosins zur Darstellung gebracht: = = = ^y'>CH . CH., .CH . C( )()11 + H, ( = ch'/^'^ ^^-^ ^'^^ OH + CO., + NH3 > • • • NH., Leuzin Cfi Hi (OH) . CH, . Isoamylalkohol. CH C( . )( )H + H., < ) = C, HJOH). CR, CH, . . ( )H + CD, + NH3 'I NH2 Tyrosin = p-()xyphenylx-aminopropion säure p-()xyphenyl-äthylalkohol = Tyrosol. Es entsteht somit immer der um ein Kohlenstoffatom ärmere Alkohol. man die Hefe auf razemische Aminosäuren einvvirken. dann wandelt sie hauptsächlich und vielleicht ausschließlich jene optisch-aktive Komponente um, die in der Natur vorkommt. Von d, 1-Leuzin wird z. B. nur 1-Leuzin in Isoamylalkohol übergeführt, die d-Komponente bleibt unverändert zurück. Wie wir schon früher erwähnt haben, werden nicht nur die Aminosäuren in die um einen Kohlenstoff ärmeren Alkohole übergeführt, sondern es werden die gleichen Alkohole auch aus den Aminen gebildet.') Diese Läßt Umwandlung läßt sich, wie folgt, darstellen: R CR, NH, + H., . . ( » =R . GH., . ()H + Nil,. p-( •xyphenyläthylamin liefert Tyrosol und Isoamyiamin Isoalkohol. Vom Alkohol amvlalkohol. Alkohf geben sich Beziehungen über den Aldehyd zur entsprechenden Säure-): R CH., .011 . —y R . C<j{ —^ R C<[ Ijj . Eine weitere Möglichkeit des Abbaues von Aminosäuren und d(;r Bildung von Alkohol ergibt sich aus der Beobachtung, daß gewisse Schimmel})ilze, z. B. Oidiiim lactis, aus Aminosäuren unter geeigneten Bedingungen optisch-aktive Oxysäuren bilden. Diese l mwandlung erfolgt nach der folgenden allgemeinen Gleichung: ') '^) Felix Ehrlich und I'. Fistsrhiwnka : Bor. d. DfiitschcMi Clieiii. (ios. 45. ]()l)(3(1912). auch M. (lU/zgenheim und H'ilh. Löf/'lcr: Bioclieni. Zeitschr. 72. 325 (19lt)) V'trl. Eiweißstoftc und ihre Bausteine. R . C'H 459 COOH + H., () = R (H COOH + NH^ . . " . ,1 1 OH NHs Aminosäure Oxysäure. So liefert 1-Tyrosin d-p-Oxyphenyl-milchsäure, d-l-Phenylalanin d-Phenyl-milehsäure und 1-Tryptophan l-Indol-niilchiäure. In allen ist die Seitenkette der aromatischen Aminosäuren, der Alaninden Milchsäurerest übergeführt worden \): drei Fällen in rest, R CH2 CH . . . ('( M Hl — R CHo >- . i . eil . C("()H 1 Oll XH.. Am inopropion Säurerest Milchsäurerest. Es könnten nun diese Oxysänren unter Kohlensäureabspaltung in Alkohol übergehen. Es würde sich in diesem Falle die Umwandlung der Aminosäuren in den um einen Kohlenstoff ärmeren Alkohol, wie folgt, vollziehen R CH COOH + . 11-, U —y R. Clh Olli. COOH— CO., —> R. CIL. oll I NH., Oxysäure Aminosäure Alkohol. EhrHch stellt sich als weitere Zwischenstufe bei der Umwandlung der Oxysäure in den Alkohol die Bildung eines Aldehyds unter Abspaltung von Ameisensäure vor. Diese müßte dann in Kohlensäure und Wasserstoff zerfallen, wobei der letztere den Aldehyd zum Alkohol reduzieren könnte: R CH (OH) COOH . . Oxvsäure —> R. ^ ^^ --^ X\n. Vorlesung. 460 Nun ist allerdings die Phenylaminoessigsäure eine Verbindung, die der Natur noch nicht aufgefunden worden ist. Daß jedoch hier ohne Zweifel nicht ein durch die Struktur der Verbindung bedingter eigenartiger in Abbau vorliegt, zeigt die Beobachtung, daß Hefe p-Oxyphenyl-milchsäure nicht oder doch nur in geringen Mengen weiter verarbeitet, während die entsprechende Ketonsäure, die p-Oxyphenyl-brenztraubensäure, leicht in p-Oxyphenyläthylalkohol übergeführt wird. Aus diesen Beobachtungen ergibt sich der folgende Weg des Abbaus einer Aminosäure zum Alkohol: OH R.CH.COOH und Eiweißstoft'e 461 Amins und nachfolgende Desaminierung Bildung des 2. ihre Bausteine. durch Hydrolyse: R CH, . (^H . COOH — CO, —y . NH., R c;H, . — NH3 —> CH, + H2 U . NH., R CH, CH, OH. . . . o. Hydrolytische Desaminierung unter Bildung von Oxysäuren und nachträgliche Abspaltung der Kohlensäure: R . CH., . CH., . . CH., . . CH COOH + H., . ( ) NH., R CH COOH — CO, . — NH, —> — >- OH R (^H., . OH. Desaniinierung unter Bildung von Keton4. Oxydative säuren und nachträgliche Abspaltung der Kohlensäure: R CH, CH (^OOH + . . . —y ( ) NH., R CH., CO COOH + 2H — CO., R CH, CH, OH. . . . . . —> . 5. Bildung von Fettsäuren durch Reduktion unter Abspaltung der NH2-Gruppe (reduktive Desaminierung): R . CH, CH . COOH -f 2 H —y R CH, CH, COOH + . . . NH3. NH, Kombination verschiedener Vorgänge mit Oxvdationen, 6. z. B.: R CH., . . CH COOH + 2 H - NH, . NH, R CH, CH, COOH + 3 0~-C0,— H, . . . —> — >- R. C^H, . COOH. Wir werden bei der Besprechung des Abbaues der Aminosäuren im tierischen Organismus auf die gleichen Fragestellungen stoßen. Wir werden denselben Wegen der Zerlegung dieser Verbindungen begegnen und erkennen, daß im Pflanzen- und Tierreich zahlreiche Zellvorgänge den gleichen Verlauf zeigen. Das Studium des Abbaues der Aminosäuren hat als allgemein wichtiges Ergebnis klargelegt, daß auch hier kein plötzlicher Zerfall bis zu den Stott'vvechselendprodukten eintritt. Der Abbau erfolgt vielmehr stufenweise. Jedes einzelne Zwischeni)rodukt kann den Ausgangspunkt zu mannigfaltigen Synthesen abgeben. Vielleicht führen alle XXII. Vorlesung. 462 Wege, die von den Aminosäuren zu einfacheren Verbindungen weisen, auch zu diesen zurück. Vielleicht ist jeder einzelne Vorgang umkehrbar! Es spricht sehr vieles dafür, daß auch die höher organisierten Pflanzenorganismen Aminosäuren in der geschilderten Weise abbauen können. Es ist in dieser Richtung von großem Interesse, daß die Rosaceen Phenyläthylalkohol unter ihren Riechstoffen enthalten. \) Auf das Vorkommen von verschiedenen Aminen im Mutterkorn usw. haben wir schon hingewiesen. 2) Hervorgehoben sei noch die Möglichkeit der Bildung von Aminoäthylalkohol Oxyäthylaminaus Serin durch Kohlensäureabspaltung \): = CH., . OH + CO., KiwcißstottV iiiul ihre Bausteine. 468 Es besteht für uns kein Zweifel, daß man von den verschiedeneu Abbaustufen der Aminosäuren aus Brücken auffinden wird, die zu allen möglichen, für die Pflanzen charakteristischen \erbindungen, wie zu der großen Klasse der Terpene. den Bestandteilen der ätherischen Öle, und der Gruppe der mannigfaltigen Farbstoffe führen. Durch Kombination der unter den Abbaustufen auftretenden Amine, Aldehyde und Alkohole mit allen möglichen von der Pflanze erzeugten Verbindungen la'fesen sich jetzt schon Zusammenhänge aller Art vermuten, doch kommt solchen Überlegungen so lange keine entscheidende Bedeutung zu, als nicht durch den Versuch entschieden werden kann, ob sie sich in die Wirklichkeit umsetzen lassen. Leider ist die höher organisierte Pflanze bis jetzt noch sehr wenig Versuchsobjekt bei Untersuchungen über den Zellstoft'wechsel gewesen. Es klaffen überall noch weite Lücken Ungezählte Probleme harren der Lösung. Die engen Beziehungen, die zwischen dem Tierreich und der Pflanzenwelt bestehen, lassen erhoffen, daß von beiden Seiten aus durch weitere Forschungen Anregungen hinüber und herüber sich ergeben werden. Schon die jetzt vorliegenden Ergebnisse zeigen deutlich, daß ein volles Verständnis des Zellstoft'wechsels nur möglich ist, w^enn Pflanzen- und Tierzelle zusammen betrachtet werden. Die Pflanze liefert dem Tier Energie und organische Verbindungen mit bestimmter Struktur. Wir müssen erfahren, wie sie entstehen, und was die Pflanze selbst mit ihnen macht, sollen wir in jedem einzelnen Falle den aus ihnen hervorgehenden Produkten über alle Zwischenstufen folgen können. Vor allen Dingen sind nur von einer sehr breiten Basis aus vergleichende Studien über den Zellstoffwechsel bei Pflanze und Tier möglich. Ähnlichen oder gleichen Vorgängen entsprechen sehr wahrscheinlich auch verwandte Züge im Zellaufbau und in den Zellfunktionen. Es gibt keine reizvollere Aufgabe, als zu erforschen, in welchen Punkten Pflanze und Tier sich gleichen, und in welchen sie ! sich unterscheiden. Vorlesung XXIII. Eiweißstoffe und ihre Bausteine. Verhalten der Eiweißstoffe im tierischen Organismus. Ihr Abbau im Magendarmkanal. Der tierische Organismus nimmt mit seiner Nahrung beständig Eidoch aus mehr oder weniger veränderten Zellen, die alle Proteine enthalten. Daneben sind in sehr geringen Mengen auch Abbaustnfen, wie Peptone und Aminosäuren, vorhanden. »Sie sind teils auf Stoffwechselvorgänge in den betrettenden Geweben zurückzuführen, zum Teil entstehen sie postmortal durch sogenannte au toly tische Vorgänge. Wir werden bald erfahren, daß a;lle Zellarten über Fermente verfügen, Während in der die Eiweiß bis zu seinen Bausteinen abbauen können. lebenden Zelle die einzelnen Fermente nur nach Bedarf ihre Wirkung entfalten, beginnen sie in der toten Zelle mit der Änderung der physikalischen Bedingungen des Zellinhaltes regellos ihre Tätigkeit zu entfalten. Dabei kommt es unter anderem auch zur Bildung von Eiweißabbaustufen. Die Zellfermente der Nahrung können auch im Verdauungskanal noch weiter wirken, solange sich Bedingungen finden, die ihrer Wirksamkeit nicht Halt gebieten. Für uns kommen sie nur insoweit in Betracht, als weiß auf, besteht sie wir Nahrungsmittel nicht in gekochter oder sonst künstlich veränderter Form aufnehmen. Die Fermente vertragen keine höheren Temperaturen. Kochen vernichtet ihre Wirksamkeit. Auch sonst sind sie gegenüber verschiedenen Einwirkungen, wie Veränderung der Reaktion sehr empfindlich. Nur beim Pflanzenfresser kommt den proteolytischen Zellfermenten eine die Verdauung in größerem Umfange unterstützende Bedeutung zu '), weil die Pflanzenzellen nicht so rasch von den Verdauungssäften durchdrungen werden, wie es beim Fleisch der Fall ist, und ferner die Herbivoren vielfach Vorrichtungen besitzen, in denen die zerkaute Speise längere Zeit bei Körpertemperatur aufbewahit werden kann, und zwar unter Bedingungen, die der Wirkung jener Fermente günstig sind. Es sei z. B. an die Einrichtungen des Wiederkäuermagens erinnert. 2) Der Pflanzenfresser nimmt mit seiner — liergmann: Ebenda Ellenberyer : Skand. Aich. f. Thysiol. 18. 306(1906). l'. W. Grimmer: Biochein. Zeitschr. 4. 80 (1907). Hans Aron und faul Klempin: Ebenda. 9. KJS (1908). •) 18. 119 -) ('1906). — Vgl. S. 100. — Kiweißstoffe iiud ihre Bausteine. 46,") besonders, wenn er Produkte verzehrt, die mit Reservestoffen beladen sind, wie Knollen, Zwiebeln, Samen, neben Eiweiß und geringen Mengen von Eiweißabbaustufen mehr oder weniger große Mengen von Asparagin und Glutamin Säureamiden aufJ) Beim Fleischfresser spielen derartige Verbindungen keine Rolle bei der Ernährung. Der tierische Organismus nimmt somit die zur Klasse der Eiweißstoffe gehörenden Verbindungen fast ausschließlich in Form von EiweiLi auf. Es gilt dies besonders für die Karnivoren und auch die Omnivoren, sofern diese ihre Nahrung hauptsächlich aus der Tierwelt beziehen. Zunächst kommen die Eiweißstoffe der Nahrung in der Mundhöhle mit dem Sekret der Speicheldrüsen in Berührung. Der Speichel enthält keine auf Eiweiß eingestellten Fermente. Die Verdauung der Proteine beginnt erst im Magen. Hier finden sich proteolytische Fermente. Ihre Wirkung wird durch gründliches Zerkauen der Speise in der Mundhöhle sehr begünstigt. Je kleiner die Partikelchen der Nahrung in den Magen kommen, um so mehr Angriffspunkte finden die Fermente. Besonders die Pflanzennahrung bedarf einer ausgiebigen Zerkleinerung, damit der Inhalt der einzelnen Zellen freigelegt wird. Sehr günstig wird die Verdauung der Proteine beim Pflanzenfresser auch durch die vorausgehende Mazeration der Pflanzenteile in den wiederholt erwähnten besonderen Abschnitten des Anfangsdarms beeinflußt. Die Erweichung der starren Hüllen der Pflanzenzellen unter dem Einfluß feuchter Wärme befördert die Auflösung der Gewebe in feinste Teilchen. Während man z. B. im Wiederkäuermagen im Inhalt des Pansens noch leicht die aufgenommenen Gewebe Hahne, Blätter usw. erkennen kann, flndet man schon im Blättermagen ein fast homogenes Gemisch feinster Teilchen, das makroskopisch nur schwer noch Besonderheiten unterscheiden läßt. Im Magen unterliegen die Eiweißstofte der Einwirkung des sauren Magensaftes. In ihm findet sich ein Ferment, Pepsin genannt, das Proteine in Peptone zerlegt. Das Pepsin wird von Zellen (Hauptzellen bestimmter Drüsen der Magenschleimhaut in Form einer unwirksamen Vorstufe, dem Pepsinzy mögen, auch Propepsin genannt, abgesondert.2) Die Überführung in das aktive Ferment besorgt die Salzsäure. Sie ist der Aktivator des zymogenen Zustandes des Pepsins. Pepsin entfaltet nur in saurer Reaktion seine Wirkung. Alkalische Reaktion vernichtet sie. Die saure Reaktion braucht nicht durch Salzsäure bedingt zu sein, andere Säuren, wie Oxalsäure, Milchsäure, Äpfelsäure usw.^), können die Salzsäure vertreten. Trotz vielfacher Bemühungen ist es noch nicht geglückt, die Wirkung des Pepsins auf Eiweiß und vor allem die Bedeutung der Säure vollständig klarzulegen. Nach den einen Autoren wirkt das Pepsin nur dann, wenn freie Säure zugegen ist), nach anderen Nahrnng, — — — — ^) Vgl. hierzu vor allem die Arbeiten von E. Schulze: Chem. 47. 507 (1906); Laudwirtsch. Jahrb. 35. 261 z. B. Zeitschr. f. physiol. (1906). ^) J. N. Lanfflei/: Journ. of. Physiol. 3. 269 (1881). ./. .V. Langley und Edkins: Ebenda. 7. 371 (1896). Vgl. auch Chapoteaut: Compt. rend. de l'Acad. des Sciences. 94. 1722 (1882). Födivissotzki: Pßügers Archiv. 39. 62 (1882). — — — Daridsohn und Dieterich: Arch. f. (Anat. u.) Physiol. 690 (1860). ) Huppert und Schätz: Pßügern Archiv. 80. 470 (1900). A. Müller: Deutsches Arch. f. kliu. Med. 94. 27 (1908). //. Davidson: Zeitschr. f. Kinderheilk. 2. L. Tobler: Ebenda. 5. 85 (1912). 420 (1911); 5. 94 (1912). B. Salge: Ebenda. 5. 111 (1912). G. Edivald: Deutsches Arch. f. klin. Med. 106. 498 (1912). Vgl. auch Ed. Zunz: Hofmeisters Beitr. 2. 435 (1902). ^) — — — Abderhalden, Physiologische Chemie. I. Teil, 5. Ana. — - — aQ XXIII. Vorlesung. 466 genügt es, wenn soviel Salzsäure vorhanden ist, um das Eiweiß zu binden. Andere Forscher dagegen Es bilden sich sogenannte Azidalbumine. l>etonen, daß die Bedeutung der Säure darin liege, daß sie daß Eiweiß zum Quellen bringt und dadurch für die Wirkung des Pepsins vorbereitend wirkt. i) l'>s ist sehr leicht möglich, daß verschiedene Momente zusammenwirken. Schon die ersten genaueren Beobachtungen über die Wirkung des Magensaftes auf Eiweiß ergaben, daß er imstande ist, koaguliertes Eiweiß zu verflüssigen bzw. in Produkte überzuführen, die in Wasser löslich sind. Beaumur hat wohl zuerst systematische Untersuchungen über die Bedeutung des Magensekretes angestellt. Er füllte Metallröhren mit Nahrung und ließ diese von einem zahmen Bussard verschlucken. Das eine Ende des Rohres war verschlossen, (las andere mit Mull bedeckt. Der Bussard bricht unverdauliche Produkte, wie Federn, Haare, Knochen usw., nach einiger Zeit aus. Auch die Metallröhren wurden nach einigem Verweilen im Magen nach außen befördert. Es zeigte sich, daß ihr Inhalt je nach der Dauer des Verweilens im Magen mehr oder weniger verflüssigt war. An Stelle von Nahrung brachte Reaumur bei späteren Versuchen Schwamm in das Metallrohr. Dieser sollte die verdauende Flüssigkeit aufsaugen. Reaumur'^) hofl'te durch -Vuspressen des Schwammes eine Flüssigkeit gewinnen zu können, mit der sich im Reagenzglas die Magensaftwirkung nachahmen ließ. Die Versuche verliefen jedoch ergebnislos. Stevens^) der ähnliche Versuche wie -ßc%Mwwr an einem Menschen anstellte, war erfolgreicher. Er konnte ferner Magensaft vom Hunde gewinnen und zeigen, daß dieser auch außerhalb des Organismus Fleisch auflöst. SpaUanzani) erweiterte diese Beobachtungen und bewies (mdgültig, daß es gelingt, den Verdauungsvorgang im Magen im Reagenzglas mittels des Sekretes der Magendrüsen nachzuahmen. Die Entdeckung des Pepsins verdanken wir Schwann^) und diejenige der freien Salzsäure im Magensaft Bidder und Schmidt^), nachdem schon vorher Tiedeniann und Gmelin') die Salzsäure als solche erkannt hatten. Es stehen zum Studium der Einwirkung des Sekretes der MagenWir können drüsen auf Eiweiß verschiedene Methoden zur Verfügung. einmal bestimmte Eiweißarten oder Gemische von solchen bestimmten Tieren verfüttern und feststellen, was aus ihnen geworden ist. Der Mageninhalt läßt sich entweder durch Aushebern gewinnen, oder es werden die VerDer suchstiere eine bestimmte Zeit nach erfolgter Fütterung getötet. Magen wird dann rasch abgebunden und sein Inhalt entleert.«) Endlich können wir Magenfisteln anlegen und aus der Fistelöflfnung den Inhalt des Magens von Zeit zu Zeit ablassen. ) ^) \gl. hierzu auch Wo. Ostwald und A.Kuhn: Kolloid. Zeitschr. 30. 234 (1922). Reaumur: M6m. de l'Acad. des Sciences. 26ß, 461 (1752). Stevens: De alimentorum concoctione. Edinburgh 1777. SpaUanzani: Expöriences sur la digestion de l'homme et de diff^rentes ospeces d'animaiix. Geneve 1783; Versuche über das Verdauungsgeschäft. Deutsch von ^) ) Michaelis. Leipzig 1785. — ) Schwann: Müllers Archiv. 90 (1836). ,Vgl. auch Eberle : Physiologie der W. Beaumont: Verdauung auf natürlichem und künstlichem Wege. Würzburg 1834. Neue Versuche und Beobachtuugeu über den Magensaft und die Physiologie der Verdauung. Deutsch von H. Luden. Leipzig 1834. ^)Bidder u. Schmidt: Die Verdauungssäfte u. der Stoffwechsel. Mitau u. Leipzig 1852. ') Tiedemann und Gmelin: Verdauung nach Versuchen. Heidelberg 1826. ) Vgl. hierzu u. A. Emil Abderhalden : Zeitschr. f. physiol. (jhem. 44. 17 (IBO-^l. — Eisveißstoff'e und ihre Baiisteinp. 4ß7 Die Untersuchung des Mageninhaltes ergab, daß die Proteine zunächst größere Mengen von Salzsäure binden. Sie quellen auf und zeigen bald ganz neue Eigenschaften. Sie werden in Wasser löslich. Es beruht diese Eigenschaft auf einer Spaltung der Proteine in einfachere Bruchstücke. Es bilden sich Peptone. Zunächst erscheinen viele Peptone, die sich aussalzen lassen, später nimmt die Menge jener Produkte zu, die beim Zufügen von Neutralsalzen in Lösung bleiben, i) Es hat sich ergeben, daß offenbar der Eiweißabbau durch Pepsin nicht bei allen Tieren gleichartig verläuft. Auch scheinen verschiedene Pepsinarten vorzukommen, doch sind unsere Kenntnisse über die Art des Eiweißabbaues durch Pepsin zurzeit noch so ungenügende, daß wir nicht in der Lage sind, anzugeben, worauf diese Unterschiede beruhen. Die Zerlegung der Proteine durch Pepsin in dialysierbare, in Wasser Abbauprodukte ist frühzeitig erkannt worden. Fraglich blieb nur, wieweit der Abbau geht. Entstehen Aminosäuren? Diese Frage wurde zunächst verschieden beantwortet. Einige Forscher wiesen solche im Chymus des Magens nach, andere vermißten sie vollständig.-) Die verschiedenen Resultate erklären sich durch folgende Momente. Einmal muß bei der Frage nach der Entstehung von Aminosäuren unter der Einwirkung des Magensaftes entweder ausgeschlossen werden, daß die zugeführte Nahrung schon solche enthält oder, falls sich Aminosäuren finden, so müssen diese ihrer Menge nach bestimmt und von den dann im Magen aufgefundenen abgezogen werden. Ferner muß darauf geachtet werden, daß nicht Aminosäuren aus dem Duodenum in den Magen gelangen. Wir haben bereits bei der Besprechung der Verdauung der Fette festgestellt'), daß bei fettreicher Nahrung häufig der Fall eintritt, daß Inhalt des Duodenums in den Magen zurückfließt. In diesem Falle können natürlich auch in jenem Darmabschnitt gebildete Abbaustufen aus Eiweiß im Magerinhalt erscheinen. Außerdem können, wenn die Reaktion des Duodenalinhaltes nicht sogleich sauer wird, die proteolytischen Fermente des Darm- und Pankreassaftes im Magen weiter wirken und Eiweiß und Peptone spalten. Schließlich muß die zum Nachweis der Aminosäuren angewandte Methode so beschatfen sein, daß nicht solche sekundär aus Peptonen gebildet werden können. lösliche Die unter Ausschluß dieser Fehlerquellen durchgeführten Versuche' haben ergeben, daß bei der Verdauung im Magen keine Amino- säuren aus den Proteinen und Peptonen abgespalten werden. Die Zerlegung der Eiweißmolekttle führt zur Bildung einfacherer Bruchstücke, die immer noch mehrere Aminosäuren gebunden enthalten. Der ') Vgl. hierzu die systematischen Untersuch uiijreu von Edgar Zun:: z. B. Bull. de TAcad. royale de m^d. de Belsrique. 30. April 1910; 27. April 1912: Internat. Beitr. z. Path. u. Ther. d. Ernährungsstonuigen. 2. H. 3. u. 4 (1910). In diesen Arheiteu liudeu sich Literaturnachweise. 2) E. Zunz: Hofmeisters Beitr. 3. 339 (1902). Emil Abderhalden: Zeitschr. f. plivsiol. Chem. 44. 17. (1905). Vtrl. ferner E. Zunz: Annales de la Soc. royale des Sciences med. et naturelles de Bru.xelles. 12. Fase. 3 (1903), 13. Fase. 8 (1904); Bull, de TAcad. royale de mM. de Belgi(iue. 19. Fase. 3 (1906), 28^ Juni (1913). Leo Langstein: Jahrh. f. Kinderheilkunde. N. F. 54. 139 (1906). Emil Abderhalden. L. Baumann, Karl Kautzsch, Kornel i\ Körest/ und E. S. London: Zeitschr. f. phvsiol. Chem. 48. 549 (1906): 51. 384 (1907); 53. 148 "(1907). — — — =) — Vgl. hierzu S. 258. 30=^ XXIII. Vorlesung. 468 Abbau kaim ohne Zweifel sehr weit gehen. Es lassen sich neben Peptonen, die die Biuretreaktion geben, auch solche abtrennen, die mit Lauge und Kupfersulfat die genannte Reaktion nicht zeigen. Man hat derartige Produkte auch abiurete genannt. Auch sie enthalten mehrere Bausteine gebunden. Es scheint, daß am Aufbau derartiger Produkte hauptsächlich die Aminosäuren Glykokoll, Phenylalanin und Prolin teilnehmen. Wir vermögen uns aus Mangel an Kenntnissen über die Konstitution des Eiweißmoleküls keine klare Vorstellung über die Art der Wirkung des Pepsins zu machen. Wir stellen uns vor, daß eine Spaltung unter Wasseraufnahme einsetzt. An welcher Stelle des Moleküls der Abbau beginnt, ist unbekannt. Jedenfalls greift das Pepsin an ganz anderer Stelle an, als das Trypsin, das proteolytische Ferment des Pankreassaftes. Nimmt man an, daß das Eiweiß aus einer langen Reihe von säurearaidartig verknüpften Aminosäuren besteht, dann muß man sich vorstellen, daß in dieser Kette Stellen vorhanden sind, an denen das Pepsin eine Trennung vermitteln kann. Vielleicht spielt dabei die Art der einzelnen Aminosäuren eine Rolle, oder Wir haben schon aber es wirkt das Pepsin auf Bindungen eigener Art. darauf hingewiesen, daß im Eiweißmolekül auch esterartige Verknüpfungen zwischen Oxysäuren und sonstigen Aminosäuren möglich sind.^) Es wäre denkbar, daß das Pepsin auf irgend eine besondere Bindungsart eingestellt Man kann sich jedoch auch vorstellen, daß im Eiweißmolekül eine ganze Reihe von Polypeptidketten unter sich in besonderer Weise verankert sind, und das Pepsin an diesen Stellen mit seiner Wirkung einsetzt. Interessant ist die schon früher erwähnte Beobachtung, daß keines der bis jetzt dargestellten Polypeptide von Pepsin abgebaut wird. Das Studium der Magensaftwirkung läßt sich noch auf einem anderen Wege erfolgreich durchführen. Es läßt sich nämlich der Magensaft in ganz reinem Zustande, d. h. ohne Beimengungen erhalten. Die gewöhnliche Magenfistel ist zur (rewinnung von reinem Magensaft nicht geeignet, weil beständig Speichel in den Magen fließt und sich mit dem Sekret der Drüsen der Magenwand vermischt. Reinen Magensaft erhält man nach den Methoden von Heidenhain -) und von Paiclow.-') Entweder kombiniert man Diese eine einfache Magenfistel mit der Durchtrennung der Speiseröhre. wird am Halse vollständig durchschnitten. Dann wird sowohl das mit der Mundhöhle zusammenhängende Ende als das in den Magen führende in die Hautwunde eingenäht. Frist das Tier, dann fällt die Speise aus der proximal gelegenen Öfthung der Speiseröhre heraus. Es kann nichts davon in den Magen gelangen. Man nennt eine solche Fütterung Scheinfütterung. Eine weitere Methode ist die folgende. Es wird vom Magen ein Teil durch Anlegung einer Naht abgetrennt. Den entstandenen Blindsack setzt man durch eine Öffnung mit einer solchen in den Bauchdecken in Verbindung und setzt eine Kanüle ein. Der Rest des Magens bleibt mit dem Ösophagus und dem Duodenum im Zusammenhang. Das Tier besitzt dann ist. ') Vgl. S. 378. Heidenhain: Physiologie der Absonderungsvorgänge. 4. Abschnitt. Haudb. d. Physiol. (herausgegeben von Hermann). .3. 179 (1881). ^) ./. /'. Fawlow: Die Arl)eit der Verdauungsdrüsen. ül)ersetzt von A. WaUJicr. .). F. Bergmann. Vgl. auch die Zusammenstellung l)ei W. N. liohlijWiesbaden 1898. E. S. London: Fottreß : Zeitschr. f. d. Ausbau d. Entwicklungslehre. 1. 12U (1907). '') — schritte der Naturwissensch. 4. 1 (1912). — Eiweißstofte uud ihre BauKteine. 469 einen kleinen, für sich abgeschlossenen, mit einer Fistel nach außen mündenden Magenteil und einen größeren Magenteil, der seine Funktionen ganz normal erfüllen kann, denn er steht mit dem Zuleitungrsrohr für die Nahin dem Ösophagus - und dem Abflußrohr dem Duodenum rung Der kleine Magen sezerniert gleichzeitig mit dem direkter Verbindung. — — — großen. Fügt man reinen Magensaft zu koaguliertem Eiweiß, z. B. zu einem hart gekochten Ei, dann beobachtet man, wenn das Gemisch bei 37" aufbewahrt wird, daß bald Teile des festen Eiweißes verschwinden. Wählt man ein Stück Eiweiß mit scharfen Kanten, dann kann man genau verwie diese abgerundet werden. Schließlich geht das ganze Eiweiß in Versucht man nunmehr Eiweiß durch Koagulation abzuscheiden, Dialysiert man ein solches Verdauungsgemisch im so gelingt dies nicht. Fergamentschlauch gegen destilliertes Wasser, dann erhält man beim Beginn des Versuches in der Außenfiüssigkeit keine die ßiuretreaktion gebenden Verbindungen. Bald tauchen jedoch solche auf. Es ist dies das Zeichen dafür, daß aus dem kolloiden Eiweiß Produkte abgespalten worden sind, die die Dialysiermembran zu durchdringen vermögen. Es sind dies folgen, Lösung. die Peptone. Derartige Versuche haben noch das folgende interessante Resultat ergeben. Wird in Magensaft eine Fibrinflocke eingetaucht, dann nimmt sie Pepsin in sich auf. ^) Es tritt eine Adsorption und damit Besonders vielleicht auch eine chemische Bindung des Fermentes ein. schön läßt sich das Eindringen von Pepsin in Eiweiß mittelst Elastins nachweisen. -) Wird ein Stückchen davon in Magensaft oder eine sonstige Pepsin enthaltende Flüssigkeit gebracht, dann erweist sich sein Inneres bald mit aktivem Pepsin beladen. Wird nämlich das Elastin aus der fermenthaltigen Flüssigkeit entfernt und dann, um das außen anhaftende Pepsin und die Salzsäure zu entfernen, gründlich abgebürstet und abgewaschen, so geht im Innern des festen Proteins die Verdauung weiter. Man kann dies dadurch beweisen, daß man gleichzeitig mehrere Stücke von Elastin in Magensaft versenkt und dann nach einiger Zeit daraus entfernt. Den einen Teil davon kocht man sofort gründlich aus und stellt mit dem Kochwasser die Biuretreaktion an. Den Rest läßt man nach erfolgter Reinigung verschieden lange Zeiten liegen und entzieht dem Innern dann durch Auskochen das gebildete Pepton. Dieses weist man mittelst der Biuretreaktion nach. Es läßt sich auf diese Weise leicht feststellen, daß je länger ein mit Pepsin „aufgeladenes" Elastinstückchen aufbewahrt um so mehr Pepton entsteht. Selbst dann, wenn man derartiges, mit Pepsin ,,geladenes" Elastin unter Bedingungen hält, die jede Pepsinwirkung sofort vernichten würden, wirkt das aufgenommene, im Innern Pepsin ist z. B. gegen Alkali des Elastins geschützte Ferment weiter. sehr empfindlich. Geringe Spuren davon verhindern seine Wirkung. Mit Pepsin geladenes Elastin zeigt auch in alkalischer Lösung so lange Abbau und Peptonbildung, als das .-Alkali das Eiweiß nicht ganz durchtränkt hat. wird, P. r. Grützner: Deutsthf med. Wochenschr. 1 (1891). Emil Abderhalden und Engen Steinbeck, Fr. W. Strauch, Franz Wachsrnuth, Otto Meyer, Karl Kiesetcetter und Fr. Friedel: Zcitschr. f ph\sinl. Chemie. 48. 293 (1910); 7J. 31;'). 449 (1911); 74. 67. 411 (1911). ') ^) XXIII. Vorlesung. 470 Diese Beobachtungen ermöglichen es, Pepsin nachzuweisen. 80 ist es auf diesem Wege z. B. geglückt, im Darminhalt aktives PepFerner wird sicher Pepsin sin in freiem Zustande aufzufinden. 1) in aktiver Form in schwer hydrolysierbaren Proteinen, wie in elastischen Es kann dann Fasern, in Bindegewebe usw. in den Darm übergeführt. dort von innen heraus die betreffenden Eiweißstoffe noch lösen, nachdem das Medium, in dem sie sich befinden, schon längst Bedingungen aufweist, die dem nicht durch die EiweißhüUc geschützten Pepsin die Wirkung nehmen würden. Erst dann, wenn der alkalisch reagierende Darm- und Pankreassaft bis in das Innere der erwähnten Proteine eingedrungen ist, hört die Pepsinwirkung auf. Es sei gleich hier erwähnt, daß das Pepsin manche Proteine rascher und leichter zerlegt, als das Trypsin. Zu diesen Proteinen gehören vor allem das Elastin, das Bindegewebe und ferner manche genuine Proteine, wie z. B. die Plasma- bzw. Serumeiweißkörper. -) Der bedeutsame Einfluß des Quellens der Eiweißstofife auf die Wirksamkeit des Pepsins beruht ohne Zweifel zum großen Teil darauf, daß dieses rascher in die gequollenen und dadurch auch gelockerten Proteingemische eindringen kann. Es werden durch die Quellung Bedingungen geschaffen, die der Aufnahme des Pepsins günstig sind. Erwähnt sei noch, daß auch das Zy mögen des Pepsins sich von Elastin aufnehmen und dann in diesem sich aktivieren . läßt. Durch Verwendung von reinem Magensaft läßt sich auch die Frage nach der Abspaltung von Aminosäuren aus Eiweiß in Angriff nehmen. Eine Fehlerquelle, die beim Tierversuch vorhanden ist, nämlich der nachträgliche Zufluß von Darminhalt, fällt weg. Selbstverständlich muß jedoch auch hier der Gehalt des Ausgangsmateriales an freien Aminosäuren in Betracht gezogen werden. Exakt durcligeführte Versuche haben ergeben, daß keine Aminosäuren auftreten, wenn man Eiweiß der Verdauung durch Es fragt sich, ob das erhaltene reinen Hu'ndemagcnsaft unterwirft. Resultat den Schluß zuläßt, daß Magensaft überhaupt keine Aminosäuren aus Eiweiß abspaltet. Man kann gegen die Versuche im Reagenzglas einen wohlbegründeten Einwand erheben. Sie unterscheiden sich in manchen Punkten von den natürlichen Verhältnissen. Einmal fügen wir zu einer bestinnnten Menge Eiweiß eine bestimmte Menge Magensaft hinzu und verfolgen dann die Spaltung bei 37". Wir wissen nicht, in welchem Verhältnis im Magen Eiweiß, Pepsin und Salzsäure zusammenwirken. Bei der Spaltung Im Magen treten neue Aminogruppen auf die Salzsäure binden können. dürfte ein so entstehender Mangel an freier Salzsäure rasch durch SekreEs kann auch von neuem Pepsin tion neuer Säure gedeckt werden. sezerniert werden. Kurz, das Gleichgewicht zwischen Subsirat und Ferment kann von Augenblick zu Augenblick in dieser oder jener Richtung verschoben werden. Beim Reagenzglasversuch bestimmen wir willkürlich die Wir vermögen nicht, sie einzustellen, weil uns noch die Bedingungen. Kenntnisse über die feineren Vorgänge bei der Magenverdauung vollständig '^j fehlen. Geben wir nachträglich noch einmal Salzsäure oder Pepsin oder ) Emil Abderhalden und Mitarbeiter: 1. Vgl. hierzu Carl Oppenheimer und ') Vgl. ') c. S. 46'J. //. Aron Zitat-). : Jlofmeisfer^ Boitr. 4. (1903). hlDiil Ahdc.rhalden, 1'!. S. London und Mitarbeiter: 1. c. 8. 467. Zitat'). "27i) Eiweißstoft'e und ihre Bausteine. 471 beides zu, dann schaffen wir wieder ganz neue Verhältnisse, die die Spaltung beschleunigen, aber auch verlangsamen bis aufheben können. Dazu kommt ein zweiter sehr wichtiger Punkt. Bei der Verdauung im Magen werden die gebildeten Peptone entfernt. Sie verlassen den Magen durch den Pylorus und gelangen in das Duodenum. Manche Forscher i) sind auch der Ansicht, daß die Magenschleimhaut Peptone aufnehmen kann. Andere Beobachter bestreiten jede Resorption von Eiweißabbauprodukten im Magen. 2) Es war bis jetzt unmöglich, eine klare Entscheidung dieser Frage herbeizuführen. Jedenfalls werden in den Magen eingeführte Aminosäuren und Polypeptide unter normalen Verhältnissen kaum von der MagenEs spricht alles dafür, daß im Magen schleimhaut aufgenommen. 3) eine Resorption von Peptonen keine Rolle spielt. Fällt somit diese Art der Entfernung von Eiweißabbaustufen wahrscheinlich ganz außer Betracht, so ist die andere, die Abgabe an das Duodenum, um so wirkSie wird nur durch die Perioden des samer, weil sie eine dauernde ist. Im Reagenzglasversuch bleiben die Abbauprodukte liegen. Sie beeinflussen die Reaktion des Mediums, in dem Nun wissen wir aus Erfahrung, die Pepsinwirkung sich vollziehen soll. daß die Abbauprodukte den Ablauf der Fermentwirkung stark beeinflussen können. Einmal muß es im Reagenzglas schließlich zu einem Gleichgewicht zwischen Substrat, Abbaustufen und Ferment kommen. Im Magen ist ein derartiger Zustand nicht oder nur ganz vorübergehend möglich, weil beständig ein Abfluß der Abbaustufen und ferner ein Zufluß von Ferment durch Sekretion stattflndet. Aus den erwähnten Gründen können wir den Versuch im ReagenzEr hat nur einen vollen Wert in glas nicht als vollwertig anerkennen. Verbindung mit Versuchen am Organismus selbst. Die Erfahrungen beider Versuche müssen sich decken, oder es muß sich bei Unterschieden ihre Ursache klar ergeben. Es wäre ganz gut denkbar, daß der Reagenzglasversuch deshalb nicht zu Aminosäuren führt, weil der Abbau aus den erwähnten Gründen zu früh zum Stillstand kommt. Da jedoch die direkte Untersuchung des Mageninhaltes und die Resultate der Reagenzglasversuche zu den gleichen Resultaten geführt haben, ist der Schluß berechtigt, daß das Pepsin die Proteine zwar zerlegt, jedoch den Abbau nicht bis zu Aminosäuren durchführt. Beim Zusatz von Auszügen aus der Magenschleimhaut, von Magensaft und auch von Organauszügen zu einer konzentrierten Peptonlösung beobachtet man unter geeigneten Bedingungen das Auftreten einer Fällung. Seit Danüewski/) diese Erscheinung zum ersten Male beobachtet hat, sind Verschlusses des Pylorus unterbrochen. — — physiol. Chemie. 47. 88 (1906). — W. Grimmer: Biochem. Zeitschr. 3. 389 Zeitschr. f. physiol. Chemie. 45. 185 (1905). ) Vgl. hierzu: Ludwig Tobler: Arthur Schcnnert und Walther Grimmer: G. Lang: Biochem. Zeitschr. 2. 225 (1906). Zeitschr. f. _ Edgard Zunz: Contribntion ä l'etude de la digestiou et de la r^sorption des proteines dans Testomac. Ann. de la Soc. royale des sciences med. et natur. de O. Folin und llenrij Lyman: The .Journ. of hiol. Chom. 12. 259 (1912). Bruxelles 1908. ) Vgl. hierzu E. S. London und W. W. Polowzowa: Zeitschr. f. physiol. Chemie. (1907). — — E. S. London und ./. S. Tschekumhc : Ebenda. 87. 314 (1913). 328 (1906). ») Emil Abderhalden, E. S. London, 0. Pr>im, Carl Vögtlin und Kornel v. Körösy Zeitschr. f. physiol. Chemie. 53. 148, 326, 334 (1907). 49. ) (Danileu'sky und) Okunew: In.-Diss. St. Petersburg 1895. XXlll. Vorlesung. 472 bis heute zahlreiche Untersuchungen unternommen worden, um diese eigen- Es ist jedoch nicht geglückt, für irgend eine der aufgestellten Hypothesen einen eindeutigen Beweis zu erbringen. Der ganze Vorgang wird vielfach für eine Synthese gehalten. Es sollen einfachere Peptone zu hoher molekularen zusammentreten, ja nach manchen Autoren soll direkt Eiweiß entstehen. Die sich bildenden Produkte sind Plasteine oder Koagulosen genannt worden. Es ist versucht worden, die Frage nach der Natur der Plasteine durch Bestimmung der freien Aminogruppen im Ausgangsmateriale und in den durch Fermente gebildeten Produkten zu entscheiden, ^j Eine Abnahme der freien Aminogruppen bei der Plasteinbildung könnte im Sinne einer Synthese gedeutet werden, doch bleibt die Möglichkeit oöen, daß anhydridartige Bindungen in einzelnen Peptonen entstehen. Auch hierbei würde die Zahl der freien NHgGruppen geringer werden. Man wird vor allen Dingen die Frage entscheiden müssen, ob ein wirklicher Fermentvorgang vorliegt, und nicht etw a durch den Zusatz der Auszüge und Fermentlösungen Bedingungen geschaflFen werden, die zur Fällung nunmehr unlöslicher Produkte führen. Ferner wird man genau feststellen müssen, ob es sich nicht bei der Bildung der Plasteine um rein physikalische Vorgänge handelt und z. B. die Größe der in Lösung vorhandenen Teilchen verändert wird und dadurch eine Art von Ausflockung oder Zusammenballung sich herausbildet. Alle diese Fragen sind deshalb so schwer zu beantworten, weil ausschließlich mit unbekannten Größen gearbeitet wird. Man läßt auf ein ganz unbekanntes Gemisch von Abbaustufen aus Eiweiß eine Fermentlösung unbekannter Zusammensetzung einwirken und erhält eine chemisch nicht genauer detinierbare Substanz. artige Reaktion aufzuklären.') Sehr lange bekannt ist eine weitere Wirkung des Magensaftes, nämlich seine Fähigkeit, Milch zur Gerinnung zu bringen. Schon im Altertum machte man von dieser Wirkung bei der Käsebereitung Gebrauch. Man verwandte allerdings nicht Magensaft, sondern die Schleimhaut des eigentlichen Magens des Kalbes. Zunächst glaubte man die Milchgerinnung auf Säurewirkung zurückführen zu können. Es ist das große Verdienst von Hammarsten^) und Alex. Schmidt), gezeigt zu haben, daß eine Fermentwirkung vorliegt. Das wirksame Prinzip ist Labferment oder Chymosin genannt worden. Es wird von den Drüsen der Magenschleimhaut in einer Vorstufe abgegeben. Die Ül)erführung in den aktiven Zustand erfolgt durch Salzsäure. M. Lawrotv: Iii -Dies. Petersburg 1897. — Sawjalotv: Pflüqer» Archiv. 85. Maria 121 (1901); 2. 411 (1902). Latorow und N. Salaskin : Zeitschr. f. physiol. Chemie. 36. 277 (1902). R. 0. Herzog: Zeitschr. f physiol. Chemie. 39. 305 (1903). A. Bat/er: Hofmrisfers Beiträge. 4. 554 J.Lukomnik: Ebenda. 9. 205 (1907). D. Lairroir: Zeitschr. f. physiol. (1903). Chemie. 56. 343 (1908). P. A. Leinne und D. D. ran Sli/ke: Biochem. Zeitschr. 13. 458 (1908): 16. 203 (1909). P. Glof/olnr : Biochem. Zeitschr. 50. 1(52 (1913). ') 171 (1901). St. — Kurajeff, Hofmeisters Beiträge. — — — 1. — — — V. Henrufues und ./. K. ('jaldhäk: Zeitschr. f. physiol. (Chemie. 71. 485 (1911); 439 (1912). ^) Vgl. O. Hammarshn: Sitzuugsl)er. der Königl. Gesellsch. d. Wissenschaften /u '-) 81. — Upsala. 1877. Vgl. die frühereu Arbeiten in Malijs Jahresbericht. 118 (1872); 135 Vgl. ferner die Literatur über die Geschichte des Labfermentes (1874); 158 (1877). bei Peters: In.-Diss. Dorpat 1894. ) — A. Schmidt: Beiträge zur Milchgeriuuung. Dorpat 1871. Kiweißstoffe und ihre Bausteine. 47 Die Milehgerinnung beruht auf (Mner Uunvundlung des in der Milch vorhandenen Eiweißkörpers Kaseinogen.i) Es bildet sieh Kasein, das als Kalksalz ausfällt. Die Wirkung des Labfermentes ist wahrscheinlich die folgende. Es verändert das in der Milch vorhandene Kaseinogen primär und führt es in ein Produkt mit neuen Eigenschaften, Kasein genannt, über. Dieses verbindet sich mit vorhandenen löslichen Kalksalzen und fällt aus. Diese Phasen in der Umwandlung des Kaseinogens lassen sich durch den folgenden Versuch verfolgen. Entfernt man aus der Milch die löslichen Kalksalze, z. B. mit Oxalsäure, so erhält man beim Zusatz von Labfermentlösung keine Fällung. Daß jedoch eine Umwandlung des genuinen Kaseinogens eingetreten ist, beweist der Umstand, daß sofort eine Fällung eintritt, sobald man dem Gemisch z. B. Kalziumchlorid zusetzt. Kaseinogen selbst gibt mit löslichen Kalksalzen keine Ausflockung.-) Es ist trotz aller Bemühungen nicht geglückt, den Vorgang der sogenannten Labgerinnung besser spricht man von einer Fällung oder Ausflockung als von Gerinnung aufzuklären. Welche Veränderung erleidet das Kaseinogen? Nach der einen Auffassung handelt es sich um eine Synthese. Es sollen mehrere Kaseinogenmoleküle zu einem noch komplizierter gebauten Produkte zusammentreten. Nach der Ansicht anderer Forscher liegt ein Abbau vor. Endlich besteht die Möglichkeit, daß das Kaseinogen im Molekül selbst eine Veränderung erleidet und sich dadurch — — Wohl die meisten Forscher stehen auf dem Standpunkte, daß das Labferment das Kaseinogen spaltet.'') Die entstehenden Abbaustufen sind durch ihre Fällbarkeit durch Kalksalze ausgezeichnet. Es lassen sich über die Art der Spaltung wahrscheinlich eine Hydrolyse und die entstehenden Spaltprodukte keine genaueren Angaben machen. Die Zahl der Möglichkeiten ist unübersehbar. Es ist denkbar, daß das Kaseinogenmolekül falls dieses überhaupt einheitlich ist! in gleichartige Bruchstücke zerfällt, die das Kasein darstellen. Es ist jedoch auch möglich, daß aus dem Kaseinogen ein Produkt abgespalten wird, das keinen Anteil an der Kaseinbildung nimmt, während der verbleibende Rest das Kasein liefert.) Da wir nicht wissen, ob das Kaseinogen einheitlich ist, ist eine Entscheidung der vorliegenden Fragen zurzeit unmöglich.') seine Eigenschaften ändern. — — — — Was geschieht mit dem Kalksalz des Kaseins? Setzen wir zu Milch Magensaft, dann erhalten wir nach kurzer Zeit die erwähnte Fällung. Halten wir das Gemisch bei 37", so beobachten wir, daß die ausgefallene Masse allmählich wieder in Lösung geht. Die genauere Analyse des Vorganges zeigt, daß das Kasein dnrch das Pepsin in Peptone ge) Die Bezeichnung Kaseinogen wird nach dem Vorschlag verschiedener Forscher an Stelle von Kasein verwendet. Der Name Kasein tritt an die Stelle von Parakasein. ) Nach Samuel Barnett ScJiri/ver \|Biochein. .Journ. 8. 152 (1914)] ist Kasein nicht ein Kalksalz, sondern eine Verbindung von Kaseinogen mit einem Ferment. ') D. D. van Shjke und A. W. liosworth sind der Ansicht, daß ein Kaseinogenmolekül zwei Moleküle Kasein liefert. The Journ. of Biol. Cheni. 14. 203 (1913). ) Sigvul Schmidt-Nielsen: Zeitschr. f. physiol. Chemie. 48. 92 (1906); HofMartin Javobi/: Binchem. Zcitsclir. 1. 53 (1906). meistern, Beiträge. 9. 322 (1907). 5) A. Kreidl Physiol. 22. 133 (1908); Pflüger^ und A. Neumunn: [Zentralbl. Archiv. 23. 523 (1908)] sind der Ansicht, daß hauptsächlich physikalische Momente beim Ausfallen des Kaseins eine Rolle spielen. Vgl. die gegenteilige Ansicht bei D. D. van Slyke und A. W. Bosworth: The Journ. of Biol. Chem. 14. 203 (1913). — i'. — XXIII. Vorlesung. 474 — Gleichzeitig wird Phosphorsäure frei ^) Kaseinogen und Kasein gehören zu den Phosphorproteiden. Es wird somit das Kasein ebenso von Pepsin weiter zerlegt, wie die übrigen Proteine. Es fehlt nicht an Forschern, die das Vorkommen eines Labfermentes bezweifeln. Es soll die Ausfällung des Kaseins als Kalksalz nur darauf beruhen, da(i Pepsin aus dem Kaseinogen Produkte bildet, die mit löslichen Kalksalzen schwer lösliche Komplexe und vielleicht chemische Verbindungen bilden. Zunächst wurde die Ansicht vertreten, daß die Pepsin- und die Labwirkung verschiedenen Gruppen ein und desselben Fermentes zukämen. 2) Selbstverständlich vermag eine solche, in absehbarer Zeit nicht beweisbare und auch nicht gut widerlegbare Ansicht den Kern der Frage nach dem Wesen der Labwirkung nicht zu treffen. Die Annahme, daß das Labferment mit Pepsin vollständig identisch sei '), bedeutet hingegen eine Arbeitshypothese. Man wird genau abmessen müssen, welche Momente für eine Identität sprechen, und welche die Annahme stützen, daß man zwei verschiedene Fermentarten anzunehmen hat. Zugunsten einer Identität beider Fermente wird angeführt, daß beide den gleichen Aktivator, nämlich Salzsäure, bzw. H-lonen hai)en, ferner werden die Wirkungen beider Fermente durch manche Einflüsse in gleicher Art gefördert bzw. gehemmt. Dazu kommen noch folgende Beobachtungen. Auch der Prankreassaft zeigt Labwirkung. Ferner finden wir im Pflanzenreich Fermente, die ganz entsprechende Wirkungen entfalten. Diese Beobachtungen lassen es begreiflich erscheinen, daß daran gedacht worden ist, die Wirkung jedes proteolytischen Fermentes führe bei der Zerlegung von Kaseinogen zu Abbaustufen, die mit Kalksalzen unlösliche Produkte ergeben. Auf der anderen Seite hat vor allem Hammarsten^) zahlreiche Beobachtungen bekanntgegeben, aus denen hervorgeht, daß man die Pepsinwirkung eines wirksamen Auszuges vernichten und die Labwirkung allein erhalten kann. Auch der umgekehrte Versuch gelingt. Wichtig ist vor allem die Beobachtung, daß Pepsin nur bei Anwesenheit von H-Ionen wirksam ist und seine Wirkung einbüßt, wenn OH-Ionen zugegen sind. Die Labwirkung ist dagegen auch im letzteren Falle nachweisbar. In ein ganz neues Licht rückt das Problem des Vorkommens oder NichtVorkommens eines besonderen Labfermentes durch die wichtige Beobachtung, daß es außer auf Kaseinogen auch noch auf andere Proteine w^rkt.^) So baut es Legumin, Muskelsyntonin und wahrscheinlich noch andere Eiweißarten ab. Nimmt man zu dieser Beobachtung noch spalten wird. Plimmer und \V. M. Bayliss: Journ. of Physiol. 33. 439 (190(i). und S. W. Farastschuk : Zeitschr. f. physiol. Chemie. 42. 415 (1904). ") W. Sairjalow: Zeitschr. f. physiol. Chemie. 46. 307 (190.'i). ./. W. Gewin: Ebeada. 54. 32 (1907/08). Vgl. auch ('. A. Pekelharing: J'ßügerH Archiv. 167. 254 (1917). ) Olaf Hammarsten: Zeitschr. f. physiol. Chemie. 56. 18 (1908); 68. 119 (1910); 74. 142 (1911); 94.104,291 (191.'i); 102.33 (1918); 121. 240, 261 (1922). Vgl. auch /rar Bang: Zeitschr. f. physiol. Chemie. 43. 358 (1904). Sigval Schmidt- Nielsen: Festschrift i\\r Olaf Hammarsten. üpsala 1906. Emil Abderhalden und F. W. Stravch: Zeitschr. f. pliysiol. Chemie. 71. 315 (1911). A. Rakoczy : Zeitschr. f. physiol. Chemie. 68. 421 (1910); 84. 329 (1913). - Vgl. auch V. Dncreschi: Archiv, di Fisiol. 5. 413 (1908). Dieser Autor berichtet, daß im Magen dos Beuteltieres kein Lab, wohl aber Pepsin sich findet. Agnes Ellen Porter: Journ, of Physiol. 42. 389 (1911). W'^. van Dam: Zeitschr. M B. n. -) ./. Ad-e.rK P. l'au'loir — — — — — — — f physiol. Chemie. 86. 77 (1913). ') 0. Hammarsten: Zeitschr. f. physiol. ('hemie. 102. 33, 115 (1918). Eiwoißstoffe und ihre Bausteiue. 475 hinzu, daß das Labferment bei einem H-lonen-Gehalt wirkt, bei dem das Pepsin unwirksam ist, dann erkennt man seine hohe Bedeutung beim Eiweißabbau ohne weiteres. Wir haben es ohne Zweifel mit vier Gruppen von proteolytischen Fermenten zu tun. Im Magen setzt zunächst bei schwach saurer Reaktion die Wirkung des Labfermentes ein. Allerdings scheint ihm keine allgemeine Eiweiß abbauende Wirkung zuzukommen. Vielmehr liegt eine spezifische Einstellung auf bestimmte Proteine vor. Darunter befindet sich auch das Kaseinogen. Aus ihm entstehen Abbaustufen, die unlösliche Kalksalze bilden. Diese werden weiter verdaut und Sobald die Reaktion des Chymus im in lösliche Peptone übergeführt. Magen stärker sauer wird, setzt die Wirkung des Pepsins ein. Nach der Überführung des Speisebreis aus dem Magen in den Darm fließen ihm alkalisch reagierende Sekrete zu. Die saure Reaktion wird abgeschwächt. jener Grad an H-lonen erreicht, bei dem das Pepsin unwirksam Das Labferment hingegen kann noch weiter Eiweiß und Peptone spalten. Schließlich werden auch ihm die Bedingungen zur Wirkung entzogen. Es setzt nun das Trypsin, ein Ferment des Pankreassaftes, ein und hierauf das Erepsin des Darmsaftes. Auf beide Fermentarten kommen wir gleich noch zurück. Das letztere ist kein ganz allgemein auf Proteine eingestelltes Ferment. Es baut in der Hauptsache Peptone und Polypeptide Wir erkennen an dieser Darstellung, daß dem Labferment bei der ab. Bald ist wird. Eiweißverdauung eine Bedeutung zukommt, die weit über das hinaus geht, was bisher von seiner Wirkung bekannt war. Es ist mit dieser Feststellung zugleich eindeutig entschieden, daß es eine besondere FermentChymosin gibt. gruppe Labferment Nicht unerwähnt wollen wir lassen, daß Ivar Bang^) beim Menschen und beim Schwein ein Labferment gewonnen hat, das sich in seiner Wirkung w^esentlich von dem aus Kälbermagen dargestellten unterscheidet. Er nannte es Parachymosin. Es ist empfindlicher gegen Alkalien als das Chymosin, dagegen beständiger gegen Hitze als das letztere. Die biologische Bedeutung der Fällung der Milch im Magen Milch ist die einzige Nahrung des Säugist ohne Zweifel die folgende. lings. Sie kommt als Flüssigkeit in den Magen. Die Beobachtung des Verhaltens von Wasser und von anderen Flüssigkeiten in diesem hat ergeben, daß solche auffallend rasch an das Duodenum abgegeben werden. Würde die Milch flüssig bleiben, dann würde sie ohne Zweifel den Magen ebenfalls rasch verlassen. Dadurch, daß das Kaseinogen in den festen Zustand übergeführt wird, ist eine umfassendere Verdauung durch das Pepsin möglich. Dazu kommt noch, daß die Kaseinflocken die geeignete Beschaffenheit haben, um sich mit Pepsin zu beladen.-) Ferner reißen sie andere Milchbestandteile, wie Fett, mit. Zu erwähnen ist noch, daß im Magen Proteide in ihre Komponenten zerlegt werden. Aus den Nukleoproteiden gehen Nukleine) hervor. = Ivar Bang: Pflüger^ Archiv. 79. 425 (1900). Emil Abderhalden tiiid /''. Friedl: Zeitschr. f. physiol. Themie. 71.449 (1911). Emil Abderhalden und Friedrich Kramm: Ebenda. 77. 462 (1912). ') Die Kerne selbst werden nach Adolf Schmidt (vfjl. Ad. Schmidt und J. Strasburger: Die Fäzes des Menschen im normalen und kranken Zustande. 5. AuÜ. 1910) vom Magensaft nicht angegriffen. Erst der Pankreassaft löst sie auf. V^gl. auch F. W. 7'. hashiuado: Ebenda. Strauch: Deutsches Archiv f. klin Med. 101 128 (1910). 104. 584 (1911). ) ^) — XXIII. Vorlesuug. 476 beobachtet man, daß der Verdauungstiüssigkeit Peptone worden, daß die Nukleoproteide zunächst in Eiweiß und Nuklein zerfallen. Das letztere wird nicht weiter umgewandelt, während das Eiweiß in Peptone übergeführt wird. Bei manchen Kernsubstanzen läßt sich gleichzeitig Abspaltung von Phosphorsäure nachweisen.!) Auch das Hämoglobin wird angegriffen. Das Hämatin wird frei und zum Teil verändert, und das Globin in Peptone Gleichzeitig auftreten. Es ist dies so in gedeutet zerlegt. Schließlich wollen wir uns noch der Frage zuwenden, ob die Magen- verdauung für die Verwertung der Proteine im tierischen (.)rganismus unentbehrlich ist. Die direkten Beobachtungen zeigen, daß das nicht der Fall ist. Tiere und Menschen können ohne Magen auskommen.-) Die Ausnützung der Proteine ist eine gute. Auch der Reagenzglasversuch zeigt, daß die Proteine von den in den Darmkanal sich ergießenden Sekreten leicht zerlegt werden. Nur Vertreter der Proteinoide Glyzinamine, wie Elastin, Bindegewebsproteine usw., werden entschieden vom Magensaft leichter angegriffen als vom Pankreas- und Darmsaft. = Die Bedeutung des Pepsins für die Verdauung der Proteine liegt nach allen Beobachtungen darin, daß aus großen Molekülen durch Abbau ungezählte kleinere entstehen. Es wird dadurch der Verdauung im Darmkanal wesentlich vorgearbeitet. Die ganze Angriffsfläche ist für die proteolytischen Fermente der Sekrete der außerordentlich stark vergrößert. Daß diese Anschauung richtig ist, beweist der Versuch im Reagenzglas. Läßt man auf Eiweiß direkt Pankreassaft einwirken, dann vollzieht sich der Abbau langsamer, als wenn man den gleichen Eiweißkörper zuerst der Wirkung von Pepsin ausgesetzt hat.») Der Unterschied in der Geschwindigkeit des Abbaus ist ohne Zweifel in Wirklichkeit viel größer, als der Reagenzglasversuch anzeigt. Der Abbau vollzieht sich im Darmkanal in viel kürzerer Zeit, weil die entstehenden Abbaustufen stets sofort durch Resorption entfernt werden. Beim Reagenzglasversuch bleibt das ganze Gemisch der Abbaustufen liegen Bald zeigt sich ihre hemmende Wirkung, und so kann ein vielleicht großer Vorsprung beim Abbau des mit Pepsin vorverdauten Eiweißes im Parallelversuch direkte Verdauung des gleichen Eiweißes mit Pankreassaft eingeholt werden. Kurzfristige Versuche, die diese Frage klar entscheiden könnten, sind schwer durchzuführen, weil dann die Abbauprodukte noch nicht quantitativ bestimmbar sind. Die Verfolgung der Zunahme der freien Aminogruppen, die uns ganz gut über den Ablauf der Spaltung unterrichtet, ist hier nicht ohne weiteres anwendbar, weil natürlich das mit Pepsin hydro- Darmdrüsen — — ) Vgl. Kmil AhderhaUlen und 7'. S. Kashiwado: Zeitschr. f. physiol. Chemie. 81. 28f) (1912). — Czerny: Beiträge zur operativen Chirurgie. Stuttgart. 141 (1878). M. Oqata u.) l'hvsiol. 89 (1883). G. CarmUo und V. Pachon : Arch. de Physiol. 5«* Serie. 6. 106 (1894). Langcnhuch : Deutsche med. Wochenschr. Nr. 52 (1894). ('. Schlatter: Korrespondenzhl. f. Schweizer Ärzte. 27. TOä (1897). A. Carrel, ^'. M. Meijer und I\ A. Lecenc Jouru. of. l'hvsiol. 26. 3(59 (1910). E. S. London und ir. F. Dagaew: Zeitschr. f. physiol. Chemie. 74. 330 (1911). ') Emil Fischer und Emil Abderhalden: Zeitschr. f. physiol. Chemie. 40. 215 (1903). - Emil Abderhalden und Alfred Gigon: Khenda. 53. 119 (1907). Emil Abderhalden und Ch. J. Valette J'ettibone: Khenda. 81. 458 (1912). Vgl. auch Carl Oppenheimer und H. Aren: Hofmeisters Beitr. 4. 279 (1903). ) Arch. f. (Anat. — — — — — : — — Eiweißstoffe und ihre Bausteine. lysierte Produkt viel 477 mehr NHo-Gruppen aufweist als das Eiweiß, von dem wir beim direkten Trypsin versuch ausgehen. Wir haben bereits festgestellt, daü der Magen immer von Zeit zu Mengen von Chymus entläßt. Dieser enthält neben den Be- Zeit geringe standteilen des Magensaftes und solchen, die noch vom Speichel herstammen, mehr oder weniger veränderten Nahrungsstoffe. Die Eiweißkörper mit wenig Ausnahmen in Peptone übergeführt und die Fette und Polysaccharide auch zum Teil gespalten. Im Duodenum setzt nun die die sind Wirkung des Sekretes der zahlreichen Drüschen seiner Schleimhaut und vor allem desjenigen der Pankreasdrüse ein. Der Pankreassaft enthält ein Fermentgemisch, das auf Eiweiß und seine Abbaustufen eingestellt ist. Es hat den Namen Trypsin erhalten. Es ist ist es zurzeit nicht möglich, eine genaue Abgrenzung der Wirkungen des Pankreassaftes auf die einzelnen Proteine und ihre Abbaustufen zu geben. Das Trypsin wird von der Pankreasdrüse in inaktivem Zustande abgegeben. Die Überführung in die wirksame P^orm erfolgt durch einen im Darmsaft enthaltenen Stotf, Enterokinase genannt. Die Natur dieser Kinase ist noch nicht aufgeklärt. 2) sicher nicht einheitlich ^), doch Der Pankreassaft enthält Fermente, die EiweißstoiTe abbauen können, somit Fermente, die zur Gruppe der Proteasen gehören. Neben diesen kommen auch solche vor, die Peptone und Polypeptide spalten. Sehr wahrscheinlich erfolgt die Hydrolyse der Eiweißabbaustufen durch besondere Fermente. 3) Von diesem Gesichtspunkte aus darf man sich unter Trypsin nicht die ganze Summe der im Pankreassaft enthaltenen, auf Eiweiß und seine Abbaustufen eingestellten Fermente vorstellen. Es muß vielmehr der Sammelname Trypsin für die eigentlichen Proteasen vorl)ehalten bleiben.) Die Wirkung des Trypsins ist eingehend studiert worden. Sie unter- von derjenigen des Pepsins. Das Trypsin zerlegt die Proteine in Peptone unter frühzeitiger Abspaltung von Aminosäuren.5) Es ist sehr schwer, den Beweis zu führen, daß diese Wirkung der Protease des Pankreassaftes zukommt. Man könnte einwenden, daß das Trypsin Peptone bildet und diese durch Peptasen, d. h. durch auf Peptone eingestellte Fermente, rasch weiter gespalten werden und dabei erst Aminosäuren in Erscheinung treten. Solange es nicht gelingt, die Wirkung der einen oder anderen Ferraentgruppe auszuschalten, sind solche Fragestellungen nicht exakt zu beantworten. Immerhin spricht das fast sofortige Auftreten von Aminosäuren bei der Einwirkung von Pankreassaft auf Eiweiß dafür, daß die Proteasen des Pankreassaftes das Eiweißmolekül an anderen Stellen angreifen als das Pepsin. Ferner wirkt das scheidet sich scharf Carl Oppenhei)ner schlägt für das Eiweiß angreifende Ferment des Pankreasden Namen Tryptase vor. Vgl. Carl Oppenheim er: Die Fermente und ihre Wirkungen. 1. 352 (1913). F. C. W. Vogel. Leipzig iyi3. ) Vgl. Horace Middleton Vernon: Biochem. .Touru. 8. 494 (1914j. Vgl. Emil Abderhalden und Andor Fodor: B^'ernientforschung. 6. 248 (1922). ) Vgl. hierzu auch W. M. Bai/liss und E. M. Starlinq: Journ. of Physiol. 29. 174 Karl Mays: Zeitschr. f. pliysiol. Chem. 38. 428 '(1903); 49. 124, 188(1901). (1903). Leo Pollak: Hofmeistern Beiträge. G. 95 (1904). K. Kiesel: P/lügem Archiv. 108. 334 (1905). ') Emil Fischer und Emil Abderhalden Zeitschr. f. physiol. Chem. 39. 81 (1903); 40. 215 (1903). ') saftes '^) — — — : XXIll. Vorlesung. 478 Ferment bei Anwesenheit von H-Ionen, während das Trypsin durch diese bald vollständig unwirksam gemacht wird. Auch die Aktivierung beider Arten von Proteasen ist eine ganz verschiedene. Pepsinzymogen wird durch Salzsäure, Trypsinzymogen durch die Enterokinase in den wirksamen Zustand gebracht. Das Studium der Wirkung des Pankreassaftes mit allen seinen auf Eiweiß und seine Abbaustufen eingestellten Fermenten ist mit großen Schwierigkeiten verknüpft, wenn man das Ziel verfolgt, das Zusammenwirken der Magen- und Darmverdauung unter den natürlichen Verhältnissen festzustellen. Der Magen entläßt immer nur kleine Mengen von Speisebrei in den Darm. Diese werden auf der großen Oberfläche des Darmes rasch in dünner Schicht ausgebreitet. Alle möglichen Sekrete vermischen sich mit den übergetretenen Produkten: Darmsaft, Pankreassaft und ferner Galle. Der Abbau der einzelnen im Chymus enthaltenen Substanzen setzt sofort ein, soweit zusammengesetzte Verbindungen vorWährend die Hydrolyse im Gange ist, erscheint ein neuer Schub liegen. von Chymus aus dem Magen und vermengt sich mit den schon vorhandenen Produkten. Gleichzeitig setzt die Resorption ein. Ferner ergießen sich neue Sekretmengen aus den verschiedenen Drüschen und Drüsen. Fortwährend wechselt das ganze Bild. Nie kommt es zu einem bleibenden Gleichgewicht. Unsere Kenntnisse über die feineren Vorgänge des Verdauungvorganges und vor allem über das Zusammenspiel der einzelnen Faktoren, die den Verlauf des Abbaus, der Sekretion und Resorption beherrschen, sind noch so unvollkommene, daß wir nur ein sehr unvollständiges Bild der Wirkung des Pankreassaftes entwerfen können. Es bezieht sich dies vor allem auch auf die quantitativen Verhältnisse. Es wäre selbstverständlich von ausschlaggebender Bedeutung, wenn wir feststellen könnten, innerhalb welcher Zeit Pankreassaft unter natürlichen Verhältnissen Eiweiß bzw. Pepton vollständig bis zu Aminosäuren abbauen kann. letztere man Tiere mit einer Eiweißart oder einem Gemisch von und tötet man sie nach verschiedenen Zeiten, dann ergibt Untersuchung des Inhaltes der einzelnen DünndarmabFüttert Proteinen, die schnitte stets, daß neben Peptonen Aminosäuren vorhanden Niemals findet man nur Aminosäuren oder nur Peptone, vorausgesetzt, daß die in den Darm sich ergießenden Sekrete ihre Wirkung entfalten konnten. Das gleiche Resultat erhält man, wenn man Tiere mit sind.i) an verschiedenen Stellen des Darmkanales verwendet. Der ausund daneben Peptone. Die letzteren Fisteln fließende Chymus enthält Aminosäuren tiberwiegen meistens stark. Man hat aus dieser Beobachtung den Schluß ableiten wollen, daß der Abbau der Proteine bzw. Peptone nicht vollständig bis zu Aminosäuren führe, es sollten vielmehr die gebildeten Peptone direkt zur Resorption gelangen. Nur ') Kühne: Verhandl. d. naturhistor.-med. Vereins zu Heidelberg. N. F. — 1. Heft 1 F. Kutscher und J. Seemann: Zeitschr. f. physiol. Chem. 34. 528 (1001); 35. (1876). 0. Cohnheim: Ebenda. 33. 451 (1901); 4». 64 (1906); 51. 415(1907). 432 (1902). Kmil Abderhalden, Emil Abderhalden: Zeitschr. f. physiol. Chem. 44. 17 (1905). E. S. London, Karl Kautzsch, L. Baumann, K. v. Körösi/ : Ebenda. 48. 549 (1906); 51. — — — — — Emil Emil Abderhalden: Ebenda. 74. 436 (1911). 384 (1907); 53. 148 (1907). Abderhalden, Wilhelm Klingemann und Theodor Fappenhusen: Ebenda. 71. 411 (1911). Emil Abderhalden: Ebenda. 78. 382 (1912). — Eiweißstoft'e und 479 ihre Baiistoino. ein relativ kleiner Teil davon soll bis zu Aminosäuren aufgespalten werden. Ein exakter Beweis für diese Folgerung liegt nicht vor. Es kann gar nicht erwartet werden, daß im Darminhalt jemals^) ausschließlich Aminosäuren auftreten. Wenn wir den Darminhalt untersuchen, unterbrechen wir die Verdauung in einem bestimmten Augenblick. Wir linden alle möglichen hochmolekulare Peptone, einfacher zusammengesetzte und Abbaustufen ferner Aminosäuren. Es ist möglich, daß der Abbau einzelner dieser zusammengesetzten Produkte vollendet war, d. h. daß diese zur Resorption gekommen wären, falls man den Chymus nicht aus dem Darm entfernt hätte. Es ist jedoch auch denkbar, daß der Abbau weiter gegangen sein würde, wenn man die Verdauung nicht künstlich unterbrochen hätte. Nun ließ sich feststellen, daß der aus dem Darm entnommene Chymus rasch peptonfrei wird und schließlich nur noch Aminosäuren aufweist, wenn man ihn bei 37° aufbewahrt. 2) Aus dieser Beobachtung könnte man den »Schluß ableiten, daß auch unter natürlichen Verhältnissen der Abbau der Peptone zu Aminosäuren führt, ehe die Resorption einsetzt. Doch ist auch da.s erwähnte Ergebnis noch lange kein Beweis dafür, daß der Abbau der Proteine und Peptone im Darmkanal ein tiefgehender sein muß, ehe die Aufnahme von Seiten der Darmwand einsetzt. Wir werden nämlich gleich erfahren, daß im Darmkanal Fermente sich finden, die Eiweiß und Peptone restlos bis zu Aminosäuren abbauen können. Infolgedessen müssen schließlich nur Aminosäuren übrig bleiben, wenn Peptone der Wirkung des Darminhaltes ausgesetzt werden. Eine Wegnahme der gebildeten höheren Abbaustufen ist nicht möglich, weil wir die Verdauung im Reagenzglas fort- — setzen! Immerhin haben die erwähnten Versuche den Beweis erbracht, daß die Fermente des Darmkanals in sehr kurzer Zeit den Abbau der Peptone zu Ende fuhren können. Wahrscheinlich würde die Hydrolyse noch viel schneller erfolgen, wenn wir die gebildeten Aminosäuren beständig aus der Verdauungsflüssigkeit entfernen könnten. Es hat sich nämlich durch . exakte Versuche beweisen lassen, daß die Aminosäuren den Abbau Polypeptiden durch peptolytische Fermente hemmen. 3) von Wo wir hinblicken, überall türmen sich Schwierigkeiten auf, sobald wir klar entscheiden sollen, wie weit der Abbau bestimmter, zusammengesetzter Nahrungsstoffe im Magen darmkanal geht, bevor die Resorption einsetzt. Jedes einzelne Ergebnis noch so sorgfältig aufgebauter Versuche ist vieldeutig. Da der Inhalt des Darmes keine Auskunft über den Umfang des Abbaus der gebildeten Peptone gibt, so könnte man daran denken, die Frage nach den zur Resorption kommenden Produkten dadurch zu entscheiden, daß man diese selbst in der Darmwand oder sonst einem Resorptionswege nachweist. Wir werden auf die Frage nach der Art der dem Blute übergebenen, resorbierten Eiweißabbaustufen noch zurückkommen, hier sei nur erwähnt, daß der Abbau der Proteine und Peptone im Darmkanal ') Theoretisch müßte man erwarten, daß ganz am Schlüsse des Verdauungsvorganges nur Aminosäuren vorhanden sind, doch läßt sich dieser Augenblick praktisch kaum feststellen. ) Emil Abderhalden und Friedrich Kramm: Zeitschr. f. ^) Emil Abderhalden und Afred Gigon: physiol. Chemie. 53. 2',A physiol. Chem. 77. 41f) (1912). (1907). Zeitschr. f. — Emil Abderhalden uad Leonor Michaelis: 52. S2G {1201). — Emil Abderhalden und Markus Guggenheim; Ebenda. 54. 331 (1908). - 480 XXIII. Vorlesung. vollzieht, i) Fortwährend setzt die Resorption ein und aufnahmefähigen Abbaustufen. Diese v^on der sehr großen Oberfläche der Wandung des Darmkanals resorbierten Stoffe werden sofort an das Blut weitergegeben. Die Gewebszellen entnehmen diesem die aufgenommenen Produkte. Es kommt unter normalen Verhältnissen nie zu Anhäufungen von Eiweißabbaustufen im Blute. Der tierische Organismus arbeitet stets in sehr großen Verdünnungen. Das erschwert den Nachweis von vom Darme aus aufgenommenen Produkten ganz außerordentlich. Dazu kommt noch, daß die von der Darmwand resorbierten .Substanzen schon in dieser weitgehend verändert werden können. Es sei daran erinnert, daß die Feststellung von Neutralfett in dem Ghylus des Ductus thoracicus und im Blute während der Fettaufnahme uns durchaus nicht berechtigt anzunehmen, daß Fett als solches zur Resorption gelangt ist. Wir wissen vielmehr, daß die Fette im Darmkanal verseift werden. In der Darmwand erfolgt dann wieder die Svnthese zu Neutralfett aus den aufgenommenen Bausteinen. Schließlich stellt sich der Verfolgung der Art der von der Darmwand resorbierten Stoffe noch eine weitere, wohl kaum überwindbare Schwierigkeit entgegen. Es ist dies die Feststellung der Quantität der aufgenommenen Produkte. Es genügt nicht, daß wir eindeutig beweisen, daß eine bestimmte Abbaustufe während der Verdauung im Blute anwesend ist. Es müßte vielmehr ihre Menge genau derjenigen entsprechen, die aus dem verfütterten Nahrungsstoff entstehen kann. Nur in diesem Falle w^äre man berechtigt, zu sagen, daß eine zusammengesetzte Verbindung in einem ganz bestimmten Grade des Abbaues dem Blute übergeben wird. Nun folgen sich Schlag auf Schlag der Abbau von Stufe zu Stufe, die Resorption der eben erst entstandenen, sehr geringen Mengen von Abbaustufen und die Übergabe an das Blut und schon sind ungezählte Zellen bereit, diesem die Nahrungsstoffe wieder zu entnehmen. An ungezählten Stellen des Darmkanals werden ununterbrochen Spuren von genügend abgebauten Produkten aufgenommen. Tötet man ein Tier in einem bestimmten Augenblicke der Verdauung, und untersucht man dann rasch sein Blut auf resorbierte Abbaustufen, dann findet man nur Spuren davon. Nieraals wird man imstande sein, ein in quantitativer Beziehung lückenloses Bild dei' zur Resorption gelangenden Abbaustufen zu erhalten. Wir werden stets auf Wahrscheinlichkeitsschlüsse angewiesen bleiben. Fassen wir zunächst zusammen, welche Befunde über den Abbau der Proteine im Magendarmkanal durch eindeutige Feststellungen sichergestellt sind. Im Magen werden die Eiweißstoffe unter Bildung von Peptonen gespalten. Es werden dabei keine Aminosäuren gebildet. Im Darmkanal wird die Hydrolyse fortgesetzt. Es treten sehr frühzeitig Aminosäuren auf. Untersucht man den Darminhalt während der Verdauung zu verschiedenen Zeiten, so findet man stets neben Peptonen auch Aminosäuren. Die Peptone können in solche, die die Biuretrcaktion geben, und solche, bei denen sie negativ ausfällt, getrennt werden. Der Befund von Aminosäuren ist sehr frühzeitig erhoben worden, ^j Er fand jedoch wenig Beachtung, weil angenommen wurde, daß normalerweise eine Abspaltung von Aminosäuren sich stufenweise beseitigt ) Zeitschr. -) Heft 3 die Vgl. hierzu Emil Abderhalden, E. S. London, Berthold Oppler, E. H. Reemlin f. physiol. Chem. 55. 447 (1908); 58. 432, 435 (1908). Vgl. Kühne: Vorhandl. d. naturhist.-med. Vereines zu Heidelberg. N. F. 1. (187f)). Kiweißstofi'e nicht stattfinde. imd ihre Bausteine. 4g Man stellte sich nämlich vor, daß die Nahrungsstofife im so weit abgebaut würden, als notwendig ist, um Magendarmkanal nur sie diffundierbar zu machen. Jeder weitere Abbau sollte einen Verlust an Energie für den Organismus bedeuten. Die Feststellung von Aminosäuren im Darminhalt glaubte man auf Bakterienwirkung zurückführen zu müssen. Diese Vorstellungen erwiesen sich als unrichtig. Der hydrolytische Abbau der zusammengesetzten Nahrungsstoffe verläuft ohne nachweisbare Wärmetönung.^) Ein Energieverlust findet bei der Spaltung der Proteine und Peptone nicht statt. Ferner wurde nachgewiesen, daß reiner Pankreassaft aus Proteinen Aminosäuren abspaltet, und endlich gelang der Nachweis, daß man nicht nur zu, sondern immer Aminosäuren im Darminhalt findet. Wie wiederholt betont worden ist, spaltet der Pankreassaft aus Eiweiß- ab und frühzeitig Aminosäuren ab. Diese Beobachtung erklärt vielleicht, weshalb man im Darminhalt stets Aminosäuren antrifft. Ihre Anwesenheit sagt an und für sich nichts über den Grad des Abbaues der Proteine aus, denn es können neben einigen wenigen Aminosäuren große Mengen von aus mehreren solchen zusammengesetzten Produkten übrig bleiben. Hier mußten zur weiteren Aufklärung des Abbaus von Eiweiß durch die Fermente des Darmkanals zunächst Untersuchungen im Reagenzglas einsetzen. Es galt festzustellen, welche Aminosäuren zuerst in Freiheit gesetzt werden. Ferner mußte verfolgt werden, ob das Trypsin imstande ist, Eiweiß vollständig in seine Bausteine aufzulösen. Zahlreiche Versuche ergaben übereinstimmend, daß die einzelnen stoffen Aminosäuren verschieden rasch abgespalten werden. Tyrosin, Tryptophan^) und auch Zystin erscheinen sehr bald in der Verdauungsflüssigkeit. Das Tyrosin zeigt sich, wenn seine Konzentration in der Verdauungsflüssigkeit groß genug ist, selbst an, wenn es in Freiheit gesetzt worden ist. Es kristallisiert infolge seiner Schwerlöslichkeit direkt aus. Das Tryptophan läßt sich mittels der Bromwasserreaktion nachweisen. Nur die freie Aminosäure gibt eine rosarote Färbung. Solange das Tryptophan mit anderen Aminosäuren verbunden ist, fällt die Brorawasserreaktion mit dem Verdauungsgemisch negativ aus. Seine Abspaltung erkennt man am Auftreten der typischen Reaktion. Sie verstärkt sich mit der Zunahme des Gehaltes der Lösung an freiem Tryptophan. Das Freiwerden des Zystins läßt sich nicht ohne weiteres erkennen, weil in den meisten Proteinen nur wenig von dieser Aminosäure enthalten ist. Man muß in diesem Falle das Zystin direkt isolieren. Die folgenden Beispiele^) zeigen, daß z. B. Tyrosin und Glutaminsäure durch Pankreassaft sehr verschieden rasch aus Eiweiß abgespalten werden.) Während die erstere Aminosäure schon nach kurzer Zeit in ihrer ganzen Menge in freiem Zustand in der Verdauungsflüssigkeit vorhanden — ») Vgl. R. V. Lengyel: Paul Hdri: Ebenda Pflügen Archiv. 115. 7 (1906). 115. 11 (19Ü6); 121. 459 (1908). E. Gräfe: Archiv f. Hygiene. 62. 21(i (1907). ) Vgl. hierzu auch Otto Fürth und Fritz Lieben: Biochem. Z. 109. 153 (1920). '') Vgl. hierzu Emil Abderhalden und Bela Reinbold: Zeitschr. f. physiol. Chemie. — — Emil Abderhalden und Karl Vöqtlin: Ebenda. 53. 284 (1905); 46. 1.59 (1905). Emil Abderhalden und Alfred Gigon: Ebenda. 53. 119 (1907). 315 (1907). 44. — ) Ob die hier geschilderten Verhältnisse für jede Proteinart zutreffen, ist natür- lich fraglich. Eiweißstoffe Es wird die Zusammensetzung und maßgebend sein. Abderhalden. Physiologische Chemie. I, Teil, ü. AaÖ. der ganze Aufbau der einzelnen gi XXIII; Vorlesung. 482 die Glutaminsäure ganz allmählich abgespalten. Die Versuche der folgenden Weise durchgeführt worden. Je 100 g Eiweiß wurden mit einer bestimmten Menge von Wasser und Pankreassaft bzw. Ti-ypsin in eine Flasche gefüllt. Zur Verhinderung der Fäulnis wurde das Gemisch mit Toluol überschichtet. Nun wurden sämtliche Flaschen gleichzeitig in einen auf 87^ erwärmten Brutschrank gebracht. An jedem ist, wird sind in Tage wurde dann eine Flasche zur Untersuchung auf den Gehalt an freiem Tyrosin und abgespaltener Glutaminsäure aus dem Brustschrank entfernt. Die Ausbeuten an den beiden Aminosäuren sind im ersten Versuche in Prozenten des Gehaltes des verwendeten Proteins an den entsprechenden Verbindungen angegeben. Im Versuche mit Kasein sind die absoluten Mengen der isolierten Aminosäuren angeführt: Versuch 1: Edestin aus Baumwollsamen: Tag 2 Tage 3 Tage 7 Tage 16 Taire 78-4 97-6 97-6 100 100 ... 43 74 10-9 311 602 3 Tage 6 Tage 9 Tage 4-40 4-66 4-88 A'M Dauer der Verdauung Abgeschiedene Menge von Tyrosin Abgeschiedene Menge von Glutaminsäure 1 Dauer des Versuches: Tag 0-75 1-53 1 1-76 901 Tage 4-20 8-65 11 Die angewandte Menge Kasein enthielt 13 Tage 17 Tage 4-50 8-80 4-46 8-90 45 g Tyrosin und Tage 4-HH 21 9-27 lOS (/ Glutaminsäure. Gleichzeitig lassen sich auch noch andere Aminosäuren, wie Alan in. i n, A s p a r a g i n s ä u r e, Ly s i n, Ar g i n i n und H i s t i d i n in der Verdauungsflüssigkeit nachweisen. Vermißt werden meist vollständig Prolin und Phenylalanin. 1) Die Resultate dieser Versuche sind nicht immer gleichartige. Vor allem zeigen sich erhebliche Unterschiede in den Mengenverhältnissen, in denen die einzelnen Aminosäuren abgeschieden werden. Ferner gelang es in einzelnen Fällen, auch Prolin und Phenylalanin aufzufinden. Diese Unterschiede sind ohne Zweifel auf die Beschaffenheit der verwendeten Trypsinpräparate zurückzuführen. Die Handelspräparate werden zum großen Teil durch Ausziehen der Pankreasdrüse dargestellt. Dabei gelangen auch Zellfermente in das Präparat und auch andere Stoffe. V a i n, L e u z 1 Manche davon hemmen die Trypsinwirkung. Es kann auch vorkommen, daß beigemischte Zellfermente Wirkungen entfalten, die dem Trypsin fremd sind. Aber auch dann, wenn man mit Pankreassaft arbeitet, ergeben sich Unterschiede. Sie sind wahrscheinlich zum großen Teil durclj den Zusatz von Darmsaft zu diesem bedingt. Er wird zugefügt, um das Trypsinzymogen mittels der Enterokinase zu aktivieren. Wir werden gleich erfahren, daß der Darmsaft über ein Ferment verfügt, das säureamidartige Verbindungen zwischen Aminosäuren löst. Verwenden wir zur Aktivierung des Zymogens des Trypsins Enterokinase, dann geben wir auch zugleicli das betreffende Ferment hinzu. Trotz der in manchen Punkten verschiedenen Resultate läßt sich als eindeutig gesichertes Ergebnis anführen, daß durch Pankreassaft 1» e- M Vgl. hierzu: Emil Fischer und Emil Abderhalden : Zeitschr. 39. 81 (1903); 40. 215 (1903). f. physiol. Chemie. Kiweißstoffe und ihre Bausteine. 48o Stimmte Aminosäuren sehr frühzeitig vollständig aus Eiweiß abgespalten werden, während andere Bausteine lang- samer folgen, und manche sich als nicht aus ihrer Bindung lösbar erweisen. Es sind im Eiweißmolekül offenbar Gruppen von Aminosäuren untereinander vereinigt, die vom Trypsin bzw. den peptolytischen Fermenten des Pankreassaftes nicht gespalten werden können. Es muß noch ein weiteres Ferment eingreifen, nämlich das schon erwähnte, im Darmsaft enthaltene. Es ist von Otto CoJinheim^) entdeckt und Erepsin genannt worden. Es löst Bindungen zwischen Aminosäuren, die den Fermenten des Pankreassaftes nicht zugänglich sind. Dieser gegen dieses letztere Ferment widerstandsfähige Rest enthält hauptsächlich Prolin und Phenylalanin. 8chon Kühne'-) war es bekannt, daß das Eiweiß von Trypsin nicht gleichmäßig abgebaut wird. Er beobachtete schon, daß ein widerstandsfähiger Rest zurückbleibt. Er nannte ihn Antipepton. Dieser der Trypsinwirkung widerstehende Rest wird von Erepsin in seine Anteile zerlegt. Ist somit das Trypsin nicht imstande, Eiweiß restlos bis zu Aminosäuren aufzuspalten, so gelingt dies, wenn Trypsin und Erepsin zusammenwirken. 3) Es dauert allerdings ziemlich lange, bis im Reagenzglasversuch der Abbau von Eiweiß durch Pankreas- und Darmsaft vollständig bis zu Aminosäuren durchgeführt ist. Bei der Beurteilung dieses Umstandes dürfen wir nicht außer acht lassen, daß der Reagenzglasversuch, wie schon wiederholt betont wurde, uns nicht vollständig über den Abbau der Proteine im Magendarmkanal aufklären kann. Wir k(>nnen mit seiner Hilfe nur die wichtige Frage entscheiden, in welcher Art der Abbau qualitativ verläuft und vor allem, ob der tierische Organismus dem Darmkanal Fermente übergibt, die die Proteine restlos bis zu den Aminosäuren abbauen können. Diese Frage muß bestimmt bejaht werden. Im Magendarmkanal sind alle Bedingungen gegeben, um den Abbau der Proteine und Peptone bis zu den einzelnen Bausteinen durchzuführen. Man könnte einwenden, daß der Abbau der Peptone bis zu den Aminosäuren so viel Zeit in Anspruch nimmt, daß er in Wirklichkeit im Darmkanal nie in vollem Umfange durchgeführt werden kann. Es ist sehr schwer abzuschätzen, wie lange jeder einzelne Teil des Chymus, der den Magen verläßt, im Darme verweilt, ehe er zur Resorption gelangt. Soviel ist jedoch sicher, daß diese Zeit eine sehr beschränkte sein muß. Betrachtet man nämlich den Darminhalt zu verschiedenen Zeiten der Verdauung, dann findet man immer nur einen relativ ganz geringen Belag von Chymus auf der Darmschleimhaut ausgebreitet. Es kommt unter normalen Verhältnissen nie zur Ansammlung größerer Mengen von Chymus. Allerdings muß bei der Abschätzung der gesamten Menge des im Darmkanal ausgebreiteten Speisebreies immer in Betracht gezogen werden, daß er eine große Oberfläche einnimmt. Vergleicht man jedoch den Inhalt des prall 1) 0. Coknheim: Zeitschr. f. pliysiol. Clieniie. 33. 451 (1901); 35. 134 (1902); 47. 28G (1906). -) Vgl. hierzu Emil Abderhalden und Peter Bona: Zeitschr. f. physiol. Chemie. Kmil Abderhalden: Ebenda. 77. 22 (1912). 405 (1910). ') M. Kühne: Vgl. auch Fr. Kutscher: Zeit]'irchoira Archiv. 39. 130 (18G7). schrift f. physiol. Chemie. 2ö. 195 (IH'.tS): 26. 110 {189S) und 28. 88 (1899); Ber. d. M. Sieafried: Zchsdu-. Deutschen Chem. Gesellsch. 33. 4357 (1899); 34. .')()4 (1900). Vd. aucli el)enda. 38. 259 (1903); 45. 252 (1905). (1899). f. physiol. Chemie. 27. 335 67. — — — — 81 XXIII. Vorlesung. 484 gefüllten Magens mit dem des Darmkanals, dann wird sofort klar, daß fortwährend Resorption stattfinden muß, denn sonst müßte man bei vollbeim Hunde etwa 6 8 Stunden nach der ständig entleertem klagen viel mehr Chymus im Dünndarm finden als dann, wenn der Mahlzeit Magen nur einen Teil seines Inhaltes abgegeben hat. Es ist dies jedoch nicht der Fall. Der Dünndarm zeigt während der Verdauung einer Mahlzeit annähernd die gleiche Füllung, wenn diese einige Zeit im Gange war. — — Die Verdauung verläuft wahrscheinlich — in den folgenden Phasen. Es verläßt jeweilen eine geringe Menge des durch den Magensaft vorverdauten Chymus den Magen. Dieser kommt sofort mit den Sekreten des Darmes Berührung. Die Wirkung des Pepsins wird bald ausgeschaltet, sofern nicht durch noch nicht gelöstes Protein geschützt ist. Länger wirkt das Labferment. ^) Schließlich werden auch ihm die Bedingungen seiner Wirksamkeit entzogen. Es setzt nun die Trypsin- und die Erepsin Wirkung ein. Bald erfolgt die Abspaltung von Aminosäuren. Gleichzeitig setzt die Resorption ein und entfernt die sich bildenden Spaltprodukte. Im Reagenzglasversuch fallen alle Regulationen weg. Wir geben Pankreassaft zu einem Eiweißkörper oder zu einem Peptongemisch und vollenden dann den Abbau durch Zugabe von Darmsaft. Wir schatten im vorneherein ganz unnatürliche Verhältnisse, weil wir das Zusanimenspiel von Trypsin und Erepsin in quantitativer Hinsicht gar nicht berücksichtigen. Ferner lassen wir die Abbauprodukte liegen. Endlich bleibt die während der Verdauung sich ausbildende Reaktion auch dann, wenn wir sie regulieren, ohne die nötige Anpassung an die vielleicht von Fall zu Fall verschiedenen Anforderungen an eine optimale Reaktion für bestimmte Fermentwirkungen. Im Darmkanal wird diese ohne Zweifel stets in bestimmter Weise eingestellt und so verschoben, daß die Ferment Wirkung sich voll entfalten kann. Es ergibt sich aus diesen großen unterschieden in den Bedingungen, unter denen der Abbau der Proteine und Peptone im Darmkanal und Reagenzglas sich vollzieht, daß wir die Zeit, die vergeht, bis im letzteren Falle Eiweiß bis zu Aminosäuren zerlegt ist, nicht auf die Verdauung unter natürlichen Verhältnissen übertragen dürfen. Für eine solche AuffassungSprechen auch die oben erwähnten Versuche, in denen gezeigt werden konnte, daß die Peptone des Darminhaltes auch außerhalb des Darmes bald fast vollständig bis zu Aminosäuren abgebaut werden. Es spielt das Mischungsverhältnis zwischen Chymus, Darm- und Pankreassaft und Galle sicher eine große Rolle beim raschen Abbau der einzelnen zusammengesetzten Nahrungsstoffe. Wir können diese Bedingungen deshalb nicht nachahmen, weil wir sie noch nicht kennen. Wir kommen somit zum Schlüsse, daß die Möglichkeit gegeben ist, daß im Darmkanal die Eiweißkörper und Peptone bis zu Aminosäuren abgebaut in es werden und im Wesentlichen Eiweißbausteine zur Resorption gelangen. M Vgl. S. 474. Vorlesung XXIV. und ihre Bausteine. Eiweißstoffe Verhalten der Eiweißstoffe im tierischen Organismus. Ihr Abbau im Darmkanal. Überblicken wir das, was wir über die Einwirkung der Proteasen des Magen-, Pankreas- und Darmsaftes auf Eiweiß wissen, so kommen daß Pepsin die Eiweißkörper in Peptone wir zum Schlüsse, zerlegt, ohne dabei Aminosäuren in Freiheit zusetzen, während das Trypsin frühzeitig solche abspaltet, jedoch nicht imstande ist, alle Bindungen zwischen den Bausteinen der Proteine zu lösen. Eine Zerlegung auch dieser Komplexe führt das Erepsin herbei. Wenn wir von Trypsin und von seiner Wirkung sprechen, und es bald Proteine, bald Peptone verschiedenster Art abbauen lassen, so folgen wir einem allgemein angenommenen Brauche. Es ist jedoch durchaus fraglich, ob das Trypsin einheitlich ist und sowohl auf die verschieden- Proteine als auch auf viele seiner Abbaustufen eingestellt ist. vielmehr sehr wahrscheinlich, daß das Trypsin eine Mischung verschiedenartiger Fermente darstellt, und teils Proteasen, teils Peptasen, d. h. Fermente, die Eiweiß, und solche, die Peptone angreifen, umfaßt.') Wichtig ist, daß im inaktiven Pankreassaft Erepsin vorkommen kann. Es scheint nicht immer zugegen zu sein. Seine Anwesenheit wurde deshalb erschlossen, weil inaktiver Pankreassaft Kasein angreifen kann, zugleich aber andere Proteine nicht abbaut. Nun besitzt das Erepsin die Eigenschaft, außer Peptonen auch Kasein abzubauen. Wahrscheinlich stellt es auch wieder ein Gemenge von Fermenten dar. Da wir zur Zeit noch keinen genauen Einblick in die Struktur der Proteine haben, d. h. wohl wissen, daß Aminosäuren säureamidartig unter einander verknüpft sind, dagegen noch nicht mit Bestimmtheit erweisen z. B. in Form von Ankönnen, ob daneben noch andere Bindungsarten kernartige Gruppierungen bilden, die zunächst von einhydridkomplexen ander gelöst werden, bevor es zum Abbau von Polypeptiden kommt, können wir über die einzelnen Vorgänge beim fermentativen Eiweißabbau nur wenig aussagen. Gut unterrichtet sind wir über den Abbau von Polyartigsten Es ist — ) Vgl. L'mil Äbihrhalden — und Antun Fodor: Fermentforscliung. 6. 248 (1922). XXIV. Vorlesung. 486 Die bisherig-en ErfahruDgen über die Hydrolyse von solchen Peptiden. mittelst peptolytischer Fermente haben ergeben, daß das zusammengesetzte ^lolekül nicht auf einmal in seine Komponenten zerlegt wird. Der Abbau stufenweise. ) Es wird zunächst am Anfang oder am Ende der Kette eine Aminosäure unter Aufnahme von Wasser abgespalten und auf diese Weise die Aminosäurekette um ein Glied gekürzt. Dann erfolgt vielmehr Abspaltung einer weiteren Aminosäure, bis schHeßlich nur noch Eiweißbausteine übrig bleiben. Für die Entscheidung der Frage, wie weit der Abbau der Peptone im Darmkanal geht, ehe die Resorption einsetzt, sind die folgenden im Keagenzglasversuch gewonnenen Befunde über den Eiweiß- und Peptonabbau durch die Fermente des Darrakanals von großer Bedeutung. Die Beobachtung, daß frühzeitig bestimmte Aminosäuren abgespalten werden, wenn man Pankreassaft auf Eiweiß oder Peptone einwirken läßt, wobei noch hochmolekulare zusammengesetzte Verbindungen zurückbleiben können, folgt die daß der Befund von Aminosäuren im Darminhalt nichts darüber aussagt, wie weit der Abbau der Peptone geht. Es kommt auf die Art der vorhandenen Aminosäuren an. Der l)eweist, Reagenzglasversuch hat gezeigt, daß bestimmte Aminosäuren, wie Prolin und Phenylalanin, erst dann zur Abspaltung gelangen, wenn der größte Teil der übrigen Aminosäuren in Freiheit gesetzt ist. Treffen wir auf jene Bausteine des Eiweißes, dann ist der Schluß berechtigt, daß ein tiefgehender Abbau stattgefunden hat. Im Darmkanal sind die Bedingungen für einen weitgehenden Abbau besonders günstige. Zuerst trifft der Chymus, der eben aus dem Magen entlassen worden ist, auf das Sekret der Brutmermhen Drüsen der Duodenalschleimhaut und auf das Pankreassekret. Hier setzt der Abbau energisch ein. Dann wird der Chymus weiter in tiefere Teile des Dünndarmes geschoben. Es wirkt jetzt vornehmlich der Darmsaft, in dem das Erepsin besonders wirksam ist. Wir müssen, falls der Abbau der Proteine und Peptone im Darmkanal ein weitgehender ist, erwarten, daß im Darminhalt alle Aminosäuren anzutreffen sind. Das ist nun in der Tat der Fall.-) Es sind alle bis jetzt bekannten Aminosäuren, soweit auf sie gefahndet wurde, im Inhalt des Dünndarms aufgefunden worden. Vor allem sind auch Prolin und Phenylalanin festgestellt worden. Würden wir den Abbau des ersten, den Magen verlassenden Chymus verfolgen können, dann wäre zu erwarten, daß man entweder nur die zuerst abspaltbaren •') Aminosäuren im Darminhalt antrefien würde oder hauptsächlich Aminosäuren, die erst bei schon weit fortgeschrittenem Abbau der Peptone erscheinen. Es käme ganz auf den Zeitpunkt der Untersuchung an. In Wirklichkeit verläuft der Abbau so schnell, daß in kurzer Zeit alle Amino- säuren zugegen sind. Außerdem wird sofort neuer Chymus nachgeschoben. M Vgl. hierzu: Kmil Abderhalden und A. H. Koelkcr, Alfred Gigon, Carl Hrahm: Zeitschr. f. phvsiol. Chemie. 51. 264 (1907); 52. 32^ (1907); 54.363 (19Ü8); 53. 251 (1907); 57. 342 (1908). ") Emil Abderhalden: Zeitschr. f. physiol. Chcm. 78. 382(1912); 81. 315 (1912); 114. 290 (1921). Vgl. über den Nachweis von Aminosäuren im Darmiiihalt auch: /''. Kutscher und J. Seemami: Zeitschr. f. physiol. Chem. 35. 432 (1902). - "A'. P. Cathcarf — und./. B. Leathes: Journ. of Physiol. 33.'4()2 (1906). ^) Neuere, noch nicht mitgeteilte Versuche bestätigen den Befund aller bis jetzt lif-kannten Aminosäuren im Darminhalt. Eiweißstoffe und ihre Baiistoine. ,487 vom Magen abgejrebeue Chymus auf einer tiefen des Abbaus, so setzt eben die Abspaltung von Aminosäuren in der {'hymusmenge ein, die den Magen später verlassen hat. Wir müssen somit stets, wenn wir Darminhalt untersuchen, alle möglichen Grade des EiweißBetindet sich der früher JStufe und Peptonabbaus nebeneinander linden. Die Untersuchung des Darm Inhaltes aufAminosäuren stützt die Annahme, daß die Proteine im Magendarmkanal tief abgebaut w^erden. Da wir jedoch aus den wiederholt angegebenen Gründen neben Aminosäuren stets Peptone antreffen und niemals nur Bausteine der Proteine, so dürfen wir aus dem Befunde jener Aminosäuren im Darminhalt, die erst in Erscheinung treten, wenn das Eiweißmolekül schon sehr weit abgebaut ist, noch nicht den Schluß ziehen, daß alle Peptone zu Aminosäuren aufgelöst werden, bevor die Resorption einsetzt. Es könnten ja fortwährend Peptone dem weiteren Abbau durch die Aufnahme in die Darmwand entzogen werden. Es wäre in diesem Falle von größtem Interesse, zu erfahren, weshalb einzelne Peptone tief abgebaut werden und andere nicht. Anzunehmen, daß der tiefgehende Abbau dem Zufall unterworfen ist und nur jene Peptone betrifft, die nicht rasch genug zur Resorption gelangen, erscheint gezwungen. Es liegt jedoch die folgende Möglichkeit vor. Der Abbau resorbierter Peptone kann in der Darmwand fortgesetzt und vollendet werden. Bekanntlich hemmen die Eiweißabbaustufen den weiteren Verlauf der Fermenthydrolyse. Durch die fortwährende Fortnahme der sich bildenden Spaltprodukte wird ein m(»glichst rascher Abbau der Peptone gewährleistet. Es würden in diesem Falle der Abbau im Darmkanal und jener in der Darmwand sich gegenseitig unterstützen. Die weitgehende Hydrolyse im Darmkanale würde als Vorarbeit der Spaltung resorbierter, noch zusammengesetzter Verbindungen in den Zellen der Darmwand zu betrachten sein. Das ganze Problem würde wesentlich klarer liegen, wenn ganz scharf und eindeutig zu entscheiden wäre, ob die Darmschleimhaut unter normalen Bedingungen Peptone aufnimmt. Es genügt durchaus nicht, festzustellen, daß bestimmte Zellen zur Aufnahme eines Stotfes gezwungen werden können, um die Frage zu entscheiden, wie ein bestimmtes Gewebe sich unter normalen Verhältnissen verhält. Es sei in dieser Beziehung z. B. auf die Niere verwiesen. Sie läßt unter normalen Bedingungen keinen Zucker in den Harn übertreten. Eine Veränderung des Gebaltes des Blutes an bestimmten Ionen und eine Änderung der Reaktion des Blutes genügt, um sie für Glukose durchlässig zu machen, ja schon ein Ansteigen des Gehaltes des Blutes an Zucker reicht aus, um diesen durch die Nierenepithelien zur Ausscheidung gelangen zu lassen. Gewiß können wir unter geeigneten Bedingungen auch Rohrzucker im Darmkanal zur Resorption bringen. Bei diesem Disaccharid ist ganz eindeutig festgestellt worden, daß es nicht ins Blut übertritt, wenn wir nicht absichtlich durch Zufuhr großer Mengen davon seinen Übergang erzwingen. )b nun der Rohrzucker unter normalen Verhältnissen bereits im Darmkanal vollständig zerfällt oder zum Teil erst in der Darmwand, ist für die Bedeutung der Hydrolyse des Rohrzuckers gleichgültig. Er ist für die Zellen des tierischen Organismus so hinge unverwertbar als seine Komponenten in ihm gebunden sind, dagegen sind diese selbst Trauben- und Fruchtzucker für diese von größter Bedeutung. ( — — XXIV. Vorlesung. 4gg Die bisher durchgeführten Versuche über die Aufnahmefähigkeit von Peptonen durch die Darmschleimhaut ergeben mit Wahrscheinlichkeit, daß der Abbau nicht bis zu Aminosäuren zu gehen braucht, sondern, daß auch zusammengesetzte Verbindungen zur Resorption gelangen können. So hat man z. B. Hunden, denen eine Darmfistel angelegt worden war, verdünnte Peptonlösungen in den Darmkanal eingeführt, i) Nach einiger Zeit wurde der Darm wieder durch die Fistel entleert, und sein Inhalt ausgespült. Der Unterschied im Stickstoffgehalt des zugeführten Produktes und desjenigen des wiedergewonnenen Darminhaltes ergibt die Menge derjenigen stickstoffhaltigen Substanzen, die resorbiert worden sind, vorausgesetzt, daß nicht durch Sekretion von Darmsaft stickstoffhaltige Verbindungen zu den durch die Fistel eingeführten hinzugekommen sind. Es lassen sich gegen derartige Versuche mancherlei Einwände erheben. Sie geben die natürlichen Verhältnisse nicht wieder. Der physikalische Zustand verdünnter Lösungen und derjenige des Chymus weichen zu sehr von einander ab. Der Chymus ist in dünner Schicht auf einer großen Oberfläche des Darmes Eine Summe von Fermenten wirkt unter ganz bestimmten ausgebreitet. Bedingungen auf ihn ein. Ohne Zweifel wird der ganze Reaktionsverlauf beständig in feinster Weise eingestellt. Spritzen wir eine Lösung mit einem Gemenge von künstlich hergestellten Abbaustufen in den Darm ein. dann schaffen wir Bedingungen, wie sie sich unter normalen Verhältnissen nie finden. Wir schließen unter Umständen die Wirkung einer ganzen Reihe von Fermenten, die zum raschen Abbau eines Peptongemisches notwendig sind, ganz aus. Die Zufuhr der Peptonlösung erfolgt in einen relativ eng Es fragt sich, ob dieser allein die ihm ohne begrenzten Darmabschnitt. jede weitere Vorbereitung übergebenen Stoffe in geeigneter Weise zerlegen und die entstandenen Produkte der Darmwand übergeben kann. Auch bei der Frage der Resorption der Fettsäuren und Seifen ergaben jene Versuche, bei denen diese Verbindungen in bestimmte Darmabschnitte eingeführt wurden, keine eindeutigen Resultate.^) Die Aufnahme der zugeführten Peptonlösung von Seiten der Darmwand war nun ungefähr eine ebenso rasche, wie wenn Aminosäuren in gelöster Form in den Darmkanal gebracht wurden. Aus diesem Ergebnis wurde der Schluß gezogen, daß Peptone als solche von der Darniwand aufgenommen werden. Diese Folgerung entbehrt jedoch der eindeutigen Begründung. Es ist nicht ausgeschlossen, daß der resorbierte Anteil der in den Darmkanal eingeführten Peptone vor der Aufnahme gespalten worden ist. Daß die in Lösung zugeführten Aminosäuren nicht sofort zur Aufnahme gelangten, kann seinen Grund darin haben, daß ungünstige, den natürlichen Verhältnissen keine Rechnung tragende Bedingungen vorlagen. Normalerweise finden wir im Darminhalt neben Peptonen immer nur sehr geringe Mengen von Aminosäuren. Dem stufenweisen Abbau geht die Resorption parallel. Niemals begegnen wir einer größeren Menge einer verdünnten Aminosäurelösung. ») Vgl. u. a. F. Nolf: Bull, de rAcad. royale de Belg. Nr. 12. 1149 (1903); Edgar Zum: Nr. 2. 153 (1904): .louru. de phys. et de patli. gön^rale. 925 (1907). //. Messerli: Biochem. Arch. de pharmacodyuamie et de th(!rapie. 15. Nr. 3 (1908). Vgl. auch O. Cohnheim : Zeitschr. f. physiol. Chem. 84. Zeitschr. 54. 446 (1913). — — — 419 (1918). '') Vgl. dazu /.. B. 0. /•. Fürth und ./. Schütz: Hofnieisten^ Beitr. 10. 462 (1907). Eiweißstoffe und ihre Bausteine. 489 Es ist ferner betont worden, daß schon deshalb nicht an einen Abi »au der Peptone bis zu Aminosäuren gedacht werden dürfe, weil weit abgebaute Peptone und manche Aminosäuren auf die Darmschleimhaut reizend wirken sollen. Nun entstehen unter normalen Verhältnissen die Aminosäuren immer nur in geringen Mengen. Die Resorption setzt sofort ein. Außerdem reizen nur bestimmte Aminosäuren, wie z. B. die Glutaminsäure, das Lysin und Arginin usw., die Darmschleimhaut etwas, jedoch erst in höherer K^onzentration. Die Reizwirkung der durch fermentativen Abbau gewonnenen Eiweißabbaustufen dürfte meistens auf Produkte bakterieller Tätigkeit zurückzuführen sein. Man findet, wenn die Verdauung nicht unter Ausschluß von Bakterien durchgeführt wird, im Verdauungsgemisch sogenannte Amine. Wir kommen auf diese Verbindungen noch zurück. Sie bilden sich auch im Darmkanal unter dem Einfluß der Darmflora. Wir kennen auch Peptone die stark reizend wirken, sobald sie in höheren Konzentrationen zugegen sind. Trotzdem werden sie beim Abbau der Proteine im Magendarmkanal gebildet. Sie entstehen jedoch immer nur in kleinsten Mengen. Der Abbau schreitet rasch fort. Können wir somit den vorliegenden Versuchen über die Resorption von Peptonen und Aminosäuren keine entscheidende Bedeutung zuerkennen ^), so müssen wir doch unbedingt mit der Möglichkeit rechnen, daß auch zusammengesetzte Abbaustufen der Proteine zur Resorption gelangen. Wenn wir alle vorliegenden Ergebnisse zusammenfassen, dann kommen wir zum Schlüsse, daß das Eiweiß derNahrung im Magendarmkanal sicher zu Peptonen und Aminosäuren abgebaut wird. Wie weit der Abbau jedes einzelnen Peptons geht, bevor die Resorption einsetzt, darübergeben die bisherigen Untersuchungen keine sicheren Anhaltspunkte. Eine direkte Beweisunmöglich, weil wir die Verdauung im Darmkanal immer nur Immer kommt aus dem Magen neuer Chymus zum bereits vorhandenen hinzu und überschwemmt den Darm mit Peptonen. Gleichzeitig arbeitet auf der anderen Seite die Resorption der Verfolgung des weiteren Schicksals der Peptone entgegen. Wir können somit nur mit Wahrscheinlichkeitsgründen und indirekten Versuchen uns ein Urteil über den Grad des Abbaus der Proteine unter natür- führung ist in bestimmten Momenten untersuchen können. Bedingungen bilden. Wichtig ist, daß der tierische Organismus in den Darmkanal Fermente abgibt, die in gemeinsamer Wirkung das Protein molekül restlos bis zu den Bausteinen zerlegen können. Von Bedeutung ist ferner der Befund aller Aminosäuren im Darminhalt. Selbst jene Aminosäuren, die bei der Verdauung im Reagenzglas spät in Erscheinung treten, sind im Darmkanal anzutreffen. lichen L. Borchardt [Zeitschr. f. physiol. Chem. 51. .ÖÜB (1907); 57. 305 (1908)] gelang Pepton aus Elastin zur Resorption zu bringen, wenn er es in größeren Mengen Dieser Befund, der übrigens von uns [Emil Abderhalden und Ernst Rilhl einführte. Zeitschr. f. physiol. Chem. 69. 301 (1910)] nicht bestätigt werden konnte, gestattet keine Schlüsse auf die Art der unter gewöhnlichen Verhältnissen zur Resorption gelangenden Das betreffende, infolge seiner Eigenschaften leicht nachweisbare Eiweißabbaustufen. Überschwemmt man den Magendarmkanal Pepton wird offenbar sehr schwer abgebaut. mit ihm, dann kann man es nach Borchardt zur Resorption bringen. ') es, ein XXIV. Vorlesung. 490 Die Diskussion des Problems nach teine und Peptone im Magendarmkanal dem Grade des Abbaues der Proeine führt uns zu der Frage, ob weitgehende Zerlegung der genannten Verbindungen aller Voraussicht nach notwendig ist? Kann der tierische Organismus seinen Geweben nicht auch mit Peptonen und Polypeptiden auskommen? Die Beantwortung dieser Frage ist außerordentlich schwierig. Wir müssen vorausschicken, daß die Eiweißstoffe der Nahrung in den Geweben ganz Einmal bildet der tierische Organismus verschiedenen Zwecken dienen. Er gibt solche mit den Sekreten nach außen beständig neue Proteine. Ferner verrät uns jedes wachsende Haar, jeder wachsende Nagel, ab. daß fortwährend Keratinsubstanzen gebildet werden. Die Galle zeigt uns, wie wir noch erfahren werden, an, daß fortwährend rote Blutkörperchen zugrunde gehen. Sie müssen ersetzt werden. Droht ein Einwandern von Mikroorganismen, oder dringt sonst etwas Fremdartiges in den Körper ein, oder bilden sich in ihm Produkte, die sich sonst nicht finden Blutgerinnsel, Thromben, feste Eiweißmassen nach Entzündungen usw. dann beobachten wir, daß ungezählte Leukozyten an die betreffende Stelle eilen. Sicher sind diese Zellen nicht alle vorher schon im Körprr vorhanden gewesen. Viele davon sind neugebildet. Schon die Eigenart vieler dieser Zellen weist auf eine Neubildung hin. Beim wachsenden Organismus kommt noch die Vermehrung von Gewebe hinzu. Bei jeder Neubildung von Eiweiß tritt uns die Frage entgegen, ob es durch Umwandlung aus bereits vorhandenem Protein entstanden sein kann oder, ob ein Autbau von einfachen und einfachsten Abbaustufen aus notwendig ist. Überblicken wir die bisherigen, eindeutig festgestellten Ergebnisse über die Verwendbarkeit bestimmter Eiweißabbaustufen zu bestimmten Zwecken, dann können wir zurzeit nur eines mit voller Bestimmtheit ausin — — daß die Aminosäuren für zahlreiche Vorgänge als Ausgangsm aterial dienen. Von ihnen aus führt der Weg zu den Kohlehydraten und zu zahlreichen anderen Verbindungen.^) Bei ihnen setzt der Abbau zu den Stoftwechselendprodukten ein. Über sie führt auch der Aufbau zu zusammengesetzten Produkten. Überall, wo wir hinblicken, Sie nehmen im Stoffwechsel die gleiche stoßen wir auf Aminosäuren. Stellung ein, wie der Traubenzucker bei den Kohlehydraten und die Fettsäuren und der Alkohol bei den Fetten. Geht nun die Synthese von Zelleiweiß oder den Proteinen de rKörperflüssigkeiten stets von Aminosäuren aus, sagen, nämhch, oder können zusammengesetzte Bruchstücke des Nahrungseiweißes zum Aufbau verschiedenartiger Proteine ver- wendet werden? Diese Frage, deren genaue Beantwortung uns einen sicheren Einblick in den Umfang des Eiweißabbaues im Darmkanal bzw. läßt sich deshalb zurzeit nicht exakt in der Darmwand ergeben würde, beantworten, weil wir über den Aufbau der einzelnen Proteine nicht genügend unterrichtet sind. Würde es gelingen, aus den verschiedenartigsten Körpereiweißstoffen und denjenigen der Nahrung Polypeptide oder sonstige Bruchstücke zu isolieren, die genau die gleiche Struktur und Konfiguration besitzen, dann könnte man sich wohl vorstellen, daß derartige Produkte kommen und nicht dem weiteren Abl)au unterliegen. Man zur Resorption ») Vgl. S. iy2. Eiweißstoft'e müßte jedoch uud ihre Bausteine. diesem Falle derartige Bruchstücke in jenseits des Darmes wieder linden können. 491 in irgend einer Form Wir können sie so lange nicht suchen, als wir über ihre mögliche Struktur nichts wissen. Vorläufig haben wir als einzige sichere Grundlage zur Entscheidung der Frage nach den Beziehungen der Eiweißstolfe der Nahrung zu denen der Gewebe des tierischen Organismus ihren Gehalt an einzelnen Aminosäuren. Die vergleichende Hydrolyse zahlreicher Proteine hat ergeben, daß sie mit wenig Ausnahmen die gleichen Aminosäuren enthalten. Es zeigen sich jedoch große Unterschiede in den Mengenverhältnissen, in denen die einzelnen Bausteine in jeder Proteinart vertreten sind. Es sind bis jetzt noch keine Proteine gefunden worden, die bei der Hydrolyse genau die gleichen Mengen der einzelnen Aminosäuren ergeben hätten. Selbst, Avenn wir Eiweißstollte antretten würden, die aus den gleichen Bausteinen aufgebaut wären und diese in den gleichen Mengenverhältnissen enthielten, so würde das. wie wir wiederholt betont haben, nichts über die Struktur und Konfiguration der betretifenden Ausgangsmaterialien aussagen. Eine gewaltige Zahl von isomeren Eiweißarten könnte das gleiche Resultat liefern, wenn wir nur die Bausteine in Betracht ziehen, die sie beim vollständigen Abbau liefern. 1) Ein einfaches Beispiel möge zeigen, daß wir uns nach den jetzigen Kenntnissen den Umbau eines Proteins in ein anderes mit verschiedener Struktur nur unter der Voraussetzung eines vollständigen Abbaus zu den einzelnen Aminosäuren vorstellen können. Wenn z. B. Glyzyl-alanin in Alanyl-glyzin übergeführt werden soll, so spalten wir die erstere Verbindung in Glykokoll und Alanin und bilden dann aus diesen Bausteinen das Dipeptid Alanyl-glyzin. In diesem einfachsten Falle könnte man auch einen anderen Weg versuchen. Es ließe sich aus Glyzyl-alanin Glyzylalaninanhydrid gewinnen. Durch Aufspaltung dieser Verbindung entsteht neben Glyzyl-alanin auch Alanyl-glyzin.-) Für alle höheren Polypeptide fehlt jede Möglichkeit einer direkten Umwandlung. Das Tripeptid Alanylglyzyl-ty rosin kann nur dann in die Verbindung Alanyl-tyrosylglyzin übergeführt werden, wenn es durch Hydrolyse in seine Bausteine zerlegt wird, und dann die Synthese aus den drei Bausteinen folgt. Handelt es sich um die Gewinnung des Tripeptides Glyzyl-tyrosyl-alanin, so würde eine Abspaltung des Alanins genügen. Glyzyl-tyrosin könnte be- stehen bleiben. Dieses Dipeptid ergibt mit Alanin verkuppelt das ge- wünschte Glyzyl-tyrosyl-alanin. Es werden sich bei verschiedenen Proteinen gewiß ebenfalls Bruchstücke finden lassen, die einzelnen davon gemeinsam sind. Manche dagegen werden überhaupt keine identischen Abbaustufen aufweisen. Bestimmen wir den Gehalt eines bestimmten Eiweißes an Aminosäuren und füttern wir dann ein Tier damit, dann ergibt uns die Analyse der verschiedensten Gewebsproteine eine Antwort darauf, ob direkte Beziehungen zu dem aufgenommenen Protein vorhanden sind. Es sei gleich hier bemerkt, daß der Versuch, die Eiweißkörper des Blutplasmas durch die Art des verfütterten Proteins 1) Vgl. S. 367. Vgl. S. 357. XXIV. Vorlesung. 492 ZU beeinflussen, ein negatives Ergebnis hatte.') Am besten wählen wir zu einem solchen Experimente einen Eiweißkörper, der sich in seiner Zusammensetzung möglichst scharf von derjenigen der Proteine des Blutplasmas unterscheidet. Ein derartiges Protein ist das (lliadin. Es enthält auffallend viel Glutaminsäure, und zwar etwa viermal so viel, wie die Plasmaeiweißkörper. Dem Versuchstier, einem Pferde, wurden zunächst beim Beginne des Versuches sechs Liter Blut entzogen. Aus diesem wurden die Serumeiweißkörper abgetrennt und ihr Gehalt an Glutaminsäure und Tyrosin festgestellt. Nach acht Tage langem Hungern wurden nochmals sechs Liter Blut abgelassen und wiederum der Gehalt an den genannten Aminosäuren in den Eiweißkörpern des Blutplasmas bestimmt. Nunmehr erhielt das Versuchstier große Mengen von Gliadin. Das einige Zeit nach erfolgter Fütterung entnommene Blut enthielt Eiweißkörper, die die gleichen Mengen Glutaminsäure und Tyrosin aufwiesen, wie die des normalen Blutes. Es war somit nicht gelungen, die Zusammensetzung der Eiweißkörper des Blutplasmas durch Verfütterung einer bestimmt zusammengesetzten Eiweißart zu beeinflussen, trotzdem infolge des sehr großen Blutverlustes das Versuchstier unzweifelhaft gezwungen worden w^ar, sein Blut rasch wieder zu ergänzen. Sehr klar liegen die Verhältnisse beim wachsenden Tier und insbesondere beim Säugling. Dieser nimmt in seiner Nahrung, der Milch, immer in engen Grenzen das gleiche Gemisch von Eiweißstoflen auf. Neben Kaseinogen finden sich in der Milch noch Albumine, Globuline und in Alkohol lösliche Eiweißstotfe. Aus diesen Proteinen bildet der rasch wachsende Organismus all die verschiedenartigen Proteine seiner (Tewebe. Es entstehen Haare, Nägel, die Eiweißkörper des Blutes, der einzelnen Zellen, der Sekrete usw. Keines dieser Proteine zeigt direkte Beziehungen zu den Eiweißkörpern der Milch. Kaseinogen finden wir nirgends im Organismus als in der sezeniierenden Brustdrüse. Die Albumine und Globuline der Gewebe und der Körperflüssigkeiten sind andere als die entsprechenden Proteine der Milch. Bildet der Organismus die Eiweißkörper der Milch, dann muß er sie neu bilden. Solange der Fötus im Mutterleibe seine verschiedenartigen Gewebe aufbaut, ist er auf die Eiweißstofte des mütterlichen Blutplasmas angewiesen. Auch diese s:cigen keine direkten Beziehungen zu den Proteinen der sich bildenden und wachsenden Organe. Wir sehen ferner beständig Blut an den Zellen der Speicheldrüsen vorbeiströmen und beobachten, daß diese nach außen Muzin abgeben, eine Eiweißart, die dem Blute nicht zukommt. Zwar soll das Plasma einen muzinartigen Körper aufweisen, doch ist es mehr als fraglich, ob er in Beziehungen zum Muzin des Speichels steht. Jedenfalls werden Müzine auch dann gebildet, wenn die Nahrung vollständig frei von solchen oder diesen ähnlichen Proteinen ist. Die Zahl der Beispiele, die beweisen, daß der tierische Organismus seine ]^roteine selbst bereitet und sie nicht direkt übernimmt, ließe sich leicht vermehren. Die gegebenen dürften genügen, um den Beweis zu liefern, daß der tierische Organismus sich bei der Bereitung der Zelll)estandteile nicht nach der Art der aufgenommenen Nahrungsstofte richtet. ) (1905). Emil Abderhalden uud Franz Samuely: — E. Abderhalden, Casimir Funk und F. Zeitschr. S. f. physiol. London: Kbenda. Chem. 46. 193 269 (1907). 51. und EiweitJstoffe ihre Bausteine 493 Die folgende Zusammenstellung VI gibt einen Überblick über das Resultat der vergleichenden Bestimmung der Zusammensetzung von Proteinen der Milch und von einzelnen Eiweißkörpern des tierischen Organismus. Eine solche Tabelle kann natürlicb nur einen sehr rohen Einblick in den Aufbau der erwähnten Proteinarten geben. Würden wir an Stelle der Zahlen für die Ausbeuten an den einzelnen Aminosäuren die Strukturformeln der einzelnen Proteine setzen können, dann würde der Vergleich sich viel eindrucksvoller gestalten und jeden Zweifel ausschließen, daß dem Umbau eines bestimmten Proteins in ein anderes ein sehr eingreifender, tiefer Abbau voraus2:ehen muß. Nahrungs- Kasein'') Glykokoll Alanin Aminovaleriansäure Leuzin Serin Zystin Asparaginsäure Glutaminsäure Lysin Arginin Phenylalanin Tyrosin Serum- Albu- min :) 1 hu i ß 3-5 . . 2-2 09 10 2-7 0-9 vorhanden 105 19-5 200 02 007 .... 12 110 öS 8-5 48 31 21 10 2-4 2M) 3-8 e b s 6 Fibrin 3-0 3-6 tHiston aus i 5-4 4-2 2-5 1-5 2-8 2-3 ll-O w e i Ü Thymusdrüse Elastiu Kerati u 0-5 3-5 260 4-7 6-6 10 1-5 0-9 21-4 71 10 15-0 1-7 4-3 Histidin Asparaginsäure .... Glutaminsäure .... Lysin Arginin Phenylalanin Tyrosin 4-4 2-5 77 10 Z\'stin 0-6 0-3 3 1 4-0 Serin 0-7 1-0 81 Glykokoll Alanin Aminovaleriansäure Leuzin 29-0 18- 100 H-2 4-2 vorhanden 0-6 2-3 45 Histidin i SerumGlobiu aus globulin Hämoglobin m n 2-5 Prolin Prolin e w e 1) s e i w e ( i eiweiß-) 11-8 vorhanden — 0-6 20 100 8-0 0-5 6-9 0-8 15-5 3-7 20 2 2 0-3 3-9 3-5 2-5 5-2 0-34 3-2 1-5 1-7 3-4 1-5 ) Emil Abderhalden: Zeitschr. f. physiol. Ghem. 44. 17 (1905); Zentralbl. f. Stoffwechsel- und Verdauungskrankheiten. 5. 647 (1904); Med. Klinik. I.Nr. 1 und 2(191)5). ^) Die Zahlen beziehen sich auf lÜO y aschefreies, bei llü" bis zur Gewichtskonstanz getrocknetes Eiweiß. ') Kaseinogen ist ähnlich zusammengesetzt. XXIV. Vorlesung:. 494 Wir sind absichtlich etwas ausführlich auf die Frage nach dem Abbau der Proteine im Magendarmkanal eingegangen. Die Erörterung die sich einer exakten Beantwortung des gestellten Problemes entgegenstellen, zeigt an einem an und für sich recht einfachen Beispiel, W'ie schwer es ist, eindeutige Beweise zu erbringen, sobald wir es mit Vorgängen im lebenden Organismus zu tun haben. Solange es sich zunächst nur um qualitative Studien handelt, können wir in vielen Fällen ganz klar sehen, sobald jedoch die Quantität ausschlaggebend wird, mehren sich die einer exakten Bestimmung entgegenstehenden störenden iMomente, Der tierische Organismus arbeitet überall mit Spuren. Ein \'organg folgt dem andern unmittelbar. Nirgends kommt es unter normalen Verhältnissen zur Anhäufung von Zwischen- und Abbaustufen im StoÖwechsel. Können wir somit zurzeit nicht mit Bestimmtheit den Grad des^ Abbaues der Proteine im Magendarmkanal abgrenzen, so ist doch soviel ganz sicher festgestellt, daß eine sehr weitgehende Zerlegung der aus den Eiweißstoffen hervorgehenden Peptone stattfindet. Dadurch wird bewirkt, daß jede einzelne Zelle Bausteine zur Verfügung hat, die in nichts mehr an die Struktur des Ausgangsmateriales erinnern. Ob nun diese Bausteine durchwegs Aminosäuren sind, oder ob auch zusammengesetzte Verbindungen als Baumaterial in Betracht kommen, ist für die ganze Fragestellung nicht der Schwierigkeiten, wesentlich. Die Hauptsache ist, daß auf direktem und indirektem Wege der Beweis geführt worden ist, daß die Proteine der Nahrung erst dann für den Organismus verwertbar sind, wenn eine weitgehende Zerlegung einge- treten ist. Die Feststellung, daß die Eiweißstoffe der Nahrung Magendarmkanal einem weitgehenden Abbau unterliegen, ehe sie zu Eiweißstoffen derGe webe werdenkönnen, setzt voraus, daß der tierische Organismus die Fähigkeit im aus den gebildeten Bruchstücken Eiweiß aufzubauen. Daß er diese Synthese wirklich durchführen kann, beweisen Ver- besitzt, suche, bei denen Tieren kein Eiweiß, sondern Peptone verabreicht wurden i), ja 0. Loeivi^) vermochte sogar mit einem Produkte, das keine Biuretreaktion mehr ergab, Hunde im Stickstoftgleichgewicht zu erhalten. W^r sprechen dann von „Stickstoffgleichgewicht", Avenn das Versuchstier gleichviel Stickstoff ausscheidet, wie es aufgenommen hat. In neuerer Zeit hat sich allerdings ergeben, daß nur sehr lange dauernde Versuche eindeutig sind. Es gelingt nämlich auch mit sehr großen Mengen von Kohlehydraten und Fetten die Stickstoffausscheidung stark einzuschränken.-^) Durch Zusatz von Ammonsalzen kann die Stickstoffbilanz ebenfalls stark ») — A. EWngcr: Vgl. z. B. Leon Blum: Zeitschr. f. phvsiol. Chem. 30. 15 (1900). Maly: Pflüger?, Archiv. 9. 585 (1874). Zeitschr. f. Biol. 15. 201 (18%). — — PUsz uud Gjiergyai: Pflügera Archiv. 10. 536 (1875). — — 2) O. Loewi: Arch. f. cxperim. Path. und Pharmak. 48. 303 (1902). Vffl. ferner Henriques und Hansen: Zeitschr. f. physiol. Chem. 43. 417 (1905); 48. 383 (1906); G. Buqlia: 49. 113(1906). V. Henriques: Ebenda. 54. 406 (1908); 60. 105(1909). Vgl. auch IL Lüthje: Pflügers Arch. 113. 547 (1906) Zeitschr. f. Biol. 57. 365 (1912). V. — ^) — Vgl. z. B. Emil Abderhalden und Paul Hirsch: Zeitschr. f. physiol. ('hcni Karl Thomas: Archiv, f. (A.nat. u.) Physiol. Suppl. 249 (1910). 136 (1912). — 80 Eiweißstoffe und ihre Bausteine. 495 beeinflußt werden, d. li. durch eine ganze Reihe von iMaßnahmen kann wenigstens für einige Zeit der Stickstoffstoffwechsel ganz wesentlich beeinflußt werden, ij Eine eindeutige Beweisführung für die Annahme einer Synthese von Eiweiß im tierischen Organismus aus Aminosäuren war erst nachdem Methoden bekannt geworden waren, die eine genaue Analyse desjenigen Produktes zuließen, das verfüttert werden sollte. Es zeigte sich nämlich, daß die Biuretreaktion eines Eiweißabbaugemisches negativ ausfallen kann, trotzdem der Abbau noch lange nicht ein voller wird ständiger ist. 2) Jetzt können wir den Abbau eines Proteins leicht zu Fütterungsversuchen zumeist mit Fermenten herbeigeführt kontrollieren, indem wir z. B. die freien Amino- bzw. Karboxylgruppen bestimmen. Finden wir, nachdem das Verdauungsprodukt mit rauchender Salzsäure gekocht worden ist, daß keine Vermehrung der betreffenden Gruppen eintritt, dann wissen wir, daß keine hydrolysierbaren, polypeptidartigen Verbindungen mehr zugegen sind. Ferner können wir die einzelnen Aminosäuren vor und nach erfolgter Hydrolyse mit Säuren bestimmen. Es sind übrigens auch Versuche mit Produkten ausgeführt worden, die durch Kochen mit Säuren aus Eiweiß bereitet worden waren. Man muß hierbei die Hydrolyse sehr vorsichtig und ganz allmählich herbeiführen, verinsbesondere das Tryptophan weil sonst einzelne Aminosäuren ändert werden. Es sind zahlreiche Versuche mit verdauten Proteinen, die möglich, — — — — nur noch aus Aminosäuren bestanden, ausgeführt worden. 3) Als Versuchstier diente der Hund. Es gelang, nicht nur Stickstoffgleichgewicht Die zu erzielen, sondern auch Zurückhaltung von Stickstoft' zu erhahen. Untersuchungen sind zum Teil an erwachsenen, zum Teil an wachsenden Ferner wurden auch solche verwendet, die Tieren ausgeführt worden. lange Zeit gehungert und infolgedessen einen großen Verlust an KörperEs gelang, wachsende und ausgehungerte Tiere gewicht erlitten hatten. zu ganz beträchtlichen Zunahmen des Körpergewichtes zu veranlassen, wenn die Nahrung auch nicht die Spur von Ei-weißstoften, Peptonen oder Polypeptiden enthielt, sondern ausschließlich aus Aminosäuren bestand. Da erstens bei vielen dieser Versuche eine bedeutende Zunahme an Körpergewicht stattgefunden hat, und die Versuche zum Teil mehrere Wochen umfaßten, so ist eindeutig bewiesen, daß der tierische Organismus Eiweiß aus Aminosäuren bilden kann. ) E. Gräfe, V. Schlüpfer, K. Turban und H. Wintz: Zcitschr. f. physiol. Chem. Ernst 485 (1912); 83. 25 (1913); 84. G9 (1913); 86. 283 (1913)! 77. 1 (1912); 78. — — Emil Abderhalden, Paul Feschek: Biochem. Zeitschr. 45. 243 (1912); 52. 275 (1913). Hirsch und Arno Ed. Lampe: Zeitschr. f. physiol. Chem. 78. 1 (1912); 80. 136, 160 (1912); 81. 323 (1912); 82. 1, 21 (1912). - Emil Abderhalden: Ebenda. 96. 1 (1915). Hans Gessler: Ebenda. 109. 280 (1920). 2) E. Abderhalden und O. J'ri/m : Zeitschr. f. physiol. Chem. 53. 320 (1907). ^) Emil Abderhalden und Peter Bona, Berthold Oppler, fJ. S. London, Josef Olinqer, Emil Messner, Heinrich Windrath, Eranz Erank, Alfred- Schilf enhelm, Oskar Frank, Fidel Glainser, Akikazu Suwa: Zeitschr. f. physiol. Chem. 42. 528 (1904); 44. — 198 (1905); 47. .397 (1906); 51. 226(1907): 52. 507 (1907); 54. 80 (1907); 57, 74, 348 (1908); 59. 35 (1909); 61. 194 (1909); 63. 158 (1910): 65. 285 (1910); 67. 405 (1910): 68. 416 (1910): 76. 22 (1912); 83. 444 (1913). XXIV. Vorlesung. 496 Es seien einige der wichtigsten Ergebnisse der Fütterungsversuche Gleichzeitig sei bemerkt, mit vollständig abgebautem Eiweiß mitgeteilt. daß auch die Kohlehydrate, Fette, Phosphatide und die Nukleoproteide in abgebautem Zustande zugegen waren. Ferner waren die anorganischen Stoffe aus ihren Bin- dungen herausgelöst. Ein junger Hund erhielt während drei Wochen vollständig abgebautes Pferdefleisch. Er nahm olO^/ an Körpergewicht zu. Ein Dachshund hungerte 17 Tage. Er verlor 1100g an Körpergewicht. Nach Verfütterung von vollständig abgebautem Fleisch wurde der Gewichtsverlust wieder ausgeglichen. Ein weiteres \'ersuchstier behielt sein Körpergewicht bei, als es 36 Tage ausschließlich die gleiche Nahrung erhielt. Schließlich sind langfristige Versuche mit vollständig abgebautem Eiweiß, bzw. Fleisch ausgeführt worden. Die Kohlehydrate waren durch Traubenzucker, die Fette durch ein Gemisch von Glyzerin und Palmitin-, Stearin- und Ölsäure und die Nukleinsäuren durch ihre Bausteine vertreten. Ferner wurden die anorganischen Nahrungsstoffe in Form von Knochenasche verabreicht. Das Versuchstier erhielt somit ausschließlich die Bausteine der Nahrungsstoffe. Drei derartige Versuche dauerten 74 Tage. Das Körpergewicht hatte bei den beiden jungen Tieren beträchtlich zugenommen (1200 und 1000 (/). Schließlich ist auch versucht worden, das vollständig abgebaute Eiweiß durch eine Mischung von reinen Aminosäuren zu ersetzen. Es gelang auch, damit annähernd Stickstoflfgleichgewicht zu erhalten, doch sind die einzelnen Versuche nicht sehr lange durchgeführt worden (8 und 7 Tage). Schließlich wurde ein Hund 100 Tage lang ausschließlich mit abgebautem Fleisch ernährt. Dem Versuche war eine Hungerperiode von 23 Tagen vorausgegangen. Das Versuchstier hatte 6700 g an Körpergewicht verloren. Am Schlüsse des lOOtägigen Versuches war das Körpergewicht um 9900^ gestiegen. Aus den eindeutigen Ergebnissen der Fütterungsversuche an Hunden daß diese Tiere sämtliche Zellbestandteile synthetisch bereiten können, wenn ihnen die Bausteine der einzelnen N ah rungs Stoffe zur Verfügung stehen. Diese Beobachtung zwingt uns, die bisherige Definition der Nahrungsstoffe zu ändern. Bisher wurden als organische Nahrungsstoffe Kohlehydrate, Fette und Ei weiß Stoffe bezeichnet. Jetzt wissen wir, daß jede einzelne Gruppe durch den Zusatz „oder ihre Bausteine" ergänzt werden muß. Es ist nicht nötig, daß wir in unserer Nahrung Eiweißstoffe aufnehmen, es genügt, wenn ihre Bausteine, die Aminosäuren, zugegen sind. Mit der Feststellung, daß es gelingt, den tierischen Organismus mit den Bausteinen der Nah rungs Stoffe zu ernähren, ist ein Problem gelöst worden, das von jeher der Traum der Naturforscher war, nämlich dasjenige der künstlichen Gewinnung der Nahrungsstoffe. ^) Da wir alle ergibt sich, Uausteine der organischen Nahrungsstoffe synthetisch bereiten können, so ) ilor Vgl. Emil Abderhalden : Zeitschr. Zellbausteine in Pflanze und Tier. J. f. physiol. Chemie. 77. 22 (1912); Synthese Springer, Berlin 1912. Eiweißstort'e und ihre Bausteine. 497 auch imstande, die Nahrung im Laboratorium synthetisch zu Allerdings erhalten wir zunächst die inaktiven Bausteine. Wir müßten sie noch in ihre optisch-aktiven Anteile zerlegen. Selbstverständlich handelt es sich nur um eine prinzipielle Lösung jenes wichtigen Problemes. Die Darstellung der einzelnen Bausteine würde viel zu teuer werden und außerdem einen großen Aufwand an Zeit und Arbeitskräften erfordern. Die Pflanze arbeitet viel billiger und vor allem auch viel zweckmäßiger. Dadurch, daß die chemische Forschung schon wiederholt bei der Bereitung von Farbstoffen, von Alkaloiden usw. der Pflanze erfolgreich Konkurrenz machte, hat sie mehr zur Lösung des Problems der Gewinnung von Nahrungsstoffen beigetragen, als sie wohl je durch die Synthese der einzelnen Bausteine leisten wird; denn dadurch, daß große Länderstrecken, die zum Anbau von Pflanzen verwendet werden, die Alkaloide, Farbstoffe sind wir bereiten. usw. liefern, frei werden, wird Land zur Anpflanzung von Getreide, von Kartoffeln usw. zur Verfügung gestellt. Die Lösung des Problems der künstlichen Darstellung der organischen Nahrungsstoffe erschien noch vor kurzem, in weiter Ferne zu liegen, glaubte man doch abwarten zu müssen, bis es dem Chemiker gelungen sei, die Struktur der Proteine, der Phosphatide und Nukleoproteide aufzuklären. Jetzt wissen wir, daß es ein ganz unnötiges Beginnen wäre, zusammengesetzte Nahrungsstoffe aufzubauen, weil sie ja doch vor ihrer Übernahme in die Gewebe im Magendarrakanal in weitgehender Weise zerlegt werden, und es wohl ganz ausgeschlossen ist, Verbindungen zu bereiten, die vom tierischen Organismus ohne Ab- und Umbau verwertet werden können. Obwohl die mitgeteilten Beobachtungen genügen, um zu beweisen, daß eine Synthese der Nahrungsstoffe möglich ist, haben wir weitere Versuche unternommen, Mäuse und Ratten mit Bausteinen der zusammengesetzten organischen Nahrungsstoffe durch längere Zeit hindurch zu ernähren, die fast alle synthetisch gewonnen worden waren. Dazu erhielten die Tiere die notwendigen Mineralstofife und Wasser. Bei einem Teil der Versuche erfolgte außerdem noch ein Zusatz einer kleinen Menge von Material, wie Hefe, Kleie, Kohl usw., das Nutramine^) enthielt. Es gelang, während längerer Zeit Stickstoffgleichgewicht zu erzielen.') Es ist selbstverständlich noch unentschieden, ob jede Tierart der gleich umfassenden Synthesen fähig ist. Wir manche Organismen Phosphatide synthetisch bereiten können, auch wenn die Bausteine nicht in der Nahrung enthalten sind.») Es ist wohl möglich, daß anderen Tieren diese Fähigkeit abgeht. Im Prinzip dürften wohl alle tierischen Organismen mit den Bausteinen der Nahrungsstoflfe auskommen. Versuche am Menschen zeigten, daß auch er vollständig abgebautes Eiweiß verwerten kann.) wissen, daß Vgl. S. 77, 107. Vgl. Emil Abderhalden: Pßügers Archiv. 195. 199 (1922). ') Vgl. S. 298. ) Vgl. Emil Abderhalden, Franz Frank und Alfred Schittenhelm : Zeitschr. f. Franz Frank und Alfred Schittenhelm: Münchener physiol. Chemie. 63. 215 (1909). med. Wochenschr. 58. Nr. 24 (1911). ») ) — A b d e rh a d I li D , Physiologische Chemi«. I.Teil, 5. Aaü. 32 XXIV. Vorlesung. 498 Es fragt sich, ob es möglich ist, Tiere dauernd mit den Bausteinen der Nahrungsstoffe zu ernähren. Ferner ist die Frage zu entscheiden, ob vollständig abgebaute Nahrungsstoffe den nicht abgebauten völlig gleichwertig sind. Die erstere Frage ist deshalb schwer zu entscheiden, weil jede Art Die der Ernährung, wenn sie gleichartig ist, Schwierigkeiten bereitet. man sie Versuchstiere verweigern schließlich die Nahrung. Ernährt unter Zwang, dann antworten sie mit Erbrechen. Es scheint ferner, daß bei dauernder Ernährung mit vollständig abgebauten Nahrungsstoffen die Darmschleimhaut schließlich geschädigt wird. Allerdings treten nach unseren Erfahrungen derartige Erscheinungen erst nach monatelangem Füttern mit den Bausteinen der Nahrungsstoffe auf. Endlich greift hier das Problem bisher noch unbekannter, in sehr kleinen Mengen notwendiger Nahrungsstoffe ein. Wir kommen darauf noch eingehend zurück. A priori sollte man erwarten, daß die Ernährung mit den Bausteinen der Nahrungsstoffe derjenigen mit den in der Natur vorkommenden zusammengesetzten Produkten nicht gleichwertig sein kann. Einmal umgehen wir dadurch, daß die Nahrungsstoffe außerhalb des Organismus vollständig gespalten werden, einen wichtigen Regulationsmechanisraus im Darmkanal, nämlich den stufen weisen Abbau. Normalerweise bilden sich im Darmkanal immer nur in Spuren. Stets folgt ihrer Bildung sofort die Resorption. Auf diese Weise wird verhindert, daß eine Überschwemmung des Organismus mit den im Darm entstehenden Abbaustuten eintritt. Es ist sehr überraschend, daß die vollständig abgebauten Nahrungsstoffe wenigstens gilt dies für die Eiweißstoffe bzw. die aus ihnen gewinnbaren Aminosäuren den nicht im abgebauten Zustande zugeführten Verbindungen gleichwertig zu sein scheinen, i) Einzelne Autoren fanden geringe Unterschiede 2), doch dürften diese sicher zum Teil darauf zurückzuführen sein, daß einzelne Bausteine im Verdauungsgeraisch sekundär verändert waren. Wird nämlich die Verdauung nicht streng steril durchgeführt, dann kommt es immer zur Bildung von Aminen aus bestimmten Aminosäuren. Nimmt die Umwandlung von Aminosäuren einen größeren Umfang an, dann wird das ganze Gemisch entwertet, wenn eine unersetzbare Aminosäure von der Veränderung betroffen wird. die einfachsten Bausteine — — Nahningsbesondere Struktur von keiner Bedeutung für jenen Organismus ist, der sich mit ihnen ernähren will, sofern er das zusammengesetzte Produkt mittels seiner Fermente zerlegen kann, hat einem ganzen Heer von Fragestellungen eine sichere Grundlage gegeben. Zunächst können wir uns die Frage vorlegen, welche Bausteine der einzelnen Nahrungsstoffe unbedingt notwendig sind, und welche entbehrt werden können. Die einzelnen Versuche können Die Feststellung, daß die zusammengesetzten stoffe durch ihre Bausteine ersetzbar sind, und ihre dadurch zu eindeutigen gemacht werden, daß wir zunächst das vollständige Gemisch der Bausteine verfüttern, dann einen bestimmten Baustein ent') Vgl. z. — Franz — Emil Ab- B. C. Michaud: Zeitschr. Frank und Alfred Schittenhelm : f. physiol. Chemie. 59. 405 (1909). Ebenda. 70. 98 (1910); 73. 157 (1911). derhalden: Ebenda. 77. 22 (1912). 2) Vgl. hierzu E. Voit und J. Zisterer: Zeitschr. f. Biol. 53. 4ri7 (1910). nnd Kiweißstotie ihre Bausteine. 499 fernen nnd schließlich den Versuch nach Zugabe des fortgenommenen Bausteines fortsetzen. Erweist sich ein bestimmter Baustein als unentbehrlich, d. h. ist das übrige Gemisch der Bausteine nicht mehr ausreichend, dann ergibt sich die Frage, ob nicht eine einfachere Abbaustufe der betreffenden Verbindung für sie eintreten kann. Auf diesem Wege ist welcher Grenze die synthetischen es möglich, zu bestimmen, bei Fähigkeiten jedes einzelnen tierischen Organismus ein Ende finden. So ist versucht worden rosin durch p-Oxyphenylbrenztraubensäure plus Ammonsalz und Phenylal anin durch Phenylbrenztraubensäure plus Ammonsalz zu ersetzen. Auch die entspechenden Amine der genannten Aminosäuren wurden auf ihr Vermögen, für diese einzutreten, untersucht. Die Ergebnisse waren negativ. Diese Untersuchungen müssen weiter ausgebaut werden. Vielleicht waren die gewählten Versuch sbedingungen nicht die richtigen. Ty Die bisherigen Versuche haben ergeben, daß.Glykokoll ent- behrlich ist. Der tierische Organismus kann diese Aminosäure in großer Menge selbst bereiten. Kasein enthält kein Glykokoll. Dieses Protein um als einzige Eiweißverbindung bzw. in Form seiner Aminosäuren Stickstoflfgleichgewicht herzustellen. Tyrosin und Phenylreicht jedoch aus, alanin^) sind unersetzbar. Dagegen können sich beide Aminosäuren gegenseitig vertreten. 2) Prolin ist offenbar entbehrlich. Der tierische Organismus bildet diese Aminosäure vielleicht aus Glutaminsäure über die Pyrrolidonkarbonsäure.3) Nicht fehlen darf das Zystin), dem eine wichtige Rolle beim Wachstum und bei den Oxydationsvorgängen in den Zellen zukommt. Auch Argini n, Lysin und Histidin sind nicht ersetzbar. &) Vielleicht können einzelne Monoaminosäuren der 6-Kohlenstoffreihe sich vertreten.^) Die Dikarbonsäuren A s p a r a g i n - und Glutaminsäure scheinen auch unentbehrliche Eiweißbausteine zu sein^). Als eine unentbehrliche Aminosäure erwies sich ferner auch das Tryptophan.'') Ernährt man z. B. Hunde oder Ratten mit vollständig abgebautem Kasein, dann läßt sich mit einer bestimmten Menge Stickstoff Stickstoffgleichgewicht herstellen. Gibt man alle Aminosäuren des Kaseins im gleichen Mengenverhältnis ohne Tryptophan, dann genügt das Amino^) Emil Abderhalden: Zeitschr. f. physiol. Chemie. 76. 1 (1915); Pßügers Arcb. 195. 199 (1922). ) Emil Abderhalden: Zeitschr. f. physiol. Chemie. 96. 1 (1915); Pßügers Archiv. — H. B. Lewis: The of biol. Chemie. 31. 363 (1917); 42. 289 (1920). — Carl 0. Johns und A. J. Türks: Ebenda. 41. 379 (1920). — Barnett Sure: Ebenda. 195. 199. (1922). 50. 103 (1922). Vgl. S. 334. Vgl. Thomas B. Osborne und Leonh. B. Mendel: The J. of biol. Chem. 25. 1 A. Akroijd und H. H. Mitchell: Ebenda. 26. 231 (1916). (1916); 26. 293 (1916). Emil Abderhalden : Pßügers Archiv. 195. F. G. Hopkins: Biochem. J. 10. 551 (1916). 199. (1922). ) Vgl. G. J. Shiple PJmil Abderhalden: Pßügers Archiv. 195. 199 (1922). und Carl P. Shernin [Journ. of the American Chem. Soc. 44. 618 (1922)] sind der Ansicht, daß der Mensch Glutamin aufl)auen kann. ") Willkock und F.G. Hopkins: Journ. of Physiol. 35. 88 (1907). Emil Abderhalden: Zeitschr. f. physiol. Chemie. 57. 348 (1908): 77. 22 (1912); 83. 444 (1913). Vgl. auch Thomas B. Osborne und Leonh. B. Mendel: The .1. of biol. Cheniie. 25. 1 (1916). ') Vgl. hiezu auch H. li. Lewis und L. E. lioot The of biol. Chem. 43. 79 (1920). =>) ) — — — — — — : 32* XXIV. Vorlesung. 500 Säuregemisch nicht mehr. Es wird bedeutend mehr Stickstoff ausgeschieden aufgenommen wurde. Vielleicht gestattet diese Beobachtung festzuEs stellen, wieviel Eiweiß der tierische Organismus täglich neu aufbaut. und das Gleiche gilt auch ist vermutet worden, daß das Tryptophan als solches im tierischen für die übrigen unersetzbaren Aminosäuren Organismus eine bedeutsame Rolle spielt und aus ihm ferner ein Produkt gebildet wird, das bei bestimmten Organfunktionen und vor allem für den normalen Ablauf des Zellstoflfwechsels unentbehrlich ist. Gewiß ist die Störung des Stickstoffgleichgewichtes beim Fehlen des Tryptophans nicht nur nach dieser Richtung zu suchen. Es wäre sonst nicht recht einzusehen, weshalb die negative Stickstoffbilanz so rasch eintritt. Offenbar liegen die Der tierische Organismus, wenigstens gilt dies Verhältnisse, wie folgt. für die untersuchten Tierarten (Hund, Maus, Ratte), vermag nicht, Tryptophan synthetisch zu bilden. Fehlt nun diese Aminosäure in der Nahrung, so vermag er aus den übrigen Bausteinen kein Zelleiweiß zu bereiten, weil zum Aufbau der Gewebsproteine Tryptophan notwendig ist. Es sind dann in gewissem Sinne alle zugeführten Bausteine wertlos, wenn man sie nach ihrer Befähigung, Eiweiß zu bilden, beurteilt, und der Organismas nicht gerade ein Protein aufzubauen hat. an dessen Aufbau Tryptophan als — — nicht beteiligt ist. Fügt man dem Aminosäuregemisch, dem man Tryptophan entzogen hat, diese Aminosäure wieder hinzu, dann ist es wieder vollwertig. Es wird nun sehr interessant sein, festzustellen, ob einfachere Abbaustufen des Tryptophans für diese Verbindung eintreten können, d. h. es wird zu prüfen sein, welches Bruchstück dieser Aminosäure zu ihrer Synthese durch den tierischen Organismus noch genügt. Es gibt nun Proteine, die, wie eine reiche Erfahrung gezeigt hat, als Nährmaterial nicht vollwertig sind. Dahin gehört z.B. die G e 1 a t i n e. Sie genügt nicht, um den tierischen Organismus dauernd im Stickstoftgleichge wicht zu halten. Nun fehlt dem Leim das Tryptophan und auch das Tyrosin. Phenylalanin ist nur in geringen Mengen zugegen. M Sollte das Fehlen der genannten beiden Aminosäuren die Ursache dafür sein, daß die Gelatine nicht vollwertig für die übrigen Eiweißstoffe eintreten kann? Es ist in der Tat gelungen, durch Zusatz der fehlenden Aminosäuren und Zugabe jener Bausteine, die in der Gelatine in nur geringen Mengen zugegen sind, vollständig abgebaute Gelatine für die Ernährung vollwertig zu machen. 2) Fassen wir alle erwähnten Beobachtungen zusammen, dann ergibt sich, daß es gelingt, Eiweiß vollständig durch seine Bausteine zu ersetzen. Der tierische Organismus hat die Fähigkeit, aus Aminosäuren Eiweiß aufzubauen und auch die übrigen mit diesem oder seinen Abbauprodukten in Zusammenhang stehenden Funktionen mit den Bausteinen der Proteine zu bestreiten. ) Die käufliche Gelatine enthält oft Tyrosin, weil ihr noch andere Proteine hcihei Stoffwechselversuchen nicht immer Rücksicht genommen gemengt sind. Darauf ist worden. Emil Abderhalden und IHmitrie Manoliv : Zeitscli. f. physiol. Chemie. 65. 'i'M\ Emil Abderhalden: Ehenda. 77. 22 (1912). Vgl. hierzu auch M. Kauffmann: Pßügers Archiv. 109. 1 (1905). — F. Rona und W. Müller: Zeitschr. f. physiol. ^) (1910). Chemie. 50." 203 (1907). Kiweißstofte und ihre Bausteine. 501 Man kann somit mittels Pepsin, Trypsin und Erepsin Eiweiß außerhalb des tierischen Organismus vollständig abbauen und damit in gewissem Sinne der ganzen Verdauung vorgreifen. Die verabreichten Abbauprodukte werden resorbiert und gerade so gut verwertet, wie wenn Eiweiß in den Magendarmkanal eingeführt worden wäre. Darf man aus diesen Tatsachen den »Schluß ziehen, daß die im Darmkanal vollständig bis zu den Aminosäuren abgebaut werden, und nur diese einfachsten Bausteine zur Resorption gelangen? Wir müssen das verneinen. Eine solche SchlußProteine folgerung wäre nicht genügend gestützt. Unsere Fragestellung war: Gelingt es. Eiweiß vollständig durch Aminosäuren zu ersetzen? Diese Frage ist bejaht worden, d. h. der tierische Organismus versagt auch dann nicht, wenn er gezwungen wird, sich ausschließlich mit den einfachsten Bausteinen zu behelfen. Die weitere Frage ist nun die, ob der tierische Organismus auch unter normalen Verhältnissen die Proteine soweit abbaut. Wir sind damit wieder bei den schon früher erwähnten SchAvierigkeiten angelangt. Sie haften jeder indirekten Beweisführung an. Wir können jedenfalls zum Ausdruck bringen, daß der Annahme einer vollständigen Zerlegung der Peptone zu Aminosäuren im Darmkanal insofern kein Hindernis entgegensteht, als der tierische Organismus seine Proteine aus Aminosäuren aufbauen kann und bei den übrigen mit dem Eiweiß bzw. seinen Abkömmlingen zusammenhängenden Vorgängen wohl ausnahmslos von diesen ausgeht. fast Die Annahme, daß der Abbau der Proteine im Darmkanal wenigstens soweit führen muß, bis jeder einzelnen Eiweißart ihre besondere Struktur genommen ist, hat eine sehr große Stütze durch biologische Experimente erhalten. i) Führen wir einen Eiweißkörper direkt in die Blutbahn oder ganz allgemein parenteral ein, dann ergeben sich Erscheinungen, die man nicht beobachtet, wenn das Protein per os gegeben wird und der Verdauung unterliegt. Wir haben bereits früher die Präzipitinbildung und das Auftreten einer Sensibilisierung (Anaphylaxie) erwähnt.-) Ferner beobachten wir regelmäßig das Auftreten von Fermenten im Blutplasma, die Eiweiß abbauen können, sobald nicht umgebautes Eiweiß direkt in die Blutbahn gelangt, ^i Diese Feststellung ist deshalb von besonderem Interesse, weil sie uns eine Erklärung dafür abgibt, daß der tierische Organismus auch parenteral zugeführte Eiweißstotfe verwerten kann.) Er holt in der Blutbahn den Abbau Hamintrger (Arteigenheit und Assimilation. Franz Deuticko, Leipzig ') Schon und Wien 1903) hat aus den vorliegenden Erfahrungen der eiffeuartigen Reaktionen des Organismus, sohald ihm Proteine parenteral zugeführt werden, den Schluß gezogen, Ein V'gl. auch Ludimar Hermann: daß die Verdauung den genannten Zweck hat. Beitrag zum Verständnis der Verdauung und Ernährung. Antrittsvorlesung 2;'). Nov. Huppert: Über die Erhaltung der Arteigen1868. Meyer und Zeller. Zürich 1869. Emil Abderhalden: Der Artenlegriff und die Artenfechaften. Joseph Koch. Prag 1896. koDStanz auf biologisch-chemischer Grundlage. Naturwiss. Rundschau. 19. Nr. 44 (1914). — — — Vgl. S. 402. Yg\. Emil Abderhalden: Abderhaldensche Reaktion. 5. Aufl. J.Springer. Berlin 1914. Hier findet sich die ganze liiteratur. Friede) Vgl. hierzu Carl Oppenheimer: Hofmeisters Beiträge. 4. 263 (1903).— Felix Lommel mann und R. Isaac : Zeitschr. f. experim. Path. u. Ther, 1. 513 (1905). Ernst Heilmr: Zeitschr f. Biol. 50. Archiv f. experim. Path. u. Pharm. 58. 50 (1907). ) ') — — C XXI V. Vorlesung. 502 nach und verwendet dann die Abbaustufen. direkt giftige Eigenschaften, Ferner zeigen viele Peptone wenn sie in die Blutbahn übergeführt werden. Manche sind allerdings wieder ganz ungiftig. Alle diese Beobachtungen im Einklang mit der Annahme, daß auch bei den Proteinen, genau so wie bei den Kohlehydraten und zum Teil auch bei den Fetten die \'erdauung den Zweck hat, nicht nur die hochmolekularen, im kolloiden Znstand befindlichen Verbindungen abzubauen, bis diffundierbare Produkte stehen entstanden sind, sondern darüber hinaus die spezifische Struktur der Nahrungsei weißstotfe zu zerstören. Es werden indifferente Abbaustufen gebildet, die dann von den Körperzellen zu den verschiedensten Zwecken verwendet werden können. Es ist noch unentschieden, ob als indifferentes Material für die Körperzellen nur Aminosäuren in Betracht kommen, oder aber auch aus mehreren Aminosäuren zusammengesetzte Verbindungen. Daß der Abbau im Darmkanal und vielleicht zum Teil erst in der Darmwand bis zu Aminosäuren führt, erscheint sehr wahrscheinlich. Einmal Geweben und vor allem nicht im ist es bisher nicht geglückt, in den Blute in einwandfreier Weise Produkte mit Peptoncharakter nachzuweisen, die sich mit Sicherheit auf von der Verdauungsprodukte zurückführen lassen. ) Dagegen sind Aminosäuren, wie wir gleich erfahren werden, zugt^gen. Für die Annahme eines sehr weitgehenden Abbaus der Eiweißkörper im Magendarmkanal sprechen, es sei dies hier nochmals hervorgehoben, die folgenden Momente. Einmal ist der Magendarmtraktus mit Fermenten ausgerüstet, die einen raschen und vollständigen Abbau der Proteine ermöglichen. Ferner findet man im Darminhalt alle Aminosäuren und vor allem auch jene, die erfahrungsgemäß erst frei werden, wenn schon der größte Teil der übrigen Aminosäuren in Freiheit gesetzt ist. Endlich ist bewiesen, daß der tierische Organismus nach Verfütterung eines Aminosäuregemisches, das alle unentbehrlichen Bausteine der Proteine enthält, gerade so gut alle mit dem Eiweißstoffwechsel in Zusammenhang stehenden Vorgänge bestreiten kann, wie wenn Eiweiß verabreicht wird. Schließlich sind die Aminosäuren jene Abbaustufen der Proteine, Darmwand aufgenommene von denen bei denen alle weiteren Fäden des Stoffwechsels ausgehen, sie auch zusammenlaufen. Ob nun die und Zerlegung der — Leonor Michaelis und Peter Bona: Pflügerv, Archiv. 12t. 163 (1908); 123. 124. 57H (1908). — W. Cramer Journ. of Physiol. 37. 146 (1908). — Harold Ringle und W. Cramer: Ebenda 37. 157 (1908). — E. Abderhalden und E. S. London: physiol. Chemie. 62. .339 (1909). — Kornel Zeitschr. Körösy: Zeitschr. physiol. Chemie. 62.68 (1909); 69.313 (1910). — Sigmund Somogi/i: Ebenda. 71. 125(1911). — E. Heilner: Zeitschr. Biologie. 50. 26 (1907). — Vgl. B. Edgar Zunz: Archiv, internal, de Physiol 11. 37 (1911). 2(5 (1907). 40() (1908): : v. f. f. r. f. ) z. A. Schitfenhelm, und W. Weichardt : Zeitschr. Therapie. 14. (509 (1612). K. Abderhalden: — f. Immunitätsforschung und exporini. f. phvsiol. Chemie. 81. 315 Zeitschr. (1912). — -) Vgl. hierzu R. Neuineister Zeitschr. f. Biologie. 24. 272 (1888). Gustav FJtnh/. Morawitz und den und Er. Knoop: Hofmeistern Beitr. 3. 120(1902). R. Dietschg: Arch. f. experim. Path. u. Pharm. 54. 88 (1905). Kmil Abderhalden und Carl Oppenheimer: Zeitschr. f. physiol. Chem. 42. 153 (1904). Emil Abderhalden, Casimir hunk und E. S. London: Ebenda. 51. 269 (1907). E. Ereund Biochem. Zeitschr. 7. 361 Emil Abderhalden: Ebenda. 8. 368 (1908). - Neuere Versuche ergaben (1908). stets die Abwesenheit von Peptonen im Blutplasma. : — — — — — : Eiweißstoffe und ihre Bausteine. 5()^ Peptone in Aminosäuren quantitativ im Darmkanal selbst erfolgt oder erst in der Darmvvand oder sonstwo vollendet wird, ist im Prinzip natürlich gleichgültig. Dadurch daß unter normalen Verhältnissen stets nur Abbaustufen des in die Blutbahn und zu den Körperzellen gelangen, die in nichts Eiweißes mehr in ihrer Struktur an diejenige des Ausgangsmateriales erinnern, wird verhindert, daß den Körperzellen beständig ganz verschiedenartige und immer wieder wechselnde Aufgaben gestellt werden. Sie erleben keine Überraschungen und sind in weitgehendem Maße von der Art der aufgenommenen Nahrung unabhängig. Es kommt nur darauf an, daß alle Bausteine des Eiweißes, die der Organismus nicht selbst bereiten kann, zur Stelle sind und jede einzelne der unentbehrlichen Aminosäuren auch in genügender Menge sich findet. Man kann sich wohl vorstellen, daß eine Aminosäure, die vom Organismus nicht synthetisch bereitet werden kann, ausschlaggebend für die Verwertbarkeit der übrigen Eiweißabbaustufen wird, wenn es sich darum handelt, Proteine aufzubauen. Die im Minimum vorhandene Aminosäure würde für die Verwendbarkeit aller übrigen bestimmend sein. Ein Aminosäuregemisch, das in seiner Zusammensetzung den in der größten Menge vorhandenen Proteinen der Gewebe am nächsten steht, müßte für den Eiweißaufbau die beste Ausnützung bieten, i) Es läßt sich jedoch kaum für alle Fälle eine bestimmte Regel aufstellen, weil ohne Zweifel die Aminosäuren ipa tierischen Organismus mancherlei Aufgaben zu erfüllen haben, und viele Beziehungen zu anderen Verbindungen angebahnt werden. Ferner wird es von Fall zu Fall darauf ankommen, welche Proteine gerade aufgebaut werden soll. Nicht unerwähnt wollen wir lassen, daß man den Durchtritt von Eiweiß und von höhermolekularen Abbaustufen, die noch den Charakter des Ausgangsmateriales tragen, durch die Darmwand erzwingen kann, wenn man namentlich genuine, an und für sich schwer angreifbare Proteine, wie Plasma- bzw. Serumeiweißkörper, Hühnereiweiß usw., in großer Menge per OS zuführt. In diesem Falle erhält man alle Erscheinungen, die auch bei parenteraler Zufuhr zu beobachten sind. Vielleicht beruhen manche nach Genuß bestimmter Nahrungsmittel bei bestimmten Personen auftretende Erscheinungen, wie z. B. die Urtikaria, auf dem Durchtritt noch ungenügend zerlegter Produkte. Vielleicht fehlt ein Ferment, das den Abbau vollendet, oder es handelt sich um sonst eine Störung bei der Verdauung oder Resorption. ) (1904). Vgl. hierzu Emil Abderhalden : Zentralbl. f. Stoftw. — L. Michaud: Zeitschr. f. Alfred Schittenhelm : Ebenda. 73. 157 (1911). 96. 1 (1915). u. V'erdaaungskrankh. 5. 647 405 U909J. Franz Frank und Emil Abderhalden : Ebenda. 77. 27 (1912); physiol. Chem. 59. — — Vorlesung XXV. Eiweißstoffe und ihre Bausteine. 9. Verhalten der Aminosäuren im Darmkanal. Die Wirkung der Darmfiora. Als wesentlichstes Ergebnis der Studien über das Verhalten der Proim Magendarmkanal haben wir kennen gelernt, daß ein weitgehender Abbau einsetzt, der zu Peptonen und Aminosäuren führt. Es wird das Nahrungseiweiß, das in irgend einer Zellart eine besondere Funktion erbesondere Zusammensetzung und füllt und dementsprechend auch eine Struktur hat, mindestens so weit zerlegt, bis alle Anklänge an die ursprüngliche Eigenart des Moleküls verschwunden sind. Vieles spricht dafür, daß der Abbau der Eiweißstoffe und Peptone vollständig bis zu Aminosäuren führt, bevor die Körperzellen mit ihren Vorgängen einsetzen. Ob nun dieser vollständige Abbau im Darmkanal selbst oder in der Darmwand und vielleicht zum Teil erst in der Leber oder den einzelnen Geweben erfolgt, ist schwer zu entscheiden. die EiweißBevor wir auf die Frage eingehen, welchen teine Weg abbauprodukte einschlagen, um zu den einzelnen Körperzellen zu gelangen und in welcher Form dieser Transport erfolgt, müssen wir noch der Tätigkeit der Darmflora gedenken. Wir haben bei den Kohlenhydraten und Fetten schon bemerkt, daß immer ein Teil der zugeführten Nahrungsstoffe von den im Darmkanal anwesenden Bakterien verwendet und dabei umgewandelt wird. Einmal bauen diese Lebewesen ihren Zelleib aus der zugeführten Nahrung auf Sie wachsen, d. h. sie vermehren sich. Außerdem brauchen auch sie Energie, und ferner formen sie Sekretstoffe, Fermente usw. aus den zur Verfügung stehenden Materialien. Es treten je nach der Zellart besondere Formen des Abbaus der Proteine auf Manche dieser Mikroorganismen werden die Peptone direkt angreifen können, andere benützen die Aminosäuren, und wieder andere sind auf Ammoniak als erstes Ausgangsmaterial angewiesen. Sie können dieses vielleicht nicht in allen Fällen selbst aus Aminosäuren bereiten. Der Abbau der Eiweißabbaustufen wird, wie Versuche im Reagenzglas bewiesen haben, durch die Menge und Art der übrigen zur Verfügung stehenden Nahrungsstoft'e nicht unerhel)lich beeinflußt. Ohne Zweifel arbeiten die einzelnen Bakterienformen gegenseitig vor, d. h. die eine Art bildet Abbaustufen, die von einer anderen weiter verwertet werden können. sich P^ivreißstofte und ihre Bausteine. ÖOf) Oft ist die Existenz der einen Or^anismenart an diejenige einer anderen geknüpft. Es kommt jedoch sicher auch vor, daü eine Bakterien art Produkte erzeugt, die einer anderen schädlich sind. Ein heftiger Kampf ums Dasein entbrennt. Immer mehr überwuchern bestimmte Mikroorganismenarten, während andere zurückgedrängt werden. Auch die Art des „Nährbodens", d. h. der Gehalt des Chymus an einzelnen Bestandteilen hat einen großen Einfluß auf die Art der Zusammensetzung der Darmtlora. Bald findet die eine Art von Mikroorganismen besonders günstige ^ erhältnisse zum Wachstum, bald eine andere. Geringfügige Änderungen in den augenblicklichen Bedingungen können die Art des Abbaus der Aminosäuren stark beeinflussen. So wird die Gegenwart von Sauerstoff die aeroben Bakterien in ihrer Wirkung unterstützen, während die anaeroben ungünstiger gestellt sind. Ist der Sauerstoff aufgebraucht, dann finden wir im Darminhalt Produkte, die vornehmlich der Tätigkeit der anaeroben Bakterien entstammen. Von diesen Gesichtspunkten ans ist es verständlich, daß die Darmflora mit der Art der Zusammensetzung der Nahrung und den durch sie geschaffenen Bedingungen wechseln kann. Hierauf beruht unzweifelhaft ein großer Teil des Erfolges einer bestimmten Diät. Sie muß allerdings über eine lange Zeit hinaus innegehalten werden, soll ein wirklicher Florawechsel eintreten. Es handelt sich bei den am Abbau der Aminosäuren beteiligten Bakterien zum größten Teil um an aerobe.^) Vor allem kommt der Bacillus putrificus in Betracht. Die gleichzeitig vorhandenen aeroben Mikroorganismen, insbesondere das Bacterium coli und lactis aerogenes, unterstützen die Tätigkeit der ersteren durch Wegnahme von Sauerstoff. Es unterliegt keinem Zweifel, daß jedoch die aeroben Bakterien manche Abbaustufe aus Aminosäuren bereiten können, die auch von den anaeroben hervorgebracht werden. Im allgemeinen sind jedoch die von den einzelnen Bakterienarten gebildeten Abbaustufen für Femer ist erwiesen, daß manche dieser Abbaudiese charakteristisch. produkte auch von den Zellen der hochorganisierten Tiere gebildet werden. Es sind keine durchgreifenden Unterschiede im Zellstoffwechsel der ver- schiedenartigsten Organismen vorhanden. Es ist von Interesse, daß der Darm des Neugeborenen keine Bakterien enthält. Sein erstes Ausscheidungsprodukt, das sog. Mekonium, ist ganz steril. Erst allmählich dringen verschiedenartige Lebe- wesen mit der Nahrung in unseren Verdauungstraktus ein. Man sollte erwarten, daß sie besonders in den Anfangsteilen des Darmkanales, in der Mundhöhle und im Magen lebhaft wachsen. Das ist jedoch nicht der Fall. Es finden sich besondere Einrichtungen, die der Entwicklung der Bakterien nicht günstig sind. Zunächst wird durch den reichen Zutritt von Luft die Entwicklung der anaeroben Bakterien ganz unterdrückt. Außerdem verweilen die Nahrungsstoffe unter normalen Verhältnissen insbesondere in der Mundhöhle nur kurze Zeit. Nur dann, wenn die Zähne Unebenheiten .und — ') Vgl. u. a. Escherich: Die Darmbakterien de.s Säuglings. Stuttgart 1886. A. Macfaydn, M. Nencki und N. Sieber: Arch. f. experim. Patb. u. Pbarm. 28. 311 (1891). Bienstoek: Zeitscbr. f. kliu. Med. 7. 1 (1884); Arcb. f. Hygiene. 36. 3.ö5 (1899); Rolh/: 39. 390 (1900); Ann. d. l'Institut Pasteur. 17. 8.^0 (1903); 20. 407 (1906). Vgl. auch D. Gerhardt: Über DarmDeutsche med. Wochenschr. Nr. 43 1733(1906). A. Ellinger: Die Chemie der Eiweißfäulnis. Ergebnisse d. Physiol. 3. I. 107 (1904). Vgl. auch F. IL Cannon: Journ. of infect. disease fäulnie. Ebenda. 6. 29. (1907). — — 29. 869 (1921). — — — XXV. Vorlesung. Ö06 vor allem kariöse Stellen aufweisen, können Speisereste liegen dann der Zersetzung durch Mikroorganismen anheimfallen. bleiben, die Eine sehr große Rolle in der Einschränkung der Bakterientätigkeit ferner der Salzsäure des Magens zugeschrieben worden, und lange Zeit galt diese Funktion als ihre vornehmste Aufgabe. Der Gehalt des ist Magensaftes an Salzsäure genügt, wie N. Sieber^) nachgewiesen hat, um Entwicklung der Bakterien zu verhindern. Schon SpallanzanP) war diese Eigenschaft des Magensaftes bekannt. Er stellte fest, daß bei einer Schlange, die eine Eidechse verschluckt hatte, die im Mageninhalt befindlichen Verdauungsprodukte nach 16 Tagen noch keinen Fäulnisgeruch zeigten. Auch fand er, daß die Fäulniserscheinungen zurücktraten, wenn er Tieren faules Fleisch in den Magen einführte. Es ging auch der vorhandene Fäulnisgeruch verloren. Doch darf man nicht erwarten, daß die Wirkung der Salzsäure sich in allen Fällen in gleicher Weise äußert oder sich gar quantitative Unterschiede in der Bildung jener Produkte aus Aminosäuren ergeben, die durch die Tätigkeit der anaeroben Bakterien bedingt sind. Die im Darmkanal sich abspielenden Vorgänge sind viel zu komplizierter Natur, als daß man derartig einfache Beziehungen erwarten darf. Es ist die Frage des Einflusses der Salzsäure auf den Abbau von Aminosäuren durch die Darmbakterien insbesondere an Hand von pathologischen Vorgängen verfolgt worden. Wir kennen Zustände, bei denen es zu einer Überproduktion von Salzsäure kommt. Hypersekretion In anderen Fällen ist im Gegenteil die Menge der sezernierten Salzsäure herabgesetzt Hyposekretion. Man hat nun erwartet, daß diese Zustände die Menge und Art der durch die Darmflora gebildeten, bestimmten Aminosäuren entstammenden Abbauprodukte beeinflussen würden. Es war dies jedoch nur zum Teil der Fall. In manchen Fällen fand man selbst bei einem vermehrten Salzsäuregehalt des Magensaftes keine Verminderung jener Abbaustufen. Umgekehrt folgte der Verminderung der Menge der sezernierten Salzsäure nicht immer eine Zunahme jener Produkte. Ja, man hat festgestellt, daß bei völligem Fehlen des Magens die Bakterien durchaus nicht größere Wirkungen als sonst zu entfalten brauchen. die — — Wir wollen einige Gründe angeben, weshalb sich die Wirkung der Salzsäure nicht unmittelbar zu offenbaren braucht. Beginnen wir mit dem letzten der erwähnten Fälle. Fehlt der Magen, dann gelangt die Speise direkt in den Darm. Gleichzeitig mit ihr wird immer auch Luft aufgenommen. Ihre Bestandteile w^erden normalerweise wohl N, 0, CO2 zum größten Teil oder auch vollständig im Magen resorbiert. Es ist dies auch der Grund, weshalb im Magen insbesondere die aeroben Bakterien zur Entwicklung gelangen, falls nicht die vorhandene Salzsäure ihre Entwicklung ausschließt. Sie selbst bilden unter den gegebenen Bedingungen keine jener Produkte, die für die anaeroben Bakterien des Darmkanales typisch sind. Sie halten sich hauptsächlich an die Kohlehydrate, deren Gärung eine besonders lebhafte ist, wenn die freie Salzsäure fehlt, wie das Auftreten von Buttersäure und Milchsäure zeigt. Die antiseptische Wirkung der Salzsäure ist somit mehr nach dieser Richtung zu suchen. Diese Be- — ') Nadina Sieber: Jourii. f. prakt. — Chcm. 19. 433 (1879). Spallanzani: Exp^riences sur la dige.stion. Trad. par Senebier. Nouvelle «dition. (ieaeve 1784. Deutsch Leipzig 1875. ^) Eiweißstoffe und ihre Bausteine. merkungen ö07 gelten auch für den Labmagen der Wiederkäuer. Daß in den und Labmagen eingeschalteten, biologisch zum drei zwischen Speiseröhre Teil zur Mundhöhle hinzuzurechnenden Magenabschnitten die (xärungs Vorgänge eine bedeutsame Rolle spielen, haben wir bei der Besprechung der Ver- dauung der Kohlehydrate bereits erwähnt. i) Im Darm hindert vorhandene Luft die Wirkung der anaeroben Bakterien. Sie können schon deshalb bei fehlendem Magen nicht ohne weiteres aufkommen. Die Salzsäure des Magens hat auch manche indirekte Wirkungen. Sie ist bei der Verdauung der Proteine beteiligt. Einmal aktiviert sie das Pepsin- und Chymosinzymogen und dann verändert sie die physikalische Ferner hat die Salzsäure noch einen Beschaffenheit der Eiweißstofte. bedeutsamen Einfluß auf die Absonderung des Pankreassaftes. Sie wirkt als Reiz und gleichzeitig setzt sie sehr wahrscheinlich einen vStoff in Freiheit, der die Pankreasdrüse zur Abgabe ihres Sekretin genannt Sekretes anregt. Endlich müssen wir noch erwähnen, daß die Salzsäure — — bei der Regelung der Abgabe des Chymus von Seiten des Magens an den Darm beteiligt ist. Die saure Reaktion des den Magen verlassenden Sekretes der bewirkt, daß der Pylorus sich schließt. Hat der Magensaft seine normale Zusammensetzung, dann werden die aufgenommenen Eiweißstoffe zum größten Teil und vielleicht ausschließlich in Form von Peptonen in den Darm übertreten. Dem Trypsin ist in weitgehendem löst einen Reflex aus, Maße vorgearbeitet. Der weitere Abbau erfolgt nun sehr rasch. Außerdem gelangen immer nur geringe Chymusmengen auf einmal in den Darmkanal. Diese können rasch auf eine große Oberfläche ausgebreitet und in die zur Resorption notwendige Form gebracht werden. Durch die Aufnahme der Abbaustufen durch die Darmwand werden sie der Wirkung der Darmflora rasch entzogen. Findet sich eine Hypersekretion. dann folgt bald einer Störung eine ganze Reihe anderer. Einmal wird die Entleerung des Magens beeinflußt. Der Pylorus öffnet sich offenbar erst dann wieder, wenn die in den Darm übergetretene Salzsäure zu einem großen Teil neutralisiert worden ist. Bei einem Zuviel an Salzsäure wird es länger als normalerweise dauern, bis Gleichzeitig wird durch die stark der Pylorus sich wieder öftnen kann. saure Reaktion im Anfangsteil des Darmes sicher die Wirkung der Fermente des Darm- und Pankreassaftes beeinflußt. Sie wirken bei schwach alkalischer bis neutraler Reaktion. Schließlich wird es darauf ankommen, in welchem den Darm gelangten Säure erfolgt. Gelangt sie bis in die tiefen Darmabschnitte, dann wird den Bakterien die Wirkung zum großen Teil sehr beschränkt sein. Ist dagegen die Salzsäure schon in den obersten Teilen des Darmkanals zum größten Teil gebunden worden, dann kann selbst bei einer hochgradigen Mehrabgabe von Salzsäure die Darmflora sich außerordentlich stark entfalten. Man darf sich nicht vorstellen, daß die saure Reaktion allein schon genügt, um die Wirkung der Darmflora einzuschränken, bzw. ganz aufzuheben. Es gehört eine gewisse Konzentration von H-Ionen dazu. Umfange die Neutralisation der in Bei einer verminderten Salzsäurebildung können sich ebenfalls sekundär alle möglichen Störungen und Folgeerscheinungen anschließen. Die Entleerung des Magens kann eine beschleunigte sein, weil der in den Darm •) Vgl. z. B. r. Tabora: Deutsches Arch. f klin. Med. 87. 254 (1908). 508 XXV. Vorlesung. Übertretende Chyraus bald in genügender Weise neutralisiert ist. Dadnrch kann der weitere Abbau im Darmkanal beeinflußt werden. Einerseits ist er dadurch begünstigt, daß die Reaktion eine für die Wirkung der Darmund Pankreasfermente günstige ist. Zugleich werden jedoch ohne Zweifel weniger tief abgebaute Produkte in den Darm übergeführt, weil der Magen sich zu rasch entleert und außerdem die Eiweißverdauung durch die ungenügende Menge der Salzsäure beschränkt ist. Viele dieser Überlegungen sind theoretischer Natur. Wir wissen, daß der tierische Organismus sich dann, wenn irgend eine Funktion beeinträchtigt ist, oft in erstaunlicher Weise den neuen Verhältnissen anpaßt. Eine Hyperazidität des Magensaftes braucht nicht eine langsame Entleerung des Magens zur Folge zu haben und umgekehrt eine Hypoazidität nicht eine rasche. Es können sich Vorgänge herausbilden, die auf lange Zeit hinaus einen annähernd normalen Ablauf der Verdauung gewährleisten. Schließlich darf bei der Frage nach der Beeinflussung der Darmflora durch die Salzsäure des Magens nie übersehen werden, daß auch die Art der Nahrung von großem Einfluß ist. Endlich ist von großer Bedeutung, daß die anaeroben Bakterien unter normalen Verhältnissen ihre Wirkung überhauj)t nur im Dickdarm und vielleicht noch dem unmittelbar benachbarten Dünndarmabschnitt in größerem Umfange entfalten. Es wird sich von Fall zu Fall fragen, ein wie großer Teil jener Produkte in diese tiefen Teile des Darmkanales gelangen, aus denen die Darmflora typische Abbaustufen bilden kann. Die Raschheit der Resorption wird auch bestimmend auf die Menge der durch die Bakterien gebildeten Verbindungen sein. Diese wird wieder von der Schnelligkeit der Verdauung beeinflußt. Es spielen somit sehr viele Momente bei der Bildung von bakteriellen Stoffwechselprodukten aus bestimmten Aminosjiuren mit. Auch der Galle ist immer wieder eine die Darmflora beschränkende Wirkung zugeschrieben worden. Man beobachtet nämlich, daß oft bei verhindertem Zufluß der Galle zum Darm der Kot auffallend stark riecht. Ferner hat man wiederholt, jedoch nicht immer, bei Ikterus einem Symptomenkomplex, der dann eintritt, wenn durch irgend welche Ursachen der Abfluß der Galle nach dem Darm behindert ist ein Ansteigen jener Produkte, wie Indoxyl, Kresol, Phenol beobachtet, die sich auf den Abbau bestimmter Aminosäuren durch Mikroorganismen zurückführen lassen. Nun wachsen Oft fehlt jedoch diese Vermehrung auch. auf der Galle die verschiedenartigsten Bakterien ganz vorzüglich. Sie bilden unter geeigneten Bedingungen auch bei Anwesenheit A^on viel Galle Indol, Kresol, Phenol usw. Das Sekret der Leber kann somit die Wirkung der Darmflora kaum wesentlich beeinflussen. Die Bedeutung der Galle ist vielmehr eine indirekte. Sie ist, wie wir bereits erfahren haben, in hervorragender Weise an der Verdauung der Fette und der Resorption der gebildeten Spaltprodukte beteiligt. Die Galle aktiviert mittels der in ihr — — enthaltenen Gallen säuren die Vorstufe der Lipase, ferner ist sie ein ausgezeichnetes Lösungsmittel für Fettsäuren und Seifen. Setzt nun der Zufluß der Galle zum Darme aus, dann ist die Verdauung der Fette sehr stark eingeschränkt. Sie bleiben zum großen Teil unzerlegt im Darme liegen und erscheinen in den Fäzes. Gleichzeitig ist auch der Abbau der Proteine und Peptone beeinträchtigt. Die Fette umhüllen nämlich Nahrungsstofle und verhindern den Fermenten dadurch rein mechanisch den Angriff. Es KiwoilSstofi'e iiml ihre Bausteine. 509 viel nngespaltene und vor allem unresorbierte Produkte in die Abschnitte des Darmkanals. Den Darmbakterien wird somit beständig eine große Menge von Nahrungsstoffen zugeführt. gelangen tieferen Eine in ihren Einzelheiten noch viel zu wenig erforschte Erscheinung daß bei Stauungen im Dünndarm der Gehalt des Harnes an Indol, Kresol usw. bedeutend ansteigt. i) Wir werden gleich erfahren, daß sehr wahrscheinlich die meisten der genannten Produkte ausschließlich im Darmkanal entstehen und nicht in den Geweben von den diese zusammensetzenden Zellen gebildet werden. Nur dann, wenn in solchen Bakterien sich ansiedeln, die jene Stoff'wechselprodukte zu bilden vermögen, entstehen auch jenseits des Darmkanals Indol, Kresol usw. Es läßt sich eine bestehende Behinderung der Vorwärtsbewegung des Darminhaltes im Dünndarm aus dem Ansteigen des Gehaltes des Harnes an den genannten Produkten direkt feststellen. Findet sich das Hindernis im Dickdarm, dann tritt zunächst keine vermehrte Bildung von Indol und von Phenolen ein. Erst dann, wenn die Stauung auf den Dünndarm übergreift, an jenen Verbindungen auf. Diese tritt ein erhöhter Gehalt des Harnes ICrscheinung ist wohl daraaf zurückzuführen, daß im allgemeinen wenig Eiweißabbauprodukte in den Dickdarm gelangen. Sie werden vorher von ist die Beobachtung, der Dünndarmschleimhaut aufgenommen. Weise haben die Versuche von Ellinger bewiesen, daß Stauungen zur Vermehrung der spezitischen, auf bestimmte Aminosäuren zurückführbaren Abbauprodukte führen. Sie schnitten Hunden Stücke aus dem Darm aus und schalteten diese in umgekehrter Richtung wieder ein, so daß also das proximale Ende des ausgeschnittenen Darmstückes mit dem distalen Teil des gesamten Darmes in Verbindung trat und umgekehrt das distale Ende mit dem mit dem Magen in Verbindung gebliebenen Darmteil. Dieses Darmstück behält nun den ursprünglichen Verlauf der Peristaltik bei und verhindert die Weiterbeförderung des Chymus bzw. des Kotes, indem es der Tätigkeit des übrigen Darmes fortwährend entgegenarbeitet. Es trat je nach der Stelle, an der das Hindernis in der Weiterbeförderung des Chymus geschaffen wurde, verschieden rasch eine starke Vermehrung des Indogehaltes des Harnes auf. In besonders überzeugender und P'iitz^} Die Feststellung, daß auch dann, wenn das Hindernis in tiefen Dünndarmabschnitten sitzt, der Indolgehalt des Harnes bald ansteigt, beweist ohne Zweifel, daß die Resorption der Eiweißabbauprodukte sich nicht in so einfacher Weise vollzieht, wie es meistens dargestellt wird. Wenn die Aufnahme der Eiweißabbaustufen nur daran geknüpft wäre, daß aus Eiweiß diffundierbare Produkte entstehen, dann wäre es schwer verständlich, weshalb bei einer Stauung der Chymus so langsam aufgenommen wird. Man sollte erwarten, daß die Resorption einen Ausgleich schaffen würde. Offenbar werden die Eiweißabbauprodukte an ganz verschiedenen Stellen des Darmkanals abgebaut. Es genügt nicht, daß Peptone entstanden sind. Der Abbau muß ohne Zweifel weiter gehen. Die Bedingungen zu einem vollständigen Abbau sind nicht überall im Darmkanal gleich günstig. Haben sich die Abbaustufen an einer Stelle angehäuft und wird dadurch der Fermentwirkung Einhalt geboten, dann kann einmal die Resorption durch Weg') ^) M. Joffe: Virchows Archiv. 70. 72 (1877). Alexander Ellinger u. Wolfgany I'rutz: Zeitschr. f. physiol. Chem. 38. 399 (1903). XXV. Vorlesung. 510 nähme einfacherer Abbaustufen regelnd eingreifen, es kann jedoch auch die Verteilung des Abbaugemisches auf verschiedene Teile des Üarmrohres den weiteren Abbau günstige Bedingungen schaffen. Eine exakte Analyse des Inhaltes der einzelnen Teile des gesamten Dünndarms wird sicher weitere Anhaltspunkte über den Grad des Abbaus der Proteine im Darmkanal und über die Bedeutung der einzelnen Darmfermente geben. für Auf Grund der Reagenzglas versuche müßte man erwarten, daß bereits im Duodenum alles Tyrosin und Tryptophan abgespalten wird. In Wirklichkeit kann man oft auch im Ileum noch Peptone antreffen, die diese Aminosäuren gebunden enthalten.^) Manchmal fehlen solche Abbaustufen auch ist nicht ausgeschlossen, daß die einzelnen Eiweißabbaustufen an verschiedenen Stellen des Darmkanales zur Aufnahme gelangen. Es liegt hier noch ein viel zu wenig berücksichtigtes Forschungsgebiet vor. Im Anschluß an die Besprechung des Abbaus von Eiweißspaltprodukten durch die Darmflora sei kurz die Frage gestreift, ob die Be- ganz. Es wohner unseres Darmes nicht auch die Verdauung der Proteine durch Abgabe von proteolytischen Fermenten unterstützen. Bis jetzt ließen sich noch keine eindeutigen Befunde erheben, die für eine Teil- nahme der Bakterien am Eiweiß- und Peptonabbau sprechen. Wir dürfen jedoch aus Beobachtungen über die Einwirkung einzelner Bakterienarten und von Gemischen solcher den Schluß ziehen, daß manche der die Darmflora zusammensetzenden Mikroorganismen lebhaft Eiweiß und Peptone zerlegen können und auf diese Weise den Abbau der genannten Verbindungen fördern. Es kommen nicht nur jene Bakterien in Betracht, die Indol, Kresol usw. bilden, ja es ist wohl möglich, daß diese überhaupt nicht am Eiweißabbau beteiligt sind. Schheßlich wollen wir noch des interessanten Umstandes gedenken, daß wir beständig Lebewesen beherbergen, die fortwährend Stoffwechselprodukte liefern, die zum Teil für unsere Körperzelleu durchaus nicht gleichgültig sind. Der Organismus muß ununterbrochen eingreifen, um diesen Verbindungen ihre schädigende Wirkung zu nehmen. Mancher Krankheitsvorgang dürfte auf chronische Vergiftung durch Stoffe zurückzuführen sein, die dauernd durch Bakterien gebildet und vielleicht nicht immer genügend entgiftet werden. Die Leber Sie fängt spielt diesen Produkten gegenüber eine bedeutungsvolle Rolle. sie ab, und verändert sie so, daß sie keinen Schaden mehr stiften können. Von Interesse ist auch, daß die Darmflora die Darmwand unter normalen Verhältnissen nicht durchdringt. Ja selbst bei Wunden braucht es durchaus nicht immer zu einer allgemeinen oder auch nur umfassenderen lokalen Infektion zu kommen. Sobald jedoch der Tod eintritt, beobachten wir, daß nunmehr die Insassen des Darmes rasch die Gewebe durchsetzen. In kurzer Zeit beginnen sie am Abbau der Zellsubstanzen teilzunehmen. Sie helfen mit, ihren Wirt, der Generationen von ihnen beherbergt hat, zu zerstören. Sie sind die ersten am Platze! bewirken die Mikroorganismen des Darmkanales keine Störungen. Sie werden zwar immer einen Teil der Nahrung verwenden, jedoch sind diese Mengen so gering, daß sie kaum Außerdem wird sicher mancher Mikroorganismus in Betracht kommen. innerhalb des Darmkanals zugrunde gehen und dann der Verdauung unterUnter ') normalen Verhältnissen Eigene Beobachtungen. Eiweißstotfe uud ihre Bausteine. 511 diesem Falle findet eine nachträgliche Verwertung der gebildeten Man hat der Synthese von Eiweiß durch Mikroorganismen insbesondere beim Pflanzenfresser eine große Bedeutung zugesprochen. Man hat nämlich gefunden, daß die Zugabe von Amiden Glutamin zur Nahrung den Eiweißstolfwechsel insofern günstig und Asparagin beeinflussen kann, als herbivore Tiere dann mit weniger P^iweiß auskommen. i) Man kann somit einen Teil des Eiweißes in der Nahrung durch diese Amide ersetzen. Man hat nun daran gedacht, daß die insbesondere beim Pflanzenfresser in großen Mengen vorhandenen Bakterien aus den Säureamiden Eiweiß aufbauen und dieses dann nachträglich vom Wirte übernommen, d. h. nach erfolgter Verdauung resorbiert wird.^) In der Tat können zahlreiche Mikroorganismen aus Säureamiden Eiweiß aufbauen, während die Zellen des tierischen Organismus mit so wenigen Grundstoifen nicht auskommen. Die Mikroorganismen desaminieren ohne Zweifel die ihnen dargebotenen Aminosäuren und Säureamide und benutzen das freigewordene Ammoniak zur Synthese. Sie bilden das Kohlenstoffgerüst der einzelnen Aminosäuren, wie wir früher schon gesehen haben, wahrscheinlich aus Kohlehydraten und den Bausteinen der Fette. Die gegebene Erklärung der Wirkung der Säureamide auf die Ernährung der Herbivoren ist zurzeit nur eine Hypothese. Es gilt dies vor allem für den Umfang und die Bedeutung des ganzen Vorganges. Es liegen nämlich noch keine diesen Vorgang quantitativ belegenden Versuche vor. Es ist auch denkbar. daß aus den Säureamiden Aminosäuren Glutaminsäure und Asparaginsänre hervorgehen, welche die aus dem zugeführten Eiweiß entstandenen Bausteine in vorteilhafter Weise ergänzen. Schließlich muß auch daran gedacht werden, daß bestimmte Aminosäuren das Ausgangsmaterial zur Synthese wichtiger Sekretstoffe und sonstiger unentbehrlicher Verbindungen bilden. Wir werden z. B. erfahren, daß das Adrenalin, ein von den Nebennieren gebildeter Stoff, nahe Beziehungen zu Aminosäuren hat, und femer werden wir im Blutfarbstoff eine Verbindung kennen lernen, die sich höchstwahrscheinlich aus solchen bildet. Endlich müssen wir stets mit der Tatsache rechnen, daß der tierische Organismus manche Aminosäure aus vorhandenen bilden kann. Ein sehr wichtiger Punkt bei der Beurteilung der Beteiligung mancher Verbindungen am Eiweißstoffwechsel ist der folgende. Wir wissen, daß aus Aminosäuren andere, für den Organismus wichtige Verbindungen hervorgehen können. Es sei z. B. an die Bildung von Zucker aus einzelnen Aminosäuren erinnert. Es braucht eine beim Abbau von Eiweiß entstandene Aminosäure jenseits des Darmes in gar keine Beziehungen zu Proteinen zu treten. Es kann der Resorption die Desaminierung sich anschließen und nunmehr die verbleibende Kohleuliegen. In Zellbestandteile statt. — — — — ') Vgl. zu diesem noch viel umstrittenen Problem: Weiske: Zeitschr. f. Biol. 17. Virchows Archiv. 94. 436 (1883). 415 (1881); 20. 279 (1884). /. Munk: Politis Zeitschr. f. Biol. 28. 492 (1891). Mauthner: Ebenda. 28. 507 (1891). S. Gabriel : Ebenda. 29. 115 (1892). C. Voit: Ebenda. 29. 125 (1892). Weiske: Ebenda. 30. 254 O. Kellner, A. Köhler, F. Barnstein, W. Zielstor ff", R. Eivert und K. Wedetneyer (1894). Ebenda. 39. 313 (339) (1900). W. Voeltz: Pflügers Archiv. 107. 360 (1905); 107. 415 (1905); 117. 541 (1907). E. Schulze: Journ. f. Landwirtschaft. 65 (1906). V. Henriques und C. Hansen: Zeitschr. f. physiol. Chem. 54. 169 (1907). Max Müller Ebenda. 117. 497 (1907); 127. 497 (1907). W. Vültz und G. Yakuwa: Ebenda. 121. 117 (1908). 0. Kellner: Ebenda. 116. 203 (1907); Journ. f. Landwirtschaft. 49 (1908). Vgl. auch W. Voeltz: Verhandlungen der phvsiol Gcsellsch. zu Berlin. 44. 4 (1919) — — — -) — — — — — — — — Vgl. 0. Hagemann: Landwirtsch. Jahrb." 20. 261 — (1891). — — XXV. Vorlesung. 512 den Kohlehydratstotfwechsel eingreifen oder sonst eine FunkIst für derartige Zwecke genügend Material zugegen, dann können unter Umständen Aminosäuren gespart werden, die zur Bildung von Eiweiß notwendig sind. Die Bestandteile der Darmflora liefern beim Abbau einzelner Aminosäuren Verbindungen, die von den Körperzellen entweder nicht mehr verwertet werden können, oder sie werden sofort nach der Resorption in eine Form gebracht, in der sie dem Zellstoffwechsel entzogen sind. Die Produkte der bakteriellen Tätigkeit aus Aminosäuren haben wir bereits besprochen, i) Wir haben gesehen, daß der Abbau ein mannigfacher sein kann. Es kimnen sich Fettsäuren bilden und ferner gewiß auch Oxysäuren. Auch Ketonsäuren dürften entstehen und Alkohole der um ein Kohlenstoffatoni ärmeren Reihe. Vor allem interessieren uns auch jene Produkte, bei deren Abbau oxydative Vorgänge eingreifen. Ferner stoßen wir im Darmkanal auf Amine, die sich auf bestimmte Aminosäuren zurückführen lassen. Aus allen aromatischen, homozyklischen Bausteinen der Eiweißstoffe und ferner aus Tryptophan und Histjdin bilden Angehörige der Darmflora charakteristische Stoffwechselprodukte. Diese werden resorbiert und der Leber Hier wird ein Teil dieser Produkte mit Schwefelsäure oder zugeführt. Glukuronsäure gepaart. Es erscheinen dann diese Verbindungen im Harn. Wir wollen nunmehr alle jene Verbindungen besprechen, die mit Sicherheit als Produkte der Einwirkung von Bakterien auf bestimmte Aminosäuren erkannt worden sind. Wir sind ihnen allen schon begegnet, als wir die Frage nach dem Abbau der einzelnen Bausteine des Eiweißes durch Bakterien beantworteten. Es erübrigt sich nur noch, festzustellen, ob der tierische Organismus die betreffenden Verbindungen verwertet, und in welcher Form er sie zur Ausscheidung bringt. Wir gehen bei der Besprechung der durch Bakterien aus Aminosäuren erzeugten Verbindungen am besten vom Harne aus. In ihm sind nämlich alle diese Produkte aufgefunden worden. Ferner hat man sie zum Teil auch in den Fäzes festgestellt. Der Harn enthält stets Parakresol und Phenol. Diese beiden Verbindungen sind zum Teil an Schwefelsäure, zum Teil an Glukuronsäure gebunden. Die Konstitution dieser Verbindungen ist die folgende: stofl'kette tion in erfiillen. Cg H, (CH3) SO3 H p- Kr esol schwefelsaure . CH . . Cß H, . . . HO.C.H H.C.-/ . . C^ H^ H C OH . . HO.C.H H.C.— I I H C OH . SO3 H . . CH CH, H C OH . Ce Hg Phenol seh wefelsäure.^) . H C OH . . I I COOH p-Kresol -glukuronsäure ') -) ') COOH Phenol-glukuron säure.') Vgl. Vorlesung XXII, S. 451 ff. Vgl. die Synthese bei E. Baumann: Zeitschr. f. physiol. Chemie. 6. 186 (1882). Carl Neuherg und \V. Neimann : Zeitschr. f. physiol. Chemie. 44. 114 (1905). KiweißstoÜ'o und ihre Baustoiiio. Verbindungen Beide Anteile spalten. So geht säure über: Ce H, . . z. lassen sich unter Ö13 Wasseraufnahme B. Phenolschwefelsäure in Phenol SO, H + IL Umgekehrt vereinigen = Ce H5 OH + OH S( . . in ihre und SchwefelH. »3 beide Anteile unter Wasseraustritt zur hat Verbindungen der letzteren Art, entsprechend ihrer Konstitution, auch Ätherschwefelsäuren genannt.^) Über die Menge ihres Vorkommens lassen sich selbstverständlich ihrer gepaarten ganzen sich Man Schwefelsäure. Entstehung nach keine bestimmten Angaben machen. Einmal kommt in Betracht, wie umfangreich die Tätigkeit jener Angehörigen der Darmflora ist, die p-Kresol und Phenol bilden können. Ferner ist der Umfang der Bildung dieser Produkte selbstverständlich in erster Linie von der Anwesenheit ihrer Ausgangsmaterialien abhängig. Die Bildung der sog. Ätherschwefelsäuren läßt sich künstlich durch Eingabe von Phenolen steigern. Verfüttert man z. B. Phenol, dann findet man im Hara Phenolschwefelsäure bzw. ein phenolschwefelsaures Salz.') Gleichzeitig ließ sich bei derartigen Versuchen feststellen, daß nicht alles eingegebene Phenol im Harn wieder erscheint. Lin Teil wird in den Geweben so verändert, daß er nicht mehr nachweisbar ist.'^j Diese Beobachtung ist sehr wichtig, weil sie zeigt, daß ohne Zweifel auch nicht alle aus dem Darme aufgenommenen Phenole quantitativ im Harne wieder erscheinen. Von Verbindungen, die nach ihrer Einführung in den Organismus in Form von Ätherschwefelsäuren im Harn ausgeschieden werden, seien erwähnt: die K r e s 1 e, CR^ C, 0, OH, T h y m 1. C3 H, (CH3) CV, H3 OH die D i x y. . . , benzole, CeHi(OH,), ferner Methvlhvdrochinon, CHj.O.C« H4.OH, Orzin, CH3 C, H3 (OH),, Pyrogallol, CrHj (0H)3, Tribromphen ol. Bro Cß Ho OH. o-Nitrophenol, NO., Cg H^ OH, p- A m n p h e n o l, . . . . i . NHo i\ H, (.)H. Protokatechusäure, HOOO C« H3 fOH)„ Tannin. Salizylamid, m- und p-Oxy ben zoesäu r e. P^in Teil dieser Ver. . . Inndungen ist meistens auch . mit Glukuronsäure verknüpft. Ferner tritt manchmal ein Teil davon, ohne vorher gekuppelt worden zu sein, in den Harn über. Wir haben früher schon bei der Besprechung der Glukuronsäure) und ihrer Paarlinge auf die interessante Tatsache hingewiesen, daß der tierische Organismus Verbindungen, die an und für sich zur Kuppelung ungeeignet so verändert, daß nunmehr die zur Bindung notwendige Gruppe vorhanden ist. Die gleiche Beobachtung ist auch bei den Ätherschwefelsäuren gemacht worden. Wenn wir z. B. Benzol verfüttern, erscheint Phenolschwefelsäure im Harn. Es ist das Benzol somit zunächst zu Phenol oxydiert und dann mit Schwefelsäure verbunden worden. sind, auch -) •') ) E. Baumann und ('. l'reiisfie: Ebenda. 3. 155 (1879). E. Baumann und E. Uertir: Ber. d. Deutschen Cheni. Ges. 9. 1747 (187(5). IL Whipple: The .1. of. l)i«)l. Chcni. 50. 499 (1922>. Vgl. K. F. Pelkan und Vgl. S. 27. C Abderhalden, Physiologische Chemie. I. Teil, 5. Aufl. 33 XXV. Vorlesung. 514 Im Menschenharn finden sich unter normalen Verhältnissen nur sehr Mengen von Phenol (O'OIT Ol (7).!) Der Harn des Pflanzenfressers enthält erheblich größere Mengen davon. 2) Der Harn des Neuge- — geringe borenen weist keine oder doch nur Spuren von Phenolen auf. 3) Interessanterweise fehlten sie auch jenen Hühnchen ganz, die steril aufgezogen worden waren.) Das p-Kresol überwiegt an Menge das Phenol des Harnes etwas. Siegfried und Zimmermann^) geben als Durchschnittswert 58"/o Kresol und 42% Phenol an. Außerdem findet man im Harn der Omnivoren und der Herbivoren regelmäßig Brenzkatechin-schwefelsäure. Im Urin der Karnivoren wurde diese Verbindung stets vermißt. Das Brenz katechin Ortho-dioxybenzol 1, 2-Dioxybenzol^), Cr H4 (OH).,, kann, wie Fütterungs versuche mit Benzol und Phenol gezeigt haben, aus diesen Verbindungen hervorgehen. Seine normale Abstammung ist ohne Zweifel die COOH, von der Prot ekatechusäur 13, 4-Dioxybenzoesäure, Cg H3 (OH) 2 Para-dioxy benzol der Pflanzennahrung. Auch Hydrochinon 1, = = e= . = = 4-Dioxy benzol, C6H4(OH)2, kommt im Harn vor, doch scheint diese Verbindung bis jetzt nur im Pferdeharn in Spuren sichergestellt zu sein. Diese beiden letzteren Phenole haben offenbar mit den durch Bakterien im Darmkanal gebildeten Produkten nichts zu tun. Wir haben sie hier kurz erwähnt, weil es sich auch um Verbindungen handelt, die als Bestandteile von Ätherschwefelsäuren auftreten können. Von großem Interesse ist das Auftreten einer Säure ini Harn der folgenden Struktur: Cß H5 CH2 . . CO NH CH2 COOH. . . . Diese Formel zeigt unschwer die Zusammensetzung dieser Verbindung an. Sie zerfällt unter Wasseraufnahme in Phenyl essigsaure und G 1 v- kokoll: OH;H Ce H5 CH2 CO . . NH . . CH2 . Phenazetursäure Synthetisch ist sie COOH^Ce H5 CH2 COOH-f- NH, CH, COOH. Glykokoll. Phenylessigsäure . . aus Phenylessigsäurechlorid . . und Glykokoll er- halten worden^): Cß H5 . CH2 . CO . Cl -h HiNH.CH^. COOH=Ce H^.CH^.CO.NH.CHo.Ci )( )H +HC1. ) A. Kassier und E. Penny: Zeitschr. Neuberq: Ebenda. 27. 123 (1899). f. physiol. Chem. 17. 139 (1892). — Cnrl 2) Z. Mmik: Archiv f. (Anat. u.) Physiol. Suppl. 23 (1880); Virchow^ .Archiv. 131. Suppl. 110 (1893). 2) Vgl. z. B. H. Senator: Zeitschr. f. physiol. Chemie. 4. 1 (1879). ) Vgl. S. 106 ff. '") M. Siegfried und R. Zimmermann: Biochein. Zeitschr. 34. 471 (1911). ') Die Zahlen 1, 2 gehen die Stellung im Benzolring an, dessen Atome liek.inatlich, wie folgt, numeriert sind ') E. Uotter: Joui-n. f. prakt. Chemie. 38 (2). 117 (1888). Eiweißstoffe iiiul 515 ihn» Baiisteiue. = Die Verbindung hat den Namen Phenazetursäure Phenyl-azetyl-glyzin erhalten. Sie tindet sich im Harn des Pferdes') und kommt auch zuweilen im Urin des Menschen vor. Nach Verfütterung von Phenylessigsäure findet man Phenazetursänre im Harn. 2) Es unterliegt keinem Zweifel, daß sie ihre Entstehung der Bildung von Phenylessigsäure verdankt, s) Diese entsteht wenigstens zum Teil im Darm. Sie wird dann nach erfolgter Resorption mit Gl y kokoll gekuppelt. Dieser letzteren Verbindung sind wir bereits begegnet. Sie nimmt am Aufbau vieler Eiweißstoife teil. Sie bindet sich noch mit anderen Säuren, wie wir bald erfahren werden. Sie spielt im tierischen Organismus die gleiche Rolle, wie die Schwefel- und Glukuronsäure. Von besonderem Interesse ist die Beobachtung, daß manche Menschen (nach eigener Feststellung nicht alle) im Gegensatz zum Affen nach Zufuhr von Phenylessigsäure Phenyl-azetyl- glutamin ausscheiden.) Im Harne finden sich auch aromatische Oxysäuren. Sie kommen unter normalen Verhältnissen nur in sehr geringen Mengen, jedoch regelmäßig vor. Sie entstehen nur zum Teil im Darmkanal. Ein Teil davon bildet sich auch in den Geweben. Das gleiche gilt wabrscheinlich auch von der Phenylessigsäure, während p-Kresol und Phenol als typische Produkte der Bakterientätigkeit gelten. Die schon erwähnten steril aufgezogenen Hühnchen schieden keine Phenole, wohl aber Oxysäuren aus. Sic finden sieh zum größten Teil frei im Harn vor. Ein kleiner Teil davon kann an Schwefelsäure gebunden sein. Die folgenden beiden ^ erbindungen sind im Harn des Menschen, des Pferdes, Kaninchens und Hundes und auch im Kloakeninhalt des Huhnes aufgefunden worden: Para-oxyphenyl-propionsäures), OH. C.iH^.CH, CHa.COOHundPara-oxyphenyl-essigsäure^j, OH. C« H, CR, COOH. Im Harne finden sich ferner auch heterozyklische Verbindungen, die sich auf die Wirkung von Bakterien zurückführen lassen. Schon lange bekannt"^) ist das Indoxyl. ''^) Es findet sich an Schwefelsäure^) und . . E. Salkowski: Ber. d. Deutschen Chem. Gesellsch. 17.3310(1884); Zeitschr f. Chem. 9. 229, 501 (1885). 2) E. und H. Salkowski: Ber. d. Deutschen Chem. Gesellsch. 12. ()ö3 (1879); Zeitschrift f. physiol. Chem. 7. 162 (1882/83). — F. Knoop: Hofmeistern^ Beitr. 7. 154 (1905). ') C. P. Sherwin und Max Helfand [The .1. of BioL Chem. 40. 17 (1919)] fanden nach Verfiitterung von p-Nitrophenylessigsäure beim Menschen keine Kuppelung, beim Hunde Ausscheidung von p-Nitropheuazetursäure im Harn, beim Huhn trat im Kloakcn) physiol. : inbalt p-Nitrophenazetornithursäure auf. ) Carl P. Sherwin:The J. of biol. Chem. 36. 309 (1918). VgLauchrar/ 1'. Sherwin, Max H'olf und W. Wolf: Ebenda 37. 113 (I9l9j. ) E. Baumann: Ber d. Deutschen Chem. Gesellsch. 12. 1450 (1879); 13. 279 - E. und h. Salkoiiski: Ebenda. //. Salkowski: Ebenda. 12. 1438 (1879). (1880). 12. 650 (1879). E. Baumann: Zeitschr. f. physiol. Chemie. 4. 304 (1880); 6. 183 — — (1882). — — Blendermann: Ebenda. 6. 234 (1882). «) Arthur Hill Hassal: Philos. Magaz. 6 (4.) 22(i (1853). -- Sicherer: Liehuß Annal. 90. 120 (1854). E. Baumann: Pßügers Archiv. 13. 304 (1876). ') Vgl. seine Gewinnung: D. Vorländer und B. Drescher: Ber. d. Deutschen Chem. Ges. 34. 1856 (1901) und 35. 1701 (1902). ) Das Indoxyl des Harns wird aucli als Indikau bezeichnet. Doch ist diese //. — Den Namen Indikau trägt niinilich auch das in bestimmten Pflanzen vorkommende Glukosid. Vgl. S. 71. ) E. Bauinann: Zeitschr. f. physiol l'ßücjer?, Archi Chemie. 1. (')7 (1877/78). 3. 291 (1870). E. Baumann und Brieger: Zeitschr. f. physiol. Cheniie. 3. 254(1879). E. Balimann und E. Herter: Ebenda. 1. 267 (1877). Bezeichnung irreführend. — — — . 33 XXV. N'oilesiiiiir. 516 an Glukuronsäure ') gebunden. Nachuntersuchungen von Stern^) wird mehr Indoxyl mit Sclnvefelsäure gepaart, während l'henol und Kresol die Bildung der Glukuronsäurepaarlinge begünstigen. Das Indoxyl geht, wie Ja/f'e^) durch konnte, aus Indol durch Oxydation hervor: CH Fütterungsversuche beweisen Kiwpiüstoffe uihI ihre Bausteiue. CH CH HC CO ("— ÜC C CH C— \ /\ \ x'\ / CH HC oYl C CH + C C NH NH '1 H., ( / CH liidigoblau. Indigoblau tritt auch und zu ab in ganz erheblichen Mengen im Schweiß auf. Es verdankt seine Entstehung der Zersetzung der Indoxylglükuronsäure. Die entsprechende Schwefelsäureverbindung ist nicht so Ganz vereinzelt ist beobachtet worden, daß der frisch gelassene Urin bereits Indigoblau enthielt. ^j Das Indigoblau ist manchmal von einem zweiten Farbstoft", dem sog. Indigorot, auch Indirubin geleicht spaltbar. nannt, begleitet 2j: CO CO / \ rn / \ C NH. C Ce H, \ / \ / H, NH Er entsteht C« gleichfalls aus Indoxyl. auch das Vorkommen von Skatox vi schwefelsaure beFerner sollen sich manche Harnfarbstoffe vom schrieben worden. Es ist jedoch Skatoxyl, dem Oxydationsprodukt des Skatols, ableiten. nicht gelungen, zwingende Beweise für diese Annahme zu erbringen. Es ist =^) Skatol = Pr H-methylindoIj, C . CH3 /\^CH \ / NH CßH^ Wunq: Festschr. f. E. Salkowski. 397. Berliu 1904. Leube: Plosz: Zeitschr. f. physiol. Chemie. 6. 504 (1882): 8. 85 (1883). liosin: Ebenda. 123. 519 (1H91). Archiv, f. pathol. Anat. 106. 418 (1886). '') Ch. Hervieu.r : Zeitschr. Vgl. speziell über das Skatolrot: Ch. Porcher und Chemie. 45. 486 (1905); Journ. de physiol. et de path. gener. 7.812(1905). f. physiol. Ch. I'orcher: Compt. rend. de l'Acad. d. Ferner über PrS-lndolkarbonsäure ') /;. ') — — — : Sciences. 148. 1210 (1909). ) Die Bezeichnung der aus der folgenden Formel vom Indolkern .sich ableitenden Verbindungen ergibt sich (.: /4\C %B l I Pr f I /'2 «In 61 V, N Die Zahlen geben die Stellung der einzelnen C-Atome lizw. des N-Atoms an. Pr bedeutet Pyrrolring und B Benzolring. Im vorliegenden Fall wird durch Pr3 augegeben, daß im Pyrrolring am Kohlenstoffatom 3 eine Substitution eingetreten ist, und zwar durch den Methyl-, bzw. bei der Indolessigsüure durch den Essigsäurerest. XXV. Vorlesung. ^[g den Fäzes nachgewiesen worden. Es wird jedoch häufig ganz Früher nahnii man an, daß Skatol die N'orstufe des Indols sei. vermißt. 1) Es hat sich jedoch gezeigt, daß für diese Annahme keine eindeutigen Beweise vorliegen. Es sprechen vielmelir alle Beobachtungen dafür, daß es Bakterien gibt, die- Skatol bilden, und andere, die direkt Indol erist in zeugen. engem Zusammenhang mit dem In Indol bzw. Indoxyl steht die Indolessigsäure-): C CH, CÖOH. . /\CH \/ NH . Ce E, Die genaue Bezeichnung dieser \'erbindung lautet lndol-Pr3-essigsäure.3) Ihre Struktur ist von A. EUinger) festgestellt worden. Sie findet sich offenbar nicht regelmäßig und nur in Spuren im Harn. Nur unter umfangreiche Zersetzungsvorgänge im pathologischen Verhältnissen angetroffen worden. ß) ist sie in großen Mengen im Harn Darmkanal Sie bildet sich, wie Fütterungsversuche mit Indoläthylamin ergeben haben, auch in den Geweben. Sie wird vielleicht nie frei, sondern stets gekuppelt ausgeschieden, und zwar scheint sie mit GlykokoU gepaart zu sein.«) Wir kommen auf diese der Phenazetursäure entsprechende Verbindung noch — — zurück.'') Auf der Gegenwart von Indolessigsäure im Harn beruht nach den Versuchen von C. A. Herter^) die Bildung von Urorosein. Dieser Farbstoff tritt auf. wenn Harn mit Schwefel- oder Salzsäure versetzt wird. Es entsteht eine rötliche bis rosarote Färbung. Die Reaktion gelingt nur dann, wenn Nitrite zugegen sind. Urorosein findet sich nicht als solches im Harn. Es entsteht erst bei der Anstellung der Reaktion. Über das Auftreten der IJroroseinreaktion liegen zum Teil widersprechende Angaben vor. Sie dürften darauf beruhen, daß zum Zustandekommen der Reaktion einmal diese sind vielleicht durch andere Indolessigsäure und ferner Nitrite notwendig sind. Oft erhält man mit ganz Oxydationsmittel ersetzbar frischem Harn keine oder nur eine geringfügige Reaktion. Der gleiche Urin zeigt jedoch, wenn er einige Tage gestanden hat, eine ganz ausgesprochene Reaktion. Diese Erscheinung erklärt sich offenbar dadurch, daß Nitrate des Harns, die ja meist in wechselnden, allerdings kleinen Mengen zugegen sind, während der Aufbewahrung des Urins durch Bakterien in — — C A. Herter: .Journ. of Ijiol. (Jhem. 4. 253 (1908). E. und //. Salkoirski: Ber. d. Deutsch. Chem. Ges. 13. 191, 2217(1881); Zeitschrift f. physiol. Chemie. 9. 13 (1885). ») Vgl. Zitat ^) auf Seite 517. ) A. FAlinqer: Ber. d Deutsch. Chem. Ges. 37. 1801 (1904). ^) CA. Herter: Journ. of biol. Chem. 4. 238, 2.53 (1908). «) Arthur James Kwins u. Patrick Plai/fair LaicUaw : The Biochem. .1. 7. 18(1913). ) ") ') Vgl. S. 523. — Vgl. auch E. //. C. A. Herter: .Tourn. of biol. Chem. 4. 239, 253 (1908). E. Salkoimki: Zeitschr. f. Steensma: Nederl. Tijdschr. voor (icneosk. 2. 425 (1904). physiol. Chemie. 42. 21G (1904). 8) — Eiweißstotie und ihre Bausteine. 519 worden sind. Ellinger'^) vermutet, daß das Triindylmethanfarb.stoffen Beziehungen hat. Nitrite übergeführt zu den L'rorosein Fassen wir das Ergebnis der Untersuchung des Harnes auf aromatische Bestandteile zusammen, dann ergibt sich, daß die folgenden Verbindungen sich auf Bakterienwirkung im Darmkanal zurückführen lassen Phenyl essigsaure (Phenazetursäurej, p-Kresol und Phenol, ferner Indol. Außerdem können auf entsprechende Vorgänge die Oxy säuren: p-Oxyphenyl-propionsäure. p-Oxyphenyl-essigsäure und Indolessigsäure zurückgeführt werden. Wir haben jedoch bereits hervorgehoben, letzteren Verbindungen auch jenseits des Darmkanals in den Geweben sich bilden können. Es ist wiederholt vermutet worden, daß auch daß diese die übrigen der genannten Verbindungen in den Körperzellen entstehen können, doch liegen bis jetzt keine einwandfreien Beweise für eine solche Annahme vor. -) Geringe Mengen von Phenolen und von Indoxyl können auch bei vollständigem Ausschluß der Nahrung ihren Ursprung in der Bakterientätigkeit des Darmes haben, ^j Es werden nämlich beständig Eiweißstofte z. B. in Form von Muzin von den zahllosen Drüschen des Verdauungstraktus abgesondert. Ferner wird auch muzinhaltiger Speichel verschluckt. Endlich können auch Bakterien selbst im Darmkanal zugrunde gehen und für andere Mikroorganismen die Quelle der genannten Verbindungen abgeben. Selbstverständlich wird man auch dann Phenole und Indoxyl im Harn antreffen, wenn jenseits des Darmes Bakterien in den Geweben ihre Tätigkeit entfalten, die die erwähnten Verbindungen zu bilden vermögen. Der Zusammenhang dieser Produkte mit der Tätigkeit bestimmter Bakterien ist durch zahlreiche experimentelle und auch klinische Beobachtungen vollständig sichergestellt. Einmal läßt sich zeigen, daß zahlreiche Bakterien aus Eiweiß, Peptonen und den gleich zu besprechenden Aminosäuren Phenole und Indol bereiten. Ferner steigern alle Bedingungen einer vermehrten Wirkung der Bakterien des Darmes die Menge der Ätherschwefelsäuren und der entsprechende Glukuronsäuren im Harn. Daß es vor allem auch auf die Art der Bakterien ankommt, l)eweisen zahlreiche Beobachtungen. So ist z. B. festgestellt worden, daß der Harn von Kaninchen nach \'erfütterung von Kartoffeln viel Indoxylschwefelsäure aufwies, während sie nach Aufnahme von Rüben ganz verschwand.) Die Menge des Phenols blieb unbeeinflußt. Bei beiden Arten der Fütterung wurden die gleichen Bakterienarten aufgefunden. Es überwog A. ElUnger und Claude Flamand: Zeitschr. f. pbysiol. Chemie. 62. 276 (1909); 0. Riesser: In.-Diss. Königsberg-Berlin (1911). 365 (1912). Vgl. dazu auch M. SchoHz : Ber. d. Deutsch. Chem. Ges. 46. 2539 (1913). ^) Vgl. die Literatur über dieses Problem: Biochemisches Handlexikon. 4. 845 ff. A. Ellinger in Ansilyse (1911) (bearbeitet von G. Zmiplrn). J. Springer. Berlin 1911. des Harns. Zweite Hälfte. 799 ff. C. W. Kreideis Verlag. Wiesbaden 1913. ^) Friedrick Müller: Mitteilung aus der Würzburger med. Klinik. 2. 341 (1881). F. Tuczek: Arch. f. Psychiatrie. 15. 784 (1885). Aler. ElUnger: Zeitschr. f. physiol. Chemie. 39. 44. (1903). B. Baumstark und L. Mohr: Arch; exper. Path. u. Ther. 3. Vgl. auch Fritz Rosen teld: Hofmeister Beitr. 5. 83 (1903). Hier finden 687(1906.) sich Angaben über die Beol)achtung von Indol im Harn bei Abwesenheit von solchem im Darminhalt. Vgl. ferner Ferdinand Blumenthal und Ernst Jacoby: Biochem. Zeitschr. 29. 472 (1910). Chüai Asayama: Acta scholae niedic. Univ. imper. Kioto. 1. 115 (1915). "1 V<rl. z. R lirrtrand: Ann. Institut I'asteiir. 27. 7i; (1913). ) — 71. 7 (1911): 78. — — — — — — — — f'. XX\. \'oil('siiug. 520 jedoch bei der Karioft'elfütterung die Menge des Bacterium coli. diesem wissen wir. daß es ein typischer Bildner von Indol ist. Die Herkunft einzelnen Verbindungen der vollständig ist klar Von ge- Phenylessigsäure stammt von Phenylalanin. p-Oxyphenylpropionsänre, p-()xyphenylessigsäure, p-Kresol und Phenol sind auf Tyrosin und Indolessigsäure. Indol und Indoxyl endlich auf Tryptophan zurückzuführen. Die Beziehungen der besprochenen stellt. Verbindungen zu den einzelnen Aminosäuren ergeben sich aus der folgenden Zusammenstelluno; ' ) CH CH HC-'^^NcH HCy AH C CR, . HC^ A^H NH2 . ('H . COOH C Phenyl-y.-a min opropion säure C CH, COOH . Phenyl- essigsaure OH . . C OH . /\ HC CR CH HC CH HC \/ C CH, . CH . CH HC NH, . —> i I \/ COOH C . CH, . CH, COOH . p-Oxyphenyl-x-amino- p-Oxyphenyl-propion- propionsäure säure C OH . HC C..OH CH I C . OH HC CH HC ru HC CH HC CH II CH HC \/ C CH, COOH C . . p-Oxyphenyl-essig- . X/ CH CH3 p-Kresol Phenol. säure NIl, CH c;h HC^ \C- C.CH, .eil .C(K>H HC,^ /CH C. CH 1 nd 1 - NH m nop r '/ - a i Hc/^Xc^^t; CH ( ) j ) i on - . CHa . ('OOH NH Indol-essiffsäure säure ') Vhor die Art dor Kntstolmiif; dieser Verliiiidiintreti vtrl. S. .^)23ff. Kiweißstofte iiud ihre Banstoiiie. 521 CH HC C CH HC C ('H CH NH Indol. Den direkten Zusammenhang der Indolessigsäure und des liidols mit Tryptophan bewiesen Versuche über die Einwirkung von Bakterien auf diese Aminosäure. ^) Daß auch im Darmkanal das Indol der gleichen Quelle entstammt, zeigen die folgenden Untersuchungen. Wurde Kaninchen Tryptophan per os gegeben, dann ließ sieh im Harn keine Vermehrung des Indoxylgehaltes feststellen. Sie trat jedoch bald in Erscheinung, als diese Aminosäure in den Dickdarm eingeführt wurde. Von ganz besonderem Interesse war die Prüfung, o!j Tryptophan, das mit Umgehung des Darmkanals direkt in die Gewebe eingeführt wird, den Gehalt des Harnes an Indoxyl beeinflußt, ^i Es war dies nicht der Fall. Diese Beobachtung stützt die Ansicht, daß in den Geweben keine Bildung von Indol aus Tryptophan erfolgt. P^s sei schließlich noch erwähnt, daß Eiweißstoflfe, denen das Tryptophan fehlt, den Gehalt des Harnes an Indoxyl nicht beeinflussen, ^l Wir kennen somit eine Anzahl von Verbindungen, die sich auf bestimmte Aminosäuren zurückführen lassen und bereits im Darmkanal so verändert werden, daß sie unzweifelhaft für mancherlei Vorgänge in den Geweben nicht mehr vollwertig sind. Ein Teil dieser veränderten Produkte wird in den Geweben weiter umgewandelt. Ein erheblicher Teil davon wird jedoch durch Verbindung mit Schwefelsäure, Glukuronsäure oder Glykokoll festgelegt und in gebundener Form zur Ausscheidung gebracht. Die Paarlinge sind verschiedener Abstammung. Die Glukuronsäure geht ohne Zweifel aas Glukose hervor. Das Glykokoll stammt entweder direkt von Eiweißstoffen ab, oder es wird, wie wir gleich noch erfahren werden, neu gebildet. Die Schwefelsäure endlich kann auf Sulfate der Nahrung zurückgeführt werden, oder aber es bildet der Schwefel des Eiweißes und insbesondere des Zystins das Ausgangsmaterial. Aus der Beobachtung, daß nach Verabreichung von großen Phenolmengen nur dann im Harn eine Vermehrung der Phenolschwefelsäure auftrat, als gleichzeitig Sultide verabreicht wurden, während Sulfate keinen Einfluß hatten, ist der Schluß gezogen worden, daß zunächst eine Bindung des Phenols an schweflige Säure und erst dann die Oxydation zu Schwefelsäure erfolgte.') Wir hätten in diesem Falle ähnliche Verhältnisse vor uns, wie bei der Glukuronsäure. die nach der Ansicht mancher Forscher ebenfalls erst nach erfolgter Kuppelung von Traul)enF. G. Hopkins- und N. W. Cole: Journ. of Physiol. 29. 451 (1<)03). Chuai A. Ellinger und M. Gentzen: Hofmeistern Beiträge. 4. 171 (1904). Asayama: Acta scholae medic. Univ. iniper. Kioto. 1. 115 (1915). 3) F. P. Underhill: Americ. .Journ. of physiol. 12. 17G (1905). ) Tauber: Archiv f. experiin. Path. und Pharniak. 36. 197 (1895); Zeits^chr. t. ') — ") physiol. Chem. 2. .SSR (1878/79). XXV. Vorlesung. 522 zucker mit der festzulegenden Verbindung entstehen soll, i) Die vorliegenden Beobachtungen reichen zu einer bestimmten Schlußfolgerung noch nicht aus. Die Bildungsstätte der gepaarten Verbindungen ist die Leber^) und vielleicht auch die Darrawand. Ob diese Organe die einzige Möglichkeit von Kuppelungen der genannten Art bieten, ist noch unentschieden. 3) Bei der Besprechung der aus Aminosäuren durch Bakterien sich bildenden Abbauprodukte haben wir die Amine kennen gelernt. vSie entstehen entweder durch Abspaltung von Kohlensäure oder von Ameisensäure bei gleichzeitiger Reduktion.) Im Darmkanal findet sich diese Art des Abbaues von Aminosäuren ebenfalls. Bis jetzt sind im Harn die folgenden Amine aufgefunden worden: Putreszin=:Tetramethylendiarain und Kadaverin Pentamethylendiamin. Das erstere stammt, wie wir früher schon festgestellt haben ^j^ von Arginin bzw. Ornithin ab. Das letztere ist auf Lysin zurückzufübren.^) Beide Amine sind nämlich der Zystinurie, beobachtet bei einer Stoffwechselanomalie, worden.'') Über den Zusammenhang der Entstehung dieser Amine und der Ausscheidung von Zystin im Harn ist nicbts bekannt. Unter normalen Verhältnissen ist man den Aminen im Harne nicht begegnet oder doch nur sehr selten. Auch im Darminhalt und den Fäzes scheinen sie nicht in nachweisbarer Menge zugegen zu sein. Wir müssen trotzdem mit der Möglichkeit rechnen, daß die Darmflora aus Aminosäuren auch Amine bereitet. Wahrscheinlich werden sie in den Geweben normalerweise umgewandelt. Vielleicht vermag der an Zystinurie Leidende nicht nur Zystin nicht abzubauen, sondern auch gebildete Amine nicht zu spalten.^) Sehr wichtig ist die Feststellung, daß in der Darmschleimhaut = (Ü-Imidazolyläthylamin nachweisbar ist.^) Es entstammt vielleicht nämlich gelungen, aus der Darmflora einen BaEs ist jedoch zillus zu isolieren, der aus Histidin dieses Amin bildet.^") auch möglich, daß die Darmzellen es bereiten. Die Beobachtung, daß mancherlei Organextrakte eine dem ß-lmidazolyläthylamin nahestehende Wirkung entfalten, hat zu der Vermutung geführt, daß dieses Amin oder dem Darmkanal. Es ist auch Yvho Hämäläinen: Skand. Archiv, f. Physiol. 30. 196 (1913). auch K. F. Pelkan und G. H. Whipple: The J. of Biol. Chem. 50. 499, ) Vgl. hierzu ^) Vgl. 513 (1922). ') sicli Vgl. hierzu Fritz Lade: Zeitschr. f. physiol. Chem. 79. 327 (1912). Hier findet, die Literatur ühcr diese Frage. Vgl. hierzu S. 459. ^) Vgl. S. 321. «) Vgl. S. 324. L. V. Udränsky und £" Baumann: Ber. d. Deutschen Chem. Gesellsch. 21. 2744, 2939 (1888); Zeitschr. f. physiol. Chem. 13. 562 (1899). Stadfhagen und L. Brieger: Virchows Archiv. 115. 490 (1889). A. E. Garrod: Inboru errors of metabolisni. Croonian lectures. 1908. - - A. E. Garrod und W. H. Hurtlei/: Journ. of Physiol. 34. 21(5 A. Loewy und C. Neubcrg: Zeitschr. f. physiol. Chem. 43. 338 (1904). (1906). ) Vgl. hierzu Fmil Abderhalden und Ernst Wertheimer : l'Jiügers Archiv. 197. ) ') — — — ( 1922). — ") G. Barger und //. JI. Dale': .lourn. of Physiol. 41. 499 (1911). Vgl. auch über ß-Imidazolyläthylamin aus Fischfieisch. IJ. Suzuki, U. Mihata, S. Otsuki, R. Junge, l\. C. BItaratkar, Y. Okuda, S. Odake, K. Yoshimura und Y. Tanaka: Journ. (3oll. Agricult. Tokio. 5. 1 (1912). '») Edmund Mellanby und F. W. Twort : Journ. of Physiol. 45. 53 (1912). Eiweißstoft'e und 523 ihre Bausteine. eine ihm nahe verwandte Verbindung in bestimmten Geweben gebildet wird. Besonders wirksam sind die Extrakte aus der Hypophyse, und es scheint, daß dieses Organ Verbindungen bereitet und sezerniert, die den» Imidazolyläthylamin nahe steheni), jedoch nicht mit ihm identisch sind.-) Über den Abbau von Aminen im tierischen Organismus unterrichten — das aus Wurde Indoläthylamin Amin durch die Leber geleitet, dann entstand Indolessigsäure. Wurde dieses Amin Hunden verfüttert, dann Sie ist ganz entsprechend auferschien im Harn Indolazetursäure. die folgenden Beobachtungen. 3) Tryptophan — bildende sich gebaut, wie die Phenylazetursäure, d. h. es hat sich die gebildete Indolessigsäure mit Glykokoll unter Wasseraustritt vereinigt: CH HC C CH.. . . (^OOH + HNH . CH., . COOH = HCy ,C\ JCR NH CH Aminoessigsäure Indolessigsäure CH HC^^Ci C CH, HCV CH . /^' CH . CO NH CH, COOH -f H, . . . NH Indolazetursäure Indolazetyl-'glyzin. Die Bildung der Indolazetursäure ist von ganz besonderem Interesse, höchstwahrscheinlich das sogenannte „Chromogen" des Uroroseins darstellt.) Ferner lehrt das Ergebnis der Verfütterung von Indoläthylamin, daß die Indolessigsäure auch über das entsprechende Amin gebildet werden kann 5): weil diese Verbindung CH HC^\CHC x. C . CH, . CH NH M ICOO H HC c.C CH Tryptophan=:Indola-am in ©Propionsäure Kr. 11 . HC CH •c^ CH CH NH, C.CH.3.CH0.NH2 + CO., CH NH Indoläthylamin Hermann Fiihner: Therap. Moiiatsh. März (1913); Deutsche med. Wochenschr. (1913). ^) Vgl. John J.Ahel und Seiko Kubota: The J. of pharm, and experim. Ther. 13. II.W. Dudley: John J. Abel unüi D.J.Macht: Ebenda. 14.279(1919). 243 (1919). M. 1. Hanke und Karl K. Koeßlcr: The J. of biolog. ehem. Ebenda. 14. 295 (1919). H. H. Dale u. H.W. J)udlei/: The J. of pharm, and experim. Ther. 43. 557 (1920). — — — — 18. 27 (1921). biochem. Journ, ') Arthur James Eivins und Patrick Flayfair Laidlaiv: The M. Guggenheim und W. Lößler: Biochem. Zeitschr. 72. 325 (1916). 18 (1913). =) Vd. hierzu S. 520. ) Vgl. hierzu' S. 518. — 7. XW Vorlesung. 524 . CH HC nC. CH, . COOH + 20 — NH, HC T.H c. NH C'H Indolessigsäure. Dali auch die Ph en y lessii>;sä ure und p-Oxypheuyl- die essigsäure in ganz entsprechender Weise aus den zugehörigen Aminen, nämlich dem Phen y lät hy ami n und dem p-Oxy phenyläthy 1amin gebildet werden können, haben direkte Versuche ergeben. Wurde Hunden per os p-O xyphenyl äthy laniin gegeben, dann erschien im 1 x y p h e n y 1 e s s i g s ä u r e. ') Das gleiche Resultat gaben Durchblutungen von Leber und lUerus. Harn p - Es ist auch möglich, daß die Umwandlung des Amins in die Säure über den entsprechenden Alkohol führt. Dieser Weg sei am Beispiel der Bildung von p-()xyphenylessigsäure aus p-Oxyphenyläthylamin unter Fortlassung etwaiger Zwischenstufen, wie z. B. des Aldehyds, dargestellt C C.ÜH HC^^\|CH OH . HC CH II I HC HC^ JcH NH2 C.CHo.CH.COOH C CH, CH, y p-Oxyj)henyl-äthylamin CO, . . Tyrosin = p-O xyphenyla-aminopropionsäure C CH . OH CH + H.,0"NH3 HC CH \, C (^H, . CHa . . OH p -OXypheny 1 äth y 1a k 1 h 1 C (JH . + 20 — EJ) CH llCi C CH, COOH p-O xyphenyl -essigsaure. . Die Amine haben alle einen mehr oder weniger ausgesprochenen Einfluß auf den tierischen Organismus. ) ») .irid //. . G. Bar<jer und H. H. Dale^) ver- A. J. Ewinn und P. J'. Laidlau-: .lourn. of Piiysiol. 41. 7H (191U). //. JI. Dale und W. K. Diron: .lonrn. of Physiol. 39. 2:') (U)09). //. Dale: Ebenda. 41. 19 (1910): 41. 499 (191U). - H. II. Dalr Laidlau- : Ebenda. 41. 318 (1910). — G. liarger und /'. /'. Kiwoißstoffe und ihro Bausteine. ö-Jfi gleichen die Wirkung der Amine mit der des Adrenalins, eines InkretSie wirken alle erregend auf das sympathische stoftes der Nebenniere. Nervensystem, i-1 midazolyläthylamin steigert z.B. vorübergehend die Schlagfolge des Herzens sehr stark. ') Es bewirkt ferner Verengerung Besonders interder Blutgefäße und damit Steigerung des Blutdruckes. essant ist die Wirkung der Amine insbesondere des Imidazolyläthylamins auf die glatte Muskulatur und im besonderen auf den Uterus. Es bewirkt kräftige Kontraktionen. Diese Beobachtung ist deshalb l)esonders bedeutungsvoll, weil dieses Amin neben anderen im Mutterkorn vorkommt und Indoläthy l.amiu wirkt ganz otfenbar dessen Wirkung mit bedingt. ähnlich. -j Ee bewirkt in Dosen von 20 mg subkutan injiziert bei Katzen heftige Krämpfe. A g m a t i n hat nur geringe Wirkungen, p -0 x y j) h e n y 1äthylamin wirkt durch Vermittlung des N. symi)athicus auf die glatte Muskulatur, während dem Imidazolyläthylamin eine direkte Wirkung zu- kommt. ^l Phenyläthylamin und p-Uxyphenyläthylamin erhöhen den Gesamtumsatz des Organismus.) Schließlich sei noch schwefelhaltiger Produkte gedacht, die Dickdarmgasen enthalten sind und ihre Entstehung ohne Zweifel in erster Linie der Einwirkung von Bakterien auf Zystin verdanken. Yj& sind dies Methy Imerkaptan. CH, SH und Schwefelwasserstoff. H.tS. Was die erstere Verbindung anbetrifft, so kann sie. wie direkte Versuche ergeben habendi, durch Bakterien aus Eiweiß und aus Zystin entstehen. Meistens ist jedoch die im Harn auftretende Verbindung in der Nahrung schon vorgebildet.''! Im Hundeharn ist ferner Diäthy Isnlfid. CiHg S C.j H5. festgestellt worden. -j Es ist wahrscheinlich auch ein Proin . . . dukt der Bakterienwirkung, 'i Außer den genannten Verbindungen der Phenol- und Indolgruppe und den schwefelhaltigen \'erbindungen finden sich ohne Zweifel im Harn noch mancherlei Produkte, die direkt oder indirekt der Darmflora entstammen. Darauf deuten manche Beobachtungen über die allmähliche Abspaltung von Indol beim Versuch, diese Verbindung zu isoHeren, hin.') Da sich die einzelnen Produkte meist nur in sehr kleiner Menge vorfinden, ist es schwer, ihre Natur festzustellen. — Vgl. auch Henry (i. Barbour und ') W. Etoins: Bloch. Zeitschr. 52. DB (1913). Edward M. Franke! Jouin. of Pharmacnl. and experim. Therap. 7. 511 (1915). — Vgl. : über weitere Wirkungen: L\ Rothlin und R. <Uindlach: Arch. interuat. Physiol. 17 .')9 (1921). Paul Schenk: Archiv f. e.xperim. Path. u. Pharm. 92. 34 (1922). 2) J\ F. Laidlaic: Biochemical Journal. 6. 141 (1911). 3) G. (^uagliariello: Zeitschr. f. Biol. 64. 2B3 (1914). Vgl. über weitere ) J. Abeiin und J. Jafi'e: Biochem. Z. 102. 39 (1920). Wirkungen M. Kuf/etia^na : Acta scholae medic. l'uiversitatis imp. in Kioto. 1. 229 (191(5). C. WohlMonatsh. f. Chemie. 10. 526 (l890i. ^) M. Nencki und A'. Sieber: C A. Ucrter : Jouru. of liii)l. (jemuth: Zeitschr. f. physiol. Chemie. 43. 4()9 (190öj. — — : — — Chem. 1. 421 (1906). — Max Rubner, M. Nencki: Archiv f. experim. Path. u. Pharm. 28. 206 (1891). F. Niemann und Staqnitta Balistreri Arcliiv f. Hygiene. 19. 136(1893). ) .;. J. Abel: "Zeitschr. f. physiol. Chem. 20. 253 (1894). Vgl. auch Carl ./. Wohlgemuth: Zeitschr. f' physiol. Chemie. 43. 469 (1905). Neuberg und Groser: Zentralbl. f. Phvsiol. 19. 316 (1905). Ch. l'or^) Vgl. M. Jaft': Archiv f. experim. Path. u. Pharm. Suppl. 299(1908). '^) : >) — — eher: C. r. de l'Acad. d. Sc. 148. 1210 (1909). XXV, Vorlesung, 526 Sicher sind uns noch nicht annähernd alle Prodakte der Täti«:keit der Darmflora bekannt. Auch die aliphatischen Aminosäuren, wie Glykokoll, Alanin, Valin, Leuzin usw., werden von Bakterien angegriffen. Es dürften unter Abspaltung von Ammoniak Fettsäuren und Oxysäuren entstehen. Auch Amine und Alkohole sind zu erwarten. Die weitere Erforschung der Wirkung der einzelnen Bakterien auf die verschiedenen Abbaustufen der Proteine und insbesondere auf die einzelnen Aminosäuren wird namentlich für die Beurteilung der sog. intestinalen Vergiftungen von größtem Werte werden. Es spricht nichts dagegen, daß bei gestörter Verdauung und namentlich bei Bedingungen, die der Darmflora eine ausgiebige Wirkung ermöglichen, Produkte im Darmkanale Es wird das namentgebildet werden, die zu Störungen führen können. lich dann der Fall sein, wenn die Bewohner des Darmkanals bereits im Dünndarm ihre Wirkung entfalten können. Im Dickdarm finden die Bakterien normalerweise nicht mehr viel Material zur Bildung der genannten Verbindungen vor, weil im Dünndarm durch Resorption bis auf geringe Reste alles aufgenommen wird, was durch die Fermente des Darmkanales in die geeignete Form gebracht ist. Auch wird die Leber, die als ein mächtiges Schutzorgan zwischen den vom Darm kommenden Blutstrom und den großen Kreislauf gelagert ist, auch dann, wenn aus dem Darm größere Mengen von Produkten bakterieller Tätigkeit zur Resorption gelangen, noch regulierend eingreifen können. Sie verbindet geeignete ProManche Verbindung wird erst abdukte mit den genannten Paarungen. gebaut und umgewandelt, bis sie zur erwähnten Synthese geeignet ist, wieder andere werden vollständig abgebaut. Versagt die Leber aus irgend einem Grunde, dann können vielleicht schon geringe Mengen von Stoflwechselprodukten der Bakterien zu schweren Schädigungen führen. Vielleicht darf die Beobachtung, daß Hunde, deren Darmblut künstlich durch Anlegung der £'cÄ:schen Fistel unter Umgehung der Leber in die Vena Cava geleitet wird, besonders leicht Vergiftungserscheinungen zeigen, wenn sie eiweißreiche Nahrung aufnehmen, im Sinne eines durch dieses Organ bewirkten Schutzes gedeutet werden, i) Die Verl)indungen Kresol, Phenol und Indol werden ganz allgemein charakteristische Stotfwechselprodukte bestimmter, den Darm bevölkernder Bakterien aufgefaßt. Ihre Bildung jenseits des Darmkanales durch Wir Gewebszellen wird im allgemeinen für ausgeschlossen betrachtet. müssen gestehen, daß eine restlose Sicherung einer solchen Annahme zur als Zeit nicht vorliegt. Im Gegenteil eröffnet der Umstand, daß Beobachtungen bekannt sind, wonach der tierische Organismus einen Teil der genannten Verbindungen in seinen Zellen abbauen kann, die M()glichkeit, daß er sie auch zu erzeugen vermag. Sie entstehen in den Zellen vielleicht nur in ganz geringen Mengen und werden sofort weiter zerlegt. Die Kuppelung der erwähnten Produkte an Schwefelsäure, Glukuronsäure und Glykokoll Vor allem kommt den ist vielleicht bestimmten Zellarten vorbehalten. Archiv f. cxperim. Patli. und ) M. Hahn, O. Massen, M. Nencki und ./. Pawlow F. Fischler: Deutsches Archiv f. klin. Med. 104. 300 (1911). Pharm. 32. IB! (1892). Dieser Autor konnte durch Einfrabc von Säure die Vergiftungserscheinungen bekämpfen, bzw. durch rechtzeitige Säurezufuhr sie verhindern. Vielleicht wirkte die Säure indirekt, indem sie die Tätigkeit der Darmtiora einschränkte. — — : Eiweißstoffe und ihre Bausteine. 527 Leberzellen diese Fähigkeit zu. Die Synthese tritt vielleicht nur dann in Funktion, wenn eine gewisse Menge an den Verbindungen Phenol, Kresol und Indol überschritten wird. Mit der eingetretenen Synthese mit den erwähnten Produkten ist die Abbaufähigkeit aufgehoben. Die vom Darm her resorbierten Abbaustufen bestimmter Aminosäuren, wie Phenol, Kresol, Indol, werden offenbar immer in der Hauptsache gekuppelt. Man wird im allgemeinen, mögen die Dinge nun liegen wie sie wollen, die im Harn auftretenden Mengen an Kresol, Phenol und Indol auf die Tätigkeit der Darmfiora beziehen dürfen; denn wenn auch die KJirperzellen die Fähigkeit besitzen sollten, jene Produkte aus bestimmten Aminosäuren zu erzeugen, so würden sie im allgemeinen doch nicht zum Vorschein kommen, weil sie dem weiteren Abbau unterliegen. Eine andere Frage ist die, ob es nicht Fälle gibt, bei denen diese Zerlegung gestört oder gehemmt ist. In diesem Falle müßte es zu einer Anhäufung dieser Verbindungen jenseits der Darmwand kommen. Die Kuppelung und die damit verbundene Entwürden nicht rasch genug einsetzen können. Es müßten Folgeerscheinungen aller Art einsetzen. Manche bisher unerklärbare Störung des Wohlbefindens läßt sich vielleicht mit derartigen Vorgängen in Zusammenhang bringen, doch liegen bisher nur Vermutungen und keine zwingenden Beweise vor. Immerhin muß der ganzen Frage der Bildung von Kresol, Phenol und Indol und vor allem auch von Aminen und andersartigen Abbauprodukten in den Zellen und der Störung des normalen giftung Ablaufs der raschen weiteren Zerlegung der einzelnen Abbaustufen erhöhtes Interesse entgegengebracht werden. , Schließlich wollen wir nicht unerwähnt lassen, daß wir ohne Zweifel mit unseren Nahrungsmitteln immer kleine Mengen der oben erwähnten Verbindungen aufnehmen. Besonders reich an Aminen ist der Käse. Aus ihm konnte z. B. Tyranin gewonnen werden. i) Histamin ist wiederholt als Bestandteil der Nahrung, z. B. im Fleisch festgestellt worden. 2) ) ') E. Winterstein and Alb.Küng: Zeitschr. f. physiol. Chemie. 59. 138 (1909). Vgl. J. Abel und Ä Kubota: .T. of Pharm. 13. 243 (1919). Vorlesung XXVI. Eiweißstoffe und ihre Hausteine. 10. Verhalten der von der Darmwand aufgenommenen Proteine jenseits des Darmkanals. Abbaustufen der Der Darminhalt enthält unter normalen N'erhältnissen Peptone und Aminosäuren. In den dem Dickdarm benachbarten Teilen des Ueums trifft man außerdem meistens auch Abbaustufen von aromatischen AminoEs ist sie wohl immer vorhanden. daß im ganzen Dünndarm beständig Abbaustufen des Eiweißes von Bakterien zu ihren Zwecken verwertet werden. Es brauchen nicht nur jene Produkte zu entstehen, die nach stattgehabter Resorption zum Teil durch Kuppelung mit Schwefelsäure, Glukuronsäure oder Glykokoll vor der weiteren Zerlegung bewahrt werden und infolgedessen im Harn zum Vorschein kommen. Es können Fettsäuren. Oxysäuren oder auch Alkohole gebildet werden, je nach der Art der Zusammensetzung der Bakterienflora und den herrschenden Bedingungen. Auf alle Fälle wird unter normalen Verhältnissen immer nur ein kleiner Teil des aufgenommenen Eiweißes von den Bewohnern des Darmes verwendet. Der bei weitem Wir größte Teil der Eiweißabbaustufen kommt direkt zur Resorption. haben bereits hervorgehoben, daß es zurzeit ganz unmöglich ist, die Frage nach der Art der zur Aufnahme gelangenden Produkte sicher zu beantworten. Unzweifelhaft werden beständig Aminosäuren aufgenommen. Daneben kann es sehr wohl auch zur Resorption von Pepsäuren an. Im Dickdarminhalt sind .sehr wahrscheinlich, tonen kommen. Wir kommen nun zu der wichtigen Frage,- was aus den von der Darmwand aufgenommenen Eiweißabbauprodukten wird. A priori man glauben, daß dieses Problem nicht allzuschwer zu lösen sei, kann man doch z. B. einem Hunde große Mengen Eiweiß zu fressen geben in (3 bis 8 Stunden und feststellen, daß innerhalb relativ kurzer Zeit das aufgenommene Material fast vollständig aus dem Darm versehwunden ist. Es ist somit nur notwendig, innerhalb dieser Zeit nachzusehen, was aus den aufgenommenen Produkten wirdi So einfach die Fragestellung sollte — — ist, so schwierig ist ihre Beantwortung! Der Abbau der Proteine und Peptone vollzieht sich im Diirmkanal stufenweise. Der vom Magen dem Darme übergebene ihre Bausloine. 529 Chymus wird auf eine durch Faltenbildang — Zotten -gewaltig;- vergrüücrte Eiwoiüstort'e uiiil Oberfläche ausgebreitet. Er wird von den Sekreten der Darmschleimhaut und der Pankreasdrüse und ferner von Galle durchtränkt. Der Abbau setzt sofort energisch ein. Gleichzeitig werden die gebildeten Abbaustufen durch die peristahischen Bewegungen des Darmes weiter geschoben. Sie kommen mit neuen Sekretmengen in Berührung. Die Bedingungen, unter denen der Abbau sich vollzieht, wechseln. Die Reaktion, die in dem oberen Teil des Darmes noch ausgesprochen sauer ist, verliert in den tieferen Abschnitten des Jejunums und im Ileum mehr und mehr den sauren Charakter. Es werden im Darme beständig Abbaustufen gebildet, die zur Resorption geeignet sind. Sie entstehen normalerweise immer nur in kleiner Menge an den verschiedensten Teilen des langen Dünndarms. Bald wird hier etwas aufgenommen, bald dort. Fortwährend strömt an den Zellen des' Darmes Blut und ferner Lymphe vorbei. Beide Flüssigkeiten sind durch die ungezählten Kapillaren, die jede einzehie Zotte versorgen, auf eine gewaltig große Oberfläche ausgebreitet. Die aufgenommenen Produkte können in den Zellen des Darmes Halt machen, sie können jedoch auch sofort an das Blut und die Lymphe weiter gegeben werden. Es ist jedoch auch möglich, daß schon innerhalb der Darmwand Veränderungen vor sich gehen. Den aufgenommenen Spuren von Abbauprodukten zu folgen, ist außerordentlich schwierig. Es bestehen nämlich alle Zellen des tierischen Organismus und auch alle Zwischensubstanzen, wie Bindegewebe usw., vornehmlich aus Eiweiß. Auch das Blut enthält große Eiweißmengen. Ferner werden im Organismus Es entstehen, wie wir bald erfahren beständig Eiweißstoffe zerlegt. werden, in den Zellen Peptone und Aminosäuren. Die letzteren werden weiter abgebaut. Zwischenprodukte im Abbau von Aminosäuren, solche selbst und ihre letzten Abbauprodukte können beständig dem Blute übergeben werden. Wie sollen wir entscheiden, ob ein im Blute gefundenes Produkt soeben vom Darme aufgenommen worden ist oder irgend einer Zelle des Körpers entstammt?! Die ganze Fragestellung würde viel einfacher zu beantworten sein, wenn von der Darmwand aus dem Chymus eigenartige, charakteristische Abbaustufen aufgenommen würden, oder wenn jenseits des Darmes Produkte entstehen würden, die sich ohne weiteres in direkte Beziehung zur Resorption von Eivveißabbaustufen bringen ließen. Endlich würde unzweifelhaft das ganze Problem nach der Art der den Körperzellen zugeführten Eiweißabbaustufen schon längst klar entschieden sein, wenn der tierische Organismus in irgend einem Organ Lagerstellen besäße, die von aufgenommenen Verbindungen, die zum Eiweiß in Beziehung stehen, gespeist würden. Wohl kann nach neueren Beobachtungen die Leber Eiweiß in gewissem Umfange speichern \), wir sind jedoch nicht imstande, wie das bei den Kohlehydraten und Fetten der Fall ist, Eiweiß in gewissen Grenzen nach Belieben zum Ansatz zu bringen. Wir finden viehnehr die zunächst überraschende Tatsache, daß mit der Zunahme der in Form von Eiweiß verfütterten Stickstofl"men}2;e bei gleichbleibender Zufuhr von stickstofl"frcien — W. ßir;/ luul ('. l'ahn») Vgl. yV. Berq: Biochcm. Zeitschr. 61. 428 (l'.)U). Hans Stiibel: Pßiif/ers Archiv. 185. 74(1920).— Bronner: Ebenda. 61."4B4 (li)14). /'. Junkersdorf: Fjlüger?, Archiv. 186. 254 (1921). — Abderhalden, Physiologische Chemie. I. Teil, .t. Aufl. 34 XXVI. Vorlesung. 530 Nahrangsstoffen — Kohlehydraten und Fetten — die Ausfuhr stickstofi- annähernd Schritt hält. Je mehr Eiweiß wir zuführen, um so mehr stickstoffhaltige Produkte erscheinen im Harn. Wir dürfen aus dieser Erscheinung nun nicht, wie dies früher oft geschehen ist, den Schluß ziehen, daß der ausgeschiedenen Stickstoffraenge ein vollständiger Verbrauch der das Eiweiß aufbauenden Bestandteile entspricht. Es hat vielmehr die exakte Untersuchung des Eiweißstoffwechsels und besonders des Verhaltens der Aminosäuren im Zellstoffwechsel ergeben, daß wir bei der Beurteilung des Eiweißumsatzes nicbt, wie es allgemein üblich war, nur die im Eiweiß aufgenonynene Menge Stickstoff der im Harn ausgeschiedenen und im Kot verbliebenen Stickstoffmenge gegenüberstellen dürfen. Es verläßt nämlich, wie wir gleich erfahren werden, nur ein relativ kleiner Teil des Kohlenstoffs des Eiweißes den Organismus mit dem Stickstoff zusammen. Der übrige Kohlenstoff tritt, wie wir bereits wiederholt hervorgehoben haben, in Beziehungen zu Kohlehydraten und Es können somit sehr wohl Teile des aufvielleicht auch zu Fetten. genommenen Eiweißes gespeichert werden, nur nmß zuvor der Stickstoff austreten und zugleich eine Umwandlung erfolgen, welche die direkte Beziehung zum Eiweiß bzw. zu seinen Bausteinen löst. haltiger Stoflfwechselendprodukte Diesem Umbau von Aminosäuren in Zucker können wir leider l)ei Untersuchungen, die den Stoffwechsel des ganzen Tieres betreffen, nicht direkt folgen. Wir können ihn nur in seiner Gesamtheit aus Stoffwechselversuchen erschließen und ihn durch Versuche am überlebenden Organ wahrscheinlich machen. Diese Umwandlung vollzieht sich sicher nicht in großem Umfange auf einmal, sondern immer nur bald hier, bald dort in kleineren Schüben.^) Daher kommt es, daß wir den Zwischenstufen der Überführung von Aminosäuren in Traubenzucker in den Geweben und auch im Blute nicht begegnen. Wird es einmal möglich sein, Spuren von einzelnen Verbindungen zu folgen, dann wird gewiß auch hier mancher Weg, den bestimmte Produkte zurücklegen, bis sie in andere übergeführt sind, klar sichtbar werden. Vorläufig vermögen uns die Beziehungen der Bausteine der Proteine zu den Kohlehydraten nichts über die Art der vom Darm aufgenommenen Eiweißabbaustufen und der zum Transport gelangenden auszusagen. Da wir die gestellte Frage nach der Art genommenen Eiweißabbaustufen und die Form, in der vom Darm aufder ihre Überführung zu den Körperzellen erfolgt, nicht eindeutig beantworten können, so bleibt uns nichts anderes übrig, als die wahrscheinlichsten Möglichkeiten zu besprechen. Zunächst müssen wir die Frage entscheiden, welchen die aufgenommenen Eiweißabbauprodukte einschlagen. Es unterliegt keinem Zweifel, daß im wesentlichen der Blutweg benutzt wird, doch findet ohne Zweifel stets auch ein Transport v^on Eiweißabkömmlingen von Seiten der Lymphe statt. 2) Weg Wir wollen nunmehr versuchen, den von der Darmwand aufgenommenen Produkten zu folgen. Zunächst interessiert uns ihr Ver) Es scheint, daß die Leber an diesen Umwandlungen besonders Ijeteiliirt ist Vgl. P. Junkersdorf: Pflüger?, Archiv. 186. 254 (1921). ) Emil Abderhalden, Arno Ed. Lampe und E. S. Ijondon: Zoitschr. f. plivsiol. Chemie. 84. 213 (1913). Kiweißstoffc und ihre Bausteine. 531 Wir haben gesehen, daß die aus dem in der Darm wand. Darmkanal aufgenommenen Bausteine der Fette in dieser wenigstens zum Teil zu Neutralfett zusammengefügt und dann in dieser Form durch des halten Sollten nicht auch Eiweißabbaustufen in den Zellen des Darmes wieder zu Eiweiß vereinigt werden? Es wäre z. B. Ductus thoracicus dem Blute zugeführt werden. die resorbierten Darmwand von bestimmten Zeliarten beständig jene Proteine gebildet Averden, die sich im Bhite und im besonderen am Blutplasma tinden. In diesem Falle kimnten wir die Zellen der Darmwand mit solchen von Drüsen vergleichen, die ja auch beständig Stoffe auf- denkbar, daß in der sie umwandeln und dann nach außen oder in die Lymph- bzw. Hhitbahn Sekret- bzw. Inkretstoffe abgeben, die eine ganz bestimmte Zusammensetzung haben. Genau ebenso könnten die Zellen der Darmwand aus dem aus dem Darmkanal aufgenommenen Gemisch von Verdauungsprodukten ein gleichartiges Gemenge von ^'erbindungen und insbesondere von Proteinen bereiten, das dann als Xährmaterial der Gewebe dem Blute übergeben würde. Auf diese Weise würden dem Blute stets quantitativ und qualitativ die gleichen Produkte zugeführt. Die Zellen des Darmes könnten bei der Synthese dieses Eiweißgemenges manchen Stoff' zurückhalten, ihn umwandeln und vielleicht aucli schon manche wichtige Syn- nehmen, these in die Wege leiten, die v^on bestimmten Aminosäuren ihren Ausgang nimmt. Bei dieser Überführung des heterogenen Gemisches von Abbaustufen in ein homogenes Material würden sicher viele Abfallstoffe entstehen. Er könnte Bald würde dieser, bald jener Baustein un verwertbar sein. umgewandelt oder zur weiteren Benutzung der Leber oder auch anderen Körperzellen zugeführt werden. Manches Eiweißabbauprodukt dient ferner vielleicht im tierischen Organismus auch ganz bestimmten Aufgaben und wird direkt übernommen. D e A n n a h m e e n e r E w e ß b d u n g in d e r D a r m w a n d konnte durch keine \' ersuche bewiesen werden. Es wurde i i i i i 1 Frage aufgeworfen, ob die Darmwand während der Resorption von Eiweißabbauprodukten einen höheren Gehalt an Eiweiß aufweist, als vor die der Resorption.^) Es ließ sich kein deutlicher Unterschied feststellen. Ein solcher fehlt vielleicht deshalb, weil die Möglichkeit besteht, daß das Ferner ist versucht gebildete Eiweiß sofort dem Blute übergeben wird. worden, eine Eiweißsynthese im überlebenden Darm festzustellen. Es wurde Eine in einen solchen ein Gemisch von Eiweißabbaustufen eingeführt. Bildung von Eiweiß war nicht nachweisbar. 2) Auch dieser Versuch schließt nicht aus, daß unter normalen Verhältnissen in der Darmwand sich eine Synthese von Plasmaproteinen vollzieht, weil ihre Bildung sich wahrscheinlich nach dem Bedarf richtet, und es sicher in hohem Maße auf die vorhandenen Bedingungen ankommt, ob ein Aufbau aus zugeführten Bausteinen eintritt. Es sei noch bemerkt, daß man selbstverständlich niciit an eine Bildung einzelner Zeliproteine in der Darmwand denken darf. Die Zellen der Darmwand können ja nicht über den Eiweißbedarf der Wohl aber ist die Möglichkeit einzelnen Körperzellen unterrichtet sein. daß vom Darme aus je nach Bedarf die Menge der Plasma- gegeben, M Etiiil Abderhalden und E. S. London: Zeitschr. f. physiol. Chem. 55. 251 (1910). Peter Rona: Biochem. Zeitschr. 46. .307 (1912). ==) 34 XXVI. N'orlesuiiij. 5;',2 zum Teil wenigstens Die erfolgreichen Versuche von Carrel^), dem es gelang, Gewebe in Blutplasma zu züchten und sie zur Zellvermehrung zu bringen, beweisen ohne Zweifel, daß die Organzellen auf Kosten der im Plasma enthaltenen Stoffe leben können. Sicher kommen auch die vorhandenen Eiweißstoffe in Betracht, denn der Gehalt des Blutes an nicht eiweißartigen, stickstoffhaltigen Produkten ist sehr gering. Leider sind bis jetzt noch keine eindeutigen Beobachtungen über eiweißstüffe ergänzt wird. Diese stellen ottenbar — das Nährmaterial der Körperzellen dar. Zunahme der explantierten. die wirkliche in Plasma gezüchteten Gewebe an neugebildetem Material gemacht worden. ist die, daß in der Darm wand die weiter verändert Ei w ei ßab bau stufen Einmal könnten etwa aufgenommene Peptone a ollständig zu Eine weitere Möglichkeit aufgenommenen werden. Aminosäuren abgebaut werden. Es könnte jedoch auch eine Umwandlung Es wäre z. B. denkbar, daß der einzelnen Aminosäuren sich vollziehen. aus ihnen Traubenzucker gebildet wird, oder sich Beziehungen zu anderen Verbindungen anbahnen. Diese Umwandlung brauchte nicht alle aufgenommenen Aminosäuren zu betreffen. Nun wissen Avir, daß der Abbau der Aminosäuren in den Geweben in der Regel in der Weise erfolgt, daß frühzeitig die Aminogruppe abgespalten wird. Es müßte somit im Falle eines umfangreicheren Abbaues von Aminosäuren in der Darmwand während der Periode der Aufnahme von Eiweißabbauprodukten aus dem Darmkanal im Blute in größerer Menge Ammoniak erscheinen. In der sich Angaben, daß während der Verdauung von Eiweißstoffen Tat linden das Blut der Pfortader einen erhöhten Gehalt an diesem aufweise. 2) Dieser Befund ist nicht eindeutig. Es kann sich auch um Ammoniak handeln, das im Darmkaual entstanden ist. Wir haben ja erfahren, daß Bakterien Ammoniak aus Eiweißbausteinen abspalten, denn nur auf diesem Wege aus den entsprechenden Amiist die Entstehung von Phenol und Indol nosäuren möglich. Daß das im Blute auftretende Ammoniak zum großen Teil durch Resorption aus dem Dickdarm in dieses gelangt, hat Folin-') durch direkte Beobachtungen über seine Herkunft gezeigt. Es liegen keine Beweise für die Annahme vor, daß D a r m w a n d resorbierte E w e ß a b b a u p r d u k t e in m m e n d e n M e n g e n über d e A m n 0rg en d w e n B e t rach t k säuren hinaus verändert werden. Selbstverständlich werden die Zellen der Darmwand genau so, wie die übrigen Organzellen beständig in der i i i i i i i bzw. Aminosäuren umsetzen. Jede einzelne wird solche ihrer Aminogruppe berauben und den Abbau weiter führen können. Es ist Eiweiß jedoch nicht erwiesen, daß die Zellen der Bedarf hinaus Aminosäuren zerlegen. Darmwand über ihren eigenen — /', Jiurroirs: Journ. Americ. med. Ass. 1379 (1910). ') Alexis Carrel und M. Vgl. weitere Literatur im Haiidb. d. biochem. Arbeitsmethoden. ,3. 836 (1912); 0. .'il9 Ferner Alber/ Oppcl: Zentralbl. f. Zoologie, ullgem. u. expcrim. Biologie. 3. (1912). — 209 (1913). ^) M. Hahn, 0. Massen, M. Nencki und ./. Pawloir: Arcliiv f. experim. Patli. u. M. Nencki, J. I'awlow und ./. Zaleski: Ebenda. 37. 26 (18'.)iii. Pharm. 32. 161 (1896). Vgl. auch <). Cohnheim: Zeitschr. f. physiol. Chemie. 33. 9 (1901); Dh 396 (1902). ') Offo Folin und VV. />rnis: .lourn. of Biol. Chem. 11. 1(')1 (1912). — — 58^ Kiweißstorti' uiul ihre Bausteine. Es bleibt nun noch die folgende Miiglichkeit. Die aus dem Darmkanal a u f g e n ni m e n c n E av e ü a b b a u s t n f e n durchwandern als solche das Darniepithel und gehen in das Blut über. i i Zunächst gelangen sie in die Pfortader und in dieser zur Leber. Es ergibt ob dieses Organ Eiweißabbaupiodukte verändert oder aber direkt durchläßt. Im einen Falle würden wir die resorbierten Eiweißabbaustufen nur im Pfortaderblut antreffen, im anderen wären sie auch im Ulnte des ül>rigen Kreislaufes zugegen. Es ist lange Zeit unermüdlich nach Aminosäuren und Peptonen im Blute geforscht worden, ohne daß es gelungen wäre, zu eindeutigen Resultaten zu gelangen. Einzig im Rinderblut wurde freies Gly kokoll aufgefunden i), doch dürfte dieser Befund kaum mit der Resorption von Aminosäuren aus dem Darmkanal in Zusammenhang stehen, weil die Eiweißkörper der Nahrung im allgemeinen nur über geringe Ulykokollmengen verfügen. Das festgestellte Glykokoll entstammt ohne Zweifel dem Zellstoftwechsel. Wir werden noch erfahren, daß der tierische Organismus diese Aminosäure in großen Mengen bilden kann. Der Umstand, daß bis vor kurzem weder Aminosäuren noch Peptone im Blute aufgefunden werden konnten, kann mehrere Ursachen haben. Eininosäur en, mal ist es Avohl möglich, daß die »Spuren von die in jedem Zeitpunkt zur Resorption gelangen, sofort sich die Frage, Am d e n G e w e b e n zugeführt und dort festgehalten werden. Wir können ja immer nur das Blut in einem bestimmten Augenblick dem Organismus entnehmen und auf diese Weise auf jene Aminosäuren fahnden, die sich eben auf dem Wege vom Darm zu der Leber bzw. zu den übrigen Geweben befinden. Es ist versucht worden, Vergleiche zwischen dem Gehalt des Blutes an Aminosäuren vor, während und nach stattgehabter Eiweißverdauung zu ziehen. Die Resultate waren nicht befriedigend. Man mußte ferner mit der Möglichkeit rechnen, daß die Aminosäuren in irgend welchen Zellen zum Transport gelangen. Es wäre ja möglich, daß weißen und auch die roten Blutkörperchen Ferner ist wiederholt die Vermutung geäußert worden, es könnten die Proteine des Blutplasmas und vielleicht auch der Lymphe Aminosäuren locker binden. Es ist Die Zellen des Blutes schwer, diese Möglichkeiten eindeutig zu prüfen. brauchen für ihren eigenen Stoftwechsel Eiweißstofle bzw. Aminosäuren. Ferner wissen wir, daß sie über Fermente verfügen, die Eiweiß und Peptone spalten können. Wie alle anderen Körperzellen werden auch die Ein roten und weißen Blutkörperchen aus Eiweiß Aminosäuren bilden. Gehalt der Zellen des Blutes an bestimmten Stoffen darf somit nicht ohne weiteres mit einer Überführung nach den Geweben in Verbindung gebracht werden. ^) die sich mit solchen beladen. ^) Die wichtigste Ursache des Versagens der bisherigen Versuche, im Blute Eiweißabbauprodukte nachzuweisen, liegt h n e Z w e i f e 1 i n d e r M e t h o d i k. Zunächst ist versucht worden, den Gehalt des Blutes an stickstoffhaltigen Produkten nicht eiweißartiger Natur im Hungerzustand und während der Verdauung von Eiweiß fest') ^) 3) A. Bitif/el: Zeitschr. f. physiol. Chemie. 57. 382 (1908). Vgl. hierzu auch A. Constanfiiio: Biochem. Zeitschr. 55. 402, 411 (1913). \gl. hierzu Emil AhderhaJdcn und //. Kiirfen: Pjlik/eri^ Archiv. 189. 311 (1921). XXVI. Vorlesung. 534 waren widerspreebend. Offenbar war die restNach Verbesserung der Methodik der Stickstoffbestimmung in kleineren Mengen von Blut und \'erwendung einer zweckmäßigen Art der Enteiweißung ist es FoJin und zustellen.!) Die Ergebnisse lose Beseitigung der Eiweißstoffe nicbt immer gelungen. Denis^) gelungen, zu zeigen, daß während der Aufnahme von Eiweißabbaustufen von seiten des Darmes im Blute die Menge jener stickstoffhaltigen Verbindungen ansteigt, dienichteiweißartigerNatursind. Ferner konnten diese Forscher durch Bestimmung einzelner stickstoffhaltiger Bestandteile des Blutes, wie des Kreatins, des Harnstoffes, des Ammoniaks, es sehr wahrscheinlich machen, daß die Vermehrung der nicht Eiweiß darstellenden Verbindungen im Blute auf eine Zunahme von Aminosäuren zu beziehen ist. Meyer und van Slijke ») konnten diesen Wahrscheinlichkeitsschluß noch dadurch stützen, daß sie nicht nur den Stickstoffgehalt des enteiweißten Blutes bestimmten, sondern auch die Menge des Am inos tick Stoffs. Wir haben früher erwähnt), daß das Eiweiß über nur wenige freie Aminogruppen verfügt. Je weiter es abgebaut wird, um so mehr Aminogruppen werden frei. Vergleicht man in einem Gemisch von Eiweißabbauprodukten das Verhältnis von Gesamtstickstoif zum Aminodann kann man beurteilen, ob es viele einfachere Eiweißabbaustufen oder gar Aminosäuren enthält. Schließlich ist es noch geglückt •'), mittels Triketohydrindenhydrat, eines Reagenzes, das mit Aminosäuren, Polypeptiden und Peptonen auch in großer Verdünnung eine Blaufärbung gibt, zu beweisen, daß während der Verdauung von Eiweiß das Blut eine deutliche Zunahme von solchen Verbindungen zeigt, die sicher nicht Eiweiß sind, denn sie dialysieren leicht. ^^ Die vorliegenden Ergebnisse zeigen eindeutig, daß während der Au fnahme von Eiweißabbauprodukten im Blute Verbindungen auftreten, die nicht Eiweiß sind, dagegen zu Eiweißabbaustufen direkte Beziehungen haben. Diese Produkte, die auch dann zu finden sind, wenn man Peptone oder direkt Aminosäuren in den Darmkanal einführt, können der Gruppe der Peptone angehören oder aber Polypeptide und endlich stiekstotf. Gustav r. Bergmann und Leo Langstein: Hofmeüters Beitr. ) Vgl. hierzu u. A.: H. Holdweg uud H. Meyer: 27 (1904). G. V. Bergmann: Ebenda. 6. 40 (1904). Ebenda. 11. 381 (1908). H. Pringle und W. Cranier: Jonrn. of Physiol. 37. 158 (1908). 12. 2) Otto Folin und W. Denis: The Journ. of biol. Chem. 11. 87, 493 (1912); Vgl. auch zu diesem Problem 0. Folin: Americ. Journ. 141 (1912): 12. 253 (1912). ot Physiol. 13. 117 (1905). Otto Folin und //. Berghmd : The J. biol. Chem. 51 Vgl. ferner Paul Ggörgy und Edgar Zunz: Journ. of biol. Chem. 21. 395 (1922). — 6. — — — ' — — 511 (1915). 3) (1912). Donald, D. van Slgke und Gustav M. Meyer: Journ. of biol. Chem. 12. 399 — Vgl. auch H. Delaunuy: Contribution ä l'ätude du röle des acides amines — E. S. London. dans l'organisme animale. A. Destout, aine et Cie., Bordeaux 1910. X. A. Dobroivolskaja uud A. 1>. Wolkotc: Zeitschr. f. plivsi(d. Chemie. 87. 325. 334 (1913). ) Vgl. S. 373 ft". ^) Emil Abderhalden und Arno Ed. Lanipr: /eitsclir. f. physiol. (Tieniie. 81. 478 (1912). ) Der l'mstand, d;iß uucli andere A'erbindungen als Eiweiß und seine Aiikömmlinge mit Triketohydrindenhydrat reagieren [vgl. W. Halle, K Lonrenstein und 7?. Piihram: Biochem. Zeitsclir. 55. 3r)7\l913); Carl Neuhrrg : Ebenda. 56. 495 (1913)]. vermag die Beweiskraft dieser Versuche niclit zu erschüttern, weil unter den angewandten Bedingungen kaum andere Produkte für die Farl»reaktion in Betracht kommen, uud außer (b^m sr-hließlich die Isolinrung von Amiuosäureu geglückt ist. Eiweißstotfe und ihro Bausteine. 535 Aminosäuren sein. Nun haben frühere Versuche schon ergeben, daß das Blutplasma unter normalen Umständen keine Produkte enthält, welche die Biuretreaktion geben und nicht eiweißartiger Natur sind. ') Auch neuere, scharfen Methoden durchgeführte Untersuchungen haben gezeigt, daß die Zunahme des Blutes an nicht eiweißartigen Produkten auf Verbindungen entfällt, die keine Biuretreaktion geben. 2) Es können somit jene stickstoffhaltigen, dialysierbaren, nicht koagulierenden, während der Resorption von Eiweißabbauprodukten an Menge zunehmenden Produkte nur noch Peptone, die die genannte Farbreaktion nicht zeigen, ferner einfacher zusammengesetzte Polypeptide und endlich Aminosäuren sein. Es t^ei gleich erwähnt, daß alle Beobachtungen dafür sprechen, daß im Blute während der Verdauung der Proteine Aminosäuren in vermehrter Menge zugegen sind. Es darf nicht verschwiegen werden, daß die vorliegenden Beobachtungen die Anwesenheit von Aminosäuren im Blute nur sehr wahrscheinlich machen, jedoch nicht direkt beweisen. Als letztes Glied der ganzen Beweisführung mußte die Darstellung von Aminosäuren aus Blut gefordert mit besonders werden. Es ist nun gelungen, durch Dialyse großer Mengen von Blut Aminosäuren nachzuweisen, und zwar sind alle bis jetzt bekannten Eiweißbausteine aus diesem in reinem Zustand isoliert worden.') Es sei gleich hervorgehoben, daß sowohl im Pfortaderblut, als auch im übrigen Blut Aminosäuren anzutreffen sind. Dagegen stößt man, wie schon oben hervorgehoben, nirgends auf Verbindungen, die die Biuretreaktion geben und nicht eiweißartiger Natur sind. Aus dieser Beobachtung geht hervor, daß entweder die im Darminhalt nachgewiesenen Peptone vor ihrer Resorption vollständig bis zu Aminosäuren oder doch zu Verbindungen abgebaut werden, die nicht mehr geben, oder es vollzieht sich dieser Darmwand. Der Leber dürfte kaum eine Rolle bei der die Biuretreaktion Abbau in der Überführung resorbierter zusammengesetzter Verbindungen in ihre Bauzukommen, denn man findet auch dann keine die Biuretreaktion gebenden Stoffe im Blute, wenn sie mittels der Eckschen Fistel ausgeschaltet worden ist.) Allerdings sind Versuche dieser Art nie ganz eindeutig, weil die Leber ja in Wirklichkeit nicht ganz aus dem Kreislauf entfernt ist. Sie steht noch mit ihm durch die Leberarterie und die Lebenvenen in Verl)indung. Immerhin müßten sich im vorliegenden Falle dann, wenn von der Darmwand Peptone ins Blut entlassen würden, im Körperblute solche nachweisen lassen, denn diese Verbindungen könnten erst dann zur Leber gelangen, nachdem sie den übrigen Kreislauf durchlaufen haben. steine Dürfen wir aus der Tatsache, daß während der Verdauung und der mit ihr parallel gehenden Resorption von .der Proteine ') Vgl. S. 503. -) Zahlreiche eigene Beobachtungen. — ./. Abel deKmif Abderhalden: Z. f. physiol. Chemie. 114. 250 (192lj. monstrierte auf dem internationalen Physiologenkongreß in Groningen (September 1913) ein Verfahren, um aus dem Blute dialysierbare Stoffe abzuscheiden. Er laßt das Blut des lebenden Tieres durch Dialysierschläuche strömen und wieder in den Körper zurückHießen. Mit diesem Verfahren ist es Abel ebenfalls geglückt, Aminosäuren im Blute nachzuweisen. Vgl. auch A Slo^se: Arch. internat. de physiol. 18. 242 (1921). ) Emil Ahder/ialdPTi. Casimir Funk und E. S. London: Zeitschr. f. physiol. Chem. •') — ."1I 279(1907). XXVI. Vorlei^uujr. 536 Eiweißabbaustiifen im Blute Aminosäuren auftreten, den Schluß ziehen, daß den Körperzellen das aufgenommene tLiweiß ausschließlich in Form von Aminosäuren zugeführt wird?^) Es ist verlockend, anzunehmen, daß eine einheitliche Form der Übermittlung der dem Eiweiß zugehiu-enden Verbindungen vorhanden ist. Es würden in dem Falle, daß nur Aminosäuren zum Transport gelangen, die Gewebe die einzelnen Eiweißbausteine nach Bedarf aufnehmen und verwenden können In der Tat ist es auch geglückt zu zeigen, daß insbesondere das Muskelgewebe während des Transportes der nicht eiweißartigen, stickstoffhaltigen Verbindungen eine beträchtliche Zunahme aa diesen Stoffen zeigt. Wir können die gestellte Frage am besten beantworten, wenn wir die strikte Beweisführung, daß die des „Eiweißtransportes" darstellen, zu stellen sind. Zunächst genügt der (lualitative Nachweis der Aminosäuren durchaus nicht, um festzustellen, ob die Gesamtheit der vom Darm aufgenommenen Eiweißabbauprodukte den Körperzellen in Form von Aminosäuren zugeführt werden. Hier können nur quantitative Untersuchungen eine eindeutige Entscheidung bringen. Man wird durch zahlreiche Versuche zu prüien haben, wie groß die Vermehrung des Blutes an Aminosäuren innerhalb bestimmter Zeiten ist. Daran anschließend wird man festzustellen haben, in welcher Zeit sich die Resorption einer bestimmten Menge von in den Darmkanal eingeführten Proteinen vollzieht. mitteilen, welche Anforderungen Aminosäuren die einzige an Form Der einfache Nachweis von Aminosäuren im Blute läßt mannigDeutungen zu. Es kimnte nämlich auch dann zu einem Übergange faltige von solchen in gebildet würden. kommen, wenn in der Darmwand Plasmaproteine, Wie die vergleichende Untersuchung der Zusammensetzung dieses der verschiedenen Proteine ergeben hat, besitzen diese zwar die gleichen Aminosäuren, jedoch tinden sich in den Mengenverhältnissen, in denen die einzelnen Bausteine vorhanden sind, große Unterschiede. Würden die Zellen der Darmwand aus dem resorbierten Gemisch von Aminosäuren und vielleicht auch von zusammengesetzten Eiweißabbaustufen bestimmte Eiweißstoffe aufbauen, dann würden ohne Zweifel viele Aminosäuren übrig bleiben, für die keine Verwendung vorhanden ist. Es würde sich die Verwendbarkeit jeder einzelnen Aminosäure nach der im Minimum vorhandenen vorausgesetzt, daß keine Umwandlung der einen Aminosäure in richten andere eintritt. 2) Ferner muß noch das folgende Bedenken beseitigt werden. Wir beobachten, daß auch im Hunger beständig im Harn stickstoffhaltige Produkte auftreten, von denen wir wissen, daß sie mit dem Eiwcißstoffwechsel in Zusammenhang stehen. Es muß nach allen Beobachtungen in den Körper-^ Zellen stets Eiweiß zum Abbau Jvommen. Wir können seine Menge auf ein' Minimum einschränken, indem wir reichlich stickstofffreie Nahrungsstoffe verfüttern. Es gelingt jedoch niemals, den Eiwcißstoffwechsel auch nur für kurze Zeit ganz zum Stillstand zu bringen. Verfüttern wir Eiweiß, dann erscheint im Harn je nach den Versuchsbedingungen und dem Zustand — ) Vgl. hierzu aiirh Joint 44. 320 (1917). ./. MkI, M. C. l'incoll's und ('. A. Rouillcr: Auicric. J. of Physiol. ^) Emil Ahdirhuldeii 647 (1904). : /ontrallil. f. Stoff wocliscl- und Verdauungskrankheiteii. 5. Ei\veißst.i)He uud ilue Bausteine. ÖH7" des Tieres eine seinem KStiekstofl'^ehalt mehr oder weniger vollständigentsprechende iMenge Stickstoff. Es fragt sich nun. ob dieser Stick-' Stoff unmittelbar auf das aufgenommene Eiweiü zurückzuführen ist oder aber, ob er wenigstens zum Teil immer Proteinen entstammt, die schon vorher in den Zellen vorhanden waren. Es spricht alles dafür, daß die Herkunft des im Harn erscheinenden Stickstoffes eine mannigfaltige ist.' Sicher werden ununterbrochen Eiweißstoffe der Körperzellen zerlegt und durch neues Eiweiß ersetzt. Es wäre denkbar, daß bei reichlichem Angel)ot von Eiweißbausteinen der Abbau innerhalb der Zellen ein gesteigerter ist. und der Ersatz durch Proteine, die aus den zugeführten Aminosäuren gebildet werden, ein besonders lebhafter ist. Sei dem nun, wie ihm wolle, ist erwiesen, daß die jedenfalls und das ist für uns die Hauptsache Körperzellen selbst Eiweiß zu Anunosäuren abbauen können. Es fragt sich nun, was aus ihnen im einzelnen Falle wird. Sie können in den Zellen selbst weiter abgebaut werden. Sie können jedoch auch ins Blut übergehen und zum Transport kommen. Diese letztere Möglichkeit führt zu der Forderung, daß in jedem einzelnen Falle ausgeschlossen wird, daß die im Blute und auch in den Geweben beobachteten Aminosäuren dem Zellstoftwechsel entstammen. Die Tatsache jedoch, daß w^ährend der Aufnahme von Eiweißabbaustufen von Seiten der Darmwand eine Vermehrung des Gehaltes des Blutes an Produkten eintritt, die wahrscheinlich zum größten Teile und vielleicht vollständig den iVminosäuren angehören, macht es im höchsten Maße wahrscheinlich, daß diese Verbindungen vom Darme aus in den Kreislauf gelangen. Es w^äre gesucht, wollte man annehmen, daß während der Resorption von Eiweißabbaustufen auch die Körperzellen in vermehrtem Maße Eiweiß zum Abbau bringen und dem Blute Aminosäuren übergeben. Immerhin muß dieser Möglichkeit gedacht werden. Überblickt man die bisherigen Ergebnisse der Erforschung des Schicksals der Eiweißstoffe im tierischen Organismus, dann ergibt sich mit größter Wahrscheinlichkeit, daß die Aminosäuren im Mittelpunkt des ganzen Eiweißstoffwechsels stehen. Über sie führt die Umwandlung eines Eiweißkörpers in einen anderen. Sie bilden den Ausgangspunkt von Synthesen aller Art. und über sie geht auch der Abbau zu den End])rodukten des Eiweißstoffwechsels. — in — Die Aminosäuren werden von den einzelnen Körperzellen mannigfaltiger Weise verwendet. Einmal dienen sie zum Aufbau Eiw^eiß. Jede Zelle enthält in der ganzen Organismenwelt mehrere Eiweißkörper. Je nach der Art der Zelle sind diese verschieden beschaffen. Jede Tierart hat eigene Eiweißstoffe und auch jedes Organ bildet in jeder Organismenart wieder, wie die chemische Untersuchung zeigt, besondere Proteine. Es ist wohl möglich, daß jede Zelle Proteine ganz verschiedener Bedeutung enthält. Es ist denkbar, daß es Eiweißkörper gibt, die ausschließlich Bausteine der Zelle sind. Andere dagegen bilden vielleicht das Ausgangsmaterial zu mannigfachen Vorgängen. Es könnte auch sein, daß in gewissem Sinne jede einzelne Zelle Keserveeiweiß enthält. Es ist nicht auf bestimmte Zellen festgelegt und wird vielleicht nach Verbrauch immer wieder ersetzt. Es tritt nicht in Erscheinung, wie das Reservefett und das Glykogen, weil es sich nicht von deni übrigen Eiweiß der Zelle abhebt. Vielleicht werden weitere Fortschritte auf dem Gebiete der Eiweißchemio von XXVI. Vorlesung. f)38 eine Unterscheidunji; der einzelnen Zellproteine ermöglichen. Vorläufig wir auf die Fermente als feinste Reagenzien auf Unterschiede im Aufbau nahverwandter Verbindungen angewiesen. Wir finden in der ganzen Organismenwelt neben den abbaufähigen Verbindungen auch die zugehörigen Fermente. Ein sehr schönes Beispiel für diese Tatsache ergeben die mannigfaltigen Glukoside der Pflanzenwelt. Stets treffen wir mit diesen auch Fermente an, das sie in ihre Bausteine zerlegen können. Es war zu erwarten, daß auch die Zellen des tierischen Organismus über Fermente verfügen, die Eivveißstoffe abbauen können. Die Fahndung auf solche hat ergeben, daß sowohl Proteasen in allen ~Körperzellen anzutreffen sind, als auch Fermente, die Peptone und Polypeptide bis zu Aminosäuren abzubauen vermögen. Würde die Annahme eines allen Körperzellen zukommenden, in gewissem Sinne für den täglichen Bedarf bestimmten Reserveeiweißes in der Form zutreffen, daß dieses Proteingemisch für jede Tierart einheitlich ist, dann wäre zu erwarten, daß alle Zellen der verschiedensten Organe über Fermente verfügen, die diese Eiweißart abbauen können. Die direkte Prüfung sind ergeben, daß jede Zellart proteolytische Fermente besitzt, die zelieigene Proteine spalten können, dagegen vermag z. B. der fermenthaltige Preßsaft aus Leberzellen nicht aus Nierenzellen stammende Eiweißstoffe zu zerlegen, i) Aus dieser Beobachtung folgt, daß offenbar jede Zellart über eigenartige Proteine verfügt und sich auf dem erwähnten Wege kein „Umsatzeiweiß", das allen Körperzellen gemeinsam ist, feststellen läßt. Es spricht vielmehr alles dafür, daß sich jede Zelle eigene Proteine bereitet. hat Die Zelle baut ohne Zweifel beständig Eiweißstoffe ab, weil sie Bruchstücke davon zur Bildung von allen möglichen Produkten braucht. Es spricht vieles dafür, daß manche Fermente in nahen Beziehungen zum Eiweiß Auch andere Sekretstoffe dürften den Proteinen verwandt sein. Ferner bedarf der tierische Organismus beständig der Aminosäuren. Je weiter die Forschung der Funktionen der einzelnen Organe vordringt, um so eindringlicher hebt sich die wichtige Tatsache hervor, daß alle untereinander in Wechselbeziehung stehen. Die gegenseitige Beeinflussung bestimmter Organe untereinander kann durch Vermittlung des Nervensystems erfolgen. Vielfach und vielleicht immer spielen auch bestimmte Inkretstoffe als Sendboten eine große Rolle. Diese werden an das Blut abgegeben und gelangen auf diesem Wege zu jenem Organ, das zu irgend einer Funktion angeregt werden soll. Diese Stoffe müssen eine ganz spezifische Struktur und Konfiguration haben und vermittelst dieser auf ein bestimmtes Substrat eingestellt sein. Dieses wird durch jene Zellart dargestellt, auf die der Inkretstoff eine Wirkung entfaltet. Wir könnten ohne diese Annahme nicht verstehen, weshalb ein bestimmter, dem Blute übergebener Stoff' nur auf eine bestimmte Zellart einwirkt und an allen anderen vorübereilt, ohne seine Wirkung geltend zu machen. Es können chemische Beziehungen maßgebend sein, es können jedoch auch physikalische eine Rolle spielen. Damit der Inkretstoff einen Einfluß ausüben kann, muß er ohne Zweifel stehen. ') Emil Abderhalden und Andor Fndor: Zeitschr. f. physiol. Chemie. 87. 220(1913). — Emil Abderhalden und hruin Schiß': Ebenda. 87. 231 (1913). — Vgl. auch Martin Jacoby: Hofmeister?, Beitr. 446 (19Ö3). — Ch. Eichet: Compt. rend. de la soc. de 3. 05. O.iß (1903). — Vgl. auch Tscherikowski: Zeitschr. biol. f. physiol. Ohouiio. 111 76(1920). Fiiweißstoffe und ihre Bausteine. 539 Vermag er das nicht, dann ist er für die beNeuere Ergebnisse machen es nun sehr wahrscheinlich, daß manche dieser Inkretstoffe sich direkt auf bestimmte Aminosäuren zurückführen lassen. Wir haben bereits erwähnt, daß in der Hypophyse sich wirksame Verbindungen finden^), die dem Imidazolyläthylaniin nahestehen. Ferner haben wir im Adrenalin eine Verbindung kennen gelernt, die nahe Beziehungen zu aromatischen Aminosäuren zeigt. Von in die Zelle eindringen. treffende Zelle wirkungslos. großem Interesse ist auch die Beobachtung, daß die Zephalopoden in ihren Speicheldrüsen p-Oxyphenyl-äthylamin bilden und nach außen abgeben. -j Werden den Zellen von außen Aminosäuren bestimmter Art zugedann wird vielleicht der Eiweißabbau in der Zelle selbst wesentlich eingeschränkt. Es scheint, daß zu den Inkretstoffen vornehmlich Aminosäuren der aromatischen Reihe \'erwendung finden. Braucht eine Zelle solche, dann werden wahrscheinlich nach erfolgtem Abbau von Eiweiß zahlreiche Aminosäuren tÜr den weiteren Eiweißstoffwechsel entwertet sein. Sie werden ihrer Aminogruppe beraubt und dann in Form von Kohlehydraten der Zelle führt, zur \'erfügung gestellt und, falls kein Bedarf an solchen ist, auch als (rjykogen oder Fett gespeichert. Vielleicht erklärt sich aus diesem Umstände, weshalb der tierische (Organismus stets beträchtliche Mengen von Stickstoff" im Harn zur Ausscheidung bringt, auch wenn ihm so viele stickstofffreie l'rodukte zur Verfügung stehen, daß er seinen ganzen Energieumsatz damit bestreiten und außerdem noch solche ablagern kann. Es muß jedoch auch hier, wie bei den Kohlehydraten, mit der Möglichkeit gerechnet werden, daß zu bestimmten Funktionen nur Aminosäuren als Energiequelle dienen können. Mit der Annahme, daß die Aminosäuren im Mittelpunkt des ganzen Eiweißstoffwechsels stehen und dieser eigentlich besser als Aminosäurestoffwechsel bezeichnet wird, ergibt sich für folgende, eigenartige Erscheinung eine verständliche Erklärung. Wenn wir einem Tiere und auch dem Menschen Eiweißstoffe zuführen und gleichzeitig Kohlehydrate und Fett geben, dann können wir bei einer bestimmten Menge der ersteren ein sog. Stickstoftgleichgewicht erreichen. Es wird gerade soviel Stickstoff ausgeschieden, wie im Eiweiß zugeführt wird. Steigern wir nun die Eiweißmenge bei gleich bleibender Zufuhr an stickstofffreien Nahrungsstoffen, dann wird nicht etwa das nun überschüssig zu betrachtende Eiweiß als solches im Organismus abgelagert, sondern es erscheint annähernd ebensoviel mehr Stickstoff' im Harn, Diese Feststellung läßt als in Form von Eiweiß mehr zugeführt wurde. sich vielleicht, wie folgt, erklärend Die Eiweißstoffe werden im Magendarmkanal weit abgebaut. Zum Transport in. der Blutbahn nach den Geals weben gelangen offenbar hauptsächlich und vielleicht ausschließlieh Aminosäuren. Diese werden den einzelnen Körperzellen zur Verfügung gestellt. Hier ergänzt eine Zelle ihren Bedarf an Eiweiß, dort verwendet eine andere eine bestimmte Aminosäure zu einem bestimmten Zwecke. Wieder andere Zellen brauchen größere Mengen von Aminosäuren zur Bildung von Sekreten und Inkreten. Es werden z. B. von ungezählten Zellen des >'erdauungs- und Muzin - gebildet und nach Kespirationstraktus l>estimrate Eiweißarten — •) Vgl. -) M. Henze: /eitsfhr. S. b23. f. physiol. Chcm. 87. .'-.1 (1918). 540 XXVI. Voilesiiug. außen abgegeben. Alle sieb in denDarnikanal ergießenden Säfte sind eiweißSchließlich bleiben Aminosäuren übrig, für die keine \'erwendung vorhanden ist. Eine Speicherung von Aminosäuren in gr()ßerem Umfang ist nicht möglich. Die einzelnen Zellen haben ihr „Umsatzeiweiß" ergänzt. Die überschüssigen Aminosäuren werden desaminiert. Der Stickstoff' erscheint in organischer Bindung im Harn. Die verbleibenden Kohlenstottketten kininen sofort über manche Zwischenstufen bis zu Kohlensäure und Wasser abgebaut werden, falls gerade Bedarf an Energie ist. Ist dies nicht der Fall, dann setzt die Umwandlung der nach der Desaminierrtng verbliebenen Kohlenstoffketten in Kohlehydrate ein. Wenn längst schon aller Stickstoff den Organismus verlassen hat, können Kohlen- und Wasserstoff" der zerlegten Aminosäuren noch im Organismus vorhanden sein und mitten im Kohlehydratstoft'wechsel stehen. Vielleicht ist auch der Kohlenstoff mit dem Wasserstoff" über die Kohlehydrate herüber in Fette übergegangen! Vielleicht tinden sich von Kohlenstott'ketten, die von Aminosäuren abstammen, auch direkte Beziehungen zu den Bausteinen der Fette. Wir können in der Tat kaum je wissen, woher die Elemente stammen, die eine bestimmte in den Geweben vorhandene Verbindung aufbauen. Wir dürfen nach dem heutigen Stand unserer Kenntnisse des Zellstoffwechsels weder den Kohlehydrat-, noch den Fett-, noch den Eiweißstoff"wechsel eng umgrenzen. Der Stoff'wechsel jeder einzelnen Art von Verbindungen greift in den anderer Stoff"e ein. Infolgedessen, das sei nochmals nachdrücklich hervorgehoben, ist es nicht angängig, den Eiweiß- bzw. Aminosäurestoff'wechsel allein von der Stickstoff bilanz aus zu beurteilen. Nur die Verfolgung des Gesamtstoff'vvechsels ergibt ein vollständiges Bild des Umsatzes von Aminosäuren und Eiweißstoff'en. Steigern wir nun die Zufuhr von Eiweiß, so stehen dem Organismus weitere Aminosäuren zur Verfügung, für die er keine direkte Verwertung hat. Speichern kann er sie nur in Form von Kohlehydraten bzw. von Fett. Es setzt die Desaminierung ein, und daran schließt sich dann der Umbau an. Im Harn erscheint eine der vermehrten Eiweißzufuhr entsprechende haltig. Zunahme des Stickstoffgehaltes. Ist diese Vorstellung richtig, dann muß man mit Eiweiß mästen können, denn jene Eiweißzufuhr, die über jenen der unbedingt notwendig ist, um Lücken im Zellstottwechsel und im Bau der Zelle auszufüllen, muß zur Vermehrung des Kohlehydrat- bzw. Fettbestandes des Organismus führen. Je höher wir den Eiweißgehalt der Nahrung halten, um so mehr muß die Stickstoff'ausscheidung im Harn ansteigen und um so mehr Kohlen stoft'ketten stehen den Zellen zu den verschiedensten Zwecken zur Verfügung. Es fragt sich nun. in welchen Zellarten die Umwandlung bestimmter Aminosäuren in Zucker erfolgt. Kann jede einzelne Körperzelle aus diesen Zucker bilden oder ist eine besondere Zellart mit dieser Aufgabe betraut? Im letzteren Falle müßten die in den einzelnen Organen frei werdenden Aminosäuren der betreff"enden Stelle zugeführt werden. Da wir jedoch, soweit unsere Kenntnisse reichen, im Blute unter normalen A'erhältnissen nie eine irgendwie in Betracht kommende Menge von diesen A'erbindungen antreffen, so müssen unzweifelhaft Momente vorhanden sein, die den Transport der Aminosäuren regeln. Wenn wir nämlich solche ins Blut einführen, dann kommt es leicht zu ihrer Ausscheidung im Urin. Eine Aminosäure, das Glykokoll. wird oft im Anteil herausgeht, Eiweißstort'e Harn angetroffen. 'j gebildet. Es Sie wird, und wie ilirt' Bausteine. selion 54[ erwähnt, in den Geweben mag sein, daß sie manchmal im Überschnß entsteht und e-; zur Hyperglyzinoplasraie kommt. Die Xiere scheidet dann das überschüssige Glykokoll aus. Es ist jedoch auch mitglich. daß die Xiere diese Aminosäure selbst zu besonderen Zwecken bereitet und dabei über das Ziel hinausschießt. Es sei daran erinnert, daß rhenylessigsäure und ferner die Benzoesäure an Glykokoll gepaart im Harn erscheinen.-) Es werden noch manche andere .Säuren auf diese Art gebunden. Wir kommen auf diese interessante Synthese noch zurück. ^Yir kennen mehrere Zustände, bei denen es zur Überschwemmung des Blutes mit Aminosäuren kommt. So beobachten wir bei Phosphorvergiftung das Auftreten von Aminosäuren im Harn.^j Ihrer Ausscheidung geht ein erhöhter Gehalt des Blutes an diesen voraus. Auch bei der akuten, gelben Leberatrophie), bei der es zu raschem Zerfall von Zellen kommt, finden wir erhebliche Mengen von Aminosäuren im Blut und gleichzeitig eine .,Amiuoazidurie". Normalerweise enthält das Blut, wie schon erwähnt, sicher nur geringe Mengen von Aminosäuren. Es müssen daher unzweifelhaft, besonders während der AufMahme von solchen von der Darm wand aus, \'orgäuge eingreifen, die verhuidern, daß der Aminosäurespiegel im Blute Ob dem Blute eine bestimmte Menge von Aminosäuren lieständig zukommt, die nur zur Zeit des Transportes von solchen vom Darm nach den Geweben ansteigt, ist noch unentschieden, jedoch nach allen Erfahrungen sehr wahrscheinlich. Eigene Beobachtungen haben ergeben, daß das Blut wohl nie frei von Aminosäuren ist. Die Regelung der Zufuhr der Aminosäuren zum Blute dürfte eine mehrfache sein. Auf der einen Seite wachen die Xieren darüber, daß ein bestimmtes Xiveau des Aminosäuregehaltes des Blutplasmas nicht übersehritten wird. Normalerweise treten sie wohl kaum in Funktion. Immerhin ist es wohl möglich, daß sie viel öfter, als wir es wissen, eingreifen. Es ist durchaus nicht gesagt, daß die Niere nur dann, wenn im Harn bestimmte Verbindungen, wie Zucker. Aminosäuren usw. erscheinen, regulierend in Tätigkeit getreten ist. Sie wird gewiß manches Produkt aus dem Blute abfangen, es zurückhalten und vielleicht nach Benutzung zu Synthesen oder in sonst einer Weise umgewandelt dem Blute zurückgeben. Weiterhin dürfte der stufenweise Abbau der Proteine und Peptone im Magendarmkanal jeder Überschwemmung des Blutes mit Aminosäuren vorbeugen. Es muß jedoch von der Darm wand oder der Leber s) aus noch ein bestimmtes Niveau überschreitet. A. Gustav Enibden und Hetterich Reese: Hofmeisters Bcitr. 7. 411 (190.")). f. physiol. Chemie. 47. 15 (1906). E'mil AbderhaMen und Alfred Schittenhelm: Zeitschr. f. physiol. Chem. 47. 339(1900). Gustaf Kinhden und Alfred Marx: Hofmeisters Beiträge. 11. 308 (1905). Franz Samuel;/: Zeitschr. f. pbvsiol. <;hem. 47. 376 (1906). J. Wohlqemuth und Varl Xeuberq : Med". Klinik Nr. 9. (1906). ) V"gl. u. — G. Forstner: Zeitschr. — — — Vgl. S. 5U, 555. ') Vgl. u. a. Emil Abderhalden — ") 464 (1903). uud Peter liergell : Zeitschr. f. physiol. Chem. 39. — Emil Abderhalden und Lewclhjs F. Barker: p:benda 42. 524 (1904 ' 1. Carl Neuberg und P. Fr. Richter: Deutsche med. Wochenschr. 30. 499 (1904). H. Gideon Wells: Jouru. of experim. Med. 9. 627 (1907). Vgl. auch Joh. Fei<jl Biochem. Zeitschr. 86. 1. 48 (1918). ) Vielleicht ist auf ein Versairen der Leber der l.'mstand zurückzuführen. dalJ bei manchen Erkrankungen nach Kingahe von Aminosäuren per os leichter solche im Harn erscheinen als hei normalen Individuen. Doch sind die vorliegenden An<;aben nocli ) — — XXVI. Vorlesung. 542 Moment eingreifen, damit nicht auf einmal große Mengen von einfachsten Bausteinen der Proteine ins Blut übertreten. Wir beobachten nämlich, daß bei der Verfütterung von vollständig bis zu Aminosäuren abgebautem Eiweiß im Harn nur sehr geringe Mengen von AminoJa selbst bei jenem Versuche, bei dem ein Hund aussäuren auftreten. schließlich mit vollständig verdautem Fleisch gefüttert worden war war der Gehalt des Kohlehydrate und Fett wurden nicht verabreicht Harns an Aminosäuren gering, i) In dfesem Falle waren große Mengen daß die von Aminosäuren aufgenommen worden. Es ist möglich, Geschwindigkeit ihrer Resorption von dem Gehalt der Darmwand an Diese selbst entledigt sich vielleicht der auf.\minosäuren abhängig ist. genommenen Abbaustufen nach Maßgabe des Gehaltes des Blutes an diesen. Entziehen sie Dieser ist wiederum abhängig vom Zuspruch der Gewebe. ihm viele Aminosäuren, so können weitere Mengen von solchen nachgeIst der Bedarf der Zellen an Aminosäuren vollständig schoben werden. gedeckt, dann werden wahrscheinlich die weiteren Aminosäuremengen, die noch zur Verfügung stehen, direkt zur Bildung von Kohlehydraten verwendet. Es spricht sehr vieles dafür, daß dieser Umbau sich in der Leber vollzieht. Dieser werden ja die resorbierten Aminosäuren auch in erster Linie zugeführt. Sie kann sehr wohl Aminosäuren zurückhalten und so den Gehalt des Blutes an solchen regeln. Da sie das Ammoniak, das bei der Abspaltung der NHg-Gruppe aus den Aminosäuren sich bildet, direkt zur Bildung von Harnstoff bzw. von Harnsäure verwenden kann, so braucht es beim Abbau von Aminosäuren in den Leberzellen nicht zum Übertritt von ein weiteres — — , Ammoniak in das Blut zu kommen. Wir müssen uns nun noch der Frage zuwenden, woher die Ei- weiß Stoffe des Blutplasmas stammen, und welche Bedeutung ihnen zukommt. Wir verfügen zurzeit über folgende Befunde. Die im Plasma sich findenden Eiweißkörper stellen ein Gemenge verschiedener Eiweißarten dar. Wir kennen Albumine und Globuline. Beide Gruppen sind auch chemisch gut charakterisiert. Die Globuline enthalten Gl y kok oll, den Albuminen fehlt diese Aminosäure. Auch sonst zeigen Es ist beide Eiweißarten große Unterschiede in der Zusammensetzung. ganz ausgeschlossen, daß Globuline in Albumine übergehen, ohne daß ein tiefgreifender Abbau vorausgeht und eine Synthese nachfolgt. Der Gehalt des Plasmas an diesen beiden Proteinen wechselt. Im Hungerzustand hat man z. B. eine Abnahme von Albuminen beobachtet. 2) Man hat auch bei verschiedenen Tierarten Unterschiede festgestellt. Ferner wird ein Einfiaß des Alters der Tiere angegeben. »j Uns interessiert hier nur die Tatsache, daß die Proteine des Plasmas einem Wechsel unterworfen sind. Entziehen wir einem Tiere Blut, dann wird dieses bald wieder ergänzt. Dabei müssen sehr vieldeutig und unsicher. Vgl. z. B. v. Jaksch : Zeitschr. f. kliu. Med. 47. 1 (1902), F. Erben: J. Ignatoirski: Zeitschr. f. physiol. Chemie. 42. 388 (1904). 50. 167 (1903). Ä. Lippstein. Hofmeistern Beiträge. 7. 527 Zeitschr. f. physiol. Chemie. 43. 320 (190.i). W. Frey: Zeitschr. f. kliu. Medizin. 72. 383 (1911). (1906). — — — — ^) Emil Abderhalden und Äkikazu Suwa: ^) Albrecht Burckhardt: Zeitschr. f. physiol. Chemie. 68. 416 (1910). Archiv Johann Lewinski: Fflügers Archiv. f. experim. Patli. 11. Pharm. 16. 100. 611 (1903). ») Vgl. J. Homer Woosley: Journ. of biol. Chemie. 14. 453 (1913). p:benda. 15. 37 (1913). 322 (1883). — C. E. Wells: Eiweißstoffe und ihre Bausteine. 54ä auch Plasmaproteine neu gebildet werden. Woher stammen diese EiweißstoffeV Welche Beziehungen unterhalten ferner die Kfirperzellen zu den Eiweißstoffen des Blutes und insbesondere zu denen des Plasmas? Im allgemeinen stellt man sich vor. daß das Blut die Nährlösung der Zellen der Gewebe darstellt. Es ergänzt seine Vorräte entweder vom Darme aus oder aber von den Reservestätten. Organische Nahrungsstoffe der Körperzellen sind in erster Linie Traubenzucker, die Bausteine der Fette und Aminosäuren. Kommen nun auch die Eiweißstoffe des Plasmaals solche in Frage? Es ist dies im höchsten Grade wahrscheinlich. \ ielleicht stellen bestimmte Plasmaeiweißkörper eine Art von Reserveeiweiß dar, das angegriffnen wird, wenn irgendwo Bedarf an Aminosäuren ist und gerade keine solchen vom Darme aus zur Verfügung gestellt sind. Die dem Plasma entnommenen Proteine würden dann wieder ergänzt, sobald Eiweiß zur Verdauung gelangt. Die Bildung dieser Proteine könnte sich sehr wohl in der Darmwand vollziehen. Ihre Bildung wäre vom Bedarf des Blutes an solchen abhängig. Diese Annahme würde erklären, weshalb man nicht ohne weiteres eine Synthese von Eiweiß in der Darmwand feststellen kann, wenn mau ein Gemisch von Abbaustufen aus solchem und insbesondere Aminosäuren in den Darm einführt. Abgesehen davon, daß der „überlebende" Darm nur sehr kurze Zeit als normal funktionierend anzusehen sind die Bedingungen zur Eiweißbildung noch gänzlich unbekannt. ist, Sie können daher im Reagenzglas versuch auch nicht nachgeahmt werden. Es ergibt sich ohne weiteres aus der Art der Darstellung unserer Kenntnisse des Verhaltens der vom Darm aus resorbierten Eiweißabbauprodukte, daß sie noch außerordentlich lückenhaft sind. Wir würden einen großen Fehler begehen, wenn wir den qualitativen Befund von Aminosäuren im Blute und die Feststellung, daß ihre Menge während der Eiweißverdauung ansteigt, jetzt schon als Beweis dafür hinstellen würden, daß vom Darm aus den Zellen ausschließlich solche zugeführt werden. Es dürfte nach den bis jetzt vorliegenden Ergebnissen das folgende Bild über das Schicksal der vom Darm aus aufgenommenen Eiweißal)bauprodukte den wirklichen Verhältnissen am besten entsprechen. Der stufenweise, durch die Wirkung der Fermente des Pankreasund Darmsaftes herbeigeführte Abbau der Peptone im Darmkanal führt zu Produkten, die zur Resorption geeignet sind. Es entstehen immer nur von Augenblick zu Augenblick kleine Mengen davon. Entweder geht der Abbau schon im Darmkanal bis zu Aminosäuren, oder er wird nach erfolgter Aufnahme von noch zusammengesetzten Produkten in der Darmwand vollendet, sofern sich keine Eiweißsynthese in dieser anschließt. Die gebildeten Aminosäuren w^erden dem Blute übergeben. In diesem werden sie der Leber zugeführt und von da ganz oder je nach dem eigenen Bedarf teilweise auf dem Blutweg den übrigen Geweben übermittelt. Alle Zellen des Organismus auch die Zellen des Blutes nehmen Aminosäuren auf. Dadurch sinkt der Gehalt des Blutes an diesen. Der Darm entläßt weitere Mengen von einfachsten Bausteinen der Proteine. Wieder greifen die Gewebszellen ein. Schließlich haben die Zellen ihren Bedarf gedeckt. Sie haben „Lücken" ausgefüllt, das Umsatzeiweiß ergänzt oder ganz ersetzt, Zellbausteine neu aufgebaut, Sekret- und Inkretstoff"e in Arbeit genommen usw. Infolgedessen werden nun dem Blute keine Aminosäuren mehr entzogen. Es müßte jetzt zu einer Anhäufung von — — X\\VI. V(ul^'sun^^ 544 solchen im Blute kommen, wenn der Zustrom keine Unterbrechung erlitte Diese erfolgt dadurch, daü entweder die Darmwand oder die Leber Aminosäuren zurückhält. Einerseits werden Proteine aufgebaut, die den ßeservcvorrat des Blutplasmas an Eiweiß ergänzen. Ferner setzt der Abbau der Aminosäuren und die Bildung von Traubenzucker und von anderen \erbindungen ein. Hierbei wird Ammoniak gebildet. Ferner haben vielleicht auch die Körperzellen bei der Bildving von »Sekret- und Inkretstoffen manche Aminosäuren ihrer Aniinogruppe beraubt. Außerdem geht der Stoffumsatz in den Zellen weiter. So kommt es, daß sehr bald der größte Teil des mit dem Eiweiß aufgenommenen Stickstoffs im Harn zur Ausscheidung gelangt. Nun ist die Resorption vollendet. Den Körperzellen noch ein geringer Vorrat an freien Aminosäuren zur Verfügung. In der Hauptsache sind sie jetzt auf Eiweiß angewiesen. Braucht irgend eine Zelle Eiweiß bzw. Aminosäuren, dann entzieht sie dem Blute Eiweiß. Sie kann dieses für ihre besonderen Zwecke nicht direkt verwenden. Sie muL) es vielmehr zuerst tief abbauen, wohl meist bis zu Aminosäuren. Auf diese Weise büßt das Plasma Eiweißstoft'e ein. Vielleicht findet ein Ersatz von den Organzellen aus statt. Diese verfügen vielleicht über Eiweiß, das nicht unmittell)ar zur Zelle hinzugehört, d. h. nicht als Baustein der Zelle zu betrachten ist. Es ist jedoch auch möglich, daß bestimmte Zellarten die Aufgabe haben, die Zusammensetzung des Plasmas nach allen Richtungen zu überwachen und zu verhindern, daß es zu einer Hyper- oder Hy po j)roteinoplasm ie und zu einer Hyper- oder Hypoaminoazidoplasmie kommt. Wir sind der Ansicht, daß der Darmwand und der Leber gemeinsam mit den Nieren eine sehr große Bedeutung in dieser Richtung zukommt. Wird nun wieder Eiweiß aufgenommen, so sind zahlreiche Bedürfnisse zu befriedigen. Es muß das verbrauchte Plasmaeiweiß ersetzt werden. Die Zellen füllen die Lücken, die im V^orrat an aufgestappeltem Fmsatzeiweiß entstanden sind, wieder aus usw. Schließlich müssen wir noch auf die Frage zurückkommen, ob jede einzelne Körperzelle Eiweiß ab- und aufbauen kann, und ferner jede Zellart zur Bildung von Glukose aus bestimmten Aminosäuren befähigt ist. Eine sichere Beantwortung dieser Fragen ist zurzeit nicht möglich. Immerhin darf aus allen Beobachtungen geschlossen werden, daß jede einzelne Gewebszelle selbständig in den Eiweißstoffwechsel eingreift und ohne Zweifel bei Bedarf auch aus Aminosäuren Zucker hervorgehen lassen kann. Dagegen scheint dann, wenn es sich darum handelt, größere Mengen von nicht verwertbaren Aminosäuren in Form von Zucker oder über diesen in Form von Fett zu speichern, insbesondere die Leber mit ihren Zellen einzugreifen. Mögfi sich nun der Eiweiß- und Aminosäurestoff'wechsel in manchen Einzelheiten auch anders abspielen, als es dargestellt wurde, so bleibt doch als wesentlichster Punkt, daß nicht das Eiweiß als solches, steht nur sondern die Aminosäuren im Mittelpunkt aller Probleme des Zel Stoffwechsels stehen. Jede Umwandlung führt über diese Verbindungen, sei es nun, daß Eiweiß aufgebaut werden soll oder bestehendes in anderes übergeführt wird. Von den Aminosäuren aus führt auch der Weg zu anderen Verbindungen. Aus diesem Grunde werden wir der Entstehung von Aminosäuren im Zellstoff'wcchsel und den sie betreffenden L'mwandlungen ein besonderes Interesse entgegenbringen. 1 Vorlesung XXVÜ. Eiweißstoffe und ihre Bausteine. 11. Die Bildung von Aminosäuren im Zeilstoffwechsel. Durch die Beobachtung, daß Aminosäuren vom Darmkanal zur Aufnahme gelangen und als solche ins Blut übertreten, ist bewiesen worden, die Zellen des tierischen Organismus ohne Zweifel ganz allgemein diese als Material zu ihren Stoffwechselvorgcängen verwenden können. "Wir daß haben früher schon den Beweis geführt, daß der tierische Organismus aus Aminosäuren Eiweiß aufbauen kann, als wir der Versuche gedachten, das Eiweiß der Nahrung durch das bei der vollständigen Hydrolyse von solchem entstehende Gemisch von Bausteinen zu ersetzen, i) Diese Untersuchungen waren von einem vollen Erfolge gekrönt. Sie zeigten, daß Aminosäuren genügen, um den ganzen Eiweißstoffwechsel aufrecht zu erhalten. Es hat sehr vieles für sich, anzunehmen, daß die Aminosäuren ganz allgemein im Mittelpunkt des ganzen Eiweißstoffwechsels stehen. In diesem Falle müssen wir uns vorstellen, daß die Zellen des tierischen Organismus nicht nur Eiweißstoffe aus Aminosäuren bilden, sondern umgekehrt auch Proteine zu den Bausteinen abbauen können. Unsere nächste Aufgabe ist nun die, Beweise dafür zu erbringen, daß die Zellen der Gewebe befähigt sind, Eiweiß unter Wasseraufnahme zu spalten. Überall, wo wir im tierischen Gewebe auf Abbau gestoßen sind, kamen Fermente in Frage. Wir werden somit auch hier zunächst festzustellen versuchen, ob die tierische Zelle über Proteasen verfügt. Das ist in der Tat der Fall. Schon die Beobachtung, daß Gewebe, das abgestorben ist, auch dann, wenn sicher keine Mikroorganismen zugegen sind, rasch in seine Bestandteile zerfällt, wobei auch bald Aminosäuren in Erscheinung treten, beweist, daß die Zellen über proteolytische Fermente verfügen. 2) Sie brechen, nachdem die Zelle endgültig die Führung der Stoffwechselvorgänge aufgegeben hat, regellos alle Zellbestandteile nieder und bauen die zusammengesetzten VerbinduDgen zu einfacheren Produkten und zum Teil zu ihren Bausteinen ab. Außer Vgl. S. 494. Vgl. E. Salkowski: Zeitschr. f. klin. Med. 17. Suppl. 77(1890). - M.Jacoby. Zeitschr. f. physiol. Chem. 30. 149 174 (1900). R. L. Benson und II. G. ^Vells : Journ. <)f biol. Chem. 8. 61 (1910). H. Wiener: Zentralbl. f. Physiol. 19. :^49 (1905). ') -) — — Abderhalden. Physiologische Chemie. I. Teil, n. .-Vufl. 35 XX VII. Vorlesung. 546 sind Fermente vorhanden, die Peptone und Polypeptide in ihre Bausteine spalten.^) Unter diesen dürfte auch das Proteasen Efepsin sich finden, vorausgesetzt, daß dieses überhaupt einheitlich ist."^) Der Nachweis der Fermente der Zellen ist aulJerordentlich erleichtert worden, seitdem Eduard Buchner uns gelehrt hat, aus Hefezellen Preßsaft zu bereiten. Die Zellen werden zunächst durch Verreiben mit Quarzsand vollständig zertrümmert. Dann wird die die Zelltrümmer enthaltende Masse mit Kieselgur vermischt. Diese hat die Eigenschaft, lebhaft Wasser aufzunehmen. Sie entzieht dem Gemenge der Zellpartikelchen den flüssigen Anteil. Nun wird der Kieselgur ihr Inhalt wieder durch Anwendung sehr abgepreßt. Derartig bereiteter bis 300 Atmosphären hoher Drucke Saft enthält fast gar keine unverletzten Zellen mehr, wohl aber Protoplasmaanteile. In genau der gleichen Weise kann man aus jedem beliebigen Organ Preßsäfte darstellen und sie auf ihren Fermentgehalt in einzelnen Fällen, prüfen. An Stelle des Preßsaftes kann man auch Mazerationssäfte) oder Auszüge mit Glyzerin nicht allgemein») verwenden. Im ersteren Falle zerhackt man das Organ und bringt es mit isotonischer Kochsalzlösung zusammen in den Brutschrank. Dabei gehen die Fermente aus den Zellen heraus und in die Kochsalzlösung hinein. auf Selbstverständlich muß man vor der Verarbeitung des Organs Fermente das Blut sorgfältig aus ihm entfernen, wenn man erfahren will, — — — — ob seine Zellen bestimmte Fermentarten enthalten. Die fermenthaltigen Auszüge oder den Preßsaft kann man nun auf Eiweiß einwirken lassen. Am besten geschieht dies in einem Dialysier- Die Bildung von Peptonen zeigt den Abbau des Proteines und damit die Anwesenheit von Proteasen an. Sie diffundieren durch die Membran hindurch und lassen sich im Dialysat z. B. mittels der Biuretreaktion erkennen. Ferner können wir einen Eiweißkörper wählen, der Tyrosin enthält. Diese sehr schwer lösliche Aminosäure fällt aus, wenn sie abgespalten wird. Oder wir benutzen ein dieser Aminosäure reiches Pepton oder ein Polypeptid und prüfen a,n eingestellte so auf die ganze der auf Eiweiß und Eiweißabbaustufen Endlich können wir auch mit Vorteil Verbindungen Fermentereihe, s) wählen, die Tryptophan enthalten. Das Auftreten der Brom was ssorreaktion zeigt an, daß diese Aminosäure in Freiheit gesetzt worden i.^t.*) Sehr überzeugende Resultate erhält man auch, wenn man ganz frische Organschnitte mit an gebundenem Tyrosin reicher Seidenpeptonlö.sung schlauch. ) Vgl. hierzu Emil Abderhalden und Peter Bona, Yutaka Teruuchi, Alfred Schittenhelm, Andrew Hunter, Filippo Lussana, Robert Heise, Eugen Steinbeck: Zeitschrift f. physiol. Chemie. 46. 176 (1905); 49. 31 (1906); 47. 4ß6 (1906); 49. 1(1906): 49. 26 (1906); 48. 537 (1906); 55. 390 (1908); 62. 136 (1909); 68. 312 (1910); 74, 409 Vgl. auch Emil Abderhalden: Die AhAerhaXdenschQ RdAkiiou. (1911); 78. 344 (1912). 5. Aufl. .1. Springer. Berlin 1922. ') H. AI. Vernon: Journ. of Physiol. 32. 33 (1904); 33. 81 (1905); Ergebnisse 147 (1910). (1. Physiol. 9. =>) Vgl. Emil Abderhalden und A. Fodor : Fermentforschung 6. 248. (1922). ) Vgl. hierzu A. v. Lebedew : Zeitschr. f. physiol. Chem. 73. 447 (1911). ') Vgl. Emil Abderhalden und Alfred Schittenhelm, Hans l'ringsheim: Zeitschr. Vgl. auch Ilaud180 (1910). f. physiol. Chem. 66. 137 (1910); 60. 421 (1909); 65. — — buch der l)iochemischen Arbeitsmethoden. 5. 575 (1911) halden). TIrban & Schwarzenberg. Berlin-Wien 1911. •) Vgl. S. 328. (bearl)eitet von Emil M>der- Eiweißstoffe und ihre Baueteiue. 547 diese Aminosäure auskristallisiert. Schließlicli kann man auch Peptone und Polypeptide in Lösung mit Preßsäften zusammenbringen und mittels eines Polarisationsapparates verfolgen, ob der Anfangsdrehung des Gemisches sich bemerkbaieine Änderung der Fall, dann beweist es, wie eindeutige Versuche macht. Ist dies ergeben haben, daß ein Abbau des verwendeten Peptons oder Polypeptide eingetreten ist. Es läßt sich durch Benützung genau bekannter Polypeptids leicht beweisen, daß der Drehungsänderung ein bestimmter Abbau entspricht.') Am zweckmäßigsten ist es, verschiedene Methoden zugleich zum Nachweis von Formenten anzuwenden, die P'.iweiß und seine zusammengesetzten Abbaustufen unter Wasseraufnahme zerlegen können. Durch zahlreiche Untersuchungen ist festgestellt worden, daß sich in allen Zellen des tierischen Organismus Fermente finden, die Eiweißstoffe bis zu Aminosäuren abzubauen vermögen. Auch die Plutkörperchen und die Blutplättchen enthalten solche Fermente.^) Sie fehlen nur dem Plasma unter normalen Verhältnissen. Sic treten jedoch bald in Erscheinung, wenn blutfremdes Eiweiß oder eine höher molekulare, zusammengesetzte Abbaustufe davon ins Blut gelangt.^) Es sind somit in den Geweben die Bedingungen zum Abbau von Proteinen gegeben. Mit dieser Feststellung dürfen wir uns nicht begnügen. Es könnte ja sein, daß diese Fermente nur in ganz bestimmten Fällen in Wirkung treten und ganz besondere Aufgaben erfüllen. Sie könnten z. B. ausschließlich der Synthese von Eiweiß aus Aminosäuren dienen. Wir müssen uns nach Ei'gebnissen umsehen, die beweisen, daß im normal ablaufenden Zellstoffwechsel Aminosäuren auftreten. bestreicht und feststellt, ob Wir sind jedesmal, wenn es sich darum handelte, Stoffwechsel- Zwischenprodukte nachzuweisen, auf große Schwierigkeiten gestoßen. Die. einzelne Zelle läßt es nie zu Anhäufungen von solchen kommen. Sie entstehen nur in Spuren und werden sofort weiter verarbeitet. Es wird auch wohl selten der Fall sein, daß eine große Anzahl von Zellen bestimmte Verbindungen gleichzeitig in der gleichen Art und genau der gleichen (^«eschwindigkeit abbauen, so daß bei einer plötzlichen Unterbrechung des Zellstoffwechsels eine bestimmte Abbaustufe nachgewiesen werden kann. Dazu kommt noch, daß es ein plötzliches Aufhören der Zelltätigkeit unter Erhaltung der natürlichen Verhältnisse nicht gibt. Die Zellen sterben langsam ab. Bereits begonnene Vorgänge werden noch weiter geführt. Während einzelne Zellen in den Geweben bereits jede Herrschaft über ihre Fermente verloren haben, arbeiten andere zum Teil wenigstens noch in geregelter Weise weiter. So kommt es, daß uns die Untersuchung frischer Gewebe auf Aminosäuren keine einheitlichen Resultate ergibt. Bald macht sich die sogenannte Autolyse geltend. Sie führt rasch zu einem ausgedehnten Abbau der Zelleiweißstoffe. Es ist nun außerordentlich schwoi-, Handbuch der biochemischen Arbeitsmethoden. 5. 575 (1911); 0. 223 (1912) Emil Abderhalden). Urban & Sehwarzeuberi,'. Berlin-Wien 1912. ') Kmil Abderhalden und H. Deetjen, Berthold Oppler, Feter Bona, Wilfred Manicaring, James Mc Lester: Zeitschr. f. physiol. Chem. 51. 334 (1907); 53. 280 (1907); ') (bearbeitet von 294 (1907); 53. 308 (1907); 55. 377 (1908); 55. 371 (1908). ^) Vgl. Etnil Abderhalden: Die Abderhaldenschc Reaktion. 5. Aufl. J. Springer. Berlin 1922. Hier findet sich die Literatur. Arbeiten von Kmil Abderhalden und Mitarbeiter (Ludwig Pincussohn, Wolfyang Weichardt, K. B. Immisch, A. Israel, J. G. Sleesicyk, E. Rathsmann, Benovar Schilling, Ernst Kämpf). 53. 3fv^ XXVIl. Vorlesiinsr. 548 wann der normale Abbau der Eiweilistoffe aufhört und die Sehr wahrscheinlich liegen die Verhältnisse so, daß die autolytischen Vorgänge an und für sich durchaus normalen Vorgängen in der lebenden Zelle entsprechen. Es wird nur der weitere Verlauf der Hydrolyse nicht mehr in bestimmter Weise geregelt, und ferner werden die entsprechenden Abbaustufen nicht in vollem festzustellen, Autolyse einsetzt. Umfang weiter verarbeitet. Endlich fehlt die Fortführung der enstandenen Verbindungen. Dadurch muß notwendigerweise der weitere Abbau der einzelnen Zellbestandteile wesentlich beeinflußt werden, so daß schließlich der Verlauf der einzelnen Fermentvorgänge in abgestorbenen Geweben uns kein getreues Bild der normalen Vorgänge mehr geben kann. Wir wollen uns nach Beobachtungen umsehen, die für eine Entstehung von Aminosäuren im Zellstoffwechsel sprechen. Wir wollen gleich bemerken, daß der Umstand, daß solche vom Darm aus zur Aufnahme gelangen, die Beurteilung der Herkunft der Bausteine der Proteine sehr erschwert. Es muß in jedem Falle ausgeschlossen werden, daß um resorbierte Aminosäuren handelt. Bei der Besprechung der Herkunft des Traubenzuckers kamen wir zu der sicheren Feststellung, daß Aminosäuren solchen liefern können. Diese Schlußfolgerung wurde zunächst nicht am normalen Organismus erhoben. Die Tatsache, daß im tierischen Organismus Glukose aus anderer Quelle als aus Kohlehydraten entstehen kann, ergab sich vielmehr aus Beobachtungen, die bei gestörtem Kohlehydratstoffwechsel erhoben worden sind. Sind keine Störungen des Eiweißstoffwechsels und insbesondere des Aminosäurestoffwechsels bekannt? Wir werden später erfahren, da(j bestimmte Drüsen, z. h. die Schilddrüse, einen bedeutungsvollen P^influß auf den pjweißstoffwechsel haben. i) Er kann beschleunigt oder verlangsamt werden. Ferner aht man bei vielen Erkrankungen und vor allem bei Infektionskrankheiten, die mit Fieber verknüpft sind, einen Einfluß auf den Eiweißumsatz beobachtet. Das Studium dieser Störungen des Eiweißstoffwechsels ergab bis jetzt im großen und ganzen nur quantitative Unterschiede. In einzelnen Fällen, so bei V'ergiftungen, z. B. auch beim Coma diabeticum und bei tiefen Narkosen, fand man Aminosäuren im Harn. 2) In anderen Fällen zeigten die in ihm auftretenden Verbindungen, daß der Abbau zwar über die Bausteine der Proteine hinausgeführt hatte, daß er jedoch verlangsamt und teilweise gehemmt war. Wir werden bald erfahren, daß der Schwefel der Proteine bzw. des Zystins unter normalen Verhältnissen zum größten Teil in Form von Schwefesäure im Harn erscheint. Nur ein kleiner Teil davon wird in reduziertem Zustand ausgeschieden. Ist der Stoffwechsel aus irgend einem Grunde geschädigt, dann beobachten wir in manchen Fällen, daß der Harn mehr von der letzteren Art von Schwefelverbindungen enthält. Lassen schon diese Beobachtungen den Schluß zu, daß mit größter Wahrscheinlichkeit der Abbau der Proteine üi)er Aminoes sich ') Nach Jlans Sfiibel bewirkt Adronaliu ein Verschwinden von gespeichertem Eiweiß [lyfiigers Archiv. 185. 74 (1920)]. — in der Leher Emil Abderhalden und ^) Mies: Münchener med. Wocheuschr. Nr. 34 (1904). Emil Abderhalden : Ebenda. E. F. Barker: Zeitschr. f. physiol. Cbem. 42. 524 (1904). C. Neuberg und //. Strauss: Berliner klin. Wochenschr. 43. 258 (190B). 44. 17 (1905). Ad. Lneiv)/: Biocliom. J. Wohlgemuth: Zeitschr. f. physiol. Chem. 44. 74 (1905). — — /citschr. 3.' 439 (1907). — Eiweißstoffc uinl ihre Buiistoiue. 549 säuren führt und diese selbst wieder stufenweise weiter abgebaut werden, können mr aus ihnen allein doch noch keine eindeutigen Rückschlüsse auf den Verlauf des normalen Eiweißstoffwechsels ziehen, weil immer noch der Einwand möglich ist, dal» die Ergebnisse von Stoffwechselversuchen, bei denen der Organismus sich unter pathologischen Bedingungen befindet, nicht auf normale Verhältnisse übertragbar sind. Nun kennen wir eine sehr interessante Stoffwechselanomalie, bei der es zur Ausscheidung einer Aminosäure, nämlich von/ystin kommt. Man spricht von einer Zy tinur ie. i) Sie ist sehr selten und tritt manchmal bei mehreren Generationen und Gliedern einer P'amilie auf. ^) Diese Anomalie ist dadurch charakterisiert, dali im Harn Zystin enthalten ist. Gleichzeitig hat man in einigen F'ällen auch Dia m ine angetroffen. 3) Ferner sind aulJer Zystin wiederholt auch andere Aminosäuren beobachtet worden. Offenbar handelt es sich bei der Zystinurie um eine mehr oder weniger vollständige Hemmung des Abbaus des Zystins. Es gibt Fälle von Zystinurie, bei denen im Harn nur Zystin angetroffen wird. In anderen hat man daneben noch Leuzin und Ty rosin festgestellt.) In den einen Fällen ist die Störung des Aminosäurestoffwechsels offenbar beschränkter als in anderen. Bei den letzteren kann man von einer Aminoazidurie so sprechen. Es handelt sich bei der teilweisen Störung des Abbaus bestimmter Aminosäuren nicht um einen etwa dem Diabetes melitus an die Seite zu stellenden Vorgang. Den betreffenden Individuen entgeht mit der Ausscheidung der betreffenden Aminosäuren Material, das noch in mannigfacher Weise verwertbar wäre, und ferner geht mit ihnen Energie verloren. Da jedoch der ganze Vorgang auf einige wenige, ja oft auf eine einzige Aminosäure beschränkt ist, so ergeben sich für den mit Zystinurie Behafteten zunächst keine schwereren und vor allem keine akuten Folgeerscheinungen. Auch die Diaminurie, d.h. die Ausscheidung von Diaminen, und zwar insbesondere von Kadaver in und Putreszin, verursacht keine Störungen. Die Zystinurie wird meistens erst dann entdeckt, wenn das Zystin Anlaß zur Bildung von Nieren- oder Blasensteinen gegeben hat. Das Zystin ist in Wasser schwer löslich. Tritt es im Harn in größerer Menge auf, dann kann es in diesem, während er noch im Nierenbecken oder der Harnblase weilt, zur Abscheidung kommen. Im Laufe der Zeit entstehen so oft ganz große Steine. Sie machen be- — ') Vgl. u. a. W. F. Loebisch: Liebigs Annaleu. 182. 231 (1876). A. Niemann: Deutsches Archiv f. klin. Med. 18. 232 (1876). Wilhelm Ebstein: Ebenda. 19. 138 Bruno Mesfer: Zeitschr. f. physiol. Chem. 14. 109 (1890). (1877); 30. 594 (1882). CajJ Arlsberc/ und Otto Folin A. Loeivy und Carl Neuberf/: Ebenda. 43. 338 (1904). Americ. Journ. of Physiol. 14. 54 (1905). Emil Abderhalden und A. Schiitenhelm: Archibald Garrod und W. H. Hurtley: Zeitschr. f. physiol. Chem. 45. 468 (1905). The Journ. of Physiol. 34, 217 (1906). A. Loeu-y und Carl Xeuberff: Biochem. ZeitCharles G. L Wolf und Philipp A. Schafer: The Journ. of schrift. 2. 438 (1907). Biol. Chem. 4. 439 (1908); 6. 337 (1909). ~ Achilles Müller : Wiener med, Wocheuschr. Nr. 37 und 38 (1911). ^) Vgl. hierzu Archibald E. Garrod: Inborn errors of metabolism. Eaucet. 4., 11,, Emil Abderhalden: Zeitschr. f. physiol. Chem. 38. 557 (1903), 18. und 25. Juli 1908. L. r. Udrdnszky und E. Baumann: Zeitschr. f. physiol. Chem. IH. 562 (1889). ) Emil Abderhalden und Alfred Schittenhelm : Zeitschr. f. physiol. Chem. 45. 468 Vgl. auch Emil Fischer und Umetaro Suzuki: Ebenda. 45, (1905); 104, 129 (1919). 405 (1905). : — — — — — '•') — — — — XXVil. Vorlesung. 5oO sonders dann Beschwerden, wenn sie die Ureter oder auch die Urethra passieren wollen und infolge ihrer Größe einmal dem Harn den Weg verlegen, und ferner durch Dehnung und zum Teil auch durch Verletzung der genannten Gebilde zu Schmerzen führen. Der Umstand, daCi manche an Zystinurie Leidende über Schmerzen in Muskeln und oft auch in Gelenken klagen, ergibt die Möglichkeit, daß es auch zur Abscheidung von Zystin in den Geweben kommen kann. In einem Fall von Zystinurie bei einem Kinde ergab die direkte Betrachtung der einzelnen Organe eine dichte Infiltration mit den charakteristischen, sechsseitigen Tafeln von Zystin. ^ Daß das bei Zystinurie zur Beobachtung gelangende Zystin auf dasjenige des Eiweißes zurückzuführen ist, hat die vergleichende Untersuchung beider Zystinarten ergeben.-) Das Zystin ist, wie schon früher erwähnt wurde, zuerst in einem Harnstein aufgefunden worden. ») In diesem Zusammenhang möchten wir die Möglichkeit betonen, daß Störung im Zystin-Zellstoffwechsel vielleicht eine tiefer gehende Bevielleicht nicht deutung hat. Es ist wohl möglich, daß in der Zystinurie eine Störung in der Bildung des von F. G. Hopkins) in allen Fällen entdeckten, aus Zystein und Glutaminsäure bestehenden, für die Oxydationsvorgänge so bedeutungsvollen Produktes vorliegt. Der von mir beol)achtete Fall von familiärer Zystinurie weist besonders auf eine solche Möglichkeit hin. Es starben drei Kiudei- unter Erscheinungen von Inanition, ohne daß eine bestimmte Ursache zu finden war. Vielleicht waren die Zellen dieser Kinder in ihrem Stoffwechsel durch das Ausbleiben des für die Oxydationsvorgänge so wesentlichen Wechselspiels zwischen Zystin und Zystein, auf das wir in Band II, Vorlesung 14, noch zurückkommen, gestört. Man wird in Zukunft Fälle von Zystinurie in der erwähnten Richtung zu untersuchen haben. Von großem Interesse ist auch, daß Mäuse und Ratten, die vollkommen zystinfrei ernährt werden, bald Störungen im [Wachstum zeigen. Die Gewebe dieser Tiere und insbesondere die Lei)er zeigen nur eine sehr schwache Reaktion auf Zystein. 5) Die Aminoazidurie und insbesondere die Zystinurie zeigen, daß der Abbau der Aminosänren in den Geweben nicht in einheitlicher Weise geregelt ist, es müßte sonst eine Störung im Abbau von Aminosäuren stets Offenbar erfolgt die weitere Veränderung der einzelneu alle betreffen. l)austeine der Proteine durch besondere Fermente, die der Struktur und Konfiguration der abzubauenden Verbindung genau angepaßt sind. Fehlen einzeln(^ dieser Fermente, oder können sie aus irgend einer Ursache ihre Wirkung nicht voll oder auch gar nicht entfalten, dann bleiben die ihnen entsprechenden Aminosäuren als solche erhalten und erscheinen infolgedessen im Harn. Auch die Diaminurie darf vielleicht als eine Störung bestimmter Art des Zellstoffwechsels aufgefaßt werden. Es ist leicht möglich, (laß. wie wir früher schon betont haben, der Abbau der Aminosäuren unter anderem auch über Amine führt. Sie gelangen unter normalen Verhältdie — — ') '^) Emil Abderhalden: Zeitschr. 1. physiol. Chcm. 38. 057 (1903) Emil Fischer und Umetaro Suzuki: Zeitschr. f. physiol. Cheni. 45. 405 (1905). — Kmü Abderhalden: Ebenda. 51. 391 (1907); 104. 129 (1919). ») ) 5) uikI Vgl. S. 318. F. G. Hopkins: The hiochemical .1. 15. 286 (1921). Emil Abderhalden: Pflügers Archiv, 195. 199. (1922) Hrnst Wfrfheinter: Elieiida. 196. (1922). F.mil Abderhalden Eiweißstoffe und ihre Bausteine. nissen nicht zur Beobachtung, weil sie 551 rasch weiter zerlegt werden. Auch vom Dannkanal aufgenommenen Amine werden sicher zum großen Teil in den Geweben abgebaut. Der un Diaminurie Leidende vermag diesen die Abliau nicht durchzuführen. Er hydrolysiert noch Arginin in Harnstoff i n V a1eriansäu re und D i a Ornithin, spaltet aus der letzteren Verbindung Kohlensäure ab und bildet Tetramethylendia min ? u t r e s z i n. Ferner führt er auf dem gleichen Wege L y s i n in P e n t a- = m = methylendiamin = Kadaver in über. Der Abbau der genannten beiden Aminosäuren bleibt auf dieser Stufe stehen, weil die Bedingungen zu seiner weiteren Durchführung fehlen. Diese Vorstellungen über die Ursache der genannten Stoffwechselanomalie sind, das sei besonders betont noch nicht eindeutig bewiesen. Vieles spricht dafür, daß die Verhältnisse nicht so einfach liegen, wie wir sie eben dargestellt haben. Wir wissen nämUch, daß das Zystin von den Leberzellen als Ausgangsmaterial zur Bildung von Taurin verwendet wird.i) Dieses verläßt die Leber in der Galle mit Chol- bzw. Desoxycholsäure gepaart. Auch der an Zystinurie Leidende bildet Taurin und scheidet dieses in Form von Taurochol- und Taurodesoxycholsäure aus. Somit muß die Möglichkeit eines Abbaus von Zystin in einem gewissen Umfang gewahrt sein. Vielleicht vermögen ihn die Leberzellen noch zu vollziehen, während andere Zellarten zum Teil oder ganz versagen. Die Störung des Zystinabbaus braucht nicht auf alle Zellen des Körpers ausgedehnt zu sein. Die Bildung von Taurin aus Z y s t i n bzw. Zystein zeigt, daß der tierische Organismus in seinen Geweben Aminosäuren unter Kohlensäureabspaltung abbauen kann. Zugleich wird das wahrscheinlich als Zwischenstufe entstehende Thioäthylamin oxydiert: S . CH2 SH CH2 . I I NH2 C • . H NHa . C I . H -h 2 H m 2 COOH CO, = NH2 C H , . I ! COOK COOH Zystin Moleküle Zystein 2 CH, .SH 30 = CH, .SO, .OH -h ' ' I I CH2 . CHj NH3 NH., . Thioäthylamin Taurin Es ist jedoch auch möglich, daß die Oxydation zuerst dann die Abspaltung von Kohlensäure folgt CH2 SH CH, SOa . NH2 C H -h HO = . . r .CR . I NH., OH CH, — VU^ . (JO2 einsetzt, SOj r NH, . . OH . " 1 ') ! COOH COOH Zystein Zysteinsäure Vel. S. .^18. Taurin. und XXVII. Vorlesung. 552 Daß Zystin und Zystein wirklich als Ausgangsmaterial für die Bildung des Taurins in Betracht kommen, beweisen Versuche an einem Hunde, dem eine Gallenblasenfistel angelegt worden war. i) Es wurde der Gehalt der ausfließenden Galle an Taurocholsäure bestimmt. Dann erhielt das Tier Zystin. Es trat keine Zunahme an der genannten Gallensäure auf. Wurde jedoch gleichzeitig mit dem Zystin auch Natriumcholat, d. h. der zur Bildung von Taurocholsäure notwendige Anteil verabreicht, dann erschien sie in vermehrter Menge in der Galle. Versuche am Kaninchen bestätigten diese Befunde. 2) Mit der Feststellung, daß ein Baustein der Proteine, nämlich das Zystin, direkte Beziehungen zu einem Bestandteil der Taurochol- bzw. Taurodesoxycholsäure hat, ist bewiesen worden, daß eine bestimmte Aminosäure das Ausgangsmaterial zur Bildung eines bestimmten Produktes des Wir müssen nun die Frage entscheiden, ob Zellstoffwechsels sein kann. das Zystin, aus dem Taurin hervorgeht, aus Eiweißstoffen der (lewebe herstammt oder aber, ob nur die im Darmkanal aufgenommene Aminosäure Verwendung findet. Es wäre denkbar, daß die Leber dem mit resorbierten Aminosäuren versehenen Blute Zystin entnimmt, um es sofort oder nach einiger Zeit in Taurin umzuwandeln. In diesem Falle würde den Körperzellen nur der nicht zur Taurinbildung verwendete Teil des in so wie so geringer Menge vorhandenen Zystins zugeführt. Würde die Taurocholsäure und die Taurodesoxycholsäure eine Verbindung darstellen, ohne die der tierische Organismus nicht auskommt, wäre sie z. B. für den Zellstoffwechsel bestimmter Organe unentbehrlich, dann wäre es verständlich, daß der tierische Organismus in erster Linie dafür Sorge trägt, daß sie gebildet wird. Schon der Umstand, daß die Gallensäuren in den Darm abgegeben werden, und daß ihre Menge sehr schwankt, macht es sehr wahrscheinlich, daß das Taurin, die Cholsäure und die Desoxycholsäure Produkte darstellen, die zu den Stoffwechselendprodukten zu rechnen sind. An dieser Auffassung ändert die Beobachtung nichts, daß die Gallensäuren als Aktivatoren der Vorstufe der Lipase eine bedeutungsvolle Rolle spielen. ^) Wir wissen nämlich, daß der tierische Organismus auch andere Stoffwechselendprodukte, wie z. B. die Kohlensäure, noch zu wichtigen Funktionen nutzbar macht. Ferner können die Taurin enthaltenden Gallensäuren durch die Glykocholsäure und Glykodesoxycholsäure in ihrer Wirkung auf das Lipasezymogen vertreten werden. Es hat mehr Wahrscheinlichkeit für sich, daß das Zystin zunächst den Gew^eben zur Verfügung gestellt wird. Ihre Zellen verwenden es zum Aufbau von Proteinen. Besonders die Keratine brauchen zu ihrer Bildung sehr viel von dieser Aminosäure. Ob sie synthetisch aus Serin oder Alanin oder einer anderen Aminosäure unter Anlagerung der Thiogruppe bereitet werden kann, wissen wir nicht. Sehr wahrscheinlich ist diese Bildungsweise nicht, denn der tierische Organismus müßte zur Bildung der Thiogruppe energische Keduktionsvorgänge durchführen. Als einziges Material für diese käme wohl die Schwefelsäure in Betracht. andere Nun wird sicher immer Zystin, das eben vom Darme resorbiert wurde, direkt zur Taurinbildung zur Verfügung stehen. Ohne Zweifel wird jedoch Bergmann: Hofmeisters Beitr. 4. 132 (1908). Wohlgemuth: Zeitschr. f. phvsiol. Cheniio. 40. ') V. ^) J. ») Vgl. 8.261. 81 (1908). Eiweißstofle und ihre Bausteine. den Leberzellen auch Zystin zugeführt, das Baustein OOH von Zelleiweili ge- worden war. Beweisend für diese Annahme ist die Tatsache, daß auch bei lang andauerndem Hunger Taurin enthaltende Gallensäuren zur Ausscheidung gelangen. Wenn auch mit der Tatsache gerechnet werden muß, daü solche nach erfolgter Ausscheidung vom Darme wieder aufgenommen und der Leber wiederum zugeführt wird, so sprechen doch alle Beobachtungen dafür, daß immer auch eine Neubildung von Taurin erfolgt. Wir werden noch eingehend') auf die Beobachtung zurückkommen, Zellarten mit lebhaftem Stoffwechsel ständig Zystein als solches oder in Verbindung mit Glutaminsäure'^) bzw. mit anderen Aminosäuren enthalten. Wir werden erfahren, daß der SH-Gruppe eine große Bedeutung Reduktionsvorgängen in den Zellen zukommt. bei Oxydations- bzw^ Zystein geht durch Oxydation leicht in Zystin und dieses wieder durch Reduktion in Zystein über. Kehren wir nunmehr zur Zystinurie zurück. Zu einer Zeit, als man annahm, daß der Abbau der Proteine im Darmkanal nicht bis zu Aminosäuren führe, sondern hochmolekulare Peptone zur Resorption gelangen und sofort wieder in Eiweiß übergehen sollten, war man geneigt, die Ausscheidung von Zystin auf seine Entstehung im Darmkanal zurückzuführen. Der Organismus sollte mit dieser Aminosäure nichts anzufangen wissen. Daß diese Vorstellung ganz unbegründet war, ergab die Feststellung, daß der normale Organismus selbst sehr große Cystinmengen abbauen kann. 3) Auch Polypeptide, an deren Aufbau Zystin beteiligt ist, ergeben eine Steigerung des Schwefelsäuregehaltes im Harn.) Der aufgenommene Schwefel gelangt fast ausschUeßlich in dieser Form zur Ausscheidung. Die Bildung von Zystin bei der Verdauung der Proteine kann somit auf keinen Fall die Ursache der Zystinurie sein. Würde ferner das Zystin nicht mehr verwertbar sein, wenn es in freiem Zustand in den Organismus gelangt, dann müßte man erwarten, daß in jenen Fällen von Zystinurie, in denen annähernd soviel Zystin im Harn zur Ausscheidung kommt, als mit der Nahrung aufgenommen wird, die Proteine der Gewebe diesen Baustein nicht besitzen. Die Bestimmung des Zystingehaltes von Geweben und insbesondere von Haaren und Nägeln bei Personen, die eine Zystinurie zeigten, ergab, daß kein Unterschied gegenüber der Zusammensetzung der entsprechenden Proteine von normalen Individuen vorhanden ist.'') daß alle Das bei der Cystinurie zur Ausscheidung gelangende Zystin dürfte somit mindestens zwei Quellen entstammen. Einmal wird solches direkt von der aus dem Darmkanal resorbierten Aminosäure abstammen können. Wird diese von den Zellen der einzelnen Organe und insbesondere auch von den Leberzellen nicht gebraucht, dann wird es ohne Zweifel durch die Nieren ausgeschieden. Ferner entsteht Zystin beim Abbau von Zellproteinen. Die Herkunft der erwähnten Aminosäure aus dieser Quelle beweist, daß im Zellstoffwechsel Aminosäuren gebildet werden. Die Aminoazidurie und Vgl. Band 2, Vorlesung 14. Vgl. S. 333. ') Vgl. u. a. //. Blum: Hofmeister» Beitr. 5. 1 (1903). Hier tiudet sich weitere Literatur. //. li. Lewis und C. H. Rothera: Journ. of Physiol. 32. 175 (1905). Lude E. Boot: The J. of Biol. Chem. 50. 303 (1922). ) Emil Abderhalden und Franz Samueli/: Zeitschr. f. physiol. Chemie. 46. 187 (190.')). Emil Abderhalden: Zeitschr. f. physiol. Chemie. 38. 557 (1903). ) ^) — — XXVII. Vorlesung. 554 Diaminurie deuten in gleicher Weise darauf hin, daß die Körperzel len Eiweiß bis zu den einfachsten Bausteinen abbauen können. Bei Hunden und Kaninchen läßt sich noch auf eine andere Weise die der Beweis erbringen, daß Zystin im Zellstoffwechsel auftritt. Verfüttert man nämlich diesen Tieren Halogenbenzol, z. B. Brombenzol, CeHg.Br, dann tritt im Harn eine schwefelhaltige Verbindung auf.i) Sie hat die folgende Struktur 2): genannt — — Merkaptursäure CH^ S Ce H, Br . . . I CH3.CO.NH.C.H I COOH Merkaptursäure. man den Aufbau dieser Verbindung mit demjenigen des dann erkennt man leicht die nahen Beziehungen, die zwischen diesen beiden Verbindungen bestehen. Das verabreichte Halogenbenzol ist mit der Thiogruppe des Zysteins in Verbindung getreten. Es hat außerdem noch eine zweite Reaktion stattgefunden. Mit der Aminogruppe hat sich ein Essigsäurerest verbunden. Die folgenden Formehi bringen die beiden Vergleicht Zysteins, Reaktionen zum Ausdruck: CH2.SH + CeHs.Br + Brombenzol I — CHa.S.CoH^.Br >\| CHs.CO.NH.C.H + 2H2O NH2C.H + HOOC.CH3 Essigsäure \| I COOH COOH Merkaptursäure. Zystein Die Bindung des aufgenommenen Halogenbenzols durch Zystein und Essigsäure ist im Prinzip ein ähnlicher Vorgang, wie die Paarung mancher Verbindungen mit Schwefelsäure und Glukuronsäure und die Kuppelung von Säuren mit Glykokoll. Es wird ohne Zweifel Zystein, das sonst in den Geweben zum weiteren Abbau gelangen würde, abgefangen. Wir erhalten damit ohne Zweifel auch Zystin bzw. Zystein, das aus Zelleiweiß hervorgegangen ist. zu Gesicht, denn es tritt auch beim hungernden Hunde die Bildung von Merkaptursäuren ein. Das Auftreten von Zystin bei der Zystinurie, die Bildung von Taurin und die Möglichkeit, große Mengen von Zystein durch d. h. künstlich Verfütterung von Halogenbenzol abzufangen und endlich die Beobachtung, eine Zysteinurie zu erzeugen daß auch beim hungernden Tiere zystinreiche Proteine, wie Haare und Nägel, gebildet werden, alle diese Feststellungen zusammen beweisen, daß im Zellstoffwechsel Aminosäuren und insbesondere Zystin bzw. Zystein entstehen. s) Es ist nach den vor- — E. Baumann und C. Preusse: Ber. d. Deutschen Chem. Gesellsch. 12.806(1879); f. physiol. Chemie. 5. 309 (1881). Vgl. ferner W. Mc Kim Marriot und C. G. Wolf: Biochem. Zeitschr. 7. 213 (1907). -) Bj. Friedmann: Hofmeistern Beitr. 4. 486 (1903). ^) Vgl. auch //. B. Letcis und D. A. Mc Ginfy : The J of hiol. Chem. 5.'{ ) Zeitschr. ('. .•549 — (1922). — Eiweißstoffe und ihre Bausteine. 555 liegenden Ergebnissen sehr unwahrscheinlich, daß Zystin allein aus vorhandenem Zelleiweiß abgespalten wird und ein an dieser Aminosäure ärmeres Protein in der Zelle zurückbleibt. Es spricht vielmehr alles dafür, daß die Loslösung einzelner Aminosäuren mit einem weitgehenden Abbau des ganzen Eiweißmoleküls verknüpft ist. Wir kennen noch zwei weitere Aminosäuren, die sich abfangen lassen, wenn man bestimmte Verbindungen verfüttert oder solche im Organismus selbst entstehen. Der einen sind wir schon begegnet, nämlich dem Gly kokoll. Wir stellten fest, daß Phenylessigsäure im Harn als Phenazetursäure erscheint.') Diese liefert bei der Hydrolyse Gly kokoll und Phenylessigsäure. Schon lange bevor diese Verbindung zur Beobachtung kam, war festgestellt worden, daß im Harne eine Verbindung vorkommt, die aus Glykokoll und Benzoesäure hervorgegangen ist. Liebic/ hatte im Jahre 1829 die Hippursäure im Harn von Pferden entdeckt. Bald darauf stellten Fre"^), Kel Ir r nnd Wöhlcr^) fest, daß verfütterte Benzoesäure nicht oder doch nur zum kleinsten Teil als solche im Urin auftritt, sondern eine Zunahme der Hippursäure zur Folge hat. Mit dieser Beobachtung war die erste Synthese im tierischen Organismus festgestellt. Hatte man bis dahin nur der Pflanzenzelle die Fähigkeit, Synthesen zu vollziehen, zuerkannt, so konnte nach der wichtigen Feststellung von Wähler bald an zahlreichen Beispielen gezeigt werden, daß auch die Zellen der Tiere aus Bausteinen zusammengesetzte Verbindungen aufzubauen vermögen. Die Entstehung der Hippursäure ergibt sich ohne weiteres aus dem Resultat ihrer Hydrolyse: ( \ H, CO NH GH, G0( )H + Hg . . . . ^ Hippursäure CV,H, C« H^ . COOK -f NH, GH., COOH . Benzoesäure . Glykokoll. .GOOH + H NH. GHa. COOH — HoO^CfiHs. CO. NH.CH^. COOH Benzoesäure Hippursäure. Glykokoll Hippursäure ist synthetisch essigsäure erhalten worden: z. B. Benz am id und Monochlor- aus CO NHa + Gl GH., COOH = C« H^ ('0 NH CHg COOH -h HCl Monochloressigsäure Hippursäure. Benzamid Ce Hg . . . . . . . . Man erhält sie ferner, indem man Glykokoll und Benzoesäure im eingeschlossenen Ptohi- 1 2 Stunden auf 160" erhitzt. Die Phenazetursäure stellt die nächste Homologe der Hippursäure dar: — C„ H5 . CO NH GH., COOH . . C^ H5 . Hippursäure GH, CO NH CH^ COOH Phenazetursäure. . . . . . Es sind noch zahlreiche Paarungen von Säuren mit Glykokoll bekannt geworden. So wurde beobachtet, daß in den Organismus von Kaninchen und Hunden eingeführte Naphtoesäuren im Harn als Naph•) Vgl. S. 514. Vre: Proc. med. and surg .louni. (1841). 3) Wilh. Keller (und Wühler): Liebigs Annaleu. 43. 108 (1843). - F. Wähler und F. Frerichs: Ebenda. 65. 33ö (1843). Vgl. weitere Literatur hei Wilhelm Wiechouski: llo/ni ei steril Beitr. 7. 204 (190.^). ^) — XXVII. 5,")6 Vorlesuiigr. tursäuren wieder erscheinen. ') Die Bildung dieser Verbindunj^en jenigen der Hippursänre ganz entsprechend: COOK + HiNH CR, COOH = C,oH,CO.NH C,oH, . . Naphtoesäuro . CH^ . ist der- COOK + H,() Naphtursiäure. Glykokoll Ebenso wird auch Salizylsiiiire mit (ilykokolP) gepaart. Es entsteht Oxyhippursäure: ()H.C6H,.COOH-hHNH.CH2.C()UH=()H.cyi4.C().NH.CHs.COOH + H.,() o-Oxyliippursäure=Salizylursäure. Glykokoll Salizylsäure auch alkylierte Benzoesäuren, wie z. B. dann als alkylierte Hippur säure zur Ausscheidung gelangen: Von Interesse ist, dali die Toluylsäure^), an Glykokoll gekuppelt werden und CH, .an,. CO OH -i- h nh . ch, cooh = . Glykokoll Toluylsäure = CH3 CeH, CO NH CH2 cooh + HaO. . . . Tolursäure. Wir haben schon der Besprechung der Glukuronsäure, die im tierischen Organismus eine ganz gleiche Rolle spielt, wie das Glykokoll, gesehen, daß die Zellen Stoffe, die an und für sich zur Kuppelung nicht geeignet sind, teils durch Oxydation, teils auch durch Reduktion und unter Umständen auch durch Kombination beider Prozesse vorbereiten. So wird Toluol) zunächst in Benzoesäure übergeführt und dann mit Glykokoll gekuppelt. Ganz ebenso verhalten sich Äthyl- und Propylbenzol. ^) Ferner wird Xylol zu Toluylsäure oxydiert. Von Interesse ist auch die Oxydation von Aldehyden zu Säuren. Als Beispiele eines solchen Vorganges seien die Überführung von Nitrobenzaldehyd in Nitrobenzoesäure und die nun erfolgende Bildung von Nitrohippursäure^) und ferner die Entstehung von Pyromykursäure=Brenzschleimsäure-glykokoll anbei geführt: NO2 Ce H, . CHO 4- O = NO2.CeH4.COOH . Nitrobezoesäure Nitrobenzaldehyd NO2 Cß H, . COOH + NHo CH . . . Ce H4 . . COOH = Glykokoll Nitrobenzoesäure NO2 g CO NH CH2 COOH + H, O. . . . Nitrohippursäure. Rudolf Cohn: Zeitschr. f. physiol. Chemie. 18. 112. (119) (1894). Bertagnini : Liebigs Annalen. 97. 248. (1856) und E. Baumann und Herfer: Zeitschr. f. physiol. Chemie. 1. 244 (253) (1877/78). ») Schnitzen und Nauni/n: Archiv f. (Anat. u.) Physiol. 352 (1867). ) E. und //. Salkou'ski: Zeitschr. f. physiol, Chemie. 7. 161 (1882/83). K. Salkowski: Ebenda. 9. 229 (1885). ') M. Nencki und P. Giucooa: Zeitschr. f. physiol. Chemie. 4. 325 (1880). ) Rudolf Cohn: Zeitschr. f. physiol. Chemie. 17. 224 (292) (1893). ») ') /'>'. — EiweiBstoffe CH = C. C<H >0 CH r:r CH iinrl Bausteine 557 CH = C CO OH + H NU . — )^ I ihr» . ('H, . COOH = >() CH = CH i a-Furfurol— Brenzschleimsäure BrenzschleimSäurealdehyd Glykokoll. CH = C.CO.NH.CH, .COOH 4- H.,0. >0 CH = CH I Brenzsch leim Sau re-gly kokoll. Von besonderem Interesse Hippursäure, denn hier gebildet werden: Cß H5 . ist die Cherführung von Benzaraid') in muß unter Wasseraufnahme zunächst Benzoesäure CO NH, + H., O =: Cß H, COOH + NH^. . . Benzoesäure Benzaraid Die Fähigkeit des Organismus. Verbindungen mit Glykokoll zu kuppeln, erstreckt sich nicht nur auf die Benzoesäure und ihre Derivate, sondern auch auf Karbonsäuren des Furan-, Thiophen- und Pyridinkerns. So wird aus Thiophenaldehyd-j zunächst Thiophensäure gebildet und diese mit Glykokoll in die Thiophenursäure verwandelt: C, H3 S . CHO + O = C, H3 S COOH C, H3 8 COOH + NH, CH, COOH = ; . Thiophenaldehyd . . . Thiophensäure C, H3 S . CO NH CH, COOH + H, 0. . . . Thiophenursäure. Die Verfolgung des Verhaltens derartiger Verbindungen im Organismus hat in mehr als einer Hinsicht großes Interesse. "Wir erhalten durch diese Untersuchungen einen klaren Einblick in die Leistungen der tierischen Zelle. Wir sehen sie mit der größten Leichtigkeit Oxydationsund auch Reduktionsvorgänge ausführen, Wasser abspalten und Wasser anlagern, je nachdem die Umstände es verlangen. Die Fähigkeit des tierischen Organismus, alle möglichen Verbindungen, die ihm mit wenig Ausnahmen normalerweise nie zugeführt werden, mit bestimmten Produkten, wie Glykokoll, Zystein, Schwefelsäure und Glukuronsäure und auch mit Harnstoff zu verknüpfen, hat zu der Frage geführt, welchen Zweck wohl diese Paarung haben mag. Es werden die einzelnen dem Organismus zum größten Teil ganz fremden Stoffe bald nach ihrem Eintritt in den Organismus in eine bestimmte Form gebracht, in dieser durch den Körper geleitet und durch die Nieren ausgeschieden. Man hat ) -) Leon r. Nenrki: Archiv f. expcrini. Path. u. l'hanuak. 1. 42U tl873). Budolf Cohn: Zeitschr. f. physiol. Chemie. 17. 281 (1893). — \g\. Kmil Fromm: Die chemischen Schutzmittel des Tierkorpors bei Vergiftungen. Karl J. Trüt)ner. Straßliurg 1V)03. S. 14 ff. und in Marceli Xencki „Opera omnia" die zahlreichen dieses Gebiet berührenden Untersuchungen dieses Forschers (Viewcg & Sohn, Braunschweig 190'i). XXVIl. Vorlesung. 558 den ganzen Vorgang als einen Schutz des tierischen Organismus gegen fremdartige Produkte aufgefaßt. Manchmal entledigt er sich ihrer durch Abbau. Ist aus irgend einem Grunde eine völUge Zerstörung des eingeführten Moleküls unmöglich, dann kommt es, wenn irgend möglich, zur Paarung. Daß in der Tat in vielen Fällen durch die Kuppelung mit den genannten Verbindungen eine schädliche Verbindung in eine unschädliche oder doch viel weniger schädliche verwandelt wird, zeigen die folgenden Ergebnisse.) Die tödliche Dosis von Phenylpropionsäure, Cg H5 CH„ CH2 COOH, Körpergewicht. Phenylpropionyl-glykokoU, ist ca. O'Qg pro Kilogramm Cg H5 CH2 CH2 CO NH CH2 COOK, bewirkt selbst in einer Menge von 15^ keine Erscheinungen. Zimtsäure ist zwar weniger giftig als Phenylpropionsäure, aber immerhin nicht ganz unwirksam. Sie wird nach erfolgter Paarung mit Glykokoll ganz ungiftig. vielfach . . . . . . . . . Hippurs äure ist immer im Harne vorhanden. Ihre Menge ist beim Karnivoren am geringsten und am größten beim Herbivoren.2) Mit der Pflanzennahrung werden stets aromatische Verbindungen aller Art aufgenommen. Soweit diese in Benzoesäure verwandelt werden können, treten Ferner trifft man im sie in Form von Hippursäure in den Harn über. Harn Phenazetursäure. Endlich haben wir schon erwähnt, daß Glykokoll die einzige Aminosäure ist, die sich häufig im Urin auch im freien Zustande vorfindet. Uns interessiert zunächst die Frage, ob der Bildung von Hippursäure im tierischen Organismus Grenzen auferlegt sind, und wo A priori ist zu erwarten, daß er nicht allzuviel Hippurdiese beginnen. säure bilden kann, denn die meisten Eiweißkörper enthalten sehr wenig Glykokoll, ja manchen, wie den Albuminen, fehlt diese Aminosäure ganz. Wir treffen nur in jenen Eiweißstoffen viel Glykokoll an, die im Organismus eine mechanische Rolle spielen. So enthalten das Elastin, das Kollagen usw. viel von diesem Eiweißbaustein. Schon die großen Hippursäuremengen, die manche Pflanzenfresser bei Aufnahme von Heu ausscheiden, erweckten Zweifel, ob ihre Bildung von der Menge des im Eiweiß zur Verfügung stehenden GlykokoUs abhängig ist. Dazu kamen dann noch die Erfahrungen, die bei Versuchen über die Hippursäurebildung nach VerEs zeigte sich, daß fütterung von Benzoesäure gemacht worden sind.^) man hungernden Tieren durch Eingabe dieser Säure große Mengen von Glykokoll entziehen kann. Nun wurden die Versuchstiere getötet und der Die erhaltene Gehalt ihres gesamten Körpers an Glykokoll festgestellt. Ausbeute an dieser Aminosäure war nicht geringer als diejenige, die Dieses Hungertiere lieferten, die keine Benzoesäure erhalten hatten.) Ergebnis beweist ') ^) (1862). in einwandfreier Weise, daß der tierische H. D. Dakin: The Journ. of Biol. Chem. 5. 413 (1909). Vgl. z. ß. Henneherg, Stohmann und Rautenberg: Liehig^ Annalen. 124. 200 Weiske, Wildt und Pfeiffer: Berichte d. Deutschen Chem. Gesellsch. 6. 1410 — (1873). — Rudolf Cohn: Wilhelm Wiechowski: Hofmeisters Beiträge. 7. 204 (1905). Adolf Magnus- Levij : Biochem. experim. Path. u. Pharm. 53. 435 (1905). Johann Letvinski: Archiv f. experim. Path. u. Pharm. 58. Zeitschr. 6. 523 (1907). 397 (1908). — W. H. Parker und Graham Lusk: Amer. Journ. of Pliysiol. 3. 472 (1900). Albert A. Epjystein und Samvel A. J. Ringer: Journ. of Biol. Chem. 10. 327 (1911). Bookman: Ebenda. 10. 327 (1911). ) Emil Abderhalden und Paul Hirsch: Zeitschr. f. physiol. Cliem. 78. 292 (1912). Vgl. auch Emil Abderhalden, Alfred Gigon und Eduard Sirauss: Ebenda. 51. 311 (1907). ') Archiv — — f. — — — Eiweißstofl'e und ihra BauBteinc. 559 Organismus große Mengen von Glykokoll selbst bereiten kann. Es ist noch unentschieden, ob seine Bildung durch Abbau von anderen Aminosäuren erfolgt, oder ob eine Synthese vorliegt. Es ist wohl möglich, Wege eingeschlagen werden. Übrigens entgeht bei sehr reichlicher Zufuhr von Benzoesäure eine geringe Menge davon der Kuppelung mit Glykokoll.') Ein kleiner Teil kann sich auch mit Glukuronsäure paaren. 2) Wir können somit den Nachweis von Glykokoll nicht unmittelbar als Beweis dafür anführen, daß diese Aminosäure als solche bei der Hydrolyse von Zellproteinen entsteht. Würde es sich feststellen lassen, daß Glykokoll beim Abbau von bestimmten Aminosäuren gebildet wird, dann wäre durch die Beobachtung der Hippursäurebildung erwiesen, daß im Zellstoffwechsel Aminosäuren gebildet werden. Es kämen ganz besonders die Versuche an hungernden Tieren in Betracht. Vielleicht erfolgt die Bildung der Hippursäure nicht immer in der gleichen Art. Es wäre z. B. auch denkbar, daß die Benzoesäure mit irgend einer aliphatischen Monoamino-monokarbonsäure verbunden und dann diese sekundär durch Abbau bis auf den GlykokoUkomplex zerlegt würde. Die Synthese von Hippursäure aus Glykokoll und Benzoesäure hat nicht nur deshalb allgemeinere Bedeutung erlangt, weil sie die erste im tierischen Organismus nachgewiesene Synthese darstellt, sondern vor allem auch deshalb, weil Bunge und Schwiedeherg'^) zum ersten- daLi beide mal diesen Aufbau als Prüfstein dafür wählten, ob derartige Vorgänge auch von bestimmten Organen dann vollzogen werden, wenn sie nicht mehr mit dem Körper in Zusammenhang stehen Diese beiden Forscher entfernten Hunden eine Niere und leiteten durch das frische Organ Blut, das ungerinnbar gemacht worden war. Es wurde durch die Nierenarterie zu- und durch die Nierenvene abgeleitet. Gleichzeitig wurde die aus dem Ureter ausfließende Flüssigkeit Harn aufgefangen. Es wurde nun festgestellt, ob das Blut und der Harn Hippursäure Dann wurden der Durchströmungsflüssigkeit Glykokoll und enthielten. Die überlebende Niere vollzog die Synthese zu Benzoesäure zugesetzt. Hippursäure. Wurde nun Benzoesäure durch die Niere geleitet, dann kam die Synthese bald zum Stillstand. Es fehlte an Glykokoll. Sie kam wieder in Gang, wenn diese Aminosäure zur Verfügung gestellt wurde. Die Synthese der Hippursäure erfolgte sowohl, wenn bei ST" gearbeitet wurd(\ als auch bei Zimmertemperatur. Für das Zustandekommen der Hippursäuresynthese haben sich die roten Blutkörperchen und die lebenden Zellen der Niere als von großer Bedeutung erwiesen. Wird das Nierengewebe durch Zerhacken zerstört oder noch besser durch Zerreiben mit Glasscherben, so findet die Kuppelung von Glykokoll und Benzoesäure anscheinend nicht mehr statt. Auch wenn die 20° abgekühlt und bei 40" wieder aufgetaut worden war, Niere auf bildete sie keine Hippursäure mehr aus den Komponenten. Ebensowenig gelang es, die Synthese zu bewirken, wenn statt des ganzen Blutes nur Blutserum durch die Niere geleitet wurde. Daß dem Sauerstoff eine wich- — — — Ther. ) Vgl. 3. 663 (1906) ^) Vgl. S. 40. ^) G. 233 (1877). V, z. B. Theodor Brtigsch und Rahel Hirsch: Zeitschr. f. experim. Path. u .1. ./. Rinqer: The Journ. of Biol. Chem. 10. 327 (1911). — Bunge und O. Schmiedeberg : Archiv f. experim. Path. 11. Fharniak (i XXV II. N'orlesung. 560 tige Rolle bei dieser Synthese zukcrmmt, geht aus den folgenden Versuchen hervor. ij Leitet man Blut durch die Niere, in dem der Sauerstoff durch Kohlenoxyd verdrängt ist, so erhält man keine Hippursäuresynthese. Es konnte den Niereuzellen auch durch Chinin die Fähigkeit genommen werden, Hippursäure zu bilden. Es sehr wahrscheinlich, daß die Synthese der Hippursäure aus und Benzoesäure unter Wasserabspaltung einem Ferment zuzu- ist (ilykokoll Man hat versucht, ein solches zu isoheren. Einige Verschreiben ist. suche, in denen es im Gegensatz zu früheren glückte, auch in zerhackter Niere die Hippursäuresynthese festzustellen ^j, lassen hoffen, da(j es gelingen wird, die Kuppelung von Glykokoll und Benzoesäure auch ohne direkte Verwendung von Organen und Zellen zu vollziehen. — — Was den Ort der Hippursäurebildung im tierischen Organismus anist zu bemerken, daß das eben Angeführte nur für Hunde gilt. Frösche bilden auch nach Entfernung der Niere und der Leber s) Hippursäure. Ebenso ließ sich bei Kaninchen nach Wegnahme der Nieren nach Eingabe von Benzoesäure reichlich Hippursäure feststellen.) Diese Beobachtung wird durch die Feststellung ergänzt, daß die Leber von Kaninchen beim Durchleiten von Benzoesäure Hippursäure liefert.^) Es ist möglich, daß beim Fleischfresser die Hippursäuresynthese eine lokalisiertere ist als beim Pflanzenfresser, weil bei ihm die Hippursäure unter normalen Verhältnissen nur in geringer Menge gebildet wird. Die Menge der vom Menschen pro Tag ausgeschiedenen Hippursäure beträgt im Mittel bei gewöhnlicher Kost 0-7 g/) Sie kann nach reichlichem Genuß von Gemüse und Obst auf mehr als 2 g ansteigen. Es ist in Erwägung gezogen worden, ob nicht die aromatischen Bausteine des Eiweißes insbesondere das Phenylalanin und das Tyrosin in den Geweben zu Benzoesäure abgebaut werden. Es scheint dies jedoch nach allen vorUegenden Beobachtungen nicht der Fall zu sein. Dagegen kann die im Darm durch Bakterien aus Phenylalanin entstehende Phenylpropionsäure in Benzoesäure übergeführt werden. betrifft, so — — Das Glykokoll findet im tierischen Organismus nicht nur zur Kuppelung der erwähnten Verbindungen Verwendung, sondern es tritt regelmäßig gepaart mit ('holsäure und Desoxycholsäure in der Galle auf. Die Glykochol- und Glykodesoxycholsäure, oder richtiger Cholylbzw. Desoxycholyl-glyzin genannt, sind entsprechend wie die Taurocholund Taurodesoxycholsäure zusammengesetzt. Beide enthalten den gleichen Paarling, nämlich die Cholsäure bzw. die Desoxycholsäure. Der Umstand, (laß beständig zur Bildung dieser Gallensäuren (jrlykokoll verwendet wird, entzieht der Hippursäurebildung bei Verabreichung von Benzoesäure und anderen zur Paarung fähigen Verbindungen diese Aminosäure zum Teil. A. Hoffmann: Archiv f. experim. Path. u. Pharmak. 7. 233 (1877). Wilhelm Kochs: rßür/ers Archiv. 20. 64 (1879). ./. K. Abelons et H. Ribaut Compt. rend. de la Soc. hiol. 1). juiii 1900. M. E. Berminzone : Bollettino acad. med. di Genua. 16. 1 (1901). ') ) — — V. Bunge und O. Schmiedeberq 1. c. S. 559. Zitat '). W. Salom'on: Zeitschr. f. physiol. Chemie. 3. 365 (1879). ') E. Friedmann und Hermann Tachau: Biochem. Zeitschr. 35. 88. (1911). ^) G. : ) «) Chem. Halluachs: Licbix/s Annalen. 106. 164 (1858). — 382 (1897). Soc. 19. 6'. Platt: Journ. of the Americ. Eiweißstoffe und ihre Bausteine. 561 Die Notwendigkeit einer unter Umständen sehr umfassenden Neubildung von Glyzin erhellt auch aus dieser Beobachtung. Interessanterweise die bil(^en eine Säure zwei Benzoylgruppen die auf, wenn ihnen Benzoesäure Vögel, ver- An ihrer Stelle tritt im Kloakeninhalt füttert wird, keine Hippursäure.'j wurde bald als Di- Sie trägt. benzoyl-ornithin = Dibenzoyl-a-£-diamino-valeriansäure erkannt. Sie ist Ornithur säure genannt worden : CH2 . CH., . CH. . CH COOH . " I I Cs H5 . NH, -f HOOC COOH + NHs . C, H, = Ornithin a,£-Diamino-valeriansäure Benzoesäure Benzoesäure i CH2 . CH2 CH, . . CH COOH + 2 H, 0. . l i C« H5 . NH CO CO NH . . . C« H, Es liegt genau die gleiche Synthese vor, wie Hippursäure aus Glykokoll und Benzoesäure: CH.' . COOH CHg =1 1 NH, + HOOC . C« n, . der Bildung von COOH NH CO . bei . C, H, Das Ornithin stammt ohne Zweifel aus dem Arginin. Es wird unter normalen Verhältnissen weiter abgebaut und kommt nur zum Vorschein, wenn es durch Eingabe von Benzoesäure abgefangen wird. In diesem Zusammenhang sei der interessanten Beobachtung gedacht, daß Menschen nach Eingabe von Phenylessigsäure Phenylazetylglut- amin im Harn ausscheiden. 2) Wir kennen nun noch eine Verbindung erscheint, der im Harn mit bestimmten Bausteinen der Proteine eine Stoffwechselanomalie, bei die Zusammenhang steht. Es ist dies die Alkaptonurie. Bei ihr treten zwar nicht Aminosäuren als solche auf, wohl aber eine Abbaustufe aus solchen, die sicher nur aus ihnen selbst und nicht aus zusammengeInfolgedessen können setzten Verbindungen hervorgegangen sein kann. wir diese Stoffwechselanomalie ebenfalls als Beweis dafür heranziehen, daß im Zellstoffwechsel aus Zelleiweiß Aminosäuren gebildet werden. Die Alkaptonurie ist im Jahre 1859 von Boedeker ) entdeckt worden. Er beobachtete, daß der Harn bei dieser Stoffwechselanomalie stark reduzierende in — ') M. Joffe: Ber. d. Deutsch. Chem. Ges. 10. 1925 (1877); 11 401 (1878). Vgl. auch ./. Yoshikawa: Zeitschr. f. physiol. Chemie. 68. 79 (1910). 2) Vgl. S. 515, Zitat ). «) Boedeker: Zeitschr. f. ration. Med. 7. 130 (1859); Liebig?, Annalen. 117 98 <1861). Abderhalden, Physiologische Chemie. I. Teil, ö. Ana. '><> XXVII. Vorlesung. 5H2 Eigenschaften besitzt und ferner beim Stehen an der Luft sich bei alkalischer Reaktion bald dunkel färbt. Schließlich wird er ganz schwarz und scheidet braune Flocken ab.-) Baedeker beobachtete ferner, daß sich das „Alkapton"«;, so wurde die den eigenartigen Eigenschaften des Harns bei Alkaptonurie zugrunde liegende Substanz genannt, mit basischem Bleiazetat ausfällen läßt. Zerlegt man das entstandene Bleisalz durch Schwefelwasserstoff, dann gelangt man zu einer kristalüsierenden Verbindung. Ihre Zusammensetzung wurde von Wolkow und E. Baumann'^) erkannt. Es handelt sich um eine 1, 4-Dioxjphenyl-3-essigsäure=:Hydrochinonessigsäure), auch Homogentisinsäure genannt : CH.OH HC <\CH HC C CH, COOH . . C.OH Homogentisinsäure. Die genannten beiden Forscher erkannten auch schon eine Mutfersubstanz der erwähnten Verbindung, nämlich das Ty rosin. Eingabe dieser Aminosäure führte nämlich nicht nur zu einer starken Vermehrung der Homogentisinsäure, sondern es entsprach die Zunahme genau der eingeführten Menge der erwähnten Verbindung. W. Falta und E. Längstem^) erhoben den gleichen Befund für Phenylalanin. Tryptophan ergab keine Zunahme der Homogentinsäure im Harn des mit Alkaptonurie Behafteten. Die nahen Beziehungen von Phenylalanin und Tyrosin zur Homogen- Formeln tisinsäure zeigen die folgenden CH HC CH HC CH C NH, \/ • C CH, CH COOH . •^2 . . ß-Phenyl-a-amino- propionsäure . OH HC CH HC CH C . OH H.C nH, \/ ^\/ C.CH,.C()OH C.OH • C CH. CH COOH . . . ß-p-Oxyphenyl-a-aminoPropionsäure 1, 4-Dioxypheny!-3essigsaure Vgl. hierzu Carl Th. Mörner: Zeitschr. f. physiol. Chemie. 69. 329 (19101. Der Name Alkapton gründet sich auf die Eigenschaft des Harns, bei Zusatz begierig vervon Alkali und Sauerstoff (Luft) sich rasch dunkel zu färben. xa:iT£iv ) ) = schlucken. 228 (1891). ) M. Wolkow und E. Baumann: Zeitschr. f. physiol. Chemie. 15 Vgl; Hier findet sich die Literatur über alle bis 1891 gemachten Beoba;;htu ngen. auch Kirk: British med. Journ. 2. 1017 (1886); Journ. of. anat. and p hysiol. 23. 69 Archihald E. Garrod und W. H. Hurtley: Journ. of physiol. 3 6. 13G (1907). (1889). G. Katsch: Münchener med. Wocheaschr. Nr. 48. S. 1337 (1918). ) Vgl. die Synthese der Homogentisinsäure bei E. Baumann und S. Fränkel: Zeitschr. f. physiol. Chemie. 20. 219 (1894). physiol. Chemie. 37. 513 (1903). ') W. Falta und Leo Langstein: Zeitschr. f. . — — — 56H Eiweißstofle und ihre Bausteine. Wir werden auf diese interessante Stoffwechselanomalie noch zurückkoramen.i) Sie hat viele Aufschlüsse über die Art des Abbaus von Aminosäuren im tierischen Organismus gebracht. Hier wollen wir sie nur insofern besprechen, als sich Beweise für die Bildung von Aminosäuren aus Zelleiweiß aus den Ergebnissen der an Personen, die eine Alkaptonurie aufweisen, angestellten Versuche ergeben haben. Das Studium der im Harn erscheinenden Menge an'Homogentisinsäure ergab, daß ein Einfluß der Art und Quantität des aufgenommenen Eiweißes unverkennbar ist.^) Je mehr Tyrosin und Phenylalanin die verabreichten Proteine besitzen, gebildet. Die mit um so mehr Homogentisinsäure wird im allgemeinen Alkaptonurie Behafteten sind ausgezeichnete Objekte zum Studium des Eiweißstoffwechsels. Wir können bei ihnen einmal das. Verhalten des mit dem Eiweiß zugeführten Stickstoffs und Schwefels verAußerdem gibt uns die Feststellung der im Xahrungseiweiß enthaltenen homozyklischen Verbindungen und der ausgeschiedenen Homogentisinsäm-e Auskunft darüber, ob Teile von Tyrosin und Phenylalanin einer Form im Organismus zurückgebUeben sind. Je mehr in irgend Anhaltspunkte wir für das Verhalten des Nahrungseiweißes im tierischen Organismus besitzen, um so eindeutiger werden die Ergebnisse. folgen. Zunächst glaubte man, daß die Bildung der Homogentisinsäure sich im Darmkanal vollziehe. Es sollte eine Bakterienwirkung vorliegen. Bald erkannte man jedoch, daß ihre Entstehung in die Gewebe verlegt werden muß. In vielen Fällen von Alkaptonurie wird offenbar unter bestimmten Bedingungen 3) überhaupt kein Phenylalanin und Tyrosin ganz abgebaut. Es bleibt die Zerlegung dieser Aminosäuren bei der Homogentisinsäure W^ürde, bevor diese in die Gewebe übertreten, aus ihnen die genannte Säure gebildet, dann müßten die Zelleiweißstoffe jener Individuen, die mit Alkaptonurie behaftet sind, frei von Phenylalanin und Tyrosin sein, oder es müßte eine Neubildung dieser Aminosäuren in den Zellen eintreten. Eine solche von aromatischen Aminosäuren ist nun nach allen bisherigen Erfahrungen sehr unwahrscheinlich. Es sei in dieser Beziehung, an die Erfahrungen mit Gelatine erinnert, mit der der tierische Organismus Ihr fehlen die aromatischen' als einziger Eiweißart nicht auskommt.) stehen. ') Vgl. Leo Lanqstein und Erich Meyer: Deutsches Arch. f. klin. Med. 78. 161. — A. E. Garrod und F. Shirley Hele: Ebenda. 33. 198 (1905) 35. 15. Dez. (1906). — A. E. Garod und Wood Clarke The Biochem. .Jouru. 2. 219 (1907). — Oscar klin. Med. 64. H. -544 (1908). — Oskar Adler:] Gross und Eduard Allard: Zeitschr. Massini: Zeitschr. Biochem. Zeitschr. 21. 5 (1909). — Emil Abderhalden und physiol. Chemie. 66. 140 (1910). — Vgl. auch die Zusammenfassung von Konrad From-. (1903). ; ./. : f. i?. f., — Ludwig Pincusherz: Über- Alkaptonurie. In.-Diss. Karl .1. Trübner. Straßburg 1908. Gotthard Söderbcrgh.\ sohn : Ergebn. d. inneren Med. und Kinderheilk. 8. 454 (1912). M. W. Scheltema: Nederl. Tijdschr. voor Nordisk Med. Arkiv. Abt. IL 1 (1915). Ratsch: Deutsches Archiv f.. Geneesk. Nr. 18. 1464 (1914); Nr. 25. 2659 (1915). klin. Med. 127. H. 3 u. 4 (1918). Emil Abderhalden und 2) Vgl. W. Falta: Archiv f. klin. Med. 81. 231 (1904). Bruno Bloch: Ebenda. Zeitschr. f. physiol. Cheni. 53. 464 (1907). bei ') Es ist von großem Interesse, daß die Ausscheidung der Homogentisinsäure Alkaptonurie zum Verschwinden gebracht werden kann, wenn in der Nahrung die Kohlehydratzufuhr stark herabgedrückt bis aufgehoben wird. Gleichzeitig zeigt sich Azetonbildung. Diese Beobachtungen von G. Katsch [Deutsches Archiv, f. klin. Med. 127. 210 (1918); 134. .59 (1920)] müssen weiter verfolgt Averden. — — — — ) Vgl. S. .500. ' 36 XXVn. Vorlesung. 5(i4 Bausteine. Erst tMue Zugabe von splchen bewirkt, daß die (lelatine anderen Proteinen gleichwertij2: wird. Ferner haben Stoffwechsel versuche ergeben, daß zwar eine der beiden homozyklischen Aminosäuren in der Nahrung fehlen darf, es werden jedoch nicht beide von den Zellen aus ersetzt. Die direkte Untersuchung von Eiweißstoffen eines an Alkaptonurie Leidenden hat ergeben, daß sie ebensoviel Tyrosin und Phenylalanin enthalten, wie die entsprechenden Proteine normaler Individuen. M Daß die Bildung der Homogentisinsäure in den Geweben konnte auch noch dadurch bewiesen werden, daß subkutan eingeführtes Glyzyl-l-tyrosin zu einer Vermehrung der Homogenerfolgt, tisinsäure führt.-) Die Alkaptonurie beweist mit voller Schärfe, daß im Zellstoffweclisel aus Eiweiß Aminosäuren entstehen. Auch das hungernde, an Alkaptonurie leidende Individuum scheidet beständig Homogentisinsäure aus. Da ihre Entstehung ohne Zweifel von den erwähnten Aminosäuren ausgeht, so müssen diese gebildet werden. Bei Verabreichung von Eiweiß in der Nahrung wird, falls seine Menge bzw. sein Gehalt an Phenylalanin und Tyrosin nicht sehr gering ist, wohl stets ein Teil der ausgeschiedenen Homogentisinsäure direkt auf die resorbierten Aminosäuren zurückzuführen sein. Ein Teil stammt jedoch sicher immer aus abgebautem Zelleiweiß. Man könnte auch hier den Einwand erheben, daß gerade die Bildung von Tyrosin und Phenylalanin im Zellstoffwechsel die Ursache der Entstehung von Homogentisinsäure sein könnte, während normalerweise diese Aminosäuren in den Geweben nicht entstehen. Diese Annahme ist jedoch ganz unhaltbar. Wir wissen, daß zugeführtes Tyrosin und Phenylalanin nicht zur Bildung von Homogentisinsäure führen, wenn nicht sehr große Mengen davon gegeben werden. 3) Es erscheinen im Harn ganz Mengen von aromatischen Verbindungen, der allergrößte Teil der verfütterten homozyklischen Aminosäuren wird vollständig über den Benzol- geringe kern hinaus abgebaut. Wir wollen noch bemerken, daß die mit Alkaptonurie behafteten Personen zumeist keine besonderen Erscheinungen zeigen. Sie wird meistens erst in den Krankenhäusern entdeckt. Das Dunkelwerden des Harns führt gewöhnlich zu ihrer Auffindung. Oft fällt den betreffenden Personen auch auf, daß ihr Ohrschmalz tiefbraun gefärbt ist. Es liegt unzweifelhaft eine ganz lokalisierte Stoffwechselanomalie vor. Sie kann verschiedene Ursachen haben. Einmal ist es denkbar, daß die Homogentisinsäure ein ganz normales Abbauprodukt der beiden homozyklischen Aminosäuren darstellt. Diese Annahme wird dadurch gestützt, daß es, wie eben erwähnt wurde, gelungen ist, bei einem Individuum, das keine Alkaptonurie aufwies, durch reichliche Aufnahme von Tyrosin Homogentisinsäure zur Ausscheidung zu bringen. Es könnte jedoch auch sein, daß die experimentell hervorgerufene Bildung von Homogentisinsäure eine Nebenreaktion beim Abbau der homozyklischen Verbindungen darstellt. Wir haben frühci- schon erwähnt, daß bei \'er- 52. ') Awu'/ ) I'Jmil Abderhalden und H'. Falta: Zeitschr. f. physiol. Chem. 39. 14B (V.M\). Abdirhdlden, Bruno Bloch und l'eter Rona Zeitschr. f. physiol. Clicin. ; 435 (1907). ') Vgl. hierzu Kmil Abderhalden: Zeitschr. f. physiol. Cheni. 77. 4.54 (1912). KiweilistdHe iiiid ihre Bausteine. 555 fütterung von allen möglichen Verbindungen in geringer Menge Abbaustufen im Harn auftreten, die ohne Zweifel nicht solche darstellen, über die der Abbau der ganzen Menge der betreffenden Substanz geht, sondern vielmehr als ein in geringem Umfange eingeschlagener Nebenweg zu betrachten sind.') Der Umstand, daß Individuen, die keine Alkaptonurie besitzen, verfütterte Homogentisinsiiure leicht vollständig abbauen, läßt es jedoch als wahrscheinlich erscheinen, daß die bei einem normalen Individuum festgestellte Bildung dieser Säure in dem Sinne zu deuten ist, daß der Abbau der homozyklischen Aminosäui'en auch normalerweise über die Homogentisinsiiure führt. Es liegt offenbar eine Hemmung in der weiteren Zerlegung dieser Bausteine der Proteine vor. Damit tritt die Alkaptonurie in die Reihe jener Stoffwechselanomalien ein. bei denen Fermente fehlen, oder sich Bedingungen finden, unter denen diese ihre Wirkung nicht entfalten können. Es ist sehr wohl möglich, daß irgend ein Organ versagt, das beim Abbau dieser Verbindungen teilzunehmen hat. Es ist z. B. denkbar, daß die Fermentvorstufe zugegen ist, es fehlt jedoch der Aktivator, der aus dieser das wirksame Ferment bereitet. Auch der umgekehrte Fall ist natürlich möglich. Es ist von größtem Interesse, daß derartige Stoff wechselanomalien vererbbar sind. Auch die Alkaptonurie tritt oft bei mehreren Oliedern der gleichen Familie auf. Interessant ist die Beobachtung, daß die sogenannte Ochronose 2) direkt oder indirekt mit der Alkaptonurie zusammenhängt. Man findet bei dieser neben anderen Veränderungen eine mehr oder weniger aus- gesprochene Schwarzfärbung der Knorpel. Es spricht vieles dafür, daß dieses Pigment auf die aromatischen Bausteine der Proteine zurückzuführen Welche Beziehungen zur Homogentisinsäurebildung bestehen, ist zurzeit noch unbekannt. Sie scheinen nicht direkter Natur zu sein. Es gibt ohne Zweifel auch Fälle von Ochronose, bei denen die Alkaptonurie fehlt. 3) Die Veränderung des Knorpels bei der Ochronose kann auch Ursache der ist. Arthritis deformans (alcaptonurica) sein.) Frage aufgeworfen, ob die Zellen des tierischen Organismus unter normalen Verhältnissen Eiweiß bis zu Aminosäuren abbauen. Zunächst konnten wir feststellen, daß in allen Zellen sich Fermente finden, die einen solchen Abbau durchführen können. Ferner Aminoazidurie, Zystinurie, Diaminurie haben wir Stoffwechselanomalien und Alkaptonurie kennen gelernt, die unzweifelhaft beweisen, daß es in den (ieweben zur Bildung von Aminosäuren aus Eiweiß kommt. Ferner ergab sich, daß es gelingt, bei Zufuhr bestimmter Verbindungen einzelne Aminosäuren abzufangen. So glückte es durch Eingabe von Halogenbenzol. Zystin bzw. Zystein festzulegen und als Merkaptursäure zur Ausscheidung zu bringen. P'erner konnte bei Vögeln Ornithin, ein Baustein des Arginins, durch Eingabe von Benzoesäure festgelegt werden. Auch die Entstehung von ließ sich beweisen, indem nach Glutamin - Amid der Glutaminsäure Eingabe von Phenylessigsäure das Kuppelungsprodukt Phenylazetylglutumin W^ir hatten die — — — 1) Vgl. hierzu S. 284. Virchow: Virchous Archiv. 37. 217 (186G). ') Vgl. u. A. Leo Langsfein: Valdemar Hofmeisters Beiträge. 4. 145 (19ü3). Foulsen: Beiträge zur path. Anat. u. zur allg. Path. 48. 346 (1910). Hier finden sich ') viele — Literaturangaben. ) Vgl. z. B. Gotthard Söderhergh: Nordish Med. Archiv. Abt. II. 1. (1915). 566 ^ XXVII.^Vorlesunj,'. Auch Glykokoll läßt sich in großen Mengen zur Ausscheidung im Harn in Form gepaarter Verbindungen bringen, doch vermag uns dieser Umstand nichts über die Bildung dieser Aminosäure aus Eiweiß auszusagen, weil sicher festgestellt werden konnte, daß sie wenigstens ist dies für die von den Zellen des tierischen Organismus neu gebildet werden kann. Wir kommen somit Säugetiere bewiesen zum Schlüsse, daß die Zellen des tierischen Organismus nicht nur die mit dem Blute ihnen zugeführten, vom Darme aufgenommenen Aminosäuren zum Ausgangspunkt aller möglichen Vorgänge machen, sondern daß sie selbst solche durch AblUT Ausscheidung" kam. — bau von Eiweiß bereiten können. — Vorlesung XXVIII. Eiweißstoffe und ihre Bausteine. 12. Der Abbau der Aminosäuren im Zellstoffwechsel. Die Endprodukte des Eiweiß- und Aminosäurestoffwechsels. Die Feststellung, daß der Abbau von Eiweißstoffen in den Zellen zu Aminosäuren führt, ist von grundlegender Bedeutung, denn wir müssen genau wissen, von welchen Produkten aus wir die Bildung der Stoffwechselendprodukte des Eiweißstoffwechsels zu verfolgen haben. Es sei schon hier festgestellt, daß beim Menschen, bei den Säugetieren, den eigent- Amphibien und, soweit unsere Kenntnisse reichen, auch den Irischen der Harnstoff das charakteristische, stickstoffhaltige Endprodukt des Aminosäurestoffwechsels darstellt. Bei den Vögeln und Reptilien und vielen Wirbellosen finden wir Harnsäure an Stelle der erwähnten Verbindung. Stellen wir diese beiden Verbindungen irgend einer Aminosäure, z. B. dem Leuzin, gegenüber, dann erkennen wir sofort, daß eine direkte Entstehung dieser Abbaustulen aus den Bausteinen der Proteine nicht denkbar ist: lichen NH., CH3 ^jj'>CH . CH2 . CH COOK . Leuzin HN i OC I HN CO i C II NH. /NH., >co C NH/ Harnsäure. c^o \NH2 Harnstoff. Eine Ausnahme macht nur das Ajginin, das durch hydrolytische Spaltung in Harnstoff und Ornithin übergeht.») Es muß unzweifelhaft bei allen übrigen Aminosäuren ein weiterer Abbau eintreten, bis Produkte entstanden sind, die Harnstoff bzw. Harn) Vgl. S. 32(\ XXVIU. Vorlesung. 568 Säure liefern können. Ist schon eine direkte Beziehung der einzehien Aminosäuren zu den genannten Stoffwechselendprodukten nicht denkbar, so kommt eine direkte Entstehung von Harnstofl' aus Eiweiß, Peptonen oder auch aus Polypeptiden erst recht nicht in Frage. Es sei als Beispiel ein Tripeptid dem Harnstoff gegenübergestellt: NHj . CHj . (^O . XH CH (CH,) CO NH . . . . . CH (C,Hg) COOH . (ilvzyl-alanyl-leuzin. /NH, \NH, Harnstoff. Es muß unzweifelhaft der Bildung der Stoffwechselendprodukte aus Eiweiß ein Abbau zu den einzelnen Bausteinen vorausgehen. Die Beobachtungen über das« Auftreten von Aminosäuren im Zellstoffwechsel geben die einfachste Erklärung der Art des Abbaus Die Frage der Bildung des Harnstoffes bzw. der Harnsäure aus Eiweiß deckt sich mit derjenigen nach der Art des Abbaus der einzelnen Aminoder zusammengesetzten Eiweißabkömmlinge. säuren. Als ein weiterer Beweis dafür, daß die Bildung von Harnstoff bzw. von Harnsäure von den Aminosäuren ausgeht, können wir die Tatsache anführen, daß verfütterte und auch mit Umgehung des Darmkanals eingeführte Aminosäuren zu Harnstoff bzw. Harnsäure abgebaut werden, i) Es erscheint der in ihnen enthaltene Stickstoff beim Hunde bis zu SOVo und mehr in Form von Harnstoff im Harne wieder. Das gleiche Resultat erhält man, M'enn an Stelle von Aminosäuren Polypeptide verfüttert werden. 2) Man muß allerdings zu derartigen Versuchen diejenigen Aminosäuren verwenden, die in der Natur vor- kommen, s) man z. B. dl-Leuzin, dann Das im Eiweiß vorkommende 1-Leuzin Verfüttert tritt im Harn d-Leuziu abgebaut worden. Die Konfiguration des d-Leuzins ist den Zellen des Organismus „fremd^. Sie sind nicht auf den Abbau dieser Verbindung, die ihnen ja unter normalen Verhältnissen nie zugeführt wird, eingerichtet. auf. der ist Wir kennen noch eine ganze Pteihe weiterer Tatsachen, die uns zu in den Geweben Eiweißkörper unter Annahme zwingen, daß ») Zeitschr. O. Schultzen and M. Neneki: Ber. d. Deutschen Chem. (iesellsch. 2. 566'{18(i9); Biol. 8. 124. (1872). M. Neneki: Ber. d. Deutschen Chem. Gesellsch. 5. f. — — Knieriem: Zeitschr. f. Biol. 10. 263 (1874). - E. Salkowski: Zeitschrift f. physiol. Chem. 4. .o4. 100 (1880). S. Salaskin und Kafh. Koxvalew.sky Ebenda: 42. 410 (1904). ) Vgl. Emil Abderhalden und Franz Samuely, l'eter Rona, Yutaka Teruurhi, Boris Bahkin, Karl Kautzsch: Zeitschr. f. physiol. Chem. 46. 17(5, 187 (1905); 47. 159,346, Vgl. auch P. A. Lerene und (J. M. Meyer: The Americ. 391 (19(J6); 48. 557 (1906). .lourn. of Physiol. 25. 214 (1909). ") J. Wohlgemuth: Ber. d. Deutschen Chem. Gesellsch. 38. 2064 (1905). M. Plaut und H. Reese: Hofmeistern Beitr. 7. 425 (1905). A. Schittenhilm und A. Katzenstein: Journ. f. experim. Path. u. Ther. 2. 560 (1906). P^mil Abderhalden und F. Samuely: Zeitschr. f. physiol. Chem. 47. 346 (1906). H. D. Dakin: Journ. of biol. Chem. 8 25 Emil Abderhalden und Artur Weil: Kbenda. 77 435 (1912). (1910). 890 (1872). \V. r. — — — — — — KiweilistofiV iiiul 569 ihn- Baiistpiuo. Wasseraufnahme gespalten werden. Wir beobachten nämlich auch dann eine Neubildung von Proteinen, wenn kein Eiwei(\ von außen zugeführt wird. Wir sehen, da(J beim hungernden Tier manche Drüsen ihre P'unktionen nicht einstellen. Die Schleimdrü sehen bilden immer noch eiweißhaltiges Sekret. Auch die Milchdrüse arbeitet weiter. Ferner wachsen die Haare und die Nägel. daß beständig rote Blutkörperchen zuKndlich beobachten wir, grunde gehen. Wir werden bald erfahren, daß der Gallenfarbstoft' Die Menge des erste ren gibt uns aus dem Blutfarbstoff hervorgeht. Da einen Einbhck in den Umfang des Zerfalls roter Blutkörperchen. das Blut annähernd den gleichen (iehalt an Hämoglobin muß der verloren gegangene Blutfarbstoff immer wieder beibehält, so ersetzt werden. Er enthält einen Eiweißanteil, das Globin. Kein Protein der (Gewebe besitzt einen dem Eiweißpaarling des Hämoglobins ähnlichen Bau. Das Globin enthält auffallend viel Histidin. Es ist nicht denkbar, daß irgend ein Protein der Zellen oder des Blutplasmas direkt in Globin Wir müssen vielmehr annehmen, daß der Überführung übergeht. eines Eiweißkörpers in einen anderen ein tiefer Abbau vorausgeht. Aus den entstandenen Spaltprodukten wird dann das neue, eigenBeim Kaseinogen der Milch liegen artige Molekül zusammengefügt. Es ist uns kein Eiweißkörper des Orgadie Verhältnisse ganz gleich. nismus bekannt, der nach geringfügigen Veränderungen in jenes Protein übergeführt werden könnte. Die Zellen der Milchdrüse müssen ohne Zweifel ihnen zugeführtes Eiweiß, sofern ein Transport von solchem überhaupt dann das Kaseinogen synthetisch stattfindet, weitgehend abbauen und bereiten. Das gleiche gilt für jeden Eiweißkörper, der in irgend welchen Zellen neu gebildet werden muß. Stehen Aminosäuren zur Verfügung, die durch Piesorption in die Blutbahn gelangt und den Körperzellen zugeführt worden sind, dann werden diese als Baumaterial verwendet. Sind jedoch keine zur solchen Verfügung, dann müssen sich durch Abbau von Eiweiß die Zellen diese selbst bereiten. Es ist wohl möglich, daß dann, wenn eine bestimmte Zellart Eiweiß für eine andere liefert, sie dieses zunächst selbst spaltet und die Abbaustufen dem Blute übergibt. Ein sehr schönes Beispiel der Umwandlung von Proteinen im tieribeim Lachs beobachtet. schen Organismus hat Friedrich Miescher^) Dieser Fisch wandert bekanntlich während seiner Laichzeit aus dem Meer ins Süßwasser, so auch in den Rhein. Von der J]inwanderung des Lachses in die Flüsse bis zur Abgabe der Geschlechtsprodukte nimmt er keine Nahrung auf. Dies war schon Barfurth-) und His^) bekannt. Miescher berechnet, daß der größte Teil der Lachse 6 9^2 Monate im Rhein sich und einige wenige bleiben soMonate, Teil 12 ein kleiner aufhält, Q^/o gar bis über 15 Monate. Während dieses ganzen Aufenthaltes im Süßwasser nehmen diese Tiere nichts zu sich. Stets wird ihr Magen und Daim — — ') Friedrich Mies-cher: Histochemische und physiologische Arbeiten. 2. V. C. W. Vgl. auch F. Zschokke: Der Lachs und seine Wanderungen. Vogel. Leipzig 1897. Charles W. Greene : The. .1. of Biol. Cliem. 39. Krwin Nägele. Stuttgart 1906. — — 435, 457 (1919). ») ) Barfurth: Troscheh Arch. f. Maturgesch. Jg. XLl. 1. 122 (1875). Tlis: Untersuchungen über das Ki und die Entwicklung bei Knochentisciien. Leipzig. 24 (1873). XXVIII.- Vorlesung. f)70 Miescher hat auch festgestellt, daß die Verdauungsdrüsen keine wirksamen Säfte abgeben. Betrachtet man den Lachs auf seiner Wanderung, dann sieht man eigentümliche Wandlungen seiner Körperbeschaffenheit und seines gesamten Aussehens sich vollziehen. Der in das Süßwasser übergehende Lachs ist nicht geschlechtsreif. Seine Geschlechtsorgane sind noch wenig entwickelt. Ausgestattet mit einer kräftigen Rumpfmuskulatur, überwindet er alle Strömungen des Rheins, ja sogar die Stromschnellen. Vergleicht man den eben eingewanderten Lachs mit dem kurz vor der Laichzeit befindlichen, dann glaubt man zwei ganz verschiedene Fischleer gefunden, ja, Der große Rumpfmuskel ist zusammenarten vor sich zu haben. geschrumpft, die Geschlechtsorgane dagegen haben stark an Masse zugenommen. Beide Vorgänge gehen parallel. So sah Miescher z. B. das Gewicht der Eierstöcke des Lachses von 04 g bis auf 15 </ anwachsen. Gleichzeitig ließ sich ein Sinken der Trockensubstanz und des Eiweißgehaltes des Rumpf muskels feststellen, wie die folgenden Mittelzahlen zeigen: Millimetern Körpergewicht in 872 886 879 9305 8953 7428 Länge in Mittel Monat März Mittel Juli und August Mittel November u. Dezember . . . . Gramm Gehalt des KnmpfEierstock in muskels an Prozenten des KörperTrockengewichtes Eiweiß in substanz in Prozenten Prozenten 0U61 18-55 4-78 17-5 13-2 33(j 26-8 18-5 Der Eiweißverlust, den der große Rumpfmuskel erleidet, wird offenbar zum Teil zum Aufbau der Geschlechtsdrüsen seits der Samenzellen - — einerseits der Eier, andrer- — verwendet. Dies bestätigt auch die direkte Beobach- tung. Die mikroskopische Untersuchung der Eierstöcke und der Hoden ergibt die lebhaftesten Wachstums- und Umbildungsvorgänge. Gleichzeitig zeigt der große Rumpfmuskel alle Zeichen einer weitgehenden Auflösung seines aufgespeicherten Materials und sogar des Inhalts seiner Zellen, der Muskelfasern. Sie selbst gehen nicht zugrunde. Sie geben nur alles her, was sie entbehren können, um, sobald der Lachs in das Meer zurückkehrt, wieder ihren früheren Bestand herzustellen. Der große Rumpfmuskel bestreitet den ganzen Stoffumsatz des hungernden Lachses, während die übrige Muskulatur des Körpers keinerlei Veränderungen zeigt, die auf eine Stoff Wanderung hindeuten. In welcher Form der Transport bewerkstelligt wird, ist noch unaufgeklärt. Miescher beschreibt das Auftreten von Fetttröpfchenreihen zwischen den Muskelfibrillen. Ihre Menge kann derartig zunehmen, daß die ganze Muskelfaser undurchsichtig wird. Offenbar macht sich hier die Vorbereitung zum Fetttransport bemerkbar. Neben Eiweiß, Fetten und Kohlehydraten müssen auch die übrigen Stoffe, wie Cholesterin usw. und die zum Aufbau der Kerne nötigen Substanzen, dem großen Rumpfmuskel entnommen werden. So sehen wir hier eine Stoffwanderung und -Umwandlung von gewaltigem Ausmaße vor uns. Unzweifelhaft muß eine Verfolgung dieses interessanten biologischen Experimentes mit den heutigen Methoden uns einen tiefen Einblick in den Umfang der vom tierischen Organismus ausführbaren Synthesen gewähren. Jede Zufuhr von außen ist Eiweißstoffe und ihre Bausteine. 571 dem Lachse abgeschnitten. Seine ganzen Geschlechtsprodukte muß er aus dem Materiale der Rumpfmuskulatur sich bilden. Es ist klar, daß eine Vergleichung des Gehaltes des Rumpfmuskels an Phosphatiden, an Nukleinsäuren usw. mit demjenigen der Geschlechtsprodukte ein eindeutiges Bild des ganzen Stoffumsatzes und der damit verbundenen chemischen Vorgänge geben muß. Auch bei den übrigen Tieren geht trotz Fehlens von Nahrungszufuhr die Bildung der Geschlechtsprodukte weiter. Ganze Zellen werden auf Kosten von Spermatozoon, Eier im Körper vorhandenen Produkten ausgerüstet. Nur eingreifende chemische können zu den für jede Zellart Analyse und Synthese \'orgänge spezifisch aufgebauten Bestandteilen führen. Wie ökonomisch der tierische Organismus mit dem seinem Körper einverleibten Material umgeht, beweist eine Beobachtung von E. Pflüger. ^) Die Larve der Geburtshelferkröte, Alytes obstetricans, ist gegen Ende Mai ausgewachsen. Sie hat dann eine Länge von 8' 1 cm erreicht. Von dieser entfallen ungefähr 3 cm auf den eigentlichen Körper. Der Rest kommt dem mächtigen Ruderschwanz zu. Ist die Larve auf diesem Entwicklungsstadium angelangt, so nimmt sie keine Nahrung mehr auf. Zu gleicher Zeit beginnt der Schwanz einzuschrumpfen. Sein Zellmaterial wird flüssig gemacht und wandert dem eigentlichen Körper zu, aus dem heraus parallel mit der Rückbildung des Schwanzes die Vorder- und — — — — Hinterbeine hervorsprossen. Welch bedeutende Umwandlungen müssen sich dem im Ruderschwanze aufgestapelten Material die Extremitäten sich voll entwickelt haben! Sobald der Schwanz völlig aufgesaugt ist, setzt die Nahrungsaufnahme wieder ein. 2) Schließlich sei noch darauf hingewiesen, daß beim hungernden Organismus nicht alle Organe gleichmäßig an Gewicht verlieren. Wir sehen, daß solche Organe, deren Funktionen für die Fortdauer des Lebens unerläßlich sind, lange Zeit ihren Bestand erhalten und offenbar zur Deckung ihres Energiebedarfes und zur Aufrechterhaltung des Zellbaues von anderen, weniger wichtigen Zellarten Material geliefert bekommen. Fassen wir alle Beobachtungen über den Eiweißstoffwechsel in den Zellen zusammen, dann kommen wir zum Schlüsse, daß er sicher über Aminosäuren führt. Auf die Frage, ob nur eine einzige Art des Abbaus der Proteine, nämlich die Hydrolyse, stattfindet, oder ob nicht vielmehr das Eiweißmolekül im Stoffwechsel auch in anderer Weise verändert werden kann, werden wir noch zurückkommen. Soviel können wir jetzt schon ausja wahrsagen, daß der weitaus größte Teil des Eiweißes, vollziehen, bis aus scheinlich das gesamte Protein über Aminosäuren zum Abgelangt. Wir haben somit bei den Proteinen ganz entsprechende Verhältnisse, wie bei den zusammengesetzten Kohlehydraten, den Fetten und Phosphatiden. Auch bei diesen Verbindungen stehen ihre Bausteine im Mittelpunkt des ganzen Zellstoffwechsels. Über sie führt der Aufbau und auch der Abbau. Wir dürfen in Übereinstimmung mit dem Abbau der Proteine im Darmkanal annehmen, daß die einzelnen Zellen zunächst bau 1) Eduard Pflüger: Pflüf/ers Archiv. 29. 78 (1892); 54. 333. (403) 19U3). Diese Erscheinung gilt gewiß nicht nur für die Larven der Geburtshelferkröte, sondern ganz allgemein für alle dieses Stadium durchmachenden Amphihienlarven. Die Larven von Rana fusca und von Rana temporaria zeigen wenigstens ein ähnliches Verhalten, nur scheint bei ihnen ein gegenseitiges Anfressen der Ruderschwänze vorzukommen. ) XXVIII. Vorlesung. 572 das Eiweiß in Peptone spalteo. (Gleichzeitig werden auch Aminosäuren frei. Es scheint, nach allen Erfahrungen, daß gewisse Aminosäuren, wie Tyrosin, Tryptophan und Cystin, frühzeitig vollständig abgespalten werden. Schließlich führt der Abbau zu immer einfacher zusammengesetzten Abbaustufen, bis endlich ausschließlich Aminosäuren entstanden sind. Es ergibt sich auch hier die Frage, ob dann, wenn aus einem bestimmten Protein ein anderes entstehen soll, der Abbau unbedingt bis zu den einfachsten Bausteinen gehen muß, oder ob nicht auch zusammengesetzte Abbaustufen, wie z. B. Polypeptide, direkt in den neuen Bau übernommen werden könuen. daß bestimmte Gruppierungen von Aminosäuren direkt Entscheiden läßt sich diese ist zuzugeben. Frage zurzeit noch nicht. Unaufgeklärt ist augenblicklich auch noch die Art der Zusammenfügung der Aminosäuren zu zusammengesetzten Produkten, Es bestehen vor allem zu Poypeptiden bis hinauf zum Eiweißmolekül. mehrere Möglichkeiten. Man könnte an die Bildung von Aldehyden aus Aminosäuren denken, die dann entweder mit einander oder mit Aminosäuren allein in Verbindung treten könnten.i) Betrachtet man den Abbau von Eiweiß und von Eiweißabkömmlingen als umkehrbaren Fermentvorgang, dann muß mit einer direkten Verkuppelung der Aminosäuren Die Möglichkeit, Verwendung finden können, Leider war es bis jetzt nicht möglich, einen gerechnet werden. Einblick in die feineren Vorgänge solcher Synthesen in Zellen zu gewinnen. Sehen wir uns nun genau so, wie bei der Frage des Abbaues der Glukose und der Bausteine der Fette um, auf welchem Wege der selbst Abbau der einzelnen Aminosäuren zu den Stoff Wechselendprodukten vor sich geht. Wir wollen zunächst die Bildung des Harnstoffs betrachten und später auf diejenige der Harnsäure eingehen. Es sei zuerst der Beobachtung von Kossei und Dakin'^) gedacht, daß der tierische Organismus über ein Ferment verfügt, das aus Arginin Harnstoff abspalten kann. Es ist Arginase genannt worden. Es kommt nur in der Leber der Säugetiere vor. Es fehlt der Leber der Vögel und Reptilien ganz.^) Die Arginase spaltet Arginin unter Wasseraufnahme unter Bildung von Harnstoff und Ornithin. Auch im Pflanzenreich findet sich Arginase.) NH2 5{}2\C MH ^ . NH CH2 CH2 CHj CH GOGH . . + HÖH . . . Arginin I NH, NH.. NH i\r,^0 + V\\^ OH^ GHo CH GOGH NH . . Harnstoff ') iirul . . Ornithin. Herrn. J'auh/: Zcitsclir. f. phvsiol. Chemie 99. 161 (1917). ~ Emil Abderhalden Hans Spinner: Ebenda. 106. 30f) (1919). A. Kossei und //. D. Dakin : Zeitschr, f. physiol. Chemie. 41. 321 (1905); 42. 181 Vgl. auch Otto Messer: und Münchener med. Wochenschr. Nr. 13(1904). Zeitschr. f. physiol. Chemie. 49. 210 (1906). — Vgl. ferner Charles Richet: Compt. rend. deia soc. de biol. 46. 525 (1894); Compt. rend. de l'Acad. des Sciences. 118. 1127 (1895). 2) — (1904) Edlbacher: Zeitschr. ») .S'. ) Vgl. hierzu A. f. physiol. Chemie. 95. 81 (1915). Chemie. 118. 267 (1922). f. physiol. Kiesel: Zeitschr. Kiwp.iÜstotlo und ihre Baustoiiip f)78 Arginin ist die einzige bis jetzt bekannte Aminosäure, aus der direkt Harnstoff abgespalten werden kann. Die Bildung des dem Ornithin und den übrigen Aminosäuren des Eiweißes entsprechenden Harnstoffs muß auf einem anderen Wege erfolgen.») Nur ein kleinei- Teil des Stoffwechselendproduktes verdankt seine erwähnten Entstehung der Wirkung der Arginase, weil jene Proteine, die sich in unserer Nahrung und in unseren Geweben finden, im allgemeinen nur über etwa ö bis lOo/o Arginin verfügen. Die Entstehung des Harnstoffs aus den einzelnen Aminosäuren ist langer Zeit Gegenstand eifrigster Forschung. Es ist nicht geglückt, sie in allen Phasen aufzuklären. Wir wollen diejenige Hypothese hier erwähnen, die durch die meisten Beobachtungen gestützt ist. Sie stammt von Nenrki^) und Sehmiedeberg. ^) Wir haben wiederholt darauf hingewiesen, daß eine Art des Abbaus der Aminosäuren mit der Abspaltung der NHo(Jruppe einsetzt. Vielleicht wird immer dieser Weg- der Zerlegung der Bausteine der Proteine eingeschlagen, es ist jedoch auch möglich, daß je nach Bedarf verschiedene Arten des Abbaus einsetzen. Die Abspaltung der Aminogruppe Desaminierung genannt kann auf verschiedene Weise erfolgen. Die NH-^-Gruppe kann durch Hydrolyse, durch Reduktion und endlich durch Oxydation entfernt werden. Die folgenden Formeln geben diese drei Arten der Desaminierung am Beispiel des Alanins wieder. Wir werden später die Frage besprechen, welche von diesen drei Arten <ler Desaminierung im tierischen Organismus die wahrscheinlichste ist. seit — CH3 . — CH COOH + H.O = CU, CH (OH) COOH + NH, . . . NH2 Alanin CH3 . Milchsäure. CH COOH + 2 H . = ( H3 CH, . . COOH + NH, I HH., Alanin Propionsäure. CH3 .CH.C()()H + =CH,.CO.COOH + NH;, NH« Alanin Brenztraubensäure. Im ersten Falle entsteht eine Oxy säure, im zweiten eine Fettsäure und im dritten eine Ke tonsäure. In allen drei Arten der Desaminierung kommt es zur Bildung von Ammoniak. Von diesem aus erfolgt nunmehr die Entstehung des Harnstoffs. Ammoniak verbindet sich mit Kohlen- säure zu kohlensaurem Amnion, und dieses geht dann unter ^) W. H. Thompson hat die interessante Beobachtung gemacht, daß Hunde nach Eingabe von Arginin zwei zeitlich getrennte Vermehrungen der Harnstoffausscheidung zeigen. Die erste entspricht dem durch Arginase in Freiheit gesetzten Harnstoff und die zweite dem aus dem Ornithin stammenden. Vgl. Journ. of Phvsiol. 32. 137 (1905); 33. 106 (1905). ^) M. Nencki: Ber. d. Deutschen Chcm. üeselisch. 5. 890 (1872). ) 0. Schmiedeberg Archiv f. exper. Path. u. Pharm. 8. 1 (1879) : XXVIII. Vorlesung. 574 Verlust von zwei Molekülen Wasser in Harnstoff iiber.O Als karbaminsaures Ammon auftreten, wie die könnte Zwischenprodukt folgenden Formeln zeigen: /O.NH, /OH /O.NH4 \0H ^O.NH» Kohlensaures Karbamin- Ammon saures Ammon C^O + 2NH, = C^O ^H.O—y — H,0 = C^O ^NH, /NH., ^NH., Harnstoff. Für diese Auffassung der Bildung von Harnstoff sprechen die folgenden Befunde. Verfüttert man Ammoniumkarbonat oder Ammonsalze von Fettsäuren, wie z. B. essigsaures Ammon usw., dann tritt der mit diesen Verbindungen eingeführte Stickstoff in Form von Harnstoff auf.^) Besonders wichtig ist die Beobachtung von v. Schröder'^), daß auch aus Ammoniumkarbonat Harnstoff entsteht, wenn dieses durch die Leber durchgeleitet wird. Interessant mit Blut die Bildung nicht erfolgt, wenn das kohlensaure Ammon in ist, daß zur Verfügung gestellt wird.) iim^erscher Lösung den Leberzellen Bedeutungsvoll ist ferner, daß im Blute und im Harne karbaminsaures Ammon festgestellt worden ist^), doch erwies sich dieser Befund nicht als eindeutig, weil bei Anwesenheit eines Ammonsalzes und eines kohlensauren Salzes, z. B. von Natriumkarbonat, sich in wässeriger Lösung Ammoniumkarbonat bildet. «) Eine sekundäre Entstehung des aufgefundenen karbaminsauren Ammons ist somit nicht ausgeschlossen. Auch beim Durchleiten von Aminosäuren durch die Leber wurde Harnstoff sichergestellt.^) Eine weitere sehr wesentliche Stütze für die Annahme einer synthetischen Bildung von Harnstoff aus Kohlensäure und Ammoniak ergeben die zahlreichen Beobachtungen über die Art des Abbaus der Aminosäuren. Es spricht alles dafür, daß zunächst Ammoniak neben einer stickstofffreien Säure entsteht. ') Vgl. zu diesem Problem auch Fr. Fichter: Zeitschr. f. Elektrochemie. 24. 41 (1918). — E. Salkowski: Zeitschr. Biol. 13. 256 (1877). — Immanuel Munk: Zeitschr. physiol. Chemie. 2. 29 (1875/79). — E. Hallervorden Archiv Zaleski: exper. Path. u. Pharm. 10. 124 (1879). — Vgl. auch M. Nencki, J. Paivlow und ) W. V. Knieriem: Zeitschr. f. Biol. 10. 203 (1874). — Ludwig Feder: Zeitschr. 1. 1 (1877). physiol. Chemie. f. f. f. : ./. f. Ebenda. 37. 26 (1895). 3) W. V. Schröder: Archiv f. exper. Path. u. Pharm. 15. 364 (1882); 19. 373 (1885). ) Antonio Clementi: Arch. di Farmac. sperim. 23. 289 (1917). John ') E. Drechsel: Journ. f. prakt. Chemie. 12. 417 (1875); 22. 476 (1880). John J. Abel und J. Abel und E. Drechsel: Archiv f. (Anat. u.) Physiol. 236 (1891). Archihald Murhead: Archiv f. exper. Path. u. Pharm. 31. 15 (1893); 32. 467 (1893). M. Hahn und M. Nencki: Ebenda. 32. 185 (1893). J.J. R. Macleod und «) P. Nolf: Zeitschr. f. physiol. Chemie. 23. 505 (1897). //. D. Haskins:' Journ. of biol. Chem. 1. 319 (1905); 12. 444 (1905). Vgl. auch ') Vgl. u. a. B. C. I'. Jansen: Journ. of biolog. Chem. 21. 557 (1915). Wilhelm Löffler: Biochem. Zeitschr. 85. 230 (1918). — — — — — Eiweißstoffe und ihre Bausteine. 575 Die Abspaltung der NHo-Gruppe wird einer besonderen F'ermentgruppe ist Desaminase genannt worden. Es sei gleich hier festgestellt, daß es bis jetzt nicht geglückt ist, dieses Ferment aus Zellen zu isolieren.') Ja, es gelingt sogar in vielen Fällen nicht, die desaminierehde Wirkung von Geweben nachzuweisen, wenn man diese zerhackt und dann dem Brei Aminosäuren zugibt. Offenbar erfolgt die Desaminierung nur anter ganz bestimmten, in der unverletzten Zelle gegebenen Bedingungen. Daß Fermente vorhanden sein müssen, die desaminieren können, lehren zahlreiche Versuche. Wir beobachten, daß viele niedere Organismen aus ganz verschiedenen Aminosäuren Bakterien, Pilze, Hefezellen usw. gleichartiges Eiweiß aufbauen. Ein Baustein genügt, um alle anderen hervorgehen zu lassen. Auch Ammonsalze genügen oft als einzige Stickstoffquelle. Offenbar verwenden die genannten Organismen nicht die einzelne Aminosäure als solche als Ausgangsmaterial zur Synthese der übrigen Bausteine des Eiweißes, sondern die NHa-Gruppe bzw. das Ammoniak. Übrigens hängt die. Frage nach dem Vorhandensein bestimmter Desaminasen eng mit derjenigen nach der Art des Abbaus der Aminosäuren zusammen. zugeschrieben. Sie — stoffs Eine weitere Stütze für die Ansicht, daß die Entstehung des Harnvon Ammoniak ausgeht, ergibt die Beobachtung, daß seine Menge direkt von derjenigen der im Organismus vorhandenen, nicht abbaufähigen Säuren abhängig ist. Der tierische Organismus hat das Bestreben, die Reaktion in seinen Geweben und Flüssigkeiten annähernd neutral zu halten, d. h. weder einen erheblichen Überschuß an H- noch an OH-Ionen zu belassen. Gegen ihm zugeführte oder in seinen Geweben entstehende Säuren kann er sich auf verschiedene Weise schützen. Einmal kann er solche durch vollständigen Abbau beseitigen. Ferner können sie Ein durch Kuppelung mit Paarungen unschädlich gemacht werden. solcher Paarung 2) und sicher der wichtigste ist das Ammoniak. Es sei daran erinnert, daß es beim Diabetes melitus häufig zur Ausscheidung von [i-Oxybuttersäure und Azetessigsäure kommt. Auf das Vorkommen dieser Säuren wurde man nicht direkt aufmerksam, sondern erst durch den auffallend hohen Ammoniakgehalt des Harns Man fragt sich, weshalb der Harn von an Diabetes Leidenden. ») Menge mit dersoviel Ammoniak enthält, und fand dann, daß seine läßt sich festGleichzeitig jenigen der genannten Säuren anstieg. stellen, daß der Gehalt eines solchen Harnes an Harnstoff ein geAm übersichtlichsten ringerer ist als unter normalen Verhältnissen. werden die Beziehungen zwischen Ammoniak und dem Harnstoff des Harnes, wenn man den Gesamtstickstoffgehalt des Harnes mit der auf Harnstoff und Ammoniak entfallenden Stickstoffmenge vergleicht. Führt man einem Tier Säure, z. B. Salzsäure zu. dann fällt die Harnstoff- Ä — — E. Abderhalden und Lang •.Hofmeister?, Beitr. 5. 321 (1904). ) Vgl. hierzu Bans Prinqsheim: Biochem. Ä. Schittenhelm: Zeitschr. f. physiol. Chemie. 49. 26 (1900). G. R. Bostock: BioF. Ehrlich: Ebenda. 18. 416 (1909). Zeitschr. 12. 15 (1908). chem. Journ. 6. 48 (1911). ) Ob das Ammoniak eine chemische Verbindung eingeht oder ausschließlich durch Neutralisation wirkt, ist für die Auffassung des Ammoniaks als „Paarung" gleichgültig. — ) — E. Stadelmann: Deutsche med. Wochenschr. 46 (1889). f. experim. Path. und Pharm 32. 372 (1894). Ä. Sirusser: Archiv — E. Münzer und XXV III. Vorlesung. öTH meuge, und es steigt, der Gehalt des Harues an Ammonniak.i) Wir können von diesen Gesichtspunkten aus das Ammoniak als wichtigen Schutzstoff des tierischen Organismus auffassen. Es neutralisiert Säuren und schützt vor Verlust an fixem Alkali. Man kann umgekehrt durch Verabreichung von Alkali die Menge dos Ammoniaks herabsetzen, ja sogar zum Verschwinden bringen. 2) Die mitgeteilten Beobachtungen machen es sehr wahrscheinlich, daü die Entstehung des Harnstoffs an das Freiwerden von Ammoniak geknüpft ist. Es ist jedoch auch möglich, daß die erwähnte Art der Entstehung von kohlensaurem Amnion und von Ammoniumkarbaminat nur einen Weg der Harnstoffbildung darstellt, und auch andere eingeschlagen werden. Zunächst ist es nicht notwendig, daß aus Ammoniak und Kohlenkohlensaures Ammon entsteht, es kann vielmehr direkt karbaminsaures Ammon gebildet w^erden. das dann unter Austritt von einem Molekül Wasser in Harnstoff übergeht. Man kann sich die Entstehung säure des Ammoniumkarbaminates auch, wie folgt, vorstellen. Wir haben früher schon erwähnt, daß Aminosäuren unter Kohlensäureabspaltung in Amine übergeführt werden. Das gleiche Ziel wird auch erreicht, wenn Ameisensäure in Freiheit gesetzt wird, s) Dieser begegnen wir stets im Harn. Sie könnte nun aminiert werden. Es würde Karbaminsäure entstehen. Diese kann in gewissem Sinne als die einfachste Aminosäure, nämlich als eine Aminoameisensäure aufgefaßt werden. Das Amid dieser Verbindung ist der Harnstoff. Es ist möglich, daß auch auf diesem Wege Harnstoff gebildet wird. Er sei am Heispiel des Alanins dargestellt: CH, . CH (JOOH + 2 H CH, . . NH, CH, -f H COOK . NH., Äthylamin Alanin Ameisensäure NH2.COOH + HaO. Aminoam eisen säure H.COÜH -i- NHs -f Ameisensäure NHa Am n i säure . NH„ CO NH, -f H., 0. COOH + NH3 m e sen sä u re a . . Harnstoff. i Oder es tritt als Zwischenprodukt das Ammonsalz der AminoameisenKarbaminsäure auf: = — — •/. Pohl iiiul 1) Friedrich Walter: Archiv f.experim. Path. u. Pharm. 7. 14H (1877). Alexander Szili: I'ßiigers Archiv. 115. 82 (190G). Münzer: Ebenda. 43. 28 (1900). H. Eppinger - Hans Eppinger: Zeitschr. f. experim. Path. u. Ther. 3. 530 (190Ü). J. Pohl: Ebenda. 18. 24 (1909). und F. Tedesko: Biocliem. /eitschr. 16. 207 (1909). ^ /•;. — — K. B. Hasselbalch: Biochem. /eitbchr. 74. 18 (1916). — W. Beckmann: Virehotvs, Archiv. 71. 504 (1884). ") E. Salkoivski und 1. Mnnk: Münzer: Archiv f. experim. Path. u. Pharm Zentralbl. f. d. med. Wissensch. 26(3 (1890). Ther. yl. Schittenhelm und A. Kaizenstein: Archiv f. experim. Path. u. 33. 193 (1894). Nelson W. Janne;/: /eitschr. f. physiol. Chemie. 76. 99 (1912). Hier 2. 541 (1905). Hndet sich weiteie Literatur — — ') V^l. S. 451. — Kiwcißstotto und ihre Bausteine. NH, .COOH + NH3 Aminoameisensäiire — H.2 O NH2 COO NH, Karbaminsaures Aminon . . = 577 NH2.COO.NH, Karbarainsaures Amnion = NH2 CO NH, . . Harnstoff. Wir können den Harnstoff auf Grund der besprochenen Bildungsweisen Diamid der Kohlensaure ameisensäure auffassen: als ein /OH Cf^O \0H /NH, -> C^O \NH., oder auch als Amid der Amino- XX VIII. Vorlesung. 578 verschiedener Substanzen, wie z. B. von Taurin ^j, Tyrosin, von Aminobenzoesäure usw. im Harn Verbindungen dieser Körper mit Harnstoff der folgenden Art: CH2 SO., . . OH CH2 SO2 . —^ I . OH I CH2 NH CO NH2 Uraminoisäthionsäure. CH2 NH, . HOOC Ce H, NHs Aminobenzoesäure . . . Taurin . —> HOOC . C« H, . . NH . (10 . NH^ Urarainobenzoesäure. Man könnte diese Beobachtungen im Sinne eines Abfangens der geCO NHg-Gruppen durch die erwähnten Verbindungen deuten. bildeten . Seitdem jedoch bekannt geworden ist, daß die Uraminosäuren sich schon beim Einengen wässeriger Lösungen von Aminosäuren und von Harnstoff bilden 2), muß ihr Nachweis aus der Reihe der Beweise für die genannte Art der Harnstoffbildung ausscheiden, es sei denn, daß eindeutig ihre primäre Entstehung erwiesen würde. ) Es sind übrigens noch andere Möglichkeiten der Bildung von Harnstoff erörtert worden.) Sie sind jedoch alle durch keine direkten Beobachtungen am Organismus bzw. an Zellen gestützt. Wir wollen sie deshalb übergehen. in der Leber gebildet. Der Harnstoff wird sicher Dieses beobachtet, daß nach schweren Schädigungen der Leber die im Harn zur Ausscheidung gelangende Harnstoffmenge zwar abnimmt, jedoch seine Bildung nicht aufgehoben ist.^) Auch Haifische, die interessanterweise in ihrem Blut und ihren Geweben auffallend viel Harnstoff aufweisen, zeigen nach Leberexstirpation keine Einschränkung in der Bildung dieses Stoffwechselendproduktes.«) Hunde mit Eckscher Fistel scheiden gleichfalls noch große Mengen von Harnstoff aus.") Diese Beobachtung ist insofern nicht eindeutig, als die Leber immer noch mit dem gesamten Kreislauf durch die Organ ist jedoch nicht die einzige Stätte seiner Bereitung. Man Leberarterie in Verbindung steht. Es könnten ihr mit dem arteriellen z. B. Ammoniak zur Bildung des Harnstoffes zuBlute Vorstufen geleitet werden.. Die Abnahme der Menge des Harnstoffes nach Schädigung oder Ausschaltung der Leber wurde zunächst im Sinne des Ausfalles eines Organes aufgefaßt, das an seiner Bildung den Hauptanteil hat. Die genauere — E. Salkowski: Virchows Archiv. 58. 460 (1873); Zeitscbr. f. pliysiol. P. Philosophow : Biochem. Zeitschr. 26. 131 (1910). 93 (1882/83). 0. Schmiedeberg: Archiv f. experim. Path. u. Rudolf Cohn: Ebenda. 17. 274(1893). Vgl. hiezu auch Carl /.. A. Schmidt und G. W. Clark: .1. of Pharm. 8. 1 (1877). biol. ehem. 50. 21 (1922). ') Chemie. Vgl. — 7. — ^) — — Vgl. S. 345. Die üraminoverbindung des Phenylalanins kristallisiert ohne Kioengeii direkt aus dem Harn aus. Sie ist hiermit als primär vorhanden zu i)etrachten. K. Sal) Vgl. z. B. Hoppe-Seyler : Physiol. Chemie. 809—810. Berlin 1881. Vgl. aucii die interessanten Beobkowski: Zeitschr. f. physiol. (Jhemie. 1. 1 (1887). achtungen von Fr. Fichter über die elektrolytische Bildung von Harnstoff. Zeitschr. f. Fr. Fichter, Karl Stutz und Fritz GriesElektrochemie. Nr. 15 (1910); 24. 41 (1918). haber: Verhandl. d. Naturforsch. Gesellsch. in Basel. 23. 222 (1912). ) — — — Vgl. W. V. Schröder: Zeitschr. f. physiol. Chemie. 14. 586 (1890). M. Hahn, 0. Massen, M. Nencki und J. Paulow : Archiv f. experim. Path. ') Pharm. W.Frey: Zeitschr. f. kl. Medizin. 72. 38.'5 (1911). ') «) z. 32. 161 B. (1892). u Eiweißstoffe und ihre ßauB(eine. 579 Analyse der nach der teilweisen oder vollständigen Ausschaltung der Leber daß die Abnahme der Harnauftretenden Verhältnisse hat gezeigt, stoffmenge eine andere Deutung zuläßt. Es treten nämlich Säuren auf, für die Ammoniak zur Neutralisation verwendet wird. Dieses wird dann der Harnstoffbildung entzogen. Aus den vorliegenden Beobachtungen ergibt sich der Schluß, daß die Leber Harnstoff bildet. Der Versuch am überlebenden Organ hat das eindeutig bewiesen. Die Harnstoffbildung läßt sich auch mikroskopisch in den Leberzellen feststellen, während der Nachweis von Harnstoff in anderen Geweben mikrochemisch mißlang. Es scheint, daß in der Leber den sogenannten Kupff'er sehen Sternzelleu eine besondere Funktion l)ei der fberleitung des gebildeten Harnstoffs an die Lymphe und ins Blut zukommt.') Harnstoff kann sehr wahrscheinlich auch von allen anderen Körperzellen erzeugt werden. Allerdings fielen bis jetzt die Versuche an anderen überlebenden Organen als der Leber negativ aus. Die Beobachtungen an Individuen mit pathologisch veränderter Leber und vor allem die Tierversuche, bei denen die Leber künstlich schwer geschädigt wurde, zwingen uns jedoch zu der Annahme, daß die verschiedensten Gewebe Harnstoff zu bilden imstande sind. 2) Die Leber wird wohl dann besonders stark in Anspruch genommen werden, wenn sie in größerem Umfange ans Aminosäuren Zucker zu bilden hat. Der Harnstoff kristallisiert in Nadeln oder in farblosen, vierseitigen, rhombischen Prismen. Er hat einen eigenartigen, an Salpeter erinnernden, kühlenden Geschmack. Er schmilzt bei 132" und löst sich in der gleichen (iewichtsmenge Wasser. Er ist auch in Alkohol löslich. Zum Nachweis Harnstoffes dient vor allem folgende Reaktion. Erhitzt man ihn in trockenen Reagenzglas, dann beobachtet man zunächst, daß er schmilzt. Steigt die Temperatur höher, dann bemerkt man bald, daß Ammoniak entweicht. Er gibt sich am Geruch zu erkennen. Ein in die Dämpfe gehaltenes rotes Lackmuspapier wird gebläut. Schließlich erstarrt des einem Masse wieder. Nimmt man die feste Masse in Wasser auf, und hinzu, dann tritt beim Zusatz von verdünnter Kupfersulfatlösung Rotviolettfärbung ein. Man nennt diese Reaktion Biuretreaktion. Sie beruht auf der Entstehung von Biuret, das aus zwei Molekülen Harnstoff unter Abspaltung von Ammoniak hervorgeht: die ganze fügt man Alkali /NH, NH C=0 \nh., /Nh; -nh3= Cf ^^^ ^nh cf \nh3 \nh, 2 Moleküle Harnstoff Biuret. Wir haben eine ganz ähnliche, in gleicher Weise angestellte Reaktion den Proteinen und Peptonen und manchen Polypeptiden kennen gelernt. Es darf nicht aus der Ähnlichkeit beider Reaktionen auf eine Übereinstimmung der sie ergebenden Produkte geschlossen werden. In der bei ») ) Erich Lescfike: Zeitscbr. f. experim. Path. u. Ther. 16. 498 (1914). Vgl. Cyrus H. Fiske und James B. Sumner: Journ. of Biol. Cliem. 18. 28f) (1914i 37 XXVIII. Vorlesung. 580 Gruppe der Proteine dürfte eine dem Biuret entsprechende Atomgruppierung vorkommen. In den Polypeptiden, die die Biuretreaktion geben, fehlt sie sicher. Neben dem Biuret entsteht immer auch Cyanursäure: nicht — n NH3 = NH, CO NHo + NH2 CO NH2 + NH2 CO NH., 3 Moleküle Harnstoff . . . . . NH CO NH CO NH CO . . . . . Cyanursäure. Harnstoff gibt Salze mit Säuren, so mit /NHo\ / CM> \ /NHoA / • I Salpetersäure C^O HNOä. mit Oxalsäure \NH./ 1. COOH usw. \| \NH,y2 COOH V aller Tiere Harnsäure. dem Eiweißstoffwechsel in Zusammenhang stehendes Produkt betrachtet worden. Es unterliegt keinem Zweifel, daß die Harnsäure bei den Vögeln, den Reptilien und vielen wirbellosen Tieren vom p]iweiß bzw. den Aminosäuren abstammt, dagegen kennen wir keine eindeutige Beobachtung, die beweisen würde, daß auch der Mensch, die Säugetiere, die Amphibien und die Fische aus Eiweißabkömmlingen die erwähnte Verbindung bereiten. Sie stammt vielmehr bei diesen Neben dem Harnstoff finden wir im Harn Sie ist früher ganz allgemein als ein mit Tieren aus anderer Quelle. Sie geht nämlich aus bestimmten Bausteinen der Nukleinsäuren, den Purinbasen, hervor. Das exakte Studium der Herkunft der Harnsäure bei den zuerst genannten Tierklassen hat ergeben, daß ein kleiner Teil der ausgeschiedenen Verbindung ebenfalls von Purinbasen aus gebildet wird. Die folgende Übersicht zeigt, daß alle Tiere eine gemeinsame Quelle der Harnsäure besitzen, nämhch die Purinbasen. Bei den Vögeln, Reptilien und vielen Wirbellosen kommen als Ausgangsmaterial außerdem noch die Aminosäuren in Betracht. Beim Menschen, den Säugetieren. Amphibien und Fischen finden wir an Stelle dieses Anteils der Harnsäure Harnstoff. Fast stets tritt neben Harnstoff und Harnsäure auch in geringen, w^echselnden Mengen Ammoniak auf. Die folgende Übersicht gibt die Herkunft der Harnsäure bei den verschiedenen Tierklassen wieder: Harnstoff -< — Aminosäuren — -< Eiweiß — Aminosäuren — Harnsäure Mensch, Säugetiere, Amphibien. Fische >- >- Vögel, Reptilien, Wirbellose Nukleinsäuren i Purinbasen i Harnsäure. Wir werden auf die Bildung der Harnsäure aus Puribasen später zurückkommen ^) und beschäftigen uns jetzt ausschließlich mit ihrer Ent') Vgl. Vorlesung XXXII. Fiiweißstoü'e uud ihre Bausteine. 581 stehung aus Eiweiß bzw. aus Aminosäuren. Sie zeigt in vielen Beziehungen Übereinstimmung mit derjenigen des Harnstoffs. Eine direkte Umwandlung irgend eines p]iweißabkömmlings bis herunter zu den Aminosäuren in Harnsäure ist so gut, wie ausgeschlossen. Die einzige Aminosäure, die mit der Harnsäure verwandte Züge zeigt, ist das Histidin. Es enthält den Imidazolkern, der auch in der Harnsäure wiederkehrt: eine große HN— CO COOH I CH.NH., I I CH— NU -11 OC I C— NH. >C0 >CH I il HN— C-NH/ CH2 C N Histidin — a-Amiuo-ß-imidazolylPropionsäure. Harnsäure. Es ist schwer, zu entscheiden, ob die Imidazolgruppe zur Bildung von Harnsäure direkt übernommen wird, weil die genannten Organismen sowieso aus Aminosäuren Harnsäure bilden und daher eine dem zugeführten Histidin entsprechende Vermehrung des Harnsäuregehaltes des Kloakeninhaltes nichts über die direkte Verwertbarkeit jener Gruppe aussagen würde. Es ist untersucht worden, ob die genannte heterozyklische Aminosäure beim Säugetier die Bildung der Harnsäure beeinflußt. 1) Es war dies nicht der Fall. Mit Ausnahme des Globins enthalten die übrigen Proteine nur geringe Mengen von Histidin, so daß selbst dann, wenn der Imidazolring direkte Verwendung finden würde, nur für einen geringen Teil der gebildeten Harnsäure die Herkunft erklärt wäre. Es sprechen alle Erfahrungen dafür, daß die Bildung der Harnsäure kein einfacher Vorgang ist, sondern eine umfassende Synthese darstellt. Es ist festgestellt Avorden, daß Vögel nach Verfütterung von Aminosäuren 2) eine der in dieser P'orm verabreichten Stickstoffmenge entsprechende Vermehrung der Harnsäure aufweisen. Ferner bewirken auch Ammonsalze^) ein Ansteigen der Harnsäurebildung. Besonders interessant ist die Beobachtung, daß auch verfütterter Harnstoff) zu einer Vermehrung der Harnsäure führt, w\ährcnd zugeführte Harnsäure selbst unverändert zur Ausscheidung gelangt. Vom Harnstoff kann nur ein bestimmter Teil zur Harnsäurebildung Verwendung finden. Führt man einen großen Überschuß davon zu, dann wird ein Teil davon unverändert au-'-geschieden.») Alle vorliegenden Beobachtungen führen zu der Annahme, daß auch im Organismus des Vogels und der Reptilien der Eiweiß- und Aminosäurenabbau ebenso verläuft, wie bei denjenigen Tieren die Harnstoff ausscheiden. Auch bei den Vögeln und Pieptilien erfolgt im Darmkanal ein tiefgehender Abbau der Proteine. Es gelangen Aminosäuren zur Resorption. Ferner entstehen solche auch aus Eiweiß im Zellstoffwechsel. Beim Abbau ») Emü Abderhalden uud //. Einbeck: Zeitschr. f. physiol. Chemie. 62. 322 (1909). E. Abderhalden, II Einbeck uud K. Kowaleivskij : Biochem. Journ. 23. 1 (1910). ^'gl. auch llarold Ackroyd uud E. G. Hopkina: Julius Schmid) Ebenda. 68. 395 (1911). Biochem. Journ. 10. 561 (1916). «) V. Knieriem: Zeitschr. f. Biol. 13. 36 (1877). 3) IV. V. Schröder: Zeitschr. f. physiol. Chemie. 2. 228 (1878). — — } Hans Jlorfit Mei/er: lu.-Diss. ^y Hugo Könifrshertr 1878. Wiener: llofvieistir^ Beitrage. 2. 42 (1902\|. XXV 111. Vorlesimg. 582 der Aminosäuren wird ferner Ammoniak gebildet. Dieses bildet das Ausgangsmaterial zur Synthese von Harnsäure. Ferner ist festgestellt worden, daß die Leber bei der Bildung der Harnsäure eine sehr wichtige Rolle spielt.i) Wird sie vollständig ausge.schaltet, dann wird nur noch eine ganz geringe Menge Harnsäure gebildet. Diese scheint nicht von Aminosäuren, sondern von Pur in blasen abzustammen. 2) An Stelle der Harnsäure tritt in großen Mengen Milchsäure im Kloakeninhalt auf. Ferner wird viel Ammoniak ausgeschieden. Es war naheliegend, diese beiden Verbindungen als die Grundsubstanzen der Harnsäurebildung aufzufassen. Bald kamen jedoch wichtige Bedenken. Je I)ie Milchsäure bewirkt als Säure ein Abfangen von Ammoniak. mehr Ammoniak durch diese festgelegt wird, um so weniger Harnsäure kann gebildet werden. Wir hätten somit ganz ähnliche Beziehungen zwischen dem Ammoniak und der Harnsäure, wie zwischen diesem und dem Harnstoff. Das Auftreten der Milchsäure konnte durch irgend eine sonstige im (xefolge der Leberausschaltung auftretende Störung des Stoffwechsels bedingt sein. Wäre die iVnsicht richtig, dali die Harnsäurebildung nur indirekt dadurch gestört ist, daß mit dem Fortfallen der Funktion der Leber auch das Material zur Harnsäurebildung fehlt, dann müßte sie sich wieder in Gang bringen lassen, wenn man Alkali zuführt und dadurch das Ammoniak zur Synthese von Harnsäure zur Verfügung stellt. Der Erfolg dieser Versuche war ein negativer. Es wurde trotz reichlicher Zufuhr von Alkali keine Harnsäure aus Ammoniak gebildet. Man darf wohl aus dem Ergebnis des erwähnten Versuches schließen, daß die Milchsäure in irgend einem Zusammenhang mit der Harnsäurebildung steht; und ferner stützen diese Untersuchungen die Ansicht, daß die lieber bei manchen Tieren für die Harnsäurebildung aus Aminosäuren unentbehrlich ist. Von dieser Grundlage aus sind die folgenden Versuche von großer Wichtigkeit. Es wurde milchsaures Amnion durch die Leber hindurchgeleitet und festgestellt, daß eine Zunahme der Harnsäure eintrat. 3) Diese Beobachtung wurde freilich bei einer Wiederholung der Versuche nicht bestätigt.) Die Versuche müssen wiederholt werden. Ferner wurde durch Fütterungsversuche gezeigt, daß bestimmte organische stickstofffreie Verbindungen die Hnrnsäurebildung beeinflussen, wenn gleichzeitig Harnstoff zugeführt wird. So erschien nach Eingabe von Milchsäure, ferner von Brenztraubensäure, Hydrakrylsäure und von Glyzerinsäure mehr Harnsäure im Kloakeninhalt. Besonders günstig wirkten die zweibasischen Säuren: Malonsäure, Tartronsäure und Mesoxalsäure. Auf Grund der Ergebnisse dieser Versuche hat Wiener^) die Vermutung ausgesprochen, daß die Harnsäurebildung im Organismus des Vogels in folgender Weise vor sich gehe. Zunächst würde aus irgend einer Quelle aus Kohlehydraten oder Aminosäuren sich Milchsäure bilden. Diese würde — — ') 2) ^) ) Arch. f. e.\perim. Path. ii. Pharm. 20. 41 (1886); 31. 214 (1893). f. pxperim. Path. n. Pharm. 24. 389 (1888). F. Kovalenskij und .S". Salaskin: Zeitsch. f. physiol. Chom. 33. 210 (1901). E. Friedmann und //. Mandel: Arch. f. experim. Palh. u. Pharm. „Schmied>iO. Minkow.Hki: V. Mach: Arch. fcfr//- Festschrift". f. 1908. H. Wiener: Hofmeister?, Beitr. physiol. Chem. 43. 497 (1905). ) 2. 42 (1902) — Vgl. dazu R. liurian: Zeitschr. Eiweißstoffe und ihre Bausteine. durch Oxydation erh.ält man aus ihr und Harnstoff Dialursäure. in Tartronsäure übergehen. zweiten Molekül Harnstoff Harnsäure: CH« I CH OH . COOH NH +30 — H, 583 Unter Wasserabspaltung gibt mit einem Diese XXVIII. Voilesuug. 584 Wir kennen nun eine ganze Anzahl von Verbindungen, die zur Synthese von Harnsäure geeignet sein könnten. Es sei daran erinnert, daß beim Abbau des Traubenzuckers die Verbindungen Methylglyoxal, Benz trau bensäure, Milchsäure und Glyzerinaldehyd als Zwischenprodukte in Frage kommen. 1) Ferner sei darauf hingewiesen, daß die gleichen Verbindungen und vor allem Methylglyoxal, Brenztraubensäure und Milchsäure auch beim Abbau von Aminosäuren entstehen können. Endlich bestehen noch Beziehungen zwischen Glyzerin. Glyzerinaldehyd und den genannten Verbindungen. Es unterliegt keinem Zweifel mehr, daß Verbindungen der Dreikohlenstoffreihe im Mittelpunkt des Zellstoffwechsels stehen und in diesen der Kohlehydrat-, Fett- und Eiweiß- bzw. Aminosäurestoffwechsel sich gegenseitig ineinander Es ist mögUch, daß Milchsäure das einzige Ausgangsmaterial verlieren. Ebenso gut können jedoch auch die anderen der Harnsäurebildung ist. Verbindungen in Betracht kommen. Vor allem vermuten wir, daß Brenztraubensäure und Methylglyoxal direkt zur Harnsäurebildung herangezogen werden können: NH. C <p^ ^^ CO + C.OH4-NH0S. 4- 3 — 4H, = CO >C0 ;C0 II NH CH2 NHo/' NH2 Harn- Methyl- Harnstoff NH. C I II I (jQ NH/ C Harnsäure. • Stoff glyoxal Wir wollen nicht unerwähnt lassen, daß die Synthese der Harnsäure auch in einer Weise erfolgen könnte, die der Bildung von Methyl im idazol aus Methylglyoxal, Ammoniak und Formaldehyd entspricht 2): Kh NH, CH, H. i OH + -F CH —h C + 3H2O NH\ iCH CHo CH NH3 Formaldehyd Methylglyoxal NX Methylimidazol. Man erkennt ohne weiteres die nahen Beziehungen dieser Verbindung der Harnsäure, doch ist es aus verschiedenen Gründen fraglich, ob der tierische Organismus diesen Weg einschlägt. Vor allem ist noch nicht Da jedoch noch kein Weg erwiesen, daß er über Formaldehyd verfügt. der Harnsäurebildung durch eindeutige Versuche festgelegt ist, so muß man nach allen möglichen Richtungen Umschau halten. zu Wir werden noch einmal auf die Harnsäure treffen, wenn wir ihre Bildung aus Purinbasen besprechen werden. Dort werden wir genauer auf ihre physikalischen Eigenschaften und vor allem auf ihre LöslichkeitsverHier sei nur erwähnt, daß sie schon im Jahre 1776 hältnisse eingehen. ») ) Vgl. S. 137. Vgl. hiezu A. Ber. d. Deutschen Chem. Gesells-cli. ÜH. 392 (190.0). Vgl. auch S. 137. Windaus und F. Knoop : 1166 (190Ö); Hofmeisters Beitr. 6. Kiweißstotte und ihre Bausteine. 585 \oü Scheele^) und von Bergmann'^) im Harn und in Blasensteinen aufgefunden worden ist. Pearson^) erkannte, daß sich in Gichtknoten HarnEndlich haben Fourcroy und Vauquelhi) festgestellt, daß säure findet. William die Exkremente der Vögel zum großen Teil solche enthalten. Prout^) erhob den gleichen Befund für den Kloakeninhalt der Boa constrictor. Die Harnsäure ist auf verschiedenem Wege synthetisch dargestellt worden. Es seien hier jene Synthesen erwähnt, die auch bei der biologischen Bildung dieser Verbindung aus einfacheren Bausteinen in Betracht kommen und uns zugleich einen Einblick in ihre Konstitution ergeben. Dem Aufbau der Harnsäure gingen zunächst Studien über ihre Abbauprodukte voraus. Wähler und Liebig ^) erhielten bei der Einwirkung von Salpetersäure auf Harnsäure Harnstoff und Alloxan: NH— CO NH— CO CO C— NH - XXVIII. Vorlesung. 586 Wasser in Oxalur säure über.i) Läßt man das Alkali länger wirken, dann entstehen unter Aufnahme eines weiteren Moleküls Wasser Oxalsäure und Harnstoff. Man kann die Parabansäure auf Grund der Ergebnisse dieser Abbauversuche als Oxalylharnstoff auffassen: NH— CO NH -CO .NH, I CO I I C— NH^ + 2 H, -j- + CO C0< NH 4- CO, ^NH, >co I II NH C-NH^ CO Parabansäure Harnsäure NH NH CO Harnstoff. CO I + CO NH H, O CO V NH COOH NHä CO Oxalursäure. Parabansäure CO NH« COOH I + i H,0 + CO COOH NH, Oxalursäure Die Struktur der I COOH I NH^ Parabansäure Harn- Oxal- stoff säure. ist auch durch die Synthese aus Oxalsäure und Harnstoff erhärtet worden: COOH NH, NH CO I I + 2H,0 CO + CO I NH, Harn- COOH NH Oxal- stoff säure Parabansäure r= CO Oxalylharnstoff. Aus Alloxan erhält man durch Oxydation NH CO CO :o NH CO Alloxan NH + ebenfalls Parabansäure: CO + CO NH CO, CO Parabansäure. Somit haben wir von der Harnsäure eine ganze Reihe von Abbaustufen kennen gelernt, die durchlaufen werden, wenn diese oxydiert wird. ') F. Wöhler und J. Liebig: lAehign Anualen. 26. 287 (1H38). Eiweißstoffe und ihre Bausteine. 587 Dem AUoxan folgt die Parabansäure. Diese geht in Oxalursäure über, und schließlich verbleiben Harnstoff und Oxalsäure. Wird die Oxydation bei alkalischer Reaktion, z. B. mit Bleisuperoxyd oder Kaliumpermanganat vollzogen, dann erhält man Allantoin 'i: NH— CH— NH NH-CO I I I I C— NH. CO + HsO- + CO COa CO I ,C0 I II NH— C— NH^ 1 NH— i I NH I OH Harnsäure Ferner sjiure in Allantoin. Einwirkung von Wasserstoffsuperoxyd auf HarnLösung Tetrakar bonimid erhalten worden); bei der ist alkalischer NH— CO I CO I C— NH — COj > CO II I \ CO + 3 I CO NH—C— NH^ NH— CO— NH Harnsäure Tetrakarbonimid. Dieses in NH-CO— NH I zerfällt dann weiter Karbonyldiharnstoff und in dieser Harnstoffs): NH— CO— NH NH, NH., — COj I NH— CO— NH CO > CO NH-CO— NH Tetrakarbonimid Karbonyldiharnstoff CO CO + Hj ^NH. + H0 — CO, ^ I CO NH,;, Moleküle Harnstoff. i. 2 Die Feststellung, daß Harnsäure durch Oxydation tion in Alloxan und weist im ersteren Falle bei saurer Reak- Allantoin übergeführt auf das Vorhandensein des Atomkomplexes bei alkalischer in wird, — •) Vgl. hiozu u.a. Claus: Ber. d. Deutschen Ghcm. Gesellsch. 7. 226 (1874). E. Sundtvick: Zeitschr. f. physiol. Chem. 41. 343 (1904). lt. Behrend: Liehig% Annalen. 333. 144 (1904); 365. 21 (1909). Vgl. auch Vorlesung XXXII. A'. — — — M. Scholtz: Ber. d. Deutschen Chem. Gesellsch. 34. 4130 (1901). Vgl. auch K. H. Walters und Louis E. M'ise: Jouru. Americ. Chem. Soc. 39. 2472 (1917). -') A. Schittenhelm uud'A'. Wiener: Zeitschr. f. physiol. Chem. 62. 100 (1909). auch Ohta Kohshi: Biochem. Zeitschr. 54. 439 (1913). •') Vgl. — XXVlll. Vorlesung. 533 N— in der Harnsänre hin. l>ie (" I I 1 i Bildung des Allantoins (.'— Ns C (^— Nschließen: läl,»t auf die (iruppierung- Eiweißstort'o iiiid ihre Bausteine. 589 NH— CO CH, I I CH NH CO . . . " . Wir C— NH. >0 CO NH — C = NH— C— NH^ Diuraminopropionsäuie Harnsäure. I NHa i CO NH., II 1 . diese Formeln nur deshalb einander gegenüber, weil erfahrungsgemäß mit derjenigen der Harnsäure und der Purine überhaupt keine besondere Vorstellung verknüpft wird. Es wird der Purinkern als gegebene Formel aufgefaßt und von ihm alle übrigen Verbindungen abgeleitet. Die mitgeteilten Ergebnisse der Abbauversuche und die erwähnten Beziehungen sollen zeigen, daß die Purine nicht für sich bestehen, sondern mit bekannten Verbindungen innig verknüpft sind. Strecker'^) beobachtete ferner die Bildung von Glykokoll, Kohlensäure und Ammoniak, als er Harnsäure im eingeschlossenen Rohr mit konzentrierter Salzsäure auf 170° erhitzte: stellen NH-CO I CO I 1 C—NHv -f- f) H., II = CH2 (NHa) COOH + 3 CO, + 3 NH^ . • >^o " " - NH— C— NH^ Harnsäure Strecker GlykokoH. betrachtete die Harnsäure entsprechend diesem Zerfall als Zyansäure gepaartes Glykokoll. Er stellte sich vor, daß die Harnsäure zunächst in Glykokoll und Zyansäure zerfalle und letztere <lann in Kohlensäure und Ammoniak: ein mit C5 H, N, O3 + 2 H2 = CH., (NH.,) COOH . -\- 3 CONH. Dieser Abbau der Harnsäure hat deshalb ein ganz besonderes Interesse erregt, weil, auf dieser Beobachtung fußend, Horhaczewski^) Harnsäure durch Zusammenschmelzen von Glykokoll und Harnstoff bei 220 230" C dargestellt hat: bis /NH. 3 CO + CH2 (NH,) COOH = ('5 H, N, 03 + 3 NH3 + 2 H.3 0. . \NH2 Beim Erhitzen des Harnstoffs entweicht Ammoniak, und es bildet sich Zyansaure, die dann auf das Glykokoll einwirken kann. Eine w^eitere Synthese gelang durch Zusammenschmelzen von Harnstoff mit Trichlormilchsäureamid»): NH2 CO NH, CO NH, . NH-CO XXVIII. Vorlesung. 590 Die Resultate der Abbaustudien und der Synthese der Harnsäure vermochten kein klares Bild der Struktur der Harnsäure zu geben. Ks blieb Emil Fischers'^) genialen, umfassenden Untersuchungen vorbehalten, nicht nur die Konstitution dieser Verbindung, sondern zugleich die der ganzen Puringruppe aufzuklären. Wir werden bei der Besprechung der Bildung der Harnsäure aus Purinbasen erkennen, welch nahe Beziehungen zwischen diesen Verbindungen bestehen. davon Zum Nachweis der Harnsäure dienen Farbreaktionen. Die bekannteste ist die sogen. Murexid probe. Wird Harnsäure mit verdünnter Sal- petersäure oder mit Chlorwasser eingedampft, dann verbleibt ein gelb bis rot gefärbter Rückstand. Gibt man zu diesem Ammoniak, dann tritt eine prachtvolle purpurrote Farbe auf. Auf Zusatz von Alkalilauge zum er- wähnten Verdampfungsrückstand erhält man eine blauviolette Färbung. Der Verlauf dieser Reaktion ist der folgende. Es entsteht aus der Harnsäure durch Oxydation Alloxantin. Dieses kann man sich aus Dialursäure und Alloxan entstanden denken: NH— CO NH— CO CO— NH CO— NH l/OH NH— CO CO— NH — CO ^H/, .1 NH— CO CO— NH Dialursäure Alloxan Alloxantin. I ! CO i I CH.OH + CO CO 1 I i -> i CO 1 I I 7C C^-^^^^^ i Läßt man Ammoniak auf Alloxantin einwirken, dann erhält mau Purpursäure und aus dieser das Ammoniumsalz, Murexid genannt): NH— CO CO—NH — l/OH CO + NH, 7C OH/, CO—NH NH— CO I CO I I i C^^^ II NH -CO —> J l/NH C^ ^C \ CO , , NH— CO Alloxantin CO— NH I C0 + 2H, O ^ I CO— NH Purpursäure. Neben den stickstoffhaltigen Verbindungen finden wir bei allen Tierarten noch solche, die Schwefel besitzen und auch zum p]iweißstoff Wechsel in Beziehung stehen. Alle Eiweißstoffe, die als Nahrungsstoffe eine Rolle spielen, enthalten Schwefel. Er gehört zum größten Teil — vielleicht bei manchen Proteinen sogar ausschließlich dem Zystin an. Diese Aminosäure enthält den Schwefel in Form einer Thiogruppe. Wir haben bereits festgestellt, daß ein Teil dieser Aminosäure zur Bildung von Taurin verwendet wird. Dieses wird mit Cholsäure bzw. Desoxycholsäure gekuppelt. Der Rest wird weiter abgebaut. Dabei wird unter normalen Verhältnissen der Schwefel zum weitaus größten Teil oxydiert. Es ist noch nicht festgestellt, ob die Thiogruppe stets im Zystinmolekül selbst oxydiert wird, und dann erst die Spaltung des Moleküls einsetzt, oder ob sie zunächst in Freiheit gesetzt und gleichzeitig oxydiert wird. Es ist wohl möglich, daß die Taurinbildung uns den Weg der weiteren Veränderung des gesamten Zystins — ') (). Emil Fischer: Untersucluingeu etc. 1. c. S. 585. Zitat -). 26. 254, 2()7, und F. Wähler: Liebig^ Annaleu, FiloUj und K. Finckh: p:benda. 333. 22 (1904). ) J. Liebig 319 (1838). Eiweißstoife und ihre Bausteiue. 591 Im Harn erscheint der Schwefel zum größten Teil in 1^'orm von Auch dann, wenn man Zystin i) verfüttert oder Polypep- zeigt. Schwefelsäure. tide«), an deren Aufbau diese Aminosäure beteiligt ist, man im findet Harn den weitaus größten Teil des zugeführten Schwefels Schwefelsäure wieder. in Form von Die Schwefelsäure tritt im Harn in zwei Formen auf. Wir können sie erkennen, indem wir den Urin z. B. mit Jiaryumhydroxyd verEs fällt sofort ein weißer Niederschlag aus. Er besteht aus setzen. Baryumsulfat. Schließlich bleibt auch bei weiterem Zusatz von Baryumhydroxyd jede Fällung aus. Nun filtrieren wir ab und versetzen das Filtrat mit Salzsäure und kochen. War ein Überschuß an Baryum zugegen, dann tritt schon nach kurzer Zeit erneut eine Fällung von Baryumsulfat auf. Wir vervollständigen sie durch weiteren Zusatz von Baryumhvdroxyd. leicht Die zuerst gefallene Schwefelsäure entspricht der freien Schwefelsäure bzw. der Sulfatschwefelsäure. Die zweite Fällung ist durch die Ätherschwefelsäure bedingt. Sie ist durch das Kochen mit Salzsäure aus ihrer Verbindung mit Kresol, Phenol oder Indoxyl abgespalten worden. Die Menge dieser Art von Schwefelsäure ist unter normalen Verhältnissen gering. Sie steigt, wie wir früher schon gesehen haben »), an, wenn im viele zur Kuppelung mit Schwefelsäure geeignete Produkte gebildet Darm werden. Untersucht man den Harn, nachdem man die beiden Formen von Schwefelsäure aus ihm entfernt hat, auf Schwefel, dann findet man noch solchen. Folgüch muß der Harn noch andere schwefelhaltige Verbindungen außer der Sulfat- und der Ätherschwefelsäure enthalten. Zu dem gleichen Resultate gelangt man, wenn man den Harn mit einem Oxydationsmittel behandelt und dadurch den gesamten vorhandenen Schwefel in Schwefelsäure überführt und nunmehr den Gehalt an Gesamtschwefel bestimmt. Wird in einer zweiten Portion des gleichen Harnes diejenige Schwefelmenge festgestellt, die der Summe der Sulfat- und der Ätherschwefelsäure entspricht, dann bleibt sie hinter der Menge des Gesamtschwefels zurück. Dieser „Fehlbetrag", der jenem Schwefel entspricht, der nicht bis zur Schwefelsäure oxydiert worden ist, ist noch nicht in allen Teilen aufgeklärt. Salkowski) hat diesen Schvvefel als „neutralen" Schwefel bezeichnet. Wir werden später erfahren, daß im Urin stets kleine Mengen von Substanzen vorkommen, die noch höher molekulare Eiweißabkömmlinge unbekannter Natur darstellen. Sie können Schwefel enthalten. Ferner kommen im Harn auch einfache Verbindungen vor, die reduzierten Schwefel aufweisen. Die Behauptung, daß im normalen Urin immer Zystiu enthalten sei, ist unbewiesen. Dagegen findet man im Harn immer geringe Mengen von Rhodan Wasserstoff, HGNS. Wir werden auf seine Herkunft noch zurückkommen. Bei manchen Tieren, z. B. bei Katzen und Hunden, findet man ') Vgl. z. muth: Zeitschr. ) ") ) B. r. n. Rothera: Jonrn. of Pbysiol. 32. 175 physiol. Chemie. 46. 81 (1907). (1905). — J. Wohlgt- f. tJmil Abderhalden und Franz Samuely : Ebenda. 46. 187 (1905). Vgl. S. 513 ff. K. Salkowski: Vircho%f>% Archiv. 58. 172 (1873). XXVIII. Vorlesung. f)92 im Harn auch Thiosulfate.i) Im Urin des Menschen fehlen sie. Erwähnt sei noch, daß im Hundeharn eine Verbindung der folgenden Zusammensetzung beobachtet worden ist 2): C2 Hb\ /CH3 Diäthyl-methyl-sulfiniumbase. Sie ist ohne Zweifel auf Zystin zurückzuführen, das offenbar zunächst Diäthylsulfid liefert. Dieses wird dann methyliert. Endlich wollen wir noch erwähnen, daß ein Teil der im Harn auf- tretenden Phosphorsäure auf Eiweißstoffe zurückzuführen ist, die, wie z.B. das Kaseinogen. Phosphor enthalten. Eine andere Form der Ausscheidung des Phosphors ist nicht bekannt. Wir hätten nunmehr alle Elemente des Eiweißes in bestimmten Stoffwechselendprodukten wieder erkannt. Es fehlen nur noch zwei wesentEndprodukte des Eiweißstoffwechsels, die dieser mit demjenigen der Kohlehydrate und Fette gemein hat. nämlich Kohlensäure und Wasser. Es sind dies die einzigen p]ndprodukte des Eiweiß- bzw. Aminosäurestoffwechsels, die den Organismus durch die Lungen verlassen. Zum Schlüsse wollen wir noch die Frage streifen, ob beim Abbau der Proteine und Aminosäuren in den Zellen die gleichen Endprodukte entstehen, wie wenn Eiweiß außerhalb des Organismus verbrannt wird. Es ist dies nicht der Fall. Bei der Verbrennung von Eiweiß oder Aminosäuren entstehen unter geeigneten Bedingungen Stickstoff. Kohlensäure und W^asser. Nur die beiden letzteren Verbindungen bilden sich auch im Organismus. Dagegen wird der Stickstoff stets zum größten Teil mit Kohlen.stoff, Wasser- und Sauerstoff zusammen ausgeschieden. Würde man den Harnstoff bzw. die Harnsäure im Reagenzglas verbrennen, dann würden Kohlensäure, Wasser und Stickstoff entstehen. Gleichzeitig kann man feststellen, daß Energie frei wird. Daraus folgt, daß der tierische Organismus nicht, wie es bei den Kohlehydraten und Fetten der Fall ist, den gesamten Energieinhalt des zugeführten Eiweißes ausnützt. Stets geht ein Teil davon je nach der Tierart in Form von Harnstoff bzw. von Harnsäure für die Zellen verloren. Wir werden später auf diese wichtige Tatsache noch zurückkommen. liche ') f. ration. 496 (1904). ^) — Schmiedeberg : Archiv der Heilkunde. 8. 422 (1867). G. Meissner: Zeitschr. Med. 31. (3). 323 (1868). - J. Wohlgeimith: Zeitschr. f. physiol. Chemie. 43. 0. C. — L. Blum: Hofmeistern Beiträge.' Neuberg und F.' Grosser: Zentralbl. 5. f. 1 (1904). Physiol. 19. 316 (1905). Vorlesung XXIX. Eiweißstott'e und ihre Bausteine. Der Abbau der Aminosäuren im Zellstoffwechsel. Neubildung von Aminosäuren. Wir haben die wesentliciisten Stoffwechseleudprodukte des EiweiÜ- Es sind dies von stickstoffhaltigen Produkten beim Menschen, den Säugetieren, den xlmphibien und Fischen der HarnITei den Vögeln, Reptilien und einigen stoff und ferner Ammoniak. Amphibien die Harnsäure, von schwefelhaltigen die beiden Formen der Schwefelsäure und der neutrale Schwefel und von phosphorhaltigen die Phosphorsäure. Ferner sind Kohlensäure und Wasser Stoffwechselendprodukte des Eiweiß- bzw. Aminosäurestoffwechsels. Unsere nächste Aufgabe ist nun die, festzustellen, welche Verbindungen zwischen den Aminosäuren und diesen tiefsten Abbaustufen stehen. Wir haben dieses Forschungsgebiet schon wiederholt betreten. Einmal beschäftigten wir uns mit Verbindungen, die beim stufenweisen Abbau von Aminosäuren entstehen, als wir der Tatsache gedachten, daß manche von ihnen Material zur Bildung von Zucker liefern. Ferner haben wir testgestellt, daß die Mikroorganismen aus Aminosäuren teils stickstoffhaltige, teils stickstofffreie Abbaustufen bilden. Auch in der Pflanze vollzieht sich ohne Zweifel fortwährend ein Abbau von Aminosäuren. Es finden sich keine durchgreifenden Unterschiede in den Stoffwechselvorgängen der Pflanzen und Tiere, wenn man von der Tatsache der Kohlenstoff-, Wasser- und Stickstoffassimilation durch die chlorophyllhaltigen Pflanzen absieht. Wir werden deshalb bei der Besprechung des Verhaltens der Aminosäuren im Zellstoffwechsel des tierischen Organismus auf manchen bereits bekannten \'organg und viele schon besprochene Verbindungen stoffwechsels kennen gelernt. stoßen. Mit der Besprechung der Abbaustufen der Aminosäuren betreten wir iebiet. Es ist mit ganz verschiedenen Methoden durchforscht worden. Einmal begegnen wir dem Stoffwechselversuch am ganzen Tiere. Es werden bestimmte \erbindungen verfüttert. Im Harn wird dann nach Produkten gefahndet, die direkte oder indirekte Beziehungen zu der eingeführten Substanz zeigen. Oder es wird einem bestimmten Organe die Frage vorgelegt, ob und ein außerordentlich interessantes und erfolgreich bearbeitetes Abderhalden, Physiologiäctia Chemie. I. Teil, ö. AuÜ. ( 38 XXIX. Voilesimg. 594: was es mit einer bestimmten Verbindung ;inzutangen Beispiel P]in weili. möge diesen Gang der Erforschung der Art des Abbaues bestimmter Aminosäuren erläutern. Wir haben bereits festgestellt, daß aus den Aminosäuren die NIL-Gruppe auf verschiedene Arten entfernt werden kann. Lange Zeit schien die hydrolytische Desaminierung der gegebene Weg des Abbaues von Aminosäuren zu sein. Dabei müssen zunächst Oxysäuren entstehen. p-Oxy Phenylalanin müüte bei dieser Art der Desaminierung p- Xyp hen y OH . 1 m Co H, NH, GH., ("H . i 1 c hs äu re 1i e fe r n OH COOH + H, . C, H, . —> — NH3 GH2 GH . Die Desaminierung unter gleichzeitiger Oxydation . ((JH) G( t( )H. . führt zu Keton- säuren. Im vorliegendem Fall würde man p-( x y p h e n y b r e n z tr au b e n s ä u r e ) 1 zu erwarten haben. NH3 OH . Gg H, . GH., . GH . G0( )H + () — XH^ — y )H G^ H, GH., GO G( ( . . . . )( )H. Welche von beiden N'erbindungen entsteht direkt aus Tyrosin'.- Durchinsbesondere von Leber - und auch Fütteblutung von Organen rungsversuche ergaben, daß Tyrosin und p-Oxyphenylbrenztraubensäure in gleicher Weise abgebaut werden, während die p-Oxyphenylmi Ichsäure sich als schwer angreifbar erwies.^) Dieses P^rgebnis macht es im höchsten Grade wahrscheinlich, daß die Desaminierung von Aminosäuren sich in der Hauptsache durch Oxydation unter Bildung von Ke ton säuren vollzieht. Dabei ist nicht ausgeschlossen, daß auch andere Arten des Abbaues sich finden. Ferner können sekundär aus Ketonsäuren durch Reduktion sich Oxysäuien bilden, doch tritt dieser Vorgang nach allen Erfahrungen höchstens in geringem Umfange ein. — Ein anderes Beispiel zur Entscheidung des Problems des Abbaues bestimmter Aminosäuren in bestimmten Organen haben wir schon früher besprochen. P]s ist dies die Frage nach der Bildung von Azetessigsäure Leuzin und I so val er i anbzw. Azeton aus einzelnen Aminosäuren. säure ergaben diese Verbindung, als sie durch die Leber hindurchgeleitet wurden. Dagegen gingen Val in und Lsobutter säure nicht in Azetessigsäure über. Aus diesem Ergebnis wurde der Schluß gezogen, daß der Abbau dieser Aminosäuren zunächst zur Abspaltung der Amino- und der Karboxylgruppe führt.-) Es entsteht aus Leuzin Isovaleriansäure und aus \'alin Isobuttersäure. Diese Fettsäuren werden nun in der früher besprochenen Weise unter [i-Oxydation und paarweiser Abspaltung von Kohlenstoff weiter fühlt der Abbau der Isovaleriansäure zu Azeton, Isobutt er säure diese Verbindung nicht liefern kann.-) /u dem gleichen Ilesultat gelangt man, wenn man annimmt, daß aus der entstandenen, um ein Kohlenstoff ärmeren Fettsäure [i-Oxybutter säure Dabei zerlegt. =5) während die hervorgeht Kotake: /eitschr. f. physiol. Chemie. 69.>()9 (l'.UO). 1) y. ') Über den Mechanismus dieser Al)spaltung vgl. weiter unten. =•) ) Vgl. S. 282 ff. Vgl. hirrzu S 198. XXIX. Vorlesiiug 596 bauen vermochte. ^j Offenbar verfügt der Organismus über mehr als einen Weg zum Abbau aromatischer Verbindungen. Es sei gleich erwähnt, daß man gegen die mit den erwähnten \'ersuchsanordnungen erhaltenen Ergebnisse Einwände erheben kann. Gegen den Fütterungsversuch läßt sich einwerfen, daß die verabreichten Substanzen bereits im Darmkanal eine Veränderung erleiden können. Die Darmflora kann sich ihrer bemächtigen und sie umwandeln. Ferner ist es leicht möglich, daß im Organismus verschiedene Organe zusammenwirken, bis aus einer Aminosäure schließlich bestimmte Abbaustufen gebildet sind. Führen wir dem Organismus eine bestimmte ^'erbindung zu, so gelangt diese vielleicht nicht zu jenen Zellen, die sonst den typischen Abbau durchführen. Es kann auch sein, daß die gebildeten Abbaustufen von sich aus bestimmte Reaktionen auslösen und dadurch den weiteren Abbau regeln. Das fällt vielleicht weg, wenn wir eine bestimmte Abbaustute auf einmal in größeren Mengen verVeränderung der verfütterten Substanzen im abreichen. Der erste Einwand Darmkanal läßt sich durch die subkutane oder intravaskuläre Verabrei<'hung der Verbindungen ausschließen. Die übrigen Bedenken bleiben bestehen. Die Versuche am überlebenden Organ verlaufen sicher nicht unter normalen Verhältnissen. Einmal führen wir eine bestimmte Verbindung, die sonst vielleicht nur in Spuren entsteht, um sofort weiter verändert oder fortgeführt zu werden, in größerer Menge zu. Ferner werden die entstandenen Abbaustufen nicht fortgeschafft. Sie werden immer wieder an den gleichen Zellen vorbeigeführt. Vielleicht kommt es durch Anreicherung einer bestimmten Abbaustufe zur Hemmung des weiteren Abbaues. Es würde unter diesen Umständen ein Stoffwechselzwischenprodukt zur Beobachtung kommen, das uns sonst entgeht. In diesem Falle würde die Abweichung von den normalen Vorgängen nur in einem Aufhalten des weiteren Abbaues bestehen. Das erhaltene Produkt wäre dagegen ein auch sonst auftretendes. Es könnte jedoch auch sein, daß der Abbau bestimmter Verbindungen in nicht normale Bahnen gedrängt würde. Vor allem könnte man bei Verbindungen, die nicht angegriffen werden, den Einwand erheben, daß es vielleicht zu einem Abbau gekommen wäre, wenn man nicht die Mitwirkung anderer Organe ausgeschlossen hätte. Die \'ersuche an einem mit Alkaptonurie behafteten Individuum sind auch nicht ohne weiteres verwertbar. Zunächst muß die Frage entschieden werden, ob die Bildung der Homogentisinsäure eine normale Stufe im Abbau der homozyklischen Aminosäuren darstellt, oder ob die Anomalie im Abbau des Phenylalanins und Tyrosins nicht vielmehr darin zu suchen ist. daß es zur Bildung der Dioxyphenylessigsäure kommt. Es wäre ja denkbar, daß eine Verbindung gebildet wird, die nur deshalb nicht weiter abgebaut wird, weil sie anormal und damit den Zellen ungewohnt ist. In diesem Falle würde den Versuchen über die \'erwertung einzelner aromatischer \'erbindungen durch den Homogentisinsäure bildenden Organismus nur eine besondere und keine allgemeine Bedeutung zukommen. Es ist immer noch nicht mit voller Schärfe entschieden, welche Stellung die Homogentisinsäure als Abbaustufe der homozyklischen Aminosäuren einnimmt. Es spricht jedoch sehr vieles dafür, daß sie ein auch im normalen Organismus auftretendes Abbauprodukt dieser Bausteine der Proteine darstellt. Abge.sehen von der Übereinstimmung der Resultate der am nor- — ') — Konrad Fromherz und Leo Iferrmannfi: Zeitschr. f. phys. Chemie 91. 194 (1914). Eiweißstoffe und ihre Bausteine. 597 malen Individuum, am überlebenden Organe und an dem mit Alkaptonurie Behafteten durchgeführten Untersuchungen über die Verwertung bestimmter aromatischer Abbaustufen des Tyrosins und Phenylalanins spricht auch dafür, daß es gelungen ist, nach \'erfütterung sehr großer Mengen von Tyrosin an ein normales Individuum Homogentisinsäure im Harn nachzuweisen. ^) Man könnte freilich daran denken, daß die beobachtete, gewissermaßen erzwungene Homogentisinsäurebildung einen Nebenweg im x\bbau des Tyrosins darstellt, doch wäre eine solche Annahme gesucht. Wir heben die Schwierigkeiten in der Deutung der einzelnen Ergebnisse der verschiedenartigen Anordnungen der Versuche absichtlich hervor, weil sie im allgemeinen viel zu wenig berücksichtigt werden. Erst dann, wenn auf verschiedenen Wegen der gleiche Befund erhoben werden kann, dürfen wir ihn als gesichert betrachten und auf die normale Zelltatigkeit übertragen. Daß z. B. der Versuch am überlebenden (Jrgan unter anderen Bedingungen vor sich geht, als im nicht isolierten Organ, beweist schon der Umstand, daß die im Oganismus befindliche Leber beständig unveränderte Aminosäuren hindurch läßt. Es wäre gewiß verkehrt, würde man, befangen durch das Ergebnis, daß z. B. die überlebende Leber Leuzin, das durch sie hindurchgeleitet wird, in Azetessigsäure bzw. Azeton überführt, schließen, daß dieses Organ auch im Organismus diesen Umbau vollzieht und kein Leuzin unverändert vorbei läßt. Die be.ständig ..normale" Leber wird bald Aminosäuren in ihre Zellen aufnehmen, bald und \'erhältnisse sie vorbeiziehen lassen, je nachdem die Bedingungen im einzelnen Ealle liegen. Das aus dem Zusammenhang mit anderen Organen herausgelüste Gewebe wird manche wichtige Regulation missen und ganz unter dem Einfluß der künstlich geschaffeneu Bedingungen stehen. Das ist für das Suchen nach bestimmten Abbaustuten von großem Vorteil, denn, wenn die Zellen, wie unter normalen ^'erhältnissen, jede Zwischenstufe nur in Spuren bilden würden, um sie auch sofort wieder weiter zu zerlegen, so würden wir nur sehr schwer zu bestimmten Resultaten gelangen. Es unterliegt keinem Zweifel, daß der Abbau der einzelnen Aminosäuren auf verschiedene Weisen erfolgen kann. Wir wollen die einzelneu Möglichkeiten betrachten und feststellen, welche Beweise für jeden einzelnen Weg vorliegen. Zunächst kann der Abbau mit \'erlust des Karbox vis einsetzen. Es entsteht ein Amin. Dieses kann, wie Fütterungs- und Durchblutungsversuche ergeben haben ^j, desaminiert und durch Oxydation in eine Säure übergeführt werden. Es sei diese Art des Abbaus am Beispiel dos Tvrosins erörtert NH NH., —^ OH C.ll^ CH^ OH C«H, CH2 ("H COOH -CO., p-Oxyphenylp-Oxyphenyl-a-aminoproprionäthylamin säure = Tyrosin > OH CeH, CH., COOH p-Oxyphenylessigsäure. . . . . — . . . . . GH., — NH, + 20 . Emil Abdirhaldcn: Zeitschr. i. physiol. Chemie. 77. 454 (1912). Otto Neuhauer: Über deu K. Sjriro: Hofmeistern Beiträge. 10. 277 (1907). Abbau der Aminosäuren im gesunden und kranken Organismus. Habilitationsschrift. A.J.Eirins und P. /'. Lr/jV//o/r.- Journ. of. Physiol. 41. F. C. W. Vogel. Leipzig 1908. 78 (1910). Vgl. auch S. 524. ') ) — XXIX. Vorlesung. Ö9S Die Überführung in die p-Oxyphenylessigsäure kann über ZwischenEs kann die Desaminierung durch Hydrolyse herbeigeführt werden, i) Es entsteht ein Alkohol. Dieser kann zum x-Mdehyd oxydiert werden und dieser endlich zur 8äuie: stufen erfolgen. NH, OH an, CH, CH, + H,()— NH3 = OH C,H, GH., GH., OH p-Oxyphenyl-äthylamin p-Oxyphenyl-äthyl. . . . . alkohol OH . G,H4 . GH., . GH2 . OH + O — H^O =: OH p-Oxyphenyl-äthylalkohol OH . C\ H, . GH, . G<;\|^ . . . = Tyrosol. GgH, GH^ . C(^ . p-Oxyphenyl-azetaldehyd. + O = OH p-Oxyphenyl-azetaldehyd . G^ H, . GH., . GOOH p-Oxyphenyl essigsaure. Es kann jedoch die Abspaltung der NHo-Gruppe auch durch Oxydation erfolgen. p]s würde in diesem Falle zunächst ein Aldehyd entstehen, der dann durch Reduktion in Alkohol übergehen könnte-): R . GH., . NH2 + O = R cS, + NH3. -0 . ,/H R GQ; + 2H = R . . GH., . OH. Alle Aminosäuren, die sich als am VKohlenstot'fatom substituiertes Alanin auffassen lassen, können genau die gleiche Art des Abbaus zeigen. So ist es z.B. möglich, daß Histidin = '/-Amin(»-;i-imidazolyl-propioii- säureüberlmidazolyl-äthylamin. Imidazolyl-äthylalkohol und Imidazolyl-azetaldehyd in Imidazoiyl-essigsäure übergeht, und ferner Tryptophan = 3c-Amino-(i-indolpropionsäure zunächst Indol-äthylamin, dann Indol-äthylalkohol, ferner Indol-azetaldehyd und schließlich Indolessigsäure liefert. Auch die übrigen Aminosäuren schlagen vielleicht beim Abbau zum Teil den gleichen Weg ein. Jedenfalls weiden auch aliphatische Amine weiter abgebaut. So liefert z. B. das dem Leuzin entsprechende Isoamylamin in der künstlich durchbluteten Leber Azetessigsäure. ^) Wir kennen eine Aminosäure, bei der die erste der genannten Abbaustufen im Stoffwechsel in Erscheinung tritt, es ist dies das Zystin bzw. Zystein. Es liefert Taurin. das als eine Äthylainin-sulfonsäure aufgefaßt werden kann: GHo SH GH.," . —> I GH NH, . . SH + 3 I GH., . NH., GH., — . ' .OH SO-, ' ! >- GH.," . NH, " " " I GOOH Zystein ') -) •') GO, Thioäthylamin Äthylamin-sulfonsäure=:Taurin. Vgl. die Arbeiteu von Feli.r Ehrlich: S. 458 tt. Vgl. Carl Neuberg imd U. Steenbock: Biochem. Zcitschi. 52. F. Sachs: Biochem. Zeitschr. 27. 27 (1910). 494 (1913) Eiweißstoffe uuil ihre Baiisteiue. 599 Schließlich sei noch daran eriimert. dali vielleicht die bei einigen Fällen von Zystinurie beobachteten Diamine Kadaverin undPutreszin ebenfalls /Aun Teil wenigstens in den (Teweben entstehen, und ihr Auftreten vielleicht auf eine Hemmung des weiteren Abbaus zurückzuführen ist. In diesem Zusammenhange sei auch der Beobachtung gedacht, daß im Sekret der Speicheldrüsen der Kephalopoden p-Oxyphenylathylamin enthalten Es entsteht ohne Zweifel in den Zellen dieser Drüsen aus Tyrosin. Für den genannten Weg des Abbaus von Aminosäuren sprechen somit mehrere Momente. Einmal die Fütterungs- und Durchblutungsversuche und dann die Feststellung von Taurin. Oregen die Annahme, daß diese Art der Zerlegung von Aminosäuren und insbesondere der aromatischen eine umfassende ist, spricht der Umstand, daß Abbaustufen entstehen, die ohne Zweifel schwer angreifbar sind. Eine weitere Art des Abbaus von Aminosäuren ist die folgende. Es wird primär die Aminogruppe unter Bildung einer Oxygruppe abgespalten. Wir können diese Art dei- Desaminierung als hydrolytische bezeichnen. I)ie folgende allgemeine Formel gibt diese Art des Abbaus wieder: ist.i) R CH . . C( )()H — NH3 = R CH COOK + Ho . . - i I OH NH.3 Früher glaubte man, daß diese Art dei- Desaminierung die gewöhnliche sei. Neuere Beobachtungen haben gezeigt, daß sie zwar eintreten kann, dalj dieser Weg des Abbaus von Aminosäure jedoch nicht häufig eingeschlagen wird. Die folgenden Beispiele zeigen, daß eine hydiolytische Desaminierung vorkommt- Es muß allerdings gleich bemerkt werden, daß stets dei' Einwand bestehen bleibt, daß die Bildung der Oxysäuren auch eine sekundäre sein kann. Es könnten z.B. zuerst durch oxydative Desaminierung Ketonsäuren sich bilden und diese dann sekundär reduziert werden. Es liegen folgende Beobachtungen vor. Nach Verfütterung von viel Alanin tritt im Harn Milchsäure auf.-) Ferner hat man nach Eingabe von '/-, ß-Diaminopropionsäure einer Aminosäure, die nicht unter den Bausteinen der Proteine sich findet im Harn Glyzerinsäure aufgefunden.') Endlich beobachtet man nach reichlicher Fütterung von Tyrosin l-p-()xyphenyl-milchsäure im Urin. — CH3 — CH, CH.XH2 -f H2O — NH3 —> CH.OH COOK COOK Alanin Milchsäure CHh CH,, — KH, — > CO CH, I I CH . NH.. + I oder + 2H — > CH OH . I COOK COOH COOK Alanin Brenztraubensäure Milchsäure. ') 3/. Herne: Zeitschr. f. pbysiol. Chemie. 87. 51 Carl Neuberg uud Leo Langsteiii (1913). Archiv f.(Anat. u.) Physiol Suppl. r)14(1913). ') Faul Mayer: Zeitschr. f. physiol. Chemie. 42. ö9 (1904). ) : XXIX. VorlesuDg. g()(( (Ho . CH, NHa . < >H 2NH3=r CH .OH CH .NH, + 2H,U I i C'OOH COOH Diaminopro- (xlyzerin- pionsäiire säure Der Abbau dieser Verbindung- könnte auch sich in den folgenden bluten vollziehen: CH., . NU., CH .NH. ' ("H., + O^NH, . NH, +2H —> — > CO I I COOH COOH lJiaminciJ)ro- Aminobreuztraubensäure pionsäure CH., . V^H NH., CH .OH + o NH, — CH.OH COOH (U)OH Aniinoglyzeiinsäure (ilyzerin- aldehvd OH CH., —y + L> H = >. CH . OH I COOH Glyzerinsäure. Selbstverständlich sind auch noch andere Möglichkeiten des Abiiaus Die hier angeführten Wege sollen nur zeigen, dall die Verhältnisse nicht so einfach zu liegen brauchen, wie die Annahme einer gegeben. Abspaltung beider Aminogruppen unter Wasseraufnahme sie zunächst darstellt. der Bildung der 1-p-Oxyphenyl-iniUhsäure aus Tyrosin Sie entsteht ohne Zweifel Verhältnisse wesentlich klarer. i) primär durch hydrolytische Desaminierung. Dieser Schluß ergibt sich aus der Beobachtung, daß nach Eingabe von p-Oxyphenyl-brenztraubensäure der Mensch d-p-Oxyphenyl-milchsäure ausscheidet.-) Würde somit die Bildung von p-Oxyphenyl-milchsäure aus Tyrosin durch sekundäre Bei die liegen Reduktion von primär entstandener Ketonsäure sich vollziehen, dann wäre Wir können somit ihre Bildung d-p-Oxyphenylmilchsäure zu erwarten. durch die folgende Formel wiedergeben ) Y. Kotake: Zeitschr. f. pliysiol. Chemie. 65. 3U7 (1910). munn: Ebenda. 6. 2.S4 (1882). — Feiner )'. Kotake: Ebenda. K.rromhfrz: Ebenda. 70. .351 (1911). 2) A. Suua: Zeitschr. f. physiol. Chemie. 72. 113 (1911). - Vfrl. auch Bh'wler«9. 409 (1910». — Kiweißstofie und ihre Bausteine. CHo Cg H, ' . . OH CH2 + H. O— NH3 I CH.NH. " (301 " — I . Cfi y . OH CH.OH . ! COOK H, I COOH p-Oxyphenyl-a-aminopropionsäure p-Oxyphenylmilchsäure. Erwähüt sei noch, daß die Bildung der 1-p-Oxyphenyl-milchsiiure bis jetzt nur unter nicht normalen Verhältnissen festgestellt werden konnte. So fand man sie nach Phosphorvergiftung M und bei der akuten gelben Leberatrophie.2) Ferner ist sie nach reichlicher Zufuhr von Tyrosin aufworden.3) Es ist möglich, daß eine Abbaustufe des Tyrosins vorliegt, die nicht zu den normalen gehört. Dafür spricht die schwere Angreifbarkeit dieser Verbindung. Es wäre jedoch auch denkbar, daß sie stets in kleineren Mengen entsteht und somit die vermehrte Bildung der p-Oxyphenyl-milchsäure keine neue Art des Abbaus, sondern nur ein in gefunden größerem Umfange eingeschlagener Nebenweg im Abbau Tyrosins des bedeuten würde. Wir kommen nun zu jener Art des Abbaus von Aminosäuren, die am eingehendsten studiert worden ist, nämlich zur oxydativen Desaminierung. Sie führt zur Bildung von Ketonsäuren: R.CH.COOH + O — NH, Als Zwischenprodukt säure auftreten: —^ R. CO. COOH. dürfte das Hydrat der entsprechenden Imino- OH K GH COOH + . . —y R C COOH — XH. —> R CO COOH. . . I . . i NH, NH2 Die Kenntnis dieses Weges des Abbaus der Aminosäuren verdanken Er zeigte, daß nach Eingabe bestimmter, unter den Eiweißspaltprodukten nicht vorkommender Aminosäuren im Harn die entsprechenden Ketonsäuren anzutreffen sind. So ergab Phenylaminoessigwir Otto XcNbauerJ) säure Phenylglyoxylsäure.^) Aus p-Oxyphenylaminoessigsäure entstand p-Oxyphenyl-glyoxylsjä ure, m-Tyrosin ergab m-Oxyphenyl- brenztrauben säure: Y. Kotake: Zeitschr. f. physiol. Chemie. 69. 414 (1910). Schulfzen nnd Ries.'i : Chai'itö-Anualeu. 15. 1 (1869). A'. haninaiiii: Zeitschr. f. physiol. Chemie. 6. 192 (1882). ') Blemtermann: Zeitschr. f. physiol. Chemie. Vgl. auch //. L). 6. 234 (1882). Dahin: The Journ. of biol. Chemie. 8. 25 (1910). ) Otto Neubauer: Deutsches Archiv f. klin. .Med. Vgl. auch 95. 211 (1909). Haiidb. d. biochem. Arbeitsmethoden. 4. 371 (1911) (bearbeitet von Otto Nenbauer). .1. Springer. Berlin 1911. H. D. Dakin: Oxydations and Reductions in the aniraal body. Longmans, Green & Cie. London 1912. Hier rindet sich die vollständige Literatur. ') Vgl. O110 Xcuhancr und Han.^ Fisrßicr: Zoitsclir. f. phvsidj. Chemie. 67, 232 ') — ) — — — (1910). XXIX. Vorlesung. 602 NH, an, .cH.cooH Phenylamino essigsaure OH . H, l'e . CH COOH . > ('. H, CO COOH . . Phenyl-glyoxvisäure OH . Ce H, . p-Oxyphenyl-glyoxyl- säure säure. CH HC^\c OH HC^\c OH . HC - . CH., . NH, iCH C t CO COOH p-Oxyphenyl-amino-essig- CH Me a . . HC. CH COOH . X y p h e n y 1 - 7. - a m n p r 0i pionsäure = m-Ty rosin Als Stütze für "CH C CH., CO COOH m-Oxyphenyl-brenz. . . traubensilure. Annahme, daß die Ke tonsäuren ein die typi- sches Abbauprodukt der Aminosäuren darsteUen. läßt sich anführen, daß bei reichlicher Verfütterung von Tyrosin p-Oxyphenyl -brenztraubensäure im Harn angetroffen wird, und ferner die Ketonsäuren ebenso leicht und in gleicherweise abgebaut werden, wie die entsprechenden Aminosäuren selbst. i) Auch die Durchblutung.'5versuche ergaben, daß die letzteren und die zugehörigen Ketonsäuren zu den gleichen Verbindungen führen. So liefert Tyrosin in der künsthch durchbluteten Leber Azeton. 2) Das gleiche Kesultat liefert p-Oxy Phenylbrenztraubensäure, während die Alkoholsäure, p-Oxyphenylmilchsäure, fast gar nicht angegriffen wird. Daß jedoch keine allgemeine Regel der Art besteht, daß Alkoholsinnen nicht abgebaut werden können, beweist, daß Phenylalanin. Phenyl-brenz- raubensäure Phenyl-milchsäure in vielen Beziehungen ein Tyrosin undPhenylalanin gehen bei den mit Alkaptonurie Behafteten in Homogentisinsäure über. Auch p-Oxyphenylbrenztraubensäure und Phenyl-brenztraubensäure liefern Dioxyphenyl-essigsäure, während p-Oxyphenyl-milchsäure zu keiner t und gleiches Verhalten zeigen. ^i Vermehrung der letzteren Verbindung führt.) Dagegen liefert Phenylmilchsäure Homogentisinsäure.^) Es fragt sich nun, wie der weitere Abbau der gebildeten Ketonsäuren vor sich geht. Es sind zahlreiche Beobachtungen bekannt geworden, die zeigen, daß beim Abbau von Aminosäuren Fettsäuren — Yushiro Kotakc : Zeitschr. f. physiol. Chem. 69. 409 (191U). A. Sinra: 113 (1911). F. Knoop: Hofmeistern Beitr. 6. 150 (1905): Zeitschr. f. physiol. Chemie. 67. 49;? (1910). -) 0. Neubauer und \V. Gross: Zeitschr. f. phvsiol. Chemie. 67. 219 (1910) /;. Sehmifz: Biochem. Zeitschr. 28. 117 (1910). ^) Vergl. auch Y. Mori ZA. physiol. Chemie 122. 22;! (1922). ) 0. Neubauer: Deutsches Arch. f. klin. Med. 95, 211 (1909). ') 0. Neubauer und [V. Folta : Zeitschr. f. physiol. Chem. 42. Hl (1904). ') Ebenda. \g\. 72. — — : und Kiweiüstoffe ihre Bausteine. ()03 eutsteheu, die um ein Kohlenstoffatom ärmer als diese sind. So geht z. B. l'hcnylaminoessigsäure zum Teil in Benzoesäure über i): NH, Cg H, ("H . . —> GOCH H5 C'e t'OOH. . Besonders interessant ist die Beobachtung, daß Y-Phenyl--/-aminohuttersäure ebenfalls Benzoesäure liefert."^) Der Übergang in diese ist allerdings bei weitem kein (juantitativer NH., —> I Ce H, CR, . CH., . . CH COOK . G, H5 COOK. . Als erste Abbaiistufe dieser Verbindung wäre die Benzyl-brenztraubensäure zu erwarten: NHo — CH ;C0( )H + () — NH. I C, H5 . CH., . CH., . >- C, H, . CH, . CH., . CO CU( )H. . Diese Verbindung wird nun offenbar zunächst unter Abspaltung von Kohlensäure in die um ein Kohlenstoffatom ärmere Fettsäure übergeführt. Dann setzt die bei den Fettsäuren übliche Art des Abbaus der Kohlenstoffkette ein. Unter Oxydation am ß-Kohlenstoffatom erfolgt die paarweise Abspaltung von Kohlenstoffatomen. Beim Abbau der Ketonsäure zu der um ein Kohlenstoffatom ärmeren Fettsäure dürften Zwischenprodukte auftreten. Es entsteht wahrscheinlich zuerst ein Aldehyd »), der dann zur Säure oxydiert wird. Der Abbau der y-Phenyl-y.-aminobuttersäure würde somit etwa folgende Abbaustufen durchlaufen NH-, C„ H5 . CH., . CH., . + CH COOH . ( y-Phenyl-a-aminobutt er säure NH., I C, Hg . CR, . CR, . C . COOH — NH3 OH Hvdrat der "-Pheuvl-x- aminobuttersäure I yr C, H, . CR, . CR CO COOH —CO., . . Benz vl-brenztrauben säure I C« H5 . CH, . CR C<jj + O . Benzyl-azetaldehyd - Vgl. weitere BefNeubaticr: Deutsches Arch. f. klin. Med. 95. 233 (1909). L. FJatoiv: Zeitschr. spielo: L. Blnm: Aldi. f. rxperim. Path. u. Phariu. 59. 290 (1908). Maas-e: Biochem. Zeitschr. 27. E. Friedmann uud 3(57 (1910). f. pliysiol. Chemie. 64. H. D. Dakin: The .[ourn. of biol. Chem. 8. 11 (1910). 97 (1910). /'. Knoop uml KrnM ') F. Knoop: Zeitschr. f. physiol. Chemie. 67. 4S9 (1910). Ifntess: Ebenda. 71. 252 (1911). ') Vgl. Carl Neuberg und //. Steenhock: Biochem. Zeitschr. 52. 494 (1913). ) 0. — — - C — XXIX. Vorlesung. ß()4 + CeH5.CH2.CHo.COOH « ß Benz vl-essigsäure=:Phenvlpropion säure Ce H5 . CH (OH) . Ca COOK + . ß Phen vi-,b-oxy Propionsäure Ce H5 . CO CR, COOH . . + H2 Benz oyl- essigsaure Ce H5 . COOH Benzoesäure — H2 Eiweißstoft'e und ihre Bausteine. 605 CH CH NH, JCH C . CHo . CH COOH + . HC CH HC CH — NHj Phenylalanin =:Phenyl-a- aminopropionsäure C CH2 . CO COOH . . Phenyl-brenztraubensäure^) C.OH HC^NcH HC^ /C . CH, COOH . C.OH Homogentisinsäure = 4-Dioxyphenyl-essigsäure = Hydrochinon1, essigsäure. C .OH C .OH HC>^NCH HC^^CH HC NH, CH C . CH., . C^H /CH HC<\ . COOH + — NH3 Tyrosin = p-Oxyphenyl-a-amino-propionsäure C . CH, . CO . COOH p-()xyphenyl-brenztraubensäure. Es folgt somit, der Abbau der Seitenkette der genannten aromatischen Aminosäuren der oben angeführten Regel. Zu erklären bleibt nur noch die Veränderung im Benzolkern. Beim Tyrosin muß die in Parastellung befindliche OH-Gruppe verschwinden. Ferner müssen in Orthound MetaStellung neue Hydroxylgruppen eintreten. Beim Phenylalanin müssen letztere ebenfalls gebildet werden. Es ist zurzeit nicht möglich, anzugeben, wo die Umwandlung im Benzolkern sich vollzieht. Dagegen kann in Übereinstimmung mit ähnlichen Vorgängen die folgende Annahme als wahrscheinlicher Weg des Umbaus des Phenol- bzw. Benzolkerns in den Hydrochinonkern bezeichnet werden, p - K r e s 1 liefert bei der Oxydation mit Caros Reagens 2) Toluhydrochinon. Dabei tritt als Zwischenstufe p-Toluchinol auf^): •) Nach B. Hemmerle' [Auü. Chim. (9). 7. 226(1917)] hat die Fheuvl-breiiztraubeuCH C (OH) COOH. eäure die Endstiuktur: Cg H^ . : ^) Sulfomonopersäure, H^SOj. ') Erich Mayer: Deutsches Arch. . f. klin. K — E. Bamberger Med. 70. 447 (1901). Vgl. auch 36. 2028 (1903). Ber. d. Deutschen Chem. Gesellsch. 28. 245 (1895); Friedmann: Hofmeister?, Beitr. 11. 304 (1908). — XXIX. Vorlesung 606 OH I C HC.^^CH HC CH EiweiOstoffe uulI ihre Bausteine. Der UmwaiidluDg' von Phenylalanin ß07 Homogentisinsäure geht wahrEs ist nämlich beobachtet worden, daß die genannte Aminosäure in der künstlich durchbluteten Leber in Tyrosin übergeführt wird.') Ferner konnte festgestellt werden, daß der scheinlich eine solche in Tyrosin in voraus. Organismus mit Phenylalanin allein auskommt, d. h. das Tyrosin in der Nahrung entbehren kann.') Für die Autfassung des normalen Abbaus der erwähnten aromatischen Aminosäuren ist es von größter Bedeutung, welche Stellung der Homogentisinsäure in der Pteihe der Abbaustufen des Phenylalanins und Tyrosins einzuräumen ist.-^) Ist sie das Produkt eines ..entStoffwechsels, oder stellt sie eine Abbaustufe dar, die auch sonst durchlaufen wird? Wir haben diese Frage bereits aufgeworfen und fest- gleisten" daß manche Gründe für die letztere Annahme sprechen. Wichtig Beobachtung, daß normale Individuen eingeführte Homogentisinsäure abbauen, während solche, die Alkaptonurie aufweisen, sie unverändert ausscheiden.) Ferner hefert die Hydrochinonessigsäure in der überlebenden Leber die gleichen x\bbaustufen. wie Tyrosin und Phenylalanin.'^) Endlich haben wir schon erwähnt, daß es geglückt ist, nach Zufuhr großer Mengen von Tyrosin bei einem nicht mit Alkaptonurie Behafteten Homogentisinsäure im Harn nachzuweisen. 6) Wir möchten nun gerne wissen, in welcher Weise die Homogentisinsäure im normalen Organismus weiter zerlegt wird. Zuletzt müssen Kohlensäure und Wasser aus ihr hervorgehen. Sicher reiht sich Abbaustufe an Abbaustufe, bis diese Produkte entstanden sind. Leider ist es noch nicht gelungen, den bis zur Homogentisinsäure wenigstens in den Hauptpunkten festgelegten Weg des Abbaus der homozyklischen Aminosäuren über den Benzolkern hinaus zu verfolgen. Es muß im weiteren Verlauf des Abbaus aromatischer Verbindungen zur Aufsprengung des Benzolringes kommen. Die einzige Beobachtung über diesen Vorgang verdanken wir JafeJ) Er stellte fest, daß Benzol in Mukonsäure^) übergeht: gestellt, ist die g()^ XXIX. Vorlesung. Schließlich erhält man. wie schon wiederholt erwähnt wurde, aus Phenylalanin und Tyrosiu und ihren Abbaustufen Azetonkörper. Damit anderer Abbauprodukte nicht ausgeist selbstverständlich die Bildung schlossen. Wir würden ohne Zweifel einen großen Fehler begehen, wenn wir den Versuchen am überlebenden Organ auch in quantitativer Richtung eine ausschlaggebende Bedeutung zuschreiben würden. Sie zeigen uns nur den Weg an. der beim Abbau einer bestimmten Verbindung eingeschlagen werden kann. Dabei bleibt vollständig unentschieden, ob nicht auch andere Bahnen benützt werden, um von bestimmten Abbaustufen aus Beziehungen zu Verbindungen anderer Körperklassen anzuknüpfen. Das Tryptophan dürfte, was die Seitenkette anbelangt, ebenso abgebaut werden, wie die homozyklischen Aminosäuren. Über die Veränderungen, die den Indolkern betreffen, wissen wir noch nichts Genaues. Vielleicht führt die folgende Beobachtung in dieser Beziehung weiter. Hunde scheiden nämlich eine eigenartige Säure aus, die mit dem Tryptophan in Zusammenhang steht. Es ist dies die Kynurensäure. Sie hat die Konstitution einer v-Oxy-chinolin-a-karbonsäure^): Eiweißstotte luid ihre liiuisteiue. 609 daU nur ein geringer Teil des Tryptophans in Form von Kynurensäure zur Ausscheidung gelangte, nicht geschlossen werden, dall nur dieser in das Chinolinderivat verwandelt wurde. Es liegt vielmehr die Möglichkeit vor, daß die Kynurensäure eine normale Abbaustufe des Tryptophans darstellt, die vielleicht auch bei anderen Tieren durchlaufen wird. In dieser Richtung ist die Beobachtung von Interesse, daß Kaninchen, die sonst nie Kynurensäure ausscheiden, solche im Harn enhielten, als iimen Tryptophan zugeführt wurde. Über die Bildungsstätte dieser interessanten Verbindung wissen wir noch nichts (Jenaues. Jedenfalls wird sie bei Hunden mit £'c^scher Fistel auch gebildet, ^j Die Entstehung der Kynurensäure aus Tryptophan ist noch nicht aufgeklärt. Man kann sich vorstellen, daß die Inciol-y.-amino-propiondarf daraus. säure zunächst durch oxydative Desaminienmg in Indol-brenztraubensäure übergeht. In der Tat liefert diese Verbindung Kynurensäure.^i Dann folgt nach der Ansicht von Ellinger-} die weitere Umwandlung im Sinne der folgenden Formeln: CH XH. HC^ \'- -C.CH.,.('H.(()UH + \ HC<^ I 0— NH, /CH /'^\ nh (:h Trypt ophan = Indol- '/-am inopropion säure CH CH hc^^c- C CH, CO . . . C— CO.CH.. HC COOH . CO . COOH I! tl HC HC \CH C NH CH CH NHo CH CO Indol-brenztraubensäure CH CO ./\c/\ CH., H( HC^^ rc^^CH, HC CO Cn CH . COOH NH, CH ^^S/C\/C.C(H)I1 CH N C OH . Hc^Xc/^XcH .^^....^^C.COOH CH N •Oxv-chinolin-a-karbonsäure = Kynurensäure. 'j Cheiii. 62. -) l-lniil Abderlialdeii, 139 ri909). .1. EUinffer und f.. Z. S. London Matsuoka: und Li(dint/ Pincusso/iH Zeitschr. Abderhalden, F'hysiologiBche Chemie. 1. Teil, ö. AuH. f. pliysiol. : /eitsclir. Cheni. f. pliysiol. 109. 2.i9 (1920). 39 XXIX. Vorlesuug. ßlQ In diesem Znsammenhange sei erwähnt, daß im Sekret der Analdrüse des amerikanischen Stinktieres z-Methylchinolin aufgefunden worden ist.M Es dürfte auch zum Tryptophan in Beziehung stehen: CH CH HC c CH HC C C.CH, CH N -/-Methylchinolin. Wir haben damit die wesentlichsten, bis jetzt bekannten Verbindungen kennen gelernt, die uns auf den Weg, auf dem sich der Abbau der Aminosäuren wohl in der Hauptsache vollzieht, hinweisen. Er läßt sich durch die folgenden allgemeinen Formeln wiedergeben: R CH, CH COOH . . . I NH, Aminosäure i OH i 11 . CH., . C . COOH NH, Hydrat der Iminosäure W CHo CO COOH Ketonsäure . . . I R CH, COOH . . Fettsäure ^ \ CO, H, Streng genommen ist diese Art des Abbaues von Aminosäitren nur homozyklischen Aminosäuren bewiesen, doch mehren sich immer mehr die Bewei.se dafür, daß auch die übrigen Bausteine des Eiweißes in ganz entsprechender Weise abgebaut werden. Besonders eingehend untersucht sind die Abbaustufen des Alan ins. Als solche sind Brenzt raubensäure und ferner Methylglyoxal festgestellt worden. Die Beziehungen dieser Verbindungen unter sich und ferner zur Milchsäure ergeben die folgenden Formeln 2): für die n. Aldrich und W. Jones: .loiirn. of exper. Med. 2. 439 (1897). hierzu S. 130 ff. uud H. I). iJakin und H. W. Dudley: Joarn, of biol. Chem. Vgl. auch F. A. Levene und G. M. Mei/er: The Jouru. of biol. Chem. 14. 55.0 (1913). ' ") 15. ./. V'gl. 475 (1913). — ' Kiweißstoffc und ihre Bunsteine. 61 1 CH, "-^^^3 CH, 3 +0— CH.NH, ^ "^1 +2H - Ho O <—^ CO Nil, i COOH COOH Brenztraubensäure Alanin=: a-Aininopropionsilure ^— CO + H. () CH.OH ^^H Methylglyoxal Milchsäure.^) Diese Verbindungen gehen ^Yechselseitig in einander über. Es sei daran erinnert, daß in diesen Verbindungen Abbaustufen des Traubenzuckers und solche aus Aminosäuren zusammentreffen. Es führen von ihnen aus auch Wege zur Glukose.-) Bemerken wollen wir noch, daß über den Abbau der heterozyklischen Aminosäuren noch wenig bekannt ist. Vom His tidin ist festgestellt, daß es in der durchbluteten Leber Azetessigsäure liefert. 3) Der Abbau zu dieser führt wahrscheinlich über die folgenden Zwischenstufen: CH— NH CH-NH >CH N il C CH. I . N C CH^ (^H >CH .\|\| + NHo " — NH3 CO I I COOH —CO, COOH Histidin = [i-Imidazolyl- S-Imidazolyl-brenz- a-aminopropionsäure CH—NH C— 1 >CH raubensäure CH3 NH,. • I CO > >C = + > CH, CH, I t NH./ I COOH (jX \h [i-Imidazoly 1- azetaldehyd ) Vgl. üVier die Bildung Azetessigsäure Harnstoff, von Milchsäure aus Bronztrauliensäure auch Gustav Kmbden und Max Oppenheimer Biochem. Zeitschr. 55. 33.') (1913). — : ^) Vgl. S. 137 ti'. H. D. Dakin und //. W. Dudle ti : The .Journ. of biol. Chem. 15. A. B. Ferner IL D. Dakin und .V. W. Janney; Ebenda. 15. 177 (1913). 127 (1913). Ringer: Ebenda. 15. 145 (1913). ) H. D. Dakin und A. J. Wakeman: The Journ. of biol. Chem. 10. 499 (1912). — — 39* XXIX. \ orlcsuiiij. ^^]'> Prolin liefert unter den «leichen Bedingungen keine Azetessigsäure. Dagegen liefert es Zucker, wie P'ütterungsversuche an mit Phlorhizin vergifteten Hunden ergaben. i) Bei der Verfolgung des Abbaus der verschiedensten \'erbindungeii sind wir wiederholt auf Reaktionen gestolien, die umkehrbar sind. Es sei z. B. an die Beziehungen der Aminosäuren zum Traubenzucker erinnert.-) Es unterliegt wohl keinem Zweifel, daß der Umbau der ersteren \'erbindungen in Glukose über Dreikohlenstoffverbindungen führt. Solche entstehen auch aus Glukose selbst und können in umgekehrter Richtung wieder zu Ferner sei an die wechselseitigen Beziehungen dieser aufgebaut werden. der [i-Oxybuttersäure und der Azetessigsäure erinnert. Die letztere kann aus ersterer sich bilden und umgekehrt kann die letztere in die erstere übergehen. 3) Es ist von größtem Interesse, daß Beobachtungen vorliegen, wonach Ketonsäuren in Aminosäuren verwandelt werden können. Die erste Beobachtung dieser Art stammt von Franz Knoop.} Er verfütterte Hunden Benzyl-brenztraubensäure und fand im Harn azetyP h e n y - 7. - a m i n o b u 1 1 e r s ä u r e 1 i e r t e 1 Cß H, . CH-, . GHj . CO . —^ COOH Cfi H, GH., . GH., . . GH., . GH . GOOH NH, Y-Phenyl-'/-aminobuttersäure Benzyl-brenztraubensäure bzw. C.H, . GH, . GH GOOH . NH GO CH, Y-Phen>l-'/-azet}iaminobuttersäure. . . Die Umwandlung einer Ketonsäure in die entsprechende Aminosäure dürfte wohl kaum direkt erfolgen. Es ist möglich, daß die Reaktion zwischen Ammoniak und der Ketonsäure zunächst zum Hydrat der Iminosäure führt, und diese dann durch Reduktion in die Aminosäure verwandelt wird: OH R . GH., . GO . G( nm + NH, —> —> R R . GH, . G . G( )0H + "211 — H.,( NIL . GH., . GH GOOH . NH2 Auf dem umgekehrten Wege sahen wir aus der Aminosäure das Hydrat der Iminosäure und daraus die Ketonsäure entstehen. Die Phenylaminobuttersäure ist bis jetzt in der Natur nicht aufgefunden worden. Man könnte daran denken, daß die Umwandlung der sicher dem Körper fremden P»enzyl-brenztraubensäure in die entsprechende Aminosäure einen \'organg darstellt, der etwa der Kuppelung körperfremder Substanzen ') H. I). hakin: The Jourii. of biol. Clieiii. Vi. :A'.] (11)13). Vgl. S. 172 ff. ') Vgl. S. 190. ) F. Knoop: /eitsclir. f. pliysiol. Cliomie. 67. 4S7 (l'.tlO). Kfrfe^s: Klionda. 71. 2.ö2 d'.)!!). ^) — /''. Knno/, und h'rn.it Kiwciüstoflc iiiiil ihre Baiisteiin'. (51;-^ mit Glykokoll, (ilukuronsäure usw. entspricht. \Veitere Versuche von Gustar Emhden^) zeigten jedoch, daß eine Reaktion ganz allgemeiner Natur vorliegt. Er fand, daß bei der Durchströmung der Leber mit Blut, dem Brenz- traubensäure zugesetzt worden war, sich d-Alanin bildete. Aus p-Oxyphenylbrenztraubensaure entstand 1-Tyrosin, aus Phenylbrenztraubensäure Phenylalanin, aus z-Keto-n-buttersäure a-Amino-nbuttersäure, aus a-Keto-n-kapronsäure -/-Amino-n-kapronsäure und endlich bildete sich Leuzin aus der entsprechenden Ketonsäure. Stets entstand findet. diejenige optisch-aktive Komponente, die sich in der Natur vorin Alanin übergeführt. Wahrscheinlich Auch Milchsäure wurde zunächst in Brenztraubensäure und dann in die Aminosäure über. vorliegenden Ergebnisse zeigen, daß der tierische Organismus Aminosäuren aus Ammoniak und stickstofffreien Verbindungen bilden kann. Diese müssen in a-Oxy- bzw. a-Ketonsäuren überführbar sein. \'on diesen Gesichtspunkten aus steht der Bildung bestimmter Aminosäuren aus Kohlehydraten nichts im Wege.^) Die wechselseitigen Beziehungen zwischen Kohlehydraten und Aminosäuren ergibt die folgende, die wesentlichsten Glieder der Umwandlung darstellende Übersicht: geht sie Die Ce H,., Oe Glukose t XXIX. Vorlesuii':. (314 Diese FeststeUungeu führen unmittelbar zu der Fragestellung, ob im Zellstoffwechsel unter normalen Verhältnissen eine praktisch in Betracht kommende Bildung von Aminosäuren aus stickstofffreien Verbindungen erfolgt. Diese Frage ist schwer zu entscheiden. Es ist sehr wohl mögUch, daß bei Mangel einzelner Aminosäuren solche Dagegen kann die Frage, ob alle Aminosäuren gebildet werden. aus Abbaustufen der Kohlehydrate und eventuell der Bausteine der Fette hervorgehen können, sicher verneint werden. Eine Bildung von Eiweiß aus Bausteinen der genannten Nahrungsstoffe und aus Ammoniak ließ sich durch direkte Stoffwechselversuche nicht erweisen. i) Ferner sei daran erinnert, daß gewisse Aminosäuren, wie z. B. Tryptophan und Tyrosin bzw. Phenylalanin nicht ersetzbar sind.^) Falls eine Synthese von Aminosäuren aus Kohlehydraten und eventuell aus Bausteinen der Fette erfolgt, dann ist diese ohne Zweifel auf einige wenige Eiweißbausteine beschränkt. Es wird stets darauf ankommen, ob die Zelle aus dem ihr zugeführten Materiale a-Ketonsäuren bereiten kann. Nun kennen wir kein aus Kohlehydraten bzw. Fetten stammendes Baumaterial, das aromatische Ketonsäuren liefern könnte. Es würden somit nie alle zur Eiweißsynthese notwendigen Bausteine sich von den stickstofffreien Nahrungsstoffen aus bilden können. Würde man jedoch den Zellen jene Aminosäuren, die sie nicht bereiten können, zur Verfügung stellen, dann wäre es denkbar, daß sie die übrigen Bausteine selbst bilden und dann zur Eiweißsynthese schreiten können. Die Synthese von Aminosäuren aus a-Ketonsäuren irgendwelcher Herkunft könnte dann von Bedeutung werden, wenn dem Organismus kein Eiweiß bzw. keine Aminosäuren zugeführt werden. Es wäre möglich, daß er unter diesen Verhältnissen entweder die unersetzlichen Aminosäuren vor dem Abbau bewahren bzw. schon in Ketonsäuren verwandelte Bausteine durch Aminierung wieder in die entsprechende Aminosäure zurückbilden würde. 3) Nun scheinen jedoch gerade die aromatischen Aminosäuren im tierischen Organismus eine besondere Rolle als Ausgangsmaterial zur Bildung von Inkretstoffen zu spielen. Wir werden gleich erfahren, daß das Adrenalin unzweifelhaft Beziehungen zum Tyrosin bzw. zum Phenylalanin besitzt. Ferner dient das Tryptophan wahrscheinlich zum Aufbau von Hämatin, dem eisenhaltigen Paarling des Blutfarbstoffs. Es sei auch noch daran erinnert, daß aus aromatischen Bausteinen abstammende Amine in Organen angetroffen worden sind. Wahrscheinlich stellen sie Verbindungen dar, die im Zellstoffwechsel unentbehrlich sind. Wenn somit die einzelne Zelle Proteine abbaut, so wird sie in vielen Fällen die Hydrolyse einleiten, um Baumaterial zur Bildung von InkretDie zur Bildung von Eiweiß unentbehrlichen Baustoffen zu gewinnen. ') Vgl. die Arbeiten von AA Grate und Mitarbeiteru und Kmil Abderhalden mit Ernst PeschecJc: Biochem. Zeitschr. faul Hirsch uud Arno Ed. Lampe ^. 495, Zitat')Alonzo Engelbert Taylor und .1. ./. Ringer: Journ. of biol. Chemie, 43. 244 (1912). Frank P. Underhül 14. 407 (1913). Frank P. Underhill: Ebenda. 15. H27, 337 (1913). und Samuel Goldschmidt : Ebenda. 15. 341 (1913). \'gl. auch Emil Abderhalden uud Einil Abderhalden, Joseph Markwalder : Zeitschr. f. physiol. Chemie. 72. 63 (1911). Alberto Furno, Erich Goebel und Panl Sfriihel: Ebenda. 74. 481 (1911). -) Vgl. S. 499 tf. Vgl. hierzu S. 499 ft. und Z-,'?«?'/ Abderhalden : Zeitschr. f. physiol. Chemie. 96. — — — '') 1 (1915). — — Eiweiüstotte und ihre Bausteiuc. 615 werden anderweitig verbraucht, so daß der Organismus wolil kaum den günstigen Fall kommen wird, Eiweiß aus Ketonsäuren aufzubauen, die aus stickstofffreien Nahrungsstoffen stammen. Die Synthese würde immer am Fehlen der aromatischen Bausteine scheitern. Es soll damit weder etwas über die Bedeutung der beobachteten Bildung von Aminosäuren aus Ketonsäuren für den Zellstoffwechsel ausgesagt werden, noch über den Umfang derartiger Vorgänge. Unsere Kenntnisse über die Bedeutung der einzelnen Aminosäuren für den Zellstoffwechsel sind noch viel zu dürftige, als daß wir jetzt schon zu einem bestimmten Schlüsse kommen könnten. Wir sind der Ansicht, daß die Aminosäuren im tierischen Organismus keinesw^egs nur als Bausteine der Proteine in Betracht kommen, sondern vielmehr als solche und in ihren Umwandlungsprodukten von größter Bedeutung für einzelne Zellfunktionen sind. Zum Schlüsse sei noch der sicher festgestellten Bildung von GlykokoU im Zellstoffwechsel gedacht. Wir haben bereits erfahren, daß seine Menge in gewissen Gremzen beliebig gesteigert werden kann, indem wir dem Organismus Substanzen zuführen, die entweder direkt /ur Verbindung mit Aminoessigsäure geeignet sind oder von den Zellen leicht in die dazu notwendige Form gebracht werden können. Die Beobachtung, daß der tierische Organismus mehr GlykokoU ausscheiden kann, als er in den ihm zur Verfügung stehenden Proteinen besitzt, läßt keine andere Deutung zu, als daß eine Neubildung dieser Aminosäure im Zellstoffwechsel erfolgt. Es fragt sich nun, ob sie synthetisch oder analytisch gebildet wird. Diese Frage ist noch unentschieden. Der Aminoessigsäure entspricht als Produkt der oxydativen Desaminierung die Glyoxylsäure. Es wäre möglich, daß diese mit Ammoniak zusammen GlykokoU Uefert. Vorläufig ließ sich diese Annahme nicht durch den direkten Versuch erhärten'): steine je in —> CH.-COOH + NH3 — H.CO. COOK CH2.COOH NH., NH.> GlykokoU =; GlykokoU. Gl'yoxylsäure Aminoessigsäure Es wäre ferner möglich, daß der Abbau der Aminosäuren je nach Bedarf so geleitet werden kann, daß GlykokoU entsteht. Man könnte z. B. an eine ß-Oxydation unter paarweiser Abspaltung von Kohlenstoffatomen denken. Die Aminosäure würde mit dem Karboxyl das Kohlenstoffatom verlieren, das die Aminogruppe trägt CH3 CH. CH, CHo . . . ; . CH COOH + H., O . — ^5 I NH. a-Aminokap ronsäure ; CH, . Ca CH, CH, OH + . . . CH., . C( )0H NH., Butylalkohol >) GlykokoU. Georg Haas: Biochein. Zeitschr. 46. 296 (1902j. >- XXIX. Vorlesuug. glg Ferner hat man daran gedacht, daß die zu kuppelnde Verbindung, B. Benzoesäure, sich zunächst mit der Aminogruppe irgend einer Aminosäure verbinden und darauf ihr Abbau erfolgen könnte^): z. CH, . CH, . CH, CHo . . CH COOH + H. () . NH.CO.CeH, Benzoy 1-a- am inokap ronsäure CH3 . CH2 . CH.. . CHo . OH + CH., COOH . I NH.CO.CßH, Butylalkohol stützt kolls Hippursäure. Keine dieser Ansichten konnte jedoch bis jetzt durch \'ersuche gewerden. Die Frage nach der Herkunft des größten Teils des Glykoist somit eine noch offene. .Nur für einen Teil davon kommen glyko- kollhaltige Proteine in Betracht. Schließüch sei daran erinnert, daß Stoffwechselversuche ergeben daß Phenylalanin Ty rosin und die letztere Aminosäure die Ferner scheint Prolin neu gebildet werden zu erstere vertreten kann. können. Es entsteht wahrscheinlich über die a-Pyrrolidonkarbonsäure aus Glutaminsäure. PJs scheint auch, daß die verschiedenen Glieder habend), der Sechskohlenstoffreihe ^). soweit sie Monoaminosäuren sind, für einander eintreten können, doch muß man mit allen Schlußfolgerungen auf diesem (iebiete sehr vorsichtig sein, weil nur sehr langfristige Versuche zu endgültigen Schlüssen berechtigen. Daß Phenylalanin in tierischen Geweben Es in Tyrosin übergeführt werden kann, haben wir bereits erwähnt.^) handelt sich somit bei dem Ersatz der erwähnten Eiweilibausteine höchstwahrscheinlich nicht um umfassendere Synthesen, sondern um eine mehr oder weniger direkte Umwandlung bereits vorhandener Aminosäuren. •) ^) ') ) A. Magmis-Levy: Biochem. Zeitschr. 6. 523, 541 (1907). Vgl. S'. 499 tf. Vgl. Emil Al,derhal(len : I'/fügers Archiv. 195. 199 (1922). Vgl. S. 499 ff. Vorlesung XXX. und ihre Bausteine. EiweilJstoffe 14. Verwendung von Aminosäuren zu Synthesen im Zellstoffweclisel. Ilire Beziehungen zu den Kohlehydraten und den Bausteinen der Fette und der Phosphatide. Stoffwechselprodul<te, die in direkten Beziehungen zu bestimmten Aminosäuren stehen. Eiweißabl<ömmlinge zusammengesetzter Natur, die in ihrem Aufbau mit i<einer der bel<annten Eiweißabbaustufen übereinstimmen. Bei der Besprechung- der Bildung von Aminosäuren im Zellstoffweehsel sind wir mehriaeh auf Verbindungen gestoßen, die aus solchen und einer zweiten Verbindung bestehen. Es liandelte sich stets um eine Synthese zwischen einer bestimmten Aminosäure und einem zweiten Produkte. Wir wollen diese Verbindungen im Zusammenhange aufführen und gleichzeitig der Frage nachgehen, ob von solchen Produkten oder den ihrer Entstehung entsprechenden Vorgängen aus sich Beziehungen zu Verbin- dungen anderer Körperklassen anbahnen lassen. Zunächst sei der Synthesen gedacht, an denen Gly kokoll beteiligt ist. Beständig erscheinen in der (Jalle Säuren, die einerseits aus Chol säure bzw. Desoxych Ölsäure und andrerseits aus Gly kokoll bestehen. Es sind dies die Glykochol- und Glykodesoxycholsäure, besser Cholylglyzin und Desoxycholyl-glyzin genannt. i) Wir können die Struktur des Cholyl-glyzins durch die folgende Formel zur Darstellung bringen: r,8H3„( );, . CO NH . . CH., . COOH -f H., = C^sHsgOs COOH + . ('holsäure Gholyl-glyzin NH., . GH., . COOH Glykokoll. Cholyl-glyzin liefert unter Wasseraufnahme seine beiden Anteile. Glykokoll wird auch zur Synthese von Hip pursäure verwendet. Ferner sind Verbindungen mit zahlreichen anderen aromatischen Säuren ') Vgl. liiorzii Vorlesung XII, S. 230tt. XXX. Vorlesimg. (ns bekannt. Es sei erinnert z. au die PhenazetursJuire=:Pheuylazetyl-glyzin B. die Paarung der Aniinoessigsäure mit körperfremden Ver- und an bindungen. \) Beim Vogel wird das Glykokoll bei derartigen Synthesen durch das dem Ar ginin entstammende Ornithin ersetzt. Das erstere wird offenbar zunächst durch Arginase in Harnstoff und in Diaminovaleriansäure == Ornithin zerlegt. Dann folgt die Synthese mit aromatischen Säuren. Die Diaminosäure kann zwei Säurereste mit ihren beiden Aminogruppen binden. Bis jetzt ist die Bildung des Dibenzoyl-ornithius Ornithursäure und ferner diejenige von Diphenylazetyl-ornithin beobachtet worden. Die letztere Verbindung wurde nach Verfiitterung von Phenylessigsäure im Organismus des Huhns gebildet-): = CH, . HOOC.CH. .C«H-, + NH-, GH., —> GH., GH NH, HOOG + . . GH., . C, U, GOGH Ornithin GH2 . 2 NH OG . Moleküle Phenylessigsäure . GH., . G0H5 + H.,0 <^gH, + ILO GH, GH., GH .NH.OG.GH., COOH Diphenylazetyl-ornithin. Weitere Verbindungen, die in ihrer Struktur den Glykokoll enthaltenden Gallensäuren entsprechen, stellen die Taurochol- bzw. Taurodesoxycholsäure dar. An Stelle des GlykokoUs findet sich das Taurin als Paarhng. Seine Entstehung' aus Zystin bzw. Zystein haben wir bereits besprochen. =) Eine weitere Synthese stellt die Bildung von Merkaptursäuren nach Eingabe von Halogenbenzol dar. Mit diesem vereinigt sich Zystein. Gleichzeitig erfolgt eine weitere Synthese, nämlich die Verknüpfung dei' Azetylgruppe mit der Aminogruppe des Zysteins. Es treten somit drei Verbindungen zusammen. Die Azetylierung stellt einen ganz entsprechenden Vorgang dar, wie die eben erwähnte Benzoylierung des GlykokoUs und des Ornithins. W'^ir haben einen weiteren Fall von Azetylierung bei der Be519 ff. ') Vgl. S. 514, -) G. Totani: Zeitselir. •') \''_'l <^ .",1«. f. plivsioi. (Jheni. 08. 75 (1910). Eiweißstoffe uud ihre Hausteine. Ö19 sprechuug der Umwandlung- der Phenyl-a.-ketobuttersiiure =^ Benzyl- brenztraubensäure in Pheny)-a-aminobuttersäure kennen gelernt') Diese Aminosäure erschien nicht in freiem Zustand im Harn, sondern Azetylverbindung. Endlich ist beobachtet worden, daß Phenylaminoessigsilure beim Durchleiten durch die Leber azetyliert wird.^j Besonders interessant wegen der eigenartigen Verwendung der Essigsäure ist die Bildung der Furfuracryisäure nach Verfütterung von als Furfurol an Hunde und Kaninchen ^j: HC —CH HC HC C.C<!! ^H + CHs.COOH — CH ii C CH = CH ii HC . . COOH + H. ü. O Es ist noch unentschieden, welche Bedeutung der Einführung der Azetylgruppe zukommt. Auch wissen wir nichts Sicheres darüber, auf w^elche Art sie gebildet wird. Man vermutet, daß der Brenztraubensäure bei der Azetylierung und gleichzeitigen Aminierung von Ketonsäuren eine Bedeutung zukommt ; R CH. CO COOH . . . XXX. Vdrlesnug. 520 Stoffwechsel entstanden ist, oder sich sekundär bei der Darstellung der Aminosäuren aus harnstoffhaltigen Flüssigkeiten gebildet hat. Die einzige Beobachtung, die für das primäre Vorkommen von Uraminosäuren spricht, ist die Uraminoverbindung des Phenylalanins, die direkt aus dem Harn herauskristallisiert.^) Schliei.'tlich noch der Karbaminosäuren Kohlensäure und Aminosäuren. auch sei bilden sich leicht aus gedacht. Es ist Sie noch im Zellstoffwechsel eine Rolle nicht festgestellt, ob derartige Verbindungen spielen. 2) seien die bis jetzt besprochenen Synthesen an je einem Beispiel gemeinsam, daß die Aminosäuren mit der Aminogruppe in Reaktion treten und sich an dieser Es Sie haben übersichtlich zusammengestellt. das alle Rest anlagert. Stelle ein Anlagerung des Kohlensäurerestes (Bildung von Karbaminosäuren): —y R CH, CH COOH . . . R V\U CH COOH . . /OU I NH.COOH + Cf=() NH.. + H, 0. . I \0H Anlagerung des Kssigsäurerestes (Azetylierung): -^ R CH, CH COOH . . . R CH. CH COOH . + H., O. . . I I NH OC NH, + HOOC CH3 . . . CH3 Anlagerung des Benzoesäurerestes (Benzoylierung): — R CH, CH C0( )H . R CH, CH COOH >- . . '1 NH2 + HOOC . . . '1 NH C, H, + H., . . 0(' . ( ). Ce H^ Anlagerung des Phenylessigsäurerestes (Phenylazetylierung): — R CH2 CH COOH . R CH2 CH COOH >- . . NH, + H( )0C CH, . . NH OC Cg H, + H, . . . . . CH., . ( ). C, H, Anlagerung des Harnstoffrestes (Bildung von Uraminosäuren): —> R CH, CH COOH . . . R CH, CH COOH . . . + NH,. ' 1 I NH, + NH., CO NH2 . NH CO . . . NH., In diesem Zusammenhange sei auch der Methylierung im tierischen Organismus gedacht, obwohl bis jetzt eine solche von Aminosäuren nicht durch die direkte Beobachtung festgestellt werden konnte. Es wäre trotzdem möglich, daß der Stickstoff der Aminogruppe mit Methylgruppen beI)iese könnten z. B. von intermediär entstandenem setzt werden kann. hakin: .loiini. of liiol. Clioni. ») //. -) Vgl. S. 346. />. 6. 2:-jr) (1909). — Vgl. auch S. Mb, 57.S. Eiweißstotte ihre Baiisteiuo. utiil 621 Methylalkohol herstammen. Die Annahme, dali im tierischen Organismus Methvlierung von Aminosäuren vorkommen kann, ist deshalb gerechtfertigt, weil er. wie früher erwähnt worden ist.i) in manchen Fällen die Bausteine der Phosphatide selbst bilden kann. Zu diesen gehört auch das Cholin. Dieses kann durch vollständige Methvlierung von Glykokoll und nachträgliche Reduktion des entstandenen Betains gebildet w-erden. Erwähnt sei, daß auch die Möglichkeit einer Abstammung des Cholins vom Serin besteht. 2) Dieses könnte zunächst durch Verlust von Kohlensäure in Oxyäthylamin übergehen. Dieses mül'tte dann methyliert werden. Die folgenden Formeln zeigen die beiden, für den tierischen Organismus in Betracht kommenden Bildungsweisen des Cholins aus Aminosäuren, wobei immer wieder des Umstandes zu gedenken ist, daß jeder einzelne Vorgang umkehrbar sein kann, und so die einzelnen Verbindungen wechselseitig mit einander verknüpft sind. CH, .COOH CHa.C'OOH A"H3 I + HO.CH3 NHo HO H( ) . . C^H.. — 2 H. O />rH... N .\CH3 CH, + 411 — Ho M)H <ilyko- H Moleküle Methyl- koll alkohol Betain CH.2.Cll2.()H /CH3 I ^CH3 ^^XH3 I H)H Cholin. CH, .OH CHo . ( )H I CH . CHo XHo XH., CH. . OH /CH3 . cH..xy;{l; ! —CO, COOH ^OH Serin=a-Amino- Oxyäthyl- [i-oxy-propion säure amin Cholin. Organismus imstande ist. Methylgruppen an beweisen die folgenden Feststellungen. Tellurige und seien ige Säure gehen in Tellur- bzw. Selenmethyl über.^j Ferner wird verfüttertes Pyridin in Form von Methylpyridylammoniumhydroxyd im Harn ausgeschieden): Daß der tierische Stickstoff anzulagern, ') (1894). Vgl. S. 225. ^) Vgl. S. 25(). ') Franz Hofmeister: Archiv f. experim. Patli. u. Pharm. 33. 198 (1894) ) Wilhelm His : Archiv f. — liudolf Colin: Zcitschr. experim. Path. u. Pharm. 22. 253 (1887): f. physiol. Chemie. 18. 112 (1894). 33. 198 XXX. Vorlesung. (522 CH CH HC CH HC HC CH HC N Pvridin CH CH \. CH, N/ \ÜH Methyl-pyridyl-ammonium hydroxyd. Interessanterweise vermögen nicht alle Tiere diese Synthese zu voll- ziehen, i) Das Vorkommen der erwähnten Verbindung im Harn des Menschen dürfte auf zugeführtes Pyridin zurückzuführen sein, das im Tabak und im Kaffee und Tee enthalten ist.-) Auch das aus Pferdeharn gewonnene 4-Methyl-pyridin3) dürfte der Nahrung entstammen bzw. Pyridin ent- sprechen, das im Tierkörper methyliert worden ist. Ferner ist beobachtet worden, daß nach Verfütterung von Nikotinsäure im Harn Trigonellin auftritt.) Wir sind dieser Verbindung bereits begegnet, als wir jene im Pflanzenreich vorkommenden Betaine besprachen, die sich auf bestimmte Aminosäuren zurückführen lassen &): CH CH HC C COOK HC ^V.CO HC CH HC . CH N N -0 CH3 Nikotinsäure=:ß-Pyridin- Betain der Nikotinsäure karbonsäure Trigonellin. Wichtig ist endlich die Beobachtung, daß im Harn von Hunden nach Phosphorvergiftung y-Trimethyl-aminobuttersäure^y-ßutyrobetain') auftritt.^) Diese Verbindung geht wahrscheinUch aus Glutaminsäure hervor, wie die folgenden Formeln zeigen: ^) Emil Abderhalden uud Carl Brahni: Zeitschr. f. pliysiol. Chcinic. 62. 133 — Emil Abderhalden, Carl Bralim uud Alfred Schittenhelm: Ebenda. 59. 32 (1909). — Z. Hoshiai: Ebenda. 62. 118 (1909). — G. Tofani und Z. Hoshiai: Ebenda. (1909). 68. 83 (1910). — M. Tomitu: Biochem. Zeitschr. 116. 48, 55 (1921). B. F. Kutscher und A. Lohmann: Zeitschr. rmigs- und üenußmitteln. 13. 177 (1907). Untersuchung von Nali- ^) Vgl. ') ) W. Achelis und F. Kutscher: Zeitschr. f. physiol. Cliemie. 52. 91 D. Ackermann: Zeitschr. f. Biol. 59. 17 (1912). z. ') Vgl. S. 442. •) Vgl. ') K. Takeda: Pflüger?, Archiv, 135. 365 (1910). Ji. f. (1907). Willstätter: Ber. d. Deutschon Chem. Gesellsch. 35. (517 (1902). Kiweißstofle und ihre Baustoinc. COOH 62n - CO., CH NH, CH, .MI, CH., CH, CH, CH, . COOH COOH Gluta'minsäure '-Aminobuttersäur e /CH3 CH. n(-CH, . CH.. OH H, I CH. CCHo CH.," OOH co- \CH, I O I v-Butyro-betain. Im Muskelextrakt ist eine Verbindung beobachtet worden, die einem ; - Trimethylamino - x -oxybuttersäure - anhydrid 1) Oxybutyrohetain entspricht 2) und vielleicht durch Oxydation aus y-Butyrobetain entsteht. Sie ist Karnitin genannt worden. ^j = CH. y CHo " i . X^CHo VCH3 3 CH., ^0 aCH.OH I CO Oxy-butyro betain. Es ist nach diesem Befunde wohl möglich, daß ^ias y-Butyrobetain einem Stoffwechselprodukt entspricht, das auch unter normalen Verhältnissen gebildet wird, und nur infolge der Störung des Stoffwechsels nach Phosphorvergiftung in Erscheinung tritt. Aus Hunde- und Pferdefleisch ist eine Substanz, Myokynin genannt, gewonnen worden, die wahrscheinlich ein Hexamethyl-ornithin darstellt.) Früher wurde Karnitin für ein v-Trimethyl-ß-oxyhutyrobetain gehalten. Vgl. S. 45:-5 und E. Engeland und F. Kutscher: Zeitschr. f. physiol. Chemie. Vgl. auch W.'Gulewitsch und R. Krimberq: Ebenda. 45. 320 (1905). 69. 281 (1910). h. Krimberq: Ebenda. 48. 412 (190(5): 49. 89 (1906); 50. 361 (1900/07); 53. 514 (1907); ferner R. Engeland: Ber. d. Deutschen Chem. Gesellsch. 43. 2705 (1910); 54. 2208 (1921). ^) Das von Kutscher, Physiol. Zbl. 19. 504 (1905), beschriebene Novain ist nacl» ') ^) — — Krimberg und Engeland (vgl. Zitat ^) identisch mit Karnitin. ) I). Ackermann: Zeitschr. f. physiol. Chemie. 69. 273 (1910). XXX. Vorlesiiug. (j24 Einstweilen verfügen wir noch über keine eindentigen Ergehnisse füj' daß Methvlierungen im tierischen Organismus unter normalen \'erhältnissen eine große Rolle spielen. Die Beobachtung, daß methylierte Produkte nur schwer angegriffen werden, spricht eher dagegen. ') die Annahme, Sehr wahrscheinlich verdankt das Adrenalin, auch Suprarenin genannt, ebenfalls der Methylierung einer ohne Zweifel aus einer aromatischen Aminosäure hervorgegangenen Verbindung seine Entstehung. Es kommt ihm die folgende Struktur zu:-'^) CH OH <^'^\' CH (OH) CH, NH • . OH . CH^. Ich c. . . . CH Adrenalin = ">. 4-l)ioxyphenyl-methylaminilthanol. Das Adrenalin wird vom Mark der Nebennieren gebildet. Es wirkt auf alle Organe, die vom N. sympathicus innerviert werden. 0000001 g dieser \'erbindung pro Kilogramm Körpergewicht bewirken noch deutliche Erhöhung des Blutdruckes. Sie wird durch Zusammenziehung der BlutgefälJemuskulatur bewirkt. Adrenalin wirkt für manche Drüsenzellen als Reiz. Es sei auch daran erinneit, daß es bei Zufuhr größerer Mengen davon zur HyperglukJimie mit nachfolgender Glukosurie kommt.) Sehr interessant ist die Beobachtung, daß das in der Natur vorkommende linksdrehende Adrenahn ) ist als die synthetisch gewonnene Razemform vergleichende Untersuchung des 1- und d-Adrenalins brachte bald die Aufklärung dieser Erscheinung. Die in der Natur nicht vorkommende d-Komponente ist nämlich im vollständig reinen Zustande wirksamer (dl-Adrenalin). Die F' Kutscher: Zeitschr. f. physiol. Chemie. 69. 26(S Arndf Physiol. 16. 452 (1911). Kolärmisch: Zentralbl. f. Physiol. 23. 'l43 (1909); Zeitschr. f. Biol. 57. 273 (1911). A'. Schuhe und G. Triet': Zeitschr. f. physiol. Chemie. 67. (Sl (1910). E. Friedmnnn Jfo/melsfers Beitr. 11. 158. 177, 194 (1908). ') <l'.llü). Vgl. dazu AckernuDui uiul — Andrlik, Velich und Slanek: Zentralbl. — f. — — -) - /;. Vgl. die Synthese bei Sfolz : Ber. d. Deutscheu Chem. Gesellsch. 37. 4149 (1904). Frier/ manu: tfofmeisteri Beitr. 6. •') 92 (1904): 8. 94 (1906). Vgl. über seine Entdeckung und Isolierung: Vulpian: Compt. rcnd. de lAcad. des — Sciences. 43. 663 (1856). G. Oliver und E. A. Schäfer: Jouru. of Physiol. 16. Proceed. of the physiol. Soc. 1 (1904): 17. Proceed. IX (1894 95). 0. r. Fürfli: Zeitschr. f. phvsiol. Chemie. 24. 142 (1S98): 26. 15 (1898/99): 29. 10.") (1900); Hofmeister?, Beitr. — — J. Abel und Crawford: John Hopkins Hosp. Bull. Xr. 7(5. .hili (1897). 243 (1901). ./. Abel: Ebenda. Xr. 90 und 91 (1898); Amer. .lourn. ot Phvsiol. 1. Milrz 1899: Zeitschr. f. physiol. Chemie. 28. 318 (1899); Ber. d. Deutschen ('hein. (iesellsch. 3«. ./. Takamine: Amer. .lourn. <if Pharmacy. 73. Xovember (1901). -1839 (1903). H. Pauli/: Ber. d. Deutschen Chem. (iesellsch. 36. 2944 (1903). Etnil Abderhalden und Peter Bergell: Ebenda. 37. 2022 (1904). Vgl. auch Albert Crairford: The use of suprarenal glauds in the physiological testing of drug plants. Washington. Government 1. — — — — printiiig oftice 1907. ) Vgl. S. 158 ff. ') F. 212" \|l<,\|,n= Gu(jqe)iheiiu : Zeitschr. ."SR.' 918 (1908).! f. — 51" in sal/saurer Lösung. [E. Abderhalden und Markus — Erauz Elächer: Ebenda. phvsiol. (Jheniio. 57. 329 (1908). Eiweißstoffe und ihre Bausteine. 620 im Vergleich ziu- Wirkung der I-Form viel weniger wirksam i), ja bei der Einwirkung auf manche Gewebe ganz unwirksam, ^j Das Adrenalin wird nicht nur in der Nebenniere gebildet. Abel^) hat die interessante Beobachtung gemacht, daß die in Jamaika vorkommende Kröte Bufo agua es in großen Mengen in den sogenannten Parotisdrüsen bereitet. Dieses Tier sondert das Adrenalin in fast b^/oiger Lösung ab. Leider ist es bis jetzt nicht gelungen, festzustellen, aus welchem Materiale die Nebennieren und die erwähnten Drüsen von Bufo agua das Adrenalin bereiten.) Wir sind auf Vermutungen angewiesen. Sehr wahrMan kann sich scheinlich kommen Tyrosin und Phenylalanin in. Frage. die Umwandlung der Seitenkette dieser Verbindungen, wie folgt, denken 5): NH, C CH, . . C . . CH COOH — CO, . — >- . GH (OH) GH., NH2 + CH3 OH — H, . . . C . GH., . GH., . — G GH >- . . NH., + ()—> (OH) GH., . . NH GH3. . Es würde somit in bekannter Art zunächst durch KohlensäureabspalB. aus Tyrosin p-Oxyphenyläthylamin entstehen. Dann folgt Oxydaund sie kann natürlich auch vorausgehen tion am [i-Kohlenstoffatom scliließlich wird die NH.,- Gruppe durch eine Methylgruppe besetzt. Gleichzeitig müssen in 3, 4-S"tellung des Benzolkernes OH-Gruppen eintreten. Das Adrenalin läßt sich durch zahlreiche Farbreaktionen feststellen. Seine saure Lösung färbt sich auf Zusatz von Eisenchlorid prachtvoll grün. Diese Farbe geht auf Zusatz von Alkali in Karminrot über. Zur Feststellung der Anwesenheit von Adrenalin sind auch biologische Methoden ausgearbeitet worden. Bringt man z. B. eine ganz verdünnte Lösung davon auf das Auge z. B. eines Frosches, dann tritt bald Erweiterung der Pupille Oder man läßt die Lösung auf Blutgefäße wirken und verfolgt ihre ein. Verengerung direkt oder indirekt dadurch, daß man die Menge des in der Zeiteinheit ausfließenden Blutes bestimmt. Erfolgt Zusammenziehung der Blutgefäße, dann nimmt sie ab. Adrenalin wird als Polyphenol leicht verändert. Seine zunächst farblosen Lösungen beginnen sich besonders bei Es tritt zunächst Rotfärbung auf. alkalischer Reaktion bald zu färben. tuug z. — — , braune bis fast schwarze Färbungen, die zum Teil Produkten entsprechen, die in Wasser schwer oder ganz unlöslich sind. Beim Studium der Wirkung von Adrenalin auf den tierischen Organismus Schließlich entstehen Vgl. Emil Abderhalden uud Ernst Gellhorn: Pflügeri, Archiv. 196. 607 (1922). Emil Vgl. hierzu Arthur Ciishni/ : The .Jouru. of Physiol. 37. IHO (1908). E. AbderAbderhalden und Franz Müller: Zei'tschr. f. physiol. Chemie. 58. 185 (1908). E. Abderhalden uud Slavu: halden und Friedrich Thies : Ebenda. 59. 22 (1909). E. Abderhalden uud Karl A'aM/^.scA: Ebenda. 61. 119 (1909). Ebenda. 59. 129 (1909). E. Abderhalden, Karl Knutzsch uud Franz Müller: Ebenda. 62. 404 (1909). ») 2) — — — Watermann — — — Ebenda. 63. 4 (1909). ./. J. Abel uud JJ. J. Macht: Journ. of pharm, and experim. therap. 3. 319 (1912). •» Eigene Versuche, die Menge des von Bufo agua horvorgebrachton Adrenalins durcli sulikutane Zufuhr von riienylalaniu und Tyrosin zu bccintlusseu, ergaben kein eiuiieutiges Resultat, auch dann nicht, wenn die erwähnten Aminosäuren in die sogenannte T'arotisdrüse direkt eingespritzt wurden. ') Vgl. hierzu auch E. Friedmann: Hofmeister>i Beitr. 8. 9.0 (1906). : •') Abderhalden, Physiologische Chemie. I.Teil, 5. Aufl. 40 XXX. Vorlesung. ^326 bestimmte Organe wird man sich die Frage vorlegen müssen, ob unverändertes Adrenalin bestimmte Erscheinungen hervorruft oder aber, ob sie aus ihm hervorgehenden Umwandlungsprodukten zukommen. bzw. auf Wir werden bei der Besprechung der Funktion der Nebennieren erBedeutung dieser so wirksamen Substanz im Haushalt des Organismus zukommt. Hier wollen wir uns mit der Feststellung begnügen, daß bestimmte Zellen des tierischen Organismus imstande sind, derartige Substanzen zu bereiten und an die Blutbahn abzugeben. Wir wissen bestimmt, daß noch viele Zellarten Stoffe bilden, die dazu bestimmt sind. in anderen Organen eine bedeutsame Rolle zu spielen. Leider kennen wir sie noch nicht genau. Beobachtungen an der Hypophyse^) sprechen dafür, daß Amine in Betracht kommen. Man dachte an das Imidazolyl-äthylamin, doch sprechen die Eigenschaften der isolierten Produkte dagegen. 2) Daß p-Oxyphenyl-äthylamin im Sekret der Speicheldrüsen der Kephalopoden vorkommt und dieses ihm seine Wirkung verdankt, haben wir schon erwähnt. 3) Es unterliegt keinem Zweifel, daß man noch manche Beziehungen von Aminosäuren zu Sekret- und Inkretstoffen finden wird. Vor allem wird man dem Tryptophan erhöhte Beachtung schenken müssen. fahren, welche Als ein interessantes Derivat des Tryptophans hat sich der Purpurist im Sekret der Hypobranchialdrüse der PurpurDieses nimmt bei Belichtung eine prachtvolle rotDer Farbstoff hat die Konstitution eines 6, 6-Di- farbstoff ergeben. Er schnecken enthalten. Färbung bromindigo.) violette an. Die nahe Verwandtschaft dieser Verbindung mit dem Tryptophan ergibt sich ohne weiteres aus seiner Formel und der früheren Feststellung über die Bildung des Indigos im Harn durch Oxydation von Indoxyl:^) CH CH HO- Br.C €— CO /\ CO— C CH C C C \/\/ CH NH C C.Br \/\X NH CH Wir haben mit der Feststellung der nahen Beziehungen bestimmte)Inkretstoffe zu Aminosäuren und der Erörterung der Umwandlung des Glykokolls in Cholin bereits Anhaltspunkte dafür gewonnen, daß die Aminosäuren außer als Bausteine des Eiweißes im tierischen Organismus noch andere Aufgaben zu erfüllen haben. Es wird dies ganz besonders klar, wenn wir der Beziehungen der Aminosäuren zu den Kohlehydraten gedenken. Wir brauchen hier nicht mehr auf deren Art einzugehen. «) Wir haben sie schon wiederholt erörtert und gesehen, daß zwar Vgl. u. a. Ilerniatin Markus Guqqenheim: Biochem. Zeitschr. 65. 189 (1914). =) ) 30. Fühner: Therap. Monatsh. März 1913. ') ^) 247 5) 8) Vgl. S. 599.' P. Friedländer : Ber. d. Deutschen Ghem. Ges. 42. 765 (1909); (1909); Ber. d. Deutschen Chem. Ges. 55. 1655 (1922). Vgl. S. 516, Vgl. hierzu S. 135 ft'. Monatsh. f. Ghcni. Eiweißstoft'e und ihre Bausteine. 627 nicht alle Aminosäuren Bausteine zur Bildung von Glukose liefern können, doch wird der größere Teil davon in diese übergeführt. Üb auch Beziehungen zu den höheren Fettsäuren bestehen, wissen wir zurzeit noch nicht. Glyzerin kann leicht aus Alanin und Serin hervorgehen und ferner aus allen Aminosäuren, die zu einer Dreikohlonstoffkette abgebaut werden können. Niedere Fettsäuren treten sicher unter den Abbaustufen der Aminosäuren auf. Wir wenden uns nun zu den wichtigsten jener Stoffwechselprodukte, ganzen Konstitution nach Beziehungen zu bestimmten Aminosäuren aufweisen, für die jedoch ein eindeutiger Beweis für ihre Zugehörigkeit zu solchen noch nicht erbracht ist. Zunächst sei Verbindungen die ihrer gedacht, die Beziehungen zum Guanidin, C=NH , haben. ^NH CH . Aus Harn sind Monomethvl-i), C==NH , undasymmetrisches \NH., /N.(CH3), Dimethylguanidin -), C=NH \NH, , isoliert worden. Die erstere Verbin- düng ist nach Phosphorvergiftung und ferner bei verbrühten Tieren in größerer Menge im Harn beobachtet worden. 3) Beide scheinen in geringen Mengen immer in diesem vorzukommen. Methylguanidin findet sich auch im frischen Muskel.^) Es ist bis jetzt nicht geglückt, die Herkunft der beiden Methylguanidine aufzuklären. Man könnte daran denken, daß sie mit dem Arginin in Zusammenhang stehen. Diese Aminosäure ist eine a-Amino-S-guanidino-valeriansäure: /NH, C^NH \nH . GH., . CH2 Guanidingruppe . GH. . CH COOH. . NH, Würde eine Spaltung dieser Aminosäure an der durch die punktierte eintreten, dann würden Guanidin und /.-Aminowenn eine Hydrolyse eintreten würde, eine a-Amino-'^- Linie bezeichneten Stelle valeriansäure, bzw. oxy valeriansäure entstehen — ) F. Kutscher und ]V. Achelis: Zeitschr. f. phjsiol. Chemie. 50. 10 (1907). Arthur James R. Kngeland: Klieuda. 57. 49 (1908). A. Lohmunn: 49. 81 (190(5). Eu'ins [Biochem. Journ. 10. 10.3 (1917)] macht darauf aufmerksam, daß Methylguauidin aus Kreatiu entsteheu kauii, weuu mau zu seinem Nachweis die Siihermethode anwendet. 2) K. Takeda: I'ßüyer?, Archiv. 115. 38ö (1910). M. llciide: Zentralbl. f. l'hysiol. — — — 25.441 (1911). «) R. Engeland: Zeitschr. f. phjsiol. Chemie. 57. 49 (1908). R. Krimberq: Zeitschr. f. physiol. Chemie. 4S. 412 (1901)). Irwitsch: Ebenda. 47. 475 (1906). J. Smorodinzew: Ebenda. •) — — Vgl. auch (f7. Cu80. 218 (1912); 87. 12 (1913). 40* XXX. Vorlesung. (528 C^NH \NH . CH., . GH., . CHo . = CR COOH +H2 . NH2 /NH., C^NH \NH2 + CH, CH-, . . CHo CH COOH. . . I I OH NH., Vorläufig fehlt für eine solche Annahme jede experimentelle Grundlage. 1) Zwei weitere, unter sich in engem Zusammenhang stehende Guanidinderivate sind das Kreatin und sein Anhydrid, das Kreatinin. Sie haben die folgende Konstitution: /NHo /IsH- C^NH C^NH \N (CH3) CH2 COOH . ^^ \N (CH3J . Kreatin . CH., . CO Kreatinin. Das Kreatin, das von Chevreul in der Fleischbrühe entdeckt worden im tierischen Organismus 2), von allen Geweben in den Skelettmuskeln in größter Menge (etwa 0'55 OAOg)'^) enthalten. In kleineren Mengen ist es in allen Organen) und auch im Blutplasma^) aufgefunden worden. Im Harn kommt es unter normalen Verhältnissen meist nur in ist, ist geringer Menge vor.^) An findet seiner Stelle man sein Anhydrid, das ) Erwähnt sei, daß aus Fleischextrakt und aus Harn vou Kutscher mehrere Verbinduiigea isoliert worden sind, die mit dem Gluanidin in Zusammenhang stehen. Ihre Struktur ist jedoch nicht eindeutig aufgeklärt, und in vielen Fällen läßt sich nicht entscheiden, ob die isolierten Verbindungen einheitlich waren. So beschreibt er z. B. einen Körper der Struktur; ^N (CH3) CH., . . GH2 . NH . C/^^Jjs Vitiatiu [Zentralbl. f. Physiol. 21. 33 (1907)]. ") Auch im Pflanzenreich kommt Kreatin vor. Vgl. Sullimn: Journ. of the Americ. Chem. Soc. 33. 2035 (1911). — Schrei/: Ebenda. 34. 99 (1912). Antonof: als — Bakteriol. I. Abteilung. 43. 209 (1907). ') Slädeler: Jahresber. d. Chemie. 543 (1857). Chemie. 87. 21 (1913). Zeitschr. f. — ./. C. Beker : Zeitschr. f. physiol. — ) Ph. Shafcr: Journ. of biol. Chem. 18. 525 (1914). V. C. Myers und M. S. Fine: Ebenda. 14. 29 (1914). S) Gregory: EbenVgl. z. B. ./. Liebig: Liebim Anualen. 62. 257, 282 (1847). Prize: Ebenda. 76. 362 da. 64. 100 (1847). Schiossberger: Ebenda. 66. 80 (1848). (1850). C. J. C. van Noogenhiiyze und JI. Verploei/h: Zeitschr. f. physiol. Chem. 46. J. C. Beker: ZeitJ. F. hyman: Journ. of biol. Chem. 5. 125 (1908). 433 (1905). schrift f. physiol. Chem. 87. 21 (1913). Vgl. u. a. Joh. Feigl: Biochem. Zeitschr. 81. 14 mg Kreatinin und 5 bis \Omg (1917). Er findet im Blute als liüchsteu Normalgehalt 2 Kreatin in 100 nw' davon. Bei Kindern ist der Gehalt geringer. (Joh. Feigl: BiocheVgl. ferner W. C. Rose, J. St. Dimmitt und IL L. mische Zeitschr. 84. 264 (1917). Bartlett: Journ. of biol. Chem. 34. 601 (1918). 0. Biesser: Z. f. physiol. Ciiem. 120. 189 (1922). ») Vgl. F. P. Cathcart: Journ. of Thysiol. 39. 811 (1908). Shaffer: Americ. Journ. of I'hysiol. 23. 1 (1908). Noiil Pnton: .lourn. of Phvsiol. 39. 485 (1910). — — — — — — — — — — — Eiweißstofte luul ihre Bausteine. Es Kreatinin. zwar ist die (329 wird sicher im Organismus aus Kreatin gebildet ^j und vorhandene Reaktion für seine Entstehung maligebend. Bei saurer Realition geht Kreatin vollständig in Kreatinin über. Bei alkalischer Reaktion vollzieht sich der umgekehrte Vorgang, jedoch unvollständig. 2) Das Kreatin kann seiner Struktur nach als a.-Methylguani- eine dinoessigsäure aufgefaßt werden. Beim Kochen mit Barytwasser zerfällt es in Harnstoff, Methylglykokoll = Sarkosin und w-eitere Zersetzungsprodukte. Es läßt sich aus Sarkosin und Zyanamid darstellen): H.NlCHa) /NH^ C^N + —y I \N (CH,) CH. CüOH Sarkosin = Methyl. Zyanamid) /NH., ('=NH . GH., . COOH Kreatin. glykokoll. Es ist trotz aller Bemühungen bis jetzt nicht gelungen, die Herkunft des Kreatins bzw. Kreatinins vollständig aufzuklären. Früher nahm man an, daß beide Verbindungen in direkten Beziehungen zu dem mit der Nahrung aufgenommenen Kreatin und Kreatinin stehen. Auch die Milch enthält Kreatin. Eine andere Quelle sollte für beide Verbindungen nicht in Frage kommen. Als wesentlichstes Resultat der jüngsten Forschungen über die Herkunft des Kreatins ist die P'eststellung zu bezeichnen, daß es in den Geweben aus anderer Quelle als aus zugeführtem Kreatin und seinem Anhydrid Auch dann, wenn keine Nahrung aufgenommen gebildet w'erden kann. wird, kommt es zur Ausscheidung von Kreatin bzw. von Kreatinin.^) Die Menge des im Harn zur Ausscheidung gelangenden Kreatinins ist für jedes Individuum auffallend konstant'"'), wenn man das mit der Nahrung zugcführte Kreatin und Kreatinin bei der Beurteilung des im Harn erscheinenden Kreatinins in Betracht zieht bzw. die Bestimmungen bei hungernden Pro Kilogramm Körpergewicht werden ungefähr Individuen ausführt. 19 30 mg Kreatinin in 24 Stunden ausgeschieden. Sehr unsicher ist immer noch, ob in den (jeweben Kreatin bzw. Kreatinin zum Abbau — ') C. A. Pekelharing 69. 395 (1910). und ('. J. ('. van Hoogcnlmyzc: Zeitschr. f. physiol. Chemie. biol. Chemie. 21. — Viktor C. Mi/ers und Morris S. Fine: Jnuru. of 383 (1915). Huhn und Georg Barkan: Zeitschr. f. IJiol. 72. 25, 305 (1920). Volhard: Sitzuiigsber. d. Miinchener Akad IL 472 (18(58). Adolf Strecker: .Tahresher. über d. Fortschritte der Chemie. 686 (1868); Zeitschr. f. Chemie. 31S (1869). ) Vgl. zur Konstitutinnsfrage. i/^/iYp Colson: .lourn. Chem. Soc. 111. 554 (1917). 5) Vgl. u. a. C. Voit: Zeitschr. f. Biol. 4. 77 (1868). G. Meissner: Zeitschr. f. rat. Medizin. 31 (3). 174 (18(58). 0. Folin: Festschrift f. Olaf llammarsfen: Upsala. K. Otio af Klerckcr: Biochem. Zeitschr. 3. 45 (1907). 1906. E. P. Cathcart Ebenda. 6. 130 (1907). •/. B. Benedict und A. R. Diefendorf: Americ. .lourn. of I'bvsiol. 18. 362 (1907). C. J. ('. J. B. Leafhes: .lourn. of Physiol. 35.' 205 (1907). van Hoogenhuijze und H. Verploegh: Zeitschr. f. physiol. Chemie. 57. 161 (1908). ") Vgl. Ä. '') J. — — — — — — — — — — Philip Shaff'cr: Americ. Journ. of Physiol. 23. 1 (1908). Mellanbi/: Journ. of Vgl. auch William C.Pose, F. 11'. /Unniiitt und Paul X. Physiol. 36. 447 (1908). Cleatham: Journ. of biol. Chem. 26. 339. 345 (1916). «) 0. Folin: Americ. Journ. of Physiol. 13. 84 (1905). Oliver E. Closson Americ. Journ. of Physiol. 16. 252 (1906). Francis Gano Benedict und Victor Cari/l Mi/ers: Ebenda. 18. 377 (1907). CJ. ('. van Hoogen/iui/ze und //. Vorploeqh: Zeitschr. f. physiol. Chemie. 57. 161 (1908); 59. 101 (1909). — — /•-'. — — ' XXX. Vorlesung. (330 kommt. Die Angabe, wonach die Leber diese Verbindungen zerlegen solli). wird bezweifelt.-) Bestimmt festgestellt ist, daß im Darmkanal Kreatin durch Bakterien abgebaut werden kann. Durch zahlreiche Untersuchungen ist festgestellt worden, daß die Bildung des Kreatins mit dem allgemeinen Eiweißstoffvvechsel nicht in direktem Zusammenhang steht. Exakte Bestimmungen des Kreatin- und Kreatiningehaltes des Harnes zeigten, daß sich zwischen diesen Verbindungen und dem Stickstoffgehalt des Harnes keine direkten Beziehungen finden. Dagegen glaubten manche Forscher aus verschiedenen Beobachtungen den Schluß ziehen zu dürfen, daß das Kreatin jenem Eiweiß entstammt, das in den Zellen zum Abbau kommt und vorher Zelleiweiß gewesen ist.») Vom Darme aus resorbierte Aminosäuren würden somit nach dieser Annahme nicht direkt zur Bildung von Kreatin Verwendung finden können. Es liegen jedoch keine eindeutigen Beweise für die Annahme eines solchen Ablaufs des Kreatinstoffwechsels vor. Er würde voraussetzen, daß der Abbau der eigentlichen Zellproteine zu anderen Produkten führt, als wenn z. B. Umsatzeiweiß zur Zerlegung kommt, oder aber es müßten am Aufbau bestimmter Gewebsproteine Atomkomplexe beteiligt sein, die bei ihrem Abbau zu Kreatin führen. In der Tat liegen Beobachtungen vor, wonach in den Muskelzellen kolloide Verbindungen sich finden sollen, aus denen sich Kreatin abspalten läßt, i) Diese sollen bei der Muskelarbeit zerfallen. Vielfach wird die Meinung vertreten, daß das Kreatin ein typisches Stoffwechselprodukt der Muskelzellen sei.^] Man hat auch die Vermutung ausgesprochen, daß es in irgend einer Weise die Muskelkontraktion beeinflusse. Es liegen jedoch keine eindeutigen Ergebnisse für eine solche Annahme vor.") Manche Forscher sind der Ansicht, daß das Kreatin ') F. GottUeh und R. Stangassiiiger: Zeitschr. f. physioi. Chemie. 52. 1 (1907); R. Statt gassinger: Ebeuda. 55. 295 (1908). Ä. Rothmann: Ebenda. 322 (1908). E. Meilanhg: Jouru. of Physioi. 36. 447 (1908). 57. 131 (1908). C. A. Pekelharing und C. J. C. van BooqenJivi/zc: Zeitschr. f. physioi. Chemie. 69. 395 (1910). Vgl. dazu Frank P. UnderhiU [E. J. Bamnann, L. Jean auch J. C. Beker: Ebeuda 87. 21 (1913). Bogert] (Journ. of biol. Chem. 27. 127, 141, 147, 151, IGl) will einen engen Zusammenhang der KreatinausscheiduDg und der sog. ,,Azidosis" gefunden haben. Nach Alkalizufuhr nahm die Kreatinmenge im Harn ab, um schließlich ganz zu verschwinden. W. Denis und A. S. Minot [J. of biol. Chem. 37.245 (1919)] bestreiten die erwähnten Beobachtungen. ^) Vgl. u. a. E. London und Bojarski: Zeitschr. f. physioi. Chemie. 62. 4(35 (1909). — ~ — 55. — — — — O. Folin und IV. Dems: Journ. of biol. Chem. 12. 141 (1912). — L. B. Mendel: Science. X. F. 29. 584 (1909). L. B. Mendel und \V. C. Rose: Chem. 10. 249 (1911). Vgl. auch Steenbock und Gross: Journ. of. biol. Chem. 36. 265 (1918). William C. Rose, J. St. IHmmift und H. L. Bartlett: Journ. of biol. Chem. 34. 601 (1918). ) R. Gottlieh und R. Stangassinger : 1. c. Diese Seite, Zitat '). ^) Vgl. R. H. Hahn: Pflüger^ Arch. 177 (1919). Max Bürger: Zeitschr. f. d. gesamte experim. Med. 9. 262 (1819). «) Vgl. u. a. A. Gregor: Zeitschr. f. physioi. Chem. 31. 98 (1900). S. Weber: Arch. f. experim. Path. u. Pharm. 58. 93 (1908). E. Mellanby: Journ. of Physioi. 36. 447 (1908). Ph. A. Shafj'er: Americ. Journ. of Physioi. 23. 1 (1908). C. A. Pekelharing und ra« •0 .Journ. of biol. — — — — — — — — Hoogenhuyze : Zeitschr. f. physioi. Chem. 64. 262 (1908). 0. r. Fürth und Karl Schwarz: Ebenda. 30. 413 (1910). C. A. Pekelharing: Zeitschr. f. physioi. Chem. 75. ]'. Scaffidi: 207 (1911). Biochem. Zeitschr. 50. 402 (1913). — Vgl. ferner ^'ictor C. Myers und Morris S. Eine: Journ. of. biol. Chem. 14. 9 (1913); 15. 183, 305 (1913); Vgl. auch Otto Folin und If^ Denis: Journ. of biol. Chem. 17. 493(1914). — 16. 1()9 (1913). 0. Riesser: Zeitschr. f. physioi. ('hem. 86. 145 (1913); Arch. f. experim. Path. u. Pharm. 80. 183 (1916). Vgl. auch J. G. D. de Barenne und D. G. ('. Terraert: Pßügers Arch. f. d. ges. Physioi. 195. 370 (1922). — — — — und Eiweißstott'e ihre Bausteiue. 631 Einfluß auf den Tonus der Muskeln habe. Gewiß nicht ohne Bedeutung ist der Umstand, daß der Kreatingehalt der Muskeln um so größer ist, je reicher sie an quergestreiften P'ibrillen sind und je rascher der Verlauf ihrer Kontraktion ist. In gleichnamigen Muskeln ist der Kreatingehalt innerhalb ein und derselben Tierart unter normalen Verhältnissen innerhalb enger Grenzen ein auffallend konstanter, i) Da wir weder wissen, aus welchen Verbindungen Kreatin entsteht, und was aus dem Teil von ihm in den Geweben wird, der zum Abbau kommt, so können wir zurzeit auch nicht erwarten, einen klaren Einblick in den Kreatin- bzw. Kreatininsto ff Wechsel zu erhalten. Es sind noch zu Vorfragen von grundlegender Bedeutung zu lösen. Es ist bei dem Stand der ganzen Forschung auf diesem Gebiet richtiger, die großen Lücken, die noch vorhanden sind, klar hervorzuheben, als sie durch bestimmte Vorstellungen rein hypothetischer Art zu überbrücken. Wir wollen uns nur noch die Frage vorlegen, welche Aminosäuren als Ausgangsmaterial für die Bildung des Kreatins in Frage kommen könnten und ferner, was für Verbindungen bei seinem Abbau zu erwarten sind. Zunächst könnte man daran denken, daß das Kreatin mit dem Arginin in Zusammenhang steht und aus diesem durch die übliche Art des Abbaues von Aminosäuren hervorgeht: viele jetzigen /NH, C^NH C^NH / / \NH CH, GH., CH. CH, CH, CH, i I CH NH, CH, . COOH — CO., . NH, NH, -f 2 XXX. Vorlesung. Ö32 Diese Art des Abbaus des Arginins ist nicht bis jetzt festgestellt. Wir kennen einstweilen nur diejenige über Ornithin und Harnstoff durch die Arginase.i) Bei dieser Zerlegung des Arginins kann selbstverstimdlich nicht an eine direkte Beziehung dieser Aminosäure zum Kreatin gedacht werden. Es ist jedoch ganz gut möglich, daß in bestimmten Zellen oder auch allgemein unter ganz bestimmten Bedingungen der Abbau des Arginins auf die oben beschriebene oder eine ähnliche Art verläuft. Dadurch würde erklärt, weshalb die Bildung des Kreatins von der Menge des zugeführten Arginins nicht ohne weiteres abhängig ist. Da nun die Leber jenes Organ ist, das Arginase enthält, so wird es verständlich, weshalb es so schwer ist, durch Fütterungsversuche mit Arginin den Beweis zu führen, daß diese Aminosäure die Muttersubstanz des Kreatins ist. Neuere Versuche machen diese Beziehung wahrscheinlich. 2) Selbst in der Leber scheint eine Kreatinbildung stattzufinden. 3) Von Interesse ist in dieser Hinsicht auch die Beobachtung, daß bei den Insekten und Krustazeen Arginin vorkommt, dagegen Kreatin ganz fehlt. Melolontha vulgaris enthält freies d-Arginin.) Jaffe hat sich die Frage vorgelegt, obGlykozyamin = Guanidinoessigsäure in Kreatin übergehen kann.^) Die Ergebnisse der Versuche verschiedener Forscher stimmen nicht überein. Die Frage ist somit noch eine offene.') zum Kreatin bzw. Eine weitere Aminosäure, die gewisse Beziehungen Kreatinin erkennen läßt, ist das Eis tidin: COOK CH NH2 . CH2 I N C %CH CH—NH Histidin = CO—NH Nc = NH + H2 CH,— N (CH,) COOK —> CH^ Kreatinin . N (CH,) Kreatin. [i-Imidazolyl- a-aminopropionsäure Der Imidazolring des Histidins könnte, wie die vorstehenden Formeln des Kreatins bilden. Erwähnt daß in diesem Fall auch die Purinbasen als Quelle für Kreatinin in zeigen, das Ausgangsmaterial zur Bildung sei, Vgl. S. 320. L. BanVgl. hierzu: \V. U. Thompson: J. of Physiol. 51. 111, 347 (1917). und J[. M. Hines: Zeitschr. f. biol. Cliera. 35. 75 (1918). ») K. Inoui/e: Zeilschr. f. phvsiol. Chem. 81. 71 (1912). ) I). Ackermann: .1. of Biol 73. 319 (1921). •') M. Joffe: Zeitschr. f. physiol. Chem. 48. 430 (1906). G. Dornet-: Ebenda. ') — 2) mann — 52. 226, 5 (1907). ) Vgl. auch die Versuche, einen Zusammenhang des Kreatins mit Guanidin Geor^/e M. Wichart: herzustellen. Achelis: Zeitschr. f. physiol. Chem. 50. 16 (1906). .Jotirn. of Physiol. 53. 480 (1920). — Eiweißstoffe und ihre Bausteine. 633 Betracht kommen würden. Sie enthalten nämlich, wie wir gleich erfahren werden, auch den Imidazolring. Es ist jedoch bis jetzt nicht gelungen, derartige Beziehungen zwischen Kreatin und dem Imidazolring durch den V^ersuch nachzuweisen. i) Man hat endlich auch daran gedacht, daß Cholin eine Muttersubstanz des Kreatins sein könnte.-) Endlich ist noch eine synthetische Bildung des Kreatins möglich. Sie könnte von GlykokoU ausgehen. Es fehlt jedoch zurzeit noch jeder Hinweis auf einen solchen Aufbau. Daß jedoch die Bildung des Kreatins für viele Zellen einen ihnen geläufigen Vorgang darstellt, beweist die Beobachtung, daß manche Bakterienarten diese Verbindung bilden.^) Was nun den Abbau des Kreatins anbetrifft, so ist es naheliegend, an eine Spaltung in Sarkosin und Harnstoff zu denken. Die erstere Verbindung ist bis jetzt in den Geweben nicht aufgefunden worden. Es wäre möglich, daß von ihr aus Cholin gebildet wird, und sie so der Beobachtung entgeht. Die einzige Feststellung genau bekannter Spaltungsprodukte aus Kreatin bzw. Kreatinin verdanken wir Ackermann.) Er fand, daß Bakterien aus Kreatinin N-Methylhydantoin bilden: /NH\^ C^NH ^\ \N . CH, . -fH^O — NH3— > /NH\ C=0 ^\ N CHc CO . . CO CH3 CH3 Kreatinin Methyl-hydantoin oder /NH\ \ Cf=NH \N CH, CO . CH3 . /NH2 +H., —y C^NH +H., \N CH., COOH . . XXX. Vorlesimg. g34 C— \n . CHo .^CO CH3 Methylhydantoin. N-Methylhydantoin kann, wie die vorstehenden Formeln zeigen, unter Wasseraufnahme und Ammoniakabspaltung aus dem Kreatinin hervorgehen. Es ist jedoch auch möglich, daß zuerst unter Wasseraufnahme Harnstoff und Sarkosin sich bilden, und daß letzteres wieder mit ersterem unter Ammoniakaustritt in Reaktion tritt. Eine weitere Möglichkeit des Abbaus des Kreatinins bzw. Kreatins könnte zu Methyl guanidin führen. Dieses könnte weiterhin die Muttersubstanz des schon erwähnten Dimethyl- guanidins sein.^) Wir kennen weiterhin zwei Verbindungen, die mit größter W^ahrauf Histidin zurückzuführen sind. Es sind dies das Karnosin und die Urokaninsäure. Die erstere Verbindung ist im Fleisch und im Fleischextrakt aufgefunden worden. Bei ihrer Spaltung werden Histidin und Aminopropionsäure erhalten.^) Die letztere scheinlichkeit ^'erbindung erwies sich mit Alanin identisch. als nicht Sie ist vielmehr eine in ß-Stellung substituierte Propionsäure: CH, . C^Ho . COOH i NH, ß-A min opropion säure. Wir sind dieser Verbindung bereits begegnet, als wir vom Abbau der Asparaginsäure durch Bakterien sprachen. 3) Diese Aminosäure geht unter Abspaltung von Kohlensäure in fi-Amino-propionsäure über: COOH - COo I CH.NH2 —^ I CH, .NH, ' " I CH2 CH2 I I COOH COOH ß-Aminopro- Asparaginsäure pionsäure. ») Vgl. S. 627. M7. Guhwitsch und Amiradzihi: Ber. d. Ueutscheu Chem. Gesellsch. 33. 1902 It. Krimberq: Ebenda. 48. 412 (1906). (1900); Zeitschr. f. physiol. Chem. 30. 565 (1900). Wl. Gulewitsch: Ebenda. 50. 204, 535 (1906/07); 73. 434 (1911); 87. 1 (1913). \^gl. auch ül)er das Vorkommen: (). r. Fürth J. Smorodinzew. Pil)enda. 87. 12 (1913). Marie Mauthner: Monatsh. und Karl Schwarz: Biochem. Zeitschr. 30. 414 (1911). ^) — — f. — Chem. 34. 269 (1913). =•) Vgl. S. 453. — — Eiweißstoffe und ihre Bausteine. 685 Das Karnosin hat die Struktur i) eines [i-Alanyl-histidins: Histidinrest HC = C HN . CH, N . CH CUOH . NH CO . \/ . CH, . CHg NHo . Ö-Alaninrest CH Im frischen Fleisch wurden etwa O'o« o Karnosin gefunden.-) Die Urokaninsäure ist im Hundeharn entdeckt worden. 3) Sie nur äußerst selten aufzutreten. Sie ist ferner beim Abbau von Histidin durch Bakterien der Coli-Typhusgruppe beobachtet worden.) Die Vermutung-, daß die im Harn von Hunden auftretende Urokaninsäure auf die Einwirkung von Darmbakterien auf Histidin zurückzuführen sei und nicht in den Geweben entstehe, konnte durch Fütterungsversuche mit der genannten Aminosäure als nicht bewiesen erwiesen werden.») Nach Hunter ^) kommt dieser Verbindung die Struktur einer [i-Imidazolylakrylsäure zu. Eine Gegenüberstellung der Formel dieser Verbindung und derjenigen des Histidins zeigt, daß die erstere durch Ammoniakabspaltung aus letzterer entstanden sein. kann: scheint CH— NH CH— NH \ CH / N C CH > N C I I CH CH, CH NH, . — NH. CH COOH COOH Histidin = ß-Imidazolyl-a-aminopropionsäure. Urokaninsäure = ß-Imidazolyl-akrylsäure. Endlich sei noch der Rhodan wasserst off säure, CNSH. gedacht. von Gscheidhn ') in geringer Menge im Harn des Menschen, von Pferden, Rindern, Hunden, Katzen und Kaninchen aufgefunden worden. Sie ist Louis Baumann und Thorsten Inf/waldsen: Jouru. of biol. Chem. 35. 263 (1918). J. 12. 402 (1918). ^) Otto r. Fürth und 'Iheodor Hryntschak : Biochem. Zeitschr. 64. 172 (1914).— W. M. Clifford: Biochem. Vgl. über die Verbreitung des Karnosins im Tierreich Journal 25. 725 (1921). M. Siegtried 3) M. Jaffe: Ber. d. Deutschen Chem. Gesellsch. 7. 1669 (1874). Zeitschr. f. physiol. Chem. 24. B99 (1898). ) Harold Raistrick : Biochem. Journ. 11. 71 (1917). 5) Y. Kotake und M. Konishi: Z. f. physiol. Chem. 122. 230 (1922). Vgl. dazu auch «) Andrew Jlunter: Journ. of biol. Chem. 11. 537 (1912). B. Barger und Ä. J. Fivins : Journ. of the Chem. Soc. 99. 2336 (1911). ') R. Gscheidlen: Tagblatt der 47. Versammlung deutscher Naturf. und Ärzte in Virchows, Archiv. 69. /. Munk: Breslau (1874): Pfitk/er?. Archiv. 14. 401 (1877). 354 (1877). ) — Vgl. auch a. Barger und Frank Tutin: Biochem. : — — — XXX. Vorlesung. 636 Sie findet sich auch im Parotis- und Submaxillarisspeichel des Menschen und mancher Tiere. Dem Speichel des Hundes und des Pferdes i) fehlt sie. Interessanterweise enthält der Magensaft beim Hunde und bei der Katze Mit dem Harn des Menschen werden im Tage Rhodanwasserstoffsiiure.^) 03— 0'05 g Rhodankalium in 24 Stunden ausgeschieden. 3) Seine etwa Menge ist bei Rauchern größer als bei Nichtrauchern.) Über den Bildungsort der Rhodanwasserstoffsäure ist noch nichts Sicheres bekannt. Es ist möglich, daß bei manchen Tieren nur die Speicheldrüsen beteiligt sind und der in verschiedenen Organen gefundene Gehalt an Rhodansalzen auf die vom Speichel aus resorbierte Verbindung zurückzuführen ist. Der Gehalt des Magensaftes beim Hund und bei der Katze an Rhodanwasserstoffsäure macht es sehr wahrscheinlich, daß die Zellen der Magendrüsen diese Verbindung bereiten können. Es bleibt jedoch die Möglichkeit, daß sie von anderen Organen gebildet und durch die Zellen des Magens nur ausgeschieden wird. Ebenso ist es natürlich auch denkbar, daß die Speicheldrüsen Rhodanwasserstoffsäure ausscheiden, die in anderen Geweben gebildet worden ist. Es ist bis jetzt unbekannt geblieben, wie die Rhodanwasserstoffsäure Der Schwefel entstammt wohl sicher dem Eiweiß bzw. dem Zystin. Die Beobachtung, daß Blausäure, Zyanide und Nitrile im Tierkörper in Rhodanide übergehen, &) macht es wahrscheinlich, daß derentsteht. artige Produkte das Ausgangsmaterial zur Bildung der Rhodanwasserstoffdarstellen. Wahrscheinlich ist der erwähnte, erhöhte (behalt des säure Speichels von Rauchern an dieser Säure auf den Blausäuregehalt des Tabakrauches zurückzuführen. Die Funktion der Rhodanide im tierischen Orga- noch unaufgeklärt. Einige Forscher sind geneigt, sie als SchutzEs wird vermutet, daß sie antiseptische Wirkungen entfalten. Während des Hungers wurde die Ausscheidung von Rhodanwasserstoffsäure stark vermindert gefunden. Ganz verschwand sie nicht.*) nismus ist aufzufassen. stoffe Zum Nachweis der Rhodanwasserstoffsäure benützt man die pracht- Farbe des Eisenrhodanids, Fe (CNS)3. Man gibt zu Speichel oder Harn verdünnte Eisenchloridlösung und beobachtet das Auftreten einer roten Färbung. Ganz eindeutig ist übrigens diese Probe nicht, weil auch andere Verbindungen des Harnes rote Eisensalze geben können. Beweisend für die Anwesenheit von Rhodanwasserstoffsäure ist nur ihre volle rote Isolierung. Damit hätten wir alle jene Verbindungen erwähnt, die direkt oder indirekt mit bestimmten Aminosäuren zusammenhängen. Sie gehen sehr wahrscheinlich aus solchen hervor, nachdem diese durch Hydrolyse aus Munk: Pflüqer^ Archiv. 61. 620 (1895). M. Nencki: Ber. d. G. Kelling: Zeitschr. f. physiol. Chem. 18. 397 (1884). Deutscheu Chem. Ges. 28. 1318 (1895). M. Nencki und N. Sieb er : Zeitschr. f. i)hysiol. ') 1. — '') — Chem. 32. 291 (1901). A. Edinger und F. Clemens: Zeitschr. f. klin. Med. •) — 59. 218 (1906). ./. Toth: (]hemiker-Ztg. 33. 1301 Fr. Kriiqer: Zeitschr. f. Biol. 37. 6 (1899). Vgl. auch A. Mayer: Deutsches Archiv f. klin. Med. 79. 209 (1904). (1909). ^) .S'. Lang: Arch. f. cxper. Path. u. Pharm. 34. 247 (1894). Vgl. dazu: Seraßno Dezani: Arch. d. Farmac. sperim. 23. 245 (1917); 24. 189 (1917); 25. 278 (1918); 28. ) — — 115 (1918). ; S. Dezani: Arch. di Farmac. sperim. 25. 83 (1918). Eiweißstoffe und ihre Bausteine. 637 Proteinen entstanden sind. Der Abbau kann sich im Dannkanal oder aber den Zellen vollzogen haben. Wir kennen nun noch eine Anzahl von Verbindungen, die nach allen bisherigen Beobachtungen sich nicht direkt von einzelnen x\niinosäuren ableiten, sondern mit hochmolekularen Eiweiß abbaustufen zusammenzuhängen scheinen. Sie finden sich beständig im Harn. Man nimmt ziemlich allgemein an, daß sie durch Oxydation von Eiweiß oder Peptonen bzw. Polypeptiden entstehen. Wir würden in diesem Fall zwei prinzipiell verschiedene Arten des Eiweißabbaues zu unterscheiden haben. Wohl der bei weitem größte Teil der Proteine wird im Zellstoffwechsel über Peptone zu Aminosäuren abgebaut. Diese werden dann in der wiederholt geschilderten Weise weiter verwandelt. Daneben wird vielleicht stets ein Teil des umgesetzten Eiweißes entweder direkt, oder nachdem Peptone entstanden sind, der Oxydation und auch anderen Vorgängen unterworfen. Es würde in diesem Falle nicht zur Bildung von Aminosäuren kommen. Es ist jedoch auch ganz gut möglich, daß die unten erwähnten in Produkte ihre Entstehung von Umwandlungsprodukten bestimmter Aminosäuren aus nehmen. Sie würden in diesem Falle durch Synthese entstehen. Vielleicht ergibt eine sorgfältige Analyse der durch die Darmbakterien erzeugten Eiweißabbau- und -Umwandlungsprodukte Hinweise auf die Herkunft der zu besprechenden Verbindungen. Es herrscht leider immer noch ein sehr großes Dunkel über den meisten dieser Produkte. Ihre Entdeckung verdanken wir Bondzynski und Gottlieb, i) Es seien die wichtigsten dieser Substanzen erwähnt und ihre elementare Zusammensetzung angegeben. Hervorheben müssen wir noch, daß für kein einziges dieser mit besonderen Namen belegten Produkte der Nachweis geführt werden konnte, daß es einheitlich ist. Es sind folgende Substanzen als Bestandteile des Harns beschrieben worden 2) Antoxyproteinsäure^): 43-21«/o C, 4-9 1 "/o H, 24-40o/o N, OGLVoS und 26-870/0O. Oxvproteinsäure): 39-H20/0 0, 5-64o/o H. Ig-OSVo N, l-12''/o S, 35-54VoO. Alloxvproteinsäure^): 41-a3VoC, 5 TOVo H, l8-55o/o N, 2-U)Vo S und 37-23% 0. Üroferrinsäure e) 45-4ö«/o C, 6-08VoH, 12-12»/oN, 3-46 "/o S. : 32-89«/o 0. Die erwähnten Verbindungen sind dadurch ausgezeichnet, daß sie Säuren sind und in Wasser lösliche und in Alkohol unlösliche Barytsalze geben. Sie lassen sich ferner durch Quecksilberazetat bei schwach alkalischer ') St. Bondzyiiski uud R. Gottlieb: Zentrall)!, f. d. med. Wissensch. 33. 577 (1S97). Bondzyiiski uud Paneh: Bull, de Pacad. des sciences de Cracovie. Octohre 1902, St. Bondztp'tski, St. Dombrou-s-ki und Ber. d. Deutschen Chera. Ges. 35. 29.')9 (1902). Fritz l'rcgl: Pflüge)-^ Archiv. und A'. I'anek: Zeitschr. f. physiol. Chem. 45. 83 (190Ö). Vgl. hierzu auch: Wilhelm Ginsberfi: Hofmeistern Bahr. 10. 411(1907). 75. 87 (1899). Moriz Weiss: W. Czernecki: Anzeig. d. Akad. d. Wissensch. zu Krakau. 400 (1910). — — St. — — — — Biochem. Zeitschr. 27. 175 (1910). ") Weitere Säuren haben Udri [Zeitschr. f. phy.si(d.Chem. 46. 1 (1905)] uud Clo'rtta [.,Urop rotsäure-'. Arch. f. e.\per. Path. u. Pharmak. 40. 29 (1897/98)] beschrieben. ') St. Bondzijn.tki, Dombroivski und I'anek: 1. c. S. 637. Zitat M. ) St. Bondzjfitski und R. Gottlieb: 1. c. S. 637, Zitat '). 5) St. liondzi/nski und Pane.k: 1. c. S. 637, Zitat ') Vgl. dazu auch «) O. Thiele: Zeitschr. f. physiol. Chemie. 37. 251 (1903). Liebermann: Ebenda. St. Bondziffifiki: Zeitschr. f. physiol. Chemie. 46. 114 (1905). 52. 129 (i907). — — XXX. Vorlesung. 638 Reaktion fällen. Die einzelnen der oben genannten Säuren unterscheiden sich durch ihr Verhalten gegenüber Schwermetallsalzen. Zur Beurteilung der Stellung dieser Produkte zum Eiweiß, zu den Peptonen und Polypeptiden ist von größter Wichtigkeit, daß sie die Biuretreaktion nicht geben. Auch die für einzelne Aminosäuren charakteristischen Farbreaktionen fallen negativ aus. Es ist bis jetzt nicht gelungen, den einwandfreien Nachweis zu führen, daß am Aufbau dieser Produkte unveränderte Aminosäuren Edlbacher 2) hat bei der Zerlegung der Oxvproteinsäure beteiligt sind.i) Harnstoff gefunden. Es hat den Anschein, als ob außer diesem kein weiterer Baustein in Frage kommt, doch müssen noch weitere Untersuchungen abgewartet werden, bevor ein endgültiges Urteil möglich ist. Unentschieden ist noch, in welcher Art und Weise die Harnstoffgruppen in der Oxyproteinsäure enthalten sind. Wahrscheinlich stehen die oben erwähnten Säuren in engem Zusammenhang und stellen vielleicht verschieden weit vorgeschrittene Zerfallsprodukte eines noch unbekannten Ausgangsmateriales dar. ^ Es ist wohl möglich, daß Studien über das Verhalten bestimmter Eiweißabbaustufen gegenüber verschiedenen Agenzien neue Wege zur Erforschung der trotz aller Bemühungen noch immer nicht ausreichend aufgeklärten Bestandteile der sogenannten Oxyproteinsäurefraktion des Harns eröffnen. Man hat versucht, Eiweißstoffe und auch Peptone auf verschiedene Arten, z. B. durch Anwendung von verschieden stark eingreifenden Oxydationsmitteln zu verändern 3) und die erhaltenen Produkte zu charakterisieren und womöglich zu identifizieren. Es ist kaum zu erwarten, daß beim jetzigen Stande der Eiweißchemie auf diesem Wege jetzt schon fruchtbringende Ergebnisse zutage gefördert werden. Ein so kompliziert gebautes, aus zahlreichen verschiedenen Bausteinen zusammengesetztes Molekül kann bei eingreifenderen chemischen Prozessen kaum zu einheitführen. mit PolyAussichtsvoller sind Versuche lichen Abbaustufen Wir gehen dabei von Verbindungen mit bekannter Struktur peptiden. aus und können dann die entstandenen Produkte mit dem Ausgangsmaterial in einen bestimmten Zusammenhang zu bringen suchen. So ist aus Glyzyl-glyzin bei der Oxydation Oxalyl-aminoessigsäure erhalten worden): CH2 CO NH . . . CH2 . COOH —> HOOC CO NH . . . CHg . COOH NH2 Glyzyl-glyzin ') Eigene Uütersuchungen machen es sehr wahrscheinlich, daß kein Aminostickder Oxj proteiusäiiie voihaudcu stoff in Oxalyl-glyzin, ist. Hdlhacher: /eitschr. f. phjsiol. Chemie. 120. 71 (1922). ^) Vgl z. B. Maly: Sitzungsbor der Kais. Akad. d. Wiss. in Wien. 91 (2). 157 St. Bondzi/ii.ski und L Zoja: Zeitschr. f. physiol. Chem. 19. 225 (1894). (1885). Otto Fr. N. Schulz: Ebenda. 29. 8() (19 )()). - G. Zickgraf: Ebenda. 41. 259 (1904). V. Fürth: Ho/mnstira Heitr. 6. "i9B (1905). J. Buraczewski und L. Kranze: Zeitschrift f. physiol. Chemie. 76. 87 (1911). ) Leo I'ollak: Hofmeistern Beitr. 7. 17 (1905). Vgl. ferner Otto FAsler: Biochcm. Zeitschr. 51. 45 (1913). 2) S. — — — — — Eiweißstoffe und ihre Bausteine. In diesem 639 Zusammenhang sei erwähnt, daß Harn von Menschen bei manchen Krankheiten mit Diazobenzolsulfosäure i) bei alkalischer Reaktion unter Bildung eines roten Farbstoffes reagiert. 2) In saurer Lösung geben manche Harne mit der erwähnten Verbindung einen gelben Farbstoff. Seine Entstehung ist auf das Vorhandensein von Urobilinogen im Harne zurückzuführen. 3) Über die Muttersubstanz des roten Farbstoffes war man lange Zeit im Unklaren. Man fahndete nach einem bestimmten Bestandteil des Harnes, der mit Diazobenzolsulfosäure in Reaktion tritt. Es zeigte sich jedoch, daß verschiedene Stoffwechselprodukte mit phenolartigem Charakter Träger der sogenannten Diazoreaktion sein können. Festgestellt wurde als eine solche Substanz Oxyindolessigsäure. ) Sie leitet sich vielleicht von Oxytryptophan ab^) oder geht aus Tryptophan hervor, wobei dieses eine Sauerstoffanlagerung im Benzolring erfahren müßte. Die Untersuchung des Harnes bei verschiedenartigen akuten und chronischen Erkrankungen unter Störung von Organfuuktionen bestimmter Art wird sicherlich noch manchen interessanten Befund bringen und Abbaustufen von Bausteinen zusammengesetzter Verbindungen zutage fördern, die sonst dem Nachweis entgehen. Im Harn scheinen noch andere, mit dem Eiweiß im Zusammenhang Verbindungen vorzukommen. So ließ sich ein schwer dialy- stehende sables Produkt abtrennen, das bei der Hydrolyse Aminosäuren liefert. ß) Auch die schon wiederholt erwähnte Chondroitinschwefelsäure'^) ist im Harn beobachtet worden. Sie wird ebenfalls von manchen Forschern mit dem Eiweiß in Zusammenhang gebracht. Es ist jedoch fraglich, ob sie ein Produkt darstellt, das von allen Körperzellen bereitet werden kann. Es spricht manche Beobachtung über das Vorkommen der Chondroitindafür, schwefelsäure in bestimmten Geweben Knorpel, Niere usw. daß sie das Produkt bestimmter Zellarten ist. — — Im Harne findet man unter bestimmten Verhältnissen nur Spuren von Eiweiß.^) Dieses entstammt den kleinen Drüschen jener der Harn Mengen von durchfließt, bis er nach außen gelangt. Enthält er Proteinen, dann liegt unter allen Umständen eine die sogenannte AlbuStörung vor. Der Eiweißgehalt des Harnes stellt nur minurie, besser allgemein Proteinurie genannt Wege, die größere — — Symptom dar. Die Eiweißausscheidung durch die Nieren kann durch ganz verschiedene Momente bedingt sein. Im Blute werden beständig große ein Eiweißmengen an jenen ») '-) Zellen der Niere vorbeigeführt, die die Aufgabe Vgl. S. 331. Paul Ehrlich: Zeitschr. f. klin. Medizin. 5. 28.i (1882); Deutsche med. Wochen419 (1884). Hans Fischer: Ha'oilit.'Ihomas: Zeitschr. f. klin. Med. 64. 247 (1911). 10. schrift. ^) — München 1912. ) Leo Hermxmns : Zeitschr. f. physiol. Chemie. 114. 79,88(1921); 122.98(1922). Schrift. 5) Vgl. S. 329. Emil Allderhalden und Fritz Preql : Zeitschr. f. physiol. Chemie. 46. 19 (1905). iV. Eb') Vgl. S. 73 und K. Ä. //. Mörner: Skaud Arch. f. Physiol. 6. 378 (1895). ) — — K.Sasaki: Ho/meis/ers Beitr. 9. 386 (1907). becke: Biochem. Zeitschr. 9. 386(1907). Ch. Föns: Ebenda. 9. 393 (1907); Arch. internat. de Physiol. 8. 393 (1909). 8) Vgl. hierzu K. A. U. Mörner: Skand. Arch. 6. 332 (1895). — XXX. Vorlesung. 540 haben, gelöste Stoffwechselendprodukte zur Ausscheidung zu bringen. Eiweiß wird unter normalen Verhältnissen nicht durchgelassen. Man nimmt allgemein an, daß seine kolloide Natur verhindert, daß es die Zellen der Harnkanälchen passiert und in den Harn übergeht. Die Verhältnisse können jedoch nicht so einfach liegen, denn wir kennen eine Art der Proteinurie, bei der die Niere sicher nicht primär beteiligt ist. Es ist dies die Ausscheidung des Bence Jonesschen Eiweißkörpers.i) Er tritt namentlich bei \'orhaudensein von Sarkomen in Knochen auf. Man erkennt ihn leicht an seinem besonderen Verhalten bei der Hitzekoagulation. Er gerinnt nämlich bei Erwärmen des Harns, um dann bei höherer Temperatur wieder in Lösung zu gehen.-) Beim Abkühlen des Harns erscheint dann wieder das koagulierte Protein. Die Untersuchung der Zusammensetzung dieses Eiweißes oder besser Eiweißgemisches ergab, daß alle Aminosäuren vorhanden sind 3), jedoch in einem Mengenverhältnis, das keinem der bis jetzt untersuchten Proteine zukommt.) Es handelt sich ohne Zweifel um Proteine, die normaler Weise im Blute nicht enthalten sind. Sie sind blutfremd und werden als fremdartige Bestandteile aus dem Körper entfernt.^) Es fragt sich nun, auf welche Art und Weise der Durchtritt des Eiweißes durch die Blutgefäßkapillaren und ferner die Epithelzellen der Harnkanälchen erfolgt. Muß dem Durchtritt ein Abbau zu diffundierbaren Produkten vorausgehen? Diese Annahme ist deshalb sehr unwahrscheinlich, weil schwer zu verstehen wäre, weshalb im Harn dann nicht diese Abbaustufen an Es müßte noch im Harn zur SynStelle von Eiweiß erscheinen würden. these von Eiweiß kommen. Ein weiteres Beispiel einer Art von Proteinurie, bei der die Nieren Eiweiß durchtreten lassen, ohne offenbar selbst direkt erkrankt zu sein, ergibt die sogenannte orthostatische Proteinurie. Bei dieser beobachten wir Eiweißausscheidung im Harn bei aufrechter Körperhaltung. Sie verschwindet beim Liegen. EndUch können wir durch sehr reichliche Zufuhr von Eiweiß Nahrungsproteine zur Resorption bringen. Das blutfremde Material wird zum Teil durch die Nieren ausgeschieden. Schließlich scheint es eine Proteinurie während der Schwangerschaft zu geben, die ebenfalls eine Ausscheidung von blutfremdem, vielleicht fötalem Eiweiß darstellt. ^j Man könnte die Frage aufwerfen, ob die Nieren nicht auch, wie beim Zucker, auf einen bestimmten Gehalt des Blutes an Eiweiß eingestellt ^) //. Bence Jones: F'hilosoph. Trausact. 1. 55 (1848); Liebigs Aunaleu. 67. 97 — W. Kühne: Zeitschr. Biol. 19. 209 (1883); 20. 40 (1884). — Huppert kliu. physiol. Chemie. 22. 501 (1897). — A. Kllinger: Deutsches Arch. Med. 62. 255 (1898). — Magnus-Levij: Zeitschr. physiol. Chemie. 30. 200 (1909). — F. W. Lamb :Proceed. of the Physiol. Soc. 29. Juui; Journ. of Physiol. 45 (1912). — Erich Krauss: Deutsches Archiv klin. Medizin. 137. 257 (1921). — F. Malengreau: (1848). Zeitschr. f. f. f. f. f. Arch. internat. de physiol. 18. 151 (1921). -) Vgl. S. 396. ^) f'Jmil Abderhalden und 0. Rostoski: Zeitschr. f. physiol. Chemie. 46. 125 (19U5). Vgl. auch Alide Grutterink und Cornelia J. de Graaff: Zeitschr. f. physiol. Chemie. 46. 47.^ (1905). F. Gowland Hopkins und Horace Savory : Journ. of Physiol. 42. 189 (1911). ) Das will allerdings bei einem Gemisch von Proteinen nicht viel sagen. ) Vgl. hierzu auch A. K. Taylor, ('. \V. Miller and J. E. Sweet: J. of biol. Chem. 29. 425 (1917). Emil Abderhalden: Zeitschr. f. physiol. Chemie. 106. 130 (1919). ) Vgl. Emil Abderh Iden : AbderhaJdensche Reaktion. 5. Autl. J. Springer, Ber- — — — lin 1922. Eiweißstoüe iiud ihre Bausteine. (;)41 und 'es eine Proteinurie infolge einer Ilyper proteinoplasmie Ferner wäre es möglich, daß die Epithelzellen der Nieren auf «inen verschiedenen Gehalt des Blutplasmas an Eiweiß eingestellt sind und manchmal Eiweiß schon dann durchtreten lassen, wenn keine Hyperproteinoplasmie besteht. Die Nierenzellen verfügen über Fermente, die zerlegen können. Vielleicht besteht eine flie verschiedensten Proteinarten wichtige Aufgabe bestimmter Zellarten der Nieren darin, dem Blute seien sie nun in einer zu großen Menge nicht zugehörende Proteine vorhanden oder seien sie an und für sich blutfremd (Heteroproteinoabzufangen und durch Abbau dem Zellstoffwechsel noch plasmie) nutzbar zu machen.M In vielen Fällen werden die Nierenzellen dieser Aufgabe gewachsen sein, in manchen wird jedoch zur raschen Entfernung Beim Abbau der dieser Proteine noch die Ausscheidung notwendig sein. Proteine in den Nierenzellen entstehen vielleicht unter manchen Umständen Abbaustufen, die für diese schädlich sind und sekundär zu Störungen in der Niere selbst Anlaß geben. Zu prüfen ist auch, ob die Durchlässigkeit von Nierenzellen für Eiweiß bestimmter Art in der gleichen Weise, wie wir es l)eim Traubenzucker kennen gelernt haben, von der Reaktion und dem lonengehalt des Blutplasmas abhängig ist. sind, i^ibt. — — Endlich kommt es auch zur Ausscheidung von Eiw^eiß im Harn, wenn die Nierenzellen geschädigt sind. Es ist außerodentlich schwer, jedem einzelnen Falle die Frage zu entscheiden, welche Erscheinungen primär sind, und welche sich sekundär entwickelt haben. Das Studium der verschiedenartigen Fälle von Proteinurie und vor allem die Ergründung ihrer Ursachen wird ohne Zweifel auch für die Beurteilung der Stellung der Niere im Eiweißstoffwechsel von großer Bedeutung werden. in Fassen wir nunmehr alles zusammen, was wir über das Verhalten Proteine im tierischen Organismus wissen. Die Eiweißstoffe der Nahrung werden im Magendarmkanal stufenweise abgebaut. Es entstehen Aminosäuren. Ob nur solche zur Resorption kommen oder auch Peptone und Polypeptide, ist nicht bekannt. Jedenfalls tritt unter normalen Verhältnissen nichts in die Blutbahn über, was seiner Struktur nach an Be- <ler aufgenommenen Proteine erinnert. Es ist daß Aminosäuren zum Transport mit dem Blute kommen. Ob daneben auch in der Darmwand bzw. in der Leber Plasmaeiweißkörper gebildet werden, wissen wir nicht. Die einzelnen Zellen verarbeiten die Aminosäuren in verschiedener Weise. Es werden daraus wertvolle Stoff- Inkret- und Sekretstoffe bereitet. Ferner wird aus wechselprodukte den zugeführten Aminowird aus Zelle Manche gebildet. ihnen Glukose säuren und pjweißstoffen Zelleiweiß aufbauen und auch direkt jene Proteine bereiten, die sie mit Sekreten nach außen abgibt. Es ist wohl möglich, daß jede Zelle Umsatzeiweiß besitzt und daneben Proteine, die zum I5au der Zolle selbst gehören. Manches spricht auch dafür, dal^ im Blutplasma Proteine kreisen, die speziell als Nahrungsstoffe der Zellen standteile der mit der Nahrung festgestellt, dienen. Die Zellen können selbst Eiweiß abbauen. Sie bilden Aminosäuren und verarbeiten diese genau so, wie die ihnen mit dem Blute zugeführten Zcitschr. f. phvsidl, ') Vgl. Emil Abderhalden und Ändor Fodor 22Q (1913). — Emil Abderhalden uud Erwin Schill': 87. 213 (l'.)13). Abderhalden, Physiologische Chemie. 1. Teil, 5. Aufl. : Cheiii. 41 87. XXX. V'orlesnng. ()42 Eiweißbausteine. Es ist dies jedoch offenbar nicht der einzige Weg des Abbaus der Eiweißstoffe. Die Feststellung der im Blute und im Harn auftretenden eigenartigen Säuren (Oxyproteinsäuren usw.) weist darauf hin, daß, wie zum vorneherein anzunehmen war, der Eiweißstoffwechsel verschiedene Bahnen kennt. Die Umwandlung eines bestimmten Eiweißstoffes in einen solchen ganz anderer Art bedingt einen weitgehenden Abbau und eine darauf folgende Synthese. Die Zellen des tierischen Organismus können aus Aminosäuren Eiweiß aufbauen. Ja sie vermögen sogar einzelne Bausteine selbst zu bereiten. Andere, wie z. B. das Tryptophan und das Tyrosin bzw. Phenylalanin, können sie hingegen nicht aufbauen. Der Abbau der einzelnen Aminosäuren zu Glukose oder anderen ^'erbindungen und zu den Stoffwechselendprodukten je nach der Tierart Harnstoff bzw. Harnsäure erfolgt stufenweise. Eine ganze Reihe von einzelneu Abbaustufen sind schon bekannt. Es gibt nicht nur einen Weg des Abbaus. Je nach Bedarf kann die Zerlegung der Aminosäuren eine verschiedene sein. Auf diesen Umstand weist uns auch der Befund von Verbindungen hin, die unzweifelhaft zu bestimmten Aminosäuren Beziehungen haben, jedoch sich nicht in den Hauptweg ihres Abbaus ein- — fügen — lassen. Im Harne treffen wir auf Verbindungen, die im Darmkanal unter der Wirkung von Bakterien entstanden und in der Leber und vielleicht auch noch in worden sind. anderen Geweben mit bestimmten Verbindungen gepaart Wir lernten als Paarlinge das Glykokoll und die Schwefel- säure kennen. Ferner erwies sich die der Glukose nahestehende Glukuronsäure als eine Verbindung, die manche der von der Darmflora erzeugten Produkte abfangen und festlegen kann. Vorlesung XXXI. Niikleoproteide. Nukleinsäuren und ihre Bausteine. Bei der Besprechung des Vorkommens der Eiweißstoffe erwähnten in den Zellen Eiweißverbindungen enthalten sind. Sie bestehen aus Eiweißstoffen und ferner einem Anteil, der in keiner direkten Beziehung zum Eiweiß steht. Eine derartige Klasse von Verbindungen wir, daß stellen die sogenannten Nukleoproteide und Pflanzenwelt außerordentlich verbreitet. dar. Sie Es gibt sind in der Tierwahrscheinlich nur wenige Zellen, die nicht Nukleoproteide enthalten. Sie sind nämlich am Aufbau der Zellkerne beteiligt. Fehlen diese, dann kommen, soweit unsere Kenntnisse reichen, auch die genannten Proteide nicht vor. Sie sind zuerst von Miescher^) aus den Kernen der Eiterzellen gewonnen worden. Später dienten die verschiedensten Organe zur Darstellung von Nukleoproteiden. Sie werden vorläufig nach ihrer Herkunft benannt. So spricht man von Pankreasnukleoproteiden, Thymusnukleoproteiden usw. Ein sehr gutes Ausgangsmaterial zur Gewinnung dieser Proteide stellen die kernhaltigen roten Blutkörperchen der Vögel, Reptilien und Amphibien dar. 2) Ferner bestehen die Spermatozoenköpfe ^) fast ganz aus Nukleoproteiden. Die Nukleoproteide stellen weiße Pulver dar. Sie lösen sich in Alkalien. In Wasser und Salzlösungen sind sie wenig löslich. Die wässerige Lösung reagiert sauer. Wir können die Nukleoproteide als solche nicht so genau charakterisieren, daß sie ohne weiteres an ihren Eigenschaften erkannt werden könnten. Vor allem vermögen wir nicht den Beweis zu führen, daß irgend ein Nukleoproteid im genuinen Zustande zur Beobachtung gekommen ist. Um sie zu gewinnen, sind meistens ziemlich eingreifende Methoden nötig. Es müssen die Kerne zerstört werden. Dann erfolgt ihre Abscheidung durch Säuren oder durch Aussalzen. Ohne Zweifel sind die Nukleoproteide hochmolekulare Verbindungen. Sie enthalten nicht nur ') Fr. MiescJier: Hoppe-Sci/lers mediz.-chem. Uutersiichuugcu. H. 4. 441 (1871). Vgl. auch die histochemischen und physiologischen Arbeiten von Friedrich Miescher. F. C. W. Vogel. Leipzig 1897. Vgl. r. Plöfiz: Hopj)e-Sei/lers mediz.-chem. Untersuchungen H. 4. 451 (1871). Ebenda. H. 4. 486 (1871). : ") F. Miescher: Verhandl. d. naturf. Gesellsch. zu Basel.. 6. 138 (1874); Arch. f. A. Kossei: Zeitschr. f. physiol. Chem. 22. experim. Path. u. Pharmak. 37. 100(1895). Arch. f. experim. Path. u. Pharm. 4.3." 57 (1900). (). Schmiedeherg: 17(5 (189G). Vgl. diMoh Richard Biirian: »gebn. d. Physiol. 5. 768 (1906). ^) - F. Hoppe-Seyler — — — 41* XXXI. Vorlesimg. (344 sondern es sind in ihnen mehrere Kiweißanteile schon bei den einfachen Eiweißkörpern ganz unmöglich, festzustellen, ob sie einheitlich sind, so stoßen wir erst recht auf große Schwierigkeiten, wenn für irgend eines der beschriebenen Xukleoproteide der Nachweis geführt werden soll, daß eine chemisch reine Verbindung vorliegt. Die Möglichkeit, daß verschiedenartige Verunreinigungen, z. B. Proteine, bei der Ausfällung der genannten Proteide mitgerissen werden, liegt nahe. Fernei- zeigen sich bei den Proteiden die gleichen Erscheinungen, wie bei den Proteinen. Auch sie können infolge ihres Gehaltes an Eiweißstoffen denaturiert werden. Dabei ändern sich natürlich die Eigenschaften der ursprünglichen Anteile des Zellkernes. Die Nukleoproteide sind in erster Linie durch ihren Gehalt an Nukleinsäuren charakterisiert. Diese besitzen ganz charakteristische Bausteine. Da es gelungen ist, aus den Nukleoproteideu durch Verdauung mit Magensaft einen von diesem nicht weiter spaltbaren Best abzuspalten, während gleichzeitig Eiweiß in Pepton übergeführt wird, so nimmt man an, daß die Nukleoproteide einen Eiweißkomplex besitzen, der locker in das ganze Molekül eingefügt ist, während ein zweiter Anteil mit der Nukleinsäure noch in Verbindung bleibt und das erwähnte, vom Magensaft übrig gelassene Produkt darstellt. Es ist Nuklein genannt worden. Vom Pankreassaft wird dieses in Nukleinsäure und Eiweiß gespalten. Dieses letztere wird dann unter Wasseraufnahme abgebaut. ein Molekül Eiweiß, War o-ebunden. es Das folgende Schema trägt diesen Beobachtungen Bechnung: Nukleoproteid Nukleoproteid Eiweiß \ / Eiweiß Nuklein oder besser Eiweiß] Nukleinsäure ' /\Nukleinsäuren Eiweiß die Zusammensetzung der Die Verhältnisse liegen nicht so einfach. Der Magensaft spaltet nicht nur Eiweißanteile ab, sondern setzt wenigstens in manchen Fällen auch Phosphorsäure in Freiheit. Es ist unbekannt, ob nur ein p]iweißmolekül bei der Einwirkung des Pepsins frei wird, oder ob vielleicht mehrere Proteinanteile am sogenannten Nuklein sitzen. Die gleiche Frage nach der Zahl der Proteinmoleküle wiederholt sich bei derjenigen nach dem Aufbau des Nukleins. Nur eines ist sicher festgestellt worden, nämlich, daß am Aufbau der Nukleoproteide basische teilnehmen. Ob jedoch auch mit Histonc und Protamine Proteine diesen wiederum saure Eiweißkörper in Verbindung stehen, und damit die Mannigfaltigkeit des Aufbaus der Nukleoproteide noch weiter gesteigert wird, wissen wir nicht. Jedenfalls kennt die Zusammensetzung der Proteide in ihrer Verschiedenartigkeit keine (u'enzen. Jede Zellart dürfte wohl über eigenartige, für sie und ihre Funktionen charakteristische Xukleol)roteide verfügen. Erwähnt sei noch, daß diese Proteide nicht aschefrei erhalten werden. Einmal enthalten sie immer Phosphorsäure. Diese gehört, wie wir gleich vernehmen werden, zu den Bausteinen der Nukleinsäuren. Daneben trifft man immer auch auf andere Aschenbestandteile und Es ist fast sicher, daß dieses Schema Nukleoproteide nicht richtig wiedergibt. — — Nukleopruteide. Xukleiiisilmcii und ihre Bausteine. (j45 iiamoiitlich auf Eisen. \'orlüufig läßt es sich nicht entscheiden, ob diese anorganischen Stoffe dem Bau der Nukleoproteide angehören, oder ob sie \'erunreinigungen darstellen. Genauer studiert sind Xukleoproteide aus der Thymusdrüse^), der Schilddrüse'^), aus Pankreas^), aus der Nebenniere), aus der Leber-'j, dem Gehirn usw.") Ferner sind aus den Köpfen der Spermatozoen^j solche dargestellt worden. Besonders die Lachsspermatozoenköpfe sind eingehend untersucht worden. Sie enthalten im entfetteten Zustand ungefähr ^'>07o Nukleinsäure, 3öVo Salmin und 2-5% anorganische Stoffe. Während, wie schon betont, die Nukleoproteide als solche im allgemeinen nicht so charakterisiert werden können, daß eine bestimmte ^'orstellung über ihren Aufbau möglich ist, so sind dagegen unsere Kenntnisse über die chemischen und physikalischen Eigenschaften der Nukleinsäuren viel bessere. Wir werden uns hauptsächlich mit diesen Verbindungen und ihren Bausteinen befassen. Über die Proteinauteile der Nukleoproteide können wir nämUch nichts aussagen, was wir noch nicht erwähnt hätten. Sie unterliegen im Magendarmkanal dem gleichen Abbau, wie die als solche aufgenommenen Proteine. Im Zellstoffwechsel erleiden sie auch die gleichen Umwandlungen. Bei den Nukleinsäuren dagegen stoßen wir auf Vei'bindungen, die zu ganz eigenartigen charakteristischen Stoffwechselprodukten führen. Die Nukleinsäuren haben, wie ihr Name saure Eigen- besagt, schaften. Ihre typischen Bausteine sind Purinbasen und Pyrimidinbasen. Ferner enthalten sie eine Kohlehydratgruppe und Phosphorsäure. Die Nukleinsäuren sind bis jetzt, als solche nicht in Kristallform erhalten worden. Sie lösen sich in Wasser und ferner in AlkaUen. Sie lassen sich aus ihren Lösungen durch Mineralsäuren abscheiden. Mit Schwermetallsalzen und Erdalkalien bilden sie in Wasser unlösliche Salze. Ehe wir zur Besprechung der Konstitution der Nukleinsäuren übergehen, dem Aufbau der einzelnen ihrer Bausteine dann die L^rage vorlegen, wie diese unter sich Wir beginnen mit der Besprechung der Purinbasen. Es wir uns und uns erst wollen befassen, mit verknüpft sind. sind bei den bisher untersuchten Nukleinsäuren zwei verschiedene Verbindungen der Klasse der Purine aufgefunden worden, nämlich das Adenin und das Guanin. Man hat sie auch als Aminopurine bezeichnet. Sie stehen in nächster Beziehung zu der bereits besprochenen Harnsäure. Sie enthalten, wie diese, den Purinkern: — ) L. Lilienfeld: /eitschr. f. physiol. Chemie. 18. 478 (1894). II'. Ihtiskamp J. Baiu/: Hofmeistcn Beitr. 4. 115. 331 (1903): ">. 317 (1904). El.euda. 34. 32 fl'901). — — F. Malengreau: La Cellule. 17. 339 (1900). A. 'Ostvuld: Zeitschr. f. physiol. Ghem. 27. 14 (1899). O. Hammarsten : Zeitschr. f. physiol. Chern. 19. 19 (1894). E. l'mber: Zeitschrift f. kliu. Med. 40. 4(54 (1900). A. Gamgie: Compt. reiid. de la soc. biol. 55. 22r> A. (iaingee uud W'.. Tones: Hofmeistens Beitr. 4. 10 (1903). (1903). ) W. Jones und G. H. Whipple: Americ. Journ. of Physiol. 7. 423 (1902). ') J. Wohlgemuth: Zeitschr. f. physiol. Chem. 37. 475 (1903); 42. 519 (1904); 44. 530 (1905). ^) A. Levene: Arch. f. Neurol. u. l'sycbopathol. 5. 1 (1899). ') Fr. Miescher: loc. cit. S. 643, Zitat '). li. Burian : Kri/elmisse iler Fhvsiol. 5. 768 (1906). -) — ) — — — XXXI. Vorlesung. 640 N=CH C (6) N(i)- I C[(6) N(3) — C(4) HC -^ (7) — I C— NH N— C— N^9) Purin. Purinkern. Seine Glieder sind von Emil Fischer ^) in der in der vorstehenden Formel angegebenen Weise numeriert worden. Bei der Bezeichnung der einzelnen Verbindungen gibt die Zahl, die den einzelnen Gruppen beigegeben diese in den Purinkern eingefügt sind. So ist ist, an, an welcher Stelle die Harnsäure nach dieser Bezeichnungsweise ein 2, 6, 8-Trioxypurin: N=C.OH NH— CO ^^ I I 0C,o, C-NH HO. C(2)C— NH oder (8))C (^^)C( OH . N— C— NH— C-NH Harnsäure. Harnsäure tautomeren Formen der Harnsäure Beide" der angeführten vorkommen.'; dürften Das Adenin ist ein 6-Aminopurin: N=C.NH. I 1 C— NH >CH N— C— Ad enin = 6-Aminopurin. HC Dem Guanin entspricht die Konstitution eines 2-Amino-6-oxy- purins: NH-CO NH., .C N=:C.OH C— NH oder NH, C . C— NH ^CH N- -C-N N- -C- VW / -N Guanin = 2-Amiuo-6-oxypurin. im Pankreasgewebe aufgefunden Das Adenin ist von A. Kossei von Nukleinsäuren auch frei im Baustein als außer findet sich Es worden. "^J ') Vgl. über die Chemie der Piiringruppe die klassischen Arbeiten von r. Baeijer und besonders von Emil Fischer. Die Arbeiten des letzteren Forschers sind zusammengefaßt in: Untersuchungen in der Puringruppe (1882— 1906) von Emil Fischer. J.Springer. Berlin 1907. Albrecht Kossei: Ber. d. Deutschen Chem. (iesellsch. 18. 79, 1928 (1885); ZeitVgl. über seine Synthese: phvsiol. Chemie. 10. 250 (1886); 12. 241 (1888). W. Traube: Emü Fischer: Ber. d. Deutschen Chem. (iesellsch. 30. 2226, 2241 (1897). Liehiqi^ Annalen. 331. 64 (1904). ') schrift f. — — Xukleoproteide. Nukleinsüurpn uikI ihre Bausteine. 547 Harn, jedoch nicht regelmäßig, ^j Auch in den Fäzes ist es enthalten. 2) Ferner wird es sicher beim Ab- und Aufbau der Nukleinsäuren in den Zellen in Erscheinung treten. Im Pflanzenreich ist man dem Adenin oft begegnet, so in den Teeblättern 3), im Zuckerrübensaft ). in Bambusschößlingen '), im Steinpilz 5) usw. Adenin kristallisiert aus verdünnten wässerigen Lösungen mit drei Molekülen Kristallwasser in langen Nadeln und aus konzentrierten Lösungen ohne Kristall Wasser. Im letzteren Falle entstehen wetzsteinförmige Kristalle.^) Adenin bildet Verbindungen mit Basen, Säuren und Salzen. So sind z. B. das Blei- und Silbersalz dargestellt worden, ferner das salzsaure und schwefelsaure Salz. Ferner ist das Pikrat zu erwähnen. Zum Nachweis des Adenins eignet sich am besten seine Umwandlung durch Erwärmen mit Zink und Salzsäure in eine Verbindung, die in neutraler oder alkalischer Lösung unter Aufnahme von Sauerstoff sich rot färbt.») Versetzt man die wässerige Lösung von Adenin mit Eisenchlorid, dann tritt Rotfärbung ein. Das Guanin ist von Unger im Guano entdeckt worden. Als BauGuanin ist im freien Zustand in verschiedenen Organen ''), ferner in den Fäzes ^2), in den Schuppen mancher Fische ^^) usw. nachgewiesen worden. Gewöhnlich erhält man das <iuanin in Form eines amorphen Pulvers. Es kann unter geeigneten Bedingungen in kugeligen oder garbenförmigen Aggregaten von Prismen und Pyramiden gewonnen werden. Es bildet, wie das Adenin, mit Basen, Säuren und Salzen Verbindungen. Adenin und Guanin gehen unter Ammoniakabspaltung in sogenannte ()xy-purine über. Aus Adenin entsteht Hypoxanthin — 6-Oxypurin. aus Guanin Xanthin 2, 6-Dioxypurin: '•) stein der Nukleinsäuren erkannte es Ä. Kossel.'^^) = N=C NH2 . C— NH —^ ^CH N— C— Adenin = 6- Aminopurin HC ') .•iV2 M. Kri'K/cr und G. Salomon : Zeitschr. f. physiol. Chemie. 24. 364 (18Ü8); 26. (1898). M. Krüger und A. Schittenhelm : Zeitschr. f. physiol. Chemie. 35. 153 (1902). M. Krüger: Zeitschr. f. physiol. Chemie. 16. 164, 329 (1892); 21. 275 (1895/96). ) K. V. Lippmann: Ber. d. Deutschen Chem. Gesellsch. 29. 2651 (1896). ) ») G. Totani: Zeitschr. physiol. Chemie. 62. 113 (1909). K. Yoshimura : Zeitschr. f. Unters, d. Nahrungs- u. Genußmittel. 20. 153 (1910). Vgl. auch K. Yoshimura und M. Kanal: Zeitschr. f. physiol. Chemie. 86. 178 (1913). ') F. 360—365" unter Zersetzung. Bei 220" sublimiert es unzersetzt. 5) ) ) Hypoxanthin gibt die gleiche Reaktion. Bodo Unger: Liebig?, Annalen. 51. 395 (1844); 58. 18 (1846); 59. 58 (1848). ") A. Kossei: Zeitschr. f. physiol. Chemie. 7. 15 (1880). Vgl. dazu auch C.PicVgl. über seine Synthese: curd: Ber. d. Deutschen Chem, Gesellsch. 7. 1714 (1874). >>'. Traube: h^mil Fischer: Ber. d. Deutschen Chem. Gesellsch. 30. 1116, 2253 (1897). Kbenda. 33. 1371 (1900). »M A. Kossei: Zeitschr. f. physiol. Chemie. 7. 19 (1882/83); 8. 406 (1883/84). »-) M. Krüger und A. Schittenhelm: Zeitschr. f. physiol. Chemie. 35. 158 (1902). ") Veit: Zeitschr. f. wissench. Zoologie. 15. 515 (1865). Bethe: Zeitschr. für physiol. Chemie. 20. 472 (1895). 8) — — — — XXXI. Vorlesuu^. 648 NH— CO N=C.()H C-NH ^CH HC /'CH N— C— NH-CO N=:C.()H . C— NH N— C— C— N Hy poxanthin — 6-().\ypnrin. H. I. NH, C C-NH HC oder C ~NH NH, .C oder / CH —> > N_C-N Guanin = 2-Amino-6-oxypuriii NH— CO N=:C OH . HO . C— NH C oder CH / NH— C— / CH N_C— I. C— NH OC Xanthin=:2, rt-Dioxypurin. H. In beiden Fällen tritt die NHg-Gruppe aus und wird durch eine Oxygruppe ersetzt. Die erste Formel (I) zeigt beim Hypoxanthin und beim Xanthin diese Umwandlung an. In der zweiten Formel (II) ist die Umlagerung dieser Laktimform in die Laktamform vollzogen. Dieser Oberführung von Aminopurinen in Oxypurine werden wir noch wiederholt begegnen, wenn wir das Verhalten der ersteren in den Geweben besprechen werden. Wir werden dann auf den Vorgang der Desaminierung eingehen. Hier sei nur erwähnt, daß man an eine hydrolytische Abspaltung der Aminogruppe denken kann: X = C . NIF, + H., O — NH, —y N = C . Oll —> HN — CO Diese Umwandlung der Aminopurine in Oxypurine wurde auch bei der Darstellung der Bausteine der Nukleinsäuren beobachtet. Man glaubte lange Zeit, daß am Aufbau dieser Verbindungen auch Oxypurine beteiligt seien, bis vor allem Sieudel den Nachweis erbrachte, daß diese sekundär aus ersteren entstehen und somit nur Adenin und Guanin Bausteine der Nukleinsäuren sind. Hypoxanthin und Xanthin sind in der Natur sehr verbreitet. Die erstere Verbindung ') ist von Scherer 2) im Herzmuskel und in der Milz entdeckt worden. Sie findet sich auch in anderen Organen, ferner im ') Vgl. ihre 2226 (1897). «) Synthese: Emil Fischer: Her. d. Deutschen - W. Traube: Liebigs Anualeu. 332. 64 (1904). Scherer: 108. 129 (1858). Liehig?. Annalen. 73. 328 (1850). Chein. Gesellsch. 30. — Vgl. auch Strecker: Ebenda. Nukleoproteidc. Xukleiusaureu uiul iliro Baiisteino. g49 Harn ^) und in den Fiizes.-) Hypoxanthin wurde ferner \ on Kassel ») beim Abbau von Nukleoproteiden erhalten. Es bildet mikroskopische Nadeln. Es in Wasser ziemlich schwer und bildet mit Basen. Säuren und löst sich Salzen Verbindungen. Das Xanthin) ist von Marcct^>) in Harnsteinen entdeckt worden. Es findet sich im Harn «) und in den Fäzes. ^) Bei der Spaltung von Nukleinsäuren wurde es von Kossei ^) aufgefunden. Es kristallisiert mit einem Molekül Wasser in farblosen, zu Drusen vereinigten, rhombischen Platten. Es löst sich sehr schwer in Wasser. Es sind Verbindungen des Xanthins mit Basen, Säuren und Salzen bekannt. Wird eine xanthinhaltige Flüssigkeit mit Chlorwasser eingedampft, dann hinterbleibt ein gelb gefärbter Rückstand. Bei höherer Temperatur wird er rot. Befeuchtet man ihn mit Ammoniak, dann tritt eine prachtvolle purpurrote Färbung auf (Murexid).") Diese Reaktion ergibt auch die Harnsäure. Neben den Aminopurinen x\deuin und Guanin sind am Aufbau der Nukleinsäuren noch Pyrimidinbasen beteiligt. Ihre Entdeckung^ verdanken wir Kossei. Es sind drei Vertreter dieser Klasse von \'erbindungen aus Nukleinsäuren gewonnen worden, nämlich das Thymin, das Zytosin und das Urazil. Sie leiten sich vom Pyrimidinkern ab. Auch in ihm sind die einzelnen Elemente mit Zahlen versehen worden, damit die Stellung der einzelnen Gruppen bei den einzelnen Verbindungen genau gekennzeichnet werden kann I I HC(2) (5)CH 11 II Der Pyrimidinkern ist auch im Purinkern enthalten, wie die Formel I Formel II stellt dar, daß im Purinkern auch der Imidazolkern vorhanden ist: zeigt. N— N— (' ("— (' C : C— I /c N— )(' N— ("— I (' I. ' N II. — ) E. Salkowski: Mrchows Archiv. 50. 19,') (1870). G. Saloiuon: Zeitschr. f. physiol. Chemie. 2. 94 (1878/79); 11. 410 (1887). ^) M. Krüger und A. Schittenhelm : Zeitschr. f. physiol. Chemie. 35. 158 (1902). A. Weintraud: Zentralbl. f. iuu. Med. 16. 453 (1895). ') A.Kossel: Zeitschr. f. physiol. Chemie. 3. 291 (1879); 5. 152 (1881); 10. 258 (1886). •) Vgl. seine Synthese bei Emil Fischer: Ber. d. Deutschen Chem. Gesellsch. 30. 2232 (1897). W. Traube: Ebenda. 33. 1371, 3043 (1900). ^) Marcet: An essay ou the chemical history and mcdical treatment of calcul disorders. London 1817. Vgl. auch Wöhler und J. Liebig: Liebigs Annalen. 26. 340 (1838). «) Sirecker: Lie%s Annalen. 102. 208 (1857);' 108. 140, 151 (1858). Scherer: Ebenda. 107. 314 (1858). - M. Krüger und G. Salomon: Zeitschr. f. physiol. Chemie. 21. 1B9 (1895). M. Stadthagen: Virchoivs Archiv. 109. 414 (1875). ') M. Krüger und A. Schittenhelm: Zeitschr. f. physiol. (^hem. 35. 161 (1902) ") A. KoRsel: Zeitschr. f. i)hysiol. Chemie. 4. 292 (1880). ») V^'l. S. 590. — — — — — 1 Nukleoproteide. Nukleinsäuren und ihre Bausteine. (551 Die Entdeckung- des Thymins verdanken wir ebenfalls A. Kossei und A. Neumann.^) Das Zytosin kristallisiert in farblosen, durchscheinenden Platten.2) Die Lösung dieser Verbindung ergibt, mit liromwasser erwärmt, auf Zusatz von Barytwasser eine Purpurfärbung. Thymin ist auch in Es bildet dendritisch oder sternförmig Kristallform erhalten worden. gruppierte kleine Plättchen. Beim vorsichtigen Erhitzen sublimiert es.») Das Urazil endlich Form feiner Nädelchen.) kristallisiert in Als weitere Bausteine der Nukleinsäuren haben wir die Phosphorsäure und Kohlehydrate erwähnt. Die Kohlehydratgruppe ist ohne Zweifel nicht einheitlich. Es sind Kohlehydrate der Sechskohlenstoffreihe und ferner Pentosen am Aufbau von Nukleinsäuren beteiligt. Die Art der ersteren konnte bis jetzt nicht mit völliger Sicherheit festgestellt werden, dagegen ist als Baustein mancher Nukleinsäuren die Pentose d-Ribose erkannt worden:'') H— C— OH H— C— OH H— C— OH 1 I CH2 . OH d-Ribose. Ob daneben noch andere Pentosen, z. B. die Xylose''), als Baustein von Nukleinsäuren in Betracht kommen, ist sehr fraglich geworden. Auf das Vorkommen von Hexosen in Nukleinsäuren ist geschlossen worden, weil bei der Spaltung von solchen mit starken Säuren Lävulinsäure, CH3 CO CH2 CH, COOH, zur Beobachtung kam. Hexosen liefern nämlich unter den gleichen Bedingungen ebenfalls diese Säure. Ferner ist es gelungen, bei der Spaltung von Nukleinsäuren eine Epizuckersäure genannte, der Zuckersäure nahe verwandte Kohlehydratsäure zu . . . . isolieren.^) A. Kossei und A. Nemnann: Ber. d. Deutschen Ghem. Gesellsch. 26. 2753 (1893); Vgl. ferner //. Steudel und A. Kossei: f. phvsiol. Chemie. 22. 188 (1896). W. Jones: Ebenda. Ebenda. 29. 3Ö3 (19Ü0). H. Steudel: Ebenda. 32. 241 (1901). Wl. Gidewitsch: Ebenda. 27. 292. 368 (1899). 29. 20 (1899). ") F. 320—325" unter Zersetzung. ^) F. 321'' unter Gasentwicklung. ) F. 325" unter Zersetzung. °) Vgl. S. 29. P. A. Levene und W. Jacobs: Ber. d. Deutschen Chem. Gesellsch. Vgl. auch F. Haiser und 42. 1198, 2102, 2469, 2474, 3247 (1909); 43. 3142 (1910). /-'. Wenzel: Mouatsh. f. Chemie. 31. 357 (1910). 8) Vgl. Carl Neilberg: Ber. d. Deutschen Chem. Gesellsch. 35. 1467 (1902). ('. Neuherg und ß. Brahn : Biochem. Zeitschr. 5. 438 (1907). ') H. Steudel: Zeitschr. f. phsyiol. Chemie. 50. 538 (1907): 52. 62(1907); 55. 4()7 ') Zcitschr. — — — — — — — (1908); 56. 212 (1908). XXXI. Vorlesung. (352 Neuerdings wird die Frage erörtert, ob der Sechskohlenstoffzucker mancher Nukleinsäuren in Beziehung zum Glukal'), einer Verbindung der Formel Ce,H,„0,, stehtJ) Nachdem wir nunmehr die Konstitution der einzelnen Bausteine der Nukleinsäuren kennen gelernt haben, ergibt sich ganz von selbst die P'rage, in welcher Weise die einzelnen Verbindungen in diesen verknüpft sind. Es ist auch hier wie bei den Proteinen versucht worden, durch stufenweisen Abbau der einzelnen Nukleinsäuren zu Produkten zu gelangen, die noch mehrere Bausteine gebunden enthalten. Das Studium derartiger Bruchstücke mußte ohne Zweifel zu Anhaltspunkten über die Struktur des Ausgaugsmateriales selbst führen. Der ganzen Forschung auf diesem Gebiete kam die Beobachtung zu Hilfe, daß einfachere, den Nukleinsäuren sehr nahe stehende Verbindungen zum Studium der Verkettung der einzelnen Bausteine zur Verfügung standen. Es handelt sich in erster Linie um die von J. L'ich/g^) im Fleisch extrakt aufgefundene Inosinsäure. Sie enthält je ein Molekül Phosphorsäure, d-Pibose und Hypoxanthin. Es ist nun geglückt), dieses Nukleotid einerseits in eine Verbindung zu zerlegen, die Phosphorsäure und d-Ribose enthält und andrerseits in eine solche, in der die Pentose mit Hypoxanthin verknüpft ist. Man kann diese aus zwei Bausteinen zusammengesetzten Produkte, da sie ein Kohlehydrat enthalten, als Glukoside auffassen und sie dementsprechend nach dem Vorschlage von Leren c Nukleoside nennen.^) Die Inosinsäure liefert bei der teilweisen Hydrolyse die Nukleoside d-Ribose-phosphorsäure und d-Ribose-hypoxanthin. Die Verbindung ist Inosin genannt worden. Zweckmäßiger ist letztere die Bezeich- nung Hypoxanthosin: Phosphorsäurerest (1-Kiboserest H //OH 0=:P^0 — CH2 \0H . H 11 III G C Cf III OH OH OH C . . A) . "H d-Ribose-phosphorsäure Vgl. Emil Fischer: Ber. d. Deutscbeu Chem. (iesellscli. 47. 19(5 (1914). Vgl. R. Feulqen: Zeitschr. f. pbysiol. Chemie. 100. 241 (1917). R. Fenlgcn G. Landmann: Ebenda. 102. 2G2 (1918). iiiul Vgl. aucb S. 20 und 27. ') J. V. Liebifi: Liebifß Annalen. 62. 317 (1847). Gregori: Ebenda. 64. 1Ü(> F. Haiser: Monatsb. f. Cbemie. 16. 190 (1895). F. Bauer: Hofmeistern (1847). Beitr. 10. 34ö (1907). C. Neuberg und B. Brahn: Ber. d. Deutschen Cbeni. Gesellscb. 5. 478 (1907). F. Ifaiser und F.'Wnizel: Ebenda. 29. 161 (1908); l)>. 147, 377 (1909): 31. 357 (1910). ) P. A. Lcvene und W. A. Jacobs: Ber. d. Deutschen dheni. (iesellscb. 42. 33f». 1198 (1909); 43. 3162 (1910): 44. 746 (1911). ^) Vgl. über die Synthese von Nukleosiden: Emil Fischer und Helfe ri eh Ber. d. Deutschen Chem. fiesollsch. 37. 210 (1914). Emil Fischer, B. Helferich und P. Ostmann: Ebenda. 53. 873 (1920). B. Ilrlicrirh und M. r. Kühlirei'n: Ebenda. 53. 17 (1920). ) — •') — — — — — - /:-'. — — Nukleoproteide. Xiikleinsiuireu uud ihre Bausteine. Hypoxanthinrest d-Riboserest H CHo (OH) C . C . H H II C . 658 OC- N— C . -XH Ht'^ OH OH CH / II ii — N d-liihose-h}'poxanthin=:Inosin = Hypoxanthosin. Noch unentschieden ist, ob >!-Stellung- verbunden aufnahme in die Kibose mit der Purinbase in 7- oder Beide Verbindungen zerfallen unter AVasser- ist.i) ihre Anteile: II OH = P^0 — CH. (' C . . ' . C C< ; + H,0 ^H . - OH OH OH OH d- Kibose- phosphorsäure H H II OH = P^OH + CH., (OH) . C C . C . . C, OH OH OH OH Phosphorsäure I CH, (OH) . C H H H d-Piibose H C . C . N— C . ! N— ' H CH, (OHj . C 1 ! i b C se- hy p . C . .^O Cf\: ^^ -X OC—NH /NH— C CH + HC ^N I i OH OH OH • II C — II Hypoxanthin. (l-i;ibose ^oinit C- Xan thin H II . CH + H, () HC^ OH OH 1 d- -XH OC- 1 Die Inosinsäure muß nach dem Ergebnis die folüende Struktur besitzen: der teilweisen Hydrolyse ') \?1. hierzu Richard Buriau : Ber. d. Deutscheu Chem. (iesellsch. 37. 696,' 708 (1934): Zeitschr. f. physinl. Chemie. 51. A'lb (1907). Ihinf: Fischer: Klienda. 60. 69 (1909). — XXXI. Vorlesuiicf. 654 OH =P— H () ( . 'H, C . C . C . I ! . N— C C HC ! ; OH OH OH OC— NH H H H 1 O CH \N— C— oder OC— NH OH =P— H H CH, . C . (^ . OH H H . C . HN— C C CH >. N— C— OH OH ()- Der Komplex Phosphorsäure-Kohlehydrat-Purin oder -Pyrimidin ist von Levene als Nukleotid bezeichnet worden. Ist er nur einmal vorhanden, dann spricht man von Mononukleotiden. Finden sich jedoch mehrere solcher Nukleotidkomplexe in einem Molekül vereinigt, dann wird das durch die Bezeichnung Polynukleotid zum Ausdruck gebracht. Kennt man die Anzahl der am Aufbau einer bestimmten Nukleinsäure beteiligten Nukleotide, dann wird man genauere Namen, wie Di-, Tri-, Tetra- usw. -nukleotide wählen. Ganz allgemein sind die Nukleinsäuren nach dieser Art der Bezeichnung als Polynukleotide aufzufassen. Ein weiteres, bis jetzt bekanntes Mononukleotid ist die Guanylsäure. Sie ist von Ivar Bang^) in der Pankreasdrüse entdeckt worden. 2) Bei ihrer Spaltung erhält man je ein Molekül Phosphorsäure, d-ßibose und Guanin. Bei der teilweisen Hydrolyse sind die beiden Glukoside d-Ribose-phosphor säure und d-liibose-guanin = Guanosin: H CH2 (OH) . C H . C . C OC H H . —OH OH N C HC —NH —C C.NHa \N— C— Guanosin. erhalten worden. 3) Der Guanylsäure muß daher eine der Inosinsäure ähnliche Konstitution zukommen), nur findet sich an Stelle des Hypoxanthins Guanin. Außerdem scheint nach neueren Ergebnissen die Bindung zwischen der d-Piibose und der Phosphorsäure eine andere als bei der Inosinsäure zu sein. Sicher wird auch bei diesen Verbindungen erst die Synthese ein endgültiges Urteil über ihre Konstitution ergeben. Bemerkt sei noch, daß ») Ivar Bang: Zeitsclir. phjsiul. Chemie. 20. 183 (1898); 31. 411 (1900). — Itar Bang und ('. A. Raaschou: Hofmeister?, Beitr. 4. 175 (1903). Vf^l. auch W. Jones und f. — — L. G. Rowntree: Journ. of biol. Chem. 4. 289 (1908). Walter ,/o»(?s;' Ebenda. 12. 31 (1912). ) Vgl. ihre Eigenschaften bei li. Feulgen: Zeitschr. f. pliysiol. Chemie. 106.249(1919). ) P. A. Levene und W. A. Jacobs: Ber. d. Deutschen Chem. (lesellsch. 42. 24()9 (1909); Biochem. Zeitschr. 28. 127 (1910). ) r. A. Levene und W. A. Jacobs: Biochem. Zeitschr. 28. 127 (1910). Vgl. auch //. Stetidel und P. Brigl: Zeitschr. f. pbysiol. Chemie. 68. 40 (1910). — Nukleoproteide. Nukleinsäuren und ihre Bausteine. 655 Guanosin in freiem Zustand aus der Pankreasdrüse gewonnen worden ist. Es kommen somit in der Natur freie Nukleoside vor. Ferner erwies sicli das in Pflanzen aufgefundene Vernin') als Guanosin. Es läßt sich durch Desaminierung in Xanthosin, d. h. eine Verbindung von d-Iiibose mit Xanthin überführen. Auch durch Fermente wird diese Umwandlung bewirkt.-) Wir haben bereits erwähnt, daß man die Nukleinsäuren als aus mehreren Nukleotiden bestehend aufgefaßt und deshalb als Polynukleotide bezeichnet hat. Daß diese Bezeichnung zurecht besteht, geht daraus hervor, daß es geglückt ist, aus Nukleinsäuren sowohl Nukleotide als Nukleoside ») zu gewinnen. Aus Hef enukleinsäure ) sind Guanosin und Ade no sin erhalten worden.^) Das letztere Nukleosid stellt eine Verbindung zwischen d-Eibose und Adenin dar. Es hat die folgende Struktur: H CH., (OH) . H H C . C C . NH, H OH OH HC I ! C=N N— C C- . . CH \N— C— Adenosin konnte durch Desaminierung in Inosin, d. h. in Hypoxanthosin. übergeführt werden. Auch Organfermente vermögen diese In der Mutterlauge der beiden Nukleoside noch Produkte, die Pyrimidinbasen gebunden enthalten. Sie Am Aufbau dieser beiden sind Zytidin und Uridin genannt worden. Umwandlung zu vollziehen.") fanden sich Verbindungen sind Zytosin bzw^ Urazil und d-Ribose beteiligt.') Über die Art der Verknüpfung der Pyrimidinbasen mit dem Kohlehydrat sind wir noch nicht genau unterrichtet. Es kommen folgende Strukturmöglichkeiten in Fraee: C CH., (OH) H H H . C C . . OH OH H ^"^-^—^ C C CO Cn 0- —NH I II 1 I oder N=^=C NH, . H H CH., (OH) . C . C H H . C . C CH CO N— -CH —OH OH Zytidin (d-Ribose-zytosin). K Schulze: Zeitschr. f. physiol. Chemie. 66. 128 (1910). r. A. Levene und W. A. Jacobs: Biochem. Zeitschr. 28. 126 (191Ü). ^) P. A. Levene und IT, A. Jacobs: Journ. of biol. Ghem. 12. 421 (1912). ) Ihr Entdecker ist Alf mann: Arch. f. (Anat. u.) Physiol. 529 (1889). Ber. d. Deutschen Clieni. Gesellsch. 42. 2474, 5) P. A. Lerene und W. A. Jacobs: 2703 (1909); 43. 3150 (1910). S. Amberg und W. Jones: 6) W. Jones: Journ. of biol. Chem. 9. 169 (1911). ') ') — Zeitschr. f. physiol. Chemie. 73. 407 (1911). ') P.A. Levene und F.B.LnForge: Ber. d. Deutschen Chem. Gesellsch. 45. 608(1912'. XXXI. Vorlesung. 656 H H H H CO XH I ! CHo (OH) C . C . . OH OH CO C (" (' . CH— NH I 1 ( oder H CH. (OH) . C H H C . H C . C . -CO OH OH 1 CH CO NH - CH Uridin (d-Ribose-urazil). Ferner ist es geglückt, aus Hefeuukleinsäure auch Verbindungen von Zytidin^) und Uridin-i mit Phosphorsäure zu gewinnen. Neben diesen Nukleotiden sind noch die folgenden isoliert worden: Adenosin-^) und Guanosinphosphorsäure.) Aus Nukleinsäure, die aus der Thymusdrüse abgetrennt worden war. ist eine Thymin-hexose-phosphorsäure isoUert worden. »i Diese Verbindung ist in ihrer Zusammensetzung nach unter die Nukleotide einzureihen. Sie liefert bei der vollständigen Spaltung Thymin, Lävuhnsäure und Phosphorsäure. Ein weiteres ans Thymusnukleinsäure isoliertes Nukleotid stellt die Zytosin-hexose-phosphorsäure dar.^) Ferner ist ein Guaninhexosid aus der gleichen Nukleinsäure abgespalten worden.^) Es kommt ihm folgende Struktur zu: H CHa (OH) . C . C OH H . C . Oll C . C OH H (' C 'N— C N -N HC \ . NH., EndHch sei noch erwähnt, daß die aus Weizenembryonen gewonnene Tritikonukleinsäure^) beider teilweisen Hydrolyse auch Nukleoside. und zwar Guano sin und Adenosin ergab.-') Ferner wurde Zy tidin gewonnen. ') P. A. Leveiic und M\ A. Jacobs: Ber. d. Deutschen Cliem. Gesellsch. 44. ./. 1 hannhauser und //. Dorf1027 (1911); Journ. of biol. Chemie. 124. 11 (1912). müller: Zeitschr. f. physiol. Chemie. 104. 65 (1918). ^) S. J. Thannhanscr und //. iJorfmüUer : Zeitschr. f. physiol. Chemie, lül). 121 1'. A. Levene: Journ. f. biol." Chemie. 33. 425 (1918); 4». 415 (1919). (1917J. Walter Jones und B. P. Kennedy: J. Pharm, and experim. Ther. 12. 253 (1918); 13. 45 (1919). ) P. A. Lerene: Journ. f. biol. Chem. 31). 77(1919i: 40. 171 (1920); 41.483 (1920). ^)P.A.Lcrene und J.A.Mandel: Ber. d. Deutschen Chem. Gesellsch. 41. 1905 (1908). ) S. J. 1 hannhauser und B. Ottmslein: Zeitschr. f. physiol. Chemie. 114. 39 (1921). ) P. A. Lerene und W.A.Jacobs: The Journ. of biol. Chem. 12. 377 (1912). Vgl. auch John A. Mandel und Eduard K. Ihtnliam: Ebenda. 11. 85 (1912). ) 'Ihoinas B. Oshorne und harte F. Harris: Zeitschr. f. physiol. Chemie. 36. 85 (1902); Annal. Report of the Connecticut agricult. experim. Station. 305 (1900); 3(55 (1901); Journ. of the Americ. Chem. Soc. 22. 379 (1900). ") P. A. fjevene und F. B. La Forqr Ber. d. Deutsclioii Chem. Gesellsch. 43. — — '') — : 31fi4 (1910). -S. Xukleoproteide. Niikleiusaureu uud ihre Bausteine. ()0 ( Aus den mitgeteilten Feststellungen gebt klar hervor, dali in der Nukleinsäure Nukleotide bekannter Struktur enthalten sind. Es bleibt nun noch die Frage, in \Yelcher Art und Weise sie in den einzelnen Nukleinsäurearten unter einander zu Polynukleotiden vereinigt sind, und welche Nukleotide und in welcher Anzahl diese im einzelnen Molekül auftreten. Auch nach dieser Richtung sind bedeutsame Fortschritte erzielt. Es ist gelungen, Hefenukleinsäure in Uridinphosphorsäure und ein Trinukleotid^ -) Von aufzuspalten. dem T/mnnhauser hat letzteren folgende Strukturformel entworfen: OC— Nil II HO II I ( ) (' C'H., . 011 Ol! HOx 0=^P 11 () . C CH., . C (' . OH/ C . HOv O CH, . 110/ . H H C C (' . I! . . N— C Adeiiyl- CH siluro HC^ I H (iuanyl- I I OH OH OH MI. N— C— 0=N ' i . I; NH.3 C— i C s;iure HC< OH H 11 . N— C . N— (- i C N (' CO (11 . Nil. Zytidin- OH OH —O phosphorsäure I NH CH Guanosyl-adenosyl -zytidyl-triphosphorsäure. Durch Hydrolyse der genannten Verbindung konnten zwei kristalProdukte erhalten werden. Das eine erwies sich als Zytidinphosphorsäure 3) und das andere als das Dinukleotid der Guanosin- lisierte adenosinphosphorsäure.) Schließlich ist es auch geglückt s), ein Purinnukleotid aus den Spaltprodukten des erwähnten Trinuklontides zu i.-^olieren. nämlich die Adenosinphosphorsäure: ') S. Ihannhauser und G. Dorfmüller: Zeitscbr. f. plivsiol. J. ( heinie. 100. 121 (1917). ") Die von Thannhauser und Dorfmüller eingeführte Bezeichnun? Tripbosphornukleinsäure für diese Verbindung erscheint mir niclit besonders glücklich gewählt zu sein. Unter Nukleinsäure verstehen wir ein Polyuuklootid. Damit sind die Pbosphorsäuremoleküle mit eingeschlossen. Man wird, um allen Mißverständnissen vorzubeugen, die Bausteingruppen mit der Angabe, wieviele Nukleotide vereinigt sind, anführen müssen. Vgl. auch .S. ./. Thaiinluiiiser und d. horlniiiller: Zeitschr. f. plnsiol. Chemie. 'i 104. 6;^ ) 4(j7 (1919). ^'. ./. Thannhauser und (j. Dorfmiiller : Ber. d. Deutschen Chem. (iesellsch. 51. (1918). ^) S. J. Thannhauser : Zeitschr. f. physiol. Chemie. 107, Vgl. ferner 157 (1919). Walter Jones und A. PJ. Richards: .lourn. of biol. Chemie. 20. 25 (1915). Walter Jones und B. E. Read: Ebenda. 29. 111, 123 (1917). P. A. Lerem: Jonrn. of biol. (.'hem. 31. 591 (1917); 33. 229, 425 (1918); 40. 415 (1919): 41 19, 4SB (1920). — Abderhalden, Physiologische Chemie. I. Teil, .'j. Aufl. — 42 XXXI. Vorlesuuff. 658 H H H H0\ I I I ) . CHo . C . H C . C NH, C- HO/ ( G=N -N— C I . . HC^ '% )H im CH II N— C— — II Sind wir auch zur Zeit über den allgemeinen Plan des Aufbaues der Nukleinsäuren ganz gut unterrichtet, so darf doch nicht übersehen werden, daß wir über die Art und Weise, wie die Nukleotide im Polynukleotid untereinander verknüpft sind, noch keine bestimmten Angaben machen können. Es stehen sich verschiedene Ansichten gegenüber. Levene^) ist der Ansicht, daß die Phosphorsäuremoleküle in den Polynukleotiden durch zweifache Veresterung mit den Kohlehydratgruppen vereinigt sind: HO 0= =P— .C.H^O., HO . C5H4N0O i =P— . C5 H, 0.3 . C, H, N., HO =P— Thannhauser '"") dagegen stellt sich vor, daß der von ihm aus der Hefenukleinsäure gewonnene Trinukleotidkomplex (vgl. S. 657) durch ätherartige Sauerstoffbrücken von Kohlehydrat zu Kohlehydrat zusammengehalten wird. Ein solcher soll dann jeweils mit einem Mononukleotid unter Anhydrierung des Phosphorsäurerestes verknüpft sein. ^) Die Konstitution des aus der Thymusdrüse isolierten Polynukleo- noch umstritten. Wie schon S. 656 erwähnt, ist es gelungen, aus ihm Nukleotide zu isolieren. Bei vorsichtiger Spaltung ist aus der Thymusnukleinsäure eine Verbindung erhalten worden, Thymosinsäure) genannt, deren Bariumsalz etw^a, wie folgt, aufgebaut sein soll :^) tides ist auch — Die Angaben von Walter ) P. A. Leccne: Joiirn. of biol. Chem. 33. 229 (1918). Jones und A. E. Richards: Jotirn. of biol. Chem. 20. 25 (191o) und Walter Jones und B.E.Read: Ebenda. 29. 111 (1917), wonach es diesen Forschern gelungen ist, aus Hefenukleinsäure Diuukleotide (Guaninzytosin- und Adeniuurazil-dinukleotid) zu isolieren, sind nicht eindeutig. Es ist möglich, daß Gemische von Mononukleotiden vorgelegen haben. Vgl. dazu P.A. Levene: Jouru. of biol. Chem, 33. 425 (1918). ) Thannhauser und P. Sachs: Zeitschr. f. pliysiol. Chemie. 109. 177 (1920). Vgl. ferner W.Jones und B. E. Read: J. of biol. Chem. 29. 111 (1917). Dieser von IL Sfeudel vorgeschlagene Name ersetzt die Bezeichnung 'rhymiusäure. Vgl. auch Stendel ) R. Feulgen: Zeitschr. f. physiol. Chemie. 101. 296 (1918). S. J. Thannhauser und B. Ottenstein S.Reiser: Ebenda. 111. 297(1921). ') •) und Ebenda. 114. 39 (1921). — Xuklooproteide. Nukleinsäuren und ihre Bausteine. 059 /Phosphorsäure— Kohlehydrat .... BaC — Kohlehydrat — Zvtosin .Phosphorsäure — Kohlehydrat — Thymin rhosphorsäure 1 Ba< Phosphorsäure — Kohlehydrat .... der Thymusnukleinsäure selbst würden in dieses Molekül noch Guanin und Adenin einzufügen sein, und zwar in Bindung mit den beiden Kohlehydratgruppen, die in obiger Formel noch nicht besetzt sind.i) In Die Nukleinsäuren sind nach dem Gewebe oder den Zellen, aus denen sie zum erstenmal isoliert worden sind, benannt worden. Es liegt auf der Hand, daß eine solche Art der Bezeichnung zu Schwierigkeiten führen muß. So begegnen wir z. B. der Angabe, daß in der Pankreasdrüse ThymusSobald die Konstitution der einzelnen Nukleinnukleinsäure vorkommt säuren aufgeklärt sein wird, wird man die bisherigen Namen fallen lassen müssen, um sie durch solche zu ersetzen, die auf die Zusammensetzung der Verbindung Bezug haben und nicht nur an ihre Herkunft erinnern. ! Wir kennen eine Thymusnukleinsäure.2) Sie ergibt bei der Spal- tung, wie schon erwähnt, eine Hexose, die erwähnten Purin- und Pyrimidinbasen und Phosphorsäure. Sie stellt ein weißes Pulver dar, das in kaltem Wasser schwer löslich ist. Dagegen ist sie in Alkalien, in Ammoniak und in Alkalikarbonat und -azetat leicht löslich. Durch Mineralsäuren läßt sich die Nukleinsäure wieder ausfällen. Mit Erdalkalien bildet sie lösliche Neutralsalze. Bei genügender Konzentration erstarren diese Lösungen zu einer Gallerte. Die aus Thymus isolierte Nukleinsäure dreht nach rechts. Wichtig ist die Beobachtung; daß sie schon bei gewöhnlicher Temperatur in saurer Lösung verändert und gespalten wird. Gegen Alkalien ist sie beständiger. Ferner ist eine Nukleinsäure Harn aus beschrieben. 3) Ihre Zu- sammensetzung und ihre Herkunft sind noch nicht genügend festgestellt. Außerdem sind noch Nukleinsäuren aus verschiedenen Organen isoliert worden.) S. 656 dargestellt, die aus Hefe mit Ausnahme der Kohlehyhrat- Eingehend untersucht ist ferner, wie dargestellte Nukleinsäure.») Sie gleichen Bausteine, ergibt wie die Thymusnukleinsäure. Sie dreht gruppe die auch nach rechts. Das an ihrem Aufbau beteiligte Kohlehydrat ist eine Pentose, und zwar die d-Iiibose. Von Nukleinsäuren der. Pf lanzenweit ist bis jetzt nur die aus Weizenkeimlingen isolierteTritikonukleinsäure") eingehender untersucht worden. Sie ist sehr schwer von Beimengungen zu befreien. Ihre Eigenschaften entsprechen in den meisten Punkten denen der Thymusnukleinsäure. Die >) E. Fciügen: Zeitschr. f. physiol. Chemie. 101. 288 (1918); 104. 2) V gl. S. (556, 658. 1 (1919). K. A. H. Monier: Skand. Arch. f. Physiol. 6. 372 (1895). Vgl. z. B. ./. A. Mandel und P. A. Levene : Zeitschr. f. physiol. Chemie. 140, 151 (1906); 49. 262 (1906); 50. 1 (1906). 3) ) 5) — Thomas B. Oshorne: The Americ. Journ. of Physiol. 9. 69 — Thomas B. Oshorne und F. W. Heiß: Ei)euda. 21. 157 (1908). «) (1903). Vgl. S. 656. Vgl. S. 656. 47. 42 (360 XXXI. Vorlesung. Tritikonukleinsäure enthält Phosphorsäure, (Tiianin. Adenin, Zytosin. Thymin d-Ribose, außerdem scheint Urazil als primärer Baustein zugegen zu sein. Wir werden bei der Besprechung des Verhaltens der Nukleinsäuren im tierischen Organismus uns hauptsächlich mit den Umwandlungen ihrer Bausteine l)eschäftigen. »Sobald wir von einer Verbindung ihre Konstitution nicht in allen Einzelheiten genau kennen, erhalten wir auf zahlreiche Fragestellungen keine genaue Antwort. Wir können zurzeit weder die Frage erörtern, in welcher Weise sich die Nukleoproteide der einzelnen Zellarten unterscheiden, und ebensowenig sind vergleichende Studien über die Eigenart einzelner Nukleinsäuren möglich. Es wiederholt sich bei allen zusammengesetzten Verbindungen das gleiche Bild. Wir sind über die Bausteine der einzelnen Substanzen genau imterrichtet. Ferner kennen wir meistens auch die Konstitution von Verbindungen, die mehrere von diesen Bausteinen enthalten. Je hoher wir jedoch in der Reihe der Abbaubzw. Aufbaustufen der betreffenden Verbindung aufsteigen, um so unsicherer werden unsere Kenntnisse. Sie versagen schlieljlich ganz, wenn wir bei jenem Produkte angelangt sind, das Bestandteil der Zellen ist. Immerhin eröffnet die Kenntnis der Zusammensetzung derartiger, aus mehreren Bestandteilen bestehenden Verbindungen aus einzelnen Bausteinen mit bekannter Konstitution tiefe Einblicke in manch'e Stoffwechselvorgänge. Vor allem können wir von den Bausteinen aus den weiteren Abbauprodukten bis zu den Stoffwechselendprodukten folgen. und Vorlesung XXXII. ^ukleoproteide. Nukleinsäuren und ihre Bausteine. Entstehung der Nukleoproteide und der Nukleinsäuren nebst ihren Bauin der Pflanzen- und Tierwelt. Ihr Verhalten im tierischen Organismus. Die StofFwechselendprodukte der Bausteine der Nukleinsäuren. Störungen des PurinstofFwechsels. steinen Die Nukleoproteide sind in der Pflanzenwelt ebenso verbreitet, wie doch die Kerne der Zellen der ganzen Organismenwelt aus Vertretern dieser Körperklasse aufgebaut. Stets finden wir Eiweilianteile mit Nukleinsäuren gepaart. Sicher bildet die Pflanze die einzelnen Bestandteile der Nukleoproteide für sich, um dann durch ihren Zusammenschluß das fertige Produkt entstehen zu lassen. Die Frage des Anfbaus des Eiweißes in der Pflanzenzelle führten wir auf das Problem der Bildung der einzelnen Aminosäuren zurück. Ebenso müssen wir die Synthese der Nukleinsäuren von derjenigen der einzelnen Bausteine aus studieren. Zwei davon sind in ihrer Herkunft leicht aufzuklären. Es sind dies die Phosphorsäure und das Kohlehydrat. Die erstere wird Das Kohlehydrat ist ohne als Phosphat mit den Wurzeln aufgenommen. Zweifel ein direktes Produkt der Kohlensäure- und Wasserassimilation. Die Pentosen könnten auch durch Abbau einer Hexose etwa über die Glukuronsäure als Zwischenstufe gebildet werden'), doch sind für diese Art der Entstehung von Zuckern mit weniger als sechs Kohlenstoffatomen in der Pflanzenwelt keine Anhaltspunkte gegeben. Es bleibt noch die Frage zu lösen, wie die übrigen liausteine der Nukleinsäuren, die Purin- und Pyrimidinbasen, gebildet werden. Es ist bis jetzt nicht geglückt, bestimmte Zwischenstufen aufzufinden, die von bestimmten N'crhiudungen aus zu den genannton Produkten hinführen. Wir müssen uns leiiler auch hier darauf beschränken, den mciglichen Weg zu kennzeichnen. Die Purinbasen enthalten den Imidazolkern. Diesem sind wir schon beim Histidin begegnet. Es ist möglich, daß diese Aminosäure zu den Purinbasen Beziehungen unterhält. Über die mögliche Bildungsweise des Imidazols unterrichten uns Beobachtungen von Ulih/ans und Knoop"-) über in der Tierwelt, sind Vgl. S. 27. Ä. Winduus und F. Kiioop: Ber. d. Dcutsclieii Chem. Gesellsch. 38. Hofmtisfer?, Beitr. 6. 5<)2 (lOnfi). Vsjl. auch S. i:'.7. ') '-) 11()() (1005); XXXII. VorlesuuE 662 Entstehung dieser Verbindung bei Belichtung von Glukose und gleichEinwirkung von Zinkhydroxydammoniak. Die Bildung des ImidGlyoxalringes aus Glukose erfolgt höchstwahrscheinlich nach azolerfolstem Abbau dieser Verbindung in folgender Weise: die zeitiger = CH. GH., + CO H NU, . H C(g = + C- NH . + C CH^N NH3 Methylglyoxal 3 H,0 2 Moleküle Formal- Ammoniak dehyd Methvlimidazol Methylglyoxal und Form aide hyd entstehen offenbar bei der Spaltung der Glukose. Sie vereinigen sich bei Anwesenheit von Ammoniak zum Imidazolring. Die beiden genannten Forscher weisen darauf hin, daß man sich die Entstehung von Purinbasen aus den gleichen Bausteinen entstanden denken kann, indem man noch ein Molekül Harnstoff an der Reaktion sich beteiligen läßt: NH, CH. C = + CO H -f . NH, + ^^0 1 NH, ^^H Harnstoff + o/Ö TT H.O\jj -f 4 = NH3 Form aide hyd 2 Moleküle A m niak Methylglyoxal m NR— CO i CO ! C— Ml II NH— C— + 6 H. O /;CH Xanthin. Das so entstandene Xanthin könnte zu den übrigen Purinbasen hinführen. Noch leichter als aus Glukose erhält man Imidazole und ferner auch Pyrrole aus Glukosamin.i) Von großem Interesse ist das Vorkommen von methy Herten Xanthinbasen im Pflanzenreich. Wir haben bereits der Besprechung der Betaine die Fähigkeit des Pflanzenorganismus, Methylgruppen anzulagern, hervorgehoben. In den Blättern und Bohnen des Kaffeebaumes, in den Früchten von Paulinia sorbilis, im Paraguay tee (Hex paraguayensis) und in den KolaKaffein.^) nüssen findet sich ein 1, 3, 7-Trimethyl-2,6-dioxypurin bei = 19. 49 und J<J. Ludwig: Zeitsclir. f. pliysiol. Chem. 121. 170 (1922). Oudr;/: Mag. Pharm. Phytochemische P^ntdeckungen. 141 (1820). Paul;/ ') //. 2) /;mm(7p; (1827)." — Nukleoproteide. Nukleinsäuren und ihre Bausteine. 668 Die Kakaobohnen und die Kolanüsse enthalten ein 3, 7-Dimethyl-2, 6dioxypurin = Theobromin.^) Endlich ist aus Tee-Extrakt das Theophyllin = 1, 3-Dimethyl-2, 6-dioxypurin isoliert worden. 2) — Die Beziehungen dieser Verbindungen zum Xanthin sich aus der folgenden Gegenüberstellung 3): 2, 6-Dioxy- purin ergeben iNH — CO CO C — N .CH, CH — X/ CH3 — eCO I CH, .N 1 sC^^NH ,C() .\ ,CH /' — ,C-,N 3NH Xunthin 1, 3, NH — CO I I CO C .N C 7-Trimethylxanthin = Kaffein — CO CO C — NH CH. .N — N €H, . ^CH — — CH3 N C N 7-Dimethylxanthin imethy Theobromin 3, 1, CH3 .N C 3-Dimethylxanthin = Theophyllin. Man kann diese Verbindungen auch den Alkaloiden zurechnen. Sie haben alle eine ausgesprochene Wirkung auf die Harnausscheidung. Sie wirken ferner alle auf das Zentralnervensystem und die quergestreifte Muskulatur ein. Es ist noch ein weiteres Alkaloid bekannt, das den Imidazolkern besitzt. Es ist dies das aus Jaborandiblättern (Pilocarpus pennatifolius) gewonnene Pilokarpiu.) Über die Entstehung der Pyrimidinbasen in der Pflanzenwelt sind keine Anhaltspunkte vorhanden. Dagegen sind in Pflanzen noch mancherlei Verbindungen aufgefunden worden, die Beziehungen zu Bausteinen der Nukleinsäuren besitzen. Sie gehören entweder dem Ab- oder Aufbau von Purinbasen an. So ist wiederholt All an toi n nachgewiesen worden. s) Ferner hat Glykolharnstoff im Rübensaft aufge0. V. Lippmann^) Hydantoin funden. Diese Verbindung steht zur Glykolsäure in der gleichen Beziehung, wie das Allantoin zur Gyloxylsäure: = ) Woskresensky : Journ. f. prakt, Chemie. 23. 394 (1841); Liebic/s Anal. 41, 125 (1842). A. Kossei: Zeitschr. f. physiol. Chemie. 13. 298 (1889). Vgl. die Synthesen dieser Verbindungen hei Emil Fischer: Untersuchungen in der Puringruppe (1882—1906). J. Springer. Berlin 1907. ) Meyer: Liebigs Ann. 204. 67 (1880). A. D. Joivett : Procoed. of the Chem. A. Pinner: Ber. d. Deutschen Chem. üesellsch. 38. 1510.2560 Soc. 21. 172 (1902). 2) ) — — (1905). ^) E. Schulze und ./. Barbiert: Journ. (1896). f. prakt. Chemie. N. F. 25. 145 (1882); — Alfred Vogl: Pharm. Post. 51. 181 (1918). Zeitschr. f. physiol. Chemie. 11. 420 (188G). «) 0. P. Lippmann: Ber. d. Deutschen Chem. Gesellsc.h. 24. 3299 (1891); 29. 2652 XXXII. Vorlesung. ÖH4 CO OH \"0 + NH CO NH., = + 2 Ho () ^-CO I CK, OH Glykolsäure + -NH Hydantoin. Harnstoff COOH NH, CO NH.3 CH., NH — CH — NH NH., /CO + A\ ^-.o _ CO + CO 2H., O NH., I I NH NH., NH C OH Harnstoff (ilyoxyl- AHantoin. Harnstoff säure Ob endlich das in Pflanzen aufgefundene Kreatinin^) mit den Purinen und insbesondere mit dem Imidazolkern in Beziehung steht, ist hier ebenso unbekannt, wie beim tierischen Organismus. Der Umstand, daß man in Pflanzen häufig freie Purinbasen, z. B. Aden in-) und auch Oxypurine angetroffen hat, beweist ohne Zweifel, daß auch in der Pflanzenzelle ein reger Ab- und Aufbau von Nukleinsäuren stattfindet. Es sind dabei die gleichen Fermente wirksam, wie im tierischen Organismus. Es treten ohne Zweifel auch die gleichen Abbaustufen auf.^) Interessant ist das Vorkommen von Nukleosiden im Pflanzenreich. Eine solche Verbindung, das Vernin), erwies sich als Guanosin»): NH — C=:0 NH, C . N C C — — OHOH O -CH / CH . C H . C . C . CHo . OH H H Vernin zerfällt bei der Hydrolyse entsprechend seiner Konstitution Guanin und in d-Ribose. Man ist noch weiteren derartigen Verbindungen auf der Spur. So gehören Vi zin und Konvizin«) hierhin.') Beide in — 1) Vgl. z. B.: //. Tscheniorutzky: Zeitschr. f. physiol. (liemic. 80. 898 (1912). 7". Kikkoji: Ebenda. 51. 201 W. Zaleski: Iwanoff: Ebenda. 39. 31 (1903). (1907). Ber. d. Deutsch. Bot. Gesellsch. 29. 146 (1911). K. ('. Teodorrsco: Compt. rend. de Tacad. des sciences. 155. 300, 054 (1912). P. de la Blanchardilrc : Zeitschr. f. physiol. Chemie. 87. 291 (1913). -) Süll i ran .lourn. of tlie Americ. ('hem. Soc. 33. 2030(1911). /v ('. Sliorn/: — — — — — : Ebenda. 34. 99 (1912). •') Ginzaburo l'otani: Zeitschr. f. physiol. Chemie. 62. 113 (1909). ) E. Schulze: Zeitschr. f. physiol. Chemie. 9. 420 (1885). E. Schulze und Bosshard: Ebenda. 10. 80. (188(5); 41. 455 (1904): 66. 128 (1910). ^) Vgl. dazu S. (554. ») Ritthausen: Journ. f. prakt. Chemie. 2. 33(5 (1160); 7. 374 (1873); 59. 480 Bitthausen und Preuss: (1899); Ber. d. Deutschen (Jhem. (iosellscli. 9. 301 (1876). Jnura. f. prakt. (Jhemie. 59. 487 (1899). ') /<;. Schulze und G. Trier: Zeitschr. f. physiol. Chemie. 70. 143 (1910); 76. 145 — — (1911). Niiklooprotcidc. NuUlciusauion iiiid iliro 665 Bausteine. N'erhindungen finden sich in VViclvensanien und im liüiiensaft.i) Vizin der Hydrolyse 2 Moleküle (Hu kose und 4, 6-Dioxy-2, 5-diamino- liefert bei pyrimidin.^) Der tierische Organismus nimmt mit seiner Nahrung stets alle BeAn eine direkte Überführung der so außerordentlich komplizierten Verbindungen in seine Zellen ist nicht zu denken. Es muß auch bei dieser Klasse von Verbindungen ohne jeden Zweifel dem Umbau ein Abbau vorausgehen. Unsere erste Aufgabe ist, festzustellen, an welchem Orte im tierischen Organismus die Zerlegung der mit der Nahrung aufgenommenen Nukleoproteide einsetzt. In der Mundhöhle findet keine chemische Veränderung der Nukleoproteide statt. Der Speichel verfügt über keine Stoffe, die einen Abbau dieser Verbindungen herbeiführen können. Im Magen dagegen verändern auf welche Art und Weise unter sie sich. Es ist noch nicht festgestellt der Wirkung des Magensaftes Eiweiß aus den Nukleoproteiden abgespalten wird. Wir wissen nur, daß ein Teil des in diesen Verbindungen gebundenen Eiweißes bereits im Magen frei wird. Es verbleibt ein unlösliches Produkt, Nuklein genannt. Das abgespaltene Protein wird von Pepsin bzw. Labferment in Pepton verwandelt und verhält sich den Fermenten des Magendarmkanals gegenüber wie jedes andere Eiweiß Das Nuklein erfährt im Darm k anal eine weitere Spaltung. Es wird der Rest des noch an die Nukleinsäure gebundenen Eiweißes abgespalten und vom Trypsin weiter zerlegt. Auch hier wissen wir noch nicht, welches Ferment die Spaltung des Nukleins in die Ei weiß an teile und die Nukleinsäure vollzieht. standteile der Nukleoproteide auf. Es interessiert uns ganz besonders, zu erfahren, w^as aus der frei gewordenen Nukleinsäure wird. Man glaubte, daß sie unverändert zur Ilesorption komme. Pieagenzglasversuche zeigten jedoch bald-^), daß zwar Pankreassaft kaum eine Wirkung auf die Nukleinsäuren hat^). Darmsaft diese Verbindungen in einfachere Komplexe zu spalten.'') Diese Wirkung kommt einem besonderen Fermente, der Nukleinazidase oder auch Polynukleotidase ß) genannt, zu. Es bildet aus der Nukleinsäure Nukleotide. Ob der Abbau über die Bildung der reine dagegen vermag der wasserlöslicher Nukleotide hinausgeht und z. B. zu Nukleosiden führt, ist 0. V. LipjJiiiaiin: Ber. d. Deutscbeii Chem. Gesellsch. 29. 2653 (lS9ü). Vgl. F. A.Levcne: .)oiirn. of Biol. Chem. 18. 305 (1914); 25. 6ü7 (1916). E. Whiterauch Emil Fischer: Ber. d. Deutschen Chem. Gesellsch. 47. 2611 (1914). stein : Zeitschr. f. physiol. Chemie. 105. 258 (1919). ") Krnil Abderhalden und Alfred Schittenhclni : Zeitschr. f. physiol. Chemie. 47. ') -) 4r)2 — (1906). — — Fmil Abderhalden ) Fritz Sachs: Zeitschr. f. physiol. ('hemie. 46. 337 (1905). /'. A. Levene und F. Mediqreceanu: The Jouru, und Alfred Schittenhelin: 1. c. Zitat). E. S. London, Alfred Schitfcnhchn und K. Wiener: of Biol. Chem. 9. 375 (1911). — — physiol. Chemie. 70. 10 (1910); 72. 459 (1911); 77. 86 (1912). Carht '') Vgl. hierzu Nakaijama: Zeitschr. f. physiol. (Chemie. 41. 348 (1904). /'. .1. Levene und F. Medigreceanu: The Journ. Foa: Arch. di Fisiol. 4. 98 (1906). of biol. Chem. 9. 375 (1911). Ks-hat sich jedoch gezeigt, daü ) Friilier sprach man von einer Xukleasc. mehrere Fermente am Abbau der Nukleinsäuren zu ihren Bausteinen beteiligt sind. Für diese B^ermeute ist der Name Nuklei nasc vorgeschlagen worden. Er scheint uns Zeitschr. f. — — zweckmäßig, weil ohne Zweifel noch Fermente aufgefunden werden dürften, die das Nuklein spalten. Diesen käme dann nach der gebräuchlichen Nomenkhitur der Name Nukleinasen /u. lieshalb nicht ganz XXXIl. Vorlesung. ßßß Über diese letzteren hinaus geht der Abbau im Darmkanal im allgemeinen offenbar nicht. Insbesondere werden keine Purin- und Pyrimidinbasen abgespalten. Man findet diese Verbindungen und insbesondere die ersteren allerdings beständig in den Fäzes. Martin Krüger und Alfred Schitteuhelm^) konnten jedoch den Beweis erbringen, dall sie zum Teil von P>akterien, zum Teil von abgestoßenen Darmepithelien herstammen und nicht auf Nukleinsäuren der Nahrung zurückzuführen sind. zweifelhaft. >) Noch fast gar nicht erforscht ist die Frage, ob die Darmflora die Bestandteile der Nukleinsäuren und diese selbst angreift, sie verändert und zu charakteristischen Produkten abbaut. Wir wissen zwar, daß Bakterien die Bausteine der Nukleinsäuren und diese selbst in mannigfaltiger Weise um- und abbauen können. 3) Es sind uns jedoch keine bestimmten Produkte bekannt, die. wie es bei manchen Aminosäuren der Fall ist, im Darmkanal durch die Einwirkung der Darmflora entstehen und dann unverändert oder nach erfolgter Kuppelung mit bestimmten Verbindungen im Harn erscheinen und sich ohne weiteres auf bestimmte Bausteine zurückführen lassen. Dieser Umstand erschwert begreiflicher Weise die Entscheidung der Frage nach dem Umfang der durch Bestandteile der Darmflora herbeigeführten Veränderungen der im Darmkanal frei werdenden Nukleinsäuren und der aus ihnen hervorgehenden Nukleotide und Nukleoside außerordentlich. Immerhin ist der Schluß berechtigt, daß unter normalen Verhältnissen kaum erhebliche Mengen von Bestandteilen der Nukleinsäuren den Darmbewohuern zum Opfer fallen.) Anders liegen die Verhältnisse, wie wir gleich erfahren werden, wenn Purinbasen verfüttert werden. Es ist zunächst auffallend, daß der Abbau der Nukleinsäuren nicht bis zu den Bausteinen führt, sondern bei zusammengesetzten Verbindungen Da jedoch manche Bestandteile der Nukleinsäuren, wie z. B. halt macht. die Purinbasen, ganz allgemein außerordentlich schwer löshch sind, so könnte eine Aufspaltung der Nukleotide und Nukleoside in ihre BestandIn der Tat hat man beobachtet, daß teile deren Resorption gefährden. nach Verfütterung von Purinbasen ganz erhebliche Mengen davon im Kot enthalten waren, ja manchmal war der größte Teil dieser Verbindungen gar nicht zur Ptesorption gelangt. ^) Ferner wird ein sehr großer Teil davon von Bakterien abgebaut und entgeht so der weiteren Verfolgung. ^) Diese — ') Vgl. auch S. J. Thannhauser: Zeitschr. f. physiol. Chemie. 91. 329 (1914). Thannhauser und G. Dorfmüller : Ebenda. 100. 121 (1917). M. Krüger und A. Schittenhelm: Zeitschr. f. physiol. Chemie. 35. 153 (1902). A. Schittenhelm und A. Schittenhelm: Deutsches Archiv f. klin. Med. 81. 423 (1904). Martin Krüger und Alfred SchittenC. Tollens: Zentralbl. f. inn. Med. Nr. 30 (1904). S. J. — '•') — — helm: Zeitschr. f. physiol. Chemie. 45. 14 (1905). — ') Vgl. hierzu A. Schittenhelm: Zeitschr. f. physiol. Chemie. 39. 199 (1903). H. Plenge: A. Schittenhelm und F.Schrötter: Ebenda. 39. 203 (1903); 41. 4 (1903). — Ebenda. 39. 190 (19Ü3). ) Vgl. E. S.London, A. Schittenhelm und K. Hiener: Zeitschr. f. physiol. Chemie. J. Thannhauser und G. J)orfmüller: Zeitschr. f. physiol. Chemie. 77. 86 (1912). A. Schittenhelm und K. IJarpuder: Zeitschr. f. d. ges. exper. Med. 102. 148 (1918). 27. 29 (1922). A. Schittenhelm: Vgl. z. B. Stadthagen: Virchotvs Arch. 109. 390 (1887). Walter Hall: Journ. of Pathol. and Arch. f. exper. Patb. u. Pharm. 47. 432 (1902). Bacteriol. 2. 246 (1905). S. ./. Ihannhauser und «) V. 0. Siren: Pflügers Archiv. 157. 582 (1914). r;. f)orfmüller: Zeitschr. f. physiol. Chemie. 102. 148 (1918). — 'S'. — — •^) — — Nukleoproteide. Nukleinsäuren und ihre Bausteine. 667 Feststellung ist zur Beurteilung von Fütterungsversuchen mit Purinbasen sehr wichtig. Man darf allerdings aus dem Verhalten verfütterter Purinbasen nicht ohne weiteres Rückschlüsse auf die normalen Verhältnisse ziehen. Es brauchten ja beim stufenweisen Abbau der Nukleinsäuren immer nur Spuren von einfachsten Bausteinen zu entstehen, die sofort zur Aufnahme gelangen könnten. Der Grund der nicht vollständigen Spaltung der Nukleinsäuren im Darmkanal ist vielleicht darin zu suchen, daß die Nukleotide und Nukleoside im Zellstoffwechsel direkt Verwendung finden können. ^ Nach allen bisherigen Erfahrungen führt, wie bereits erwähnt, der Abbau der Nukleinsäuren im Darmkanal nur bis zu Nukleotiden und Nukleosiden. Diese kommen dann zur Resorption und schlagen den Blutweg ein. i) Im Blute konnten Nukleotide nachgewiesen werden. 2) Ob solche und Nukleoside von den Zellen direkt zu Synthesen von Polynukleotiden verwendet werden oder, ob diese nur von den Bausteinen der Mononukleotide ausgehen, entzieht sich noch unserer Kenntnis. Wir wissen auch nicht, ob bei der Bildung der Nukleoproteide die Zwischenstufe Nuklein in Erscheinung tritt. Daß im tierischen Organismus beständig Nukleoproteide und insbesondere auch Nukleinsäuren gebildet werden müssen, ist schon durch den Umstand sichergestellt, daß auch das hungernde Tier stets im Harne Produkte ausscheidet, die nur aus den Bausteinen der letzteren Verbindungen hervorgegangen sein können. Die auf diese Weise stets entstehenden Lücken werden ohne Zweifel beständig durch die mit der Nahrung aufgenommenen Bausteine der Nukleinsäuren ausgefüllt. In den meisten Fällen werden sicher mehr Nukleotide und Nukleoside vom Darme übernommen, als zum Aufbau von Nukleinsäuren benötigt werden. Diese dienen vielleicht zum Teil besonderen Funktionen, zum größten Teil werden sie jedoch bald weiter abgebaut. Wir haben ohne Zweifel im Verhalten der Bausteine der Nukleinsäuren im tierischen Organismus ganz entsprechende Verhältnisse vor uns, wie bei allen übrigen organischen Nahrungsstoffen. Überall begegnen wir mannigfaltiger Verwertung der einzelnen Verbindungen. Bald wird ein aufgenommener Stoff Baustein von Zelibestandteilen, bald geht er in irgend einer Form in ein Sekret über, bald knüpft er Beziehungen zu allen möglichen anderen Verbindungen an, bald wird er auch direkt bis zu bestimmten Stoffv/echselendprodukten abgebaut. Wir stehen bei der Erörterung der Verwertung der Nukleinsäuren im tierischen Organismus den gleichen Schwierigkeiten gegenüber, wie bei den Proteinen bzw. ihren Bausteinen, den Aminosäuren. Es ist bis jetzt nicht bekannt geworden, ob es Orte gibt, an denen Nukleinsäuren oder ihre Bestandteile in irgend einer Form aufgespeichert werden können. Wahrscheinlich ist eine Ablagerung nach allen bisherigen Erfahrungen nicht. Es wäre jedoch möglich, daß jede einzelne Zelle Umsatznukleoproteide bzw. -nukleinsäuren besitzt und aulüerdem bestimmte Vertreter dieser Ivlassen von Verbindungen das eigentliche Baumaterial der Zellkerne darstellen. Leider wissen wir gar nichts Genaues über die Rolle, die die Nukleinsäuren bzw. die Nukleoproteide Es, ist uns immer noch ziemlich unbekannt, welche in der Zelle .spielen. Bedeutung dem Kern bei den Stoff Wechsel Vorgängen der Zellen zukommt. M J. Biberfeld und J.Schmid: Zeitschr. f. physiol. Chemie. 69. 292 (1909). ThannhauRfr und Czoniczcr: Zeitschr. f. physiol. Chemie. 110. 307 (1920). -) XX XU. Voilesuntr. (568 Wir wissen nur, dalJ er l)ei der Zellteilung wichtige Funktionen übernimmt und vielleicht bei der Vererbung eine besondere Bedeutung hat. Wir wollen zunächst versuchen, die Entstehung der Bausteine der Nukleinsäuren in den Zellen zu verfolgen und ferner ihren weiteren Abbau klarzustellen. Zunächst sei bemerkt, daß die verschiedenen Körperzellen Fermente besitzen, um die Nukleinsäuren stufenweise abzubauen. Zunächst werden sie durch Polynukleotidasen V) in Nukleo•fide gespalten. Diese fallen einer weiteren Zerlegung in Nukleoside und l'hosphorsäure anheim. Das diesen Abbau vollziehende Ferment ist Nukleotidase genannt worden. Die Nukleoside endlich werden durch die Nukleosidasen in ihre Anteile, nämlich in Zucker und die mit ihm verbundene Base, gespalten.-) Es ist von groüem Interesse, daß festgestellt werden konnte, daß die Nukleoside bereits vor ihrer Spaltung Veränderungen erleiden können. So kann das Nukleosid Adenosin durch Desaminierung in Hypoxanthosin Inosin übergeführt werden. Aus Guanosin entsteht in entsprechender = Weise Xanthosin. Das folgende Schema soll die Stufen wiedergeben, die durchlaufen wenn Nukleinsäuren Polynukleotide in ihre Bausteine zerlegt werden (luanin Phosphorsäure Kohlehvdrat = werden, — r Phosphorsäurc — Kohlehvdrat — Adenin Zytosin Phosphorsäurc — Kohlehydrat Phosphorsäurc — Kohlehydrat Urazil — I i" I 1 I — Kohlehydrat ~ Thymin Nukleinsäure = Polynukleotid. Phosphorsäure Das Polynukleotid zerfällt unter der Wirkung der Polynukleotidase unter Wasseraufnahme in die Nukleotide: Phosphorsäure Phosphorsäurc l'hosphorsäure Phosphorsäure Phosphorsäurc — Kohlehydrat Kohlehydrat Kohlehydrat Kohlehydrat Kohlehydrat Die Nukleotide werden und Nukleoside gespalten: — Guanin Adenin — Zytosin Urazil Thymin ) J ; \| } Guanylsäure Adenylsäure Zytidinphosphorsäure Uridinphosphorsäure Thymidinphosphorsäure. durch Nnkleotidasen in Phosphorsäure — 1) Vgl. hierzu Aniki: Zeitschr. f. physiol. Cheinie. 38. S4 (1903). Ä. Schitte/iJmtschenko: Sachs: Ebenda. 46. :-^37 (1905). /idm: Kbonda. 42. 2.')! (liKj4). Biochom. Zeitschr. 31. 337 ('1911). - P. de la Blanchardiere: Zoitschr. f. physiol. Cheui. — — 87. 291 (1913). Vgl. zur Feststellung dieser F^enacnte: 1'. A. Lerene und /'. Medifjrea anu: 375, 389 (1911). S. Ambou/ und W. Jones: Zeitschr. f. n alter Jones: Journ.'of Biol. Chem. 9. 129, 169 physiol. Chemie. 73. 407 (1911). /'. A. Jjfvene. IC. A. Jarohs und F. Medigreceanii : Khciida. 11. 371 (1912). (1911). '-) .louru. of Biol. — Chem. 3. (iä. — — Xukleoproteide. iS'uklciusauieii und ihre Bausteine <Tuanosin Kohlehydrat können — Guanin durch Adenosin Kohlehydrat Des- aminierung — Adenin 669 Kohlelivdiat Xanthin Xanthosin Kohlehydrat — Hypoxanthin. üborg-ehen in Ilypoxaiithosin = Inosin Zytidin Kohlehydrat Zytosin l'ridin Kohlehydrat Urazil Kohlehydrat Thyniin Die Xukleoside endlich werden durch die Xukleosidasen in ihre Bausteine zerlegt. Adenosin liefert Adenin und Kohlehydrat, Guanosin Guanin und Kohlehydrat, Hypoxanthosin Hypoxanthin und Kohlehydrat und Xanthosin Xanthin und Kohlehydrat. Während die Bildung dieser Bausteine der Xukleinsäuren genau festgestellt worden ist. wissen wir noch nichts Sicheres über das Verhalten derjenigen Xukleoside. an deren Aufbau Pyrimidinbasen beteiligt sind. Wahrscheinlich werden sie durch Fermente besonderer Art ebenfalls in ihre Bausteine zerlegt. W^as nun das weitere Schicksal der einzelnen Bausteine der Xukleinsäuren anbetrifft, so sind wir bis auf die Pyrimidinbasen gut unterrichtet. Über das Verhalten der letzteren im Zellstoffwechsel herrscht noch immer vollständiges Dunkel.') Man könnte daran denken, daß sie in irgend einer Beziehung zu den S. 185 erwähnten noch unbekannten Xahrungsstoffen mit besonderer Wirkung stehen. Die Phosphor säure kann als Baustein von Zellen dienen. Sie kann z. B. an der Synthese von Phosphatiden teilnehmen. Sie kann jedoch auch als überflüssiger Baustein durch die Xieren ausgeschieden werden. Das Kohlehydrat dürfte sich in seinem weiteren Schicksal in das der übrigen Vertreter dieser Klasse einreihen. Sehr genau verfolgt ist das Verhalten der beiden Purinbasen Aden in und Guanin in den Geweben. Horhaczewskl^) stellte fest, daß Organauszüge oder Organbreie von Säugetieren aus den in ihnen enthaltenen Xukleoproteiden durch Abbau ganz verschiedene Produkte liefern, je nachdem man der Luft den Zutritt gestattet oder verwehrt. Im ersteren Fall trat Harnsäure auf, im letzteren wurden bestimmte Oxypurine gefunden. Wir sind jetzt imstande, das Ergebnis dieser Versuche ganz klar zu übersehen. In beiden Versuchen mit und ohne Luft- bzw. Sauerstoffzufuhr sind durch die oben geschilderten Fermente Purinbasen aus den vorhandenen Xukleoproteiden bzw. den aus ihnen abgespaltenen Xukleinsäuren gebildet worden. Ist Sauerstoff zugegen, so entsteht Harnsäure, fehlt er, dann erfolgt kein so weitgehender Abbau der genannten Basen. Dali in der Tat ein direkter Zusammenhan»' f. phvsiol. Chemie. ;{2. 280 (19U1). Horbaczewski : Monatshefte f. Chemie. 10. (524 (1S89) 12. 221 (1891). Vgl. W. auch F. Giacosa: Atti rt. K. Acc. delhi Szienze di Toiino. 25. 726 (1891). Spitzer: Pfiiiffers Archiv. 76. 192 (1889). Iha/o K iener: Verhandl. d. XV'II. Kongr. f. inn. Medizin. (562 (1899) und Arch. f. experim. Path. u. Pharmak. 42. 'Mb (1899). ') Vgl. 11. Stcude/: Zeitschr. ^) J. — — XXXII. Vorlesung. 670 der genannten Art zwischen den Purinbasen und der Harnsäure besteht, bewiesen in eindeutiger Weise vor allem Alfred Schittenhelm ^) und Walter Jones.") Diese beiden Forscher zeigten, daß die einzelnen Gewebe über Fermente verfügen, die Adenin und Guanin in charakteristischer Weise verändern. Es ist schließlich auch geglückt, die Fermente von den Zellen abzutrennen. Das wichtigste Ergebnis dieser Versuche ist zunächst, daß die erwähnten Fermente aus xldenin Hypoxanthin bilden. Dieses Erscheinung, wenn unter Sauerstoffausschluß gearbeitet wird. Ist zugegen, dann treffen wir außerdem Xanthin und schließli{:h Harnsäure an. Gehen wir von Guanin aus, dann gelangen wir bei Luftausschluß zu Xanthin und bei Zutritt von Sauerstoff zu Harnsäure. Die folgenden Formeln erläutern diese Überführungen der beiden Aminopurine in die entsprechenden Oxypurine und in die Harnsäure: tritt in solcher N=C.NH, + HoO — NH3 C— NH /^« N— C— X Adenin — 6-Aminopurin NH— CO HC II i N=:C . OH C— NH HC HC / CH C— NH >CH N— C-X X + N C Hypoxanthin = 6-Oxypurin. XH— CO NH— CO HO C . X C— XH / —y CH C-XH OC \ XH— C— C— X / CH +0 —> Xanthin — 2,6-Dioxypurin. ^) Alfred Schittenhelm: Zeitschr. f. physiol. Chemie. 42. 251 (1904); 43. 228 Alfred Schittenhelm und (1904); 45. 121, 152(1905); 45. 161 (1905); 46. 354 (1905). Julius Schmid: Ebenda. 50. 30 (1906); Zeitscbr. f. cxperim. Tath. u. Tlierapie. 4. 424, Werner Künzel und A. Schittenhelm: Ebenda. 5. 489, 393 (1908). 432 (1907). — — — — Alfred Schittenhelm Alfred Schittenhelm: Zeitschr. f. physiol. Chemie. ü(j. 53 (1910). und Karl Wiener: Ebenda. 77. 77 (1912). «) Walter Jones: Zeitscbr. f. physiol. Chemie. 41. 101 (1904); 42. 35 (1904). Walter Jones und ('. L. Partridqe: Ebenda. 42. 343. (1904). -— Walter Jones und Walter Jones: Ebenda. 45. 82 (1905). M. C. Winternitz: Ebenda. 44. 1 (1905). Walter Jones und C. li.Austrian: Ebenda. 48. 110 (1906); Journ. of Biol. Chcniistry. 3. Vgl. auch Lafaijette B. Mendel and I'hilij) H. Mitchell: The Amer. .lourn. 227 (1907). A. E. Austin: Journ. of Med. Research. 16. 71 (1907). of Physiol. 20. 97 (1907). Samuel Amberg und Walter Walter Jones: Journ. of Biol. Chem. 9. 129 (1911). Jones: Ebenda. 10. 81 (1911); Zeitschr. f. physiol. Chemie. 73. 407 (191J). — — — — — — — Nukleoproteide. Nukleiusäureu iiud ihre Bausteiue. NH— CO «71 NH -CO OC C— NH NH— / C— C C— NH OC . OH )C0 NH— C-NH Harnsäure = 2, 6, 8-Trioxypurin NH— CO Harnsäure C— NH ^CH + H. C— N Guaniii = 2Amino- NHo C . --NH3 6-oxypurin NH— CO NH -CO OH.C C-NH OC C-NH >CH N NH-C— C— / CH + Xanthin — 2, 6-Dioxypurin. Aus der gegebenen Darstellung ersieht man, daß dieBildungvon Hypoxanthin aus Adenin und diejenige von Xanthin aus Guanin in vollständig entsprechender Weise vor sich gehen. Es handelt sich offenbar um eine hydrolytische Desaminierung. Der weitere erfolgt unter Oxydation. Er bleibt aus, wenn kein Sauerstoff zugeführt wird. Hypoxanthin geht zunächst durch Oxydation in Xanthin über. Dieses wird dann weiter zu Harnsäure oxydiert. In den obigen Formeln ist angeführt, wie die Aminogruppe zunächst durch die OH-Gruppe ersetzt wird und sich dann durch Umlagerung einerseits die NH-Gruppe und andrerseits die CO-Gruppe bildet. Ebenso sind der Sauerstoffeintritt und die Umlagerung in beiden Phasen angegeben. Dazu ist zu bemerken, daß wir natürlich nicht wissen, in welcher Form die einzelnen Verbindungen in Wirklichkeit vorkommen. Es ist wohl möglich, daß sie beide im Orga- Abbau nismus vorhanden sind. Nach Beobachtungen von Thanriliauser und Ottenüber den Abbau von Nukleosiden durch Leberauszug scheinen diese gleichzeitig desaminiert und in ihre Anteile gespalten zu werden.^) ste'm Es ist von großem Interesse, daß der Mensch und die anthropoiden Affen Harnsäure ausscheiden, während, wie besonders durch die Untersuchungen von Wiechowski 2) gezeigt worden ist, viele Tiere, so der Hund, \) S. J. Thannhauser und B. Oitenstein: Zeitschr. für physiol. Chemie. 114. 17 (1921). ^) Wilhelm Wiechowski: Hofmeistern 431 (1910). Beitr. 9. 295 (1907); Biocheru. Zeitschr. 2.). XXXIl. Vorles^l)l,^ 672 an ihrer Stelle Allantoin bereiten. Abbauprodukt der Harnsäure zu betrachten: die uicht-anthropoiden Affen ij usw.. Diese Verbindung- ist als NH— CO I i CO C-NII ^CO NH_C— NH llai'nsänrc NH.3 OH^) CO C— NH H3) >H— C—NH oder .CO / XH — NH CO l CO C- NH— C— NH OH Allantoin. Die Überführung von Harnsäure in Allantoin wird einem oxydierenden Ferment, Urikase oder urikolytisches Ferment genannt, zugeschrieben.' Das Allantoin. das seiner Bildung aus Glyoxylsäure und zwei Molekülen Harnstoff entsprechend'') auch als Glyoxyldiureid bezeichnet werden kann, ist von Vauquelin und Buniva im Jahre 1791) in der Amnionflüssigkeit von Kühen und bald darauf von Lassa igne^) in der AUantoisflüssigkeit entdeckt worden. Wälder') fand das Allantoin später im Harn säugender Kälber. Durch zahlreiche F^ütterungsversuche konnte die Beziehung des Allantoins zu den Purinbasen und insbesondere zu der Harnsäure festgestellt werden.^) Schließlich wurde auch durch Versuche am überlebenden Organ //. />. ) Ainh-pw Iluntcr und Maurice IL (Hvens: .Tourn. of biol. Chem. 2) Vgl. Lafmiettv, B. Mendel und //. ^) R.Behrend: Liebiy^ Annaleu. IJ. Dakin 333. 144 13. 371 (1921)Journ. of biol. Chom. 7. 153 (lUlO)(1904); 365. 21 (1909): vgl. auch : Dakin: J. (^hem. See. 107. 434 (1915). — Nederl. Tijdschr. voor Geneeskunde. 2. 26S (1860). Zeutralbl. f. Physiol. 18. (i9fi (1905). .(. /-;. Austin: .lourn. of Medical Research. 15. 309 (1906); 16. 71 (1907). Werner Kiinzel und A. Schittenhel in : Zeitschr. f. experim. Path. u. Ther. 5. 389 (1908). W. Wiechowski und //. Wiener: Hofmeisters Beitr. 9. 247 (1907). -- Vgl. auch Vittorio ScaX/idi: Biochem. Zeitschr. 18." 506 (1909); 25. 415 (1910). F. Batelli und L.Stern: Biochem. Zeitschr. 10. 219 (1909). 5) Vgl. S. 664. ) Lassaigne: Annales de (Jhim. et de Physi(\|ue. 17. 301 (1821). ') F. Wähler: Liebi</a Annalen. 70. 229 (1849); 88. 100 (1853). ") E. Salkowski: Ber. d. Deutschen Chem. Gesellsch. 9. 719 (1876): Zeitschr. für physiol. Chemie. 35. 495 (1902); Zentralbl. f. d. med. Wissensch. 36. 929 (1898). (>. Minkouski: Ebenda. 19. Nr. 19 (1898): Arch. f. experim. Path. u. Pharm. 41. 393 (1898). Theodor Cohn: Zeitschr. f. physiol. Chemie. 25. 507 (1898). ./. Pohl: Ebenda. 48. 367 Lafayette B. Mendel und Benjamin White: American Journ. of Phvsiol. 12. (1902). F. P. Underhill und ./. S. Kleiner: Journ. of biol. Chem. 4. 165 ("1907). 85 (1895). ir. Wiechowski: Hofmeister':^ Beitr. 11. 109 (1908). A. Schittenhehn und /'. Srisser: Zeit<fhr. f. oxporiiii. l'ath. ii. Ther. 9. 295 (1907). ) Hur/o Vgl. hierzu Stockvis: Wimer: Arch. f. experim. Path. u. Pharm. 42. 375 (1889); — — — — — — — — — — — 673 Nukleoproteide. Nukleinsäuren timl ihre Bausteine. XH-CH-NH 1 1 i XXXII. Vorlesung. 674 NH— CO NH— CO NH, C . N C— NH >CH C N — Nukleoproteide. Nukleinsäureu und ihre Bausteine. 675 l-Methylxanthin^), das 1, 7-Dimethyl-xanthin = Paraxanthin») und das 7-Methylxanthin = Heteroxanthin) beobachtet worden. Ferner hat man ;-J-Methylxanthin nach Einnahme von guanini), das methylierten Xanthinen (TheophyUin, Theobromin, Kaffein) festgestellt. Die folgenden Formeln geben die Struktur dieser Verbindungen wieder: NH— CO NH, .C C CH, -N . . N— CO OC CH3 >CH C— NH C— CH / HN—C— 7-Methyl-2-amino-6-oxypurin = Epiguanin purin = 1-Methylxanthin N CH, . l-Methyl-2, 6-dioxy- N— CO OC NH— CO N C- . OC CH3 >CH HX—C— HN 7-Dimethyi-2, 6-dioxypurin = 1, 7-dimethylxanthin = Paraxauthin C— X CH3 >CH C— X . 7-Methyl-2, 6-dioxypurin 7-Methylxanthin = Heteroxanthin. 1, = HX— CO C— NH ^CH CH3 N— C-X 3-Methyl-2, 6-dioxypurin = 8-Methylxanthin. OC . Die Herkunft der methylierten Amino- und Oxypurine ist zum Teil So liefert Theobromin 8, 7-Dimethyl-2, 6-dioxypurin 7-j\lethylxanthin.») Daneben tritt auch 3-Methylxanthin auf. Diese letztere Verbindung wird auch im Harn angetroffen, wenn man Theo- = festgestellt. = phyllin ') o-Dimethyl-2, 6-dioxypurin verfüttert.'') Xach Eingabe M. Krüger und Wtdfj': Verhaudl. d. phvsiol. GesLdkch. zu Berlin. 27. Juli 1894 M. Krüyer und G. Salomon; Zeitschr. f. pbysiol. Chemie. 24. 387 (1898); (1893/94). 26. 1, — ö89 (l>^98/99). ^) M. Krüger und G. Salomon; Zeitschr. f. physiol. Chemie. 24. 364 (1898); 26. 358 (1898/99). Thndichum: Zeitschr. f. phvsiol. Chemie. 11. 415 (1887); Couipt. reud de l'acad. G. Salomon: Arch. f. (Auat. u.) IMiysiol. 426 (1882); des Sciences. 106. 1805 (1888). Ber. d. Deutschen Chem. Gesellsch. 16. 195 (1883); l'ircliows Archiv. 125. 554 (1891). 24. 364 (1598); 26. ^) M. Krüger und G. Salomon: Zeitschr. f. physiol. Chemie. 358 (1898/99). •') M. Krüger und J. Schmidt: Zeitschr. f. physiol. Chemie. 36. 1 (1902). ^) St.Bondzijnski und H.Gotilieb: Arcli. f. experini. Path. u. Pharm. H6. 45 (1895); M. Krüger und /'. Schmidt: Berichte d. Deutschen Chem. Gesellsch. 28. 1117 (1895). Ebrnda. 32. 2677 (1899). M. Krüyer und ./. Schmid: Arch. f. experim. Path. u. Pharm. '') — — 45. — 259 (1901). 43* XXXll. Vorlesung. fj76 = von Kaffeiii 1, o, 7-Trimethyl-2, 6-dioxypurin findet man im Harn von Kaninchen Paraxanthin und feiner 1- und 7-Methylxanthin.M Daneben tritt unverändertes Kaff ein auf. Hunde bereiten aus Kaffein Theobromin 3-Methylxantliin und o, 7-I)imetliylxantliin.2) Aus diesen Beobachtungen geht hervor, daß der tierische Organis- mus imstande ist, Methylgruppen abzuspalten. Es ist auch gelungen, aus Organbrei zugesetzten, mehrfach methylierten Xanthinbasen Verbindungen mit weniger Methylgruppen zu erhalten. ^j Es ist von. größtem Interesse, daß die einzelnen Tierarten nach Eingabe von Methylpurinen ganz verschiedene Mengen an den einzelnen Abbaustufen im Harn zur Ausscheidung bringen. So enthielt der Harn vom Hunde nach Eingabe von Theobromin 5ro57o unveränderte Substanz, 2"89'/'o 3-Methyl-xanthin Beim Kaninchen fanden sich nach Ver"-Methyl-xanthin. und U62' fütterung von Theobromin 16'05Vo unveränderte Verbindung, 14'Hlo/o ''^Methyl-xanthin und O'OlVo 3-Methyl-xanthin im Harn. Im Urin vom Menschen wurden nach Eingabe des gleichen Methylxanthins 16'3Vo 7-MethylSelbstverständlich xanthin und SößVo 3-Methyl-xanthin aufgefunden.) haben die angeführten Zahlen keinen absoluten Wert, weil sicher je nach den gerade vorliegenden Bedingungen im einzelnen Organismus der Abbau Auch der zugeführten Verbindungen ein quantitativ verschiedener ist. dürften die Resorption der verfütterten Produkte und eventuelle Umwandlungen durch die Darmflora nicht gleichmäßige gewesen sein. Doch sind die Unterschiede zwischen den einzelnen Tierarten so groß, daß ohne Zweifel ein für die einzelne Art charakteristischer Abbau des verabreichten 3, 7-Dimethylxanthins vorliegt. Wahrscheinlich ist die Entmethylierung eines Teiles der zugetührten Methylpurine eine vollständige. Ob es bis zur Bildung von Harnsäure bzw. von Allantoin kommt ist noch unentschieden. &) Sicher sind nicht alle methylierten Purinbasen des Harns auf in der Nahrung enthaltene Methylverbindungen dieser Reihe zurückzuführen. Vielmehr vermag der Organismus offenbar auch Purinbasen zu methylieren. Es gilt dies ohne Zweifel für das Methyl-guanin. Ferner ist das Auftreten von Heteroxanthin beobachtet worden, als nur Fleisch verfüttert wurde. •^) bau Wir haben bereits hervorgehoben, daß bei den einen Tieren der AbHarnsäure im wesentlichen bei der Harnsäure stehen bleibt, der in der Hauptsache Allantoin liefern.^) Ferner haben wir gesehen, daß methylierte Xanthine bei verschiedenen Tierarten verschieden abgebaut werden. Nun gibt auch eine vergleichende Betrachtung des Purinbasengehaltes des Harns ganz charakteristische Unterschiede. Beim während andere 2) M. Krüger: Ber. d. Deutscheu Cliem. Gcsellsch. 32. 3336 (1899). J/. Krüger: Ber. d. Deutschen Chem. Gesellsch. 32. 2318 (1899). 3) ./. 1) Sclimid: Zeit.schr. f. physiol. Cliemie. 67, 155 (1910). M. Krüger und .7. Schmid: Ber. d. Deutschen Chem. Gesellsch. 32. 2677 (1899); Arch. f. experim.'Path. u. Pharm. 45. 295 (1901). ^) 0. Minkowski: Arch. f. experim. l'hat. u. l'harm. 41. 375 (1898). R. Buriari und H. Schur; rjlügers Archiv. 8f». 241 (1900); 87. 239 (1901). — M. Krüger und - J. Schmid: Eheuda. 67. J. Schmid: /eiti^chr.' f physiol. Chemie. 32. 104 (1901). ) — 155 (1910). — Zeitschr. f. physiol. Chemie. 11. 413 (1887). G. Salotnon und ) G.Salomon: Carl Neuberg: Salkoicski-Featachr. 37. Berlin 1894. ') U'.' Hicchoirski: Prager med. Wochenschr. 37. Nr. 22 (1912). Xukleoproteide. Nukleinsäuren und ihre Bausteine. 677 Menschen findet man hauptsächlich Xanthin. Daneben geringe Mengen von Hypoxanthin und Spuren von Aminopurinen. Ob das in sehr geringer Menge vorhandene Allantoin in direkter Beziehung zum Abbau von Purinbasen steht oder nicht vielmehr vorgebildet in der Nahrung enthalten war, ist noch unentschieden. Bei den anthropoiden Affen finden sich ganz ähnliche Verhältnisse wie beim Menschen, während die übrigen Vertreter dieser Tierklasse den Allantoin bildenden Tieren nahestehen. Übrigens ist der Gehalt des Harnes an Purinbasen sehr gering (Oln— O^^r Stickstoff in dieser Form). Bei den Tieren, die Allantoin bilden, finden wir solche bei denen daneben relativ viel Harnsäure vorkommt. Die Purinbasen treten an Menge ganz zurück. Beim Hund, beim Kaninchen und Rind-j zeigt der Harn z. B. eine solche Verteilung der Purinbasenabkömmlinge. Beim Schwein und Pferd dagegen überwiegt die Menge der Purinbasen über die Harnsäureraenge. Bei ersterem fehlt Guanin ganz. 3) Es ist von groliem Interesse, daß bei diesen Tieren Ablagerungen von Guanin in Gelenken, in der Leber und in Muskeln beobachtet worden sind.) Ferner wurde in einem solchen Falle auch Guanin im Harne aufgefunden. &) Offenbar tehlt in diesen Fällen man hat geradezu von einer Guanin gi cht gesprochen jenes Ferment, das die erwähnte Purinbase abbauen kann. Da offenbar gleichzeitig Adenin ganz gut angegriffen und in seine typischen Abbaustufen zerlegt wird, ist wohl der Schluß berechtigt, daß die Desaminierung von Guanin und von Adenin nicht durch das gleiche Ferment erfolgt. Dafür sprechen auch die Beobachtungen von Jones^), daß nicht jedes Organ Adenin und Guanin abbauen kann. Manche greifen nur Guanin an. andere wieder nur Adenin und endlich manche auch beide Aminopurine. Man hat daher von einer Adenase und einer Guanase gesprochen. Es ist möglich, daß auch diejenigen Fermente, die Hypoxanthin in Xanthin und dieses in Harnsäure überführen, besonderer Art sind und wiederum ein besonderes Ferment die Xukleoside Adenosin und Guanosin in die entsprechenden oxydierten \'erbindungen Hypoxanthosin und Xanthosin überführt. Wenn wir nunmehr alles zusammenfassen, dann ergibt sich ein ganz — — : Abbaus der Nukleinsäuren im tierischen Organismus. Er erfolgt über die Nukleotide zu den Nukleosiden, w^obei gleichklares Bild des Phosphorsäure abgespalten wird. Diese letzteren Verbindungen zerdann in ihre Bausteine. Es entstehen Purin- und Pyrimidinbasen und ferner Kohlehydratmoleküle. Wahrscheinlich erfordert jede Art der Nukleotide und Nukleoside eigenartige Fermente. Die Purinbasen werden dann weiter über die Oxypurine bis zur Harnsäure und bei den meisten Tieren darüber hinaus zu Allantoin abgebaut. zeitig fallen — Andren>) Vgl. z B. H. G. Wells: Jouru. of biul. Chem. 7. 171 (1909 10). Hunter und Maurire H. Givens: Ebenda. 13. 371 (1912). ) A. Schittenhelm und E. Bendix: Zeitsclir. f. phvsiol. Chemie. 48. 140 (1906). — A. Schittenhelm: Ebenda. 46. Sö4 (1905). A. Schittenhelm: Zeitschr. f. physiol. Chemie. 66. n3 (1901). G. Salomon: R. Virchoic: Virchous Archiv. 35. 358 (1860); 31. 147 (1866). Ebenda. 97. 360 (1884). W. Mmdelsohn: The Americ. Jouru. of medical Science. 109 Vgl. auch .4. Schittenhelm und E. Bendix: Zeitschr. f. physiol. Chemie. 48. (1888). 140 (1906\ ) Pecile: Liebigs Annalen. 183. 141 (1876). ') ) — «) — Vgl. die Literatur S. 670, Zitat -). — XXXII. Vorlesung. 678 die Es fragt sich nun, ob die Purinbasen die einzige Quelle für Bildung der Harnsäure bzw. des Allantoins darstellen. Lange Zeit glaubte man, daß in der ganzen Tierreihe auch die Eiweißstoffe zu den genannten Abbaustufen führen. Jetzt wissen wir ganz bestimmt, daß dies nur bei den Vögeln und Reptilien stets der Fall ist. Bei den Säugetieren, den meisten Amphibien und den Fischen bestehen keine direkten Beziehungen der Harnsäure bzw. des Allantoins zum Eiweißstoffwechsel. In der ganzen Tierreihe liefern dagegen die Purinbasen Harnsäure bzw. Allantoin. Es gilt dies für jede einzelne Art. Dazu kommt dann bei den Vögeln und Reptilien als eine für diese Tieraiten charakteristische Quelle für Harnsäure das Eiweiß bzw. die Aminosäuren. Wie bei allen bisher besprochenen Verbindungen müssen wir auch hier die Frage nach den Beziehungen der Bausteine der Nukleinsiiuren zu anderen Substanzen erörtern. Ist eine Synthese von Nukleinsäuren aus Kohlehydraten, Fetten und Eiweißstoffen bzw. ihren Bausteinen möglichV Knüpfen ferner die typischen Bausteine der Nukleinsäuren, die Purin- und Pyrimidinbasen, Beziehungen zu Vertretern der genannten Klassen von Verbindungen anV Wir wollen mit der letzteren Fragestellung beginnen. So weit unsere Kenntnisse reichen, wandeln sich die im Organismus entstehenden und ihm zugeführten Purinbasen in keine Verbindungen um, die zur Synthese von anderen Stoffen dienen könnten. Sie scheinen vielmehr in ihrer Gesamtheit Stufe um Stufe bis zu den Stoffwechselendprodukten abgebaut zu werden. Was aus den Pyrimidinbasen wird, wissen wir zurzeit noch nicht. Es wäre denkbar, daü sie aufgespalten werden, und die entstandenen Bruchstücke in irgend einer Weise noch Verwendung finden. Viel besser sind wir über die Frage der Synthese der Nukleinsäurebausteine und insbesondere der Purin- und Pyrimidinbasen im tierischen Organismus aus Material, das keine direkten Beziehungen zu diesen Verbindungen besitzt, unterrichtet. Miescher^) hat festgestellt, daß Lachse, die sich monatelang im Süßwasser aufhalten, ohne Nahrung zu sich zu nehmen, große Mengen von Spermatozoon bilden. Diese bestehen zum größten Teil aus Nukleoproteiden. Ferner ist beobachtet worden, daß bebrütete Hühnereier Purinbasen neu bildrn können.'^) Die Fähigkeit der Neubildung scheint allerdings nur eine zeitlich beschränkte zu sein. Sie soll am 17. Bebrütungstage aufhören. .\uch im Ei des Seidenspinners ist Synthese von Purinbasen beobachtet worden. 3) Zweifelhaft in ihrer Deutung sind jene Versuche geworden, die mit Milch als Nahrung durchgeführt worden sind.) Die Annahme, daß dieses Nahrungsmittel völlig frei von Purinbasen sein soll, hat sich nicht bestätigt.^) Zwei Bausteine kann die tierische Zelle ohne Zweifel leicht zur Synthese von Nukleinsäuren zur Verfügung stellen. Es sind dies die PhosFr. Miescher: Arcli. f. experim. Patli. u. Pharmak. 37. 100 (1896). A. Kossei: Zeitschr. f. physiol. (Jhemie. 10. '248 (1886). Lafayette B. Mendel und dharles S. Leacemvorth : Amer. Journ. of Physiol. 21. 77 (1908). L. S. Fridericia: Skand. Arch. f. Physiol. 26. 1 (1912). •') .1. Tichomirojf: Zeitschr. f. physiol. Ciicmic. 9. 618 (1885). ^) Vgl. Richard liurian und Heinrich Schur: Zeitschr. f. physiol. Chemie. 23. 1) — 2) 55 (1897). 5) Carl Voee/diu und Carl P. Sheriviii : Jouru. of biol. — (hcm. 33. 145 (1918). Nukleoproteide. Nukleinsäuren und ihre Bausteine. (379 phorsäure und das Kohlehydrat. Die erstere nimmt sie mit der Nahrung in P'orra von Phosphaten auf. Sie kann auch aus Phosphatiden und den phosphorhaltigen Proteinen gebildet werden. Das Kohlehydrat dürfte aus solchen hervorgehen oder aber synthetisch z. B. aus desaminierten Aminosäuren gebildet werden. Aus welchem Material die Pyrimidin- und Purinbasen hervorgehen, wissen wir nicht. Es kommen dieselben Möglichkeiten in Betracht, die für die Pflanzenzelle Seite 661 ff. erörtert worden sind. Der Versuch, Histidin in Beziehungen zu den Purinbasen zu bringen, hatte keinen Erfolg. i) Hunde, denen große Mengen dieser Aminosäuren gegeben wurden, zeigten keine Vermehrung der Allantoinausscheidung. Für die ganze Auffassung der Herkunft der im Harn erscheinenden Abkömmlinge der Purinbasen ist die Entscheidung der Frage, ob beständig synthetisch Purinbasen gebildet werden können, von der allergrößten Bedeutung. Man ist geneigt, die im Urin auftretenden Stoff wechselelendprodukte, die sich auf Purinbasen zurückführen lassen, insbesondere bei den Säugetieren und dem Menschen nur auf solche zu beziehen, die entweder in den Geweben schon vorhanden sind oder aber von außen zugeführt werden. Man spricht von einem Purinstoffwechsel und will damit zum Ausdruck bringen, daß dieser in sich abgeschlossen sei. Es kann diese Auffassung auf das ganze Tierreich übertragen werden, nur vermögen wir bei jenen Organismen, die auch aus Aminosäuren Harnsäure bereiten, den Purinstoffwechsel nicht so scharf vom Eiweiß- bzw. Aminosäurestoffwechsel zu trennen, wie bei denjenigen Tieren, die aus diesen Harnstoff und nur aus Purinbasen Harnsäure bilden. Es ist zurzeit unmöglich^ die Frage zu entscheiden, ob tatsächlich der Purinstoffwechsel in einem fest geschlossenen Kreise abläuft. Es müßte in diesem Falle jede Neubildung von Purinbasen ausgeschlossen sein.^) Wir kennen zurzeit wenig Analogien für die Annahme, daß tierische Zellen eine bestimmte synthetische Fähigkeit nur um sie dann für immer erlöschen zu lassen. Wohl aber kennen wir zahlreiche Beispiele dafür, daß eine solche nur in bestimmten Zeiten in Funktion tritt. Es sei z. B. an die Bildung der Milchbestandteile erinnert. Die Zellen der Milchdrüse können jahrzehntelang ruhen. Erst dann, wenn ein sich entwickelnder Fötus bzw. eine Plazenta zugegen ist, zeitweise besitzen, nehmen sie die Herstellung der charakteristischen Bestandteile der Milch auf. So könnte es sich auch mit der Synthese von Purinbasen verhalten. Solange kein Bedarf an solchen besteht und diese in genügender Menge von außen zugeführt werden, findet vielleicht keine Neubildung von solchen statt. Im allgemeinen dürften die Purinbasen aus folgenden Quellen stammen. Mit der Nahrung werden normalerweise immer Purinbasen bzw. Verbindungen zugeführt, die diese gebunden enthalten. Es wird nun im einzelnen Falle darauf ankommen, ob einzelne Zellen diese Bausteine zu bestimmten Zwecken verwenden können. Der Rest wird weiter zerlegt. Beständig werden in den Zellen Nukleinsäuren abgebaut und aus diesen ihre Bausteine gebildet. Die Verhältnisse liegen offenbar im Prinzip genau gleich, wie bei M Emil Abderhalden und llans Kinheck : Zeitsclir. f. pliysiol. Chemie. 62. 322 K. Kowalewski : Biochem. Zeitschr. 23. 1 (1909). — Emil Abderhalden, H. Ackroijd H. Einbeck und J. Schmid: Zeitschr. f. physiol. Chemie. 68. 395 (1911). und F.G. Hopkins: The Biochem. J. 10. 551 (1916). 2) Vgl. hierzu G. Kollmann: Biochem. Zeitschr. 123. 235 (1921). (1909). — — XXXII. Vorlesung. (3;^Q den Eiweißstoffeu. Auch von diesen wird immer ein Teil abgebaut. Nie A mau im Hungerzustand den Harn stickstofffrei. Stets sind di e b" baustufen der Proteine und der Nukleinsäuren in den Geweben anzutreffen. Wahrscheinlich verwendet die Zelle Teile der Nukleoproteide und vor allem der Nukleinsäuren zur Bildung von Sekret- und Inkretstoffen und vielleicht auch von Fermenten. Jedenfalls müssen beständig Lücken im Gehalt der einzelnen Zellen an Bausteinen der Nukleinsäuren entstehen. Diese können nur von der zugeführten Nahrung aus ausgefüllt werden, wenn nicht eine Neubildung von Bausteinen der Nukleinsäuren im Organismus selbst erfolgt, oder von anderen Zellen Material zur Verfügung gestellt werden kann. Es ist nicht sehr wahrscheinlich, daß der tierische Organismus ihm zugeführte Bausteine verschmäht, und es vorzieht, die sicher sehr komplizierte Svnthese durchzuführen, um zu denselben Produkten zu gelangen. Läßt man den tierischen Organismus hungern, dann beobachtet man, daß beständig im Harn Abkömmlinge von Purinbasen auftreten. Interessanterweise ist die Menge der Purinbasen im Harn annähernd konstant, und zwar scheidet jedes Individuum eine ganz bestimmte Menge dieser Substanzen aus. Daraus darf gefolgert werden, daß im Zellstoffwechsel Tag für Tag in engen Grenzen gleiche Mengen von Nukleoproteiden bzw. Nukleinsäuren oder noch genauer ausgedrückt von Purinbasen zum Umsatz kommen. Man hat von einer endogenen Abkunft dieses Anteils von Abkömmlingen der Purinbasen gesprochen. ^) Wenn man nun in der Nahrung solche in genügender Menge zuführt, 2) dann steigt der Gehalt des Harns an Stoffwechselendprodukten an, die sich auf Purinbasen zurückführen lassen. Dieses Plus an diesen Verbindungen hat man als exogenen Anteil der im Harn erscheinenden, mit den Purinbasen in Zusammenhang stehenden Produkte bezeichnet. Uns scheint diese scharfe Abgrenzung einer endogenen und exogenen Abkunft von Stoffwechselprodukten wenig gerechtfertigt. 3) Man müßte sie denn schon auf den gesamten Stoffwechsel übertragen. LTnzweifelhaft werden mit der Nahrung aufgenommene Purinbasen auch Bestandteil von Zellen. Es reihen sich damit gewiß immer solche in den „endogenen" Anteil der Purinbasen ein. Wir können wohl rein äußerlich eine Trennung von Stoffen, die aus zelleigenen Produkten hervorgehen, von solchen, die im Überschuß vom Darme aus zugeführt werden, durchführen, doch dürfen wir derartige Begriffe nicht auf die Stoffwechselvorgänge als solche übertragen. Sie können leicht zu Mißverständnissen Es führen und sagen entschieden mehr aus, als wir eigentlich wissen. der Abbau stufen verschiedenen daß die werden, übersehen auch nicht darf Purinbasen in den Zellen vielleicht ganz besondere Funktionen erfüllen. Man darf sie nicht einfach als unnütze Schlacken betrachten. Schließlich hat eine wichtige Fragestellung bei der Besprechung der Frage nach der Herkunft der Harnsäure bzw. des AUantoins viel zu wenig Berücksichtigung gefunden, nämlich diejenige nach dem weiteren Schicksal dieser Vorbin- findet ») (1901). Richard Bunan und Heinrich Schur: Pflügerf^ Archiv. 80. 241 (1900); 87. 239 — Elbert W. liockwood: Aiueric. Jouru. of I^hysiol. 12. 38 (1905). - Schreiber und Waldvogel: Arch. f. oxperim. Path. u. Pharmak. 32. 69 (1899). den Gehalt einiger Nahrungsmittel an Purinbasen: liessau und Th. Brugsch und Hesse: Med. Klinik. Therapeut. Monatshefte. 116(1910). TA. V. Fellenberg: Biochem. Zeitschr. 88. 323 (1918). 623 (1910). ') Vgl. zu dieser F'rage auch M. Kikuchi: .1. of Biochem. 1. S3 (19221. W. C. Rose: J. <.f bin], (hcm. 48. .')68. 57ö (1921). 2) Vgl. über J. ScA/w/d; — — — Nuk leopiotcide. Nukleinsäuren und ihre Bausteine. ßgl Geht der Abbau der Purinbasen beim Menschen und den anthropoiden Affen wirklich nicht über die Harnsäure hinaus? Ist dasAllantoin in der Tat das letzte Stoff Wechselprodukt, das bei den übrigen Tieren aus Purinbasen gebildet werden kann? dangen. Was die Harnsiiure anbetrifft, so steht zurzeit Meinung gegen Meinung, i) Dagegen verfügen wir beim AUantoin über Beobachtungen, die zeigen, daß beim Hunde vielleicht ein weiterer Abbau dieser Verbindung möglich Es wurde nach Verfütterung von AUantoin, von Inosin und von ist.2) Nukleinsäure ein sehr erheblicher Teil des im Harn zu erwartenden Allantoins vermißt. Ganz eindeutig sind diese Versuche allerdings nicht weil sich die Ergebnisse auf Fütterungs versuche stützen und gegen diese der Einwand erhoben werden kann, daß die Üarmflora vielleicht weitgehende Umwandlungen der zugeführten Produkte bewirkt hat. Sollte es sich bewahrheiten, daß AUantoin nicht die letzte Abbaustufe der Purinbasen darstellt, dann sind natürlich auch Schwankungen im Umfange des weiteren Abbaus dieser Verbindung denkbar. ») Es vAirden sich jedenfalls die Beziehungen zwischen den Zell- und Nahrungspurinen einerseits und den Abkömmlingen der Purinbasen des Harns nicht so klar abgrenzen lassen, wenn immer ein wechselnder Teil davon über das AUantoin hinaus zerlegt würde. Immer wieder hat man den Versuch unternommen, ganz bestimmte Zellarten mit dem sog. Purinstoffwechsel in Verbindung zu bringen. Zunächst sollten nur die Leukozyten) an der Bildung der Abkömmlinge der Purinbasen beteiligt sein. Dann dachte man vor allem an die Muskulatur.^) Endlich brachte man die Entstehung der Stoffwechselendprodukte der Purinbasen mit der Tätigkeit der Verdauungsdrüsen ") in Zusammenhang. Es ist nun wohl möglich, daß bestimmte Zellen mehr Purinbasen hervorbringen als andere. So soll z. B. die Leber großen Anteil an der Bildung von Abkömmlingen der Purinbasen^) haben. Ja, es wird ihr sogar eine zentrale Stellung im Purinstoffwechsel zugeschrieben. Möge dem sein, wie ihm wolle, jedenfalls hat jede einzelne Zelle — 1) F. Frank und A. Schitfenhelm Zeitschr. f. physiol. Clieniie. 63. 243 (1909). Th. Brtigsch und A. Schitfenhelm : Zeitschr. i'. experim. Path. u. Therapie. 4. 480 (1907). l.afaycfte, Vgl. auch L.B.Mendel und B. White: Amer. Journ. of Physiol. 12. 85 (1904). B. Mendel und Frnest W. Brown: The Journ. of the Amer. Med. Assoc. 49. 896 (1907). Wilhelm Wiechoivski : Arch. f. exper. Path. u. Pharmak. 60. 185 (1909); Biochem. Zeitschr. Vgl. auch 0. Loewi: Arch. f. exper. Path. u. Pharmak. 44. 21 (1900). 25. 431 (1910). W. WieF. Soefbeer und ./. Ibrahim: Zeitschr. f. physiol. Ciiemie. 35. 1 (1902). W. Lewinthal : 'A^it^^oXw. f. physiol. Chemie. choivski: Biochem. Zeitschr. 25. 431 (1910). "//. G. Wells: Moris S. Fine: Journ. f. hiol. Chem. 23. 471 (1915). 77. 273 (1912). S.J. Thannhauser und G. Czoniczer: Deutsches Arch f. kliu. El)enda. 26. 319 (1911). A. Schittenhelm und K. Earpiider: Zeitschr. f. d. gas. experim. Med. 135. 224 (1921). Medizin. 27. 14, 34, 43 (1922). ) P. A. Levene und F. Mediqreceanu : Amer. Journ. of Physiol. 27. 438 (1911). ) Beim Schweine scheint allerdings das AUantoin ein Endprodukt des Stoffwechsels darzustellen. Vgl. A. Schittenhelm: Zeitschr. f. phvsiol. Chemie. 66. 53 (1910). ) J. Horbaczewski: Monatshefte f. Chemie. 10. 624 (188'.»); 12. 221 (1891). I'. O. Siren ^) Richard Hurian: Zeitschr. f. physiol. Chemie. 43. 532 (1905). Arch. f. Physiol. 18. 177 (1906). «) Fra»z Mare.i: Monatshefte f. Chemie. 13. 101 (1892): l'fUh/ers Archiv. 134. Franz Franz Smetdnka: Ebenda. 138. 217 (1911); 149. 287 (1912). 59 (1910). Mares: Pflügers Archiv. 149. 275 (1912). ') Vgl. Hans Rosenhrrc): Zeitschr. f. experim. l'ath. u. Therap. 14. 245 (1913). .- — — — — — — ~ — — — — — — — XXXII. Vorlesuug. (382 einen Purinstoff Wechsel für sich. Da einzelne davon den Abbau der Purinbasen nicht zu Ende führen können, so müssen wir annehmen, daß bestimmte, nicht weiter veränderbare Abbaustufen solchen Zellen zugeführt werden, die über jene Fermente verfügen, die der ersteren Zellart fehlen. Vielleicht erklärt sich das Vorkommen von Purinbasen im Urin zum Teil dadurch, daß nicht alle Zellen ihren Abbau zu Ende führen können und sich nicht immer die Gelegenheit findet, ihn in anderen Zellen zu vollenden. Zum Schlüsse wollen wir uns noch nach Anomalien im Purinstoff Wechsel umsehen. Es ist klar, daß dann, wenn aus irgend einem Grunde Kernsubstanzen in vermehrtem Maße zerfallen, auch der Gehalt des Harns an Produkten, die auf die Purine zu beziehen sind, ansteigt. So findet man z. B. bei Leukämie, einer Erkrankung, bei der die Zahl der weißen Blutkörperchen sehr stark gesteigert ist und auch viele davon zugrunde gehen, eine erhöhte Ausfuhr von Purinbasen und ihren Abbauprodukten. Einer Anomalie des Purinstoffwechsels haben wir bereits gedacht, nämüch der sog. Guaningicht des Schweines. Wir haben als Erklärung für die interessante Beobachtung, daß bei dieser Guanin im Harn und ferner in den Geweben auftritt, angeführt, daß wahrscheinlich die Guanase nicht in Funktion tritt. Wir können diese Stoff wechselanomalie der Aminoazidurie mit den Spezialfällen der Diaminurie und der Cystinurie an die Seite stellen. Auch die Ausscheidung der Homogentisinsäure bei der Alkaptonurie hat wahrscheinlich eine ähnliche Ursache, wie Guaninurie. Der Abbau ist in allen Fällen aufgehalten, weil die Fermente fehlen oder nicht wirken können, die sonst den weiteren Abbau die besorgen. Beim Menschen gibt es eine Harnsäuregicht.^) Wir finden Ab- scheidungen von Harnsäure in bestimmten Geweben. 2) Bevorzugt für diese ist ganz besonders das Knorpelgewebe. 3) Eine Zeit lang schien es, als ob sich die erwähnte Erkrankung ohne weiteres als Anomalie des Purinstoffwechsels erklären lasse. Man dachte daran, daß zunächst die Umwandlung der Aminopurine in Oxypurine und in Harnsäure stark verlangsamt sei, und endlich die Urikolyse entweder gar keine oder doch nur eine mangelhafte Wirkung entfalte. Es sollte dadurch zur Zurückhaltung der Abbaustufen der Purinbasen und schließlich zu einer Überschwemmung des Blutes und der Gewebe mit Harnsäure kommen. Nun werden wir gleich erfahren, daß die Harnsäure sehr schwer löslich ist. Ihre Salze lösen sich etwas leichter. Im Blute kommt nur das Mononatriumsalz der Harnsäure vor. ) Erreicht der Gehalt einer Flüssigkeit an Harnsäure einen gewissen Grad von Sättigung, dann kommt es zu ihrer Abscheidung. In der Tat ~ Vgl. hierzu O.Minkowski: Die Gicht. Noihnageh HaiuRmch. 1903. C. v. Noorder Pathol. des Stoffwechsels. Hirschwald. Berlin 1906. Ebstein: Natur und Behandlung der Gicht. J. F. Bergmann. Wiesbaden 1906. Th. Brugsch und A. Sehittenhelm: Der Nnkleinstoütwechsel und seine Störungen. G. Fischer. Jena 1910. ^) Vgl. dazu die Beobachtungen von Theodor Brugsch und A. Sehittenhelm : Zeitschrift f. experim. Path. u. Ther. 4. .'i32 (1909). ^) Vgl. dazu ./. J. Loghein: Zentralbl. f. d. ges. Physiol. u. Path. d. Stoffw. N. F. Roberts: Brit. med. Journ. 18. June 25. 2. u. 3. Jahrg. (1892). Vgl. 2. 244 (1907). dazu auch F. Gudzent: Zeitschr. f. physiol. Chemie. 63. 455 (1909). A. Almaqia Hofmeistern Beitr. 7. 466 (1906). ) Vgl. F. Gudzent: Zeitschr. f. physiol. Chemie. 63. 455 (1909). ') den: Handbuch — — — — — Nukleoproteide. Nukleinsäureu uud ihre Bausteiue. 688 hat man im Blute von an Gicht Leidenden wiederholt eine VermehrungInsbesondere unmitteli3ar vor und während der Harnsäure gefunden. i) eines sogenannten Anfalles ist meistens eine Hyperurikoplasmie gefunden w^orden. Interessanter Weise ließ sich feststellen, daß die Gelenkflüssigkeit bei Gichtikern einen auffallend hohen Gehalt an Harnsäure hat. Dem Versuche, das Zustandekommen der Harnsäureabscheidung in Geweben und das Zustandekommen der Gichtanfälle dabei linden wir — bestimmte Gelenke (meistens das Großzehengelenk) stark gerötet und geschw^oUen; der Patient klagt über heftige Schmerzen; es liegt eine Entzündung vor von einer Überschwemmung des Organismus mit Harnsäure abhängig zu machen, stehen nun viele Bedenken entgegen. Zunächst zeigt die Erfahrung, daß die Niere große Mengen von Harnsäure auszuscheiden vermag, wenn das Blut mit solcher überschwemmt wird. Man kann experimentell durch Verfütterung von Purinbasen und auch durch Verabreichung von Gewebe, das reich an Nukleoproteiden ist, z. B. von Thymusgewebe, eine Hyperurikoplasmie hervorrufen.-) Man kann sie als eine alimentäre bezeichnen. Sie führt unter normalen Verhältnissen zu keinen Störungen. Die Niere zeigt eine vermehrte Ausscheidung der Harnsäure. Sie entfernt den Überschuß an dieser aus dem Blute. Es kann auch dann zu einer Hyperurikoplasmie kommen, wenn an Purinbasen reiche Zellen, z. B. Leukozyten, in großer Menge zerfallen. Es ist dies bei der Leukämie, der Pneumonie usw. der Fall. ^) Es w^äre auffallend, wenn die Nieren bei der Gicht dieser Aufgabe nicht gerecht werden sollten. Sollte eine funktionelle Störung jener Zellen der Harnkanälchen vorliegen, die Harnsäure auszuscheiden haben ? Es braucht sich eine solche nicht immer durch eine erkennbare morphologische Veränderung der betreffenden Zellarten kund zu tun ! ) Man glaubte, wie schon oben erwähnt, die ganze Störung des Purinstoffwechsels bei der Gicht restlos durch den Umstand erklären zu können, daß der fermentative Abbau der Purinbasen und vor allem die Zerlegung der Harnsäure gestört sei. Diese Annahme steht und fällt mit der eindeutigen Entscheidung der Frage, ob Gewebe des Menschen Harnsäure abzubauen vermögen. Sie ist, wie S. 681 erw-ähnt, noch offen. Erwähnt sei noch, daß daran gedacht \Yorden ist, es könnte die Leber im Purinstoffwechsel eine ähnliche Rolle spielen, wie im Kohlehydratstoffwechsel. 6) Sie soll Purinbasen speichern. Ferner soll in der Medulla oblongata ein Zentrum vorhanden sein, das den Purinumsatz der Leber regelt. "'>) 1) A. B. Garrod: Medical-chirurg. Transact. Loudou. 37. 49 (1884). -— Bruno Theodor Brugsch und Alfred Bloch: Zeitschr. f. physiol. Chemie. 51. 472 (1907). P. Salecker: Schittenhelm: Zeitschr. f. experim. Path. n. Ther. 4. 438, 44G (1907). Vgl. auch Robert Bass uud R. lierzhery: Deutsches Arch. f. klin. Med. 95. 353(1909). Gudzent : Zeitschr. f. klin. Medizin. 90. ^r. 3'4 Arch. f. klin. Med. 119. 482 (1918). — — — — (1921). 2) 3) ) ^) (1907). (1910). Vgl. z. B. W. Weintraud: Wiener klin. Rundschau. 10. 3, 21 (1896). Vgl. hierzu z. B. Linser und Sick: Arch. f. klin. Med. 89. 413 (1907). Vgl. hierzu auch S. J. Thannhauser: Hab.-Sclirift. München 1917. Vgl. Th. Brugsch und A. Schittenhelm: Zeitschr. f. exper. l'ath. u. Ther. 4. 551 F. Umher und //. Rcfzluf: Ber. d. 27. Kongresses f. innere Medizin. 436 P. A. Levene und Leo Kristeller: Journ. f. experim. Med. 16. 303 (1912). Th. Brugsch und //. Poscnberg: Zeitschr. f. experim. l'ath. u. 'I'her. 14. 1 (1913). — — «) XXXII. Vorlesung. (384 Da diese Vorstellung noch nicht ausreichend begründet erscheint, wollen wir nicht auf die Folgerungen eingehen, die aus der Annahme eines Purinoder Harnsäurezentrums für die Entstehung der Symptome der Gicht gezogen worden sind.') Die Störung im Purinstoffwechsel bei der Gicht 'kommt natürlich auch in der Zusammensetzung des Harns zum Ausdruck. 2) Der Harn enthält namentlich zur Zeit des Gichtanfalles weniger Harnsäure als unter normalen Verhältnissen. Die Harnsäureausscheidung ist verschleppt. Dagegen ist der Gehalt des Harns infolge der verlangsamten Umwandlung der Purinbasen in Harnsäure relativ reich an diesen. Schließlich wollen wir noch kurz erwähnen, daß man daran gedacht hat, daß die Harnsäure unter normalen Verhältnissen nicht frei im Blute kreist, sondern in gebundenem Zustande.^) Bei der Gicht sollte diese Bindung gestört sein. Es ließen sich jedoch auch für diese Annahme keine eindeutigen Beweise erbringen. Dagegen ist die Beobachtung wichtig, daß die Harnsäure bei Gegenwart von Eiweiß in Wasser viel leichter löslich ist.) Es handelt sich vielleicht um Adsorptionserscheinungen. ^) Endlich sei noch darauf hingewiesen, daß daran gedacht worden ist ß), daß die Harnsäure in tautomeren Formen vorkommt und bei der Gicht vielleicht jene Strukturform in Erscheinung tritt, die ein schwerer lösliches Salz bildet.') Es sind nach Emil Fischer^) die folgenden beiden Formen der Harnsäure möglich: N^C.OH NH-CO IIC— NH II OH C CO . C— NH >C...H N— C-N Lak timform der Harnsäure. hat wenig für sich. Man könnte mit dem annehmen, daß beim Diabetes der Traubenzucker einer anderen Form zugegen ist. als normalerweise. Außerdem müßte Eine solche gleichen Rechte in ') Annahme auch Vgl. hierzu auch Th. Brugsch und ,7. liother: Klinische Wochenschr. 1. 1729 (1922). 1495, — 2) Vgl. u. a. M. Kaufmann und L. Mohr: Archiv f. klin. Med. 74. 586 (1902). Bruno Bloch: Deutsches Archiv f. kliu. Med. 83. 499 (19(35). Theodor Brugsch: Zeitschr. f. experim. Path. u. Ther. 2. 619 (1906). — Th. Brugsch und A. Schiftenhelm — Zeitschr. f. experim. Path. u. Ther. 4. 480 (1907); ebenda. 4. 1 (1907). ^) 0. Minkowski: Th. Brugsch: Zeitschr. f. experim. Die Gicht. Wien 1903. Path. u. Ther. 6. 282 (1909). A. Schiff enhelm: Ebenda. 7. 110 (1910). Max Dohrn: Zeitschr. f. kliu. Med. 74. 445 (1912). Zeitschr. f. physiol. Chemie. 86. 130 Vgl. dazu auch O. Minkowski: Ebenda. 88. 159 (1913). Mathieu-Fierre (1913). Weil und Ch. 0. Guillaumin: Compt. rend. de la soc. de biol. 86. 242, 319, 659 (1922). ^j H. Bechhold und J. Ziegler: Biochem. Zeitschr. 64. 471 (1914). Vgl. auch H. Schade: Zeitschr. f. klin. Medizin. 93. Heft 1/3 (1922). ^) Vgl. hiezu H. Harpuder: Z. f. die gesamte experim. Medizin. 29. 208 (1922). ) F\ Gudzent: Deutsche med. Wochensclir. 21. 219 (1909); Zeitschr. f. physiol. Chemie. 60. 38 (1909): 63. 4.55 (1909). ") Das unstabilere Salz (Laktamforni) ist löslicher (in 100 c«'' Blutplasma lösen sich 18"4 w// Mononatrinmsalz) als die stal)ile Verbindung (Laktimform) (es lösen sich davon nur 8"3 mg in der gleichen Menge Plasma). «) Emil Fischer: Ber. d. Deutschen (;hem. Gesellsch. 32. 435 (1899). — — — — — — — Mukleoproteide. Mukleiusäuieii und üirc Bausteine gjKÖ die erwähnte Annahme auch zu Hilfshypothesen greifen und zu erklären ver- suchen, weshalb die eine Form bevorzugt sein sollte, und weshalb die Nieren Harnsäure nicht in genügender Weise aus dem Blute entfernen. Die Ergebnisse der Studien über den Purinstoffwechsel bei der Gicht vermögen zurzeit, wie schon betont, unsere Kenntnisse des normalen Ablaufes des genannten Stoffwechsels nicht zu fördern, i) Es scheint vielmehr, als ob die Gicht im wesentlichen gar keine primäre Störung im Abbau der Puriubasen und ihrer Bildung darstelle, sondern vielmehr die Entfernung der Harnsäure aus den Zellen und dem Blute in irgend einer Weise gestört ist. Es könnte ganz gut sein, daß die Gegenwart größerer Mengen von Harnsäure für die Verlangsamung des Abbaues der Purinbasen verantwortlich ist, d. h. daß diese Erscheinung sekundärer Natur ist. Es ist auch möglich, daß die Harnsäure bei der beobachteten Ansammlung in der Gelenkflüssigkeit nicht primär in Frage kommt, vielmehr könnte eine Veränderung im Gelenke selbst ihre vermehrte Anwesenheit bedingen. -) Jedenfalls ist die Aufklärung des Wesens der Gicht noch weit von ihrem Ziel entfernt. Erwähnen wollen wir noch, daß die Harnsäure als Bestandteil von Harnsteinen auftreten kann. Besonders interessant sind jene Steine, an die deren Zusammensetzung Xanthin beteiligt ist. Es wäre von größtem beim \'orkommen von Xanthinsteinen zu forschen, ob eine Anomalie im Abbau der Purinbasen und insbesondere des Xanthins vorliegt. Es wäre ja denkbar, daß die Umwandlung des Xanthins in Harnsäure nicht erfolgt. In diesem Falle dürfte allerdings keine Harnsäure aus den Purinbasen entstehen, w^enn nicht bei der Umwandlung von Adenin und Guanin die Oxydation zuerst einsetzt und erst dann die Hesaminierung erfolgt. ^) Interesse, Das Studium der Gicht hat immer wieder zu der Frage nach den Löslichkeitsverhältnissen und den Eigenschaften der Harnsäure Die reine Verbindung bildet mikroskopische, rhombische, durchsichtige Täfelchen. Sie löst sich sehr schwer in Wasser. Bei 18" löst sich ein geführt. Teil Harnsäure in o9-430 Teilen Wasser) und bei oT" in 15505 Teilen. ^j Von großem Einfluß auf die Löslichkeit der Harnsäure ist die Wasserstoffionenkonzentration.ßj Die Harnsäure ist in wässeriger Lösung eine einbasische Säure. Sie dissoziiert nämlich ein Wasserstoff-Ion ab. Sie ist eine stärkere als die Kohlensäure, HCO. H. und als das primäre Natriumphosphat, NaHPOi H. In wässeriger Lösung finden sich nur primäre, einfachsaure Salze. Nur unter besonderen Umständen, z. B. bei Anwesenheit von starken Basen, kann die Harnsäure noch ein zweites H-Ion Säure . . Es können sich dann sekundäre Urate bilden. Sie sind und setzen sich in wässeriger Lösung sofort wieder in das primäre Salz um. Die folgenden Formeln veranschaulichen diese „stufenweise- Dissoziation der Harnsäure und die Bildung der beiden Reihen von Salzen: abdissoziieren. nicht 61. beständig ) Vgl. hierzu auch •/. R. Müller und Walter Jones-: Zcitschr. f. physiol. Chemie. 395 (1909). ) Bobert ßass und li. Jlerzbert/: Deutsches Arch. f. kliu. Med. 119. 482 (1918). Vgl. S. 070 und 671. ) W. His und Th. Paul: Zeitschr. f. plivsiol. Chemie. 31. 1, 64 (1900i. 5) F. Gudzent: Ebenda. 56. l.öO (1908);' 60. 25. 38 (1909). «) A. Jung: Helvet. chim. acta. 5. 687 (1922). =>) XXXII. Vorlesung. 536 C5 H, N, O3 Harnsäure / aH3N4 0r+ H+ C.H^N.O^ .Na Primäres Natriumsalz \ C5H2N,Or+ h++ h + CVH2N, 03_Na2 Sekundäres Natriumsalz. Das erstere Salz, das Mononatriumurat, kommt allein im tierischen Organismus vor. 1) Es ist in Lösung stark dissoziiert. Das Blutplasma und wahrscheinlich auch die Zellen können mehr Mononatriumurat aufnehmen, Offenbar spielen die Kolloide als eine entsprechende Menge von Wasser. eine große Rolle beim in Lösung halten des genannten Salzes. 2) Auch im Harn dürften durch solche bedingte, erhöhte Löslichkeitsverhältnisse vor- handen sein. 3) Im Harn kommt es oft zur Abscheidung von Harnsäure. Sie hat die Eigenschaft, Farbstoffe mitzureißen. Deshalb ist das Sediment, das der Harn rosarot gefärbt (Sedimentum lateritiiim). ) Meistens Abscheidung freie Harnsäure und daneben wechselnde Mengen von Mononatriumurat. 0) Die Bildung der freien Harnsäure erfolgt offenbar erst im Harn. Die Nieren scheiden sie wohl in ihrer Gesamtheit absetzt, oft gelb bis enthält diese Sak aus. e) als Vgl. F. Gudzent: Zeits.chr. f. phvsiol. Chemie. 63. 455 (1909). H. Bechhold und ./. Ziegler: Biochem. Zeitschr. 20. 189 (1909); 24. 146 (1910); F. Gudzent: Ebenda. 23. 275 (1909). 64. 471 (1914). L. Lichtwitz und ^) L. Lichtwitz: Zeitschr. f. physiol. Chemie. 64. 144 (1910). 1) 2) — — Bosenbach: Ebenda. 61. 112 (1909). ) Die gelbe Farbe ist durch Urochrom undUrobiliu bedingt. Die rote rührt von Uroerythrin her. ^) Vgl. hierzu W. E. Ringer: Zeitschr. f. physiol. Chemie. 75. 13 (1911). Budolf Kohler: Ebenda. 70. 360 (1911); 72. 169 (1912); 88. 259 (1913). ) Vgl. weitere Einzelheiten in der Vorlesung über Harn. (). — Vorlesung XXXIII. Chlorophyll und Hämoglobin. Ihre Herkunft und ihr Verhalten im tierischen Organismus. Die Beziehungen des Hämatins zum Gallenfarbstoff und zum Urobilin. Sonstige Farbstoffe. Blatt- und Blutfarbstoff. Eiue weitere Klasse von Proteiden stellt die Gruppe der Blutfarbstoffe dar. Sie bestehen aus einem Eiweißanteil und einer Komponente, die mit den Proteinen in keinen unmittelbaren Beziehungen steht. Un^ interessiert hier am meisten der Blutfarbstoff der Wirbeltiere, das Hämoglobin. Es liefert bei der Spaltung einen an Histidin reichen Eiweiükörper, das Globin, und eine eisenhaltige Verbindung. Diese hat den Xameii fläraochromogen erhalten.') Das Hämoglobin findet sich als Bestandteil der roten Blutkörperchen. Es kommt ihm die wichtige Funktion zu. Sauerstoff zu binden. Dibei entsteht Oxyhämoglobin. Wir werden später erfahren, daij das Blut nur eine geringe Menge von Sauerstoff zu lösen vermag. Wären keine besonderen Einrichtungen vorhanden, dann würde die Versorgung der Gewebe mit Sauerstoff eine außerordentlich beschränkte sein. Die Zellen brauchen diesen wichtigen Nahrungsstoff vor allen Dingen, um sich die in den organischen Verbindungen vorhandene Energie zu erschließen. In ganz besonders hohem Maße ist der Warmblüter auf Sauerstoff angewiesen, muß er doch ununterbrochen Energie zur \'erfügung haben, um die Körpertemperatur auf bestimmter Höhe zu erhalten. Dadurch, daß im Blute Hämoglobin zugegen ist, das Sauerstoff binden kann, verfügt der tierische Organismus über die Möglichkeit, große Mengen von Sauerstoff aufzunehmen und für die Gewebe zur Verfügung zu halten. Die Bindung zwischen Hämoglobin und Sauerstoff ist nämlich keine feste. Das Oxyhämoglobin zerfällt leicht in Hämoglobin und Sauerstoff. Solange die.ser gebunden ist, hat er keinen Einfluß auf die Sauerstoffspannung im Blute. Erst dann, wenn er frei wird und sich im gelösten Zustande im Blute bzw. in Sauerstoffdruck. stoffs vor. den Geweben findet, beeinflußt er den vorhandenen Wir werden später bei der Besprechung der Bedeutung des Sauerfür den Organismus ausführlich auf die physikalischen Eigen- ') Dieser Name ist niclit sehr glücklich gewählt, denn es liegt kein „Chromogen" Die Ausdrücke Oxyhämatin für Humatin und Hämatin für Hiimochromogei wären viel klarer. XXXIII. Vorlesung. ßg3 Schäften des Hämoglobins und Oxyhämoglobins eingehen. Wir werden dann erfahren, dal) Hämoglobin nnd .Sauerstoff sicli in einem bestimmten Mengenverhältnis binden. Ferner werden wir sehen, daß an Stelle des Sauerstoffes andere Gase, wie Kohlenoxyd und Stickoxyd, Verbindungen mit Hämoglobin eingehen können. Hämoglobin und Oxyhämoglobin lassen sich in Kristallen zur Ab- scheiduDg bringen. Das Oxyhämoglobin des Eichhörnchens, der Maus in sechsseitigen Tafeln des hexagonalen Systems, das der übrigen Tierarten in Nadeln, Prismen, Tetraedern oder Tafeln des rhombischen Systems. Man glaubte die verschiedene Kristallform als Anzeichen von Verschiedenheiten in der Zusammensetzung der von verschiedenen Tieren herstammenden Oxyhämoglobinarten auffassen zu dürfen. Es zeigte sich jedoch, daß z. B. das gewöhnlich im hexagonalen System kristallisierende Oxyhämoglobin des Eichhörnchenblutes nach wiederholtem Umkristallisieren Nadeln und Tetraeder des rhombischen Systems Die Löslichkeit der Oxyhämoglobine verschiedener Tierarten in liefert. 1) Wasser ist eine verschiedene. So ist z. B. das Oxyhämoglobin des Hundes schwerer löslich als das der Katze. 2) Leicht löslich und deshalb auch schwerer darstellbar sind die Oxyhämoglobine des Menschen-, Rinder- und Schweineblutes. Man hat versucht, aus der elementaren Zusammensetzung der Blutfarbstoffe verschiedener Tierarten Schlüsse auf ihre Identität bzw. Verschiedenheit zu ziehen. Wir geben einige dieser Analysen in der folgenden Tabelle wieder, möchten jedoch nicht versäumen, auch an dieser Stelle ausdrücklich zu betonen, daß bei so komplizierten Verbindungen die Ergebnisse von Elementaranalysen nach keiner Richtung hin etwas über eine etwaige Identität beweisen. und des Hamsters kristallisiert =^) Elementaranalysen des Oxyhämoglobins Blatt- uud^Blutfarbstoff. Chlorophyll uud Hämoglobin usw. (5^9 Wir können diesen Analysen entnehmen, daß das Oxyhämoglohin und Fe enthält, während im der Säugetiere die Elemente C, H, N, S, Vogeloxvhämoglobin außerdem noch Phosphor aufgefunden worden ist. Der Phosphorgehalt des Oxyhämoglobins des Hühnerblutes ist jedoch ohne Zweifel auf eine Verunreinigung zurückzuführen. Es ist nämlich gelungen, das Oxyhämoglohin des Gänseblutes phosphorfrei zu gewinnen^), nachdem früher auch bei diesem ein Phosphorgehalt festgestellt worden war. Der Phosphor ist sehr wahrscheinlich auf beigemengte Nukleoproteide oder Nukleinsäuren zurückzuführen. Die roten Blutkörperchen der \'ögel enthalten nämlich Kerne und damit auch die genannten phosphorh altigen Bestandteile. Den roten Blutkörperchen der Säugetiere dagegen fehlen die Kerne. Darauf ist es zurückzuführen, daß das von ihnen gewonnene )xyhämoglobin ohne weiteres frei von Phosphor erhalten wird. Die Beobachtung, daß Oxyhämoglobin mit Verunreinigungen beladen kristallisieren kann, zeigt immer wieder aufs neue, daß die Kristallbildung an und für sich bei so kompliziert gebauten Körpern nichts über ihre Reinheit auszusagen braucht. Wie weit eine Reinigung der Kristalle durch Umlösen möglich ist, ist schwer zu sagen. Es besteht nämlich die Gefahr, daß sie bei mehrfachem Umkristallisieren Änderungen in ihren Eigenschaften erfahren. Wir dürfen nie außer acht lassen, daß das Oxyhämoglobin Eiweiß enthält, das leicht Zustandsänderungen erleidet. Diese können dem ganzen Proteid andere Eigenschaften erteilen und z. B. auch das Gasbinduugsvermögen beEndlich ist beobachtet worden, daß das Oxyhämoglobin die einflussen. Neigung hat, in eine vielleicht isomere Verbindung überzugehen, die den Sauerstoff fest gebunden enthält. Sie ist Methämoglobin genannt worden. 2) Es ist klar, daß schon geringe Mengen dieser Verbindung das Resultat der Bestimmung des vom Oxyhämoglobin gebundenen Sauerstoffes beeinflussen müssen, denn das Methämoglohin gibt seinen Sauerstoff nicht ( mehr ab. Wir werden später erfaliren, daß es für das Verständnis der Bindung des Sauerstoffes an das Hämoglobin und die Spaltung von Oxyhämoglobin in Sauerstoff und Hämoglobin von größter Bedeutung ist, genau zu wissen, Ferner möchten wir gerne wieviel Sauerstoff das letztere binden kann. wissen, ob das Blut jeder Tierart nur ein bestimmtes Hämoglobin besitzt. Endlich ist die Möglichkeit gegeben, daß in der ganzen Tierreihe überhaupt nur eine Art von Hämoglobin vorkommt. In vielen Eigenschaften stimmen alle Oxyhämoglobinarten der verschiedensten Tiere überein. So /eigen sie alle das gleiche oder doch ein sehr ähnliches AbsorptionsWir Andrerseits sind doch gewisse Unterschiede vorhanden. spektrum. haben schon der verschiedenen Löslichkeit gedacht. Vorläufig wäre es müßig, dieser Frage weiter nachzugehen. Dazu sind unsere Kenntnisse über den Bau des Oxyhämoglobins viel zu geringe. Es ist ganz gut möglich daß das Globin einen artspezifischen Aufbau hat und dadurch jeder Tierart auch ein besonderes Oxvhämoglobin zukommt. Einheitlich scheint in der — KmH ') Vgl. hierzu Zeitt^chr. f. pliysiol. Chemie. 18. 57 (1894). Y. Inoko Abderhalden und Florentin Mediyreceanu: Kbeuda. 51). Kiö (19'J9). (Hi2ü). SC) 2) Vgl. Fh. Ellinf/er: Zeitschr. f. physiol. Chemie. 111 u. a. IV.Heubner: Archiv f. ex.perim. Pathol. u. Pharm. 72. 241 (1913). : — Abderhald« 11 . Physiologiäche Chemie. I. l'ail, 5. Aufl 44 XXXIII. Vorlesung. 690 ganzen Tierreihe die zweite Komponente des Blutfarbstoffes zu sein, nämlich das Hämochromogen bzw. dessen Sauerstoffverbindung, das Hämatin.i) Das reduzierte Oxyhämoglobin, das Hämoglobin, kristallisiert ebenfalls. Es bildet lange, dunkelrote, doppelbrechende Nadeln, rhombische Prismen und holoedrische Tafeln. Die Kristalle des Hämoglobins lösen sich in Wasser leichter als die des Oxyhämoglobins. Sie bilden dichroitische Lösungen. In dicker Schicht sehen sie dunkelkirschrot in dünner grünlich aus. Den Eiweißpaarling des Blutfarbstoffes haben wir bereits besprochen. Er gehört zu den basischen Proteinen. Sein auffallend hoher Gehalt an Histidin ist es hauptsächlich, der ihm die basischen Eigenschaften verleiht. Noch nicht begegnet sind wir bisher dem nichteiweißartigen Paarling des Hämoglobins bzw. Oxyhämoglobins. Schon der Umstand, daß wir zwei verschiedene ^>rbindungen nannten, als wir von diesem Paarling beim Hämoglobin und Oxyhämoglobin sprachen, weist darauf hin, daß offenbar ihm die Bindung des Sauerstoffs zukommt. Dem ist in der Tat Zunächst beobachtete man, daß die roten Blutkörperchen Sauerstoff so. binden können. Dann lokalisierte man diese Eigenschaft auf den in ihnen enthaltenen roten Farbstoff. Man fand, daß dieser ebensoviel Sauerstoff festhalten kann, wie die der betreffenden Hämoglobinmenge entsprechende Anzahl roter Blutkörperchen. Weiterhin wurde dann "gezeigt, daß das Hämochromogen genau so viel Sauerstoff binden kann, als die ihm ent- Damit ist sichergestellt, daß dem Hämochromogen die Funktion, Sauerstoff zu binden, zukommt. .sprechende Menge Hämoglobin. Es wäre von allergrößtem Interesse, wenn wir genau wissen würden, Art und Weise der Sauerstoff und auch die anderen Gase, die vom Hämochromogen gebunden werden können, mit diesem verankert in welcher sind. die Da es Eisen enthält, dachte man .schon frühzeitig daran, daß diesem Funktion blick in zufällt, die betreffenden die Bindungsverhältnisse des Gase zu binden. Einen klaren EinHämochromogens und des Sauer- werden wir ohne Zweifel erst erhalten, wenn die Konstitution der erstereu Verbindung vollständig klargestellt ist, und wir vor allem auch genau wissen, wie das^Eisen in das ganze Molekül eingefügt ist. Es ist vielleicht im Hämoglobin in der Ferro- und im ^_ Oxyhämoglobin in der stoffs Ferriform vorhanden.-) Das Hämochromogen geht unter Sauer.stoffaufnahme in Hämatiii Zerlegen wir Oxyhämoglobin in seine Anteile, dann erhalten wir direkt Hämatin. Das gleiche ist der Fall, wenn -Hämoglobin unter Luftzutritt gespalten wird. Will man zum Hämochromogen gelangen, dann muß man jeden Zutritt von Sauerstoff vermeiden. Der Blutfarbstoff läßt sich sehr leicht in seine Bestandteile zerlegen. Schon Essigsäure veranlaßt übei". Abscheiduug des des Hämoglobins in die eisenhaltigen Paarlings. Diese leichte Spaltbarkeit Anteile weist darauf hin, daß Globin und seine Vgl. hierzu u. a. (f. Dilliny : Atlas der Kristallfoniieu iiiid der AbsorptionsV) bäiider der Häniochromogeue. F. Enite. Stuttgart 1910. ") Wilhelm Manchot: Zeitschr. f. physiol. Chemie. 70. 230 (1910): Liebigs Anoal. \V. Manchot u. F. mittner: Ebenda. 372. 153 (1910). 372. 179(1910). W. Manchot: Biofhein. Zeitsehr. 43. 4,SH (1912): Bor. d. Deutschen Cheiii.Gesellsch. 45. 28ß9 (19121 — — Blatt- uucl Blutfaihstott. (Jhloropliyll und Hämogloliiii usw. f391 Hämochromogen nicht in fester chemischer Hindiing vereinigt sind, vielmehr dürfte das letztere von der Eiweißkomponente adsorbiert sein. Man erhält aus 100«7 Blutfarbstoff ungefähr 4f/ Hämochromogen bzw. Hämatin.') Der Rest auf das Globin. entfällt Das Hämatin ist seit vielen Jahren Gegenstand eingehender Untersuchungen. Es ist geglückt, es in verschiedene Verbindungen überzuführen, die ihm noch sehr nahe stehen, und ferner Abbaustufen zu erhalten, die in ihrer Struktur vollständig aufgeklärt sind. Es schien zunächst, als würde die Konstitution des Hämatins bald aufgeklärt sein. Man durfte um so eher an eine rasche Erweiterung unserer Kenntnisse des Aufbaus der genannten Verbindung denken, weil ein anderer Farbstoff ihm sehr nahe zu stehen schien, dessen Erforschung gleichzeitig mit großem Erfolg in Angriff genominen worden war. Es ist dies der Blattfarbstoff, das Chlorophyll. Aus manchen Befunden hatte man geschlossen, dalo Hämatin und Chlorophyll nahe verwandt seien, ja man glaubte, da(i beide einen ganz ähnlichen, wenn nicht zum Teil identischen Bau besäßen. 2) Wir wollen daß die neueren Untersuchungen diese Vorstellung nur teilweise stützen. Gewiß sind gemeinsame Züge in beiden Verbindungen vorhanden, doch zeigen die fertigen Verbindungen große Unterschiede.^) Wir werden gleich erfahren, daß man Ijeim oxydativen und reduktiven Abbau des Hämatins und des Chlorophylls auf Verbindungen gestoßen ist Wir müssen die sich als sehr ähnlich und zum Teil identisch erwiesen. uns jedoch bei jedem Abbauprodukt, das durch eingreifende Maßnahmen aus einem kompliziert gebauten, zusammengesetzten Molekül gewonnen wird, die Frage vorlegen, ob man es als unverändertes Bruchstück der ursprünglichen Verbindung auffassen darf. Wir berühren damit den wundesten Punkt der ganzen Forschung auf dem Gebiete des Hämatins und Wir müssen uns bei jedem einzelnen Umwandluugsdes Chlorophylls. produkt und jeder Abbaustufe fragen, ob noch direkte Beziehungen zum Ausgangsmaterial vorhanden sind. Es müssen sekundäre Veränderungen ausgeschlossen werden. Ergibt eine bestimmte Art des Eingriffs in ein -Molekül Änderungen der Konstitution der entstehenden Produkte, dann können verhängnisvolle Trugschlüsse auf bestimmte Befunde aufgebaut werden, wenn nicht rechtzeitig erkannt wird, welcher Vorgang vom neuen Ausgangsmaterial zur neuen Verbindung führt. gleich bemerken, ' Es ist hier nicht der Ort, die Entwicklung der Erforschung des Aufbaus des Hämatins wiederzugeben, und ebensowenig können wir hier auf alle erhaltenen Ergebnisse eingehen. Sie gehören einstweilen zum größten Teil noch vor das Forum der Forscher. In manchen Punkten bestehen noch Meinungsverschiedenheiten. Wir können gleich vorausschicken, daß zurzeit die Konstitution des Hämatins noch nicht völlig aufgeklärt ist. Da^iicgen M Fr. X. Schulz: Zeitschr. f. physiol. Chemie. 24. 449 (1898). E. Schunck uud L. Marchlewski; Liebu/s Annalen. 278. 329 (1894); 284. 81 L. Marchlewski : Bnll. de l'Acad. des (1890); 288. 209 (189f)): 290. 306 (1896). Sciences du Gracovie. Math, uud uaturw. Klasse. Jauuar und April (1902); Jouru. f. M. Nencki und praktische Chemie. 65. U',1 (1902); Biochem. Zeitschr. 3. 320 (1907). •/. Zaleski; Zeitschr. J. Zaleski: Ber. d. Deutscheu Chem. Gesellsch. 34. 997 (1901). f. physiol. Chemie. 37. 54 (1902/03). ^) Vgl. dazu Bichard WiUstäfter uud Max Fischer: Zeitschr. f. phvsiol. Chemie. -) — — 87. — 423 (1913). 44* XXXIII. Vorlesung. 692 die Forschung dank der Bemühungen von Küster ^), Piloty 2), Hans Fischer^) und vor allem von WUhtätter ^) so weit vorgedrungen, daß sich eine Formel für das Hämatin entwerfen läßt, die ein ungefähres Bild der Anordnung der einzelnen Gruppen im Molekül wiedergibt») ist Aus Hämatin läßt sich leicht eine ihm sehr nahestehende Verbindung, Hämin genannt, gewinnen. Sie ist das wichtigste Ausgangsmaterial für Forschungen über die Konstitution des Hämatins geworden. Man erhält Hämin, wenn man das letztere oder auch Blutfarbstoff mit Salzsäure erwärmt. Man kann es auch direkt aus Blut gewinnen, indem man dieses eintrocknet und den Rückstand mit etwas Kochsalz und Eisessig vorsichtig erwärmt. Das Hämin kristallisiert dann in rhombischen Tafeln und Prismen. Man nennt diese Art der Gewinnung von Häminkristallen, die zur Erkennung von Blut vorgenommen wird, die l'cicJimannsche Häminprobe. ") Je nach der Art der Darstellung des Hämins erhält man verschiedene Verbindungen, die wahrscheinlich ihre besonderen Eigenschaften und ihre verschiedene Zusammensetzung dem Umstände verdanken, daß Lösungsmittel mit gebunden oder doch eingeschlossen wird.^j In manchen Fällen ist das Verfahren der Darstellung der Häminkristalle ein so eingreifendes gewesen, daß ohne Zweifel sekundäre Veränderungen die Folge waren. s) Es sind William Küster: Ber. d. Deutschen Chem. Gesellsch. 27. 572 (1894); 29. 821 Zeitschr. f. physiol. Chem. 28. 1 (1899); 29. 185 (1900): Ber. d. Deutschen Chem. Gesellsch. 32. 678 (1899): 33. 3021 (1900); 35. 1268 (1902); 35. 2948(1902); Liebigs Anna-len. 315.174(1900): Zeitschr. f. physiol. Chemie. 40.391, 423 (1904); 44. 391 (1906); Liebigs Annaleu. 345. 1 (1906); 346. 1 (1906); Zeitschr. für physiol. Chemie. 54. .501 (1908): 55. 505(1908): Ber. d. Deutschen Chem. Gesellsch. William Küster und A. Greiner: Zeitschr. f. physiol. Chemie. 40. 2017, 2021 (1907). 86. 185 (1913). —William Küster u. Paul Deihle: Zeitschr. f.physiol. Chem. 86. 51 (1913). ) 0. Pilofy und S. Merzhacher : Ber. d. Deutschen Chem. Gesellsch. 42. 3253, 0. Pilott/: Liebigs 0. Piloti/ und E. Quifmami: Ebenda. 42. 4693 (1909). 3258 (1909). Annalen. 366. 237 (1909); Ber. d. Deutschen Chem. Gesellsch. 43. 489 (1910); Liebigs Oscar Piloti/ und Edmund Dormann: Ebenda. 388. 314 Annalen. 377. 314 (1910). O.Piltoi/ und Sieqfried (1912); Ber. d. Deutschen Chem. Gesellsch. 46. 1002 (1913). J. Thannhauser: Ebenda. 390. 191 (1912). 0. Piloty und Josef Stock: Ebenda. 392. 0. Pilottj, H. Fink, K. Wilke, E. Dormann, 215 (1912); ebenda. 46. 1008 (1913). P. Hirsch: Ebenda. 45. 2495, 2586, 2592, 2.595 (1912). ^) Hans Fischer und p]. Bartholomäus Ber. d. Deutschen Chem. Gesellsch. 44. 3313 (1911); Zeitschr. f. physiol. Chemie. 77. 185 (1912); 81. 6 (1912); Ber. d. Deutschen Hans Fischer und E.BartholoChem. Gesellsch. 45. 466, 1315, 1919, 1979 (1912). Hans Fischer und Jmandtis Hahn: mäus: Zeitschr. f. physiol. Chemie. 83. 50 (1913). Ebenda. 84. 264(1913). H.Fischer, E. Bartholomäus und H.Röse: Ebenda. 84. 262 Hans Fischer und Heinrich Böse: Ebenda. 87. 38 (1913). H.Fischer und (1913). H. Fischer und Heinrich Rö.'^e: Ebenda. E. Bartholomäus: Ebenda. 87. 255 (1913). 1) 30. 105 (1897); (1896;; — — — — — — — : — — — — — — 88. 9 (1913). — ) Richard Willstätter : Liebigs Annalen. 358. 205 (1908). Richard Willstätter und Max Fischer: Zeitschr. f. physiol. Chemie. 87. 423 (1913). Zu allen Formeln der Hämatiureihe ist zu bemerken, daß Willstätter die Anzahl der Kohlenstoffatomc zu 33 annimmt, während Küster u. A. mit 34 rechnen. Wir haben uns der letzteren Meinung angeschlossen. ») L. T. S. Teichmann: Zeitschr. f. rationelle Medizin. N. F. 3. 375 (1853); 8. 141 '") (1857). ') Die Annahme, daß Hämin Essigsäure gebunden enthalte und somit eine Azetylverbindung sei, ist dadurch wiederlegt worden, daß man ohne Verwendung von Essigsäure das gleiche Hämin erhält. Vgl. ./. Heptner und L.Marchlewski: Zeitschr. f. physiol. Chemie. A.c. Siewert: Archiv f. experim. Path. u. Pharmak. 58. 386 (1905). 42. 65 (1904). ') Vgl. zur Frage der Zusammensetzung des Hämins: M. Nenrki und N. Sieher: Ber. d. Deutschen Chem. Gesellsch. 17. 2267 (1884). Arch. f. expeiitn M. Cloetta - : Blatt- und BlutfarbstoÖ. Chlorophyll und Hämoglobin usw. 693 Methoden zur Darstellung des Hämins ausgearbeitet, die zu einem Produkt von immer gleicher Zusammensetzung führen. Nach William Kilsfer^) kommt dem Hämin die folgende Konstitutions- jetzt formel zu: H C— C-CH = CH., HOOC -CH2— CH2— C=C N^ >N = c' H3 c- c C— CH, C \ CH HC Fe~Cl C— C— CH, HOOC— CH,— CH,— C=C n/ C— C— CH=:CH., H,C— C=C \c i H Hainiii. C,, H3., Ü4 Ni . FeCl. Ersetzen wir in der obigen Formel die Gruppe — Fe — durch — Fe I 1 OH Cl dann entspricht sie der angenommenen Konstitution des Hämatins.^) Die Betrachtung der oben wiedergegebenen Formel zeigt, daß im Hämin bzw. Hämatin vier Pvrrolkerne enthalten sind: CH=:rCH \)NH CH=:CH Pyrrol. Die vier Pyrrolringe sind folgt, unter sich verbunden — nach Willstätter und Max Fischer, wie Path. u. Pharmak. 36. :-^49 \|1895). K. A. H. Mörrier : Nord. med. Arch Nr. 14. 1 u. 26 M. Nencki und J. Zaleski: Zeitschr. (1897); Zeitschr. f. physiol. Chemie. 41. 542 (1904). f. physiol. Chemie. 30. 384 (1900). R. v. Zcijnek: Zeitschr. f. physiol. Chemie. 30. 128 A. V. Sieu-ert: Arch. f. experim. Path. u. Pharmak. 58. 386 (1905). (1900). Vgl. ferner die Arbeiten der S. 692 genannten Forscher. ') Vgl. hierzu William Küster: Zeitschr. f. physiol. Chemie. 110. 9.^ (1920). auch 121. 121, 135 (1922). — - — — — 2) Vgl. W. Küster: Zeitschr. f. physiol. Chemie. 40. 391 (1903). XXXIII. Vorlesuug 594 CH— CH CH— CH Nn N^ II C— CH I j CH— C=CH CHr^C NH<^ IV .NH III I CH=CH CH=CH Läßt man auf Hämatin bzw. Hämin wässerige Brom Wasserstoff saure einwirken, dann erhält man ein Dihydrobromid, Ca^HsgOiNiFeßr 2HBr, das schön kristallisiert. Bei weiterer Einwirkung der Säure erfolgt Um. in Hämatoporphyrin 1), Cä^HggOgNi. Diese Verbindung unterscheidet sich schon dadurch scharf vom Ausgangsmaterial, daß sie kein Eisen mehr enthält. Bei der Bildung des Hämatoporphyrins dürften sich nach E. Wülstäfter und Max Fischer ^) die Brücken von den zwei Pyrrolstickstoffen loslösen, worauf sich die mittlere Gruppe wandlung /C C\ in /C C\ umwandelt. Die Entstehung des Hämatoporphyrins aus : . Hämin in zwei Stufen läßt sich durch folgenden die beiden Formeln wiedergeben C3,H3,0,N, Cs^Hg.O.N, . . FeCl + 4HBr = FeClBr. + CuH3,0,N, 2HBr + 2H2O - 2 HBr -f . 2HBr C3,H3eO,N, (OH).,. Die letztere Formel bringt zum Ausdruck, daß das Hämatoporphyrin zwei besitzt. Sie sind die Ursache, daß dieses Äther und Ester bilden kann. Entsprechend dem Gehalt von zwei Karboxylgruppen ist Hämatoporphyrin eine zweisäurige Base. alkoholische Hydroxyle Das Hämatoporphyrin entsteht auch im tierischen Organismus aus Im normalen Harn findet es sich nur in Spuren. In größeren Mengen tritt es bei Fieber 3) und vor allem nach Sulfonal- und Trionalvergiftung auf.) Es soll ferner bei manchen wirbellosen Tieren vorkommen. ^j Von großem Interesse ist die Beobachtung, daß das Hämatoporphyrin ein ausgezeichneter optischer Sensibilisator ist.') Fügt man z. B. eine Lösung dieser Verbindung zü einer Kultur von Paramäzien (Infusorien), dann beobachtet man, solange sie unbelichtet ist, keine Erscheinungen. Sobald jedoch Licht zutreten kann, gehen die Tierchen zugrunde. Auch Hämatin. Vgl. M. Nencki uad ./. Zale.^ki : Zeitschr. f. i)livsiol. Chemie. 30. 423 (1900). Vgl. auch Ch. Mac Mimn: Journ. of Physiol. 10. 71 (1890): 11. 13 (1890). A. E. Garrod: Jouru. of Physiol. F. G. Hopkins: Guvs Hospital Keports. 359 (1883). H. Günther: Deutsches Archiv f. klin. Med. 105. 89 15. 108 (1893); 17. '349 (1899). E. Salkotvski: (1911). -^ 0. Hamniursten: Upsala Läkaref. förhandl. 26. 259 (1891). ') =) — — — — Zeitschr. f. physiol. Chemie. 15. 286 (1891.) ») A. Garrod: Journ. of Path. and Bact. Oktober (1892). E. Salkoirski: \ B. SfockiHs: Nederl. Tijdschr. voor Geneesk. 2. 409 (1889). A. Käst und Th. Weiss: Berliner klin. Zeitsclir. f. physiol. Cliemie. 15. 28() (1891). — — Wochenschr. ^) ") 14. fi21 (189(5). Ch. Mac Munn: Journ. of Physiol. 8. 384 (1887). W.Hausmann: Wiener klin. Wochenschr. 21. 1027(1908); Biochem. Zeitsciir. 275 (1909); Fortschritte der Naturwissenschaften. 6. 243 (1912). — Vgl. ferner S. (56. Blatt- uud BlutfarbstotV. ( hlorophyll und Hämoglobin usw. (395 Warmblüter zeigen schwere Erscheinungen, wenn ihnen llamatoporphyrin ' dem Licht ausgesetzt werden. Spritzt man z. B. einer Maus eine ganz geringe Menge von Hämatoporphyrin ein, eingespritzt wird und sie gleichzeitig bleibt sie ganz munter, wenn sie im Dunkeln gehalten wird. Sobald das Tier dem IJcht ausgesetzt wird es genügt diffuses Tageslicht so beginnt es an der Injektionsstelle sich lebhaft zu kratzen. Es wälzt sich bald umher. Alles deutet darauf hin. daß äußerst lebhafte Reizerscheinungen vorhanden sind. Nach einiger Zeit wird das Tier ruhiger. Es hegt meistens teilnahmslos da, schließlich geht es zugrunde. Vollzieht sich der ganze Symptomenkomplex langsamer, dann beobachtet man ausgedehnte Ödeme. dann — — Man könnte zunächst daran denken, daß durch das Licht das Hämatoporphyrin umgewandelt M-ird und dabei giftig wirkende Substanzen entstehen. Diese Möglichkeit läßt sich durch den folgenden Versuch mit größter Wahrscheinlichkeit i) experimentell ausschließen. Man belichtet eine Lösung von Hämatoporphyrin und spritzt dann einem im Dunkeln gehaltenen Tiere etwas davon ein. Es zeigen sich keine Erscheinungen. Sie treten erst bei Belichtung des Tieres auf. 2) Im pathologischen Harn ist ganz vereinzelt noch ein weiteres Porphyrin aufgefunden worden. Es führt vorläufig den Namen Urin porphyrin. Seine Konstitution ist noch nicht aufgeklärt. •') Im Kot kommt ebenfalls ein Porphyrin vor.) Es hat den Namen Kotporphyrin erhalten. Es enthält drei, das Ürinporphyrin dagegen sieben Karboxylgruppen. Beide sind nahe verwandt. Es ist auch gelungen, )hne Zweifel ist dieses das das letztere in Kotporphyrin überzuführen. ') primäre Produkt, das im Organismus durch Karboxylierung in das Urinporphyrin übergeführt wird. Beide Porphyrine wirken auf weiße Mäuse stark sensibilisierend. Werden die Versuchstiere im Dunkeln aufbewahrt, dann erweist sich das Kotporphyrin doppelt so wirksam als das Urinporphyrin. Bei Belichtung ist umgekehrt das letztere giftiger, ß) Läßt man auf Hämatin bzw. Hämin Jodwasserstoffsäure und Jodphosphonium, d. h. ein energisches Reduktionsmittel einwirken, dann erhält man das Mesoporph yrin.') Es enthält zwei Karboxylgruppen. ( ') Mau könnte daran denken, daß das in den Geweben befindliche Hämatoporphyrin von Lichtstrahlen anders beeinflußt wird, als das in Lösung befindliche. Doch ist eine solche Annahme nicht sehr wahrscheinlich. -) Es sei darauf hingewiesen, daß unter pathologischen Verbältnissen wiederholt im Blute Hämatin aufgefunden worden ist. Es tritt dann auch im Harn auf. Vgl. z. B. O. Schlimm: Zeitschr. f. physiol. Chemie. Z. f. Geburtsh. u. 97. 32 (1916). — //. Brii/t und Gynäk. 80. 145 (1916). <>. ScfiKmm: — — ') Hans Fischer: Zeitschr. f. physiol. Chemie. 95. 34 (1915). Vgl. auch Ich habe den Schumin: Ebenda. 96. 183 (1915); 98. 123 (1916): 105. 158 (1919). gleichen Farbstoff bei einem Soldaten beobachtet, der weitgehende Nervendegenerationen bei der Sektion aufwies. Vgl. Zeitschr. f. physiol. Chemie. 106. 178 (1919).— G. Grund: (). Zbl. f. innere Medizin. 40. 1 (1919). ) Hans Fischer: Zeitschr. f. physiol. Chemie. 96. 148 (1915). FUinuer und Otto Rießer: Ebenda. 98'. 1 (1916). '") Hans Fischer: Zeitschr. f. phvsiol. Chemie. 97. 109 (1917). — Vgl. auch — Ale.i-. Vgl. auch 97. 148 (1917); 98. 14, 78 (1917). ) Vgl. weitere Versuche über die sensibilisierende Wirkung von Porphyrinen: Hans Fischer H'alther Hausmann: Biochem. Zeitschr. 67. 309(1914); 77. 2()S (1916). und G. A V. Kemnitz: Zeitschr. f. physiol. Chemie. 96. 309 (1916). ') Vgl. M. Nencki und ./. Zaleski: Zeitschr. f. physiol. Chemie. 34. 997 (1901), W. Küs/er: Ber. d. Deutschon Chem Gesellsch. 45. 37 54 (1902); 43. 11 ll904). — — XXXIII. Vorlesung. 696 H'. Küster schreibt ihm folgende Struktur zu: — CHs C—C H2 C H, C H3C — C -^ CH \V- — H, C — C H( )(JC - H. C C— CH C H, C // C C = C — CH, C = C CH. CH, — CUOH NH NHc H, C — C CH, X.3 V CHg Mesoporphyrin. Aus Hämatoporphyrin ist eine dem Mesoporphyrin nahestehende Verbindung gewonnen worden. poiphyrin erhalten. 1) Sie hat den offenbar sehr Namen Hämo- Sie ist zweibasisch: CH = CH CH, C — CH \ c- — CHo — C — C \c HOOC — CH, — CH, C = C< CH C CHo / C )C NH CH, -C / N = C— CH, — CH, — COCH NH —C=C C=C — CH, CHg CHg Hämoporphyrin. Das Hämophorphyrin interessiert uns in besonders hohem Maße, weil aus ihm durch Abspaltung der beiden Karboxyle (oder bei Annahme von C34 eines Karboxyls und einer Essigsäuregruppe 2) ein Porphyrin gewonnen — O. l'iloiy und Fink: Eer. 193Ö (1912j; Zeitschr. f. physiol. Chemie. 82. 463 (1912). Hans Fischer und F. Meyer-Befz: Deutschen Chem. Gos^ellsch. 45. 2495 (1912). (1. Zeitschr. f. physiol. Chemie. 82. 96 (1912). W. Küster; Zeitschr. f. physiol. Chemie. Hans Fischer, F. Bartholomäus und H. Rose: Zeitschr. f. phvsiol. 1913). 86. 51 Chemie. 84. 2ü2 (1913). ») R. Willstäfter und Max Fischer: Zitat ). 1. c. S. 692, ) Der obigen Strukturfoniicl ist die Grundformel C33 Hjg 0^ N^ zugrunde gelebt. Bei Cj^ müßte ein Karboxyl durch dio Gruppe: CH^ COOH ersetzt werden. — — — . Blatt- und Blutfarbstofi'. Chlorophyll und Hämoglobin usw. ß97 worden ist, das keine sauren Eigenschaften mehr besitzt und sich tisch als iden- mit dem aus Chlorophyll erhaltenen Ätioporphyrin erwiesen hat. i) Durch weitere Reduktion des oben erwähnten Mesoporphyrins gelangt man zum Mesoporphyrinogen. Es ist auch gelungen, durch Reduktion unter Anwendung von Pyridin dem Mesoporphyrin entspricht, jedoch noch Eisen enthält. Es ist Mesohämatin genannt worden. Seine Chlorverbindung heißt in Analogie zu den Beziehungen zwischen Hämatin und Hämin Mesohämin. 2) Seine Zusammensetzung ist die folgende: C34 Ho^ O4 N4 Fe Cl. Dem Mesohämatin entspricht die Formel C'34 Hgg O4 N4 Fe OH. ein Produkt zu gewinnen, das . . . Durch weitere Spaltung und Reduktion von Hämatin, Hämin, Hämatound Mesoporphyrin gelangt man zu Abbaustufen, die unser Interesse deshalb in besonders hohem Maße gefangen nehmen, weil bei der (Xxydation ') und Reduktion) von Spaltprodukten des Chlorophylls, auf das wir noch zurückkommen, Verbindungen erhalten werden, die diesen vollständig entsprechen. Bei der Oxydation sind gewonnen worden: porphyrin CH3 C = C II'" = = . C CH., . CH3 . CH, C rr. C CH, CH2 . . . COOH I =C C NH C r= o NH Imid der dreibasischen Hämatinsäure. Methyl-äthyl-maleinimid Bei der . " Reduktion der Porphyrine sind vier verschiedene Pyrrol- abkömmlinge erhalten worden: CH3 C . — C. CH, CHs " CH, C . . - C . CK, CR, " II II CH3.C II II CH3.C C.CH3 CH NH NH Isohämopyrrol Phyllopyrrol ') R.Willstütter; Liebig% Aonalen. 396. 186 (1913;: 400. 182 (1913). Zeitschr. physiol. Chemie. 87. 494 (1913j. ^) Vgl. Uanfi Fischer und Heinrich Rose: Zeitschr. f. physiol. Chemie. 88. 9 (1913). =•) W. Küster; Zeitschr. f. physiol. Chemie. 28. 1 (1899): 29. 185 (1900); 44. 391 (19U5); 54. 501 (1908); 61. 164 (1909); Liebig?, Annalen. 315. 174 (1900); Her. d. R. Willstätter und Y.Asahina: Liebige, Deutschen Chem. Gesellsch. 40. 2017 (1907). f. — Annalen. 373. 227 (1910). ) R. Willstätter und Y. Asahina ; Liebigi Ann?ilen. 385. 188(1911); Hans Fischer und E. Bartholomäus: Ber. d. Deutschen Chem". Gesellsch. 44. 3313 (1911): 45. 466, 1979 Hans Fischer (1912); Zeitschr. f. physiol. Chemie. 77. 185 (1912) und 80. 6 (1912). 0. Piloty: Liebigs Annalen. 366. Her. d. Deutschen Chem. Gesellsch. 48. 401 (1915). 237 (1909); 377. 314(1910); 0. P(7o^/ und ^j^Z/wa«».- Ber. d. Deutscheu Chem. Gesellsch. 42. 4693 (1909): 0. Piloti/ \im\ J. Stock: Liebiqs Annaleu. 392. 215 (1912); Ber. d. Deutschen Chem'. Gesellsch. 46. 1008 (1911): 0. J'iloty und K. Wilke: Ebcuda. 46. 1597 L. Knorr und K. Hess: Ber. d. Deutschen Chem. Gesellsch. 44. 27.58 (1911) (1913). und 45. 2626 (1912). — — — XXXIII. Vorlesuug. 698 CH3 C . — C CE, CH3 . CH3 C , . —C . GH., . CH, -11 II II II HC HC C.CH3 \/ NH CH \/ NH Kryptopyrrol 3-Methyl-4-äthyl-p\ rrol. Alle vier Verbindungen sind ß ß'-Methyl-äthyl-pyrrole i d. 1) a1eini ethy1-ä thy1- der Oxydation M und liefern bei m m Wir haben aus dem vorliegenden, sehr umfangreichen Materiale über die Erforschung des Aufbaus des Häraatins diejenigen Ergebnisse heraus- am besten gesichert sind und zugleich wenigstens den Hauptzügen über die Konstitution des eisenhaltigen Paarlings des Blutfarbstoffes unterrichten. Fassen wir die zuletzt erwähnten Ergebnisse zusammen, so können wir aus ihnen folgendes herauslesen. Die Oxydation des Hämins und seiner nächsten Abbaustufen hat zu Verbindungen geführt, die sich als Abkömmlinge der Maleinsäure erwiesen haben. Sie müssen daher aus Pyrrolkernen des Hämatins hervorgegangen sein, und zwar aus zwei solchen, wie die quantitativen Versuche einsvandfrei ergehen haben. Die Entstehung der beobachteten Säuren führt zum Schluß, daß die ß- Stellungen dieser Kerne durch Substituenten besetzt waren und somit für die Verkettung im Gesamtmolekül nur die a-Stellnngen in Frage kommen. Die Reduktion führt zu den Mutterstoffen der Hämatinsäuren sie sind als Phono- und Isophonopyrrol-karbonsäure bezeichnet worden. Außerdem entstehen die oben angeführten substituierten Pyrrole. Sie entstammen der zweiten Hälfte des Häminmoleküls. Auch hier decken sich die isolierten Mengen dieser Verbindungen mit dieser Annahme. Es sind vier Pyrrolringe im Hämatinmolekül vorhanden, unbewiesen ist zurzeit nur noch die Art ihrer Verknüpfung. gegriffen, die zurzeit in — Das Hämoglobin ist nicht der einzige Blutfarbstoff, den wir kennen. Manche wirbellosen Tiere besitzen besondere sog. Atmungspigmente. So findet man bei den Kephalopoden, manchen Lameliibranchiaten, Gastropoden, Krustazeen und Arachniden einen kupferhaltigen Blutfarbstoff. Hämozvanin genannt. 2) Seine Analvse ergab: 53-66"/o ^'^ 7-32«/o H, l6-09Vo N, O-SSVo Cu, 0-86Vo S und 21-67« « 0. ») Es ist noch nicht sicher festgestellt, ob es nur ein Hämocyanin oder mehrere Farbstoffe Anteil dieser Art gibt. ) noch Es besteht aus Eiweiß und einem kupferhaltigen Natur. Es kann Sauerstoff binden und in unbekannter W. Küster: Zeitschr. f. physiol. Chemie. 28. 1 (1899); 29. 185 (1900): 44. 391 (1905): 54. 501 (1908): 61. 164 (1909); Liebigs Ann. 315.174 (1900); Ber. d. Deutschen Chem. Gesellsch. 40. 2017 (1907). M. Will'sfäiter und Y. Asahina: Lie.bigs Ann. 373. 227 (1910). =) L. Fredericq: Bull, de TAcad. Royale de Belg. (2). 46. (1878); 47. 409 (1879). ( F. W. Krukenberg: Zentralbl. f. med. Wissensch. Nr. 23 (1880j. A. B. Griffith: €. r. de l'Aead. des Sciences. 114.496 (1892). C. Ciienof: Ebenda. 115. 669 (1892). ir. J). Halliburton: Journ. of Thysiol. 6. 300 (1885). - Ch. Dherr : ('. r. de TAcad. des Sc. 146. 784 (1908); C. r. de la Soc. de Biol. 64. 788 (1908). •^) A. B. Griffith: C. r. de PAcad. des Sc. 114. 496 (1892). M. Nenze: Zeitschr. f. physiol. Chemie 33. 370 (1901). ) ('. L. Aisberg und E. J). Clark: .lourn. of biol. (Jhemistrv. 8. 1 (1950). Krnst riiili/jpi: Zeitschr. f. phvsiol. Chemie. 104. 88 (1919). — — — — — — — Blatt- und Blntfarbstolt". Chlorophyll und Hanidglohiii usw. 699 Oxyhämozyanin übergehen. Dieses kristallisiert in doppelbrechenden Prismen. Das Hämozyanin ist farblos. Die iSauerstoffverbindung ergibt eine blaue Lösung. Ferner hat man im Dlute von Pinna scjuamosa eine Verbindung aufgefunden, die Mangan enthält. Sie ist Pinnaglobiu genannt worden.') Bei zahlreichen Avertebraten sind sogenannte Achroglobine beschrieben worden. 2) Auch sie sollen den respiratorischen Pigmenten zugehören. Endlich seien noch genannt das Echinochrora, ») ein Farbstoff, der in der Periviszeralflüssigkeit von Echinoiden vorkommt, das Hämerythrin \i. der rote Farbstoff der perienteritischen Flüssigkeit einiger Würmer, das Chlorcuorinä), ein grüner Farbstoff mancher Chätopoden, das Aktinohämatin"), ein bei Aktinien vorkommender Farbstoff, die Histohämatine^), die bei zahlreichen Avertebraten im Integument angetroffen worden sind, usw. Alle diese Farbstoffe sollen in ihrem Bau Beziehungen zum Hämatiii bzw. Hämoglobin haben, doch ist bei keinem einzigen dieser Produkte ein Bew-eis für diese Annahme erbracht. Schließlich wollen wir noch erwähnen, daß der Farbstoff der Muskeln. Myochrom«) genannt, mit ihm identisch ist. dem Hämoglobin sehr nahe steht und vielleicht Im Anschluß an die Besprechung des Blutfarbstoffes, der, wie beAufgabe hat, einen umfassenden Sauerstofftransport zu ermöglichen, wollen wir noch jenes Farbstoffes gedenken, der die Pflanze befähigt, Kohlensäure und Wasser zu organischen Verbindungen zusammenzufügen. Es ist dies der Blattfarbstoff und insbesondere das Chlorophyll. Die ihn führenden Zellen, die Chloroplasten. enthalten vier Farbstoffe, nämlich die Chlorophyllkomponente a, C,^5 H-., Or, X4 Mg, die Chlorophyllkomponente b, Cgg H^o <A> N^ Mg, das Karotin, Cio H^e und das Xanthophyll, C^o H-.c O«.^) Das Karotin ist ein ungesättigter Kohlenwasserstoff. Es kristallisiert in Rhomboedern und in rhombenförmigen. reits betont, die . . fast (\|uadratischen Täfelchen. Die Kristalle zeigen einen lebhaften, bald A. B. Griftith: C. r. de l'Acad. des Sc 114. 840 (1892). A.B. Grifßlh: C. r. de TAcad. des Sc. 115. 259. 474, 738 (1892); 116. 1206 (1892). ") A. B. Griffith: C. r. de l'Acad. des Sc. 115. 419 (1892). Mac Mtinn: Quarterlv •louru. of microsc. Sciences. 25. 469 (188.Ö): 30. 70 (1892). )-A. B. Grityith: C. r. de PAcad. des Sc. 115. 419. 669 (1892). ^)Ray Lankesfer: Journ. of Anat. aud Physiol 3. 119 (1870). «) Moseleir Quart. Journ. of microsc. Sc. 13. 143 (1873). ') Mac. Munn: Philosoph. Transactions. 176. 641 (1885); 177. 267 (1881). 8) K. A. H. Mörnrr: Nordisk. Med. Archiv. Nr. 2. (1897). Mac Mimn: Jouru. of Physiol. 5. 24 (1885); 8. 51 (1887). ^) Vgl. hierzu Handbuch der biochemischen Arbeitsmethoden. 2. 671 (1910). (Bearbeitet von Richard H'iUs/äfter.) Urbau & Schwarzenberg. Berliu-Wien. 1910. Biochemischee Handlexikon. 6. 1 (1911). (Bearbeitet von Richard WiJlstätter.) .1. Sprini^er. Berlin~'1911. Richard U ill.'^fätfer und Arfliui Sfoll : Untersuchungen über ChloroJulius Springer. Berlin 1913. phyll. Vgl. auch L. Marchlewski: Die Chemie dos Chlorophylls. Leopold Voss. Hamburg- l^eipzig 1895. Von einzelnen Arbeiten seien iiocii genannt: A'. Willutütter, Ferd. Hocheder, \i'. Mief/, Adolf PjatDiensfiel. Mex Benz, Ernst 'Hug, Arthur Stoll, Mar hier, Max IJtzinger, Lennart Forsen, Yasuhiko Asahina: Liehicß Anualen. 350. 48 (1916); 354. 505 (1907); 355. 1 (19!)7); 358. 205. 267 (1908); 371.1(1909); 380. 148, 154, 177 (1911); 382. 129 (1911); 387. 317 (1912); 100. 269 (1912); 396. 180 (1913); Ber, d. neutschen Chem. Ges. 44. 3707 (1911). 'I 2) — — — — — — — XXXIII. Vorlesung. 700 kupterigen, bald blauen Oberflächenglanz. ist noch nicht aufgeklärt. i) Die Konstitution des Karotins Das Xanthophyll bildet längliche Täf eichen und Prismen. Die Kristalle sind pleochromatisch und zeigen stahlblauen Glanz. Auch diese Verbindung ist in ihrem Aufbau noch wenig aufgeklärt. Seine Zu- sammensetzung deutet auf ein oxydiertes Karotin hin. Viel besser sind wir über die Struktur der beiden Chlorophyllarten a und h unterrichtet. Eine ganze Reihe von Forschern haben sich mit der Erforschung der Konstitution dieser wichtigen Verbindungen befalit. Es seien genannt Schunk, Marchleivski Nencki und Tswett. Den Hauptfortschritt in der Erkenntnis der Zusammensetzung des Chlorophylls verdanken wir Willstätter. Ihm ist es gelungen, die vier genannten Farbstoffanteile der Chloroplasten zu trennen und genau zu charakterisieren. Das Chlorophyll enthält Magnesium. Dieses Element ist ein Baustein des Chlorophyllmoleküls. Chlorophyll hinterläßt bei der Verbrennung Sie besteht aus reiner Magnesia. Das Chlorophyll ist ein 4-5<'/o Asche. Derivat einer Trikarbonsäure. Ein Karboxyl ist mit Phytol und eines mit Methylalkohol verestert. Phytol ist ein ungesättigter, primärer Alkohol der Fettreihe mit offener Kette von Kohlenstoffatomen. Die Kohlenstoffkette ist stark verzweigt.-) Phytol hat die Zusammensetzung C.q H39.OH2.-O Es macht etwa ein Drittel des Chlorophyllmoleküls aus. , Chlorophyll a und b sind mikrokristallinisch. Chlorophyll a bildet ein blauschwarzes Pulver. Beim Verreiben nimmt es einen stahlblauen Glanz an. Die b-Form ist dunkelgrün bis grünschwarz. Die Lösung des Chlorophylls a in Äthylalkohol ist blaugrün, tiefrot fluoreszierend. Die konzentrierte, ätherische Lösung ist blau. Beim Verdünnen wird die Farbe mehr grünlich. Chlorophyll b gibt mit Alkohol eine mattgrüne, mit Äther eine leuchtend grün gefärbte Lösung. Beide Anteile zeigen zum Teil verschiedene Eigenschaften und haben eine verschiedene Zusammensetzung. Während man früher annahm, daß die Zahl der Chlorophyllarten eine sehr große sei, hat Willstätter beweisen können, daß wahrscheinlich im ganzen Pflanzenreich der gleiche Farbstoff an der Assimilation von Kohlensäure und Wasser beteiligt ist. Von großem Interesse ist die Auffindung eines Fermentes in den chlorophyllführenden Pflanzenorganen, das ChlorophyU spalten kann. Es ist Chlorophyllase genannt worden.) Dieses Ferment löst die esterartige Bindung zwischen dem Phytol und dem Karboxyl, das es besetzt hält. Es handelt sich um eine Hydrolyse, die in Analogie mit der Verseifung von Fetten durch die Lipase zu setzen ist. Läßt man die Chlorophyllase in alkoholischer Lösung auf Chlorophyll einwirken, dann folgt der Abspaltung des Phytols eine Anlagerung desjenigen Alkohols, den man als Lösungsmittel verwendet hat. /.. Marchleivski: Biochem. Zoitsehr. 42. 234 (1912). — B. A. Jacobsohn und L. March- leivski: Bull, de l'Ac. des sciences de Cracovie. Classes des sciences Februar 1912. — Iswett: Biochem. Zeitschr. 35. 433 (1911) ') Vgl. Heinrich ^) Vgl. dazu: li. II. math et uature. A. Escher: In.-Diss. Zürich 1909. Erwin W. Mayer und Ernst Uüni; Liebig?, Annaleu. Willstätter, 378. 73 (1910). ^) 418. Richard Willstätter, 0. Schuppti und Erivin W. Mayer: Liebig^ Annalen. 121 (1919). ) Richard Willstätter und Arthur Stoll : Liebic/» Annalen. 378. 4 (1910). Blatt- uüd Blutfarbstoff. Chlorophyll uud Hämoglobin iisw 701 Die folgenden Formeln ergeben den Verlauf der einzelnen Reaktionen Als Alkohol sei der Äthylalkohol gewählt. 1. Hydrolyse in wässeriger Lösung Methyl- gruppe Phytol- gruppe Chlorophyll C3. H30 ON, Mg<^o[]j^^^^ + Co H3, Chlorophyllid 2. .. ( )H Phytol. Spaltung in äthylalkoholischer Lösung (Alkoholyse^: C3. H30 ON, C,, H30 ON, Mg>^^^ Mg<coo : g\|J^H3, + : cffl, + C^o H3« ^^-^ Äthylchlorophyllid H^ • ^^H . + OH. Phytol. Bei der Besprechung der Spaltung der Glyzeride durch Lipase hatten wir erwähnt, daß, es gelungen ist, Neutralfette aus den Bausteinen Glyzerin und Fettsäuren zu erhalten, wenn man diese in hoher Konzentration der Wirkung des genannten Fermentes überläßt. Es ist von großem Interesse, daß es möglich ist, Chlorophyll aus Phytol und dem nach seiner Abspaltung verbUebenen, Chlorophyllid genannten, Reste aufzubauen, M'enn man die Chlorophyllase auf dieses Gemisch einwirken läßt:») C32 H30 ON, Mg<^Q^fi ^"^ + OH Chlorophyllid /. TT ,-^x^ . t ,0 H3,, - n, —^ Phytol ^r /COOCHj C3,H3oON,Mg<(.ooc.,„H3o. Aus dem Chlorophyll läßt sich das Magnesium durch vorsichtige Behandlung mit Oxalsäure entfernen. Es entsteht das Phäophytin.^) An Stelle des Magnesiums tritt Wasserstoff. Im übrigen wird die Struktur des Moleküls nicht verändert. Durch Abspaltung des Phytols gelangt man zum Phäophorbid. Chlorophyll a und b liefern verschiedene Chlorophyllide, Phäophytine und Phäophorbide. Die letzteren Verbindungen kann man aus dem Chlorophyllid auch direkt unter Abspaltung von Magnesium gewinnen. Die folgenden Formeln unterrichten über die geschilderte Art des Abbaus des Chlorophylls: ') ) B. Willstätter und A. Stoll: lAebigi Annaleu. 380. 148 (1911). R. Willstätter und Ferd. Hocheder: Ebenda. 354. 205 1907). H. WiUstättir, Ferd. Hocheder und Ernst Hug : Ebenda. 371. 1 (1909). XXXIII. N'orlesuug. JQ-} Mg N, C32 H30 — COOCH3 —('()(). C ) C'oo H3, Chlorophyll. durch (,'hlorophyllase ^^ COOCH3 — COOH + C20 H39 OH Mg N4 C3., H30 O . Phytol Chlorophyllid — Mg + 2 H ^ N, ( V2 H32 O durch verdünnte Säure — COO CH3 — COOK. . Phäorphorbid. — COO CH3 - COO Coo H3, Mg N4 C30 H30 . . Chlorophyll — Mg+2H mit verdünnter Säure ' N4 C32 H32 — COO CH3 - COO C,o Hg, - . . Phäophytin. f Ha N, C32 H3., O — COO CH3 — COOH; + C20 H3. OH . . Phäophorbid Phytol. Durch i\.lkali lassen sich' Chlorophyll und auch die erwähnten Abkömmlinge weiter spalten. Es tritt dabei nicht nur Hydrolyse ein, sondern kommt zu sekundären Veränderungen. Chlorophyll liefert mit Alkali Iso-chlorophyllin. Dieses geht bei der Einwirkung von Säure in Phytochlorin über: es - COO CH3 Chlorophyll MgN, C32 H3,, . ' Mg N, C32 H30 - COO C20 H3, . durch Alkali — COOH — COOH Iso-chlorophyllin — Mg + 2 H yr ^'4 <^ 32 H32 durch Säure — COOH — COOH Phytochlorin. Uns interessiert hier ganz besonders der Abbau des Chlorophylls zu Durch Einwirkung von AlkaHen sind die sog. Phylline und Porphyririe erhalten worden.^) Die ersteren sind Karbonsäuren. Sie enthalten noch das Magnesium. Die letzteren stellen ebenfalls Karbonsäuren dar, doch fehlt ihnen das Magnesium. Sie entstehen aus den Phyllinen durch Abspaltung dieses Elementes. tieferen Spaltprodukten. ) B. Wülstätter und Adolf P/annenisfiel: Aiinalen der Chemie. 358. 205 — R .WillsUiUer und Hermann 'Fri'tzscfie: Ebenda. 371. 3.S (1909). (11)07). Blatt- uutl Bliitfarl)stoff. t'hloioph\ 11 und Hämoglobin usw. 70:3 Die Oxydation dieser Abbauprodukte führt, wie S. 697 erwähnt, zu \'erbindungen, die zu solchen nahe "Beziehungen besitzen, die wir bereits als Abbaustufen des Hämatins und seiner Abkömmlinge kennen gelernt haben. Phylloporphyrin ergibt eine Verbindung, die mit dem Imid der dreibasischen Hämatinsäure identisch ist. Ferner sind bei der Reduktion von Chlorophyllabkömmlingen Verbindungen gewonnen worden, die Anteilen des Hämopyrrolgemisches entsprechen 1), das bei der Einwirkung von Reduktionsmitteln auf Hämatin bzw.Hämin erhalten wird (vgl. S. 697 Endlich sei noch erwähnt, daß es gelungen ist, Chlorophyll zur karboxylfreien Stammsubstanz dem Ätiophyllin, C3, H;^4N4 Mg, und Ätioporphyrin, C31H36N4, abzubauen. Die letztere Verbindung entsteht aus ersterer unter Austritt des Magnesiums. Sie ist mit dem durch Abspaltung der beiden Karboxyle aus Hämoporphyrin hervorgehenden Porphyrin identisch (vgl. S. 696). WiUstätter und A. Sfoll geben diesen Verbindungen die folgenden Formeln 2): CH=('H CH3 CH3 . GH., . C -CH C- . V N^ C . CH, CH3 . . C= \ / C CH y ^c ('H3 (' C- C CH, CH3 C= C.CH, . . -Mg 1 i CH3 Ätiophyllin. 3) CH=CH CH3 C- . \N CH3 . CH, . C — / c -CH N' CH -C ^C CH3 . CH. . C CHo C= . \NH CH, C . =c . CH3 HN C CH3 c= . CH3 CH3 Ätioporphyrin. Richard WiUstätter und Yasuhiko Asahina Her. il. Deutschen Chem. Ge3707 (1911) und S. 697. ') Vgl. William Küster: Zeitschr. f. physiol. Chemie. HO. 93 (1920). ) Die punktierten Linien stellen Partialvalenzen im Sinne von A. Werner dar. Vg. A. ferner: Neuere Anschauungen auf dem Gel)ipte der Chemie. 5. Auflage. F. Vieweg, Braunschweig 1913. ) Vgl. sellsch. 44. : XXXIII. Vorlesung. 704 Wir erkennen in diesen Formeln ohne weiteres wieder vier Pyrrolbeim Humatin. Eine recht nahe Verwandtschaft von Teilstücken des Chlorophylls und des Hämatins ist nach den bisherigen Ergebnissen der Erforschung dieser beiden Verbindungen ohne Zweifel vorhanden. Daraus darf nun nicht, wie es früher geschehen ist, auf irgend welche Analogien in der Funktion dieser Verbindungen geschlossen werden. Ebenso wenig ist es angängig, Kückschlüsse auf die Beziehungen des Hämatins zum Chlorophyll und umgekehrt zu ziehen. Das fertige Chlorophyll ist vom Hämatin ganz verschieden. Der Eintritt der Phytolgruppe ist in gewissem Sinne der Beziehung des Hämatins zum Globin vergleichbar. Doch sind die Eigenschaften des Blutfarbstoffs und des Blattfarbstoffs ganz verschiedene. Vielleicht ergeben sich aber gemeinsame Punkte in der Entstehung beider Verbindungen. Leider sind wir wieder über die Bildung des Blattfarbstoffs in der Pflanze, noch über die des Blutfarbstoffs bzw. des Hämatins im tierischen Organismus unterrichtet. Es ist w^ohl möglich, daß zunächst Pflanzen- und Tierzellen dieselben Bausteine als Ausgangsmaterial zur Darstellung des respiratorischen Pigmentes bereiten und sich erst im Verlaufe der weiteren Synthese die Unterschiede entwickeln. Wir dürfen jedoch auf derartige Erscheinungen kein zu großes Gewicht legen, denn wir wissen, daß die Pflanzen- und Tierzellen überhaupt in allen prinzipiellen Punkten übereinstimmende Funktionen und die diesen entsprechenden Zellbestandteile zeigen. Wir treffen hüben und drüben entsprechend gebaute Kohlehydrate, Fette, Phosphatide, Proteine und Nukleoproteide an. Es sind die gleichen Bausteine vorhanden, und doch sind die fertigen Zellbestandteile ganz eigener Art, weil sie die einzelnen Verbindungen in verschiedener Anordnung und vielleicht auch in verschiedener Bindung enthalten. Außerdem wechselt die Menge der einzelnen Bausteine. Bei dem jetzigen Stande unserer Kenntnisse über den Aufbau des Chlorophylls vermögen wir der Entstehung dieses wichtigen Farbstoffes in den Zellen der Pflanzen nicht zu folgen. Wir können nur mit großer Wahrscheinlichkeit der Vermutung Ausdruck geben, daß auch hier die Synthese von einfacheren Produkten aus ihren Ausgang nimmt. Zuniichst dürften wohl die beiden Anteile des Chlorophylls, das Phytol und das ringe, wie Chlorophyllid. für sich gebildet werden. Ihre Vereinigung, die die Chlorophyllase vollzieht, führt dann zum Chlorophyll. In welchem Stadium der Chlorophyllsynthese das Magnesium in das Molekül eingefügt wird, wissen wir nicht. Einstweilen können wir das Phytol mit keiner anderen, bekannten Verbindung in Beziehung bringen. Dagegen lassen die Pyrrolkerne, die am Aufbau der Chlorophyllide beteiligt sind, Vermutungen über ihre Herkunft zu. Wir haben nämlich bei der Besprechung der Aminosäuren einige kennen gelernt, die eine dem Pyrrol verwandte Struktur besitzen. Es seien jene Bausteine der Eiweißstoffe hier angeführt, die als Ausgangsmaterial zur Synthese des „Pyrrolringgerüstes" des Chlorophylls in Betracht kommen können. Ihre Struktur sei mit derjenigen des Pyrrols verglichen: CH II CH il CH CH \NU X Pyrrol II CH2 CH., CHa CHa \ NH / Pyrrolidin. Blatt- und CH,- Blutfarbstoff". Chlorophyll und Hämoglobiu usw. HO.CH -CH, 1 CH, I CH GOOH CH, i €H GOCH CHj . 705 . \NH/ NH r-Pyrrolid in karbonsäure = Prolin y-Oxy-a-pyrrolidinkarbonsäure = OxyproHn CH, CH, ! I CO \ CH, NH / PyrroUdon COOH t CH NH, . — H, CH, I CH, COOH Glutaminsäure GOOH CH NH CH,- . I -CH, } I GH, €H COOH CO oder . 1 NH, CH, { GOPvrrolidonkarbonsäure GH NHs HC^^G- G HC CH GH . GH., . CH GOOH . NH Pyrrolkern Tryptophan = a- Ami no-,b-indol-prop ionsäure. Prolin und OxyproHn enthalten den Pyrrolidinkern. üie Glu- taminsäure kann unter Wasseraustritt in ein Derivat des Pyrrolidonrin^^es übergehen. Im Tryptophan endlich finden wir den Pyrrolkern vorgebildet. Wir können das Indol als Benzopyrrol betrachten. SchlieliA Ijdor h al Jon. riiysiologifcli« Cherain. I. Tail. h. Aufl. 45 XXXIII. Vorlesnng. 706 lieh kommt auch noch das Glukosamin in Frage, das leicht in Pyrrol- derivate übergeht. ^) Es ist nun nicht sehr wahrscheinlich, daß die Pflanze zunächst eine dieser Aminosäuren und insbesondere das Tryptophan darstellt, um dann auf diesem Umwege zu jenen Pyrrolringen zu gelangen, die den Kern des Chlorophyllmoleküls bilden. Uns interessiert hier nur die Tatsache, daß die Pflanzenzelle den Pyrrolring mehrfach verwendet und ihn ohne Zweifel leicht bereiten kann. Es unterliegt keinem Zweifel, daß mit der völligen Aufklärung der Struktur des Chlorophylls und der Kenntnis aller seiner Abbaustufen auch Anhaltspunkte für ein erfolgreiches Studium der Synthese dieser Verbindung in der Pflanzenwelt gewonnen werden. 2) Es ist ganz selbstverständlich, daß man seit der Feststellung, daß im Chlorophyll und im Hämatin sich entsprechende Atomgruppen finden, daran gedacht hat, es könnte das mit der Nahrung aufgenommene Chlorophyll das Ausgangsmaterial zur Bildung des letzteren abgeben. Leider sind unsere Kenntnisse über das Verhalten des Chlorophylls im Magendarmkanal noch sehr dürftige.») Es wird wohl zur Abspaltung des Phytols kommen. Auch das Magnesium wird wahrscheinlich in Freiheit gesetzt. Was aber aus dem Reste des Chlorophyllmolekülls wird, wissen wir nicht. Es wäre von größtem Interesse zu erfahren, ob der tierische Organismus Bausteine des Chlorophylls direkt oder auch nach erfolgtem Umbau verwerten kann. Aus dem Umstände, daß im Kote Chlorophyll und daraus hervorgegangene Umwandlungsprodukte enthalten sind, darf nicht geschlossen werden, daß es und seine Abkömmlinge im tierischen Organismus keine Verwertung finden. Es ist ganz gut möglich, daß ein Teil des aufgenommenen Chlorophylls von den tierischen Zellen verwertet wird. Ebensowenig, wie über das Verhalten des Chlorophylls im tierischen Organismus sind wir über die Beziehungen des mit der Nahrung aufgenommenen Blutfarbstoffs zu dem im Blute enthaltenen unterrichtet. Wir wissen nur, daß das Hämoglobin schon im Magen verändert wird. Die Salzsäure des Magensaftes wirkt spaltend auf die Globin-hämochromogen- bzw. Globin-häma tili Verbindung. Es entsteht Hämochromogen bzw. Hämatin und daneben Globin, das ohne Zweifel vom Pepsin rasch in Peptone übergeführt wird. Ob schon Eisen im Magen aus dem Hämochromogen bzw. Hämatin abgespalten wird, ist noch nicht einwandfrei geprüft, dagegen wissen wir, daß im Darmkanal bereits ein Abbau zu eisenfreien Produkten erfolgt. ) Auch hier stehen wir noch vor zahllosen ungelösten Fragestellungen. Wir wissen nicht, wie weit der Abbau des Hämochromogens bzw. Hämatins bzw. des wahrscheinlich sich bildenden Hämatoporphyrins geht. ^) Ferner i.st uns Vgl. JI. I'cmly und E. Ludwig: Zeitsclir. f. physiol. Chem. 121. 170 (VMl). Vgl. auch B.'Oddo und G. Pollacci: Gazz. chim. ital. 50. I. 54 (11)19). ä) Vgl. L. Marchlewski: Zeitschr. f. Biol. 41. 33 (1904). ) Vgl. hierzu E. Abderhalden: Zeitschr. f. Biol. 39. 113 (1899). Emil Ahderhalden und Rud. llanslian: Zeitschr. f. physiol. Chemie. 80. 113 (1912). ) Es ist möglich, daß das im Harn aufgefundene Hämatin und ferner das Hämatoporphyrin als Produkte aufzufassen sind, die der weiteren Umwandlun.: in den Gewehen entgangen sind. Beide Verhindungen stammen jedoch mögliciier Weise auch direkt von der aufgenommenen Nahrung her. Wahrend die letztere Verhindnng Btets im Harn enthalten sein soll, ist das Hämatin selten anzutreffen. Vgl. z. B. Saillrf ') ^) - Blatt- und Blutfarbstoff. Chlorophyll und Hiimoglohin usw. 707 unbekannt, in welchem Umfange die Resorption einsetzt, und was aus den resorbierten Produkten wird. Es ist sehr wohl möglich, daß der tierische Organismus Bestandteile des mit der Nahrung aufgenommenen Hämobzw. Hämatins in bestimmter Weise verwertet. Wenn di(\s der Fall wäre, dann müßte die tierische Zelle in der Lage sein, Pyrrolkerne zu bilden. Vielleicht ist das Tryptophan ein direktes Baumaterial zur Bildung von Hämochromogen. Ob auch der Pyrrolidin- und der Pyrrolidonring von der tierischen Zelle zum Pyrrolring umgebaut werden können, wissen wir nicht. chromogens nicht Es sei vorausgeschickt, daß das Plämochromogen bzw. Humatin in Beziehung zum Gallenfarbstoff steht. Dieser ergießt sich mit der Galle in den Darm. Wir können sie durch Anlegung einer Fistel nach außen ableiten und dann einmal die in 24 Stunden gebildete Galle genau messen und ferner ihren Gehalt an Gallenfarbstoff bestimmen. Da wir keine andere Quelle für diesen kennen, als den eisenhaltigen Paarling des Hämoglobins, und wir ferner wissen, in welcher Beziehung er zu diesem letzteren steht, so läßt sich leicht berechnen, wieviel Hämochromogen bzw. Hämatin in 24 Stunden in Gallenfarbstoff übergeht. Derartige Bestimmungen haben ergeben 1), daß der Mensch im Tag etwa 05/7 Gallenfarbstoff ^^^ Darme zuführt. Dieser Menge entsprechen ungefähr Obg Hämatin. Wir können weiter berechnen, wieviel (Jxyhämoglobin notwendig ist, um diese Menge von Hämatin zu liefern. Wir haben nämlich früher festgestellt, daß der Blutfarbstoff bei der Spaltung etwa 4"/o Hämatin liefert. 2) Somit entsprechen 05 <7 Hämatin l^-bg Oxyhämoglobin. Diese Menge Hämoglobin muß ohne Zweifel fortwährend ersetzt werden. Wir schließen das daraus, daß der Gehalt des Blutes an Hämoglobin bei gesunden Individuen annähernd konstant bleibt. Wenn wir ferner ein Tier längere Zeit hungern lassen, dann ist zwar die Bildung der Galle eingeschränkt, jedoch nicht aufgehoben. Immer gehen rote Blutkörperchen zugrunde. Es wird Hämoglobin frei, das wohl in den meisten Fällen zum Abbau kommt. Eine erhebliche Abnahme des Hämoglobingehaltes des Blutes ergibt sich selbst nach recht langen Hungerperioden nicht. W^ir können ferner einem Tiere große JMengen von Blut und damit von Hämoglobin entziehen. Selbst dann, wenn jede Nahrungszufuhr fehlt, kommt es wieder zur Neubildung von roten Blutkörperchen mit allen ihren Bestandteilen. Schließlich sei auch noch auf den Säugling hingewiesen, der in seiner Nahrung keine vorgebildeten Bausteine zur Bildung von Hämochromogen aufnimmt. Er wächst sehr rasch und baut fortwährend Blutfarbstoff auf. Allerdings scheint er P.nuniäterialien zur Bildung von solchen vom mütterlichen Organismus •- (ii die Plazenta übernommen zu haben 3), denn erst bei lange fort^visetzter Ernährung mit Milch tritt allmählich eine bedeutende \'erarmung des Blutes au Hämoglobin auf, während innerhalb der normalen Zeit der ausschließ- — 0. Neubauer: Arch. 400 (18i)4); Revue de med. 16. 542 (18UG). Archibald E. Garrod: .lourn. of Physiol. experim. l'ath. u. Pharm. 43. 456 (1900). 13. 610 (1893); 17. 350 (1894). ) Vgl. hierzu Edward H. Goodmann : Ho/meisten Beitr. 9. 91 (1907). Vfrl. auch Th. Urugsch und K. Jietzla(f: Zeitschr. f. e.xpcrim. l'atli. u. Ther. 11. 508 (191 L'i. Bull. de thöreapie. f. — — ^) Vgl. 's. 691. 3) Emil Abderhalden: Zeitschr. f. physiol. Chemie. 34. 500 (1902). 45* XXXIII. Vorlesung. 708 Ernährung mit Milch liehen — also während der Säuglingsperiode -^ der Häraoglobingehalt zwar im Verhältnis zum rasch zunehmenden Körpergewicht sinkt, jedoch seiner absoluten Menge nach zunimmt, i) Nun hat Bunge festgestellt 2), daß die Milch sehr arm an Eisen ist. Es wäre denkr bar, daß Eisenmangel der Bildung von Hämoglobin Schranken setzt. Allein es hat sich gezeigt, daß offenbar auch noch andere Baumaterialien fehlen, wenigstens kann man mit Milch und Eisen allein auf die Dauer nicht den normalen Gehalt des Blutes an Blutfarbstoff aufrecht erhalten. «) In besonders eindringlicher Weise tritt uns die Synthese des Blut- farbstoffs bei der Bildung der roten Blutkörperchen im Vogelembrjo ent- gegen. Während man beim Säugetier an eine Übertragung von vorbereiteten Bausteinen von der Mutter auf den Fötus denken kann, muß das Vogelei beständig in sich alle Materialien zur Hämochromogen- und Hämoglobinbildung zur Verfügung haben. Ist Befruchtung erfolgt, dann fügen sich bestimmte Verbindungen zum roten Blutfarbstoff zusammen. Man hat aus Eiern ein sog. Hämatogen isoliert, das eine ähnliche Zusammensetzung wie das Hämoglobin haben soll.) Es ist jedoch die vorliegende Beweisführung für diese Annahme ganz ungenügend, weil sie sich nur auf die Ergebnisse von Elementaranalysen stützt, und bekanntlich solche bei so komMan müßte auf Verpliziert gebauten Molekülen gar nichts besagen. bindungen fahnden, die zu irgend welchen Abbaustufen des Hämochromogeus bzw. Hämatins in Beziehung stehen. Es liegt hier ein hochinteressantes und sicher an Früchten reiches Arbeitsgebiet vor. globin Wenn wir alles zusammenfassen, was wir über die Bildung von Hämoim tierischen Organismus wissen, dann kommen wir zum Schlüsse, daß ohne jeden Zweifel fortwährend eine Synthese von Blutfarbstoff und damit von Hämatin erfolgt. Über die Herkunft der einzelnen zum Aufbau der letzteren Verbindung notwendigen Bausteine können wir zurzeit gar nichts Bestimmtes aussagen. Es scheint nach allen Erfahrungen, daß der tierische Organismus nicht unbedingt auf in der Nahrung vorgebildete anAbbaustufen des Chlorophylls und des Hämatins Materialien gewiesen ist, sondern die Synthese des Hämatins aus einfacheren Baumaterialien vollziehen kann. Vielleicht wird das Tryptophan dazu verwendet. Auch die übrigen S. 704 und 705 genannten Verbindungen kommen als Baumaterial in Betracht. — — Viel besser als über die Bildung des Hämatins im Organismus sind wir über seinen Abbau unterrichtet. Er führt, wie schon erwähnt, zum Gallenfarbstoff, und zwar zum Bilirubin. Subkutan zugeführtes in Hämatin erscheint fast vollständig in Form von Gallenfarbstoffen Es ist sicher festgestellt worden, daß diese Umwandlung der Galle, ß) Vgl. hierzu die Vorlesung liber Prisen, Bd. II. Vorlesung 2. G. V. Bunge: Zeitschr. f. physiol. Chemie. 16. 173 (1892); 17. 63 (1893); Zeitschrift f. Biol. 45. .^)32 (1901). ') Vgl. z. B. Emil Abderhalden: Zeitschr. f. Biol. 39. 113, 193, 483 (1909). ) G. V. Bunge: Zeitschr. f. physiol. Chemie. 9. 49 (1884). L. Ilugonnenq und Albert Morel: Compt. rend. de l'acad. des scienc. 140. 1065 (1905); Journ. de physiol. ') «) — et 15. de pathol. g6n6r. 747 (1891). ) 8. 391 (190()). — Vgl. auch G. Walter: Zeitschr. f physiol. Chemie. Th. Brtigsch und Joshirnoto: Zeitschr. f. experim. Path. u. Ther. 8. 739 (1911). Blatt- und Blutfarbstoft. Chlorophyll und Hämoglobin usw. 709 von Hämatin in Bilirubin sich in der Leber vollzieht, ^j Sie ist jedoch der einzige Ort der Gallenfarbstoffbildung, vielmehr sollen unter besonderen Verhältnissen auch andere Zellarten Bilirubin bilden können. 2) Man hat z. B. beobachtet, daß bei Blutergüssen an Ort und Stelle Kristalle auftreten, die mit Bilirubin identisch sind, »j Auch in der Milz soll Gallenfarbstoff entstehen können. ) Ferner findet man in der Plazenta von Hunden Farbstoffe, die in engen Beziehungen zum Gallenfarbstoff zu stehen scheinen. ^) Weitere Untersuchungen müssen zeigen, ob es tatsächlich eine nichthepatogene Gallenfarbstoffbildung gibt.«) vielleicht nicht Das Bilirubin'^) kristaUisiert aus heißem Dimethylanilin in großen, rhombischen Säulen. Es ist übrigens nicht einheitlich, es besteht vielmehr aus einem Gemisch von zwei Modifikationen. ^) Das Bilirubin unterscheidet sich zunächst vom Hämatin durch das Fehlen des Eisens. Seine empirische Formel Cs, Hgg N^ Og weist verglichen mit der des Hämatins auf eine oxydative Veränderung des letzteren Moleküls beim Übergang in Bilirubin hin. Die Umwandlung ist keine einfache. Es finden vielmehr Umlagerungen statt. William Küster'^) gibt ihm die folgende, den Tribreiten, phenylmethanfarbstoffen ähnliche Konstitution C=:C— CH, C= \ GH, — CHa -^N N< C = C— CH, CH,— C= OH CH,— C=G- . CHj =C / \NH Nf-CH,-CH, \ / OH . CH, COOH C -C= HOOC CHg— CH,-C=C . C=—— C-CH, ') Vgl. hierzu Hans Stern: Arch. f. exper. Patb. u. Pharmak. 19. 39 (1885). 0. Minkowski und B.Naunyn: Ebenda. 21. 1 (1886). Stadelmann : Ebenda. 15. 337 (1882); 27. 93 (1890). ») Vgl. z.B. M. Afanasiew: Zeitschr. f. klin. Med. 6. 291 (1883). J.Latschenherger: Sitzungsber. d. Wiener Akad., math.-natursv. Klasse. 97. Abt. IIb. 15 (1888). — E . — Vgl. z.B. B. Virchow: Virchows Archiv. 1. 379, 407 (1847). Vgl. zu diesen Fragen besonders: A. A. Hymans van den Bergh und ./. SnappeiBerliner klin. Wochenschr. 52. 1081 (1916); A. A. Hymans van den Bergh: Der Gallenfarbstoff im Blute. Leipzig. Johann Arabrosius Barth. 1918. G.Lepchne: Münchenor ») ) — med. Wochenschr. 66. 619 (1919). ») ) C. Etti: Jahresber. f. Tierchemie. 1. 233 (1871); 2. 287 (1872). Vgl. auch Hans Fischer und F. Reindel: Münchener med. Wochenschr. Vgl. 69 Nr. 41, 1451 (1922). ') Vgl. Handbuch der biochemischen Arbeitsmethoden. 2. 635 (1910). (Bearbeitet Biochem. Handvon William Küster.) Urban & Schwarzenberg. Berlin-Wien 1910. lexikon. 6. 277 (1911). (Bearbeitet von B^la v. Reinbold.) J.Springer. Berlin 1911. ") Vgl. William Küster, H. Bauer, K. Reihling und A. Schwaderer : Zeitschr. für — physiol. Chemie. 94. 136 (1915). ») William Küster: Zeitschr. f. physiol. Chemie. 99. 86 (97) (1917). 121. 80. 94. 110 (1922). — Vgl. auch XXXIII. Vorlesuüg. 710 Nach dieser Formel ist Bilirubin als Pyridyl-pyrryl-pyrryl-pyrrolenraethan aufzufassen. Die gegebene Konstitutionsformel gibt eine Erklärung für das Bestehen von Modifikationen und ferner für die beobachtete Umlagerungsfähigkeit. Reduziert man Bilirubin in alkalischer'^Lösung mit Wasserstoff und in kolloidem Zustand befindlichem Palladium, so erhält man Mesobilirubin.i) C33 H40 Og N4. Reduziert man mit Natrium amalgam., so gelangt man zu Mesobilirubinogen,^) C33 H44 Og N4. 3) Dieses hat sich mit dem Urobilinogen, der Muttersubstanz des Urobilins, als identisch erwiesen.) In diesem Zusammenhang ist von größtem Interesse, daß Hämin bei gelinder Reduktion unter Abspaltung von Eisen Mesoporphyrin liefert, Dieses kann durch weitere Reduktion in das farblose Porphyrinogen übergeführt werden. Stellt man diesem Befund gegenüber, daß man durch Reduktion von Bilirubin Mesobilirubin und durch weitere Reduktion Mesobilirubinogen und umgekehrt durch Oxydation der letzteren Verbindung Mesobilirubin erhält, so erkennt man, daß für Bilirubin, Mesobilirubin und Mesobilirubinogen, die alle drei die gleiche Molekulargröße haben, die gleichen Beziehungen bestehen, wie zwischen Mesoporphyrin und Porphyrinogen. ^) Sehr interessant ist, daß Mesobilirubinogen unter pathologischen Verhältnissen auch im Harn gefunden worden ist.«) Die Aufspaltung des Bilirubins unter Reduktion mittels EisessigJodwasserstoff führt zu einem zweikernigen Pyrrolderivat C17 Hoi O3 Ng, Bilirubinsäure ') genannt. Durch Oxydation geht diese unter Verlust von zwei Wasserstoff atomen in eine gefärbte Substanz, die Xanthobili- rubinsäure über. Diese zerfällt weiter Imid der Methyl-äthylmaleinsäure. CH3 .'C— . CH., \| . ÜH. . COOH in Hämatinsäure^) und das CH3 . C^=C GH., OH3 . . ^ " I o=c C==0 \/ NH ! I 0=C c=o VNH Imid der dreibasischen Hämatinsäure Methyl-äthyl-maleinimid. Hans Fischer und Mitarbeiter geben der Bilirubinsäure die folgende Strukturformel: 9) H. Fischer: Zeitschr. f. Biol. 65. 172 (1914). H. Fischer uud /'. Mayer: Zeitschr. f. pbysiol, Chemie. 75. 342 (1911'. W. Küster: Ebenda. 82. 463 (1912). '^) Hans Fischer: Zeitschr. f. physiol. Chemie. 73. 204 (1911). ) Vgl. hierzu auch Th. Brugsch uud K. Retzlaf: Zeitschr. f. exper. Path. u. Thor. Über deu Nachweis: D. Ciiarnas: Biochem. Zeitschr. 20. 414 (1909). 11. .508 (1912). 47. 2330 (1914): Zeitschr. 5) Hans Fischer: Ber. d. Deutschen Chem. Gesellsch. 65. 163 (1914). t. Biol. «) Vgl. hierzu William Küster : Liebigs Annalen. 315. 186 (1901); Ber. d. Deutschen Chem. Gesellsch. 35. 1268(1902); Zeitschr. f. physiol. Chem. 47.294(190(5); 82. 4(53 (1912), der zuerst den Zusammenhang zwischen Ililmatiu uud Bilirubin erkannt hat. ') Hans Fischer und F. Meyer-.Belz: Zeitschr. f. physinl. Chemie. 82. 232 (1911). Vgl. auch Münchener med. Wochenschr. Nr. 15 (1912). 0. rUotg «) H. Fischer: Ber. d. Deutschen Chem. (iesellsch. 45. 1979 (1912). Liebigs Annalen. 390. 191 (1912) ^) Hans Fischer, S. Bartholomäus und H, Böse: Zeitschr. f. physiol. Chemie. 84 Vgl. weitere 2(j7 (1913) und Hans Fischer und H. Böse: Ebenda. 89. 2.o5 (1914). ') ") — — - — — und Blutfarbstoft'. Chlorophyll und Hämoglobin usw. Blatt- CH3 HO . . C CHg C C C, H5 „ . I! i C C . C; . GH., ^ ' C C ^ Sie erklärt die weiteren CHj . COOK ' !! NH . 711 li C CH, . NH Umwandlungen zwanglos. Durch energische man aus ihr neben wenig Phonopyrrolkarbonsäure 1) die Isophonopyrrolkarbonsäure und sehr geringe Mengen KryptoReduktion erhält pyrrol. ^) aus Die mitgeteilten stufen ergeben ohne dem Bilirubin gewonnenen Abbaudie nahen Beziehungen zum w^eiteres Hämatin. Ferner ist eindeutig erwiesen, daß der Gallenfarbstoff direkte Beziehungen zum Mesobilirubinogen bzw. Urobilinogen und damit zum Urobilin hat. ^O Wir haben somit eine ganze Kette von Umwandlungsprodukten des Hämatins vor uns. Es ist nun von größtem Interesse, daß Urobilinogen und Urobilin auch im tierischen Organismus in Erscheinung treten. Man hat geforscht. eifrig nach dem Entstehungsort dieser Verbindungen Man glaubte zunächst, daß diese Verbindungen in den Geweben sehr entstehen können. Es scheint jedoch, daß die Ansicht von Friedrich Müller^} wonach die genannten Verbindungen ihre Bildung der Tätigkeit der Darmflora verdanken, trotz mancher gegenteiliger Behauptungen recht In diesem Falle würde der Abbau des Hämatins im tierischen Organismus nicht über den Gallenfarbstoff hinausgehen. behält. Urobilin wäre dann nicht als ein Stoffwechselprodukt der tierischen Zellen aufzufassen. Das im Darm gebildete Urobilin verbleibt zum Teil im Kot, zum Teil wird es resorbiert. Im letzteren Falle wird ein Teil davon wieder von neuem durch die Galle dem Darmkanal zugeführt. Es heß sich auch daß nach Verfütterung verschiedener Pyrrolderivate Urobilinogen Dieses gibt mit p-Dimethylaminobenzaldehyd: zeigen, gebildet wurde. ^j ~ Vgl. auch physiol. Chemie. 99. 86 (1917). Deutscheu (Micm. Gescllsch. 45. 1579 (1912). Vgl. dazu auch 0. Pihfi/ und S. J. Thannhaufier : Ber. d. Peutscheu Chem. Gesellsch. Vul- auch Hansi Fisscher und Marianne Herrniann : Zeitschr. f. 45. 2393 (1912). physiol. Chem. 122. 1 (^1922). Kiuzelheiteij bei W. Küsler: Zeitschr. Ihins Fischer und Heinrich Rose: Ber. — t. d. — M Vgl. rilotij: Li>^//.9S Auiialcu. 377. 314 (1910); //. Fischer und .V. BartitolomäusB.r. d. Deutsclicu Chem. Gesellsch. 45. 13iri (1912); H. Fischer uud F. Meijer-Betz: Zeitschr. f. phvsiol. Chemie. 82. 9B (1912); //. Fischer und //. liösc : Ebenda. 82. 391 Vgl auch William Küster: Zeitschr. f. physiol. Chemie. 82. 403 (1912). (1912). — II. Fischer und JI. liäs^ : Ber. d. Deutschen Chem. Gesellsch. 45. 3274 (1912); 439 (1913): Zeitschr. f. physiol. Chemie. 89. 25ö, 268 (1914). ^) Vgl, dazu auch Scherer: Liebiffn Aunalen. 57. 180 (1846). -) 4«. ) 1892. Friedrich Müller: Jahresber. d. Schlesischen Ges. — Vgl. dazu auch Wilhelm llilclebrandt: Zeitschr. f. f. vaterläud. Kultur. Breslau Medizin. 59. H. 2—4 klin. Nr. 14 Deutsche med. Wochenschr. Nr. 12. Xr. IT) (1905); Münchener med. Wochenschr. und 15 (1909). ^) M. Jaffe hat es zuerst aus Harn gewonnen (Zentralbl. f. die mcdiz. Wissen- schaft, 243 (1868); 177 (1869); (19(J6); Virchon-9, Archiv. 47. 405 (1869). XXXIll. VoriesuDjr. 712 C.N(CH3)2 CH CH il I CH CH \ C^^ eine prachtvolle Rotfärbung {Ehrlichsche. Reaktion i). Es besteht immer noch eine große Lücke in der Erforschung der Beziehungen des Hämatins zum Gallenfarbstoff. Es ist dies die eindeutige Feststellung, daß die erstere Verbindung nur auf diesem Wege zum Abbau gelangt. Es spricht vieles für eine derartige Annahme. Sie ist jedoch noch nicht scharf bewiesen. Ferner fehlen noch Einblicke in das weitere Schicksal des aus dem Darm resorbierten Urobilins. Wir wissen, daß es in den Harn übergehen kann und auch auf dem Wege der Galle in den Darm zurückkehrt, dagegen ist es schwer, diesen Kreislauf des Urobilins eindeutig quantitativ zu verfolgen. Es ist einstweilen nicht ausgeschlossen, daß Urobilin und Urobilinogen von den tierischen Zellen zum Teil verwertet werden. Der Gallenfarbstoff kann leicht erkannt werden. Er erteilt der Galle die Farbe. Er wird in ihr vor allem durch die Gallensäuren in kolloider Lösung gehalten. 2) Meist enthält die Galle nicht nur Bilirubin, sondern auch aus ihm hervorgegangene Umwandlungsprodukte. So finden wir fast beständig das Biliverdin. ») Es ist ein Oxydationsprodukt des Bilirubins. Das Biliverdin bewirkt die grüne Farbe der Galle. Oberwiegt .seine Menge, dann zeigt die Galle eine olivengrüne Färbung, wenn dagegen mehr Bilirubin zugegen ist, dann finden sich rote bzw. braune Farbtöne. Jede Tierart hat im allgemeinen eine bestimmt gefärbte Galle. Eine ganze Skala von verschiedenen Oxydationsstufen erhält man aus Bilirubin, wenn man seine Lösung mit rauchender Salpetersäure oxydiert. Man sieht rote, rotgelbe, grüne, blaue und violette Farbtöne auftreten. Diese farbigen Umwandlungsprodukte liegen der sog. G melin sehen Reaktion auf Gallenfarbstoffe zugrunde. Man unterschichtet die Gallenfarbstoff enthaltende Flüssigkeit vorsichtig mit konzentrierter Salpetersäure, die etwas salpetrige Säure enthält. An der Berührungsstelle beider Schichten treten dann farbige Ringe auf. Man kann den Gallenfarbstoff auch vor der Anstellung der Probe mit Chloroform ausziehen und dann die Salpetersäure zu dieser Lösung zufügen. Man hat den einzelnen Oxydationsstufen besondere Namen zugelegt, doch ist es fraglich, ob einheitliche Verbindungen zur Beobachtung gelangt sind. So hat man z.B. ein Cholecyanin ) und Choletelin •') unterschieden. — ') P. Ehrlich: Mediz. Woche. 1901. Vgl. auch O. Neubauer: Sitziingsber. iL Gesellsch. f. Morph, u. Physiol. München 1903. '') Hans Fischer: Zeitschr. f. physiol. Chemie. 73. 204 (1911). ^) Vgl. seine Darstellung aus Bilirubin Städeler: Vierteljahrsschr. d. Züriclier Naturforsch. Ges. 8. 1 (1863). A. .Tolles: Pßilger^ Archiv. 75. 46 (1899); Zeitschr. f. physiol. Chemie. 27. 83 (1899). — — A. Heynsius und 241 nSÖS). 520 (1871). R. Mali/: Sitzungsber. der Wiener Akad. (math.-naturw. Kl.). 57. Abh. 2 (188öj; A. Heynsius und J. F. F. Campbell: PfiüfferH Archiv. 4. 497 0871). 59 (1869). ') .7. /''. M. Joffe: Zentralbl. f. d. mediz. Wisseusch. F. Campbell: I'/liigers Archiv. 4. =>) — Blatt- und Blutfarbstoff. Chlorophyll und Hämoglobin, usw. 71;', Der Gallenfarbstoff tritt manchmal in großen Mengen in den sog. Gallensteinen auf. Es gibt insbesondere beim Rinde fast reine Bilirubinsteine. Derartige Konkremente können auch Biliverdin enthalten. Man hat und in Gallensteinen noch andere Farbstoffe gefunden. Es das Biliprasin, 1) das Gholeprasin,^) das Bilifuszin^) und endlich das Bilipurpurin) erwähnt. Das letztere ist identisch mit dem Phylloerythrin. 5; Es wird angenommen, daß diese Verbindung im Darmkanal aus dem Chlorophyll hervorgeht, zur Resorption gelangt und dann durch die Galle wieder in den Darm zurückkehrt. Es bleibt späteren Untersuchungen vorbehalten, zu erweisen, ob die genannten Verbindungen wirklich einheitliche Substanzen darstellen, und in welchen Beziehungen sie zum Bilirubin stehen. So ist z. B. das sog. Hydrobilirubin bereits als ein Gemisch erkannt worden.^) Soviel scheint ganz sicher zu sein, daß einzig und allein Bilirubin ein primäres Stoffwechselprodukt der Leberzellen darstellt. Es sei noch erwähnt, daß dann, wenn der Abfluß der Galle nach dem Darme aus irgend einer Ursache Verlegung des Gallengangs durch Schwellung seiner Schleimhaut, durch ein Konkrement, einen Tumor usw. erschwert oder verhindert ist, es zu einem ohne weiteres in die Augen fallenden Symptomenkomplex kommt. Die Haut und besonders auch die Konjunktiven nehmen eine gelbe Farbe an. Sie ist durch Gallenfarbstoff bedingt. Die Galle staut sich. Die Bildung der Gallenbestandteile durch die Leberzellen erfolgt weiter. „Diese laufen schließlich gewissermaßen über". weil der normale Abfluß fehlt. Die Lymphbahnen tragen die Bestandteile der Galle dem Blute zu. Das Blutplasma wird gelb gefärbt") Es kommt dann bald zur Ausscheidung von Gallenfarbstoff in den Geweben und auch in der Haut. Der Urin zeigt gleichfalls eine tiefe, gelbe Färbung. in der Galle !«!eien — Schüttelt man ihn, dann tritt — gelb gefärbter Schaum auf. Auch Gallen- säuren finden sich im Blutplasma. Ihre Anwesenheit in diesem macht sich in einer starken Verlangsamung der Herztätigkeit geltend. Es kommt zui' Erscheinung des seltenen Pulses infolge der Einwirkung der erwähnten Säuren auf das Herz. Dauert die Behinderung des Gallenabflusses nach dem Darme längere Zeit an, dann kommt es auch zu einer Hypercholesterinoplasmie.®) Man nennt den ganzen Symptomenkomplex Ikterus. Im Anschluß an die besprochenen Farbstoffe wollen wir ganz kurz noch gefärbter Verbindungen gedenken die im Tierreich anzutreffen jedoch unseres Wissens nichts mit dem Sauerstofftransport zu tun einiger sind, ') A. Dastre •') ») , Städeler: Vierteljahrsschr. d. Naturforsch. Ges. in Zürich. 8. und N. Floresco : C. r. de la Soc. de biol. 49. 306, 513 (1897). 1 (1863). W. Küster: Zeitschr. f. physiol. Chemie. 17. 294 (1906); 94. 163 (1915). 132. 323 (1864). — L. r. Zumbusch: Zeitschr. Städeler: Liebig?, Aunalen. — f. physiol. Chemie. 31. 446 (1900). ) W. F. Löbisch und M. Tischler: Monatsh. f. Chemie. 24. 335 (1903). Ch. Marc Munn: Joum. of Physiol. 6. 22 (1885). «) L. Marrhlewski: Zeitschr. f. physiol. Chemie. 48. 207 (1904); 45. 466 (1905). Vgl. dazu auch E. Schunek und L. Marchlewski : Liebig^ Annalen. 278. 329 (1894); 284. 81 (1895); 288. 209 (1895); 290. 306 (1896). L. Marrhletcski : Zeitschr. f. prakt. Chemie. Hans Fischer: Zeitschr. f. phvsiol 65. 161 (1902); Biochem. Zeitschr. 3. 320 (1907). — — — — Chemie. 96. 293 (1916). ") Hans Fischer: Zeitschr. f. physiol. Chemie. 73. 204 (1911). ') Vgl. u. a. J. Feigl und E. Querner: Zeitschr. f. d. gesamte experim. Med. 153 (1919). 8) Jvar Bang: Biochem. Zeitschr. 91. 122 (1918). {'. XXXUL Vorlesung, 714 haben und auch nicht als Sauerstoffspeicherer in Frage kommen. Wir haben bereits Mher hervorgehoben, daß die Pflanzen eine außerordentlich große Anzahl verschiedenartiger Farbstoffe hervorbringen. Von einem großem Teil davon kennen wir die Konstitution.^) Von den bei den Tieren vorkommenden Farbstoffen ist zumeist nicht viel mehr als der Name bekannt. Nur bei einigen wenigen können wir wenigstens die Zugehörigkeit zu einer bestimmten Klasse von Verbindungen angeben. Wir kennen eine ganze Anzahl von schwarz oder braun gefärbten Farbstoffen. Solche finden sich in den Haaren, in der Haut (insbesondere der dunkel gefärbten Rasseh), in den Pigmentzellen der Uvea usw.) Man hat diese Faibstoffe als Melanine bezeichnet. 3) Von keinem dieser Produkte können wir etwas über seine Zusammensetzung aussagen. Sie treten zumeist in sehr geringen Mengen auf. Ferner sind sie schwer von Beimengungen zu trennen, und endlich sind sie sehr widerstandsfähig. Ab und zu kommt es zu größeren Ansammlungen von derartigen Pigmenten. So trifft man bei den unechten Schimmeln häufig große Geschwülste in den Muskeln an, die ganz mit einem tintenartigen Saft erfüllt sind. Man hat den Eindruck, als wäre bei diesen Tieren das in der Haut nach der Geburt noch vorhandene Pigment zu diesen Stellen hingewandert. Das Pigment ist Hippomelanin genannt worden.) Bei seiner oxydativen Spaltung konnte Gu anidin gewonnen werden.^) Sie können durch intensive Bestrahlung stark an Menge zunehmen. Auch die Melanosarkome des Menschen und der Ab und zu Tiere enthalten auffallend viel Pigment (Sarkomelanin).^) kommt es bei melanotischen Neubildungen zur Ausscheidung von Pigment im Urin. 7) Manchmal ist der frisch gelassene Harn bereits dunkel bis ^) ) Vgl. S. 71. Vgl. über da,s Melauiu der Ch orioidea: Landolt: Zeitschr. f. physiol. Chem. — Über das Fuszin (Melauiu der Retina): Kühne und Sewell: UnterHaarsuchungen aus dem physiol. Institut der Univers. Heidelberg. 3. 221 (1883). Spiegier: melanine: N. Sieber: Archiv, f. experim. Path. u. Pharm. 20. 3G4 (1886). Boss Aiken Gortner: Journ. of Biol. Chem. Ö. Hofmeisters Beitr. 10. 253 (1907). Helmann: Arch. Hugo Fasal : Biochcm. Zeitschr. 55. 393 (1913). 341 (1910). 28. 192 (1899). — — — internat. de pharm". 12. 271 (1904). — — — H.Epjnnger: Biochera. Zeitschr. 28. 181 (1910). — — Hautmelauin: J. Abel und Davis: Journ. of experim. Med. 1. 361 (1896). den Ursprung des melanotischen Pigments der Haut und des Auges. Werner Klinkhardt. Leipzig 1908. Mit viel Literatur. ä) Vgl. u. a. 0. V. Fürth: Zeutralbl. f. allg. Pathol. u. pathol. Anat. 15. 617 (1904). Vergleichende chemische Physiologie der niederen Tiere. G. Fischer. Jena 1903. Biochem. Handlexikon. 6. 293 (1911). (Bearbeitet von Franz Samuely.) J.Springer. Berlin 1911. ) Berdez und M. Nencki: Arch. f. experim. Path. u.' Pharm. 20. 346 (1886). O. v. Fürth und E. Jerusalem: JJ. Nencki und iV. Sieber: Ebenda. 24. 17 (1887). E. Meirotvsky : t)ber — — Hofmeister?, Beitr. 10. 131 (1907). — Peter Ro na und Otto Riesser: Zeitschr. Chem. 57. 143 (1908); 61. 12 (1909). comparee. 17.— 23. Oktober 1912. f. physiol. — Maurice Piettre: Congr^s internat. de Pathol. — J. Adler- Herzmark: Biochem. Zeitschr. 49. 130 (1913). 5) «) Otto Riesser und J'eter Rona: Zeitsohr. f. physiol. Chem. 10J. 16 (1920). K.A.H.Mörner: Zeitschr. f. physiol.'.Chem. 11. 66 (1886). — O. Schmiedeberg: — Arch. f. experim. Path. u. Pharm. 39. 1 (1897). E. Zdarek und R. i\ Zeynek: Zeitschr. - //. Wolff: Hofmeisters Beitr. 5. 476 (1904). f. physiol. Chemie. 36. 493 (1902). ') Zeller: r.Jaksch: Zeitschr. f. physiol. Arch. f. klin. Chir. 29. 245' (1883). Chemie. 13. 385 (1889). E. Zdarek: Zeitschr. f. Heilkunde u. Path. 23. 379 (1912). K. A. H. Monier: Zeitschr. f. pliysiol. Chemie. 11. 66 (1887). A. Pribram und GangJ: Berdez und M. Nencki: Archiv für hofner-: Prager V'ierteljahrsschr. 88. 16 (1865). experim. Path. u. Pharm. 20. 24»; (1886). D. Ilelman: Arch. internat. de pharm, tet de th(5rap. 12. 271 (1903). — — — — Blatt- und Blutfarbstoff. Chlorophyll und Hämoglobin usw. 715 entsteht Meistens jedoch der Farbstoff erst nach Man findet bei den Melaninen die Oxydation des Harns. Elemente C, H, N, S und 0. Es spricht vieles dafür, daß sie aus Eiweißbausteinen hervorgehen. Tryptophan, Prolin, Oxyprolin und auch Tyrosin und Glukosamin sind geeignete Ausgangsmaterialien zur Bildung von P'arbstoffen. Wir haben schon erwähnt, daß es Fermente gibt, die Tyrosin in einen Farbstoff und schließlich in ein braunes Pigment verwandeln, ^j Tryptophan zeigt auch große Neigung zur Farbstoffbildung. Eine Umwandlung von Tyrosin in einen Farbstoff nimmt man auch bei der Bildung des Sepiaschwarzes 2), des Sekretes der Tintenfische, an. Es ist auch an das 3, 4-Dioxyphenylalanin als Ausgangsmaterial für Pigmente gedacht worden. ^) schwarz gefärbt. erfolgter Eine große Gruppe von Farbstoffen stellen die sogenannten LipoSie finden sich in der Pflanzen- und Tierwelt in großer Zahl. Über ihren Bau läßt sich zurzeit nichts aussagen. Aufgeklärt ist dagegen die Natur einer ganzen Reihe von gelben, zum Teil mit besonderen Namen, wie z. B. Lutein ^) belegten Farbstoffen. Es hat sich herausgestellt, daß z. B. der gelbe Farbstoff des Blutserums, des Fettes, des Eidotters in engstem Zusammenhang mit der Nahrung steht, und zwar mit dem Xantophyll und dem Karotin der Pflanzennahrung. Das erstere ist im Hühnereigelb nachgewiesen worden. Das letztere findet sich im Blutplasma und im Fett, ß) Auch aus dem Corpus luteum ist ein Farbstoff erhalten worden, der dieser Reihe angehört. ') Der enge Zusammenhang der erwähnten gelben Farbstoffe mit den Blattfarbstoffen der Nahrung ließ sich direkt feststellen. Beim Diabetes ist wiederholt eine eigenartige kanariengelbe Farbe der Haut beobachtet worden. ^) Man hat von einer Xanthosis diabetica gesprochen. Diese Erscheinung ist in der letzten Zeit in Deutschland häufiger geworden. Der Umstand, daß reichein liche Zufuhr von grünem Gemüse und vor allem von Mohrrüben wesentlicher Bestandteil der jetzigen Kost! mit dem Auftreten der Xanthosis parallel geht und diese mit dem Übergang zu anderer Nahrung sich vermindert, darf wohl als ein Beweis für den erwähnten engen Zusammenhang der genannten Farbstoffe angesehen werden.'-) chrome) dar. — — M Vgl. S. .'526. - Vgl. auch Boss Aiken Gor/ner: Biochein. Bull. 1. 201 (1911); Aineric. Naturalist. 743 (1911). L. C. MaiUord: Genese de matieres protöiques et dos iuatieres himiiques. Masson et Cie. Paris 1913 (S. 416). 24. 21 (1887). -) M. Nencki und .Y. Sieher: Arch. f. cxperim. Path. u. Pharm. <>. r. Fürth und Schneider: Hofmeister?, Beitr. 1. 241 (1901). ^) Vgl. Bruno Bloch und F. Ryhner: Zeitschr. f. d. ges. experim. Med. 5. 179 (1917). \ \g\. B. Krukenherij : Vergleichende physiologische Studien. 1. 11. 111 (1880); — — 2. III. SC), 108 (1882). '•) W. Halliburton: .Jouru. ^) Vgl. hierzu of Physiol. 7. 324 (1886). auch A. A. Hynians ran den Bergh und J. Snapper: Deutsches li. Wilhlätter und H.H. Escher : Zeitschr. f. physiol. Arch. t. klin. Med. 110 (1913). l.rroy S. Bahner: .Journ. of biol. Chem. 23. 261 (1915); 27. Chemie. 76. 214 (1912). — — 27 (1916). ') Heinrich II. Escher: Zeitschr. f. physiol. Chemie. 83. 198 (1913). Umher: Berliner «) Vgl. von Noorden: Zuckerkrankheit. VI. AuH. 181 (1912). klin. Wochenschr. Nr. 30 (1916). ) M. Bürger und A. Reinhart: Zeitschr. f. d. ges. experim. Med. 7. 119 (1918). W.Stölizner: P^benda. Nr. 15, ./.Laupe: Münchener med. Wochenschr. Nr. 12 (1919). — 419 (1919). — — H.Salomon: Wiener klin. Wochenschr. 32. 496 (1919). ^ Vgl. auch 7-u XXXIII. Vorlesung. 716 den Purpurfarbstoffen Unter stoßen wir auf einen, der von Murex brandaris gebildet wird, und dessen Konstitution vollständig aufgeklärt ihm ist. Wir haben seiner die Konstitution eines 6, früher schon gedacht und angeführt, daß 6-Dibromindigos zukommt.') Von den übrigen Farbstoffen erregt das Turazin^) noch unser besonderes Interesse. Es findet sich als rotvioletter Farbstoff in den farbigen Federn von Turacus, Gallirex und Musophaga. Dieser Farbstoff ist durch einen hohen Gehalt an Kupfer ausgezeichnet. Ferner wollen wir noch die Kochen illefarbstoffe anführen. Sie werden aus den getrockneten, ungeflügelten Weibchen der Schildlaus, Coccus cacti coccineliferi, gewonnen und enthalten die Karminsäure, C^aHgjOiä. ') Von großem Interesse ist die Feststellung, daß manche Farbstoffe der Schmetterlinge enge Beziehungen zu Verbindungen der Purinreihe besitzen.) Auch bei niederen Wirbeltieren, z. B. Fröschen, hat man „Purinpigmente" (Guanin) festgestellt. &) Eingehend untersucht worden ist ferner der von Boll entdeckte Farbstoff der Netzhaut, der Sehpurpur. «) Er wird von Licht rasch ausgebleicht. Er löst sich in Galle und kann der Netzhaut damit entzogen werden. Bis jetzt ist über seine chemische Natur ebensowenig bekannt, wie über seine Beziehungen zum Sehakt. Schließlich sei noch der Harnfarbstoffe Urochrom, ürorosein^ und Uroerythrin^) gedacht. Der letztere Farbstoff verleiht dem Sediment des Harnes seine rote Farbe. Seine Herkunft ist noch unaufgeklärt. Strittig ist auch die Herkunft des gelben Harnfarbstoffes Urochrom.^) ganzen Frage llymuns van den Bergh und P. Müller (J. Broekmtyer) : Biochem. Interessant ist das Zusammentreffen von Cholesterinestern mit gelbem Farbstoff in den Xanthome genannten Geschwülsten. iler — Zeitschr. 108. 279 (1920). Vgl. S. 626. ') Church: Chemical News. 19. 265 (1869); 65. 218 (1892): Philosoph. TransB. Krukenberg : Vergleichende physiol. Studien. 1. V. 7.'> actious. 183. 511 (1892). P. P. Laidlaw: Journ. of Physiol. 31. 464 (1904). (1880). C. Liebermann, Hörnig und F. Wiedermann: Ber. d: Deutscheu Chem. GesellO.Dimroth: Ebenda. 42. 1611 (1909); 43. 1387 (1910). schaft. 33. 149 (1900). Über ihre Konstitution vgl. 0. Dimroth, W. Scheurer und St. Goldschmidt: Liebigs Ann. 399. 1 (1913). C. Liebermann und Hans Liebermann : Ber. d. Deutschen Chem. Gesellschaft. 47. 1213 (1914). ) F.G.Hopkins: Chem. News. 60. 57 (1899); Philosoph. Transactions. 186. »;61 (1894). Griffiths: Compt. rend. de l'acad. des sciences. 115. 9.^8 (1892). ») J. Millot: C. r. ßoc. de biol. 87. 63 (1922). ') — — '') - — — — •) Boll: Sitzungsber. d. Berliner Akademie der Wissensch. S.November 1876. — W.Kühne- Untersuchungen des Heidelberger phvsiol. Instituts. 1. 515 (1878); für Biol. 32. 21 (1895). — P. Trendelenburg: Arch. f. (Anat. n.) Phvsiol. Zeitschr. (Suppl.) 228 (1904). ') Vgl. S. 518 Zentralbl. — A. Garrod: Journ. (»t f. d. med. Wissensch. 705 (1892). iV^. .ßorrt««; Journ. Pharmac. et Chim. 439 (1845). [7.] 16. 45 (1917). «) M. Weiss: Biochem. Zeitschr. 30. 333 (1910); 81. 342 (1917); 102. 228 (1920) Vgl. u. a. Thudichum: Journ. f. prakt. Chemie. 104. 257 (1864); Journ. of the chem Archibald E. Garrod: soc. 13 (2). 397, 401 (1875); Chem. News. 68. 275 (1893). Proceed. of the Royal Soc. 56. 394 (1894). Ottorino Bocchi: Hofmeisters Beitrag 11. 79 St. Dombrowski: Zeitschr. f. physiol. Chem. 45. 390 (1908). —J. Browinski (1907). und St. Dombrowski : Journ. de physiol. et de path. g6n4rale. 819 (1908). H. Hohlweg: Biochem. Zeitschr. 13. 199 (1908). K. E. Salomonsen: Ebenda. 13. 205 (1908). ') Zoja: Physiol. 17. — — — — — — Blatt- und Blutfarbstoff. Chlorophyll und Hämoglobin usw. 717 Er soll durch Oxydation aus der Vorstufe Urochromogen hervorgehen. Es wird vermutet, daß das Urochrom in Beziehung zum Chlorophyll steht. 1) Wir sehen eine große Fülle von gefärbten Verbindungen vor uns, dem Zellstoffwechsel entstammen und ohne Zweifel im Organis- die alle mus eine ganz bestimmte Rolle spielen. Leider vermögen wir nur mit ganz wenigen Ausnahmen etwas über ihren Bau, ihre Herkunft und ihio Funktion auszusagen. ') Vgl. u. a. H. E. Roaf: Biochem. .). 15. 687 (1921). Sachverzeichnis. (Die Ziffern bedeuten die Seitenzahlen. Abfangeverfahren zum Stu- dium des Zuckerabbaus Albumosen, aussalzbare und Ätiophyllni 703. nicht aussalzbare 351. Aldehyde, Einfluß auf alko- Agar-Agar 56. Agmatin, Vorkommen in Pflanzen 438. Wirkung 525. Akonitin 445. Akrolein 211. 128 ff. — Abomasus 101. Accipenserin 398. Achroglobine 699. Acrylsäure 650. Actinohämatiu 699. Adenase 677. Adenin 645. Abbau 670 ff. Akrose 19. Aktinohämatin 699. Aleuronkristalle 392. Aktivitä.t, optische 19. Alizarin 71, 78. Alkaliseifen 216. Alkaloide 444. Alimentäre Glukosurie 151. stoffe 77, 107. — asymmetrische Synthese Alanin 21, 315. Adenosin 655. Adenosinphosphorsäure 656. Adipocire 295. Adipositas 296. — Abbaustufen 599. — Bildung von Brenztraubensäure aus mittelst 91. — Bedeutunji für die Pflan: 133, 611. — Entstehung von — aus Brenztraubensäure 613. Adonin 70. Adonit 30. — Bildung in Pflanzen 438. — Konfiguration 316. 158, 525. 614, 624 ff. — Beziehung zum Kohlehydratstoffwechsel 156, 158 ff. Glukosurie 158. Nachweis 625. Äpfelsäure, Beziehung zur Milchsäure 132. Quelle für Glukose 208. Äther, Glukosurie 158. — — — Ätherische Öle 463. Ätherschwefelsä.uren 513. im Harn 591. Bildung in Äthylalkohol, der Leber 143. — — Bildung beim Zucker- abbau 125 ff. Äthylaminsulfonsä-ure 598. Äthylbenzol, Verhalten im Organismus 556. Äthylchlorophyllid 701. Äthylenoxyd 243. holische Gärung 132. Aldehydmutase 254. Aldohexosen 16. Aldol 187, 194. Aldosen 16. 35. Akzessorische Nahrungs- — — in Pflanzen 647. — Vorkommen 646. Adonitin 70. Adrenalin 156, Ätiopaphvrin 697. — Quelle für Glukose 205, 208. zen 450 ff. Alkaptonurie 561. 595. Alkohol, Verfettung 294. Alkoholische Gärung 35. 108. AUantoin 587. — in Pflanzen 663. — Bildung 672. 6114,' ß-Alanin 453. Alaninamid 588. Alanyl-glyzin 354. d-Alanyl-d-alanin 361. d-Alanyl-glyzin 392. d-Alanyl-glyzin-anhydrid 357 Alanvl-glyzvl-glyzin 358, 359. Alan vl-glyzvl-leuzyl valin 359." • Alanyl - glyzyl - leuzyl - valylglyzyl-alanyl- glyzyl-leuzyl-glyzyl-1-tyrosin 359. d-Alanyl-ülyzvl-1-tyrosin " 398. ß-Alanyl-histidin 635. Alanyl-leuzin 354. d-Aianyl-1-leuzin 364. Albumine 395. Albumosen 351. Allesfresser 78. Allose, 1- und d- 25. AUosan 585. 590. Alloxantin 590. Alloxyproteinsäure 637. Almen-Nvlandersche Prohe 32. Altrose, 1- und d- 25. Alytes obst'etricans, Eiweißumbau 571. Amandin 396, Ameisensäure 13, 212. — als Abbaupiodukt 460. Amine, Bildung 452, 522. — im Harn 522. Aminoäthylalkohol 241, 462. Aminoaldehyde 462. Aminoameisensäure 313. 576. Aminoazetaldehyd 256, 347. 462. Sachverzeichnis. 720 Aminoazidurie 549, 553. Aminobenzoesäare, Fütterungsversuch mit 578. Aminobernsteinsäure 333. Aminobrenztraubensäure 600. K-Aminobuttersäure 318. — Abbaa 193. y-Aminobattersäure 455, 623. Aminobutylenguanidiii 439. 2-Amino-6, 8-dioxypurin 673. 6-Amino-2, 8-dioxypurin 673. Atninoessigsäure 21, 313. Kupfersalz 313. Aminoglutarsäure 333. — d-Aminoglyzerin 23. 1-Aminoglyzerin 23. Aminoglyzerinsäure 600. Aininoglyzerose 410. Aminogruppen, Bestimmung 374 ff. a-Ämino-ß-dimethyl-propionsäure 322. a-Amino - 5 - guanidino-n-valeriansäure 320. «-Amino-ß-imidazolyl- Propionsäure 330. a-Amino-ß-indol- Propionsäure 323. a- Amino-isobutylessigsäure zu Azeton 192, 193. a-Arainoisovaleriansäure 319. Abbau 192, 595. — a-Amino-n-kapronsäure323. a-Aminokapronsäure, Abbau 193, 615. 5;-Araino-ß-methyl-ß-äthylpropionsäure 322. a-Amino-ß -methyl - Propionsäure 319. a-Amino- ß -oxypropionsäure 316. a-.\mino-ß-p-oxyphenyl-propionsäure 326. 2-Amino-6-oxypurin 646, 648. 6-Amino-2-oxypyrimidiu 650. a-Amino-5- oxyvaleriansäure 332. p-Aminophenol 513. a-Amino-ß-phenylpropionsäure 324. c-Amino-propionsäure 21, 315. — — Aminosäuren 312 ff. Abbau durch Bakterien Amylane 56. Amylchlorid 22. Amylnitrit, Glukosurie 15R, 458. Ämylolytische Fermente 99. der Leber 147. — — Amylopektin 58. Amylose 58. und ß-Amyrin 226. Anaphylaxie 402, 501. Anhalin 444. Anhydro-arabino- galaktosegalakturonsäure 57. Anhydrotrifruktosekomplexe 55. Anoxybiose 139. ff. Darmkanal im 504 ff. — Abbau im Zellstoffwechsel 70. a- Aminosäurechloride 359. Aminosäureester 349. — 451 — Abbau Amygdalin d-Amylalkohol 323. Amylum 57. 6-Aminopurin 646. Abbau 670 ff. Aminopurine 645. Aminosäurealdehyde 346. 572 ff., 593 ff. — Art des Abbaus 593 — Bildung in Pflanzen 405 — Bildung aus Eiweiß ff. ff. durch Pankreas- und Darmsaft 478 ff. Bildung im Zelistoffwechsel 545 ff. im Blute 533 ff. — — — Einteilung 344. — Nachweis 348 374 — Nachweis im Darminhalt 478. — Neubildung 499, 612 — Quellen für Glukose 172, 177, 186, 200,203. — Prüfung ihrer Wertigkeit 499 — Schema des Abbaus 610. — Schema der Bildung 613. — Titration 376. — Übersicht über ihren Abbau 460 — Rolle im Zellstoffwechsel 538 — Zusammenstellung 340 bis 344. — Zusammenstellung der ff., 322. — Beziehung a-Animo-propionsäure, Bildung von Brenztraubensäure aus 133. Quelle für Glukose 205. 1-a-Aminopropionsäure, Beziehung zurß-Oxybuttersäure 191. ß-Aminopropionsäure 634. a-Amino- ß - propyl - Propionsäure 323. ff. ff. ff. Anpassung Kuppelung der Aminogruppen 620. a-Amino-ß-thiopropionsäure 317. a-Aminovaleriansäure, Ab- bau 193. 5-Aminovaleriansäure 454. Ammoniak als Bestandteil von Proteinen 338. Ammoniumkarbonat 574. Ampholyte 312. Blattfarb- Apiin 41. Apiose 41. Arabinose 28, 29. Arabinoside 68. Arabit 30. Arabonsäure 30. Arachinsäure 212, 309. Arachnolysin 304. Arbeit,Einfluß auf Glykogeugehalt der Muskeln 119. Arbutin 68. — methyliertes 68. Arekaidin 446. Arekolin 447. Arginase 320, 572. Arginin 320. Harnstoffbildung aus — 572. — Quelle für Glukose 208. — Vorkommen in Pflanzen 436. ff. ff. des an Strahlenart 83. Anthocyano 71. Antiketogene Körper 190. Antimon, Verfettung 294. Antoxyproteinsäure 637. stoffs — seine Bedeutung 499. — Beziehung zum Kreatin 631. Arsen, Glukosurie 158. Verfettung 294. Artfremdes Fett 277. — Arthritis deformans 565. Aschamin 246. Asparagin 335. — Bedeutung als Nahrungsstoff 511. — Bedeutung für Pflanzen 435 ff. Asparaginsäure 333. Quelle für Glukose 208. — — seine Bedeutung 499. SachverzeicLnis. 21 Asparagyl-diglyzin 355. Benzoylessigsäure 282, 604. Asparagyl-monoglyzin 355. Assimilation von Kohlen- Benzoyl-glukuronsäure 40. Benzoyl-glykokoU 314. säure und Wasser 80 Assimilationsgrcnze für Benzoyl-glyzin 314. ff. Kohlehydrate 151. Assimilationsquotient 85. Assimilatorische Leistung 85. Assimilatorischer Koeffizient 84. Asvm metrische Synthesen " 85 ff., 89. .Asymmetrisches Kohlen" Stoffatom 20. Atherosklerose 306. Atropin 445, 448. Aufzucht, sterile 10(1 ft. 311, 386. Autolyse 464, 547. Azetaldehyd 128. — Vorkommen im Blut und Harn 134. 187. — Beziehung zurAzetessigsäure 194. Azetessigsäure 189. 283. Bildung aus Brenztrau- — bensäure 135. — Abbau 284. Azeton 15, 189, 283. 595. — Glukosurie 158. Azetonkörper 189. Azetonoplasmie 196, 202. Azetonurie 190. Azetophenon 283. Azetyl-benzol 283. Azetylcholin 244. Azetylierung 618 ff. Azetyl-glukosamin 42. Azetyl-propionsäure 33. Azi-nitromethan 415. B. Bacterium thermo 83. Bakterien 505. — denitrifizierende 419. — nitrifizierende 413. — stickstoffbindende 422. — thermophile 426! Bakteroidgewebe 425. Bence- Jonesscher Eiweißkörper 396, 640. Benzaldehyd 129. Benzamid, Verhalten im (Jrganismus 557. Benzoesäure, Bildung 282. Benzol, Abbau 514, 607. Benzoyl-a-aminokapronsäure 616. 557. BrenzschleimsäureglykokoU Benzoylierung 619 ff. Benzylalkohol 459. Benzylazetaldehyd 603. Benzyl-brenztraubensäure, 557. d-Brom-chlor-propan-2-ol 23. d- Aminierung 603, 612. Bernsteinsäure 454. Quelle für Glukose 208. Betain 244. Möglichkeit der Bildung — — 621. Betaine 440 Betonizin 442. Bezoarsteine 236. Biliansäure 231. 316. Bufo agua, Adrenalin 625. Bufotalein 236. Bufotalin 236. Bufotoxin 237. Butter 309. Buttersäure 191, 212,. 253. Buttersäuregärung 35, 108. Butylalkohol 615. Y-Butyrobetain 622, 623. Byssus 399. Bilifuszin 713. Biliprasin 713. Bilipurpurin 713. Bilirubin 708. Bilirubinsäure 710. Biliverdin 712. Biologische Methode 402. Biologisches Gesetz über Zellproteine 382. Cannizzarosche Reaktion 131, 253. Carnaubasäure 309. Carnavtbylalkohol 309. Carnivore 78. Carotin 82. Biosen 16. Biuret 579. Biuretreaktion 384, 579. Cerealose 50. Cerebron 38, 249. Cerebronsäure 249. Blättermagen 101. Blatt farbstoff 699 ff. Blausäure, Vorkommen Pflanzen 412. in Aminosäuregehalt 533 ff Blutfarbstoff 687 ff. Herkunft 706. Blutkörperchen, rote, Zuckergehalt 145. Blutzucker 144. Bohnenstärke 58. Bombicesterin 225. Blut, — Bornesit 43. Böttchersche Probe 32. Brenzkatechin 514. Brenzkatechinschwefelsänre Cerebroside 249. Cerotinsäure 309. Cerotinsäure-cerylester 224. Cerylalkohol 309. Cetin 224. Cetylalkohol 213, 224. Cheiranthin 70. Chemie, Bedeutung für Physiologie 3. Chenocholsäure 236. Chenocholyltaurin 236. Chinaalkaloide 449. asymmetrische Synthese mittelst 91. Chinin 449. asymmetrische Synthese Chinidin, — 514. mittelst 91. Brenztraubensäure 127. aus Alanin 573. als sekundäres Abbauprodukt aus Eiweiß 374. Beziehung zur Glukose — — — 205. — Beziehung zur Harnsäure 582. — kein Glykogenbildner 139. — liefert Alanin 613. Brenztraubensäurealdehyd, Beziehung zur Glukose 206. Abderhalden. Physiologigche Chemie, und l-Brompropionsänre Butterfett 221. ff'. Ausflockung 311. Aussalznng von Proteinen Brenzschleimsäure 557. Brenzschleimsäurealdehyd f. Teil, 6. Anfl. Chinol 606. Chinolin 445, 449. — Nomenklatur 608. Chinovose 30. Chitin 41. Chloral, Glukosurie 158. Chloralhydrat 123. Chlorcruorin 699. Chloroform, Glukosurie 158. Verfettung 294. Chlorophyll 80, 81, 699 ff. Herkunft 704. Chlorophyllase 700. Cldorophyllid 701. — — 46 722 Sachverzeichnis. Chlorophyllformaldehydper- oxyd 86. Chlorophyllkomponente a und b 599. Choladienkarbonsäure 231. Cholan 231. Cholankarbonsäure 232. Cholansäure 232. Cholatrienkarbonsäure 232. Cholecyanin 712. Choleprasin 713. Cholestan 227. ß-Cholestanol 227. Cholestenon 227. Cholgsterin 213, 225 ff. Konstitution 229. Beziehung zur Chol- bzw. Desoxycholsäure 232, — — Cyclosen 43. — seine Bedeutung 553. Cystein 235, 317, 551. Cysteinsänre 235,318, 551. Cystin 317. Bildung in Pflanzen 407. Quelle für Glukose 208. Unentbehrlichkeit 499. Cyklogallipharsäure 213. Cystinsteine 549. Cystinurie 318, 530, 549. Cytidin 655. Cytidin-phosphorsäure 656, — — — — Choletelin 712. steine 269. Darmmazin 3 »7. Bedeutung für Kohlehvd ratverdauung Darmsaft, 103. — Wirkung auf Eiweiß 477 ff. Darmwand, vielleicht dungsstätte Bil- gepaarter Verbindungen 522. Dehydro- cholsäure 231. Dehydro- desoxycholsäure 231. Cholyl-glyzin 234, 617. Cholyl-taurin 235. Delphinidin 71, Delphinin 71. Chondroitinschwefelsäure Denaturierung der Proteine — Konstitution 337. 389. Denitrifizierende Chondromukoid 397. Chondrosamin 43, 73. Chymosin 472 ff. 419 ff. Desaminieruug 601. Desoxycholsäure Ciliansäure 232. Cinchonin 449. Colamin 241. Colchicin 446. Conliydrin 446. a-Coniin 446. . ö-Diketopiperazine 357 5-Dijodtyrosin 327. Diketone, Einfluß auf alkd holische Gärung 132. p-Dimethylaminobetizal2, Bakterien 231, deliyd 711. 233, 617. Dimethylbetain des Prolins 232. 1, Deuteroalbumosen 351. 3, Dextrine 59, 63. in — kristallisierte 59. — Beziehung zum Glykogen 411. 3-Dimethyl-2, 6-dioxypurin 663. 7-Dimethyl-2. 6-dioxypurin 663. Dimethyl-guanid in, asymme- Dextrose 38. trisches 627, 634. 139. propenylamin 245. Azetessigsäure 286. Cuorin 247. Curare, Glukosurie 158. Cyanhydrinsynthese 26. Cyanidin 71. Cyanin 71. Cyanursäure .ö80, 589. Digitonin-cholesterin verbin dang 226, 305. Diglyzeride 218. Diglyzeridphosphorsäurt' ^^'^ Diglyzyl-glyzin 376. Diglyzyl-lysin 356. Diglyzyl-lysyl-glyzin 356. Dihydrocholesterin 227. Desoxycholylglyzin 234, 617. 715. Übergang d-Dibutyrin 23. I-Dibutyrin 23. a, a-Dichlorhydrin 215. Dierucin 218. Digitalisglukoside 70. Digitonin 38. Digitonincholesterid 305. 3, — Beziehung zu Cholesterin Corpus luteum, Farbstoff Ootonsäure, Di-(a-amino-ß-thiopropionsäure) 317. a-5-Diamino-valeriansäure — Darmflora 104. Wirkung auf Aminosäuren 504 ff. -^ Wirkung auf Fettbau- 43, 73, 639. Ab- bau 599. Diastase 50, 99. der Leber 148. Diazeturie 190. Diazobenzolsulfosäure 331. Diazoreaktion 639. Dibenzoyl-a-c-diaminovaleriansäure 561. Dibenzoyl-ornithiü 561. 6, 6-Dibromindigo 626. 676. Cholesterinnachweis 226. — Beziehung zu Cholesterin 232. — Struktur 231. 247. Diamino-propionsäure, Cytosinhexosephosphorsäure 656. Dambonit 44. gung 300. Cliolsäure 230. 324. Diamino-monophosphatide 320. D. — — Organismus 298. — Möglichkeiten der Bildung 621. — Einfluß auf Darmbewe- £-Diamino-n-kapronsäure Diaminurie 549, 554. 303. Cholesterinester 225. Choiin 240. Bildung in Pflanzen 243. Bildung im tierischen a, 657. 303. 301 ff. — kompensatorische 144. Diamine bei Cystinurie 549. Cytosin 650. — Einfluß auf Hämolyse Cholesterinstoffwechsel Dialursäure 583. Dialyse 311. Dextroseabbau 123 ff. Diabetes melitus 178 ff. Lipoplasmie bei 275. — oxyprolin 442. 1, 7-Dimetliyl-xanthin 675. 6()3. Diäthyl-methylsulfiniumbase 3-7-Dimethyl-xanthin 663. Di -ß- naphtalin-sulfo-1 vrosin 592. Diäthvlsulfid Dinukleotid 657. 371. .52.") yachverzeichnis. Dioleostearin 217. Diosen 16. 78. 1, 2-Dioxyanthrachinon Dioxyazetoa 130. 4-Dioxybenzoesäure 514. ;}, 1, 2-Dioxybenzol 514. 1, 3-Dioxybenzol 32. 1, 4-Dioxybenzol 514. Dioxybsnzole 513. 5-diaminopy4, 6-Dioxy-2, rimidin 665. Dioxyfettsäuren 213. 3, 4-Dioxy-phenylalanin 325. 1, 4-Dioxy-phenyl-3-essigsäure 562, 595. 3, 4-Dioxyphenyl-methyl- aminäthanol 624. 2, ff. 6-Dioxypurin, Abbau 670 ff. 2, Eiweißstoffe, Definition 312. — Eigenschaften 383 — Einteilung 401. — Fällungsreaktionen 386. — Farbreaktionen 384. — kristallisierte 390. — Molekulargewicht 3J2 — Struktur 384. — vergleichende Hydrolyse ff. 3134, ff. von 492 ff. Eiweißstoffwechsel der Pflanzen 434 Eiweißsynthese im Tierkörper 494 ff. Ei weiß Verdauung 464 ff. Elastin 399. — Gehalt an Animosäuren , 6-Dioxypurin 647. — Abbau 670 6Dioxypyrimidin 650. Dioxystearinsäure 213. 493. Emulsin, asymmetrische Synthese mittels 91. Emulsion 210. Bedeutung für Verdau- — ung 259. Dipentosamin 65. Diphenyl-azetyl-ornithin 618. Disaccharide 46 ff. Disulfide, Einfluß auf alkoholische Gärung 132. Dinraminopropionsäure 589. Döglingsäure 212. Drei kohlenstoffverbi n düngen, Bedeutung für Zellstoffwechsel 194. Ductus thoracicus, Bahn für Pankreasinkret? 175. Dulcit 36. E. Ecgonin 449. Echinochrom 699. Ecksche P'istel 526. Edestin, Molekulargewicht 393. Edestine 396. Ehrlichsche Reaktion 712. Eichhörnchen, Oxyhämoglobin 688. •iH Enterokinase 477. d-Epibromhydrin 23. l-Epiclilor!iydrin 23. Epigaanin 675. d-Epihydrinalkohol 23. 1-Epihydrinalkohol 23. Epizuckersäure 651. Erepsin 482. in den Zellen 590. Ergosterin 226. Ergothionein 411. Eierglobuiin 395. Eisensalze, als Katalysatoren 87. Eiweißabbau im Darmkanal Bedeutung des 477 ff. Eiweißneubildung 569 ff. Eiweißphosphate 394. Eiweißstoffe 310 ff, — Aussalzmetiiode zu ilirer Darstellung 311, 3S6 ff. Bildung in Pflanzen 405ff. — Stoffwechselendpro- dukte 281. Fettbildung aus Aminosäuren ? 291 ff. aus Zucker 117, 209. — Fettdepots 220. Beziehungen zu anderen Nahrungsstoffen 276 ff Fette, — Bildung in Pflanzen 252 — Löslichkeit 210. — optisch-aktive 217. — Resorbierbarkeit verschiedener 266. — Resorptionsweg 265. — Beziehung zu den Azeff. ton körpern 195. Fettgewebe 220. Fettnachweis, Methodik 294. Fettresorption 263. Fettsäure-äthylester 266. glykolester 266. Fettsäuren, Abbau 191, 282. gesättigte 212. mit verzweigter Kohlenstoff l^ette, Abbau 287. ungesättigte 212. — — — — — ungesättigte, Bildung 285. — aus Aminosäru-en 454 — Fettstoffe Erstarrungspunkt Fettsucht 296. Fettsynthese 214 ff. in der Darm wand einiger Fettarten 220. Erukasäure 212, 277. Erythrit 14. Erythritsäure 14. Erythrose 14. Essigsäure 131, 212. aus GlykokoU 454. Essigsäurecholinester 301 — 210 ff. — Fettverdauung 258 2(i3. ff'. Fettzellen 220. Fibrin 396. — Gehalt an Aminosäuren 493. Fibrinogen 396. Fistel, Anlegung einer 97. Flavone 71. Estermethode 349. Fieisciifresser 78. Euphorbon 226. Fluoi'eszierende Stoffe 88. Exzelsin 396. Formaldehyd 13, 16, 18,31. — erstes Assimilationsprodukt 89. Eieralbumin 395. — kristallisiertes 388. — Molekulargewicht 393. Fett, Fadenpilze, stickstoffbin- dende 423. Farbreaktionen auf Zucker 31. Fäulnisbakterien 457. Fehlingsche Probe 32. Fellinsäure 235. Fessolungs- Glukosurie 158. Fett, freies und gebundenes Formhydroxiimsäure 415. Formoltitration 376. Formylhydroperoxyd 86. Fruchtzucker 17, 37. Fruktose 17, 37. — Glukosebildner 207. Fruktosediphosphorsäure 125. 278 ff. Fraktosurie 37. leiter 220, 280. Fukose 30. Fumarsäure, Quelle für Gin- — als sclilecliter Wärme- — Forma Idehydperoxyd 86. Formamid 415. als Reservestoff 219. kose 208. 46* Sachverzeichnis. 7-24 Fungisterin 226. Furanaldehyd 31. Furfurakrylsäure 288, 619. Glukolytische Fermente 122. Furfurol 31. Verhalten — im Organis- mus 288, 557, 619. Fuselöl 458. Fuszin 714. Glukonsäure 28, 36. Glukoproteide 395. Glukosamin 41 ff., 336, 411. Beziehung zu Pyrrol 662. — G. Gadoleinsäure 212. Gänsefett 221. Gärung des Zuckers 35. Gärungsmilchsäure 35. Galaktane 38, 56. Gaiaktid 36. Galaktonsäure 36. Galaktose 36, 38. Baustein des Zerebrons — 249. — Bausteine von Phospha- tiden 248. d-Galaktose, Glukosebildner 207. 1- und d-Galaktose 25. Galaktoside 68. Galaktosurie 38. Galakturonsäure 56. Galle, Aktivator des Lipase- — Glukal 26, 652. Glukit 36. Glukolyse 122. zymogens 261. Bedeutung für Fett- resorption 268. löst Seifen 268. Gallenfarbstoff 708 ff. Menge pro Tag 707. Gallenmuzin 397. — — Gallensäuren 230 ff. Aktivatoren des Lipase- — zymogens 261. Gallensteine 227, 713. Gans, Oxyhämoglobin 688. Gaultherin 70. Geburtshelferkröte, umbau 571. Gelatine 399. Nährwert 500. Gentiano'se 52. Eiwreiß- im — a-Glukose 67. ß-Glukose 67. 1- und d-Glukose 24. Glukoside 66 ff. — a- 66. — ß- 66. Glukuron 39. Glukuronsäure 27, 38. — Frage, ob Ai)baustufe der Glukose 124. Glukiironsäurepaarlinge 39, 122. Glutamin 335. Bedeutung als Nahrungs- — stoff 511. — Bedeutung für Pflanzen 435. Glutaminsäure 333. Quelle für Glukose 208. seine Bedeutung 499. — — Gluteline 396. Glutenin 396. Glutin 399. Glykocholsäure 230, 617. Glyko-desoxycholsäure 230, 617. Glykogen 62. — Abbau 63. — Bedeutung als Reserve— Harn 591. Gesetz der Erhaltung der Energie 75. der Erhaltung des Stoffes — 75. — biologisches 382. Gicht 682. Gliadin 396. Globin 399, 690. — Gehalt an Aminosäuren 493. Globulin, Molekulargewicht 393. Globuline 396. — 207. Glykolaldehyd 14. Glykogenbildner 139. Quelle für Glukose 207. Glykolaldehyddikarbonsäure, Glykogenbildner — — 139. Glykolliarnstoff 663. Glykolose 14, 244. Glykolsäure 14. aus Betain 440. kein Glykogenbildner — — 139. Glukosurie 151. Glukothionsäure 72. kohlehydrat 114 ff. Glykogenbildner 139. Glykokoll 21, 313 ff Kupfersalz 313. — Gesamtschwefel Glukosaminsäure 411. Glukosazon 33. Glukose 36, 38. Abbau 120 ff. Glykokollesterchlorhydrat 314. Glykokollkarbonsäure 346. Glykol 14, 244. Glykogenbildner 139, — seine Bedeutung 499, 559. — Möglichkeiten seiner Bildung 615. — -Neutralsalzverbindungen 314. — Quelle für Glukose 208. — Verwendung zu Synthesen 617 — Vorkommen im Harn ff. 540. — Vorkommen im Blut 533. — Baustein von Gallensäuven 560. — Beziehung zu Betain 245. Glykolyldiharnstoff 673. Glykozyamin 631, 632. Glyoxalase 134, 206. Glyoxyldiureid 672. Glyoxylsäure 285, 615. kein Glykogenbildner 139. Glyoxylsäureprobe 328. Glyzeride 214. Glyzerin 14. Abbau 281. Baustein von Fetten 211. — — — — Beziehung zur Glukose 205, 207, 208. — Bildung bei der alkoholischen Gärung 128. — Bildung in Pflanzen 252. Glyzerinaldehyd 600. Bildung beim Glukose- — abbau 130, 133. — Glykogenbildner 139. — Quelle für Glukose? 207. Glyzerinsäure 14. — Beziehung zur Harnsäure 582. — aus Diaminopropionsäure 599, 600. — Glykogenbildner 139. — Beziehung zur Glukose 207. Glyzerose 14, 19. — Beziehung zu Glyzerin 252. Glyzerylphosphorsäure 240. a- und ß-Glyzerylphosphorsäure 242. — optisch-aktive 242. Glyzin 313. Glyzinamine 398. Glyzinanhydrid 357. Glyzinin 396. Glyzyl-alanin 354, 357. Glyzyl-d-alanin 363. Glyzyl-alanin-anhydrid 357. Sachverzeichnis. Glyzyl-d-alanin-anhydrid 363. Glyzyl-d-alanyl-glyzyl-1-tyrosin 370. ülyzyl-alanyl-seryl-leuzyltyrosin 355. Glyzyl-asparaginsäure 355. Glyzyl-asparagyl-diglyzin 355. Glyzyl-glyzin 353, 357. Oxydation 639. Glyzyl-Meuzin 364. Glyzyl - 1 - leuzin - anhydrid 864. Glyzyl-1-phenyIalanin 364. Glyzyl-1-prolin-anhydrid 364. Glyzyl-serinester 380. Glyzyl- 1-tyrosin 364. Glyzyl-1-tvrosin-anhydrid 364. Glyzyl-d-valin-anhydrid 364. Gmeiinsche Reaktion 712. Gorgonin 398. Guanase 677. Guanidin 320, 714. — — Vorkommen in Pflanzen 436. y-Guanidinobuttersäure 631 Guanidinoessigsäure 631. Gnanin 645. Abbau 670 ff. — — Vorkommen 647. Guaningicht 677. Guaninhexosid 656. Guaninurie 677. Guano 428. Guanosin 654, 664. Guanosin-adenosin-phosphorsäure 657. Gnanosinphosphorsäure 656. Guanosyl- adenosyl - zytidyltriphosphorsäure 657. Guanylsäure 657. Gulose, 1- und d-, 24. Gummi, tierischer 56, 65. Gummisubstanzen 56. H. Haarmelanine 714. Hämatin 690 ff. Hämatinsäure 697, 710. Hämatogen 708. Hämatoporphyrin 694. Hämerythrin 699. Hilmin 692. Häminprobe 692. Hämochromogen 690 tf. Hämozyanin 698. Hämoglobin 390,687 ff. 725 Hämoglobin, Herkunft 704 ff. Verhalten im Magen 706. — Hippursäure im Harn 558. Histamin 438, 527. Hämokonien 273. Histidin 330. Hämolyse 304. Hämolysin 304. Hämoporphyrin 696. — Bausteine — Abbaustufen 611, 632. des Karno- sins 634, 635. — Quelle für Glukose Haferstärke 58. Halogenbenz,ol, Verfütterung 618. Hammeltalg 221. Ablagerung 277. Harn, Nukleinsäure des 659. Harnfarbstoffe 716. Harnglobuline 396. Harnmukoid 397. — Harnsäure, Bildung 671 ff. Eigenschaften 685. Laktani- und Laktimform 684. Histohä-T atine 699. Histol 458. Harnsäure aus Aminosäuren 580 ff. aus Purinbasen 646 ff. Histone 397. Hitzekoagulation 311. Homoazide Fette 222. — — — Salze 682. Histon aus Thymusdrüse, Gehalt an Aminosäuren 493. — — Bildungsmöglichkeiten 679 — Synthese 589. ff. Harnsäuregicht 682. Harnsteine 685. Harnstoff aus Aminosäuren 567 ff Möglichkeiten der Bildungsweise 567 ff. — — Salze 580. Hautmelanin 714. ? 208. — Quelle für Harnsäure? 581. — Quelle für Azetessigsäure 611. — Quelle für Kreatin? 632. — Quelle für Urokaninsäure 634. — seine Bedeutung 499. * Homogentisinsäure 562, 595. Bildungsalt 595 ff., 604 ff. Hordenin 396, 443. — Huhn, Oxyhämoglobin 688. Hühnerfett 221. Huminsubstanzen 33, 65. 338. Hund, Oxyhämoglobin 688. Hundefett 221. Hunger, Eiweißumbau 616. — -Glukosurie 164. — Lipoplasmie bei 275^ Hefenukleinsäure 657 ff. Hemizellulosen 56. Hepatogene Glukosurie 155. Heptosen 16. Heptylsäure 212. Herbivore 78. Hydantoin 346, 663. Hydrakrylsäure, Beziehung zur Harnsäure 582. Hydrazone 33. Hyalomukoid 397. Heteroalbumosen 351. Hydrobilirubin 713. Heteroazide Fette 222. Heteroproteinoplasmie 641. Heteroxanthin 675. Hydrochinon 68, 514. Hydrochinonbrenztrauben- Hexaamylose 59, 99. Hvdrochinonessigsäure 562, Hexamethylornithin 623. Hexaoxy-hexahy d robenzol säure 606. " 595, 606. Hydrokephalin 247. Hexonbasen 366. Hydrolezithin 247. Hydrouracil 650. Hexosediphosphorsiiure 74. Hexosediphosphorsäureester Hydroxyconiin 446. Hyocholsäure 235. 44. Hexoside 68. Hyoscyamin 448. Hypaphorin 441. Hvperaminoazidoplasmie Hilfsdisziplinen der Physiologie 2 ff. Hippokoprosterin 230, 270. 544. Hypercholesterinoplasniie 306, 713. Hippomelanin 714. Hippursäure 282, 314. 353, 555 ff, 604. Hyperglukoplasmie 151. Hyperglyzinoplasmie 541. Hvperproteinoplasmie 544, 74. Hexosen 16 — -Bildung, Ort der 559. — Svnthese 79. " " 641. Hyperurikoplasmie 683. Sachverzeichnis. 726 Hypoaminoazidoplasmie 544. Hypophyse, Einfluß auf Fettstofi'wechsel 2:'97. — Beziehung zum Kohlehydratstoffwechsel 141, 16S, 183. — Bildung von Stoffen, die dem ß-Imidazolyläthylamin nahe stehen 523, 539, 626. Hypoproteinoplasmie 651. Hypothesen 4. Hypoxanthin 647. Abbau 670 ff. Bildung aus Adenin 670. — — — Vorkommen 649. Hypoxanthosin 652, 655. Indolessigsäure 457, 518, 520, 523, 524, 598. Pr-3-Iiidolkarbonsäure 517. Indol-Pr-S-essigsäure 518. Indolkern, Bezeichnung 517. I-Indolmilchsjiure 459. Indolpropionsäure 494, 457. Iiidoxyl 71, 557. Indoxylglukuronsäure 40, 516. Indoxylschvrefelsäure 516. Inkrete 161. Inosin 653, 655. Inosinsäure 652. Struktur 652. Idose, und d-, 24. 1- Ikterus 508, 713. Imidazolyläthylalkohol 458. Imidazolyläthylamin 438, 522. — in der Darmschleimhaut 522. — Vorkommen in Pflanzen 438. — Wirkung 525. ß-lmidazolyl-akrylsäure 635. ß-Imidazolyl-azetaldehyd '611. ß-Imidazol yl- bren ztrauben säure 611. Imidazolyl-essigsäure 598. Imidazolvl-propionsäure " 454. Indigo 71, 437, 516. Indigoblau 516 ff. Indijiorot 517. Indigotin 71. Indikan 71, 437. Indirubin 517. Indol 457, 521. im Harn 509. — — Vorkommen in Pflanzen 487. Indol-äthyl-nlkoliol 458, 598. Inuol-äthyl-amin 452, 523, 598. Quelle von liidole^sigsäure 598. — — Wirkung 525. Indol-azetaldehyd 598. liidolazetursäure 523. Indolazetyl-glyzin 523. 1 ndoi-brenztraubensäure 609. — bei Cystinurie 549. 599. — in Harn 547. — Vorkommen in Pflanzen Inosit 43. Inosit-dimethylester 44. Kalziumcyanamid 430. Inosithexaphosphorsäure- Kalziumion, Antagonist des Inositmethylester 44. Ichthuline 400. Idäin 71. K. Kadaverin 324, 438, 522. 438. Kaffein 662. Kalkseife 217. — ester 44. I. Jodgoriiosiiure 327. Jodreaktion 58. Jodzahl 221. Juglansin 396. Inositpentapho jphorsL'; ureester 44. Intraperitoneale Zutiihr 96 Intravasculäre Zufuhr 96. Inulin 55. Inversion 48. Invertin 103. Natriumions 157. Kalziumkarbid 430. Kaprin:=äure 212, 309. Kapronsäure 212, 309. — Abbau 191. Kaprylsäure 212, 309. Karamel 33. Karbaminsäure Invertzucker 48. Isoamylalkohol 323, 458. Isoamylamin, Vorkommen in Pflanzen 439. Karbaminsaures Isobiliansäure 231. Isobuttersäure, Abbau 287. ; liefert kein Azeton 595. Isobutylalkohol 458. Quelle für Glukose 207. Isobutylamin 452. Isobutylessigsäure aus Leuzin 454. Isochinolin 445, 449. Iso-chlorophyllin 702. Isocholansäure 232. IsoCholesterin 225, 230, 309. Isohämopyrrol 697. Isolaktose 47. Isoleuzin 321. Quelle für Glukose? 208. Synthese 323. Isoleuzyl-valinanhydrid 364. Isomaltose 47. Karbide 430. — — — — Isophonopyrrol-karbonsäure 698, 711. Isopropylalkohol 15. Isorhodeose 30. Isovaleraldehyd 323. Isovaleriansäure, Abbau zu Azeton 193, 641. - aus Valin 454. J. Jekorin 73, 246. Jekorinsäure 212. 313, 435. 620. Ammon 574, 577. Karbamino-bernstein säure 346. Karboligase 129, 255. Karbonate, Rolle als Reguder Reaktion latoren 196. Karbonylformel des Zuckers 24. Karbonyldiharnstoff 587. Karboxylase 127. Karmin äure 716. Karnaubasäure 212, 309. Karnaubylalkohol 213. Karnitin 453. Karnosin (534. Karotin 82, 699. Kartoffelstärke 58. Kasein 473. — Gehalt an Aminosäuren 493. Kaseinogen 473. Kastanienstärke 58. Kastration, Einfluß auf Fettstoffwechsel 297. Katalysator 87. Katze, Oxyhämoglobin 688. Kauakt 97. Keimfreie Aufzucht Kephalin 246. Kephalinsäure 246. Kerasin 249, 250. Kerasinsäure 268. 106 ff. Sachverzeichnis. Keratin, Gehalt an säui-en 493. Keratine 399. Amino- i-Ketö-n-buttersäure liefert a-Amino-n-buttersäure 613. Ketoglntarsäure 455. Ketohexüse 17. a-Keto-n-kapronsäure liefert Konfiguration, Bedeutung für Durchlässigkeit der Niere 165. Konglutin 396. Konvizin 664. Koprosterin 225, 229, 270. Kork säure 237. Korneamukoid 397. Korylin 396. a - Amino-n-kapronsäure Kotporphyrin 613. Krappfarbstoff 71, 78. Kreatin 628. Ketone, Einfluß auf alkoholische Gärung 132. Ketosen 16, 35. 27 09.'). 85. — Abbau 633. — Herkunft 629 — Möglichkeiten der Bilff. Kleister 58. Klupein 398. dung 631 ff. Knoblauchkröte, keimfreie Aufzucht 107. Knöilchenbakterien 423. Knoopsche Regel 282. Koagulation 336. Koagulosen 472. Kobragift, Hämolvse 304. Kochenillefarbstoff 716. Kochsalzglukosnrie 157. Körperfett, Abhängigkeit von Nahrungsfett 277. Koeffizient, assimilator. 91. Koferment 126. Kohlehydratabbau 121 ff. — Überblick 137. Kohlehydrate 12 — stickstoffhaltige 41. — Struktur 13 Kreatinin 628, 664. Kreislauf des Stickstoffs 422. Kreislauf von Stoff- und Energie 92 ff'. Kresol 456, 555, p-Kresol 520. — im Harn 509. glakuronsäure 512. — Synthese von kohlenstoffreicheren 26. — Abbau in den Geweben 121. — Verhalten im Organismus ff. Kohlehydratnachweis 32. Kohlenlager 92. Kohlenoxydhämoglobin 688. Kohlenoxydvergiftung, Glukosurie 158. Kohlensäure, Glukosurie 158. Kohlensäureverbindung des Chlorophylls 85. Kohlenstoffatom, asymmetrisches 20. Koilin 399. Kokain 445, 448 Kollagen 399. Kolloide 311. Koma diabeticum 197. Konchiolin 399. Konfiguration der Zucker 24, 25. Lenkosin 395. Leukozyten, Rolle beim Abbau von Purinbasen 681. — Rolle bei Fettresorption 262. Leuzin 322. — Abbau zü Azeton 213, 595. — Qaelle für Glukose? 208. — Synthese 323. I-Leazinimid 374. Leuzyl-alanin 355. Krotonsäure 286. Kryptopyrrol 698, 711. Kynurensäure 608 Kyrine 352. 1-Leuzyl-d-alanin 364. Leuzyl-alanyl-glyzyl-glyzin 359. 1-Leuzyl-d-glataminsäure L. 364. 1-Leuzyl-glyzin 364. 1-Leuzyl-glyzyl-d-alanin A bbau durch Fermente 369. I-Leuzyl- trigly zyl - 1 - leuzy 1triglyzyl - 1 - leuzyl - oktaglyzyl-glyzin 381. , Labferment 472 ff. Labmagen 101. Lachs, Eiweißumbau 569 ff'. Lävulin säure 33, 651, 656. Lävulose 37. Verwertung beim Diabe- — 1- Leuzyl - triglyzyl - 1 - leuzyltriglyzyl - 1 - leuzyl-trigly- zyl-l-leuzyl-penta-glyzvlglyzin 381. tiker 187. 95. — Transport im Organismus 111 — Umbildung in Fett 117. Lentoglobulin 396. Leuchtbakterien 84. licukämie 682. schwefelsaure 512. Kropf, Funktion 108. ff. ff. Leber, Verhalten bei Pankreasexstirpation 170. Leberatrophie, akute, gelbe 5il, 601. Legumelin 395. Legumin 396. Leichenwachs 295. Leim 399. Leimsüß 313. Leistung, assimilatorische Laktase 103. Laktazidogen 125. Laktobiose 49. Laktoglobulin 396. Laktose 49. Langerhanssche Inseln 176. Lanolin 225, 308. Resorbierbarkeitdes267. — Lanopalminsäure 309. Lanozerinsäure 213, 309. Laurinsäure 212. lieber, Bildungsort des Harn- säure aus Aminosäuren 581. Bildungsstätte der Azetonkörper 195, 397. Bildungsstätte der ge- paarten 1-Leuzyl-d-valinanhydrid 374. Lezithide 246. Lezithin 238 ff". Bildung in Pflanzen 255. Verdauung 270. Lezithinsynthese im tierischen Organismus 298 — — ff". Lichtchemische Wirkungen 87. stoffs 574. — Bildungsort der Harn- - Leuzvl-tvrosin-anhydrid 357." Verbindungen 522. — Gehalt an Glykogen 115 ff. Liebermann-Burchards Reaktion 226. Lignin 62. Lignozerinsäure 247, 24Q. Linolsäure 212. Lipase 218, 258. Lipasezymogen 260. Lipochrome 715. Lipoide 307. Sachverzeichnis. 728 Lipolytische Fermente 218. Melanosarkome 714. Lithobilinsäure 236. Lithocholsäure 230, 234. Lithofellinsäure 236. Lösungsmittel für Fette 210. Lupeol 226. Melibiose 60. Melitriose 52. Lupeose 52. Methylpentosen 30. 4-Methylpyridin 622. Methylpyridylammoiiiuiii- Melanoidine 388. Lipoplasmie 275. Menschen fett 221. Mei'kaptursäuren 554, 618. Mesobilirubin 710. Mesobilirubinogen 710. Mesohämatin 697. Mesohämin 697. Luteiii 715. Lycoperdin 43. Lycoperdon 43. Lysin 324. Quelle für Glukose? 208. — — seine Bedeutung 499. Mesoxalsäure 585. Beziehung zurHarnsäure — 582. Lysozithin 304. Lysyl-glyzin 356. Mesoxalylharnstoff 585. Mesoporphyrin 695, 710. Mesoporphyrinogen 697. d-Lyxohexosamin 336. Methämoglobin 689. M. 101. 468 ff. Magenverdauung des Eiweißes, Bedeutung 472. — der Fette 259. — der Proteine 465 fi'. Magnesiumgehalt des Chlo- Milchgerinnung 472 697, 710. d-Methyl-äthyl-propionsäure 454. 3-Methyl-4-äthylpyrrol 698. Methylbetain der Nikotinsäure 442. a-Methyl-chinolin 610. Methylconiin 446. N-Methylconiin 446. Maltobiose 50. Maltose 50. l-Methyl-3,5-dioxybenzol31 5-Methyl-2, 6-dioxypryimidin 650. Methylfarfurol 31. a-Methyl-d-glukosid 67. ß-Methyl-d-glukosid 67. Methylgly kokoll 629. Methylglyoxal, Bildung beim Malzzucker 50. Mandelsäure 90. — Beziehung zur Glukose rophylls 82, 701. Maisglutenin 396. Malleinsänre 698. Malonsäure, Beziehung zur Harnsäure 582. Glukoseabbau 130, 134. Mandelsäurenitril 90. Mandelnitrilglukosid 70. d-Mandelnitril-ß-glukosid71 l-Mandelnitril-ß-glukosid71. Mannane 56. Mannit 36. Mannogalaktane 56. d-Mannoheptid 16. d-Mannoketoheptose Mannosäure 36. — Beziehung • Mannose 36. d- und 1-Mannose 24. Mannozuckersäure 36. Mazerationssaft 54G. Meerschweinchen, Oxyiiiimoglobin 788. Meibomsche Drüsen, fetthaltiges Sekret 309. — 259. ft'. Milchglobulin 395. Milchsäure aus Alanin 599. Beziehung zur Äpfelsäure 132. Beziehung zur Gluko.se — — 206. — Beziehung zur Harn- säure 582. — Spaltung bei BeliohtunL!: 87. — liefert Alanin 613. d-Milchsäure, Glykogenbildner 139, 207. aus Traubenzucker 125, — 130. Milchsäuregärung 35, 108. Milchzucker 49. Millons Reagens 326. Milz, Beziehung zum Kohlehydratstottwechsel 163. Molisch-Udränskvsche Re- aktion 32. 206. zur Harn- säure 584. Methylguanidin 634. a-Methyl-guanidinoessigsäure 629 ff. 7-Methyl-guanin 676. 16. Mikrochemie 136. Milchalbumin 395. Pflanzen 439. Methyl-äthylmaleinimid 697, Magensaft, — Wirkung auf Eiweiß tinsäure-methylester446. Methyltyrosin 443. 5-Methyl-urazil 650. 1-Methylxanthin 675. 3-Methylxanthin 675. 7-Methylxanthin 675. Mezcalin 444. Milchdrüse, Fett 309. Milchfett 295. Verhalten bei Emulsion Methyl-äthylmaleinsäure Bedeutung für Kohlehydratverdauung N- Methyl - tetra - hydroniko- Methylalkohol 13, 700. Methylamin, Vorkommen in 710. Magenmuzin 397. hydroxyd 621. N-Methyl-tetra-hydronikiptinsäui'e 446. Methylhydantoin 633. Methylhydantoinsäure 633. Methyl-hydrochinon 513. Methylierung von Aminosäure 620 ff. Methylimidazol 137, 330, 584. Pr-3-Methylindül 517. Mekonium 505. ß-Methylindol, Vorkommen in Pflanzen 437. Methylmerkaptan 525. Methylornithin 324. Melanine 404, 714. Methylpentosane 55. Monoamino- di - phosphatide 247. Monoamino-nionophosphatide 247. Monoazetylglukosamin 42. d-Monobromhydrin 23 d-Monobutyrin 23. 1-Monobutyrin 23. a-Monochlorhydrin 214. 1-Monochlorhydrin 23. Monoglyzeride 218. Monoglyzyllysin 356. Monolezithide 242. Monomethylguanidin im Harn 627. Mononaplitalinsulfotyrosin 370. Mononatriumurat 686. Mononitrotyrosin 326. Sachverzeichnis. 729 Mononukleotide 654. Mono-oxyfettsäuren 212. ß-Naphtalinaulfo-tyrosylglyzyl-alanin 372. Monopalmityllezithin 304. Naphtoesäuren Verhalten im Organismus 556. Monosen 16. a-Monostearinsäureglyzerinester 214. Mooresche Probe 32. Morphium, Glukosurie 158. Mucoitin 73. Mucoitinschwefelsäure 42, 78. — Konstitution 337. Mukoide 397. Mukonsäure 607. Murexidprobe 590. , Naphtoresorcin 41. «-Naphtol 41. Reaktion 339. Muskelarbeit, Beziehung zum Kohlehydratstoffwechsel 115, 118, 152. Muskeln, Glykogenspeicher 117. Muskelzucker 43. Müzine 397. Nuklein 644. — Verhalten gegen Glukosurie 157. Nebenniere, Adrenalin 158, 624. — Beziehung zum Cholesterinstoffwechsel 302. — Beziehung zum Kohlehydratstoffwechsel 158 ff., 183. 141, — Beziehung zur Pankreasdrüse 171. Nebenschilddrüsen Beziehung zum Kohlehydrat, stoffwechsel 163. Nukleinazidase 665. Nukleinsäuren 643 ff. Bausteine 645 fi'. Bildung in Pflanzen 661 ff als Glukoside 652. — — — — Schema des Abbaues 668. — Verdauung 666 Nukleoproteide 399, 643 — Aufbau 665 — Verhalten gegen Magensaft 644, 665. — Verdauung 665 ff'. ff. ff. ff". Nukleosidasen 668. Nukleoside 652. — in Pflanzen 664. Neottin 248. Nervi splanchnici, Beziehung Nukleotidasen 668. Nukleotide 654. zum Zuckerzentrum 154. Nervus vagus, Beziehung zum Zuckerzentrum 154. Nutramine 77, 107. Myogen 396. Myokynin 623. Myosin 396. Netzmagen 100. Myricylalkohol 213. Myristinsäure 212, 309. ot-Myristinsäureester des Neurin 245. Neurokeratin 399. Myeline 247. Myochrom 699. Neutraler Schwefel im N. Nahrungsfett, Einfluß auf Körperfett 277. ß-Naphtalinsulfo-alanyl-glyzin 366. ß-Naphtalinsulfo-alanyl-glyzyl-ty rosin 371. ß-Naphtalinsulfo-alanyl-tyrosyl-glyzin 371. ß-Naphtalinsulfoglyzin 365. ß-Naphtalinsulfo-glyzyl - alanin 365. ß-Naphtalinsulfo-glyzyl - alanyl-leuzyl-tyrosin 365. ß-Naphtalinsulfo-glyzyl- alanyl-tyrosin 371. ß-Naphtalinsulfo-glyzyl-tyrosyl-alanin 371. ß-Naphtalinsulfo-tyrosylalanyl-glyzin 372. 0. Neutralfett 221. Nichtkolloide 311. Obesitas 296. Ochronose 565. Öle 219. Ölsäure 212. Niere, Ort der Hippursäure- Önidin 71. bildung 559 ff. Verhalten bei Phlorhizinzufuhr 200. Retentionsvermögen für Glukose 164. Nikotin 442. Nikotin säure 442. Verhalten im Organis- Önin 71. Ohrschmalzdrüsen, fetthaltiges Sekret 309. Harn 591. a-Monochlorhydrins 215. a-Myristinsäure-ß-stearinsäureester des a-Monochlorhydrins 215. a-Myristin-ß-stearin 215. Myrtillidin 71. Myrtillin 71. Mytilit 45. Paii- kreassaft 665. Verdauung 665 ff. Naphtursäure 556. Natriumion, Beziehung zur Muscarin 246. Norleuzin 323. — — — mus 622. Ninhydrin 314. Nitride 430. Nitrierung von Proteinen 376. Nitrifikation 413. Nitrifizierende Bakterien 413. Nitrobenzaldehyd, Verhalten im Organismus 556. Nitrobenzoesäure 556. Nitrobenzol, Glukosurie 158. Nitrobenzylalkohol 123. Nitrohippursäure 556. o-Nitrophenol 513. Nitrosomethylalkohol 415. Nitrosylkalium 415. Nitrotoluol 123. Oktaazetyl-zellobiose 61 Oktadezylalkohol 213. 224. Oktylsäure 212. Oleo-palmito-butyrin 218. Omasus 101. Omnivore 78. Onuphin 398. Optische Aktivität 19. — Methode 370. Ornithin 320. 561. Quelle für Glukose 208. Ornithursäure 561. Orthostatische Proteinurie — 640. Oryzenin 396. Orzin 31, 41, 513. Osazone 33. Osseomukoid 397. Ovarialkystome (Müzine) 397. Ovokeratin 399. Ovomukoid 397. Ovomuzin 397. Oxalsäure 14. 586. 7 HO Sachverzeichnis. Oxalursäure 586. Oxalyl-aminoessigsäure 638. Oxalylharnstoff 586. Oxamid 415. Oxyäthylamin 241, 246, 298, 462. m-Oxybenzoesäure 513. p-Oxybenzoesäure 457, 515. ß-Oxybuttersäure 189. Entstehung aus Fettsäure 282 ff. Oxybutyrobetain 623. ß- Oxy-chinolin - a - karbon- Oxyphenylmilchsäure, Quelle von Homogentin säure 595. d-p-Oxyphenylmilchsäure 495, 600. 1-p-Oxyphenylmilchsäure 599. p-Oxyphenylpropionsäure 68, 199, 454, — säure 608. Oxycholesterine 230, 309. ß-Oxydation der Fettsäuren 456, 515, 520. Oxyprolin 332. Oxyprolinbetain 442. Oxyproteinsäuren 637. Oxypurine 647. 6-Oxypurin 647. Oxydoformel der Zucker 24. Y-Oxy-pyrrolidin-«-karbonsäure 332. Oxystachydrine 476. Oxyfettsäuren 212. Oxytryptophan 329. 191, 282. Oxyglutaminsäure 334. Oxyhämocyanin 699. Oxyhämoglobin 687 ff. — Zusammensetzung 688. säure 648. p-Oxypheny läthy lal koho 1 458, 524, 598. p-Oxyphenyläthylamin 327, 438, 443, 597, 598. — bei Cephalopoden 599, 626. — im Käse 527. — Vorkommen in Pflanzen 438. — Wirkung 525. p-Oxyphenylazetaldehyd 598. ni - Oxyphenylbrenztrauben- säure 601. p-Oxyphenylbrenztraubensäare 594, 595 liefert Azeton 602. liefert Homogentisinsäure 595 ff'. fi". — — — liefert Tyrosin 613. p-Oxyphenyl - dimethyläthylamin 443. p-Oxyphenylessigsäure 456, 515, 520, 566, 644. p-Oxyphenylglvoxylsäure 601. p-Oxyphenyl-a-methylamino-propionsäure 434. p-Oxyphenylmilchsäure 459, 594, 595. kommen in Pflanzen 438. Pentan 22. Pentasaccharide 53. Pentosane 55, 107. Pentosen 16, 29. Farbreaktionen 31. Pentoside 68. Pentosurie 29. Pepsin 465. Pepsin, aktives, im Darm- — inhalt 470. — Einwirkung auf Polypeptide 380. Pepsinzymogen 465. Peptasen 378. Peptide 356. Peptolytische Fermente 378. Peptone 351, 377. Entstehung im Magen — 465. — Kristalle 688. — Molekulargewicht 393. o-Oxyhippursäure 556. Oxyindolessigsäure 639 ß-Oxymethylerythrose 41. Oxymethylfurfurol 33. p-Oxyphenylalanin 326. p-Oxyphenylaminoesäig- Pentamethylendiamin, Vor- Perameisensäure 86. Perkaglobulin 396. Palmitinsäure 212. Perseit 16. Palmitinsäurecetylester 224. Palmitinsäurecholesterinester 225. Palmitinsäuremyricylester 224. Palmito-distearin 217. Palmito-olestearin 218. Pankreasdrüse, Beziehung zum Kohlehydratstoffwechsel 141, 168ff. Beziehung zur Leber 171. Petroleum 93. — Pankreasnukleoproteide 643. Pankreassaft, Bedeutung für Kohlehydratverdau-— ung 103. Wirkung Pettenkofersche Reaktion 234. Pferd, Oxyhämoglobin 688. Pferdefett 221. Pflanzen, Eiweißstoffwechsel 434. synthetische Leistungen 433. Pflanzenfresser 78. Pflanzenkristalloide aus Eiweiß 392. — Pflanzenschleime 56. Phäophorbid 701. Phäophytin 85, 701. Pharmakologie, Beziehung auf Eiweiß 477 ff. Pansen 100. Papaverin 449. Parabansäure 585. zur Physiologie Phaseolin 396. 7. Phenazetursäure 514, 555, Parabiose 174. Parach^-mosin 475. Paraffin, Verhalten im Darm Phenol- glukuronsäure 40, 267. Paramuzin 397. Paraxanthin 675. Parasiten 417. Parenterale Zufuhr 96. Pathologie, Beziehung zur Physiologie 6. Pektinsäure 56. Pektinsäuremethylester 56. Pektinstoffo 56. Pontamethylendiamin 324, 522. — im Harn 564. 618. Phenol 456, 520. — im Harn 554. 554. Phenolschwefelsäure 554. Phenosol 458. Phenyläthylalkohol 458. — Vorkommen 362. Phenyläthylamin 325, 524. Phenylalanin 324. für Glukose V Quelle — 208. — Quelle für Homogentisinsäure 563. ^ vertritt Tyrosin 499. 1-Phenylalanyl-d-alaninanhydrid 874. Sachverzeichnis. Phenyl-a-aminobuttersäure 612. y-Phenyl-a-aniinobuttersäure, Abbaustufen 603. liefert Benzoesäure 603. — Phenylaminoessigsäure 459, 603, 619. — liefert Benzoesäure 603. Phenylazetaldehyd 325. y-Phenyl-a-azetyl-aminobuttersäure 612. Plienyl-azetyl-glutamin 335, 515, 561. Phenylazetylglyzin 515,618. Phenylazetylierung 618. Phenylazetylkarbinol 1 29, 254. Piienylbenzopyrylium 72. - Phenylbrenztraubenalkohol 129. Phenylbrenztraubensäure liefert Homogentisinsäure 602. liefert Phenylalanin 613. Phenylbuttersäure 282. Phenylessigsäure 335, 456, 1 . — 520, 618. Phenylglyoxylsäure — Harn 627. — 1-p-OxyphenyImilchsäure im Harn 601. Phenylhydrazin 33. Plicnyllactimid 325. d-Phenylmilchsäure 459. Phcnylmilchsäure liefert Homogentisinsäure 602. Phenvl-ß-oxy- Propionsäure 282, Phylline 702. der Zellen 590. Proteasen 312, 477, 546. Proteide 394. — Pliylloerythrin 713. Phylloporphyrin 703. Phyllopyrrol 697. — Verdauung 475. Physik, Bedeutung für Physiologie 3. Physikalische Chemie, Bedeutung für Physio2. Physiologie, Definition 1 405 a-Phocaecholsäure 236. fi-Phocaecholsäure 236. a-Phocaetaurocholsäure236. ß-Phocaetaurocholsäure 236. Phonopvrrolkarbonsäure 698,^711. Rolle als Regulatoren der Reaktion 196. Phosphatide 238. Bildung in Pflanzen 255. Verdauung 270. Phosphatidoproteide 400. Plasmaalbumin 391. ff'. Polynukleotidase 665, 668. Polynukleotide 654. Abbaues 668. Proteinsynthese im Tier- körper 494 ff. Proteinurie 639. orthostatische 640. — Proteinverdauung 464 Proteolytische Fermente fl'. durch Permente 368 ff. — beim partiellen Abbau von Proteinen gewonnen 363 ff". — als Bestandteile von Peptonen 366 — aussalzbare 360. — Synthese 353 ff. ft". — Zahl der Isomeren 367. Pliosphate, Polyproteine 394. Polysaccharide 17, 4(), 54 ff". Porphyrine 702 ff — — Porphyrinogen 710. Präzipitinbildung 402 Prolamine 396 Prolin 331. fl'. ff. Pinnaglobin 699. des — einfache 394. — zusammengesetzte 394. — Aussalzmethode zu ihrer — Definition 312, 388. — Eigenschaften 383 — Einteilung 394, 401. — Fällungsreaktionen 494. — Farbreaktionen 384. — kristallisierte 390. — Molekulargewicht 392 Proteinoide 398. — Verdauung 476. Phystosterine 225. Pigmente 404. Pilokarpin 663. — Schema Proteinabbau im Darmkaual, Bedeutung des 500 ff. Proteine 310 ff. Darstellung 311, 386. Proteine, Bildung in Pflanzen Polypeptidasen 378. Polypeptide, Abbau 199. Glukose 207. Propylbenzol, Verhalten im Organismus 556. d-Propylenglykol, Beziehung zur ß-Oxybuttersäure Protagon 249. Protalbumosen 351. Protamine 397. Phlorogluzinglukosid Phlorhizin 68, 199. Phlorhizinglnkosurie 199. Phlorhizinglukuronsäure Propionsäureätliylester 216. Propionsäurecholinester 301 Propylaikohol, Quelle für Phrenosin 249. Phycocyan 394. Phycoerythrin 394. Plasmaglo1)ulin 391 Plasteine 472. Polylezithide 242. 199. — Abbau 193. — aus Alanin 454, 573. d-, a-, n-Propylpiperidin446. 283, 454, 456. Phenylvaleriansäure, Abbau 282. Phloretin 68, 192. Phloretinsäure 68, 199, 222. Phlorin 68, 199. Phlorogluzin 31, 41, 199. 68, 364. 191. Wirkungen 88. Piperidin 445. 283, 284. 1-Prolyl-l-phenylalanin Propepsin 465. Propionsäure 212. Photodynamische Phytin 44. Phytochlorin 702. Phytol 700. 601. Phenylpropionsäure Phosphatidsynthesen im tierischen Organismus 298 ff. Phosphor, Glukosurie 158. Phosphorproteide 399. Phosphorproteine 394. Phosphorsäure im Harn, Herkunft 592. Phosphorvergiftung, Aminosäuren im Harn 292, 541 Guanidinderivate im logie 459, 731 312. Protokatechusäure 513 Protone 398. Prunolaurasin 71. Psalterium 101. Pseudocholestan 232. Pseudomuzin 397. Ptyalose 50. Purin 646. Purinbasen 645 ff'. — Abbau im tierischen Organismus 669 ft. — Bildung in Pflanzen 661 ff. Frage der Neubildung 678 ff. Sachverzeichnis. 32 Purinbasenabbau, Fermente 669 if. Purinstoffwechsel 669 ft. Purpurfarbstoff 626, 716. Purpuvsäure 590. Resorptionsweg der Fette 269. Resorzin 32. Respiratorischer Quotient 85, 211. — — Putreszin 321, 522. bei Cystinurie 522, 549, 599. — beim Diabetes Retikulin 399. — im Harn 522. Retikulum 100. Rhamninose 52. Pyridin 445, 480, 621. J3-Pyridinkarbonsäure 442, Rhamnose 30. Rhodan wasserstoffsaure 591, 622. 184. Sarkosin 629. Scheinfütterung 468. Schichtung des Mageninhaltes 101. Schilddrüse, Beziehung zum Kohlehydratstoff'wechsel 635. a-Py r idyl- ß-N-methyl-py rro- Saponine 72, 226. Saprophyten 417. Sarkomelanin 714. — Bildung 636. — Nachweis 636. 141, 163, 183. — Einfluß auf Eiweißstottwechsel 548. — Einfluß auf Fettstoft- Pyridyl - pyrryl-pyrryl-pyrolen-methan 710. Rhodeose 30. wechsel 297. Schleimsäure 36, Ribit 30. Sclilundrinne 101. Pyrimidinbasen 649 ö. Bildung in Pflanzen 661 ff. Ribonsäure 30. Schmelzpunkt einiger Fett- lidin 447. — Pyrogallol 513. Pyromykursäure 556. Pyrrol 445, 693. Pyrrolidin 445, 704. a-Pyrrolidinkarbonsäure 331. Pyrrolidonkarbonsäure 334. Pyrrolin 445. d-Ribose 29, 651. d-Ribosecytosin 655. d-Riboseguanin 654. d-Ribosehypoxanthin 653. d-Ribosephosphorsäure 652. d-Riboseurazil 656. Ribo-trioxyglutarsäure 30. Rind, Oxyhämoglobin 688. Rindertalg 221. Rizin 395. Rizinolsäure 213. Roggenstärke 58. Rohrzucker 48 Rüböl, Ablagerung 277. Ruberythrinsäure 71, 78. Quercetin 71. Quercitrin 71. Quotient D N 186, 204. 211. respiratorischer, — beim Diabetes melitus 184. — respiratorischer bei Fettabbau 112. Schwefelsäure, gepaarte 5 13. Schwefelwasserstoff 525. Schwein, Oxyhämoglobin Scyllit 45. a- Saccharase 103. Saccharide 17. Saccharobiose 48. Saccharose 48 Sapotoxin 38. Sauerstoffmangel , Glukos- urie 158. Säureamide 360. Bedeutung als Nahrungs- — Raffinose 52. Abbau 60. — stoff 465. der Fette 221. Rastvorstellnngen 4. Ratanhin 443. Reaktion des Blutes 196. Reduktionsproben auf Zucker 32. Sahidin 248. Salizylamid 513. Salizylsäure, Verhalten — 87. 183. Reserveeiweiß 537. Reservekohiehydrate 57, 114. Reservezellulosen 56. Resorbierbarkeit verschiedener Fette 267. Probe auf Cholesterin 226. Salmin 378. Salpeter, Stickstoftquelle für Pflanzen 405 ff. Salpeterlager 428. Salzsäure als Aktivator 465. Sambunigrin 71. Saponin, Hämolyse 304. 694 ff. Sepiaschwarz 715. — Beziehung zu Cliolin 256. im Organismus 556. Bildung aus Benzoesäure Salkowskische des Diabetes Sedolieptose 16. Sehpurpur 716. Seidenfibroin 399. Seidenleim 399. Seifen 216. Sekretin 507. Selenige Säure, Verhalten im Organismus 621. Selenmethyl 621. Sensibilisierung 402, 88, Serin 316. Salizylursäure 556. Reisstärke 58. und ,j-Scymnol 236. Scymnolschwefelsäuren 236. ff'. R. Form 591. Schwefelausscheidung im Harn 590 ff'. Schwefelbakterien 416. Schwefelbleiprobe 318. 688. — respiratorisciier 120, Renale Schutzstoffe 123. Schwefel, neutraler im Harn Schweinefett 221. : Ranzigwerden arten 221. Schock 402. ff". Q. .50. — Bildung in Pflanzen 407. — Quelle für Glukose 208. Serizin 399. Serosani uzin 397. Serumalbumin 395. — Molekulargewicht 393. — Gehalt an Aminosäuren 493. Serumglobulin 395. Gehalt an Aminosäuren — 493. Serummukoid Silurin 398. Sinapin 445. 397. Sacliverzeichnis. Sinapinsäure 445. Sitosterin 226. Skatol 457, 517. — Vorkommen in Pflanzen 437. Skatolrot 517. Skatoxylschwefelsäure 517. Skombrin 398. Sorbose 37. Wirkung anf Steinkohle Quelle Stickstoff 429. Sterile als Aufzucht 106 Sterine 213, 225 für ff. ff. Organismus 302. Stickoxydhämoglobin 688. Kreislauf 422. StickstoffbindendeBakterien 422. Stickstoffbindung, künstStickstoff, ' 430 1- und d-. 25. ff'. 658. Thymusnukleoproteide 645. Thyreoglobulin 396. Thyroxin 330. p-Toluchinol 605. Toluhydrochinon 605. im Tannin 513. Tannine 72. Toluol, Tartronsäure 14. Beziehung zur Harn- Tolursäure 556. Toluylsäure, Verhalten V^erhalten Or- ganismus 556. im Organismus 556. Toxikologie, Beziehung zur Physiologie mit 578. Taurochenocholsäure 236. Taurocholsäure 230, 618. Taurodesoxvcholsäure 230, 7. Traubenzucker 38. Traubenzuckerabbau 122ff. Entstehung aus Nichtkohlehvdraten 172, 187, — 200. 618. — Verhalten im Magen daimkanal 270. — \'erhalten im tierischen liche Talose, Teichmannsclie Härninprobe a-Stearo-ot, J3-dipalmitin 218. auf 659. — Fütterungsversuch setzung .58. Stearinsäure 212. Stearo-dipalmitin 217. 513. Thymosinsäure 658. Thymusdrüse, Einfluß Thymusnukleinsäure säure 582. Taurin 230, 318, 551, 578. Zusammen- säure 656. Thymol T. — — Abbau 59. — Bildung in Pflanzen 81. Thymin-hexose-phosphor- Fettst.offwechsel 297. Tanazeton 123. Kohlehydrate 97. Sphingomyelin 247. Sphingosin 247, 249. Spirographin 398. Spongin 398. Spongosterin 226. Stachydrin 441. Stachyose 52. Stärke 57 if. StJirkearten, Suprarenin 624 ff. Surinamin 443. Symbiose 77. Synthesen im tierischen Organismus 79. Talg 295. Talgdrüsen, fetthaltiges Sekret 309. Sonnenenergie 80. Sorbit 36. Speichel, 733 — Transportzucker 142. Trehalose 48. 692. Tellurige Säure, Verhalten im Organismus 621. Tellurmethyl 621. Tendomukoid 397. Terpene 463. Tetraamylose 59, 90. Tetrakarbonimid 587. Tetramethvlendiamin 321, 444, 522. — im Harn 522. Tetramethylputreszin 444. Tetramethoxy-benzyl-isochi- Triamino - monophosphatide 248. Triamino-diphosphatide248. Triazetin 216. Tribomphenol 513. Tributyrin 216. Trichloräthvlalkohol 123. Trichlorhydrin 213. Trichlormilchsäureamid 589. Triglyzeride, Einteilung 222. Trigonellin 442,622. Triindylmethanfarbstoffe nolin 449. 519. Tetrasaccharide 52. Tetrosen 16. Triketohydrindenhydrat Stickstoffdüngung 428. Theobromin 663. Stickstoffgleichgewicht 494. Stigmasterin 226. Theophyllin 663. Theorien 4. 3, 4, 5-Trimethoxyphenyl- Stoffwechselendprodukte ans Kohlehydraten 120. Strahlenarten, die bei der Kohlensäureassimilation Therapie, experim.entelle, Beziehung zur Physio- äthvlamin 444. Trimethylamin 243, 301. wirksam sind 82. Strophantin 70. Strychnin 449. Glukosurie 158, 203. Einfluß auf Reflexerreg- — — Y-Trimethylaminobutter- logie 7. Thermopliile Bakterien 426. Thioäthylamin 551, 598. a-Thiomilchsäure 374. Thiophenaldehyd, Verhalten im Organismus 557. Thiophensäure 557. Thiophenursäure 557. barkeit 115. Sturin 378. Suberylarginin 237. Thiosulfate im Subkutane Zufuhr 96. Thujon 123. Sublimat, Glukosurie 158. Submaxillaris-muzin 397. Tliujonhydrat 123. Sulfatide 251. Sulfatschwefelsüure 591. 314. Trilaurin 216. Harn 592. Thujonhydratglukuronsäure 123. Thymin 650. säure 622. Y-Trimethylamino-a-oxybuttersäure-anhvdrid 623. Trimethylbetain des Histidins 441. Trimethylbetain des Thio- histidins 441. Trimethylbetain des Tryto- phans 441. Y-Trimethyl-butyrobetain 453. 1, 3, 7-Trimethyl-2, purin 662. 6-dioxy- Sachverzeichnis. 734 Trimethyl -oxyäthyl - ammo- Uranylsulfat als Katalysator niumhydroxyd 243. 87. Y-Trimethyl-a-oxybutyrobetain 453. Trimethyl -vinylammonium- Urazil 649. hydroxyd 245. Trimy ristin 216. Trinükleotid 657. — Synthese 650. Urease 436. üreinpropansäure 345. üridin 655. Uridinphosphorsäure 656, Triosen 16. 1,3, 5-Trioxybenzol 31. Trioxyglutarsäure 30. 2, 6, 8-trioxypurin, Bildung 670. — Resorption 266. zwiWechselbeziehungen schen Pflanze und Tier 93. Weinsäure 14. Weizenstärke 58. Whartonsche Sülze 397. Wiederkäuermagen 100. Wollfett. Zusammensetzung 657. Triolein 216. Walrat 224. ürikase 672. ürikolytisches Ferment 672. Urinporphyrin 695. Urobilin 710, Urobilinogen 710. ms. X. ürochloraisäure 123. Tripalmitin 216. Trisaccharide 52. Urochrom 716. Urochromogen 716. Tristearin 216. Xanthelasma 306. Xanthin 647. Abbau 670 ff. — — Vorkommen und Nach- Triticonukleinsäure 656, 659. Uroerythrin 716. Trommersche Probe 32. Urokaninsäure 635. Tropasäure 445, 448. Tropasäure-tropinester 448. Tropin 448. Trypsin 477 ff. ürorosein 518, 523, 716. Tryptophan 328.^ Abbaustufen 598. Abbau zu Kvnurensäure — — 608. — Quelle für Glukose? 208. — Unersetzbarkeit 499. Tryptophol 458. Tunicin 61. Turazin 716. Urofemnsäure 637. weis 649. V. Valeraldehyd 323. Valeriansäure 212, 455. — Abbau 193. — Abbau 192. — liefert kein Azelon 595. — Quelle für Glukose? 208. Valin 319. Verdauung derKolilehydrate Turizin 442. Tyramin 438, 527. Tyrosin 326. 99. — Abbaustufen 597 — durch Phenylalanin vertretbar 499. — liefert Azeton 602. — Quelle für Glukose? 208. — Quelle für Homogentiff. sinsäure 563. m-Ty rosin 601. Tyrosinase 32.7. Tyrosol 458, 598. — Vorkommen in Pflanzen 440. BezieVeidauungsdrüsen, liung zur Bildung von Purinbasen 681. Verfettung nach Vergiftungen 292 ff. Vergiftungen, intestinale 526. Vernin 655, 664. Vernix caseosa, fetthaltiges Sekret 309. Ver eifung 216. Vicianose 46. Vignin 396. Vitamine 77, 107. Vitell ne 400. Vizilin 396. Überempfindlichkeit 402. ümsatzeiweiß 538. ümsatznukleinsäuren 667. ümsatznukleoproteide 667. üraminobenzoösäure 578. üraminoisaethionsäure 578. a-Uraminopropionsäure 345. Uraminosäuren 345, 619. üransalze, Glukosurie 158. Xylonsäure 30. Xylose 27, 29, 30. Xyloside 68. Xylo-trioxyglutarsäure 30. z. Zein 396. Nukleoproteide Zellkerne, der 643. Zellobiuse 105. Zellobiose 51, 61. Zellproteasen 538. Zellulose 61. 103. Zellulosenitrate 61. Zerotinsäure 309. Zervlalkohol 3U9. Zetylalkohol 224. Zimtsäure 282. Zoosterine 225. Vizin 664. Volemit 16. Vormagen 100. w. Zuckeiabbau 121 fl. /uckerarten 12. Wachse 224. Wärmehaushalt, Xantliobilirubinsäure 711. Xantliome 306. Xanthopliyll 82, 699. Xanlhoproteinreaktion 325. Xanthosin 655. Xanthosis diabetica 715. Xylit 30. Xyloketose 30. Xylol, Verhalten im Organismus 556. — Abbau 61. — Abbau im Darmkänal Vitiatin 628. u. Xanthinbasen, metl)ylierte 662 ff. Xanthinsteine 685. Funktion des Fettes bei 280. Zuckcr;;eh:ilt des Blutes 145. ZuckerlKirnruhr 178 ff. SachverzeichHis Zuckernachweis 32. Znckersänre 36. Zyanursäure 580, 589. Zystin, Zyklopterin 398. 499, 553. Zystinurie 318, 522, 549, 599. Zytidin 655. Zuckerstich 153. Zymase 126. Zuckerzentrum 153. Zustand sänderungen Zyprinine 398. Zystein 317, 551. Zysteinsäure 235, 318, 551. Zystin 317. Proteine 389. Zyanamid 430, 629. der 735 seine Bedeutung Zytidinphosphorsäure 656 657. Zytösin 6.50 ü. Berichtigungen. Die Seite 22, oben, gemachte Bemerkung, wonach es noch nicht gehingen ist, von d- und 1-Alanin festzustellen, ist überholt. Vgl. hierzu S. 316. S. 206 muß es in der zweiten Formelreihe statt Metyhylglyoxal heißen Methylglyoxal die Konfiguration Druck von Gottlieb GistPl & Cie., Wien. III.. 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https://openalex.org/W2195062255	https://europepmc.org/articles/pmc4684333?pdf=render	English	null	The Drosophila Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of spookier	PLOS genetics	2,015	cc-by	15,557	The Drosophila Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of spookier Tatsuya Komura-Kawa1, Keiko Hirota1,2, Yuko Shimada-Niwa2, Rieko Yamauchi1,2, MaryJane Shimell3, Tetsuro Shinoda4, Akiyoshi Fukamizu1,2, Michael B. O’Connor3, Ryusuke Niwa1,5* 1 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan, 2 Life Science Center, Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki, Japan, 3 Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America, 4 National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan, 5 PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan * ryusuke-niwa@umin.ac.jp RESEARCH ARTICLE * ryusuke-niwa@umin.ac.jp Abstract Citation: Komura-Kawa T, Hirota K, Shimada-Niwa Y, Yamauchi R, Shimell M, Shinoda T, et al. (2015) The Drosophila Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of spookier. PLoS Genet 11(12): e1005712. doi:10.1371/journal.pgen.1005712 Steroid hormones are crucial for many biological events in multicellular organisms. In insects, the principal steroid hormones are ecdysteroids, which play essential roles in regulating molting and metamorphosis. During larval and pupal development, ecdysteroids are synthesized in the prothoracic gland (PG) from dietary cholesterol via a series of hydroxylation and oxidation steps. The expression of all but one of the known ecdysteroid biosynthetic enzymes is restricted to the PG, but the transcriptional regulatory networks responsible for generating such exquisite tissue-specific regulation is only beginning to be elucidated. Here, we report identification and characterization of the C2H2-type zinc finger transcription factor Ouija board (Ouib) necessary for ecdysteroid production in the PG in the fruit fly Drosophila melanogaster. Expression of ouib is predominantly limited to the PG, and genetic null mutants of ouib result in larval developmental arrest that can be rescued by administrating an active ecdysteroid. Interestingly, ouib mutant animals exhibit a strong reduction in the expression of one ecdysteroid biosynthetic enzyme, spookier. Using a cell culture-based luciferase reporter assay, Ouib protein stimulates transcription of spok by binding to a specific ~15 bp response element in the spok PG enhancer element. Most remarkable, the developmental arrest phenotype of ouib mutants is rescued by over- expression of a functionally-equivalent paralog of spookier. These observations imply that the main biological function of Ouib is to specifically regulate spookier transcription during Drosophila development. Editor: Liliane Schoofs, K.U.Leuven, BELGIUM Received: May 17, 2015 Accepted: November 8, 2015 Published: December 10, 2015 Editor: Liliane Schoofs, K.U.Leuven, BELGIUM Received: May 17, 2015 Accepted: November 8, 2015 Published: December 10, 2015 Copyright: © 2015 Komura-Kawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Published: December 10, 2015 Copyright: © 2015 Komura-Kawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Drosophila Steroidogenic Transcription Factor Ouija Board Competing Interests: The authors have declared that no competing interests exist. Author Summary Steroid hormones are crucial for development and reproduction in multicellular organ- isms. The spatially-restricted expression of almost all steroid biosynthesis genes is key to the specialization of steroid producing cells. In the last decade, insects have become the focus for research on the biosynthesis of the principal steroid hormones, ecdysteroids. However, the transcriptional regulatory mechanisms controlling the ecdysteroid biosyn- thesis genes are largely unknown. Here we show that a novel zinc finger transcription fac- tor Ouija board (Ouib) is essential for activating the expression of one ecdysteroid biosynthesis gene, spookier, in the ecdysteroid producing cells. Ouib is the first inverte- brate transcription factor that is predominantly expressed in the steroidogenic organs and essential for development via inducing expression of the steroidogenic gene. In addition, this is the first report showing the catalytic step-specific control of steroid hormone bio- synthesis through transcriptional regulation. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency http://www.jst.go.jp/kisoken/presto/en/index.html; The Naito Foundation; https://www.naito-f.or.jp/en/ and by the National Institutes of Health, USA; grant number R01 GM093301; http://grants.nih.gov/grants/funding/ r01.htm. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 26 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Drosophila Steroidogenic Transcription Factor Ouija Board Previous studies have identified multiple transcription factors essential for ecdysteroido- genic functions in the PG. For example, the Ecdysone receptor-Ultraspiracle complex and sev- eral other ecdysteroid-regulated transcription factors such as βFTZ-F1, Broad, E75A and DHR4 are involved in both forward and feedback regulation of cyclic ecdysteroid production [28–35]. Ecdysteroid biosynthesis is also transcriptionally regulated by other factors including Without children [36], Molting defective (Mld) [37], the CncC-dKeap1 complex [38], Ventral veins lacking (Vvl) [39,40], Knirps [39] and FOXO [35]. Importantly, it has been reported that Broad, CncC, dKeap1, Vvl and Knirps directly bind to enhancer regions of some ecdysteroido- genic enzyme genes [31,33,38,39]. However, it should be noted that, unlike vertebrate Ad4BP/SF-1 and LHR-1, all of the iden- tified steroidogenic transcription factors in insects are highly expressed not only in the PG, but also in many other non-ecdysteroidogenic tissues. Furthermore, some of these transcription factors have important functions other than ecdysteroid biosynthesis. For example, FOXO is well characterized as the primary transcriptional mediator of the insulin/insulin-like peptide signaling pathway in almost all cells [41]. Similarly, the CncC-dKeap1 complex is known to regulate xenobiotic responses [42] while Vvl and Knirps play key roles in cellular differentia- tion and morphogenesis of several tissues during embryogenesis including the PG (i.e. [43,44]). More notably, βFTZ-F1, the insect homolog of vertebrate Ad4BP/SF-1, plays a crucial role in ecdysteroid-dependent transcriptional cascades in not only the PG but also many other tissues [4,9]. h b d l h ll h d f l h d [43,44]). More notably, βFTZ-F1, the insect homolog of vertebrate Ad4BP/SF-1, plays a crucial role in ecdysteroid-dependent transcriptional cascades in not only the PG but also many other tissues [4,9]. In contrast to the broad roles that all these steroidogenic factors play in other tissues during development, we describe here a much more specific role for the transcription factor coded by the gene ouija board (ouib). Ouib is a C2H2-type zinc finger transcription factor, that is specifi- cally expressed in the Drosophila PG and our genetic analysis clearly demonstrates that ouib is only essential for the expression of Spookier (Spok), a potential rate-limiting enzyme in the ecdysone biosynthetic pathway. Most remarkable, however, is that spok appears to be the essential target of ouib since resupply of a Spok paralog in PG tissue rescues ouib mutants to viability. CG11762/ouija board is predominantly expressed in the prothoracic gland during embryonic and larval development We identified CG11762, designated ouija board (ouib), as a gene predominantly expressed in the PG primordia in the embryonic in situ gene expression pattern database of the Berkeley Drosophila Genome Project Experiment ID RT01107 [45]. We confirmed the PG restricted expression of ouib in embryos using RNA in situ hybridization (Figs 1A, 1B and S1). Addi- tional RNA in situ hybridization and quantitative reverse-transcription (qRT)-PCR experi- ments revealed that ouib is also predominantly expressed in the ring gland including the PG cells during larval development (Fig 1C, 1D and 1E). These results suggest that ouib may be involved in ecdysteroid biosynthesis. Since orthologs of ouib and spok are found only in Drosophiladae genomes, this study also suggests a presence of insect clade-specific transcriptional regulatory mechanisms of ecdy- sone biosynthesis. Introduction Steroid hormones are responsible for the coordination and regulation of many biological events during development of multicellular organisms. In all species, steroid hormones are syn- thesized from cholesterol and/or other phytosterols by multiple steroidogenic enzymes, epito- mized by the members of the steroidogenic cytochrome P450 monooxygenases. High-level steroid hormone biosynthesis generally occurs in specialized steroidogenic tissues. Thus, an important condition for achieving tissue-specificity of steroid biosynthesis is providing a regu- latory mechanism that ensures tissue-specific expression of the steroidogenic enzyme genes. In vertebrates, major sites of steroid hormone biosynthesis are the adrenal cortex, gonads and placenta, that express steroidogenic enzyme genes such as Cyp11a1, P450c17a, 3β-HSD and 17β-HSD [1]. Key transcriptional regulators for these genes are the orphan nuclear recep- tors NR5A1 and NR5A2, also known as Ad4BP/Steroidogenic Factor 1 (SF-1) and Liver receptor homolog-1 (LRH-1), respectively [2–5]. Ad4BP/SF-1 and LRH-1 are predominantly expressed in the steroidogenic cells. A collective body of previous studies has established that Ad4BP/SF-1 controls steroid hormone biosynthesis through the transcriptional regulation of all steroidogenic genes [3,5]. Moreover, forced expression of this gene is sufficient to differenti- ate embryonic stem cells and human induced pluripotent stem cells into the steroidogenic cells [6,7] and to induce ectopic adrenal formation [8], indicating that Ad4BP/SF-1 acts as a master regulator for steroid hormone biosynthesis in vertebrates. In insects, the principal steroid hormones are ecdysteroids, including ecdysone and its active derivative 20-hydroxyecdysone (20E), which plays pivotal roles in controlling a number of developmental and physiological events, especially in guiding transition from one developmen- tal stage to the next via molting and metamorphosis [9–13]. During larval and pupal develop- ment, ecdysone is synthesized from dietary cholesterol in a specialized endocrine organ called the prothoracic gland (PG). After release from the PG, ecdysone is converted to 20E in the peripheral tissues through the action of Shade, the terminal P450 monoxygenase in the biosyn- thetic pathway [14]. In the last 15 years, a number of genes encoding essential ecdysteroido- genic enzymes acting in the PG have been identified and characterized, including noppera-bo [15–17], neverland (nvd) [18,19], Cyp307a1/spook (spo) [20,21], Cyp307a2/spookier (spok) [21], non-molting glossy/shroud (sro) [22], Cyp306a1/phantom (phm) [23,24], Cyp302a1/disem- bodied (dib) [25,26] and Cyp315a1/shadow (sad) [26]. All of these enzymes (except nvd and spok) are collectively referred to as the Halloween genes [13,27]. 2 / 26 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Drosophila Steroidogenic Transcription Factor Ouija Board Fig 1. Expression analysis of ouib in Drosophila larva and embryo. (A, B) RNA in situ hybridization of stage 14 (A) and stage 16 (B) embryos with the ouib antisense RNA probe. Dorsal views are shown. ouib signal was detected in the primordia of PG cells (arrows). An image with sense RNA probe is shown in S1 Fig (C D) in situ hybridization of third instar larval brain ring gland complexes with the ouib antisense (C) and sense (D) RNA probes ouib signal Fig 1. Expression analysis of ouib in Drosophila larva and embryo. (A, B) RNA in situ hybridization of stage 14 (A) and stage 16 (B) embryos with the ouib antisense RNA probe. Dorsal views are shown. ouib signal was detected in the primordia of PG cells (arrows). An image with sense RNA probe is shown in S1 Fig (C, D) in situ hybridization of third instar larval brain-ring gland complexes with the ouib antisense (C) and sense (D) RNA probes. ouib signal is detected in the ring gland including the PG cells (arrow). (E) The expression levels of ouib in several tissues quantified by qRT-PCR (N = 3). Total RNA was prepared from wandering third instar larvae. BR, brain; RG, ring gland; ID, imaginal disc; IN, intestine; FB, fat body; SG, salivary gland. Error bars indicate the s.e.m. d i 10 1371/j l 1005712 001 Fig 1. Expression analysis of ouib in Drosophila larva and embryo. (A, B) RNA in situ hybridization of stage 14 (A) and stage 16 (B) embryos with the ouib antisense RNA probe. Dorsal views are shown. ouib signal was detected in the primordia of PG cells (arrows). An image with sense RNA probe is shown in S1 Fig (C, D) in situ hybridization of third instar larval brain-ring gland complexes with the ouib antisense (C) and sense (D) RNA probes. ouib signal is detected in the ring gland including the PG cells (arrow). (E) The expression levels of ouib in several tissues quantified by qRT-PCR (N = 3). Total RNA was prepared from wandering third instar larvae. BR, brain; RG, ring gland; ID, imaginal disc; IN, intestine; FB, fat body; SG, salivary gland. Error bars indicate the s.e.m. ouija board encodes a ZAD-C2H2 zinc-finger protein and is conserved only in Drosophilidae species The predicted open reading frame of ouib encodes a protein that belongs to the family of the zinc-finger associated domain (ZAD) containing C2H2 zinc-finger proteins (ZFPs) [46,47]. PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 3 / 26 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Drosophila Steroidogenic Transcription Factor Ouija Board Fig 2. Generation of ouib mutant alleles by CRISPR/Cas9 system. (A) A schema of the ouib gene showing the sgRNA target sites. Exons are shown as black boxes, the transcriptional start site as arrow, and sgRNA target sites as black triangles. (B) Sequences of sgRNA target sites. The 20 bp target sequence corresponding to each target site is indicated in orange, along with the neighboring NGG protospacer adjacent motif (PAM) in pink and the cleavage site of Cas9 is shown as black triangles. (C) Predicted protein structures of ouib alleles. Ouib29 and Ouib74 are composed of 29 and 74 amino acids, respectively. Fig 2. Generation of ouib mutant alleles by CRISPR/Cas9 system. (A) A schema of the ouib gene showing the sgRNA target sites. Exons are shown as black boxes, the transcriptional start site as arrow, and sgRNA target sites as black triangles. (B) Sequences of sgRNA target sites. The 20 bp target sequence corresponding to each target site is indicated in orange, along with the neighboring NGG protospacer adjacent motif (PAM) in pink and the cleavage site of Cas9 is shown as black triangles. (C) Predicted protein structures of ouib alleles. Ouib29 and Ouib74 are composed of 29 and 74 amino acids, respectively Fig 2. Generation of ouib mutant alleles by CRISPR/Cas9 system. (A) A schema of the ouib gene showing the sgRNA target sites. Exons are shown as black boxes, the transcriptional start site as arrow, and sgRNA target sites as black triangles. (B) Sequences of sgRNA target sites. The 20 bp target sequence corresponding to each target site is indicated in orange, along with the neighboring NGG protospacer adjacent motif (PAM) in pink and the cleavage site of Cas9 is shown as black triangles. (C) Predicted protein structures of ouib alleles. Ouib29 and Ouib74 are composed of 29 and 74 amino acids, respectively doi:10.1371/journal.pgen.1005712.g002 orthologs of ouib in the mosquito species Aedes aegypti, Anopheles gambiae and Culex quinque- fasciatus. However, a reciprocal BLAST search does not support the idea that the mosquito genomes have true ouib orthologs. In addition, a standard BLAST search did not detect any orthologous counterparts of ouib in any organisms other than Drosophilidae species. This is consistent with the previous report [47], that no orthologs of ouib are found in genomes of the silkworm Bombyx mori and the beetle Tribolium castatenum. Taken together, these results sug- gest that ouib is a Drosophilidae-specific ecdysteroidogenic component. doi:10.1371/journal.pgen.1005712.g001 The ZAD-ZFP family constitutes the largest subgroup of C2H2 ZFPs especially in insect spe- cies, and are characterized by an N-terminal ZAD consisting of 75 amino acid residues that are thought to serve as a protein-protein interaction domain [48]. In D. melanogaster, there are 98 independent loci encoding ZAD-ZFPs [47]. At least some of ZAD-ZFPs are thought to act as transcription factors, since several of them have been reported to bind DNA [49,50]. Nota- bly, 5 paralogs of ouib are duplicated at the 85A9 cytological position of the third chromosome in D. melanogaster genome (S2 Fig), and one of the paralogs designated M1BP codes for a gen- eral transcription factor [51], raising the possibility that Ouib acts as a transcription factor in the PG. Orthologs of ouib are found in genomes of 11 other Drosphilidae species (S1 Table) [52]. FlyBase (http://flybase.org/reports/FBgn0037618.html) also indicates the presence of potential PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 4 / 26 The ouija board loss-of-function phenotype is due to ecdysteroid deficiency We next examined whether the larval arrest and lethality phenotype of ouib mutant animals was due to the loss of ecdysteroids. An ELISA assay revealed that the ecdysteroid titer in ouib29/ouib74 transheterozygotes was significantly reduced compared to control animals (Fig 3C). Consistent with this observation, the expression of E75A, which is an early ecdysteroid- inducible gene, was greatly reduced in ouib29/ouib74 transheterozygotes (Fig 3D). Moreover, when ouib29/ouib74 animals or ouib74/Df animals were fed yeast paste containing 20E after hatching, they molted to the second instar larval stage or later, as judged by the anterior spirac- ular morphologies (Table 1). These results suggest that loss of ouib mutant phenotype is due to ecdysteroid deficiency and that ouib regulates ecdysteroid production in the PG during normal development. Drosophila Steroidogenic Transcription Factor Ouija Board led to premature stop codons in the putative coding sequence of ouib, eliminating all 5 zinc-fin- ger domains in the C-terminal region of Ouib (Fig 2C). Embryos transheterozygous for ouib29/ouib74 completed embryogenesis, hatched normally, and showed no apparent morphological defects after hatching. However, ouib29/ouib74 trans- heterozygotes arrested development in the first instar larval stage and, even 108 hours after egg laying (AEL) or later, never molted into second instars and (Fig 3A and 3B). Eventually all ouib29/ouib74 transheterozygous animals died by 144 hours AEL retaining the first instar larva- type morphology. In contrast, the majority of control ouib29/+ or ouib74/+ heterozygous ani- mals became pupae (Fig 3A and 3B) by this time. To rule out the possibility that the observed phenotype was due to off-target mutations by CRISPR/Cas9 system, we combined ouib29 or ouib74 allele with a deficiency (Df) line that deletes a genomic region containing ouib locus. Similar to ouib29/ouib74 transheterozygotes, ouib29/Df or ouib74/Df animals died in the first instar stage, while +/Df animals were fully viable. This result provides evidence that ouib locus is responsible for the lethal phenotype. These results demonstrate that Ouib is essential for lar- val development. PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 ouija board is essential for larval development To assess the in vivo functional importance of ouib, we generated ouib loss-of-function alleles by a CRISPR/Cas9-dependent genome editing technology [53]. We succeeded in isolating two independent mutant alleles, ouib29 and ouib74, each of which had a small deletion induced by different CRISPR single guide RNAs (sgRNAs; Fig 2A and 2B). Both ouib29 and ouib74 alleles 5 / 26 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Loss of ouija board strongly reduces the expression of one ecdysone biosynthetic enzyme gene spookier As described above, we expect that Ouib acts as a transcription factor. Considering the spatial expression pattern and the loss-of-function phenotype of ouib, we wondered whether loss of ouib resulted in changes in the expression levels of any ecdysteroidogenic genes in the PG. To address this issue, we conducted qRT-PCR experiment to examine expression levels of 6 ecdys- teroidogenic genes in the first instar larvae of control and ouib29/ouib74 transheterozygotes. Among the 6 genes, the expression of one gene Cyp307a2/spok was drastically reduced in ouib29/ouib74 transheterozygotes as compared to control animals (Fig 4A). An immunohistolo- gical analysis using anti-Spok antibody also revealed a strong decrease of Spok protein level in ouib29/ouib74 larvae compared to control animals, but not that of the Sro protein, another ecdy- sone biosynthetic enzyme expressed in the PG. (Fig 4B). We also found that expression of Cyp302a1/dib and Cyp315a1/sad, two other ecdysone biosynthetic P450 genes, were also lower than in ouib29/ouib74 animals compared to control animals, but their reduction was just on the threshold of significance (Fig 4A). On the basis of the observation that the mutants cannot induce the expression of “spookier,” we named CG11762 “ouija board” since this is an instru- ment for calling ghosts in western countries. We also examined whether there was a correlation between expression of ouib and spok dur- ing larval development. Overall the expression of both genes was relatively low during early 6 / 26 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Drosophila Steroidogenic Transcription Factor Ouija Board Table 1. Rescue of ouib null mutant animals by oral administration of sterols and ecdysteroids. Ecdy-steroid Number of dead animals at each stage Total number of animals 1st instar 2nd instar and later ouib29/ouib74 ouib74/Df ouib29/ouib74 ouib74/Df ouib29/ouib74 ouib74/Df None 47 36 0 0 47 36 C 48 - 0 - 48 - 7dC 46 - 0 - 46 - 5βkd 1 - 43 - 44 - 20E 0 39 47 24 47 63 The ﬁrst instar larvae of ouib29/ouib74 or ouib74/Df animals were collected 36 hours AEL, and then fed yeast pastes containing 0.5% (w/w) each steroid. The number of dead animals at each stage was counted. C, cholesterol; 7dC, 7-dehydrocholesterol; 5βkd, 5β-ketodiol; 20E, 20-hydroxyecdysone; -, not determined. Table 1. Rescue of ouib null mutant animals by oral administration of sterols and ecdysteroids. Ecdy-steroid Number of dead animals at each stage Total number of animals 1st instar 2nd instar and later ouib29/ouib74 ouib74/Df ouib29/ouib74 ouib74/Df ouib29/ouib74 ouib74/Df None 47 36 0 0 47 36 C 48 - 0 - 48 - 7dC 46 - 0 - 46 - 5βkd 1 - 43 - 44 - 20E 0 39 47 24 47 63 The ﬁrst instar larvae of ouib29/ouib74 or ouib74/Df animals were collected 36 hours AEL, and then fed yeast pastes containing 0.5% (w/w) each steroid. The number of dead animals at each stage was counted. C, cholesterol; 7dC, 7-dehydrocholesterol; 5βkd, 5β-ketodiol; 20E, 20-hydroxyecdysone; -, not determined. Table 1. Rescue of ouib null mutant animals by oral administration of sterols and ecdysteroids. The ﬁrst instar larvae of ouib29/ouib74 or ouib74/Df animals were collected 36 hours AEL, and then fed yeast pastes containing 0.5% (w/w) each steroid. The number of dead animals at each stage was counted. C, cholesterol; 7dC, 7-dehydrocholesterol; 5βkd, 5β-ketodiol; 20E, 20-hydroxyecdysone; -, not determined. The ﬁrst instar larvae of ouib29/ouib74 or ouib74/Df animals were collected 36 hours AEL, and then fed yeast pastes containing 0.5% (w/w) each steroid. The number of dead animals at each stage was counted. C, cholesterol; 7dC, 7-dehydrocholesterol; 5βkd, 5β-ketodiol; 20E, 20-hydroxyecdysone; -, not determined. doi:10.1371/journal.pgen.1005712.t001 stages and gradually became higher in late stages. We also found that the temporal expression profile of ouib closely correlates to that of spok in the late third instar stage (Fig 4C). ouija board plays an essential role in the “Black Box” where spookier has a crucial function A previous study reported that Spok plays a crucial role in the “Black Box”, which consists of the conversion steps from 7-dehydrocholesterol (7dC) to 5β-ketodiol (5βkd) in the ecdysteroid biosynthetic pathway [21]. The participation of Spok in the “Black box” reactions was inferred by the observation that the larval arrest phenotype of spok RNAi animals was rescued by oral administration of 5βkd, but not 7dC or the most upstream precursor cholesterol [21]. Indeed, the same tendency as observed in spok RNAi animals was found in ouib loss-of-function ani- mals. When ouib29/ouib74 transheterozygotes were fed yeast paste supplemented with choles- terol or 7dC, the larvae still arrested at the first instar larval stage (Fig 4D and Table 1). In contrast, we found that the first instar larval arrest phenotype of ouib29/ouib74 transheterozy- gotes was rescued when the animals were fed yeast paste supplemented with 5βkd (Fig 4D and Table 1). These results suggest that loss of ouib function specifically impairs the catalytic con- version that takes place during the “Black Box” reactions. These results also imply that the moderate reduction seen in dib and sad expression does not contribute in a major way to the ouib mutant phenotype. Drosophila Steroidogenic Transcription Factor Ouija Board Fig 3. Larval lethality and developmental arrest phenotype of ouib mutant larvae. (A) The survival rate and developmental progression of control and ouib mutant animals (N = 50~100). (B) Comparison of body size and developmental stage between control (right and middle) and ouib mutant (left) at 108 hours AEL. Control animals became third instar larvae, while ouib mutant animals were first instar larvae. Scale bar: 1 mm. (C) Ecdysteroid levels in control and ouib mutant first instar larvae at 12 hours AH measured by ELISA (N = 4). (D) Ecdysteroid levels, as measured by the ecdysone inducible gene E75A, in control and ouib mutant first instar larvae at 36 hours AEL measured by qRT-PCR (N = 3). Error bars indicate s.e.m. ; P<0.01 with Student’s t-test. doi:10.1371/journal.pgen.1005712.g003 Fig 3. Larval lethality and developmental arrest phenotype of ouib mutant larvae. (A) The survival rate and developmental progression of control and ouib mutant animals (N = 50~100). (B) Comparison of body size and developmental stage between control (right and middle) and ouib mutant (left) at 108 hours AEL. Control animals became third instar larvae, while ouib mutant animals were first instar larvae. Scale bar: 1 mm. (C) Ecdysteroid levels in control and ouib mutant first instar larvae at 12 hours AH measured by ELISA (N = 4). (D) Ecdysteroid levels, as measured by the ecdysone inducible gene E75A, in control and ouib mutant first instar larvae at 36 hours AEL measured by qRT-PCR (N = 3). Error bars indicate s.e.m. ; P<0.01 with Student’s t-test. doi:10.1371/journal.pgen.1005712.g003 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 7 / 26 Curiously, the temporal expression profile did not always correlates to the dynamics of ecdysteroid titer during the third instar stage (S3 Fig). For example, ouib expression did not increase prior to white prepupal stage, when the level of ecdysteroid titer was high. This result suggests that the ouib-spok coordinated transcriptional relationship does not fully account for the temporal dynamics of ecdysteroid biosynthesis during development. Drosophila Steroidogenic Transcription Factor Ouija Board Fig 4. Expression analysis of ecdysteroidogenic genes and feeding rescue experiment in ouib mutant larvae. (A) The expression levels of ecdysteroidogenic genes in control and ouib mutant first instar larvae at 38 hours AEL measured by qRT-PCR (N = 3). (B) Immunostaining of the PG cells from control and ouib mutant first instar larvae at 36 hours AEL with antibodies against Spok (green) and Sro (magenta). Scale bar: 25 μm. (C) The transcriptional expression profiles of ouib and spok in w1118 during larval development measured by qRT-PCR (N = 3). The solid line and dashed lines indicate the expression levels of ouib and spok, respectively. (D) Feeding rescue experiments for ouib mutant (ouib29/ouib74) larvae. Mutant animals fed 5β- ketodiol (5βkd) and 20-hydroxyecdysone (20E) became third instar larvae, while animals fed cholesterol (C), 7-dehydrocholesterol (7dC) and none remained first instar larvae. The lethal stages in each experimental condition were scored and shown in Table 2. Scale bar: 1 mm. Error bars indicate s.e.m. ; P<0.05, *; P<0.01 with Student’s t-test. Fig 4. Expression analysis of ecdysteroidogenic genes and feeding rescue experiment in ouib mutant larvae. (A) The expression levels of ecdysteroidogenic genes in control and ouib mutant first instar larvae at 38 hours AEL measured by qRT-PCR (N = 3). (B) Immunostaining of the PG cells from control and ouib mutant first instar larvae at 36 hours AEL with antibodies against Spok (green) and Sro (magenta). Scale bar: 25 μm. (C) The transcriptional expression profiles of ouib and spok in w1118 during larval development measured by qRT-PCR (N = 3). The solid line and dashed lines indicate the expression levels of ouib and spok, respectively. (D) Feeding rescue experiments for ouib mutant (ouib29/ouib74) larvae. Mutant animals fed 5β- ketodiol (5βkd) and 20-hydroxyecdysone (20E) became third instar larvae, while animals fed cholesterol (C), 7-dehydrocholesterol (7dC) and none remained first instar larvae. The lethal stages in each experimental condition were scored and shown in Table 2. Scale bar: 1 mm. Error bars indicate s.e.m. ; P<0.05, *; P<0.01 with Student’s t-test. doi:10.1371/journal.pgen.1005712.g004 and thus these constructs were not suitable for our experimental purpose. Therefore, we decided to examine whether the ouib mutant phenotype could be rescued by forced expression of Cyp307a1/spo, a paralog of spok that appears to provide the same enzymatic activity but only in embryos and in the follicular cells of the ovary [20,21]. The ouija board mutant phenotype is due to loss of spookier expression in the prothoracic gland In addition to the feeding rescue experiment, we examined whether the ouib mutant phenotype was rescued by forced expression of spok using GAL4-UAS binary gene expression system. We first established UAS-spok transgenic strains to drive spok expression in the PG cells under con- trol of phm-GAL4#22 driver. However, for an unknown reason, none of our UAS-spok trans- genes was expressed in the PG of first and second instar larvae with the phm-GAL4#22 driver 8 / 26 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Identification of Ouija board-response element in the spookier enhancer region To address whether Ouib protein acts directly on the spok enhancer region to induce spok expression, we initially searched for an Ouib-response element in the enhancer/promoter region of spok. We first identified a ~1.4 kb genomic region upstream of the spok coding sequence that was sufficient to mimic the expression of spok in the PG when fused to a GFP reporter (Figs 5A and S4). The GFP expression driven by the ~1.4 kb spok enhancer region was almost completely abolished in ouib29/ouib74 transheterozygotes (Fig 5A), suggesting that the ~1.4 kb element contains a Ouib-response element. p In order to identify the cis-regulatory element(s) responsible for the Ouib-mediated control of spok expression, we conducted a promoter/enhancer characterization analysis in a heterolo- gous cell culture system. We generated DNA constructs carrying the upstream region of spok fused with a luciferase (luc) gene cassette and then transfected Drosophila Schneider 2 (S2) cells using these DNA constructs with or without a plasmid for overexpressing FLAG-ouib. We identified a 300 bp genomic region corresponding to the region from -331 bp to -32 bp upstream of the ATG start codon of spok that drives expression of the luc reporter in S2 cells in an Ouib-dependent manner (S5 Fig). The 300 bp region was also sufficient to drive expression of a GFP reporter in the PG cells (S4 Fig). To narrow down the element(s) responsible for the Ouib-dependent expression of spok, we tested several constructs carrying the upstream region of spok with a range of deletions within the 300 bp region (S5 Fig). We first generated the dele- tion constructs in 50 bp increments from 5´ terminus of the 300 bp region and found that the region from -181 to -131 bp was crucial for the Ouib-dependent luc reporter activity (S5 Fig). We then generated the deletion constructs in 10 bp increments from 5´ terminus of the -181 to -32 region. The construct carrying the -151 to -32 region did not show any induction in luc reporter activity even in the presence of Ouib (Fig 5B). The construct carrying the longer 10 bp 5´ extension (-161 to -32) still retained statistical significant Ouib-dependent luc reporter activ- ity. However, the fold induction of luc reporter activity with the -161 to -32 region was slightly reduced as compared to the -171 to -32 region or longer (Fig 5B). We confirmed that spo was function- ally equivalent to spok in vivo, as spo overexpression rescued the first instar larval arrest pheno- type of spok RNAi animals (S2 Table). Indeed, spo overexpression in the PG rescued the larval arrest phenotype of ouib29/ouib74 transheterozygotes, and some of the animals grew up to the adult stage (Table 2). These results strongly suggest that the developmental arrest phenotype of ouib mutant is due solely to loss of spok expression in the PG. Our data therefore support the idea that Ouib is a special transcription factor primarily required for inducing expression of one biosynthetic gene spok, and no other essential gene during development. and thus these constructs were not suitable for our experimental purpose. Therefore, we decided to examine whether the ouib mutant phenotype could be rescued by forced expression of Cyp307a1/spo, a paralog of spok that appears to provide the same enzymatic activity but only in embryos and in the follicular cells of the ovary [20,21]. We confirmed that spo was function- ally equivalent to spok in vivo, as spo overexpression rescued the first instar larval arrest pheno- type of spok RNAi animals (S2 Table). Indeed, spo overexpression in the PG rescued the larval arrest phenotype of ouib29/ouib74 transheterozygotes, and some of the animals grew up to the adult stage (Table 2). These results strongly suggest that the developmental arrest phenotype of ouib mutant is due solely to loss of spok expression in the PG. Our data therefore support the idea that Ouib is a special transcription factor primarily required for inducing expression of one biosynthetic gene spok, and no other essential gene during development. 9 / 26 Drosophila Steroidogenic Transcription Factor Ouija Board Table 2. Rescue of ouib mutant animals by spo overexpression in the PG. Transgenes Number of ouib29/ouib74 adults Tb 3rd instar larvae Pupae Adults phm-GAL4#22, UAS-spo + 169 (100%) 124 (73.4%) 29 (17.2%) - 246 n.d. n.d. phm-GAL4#22 + 0 0 0 - 85 n.d. n.d. UAS-spo + 0 0 0 - 84 n.d. n.d. The number of ouib29/ouib74 animals that grew up to the third instar larval, pupal and adult stages was scored. Tb+ indicates ouib29/ouib74 animals, while Tb- indicates ouib29/TM6 or ouib74/TM6 animals from the parental strains in the same experimental batches. Tb- animals can be used as internal controls. Values in parentheses indicate the percentage of animals that survive to the pupal and adult stages. The animals were fed standard cornmeal food without any steroidal supplements. Detailed genetic crosses for this experiment are described in Materials and Methods. n.d., not determined. Table 2. Rescue of ouib mutant animals by spo overexpression in the PG. The number of ouib29/ouib74 animals that grew up to the third instar larval, pupal and adult stages was scored. Tb+ indicates ouib29/ouib74 animals, while Tb- indicates ouib29/TM6 or ouib74/TM6 animals from the parental strains in the same experimental batches. Tb- animals can be used as internal controls. Values in parentheses indicate the percentage of animals that survive to the pupal and adult stages. The animals were fed standard cornmeal food without any steroidal supplements. Detailed genetic crosses for this experiment are described in Materials and Methods. n.d., not determined. The number of ouib29/ouib74 animals that grew up to the third instar larval, pupal and adult stages was scored. Tb+ indicates ouib29/ouib74 animals, while Tb- indicates ouib29/TM6 or ouib74/TM6 animals from the parental strains in the same experimental batches. Tb- animals can be used as internal controls. Values in parentheses indicate the percentage of animals that survive to the pupal and adult stages. The animals were fed standard cornmeal food without any steroidal supplements. Detailed genetic crosses for this experiment are described in Materials and Methods. n.d., not determined. doi:10.1371/journal.pgen.1005712.t002 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Drosophila Steroidogenic Transcription Factor Ouija Board Fig 5. Transcriptional activity of Ouib for the upstream element of spok. (A) Fluorescence images of the PG cells from control and ouib mutant larvae with spok enhancer/promoter-driven nuclear localized-GFP construct (spok>GFP) at 36 hours AEL. PG cells are immunostained with antibody against Sro (magenta). Scale bar: 20 μm. (B) Luciferase reporter assay with plasmids containing the series of upstream elements of spok. Numbers indicate the distance from the translation initiation site (+1) of spok. The red bar indicates the Ouib-response element (-166 to -152). The white box represents the coding regions. Reporter activities of progressive deletion constructs are shown right (N = 3). The GFP expression plasmid was used as a negative control. (C) Schematic representation of the location of Ouib-response element (-166 to -152) in the spok enhancer/promoter region. The nucleotide sequence is shown below the cartoon of spok gene structure. The bar and box with red color indicate the 15 bp Ouib-response element. The black bar and the box indicate the enhancer/ promoter and coding region, respectively. (D) Luciferase reporter assay with plasmids containing the 15 bp transversion mutation in the -166 to -152 region of the 300 bp upstream element of spok (N = 3). The GFP expression plasmid was used as a negative control. Error bars indicate s.e.m. ; P<0.05, *; P<0.01 with Student’s t-test. (A) Fluorescence images of the PG cells from control and ouib mutant larvae GFP) at 36 hours AEL. PG cells are immunostained with antibody against Sro taining the series of upstream elements of spok. Numbers indicate the distance response element (-166 to -152). The white box represents the coding regions. The GFP expression plasmid was used as a negative control. (C) Schematic spok enhancer/promoter region. The nucleotide sequence is shown below the bp Ouib-response element. The black bar and the box indicate the enhancer/ plasmids containing the 15 bp transversion mutation in the -166 to -152 region of as used as a negative control. Error bars indicate s.e.m. ; P<0.05, *; P<0.01 Fig 5. Transcriptional activity of Ouib for the upstream element of spok. (A) Fluorescence images of the PG cells from control and ouib mutant larvae with spok enhancer/promoter-driven nuclear localized-GFP construct (spok>GFP) at 36 hours AEL. PG cells are immunostained with antibody against Sro (magenta). Scale bar: 20 μm. Identification of Ouija board-response element in the spookier enhancer region From these results, we hypothesized that the Ouib-response element lay between -166 to -152 bps (Fig 5C). To clarify the importance of this 15 bp region for Ouib-dependent control of gene expression, we intro- duced transversion mutations of the entire 15 bp sequence. This mutated construct exhibited no luc reporter induction in the presence of Ouib upon transfection into S2 cells (Fig 5D). We PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 10 / 26 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 (B) Luciferase reporter assay with plasmids containing the series of upstream elements of spok. Numbers indicate the distance from the translation initiation site (+1) of spok. The red bar indicates the Ouib-response element (-166 to -152). The white box represents the coding regions. Reporter activities of progressive deletion constructs are shown right (N = 3). The GFP expression plasmid was used as a negative control. (C) Schematic representation of the location of Ouib-response element (-166 to -152) in the spok enhancer/promoter region. The nucleotide sequence is shown below the cartoon of spok gene structure. The bar and box with red color indicate the 15 bp Ouib-response element. The black bar and the box indicate the enhancer/ promoter and coding region, respectively. (D) Luciferase reporter assay with plasmids containing the 15 bp transversion mutation in the -166 to -152 region of the 300 bp upstream element of spok (N = 3). The GFP expression plasmid was used as a negative control. Error bars indicate s.e.m. ; P<0.05, *; P<0.01 with Student’s t-test. doi:10.1371/journal.pgen.1005712.g005 also conducted subsequent reporter assays using constructs carrying various mutations in the 15 bp sequence. None of the constructs carrying any of several 3 bp substitutions within the 15bp sequence eliminated the responsive to Ouib (S6 Fig). Therefore, we conclude that Ouib binding tolerates degeneracy throughout the 15 bp sequence (5´-AGCTTTATTATTTAG-3´). We also examined the evolutionary conservation of the Ouib-response elements in putative spok enhancer regions in 12 Drosophilide species whose genome sequences have been deter- mined [52]. EMBOSS Matcher, an algorithm to identify local similarities between two sequences [54], found sequence motifs similar to the D. melanogster Ouib-response element in almost all of the Drosophilidae species (S7 Fig). In particular, the D. yakuba putative spok enhancer contains exactly the same 15 bp sequence motif. In addition, in the species belonging also conducted subsequent reporter assays using constructs carrying various mutations in the 15 bp sequence. None of the constructs carrying any of several 3 bp substitutions within the 15bp sequence eliminated the responsive to Ouib (S6 Fig). Therefore, we conclude that Ouib binding tolerates degeneracy throughout the 15 bp sequence (5´-AGCTTTATTATTTAG-3´). We also examined the evolutionary conservation of the Ouib-response elements in putative spok enhancer regions in 12 Drosophilide species whose genome sequences have been deter- mined [52]. ouija board transcript level is not affected by PTTH signaling Previous studies found that the increase of spok expression in the late third instar larvae is posi- tively controlled by prothoracicotropic hormone (PTTH) [55,56]. We therefore examined whether ouib expression changed in response to down regulation of PTTH signaling. However, when the levels of the PTTH receptor gene torso were knocked down in the PG by RNAi, we observed no change in ouib expression, suggesting that PTTH regulation of spok is not medi- ated through ouib (S8 Fig). Ouija board physically associates with the Ouija board-response element We sought to further establish if Ouib binds directly to the Ouib-response element by perform- ing a DNA/protein binding assay. We first examined the physical interaction between the Ouib-response element sequence and Ouib protein by an ABCD assay, which uses biotin con- jugated, double-stranded oligonucleotides containing the Ouib-response element sequences. Nuclear extracts obtained from S2 cells expressing FLAG-ouib were mixed with the biotin- labeled oligonucleotide, and then the protein-oligonucleotide complexes were pulled down using streptavidin beads. We found that FLAG-Ouib protein bound strongly to the wild type Ouib-response element probe, but not to the mutated probe (Fig 6A). In the control experi- ments, a biotinylated probe corresponding to M1BP (another ZAD-ZFP homolog of Ouib) binding sequence in the enhancer of smoothened locus [51] did not efficiently precipitate FLA- G-Ouib. Conversely, FLAG-M1BP protein did not bind to the Ouib-response element, while it bound to M1BP binding element (Fig 6A). To exclude the possibility that FLAG-Ouib protein isolated from cultured cells, indirectly associated with the probe through a complex containing some other endogenous transcription factor unrelated to Ouib, we prepared an E. coli produced recombinant protein containing the C-terminal 5 zinc finger domains (Ouib-Zf), and performed electrophoretic mobility shift assays (EMSAs) between the recombinant protein and the 15 bp Ouib-response element. We utilized 45 bp radiolabeled DNA probes, whose sequences corresponded to the spok enhancer region containing the 15 bp Ouib-response element. We found that the wild type oligonucleo- tide probes formed DNA/protein complexes with GST-Ouib-Zf, but not with GST alone (Fig 6B and 6C). In contrast, such DNA/protein complexes were not detected when radiolabeled mutated Ouib-response element sequences or sequences corresponding to the M1BP site [51] (Fig 6D) were used as probes, thereby confirming the specificity of the binding. Moreover, the complexes with the wild type 45 bp probes were outcompeted by unlabeled 45 bp DNA probes with the wild type Ouib-response element sequences, but not by the unlabeled mutated DNA probes or by the unlabeled M1BP probes (Fig 6B and 6C). Taken together, these findings strongly support the idea that Ouib specifically regulates spok transcription by direct binding to the Ouib-response element in the spok enhancer. Drosophila Steroidogenic Transcription Factor Ouija Board to the subgenus Sophophora, which includes D. melanogaster, the Ouib-response element-like motifs are found in proximity (within 500 bp) to the spok coding region (S7 Fig). These data suggest that Ouib-like response elements are also evolutionarily conserved to some degree. EMBOSS Matcher, an algorithm to identify local similarities between two sequences [54] found sequence motifs similar to the D melanogster Ouib response element in also conducted subsequent reporter assays using constructs carrying various mutations in the 15 bp sequence. None of the constructs carrying any of several 3 bp substitutions within the 15bp sequence eliminated the responsive to Ouib (S6 Fig). Therefore, we conclude that Ouib binding tolerates degeneracy throughout the 15 bp sequence (5´-AGCTTTATTATTTAG-3´). We also examined the evolutionary conservation of the Ouib-response elements in putative spok enhancer regions in 12 Drosophilide species whose genome sequences have been deter- mined [52]. EMBOSS Matcher, an algorithm to identify local similarities between two sequences [54], found sequence motifs similar to the D. melanogster Ouib-response element in almost all of the Drosophilidae species (S7 Fig). In particular, the D. yakuba putative spok enhancer contains exactly the same 15 bp sequence motif. In addition, in the species belonging PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 11 / 26 Discussion In this study, we have demonstrated that the ZAD-ZFP Ouib is required for ecdysteroid bio- synthesis in the PG during D. melanogaster development. The following points summarize our finding. First, ouib is predominantly expressed in the PG during embryonic and larval stages. Second, ouib null mutants exhibit early (first instar) larval developmental arrest due to a low PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 12 / 26 Drosophila Steroidogenic Transcription Factor Ouija Board Fig 6. DNA-Binding analysis of Ouib for the upstream element of spok. (A) ABCD assay with nuclear extracts from S2 cells and avidin-biotin-conjugated double-stranded DNA probes. After pull-down, proteins were detected by western blotting using anti-FLAG antibody. (B) EMSA using recombinant proteins of GST alone or GST-fusion zinc finger domains of Ouib (amino acids 150–313) with 32P-labeled double-stranded oligonucleotide probes containing the wild type 15 bp Ouib-response element (ORE). The x100 and x200 amounts of the non-labeled probes of the wild type ORE (ORE WT), the mutated ORE (ORE Mut) and the wild type M1BP-response element (MRE WT) were used as cold competitors. (C) Densitometric analysis of the EMSA band radioactive intensities in the same experimental condition as B with 3 independent replicates. Average radioactivity of the ORE/GST-Ouib-Zf complex is set as 100%. Note that the complexes with the ORE were outcompeted by the unlabeled ORE WT probe, but not by the unlabeled MRE WT probe. P<0.05 and **P<0.01 by Tukey's multiple comparisons test. n.s., not significant. (D) EMSA using recombinant proteins of GST-fusion zinc finger domains of Ouib (amino acids 150–313) with 32P-labeled double-stranded oligonucleotide probes of ORE WT, ORE Mut and MRE WT. doi:10.1371/journal.pgen.1005712.g006 ecdysteroid titer. Third, the larval arrest phenotype is caused by a failure of spok expression in the PG, and is rescued by sole overexpression of a spok paralog. Finally, a specific Ouib- response element that binds Ouib was identified in the enhancer region of spok. Our study reports on the discovery of the first invertebrate tissue-specific, steroidogenic transcription factor. ouib mutants exhibit a drastic reduction of spok expression. However, we point out that ouib mutants also show a mild statistically-significant reduction of dib and sad (Fig 4A). In fact, while no DNA sequences exactly matching the spok Ouib-response element (5´-AGCTT TATTATTTAG-3´) are found elsewhere in D. melanogaster genome, a number of degenerate sequences do exist in the genome, including the regions upstream of ecdysteroidogenic gene coding regions (S9 Fig). Considering the fact that the luciferase constructs carrying any of sev- eral 3 bp substitutions within the 15bp sequence are still responsive to Ouib (S6 Fig), we cannot completely rule out the possibility that Ouib is also involved in direct transcriptional regulation of genes other than spok, particularly dib and sad. Drosophila Steroidogenic Transcription Factor Ouija Board Drosophila Steroidogenic Transcription Factor Ouija Boa PLOS Genetics \| DOI:10 1371/journal pgen 1005712 December 10 2015 13 / 2 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 13 / 26 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Drosophila Steroidogenic Transcription Factor Ouija Board limiting the food source of Drosophila pachea to a single species of cactus [60]. Further assess- ment of the biological and evolutionary roles of ouib and spok will require determining which transcription factors are involved in the transcriptional regulation of D. melanogaster spo and Cyp307a genes in other insects. Since there are many divergent ZAD-ZFP genes in each insect genome and they are expanded in insect lineage-specific manner [47], it is possible that a dif- ferent ZAD-ZFP gene whose primary structure is not orthologous to ouib could be a transcrip- tion factor for other Cyp307a genes. Regarding the evolutionarily aspect of ouib, it is important to recognize that spok expression is regulated by another ZAD-ZFP called Molting defective (Mld) [21,37,39]. Interestingly, just like ouib, mld genes are also found only in genomes of Drosophilidae but not other insects [21,37]. In contrast to ouib, Mld does not appear to be specific for the regulation of spok expres- sion. First, Mld, unlike ouib, is expressed in several other tissues during development besides the PG [37]. Second, Mld is essential for regulating expression Nvd as well as spok and perhaps other genes [39]. Third and most important, the mld loss–of-function phenotype is not rescued by overexpressing either spo or spok [21]. Therefore, Ouib and Mld overlap in their regulation of spok expression, but also have distinct functions during development. While it is still unclear whether Mld is a transcription factor, it would be intriguing to examine a functional relation- ship between Ouib and Mld for induction of spok expression in the PG. According to our qRT-PCR data, it is less likely that Mld controls ouib expression in the PG (S10 Fig). Another question to be answered is how ouib expression is regulated during larval develop- ment. As shown above, it does not seem to be by PTTH. However, recent work as shown that spok and other ecdysteroidogenic enzyme genes are also influenced by humoral factors such as TGFβ/Activin [61] and monoaminergic tropic factors [62,63]. It will be interesting to deter- mine whether these factors affect spok expression in the PG through modulation of ouib levels. An additional significant aspect of this work is to provide the first evidence for the existence of a catalytic step-specific transcriptional regulation of steroid hormone biosynthesis in organ- isms. PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Nevertheless, our results indicate that the impairment of expression of dib and sad seems not to contribute to the ouib phenotype in a major way. First, the arrest during the first instar larval stage of ouib mutants is rescued by oral administration of 5βkd. Since Dib and Sad play roles in the terminal hydroxylation steps down- stream of the conversion of 5βkd to ecdysone [23,24,26], this finding suggests that the enzy- matic levels of Dib and Sad are still sufficient to make functional levels of ecdysone. Second and more importantly, the first instar larval arrest phenotype of ouib mutants is rescued by a sole overexpression of spo, which is functionally equivalent to spok. Therefore, in addition to the PG specificity, we would argue that the key additional feature of Ouib is its specific role in spok expression. To further clarify the extent to which Ouib regulates other genes, and the functional importance of these genes, will require additional studies including transcriptome analysis/ChIP-seq analysis together with eventual mutational analysis of any identified targets. y y g y y g Curiously, the presence of ouib only in the Drosophilidae genomes is concordant with the Drosophilidae-specific duplication of Cyp307a P450 subfamily. While members of the Cyp307 P450 subfamily, which includes spok, are found in all arthropod species examined so far [20,21,57,58], Drosophilidae Cyp307 genes have been duplicated within the Drosophila radia- tion [21,59]. In the case of D. melanogaster, the duplicated Cyp307 genes are Cyp307a1/spo and Cyp307a2/spok, which are sub-functionally divergent in terms of gene expression pattern; spo is expressed in early embryogenesis and oogenesis, while spok is expressed in the PG cells in late embryogenesis as well as the larval and pupal stages [20,21]. Our data demonstrate that the spatiotemporal expression pattern of ouib closely matches that of spok but not spo. Notably, neither ouib nor spok transcripts are detected in embryonic stages 5–9 when the embryonic ecdysteroid titer is maximal [21,45], indicating that these genes do not contribute to producing embryonic ecdysteroids. Therefore, an acquisition of ouib might be a critical event for the sub- functionalization of two Cyp307 genes by changing the regulation of their expression during the Drosophilidae evolution. In terms of evolution, it is worth mentioning that there is a case where evolution changed the activity of a single ecdysteroidogenic enzyme (Nvd) dramatically PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 14 / 26 Drosophila Steroidogenic Transcription Factor Ouija Board Materials and Methods Drosophila strains Drosophila melanogaster flies were reared on standard agar-cornmeal medium at 25°C under a 12:12 h light/dark cycle. w1118, yw and Oregon R were used as the wild type strain. phm– GAL4#22 [55] and w; UAS-dicer2; phm-GAL4#22/TM6 Ubi-GFP was used as the strain to drive forced gene expression in the PG. UAS-spo [20] and UAS-spok-IR [21] transgenic flies were obtained from Hiroshi Kataoka (The University of Tokyo) and Hajime Ono (Kyoto Univer- sity), respectively. y1 v1 nos-phiC31; attP40, v1 and y2 cho2 v1; attP40{nos-Cas9}/CyO [53] were obtained from National Institute of Genetics, Japan. The w; snaSco/CyO; P{w+mC = tubP- GAL80ts}7 (stock number #130453) and w1118; Df(3R)ED5330/TM6C Sb1, a deficiency strain that deletes a genomic region including the ouib locus (stock umber #150241) [67], were obtained from Drosophila Genetic Resource Center. UAS-torso-IR (stock number #101154) and UAS-mld-IR (stock number #17329) were obtained from the Vienna Drosophila RNAi center. in situ RNA hybridization Digoxygenin (DIG)-labeled antisense RNA probes were synthesized using DIG RNA labeling mix (Roche) and T3 and T7 RNA polymerase (Fermentas). To generate the ouib probe, the ouib ORF was amplified by PCR with cDNA derived from whole bodies of Oregon R larvae and the primers described in S3 Table. PCR product was inserted into SmaI-digested pBluescript II SK (-), and then used as the templates for synthesizing RNA probes. Fixation, hybridization and detection were performed as [23,68]. Quantitative reverse transcription (qRT)-PCR RNA was isolated using the RNAiso Plus reagent (TaKaRa). Genomic DNA digestion and cDNA synthesis were performed using the ReverTra Ace qPCR RT Kit (TOYOBO). qRT-PCR was performed using the THUNDERBIRD SYBR qPCR Mix (TOYOBO) or Universal SYBR Select Master Mix (Applied Biosystems) with a Thermal Cycler Dice TP800 or TP870 system (TaKaRa). Serial dilutions of a plasmid containing the ORF of each gene were used as a stan- dard. The expression levels of the target genes were normalized to an endogenous control ribo- somal protein 49 (rp49) in the same sample. The primers for quantifying D. melanogaster ouib and E75A are described in S3 Table. Primers amplifying nvd, sro, spok, phm, dib, sad and rp49 were previously described [22,55]. PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Whereas the substrate of Spok and its product have not yet been identified, Spok appears to play a crucial role in the “Black Box” step of ecdysteroid biosynthetic pathway, and it is a strong candidate for acting as a rate-limiting enzyme in the pathway [10,21,64]. Interestingly, a recent study has reported that pre-mRNA splicing of spok, but not any other ecdysteroidogenic genes expressed in the PG, seems to specifically depend on a protein encoded by ecdysoneless (ecd), whose mutant phenotype includes ecdysteroid deficiency [65]. Thus, a rate-limiting step of ecdysteroid biosynthesis catalyzed by Spok could be under tight control by both specific transcriptional and post-transcriptional mechanisms. Currently, it is unknown whether such catalytic-specific transcriptional and/or posttranscriptional mechanisms also exist in other organisms including vertebrates. Similar to ecdysteroids, vertebrate steroid hormones are synthesized via several intermedi- ates by multiple steroidogenic enzymes. Among them, the rate-limiting step in vertebrate ste- roid hormone productions is the delivery of substrate cholesterol from the outer mitochondrial membrane to the inner one and the subsequent conversion of cholesterol to pregnenolone by CYP11A1. It is attractive to hypothesize that the rate-limiting step in vertebrate steroid hor- mone biosynthesis is also specifically regulated by unidentified transcriptional and/or splicing regulator(s). Whereas no apparent orthologs of ouib are found in vertebrates, their genomes possess a ZAD-ZFP gene called ZFP276, which is a tumor suppressor gene [66]. Interestingly a ecd ortholog is also found in humans and may also contribute to the malignancy of certain tumor types [65]. It would be worth examining roles of these genes in steroid hormone biosyn- thesis in vertebrates. PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 15 / 26 Drosophila Steroidogenic Transcription Factor Ouija Board UAS vectors, overexpression of genes and generation of transgenic strains The GAL4-UAS system [69] was used to overexpress genes in D. melanogaster. To generate pUAST vector to overexpress ouib, specific primers including a sequence coding FLAG tag at N terminal were used for PCR to add EcoRI and XbaI sites to the 5´ and 3´ ends, respectively (S3 Table). Template cDNAs were reverse transcribed using total RNA of the ring gland from D. melanogaster using ReverTra Ace qPCR RT Kit (TOYOBO). PCR was performed using KOD Plus Neo (TOYOBO). The amplified CDS region of ouib was digested with EcoRI and XbaI, and then ligated into a pWALIUM10-moe vector [70]. Transformants were established by BestGene, Inc. Generation of the ouib alleles Generation of the ouib allele was carried out by CRISPR/Cas9 system using the pBFv-U6.2 vec- tor [53] provided by the National Institute of Genetics, Japan. We selected 2 independent target sites (target#1 and target#2 as shown in Fig 2). To minimize off-target effects of CRISPR/Cas9 system, we confirmed by BLAST search that no 15 nucleotide stretches within the selected tar- get sequence (23 nucleotides including PAM motif) matched any other sequence on the 3rd chromosome. Sense and antisense oligonucleotides corresponding to sgRNA target sequences (S3 Table) were annealed and inserted into BbsI-digested pBFv-U6.2 vector. The ouib sgRNA vectors were injected into the embryos of the y1 v1 nos-phiC31; attP40 strain. The nos-Cas9- based gene targeting was carried out as previously described [53]. Males carrying nos-Cas9 and a sgRNA transgene were crossed to wild-type flies by mass mating. From their progeny, 10 and 50 single males for the target#1 and target#2 sites, respectively, were isolated. Each male was crossed with w; TM3 Sb/TM6 Tb females and then the independent isogenized strains were established. Among them, we surveyed the strains showing homozygous lethality and eventually 1 target#1 and 29 target#2 lethal strains were selected. To confirm indel mutations at ouib locus in each strain, we performed the T7EI assay as previously described [53]. In this assay, genome DNA from the heterozygous adults of each strain was extracted as previously described [53]. To amplify the DNA fragment including Cas9 target sites, PCR was conducted with KOD FX Neo (TOYOBO), the extracted genome DNA, and the primers listed in S3 Table [53]. The PCR products were treated with T7 endonuclease (NEB). The reacted samples were analyzed by agarose gel electrophoresis. Out of 30 total candidate strains, 1 target#1 and 8 tar- get#2 strains were selected as candidate flies possessing indel mutations in ouib region. The PCR products from the 9 strains were subcloned into a SmaI-digested pBluescript II (Promega) and then sequenced with T3 and T7 primers. We detected small deletions in 8 out of the 9 strains. The minimal and maximal deletion sizes were 1 bp and 13 bp, respectively. We chose 1 strain for each target sites for further analyses and renamed them ouib29 and ouib74, both of which caused frameshift mutations for ouib locus (Fig 2). Immunostaining Tissue dissections were performed in PBS followed by fixation in 4% PFA for 20 minutes at room temperature. For this study, the following primary antibodies were: mouse anti-FLAG M5 (1:1,000) (Sigma); rabbit anti-Phm (1:200) [30], guinea pig anti-Spok (1:200) [61]; guinea pig anti-Sro (1:1,000) [62]. Tissues were incubated over night with primary antibodies at 4°C. Fluorescent conjugated secondary antibodies used in this study, goat anti-mouse Alexa Fluor 488, goat anti-guinea pig Alexa Fluor 488, goat anti-rabbit Alexa Fluor 555 and goat anti- guinea pig Alexa Fluor 555, were purchased from Life Technologies. Secondary antibodies were diluted 1:500 and incubated for 1 hour at room temperature. Confocal images were cap- tured using Carl Zeiss LSM 700 laser scanning microscope. PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 16 / 26 Drosophila Steroidogenic Transcription Factor Ouija Board Genetic rescue experiments with ouib and spo For the rescue experiments of ouib mutant by ouib overexpression, ouib29 phm-GAL4#22/TM3 Act-GFP was established by chromosomal recombination. The flies of UAS-FLAG-ouib-1M; ouib74/TM6 Ubi-GFP were crossed with the flies of ouib29 phm-GAL4#22/TM3 Act-GFP, the flies of ouib74/TM3 Act-GFP were crossed with the flies of ouib29 phm-GAL4#22/TM3 Act-GFP, and the flies of UAS-FLAG-ouib-1M; ouib74/TM6 Ubi-GFP were crossed with the flies of ouib29/TM3 Act-GFP. Eggs were laid on grape plates with yeast pastes at 25°C for 12 hours. At 36 hours AEL, 50 hatched GFP negative (UAS-FLAG-ouib-1M/+; ouib29 phm-GAL4#22/ouib74, ouib29 phm-GAL4#22/ouib74 and UAS-FLAG-ouib-1M/+; ouib29/ouib74) first instar larvae were transferred to the standard agar-cornmeal medium. Developmental stages were scored 108 hours AEL by tracheal morphology as previously described [22]. For the rescue experiments of spok RNAi by spo overexpression, UAS-spok-IR UAS-spo was established by chromosomal recombination on third chromosome. The flies of UAS-spok-IR UAS-spo strain was crossed with w; UAS-dicer2; phm-GAL4#22/TM6 Ubi-GFP flies. Eggs were laid on standard agar-cornmeal medium at 25°C for 24 hours. After 7 days, developmental stages of the animals on the wall were scored by presence of TM6 balancer. For the rescue experiments of ouib mutant by spo overexpression, Roi/CyO; ouib29 phm- GAL4#22/TM6, Roi/CyO; ouib29 UAS-spo/TM6 and Roi/CyO; ouib74 UAS-spo/TM6 were estab- lished by chromosomal recombination on third chromosome. The flies of Roi/CyO; ouib29 phm-GAL4#22/TM6 were crossed with Roi/CyO; ouib74 UAS-spo/TM6, the flies of Roi/CyO; ouib29 phm-GAL4#22/TM6 were crossed with Roi/CyO; ouib74/TM6 and Roi/CyO; ouib74/TM6 were crossed with Roi/CyO; ouib29 UAS-spo/TM6. Eggs were laid on standard agar-cornmeal medium at 25°C for 24 hours. After 7 days, developmental stages of the animals on the wall were scored by presence of TM6 balancer. Feeding rescue experiments with ecdysteroids and intermediates For the rescue experiments, 20 mg of dry yeast was mixed with 38 μl H2O and 2 μl ethanol or supplemented with 2 μl of the following sterols dissolved in ethanol: cholesterol (Wako; 150 mg/ml), 7-dehydrocholesterol (Sigma; 150 mg/ml), 5β-ketodiol (kindly gifted from Yoshi- nori Fujimoto, Tokyo Institute of Technology; 150 mg/ml) and 20-hydroxyecdysone (Sigma; 50 mg/ml). We crossed ouib29/TM3 Ser1 GMR2 Act-GFP flies with ouib74/TM3 Ser1 GMR2 Act-GFP flies. Eggs were laid on grape plates with yeast pastes at 25°C for 12 hours. At 36 hours AEL, 50 hatched GFP negative (ouib29/ouib74) first instar larvae were transferred to the yeast paste on grape plates and kept at 25°C. Every 24 hours, developmental stages were scored by tracheal morphology as previously described [22]. PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Scoring of developmental progression of ouib mutants ouib29/TM3 Act-GFP flies, ouib74/TM3 Act-GFP flies and w1118 flies were crossed each other. Eggs were laid on grape plates with yeast pastes at 25°C for 8 hours. 36 hours AEL, 100 hatched GFP negative (ouib29/+, ouib74/+ and ouib29/ouib74) first instar larvae were transferred into vials with standard cornmeal food (25 animals per vial). Every 24 hours, developmental stages were scored by tracheal morphology as previously described [22]. PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 17 / 26 Transfection and luciferase reporter assays S2 cells were seeded in 1 ml Schneider’s Drosophila Medium (GIBCO) in a 24-well plate (grei- ner bio-one) 1 day before transfection. Transfection of S2 cells was performed using the Effectene Transfection Reagent (Qiagen). GFP-pUAST [23] and FLAG-ouib-pWALIUM10- moe plasmids were transfected, respectively, along with the Actin5C-GAL4 construct (a gift from Yasushi Hiromi, National Institute of Genetics) and the luciferase reporter plasmids. The Copia Renilla Control plasmid (addgene; #38093) [72] was used as the reference. The cells were incubated for 2 days after transfection. Then they were processed by using the Dual-Lucif- erase Reporter Assay System (Promega) in accordance with the manufacturer’s instructions and were analyzed with Flash’n glow LB 955 (Berthold Technologies). GFP reporter assay To generate the spok>GFP reporter construct, a ~1.4 kb fragment immediately upstream of the spok transcription unit was amplified from yw genomic DNA using the primers 1.45spok- p_F and 1.45spok-p_R (S3 Table). This fragment was first subcloned into the pCR2.1-TOPO vector (Life Technologies) and then removed as an EcoRI fragment and cloned into the Dro- sophila transformation vector pH-Stinger [71]. To refine the location of the PG enhancer, seven 250–300 bp overlapping fragments that covered the entire 1.4 kb fragment were derived through PCR and each cloned into hH- Stinger. The only fragment that gave expression in the PG of transgenic animals was the ~300 bp fragment immediately upstream of the transcrip- tional start site. This fragment was generated using the primers 300spok-p_F and 300spok-p_R (S3 Table). Transgenic lines were generated through standard means using a w1118 host back- ground. The GFP reporter strains of spok>GFP; ouib29/TM6 Ubi-GFP and spok>GFP; ouib74/ TM6 Ubi-GFP were established and crossed each other. Eggs were laid on grape plates with yeast pastes at 25°C for 4 hours. The first instar larvae were dissected 36 hours AEL and immunostained. Construction of luciferase reporter plasmids The upstream regions of spok were amplified from Oregon R genomic DNA by specific primers to add SacI and BglII sites to the 5´ and 3´ ends, respectively. PCR was performed using KOD Plus Neo (TOYOBO). The amplified upstream regions of spok were digested with SacI and BglII, and then ligated into a pGL3-Basic vector luciferase reporter plasmid (Promega). Reporter plasmids carrying mutated regions were constructed from the pGL3-Basic plasmid containing WT upstream 300 bp region by inverse PCR. The primers for PCR are listed in S3 Table. Drosophila Steroidogenic Transcription Factor Ouija Board generated using 20E (Sigma). Absorbance was measured at 415 nm on a plate reader, Multis- kan GO (Thermo Scientific) using the SkanIt Software 3.2 (Thermo Scientific). Ecdysteroid measurement ouib29/TM3 Ser1 GMR2 Act-GFP flies and w1118 flies were crossed with ouib74/TM3 Ser1 GMR2 Act-GFP flies. Eggs were laid on grape plates with yeast pastes at 25°C and the hatched larvae were cleared. After 8 hours, GFP negative (ouib74/+ and ouib29/ouib74) first instar larvae were transferred into vials with standard cornmeal food. At 12 hours AH, whole larvae were rinsed in water and homogenized in 50 μl methanol and supernatant was collected following centrifu- gation at 14,000 rpm at 4°C. The remaining tissue was re-extracted in 50 μl methanol over night at 4°C. The supernatants were evaporated using a EYELA CVE-2000 (Tokyo Rikakikai) and redissolved in 50 μl EIA buffer [0.1 M PBS/0.1% BSA, 0.4 M NaCl, 1 mM EDTA and 0.01% NaN3]. ELISA was performed according to manufacturer’s instructions using 20-Hydro- xyecdysone EIA Antiserum, 20-Hydroxyecdysone AChE Tracer and Ellman’s Reagent (Cay- man Chemical) that detects 20-hydroxyecdysone with the same affinity. Standard curves were PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 18 / 26 Preparation of recombinant proteins in E coli GST proteins fused with or without 150–313 amino acid residues of Ouib (GST-Ouib-Zf) con- taining 5 zinc finger domains were expressed using pGEX-4T-3 vector system (GE Healthcare) in Escherichia coli BL-21 strain. E. coli cells were harvested and crashed with sonication. GST alone and GST-Ouib-Zf were purified from the supernatant with AKTA start equipped with GSTrap affinity column (GE Healthcare). Electrophoretic mobility shift assay (EMSA) Electrophoretic mobility shift assay was conducted as previously described [74,75]. 45 bp dou- ble-stranded oligonucleotide probes containing wild type M1BP binding site, wild type and mutated (transversion) Ouib response element were prepared by annealing single-strand oligo- nucleotides listed in S3 Table. The wild type M1BP binding site was derived from the smooth- ened promoter [51]. Double-stranded DNA fragment was end-labeled by using T4 polynucleotide kinase (TOYOBO) and [γ-32P]ATP. GST or GST-Ouib fusion proteins (400 ng) were incubated for 30 min at 4°C in the reaction mixture [12 mM Hepes, pH 7.9, 1 mM dithiothreitol, 1 mM EDTA, 60 mM KCl, 4 mM MgCl2, 2 mM ZnSO4, 50 ng/ul poly(dI-dC), 1 mg/ml BSA and 12% Glycerol] in the presence or absence of 100–200-fold molar excess of specific double-stranded competitor DNA. A radiolabeled DNA probe (0.3 ng, 40,000 cpm) was added, and the incubation was continued for 20 min at 4°C. The incubation mixture was directly loaded on a 5% non-denaturing polyacrylamide gel in 1 × TBE buffer [89 mM Tris- HCl, pH 8.0, 89 mM boric acid, and 2 mM EDTA], and electrophoresed at 4°C with buffer cir- culation. The gels were dried and analyzed with a bio-imaging analyzer Typhoon 8600 (Amer- sham Pharmacia Biotech Inc). The competitor oligonucleotides used are listed in the S3 Table. Avidin-Biotin-Conjugated DNA-Binding (ABCD) assay Preparation of S2 cell nuclear extracts is described in the Supplemental Materials. ABCD assay was conducted essentially as previously described [73]. Biotin-labeled DNA probes were pur- chased from Life Technologies. The probes were incubated with Dynabeads M-280 Streptavi- din (Life Technologies) at room temperature for 15 min. DNA-beads complexes were mixed with nuclear extracts and ABCD Binding Buffer [50 mM Hepes pH 7.9, 150 mM NaCl, 0.5% Triton X-100, 20 ng/μl poly(dI/dC)], and incubated at 4°C for 1 hour. After incubation, the beads were washed with ABCD Binding Buffer. The biotin-labeled oligonucleotides are listed in S3 Table. Western blotting Samples were boiled with SDS sample buffer [150 mM Tris-HCl pH 6.8, 0.6% SDS, 15% glyc- erol, 0.009 mg/μl Bromophenol blue, 5% 2-mercaptoethanol and 1 unit Complete Mini (Roche)] for 5 min, and loaded on 12% polyacrylamide gel followed by transfer onto PVDF membrane (GE Healthcare). Anti-FLAG M5 monoclonal antibody (1:1,000; Sigma) was used for primary antibody and ECL Peroxidase labeled anti-mouse antibody (1:10,000; GE Health- care) was used for secondary antibody. The band was detected by ECL Ultra Lumigen TMA-6 (GE Healthcare) and Ez-Capture MG (ATTO). Drosophila Steroidogenic Transcription Factor Ouija Board Preparation of S2 cell nuclear extracts S2 cells overexpressing FLAG-ouib or FLAG-M1BP were collected and washed with TBS. Cells were then centrifuged at 4000 g at 4°C for 5 min. The pellet was suspended and vortexed with 400 μl Buffer A [10 mM Hepes pH 7.9, 10 mM KCl, 1 mM DTT and 1 unit Complete Mini (Roche)] and 25 μl 10% NP-40. Then sample was centrifuged at 1500 g at 4°C for 5 min. The pellet was suspended and vortexed with 50 μl Buffer C [20 mM Hepes pH7.9, 400 mM NaCl, 2 mM MgSO4, 1mM DTT and Complete Mini (Roche)], then shaked at 4°C for 30 min. After shaking, sample was centrifuged at 14,000 rpm at 4°C for 5 min and supernatant was collected. PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 19 / 26 Drosophila Steroidogenic Transcription Factor Ouija Board putative proteins as a query is most similar to that of D. melanogaster CG11762. (PDF) S2 Table. Rescue of spok RNAi animals by spo overexpression in the PG. The number of spok RNAi animals that grew up to the 3rd instar larval stage or later stage was scored. Detailed genetic crosses for this experiment are described in Materials and Methods. The animals were fed standard cornmeal food without any steroidal supplements. Values in parentheses indicate the number of control non-RNAi progeny from the parental strains in the same experimental batches. (PDF) S3 Table. List of primers/oligonucleotide DNAs used in this study. Small letters indicate the restriction enzyme recognition sequences. Under lines indicate the transversion mutation sequences. Asterisks indicate 5´ biotinylation. (PDF) S1 Fig. RNA in situ hybridization using embryos with antisense and sense ouib RNA probes. Dorsal views are shown. (A) Signals with antisense probe. (B) Signals with sense probe. Arrows indicate positions of the PG primordia. Scale bar: 100 μm. (PDF) S2 Fig. The genomic structure of ouija board (CG11762) and surrounding genes. The data are derived from the FlyBase GBrowse website (http://flybase.org/cgi-bin/gbrowse2/dmel/? Search=1;name=FBgn0037618). Numbers indicate the nucleotide positions at the 85A9 cytological position of the chromosome 3R scaffold. Boxed arrows represent gene spans and their directions. The 5 ZAD-ZNF genes are colored by magenta. (PDF) S3 Fig. The temporal expression profiles of ouib expression and ecdysteroid levels during the third instar larval development. ouib expression and ecdysteroid levels in w1118 during the 3rd instar stage measured by qRT-PCR (N = 3) and ELISA (N = 4). The blue line indicates the relative expression level of ouib, normalized to the level of 0–6 hours after L2-L3 molting (0–6 hr A3L). Error bars indicate the s. e. m. (PDF) S4 Fig. GFP expression driven by the spok enhancer. (A, B) Phase-contrast (left) and fluores- cence (right) images of the 108 hours AEL 3rd instar larval brain-ring gland complexes with spok>GFP construct. The PG cells were immunostained with anti-Sro antibody (magenta). The spok>GFP constructs contain 1.45 kbp (A) and 300 bp (B) enhancer regiosn of spok, respectively. Scale bar: 100 μm. (PDF) S4 Fig. GFP expression driven by the spok enhancer. (A, B) Phase-contrast (left) and fluores- cence (right) images of the 108 hours AEL 3rd instar larval brain-ring gland complexes with spok>GFP construct. The PG cells were immunostained with anti-Sro antibody (magenta). Supporting Information S1 Table. Drosophilidae orthologs of ouija board. The orthologs of ouija board in 12 Droso- philidae species are described in the FlyBase website (http://flybase.org/reports/FBgn0209782. html). We confirmed by the BLAST search that the amino acid sequence of any of these 20 / 26 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Drosophila Steroidogenic Transcription Factor Ouija Board and the s. e. m., respectively, of three independent experiments. ; P<0.01 by Student’s t-test. (PDF) S7 Fig. The evolutionary conservation of sequences similar to Ouib-response element in putative spok enhancer/promoter regions of 12 Drosophilidae species. EMBOSS Matcher [54] was used to search for sequences similar to the D. melanogaster Ouib response element (15 bp) within the 1 kb regions upstream of the translation initiation site of the spok loci from 12 Drosophilidae species. Numbers before and after nucleotide sequences indicate the distance from the translation initiation site of spok. Parentheses indicate numbers of identical matches to D. melanogaster Ouib response element. “S” and “D” indicate the subgenera Sophophora and Dro- sophila, respectively. spok genes are DG27210 (D. simulans #1), GD27133 (D. simulans #2), GD28291 (D. simulans #3), GM22791 (D. sechellia), GG16659 (D. erecta), GE19452 (D. yakuba), GF20000 (D. ananassae), GA31537 (D. pseudoobscura), GL21970 (D. persimilis), GK19177 (D. willistoni), GI23968 (D. mojavensis), GH21174 (D. grimshawi) and GJ26360 (D. virilis). Note that a BLAST search using D. melanogaster Spok protein sequence as a query hit 3 D. simulans spok candidate genes. Also note that the BLAST search hit only one spo/spok family gene in D. grim- shawi genome and thus it is not faithfully judged if GH21174 is orthologous to spo or spok. (PDF) S8 Fig. Expression level of ouib in the PGs of control and torso RNAi third instar larvae. Amounts of ouib mRNAs were measured by qRT-PCR. phm>+ and phm>torso-IR indicate w1118; +/+; phm-GAL4#22/+ and w1118; UAS-torso-IR/+; phm-GAL4#22/+, respectively. RNA samples were collected 140 hours after egg laying. Bars and error bars represent the average and the s. e. m., respectively, of three biological replicates. n.s. means P>0.05 by Student’s t- test. (PDF) S9 Fig. Sequences similar to Ouib-response element in putative enhancers/promoters of ecdysteroidogenic genes in D. melanogaster. EMBOSS Matcher [54] was used to search for sequences similar to D. melanogaster Ouib response element (15 bp) within the putative enhancer/promoter regions of D. melanogaster ecdysteroidogenic enzyme genes. Numbers before and after nucleotide sequences indicate the distance from the translation initiation site of each gene. Parentheses indicate numbers of identical matches to D. melanogaster Ouib response element. Except for phm, a enhancer/promoter region was defined as a genomic region between the translation initiation site of each ecdysteroidogenic enzyme gene and the 3´ end of a gene next to the enzymatic gene. A phm enhancer/promoter is a -500 to -1 region as previously characterized [31]. S10 Fig. Expression level of spok and ouib in the PGs of control and mld RNAi first instar larvae. Amounts of spok and ouib mRNAs were measured by qRT-PCR. phm>dicer2 and phm>dicer2+mld-IR indicate w1118; UAS-dicer2/+; phm-GAL4#22/+ and w1118; UAS-dicer2/ UAS-mld-IR; phm-GAL4#22/+, respectively. RNA samples were collected 36 hours after egg laying. Bars and error bars represent the average and the s. e. m., respectively, of three biologi- cal replicates. and n.s. mean P<0.01 and P>0.05 by Student’s t-test, respectively. (PDF) The spok>GFP constructs contain 1.45 kbp (A) and 300 bp (B) enhancer regiosn of spok, respectively. Scale bar: 100 μm. (PDF) S5 Fig. Luciferase reporter assay with plasmids containing the series of upstream elements of spok. Numbers indicate the distance from the translation initiation site (+1) of spok, and white box represents the coding region of spok. Luc reporter activities of progressive deletion constructs are shown in right. Bars and error bars represent the average and the s. e. m., respec- tively, of three independent experiments. ; P<0.01 by Student’s t-test. (PDF) S5 Fig. Luciferase reporter assay with plasmids containing the series of upstream elements of spok. Numbers indicate the distance from the translation initiation site (+1) of spok, and white box represents the coding region of spok. Luc reporter activities of progressive deletion constructs are shown in right. Bars and error bars represent the average and the s. e. m., respec- tively, of three independent experiments. ; P<0.01 by Student’s t-test. (PDF) S5 Fig. Luciferase reporter assay with plasmids containing the series of upstream elements of spok. Numbers indicate the distance from the translation initiation site (+1) of spok, and white box represents the coding region of spok. Luc reporter activities of progressive deletion constructs are shown in right. Bars and error bars represent the average and the s. e. m., respec- tively, of three independent experiments. ; P<0.01 by Student’s t-test. (PDF) S6 Fig. Luciferase reporter assay with plasmids containing the triplet transversion muta- tions in the -166 to -152 region of the 300 bp upstream element of spok. The introduced transversion mutations in the 1’, 2’, 3’, 4’ and 5’ constructs are shown in the top. The GFP expression plasmid was used as a negative control. Bars and error bars represent the average S6 Fig. Luciferase reporter assay with plasmids containing the triplet transversion muta- tions in the -166 to -152 region of the 300 bp upstream element of spok. The introduced transversion mutations in the 1’, 2’, 3’, 4’ and 5’ constructs are shown in the top. The GFP expression plasmid was used as a negative control. Bars and error bars represent the average PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 21 / 26 References 1. Lavoie HA, King SR. Transcriptional regulation of steroidogenic genes: STARD1, CYP11A1 and HSD3B. Exp Biol Med. 2009; 234: 880–907. 2. Parker KL, Schimmer BP. Steroidogenic factor 1: a key determinant of endocrine development and function. Endocr Rev. 1997; 18: 361–377. PMID: 9183568 3. Parker KL, Rice DA, Lala DS, Ikeda Y, Luo X, Wong M, et al. Steroidogenic factor 1: an essential media- tor of endocrine development. Recent Prog Horm Res. 2002; 57: 19–36. PMID: 12017543 4. Fayard E, Auwerx J, Schoonjans K. LRH-1: An orphan nuclear receptor involved in development, metabolism and steroidogenesis. Trends Cell Biol. 2004; 14: 250–260. PMID: 15130581 5. Morohashi K, Baba T, Tanaka M. Steroid hormones and the development of reproductive organs. Sex Dev. 2013; 7: 61–79. doi: 10.1159/000342272 PMID: 22986257 6. Miyamoto K, Yazawa T, Mizutani T, Imamichi Y, Kawabe S, Kanno M, et al. Stem cell differentiation into steroidogenic cell lineages by NR5A family. Mol Cell Endocrinol. 2011; 336: 123–126. doi: 10. 1016/j.mce.2010.11.031 PMID: 21134412 7. Sonoyama T, Sone M, Honda K, Taura D, Kojima K, Inuzuka M, et al. Differentiation of human embry- onic stem cells and human induced pluripotent stem cells into steroid-producing cells. Endocrinology. 2012; 153: 4336–4345. doi: 10.1210/en.2012-1060 PMID: 22778223 8. Zubair M, Oka S, Parker K, Morohashi K. Transgenic expression of Ad4BP/SF-1 in fetal adrenal pro- genitor cells leads to ectopic adrenal formation. Mol Endocrinol. 2009; 23: 1657–1667. doi: 10.1210/ me.2009-0055 PMID: 19628584 9. Thummel CS. Molecular Mechanisms of Developmental Timing in C. elegans and Drosophila. Dev Cell. 2001; 1: 453–465. PMID: 11703937 10. Gilbert LI, Rybczynski R, Warren JT. Control and biochemical nature of the ecdysteroidogenic pathway. Annu Rev Entomol. 2002; 47: 883–916. PMID: 11729094 11. Yamanaka N, Rewitz KF, O’Connor MB. Ecdysone control of developmental transitions: lessons from Drosophila research. Annu Rev Entomol. 2013; 58: 497–516. doi: 10.1146/annurev-ento-120811- 153608 PMID: 23072462 12. Niwa YS, Niwa R. Neural control of steroid hormone biosynthesis during development in the fruit fly Drosophila melanogaster. Genes Genet Syst. 2014; 89: 27–34. PMID: 24817759 13. Niwa R, Niwa YS. Enzymes for ecdysteroid biosynthesis: their biological functions in insects and beyond. Biosci Biotechnol Biochem. 2014; 78: 1283–1292. doi: 10.1080/09168451.2014.942250 PMID: 25130728 14. Petryk A, Warren JT, Marqués G, Jarcho MP, Gilbert LI, Kahler J, et al. Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydro- xyecdysone. Drosophila Steroidogenic Transcription Factor Ouija Board Hajime Ono, the Drosophila Genetic Resource Center in Kyoto, the National Institute of Genetics, the Vienna Drosophila RNAi Center and the Developmental Studies Hybridoma Bank for stocks and reagents; and Aya Fukuda, Barry M. Honda, Marek Jindra, Takao Naga- numa, Kazushige Nagaosa, Koji Nakamura, Yoshinobu Nakanishi, Kim F. Rewitz, and Yuichi Shima for helpful discussion and critical reading of the manuscript. Author Contributions Conceived and designed the experiments: TKK KH YSN MBO RN. Performed the experi- ments: TKK KH YSN RY MS RN. Analyzed the data: TKK KH YSN RY MBO RN. Contributed reagents/materials/analysis tools: TKK KH YSN MS TS AF MBO RN. Wrote the paper: TKK KH YSN MBO RN. PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Acknowledgments We thank Yosuke Umei for his technical assistance; Tomoki Chiba and Fuminori Tsuruta for use of their luminometer; Yoshinori Fujimoto, Hiroshi Kataoka, Yasushi Hiromi, Shu Kondo, 22 / 26 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Drosophila Steroidogenic Transcription Factor Ouija Board 17. Enya S, Daimon T, Igarashi F, Kataoka H, Uchibori M, Sezutsu H, et al. The silkworm glutathione S- transferase gene noppera-bo is required for ecdysteroid biosynthesis and larval development. Insect Biochem Mol Biol. 2015; 61: 1–7. doi: 10.1016/j.ibmb.2015.04.001 PMID: 25881968 18. Yoshiyama T, Namiki T, Mita K, Kataoka H, Niwa R. Neverland is an evolutionally conserved Rieske- domain protein that is essential for ecdysone synthesis and insect growth. Development. 2006; 133: 2565–2574. PMID: 16763204 19. Yoshiyama-Yanagawa T, Enya S, Shimada-Niwa Y, Yaguchi S, Haramoto Y, Matsuya T, et al. The con- served rieske oxygenase DAF-36/Neverland is a novel cholesterol-metabolizing enzyme. J Biol Chem. 2011; 286: 25756–25762. doi: 10.1074/jbc.M111.244384 PMID: 21632547 20. Namiki T, Niwa R, Sakudoh T, Shirai K-I, Takeuchi H, Kataoka H. Cytochrome P450 CYP307A1/ Spook: a regulator for ecdysone synthesis in insects. Biochem Biophys Res Commun. 2005; 337: 367– 374. PMID: 16188237 21. Ono H, Rewitz KF, Shinoda T, Itoyama K, Petryk A, Rybczynski R, et al. Spook and Spookier code for stage-specific components of the ecdysone biosynthetic pathway in Diptera. Dev Biol. 2006; 298: 555– 570. PMID: 16949568 22. Niwa R, Namiki T, Ito K, Shimada-Niwa Y, Kiuchi M, Kawaoka S, et al. Non-molting glossy/shroud encodes a short-chain dehydrogenase/reductase that functions in the “Black Box” of the ecdysteroid biosynthesis pathway. Development. 2010; 137: 1991–1999. doi: 10.1242/dev.045641 PMID: 20501590 23. Niwa R, Matsuda T, Yoshiyama T, Namiki T, Mita K, Fujimoto Y, et al. CYP306A1, a cytochrome P450 enzyme, is essential for ecdysteroid biosynthesis in the prothoracic glands of Bombyx and Drosophila. J Biol Chem. 2004; 279: 35942–35949. PMID: 15197185 24. Warren JT, Petryk A, Marqués G, Parvy J-P, Shinoda T, Itoyama K, et al. Phantom encodes the 25- hydroxylase of Drosophila melanogaster and Bombyx mori: a P450 enzyme critical in ecdysone biosyn- thesis. Insect Biochem Mol Biol. 2004; 34: 991–1010. PMID: 15350618 25. Chávez VM, Marqués G, Delbecque JP, Kobayashi K, Hollingsworth M, Burr J, et al. The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels. Development. 2000; 127: 4115–4126. PMID: 10976044 26. Warren JT, Petryk A, Marque G, Jarcho M, Parvy J-P, Dauphin-villemant C, et al. Molecular and bio- chemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophila mela- nogaster. Proc Natl Acad Sci U S A. 2002; 99: 11043–11048. PMID: 12177427 27. PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 References Proc Natl Acad Sci U S A. 2003; 100: 13773–13778. PMID: 14610274 15. Enya S, Ameku T, Igarashi F, Iga M, Kataoka H, Shinoda T, et al. A Halloween gene noppera-bo encodes a glutathione S-transferase essential for ecdysteroid biosynthesis via regulating the behaviour of cholesterol in Drosophila. Sci Rep. 2014; 4: 6586. doi: 10.1038/srep06586 PMID: 25300303 16. Chanut-delalande H, Hashimoto Y, Pelissier-monier A, Spokony R, Dib A, Kondo T, et al. Pri peptides are mediators of ecdysone for the temporal control of development. Nat Cell Biol. 2014; 16: 1035– 1044. doi: 10.1038/ncb3052 PMID: 25344753 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 23 / 26 Drosophila Steroidogenic Transcription Factor Ouija Board 37. Neubueser D, Warren JT, Gilbert LI, Cohen SM. molting defective is required for ecdysone biosynthe- sis. Dev Biol. 2005; 280: 362–372. PMID: 15882578 38. Deng H, Kerppola TK. Regulation of Drosophila metamorphosis by xenobiotic response regulators. PLoS Genet. 2013; 9: e1003263. doi: 10.1371/journal.pgen.1003263 PMID: 23408904 39. Danielsen ET, Moeller ME, Dorry E, Komura-Kawa T, Fujimoto Y, Troelsen JT, et al. Transcriptional control of steroid biosynthesis genes in the Drosophila prothoracic gland by Ventral veins lacking and Knirps. PLoS Genet. 2014; 10: e1004343. doi: 10.1371/journal.pgen.1004343 PMID: 24945799 40. Cheng C, Ko A, Chaieb L, Koyama T, Sarwar P, Mirth CK, et al. The POU Factor Ventral veins lacking/ Drifter directs the timing of metamorphosis through ecdysteroid and juvenile hormone signaling. PLoS Genet. 2014; 10: e1004425. doi: 10.1371/journal.pgen.1004425 PMID: 24945490 41. Puig O, Mattila J. Understanding Forkhead box class O function: lessons from Drosophila melanoga- ster. Antioxid Redox Signal. 2011; 14: 635–647. doi: 10.1089/ars.2010.3407 PMID: 20618068 42. Misra JR, Horner MA, Lam G, Thummel CS. Transcriptional regulation of xenobiotic detoxification in Drosophila. Genes Dev. 2011; 25: 1796–1806. doi: 10.1101/gad.17280911 PMID: 21896655 43. Chung S, Chavez C, Andrew DJ. Trachealess (Trh) regulates all tracheal genes during Drosophila embryogenesis. Dev Biol. 2011; 360: 160–172. doi: 10.1016/j.ydbio.2011.09.014 PMID: 21963537 44. Sánchez-Higueras C, Sotillos S, Castelli-Gair Hombría J. Common origin of insect trachea and endo- crine organs from a segmentally repeated precursor. Curr Biol. 2014; 24: 76–81. doi: 10.1016/j.cub. 2013.11.010 PMID: 24332544 45. Fisher B, Weiszmann R, Frise E, Hammonds A, Tomancak P, Beaton A, et al. BDGP insitu homepage. 2012;http://insitu.fruitfly.org/cgi-bin/ex/insitu.pl 46. Chung HR, Schäfer U, Jäckle H, Böhm S. Genomic expansion and clustering of ZAD-containing C2H2 zinc-finger genes in Drosophila. EMBO Rep. 2002; 3: 1158–1162. PMID: 12446571 47. Chung H-R, Löhr U, Jäckle H. Lineage-specific expansion of the zinc finger associated domain ZAD. Mol Biol Evol. 2007; 24: 1934–1943. PMID: 17569752 48. Jauch R, Bourenkov GP, Chung HR, Urlaub H, Reidt U, Jäckle H, et al. The zinc finger-associated domain of the Drosophila transcription factor Grauzone is a novel zinc-coordinating protein-protein interaction module. Structure. 2003; 11: 1393–1402. PMID: 14604529 49. Payre F, Vincent A. Genomic targets of the serendipity β and δ zinc finger proteins and their respective DNA recognition sites. EMBO J. 1991; 10: 2533–2541. PMID: 1868833 50. Krystel J, Ayyanathan K. PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Rewitz KF, Rybczynski R, Warren JT, Gilbert LI. The Halloween genes code for cytochrome P450 enzymes mediating synthesis of the insect moulting hormone. Biochem Soc Trans. 2006; 34: 1256– 1260. PMID: 17073797 28. Bialecki M, Shilton A, Fichtenberg C, Segraves WA, Thummel CS. Loss of the ecdysteroid-inducible E75A orphan nuclear receptor uncouples molting from metamorphosis in Drosophila. Dev Cell. 2002; 3: 209–220. PMID: 12194852 29. Ou Q, Magico A, King-Jones K. Nuclear receptor DHR4 controls the timing of steroid hormone pulses during Drosophila development. PLoS Biol. 2011; 9: e1001160. doi: 10.1371/journal.pbio.1001160 PMID: 21980261 30. Parvy J-P, Blais C, Bernard F, Warren JT, Petryk A, Gilbert LI, et al. A role for βFTZ-F1 in regulating ecdysteroid titers during post-embryonic development in Drosophila melanogaster. Dev Biol. 2005; 282: 84–94. PMID: 15936331 31. Moeller ME, Danielsen ET, Herder R, O’Connor MB, Rewitz KF. Dynamic feedback circuits function as a switch for shaping a maturation-inducing steroid pulse in Drosophila. Development. 2013; 140: 4730– 4739. doi: 10.1242/dev.099739 PMID: 24173800 32. Parvy J-P, Wang P, Garrido D, Maria A, Blais C, Poidevin M, et al. Forward and feedback regulation of cyclic steroid production in Drosophila melanogaster. Development. 2014; 141: 3955–3965. doi: 10. 1242/dev.102020 PMID: 25252945 33. Xiang Y, Liu Z, Huang X. Br regulates the expression of the ecdysone biosynthesis gene npc1. Dev Biol. 2010; 344: 800–808. doi: 10.1016/j.ydbio.2010.05.510 PMID: 20621708 34. Talamillo A, Herboso L, Pirone L, Pérez C, González M, Sánchez J, et al. Scavenger receptors mediate the role of SUMO and Ftz-f1 in Drosophila steroidogenesis. PLoS Genet. 2013; 9: e1003473. doi: 10. 1371/journal.pgen.1003473 PMID: 23637637 35. Koyama T, Rodrigues MA, Athanasiadis A, Shingleton AW, Mirth CK. Nutritional control of body size through FoxO-Ultraspiracle mediated ecdysone biosynthesis. Elife. 2014; 3: e03091. 36. Wismar J, Habtemichael N, Warren JT, Dai JD, Gilbert LI, Gateff E. The mutation without childrenrgl causes ecdysteroid deficiency in third-instar larvae of Drosophila melanogaster. Dev Biol. 2000; 226: 1–17. PMID: 10993670 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 24 / 26 Drosophila Steroidogenic Transcription Factor Ouija Board 60. Lang M, Murat S, Clark AG, Gouppil G, Blais C, Matzkin LM, et al. Mutations in the neverland gene turned Drosophila pachea into an obligate specialist species. Science. 2012; 337: 1658–1661. PMID: 23019649 61. Gibbens YY, Warren JT, Gilbert LI, O’Connor MB. Neuroendocrine regulation of Drosophila metamor- phosis requires TGFβ/Activin signaling. Development. 2011; 138: 2693–2703. doi: 10.1242/dev. 063412 PMID: 21613324 62. Shimada-Niwa Y, Niwa R. Serotonergic neurons respond to nutrients and regulate the timing of steroid hormone biosynthesis in Drosophila. Nat Commun. 2014; 5: 5778. doi: 10.1038/ncomms6778 PMID: 25502946 63. Ohhara Y, Shimada-Niwa Y, Niwa R, Kayashima, Hayashi Y, Akagi K, et al. Autocrine regulation of ecdysone synthesis by β3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis. Proc Natl Acad Sci U S A. 2015; 112: 1452–1457. doi: 10.1073/pnas.1414966112 PMID: 25605909 64. Rewitz KF, Larsen MR, Lobner-Olesen A, Rybczynski R, O’Connor MB, Gilbert LI. A phosphoproteo- mics approach to elucidate neuropeptide signal transduction controlling insect metamorphosis. Insect Biochem Mol Biol. 2009; 39: 475–483. doi: 10.1016/j.ibmb.2009.04.005 PMID: 19422916 65. Claudius A-K, Romani P, Lamkemeyer T, Jindra M, Uhlirova M. Unexpected role of the steroid-defi- ciency protein ecdysoneless in pre-mRNA splicing. PLoS Genet. 2014; 10: e1004287. doi: 10.1371/ journal.pgen.1004287 PMID: 24722212 66. Wong JCY, Gokgoz N, Alon N, Andrulis IL, Buchwald M. Cloning and mutation analysis of ZFP276 as a candidate tumor suppressor in breast cancer. J Hum Genet. 2003; 48: 668–671. PMID: 14605947 67. Ryder E, Blows F, Ashburner M, Bautista-Llacer R, Coulson D, Drummond J, et al. The DrosDel collec- tion: A set of P-element insertions for generating custom chromosomal aberrations in Drosophila mela- nogaster. Genetics. 2004; 167: 797–813. PMID: 15238529 68. Lehmann R, Tautz D. Chapter 30 In situ hybridization to RNA. Methods Cell Biol. 1994; 44: 575–598. PMID: 7535885 69. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dom- inant phenotypes. Development. 1993; 415: 401–415. 70. Ni J-Q, Zhou R, Czech B, Liu L-P, Holderbaum L, Yang-Zhou D, et al. A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nat Methods. 2011; 8: 405–407. doi: 10.1038/nmeth.1592 PMID: 21460824 71. Barolo S, Carver LA, Posakony JW. GFP and β-galactosidase transformation vectors for promoter/ enhancer analysis in Drosophila. Biotechniques. 2000; 29: 726–732. PMID: 11056799 72. Lum L, Yao S, Mozer B, Rovescalli A, Von Kessler D, Nirenberg M, et al. PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Global analysis of target genes of 21 members of the ZAD transcription factor family in Drosophila melanogaster. Gene. 2013; 512: 373–382. doi: 10.1016/j.gene.2012.09.114 PMID: 23085320 51. Li J, Gilmour DS. Distinct mechanisms of transcriptional pausing orchestrated by GAGA factor and M1BP, a novel transcription factor. EMBO J. 2013; 32: 1829–1841. doi: 10.1038/emboj.2013.111 PMID: 23708796 52. Clark AG, Eisen MB, Smith DR, Bergman CM, Oliver B, Markow TA, et al. Evolution of genes and genomes on the Drosophila phylogeny. Nature. 2007; 450: 203–218. PMID: 17994087 53. Kondo S, Ueda R. Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics. 2013; 195: 715–721. doi: 10.1534/genetics.113.156737 PMID: 24002648 54. McWilliam H, Li W, Uludag M, Squizzato S, Park YM, Buso N, et al. Analysis tool web services from the EMBL-EBI. Nucleic Acids Res. 2013; 41. 55. McBrayer Z, Ono H, Shimell M, Parvy J-P, Beckstead RB, Warren JT, et al. Prothoracicotropic hormone regulates developmental timing and body size in Drosophila. Dev Cell. 2007; 13: 857–871. PMID: 18061567 56. Rewitz KF, Yamanaka N, Gilbert LI, Connor MBO. The insect neuropeptide PTTH activates receptor tyrosine kinase torso to initiate metamorphosis. Science. 2009; 326: 1403–1406. doi: 10.1126/science. 1176450 PMID: 19965758 57. Rewitz KF, O’Connor MB, Gilbert LI. Molecular evolution of the insect Halloween family of cytochrome P450s: Phylogeny, gene organization and functional conservation. Insect Biochem Mol Biol. 2007; 37: 741–753. PMID: 17628274 58. Rewitz KF, Gilbert LI. Daphnia Halloween genes that encode cytochrome P450s mediating the synthe- sis of the arthropod molting hormone: evolutionary implications. BMC Evol Biol. 2008; 8: 60. doi: 10. 1186/1471-2148-8-60 PMID: 18298845 59. Sztal T, Chung H, Gramzow L, Daborn PJ, Batterham P, Robin C. Two independent duplications form- ing the Cyp307a genes in Drosophila. Insect Biochem Mol Biol. 2007; 37: 1044–1053. PMID: 17785192 25 / 26 PLOS Genetics \| DOI:10.1371/journal.pgen.1005712 December 10, 2015 Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science. 2003; 299: 2039–2045. PMID: 12663920 73. Matsuzaki H, Daitoku H, Hatta M, Aoyama H, Yoshimochi K, Fukamizu A. Acetylation of Foxo1 alters its DNA-binding ability and sensitivity to phosphorylation. Proc Natl Acad Sci U S A. 2005; 102: 11278– 11283. PMID: 16076959 74. Yanai K, Hirota K, Taniguchi-Yanai K, Shigematsu Y, Shimamoto Y, Saito T, et al. Regulated expres- sion of human angiotensinogen gene by hepatocyte nuclear factor 4 and chicken ovalbumin upstream promoter-transcription factor. J Biol Chem. 1999; 274: 34605–34612. PMID: 10574924 75. Shimotsuma M, Okamura E, Matsuzaki H, Fukamizu A, Tanimoto K. DNase I hypersensitivity and ε-glo- bin transcriptional enhancement are separable in locus control region (LCR) HS1 mutant human β-glo- bin YAC transgenic mice. J Biol Chem. 2010; 285: 14495–14503. doi: 10.1074/jbc.M110.116525 PMID: 20231293 26 / 26
https://openalex.org/W2833439083	https://hal.archives-ouvertes.fr/hal-02326205/document	English	null	Cholinergic-mediated coordination of rhythmic sympathetic and motor activities in the newborn rat spinal cord	PLoS biology	2,018	cc-by	13,127	RESEARCH ARTICLE Cholinergic-mediated coordination of rhythmic sympathetic and motor activities in the newborn rat spinal cord Me´lissa Sourioux, Sandrine S. Bertrand‡, Jean-Rene´ Cazalets‡ Universite´ de Bordeaux, CNRS UMR 5287, Bordeaux, France ‡ These authors are joint senior authors on this work. ‡ These authors are joint senior authors on this work. * jean-rene.cazalets@u-bordeaux.fr (JRC); sandrine.bertrand@u-bordeaux.fr (SSB) ‡ These authors are joint senior authors on this work. * jean-rene.cazalets@u-bordeaux.fr (JRC); sandrine.bertrand@u-bordeaux.fr (SSB) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Sourioux M, Bertrand SS, Cazalets J-R (2018) Cholinergic-mediated coordination of rhythmic sympathetic and motor activities in the newborn rat spinal cord. PLoS Biol 16(7): e2005460. https://doi.org/10.1371/journal. pbio.2005460 Academic Editor: Pascal Legendre, Institut de Biologie Paris-Seine, France Received: January 22, 2018 Accepted: June 22, 2018 Published: July 9, 2018 Copyright: © 2018 Sourioux et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Here, we investigated intrinsic spinal cord mechanisms underlying the physiological require- ment for autonomic and somatic motor system coupling. Using an in vitro spinal cord prepa- ration from newborn rat, we demonstrate that the specific activation of muscarinic cholinergic receptors (mAchRs) (with oxotremorine) triggers a slow burst rhythm in thoracic spinal segments, thereby revealing a rhythmogenic capability in this cord region. Whereas axial motoneurons (MNs) were rhythmically activated during both locomotor activity and oxotremorine-induced bursting, intermediolateral sympathetic preganglionic neurons (IML SPNs) exhibited rhythmicity solely in the presence of oxotremorine. This somato-sympa- thetic synaptic drive shared by MNs and IML SPNs could both merge with and modulate the locomotor synaptic drive produced by the lumbar motor networks. This study thus sheds new light on the coupling between somatic and sympathetic systems and suggests that an intraspinal network that may be conditionally activated under propriospinal cholinergic con- trol constitutes at least part of the synchronizing mechanism. Introduction Locomotion, or any other form of physical activity, mobilizes not only the motor nervous sys- tem but also the autonomic nervous system, which governs body homeostatic processes such as blood pressure control and respiratory frequency. These autonomic responses that confront metabolic expenditure during exercise rely mostly on a functional coupling between the sym- pathetic and somatic motor systems. However, the neural substrate for this coupling remains enigmatic. Although the crucial role of supraspinal structures in this adaptive process, such as the rostral ventrolateral medulla, the hypothalamus, and the nucleus of the solitary tract, has been established [1,2], the involvement of intrinsic spinal mechanisms, albeit acknowledged, remains unclear (for review, see [3]). Significantly, several studies have reported that an intraspinal coordination between sympathetic and motor outflow may still occur in newborn [4] and adult rats [5] and mice [6] after removal of supraspinal influences by spinal cord sectioning. The cells responsible for the spinal sympathetic outflow are the sympathetic preganglionic neurons (SPNs), which in turn innervate postganglionic sympathetic neurons. Studies on SPN membrane properties have revealed that these cells can express spontaneous rhythmic burst activity at frequencies ranging from 0.1 to 10 Hz. Each of these frequencies has been related to a specific physiological rhythm such as the cardiac and respiratory rhythms or low frequency changes in blood pressure [7–9]. Intracellular recordings from in vitro preparations or anes- thetized animals have also revealed that different SPN rhythmic activity patterns can be elicited under various conditions, relying mainly on neuromodulatory controlling influences [8,10– 17]. Similarly, spinal motoneurons (MNs) also fire rhythmic bursts of action potentials when engaged in locomotor activity and, as for SPNs, this rhythmogenicity is initiated and regulated mainly by neuromodulatory pathways [18,19]. Other than extrinsic neuromodulatory influences originating from supraspinal centers, an intrinsic spinal neuromodulatory influence involving acetylcholine (Ach) has been shown to play an important role in controlling both the spinal motor network and sympathetic neuron activity [20–28]. In addition to SPNs and MNs that use Ach as their neurotransmitter, thoracic segments are enriched in cholinergic interneurons and show a dense expression of muscarinic cholinergic receptors (mAchRs) [29,30]. The question then arises as to the precise role of these Ach-releasing interneurons in the thoracic spinal cord region. Oxotremorine-induced somato-sympathetic coupling action potential threshold; AP5, 2-amino-5- phosphonovalerate; DNQX, 6, 7-dinitroquinoxaline- 2,3-dione; IML SPN, intermediolateral sympathetic preganglionic neurons; mAchR, muscarinic cholinergic receptor; MN, motoneuron; NMDA, N- methyl-D-aspartate; P, postnatal day; RMP, resting membrane potential; SCI, spinal cord injury; SPN, sympathetic preganglionic neuron; SSD, somato- sympathetic drive; 5-HT, serotonin. coupling mechanism we describe here underlies part of the vascular changes needed to maintain adequate muscle oxygenation during locomotion. Introduction In the present study, by using the in vitro isolated spinal cord preparation of newborn rats, we show that an activation of mAchRs unmasks rhythmogenic capabilities of the spinal tho- racic segments and a common synaptic drive to SPNs and axial MNs. This thoracic mAchR- induced rhythmicity merges with the locomotor activity expressed in lumbar spinal networks and modulates the latter’s expression. We therefore propose that thoracic networks sensitive to mAchR-mediated cholinergic influences may act as an important intrinsic substrate for the coupling between spinal motor and sympathetic activities. Author summary Physical movements require mobilization of animals’ autonomic nervous system, in order to maintain stable bodily functions while matching the increasing physiological demand. These autonomic responses rely on a coupling between the sympathetic and somatic ner- vous systems, although how this coupling occurs remains unresolved. To address this issue, we used the in vitro isolated spinal cord preparation from newborn rats, which can be kept alive up to 12 h and generate locomotion-like activity. We found that the stimula- tion of the muscarinic cholinergic receptors specifically activates intraspinal neural net- works generating a slow motor rhythm. During this slow rhythm, recordings from sympathetic neurons (the cells responsible for the spinal sympathetic output) and somatic motoneurons (responsible for skeletal muscle activity) reveal that both cell types receive a common synaptic input that results in a coupling of sympathetic and rhythmic locomotor activities. This shared somato-sympathetic drive could merge with and modulate the loco- motor synaptic drive produced by the lumbar motor networks. We propose that the Data Availability Statement: All relevant data are within the paper and its Supporting information files. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Abbreviations: Ach, acetylcholine; AchE, acetylcholinesterase; aCSF, artificial cerebrospinal fluid; AHP, after-spike hyperpolarization; AP Th, Abbreviations: Ach, acetylcholine; AchE, acetylcholinesterase; aCSF, artificial cerebrospinal fluid; AHP, after-spike hyperpolarization; AP Th, PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 1 / 21 Oxotremorine-induced somato-sympathetic coupling oxotremorine was bath-applied on the whole thoracolumbar spinal cord. As previously described in such preparations, a mixture of N-methyl-D-aspartate (NMDA) and serotonin (5-HT)-induced rhythmic locomotor-related activity was recorded from both lumbar and tho- racic ventral roots (Fig 1A1, 2), with a mean period of 3.2 ± 0.1 s, n = 31 (Fig 1C) [31,32]. This fictive locomotion was characterized by right-left and extensor-flexor alternations of bursts of action potentials monitored from the L2 and L5 ventral root bursts, respectively (Fig 1E) [33]. In contrast, the bath-application of 10 μM oxotremorine on the whole spinal cord elicited a slow rhythmic activity in the thoracic and lumbar region, with a mean period of 21.7 ± 1.3 s, n = 26 (Fig 1B1, 2 and Fig 1C). This oxotremorine-induced rhythm consisted of right and left slow alternating bursts of action potentials, but with no alternation between the flexor and extensor units in the majority of the preparations tested (Figs 1E and 2D). Oxotremorine also was more potent in exciting thoracic segments than the NMDA/5-HT cocktail. Indeed, the amplitude of the thoracic bursts was significantly increased in the presence of oxotremorine compared to NMDA/5-HT, while L2 burst amplitudes were not significantly increased in the same conditions (Fig 1D). Fig 1. Oxotremorine-induced rhythmic bursting in the isolated newborn rat spinal cord. (A) Representative extracellular recordings from different right (r) and left (l) thoracic (T) and lumbar (L) ventral roots in the presence of NMDA/5-HT on the whole thoracolumbar spinal cord at slow (A1) and faster timescales (A2). (B) Equivalent recording setup in the same preparation with 10 μM oxotremorine bath-applied to the whole thoracolumbar spinal cord. (C) Box plots of the mean cycle period under NMDA/5-HT (blue boxes) or oxotremorine (Oxo, black boxes). (D) Box plots of normalized burst activity recorded in the presence of NMDA/5-HT (blue boxes) or oxotremorine (Oxo, black boxes). Motor burst amplitudes were normalized to the mean NMDA/5-HT burst amplitude computed in each experiment. (E) Polar graphs of rL2/lL2 and rL2/rL5 burst phase relationships under NMDA/5-HT (blue circles) or 10 μM Oxo (open circles) generated from the experiments presented in A and B. 0, in phase; 0.5, antiphase. The direction and length of vectors (blue line) indicate phase means and dispersions, respectively. ⇤p < 0.05. Numbers in boxes indicate numbers of preparations tested. PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 Muscarinic activation of thoracolumbar networks To assess the effect of cholinergic system activation on thoracic segments, we used oxo- tremorine, a nonselective cholinergic muscarinic agonist. In a first series of experiments, PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 2 / 21 l, left; L, lumbar; NMDA, N-methyl-D-aspartate; ns, nonsignificantly different; Oxo, oxotremorine; r, right; T, thoracic; 5-HT, serotonin. https //doi org/10 1371/journal pbio 2005460 g001 Fig 1. Oxotremorine-induced rhythmic bursting in the isolated newborn rat spinal cord. (A) Representative nduced rhythmic bursting in the isolated newborn rat spinal cord. (A) Representative Fig 1. Oxotremorine-induced rhythmic bursting in the isolated newborn rat spinal cord. (A) Representative extracellular recordings from different right (r) and left (l) thoracic (T) and lumbar (L) ventral roots in the presence of Fig 1. Oxotremorine-induced rhythmic bursting in the isolated newborn rat spinal cord. (A) Representative extracellular recordings from different right (r) and left (l) thoracic (T) and lumbar (L) ventral roots in the presence of NMDA/5-HT on the whole thoracolumbar spinal cord at slow (A1) and faster timescales (A2). (B) Equivalent recording setup in the same preparation with 10 μM oxotremorine bath-applied to the whole thoracolumbar spinal cord. (C) Box plots of the mean cycle period under NMDA/5-HT (blue boxes) or oxotremorine (Oxo, black boxes). (D) Box plots of normalized burst activity recorded in the presence of NMDA/5-HT (blue boxes) or oxotremorine (Oxo, black boxes). Motor burst amplitudes were normalized to the mean NMDA/5-HT burst amplitude computed in each experiment. (E) Polar graphs of rL2/lL2 and rL2/rL5 burst phase relationships under NMDA/5-HT (blue circles) or 10 μM Oxo (open circles) generated from the experiments presented in A and B. 0, in phase; 0.5, antiphase. The direction and length of vectors (blue line) indicate phase means and dispersions, respectively. ⇤p < 0.05. Numbers in boxes indicate numbers of preparations tested. l, left; L, lumbar; NMDA, N-methyl-D-aspartate; ns, nonsignificantly different; Oxo, oxotremorine; r, right; T, thoracic; 5-HT, serotonin. https://doi.org/10.1371/journal.pbio.2005460.g001 https://doi.org/10.1371/journal.pbio.2005460.g001 3 / 21 PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 Oxotremorine-induced somato-sympathetic coupling This oxotremorine-induced rhythm was consistently observed at concentrations ranging from 0.5 to 100 μM (Fig 2A1–5). Increasing the oxotremorine concentration up to 10 μM pro- gressively decreased the cycle period of thoracic motor bursts (Fig 2B) and increased their amplitude (Fig 2C). Furthermore, there was a switch from an in-phase activity recorded from the left and right segments at the lowest concentrations (0.5 and 1 μM) to an out-of-phase activity under 10 μM (Fig 2D). Five micromolar oxotremorine appears as an intermediate con- centration, with in-phase left and right motor bursts observed in 55% of the preparations tested and alternating motor bursts in the remaining ones (n = 9) (Fig 2D). Increasing oxotre- morine up to 100 μM seemed to become less effective as the induced motor activity exhibited an increased period, compared to that with 5 and 10 μM oxotremorine (Fig 2B), and a disorga- nized motor burst expression (Fig 2D). The various patterns elicited when raising the oxotre- morine concentration could be linked to the progressive increase in the excitation state of spinal networks and recruitment of spinal neurons [34,35]. It has been previously shown that activation of the cholinergic system by inhibiting acetyl- cholinesterase (AchE), the enzyme responsible for Ach degradation with physostigmine, neo- stigmine, or edrophodium, elicits locomotor-like activities in lumbar ventral roots in the isolated spinal cord preparation from newborn rat [21,24,25] and that this rhythm is mediated Fig 2. Dose-dependent effects of oxotremorine. (A) Recordings from right thoracic 11 and left thoracic 12 (rT11, lT12) ventral roots in the presence of increasing concentrations of oxotremorine bath-applied to the same whole thoracolumbar cord preparation (A1–A5). (B, C) Box plots of burst cycle period (B) and normalized amplitude (C) at different oxotremorine concentrations. Motor burst amplitudes were normalized to the mean burst amplitude computed in the presence of 0.5 μM oxotremorine in each experiment. (D) Polar graphs of the right and left alternation of thoracic motor bursts (Pop ϕ rTh-lTh) computed under increasing concentrations of oxotremorine (Oxo). 0, in phase; 0.5, antiphase. The direction and length of vectors (blue lines) indicate phase means and dispersions, respectively. Each dot on polar graphs represents one experiment. ⇤p < 0.05. l, left; n, number of preparations; Oxo, oxotremorine; Pop, population; Pop ϕ rTh-lTh, right and left alternation of thoracic motor bursts; r, right; T, thoracic. Oxotremorine-induced somato-sympathetic coupling Fig 3. Acetylcholinesterase inhibitors elicit locomotor-like spinal cord activity. Representative extracellular recordings from left thoracic 11, right thoracic 12, and right lumbar 2 (lT11, rT12, rL2) ventral roots in the presence of oxotremorine (A) or with the acetylcholinesterase inhibitors neostigmine (B) or physostigmine (C) bath-applied on the whole thoracolumbar spinal cord. l, left; L, lumber; r, right; T, thoracic. https://doi.org/10.1371/journal.pbio.2005460.g003 Fig 3. Acetylcholinesterase inhibitors elicit locomotor-like spinal cord activity. Representative extracellular recordings from left thoracic 11, right thoracic 12, and right lumbar 2 (lT11, rT12, rL2) ventral roots in the presence of oxotremorine (A) or with the acetylcholinesterase inhibitors neostigmine (B) or physostigmine (C) bath-applied on the whole thoracolumbar spinal cord. l, left; L, lumber; r, right; T, thoracic. https://doi.org/10.1371/journal.pbio.2005460.g003 https://doi.org/10.1371/journal.pbio.2005460.g003 through mAchR activation [27]. Therefore, in a next step, we compared the effects of oxotre- morine (Fig 3A) with those of the AchE inhibitors neostigmine and physostigmine. In our experimental conditions, neither inhibitor mimicked the effects of oxotremorine (Fig 3B and 3C). Rather, their superfusion transiently elicited episodes of fast locomotor-like rhythmicity, as previously described [24,25,27], instead of the slow non-locomotor rhythm observed here in the presence of oxotremorine. The mean period computed in the presence of neostigmine or physostigmine was 3 ± 0.3 s (n = 5 preparations tested in the presence of neostigmine and n = 2 in the presence of physostigmine). The results obtained when oxotremorine was bath-applied to the whole thoracolumbar spi- nal cord (Fig 1D) suggested that the thoracic and lumbar spinal levels may respond differen- tially to oxotremorine. To test whether the thoracic and lumbar segments exhibit similar responses to mAchR activation by oxotremorine, we used a partitioned spinal cord (see Mate- rial and methods) in which the thoracic and lumbar compartments could be independently superfused (Fig 4A1 and 4B1). Bath-application of oxotremorine to the thoracic segments alone induced a rhythmic burst pattern that was recorded from the various thoracic segments but also from lumbar roots (Fig 4A2). This thoracolumbar propagation was observed in 24 of 28 preparations tested. In contrast, and as previously shown [32], the bath-application of NMDA/5-HT to the thoracic segments alone did not induce any rhythmic activity (n = 10; Fig 4A3) in these experimental conditions. PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 htt //d i /10 1371/j l bi 2005460 002 ependent effects of oxotremorine. (A) Recordings from right thoracic 11 and left thoracic 12 (rT11, lT12) Fig 2. Dose-dependent effects of oxotremorine. (A) Recordings from right thoracic 11 and left thoracic 12 (rT11, lT12) ventral roots in the presence of increasing concentrations of oxotremorine bath-applied to the same whole thoracolumbar cord preparation (A1–A5). (B, C) Box plots of burst cycle period (B) and normalized amplitude (C) at different oxotremorine concentrations. Motor burst amplitudes were normalized to the mean burst amplitude computed in the presence of 0.5 μM oxotremorine in each experiment. (D) Polar graphs of the right and left alternation of thoracic motor bursts (Pop ϕ rTh-lTh) computed under increasing concentrations of oxotremorine (Oxo). 0, in phase; 0.5, antiphase. The direction and length of vectors (blue lines) indicate phase means and dispersions, respectively. Each dot on polar graphs represents one experiment. ⇤p < 0.05. l, left; n, number of preparations; Oxo, oxotremorine; Pop, population; Pop ϕ rTh-lTh, right and left alternation of thoracic motor bursts; r, right; T, thoracic. Fig 2. Dose-dependent effects of oxotremorine. (A) Recordings from right thoracic 11 and left thoracic 12 (rT11, lT12) ventral roots in the presence of increasing concentrations of oxotremorine bath-applied to the same whole thoracolumbar cord preparation (A1–A5). (B, C) Box plots of burst cycle period (B) and normalized amplitude (C) at different oxotremorine concentrations. Motor burst amplitudes were normalized to the mean burst amplitude computed in the presence of 0.5 μM oxotremorine in each experiment. (D) Polar graphs of the right and left alternation of thoracic motor bursts (Pop ϕ rTh-lTh) computed under increasing concentrations of oxotremorine (Oxo). 0, in phase; 0.5, antiphase. The direction and length of vectors (blue lines) indicate phase means and dispersions, respectively. Each dot on polar graphs represents one experiment. ⇤p < 0.05. l, left; n, number of preparations; Oxo, oxotremorine; Pop, population; Pop ϕ rTh-lTh, right and left alternation of thoracic motor bursts; r, right; T, thoracic. https://doi.org/10.1371/journal.pbio.2005460.g002 https://doi.org/10.1371/journal.pbio.2005460.g002 PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 4 / 21 When the bath-application of oxotremorine was now restricted to the lumbar spinal cord, rhythmic bursting was recorded solely from the lumbar ventral roots, without any propagation to the thoracic segments (n = 10; Fig 4B2). The same lack of lumbo-thoracic propagation was observed when oxotremorine was replaced by PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 5 / 21 Oxotremorine-induced somato-sympathetic coupling Fig 4. Differential sensitivity of thoracic and lumbar segments to oxotremorine and NMDA/5-HT. (A, B) Diagrams of the experimental setup for two different preparations. In both cases, a Vaseline wall (black bar) was placed at the T13 level (A1, B1). Representative recordings from left thoracic 9 (lT9), lumbar 2 (lL2), and right T10 (rT10), L2 (rL2), and L5 (rL5) ventral roots (black dots in A1 and B1) in the presence of oxotremorine in either the thoracic (A2) or lumbar compartments (B2). Same preparations in the presence of NMDA/5-HT in either the thoracic (A3) or lumbar compartments (B3). l, left; L, lumbar; NMDA, N-methyl-D-aspartate; r, right; T, thoracic; 5-HT, serotonin. https://doi.org/10.1371/journal.pbio.2005460.g004 Fig 4. Differential sensitivity of thoracic and lumbar segments to oxotremorine and NMDA/5-HT. (A, B) Diagrams of the experimental setup for two different preparations. In both cases, a Vaseline wall (black bar) was placed at the T13 level (A1, B1). Representative recordings from left thoracic 9 (lT9), lumbar 2 (lL2), and right T10 (rT10), L2 (rL2), and L5 (rL5) ventral roots (black dots in A1 and B1) in the presence of oxotremorine in either the thoracic (A2) or lumbar compartments (B2). Same preparations in the presence of NMDA/5-HT in either the thoracic (A3) or lumbar compartments (B3). l, left; L, lumbar; NMDA, N-methyl-D-aspartate; r, right; T, thoracic; 5-HT, serotonin. https://doi.org/10.1371/journal.pbio.2005460.g004 https://doi.org/10.1371/journal.pbio.2005460.g004 https://doi.org/10.1371/journal.pbio.2005460.g004 NMDA/5-HT in the caudal compartment, with the recorded locomotor-like activity always restricted to the lumbar segments only (n = 13; Fig 4B3). It should also be noted that, using transverse sections of the thoracic spinal cord, we determined the number of thoracic seg- ments sufficient to produce the oxotremorine-induced rhythm. In five preparations, we observed that, regardless of the thoracic level, spinal pieces of at least three thoracic segments were sufficient to generate bilateral alternating rhythmic activity in the presence of 10 μM oxo- tremorine. PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 Oxotremorine-induced somato-sympathetic coupling Fig 5. Effects of cholinergic muscarinic antagonists and agonists on the oxotremorine-induced rhythm. (A) Extracellular recordings from right (r) and left (l) thoracic (T) or lumbar (L) ventral roots in the presence of oxotremorine alone and after concomitant bath-application of oxotremorine and the M1 receptor antagonist pirenzepine (A1), or the M2 receptor antagonist AF-DX 116 (A2), or the M3 receptor antagonist 4-DAMP (A3) or the M4 antagonist tropicamide (A4). (B) The M1 agonists McN-A 343 (B1) and xanomeline (B2) failed to induce any activity. (C) Slow bursting activity elicited by bath-application of the M2 receptor agonist arecaidine at 10 μM (C1) and 100 μM (C2). l, left; L, lumbar; NMDA, N-methyl-D-aspartate; Oxo., oxotremorine; r, right; T, thoracic. htt //d i /10 1371/j l bi 2005460 005 Fig 5. Effects of cholinergic muscarinic antagonists and agonists on the oxotremorine-induced rhythm. (A) Extracellular recordings from right (r) and left (l) thoracic (T) or lumbar (L) ventral roots in the presence of oxotremorine alone and after concomitant bath-application of oxotremorine and the M1 receptor antagonist pirenzepine (A1), or the M2 receptor antagonist AF-DX 116 (A2), or the M3 receptor antagonist 4-DAMP (A3) or the M4 antagonist tropicamide (A4). (B) The M1 agonists McN-A 343 (B1) and xanomeline (B2) failed to induce any activity. (C) Slow bursting activity elicited by bath-application of the M2 receptor agonist arecaidine at 10 μM (C1) and 100 μM (C2). l, left; L, lumbar; NMDA, N-methyl-D-aspartate; Oxo., oxotremorine; r, right; T, thoracic. htt //d i /10 1371/j l bi 2005460 005 motor activities in the spinal cord through the activation of the M1, M2, M3, and M4 receptors (see Discussion). PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 Altogether, these results suggest that the thoracic spinal cord exhibits a distinct sen- sitivity to oxotremorine compared to the lumbar spinal cord and that thoracic segments are endowed with specific rhythmogenic capabilities that can be unveiled by mAchR activation. In a next step, we assessed whether and which of the four classes of mAchR subtypes pres- ent in the spinal cord [36] were involved in the genesis of the oxotremorine-induced rhythm. To this end, after a first control bath-application of oxotremorine, receptor subtype antago- nists [37] were preincubated on the in vitro spinal cord preparation before their co-application with oxotremorine. An example of the blockade of the oxotremorine-induced rhythm by the presence of the antagonist of the M1 receptor subtype (pirenzepine 1–10 μM, n = 6) is pro- vided in Fig 5A1. A similar blockade was observed with antagonists of the M2 (AF-DX 116, 10 μM, n = 8; Fig 5A2), M3 (4-DAMP, 0.5–1 μM, n = 4; Fig 5A3) and M4 (tropicamide 1 μM, n = 2; Fig 5A4) receptors. At concentrations lower than 1 μM, M1 and M4 antagonists did not suppress the rhythm (n = 4). Lower concentrations of M2 antagonist also failed to suppress rhythmic activity (AF-DX 116, 0.5 μM, n = 2 and 1 μM, n = 2). The effects of selective agonists for M1 and M2 receptors were also tested. M1 agonists (xanomeline and McN-A343) bath- applied to the whole thoracolumbar spinal cord did not elicit any activity at concentrations ranging from 10 to 100 μM (n = 2; Fig 5B1, 2). Bath-application of the M2 receptor agonist are- caidine only partly reproduced the oxotremorine effects and induced a weak rhythmic activity, even at high concentration (n = 2, Fig 5C1, 2). These data suggest that oxotremorine triggers PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 6 / 21 Slow and fast rhythmic bursting in MNs and IML SPNs (A) Schematics of the experimental setup to record MNs and IML SPNs. The cut end of the thoracic spinal cord was upturned on a Sylgard wedge (A1; see Methods) to record from single thoracic MNs (A2) or IML SPNs (A3) identified by their different locations and antidromic action potential firing (AD MN or AD IML SPN) in response to ventral root stimulation of the same segment. (B) Left and right lumbar 2 (lL2, rL2) and right thoracic (rT10) ventral root recordings along with an intracellularly recorded rT10 MN in the presence of NMDA/5-HT (B1) or oxotremorine (B2) on the whole thoracolumbar spinal cord. The mixed cross-coherence maps (CXWT) in lower B1 and B2 show the correlation between the recorded MN’s membrane potential oscillations and rL2 activity under NMDA/5-HT (B1) or oxotremorine (B2). (C) Same display arrangement as in B for an intracellularly recorded rT7 IML SPN (rT7 IML). Note that the IML SPN was not driven by the NMDA/5-HT-induced rhythm (C1) but was strongly activated by the oxotremorine-induced rhythm (C2). AD, antidromic; CXWT, mixed cross-coherence map; IML SPN, intermediolateral sympathetic preganglionic neuron; l, left; L, lumbar; MN, motoneuron; NMDA, N-methyl-D- aspartate; T, thoracic; VR stim, ventral root stimulation; 5-HT, serotonin. h //d i /10 13 1/j l bi 200 460 006 Fig 6. The oxotremorine-induced rhythm drives bursting in both axial MNs and IML SPNs. (A) Schematics of the experimental setup to record MNs and IML SPNs. The cut end of the thoracic spinal cord was upturned on a Sylgard wedge (A1; see Methods) to record from single thoracic MNs (A2) or IML SPNs (A3) identified by their different locations and antidromic action potential firing (AD MN or AD IML SPN) in response to ventral root stimulation of the same segment. (B) Left and right lumbar 2 (lL2, rL2) and right thoracic (rT10) ventral root recordings along with an intracellularly recorded rT10 MN in the presence of NMDA/5-HT (B1) or oxotremorine (B2) on the whole thoracolumbar spinal cord. The mixed cross-coherence maps (CXWT) in lower B1 and B2 show the correlation between the recorded MN’s membrane potential oscillations and rL2 activity under NMDA/5-HT (B1) or oxotremorine (B2). (C) Same display arrangement as in B for an intracellularly recorded rT7 IML SPN (rT7 IML). Note that the IML SPN was not driven by the NMDA/5-HT-induced rhythm (C1) but was strongly activated by the oxotremorine-induced rhythm (C2). Slow and fast rhythmic bursting in MNs and IML SPNs Ventral roots not only carry axons of somatic MNs all along the spinal cord but also axons of SPNs from thoracic 1 to lumbar 3 segments in rats. Consequently, extracellular recordings from these latter ventral roots do not permit somatic and sympathetic axonal activity to be dis- tinguished. To assess whether axial MNs and/or SPNs are activated during pharmacological activation of the thoracolumbar spinal cord with NMDA/5-HT or oxotremorine, intracellular recordings from these two cell types were then performed in whole cord preparations (Fig 6A1), in which the MNs (Fig 6A2) or SPNs located in the IML (intermediolateral sympathetic preganglionic neurons [IML SPNs]; Fig 6A3) were identified by ventral root antidromic stimu- lation. In the majority of recorded thoracic MNs, the bath-application of NMDA/5-HT elicited locomotor-related activity with action potentials superimposed on phasic depolarizations dur- ing locomotor cycles (Fig 6B1) [32]. Wavelet transform analysis was conducted to assess whether the extracellular and intracellular activities recorded from the ventral roots and from MNs, respectively, were linked. The mixed cross-coherence map (bottom panel, Fig 6B1) shows that the MN membrane potential oscillations were significantly correlated to the lumbar L2 activity in a frequency band of 0.4 ± 0.02 Hz. In the presence of oxotremorine on the whole thoracolumbar spinal cord, intracellular recordings revealed phasic depolarizations in MNs that were significantly correlated to the lumbar L2 activity (n = 22/30 recorded MNs; Fig 6B2). In this sequence, because of the irregular nature of the oxotremorine-induced activity, the fre- quency band ranged from 0.06 to 0.03 Hz. In contrast, in almost all the IML SPNs tested, no detectable variations in their membrane potential (n = 16/18 recorded IML SPNs) were observed in time with locomotor-like activity induced by the bath-application of NMDA- 5-HT (top panel, Fig 6C1). The mixed cross-coherence map (bottom panel, Fig 6C1) con- firmed that the IML SPN activity was not correlated to NMDA/5-HT-induced fictive locomo- tion. In contrast, in the presence of oxotremorine, IML SPNs exhibited strong rhythmic burst activity associated with large membrane potential oscillations in correlation with the slow bursting activity recorded from both thoracic and lumbar segments (n = 21/25 recorded IML SPNs, Fig 6C2). Wavelet analysis confirmed that in the presence of oxotremorine, IML SPN PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 7 / 21 Oxotremorine-induced somato-sympathetic coupling Fig 6. The oxotremorine-induced rhythm drives bursting in both axial MNs and IML SPNs. PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 Slow and fast rhythmic bursting in MNs and IML SPNs AD, antidromic; CXWT, mixed cross-coherence map; IML SPN, intermediolateral sympathetic preganglionic neuron; l, left; L, lumbar; MN, motoneuron; NMDA, N-methyl-D- aspartate; T, thoracic; VR stim, ventral root stimulation; 5-HT, serotonin. https://doi.org/10.1371/journal.pbio.2005460.g006 https://doi.org/10.1371/journal.pbio.2005460.g006 activity was significantly correlated to the extracellularly recorded neuronal activity in the fre- quency band of 0.03 ± 0.01 Hz (bottom panel, Fig 6C2). q y p g In conclusion, almost all the MNs and IML SPN neurons tested in these different experi- mental conditions exhibited similar rhythmic behavior: MNs (73%) were rhythmically acti- vated by both NMDA/5-HT and oxotremorine pharmacological stimulation while IML SPNs (84%) were rhythmically activated solely in the presence of oxotremorine. We call the synaptic command shared by both axial MNs and IML SPNs, in the presence of oxotremorine, the somato-sympathetic drive (SSD). PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 8 / 21 Oxotremorine-induced somato-sympathetic coupling Fig 7. Electrophysiological properties of thoracic MNs and IML SPNs. (A) Box plots of the mean resting membrane potential (Rin) of MNs (blue box) and IML SPNs (black box). (B) Representative traces of the AHP recorded from a MN (blue trace) and an IML SPN (black trace) in response to brief depolarizing current injection (bottom panel) (B1). Box plots of the mean absolute AHP amplitudes (B2) and AHP half decay times (B3) in MNs (blue boxes) and IML SPNs (black boxes). ⇤p < 0.05; numbers in boxes indicate numbers of neurons tested. (C) Membrane potential responses (C1, C3) and firing rate measurements (C2, C4) for two neurons exhibiting linear (type 1, C1, 2) or adaptive discharge (type 2, C3, 4) in response to triangular ramp depolarization (bottom traces in C1, C2). Numbers of MNs (blue bar) and IML SPNs (black bar) expressing type 1 or type 2 firing behavior during triangular current ramps (C5). (D) Representative response of a MN (blue trace) and an IML SPN (black trace) to negative current injections (bottom panel). Note the expression in both neuron subtypes of a hyperpolarization-evoked sag potential and a small post-hyperpolarization rebound. AHP, spike after-hyperpolarization; Depol, depolarization phase; IML SPN, intermediolateral sympathetic preganglionic neuron; MN, motoneuron; Repol, repolarization phase; Rin, resting membrane potential. Fig 7. Electrophysiological properties of thoracic MNs and IML SPNs. (A) Box plots of the mean resting membrane Fig 7. Electrophysiological properties of thoracic MNs and IML SPNs. Slow and fast rhythmic bursting in MNs and IML SPNs (A) Box plots of the mean resting membrane potential (Rin) of MNs (blue box) and IML SPNs (black box). (B) Representative traces of the AHP recorded from a MN (blue trace) and an IML SPN (black trace) in response to brief depolarizing current injection (bottom panel) (B1). Box plots of the mean absolute AHP amplitudes (B2) and AHP half decay times (B3) in MNs (blue boxes) and IML SPNs (black boxes). ⇤p < 0.05; numbers in boxes indicate numbers of neurons tested. (C) Membrane potential responses (C1, C3) and firing rate measurements (C2, C4) for two neurons exhibiting linear (type 1, C1, 2) or adaptive discharge (type 2, C3, 4) in response to triangular ramp depolarization (bottom traces in C1, C2). Numbers of MNs (blue bar) and IML SPNs (black bar) expressing type 1 or type 2 firing behavior during triangular current ramps (C5). (D) Representative response of a MN (blue trace) and an IML SPN (black trace) to negative current injections (bottom panel). Note the expression in both neuron subtypes of a hyperpolarization-evoked sag potential and a small post-hyperpolarization rebound. AHP, spike after-hyperpolarization; Depol, depolarization phase; IML SPN, intermediolateral sympathetic preganglionic neuron; MN, motoneuron; Repol, repolarization phase; Rin, resting membrane potential. Fig 7. Electrophysiological properties of thoracic MNs and IML SPNs. (A) Box plots of the mean resting membrane potential (Rin) of MNs (blue box) and IML SPNs (black box). (B) Representative traces of the AHP recorded from a MN (blue trace) and an IML SPN (black trace) in response to brief depolarizing current injection (bottom panel) (B1). Box plots of the mean absolute AHP amplitudes (B2) and AHP half decay times (B3) in MNs (blue boxes) and IML SPNs (black boxes). ⇤p < 0.05; numbers in boxes indicate numbers of neurons tested. (C) Membrane potential responses (C1, C3) and firing rate measurements (C2, C4) for two neurons exhibiting linear (type 1, C1, 2) or adaptive discharge (type 2, C3, 4) in response to triangular ramp depolarization (bottom traces in C1, C2). Numbers of MNs (blue bar) and IML SPNs (black bar) expressing type 1 or type 2 firing behavior during triangular current ramps (C5). (D) Representative response of a MN (blue trace) and an IML SPN (black trace) to negative current injections (bottom panel). Note the expression in both neuron subtypes of a hyperpolarization-evoked sag potential and a small post-hyperpolarization rebound. PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 Slow and fast rhythmic bursting in MNs and IML SPNs AHP, spike after-hyperpolarization; Depol, depolarization phase; IML SPN, intermediolateral sympathetic preganglionic neuron; MN, motoneuron; Repol, repolarization phase; Rin, resting membrane potential. https://doi.org/10.1371/journal.pbio.2005460.g007 https://doi.org/10.1371/journal.pbio.2005460.g007 Because axial MNs and IML SPNs responded differently to the pharmacological conditions tested, we investigated whether the two subtypes are endowed with different electrophysiologi- cal properties that could account for their differing responses (Fig 7). In the absence of phar- macological stimulation, resting membrane potential (RMP) and action potential threshold (AP Th) values were similar between axial MNs (RMP: −61.1 ± 1.2 mV, AP Th: −51 ± 0.7 mV, n = 36) and IML SPNs (RMP: −60.4 ± 1.2 mV, AP Th: −49.3 ± 0.9 mV, n = 24). However, IML SPNs had a significantly higher membrane input resistance (Fig 7A) and a significantly larger and longer after-spike hyperpolarization (AHP) compared to axial MNs (Fig 7B1–3). Using triangular current pulses and the functional categorization established by Button and collabo- rators [38], we found that the frequency-current relationships of both axial MNs and IML SPNs could be classified as type 1 (linear, Fig 7C1, 2) or type 2 (adapting, Fig 7C3, 4), with the proportions of these two firing patterns not being significantly different between axial MNs and IML SPNs (Fig 7C5). Finally, we found that both axial MNs and IML SPNs exhibited a sag potential and a post-hyperpolarization rebound in response to hyperpolarizing current steps (Fig 7D). Altogether, these results thus indicate that axial MNs and IML SPNs exhibit basic membrane properties that are relatively similar. In a next series of experiments, we investigated the nature of the SSD received by thoracic neurons during the oxotremorine-induced rhythm. In the presence of NMDA and non- NMDA receptor antagonists (10 μM 2-amino-5-phosphonovalerate [AP5] and 10 μM 6, PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 9 / 21 Oxotremorine-induced somato-sympathetic coupling Fig 8. Glutamatergic interneurons are involved in the somato-sympathetic synaptic drive induced by oxotremorine. (A) Recordings from left lumbar 2 (lL2), right L2 (rL2), and right thoracic (rT8) ventral roots and an rT8 MN in the presence of oxotremorine alone (A1) or in combination with the NMDA and non-NMDA receptor antagonists, DNQX and AP5, respectively (A2). (B) A different preparation with lumbar and thoracic (lL2, rL2, rT5) ventral root recordings together with an intracellularly recorded rT5 IML SPN under oxotremorine alone (B1), then oxotremorine plus the glutamatergic receptor antagonists (B2). Slow and fast rhythmic bursting in MNs and IML SPNs This suppressive effect, which was consistently observed in the 10 preparations tested, suggested that the spinal net- work activated by oxotremorine involves glutamatergic neuronal relays that are upstream from MNs and IML SPNs. 7-dinitroquinoxaline-2,3-dione [DNQX], respectively), we observed the disappearance of oxo- tremorine-induced rhythmic bursting in both recorded ventral roots (Fig 8A and 8B) and intracellularly recorded axial MNs (Fig 8A) and IML SPNs (Fig 8B). This suppressive effect, which was consistently observed in the 10 preparations tested, suggested that the spinal net- work activated by oxotremorine involves glutamatergic neuronal relays that are upstream from MNs and IML SPNs. We then assessed whether glycinergic or GABAergic synaptic inputs also participate in the SSD drive, because bursts of inhibitory postsynaptic potentials could be clearly observed (and even reversed) in axial MNs (Fig 9A1) and IML SPNs (Fig 9A2) during the oxotremorine- induced rhythm. However, the glycinergic and/or GABAergic nature of this synaptic drive was difficult to determine, because the addition of strychnine (1 μM) and/or gabazine (1 μM) (antagonists of the glycinergic and GABAergic receptors, respectively) to the oxotremorine- containing saline completely altered the pattern of the elicited rhythm (n = 4, Fig 9B) and led to the expression of disinhibited, large amplitude bursting [39]. Nonetheless, together our data suggest that the SSD received by both axial MNs and IML SPNs following mAchR activation involves glutamatergic excitatory neurons and should also implicate inhibitory interneurons. Slow and fast rhythmic bursting in MNs and IML SPNs AP5, 2-amino-5-phosphonovalerate; DNQX, 6, 7-dinitroquinoxaline-2,3-dione; IML SPN, intermediolateral sympathetic preganglionic neuron; l, left; L, lumbar; MN, motoneuron; NMDA, N-methyl-D-aspartate; r, right; T, thoracic. https://doi.org/10.1371/journal.pbio.2005460.g008 Fig 8. Glutamatergic interneurons are involved in the somato-sympathetic synaptic drive induced by Fig 8. Glutamatergic interneurons are involved in the somato-sympathetic synaptic drive induced by oxotremorine. (A) Recordings from left lumbar 2 (lL2), right L2 (rL2), and right thoracic (rT8) ventral roots and an rT8 MN in the presence of oxotremorine alone (A1) or in combination with the NMDA and non-NMDA receptor antagonists, DNQX and AP5, respectively (A2). (B) A different preparation with lumbar and thoracic (lL2, rL2, rT5) ventral root recordings together with an intracellularly recorded rT5 IML SPN under oxotremorine alone (B1), then oxotremorine plus the glutamatergic receptor antagonists (B2). AP5, 2-amino-5-phosphonovalerate; DNQX, 6, 7-dinitroquinoxaline-2,3-dione; IML SPN, intermediolateral sympathetic preganglionic neuron; l, left; L, lumbar; MN, motoneuron; NMDA, N-methyl-D-aspartate; r, right; T, thoracic. https://doi.org/10.1371/journal.pbio.2005460.g008 Fig 8. Glutamatergic interneurons are involved in the somato-sympathetic synaptic drive induced by oxotremorine. (A) Recordings from left lumbar 2 (lL2), right L2 (rL2), and right thoracic (rT8) ventral roots and an rT8 MN in the presence of oxotremorine alone (A1) or in combination with the NMDA and non-NMDA receptor antagonists, DNQX and AP5, respectively (A2). (B) A different preparation with lumbar and thoracic (lL2, rL2, rT5) ventral root recordings together with an intracellularly recorded rT5 IML SPN under oxotremorine alone (B1), then oxotremorine plus the glutamatergic receptor antagonists (B2). AP5, 2-amino-5-phosphonovalerate; DNQX, 6, 7-dinitroquinoxaline-2,3-dione; IML SPN, intermediolateral sympathetic preganglionic neuron; l, left; L, lumbar; MN, motoneuron; NMDA, N-methyl-D-aspartate; r, right; T, thoracic. https://doi.org/10.1371/journal.pbio.2005460.g008 oxotremorine. (A) Recordings from left lumbar 2 (lL2), right L2 (rL2), and right thoracic (rT8) ventral roots and an rT8 MN in the presence of oxotremorine alone (A1) or in combination with the NMDA and non-NMDA receptor antagonists, DNQX and AP5, respectively (A2). (B) A different preparation with lumbar and thoracic (lL2, rL2, rT5) ventral root recordings together with an intracellularly recorded rT5 IML SPN under oxotremorine alone (B1), then oxotremorine plus the glutamatergic receptor antagonists (B2). AP5, 2-amino-5-phosphonovalerate; DNQX, 6, 7-dinitroquinoxaline-2,3-dione; IML SPN, intermediolateral sympathetic preganglionic neuron; l, left; L, lumbar; MN, motoneuron; NMDA, N-methyl-D-aspartate; r, right; T, thoracic. https://doi.org/10.1371/journal.pbio.2005460.g008 7-dinitroquinoxaline-2,3-dione [DNQX], respectively), we observed the disappearance of oxo- tremorine-induced rhythmic bursting in both recorded ventral roots (Fig 8A and 8B) and intracellularly recorded axial MNs (Fig 8A) and IML SPNs (Fig 8B). PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 Functional coupling between somatic and sympathetic activities The above data show that mAchR activation with oxotremorine constitutes a means to unmask the rhythmogenic capabilities of thoracic segments and to recruit both axial MN and IML SPN activity. The question then arises as to the functional consequences of this activation of tho- racic networks during the expression of fictive locomotion in the lumbar part of the spinal cord. Fig 10A1 shows representative recordings from a compartmentalized preparation of typ- ical locomotor-like activity induced by bath-applied NMDA/5-HT to the lumbar area only. The subsequent addition of oxotremorine specifically to the thoracic region (Fig 10A2) trig- gered independent slow bursting activity in the thoracic ventral roots. This slower thoracic burst rhythm was also observed in lumbar ventral roots, where it could be seen to regularly PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 10 / 21 Oxotremorine-induced somato-sympathetic coupling Fig 9. Inhibitory component of the somato-sympathetic drive induced by oxotremorine. (A) Recordings from a right thoracic (rT13) ventral root and a rT10 MN at two different membrane potentials during the bath-application of 10 μM oxotremorine (A1). Note the reversed inhibitory synaptic drive at −92 mV compared to −60 mV. Recordings from an rT10 ventral root and a rT9 IML under oxotremorine (A2). (B) Same MN as in A1 in the presence of oxotremorine and the GABAergic and glycinergic receptor antagonists, gabazine and strychnine (1 μM each), respectively. The gray panels in each case present an enlarged section of the intracellular recording to highlight the inhibitory postsynaptic potentials received by the MN and IML in the oxotremorine condition and their disappearance during the disinhibited rhythm with large spike bursts induced by oxotremorine in the presence of the strychnine/ gabazine cocktail. Vertical calibration bars: 5 mV. IML, intermediolateral neuron; l, left; MN, motoneuron; r, right; T, thoracic. https://doi org/10 1371/journal pbio 2005460 g009 Fig 9. Inhibitory component of the somato-sympathetic drive induced by oxotremorine. (A) Recordings from a right thoracic (rT13) ventral root and a rT10 MN at two different membrane potentials during the bath-application of 10 μM oxotremorine (A1). Note the reversed inhibitory synaptic drive at −92 mV compared to −60 mV. Recordings from an rT10 ventral root and a rT9 IML under oxotremorine (A2). (B) Same MN as in A1 in the presence of oxotremorine and the GABAergic and glycinergic receptor antagonists, gabazine and strychnine (1 μM each), respectively. Functional coupling between somatic and sympathetic activities The gray panels in each case present an enlarged section of the intracellular recording to highlight the inhibitory postsynaptic potentials received by the MN and IML in the oxotremorine condition and their disappearance during the disinhibited rhythm with large spike bursts induced by oxotremorine in the presence of the strychnine/ gabazine cocktail. Vertical calibration bars: 5 mV. IML, intermediolateral neuron; l, left; MN, motoneuron; r, right; T, thoracic. https://doi.org/10.1371/journal.pbio.2005460.g009 disrupt the ongoing faster locomotor rhythm (Fig 10A2) (n = 4/5 tested preparations). The gray columns in Fig 10A2 overlay the motor bursts recorded from the right T8 (rT8) ventral root, which alternated with left T10 bursts. As can be seen in the plots of Fig 10A3, which dis- play the cycle-by-cycle amplitudes of L2 locomotor-like bursts before (blue dots; see Fig 10A1) and during the additional slower rhythm triggered by oxotremorine applied to the thoracic cord (black dots; see Fig 10A2), L2 burst amplitudes were strongly decreased throughout the occurrence of each slow burst in the ipsilateral thoracic segments (see red ellipses in Fig 10A3). In contrast, when oxotremorine was applied with NMDA/5-HT to the lumbar compartment only (Fig 10B), the structure and regularity of the fictive locomotor rhythm remained unal- tered, although in 9 out of 12 tested preparations, an overall increase in lumbar burst ampli- tudes consistent with a previously described direct depolarizing effect of oxotremorine on lumbar MNs was observed [37,40]. Moreover, whether oxotremorine was applied either to the thoracic or lumbar cord region, or to both, the mean cycle periods of the ongoing fictive loco- motor bursting were not significantly modified (Fig 10C). disrupt the ongoing faster locomotor rhythm (Fig 10A2) (n = 4/5 tested preparations). The gray columns in Fig 10A2 overlay the motor bursts recorded from the right T8 (rT8) ventral root, which alternated with left T10 bursts. As can be seen in the plots of Fig 10A3, which dis- play the cycle-by-cycle amplitudes of L2 locomotor-like bursts before (blue dots; see Fig 10A1) and during the additional slower rhythm triggered by oxotremorine applied to the thoracic cord (black dots; see Fig 10A2), L2 burst amplitudes were strongly decreased throughout the occurrence of each slow burst in the ipsilateral thoracic segments (see red ellipses in Fig 10A3). PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 Functional coupling between somatic and sympathetic activities In contrast, when oxotremorine was applied with NMDA/5-HT to the lumbar compartment only (Fig 10B), the structure and regularity of the fictive locomotor rhythm remained unal- tered, although in 9 out of 12 tested preparations, an overall increase in lumbar burst ampli- tudes consistent with a previously described direct depolarizing effect of oxotremorine on lumbar MNs was observed [37,40]. Moreover, whether oxotremorine was applied either to the thoracic or lumbar cord region, or to both, the mean cycle periods of the ongoing fictive loco- motor bursting were not significantly modified (Fig 10C). Because the axons of both MNs and IML SPNs are conveyed in ventral roots until the L3 segment, we wondered whether the two rhythms expressed in lumbar ventral roots in vitro could somehow be linked to the differential activation of these two neuronal subtypes in vivo. To address this possibility, we made intracellular recordings from thoracic MNs (n = 7) under PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 11 / 21 Oxotremorine-induced somato-sympathetic coupling Fig 10. Different influences of oxotremorine-induced rhythmicity in thoracic segments and on lumbar fictive locomotion. (A) In a Vaseline partitioned spinal cord (at the T13 level), bath-application of NMDA/5-HT to the lumbar compartment elicited locomotor-like activity recorded from lumbar ventral roots (A1). Ten micromolar oxotremorine then added to the thoracic compartment induced slow alternating rhythmic bursting in right and left thoracic ventral roots and a strong modulation of the lumbar locomotor-like activity (A2). Cycle-by-cycle plots of burst amplitude in right and left L2 ventral roots (A3). Note that whenever a large burst occurred at the thoracic level, the amplitude of ongoing faster locomotor-like bursts in the ipsilateral lumbar ventral root was strongly decreased (red ovals). (B) The simultaneous bath-application of NMDA/5-HT and oxotremorine to the lumbar compartment failed to trigger slow rhythmic motor bursting in thoracic segments. A and B are from different experiments. (C) Bar plots of the cycle period of fictive locomotion in the different pharmacological conditions tested. aCSF, artificial cerebrospinal fluid; Amp, amplitude; l, left; L, lumbar; Oxo., oxotremorine; NMDA, N-methyl-D-aspartate; r, right; T, thoracic; 5-HT, serotonin. Fig 10. Different influences of oxotremorine-induced rhythmicity in thoracic segments and on lumbar fictive locomotion. (A) In a Vaseline partitioned spinal cord (at the T13 level), bath-application of NMDA/5-HT to the lumbar compartment elicited locomotor-like activity recorded from lumbar ventral roots (A1). Functional coupling between somatic and sympathetic activities Ten micromolar oxotremorine then added to the thoracic compartment induced slow alternating rhythmic bursting in right and left thoracic ventral roots and a strong modulation of the lumbar locomotor-like activity (A2). Cycle-by-cycle plots of burst amplitude in right and left L2 ventral roots (A3). Note that whenever a large burst occurred at the thoracic level, the amplitude of ongoing faster locomotor-like bursts in the ipsilateral lumbar ventral root was strongly decreased (red ovals). (B) The simultaneous bath-application of NMDA/5-HT and oxotremorine to the lumbar compartment failed to trigger slow rhythmic motor bursting in thoracic segments. A and B are from different experiments. (C) Bar plots of the cycle period of fictive locomotion in the different pharmacological conditions tested. aCSF, artificial cerebrospinal fluid; Amp, amplitude; l, left; L, lumbar; Oxo., oxotremorine; NMDA, N-methyl-D-aspartate; r, right; T, thoracic; 5-HT, serotonin. https://doi.org/10.1371/journal.pbio.2005460.g010 the different neuromodulatory conditions using the same experimental procedures as illus- trated in Fig 6. During bath-application of NMDA/5-HT to whole cord, as seen in Fig 6A1, the membrane potential of a recorded thoracic MN underwent rhythmic fluctuations correlated with the locomotor-like bursts monitored extracellularly in the L2 ventral root (Fig 11A; mean cycle period 2.4 ± 0.1 s, n = 30 cycles). After washout with normal saline in the same prepara- tion, the bath-application of oxotremorine alone elicited slow rhythmic oscillations in mem- brane potential correlated with the slow extracellular activity now expressed in the L2 ventral root (Fig 11B; see also Fig 6B2). When both the locomotor-like and slow bursting pattern were simultaneously elicited by the conjoint bath-application of NMA/5-HT and oxotremorine, respectively, a synaptic merging of the two motor rhythms with the superimposition of both fast locomotor-like drive potentials and slow membrane potential oscillations was observed (Fig 11C). Altogether, these data thus indicate that the oxotremorine-induced bursting gener- ated by thoracic networks is able to impose its slower motor-related rhythm on the locomotor activity generated by lumbar networks, resulting in a combined functional coupling of the two motor patterns. the different neuromodulatory conditions using the same experimental procedures as illus- trated in Fig 6. During bath-application of NMDA/5-HT to whole cord, as seen in Fig 6A1, the membrane potential of a recorded thoracic MN underwent rhythmic fluctuations correlated with the locomotor-like bursts monitored extracellularly in the L2 ventral root (Fig 11A; mean cycle period 2.4 ± 0.1 s, n = 30 cycles). PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 Functional coupling between somatic and sympathetic activities After washout with normal saline in the same prepara- tion, the bath-application of oxotremorine alone elicited slow rhythmic oscillations in mem- brane potential correlated with the slow extracellular activity now expressed in the L2 ventral root (Fig 11B; see also Fig 6B2). When both the locomotor-like and slow bursting pattern were simultaneously elicited by the conjoint bath-application of NMA/5-HT and oxotremorine, respectively, a synaptic merging of the two motor rhythms with the superimposition of both fast locomotor-like drive potentials and slow membrane potential oscillations was observed (Fig 11C). Altogether, these data thus indicate that the oxotremorine-induced bursting gener- ated by thoracic networks is able to impose its slower motor-related rhythm on the locomotor activity generated by lumbar networks, resulting in a combined functional coupling of the two motor patterns. PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 12 / 21 Oxotremorine-induced somato-sympathetic coupling Fig 11. Oxotremorine-induced synaptic merging of slow and fast motor rhythms. Recordings from a left lumbar 2 (lL2) ventral root and a right thoracic (rT10) MN during bath-application to the whole thoracolumbar spinal cord of NMDA/5-HT alone (A), 10 μM oxotremorine alone (B), and NMDA/5-HT plus oxotremorine (C). The conjoint presence of oxotremorine + NMDA/5-HT produced a merging of the slow and fast motor rhythms. l, left; L, lumbar; MN, motoneuron; NMDA, N-methyl-D-aspartate; T, thoracic; 5-HT, serotonin. htt //d i /10 1371/j l bi 2005460 011 Fig 11. Oxotremorine-induced synaptic merging of slow and fast motor rhythms. Recordings from a left lumbar 2 (lL2) ventral root and a right thoracic (rT10) MN during bath-application to the whole thoracolumbar spinal cord of NMDA/5-HT alone (A), 10 μM oxotremorine alone (B), and NMDA/5-HT plus oxotremorine (C). The conjoint presence of oxotremorine + NMDA/5-HT produced a merging of the slow and fast motor rhythms. l, left; L, lumbar; MN, motoneuron; NMDA, N-methyl-D-aspartate; T, thoracic; 5-HT, serotonin. Fig 11. Oxotremorine-induced synaptic merging of slow and fast motor rhythms. Recordings from a left lumbar 2 (lL2) ventral root and a right thoracic (rT10) MN during bath-application to the whole thoracolumbar spinal cord of NMDA/5-HT alone (A), 10 μM oxotremorine alone (B), and NMDA/5-HT plus oxotremorine (C). The conjoint presence of oxotremorine + NMDA/5-HT produced a merging of the slow and fast motor rhythms. l, left; L, lumbar; MN, motoneuron; NMDA, N-methyl-D-aspartate; T, thoracic; 5-HT, serotonin. https://doi.org/10.1371/journal.pbio.2005460.g011 https://doi.org/10.1371/journal.pbio.2005460.g011 https://doi.org/10.1371/journal.pbio.2005460.g011 Cholinergic activation of MNs and IML SPNs Ach plays a ubiquitous role in the control of motor and sympathetic outflow. As a neurotrans- mitter utilized by both the MNs and SPNs, it is engaged in all autonomic ganglia, neuromuscu- lar junctions, and also at many autonomically innervated organs. Furthermore, both MNs and IML SPNs are themselves targeted by cholinergic synapses arising from the cholinergic pro- priospinal system [20,22,24,29,37,41,43]. The data presented here suggest that the rhythmic activity observed following mAchR activation results from concomitant network and direct effects of oxotremorine on SPNs and MNs. Although the sympathetic nervous system is still maturing at the age studied here [44], Zimmerman and Hochman (2010) previously demon- strated that the membrane properties of IML SPNs are specified as early as P3 and that the overall functional features of SPNs are already mostly in place in the neonate [45]. The present study is the first to provide a direct comparison of the membrane properties of MNs and IML SPNs in similar experimental conditions and reveal no striking differences between these two neuronal subtypes. Oxotremorine-induced somato-sympathetic coupling In contrast to the majority of known neuromodulatory systems capable of activating or controlling the motor spinal networks, the cholinergic system does not originate from extrinsic spinal sources but is thought to be completely intrinsic to the spinal cord. While the role of Ach in influencing spinal motor network activities is recognized [21,23,24,26–28,41], its con- tribution to the generation of the mammalian locomotor rhythm is still debatable [42]. Some of the effects of the cholinergic propriospinal system were also observed in the isolated spinal cord of rodents through the use of AchE inhibitors (such as edrophonium, neostigmine, and physostigmine) that prevent Ach degradation and so enhance the spinal endogenous content of spontaneously released Ach [24,27,42] and increase the potency of bath-applied Ach [21]. In the in vitro spinal cord preparation, AchE inhibitors induce episodes of locomotor-like activity that can be blocked by M2 and M3 muscarinic antagonists [27]. In our experimental conditions with the newborn rat preparation, AchE inhibitors only triggered short bouts of locomotor-like activity. In addition, we observed that the bath-application of oxotremorine systematically and uniquely induced a slow motor rhythm devoid of extensor-flexor alterna- tion, instead of a recognizable locomotor-like activity. Interestingly, this slow motor pattern has been reported in previous studies (see, for example, Fig 1 in Jordan and colleagues, 2014; Fig 1B in Anglister and colleagues, 2008). However, these authors did not describe this slow rhythm and did not test the effects of cholinergic muscarinic agonists. Why do cholinergic muscarinic agonists only partly reproduce the activating effects of AchE inhibitors? Even if muscarinic antagonists abolish the action of AchE inhibitors [27], it cannot be ruled out that either nicotinic receptors are involved or a more localized activation of mAchRs are required to induce locomotor-like activity. Alternately, this may be due to weak potency of available selective mAchR agonists (Ishii and Kurashi, 2006) because the different agonists tested either partly reproduced oxotremorine action or failed to elicit any motor activity. Motor rhythms generated by muscarinic agonists in the spinal cord In the present report, we show that the thoracic segments of the newborn rat spinal cord exhibit a preferential sensitivity to mAchR activation compared to the rhythmogenic circuitry in the lumbar segments, leading to the expression of a slow and robust bursting activity. To the best of our knowledge, this is the first demonstration that thoracic networks are endowed with such an independent rhythmogenic capability. PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 13 / 21 PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 Oxotremorine-induced somato-sympathetic coupling outputs were generated by central neural networks capable of rhythm generation, rather than by pools of interneurons driven by afferent inputs (for review, see [8]). Interestingly, a similar debate emerged at the same time regarding the central generation of motor patterns [46,47]. Double retrograde labeling has identified neurons in the pontomedullary reticular forma- tion, the pedunculopontine tegmental nucleus, and lateral hypothalamus that have been classi- fied as central commanders of both autonomic and motor outflow [48,49]. Some reports have also postulated the existence of a decentralized control system that would reside in the spinal cord [3,5,6,50]. The first evidence for a coupling between somatic and sympathetic outflow in the thoracolumbar spinal cord was provided by Chizh and colleagues [6]. Using an arterially perfused trunk–hindquarter preparation of adult mouse, these authors found that at rest, when only tonic background activity was produced, or during NMDA-induced rhythmic activity, sympathetic and somatic motor output could become synchronized. Subsequently, Goodchild and colleagues (2008) showed that an independent supraspinal coupling exists between multiple sympathetic and motor outflows in the adult rat spinal cord in vivo. These authors proposed that this coupling allows a coordination of activity between the different out- flows when hyper-excitation occurs [5]. Here, we propose that an intraspinal rhythmogenic network, whose activation is conditional upon the cholinergic propriospinal system, is likely to be responsible for this synchronizing process. The neuronal substrate of such a network remains elusive (for reviews, see [3,51]). Our data indicate that glutamatergic neurons are part of the local network unmasked by oxotremorine. Potential candidates for the inhibitory com- ponent of the SSD is a subset of GABAergic interneurons located around the central canal, which has been previously shown to inhibit SPNs [51,52]. Therefore, further investigation will be needed to assess the different cholinergic, glutamatergic, and inhibitory neurons that com- pose the rhythmogenic network activated by oxotremorine. Here, we also found that mAchR activation leads to a superposition of the thoracic and lumbar rhythms induced by the presence of oxotremorine and NMDA/5-HT, respectively. Comparable reconfigurations of motor outputs have been observed in both invertebrate [53] and vertebrate [54] central pattern-generating systems that have been proposed as the basis for the functional flexibility of motor system output. Intraspinal interactions between somatic and sympathetic neuronal activities MNs as well as SPNs must integrate inputs from both descending and sensory systems that shape the output of the somatic and sympathetic nervous systems. The classical view that has emerged over the past century is that the background activity of SPNs is controlled by suprasp- inal neuronal networks whose random and diffuse outputs were synchronized by rhythmic inputs arising from cardiovascular and respiratory origins (for review, see [8]). This view was challenged from the late 1970s by the hypothesis that the various rhythmic sympathetic PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 14 / 21 PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 In vitro isolated spinal cord preparation Rat pups were anesthetized with isofluorane until reflexes could no longer be elicited in response to tail or toe pinching. Animals were decapitated, and the skin of the back was removed before preparations were placed dorsal side up in a dissecting chamber. A laminectomy was performed to expose the spinal cord, which was carefully dissected free under a binocular microscope. Dis- section and recording procedures were conducted under continuous superperfusion with artificial cerebrospinal fluid (aCSF) equilibrated with 95% O2/5% CO2, adjusted to pH 7.4 at room temper- ature (24–26˚C) and containing the following (in mM): 130 NaCl, 3 KCl, 2.5 CaCl2, 1.3 MgSO4, 0.58 NaH2PO4, 25 NaHCO3, and 10 glucose. Spinal cords were sectioned at the T1 level at the beginning of the experiment. In some experiments, the spinal cord was artificially partitioned using Vaseline walls, as previously described [62], to restrict the bath-application of pharmacologi- cal agents to specific segmental regions. The watertightness of the barriers was systematically checked at the end of each experiment by observing the movements of methylene blue added to the bathing medium on one side of the Vaseline wall(s). Ethics statement Experiments were conducted in vitro on isolated spinal cords from newborn Sprague Dawley rats of either sex, aged from postnatal day 0 (P0) to P5 (n = 108 preparations). All procedures were conducted in accordance with the local ethics committee of the University of Bordeaux and the European Committee Council Directive (approval number 2016012716035720). All efforts were made to minimize animal suffering and reduce the number of animals used. Oxotremorine-induced somato-sympathetic coupling involved in respiratory-related discharge concomitant with hind limb muscle activity (as shown by Wienecke and colleagues, 2015). This would require a new experimental paradigm because the isolated spinal cord does not allow us to monitor the respiratory activity that is generated in the brain stem [59]. The frequency range of the oxotremorine-induced rhythm found in the present study is compatible with the frequency of the Mayer waves because, in our experimental conditions, the cycle period of the oxotremorine-induced activity ranged from 13 to 30 s (average period, 21.7 s; Fig 1D1). In our in vitro conditions, however, it has been shown that the locomotor period decreases by more than 50% when the temperature is increased from 25˚C (the temperature at which recordings are made) to 35˚C [60]. On this basis, therefore, at such higher temperatures corresponding to those in the intact animal, the cycle period of the oxotremorine-induced rhythm would be expected to be less than 10 s. An important wider implication of the present findings relates to overcoming one of the major problems for people suffering from spinal cord injury (SCI): severe cardiovascular disturbances that contribute to 40% of deaths [61]. The existence of an intraspinal coupling mechanism and the possibility to activate coordinated somatic and sympathetic activities through pharmacological means thus raises the prospect for mitigating vascular dysfunctions after SCI. Functional importance of intraspinal coupling between somatic and sympathetic activities Does the slow oxotremorine-induced rhythm have a specific physiological relevance? In physi- ological conditions, SPNs are rhythmically active in a frequency band ranging from 0.1 to 10 Hz [55]. The slow sympathetic neuronal activities have been related to cardiovascular control [7] and rhythmic changes in arterial pressure (corresponding to the ‘‘10-s rhythm” in humans). In the context of the present study, these so-called “Mayer waves” are of particular interest (for review, see [56]). From their first description, these activity oscillations were referred to as vasomotor waves and were proposed to provide an indirect measure of efferent sympathetic nervous activity. In several species, including humans, Mayer waves are modu- lated in situations that result in sympathetic activation and parallel the mean level of sympa- thetic nervous activity [57]. The relationship between the Mayer waves and locomotor (or respiratory) rhythms has been recently explored in decerebrate cats under neuromuscular blockade [58]. These authors reported that the occurrence of Mayer waves was frequently related to the initiation of episodes of fictive locomotion and to variations in the extracellular locomotor burst amplitude. Furthermore, the occurrence of Mayer waves also matched epi- sodes of entrainment between the respiratory and the locomotor rhythms. In the present study, we could not assess whether the intraspinal network revealed by oxotremorine is also PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 15 / 21 Statistics Statistical analyses of raw data were conducted using GraphPad Prism software. Because of some relatively small samples, nonparametric tests were used for all analyses performed. In the text, all data are expressed as means ± SEM. In the figures, the box plots display the distribu- tion of data based on the minimum, first quartile, median, third quartile, and maximum. Asterisks in the figures indicate statistical significance (p < 0.05). Wilcoxon or Mann–Whitney tests were used when applicable. The coupling between left and right activities was analyzed using circular statistics, using the Oriana Software (Kovach Computing Services). Oxotremorine-induced somato-sympathetic coupling chosen thoracic level using fine scissors (MC-26B, Moria). To maintain the sectioned surface of the cord face-upwards and facilitate microelectrode positioning, the cut end of the spinal cord was positioned on a Sylgard wedge (see Fig 6A1). SPNs and MNs were targeted on the basis of their location in the transverse plane (see Fig 6A) and subsequently identified by recording their antidromic action potentials in response to electrical ventral root stimulation of the same segment (see Fig 6A2–6A3). A liquid junction potential of +12 mV was experimentally determined [63] and records were corrected for this potential. Series resistance was monitored throughout the experiments and was not compensated. Data were discarded if series resistance varied more than ±20% of the initial value. Motor activities were recorded extracellularly from various spinal cord ventral roots using glass suction electrodes. Recorded activity was amplified using custom-made amplifiers. The recordings were digitized using an analog-to-digital interface (Heka Elektronik, Germany) driven by Axograph software (Axograph, Australia). Raw signals were high-pass filtered (50 Hz), rectified, and integrated before analysis. Locomotor burst parameters were computed using custom-made routines written in Matlab (Mathworks, France). Mean cycle periods were computed using an L2 ventral root as the reference because it invariably exhibited the best sig- nal-to-noise ratio. Wavelet transform analyses [64] were performed using a Matlab wavelet coherence package provided by Aslak Grinsted (http://noc.ac.uk/using-science/crosswavelet- wavelet-coherence). Of particular interest was the extraction of the common power, correla- tion, and phase relationship between two simultaneously acquired signals of the cross wavelet transform and wavelet coherence [65,66]. A detailed explanation of the wavelet-based method- ology used in the present work has been previously reported [32]. Pharmacology Episodes of locomotor-like activity were elicited by exogenous bath-application of a mixture of 7.5 μM NMDA and 15 μM 5-HT [67]. All drugs were obtained from ABCAM. AF-DX 116 (M2 antagonist), 4-DAMP (M3 antagonist), and tropicamide (M4 antagonist) were diluted in DMSO at concentration less than 0.1% [68]. All other drugs were prepared as stock solutions in aCSF. Drugs were bath-applied using a peristaltic pump (flow rate 4 mL/min; recording chamber volume 4 mL). The effects of the drugs were monitored from 5–10 min after reaching the recording dish (i.e., the time estimated for a total replacement of the bathing saline and diffu- sion into the tissue). Electrophysiological recordings and analysis Whole cell patch-clamp recordings from neurons were obtained with a Multiclamp 700B amplifier (Molecular Devices). Patch-clamp glass microelectrodes (4–7 MO) were filled with the following solution (in mM): 120 K-gluconate, 20 KCl, 0.1 MgCl2, 1 EGTA, 10 HEPES, 0.1 CaCl2, 0.1 GTP, 0.2 cAMP, 0.1 leupeptin, 77 D-mannitol, 3 Na2-ATP, pH 7.3. Intracellular recordings from MNs and SPNs were made according to a protocol developed in a previous study [32]. To access thoracic neurons, a transverse section of the spinal cord was made at a PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 16 / 21 References 1. Saper CB. The Central Autonomic Nervous System: Conscious Visceral Perception and Autonomic Pattern Generation. Annu Rev Neurosci. 2002; 25: 433–469. https://doi.org/10.1146/annurev.neuro.25. 032502.111311 PMID: 12052916 2. Guyenet PG. The sympathetic control of blood pressure. Nat Rev Neurosci. 2006; 7: 335–346. https:// doi.org/10.1038/nrn1902 PMID: 16760914 3. Deuchars SA. Multi-tasking in the spinal cord—do “sympathetic” interneurones work harder than we give them credit for? J Physiol (Lond). 2007; 580: 723–729. https://doi.org/10.1113/jphysiol.2007. 129429 PMID: 17347266 4. Su CK. Rhythmic sympathetic nerve discharges in an in vitro neonatal rat brain stem-spinal cord prepa- ration. J Appl Physiol. 1999; 87: 1066–1074. https://doi.org/10.1152/jappl.1999.87.3.1066 PMID: 10484578 5. Goodchild AK, van Deurzen BTM, Hildreth CM, Pilowsky PM. Control of sympathetic, respiratory and somatomotor outflow by an intraspinal pattern generator. Clin Exp Pharmacol Physiol. 2008; 35: 447– 453. https://doi.org/10.1111/j.1440-1681.2008.04913.x PMID: 18307739 6. Chizh BA, Headley PM, Paton JF. Coupling of sympathetic and somatic motor outflows from the spinal cord in a perfused preparation of adult mouse in vitro. J Physiol (Lond). 1998; 508 (Pt 3): 907–918. 7. Malpas SC. The rhythmicity of sympathetic nerve activity. Prog Neurobiol. 1998; 56: 65–96. PMID: 9723131 8. Coote JH. Landmarks in understanding the central nervous control of the cardiovascular system. Exp Physiol. 2007; 92: 3–18. https://doi.org/10.1113/expphysiol.2006.035378 PMID: 17030558 9. Deuchars SA, Spyer KM, Brooks PA, Gilbey MP. A study of sympathetic preganglionic neuronal activity in a neonatal rat brainstem-spinal cord preparation. J Auton Nerv Syst. 1995; 52: 51–63. PMID: 7782569 10. Spanswick D, Logan SD. Spontaneous rhythmic activity in the intermediolateral cell nucleus of the neo- nate rat thoracolumbar spinal cord in vitro. Neuroscience. 1990; 39: 395–403. PMID: 2087263 11. Pickering AE, Spanswick D, Logan SD. 5-Hydoxytryptamine evokes depolarizations and membrane potential oscillations in rat sympathetic preganglionic neurones. J Physiol (Lond). 1994; 480 (Pt 1): 109–121. 12. Su CK. Intraspinal amino acid neurotransmitter activities are involved in the generation of rhythmic sym- pathetic nerve discharge in newborn rat spinal cord. Brain Res. 2001; 904: 112–125. PMID: 11516417 13. Marina N, Taheri M, Gilbey MP. Generation of a physiological sympathetic motor rhythm in the rat fol- lowing spinal application of 5-HT. J Physiol (Lond). 2006; 571: 441–450. https://doi.org/10.1113/ jphysiol.2005.100677 PMID: 16396930 14. Thomson LM, Zeng J, Terman GW. An N-methyl-D-aspartate receptor mediated large, low-frequency, spontaneous excitatory postsynaptic current in neonatal rat spinal dorsal horn neurons. Neuroscience. 2006; 141: 1489–1501. https://doi.org/10.1016/j.neuroscience.2006.04.049 PMID: 16750886 15. Pierce ML, Deuchars J, Deuchars SA. Author Contributions Conceptualization: Me´lissa Sourioux, Sandrine S. Bertrand, Jean-Rene´ Cazalets. Data curation: Me´lissa Sourioux, Sandrine S. Bertrand. Formal analysis: Me´lissa Sourioux. Investigation: Me´lissa Sourioux, Sandrine S. Bertrand, Jean-Rene´ Cazalets. Investigation: Me´lissa Sourioux, Sandrine S. Bertrand, Jean-Rene´ Cazalets. Project administration: Sandrine S. Bertrand, Jean-Rene´ Cazalets. Project administration: Sandrine S. Bertrand, Jean-Rene´ Cazalets. Supervision: Sandrine S. Bertrand, Jean-Rene´ Cazalets. Writing – original draft: Me´lissa Sourioux, Sandrine S. Bertrand, Jean-Rene´ Cazalets. Writing – review & editing: Me´lissa Sourioux, Sandrine S. Bertrand, Jean-Rene´ Cazalets. Acknowledgments The authors thank Aslak Grinsted for providing a Matlab wavelet coherence package (http:// noc.ac.uk/using-science/crosswavelet-wavelet-coherence) and Dr. G. Barrière for assistance with wavelet analysis. 17 / 21 PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 Oxotremorine-induced somato-sympathetic coupling Author Contributions Conceptualization: Me´lissa Sourioux, Sandrine S. Bertrand, Jean-Rene´ Cazalets. Data curation: Me´lissa Sourioux, Sandrine S. Bertrand. Formal analysis: Me´lissa Sourioux. Investigation: Me´lissa Sourioux, Sandrine S. Bertrand, Jean-Rene´ Cazalets. Project administration: Sandrine S. Bertrand, Jean-Rene´ Cazalets. Supervision: Sandrine S. Bertrand, Jean-Rene´ Cazalets. Writing – original draft: Me´lissa Sourioux, Sandrine S. Bertrand, Jean-Rene´ Cazalets. Writing – review & editing: Me´lissa Sourioux, Sandrine S. Bertrand, Jean-Rene´ Cazalets. PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 Oxotremorine-induced somato-sympathetic coupling 17. Marina N, Becker DL, Gilbey MP. Immunohistochemical detection of connexin36 in sympathetic pre- ganglionic and somatic motoneurons in the adult rat. Peripheral projections of NESP55 containing neu- rons in the rat sympathetic ganglia. Elsevier B.V; 2008; 139: 15–23. https://doi.org/10.1016/j.autneu. 2007.12.004 PMID: 18280223 18. Rossignol S, Chau C, Brustein E, Giroux N, Bouyer L, Barbeau H, et al. Pharmacological Activation and Modulation of the Central Pattern Generator for Locomotion in the Cata. Ann NY Acad Sci. Blackwell Publishing Ltd; 1998; 860: 346–359. https://doi.org/10.1111/j.1749-6632.1998.tb09061.x PMID: 9928324 19. Miles GB, Sillar KT. Neuromodulation of Vertebrate Locomotor Control Networks. Physiology. 2011; 26: 393–411. https://doi.org/10.1152/physiol.00013.2011 PMID: 22170958 20. Yoshimura M, Nishi S. Intracellular recordings from lateral horn cells fo the spinal cord in vitro. J Auton Nerv Syst. 1982; 6: 5–11. PMID: 6290560 21. Cowley KC, Schmidt BJ. A comparison of motor patterns induced by N-methyl-D-aspartate, acetylcho- line and serotonin in the in vitro neonatal rat spinal cord. Neurosci Lett. 1994; 171: 147–150. PMID: 8084477 22. Gibson IC, Logan SD. Effects of muscarinic receptor stimulation of sympathetic preganglionic neurones of neonatal rat spinal cord in vitro. Neuropharmacology. 1995; 34: 309–318. PMID: 7630486 23. Huang A, Noga B, Carr P, Fedirchuk B, Jordan L. Spinal Cholinergic Neurons Activated During Locomo- tion: Localization and Electrophysiological Characterization. J Neurophysiol. 2000; 83: 3537. https://doi. org/10.1152/jn.2000.83.6.3537 PMID: 10848569 24. Miles GB, Hartley R, Todd AJ, Brownstone RM. Spinal cholinergic interneurons regulate the excitability of motoneurons during locomotion. Proc Natl Acad Sci USA. 2007; 104: 2448–2453. https://doi.org/10. 1073/pnas.0611134104 PMID: 17287343 25. Anglister L, Etlin A, Finkel E, Durrant AR, Lev-Tov A. Cholinesterases in development and disease. Chem Biol Interact. 2008; 175: 92–100. https://doi.org/10.1016/j.cbi.2008.04.046 PMID: 18571632 26. Zagoraiou L, Akay T, Martin JF, Brownstone RM, Jessell TM, Miles GB. A cluster of cholinergic premo- tor interneurons modulates mouse locomotor activity. Neuron. 2009; 64: 645–662. https://doi.org/10. 1016/j.neuron.2009.10.017 PMID: 20005822 27. Jordan LM, McVagh JR, Noga BR, Cabaj AM, Majczyński H, S¯awińska U, et al. Cholinergic mecha- nisms in spinal locomotion-potential target for rehabilitation approaches. Front Neural Circuits. 2014; 8: 132. https://doi.org/10.3389/fncir.2014.00132 PMID: 25414645 28. Tillakaratne NJK, Duru P, Fujino H, Zhong H, Xiao MS, Edgerton VR, et al. Identification of interneurons activated at different inclines during treadmill locomotion in adult rats. J Neurosci Res. 2014; 92: 1714– 1722. https://doi.org/10.1002/jnr.23437 PMID: 24975393 29. Barber RP, Phelps PE, Houser CR, Crawford GD, Salvaterra PM, Vaughn JE. PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 References Spontaneous rhythmogenic capabilities of sympathetic neuronal assemblies in the rat spinal cord slice. 2010; 170: 827–838. https://doi.org/10.1016/j.neuroscience. 2010.07.007 PMID: 20650307 16. Shen E, Dun NJ. Neonate rat sympathetic preganglionic neurons intracellularly labelled with lucifer yel- low in thin spinal cord slices. J Auton Nerv Syst. 1990; 29: 247–254. PMID: 2341699 PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 18 / 21 Oxotremorine-induced somato-sympathetic coupling 38. Button DC, Gardiner K, Marqueste T, Gardiner PF. Frequency-current relationships of rat hindlimb -motoneurones. J Physiol (Lond). 2006; 573: 663–677. https://doi.org/10.1113/jphysiol.2006.107292 PMID: 16613880 39. Bracci E, Ballerini L, Nistri A. Localization of rhythmogenic networks responsible for spontaneous bursts induced by strychnine and bicuculline in the rat isolated spinal cord. J Neurosci. 1996; 16: 7063–7076. PMID: 8824342 40. Milan L, Courtand G, Cardoit L, Masmejean F, Barrière G, Cazalets J-R, et al. Age-Related Changes in Pre- and Postsynaptic Partners of the Cholinergic C-Boutons in Wild-Type and SOD1G93A Lumbar Motoneurons. Raoul C, editor. PLoS ONE. 2015; 10: e0135525–21. https://doi.org/10.1371/journal. pone.0135525 PMID: 26305672 41. Deardorff AS, Romer SH, Sonner PM, Fyffe REW. Swimming against the tide: investigations of the C- bouton synapse. Front Neural Circuits. 2014; 8: 106. https://doi.org/10.3389/fncir.2014.00106 PMID: 25278842 42. Anglister L, Cherniak M, Lev-Tov A. Ascending pathways that mediate cholinergic modulation of lumbar motor activity. J Neurochem. 2017; 142 Suppl 2: 82–89. https://doi.org/10.1111/jnc.14065 PMID: 28791705 43. Sherriff FE, Henderson Z, Morrison JFB. Further evidence for the absence of a descending cholinergic projection from the brainstem to the spinal cord in the rat. Neurosci Lett. 1991; 128: 52–56. https://doi. org/10.1016/0304-3940(91)90758-l PMID: 1717898 44. Black IB. Regulation of autonomic development. Annu Rev Neurosci. 1978; 1: 183–214. https://doi.org/ 10.1146/annurev.ne.01.030178.001151 PMID: 386901 45. Zimmerman A, Hochman S. Heterogeneity of Membrane Properties in Sympathetic Preganglionic Neu- rons of Neonatal Mice: Evidence of Four Subpopulations in the Intermediolateral Nucleus. J Neurophy- siol. 2010; 103: 490–498. https://doi.org/10.1152/jn.00622.2009 PMID: 19923248 46. Rossignol S, Dubuc R. Spinal pattern generation. Curr Opin Neurobiol. 1994; 4: 894–902. PMID: 7888774 47. Marder E, Bucher D. Central pattern generators and the control of rhythmic movements. Current Biol- ogy. 2001; 11: R986–96. PMID: 11728329 48. Kerman IA, Enquist LW, Watson SJ, Yates BJ. Brainstem substrates of sympatho-motor circuitry identi- fied using trans-synaptic tracing with pseudorabies virus recombinants. J Neurosci. 2003; 23: 4657– 4666. PMID: 12805305 49. Krout KE, Mettenleiter TC, Loewy AD. Single cns neurons link both central motor and cardiosympathetic systems: a double-virus tracing study. Neuroscience. 2003; 118: 853–866. https://doi.org/10.1016/ s0306-4522(02)00997-1 PMID: 12710992 50. Su CK. Rhythmic sympathetic nerve discharges in an in vitro neonatal rat brain stem-spinal cord prepa- ration. J Appl Physiol. 1999; 87: 1066–1074. https://doi.org/10.1152/jappl.1999.87.3.1066 PMID: 10484578 51. Deuchars SA. How sympathetic are your spinal cord circuits? Exp Physiol. 2015; 100: 365–371. https:// doi.org/10.1113/EP085031 PMID: 25655449 52. Wang L, Spary E, Deuchars J, Deuchars SA. PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 The morphology and dis- tribution of neurons containing choline acetyltransferase in the adult rat spinal cord: an immunocyto- chemical study. J Comp Neurol. 1984; 229: 329–346. https://doi.org/10.1002/cne.902290305 PMID: 6389613 30. Kayaalp SO, Neff NH. Regional distribution of cholinergic muscarinic receptors in spinal cord. Brain Res. 1980; 196: 429–436. https://doi.org/10.1016/0006-8993(78)91047-8 PMID: 7397537 31. Falgairolle M, Cazalets J-R. Metachronal coupling between spinal neuronal networks during locomotor activity in newborn rat. J Physiol (Lond). Blackwell Publishing Ltd; 2007; 580: 87–102. https://doi.org/ 10.1113/jphysiol.2006.115709 PMID: 17185345 32. Beliez L, Barrière G, Bertrand SS, Cazalets JR. Origin of Thoracic Spinal Network Activity during Loco- motor-Like Activity in the Neonatal Rat. J Neurosci. 2015; 35: 6117–6130. https://doi.org/10.1523/ JNEUROSCI.4145-14.2015 PMID: 25878284 33. Cazalets JR, Sqalli-Houssaini Y, Clarac F. Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat. J Physiol (Lond). 1992; 455: 187–204. 34. Sharples SA. Dopamine Pumping Up Spinal Locomotor Network Function. J Neurosci. 2017; 37: 3103– 3105. https://doi.org/10.1523/JNEUROSCI.0019-17.2017 PMID: 28330979 35. Marder E, O’Leary T, Shruti S. Neuromodulation of circuits with variable parameters: single neurons and small circuits reveal principles of state-dependent and robust neuromodulation. Annu Rev Neu- rosci. 2014; 37: 329–346. https://doi.org/10.1146/annurev-neuro-071013-013958 PMID: 25032499 36. Wilson JM, Rempel J, Brownstone RM. Postnatal development of cholinergic synapses on mouse spi- nal motoneurons. J Comp Neurol. 2004; 474: 13–23. https://doi.org/10.1002/cne.20089 PMID: 15156576 37. Bertrand SS, Cazalets J-R. Cholinergic partition cells and lamina x neurons induce a muscarinic-depen- dent short-term potentiation of commissural glutamatergic inputs in lumbar motoneurons. Front Neural Circuits. 2011; 5: 15. https://doi.org/10.3389/fncir.2011.00015 PMID: 22069380 PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 19 / 21 PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 Tonic GABAergic inhibition of sympathetic preganglionic neurons: a novel substrate for sympathetic control. J Neurosci. 2008; 28: 12445–12452. https://doi.org/ 10.1523/JNEUROSCI.2951-08.2008 PMID: 19020037 53. Hooper SL, Moulins M. Switching of a neuron from one network to another by sensory-induced changes in membrane properties. Science. 1989; 244: 1587–1589. PMID: 2740903 54. Gestreau C, Gre´lot L, Bianchi AL. Activity of respiratory laryngeal motoneurons during fictive coughing and swallowing. Exp Brain Res. 2000; 130: 27–34. PMID: 10638438 55. Deuchars SA, Lall VK. Sympathetic preganglionic neurons: properties and inputs. Comprehensive Physiology. 2015; 5: 829–869. https://doi.org/10.1002/cphy.c140020 PMID: 25880515 56. Julien C. The enigma of Mayer waves: Facts and models. Cardiovasc Res. 2006; 70: 12–21. https://doi. org/10.1016/j.cardiores.2005.11.008 PMID: 16360130 57. Julien C, Malpas SC, Stauss HM. Sympathetic modulation of blood pressure variability. Journal of Hypertension. 2001; 19: 1707–1712. https://doi.org/10.1097/00004872-200110000-00002 PMID: 11593088 58. Wienecke J, Enrı´quez Denton M, Stecina K, Kirkwood PA, Hultborn H. Modulation of spontaneous loco- motor and respiratory drives to hindlimb motoneurons temporally related to sympathetic drives as revealed by Mayer waves. Front Neural Circuits. 2015; 9: 1. https://doi.org/10.3389/fncir.2015.00001 PMID: 25713515 59. Mellen NM, Thoby-Brisson M. Respiratory circuits: development, function and models. Curr Opin Neu- robiol. 2012; 22: 676–685. https://doi.org/10.1016/j.conb.2012.01.001 PMID: 22281058 PLOS Biology \| https://doi.org/10.1371/journal.pbio.2005460 July 9, 2018 20 / 21 Oxotremorine-induced somato-sympathetic coupling 60. Sqalli-Houssaini Y, Cazalets JR, Fabre JC, Clarac F. A cooling/heating system for use with in vitro prep- arations: study of temperature effects on newborn rat rhythmic activities. J Neurosci Meth. 1991; 39: 131–139. 61. Garshick E, Kelley A, Cohen SA, Garrison A, Tun CG, Gagnon D, et al. A prospective assessment of mortality in chronic spinal cord injury. Spinal Cord. 2005; 43: 408–416. https://doi.org/10.1038/sj.sc. 3101729 PMID: 15711609 62. Cazalets J, Borde M, Clarac F. The synaptic drive from the spinal locomotor network to motoneurons in the newborn rat. J Neurosci. 1996; 16: 298. PMID: 8613795 63. Neher E. Correction for liquid junction potentials in patch clamp experiments. Meth Enzymol. 1992; 207: 123–131. PMID: 1528115 64. Mor Y, Lev-Tov A. Analysis of rhythmic patterns produced by spinal neural networks. J Neurophysiol. 2007; 98: 2807–2817. https://doi.org/10.1152/jn.00740.2007 PMID: 17715187 65. Torrence C, Compo GP. A practical guide to wavelet analysis. Bulletin of the American Meteorological society. 1998; 79: 61–78. 66. Grinsted A, Moore JC, Jevrejeva S. Application of the cross wavelet transform and wavelet coherence to geophysical time series. Nonlinear processes in geophysics. Copernicus GmbH; 2004; 11: 561–566. 67. Sqalli-Houssaini Y, Cazalets JR, Clarac F. Oscillatory properties of the central pattern generator for locomotion in neonatal rats. J Neurophysiol. 1993; 70: 803–813. https://doi.org/10.1152/jn.1993.70.2. 803 PMID: 8410173 68. Kubota K, Fujibayashi K, Saito K. Enhancing effect of dimethyl sulfoxide on nociceptive transmission in isolated spinal cord of newborn rat. European Journal of Pharmacology. 1998; 351: 173–179. PMID: 9687000 21 / 21
https://openalex.org/W3015994505	https://www.mdpi.com/2072-6694/12/4/910/pdf?version=1586511064	English	null	Analysis of BRCA1 and RAD51C Promoter Methylation in Italian Families at High-Risk of Breast and Ovarian Cancer	Cancers	2,020	cc-by	5,373	Received: 27 February 2020; Accepted: 4 April 2020; Published: 8 April 2020 Abstract: Previous studies on breast and ovarian carcinoma (BC and OC) revealed constitutional BRCA1 and RAD51C promoter hypermethylation as epigenetic alterations leading to tumor predisposition. Nevertheless, the impact of epimutations at these genes is still debated. One hundred and eight women aﬀected by BC, OC, or both and considered at very high risk of carrying BRCA1 germline mutations were studied. All samples were negative for pathogenic variants or variants of uncertain signiﬁcance at BRCA testing. Quantitative BRCA1 and RAD51C promoter methylation analyses were performed by Epityper mass spectrometry on peripheral blood samples and results were compared with those in controls. All the 108 analyzed cases showed methylation levels at the BRCA1/RAD51C promoter comparable with controls. Mean methylation levels (± stdev) at the BRCA1 promoter were 4.3% (± 1.4%) and 4.4% (± 1.4%) in controls and patients, respectively (p > 0.05; t-test); mean methylation levels (± stdev) at the RAD51C promoter were 4.3% (± 0.9%) and 3.7% (± 0.9%) in controls and patients, respectively (p > 0.05; t-test). Based on these observations; the analysis of constitutional methylation at promoters of these genes does not seem to substantially improve the deﬁnition of cancer risks in patients. These data support the idea that epimutations represent a very rare event in high-risk BC/OC populations. Keywords: germline epigenetic defects; BRCA1; RAD51C; promoter methylation; breast carcinoma; ovarian carcinoma cancers cancers cancers Cancers 2020, 12, 910; doi:10.3390/cancers12040910 www.mdpi.com/journal/cancers Article Silvia Tabano 1,2,† , Jacopo Azzollini 3,† , Chiara Pesenti 4 , Sara Lovati 2, Jole Costanza 5 , Laura Fontana 5 , Bernard Peissel 3, Monica Miozzo 1,5 and Siranoush Manoukian 3,* ia Tabano 1,2,† , Jacopo Azzollini 3,† , Chiara Pesenti 4 , Sara Lovati 2, Jole Costanza 5 , ra Fontana 5 , Bernard Peissel 3, Monica Miozzo 1,5 and Siranoush Manoukian 3,* 1 Medical Genetics, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, 20122 Milano, Italy; silvia.tabano@unimi.it (S.T.); monica.miozzo@unimi.it (M.M.) 1 Medical Genetics, Department of Pathophysiology and Transplantation, Università degli Studi di M 20122 Milano, Italy; silvia.tabano@unimi.it (S.T.); monica.miozzo@unimi.it (M.M.) 1 Medical Genetics, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, 20122 Milano, Italy; silvia.tabano@unimi.it (S.T.); monica.miozzo@unimi.it (M.M.) 2 Laboratory of Medical Genetics, IRCCS Ca’ Granda, Ospedale Maggiore Policlinico Milano, 20122 Milano, Italy; sara lovati@studenti unimi it y 2 Laboratory of Medical Genetics, IRCCS Ca’ Granda, Ospedale Maggiore Policlinico Milano, 20122 M Italy; sara.lovati@studenti.unimi.it 3 Unit of Medical Genetics, Department of Medical Oncology and Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milano, Italy; Jacopo.Azzollini@istitutotumori.mi.it (J.A.); bernard.peissel@istitutotumori.mi.it (B.P.) 3 Unit of Medical Genetics, Department of Medical Oncology and Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milano, Italy; Jacopo.Azzollini@istitutotumori.mi.it (J.A.); bernard.peissel@istitutotumori.mi.it (B.P.) 4 Oncology Department, Istituto di Ricerche Farmacologiche Mario Negri, 20156 Milano, Italy; chiara.pesenti@marionegri.it 4 Oncology Department, Istituto di Ricerche Farmacologiche Mario Negri, 20156 Milano, Italy; chiara.pesenti@marionegri.it 5 Unit of Research Laboratories coordination, IRCCS Ca’ Granda, Ospedale Maggiore Policlinico Milano 20122 Milano, Italy; jole.costanza@policlinico.mi.it (J.C.); laura.fontana@unimi.it (L.F.) it of Research Laboratories coordination, IRCCS Ca Granda, Ospedale Maggiore Policlinico Milano, 122 Milano, Italy; jole.costanza@policlinico.mi.it (J.C.); laura.fontana@unimi.it (L.F.) * Correspondence: siranoush.manoukian@istitutotumori.mi.it † These authors contributed equally to this work. 1. Introduction Epigenetic alterations, i.e., epimutations, are an emerging mechanism that plays a pivotal role in carcinogenesis. Several studies demonstrated that defects in epigenetic markers, such as modiﬁcations of CpG methylation at gene promoters, result in transcriptional silencing of active genes or activation of silent genes, causing eﬀects similar to those of gene mutations. Particularly, DNA methylation at CpG sites located in promoter regions of tumor-suppressor genes is a well-described Cancers 2020, 12, 910; doi:10.3390/cancers12040910 www.mdpi.com/journal/cancers 2 of 8 Cancers 2020, 12, 910 event that could be present: (i) only in tumor cells, i.e., somatic epimutations, (ii) at the germline level, with evidence of inheritance, i.e., linked to cis-acting mutations [1,2] or, ﬁnally, (iii) in multiple tissues of diﬀerent embryonic origin with no evidence of inheritance and often in a mosaic state, i.e., constitutional epimutations. Regarding breast and ovarian cancer (BC and OC), mutations in BRCA1, along with BRCA2 and few other genes, are responsible for only a fraction of familial cases. Moreover, somatic epimutations in these genes are nowadays largely studied to understand not only their involvement in tumorigenesis but also their eﬀects on therapy response, such as at cisplatin and PARPi [3,4]. The study of germline and constitutional epimutations in BC and OC related genes is a relatively novel research ﬁeld. Recently, Evans and colleagues reported the ﬁrst case of germline BRCA1 methylation in two families aﬀected by Hereditary Breast and Ovarian Cancer Syndrome. The BRCA1 promoter resulted hemi-methylation in all cells and it was linked to a cis-acting 5’UTR promoter variant [5]. Constitutional BRCA1 methylation was analyzed by diﬀerent groups with multiple technical approaches and it has been demonstrated to be associated with an increased risk of developing BC and/or OC [6–11]. Indeed, constitutional BRCA1 methylation has been related to early onset of the disease and to basal-like phenotypes in case of BC, especially the triple negative (TN) subtype, and high-grade serous histotypes in the case of OC. Along with BRCA1, other BC/OC-related genes have been investigated. Among these, RAD51C was found constitutively hypermethylated in sporadic BC, as showed by Hansmann and colleagues [10], who identiﬁed hypermethylation at both BRCA1 and RAD51C promoters in 1.4% and 0.5% of high-risk cancer patients, respectively. The authors noted that epimutations were present in 5.5%, (2/37) sporadic early-onset BC patients, 0.9% (4/460) familial BC patients, and in 10% (4/39) of OC patients. 1. Introduction Moreover, epimutations were conﬁned to one of the two parental alleles always at the mosaic level in tissues of diﬀerent embryonic origins. This indicated that they occurred in a single cell relatively early during the embryonic development. This model was further supported by recent studies demonstrating that the majority of constitutional BRCA1 methylation events arose early during development and seemed to remain quite stable during life and potentially were inherited from mother to daughter [12,13]. This novel hypothesis of inheritance is controversial and needs further studies to be completely elucidated [13]. Apart from the etiopathogenesis of this phenomenon, its prevalence is still debated. Depending on the studies, it ranges from less than 1% to 15%. This lack of homogeneity might be due to diﬀerent composition of the patients’ cohorts as well as to the diﬀerent nature of the technical approaches applied to proﬁle methylation, which were not always quantitative. Deﬁning the prevalence of constitutional epimutations in BC and OC might improve screening strategies for identifying women at increased risk who may beneﬁt from tailored preventive options. Our group recently reported constitutional BRCA1 methylation in one out of 154 selected isolated early-onset BC patients, exploiting an accurate quantitative approach and establishing strict analysis parameters [11]. Based on the previous ﬁndings, we tried to clarify the prevalence of constitutional epimutations in BC and OC predisposition by analyzing the methylation status of BRCA1 and RAD51C promoters in 108 women aﬀected by BC and/or OC, with high a priori risk of harboring pathogenetic variants at BRCA1 and who tested negative for BRCA sequence variants. BRCA1 and RAD51 Methylation Levels As shown in Figure 1a, none of the 108 analyzed cases exceeded the ﬁxed minimum hypermethylation threshold deﬁned in controls (threshold values were 13.6% and 12.1% for BRCA1 and RAD51C, respectively). In detail, mean methylation levels (± stdev) of the BRCA1 promoter region were 4.3% (± 1.4%) and 4.4% (± 1.4%) in controls and patients, respectively (p > 0.05, t-test); mean methylation levels (± stdev) of the RAD51C promoter region were 4.3% (± 0.9%) and 3.7% (± 0.9%) in Cancers 2020, 12, 910 3 of 8 controls and patients, respectively (p > 0.05, t-test). These overall data are consistent with the absence, in the investigated population, of germline or constitutive mosaic epigenetic alterations, resulting in BRCA1 or RAD51C promoter hypermethylation. Cancers 2020, 12, x 3 of 8 b i th i ti t d l ti f li tit ti i i ti lt ti We also investigated possible diﬀerences in methylation levels among clinical subgroups, without ﬁnding signiﬁcant diﬀerences. In particular, patients aﬀected with BC-only showed methylation levels at BRCA1 and RAD51C comparable with those aﬀected with OC-only (p > 0.05, t-test). In detail, BRCA1 mean methylation was 4.3% (± 0.93%) and 3.9% (± 0.97%) in patients with BC- or OC-only, respectively. RAD51C mean methylation was 3.7% (± 0.91%) and 3.5% (± 0.56%) in patients with BC- or OC-only, respectively (Figure 1b). No diﬀerences were noted among diﬀerent histological subtypes of both BC and OC, albeit the number of cases in each subgroup might be too small to highlight slight discrepancies. absence, in the investigated population, of germline or constitutive mosaic epigenetic alterations, resulting in BRCA1 or RAD51C promoter hypermethylation. We also investigated possible differences in methylation levels among clinical subgroups, without finding significant differences. In particular, patients affected with BC-only showed methylation levels at BRCA1 and RAD51C comparable with those affected with OC-only (p > 0.05, t- test). In detail, BRCA1 mean methylation was 4.3% (± 0.93%) and 3.9% (± 0.97%) in patients with BC- or OC-only, respectively. RAD51C mean methylation was 3.7% (± 0.91%) and 3.5% (± 0.56%) in patients with BC- or OC-only, respectively (Figure 1b). No differences were noted among different histological subtypes of both BC and OC, albeit the number of cases in each subgroup might be too small to highlight slight discrepancies. Figure 1. Methylation levels at BRCA1 and RAD51C promoters in controls and patients. BRCA1 and RAD51 Methylation Levels (a) Mean methylation levels at BRCA1 and RAD51C promoters measured in 60 controls (left panel) and 108 patients (right panel). In the right panel, the red, green, and blue dashed lines indicate the thresholds used for the identification of hypermethylated cases obtained by adding the prefixed value of 0.15, 0.1, and 0.05, respectively, to the one-sided 95% bootstrap confidence interval of the controls’ mean; (b) mean methylation levels at BRCA1 and RAD51C promoters in samples stratified according to cancer localization (breast cancer (BC) only vs. ovarian cancer (OC) only). 3. Discussion Figure 1. Methylation levels at BRCA1 and RAD51C promoters in controls and patients. (a) Mean methylation levels at BRCA1 and RAD51C promoters measured in 60 controls (left panel) and 108 patients (right panel). In the right panel, the red, green, and blue dashed lines indicate the thresholds used for the identiﬁcation of hypermethylated cases obtained by adding the preﬁxed value of 0.15, 0.1, and 0.05, respectively, to the one-sided 95% bootstrap conﬁdence interval of the controls’ mean; (b) mean methylation levels at BRCA1 and RAD51C promoters in samples stratiﬁed according to cancer localization (breast cancer (BC) only vs. ovarian cancer (OC) only). Figure 1. Methylation levels at BRCA1 and RAD51C promoters in controls and patients. (a) Mean methylation levels at BRCA1 and RAD51C promoters measured in 60 controls (left panel) and 108 patients (right panel). In the right panel, the red, green, and blue dashed lines indicate the thresholds used for the identification of hypermethylated cases obtained by adding the prefixed value of 0.15, 0.1, and 0.05, respectively, to the one-sided 95% bootstrap confidence interval of the controls’ mean; (b) mean methylation levels at BRCA1 and RAD51C promoters in samples stratified according to cancer localization (breast cancer (BC) only vs. ovarian cancer (OC) only). 3. Discussion Figure 1. Methylation levels at BRCA1 and RAD51C promoters in controls and patients. (a) Mean methylation levels at BRCA1 and RAD51C promoters measured in 60 controls (left panel) and 108 patients (right panel). In the right panel, the red, green, and blue dashed lines indicate the thresholds used for the identiﬁcation of hypermethylated cases obtained by adding the preﬁxed value of 0.15, 0.1, and 0.05, respectively, to the one-sided 95% bootstrap conﬁdence interval of the controls’ mean; (b) mean methylation levels at BRCA1 and RAD51C promoters in samples stratiﬁed according to cancer localization (breast cancer (BC) only vs. ovarian cancer (OC) only). Th 3. Discussion RAD51C genes in peripheral blood lymphocytes from women affected by BC and/or OC who tested negative at the BRCA analysis, although they were at high risk to harbor pathogenic variants. The rationale of the study was based on previous findings on the possible influence of germline hypermethylation of cancer predisposing gene promoters [1,2] in a few conditions including breast cancer [14]. The BRCA1 and RAD51C genes were selected as potential candidate genes based on previous studies and on the phenotype of patients included in the study. While BRCA1 is the most recognized predisposing gene for BC and OC, RAD51C was first identified as a putative cancer-predisposing This study evaluated the presence of DNA hypermethylation at promoter regions of BRCA1 and RAD51C genes in peripheral blood lymphocytes from women aﬀected by BC and/or OC who tested negative at the BRCA analysis, although they were at high risk to harbor pathogenic variants. The rationale of the study was based on previous ﬁndings on the possible inﬂuence of germline hypermethylation of cancer predisposing gene promoters [1,2] in a few conditions including breast cancer [14]. p p g g p p p g The BRCA1 and RAD51C genes were selected as potential candidate genes based on previous studies and on the phenotype of patients included in the study. While BRCA1 is the most recognized predisposing gene for BC and OC, RAD51C was ﬁrst identiﬁed as a putative cancer-predisposing gene in BC/OC families in 2010 [15]. Subsequently, it was conﬁrmed to be associated with an increased Cancers 2020, 12, 910 4 of 8 risk of OC and, only recently, of BC, in particular of the TN type [16,17]. Moreover, to date somatic epimutations in both BC [5,10,18,19] and OC [10,19,20] have been identiﬁed only at these two genes. risk of OC and, only recently, of BC, in particular of the TN type [16,17]. Moreover, to date somatic epimutations in both BC [5,10,18,19] and OC [10,19,20] have been identiﬁed only at these two genes. In our study, none of the patients exhibited increased methylation levels at BRCA1 and RAD51C In our study, none of the patients exhibited increased methylation levels at BRCA1 and RAD51C promoters, with respect to the control population. No statistical diﬀerences were detected among speciﬁc subgroups, in particular patients aﬀected with BC only and those aﬀected with OC only. 4. Materials and Methods 4.1. Study Subjects Th 3. Discussion Although this result might be burdened by the limited size of our cohort, our approach, based on highly selective inclusion criteria, should have maximized the chances of identifying potentially causative epimutations. Therefore, we hypothesize that epimutations, either germline or constitutional, represent a very rare event in women with BC and/or OC who tested negative at the BRCA analysis, although they were at high risk to harbor pathogenic variants. This ﬁnding also supports previous reports [21,22], highlighting the absence of germline epimutations at BRCA1 or other BC/OC-predisposing genes in similar cohorts. Moreover, this result is in line with our previous study on isolated early-onset BC patients, which demonstrated the extremely low frequency of constitutional epiphenomena in a diﬀerent subgroup of patients considered to be at increased risk to carry cancer predisposing alterations [11]. In the attempt to clarify the importance of epimutations, instead of genetic variants, in cancer predisposition genes, a number of technical limitations should be taken into account: (i) blood might not be the ideal tissue, since the hypermethylation could be present in a mosaic condition and blood may be not involved in the alteration; (ii) the sensitivity of the methylation detection may not completely exclude the presence of constitutional mosaic epimutations. In our analyses the lower threshold of hypermethylation was relatively high (i.e., +0.5 above the one-sided 95% bootstrap conﬁdence interval) and archival tumor samples were not available for this study, therefore we were able to investigate germline epimutations though we could not completely rule out the presence of mosaic epimutations. Nevertheless, a mosaic hypermethylation would not ﬁt in with the family history of most of our patients, who reported multiple BC/OC aﬀected family members. Additional factors, not assessed in the present study, including age or chemotherapy treatment, might aﬀect methylation levels in blood. Notwithstanding, we previously investigated some of these factors on a cohort of isolated early onset BC patients and found that blood methylation at these loci was stable regardless of age at blood withdrawal or previous chemotherapy [11]. 4.1. Study Subjects A total of 108 patients were selected among individuals who underwent genetic counseling and testing at Fondazione IRCCS Istituto Nazionale dei Tumori di Milano (INT). All patients enrolled in the study were aﬀected by either BC, OC, or both and were considered at very high risk of carrying a BRCA1 pathogenic variants based on personal and/or family history. Patients who fulﬁlled one of the four criteria reported in Table 1 were included in the study. Table 1. Study cohort composition following the reported inclusion criteria. BC, breast cancer; OC, ovarian cancer; TNBC, triple negative breast cancer. Table 1. Study cohort composition following the reported inclusion criteria. BC, breast cancer; OC, ovarian cancer; TNBC, triple negative breast cancer. Table 1. Study cohort composition following the reported inclusion criteria. BC, breast cancer; OC, ovarian cancer; TNBC, triple negative breast cancer. Inclusion Criteria n. Patients (1) BC < 50 years + high mutation probability 73 (2) OC any age + high mutation probability 19 (3) TNBC < 55 years + high mutation probability 10 (4) BC < 50 years/TNBC any age + OC any age 6 All the BC cases were either invasive or ductal carcinomas in situ (DCIS). All the OC included in the analysis were not of the mucinous type or low grade. A “high mutation probability” was deﬁned as ≥30% according to the BRCAPRO model (CaGene software version 5.1; PMID 9443863, http://www4.utsouthwestern.edu/breasthealth/cagene/) or either ≥30% for BRCA1/BRCA2 or ≥60% for Inclusion Criteria n. Patients (1) BC < 50 years + high mutation probability 73 (2) OC any age + high mutation probability 19 (3) TNBC < 55 years + high mutation probability 10 (4) BC < 50 years/TNBC any age + OC any age 6 All the BC cases were either invasive or ductal carcinomas in situ (DCIS). All the OC included in the analysis were not of the mucinous type or low grade. A “high mutation probability” was deﬁned as ≥30% according to the BRCAPRO model (CaGene software version 5.1; PMID 9443863, http://www4.utsouthwestern.edu/breasthealth/cagene/) or either ≥30% for BRCA1/BRCA2 or ≥60% for 5 of 8 Cancers 2020, 12, 910 BRCA1/BRCA2 plus a hypothetical third gene, which would account for all the other BC-associated genes in BRCA-negative families, according to the case-only-study, COS software [23]. All the selected patients were negative for pathogenic variants (including large deletions/duplications) or variants of uncertain signiﬁcance (VUS) in BRCA1 and BRCA2. 4.3. Epityper Mass Spectrometry Methylation analyses were performed according to the procedure implemented by our group in a previous study [11]. MassARRAY® EpiTYPER platform, with MassCleave settings, was used to determine methylation levels of BRCA1 and RAD51C promoter regions. The BRCA1 and RAD51C PCR primers were the same as the previous study and ampliﬁed about 400 bp of the promoters [11]. As previously described [11], two human commercial control DNA samples were used as methylated and non-methylated control samples, Human HCT116 DKO Methylated DNA and Human HCT116 DKO Non-Methylated DNA (Zymo research corporation). 4.2. DNA Extraction and Bisulﬁte Conversion Genomic DNA was puriﬁed from PBLs using the MagCore Super instrument and the MagCore Genomic DNA Whole Blood Kit (Diatech Lab Line, JEsi, Italy, CE-IVD). Bisulﬁte conversion was conducted on 500 ng of DNA using the EZ Direct DNA Methylation Kit (Zymo Research, Irvine, CA, USA), following the manufacturer’s instructions. 4.1. Study Subjects BC, breast cancer; OC, ovarian cancer; SD, standard deviation; N.A., data not available. cancer; SD, standard deviation; N.A., data not available. Ovarian Cancer Features OC-Only Patients BC + OC Patients No. 19 6 OC Age (Years) Mean ± SD 58 ± 12 57 ± 12 Median 61 54.5 Range 32–76 40–78 OC Histotype Serous 12 (63%) 3 (50%) Endometrioid 3 (16%) 3 (50%) Undiﬀerentiated 3 (16%) 0 Clear cell 1 (5%) 0 OC Grade II 5 (26%) 4 (67%) III 14 (74%) 2 (33%) OC Stage 1 1 (5%) 2 (33%) 2 1 (5%) 0 3 5 (26%) 2 (33%) 4 2 (11%) 0 N.A. 10 (53%) 2 (33%) The analysis was also carried out in a control population of 60 women, selected among healthy blood donors at the Immunohematology and Transfusion Medicine Service of our Institution. The age at blood withdrawal in controls ranged from 21 to 59 years (median 41). All participants provided a signed informed consent for the use of their biological samples and data for research purposes. The investigations were conducted in accordance with the Declaration of Helsinki and the study was approved by the Ethics Committee of INT (approval code INT 171/15). 4.2. DNA Extraction and Bisulﬁte Conversion 4.1. Study Subjects All patients underwent the methylation analysis on peripheral blood leukocytes (PBLs). Eighty-nine patients developed BC, with a median age at onset of 34 years (range 24–71 years). Among these women, 11 developed ipsilateral BC (median age 40, range 36–61), 23 contralateral BC (median age 45, range 33–65), and 6 OC (median age 54.5, range 40–78). Among the 19 patients who developed OC, but not BC, the median age at onset was 61 (range 32–76). The age at blood withdrawal ranged from 27 to 78 years (median 46). The main histopathological features of BC and OC cases included in the study are reported in Tables 2 and 3, respectively. Table 2. Main features of breast cancer cases in the study cohort. BC, breast cancer; OC, ovarian cancer; TN, triple negative; ER, estrogen receptor, PgR, progesterone receptor; SD, standard deviation; N.A., data not available; * not in-situ lobular carcinoma. Table 2. Main features of breast cancer cases in the study cohort. BC, breast cancer; OC, ovarian cancer; TN, triple negative; ER, estrogen receptor, PgR, progesterone receptor; SD, standard deviation; N.A., data not available; * not in-situ lobular carcinoma. Breast Cancer Features BC-Only Patients BC+OC Patients No. 83 6 1st BC age (years) Mean ± SD 35 ± 6 46 ± 13 Median 33 43 Range 24–51 36–71 1st BC Invasive Yes 77 (93%) 5 (83%) No (CDIS) 2 (2%) 1 (17%) N.A.* 4 (5%) 0 1st BC Histotype Ductal 66 (79%) 5 (83%) Lobular 4 (5%) 0 Mixed 3 (4%) 1 (17%) Other 7 (8%) 0 N.A. 3 (4%) 0 1st BC Grade I 7 (8%) 0 II 18 (22%) 1 (17%) III 30 (36%) 1 (17%) N.A. 28 (34%) 4 (67%) 1st BC pT Is 1 (1%) 1 (17%) 1 35 (42%) 1 (17%) 2 15 (18%) 3 3 2 (2,5%) 0 4 2 (2,5%) 0 N.A. 28 (34%) 1 (17%) 1st BC ER Pos 39 (47%) 1 (17%) Neg 26 (31%) 1 (17%) N.A. 18 (22%) 4 (67%) 1st BC PgR Pos 38 (46%) 0 Neg 27 (32%) 2 (33%) N.A. 18 (22%) 4 (67%) 1st BC HER2 Pos 9 (11%) 0 Neg 29 (35%) 2 (33%) N.A. 45 (54%) 4 (67%) 1st BC TN Yes 10 (12%) 1 (17%) 6 of 8 Cancers 2020, 12, 910 Table 3. Main features of ovarian cancer cases in the study cohort. References 1. Peltomäki, P. Epigenetic mechanisms in the pathogenesis of Lynch syndrome. Clin. Genet. 2014, 85, 403–412. [CrossRef] [PubMed] 1. Peltomäki, P. Epigenetic mechanisms in the pathogenesis of Lynch syndrome. Clin. Genet. 2014, 85, 403–412. [CrossRef] [PubMed] 2. Castillejo, A.; Hernández-Illán, E.; Rodriguez-Soler, M.; Pérez-Carbonell, L.; Egoavil, C.; Barberá, V.M.; Castillejo, M.I.; Guarinos, C.; Martínez-de-Dueñas, E.; Juan, M.J.; et al. Prevalence of MLH1 constitutional epimutations as a cause of Lynch syndrome in unselected versus selected consecutive series of patients with colorectal cancer. J. Med. Genet. 2015, 52, 498–502. [CrossRef] [PubMed] 2. Castillejo, A.; Hernández-Illán, E.; Rodriguez-Soler, M.; Pérez-Carbonell, L.; Egoavil, C.; Barberá, V.M.; Castillejo, M.I.; Guarinos, C.; Martínez-de-Dueñas, E.; Juan, M.J.; et al. Prevalence of MLH1 constitutional epimutations as a cause of Lynch syndrome in unselected versus selected consecutive series of patients with colorectal cancer. J. Med. Genet. 2015, 52, 498–502. [CrossRef] [PubMed] 3. Veeck, J.; Ropero, S.; Setien, F.; Gonzalez-Suarez, E.; Osorio, A.; Benitez, J.; Herman, J.G.; Esteller, M. BRCA1 CpG island hypermethylation predicts sensitivity to poly(adenosine diphosphate) ribose polymerase inhibitors. J. Clin. Oncol. 2010, 28, e563–e564. [CrossRef] [PubMed] 3. Veeck, J.; Ropero, S.; Setien, F.; Gonzalez-Suarez, E.; Osorio, A.; Benitez, J.; Herman, J.G.; Esteller, M. BRCA1 CpG island hypermethylation predicts sensitivity to poly(adenosine diphosphate) ribose polymerase inhibitors. J. Clin. Oncol. 2010, 28, e563–e564. [CrossRef] [PubMed] 4. Xu, Y.; Diao, L.; Chen, Y.; Liu, Y.; Wang, C.; Ouyang, T.; Li, J.; Wang, T.; Fan, Z.; Fan, T.; et al. Promoter methylation of BRCA1 in triple-negative breast cancer predicts sensitivity to adjuvant chemotherapy. Ann. Oncol. 2013, 24, 1498–1505. [CrossRef] 4. Xu, Y.; Diao, L.; Chen, Y.; Liu, Y.; Wang, C.; Ouyang, T.; Li, J.; Wang, T.; Fan, Z.; Fan, T.; et al. Promoter methylation of BRCA1 in triple-negative breast cancer predicts sensitivity to adjuvant chemotherapy. Ann. Oncol. 2013, 24, 1498–1505. [CrossRef] 5. Evans, D.G.R.; van Veen, E.M.; Byers, H.J.; Wallace, A.J.; Ellingford, J.M.; Beaman, G.; Santoyo-Lopez, J.; Aitman, T.J.; Eccles, D.M.; Lalloo, F.I.; et al. A Dominantly Inherited 5′ UTR Variant Causing Methylation- Associated Silencing of BRCA1 as a Cause of Breast and Ovarian Cancer. Am. J. Hum. Genet. 2018, 103, 213–220. [CrossRef] 6. Matros, E.; Wang, Z.C.; Lodeiro, G.; Miron, A.; Iglehart, J.D.; Richardson, A.L. BRCA1 promoter methylation in sporadic breast tumors: Relationship to gene expression proﬁles. Breast Cancer Res. Treat. 2005, 91, 179–186. [CrossRef] 7. Iwamoto, T.; Yamamoto, N.; Taguchi, T.; Tamaki, Y.; Noguchi, S. 4.4. Statistical Pipeline Methylation data in the patients’ cohort were analyzed and classiﬁed as hypo- or hyper-methylated by comparison with methylation levels obtained in a control population, composed of 60 healthy women, as reported in Azzollini et al., 2019. Brieﬂy, the mean methylation level of the selected CpGs within each promoter (9 CpG sites for BRCA1 and 11 for RAD51C) was calculated. A thousand 7 of 8 Cancers 2020, 12, 910 bootstrap samples were generated based on the distribution of the mean methylation levels in controls and the one-sided 95% percentile bootstrap conﬁdence interval (bCI) of the controls’ mean was derived [11]. Cases were considered hypermethylated when their mean promoter methylation exceeded the one-sided 95% bCI by 0.05. 5. Conclusions Considering all the above, this study conﬁrmed that, in our cohorts, both germline and constitutional epimutations represent a very rare event in BC and OC carcinogenesis. Based on these observations, although additional data are needed to better characterize the prevalence of this phenomenon, the analysis of constitutional methylation at promoters of known cancer-predisposing genes does not seem to signiﬁcantly improve the deﬁnition of cancer risk. Author Contributions: Conceptualization: S.T., J.A., M.M. and S.M.; Methodology: S.T., J.A., C.P., S.L. and M.M.; Validation: S.T., C.P., J.C. and L.F.; Investigation: J.A., C.P., S.L., B.P., L.F. and S.M.; Formal Analysis: S.T., J.A. and J.C.; Resources, M.M. and S.M.; Writing—Original Draft Preparation: S.T., J.A., and C.P.; Writing—Review & Editing: S.L., J.C., L.F., B.P., M.M. and S.M.; Supervision: M.M. and S.M.; Project Administration and Funding Acquisition: M.M. and S.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Initiative “Prove Aperte” (Filarmonica della Scala of Milan in collaboration with UniCredit Foundation) to S.M. and by the Ricerca Corrente 2019, RC2019-06007 (Scientiﬁc Direction, IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milano) to M.M. The APC was funded by the Unit of Research Laboratories Coordination, Scientiﬁc Direction, IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milano. Acknowledgments: We thank Filarmonica della Scala of Milan and UniCredit Foundation for supporting the study and Benedetta Beltrami for her support in clinical data collection. Conﬂicts of Interest: The authors declare the absence of conﬂicts of interest. References BRCA1 promoter methylation in peripheral blood cells is associated with increased risk of breast cancer with BRCA1 promoter methylation. Breast Cancer Res. Treat. 2011, 129, 69–77. [CrossRef] 8. Kontorovich, T.; Cohen, Y.; Nir, U.; Friedman, E. Promoter methylation patterns of ATM, ATR, BRCA1, BRCA2 and p53 as putative cancer risk modiﬁers in Jewish BRCA1/BRCA2 mutation carriers. Breast Cancer Res. Treat. 2009, 116, 195–200. [CrossRef] 8 of 8 Cancers 2020, 12, 910 9. Wong, E.M.; Southey, M.C.; Fox, S.B.; Brown, M.A.; Dowty, J.G.; Jenkins, M.A.; Giles, G.G.; Hopper, J.L.; Dobrovic, A. Constitutional methylation of the BRCA1 promoter is speciﬁcally associated with BRCA1 mutation-associated pathology in early-onset breast cancer. Cancer Prev. Res. (Phila) 2011, 4, 23–33. [CrossRef] 10. Hansmann, T.; Pliushch, G.; Leubner, M.; Kroll, P.; Endt, D.; Gehrig, A.; Preisler-Adams, S.; Wieacker, P.; Haaf, T. Constitutive promoter methylation of BRCA1 and RAD51C in patients with familial ovarian cancer and early-onset sporadic breast cancer. Hum. Mol. Genet. 2012, 21, 4669–4679. [CrossRef] 11. Azzollini, J.; Pesenti, C.; Pizzamiglio, S.; Fontana, L.; Guarino, C.; Peissel, B.; Plebani, M.; Tabano, S.; Sirchia, S.M.; Colapietro, P.; et al. Constitutive BRCA1 Promoter Hypermethylation Can Be a Predisposing Event in Isolated Early-Onset Breast Cancer. Cancers 2019, 11, 58. [CrossRef] [PubMed] 12. Lønning, P.E.; Berge, E.O.; Bjørnslett, M.; Minsaas, L.; Chrisanthar, R.; Høberg-Vetti, H.; Dulary, C.; Busato, F.; Bjørneklett, S.; Eriksen, C.; et al. White Blood Cell BRCA1 Promoter Methylation Status and Ovarian Cancer Risk. Ann. Intern. Med. 2018, 168, 326–334. [CrossRef] [PubMed] 13. Al-Moghrabi, N.; Al-Showimi, M.; Al-Yousef, N.; Al-Shahrani, B.; Karakas, B.; Alghofaili, L.; Almubarak, H.; Madkhali, S.; Al Humaidan, H. Methylation of BRCA1 and MGMT genes in white blood cells are transmitted from mothers to daughters. Clin. Epigenetics. 2018, 10, 99. [CrossRef] g p g 14. Lønning, P.E.; Eikesdal, H.P.; Løes, I.M.; Knappskog, S. Constitutional Mosaic Epimutations—A hidden cause of cancer? Cell Stress 2019, 3, 118–135. [CrossRef] 15. Meindl, A.; Hellebrand, H.; Wiek, C.; Erven, V.; Wappenschmidt, B.; Niederacher, D.; Freund, M.; Lichtner, P.; Hartmann, L.; Schaal, H.; et al. Germline mutations in breast and ovarian cancer pedigrees establish RAD51C as a human cancer susceptibility gene. Nat. Genet. 2010, 42, 410–414. [CrossRef] [PubMed] 16. Song, H.; Dicks, E.; Ramus, S.J.; Tyrer, J.P.; Intermaggio, M.P.; Hayward, J.; Edlund, C.K.; Conti, D.; Harrington, P.; Fraser, L.; et al. Contribution of Germline Mutations in the RAD51B, RAD51C, and RAD51D Genes to Ovarian Cancer in the Population. J. Clin. Oncol. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References 2015, 33, 2901–2907. [CrossRef] 17. Li, N.; McInerny, S.; Zethoven, M.; Cheasley, D.; Lim, B.W.X.; Rowley, S.M.; Devereux, L.; Grewal, N.; Ahmadloo, S.; Byrne, D.; et al. Combined Tumor Sequencing and Case-Control Analyses of RAD51C in Breast Cancer. J. Natl. Cancer Inst. 2019, 111, 1332–1338. [CrossRef] 18. Manié, E.; Popova, T.; Battistella, A.; Tarabeux, J.; Caux-Moncoutier, V.; Golmard, L.; Smith, N.K.; Mueller, C.R.; Mariani, O.; Sigal-Zafrani, B.; et al. Genomic hallmarks of homologous recombination deﬁciency in invasive breast carcinomas. Int. J. Cancer. 2016, 138, 891–900. [CrossRef] 19. Cunningham, J.M.; Cicek, M.S.; Larson, N.B.; Davila, J.; Wang, C.; Larson, M.C.; Song, H.; Dicks, E.M.; Harrington, P.; Wick, M.; et al. Clinical characteristics of ovarian cancer classiﬁed by BRCA1, BRCA2, and RAD51C status. Sci. Rep. 2014, 4, 4026. [CrossRef] 20. Bernards, S.S.; Pennington, K.P.; Harrell, M.I.; Agnew, K.J.; Garcia, R.L.; Norquist, B.M.; Swisher, E.M. Clinical characteristics and outcomes of patients with BRCA1 or RAD51C methylated versus mutated ovarian carcinoma. Gynecol. Oncol. 2018, 148, 281–285. [CrossRef] 21. Rodríguez-Balada, M.; Roig, B.; Melé, M.; Salvat, M.; Martorell, L.; Borràs, J.; Gumà, J. Germline promoter hypermethylation in BRCA1 and BRCA2 genes is not present in hereditary breast cancer patients. Clin. Transl. Oncol. 2018, 20, 1226–1231. [CrossRef] [PubMed] 22. Staaf, J.; Glodzik, D.; Bosch, A.; Vallon-Christersson, J.; Reuterswärd, C.; Häkkinen, J.; Degasperi, A.; Amarante, T.D.; Saal, L.H.; Hegardt, C.; et al. Whole-genome sequencing of triple-negative breast cancers in a population-based clinical study. Nat. Med. 2019, 25, 1526–1533. [CrossRef] [PubMed] 23. Berrino, J.; Berrino, F.; Francisci, S.; Peissel, B.; Azzollini, J.; Pensotti, V.; Radice, P.; Pasanisi, P.; Manoukian, S. Estimate of the penetrance of BRCA mutation and the COS software for the assessment of BRCA mutation probability. Fam. Cancer 2015, 14, 117–128. [CrossRef] [PubMed] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
W4220798372.txt	https://papiro.unizar.es/ojs/index.php/ond/article/download/6153/5370	fr		Les contes des Grimm en France au XXIème siècle : quand le conte devient album	Ondina ondine/Ondina, ondine	2,022	cc-by	6,654	Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Les contes des Grimm en France au XXIe siècle : quand le conte devient album LOS CUENTOS DE LOS GRIMM EN FRANCIA EN EL SIGLO XXI: CUANDO EL CUENTO SE CONVIERTE EN ÁLBUM Christiane CONNAN-PINTADO Université de Bordeaux christiane.connan-pintado@orange.fr Resumen Los cuentos de los Grimm se publicaron primero en colecciones, y luego los más famosos se publicaron en formato de álbum. Este fenómeno se acentuó en la segunda mitad del siglo xx, en los niveles más prestigiosos. Aquí hacemos un análisis de los retos de este enfoque desde principios del siglo xxi, cuando los artistas acompañan los cuentos con nuevas imágenes que consiguen no traicionar el propósito de los textos fuente y, al mismo tiempo, renovar su lectura de forma original. Palabras clave: Ilustración, Grimm, Francia, artista, álbum. LES CONTES DES GRIMM EN FRANCE AU XXIe SIÈCLE : QUAND LE CONTE DEVIENT ALBUM Résumé Les contes des Grimm sont d’abord publiés en recueils, puis les plus célèbres d’entre eux font l’objet d’une publication en singleton sous le format de l’album. Ce phénomène s’accentue dans la deuxième moitié du xxe siècle, à tous les échelons de la légitimité. Nous faisons ici le point sur les enjeux de cette démarche depuis le début du xxie siècle, lorsque des artistes accompagnent les contes de nouvelles images qui parviennent à ne pas trahir le propos des textes sources tout en renouvelant leur lecture de manière originale. ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 29 30 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Christiane Connan-Pintado Mots-clés: Illustration, Grimm, France, artiste, album THE GRIMMS’ TALES IN FRANCE IN THE 21st CENTURY: WHEN THE TALE BECOMES A PICTUREBOOK Abstract The Grimm’s tales were first published in collections, and then the most famous of them were published as single-tale picture-books. This phenomenon increased in the second half of the 20th century, at all levels of legitimacy. Here we take stock of the challenges of this trend since the beginning of the 21st century, when artists illustrated the tales with new pictures that manage not to betray the meaning of the source texts while renewing their reading in an original way. Keywords: Illustration, Grimm, France, artist, picturebook D’abord fixé par le passage de l’oral à l’écrit, le conte se voit soumis à une tout autre sorte de transformation et de figement lorsqu’il entre dans le monde de l’image, et en l’espèce dans le cadre spécifique de l’album, qui détermine un format et un équilibre à construire pour négocier dans sa mise en page le voisinage et l’interaction du texte et de l’image. Cette conversion générique engage fortement l’éditeur et l’artiste dans la création d’une œuvre originale qui ambitionne à la fois de servir, de rafraîchir, voire de renouveler la lecture d’un texte bien connu. Qu’un conte soit prélevé dans un recueil pour être publié en singleton sous le format de l’album dépend de sa célébrité et conditionne fortement sa réception. En général, l’éditeur passe commande à un artiste pour illustrer un conte précis, comme le firent au début des années 1980 Rita Marshall et Étienne Delessert avec la collection « Il était une fois » chez Grasset en retenant 20 titres parmi ceux que Bruno Bettelheim venait de mettre en valeur dans The Uses of Enchantment (Bettelheim, 1976) (Connan-Pintado, 2013). Mais l’impulsion peut venir de l’artiste lui-même, amoureux des contes et désireux de s’approprier leur univers pour le traduire en images : c’est de son propre mouvement que Jean Claverie a illustré la plupart des contes de Perrault et certains contes des Grimm, d’Andersen, de Bechstein… Plus récemment, Benjamin Lacombe confie sa prédilection pour les contes classiques ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 31 Les contes des Grimm en France au XXIe siècle : quand le conte devient album qui soumettent l’artiste à un défi graphique afin de conjuguer, ou de confronter, le respect du texte source et la liberté créative1. Alors que celui qui illustre un recueil ne peut consacrer que peu d’images, parfois une seule, à chaque conte, et doit donc élire une scène propre à le représenter – par exemple Shaun Tan illustrant les contes des Grimm adaptés par Philip Pullman –, celui qui convertit un conte en album dispose en revanche de toute latitude pour en explorer les péripéties et leurs résonances, au gré de son interprétation personnelle. À lui de tenter de se démarquer de la longue liste des illustrations antérieures qui ont donné forme et couleur à l’univers des contes et profondément touché l’imagination des lecteurs. Sans doute lui faut-il faire preuve à la fois de modestie et d’audace pour prendre sa place et apposer sa propre empreinte. En quête des rééditions des Contes des Grimm en France sous le format de l’album, et après avoir fait le point sur l’offre éditoriale dans sa diversité au cours des deux premières décennies du XXIe siècle, nous en retiendrons la frange la mieux légitimée pour détailler les choix graphiques opérés par les artistes avant de porter notre attention sur quelques exemples singuliers. Présence des contes des Grimm sous le format de l’album dans l’édition française Une consultation du catalogue de la BnF à partir des mots « conte » et « Grimm » (448 occurrences) permet, une fois retirés les recueils et les intrus, de donner la mesure de cette production : 337 titres qui englobent les nouvelles éditions et les rééditions en singleton de 2000 à 2019. Les albums qui reprennent un conte des Grimm en singleton sont donc très nombreux, à tous les échelons de la légitimité, incluant la mise en livre des dessins animés. Cette rapide recherche nous permet de classer les titres mis en exergue et de donner la mesure de leur fréquence. 1https://actualitte.com/article/9899/interviews/les-contes-illustres-la-collection-revee-de-benjamin-lacombe 20/12/2019 [consulté le 24/10/2021] ISSNe: 2605-0285 Publié le Recibido: 09/11/2021 Aceptado: 22/12/2021 32 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Christiane Connan-Pintado Nombre de titres Titre du conte et numéro du Kinder-und Hausmärchen « Blanche-Neige » (KHM 53) 61 57 53 26 21 16 13 9 8 7 5 4 « Hansel et Gretel » (KHM 15) « Le Petit Chaperon rouge » (KHM 26) « Les Musiciens de Brême » (KHM 27) « Le Loup et les sept chevreaux » (KHM 5) « La Belle au bois dormant » (KHM 50) « Raiponce » (KHM 12) « Le vaillant petit tailleur » (KHM 20) « Le roi-grenouille » (KHM 1) « Tom Pouce » (KHM 37) « Neige-blanche et Rose-rouge » (KHM 161) ; « Rumpelstilkin » (KHM 55) « Les sept corbeaux » (KHM 25) ; « Dame Hölle » (KHM 24) ; « Le pêcheur et sa femme (KKHM 19) « Hans la chance » (KHM 83) ; « Les lutins et le cordonnier » (KHM 39) ; « Le roitelet » (KHM 171) ; « Jorinde et Joringel » (KHM 69) « Cendrillon » (KHM 21) ; « Les six serviteurs » (KHM 34) ; « L’oiseau d’Ourdi » (KHM 46); « La reine des abeilles » (KHM 62) ; « Les six cygnes » (KHM 49) ; « Les trois cheveux d’or du diable » (KHM 29) « Les trois plumes » (KHM 63) ; « Hans-mon-hérisson » (KHM 108) ; « Les trois fileuses » (KHM 14) ; « Le Roi Bec de grive » (KHM 52) ; « Celui qui ne connaissait pas la peur » (KHM 4) ; « L’oie d’or » (KHM 64) ; « L’oiseau d’or » (KHM 57); « Le Maître voleur » (KHM 192) ; Les douze frères » (KHM 9) ; « La mort marraine » (KHM 44) ; « Le tailleur et ses trois fils » (KHM 36) ; « Le taler des étoiles » (KHM 153) ; « La gardeuse d’oies » (KHM 89) ; « La jeune fille sans mains » (KHM 31) ; « Le conte du genévrier » (KHM 47) ; « L’homme à la peau d’ours » (KHM 101) ; « Le lièvre et le hérisson » (KHM 187) ; « La clé d’or » (KHM 200) 3 2 1 Tableau 1 des contes des Grimm les plus représentés d’après le catalogue général de la BnF On repère 44 titres différents, soit environ un cinquième des Kinder- und Hausmärchen2. La composition de ce corpus restreint confirme que ce sont toujours les mêmes contes qui sont réédités et réillustrés, et ce constat sur l’édition française est sans doute généralisable aux différents pays européens, voire occidentaux3. En tête de liste prend place le trio attendu : 61 titres pour « BlancheNeige », 57 pour « Hansel et Gretel » et 53 pour « Le Petit Chaperon rouge ». Nettement moins représentés viennent ensuite « Les Musiciens de Brême » (26), « Le Loup et les sept chevreaux » (21), « La Belle au bois dormant » (16) et « Raiponce » (13), ce dernier titre ayant bénéficié de l’impulsion donnée par le film de Disney en 2010, alors qu’il était fort rare auparavant dans l’édition française 2 Nous nous appuyons sur la traduction exhaustive du corpus par Natacha Rimasson-Fertin en 2009 : 229 titres incluant les contes supprimés. 3 Et même au-delà de l’Occident : Grimm’s manga de Kei Ishiyama, publié en 2011 à Hambourg (Tokyo, 2007) reprend « Le petit chaperon rouge », « Hansel et Gretel » et « Rapunzel », mais aussi deux titres moins connus : « Les douze chasseurs » et « Les deux frères ». ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Les contes des Grimm en France au XXIe siècle : quand le conte devient album (Connan-Pintado, 2017). Enfin, 18 contes sont présents de 2 à 9 fois, et 18 autres une seule fois. Parmi ces hapax, certains n’avaient jamais fait l’objet d’une publication isolée auparavant, aussi peut-on avancer l’hypothèse que le coup de projecteur donné par les célébrations du bicentenaire des Kinderund Hausmärchen en 2012 a incité les éditeurs à rechercher et à mettre en valeur des textes moins connus en France, seulement présents jusque-là au sein de quelques rares recueils, tels « Hans-monhérisson », « Le conte du genévrier », « Le Lièvre et le hérisson ». Certains éditeurs se sont fait une spécialité de ces éditions illustrées, tels Minédition (qui succède à NordSud en 2004) et le Genévrier (dans ses collections « Ivoire » et « Caldecott »). Parmi les albums illustrés par les artistes les plus fameux, beaucoup sont des importations et des rééditions d’ouvrages publiés au siècle dernier, comme ceux de Paul O. Zélinsky (Hansel et Gretel, Raiponce, Rumpelstiltskin), Gennady Spirin (NeigeBlanche et Rose-Rouge) ou Paul Galdone (Le Petit Chaperon rouge). La création contemporaine trouve à s’exercer dans le secteur de l’album à travers nombre de ses sous-genres, comme l’album sans texte dont le récit se fait purement visuel (Hansel et Gretel, par Rascal). Tous les types de techniques sont utilisés et on note aujourd’hui un retour de la tradition des ombres chinoises et des découpages, une forme qui sied particulièrement à l’universalité et à la stylisation des personnages de contes : Sybil Schenker publie Le Petit Chaperon rouge, Hansel et Gretel et Le Roi-Grenouille ; Emmanuel Fornage Hansel et Gretel. Chaque créateur trouve à exercer ses techniques de prédilection, caractéristiques de son univers d’auteur, comme les papiers déchirés de Sara pour illustrer Blancheneige et Le Roi-Grenouille. Parmi les innombrables propositions iconographiques qui se succèdent pour se colleter aux contes les plus célèbres, sans doute certains de ces albums passeront-ils à la postérité, car ils sont porteurs d’« illustrations qui ont la force de créations s’arrachant au temps, au périssable, au commun » (Van der Linden, 2012, p. 4). On mentionnera ceux de Susanne Janssen et Lorenzo Mattotti qui ont interprété « Hansel et Gretel » en ce début de siècle, à la suite d’Anthony Browne – dont l’album publié en 1981 au Royaume Uni n’est entré en France qu’en 2001 – ou encore le Blanche-Neige de Benjamin Lacombe, toutes créations qui ont franchi les frontières et retenu l’attention de la critique : ainsi, ce dernier titre est étudié par Catherine Tauveron (2013, pp.101-105), le Hansel et Gretel de Susanne Janssen par Anna Castagnioli (2009), les illustrations de Sara et celles de Gilles Rapaport pour Le Conte du genévrier par Catherine D’Humières (2020 et 2017), le « Blancheneige » de Sara par Ghislaine Chagrot (2018). ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 33 34 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Christiane Connan-Pintado Nous proposons de nous attacher plus précisément à trois albums récents et de regarder de plus près leur approche de l’illustration, soit pour mettre en lumière des contes relativement peu connus en France, « Les trois cheveux d’or du diable » et « La clé d’or », soit au contraire pour reprendre l’un des plus célèbres, « Le Petit Chaperon rouge ». Pour leurs créateurs, il ne s’agit pas obligatoirement d’une commande d’éditeur, mais d’un choix d’auteur, qui donne accès à un « texte de lecteur » (Mazauric et al., 2012) singulier, celui d’un artiste qui investit l’œuvre source et l’interprète par ses moyens graphiques, donne forme visuelle à sa lecture et nous invite à épouser son point de vue pour relire le conte. L’illustration pour éclairer des contes restés dans l’ombre Le dernier conte des Kinder- und Hausmärchen, « La clé d’or » (KHM 200) a constamment occupé cette place ultime, au fur et à mesure des éditions qui se sont succédé du vivant des frères Grimm, de 1812 à 1857. Ils ont ainsi marqué leur volonté de placer ce texte en fin de recueil, alors qu’il pourrait paraître peu conclusif, mais qu’il ouvre au contraire à l’infini le territoire imaginaire des contes. Son explicit se signale en effet par une intervention narratoriale, quand il s’interrompt au moment même où s’ouvre le couvercle d’un coffre mystérieux dont le contenu n’est pas révélé au lecteur : « Il donna un tour de clé, et à présent, il nous faut attendre qu’il finisse d’ouvrir la serrure et qu’il soulève le couvercle pour savoir quelles merveilles étaient contenues dans le coffret » (Grimm, 2009, t. 2, p. 473). Pour la première fois en 2006, ce conte souvent oublié dans les recueils français (Connan-Pintado, 2013, pp. 385 et 410) est mis en valeur dans un album de 26 pages, qui dilate le bref texte des Grimm au profit d’une aventure essentiellement visuelle. On le doit à Joseph Vernot, jeune artiste qui se consacre pour l’instant exclusivement à l’illustration des contes et déclare s’inscrire dans cette tradition née au XIXe siècle : Mes illustrations sont marquées par la nostalgie et tentent modestement de faire revivre l’Âge d’Or de l’illustration lié à la révolution de l’imprimerie et qui a vu des artistes tels que Edmund Dulac, Arthur Rackham, Harry Clarke, Kay Nielsen ou Aubrey Beardsley faire du livre d’étrennes destiné aux enfants un objet d’art à part entière. Ma passion pour le mouvement Arts & Crafts, l’Art Nouveau ainsi que les arts décoratifs islamiques, japonais ou russes complètent d’égale façon ce souhait de remettre au goût du jour l’Art de l’Enchantement4 … 4 https://www.crl-franche-comte.fr/auteurs/joseph-vernot-0, [consulté le 20/02/2019]. ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Les contes des Grimm en France au XXIe siècle : quand le conte devient album L’album se signale d’abord par son format très allongé (18.5 X 34 cm) qui met en valeur la clé baroque de la couverture. Illustration 1. Joseph Vernot © Chocolat ! Jeunesse Au sein de l’album, Joseph Vernot emprunte à la tradition allemande de Käthe Reine et Dora Polster la technique des ombres chinoises qui convient si bien aux contes car elle fait de leurs minces personnages de simples silhouettes. Comme l’écrit Noel Daniel, l’éditrice du recueil publié chez Taschen : « Sortes de tests de Rorschach, elles [les silhouettes] nous permettent de projeter sur elles nos peurs, nos espoirs, nos rêves » (Daniel, 2011, p. 17). La palette chromatique de Joseph Vernot ne se borne pas au noir et blanc, mais reprend aussi les couleurs vert et or de la couverture. Chaque double page convoque un ou plusieurs animaux – oiseaux, poissons, mammifères – dessinés avec une précision documentaire et les pages de garde reprennent, comme en motif de tapisserie, à la manière des Arts & Crafts admirés par l’artiste, de petits rouges-gorges posés sur une branche. ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 35 36 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Christiane Connan-Pintado Illustration 2. Joseph Vernot © Chocolat ! Jeunesse Illustration 3. Joseph Vernot © Chocolat ! Jeunesse ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Les contes des Grimm en France au XXIe siècle : quand le conte devient album Dans ces témoins silencieux et attentifs d’une aventure inscrite au sein de la nature, nous pouvons voir aussi un clin d’œil à la naturliteratur célébrée par les frères Grimm. Ici encore la notion de « texte du lecteur » s’avère heuristique, car l’imagination de l’artiste comble les ellipses, construit un décor, introduit des scènes complémentaires, se livre à des « broderies » dignes d’un conteur pour jouer sa partition. Nous avons bien affaire à un iconotexte dans lequel se noue un dialogue entre le texte et l’image : à chacune des phrases correspond une ou deux doubles pages où se plante un décor, s’animent les personnages, se greffent des actions qui développent les potentialités de la situation. Illustration 4. Joseph Vernot © Chocolat ! Jeunesse Par exemple, si la découverte de la clé dans la neige est conforme à ce que dit le conte, elle est suivie d’une aventure imaginée par l’illustrateur : la quête du jeune garçon racontée exclusivement par l’image pendant deux doubles pages le conduit à explorer un gouffre dans lequel il trouve en effet le coffret ; de la même façon, la recherche de la serrure occupe deux autres doubles pages pendant lesquelles il plonge le coffret dans l’eau pour vérifier son étanchéité. ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 37 38 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Christiane Connan-Pintado Illustration 5. Joseph Vernot © Chocolat ! Jeunesse Comme l’écrit Catherine Tauveron, empruntant à Richard Saint-Gelais la notion de « fiction transfuge » (Saint-Gelais, 2011), ces moments « s’inscrivent dans les zones d’indétermination de la narration, explorent l’incomplétude constitutive des récits sources » (Tauveron, 2013, p. 110). Quant au dénouement, si intrigant pour le lecteur, et si gratifiant pour ceux que réjouit l’indécidabilité de la fiction (Connan-Pintado, 2017), il ménage le suspense : le jeune garçon ouvrant le coffret est montré de dos, assis parmi les arbres, et seuls les oiseaux posés sur une branche au-dessus de lui sont en mesure porter un regard sur son contenu. L’album se clôt en quatrième de couverture par un détail qui ne manque pas d’humour : en son centre, une minuscule serrure dorée contraste avec la majestueuse clé ornementée qui était affichée en première de couverture. L’album, comme le conte, comme le recueil, se referment sur la magie et le mystère de leur secret. Avec ses images délicatement poétiques et humoristiques, Joseph Vernot a su contextualiser la mince aventure, l’enrichir d’épisodes inédits, tout en respectant la dimension merveilleuse et le caractère crypté du conte. Présent dans certains des recueils publiés en France depuis le milieu du XIXe siècle (ConnanPintado, 2013, pp. 312 et 390), le KHM 29, « Les trois cheveux d’or du diable » a fait l’objet de quelques publications isolées : un livre d’images illustré par l’affichiste Henri Thériet en 1948, un document multisupport (brochure et cassette audio) sous l’égide de Marlène Jobert en 1995 et une ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Les contes des Grimm en France au XXIe siècle : quand le conte devient album bande dessinée signée Cécile Chicault en 2004. Ce dernier titre figure sur la liste de littérature de référence du ministère de l’éducation nationale (Eduscol, 2013), ce qui légitime le succès de ce conte facétieux dans lequel un jeune garçon « né coiffé » accomplit plusieurs épreuves qui l’affrontent à un roi malveillant et au redoutable personnage éponyme. Il a retenu l’attention de Juliette Binet, une jeune artiste qui s’est fait connaître en ce début de XXIe siècle par ses albums minimalistes, parfois sans texte, publiés chez des éditeurs exigeants (Autrement, Albin Michel, Le Rouergue, Gallimard). On retrouve dans Les trois cheveux d’or du diable les traits délicats et les couleurs pastel qui caractérisent son style graphique à travers la fine typographie du texte de couleur bleue et les images comme détourées qui se détachent sur le même fond pâle. Les représentations du décor et des personnages convoquent nombre de références, plus ou moins marquées, à l’histoire de l’art ou à l’histoire de l’album. Juliette Binet nous a confié5 être à l’initiative de l’illustration de ce conte, avec le projet de représenter le diable comme une sorte de Saint-Jérôme au désert, dans les rochers, et pour le plaisir de le vêtir d’un simple pelarzonium, comme le Christ dans les crucifixions de la peinture flamande6. En première de couverture, la silhouette mince et pâle du diable se détache devant un chaos de rochers sombres. Cette représentation inhabituelle se distingue nettement de celle que réalise Gilles Rapaport dans Les trois poils de la barbe d’or du diable, pour illustrer la version bretonne du conte par Luzel, avec un diable géant conforme aux stéréotypes de l’iconographie : rouge, cornu, avec une queue fourchue. Le choix du cadre rupestre nous rappelle, par un effet d’interlecture, l’album de Grégoire Solotareff, Le diable des rochers, dans lequel un enfant stigmatisé par sa différence se réfugie loin du monde, dans la nature sauvage. C’est un diable bien peu conventionnel que celui de Juliette Binet, sans cornes ni sabots, avec sa crinière blonde qui court le long de son dos nu, et son pélarzonium rouge en partie dénoué qui vole au vent derrière lui7. C’est donc dans ce cache-sexe précaire que se niche de façon bien dérisoire le rouge satanique. En dépit de sa mine renfrognée et de ses extrémités griffues, le personnage est plus risible qu’inquiétant, en accord avec son rôle de dupe dans le conte : en effet, le jeune héros parvient non seulement à échapper à sa colère, mais il profite de sa clairvoyance pour lui extorquer la solution des 5 Nous remercions chaleureusement l’artiste pour les informations qu’elle nous a transmises en réponse à nos questions dans son courrier électronique du 10/09/2019 à 09h48. 6 Ibid., « J’ai aussi souhaité qu’il soit effrayant et ridicule à la fois, l’humour -si l’on peut considérer cela comme de l’humour- se cache peut-être dans les coiffures, et les habits. » 7 Ibid., « Il m’a semblé idéal et machiavélique, que le diable ait emprunté son slip au christ, je le lui ai tout de même fait rouge ». On peut voir sur le site de l’artiste toute une série de dessins de pelarzoniums également soulevés et ondulant sous l’effet d’un courant d’air https://juliettebinet.fr/dessins-perizoniums [consulté le 25/10/2021]. ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 39 40 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Christiane Connan-Pintado problèmes qu’il doit résoudre s’il veut devenir roi. Juliette Binet mêle les époques, comme les tenues des personnages : celle du roi connote le Moyen Orient, les gardiens portent des sortes de toges plissées, en contraste avec les vêtements contemporains du héros auquel le jeune lecteur pourra s’identifier ; les décors stylisés figurent la nature – rochers, forêt, rivière sinueuse dont le dessin rappelle celle de Natalie Parain dans Baba-Yaga – et la ville géométrique agrémentée d’ornements architecturaux de fantaisie. Ce brassage non réaliste d’éléments hétérogènes peut représenter l’universalité du conte qui concerne toutes les aires géoculturelles depuis la nuit des temps. Plusieurs doubles pages sans texte ralentissent le récit et invitent le lecteur à s’interroger sur la quête du personnage et sur les mystères affrontés. Ainsi, Juliette Binet fait entrer le conte des Grimm dans son univers graphique aux couleurs douces, son monde un peu étrange, comme en suspens sur la page blanche, avec ses images elliptiques et troublantes qui forcent l’attention et invitent à mieux regarder pour mieux lire un conte dont le héros est né sous une bonne étoile, comme l’annoncent ces étranges pages de garde roses, semées de formes blanches irrégulières, annonçant celle qui entoure l’enfant « né coiffé », à l’incipit. L’illustration pour renouveler la lecture du « Petit Chaperon rouge » D’origine polonaise, Joanna Consejo travaille en France et a également illustré en 2011 « Les cygnes sauvages » pour les éditions Notari, qui se distinguent souvent par leurs choix audacieux et innovants. Il s’agit cette fois d’une commande et l’album regroupe les versions de Perrault et des Grimm, qui se succèdent dans l’album : la première en texte intégral, moralité comprise ; la seconde dans la traduction de Charles Deulin – toute première traduction française (Ed. Dentu, 1878) – qui complète le conte avec l’épisode dans lequel grand-mère et chaperon se débarrassent du loup entré par la cheminée. Les deux versions du texte se succèdent, dotée chacune d’une page de titre, mais l’illustration enjambe cette frontière sans différence de style ni de tonalité. Joanna Consejo entérine ainsi un phénomène souvent observé dans l’édition pour la jeunesse comme dans la mémoire des lecteurs : l’écheveau des contes8 devient si inextricable que les deux versions tissées ensemble finissent par n’en former qu’une seule. L’originalité de l’illustration ne s’impose pas d’emblée car elle peut paraître désuète, par son dessin au crayon noir marqué de quelques indices colorés en rouge comme la robe de la fillette. La 8 Tel est le titre du chapitre 5 de Fortune des Contes des Grimm en France… ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Les contes des Grimm en France au XXIe siècle : quand le conte devient album palette chromatique dominante reprend la triade noir-blanc-rouge traditionnellement dévolue à ce conte, à l’exception de quelques pages au pastel vert pour peindre certains éléments végétaux. Faute de pouvoir détailler l’analyse de cet album assez épais (60 pages), l’originalité de ses cadrages, de ses effets d’échelle, de son usage de l’ellipse, on soulignera cependant la part considérable accordée au paysage : six doubles pages au début et à la fin, incluant les pages de garde, immergent le regard dans la forêt profonde qu’il faut regarder attentivement pour ne pas manquer un détail signifiant. On découvre ainsi au début de l’album que le point de vue en surplomb sur un vaste paysage est celui du loup dont la tête est montrée de dos, à peine visible, tout en bas de la double page. L’option graphique la plus étonnante consiste à instaurer une relation ludique entre la fillette et le loup, à partir du motif d’un fil rouge qui n’est d’abord qu’un ruban dans la chevelure de la fillette, puis court et ondule tout au long de l’album. Ce parti-pris s’affiche dès la première de couverture, car la relation entre les deux personnages s’effectue sous le signe du jeu, à commencer par un jeu de cache-cache qui se poursuit un peu plus loin : la fillette cache ses yeux, ou se cache sous un buisson. L’image la plus surprenante est celle où sur le fond blanc de la double page, la fillette et le loup se livrent joyeusement à une « course en sac ». Joanna Consejo décline de façon troublante cette dimension ludique, lorsqu’elle lui donne, dans certaines scènes, un caractère sensuel qui ne manque pas de rappeler les gravures de Doré, dans la proximité soulignée entre les deux personnages : l’enfant tient le loup dans ses bras pour cacher ses yeux, le loup tresse les cheveux de la fillette. Dans une interview disponible sur youtube (2017), Joanna Consejo commente sa vision du conte : elle a voulu aller plus loin que la simple notion de conte d’avertissement, en présentant la rencontre du loup et de la fillette comme une rencontre amoureuse, celle d’un couple impossible mais dont les deux partenaires désirent se rapprocher l’un de l’autre. Aussi leur donne-t-elle le temps de jouer ensemble, même si le dénouement tragique est inéluctable. La couleur rouge, qui sert de fil conducteur, se matérialise en fil de laine rouge, d’abord destiné à lier les cheveux de la fillette, puis tendu d’une page à l’autre. Il sert à relier les deux personnages qui le tiennent en main dans la fiction par exemple lorsqu’ils enroulent des pelotes ou des écheveaux de laine (on pense à la quenouille de la fileuse dans le frontispice de Clouzier pour les Histoires ou contes du temps passé de Perrault). On retrouve aussi ce fil à la cheville de la fillette (et l’on se souvient alors des versions populaires, comme la version nivernaise, dans laquelle le loup attache la cheville de la ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 41 42 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Christiane Connan-Pintado fillette qui demande à quitter le lit, sous prétexte d’un besoin pressant). Le fil sert à museler le loup, à coudre son ventre rempli de pierres, et il devient même le fil de sa vie, lorsque le Chaperon s’érige en troisième Parque, avec sa paire de ciseaux, au moment où le chasseur et la grand-mère s’apprêtent à jeter le loup dans le puits. Il s’agit de plus d’un fil métaphorique qui reprend le cours du conte éternellement raconté, et relie les différentes versions du conte juxtaposées ici. Enfin, le fil rouge prend une dimension métanarrative au dénouement quand la fillette assise dans une clairière brode une scène qui met en abyme le conte du Petit Chaperon rouge, celui qui vient de nous être conté dans le jeu du texte et de l’image. Ce travail de facture minutieuse et ludique qui témoigne d’une subtile maîtrise graphique et d’un réjouissant esprit d’enfance, est aussi un travail lettré, qui sait mettre en perspective les versions et les lectures d’un conte qui a marqué l’imagination – ainsi que le monde de l’édition – depuis trois siècles. On peut reprendre pour cet album les mots de Sophie Van der Linden à propos de l’illustration des Cygnes sauvages par Joanna Consejo : pour elle, l’artiste donne « une habile leçon d’illustration » et « son style – apparemment désuet, très estompé dans ses couleurs – s’affirme pourtant avec force dans sa dimension interprétative, auprès du lecteur attentif. […] Rarement un illustrateur avait fait de son style même une telle partition de sens pour le lecteur » (Van der Linden, 2012, p. 17). La transmission de sa vision par le sujet lecteur artiste au lecteur récepteur assure la vitalité d’un conte qui continue à nous interpeller par-delà les siècles et les cultures, à travers les nouvelles images qu’il inspire. Aujourd’hui où les contes des Grimm, comme tous les contes, sont si souvent publiés sous le format de l’album, il faudrait peut-être désigner ces ouvrages du nom d’album-conte, ou de contealbum, tant les deux genres, contenu et contenant, support et matière, image et texte, s’intriquent jusqu’à devenir indissociables. Dans le même temps, les illustrations se succèdent et s’accumulent au fil du temps pour les contes les plus connus, inlassablement revisités par les artistes de l’image qui proposent de nouvelles transpositions iconographiques sensibles à l’air du temps, passibles d’une lecture sociopoétique (Montandon, 2016), ou marquées par un style personnel. Dans la mesure où l’illustration intervient a posteriori pour accompagner le texte du conte, il arrive que son interaction avec l’image soit contestée au point que certains chercheurs interdisent alors à l’album d’accéder au statut d’iconotexte. Telle est la position d’Isabelle Nières-Chevrel pour qui « Les éditions illustrées de contes traditionnels ou de contes lettrés sont des albums au sens éditorial du terme, mais ce ne ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Les contes des Grimm en France au XXIe siècle : quand le conte devient album sont pas des iconotextes » (2012, p.18). Le dialogue entre texte et image apparaît pourtant patent lorsque l’interprétation iconographique « non seulement ne recouvre pas le texte-source mais, dans le meilleur des cas, bien au contraire, le découvre » (Tauveron, 2013, p. 94). Les quelques exemples qui précèdent attestent en effet que l’image se montre capable d’instaurer avec le texte un dialogue propre à renouveler l’approche du conte, pour le maintenir encore et toujours vivant, sous un éclairage inédit, ce qui renvoie au sens étymologique du beau mot d’« illustration ». Bibliographie Albums (dans l’ordre alphabétique des noms d’illustrateurs) Binet, J. (2015). Les trois cheveux d’or du diable, trad. et adapt. de l’éditeur. Paris : coll. Giboulées, Gallimard jeunesse. Browne, A. (2001). Hansel et Gretel. Paris : Kaléidoscope, 2001 [Londres, Walker Books, 1981]. Chicault, C. (2004). Le diable aux trois cheveux d’or. Paris : Delcourt. Consejo, J. (2015). Le Petit Chaperon rouge, trad. Charles Deulin. Genève : coll. L’oiseau sur le rhino, section Les huppes. Fornage, E. (2016). Hansel et Gretel. Paris : Circonflexe. Galdone, P. (2012). Le Petit Chaperon rouge. Paris : Circonflexe. Guittard, B. (2005). Hans-mon-hérisson trad. Armel Guerne, Lectoure, coll. « Le Bestiaire du Capucin », Éd. Le Capucin, 2005. Janssen, S. (2007), trad. S. Janssen et C. Bruel. Hansel et Gretel. Paris : Être. Janssen, S. (2002). Le Petit Chaperon rouge. Paris : Seuil jeunesse [Munich : Carl Hanser Verlag] Jobert, M. (1995). Le diable et ses trois cheveux d’or. Paris : coll. Les plus beaux contes du monde, Atlas. Lacombe, B. (2010) trad. et adapt. S. Kabok. Blanche-Neige de Benjamin. Toulouse : Milan. Lauströer, J. (2016). Le lièvre et le hérisson, trad. Julie Duteil, Paris : Minedition. Grimm, J. et W., trad. Rimasson-Fertin (2009). Contes pour les enfants et la maison, Paris : coll. « Merveilleux », José Corti, n° 40, 2 vol. Mattotti, L. (2009). Hansel et Gretel. trad. Mourlevat J.-C. Paris : Gallimard jeunesse. ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 43 44 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Christiane Connan-Pintado Parain, N. (2010). Baba Yaga. Paris : Memo [Père Castor : 1932] Pullman, P. ill. Tan, S. (2014). Contes de Grimm. Paris : Gallimard. Rapaport (2014), Luzel F.-M.. G. Les trois poils de la barbe d’or du diable. Paris : coll. Ivoire, Le Genévrier. Rapaport, G. (2012). Le conte du genévrier, trad. Armel Guerne, Paris: Le Genévrier, « Ivoire ». Rascal (2015). Hansel et Gretel. Paris : L’École des loisirs. Sara (2014). Blancheneige. Paris : coll. Ivoire, Le Genévrier. ––– (2013). Le Roi-Grenouille. Paris : coll. Ivoire, Le Genévrier. Schenker, S. (2018). Le Roi-Grenouille. Paris : Minedition. ––– (2014). Hansel et Gretel. Paris : Minedition. ––– (2011). Le Roi grenouille. Paris : Minedition. Solotareff, G. (1993). Le diable des rochers. Paris : L’École desloisirs. Spirin, G. (1995). Neige-Blanche et Rose-Rouge. Paris : Gautier-Languereau. Thiriet, H. (1948). Les trois cheveux d’or du diable. Paris : N. Fortin et fils. Vernot, J. (2006). La clé d’or, trad. et adapt. R. Baud. Bresilley : Éditions Chocolat Jeunesse. Zelinsky, P. O. (2016). Hansel et Gretel. Paris : coll. Ivoire, Le Genévrier. ––– (2011). Grigrigredinmenufretin (Rumpelstiltskin). Paris : coll. Ivoire, Le Genévrier. ––– (2011). Raiponce (Rapunzel). Paris : coll. Ivoire, Le Genévrier. Études Bettelheim, B. (1976). Psychanalyse des contes de fées, trad. T. Carlier. Paris : coll. Réponses, Robert Laffont [The Uses of Enchantment]. Castagnoli, A. (2009). Susanne Janssen, Hansel et Gretel, éditions Être, 2007 , dans La Revue des livres pour enfants, n° 246, avril 2009, http://lajoieparleslivres.bnf.fr Chagrot G. (2018). Comment Sara révèle «Blancheneige» . La Revue des Livres pour enfants, n° 301, juin 2018, pp. 172-179. Connan-Pintado, C. (2017). Au fil des mots et des motifs : «Raiponce» en France dans l’édition pour la jeunesse , dans Peyrache-Leborgne D.(dir.), Vies et Métamorphoses des contes de Grimm : traductions, réception, adaptations. Rennes, coll. « Interférences », Presses universitaires de ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Les contes des Grimm en France au XXIe siècle : quand le conte devient album Rennes, pp. 127-143. ––– (2017). «La clé d’or» des frères Grimm : une clé pour écrire et/ou pour lire ? , dans F. Le Goff et M.-J. Fourtanier (dir.), Les formes plurielles des écritures de la réception. Namur : coll. « Diptyque », Presses universitaires de Namur, vol. II, pp. 46-60. ––– (2013). Relectures iconographiques. La collection «Il était une fois» chez Grasset-Monsieur Chat , dans C. Connan-Pintado et C. Tauveron, Fortune des Contes des Grimm en France. Formes et enjeux des rééditions, reformulations, réécritures dans la littérature de jeunesse. ClermontFerrand : coll. « Mythographies et sociétés », Presses Universitaires Blaise Pascal. D’Humières, C. (2020). Déchirure, peinture et relecture. Interprétations chromatiques de trois contes patrimoniaux illustrés par Sara dans Connan-Pintado et G. Béhotéguy (dir.) Littérature de jeunesse au présent 2. Genres graphiques en question(s). Bordeaux : coll. « Études sur le livre de jeunesse », Presses universitaires de Bordeaux, pp. 207-219. ––– (2017). Reconfigurations iconographiques du Conte du Genévrier à travers les visions artistiques d’Alejandra Acosta et de Gilles Rapaport , dans D. Peyrache-Leborgne (dir.) Vies et métamorphoses des contes de Grimm. Traductions, réception, adaptations. Rennes : coll. Interférences, Presses universitaires de Rennes, pp. 169-179. Montandon, A. (2016). Sociopoétique , Sociopoétiques, n° 1, « Mythes, contes et sociopoétique », Maison des Sciences de l’Homme, Université Clermont Auvergne, [en ligne] https://revuesmsh.uca.fr/sociopoetiques/index.php?id=640 Nières-Chevrel, I. (2012). L’album, le mot, la chose , dans I. Cani, N. Chabrol-Gagne, C. D’Humières, L’album le parti-pris des images. Clermont-Ferrand : Presses universitaires Blaise Pascal, p. 15-20. Noel, D. (2011). Introduction , Les Contes des Frères Grimm, trad. N. Rimasson-Fertin. Cologne : Taschen. Saint-Gelais, R. (2011). Fictions transfuges. La transfictionnalité et ses enjeux. Paris : coll. Poétique, éditions du Seuil. Tauveron, C. (2013). Les Contes des Grimm : typologie et enjeux de leurs reformulations dans la littérature de jeunesse , dans C. Connan-Pintado et C. Tauveron, Fortune des Contes des Grimm en France. Formes et enjeux des rééditions, reformulations, réécritures dans la littérature de ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021 45 46 Ondina/Ondine. Revista de Literatura Comparada Infantil y Juvenil. Investigación en Educación 7 (2021): 29 - 46 Christiane Connan-Pintado jeunesse. Clermont-Ferrand : coll. « Mythographies et sociétés », Presses Universitaires Blaise Pascal, pp. 91-183. Van der Linden, S. (2012). Exercices de style , Hors-cadre(s). Observatoire de l’album et des littératures graphiques, « L’illustration des contes », n° 9, oct. 2011-mars 2012, pp. 4-5. Van der Linden, S. (2012). Le livre de conte illustré : le défi de la modernité , Hors-cadre(s). Observatoire de l’album et des littératures graphiques, « L’illustration des contes », n° 9, oct. 2011-mars 2012, pp. 14-17. Sitographie Site de Juliette Binet https://juliettebinet.fr/livres-les-trois-cheveux-d-or-du-diable Site des éditions Chocolat jeunesse https://chocolat-jeunesse.com/livre_jeunesse_la_cle_d-or.php Blog de Joanna Consejo http://joannaconcejo.blogspot.com/ Site de Corse infos http://www.corsenetinfos.corsica Entretien de Joanna Consejo à Bastia https://www.youtube.com/watch?v=vjgSqo7l71Y « Il était une fois Le Petit Chaperon rouge », entretien du 11 avril 2017 lors de l’exposition des originaux de l’album à Bastia lors de la 24ème édition des Rencontres de la Bande Dessinée et de l’Illustration. C. Deulin https://gallica.bnf.fr/ark:/12148/bpt6k5805750s/f183.item traduction du « Petit Chaperon rouge » Eduscolhttps://cache.media.eduscol.education.fr/file/Litterature/80/6/LISTE_DE_ReFeRENCE_ CYCLE_2_2013_238806.pdf Entretien avec Benjamin Lacombe https://actualitte.com/article/9899/interviews/les-contes-illustresla-collection-revee-de-benjamin-lacombe Publié le 20/12/2019 Revue de sociopoétique https://revues-msh.uca.fr/sociopoetiques/index.php?id=640 ISSNe: 2605-0285 Recibido: 09/11/2021 Aceptado: 22/12/2021
https://openalex.org/W4307047926	https://journalofethnicfoods.biomedcentral.com/counter/pdf/10.1186/s42779-022-00156-1	English	null	Vegetarian ethnic foods of South India: review on the influence of traditional knowledge	Journal of ethnic foods	2,022	cc-by	14,593	© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Journal of Ethnic Foods Journal of Ethnic Foods Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 https://doi.org/10.1186/s42779-022-00156-1 Journal of Ethnic Foods Abstract South Indian cultures are diverse and unique amongst Indian traditions. In spite of many changes in Indian traditions over generations, South Indian states seem to have maintained a great extent of similarity with reference to veg- etarian ethnic food habits and the reason behind is not convincingly known. Hindu traditional texts have extensive mention of the ethnic origins of many cultural practices prevailing in India and the present review aims to explore the different vegetarian ethnic foods of South India and also look into the influential role of food related ideologies mentioned in the traditional texts. Ethnographic study data about the prevailing vegetarian foods of the states were generated using multiple tools and presented. It is observed that there is a great extent of similarity amongst the vari- eties of vegetarian foods being prepared in Hindu communities of South India. Our study also highlights the strong influential role of tradition in evolution of vegetarian foods prevailing even today in South India. Keywords: South India, Hinduism, Vegetarian ethnic foods, Philosophical perspective 6,35,748 km2) comprises of a high percentage of forest cover viz., 20.31% in TN, 20.19% in KA, 54.7% in KL, 18.28% in AP and 18.93% in TG; which enriches the food heritage of South India [2]. The states are also major con- tributors of agricultural economy of India and nearly 48% of its population engage in agriculture. Paddy, sorghum, pearl millet, pulses, chilli and ragi are the major crops cultivated in the South India. The South Indian states are also predominant spice-producing Indian states [3] and thus cuisines here are relatively more flavoured and spicy when compared to other Indian cuisines because of the extensive usage of spices such as ginger, paprika, corian- der, cinnamon, tamarind, pepper and cumin seeds [4]. With reference to the dietary practices, reports show that approximately 30% of South Indian people are vegetar- ians [5] and they generally belong to either of the Brah- min, Arya Vysya, Lingayat and Nambudiri communities under Hinduism. Vegetarian ethnic foods of South India: review on the influence of traditional knowledge Srinidhi K. Parthasarathi1, Ananda Vardhan Hebbani2* and Padma Priya Dharmavaram De Correspondence: h.anandavardhan@gmail.com Introduction South India represented by five major Indian states viz., Tamil Nadu (TN), Karnataka (KA), Kerala (KL), Andhra Pradesh (AP) and Telangana (TG); is well known for its rich cultural heritage and food is one of the major con- tributors to its richness. Traditions and food are highly inter-influential throughout India, more so in South India. Based on the historical evidences, it is clear that the Dravidian civilization of South India is much more primitive (flourished approximately 4500 years ago) in comparison to the rest of Indian civilizations [1] and South Indian cuisines seemingly continue to retain many of the ancient Dravidian food culture elements. According to India State of Forest Report (IFSR 2021), South Indian states (total geographical area of © The Author(s) 2022. Open Access This art permits use, sharing, adaptation, distribution original author(s) and the source, provide a l other third party material in this article are in to the material. If material is not included in t regulation or exceeds the permitted use, yo licence, visit http://creativecommons.org/lic Correspondence: h.anandavardhan@gmail.com 2 Department of Biochemistry, Indian Academy Degree College (Autonomous), Bengaluru, India Full list of author information is available at the end of the article South Indian vegetarian cuisines seem to be unaf- fected by other cultural influences and people continue 2 Department of Biochemistry, Indian Academy Degree College (Autonomous), Bengaluru, India Full list of author information is available at the end of the article 2 Department of Biochemistry, Indian Academy Degree College (Autonomous), Bengaluru, India Full list of author information is available at the end of the article Page 2 of 19 Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods to prefer eating foods in the old traditional ways. Rice is the predominant component of a typical South Indian vegetarian meal since it is grown in large quan- tities in all the states of South India and people over generations are evolved with a natural affinity for rice. Moreover, since climate of South India is generally hot and humid throughout the year, it becomes congen- ial for assimilation and digestion of rice based foods. Review of literature Globally, there has been a consistent rise in ethnic food research, because of the growing demand for tradi- tional foods and also because of the increased opinion that ethnic foods are the best sustainable alternatives to provide healthy food to the world’s population in future [7]. Indian ethnic foods are highly diverse and research clearly reveals that they are strongly influ- enced by culture, religion and traditional knowledge systems like Ayurveda [6, 8–11]. It is also known that many of the food ingredients used in Indian diets (viz., rice, salt, sugar, jaggery, mustard, turmeric etc.) are being mentioned in many pre-historic traditional texts like Vedas, Upanishads, Bhagavad Gita and Mahab- harata [12]. There has been a considerable amount of literature available predominantly highlighting the ethnic fermented foods and beverages of various parts of North, East and West India and the key observa- tions are being summarized in Table 1. Similarly South Indian fermented foods and beverages with specific ethnic names are elaborated in Table 2. Additionally non-fermented ethnic food cultures of Sikkim [25] and Chhattisgarh [26] are also being exclusively studied and presented. Thorough overview of existing stud- ies on ethnic foods in India clearly indicates a scope to additionally understand Indian ethnic food culture from a traditional perspective, so that the influential role of traditional knowledge on the food practices could be established. Introduction Additionally, a rich repertoire of ingredients and addi- tives used for rest of the South Indian dishes viz., veg- etables (both raw form or cooked), cereals, pulses, tamarind juice, curds, jaggery etc., makes the food more of a functional neutraceutical which is a unique feature of South Indian vegetarian cuisine. Thorough observation of the similarity that exists between vari- ous vegetarian South Indian dishes across the states (both in terms of the used ingredients or preparations procedures) clearly gives a clue about a sort of bind- ing phenomenon behind their evolution. There is an increased necessity for understanding the ethnic ori- gins of foods through a traditional perspective as it helps in knowing the population better. This ultimately would help in developing more practical healthcare regimens through scientifically relevant dietary modi- fications. Literature clearly suggests that traditional knowledge systems serve as strong repositories for understanding the evolutionary lineage of any age old practice [6] and this review attempts to understand the same especially for the vegetarian ethnic food prac- tices of South India. Telangana and Andhra Pradesh Study design, data sources and collection processi Study design, data sources and collection process To understand food culture of the five South Indian states (Fig. 1), qualitative ethnographic studies were conducted as per standard fieldwork procedures using various methods viz., semi-structured interviews, direct observations and informal conversations [27]. Since the sources of generating authentic information for the pre- sent study is very limited (accounting to the fact that a mere 30% of South Indian population are vegetarians), studies were designed to cover all the prominent loca- tions of all the five states where the ethnic groups of Brahmins, Arya Vysyas, Lingayats and Nambudiris would be concentrated and also living in the same locality from atleast 4–5 generations. Additionally, since most of the South Indian vegetarian foods are associated with one or the other religious activities, where in they are offered Review of literature There is not much of a work done with this objective and the present work is an attempt Table 1 Fermented ethnic food categories of North, East and West India Title Process involved Food category (summarized from all the studies) References Ethnic fermented foods and beverages of India: science history and culture Foods and beverages derived from natural fermentation Fermented • Rice, legumes and cereal foods • Milk foods • Non-soy bean legume foods • Rice-legume mixture foods • Soy-bean foods • Bamboo shoot foods • Vegetable foods • Sundried/smoked fish products • Sundried/smoked meat products • Beverages • Tea/crabs/fruits etc [13] An overview on ethnic fermented food and beverages of India: Interplay of microbes, immunity and nutrition [14] Diversity of traditional and fermented foods of the Seven Sister states of India and their nutritional and nutraceutical potential: a review [15] Folk to functional: an explorative overview of rice-based fermented foods and beverages in India [16] Naturally fermented ethnic soybean foods of India [17] Traditional Indian fermented foods: a rich source of lactic acid bacteria [18] Dietary culture and antiquity of the Himalayan fermented foods and alcoholic fermented beverages [19] Fermented foods and beverages of Mizoram [20] Table 1 Fermented ethnic food categories of North, East and West India Title Process involved Food category (summarized from all the studies) References Fermented foods and beverages of Mizoram Parthasarathi et al. Review of literature Journal of Ethnic Foods (2022) 9:42 Page 3 of 19 Table 2 Fermented ethnic food items prepared in South India Table 2 Fermented ethnic food items prepared in South India Title Regional area Process involved Food items References Ethnic fermented foods and beverages of Tamil Nadu Tamil Nadu Foods and beverages derived from natural fermentation • Fermented rice (pazhayasadham) • Idli • Dosa/Dosai • Uthappam • Appam • Koozh • Dahi/Thayir • Dahi rice/Thayir sadham • Moru/Butter milk • Kallu/Toddy [13, 21, 22] Ethnic fermented foods and beverages of Karnataka Karnataka • Idli • Dosa • Adai Dosa • Sannas • Ambali • Pickle • Sandigie • Dahi • Butter milk • Ginna • Lassi • Neera • Palm Vinegar • Toddy • Cashew apple wine • Jamun wine • Kanji • Wine [13, 23] Ethnic fermented foods and beverages of Telangana and Andhra Pradesh Telangana and Andhra Pradesh • Idli • Dosa • Uttapam • Vada • Ambali • Taravani/kali • Dahi • Butter milk • Toddy/Kallu [13, 24] Table 2 Fermented ethnic food items prepared in South India Title Regional area Process involved Food items References Ethnic fermented foods and beverages of Tamil Nadu Tamil Nadu Foods and beverages derived from natural fermentation • Fermented rice (pazhayasadham) • Idli • Dosa/Dosai • Uthappam • Appam • Koozh • Dahi/Thayir • Dahi rice/Thayir sadham • Moru/Butter milk • Kallu/Toddy [13, 21, 22] Ethnic fermented foods and beverages of Karnataka Karnataka • Idli • Dosa • Adai Dosa • Sannas • Ambali • Pickle • Sandigie • Dahi • Butter milk • Ginna • Lassi • Neera • Palm Vinegar • Toddy • Cashew apple wine • Jamun wine • Kanji • Wine [13, 23] Ethnic fermented foods and beverages of Telangana and Andhra Pradesh Telangana and Andhra Pradesh • Idli • Dosa • Uttapam • Vada • Ambali • Taravani/kali • Dahi • Butter milk • Toddy/Kallu [13, 24] Ethnic fermented foods and beverages of Tamil Nadu Tamil Nadu Ethnic fermented foods and beverages of Karnataka Karnataka Ethnic fermented foods and beverages of Telangana and Andhra Pradesh to understand the same particularly for the vegetarian ethnic foods of South India. Ethnic fermented foods and beverages of Telangana and Andhra Pradesh Materials and Methods South Indian vegetarian traditional preparations (especially served during a typical meal), their names across all the South Indian states, their brief prepara- tion procedures and health benefits are being reviewed and presented. Additionally the food related ideologies (either in the form of sayings, precepts and prescrip- tions) mentioned in some predominant Indian tra- ditional texts with a particular interest on ideologies presented by South Indian philosophers and theologi- ans is also being discussed to establish the envisaged connect between traditions and practices. Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods Page 4 of 19 (2022) 9:42 Fig. 1 Geographical location of South Indian states Fig. 1 Geographical location of South Indian states Fig. procedures (Q2) and foods being offered (prasadam) to god before consumption (Q3). In the case of interviews conducted at temples Q1 was to know the religious tradi- tion being followed in the temple and Q2 was to know the foods being prepared as an offering to God. to god (as prasadam), religious heads and priests at the Hindu temples were also interviewed informally during the study. Empirical evidences were generated by direct observa- tions, which were conducted in-between October 2020 and November 2021. While many semi-structured inter- views and informal conversations were initially done over phone during the above mentioned period (keeping in view the state wise restrictions during COVID 19), physi- cal informal conversations and semi-structured inter- views by site visits were conducted between November 2021 and February 2022. b. Rice based sweets • Tirukannamadai: Tirukannamadai is a dessert prepared by sautéing red rice in ghee and finally mixed with molasses (Fig. 2e). The dish is gener- ally preferred as a side dish for a meal especially during festivals. • Appam: Appam is a sweet generally prepared during festivals or even eaten as an evening snack (Fig. 2f). Rice flour is mixed with jaggery syrup and kneaded to make a batter. Later, the batter is poured as small cakes on a pan and fried using ghee. 1. Rice based foods: Rice is the predominant item in any South Indian platter, which is generally cooked and used as a base for mixing and eating different vari- eties of other dishes ranging in tastes from bland to spicy to hot. Additionally rice based sweets are also prepared especially during festivals. Not just being a staple food, rice is also known to be a low fat diet; rich in carbohydrates, proteins, vitamins and min- erals [29]. Moreover, apart from the general white rice, there exists an extent of flexibility with refer- ence to the other varieties of rice (pigmented rice), with much more medicinal benefits, that are gen- erally used in South Indian dishes [30]. The gen- eral improvisations done with rice and the resultant South Indian vegetarian foods are as follows 1. Rice based foods: Rice is the predominant item in any South Indian platter, which is generally cooked and used as a base for mixing and eating different vari- eties of other dishes ranging in tastes from bland to spicy to hot. Additionally rice based sweets are also prepared especially during festivals. Not just being a staple food, rice is also known to be a low fat diet; rich in carbohydrates, proteins, vitamins and min- erals [29]. Moreover, apart from the general white rice, there exists an extent of flexibility with refer- ence to the other varieties of rice (pigmented rice), with much more medicinal benefits, that are gen- erally used in South Indian dishes [30]. The gen- eral improvisations done with rice and the resultant South Indian vegetarian foods are as follows • Atirasam: Atirasam is a very traditional sweet generally prepared during festivals and composi- tion wise it is a richer version of Appam men- tioned above (Fig. 2g). Results and discussionh The empirical data for the religious traditions (Q1) of the respondents seem to be clearly falling into either of the Brahmin (Vaishnava, Smartha, Niyogi, Badaganadu, Madhwa and Havyak communities) Arya Vysya, Lingay- ath or Nambudiri (Nair) ethnic groups across the five dif- ferent states. Results of the foods with vernacular names across the states and their overall preparation procedures (Q2) is being presented below. b. Rice based sweets For Atirasam, rice flour is mixed with jaggery to prepare dough with uniform consistency, which is rolled into round shaped cakes, sprinkled with sesame seeds and deep fried in ghee. • Sidai: Sidai is prepared both as a sweet or a savory by using fried rice flour, either mixed with jaggery syrup or cumin seeds respectively (Fig. 2h). The mixture is rolled over lightly sau- teed sesame seeds into balls and deep fried in oil/ghee. Data analysis d h Based on the demographic fact that 30% of South Indian population (TN—2.35%, 21.1%—KA, 3.00%—KL, 1.75%—AP and 1.30%—TG) were vegetarian, a sample size of 140 was fixed to generate a data with 95% confi- dence level, 5% margin of error and representing 10% of the total vegetarian population. A total of 142 (42% were male and 58% were female—mean age 58.2 years) responses were obtained through semi-structured inter- views and informal conversations (Participant pro- files presented in Additional file 1: Appendix 1). Direct observational studies that were conducted at multiple instances by all the authors prior to the onsite/online To ensure privacy and comfort for the participants, all the semi-structured and informal conversations were conducted with their voluntary consent and at their leisure times either at homes or temples (when there wouldn’t be much crowd). The questions were basically to know the religious traditions being practiced in their families (Q1), their food preferences and preparation Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods Page 5 of 19 hot (Fig. 2c). It is prepared by cooking rice with green lentils and finally seasoned with generous amounts of cumin seeds, pepper corns, cashew nuts sautéed in ghee.. conversations gave a better comprehensive picture of the actual area to be subsequently concentrated to generate authentic data for this study. The resultant empirical data is being scrutinized and analysed using standard constant comparison method, inductive analysis and triangulation approaches by thorough recheck—revision of informal questions to be asked, result analysis and interpretation [28]. conversations gave a better comprehensive picture of the actual area to be subsequently concentrated to generate authentic data for this study. The resultant empirical data is being scrutinized and analysed using standard constant comparison method, inductive analysis and triangulation approaches by thorough recheck—revision of informal questions to be asked, result analysis and interpretation [28]. • Dadhyodanam: Dadhyodanam is special curd rice prepared on a regular basis and consumed at the end of a sumptuous meal (Fig. 2d). Cooked and slightly cooled rice is mixed with thick curds until a semisolid consistency and then seasoned to prepare the dish. a. Plain rice and variants Also, being a rich source of potassium, iron, serotonin and vitamin A, banana becomes a wholesome food dessert ingredient in South Indian meals [63]. Additionally honey that is used as a topic on the dish is also a well proven to be rich resource of many flavonoids and phenolic acids which confer it with an array of health benefits [64]. ab Tirattupal (TN)/Palkatti (KL)/Kova (KA, TG, AP): Milk is considered a complete food since it is rich in proteins, minerals like calcium and vitamins like A, B1, B2 and B12. While normal boiling of milk ensures its safety for human consumption and also offers many health benefits [65]; prolonged boiling alters the milks organoleptic features to a sweet and thick protein coagulated mass, which is a preferred form of milk for this dish. Moreover, addition of raw cardamom and pistachios makes the dish rich flavoured and healthy too, up to a limited quantity. Both cardamom and pistachios are proven to possess many health benefits. Cardamom is a major cultivated spice in South India and is known for its aroma due to the rich presence of volatile oils such as α-terpinyl acetate and 1,8-cineole. The oils also are proven to relieve conditions such as bronchitis, depression and other infections [66]. Similarly, pistachios having high levels of unsaturated fatty acids, potassium and tocopherols possess antioxidant and anti-inflammatory properties [67] (See figure on next page.) Fig. 2 a Nei sadam (TN)/Tuppada anna (KA)/Nei chorru (KL)/Neyyannam (TG, AP): Apart from the medicinal values rice possess, ghee that is used in Nei sadam also possess many proven antioxidant and protective activities [31]. b Puliyodharai or Puliyogharai (TN, KL)/Puliyogare (KA)/Pulihora (TG, AP): Tamarind juice, the major ingredient of this dish, is proven to possess many health benefits especially aiding gastrointestinal tract for easy food processing [32]. c Pongal (name continues in all five states): Green lentils used in the dish are known sources of many phytochemicals conferring them with many medicinal properties [33]. Similarly cumin seeds and pepper corns are potent neutraceuticals which possess many medicinal values, more importantly antioxidant, antidiabetics and anti-inflammatory properties [34]. d Dadhyodanam (name continues in all five states): Curd, the predominant ingredient of the dish is a known source of calcium and probiotics [35]. a. Plain rice and variants e Tirukannamadai (or) Tirukannamadu (TN), Nei Payasam (KL)/Aravani (KA, AP): The dish possesses many health benefits since red rice is proven to have antioxidant, antidiabetic and antiproliferative activities [36–38]. Similarly molasses is also proven to possess antioxidant, anti obesity, anti microbial and anti cancer potentials [39]. f Appam (TN, KA, TG, AP)/Unni Appam (KL): Apart from the medicinal values rice possesses, jaggery is proven to possess many micronutrients which have medicinal properties such as anticarcinogenicity and antitoxicity [40]. g Atirasam (TN, KL)/Kajjaya (KA)/Ariselu (AP, TG): Apart from the medicinal values of rice and jaggery, sesame seeds topped on the dish are known to be rich in oils with high levels of unsaturated fatty acids and many other micronutrients, which confer many medicinal properties. Sesame oil is known to decrease lipid peroxidation and increase antioxidant status of the body when consumed [41]. h Sidai (or) Seedai (TN)/Cheedai (KL, KA)/Undalu (TG, AP): Medicinal values of all the ingredients used in the dish are mentioned above. i Tirukannamudu (TN)/Pal Payasam (KL)/Paramanna (KA)/Paramannam (TG, AP): The dry fruits used for garnishing the dish brings an additional neutraceutical property to the dish viz., cashew being rich in magnesium and calcium is known to support healthy muscles and gums, while raisins being rich in calcium and boron is known to aid in maintenance of bone, eye and dental health [42]. j Sukhiyan (or) Sugiyan (TN, KL)/Sukkinunde (KA)/Purnalu (TG, AP): Green gram used in the dish is a protein rich resource with high dietary fibre, vitamin and mineral contents; which confer the ingredient with many medicinal values like antioxidant and hypolipidemic activities [43]. k Varuval (TN)/Varavu (KL)/Vadiyam (KA, TG, AP): Raw banana is proven to possess higher amounts of functional ingredients such as dietary fibre and total starch, which impart better nutritive values in comparison to the ripened banana [44]. Similarly, jackfruit, being a rich resource of many vitamins, minerals and carotenoids in particular, is proved to confer protections against many chronic conditions including cancer, hypertension and coronary heart disease [45]. l Vadam and Vatral (TN, KL), Sandige (KA), vadiyam (TG, AP): Sago grains being very rich in carbohydrates, turns translucent and spongy after cooking, because of which it can become a base material like rice to prepare a snack and is preferred to be munched along with other spicy foods. a. Plain rice and variants m Aviyal (TN, KL)/Majjigae Huli (KA)/Majjiiga Pulusu (TG, AP): Grated coconut predominates the taste while consuming Avial and is known to possess many health benefits like cardioprotection [46] and being high in fibre also aids weight loss and digestion health [47, 48]. n Pulippu koottu (TN)/Puliserry (KL)/Gojju (KA, TG, AP): While tamarind concentrates health benefits are already mentioned above, high mineral content of the gourd family vegetables is understood to confer significant prebiotic ability [49]. o Poritta koottu (or) Poricha (TN, KL)/Playa (KA)/Vepudu (TG, AP): Along with the other ingredients for which the medicinal values are mentioned above, green legumes that are predominantly seen in the dish are well proven low-GI foods and are also hypocholesterolaemic [50]. p Puli kariamudu (or) Puli Poriyal (TN, KL), Gojju (KA, TG, AP): The dish is semisolid in its consistency and is protein, fibre rich and thus very healthy. Due to its tangy sour taste because of tamarind concentrate, the dish is generally slurped in little quantities along with other foods. Health benefits of all the ingredients used in the dish is mentioned above. q Kariamudu (or) Kuzhambu (TN)/Podduthol (KL)/Palya (KA)/Koora (TG, AP): The dish is generally dry and is also rich in proteins and fibres. Health benefits of all the ingredients used in the dish is mentioned above. r Paruppu usili (TN), Parippu (KL), Bele playa (KA)/Koora (TG, AP): All the lentils are generally known to be rich resources of many bioactive peptides and thus have potential health benefits such as anticarcinogenic, hypocholesterolaemic and antidiabetic effects [51]. s Kulumbu (or) Kolambu (TN)/Huli (KA)/Sambar (KL, TG, AP): Pigeon peas which make the predominant ingredient of Kulumbu is well proven not just as a protein and carbohydrate rich nutrient, but also highly medicinal due to the rich presence of multiple polyphenols and flavonoids. The phytochemicals of pigeon peas is proven to be anti-oxidant and anti-inflammatory in nature conferring many health benefits such as hepaprotective, hypoglycemic and even cancer prevention properties [52]. Moreover the major components used for seasoning are also highly therapeutic viz., coriander possessing antioxidant, anticonvulsant, antidiabetic and antihelmentic properties [53] and asafoetida which is antidiabetic, hypolipedimic, anti-helminthic, anti-metastatic, anti-diarrhoeal and even neuroprotective [54]. a. Plain rice and variants t Oorugaai (TN)/Uppilittat (KL)/uppinakayi (KA)/Uragaya (TG, AP): Lactic acid bacteria contributing for the pickling process are also known to possess probiotics features and thus confer health benefits such as protection of GI tract from infections, prevention of urogenital infections, increasing digestion capacity, hypocholesterolaemic and suppression of cancer [55]. u Paruppu Avial (or) Paruppu Avial (TN, KL)/Sundal (KA)/Guggillu (TG, AP): The dish is a rich source of calories owing to the fact that the ingredients are proven to possess high nutritional value [56, 57]. v Satramudu (or) rasam (or) chaaru (or) saaru in all five states: Being rich in multiple ingredients the dish is proved to possess antipyretic, hypoglycaemic and antimicrobial properties. Also rasam is proven to be a best cure for anaemia and also reported to be of use in increasing lactation [58]. Spices used in rasam are also reported to possibly regulate immunity by interfering with many inflammatory factors and confer protection against COVID-19 disease [59]. Owing to its prophylactic immunobooster properties, there also had been a reported rise in number of outlets selling readymade rasam or rasam powder during peak times of COVID-19 infections worldwide. w Appalam (TN)/Pappadam (KL)/Happala (KA)/Appadam (TG, AP): Lentils the major ingredient of the dish, are known protein rich source which is low on fat. Theyare proven to reduce the risks of diabetes, obesity and coronary heart disease [60]. x Vadai (TN)/Vada (KA, KL, TG, AP): Black gram, the major component of this dish is an established protein and dietary fibre rich food. Flour of black gram is enriched with multiple phenolic acids conferring anti-diabetic and antioxidant properties [61]. y Tayir vadai (TN, KL)/Masuru vada (KA), Perugu Vada (TG, AP): While the health benefits of black gram used in preparing vadai is already explained above, curds that are used additionally makes the dish a good neutraceutical rich functional food. z Laddu (name continues in all five states): They are rich resources of carbohydrates, proteins, vitamins and unsaturated fatty acids; with many health benefits viz., cardioprotective, antidiabetic and anticancer potentials [62]. aa Thaen palankal (TN, KL)/Rasayana (KA)/ Rasavali (TG, AP): Ripened banana, the major fruit widely preferred for the salad is a rich resource of phenolics, carotenoids, flavonoids and biogenic amines which confer the fruit with multiple health benefits like antioxidant, antitumor, hypoglycaemic and hypocholesterolaemic activities. a. Plain rice and variants • Nei sadam: Nei sadam is the main ingredient of any South Indian meal (Fig. 2a). Rice grains are cleaned and steam cooked to form a flowery white dish with great aroma. The dish is gener- ally topped with ghee and consumed as such or along with other dishes. • Tirukannamudu: Tirukannamudu is a dessert prepared to be served along with meals during festivals or even eaten regularly as an independ- ent snack (Fig. 2i). The dish is prepared by boil- ing rice in milk along with jaggery and ghee. The dish is finally garnished by layering it with ghee fried cashew nuts and raisins. g • Puliyodharai: Puliyodharai is one of the fre- quently consumed dish both during festivals and on regular basis (Fig. 2b). Rice is cooked and mixed with gravy enriched with a thick season- ing of lentils, chillies, groundnuts, curry leaves, asafoetida, thick tamarind juice, jaggery and dried grated coconut.. • Sukhiyan: Sukhiyan is another traditional sweet prepared during festivals (Fig. 2j). Overnight soaked green gram is ground into a semi smooth viscous paste and ground jaggery is mixed with it and the kneaded paste is shaped into balls, dipped in rice batter and deep fried in ghee. g • Pongal: Pongal is the most frequently prepared breakfast and is generally consumed steaming Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods Page 6 of 19 (See figure on next page.) Fig. 2 a Nei sadam (TN)/Tuppada anna (KA)/Nei chorru (KL)/Neyyannam (TG, AP): Apart from the medicinal values rice possess, ghee that is used in Nei sadam also possess many proven antioxidant and protective activities [31]. b Puliyodharai or Puliyogharai (TN, KL)/Puliyogare (KA)/Pulihora (TG, AP): Tamarind juice, the major ingredient of this dish, is proven to possess many health benefits especially aiding gastrointestinal tract for easy food processing [32]. c Pongal (name continues in all five states): Green lentils used in the dish are known sources of many phytochemicals conferring them with many medicinal properties [33]. Similarly cumin seeds and pepper corns are potent neutraceuticals which possess many medicinal values, more importantly antioxidant, antidiabetics and anti-inflammatory properties [34]. d Dadhyodanam (name continues in all five states): Curd, the predominant ingredient of the dish is a known source of calcium and probiotics [35]. a. Plain rice and variants e Tirukannamadai (or) Tirukannamadu (TN), Nei Payasam (KL)/Aravani (KA, AP): The dish possesses many health benefits since red rice is proven to have antioxidant, antidiabetic and antiproliferative activities [36–38]. Similarly molasses is also proven to possess antioxidant, anti obesity, anti microbial and anti cancer potentials [39]. f Appam (TN, KA, TG, AP)/Unni Appam (KL): Apart from the medicinal values rice possesses, jaggery is proven to possess many micronutrients which have medicinal properties such as anticarcinogenicity and antitoxicity [40]. g Atirasam (TN, KL)/Kajjaya (KA)/Ariselu (AP, TG): Apart from the medicinal values of rice and jaggery, sesame seeds topped on the dish are known to be rich in oils with high levels of unsaturated fatty acids and many other micronutrients, which confer many medicinal properties. Sesame oil is known to decrease lipid peroxidation and increase antioxidant status of the body when consumed [41]. h Sidai (or) Seedai (TN)/Cheedai (KL, KA)/Undalu (TG, AP): Medicinal values of all the ingredients used in the dish are mentioned above. i Tirukannamudu (TN)/Pal Payasam (KL)/Paramanna (KA)/Paramannam (TG, AP): The dry fruits used for garnishing the dish brings an additional neutraceutical property to the dish viz., cashew being rich in magnesium and calcium is known to support healthy muscles and gums, while raisins being rich in calcium and boron is known to aid in maintenance of bone, eye and dental health [42]. j Sukhiyan (or) Sugiyan (TN, KL)/Sukkinunde (KA)/Purnalu (TG, AP): Green gram used in the dish is a protein rich resource with high dietary fibre, vitamin and mineral contents; which confer the ingredient with many medicinal values like antioxidant and hypolipidemic activities [43]. k Varuval (TN)/Varavu (KL)/Vadiyam (KA, TG, AP): Raw banana is proven to possess higher amounts of functional ingredients such as dietary fibre and total starch, which impart better nutritive values in comparison to the ripened banana [44]. Similarly, jackfruit, being a rich resource of many vitamins, minerals and carotenoids in particular, is proved to confer protections against many chronic conditions including cancer, hypertension and coronary heart disease [45]. l Vadam and Vatral (TN, KL), Sandige (KA), vadiyam (TG, AP): Sago grains being very rich in carbohydrates, turns translucent and spongy after cooking, because of which it can become a base material like rice to prepare a snack and is preferred to be munched along with other spicy foods. a. Plain rice and variants m Aviyal (TN, KL)/Majjigae Huli (KA)/Majjiiga Pulusu (TG, AP): Grated coconut predominates the taste while consuming Avial and is known to possess many health benefits like cardioprotection [46] and being high in fibre also aids weight loss and digestion health [47, 48]. n Pulippu koottu (TN)/Puliserry (KL)/Gojju (KA, TG, AP): While tamarind concentrates health benefits are already mentioned above, high mineral content of the gourd family vegetables is understood to confer significant prebiotic ability [49]. o Poritta koottu (or) Poricha (TN, KL)/Playa (KA)/Vepudu (TG, AP): Along with the other ingredients for which the medicinal values are mentioned above, green legumes that are predominantly seen in the dish are well proven low-GI foods and are also hypocholesterolaemic [50]. p Puli kariamudu (or) Puli Poriyal (TN, KL), Gojju (KA, TG, AP): The dish is semisolid in its consistency and is protein, fibre rich and thus very healthy. Due to its tangy sour taste because of tamarind concentrate, the dish is generally slurped in little quantities along with other foods. Health benefits of all the ingredients used in the dish is mentioned above. q Kariamudu (or) Kuzhambu (TN)/Podduthol (KL)/Palya (KA)/Koora (TG, AP): The dish is generally dry and is also rich in proteins and fibres. Health benefits of all the ingredients used in the dish is mentioned above. r Paruppu usili (TN), Parippu (KL), Bele playa (KA)/Koora (TG, AP): All the lentils are generally known to be rich resources of many bioactive peptides and thus have potential health benefits such as anticarcinogenic, hypocholesterolaemic and antidiabetic effects [51]. s Kulumbu (or) Kolambu (TN)/Huli (KA)/Sambar (KL, TG, AP): Pigeon peas which make the predominant ingredient of Kulumbu is well proven not just as a protein and carbohydrate rich nutrient, but also highly medicinal due to the rich presence of multiple polyphenols and flavonoids. The phytochemicals of pigeon peas is proven to be anti-oxidant and anti-inflammatory in nature conferring many health benefits such as hepaprotective, hypoglycemic and even cancer prevention properties [52]. Moreover the major components used for seasoning are also highly therapeutic viz., coriander possessing antioxidant, anticonvulsant, antidiabetic and antihelmentic properties [53] and asafoetida which is antidiabetic, hypolipedimic, anti-helminthic, anti-metastatic, anti-diarrhoeal and even neuroprotective [54]. a. Plain rice and variants t Oorugaai (TN)/Uppilittat (KL)/uppinakayi (KA)/Uragaya (TG, AP): Lactic acid bacteria contributing for the pickling process are also known to possess probiotics features and thus confer health benefits such as protection of GI tract from infections, prevention of urogenital infections, increasing digestion capacity, hypocholesterolaemic and suppression of cancer [55]. u Paruppu Avial (or) Paruppu Avial (TN, KL)/Sundal (KA)/Guggillu (TG, AP): The dish is a rich source of calories owing to the fact that the ingredients are proven to possess high nutritional value [56, 57]. v Satramudu (or) rasam (or) chaaru (or) saaru in all five states: Being rich in multiple ingredients the dish is proved to possess antipyretic, hypoglycaemic and antimicrobial properties. Also rasam is proven to be a best cure for anaemia and also reported to be of use in increasing lactation [58]. Spices used in rasam are also reported to possibly regulate immunity by interfering with many inflammatory factors and confer protection against COVID-19 disease [59]. Owing to its prophylactic immunobooster properties, there also had been a reported rise in number of outlets selling readymade rasam or rasam powder during peak times of COVID-19 infections worldwide. w Appalam (TN)/Pappadam (KL)/Happala (KA)/Appadam (TG, AP): Lentils the major ingredient of the dish, are known protein rich source which is low on fat. Theyare proven to reduce the risks of diabetes, obesity and coronary heart disease [60]. x Vadai (TN)/Vada (KA, KL, TG, AP): Black gram, the major component of this dish is an established protein and dietary fibre rich food. Flour of black gram is enriched with multiple phenolic acids conferring anti-diabetic and antioxidant properties [61]. y Tayir vadai (TN, KL)/Masuru vada (KA), Perugu Vada (TG, AP): While the health benefits of black gram used in preparing vadai is already explained above, curds that are used additionally makes the dish a good neutraceutical rich functional food. z Laddu (name continues in all five states): They are rich resources of carbohydrates, proteins, vitamins and unsaturated fatty acids; with many health benefits viz., cardioprotective, antidiabetic and anticancer potentials [62]. aa Thaen palankal (TN, KL)/Rasayana (KA)/ Rasavali (TG, AP): Ripened banana, the major fruit widely preferred for the salad is a rich resource of phenolics, carotenoids, flavonoids and biogenic amines which confer the fruit with multiple health benefits like antioxidant, antitumor, hypoglycaemic and hypocholesterolaemic activities. c. Rice based savouries: a. Vegetable based gravies and soups: a. Plain rice and variants Also, being a rich source of potassium, iron, serotonin and vitamin A, banana becomes a wholesome food dessert ingredient in South Indian meals [63]. Additionally honey that is used as a topic on the dish is also a well proven to be rich resource of many flavonoids and phenolic acids which confer it with an array of health benefits [64]. ab Tirattupal (TN)/Palkatti (KL)/Kova (KA, TG, AP): Milk is considered a complete food since it is rich in proteins, minerals like calcium and vitamins like A, B1, B2 and B12. While normal boiling of milk ensures its safety for human consumption and also offers many health benefits [65]; prolonged boiling alters the milks organoleptic features to a sweet and thick protein coagulated mass, which is a preferred form of milk for this dish. Moreover, addition of raw cardamom and pistachios makes the dish rich flavoured and healthy too, up to a limited quantity. Both cardamom and pistachios are proven to possess many health benefits. Cardamom is a major cultivated spice in South India and is known for its aroma due to the rich presence of volatile oils such as α-terpinyl acetate and 1,8-cineole. The oils also are proven to relieve conditions such as bronchitis, depression and other infections [66]. Similarly, pistachios having high levels of unsaturated fatty acids, potassium and tocopherols possess antioxidant and anti-inflammatory properties [67] Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods Page 7 of 19 (See legend on previous page.) Fig. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods Page 8 of 19 Fig. 2 continued Fig. 2 continued Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods Page 9 of 19 Fig. 2 continued Fig 2 continued Fig. 2 continued Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods Page 10 of 19 Fig. 2 continued Fig. 2 continued Fig. 2 continued Fig. 2 continued Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods Page 11 of 19 Fig. 2 continued Fig. 2 continued Fig. 2 continued 2. Vegetable based foods c. Rice based savouries: 3. Other Cereal and Pulses based foods: c. Cereal and Pulses based savoury: c. Cereal and Pulses based savoury: • Appalam: Appalam is also a similarly used snack as mentioned above (Fig. 2w). Dough of lentil flour is rolled into thin wafers and dried. Just before consumption the wafers are deep fried in oil. • Kulumbu: Kulumbu is an authenticated semi solid dish consumed with rice (Fig. 2s). Yellow split pigeon peas is cooked like a thick sauce and mixed with boiled/fried vegetables and further boiled with a powder (fried and ground lentils, red chillies, coriander seeds and dried coconut). The content is finally seasoned along with a sprinkle of asafoetida. The dish is a compulsory ingredient of a typical South Indian vegetarian meal and is generally preferred to be consumed hot. • Vadai: Vadai is a widely and regularly consumed breakfast/lunch dish, generally eaten along with Idali—Dish 8 (Fig. 2x). Pre-soaked ground black gram is made into plain batter or with grated chillies and coconut and deep fried like a dough- nut. • Tayir vadai: Tayir Vadai is also a widely con- sumed breakfast/lunch item and it is a Vadai (Fig. 2y), post soaked in curds, garnished with curry leaves and coriander. b. Vegetable based fermented pickles: a. Cereal and Pulses based starters: a. Cereal and Pulses based starters: a. Cereal and Pulses based starters: • Paruppu Avial: Paruppu Avial is a dish prepared with pre-soaked pulses boiled with salt and seasoned (Fig. 2u). Two varieties of pulses can be used for the preparation i.e., green gram or chick peas. Generally the dish is preferred either as a snack or a side dish during a meal, especially during festivals. • Poritta koottu: Poritta koottu is a relatively dry version of a side dish as mentioned above, gen- erally consumed along with rice (Fig. 2o). Here, green legumes or gourd vegetables are mixed with lentils and grated coconut and the mixture is fried or sauteed to make the dish. • Puli kariamudu: Puli kariamudu is also a regu- larly consumed side dish during a meal (Fig. 2p). Vegetables like okra, raw brinjal or raw banana is mixed with spices, cooked with tamarind con- centrate and seasoned to prepare the dish. b. Cereal and Pulses based appetizer: • Satramudu: Satramudu is considered a healthy appetising dish consumed along with rice or directly slurped like any typical soup (Fig. 2v). Thin and delicate supernatant on the cooked yellow split pigeon peas, is collected and boiled with spicy powdered mix of cumin seeds, fenu- greek seeds and pepper corn. The mixture is finally seasoned along with a sprinkle of asafoet- ida. Rasam is considered as the most popular South Indian traditional foods and is considered very ideal recipe following the principles laid by Ayurveda, the Indian system of medicine. • Kariamudu: Kariamudu is also a very frequently prepared side dish, generally consumed along with rice (Fig. 2q). Here, plain steamed vegeta- bles are mixed with spices, fresh coconut and seasoned. • Paruppu usili: Paaruppu usili is a very soft side dish prepared using lentils which is cooked and made into a paste and then mixed with cooked vegetable and seasoned (Fig. 2r). The dish is pre- ferred to be consumed along with rice during a meal. Out of different varieties of lentils, tradi- tional South Indian food recipes generally use green gram, black gram and horse gram to pre- pare Paruppu usili. a. Vegetable based gravies and soups: • Varuval: Varuval is a dried side dish eaten along with rice mixed with spicy items (Fig. 2k). They are generally prepared and stored because of a reasonably good shelf life. Raw banana or yams or Jack fruit, are cut into fine slices, sun dried and then deep fried in oil and finally garnished with salt and dry chilli powder. • Aviyal: Avial is a traditional south Indian dish prepared for all types of special festive occa- sions, generally eaten along with rice (Fig. 2m). The dish is prepared by using boiled vegetables mixed with grated coconut and thick curd mak- ing it a specially flavoured dish. • Vadam and Vatral: Vadam and Vatral are also similarly used snacks as mentioned above (Fig. 2l). Rice flour or sago grains are cooked to a thick, paste like consistency and poured into small circles and dried under direct sunlight. Just before consumption the dried wafers are deep fried in oil. l • Pulippu koottu: Pulippu kutu is a regularly pre- pared side dish generally preferred to be con- sumed along with rice (Fig. 2n). For the dish, gourd vegetables (bitter gourd, snake gourd, bottle gourd or ridge gourd) is cooked along with soaked tamarind concentrate and finally Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods Page 12 of 19 seasoned with lentils and red chillies to make a gravy. seasoned with lentils and red chillies to make a gravy. Foods according to Indian Hindu traditional textsh Foods according to Indian Hindu traditional textsh The ancient Indian Hindu wisdom gives insightful refer- ences about different categories of food and their effects on human beings. Hindu philosophical knowledge, with its roots in the Vedas, is being perceived and branched out as six schools of philosophy viz. Sankhya, Yoga, Nyaya, Vaisheshika, Mimamsa and Vedanta. As per these six schools of philosophy, all knowable things are divided into two kinds ie. the means of knowledge (Pramana) and the object of knowledge (Prameya). The objects of knowledge are classified into substance (Dravya) and non-substance (Adravya). Dravya- the substance is further categorized as material (Jada) and immaterial (Ajada). Jada– the material component is formed by Prakruthi along with time, whereas the Purusha (indi- vidual soul) forms the immaterial component [68, 69]. The Sankhya system propounded by Kapila delineates that the creation is made of two interdependent reali- ties, the Prakruthi and the Purusha. Prakruthi or the material component has been created with three innate dispositions viz., Sattva (Goodness), Rajas (passion or activity) and Tamas (darkness or inertia); which have their effect on all the materials or substances including food and mind [70, 71]. Foods can thus be classified into three categories viz. Sattvik, Rajasic and Tamasic foods and this categorization method also has a deep-rooted reference in the universally accepted song of the lord “Bhagavad Gita” verse 17.7 [72–74]. Literature suggests that Sattvik foods like vegetable, fruits, nuts and whole grains are non-irritating to the stomach and induce calm- ness and nobility to the person consuming by increasing energy of mind; Rajasic foods like meat, eggs, fish, spices, • Thaen palankal: Thaen palankal is fruit salad topped with honey or sugar (Fig. 2aa). The dish is frequently consumed along with a meal or as a sup- plement during rituals, when people do fasting. 5. Milk/milk product based foods • Tirattupal: Tiruttupal is concentrated milk sweet consumed occasionally (Fig. 2ab). Cream milk is boiled for a prolonged period of time to reduce into a thick consistent gel, with a midway addition of sugar. The gel is poured into the required shape while hot and seasoned with a sprinkle of carda- mom and slices of pistachios. Responses also clearly indicate that the above men- tioned foods had been in existence in the respondent families from the past four to five generations. d. Cereal and Pulses based sweets: d. Cereal and Pulses based sweets: • Oorugaai: Oorugaai are premade fermented pickles consumed as a side dish in very min- ute amounts, along with any rice mixed foods (Fig. 2t). Tingling nature of the dish is due to the pickling process that happens on the ingredients over time. Freshly cut ginger, mango or slices of lime are mixed with gingelly oil, turmeric, dry red chilli powder, fenugreek powder, mustard powder and salt. The mixture is stored in air- tight porcelain jars for atleast 1–2 weeks and then consumed. • Laddu: Laddu is a ball shaped traditional sweet prepared during auspicious Hindu occasions (Fig. 2z). Gram flour is mixed with water to a semi liquid consistency and poured over a porous filter to form small sized droplets into boiling oil and deep fried. Handful size of fried and dried droplets are bonded together with thick sugar syrup and rolled into small balls. Garnishing of the balls with cloves, raisins and cashew nuts, gives an additional flavour and taste. Chickpea, the primary source to prepare gram flour is a vastly grown and consumed crop in Asia. 3. Other Cereal and Pulses based foods: Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods Page 13 of 19 4. Fruit based foodsh 4. Fruit based foodsh Foods according to Indian Hindu traditional textsh Responses to Q3 clearly gives a hint that atleast 80% of the foods appear to be offered to god as prasadam (Data presented in Additional file 1: Appendix 1). Moreover, responses from the semi-structured interviews conducted at tem- ples confirm that many foods prepared at temples are mostly the same across all the five different states, with only a vernacular difference of names. Results of the present study thus clearly gives a hint about the strong association between food practices and religious tradi- tions across all the five South Indian states, which is fur- ther established by looking into the phenomenon of food according to Indian Hindu traditional knowledge system. Since origination of any food practise though predomi- nantly are influenced by climatic conditions and avail- ability of culinary ingredients, role of traditional beliefs is undeniable. Food-sheath Vital-sheath Mental-sheath Intellectual-sheath Bliss-sheath SOUL Physical Body Subtle Body Casual Body GUT-BRAIN AXIS Fig. 3 Layers around the soul and its relation with phenomenon of Gut-Brain Axis GUT-BRAIN AXIS Fig. 3 Layers around the soul and its relation with phenomenon of Gut-Brain Axis Fig. 3 Layers around the soul and its relation with phenomenon of Gut-Brain Axis Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods Page 14 of 19 Page 14 of 19 onions, garlic, hot peppers and pickles confer emotional, passionate, restless qualities like anger, delusion, fanta- sies and egotism; and Tamasic foods like leftovers, stale, overripe, spoiled foods are considered to produce nega- tive emotions like pessimism, laziness and doubt [75]. As per Chandogya Upanishad 6.5.4, while quality of food which is being consumed is known to influence the mind, people with the innate dispositions of Sattva, Rajas and Tamas, seems to prefer the foods which are associated with their respective innate dispositions [76]. Gita 17.10. Further it’s also stated in Bhagavad Gita that consumption of food with the quality of goodness helps in cleansing the excess impurities of mind and thus make it become pure; whereas consumption of foods with the quality of passion arises an increased grief and consump- tion of foods with the quality of ignorance arises the lev- els of ignorance and inertia—Bhagavad Gita 14.16 [77]. Foods according to Indian Hindu traditional textsh Divyaprabandham, the ancient Tamil text of sacred verses in praise of the Lord, states that the disposition of food is also changed by the disposition of the per- son who is either cooking it or is in contact with it [79]. The famous experiment carried out by Masuru Emoto on water and cooked rice, scientifically showed the same phenomenon, wherein the effect of an individual’s intention on water was tested through a double-blind test where a group of 2000 people focused positive intentions on water samples located inside an elec- tromagnetic shielded room and similar water samples were set aside for controls. Observations adjudged by hundred neutral observers, clearly indicated that the ice crystals formed from the water exposed to posi- tive intentions got higher scores for aesthetic appeal in comparison to the control water samples [80]. The Anna Sukta, literally meaning hymn to food, con- tained in the Taittiriya Brahmana and also in the Rig- veda samhita states that the person who shares food obtains the same from the nature and person who does not share the food is devoured by the same food he partakes. It is also mentioned in Anna Sukta that the food forsakes a person who eats without giving and will always remain with a person who shares the food. Moreover, it is believed that the food given to the celes- tials, demigods and other human beings is set apart for the giver in this world and beyond [81]. Dietary regi- men mentioned in ancient Indic texts considers food not just as physical nourishment but also as a means to realize the true function and purpose of life. As per Chandogya Upanisad (7–26-2) eating of pure form of food is important to lead a healthy life for which mod- eration in eating habits has been mentioned in Yoga- tattva Upanisad. While moderate eating is defined as Yama (control) and refraining from killing to eat is defined as Niyama (the rule). According to the Bhaga- vad Gita, over-eating is mentioned as a disqualifying habit for practicing yoga, which further transforms into an old saying of “one meal a day – man is a yogi (the balanced man), two meals a day – man is a bhogi (enjoyer) and three meals a day – man is a rogi (the sick man)” [82]. Foods according to Indian Hindu traditional textsh S i V d t D ik ’ Ah Ni i l b t Hindu philosophy clearly states that individual soul (jiva) is enclosed within five-sheaths termed the Kosas viz., food-sheath (annamaya kosa), vital-sheath (pran- amaya kosa), mental-sheath (manomaya kosa), intel- lectual-sheath (vijnanamaya kosa) and the bliss-sheath (anandamaya kosa) [77]. This clearly appears to have a correlation with the modern phenomenon of gut-brain axis, wherein the food-sheath can physiologically be considered as the digestive system and the other sheaths would constitute the different neuroendocrinal compo- nents ultimately culminating at brain. The phenomenon is elaborated for the ease of understanding in Fig. 3. According to Paramahamsa Yogananda, the famous Indian theologian and yoga guru; diet affects the con- sumers psychological disposition, as it has a bearing on his/her state of mind both favourably and unfavourably. It is also being said that the food one consumes has a relationship with the mind and thus eating the right kind of food is a must for maintaining a healthy body and brain [78]. Since the dispositions of mind are believed to have got by their past experiences which cause impres- sions called vasanas, a person with innate disposition (vasana) of Sattva prefers to have saatvic food, a person with Rajas innate disposition prefers rajasic food and a person with a innate disposition of Tamas would pre- fer Tamasic food—Bhagavad Gita 17.7 [77]. Bhagavad Gita states that foods which aid longevity, knowledge, strength, health, happiness and love are preferred by peo- ple who are with a disposition of Sattva (goodness); and such foods would be full of juicy essences, made of fresh ghee or oil; thus highly nourishing to the mind and natu- rally tasteful—Bhagavad Gita 17.8. Foods that are very bitter, very sour, very salty, very hot, that cause burning sensation to the body, that dry up the body, that is burn- ing hot; are the food items generally liked by those who are with disposition of Rajas (passion); and such foods cause mental stress, bodily suffering, pain and diseases— Bhagavad Gita 17.9. Foods according to Indian Hindu traditional textsh Foods that are cooked and kept for a long time, which have lost their natural taste, which have started to smell bad, which have become old, which is left over after consumption; are the foods liked by those with the disposition of Tamas (ignorance or inertia); and such foods also cause serious illnesses to the body—Bhagavad Sri Vedanta Desika’s Ahara Niyama is an elaborate discussion about food, majorly about its hygiene, and also gives a list of foods (fruits, vegetables, dried food) and waters that are forbidden from consumption ex. Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Page 15 of 19 Parthasarathi et al. Journal of Ethnic Foods Page 15 of 19 foods that over-stimulate (such as garlic, onions, rad- ish, drumsticks, varieties of guards, greens, and mush- rooms) cause an imbalance between the mind and body are forbidden from consumption. Ahara Niyama also gives a strict dietary code also including a list of constraints in the way food is being cooked and the machinery used for cooking. The texts of Ahara Niy- ama also have mentioned on “transformed foods” like Appam (puffed rice balls) and Murukku (a fried len- til pretzel): which are to be consumed in a short time after cooking preferably suitable during the pilgrimage. It is known that sweets like Appam/Attirasam, made out of jaggery, ground-nut, and ghee, are considered as wholesome and sattvic indigenous and ethnic South Indian foods [79]. The Materia Medica (Padarthaguna Chintamani) of Tamil Siddhars, compiled between the ninth and fourteenth centuries gives an extensive list of food-related terminologies, more specifically about the culinary practices like effects of cooked rice in combi- nation with ghee, milk, curds, buttermilk, or tamarind. It also confirms garlic as a cure for skin diseases and phlegmatic conditions [83]. of partaking food and are generally intended to be fol- lowed while procuring, processing, preparing and con- suming foods. The text primarily lists many forbidden foods and unhygienic culinary practices, with an inten- tion to help people lead a life with spiritual discipline [79]. Foods according to Indian Hindu traditional textsh Sri Vedanta Desika is recorded to have been born in South India (Tamil Nadu), and also because of the fact that there is a lot of traditional similarity (especially amongst the Vegetarian Hindu community) across all the South Indian states, the present study is being designed to look at the ethnic foods of the geographical locations as shown in Fig. 1. Since ethnic foods are unique because of their deep rooted history basically constituted of tra- ditional knowledge systems of South India, it is recom- mended that understanding the cultural roots would help in understanding food practices better, which would fur- ther help in understanding the population better. Ethnographic studies done on the South Indian veg- etarian foods in the present work, clearly indicates that the foods are perfect blend of all the tastes i.e., sweet, sour, salty, bitter and savoury. Moreover since the num- ber of ingredients used to prepare a South Indian ethnic food is very high, food preparation process invariably needs to be a well calculated practice. All the aforemen- tioned foods are also mentioned in Sri Vedanta Desika’s Ahara Niyama and other Indic traditional texts which explain about its age old lineage. Most of the foods are consumed even today across all the five states of South India, with some slight modifications of cooking method- ologies. Though there appears an evident linguistic dif- ference with reference to the names of ethnic foods, we found that there are negligible differences with reference to its culinary practices. Presently, few of the aforemen- tioned food items are even being commercialized and sold in food outlets under the name of traditional authen- tic South Indian foods [86]. Though recipes for almost all the items are being there in wide publicity from many years, due to a gradual change in the mind set of the younger generations and also changing lifestyle demands, there is an increased preference towards readymade junk foods leading to an increased incidences of health upsets especially in the youth. Thus there is necessity for educat- ing society about cultural roots and importance of eth- nic foods. Moreover with the passing generations there is also a danger of losing the right culinary knowledge and present article is also an informative document in that direction. Similar such evidences are also being explained and drawn by earlier researches on different dimen- sions of traditional foods [87]. Foods according to Indian Hindu traditional textsh Earlier studies state that South Asian countries such as Bangladesh, India, Nepal, Pakistan and Sri Lanka possess a wide variety of ethnic dishes that are nutrient dense [88]. When analyzed and compared with Indian vegetarian ethnic food cultures, Influence of Hindu traditional texts on South Indian ethnic foods Based on the aforementioned explanations of the promi- nent Hindu traditional texts, it is very clear that food should be a means to primarily lead a healthy and holis- tic life. Also, according to the ancient Indic texts, food is a sacred entity and thus termed as “Annam Parabrahma Swaroopam” which translates to “food is the form of the greatest creator and symbolizes divine universal con- sciousness” and also “Annam Bhootanama Jyeshtam tas- mat annam sarvaushadam ucyate” which translates to “food verily is the eldest born of beings and therefore it is the healing herb for all’ [84, 85]. Needless to say, food is thus given a very sacred position in ancient Hindu cul- ture and its consumption had a hidden meaning, which later transformed into traditions. Since ethnic foods pri- marily consider the geographical availability of resources as a primary requirement and food preparations are sup- posed to be economically viable, invariably there would have been a traditional knowledge system behind their evolution. Traditional knowledge systems thus would have acted as guidelines and influenced the evolution of ethnic foods. In the present work, we reviewed the classical Indic traditional text “Ahara Niyama” written by an ancient Indian theologian, poet and exponent of vaisnavism tradition, Sri Vedanta Desika (1268-1369), who pro- posed various food precepts, which in accordance to our knowledge are thought provoking and highly contem- porary. Food precepts are in general, food related rules Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Page 16 of 19 Page 16 of 19 Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods Srilanka also appears to possess rice, curry, cereal, grain and vegetable (fermented) based diets in the vegetarian foods. Additionally, traditional sweets made from rice, wheat and other cereals appear to be a similar feature to that of South Indian ethnic food tradition [89, 90]. Simi- larly, Pakistan is also observed to share common ethnic and traditional food practices with India and Afghanistan [88]. Fermented cereal based, dairy based, fruit/vegeta- ble based foods are being reported as traditional ethnic foods of Pakistan [91]. Bangladeshis vegetarian ethnic foods also seem to be predominated by rice, wheat, green leafy vegetables, sweets, fruits, fermented beverages and pickles [92, 93]. Conclusion While food is certainly a person’s choice, it is known to be influenced by many factors viz., biological (hunger, appetite and taste), economical (cost, income and avail- ability), physical (access, cooking skill and time), social (culture, tradition and meal pattern) and psychological (mood, stress and guilt) [103]. However, ethnic origin of a population seems to be a very strong influencing fac- tor while looking at the phenomenon of food choices and there has been a steady rise in the research on food con- sumption traditions worldwide. Since culture is known to be a pivotal influential factor for lives, food consumption worldwide is tightly associated with cultures and tradi- tions. Understanding and researching on ethnic foods is thus an important field of confluence and also need of hour. In further enhancing our understanding on ethnic foods and making them into a globally acceptable phe- nomenon, understanding their origin and the associated cultural backgrounds is highly warranted. Further, the developed knowledge has to be integrated with the sci- entific ideologies to make it more viable and acceptable. Though it is a general opinion that traditional practices are orthodox, not all of them can be rejected. In real- ity, traditional practices have been proven to be the best alternatives since they have formidable scientific rel- evance [104]. Moreover, in-depth understanding of tra- ditional knowledge systems, undoubtedly would help life science researchers, in particular, because of the increas- ing demand for their research outputs in dealing with many health threat conditions, more evident from the current global pandemic—COVID-19 disease. Religion is opined to account for 70% of human activi- ties in South Asian Countries [98] and South Asia is reported to be having more vegetarian food choices because high percentages of Indians are pure vegetar- ians [99]. As opined by earlier researchers, food rules and laws (taboos) are influenced by religious beliefs/customs, which in turn are influenced by geographical location, environmental factors and availability of raw material for food preparation [100]. Having similarity of these factors would be a reason for most of the South Asian countries to have almost common ethnic food practices. However individual traditional beliefs across the countries would certainly influence minor variations of their ethnic foods. Influence of Hindu traditional texts on South Indian ethnic foods Similarly, main vegetarian meal of Nepal also seems to have rice with pulses, vegetable curry, milk, curd and pickle. Additionally, as a part of traditional practice, fermented foods from split black gram/green gram, wheat/rice floor, soybean, fruits, vegetables and milk are also being reported in Nepal [94, 95]. Apart from South Asian countries, many other Asian countries are also reported to possess rich variety of ethnic food tradi- tions. Cereal and milk based ethnic foods of Kyrgyzsthan [96] and over 100 varieties of ethnic rice based foods and desserts of Iran [97] seems to possess many similari- ties with South Asian ethnic foods especially in terms of ingredient usage and also appearance. Conclusion Similar to our observation in the present study, influ- ence of an ancient text “Mahavamsa” on food practices of ethnic group of “Vedda” people of Srilanka [101] and reports of traditional Islamic texts being used as a knowl- edge resource to know that some vegetarian foods (dates, grapes, figs, pomegranate, cereal powder blend i.e., sattu, melons, carrots, pumpkins, lentils, powdered pulses and seeds i.e., qawoot and powdered sesame i.e., savigh and quince) are useful to treat male fertility related prob- lems [102] strengthens our hypothesis of the influential role traditional knowledge systems play on the evolution of ethnic food cultures. However more in depth analysis would help understand similar such traditions probably associated with the ethnic food cultures of other South Asian countries Ethnic foods are strongly influenced by the cultural setup of a particular region and the knowledge is gener- ally transferred from generations, which subsequently becomes a tradition over time. In very early generations, since the general knowledge and know-how over several aspects were low, dependency on some form of tradi- tional knowledge system seems to have been an invariant option. Food practices undoubtedly are thus a part of that knowledge flow, which obviously will have a deep rooted connection in culture. Present work shows that traditional Indic texts has many food laws being laid and also constituted relevant food formulations, which perhaps would have been a necessity to suit to the then environmental conditions and people’s socio-economic status. However, since the traditions are carried from generations to generations, their unchanged existence even to date speaks about the traditional bind- ing people evolve with. Though the present work only looks at one particular culture i.e., Hinduism, the aspect seems to have relevance in vast array of other cultures too. Moreover, the fact that there is a traditional continuation Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Parthasarathi et al. Journal of Ethnic Foods Page 17 of 19 Page 17 of 19 of differential usage of the food items across all the south Indian states, exactly as meant by Sri Vedanta Desika’s Ahara Niyama, is a very thought provoking observation. Availability of data and materials All the data generated during this study are included within the manuscript. Additional supporting materials are available in the Additional file 1: Appen- dix 1 and Additional file 2: Appendix 2. All the photographs are authors own contribution. In our perspective, this study is a novel attempt clearly highlighting the importance of inculcating a different scien- tific acumen in understanding the domain of food. A thor- ough understanding of our theological and philosophical knowledge base would make the acumen transform into a holistic approach. Holistic approach of both science and phi- losophy would in turn help us revive our traditional ethnic food practices which ultimately help us in a better disease diagnosis—treatment regimens. This thought process con- fluence is need of the hour, given the uncertain health related threats present world is facing, where right kind of food cer- tainly has an ability to bring right changes in one’s health. References 1. Masson-Ousel P, Stern P, Willman-Grabowska H. Ancient India and Indian civilization. London: Routledge; 2013. Indian civilization. London: Routledge; 2013. 2. India State of Forest Report, Forest Survey of India, Ministry of Environ- ment, Forest and Climate Change. Government of India; 2021. 3. Farooqi AA, Sreeramu BS, Srinivasappa KN. Cultivation of spice crops. Madison: Universities Press; 2005. 4. Sugasini D, Yalagala PC, Kavitha B, Kasthuri T, Vijayalakshmi Y, Kumar PK, Kumar S. Indian culinary ethnic spices uses in foods are palate of paradise. Acta Sci Nutr Health. 2018;2(8):22–8. 5. https://indianexpress.com/article/lifestyle/food-wine/indians-love- meat-of-all-kinds-thats-what-an-rgi-survey-says-2850992-foodie/ Conclusion The order in which items are served and consumed follow a certain pattern, with optional exclusion of some items, which is predominantly decided by the occasion for which the items are prepared, psychological status of the person eating, prevailing climatic conditions and physiological status (assimilating capacity of digestive system). Thus, ethnic foods are understood to be prepared very carefully, so that they not only suffice the bodily nutritional demand but also can act as a natural medicament. Present theoret- ical review of the medicinal values all the food ingredients used in the preparation of South Indian vegetarian foods possess clearly proves this. Author contributions SKP contributed in providing the vast literature of Indic traditional texts and ideated the work. AVH contributed towards the overall planning of work and preparation of manuscript. DDPP majorly contributed towards conduction of ethnographic surveys, compilation of the data and also prepared few of the presented ethnic foods herself and gave photographs. All authors read and approved the final manuscript. Acknowledgements Authors acknowledge all the survey respondents and IAET trust for providing the platform to work. Funding The work is carried forward by the faculty-researchers seed grant given by Indian Academy Educational Trust (IAET), Bangalore. No external fund was received for this project work. Competing interests A h d h Authors do not have any known competing financial interests or personal relationships that could appear to influence the work reported in this paper. Limitation of the study and future scope Present study is a micro-level understanding of only the vegetarian ethnic foods of South India, which is a limita- tion. However, the study sets a foundation on the strong influential role of religious traditions on the ethnic foods of a particular region. Similar such studies are possible even with non-vegetarian food practices across South India. The study opens up a window to further investigate and under- stand the Asian food cultural landscape more closely in an ethnographic perspective. Author details 1 Department of Management Studies, IA School of Management Studies, Ben- galuru, India. 2 Department of Biochemistry, Indian Academy Degree College (Autonomous), Bengaluru, India. 3 Department of Basic Sciences, New Horizon College of Engineering, Bengaluru, India. Received: 5 May 2022 Accepted: 10 October 2022 Received: 5 May 2022 Accepted: 10 October 2022 Supplementary Information Fiber and prebiotics: mechanisms and health benefits. Nutri- ents. 2013;5(4):1417–35. 49. Sreenivas KM, Lele SS. Prebiotic activity of gourd family vegetable fibres using in vitro fermentation. Food Biosci. 2013;1:26–30. 22. Antony U, Ilango S, Chelliah R, Ramakrishnan SR, Ravichandran K. Ethnic fermented foods and beverages of Tamil Nadu. In: Ethnic fermented foods and beverages of India: science history and culture. Singapore: Springer; 2020. p. 539–560. 49. Sreenivas KM, Lele SS. Prebiotic activity of gourd family vegetable fibres using in vitro fermentation. Food Biosci. 2013;1:26–30. 50. Trinidad TP, Mallillin AC, Loyola AS, Sagum RS, Encabo RR. The potential health benefits of legumes as a good source of dietary fibre. Br J Nutr. 2010;103(4):569–74. g 23. Baruah R, Appaiah KA, Halami PM. Ethnic fermented foods and bever- ages of Karnataka. In: Ethnic fermented foods and beverages of India: science history and culture. Singapore: Springer; 2020. p. 209–230. 51. Faris MEAIE, Takruri HR, Issa AY. Role of lentils (Lens culinaris L) in human health and nutrition: a review. Mediterr J Nutr Metab. 2013;6(1):3–16. 24. Palika R, Dasi T, Kulkarni B, Pullakhandam R. Ethnic fermented foods and beverages of Telangana and Andhra Pradesh. In: Ethnic fermented foods and beverages of India: science history and culture. Singapore: Springer; 2020. p. 561–582. 52. Talari A, Shakappa D. Role of pigeon pea (Cajanus cajan L.) in human nutrition and health: a review. Asian J Dairy Food Res. 2018;37(3):212–20. 53. Nadeem M, Anjum FM, Khan MI, Tehseen S, El‐Ghorab A, Sultan JI. Nutritional and medicinal aspects of coriander (Coriandrum sativum L): a review. Br Food J. 2013. 25. Tamang JP, Thapa N. Some nonfermented ethnic foods of Sikkim in India. J Ethnic Foods. 2014;1(1):29–33. 26. Shukla A. Ethnic food culture of Chhattisgarh state of India. J Ethnic Foods. 2021;8(1):1–16. 54. Shahrajabian MH, Sun W, Soleymani A, Khoshkaram M, Cheng Q. Asafoetida, god’s food, a natural medicine. Pharmacogn Commun. 2021;11(1):36–9. 27. O’reilly K. Ethnographic methods. London: Routledge; 2012.i 28. Phillippi J, Lauderdale J. A guide to field notes for qualitative research: context and conversation. Qual Health Res. 2018;28(3):381–8. 55. Behera SS, El Sheikha AF, Hammami R, Kumar A. Traditionally fermented pickles: How the microbial diversity associated with their nutritional and health benefits? J Funct Foods. 2020;70:103971. 29. Chaudhari PR, Tamrakar N, Singh L, Tandon A, Sharma D. Rice nutritional and medicinal properties: a review article. J Pharmacogn Phytochem. 2018;7(2):150–6. 56. Rachwa-Rosiak D, Nebesny E, Budryn G. Supplementary Information 6. Mishra A. Traditional methods of food habits and dietary prepara- tions in Ayurveda—the Indian system of medicine. J Ethnic Foods. 2019;6(1):1–9. The online version contains supplementary material available at https://doi. org/10.1186/s42779-022-00156-1. 7. Chen Y, Michalak M, Agellon LB. Focus: nutrition and food science: importance of nutrients and nutrient metabolism on human health. Yale J Biol Med. 2018;91(2):95. Additional file 1: Appendix 1: Data procurement and analysis methodol- ogy employed for the ethnographic study presented in the manuscript. Table S1: Semi structured interviews conducted in-person at Temples on the traditional food preparations offered as Prasadam to the God. Table S2: Semi structured interviews and informal conversations con- ducted in-person or over telephone with individual Brahmin, Arya Vysya, Lingayath and Namboodiri families on their traditional food beliefs and practices. 8. Cohen AB. You can learn a lot about religion from food. Curr Opin Psychol. 2021;40:1–5. 9. Wagh K, Bhalerao S, Traditional foods, Ayurveda, and diet. In: Nutritional and health aspects of food in South Asian countries. Academic Press; 2020. 10. Tamang JP. Indian dietary culture. J Ethnic Foods. 2016;3(4):243–5. 11. Vecchio MG, Paramesh EC, Paramesh H, Loganes C, Ballali S, Gafare CE, et al. Types of food and nutrient intake in India: a literature review. Indian J Pediatr. 2014;81(1):17–22. Additional file 2: Appendix 2: Few photographs of Semi-structured interviews conducted at Temples (Photos taken only with the consent of the respondents) Additional file 2: Appendix 2: Few photographs of Semi-structured interviews conducted at Temples (Photos taken only with the consent of the respondents) Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Page 18 of 19 Page 18 of 19 Parthasarathi et al. Journal of Ethnic Foods 12. Sarkar P, Dh LK, Dhumal C, Panigrahi SS, Choudhary R. Traditional and ayurvedic foods of Indian origin. J Ethnic Foods. 2015;2(3):97–109. 38. Ghasemzadeh A, Karbalaii MT, Jaafar HZ, Rahmat A. Phytochemical constituents, antioxidant activity, and antiproliferative properties of black, red, and brown rice bran. Chem Cent J. 2018;12(1):1–13. 13. Tamang JP, editor. Ethnic fermented foods and beverages of India: sci- ence history and culture. Berlin: Springer Nature; 2020. 39. Jamir L, Kumar V, Kaur J, Kumar S, Singh H. Composition, valorization and therapeutical potential of molasses: a critical review. Environ Tech- nol Rev. 2021;10(1):131–42. 14. Sarma U, Gupta S. An overview on ethnic fermented food and bever- ages of India: interplay of microbes, immunity and nutrition. Nutr Health. 2022. https://doi.org/10.1177/02601060221085138. 40. 63. Singh B, Singh JP, Kaur A, Singh N. Bioactive compounds in banana and their associated health benefits—a review. Food Chem. 2016;206:1–11. Supplementary Information Kumar A, Singh S. The benefit of Indian jaggery over sugar on human health. In: Dietary sugar, salt and fat in human health. Academic Press; 2020. p. 347–359. 15. Das G, Patra JK, Singdevsachan SK, Gouda S, Shin HS. Diversity of traditional and fermented foods of the Seven Sister states of India and their nutritional and nutraceutical potential: a review. Front Life Sci. 2016;9(4):292–312. 41. Elleuch M, Bedigian D, Zitoun A. Sesame (Sesamum indicum L) seeds in food, nutrition, and health. In: Nuts and seeds in health and disease prevention. Academic Press; 2011. p. 1029–1036. 16. Ray M, Ghosh K, Singh S, Mondal KC. Folk to functional: an explora- tive overview of rice-based fermented foods and beverages in India. J Ethnic Foods. 2016;3(1):5–18. 42. Alasalvar C, Salvadó JS, Ros E. Bioactives and health benefits of nuts and dried fruits. Food Chem. 2020;314:126192. 17. Tamang JP. Naturally fermented ethnic soybean foods of India. J Ethnic Foods. 2015;2(1):8–17. 43. Mekkara nikarthil Sudhakaran S, Bukkan DS. A review on nutritional composition, antinutritional components and health benefits of green gram (Vigna radiata (L.) Wilczek). J Food Biochem. 2021;45(6):e13743-1. 18. Satish Kumar R, Kanmani P, Yuvaraj N, Paari KA, Pattukumar V, Arul V. Traditional Indian fermented foods: a rich source of lactic acid bacteria. Int J Food Sci Nutr. 2013;64(4):415–28. 44. Netshiheni RK, Omolola AO, Anyasi TA, Jideani AI. Banana bioactives: absorption, utilisation and health benefits. In: Banana nutrition-function and processing kinetics. IntechOpen; 2019. 19. Tamang JP. Dietary culture and antiquity of the Himalayan fer- mented foods and alcoholic fermented beverages. J Ethnic Foods. 2022;9(1):1–18. 45. Swami SB, Thakor NJ, Haldankar PM, Kalse SB. Jackfruit and its many functional components as related to human health: a review. Compre- hens Rev Food Sci Food Saf. 2012;11(6):565–76. 20. Thanzami K, Lalhlenmawia H. Ethnic fermented foods and beverages of Mizoram. In: Ethnic fermented foods and beverages of India: science history and culture. Singapore: Springer; 2020. p. 435–457. 46. Hajar R. Risk factors for coronary artery disease: historical perspectives. Heart Views. 2017;18(3):109.i 21. Latha S, Hemamalini A, Kumar SD, Arulmozhi M, Dhanasekaran D. Ethnic probiotic foods of South India and their health benefits. In: Fermented food products. CRC Press; 2019. p. 77–92. 47. Hervik AK, Svihus B. The role of fiber in energy balance. J Nutr Metab. 2019;2019:1–11. 48. Slavin J. Fiber and prebiotics: mechanisms and health benefits. Nutri- ents. 2013;5(4):1417–35. 48. Slavin J. Supplementary Information Chickpeas—composition, nutritional value, health benefits, application to bread and snacks: a review. Crit Rev Food Sci Nutr. 2015;55(8):1137–45. 30. Rathna Priya TS, Eliazer Nelson ARL, Ravichandran K, Antony U. Nutri- tional and functional properties of coloured rice varieties of South India: a review. J Ethnic Foods. 2019;6(1):1–11. 57. Wallace TC, Murray R, Zelman KM. The nutritional value and health benefits of chickpeas and hummus. Nutrients. 2016;8(12):766. 31. Sindhuja S, Prakruthi M, Manasa R, Shivananjappa M. Health benefits of ghee (clarified butter)—a review from ayurvedic perspective. IP J Nutr Metab Health Sci. 2020;3(3):64–72. 58. Devarajan A, Mohanmarugaraja MK. A comprehensive review on Rasam: a South Indian traditional functional food. Pharmacogn Rev. 2017;11(22):73. 32. Rao YS, Mathew KM. Tamarind. In: Handbook of herbs and spices. Woodhead Publishing; 2012. p. 512–533. 59. Elengoe A. Indian spices boost the immune system against covid- 19. Ann Univ Dunarea de Jos Galati Fascicle VI-Food Technol. 2020;44(2):189–206. 33. Vijayakumar V. Nutraceutical legumes: a brief review on the nutritional and medicinal values of legumes. Sustain Agric Rev. 2021;51:1–28. 34. De, A. K., & De, M. Functional and therapeutic applications of some important spices. In The role of functional food security in global health. Academic Press. 2019:499–510. 60. Chelladurai V, Erkinbaev C. Lentils. In: Pulses. Cham: Springer; 2020. p. 129–143. 61. Girish TK, Pratape VM, Rao UP. Nutrient distribution, phenolic acid composition, antioxidant and alpha-glucosidase inhibitory potentials of black gram (Vigna mungo L.) and its milled by-products. Food Res Int. 2012;46(1):370–7. 35. Kukkupuni SK, Shashikumar A, Venkatasubramanian P. Fermented milk products: probiotics of ayurveda. J Med Nutr Nutraceuticals. 2015;4(1):14. 36. Bhat FM, Riar CS. Health benefits of traditional rice varieties of temper- ate regions. Med Aromat Plants. 2015;4:198. 62. Jukanti AK, Gaur PM, Gowda CLL, Chibbar RN. Nutritional quality and health benefits of chickpea (Cicer arietinum L.): a review. Br J Nutr. 2012;108(S1):S11–26. 37. Boue SM, Daigle KW, Chen MH, Cao H, Heiman ML. Antidiabetic poten- tial of purple and red rice (Oryza sativa L.) bran extracts. J Agric Food Chem. 2016;64(26):5345–53. 63. Singh B, Singh JP, Kaur A, Singh N. Bioactive compounds in banana and their associated health benefits—a review. Food Chem. 2016;206:1–11. Parthasarathi et al. Journal of Ethnic Foods (2022) 9:42 Page 19 of 19 Page 19 of 19 64. Cianciosi D, Forbes-Hernández TY, Afrin S, Gasparrini M, Reboredo- Rodriguez P, Manna PP, et al. Phenolic compounds in honey and their associated health benefits: a review. Molecules. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 79. Srinivasan P. Ahara-Niyama: the Srivaisnava dietary regimen. Res: Anthropol Aesthet. 2000;37(1):186–204. 80. Radin D, Hayssen G, Emoto M, Kizu T. Double-blind test of the effects of distant intention on water crystal formation. Explore. 2006;2(5):408–11. https://doi.org/10.1016/j.explore.2006.06.004. 81. Pushpendra K. Taitireeya Brahmanam, vol 2. Nag Publishers, ISBN: 81-7081-397-2. 1998. 82. Ramanujan AK, Khare RS. Food for thought: toward an anthology of Hindu food-images. In: The eternal food: gastronomic ideas and experi- ences of Hindus and Buddhists; 1992. p. 221–50. 83. Thas JJ. Siddha medicine—background and principles and the applica- tion for skin diseases. Clin Dermatol. 2008;26(1):62–78. 84. Shukla S, Settee P. Synthesis: revitalizing the past, nourishing the present, and feeding the future. In: Indigenous food systems: concepts, cases, and conversations; 2020. p. 269–284. 85. Raju MSVK. Medical nutrition in mental health and disorders. Indian J Psychiatry. 2017;59(2):143. 85. Raju MSVK. Medical nutrition in mental health and disorders. Indian J Psychiatry. 2017;59(2):143. 86. Prakash V. Introduction: the importance of traditional and ethnic food in the context of food safety, harmonization, and regulations. In: Regulating safety of traditional and ethnic foods. Academic Press; 2016. p. 1–6. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 87. Rocillo-Aquino Z, Cervantes-Escoto F, Leos-Rodríguez JA, et al. Supplementary Information Religious ethnic food. J Ethnic Foods. 2015;2:45–6. https://doi.org/10.1016/j.jef.2015.05.001. 101. Mihiranie S, Jayasinghe JK, Jayasinghe CV, Wanasundara JP. Indigenous and traditional foods of Sri Lanka. J Ethnic Foods. 2020;7(1):1–19. 74. Ranganathananda Swami, Universal Message of the Bhagavad Gita (Vol 3). Advaita Ashrama Kolkata, 2000; ISBN 81-7505-213-9. 102. Dehghani-Bidgoli R. Vegetarian food recommendation in the traditional Islamic teachings to increase the fertility of men. J Ethnic Foods. 2019;6(1):1–7. 75. Ramos-Jiménez A, Wall-Medrano A, Corona-Hernández RI, Hernández- Torres RP. Yoga, bioenergetics and eating behaviors: a conceptual review. Int J Yoga. 2015;8(2):89–95. https://doi.org/10.4103/0973-6131. 158469. 103. Bellisle F. Food choice, appetite and physical activity. Public Health Nutr. 1999;2(3a):357–61. 104. Reyes-García V. The relevance of traditional knowledge systems for ethnopharmacological research: theoretical and methodological con- tributions. J Ethnobiol Ethnomed. 2010;6(1):1–12. 76. Samskaras SM. The Vedic perspective of nutrition and learning. In: Althans B, Bilstein J, editors. Essen-Bildung-Konsum. Wiesbaden: Springer; 2016. https://doi.org/10.1007/978-3-658-01543-5_11. p g p g 77. Sivananda S. All about hinduism. Rishikesh: Divine Life Society; 1977. 78. Shankari U. Brahmin, king and bhakta in a temple in Tamil Nadu. Con- trib Indian Sociol. 1984;18(2):169–87. Supplementary Information 2018;23(9):2322. 91. Akhtar S, Hussain M, Ismail T, Riaz M. Ethnic fermented foods of Pakistan. In: Ethnic fermented foods and alcoholic beverages of Asia. New Delhi: Springer; 2016. p. 119–137. i 65. Melini F, Melini V, Luziatelli F, Ruzzi M. Raw and heat-treated milk: from public health risks to nutritional quality. Beverages. 2017;3(4):54. 92. Alam SMN, Naser MN. Role of traditional foods of Bangladesh in reaching-out of nutrition. In: Nutritional and health aspects of food in South Asian countries. Academic Press; 2020. pp. 217–235. 66. Singletary K. Cardamom: potential health benefits. Nutr Today. 2022;57(1):38–49. 93. Hossain M, Kabir Y. Ethnic fermented foods and beverages of Bangla- desh. In: Ethnic fermented foods and alcoholic beverages of Asia. New Delhi: Springer; 2016. p. 73–89. 67. Terzo S, Baldassano S, Caldara GF, Ferrantelli V, Lo Dico G, Mulè F, Amato A. Health benefits of pistachios consumption. Nat Prod Res. 2019;33(5):715–26. 94. Khadka DB, Lama JP. Traditional fermented food of Nepal and their nutritional and nutraceutical potential. In: Nutritional and health aspects of food in South Asian countries. 2020; p. 165–194. 68. Bhugra D. Hinduism and Ayurveda: implications for managing mental health. In: Psychiatry and religion. Routledge; 2013. p. 113–127. 95. Dahal NR, Karki TB, Swamylingappa B, Li QI, Gu G. Traditional foods and beverages of Nepal: a review. Food Rev Intl. 2005;21(1):1–25. 69. Adidevananda Swami. Yatindramatadipika, Ramakrishna Math. 1949. ISBN 81-7120-508-9. 70. Jacobsen, K. A. Kapila, Founder of Sāṃkhya and Avatāra of Viṣṇu: With a Translation of Kapilāsurisaṃvāda. 2008. 96. Smanalieva J, Iskakova J, Musulmanova M. Milk-and cereal-based Kyrgyz ethnic foods. Int J Gastronomy Food Sci. 2022;29:100507. 71. Larson GJ. Classical Sāṃkhya: an interpretation of its history and mean- ing. New Delhi: Motilal Banarsidass Publication; 2001. 97. Karizaki VM. Ethnic and traditional Iranian rice-based foods. J Ethnic Foods. 2016;3(2):124–34. 98. Alsdorf L. The history of vegetarianism and cow-veneration in India. London: Routledge; 2010. 72. Beri K, Menon V, Guzman E, Chapa C, Patel R, Shariff MA, Kasubhai M. The effect of living a ’yogic lifestyle’ on stress response and self-image in healthcare professionals: a pilot study. Future Sci. 2020;6(6):FSO473. https://doi.org/10.2144/fsoa-2019-0154. 99. Josiam B, Sohail MS, Monteiro P. Curry cuisine: perceptions of Indian restaurants in Malaysia. Tour Int J Tour. 2007;2(2):25–38. g 73. Dhanya S, Ramesh NV, Mishra A. Traditional methods of food habits and dietary preparations in Ayurveda—the Indian system of medicine. J Ethnic foods. 2019;6:1. https://doi.org/10.1186/s42779-019-0016-4. 100. Kwon D, Tamang J. Publisher’s Note What is a traditional food? Conceptual evolution from four dimensions. J Ethn Food. 2021;8:38. https://doi.org/10.1186/s42779-021-00113-4. 88. Smarta RB. Lifecycle stages for food safety of traditional foods. In: Nutri- tional and health aspects of food in South Asian countries. Academic Press; 2020. p. 291–295. 89. Waisundara VY. Traditional functional food of Sri Lanka and their health significance. In: Nutritional and health aspects of food in South Asian Countries. Academic Press; 2020. p. 143–158. 90. Liyanage CJ (2020) Traditional and ethnic foods of Sri Lanka-safety aspects. In: Nutritional and health aspects of food in South Asian Coun- tries. Academic Press; 2020. p. 127–141. 90. Liyanage CJ (2020) Traditional and ethnic foods of Sri Lanka-safety aspects. In: Nutritional and health aspects of food in South Asian Coun- tries. Academic Press; 2020. p. 127–141.
https://openalex.org/W3037510981	http://periodicos.ufc.br/revistademedicinadaufc/article/download/40342/100230	Portuguese	null	Icterícia neonatal e seus fatores perinatais associados: perfil dos recém-nascidos internados em uma unidade de terapia intensiva neonatal de maternidade de referência terciária no município de Fortaleza – Ceará	Revista de Medicina da UFC/Revista de Medicina da Universidade Federal do Ceará	2,020	cc-by	4,195	RESUMO Objetivos: conhecer o perfil de recém-nascidos (RN) internados em unidade de terapia intensiva (UTIN), no Serviço de Neonatologia de uma maternidade pública terciária do município de Fortaleza, que apresentaram icterícia. Metodologia: estudo transversal, prospectivo, sendo os dados coletados nos prontuários de 01 de fevereiro a 31 de julho de 2018, utilizando questionário simples. As análises estatísticas foram realizadas no programa software R 3.3.1 e Microsoft Excel® 2016. Resultados: os dados revelaram que 70,7% dos RN apresentaram icterícia significativa e 84,5% tinham idade gestacional menor que 35 semanas. O sexo masculino apresentou-se em 58,2% dos casos. Em 65,3% o tempo de clampeamento do cordão umbilical foi menor que 1 minuto. A taxa de sepse precoce foi de 68% e a de sepse tardia de 35,9%. A hemorragia peri-intraventricular (HPIV) esteve presente em 37,4% da amostra. O índice de policitemia foi de 1%. Pré-eclâmpsia foi fator protetor para icterícia significativa. Fototerapia foi realizada em todos os RN e 5,1% necessitaram de exsanguíneotransfusão. Conclusões: a prematuridade abaixo de 35 semanas foi o principal fator associado a icterícia de alto risco e esforços obstétricos e neonatais voltados para a sua prevenção são necessários no sentido de melhorar esse quadro e seus fatores relacionados. alavras-chave: Icterícia neonatal. Unidades de Terapia Intensiva Neonatal. Fototerapia. Recém-nascido. Neonatal jaundice and its associated perinatal factors: profile of newborns hospitalized in a neonatal intensive care unit of a tertiary reference neonatal clinic in the municipatility of Fortaleza – Ceará Rafaela Loiola de Carvalho1. Maria Francielze Holanda Lavor1,2. 1 Universidade Federal do Ceará (UFC), Maternidade Escola Assis Chateaubriand (MEAC), Fortaleza, Ceará, Brasil. 2 Instituto Doutor José Frota, Fortaleza, Ceará, Brasil. 1 Universidade Federal do Ceará (UFC), Maternidade Escola Assis Chateaubriand (MEAC), Fortaleza, Ceará, Brasil. 2 Instituto Doutor José Frota, Fortaleza, Ceará, Brasil. Autor correspondente: Rafaela Loiola de Carvalho, Rua Coronel Nunes de Melo, sem número, Rodolfo Teófilo, Fortaleza, Ceará. CEP: 60430-270. Telefone: +55 85 3366-8528. E-mail: carvrafaela@hotmail.com Conflito de interesses: Não há qualquer conflito de interesses por parte de qualquer um dos autores. Recebido em: 12 Fev 2019; Revisado em: 13 Jun 2019; Aceito em: 15 Jun 2019. 11 11 doi: 10.20513/2447-6595.2020v60n2p11-17 ARTIGO ORIGINAL INTRODUÇÃO de DMG e de pré-eclâmpsia), ao RN (APGAR no primeiro minuto, sexo, idade gestacional, peso de nascimento, classificação do recém-nascido de acordo com a IG e com o peso de nascimento, tipagem sanguínea, coombs direto, tempo de eliminação de mecônio) e à assistência (tempo de clampeamento do cordão umbilical, tempo de jejum e de nutrição parenteral, perda de peso nas primeiras 72 horas de vida, valor de bilirrubina total (BT) antes de 24 horas de vida, entre 24 e 72 horas, entre 5 e 7 e após 7 dias de vida, necessidade de fototerapia e de exsanguíneotransfusão, tempo de internação em UTIN, desfecho final e presença de patologias como síndrome do desconforto respiratório (SDR), sepse precoce e tardia, cardiopatias, policitemia e hemorragia peri-intraventricular (HPIV), além de alterações como hipotermia e hipertermia e hematomas ou equimoses). A icterícia é uma condição médica comum no período neonatal, sendo a principal causa de reinternação na primeira semana de vida.1 É considerada “fisiológica” quando ocorre após as primeiras 24h de vida, sendo vista como patológica antes desse período ou quando permanece por períodos prolongados. Quando patológica e associada a altos níveis de bilirrubina, pode levar a um quadro grave de encefalopatia, o Kernicterus, ou até mesmo a óbito, se não for adequadamente tratada.2 Diversos fatores maternos e perinatais estão associados ao surgimento de icterícia neonatal. Dentre eles destacam- se idade gestacional (IG), sexo do recém-nascido (RN), diabetes mellitus gestacional (DMG), pré-eclâmpsia, tempo de clampeamento do cordão umbilical, eliminação tardia de mecônio, nutrição enteral tardia e perda de peso importante na primeira semana de vida. Algumas situações geram o aumento da circulação êntero-hepática, como jejum prolongado e anomalias gastrointestinais (processos obstrutivos, estenose hipertrófica do piloro) e favorecem a hiperbilirrubinemia. Ainda se destacam como fatores importantes no surgimento dessa condição as doenças hemolíticas (incompatibilidades ABO e Rh) e a presença de coleções sanguíneas extravasculares (cefalohematoma, bossa serossanguínea e equimoses).3 A IG em semanas foi subdividida nas seguintes categorias: <28, de 28 a 29, de 30 a 31, de 32 a 33, de 34 a menores que 35 e maiores ou iguais a 35 semanas. ABSTRACT Objectives: the main objective was to know the profile of newborns (NB) hospitalized in a neonatal intensive care unit (NICU) in a tertiary reference Neonatal Clinic in the municipality of Fortaleza who presented neonatal jaundice. Methodology: it was a cross-sectional study, prospective, conducted during February-July 2018 through the use of a simple questionnaire. The data were analyzed with software R 3.3.1 e Microsoft Excel® 2016. Results: it was noted that 70,7% of NB presented significant neonatal jaundice, 84,5% had gestational age less than 35 weeks. The majority of NB studied was male (58,2%). Umbilical cord clamping time less than 1 minute represented 65,3% of cases studied. The rate of early and late sepsis was 68% e 35,9%, respectively. Intracranial hemorrhage (IH) was associated to 37,4% of NB and polycythaemia to 1%. Pre-eclampsia was protective factor for severe hyperbilirubinemia. All babies were submitted to phototherapy and 5,1% needed exchange transfusion. Conclusions: the main factor of significant neonatal jaundice is premature birth under 35 weeks of gestacional age. Obstetrics and neonatal efforts are necessary to avoid it. Keywords: Neonatal jaundice. Neonatal Intensive Care Units. Phototherapy. Newborn. Autor correspondente: Rafaela Loiola de Carvalho, Rua Coronel Nunes de Melo, sem número, Rodolfo Teófilo, Fortaleza, Ceará. CEP: 60430-270. Telefone: +55 85 3366-8528. E-mail: carvrafaela@hotmail.com Conflito de interesses: Não há qualquer conflito de interesses por parte de qualquer um dos autores. Recebido em: 12 Fev 2019; Revisado em: 13 Jun 2019; Aceito em: 15 Jun 2019. Rev Med UFC. 2020;60(2):11-17. Rev Med UFC. 2020;60(2):11-17. 12 Perfil de bebês ictéricos em UTIN INTRODUÇÃO Foram selecionados como apresentando icterícia significativa, os RN que tinham valores de BT correspondentes aos pontos de corte para uso de fototerapia considerados de alto risco de acordo com a IG e a idade de nascimento, conforme dados presentes nos gráficos de Bhutani naqueles com IG maior ou igual a 35 semanas e nas tabelas da Academia Americana de Pediatria para IG menor que 35 semanas (TABELA 1).6,7 O tratamento baseia-se em fototerapia e exsanguíneotransfusão, quando há predominância da bilirrubina indireta, sendo realizado a partir de dados retirados de gráficos e tabelas, que variam de acordo com a IG e a idade do RN.4 Tabela 1. Presença de icterícia significativa de acordo com valores de BT, IG e idade do RN.6,7 Idade gestacional* BT com menos de 24 horas BT de 24 a 72 horas BT de 5 a 7 dias BT >ou=7 dias < 28 semanas 5 5 5 5 28 a 29 semanas 6 6 6 6 30 a 31 semanas 8 8 8 8 32 a 33 semanas 10 10 10 10 34 a <35 semanas 12 12 12 12 35-37 semanas 8 8 - 13,5+ 15 15 >ou= 38 semanas 10 10-15,5§ 18 18 Tabela 1. Presença de icterícia significativa de acordo com valores de BT, IG e idade do RN.6,7 No contexto do presente estudo, é importante conhecer a classificação dos RN de acordo com a IG ao nascimento. A divisão apresenta-se da seguinte forma: pré-termos, prematuros extremos, muito prematuros, prematuros moderados e tardios, que correspondem a menores que 37 semanas, menores que 28 semanas, de 28 a 31 semanas e 6 dias, de 32 a 33 semanas e 6 dias e de 34 a 36 semanas e 6 dias, respectivamente.5 Diante do exposto, o estudo teve o objetivo de conhecer o perfil dos RN internados em unidade de terapia intensiva (UTIN) do Serviço de Neonatologia de uma maternidade pública terciária do município de Fortaleza-CE, no período de 01 de fevereiro a 31 de julho de 2018, que apresentaram icterícia. RESULTADOS Foram selecionados para o estudo 163 pacientes internados na UTIN da MEAC, sendo excluídos 23 RN, pois estes foram transferidos para outros hospitais. Dos 140 restantes, 70,7% apresentaram icterícia significativa. Desses, 84,5% tinham menos que 35 semanas, sendo 21,2% pré-termos extremos, 26,3% muito prematuros, 30,3% com prematuridade moderada e 13,1% tardia. Apenas 8,1% eram a termo (FIGURA 1). O sexo predominante foi o masculino (58,2%), sendo 40% RN do sexo feminino e 1,4% dos bebês com sexo indeterminado por genitália ambígua. O presente estudo foi avaliado pelo Comitê de Ética em Pesquisa envolvendo seres humanos da Maternidade Escola da Universidade Federal do Ceará e aprovado através do parecer número 2.510.991. Figura 1. Estratificação dos pacientes estudados. Figura 1. Estratificação dos pacientes estudados. O peso do nascimento foi maior que 2500 gramas (g) em 19,3% dos RN. Apresentaram peso abaixo de 2500g 80,7% dos bebês, estando abaixo de 1500g e de 1000g 42,8% e 21,4%, respectivamente. Das patologias apresentadas pelos RN durante a internação, destacaram-se a síndrome do desconforto respiratório (SDR), presente em 73,8%, além da sepse precoce e tardia, com 68% e 35,9%, respectivamente. Problemas cardíacos representaram 38,8% dos casos. A hipotermia foi outro fator de destaque nos pacientes estudados, tendo uma prevalência de 90,3%, seguida da hipertermia, equivalente a 68% da amostra. A principal via de parto foi a cesárea, em 61,6% dos casos, por indicação materna ou fetal. Pré-eclâmpsia grave (PEG) e DMG, foram as principais causas maternas, com 31,1% e 15,5% respectivamente. Relacionando o peso com a IG, 82,8% dos RN eram adequados para a idade gestacional (AIG). O escore APGAR no primeiro minuto de vida foi menor que 7 em 44,4% dos RN. O tempo de clampeamento do cordão umbilical foi menor que 1 minuto em 65,3%, entre 1 e 3 minutos em 33,7% e maior que 3 minutos em 1% da amostra. Dos RN em análise, a presença de hematomas ou equimoses ocorreu em apenas 10,1% dos casos e a hemorragia peri- intraventricular (HPIV) em 37,4% da amostra. A policitemia apresentou-se somente em 1% dos pacientes. A eliminação tardia de mecônio (após 48 horas de vida) ocorreu em 21,4% dos casos. O tempo de jejum por mais de 48 horas representou 26,2% da amostra, ficando 33% entre 24 e 48 horas e 40,8% com início da dieta nas primeiras 24 horas de vida. MATERIAL E MÉTODOS Estratificação da IG de acordo com o gráfico de Bhutani para IG>ou=35semanas e com a tabela da Academia Americana de Pediatria para IG<35semanas com delimitação dos valores de BT com pontos de corte para uso de fototerapia considerados de alto risco (correspondentes aos portadores de icterícia significativa). É um estudo transversal, prospectivo, onde os dados foram coletados nos prontuários, através de questionário simples. A população do estudo incluiu recém-nascidos internados na UTIN da Maternidade Escola Assis Chateaubriand (MEAC). Foram elegíveis todos os prontuários de neonatos que nasceram na MEAC e estiveram internados na UTIN no período de 01 de fevereiro a 31 de julho de 2018. Excluíram‑se os pacientes que foram transferidos durante o processo de coleta de dados. +BT com 24h de vida: >8mg/dL, BT com 36h: >9,5mg/dL; BT com 48h: >11,5mg/dL e BT com 72h: >13,5mg/dL §BT com 24h de vida: >10mg/dL, BT com 36h: >11,5mg/dL; BT com 48h: >13mg/dL e BT com 72h: >15,5mg/dL Na análise das variáveis numéricas, os dados foram avaliados em médias e percentis. Nas variáveis categóricas, expostos em frequência e taxa de prevalência, de modo a investigar Para a captação dos dados, foi utilizada uma ficha de coleta, elaborada pela própria pesquisadora que continha variáveis relativas à mãe (tipo de parto, tipagem sanguínea, presença Rev Med UFC. 2020;60(2):11-17. Rev Med UFC. 2020;60(2):11-17. 13 Perfil de bebês ictéricos em UTIN associações entre a icterícia neonatal e os seus fatores associados. Adotou-se um nível de significância de 5%. Na investigação da associação entre as variáveis categóricas utilizou-se o teste de qui-quadrado de Pearson e o exato de Fisher. As análises estatísticas foram realizadas utilizando os programas estatísticos software R 3.3.1, JAMOVI 0.9.5.12 e o Microsoft Excel® 2016. RESULTADOS O início da nutrição parenteral precoce (menos de 24 horas de vida) aconteceu em 60,3% dos RN e o seu uso durou em média 14,3 dias. A perda de peso nas primeiras 72 horas após o nascimento foi menor que 7% em 46,5% dos pacientes e maior que 7% em 53,5%. A tipagem sanguínea materna mais prevalente foi a O positivo (45,6%), seguida da A positivo (35,9%). Tipagens com Rh negativo somaram 5,8% (A negativo 1,9% e O negativo 3,9%). Já em relação a tipagem dos RN, a A positivo e a O positivo tiveram a mesma prevalência, representando 37,4% cada uma. O Coombs direto foi positivo em apenas 5,1% dos casos. Rev Med UFC. 2020;60(2):11-17. Rev Med UFC. 2020;60(2):11-17. 14 Perfil de bebês ictéricos em UTIN O tempo médio de internação na UTI neonatal foi de 21,3 dias. O desfecho dos bebês foi alta hospitalar em 87,9% dos casos e óbito em 12,1%. Quanto ao tratamento, 100% apresentaram icterícia às custas de bilirrubina indireta e foram submetidos à fototerapia e 5,1% realizaram exsanguíneotransfusão. Em 2,9% dos casos, houve aumento importante da bilirrubina direta, necessitando de investigação adicional. A TABELA 2 mostra os dados estatísticos comparativos da icterícia significativa com os fatores maternos e neonatais. Variáveis Icterícia significativa N (%) Icteríca não significativa N (%) p IG <0,012 >ou= 35s 16 (15,5%) 24 (64,9%) < 35s 87 (84,5%) 13 (35,1%) Sexo <0.638 Feminino 41 (41,8%) 15 (37,5%) Masculino 57 (58,2%) 25 (62,5%) Tipo de parto <0.112 Vaginal 38 (38,4%) 10 (24,4%) Cesáreo 61 (61,6%) 31 (75,6%) Peso ao nascimento <0.543 >2500g 16 (16,2%) 11 (26,8%) 2500-1500g 39 (39,4%) 14 (34,1%) 1500-1000g 23 (23,2%) 8 (19,5%) <1000g 21 (21,2%) 8 (19,5%) Tempo de clampeamento do cordão umbilical <0.574 <1 min 64 (65,3%) 30 (73,2%) 1-3min 33 (33,7%) 11 (26,8%) >3 min 1 (1,0%) 0 (0%) APGAR no primeiro minuto <0.837 <7 44 (44,4%) 19 (46,3%) >7 55 (55,6%) 22 (53,7%) Peso de acordo com a IG <0.572 AIG 82 (82,8%) 30 (75,0%) PIG 12 (12,1%) 7 (17,5%) GIG 5 (5,1%) 3 (7,5%) DMG+ 16 (15,5%) 9 (24,3%) <0.316 Pré-eclâmpsia 32 (31,1%) 19 (51,4%) <0.028* Sepse Precoce 70 (68,0%) 22 (59,5%) <0.420 Sepse tardia 37 (35,9%) 14 (37,8%) <0.844 SDR§ 76 (73,8%) 27 (73%) <0.923 Cardiopatia 40 (38,8%) 11 (29,7%) <0.324 Hipotermia 93 (90,3%) 32 (86,5%) <0.521 Hipertermia 70 (68,0%) 25 (67,6%) <0.965 Policitemia 1 (1,0%) 0 (0%) <0.518 Tabela 2. RESULTADOS Dados comparativos de icterícia significativa com as variáveis maternas e neonatais. C ti Tabela 2. Dados comparativos de icterícia significativa com as variáveis maternas e neonatais. Rev Med UFC. 2020;60(2):11-17. 15 Perfil de bebês ictéricos em UTIN Variáveis Icterícia significativa N (%) Icteríca não significativa N (%) p Eliminação de mecônio <0.973 <48h 81 (78,6%) 29 (78,4%) >48h 22 (21,4%) 8 (21,6%) Tempo de dieta zero <0.152 <24h 42 (40,8%) 14 (37,8%) 24-48h 34 (33,0%) 18 (48,6%) >48h 27 (26,2%) 5 (13,5%) ABO-RH materno <0.543 O+ 47 (45,6%) 22 (59,5%) O- 4 (3,9%) 0 (0%) A- 2 (1,9%) 1 (2,7%) A+ 37 (35,9%) 12 (32,4%) ABO-RH do RN <0.847 O+ 37 (37,4%) 17 (41,5%) A+ 37 (37,4%) 17 (41,5%) A- 1 (1,0%) 0 (0%) B+ 13 (13,1%) 3 (7,3%) B- 1 (1,0%) 0 (0%) Coombs direto 5 (5,1%) 0 (0%) <0.824 Duração da NPT** <0.372 < 7 dias 49 (81,7%) 17 (81%) > 7 dias 13 (20,6%) 4 (19%) Perda Percentual de peso nas primeiras 72h <0.438 <7% 46 (46,5%) 22 (53,7%) >7% 53 (53,5%) 19 (46,3%) Hematomas e equimoses 10 (10,1%) 3 (7,3%) <0.606 HPIV 37 (37,4%) 16 (37%) <0.855 Fototerapia 99 (70,7%) 41 (28,3%) - Exsanguíneotransfusão 5 (5,1%) 0 (0%) <0.143 Tabela 2. Dados comparativos de icterícia significativa com as variáveis maternas e neonatais. Conclusão. * p estatisticamente significante; + Diabetes Mellitus Getacional; § Síndrome do Desconforto Respiratório; **Nutrição Parenteral. Tabela 2. Dados comparativos de icterícia significativa com as variáveis maternas e neonatais. Exsanguíneotransfusão DISCUSSÃO Todos os achados não foram estatisticamente significantes.17 Estudos mostram que o clampeamento tardio do cordão umbilical leva a maiores índices de icterícia neonatal. Pesquisa realizada em São Paulo, no Hospital Geral de Grajaú, mostrou que o clampeamento precoce foi fator protetor para icterícia, apresentando-se em 73,8% dos RN que realizaram esse procedimento.14 O presente estudo apresentou valor absoluto semelhante (65,3%), porém sem significância clínica estatisticamente (p<0.574), já que, mesmo com o cordão clampeado precocemente, os RN tiveram icterícia significativa.7 O percentual de perda de peso maior ou igual a 7%, fator de risco sabidamente associado a hiperbilirrubinemia, foi praticamente semelhante ao menor que 7%, o que estatisticamente não demonstrou significância clínica (p<0.438).2 A tipagem sanguínea materna mais prevalente foi a O positivo, seguido pela A positivo, achados concordantes com dois estudos (Keren et al. e Enk et al.). Já quanto aos RNs, as tipagens sanguíneas principais foram A positivo e O positivo com mesmo percentual (37,4%). O Coombs direto (CD) foi positivo numa minoria dos casos (5,1%), sendo todos associados a incompatibilidade ABO e com IG maior ou igual a 35 semanas. Maisels et al. encontraram 22% dos pacientes com CD positivo, divergindo do achado do presente estudo, porém Enk et al. encontraram valor aproximado (2,7%).13,15,16 A análise estatística não mostrou significância clínica (p<0.824). Todos os RN foram submetidos a tratamento com fototerapia com o objetivo de evitar a encefalopatia bilirrubínica, forma grave da doença, e um pequeno percentual realizou exsanguíneotransfusão (5,1%), sendo exatamente aqueles que tinham CD positivo e incompatibilidade ABO.2 Enk et al encontraram uma taxa de fototerapia de 97,3% e de exsanguíneotransfusão de 1,4%, em concordância com os achados do presente estudo.12 O tempo de internação hospitalar e o seu desfecho foram influenciados por outras enfermidades além da icterícia, não apresentando esses dados significância estatística. A sepse, fator associado à icterícia por alterar a capacidade de ligação da bilirrubina à albumina, se mostrou com alta prevalência na amostra analisada (68% precoce e 35,9% tardia) porém sem apresentar significância estatística (p<0.420 para sepse precoce e p<0.844 para tardia), mostrando discordância com estudo realizado por Taheri et al., que só encontrou 11,7% dos casos (soma total).6,11 A diferença provavelmente se deve ao fato de que no presente estudo foi considerada a sepse com diagnóstico clínico, enquanto Taheri et al. utilizaram apenas aquela amostra com hemocultura positiva. DISCUSSÃO terciário de Porto Alegre e em hospital de Tehran revelaram peso médio de 3054g e de 1950g, respectivamente, em RN com icterícia significativa.11 Porém o peso ao nascimento não apresentou significância estatística (p<0.543). A icterícia neonatal ocorre em 60% dos RN a termo e em 80% dos pré-termos. A maior frequência no prematuro decorre da imaturidade dos sistemas e do início da alimentação enteral não tão precocemente, o que aumenta a circulação êntero- hepática.8,9 De acordo com a literatura, encontrou-se a maior prevalência da icterícia em pré-termos, 84,5% da amostra analisada, apresentando significância estatística (p<0.012) e com uma chance 2,95 vezes maior desses RN apresentarem essa doença (IC=1.95-6.96). Estudo de Bhutani et al. encontrou resultado semelhante.10 Comparando-se o sexo mais prevalente encontrado no estudo, o masculino (58,2%), os resultados foram semelhantes ao de outras duas pesquisas, citadas acima (52,7% em hospital terciário de Porto Alegre e 56,4% em hospital de Tehran), porém estatisticamente sem significância (p<0.638).11,12 O parto cesáreo representou a maior parte dos casos, o que concordou com estudo realizado em Porto Alegre, porém não se mostrou significante estatisticamente (p<0.112).12 As O peso médio dos bebês foi de 1761g. Estudos em hospital Rev Med UFC. 2020;60(2):11-17. Rev Med UFC. 2020;60(2):11-17. 16 Perfil de bebês ictéricos em UTIN apesentaram significância estatística, com p<0.606 e p<0.855, respectivamente, diferente do que há descrito na literatura.3 apesentaram significância estatística, com p<0.606 e p<0.855, respectivamente, diferente do que há descrito na literatura.3 principais condições maternas foram PEG e DMG, fatores sabidamente associados a icterícia, porém, no presente estudo, a hipertensão arterial mostrou-se um fator protetor (p<0.028), representando 2,3 vezes menos chances de desenvolver a doença (IC=0.198-0.920). Isso diverge do estudo de Marra et al. em que a pré-eclâmpsia esteve associada a icterícia.13 Já a relação da icterícia neonatal com a DMG não se mostrou significativamente estatísca.7 A eliminação precoce de mecônio (com menos de 48 horas de vida) está associada a menores níveis de bilirrubina. Esse achado foi o mais encontrado nos RN do presente estudo, porém estes apresentaram icterícia significativa, o que discorda da literatura. O achado não apresentou relevância estatística.17 Outros aspectos demonstrados foram o início tardio da nutrição enteral (após 24 horas de vida) e o uso prolongado de nutrição parenteral associados a altos níveis de bilirrubina, sendo estes dados concordantes com os encontrados na literatura. DISCUSSÃO O estudo demonstrou que os prematuros menores que 35 semanas têm maior risco de desenvolver icterícia significativa, o que corrobora com a literatura. Outro achado com significância estatística, porém divergente do que já existe descrito, foi a hipertensão materna como fator protetor. Os demais fatores perinatais associados, conhecidos influenciadores da hiperbilirrubinemia, não se mostraram relevantes nos achados aqui presentes. Conclui-se que a prematuridade abaixo de 35 semanas é o principal fator associado a icterícia de alto risco e esforços obstétricos e neonatais voltados para a sua prevenção são necessários no sentido de melhorar esse quadro e seus fatores relacionados. A presença de equimoses e hematomas também foram comparadas com icterícia significativa, já que também são fatores sabidamente relacionados ao alto risco da doença, mas não REFERÊNCIAS 1. Canadian Paediatric Society. Guidelines for detection, management and prevention of hyperbilirubinemia in term and late preterm newborn infants (35 or more week’s gestation) – summary. Paediatr Child Health. 2007;12(5):401-18. 4. Procianoy RS, Silveira RC, Pereira MR, Ribeiro MA, Grossi SP, Boher MA, et al. Problemas comuns no alojamento conjunto. In: Nader SS, Pereira DN. Atenção integral ao recém-nascido: guia de supervisão de saúde. Porto Alegre: Artmed; 2004. p.105-9. 2. Bhutani VK, Johnson L. Prevenção de hiperbilirrubinemia neonatal grave em lactentes saudáveis com 35 ou mais semanas de gestação: implante de uma abordagem sistemática. J Pediatr. 2007;83(4):289-93. 2. Bhutani VK, Johnson L. Prevenção de hiperbilirrubinemia neonatal grave em lactentes saudáveis com 35 ou mais semanas de gestação: implante de uma abordagem sistemática. J Pediatr. 2007;83(4):289-93. 5. American Academy of Pediatrics; The American College of Obstetricians and Gynecologists. Guidelines for Perinatal Care. 8. ed. Itasca: American Academy of Pediatrics; Washington: The American College of Obstetricians and Gynecologists; 2017. 3. Araújo BF. Manejo da icterícia no recém-nascido. In: Ferreira JP, organizador. Pediatria: diagnóstico e tratamento. Porto Alegre: Artmed; 2005. p.39-45. 3. Araújo BF. Manejo da icterícia no recém-nascido. In: Ferreira JP, organizador. Pediatria: diagnóstico e tratamento. Porto Alegre: Artmed; 2005. p.39-45. 6. Bhutani VK, Johnson L, Sivieri EM. Predictive ability of a presdicharge hour-specific serum bilirrubin for subsquent significant Rev Med UFC. 2020;60(2):11-17. Rev Med UFC. 2020;60(2):11-17. 17 Perfil de bebês ictéricos em UTIN 17. Bertini G, Dani C, Pezzatti M, Rubaltelli FF. Prevention of bilirubin encephalopathy. Biol Neonate. 2001;79(3-4):219-23. hyperbilirrubinemia in health term and near-term newborn. Pediatrics. 1999;103(1):6-14. hyperbilirrubinemia in health term and near-term newborn. Pediatrics. 1999;103(1):6-14. HS, et al. Icterícia como causa de internação neonatal: a experiência em um serviço terciário de Porto Alegre, RS. Revista da AMRIGS. 2009;53(4):361-7. hyperbilirrubinemia in health term and near-term newborn. Pediatrics. 1999;103(1):6-14. HS, et al. Icterícia como causa de internação neonatal: a experiência em um serviço terciário de Porto Alegre, RS. Revista da AMRIGS. 2009;53(4):361-7. 7. American Academy of Pediatrics. Management of hyperbilirrubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics. 2004;114(1):297-316. 13. Marra NB, Nascimento DW, Sousa FL, Paltronieri MR, Guidoni RG, Toledo SF, et al. Prematuridade eletiva e as suas repercussões perinatais nas síndromes hipertensivas da gestação. UNILUS Ensino Pesqui. 2016;13(32):26-32. 8. Watchko JF, Maisels MJ. Jaundice in low birthweight infants: pathobiology and outcome. Arch Dis Child Fetal Neonatal Ed. 2003;88(6):455-8. 14. Sarli YO. Associação entre tempo de clampeamento do cordão umbilical e icterícia neonatal precoce em recém-nascidos a termo [Internet]. São Paulo: Universidade Santo Amaro; 2018 [acesso em: 15 jan 2019]. Disponível em: http://dspace.unisa.br/ handle/123456789/183 9. Almeida MF, Nader PJ, Draque CM. Icterícia neonatal. In: Lopez FA, Campos D Jr, editores. Tratado de Pediatria. 2. ed. São Paulo: Manole; 2010. p. 1515-26. 10. Bhutani VK, Stark AR, Lazzeroni LC, Poland R, Gourley GR, Kazmierczak S, et al. Predischarge screening for severe neonatal hyperbilirubinemia identifies infants who need phototherapy. J Pediatr. 2013;162(3):477-82. 15. Keren R, Luan X, Friedman S, Saddlemire S, Cnaan A, Bhutani VK. A comparison of alternative risk-assessment strategies for predicting significant neonatal hyperbilirrubinemia in term and near- term infants. Pediatrics. 2008;121(1):170-9. 11. Taheri PA, Sadeghi M, Sajjadian N. Severe neonatal hyperbilirubinemia leading to exchange transfusion. Med J Islam Repub Iran. 2014;28(1):64. 16. Maisels MJ, Kring E. Rebound in serum bilirubin level following intensive phototherapy. Arch Pediatr Adolesc Med. 2002;156(7):669-72. 17. Bertini G, Dani C, Pezzatti M, Rubaltelli FF. Prevention of bilirubin encephalopathy. Biol Neonate. 2001;79(3-4):219-23. 12. Enk I, Abegg MP, Alves RJ, Stringhani F, Campos JF, Menezes Como citar: Carvalho RL, Lavor MF. Icterícia neonatal e seus fatores perinatais associados: perfil dos recém-nascidos internados em uma unidade de terapia intensiva neonatal de maternidade de referência terciária no município de Fortaleza – Ceará. Rev Med UFC. 2020 abr-jun;60(2):11-17. Como citar: Carvalho RL, Lavor MF. Icterícia neonatal e seus fatores perinatais associados: perfil dos recém-nascidos internados em uma unidade de terapia intensiva neonatal de maternidade de referência terciária no município de Fortaleza – Ceará. hyperbilirrubinemia in health term and near-term newborn. Pediatrics. 1999;103(1):6-14. Rev Med UFC. 2020 abr-jun;60(2):11-17. Rev Med UFC. 2020;60(2):11-17. Rev Med UFC. 2020;60(2):11-17.
https://openalex.org/W2799773035	https://europepmc.org/articles/pmc5940182?pdf=render	English	null	Fn3 proteins engineered to recognize tumor biomarker mesothelin internalize upon binding	PloS one	2,018	cc-by	11,203	Fn3 proteins engineered to recognize tumor biomarker mesothelin internalize upon Allison R. Sirois1,2, Daniela A. Deny3, Samantha R. Baierl2, Katia S. George3, Sarah J. Moore1,2,4* 1 Molecular and Cellular Biology Program, University of Massachusetts, Amherst, Amherst, Massachusetts, United States of America, 2 Picker Engineering Program, Smith College, Northampton, Massachusetts, United States of America, 3 Department of Biochemistry, Smith College, Northampton, Massachusetts, United States of America, 4 Department of Biological Sciences, Smith College, Northampton, Massachusetts, United States of America 1 Molecular and Cellular Biology Program, University of Massachusetts, Amherst, Amherst, Massachusetts, United States of America, 2 Picker Engineering Program, Smith College, Northampton, Massachusetts, United States of America, 3 Department of Biochemistry, Smith College, Northampton, Massachusetts, United States of America, 4 Department of Biological Sciences, Smith College, Northampton, Massachusetts, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * sjmoore@smith.edu * sjmoore@smith.edu Editor: Ashley Maurice Buckle, Monash University, AUSTRALIA Copyright: © 2018 Sirois et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by National Institutes of Health, National Cancer Institute (www.cancer.gov) grant R15CA198927-01 to S.J. M. A.R.S. was supported by a Smith College Wilens Teaching Fellowship and by a Schultz Foundation Research Fellowship. K.S.G. received a Schultz Foundation Undergraduate Research Fellowship. The funders had no role in study OPEN ACCESS Citation: Sirois AR, Deny DA, Baierl SR, George KS, Moore SJ (2018) Fn3 proteins engineered to recognize tumor biomarker mesothelin internalize upon binding. PLoS ONE 13(5): e0197029. https:// doi.org/10.1371/journal.pone.0197029 Editor: Ashley Maurice Buckle, Monash University, AUSTRALIA RESEARCH ARTICLE Abstract Mesothelin is a cell surface protein that is overexpressed in numerous cancers, including breast, ovarian, lung, liver, and pancreatic tumors. Aberrant expression of mesothelin has been shown to promote tumor progression and metastasis through interaction with estab- lished tumor biomarker CA125. Therefore, molecules that specifically bind to mesothelin have potential therapeutic and diagnostic applications. However, no mesothelin-targeting molecules are currently approved for routine clinical use. While antibodies that target mesothelin are in development, some clinical applications may require a targeting molecule with an alternative protein fold. For example, non-antibody proteins are more suitable for molecular imaging and may facilitate diverse chemical conjugation strategies to create drug delivery complexes. In this work, we engineered variants of the fibronectin type III domain (Fn3) non-antibody protein scaffold to bind to mesothelin with high affinity, using directed evolution and yeast surface display. Lead engineered Fn3 variants were solubly produced and purified from bacterial culture at high yield. Upon specific binding to mesothelin on human cancer cell lines, the engineered Fn3 proteins internalized and co-localized to early endosomes. To our knowledge, this is the first report of non-antibody proteins engineered to bind mesothelin. The results validate that non-antibody proteins can be engineered to bind to tumor biomarker mesothelin, and encourage the continued development of engineered variants for applications such as targeted diagnostics and therapeutics. Engineered mesothelin-binding Fn3 proteins fewer side effects compared to traditional chemotherapies [2]. Currently, over 75 targeted ther- apies are approved for clinical use as essential treatments for a variety of malignancies [3,4]. For many cancers, however, targeted therapeutics are not yet available, and it is imperative to develop targeted therapies for patients who do not currently have this treatment option. Fur- thermore, it has been recognized that a targeted therapy is only effective when a patient’s tumor expresses the molecular target; therefore, companion diagnostics, including molecular imaging agents, are a critical component for developing targeted therapies [5]. design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Mesothelin (MSLN) is a cell surface protein shown to be overexpressed in many ovarian [6–8], breast [8–10], pancreatic [11–14], liver [15], and lung [16–18] tumors, among others [19], with limited expression in healthy tissues [20]. MSLN has been shown to bind with estab- lished cell surface tumor marker MUC16, also known as CA125, leading to increased tumor cell proliferation and metastasis [8,12,21]. Promising results from ongoing efforts in pre-clini- cal and clinical trials to target MSLN with antibody and antibody derivatives for therapy dem- onstrate the promise of MSLN-targeting methods [16,17,22]. However, no MSLN-targeting agents have thus far received approval from the US Food and Drug Administration (FDA). Directed evolution by yeast surface display (YSD) has been used extensively in protein engi- neering to improve the molecular recognition, biophysical, and catalytic properties of target proteins [23–25]. Directed evolution relies on the generation of mutant libraries followed by identification of mutants with improvements in a desired phenotype by high-throughput screening and selection. The YSD platform offers unique advantages over other directed evolu- tion display formats, including the ability to incorporate post-translational modifications such as glycosylation and disulfide bonds, eukaryotic protein quality control processes, and compat- ibility with fluorescent-activated cell sorting (FACS) for quantitative discrimination between protein variants. YSD has been used for a wide range of protein classes for a variety of applica- tions, including affinity maturation [26,27], improving thermal stability [28], selecting against cell-based targets [29–31], and epitope mapping [32,33]. While antibodies are widely used for a variety of research and clinical indications, non-anti- body protein scaffolds are being developed for research, biotechnology, and medical applica- tions where the inherent properties of antibodies may be limiting. Introduction In recent years, the focus in cancer drug development has shifted from relatively non-specific cytotoxic agents, to selective, rationally designed, and mechanism-based therapies [1]. Tar- geted cancer compounds, which are designed to inhibit specific molecular targets or molecular pathways critical for tumor growth and maintenance, are associated with greater efficacy and 1 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 (C) Fn3 proteins that bind cell surface protein MSLN have numerous potential clinical applications, such as through diagnostic imaging, internalization for drug delivery, and metastatic reduction by blocking MSLN-MUC16 interactions. Stars represent conjugated imaging or therapeutic molecules. https://doi.org/10.1371/journal.pone.0197029.g001 Fig 1. Approach to engineering Fn3 proteins to recognize tumor biomarker MSLN for diagnostic and therapeutic applications. (A) The tenth domain of human fibronectin type III (Fn3) (PDB 1TTG) is a highly stable protein structure with three loops (BC, DE, and FG) broadly tolerant of mutation to confer novel binding properties. Structure was rendered in PyMOL. (B) We employed a previously developed hydrophilic Fn3 yeast surface display library [56] that incorporates a range of loop lengths and biased amino acid composition to mimic the diversity of naturally occurring antibody complementarity-determining regions. (C) Fn3 proteins that bind cell surface protein MSLN have numerous potential clinical applications, such as through diagnostic imaging, internalization for drug delivery, and metastatic reduction by blocking MSLN-MUC16 interactions. Stars represent conjugated imaging or therapeutic molecules. Fig 1. Approach to engineering Fn3 proteins to recognize tumor biomarker MSLN for diagnostic and therapeutic applications. (A) The tenth domain of human fibronectin type III (Fn3) (PDB 1TTG) is a highly stable protein structure with three loops (BC, DE, and FG) broadly tolerant of mutation to confer novel binding properties. Structure was rendered in PyMOL. (B) We employed a previously developed hydrophilic Fn3 yeast surface display library [56] that incorporates a range of loop lengths and biased amino acid composition to mimic the diversity of naturally occurring antibody complementarity-determining regions. (C) Fn3 proteins that bind cell surface protein MSLN have numerous potential clinical applications, such as through diagnostic imaging, internalization for drug delivery, and metastatic reduction by blocking MSLN-MUC16 interactions. Stars represent conjugated imaging or therapeutic molecules. https://doi.org/10.1371/journal.pone.0197029.g001 vascular endothelial growth factor receptor 2 (VEGFR-2) has advanced to Phase II clinical tri- als, demonstrating the protein scaffold’s promise as a targeted therapeutic [51,52]. While engineered Fn3 clones have high affinity for their targets, some engineered variants have also exhibited oligomeric states or instability [53,54]. Hackel and colleagues demon- strated that an Fn3 YSD library engineered using loop length diversity and recursive mutagen- esis could yield highly stable variants that recognized a variety of targets with high affinity [55]. Woldring et al. For example, oncological molecular imaging allows clinicians to non-invasively obtain information such as a tumor’s molecular behavior and a patient’s response to treatment [34]. An optimal molecular imaging agent should efficiently localize to the tumor, while rapidly clearing from non-target tissues and organs [35]. Unfortunately, because of their large size and long clearance half-life, anti- bodies tend to produce undesirable images with high background signals and low contrast [36]. The complex structure of antibodies also poses many challenges when developing chemi- cal strategies for conjugating polymers or drugs for drug delivery applications, such as in the development of antibody-drug conjugates [37]. Efforts to engineer non-antibody, alternative protein scaffolds for molecular recognition have led to binding affinities and specificities once thought to be unique to antibodies [38–43]. Here, we report the engineering of MSLN-binding proteins based on the non-antibody scaf- fold Fn3, derived from the tenth domain of human fibronectin type III. The hydrophobic core of the immunoglobulin-like fold of Fn3 provides a stable framework structure and high ther- mostability (Tm = 88˚ C), while the solvent exposed loops of Fn3 are amenable to high diversi- fication (Fig 1A) [44,45]. The Fn3 scaffold has shown great versatility for its ability to be engineered to recognize a variety of targets including ubiquitin [44], epidermal growth factor receptor (EGFR) [46], carcinoembryonic antigen (CEA) [47], human Fc gamma receptors [46], and Abelson kinase Src homology 2 (Abl SH2) domain [48]. Further, engineered Fn3 var- iants have recently been used for molecular imaging applications, demonstrating the potential of this scaffold as a molecular diagnostic [49,50]. An Fn3 protein that is an antagonist of PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 2 / 19 Engineered mesothelin-binding Fn3 proteins Fig 1. Approach to engineering Fn3 proteins to recognize tumor biomarker MSLN for diagnostic and therapeutic applications. (A) The tenth domain of human fibronectin type III (Fn3) (PDB 1TTG) is a highly stable protein structure with three loops (BC, DE, and FG) broadly tolerant of mutation to confer novel binding properties. Structure was rendered in PyMOL. (B) We employed a previously developed hydrophilic Fn3 yeast surface display library [56] that incorporates a range of loop lengths and biased amino acid composition to mimic the diversity of naturally occurring antibody complementarity-determining regions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 Reagents and cell lines PBSA buffer was composed of phosphate buffered saline (PBS) and 0.1% bovine serum albu- min (BSA). Escherichia coli (E. coli) XL1-Blue Supercompetent cells and E. coli BL21(DE3) cells were purchased from Agilent Technologies and New England Biolabs, respectively. The Gr2 YSD library (generously provided by B. Hackel, University of Minnesota) was grown in selec- tive SD-CAA media containing 20 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids, 5 g/L casamino acids, 7.4 g/L citric acid monohydrate, 10.4 g/L sodium citrate, pH 4.5. SG-CAA media for yeast induction contained 18 g/L galactose, 2 g/L dextrose, 6.7 g/L yeast nitrogen base without amino acids, 5 g/L casamino acids, 5.4 g/L Na2HPO4, 8.6 g/L NaH2- PO4H2O, pH 6.0. A431/H9 cells (gift of M. Ho, National Cancer Institute, 2016) [59] were cul- tured in RPMI-1640 (Gibco) supplemented with 10% FBS, 1% penicillin-streptomycin and 700 μg/mL Geneticin selective antibiotic (G418) (Thermo Fisher). KB-3-1 cells (gift of M. Got- tesman, National Cancer Institute, 2016) [60], and MCF-7 cells (ATCC #HTB-22, gift of S. Peyton, UMass Amherst, 2017) were cultured in DMEM (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin. Engineered mesothelin-binding Fn3 proteins Here, we report the engineering of Fn3 variants that bind with high affinity to the MSLN tumor cell surface protein, beginning with the naïve fibronectin YSD Gr2 library. The binding interaction of MSLN and MUC16 is facilitated by non-covalent interactions between the many carbohydrate chains decorating the surface of MUC16 and a minimal binding domain of approximately 64 amino acids on MSLN [58]. Therefore, there is no known native polypeptide sequence that recognizes MSLN, necessitating the use of a naïve protein library as an initial point for our studies. To our knowledge, this is the first non-antibody protein engineered to bind MSLN. The engineered Fn3 variants were expressed and purified at high yields (~10 mg/ L) using bacterial culture. Soluble Fn3 variants demonstrated high-affinity binding to tumor cells positive for MSLN expression, and were internalized into tumor cells upon binding. The work described here validates the engineered binding proteins for further development as tar- geted therapeutics and companion molecular imaging agents (Fig 1C). PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 developed a second generation YSD Fn3 library by incorporating amino acid distributions that recapitulated binding antigens found in nature, which they termed the Gr2 library [56] (Fig 1B). The Gr2 library also incorporated Fn3 framework mutations that increased variant hydrophilicity towards the goal of more desirable in vivo biodistribution for molecular imaging applications [57]. Therefore, the Gr2 Fn3 library is as large in diversity as its parent library, and is designed to be a higher quality protein library. The sequence space sampled is biased toward sequences that are likely to be more successful for identifying high affinity binding variants and for applications in molecular imaging. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 3 / 19 Maturation and evolution of mesothelin binders The naïve Gr2 library (2.8 x 109 diversity), in which EBY100 yeast cells were transformed with the pCT surface display vector encoding for Fn3 variants [56], was sorted and affinity matured generally as previously described [61]. Briefly, the induced library was sorted twice by mag- netic bead selection with depletion of non-specific binders using Dynabeads Biotin Binder magnetic beads (Life Technologies). This step served as a negative selection by depleting yeast that displayed Fn3 binders to bare beads or streptavidin. The negative sort was followed by enrichment of specific binding variants by magnetic beads functionalized with biotinylated Fc-tagged recombinant human MSLN (Acro Biosystems #MSN-H826x). The magnetic sorts were followed by a fluorescent-activated cell sorting (FACS) selection for full-length clones using an antibody against the C-terminal c-myc epitope tag (clone 9E10, Life Technologies, 1:50) and a goat anti-mouse phycoerythrin (PE) conjugate (Sigma #P9670, 1:25). Full-length clones were induced and incubated with a chicken anti-c-myc antibody (Gallus Immunotech #ACMYC, 1:330) and the biotinylated Fc-tagged MSLN. To increase the sorting stringency, concentrations of MSLN were decreased over sorting rounds from 300 nM in the first genera- tion sorting to 10 nM by the fourth sort of the second generation library. Cells were washed and incubated with a goat anti-chicken Alexa Fluor 647 (AF647) conjugate (Thermo Fisher #A-21449, 1:250) and either Alexa Fluor 488 (AF488)-conjugated streptavidin (Thermo Fisher PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 4 / 19 Engineered mesothelin-binding Fn3 proteins #S11223, 1:700) to detect the biotin molecules of the biotinylated Fc-tagged MSLN, or a goat anti-human IgG Fc FITC conjugate (Thermo Fisher #A18830, 1:500) to detect the human Fc domain of the biotinylated Fc-tagged MSLN. Alternating between the two sorting detection methods served to minimize the likelihood of engineering Fn3 variants that bound streptavi- din. Cells were washed and double-positive yeast cells were collected on a BD BioSciences FACSAria II. Four iterative rounds of enrichment were performed. Plasmid DNA from the enriched library was recovered using a Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research) following manufacturer’s protocol, transformed into bacteria, and individual clones were sequenced by standard Sanger DNA sequencing methods. Plasmid DNA was subse- quently mutated by error-prone PCR of either the entire Fn3 gene or the paratope loops using nucleotide analogues, 8-oxo-2’-deoxyguanosine-5’-triphosphate (8-oxo-dGTP) (TriLink Bio- technologies) and 2’deoxy-p-nucleoside-5’-triphosphate (dPTP) (TriLink Biotechnologies) [62]. All error prone PCR reactions were conducted using primers previously reported [56]. Binding affinity measurements of yeast surface displayed variants Plasmids for Fn3 variants 1.4.1 and 2.4.1, as well as wild type Fn3 (Fn3 WT), were transformed into EBY100 yeast using the Frozen-EZ Yeast Transformation Kit II (Zymo Research) follow- ing manufacturer’s protocol. Yeast were grown in SD-CAA media at 30˚C and induced with SG-CAA media at 20˚C with aeration. Aliquots of 106 yeast cells were simultaneously labeled with 9E10 mouse anti-c-myc antibody (1:50) and a range of concentrations of either biotiny- lated MSLN-Fc or biotinylated Fc fragment in a total volume of 50 μL PBSA and incubated for 45 minutes with gentle rotation at 23˚C. Cells were washed with PBSA and then incubated with a goat anti-mouse PE (1:25) and streptavidin-Alexa Fluor 488 (1:700) for 20 min with gentle rotation on ice in a total volume of 25 μL PBSA, protected from light. Cells were washed with PBSA, pelleted, and resuspended in PBSA for analysis on an EMD Millipore Guava easy- Cyte flow cytometer. Mean fluorescence intensity for MSLN binding was determined for yeast cells displaying full length protein using InCyte software (EMD Millipore). Data was plotted and fit with a sigmoidal curve using KaleidaGraph software (Synergy). Dissociation constants (KD) were determined as the half-maximal value of the sigmoidal fit for three separate experi- ments for each protein variant, and the mean and standard deviation for the KD values are reported. Maturation and evolution of mesothelin binders Reaction components and cycling conditions were identical to those previously described [61] with the following exceptions: Standard Taq (Mg-free) Reaction Buffer (New England Biolabs) was substituted as the reaction buffer and MgCl2 (New England Biolabs, 1.5mM) was added to each reaction. All error prone PCR reactions were conducted as both 10 and 20 cycle reactions to vary the extent of mutagenesis. Mutated plasmid DNA was then amplified and reintroduced into yeast by electroporation with homologous recombination [61]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 Alexa Fluor-488 dye conjugation Pure, folded Fn3 proteins (1 mg/mL) were incubated with AF488 tetrafluorophenyl ester (Thermo Fisher) in a 0.1 M sodium bicarbonate solution, pH 8.3, at an 8:1 dye/protein molar ratio for 1 hr at 23˚C with rotation and protected from light. The resulting AF488-labeled 1.4.1 protein was purified by extensive buffer exchange with PBS using a 3 kDa centrifugal filter unit. Again, because of likely adsorption onto the membrane of the centrifugal filter unit, AF488-labeled 2.4.1 protein was purified with an alternative method, using fluorescent dye removal columns (Thermo Fisher #22858), according to manufacturer’s protocol. Concentra- tions and degree of labeling (DOL) were determined using UV-Vis spectroscopy, measuring dye absorption at 494 nm (ε = 71,000 cm-1 M-1). Engineered Fn3 protein production and purification Engineered Fn3 genes 1.4.1 and 2.4.1 were cloned into the NheI and BamHI sites of a pET24b (+) expression vector modified to include a C-terminal His6-KGSGK tag [61] (provided by B. Hackel, University of Minnesota) and expressed in BL21(DE3) E. coli. Cultures were grown in LB media at 37˚C to an optical density at 600 nm of 1.0 before induction with 0.5 mM Isopro- pyl-β-D-thiogalactopyranoside (IPTG). Fn3 proteins were induced for 3 h at 30˚C. Cells were harvested by centrifugation for 15 min at 3,200g, and resuspended in lysis buffer (35 mM Na2HPO4dibasic, 15 mM Na2HPO4monobasic, 500 mM NaCl, 5 mM CHAPS, 25 mM imid- azole, 5% glycerol) supplemented with protease inhibitor (cOmplete, Roche). Cells were incu- bated on ice for 30 min, lysed by repeated freezing and thawing, then centrifuged at 12,000g PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 5 / 19 Engineered mesothelin-binding Fn3 proteins for 10 min and passed through a 0.45-micron filter. Fn3 proteins were purified by nickel affin- ity chromatography with Ni-NTA agarose resin (Thermo Fisher). Fn3 variant 1.4.1 was further purified by size exclusion chromatography (SEC) on a Superdex 75 10/300 column (GE Healthcare Life Sciences). Fractions of interest were pooled and concentrated with a centrifu- gal filter unit with a 3 kDa molecular weight cutoff (EMD Millipore). Due to likely nonspecific adsorption onto our SEC column, Fn3 variant 2.4.1 was alternatively further purified by reversed phase high-performance liquid chromatography (HPLC) on a Hypersil ODS C18 col- umn (Thermo Fisher) using a linear gradient of 90% acetonitrile in water containing 0.1% tri- fluoroacetic acid. Fn3 variant 2.4.1 fractions were pooled, lyophilized, and resuspended. All protein samples were analyzed by SDS-PAGE on a BioRad ChemiDoc MP imaging system using Image Lab 6.0 software (BioRad). Binding affinity measurements of soluble Fn3 protein for MSLN-positive tumor cells A431/H9 and MCF-7 cells were cultured to 80% confluency, as described above, and detached by 0.25% trypsin-EDTA (Gibco). Aliquots of 105 cells were washed and pelleted at 200g for 5 min at 4˚C. MSLN expression was detected by a mouse anti-MSLN antibody (clone K1, Abcam, 1:50) and a goat anti-mouse PE conjugate (1:25). Cells were incubated with a range of concentrations of AF488-labeled 1.4.1 and 2.4.1 in a total volume of 25 μL PBSA for 1 h at 23˚C with rotation and protected from light. Cells were washed and pelleted as above, resus- pended with ice cold PBSA, and fluorescence was analyzed using a Guava easyCyte flow cytometer. Mean fluorescence intensities for Fn3 variant binding were determined using InCyte software. Data was plotted and fit with a sigmoidal curve using KaleidaGraph software. Dissociation constants (KD) were determined as the half-maximal value of the sigmoidal fit for three separate experiments, and the mean and standard deviation for the KD are reported. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 MSLN-binding Fn3 proteins engineered using yeast surface display and directed evolution Yeast surface display has been previously shown to be a robust method for engineering pro- teins with improved biophysical, catalytic, and molecular recognition properties [23–25,63]. To engineer MSLN-binding proteins, a naïve Gr2 YSD library of 2.8 x 109 variants was screened. Fn3 expression levels were monitored using an antibody to the terminal c-myc epi- tope tag. Following two rounds of MACS and a FACS sort for full-length expression to elimi- nate truncated protein variants, four iterative rounds of dual-color FACS were performed for binding to MSLN normalized by full length protein expression. An enriched population of var- iants was isolated that demonstrated selective affinity to MSLN, compared to no visible bind- ing in the unsorted naïve library (Fig 2). This enriched population of Fn3 variants was then subjected to a single round of mutagenesis and transformed back into yeast for further enrich- ment and selection as a second generation library. An enriching population of yeast cells dis- playing full length protein that bound MSLN was observed throughout rounds of sorting (Fig 2). We note that our initial efforts to engineer Fn3 variants to bind to a small, 64-amino acid domain of MSLN responsible for binding to MUC16 and using an earlier variation of the Fn3 library were unsuccessful, resulting only in variants that bound to the streptavidin secondary reagent and no engineered variants that bound to the MSLN minimal binding domain. To overcome this challenge, we changed our target reagent from this small domain of MSLN to a full-length extracellular domain of MSLN to provide additional surface topology that Fn3 vari- ants could interact with, and alternated sorting detection methods to only use streptavidin as a reagent in some sort rounds, thereby limiting the likelihood of engineering streptavidin binders. From E. coli transformed with plasmids obtained from the engineered first and second gen- eration libraries of enriched MSLN-binding Fn3 variants, 30 independent clones from each generation were randomly chosen and sequenced. Following four rounds of dual-color FACS sorting of the first generation library, there were 10 unique sequences. Of those sequences, one unique clone dominated, representing 18 of the 30 clones sequenced, which we refer to as clone 1.4.1, denoting the first generation library, with four rounds of sorting by FACS, clone number one. Following four rounds of sorting of the second generation library, a second unique clone, variant 2.4.1, emerged. Imaging flow cytometry KB-3-1, A431/H9, and MCF-7 cells were cultured and harvested as described above. Aliquots of 2.5 x 106 cells were washed, pelleted, and incubated with AF488-labeled 1.4.1 or 2.4.1 (1 μM) in a total volume of 25 μL for 1 hr at either 23˚C or 37˚C with rotation and protected from light. Cells were washed with ice cold PBSA and pelleted as above. Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at 23˚C followed by permeabilization with 0.2% Tween 20 in PBS (PBST) for 20 min at 23˚C. Cells were washed twice and pelleted then incubated with an AF647-conjugated rabbit anti-EEA1 antibody (Abcam #196186, 1:50) in a total volume of 50 μL PBST for 30 min at 23˚C with rotation and protected from light. EEA1 is an early endosomal marker. Cells were washed, pelleted, and resuspended in 100 μL PBSA. Images were acquired on an Amnis ImageStream X Mark II (EMD Millipore) with a 40X PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 6 / 19 Engineered mesothelin-binding Fn3 proteins magnification. Collected data (5000 images) were analyzed with IDEAS 6.2 software (EMD Millipore). A compensation matrix was created using single color controls acquired with the brightfield laser turned off. Cells were gated for focused cells with the Gradient RMS feature and for single cells with the area and aspect ratio. Co-localization quantification was deter- mined by the Bright Detail Similarity (BDS) metric in IDEAS software. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 MSLN-binding Fn3 proteins engineered using yeast surface display and directed evolution Fn3 variants 1.4.1 and 2.4.1 differ only by their FG loop and incorporate a single K63N framework mutation compared to the library wildtype frame- work sequence (Fig 3A). Plasmids for all unique clones were transformed back into EBY100 yeast and their specific binding to 200 nM MSLN was assessed by flow cytometry. Clones 1.4.1 and 2.4.1 had substantially greater binding to MSLN compared to all other variants, and were subsequently selected for further study. To measure the binding affinity of these two clones for soluble MSLN extracellular domain, titration binding assays with the Fn3 variants expressed on the surface of yeast were performed (Fig 3B). Clone 1.4.1 exhibited a binding affinity of KD = 700 ± 300 nM, and clone 2.4.1 exhib- ited a binding affinity of KD = 290 ± 40 nM, while Fn3 WT displayed no specific binding to MSLN. Furthermore, clones 1.4.1 and 2.4.1 exhibited no binding to a biotinylated, Fc fragment PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 7 / 19 Engineered mesothelin-binding Fn3 proteins Fig 2. Directed evolution of a naïve yeast surface display library yielded Fn3 variants that bind soluble MSLN. We started with a naïve yeast surface display library with 2.8 x 109 variants of the Fn3 non-antibody scaffold. The library was sorted for full-length protein expression, detected by an antibody to a terminal c-myc epitope tag, and binding to MSLN using MACS and FACS. Red polygon indicates example cell population collected for further enrichment and analysis. Additional diversity was introduced into the enriched library through a single round of mutagenic PCR and sorting of this second generation library resulted in further enrichment for MSLN binding variants. A double-negative population of yeast cells is characteristic of yeast surface display. https://doi.org/10.1371/journal.pone.0197029.g002 Fig 2. Directed evolution of a naïve yeast surface display library yielded Fn3 variants that bind soluble MSLN. We started with a naïve yeast surface display library with 2.8 x 109 variants of the Fn3 non-antibody scaffold. The library was sorted for full-length protein expression, detected by an antibody to a terminal c-myc epitope tag, and binding to MSLN using MACS and FACS. Red polygon indicates example cell population collected for further enrichment and analysis. Additional diversity was introduced into the enriched library through a single round of mutagenic PCR and sorting of this second generation library resulted in further enrichment for MSLN binding variants. MSLN-binding Fn3 proteins engineered using yeast surface display and directed evolution A double-negative population of yeast cells is characteristic of yeast surface display. Fig 2. Directed evolution of a naïve yeast surface display library yielded Fn3 variants that bind soluble MSLN. We started with a naïve yeast surface display library with 2.8 x 109 variants of the Fn3 non-antibody scaffold. The library was sorted for full-length protein expression, detected by an antibody to a terminal c-myc epitope tag, and binding to MSLN using MACS and FACS. Red polygon indicates example cell population collected for further enrichment and analysis. Additional diversity was introduced into the enriched library through a single round of mutagenic PCR and sorting of this second generation library resulted in further enrichment for MSLN binding variants. A double-negative population of yeast cells is characteristic of yeast surface display. https://doi.org/10.1371/journal.pone.0197029.g002 alone, demonstrating their specific binding interaction with MSLN (Fig 3C). While further rounds of directed evolution to obtain higher affinity clones will be necessary for eventual clin- ical translation, we sought to characterize these two variants to learn more about their interac- tion with tumor cells expressing MSLN, to inform further engineering of Fn3 clones to recognize MSLN. Engineered Fn3 proteins were recombinantly produced To further develop and characterize the engineered Fn3 proteins for future diagnostic and therapeutic applications, lead variants were solubly expressed and purified. Fn3 variants 1.4.1 and 2.4.1 were expressed in bacteria with a C-terminal hexahistidine tag and purified by nickel affinity chromatography and either SEC or HPLC. Chromatograms indicated protein elution at the expected retention times for Fn3 variant 1.4.1 on SEC (Fig 4A) and Fn3 variant 2.4.1 on HPLC (Fig 2B). Analysis by SDS-PAGE confirmed high purity > 99% for 1.4.1 and 2.4.1 and (Fig 4C), with routine yields of ~ 10 mg/L. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 Engineered mesothelin-binding Fn3 proteins Fig 3. Yeast displayed Fn3 variants 1.4.1 and 2.4.1 bound specifically to tumor biomarker MSLN. (A) Two dominant Fn3 variants, 1.4.1 and 2.4.1, were recovered from a first generation and second generation Fn3 library, respectively. (B) Individual clones and Fn3 WT were displayed on the surface of yeast and incubated with a range of concentrations of soluble MSLN. Experimental triplicate data were collected, and the dissociation constant is reported as the mean and standard deviation of the KD values calculated for each replicate. A representative binding curve is shown for each variant. (C) Individual clones were displayed on the surface of yeast and incubated with a range of concentrations of a biotinylated, Fc fragment. Experimental triplicate data were collected. A representative curve is shown for each variant. Fig 3. Yeast displayed Fn3 variants 1.4.1 and 2.4.1 bound specifically to tumor biomarker MSLN. (A) Two dominant Fn3 variants, 1.4.1 and 2.4.1, were recovered from a first generation and second generation Fn3 library, respectively. (B) Individual clones and Fn3 WT were displayed on the surface of yeast and incubated with a range of concentrations of soluble MSLN. Experimental triplicate data were collected, and the dissociation constant is reported as the mean and standard deviation of the KD values calculated for each replicate. A representative binding curve is shown for each variant. (C) Individual clones were displayed on the surface of yeast and incubated with a range of concentrations of a biotinylated, Fc fragment. Experimental triplicate data were collected. A representative curve is shown for each variant. https://doi.org/10.1371/journal.pone.0197029.g003 on the surface of A431/H9 cells compared to the MCF-7 cell line expected to be negative for human MSLN (Fig 5A). Direct equilibrium binding titrations of the engineered 1.4.1 and 2.4.1 Fn3 variants on the A431/H9 and MCF-7 carcinoma cells were performed (Fig 5B). The Fn3 variants were directly conjugated to Alexa Fluor 488 and incubated over a range of concentrations with cells for 1 h at 23˚C and analyzed by flow cytometry. Equilibrium binding constant (KD) values were obtained by fitting plots of AF488-labeled 1.4.1 and AF488-labeled 2.4.1 concentrations versus the mean fluorescence intensity. Consistent with the yeast surface display binding data, Fn3 variant 1.4.1 bound to A431/H9 cells with a binding affinity of KD = 510 ± 90 nM, while Fn3 variant 2.4.1 bound to A431/H9 cells with a binding affinity of KD = 440 ± 30 nM. Neither Fn3 variant displayed binding to the MSLN-negative MCF-7 cell line, with only expected, non-spe- cific binding observed at the highest concentrations analyzed. Soluble engineered Fn3 variants bound tumor cells expressing MSLN We established a tumor cell binding assay to measure the binding affinities of soluble engi- neered Fn3 variants for MSLN-expressing cancer cells. The A431/H9 cell line is an A431 (human epidermoid carcinoma) cell line transfected to stably overexpress MSLN on its surface [16]. MCF-7 is a human breast cancer cell line reported not to express MSLN on its surface [7]. Using an anti-MSLN antibody (clone K1, Abcam), high levels of MSLN were confirmed PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 8 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 Fn3 variants co-localized to early endosomes following binding to MSLN Future application of an engineered Fn3 variant for drug delivery to cancer cells could benefit from the internalization of the targeting molecule upon target binding to effectively deliver a conjugated payload into the cells. Mesothelin has been previously reported to efficiently inter- nalize [64–66]. Using imaging flow cytometry, we sought to assess whether engineered Fn3 variants 1.4.1 and 2.4.1 could be internalized into cancer cells following binding to surface MSLN. The KB-3-1 cell line is a human cervical carcinoma cell line reported to express MSLN on its surface [67]. Using an anti-MSLN antibody (clone K1) and a PE-conjugated secondary PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 9 / 19 Engineered mesothelin-binding Fn3 proteins Fig 4. Production and characterization of selected Fn3 variants. Engineered Fn3 clones 1.4.1 and 2.4.1 were expressed in bacteria with a C-terminal hexahistidine tag and a short peptide tag containing GKSK residues for later bioconjugation chemistry. (A) Fn3 protein 1.4.1 was purified by nickel affinity chromatography followed by SEC, demonstrating desired product with retention time of ~ 42 min. (B) Fn3 protein 2.4.1 was purified by nickel affinity chromatography followed by HPLC, demonstrating desired product with retention time of ~30 min. (C) Proteins were purified to high purity > 99% as analyzed by SDS-PAGE. Yields of Fn3 protein production were routinely ~ 10 mg/L. https://doi.org/10.1371/journal.pone.0197029.g004 Fig 4. Production and characterization of selected Fn3 variants. Engineered Fn3 clones 1.4.1 and 2.4.1 were d i b i i h C i l h hi idi d h id i i GKSK id f l Fig 4. Production and characterization of selected Fn3 variants. Engineered Fn3 clones 1.4.1 and 2.4.1 were expressed in bacteria with a C-terminal hexahistidine tag and a short peptide tag containing GKSK residues for later bioconjugation chemistry. (A) Fn3 protein 1.4.1 was purified by nickel affinity chromatography followed by SEC, demonstrating desired product with retention time of ~ 42 min. (B) Fn3 protein 2.4.1 was purified by nickel affinity chromatography followed by HPLC, demonstrating desired product with retention time of ~30 min. (C) Proteins were purified to high purity > 99% as analyzed by SDS-PAGE. Yields of Fn3 protein production were routinely ~ 10 mg/L. https://doi.org/10.1371/journal.pone.0197029.g004 https://doi.org/10.1371/journal.pone.0197029.g004 antibody, MSLN was confirmed on the surface of KB-3-1 cells while no MSLN expression was detected on the surface of the MCF-7 cell line (Fig 6A and 6B). PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 Fn3 variants co-localized to early endosomes following binding to MSLN ( g ) KB-3-1 cells and MCF-7 cells were incubated with either AF488-labeled 1.4.1 (Fig 6C) or 2.4.1 (Fig 6D) at 37˚C and then fixed and permeabilized before incubation with an AF647-conjugated antibody against the early endosomal marker, EEA1. Co-localization of EEA1 antibody and engineered variants was quantified using the Bright Detail Similarity (BDS) metric, which uses a modified Pearson’s correlation coefficient to quantify the degree of similarity between the AF488-labeled 1.4.1 or 2.4.1 image and the AF647-EEA1 endoso- mal image. Cells with increased AF488-labeled 1.4.1 or 2.4.1 trafficking to early endosomes have higher similarity values as a result of greater co-localization of the two fluorescent chan- nel signals. Imaging flow cytometry data demonstrated that when KB-3-1 cells were incu- bated with AF488-labeled 1.4.1 (Fig 6C, top) or 2.4.1 (Fig 6D, top), the AF488-labeled 1.4.1 and 2.4.1 was internalized and co-localized with early endosomes with a BDS = 1.31 and 0.919, respectively, for 5000 cells. Further, efficient binding and subsequent internalization is not observed when AF488-labeled 1.4.1 (Fig 6C, bottom) or 2.4.1 (Fig 6D, bottom) is incu- bated with the MSLN-negative MCF-7 cell line. BDS values were not determined for the neg- ative control cell line as this metric requires a substantial population of double positive cells, which was not present for the negative control cell line. In an additional imaging flow cytom- etry study, MSLN was again confirmed on the surface of KB-3-1 and A431/H9 cells, and AF488-labeled 1.4.1 internalized and co-localized with early endosomes when the experi- ment was conducted at 23˚C (S1 Fig). PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 10 / 19 Engineered mesothelin-binding Fn3 proteins Fig 5. Engineered Fn3 protein variants bound cancer cells expressing MSLN. A431/H9 cells, epidermoid carcinoma cells transfected to express high levels of MSLN, and MCF-7 cells, breast cancer cells lacking surface MSLN, were used in all binding assays. (A) Analysis by flow cytometry confirms MSLN presence on the surface of A431/H9 cells as detected by an anti-MLSN antibody. The MCF-7 cell line does not express MSLN. (B) Fn3 variants 1.4.1 and 2.4.1 were isolated and binding to MSLN was measured using equilibrium binding assays. A431/H9 and MCF-7 cells were incubated with a range of concentrations of soluble fluorescently labeled 1.4.1 or 2.4.1. The assays were performed in experimental triplicate. Data from each replicate were fit to a sigmoidal curve, and a KD value was calculated for each replicate. Fn3 variants co-localized to early endosomes following binding to MSLN The KD is reported as the mean +/- standard deviation. A representative binding curve of each clone for both cell lines is shown. https://doi org/10 1371/journal pone 0197029 g005 Fig 5. Engineered Fn3 protein variants bound cancer cells expressing MSLN. A431/H9 cells, ep Fig 5. Engineered Fn3 protein variants bound cancer cells expressing MSLN. A431/H9 cells, epidermoid carcinoma cells transfected to express high levels of MSLN, and MCF-7 cells, breast cancer cells lacking surface MSLN, were used in all binding assays. (A) Analysis by flow cytometry confirms MSLN presence on the surface of A431/H9 cells as detected by an anti-MLSN antibody. The MCF-7 cell line does not express MSLN. (B) Fn3 variants 1.4.1 and 2.4.1 were isolated and binding to MSLN was measured using equilibrium binding assays. A431/H9 and MCF-7 cells were incubated with a range of concentrations of soluble fluorescently labeled 1.4.1 or 2.4.1. The assays were performed in experimental triplicate. Data from each replicate were fit to a sigmoidal curve, and a KD value was calculated for each replicate. The KD is reported as the mean +/- standard deviation. A representative binding curve of each clone for both cell lines is shown. https://doi.org/10.1371/journal.pone.0197029.g005 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 Discussion Mesothelin has broad potential as a novel tumor target for both diagnosis and therapy, yet no MSLN-targeting molecules are currently FDA approved. Thus, there remains critical need for MSLN-targeting therapeutics and for molecular diagnostics that can identify patients who are most likely to respond to such therapies. In this work, we used directed evolution and a yeast surface display library to engineer Fn3 variants that bind specifically to MSLN, for future appli- cation in diagnosis and therapy. Variants 1.4.1 and 2.4.1 demonstrate specific affinity for the MSLN tumor marker present on the surface of tumor cells, and, upon MSLN binding, are internalized and co-localize with early endosomes. Internalization could be valuable for deliv- ery of cytotoxic molecules conjugated to engineered Fn3 variants into tumor cells. The work described here validates our approach for engineering MSLN-binding variants, using yeast surface displayed Fn3 libraries and directed evolution. The results demonstrating specific binding to, and internalization into, a tumor cell line encourage further engineering of higher affinity variants towards clinical applications. To our knowledge, this is the first report of a non-antibody protein engineered to bind MSLN. In an initial protein engineering strategy, we sought to engineer Fn3 variants that were selected to bind to a 64-amino acid domain of MSLN previously reported to be the minimal domain for binding to MUC16 [58]. It was expected that an Fn3 variant that targeted this binding domain would likely block MSLN and MUC16 interactions, enhancing therapeutic activity of such Fn3 variants. MSLN and MUC16 binding has been reported to enhance tumor cell proliferation and metastasis [8,12,21]. This initial protein engineering strategy did not suc- cessfully yield MSLN binding variants, potentially due to insufficient binding topography on the 64-amino acid domain, and, instead, resulted in Fn3 variants that bound the streptavidin secondary reagent. While we had attempted to prevent selecting streptavidin-binding variants PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 11 / 19 Engineered mesothelin-binding Fn3 proteins Fig 6. Engineered Fn3 protein variants 1.4.1 and 2.4.1 localized to early endosomes upon binding MSLN. Analysis by (A) flow cytometry and (B) imaging flow cytometry confirms MSLN presence on the surface of KB-3-1 cells compared to the MSLN-negative MCF-7 cells, as detected by an anti-MSLN antibody. (C, D) KB-3-1 cells (top) internalize AF488-labeled 1.4.1 (C) and AF488-labeled 2.4.1 (D), while MCF-7 cells show no specific binding or internalization (C bottom, D bottom). PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 Discussion for AF488-1.4.1 and AF488-2.4.1, respectively. BDS values are not quantifiable for the negative control cell line, due to insufficient fraction of negative control cell population staining for binding or internalization of engineered protein variants. https://doi.org/10.1371/journal.pone.0197029.g006 https://doi.org/10.1371/journal.pone.0197029.g006 by using negative magnetic sorts, this method is not always adequate to influence the selection towards the desired interaction with target protein. Because naïve libraries are not based on pre-existing binding interactions, such libraries display no initial bias toward a specific target molecule or epitope [68]. We were ultimately successful in engineering Fn3 variants that bound to our target protein by using the full-length extracellular domain of MSLN and by avoiding the use of streptavidin in some FACS rounds. Determining the epitopes on MSLN that the engineered variants bind is of interest to further evaluate therapeutic potential of the Fn3 proteins. Diagnostic molecular imaging is one promising application for non-antibody proteins engi- neered to bind MSLN positive tumors. While antibodies can be engineered to bind to a variety of targets with high affinities, their large size and slow clearance from circulation can often result in low contrast images [69]. Instead, non-antibody scaffolds have been explored and have demonstrated promising results in preclinical and clinical evaluations [70]. Recently, Fn3 proteins engineered to bind EGFR and EphA2 have been shown to identify tumors expressing their respective molecular target in murine molecular imaging models [50,71,72]. The cystine- knot, or knottin, protein scaffold has also been engineered for tumor targeting applications and has shown promise for molecular imaging in pre-clinical studies targeting tumors and tumor vasculature expressing integrins [73–77]. Likewise, affibodies and DARPins engineered to bind human epidermal growth factor 2 (HER2) or EGFR have been used to image tumor xenografts in mice [78–80]. Recently, a novel Gp2 scaffold has been developed for molecular imaging of EGFR [81]. In each of these studies, the imaging agents were proteins engineered to have picomolar to single-digit nanomolar dissociation constants for their targets, and the importance of this high affinity for tumor targeting applications is further supported by theo- retical modeling [82], motivating additional rounds of mutagenesis and directed evolution for our engineered proteins targeting the novel tumor target MSLN. There is also sustained interest around using engineered proteins as drug delivery agents, such as by conjugating cytotoxic molecules or polymeric systems to proteins that recognize a tumor biomarker [83,84]. Discussion Endosomes are detected by an AF647-conjuated antibody recognizing the EAA1 early endosomal marker. Yellow in the merged images indicate co-localization between AF488-1.4.1 or AF488-2.4.1 anti-MSLN engineered proteins (green) and EEA1 (red). Original magnification 40X. Co-localization is quantified by the Bright Detail Similarity (BDS) metric, with values near 1 indicating co-localization. KB-3-1 BDS = 1.31 and 0.919 Fig 6. Engineered Fn3 protein variants 1.4.1 and 2.4.1 localized to early endosomes upon binding MSLN. Analysis by (A) flow cytometry and (B) imaging flow cytometry confirms MSLN presence on the surface of KB-3-1 cells compared to the MSLN-negative MCF-7 cells, as detected by an anti-MSLN antibody. (C, D) KB-3-1 cells (top) internalize AF488-labeled 1.4.1 (C) and AF488-labeled 2.4.1 (D), while MCF-7 cells show no specific binding or internalization (C bottom, D bottom). Endosomes are detected by an AF647-conjuated antibody recognizing the EAA1 early endosomal marker. Yellow in the merged images indicate co-localization between AF488-1.4.1 or AF488-2.4.1 anti-MSLN engineered proteins (green) and EEA1 (red). Original magnification 40X. Co-localization is quantified by the Bright Detail Similarity (BDS) metric, with values near 1 indicating co-localization. KB-3-1 BDS = 1.31 and 0.919 Fig 6. Engineered Fn3 protein variants 1.4.1 and 2.4.1 localized to early endosomes upon binding MSLN. Analysis by (A) flow cytometry and (B) imaging flow cytometry confirms MSLN presence on the surface of KB-3-1 cells compared to the MSLN-negative MCF-7 cells, as detected by an anti-MSLN antibody. (C, D) KB-3-1 cells (top) internalize AF488-labeled 1.4.1 (C) and AF488-labeled 2.4.1 (D), while MCF-7 cells show no specific binding or internalization (C bottom, D bottom). Endosomes are detected by an AF647-conjuated antibody recognizing the EAA1 early endosomal marker. Yellow in the merged images indicate co-localization between AF488-1.4.1 or AF488-2.4.1 anti-MSLN engineered proteins (green) and EEA1 (red). Original magnification 40X. Co-localization is quantified by the Bright Detail Similarity (BDS) metric, with values near 1 indicating co-localization. KB-3-1 BDS = 1.31 and 0.919 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 12 / 19 Engineered mesothelin-binding Fn3 proteins for AF488-1.4.1 and AF488-2.4.1, respectively. BDS values are not quantifiable for the negative control cell line, due to insufficient fraction of negative control cell population staining for binding or internalization of engineered protein variants. for AF488-1.4.1 and AF488-2.4.1, respectively. BDS values are not quantifiable for the negative control cell line, due to insufficient fraction of negative control cell population staining for binding or internalization of engineered protein variants. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 Supporting information S1 Fig. Engineered Fn3 protein variant 1.4.1 localized to early endosomes in KB-3-1 and A431/H9 cells upon binding MSLN. (A) Analysis by imaging flow cytometry confirms MSLN on the surface of KB-3-1 (top) and A431/H9 (bottom) cells as detected by an anti-MSLN anti- body. (B) KB-3-1 (top) and A431/H9 (bottom) cells were incubated with AF488-1.4.1 at 23˚C for 1 hr. Cells were fixed and permeabilized, then incubated with an AF647-conjugated anti- body directed against the early endosomal marker EEA1. Yellow in the merged image indicates co-localization between AF488-1.4.1 anti-MSLN engineered protein (green) and EEA1 (red). Original magnification 40X. Quantification of co-localization for KB-3-1 and A431/H9 as measured by BDS was 0.904 and 0.857, respectively. (PDF) Discussion Current drug delivery strategies, such as antibody-drug conjugates (ADCs), take advantage of the specificity of antibodies to selectively deliver cytotoxic drugs to antigen-expressing cancer cells [85]. ADCs, including Adcentris1 (Seattle Genetics) [86] and Kadcyla1 (Genentech)[87], have received FDA approval for targeted treatment of relapsed Hodgkin Lymphoma and Her-2 positive breast cancer, respectively. ADCs are comprised of a targeting antibody, a stable linker with acid labile bonds, and the cytotoxic payload [88]. Upon antigen recognition and binding, the ADC is internalized via receptor-mediated endocytosis and trafficked through endosomal vesicles to the lysosome [89]. The low pH of the lysosome will trigger degradation of the antibody and hydrolysis of the linker, thereby releasing the drug to exert its cytotoxic effect [85]. Dose-limiting toxicities, however, can limit penetration of ADCs into solid tumors, whereas small non-antibody scaffolds may be advantageous by effi- ciently delivering cytotoxic payloads deep within a tumor while maintaining rapid clearance from circulation [90]. The observed internalization of the MSLN-targeting Fn3 variant is intriguing toward the goal of delivering a payload across the membrane of MSLN-positive tumor cells. Further understanding of the trafficking of engineered proteins that bind MSLN will inform development of anti-MSLN therapeutic strategies. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 13 / 19 Engineered mesothelin-binding Fn3 proteins In summary, we demonstrate that the Fn3 protein scaffold is suitable for engineering target- ing molecules for the underdeveloped tumor target MSLN. To our knowledge, this is the first report of a non-antibody protein engineered to bind MSLN. Our data demonstrating specific binding of the engineered variants to tumor cells positive for MSLN, followed by subsequent internalization of the engineered Fn3 proteins, establishes the potential for further develop- ment of MSLN-targeting Fn3 proteins for a variety of clinically relevant applications in diag- nosis and therapy. Acknowledgments We are extremely grateful to Dr. Benjamin Hackel (University of Minnesota—Twin Cities) for the Gr2 Fn3 library, the Fn3 loop vector, and the pEThk plasmid, and to Dr. Hackel, Dr. Dan- iel Woldring, and Dr. Lawrence Stern (University of Minnesota) for insightful conversations about the Gr2 library. We are grateful to Dr. K. Dane Wittrup (Massachusetts Institute of Technology) for yeast surface display strains. We would like to acknowledge Dr. Mitchell Ho and Dr. Michael Gottesman (National Cancer Institute) and Dr. Shelly Peyton (University of Massachuetts Amherst) for providing the A431/H9, KB-3-1, and MCF-7 cell lines, respectively. We would like to acknowledge Dr. Amy Burnside (University of Massachusetts Amherst, Flow Cytometry Core Facility) for providing expertise in cell sorting. References 1. Arruebo M, Vilaboa N, Sa´ez-Gutierrez B, Lambea J, Tres A, Valladares M, et al. Assessment of the evo- lution of cancer treatment therapies. Cancers (Basel). 2011; 3: 3279–3330. https://doi.org/10.3390/ cancers3033279 PMID: 24212956 2. de Bono JS, Ashworth A. Translating cancer research into targeted therapeutics. Nature. 2010; 467: 543–549. https://doi.org/10.1038/nature09339 PMID: 20882008 3. Huang M, Shen A, Ding J, Geng M. Molecularly targeted cancer therapy: Some lessons from the past decade. Trends Pharmacol Sci. 2014; 35: 41–50. https://doi.org/10.1016/j.tips.2013.11.004 PMID: 24361003 4. Abramson RG. Overview of Targeted Therapies for Cancer. In: My Cancer Genome [Internet]. 2017. https://www.mycancergenome.org/content/molecular-medicine/overview-of-targeted-therapies-for- cancer/ 5. Zieba A, Grannas K, So¨derberg O, Gullberg M, Nilsson M, Landegren U. Molecular tools for companion diagnostics. N Biotechnol. 2012; 29: 634–640. https://doi.org/10.1016/j.nbt.2012.05.004 PMID: 22634023 6. Chang K, Pastan I, Willingham MC. Isolation and characterization of monoclonal antibody, K1, reactive with ovarian cancers and normal mesothelium. Int J Cancer. 1992; 50: 373–381. https://doi.org/10. 1002/ijc.2910500308 PMID: 1735605 7. Chang K, Pastan I. Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. PNAS. 1996; 93: 136–140. https://doi.org/10.1073/pnas.93.1. 136 PMID: 8552591 8. Rump A, Morikawa Y, Minami S, Umesaki N, Takeuchi M, Miyajima A. Binding of Ovarian Cancer Anti- gen Cell Adhesion Binding of Ovarian Cancer Antigen CA125 / MUC16 to Mesothelin Mediates Cell Adhesion. J Biol Chem. 2004; 279: 9190–9198. 9. Tchou J, Wang LC, Selven B, Zhang H, Conejo-Garcia J, Borghaei H, et al. Mesothelin, a novel immu- notherapy target for triple negative breast cancer. Breast Cancer Res Treat. 2012; 133: 799–804. https://doi.org/10.1007/s10549-012-2018-4 PMID: 22418702 10. Uehara N, Matsuoka Y, Tsubura A. Mesothelin promotes anchorage-independent growth and prevents anoikis via extracellular signal-regulated kinase signaling pathway in human breast cancer cells. Mol Cancer Res. 2008; 6: 186–193. https://doi.org/10.1158/1541-7786.MCR-07-0254 PMID: 18245228 11. Shimizu A, Hirono S, Tani M, Kawai M, Okada KI, Miyazawa M, et al. Coexpression of MUC16 and mesothelin is related to the invasion process in pancreatic ductal adenocarcinoma. Cancer Sci. 2012; 103: 739–746. https://doi.org/10.1111/j.1349-7006.2012.02214.x PMID: 22320398 12. Chen S-H, Hung W-C, Wang P, Paul C, Konstantopoulos K. Mesothelin binding to CA125/MUC16 pro- motes pancreatic cancer cell motility and invasion via MMP-7 activation. Sci Rep. 2013; 3: 1870. https:// doi.org/10.1038/srep01870 PMID: 23694968 13. Argani P, Iacobuzio-donahue C, Ryu B, Rosty C, Goggins M, Wilentz RE, et al. Author Contributions Conceptualization: Allison R. Sirois, Daniela A. Deny, Samantha R. Baierl, Katia S. George, Sarah J. Moore. Conceptualization: Allison R. Sirois, Daniela A. Deny, Samantha R. Baierl, Katia S. George, Sarah J. Moore. Formal analysis: Allison R. Sirois, Sarah J. Moore. Funding acquisition: Sarah J. Moore. Funding acquisition: Sarah J. Moore. Investigation: Allison R. Sirois, Daniela A. Deny, Samantha R. Baierl. Methodology: Allison R. Sirois, Daniela A. Deny, Samantha R. Baierl, Katia S. George, Sarah J. Moore. Supervision: Sarah J. Moore. Supervision: Sarah J. Moore. Visualization: Allison R. Sirois, Sarah J. Moore. Writing – original draft: Allison R. Sirois, Sarah J. Moore. Writing – review & editing: Allison R. Sirois, Daniela A. Deny, Samantha R. Baierl, Katia S. George, Sarah J. Moore. 14 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 Engineered mesothelin-binding Fn3 proteins PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 References https://doi.org/10.1038/nbt0697-553 PMID: 9181578 24. Gera N, Hussain M, Rao BM. Protein selection using yeast surface display. Methods. 2013; 60: 15–26. https://doi.org/10.1016/j.ymeth.2012.03.014 PMID: 22465794 25. Cherf GM, Cochran JR. Applications of yeast surface display for protein engineering. Methods Mol Biol. 2015; 1319: 155–175. https://doi.org/10.1007/978-1-4939-2748-7_8 PMID: 26060074 26. Boder ET, Midelfort KS, Wittrup KD. Directed evolution of antibody fragments with monovalent femto- molar antigen-binding affinity. Proc Natl Acad Sci U S A. 2000; 97: 10701–10705. https://doi.org/10. 1073/pnas.170297297 PMID: 10984501 27. Colby DW, Kellog BA, Graff CP, Yeung YA, Swers JS, Wittrup K. Engineering antibody affinity by yeast surface display. Methods Enzymol. 2004; 388: 348–358. https://doi.org/10.1016/S0076-6879(04) 88027-3 PMID: 15289082 28. Traxylmayr MW, Shusta EV. Directed evolution of protein thermal stability using yeast surface display. Methods Mol Biol. 2017; 1575: 45–65. https://doi.org/10.1007/978-1-4939-6857-2_4 PMID: 28255874 29. Xin XW, Shusta EV. The use of scFv-displaying yeast in mammalian cell surface selections. J Immunol Methods. 2005; 304: 30–42. https://doi.org/10.1016/j.jim.2005.05.006 PMID: 16099466 30. Stern LA, Schrack IA, Johnson SM, Deshpande A, Bennett NR, Harasymiw LA, et al. Geometry and expression enhance enrichment of functional yeast-displayed ligands via cell panning. Biotechnol Bioeng. 2016; 113: 2328–2341. https://doi.org/10.1002/bit.26001 PMID: 27144954 31. Stern LA, Csizmar CM, Woldring DR, Wagner CR, Hackel BJ. Titratable avidity reduction enhances affinity discrimination in mammalian cellular selections of yeast-displayed ligands. ACS Comb Sci. 2017; https://doi.org/10.1021/acscombsci.6b00191 PMID: 28322543 32. Chao G, Cochran JR, Dane Wittrup K. Fine epitope mapping of anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display. J Mol Biol. 2004; 342: 539–550. https://doi.org/10.1016/j.jmb.2004.07.053 PMID: 15327953 33. Levy R, Forsyth C, LaPorte S, Geren I, Smith L, Marks J. Fine and domain-level epitope mapping of bot- ulinum neurotoxin type A neutralizing antibodies by yeast surface display. J Mol Biol. 2007; 365: 196– 210. https://doi.org/10.1016/j.jmb.2006.09.084 PMID: 17059824 34. Mankoff DA. A definition of molecular imaging. J Nucl Med. 2007; 48: 18N, 21N. 35. Friedman M, Ståhl S. Engineered affinity proteins for tumour-targeting applications. Biotechnol Appl Biochem. 2009; 53: 1–29. https://doi.org/10.1042/BA20080287 PMID: 19341363 36. Wu AM. Engineered antibodies for molecular imaging of cancer. Methods. 2014; 65: 139–147. https:// doi.org/10.1016/j.ymeth.2013.09.015 PMID: 24091005 37. Gordon MR, Canakci M, Li L, Zhuang J, Osborne B, Thayumanavan S. Field guide to challenges and opportunities in antibody −drug conjugates for chemists. Bioconjug Chem. 2015; 26: 2198–2215. https://doi.org/10.1021/acs.bioconjchem.5b00399 PMID: 26308881 38. Nord K, Gunneriusson E, Ringdahl J, Ståhl S, Uhle´n M, Nygren P. References Mesothelin Is overex- pressed in the vast majority of ductal adenocarcinomas of the pancreas: Identification of a new pancre- atic cancer marker by serial analysis of gene expression (SAGE). Clin Cancer Res. 2001; 7: 3862– 3868. PMID: 11751476 14. Glass JP, Parasher G, Arias-Pulido H, Donohue R, Prossnitz ER, Cerilli LA. Mesothelin and GPR30 staining among a spectrum of pancreatic epithelial neoplasms. Int J Surg Pathol. 2011; 19: 588–596. https://doi.org/10.1177/1066896911409575 PMID: 21632639 15. Yu L, Feng M, Kim H, Phung Y, Kleiner DE, Gores GJ, et al. Mesothelin as a potential therapeutic target in human cholangiocarcinoma. J Cancer. 2010; 1: 141–149. PMID: 20922056 16. Ho M, Feng M, Fisher RJ, Rader C, Pastan I. A novel high-affinity human monoclonal antibody to mesothelin. Int J Cancer. 2011; 128: 2020–2030. https://doi.org/10.1002/ijc.25557 PMID: 20635390 17. Feng Y, Xiao X, Zhu Z, Streaker E, Ho M, Pastan I, et al. A novel human monoclonal antibody that binds with high affinity to mesothelin-expressing cells and kills them by antibody-dependent cell-mediated cytotoxicity. Mol Cancer Ther. 2009; 8: 1113–1118. https://doi.org/10.1158/1535-7163.MCT-08-0945 PMID: 19417159 18. Kachala SS, Bograd AJ, Villena-Vargas J, Suzuki K, Servais EL, Kadota K, et al. Mesothelin overex- pression is a marker of tumor aggressiveness and is associated with reduced recurrence-free and over- all survival in early-stage lung adenocarcinoma. Clin Cancer Res. 2014; 20: 1020–1028. https://doi.org/ 10.1158/1078-0432.CCR-13-1862 PMID: 24334761 19. Thomas A, Chen Y, Steinberg SM, Luo J, Pack S, Abdullaev Z, et al. High mesothelin expression in advanced lung adenocarcinoma is associated with KRAS mutations and a poor prognosis. Oncotarget. 2015; 6: 11694–11703. https://doi.org/10.18632/oncotarget.3429 PMID: 26028668 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 15 / 19 Engineered mesothelin-binding Fn3 proteins 20. Hassan R, Bera T, Pastan I. Mesothelin: A new target for immunotherapy. Clin Cancer Res. 2004; 10: 3937–3942. https://doi.org/10.1158/1078-0432.CCR-03-0801 PMID: 15217923 21. Gubbels JAA, Belisle J, Onda M, Rancourt C, Migneault M, Ho M, et al. Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors. Mol Cancer. 2006; 5: 50. https://doi.org/10.1186/1476-4598-5-50 PMID: 17067392 22. Tang Z, Feng M, Gao W, Phung Y, Chen W, Chaudhary A, et al. A human single-domain antibody elicits potent antitumor activity by targeting an epitope in mesothelin close to the cancer cell surface. Mol Can- cer Ther. 2013; 12: 416–426. https://doi.org/10.1158/1535-7163.MCT-12-0731 PMID: 23371858 23. Boder ET, Wittrup KD. Yeast surface display for screening combinatorial polypeptide libraries. Nat Bio- technol. 1997; 15: 553–557. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 References Binding proteins selected from com- binatorial libraries of an alpha-helical bacterial receptor domain. Nat Biotechnol. 1997; 15: 772–777. https://doi.org/10.1038/nbt0897-772 PMID: 9255793 39. Pham T-N, Koide A, Koide S. A stable single-layer bold beta-sheet without a hydrophobic core. Nat Struct Biol. 1998; 5: 115–119. https://doi.org/10.1038/nsb0298-115 PMID: 9461076 40. Lo¨fblom J, Feldwisch J, Tolmachev V, Carlsson J, Ståhl S, Frejd F. Affibody molecules: Engineered pro- teins for therapeutic, diagnostic and biotechnological applications. FEBS Lett. 2010; 584: 2670–2680. https://doi.org/10.1016/j.febslet.2010.04.014 PMID: 20388508 41. Kolmar H. Natural and engineered cystine knot miniproteins for diagnostic and therapeutic applications. Curr Pharmarceutical Des. 2011; 17: 4329–436. 42. Hausammann S, Vogel M, Hovinga JAK, Lacroix S, Stadler BM, Horn MP. Designed Ankyrin Repeat Proteins: A new approach to mimic complex antigens for diagnostic purposes? PLoS One. 2013; 8: e60688. https://doi.org/10.1371/journal.pone.0060688 PMID: 23626669 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 16 / 19 Engineered mesothelin-binding Fn3 proteins 43. Rasool M, Malik A, Hussain M, Haq K, Butt K, Ashraf M, et al. DARPins bioengineering and its theranos- tic approaches: Emerging trends in protein engineering. Curr Pharmarceutical Des. 2017; 23: 1610– 1615. https://doi.org/10.2174/1381612822666161208121829 PMID: 27928961 44. Koide A, Bailey CW, Huang X, Koide S. The fibronectin type III domain as a scaffold for novel binding proteins. J Mol Biol. 1998; 284: 1141–1151. https://doi.org/10.1006/jmbi.1998.2238 PMID: 9837732 45. Koide S, Koide A, Lipovsˇek D. Target-Binding Proteins Based on the 10th Human Fibronectin Type III Domain (10Fn3). Methods Enzymol. 2012; 503: 135–156. https://doi.org/10.1016/B978-0-12-396962- 0.00006-9 PMID: 22230568 46. Hackel BJ, Ackerman ME, Howland SW, Wittrup KD. Stability and CDR composition biases enrich binder functionality landscapes. J Mol Biol. 2010; 401: 84–96. https://doi.org/10.1016/j.jmb.2010.06. 004 PMID: 20540948 47. Pirie CM, Hackel BJ, Rosenblum MG, Wittrup KD. Convergent potency of internalized gelonin immuno- toxins across varied cell lines, antigens, and targeting moieties. J Biol Chem. 2011; 286: 4165–4172. https://doi.org/10.1074/jbc.M110.186973 PMID: 21138845 48. Wojcik J, Hantschel O, Grebien F, Kaupe I, Bennett KL, Barkinge J, et al. A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain. Nat Struct Mol Biol. 2010; 17: 519–527. https://doi.org/ 10.1038/nsmb.1793 PMID: 20357770 49. Natarajan A, Hackel BJ, Gambhir SS. A novel engineered anti-CD20 tracer enables early time PET imaging in a humanized transgenic mouse model of b-cell non-hodgkins lymphoma. Clin Cancer Res. 2013; 19: 6820–6829. https://doi.org/10.1158/1078-0432.CCR-13-0626 PMID: 24097872 50. Hackel BJ, Kimura RH, Gambhir SS. Use of 64Cu-labeled fibronectin domain with EGFR- overexpres- sing tumor xenograft: Molecular Imaging. Radiology. References 2012; 263: 179–188. https://doi.org/10.1148/ radiol.12111504 PMID: 22344401 51. Getmanova E V, Chen Y, Bloom L, Gokemeijer J, Shamah S, Warikoo V, et al. Antagonists to human and mouse vascular endothelial growth factor receptor 2 generated by directed protein evolution In vitro. Chem Biol. 2006; 13: 549–556. https://doi.org/10.1016/j.chembiol.2005.12.009 PMID: 16720276 52. Bloom L, Calabro V. FN3: a new protein scaffold reaches the clinic. Drug Discov Today. 2009; 14: 949– 955. https://doi.org/10.1016/j.drudis.2009.06.007 PMID: 19576999 53. Parker MH, Chen Y, Danehy F, Dufu K. Antibody mimics based on human fibronectin type three domain engineered for thermostability and high-affinity binding to vascular endothelial growth factor receptor two. Protein Eng Des Sel. 2005; 18: 435–444. https://doi.org/10.1093/protein/gzi050 PMID: 16087651 54. Xu L, Aha P, Gu K, Kuimelis RG, Kurz M, Lam T, et al. Directed evolution of high-affinity antibody mim- ics using mRNA display. Chem Biol. 2002; 9: 933–942. https://doi.org/10.1016/S1074-5521(02)00187- 4 PMID: 12204693 55. Hackel BJ, Kapila A, Wittrup KD. Picomolar affinity fibronectin domains engineered utilizing loop length diversity, recursive mutagenesis, and loop shuffling. J Mol Biol. 2008; 381: 1238–1252. https://doi.org/ 10.1016/j.jmb.2008.06.051 PMID: 18602401 56. Woldring DR, Holec P V, Zhou H, Hackel BJ. High-throughput ligand discovery reveals a sitewise gradi- ent of diversity in broadly evolved hydrophilic fibronectin domains. PLoS One. 2015; 10: 1–29. https:// doi.org/10.1371/journal.pone.0138956 PMID: 26383268 57. Hackel BJ, Sathirachinda A, Gambhir SS. Designed hydrophilic and charge mutations of the fibronectin domain: towards tailored protein biodistribution. Protein. 2012; 25: 639–647. https://doi.org/10.1093/ protein/gzs036 PMID: 22691700 58. Kaneko O, Gong L, Zhang J, Hansen JK, Hassan R, Lee B, et al. A binding domain on mesothelin for CA125/MUC16. J Biol Chem. 2009; 284: 3739–3749. https://doi.org/10.1074/jbc.M806776200 PMID: 19075018 59. Ho M, Hassan R, Zhang J, Wang Q, Onda M, Bera T, et al. Humoral immune response to mesothelin in mesothelioma and ovarian cancer patients. Clin Cancer Res. 2005; 11: 3814–3820. https://doi.org/10. 1158/1078-0432.CCR-04-2304 PMID: 15897581 60. Shen DW, Cardarelli C, Hwang J, Cornwell M, Richert N, Ishii S, et al. Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins. J Biol Chem. 1986; 261: 7762–7770. PMID: 3711108 61. Chen TF, De Picciotto S, Hackel BJ, Wittrup KD. Engineering fibronectin-based binding proteins by yeast surface display. Methods Enzymol. 2013; 523: 303–326. https://doi.org/10.1016/B978-0-12- 394292-0.00014-X PMID: 23422436 62. Zaccolo M, Williams DM, Brown DM, Gherardi E. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 References An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues. J Mol Biol. 1996; 255: 589–603. https:// doi.org/10.1006/jmbi.1996.0049 PMID: 8568899 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 17 / 19 Engineered mesothelin-binding Fn3 proteins 63. Lutz S. Beyond directed evolution-semi-rational protein engineering and design. Curr Opin Biotechnol. 2010; 21: 734–743. https://doi.org/10.1016/j.copbio.2010.08.011 PMID: 20869867 64. Zhang Y, Pastan I. Modulating mesothelin shedding to improve therapy. Oncotarget. 2012; 3: 114–115. https://doi.org/10.18632/oncotarget.445 PMID: 22337812 65. Zhao X-Y, Subramanyam B, Sarapa N, Golfier S, Dinter H. Novel antibody therapeutics targeting mesothelin in solid tumors. Clin Cancer Drugs. 2016; 3: 76–86. https://doi.org/10.2174/ 2212697X03666160218215744 PMID: 27853672 66. Hassan R, Ho M. Mesothelin targeted cancer immunotherapy. Eur J Cancer. 2008; 44: 46–53. https:// doi.org/10.1016/j.ejca.2007.08.028 PMID: 17945478 67. Zhang Y, Xiang L, Hassan R, Pastan I. Immunotoxin and Taxol synergy results from a decrease in shed mesothelin levels in the extracellular space of tumors. PNAS. 2007; 104: 17099–17104. https://doi.org/ 10.1073/pnas.0708101104 PMID: 17940013 68. Ackerman M, Levary D, Tobon G, Hackel B, Orcutt KD. Highly Avid Magnetic Bead Capture: An Effi- cient Selection Method for de novo Protein Engineering Utilizing Yeast Surface Display. Biotechnol Bioeng. 2009; 25: 774–783. 69. Stern LA, Case BC, Hackel BJ. Alternative Non-Antibody Protein Scaffolds for Molecular Imaging of Cancer. Curr Opin Chem Eng. 2013; 2. https://doi.org/10.1016/j.coche.2013.08.009 PMID: 24358455 70. Miao Z, Levi J, Cheng Z. Protein scaffold-based molecular probes for cancer molecular imaging. Amino Acids. 2011; 41: 1037–1047. https://doi.org/10.1007/s00726-010-0503-9 PMID: 20174842 71. Abou-elkacem L, Wilson KE, Johnson SM, Chowdhury SM, Bachawal S. Ultrasound molecular imaging of the breast cancer neovasculature using engineered fibronectin scaffold ligands : A novel class of tar- geted contrast ultrasound agent. Theranostics. 2016; 6: 1740–1752. https://doi.org/10.7150/thno. 15169 PMID: 27570547 72. Park S, Park S, Kim D, Pyo A, Kimura RH, Sathirachinda A, et al. Isolation and characterization of a monobody with a fibronectin domain III scaffold that specifically binds EphA2. PLoS One. 2015; 10: e0132976. https://doi.org/10.1371/journal.pone.0132976 PMID: 26177208 73. Kimura R, Levin A, Cochran F, Cochran JR. Engineered cystine knot peptides that bind alphavbeta3, alphavbeta5, and alpha5beta1 integrins with low-nanomolar affinity. Proteins. 2009; 77: 359–369. https://doi.org/10.1002/prot.22441 PMID: 19452550 74. Kimura RH, Cheng Z, Gambhir SS, Cochran JR. Engineered knottin peptides: a new class of agents for imaging integrin expression in living subjects. Cancer Res. 2009; 69: 2435–2442. https://doi.org/10. 1158/0008-5472.CAN-08-2495 PMID: 19276378 75. References Moore SJ, Hayden Gephart M, Bergen J, Su Y, Rayburn H, Scott M, et al. Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma. PNAS. 2013; 110: 14598–14603. https://doi.org/10.1073/pnas.1311333110 PMID: 23950221 76. Moore SJ, Leung C, Norton H, Cochran JR. Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging. PLoS One. 2013; 8: 260498. https://doi.org/10. 1371/journal.pone.0060498 PMID: 23573262 77. Kimura RH, Teed R, Hackel BJ, Pysz MA, Chuang CZ, Sathirachinda A, et al. Pharmacokinetically sta- bilized cystine knot peptides that bind alpha-v-beta-6 integrin with single-digit nanomolar affinities for detection of pancreatic cancer. Clin Cancer Res. 2012; 18: 839–849. https://doi.org/10.1158/1078- 0432.CCR-11-1116 PMID: 22173551 78. Orlova A, Magnusson M, Eriksson TL, Nilsson M, Larsson B, Ho¨ide´n-Guthenberg I, et al. Tumor imag- ing using a picomolar affinity HER2 binding affibody molecule. Cancer Res. 2006; 66: 4339–4348. https://doi.org/10.1158/0008-5472.CAN-05-3521 PMID: 16618759 79. Tolmachev V, Friedman M, Sandstro¨m M, Eriksson TL, Rosik D, Hodik M, et al. Affibody molecules for epidermal growth factor receptor targeting in vivo: aspects of dimerization and labeling chemistry. J Nucl Med. 2009; 50: 274–283. https://doi.org/10.2967/jnumed.108.055525 PMID: 19164241 80. Zahnd C, Kawe M, Stumpp MT, de Pasquale C, Tamaskovic R, Nagy-Davidescu G, et al. Efficient tumor targeting with high affinity designed ankryin repeat proteins: effects of affinity and molecular size. Cancer Res. 2010; 70: 1595–1605. https://doi.org/10.1158/0008-5472.CAN-09-2724 PMID: 20124480 81. Kruziki MA, Case BA, Chan JY, Zudock EJ, Woldring DR, Douglas Y, et al. 64Cu-labeled Gp2 domain for PET imaging of Epidermal Growth Factor Receptor. Mol Pharm. 2016; 13: 3747–3755. https://doi. org/10.1021/acs.molpharmaceut.6b00538 PMID: 27696863 82. Zhang L, Bhatnagar S, Deschenes E, Thurber GM. Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design. Sci Rep. 2016; 6. https://doi.org/10.1038/srep25424 PMID: 27147293 83. Binz HK, Amstutz P, Pluckthun A. Engineering novel binding proteins from nonimmunoglobulin domains. Nat Biotechnol. 2005; 23: 1257–1268. https://doi.org/10.1038/nbt1127 PMID: 16211069 PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 18 / 19 Engineered mesothelin-binding Fn3 proteins 84. Skerra A. Alternative non-antibody scaffolds for molecular recognition. Curr Opin Biotechnol. 2007; 18: 295–304. https://doi.org/10.1016/j.copbio.2007.04.010 PMID: 17643280 85. Perez HL, Cardarelli PM, Deshpande S, Gangwar S, Schroeder GM, Vite GD, et al. Antibody—drug conjugates: current status and future directions. Drug Discov Today. 2014; 19: 869–881. https://doi.org/ 10.1016/j.drudis.2013.11.004 PMID: 24239727 86. Berger GK, Mcbride A, Lawson S, Royball K, Yun S, Gee K, et al. Brentuximab vedotin for treatment of non-Hodgkin lymphomas: A systematic review. PLOS ONE \| https://doi.org/10.1371/journal.pone.0197029 May 8, 2018 References Crit Rev Oncol Hematol. 2017; 109: 42–50. https://doi. org/10.1016/j.critrevonc.2016.11.009 PMID: 28010897 87. Verma S, Miles D, Gianni L, Krop I, Welslau M, Baselga J, et al. Trastuzumab emtansine for HER2-posi- tive advanced breast cancer. N Engl J Med. 2012; 367: 1783–1791. https://doi.org/10.1056/ NEJMoa1209124 PMID: 23020162 88. Sievers EL, Senter PD. Antibody-drug conjugates in cancer therapy. Annu Rev Med. 2014; 64: 15–29. 89. Schrama D. Antibody targeted drugs as cancer therapeutics. Nat Rev Drug Discov. 2006; 5: 147–159. https://doi.org/10.1038/nrd1957 PMID: 16424916 90. Vazquez-lombardi R, Phan TG, Zimmermann C, Lowe D, Jermutus L, Christ D. Challenges and oppor- tunities for non-antibody scaffold drugs. Drug Discov Today. 2015; 20: 1271–1283. https://doi.org/10. 1016/j.drudis.2015.09.004 PMID: 26360055 19 / 19
https://openalex.org/W4249246480	https://repository.publisso.de/resource/frl:6414015/data	English	null	Beverage consumption among U.S. children aged 0–24 months: National Health And Nutrition Examination Survey (NHANES)	Journal of nutrition & intermediary metabolism	2,017	cc-by	12,388	Carley A. Grimes 1, Ewa A. Szymlek-Gay 1 and Theresa A. Nicklas 2,* 1 Deakin University, Institute for Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Locked Bag 20000, Waurn Ponds, Geelong VIC 3000, Australia; carley.grimes@deakin.edu.au (C.A.G.); ewa.szymlekgay@deakin.edu.au (E.A.S.-G.) 2 Children’s Nutrition Research Center, Baylor College of Medicine, 1100 Bates Ave, Houston, TX 77030, USA * Correspondence: tnickals@bcm.edu; Tel.: +61-3-9244-6017 Received: 17 January 2017; Accepted: 2 March 2017; Published: 13 March 2017 1 Deakin University, Institute for Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Locked Bag 20000, Waurn Ponds, Geelong VIC 3000, Australia; carley.grimes@deakin.edu.au (C.A.G.); ewa.szymlekgay@deakin.edu.au (E.A.S.-G.) 2 Children’s Nutrition Research Center, Baylor College of Medicine, 1100 Bates Ave, Houston, TX 77030, USA * Correspondence: tnickals@bcm.edu; Tel.: +61-3-9244-6017 Received: 17 January 2017; Accepted: 2 March 2017; Published: 13 March 2017 Abstract: Data on beverage consumption patterns in early life are limited. The aim of this study was to describe beverage consumption by sociodemographic characteristics, along with water intake and sources of water among U.S. children aged 0–24 months. Data from 2740 children in the 2005–2012 NHANES were analysed. Food intake was determined via one 24-h dietary recall. Beverages were categorised according to What We Eat In America groups. Poverty–Income ratio was used to deﬁne household income. During infancy (0–5.9 months and 6–11.9 months) infant formulas were the most commonly consumed beverage, 74.1% and 78.6% of children consuming, respectively. Comparatively fewer children, 41.6% and 24.3%, consumed breast milk. In toddlers (12–24 months), the most commonly consumed beverages were plain milk (83.6% of children consuming), water (68.6%), 100% fruit juice (51.8%) and sweetened beverages (31.2%). Non-Hispanic black and Mexican-American children were more likely to consume sweetened beverages, 100% fruit juice and infant formula than Non-Hispanic white children. Children from lower income households were more likely to consume sweetened beverages and 100% fruit juice and less likely to consume breast milk than children from higher income households. Total water intake increased with age and the contribution of water from food and beverage sources was ~20% and ~80% for all children, respectively. Disparities in beverage consumption by race/ethnicity and income level are apparent in early life. Keywords: NHANES; infant; toddler; beverage intake; water intake nutrients nutrients nutrients nutrients Beverage Consumption among U.S. Children Aged 0–24 Months: National Health and Nutrition Examination Survey (NHANES) Carley A. Grimes 1, Ewa A. Szymlek-Gay 1 and Theresa A. Nicklas 2,* 1. Introduction Beverages are an important source of energy and micronutrients during the ﬁrst two years of life [1,2]. Over time, beverage consumption patterns among U.S. children 0–5 years have shifted [3]. In 2001–2006 fewer children drank milk and more children drank 100% fruit juice, compared to earlier periods of 1976–1980; 1988–1994. Although there was no change in the consumption of fruit drinks and soft drinks during this period, the consumption of these beverages was relatively common, consumed by about a third of all children [3]. The American Academy of Pediatrics recommends infants drink breastmilk, or if this is not possible, formula for the ﬁrst year of life and the introduction of 100% fruit juice should be avoided. If 100% fruit juice is introduced this should be after 6–9 months and limited to no more than 4–6 ounces per day (118–177 mL/day). The introduction of sweetened beverages should also be avoided in the ﬁrst two years of life, and toddlers should be encouraged to drink water and milk [4]. There are limited data on beverage intake among U.S. infants and toddlers. Earlier reports come from the 1994–1996, 1998 Continuing Survey of Food Intakes by Individuals (CSFII) [5], the 2002 [2,6] Nutrients 2017, 9, 264; doi:10.3390/nu9030264 www.mdpi.com/journal/nutrients www.mdpi.com/journal/nutrients 2 of 18 Nutrients 2017, 9, 264 and 2008 [7] Feeding Infants and Toddlers Study (FITS) and trend analysis of NHANES data spanning up to 2006 [3]. Some of these reports have excluded participants who were breastfed [3,5]. It is important to understand beverage habits in early life, as this is when food preferences are developed [8]. Furthermore, there is some evidence to suggest that food and beverage habits may track from infancy into early childhood [9–11]. The primary purpose of this paper was to describe beverage consumption among U.S. infants and toddlers by sex, age group, race/ethnicity, and poverty–income ratio. As beverages contribute substantially (about 80%) [12] to daily water intakes we also report on water intake from beverage categories and food sources (i.e., moisture in food) as well as total daily water intake. Information on total water intake and sources of water in the diet is particularly relevant for this age group. Infants are susceptible to dehydration because they have a large surface area to volume ratio, which leads to greater water loss, limited ability to excrete renal solutes and inability to communicate thirst [12]. 1. Introduction Whilst national data on water intake among children aged 4–12 years have been documented [13], no recent information concerning infants and toddlers is available. The ﬁndings from this study will provide information on beverage consumption during the ﬁrst two years of life and can be used to inform the U.S. 2020 Dietary Guidelines for Infants and Toddlers [14]. In addition, understanding sociodemographic differences in beverage patterns in early life is necessary to identify groups at risk of poor beverage choices and support the development of targeted strategies to improve infant feeding practices. 2.2. Measures The study child’s proxy self-reported information on the age, sex and race/ethnic group of the child. Race/ethnic group was deﬁned according to the NHANES categories and included non-Hispanic white (Non-HW), non-Hispanic black (Non-HB), Mexican-American (MA), Non-Hispanic Asian, other Hispanic or other race/mixed race. Due to small numbers in the Non-Hispanic Asian, other Hispanic and other/mixed race categories these participants were grouped for analysis e.g., Non-Hispanic Asians, other Hispanic, other race/mixed race (Non-HA/OH/OR). Proxy-reported household income was used by the NCHS to calculate the poverty–income ratio (PIR). PIR represents the ratio of household income to poverty threshold. A ratio <1.00 indicates household income is below the poverty line threshold, whereas values >1.00 indicates income above the poverty level. Participants were grouped into the following PIR categories <1.0, 1.0–1.99, 2.0–3.49, ≥3.5 which are consistent with those used in previous analyses [13] (e.g., a ratio of 3.5 indicates that income was 350% above the poverty threshold). 2.1. Study Design and Participants This study used data from the U.S. 2005–2012 National Health and Nutrition Examination Survey (NHANES) [15]. The NHANES is a cross-sectional survey which utilizes a complex, multistage, probability sampling design. Approval for the study was granted by the National Center for Health Statistics (NCHS) ethics review board [16] and parental/guardian consent was obtained for all participants. Further details of the methodology are reported elsewhere [17]. This analysis includes participants aged from birth to 23.9 months. A total of 2857 children agreed to participate, this analysis includes those children who completed a 24-h dietary recall which was deemed as reliable by NCHS (n = 2740). Data from four survey cycles (2005–2006, 2007–2008, 2009–2010, and 2011–2012) were combined. As this was a secondary data analysis which lacked personal identiﬁers, this study did not require additional institutional review. 2.3. Dietary Intake Dietary intake was assessed via one face-to-face 24-h dietary recall. Given the age of the participants this was completed by the proxy (i.e., the parent or guardian of the child). Trained 3 of 18 Nutrients 2017, 9, 264 interviewers administered the dietary recall using the United States Department of Agriculture (USDA) Automated Multiple–Pass Method [18]. Dietary recalls were completed across all days of the week and throughout the year, capturing all seasons. Participants with dietary recall data that were deemed unreliable by the NCHS were excluded (n = 51). The What We Eat In America (WWEIA) food group classiﬁcation system was used to classify beverages [19]. In this system beverages are grouped within nine major categories (“milk”, “ﬂavoured milk”, “dairy drinks and substitutes”, “100% juice”, “diet beverages”, “sweetened beverages”, “coffee and tea”, “plain water”, “ﬂavoured or enhanced water”, “baby beverages”, “infant formula” and “breast milk”). A complete list of all foods that fall under each beverage category is provided in Supplementary File S1. The “milk” category includes non-ﬂavoured whole, reduced fat, low fat and non-fat milk varieties. As per the WWEIA “milk” category deﬁnition, milk that was consumed in combination with food (e.g., cereal) has been reported here as a beverage. Of note, the majority (87%) of occurrences of milk consumed were done so as a beverage, with the remaining 13% consumed as an addition to cereal. We have also reported separately on intakes of “whole milk” and “reduced, low or non-fat milk”. All ﬂavoured milks (whole and reduced fat varieties) fall under the category “ﬂavoured milk”. “Dairy drink and substitutes” includes milkshakes and milk substitutes. The “100% juice” category includes 100% fruit and vegetable juices, of note in this analysis only nine children (0.4%) consumed 100% vegetable juice hence this category primarily reﬂects 100% fruit juice consumption. The term “100% fruit juice” will be used throughout the manuscript to describe this category. “Diet beverages” includes diet soft drinks, sports and energy drinks. “Sweetened beverages” includes sweetened soft drinks, fruit drinks, energy drinks and nutritional beverages. “Coffee and tea” includes sweetened and unsweetened, caffeinated and decaffeinated varieties. ”Plain water” includes tap water, bottled water and water consumed as an ingredient. Similar to milk, most instances (77%) of water consumed as an ingredient related to the addition of water to a beverage (e.g., water added to fruit juice) therefore water consumed as an ingredient has been categorised as a beverage. 2.3. Dietary Intake We also report on the contribution of water 4 of 18 Nutrients 2017, 9, 264 from food sources and beverage sources. Water from beverage sources represents the total amount of water that came from beverages as per our deﬁnition of beverages outlined above. Water from food sources represents all other sources, this includes water found in stock/broth that is coded under the WWEIA “Soup” category. 2.4. Statistical Analysis Descriptive statistics (% weighting and standard error (SE) or mean and (SE)) were used to describe the proportion of participants consuming each type of beverage, mean beverage intake and mean intake of water from beverage and food sources. For beverage consumption per capita and per consumer mean intakes are reported. Results are reported by sex, age group (0–5.9 months, 6.0–11.9 months, 12.0–23.9 months), race/ethnic group and PIR level. Chi-square test was used to assess the association between sociodemographic characteristics and the consumption of each beverage type. To further examine between group differences logistic regression was used with Bonferroni adjustment for multiple comparisons. Differences in beverage intake (g/day) across sub-groups were assessed within the whole group (per capita intake) as well as within consumers only. This was done using the Wald F-test to assess overall group difference in mean intake (g/day) for each beverage category; where a signiﬁcant group effect (p < 0.05) was observed an independent T-test with Bonferroni adjustment for multiple comparisons was used to assess differences between each sub-group. All analyses accounted for the complex survey design of NHANES and incorporated a combined 8-year dietary day one sample weight, which accounts for non-response and weekday of the dietary recall [23]. STATA/SE 14 software (StataCorp, College Station, TX, USA) was used to complete analyses. 2.3. Dietary Intake “Flavoured or enhanced water” includes ﬂavoured or carbonated water and enhanced or fortiﬁed water. Baby beverages include products that are speciﬁcally marketed as baby beverage products within the U.S. food supply. This category consists of two sub-categories, “baby juice” and “baby water”. “Baby water” includes puriﬁed and distilled water and in this analysis these beverages were combined with the major “plain water” category. “Baby juice” includes 100% juice products and one fruit drink (40% juice), in this analysis we combined the 100% baby juices with the major “100% fruit juice” category and the fruit drink with the “sweetened beverages” category. With regards to the consumption of breast milk, the proxy only reported the frequency of feeds during the 24-h recall period and no information on the duration or quantity of feed was recorded. To quantify the amount of breast milk consumed we used a method consistent with past research [20,21] Participants who were exclusively breastfed were allocated a standard reference value of 780 mL/day of breast milk if aged 0–5.9 months and 600 mL/day of breast milk if aged 6.0–11.9 months. If the participant was partially breastfed the amount of breast milk allocated was 780 mL/day minus the total amount of “other milks (mL/day)” consumed on the day of the recall if aged 0–5.9 months; or 600 mL/day minus the total amount of “other milks (mL/day)” consumed if aged 6–11.9 months. “Other milks” included infant formula, cow’s/goat’s milk, ﬂavoured milk or soy/rice milk. If the total daily volume of “other milks” exceeded the age speciﬁc daily reference value (i.e., 780 mL or 600 mL), the subject was allocated 89 mL of breast milk per reported feeding occasion. In children aged 12–17.9 months and 18–23.9 months, the total daily amount of breast milk was calculated as 89 mL or 59 mL, respectively, for every reported feeding occasion. The nutrient content of breast milk was obtained from the USDA NNDSR 26 [22]. To calculate nutrient intakes NHANES uses the USDA’s Food and Nutrient Database for Dietary Studies [22], which links to food composition data from the USDA National Nutrient Database for Standard Reference [19]. In this analysis we report on daily intake of water (g/day). This includes water (i.e., moisture) that is inherently found in food (e.g., water found in fruits, vegetables, meat, milk, fruit juice) as well as water consumed as a beverage. 3. Results With regards to infant formula, both MA and Non-HB children were more likely to consume this compared to Non-HW children (Figure 3) and Non-HB children were found to consume greater per capita amounts of infant formula compared to Non-HW (Table 3). Non-HB children were less likely to consume breast milk than all other race/ethnic groups (Figure 3) and average per capita intake was also lower compared to all other race/ethnic groups (Table 3, per capita intake). p y g p ( g ) difference in the per capita amount consumed (Table 3). With regards to infant formula, both MA and Non‐HB children were more likely to consume this compared to Non‐HW children (Figure 3) and Non‐HB children were found to consume greater per capita amounts of infant formula compared to Non‐HW (Table 3). Non‐HB children were less likely to consume breast milk than all other race/ethnic groups (Figure 3) and average per capita intake was also lower compared to all other race/ethnic groups (Table 3, per capita intake). Figure 1. Proportion (%) of children consuming each type of beverage by age group (n = 2740) 1,2,3,4,5. 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥30% [24]; 2 Chi‐square test to assess difference in proportions, * p < 0.01, ** p < 0.001. Where significant, logistic regression with Bonferroni adjustment for multiple comparisons was used to assess differences between sub‐groups; a 0–5.9 months vs. 6.0–11.9 months p < 0.05; b 0–5.9 months vs. 12.0– 23.9 months p < 0.05; c 6.0–11.9 months vs. 12.0–23.9 months p < 0.05; 3 As only n = 1 consumer of milk in 0–5.9 months, sub‐group differences only assessed between 6.0–11.9 months and 12.0–23.9 months; 4 As n = 0 consumers in 0–5.9 months for flavoured milk, dairy drinks and substitutes, diet beverages and flavoured or enhanced water, differences in intake only assessed in 6.0–11.9 months and 12.0– 23.9 months; 5 On the day of the 24‐h dietary recall there were 2544 instances of plain water consumed across 1526 participants. 3. Results Of the 2740 participants, 51.1% were boys and the average age (SE) was 11.6 (0.2) months. The sample was comprised of 54.1% non-Hispanic white (Non-HW), 13.4% non-Hispanic Black (Non-HB), 17.8% Mexican-American (MA) and 14.7% Non-Hispanic Asian, other Hispanic and other/mixed race (Non-HA/OH/OR). There were marked differences in beverage consumption patterns by age group (Figure 1, Table 1). About three quarters of infants 0–5.9 months consumed infant formula, whereas less than half consumed breast milk. Other beverages consumed at this age included plain water and 100% fruit juice. Among infants 6.0–11.9 months, infant formula remained the most commonly consumed beverage (79% consumers) and far fewer children in this age group consumed breast milk (24%). At this age a greater number of children reported consumption of 100% fruit juice (38%) and plain water (61%); and the introduction of milk (11% consumers) and sweetened beverages (6% consumers) was also apparent. Among toddlers 12.0–23.9 months the pattern continued whereby more children consumed 100% fruit juice (57%) and sweetened beverages (32%). The majority (80%) of toddlers consumed milk, with 63% consuming whole milk and 25% consuming reduced, low or non-fat varieties. Overall, intake of most beverages, was greatest in toddlers compared to younger age groups (i.e., milk, ﬂavoured milk, dairy drinks and substitutes, 100% fruit juice, diet beverages, sweetened beverages, coffee and tea, plain water), with the exception of infant formula and breast milk where intake decreased with age (Table 1). Beverage consumption patterns did not differ by boys and girls (Figure 2, Table 2) however there were differences by race/ethnicity (Figure 3, Table 3) and PIR (Figure 4, Table 4). Compared to Non-HW children, all other race/ethnic groups were more likely to consume 100% fruit juice (Figure 3). With regards to per capita intake of 100% fruit juice, Non-HB children consumed about 60 g/day more than Non-HW and MA children (Table 3, per capita intake). While both Non-HB and MA children were more likely to consume sweetened beverages than Non-HW children (Figure 3), the per capita amount of sweetened beverages consumed was only greater in Non-HB children compared to Non-HW (Table 3, per capita intake). Although there was an overall association between plain water consumption and race/ethnicity there were no between-group differences (Figure 3) and no difference 5 of 18 mpared n plain Nutrients 2017, 9, 264 per capita amoun to Non HW (Tab in the per capita amount consumed (Table 3). 3. Results n = 1700 (66.8%) of these instances were consumed in isolation, n = 650 (25.6%) were consumed as an addition to a beverage (e.g., water added to fruit juice), 7.3% instances were combined as an addition to cereal (e.g., dry, instant, rice cereal, baby food) and 0.3% were consumed with other food additions (e.g., added to fruit puree). Figure 1. Proportion (%) of children consuming each type of beverage by age group (n = 2740) 1,2,3,4,5. 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥30% [24]; 2 Chi-square test to assess difference in proportions, * p < 0.01, ** p < 0.001. Where signiﬁcant, logistic regression with Bonferroni adjustment for multiple comparisons was used to assess differences between sub-groups; a 0–5.9 months vs. 6.0–11.9 months p < 0.05; b 0–5.9 months vs. 12.0–23.9 months p < 0.05; c 6.0–11.9 months vs. 12.0–23.9 months p < 0.05; 3 As only n = 1 consumer of milk in 0–5.9 months, sub-group differences only assessed between 6.0–11.9 months and 12.0–23.9 months; 4 As n = 0 consumers in 0–5.9 months for ﬂavoured milk, dairy drinks and substitutes, diet beverages and ﬂavoured or enhanced water, differences in intake only assessed in 6.0–11.9 months and 12.0–23.9 months; 5 On the day of the 24-h dietary recall there were 2544 instances of plain water consumed across 1526 participants. n = 1700 (66.8%) of these instances were consumed in isolation, n = 650 (25.6%) were consumed as an addition to a beverage (e.g., water added to fruit juice), 7.3% instances were combined as an addition to cereal (e.g., dry, instant, rice cereal, baby food) and 0.3% were consumed with other food additions (e.g., added to fruit puree). Figure 1. Proportion (%) of children consuming each type of beverage by age group (n = 2740) 1,2,3,4,5. 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥30% [24]; 2 Chi‐square test to assess difference in proportions, * p < 0.01, ** p < 0.001. Where significant, logistic regression with Bonferroni adjustment for multiple comparisons was used to assess differences between sub‐groups; a 0–5.9 months vs. 6.0–11.9 months p < 0.05; b 0–5.9 months vs. 12.0– 23.9 months p < 0.05; c 6.0–11.9 months vs. 3. Results 12.0–23.9 months p < 0.05; 3 As only n = 1 consumer of milk in 0–5.9 months, sub‐group differences only assessed between 6.0–11.9 months and 12.0–23.9 months; 4 As n = 0 consumers in 0–5.9 months for flavoured milk, dairy drinks and substitutes, diet beverages and flavoured or enhanced water, differences in intake only assessed in 6.0–11.9 months and 12.0– 23.9 months; 5 On the day of the 24‐h dietary recall there were 2544 instances of plain water consumed across 1526 participants. n = 1700 (66.8%) of these instances were consumed in isolation, n = 650 (25.6%) were consumed as an addition to a beverage (e.g., water added to fruit juice), 7.3% instances were combined as an addition to cereal (e.g., dry, instant, rice cereal, baby food) and 0.3% were consumed with other food additions (e.g., added to fruit puree). Figure 1. Proportion (%) of children consuming each type of beverage by age group (n = 2740) 1,2,3,4,5. 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥30% [24]; 2 Chi-square test to assess difference in proportions, * p < 0.01, ** p < 0.001. Where signiﬁcant, logistic regression with Bonferroni adjustment for multiple comparisons was used to assess differences between sub-groups; a 0–5.9 months vs. 6.0–11.9 months p < 0.05; b 0–5.9 months vs. 12.0–23.9 months p < 0.05; c 6.0–11.9 months vs. 12.0–23.9 months p < 0.05; 3 As only n = 1 consumer of milk in 0–5.9 months, sub-group differences only assessed between 6.0–11.9 months and 12.0–23.9 months; 4 As n = 0 consumers in 0–5.9 months for ﬂavoured milk, dairy drinks and substitutes, diet beverages and ﬂavoured or enhanced water, differences in intake only assessed in 6.0–11.9 months and 12.0–23.9 months; 5 On the day of the 24-h dietary recall there were 2544 instances of plain water consumed across 1526 participants. n = 1700 (66.8%) of these instances were consumed in isolation, n = 650 (25.6%) were consumed as an addition to a beverage (e.g., water added to fruit juice), 7.3% instances were combined as an addition to cereal (e.g., dry, instant, rice cereal, baby food) and 0.3% were consumed with other food additions (e.g., added to fruit puree). 6 of 18 Nutrients 2017, 9, 264 Table 1. Beverage consumption by age group (n = 2740). Table 1. Beverage consumption by age group (n = 2740). 3. Results Beverage Category Per Capita Mean Intake ± SE (g/Day) Per Consumer Mean Intake ± SE (g/Day) 0–5.9 Months 6.0–11.9 Months 12.0–23.9 Months p-Value 1 0–5.9 Months 6.0–11.9 Months 12.0–23.9 Months p-Value 1 Milk 2 0 0 46 ± 8 472 ± 14.4 <0.001 7.6 †† 426 ± 41 565 ± 14 <0.001 Milk, whole 2 0 ± 0 35 ± 7 363 ± 12.7 <0.001 7.6 †† 460 ± 48 572 ± 15 0.03 Milk, reduced, low or non-fat 3 0 11 ± 4 109 ± 9.1 <0.001 n/a 325 ± 76 446 ± 26 0.14 3 Flavoured milk 3 0 1 ± 0.3 † 20 ± 4.8 <0.001 n/a 95 ± 29 † 363 ± 59 <0.001 Dairy drinks & substitutes 3 0 4 ± 2 † 21 ± 4.8 0.03 n/a 234 ± 105 †,c 542 ± 79 0.04 100% fruit juice 6 ± 1 a,b 53 ± 4 c 158 ± 10.0 <0.001 99 ± 12 a,b 138 ± 7 c 278 ± 13 <0.001 Diet beverages 3 0 0.7 ± 0.4 † 6 ± 1.3 <0.001 n/a 22 ± 17 † 183 ± 24 <0.001 Sweetened beverages 2 ± 1 †,a,b 10 ± 2 †,c 94 ± 8.0 <0.001 155 ± 74 † 163 ± 23 c 296 ± 20 <0.001 Coffee & tea 1 ± 0.5 †,b 2 ± 1 †,c 15 ± 3.6 <0.001 132 ± 33 77 ± 14 c 223 ± 44 0.01 Plain water 15 ± 2 a,b 87 ± 6 c 215 ± 9.6 <0.001 75 ± 10 a,b 143 ± 10 c 308 ± 10 <0.001 Flavoured or enhanced water 3 0 0 ± 0.4 † 4 ± 2.1 † 0.07 n/a 87 ± 16 236 ± 52 0.01 Infant formulas 618 ± 23 b 615 ± 20 c 38 ± 6.8 <0.001 834 ± 17 b 783 ± 15 c 591 ± 48 <0.001 Breast milk 264 ± 18 a,b 119 ± 11 c 23 ± 4.0 <0.001 635 ± 17 a,b 492 ± 15 c 304 ± 33 <0.001 Total beverage intake 905 ± 10 b 938 ± 13 c 1065 ± 18.3 <0.001 n/a n/a n/a 1 Wald F-test to assess overall group difference in per capita or consumer mean intake (g/day); where signiﬁcant group effect was found, Independent T-test with Bonferroni adjustment for multiple comparisons was used to assess differences between each sub-group. 1 Wald F-test to assess overall group difference in per capita or consumer mean intake (g/day); where signiﬁcant group effect was found, Independent T-test with Bonferroni adjustment for multiple comparisons was used to assess differences between each sub-group. Sub-group differences indicated by: a 0–5.9 months vs. 6.0–11.9 months p < 0.05; b 0–5.9 months vs. 12.0–23.9 months p < 0.05; c 6.0–11.9 months vs. 12.0–23.9 months p < 0.05; 2 As only n = 1 consumer in 0–5.9 months, differences in intake only assessed in 6.0–11.9 months and 12.0–23.9 months; 3 As n = 0 consumers in 0–5.9 months, differences in intake only assessed in 6.0–11.9 months and 12.0–23.9 months; † Data are statistically unreliable, relative standard error ≥30% [24]; †† No SE calculated as only 1 consumer; n/a represent not applicable as not possible to calculate mean intake as there were no consumers of the beverage category. Nonsensical to calculate total beverage intake as the number of consumers varies across each beverage category. 3. Results Those bolded in red are statistically unreliable, relative standard error ≥30% [24]; 2 Chi-square test to assess difference in proportions. No signiﬁcant differences between boys and girls for any beverage category. Figure 2. Proportion (%) of children consuming each type of beverage by sex (n = 2740)1,2; 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥30% [24]; 2 Chi‐square test to assess difference in proportions. No significant differences between boys and girls for any beverage category. Figure 2. Proportion (%) of children consuming each type of beverage by sex (n = 2740)1,2; 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥30% [24]; 2 Chi-square test to assess difference in proportions. No signiﬁcant differences between boys and girls for any beverage category. The proportion of children consuming 100% fruit juice, sweetened beverages, infant formulas and breast milk differed across PIR groups (Figure 4). Children in the lowest income households (i.e., PIR < 1.0) were more likely to consume 100% fruit juice and sweetened beverages; and less likely to consume breast milk than those children living in the highest income households (i.e., PIR ≥ 3.5) (Figure 4). Compared to children from the highest income households (i.e., PIR ≥ 3.5) the per capita amount of 100% fruit juice and sweetened beverages consumed was greater among children from the lowest income households (i.e., PIR < 1.0), whereas the per capita amount of breastmilk consumed was lower (Table 4). The pattern for infant formula was less clear, whereby those children in the lowest household income group (i.e., PIR < 1.0) were more likely to be consumers of formula than those in the second highest household income groups (i.e., PIR 2.0–3.49), but there was no difference with those in the highest household income group (i.e., PIR ≥ 3.5) (Figure 4). With respects to per capita quantities consumed, intakes were greater in the lowest household income group (i.e., PIR < 1.0) compared to the top two household income groups (i.e., PIR 2.0–3.49 and PIR ≥ 3.5) (Table 4). The proportion of children consuming 100% fruit juice, sweetened beverages, infant formulas and breast milk differed across PIR groups (Figure 4). 3. Results Sub-group differences indicated by: a 0–5.9 months vs. 6.0–11.9 months p < 0.05; b 0–5.9 months vs. 12.0–23.9 months p < 0.05; c 6.0–11.9 months vs. 12.0–23.9 months p < 0.05; 2 As only n = 1 consumer in 0–5.9 months, differences in intake only assessed in 6.0–11.9 months and 12.0–23.9 months; 3 As n = 0 consumers in 0–5.9 months, differences in intake only assessed in 6.0–11.9 months and 12.0–23.9 months; † Data are statistically unreliable, relative standard error ≥30% [24]; †† No SE calculated as only 1 consumer; n/a represent not applicable as not possible to calculate mean intake as there were no consumers of the beverage category. Nonsensical to calculate total beverage intake as the number of consumers varies across each beverage category. Per Capita Mean Intake ± SE (g/Day) Per Consumer Mean Intake ± SE (g/Day) 1 Wald F-test to assess overall group difference in per capita or consumer mean intake (g/day); where signiﬁcant group effect was found, Independent T-test with Bonferroni adjustment for multiple comparisons was used to assess differences between each sub-group. Sub-group differences indicated by: a 0–5.9 months vs. 6.0–11.9 months p < 0.05; b 0–5.9 months vs. 12.0–23.9 months p < 0.05; c 6.0–11.9 months vs. 12.0–23.9 months p < 0.05; 2 As only n = 1 consumer in 0–5.9 months, differences in intake only assessed in 6.0–11.9 months and 12.0–23.9 months; 3 As n = 0 consumers in 0–5.9 months, differences in intake only assessed in 6.0–11.9 months and 12.0–23.9 months; † Data are statistically unreliable, relative standard error ≥30% [24]; †† No SE calculated as only 1 consumer; n/a represent not applicable as not possible to calculate mean intake as there were no consumers of the beverage category. Nonsensical to calculate total beverage intake as the number of consumers varies across each beverage category. 7 of 18 7 of 17 Nutrients 2017, 9, 264 Nutrients 2017 9 264 Figure 2. Proportion (%) of children consuming each type of beverage by sex (n = 2740)1,2; 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥30% [24]; 2 Chi‐square test to assess difference in proportions. No significant differences between boys and girls for any beverage category. Figure 2. Proportion (%) of children consuming each type of beverage by sex (n = 2740)1,2; 1 Error bars represent ± 1 SE. 3. Results Children in the lowest income households (i.e., PIR < 1.0) were more likely to consume 100% fruit juice and sweetened beverages; and less likely to consume breast milk than those children living in the highest income households (i.e., PIR ≥3.5) (Figure 4). Compared to children from the highest income households (i.e., PIR ≥3.5) the per capita amount of 100% fruit juice and sweetened beverages consumed was greater among children from the lowest income households (i.e., PIR < 1.0), whereas the per capita amount of breastmilk consumed was lower (Table 4). The pattern for infant formula was less clear, whereby those children in the lowest household income group (i.e., PIR < 1.0) were more likely to be consumers of formula than those in the second highest household income groups (i.e., PIR 2.0–3.49), but there was no difference with those in the highest household income group (i.e., PIR ≥3.5) (Figure 4). With respects to per capita quantities consumed, intakes were greater in the lowest household income group (i.e., PIR < 1.0) compared to the top two household income groups (i.e., PIR 2.0–3.49 and PIR ≥3.5) (Table 4). 8 of 18 Nutrients 2017, 9, 264 Table 2. Beverage consumption by sex (n = 2740). Table 2. Beverage consumption by sex (n = 2740). 3. Results Beverage Category Per Capita Mean Intake ± SE (g/Day) Per Consumer Mean Intake ± SE (g/Day) Boys Girls p-Value 1 Boys Girls p-Value 1 Milk 245 ± 13.2 249 ± 13.2 0.82 571 ± 18 541 ± 16 0.19 Milk, whole 188 ± 11.5 192 ± 11.3 0.83 572 ± 20 558 ± 17 0.57 Milk, reduced, low or non-fat 57 ± 6.7 58 ± 7.2 0.94 475 ± 37 405 ± 30 0.13 Flavoured milk 10 ± 4.1 † 10 ± 2.8 0.96 314 ± 88 383 ± 64 0.53 Dairy drinks & substitutes 11 ± 3.2 † 12 ± 3.8 † 0.72 392 ± 81 598 ± 98 0.13 100% fruit juice 91 ± 6.9 97 ± 7.7 0.57 236 ± 13 235 ± 13 0.95 Diet beverages 2 ± 0.7 † 4 ± 1.4 † 0.17 122 ± 29 216 ± 44 0.16 Sweetened beverages 55 ± 6.3 44 ± 4.4 0.15 302 ± 25 258 ± 24 0.23 Coffee & tea 9 ± 3.3 † 7 ± 1.7 0.66 219 ± 73 173 ± 24 0.56 Plain water 132 ± 8.2 136 ± 7.0 0.70 240 ± 11 † 240 ± 11 0.98 Flavoured or enhanced water 3 ± 2.0 † 1 ± 0.6 † 0.23 224 ± 58 194 ± 76 † 0.76 Infant formulas 335 ± 15.9 322 ± 14.8 0.51 800 ± 17 778 ± 17 0.36 Breast milk 102 ± 9.0 108 ± 8.1 0.69 526 ± 20 526 ± 17 0.99 Total beverage intake 996 ± 13.6 991 ± 14.1 0.79 n/a n/a 1 Independent T-test to assess difference in per capita or consumer mean intake (g/day) by sex; † Data are statistically unreliable, relative standard error ≥30% [24]; n/a represent not applicable as nonsensical to calculate total beverage intake as the number of consumers varies across each beverage category. Per Capita Mean Intake ± SE (g/Day) 1 Independent T-test to assess difference in per capita or consumer mean intake (g/day) by sex; † Data are statistically unreliable, relative standard error ≥30% [24]; n/a represent not applicable as nonsensical to calculate total beverage intake as the number of consumers varies across each beverage category. Table 3. Beverage consumption by race/ethnic group (n = 2740). Table 3. Beverage consumption by race/ethnic group (n = 2740). Table 3. Beverage consumption by race/ethnic group (n 2740). 3. Results Beverage Category Per Capita Mean Intake ± SE (g/Day) Per Consumer Mean Intake ± SE (g/Day) Non-HW Non-HB MA Non-HA/OH/OR p-Value 1 Non-HW Non-HB MA Non-HA/OH/OR p-Value 1 Milk 246 ± 15 210 ± 18 271 ± 15 255 ± 19 0.08 574 ± 20 511 ± 26 563 ± 24 522 ± 30 0.08 Milk, whole 189 ± 13 167 ± 17 210 ± 16 190 ± 21 0.33 573 ± 22 546 ± 36 566 ± 30 554 ± 36 0.86 Milk, reduced, low or non-fat 57 ± 8 43 ± 7 61 ± 8 65 ± 12 0.34 453 ± 38 329 ± 39 b 502 ± 40 412 ± 45 0.02 Flavoured milk 12 ± 4 † 4 ± 2 † 11 ± 4 † 7 ± 4 † 0.16 357 ± 82 344 ± 88 396 ± 119 † 238 ± 29 0.28 Dairy drinks & substitutes 16 ± 4 5 ± 3 † 3 ± 2 † 10 ± 6 † 0.06 491 ± 78 340 ± 82 404 ± 184 † 559 ± 220 † 0.54 100% fruit juice 80 ± 7 a 146 ± 17 b 84 ± 7 111 ± 13 0.001 230 ± 14 292 ± 25 b 189 ± 10 249 ± 23 0.01 Diet beverages 3 ± 1 2 ± 1 † 3 ± 1 † 2 ± 2 † 0.85 197 ± 41 135 ± 38 160 ± 53 † 141 ± 49 † 0.63 Sweetened beverages 38 ± 7 a 73 ± 8 69 ± 11 50 ± 14 0.001 256 ± 37 320 ± 23 262 ± 37 361 ± 76 0.51 Coffee & tea 9.0 ± 3 11 ± 3 † 7 ± 2 6 ± 2 † 0.62 236 ± 71 233 ± 55 149 ± 26 118 ± 23 0.02 Plain water 102 ± 7 122 ± 10 108 ± 7 129 ± 12 0.52 243 ± 12 249 ± 14 208 ± 11 222 ± 14 0.05 Flavoured or enhanced water 4 ± 2 0.3 ± 0.3 † 2 ± 0.8 † 0.1 ± 0.1 † 0.11 226 ± 63 237 †† 186 ± 41 e 46 †† 0.001 Infant formulas 296 ± 17 a 404 ± 25 363 ± 15 338 ± 21 0.003 765 ± 20 a 859 ± 27 b 767 ± 20 832 ± 18 <0.01 Breast milk 126 ± 9 a 48 ± 9 b,c 91 ± 9 99 ± 15 <0.001 535 ± 18 486 ± 31 483 ± 21 558 ± 27 0.19 Total beverage intake 932 ± 17 a 1025 ± 22 1010 ± 18 1008 ± 22 0.01 n/a n/a n/a n/a 1 Wald F-test to assess overall group difference in per capita or consumer mean intake (g/day); where signiﬁcant group effect was found, Independent T-test with Bonferroni adjustment for multiple comparisons was used to assess differences between each sub-group. 3. Results Sub-group differences indicated by: a Non-HW vs. Non-HB p < 0.05; b Non-HB vs. MA p < 0.05; c Non-HB vs. Non-HA/OH/OR p < 0.05; d Non-HW vs. MA p < 0.05; e MA vs. Non-HA/OH/OR p < 0.05; † Data are statistically unreliable, relative standard error ≥30% [24]; †† No SE calculated as only 1 consumer; n/a represent not applicable as nonsensical to calculate total beverage intake as the number of consumers varies across each beverage category. Wald F test to assess overall group difference in per capita or consumer mean intake (g/day); where signiﬁcant group effect was found, Independent T test with Bonferroni adjustment for multiple comparisons was used to assess differences between each sub-group. Sub-group differences indicated by: a Non-HW vs. Non-HB p < 0.05; b Non-HB vs. MA p < 0.05; c Non-HB vs. Non-HA/OH/OR p < 0.05; d Non-HW vs. MA p < 0.05; e MA vs. Non-HA/OH/OR p < 0.05; † Data are statistically unreliable, relative standard error ≥30% [24]; †† No SE calculated as only 1 consumer; n/a represent not applicable as nonsensical to calculate total beverage intake as the number of consumers varies across each beverage category. 9 of 18 Nutrients 2017, 9, 264 Table 4. Beverage consumption by poverty–income ratio (n = 2554) 1,2. Table 4. Beverage consumption by poverty–income ratio (n = 2554) 1,2. 3. Results Beverage Category Per Capita Mean Intake ± SE (g/Day) Per Consumer Mean Intake ± SE (g/Day) PIR < 1.0 PIR1.0–1.99 PIR 2.0–3.49 PIR ≥3.5 p-Value 2 PIR < 1.0 PIR1.0–1.99 PIR2.0–3.49 PIR ≥3.5 p-Value 2 Milk 256 ± 16 231 ± 16 267 ± 25 245 ± 27 0.62 569 ± 22 538 ± 25 556 ± 37 565 ± 30 0.86 Milk, whole 184 ± 12 177 ± 16 200 ± 21 202 ± 24 0.77 592 ± 23 570 ± 29 543 ± 39 569 ± 29 0.70 Milk, reduced, low or non-fat 71 ± 11 54 ± 8 68 ± 16 43 ± 9 0.21 462 ± 50 434 ± 41 513 ± 56 353 ± 42 0.14 Flavoured milk 7 ± 3 11 ± 3 † 16 ± 9 † 7 ± 4 † 0.65 351 ± 105 † 240 ± 43 483 ± 141 368 ± 133 † 0.31 Dairy drinks & substitutes 12 ± 5 † 7 ± 4 † 15 ± 7 † 12 ± 6 † 0.73 531 ± 95 492 ± 89 489 ± 133 485 ± 155 † 0.99 100% fruit juice 108 ± 9 a 118 ± 12 c 99 ± 13 d 54 ± 8 0.001 246 ± 15 249 ± 19 240 ± 25 202 ± 23 0.46 Diet beverages 4 ± 1 3 ± 2 † 5 ± 2 † 2 ± 1 † 0.49 124 ± 29 a 170 ± 59 † 233 ± 63 301 ± 47 0.02 Sweetened beverages 71 ± 8 a 51 ± 10 c 57 ± 16 18 ± 5 <0.001 304 ± 23 a 275 ± 43 305 ± 72 178 ± 26 <0.01 Coffee & tea 13 ± 3 a 5 ± 2 † 14 ± 8 † 2 ± 1 † 0.004 182 ± 29 128 ± 38 † 308 ± 138 † 119 ± 27 † 0.32 Plain water 109 ± 7 96 ± 7 121 ± 12 116 ± 11 0.50 230 ± 11 216 ± 13 e 258 ± 19 234 ± 17 0.33 Flavoured or enhanced water 2 ± 1 † 2.0 ± 1 † 4 ± 4 † 2 ± 0.9 † 0.93 170 ± 62 † 147 ± 34 414 ± 21 d 169 ± 55 † 0.001 Infant formulas 397 ± 21 a,b 359 ± 20 e 252 ± 19 291 ± 29 <0.001 848 ± 20 a,b 817 ± 17 c 755 ± 28 726 ± 31 <0.01 Breast milk 64 ± 7.0 a,b 90 ± 12 126 ± 15 135 ± 17 <0.001 505 ± 26 504 ± 30 584 ± 25 503 ± 27 0.09 Total beverage intake 1042 ± 16 a 973 ± 18 c 975 ± 33 834 ± 19 <0.001 n/a n/a n/a n/a 1 Includes those participants with data for poverty–income ratio; 2 Wald F-test to assess overall group difference in per capita or consumer mean intake (g/day); where signiﬁcant group effect was found, Independent T-test with Bonferroni adjustment for multiple comparisons was used to assess differences between each sub-group. 1 Includes those participants with data for poverty–income ratio; 2 Wald F-test to assess overall group difference in per capita or consumer mean intake (g/day); where signiﬁcant group effect was found, Independent T-test with Bonferroni adjustment for multiple comparisons was used to assess differences between each sub-group. Sub-group differences indicated by: a PIR <1.0 vs. PIR ≥3.5, p < 0.05; b PIR < 1.0 vs. PIR 2.0–3.49, p < 0.05; c PIR 1.00–1.99 vs. PIR ≥3.5, p < 0.05; d PIR 2.00–3.49 vs. PIR ≥3.5, p < 0.05; e PIR 1.00–1.99 vs. PIR 2.0–3.49, p < 0.05; † Data are statistically unreliable, relative standard error ≥30% [24]; †† No SE calculated as only 1 consumer; n/a represent not applicable as nonsensical to calculate total beverage intake as the number of consumers varies across each beverage category. 3. Results Sub-group differences indicated by: a PIR <1.0 vs. PIR ≥3.5, p < 0.05; b PIR < 1.0 vs. PIR 2.0–3.49, p < 0.05; c PIR 1.00–1.99 vs. PIR ≥3.5, p < 0.05; d PIR 2.00–3.49 vs. PIR ≥3.5, p < 0.05; e PIR 1.00–1.99 vs. PIR 2.0–3.49, p < 0.05; † Data are statistically unreliable, relative standard error ≥30% [24]; †† No SE calculated as only 1 consumer; n/a represent not applicable as nonsensical to calculate total beverage intake as the number of consumers varies across each beverage category. Per Capita Mean Intake ± SE (g/Day) Per Consumer Mean Intake ± SE (g/Day) 1 Includes those participants with data for poverty–income ratio; 2 Wald F-test to assess overall group difference in per capita or consumer mean intake (g/day); where signiﬁcant group effect was found, Independent T-test with Bonferroni adjustment for multiple comparisons was used to assess differences between each sub-group. Sub-group differences indicated by: a PIR <1.0 vs. PIR ≥3.5, p < 0.05; b PIR < 1.0 vs. PIR 2.0–3.49, p < 0.05; c PIR 1.00–1.99 vs. PIR ≥3.5, p < 0.05; d PIR 2.00–3.49 vs. PIR ≥3.5, p < 0.05; e PIR 1.00–1.99 vs. PIR 2.0–3.49, p < 0.05; † Data are statistically unreliable, relative standard error ≥30% [24]; †† No SE calculated as only 1 consumer; n/a represent not applicable as nonsensical to calculate total beverage intake as the number of consumers varies across each beverage category. 10 of 18 Nutrients 2017, 9, 264 Figure 3. Proportion (%) of children consuming each type of beverage by race/ethnic group (n = 2740) 1,2. 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥ 30% [24]; 2 Chi‐square test to assess difference in proportions * p < 0.01, ** p < 0.001. Where significant, logistic regression with Bonferroni adjustment for multiple comparisons was used to assess differences between sub‐groups; a Non‐HW vs Non‐HB p < 0 05; b Non‐HW vs MA p < 0 05; c Non‐ Figure 3. Proportion (%) of children consuming each type of beverage by race/ethnic group (n = 2740) 1,2. 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥30% [24]; 2 Chi-square test to assess difference in proportions * p < 0.01, ** p < 0.001. 3. Results Where signiﬁcant, logistic regression with Bonferroni adjustment for multiple comparisons was used to assess differences between sub-groups; a Non-HW vs. Non-HB p < 0.05; b Non-HW vs. MA p < 0.05; c Non-HW vs. Non-HA/OH/OR p < 0.05; d Non-HB vs. MA p < 0.05; e Non-HB vs. Non-HA/OH/OR p < 0.05; f MA vs. Non-HA/OH/OR p < 0.05. Figure 3. Proportion (%) of children consuming each type of beverage by race/ethnic group (n = 2740) 1,2. 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥ 30% [24]; 2 Chi‐square test to assess difference in proportions * p < 0.01, ** p < 0.001. Where significant, logistic regression with Bonferroni adjustment for multiple comparisons was used to assess Figure 3. Proportion (%) of children consuming each type of beverage by race/ethnic group (n = 2740) 1,2. 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥30% [24]; 2 Chi-square test to assess difference in proportions * p < 0.01, ** p < 0.001. Where signiﬁcant, logistic regression with Bonferroni adjustment for multiple comparisons was used to assess differences between sub-groups; a Non-HW vs. Non-HB p < 0.05; b Non-HW vs. MA p < 0.05; c Non-HW vs. Non-HA/OH/OR p < 0.05; d Non-HB vs. MA p < 0.05; e Non-HB vs. Non-HA/OH/OR p < 0.05; f MA vs. Non-HA/OH/OR p < 0.05. 11 of 18 Nutrients 2017, 9, 264 Figure 4. Proportion (%) of children consuming each type of beverage by poverty–income ratio (n = Figure 4. Proportion (%) of children consuming each type of beverage by poverty–income ratio (n = 2554) 1,2,3. 1 Error bars represent ± 1 SE. Those bolded i 2 Figure 4. Proportion (%) of children consuming each type of beverage by poverty–income ratio (n = 2554) 1,2,3. 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥ 30% [24]; 2 Chi‐square test to assess difference in proportions * p < 0.01, ** p < 0.001. Where significant, logistic regression with Bonferroni adjustment for multiple comparisons was used to assess differences between sub‐groups; a PIR < 1 0 vs PIR ≥3 5 p < 0 05; b PIR < 1 0 vs PIR 2 0–3 49 Figure 4. Nutrients 2017, 9, 264 Nutrients 2017, 9, 264 Daily intake of water from beverage and food sources by age group is shown in Figure 5. Total water intake was greater among older children with intakes of 809 ± 8 g/day, 1049 ± 14 g/day and 1278 ± 19 g/day among 0–5.9 months, 6.0–11.9 months and 12–24 months, respectively (p < 0.001). The contribution of daily water from food and beverage sources for each age group was 0–5.9 months: 1.8% from food and 98.2% from beverages; 6.0–11.9 months: 20.6% from food and 79.4% from beverages; 12.0–23.9 months 24.4% from food and 75.6% from beverages. In infants 0–5.9 months and 6.0–11.9 months the principal dietary source of water was infant formulas, whereas for toddlers the major dietary source of water was milk, followed by food sources and plain water (Figure 5). There was no difference in daily water intake among boys and girls, 1114 ± 17 g/day and 1100 ± 15 g/day, respectively (p = 0.56). Likewise, the amount of water from food and beverage sources was similar with about 20% coming from food sources and 80% from beverage sources. This was the same for race/ethnicity, whereby there were no differences in total water intake and a similar contribution came from food and beverage sources. Figure 6 shows the amount of water from food and beverage sources by household income level. Total water intake did differ by household income level; intakes were higher in children from the lowest income households (1115 ± 17 g/day), compared to those children in the highest (1044 ± 23 g/day, p < 0.01). Figure 5. Per capita daily intake of water (g/day) from beverage and food sources by age group (n = 2740) 1. 1 Wald F-test to assess overall group difference in daily mean intake (g/day) of water p < 0.001; Independent T-test with Bonferroni adjustment for multiple comparisons was used to assess differences between each sub-group. Sub-group differences indicated by: a 0–5.9 months vs. 6.0–11.9 months p < 0.05; b 0–5.9 months vs. 12.0–23.9 months p < 0.05; c 6.0–11.9 months vs. 12.0–23.9 months p < 0.05. Figure 5. Per capita daily intake of water (g/day) from beverage and food sources by age group (n = 2740) 1. 3. Results Proportion (%) of children consuming each type of beverage by poverty–income ratio (n = 2554) 1,2,3. 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥30% [24]; 2 Chi-square test to assess difference in proportions * p < 0.01, ** p < 0.001. Where signiﬁcant, logistic regression with Bonferroni adjustment for multiple comparisons was used to assess differences between sub-groups; a PIR < 1.0 vs. PIR ≥3.5, p < 0.05; b PIR < 1.0 vs. PIR 2.0–3.49, p < 0.05; c PIR 1.00–1.99 vs. PIR 2.0–3.49, p < 0.05; d PIR 1.00–1.99 vs. PIR ≥3.5, p < 0.05; e PIR 2.00–3.49 vs. PIR ≥3.5, p < 0.05; 3 Includes those participants with data for PIR. Figure 4. Proportion (%) of children consuming each type of beverage by poverty–income ratio (n = 2554) 1,2,3. 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥ 30% [24]; 2 Chi‐square test to assess difference in proportions * p < 0.01, ** p < 0.001. Where significant, logistic regression with Bonferroni adjustment for multiple comparisons was used to a e diffe e e bet ee ub ou a PIR < 1 0 PIR ≥3 5 < 0 05 b PIR < 1 0 PIR 2 0 3 49 Figure 4. Proportion (%) of children consuming each type of beverage by poverty–income ratio (n = 2554) 1,2,3. 1 Error bars represent ± 1 SE. Those bolded in red are statistically unreliable, relative standard error ≥30% [24]; 2 Chi-square test to assess difference in proportions * p < 0.01, ** p < 0.001. Where signiﬁcant, logistic regression with Bonferroni adjustment for multiple comparisons was used to assess differences between sub-groups; a PIR < 1.0 vs. PIR ≥3.5, p < 0.05; b PIR < 1.0 vs. PIR 2.0–3.49, p < 0.05; c PIR 1.00–1.99 vs. PIR 2.0–3.49, p < 0.05; d PIR 1.00–1.99 vs. PIR ≥3.5, p < 0.05; e PIR 2.00–3.49 vs. PIR ≥3.5, p < 0.05; 3 Includes those participants with data for PIR. 12 of 18 12 of 18 Nutrients 2017, 9, 264 1 Wald F-test to assess overall group difference in daily mean intake (g/day) of water p < 0.001; Independent T-test with Bonferroni adjustment for multiple comparisons was used to assess differences between each sub-group. Sub-group differences indicated by: a 0–5.9 months vs. 6.0–11.9 months p < 0.05; b 0–5.9 months vs. 12.0–23.9 months p < 0.05; c 6.0–11.9 months vs. 12.0–23.9 months p < 0.05. 13 of 18 Nutrients 2017, 9, 264 Figure 6. Per capita daily intake of water (g/day) from beverage and food sources by poverty–Income ratio (n = 2554) 1. 1 Wald F-test to assess overall group difference in daily mean intake (g/day) of water p < 0.01; Independent T-test with Bonferroni adjustment for multiple comparisons was used to assess differences between each sub-group. Sub-group differences indicated by: a PIR < 1.0 vs. PIR ≥3.5, p < 0.05. Figure 6. Per capita daily intake of water (g/day) from beverage and food sources by poverty–Income ratio (n = 2554) 1. 1 Wald F-test to assess overall group difference in daily mean intake (g/day) of water p < 0.01; Independent T-test with Bonferroni adjustment for multiple comparisons was used to assess differences between each sub-group. Sub-group differences indicated by: a PIR < 1.0 vs. PIR ≥3.5, p < 0.05. 4. Discussion This study provides an update on beverage consumption patterns among a nationally representative sample of U.S. infants and toddlers. Whilst some positive consumption patterns were observed, such as plain milk and plain water being the most popular beverage choices among toddlers, some less desirable patterns, such as the tendency for the consumption of infant formula compared to breastmilk and the introduction of sweetened beverages were also noted, some of which were more common among lower socioeconomic and particular race/ethnic sub-groups. During the ﬁrst year of life, the focus of infant feeding relates to exclusive breastfeeding until about 6 months of age, at which point solid foods are introduced to complement breast feeding [4]. Where breastfeeding is not possible, infant formula is recommended [4]. Consistent with national reports for breastfeeding rates [25] the proportion of infants being breastfed on the day of the dietary recall decreased with age from 42% at 0–5.9 months to 24% at 6.0–11.9 months. For those aged 0–5.9 months, rates (42% consumers) are comparable to those reported in the 2008 FITS study (e.g., 42% of 4–5.9 months) yet for those aged 6.0–11.9 months consumption rates (24% consumers) are lower (e.g., 33% 6.0–11.9 months participating in 2008 FITS) [26]. We found breastfeeding differed by race/ethnicity and household income, whereby Non-HB children and those in the lowest income household groups were least likely to breastfed. Following a similar pattern Non-HB children were more likely to consume infant formulas. Barriers to breastfeeding among low income and ethnic minorities relate to lack of access to maternal information to support breastfeeding, literacy and language constraints, lack of partner support, perception that formula feeding is advantageous, early return to work and unsupportive work environments [27,28]. As breastfeeding provides a multitude of short term and long term health beneﬁts to the child and mother [29] it is important that strategies which promote and support breastfeeding are appropriate for all race/ethnic and socioeconomic groups. 1 Consistent with ﬁndings from the 2008 FITS [7] we found the consumption of sweetened beverages was low among infants aged 6–11.9 months (6% consumers) however by 1–2 years almost a third (32%) Nutrients 2017, 9, 264 14 of 18 of toddlers consumed these beverages. Previous reports from a cohort of Australian infants showed tracking of sugar-sweetened beverage consumption from 9–18 months of age [9]. 4. Discussion In the US, by the age of 2–11 years 66% of children reported consumption of sugar-sweetened beverages [30]. It is not known if sweetened beverage consumption tracks over childhood however, it is apparent that for a third of U.S. toddlers consumption of these nutrient-poor beverages begins early in life. Importantly, due to their poor nutritional quality the introduction of sweetened beverages should be discouraged [4]. Instead, healthy beverage options should be encouraged, which include breastmilk or formula during the ﬁrst year of life, followed by the inclusion of water, milk and limited quantities (118–177 mL/day) of 100% fruit juice from age one [4]. Among toddlers it is reassuring to see that the majority were consumers of water and milk. Milk is the top source of both calcium and vitamin D among U.S. toddlers aged 12.0–23.9 months [1]. A recent analysis of NHANES 2009–2012 data indicated that although toddlers of this age are meeting dietary recommendations for calcium, vitamin D may be a ‘shortfall’ nutrient with 74% with intakes, calculated from food and beverage sources only (i.e., no supplements), below the Estimated Average Requirement [31]. The majority (94%) of infants aged 0–5.9 months did not consume 100% fruit juice, whereas consumption was more common among those aged 6–11.9 months (38%) and 12.0–23.9 months (57%). The American Academy of Pediatrics states that the introduction of 100% fruit juice should be avoided until the child is a toddler [4]. However, if 100% fruit juice is introduced it is advised that parents wait until the child is 6–9 months and only be provided in limited quantities of 118–177 mL/day [4] which is equivalent to 123–184 g/day (assuming a juice density of 1.04 g/mL) [32]. Similarly, intake among toddlers should also be limited to 118–177 mL/day [4]. We found the per capita quantity of 100% fruit juice was in agreement with American Academy of Pediatrics recommendations (i.e., 53 g/day and 158 g/day among 6.0–11.9 months and 12.0–23.9 months, respectively). Among consumers only intakes were higher, 163 g/day and 296 g/day, among 6.0–11.9 months and 12.0–23.9 months, respectively, indicating that among those toddlers (57%) consuming 100% fruit juice intakes may be in excess of American Academy of Pediatrics recommendations (Supplementary Table S1). y pp y National trends indicate that among toddlers aged 1–2 years the proportion who consumed 100% fruit juice signiﬁcantly increased between 1999–1994 (41%) and 2001–2006 (63%) [3]. 4. Discussion Whilst limited quantities of 100% fruit juice (118–177 mL/day) can provide some nutritional value to the diets of children aged two years and above [33,34], there are some concerns that higher intake may relate to increased risk of dental caries and the contribution of excess calories to the diet [35]. Previous analyses within this same cohort revealed that among toddlers 100% fruit juice accounted for 6% of daily energy intake, ranking them as the 2nd source of energy [1]. Among older children aged 2–5 years participating in the 1999–2002 NHANES greater intake of 100% fruit juice was associated with higher daily energy intake [36], however there was no relationship with weight status [36]. A recent systematic review among children aged 1–18 years found no association between 100% fruit juice consumption and weight status when total energy intake was controlled for, however in those studies that did not adjust for total energy intake a positive association was reported [37]. In a prospective study of U.S. infants greater intake of 100% fruit juice at one year was associated with higher BMI z-scores at three and seven years of age [11]. This study also found that 100% fruit juice intake during infancy predicted 100% fruit juice intake at both time points in early childhood [11]. In children aged two years and over some studies have shown no association between 100% fruit juice consumption and weight outcomes [38–40] and instead shown that 100% fruit juice provides valuable short fall nutrients in the diets of children. Intake of 100% fruit juice should be monitored during early life. Race/ethnic and socioeconomic disparities in the consumption of 100% fruit juice and sweetened beverages were apparent, whereby children of low income households and those of MA and Non-HB ethnic groups were more likely to be consumers. Similar demographic differences have been reported for 100% fruit juice consumption among U.S. infants [11] and for sweetened beverage consumption among U.S. toddlers [41] and adolescents [42]. Among children, parental modelling and home availability have been shown to be important determinants of soft drink consumption [43]. Given 15 of 18 Nutrients 2017, 9, 264 the dependency of young children on parents for beverage choices, such factors may be important in explaining socioeconomic differences in beverage intake. 4. Discussion Given the well documented race/ethnic disparities that relate to childhood obesity, which begin as early as the preschool years [44], it is essential that strategies and interventions related to early life feeding practices, which include the promotion of healthy beverage options (as previously outlined above), reach all sub-groups of the population. As expected, total daily water intake increased across age groups 809 g/day, 1049 g/day and 1278 g/day among 0–5.9 months, 6.0, 11.9 months and 12.0–23.9 months, respectively, and the contribution of water from food sources also increased (e.g., 1.8%, 21.8% and 24.4%, respectively), reﬂecting the shift to complementary feeding practices. The only demographic variable that was related to total water intake, was household income, with the highest intakes reported in those children from the lowest income households. This may be explained by overall greater intakes of sweetened beverages and infant formula among this group, both of which have a high water content. The Adequate Intake (AI) for total water for infants 0–6 months is 700 g/day [12]. This recommendation is based on the average amount of breastmilk consumed and its corresponding water content. For 7–12 month olds the AI increases to 800 g/day, accounting for water from complementary foods and beverages and for age 1–3 years the AI is 1300 g/day [12]. Thus, reported group average intakes of total water meet or exceed the recommended AIs for each age group in our study, which indicates that the prevalence of inadequate water intake is likely to be low in U.S. infants and toddlers. This study has a number of limitations. Firstly, the use of proxy-reported 24-h dietary recalls to measure food intake is subject to reporting errors. Furthermore, information on the amount of breast milk consumed by breastfed infants was not available and instead established methods [1,20,21] were used to estimate breast milk intake. We grouped beverages according to the WWEIA beverage categories and did not separate beverages which may have been consumed in combination with a food (e.g., milk and cereal). This is likely to have minimal impact on the results as the consumption of beverages with food was relatively infrequent. The race/ethnic group ‘Non-Hispanic Asians’ was only oversampled since 2011–2012, as such the low numbers within this group did not allow for comparisons and instead were combined with the Other Hispanic and Other race/mixed-race groups. 4. Discussion Strengths of this analysis include the use of data from NHANES, which is a nationally representative study which includes the collection of dietary data throughout all seasons of the year. Furthermore, the combination of four survey cycles provided an adequate sample size which enabled comparison of beverage intake across sociodemographic groups. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions In conclusion, the ﬁndings from this study indicate that important sociodemographic differences in beverage consumption patterns are apparent from early on in life. Strategies that seek to improve the quality of beverages consumed by U.S. infants and toddlers are needed, with a focus on encouraging uptake and continuation of breastfeeding during the ﬁrst year of life along with beverage choices which are in line with dietary recommendations for toddlers. Additional longitudinal studies are needed to monitor beverage consumption patterns during early life. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/9/3/264/s1, File S1: WWEIA Beverage List, Table S1: Overview of American Academy of Pediatrics (AAP) Infant and Toddler Feeding Guidelines [4] and intakes of 100% juice and sweetened beverages among U.S infants and toddlers aged 0–23.9 months. Acknowledgments: C.A.G. is currently supported by an Alfred Deakin Postdoctoral Fellowship (Project ID: RM 0000031099) at the time of the work she was supported by a National Heart Foundation of Australia Postdoctoral Fellowship (Award ID: 100155). C.A.G. received funding by the National Heart Foundation of Australia (Collaboration and Exchange Award No.: 100734) to support travel to the U.S. to complete this work. Author Contributions: C.A.G., E.A.S.-G. and T.A.N. developed the research question and data analysis plan, C.A.G. analyzed data, C.A.G. wrote the manuscript, E.A.S.-G. and T.A.N. helped with data interpretation and revisions of the manuscript. All authors read and approved the ﬁnal manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 16 of 18 16 of 18 Nutrients 2017, 9, 264 References 1. Grimes, C.A.; Szymlek-Gay, E.A.; Campbell, K.J.; Nicklas, T.A. Food Sources of Total Energy and Nutrients among U.S. Infants and Toddlers: National Health and Nutrition Examination Survey 2005–2012. Nutrients 2015, 7, 6797–6836. [CrossRef] [PubMed] 1. Grimes, C.A.; Szymlek-Gay, E.A.; Campbell, K.J.; Nicklas, T.A. Food Sources of Total Energy and Nutrients among U.S. Infants and Toddlers: National Health and Nutrition Examination Survey 2005–2012. Nutrients 2015, 7, 6797–6836. [CrossRef] [PubMed] 2. Skinner, J.D.; Ziegler, P.; Ponza, M. Transitions in infants’ and toddlers’ beverage patterns. J. Am. Diet. Assoc. 2004, 104, s45–s50. [CrossRef] [PubMed] 2. Skinner, J.D.; Ziegler, P.; Ponza, M. Transitions in infants’ and toddlers’ beverage patterns. J. Am. Diet. Assoc. 2004, 104, s45–s50. [CrossRef] [PubMed] 3. Fulgoni, V.L.; Quann, E.E. National trends in beverage consumption in children from birth to 5 years: Analysis of NHANES across three decades. Nutr. J. 2012, 11, 92. [CrossRef] [PubMed] 3. Fulgoni, V.L.; Quann, E.E. National trends in beverage consumption in children from birth to 5 years: Analysis of NHANES across three decades. Nutr. J. 2012, 11, 92. [CrossRef] [PubMed] 4. American Academy of Pediatrics. Food and Feeding—Age Speciﬁc Content. Available online: https://www.aap.org/en-us/advocacy-and-policy/aap-health-initiatives/HALF-Implementation- Guide/Age-Speciﬁc-Content/Pages/Age-Speciﬁc-Content.aspx (accessed on 6 October 2016). 4. American Academy of Pediatrics. Food and Feeding—Age Speciﬁc Content. Available online: https://www.aap.org/en-us/advocacy-and-policy/aap-health-initiatives/HALF-Implementation- Guide/Age-Speciﬁc-Content/Pages/Age-Speciﬁc-Content.aspx (accessed on 6 October 2016). 5. Rampersaud, G.C.; Bailey, L.B.; Kauwell, G.P. National survey beverage consumption data for children and adolescents indicate the need to encourage a shift toward more nutritive beverages. J. Am. Diet. Assoc. 2003, 103, 97–100. [CrossRef] [PubMed] 5. Rampersaud, G.C.; Bailey, L.B.; Kauwell, G.P. National survey beverage consumption data for children and adolescents indicate the need to encourage a shift toward more nutritive beverages. J. Am. Diet. Assoc. 2003, 103, 97–100. [CrossRef] [PubMed] 6. Fox, M.K.; Pac, S.; Devaney, B.; Jankowski, L. Feeding Infants and Toddlers Study: What Foods Are Infants and Toddlers Eating? J. Am. Diet. Assoc. 2004, 104, s22–s30. [CrossRef] [PubMed] 7. Siega-Riz, A.M.; Kinlaw, A.; Deming, D.M.; Reidy, K.C. New ﬁndings from the Feeding Infants and Toddlers Study 2008. Nestle Nutr. Workshop Ser. Pediatr. Program 2011, 68, 83–100. [PubMed] 8. Schwartz, C.; Scholtens, P.A.; Lalanne, A.; Weenen, H.; Nicklaus, S. Development of healthy eating habits early in life. Review of recent evidence and selected guidelines. Appetite 2011, 57, 796–807. [CrossRef] [PubMed] 9. Lioret, S.; McNaughton, S.A.; Spence, A.C.; Crawford, D.; Campbell, K.J. Tracking of dietary intakes in early childhood: The Melbourne InFANT Program. Eur. J. Clin. References Sodium and potassium intakes among US inf and preschool children, 2003–2010. Am. J. Clin. Nutr. 2013, 98, 113–122. [CrossRef] [PubMed] 22. U.S. Department of Agriculture, Agricultural Research Service. USDA Food and Nutrient Database for Dietary Studies. Available online: http://www.webcitation.org/6ZVymUPd3 (accessed on 23 March 2015). 23. Centers for Disease Control and Prevention, National Center for Health Statistics. National Health and Nutrition Examination Survey: Analytic Guidelines, 2011–2012. Available online: http://www.webcitation. org/6ZVy7rMoa (accessed on 22 April 2015). 24. National Health and Nutrition Examination Survey: Analytical Guidelines 1999–2010. Available online: http://www.cdc.gov/nchs/data/series/sr_02/sr02_161.pdf (accessed on 9 December 2016). 25. Centers for Disease Control and Prevention. Breastfeeding Report Card United States 2014; Centers for Disease Control and Prevention: Atlanta, GA, USA, 2014. 26. Siega-Riz, A.M.; Deming, D.M.; Reidy, K.C.; Fox, M.K.; Condon, E.; Briefel, R.R. Food consumption patterns of infants and toddlers: Where are we now? J. Am. Diet. Assoc. 2010, 110, S38–S51. [CrossRef] [PubMed] 27. Centers for Disease Control and Prevention. Progress in Increasing Breastfeeding and Reducing Racial/Ethnic Differences—United States, 2000–2008 Births. Morb. Mortal. Wkly. Rep. (MMWR) 2013, 26. Siega-Riz, A.M.; Deming, D.M.; Reidy, K.C.; Fox, M.K.; Condon, E.; Briefel, R.R. Food consumption patterns of infants and toddlers: Where are we now? J. Am. Diet. Assoc. 2010, 110, S38–S51. [CrossRef] [PubMed] 27. Centers for Disease Control and Prevention. Progress in Increasing Breastfeeding and Reducing Racial/Ethnic Differences—United States, 2000–2008 Births. Morb. Mortal. Wkly. Rep. (MMWR) 2013, 62, 77–80. 28. Jones, K.M.; Power, M.L.; Queenan, J.T.; Schulkin, J. Racial and ethnic disparities in breastfeeding. Breastfeed. Med. 2015, 10, 186–196. [CrossRef] [PubMed] 29. American Academy of Pediatrics. Breastfeeding and the Use of Human Milk. Pediatrics 2012, 129, e8 29. American Academy of Pediatrics. Breastfeeding and the Use of Human Milk. Pediatrics 2012, 129, e827–e841. 30. Han, E.; Powell, L.M. Consumption patterns of sugar-sweetened beverages in the United States. J. Acad. Nutr. Diet. 2013, 113, 43–53. [CrossRef] [PubMed] 30. Han, E.; Powell, L.M. Consumption patterns of sugar-sweetened beverages in the United States. J. Acad. Nutr. Diet. 2013, 113, 43–53. [CrossRef] [PubMed] 31. Ahluwalia, N.; Herrick, K.A.; Rossen, L.M.; Rhodes, D.; Kit, B.; Moshfegh, A.; Dodd, K.W. Usual nutrient intakes of US infants and toddlers generally meet or exceed Dietary Reference Intakes: Findings from NHANES 2009–2012. Am. J. Clin. Nutr. 2016, 104, 1167–1174. [CrossRef] [PubMed] 32. Food and Agriculture Organization of the United Nations (FAO). FAO/INFOODS Databases Density Database Version 2.0. Available online: http://www.fao.org/docrep/017/ap815e/ap815e.pdf (accessed on 16 December 2017). 33. References Nutr. 2013, 67, 275–281. [CrossRef] [PubMed] 10. Park, S.; Pan, L.; Sherry, B.; Li, R. The association of sugar-sweetened beverage intake during infancy with sugar-sweetened beverage intake at 6 years of age. Pediatrics 2014, 134, S56–S62. [CrossRef] [PubMed] 11. Sonneville, K.R.; Long, M.W.; Rifas-Shiman, S.L.; Kleinman, K.; Gillman, M.W.; Taveras, E.M. Juice and water intake in infancy and later beverage intake and adiposity: Could juice be a gateway drink? Obesity 2015, 23, 170–176. [CrossRef] [PubMed] 12. Institute of Medicine. Dietary Reference Intakes for Water, Potassium, Sodium, Chloride and Sulfate; The National Academies Press: Washington, DC, USA, 2004. 13. Drewnowski, D.; Rehm, C.D.; Constant, F. Water and beverage consumption among children age 4–13y in the United States: Analyses of 2005–2010 NHANES data. Nutr. J. 2013, 12, 85. [CrossRef] [PubMed] 14. Raiten, D.; Raghavan, R.; Porter, A.; Obbagy, J.E.; Spahn, J. Executive summary: Evaluating the evidence base to support the inclusion of infants and children from birth to 24 mo of age in the Dietary Guidelines for Americans—“The B-24 Project”. Am. J. Clin. Nutr. 2014, 99, 663S–691S. [CrossRef] [PubMed] 15. National Health and Nutrition Examination Survey Data. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention: Hyattsville, MD, USA, (2005–2012). Available online: https: //wwwn.cdc.gov/nchs/nhanes/Default.aspx (accessed on 23 March 2015). 16. Centers for Disease Control and Prevention, National Center for Health Statistics. National Health and Nutrition Examination Survey Questionnaires, Datasets, and Related Documentation. Available online: http://www.webcitation.org/6ZVyAVRTi (accessed on 25 May 2015). 17. Centers for Disease Control and Prevention, National Center for Health Statistics. National Health and Nutrition Examination Survey, Survey Methods and Analytic Guidelines. Available online: http://www. webcitation.org/6ZVxsoxgr (accessed on 23 March 2015). 18. Centers for Disease Control and Prevention, National Center for Health Statistics. National Health and Nutrition Examination Survey MEC In-Person Dietary Interviewers Procedures Manual. Available online: http://www.webcitation.org/6ZVxoRqpy (accessed on 23 March 2015). 19. U.S. Department of Agriculture, Agricultural Research Service. What We Eat in America Food Categories 2009–2010. Available online: http://www.webcitation.org/6ZVxl7Nr3 (accessed on 23 March 2015). 17 of 18 17 of 18 Nutrients 2017, 9, 264 20. Briefel, R.R.; Kalb, L.M.; Condon, E.; Deming, D.M.; Clusen, N.A.; Fox, M.K.; Harnack, L.; Gemmill, E.; Stevens, M.; Reidy, K.C. The Feeding Infants and Toddlers Study 2008: Study design and methods. J. Am. Diet. Assoc. 2010, 110, S16–S26. [CrossRef] [PubMed] 1. Tian, N.; Zhang, Z.; Loustalot, F.; Yang, Q.; Cogswell, M.E. 40. O’Neil, C.; Nicklas, T. A Review of the Relationship between 100% Fruit Juice Consumption and Weight in Children and Adolescents. Am. J. Lifestyle Med. 2008, 2, 315–354. [CrossRef] References Nicklas, T.A.; O’Neil, C.E.; Fulgoni, V.L. Consumption of 100% Fruit Juice is Associated with Better Nutrient Intake and Diet Quality but not with Weight Status in Children: NHANES 2007–2010. Int. J. Child Health Nutr. 2015, 4, 112–121. [CrossRef] 34. U.S. 2015 Dietary Guidelines Advisory Committee. Scientiﬁc Report of the 2015 Dietary Guidelines Advisory Committee. Available online: http://www.webcitation.org/6ZVyJBo45 (accessed on 15 March 2015). 35. American Academy of Pediatrics Committee on Nutrition. The Use and Misuse of Fruit Juice in Pediatrics. Pediatrics 2001, 107, 1210–1213. 36. O’Connor, T.; Yang, S.-J.; Nicklas, T.A. Beverage Intake Among Preschool Children and Its Effect on Weight Status. Pediatrics 2006, 118, e1013. 37. Crowe-White, K.; O’Neil, C.E.; Parrott, J.S.; Benson-Davies, S.; Droke, E.; Gutschall, M.; Stote, K.S.; Wolfram, T.; Ziegler, P. Impact of 100% Fruit Juice Consumption on Diet and Weight Status of Children: An Evidence-based Review. Crit. Rev. Food Sci. Nutr. 2016, 56, 871–884. [CrossRef] [PubMed] 38. O’Neil, C.; Nicklas, T.; Rampersaud, G.; Fulgoni, V.R. One hundred percent orange juice consumption is associated with better diet quality, improved nutrient adequacy, and no increased risk for overweight/obesity in children. Nutr. Res. 2011, 31, 673–682. [CrossRef] [PubMed] 39. Nicklas, T.A.; O’Neil, C.E.; Kleinman, R. Association between 100% juice consumption and nutrient intake and weight of children aged 2 to 11 years. Arch. Pediatr. Adolesc. Med. 2008, 162, 557–565. [CrossRef] [PubMed] 40. O’Neil, C.; Nicklas, T. A Review of the Relationship between 100% Fruit Juice Consumption and Weight in Children and Adolescents. Am. J. Lifestyle Med. 2008, 2, 315–354. [CrossRef] Nutrients 2017, 9, 264 18 of 18 18 of 18 41. Taveras, E.M.; Gillman, M.W.; Kleinman, K.; Rich-Edwards, J.W.; Rifas-Shiman, S.L. Racial/ethnic differences in early-life risk factors for childhood obesity. Pediatrics 2010, 125, 686–695. [CrossRef] [PubMed] 42. Brener, N.D.; Merlo, C.; Eaton, D.; Kann, L. Beverage Consumption Among High School Students - United States, 2010. Morb. Mortal. Wkly. Rep. (MMWR) 2011, 60, 778–780. y p 43. Grimm, G.C.; Harnack, L.; Story, M. Factors associated with soft drink consumption in school-aged children. J. Am. Diet. Assoc. 2004, 104, 1244–1249. [CrossRef] [PubMed] 43. Grimm, G.C.; Harnack, L.; Story, M. Factors associated with soft drink consumption in school-aged children. J. Am. Diet. Assoc. 2004, 104, 1244–1249. [CrossRef] [PubMed] 44. Dixon, B.; Pena, M.M.; Taveras, E.M. Lifecourse approach to racial/ethnic disparities in childhood obesity. Adv. Nutr. 2012, 3, 73–82. [CrossRef] [PubMed] 44. Dixon, B.; Pena, M.M.; Taveras, E.M. 2. Brener, N.D.; Merlo, C.; Eaton, D.; Kann, L. Beverage Consumption Among High School Students - Un States, 2010. Morb. Mortal. Wkly. Rep. (MMWR) 2011, 60, 778–780. References Lifecourse approach to racial/ethnic disparities in childhood obesity. Adv. Nutr. 2012, 3, 73–82. [CrossRef] [PubMed] © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2102116876	https://bmcpsychiatry.biomedcentral.com/counter/pdf/10.1186/s12888-015-0503-0	English	null	The validity of the Patient Health Questionnaire for screening depression in chronic care patients in primary health care in South Africa	BMC psychiatry	2,015	cc-by	7,323	RESEARCH ARTICLE Open Access Bhana et al. BMC Psychiatry (2015) 15:118 DOI 10.1186/s12888-015-0503-0 Bhana et al. BMC Psychiatry (2015) 15:118 DOI 10.1186/s12888-015-0503-0 © 2015 Bhana et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: People with chronic health conditions are known to have a higher prevalence of depressive disorder. The Patient Health Questionnaire (PHQ-9) is a widely-used screening tool for depression which has not yet been validated for use on chronic care patients in South Africa. Methods: A sample of 676 chronic care patients attending two primary health facilities in North West Province, South Africa were administered the PHQ-9 by field workers and a diagnostic interview (the Structured Clinical Interview for DSM-IV) (SCID) by clinical psychologists. The PHQ-9 and the PHQ-2 were evaluated against the SCID, as well as for sub-samples of patients who were being treated for HIV infection and for hypertension. Results: Using the SCID, 11.4 % of patients had major depressive disorder. The internal consistency estimate for the PHQ-9 was 0.76, with an area under the receiver operator curve (AUROC) of 0.85 (95 % CI 0.82–0.88), which was higher than the AURUC for the PHQ-2 (0.76, 95 % CI 0.73–0.79). Using a cut-point of 9, the PHQ-9 has sensitivity of 51 % and specificity of 94 %. The PHQ-9 AUROC for the sub-samples of patients with HIV and with hypertension were comparable (0.85 and 0.86, respectively). Conclusions: The PHQ-9 is useful as a screening tool for depression among patients receiving treatment for chronic care in a public health facility. Keywords: Questionnaire (PHQ-9), Validation studies, Depressive disorder, Chronic disease, South Africa, Mass screening, Diagnosis [6, 7]. The WHO World Mental Health Survey in developed and developing countries [8] and the review of the evidence by the National Institute for Health and Clinical Excellence (NICE) guidance for depression in adults with a chronic physical health problem reports similar findings [9]. The validity of the Patient Health Questionnaire for screening depression in chronic care patients in primary health care in South Africa Arvin Bhana1, Sujit D Rathod2, One Selohilwe1, Tasneem Kathree1 and Inge Petersen1 Correspondence: arvin.bhana@gmail.com 1School of Applied Human Sciences, University of KwaZulu-Natal, Durban, South Africa Full list of author information is available at the end of the article Background An estimated 14 % of the global burden of disease can be attributed to neuropsychiatric disorders, primarily related to the disabling nature of depression and other common mental disorders [1, 2]. Depression has also been found to be co-morbid with a range of chronic diseases, including HIV/AIDS [3, 4] cardiovascular disorder (CVD) and diabetes [1, 5]. Large studies have shown that patients with chronic obstructive pulmonary disease, chronic renal failure and cerebrovascular disease compared with age-matched healthy controls were almost three times more likely to have depression, and twice as likely for patients with diabetes or hypertension The co-morbidity of depression with chronic condi- tions is a public health concern due to their mutual reinforcement and synergistic clinical effects [7, 10, 11]. Evidence for the reciprocal relationship between depres- sion and chronic physical health problems suggests a number of causal pathways, including emotional distress and poor sleep due to pain [12], the prospect of disabil- ity, [1] and changes in allostatic load whereby the ability of the body to adapt may be compromised due to ongoing tissue damage and degenerative changes [9]. Similarly, depression can also contribute to the * Correspondence: arvin.bhana@gmail.com 1School of Applied Human Sciences, University of KwaZulu-Natal, Durban, South Africa Full list of author information is available at the end of the article Page 2 of 9 Bhana et al. BMC Psychiatry (2015) 15:118 development of physical health problems. Systematic reviews of 11 prospective cohort studies in health popula- tions reveal that depression predicts later development of coronary heart disease [13] and stroke. Furthermore, major depressive disorder (MDD) can act as a precipitant for HIV infection as well be a consequence of HIV infec- tion [14, 15]. One third of HIV-infected patients were found to be depressed in a recent screening study for depression in a high HIV burden primary health care clinic in South Africa study [16]. The burden of disease in South Africa is in the process of profound health transi- tions where communicable and non-communicable disorders coexist, and where chronic conditions such as cardiovascular disease, type 2 diabetes, cancer, chronic lung disease and depression are all predicted to increase substantially [17]. have examined the PHQ-9 against a criterion standard. The PHQ-9 successfully screened post-stroke depression patients with operating characteristics similar or superior to other depression measures [29]. Background Similar findings are noted in the Heart and Soul study of depression among patients with coronary heart disease [30]. A meta-analysis that included chronic care condi- tions involving patients from cardiology, renal dialyses, brain injury and stroke facilities as well as general medical outpatients found the PHQ-9 acceptable as a diagnostic screening tool for major depression [31]. In sub-Saharan Africa, only a few studies have estab- lished the criterion validity of the PHQ-9 as a screening tool for depression [16, 26, 32, 33]. A meta-analysis establishing the range of optimal cut-off scores for diag- nosing depression with the PHQ-9 notes that optimal cut-off scores range between 8 and 11 [34]. In low and middle income contexts, a positive screen for depression was defined as a score of ≥10 [32, 35]. Treatment of depression is vital to improved adher- ence, social functioning and the disease course of chronic conditions [5, 14, 18, 19]. Early recognition and appropriate management of depression has the potential to improve adherence and impact on the social function- ing and the disease progression of chronic conditions, thereby enhancing quality of life [5, 9, 18–20]. This study aimed to validate the PHQ-9 as a screening tool for depression among chronic care patients attend- ing two public primary health care facilities in South Africa. As a secondary objective, we validated the PHQ- 2 - a subset of two questions drawn from the PHQ-9 – which is a simpler screening option that has been validated elsewhere [36]. The PHQ-2 has not been vali- dated against a gold standard in sub-Saharan Africa, though it has been recommended as a valid and reliable tool for use in resource-constrained settings [28]. While identifying depressive disorder in primary care is recognized as important to effective treatment, only about half of patients with depressive disorder are detected by regular health care providers in high-income settings [21, 22]. In South Africa, this gap is far greater, with only one in four people with a common mental disorder receiving treatment [23]. With HIV transition- ing to a chronic condition as a function of the roll-out of anti-retroviral treatment (ART) in South Africa, as well as the rising burden of NCDs (Non Communicable Diseases), which includes mental disorders [17], there is an imperative to integrate the treatment of depression with chronic disease management. Setting and participants The study was conducted in two primary health care clinics in the North West province of South Africa. The two clinics are pilot sites for a national Department of Health model for integrated chronic disease manage- ment, which adopts the collaborative chronic care model and services all chronic care patients at one service point [37]. In the context of busy primary health care (PHC) clinics, the use of valid screening tools that facilitate assessment of depression by non-specialists would allow for greater depression detection and care, as well as being in line with the task-sharing model that has been embraced by the recent National Mental Health Policy Framework and Strategic Action Plan (2013–2020) in South Africa [24]. The Patients Health Questionnaire (PHQ-9) is a brief diagnostic and severity measure of depression, which has been widely used in research and clinical practice but has not been validated for use in South Africa on chronic care patients. Assessments Th P Q The PHQ-9 asks patients to rate how often they were bothered by specific problems over the last two weeks. Each item is scored from 0 to 3 (0 = not at all; 1 = several days; 2 = more than half the days; and 3 = nearly every day) [36]. All nine items in the PHQ-9 are derived from DSM-IV criteria relevant for diagnosis of a current depres- sive episode [21]. The PHQ-2 is comprised of the first two questions of the PHQ-9, namely whether the patient has depressed mood and loss of interest (anhedonia). The PHQ-9 asks patients to rate how often they were bothered by specific problems over the last two weeks. Each item is scored from 0 to 3 according to the frequency of the prob- lem. After a piloting process we adapted the response set to improve respondent understanding, such that “several days” was understood to be 1–7 days, “half the days” was understood to be 8–11 days and “nearly every day” was understood to be 12–14 days, as was the case in other validity studies in Africa [16, 26]. The PHQ-9 was trans- lated into seTswana by a seTswana-speaking mental health professional and then back-translated by an independent seTswana-speaking clinical psychologist using the method- ology described by Brislin [40] (Additional file 1). Due to time considerations and the logistics of administering the PHQ-9 twice to a clinic population, test-retest reliability was not done. Inter-rater reliability was also not com- pleted for the same reasons. Research assistants who administered the PHQ-9 were supervised by mid-level psychological counsellors with 4-year Bachelor’s degrees in psychology who were fluent in seTswana and trained by the seTswana-speaking clin- ical psychologist in the administration of the PHQ-9. These research assistants then orally administered the PHQ-9 screening tool, and entered the participant’s responses in a questionnaire application programmed onto a mobile handheld device [38]. In addition to the PHQ-9 screening instrument, the interview contained questions on socio-demographic characteristics, eco- nomic status, chronic care services received at the clinic, alcohol use and disability status as part of the larger study. Immediately after the conclusion of the screening interview, the research assistant directed the participant to another private consultation room for the diagnostic interview with a clinical psychologist. The research assistant did not appraise the participant or the psych- ologist of the participant’s PHQ-9 screening score. Assessments Th P Q In each clinic, a clinical psychologist conducted the Structured Clinical Interview for DSM-IV (SCID) [39] diagnostic interview for a current episode of depressive disorder. One psychologist was fluent in both study languages, while the other was assisted by a seTswana- speaking mid-level trained psychological counsellor to conduct the diagnostic interviews. Most patients chose to have the interviews in seTswana with only a few who chose to be interviewed in English. At the conclusion of the diagnostic interview, any patient who 1) expressed suicidality (i.e. thoughts, plans, actions); or 2) was judged to have severe symptoms of depression by the clinical psychologist, was asked by the research assistant to pro- vide consent for a referral to the consulting nurse in the clinic. The gold standard diagnostic interview was the de- pression module of the SCID-I to assess the participants for the presence or absence of major depressive disorder. The SCID is a semi-structured interview administered by a trained clinician who assesses a respondent for the presence or absence of a mental health disorder. At the time of the study, the more recent version of the SCID was not available. Nevertheless, the criteria for depres- sion remains the same in the DSM-V as the DSM-IV version. According to the American Psychiatric Associ- ation “neither the core criterion symptoms applied to the diagnosis of major depressive episode nor the requisite duration of at least 2 weeks has changed from DSM-IV. Criterion A for a major depressive episode in DSM-V is identical to that of DSM-IV, as is the requirement for clinically significant distress or impairment in social, occupational, or other important areas of life” (p. 4) [41]. Ethical approval for the validation study was obtained from the University of KwaZulu-Natal Biomedical Re- search Ethics Committee (BE271/13). Ethical approval for the larger study was obtained from the University of Study procedures Thi lid i This validation study was nested within a larger facility detection survey designed to assess the detection and treatment of depression and alcohol use disorders by health care providers for adult attendees of primary health clinics. Patients who had come to the chronic care clinic were recruited from the consultation waiting room before their consultation with a clinician. In the waiting room, a field worker asked for volunteers to take part in a survey on depression and alcohol use disorders. The field worker directed interested individuals to a research assistant in a private consultation room. The research assistants who were recruited from the local communities had completed secondary school, were While there are numerous studies that report on the validity of the PHQ-9 in relation to various chronic illnesses, most of these studies fail to use an appropriate reference standard to evaluate the performance of the PHQ-9′s performance in identifying depression [25–28]. Only a few studies involving patients in chronic care Bhana et al. BMC Psychiatry (2015) 15:118 Page 3 of 9 Page 3 of 9 Bhana et al. BMC Psychiatry (2015) 15:118 KwaZulu-Natal Biomedical Research Ethics Committee (BE400/13), and the University of Cape Town, Faculty of Health Sciences, Human Research Ethics Committee (412/2011), and the World Health Organization Research Ethics Review Committee (RPC497). fluent in seTswana and English, and were trained in administering the PHQ-9 by a clinical psychologist. The research assistant assessed the patients for inclusion criteria, which were: age 18 years or older, clinic attend- ance for routine chronic disease services (e.g., HIV, hypertension, diabetes) and ability to comprehend and complete study components in seTswana or English. Exclusion criteria included incapacity to provide informed consent (e.g., less than 18 years of age, presence of severe intellectual disability or currently experiencing an acute medical issue, or in treatment for major depression). Eligible patients provided written informed consent to participate in the validation study. Patients with low levels of literacy could sign the informed consent form with an “X” after discussing the study with a study supervisor, and having the consent form read out to them. KwaZulu-Natal Biomedical Research Ethics Committee (BE400/13), and the University of Cape Town, Faculty of Health Sciences, Human Research Ethics Committee (412/2011), and the World Health Organization Research Ethics Review Committee (RPC497). Data analyses Fi d y First, we described the socio-demographic and clinical characteristics of participants recruited for this valid- ation study and proportion of patients receiving a depression diagnosis on the SCID. We evaluated the internal consistency of the PHQ-9 by calculating the Cronbach alpha. Next, we used the screening scores from the PHQ-9 interview to construct a receiver oper- ating characteristic (ROC) curve and calculated the area under the ROC (AUROC) using the SCID as the gold standard. The AUROC provides a summary measure of a screening tool’s sensitivity and specificity, relative to the gold standard diagnostic, across the entire range of screening scores. An AUROC score of 0.5 is consistent with a screening tool that is no better than chance, and a score of 1.0 indicates a perfectly accurate screening tool. We also calculated the AUROC for the PHQ2 and compared it to the AUROC for the PHQ-9. To explore potential heterogeneity of the validity of the PHQ-9, we also repeated the AUROC analysis with two (overlapping) subsets of participants who reported that their clinic attendance was for ongoing care with HIV infection and with hypertension. For all AUROC calculations, we report exact binomial 95 % confidence intervals (95 % CI). Table 1 Demographic and clinical characteristics chronic care patients (n = 676) in North West Province, South Africa Description n (%) Sex Male 169 (25.0) Female 507 (75.0) Age (years) Median 47 IQR 37–56.5 Range 18–88 Education None 52 (7.7) Grades 1–5 135 (20.0) Grades 6–12 478 (70.7) >12 years 11 (1.6) Chronic care servicea HIV/AIDS 413 (61.1) Hypertension 345 (51.0) Diabetes 63 (9.3) Tuberculosis 33 (4.9) Asthma 24 (3.6) Epilepsy 23 (3.4) Arthritis 23 (3.4) Other Mental Healthb 17 (2.5) Heart problems 6 (0.9) Chronic obstructed pulmonary disorder 2 (0.30) Number of chronic care services 1 399 (59.0) 2 237 (35.1) 3 38 (5.3) > = 4 2 (0.3) IQR interquartile range aParticipants can report >1 chronic service, total is >100 % bFor example Schizophrenia and Bipolar Mood Disorders We completed all analyses using Stata 13.1 (StataCorp, College Station, USA); we used the ‘roctab’ command for analysis of the individual AUCs, and ‘roccomp’ for the comparison of the PHQ-9 and the PHQ-2. Sample characteristics Out of a total of 1321 eligible patients who participated in the larger facility survey, 1025 patients were attendees Bhana et al. BMC Psychiatry (2015) 15:118 Page 4 of 9 of the clinics where the validation study was conducted. Of these patients, a sub-sample of 676 patients partici- pated in the validation study between February and April 2014. Participants who consented to the general study also consented to the PHQ9 validation study. The sub-sample size was limited by the availability of the clinical psychologists to conduct diagnostic interviews. Overall, there were 233 refusals to the larger study. Table 1 reflects the demographic characteristics of the 676 participants who completed both the SCID and PHQ-9 interviews. The participants were predominantly female (75.0 %), with mean age of 47.1 years (SD 13.1). Most participants (72.4 %) had completed 6 or more years of primary education. Participants could report multiple conditions for which they had been diagnosed and for which they were receiving services. The most common conditions reported were HIV infection (61.1 %), hypertension (51.0 %), diabetes (9.3 %), and tuberculosis (4.9 %). Of the 413 participants diagnosed with HIV and 345 patients diagnosed with hypertension, 118 participants were found to be co-morbid. IQR interquartile range aParticipants can report >1 chronic service, total is >100 % b p p , bFor example, Schizophrenia and Bipolar Mood Disorders IQR interquartile range Descriptive results In the SCID diagnostic interview, more than one in ten (11.47 %) participants were diagnosed as currently experiencing a major depressive episode. The mean PHQ- 9 score for those who were SCID-positive (PHQ-9 score 9.4, SD 5.3) was substantially higher than for those who were SCID-negative (PHQ-9 score 3.2, SD 3.1). For those with an HIV diagnosis, 12.6 % were SCID-positive, while 9.9 % of those with hypertension were also SCID-positive. No participant experienced any adverse events during either interview. The study psychologist referred 69 participants to the clinic on-call nurse, following disclosure of suicidal ideation, planning or attempts (Table 2). Internal consistency of the PHQ-9 revealed a Cronbach alpha of 0.76. Bhana et al. BMC Psychiatry (2015) 15:118 Page 5 of 9 Page 5 of 9 Table 2 Performance of the PHQ-9 and PHQ-2 to detect major depressive disorder among chronic care patients (n = 676) PHQ-9 PHQ-2 Cut point Sensitivity (%) Specificity (%) Correctly classified (%) LR + a LR-b Sensitivity (%) Specificity (%) Correctly classified (%) (≥0) 100.0 0.0 11.4 1.000 100.0 0.0 11.4 (≥1) 100.0 21.4 30.3 1.2718 0.0000 80.5 57.9 60.5 (≥2) 97.4 37.1 43.9 1.5476 0.0701 59.7 83.5 80.8 (≥3) 90.9 52.4 56.8 1.9107 0.1734 39.0 91.7 85.7 (≥4) 87.0 63.3 66.0 2.3691 0.2053 19.5 97.8 88.9 (≥5) 80.5 71.8 72.8 2.8539 0.2714 9.1 99.3 89.1 (≥6) 71.4 79.1 78.2 3.4229 0.3611 0.0 100.0 88.6 (≥7) 64.9 85.6 83.3 4.5228 0.4094 (≥8) 53.2 90.6 86.4 5.6955 0.5157 (≥9) 49.3 93.7 88.6 7.7792 0.5408 (≥10) 41.6 96.5 90.2 11.8540 0.6056 (≥11) 36.4 98.2 91.1 19.8016 0.6483 (≥12) 32.5 98.5 91.0 21.6090 0.6856 (≥13) 26.0 98.5 90.2 17.2872 0.7516 (≥14) 24.7 98.8 90.4 21.1150 0.7622 (≥15) 19.5 99.5 90.4 38.8963 0.8092 (≥16) 14.3 99.7 89.9 42.7863 0.8600 (≥17) 9.1 99.7 89.4 27.2276 0.9121 (≥19) 9.1 99.4 89.5 54.4541 0.9106 (≥20) 3.9 99.8 88.9 23.3375 0.9626 (≥21) 2.6 99.8 88.8 15.5583 0.9757 (≥22) 1.3 100.0 88.8 0.9870 (≥22) 0.0 100.0 88.6 1.0000 The SCID depression module was used as the diagnostic reference standard aLikelihood Ratio Positive bLikelihood Ratio Negative The SCID depression module was used as the diagnostic reference standard aLikelihood Ratio Positive bLikelihood Ratio Negative Fig. 1 Receiver operating characteristic (ROC) curve for PHQ-9 Fig. 1 Receiver operating characteristic (ROC) curve for PHQ-9 Bhana et al. BMC Psychiatry (2015) 15:118 Page 6 of 9 Fig. Descriptive results Error 95 % Confidence Interval 345 0.8596 0.0309 0.82 0.90 Table 3 Item-level discrimination PHQ-9 (N = 676) Item Item-test correlation Item-rest correlation Average Inter-item covariance Item Alpha Phq1 0.578 0.429 0.144 0.734 Phq2 0.649 0.491 0.133 0.724 Phq3 0.619 0.440 0.135 0.734 Phq4 0.655 0.509 0.133 0.721 Phq5 0.620 0.451 0.135 0.732 Phq6 0.602 0.465 0.142 0.723 Phq7 0.587 0.466 0.147 0.730 Phq8 0.347 0.250 0.170 0.757 Phq9 0.546 0.420 0.150 0.736 Overall 0.143 0.756 Bhana et al. BMC Psychiatry (2015) 15:118 Page 7 of 9 Table 4 Performance of the PHQ-9 in Detecting Depression in HIV positive patients (n = 413) in North West Province, South Africa Cut-point Sensitivity % Specificity % Percent correctly classified LR+ LR- (≥0) 100.00 0.00 12.59 1.000 (≥1) 100.00 21.05 30.99 1.2667 0.0000 (≥2) 98.08 33.80 41.89 1.4814 0.0569 (≥3) 88.46 49.31 54.24 1.7451 0.2340 (≥4) 84.26 61.22 64.16 2.1819 0.2513 (≥5) 82.69 68.42 70.22 2.6186 0.2530 (≥6) 76.92 77.29 77.24 3.3865 0.2986 (≥7) 71.15 83.93 82.32 4.4287 0.3437 (≥8) 57.69 88.92 84.99 5.2067 0.4758 (≥9) 57.69 92.80 88.38 8.0104 0.4559 (≥10) 48.08 95.57 89.59 10.4874 0.5433 (≥11) 40.38 97.51 90.31 16.1987 0.6114 (≥12) 36.54 98.06 90.31 18.8434 0.6472 (≥13) 28.85 98.06 89.35 14.8764 0.7256 (≥14) 26.92 98.61 89.59 19.4385 0.7410 (≥15) 23.08 99.45 89.83 41.6359 0.7735 (≥16) 17.31 99.72 89.35 62.4808 0.8292 (≥19) 11.54 99.72 88.62 41.6539 0.8871 (≥20) 5.77 99.72 87.89 20.8269 0.9449 (≥21) 3 85 99 72 87 65 13 8846 0 9642 Table 3 Item-level discrimination PHQ-9 (N = 676) Item Item-test correlation Item-rest correlation Average Inter-item covariance Item Alpha Phq1 0.578 0.429 0.144 0.734 Phq2 0.649 0.491 0.133 0.724 Phq3 0.619 0.440 0.135 0.734 Phq4 0.655 0.509 0.133 0.721 Phq5 0.620 0.451 0.135 0.732 Phq6 0.602 0.465 0.142 0.723 Phq7 0.587 0.466 0.147 0.730 Phq8 0.347 0.250 0.170 0.757 Phq9 0.546 0.420 0.150 0.736 Overall 0.143 0.756 Table 3 Item-level discrimination PHQ-9 (N = 676) Item Item-test correlation Item-rest correlation Average Inter-item covariance Item Alpha Phq1 0.578 0.429 0.144 0.734 Phq2 0.649 0.491 0.133 0.724 Phq3 0.619 0.440 0.135 0.734 Phq4 0.655 0.509 0.133 0.721 Phq5 0.620 0.451 0.135 0.732 Phq6 0.602 0.465 0.142 0.723 Phq7 0.587 0.466 0.147 0.730 Phq8 0.347 0.250 0.170 0.757 Phq9 0.546 0.420 0.150 0.736 Overall 0.143 0.756 Table 3 Item-level discrimination PHQ-9 (N = 676) specificity of 84 %, correctly classifying 81 % of the popu- lation at this level (Figs. Descriptive results 1, 2 and 3, Tables 3, 4 and 5). Descriptive results 2 Receiver operating characteristic (ROC) curve for PHQ-2 Sensitivity and specificity of the PHQ-9 and PHQ-2 of ≥5, with a maximum value of sensitivity- (1-specificity) of 52.3 %. y p y With the full study sample, the PHQ-9 showed reason- ably high validity (AUROC 0.85, 95 % CI 0.82–0.88). With a cut point of ≥9, the PHQ-9 had sensitivity of 49 % and specificity of 94 %. The likelihood ratio of a person testing positive for depression was seven times more likely at this cut point (7.7792). The Overall Cor- rect Classification (OCC) rate of 0.886 was also the best at a cutoff of 9 and above. With this cut point, the positive and negative predictive values were 50 % and 94 %, respect- ively. Youden’s Index (Youden’s J) identifies a cut point The validity of the PHQ-9 was similar for the subsam- ples of 413 patients receiving services for HIV infection (AUROC 0.85, 95 % CI 0.81–0.88) and the 345 patients receiving services for hypertension (AUROC 0.85, 95 % CI 0.82–0.90). While the PHQ-2 appeared to be a valid screening tool (AUROC 0.76, 95 % CI 0.73–0.79), its AUROC was sig- nificantly lower than for the PHQ-9 (p < 0.0001). With a cut point of ≥2, the PHQ-2 had sensitivity of 60 % and Fig. 3 Receiver operating characteristic (ROC) curve comparing PHQ-9 to PHQ-2 Bhana et al. BMC Psychiatry (2015) 15:118 Page 7 of 9 Page 7 of 9 specificity of 84 %, correctly classifying 81 % of the popu- lation at this level (Figs. 1, 2 and 3, Tables 3, 4 and 5). Discussion The health care burden associated with the burgeoning chronic care population in South Africa makes this a timely and important focus as individuals with depres- sion are less likely to be treatment adherent or engage in health enhancing behavior change to promote healthy lifestyles [5, 18–20]. Given that identifying depression in chronic care patients can be a diagnostic challenge in busy clinics, a short and valid screening tool can assist in the identification of patients with depression. A meta-analysis establishing the range of optimal cut- off scores for diagnosing depression with the PHQ-9 notes that optimal cut-off scores range between 8 and 11 [34]. Descriptive results In this study, a cut-point of ≥9 yielded a fairly low sensitivity (49 %) in comparison to sensitivity indices of 78.7 % and 89.6 %, respectively in other similar valid- ity studies in sub-Saharan Africa [16, 32], However, higher specificity (94 %) was noted in the present study Table 4 Performance of the PHQ-9 in Detecting Depression in HIV positive patients (n = 413) in North West Province, South Africa Cut-point Sensitivity % Specificity % Percent correctly classified LR+ LR- (≥0) 100.00 0.00 12.59 1.000 (≥1) 100.00 21.05 30.99 1.2667 0.0000 (≥2) 98.08 33.80 41.89 1.4814 0.0569 (≥3) 88.46 49.31 54.24 1.7451 0.2340 (≥4) 84.26 61.22 64.16 2.1819 0.2513 (≥5) 82.69 68.42 70.22 2.6186 0.2530 (≥6) 76.92 77.29 77.24 3.3865 0.2986 (≥7) 71.15 83.93 82.32 4.4287 0.3437 (≥8) 57.69 88.92 84.99 5.2067 0.4758 (≥9) 57.69 92.80 88.38 8.0104 0.4559 (≥10) 48.08 95.57 89.59 10.4874 0.5433 (≥11) 40.38 97.51 90.31 16.1987 0.6114 (≥12) 36.54 98.06 90.31 18.8434 0.6472 (≥13) 28.85 98.06 89.35 14.8764 0.7256 (≥14) 26.92 98.61 89.59 19.4385 0.7410 (≥15) 23.08 99.45 89.83 41.6359 0.7735 (≥16) 17.31 99.72 89.35 62.4808 0.8292 (≥19) 11.54 99.72 88.62 41.6539 0.8871 (≥20) 5.77 99.72 87.89 20.8269 0.9449 (≥21) 3.85 99.72 87.65 13.8846 0.9642 (≥22) 1.92 100 87.65 0.9808 (≥22) 0.00 100 87.41 1.0000 Observations ROC Area under the curve Std. Error 95 % Confidence Interval 413 0.8488 0.0293 0.81 0.88 Table 5 Performance of the PHQ-9 in Detecting Depression in Patients with hypertension (n = 345) in North West Province, South Africa Cut-point Sensitivity % Specificity % Percent correctly classified LR+ LR- (≥0) 100.00 0.00 9.86 1.000 (≥1) 100.00 20.58 28.41 1.2591 0.0000 (≥2) 97.06 37.03 43.19 1.5408 0.0789 (≥3) 97.06 51.77 56.23 2.0124 0.0568 (≥4) 91.18 62.38 65.22 2.4236 0.1414 (≥5) 79.41 72.35 73.04 2.8718 0.2846 (≥6) 67.65 80.39 79.13 3.4489 0.4025 (≥7) 64.71 85.85 83.77 4.5735 0.4111 (≥8) 52.94 91.96 88.12 6.5859 0.5117 (≥9) 44.12 94.53 89.57 8.0709 0.5911 (≥10) 38.42 98.07 92.17 19.8186 0.6298 (≥11) 32.35 99.36 92.75 50.3086 0.6808 (≥12) 29.41 99.36 92.46 45.7351 0.7105 (≥14) 23.53 99.36 91.88 36.5881 0.7697 (≥15) 17.65 99.68 91.59 51.8828 0.8262 (≥16) 11.76 99.68 91.01 36.5885 0.8852 (≥17) 8.82 99.68 90.72 27.4414 0.9147 (≥19) 8.82 100 91.01 0.9118 (≥20) 2.94 100 90.43 0.9706 (≥20) 0.00 100 90.14 0.9706 Observations ROC Area under the curve Std. Authors’ contributions AB helped conceptualize the study, wrote the first and final draft and edited all interim drafts. SDR helped conceptualize the study, provided oversight over data management, conducted the analysis and contributed to drafting the paper. OS oversaw the data collection and management and contributed to drafting of the paper. TK helped with data management and contributed to drafting the paper. IP helped conceptualize the study, wrote the protocol, provided oversight over data collection and management and contributed to the drafting of the paper. All authors read and approved the final manuscript. The strength of this study is that it is one of the few studies to consider the validity of the PHQ-9 in sub- Saharan Africa, and the first to validate the PHQ-9 or the PHQ-2 for a chronic care population in South Africa. Limitations of this study include that the partici- pants were drawn from only two clinics in North West province, and were predominantly female. Therefore, it may not be possible to generalize our results to other populations. We were unable to establish test-retest reliability due to time considerations and the burden it would impose on the public health clinics. On the same basis, we were unable to establish inter-rater reliability of administration of the gold standard instrument. For logistic reasons we were also unable to randomize the order of interviews as participants with depression would be more likely to be detected in the diagnostic interview than in the screening interview. Further, a willingness to disclose feelings of distress in the diagnos- tic interview may have been heightened after completing the screening interview. This ‘order’ effect may have biased our AUROC results to be lower than the true values. Discussion The health care burden associated with the burgeoning chronic care population in South Africa makes this a timely and important focus as individuals with depres- sion are less likely to be treatment adherent or engage in health enhancing behavior change to promote healthy lifestyles [5, 18–20]. Given that identifying depression in chronic care patients can be a diagnostic challenge in busy clinics, a short and valid screening tool can assist in the identification of patients with depression. A meta-analysis establishing the range of optimal cut- off scores for diagnosing depression with the PHQ-9 notes that optimal cut-off scores range between 8 and 11 [34]. In this study, a cut-point of ≥9 yielded a fairly low sensitivity (49 %) in comparison to sensitivity indices of 78.7 % and 89.6 %, respectively in other similar valid- ity studies in sub-Saharan Africa [16, 32], However, higher specificity (94 %) was noted in the present study Bhana et al. BMC Psychiatry (2015) 15:118 Page 8 of 9 relative to the only other South African study (83.4 %) [16] and was the same as that found for HIV patients in Cameroon [26]. A prospective study of psychosocial factors and health outcomes in patients with a diagnosis of coronary heart disease also showed similar sensitivity and specificity to the present study [30]. understanding of depression as a legitimate disorder and recognition of depressive symptoms are likely to respond to screening tools such as the PHQ-9. Competing interests Competing interests The authors declare they have no competing interests. Received: 1 October 2014 Accepted: 18 May 2015 Received: 1 October 2014 Accepted: 18 May 2015 Received: 1 October 2014 Accepted: 18 May 2015 References 1. Prince M, Patel V, Saxena S, Maj M, Maselko J, Phillips MR, et al. No health without mental health. Lancet. 2007;370(9590):859–77. 2. Murray CJ, Vos T, Lozano R, Naghavi M, Flaxman AD, Michaud C, et al. Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990–2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012;380(9859):2197–223. y 3. Brandt R. The mental health of people living with HIV/AIDS in a systematic review. Afr J AIDS Res. 2009;8(2):123–33. 3. Brandt R. The mental health of people living with HIV/AIDS in Africa: a systematic review. Afr J AIDS Res. 2009;8(2):123–33. 4. Owe-Larsson B, Sall L, Salamon E, Allgulander C. HIV infection and psychiatric illness. Afr J Psychiatry. 2009;12(2):115–28. l b h ll h h y 4. Owe-Larsson B, Sall L, Salamon E, Allgulander C. HIV infection and psychiatric illness. Afr J Psychiatry. 2009;12(2):115–28. 5. Ziegelstein RC, Fauerbach JA, Stevens SS, Romanelli J, Richter DP, Bush DE. Patients with depression are less likely to follow recommendations to reduce cardiac risk during recovery from a myocardial infarction. Arch Intern Med. 2000;160(12):1818–23. Acknowledgements Our grateful thanks to the patients for their enduring patience and participation in the various phases of the assessments. We also wish to thank the data collection team, Palesa Mothibedi, Primrose Mpahane, Rothea Kruger, Emily Lesekele, Mirriam Maphai, Sammy Otsheleng and their respective teams. This study is an output of the PRogramme for Improving Mental health carE (PRIME). The material has been funded by UK aid from the UK Government, however the views expressed do not necessarily reflect the UK Government’s official policies. Author details 1 1School of Applied Human Sciences, University of KwaZulu-Natal, Durban, South Africa. 2Department of Population Health, London School of Hygiene and Tropical Medicine, London, UK. Additional file Additional file 1: PHQ-9 and treatment{ TC “13 items; (c) Pfizer 1999; History question adapted from MINI” \l 5 n }. Similar to the PHQ-9, the PHQ-2 had lower sensitivity (60 %) than specificity (84 %) and may be explained in the same way as that for the PHQ-9 given that these data are drawn from the same sample. The PHQ-2 remains a valid option for use, particularly in time- constrained settings. The trade-off between sensitivity and specificity using the PHQ-2 is more substantial than for the PHQ-9. Selecting a high cut-off score when using the PHQ-2 would enable clinicians to screen a large number of patients, but then to refer a relatively modest number of patients – who are likely true cases - for confirmation of a diagnosis of depression. Competing interests The authors declare they have no competing interests. Conclusions The brevity of the scale, ease of administration and its concurrent validity with the SCID suggests that the PHQ-9 can be a valuable instrument for identifying co- morbid depression in patients with chronic conditions using a cut-point of ≥9. It is possible that the abbreviated PHQ-2 could be used in a busy primary care clinic, but would need to be followed up with a full assessment. Identification is the first step towards closing the treatment gap for depression and advancing the agenda of integrated chronic disease management in South African public health facilities. Further research is needed in understanding how patients with low levels of 6. Egede LE. Major depression in individuals with chronic medical disorders: prevalence, correlates and association with health resource utilization, lost productivity and functional disability. Gen Hosp Psychiatry. 2007;29(5):409–16. 7. Moussavi S, Chatterji S, Verdes E, Tandon A, Patel V, Ustun B. Depression, chronic diseases, and decrements in health: results from the World Health Surveys. Lancet. 2007;370(9590):851–8. 6. Egede LE. Major depression in individuals with chronic medical disorders: prevalence, correlates and association with health resource utilization, lost productivity and functional disability. Gen Hosp Psychiatry. 2007;29(5):409–16. 7. Moussavi S, Chatterji S, Verdes E, Tandon A, Patel V, Ustun B. Depression, chronic diseases, and decrements in health: results from the World Health Surveys. Lancet. 2007;370(9590):851–8. 6. Egede LE. Major depression in individuals with chronic medical disorders: prevalence, correlates and association with health resource utilization, lost productivity and functional disability. Gen Hosp Psychiatry. 2007;29(5):409–16. 6. Egede LE. Major depression in individuals with chronic medical disorders: prevalence, correlates and association with health resource utilization, lost productivity and functional disability. Gen Hosp Psychiatry. 2007;29(5):409–16. 7. Moussavi S, Chatterji S, Verdes E, Tandon A, Patel V, Ustun B. Depression, chronic diseases, and decrements in health: results from the World Health Surveys. Lancet. 2007;370(9590):851–8. 6. Egede LE. Major depression in individuals with chronic medical disorders: prevalence, correlates and association with health resource utilization, lost productivity and functional disability. Gen Hosp Psychiatry. 2007;29(5):409–16. 7. Moussavi S, Chatterji S, Verdes E, Tandon A, Patel V, Ustun B. Depression, productivity and functional disability. Gen Hosp Psychiatry. 2007;29(5):409–16. 7. Moussavi S, Chatterji S, Verdes E, Tandon A, Patel V, Ustun B. Depression, chronic diseases, and decrements in health: results from the World Health Surveys. Lancet. 2007;370(9590):851–8. 8. von Korff M, Scott K, Gureje O, editors. Global Perspectives on Mental– Physical Comorbidity in the WHO Mental Health Surveys. 8. von Korff M, Scott K, Gureje O, editors. Global Perspectives on Mental– Physical Comorbidity in the WHO Mental Health Surveys. Cambridge: Cambridge University Press; 2009. 9. National Collaborating Centre for Mental H. National Institute for Health and Clinical Excellence: Guidance. In: Depression in Adults with a Chronic Physical Health Problem: Treatment and Management. edn. Leicester (UK): British Psychological Society The British Psychological Society & The Royal College of Psychiatrists; 2010. Conclusions Cambridge: Cambridge University Press; 2009. 8. von Korff M, Scott K, Gureje O, editors. Global Perspectives on Mental– Physical Comorbidity in the WHO Mental Health Surveys. Cambridge: Cambridge University Press; 2009. 9. National Collaborating Centre for Mental H. National Institute for Health and Clinical Excellence: Guidance. In: Depression in Adults with a Chronic Physical Health Problem: Treatment and Management. edn. Leicester (UK): British Psychological Society The British Psychological Society & The Royal College of Psychiatrists; 2010. 9. National Collaborating Centre for Mental H. National Institute for Health and Clinical Excellence: Guidance. In: Depression in Adults with a Chronic Physical Health Problem: Treatment and Management. edn. Leicester (UK): British Psychological Society The British Psychological Society & The Royal College of Psychiatrists; 2010. Page 9 of 9 Page 9 of 9 Bhana et al. BMC Psychiatry (2015) 15:118 33. Weobong B, Akpalu B, Doku V, Owusu-Agyei S, Hurt L, Kirkwood B, et al. The comparative validity of screening scales for postnatal common mental disorder in Kintampo. Ghana J Affect Disord. 2009;113(1–2):109–17. 10. Thombs BD, Ziegelstein RC, Whooley MA. Optimizing detection of major depression among patients with coronary artery disease using the patient health questionnaire: data from the heart and soul study. J Gen Intern Med. 2008;23(12):2014–7. 34. Manea L, Gilbody S, McMillan D. Optimal cut-off score for diagnosing depression with the Patient Health Questionnaire (PHQ-9): a meta-analysis. CMAJ. 2012;184(3):E191–6. 11. Breuer E, Myer L, Struthers H, Joska J. HV/AIDS and mental health research in sub-Saharan Africa: a systematic review. Afr J AIDS Res. 2011;10(2):101–22. 35. Akena D, Joska J, Obuku EA, Amos T, Musisi S, Stein DJ. Comparing the accuracy of brief versus long depression screening instruments which have been validated in low and middle income countries: a systematic review. BMC Psychiatry. 2012;12:187. 12. Dworkin SF, Von Korff M, LeResche L. Multiple pains and psychiatric disturbance. An epidemiologic investigation. Arch Gen Psychiatry. 1990;47(3):239–44. 13. Nicholson A, Kuper H, Hemingway H. Depression as an aetiologic and prognostic factor in coronary heart disease: a meta-analysis of 6362 events among 146 538 participants in 54 observational studies. Eur Heart J. 2006;27(23):2763–74. 36. Kroenke K, Spitzer RL, Williams JB, Lowe B. The patient health questionnaire somatic, anxiety, and depressive symptom scales: a systematic review. Gen Hosp Psychiatry. 2010;32(4):345–59. 37. Wagner EH, Austin BT, Von Korff M. Organizing chronic illness. Milbank Q. 1996;74(4):511–44. 14. Conclusions Simoni JM, Safren SA, Manhart LE, Lyda K, Grossman CI, Rao D, et al. Challenges in addressing depression in HIV research: assessment, cultural context, and methods. AIDS Behav. 2011;15(2):376–88. chronic illness. Milbank Q. 1996;74(4):511–44. 38. Mobenzi. [http://mobenzi.com/] 15. Smit J, Myer L, Middelkoop K, Seedat S, Wood R, Bekker LG, et al. Mental health and sexual risk behaviours in a South African township: a community-based cross-sectional study. Public Health. 2006;120(6):534–42. 39. First MB, Spitzer RL, Williams JBW, Gibbon M. Structured Clinical Interview for DSM-IV (SCID). Washington, DC: American Psychiatric Association; 1995. 40. Brislin RW. Back-translation for cross-cultural research. J Cross-Cult Psychol. 1970;1(3):185–216. 16. Cholera R, Gaynes BN, Pence BW, Bassett J, Qangule N, Macphail C, et al. Validity of the patient health questionnaire-9 to screen for depression in a high-HIV burden primary healthcare clinic in Johannesburg, South Africa. J Affect Disord. 2014;167:160–6. 41. Highlights of changes from DSM-IV-TR to DSM-5. [http://www.dsm5.org/ Documents/changes%20from%20dsm-iv-tr%20to%20dsm-5.pdf] 17. Mayosi BM, Flisher AJ, Lalloo UG, Sitas F, Tollman S, Bradshaw D. The burden of non-communicable diseases in South Africa. Lancet. 2009;374:934–47. 18. Gonzalez JS, Batchelder AW, Psaros C, Safren SA. Depression and HIV/AIDS treatment nonadherence: a review and meta-analysis. J Acquir Immune Defic Syndr. 2011;58(2):181–7. 19. Starace F, Ammassari A, Trotta MP, Murri R, De Longis P, Izzo C, et al. Depression is a risk factor for suboptimal adherence to highly active antiretroviral therapy. J Acquir Immune Defic Syndr. 2002;31 Suppl 3:S136–9. 20. Leserman J. Role of depression, stress, and trauma in HIV disease progression. Psychosom Med. 2008;70(5):539–45. 21. Wittkampf K, van Ravesteijn H, Baas K, van de Hoogen H, Schene A, Bindels P, et al. The accuracy of Patient Health Questionnaire-9 in detecting depres- sion and measuring depression severity in high-risk groups in primary care. Gen Hosp Psychiatry. 2009;31(5):451–9. 22. Gilbody S, Richards D, Barkham M. Diagnosing depression in primary care using self-completed instruments: UK validation of PHQ-9 and CORE-OM. Br J Gen Pract. 2007;57(541):650–2. 23. Seedat S, Williams DR, Herman AA, Moomal H, Williams SL, Jackson PB, et al. Mental health service use among South Africans for mood, anxiety and substance use disorders. S Afr Med J. 2009;99(5 Pt 2):346–52. 24. Department of Health. National Mental Health Policy Framework and Strategic Plan 2013–2020. Pretoria: Department of Health; 2013. 25. Wu SF. Rapid screening of psychological well-being of patients with chronic illness: reliability and validity test on WHO-5 and PHQ-9 scales. Depress Res Treat. 2014;2014:239490. Conclusions 26. Pence BW, Gaynes BN, Atashili J, O’Donnell JK, Tayong G, Kats D, et al. Validity of an interviewer-administered patient health questionnaire-9 to screen for depression in HIV-infected patients in Cameroon. J Affect Disord. 2012;143(1–3):208–13. 27. Monahan PO, Shacham E, Reece M, Kroenke K, Ong’or WO, Omollo O, et al. Validity/reliability of PHQ-9 and PHQ-2 depression scales among adults living with HIV/AIDS in western Kenya. J Gen Intern Med. 2009;24(2):189–97. 28. Omoro SA, Fann JR, Weymuller EA, Macharia IM, Yueh B. Swahili translation and validation of the Patient Health Questionnaire-9 depression scale in the Kenyan head and neck cancer patient population. Int J Psychiatry Med. 2006;36(3):367–81. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 29. Williams LS, Brizendine EJ, Plue L, Bakas T, Tu W, Hendrie H, et al. Performance of the PHQ-9 as a screening tool for depression after stroke. Stroke. 2005;36(3):635–8. • Convenient online submission 30. McManus D, Pipkin SS, Whooley MA. Screening for depression in patients with coronary heart disease (data from the Heart and Soul Study). Am J Cardiol. 2005;96(8):1076–81. 31. Gilbody S, Richards D, Brealey S, Hewitt C. Screening for depression in medical settings with the patient health questionnaire (PHQ): a diagnostic meta-analysis. J Gen Intern Med. 2007;22(11):1596–602. 31. Gilbody S, Richards D, Brealey S, Hewitt C. Conclusions Screening for depression in medical settings with the patient health questionnaire (PHQ): a diagnostic meta-analysis. J Gen Intern Med. 2007;22(11):1596–602. 32. Adewuya AO, Ola BA, Afolabi OO. Validity of the patient health questionnaire (PHQ-9) as a screening tool for depression amongst Nigerian university students. J Affect Disord. 2006;96(1–2):89–93. 32. Adewuya AO, Ola BA, Afolabi OO. Validity of the patient health questionnaire (PHQ-9) as a screening tool for depression amongst Nigerian university students. J Affect Disord. 2006;96(1–2):89–93.
https://openalex.org/W2310737822	http://pure-oai.bham.ac.uk/ws/files/27970601/220_1408_3_PB.pdf	English	null	Influence of reduction conditions of NiO on its mechanical and electrical properties	Journal of electrochemical science and engineering	2,016	cc-by	5,860	Influence of reduction conditions of NiO on its mechanical and electrical properties p p Brodnikovskyi, Yehor; Vasyliv, Bogdan; Podhurska, Viktoriya; Andrzejczuk, Mariusz; McDonald, Nikkia; Kyrpa, Oleksandr; Ostash, Orest; Vasylyev, Oleksandr; Steinberger- Wilckens, Robert; Lewandowska, Malgorzata DOI: 10.5599/jese.220 Document Version Publisher's PDF, also known as Version of record Document Version Publisher's PDF, also known as Version of record Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Brodnikovskyi, Y, Vasyliv, B, Podhurska, V, Andrzejczuk, M, McDonald, N, Kyrpa, O, Ostash, O, Vasylyev, O, Steinberger-Wilckens, R & Lewandowska, M 2016, 'Influence of reduction conditions of NiO on its mechanical and electrical properties', Journal of Electrochemical Science and Engineering, vol. 6, no. 1, pp. 113-121. https://doi.org/10.5599/jese.220 Link to publication on Research at Birmingham portal General rights U l li General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. 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University of Birmingham University of Birmingham Citation for published version (Harvard): Brodnikovskyi, Y, Vasyliv, B, Podhurska, V, Andrzejczuk, M, McDonald, N, Kyrpa, O, Ostash, O, Vasylyev, O, Steinberger-Wilckens, R & Lewandowska, M 2016, 'Influence of reduction conditions of NiO on its mechanical and electrical properties', Journal of Electrochemical Science and Engineering, vol. 6, no. 1, pp. 113-121. https://doi.org/10.5599/jese.220 Open Access : : ISSN 1847-9286 www.jESE-online.org Original scientific paper Influence of reduction conditions of NiO on its mechanical and electrical properties Yehor Brodnikovskyi, Bogdan Vasyliv, Viktoriya Podhurska, Mariusz Andrzejczuk, Nikkia McDonald, Oleksandr Kyrpa, Orest Ostash, Oleksandr Vasylyev, Robert Steinberger-Wilckens*, Malgorzata Lewandowska Frantsevich Institute for Problems of Materials Science, 3 Krzhyzhanovsky Str., Kyiv, 03680, Ukraine Yehor Brodnikovskyi, Bogdan Vasyliv, Viktoriya Podhurska, Mariusz Andrzejczuk, Nikkia McDonald, Oleksandr Kyrpa, Orest Ostash, Oleksandr Vasylyev, Robert Steinberger-Wilckens*, Malgorzata Lewandowska Frantsevich Institute for Problems of Materials Science, 3 Krzhyzhanovsky Str., Kyiv, 03680, Ukraine Karpenko Physico-Mechanical Institute, 5, Naukova Str., Lviv, 79060, Ukraine Warsaw University of Technology, Faculty of Materials Science and Engineering, Wołoska Str., 141, 02-507 Warsaw, Poland Yehor Brodnikovskyi, Bogdan Vasyliv, Viktoriya Podhurska, Mariusz Andrzejczuk, Nikkia McDonald*, Oleksandr Kyrpa, Orest Ostash, Oleksandr Vasylyev, Robert Steinberger-Wilckens*, Malgorzata Lewandowska Frantsevich Institute for Problems of Materials Science, 3 Krzhyzhanovsky Str., Kyiv, 03680, Ukraine Karpenko Physico-Mechanical Institute, 5, Naukova Str., Lviv, 79060, Ukraine Warsaw University of Technology, Faculty of Materials Science and Engineering, Wołoska Str., 141, 02-507 Warsaw, Poland **Centre for Fuel Cell and Hydrogen Research, School of Chemical Engineering, University of Birmingham, Birmingham, B15 2TT, United Kingdom Corresponding Author: bregor@ukr.net; Tel.: +380-44-424-0294; Fax: +1-111-111-112 Received: September 16, 2015; Revised: December 10, 2015; Accepted: February 10, 2016 Corresponding Author: bregor@ukr.net; Tel.: +380-44-424-0294; Fax: +1-111-111-112 Received: September 16, 2015; Revised: December 10, 2015; Accepted: February 10, 2016 Take down policy Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to the work immediately and investigate. Download date: 24. Oct. 2024 J. Electrochem. Sci. Eng. 6(1) (2016) 113-121; doi: 10.5599/jese.220 Open Access : : ISSN 1847-9286 www.jESE-online.org Open Access : : ISSN 1847-9286 www.jESE-online.org Introduction Solid Oxide Fuel Cells (SOFCs) are energy conversion devices that convert chemical energy into electrical energy with higher conversion efficiencies and lower greenhouse gas emissions than conventional heat engines [1]. Traditional materials used in the manufacture of SOFCs include Ni- YSZ cermet for the anode, YSZ (8-10 mol. % Yttria Stabilized Zirconia) for the electrolyte and LSM- YSZ (Lanthanum Strontium Manganite – YSZ composite) for the cathode. These SOFCs typically operate on H2 at temperatures of 700-850 °C. Literature delivers vast information regarding SOFC component requirements [1]. Since this paper discusses research centered on anode material properties, anode component requirements will be the main focus. Ni has proven to be the best material for use in SOFC anodes due to its high catalytic activity for hydrogen oxidation, absence of undesirable chemical interactions with YSZ and good electronic conductivity, making Ni-YSZ the anode material of choice [2]. For simplification of anode manufacturing, NiO powder is typically used instead of Ni metal as Ni metal melts at temperatures commonly used for cell sintering. Because NiO does not form solid solutions with YSZ, the NiO-YSZ composite is reduced to a Ni-YSZ cermet during cell operation [3]. The reduction kinetics of NiO powders have been thoroughly studied [4-5], also it is known that the reduction of NiO to Ni is accompanied by volume and structural changes which influence the physical and mechanical properties of the anode [6-8]. It is clear that the transformation of NiO to Ni during reduction brings the changes into the anode structure and determines its final properties. To understand the impact of Ni phase transformations during reduction on the creation of Ni-metal networks within the anode composite the study of NiO properties after reduction are required. Moreover, normal SOFC start-up/shut down procedures where fuel flow is interrupted and system temperature is reduced may result in oxidation and reduction of Ni, which further greatly influences anode microstructure. This process, commonly referred to as RedOx cycling, is considered unfavorable by some researchers [8] due to drastic volume changes of the Ni phase, cracking and reduced strength of anode. While other researchers show RedOx cycling improves anode electrical and mechanical properties [9], at least initially. There is insufficient data to determine whether RedOx cycling improves or impairs the physical properties (electrical conductivity, mechanical strength) of Ni-YSZ anodes. Introduction To develop a better understanding of the influence of nickel phase on structure formation and properties of SOFC anode, it is important to study the microstructural changes of NiO samples and their properties before and after reduction and RedOx cycling. This work continues the previous study [10] and represents more details concerning formation of NiO structure under variable reduction conditions. The relationships between mechanical, electrical properties of treated NiO and its structure were established. Abstract Yttria stabilized zirconia with a nickel catalyst (Ni-YSZ) is the most developed, widely used cermet anode for manufacturing Solid Oxide Fuel Cells (SOFCs). Its electro-catalytic properties, mechanical durability and performance stability in hydrogen-rich environments makes it the state of the art fuel electrode for SOFCs. During the reduction stage in initial SOFC operation, the virgin anode material, a NiO-YSZ mixture, is reduced to Ni-YSZ. The volume decrease associated with the change from NiO-YSZ to Ni-YSZ creates voids and causes structural changes, which can influence the physical properties of the anode. In this work, the structural, mechanical and electrical properties of NiO samples before and after reduction in pure H2 and a mixture of 5 vol. % H2-Ar were studied. The NiO to Ni phase transformations that occur in the anode under reducing and Reduction-Oxidation (RedOx) cycling conditions and the impact on cell microstruc- ture, strength and electrical conductivity have been examined. Results show that the RedOx treatment of the NiO samples influence on their properties controversially, due to structural transformation (formation of large amount of fine pores) of the reduced Ni. It strengthened the treated samples yielding the highest mechanical strength values of 25.7 MPa, but from another side it is resulting in lowest electrical conductivity value of 1.9×105 S m-1 among all reduced samples. The results of this investigation shows that reduction conditions of NiO is a powerful tool for influence on properties of the anode substrate. Keywords NiO, Ni, RedOx, SOFC anode Keywords NiO, Ni, RedOx, SOFC anode doi:10.5599/jese.220 113 J. Electrochem. Sci. Eng. 6(1) (2016) 113-121 MECHANICAL AND ELECTRICAL PROPERTIES OF NiO Experimental In this study commercial NiO powder (d50 = ~300 nm, Donetsk Chemical Reagent Plant, Ukraine) was used. The NiO powder was wet ball milled in alcohol using ZrO2 media balls for 24 h and left to dry in air. The powder was passed through a 100 mesh sieve to achieve homogeneity and a narrow particle size distribution. Chromatographic thermal desorption (Sorbtometr P2, N2 - gas adsorbate, Ar – gas carrier) and laser granulometry (SK Laser Micron Sizer PRO-7000) studies were carried out to determine powder surface area and agglomerate sizes. The size of the initial particles (crystallites) was estimated from TEM images of the powder. Reduction and RedOx cycling studies were performed on 25 mm diameter and 1.5-2 mm thick disc shaped samples that were pressed uniaxially at 20 MPa and then sintered at 1400 °C for 2 h in air using a VMK 1600 114 Y. Brodnikovskyi et al. J. Electrochem. Sci. Eng. 6(1) (2016) 113-121 J. Electrochem. Sci. Eng. 6(1) (2016) 113-121 J. Electrochem. Sci. Eng. 6(1) (2016) 113-121 Linn High Therm furnace (Germany). To improve compaction of as-pressed samples, polyvinyl alcohol was used as a plasticizer. The porosity of the sintered NiO samples was measured by means of the Archimedes method. All NiO samples were divided into three groups and their reduction was carried out under three different testing conditions. The first group was reduced by holding the samples in high purity hydrogen (99.99 vol. % H2) at 600 °C for 4h. The second group was reduced by holding the samples in a 5 vol. % H2-Ar fuel mixture at 600 °C for 4h and the last group was exposed to 5 RedOx cycles. Each RedOx cycle consisted of an initial 1h dwell at 600°C in the 5 vol. % H2-Ar fuel mixture followed by a 1 h dwell at 600 °C in air and ending with a 1h hold at 600 °C in the 5 vol. % H2-Ar fuel mixture [9]. 600 °C for the reduction of NiO samples was chosen as temperatures above 600°C can promote not only the sintering of a reduced highly porous Ni-metal layer but also decrease the kinetics of the reduction process due to mass transfer limitations [5]. Experimental It was shown that tempera- tures within the 550-600 °C range are most effective for the reduction of NiO powders and that the reduction of NiO compacts at 600 °C preserves the newly formed pores inside the Ni-grains. Resistance measurements of the reduced samples were obtained by employing the DC 4-pt probe lateral method in air at 25°C. Mechanical strength tests for the non-reduced and reduced samples were performed by using the ring-on-ring biaxial bending method at room temperature [11]. Micrographs of the NiO grain shape and size were obtained using a TEM JEM100СXІІ, the cross- sectional microstructures of the reduced and non-reduced NiO samples were compared using a Superprobe 733 SEM (JEOL, Japan), and a high-resolution NB5000 dual-beam system microscope (Hitachi High Technologies Corporation, Japan) was used for internal structure characterization. 3D image analysis of the treated NiO was carried out using focused ion beam (FIB-SEM) microscopy. NiO porosity and post-reduction volume phase changes were calculated via estimation of surface area of different NiO phases contained within the SEM sample images by means of GIMP and ImageJ software. Microstructure The microstructure of the NiO samples in the non-reduced state is presented in Figure 2a. It can be seen that the sample consists of dense grains without visible defects along the boundaries and surfaces of the grains. The SEM image in Figure 2b shows a sample reduced in the 5-vol. % H2-Ar mixture at 600 °C for 4 h which has fine pores along the grain boundaries and no visible changes in grain surfaces. It appears that the reduction of NiO in the 5 vol. % H2-Ar mixture mostly effected on its grain boundaries. It is well known that the areas nearby grain contacts has the higher surface energy comparing to the grain surfaces what, for example, initiate diffusion processes and neck growth between grains during sintering of the powders compacts [12]. Thus, observed in Figure 2b structural changes of grain contacts can be explained by the aspiration of the material (NiO) to decrease its surface energy of the grain contacts via reduction of NiO to Ni. In other words, the grain boundary sites are more energy favored over grain surfaces for carrying out reduction processes. Also, it is known that the reduction of NiO in a hydrogen-containing medium begins from the nucleation of metallic clusters which then grow into crystallites at a near linear rate and this occurs at the interface between NiO and reduced porous Ni [4,5]. It means that the newly formed interface between NiO and reduced Ni is also more energy preferable site for further carrying out reduction processes comparing with the non-reduced NiO surface. Due to this, the primary reduction of the grain contacts results in further facilitation of the reduction front spreading in the direction from grains contacts to the grain cores due to already formed metallic Ni. This conclusion is in a good agreement with the FIB section of the reduced NiO samples in the 5 vol% H2-Ar mixture pictured in Figure 3a, which reveals much reduced grain contacts compared with the grain surfaces. As can also be seen in Figure 3a, as in Figure 2b, the NiO grains were only partly reduced - the core remained unaffected. The SEM micrograph obtained in BSE mode (Figure 3a), created a contrast in the imaging allowing a clear distinction between the Ni and NiO phases. The thickness of an outer Ni-metal rim surrounding the NiO grains is less than 400 nm. Powder characterization A summary of the characterized NiO powder is presented in Table 1. It can be seen that the powder has a low surface area of 3 m2 g-1 and consists of big agglomerates with an average size of 12.9 µm. Figure 1 represents the general view of the initial particles of the NiO powder. NiO samples made from this powder were highly porous with an average porosity of 40 ± 4 %. Table 1. Properties of NiO powder. Initial particles size, nm Agglomerate size, µm specific surface area, m2 g-1 100-150 12.9 3 Figure 1. TEM image of NiO powder Figure 1. TEM image of NiO powder doi:10.5599/jese.220 115 J. Electrochem. Sci. Eng. 6(1) (2016) 113-121 MECHANICAL AND ELECTRICAL PROPERTIES OF NiO Microstructure From Figure 3a it is possible to estimate the specific area of the Ni-metal rim relative to the total area of the grains. This estimation indicates that the Ni-metal rim is 11 % of the whole NiO volume. Thus, only about 11 % of the NiO phase was reduced to Ni in a mixture of 5 vol. % H2-Ar. The partial reduction of the NiO samples can be explained by insufficient contact between H2 (reducing agent) and the NiO surface due to the low content of H2 in the fuel and/or insufficient dwell time and/or too low operating temperature. These findings are in good agreement with the results of previous studies [13]. The detection of the reduction front nucleation and its spreading inside the grains of NiO helps to better understand the kinetics of the NiO reduction process. Nowadays, in order to explain the kinetics of the reduction process of NiO, the shrinking core model and the grain model employed by Szekely et al. [14,15] are generally used. The shrinking core model assumes that during reduction, the solid NiO particle consists of an un-reacted core (NiO), shelled by a uniform layer of the reaction product (Ni-metal rim). As the reaction proceeds, this layer thickens and the un- reacted core shrinks. In the grain model, a particle is considered an agglomerate of the individual grains. The reduction of such particles occurs as the reduction of individual grains, proceeding non-uniformly and causing reduction fronts to appear at different sites [4]. In the case of highly porous NiO samples, the presence of brachiate porous channels facilitates the reduction according to the grain model, following further clarification. Firstly, the appearance of the nucleation of the reduction front is not as random as the grain model suggests [4]. The 116 Y. Brodnikovskyi et al. J. Electrochem. Sci. Eng. 6(1) (2016) 113-121 J. Electrochem. Sci. Eng. 6(1) (2016) 113-121 reduction begins from specific sites with the highest surface energy such as grain contacts. It is possible to assume that the activity of these sites can be estimated via the dihedral angle between two given grains in the same way as for a sintering process [12]. Secondly, the direction of the reduction front spreading from grain contacts to the grain cores is not observed as a gradual reduction of individual NiO grains uniformly from grain surfaces to the cores as suggested by the shrinking core model. Microstructure Figure 2. Typical SEM images of fracture surfaces of NiO samples before (a) and after reduction in different conditions: b – 5 vol. % H2-Ar; c – pure H2; d – RedOx Figure 2. Typical SEM images of fracture surfaces of NiO samples before (a) and after reduction in different conditions: b – 5 vol. % H2-Ar; c – pure H2; d – RedOx Figure 3. SEM images of NiO sample after (a) reduction in mixture 5 % H2-Ar at 600 °C; (b) reduction in pure H2 at 600 °C; (c) RedOx in mixture 5 vol. % H2-Ar at 600 °C Figure 3. SEM images of NiO sample after (a) reduction in mixture 5 % H2-Ar at 600 °C; (b) reduction in pure H2 at 600 °C; (c) RedOx in mixture 5 vol. % H2-Ar at 600 °C The full conversion of NiO into Ni was achieved during reduction of the NiO samples in pure H2. This leads to a change in the grain surface as it becomes pitted with holes as seen it in Fig. 2c. The observed pits are most likely caused by the volumetric change of NiO to Ni during reduction. Research shows [7], that under reducing conditions, the physical dimensions (size/shape) of the initial NiO particles essentially stay the same but the volume changes of NiO-Ni occur by means of 117 doi:10.5599/jese.220 J. Electrochem. Sci. Eng. 6(1) (2016) 113-121 MECHANICAL AND ELECTRICAL PROPERTIES OF NiO pore formation inside the particles. The FIB sections of the NiO samples reduced in pure H2 at 600 oC reveal a sponge-like Ni structure with pore sizes in the range of 50 to 500 nm as presented in Figure 3b. These results are also in agreement with previously reported findings [7,13]. Correlating the specific area of the newly formed pores to the specific area of the grains could give an approximation of the apparent porosity of the post-reduced NiO samples. So, the volume of the inner pores is 30% relatively to the volume of the grains. The initial porosity of the samples was 40 % and the volume of NiO phase was 60%, accordingly. After reduction of NiO to Ni 30% of solid phase became the newly formed pores. This 30 % from the initial solid phase NiO is 18 % of additional porosity to the initial porosity value 40 %. Microstructure Thus, the total porosity of NiO sample after its reduction in pure H2 is about 58 %. The use of the 5 vol. % H2-Ar fuel mixture for the reduction of the NiO compacts was chosen to monitor the steps of the reduction process to better understand the kinetics of NiO reduction. While the use of the 5 vol. % H2-Ar gas mixture is not effective for full reduction of NiO to Ni-metal, it is very useful for studying the microstructural changes in NiO during reduction. Thus, this gas mixture was used to study the influence of RedOx treatment on the properties of NiO samples. An initial view of the SEM images of the RedOx treated samples pictured in Figure 2d, show peculiar changes on the surface of the grains when compared against samples in the initial state and those reduced in the 5 vol. % H2-Ar gas mixture as displayed in Figures 2a and b respectively. The edges of the grains became smooth while their surfaces became corrugated without the presence of big pitting holes as observed for samples reduced in pure H2. More detailed analysis of the RedOx treated samples reveal the transformation of the grain’s surface structure as pictured in Figure 3c. The formed Ni-metal rim contained a lot of fine pores and resembled a sponge-like structure, while the cores of grains remain unreduced. The thickness of the Ni-rim of the RedOx treated samples was thicker than what was observed in a single reduction in the 5 vol. % H2-Ar gas mixture. From Figure 3c the specific area of the Ni-metal rim relatively to the total area of the grains was estimated. This estimation indicates that Ni-metal rim is 19 % of total grain area. This is practically twice as much than what was seen during a single reduction treatment in the 5 vol. % H2-Ar gas mixture. The appearance of a larger volume of reduced Ni-layer in NiO samples after RedOx treatment, which includes oxidation cycles, can be explained by the creation of a porous structure on the Ni layer surface. This finding suggests that following each reduction cycle more volume of material is reduced due to an increase in the available sample area because of the presence of pores created during RedOx cycles. Practically, the RedOx treatment actualizes the step by step reduction of NiO increasing the thickness and volume of the reduced Ni-layer. Microstructure Moreover, comparing the structures of NiO samples pictured in Figures 3a and 3c makes it possible to observe the dynamics of the propagation of the reduction front. It is noticeable that the thickness of the reduced Ni-layer in a grains contact zone is thicker than the reduced Ni-layer from the surface of the pore. This shows that the reduction of NiO compacts occurs in regions where there is grain-to-grain contact and these sites are a priority zone for NiO reduction. RedOx treatment has an even stronger influence on the formation of Ni-to-Ni intergranular contact than what has been observed during a single reduction in the 5 vol. % H2-Ar gas mixture and pure H2. The difference in contacting between NiO grains after RedOx treatment and a single reduction in H2 is clearly seen in the SEM images of their surfaces pictured in Figures 4a and 4b respectively. In the case of a single reduction, the angularity of the grains are visible much like what is observed for unreduced NiO in Figure 2a but after RedOx treatment, the grains have 118 J. Electrochem. Sci. Eng. 6(1) (2016) 113-121 Y. Brodnikovskyi et al. smoother edges yielding a more rounded shape which serves to expand the contact area between neighboring grains as observed in Figure 4b. Figure 4. Typical SEM images of surfaces of NiO samples after RedOx treatment (a) and after single reduction in pure H2 (b) b a b a b a a Figure 4. Typical SEM images of surfaces of NiO samples after RedOx treatment (a) and after single reduction in pure H2 (b) Electrical conductivity Depending on the selected reduction medium the NiO samples were reduced to a varying degree. The mixture of Ar with 5-vol. % H2 can reduced NiO samples only partially. Only about 11 % of NiO was reduced to the Ni-metal as it was introduced above in paragraph 3.2. The grain surfaces and grain contacts were reduced to Ni-metal phase, but the grain cores left unreduced in NiO phase. NiO phase is a bad electronic conductor and limits the conductivity of the whole sample. Thus, only the reduced Ni-metal rim, a thin layer of the grain surfaces and grain contacts, can react as electronic conductor in this sample. Due to this, NiO samples reduced in the mixture 5 vol% H2-Ar had relatively low electrical conductivity of 3.3·105 S cm-1 as listed in Table 2. Despite of newly formed porosity during reduction and a quite high value of total porosity of 58% these samples had the highest electrical conductivity 2.7·106 S cm-1. This can be explained by the creation of the highest cross-sectional area of the conductor (Ni-metal) as a result of full transformation of the ceramic NiO sample into a Ni-metal sample. For RedOx treatment, a mixture of 5 vol% H2-Ar was used. Only partial reduction of NiO was observed in the treated samples. The reason is the same as in the case of single reduction of NiO in a mixture 5 vol% H2-Ar. As introduced in paragraph 3.2 above, RedOx treatment allowed reducing more amount of NiO (about 19 %) when comparing with single reduction in a mixture – which was about 11 %. Due to the limitation of electronic conduction (Ni-metal phase) it was expected that these samples had much lower conductivity than the samples reduced in pure H2, but they represented the lowest electrical conductivity of 1.9·105 S m-1 as listed in Table 2. In spite of the big difference in reduced Ni amount (practically double), a 42 % decrease in the electrical conductivity from 3.3·105 to 1.9·105 S m-1 for the NiO samples under RedOx treatment compared to the single gas mixture reduced samples was observed. This can be explained by newly formed fine porosity in the Ni-metal layer as seen in Figure 3c. Newly formed fine porosity as non- electronically conducting phase decreases the total conductivity of the Ni-rim due to thinning of the cross-sectional area of the conductor (Ni). Mechanical strength The results of this investigation on the mechanical strength of NiO samples before and after reduction in the aforementioned testing environments are summarized in Table 2. It can be seen that the mechanical strength of the samples increase after reduction and RedOx treatment when compared against the initial state. Table 2. Properties of NiO samples after reduction in different conditions [10]. State of NiO samples Strength, MPa Electric conductivity, S m-1 Initial state 13.3 – After reduction in 5 vol. % H2-Ar 15.9 3.3·105 After reduction in pure H2 18.8 2.7·106 After RedOx treatment 25.7 1.9·105 Table 2. Properties of NiO samples after reduction in different conditions [10]. Table 2. Properties of NiO samples after reduction in different con An increase in the mechanical strength of the 5 vol% H2-Ar reduced samples (15.9 MPa) when compared against samples in their initial non-reduced state (13.3 MPa) can only be related to the formation of Ni-Ni grain contacts what was discussed in paragraph 3.2. The presence of the ductile Ni metal component in the grain contacts improved the strength of entire sample. When examining sample mechanical strength, it is clear that Ni-Ni contacting yields higher values than NiO-NiO contacting. Despite the newly formed porosity (see paragraph 3.2), the strength (18.8 MPa) of the anode samples reduced in pure H2 was also relatively higher than that in the initial state as indicated in Table 2. Such behavior can be explained by full transformation of NiO to Ni-metal and as result appearance of plasticity in the samples. The strength of RedOx treated samples (25.7 MPa) is the highest compared with other samples. This obvious difference in strength can be explained by structural changes in the NiO samples after RedOx treatment. The newly formed fine porosity was observed in the Ni-rim of the RedOx treated samples as Figure 3c shows. It is seen that reduced grain contacts are very porous too. These fine pores complicate the passage of cracks through grain contacts and thus make them stronger. The close examination of the microstructure of the fracture surfaces picture in Figure 2 indicates the 119 doi:10.5599/jese.220 J. Electrochem. Sci. Eng. 6(1) (2016) 113-121 MECHANICAL AND ELECTRICAL PROPERTIES OF NiO cleavage facets only in RedOx treated samples (Figure 2d). The sizes of these facets are proportional to the sizes of the grains of the sample. Mechanical strength This implies that the sample fracture permeates through the bodies of the non-reduced NiO grains. It is evident that formed porous Ni- Ni grain contacts are stronger over the dense NiO grain bodies (core). Electrical conductivity In case of single reduction in the gas mixture the visible porosity in the Ni-rim was not observed as Figure 3a showed. Thus, a thin and dense Ni-rim formed after single reduction in gas mixture provides higher electrical conductivity compared with the thicker and highly porous Ni-rim formed after RedOx treatment. Conclusion Results show that the RedOx treatment of the NiO samples provides the formation of a large number of fine pores in the reduced Ni-metal layer. These newly formed fine pores influence on the properties of the NiO samples controversially. On one side, it strengthened intergranular contacts yielding the highest mechanical strength values of 25.7 MPa and, on the other side, resulted in the lowest electrical conductivity value of 1.9 . 105 S m-1 among all reduced samples. The mechanical strength of the NiO samples after reduction in pure H2 and 5 vol. % H2-Ar were 18.8 and 15.9 MPa respectively, while the strength of NiO samples before reduction (initial state) was 13.3 MPa. The electrical conductivity of reduced NiO in 5 vol. % H2-Ar (3.3 · 105 S m-1) was The mechanical strength of the NiO samples after reduction in pure H2 and 5 vol. % H2-Ar were 18.8 and 15.9 MPa respectively, while the strength of NiO samples before reduction (initial state) was 13.3 MPa. The electrical conductivity of reduced NiO in 5 vol. % H2-Ar (3.3 · 105 S m-1) was 120 Y. Brodnikovskyi et al. J. Electrochem. Sci. Eng. 6(1) (2016) 113-121 J. Electrochem. Sci. Eng. 6(1) (2016) 113-121 J. Electrochem. Sci. Eng. 6(1) (2016) 113-121 lower than the electrical conductivity of reduced NiO in pure H2 (2.7 106 S m-1) due to incomplete reduction of NiO. These results demonstrate the large influence the reducing conditions have on the physical properties of the Ni-phase component of Ni-based anodes and, thus, the performance of SOFC cells. Reducing conditions in start-up of virgin cells and conditioning by careful choice of operating conditions and/or planned initiating RedOx cycles can greatly predefine cell performance. It was also found that during reduction of the porous NiO samples, the nucleation of the reduction front begins in the intergranular contact zone and then spreads from there to the grain core. It was shown that this model of reduction front nucleation and spreading is more preferable than the assumption of uniform reduction as proposed in the shrinking core model. These results support the application of grain model NiO reduction kinetics for NiO agglomerates and improves the understanding of the behavior of reduction fronts throughout SOFC anodes. References [1] N.Q. Minh, Solid State Ionics 174 (2004) 271 – 277. [1] N.Q. Minh, Solid State Ionics 174 (2004) 271 – 277. [1] N.Q. Minh, Solid State Ionics 174 (2004) 271 – 277. [2] A. Atkinson, S. Barnett, R. J. Gorte, J. T. S. Irvine, A.J. McEvoy, M. Mogensen, S.C. Singhal, J. Vohs, Nature 3 (2004) 17–27. [3] R.F. Martins, M.C. Brant, R.Z. Dominques, Materials Research Bulletin 4 (2009) 451–456 [4] G. Plascenciaa,T. Utigard, Chemical Engineering Science 64 (2009) 3879–3888. [5] T. A. Utigard, M. Wu, G. Plascencia, T. Marin, Chemical Engineering Science 60 (2005) 2061 – 2068. [5] T. A. Utigard, M. Wu, G. Plascencia, T. Marin, Chemical Engineering Science 60 (2005) 2061 – 2068. [6] D. Sarantaridis, A. Atkinson, Journal of Fuel Cells 3 (2007) 246–258. [7] D. Waldbillig, A. Wood, D.G. Ivey, Journal of Power Sources 145 (2005) 206–21 [8] M. Ettler, H. Timmermann, J. Malzbender, A. Weber, N.H. Menzler, Journal of Power Sources 195 (2010) 5452–5467. [8] M. Ettler, H. Timmermann, J. Malzbender, A. Weber, N.H. Menzler, Journal of Power Sources 195 (2010) 5452–5467. [9] O. P. Ostash, B. D. Vasyliv, V. Podhurs’ka, O. D. Vasyl’ev, E. M. Brodnikovs’kyi, L. M. Ushkalov, Journal of Materials Science 46 (2011) 653–658. [9] O. P. Ostash, B. D. Vasyliv, V. Podhurs’ka, O. D. Vasyl’ev, E. M. Brodnikovs’kyi, L. M. Ushkalov, Journal of Materials Science 46 (2011) 653–658. [9] O. P. Ostash, B. D. Vasyliv, V. Podhurs’ka, O. D. Vasyl’ev, E. M Ushkalov, Journal of Materials Science 46 (2011) 653–658. [10] V.Ya. Podhurs’ka, B.D. Vasyliv, O.P. Ostash O. D. Vasyl’ev, E. M. Brodnikovs’kyi, Journal of Materials Science 49 (2014) 805 – 811. [10] V.Ya. Podhurs’ka, B.D. Vasyliv, O.P. Ostash O. D. Vasyl’ev, E. M. Brodnikovs’kyi, Journal of Materials Science 49 (2014) 805 – 811. [11] M. Radovic, E. Lara-Curzio, Acta Materialia Journal 52 (2004) 5747–5756. [11] M. Radovic, E. Lara-Curzio, Acta Materialia Journal 52 (2004) 5747–5756. [12] F.F. Lange, Journal of the European Ceramic Society 28 (2008) 1509-1516. [13] M. Andrzejczuk, O. Vasylyev, I. Brodnikovskyi, V. Podhurska, B. Vasyliv, O. Ostash, M. Lewandowska, K.J. Kurzydłowski, Materials Characterization 87 (2014) 159-165. [13] M. Andrzejczuk, O. Vasylyev, I. Brodnikovskyi, V. Podhurska, B. Vasyliv, O. Ostash, M. Lewandowska, K.J. Kurzydłowski, Materials Characterization 87 (2014) 159-165. [14] V. J.Szekely, J. W.Evans, H. Y. Soh, Gas Solid Reactions, Academic Press, New York, USA, 1976, 372p. [14] V. J.Szekely, J. W.Evans, H. Y. Conclusion Acknowledgments: the authors are grateful to the European FP7 NANOMAT-EPC project ”Deployment of Societally Beneficial Nano- and Material Technologies in European Partnership Countries” # 608906, the National Academy of Science of Ukraine, their projects “Hydrogen for alternative energetic and advanced technologies application” and "SOFC structural optimization based on consideration of interdiffusion at manufacturing and operation" for their respective support. © 2016 by the authors; licensee IAPC, Zagreb, Croatia. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/) References Soh, Gas Solid Reactions, Academic Press, New York, USA, 1976, 372p. [15] J. Szekely, J. W. Evans, Metallurgical Transactions. 2 (1971) 1699-1710. [15] J. Szekely, J. W. Evans, Metallurgical Transactions. 2 (1971) 1699-1710. © 2016 by the authors; licensee IAPC, Zagreb, Croatia. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/) 121 doi:10.5599/jese.220
https://openalex.org/W2161336117	https://discovery.ucl.ac.uk/1423648/1/c3ce41677_harding_13.pdf	English	null	Oriented crystal growth on organic monolayers	CrystEngComm	2,014	cc-by	7,839	2 Mechanisms for nucleation and growth on substrates One of the remarkable features of biomineralisation is the ability of the organic vesicle within which the mineral grows to control the orientation of the growing crystal. Natural sys- tems are highly complex; hence there has been a search for model systems that can reproduce this orientation control but are easier to perform controlled simulations and experi- ments on, and hence to understand. In their review, Song and Cölfen1 identified a number of systems that can control mineral orientation including Langmuir and self-assembled monolayers, latexes, colloidal crystals and insoluble scaffolds such as sea-urchin spine replicas or viruses. The review of Fricke and Volmer2 gives an extended discussion of experi- mental work on growth of calcium carbonate below insoluble monolayers (whether of surfactants or macrocyclic amphi- philes) and the insight this can give to the biomineralisation process. A number of parameters that could control the cho- sen orientation of the growing crystal have been investigated: epitaxial mismatch, charge density of the monolayer, tilt angle of the chains, nature of the substrate and the effects of the solution. We shall begin this review with a brief discus- sion of nucleation theories as background and then consider the experimental work that has been done in the area before turning to the use of simulations to understand how organised organic substrates control crystal growth. Theories and models of crystal nucleation and growth on substrates face similar challenges to those of homogeneous nucleation and growth. There are numerous possible mecha- nisms and pathways involved in going from isolated ions in supersaturated solution to a solid crystal and the dominating pathway depends on many factors. The most popular and successful model of crystallisation from supersaturated solution is classical nucleation theory (CNT), which is based on the idea that crystal nuclei form from fluctuations of ion concentrations in the solute, but these nuclei are unstable below a critical size. Adding an ion to a nucleus or cluster costs energy, due to the increased interfacial area, but this is offset by the energy gain from increasing the volume of the crystal. The critical nucleus size is reached when increasing the cluster size results in a net energy gain. The size of the critical nucleus, and hence the free energy barrier to nucleation, depends on the interfa- cial energy and the chemical potential difference of the crystallising species between the solid and the solvent. a Department of Materials Science and Engineering, University of Sheffield, Sheffield, UK. E-mail: j.harding@sheffield.ac.uk; Fax: +44 114 25943; Tel: +44 114 25957 b Department of Physics and Astronomy, University College London, London, UK. E-mail: d.duffy@ucl.ac.uk; Tel: +44 2076 793032 This journal is © The Royal Society of Chemistry 2014 CrystEngComm HIGHLIGHT View Article Online View Journal \| View Issue CrystEngComm HIGHLIGHT View Article Online View Journal \| View Issue iew Article Online Open Access Article. Published on 15 October 2013. Downloaded on 02/07/2015 16:54:00. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. J. H. Harding,*a C. L. Freemana and D. M. Duffyb Cite this: CrystEngComm, 2014, 16, 1430 Ordered organic substrates influence the crystallisation of minerals and different crystal morphologies and polymorphs can be stabilised by varying the properties of the substrates. The mechanisms behind this crystallisation control are not always apparent; however in recent years results from molecular modelling studies have led to an increased level of understanding. We present a review of the experimental evidence for crystallisation control by organic self-assembled monolayers and discuss the modelling methods that have been used to study these effects. We give an overview of the contribution modelling has made to the field of mineral crystallisation on organic substrates. The focus is on calcium carbonate because of its importance as a biomineral and, consequently, the large number of experimental and modelling studies that have been performed for this mineral. Open Access Article. Published on 15 October 2013. Downloaded on 02/07/2015 16:54:00. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2 Schematic showing various crystallisation mechanisms on surfaces. Circles represent ions, rectangles represent crystals and irregular shapes represent an amorphous phase. (i) shows crystal nucleation on the substrate followed by ion-by-ion addition. (ii) shows an amorphous cluster forming in solution followed by deposition on the substrate and an amorphous to crystalline transition (green arrows). In (iii) the amorphous cluster on the substrate crystallises via dissolution and re-precipitation. (iv) represents crystal growth via oriented attach- ment (yellow arrow). The possible mechanisms and pathways for crystallisation on substrates are more numerous and complex than those available to homogeneous nucleation (Fig. 2). Analogous to classical nucleation in a homogeneous solution, a critical cluster may form on the substrate and grow by ion addition.7 Amorphous clusters may form in solution and undergo an amorphous to crystalline transition after coming into con- tact with the substrate. The amorphous clusters may aggre- gate on the substrate, or form a continuous amorphous film, and then transform to a single crystal or polycrystalline film.8 In a process analogous to oriented attachment in bulk solution, small crystalline clusters may nucleate on the sub- strate and then aggregate all over the substrate through a diffusion mechanism. 2 Mechanisms for nucleation and growth on substrates Nucle- ation on a substrate is favoured when the interfacial energy is reduced by the crystal-substrate interaction as this lowers the size of the critical nucleus and, hence, the nucleation bar- rier. As the nucleation rate depends exponentially on the free energy barrier, a small reduction in the interfacial energy, due to favourable crystal substrate interactions, results in a significant enhancement of nucleation on the substrate com- pared to bulk solution. There are numerous approximations involved in classical nucleation theory, for example the calcu- lation of the surface and bulk energies may not give the values appropriate for small clusters, therefore numerical calculations of nucleation rates are often out by many orders This journal is © The Royal Society of Chemistry 2014 1430 \| CrystEngComm, 2014, 16, 1430–1438 Fig. 2 Schematic showing various crystallisation mechanisms on surfaces. Circles represent ions, rectangles represent crystals and irregular shapes represent an amorphous phase. (i) shows crystal nucleation on the substrate followed by ion-by-ion addition. (ii) shows an amorphous cluster forming in solution followed by deposition on the substrate and an amorphous to crystalline transition (green arrows). In (iii) the amorphous cluster on the substrate crystallises via dissolution and re-precipitation. (iv) represents crystal growth via oriented attach- ment (yellow arrow). Highlight View Article Online View Article Online CrystEngComm of magnitude. Nevertheless the concepts remain valid, partic- ularly if it is recognised that the chemical potential is not necessarily a monotonic function of the cluster size,4 and that several intermediate steps may be involved (Fig. 1). Recently there has been much discussion about non- classical nucleation,5 which is generally taken to mean that nucleation occurs via the aggregation of small clusters, rather than by the addition of single ions or formula units to a sub-critical cluster. The aggregating clusters may be amorphous (ACC in the figure) or crystalline. In the former case the amorphous clusters may undergo an amorphous to crystalline transition when the cluster reaches a critical size. The latter process is referred to as oriented attach- ment and this can result in the formation of mesocrystals,6 which are single crystals that retain some internal structure related to the subunits from which the macroscopic crystal is formed. This journal is © The Royal Society of Chemistry 2014 Open Access Article. Published on 15 October 2013. Downloaded on 02/07/2015 16:54:00. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Most of the studies on crystallisation (like those cited above) have used calcium carbonate. However, a number of other minerals have been considered including stron- tium carbonate,19 zinc oxide,20 barium sulphate,21 calcium phosphate,22 hydroxyapatite23 and hydrated iron oxides (lepidocrocite24 and goethite25). For a discussion of the use of SAMs to control the crystallisation of organic mole- cules, see the review of Singh et al.26 The length of the alkane chain can affect many properties of SAMs. Of particular importance are the so-called “odd– even” effects – the observation that SAMs with carbon chains having an odd number of carbon atoms (–S–C2n+1COO−) have different structures or properties to those with chains having an even number of carbon atoms (–S–C2nCOO−). A full review of the extensive literature can be found in.27 Our particular concern is how the alternation between odd and even SAMs can affect the ability of a SAM to control the crystallisation profoundly. Aizenberg and coworkers28 have observed that SAMs with an odd number of carbons in the chains (hereafter denoted odd SAMs) have a strong orientating effect, producing crystallisation in the (012) nucleating plane. On the other hand, even SAMs have a much weaker orienting effect and, if nucleation is observed, it tends to be in the (11m) plane where m is about three. OH functional groups can stabilise the amorphous phase.29 In the presence of free water, this transforms to calcite through a dissolution– reprecipitation mechanism. A more controllable transforma- tion can be achieved using a SAM with carboxylate functional groups. Foreign molecules and ions can be introduced into the amorphous calcium carbonate (ACC) and some become incorporated into the crystalline calcite. It has been suggested that this can be used to make nanostructures using complex patterned quasi-2D systems.30 Studies of crystallisation of calcium carbonate on SAMs15 demonstrated that epitaxial matching was not necessary for orientational control. Aizenberg et al.15 also demonstrated that some functional groups (OH, COO−and SO3 2−) give strong orientational control whereas others (PO3 2−) do not. Methyl termination, indeed, inhibits crystallisation. When solutions are replaced by an agarose gel16 the SAM still con- trols the orientation, but the aspect ratio (ratio of length to Fig. 3 16-Mercaptohexadecanoic acid (MHA) and its degrees of freedom. The carboxylic acid endgroup is free to rotate about γ. Reprinted with permission from A. M. Travaille, L. Kaptijn, P. Verwer, B. Hulsken, J. A. A. W. Elemans, R. J. This journal is © The Royal Society of Chemistry 2014 3 The experimental position There have been several major reviews on the experimental work on the use of monolayers to control crystal growth. Fricke and Volmer2 have discussed the work on calcium car- bonate in detail. Song and Cölfen1 consider a number of sys- tems in the context of additive control of crystal growth. We therefore shall summarise only the major points here. Most work has considered that the ability of organic monolayers to control crystal growth should be explained as some kind of templating effect. Early work9 used Langmuir monolayers as the templating agent for crystallisation. Since it was initially assumed that epitaxial matching was an essential part of the templating mechanism, the great flexibility of these layers to change their lattice parameters (provided this did not require great compression) was a perceived advantage. The degree of orientational control, however, is fairly low. Stripe et al.10 have used mixed monolayers to suggest that it is the average templating lattice that matters, not a detailed stereochemical fit. They find no effect of molecular tilt angle. Work from Volkmer and co-workers on Langmuir monolayers2,11 empha- sized the importance of the charge density at the monolayer In the next section we will review the experimental data in support of all the various mechanisms for crystallisation on organic substrates and the influence that substrates have on crystal polymorph and orientation. We will then review the theory and modelling techniques that have been used to study crystallisation on ordered organic substrates and the contribution these methods are making to the understanding of the influence of organic substrates on crystallisation processes. Fig. 1 Schematic showing the free energy variation for a variety of possible pathways for crystallisation from ions in solution. DOLLOP (Dynamically Ordered Liquid-Like Oxyanion Polymer) refers to the prenucleation clusters identified in ref. 3, (image courtesy of R. Darkins). Fig. 1 Schematic showing the free energy variation for a variety of possible pathways for crystallisation from ions in solution. DOLLOP (Dynamically Ordered Liquid-Like Oxyanion Polymer) refers to the prenucleation clusters identified in ref. 3, (image courtesy of R. Darkins). Fig. 1 Schematic showing the free energy variation for a variety of possible pathways for crystallisation from ions in solution. DOLLOP (Dynamically Ordered Liquid-Like Oxyanion Polymer) refers to the prenucleation clusters identified in ref. 3, (image courtesy of R. Darkins). 3 The experimental position CrystEngComm, 2014, 16, 1430–1438 \| 1431 This journal is © The Royal Society of Chemistry 2014 View Article Online Highlight CrystEngComm CrystEngComm surface rather than epitaxial matching in controlling which phase of calcium carbonate is formed. The role of pre- nucleation clusters has been emphasized, with cryo-TEM suggesting that the initial stages of formation of CaCO3 on monolayers12 involves such species. Aggregation of pre- nucleation clusters has also been observed in the crystalliza- tion of calcium phosphate.13 width) is reduced – probably a consequence of the incorpora- tion of agarose fibres into the crystal. The use of monocrys- talline mica substrates can increase the orienting effect of SAMs by suppressing the microdomain structure of the gold films evaporated onto them, producing a SAM that is ordered over long distances.17 Moreover, the observed control is dif- ferent from that seen for Langmuir monolayers. In that case orientation in the (100) and (001) directions was observed.18 These authors ascribed these effects to the symmetry of the functional head-group. A detailed review of the preparation and use of self assem- bled monolayers (SAMs) is given in ref. 14. Most of the SAMs used in mineral ( ) 3 3 30   R crystallisation are deposited on gold or silver substrates, most commonly on the Au (111) surface, using thiolates. For a high-coverage thiol array, the bonding habit is usually where the sulphur atoms are in the hollows of the gold lattice. The perfect surface unit cell of the alkane chains then has the symmetry c(4 × 2). The alkane chains are nearly all in the trans configuration and the chain is tilted, typically at a canting angle (α) of 30° and rotation angle (β) of 50° (see Fig. 3 for definitions). In practice, SAMs contain defects. These can be caused by metal defects in the substrate, grain boundaries or steps. Complex phase changes are possible with the introduction of gauche conformers into the system. Also, particularly for short chain lengths, the SAM can be disordered. In the simulations that we shall discuss, these complications are ignored since they usually occur on larger length-scales than those attainable by atomistic simulation. Their presence, however, should be considered when comparing with experiment. Open Access Article. Published on 15 October 2013. Downloaded on 02/07/2015 16:54:00. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. M. Nolte and H. van Kempen, J. Amer. Chem. Soc., 2003, 125, 11571.43 ©2003 American Chemical Society. Direct effect of the solution can also affect the crystallisation. Aizenberg and co-workers31 point out that the order in which the calcium and carbonate containing solutions are added determines the efficacy of the monolayer – adding the calcium solution first greatly strengthens the orientating effect. Different concentrations of calcium ion affect the size, shape and orientation of calcite crystals on carboxylate monolayers.32 On alcohol terminated monolayers, low concen- trations of calcium give vaterite, whereas high concentrations give calcite rhombs in the standard (104) nucleating plane. Fig. 3 16-Mercaptohexadecanoic acid (MHA) and its degrees of freedom. The carboxylic acid endgroup is free to rotate about γ. Reprinted with permission from A. M. Travaille, L. Kaptijn, P. Verwer, B. Hulsken, J. A. A. W. Elemans, R. J. M. Nolte and H. van Kempen, J. Amer. Chem. Soc., 2003, 125, 11571.43 ©2003 American Chemical Society. A considerable body of work has emphasised the impor- tance of monolayer flexibility for both Langmuir monolayers and SAMs. Popescu et al.33 have prepared a set of Langmuir This journal is © The Royal Society of Chemistry 2014 1432 \| CrystEngComm, 2014, 16, 1430–1438 View Article Online View Article Online View Article Online Highlight CrystEngComm to the ratio of the difference between the surface/crystal and surface/fluid interfacial free energies to the crystal/fluid inter- facial free energy. to the ratio of the difference between the surface/crystal and surface/fluid interfacial free energies to the crystal/fluid inter- facial free energy. monolayers where they have systematically changed the den- sity of the surfactant monolayers. Although all the mono- layers can nucleate calcite, only those that are flexible enough to allow the rearrangement of the molecules can affect the morphology. Lee et al.34 have prepared SAMs of varying flexibility by using mercaptodecyl benzoic acids with different placing of the functional carboxylate group on the aryl ring with respect to the alkyl chain. Only the para case produces oriented growth. This is the configuration that shows by far the greatest flexibility due to the reduced hydro- gen bonding of this isomer.35 The crystal and monolayer (in effect) template one another to find the most stable inter- facial structure; this is so-called cooperative crystallisation. 4.1 Classical theory of heterogeneous nucleation Here γc and γs are the surface free energies of the crystal and substrate respectively and βsc is the interfacial binding free energy, which is the free energy (per unit area) gained by joining the crystal and the substrate. βsc is, generally, negative and large negative values are representative of strong interac- tions between the crystal and the substrate. In the case where the substrate is a monolayer this definition is invalid as we cannot define the surface energy of the monolayer – the monolayer is already a surface. However if we consider only the difference between the interfacial free energies γsc −γsf the problematic monolayer surface energy cancels. We can, therefore, express m in terms of well-defined parameters. The effect of a substrate on the nucleation of a solid from a fluid can be understood in terms of CNT (see, for example, Liu et al.7). The free energy change (ΔG) due to the formation of a nucleus of volume Vs in a bulk fluid is:    G V S   s cf cf  (1) (1) Here Δμ is the chemical potential, Ω is the atomic volume, γcf is the crystal/fluid interfacial free energy and Scf is the sur- face area of the crystal nucleus. For nucleation on a substrate we need to adjust the interfacial energy term to take account of the part of the crystal that is in contact with the substrate: m                   sc sf cf c f sc sf c f cf (5) (5)   G V S S           s cf cf sc sf sc  (2) (2) The enthalpic component of the surface and interfacial binding free energies can be calculated using molecular dynamics,39 at least when the surfaces are charge neutral and there is good epitaxial match between the substrate and the crystal. Complications arise when we consider polar crystal surfaces nucleating on ionised substrates. In the absence of Here γsc is the substrate/crystal interfacial free energy, γsf is the substrate/fluid interfacial free energy and Ssc is the area of the crystal in contact with the substrate. 4 Modelling studies The gulf between the timescales that can be modelled by atomistic simulations (nanoseconds) and the timescales required for nucleation (minutes to hours) generally inhibits the direct simulation of nucleation events. Understanding can, however, be greatly enhanced by employing simulation methods to calculate relevant interfacial and surface ener- gies. Accelerated modelling methods, such as metadynamics for exploring nucleation pathways36 and Kinetic Monte Carlo methods for modelling growth,37 have also contributed much to the understanding of the fundamental mechanisms. Here we present the modified CNT for heterogeneous nucleation to highlight the relevant interfacial energies that can be calcu- lated using atomistic simulations. We then present a review of the high temperature molecular dynamics and metadynamics simulations that have been used to model crystallisation of amorphous calcium carbonate on self-assembled monolayers. Under certain conditions the enthalpic components of the interfacial free energies in eqn (2) and (3) (γcf, γsc, γsf) can be calculated from atomistic simulations. This is straight forward when the substrate is the surface of a 3-dimensional material as the interfacial free energy (γsc) is defined uniquely as: γsc = γc + γs + βsc (4) (4) This journal is © The Royal Society of Chemistry 2014 Open Access Article. Published on 15 October 2013. Downloaded on 02/07/2015 16:54:00. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. m        sc sf cf (3) (3) m is close to +1 for crystals that bind strongly to the substrate, in which case the cluster will be thin with a low contact angle. For weak crystal/surface interactions, m is close to −1 and the spherical cluster just touches the surface, resulting in a heterogeneous nucleation rate close to the homogeneous nucleation rate. The ratio of the heterogeneous to homogenous nucleation barriers, and thus the nucleation rate, increases with increasing m. The case of facetted clusters is more complex as the crystal/fluid area is a function, not only of the height of the nucleus above the surface, but also of the orientation of the crystal facet in contact with the sub- strate. Travaille et al.38 have calculated the contact area for different ranges of the interfacial energies for the particular case of calcite with a {012} surface in contact with the sub- strate and low energy {104} surfaces in contact with the fluid. They demonstrated that the spherical cap approximation was reasonable, at least for this particular case. 4.2 High temperature molecular dynamics In general, the symmetry of the crystal surface will not be the same as the substrate, therefore the epitaxial match will be better in some directions than others. Experimentally, this anisotropic epitaxial mismatch results in the growth of elon- gated crystals (Fig. 4).43 Indeed more mature crystals grow off the substrate such that the area in contact with the sub- strate remains constant as the crystal grows. This suggests that at some point during the growth, the unfavourable epi- taxial strain energy dominates the favourable substrate/crys- tal energy. Pokroy and Aizenberg44 modelled this effect by extending the Hartman–Perdok growth model45 with an addi- tional strain energy term to take account of misfit strain. As the misfit strain is, in general, anisotropic; the growth rates in different directions will be different, resulting in elongated crystals. Pokroy et al. calculated the differential growth rate by considering the strain to be accommodated by elastic deformation of the crystal and substrate. Large mismatch or Modelling the interfacial interactions and energies is one method available to computer simulation. More recently, to understand the morphology control of organic substrates better, modellers have turned to direct simulation of the crystallisation process. By its very nature, crystallisation will require the system to traverse a significant energy barrier, large enough to make it a rare event. These rare event pro- cesses are not seen in standard molecular dynamics where the timescale of the simulation (100 ns or less) does not generate a true time average of the system and allow for the necessary exploration of an entire energy surface. Therefore it is essential for modellers to devise methods to accelerate the exploration of the energy surface. A range of methods exist48 – mostly these involve some form of path sampling where the simulation is forced to remain close to a particular route by an energy biasing. This process can be very effective but only when that pathway is already reasonably well characterised. Unfortunately the actual atomic process of crystallisation is poorly understood and this type of path sampling could potentially dramatically alter the actual process that we wish to sample. Therefore methods that require less specific biasing of the simulation are needed. Fig. 4 Calcite crystals grown on a SAM of MHA. Note the elongation of the more mature crystals. Reprinted with permission from A. M. Travaille, L. Kaptijn, P. Verwer, B. Hulsken, J. A. A. W. Elemans, R. Open Access Article. Published on 15 October 2013. Downloaded on 02/07/2015 16:54:00. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. From a modelling perspective, the computational surface unit cell of the substrate and the crystal must be identical, therefore any lattice mismatch must be accommodated by adjusting the surface lattice parameters of the crystal, the sub- strate, or both. The induced strain energy can be cancelled by calculating the bulk reference energies using the same com- putational cells. For large lattice mismatch the misfit can be accommodated by line defects or misfit dislocations and, again, overall charge neutrality must be maintained. In the case of calcium carbonate it is possible that bicarbonate ions may play a role in charge neutralisation.42 In summary, atomistic modelling can contribute to the understanding of nucleation and growth on substrates through the calculation of the various interfacial energies associated with the free energy barrier and critical nucleus. These do not provide absolute nucleation rates but they can be used to predict which crystal faces will be favoured on particular substrates. This journal is © The Royal Society of Chemistry 2014 4.1 Classical theory of heterogeneous nucleation When the nucleus is a spherical cap, the position of intersection of the sphere with the surface (the cosine of the contact angle, m) is related CrystEngComm, 2014, 16, 1430–1438 \| 1433 This journal is © The Royal Society of Chemistry 2014 View Article Online Highlight CrystEngComm large interfacial area would, however, induce excessive strains therefore one might expect the misfit to be accommodated by defects such as misfit dislocations. These dislocations would also cost energy and result in the same anisotropic growth as that predicted by the elastic strain model and, indeed, result in a reduced footprint on the substrate.46 Modelling the interface with the growing crystal raises further questions as the state of the system before crystallisation is generally unknown – i.e. are intermediates present at the interface? Simulations have attempted to calculate the inter- facial energy with different disordered calcium carbonate structures47 showing a potential preference for particular SAM structures. reconstruction or neutralising species, the separated crystal and substrate have infinite energy so the challenge is then to obtain a meaningful definition of the interfacial energy that corresponds to the parameter that is relevant to the nucle- ation event. If we assume that the reference state (before the formation of the nucleus) is the Ca2+ and CO3 2−ions in solu- tion with sufficient Ca2+ ions adsorbed on the substrate to neutralise the charge, we can determine the interfacial energy by calculating the surface energy of the polar surface by reducing the charge density of the outer layer of the slabs by half,40 thereby quenching the dipole.41 4.2 High temperature molecular dynamics J. M. Nolte and H. van Kempen, J. Amer. Chem. Soc. 2003, 125, 11571.43 ©2003 American Chemical Society. The most obvious mechanism to push a simulation over an energy barrier associated with crystallisation is to increase the temperature and hence the energy in the system. Duffy and Harding demonstrated this approach for CaCO3 on the SAM system.49 Although this approach works in many inor- ganic systems, increasing the temperature has the drawback that the monolayer molecules must be frozen as their struc- ture will not survive the high temperatures. The simulations used a melted CaCO3 cluster on charged odd and even mono- layers. The cations and anions could clearly be seen ordering into layers of alternating charge. Within these layers some Fig. 4 Calcite crystals grown on a SAM of MHA. Note the elongation of the more mature crystals. Reprinted with permission from A. M. Travaille, L. Kaptijn, P. Verwer, B. Hulsken, J. A. A. W. Elemans, R. J. M. Nolte and H. van Kempen, J. Amer. Chem. Soc. 2003, 125, 11571.43 ©2003 American Chemical Society. This journal is © The Royal Society of Chemistry 2014 1434 \| CrystEngComm, 2014, 16, 1430–1438 View Article Online CrystEngComm Highlight further ordering could be observed particularly on the even monolayer with the radial distribution function suggesting a calcite-like structure being formed and with a (001) surface. Further analysis of this temperature-based crystallisation50 confirmed the (001) surface. Artificial rearrangement of the head-groups was performed and it was found that the only way to induce a surface other than the (001) surface was to completely alter the organisation of the head-groups to match the (012) calcite surface (Fig. 5). These temperature- based simulations with frozen monolayers suggested that the predicted experimental structure of the monolayer that matches the (001) calcite surface so well was only able to induce the (001) calcite surface in disagreement with experimental results. are systematically added to a cluster and with each addition a range of potential sites is explored to identify the lowest energy addition. This whole system is then minimised before proceeding to the next addition. They utilised this methodology to explore calcium phosphate addition to an analogue of a collagen molecule.53 Exploring how this molecule influences the growth of calcium phosphate has obvious implications for bone development. 4.3 Accelerated simulation methods The two studies discussed in the previous section clearly highlighted that a frozen monolayer approach is not able to represent the crystallisation of calcite on the SAMs correctly. It could be expected that in many cases the flexibility and mobility of the organic substrate may play a crucial role in the crystallisation process. Therefore further methods that can include this have been explored. g y For organic substrate control this method was first used by Quigley et al.55 to model the crystallisation of calcite on SAMs. They considered a large matrix of different configura- tions varying the length of the monolayer chains, their ionisation and the presence of water molecules. Their simula- tions observed the formation of the (012) calcite surface on the even SAMs as experimentally reported which suggested in agreement with the earlier high temperature studies that the flexibility of the substrate was a crucial factor. Fig. 6 shows the results of metadynamics simulations of the crystallisation of ACC on an MHA SAM. When the substrate is frozen the crystal grows in the (001) orientation (Fig. 6a) whereas on the flexible substrate the (012) orientation is formed (Fig. 6b). In the (012) orientated structure, distortion of the SAM ensures localized areas of good match interspersed with areas of poor substrate/crystal epitaxy. We have recently shown that the (001) oriented structure has lower energy than the (012) structure, which indicates that kinetic factors dominate the orientation selection. Although metadynamics allows us to explore the system with a limited amount of biasing it still imposes important restrictions on our study. The bias within the simulation is applied to all the atoms of interest so all the Ca2+ and CO3 2−ions are being encouraged to order across the whole disordered cluster. Obviously crystallisation is generally a local event and not a global one but we are apply- ing a global biasing. In addition, metadynamics is limited by its order parameters and therefore we must be able to One methodology employed by Kawska et al.52 was to use a Monte Carlo type approach within a docking protocol. Ions Fig. 5 Shows the formation of the (A) (001) calcite and (B) (012) calcite surfaces depending on the selected frozen structure of the SAM. Figure reproduced with permission from C. L. Freeman, J. H. Harding and D. M. Duffy, Langmuir, 2008, 24, 9607.50 ©American Chemical Society. Fig. Open Access Article. Published on 15 October 2013. Downloaded on 02/07/2015 16:54:00. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. High temperature simulations have also been used to explore calcite crystallisation on calcite substrates and have suggested that the growth rates of calcite surfaces do not follow a simple pattern based on their surface energies.47,51 These results suggest that the growing crystal may have pre- ferred orientations that will be sought out while in contact with the substrate. Metadynamics54 is another methodology to tackle the issue of crystallisation. At its core, metadynamics defines a system by a series of order parameters, which for the case of crystallisation will be the spacing and orientation of the ions (i.e. structure factors). As the simulation runs, a record is kept of the order parameters for each configuration sampled and then a bias is applied to the system to encourage it not to return to these specific order parameter values. Via this approach the system is forced to explore more and more rare events, eventually leading to a crystallisation process. The clear advantage of this method is that it does not fundamentally force the system to follow a particular pathway nor does it alter the ensemble conditions (i.e. temperature). The simulation, is however, massively controlled by the choice of order parame- ter and the rate of biasing which must be carefully selected. This journal is © The Royal Society of Chemistry 2014 CrystEngComm, 2014, 16, 1430–1438 \| 1435 4.2 High temperature molecular dynamics This method does not actually encourage crystallisation and during the simulations only a small degree of order was observed to occur within the cluster formed. Without any actual direct encouragement of crystallisation the method remains largely restricted to analysing aggrega- tion. This obviously may be a crucial step on the pathway to the final crystal. 4.3 Accelerated simulation methods 5 Shows the formation of the (A) (001) calcite and (B) (012) calcite surfaces depending on the selected frozen structure of the SAM. Figure reproduced with permission from C. L. Freeman, J. H. Harding and D. M. Duffy, Langmuir, 2008, 24, 9607.50 ©American Chemical Society. CrystEngComm, 2014, 16, 1430–1438 \| 1435 This journal is © The Royal Society of Chemistry 2014 1435 View Article Online Highlight Highlight Fig. 6 Structures formed by the crystallisation of ACC on MHA SA using metadynamics. In (a) the SAM is frozen and the crystal forms the (001) orientation, whereas in (b) the substrate molecules are f to move and the crystal forms in an (012) orientation (image court of A. S. Côté and A. Travis). Highlight Open Access Article. Published on 15 October 2013. Downloaded on 02/07/2015 16:54:00. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. CrystEngComm CrystEngComm ight the substrate – ensuring that different regions of the substrate promote the growth of different orientations or polymorphs of a given material or even different materials. A limitation on the structures produced has been the imperfections found in the monolayer, whether produced by defects in the underlying metal substrate or disorder in the monolayer itself. Although most work has been done on calcium carbonate, it is clear that the process is applicable to a wide range of minerals and also to organic molecules. This is also known in biological systems, in particular the formation of nacre. Open Access Article. Published on 15 October 2013. Downloaded on 02/07/2015 16:54:00. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Ideas concerning the mechanism have changed consider- ably with contributions from both simulation and experiment. The original suggestion that control was due to epitaxial matching proved to be far too simple, neglecting the fact that an interface involving a flexible substrate allows many possi- bilities of evading the requirements of strict geometrical epi- taxy. Moreover, the best epitaxial match need not be the interface that is the most stable. This is shown by the rarity of formation of the (001) orientation – which has an excellent epitaxial match – compared to the (012) orientation where the epitaxial matching is much poorer. Considerations of electro- statics are, however, important as Volkmer and coworkers2,11 have argued from the experimental data on the systems they have studied and as simulations have born out. 4.3 Accelerated simulation methods Much work remains to be done on the mechanism of for- mation. It is clear that an amorphous phase is part of the process of formation. However whether the crystalline phase is obtained by direct transformation or by a dissolution– reprecipitation mechanism depends on circumstances. If direct transformation occurs, there is the further question of how and when the water is removed from the amorphous phase (which is always hydrated, at least initially) to produce the anhydrous vaterite, aragonite or calcite phases. Also, where direct transformation occurs, if the cation or anion is highly asymmetric (as is the case for calcium carbonate), the initial distribution of the orientation of the asymmetric ion can play an important part in determining the final morphology of the growing crystal. Fig. 6 Structures formed by the crystallisation of ACC on MHA SAMs using metadynamics. In (a) the SAM is frozen and the crystal forms in the (001) orientation, whereas in (b) the substrate molecules are free to move and the crystal forms in an (012) orientation (image courtesy of A. S. Côté and A. Travis). Since so much of the experimental work has been done on the calcium carbonate system, an important question is how specific the conclusions are to that material. As noted above, studies have been performed on other systems but to nothing like the same extent as for calcium carbonate. Also, calcium carbonate remains the only system where extensive simula- tions have been performed. In this system, close collabora- tion between experimentalists and simulators has enabled a detailed picture to be developed of the role of SAMs in con- trolling crystal orientation. However, if SAMs are to fulfil their potential for applications, there needs to be more work to see whether the lessons learned from this (comparatively) well-understood system are applicable more generally. design parameters that will sample what we need, which can be challenging. 5 Summary and conclusions Much effort (both theoretical and experimental) has been put into understanding how self-assembled monolayers can control crystallisation. This is both because these systems have been considered as model systems for biomineralisation and in their own right as nanostructures and the foundation of nanostructures. The ability to make SAMs that contain patterns of different head-groups suggests that it is possible to grow complex structures and composites by careful design of This journal is © The Royal Society of Chemistry 2014 References 20 J. W. P. Hsu, M. W. Clift and L. N. Brewers, Langmuir, 2008, 24, 5375. 1 R.-Q. Song and H. Cölfen, CrystEngComm, 2011, 13, 1249. 21 H. Eun and Y. Amasuma, Anal. Chim. Acta, 1998, 375, 155. 2 M. Fricke and D. Volmer, Top. Curr. Chem., 2007, 270, 1. 22 Q. Liu, J. Ding, F. K. Mante, S. L. Wunder and G. R. Baran, Biomaterials, 2002, 23, 3103. 3 R. Demichelis, P. Raiteri, J. D. Gale, D. Quigley and D. Gebauer, Nat. Commun., 2011, 2, 590. 23 D. P. Liu, P. Majewski, B. K. O'Neill, Y. Ngothai and C. B. Colby, J. Biomed. Mater. Res., Part A, 2006, 77, 763. 4 W. J. E. M. Habraken, J. Tao, L. J. Brylka, H. Friedrich, L. Bertinetti, A. S. Schenk, A. Verch, V. Dmitrovic, P. H. H. Bomans, P. M. Frederik, J. Laven, P. van der Schoot, 4 W. J. E. M. Habraken, J. Tao, L. J. Brylka, H. Friedrich, L. Bertinetti, A. S. Schenk, A. Verch, V. Dmitrovic, P. H. H. Bomans, P. M. Frederik, J. Laven, P. van der Schoot, B. Aichmayer, G. de With, J. J. DeYoreo and N. A. J. M. Sommerdijk, Nature Comm., 2013, 4, 1507. 24 M. Nagtegaal, P. Stroeve and W. Tremel, Thin Solid Films, 1998, 327, 571. 25 B. J. Tarasevich, P. C. Rieke and J. Liu, Chem. Mater., 1996, 8, 292. 5 D. Erdemir, A. Y. Lee and A. S. Myerson, Acc. Chem. Res., 2009, 42, 621; D. Gebauer, A. Volkel and H. Cölfen, Science, 2008, 322, 1819; J. J. de Yoreo, Nat. Mater., 2013, 12, 284; D. Kashchiev, P. G. Vekilov and A. B. Kolomeisky, J. Chem. Phys., 2005, 122, 244706; B Mutaftschiev, The atomistic nature of crystal growth, Springer-Verlag, 2001; S. Whitlam, J. Chem. Phys., 2010, 132, 94901; D. Gebauer and H. Cölfen, Nano Today, 2011, 6, 564. 26 A. Singh, I. S. Lee, K. Kim and A. S. Myerson, CrystEngComm, 2011, 13, 24. 27 F. Tao and S. L. Bernasek, Chem. Rev., 2007, 107, 1408. 28 Y. J. Han and J. Aizenberg, Angew. Chem., Int. Ed., 2003, 42, 3668. 29 T. Y.-J. Han and J. Aizenberg, Chem. Mater., 2008, 20, 1064. 30 J. Aizenberg, D. A. Muller, J. L. Grazul and D. R. Hamann, Science, 2003, 299, 1205. 31 J. Aizenberg, A. J. Black and G. M. Whitesides, J. Am. Chem. Soc., 1999, 121, 4500. 6 H. References Cölfen and M. Antonietti, Angew. Chem., Int. Ed., 2005, 44, 5576; J. Fang, B. Ding and H. Cölfen, Chem. Soc. Rev., 2011, 40, 5347; R.-Q. Song and H. Cölfen, Adv. Mater., 2012, 22, 1301. 32 H. Deng, X.-M. Wang, C. Du, X.-C. Shen and F.-Z. Cui, CrystEngComm, 2012, 14, 6647. 7 X. Y. Liu and S. W. Lim, J. Am. Chem. Soc., 2003, 125, 888J. J. De Yoreo and P. Vekilov, Principles of crystal nucleation and growth, in Biomineralization., ed. P. M. Dove, J. J. de Yoreo and S. Weiner, Mineral Soc Am, Washington, DC, 2003, pp. 57–93. 33 D. Popescu, M. M. J. Smulders, B. P. Pichon, N. Chebotareva, S.-Y. Kwak, O. L. J. van Asselen, R. P. Sijbesma, E. DiMasi and N. A. J. M. Sommerdijk, J. Am. Chem. Soc., 2007, 129, 4058. 34 J. R. I. Lee, T. Y.-J. Han, T. M. Willey, D. Wang, R. W. Meulenberg, J. Nilsson, P. M. Dove, L. J. Terminello, T. van Buuren and J. J. De Yoreo, J. Am. Chem. Soc., 2007, 129, 10370. 8 A. Dey, G. de With and N. A. J. M. Sommerdijk, Chem. Soc. Rev., 2010, 39, 381. 35 A. S. Côté, C. L. Freeman, R. Darkins and D. M. Duffy, J. Phys. Chem. C, 2013, 117, 7148. 9 B. R. Heywood and S. Mann, Chem. Mater., 1994, 6, 311. 10 B. Stripe, S. Uysal, B. Lin, M. Meron and P. Duttaz, Langmuir, 2012, 28, 572. 36 D. Quigley, C. L. Freeman, J. H. Harding and P. M. Rodger, J. Chem. Phys., 2011, 134, 044703. 11 F. F. Amos, D. M. Sharbaugh, D. R. Talham, L. B. Gower, M. Fricke and D. Volkmer, Langmuir, 2007, 23, 1998; M. Fricke, D. Volkmer, C. E. Krill III, M. Kellermann and A. Hirsch, Cryst. Growth Des., 2006, 6, 1120. 37 J. M. McCoy and J. P. LaFemina, Surf. Sci., 1997, 373, 2889; R. E. Williford, D. R. Baer, J. E. Amonette and A. S. Lea, J. Cryst. Growth, 2004, 262, 503. 38 A. M. Travaille, E. G. A. Steijven, H. Meekes and H. van Kempen, J. Phys. Chem. B, 2005, 109, 5618. 12 E. M. Pouget, P. H. H. Bomans, J. A. C. M. Goos, P. M. Frederik, G. de With and N. A. J. M. Sommerdijk, Science, 2009, 323, 1455. 39 D. M. Duffy and J. H. Harding, Langmuir, 2004, 20, 7630. 13 A. Dey, P. H. H. Acknowledgements This work was supported by the UK Engineering and Physical Sciences Research Council [grant number EP/I001514/1]. This This journal is © The Royal Society of Chemistry 2014 1436 \| CrystEngComm, 2014, 16, 1430–1438 View Article Online CrystEngComm 16 H. Li and L. A. Estroff, J. Am. Chem. Soc., 2001, 129, 5460. Programme Grant funds the Materials Interface with Biology (MIB) consortium. Via our membership of the UK's HPC Materials Chemistry Consortium, which is funded by EPSRC (EP/F067496), this work made use of the facilities of HECToR, the UK's national high-performance computing service, which is provided by UoE HPCx Ltd at the University of Edinburgh, Cray Inc and NAG Ltd, and funded by the Office of Science and Technology through EPSRC's High End Computing Programme. 17 A. M. Travaille, J. J. J. M. Donners, J. W. Gerritsen, N. A. J. M. Sommerdijk, R. J. M. Nolte and H. van Kempen, Adv. Mater., 2002, 14, 492. 18 S. Mann, B. R. Heywood, S. Rajam and J. B. A. Walker, J. Phys. D: Appl. Phys., 1991, 242, 154; B. R. Heywood and S. Mann, Chem. Mater., 1994, 6, 311. 19 J. Kuther, G. Nelles, R. Seshadri, M. Schaub, H. J. Butt and W. Tremel, Chem.–Eur. J., 1998, 4, 1521. References Bomans, F. A. Müller, J. Will, P. M. Frederik, G. de With and N. A. J. M. Sommerdijk, Nat. Mater., 2010, 9, 1010. 40 D. M. Duffy and J. H. Harding, Langmuir, 2004, 20, 7637. 41 P. W. Tasker, J. Phys. C: Solid State Phys., 1979, 12, 4977. 42 D. M. Duffy, A. M. Travaille, H. van Kempen and J. H. Harding, J. Phys. Chem. B, 2005, 109, 5713. 14 J. C. Love, L. A. Estroff, J. K. Kriebel, R. G. Nuzzo and G. M. Whitesides, Chem. Rev., 2005, 105, 1103. 43 A. M. Travaille, L. Kaptijn, P. Verwer, B. Hulsken, J. A. A. W. Elemans, R. J. M. Nolte and H. van Kempen, J. Am. Chem. Soc., 2003, 125, 11571. 15 J. Aizenberg, A. J. Black and G. M. Whitesides, J. Am. Chem. Soc., 1999, 121, 4500. CrystEngComm, 2014, 16, 1430–1438 \| 1437 This journal is © The Royal Society of Chemistry 2014 View Article Online Highlight CrystEngComm CrystEngComm 50 C. L. Freeman, J. H. Harding and D. M. Duffy, Langmuir, 2008, 24, 9607. 44 B. Pokroy and J. Aizenberg, CrystEngComm, 2007, 9, 1219. 45 P. Hartman and W. G. Perdok, Acta Crystallogr., 1955, 8, 49. 45 P. Hartman and W. G. Perdok, Acta Crystal 51 R. Darkins, A. S. Cote, C. L. Freeman and D. M. Duffy, J. Cryst. Growth, 2013, 367, 110. 46 C. R. Henry, Prog. Surf. Sci., 2005, 80, 92. 47 C. L. Freeman, Q. Hu, M. H. Nielsen, J. Tao, J. J. De Yoreo and J H Harding J Phys Chem 2013 117 5154 47 C. L. Freeman, Q. Hu, M. H. Nielsen, J. Tao, J. J. and J. H. Harding, J. Phys. Chem., 2013, 117, 5154. 52 A. Kawska, J. Brickmann, R. Kniep, O. Hochrein and D. Zahn, J. Chem. Phys., 2006, 124, 024513. 48 Examples include: Umbrella sampling, G. M. Torrie and J. P. Valleau, J. Comput. Phys., 1977, 23, 187; Temperature accelerated dynamics: A. R. Sorensen and A. F. Voter, J. Chem. Phys., 2000, 112, 9599; Parallel Tempering: R. H. Swendsen and J. S. Wang, Phys. Rev. Lett., 1986, 198657, 2607. 53 A. Kawska, O. Hochrein, J. Brickmann, R. Kniep and D. Zahn, Angew. Chem., Int. Ed., 2008, 47, 4982. 54 A. Laio and M. Parrinello, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 12562. 55 D. Quigley, P. M. Rodger, C. L. Freeman, J. H. Harding and D. M. This journal is © The Royal Society of Chemistry 2014 49 D. M. Duffy and J. H. Harding, Surf. Sci., 2005, 595, 151. 1438 \| CrystEngComm, 2014, 16, 1430–1438 References Duffy, J. Chem. Phys., 2009, 131, 094703. 49 D. M. Duffy and J. H. Harding, Surf. Sci., 2005, 595, 151. 1438 \| CrystEngComm, 2014, 16, 1430–1438
https://openalex.org/W2603287241	https://europepmc.org/articles/pmc5368604?pdf=render	English	null	Nine phenylethanoid glycosides from Magnolia officinalis var. biloba fruits and their protective effects against free radical-induced oxidative damage	Scientific reports	2,017	cc-by	10,183	Lanlan Ge1, Wenhui Zhang1, Gao Zhou1, Bingxin Ma1, Qigui Mo1, Yuxin Chen1 & Youwei Wang1,2 Lanlan Ge1, Wenhui Zhang1, Gao Zhou1, Bingxin Ma1, Qigui Mo1, Yuxin Chen1 & Youwei Wang1,2 To systematically study the chemical constituents in Magnolia officinalis var. biloba fruits, nine phenylethanoid glycosides were isolated by solvent extraction, silica gel, and preparative high- performance liquid chromatography (HPLC). Their structures were elucidated by 1D and 2D NMR analyses, including COSY, HMQC and HMBC correlations, and HPLC analysis of sugar residue. Nine phenylethanoid glycosides, namely, magnoloside Ia (1), magnoloside Ic (2), crassifolioside (3), magnoloside Ib (4), magnoloside IIIa (5), magnoloside IVa (6), magnoloside IIa (7), magnoloside IIb (8) and magnoloside Va (9), were first isolated from the n-butanol fraction of Magnolia officinalis var. biloba fruits alcohol extract. Free radical scavenging activities of the nine phenylethanoid glycosides were assessed using the DPPH, ABTS, and superoxide anion radical scavenging assays. Simultaneously, protective effects of all compounds against free radical-induced oxidative damage were evaluated by two different kinds of mitochondrial damage model. The protective effects were assessed by mitochondrial swelling, the formations of malondialdehyde (MDA) and lipid hydroperoxide (LOOH), the activities of catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD). All phenylethanoid glycosides showed significant protective effects. Magnolia bark extracts are derived from Magnolia officinalis Rehd. et Wils. (M. officinalis) or Magnolia offic- inalis Rehd. et Wils. var. biloba Rehd. et Wils. (M. officinalis var. biloba). They have been used as traditional Chinese medicine (TCM) for treating abdominal distention, nausea, vomiting, dyspepsia, cough, and asthma1. Modern pharmacological research showed that magnolia bark extracts have effective antibacterial2, antioxidant3,4, anti-inflammatory5,6, anti-anxiety7, anti-gastric ulcer8, antitumor9, neuroprotective10, and cardiovascular protec- tion11 activities. The bioactive components of magnolia bark extracts include mainly neolignans, lignans, sesqui- terpenes, alkaloids, and phenylethanoid glycosides12–16. Recently, magnolia bark extracts have been recognized as a food additive worldwide. In 2002, the Ministry of Health of the People’s Republic of China passed the notice to further standardize the management of health food raw materials, including M. officinalis and M. officinalis var. biloba barks, into healthy food materials17. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1Institute of TCM and Natural Products, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, P. R. China. 2MOE Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Wuhan University, Wuhan 430072, P. R. China. Correspondence and requests for materials should be addressed to Y.W. (email: wyw@whu.edu.cn) Nine phenylethanoid glycosides from Magnolia officinalis var. biloba fruits and their protective effects against free radical-induced oxidative damage received: 08 November 2016 accepted: 23 February 2017 Published: 28 March 2017 Lanlan Ge1, Wenhui Zhang1, Gao Zhou1, Bingxin Ma1, Qigui Mo1, Yuxin Chen1 & Youwei Wang1,2 In 2008, magnolia bark extracts achieved GRAS status in the United States and were allowed to be added as an ingredient to chewing gums and mints sold in the United States; In 2009, magnolia bark extracts were approved as a new ingredient of food resource by the United Kingdom and European Union; In 2010, the United Kingdom authorities officially approved and specified that the maximum addition amount of magnolia bark extracts in gum and mints is 0.2%18,19. Michael et al. also confirmed that compressed mints and chewing gum containing bark extracts are effective against bacteria responsible for oral malodor20. These policies marked the transformation of magnolia bark extracts from TCM to food and dramatically expanded their application. China is the only country to cultivate M. officinalis and M. officinalis var. biloba (the plant area of M. officinalis var. biloba 1Institute of TCM and Natural Products, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, P. R. China. 2MOE Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Wuhan University, Wuhan 430072, P. R. China. Correspondence and requests for materials should be addressed to Y.W. (email: wyw@whu.edu.cn) Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Chemical structure of all identified phenylethanoid glycosides in M. officinalis var. biloba fruits. Figure 1. Chemical structure of all identified phenylethanoid glycosides in M. officinalis var. biloba frui is wider than that of M. officinalis)21, which are obtained largely by cutting down trees and peeling off their barks. However, this destructive utilization pattern is unsuitable for ecological and environmental protection. Therefore, a sustainable utilization pattern must be established to change this situation. M. officinalis and M. officinalis var. biloba fruits, which are nutritious reproductive organs of these plants, can be harvested every year. However, whether their fruits can be used as an alternative food resource remains unknown.fi is wider than that of M. officinalis)21, which are obtained largely by cutting down trees and peeling off their barks. However, this destructive utilization pattern is unsuitable for ecological and environmental protection. Therefore, a sustainable utilization pattern must be established to change this situation. M. officinalis and M. officinalis var. biloba fruits, which are nutritious reproductive organs of these plants, can be harvested every year. However, whether their fruits can be used as an alternative food resource remains unknown.fi We had already reported that phenylethanoid glycosides were isolated from M. officinalis var. biloba fruits in 201522. Lanlan Ge1, Wenhui Zhang1, Gao Zhou1, Bingxin Ma1, Qigui Mo1, Yuxin Chen1 & Youwei Wang1,2 Phenylethanoid glycosides, which exist mostly in Orobanchaceae plants23,24, have been demonstrated to possess diverse biological activities, such as antioxidant, anti-inflammatory, antibacterial, antiviral, antitumor, neuroprotective, and immunomodulatory effects25–31. However, whether the phenylethanoid glycosides isolated from M. officinalis var. biloba fruits have the same effects as those isolated from Orobanchaceae plants needs to be elucidated. Moreover, we have not validated whether the analogous components from their fruits can be associated with the traditional functions of magnolia bark extracts. Therefore, more studies are needed to further understand the function of phenylethanoid glycosides. p y g y In view of the more abundant resources of M. officinalis var. biloba, we focused on the n-butanol fraction of the alcohol extract from M. officinalis var. biloba fruits and isolated nine phenylethanoid glycosides (Fig. 1). The iso- lation and structural elucidation of these nine phenylethanoid glycosides, as well as their free radical scavenging activities, are reported in this paper. Subsequently, their protective effects against free radical-induced oxidative damage in two different kinds of mitochondrial damage model were also evaluated. Results and Disscussioni Results and Disscussion Structural identification of phenylethanoid glycosides. Compounds 1–9 were identified as pheny- lethanoid glycosides. Their structures were characterized by the phenylpropionyl group and benzene ethanol group through ester linkage and glycoside bond connected to the central sugar, respectively. g p g g g y g p y Compound 2 was isolated as a light yellow amorphous powder. Its molecular formula was C29H36O15, which was deduced from the molecular ion peak at m/z 623.1999[M-H]− (calcd. C29H35O15, 623.1981) by HR-ESI-MS and supported by the 13C NMR spectral data. The IR spectrum of 2 displaced the characteristic absorption bands for the hydroxyl group (3410 cm−1), conjugated carbonyl group (1687 cm−1), aromatic rings (1604 and 1516 cm−1), and glycosidic group (816 cm−1). Results and Disscussioni of trans-olefinic proton signals at δH 7.63 (1H, d, J = 15.9 Hz), 6.33 (1H, d, J = 15.9 Hz), and benzylic methylene proton signal at δH 2.80 (2 H, t, J = 7.4 Hz), which were assigned to δC 115.30 (C-2′), 123.20 (C-6′), 116.45 (C-5′), 117.25 (C-2), 116.59 (C-5), 121.41 (C-6), 147.72 (C-7′), 114.71 (C-8′), and 36.73 (C-7), respectively, via HMQC correlation. Additionally, two anomeric proton resonances appeared at δH 4.85 (1H, d, J = 8.0 Hz) and 4.90 (1H, s), which correlated with signals at δC 100.47 (C-1″) and 97.91 (C-1″′) in the HMQC spectrum, respectively. The 1H-NMR spectrum also showed the presence of a methyl group at δH 1.29 (3H, d, J = 6.0 Hz), which indicated the presence of rhamnose in the compound. A series of signals in 1H-NMR (CD3OD, 400 MHz) at δH 4.85 (1H, d, J = 8.0 Hz), 3.47 (1H, m), 4.51 (1H, s), 4.84 (1H, m), 3.60 (1H, m), 3.62 (1H, m), and 3.79 (1H, m) and 13C NMR (CD3OD, 100 MHz) at δC 100.47 (C-1″), 73.19 (C-2″), 66.45 (C-3″), 70.57 (C-4″), 74.53 (C-5″), and 62.37 (C-6″ ) indicated a rare β-allopyranosyl unit. Moreover, this unit was confirmed by comparing the NMR features of those of magnolosides A, B, C, D, and E16,32,33 isolated from M. officinalis barks or M. obovata barks, which also contained allopyranose. Acid hydrolysate of compound 2 contained monosaccharide components, which were identified as D-allose and L-rhamnose by HPLC analysis. In the HMBC relationship of 2 (Fig. 2), the correlations between H-1″ (δH 4.85) and C-8 (72.30) showed that the phenylethanol moiety was linked to C-1″ (All-C-1″); the cross peak between H-4″ (δH 4.84) and C-9′ (168.14) showed that the trans-caffeoyl moiety was linked to C-4″ Table 1. H NMR (CD3OD, 400 MHz) data of compounds 2, 8, and 3 (δH, dppm). of trans-olefinic proton signals at δH 7.63 (1H, d, J = 15.9 Hz), 6.33 (1H, d, J = 15.9 Hz), and benzylic methylene proton signal at δH 2.80 (2 H, t, J = 7.4 Hz), which were assigned to δC 115.30 (C-2′), 123.20 (C-6′), 116.45 (C-5′), 117.25 (C-2), 116.59 (C-5), 121.41 (C-6), 147.72 (C-7′), 114.71 (C-8′), and 36.73 (C-7), respectively, via HMQC correlation. of trans-olefinic proton signals at δH 7.63 (1H, d, J = 15.9 Hz), 6.33 (1H, d, J = 15.9 Hz), and benzylic methylene proton signal at δH 2.80 (2 H, t, J = 7.4 Hz), which were assigned to δC 115.30 (C-2′), 123.20 (C-6′), 116.45 (C-5′), 117.25 (C-2), 116.59 (C-5), 121.41 (C-6), 147.72 (C-7′), 114.71 (C-8′), and 36.73 (C-7), respectively, via HMQC correlation. Additionally, two anomeric proton resonances appeared at δH 4.85 (1H, d, J = 8.0 Hz) and 4.90 (1H, s), which correlated with signals at δC 100.47 (C-1″) and 97.91 (C-1″′) in the HMQC spectrum, respectively. The 1H-NMR spectrum also showed the presence of a methyl group at δH 1.29 (3H, d, J = 6.0 Hz), which indicated the presence of rhamnose in the compound. A series of signals in 1H-NMR (CD3OD, 400 MHz) at δH 4.85 (1H, d, J = 8.0 Hz), 3.47 (1H, m), 4.51 (1H, s), 4.84 (1H, m), 3.60 (1H, m), 3.62 (1H, m), and 3.79 (1H, m) and 13C NMR (CD3OD, 100 MHz) at δC 100.47 (C-1″), 73.19 (C-2″), 66.45 (C-3″), 70.57 (C-4″), 74.53 (C-5″), and 62.37 (C-6″ ) indicated a rare β-allopyranosyl unit. Moreover, this unit was confirmed by comparing the NMR features of those of magnolosides A, B, C, D, and E16,32,33 isolated from M. officinalis barks or M. obovata barks, which also contained allopyranose. Acid hydrolysate of compound 2 contained monosaccharide components, which were identified as D-allose and L-rhamnose by HPLC analysis. In the HMBC relationship of 2 (Fig. 2), the correlations between H-1″ (δH 4.85) and C-8 (72.30) showed that the phenylethanol moiety was linked to C-1″ (All-C-1″); the cross peak between H-4″ (δH 4.84) and C-9′ (168.14) showed that the trans-caffeoyl moiety was linked to C-4″ Results and Disscussioni The UV spectrum obtained the maximum absorption at 206, 291, and 330 nm.h The 1H-NMR and 13C NMR spectra of 2 (Tables 1, 2 and supplementary information) exhibited characteristic signals arising from the trans-caffeoyl group and 3,4-dihydroxy phenethyl alcohol group showing two sets of ABX-type signals caused by aromatic protons at δH 7.08 (1H, d, J = 2.0 Hz), 6.97 (1H, dd, J = 2.0, 8.0 Hz), 6.80 (1H, d, J = 8.0 Hz) and δH 6.73 (1H, d, J = 2.0 Hz), 6.71 (1H, d, J = 8.0 Hz), 6.58 (1H, dd, J = 2.0, 8.0 Hz), a pair Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 2 www.nature.com/scientificreports/ Position 1H-NMR 2 8 3 Aglycone 1 — — — 2 6.73 (1H,d,2.0) 6.74 (1H,d,2.0) 6.71 (1H,d,2.0) 3 — — — 4 — — — 5 6.71 (1H,d,8.0) 6.71 (1H,d,8.0) 6.69 (1H,d,8.0) 6 6.58 (1H,dd,2.0,8.0) 6.60 (1H,dd,2.0,8.0) 6.58 (1H,dd,2.0,8.0) 7 2.80 (2 H,t,7.4) 2.80 (2 H,t,7.4), 2.84 (2 H,t,8.0), 8 3.94 (1H,m);3.77 (1H,m) 3.65 (1H,m);3.76 (1H,m) 3.69 (1H,m);3.91 (1H,m) Caffeoyl 1′ — — — 2′ 7.08 (1H,d,2.0) 7.09 (1H,d,2.0) 7.07 (1H,d,2.0) 3′ — — — 4′ — — — 5′ 6.80 (1H,d,8.0) 6.81 (1H,d,8.0) 6.80 (1H,d,8.0) 6′ 6.97 (1H,dd,2.0,8.0) 6.97 (1H,dd,2.0,8.0) 6.97 (1H,dd,2.0,8.0) 7′ 7.63 (1H,d,15.9) 7.63 (1H,d,15.9) 7.60 (1H,d,15.9) 8′ 6.33 (1H,d,15.9) 6.33 (1H,d,15.9) 6.30 (1H,d,15.9) 9′ — — — Allose Glucose 1″ 4.85 (1H,d,8.0) 4.85 (1H,d,8.0) 4.51 (1H,d,7.7) 2″ 3.47 (1H,m) 3.46 (1H,m) 3.97 (1H,m) 3″ 4.51 (1H,s) 4.51 (1H,s) 3.57 (1H,m) 4″ 4.84 (1H,m) 4.92 (1H,m) 4.96 (1H,m) 5″ 3.60 (1H,m) 3.22 (1H,m) 3.51 (1H,m) 6″ 3.62 (1H,m); 3.79 (1H,m) 3.67 (1H,m); 4.01 (1H,m) 3.52 (1H,m); 3.63 (1H,m) 2″-Rhamnose 1″′ 4.90 (1H,s) 4.89 (1H,s) 4.96 (1H,s) 2″′ 3.68 (1H,m) 4.02 (1H,m) 3.46 (1H,m) 3″′ 3.61 (1H,m) 4.24 (1H,m) 3.84 (1H,m) 4″′ 3.36 (1H,m) 3.93 (1H,m) 3.32 (1H,m) 5″′ 4.04 (1H,m) 3.67 (1H,m) 4.05 (1H,m) 6″′ 1.29 (3H,d,6.0) 1.30 (3H,d,6.0) 1.14 (3H,d,6.0) 6″-Glucose 3″-Rhamnose 1″″ 4.30 (1H,d,7.7) 5.01 (1H,s) 2″″ 3.60 (1H,m) 3.46 (1H,m) 3″″ 3.25 (1H,m) 3.84 (1H,m) 4″″ 3.32 (1H,m) 3.32 (1H,m) 5″″ 3.35 (1H,m) 4.05 (1H,m) 6″″ 3.64 (1H,m); 3.86 (1H,m) 1.29 (3H,d,6.0) T bl 1 1H NMR (CD OD 400MH ) d t f d 2 8 d 3 (δ d ) Table 1. 1H-NMR (CD3OD, 400 MHz) data of compounds 2, 8, and 3 (δH, dppm). Results and Disscussioni 13C-NMR (CD3OD, 100 MHz) data of compounds 2, 8, and 3 (δC, dppm). (All-C-4″); and the long-range correlations between H-1″′ (δH 4.90) and C-2″ (73.19) (All-C-2″) showed that the rhamnose moiety was linked to C-2″ (All-C-2″). (All-C-4″); and the long-range correlations between H-1″′ (δH 4.90) and C-2″ (73.19) (All-C-2″) showed that the rhamnose moiety was linked to C-2″ (All-C-2″). y y yf y y Compound 3 was obtained as a canary yellow amorphous powder. The HR-ESI-MS data showed an accurate [M-H]− ion at m/z 769.2668 (calcd. for C35H45O19, 769.2654), in accordance with the empirical molecular formula of C35H46O19. The IR spectrum of 3 displaced characteristic absorption bands for the hydroxyl group (3402 cm−1), conjugated carbonyl group (1699 cm−1), aromatic rings (1603 and 1517 cm−1), and glycosidic group (814 cm−1). The UV spectrum obtained the maximum absorption at 206, 280, and 330 nm.h y y yf y y Compound 3 was obtained as a canary yellow amorphous powder. The HR-ESI-MS data showed an accurate [M-H]− ion at m/z 769.2668 (calcd. for C35H45O19, 769.2654), in accordance with the empirical molecular formula of C35H46O19. The IR spectrum of 3 displaced characteristic absorption bands for the hydroxyl group (3402 cm−1), conjugated carbonyl group (1699 cm−1), aromatic rings (1603 and 1517 cm−1), and glycosidic group (814 cm−1). The UV spectrum obtained the maximum absorption at 206, 280, and 330 nm.h f Compound 3 was obtained as a canary yellow amorphous powder. The HR-ESI-MS data showed an accurate [M-H]− ion at m/z 769.2668 (calcd. for C35H45O19, 769.2654), in accordance with the empirical molecular formula of C35H46O19. The IR spectrum of 3 displaced characteristic absorption bands for the hydroxyl group (3402 cm−1), conjugated carbonyl group (1699 cm−1), aromatic rings (1603 and 1517 cm−1), and glycosidic group (814 cm−1). The UV spectrum obtained the maximum absorption at 206, 280, and 330 nm.h h p p , , The 1H-NMR and 13C NMR spectra of 3 (Tables 1, 2 and supplementary information) exhibited the same char- acteristic signals to prove the existence of the trans-caffeoyl group and 3,4-dihydroxy phenethyl alcohol group as compound 2. By contrast, three sugar anomeric protons were observed at δH 5.01 (1H, s), 4.96 (1H, s), and 4.51 (1H, d, J = 7.7 Hz) and resonated at δC 102.32 (C-1″″), 103.07 (C-1″′), and 102.46 (C-1′) in 13C NMR, respectively. They also could be supported in the HMQC spectrum. Results and Disscussioni Additionally, two anomeric proton resonances appeared at δH 4.85 (1H, d, J = 8.0 Hz) and 4.90 (1H, s), which correlated with signals at δC 100.47 (C-1″) and 97.91 (C-1″′) in the HMQC spectrum, respectively. The 1H-NMR spectrum also showed the presence of a methyl group at δH 1.29 (3H, d, J = 6.0 Hz), which indicated the presence of rhamnose in the compound. A series of signals in 1H-NMR (CD3OD, 400 MHz) at δH 4.85 (1H, d, J = 8.0 Hz), 3.47 (1H, m), 4.51 (1H, s), 4.84 (1H, m), 3.60 (1H, m), 3.62 (1H, m), and 3.79 (1H, m) and 13C NMR (CD3OD, 100 MHz) at δC 100.47 (C-1″), 73.19 (C-2″), 66.45 (C-3″), 70.57 (C-4″), 74.53 (C-5″), and 62.37 (C-6″ ) indicated a rare β-allopyranosyl unit. Moreover, this unit was confirmed by comparing the NMR features of those of magnolosides A, B, C, D, and E16,32,33 isolated from M. officinalis barks or M. obovata barks, which also contained allopyranose. Acid hydrolysate of compound 2 contained monosaccharide components, which were identified as D-allose and L-rhamnose by HPLC analysis. In the HMBC relationship of 2 (Fig. 2), the correlations between H-1″ (δH 4.85) and C-8 (72.30) showed that the phenylethanol moiety was linked to C-1″ (All-C-1″); the cross peak between H-4″ (δH 4.84) and C-9′ (168.14) showed that the trans-caffeoyl moiety was linked to C-4″ Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 3 www.nature.com/scientificreports/ Position 13C-NMR 2 8 3 Aglycone 1 131.68 131.67 130.70 2 117.25 116.63 115.87 3 146.82 146.80 146.32 4 144.61 144.59 144.17 5 116.59 117.31 116.64 6 121.41 121.51 120.78 7 36.73 36.72 36.04 8 72.30 72.45 71.86 Caffeoyl 1′ 127.70 127.68 127.17 2′ 115.30 115.37 114.74 3′ 146.01 145.97 145.61 4′ 149.75 149.77 149.26 5′ 116.45 116.53 116.04 6′ 123.20 123.33 122.70 7′ 147.72 147.96 147.38 8′ 114.71 114.66 114.47 9′ 168.14 168.23 167.91 Allose Glucose 1″ 100.47 100.50 102.46 2″ 73.19 73.97 82.68 3″ 66.45 66.42 80.43 4″ 70.57 70.64 69.83 5″ 74.53 75.03 75.50 6″ 62.37 69.62 61.88 2″-Rhamnose 1″′ 97.91 97.91 103.07 2″′ 72.37 72.19 72.01 3″′ 72.22 72.25 71.71 4″′ 73.99 72.36 73.33 5″′ 69.99 70.01 70.71 6″′ 18.04 18.09 17.91 6″-Glucose 3″-Rhamnose 1″″ 104.92 102.32 2″″ 74.41 71.80 3″″ 77.78 71.49 4″″ 71.48 73.18 5″″ 77.90 70.43 6″″ 62.67 17.69 Table 2 13C NMR (CD OD 100MHz) data of compou Table 2. y ( ) In light of all the above considerations, the structure of 2 was revealed as 2-(3, 4-dihydroxyphenyl) ethyl-O-a-L-rhamnopyranosyl-(1 → 2)-(4-O-trans-caffeoyl)-β-D-allopyranoside.h All-C-4″); and the long-range correlations between H-1″′ (δH 4.90) and C-2″ (73.19) (All-C-2″) showed that the hamnose moiety was linked to C-2″ (All-C-2″). www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Key HMBC correlations (H → C) of compounds 2, 8, and 3. Figure 2. Key HMBC correlations (H → C) of compounds 2, 8, and 3. Figure 2. Key HMBC correlations (H → C) of compounds 2, 8, and 3. Figure 2. Key HMBC correlations (H → C) of compounds 2, 8, and 3. rhamnoses. A series of signals in 1H-NMR (CD3OD, 400 MHz) at δH 4.51 (1H, d, J = 7.7 Hz), 3.97 (1H, m), 3.57 (1H, m), 4.96 (1H, m), 3.51 (1H, m), 3.52 (1H, m), and 3.63 (1H, m) and in 13C NMR (CD3OD, 100 MHz) at δC 102.46 (C-1″), 82.68 (C-2″), 80.43 (C-3″), 69.83 (C-4″), 75.50 (C-5″), and 61.88 (C-6″) showed that the central sugar was β-glucose. The acid hydrolysate of compound 3 further confirmed the existence of D-glucose and L-rhamnose, which were identified by HPLC analysis. In the HMBC relationship of 3 (Fig. 2), the correlations between H-1″ (δH 4.51) and C-8 (δC 71.86) showed that the phenylethanol moiety was linked to C-1″ (Glc-C-1″); the cross peak between H-4″ (δH 4.96) and C-9′ (δC 167.91) showed that the trans-caffeoyl moiety was linked to C-4″ (Glc-C-4″); and the long-range correlations between H-1″′ (δH 4.96) to C-2″ (δC 82.68) (Glc-C-2″) and H-1″″ (δH 5.01) to C-3″ (δC 80.43) (Glc-C-3″) unambiguously showed that the two rhamnose moieties were linked at C-2″ and C-3″, respectively.i p y All these results confirmed the structure of compound 3 as 2-(3, 4-dihydroxyphenyl)-ethyl-O-a-L-rhamnopyranosy (1 → 2)-[a-L-rhamnopyranosyl-(1 → 3)]-(4-O-trans-caffeoyl)-β-D-glucopyranoside, named crassifolioside34.h Compound 8 was obtained as a light yellow amorphous powder. The negative-ion quasi-molecular ion peak in its HRESI-MS data at m/z 785.2520 [M-H]− (calcd. for C35H45O20, 785.2510) together with the 13C NMR spec- tral data suggested a molecular formula of C35H46O20. The IR spectrum of 8 displaced characteristic absorption bands for the hydroxyl group (3234 cm−1), conjugated carbonyl group (1692 cm−1), aromatic rings (1604 and 1518 cm−1), and glycosidic group (813 cm−1). The UV spectrum obtained the maximum absorption at 205, 280, and 330 nm. Comparison of the 1H-NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) spectra of 8 (Tables 1, 2 and supplementary information) with those of 2 suggested that compound 8 contained one more sugar group than 2. Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 Results and Disscussioni Two methyl groups at δH 1.14 (3H, d, J = 6.0 Hz) and 1.29 (3H, d, J = 6.0 Hz), as well as δC 17.91 (C-6″′) and 17.69 (C-6″″), indicated that compound 3 may contain two h p p The 1H-NMR and 13C NMR spectra of 3 (Tables 1, 2 and supplementary information) exhibited the same char- acteristic signals to prove the existence of the trans-caffeoyl group and 3,4-dihydroxy phenethyl alcohol group as compound 2. By contrast, three sugar anomeric protons were observed at δH 5.01 (1H, s), 4.96 (1H, s), and 4.51 (1H, d, J = 7.7 Hz) and resonated at δC 102.32 (C-1″″), 103.07 (C-1″′), and 102.46 (C-1′) in 13C NMR, respectively. They also could be supported in the HMQC spectrum. Two methyl groups at δH 1.14 (3H, d, J = 6.0 Hz) and 1.29 (3H, d, J = 6.0 Hz), as well as δC 17.91 (C-6″′) and 17.69 (C-6″″), indicated that compound 3 may contain two Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 4 www.nature.com/scientificreports/ The extra series of sugar signals in 1H-NMR at δH 4.30 (1H, d, J = 7.7 Hz), 3.60 (1H, m), 3.25 (1H, m), 3.32 (1H, m), 3.35 (1H, m), 3.64 (1H, m), and 3.86 (1H, m) and in 13C NMR at δC 104.92 (C-1″″), 74.41 (C-2″″), 77.78 (C-3″″), 71.48 (C-4″″), 77.90 (C-5″″), and 62.67 (C-6″″) deduced that the extra sugar group was β-glucose. Acid hydrolysis of 8 further gave D-allose, D-glucose, and L-rhamnose as sugar residues by HPLC analysis. The 7.25 ppm down-field shift of C-6″ in the 13C NMR spectrum indicated the attachment of the extra sugar group to C-6″ of the central sugar. This finding was also supported by the HMBC correlation (Fig. 2) of H-1″″ (δH 4.30) and C-6″ (δC 69.62). ( C ) Based on the above evidence, compound 8 was established as 2-(3, 4-dihydroxyphenyl)-ethyl-O- a-L-rhamnopyranosyl-(1 → 2)-[β-D-glucopyranosyl-(1 → 6)]-(4-O-trans-caffeoyl)-β-D-allopyranoside. f Compounds 5 and 6 were isolated as yellow amorphous powder, with the molecular formula of C29H36O14, as deduced from the [M-H]− peak at m/z 607.2011 (calcd. for C29H35O14, 607.2021) and 607.2009 (calcd. for C29H35O14, 607.2021) via HR-ESI-MS and supported by the 13C NMR spectral data. The 1H-NMR and 13C NMR spectra of 5 (Table 3 and supplementary information) were very similar to those of compound 1 (magnoloside A), except for the signals caused by the 4-hydroxyphenylethyl alcohol group {ortho-coupled A2B2-type aromatic pro- tons [δH 7.10 (2 H, d, J = 2.0 Hz), 6.71 (2 H, d, J = 8.0 Hz)] and two methylenes [δH 2.85 (2 H, t, J = 8.0 Hz), 3.67 (2 H, m)]}, which was also supported by the literature35. www.nature.com/scientificreports/ Comparison of the NMR data of 6 (Table 3 and supplementary information) with those of compound 1 (magnoloside A) revealed that the caffeoyl group of magnoloside A was replaced by the trans-p-coumaroyl group {ortho-coupled A2B2-type aromatic protons [δH 7.51 (2 H, d, J = 8.0 Hz), Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 5 www.nature.com/scientificreports/ Position 5 6 δH δC δH δC Aglycone 1 — 130.80 — 131.60 2 7.10 (1H,d,2.0) 131.02 6.72 (1H,d,2.0) 117.17 3 6.71 (1H,d,8.0) 116.18 — 146.91 4 — 156.74 — 144.62 5 6.71 (1H,d,8.0) 116.18 6.69 (1H,d,8.0) 116.37 6 7.10 (1H,d,2.0) 131.02 6.59 (1H,dd,2.0,8.0) 121.37 7 2.85 (2 H,t,8.0) 36.57 2.79 (2 H,t,8.0) 36.75 8 3.67 (2 H,m) 72.19 3.67 (2 H,m) 72.15 Caffeoyl Coumaroyl 1′ — 127.82 — 127.24 2′ 7.08 (1H, d,2.0) 115.31 7.51 (1H,d,8.0) 131.30 3′ — 146.84 6.84 (1H,d,8.0) 116.88 4′ — 149.66 — 161.30 5′ 6.81 (1H,d,8.0) 116.58 6.84 (1H,d,8.0) 116.88 6′ 6.99 (1H,dd,2.0,8.0) 123.13 7.51 (1H,d,8.0) 131.30 7′ 7.61 (1H,d,15.9) 147.33 7.68 (1H,d,15.9) 146.04 8′ 6.38 (1H,d,15.9) 115.18 6.45 (1H,d,15.9) 115.23 9′ — 168.93 — 168.89 Allose 1″ 4.76 (1H,d,8.0) 100.86 4.76 (1H,d,8.0) 100.80 2″ 3.65 (1H,m) 73.86 3.65 (1H,m) 73.89 3″ 5.75 (1H,t,3.0) 71.22 5.75 (1H,t,3.0) 69.95 4″ 3.76 (1H,m) 67.17 3.76 (1H,m) 67.15 5″ 3.82 (1H,m) 76.06 3.82 (1H,m) 76.03 6″ 3.69 (1H,m); 3.92 (1H,m) 62.74 3.70 (1H,m); 3.92 (1H,m) 62.71 Rhamnose 1″′ 4.94 (1H,s) 98.49 4.95 (1H,s) 98.44 2″′ 3.6–3.8 (1H,m) 72.31 3.6–3.8 (1H,m) 72.25 3″′ 3.6–3.8 (1H,m) 72.05 3.6–3.8 (1H,m) 72.02 4″′ 3.42 (1H,m) 73.80 3.42 (1H,m) 73.79 5″′ 4.02 (1H,m) 69.99 4.02 (1H,m) 71.19 6″′ 1.25 (3H,d,6.0) 18.07 1.26 (3H,d,6.0) 18.02 Table 3. 1H-NMR (CD3OD, 400 MHz) and 13C-NMR (CD3OD, 100 MHz) data of compounds 5 and 6. 6.84 (2 H, d, J = 8.0 Hz)] and trans-olefinic protons [7.68 (1H, d, J = 15.9 Hz), 6.45 (1H, d, J = 15.9 Hz)]}, which was also supported by the literature25. Furthermore, the HMBC correlation of 5 and 6 (Fig. 3) also showed sim- ilar modes of long-range correlations with 1. Therefore, the structures of compounds 5 and 6 were elucidated as 2-(4-hydroxyphenyl)-ethyl-O-a-L-rhamnopyranosyl-(1 → 2)-(3-O-trans-caffeoyl)-β-D-allopyranoside and 2-(3, 4-dihydroxyphenyl)-ethyl-O-a-L-rhamnopyranosyl-(1 → 2)-(3-O-trans-coumaroyl)-β-D-allopyranoside, respectively. 6.84 (2 H, d, J = 8.0 Hz)] and trans-olefinic protons [7.68 (1H, d, J = 15.9 Hz), 6.45 (1H, d, J = 15.9 Hz)]}, which was also supported by the literature25. Furthermore, the HMBC correlation of 5 and 6 (Fig. www.nature.com/scientificreports/ Compound IC50 (μM) Kb ( × 10−4 A/s) DPPH ABTS Superoxide anion 1 11.79 ± 0.57 3.52 ± 0.11 10.37 ± 0.33 2 12.99 ± 0.48 3.53 ± 0.28 10.68 ± 0.49 3 21.38 ± 0.52 3.28 ± 0.35 11.57 ± 0.17 4 16.23 ± 0.16 3.75 ± 0.11 10.50 ± 0.18 5 32.18 ± 0.97 4.16 ± 0.25 11.22 ± 0.23 6 35.17 ± 0.22 5.09 ± 0.07 9.84 ± 0.38 7 22.94 ± 0.26 4.61 ± 0.10 9.41 ± 0.77 8 24.62 ± 0.15 4.78 ± 0.08 9.07 ± 0.76 9 20.99 ± 0.50 6.23 ± 0.06 8.69 ± 0.70 Vc 40.94 ± 0.78 11.51 ± 1.05 0.09 ± 0.02 BHT 89.94 ± 4.57 7.29 ± 0.33 10.40 ± 0.55 control — — 15.35 ± 0.19 Table 4. Radical scavenging activities of phenylethanoid glycosides from M. officinalis var. biloba fruits. Values were the means ± SD, n = 3; Vc is ascorbic acid; BHT is butylated hydroxytoluene. Compound IC50 (μM) Kb ( × 10−4 A/s) DPPH ABTS Superoxide anion 1 11.79 ± 0.57 3.52 ± 0.11 10.37 ± 0.33 2 12.99 ± 0.48 3.53 ± 0.28 10.68 ± 0.49 3 21.38 ± 0.52 3.28 ± 0.35 11.57 ± 0.17 4 16.23 ± 0.16 3.75 ± 0.11 10.50 ± 0.18 5 32.18 ± 0.97 4.16 ± 0.25 11.22 ± 0.23 6 35.17 ± 0.22 5.09 ± 0.07 9.84 ± 0.38 7 22.94 ± 0.26 4.61 ± 0.10 9.41 ± 0.77 8 24.62 ± 0.15 4.78 ± 0.08 9.07 ± 0.76 9 20.99 ± 0.50 6.23 ± 0.06 8.69 ± 0.70 Vc 40.94 ± 0.78 11.51 ± 1.05 0.09 ± 0.02 BHT 89.94 ± 4.57 7.29 ± 0.33 10.40 ± 0.55 control — — 15.35 ± 0.19 Table 4. Radical scavenging activities of phenylethanoid glycosides from M. officinalis var. biloba fruits. Values were the means ± SD, n = 3; Vc is ascorbic acid; BHT is butylated hydroxytoluene. Table 4. Radical scavenging activities of phenylethanoid glycosides from M. officinalis var. biloba fruits Values were the means ± SD, n = 3; Vc is ascorbic acid; BHT is butylated hydroxytoluene. Table 4. Radical scavenging activities of phenylethanoid glycosides from M. officinalis var. biloba fruits. Values were the means ± SD, n = 3; Vc is ascorbic acid; BHT is butylated hydroxytoluene. phenylethanoid glycosides are summarized in Table 4. www.nature.com/scientificreports/ 3) also showed sim- ilar modes of long-range correlations with 1. Therefore, the structures of compounds 5 and 6 were elucidated as 2-(4-hydroxyphenyl)-ethyl-O-a-L-rhamnopyranosyl-(1 → 2)-(3-O-trans-caffeoyl)-β-D-allopyranoside and 2-(3, 4-dihydroxyphenyl)-ethyl-O-a-L-rhamnopyranosyl-(1 → 2)-(3-O-trans-coumaroyl)-β-D-allopyranoside, respectively. p y The other compounds were identified as magnoloside A (1), magnoloside B (7), magnoloside D (4), and magnoloside E (9) by comparing their 1H-NMR and 13C NMR data with those reported in the literature16. All compounds were obtained from M. officinalis var. biloba fruits for the first time. ffii In the last few years, some confusing nomenclatures were found in the original articles36,37. For example, com- pound 2 and compound 8, mentioned in this article, were both given the same nomenclature as magnoloside F though they actually posses different structures, while magnoloside F36 and magnoloside M37 were characterized by the same structure (compound 2, mentioned in this article) but different nomenclature. In this manuscript, we propose a reasonable rule of nomenclature in view of the rich structure type of phenylethanoid glycosides. Thus, the confusion and ambiguity caused by two research groups could be clarified. Different types of pheny- lethanoid glycosides are numberd with Roman numerals (I, II, III, IV, V…), and isomers with subscripts a, b, c… are distinguished. Therefore, compounds 1, 2, 4, 5, 6, 7, 8 and 9 were renamed magnoloside Ia (old name was magnoloside A32), magnoloside Ic (old names were magnoloside F31 and magnoloside M37), magnoloside Ib (old name was magnoloside D16), magnoloside IIIa (old name was magnoloside H36), magnoloside IVa (old name was magnoloside G36), magnoloside IIa (old name was magnoloside B33), magnoloside IIb (old name was magnoloside F37), magnoloside Va (old name was magnoloside E16). Free radical scavenging activities and their structure–activity relationship. In vitro DPPH rad- ical scavenging, ABTS radical scavenging, and superoxide anion radical scavenging activities of the isolated Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 6 www.nature.com/scientificreports/ Figure 3. Key HMBC correlations (H → C) of compounds 5 and 6. Figure 3. Key HMBC correlations (H → C) of compounds 5 and 6. www.nature.com/scientificreports/ Symbols represent statistical significance. *p < 0.001, compared with the model group; ###p < 0.001, compared with the control group. BHT is butylated hydroxytoluene. Moreover, compared with the other seven compounds, compounds 5 and 6 belonged to phenylethanoid glyco- ides with two adjacent phenolic groups only in one side, so they exhibited poor activity.h j p g p y y p y The ABTS radical scavenging assay demonstrated that compounds 1, 2, and 4 also exhibited good ABTS radi- cal scavenging activity, but the IC50 value (6.23 ± 0.06 μM) of compound 9 was large. This result was different from that of the DPPH radical scavenging assay, indicating that the apiose group in compound 9 produced a negative effect on the ABTS radical scavenging assay. By contrast, the ABTS radical scavenging ability of compound 3 was far better than that of compounds 7 and 8, which could be due to the glucose group in compound 3. The super- oxide anion radical scavenging assay showed that the activity of compound 9 was the best, whereas the activity of compound 3 was the worst. These completely opposite results may be due to the fact that the apiose group in compound 9 enhanced the influence on the superoxide anion radical scavenging assay, whereas the glucose group in compound 3 minimized this influence. These different results obtained from three assays may be explained by the various mechanisms of these assays, suggesting that combined assay methods should be adopted in the screening and evaluation of bioactive compounds from natural materials.fl g p Our experimental results also showed that the substitution position of the caffeoyl group also influenced free radical scavenging activity. By analyzing the structure of similar compounds and experimental results, we found that the free radical scavenging activity of 3-caffeoyl substitution was optimal, followed by 4-caffeoyl substitution and 6-caffeoyl substitution. This conclusion could be supported by the order of activity as follows: 1 > 2 > 4, 7 > 8. Protective effects against free radical-induced oxidative damage. Repeated ultraviolet B (UVB) exposure or constant treated with Fe2+/H2O2 can produce reactive oxygen species (ROS), which lead to various adverse effects on the body tissues40,41. The free radicals could attack on the fatty acid component of membrane lipids, and then resulting in mitochondrial damage and lipid peroxidation. These facts have been previously demonstrated in mitochondria42. www.nature.com/scientificreports/ Therefore, UVB-induced oxidative damage model and Fe2+/H2O2-induced oxi- dative damage model are classical mitochondria models which are used to measure the protective effect against free radical-induced oxidative damage. g When mitochondria are damaged, swelling will occur; thus, the value of A520 will be reduced43. Malondialdehyde (MDA) and lipid hydroperoxide (LOOH) are two relatively unstable products of lipid perox- idation, which is a process where ROS degrade polyunsaturated fatty acids41. These toxic products could cause toxic stress in mitochondria, accelerate further oxidative damage44. Antioxidant enzymes, such as catalase (CAT), glutathione reductase (GR), and superoxide dismutase (SOD) can confer protection against oxidative stress and tissue damage. These enzymes are critical for defense against the harmful effects of ROS and free radicals in biological systems45. In this study, compounds 1–9 were investigated for their protective effects against free radical-induced oxidative damage.i g In mitochondrial damage model caused by UVB (Fig. 4), the model group showed a significant decrease in mitochondrial swelling assay, and the change was significantly reversed during treatment with test compounds (p < 0.001). The A532 value and A560 value of the MDA (0.075 ± 0.001) and LOOH (0.097 ± 0.001) were signifi- cantly increased in the model group compared with the control group (p < 0.001). However, the test compound groups reduced the MDA and LOOH level and showed significant effects (p < 0.001). In mitochondrial damage model caused by Fe2+/H2O2, compared with the control group, the model group showed a significant decrease in CAT, GR and SOD levels, together with a significant increase in the level of MDA and LOOH (Fig. 5). Overall, these changes were significantly reversed during treatment with test compounds. However, the level of improve- ment of some group was not significant (p > 0.05).f i Comparing the above experimental results, we found that protective effect of compounds containing two pair of two adjacent phenolic groups was optimal, followed by compounds containing one pair of two adjacent phenolic groups (such as compounds 5 and 6). Furthermore, compounds with two sugars were best. These results were consistent with the experimental results of free radical scavenging assays. Therefore, we could speculate that the protective effects of the nine phenylethanoid glycosides against free radical-induced oxidative damage were attributed to their radical scavenging activity, which was caused by the number of two adjacent phenolic groups. Potential of M. officinalis var. biloba fruits as a promising functional alternative food resource. www.nature.com/scientificreports/ The table illustrates that all the isolated phenylethanoid glycosides showed excellent free radical scavenging activity. The analysis of the structure–activity relationship of these phenylethanoid glycosides in the free radical scavenging activity assay suggested that the presence of two adjacent phenolic groups in the molecule resulted in strong free radical scavenging activity. The more two adja- cent phenolic groups were, the stronger the free radical scavenging activity was. Meanwhile, all isolated pheny- lethanoid glycosides were cinnamic acid derivatives, which contain α,β-conjugated unsaturated ester structures, thereby increasing benzene ring plane conjugation and allowing electron delocalization to stabilize free radicals. This conclusion was well supported by previous reports38,39.i h In the DPPH radical scavenging assay, compound 1 and its structural analogs (2, 4, and 9) showed signifi- cant DPPH radical scavenging activity with far smaller IC50 values (11.79 ± 0.57, 12.99 ± 0.48, 16.23 ± 0.16, and 20.99 ± 0.50 μM, respectively) than the positive controls, such as VC (40.94 ± 0.78 μM) and BHT (89.94 ± 4.57 μ M). Compounds 3, 7, and 8 expressed inferior activity compared with the above four compounds because their IC50 values (21.38 ± 0.52, 22.94 ± 0.26, and 24.62 ± 0.15 μM, respectively) were near the value of VC. Moreover, Compound 5 and its structural isomer 6 showed large IC50 values (32.18 ± 0.97 and 35.17 ± 0.22 μM, respec- tively). Some observations could be made according to the above results. Compounds 3, 7, and 8 possessed larger steric hindrance because they contained three sugars, whereas compounds 1, 2, 4, and 9 only contained two sugars. The increased steric hindrance effect of compounds 3, 7, and 8 prevented them from easily approaching the free radicals, so their DPPH radical scavenging capacity was relatively weak than compounds 1, 2, 4, and 9. Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Protective activity of nine compounds against UVB-induced formation of mitochondrial swelling degree (A), MDA (B), and LOOH (C) in rat liver mitochondria. Data are presented as the mean ± SD (n = 3). Symbols represent statistical significance. p < 0.001, compared with the model group; ###p < 0.001, compared with the control group. BHT is butylated hydroxytoluene. Figure 4. Protective activity of nine compounds against UVB-induced formation of mitochondrial swelling degree (A), MDA (B), and LOOH (C) in rat liver mitochondria. Data are presented as the mean ± SD (n = 3). Methods l Plant material. M. officinalis var. biloba fruits were collected from the mountain area in Enshi (N: 30°17′ 51.56″, E: 109°28′27.33″; A: 1,545 m), Hubei Province, China, and identified by Prof. Youwei Wang (Wuhan University, Wuhan, China). A voucher specimen (No. 14610) was deposited at the Traditional Chinese Medicine Specimen Museum, School of Pharmaceutical Science, Wuhan University, China. Chemical and reagents. 2,2′-Azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) was procured from Fluka (Menlo Park, CA, USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), high-performance liquid chro- matography (HPLC)-grade methanol, and acetonitrile were purchased from Sigma–Aldrich Co. (St. Louis, USA). D-Allose (D1218014), L-rhamnose (B1504021), L-cysteine methyl ester hydrochlorides (G14118068), and 2-thiobarbituric acid (C1510117) were purchased from Aladdin Industrial Corporation (Shanghai, China). Diagnostic kits for CAT, GR, and SOD were purchased from Nanjing Jiancheng Technology Co. Ltd. (Nanjing, China). Ascorbic acid (Vc) (F20060609), ammonium iron (II) sulfate hexahydrate (20120215), butylated hydrox- ytoluene (BHT) (20121211), D-glucose (20140724), dodecyl sodium sulfate (20130625), hydrogen peroxide 30% (20150911), iron (II) sulfate heptahydrate (20100506), methanol-d4 (20140519), xylenol orange (20100826), and other analytical chemicals and reagents were purchased from Sinopharm Chemical Regent Co. Ltd. (Shanghai, China). Instrumentation. All NMR spectra (1H NMR13,C NMR, H-H COSY, HMQC, and HMBC spectra) were obtained on a Bruker DPX-400 spectrometer using standard Bruker pulse programs (Bruker BioSpin GmbH, Rheinstetten, Germany) with solvent peak as references. HR-ESI-MS data were obtained on a Bruker Daltonic micrOTOF-Q II MS spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). UV absorption spec- tra were recorded on a UV-2600 ultraviolet and visible spectrophotometer (Shimadzu, Tokyo, Japan) from 200 nm to 900 nm. Infrared (IR) spectra were recorded on a Nicolet Nexus 470 spectrometer (Nicolet, Madison, American) using the compounds in the KBr pellet form. Preparative HPLC was carried out on a LabAllance Model 201 HPLC system (LabAllance, Tianjin, China) and a Kromasil C18 column preparative column (10 μm, 250 × 30 mm, Kromasil, Stockholm, Sweden). Preparation of plant extracts. The dried and powdered M. officinalis var. biloba fruits (5.1 kg, size less than 0.25 mm) were extracted three times using 10 volumes of 70% alcohol (50 L) for 3 h at reflux. The filtrate was combined and concentrated under vacuum to yield a dried alcohol extract (804.9 g). www.nature.com/scientificreports/ Oral malodor is a major social and psychological problem that affects the majority of the general population20. Oral malodor is divided into two kinds, namely, pathological and physiological. Pathological oral malodor is mainly caused by oral diseases, such as caries. Physiological oral malodor is the result of lipid peroxi- dation from gastrointestinal food debris. Studies2,4,20 have proven that the antioxidant and antimicrobial activities Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 8 www.nature.com/scientificreports/ Figure 5. Protective activity of nine compounds against Fe2+/H2O2-induced formation of MDA (A), LOOH (B), CAT (C), GR (D) and SOD (E) in rat liver mitochondria. Data are presented as the mean ± SD (n = 3). Symbols represent statistical significance. p < 0.001, compared with the model group; p < 0.01, compared with the model group; p < 0.05, compared with the model group; ###p < 0.001, compared with the control group. BHT is butylated hydroxytoluene. Figure 5. Protective activity of nine compounds against Fe2+/H2O2-induced formation of MDA (A), LOOH (B), CAT (C), GR (D) and SOD (E) in rat liver mitochondria. Data are presented as the mean ± SD (n = 3). Symbols represent statistical significance. p < 0.001, compared with the model group; p < 0.01, compared with the model group; *p < 0.05, compared with the model group; ###p < 0.001, compared with the control group. BHT is butylated hydroxytoluene. of magnolol and honokiol are responsible for oral malodor combat. In the present study, phenylethanoid glyco- sides were demonstrated to have free radical scavenging activities and inhibitory effects of lipid peroxidation. Thus, phenylethanoid glycosides could assist in improving the condition of physiological oral malodor. M. offic- inalis var. biloba fruits should be used as an alternative food resource added in gum and mints. More studies are necessary to further understand the action mechanism of phenylethanoid glycosides. These studies should also add further value to this by-product of M. officinalis var. biloba fruits. Methods l The alcohol extract was suspended in distilled water (10 L) and then successively partitioned three times with chloroform (20 L), ethyl acetate (10 L), and n-butanol alcohol (10 L), which yielded the chloroform fraction (398.4 g), ethyl acetate fraction (180.8 g), and n-butanol fraction (63.4 g), respectively. All fractions were stored at −20 °C until further use. Isolation and purification procedures of phenylethanoid glycosides. The n-butanol fraction (20 g) prepared from the previously described steps was further isolated on a preparative HPLC system. Each chroma- tographic run was carried out at a flow rate of 20 mL/min with a binary mobile phase consisting of methanol (A) Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 9 www.nature.com/scientificreports/ and 0.1% formic acid (B) using a step gradient profile. The gradient started with 10% A, was varied to 35% A at 10 min, 46% A at 40 min, 48% A at 43 min, 100% A at 50 min, and 100% A isocratic for 10 min, and decreased to 10% A in 0.1 min. After re-equilibration at 10% A for 12 min, the next sample was injected. The temperature of the column oven was 25 °C, and 100 μL (300 mg/mL) was injected into the system every time. The peaks adsorbed at 315 nm were recorded. Compounds 1 (797.7 mg), 2 (226.4 mg), 3 (195.9 mg), and 4 (298.3 mg) were obtained at the retention times of 22.4, 32.6, 35.3, and 41.7 min, respectively. The subfraction that was collected at a retention time from 27 min to 29 min was further applied to the preparative HPLC system, which was eluted isocratically with 18% acetonitrile in water (containing 0.1% formic acid) at a flow rate of 20 mL/min. Compounds 5 (56.0 mg) and 6 (34.1 mg) were then purified. Simultaneously, the subfraction obtained at a retention time of 19.3 min was subjected to the preparative HPLC system, which was eluted isocratically with 26% methanol in water at a flow rate of 20 mL/min. Compounds 7 (37.7 mg) and 8 (52.7 mg) were then obtained. The subfraction obtained at a retention time of 38.4 min was further isolated over the preparative HPLC system, which was eluted isocratically with 43% methanol in water at a flow rate of 20 mL/min to yield compound 9 (35.9 mg). Acid hydrolysis and sugar analysis in glycosides. Methods l Sugar analysis of compounds 2, 3, 5, 6 and 8 was carried out according to the method described by previous studies46,47. In brief, compounds 2, 3, 5, 6 and 8 (each 4.0 mg) were hydrolyzed by heating in 4 M CF3COOH (4 mL) at 95 °C for 4 h. After cooling, the solution was extracted with CH2Cl2 (3 × 2 mL). The water layer was concentrated by reducing pressure and dried by vacuum. Anhydrous pyridine (0.5 mL) and L-cysteine methyl ester hydrochloride (2 mg) were added and reacted at 60 °C for 1 h. After cooling to room temperature, o-tolyl isothiocyanate (10 μL) was added to the mixture and further heated at 60 °C for 1 h. The reaction mixture was directly analyzed by reversed-phase HPLC using a Diamonsil C18 analytical column (5 μm, 250 × 4.6 mm), which was eluted isocratically with 25% acetonitrile in water (con- taining 0.1% formic acid) at a flow rate of 1 mL/min. The temperature of the column oven was 25 °C, and 20 μ L was injected into the system every time. The UV spectra were collected at 250 nm. Peaks of the hydrolysate of compounds 2, 3, 5, 6 and 8 were identified by comparing the retention times of authentic samples of D-allose (tR = 19.330), D-glucose (tR = 18.566), and L-rhamnose (tR = 33.411) after simultaneous treatment under the same conditions. DPPH and ABTS radical scavenging assays. DPPH and ABTS, two relatively stable free radical compounds widely used to test free radical scavenging activity, were determined using a previously described method48,49. Radical scavenging activity was calculated using the equation: radical scavenging activity (%) = [(A0 − A)/A0] × 100 (where A0 is the absorbance of the control, and A is the absorbance of the test sample). VC and BHT with the same concentrations as the samples were used as positive controls. Superoxide anion radical (O2 −) scavenging assay. The superoxide anion radical is the most common free radical generated in vivo. The capacity of each compound to scavenge superoxide radicals was examined by a pyrogallol auto-oxidation system50. Pyrogallic acid can auto-oxidize under alkaline conditions to produce O2 − directly, and the rate constant of this auto-oxidation reaction is dependent on the O2 − concentration. If the test compound can scavenge O2 −, the auto-oxidation reaction can slow down significantly. Methods l The auto-oxidation rate constant (Kb) of pyrogallic acid was calculated from the curve of A325 nm versus time. VC and BHT were used for comparative purposes. Simultaneously, the Kb value (10−4 A/s) of the blank control was recorded. Test animals. Sprague–Dawley (SD) rats (200 ± 20 g) were purchased from the Laboratory Animal Center of Wuhan University, Wuhan, China. All the rats were housed under regulated conditions of 12 h light/12 h dark cycles, 25 ± 2 °C, and 30%–60% relative humidity. They were fed with a standard pellet diet (rat feed, purchased from the Laboratory Animal Center of Wuhan University, Wuhan, China) and unlimited water. The animals were allowed to acclimatize to the room conditions for 2 days. All experimental procedures were approved by the Institutional Animal Ethical Committee of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Wuhan University in Wuhan, China. The experimental methods were performed in accordance with “Regulations for the Administration of Affairs Concerning Experimental Animals” and “Guiding Opinions of Treating Experimental Animals with Good Ethics”, which have been formally released and imple- mented by Chinese Government. Preparation of mitochondria. Mitochondria were isolated from SD rats according to the method described by previous studies45. All procedures were carried out at 4 °C. Mitochondrial damage model caused by UVB. The experiment consisted of the control group, model group, BHT group, and test sample group. Each group comprised the following set up: for the control group, 0.5 mL of mitochondrial protein + 0.1 mL of phosphate buffer (0.05 M, pH 7.4); for the model group, 0.5 mL of mitochondrial protein + 0.1 mL of phosphate buffer (0.05 M, pH 7.4) + UVB; for the BHT group, 0.5 mL of mitochondrial protein + 0.1 mL of BHT (12.5 μM, in phosphate buffer) + UVB; and for the test compound group, 0.5 mL of mitochondrial protein + 0.1 mL of test sample (12.5 μM, in phosphate buffer) + UVB. The model, BHT, and test compound groups were irradiated for 4 h with a 20 W UVB lamp (TL/12RS, Philips) from a 15 cm dis- tance. The control group was kept at 37 °C for 4 h without irradiation. Mitochondrial damage model caused by Fe2+/H2O2. The experiment consisted of the control group, model group, BHT group, and test sample group. References Yan, R. Y., Liu, H. L., Zhang, J. Y. & Yang B. Phenolic glycosides and other constituents from the bark of Magnolia officinalis. J. Asian Nat. Prod. Res. 16, 400–405 (2014).fi 15. Guo, Z. F. et al. A novel aporphine alkaloid from Magnolia officinalis. Fitoterapia 82, 637–641 (2011). 15. Guo, Z. F. et al. A novel aporphine alkaloid from Magnolia officinalis. Fitoterapia 82, 637–641 (2011).fi fi 6. Yu, S. X., Yan, R. Y., Liang, R. X., Wang, W. & Yang, B. Bioactive polar compounds from stem bark of Magnolia officinalis. Fitoterapia 83, 356–361 (2012).h 7. National health and family planning commission of the people’s republic of China. The Ministry of Health informed about further standardize health food raw materials management 51 (2002). f g 8. College ter Beoordeling van Geneesmiddelen. Magnolia bark extract http://www.cbg-meb.nl/documenten/rapporten/2010/09/25 nv-extract-van-magnoliabast (2010). 18. College ter Beoordeling van Geneesmiddelen. Magnolia bark extract http://www.cbg-meb.nl/documenten/rapporten/2010/09/25/ nv-extract-van-magnoliabast (2010). 19 Advisory Committee on Novel Foods and Processes (ACNFP) Secretariat Food Standards Agency Initial opinion: Magnolia bark nv-extract-van-magnoliabast (2010). 19. Advisory Committee on Novel Foods and Processes (ACNFP) Secretariat Food Standards Agency. Initial opinion: Magnolia bark supercritical carbon dioxide extract as a food ingredient Magnolia bark extract EC No 107 http://acnfp food gov uk/assess/ g 9. Advisory Committee on Novel Foods and Processes (ACNFP) Secretariat Food Standards Agency. Initial opinion: Magnolia bark supercritical carbon dioxide extract as a food ingredient. Magnolia bark extract. EC No. 107 http://acnfp.food.gov.uk/assess fullapplics/magnoliabarkextract (2011).f pp g 20. Greenberg, M., Urnezis, P. & Tian, M. Compressed mints and chewing gum containing magnolia bark extract are effective against bacteria responsible for oral malodor. J. Agric. Food Chem. 55, 9465–9469 (2007). p g 21. He, J. S. et al. Population structure and genetic diversity distribution in wild and cultivated populations of the traditional Ch medicinal plant Magnolia officinalis subsp. biloba (Magnoliaceae). Genetica 135, 233–243 (2009). 21. He, J. S. et al. Population structure and genetic diversity distribution in wild and cultivated populations of the traditional Chinese medicinal plant Magnolia officinalis subsp. biloba (Magnoliaceae). Genetica 135, 233–243 (2009).fii , J p g y p p medicinal plant Magnolia officinalis subsp. biloba (Magnoliaceae). Genetica 135, 233–243 (2009).fii p g ffi p 22. Wang, Y. W. et al. New phenylethanoid glycosides from of Pacific Basin Societies(Hawaii, USA) 2082 (2015).i g ffi g 22. Wang, Y. W. et al. New phenylethanoid glycosides from Magnolia officinalis fruits. References 1. Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic China, 2015 (China Medical Science Press: Beijing China), 251–252 (2015).f 2. Greenberg, M., Dodds, M. & Tian, M. Naturally occurring phenolic antibacterial compounds show effectiveness against oral bacteria by a quantitative structure-activity relationship study. J. Agric. Food. Chem. 56, 11151–11156 (2008). y q y p y g 3. Tang, S. Y. et al. Characterization of antioxidant and antiglycation properties and isolation of active ingredients from tradit chinese medicines. Free Radical Bio. Med. 36, 1575–1587 (2001). 4. Zhao, C. & Liu, Z. Q. Comparison of antioxidant abilities of magnolol and honokiol to scavenge radicals and to protect DNA Biochimie 93, 1755–1760 (2011).l 5. Chao, L. K. et al. Anti-inflammatory bioactivities of honokiol through inhibition of protein kinase C, mitogen-activated protein kinase, and the NF-kappaB pathway to reduce LPS-induced TNF alpha and NO expression. J. Agric. Food. Chem. 58, 3472–3428 (2010).fil 6. Kuo, W. L., Chung, C. Y., Hwang, T. L. & Chen, J. J. Biphenyl-type neolignans from Magnolia officinalis and their anti-inflammatory activities. Phytochemistry 85, 153–160 (2013).i y y 7. Maruyama, Y., Kuribara, H., Morita, M., Yuzurihara, M. & Weintraub, S. T. Identification of magnolol and honokiol as anxiolytic agents in extracts of saiboku-to, an oriental herbal medicine. J. Nat. Prod. 61, 135–138 (1998). g 8. Watanabe, K. Pharmacology of magnolia bark with special reference to gastrointestinal functions. Gendai Toyo Igaku 7, 54–59 (1986).hi 9. Liu, Y. et al. The natural compound magnolos inhibits invasion and exhibits potential in hunman breast cancer therapy. Scientific Reports 3, 3098–3105 (2013). p 10. Lin, Y. R., Chen, H. H., Ko, C. H. & Chan, M. H. Neuroprotective activity of honokiol and magnolol in cerebellar granule cell damage. Eur. J. Pharmacol. 537, 64–69 (2006).if g 1. Jennifer, H., Ho, C. & Hong, C. Y. Cardiovascular protection of magnolol: cell-type specificity and dose-related effects. J. Biomed. Sci 19, 70–78 (2012). 19, 70 78 (2012). 2 Zhang, B B , Wang, H & Chen, S Z Quantification and structural identification of related phenolic compounds in the raw 2. Zhang, B. B., Wang, H. & Chen, S. Z. Quantification and structural identification of related phenolic compounds in the raw medicinal material honokiol. J.Chi.Pharm. Sci. 22, 420–426 (2013). 3. Yahara, S., Nishiyori, T., Kohda, A., Nohara, T. & Nishioka, I. Isolation and charaterization of phenolic compounds from Magnoliae Cortex produced in China. Chem. Pharm. Bull. 39, 2024–2036 (1991).fi p 4. Methods l Each group comprised the following set up: for the control group, 0.5 mL of mitochondrial protein + 0.5 mL of phosphate buffer (0.05 M, pH 7.4); for the model group, 0.5 mL of mitochondrial protein + 0.5 mL of phosphate buffer (0.05 M, pH 7.4) + 30 μL FeSO4(10 mM) + 20 μ L H2O2 (65 mM); for the BHT group, 0.5 mL of mitochondrial protein + 0.5 mL of BHT (12.5 μM, in phosphate Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 10 www.nature.com/scientificreports/ buffer) + 30 μL FeSO4(10 mM) + 20 μL H2O2 (65 mM); and for the test compound group, 0.5 mL of mitochondrial protein + 0.1 mL of test sample (12.5 μM, in phosphate buffer) + 30 μL FeSO4(10 mM) + 20 μL H2O2 (65 mM). All experiment groups were kept at 37 °C for 1 h. buffer) + 30 μL FeSO4(10 mM) + 20 μL H2O2 (65 mM); and for the test compound group, 0.5 mL of mitochondrial protein + 0.1 mL of test sample (12.5 μM, in phosphate buffer) + 30 μL FeSO4(10 mM) + 20 μL H2O2 (65 mM). All experiment groups were kept at 37 °C for 1 h. Measurement of biochemical indicators. The values of A520 were used to evaluate the mitochondrial swelling degree43. MDA assay was evaluated using the thiobarbituric acid method45. LOOH assay was measured using a modified version of a previously reported method45. CAT, GR and SOD activities in mitochondrial pro- tein were measured by commercial kits purchased from Nanjing Jiancheng Technology Co. Ltd. (Nanjing, China). Statistical analysis. All experiments were carried out in triplicate, and the results were reported as the mean ± standard deviation (n = 3). The data were analyzed using one-way ANOVA. Statistically significant effects were analyzed, and the means were also compared using least-significant difference (LSD) test. Statistical signif- icance was determined at p < 0.05. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 2. Hasegawa, T., Fukuyama, Y., Yamada, T. & Nakagawa, K. Isolation and structure of magnoloside A, a new phenylpropanoid glycosides from Magnolia obovata Thunb. Chem. Lett. 17, 163–166 (1988). g y gh 3. Hasegawa, T., Fukuyama, Y., Yamada, T. & Nakagawa, K. Structures of magnoloside B and C. Novel phenylpropanoid glycosides with allopyranose as core the sugar unit. Chem. Pharm. Bull. 36, 1245–1248 (1988).l py g ( ) 4. Porter, E. A. et al. Phenylethanoid glycosides in tepals of Magnolia salicifoliaand their occurrence in flowers of Magnoliaceae Phytochemistry 117, 185–193 (2015). y y 35. Yoshikawa, M. et al. Phenylethanoid oligoglycosides and acylated oligosugars with vasorelaxant activity from Cistanche tubu Bioorg. Med. Chem. 14, 7468–7475 (2006). g ( ) 36. Seo, K. H. et al. Phenylethanoid glycosides from the fruits of Magnolia obovata, Chem. Nat. Compd+. 51, 660–665 (2015). 37. Xue, Z. Z., Yan, R. Y. & Yang, B. Phenylethanoid glycosides and phenolic glycosides from stem bark of Magnolia offici Phytochemistry 127, 50–62 (2016).iif y y 38. Es-Safi, N. E., Kollmann, A., Khlifi, S. & Ducrot, P. H. Antioxidative effect of compounds isolated from Globularia alypum L. structure–activity relationship. LWT - Food Sci. Technol. 40, 1246–1252 (2007). y p 9. Wu, A. Z., Lin, C. Z. & Zhu, C. C. Progress in structure-activity relationship of phenylethanoid glycosides. Nat. Prod. Res. Dev. 25 862–865 (2013). 0. Park, H. M. et al. Extract of Punica granatum inhibits skin photoaging induced by UVB irradiation. Int. J. Dermatol. 49, 276–282 (2010).ffi ( ) 41. Oboh, G., Akinyemi, A. J. & Ademiluyi, A. O. Antioxidant and inhibitory effect of red ginger (Zingiber officinale var. Rubra) and white ginger (Zingiber officinale Roscoe) on Fe2+ induced lipid peroxidation in rat brain in vitro. Exp. Toxicol. Pathol. 64, 31–36 (2012). ( ) 42. Bhattacharya, S., Kamat, J. P., Bandyopadhyay, S. K. & Chattopadhyay, S. Comparative inhibitory properties of some Indian medicinal plant extracts against photosensitization-induced lipid damage. Food Chem. 113, 975–979 (2009). p g p p g 43. Hunter, F. E. et al. Swelling and lysis of rat liver minochondria induced by ferrous ions. J. Biol. Chem. 238, 828–835 (1963).l 44. Zhu, L. et al. Coptis chinensis inflorescence extract protection against ultraviolet-B-induced phototoxicity, and HPLC-MS analysis of its chemical composition. Food Chem. Toxicol. 50, 2584–2588 (2012). of its chemical composition. Food Chem. Toxicol. 50, 2584–258 45. Huang, B. et al. www.nature.com/scientificreports/ In vitro and in vivo evaluation of inhibition activity of lotus (Nelumbo nucifera Gaertn.) leaves against ultraviolet B-induced phototoxicity. J. Photoch. Photobio. B 121, 1–5 (2013). p y 6. Tanana, T., Nakashima, T., Ueda, T., Tomii, K. & Kouno, I. Facile discrimination of aldose enantiomers by reversed-phase HPLC Chem. Pharm. Bull. 55, 899–901 (2007). 7. Tian, E. L., Yang, G. Z., Mei, Z. N. & Chen, Y. Chemical constituents from stems of Glycosmis pentaphylla. Chin. Tradit. Herb. Drug 45, 1358–1362 (2014).f 48. Mawalagedera, S. M. M. R., Ou, Z. Q., McDowell, A. & Gould, K. L. Effects of boiling and in vitro gastrointestinal digestion on the antioxidant activity of Sonchus oleraceus leaves. Food Funct. 7, 1515–1522 (2016). antioxidant activity of Sonchus oleraceus leaves. Food Funct. 7, 15 y 9. Cristina, R. C. & Rudolf, J. Free radical scavenging (antioxidant activity) of natural dissolved organic matter. Mar. Chem. 177 668–676 (2015). 50. Zhou, G., Chen, Y. X., Liu, S., Yao, X. C. & Wang, Y. W. In vitro and in vivo hepatoprotective and antioxidant activity of etha extract from Meconopsis integrifolia (Maxim.) Franch. J. Ethnopharmacol. 148, 664–670 (2013). References P of Pacific Basin Societies(Hawaii, USA) 2082 (2015).i g ffi g 2. Wang, Y. W. et al. New phenylethanoid glycosides from Magnolia officinalis fruits. Pacifichem 2015, International Chemical Congress of Pacific Basin Societies(Hawaii, USA) 2082 (2015).i fi 22. Wang, Y. W. et al. New phenylethanoid glycosides from Mag i 23. Li, L. et al. Isolation and purification of phenylethanoid glycosides from Cistanche deserticola by high-speed counter-current chromatography. Food Chem. 108, 702–710 (2008).l g p y 24. Nan, Z. D. et al. Phenylethanoid glycosides with anti-inflammatory activities from the stems of Cistanche deserticola cultured in Tarim desert. Fitoterapia 89, 167–174 (2013). p 5. Morikawa, T. et al. Acylated phenylethanoid oligoglycosides with hepatoprotective activity from the desert plant Cistanche tubulosa Bioorg. Med. Chem. 18, 1882–1890 (2010).f g 26. Song, X. et al. Immunomodulatory effects of crude phenylethanoid glycosides from Ligustrum purpurascens. J. Ethnopharmacol. 144, 584–591 (2010).l 7. Liu, Y. L. et al. Antimicrobial, anti-inflammatory activities and toxicology of phenylethanoid glycosides from Monochasma savatier Franch. ex Maxim. J. Ethnopharmacol. 149, 431–437 (2013). p 8. Bardakci, H. et al. Antioxidant activities of several Scutellaria taxa and bioactive phytoconstituents from Scutellaria hastifolia L. Ind Crop. Prod. 77, 196–203 (2015). p 29. Marino, S. D. et al. Antioxidant activity of phenolic and phenylethanoid glycosides from Teucrium polium L. Food Chem. 133, 21–28 (2012). 30. Liu, Y. G. et al. Synthetic phenylethanoid glycoside derivatives as potent neuroprotective agents. Eur. J. Med. Chem. 95, 313–323 (2015).i Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 11 Author Contributions L.L.G. and Y.W.W. designed research and wrote the manuscript; L.L.G., W.H.Z., G.Z., B.X.M., Q.G.M. and Y.X.C. contributed experimental data and analysis; All authors reviewed the manuscript. Acknowledgements g This work was supported by the Project of the National Twelve-Five Year Research Program of China (2011BAI06B06; 2012BAI29B03), the Commonweal Specialized Research Fund of China Agriculture (201103016), and the Nanjing 321 plan for Bringing in technological leading talents (2013A12011). Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing Interests: The authors declare no competing financial interests. How to cite this article: Ge, L. et al. Nine phenylethanoid glycosides from Magnolia officinalis var. biloba fruits and their protective effects against free radical-induced oxidative damage. Sci. Rep. 7, 45342; doi: 10.1038/ srep45342 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 7:45342 \| DOI: 10.1038/srep45342 12
https://openalex.org/W3011006851	https://europepmc.org/articles/pmc7139477?pdf=render	English	null	Ellagic Acid and Urolithins A and B Differentially Regulate Fat Accumulation and Inflammation in 3T3-L1 Adipocytes While Not Affecting Adipogenesis and Insulin Sensitivity	International journal of molecular sciences	2,020	cc-by	13,886	Received: 14 December 2019; Accepted: 11 March 2020; Published: 18 March 2020 Abstract: Ellagic acid (EA) is a component of ellagitannins, present in crops such as pecans, walnuts, and many berries, which metabolized by the gut microbiota forms urolithins A, B, C, or D. In this study, ellagic acid, as well as urolithins A and B, were tested on 3T3-L1 preadipocytes for diﬀerentiation and lipid accumulation. In addition, inﬂammation was studied in mature adipocytes challenged with lipopolysaccharide (LPS). Results indicated that EA and urolithins A and B did not aﬀect diﬀerentiation (adipogenesis) and only EA and urolithin A attenuated lipid accumulation (lipogenesis), which seemed to be through gene regulation of glucose transporter type 4 (GLUT4) and adiponectin. On the other hand, gene expression of cytokines and proteins associated with the inﬂammation process indicate that urolithins and EA diﬀerentially inhibit tumor necrosis factor alpha (TNFα), inducible nitric oxide synthase (iNOS), interleukin 6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Urolithins A and B were found to reduce nuclear levels of phosphorylated nuclear factor κB (p-NF-κB), whereas all treatments showed expression of nuclear phosphorylated protein kinase B (p-AKT) in challenged LPS cells when treated with insulin, indicating the fact that adipocytes remained insulin sensitive. In general, urolithin A is a compound able to reduce lipid accumulation, without aﬀecting the protein expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer binding protein-α (c/EBPα), and PPARα, whereas EA and urolithin B were found to enhance PPARγ and c/EBPα protein expressions as well as fatty acid (FA) oxidation, and diﬀerentially aﬀected lipid accumulation. Keywords: urolithins A and B; ellagic acid; adipogenesis; lipid metabolism; inﬂammation; insulin sensitivity; 3T3-L1 adipocytes Ellagic Acid and Urolithins A and B Diﬀerentially Regulate Fat Accumulation and Inﬂammation in 3T3-L1 Adipocytes While Not Aﬀecting Adipogenesis and Insulin Sensitivity Luis Cisneros-Zevallos 1,2,, Woo Young Bang 1 and Claudia Delgadillo-Puga 3 Luis Cisneros-Zevallos 1,2,, Woo Young Bang 1 and Claudia Delgadillo-Puga 3 1 Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843-2133, USA; wybang@gmail.com 1 Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843-2133, USA; wybang@gmail.com 2 Department of Nutrition and Food Science, Texas A&M University, College Station, TX 77843, USA 3 Departamento de Nutrición Animal Dr. Fernando Pérez-Gil Romo, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), CDMX 14080, Mexico; claudia.delgadillop@incmnsz.mx * Correspondence: lcisnero@tamu.edu; Tel.: +1-979-845-3244 2 Department of Nutrition and Food Science, Texas A&M University, College Station, TX 77843, USA 3 Departamento de Nutrición Animal Dr. Fernando Pérez-Gil Romo, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), CDMX 14080, Mexico; claudia.delgadillop@incmnsz.mx * Correspondence: lcisnero@tamu.edu; Tel.: +1-979-845-3244 International Journal of Molecular Sciences International Journal of Molecular Sciences 1. Introduction Ellagic acid (EA) is present in plants in the form of hydrozable tannins called ellagitannins with protective roles in the plant cell and possibly regulating plant metabolism. Diﬀerent sources of ellagic acid include pecans, walnuts, and many berries [1–3]. Ellagic acid is extensively metabolized by the gut microbiota to yield the hydroxy-dibenzopyranone derivatives, used to form urolithins A, B, C, or D. Urolithins (Uro) are bioavailable, and they can reach a concentration as high as a 0.5–18.5 µM level as glucuronide derivatives in plasma after consuming 1L of pomegranate juice daily [4,5], or up to 185 µM after consuming 200g of walnuts [6]. However, there are urolithin non-producer Int. J. Mol. Sci. 2020, 21, 2086; doi:10.3390/ijms21062086 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 2086 2 of 16 individuals (metabotype 0), as well as urolithin producer individuals metabotypes A (mainly urolithin A derivatives) or metabotype B (mainly urolithin B and A derivatives). Diﬀerences in intestinal microbiota have been associated with the interindividual variability, which seems also correlated with health status and predisposition to chronic diseases (e.g., metabolic syndrome) [7]. Several studies have indicated that ellagic acid and its derivative urolithins possess beneﬁcial eﬀects against prostate and colon cancer and exert some proestrogenic/antiestrogenic eﬀects [8–10], as well as having anti-inﬂammatory activity in colon ﬁbroblasts and protection against oxidative stress [11,12]. Other reports have shown antiatherogenic eﬀects in human umbilical vein endothelial cells [13] and neuroprotective eﬀects against Alzheimer’s disease in in vitro studies [14]. However, these compounds (EA and Uro) as potential anti-obesity treatments have started to acquire attention in recent years. Obesity is a complex disorder with multiple causes, including both genetic and environmental factors, and is associated with the risk of insulin resistance (type 2 diabetes), fatty liver disease, hypertension, and atherosclerosis [15]. y yp Over the past years, in vitro systems have been used to deﬁne the transcriptional events regulating preadipocyte diﬀerentiation (adipogenesis) and adipocyte function; 3T3-L1 being the most frequently employed cell line [16]. During the diﬀerentiation process of preadipocytes into adipocytes, an elaborate network of transcription factors coordinates the expression of hundreds of speciﬁc proteins. At the center of this elaborate network are speciﬁc adipogenic factors such as peroxisome proliferator-activated receptor-γ (PPARγ) and the CCAAT/enhancer binding protein-α (c/EBPα). Both transcription factors are key to successfully complete the entire diﬀerentiation process. 1. Introduction PPARγ is a member of the nuclear–receptor superfamily—its expression induces growth arrest and initiates adipogenesis in ﬁbroblasts. It is also required for maintenance of the diﬀerentiated state [17], and in particular is considered the master regulator of adipogenesis; without it, precursor cells are incapable of expressing any known aspect of the adipocyte phenotype [18]. On the other hand, cells deﬁcient in c/EBPα are capable of adipocyte diﬀerentiation; however, these c/EBPα-deﬁcient cells are insulin resistant [19,20]. More recently, data from a variety of knockout mice have conﬁrmed these in vitro studies, showing that many components of this network are required regulators of adipocyte development and function. Another transcription factor member of nuclear-receptor superfamily, PPARα, plays an important role in fatty acid oxidation in liver and skeletal muscle, and it is reported that PPARα activators may aﬀect adipose tissue metabolism and the activation of both mitochondrial and peroxisomal β-oxidation [21,22]. The objective of this study was to determine the eﬀect of ellagic acid and derived gut microbial metabolites urolithins A and B (Figure 1A) on adipocyte diﬀerentiation in murine 3T3-L1 ﬁbroblasts and fat accumulation. The study also examined the eﬀect on PPARγ, PPARα, and c/EBPα, and genes associated with lipogenic and lipolytic enzymes in order to provide basic data for the elucidation of the mode of action. In addition, we reported the anti-inﬂammatory eﬀects of these compounds on lipopolysaccharide (LPS)-challenged adipocytes and insulin-sensitizing activities. This work is based on a report by Cisneros-Zevallos and Bang [23], which, to the best of our knowledge, was the ﬁrst study on ellagic acid-derived gut microbial metabolites urolithins A and B’s eﬀects on adipocytes. 3 of 16 ARγ and Int. J. Mol. Sci. 2020, 21, 2086 or enhanced lipolytic e /EBP i Figure 1. The effect of ellagic acid, urolithin A, and urolithin B on cell viability and intracellular triglyceride content in preadipocytes and mature adipocytes. (A) Chemical structures of ellagic acid and its derivatives urolithins A and B. (B) Cell viability of 3T3-L1 preadipocytes in control samples (0.025% dimethyl sulfoxide (DMSO)) and after 24 h treatment with 25 μM urolithin A, urolithin B, and ellagic acid. The values represent the mean ± SD (n = 3) of three independent experiments conducted in duplicate. (C) Intracellular lipid accumulation in 3T3-L1 mature adipocytes after 8-day treatment with 25 μM of urolithin A, urolithin B, ellagic acid, and control (0.025% DMSO). 1. Introduction The values represent the mean ± SD (n = 3) of three independent experiments conducted in duplicate. (C) Intracellular lipid accumulation in 3T3-L1 mature adipocytes after 8-day treatment with 25 µM of urolithin A, urolithin B, ellagic acid, and control (0.025% DMSO). Data are means ± SD (n = 3) of three independent experiments conducted in triplicate. Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. Fi 1 Th ff t f ll i id lithi A d lithi B ll i bilit d i t ll l Figure 1. The eﬀect of ellagic acid, urolithin A, and urolithin B on cell viability and intracellular 1. Introduction Data are means ± SD (n = 3) of three independent experiments conducted in triplicate. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 1. The eﬀect of ellagic acid, urolithin A, and urolithin B on cell viability and intracellular triglyceride content in preadipocytes and mature adipocytes. (A) Chemical structures of ellagic acid and its derivatives urolithins A and B. (B) Cell viability of 3T3-L1 preadipocytes in control samples (0.025% dimethyl sulfoxide (DMSO)) and after 24 h treatment with 25 µM urolithin A, urolithin B, and ellagic acid. The values represent the mean ± SD (n = 3) of three independent experiments conducted in duplicate. (C) Intracellular lipid accumulation in 3T3-L1 mature adipocytes after 8-day treatment with 25 µM of urolithin A, urolithin B, ellagic acid, and control (0.025% DMSO). Data are means ± SD (n = 3) of three independent experiments conducted in triplicate. Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. Figure 1. The effect of ellagic acid, urolithin A, and urolithin B on cell viability and intracellular triglyceride content in preadipocytes and mature adipocytes. (A) Chemical structures of ellagic acid and its derivatives urolithins A and B. (B) Cell viability of 3T3-L1 preadipocytes in control samples (0.025% dimethyl sulfoxide (DMSO)) and after 24 h treatment with 25 μM urolithin A, urolithin B, and ellagic acid. The values represent the mean ± SD (n = 3) of three independent experiments conducted in duplicate. (C) Intracellular lipid accumulation in 3T3-L1 mature adipocytes after 8-day treatment with 25 μM of urolithin A, urolithin B, ellagic acid, and control (0.025% DMSO). Data are means ± SD (n = 3) of three independent experiments conducted in triplicate. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test Figure 1. The eﬀect of ellagic acid, urolithin A, and urolithin B on cell viability and intracellular triglyceride content in preadipocytes and mature adipocytes. (A) Chemical structures of ellagic acid and its derivatives urolithins A and B. (B) Cell viability of 3T3-L1 preadipocytes in control samples (0.025% dimethyl sulfoxide (DMSO)) and after 24 h treatment with 25 µM urolithin A, urolithin B, and ellagic acid. 2.3. Effect of Ellagic Acid and Urolithins A and B on Transcription Differentiation 2.1. Eﬀect of Ellagic Acid and Urolithins A and B on Cell Viability In the early stage of differentiation, preadipocyte factor 1 (PREF-1) is an important transcription factor present only in preadipocytes, and the overexpression of this marker has an inhibitory effect on the differentiation process. On the other hand, PPARγ is a key transcription factor to conduct the differentiation process in preadipocytes, and the expression of this marker starts when differentiation is triggered, being maintained during the whole life of mature adipocytes. Accordingly, PREF-1 To study the eﬀect on cell viability, 3T3-L1 preadipocytes were treated with ellagic acid and urolithins at 25 µM for 24 h; then the MTS assay was performed. No signiﬁcant reductions were observed with respect to the control after the treatments with ellagic acid and urolithins A and B (Figure 1B). These results showed that these compounds did not aﬀect cell viability in preadipocytes. Similar results were reported in human colonic ﬁbroblasts by González-Sarrías et al. [12]. decreased for controls and all treated cells, whereas PPARγ was expressed f (Figures 2 and 3), confirming that adipogenesis was not affected by ellagic acid a 2.2. Eﬀect of Ellagic Acid and Urolithins A and B on Intracellular Triglyceride Content Eﬀect of ellagic acid, urolithin A, and urolithin B over preadipocyte factor 1 (PREF-1) gene expression at day 4 after diﬀerentiation induction. 3T3-L1 cells were treated with 25 µM of each compound. For control samples, 0.025% DMSO was used. The values represent the mean ± SD (n = 3) of three independent experiments. Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. Figure 2. Effect of ellagic acid, urolithin A, and urolithin B over preadipocyte factor 1 (PREF-1) gene expression at day 4 after differentiation induction. 3T3-L1 cells were treated with 25 μM of each compound. For control samples, 0.025% DMSO was used. The values represent the mean ± SD (n = 3) of three independent experiments. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 2. Effect of ellagic acid, urolithin A, and urolithin B over preadipocyte factor 1 (PREF-1) gene expression at day 4 after differentiation induction. 3T3-L1 cells were treated with 25 μM of each compound. For control samples, 0.025% DMSO was used. The values represent the mean ± SD (n = 3) of three independent experiments. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 2. Eﬀect of ellagic acid, urolithin A, and urolithin B over preadipocyte factor 1 (PREF-1) gene expression at day 4 after diﬀerentiation induction. 3T3-L1 cells were treated with 25 µM of each compound. For control samples, 0.025% DMSO was used. The values represent the mean ± SD (n = 3) of three independent experiments. Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. Figure 2. Effect of ellagic acid, urolithin A, and urolithin B over preadipocyte factor 1 (PREF-1) gene expression at day 4 after differentiation induction. 3T3-L1 cells were treated with 25 μM of each compound. For control samples, 0.025% DMSO was used. The values represent the mean ± SD (n = 3) of three independent experiments. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 3. decreased for controls and all treated cells, whereas PPARγ was expressed f (Figures 2 and 3), confirming that adipogenesis was not affected by ellagic acid a 2.2. Eﬀect of Ellagic Acid and Urolithins A and B on Intracellular Triglyceride Content ( g ), g p g y g B. To investigate eﬀects of ellagic acid and derived metabolites, urolithins A and B, on intracellular triglyceride accumulation during the diﬀerentiation of preadipocytes into fully mature adipocytes (day 8), cells were cultured with these compounds at a concentration of 25 µM from day 0 to day 8. AdipoRed assay was performed to measure the amount of intracellular triglycerides. We found a signiﬁcant decrease over intracellular triglyceride accumulation with ellagic acid and urolithin A (p < 0.05), whereas with urolithin B, a signiﬁcant increase was observed (Figure 1C). These results showed a diﬀerential eﬀect of both urolithins, likely due to diﬀerences between their chemical structures (Figure 1A). Moreover, this result could have been due to anti-adipogenic, anti-lipogenic, or enhanced lipolytic eﬀects of ellagic acid and urolithin A. To answer this question, PPARγ and c/EBPα protein expression was measured. Int. J. Mol. Sci. 2020, 21, 2086 4 of 16 2.3. Eﬀect of Ellagic Acid and Urolithins A and B on Transcription Factors Related to Adipocyte Diﬀerentiation 2.3. Eﬀect of Ellagic Acid and Urolithins A and B on Transcription Factors Related to Adipocyte Diﬀerentiation In the early stage of diﬀerentiation, preadipocyte factor 1 (PREF-1) is an important transcription factor present only in preadipocytes, and the overexpression of this marker has an inhibitory eﬀect on the diﬀerentiation process. On the other hand, PPARγ is a key transcription factor to conduct the diﬀerentiation process in preadipocytes, and the expression of this marker starts when diﬀerentiation is triggered, being maintained during the whole life of mature adipocytes. Accordingly, PREF-1 decreased for controls and all treated cells, whereas PPARγ was expressed for all cells at day 4 (Figures 2 and 3), conﬁrming that adipogenesis was not aﬀected by ellagic acid and urolithins A and B. Int. J. Mol. Sci. 2020, 21, x 4 of 16 Int. J. Mol. Sci. 2020, 21, x 4 of 16 Figure 2. Effect of ellagic acid, urolithin A, and urolithin B over preadipocyte factor 1 (PREF-1) gene expression at day 4 after differentiation induction. 3T3-L1 cells were treated with 25 μM of each compound. For control samples, 0.025% DMSO was used. The values represent the mean ± SD (n = 3) of three independent experiments. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 2. decreased for controls and all treated cells, whereas PPARγ was expressed f (Figures 2 and 3), confirming that adipogenesis was not affected by ellagic acid a 2.2. Eﬀect of Ellagic Acid and Urolithins A and B on Intracellular Triglyceride Content Effect of ellagic acid, urolithin A, and urolithin B over PPARγ protein expression at day 4 after differentiation induction. 3T3-L1 cells were treated with 25 μM of each compound. For control samples, 0.025% DMSO was used. The values represent the mean ± SD (n = 3) of three independent experiments. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 3. Effect of ellagic acid, urolithin A, and urolithin B over PPARγ protein expression at day 4 after differentiation induction. 3T3-L1 cells were treated with 25 μM of each compound. For control samples, 0.025% DMSO was used. The values represent the mean ± SD (n = 3) of three independent experiments. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 3. Eﬀect of ellagic acid, urolithin A, and urolithin B over PPARγ protein expression at day 4 after diﬀerentiation induction. 3T3-L1 cells were treated with 25 µM of each compound. For control samples, 0.025% DMSO was used. The values represent the mean ± SD (n = 3) of three independent experiments. Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. Figure 3. Effect of ellagic acid, urolithin A, and urolithin B over PPARγ protein expression at day 4 after differentiation induction. 3T3-L1 cells were treated with 25 μM of each compound. For control samples, 0.025% DMSO was used. The values represent the mean ± SD (n = 3) of three independent experiments. Different letters among bars denote significant changes among treatments and control ( 0 05) f d b ANOVA d T k ’ h Figure 3. Effect of ellagic acid, urolithin A, and urolithin B over PPARγ protein expression at day 4 after differentiation induction. 3T3-L1 cells were treated with 25 μM of each compound. For control samples, 0.025% DMSO was used. The values represent the mean ± SD (n = 3) of three independent experiments. Different letters among bars denote significant changes among treatments and control (p ≤0 05) performed by ANOVA and Tukey’s post hoc test Figure 3. Eﬀect of ellagic acid, urolithin A, and urolithin B over PPARγ protein expression at day 4 after diﬀerentiation induction. 3T3-L1 cells were treated with 25 µM of each compound. decreased for controls and all treated cells, whereas PPARγ was expressed f (Figures 2 and 3), confirming that adipogenesis was not affected by ellagic acid a 2.2. Eﬀect of Ellagic Acid and Urolithins A and B on Intracellular Triglyceride Content For control samples, 0.025% DMSO was used. The values represent the mean ± SD (n = 3) of three independent experiments. Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. Int. J. Mol. Sci. 2020, 21, 2086 5 of 16 On the late stage, diﬀerentiation of cultured 3T3-L1 preadipocytes into adipocytes is accompanied by a dramatic rise in the rate of transcription of adipose-speciﬁc proteins, like PPARγ and c/EBPα [24]. On mature adipocytes (day 8), we found that 25 µM treatments with ellagic acid and urolithin B signiﬁcantly up-regulated protein expression levels of PPARγ and c/EBPα. No signiﬁcant changes compared with the control were observed for PPARγ and c/EBPα protein expression in cells treated with urolithin A (Figure 4). PPARγ has been proposed as the master regulator of adipogenesis, which is supported by evidence from both in vivo and in vitro studies. PPARγ can induce adipocyte diﬀerentiation in c/EBPα-deﬁcient mouse embryonic ﬁbroblasts, whereas c/EBPα is incapable of inducing the adipogenic program in the absence of PPARγ [18]. This observation suggests that c/EBPα and PPARγ participate in a single pathway of adipose development, in which PPARγ is the most important transcription factor. Several studies have shown that the activation of PPARγ in adipocytes can induce insulin-sensitization and restore the insulin-mediated glucose uptake, storage, and metabolism [25]. In contrast, Armoni et al. [26] reported that PPARγ represses glucose transporter type 4 (GLUT4). Moreover, PPARγ is able to activate c/EBPα, and c/EBPα is required for the induction of insulin sensitivity in adipocytes. This suggests that the insulin sensitization induced by PPARγ is in part mediated by c/EBPα activation. On the other hand, in addition to controlling insulin action, c/EBPα is required for maintaining expression of PPARγ in the mature fat cell [27], and both are considered key regulators in fatty acid biosynthesis and accumulation [28]. On the basis of the intracellular triglyceride accumulation results, we were expecting a decrease in c/EBPα and PPARγ protein expression after the treatments with ellagic acid and urolithin A. However, the present results did not show a clear relationship between these markers and the content of intracellular triglycerides. This fact suggests that these compounds could be modifying pathways related with PPARα and lipo-oxidation. Int. J. Mol. Sci. 2020, 21, x 5 of 16 development, in which PPARγ is the most important transcription factor. decreased for controls and all treated cells, whereas PPARγ was expressed f (Figures 2 and 3), confirming that adipogenesis was not affected by ellagic acid a 2.2. Eﬀect of Ellagic Acid and Urolithins A and B on Intracellular Triglyceride Content Effect of ellagic acid, urolithin A, and urolithin B in the protein expression of PPARγ ( Figure 4. Eﬀect of ellagic acid, urolithin A, and urolithin B in the protein expression of PPARγ (A) an Figure 4. Effect of ellagic acid, urolithin A, and urolithin B in the protein expression of PPARγ (A) and CCAAT-enhancer-binding protein alpha (c/EBPα) (B) in 3T3-L1 adipocytes. The protein expression of PPARγ and c/EBPα were determined by Western blot after 8 days of treatment with 25 µM of the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 4. Eﬀect of ellagic acid, urolithin A, and urolithin B in the protein expression of PPARγ (A) and CCAAT-enhancer-binding protein alpha (c/EBPα) (B) in 3T3-L1 adipocytes. The protein expression of PPARγ and c/EBPα were determined by Western blot after 8 days of treatment with 25 µM of the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. Figure 4. Effect of ellagic acid, urolithin A, and urolithin B in the protein expression of PPARγ (A) and CCAAT-enhancer-binding protein alpha (c/EBPα) (B) in 3T3-L1 adipocytes. The protein expression of PPARγ and c/EBPα were determined by Western blot after 8 days of treatment with 25 µM of the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 4. Eﬀect of ellagic acid, urolithin A, and urolithin B in the protein expression of PPARγ (A) and CCAAT-enhancer-binding protein alpha (c/EBPα) (B) in 3T3-L1 adipocytes. The protein expression of PPARγ and c/EBPα were determined by Western blot after 8 days of treatment with 25 µM of the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. decreased for controls and all treated cells, whereas PPARγ was expressed f (Figures 2 and 3), confirming that adipogenesis was not affected by ellagic acid a 2.2. Eﬀect of Ellagic Acid and Urolithins A and B on Intracellular Triglyceride Content Several studies have shown that the activation of PPARγ in adipocytes can induce insulin-sensitization and restore the insulin- mediated glucose uptake, storage, and metabolism [25]. In contrast, Armoni et al. [26] reported that PPARγ represses glucose transporter type 4 (GLUT4). Moreover, PPARγ is able to activate c/EBPα, and c/EBPα is required for the induction of insulin sensitivity in adipocytes. This suggests that the insulin sensitization induced by PPARγ is in part mediated by c/EBPα activation. On the other hand, in addition to controlling insulin action, c/EBPα is required for maintaining expression of PPARγ in the mature fat cell [27], and both are considered key regulators in fatty acid biosynthesis and accumulation [28]. On the basis of the intracellular triglyceride accumulation results, we were expecting a decrease in c/EBPα and PPARγ protein expression after the treatments with ellagic acid and urolithin A. However, the present results did not show a clear relationship between these markers and the content of intracellular triglycerides. This fact suggests that these compounds could be modifying pathways related with PPARα and lipo-oxidation y g p y p Figure 4. Effect of ellagic acid, urolithin A, and urolithin B in the protein expression of PPARγ (A) and CCAAT-enhancer-binding protein alpha (c/EBPα) (B) in 3T3-L1 adipocytes. The protein expression of PPARγ and c/EBPα were determined by Western blot after 8 days of treatment with 25 µM of the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 4. Eﬀect of ellagic acid, urolithin A, and urolithin B in the protein expression of PPARγ (A) and CCAAT-enhancer-binding protein alpha (c/EBPα) (B) in 3T3-L1 adipocytes. The protein expression of PPARγ and c/EBPα were determined by Western blot after 8 days of treatment with 25 µM of the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. 4 Eﬀect of Ellagic Acid and Urolithins A and B on PPARα and Glycerol Release Figure 4. decreased for controls and all treated cells, whereas PPARγ was expressed f (Figures 2 and 3), confirming that adipogenesis was not affected by ellagic acid a 2.2. Eﬀect of Ellagic Acid and Urolithins A and B on Intracellular Triglyceride Content The protein expression of PPARα was determine by Western blot after 8 days of treatment with 25 µM of the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. (B) Shows the concentration of glycerol released into the culture medium determined by spectrophotometry at 540 nm. Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. Figure 5. Effect of ellagic acid, urolithin A, and urolithin B in the protein expression of peroxisome proliferator-activated receptor-α (PPARα) (A) and measurement of glycerol released into the culture medium of 3T3-L1 adipocytes (B). The protein expression of PPARα was determine by Western blot after 8 days of treatment with 25 µM of the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. (B) Shows the concentration of glycerol released into the culture medium determined by spectrophotometry at 540 nm. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 5. Eﬀect of ellagic acid, urolithin A, and urolithin B in the protein expression of peroxisome proliferator-activated receptor-α (PPARα) (A) and measurement of glycerol released into the culture medium of 3T3-L1 adipocytes (B). The protein expression of PPARα was determine by Western blot after 8 days of treatment with 25 µM of the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. (B) Shows the concentration of glycerol released into the culture medium determined by spectrophotometry at 540 nm. Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. 2.5. Effect of Ellagic Acid and Urolithins A and B on Adipokines, Lipogenesis, and Lipolysis Fat accumulation is the result of the balance between lipogenesis and lipolysis events. Thus, we herein report the activities of different key players as affected by the treatments. Initially, we measured the gene expression of GLUT4 and regulatory adipokines adiponectin and leptin. decreased for controls and all treated cells, whereas PPARγ was expressed f (Figures 2 and 3), confirming that adipogenesis was not affected by ellagic acid a 2.2. Eﬀect of Ellagic Acid and Urolithins A and B on Intracellular Triglyceride Content Results showed a similar pattern among GLUT4 and adiponectin gene expressions with down-regulations for ellagic acid and urolithin A and no effects by urolithin B (Figure 6A,B) similar to the trend observed for fat accumulation (Figure 1C). On the other hand, neither of the compounds affected leptin gene expression with the exception of urolithin B at a higher dose (Figure 6C). Furthermore, because PPARγ and c/EBPα regulate the genes involved in adipogenesis and lipogenesis, including adipocyte protein 2 (AP2), fatty acid synthase (FASN), stearoyl-CoA desaturase-1 (SCD1), and acetyl-CoA carboxylase-1 and 2 (ACC1 and ACC2), we measured their effects by ellagic acid and urolithins. Results indicated that urolithin A reduced mRNA levels of lipogenic genes AP2, ACC1, ACC2, FASN, and SCD1, whereas urolithin B down-regulated these genes with the exception of AP2 and SCD1. On the other hand, ellagic acid differentially affected these genes by down-regulating ACC1 and FASN, not affecting AP2, and up-regulating ACC2 and SCD1 (Figure S1). PPARα is a dietary lipid sensor, whose activation results in hypolipidemic eﬀects in vivo. In addition, its activation promotes both adipocyte diﬀerentiation and FA oxidation in 3T3-L1 adipocytes [29]. Our results did not show a direct relationship between PPARα activation and increased FA oxidation, suggesting that ellagic acid and urolithin B do not have PPARα agonist action, despite non-signiﬁcant increased PPARα levels. PPARα activators are able to attenuate adiposity in animal models of obesity and type 2 diabetes mellitus [31,32], and PPARα agonists such as “ﬁbrates” decrease circulating lipid levels and are commonly used to treat hyperlipidemia and other dyslipidemic states [31]. On the basis of the present ﬁndings, ellagic acid and urolithin B could be explored in the future as a natural alternative to ﬁbrates, but further work is needed to conﬁrm the mode of action of these compounds. In summary, in this part of the study, we found that urolithin A is a compound able to reduce intracellular triglyceride accumulation without aﬀecting the protein expression of PPARγ, c/EBPα, and PPARα, as well as FA oxidation. On the other hand, we found that ellagic acid and urolithin B have a similar eﬀect over the protein expression of PPARγ, c/EBPα, and PPARα, as well as FA oxidation, despite its diﬀerentiated eﬀect over intracellular triglyceride accumulation. The present results suggest that these compounds probably act directly over enzymes related with FA oxidation or glucose uptake and metabolism. decreased for controls and all treated cells, whereas PPARγ was expressed f (Figures 2 and 3), confirming that adipogenesis was not affected by ellagic acid a 2.2. Eﬀect of Ellagic Acid and Urolithins A and B on Intracellular Triglyceride Content Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. 2 4 Effect of Ellagic Acid and Urolithins A and B on PPARα and Glycerol Release 2.4. Eﬀect of Ellagic Acid and Urolithins A and B on PPARα and Glycerol Release 2.4. Effect of Ellagic Acid and Urolithins A and B on PPARα and Glycerol Release After analyzing the effect on intracellular triglyceride content and adipocyte differentiation of ellagic acid and urolithins, we evaluated if these compounds modified the amount of glycerol released and protein expression of PPARα. In the adipose tissue, triglycerides stored in the intracellular lipid droplets can be hydrolyzed into free fatty acids (FA) and glycerol which are After analyzing the eﬀect on intracellular triglyceride content and adipocyte diﬀerentiation of ellagic acid and urolithins, we evaluated if these compounds modiﬁed the amount of glycerol released and protein expression of PPARα. In the adipose tissue, triglycerides stored in the intracellular lipid droplets can be hydrolyzed into free fatty acids (FA) and glycerol, which are subsequently released Int. J. Mol. Sci. 2020, 21, 2086 the mode of action of th A is a compound able t 6 of 16 urolithin protein into the surrounding environment. Several enzymes responsible of this process are regulated by PPARα [29,30]. After treatments with ellagic acid and urolithins A and B in adipocytes from day 0 to day 8, we found that neither treatment aﬀected signiﬁcantly the protein expression level of PPARα (Figure 5A), however, the concentration of glycerol released into the culture medium was signiﬁcantly increased with ellagic acid and urolithin B, but not urolithin A (Figure 5B). that ellagic acid and urolithin B have a similar effect over the protein expression of PPARγ, c/EBPα, and PPARαas well as FA oxidation, despite its differentiated effect over intracellular triglyceride accumulation. The present results suggest that these compounds probably act directly over enzymes related with FA oxidation or glucose uptake and metabolism. In the following section, studies on lipogenesis and lipolysis were conducted to understand the different mode of action for these o ou d p Figure 5. Effect of ellagic acid, urolithin A, and urolithin B in the protein expression of peroxisome proliferator-activated receptor-α (PPARα) (A) and measurement of glycerol released into the culture medium of 3T3-L1 adipocytes (B). decreased for controls and all treated cells, whereas PPARγ was expressed f (Figures 2 and 3), confirming that adipogenesis was not affected by ellagic acid a 2.2. Eﬀect of Ellagic Acid and Urolithins A and B on Intracellular Triglyceride Content In the following section, studies on lipogenesis and lipolysis were conducted to understand the diﬀerent mode of action for these compounds. decreased for controls and all treated cells, whereas PPARγ was expressed f (Figures 2 and 3), confirming that adipogenesis was not affected by ellagic acid a 2.2. Eﬀect of Ellagic Acid and Urolithins A and B on Intracellular Triglyceride Content The protein expression of PPARα was determine by Western blot after 8 days of treatment with 25 µM of the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. (B) Shows the concentration of glycerol released into the culture medium determined by spectrophotometry at 540 nm. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 5. Eﬀect of ellagic acid, urolithin A, and urolithin B in the protein expression of peroxisome proliferator-activated receptor-α (PPARα) (A) and measurement of glycerol released into the culture medium of 3T3-L1 adipocytes (B). The protein expression of PPARα was determine by Western blot after 8 days of treatment with 25 µM of the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. (B) Shows the concentration of glycerol released into the culture medium determined by spectrophotometry at 540 nm. Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. Figure 5. Effect of ellagic acid, urolithin A, and urolithin B in the protein expression of peroxisome Figure 5. Eﬀect of ellagic acid, urolithin A, and urolithin B in the protein expression of peroxisome Figure 5. Effect of ellagic acid, urolithin A, and urolithin B in the protein expression of peroxisome proliferator-activated receptor-α (PPARα) (A) and measurement of glycerol released into the culture medium of 3T3-L1 adipocytes (B). The protein expression of PPARα was determine by Western blot after 8 days of treatment with 25 µM of the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. (B) Shows the concentration of glycerol released into the culture medium determined by spectrophotometry at 540 nm. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 5. Eﬀect of ellagic acid, urolithin A, and urolithin B in the protein expression of peroxisome proliferator-activated receptor-α (PPARα) (A) and measurement of glycerol released into the culture medium of 3T3-L1 adipocytes (B). 2.5. Eﬀect of Ellagic Acid and Urolithins A and B on Adipokines, Lipogenesis, and Lipolysis Fat accumulation is the result of the balance between lipogenesis and lipolysis events. Thus, we herein report the activities of diﬀerent key players as aﬀected by the treatments. Initially, we measured the gene expression of GLUT4 and regulatory adipokines adiponectin and leptin. Results showed a similar pattern among GLUT4 and adiponectin gene expressions with down-regulations for ellagic acid and urolithin A and no eﬀects by urolithin B (Figure 6A,B) similar to the trend observed 7 of 16 Int. J. Mol. Sci. 2020, 21, 2086 for fat accumulation (Figure 1C). On the other hand, neither of the compounds aﬀected leptin gene expression with the exception of urolithin B at a higher dose (Figure 6C). Int. J. Mol. Sci. 2020, 21, x 7 of 16 Figure 6. Effect of ellagic acid, urolithin A, and urolithin B on the gene expression of glucose transporter type 4 (GLUT4), adiponectin, and leptin in 3T3-L1 adipocytes. The gene expression of GLUT4 with treatments at 25 µM (A), and gene expressions of adiponectin (B) and leptin with treatments at 25 and 50 µM (C) was determined by RT-PCR after 8 days of treatment with the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. Different letters among bars denote significant changes among treatments and control (p ≤ 0.05) performed by ANOVA and Tukey’s post hoc test. Figure 6. Eﬀect of ellagic acid, urolithin A, and urolithin B on the gene expression of glucose transporter type 4 (GLUT4), adiponectin, and leptin in 3T3-L1 adipocytes. The gene expression of GLUT4 with treatments at 25 µM (A), and gene expressions of adiponectin (B) and leptin with treatments at 25 and 50 µM (C) was determined by RT-PCR after 8 days of treatment with the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. F th b PPAR d /EBP l t th i l d i di i Figure 6. Effect of ellagic acid, urolithin A, and urolithin B on the gene expression of glucose transporter type 4 (GLUT4), adiponectin, and leptin in 3T3-L1 adipocytes. 2.5. Eﬀect of Ellagic Acid and Urolithins A and B on Adipokines, Lipogenesis, and Lipolysis Lipolysis regulates adipose tissue weight and obesity through enzymes that catabolize triglycerides, including hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), and lipid droplet-associated protein (perilipin). In mature adipocytes, urolithins A and B and ellagic acid decreased the transcriptional activities of these genes involved in lipolysis and the oxidative pathways, with the exception of ATGL for urolithin B and perilipin for ellagic acid (Figure S1). p p p g g Accordingly, these results suggest that lipid accumulation by ellagic acid and urolithins A and B is likely due to the regulation of the GLUT4 gene, which might be associated to the regulatory eﬀects of adiponectin [33]. Thus, a decrease in GLUT4 leads to a decrease in glucose transport into the cells from the extracellular environment, reducing a key substrate for triglyceride biosynthesis [17]. In addition to the limitation of the key substrate necessary for fat biosynthesis, the diﬀerential eﬀects of ellagic acid and urolithins A and B on gene expression of enzymes involved in lipogenesis and lipolysis, as observed above, will determine the rate kinetics of lipogenesis (kLg) and lipolysis (kLs), ultimately deﬁning the fat accumulation in mature adipocytes. Thus, for ellagic acid and urolithin A, treatments that showed a reduction in fat accumulation the rate kinetics would be kLs > kLg, whereas for the urolithin B treatment associated with fat accumulation, the rate kinetics would be kLg > kLs. Further studies are needed to understand how ellagic acid and urolithn A regulate adiponectin levels and thus GLUT4, as well as the possible alternative mode of action to PPARγ that takes place. 2.6. Eﬀect of Ellagic Acid and Urolithins A and B on LPS-Induced Inﬂammation and Insulin Sensitivi Mature adipocytes challenged with LPS showed a signiﬁcant increase in pro-inﬂammatory genes including tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and monocyte chemoattractant protein-1 (MCP-1) while not aﬀecting cyclooxygenase 2 (COX-2) (Figure S2). When LPS-challenged mature adipocytes were treated previously with urolithins A and B and ellagic acid, we observed a diﬀerential response, with down regulations of gene expressions for TNFα by urolithin A and ellagic acid, down regulations of iNOS for urolithins A and B and ellagic acid, and down regulation of IL-6 and MCP-1 only for ellagic acid. Treatments did not have major eﬀects on COX-2. 2.5. Eﬀect of Ellagic Acid and Urolithins A and B on Adipokines, Lipogenesis, and Lipolysis The gene expression of GLUT4 with treatments at 25 µM (A), and gene expressions of adiponectin (B) and leptin with treatments at 25 and 50 µM (C) was determined by RT-PCR after 8 days of treatment with the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. Different letters among bars denote significant changes t t t d t l ( ≤0 05) f d b ANOVA d T k ’ t h t t Figure 6. Eﬀect of ellagic acid, urolithin A, and urolithin B on the gene expression of glucose transporter type 4 (GLUT4), adiponectin, and leptin in 3T3-L1 adipocytes. The gene expression of GLUT4 with treatments at 25 µM (A), and gene expressions of adiponectin (B) and leptin with treatments at 25 and 50 µM (C) was determined by RT-PCR after 8 days of treatment with the compounds, as described in the Materials and Methods section. The values represent the mean ± SD (n = 3) of three independent experiments. Diﬀerent letters among bars denote signiﬁcant changes among treatments and control (p ≤0.05) performed by ANOVA and Tukey’s post hoc test. Lipolysis regulates adipose tissue weight and obesity through enzymes that catabolize triglycerides, including hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), and lipid droplet-associated protein (perilipin). In mature adipocytes, urolithins A and B and ellagic acid decreased the transcriptional activities of these genes involved in lipolysis and the oxidative pathways, with the exception of ATGL for urolithin B and perilipin for ellagic acid (Figure S1). Furthermore, because PPARγ and c/EBPα regulate the genes involved in adipogenesis and lipogenesis, including adipocyte protein 2 (AP2), fatty acid synthase (FASN), stearoyl-CoA desaturase-1 (SCD1), and acetyl-CoA carboxylase-1 and 2 (ACC1 and ACC2), we measured their eﬀects by ellagic acid and urolithins. Results indicated that urolithin A reduced mRNA levels of lipogenic genes AP2, ACC1, ACC2, FASN, and SCD1, whereas urolithin B down-regulated these genes with the exception of Int. J. Mol. Sci. 2020, 21, 2086 8 of 16 AP2 and SCD1. On the other hand, ellagic acid diﬀerentially aﬀected these genes by down-regulating ACC1 and FASN, not aﬀecting AP2, and up-regulating ACC2 and SCD1 (Figure S1). AP2 and SCD1. On the other hand, ellagic acid diﬀerentially aﬀected these genes by down-regulating ACC1 and FASN, not aﬀecting AP2, and up-regulating ACC2 and SCD1 (Figure S1). 2.5. Eﬀect of Ellagic Acid and Urolithins A and B on Adipokines, Lipogenesis, and Lipolysis To have an insight of the mode of action of the diﬀerential anti-inﬂammatory response observed, we evaluated the protein expression of transcription factor nuclear factor κB (NF-κB). Western blot assays showed a decrease in protein expression of phosphorylated nuclear factor κB (p-NF-κB) in nuclear extracts for urolithins A and B on adipocytes challenged with or without LPS, whereas for ellagic acid, this eﬀect was only seen in non-LPS-challenged cells (Figure 7A). Accordingly, the anti-inﬂammatory properties observed for urolithins A and B and ellagic acid might be mediated by regulation of additional transcriptional factors besides p-NF-κB, for instance activator protein 1 (AP1); however, further studies are recommended to conﬁrm this. In addition, we measured the eﬀects of ellagic acid and urolithins A and B on phosphorylated protein kinase B (p-AKT) under LPS-induced inﬂammation in adipocytes. Results indicated that urolithins A and B and ellagic acid-treated adipocytes showed expression of p-AKT in nuclear extracts under LPS challenge and stimulated by insulin (Figure 7B). This insulin response conﬁrmed that ellagic acid and metabolites urolithins A and B do not aﬀect insulin sensitivity in adipocytes. 9 of 16 hins are Int. J. Mol. Sci. 2020, 21, 2086 obese mice, and thus a ponsible for this effect. p Figure 7. Ellagic acid and urolithins A and B effects on protein expression of transcription factors nuclear factor κB (NF-κB) and protein kinase B (AKT) associated with inflammation and insulin sensitivity in 3T3-L1 adipocytes at day 8 treated with compounds for 24 h and then exposed for 1 h to lipopolysaccharide (LPS; 100ng/mL). Western blot assays for nuclear phosphorylated nuclear factor κB (p-NF-κB; nuclear extract) and cytosolic NF-κB (cytosolic extract) at 25 µM treated adipocytes challenged with or without LPS (A) and nuclear phosphorylated AKT (p-AKT; nuclear extract) under LPS-induced inflammation in mature adipocytes with or without insulin (10 µg/mL) (B), as described in the Materials and Methods section Figure 7. Ellagic acid and urolithins A and B eﬀects on protein expression of transcription factors nuclear factor κB (NF-κB) and protein kinase B (AKT) associated with inﬂammation and insulin sensitivity in 3T3-L1 adipocytes at day 8 treated with compounds for 24 h and then exposed for 1 h to lipopolysaccharide (LPS; 100ng/mL). 2.5. Eﬀect of Ellagic Acid and Urolithins A and B on Adipokines, Lipogenesis, and Lipolysis Western blot assays for nuclear phosphorylated nuclear factor κB (p-NF-κB; nuclear extract) and cytosolic NF-κB (cytosolic extract) at 25 µM treated adipocytes challenged with or without LPS (A) and nuclear phosphorylated AKT (p-AKT; nuclear extract) under LPS-induced inﬂammation in mature adipocytes with or without insulin (10 µg/mL) (B), as described in the Materials and Methods section. 3. Materials and Methods 3.1. Chemicals The following chemicals were used in the experiments: 3-isobutyl-1-methylxanthine, dexamethasone, insulin, Dulbecco’s modified Eagle’s medium, fetal bovine serum (FBS), trypsin- EDTA, and protease inhibitor cocktail were purchased from Sigma (St. Louis, MO, USA). D-glucose was obtained from Acros Organics (Fair Lawn, NJ, USA). Sodium bicarbonate was purchased from Mallinckrodt Chemicals (Phillipsburg, NJ, USA). Murine 3T3-L1 preadipocytes and dimethyl sulfoxide (DMSO) were acquired from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Penicillin-streptomycin was bought from Invitrogen (Carslbad, CA, USA). Urolithins A and B were manufactured by Kylolab (Ceuti, Spain). Ellagic acid was from MP Biomedicals (Solon, OH, USA). Cell lysis buffer was obtained from Cell Signaling Technology (Danvers, MA, USA). Sodium dodecyl sulfate solution, 30% acrylamide/bisacrylamide solution, N,N,N’,N’- tetramethylethylenediamine (TEMED), ammonium persulfate, Tween 20, and Precision Plus Protein marker were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Laemmli’s loading buffer was acquired from Fermentas Inc. (Glen Burnie, MD, USA). Polyvinylidene fluoride (PVDF) membranes were obtained from Millipore Corp. (Billerica, MA, USA). Antibodies for PPARγ (sc- 7196), c/EBPα (sc-61), PPARα (sc-9000), p-NF-κB (sc-33039), p-AKT, and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody for NF-κB (C22B4) was obtained from Cell Signaling Technology (Danvers, MA, USA). Goat anti-rabbit-horseradish Taken together, our results with murine cells indicate that EA and urolithin A may work potentially in diﬀerent fronts by attenuating lipogenesis and fat accumulation without aﬀecting adipogenesis, whereas only EA and urolithin B enhanced FA oxidation. On the other hand, EA and urolithins A and B diﬀerentially attenuated inﬂammation in mature adipocytes while not aﬀecting insulin sensitivity. Other studies with primary human adipocytes later conﬁrmed that urolithin A reduced fat accumulation but mainly through inhibition of adipogenesis and enhanced FA oxidation [34]. 2.5. Eﬀect of Ellagic Acid and Urolithins A and B on Adipokines, Lipogenesis, and Lipolysis Western blot assays for nuclear phosphorylated nuclear factor κB (p-NF-κB; nuclear extract) and cytosolic NF-κB (cytosolic extract) at 25 µM treated adipocytes challenged with or without LPS (A) and nuclear phosphorylated AKT (p-AKT; nuclear extract) under LPS-induced inﬂammation in mature adipocytes with or without insulin (10 µg/mL) (B), as described in the Materials and Methods section. Figure 7. Ellagic acid and urolithins A and B eﬀects on protein expression of transcription factors Figure 7. Ellagic acid and urolithins A and B effects on protein expression of transcription factors nuclear factor κB (NF-κB) and protein kinase B (AKT) associated with inflammation and insulin sensitivity in 3T3-L1 adipocytes at day 8 treated with compounds for 24 h and then exposed for 1 h to lipopolysaccharide (LPS; 100ng/mL). Western blot assays for nuclear phosphorylated nuclear factor κB (p-NF-κB; nuclear extract) and cytosolic NF-κB (cytosolic extract) at 25 µM treated adipocytes challenged with or without LPS (A) and nuclear phosphorylated AKT (p-AKT; nuclear extract) under LPS-induced inflammation in mature adipocytes with or without insulin (10 µg/mL) (B), as described Figure 7. Ellagic acid and urolithins A and B eﬀects on protein expression of transcription factors nuclear factor κB (NF-κB) and protein kinase B (AKT) associated with inﬂammation and insulin sensitivity in 3T3-L1 adipocytes at day 8 treated with compounds for 24 h and then exposed for 1 h to lipopolysaccharide (LPS; 100ng/mL). Western blot assays for nuclear phosphorylated nuclear factor κB (p-NF-κB; nuclear extract) and cytosolic NF-κB (cytosolic extract) at 25 µM treated adipocytes challenged with or without LPS (A) and nuclear phosphorylated AKT (p-AKT; nuclear extract) under LPS-induced inﬂammation in mature adipocytes with or without insulin (10 µg/mL) (B), as described in the Materials and Methods section. sensitivity in 3T3-L1 adipocytes at day 8 treated with compounds for 24 h and then exposed for 1 h to lipopolysaccharide (LPS; 100ng/mL). Western blot assays for nuclear phosphorylated nuclear factor κB (p-NF-κB; nuclear extract) and cytosolic NF-κB (cytosolic extract) at 25 µM treated adipocytes challenged with or without LPS (A) and nuclear phosphorylated AKT (p-AKT; nuclear extract) under LPS-induced inflammation in mature adipocytes with or without insulin (10 µg/mL) (B), as described lipopolysaccharide (LPS; 100ng/mL). 3.1. Chemicals The following chemicals were used in the experiments: 3-isobutyl-1-methylxanthine, dexamethasone, insulin, Dulbecco’s modiﬁed Eagle’s medium, fetal bovine serum (FBS), trypsin-EDTA, and protease inhibitor cocktail were purchased from Sigma (St. Louis, MO, USA). D-glucose was obtained from Acros Organics (Fair Lawn, NJ, USA). Sodium bicarbonate was purchased from Mallinckrodt Chemicals (Phillipsburg, NJ, USA). Murine 3T3-L1 preadipocytes and dimethyl sulfoxide (DMSO) were acquired from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Penicillin-streptomycin was bought from Invitrogen (Carslbad, CA, USA). Urolithins A and B were manufactured by Kylolab (Ceuti, Spain). Ellagic acid was from MP Biomedicals (Solon, OH, USA). Cell lysis buﬀer was obtained from Cell Signaling Technology (Danvers, MA, USA). Sodium dodecyl sulfate solution, 30% acrylamide/bisacrylamide solution, N,N,N′,N′-tetramethylethylenediamine (TEMED), ammonium persulfate, Tween 20, and Precision Plus Protein marker were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Laemmli’s loading buﬀer was acquired from Fermentas Inc. (Glen Burnie, MD, USA). Polyvinylidene ﬂuoride (PVDF) membranes were obtained from Millipore Corp. (Billerica, MA, USA). Antibodies for PPARγ (sc-7196), c/EBPα (sc-61), PPARα (sc-9000), p-NF-κB (sc-33039), p-AKT, and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody for NF-κB (C22B4) was obtained from Cell Signaling Technology (Danvers, MA, USA). Goat anti-rabbit-horseradish peroxidase (HRP) polyclonal secondary antibody (A120-101P) was obtained from Bethyl Laboratories (Montgomery, TX, USA). 3.2. Cell Culture Murine 3T3-L1 preadipocytes (ATCC, Manassas, VA, USA) were maintained in high glucose Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO, USA), penicillin (100 unit/mL), and streptomycin (100 µg/mL), and incubated under humidiﬁed atmosphere at 37 ◦C and 5% CO2. 2.5. Eﬀect of Ellagic Acid and Urolithins A and B on Adipokines, Lipogenesis, and Lipolysis Nevertheless, considering the existant interindividual variability to produce urolithins and their diﬀerent predispositions to chronic diseases, such as the metabolic syndrome [7], the present results support the idea that metabotype A individuals (urolithin A producers) may beneﬁt from this dual role of urolithin A of reduced fat accumulation and decreased inﬂammation, compared to metabotype B individuals (mainly urolithin B producers) and metabotype 0 individuals (nonproducers), who are more susceptible to the metabolic syndrome. Age is considered the key factor in determining the gut microbiota involved in ellagic tannin–ellagic acid metabolism and ultimately the urolithin metabotypes, where younger individuals are type A and older individuals are mainly type B, with type 0 unaltered with age [35]. This opens the possibility of designing tailored diets and nutrition, as well as perhaps the microbiota of individuals. However, studies are still in need to better understand the speciﬁc roles of EA and derived urolithins in future works, or even to elucidate previous reports where EA might be the bioactive compound or alternatively the derived gut microbial urolithins. For instance, Yoshimura et al. [36] reported that EA improved hepatic steatosis and lipid composition through reduction of adipokine resistin and activation of PPARα in obese mice, and thus a next step would be to understand if EA or the derived urolithins are responsible for this eﬀect. 10 of 16 Int. J. Mol. Sci. 2020, 21, 2086 10 of 16 3.3. Cell Viability Assay Cell viability on murine 3T3-L1 preadipocytes was determined using MTS assay (Promega Corp., Madison, WI, USA), according to the manufacturer’s instructions. The cells were seeded at a density of 7500 cells per well in a 96-well plate and incubated with 10% FBS/DMEM medium for 24 h. Treatments with ellagic acid, urolithin A, and urolithin B were added at a concentration of 25 µM in DMSO. Final DMSO concentration in the culture medium was 0.025%; DMSO was added at this percentage as control. Cell viability was measured at 24 h; absorbance was measured at 490 nm in a microplate reader (Synergy HT, Bio-Tek Instruments, Inc., Winooski, VT, USA). 3.4. Cell Diﬀerentiation and Treatments for Adipogenesis Assays Murine 3T3-L1 cells were seeded at a density of 10,000 cells per well in 6-well plates. Preadipocytes were induced to diﬀerentiation 2 days after they reached 100% conﬂuency (day 0). Growth medium was supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 µM dexamethasone, and 10 µg/mL insulin, for 48 h. Additionally, on day 0, treatments of ellagic acid, urolithin A, and urolithin B were added to reach a ﬁnal concentration of 25 µM, and diluted in DMSO. At day 2, medium was replaced with fresh medium supplemented with insulin (10 µg/mL) for 2 additional days, until day 4. After day 4, 10% FBS/DMEM medium was replaced every 2 days until >90% fully mature adipocytes were reached (day 8). Int. J. Mol. Sci. 2020, 21, 2086 11 of 16 11 of 16 3.7. Ellagic Acid and Urolithins A and B on LPS-Induced Inﬂammation and Insulin Sensitivity The eﬀect of ellagic acid and urolithins A and B on the activation of p-NF-κB and the gene expression of several inﬂammation markers including TNFα, IL-6, COX-2, iNOS, and MCP-1 was studied in mature adipocytes (day 8). Fully mature adipocytes (obtained as described above) were exposed for 24 h to 25 and 50 µM of ellagic acid, and urolithins A and B, and then treated with LPS (100 ng/mL) for 1 h to induce the inﬂammatory response. To test insulin sensitivity, mature adipocytes were obtained as described above and treated with ellagic acid and urolithins A and B with 25 µM for 24 h, where they were then exposed to LPS (100 ng/mL) and insulin (10 µg/mL) for 1 h. Protein and mRNA samples were collected and stored at −80 ◦C until used. 3.5. Quantiﬁcation of Lipid Content Intracellular lipid content was measured using a commercially available AdipoRed kit (AdipoRed, Lonza Wakersville, Inc., Wakersville, MD, USA). AdipoRed is a solution of the Nile Red stain, which ﬂuoresces and enables the quantiﬁcation of intracellular lipid droplets. Preadipocytes were diﬀerentiated and treated following the method previously described. Brieﬂy, on day 8, cells were washed with 2 mL of phosphate-buﬀered saline (PBS, pH 7.4); then, 5 mL PBS was left per well and 140 µL AdipoRed was added, the plate was left at 37 ◦C for 10 min, and ﬂuorescence readings were measured by well scanning with excitation at 485 nm and emission at 560 nm in a plate reader (Synergy HT, Bio-Tek Instruments, Inc., Winooski, VT, USA). Measurements were expressed as relative ﬂuorescence units (RFU). 3.6. Quantiﬁcation of Glycerol Release The amount of glycerol released into the culture medium was determined using the Glycerol Cell-Based Assay Kit of Cayman Chemical Company (item 10011725, Ann Arbor, MI, USA), following the manufacturer’s instructions. In this assay, the amount of glycerol released into the medium is proportional to the triglyceride/fatty acid cycling rate. To prepare the cells for this assay, we seeded 3T3-L1 preadipocytes at a density of 8500 cells per well in 12-well plates. Two days after cells reached 100% conﬂuency (day 0), we induced diﬀerentiation, following the method described previously. Ellagic acid, urolithin A, and urolithin B were co-incubated with the cells at a concentration of 25 µM from day 0 to day 8. At day 8, the cells were washed and the glycerol cell-based assay was performed. The concentration of glycerol released was determined by absorbance at 540 nm in a plate reader (Synergy HT, Bio-Tek Instruments, Inc., Winooski, VT, USA). 3.8. Western Blot Analysis Cells were lysed on ice using cell lysis buﬀer (Cell Signaling Technology, Danvers, MA, USA) supplemented with protease and phosphatase inhibitors, following the manufacturer’s instructions. Brieﬂy, cells were then scraped, left at −80 ◦C overnight, centrifuged at 14,000 rpm at 4 ◦C, and the supernatant was stored at −80 ◦C. The protein concentration was determined using the BCA Protein Assay Kit (Pierce, Thermo Fisher Scientiﬁc, Inc., Rockford, IL, USA). Cell protein from nuclear and cytosolic extract was obtained with a nuclear extraction kit (Cayman Chemical, item 10009277, Ann Arbor, MI, USA) following the manufacturer’s instructions. In our experiments, 40 µg of protein was loaded. Equal amounts of proteins were separated by SDS-polyacrylamide gels and then electrophoretically transferred from the gel onto a PVDF membrane (Millipore, Bedford, MA, USA). The membranes were then blocked with 5% non-fat milk in Tris-buﬀered saline with 1% Tween-20 (TBS-T) for 1 h with gentle shaking; washing four times (5 min) with TBS-T was performed consecutively. Membranes were then incubated with a speciﬁc primary antibody against PPARγ (1:1000), PPARα (1:5000), c/EBPα (1:7000), p-NF-κB (1:2000), NF-κB (1:1500), and p-AKT (1:1000). For internal control, membranes were incubated with a conjugated HRP primary antibody against β-actin at a dilution of 1:10,000. The membranes were washed four times (5 min) with TBS-T and incubated for 1 h with the 12 of 16 12 of 16 Int. J. Mol. Sci. 2020, 21, 2086 secondary antibody conjugated with horseradish peroxidase (HRP) at 1:30,000 dilutions. The blots were developed using a SuperSignal West Femto enhanced chemiluminescence (ECL) Western blotting detection kit (Pierce, Thermo Fisher Scientiﬁc, Inc., Rockford, IL, USA) after signals were captured by CCD Camera (Cascade II:512, Photometric, Tucson, AZ, USA) using the Win View/32 software (Version 2.5, Princeton Instruments, Trento, NJ, USA). The blot images were quantitated by densitometry using the Image J software (NIH, Bethesda, MD, USA). 3.9. Real-Time Quantitative RT-PCR One microgram of RNA was reverse-transcribed into cDNA using the SuperScript III ﬁrst-strand synthesis supermix (item 18080-004, Invitrogen, Carlsbad, CA, USA), following the manufacturers protocol. The real-time PCR for leptin was quantiﬁed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), following the manufacturer’s instructions. DNA ampliﬁcation was carried out using a 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Real time PCR reaction was performed in a 20 µL volume containing Power SYBR Green PCR Master Mix and 25 ng cDNA, and 0.3 µM primers mouse leptin 5’-GAGACCCCTGTGTCGGTTC-3’ (forward) and 5’-CTGCGTGTGTGAAATGTCATTG-3’ (reverse); mouse β-actin 5’-CCCAGGCATTGCTGACAGG-3’ (forward) and 5’-TGGAAGGTGGACAGTGAGGC-3’ (reverse); mouse ACC1 5′-GGATGGTTTGGCCTTTCACA-3′ (forward) and 5′-TTTTCTTTCTGTCTCGACCTTGTTT-3′ (reverse); mouse ACC2 5′-ACAGAGATTTCACCGTTGCGT-3′ (forward) and 5′-CGCAGCGATGCCATTGT-3′ (reverse); mouse AP2 5′-CTTCAAACTGGGCGTGAA-3′ (forward) and 5′-CTAGGGTTATGATGCTCTTCACCTT-3′ (reverse); mouse FASN 5′-GGCTCAGCATGGTCGCTT-3′ (forward) and 5′-CTCCCGCCAGCTGTCATT-3′ (reverse); mouse GLUT4 5′-TTGGTACCTACGCTTTGCAGC-3′ (forward) and 5′-CGGTTAGAGCGCATCAGTCTC-3′ (reverse); mouse HSL 5′-GCAAGATCAAAGCCTCAGCG-3′ (forward) and 5′-GCCATATTGTCTTCTGCGAGTGT-3′ (reverse); mouse ATGL 5′-GTCCTTCACCATCCGCTTGTT-3′ (forward) and 5′-CTCTTGGCCCTCATCACCAG-3′ (reverse); mouse SCD1 5′-ATCGCCTCTGGAGCCACAC-3′ (forward) and 5′-ACACGTCATTCTGGAACGCC-3′ (reverse); mouse perilipin 5′-GGTACACTATGTGCCGCTTCC-3′ (forward) and 5′-CTTTGCGCTCCGCCTCT-3′ (reverse); mouse PREF-1 5′-CAG CGGCTATGGGCTCACCT-3′ (forward) and 5′-TGTTGCTCGGGCTGCTGAA-3′ (reverse); mouse TNFα 5’-ACTGGCAGAAGAGGCACTCC-3’ (forward) and 5’-CGATCACCCCGAAGTTCA-3’ (reverse); mouse IL-6 5’-TGACAACCACGG CCTTCCCT-3’ (forward) and 5’-AGCCTCCGACTTGTGAAGTGGT-3’ (reverse); mouse COX-2 5’-ACATCGATGTCATGGAACTG-3’ (forward) and 5’-GGACACCCCTTCACATTATT-3’ (reverse); mouse iNOS 5’-ACATCGACCCGTCCACAGTAT-3’ (forward) and 5’-CAGAGGGGTAGGCTTGTCTC-3’ (reverse); mouse MCP-1 5’-CAGCCAGATGCAATCAATGC-3’ (forward) and 5’-GTGGTCCATGGAATCCTGAA-3’ (reverse) were provided by Integrated DNA Technologies (IDT, Coralville, IA). After 10 min polymerase activation at 95 ◦C, 40 cycles with 95◦C for 15 s (denaturation) and 60 ◦C for 1 min (annealing/extension) were performed. Fluorescence was measured at the end of the 60 ◦C extension period. The relative expression of genes was normalized using β-actin, and was calculated following the comparative Ct method (∆∆Ct), also known as the 2−∆∆Ct method [37]. 3 10 Statistical Analysis 3.9. Real-Time Quantitative RT-PCR Total RNA was extracted from 3T3-L1 mature adipocyte controls and treatment at 25 and 50 µM with ellagic acid and urolithins A and B using the RNeasy mini kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s instructions. RNA concentration was measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Willmington, DE, USA). One microgram of RNA was reverse-transcribed into cDNA using the SuperScript III ﬁrst-strand synthesis supermix (item 18080-004, Invitrogen, Carlsbad, CA, USA), following the manufacturers protocol. The real-time PCR for leptin was quantiﬁed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), following the manufacturer’s instructions. DNA ampliﬁcation was carried out using a 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Real time PCR reaction was performed in a 20 µL volume containing Power SYBR Green PCR Master Mix and 25 ng cDNA, and 0.3 µM primers mouse leptin 5’-GAGACCCCTGTGTCGGTTC-3’ (forward) and 5’-CTGCGTGTGTGAAATGTCATTG-3’ (reverse); mouse β-actin 5’-CCCAGGCATTGCTGACAGG-3’ (forward) and 5’-TGGAAGGTGGACAGTGAGGC-3’ (reverse); mouse ACC1 5′-GGATGGTTTGGCCTTTCACA-3′ (forward) and 5′-TTTTCTTTCTGTCTCGACCTTGTTT-3′ (reverse); mouse ACC2 5′-ACAGAGATTTCACCGTTGCGT-3′ (forward) and 5′-CGCAGCGATGCCATTGT-3′ (reverse); mouse AP2 5′-CTTCAAACTGGGCGTGAA-3′ (forward) and 5′-CTAGGGTTATGATGCTCTTCACCTT-3′ (reverse); mouse FASN 5′-GGCTCAGCATGGTCGCTT-3′ (forward) and 5′-CTCCCGCCAGCTGTCATT-3′ (reverse); mouse GLUT4 5′-TTGGTACCTACGCTTTGCAGC-3′ (forward) and 5′-CGGTTAGAGCGCATCAGTCTC-3′ (reverse); mouse HSL 5′-GCAAGATCAAAGCCTCAGCG-3′ (forward) and 5′-GCCATATTGTCTTCTGCGAGTGT-3′ (reverse); mouse ATGL 5′-GTCCTTCACCATCCGCTTGTT-3′ (forward) and 5′-CTCTTGGCCCTCATCACCAG-3′ (reverse); mouse SCD1 5′-ATCGCCTCTGGAGCCACAC-3′ (forward) and 5′-ACACGTCATTCTGGAACGCC-3′ (reverse); mouse perilipin 5′-GGTACACTATGTGCCGCTTCC-3′ (forward) and 5′-CTTTGCGCTCCGCCTCT-3′ (reverse); mouse PREF-1 5′-CAG CGGCTATGGGCTCACCT-3′ (forward) and 5′-TGTTGCTCGGGCTGCTGAA-3′ (reverse); mouse TNFα 5’-ACTGGCAGAAGAGGCACTCC-3’ (forward) and 5’-CGATCACCCCGAAGTTCA-3’ (reverse); mouse IL-6 5’-TGACAACCACGG CCTTCCCT-3’ (forward) and 5’-AGCCTCCGACTTGTGAAGTGGT-3’ (reverse); mouse COX-2 5’-ACATCGATGTCATGGAACTG-3’ (forward) and 5’-GGACACCCCTTCACATTATT-3’ (reverse); mouse iNOS 5’-ACATCGACCCGTCCACAGTAT-3’ (forward) and 5’-CAGAGGGGTAGGCTTGTCTC-3’ (reverse); mouse MCP-1 5’-CAGCCAGATGCAATCAATGC-3’ (forward) and 5’-GTGGTCCATGGAATCCTGAA-3’ (reverse) were provided by Integrated DNA Technologies (IDT, Coralville, IA). After 10 min polymerase activation at 95 ◦C, 40 cycles with 95◦C for 15 s (denaturation) and 60 ◦C for 1 min (annealing/extension) were performed. Fluorescence was measured at the end of the 60 ◦C extension period. The relative expression of genes was normalized using β-actin, and was calculated following the comparative Ct method (∆∆Ct), also known as the 2−∆∆Ct method [37]. Total RNA was extracted from 3T3-L1 mature adipocyte controls and treatment at 25 and 50 µM with ellagic acid and urolithins A and B using the RNeasy mini kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s instructions. RNA concentration was measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Willmington, DE, USA). 4. Conclusions Ellagic acid and urolithins A and B did not aﬀect adipogenesis, however, they diﬀerentially aﬀected lipid accumulation. Although urolithin A and ellagic acid decreased fat accumulation in adipocytes, urolithin B showed similar lipid accumulation to that of controls. This diﬀerential response seemed to be associated with the regulation of GLUT4 gene expression and associated changes in adiponectin that followed similar trends. In addition, we report that ellagic acid and urolithins A and B showed anti-inﬂammatory properties in LPS-challenged mature adipocytes, and this response was diﬀerentially observed in gene expressions of pro-inﬂammatory markers including TNF-α, IL-6, iNOS, and MCP-1. This response was in part associated with a decrease in nuclear transcription factor p-NF-κB and possibly to the regulation of other transcription factors, for instance, AP1. Furthermore, we report that nuclear transcription factor p-AKT was expressed in mature adipocytes treated with ellagic acid and urolithins A and B when challenged with LPS and exposed to insulin, conﬁrming the idea that mature adipocytes are sensitive to insulin. In general, our study gives insight into the mode of action of how ellagic acid and derived gut microbial metabolites urolithins A and B diﬀerentially attenuate lipid accumulation and inﬂammation in mature adipocytes (Figure 8). This information is relevant for crops such as pecans and others that contain high levels of ellagitannins [38]. Further eﬀorts are recommended for in in vivo studies to conﬁrm these results. Int. J. Mol. Sci. 2020, 21, x 13 of 16 Figure 8. Proposed model of modulation of lipogenesis and inflammation by ellagic acid and derived gut microbial metabolites urolithins A and B in adipocytes. Ellagic acid (EA) and urolithins A and B did not affect adipogenesis in 3T3-L1 cells, where PREF-1 decreased and PPARγ and c/EBPα increased for all treatments. During lipogenesis/lipolysis, only EA and urolithin A reduced fat accumulation in mature adipocytes attenuating GLUT4 gene expression and adiponectin. In LPS- challenged cells, EA and urolithins A and B differentially ameliorated inflammation through down- regulation of transcription factor p-NF-κB and pro-inflammatory genes while not affecting insulin sensitivity, depicted by an increase in protein expression of transcription factor p-AKT when exposed Figure 8. Proposed model of modulation of lipogenesis and inﬂammation by ellagic acid and derived gut microbial metabolites urolithins A and B in adipocytes. Ellagic acid (EA) and urolithins A and B did not aﬀect adipogenesis in 3T3-L1 cells, where PREF-1 decreased and PPARγ and c/EBPα increased for all treatments. 3.10. Statistical Analysis Data represent the mean ± SD for cell viability assay and all the other markers measured related with adipogenesis, lipogenesis, lipolysis, and inﬂammation. Statistical signiﬁcance was assessed by ANOVA and separation of means by Tukey’s post hoc test. Diﬀerences were considered signiﬁcant Int. J. Mol. Sci. 2020, 21, 2086 13 of 16 13 of 16 when p ≤0.05. Tests were conducted using JMP, Version 15 (SAS Institute Inc., Cary, NC, USA, 1989–2019). when p ≤0.05. Tests were conducted using JMP, Version 15 (SAS Institute Inc., Cary, NC, USA, 1989–2019). 4. Conclusions During lipogenesis/lipolysis, only EA and urolithin A reduced fat accumulation in mature adipocytes attenuating GLUT4 gene expression and adiponectin. In LPS-challenged cells, EA and urolithins A and B diﬀerentially ameliorated inﬂammation through down-regulation of transcription factor p-NF-κB and pro-inﬂammatory genes while not aﬀecting insulin sensitivity, depicted by an increase in protein expression of transcription factor p-AKT when exposed to insulin. Figure 8. Proposed model of modulation of lipogenesis and inflammation by ellagic acid and derived Figure 8. Proposed model of modulation of lipogenesis and inﬂammation by ellagic acid and derived Figure 8. Proposed model of modulation of lipogenesis and inflammation by ellagic acid and derived gut microbial metabolites urolithins A and B in adipocytes. Ellagic acid (EA) and urolithins A and B did not affect adipogenesis in 3T3-L1 cells, where PREF-1 decreased and PPARγ and c/EBPα increased for all treatments. During lipogenesis/lipolysis, only EA and urolithin A reduced fat accumulation in mature adipocytes attenuating GLUT4 gene expression and adiponectin. In LPS- challenged cells, EA and urolithins A and B differentially ameliorated inflammation through down- regulation of transcription factor p-NF-κB and pro-inflammatory genes while not affecting insulin sensitivity, depicted by an increase in protein expression of transcription factor p-AKT when exposed Figure 8. Proposed model of modulation of lipogenesis and inﬂammation by ellagic acid and derived gut microbial metabolites urolithins A and B in adipocytes. Ellagic acid (EA) and urolithins A and B did not aﬀect adipogenesis in 3T3-L1 cells, where PREF-1 decreased and PPARγ and c/EBPα increased for all treatments. During lipogenesis/lipolysis, only EA and urolithin A reduced fat accumulation in mature adipocytes attenuating GLUT4 gene expression and adiponectin. In LPS-challenged cells, EA and urolithins A and B diﬀerentially ameliorated inﬂammation through down-regulation of transcription factor p-NF-κB and pro-inﬂammatory genes while not aﬀecting insulin sensitivity, depicted by an increase in protein expression of transcription factor p-AKT when exposed to insulin. Figure 8. Proposed model of modulation of lipogenesis and inflammation by ellagic acid and derived gut microbial metabolites urolithins A and B in adipocytes. Ellagic acid (EA) and urolithins A and B did not affect adipogenesis in 3T3-L1 cells, where PREF-1 decreased and PPARγ and c/EBPα increased for all treatments. During lipogenesis/lipolysis, only EA and urolithin A reduced fat accumulation in mature adipocytes attenuating GLUT4 gene expression and adiponectin. 4. Conclusions In LPS- challenged cells, EA and urolithins A and B differentially ameliorated inflammation through down- regulation of transcription factor p-NF-κB and pro-inflammatory genes while not affecting insulin sensitivity, depicted by an increase in protein expression of transcription factor p-AKT when exposed Figure 8. Proposed model of modulation of lipogenesis and inﬂammation by ellagic acid and derived gut microbial metabolites urolithins A and B in adipocytes. Ellagic acid (EA) and urolithins A and B did not aﬀect adipogenesis in 3T3-L1 cells, where PREF-1 decreased and PPARγ and c/EBPα increased for all treatments. During lipogenesis/lipolysis, only EA and urolithin A reduced fat accumulation in mature adipocytes attenuating GLUT4 gene expression and adiponectin. In LPS-challenged cells, EA and urolithins A and B diﬀerentially ameliorated inﬂammation through down-regulation of transcription factor p-NF-κB and pro-inﬂammatory genes while not aﬀecting insulin sensitivity, depicted by an increase in protein expression of transcription factor p-AKT when exposed to insulin. Supplementary Materials: Supplementary materials can be found at www mdpi com/xxx/s1 Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/6/2086/ s1 support from P. Castillo. C fli t f I t t Th th d l fli t f i t t Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Marwan, A.G.; Nagel, C.W. Characterization of cranberry benzoates and their antimicrobial properties. J. Food Sci. 1986, 51, 1069–1070. [CrossRef] 1. Marwan, A.G.; Nagel, C.W. Characterization of cranberry benzoates and their antimicrobial properties. J. Food Sci. 1986, 51, 1069–1070. [CrossRef] 2. Daniel, E.M.; Krupnick, A.S.; Heur, Y.H.; Blinzler, A.J.; Nims, R.W.; Stoner, G.D. Extraction, stability and quantitation of ellagic acid in various fruits and nuts. J. Food Compos. Anal. 1989, 2, 338–349. [CrossRef] 2. Daniel, E.M.; Krupnick, A.S.; Heur, Y.H.; Blinzler, A.J.; Nims, R.W.; Stoner, G.D. Extraction, stability and quantitation of ellagic acid in various fruits and nuts. J. Food Compos. Anal. 1989, 2, 338–349. [CrossRef] 2. Daniel, E.M.; Krupnick, A.S.; Heur, Y.H.; Blinzler, A.J.; Nims, R.W.; Stoner, G.D. Extraction, stability and quantitation of ellagic acid in various fruits and nuts. J. Food Compos. Anal. 1989, 2, 338–349. [CrossRef] 3. Chen, H.; Zuo, Y.; Deng, Y. Separation and determination of ﬂavonoids and other phenolic compounds in 2. Daniel, E.M.; Krupnick, A.S.; Heur, Y.H.; Blinzler, A.J.; Nims, R.W.; Stoner, G.D. Extraction, stability and quantitation of ellagic acid in various fruits and nuts. J. Food Compos. Anal. 1989, 2, 338–349. [CrossRef] 3. Chen, H.; Zuo, Y.; Deng, Y. Separation and determination of ﬂavonoids and other phenolic compounds in cranberry juice by high-performance liquid chromatography. J. Chromatogr. A 2001, 913, 387–395. [CrossRef] 4. Espín, J.C.; Larrosa, M.; García-Conesa, M.T.; Tomás-Barberán, F. Biological signiﬁcance of urolithins, the gut microbial ellagic acid-derived metabolites: The evidence so far. Evid-Based Compl. Alt. 2013, 2013, 270418. [CrossRef] [PubMed] quantitation of ellagic acid in various fruits and nuts. J. Food Compos. Anal. 1989, 2, 338–349. [CrossRef] 3. Chen, H.; Zuo, Y.; Deng, Y. Separation and determination of ﬂavonoids and other phenolic compounds in cranberry juice by high-performance liquid chromatography. J. Chromatogr. A 2001, 913, 387–395. [CrossRef] 4. Espín, J.C.; Larrosa, M.; García-Conesa, M.T.; Tomás-Barberán, F. Biological signiﬁcance of urolithins, the gut 3. Chen, H.; Zuo, Y.; Deng, Y. Separation and determination of ﬂavonoids and other phenolic compounds in cranberry juice by high-performance liquid chromatography. J. Chromatogr. A 2001, 913, 387–395. [CrossRef] 3. Chen, H.; Zuo, Y.; Deng, Y. Separation and determination of ﬂavonoids and other phenolic compounds in cranberry juice by high-performance liquid chromatography. J. Chromatogr. A 2001, 913, 387–395. [CrossRef] y j y g p q g p y g 4. Espín, J.C.; Larrosa, M.; García-Conesa, M.T.; Tomás-Barberán, F. Supplementary Materials: Supplementary materials can be found at www.mdpi.com/xxx/s1. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/6/2086/ s1. Author Contributions: Conceptualization; L.C.Z.; writing—review and editing, L.C.Z., Y.B.W., C.D-P. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization; L.C.-Z.; writing—review and editing, L.C.-Z., W.Y.B., C.D.-P. All authors have read and agreed to the published version of the manuscript. p p Funding: This research was funded by the Texas Department of Agriculture (TDA). Funding: This research was funded by the Texas Department of Agriculture (TDA). g y p g ( ) Acknowledgments: We would like to acknowledge funding from the Texas Pecan Board. We thank the technical support from P Castillo Acknowledgments: We would like to acknowledge funding from the Texas Pecan Board. We thank the technical support from P. Castillo. support from P. Castillo. fli f I h h d l fl f Conﬂicts of Interest: The authors declare no conﬂict of interest. 14 of 16 Int. J. Mol. Sci. 2020, 21, 2086 Abbreviations ACC1 acetyl-CoA carboxylase 1 ACC2 acetyl-CoA carboxylase 2 AKT protein kinase B AP1 activator protein 1 AP2 adipocyte protein 2 ATGL adipose triglyceride lipase c/EBPα CCAAT-enhancer-binding protein alpha COX-2 cyclooxygenase 2 DMSO dimethyl sulfoxide EA ellagic acid FASN fatty acid synthase GLUT4 glucose transporter type 4 HSL hormone sensitive lipase IL-6 interleukin 6 iNOS inducible nitric oxide synthase LPS lipopolysaccharide MCP-1 monocyte chemoattractant protein-1 NF-κB nuclear factor κB p-AKT phosphorylated protein kinase B p-NF-κB phosphorylated nuclear factor κB PPARα peroxisome proliferator-activated receptor alpha PPARγ peroxisome proliferator-activated receptor gamma SCD1 stearoyl-CoA desaturase TNFα tumor necrosis factor alpha Uro urolithin References Biological signiﬁcance of urolithins, the gut microbial ellagic acid-derived metabolites: The evidence so far. Evid-Based Compl. Alt. 2013, 2013, 270418. [CrossRef] [PubMed] 4. Espín, J.C.; Larrosa, M.; García-Conesa, M.T.; Tomás-Barberán, F. Biological signiﬁcance of urolithins, the gut microbial ellagic acid-derived metabolites: The evidence so far. Evid-Based Compl. Alt. 2013, 2013, 270418. [CrossRef] [PubMed] 5. Cerdá, B.; Espín, J.C.; Parra, S.; Martínez, P.; Tomás-Barberán, F.A. The potent in vitro antioxidant ellagitannins from pomegranate juice are metabolized into bioavailable but poor antioxidant hydroxy-6H-dibenzopyran-6-one derivatives by the colonic microﬂora of healthy humans. Eur. J. Nutr. 2004, 43, 205–220. 5. Cerdá, B.; Espín, J.C.; Parra, S.; Martínez, P.; Tomás-Barberán, F.A. The potent in vitro antioxidant ellagitannins from pomegranate juice are metabolized into bioavailable but poor antioxidant hydroxy-6H-dibenzopyran-6-one derivatives by the colonic microﬂora of healthy humans. Eur. J. Nutr. 2004, 43, 205–220. 6. Pfundstein, B.; Haubner, R.; Würtele, G.; Gehres, N.; Cornelia, M.; Owen, R.W.U. Pilot walnut intervention study of urolithin bioavailability in human volunteers. J. Agric. Food Chem. 2014, 62, 10264–10273. [CrossRef] 7. Tomás-Barberán, F.A.; González-Sarrías, A.; García-Villalba, R.; Núñez-Sánchez, M.A.; Selma, M.V.; García-Conesa, M.T.; Espín, J.C. Urolithins, the rescue of “old” metabolites to understand a “new” concept: Metabotypes as a nexus among phenolic metabolism, microbiota dysbiosis, and host health status. Mol. Nutr. Food Res. 2017, 61, 1500901. [CrossRef] 8. Seeram, N.P.; Aronson, W.J.; Zhang, Y.; Henning, S.M.; Moro, A.; Lee, R.; Sartippour, M.; Harris, D.M.; Rettig, M.; Suchard, M.A.; et al. Pomegranate ellagitannin-derived metabolites inhibit prostate cancer growth and localize to the mouse prostate gland. J. Agric. Food Chem. 2007, 55, 7732–7737. [CrossRef] Int. J. Mol. Sci. 2020, 21, 2086 15 of 16 9. Larrosa, M.; Tomás-Barberán, F.A.; Espín, J.C. The dietary hydrolysable tannin punicalagin releases ellagic acid that induces apoptosis in human colon adenocarcinoma Caco-2 cells by using the mitochondrial pathway. J. Nutr. Biochem. 2006, 17, 611–625. [CrossRef] 10. Larrosa, M.; González-Sarras, A.; García-Conesa, M.T.; Tomás-Barberán, F.A.; Espín, J.C. Urolithins, ellagic-acid derived metabolites produced by human colonic microﬂora, exhibit estrogenic and antiestrogenic activities. J. Agric. Food Chem. 2006, 54, 1611–1620. [CrossRef] 11. Bialonska, D.; Kasimsetty, S.G.; Khan, S.I.; Ferreira, D. Urolithins, intestinal microbial metabolites of pomegranate ellagitannins, exhibit potent antioxidant activity in a cell-based assay. J. Agric. Food Chem. 2010, 57, 10181–10186. [CrossRef] [PubMed] 12. González-Sarrías, A.; Larrosa, M.; Tomás-Barberán, F.A.; Dolara, P.; Espín, J.C. NF-κB dependent anti-inﬂammatory activity of urolithins, gut microbiota ellagic acid-derived metabolites, in human colonic ﬁbroblasts. Brit. J. References Nutr. 2010, 26, 1–10. [CrossRef] [PubMed] 13. Mele, L.; Mena, P.; Piemontese, A.; Marino, V.; López-Gutiérrez, N.; Bernini, F.; Brighenti, F.; Zanotti, I.; Del Rio, D. Antiatherogenic eﬀects of ellagic acid and urolithins in vitro. Arch. Biochem. Biophys. 2016, 599, 42–50. [CrossRef] [PubMed] 14. Yuan, T.; Ma, H.; Liu, W.; Niesen, D.B.; Shah, N.; Crews, R.; Rose, K.N.; Vattem, D.A.; Seeram, N.P. Pomegranate’s neuroprotective eﬀects against Alzheimer’s disease are mediated by urolithins, its ellagitannin-gut microbial derived metabolites. ACS Chem. Neurosci. 2016, 20, 26–33. [CrossRef] 15. Hotamisligil, G.S. Inﬂammation and metabolic disorders. Nature 2006, 444, 860–867. [CrossRef] 16. Green, H.; Kehinde, O. An established preadipose cell line and its diﬀerentiation in culture II. Factors aﬀecting adipose conversion. Cell 1975, 5, 19–27. [CrossRef] 17. Wong-a-nan, N.; Inthanon, K.; Saiai, A.; Inta, A.; Nimlamool, W.; Chomdej, S.; Kittakoop, P.; Wongkham, W. Lipogenesis inhibition and adipogenesis regulation via PPARγ pathway in 3T3-L1 cells by Zingiber cassumunar Roxb. rhizome extracts. EJBAS 2018, 5, 289–297. [CrossRef] 18. Rosen, E.D.; Hsu, C.H.; Wang, X.; Sakai, S.; Freeman, M.W.; Gonzalez, F.J.; Spiegelman, B.M. C/EBPalpha induces adipogenesis through PPARgamma: A uniﬁed pathway. Genes Dev. 2002, 16, 22–26. [CrossRef] 19. El-Jack, A.K.; Hamm, J.K.; Pilch, P.F.; Farmer, S.R. Reconstitution of insulin-sensitive glucose transport in ﬁbroblasts requires expression of both PPARγ and C/EBPα. J. Biol. Chem. 1999, 274, 7946–7951. [CrossRef] 20. Wu, Z.; Rosen, E.D.; Brun, R.; Hauser, S.; Adelmont, G.; Troy, A.E.; McKeon, C.; Darlington, G.J.; Spiegelman, B.M. Cross-regulation of C/EBPα and PPARγ controls the transcriptional pathway of adipogenesis and insulin sensitivity. Mol. Cell 1999, 3, 151–158. [CrossRef] 21. Cabrero, A.; Alegret, M.; Sánchez, R.M.; Adzet, T.; Laguna, J.C.; Vázquez, M. Bezaﬁbrate reduces mRNA levels of adipocyte markers and increases fatty acid oxidation in primary culture of adipocytes. Diabetes 2001, 50, 1883–1890. [CrossRef] [PubMed] 22. Vazquez, M.; Roglans, N.; Cabrero, A.; Rodríguez, C.; Adzet, T.; Alegret, M.; Sanchez, R.M.; Laguna, J.C. Bezaﬁbrate induces Acyl-CoA oxidase mRNA levels and fatty acid peroxisomal beta-oxidation in rat white adipose tissue. Mol. Cell Biochem. 2001, 216, 71–78. [CrossRef] [PubMed] 23. Cisneros-Zevallos, L.; Bang, W.Y. Modulation of Adipogenesis and Inﬂammation in Adipocytes by Ellagic Acid and Metabolites Urolithins A and B; AGFD 93, American Chemical Society Annual Meeting Abstracts, Cornucopia; American Chemical Society: Washington, DC, USA, 2013; Volume 45, Available online: http://agfd.sites.acs.org/cornucopia13F.pdf (accessed on 10 March 2020). 24. Kaestner, K.H.; Christy, R.J.; Lane, M.D. References CCAAT/enhancer binding protein gene promoter: Binding of nuclear factors during diﬀerentiation of 3T3-L1 preadipocytes. Proc. Natl. Acad. Sci. USA 1991, 88, 2593–2597. 25. Wu, Z.; Xie, Y.; Morrison, R.F.; Bucher, N.L.R.; Farmer, S.R. PPARγ induces the insulin-dependent glucose transporter GLUT4 in the absence of C/EBPα during the conversion of 3T3 ﬁbroblasts into adipocytes. J. Clin. Invest. 1998, 101, 22–32. [CrossRef] [PubMed] 26. Armoni, M.; Kritz, N.; Harel, C.; Bar-Yoseph, F.; Chen, H.; Quon, M.J.; Karnieli, E. Peroxisome proliferator-activated receptor-γ represses GLUT4 promoter activity in primary adipocytes, and rosiglitazone alleviates this eﬀect. J. Biol. Chem. 2003, 278, 30614–30623. [CrossRef] [PubMed] 27. Madsen, M.S.; Siersbaek, R.; Boergesen, M.; Nielsen, R.; Mandrup, S. Peroxisome proliferator-activated receptor γ and C/EBPα synergistically activate key metabolic adipocyte genes by assisted loading. Mol. Cell Biol. 2014, 34, 939–954. [CrossRef] Int. J. Mol. Sci. 2020, 21, 2086 16 of 16 16 of 16 28. Park, S.K. Raspberry ketone, a naturally occurring phenolic compound, inhibits adipogenic and lipogenic gene expression in 3T3-L1 adipocytes. Pharm. Biol. 2015, 53, 870–875. [CrossRef] 29. Goto, T.; Lee, J.Y.; Teraminami, A.; Kim, Y.; Hirai, S.; Uemura, T.; Inoue, H.; Takahashi, N.; Kawada, T. Activation of peroxisome proliferator-activated receptor-alpha stimulates both diﬀerentiation and fatty acid oxidation in adipocytes. J. Lipid Res. 2011, 52, 873–884. [CrossRef] 30. Wang, W.; Lin, Q.; Lin, R.; Zhang, J.; Ren, F.; Zhang, J.; Ji, M.; Li, Y. PPARα agonist fenoﬁbrate attenuates TNF-α-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway. Expl. Cell Res. 2013, 319, 1523–1533. [CrossRef] 31. Guerre-Millo, M.; Gervois, P.; Raspé, E.; Madsen, L.; Poulain, P.; Derudas, B.; Herbert, J.-M.; Winegar, D.A.; Willson, T.M.; Fruchart, J.-C.; et al. Peroxisome proliferator-activated receptor α activators improve insulin sensitivity and reduce adiposity. J. Biol. Chem. 2000, 275, 16638–16642. [CrossRef] 32. Fu, Y.; Luo, N.; Klein, R.L.; Garvey, W.T. Adiponectin promotes adipocyte diﬀerentiation, insulin sensitivity, and lipid accumulation. J. Lipid Res. 2005, 46, 1369–1379. [CrossRef] [PubMed] 33. Tsuchida, A.; Yamauchi, T.; Takekawa, S.; Hada, Y.; Ito, Y.; Maki, T.; Kadowaki, T. Peroxisome proliferator–activated receptor (PPAR) α activation increases adiponectin receptors and reduces obesity-related inﬂammation in adipose tissue comparison of activation of PPAR α, PPAR γ, and their combination. Diabetes 2005, 54, 3358–3370. [CrossRef] [PubMed] 34. Kang, I.; Kim, Y.; Tomás-Barberán, F.A.; Espín, J.C.; Chung, S. Urolithin A, C, and D, but not iso-urolithin A and urolithin B. attenuate triglyceride accumulation in human cultures of adipocytes and hepatocytes. Mol. Nutr. References Food Res. 2016, 60, 1129–1138. [CrossRef] [PubMed] 35. Cortés-Martín, A.; García-Villalba, R.; González-Sarrías, A.; Romo-Vaquero, M.; Loria-Kohen, V.; Ramírez-de-Molina, A.; Tomás-Barberán, F.A.; Selma, M.V.; Espín, J.C. The gut microbiota urolithin metabotypes revisited: The human metabolism of ellagic acid is mainly determined by aging. Food Funct. 2018, 9, 4100–4106. [CrossRef] [PubMed] 36. Yoshimura, Y.; Nishii, S.; Zaima, N.; Moriyama, T.; Kawamura, Y. Ellagic acid improves hepatic steatosis and serum lipid composition through reduction of serum resistin levels and transcriptional activation of hepatic PPARα in obese, diabetic KK-A(y) mice. Biochem. Biophys. Res. Commun. 2013, 434, 486–491. [CrossRef] [PubMed] 37. Livak, K.J.; Schmittgen, T.D. Analysis of the relative gene expression data using real-time quantitative PCR and the 2-DDCt method. Methods 2001, 25, 402–408. [CrossRef] 38. Ortiz-Quesda, A.G.; Lombardini, L.; Cisneros-Zevallos, L. Antioxidants in pecan nut cultivars [Carya illinoinensis (Wangenh.) K. Koch]. In Nuts and Seeds in Health and Disease Prevention; Preedy, V., Watson, R., Eds.; Academic Press: Cambridge, MA, USA, 2011; Chapter 104; pp. 881–889. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2320485936	https://www.biodiversitylibrary.org/partpdf/53904	English	null	XII.—<i>A contribution to the characteristic of corals of the group Rugosa</i>	Annals & magazine of natural history	1,904	public-domain	1,660	* N. Yakovleff, “Fauna of the upper Portion of the Palzozoic Deposits of the Donetz Basin,” Transactions of the Geological Committee, new series, no. 12 (1903). f. N. Yakovleff on the Characteristic of f. N. Yakovleff on the Characteristic of 114 foveation ; apical half of membrane bronzy brown ; abdomen beneath orange-red. Long. 6 mm. g Hab. Ternate (J. J. Walker, Brit. Mus.). g Hab. Ternate (J. J. Walker, Brit. Mus.). g Hab. Ternate (J. J. Walker, Brit. Mus.). Sabellicus apicifer. Capsus apicifer, Walk. Cat. Het. vi. p. 124. n. 293 (1878), Hab. Celebes: Makian (Brit. Mus.). Type in bad condition. SABELLICUS, gen. nov. Resembling Dercocoris, from which it differs principally by the structure of the antenne. Head elongately depressed in front of insertion of antenne, of which the first joint is as long or a little longer than the head, prominently incrassated, and sometimes compressed from immediately beyond base, somewhat longly marginally pilose, with a distinct spur on outer side of apex; second joint much longer than first, slender at base and regularly and moderately incrassated towards apex ; remaining joints mutilated in type. yes large, almost touching anterior margin of pronotum. Pronotum with the basal margin about twice as broad as anterior margin, with a distinct pronotal collar, and with the posterior angles sub- tuberculous ; rostrum reaching the intermediate coxe; cuneus slightly longer than broad, the fracture profound ; anterior legs robust, the tibiae moderately incrassate; intermediate and posterior legs mutilated in type. Sabellicus apicifer. Capsus apicifer, Walk. Cat. Het. vi. p. 124. n. 293 (1878), Hab. Celebes: Makian (Brit. Mus.). Type in bad condition. Sabellicus apicifer. XII.—A Contribution to the Characteristic of Corals of the Group Rugosa. By Prof. N. YAKOVLEFF. WHILE engaged in investigating the Upper Paleozoic coral Lophophyllum proliferum, regarding which there have lately Sabellicus sordidus. Lopus sordidus, Walk. Cat. Het. vi. p. 57. n. 29 (1873). Leptomerocoris antennatus, Walk. loe. cit. p. 145, n. 109. Lopus sordidus, Walk. Cat. Het. vi. p. 57. n. 29 (1873). Leptomerocoris antennatus, Walk. loe. cit. p. 145, n. 109. XII.—A Contribution to the Characteristic of Corals of the Group Rugosa. By Prof. N. YAKOVLEFF. WHILE engaged in investigating the Upper Paleozoic coral Lophophyllum proliferum, regarding which there have lately Corals of the Group 115 been published the interesting researches of Duerden , I had, in the first place, the opportunity of verifying the results of Duerden’s labours, which are of certain importance in estab- lishing the general characteristics of the Rugosa, and, secondly, of adding a few data to these characteristics. As is known, the distinguishing feature of the Rugosa is considered to be the fact that they possess four primary septa, of which two—the main septum and the counter septum—are in the plane of symmetry of the coral, and the other two— the alar septa—on either side of the plane. Besides, in the quadrants between the primary septa the secondary septa are arranged pinnately as regards the main septum in the quadrants which adjoin it, and parallel with regard to the counter septum in the counter quadrants. The septa belong to two cycles, of which one consists of large and the other of small septa. It is interesting to observe the way in which the septa are developed in the coral. As proved by Duerden, the septa of one cycle—the small ones—appear comparatively late, simultaneously, and at a certain height. As to the septa of the other cycle—the prin- cipal ones—their mode of development has led Duerden to approximate the Rugosa to the group of now living Actinia— Zoanthee,—their development precluding the possibility of approximating them, as is generally done, to the Hexacoralla, which form a skeleton, and of regarding the former as the progenitors of the latter. The section (fig. A, p. 116) nearest to the pointed end of the coral is 2°1 mm. in diameter, and represents twelve septa, of which (according to Duerden) are to be regarded as primary not four, as usually accepted for the Rugosa, but six septa, which are marked in the figure by the cipher I. J. E. Duerden, “On the Relationships of the Rugosa to the living Zoanthee,” Ann, & Mag. Nat. Hist., May 1902, p. 381. Sabellicus sordidus. Four of them are: the main septum I (H), the counter septum I (G), and the alar septa I (S)—the two remaining septa I being situated next to the counter septum, and forming with it interseptal chambers in which (and exclusively in them) no new septa of the same cycle are developed. Comparing figures A and B, we notice that the difference between them is but slight, consisting chiefly in this, that the main septum in the Russian specimen is situated on the convex side of the coral, and in the American on the concave side. ‘The same coral in both specimens is bent in an opposite direction. The observed relationship between the degree of development of the main septum and the counter septum in a radial direction gt orals of the Group Rugosa. 116 is in all probability owing to the direction of the bend; the primary septum on the concave side (in the Russian specimens the counter septum and in the American specimens the main septum) ?s short and the primary septum on the convex side is long (figs. Aand B). It seems to me that, with regard both to A B Iq) 1/24) B 1/24) A Iq) A and B, the corresponding sections of the Russian and American specimens (the latter after Duerden, modified as regards the main septum and the counter septum, see below, at end) of Lopho- phyllum proliferum ; the portions of the sections turned upwards lie on the convex side of the coral. The primary interseptal chambers in which no new septa are formed are striated. I, I (H), 1 (G), I (5), the primary septa; 1, 2, the later principal septa. the main septum and the counter septum, the fact may be easily explained by supposing that the bend of the coral on the concave side causes a contraction, affording less space for the development of the septa than on the convex side; the former is characterized by contraction, the latter by distention. This assumption is strengthened by another peculiarity of the coral, viz. that of the four primary interseptal chambers, in which the successive principal septa are generally deve- loped, the two situated nearest to the convex side develop the septa more rapidly (in greater number). These chambers are not the same in the Russian and American specimens: in the former (fig. Sabellicus sordidus. A) they are contiguous with the main septum, in the latter they are separated from it as well as from the counter septum by other primary chambers. In examining the two specimens (figs. A and B) we must also notice that in two of the four primary chambers no new septa are formed—invariably in those primary chambers which On the Distribution of Marine An 17 adjoin the counter septum,—either on the convex or concave side, and whether it be long or short. We thus arrive at a more complete definition of the primary counter septum: it is that (L) dn relation to which the con- tiqguous septa are arranged tn a parallel direction, and (2) which has adjoining primary interseptal chambers, “containing no secondary principal septa. Duerden is not correct in stating that the main septum and the counter septum lie respectively on the convex and on the concave side of the coral independently of the arrangement of the contiguous septa. This very arrangement has been regarded by ‘paleontologists as characteristic of the primary septa, and, as will be seen from the above, it is more permanent ‘than has hitherto been known. XIITI.—On the Distribution of Marine Animals * By Prof. M‘Intosu, M.D., LL.D., F.R.S., &e. THE distribution of land-animals is a subject which has always been fraught with deep interest to naturalists—more especially as certain regions are characterized by the forms inhabiting them. Thus it would be anomalous to find, for instance, a marsupial in Africa, an armadillo or a sloth (Bradypus) in Asia, or a stag in Australia. The chief barriers, moreover, to the general distribution of such forms have been mountain- chains, deep tracts of the sea, barren regions such as the great deserts, and the vicissitudes of temperature. Yet certain aerial forms, such as the bats, are more or less cosmopolitan, and the shrews, the pigs, and the mice are almost so. In weighing the statement, however, that the distribution of certain of these forms, such as the pigs, has been extended by their swimming powers across arms of the sea, it has to be borne in mind that even marine animals do not always avail themselves of the lines of migra- tion at their disposal. * Notes of an Introductory Lecture, 16th October, 1903, Sponsored by Smithsonian Sabellicus sordidus. As three fourths of the surface of the globe are composed of water—for the most part continuous throughout—a vast field exists for the distribution, under natural conditions, of its inhabitants, from mammals to Protozoa, Pelagic types may thus range from pole to pole and from the eastern shore of the Isthmus of Panama round the world to the western. * Notes of an Introductory Lecture, 16th October, 1903, This file was generated 31 March 2024 at 20:51 UTC Yakovleff, N. 1904. "XII.—A contribution to the characteristic of corals of the group Rugosa." The Annals and magazine of natural history; zoology, botany, and geology 13, 114–117. https://doi.org/10.1080/00222930408562445. View This Item Online: https://www.biodiversitylibrary.org/item/54611 DOI: https://doi.org/10.1080/00222930408562445 Permalink: https://www.biodiversitylibrary.org/partpdf/53904 Holding Institution Smithsonian Libraries and Archives Copyright & Reuse Copyright & Reuse Copyright Status: Public domain. The BHL considers that this work is no longer under copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's largest open access digital library for biodiversity literature and archives. Visit BHL at https://www.biodiversitylibrary.org. This file was generated 31 March 2024 at 20:51 UTC
https://openalex.org/W2695687054	https://www.frontiersin.org/articles/10.3389/fendo.2017.00139/pdf?isPublishedV2=False	English	null	Psoriasis, Psoriatic Arthritis, and Thyroid Autoimmunity	Frontiers in endocrinology	2,017	cc-by	4,409	Abbreviations: AITDs, autoimmune thyroid diseases; AT, autoimmune thyroiditis; AbTg, anti-thyroglobulin antibodies; AbTPO, antithyroid peroxidase antibodies; FT4, free thyroxine; IBD, inflammatory bowel disease; IFN, interferon; IL, inter- leukin; JIA, juvenile idiopathic arthritis; PsA, psoriatic arthritis; PsO, psoriasis; RA, rheumatoid arthritis; RF, rheumatoid factor; TD, thyroid dysfunction; TSH, thyroid-stimulating hormone. Keywords: psoriasis, psoriatic arthritis, autoimmune thyroiditis, hypothyroidism, CXCL10, AbTPO, anti- thyroglobulin antibodies Correspondence: Ilaria Ruffilli Specialty section: This article was submitted to Thyroid Endocrinology, a section of the journal Frontiers in Endocrinology Received: 13 April 2017 Accepted: 06 June 2017 Published: 19 June 2017 Specialty section: This article was submitted to Thyroid Endocrinology, a section of the journal Frontiers in Endocrinology Psoriasis, Psoriatic Arthritis, and Thyroid Autoimmunity Ilaria Ruffilli 1, Francesca Ragusa1, Salvatore Benvenga 2,3,4, Roberto Vita 2, Alessandro Antonelli 1, Poupak Fallahi 1 and Silvia Martina Ferrari 1 1 Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy, 2 Department of Clinical and Experimental Medicine, University of Messina School of Medicine, Messina, Italy, 3 Master Program of Childhood, Adolescence and Women’s Endocrine Health, University of Messina School of Medicine, Messina, Italy, 4 Interdepartmental Program of Molecular & Clinical Endocrinology, and Women’s Endocrine Health, University Hospital, Messina, Italy Psoriasis (PsO) is a chronic relapsing/remitting autoimmune skin disease, associated with an increased risk of other autoimmune disorders. Psoriatic arthritis (PsA) is a chronic inflammatory arthritis occurring approximately in 30% of PsO patients. Sporadic cases of association between PsO and autoimmune thyroid disorders (AITDs) have been reported. However, two different recent studies did not find any association between them. In patients with PsO and PsA, an association with AITD has been shown by most of the studies in adults, but not in the juvenile form. In PsA women and men, thyroid autoim- munity [positive antithyroid peroxidase (AbTPO) antibodies, hypoechoic thyroid pattern] and subclinical hypothyroidism were more prevalent than in the general population. An association has been shown also in patients with PsO, arthritis, and inflammatory bowel disease, who have more frequently AITD. A Th1 immune predominance has been shown in early PsO, and PsA, with high serum CXCL10 (Th1 prototype chemokine), overall in the presence of autoimmune thyroiditis. This Th1 immune predominance might be the immunopathogenetic base of the association of these disorders. A raised incidence of new cases of hypothyroidism, thyroid dysfunction, positive AbTPO, and appearance of a hypoechoic thyroid pattern in PsA patients, especially in women, has been shown recently, suggesting to evaluate AbTPO levels, thyroid function, and thyroid ultrasound, especially in PsA women. Thyroid function follow-up and suitable treatments should be performed regularly in PsA female patients at high risk (thyroid-stimulating hormone within the normal range but at the higher limit, positive AbTPO, hypoechoic, and small thyroid). INTRODUCTION Received: 13 April 2017 Accepted: 06 June 2017 Published: 19 June 2017 Psoriasis (PsO) (1) affects about 2–4% of the population (2); it is a chronic relapsing/remitting autoimmune skin disease (1) and presents with itchy red, scaly patches, papules, and plaques, with different severity, from localized patches to general body coverage. PsO is classified in five types: plaque, guttate, inverse, pustular, and erythrodermic (3). These lesions are usually evident on the skin Mini Review Mini Review published: 19 June 2017 doi: 10.3389/fendo.2017.00139 Reviewed by: Maria Brito, Reviewed by: Maria Brito, Mount Sinai Health System, United States Tony Weetman, University of Sheffield, United Kingdom Edited by: Terry Francis Davies, Icahn School of Medicine at Mount Sinai, United States Reviewed by: Maria Brito, Mount Sinai Health System, United States Tony Weetman, University of Sheffield, United Kingdom *Correspondence: Ilaria Ruffilli ilaria.ruffilli@gmail.com Edited by: Terry Francis Davies, Icahn School of Medicine at Mount Sinai, United States Citation: Ruffilli I, Ragusa F, Benvenga S, Vita R, Antonelli A, Fallahi P and Ferrari SM (2017) Psoriasis, Psoriatic Arthritis, and Thyroid Autoimmunity. Front. Endocrinol. 8:139. doi: 10.3389/fendo.2017.00139 June 2017 \| Volume 8 \| Article 139 Frontiers in Endocrinology \| www.frontiersin.org 1 PsO, Arthritis, Thyroid Autoimmunity Ruffilli et al. anti-thyroglobulin antibodies (AbTg) in 42 patients with PsA, versus 52 normal subjects, as controls. The average thyroid volume, measured at ultrasounds, was increased in compari- son to controls. Patients with PsA had a raised prevalence of anti-microsome antibodies; thyroid involvement was confined to patients with active disease. These results suggested a signifi- cant thyroid involvement in PsA patients. However, the study was basically a retrospective study, and it did not specify the selection criteria of the patients (15). of elbows and knees, and also on scalp, palms of hands, and soles of feet. Psoriatic nail dystrophy is usually present in fingernails and toenails and can be an isolated sign. A genetic predisposition is very important in the pathogenesis of PsO; however, environ- mental factors can activate the disease (1).h anti-thyroglobulin antibodies (AbTg) in 42 patients with PsA, versus 52 normal subjects, as controls. The average thyroid volume, measured at ultrasounds, was increased in compari- son to controls. Patients with PsA had a raised prevalence of anti-microsome antibodies; thyroid involvement was confined to patients with active disease. These results suggested a signifi- cant thyroid involvement in PsA patients. However, the study was basically a retrospective study, and it did not specify the selection criteria of the patients (15). The skin epidermal layer grows rapidly in PsO (4), determin- ing an abnormal production and an excess of skin cells (5), that are replaced in 3–5 days in PsO (while commonly every 28–30 days) (6). These events are probably induced by the pre- mature keratinocytes maturation, induced by the inflammatory cascade in the dermis (7). The immune competent cells go from dermis to epidermis and release different inflammatory cytokines [interleukin (IL)-1β, interferon (IFN)-γ, tumor necrosis factor-α, chemokine (C-X-C motif) ligand (CXCL)10, IL-6, IL-22] (8). In PsO, DNA can stimulate the dendritic cells, to produce IFN-α. Citation: The secretion of such inflammatory cytokines leads to stimulate the proliferation of keratinocytes (8).l A second study evaluated the prevalence of thyroid disorders in PsA patients, conducting a complete thyroid work-out in 80 PsA patients, versus control subjects extracted (1:5) from the general population (matched by age and gender), and 112 patients with rheumatoid arthrtitis (RA) (with similar iodine intake). PsA women had significantly more frequently a hypoechoic thyroid pattern, antithyroid peroxidase antibodies (AbTPO), and sub- clinical hypothyroidism than control women, with a frequency comparable to that in RA patients (hypoechoic thyroid 31, 16, and 36%; positive AbTPO titer 28, 12, and 31%; subclinical hypothy- roidism 25, 8, and 12%, respectively). PsA and RA men showed more frequently hypoechoic thyroid pattern and positive AbTPO than control subjects (hypoechoic thyroid 16, 10, and 3%; posi- tive AbTPO titer 14, 5, and 2%, respectively). PsA patients with subclinical hypothyroidism had a longer disease duration (years; 19 ± 15 versus 11 ± 8, p = 0.03) and polyarticular involvement (p ≤ 0.05) than euthyroid PsA patients. Therefore, a significantly higher prevalence of thyroid autoimmunity (positive AbTPO, hypoechoic thyroid pattern) in PsA men and women, and of subclinical hypothyroidism in PsA women, than in the general population were evidenced (12). Conversely, a subsequent study investigated the frequency of rheumatic diseases in 65 patients (56 F, 9 M), suffering from AITD; antinuclear antibody and rheumatoid factor levels were also measured. Various rheumatic disorders were detected in 40 (62%) of patients with AITD: the most frequent were fibromyalgia, osteoarthritis, keratocon- junctivitis sicca, and xerostomia. Autoimmune diseases were detected in 10 patients with AITD, and among them also PsO and PsA (19). A further study (20) evaluated the frequency of AITD in 80 children with juvenile idiopathic arthritis (JIA) (27 Psoriatic arthritis (PsA) is a chronic inflammatory arthritis that has a variable clinical presentation and occurs approximately in 30% of PsO patients (7, 9, 10). PsA typically affects the joints of the fingers and toes, and it is characterized by a painful inflamma- tion of the joints and surrounding connective tissue. This process results in a sausage-shaped swelling of the fingers and toes called dactylitis (9). PsA can also affect the hips, spine (spondylitis), knees, and sacroiliac joint (sacroiliitis), and any other joint (11). Dermatologic manifestations of PsO appear before the manifesta- tions of arthritis in about 75% of patients (10). Citation: Psoriasis is associated with an increased risk of other auto- immune disorders like ulcerative colitis, Crohn’s disease, and autoimmune thyroiditis (AT) too (12).i Here, we review the scientific literature about PsO, PsA, and the possible association with autoimmune thyroid disorders (AITDs). June 2017 \| Volume 8 \| Article 139 PsO, PsA, AND AITD Patients with both PsO and IBD (versus PsO-only) had significantly higher rates of hepatitis (6.2 versus 0.7%), AT (6.8 versus 2.1%), and diabetes (26.77 versus 11.0%), and 60 (41.1%) were diagnosed with seronegative arthritis, suggesting that patients with both PsO and IBD have more frequently AITD and arthritis (21).h The prevalence of 12 autoimmune diseases was also evalu- ated in 25,885 people extracted from the general population in Sardinia (22). A high prevalence was observed for RA, ulcerative colitis, Crohn’s disease, type 1 diabetes, systemic lupus erythe- matosus, celiac disease, myasthenia gravis, systemic sclerosis, multiple sclerosis, Sjogren’s syndrome, PsO/PsA (939 cases), and AT (2,619 cases). The statistical analysis of the comorbidity of autoimmune diseases shows that the number of people with more than one autoimmune disease was significantly higher than the expected number, both in women and men (22). CXCL10 is a determinant chemoattractant for neutrophils, and an elevated infiltration and microabscess formation by neutrophils is a characteristic PsO feature. Different papers have shown a critical pathogenic role of neutrophils in PsO, particu- larly in the first phases, leading to hypothesize that blocking neutrophil function could have therapeutic effectiveness in this disease (23, 29, 30). Also, in PsA patients, high levels of CXCL10 are observed in synovial fluid, and in circulation. Th1 cells immune predomi- nance has been also shown at the beginning of the disease, with a subsequent later decline in long-lasting PsO or PsA, suggesting a shift from Th1 to Th2 immune response in long duration diseases (31–33). Another study (17) evaluated prospectively 114 PsO patients with disease duration of 5–38 years, 30 of them with PsA, in comparison with 286 age- and body mass index-matched sub- jects. No difference in the prevalence of AT between PsO patients and controls (20.2 versus 19.6%) was present. The prevalence of AT in male and female PsO patients was similar (9.6 and 10.5%, respectively) unlike the increased, as expected, prevalence in female versus male controls (14.7 versus 4.9%). Detected cases with hypothyroidism due to AT were similar in PsO patients and controls (7.9 and 7.0%, respectively). However, the number of patients with PsA was low (23) and not sufficient to a reliable evaluation of AITD in these last patients (17). PsO, PsA, AND AITD Few case reports initially reported an association of PsO and Hashimoto’s thyroiditis (13, 14) (Table 1). A first system- atic study by Bianchi et al. (15) evaluated thyroid volume and function and the prevalence of anti-microsome and Table 1 \| Prevalence of thyroid autoimmunity in psoriasis (PsO), or psoriatic arthritis (PsA) patients, versus controls, in the studies that have an internal control group. Reference PsO patients (n) Autoimmune thyroid disorder (AITD)% in PsO patients Controls (n) AITD% in controls P Antonelli et al. (12) 80 with PsA 12/36 of F patients (33%); 11/44 of M patients (25%) 112 patients with rheumatoid arthritis; 400 control subjects 33/180 of F controls (18%); 10/220 of M controls (5%) 0.0001 Bianchi et al. (15) 42 with PsA AbTg prevalence 5%; anti-microsome antibodies prevalence 14% 52 AbTg prevalence 3%; anti-microsome antibodies prevalence 0% <0.05 Gul et al. (16) 105 6 patients had increased AbTPO (6%); 8 patients had increased AbTg (8%); 3 patients had both increased AbTPO and AbTg (3%) 96 with tinea pedis AbTPO levels were increased in 6 subjects (6%), AbTg levels were increased in 11 subjects (11%) and both of them were increased in 6 subjects (6%) NS Vassilatou et al. (17) 114 (30 of them with PsA) Prevalence of autoimmune thyroiditis (AT) 20.2% 286 Prevalence of AT 19.6% NS Fallahi et al. (18) 97 with PsA 34% thyroid autoimmunity 97 15% thyroid autoimmunity 0.002 AbTg, anti-thyroglobulin antibodies; AbTPO, anti-thyroperoxidase antibodies; F, females; M, males; NS, not significant. June 2017 \| Volume 8 \| Article 139 2 PsO, Arthritis, Thyroid Autoimmunity Ruffilli et al. random sample of the general population, with similar iodine intake. The results of our study demonstrated a significant increase of the prevalence of PsA in AT patients (24). oligoarticular, 26 polyarticular, 17 enthesitis-related, 6 systemic, and 4 PsA), versus 81 healthy control subjects, matched by age and gender. AITD were found in four patients in the JIA group (5%). No significant difference between the study and control groups was observed in the frequency of circulating antithyroid antibodies, or AT, suggesting that in JIA there is no association with AITD (20). A more recent study (18) aimed to assess the incidence of new cases of clinical and subclinical thyroid dysfunction (TD) in a broad group of PsA patients versus a control group, matched by age and gender with a similar iodine intake. PsO, PsA, AND AITD PsA patients with TD were excluded first, and new cases of thyroid disorders were evaluated in 97 PsA patients and 97 matched controls (median follow-up of 74 months in PsA versus 92 in controls). A raised rate of new cases of hypothyroidism, TD, positive AbTPO, and appearance of a small hypoechoic thyroid pattern in PsA, espe- cially in female gender, compared to controls has been evidenced. Risk factors in female gender for the development of TD were TSH within the normal range but at the higher limit, positive AbTPO, and small thyroid volume (18). A further study evaluated thyroid autoimmunity in 105 patients with PsO (without PsA), versus 96 sex- and age- matching controls (with tinea pedis). The levels of free thyroxine (FT4) resulted significantly increased in the PsO group; however, AbTPO and AbTg levels were not significantly different between the two groups. The study showed that the serum FT4 levels can increase in psoriatic patients. However, thyroid-stimulating hormone (TSH), FT4, or FT3 were not reported. Furthermore, an increase of FT4 should be related to TSH decreased levels, however any correlation was reported (16).hl y Interferon-γ and Th1 cytokines/chemokines are involved in the pathogenesis of PsO. Activated T cells and HLA-DR keratinocytes have been shown in active plaques (25). It has been shown that CXCL10, the Th1 prototype chemokine, and its receptor (CXCR)3 are present in keratinocytes and in the dermal infiltrate derived from active psoriatic plaques and that effective treatment of active plaques decreased the expression of CXCL10. Elevated circulating CXCL10 has been shown in PsO patients (25–28). The cellular infiltrate in acute plaques is constituted by 5–8% CD3(−)CD56(+) NK cells, as indicated by immunohisto- chemical techniques, especially localized in the mid and papillary dermis. NK lymphocyte migration toward CXCL10 is involved in the pathogenesis of PsO (27). The association between PsO and inflammatory bowel disease (IBD) has been previously reported, even if potential associated comorbidities are not clear. A study (21) examined comorbidities in 146 patients diagnosed with both PsO and IBD, in comparison with those diagnosed with PsO-only (146, matched by gender, ethnicity, and age). Frontiers in Endocrinology \| www.frontiersin.org REFERENCES 13. Molta CT, Khan MA, Aponte CJ, Reynolds TL, Macintyre SS. Familial occur- rence of systemic sclerosis, rheumatoid arthritis and other immunological disorders: report of two kindreds with study of HLA antigens and review of the literature. Clin Exp Rheumatol (1989) 7:229–36. 1. Menter A, Gottlieb A, Feldman SR, Van Voorhees AS, Leonardi CL, Gordon KB, et al. Guidelines of care for the management of psoriasis and psoriatic arthri- tis: Section 1. Overview of psoriasis and guidelines of care for the treatment of psoriasis with biologics. J Am Acad Dermatol (2008) 58:826–50. doi:10.1016/j. jaad.2008.02.039 14. Nogita T, Aramoto Y, Terajima S, Akimoto K, Kawashima M, Hidano A, et al. The coexistence of psoriasis vulgaris, Sjögren’s syndrome, and Hashimoto’s thy- roiditis. J Dermatol (1992) 19:302–5. doi:10.1111/j.1346-8138.1992.tb03229.x 2. Parisi R, Symmons DP, Griffiths CE, Ashcroft DM. Identification and Management of Psoriasis and Associated ComorbidiTy (IMPACT) project team. Global epidemiology of psoriasis: a systematic review of incidence and prevalence. J Invest Dermatol (2013) 133:377–85. doi:10.1038/jid.2012.339 15. Bianchi G, Marchesini G, Zoli M, Falasconi MC, Iervese T, Vecchi F, et al. Thyroid involvement in chronic inflammatory rheumatological disorders. Clin Rheumatol (1993) 12:479–84. doi:10.1007/BF02231775 16. Gul U, Gonul M, Kaya I, Aslan E. Autoimmune thyroid disorders in patients with psoriasis. Eur J Dermatol (2009) 9:221–3. doi:10.1684/ejd.2009.0632 3. Sima J. Dermatology: Illustrated Study Guide and Comprehensive Board Review. New York: Springer International Publishing (2012).l 17. Vassilatou E, Papadavid E, Papastamatakis P, Alexakos D, Koumaki D, Katsimbri P, et al. No association of psoriasis with autoimmune thyroiditis. J Eur Acad Dermatol Venereol (2017) 31:102–6. doi:10.1111/jdv.13767 fi 4. Ouyang W. Distinct roles of IL-22 in human psoriasis and inflammatory bowel disease. Cytokine Growth Factor Rev (2010) 21:435–41. doi:10.1016/j. cytogfr.2010.10.007 18. Fallahi P, Ferrari SM, Ruffilli I, Elia G, Miccoli M, Sedie AD, et al. Increased incidence of autoimmune thyroid disorders in patients with psoriatic arthritis: a longitudinal follow-up study. Immunol Res (2017) 65:681–6. doi:10.1007/s12026-017-8900-8 5. Raychaudhuri SK, Maverakis E, Raychaudhuri SP. Diagnosis and classification of psoriasis. Autoimmun Rev (2014) 13:490–5. doi:10.1016/j.autrev.2014. 01.008 6. Parrish L. Psoriasis: symptoms, treatments and its impact on quality of life. Br J Community Nurs (2012) 17:524, 526, 528. doi:10.12968/bjcn.2012.17.11.524 19. Soy M, Guldiken S, Arikan E, Altun BU, Tugrul A. Frequency of rheumatic diseases in patients with autoimmune thyroid disease. Rheumatol Int (2007) 27:575–7. doi:10.1007/s00296-006-0263-8 h 7. Palfreeman AC, McNamee KE, McCann FE. PsO, PsA, AND AITD Also, AITD are Th1 immune-mediated autoimmune disor- ders in which Th1 lymphocytes, IFN-γ, and IFN-γ dependent chemokines (CXCL9, CXCL10, CXCL11) play an important role (34, 35).h Serum levels of CXCL10 (the Th1 prototype chemokine) and CCL2 (the Th2 prototype chemokine) were measured in 37 patients with PsA without AT (PsA) and 28 with AT (PsA + AT), and in gender- and age-matched controls. The results of the study demonstrated higher circulating CXCL10 and CCL2 in PsA patients than in control subjects. Furthermore, serum CXCL10 (but not CCL2) levels in PsA patients were significantly higher in the presence of AT (36). These data suggested that a Th1 immune predominace, both in PsA such as in AT, might be the immunopathogenetic base of the association of these diseases. Conversely, a subsequent study evaluated prospectively the prevalence of other autoimmune disorders in outpatient clinic in 3,069 consecutive patients with diagnosed chronic AT, with respect to two age- and sex-matched control groups: (a) a control group of 1,023 subjects, extracted from a random sample of the general population without thyroid disorders and (b) 1,023 patients with non-toxic multinodular goiter drawn by the same June 2017 \| Volume 8 \| Article 139 3 PsO, Arthritis, Thyroid Autoimmunity Ruffilli et al. AUTHOR CONTRIBUTIONS A Th1 immune predominance has been shown in early PsO, and PsA, such as in AT, with high circulating levels of the Th1 prototype chemokine CXCL10 overall in the presence of the association with AT. These data suggest that this Th1 immune predominance might be the immunopathogenetic base of the association of these disorders. IR, FR, SB, RV, AA, PF, and SMF gave substantial contribution in the conception and design of the work, and in writing the paper; gave the final approval of the version to be published; agreed to be accountable for all aspects of the work in ensuring that ques- tions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. AA and SB revised it critically for important intellectual content. A very recent longitudinal study in PsA patients has shown a raised incidence of new cases of hypothyroidism, TD, CONCLUSION positive AbTPO, and appearance of a small and hypoechoic thyroid in PsA, especially in female gender, compared to controls. Risk factors in female gender for the development of TD are TSH within the normal range but at the higher limit, positive AbTPO, and small thyroid volume, suggesting to evaluate AbTPO levels, thyroid function, and thyroid ultra- sound, especially in PsA women. Thyroid function follow-up and suitable treatments should be performed regularly in PsA female patients at high risk TSH within the normal range but at the higher limit, positive AbTPO, hypoechoic, and small thyroid. Psoriasis is associated with an increased risk of other autoim- mune disorders like ulcerative colitis, Crohn’s disease, and celiac disease (37). Sporadic cases of association of PsO and AITD have been reported. However, two different recent studies did not find any association between PsO and AITD.l Psoriatic arthritis is a chronic inflammatory arthritis that occurs approximately in 30% of PsO patients. In patients with PsO and PsA, an association with AITD has been shown by most of the studies in adults, but not in the juvenile form. In PsA women and men, thyroid autoimmunity (positive AbTPO antibodies, hypoechoic thyroid pattern) and subclinical hypothyroidism were more prevalent than in the general population. An associa- tion has been shown also in patients with PsO, arthritis, and IBD who have more frequently AITD.h However, studies in larger number of patients are required to evaluate if routine thyroid screening could be beneficial for PsO patients. REFERENCES New developments in the man- agement of psoriasis and psoriatic arthritis: a focus on apremilast. Drug Des Devel Ther (2013) 7:201–10. doi:10.2147/DDDT.S32713 20. Unsal E, Oren O, Salar K, Makay B, Abaci A, Ozhan B, et al. The frequency of autoimmune thyroid disorders in juvenile idiopathic arthritis. Turk J Pediatr (2008) 50:462–5. h 8. Nestle FO, Kaplan DH, Barker J. Psoriasis. N Engl J Med (2009) 361:496–509. doi:10.1056/NEJMra0804595 21. Binus AM, Han J, Qamar AA, Mody EA, Holt EW, Qureshi AA. Associated comorbidities in psoriasis and inflammatory bowel disease. J Eur Acad Dermatol Venereol (2012) 26:644–50. doi:10.1111/j.1468-3083.2011.04153.x 9. Durham LE, Taams LS, Kirkham BW. Psoriatic arthritis. Br J Hosp Med (Lond) (2016) 77:C102–8. doi:10.12968/hmed.2016.77.7.C102 10. Goldenstein-Schainberg C, Favarato MH, Ranza R. Current and relevant con- cepts in psoriatic arthritis. Rev Bras Reumatol (2012) 52:98–106. doi:10.1590/ S0482-50042012000100010 22. Sardu C, Cocco E, Mereu A, Massa R, Cuccu A, Marrosu MG, et al. Population based study of 12 autoimmune diseases in Sardinia, Italy: prevalence and comorbidity. PLoS One (2012) 7:e32487. doi:10.1371/journal.pone.0032487 fi 11. Krawczyk-Wasielewska A, Skorupska E, Samborski W. Sacroiliac joint pain as an important element of psoriatic arthritis diagnosis. Postepy Dermatol Alergol (2013) 30:108–12. doi:10.5114/pdia.2013.34161 23. Ferrari SM, Ruffilli I, Colaci M, Antonelli A, Ferri C, Fallahi P. CXCL10 in psoriasis. Adv Med Sci (2015) 60:349–54. doi:10.1016/j.advms.2015.07.011 fih 24. Fallahi P, Ferrari SM, Ruffilli I, Elia G, Biricotti M, Vita R, et al. The associa- tion of other autoimmune diseases in patients with autoimmune thyroiditis: review of the literature and report of a large series of patients. Autoimmun Rev (2016) 15:1125–8. doi:10.1016/j.autrev.2016.09.009 12. Antonelli A, Delle Sedie A, Fallahi P, Ferrari SM, Maccheroni M, Ferrannini E, et al. High prevalence of thyroid autoimmunity and hypothy- roidism in patients with psoriatic arthritis. J Rheumatol (2006) 33:2026–8. June 2017 \| Volume 8 \| Article 139 Frontiers in Endocrinology \| www.frontiersin.org 4 Ruffilli et al. PsO, Arthritis, Thyroid Autoimmunity 25. Gottlieb AB, Luster AD, Posnett DN, Carter DM. Detection of a gamma interferon-induced protein IP-10 in psoriatic plaques. J Exp Med (1988) 168: 941–8. doi:10.1084/jem.168.3.941 34. Antonelli A, Ferrari SM, Corrado A, Di Domenicantonio A, Fallahi P. Autoimmune thyroid disorders. Autoimmun Rev (2015) 14:174–80. doi:10.1016/j.autrev.2014.10.016 26. Boorsma DM, Flier J, Sampat S, Ottevanger C, de Haan P, Hooft L, et al. Chemokine IP-10 expression in cultured human keratinocytes. Arch Dermatol Res (1998) 290:335–41. doi:10.1007/s004030050314 35. REFERENCES Antonelli A, Ferrari SM, Frascerra S, Pupilli C, Mancusi C, Metelli MR, et al. CXCL9 and CXCL11 chemokines modulation by peroxisome proliferator- activated receptor-alpha agonists secretion in Graves’ and normal thyrocytes. J Clin Endocrinol Metab (2010) 95:E413–20. doi:10.1210/jc.2010-0923 27. Ottaviani C, Nasorri F, Bedini C, de Pità O, Girolomoni G, Cavani A. CD56brightCD16(-) NK cells accumulate in psoriatic skin in response to CXCL10 and CCL5 and exacerbate skin inflammation. Eur J Immunol (2006) 36:118–28. doi:10.1002/eji.200535243 36. Antonelli A, Fallahi P, Delle Sedie A, Ferrari SM, Maccheroni M, Bombardieri S, et al. High values of alpha (CXCL10) and beta (CCL2) circulating chemokines in patients with psoriatic arthritis, in presence or absence of autoimmune thyroiditis. Autoimmunity (2008) 41:537–42. doi:10.1080/08916930802170401 28. Deeva I, Mariani S, De Luca C, Pacifico V, Leoni L, Raskovic D, et al. Wide- spectrum profile of inflammatory mediators in the plasma and scales of patients with psoriatic disease. Cytokine (2010) 49:163–70. doi:10.1016/j. cyto.2009.09.014 37. Bhatia BK, Millsop JW, Debbaneh M, Koo J, Linos E, Liao W. Diet and psoriasis, part II: celiac disease and role of a gluten-free diet. J Am Acad Dermatol (2014) 71:350–8. doi:10.1016/j.jaad.2014.03.017 29. Naik HB, Cowen EW. Autoinflammatory pustular neutrophilic diseases. Dermatol Clin (2013) 31:405–25. doi:10.1016/j.det.2013.04.001 30. Christophers E, Metzler G, Röcken M. Bimodal immune activation in psoria- sis. Br J Dermatol (2014) 170:59–65. doi:10.1111/bjd.12631 Conflict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 31. Proost P, Struyf S, Loos T, Gouwy M, Schutyser E, Conings R, et al. Coexpression and interaction of CXCL10 and CD26 in mesenchymal cells by synergising inflammatory cytokines: CXCL8 and CXCL10 are discriminative markers for autoimmune arthropathies. Arthritis Res Ther (2006) 8:R107. doi:10.1186/ar1997 The reviewer, MB, and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review. 32. Lande R, Giacomini E, Serafini B, Rosicarelli B, Sebastiani GD, Minisola G, et al. Characterization and recruitment of plasmacytoid dendritic cells in synovial fluid and tissue of patients with chronic inflammatory arthri- tis. J Immunol (2004) 173:2815–24. doi:10.4049/jimmunol.173.4.2815 Copyright © 2017 Ruffilli, Ragusa, Benvenga, Vita, Antonelli, Fallahi and Ferrari. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Endocrinology \| www.frontiersin.org REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 33. Antonelli A, Fallahi P, Delle Sedie A, Ferrari SM, Maccheroni M, Bombardieri S, et al. High values of Th1 (CXCL10) and Th2 (CCL2) chemo- kines in patients with psoriatic arthritis. Clin Exp Rheumatol (2009) 27:22–7. June 2017 \| Volume 8 \| Article 139 Frontiers in Endocrinology \| www.frontiersin.org 5
https://openalex.org/W2784935857	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0191357&type=printable	English	null	Risk preferences impose a hidden distortion on measures of choice impulsivity	PloS one	2,018	cc-by	11,215	Risk preferences impose a hidden distortion on measures of choice impulsivity Silvia Lopez-Guzman1, Anna B. Konova1, Kenway Louie1, Paul W. Glimcher1,2 1 Center for Neural Science, New York University, New York, United States of America, 2 Institute for the Study of Decision Making, New York University, New York, United States of America silvia.lopez@nyu.edu * silvia.lopez@nyu.edu a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Editor: Pablo Brañas-Garza, Middlesex University, UNITED KINGDOM UNITED KINGDOM Received: August 10, 2017 Accepted: January 3, 2018 Published: January 26, 2018 Copyright: © 2018 Lopez-Guzman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. UNITED KINGDOM Received: August 10, 2017 Accepted: January 3, 2018 Published: January 26, 2018 Copyright: © 2018 Lopez-Guzman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2018 Lopez-Guzman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The data set and code can be found on figshare: https://doi.org/10. 6084/m9.figshare.5768340.v1. Funding: PWG received a grant from the National Institute on Drug Abuse, https://www.drugabuse. gov/, number:5R01DA038063; ABK received the Ruth L. Kirschstein National Service Award (NRSA) from the National Institute on Drug Abuse, https:// www.drugabuse.gov/, number:F32DA039648; SLG received a grant from the US-Colombian Fulbright Commission, http://www.fulbright.edu.co/, (no number). The funders had no role in study design, Abstract Measuring temporal discounting through the use of intertemporal choice tasks is now the gold standard method for quantifying human choice impulsivity (impatience) in neurosci- ence, psychology, behavioral economics, public health and computational psychiatry. A recent area of growing interest is individual differences in discounting levels, as these may predispose to (or protect from) mental health disorders, addictive behaviors, and other dis- eases. At the same time, more and more studies have been dedicated to the quantification of individual attitudes towards risk, which have been measured in many clinical and non-clin- ical populations using closely related techniques. Economists have pointed to interactions between measurements of time preferences and risk preferences that may distort estima- tions of the discount rate. However, although becoming standard practice in economics, dis- count rates and risk preferences are rarely measured simultaneously in the same subjects in other fields, and the magnitude of the imposed distortion is unknown in the assessment of individual differences. Here, we show that standard models of temporal discounting —such as a hyperbolic discounting model widely present in the literature which fails to account for risk attitudes in the estimation of discount rates— result in a large and systematic pattern of bias in estimated discounting parameters. This can lead to the spurious attribution of differ- ences in impulsivity between individuals when in fact differences in risk attitudes account for observed behavioral differences. We advance a model which, when applied to standard choice tasks typically used in psychology and neuroscience, provides both a better fit to the data and successfully de-correlates risk and impulsivity parameters. This results in mea- sures that are more accurate and thus of greater utility to the many fields interested in indi- vidual differences in impulsivity. OPEN ACCESS Citation: Lopez-Guzman S, Konova AB, Louie K, Glimcher PW (2018) Risk preferences impose a hidden distortion on measures of choice impulsivity. PLoS ONE 13(1): e0191357. https:// doi.org/10.1371/journal.pone.0191357 Editor: Pablo Brañas-Garza, Middlesex University, UNITED KINGDOM RESEARCH ARTICLE Risk distorts impulsivity subjective value is captured in models with diverse functional forms, with time preference typi- cally summarized as a discount rate or discount parameter. The two most widely used func- tional forms are the exponential and the hyperbolic classes of models. Exponential discounting is derived from economic theory and assumes a constant rate of discounting at every time period. Hyperbolic forms are favored in psychology and neuroscience, as they can fit empirical data better [1, 2], exhibiting steeper discounting for near-future outcomes and shallower dis- counting of far-future payoffs, an inconsistency referred to as “present bias” [3]. More recently, psychologists have related this time preference to the multidimensional construct of impulsiv- ity [4, 5]. Scholars of impulsivity have converged to a taxonomy that divides it into action and choice impulsivity, and officially consider the measurement of discounting as a quantitative assessment of the latter [6]. We note that for economics, impulsivity is not directly equated to elevated discounting. For this field, when discounting is constant (such as in exponential dis- counting) the discount rate reflects consistent impatient preferences. By contrast, in hyperbolic discounting the discount parameter may reflect inconsistencies in these preferences (present bias) and therefore reflect what economists consider impulsivity. In this paper, we attempt to reconcile these insights from economics and bring them to the attention of fields like psychol- ogy, neuroscience, and psychiatry. data collection and analysis, decision to publish, or preparation of the manuscript. data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. With hundreds of publications a year focused on delay discounting, these measurements have now been performed on a wide variety of healthy and clinical populations under many different conditions. Examples of the prevalence of this measure in the literature include large studies and meta-analyses in healthy volunteers [7–13], as well as case-control studies in a vari- ety of patient populations with substance use disorders [14–16], anorexia [17], obesity [18], personality disorders [19], ADHD [20], and anxiety [21–23]. Adding to this growing literature, several groups have investigated the neural basis of temporal discounting [24–29] and others have explored the effect of behavioral or neural manipulations on temporal discounting in healthy volunteers [30–33]. Introduction Time preference, or the preference of typical humans for immediate over delayed rewards, has long been a subject of study in economics, finance, neuroscience, and psychology. Temporal discounting describes this preference mathematically by quantifying how the subjective value of a payoff decreases as the time to its receipt increases. This delay-dependent decrease in 1 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Risk distorts impulsivity studies in the neuroscience and psychology fields, even when both types of preferences are measured in the same individual. Further, because in economics the focus is rarely on the employment of these measures for the study of individual differences, the size of the impact of risk preference on individual discount rate estimates has not been well characterized. In this study, we investigated how individuals’ risk attitudes bias the estimation of their dis- count rates. As our goal is to propose this methodology to the fields that do not already employ it, we used simple standard binary choice tasks widely used in the psychology and neurosci- ence literature [24, 25, 54, 55] in a real-world non-expert sample. We hypothesized that a pro- cedure that estimated individual subject temporal discounting rates, but that also incorporated independently estimated risk attitude parameters, would outperform standard (economic) tools for estimating temporal discounting rates. We found that in our community sample of subjects, where there was a wide diversity of individual risk preferences, our approach showed superior performance in capturing individual intertemporal choice behavior. Unlike previous studies that employed similar methods but that focused on population-level discount rate esti- mates, here we focused on individually estimated parameters. We found that the standard approach introduces a systematic pattern of bias that distorts individual discount rate esti- mates. We conclude that ignoring individuals’ risk attitudes when measuring temporal dis- count rates can significantly impact interpretations about their degree of choice impulsivity. https://doi.org/10.1371/journal.pone.0191357.t001 Values are presented as mean (± 1 standard deviation) unless they are percentages. Race is divided into Caucasian (C) and African American (AA). No other races were present in our sample. Ethnicity is divided into Hispanic and non-Hispanic. The education and income of our sample match median education level and personal income level adjusted for educational attainment in the United States. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 In parallel to using measures of temporal discounting to assess impulsivity, there has been growing interest in the quantitative assessment of risk attitudes by measuring formal risk pref- erences, that is, an individual’s general proneness to or avoidance of risky prospects, in a wide array of populations [34–37]. In expected utility theory and many other cardinal economic theories of choice, risk attitude is associated with the curvature of the utility function. This function can be interpreted as the mapping from the objective amount of a good (or money) to the subjective value derived from obtaining it. In these theories, this function is related to choices over probabilistic outcomes [38, 39]. When a subject’s utility function is linear, she chooses between lotteries as if maximizing expected value (choosing the option for which the product of the value of the prize and its probability is highest); this is often referred to as risk neutrality. When the utility function is concave, subjects are risk averse; when it is convex, sub- jects are risk seeking. Many studies have concluded that there is great diversity in risk prefer- ence and that risk attitudes are affected by age, context and even physiological states like menstrual cycle phase [40–45]. More recently, economists have examined how risk attitudes might introduce possible con- founds to the empirical measurement of discount rates [46–48]. The key ideas in this literature are: first, that because the future is inherently uncertain, risk attitude must play a role when evaluating future prospects irrespective of their time preferences [49, 50]; and second, that the preference for smaller-sooner rewards may be driven by either impatience (what in psychology is considered the “choice impulsivity” dimension of impulsivity) or by diminishing marginal utility as captured by nonlinearities in the utility function [51, 52]. Although accounting for risk preferences when estimating discount rates is of growing importance in the economics and management fields, with few exceptions [26, 53], it is not a practice that has fully impacted 2 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Intertemporal choice (ITC) task The task was a two alternative forced choice task consisting of 102 trials that presented two options, one monetary reward to be received on that day and one monetary reward to be received with variable delay (in days). On each trial, both the fixed immediate and delayed options were presented side by side on the screen. The range of rewards across both periods went from $2 to $66. The immediate reward was either $2, $5 or $15, and the delayed reward was always a larger amount in the following increment levels: for trials with $2 immediate reward, +$5, +$10, +$20, +$40, +$64; for trials with $5 immediate reward, +$5, +$10, +$20, + $40, +$60; and for trials with $15 immediate reward, +$5, +$10, +$20, +$40, +$50. The actual delayed alternative presented was the result of the exact given increment level or plus or minus $1. Possible delays were 5, 10, 30, 60, 90, 120 and 150 days. The actual delay presented corre- sponded to the stated delays in days or plus or minus one day. For example, one of the trials was a choice between $5 today and $66 in 89 days. The selected choice set allowed for a very distributed investigation of the space to ensure our ability to estimate very high or very low dis- count rates with equivalent precision. Risk attitude (RA) task The task consisted of 64 lottery choices in the gain domain. Each trial involved a choice between a fixed amount of money ($5 for sure) and a lottery with the probability level associ- ated with winning a (usually higher) amount changing from trial to trial. Each lottery had two possible outcomes: $v or $0. The exact amounts of v were: $5, $6, $7, $8, $9, $10, $12, $14, $16, $18, $20, $23, $26, $30, $34, $39, $44, $50, $57, and $66. We used three winning probabilities, p, 25%, 50%, and 75%. In this case, each lottery can be fully described by v and p. Each amount v was presented with each probability level once in random order over 4 blocks of 16 trials. In addition, 4 “catch” trials were included at the start of each block. These trials always presented a choice between $5 for sure vs. 50% chance of $4 or $0. Thus, these trials in addition to trials that offered risky lotteries where v was $5 (10 in total), allowed us to assess the frequency of first order stochastic dominance violations, that is, whether subjects chose the objectively worse of the two options. We considered two or more of such violations as evidence that we could not reliably model subjects’ choices with a monotonic utility function. Both the fixed $5 and the lottery were presented side by side on the screen. Subjects were told that each lottery image represents a physical paper bag that contains 100 poker chips, some red and some blue. Subjects were told the precise number of red and blue chips in the bag by explicitly showing the number and by coloring parts of the image according to the proportion of red and blue chips. Materials and methods Subjects All participants gave written informed consent in accordance with the procedures of the Uni- versity Committee on Activities Involving Human Subjects of New York University and the Institutional Review Board of the New York University School of Medicine, which approved this study. We recruited 56 medically healthy participants (11 women) from the general com- munity (via flyers, internet advertisement and word-of mouth) without significant history of substance use or psychiatric illness. Subjects’ demographic information including average edu- cation level, income level, employment and race and ethnicity breakdown is presented in Table 1. Table 1. Sample demographics. Gender (% males) 73.3% Age (years) 44.04 (12.4) Nonverbal I.Q. 91 (2.5) Education (years) 13.8 (2.0) Unemployed (%) 14% Income (monthly $) 1770.49 (263.73) Race (% C—% AA) 47.6—52.4 Ethnicity (% Hispanic) 14.3 Table 1. Sample demographics. Values are presented as mean (± 1 standard deviation) unless they are percentages. Race is divided into Caucasian (C) and African American (AA). No other races were present in our sample. Ethnicity is divided into Hispanic and non-Hispanic. The education and income of our sample match median education level and personal income level adjusted for educational attainment in the United States. Values are presented as mean (± 1 standard deviation) unless they are percentages. Race is divided into Caucasian (C) and African American (AA). No other races were present in our sample. Ethnicity is divided into Hispanic and non-Hispanic. The education and income of our sample match median education level and personal income level adjusted for educational attainment in the United States. https://doi.org/10.1371/journal.pone.0191357.t001 3 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Risk distorts impulsivity Session description After collecting pertinent contact and demographic information, subjects completed the inter- temporal choice task and the risk task. The order of the tasks was randomized across subjects and sessions. Both tasks were computerized (Psychtoolbox for MatLab and e-prime 2.0) and were completed in a private testing room. Subjects were given extensive instructions as well as some practice trials to ensure they understood the tasks fully before beginning. Subjects com- pleted 2 sessions separated by at least one week. RA task analysis To quantify technical risk attitude for each session, we used a power utility model to fit the choice data from the RA task as we have described previously [40, 41, 43, 54, 56]. In this model, the utility (U) of each option (safe or lottery) is given by: UðoptionÞ ¼ pva ð1Þ ð1Þ where v is the dollar amount, p is the probability of winning, and α is the curvature of the util- ity function which serves as a subject-specific measure of technical risk attitude. A subject whose α = 1 has a linear utility function and is thus risk neutral. A subject whose α > 1 has a convex utility function and is thus risk-seeking (reflecting a tolerance of risk and uncertainty). A subject whose α < 1 has a concave utility function and is thus risk-averse (reflecting a dis- taste for risk and uncertainty). Using maximum likelihood estimation in MATLAB, we fit a single logistic function to the trial-by-trial choice data of each subject: PrðlotteryÞ ¼ 1 = ð1 þ egðUlottery UsafeÞÞ ð2Þ ð2Þ where Pr is the probability that the subject chose the lottery option on a given trial, Usafe and Ulottery are the utilities (subjective values) of the safe and lottery options, respectively, and γ is the slope of the logistic function, which is a second subject-specific parameter. The parameter γ captures the stochasticity (as it is related to the randomness of the choice data). Incentive compatibility and payment We compensated subject participation with a $10 fee and a bonus. At the end of the session, one choice from either the ITC or RA task was randomly selected to determine this bonus. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 4 / 18 Risk distorts impulsivity This ensures that subjects’ decisions were incentive compatible: they do not know which choice will count so their best strategy is to treat each one as if it were the one that counts. Pay- ment for both the participation fee and the bonus was made via money order in the following way: subjects received a code via a text message to their phone on the day the payment was due, to prevent against subjects forgetting payments, to claim their bonus at their convenience. Critically, because all payments were made this way we introduced no differences in the trans- action costs for different types of payments (participation fee, RA task payment, ITC immedi- ate payment or ITC delayed payment). For ITC delayed payments, subjects received the code on the date corresponding to the delay for that chosen option. Risk distorts impulsivity reference in the text. In this model, the utility (U) of each option is given by: eference in the text. In this model, the utility (U) of each option is given by: UðoptionÞ ¼ vekd ð5Þ ð5Þ The fourth model was a normative exponential discounting model that took into account the curvature of the utility function again by using α. We call this model Nonlinear utility Expo- nential discounting (NLE) for ease of reference in the text. In this model, the utility (U) of each option is given by: UðoptionÞ ¼ vaekd ð6Þ ð6Þ Using maximum likelihood estimation in MATLAB, we fit a single logistic function to the trial-by-trial choice data of each subject: Using maximum likelihood estimation in MATLAB, we fit a single logistic function to the trial-by-trial choice data of each subject: PrðdelayedÞ ¼ 1 = ð1 þ ebðUdelayed UimmediateÞÞ ð7Þ ð7Þ where Pr is the probability that the subject chose the delayed option on a given trial, Uimmediate and Udelayed are the utilities (subjective values) of the immediate and delayed options, respec- tively, and β is the slope of the logistic function, which is another subject-specific parameter. The parameter β captures the stochasticity (as it is related to the randomness of the choice data). where Pr is the probability that the subject chose the delayed option on a given trial, Uimmediate and Udelayed are the utilities (subjective values) of the immediate and delayed options, respec- tively, and β is the slope of the logistic function, which is another subject-specific parameter. The parameter β captures the stochasticity (as it is related to the randomness of the choice data). Model comparison The models were evaluated by comparison of their cross-validated log likelihoods: we com- puted the log likelihood by leave-one-out cross-validation, fitting the model to the data from all the trials except for one. This process was repeated iteratively for each of the trials and the likelihoods were added to compute the final log likelihood. We chose this leave-one-out method to avoid discarding too much data from the estimation process as there were no repli- cates of the trials and the indifference point location could be hard to resolve for some sessions (see S1 Fig for a description and rationale of the choice set.) PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 ITC task analysis We applied four models to quantify subject time preferences. The first model was a non-nor- mative hyperbolic discounting model [57], which assumes an underlying linear utility. We call this model Linear utility Hyperbolic discounting (LH) for ease of reference in the text. In this model, the utility (U) of each option (immediate or delayed) is given by: UðoptionÞ ¼ v = ð1 þ kdÞ ð3Þ ð3Þ where v is the dollar amount of the option, d is the delay to the delivery of v (which is 0 for the immediate option), and κ is the discount parameter which serves as a subject-specific measure of impulsivity. The second model was a non-normative hyperbolic discounting model that took into account the curvature of the utility function as estimated by Eq (1), captured in the parameter α. We call this model Nonlinear utility Hyperbolic discounting (NLH) for ease of reference in the text. In this model, the utility (U) of each option is given by: UðoptionÞ ¼ va = ð1 þ kdÞ ð4Þ ð4Þ The third model was a normative exponential discounting model which assumes an underly- ing linear utility. We call this model Linear utility Exponential discounting (LE) for ease of PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 5 / 18 Results A (top): RA task design, the safe and lottery options are simultaneously displayed Fig 1. RA and ITC task design. A (top): RA task design, the safe Fig 1. RA and ITC task design. A (top): RA task design, the safe and lottery options are simultaneously displayed during the decision phase. A green dot cues the response time. A yellow square provides feedback on the choice entered. A variable inter-trial interval (ITI) follows. A (bottom): ITC task design, the immediate and delayed options simultaneously displayed during the decision phase. The offer disappears and a white dot cues the response time. A white check mark provides feedback on the choice entered. A variable ITI ensues. For a description of the choice set (see S1A and S1B Fig). B (left): distribution of natural logarithm of risk attitude parameter (ln(α)) across all subjects and all sessions. B (right): distribution of natural logarithm of discount parameter (ln(κ)) estimated from the LH model across all subjects and all sessions. Fig 1. RA and ITC task design. A (top): RA task design, the safe and lottery options are simultaneously displayed during the decision phase. A green dot cues the response time. A yellow square provides feedback on the choice entered. A variable inter-trial interval (ITI) follows. A (bottom): ITC task design, the immediate and delayed options simultaneously displayed during the decision phase. The offer disappears and a white dot cues the response time. A white check mark provides feedback on the choice entered. A variable ITI ensues. For a description of the choice set (see S1A and S1B Fig). B (left): distribution of natural logarithm of risk attitude parameter (ln(α)) across all subjects and all sessions. B (right): distribution of natural logarithm of discount parameter (ln(κ)) estimated from the LH model across all subjects and all sessions. https://doi.org/10.1371/journal.pone.0191357.g001 https://doi.org/10.1371/journal.pone.0191357.g001 Diversity in risk preferences We found that our sample exhibited very diverse risk preferences. We fit the power utility model shown in Eq (1) to the RA task choice data from each session using the softmax decision rule in Eq (2). We estimated a subject and session-specific RA parameter (α). This parameter ranged from 0.1 to 2.173, as shown in Fig 1B (note that values in those distributions are natural log-transformed). The average RA parameter (α) was 0.7508, and the median was 0.6087. Note that although on average our sample exhibited risk aversion (α < 1), individual risk prefer- ences were heterogenous: there was a wide range of α values with many subjects deviating far from risk neutrality (α = 1, or 0 in the log space shown in Fig 1B). Results By design, our subjects exhibited a wide demographic diversity and were representative of the general urban population (see Table 1). None of our subjects were students or had any advanced knowledge of finance or previous experience with the type of tasks used in this study. Subjects performed two decision-making tasks during each session, an intertemporal choice (ITC) task and a risk attitude (RA) task (see Fig 1A). We recruited 56 subjects to com- plete 2 identical testing sessions to allow us to assess test-retest reliability. We found that for our tools and subject sample, reliability was high (intraclass correlation coefficient for risk atti- tude and discount parameters r > 0.54, p < 0.05). For our analyses, we excluded any sessions for which the goodness-of-fit (R2) for the risk attitude parameter estimation was lower than 0.4. We also excluded sessions in which subjects always selected one of the options in the ITC task as this makes it impossible to resolve their indifference point with our choice set. After these exclusions, we analyzed a total of 78 sessions (2 sessions for each of 39 subjects). In most of the following analyses, each session is regarded as a separate data point. Since we do not per- form group comparisons between subjects (all comparisons are of different model fits within subjects), we note that there are no statistical implications from treating the data in this manner. 6 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Risk distorts impulsivity Fig 1. RA and ITC task design. A (top): RA task design, the safe and lottery options are simultaneously displayed during the decision phase. A green dot cues the response time. A yellow square provides feedback on the choice entered. A variable inter-trial interval (ITI) follows. A (bottom): ITC task design, the immediate and delayed options simultaneously displayed during the decision phase. The offer disappears and a white dot cues the response time. A white check mark provides feedback on the choice entered. A variable ITI ensues. For a description of the choice set (see S1A and S1B Fig). B (left): distribution of natural logarithm of risk attitude parameter (ln(α)) across all subjects and all sessions. B (right): distribution of natural logarithm of discount parameter (ln(κ)) estimated from the LH model across all subjects and all sessions. https://doi org/10 1371/journal pone 0191357 g001 Fig 1. RA and ITC task design. Taking risk preferences into account improves the fit to ITC choice data We used maximum-likelihood estimation to fit four different models to each individual ses- sion ITC choice data: two hyperbolic discounting models and two exponential discounting models. We focused on these two classes of models because they are the most prevalent in the literature. Exponential discounting provides a normative account of discounting grounded in discounted utility theory. Hyperbolic discounting often provides a better fit to behavioral human and animal data [3, 58]. Of the two hyperbolic models, one did not take risk attitude into account (assumed a linear utility function, LH) as in Eq (3) and the other (NLH) used the estimated risk attitude (the utility function curvature parameter α from Eq (1)) as in Eq (4). Similarly, for the exponential type, one model assumed a linear utility function (LE) as in Eq (5), and the other (NLE) used the α as shown in Eq (6). To evaluate these four models, we com- pared their cross-validated log likelihoods (LL). Note that all four models have the same num- ber of free parameters because the α parameter in the models that incorporated a risk attitude estimate (NLH and NLE) was fixed, taken from the maximum likelihood estimation procedure performed on independent data from the RA task (Eq (1)). We employed cross-validation to avoid over-fitting by iteratively fitting the model on all trials but one and computing the log likelihood of the model for the left-out trial (see Materials and methods section). We found that the LL was higher for the model that included an independent estimate of risk when estimating a hyperbolic discount parameter (NLH) than for the model that did not (LH), for the majority of the sessions in our data set (Fig 2A). Similarly, we found that the model that included an independent estimate of risk when estimating an exponential discount parameter (NLE) had a higher LL than did a model that omitted this term (LE) for the majority of the sessions in our data set (Fig 2B). Interestingly, the two models that included an estimate of risk attitude (NLH and NLE) performed similarly well, suggesting that in our data good- ness-of-fit does not rely on exponential or hyperbolic assumptions (Fig 2C). AIC and BIC scores were also compared and yielded similar results to our cross-validated LL comparison (see S2 Fig). Diversity in time preferences To evaluate the diversity of time preferences in our sample, we first ignored risk preferences and estimated discount parameters using standard methods (effectively assuming risk neutral- ity in all subjects.) To do so, we fit the “Linear utility Hyperbolic discounting” model (LH) shown in Eq (3) to each session’s ITC choice data using the softmax decision rule in Eq (7). 7 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Risk distorts impulsivity We estimated a subject and session-specific ITC discount parameter (κ). The distribution of subject discount parameters is shown in Fig 1B (note that values in those distributions are nat- ural log-transformed). Values ranged from 0.001 (equivalent to the discounting of 2.9% of the reward’s value after a delay of one month) to 6.4 (equivalent to the discounting of 99.5% of the reward’s value after one month). The mean κ was 0.3139 and the median was 0.0499 (equiva- lent to the loss of 60% of the reward’s value after one month). PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Taking risk preferences into account improves the fit to ITC choice data B: LL comparison of model LE against model NLE. Panel C, LL comparison of model NLE against model NLH. Shaded areas for panels A and B correspond to sessions for which the nonlinear utility models fit the data better than the linear utility models. C: Shaded area corresponds to sessions for which the NLH model fit the data better than the NLE model. D: average difference in LL across all sessions between model NLH and model LH (dark color), between model NLE and model LE (intermediate color) and, between model NLH and model NLE (light color), black bars indicate S.E.M. https://doi.org/10.1371/journal.pone.0191357.g002 generated from NLH itself and not from the other models. We therefore focus on the NLH model’s superiority for the rest of our analyses. Taking risk preferences into account improves the fit to ITC choice data We note that for both the hyperbolic and exponential forms, the advantage in goodness-of-fit of the models that account for risk (NLH relative to LH and NLE relative to LE) was correlated with the discount parameter. However, this correlation was not only car- ried by outliers as the rank-ordered Spearman coefficient was significant, rho = 0.5, p < 0.001 for the exponential form and rho = 0.52, p < 0.001 for the hyperbolic form. This means that the advantage of NLE and NLH was not only true for impatient outliers. To evaluate the overall performance of the models, we computed the average difference in LL across all sessions and subjects of the LH, LE, and NLE models relative to the LL of the NLH and NLE models (Fig 2D and for all possible comparisons between models see S3 Fig). Across all the data, the risk-incorporating models (NLH and NLE) show overall significantly superior performance than models that did not include risk (LH and LE). The difference in goodness-of-fit between NLH and NLE was not significant. However, when we simulated data generated from each model, NLE proved to be less distinguishable from the other models than NLH (see S2 Fig); the NLE model fits were equally good for data generated from NLE itself as for data generated from other models, whereas NLH fits were significantly better for data 8 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Risk distorts impulsivity Fig 2. Model comparison. A: cross-validated log likelihood (LL) comparison of model LH against model NLH. Each dot corresponds to data from a single subject’s session. B: LL comparison of model LE against model NLE. Panel C, LL comparison of model NLE against model NLH. Shaded areas for panels A and B correspond to sessions for which the nonlinear utility models fit the data better than the linear utility models. C: Shaded area corresponds to sessions for which the NLH model fit the data better than the NLE model. D: average difference in LL across all sessions between model NLH and model LH (dark color), between model NLE and model LE (intermediate color) and, between model NLH and model NLE (light color), black bars indicate S.E.M. https://doi.org/10.1371/journal.pone.0191357.g002 Fig 2. Model comparison. A: cross-validated log likelihood (LL) comparison of model LH against model NLH. Each dot corresponds to data from a single subject’s session. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Failing to measure risk attitude systematically deviates estimates of discount parameter Having established that model NLH provides a better account of the data than models that do not incorporate risk attitude, we next examined the magnitude and direction of the misestima- tion when risk preference was ignored. We compared the individual discount parameter esti- mates (κ) obtained from model LH (the most commonly used model in the psychology literature) and those obtained with model NLH. We found that the two discount parameter estimates indeed differed for most of our subjects (Fig 3A): for the large majority of subject ses- sions the discount parameter from model LH was higher than the discount parameter from model NLH. We next tested whether the difference in estimated discount parameters depended systematically on risk preference (captured by the α parameter). We simulated 100 data sets using our NLH model with values for the risk parameter samples drawn from a uni- form distribution within the range of our RA task choice set. This allowed us to cover the space of α fully, given that in our sample (as in most reported studied subjects in the literature) PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 9 / 18 Risk distorts impulsivity Fig 3. A systematic bias in discount parameters. A: comparison of estimated discount parameters from model LH against model NLH for each subject’s sessions presented as natural logarithm of discount parameter (ln(κ)). B: discount parameter bias computed as difference between the natural log of estimated parameters from model LH against model NLH (ln(κ)LH −ln(κ)NLH), plotted as a function of the corresponding risk attitude parameter (α), dark dots represent data from each of our subjects’ sessions, gray dots represent simulated data. C: difference of goodness of fit (LL from NLH—LL from LH) between NLH and LH model as a function of the absolute value of the natural logarithm of α (\|ln(α)\|), risk neutrality here is 0 and any value above 0 is either risk averse or risk seeking. Spearman correlation: rho = 0.297, p < 0.01. Shaded area corresponds to the sessions for which model NLH fit the data better than model LH. D: correlation between the natural logarithm of the risk attitude parameter (ln(α)) and the natural logarithm of the discount parameter and model NLH (dark color). https://doi.org/10.1371/journal.pone.0191357.g003 Fig 3. A systematic bias in discount parameters. A: comparison of estimated discount parameters from mode Fig 3. A systematic bias in discount parameters. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Discussion We show here that 1) a temporal discounting model that incorporates an individual out-of- sample estimate for risk attitude outperforms models that assume risk neutrality (Fig 2), and 2) that failing to incorporate this risk attitude estimate leads to systematically biased discount parameters that entangle risk attitude and choice impulsivity (as in Fig 3); risk-averse subjects appear more impulsive than they are. The finding that risk attitude interacts with intertemporal preference is not unexpected. Recent advances in economic research focusing on time preferences have been prompted by the potential confounding influence of non-linear utility upon estimates of discount parameter [59]. Studies devoted to this issue have applied either joint elicitation techniques of the kind used in our study (where risk preferences and time preferences are estimated from separate choice tasks) or methods where both utility and discounting are elicited from intertemporal choices [60, 61]. There is still no theoretical consensus among economists on the correct inter- pretation of the relationship between utility for risk and instantaneous utility for time [62] and, for some, subjective valuation of temporal payoffs may differ from that of risky ones [60, 63–65], but see [66]. However, from an empirical point of view, our study contributes to the growing number of reports that indicate there is a systematic bias in the discount parameter estimates when risk attitude is ignored. This dependence can be observed even in simple and widely employed binary choice tasks and at the individual subject level. y p y y j While we recapitulate the results reported by previous studies coming from economics and finance, the novel contribution of our work lies in two keys aspects: First, unlike previous stud- ies we do not aggregate the subject data and estimate a single group parameter for the entire sample. Instead, we capitalize on the diversity of individual preferences by independently esti- mating a risk aversion parameter and a discount parameter for each subject’s session. This diversity is the result of our rich community sample which is representative of the average urban dweller. With some notable exceptions [51], the majority of previous studies on this topic have been performed with expert samples such as economics or finance students, which may lack the diversity in demographics that would allow for inferences about how these deci- sions are made by typical people. Risk distorts impulsivity Importantly, the NLH model also resulted in the orthogonalization of the risk attitude parame- ter α and the discount parameter κ, while α and κ derived from model LH were significantly neg- atively correlated (Fig 3D). For model LH, the Pearson correlation coefficient was r = -0.451, p = 0.0001. Conversely, the correlation between α and the κ derived from model NLH was not significant (Pearson’s r = -0.02, p = 0.861). Taken together, these results show that the NLH model yields a discount parameter that is not only more precise but also allows for κ and α to reflect distinct aspects of decision-making rather than being conflated as is the case with esti- mates derived from the traditional model LH. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Failing to measure risk attitude systematically deviates estimates of discount parameter A: comparison of estimated discount parameters from model LH against model NLH for each subject’s sessions presented as natural logarithm of discount parameter (ln(κ)). B: discount parameter bias computed as difference between the natural log of estimated parameters from model LH against model NLH (ln(κ)LH −ln(κ)NLH), plotted as a function of the corresponding risk attitude parameter (α), dark dots represent data from each of our subjects’ sessions, gray dots represent simulated data. C: difference of goodness of fit (LL from NLH—LL from LH) between NLH and LH model as a function of the absolute value of the natural logarithm of α (\|ln(α)\|), risk neutrality here is 0 and any value above 0 is either risk averse or risk seeking. Spearman correlation: rho = 0.297, p < 0.01. Shaded area corresponds to the sessions for which model NLH fit the data better than model LH. D: correlation between the natural logarithm of the risk attitude parameter (ln(α)) and the natural logarithm of the discount parameter and model NLH (dark color). https://doi.org/10.1371/journal.pone.0191357.g003 most subjects are risk averse (α < 1). We observed that the difference in discount parameter estimates obtained for these synthetic data sets (κ from LH—κ from NLH) led to a systematic shift in the estimated discount parameter as a function of the risk preference parameter (α) (see gray points in Fig 3B). We also computed the difference in discount parameter estimates from both models for our subject data and saw the same trend we observed for simulated data (see dark points in Fig 3B): When the α parameter is less than 1 (risk aversion) the κ parameter estimated by LH is higher than that of NLH. Thus, the standard method employed by most studies leads errone- ously to the conclusion that risk-averse individuals are more impulsive than they really are. Con- versely, when the α parameter is higher than 1 (risk tolerant) the κ parameter estimated by LH is lower than that of NLH: risk-seeking individuals appear to be less impulsive than they really are. Logically, when α = 1 NLH and LH converge to the same functional form and the discount parameter estimates are identical. Further, the larger the deviation from risk neutrality, the more model NLH outperforms LH, suggesting that any individuals with non-neutral risk pref- erences are much better captured by our model NLH (Fig 3C). 10 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Risk distorts impulsivity any higher education (in our sample the average education level is 13.8 years, see Table 1) and without any sophisticated understanding of interest rates and finance in general. To establish whether the diversity in risk preferences has implications for how well models of temporal discounting fit individual subject choice data, we compared four models that are used in this growing literature. Two of these models assume that subjects are risk neutral: the linear hyperbolic (LH) and exponential (LE) models. The two other models incorporate sub- ject-specific risk attitudes, that is, the curvature of the individual’s utility function. The LH model is the most widely used in the psychology and neuroscience literature to parametrically investigate discounting as a measure of impulsivity. It has been shown to fit behavioral data better than the normative exponential (LE) alternative, which often fails to fit especially highly impulsive subjects’ data. The superiority of LH over LE has usually been linked to the fact that subjects exhibit a “present bias”, that is, the tendency to give stronger weight to payoffs that are closer to the present time when considering trade-offs between two future moments. As such, subjects’ preferences have been reported to be better modeled by a hyperbolic rather than an exponential decay function. We recapitulated that result in our sample but found that our NLH model fit the intertemporal data better than both LH and LE across most of our subjects and sessions, suggesting taking risk into account is important regardless of where one sits in the exponential versus hyperbolic debate. Interestingly, NLH and NLE had similar perfor- mance. Consistent with previous studies [51], accounting for risk in the exponential form (NLE) resulted in a significantly better fit than the hyperbolic form that did not (LH) (see S3 Fig), which suggests that discounting becomes more constant when risk is considered. However, NLE seems to be a less precise model at distinguishing between LE and NLE gener- ated data, while our model NLH was more specific (Fig 2 and S2 Fig). This means that NLE is too flexible, fitting data equally well even when it comes from different generative models. By contrast, NLH was a more selective model, a feature that may be more desirable for fields such as neuroscience that seek the true (neural) generative mechanism behind the behavior exhib- ited by the decision maker. Discussion If not enough variance in risk preferences is achieved in the studied sample, it is possible the effect of risk may not be fully observable. We found wide vari- ability in parametric risk attitudes in our sample, despite the mean not being much different from what has been previously reported in the literature. Furthermore, we simulated data with higher risk-seeking preferences to explore what the bias in the discount parameter would be in that direction. Although not often seen in healthy volunteers, risk-seeking behavior is more prevalent in psychiatric conditions [67–72], making this bias relevant for these types of studies. Second, we employed widely used binary choice tasks that can easily be completed by any sub- ject and do not require any explicit knowledge of finance. We believe this is important if these assessments are to be deployed across different types of populations, including those without PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 11 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Risk distorts impulsivity that temporal discounting could provide a behavioral marker for psychiatric diagnosis and prognosis [75, 76]. Transforming neural measurements of temporal discounting into a valid biomarker of clinical utility for psychiatry supposes that it accurately measures the intended biological and behavioral process [77, 78]. Our study shows that incorporating risk attitude is critical to delineating this process with precision. that temporal discounting could provide a behavioral marker for psychiatric diagnosis and prognosis [75, 76]. Transforming neural measurements of temporal discounting into a valid biomarker of clinical utility for psychiatry supposes that it accurately measures the intended biological and behavioral process [77, 78]. Our study shows that incorporating risk attitude is critical to delineating this process with precision. As shown in Fig 2 (and S3 Fig) we found a clear benefit of taking risk preferences into account. While several psychiatric disorders are characterized by exhibiting extreme attitudes toward taking risks, most clinical studies that have examined impulsivity in these populations have not simultaneously assessed risk preference and have not done so in an incentive-com- patible manner. For example, an implication of doing so could be that differences in discount- ing could be underestimated if in fact there are larger differences in risk preferences, e.g. if the control sample is significantly more risk averse than the clinical sample. Similarly, differences in risk preferences could be interpreted as differences in discounting. A few psychological studies in problem gamblers and alcoholics have performed both measures of temporal and probability discounting [79, 80]. Although these studies employ tasks very similar to our RA task for the estimation of probability discounting rates, they have not explicitly resolved the curvature of the utility function and therefore have not corrected for it in their estimation of discount rates. This would prove fairly easy to do using the methodology we propose here. The impact of ignoring risk preferences may reach beyond studies of clinical populations. In the social decision-making field for example, studies have suggested that discounting may be related to lack of cooperation [81] and to the willingness to punish other free-riders and non- cooperators [82, 83]. However, few of these studies have controlled for risk preferences, which may be important given that these seem to also correlate with individuals’ willingness to coop- erate [84]. Temporal discounting is considered to be one of the many dimensions of the impulsivity personality construct. In the psychology literature, risk-seeking aspects of personality are often included in descriptions of impulsivity but there have been many attempts to separate these dimensions. Our approach results in the decorrelation of risk preference and time preference parameters (Fig 3) and allows for these parameters to reflect different aspects of decision-mak- ing and personality. We believe this separation could result in discount parameter estimates that may be more meaningful than those obtained using traditional methods. We propose a methodology that better resolves individual estimates of impulsivity and that could be easily incorporated in the growing literature on impulsivity by researchers interested in individual differences and behavioral phenotyping in clinical samples. We have shown that ignoring risk attitude results in a systematic bias in the discount parameter estimates (Fig 3). For a large range of α values close to 1, LH and NLH do not per- form differently, but the more risk averse or the more risk-seeking an individual, the more our approach outperforms the traditional approach. One clear implication of using the LH model in subjects that exhibit widely varying risk attitudes is that discount parameter estimates may be biased. We showed that when subjects are risk averse, the LH model returns a higher κ esti- mate value than NLH and conversely, when subjects are risk-seeking, LH underestimates the discount parameter. If one were comparing risk-averse individuals (e.g., the average healthy person) to less risk averse individuals (e.g., problem gamblers) the standard approach might lead to conclusions about differences in choice impulsivity between these groups that might be (perhaps better) explained by differences in risk attitude. Some studies have already begun to suggest that reported differences in discount rates between substance users and controls may be inflated by the use of the LH model [73]. This could also prove relevant for the comparison between patients with anxiety disorders (who have been reported to be highly risk-averse [74]) and healthy controls. Of particular interest to us is the fact that investigations into the neural correlates of utility (or subjective value) and of intertemporal choice have also shown that the shape of this func- tion is important when computing discounted subjective value [26]. We found that the NLH model not only fits behavioral data better but also is the most specific model (see S2 Fig), sug- gesting that the NLH model could be the best approximation to the brain’s generating model, a question that remains to be tested in future studies. This is relevant for any study that attempts to tie time preference behavior to neural activity. For instance, it has been suggested PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 12 / 18 Supporting information S1 Fig. Choice set space and example subject’s choices. A) Visualization of the ITC task trial space. Each dot is a trial composed of an offered immediate (no delay) monetary amount, a larger monetary amount to be delivered with a delay, and the delay to its delivery (in days). Shaded plains correspond to trials where the immediate monetary amount is the same, B) The same trial space displaying the choices made by an example subject. Blue dots correspond to the trials where the subject chose the immediate payment option and pink dots correspond to the trials where the subject chose the delayed payment. The boundary between pink and blue dots reflects the location of the indifference points in the space. Shaded plains correspond to trials where the immediate monetary amount is the same. (TIF) S2 Fig. Model recovery analysis. Synthetic datasets were generated from each of the four models, LH, NLH, LE and NLE and then fitted with each model. Each cell in this matrix PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 13 / 18 Risk distorts impulsivity represents: AIC Fitted model—AIC True model. This difference equals 0 when the data gener- ated from a model is fit with the same generative model. The larger the difference the worse the fitted model’s performance with respect to the generative model. Model LH is not a very discriminative model, models LE and NLE are especially bad a discriminating between each other. Out of the four models, model NLH is the most discriminative and the only that is sig- nificantly superior in goodness-of-fit to all other ones. (TIF) S3 Fig. Cross comparison of all models. Cells indicate medians and 95% CI of bootstrapped log likelihood (LL) score differences. A positive median (in red) indicates that the model in the corresponding row had a higher score (better fit) than the model in the corresponding col- umn. (TIF) S3 Fig. Cross comparison of all models. Cells indicate medians and 95% CI of bootstrapped log likelihood (LL) score differences. A positive median (in red) indicates that the model in the corresponding row had a higher score (better fit) than the model in the corresponding col- umn. (TIF) Author Contributions Conceptualization: Silvia Lopez-Guzman, Paul W. Glimcher. Data curation: Silvia Lopez-Guzman, Anna B. Konova. Data curation: Silvia Lopez-Guzman, Anna B. Konova. Formal analysis: Silvia Lopez-Guzman. Investigation: Silvia Lopez-Guzman, Anna B. Konova, Kenway Louie, Paul W. Glimcher. Investigation: Silvia Lopez-Guzman, Anna B. Konova, Kenway Louie, Paul W. Glimcher. Methodology: Silvia Lopez-Guzman, Paul W. Glimcher. Methodology: Silvia Lopez-Guzman, Paul W. Glimcher. Methodology: Silvia Lopez-Guzman, Paul W. Glimcher. Writing – original draft: Silvia Lopez-Guzman. Writing – original draft: Silvia Lopez-Guzman. Writing – review & editing: Silvia Lopez-Guzman, Anna B. Konova, Kenway Louie, Paul W. Glimcher. Writing – review & editing: Silvia Lopez-Guzman, Anna B. Konova, Kenway Louie, Paul W. Glimcher. Acknowledgments The authors wish to acknowledge Jeff Dennison, John Messinger, Adelya Urmanche and Yuqi Yan for their help with the majority of the data collection. They would also like to thank Jan Zimmermann, Christopher Steverson and Will Adler for their invaluable input in analysis and interpretation. S.L.G. was supported by a fellowship from the U.S. Fulbright Commission and the Colombian government. A.B.K. was supported by a Ruth L. Kirschstein National Service Award (NRSA) from the National Institute on Drug Abuse (F32DA039648). This project was supported by a National Institute on Drug Abuse grant (5R01DA038063). PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 References 1. Myerson J, Green L. Discounting of delayed rewards: Models of individual choice. Journal of the Experi- mental Analysis of Behavior. 1995; 64(3):263–276. https://doi.org/10.1901/jeab.1995.64-263 PMID: 16812772 2. Benhabib J, Bisin A, Schotter A. Present-bias, quasi-hyperbolic discounting, and fixed costs. Games and Economic Behavior. 2010; 69(2):205–223. https://doi.org/10.1016/j.geb.2009.11.003 3. Green L, Fristoe N, Myerson J. Temporal discounting and preference reversals in choice between delayed outcomes. Psychonomic Bulletin & Review. 1994; 1(3):383–389. https://doi.org/10.3758/ BF03213979 4. Baumann AA, Odum AL. Impulsivity, risk taking, and timing. Behav Processes. 2012; 90(3):408–14. https://doi.org/10.1016/j.beproc.2012.04.005 PMID: 22542458 5. Stevens JR. The Many Faces of Impulsivity. In: Impulsivity. Nebraska Symposium on Motivation. Springer, Cham; 2017. p. 1–6. Available from: https://link.springer.com/chapter/10.1007/978-3-319- 51721-6_1. 14 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Risk distorts impulsivity 6. Hamilton KR, Mitchell MR, Wing VC, Balodis IM, Bickel WK, Fillmore M, et al. Choice impulsivity: Defini- tions, measurement issues, and clinical implications. Personality Disorders. 2015; 6(2):182–198. https://doi.org/10.1037/per0000099 PMID: 25867841 7. Silverman IW. Gender Differences in Delay of Gratification: A Meta-Analysis. Sex Roles. 2003; 49(9- 10):451–463. https://doi.org/10.1023/A:1025872421115 8. de Wit H, Flory JD, Acheson A, McCloskey M, Manuck SB. IQ and nonplanning impulsivity are indepen- dently associated with delay discounting in middle-aged adults. Personality and Individual Differences. 2007; 42(1):111–121. https://doi.org/10.1016/j.paid.2006.06.026 9. Steel P. The nature of procrastination: A meta-analytic and theoretical review of quintessential self-reg- ulatory failure. Psychological Bulletin. 2007; 133(1):65–94. https://doi.org/10.1037/0033-2909.133.1.65 PMID: 17201571 10. Cross CP, Copping LT, Campbell A. Sex differences in impulsivity: A meta-analysis. Psychological Bul- letin. 2011; 137(1):97–130. https://doi.org/10.1037/a0021591 PMID: 21219058 11. Fields SA, Lange K, Ramos A, Thamotharan S, Rassu F. The relationship between stress and delay discounting: a meta-analytic review. Behavioural Pharmacology. 2014; p. 1. https://doi.org/10.1097/ FBP.0000000000000044 12. Wesley MJ, Bickel WK. Remember the future II: meta-analyses and functional overlap of working mem- ory and delay discounting. Biol Psychiatry. 2014; 75(6):435–48. https://doi.org/10.1016/j.biopsych. 2013.08.008 PMID: 24041504 13. Perales JC, Verdejo-Garcia A, Moya M, Lozano O, Perez-Garcia M. Bright and dark sides of impulsivity: performance of women with high and low trait impulsivity on neuropsychological tasks. Journal of Clini- cal and Experimental Neuropsychology. 2009; 31(8):927–944. https://doi.org/10.1080/ 13803390902758793 PMID: 19358009 14. Amlung M, Vedelago L, Acker J, Balodis I, MacKillop J. Steep delay discounting and addictive behavior: a meta-analysis of continuous associations. Addiction. 2017; 112(1):51–62. https://doi.org/10.1111/ add.13535 PMID: 27450931 15. Contreras-Rodrı´guez O, Albein-Urios N, Vilar-Lo´pez R, Perales JC, Martı´nez-Gonzalez JM, Ferna´n- dez-Serrano MJ, et al. References https://doi.org/10.1016/j.tics.2011.03.002 PMID: 21497544 28. Sripada CS, Gonzalez R, Phan KL, Liberzon I. The neural correlates of intertemporal decision-making: contributions of subjective value, stimulus type, and trait impulsivity. Hum Brain Mapp. 2011; 32 (10):1637–48. https://doi.org/10.1002/hbm.21136 PMID: 20886577 29. Volkow ND, Baler RD. NOW vs LATER brain circuits: implications for obesity and addiction. Trends in Neurosciences. 2015; 38(6):345–352. https://doi.org/10.1016/j.tins.2015.04.002 PMID: 25959611 30. Cho SS, Ko JH, Pellecchia G, Van Eimeren T, Cilia R, Strafella AP. Continuous theta burst stimulation of right dorsolateral prefrontal cortex induces changes in impulsivity level. Brain Stimulation. 2010; 3 (3):170–176. https://doi.org/10.1016/j.brs.2009.10.002 PMID: 20633446 31. Figner B, Knoch D, Johnson EJ, Krosch AR, Lisanby SH, Fehr E, et al. Lateral prefrontal cortex and self-control in intertemporal choice. Nature Neuroscience. 2010; 13(5):538–539. https://doi.org/10. 1038/nn.2516 PMID: 20348919 32. Koffarnus MN, Jarmolowicz DP, Mueller ET, Bickel WK. Changing delay discounting in the light of the competing neurobehavioral decision systems theory: a review. J Exp Anal Behav. 2013; 99(1):32–57. https://doi.org/10.1002/jeab.2 PMID: 23344987 33. Pickens CL, Airavaara M, Theberge F, Fanous S, Hope BT, Shaham Y. Neurobiology of the incubation of drug craving. Trends Neurosci. 2011; 34(8):411–20. https://doi.org/10.1016/j.tins.2011.06.001 PMID: 21764143 34. Cardenas JC, Carpenter J. Risk attitudes and economic well-being in Latin America. Journal of Devel- opment Economics. 2013; 103:52–61. https://doi.org/10.1016/j.jdeveco.2013.01.008 35. Kimball MS, Sahm CR, Shapiro MD. Risk Preferences in the PSID: Individual Imputations and Family Covariation. The American economic review. 2009; 99(2):363–368. https://doi.org/10.1257/aer.99.2. 363 PMID: 21132079 36. Reyna VF, Farley F. Risk and Rationality in Adolescent Decision Making: Implications for Theory, Prac- tice, and Public Policy. Psychological Science in the Public Interest. 2006; 7(1):1–44. https://doi.org/10. 1111/j.1529-1006.2006.00026.x PMID: 26158695 37. von Gaudecker HM, van Soest A, Wengstrom E. Heterogeneity in Risky Choice Behavior in a Broad Population. American Economic Review. 2011; 101(2):664–694. https://doi.org/10.1257/aer.101.2.664 38. Neumann Jv, Morgenstern O. Theory of Games and Economic Behavior (2007 Commemorative Edi- tion). Princeton University Press; 1944. 39. Kahneman D, Tversky A. Prospect Theory: An Analysis of Decision under Risk. Econometrica. 1979; 47(2):263–291. https://doi.org/10.2307/1914185 40. Gilaie-Dotan S, Tymula A, Cooper N, Kable JW, Glimcher PW, Levy I. Neuroanatomy Predicts Individ- ual Risk Attitudes. Journal of Neuroscience. 2014; 34(37):12394–12401. https://doi.org/10.1523/ JNEUROSCI.1600-14.2014 PMID: 25209279 41. Grubb MA, Tymula A, Gilaie-Dotan S, Glimcher PW, Levy I. Neuroanatomy accounts for age-related changes in risk preferences. Nature Communications. 2016; 7:13822. https://doi.org/10.1038/ ncomms13822 PMID: 27959326 42. Holt CA, Laury, Susan K. Risk Aversion and Incentive Effects. American Economic Review. 2002; 92 (5):1644–1655. https://doi.org/10.1257/000282802762024700 43. References Increased corticolimbic connectivity in cocaine dependence versus pathological gambling is associated with drug severity and emotion-related impulsivity. Addiction Biology. 2016; 21 (3):709–718. https://doi.org/10.1111/adb.12242 PMID: 25818325 16. Contreras-Rodrı´guez O, Albein-Urios N, Perales JC, Martı´nez-Gonzalez JM, Vilar-Lo´pez R, Ferna´n- dez-Serrano MJ, et al. Cocaine-specific neuroplasticity in the ventral striatum network is linked to delay discounting and drug relapse. Addiction (Abingdon, England). 2015; 110(12):1953–1962. https://doi. org/10.1111/add.13076 17. Steinglass JE, Figner B, Berkowitz S, Simpson HB, Weber EU, Walsh BT. Increased Capacity to Delay Reward in Anorexia Nervosa. Journal of the International Neuropsychological Society. 2012; 18 (4):773–780. https://doi.org/10.1017/S1355617712000446 PMID: 22591835 18. Amlung M, Petker T, Jackson J, Balodis I, MacKillop J. Steep discounting of delayed monetary and food rewards in obesity: a meta-analysis. Psychological Medicine. 2016; 46(11):2423–2434. https://doi.org/ 10.1017/S0033291716000866 PMID: 27299672 19. Andrade LF, Riven L, Petry NM. Associations between Antisocial Personality Disorder and Sex on Dis- counting Rates. The Psychological Record. 2014; 64(4):639–644. https://doi.org/10.1007/s40732-014- 0085-0 PMID: 25364042 20. Jackson JNS, MacKillop J. Attention-Deficit/Hyperactivity Disorder and Monetary Delay Discounting: A Meta-Analysis of Case-Control Studies. Biological Psychiatry: Cognitive Neuroscience and Neuroimag- ing. 2016; 1(4):316–325. https://doi.org/10.1016/j.bpsc.2016.01.007 21. Bickel WK, Jarmolowicz DP, Mueller ET, Koffarnus MN, Gatchalian KM. Excessive discounting of delayed reinforcers as a trans-disease process contributing to addiction and other disease-related vul- nerabilities: Emerging evidence. Pharmacology & Therapeutics. 2012; 134(3):287–297. https://doi.org/ 10.1016/j.pharmthera.2012.02.004 22. Story GW, Vlaev I, Seymour B, Darzi A, Dolan RJ. Does temporal discounting explain unhealthy behav- ior? A systematic review and reinforcement learning perspective. Front Behav Neurosci. 2014; 8:76. https://doi.org/10.3389/fnbeh.2014.00076 PMID: 24659960 23. Steinglass JE, Lempert KM, Choo TH, Kimeldorf MB, Wall M, Walsh BT, et al. Temporal discounting across three psychiatric disorders: Anorexia nervosa, obsessive compulsive disorder, and social anxi- ety disorder. Depression and Anxiety. 2016; p. 1–11. https://doi.org/10.1002/da.22586 24. Kable JW, Glimcher PW. The neural correlates of subjective value during intertemporal choice. Nat Neurosci. 2007; 10(12):1625–33. https://doi.org/10.1038/nn2007 PMID: 17982449 15 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Risk distorts impulsivity 25. McClure SM, Laibson DI, Loewenstein G, Cohen JD. Separate neural systems value immediate and delayed monetary rewards. Science. 2004; 306(5695):503–7. https://doi.org/10.1126/science.1100907 PMID: 15486304 26. Pine A, Seymour B, Roiser JP, Bossaerts P, Friston KJ, Curran HV, et al. Encoding of marginal utility across time in the human brain. J Neurosci. 2009; 29(30):9575–81. https://doi.org/10.1523/ JNEUROSCI.1126-09.2009 PMID: 19641120 27. Peters J, Bu¨chel C. The neural mechanisms of inter-temporal decision-making: understanding variabil- ity. Trends Cogn Sci. 2011; 15(5):227–39. References Lazzaro SC, Rutledge RB, Burghart DR, Glimcher PW. The Impact of Menstrual Cycle Phase on Eco- nomic Choice and Rationality. PLoS ONE. 2016; 11(1):e0144080. https://doi.org/10.1371/journal.pone. 0144080 PMID: 26824245 44. Levy I, Snell J, Nelson AJ, Rustichini A, Glimcher PW. Neural representation of subjective value under risk and ambiguity. J Neurophysiol. 2010; 103(2):1036–47. https://doi.org/10.1152/jn.00853.2009 PMID: 20032238 45. Tymula A, Belmaker LAR, Ruderman L, Glimcher PW, Levy I. Like cognitive function, decision making across the life span shows profound age-related changes. Proceedings of the National Academy of Sci- ences. 2013; 110(42):17143–17148. https://doi.org/10.1073/pnas.1309909110 46. Chapman GB. Temporal discounting and utility for health and money. Journal of Experimental Psychol- ogy: Learning, Memory, and Cognition. 1996; 22(3):771–791. https://doi.org/10.1037/0278-7393.22.3. 771 PMID: 8656156 16 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Risk distorts impulsivity 47. Frederick S, Loewenstein G, O’Donoghue T. Time Discounting and Time Preference: A Critical Review. Journal of Economic Literature. 2002; 40(2):351–401. https://doi.org/10.1257/002205102320161311 48. Loewenstein G, Prelec D. Anomalies in Intertemporal Choice: Evidence and an Interpretation. The Quarterly Journal of Economics. 1992; 107(2):573–597. https://doi.org/10.2307/2118482 49. Epper T, Fehr-Duda H. The missing link: Unifying risk taking and time discounting. Department of Eco- nomics—University of Zurich; 2012. 096. Available from: https://ideas.repec.org/p/zur/econwp/096.html. 50. Halevy Y. Strotz Meets Allais: Diminishing Impatience and the Certainty Effect. American Economic Review. 2008; 98(3):1145–1162. https://doi.org/10.1257/aer.98.3.1145 51. Andersen S, Harrison GW, Lau MI, Rutstro¨m EE. Eliciting Risk and Time Preferences. Econometrica. 2008; 76(3):583–618. https://doi.org/10.1111/j.1468-0262.2008.00848.x 52. Ferecatu A, O¨ nc¸u¨ler A. Heterogeneous risk and time preferences. Journal of Risk and Uncertainty. 2016; p. 1–28. https://doi.org/10.1007/s11166-016-9243-x 53. Killeen PR. Models of ADHD: Five ways smaller sooner is better. Journal of Neuroscience Methods. 2015; 252:2–13. https://doi.org/10.1016/j.jneumeth.2015.01.011 PMID: 25597911 54. Levy I, Rosenberg Belmaker L, Manson K, Tymula A, Glimcher PW. Measuring the subjective value of risky and ambiguous options using experimental economics and functional MRI methods. Journal of Visualized Experiments: JoVE. 2012;(67):e3724. https://doi.org/10.3791/3724 PMID: 23022992 55. Weber EU, Johnson EJ, Milch KF, Chang H, Brodscholl JC, Goldstein DG. Asymmetric Discounting in Intertemporal Choice: A Query-Theory Account. Psychological Science. 2007; 18(6):516–523. https:// doi.org/10.1111/j.1467-9280.2007.01932.x PMID: 17576265 56. Levy DJ, Thavikulwat AC, Glimcher PW. State dependent valuation: the effect of deprivation on risk preferences. PLoS ONE. 2013; 8(1):e53978. https://doi.org/10.1371/journal.pone.0053978 PMID: 23358126 57. Mazur JE. An adjusting procedure for studying delayed reinforcement. In: Commons ML, Mazur JE, Nevin JA, Rachlin H, editors. The Effect of Delay and of Intervening Events on Reinforcement Value, Quantitative Analyses of Behavior. References Hillsdale, New Jersey: Erlbaum; 1987. p. 55–73. 58. Ainslie GW. Impulse control in pigeons. J Exp Anal Behav. 1974; 21(3):485–9. https://doi.org/10.1901/ jeab.1974.21-485 PMID: 16811760 59. Cheung SL. Recent developments in the experimental elicitation of time preference. Journal of Behav- ioral and Experimental Finance. 2016; 11:1–8. https://doi.org/10.1016/j.jbef.2016.04.001 60. Abdellaoui M, Bleichrodt H, l’Haridon O, Paraschiv C. Is There One Unifying Concept of Utility?An Experimental Comparison of Utility Under Risk and Utility Over Time. Management Science. 2013; 59 (9):2153–2169. https://doi.org/10.1287/mnsc.1120.1690 61. Andreoni J, Sprenger C. Estimating Time Preferences from Convex Budgets. American Economic Review. 2012; 102(7):3333–3356. https://doi.org/10.1257/aer.102.7.3333 62. Attema AE, Bleichrodt H, Gao Y, Huang Z, Wakker PP. Measuring Discounting without Measuring Util- ity. American Economic Review. 2016; 106(6):1476–1494. https://doi.org/10.1257/aer.20150208 63. Andreoni J, Sprenger C. Risk Preferences Are Not Time Preferences. American Economic Review. 2012; 102(7):3357–3376. https://doi.org/10.1257/aer.102.7.3357 64. Cheung SL. Eliciting utility curvature and time preference. University of Sydney, School of Economics; 2015. 2015-01. Available from: https://ideas.repec.org/p/syd/wpaper/2015-01.html. 65. Luckman A, Donkin C, Newell BR. People Wait Longer when the Alternative is Risky: The Relation Between Preferences in Risky and Inter-temporal Choice. Journal of Behavioral Decision Making. 2017; p. n/a–n/a. https://doi.org/10.1002/bdm.2025 66. Luckman A, Donkin C, Newell BR. Can a single model account for both risky choices and inter-temporal choices? Testing the assumptions underlying models of risky inter-temporal choice. Psychonomic Bul- letin & Review. 2017; p. 1–8. https://doi.org/10.3758/s13423-017-1330-8 67. Paulus MP. Decision-making dysfunctions in psychiatry–altered homeostatic processing? Science (New York, NY). 2007; 318(5850):602–606. https://doi.org/10.1126/science.1142997 68. Schutter DJLG, Bokhoven Iv, Vanderschuren LJMJ, Lochman JE, Matthys W. Risky Decision Making in Substance Dependent Adolescents with a Disruptive Behavior Disorder. Journal of Abnormal Child Psy- chology. 2011; 39(3):333–339. https://doi.org/10.1007/s10802-010-9475-1 PMID: 21153697 69. Matthies S, Philipsen A, Svaldi J. Risky decision making in adults with ADHD. Journal of Behavior Ther- apy and Experimental Psychiatry. 2012; 43(3):938–946. https://doi.org/10.1016/j.jbtep.2012.02.002 PMID: 22445935 70. Verdejo-Garcia A, Chong TTJ, Stout JC, Yu¨cel M, London ED. Stages of dysfunctional decision-making in addiction. Pharmacology Biochemistry and Behavior. 2017; https://doi.org/10.1016/j.pbb.2017.02. 003 17 / 18 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 Risk distorts impulsivity 71. Fishbein DH, Eldreth DL, Hyde C, Matochik JA, London ED, Contoreggi C, et al. Risky decision making and the anterior cingulate cortex in abstinent drug abusers and nonusers. Cognitive Brain Research. 2005; 23(1):119–136. https://doi.org/10.1016/j.cogbrainres.2004.12.010 PMID: 15795139 72. Euteneuer F, Schaefer F, Stuermer R, Boucsein W, Timmermann L, Barbe MT, et al. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191357 January 26, 2018 References Dissociation of decision-making under ambiguity and decision-making under risk in patients with Parkinson’s disease: a neuropsychological and psychophysiological study. Neuropsychologia. 2009; 47(13):2882–2890. https://doi.org/10.1016/j.neuropsychologia.2009.06.014 PMID: 19545579 73. Gu L, Lim KO, Specker SM, Raymond N, MacDonald AW III. Valuation Bias of Reward in Long-Term Cocaine Users: The specificity of decision-making biases in cocaine addiction. Acta Psychopathologica. 2015; 1(1). 74. Hartley CA, Phelps EA. Anxiety and Decision-Making. Biological psychiatry. 2012; 72(2). https://doi. org/10.1016/j.biopsych.2011.12.027 PMID: 22325982 75. Bickel WK, Koffarnus MN, Moody L, Wilson AG. The behavioral- and neuro-economic process of tem- poral discounting: A candidate behavioral marker of addiction. Neuropharmacology. 2014; 76 Pt B:518– 27. https://doi.org/10.1016/j.neuropharm.2013.06.013 PMID: 23806805 76. Story GW, Moutoussis M, Dolan RJ. A Computational Analysis of Aberrant Delay Discounting in Psychi- atric Disorders. Frontiers in Psychology. 2016; 6. https://doi.org/10.3389/fpsyg.2015.01948 PMID: 26793131 77. Abi-Dargham A, Horga G. The search for imaging biomarkers in psychiatric disorders. Nature Medicine. 2016; 22(11):1248–1255. https://doi.org/10.1038/nm.4190 PMID: 27783066 78. Ahn WY, Busemeyer JR. Challenges and promises for translating computational tools into clinical prac- tice. Current Opinion in Behavioral Sciences. 2016; 11:1–7. https://doi.org/10.1016/j.cobeha.2016.02. 001 PMID: 27104211 79. Miedl SF, Peters J, Bu¨chel C. Altered Neural Reward Representations in Pathological Gamblers Revealed by Delay and Probability Discounting. Archives of General Psychiatry. 2012; 69(2):177–186. https://doi.org/10.1001/archgenpsychiatry.2011.1552 PMID: 22310505 80. Richards JB, Zhang L, Mitchell SH, de Wit H. Delay or probability discounting in a model of impulsive behavior: effect of alcohol. Journal of the Experimental Analysis of Behavior. 1999; 71(2):121–143. https://doi.org/10.1901/jeab.1999.71-121 PMID: 10220927 81. Curry OS, Price ME, Price JG. Patience is a virtue: Cooperative people have lower discount rates. Per- sonality and Individual Differences. 2008; 44(3):780–785. https://doi.org/10.1016/j.paid.2007.09.023 82. Crockett MJ, Clark L, Lieberman MD, Tabibnia G, Robbins TW. Impulsive choice and altruistic punish- ment are correlated and increase in tandem with serotonin depletion. Emotion (Washington, DC). 2010; 10(6):855–862. https://doi.org/10.1037/a0019861 83. Espı´n AM, Brañas-Garza P, Herrmann B, Gamella JF. Patient and impatient punishers of free-riders. Proceedings of the Royal Society of London B: Biological Sciences. 2012; 279(1749):4923–4928. https://doi.org/10.1098/rspb.2012.2043 84. Espı´n AM, Exadaktylos F, Herrmann B, Brañas-Garza P. Short- and long-run goals in ultimatum bar- gaining: impatience predicts spite-based behavior. Frontiers in Behavioral Neuroscience. 2015; 9. https://doi.org/10.3389/fnbeh.2015.00214 18 / 18
https://openalex.org/W2980879484	https://zenodo.org/records/4022306/files/1910.07791.pdf	English	null	Coherence transfer in an akinetic swept source OCT laser with optical feedback	Optics letters/Optics index	2,019	cc-by	6,145	Coherence transfer in an akinetic swept source OCT laser with optical feedback †, A. KOVALEV4,†, N. REBROVA2,3, K. GRIGORENKO4, E. VIKTOROV4, AND G. 7791v1 [physics.optics] 17 Oct 2019 arXiv:1910.07791v1 [physics.optics] 17 Oct 2019 1Université Côte d’Azur, CNRS, INPHYNI, Nice, France 2Centre for Advanced Photonics and Process Analysis and Department of Physical Sciences, Cork Institute of Technology, Cork, Ireland 3Tyndall National Institute, University College Cork, Cork, Ireland 4ITMO University, Saint Petersburg, Russia †These authors contributed equally to this Letter. Corresponding author: svetlana.slepneva@inphyni.cnrs.fr 1Université Côte d’Azur, CNRS, INPHYNI, Nice, France 2Centre for Advanced Photonics and Process Analysis and Department of Physical Sciences, Cork Institute of Technology, Cork, Ireland 3Tyndall National Institute, University College Cork, Cork, Ireland 4ITMO University, Saint Petersburg, Russia †These authors contributed equally to this Letter. *Corresponding author: svetlana.slepneva@inphyni.cnrs.fr arXiv:1910.07791v1 [physics.optics] 17 Oct Compiled October 18, 2019 Compiled October 18, 2019 We theoretically investigate the inﬂuence of optical feedback onto the dynamics of a semiconductor swept source laser. In particular, we show that optical feed- back can be used to lock the phase of the successive las- ing modes of a multi-section semiconductor laser com- monly used for Optical Coherence Tomography (OCT) applications. We also identify two different regimes called sliding frequency self-mixing and sliding fre- quency mode-locking. The second regime demon- strates sub-nanosecond sliding frequency pulses for non-linear optics applications. © 2019 Optical Society of America ence length of akinetic SG-DBR lasers by locking the successive longitudinal sweeping modes. Our study targets the laser con- ﬁguration based on akinetic multi-section technology, similar to that presented in [8]. The advantage of this swept source is its stability, high speed and potential to be integrated into a compact device. Such lasers have been used as widely tunable lasers in the telecom industry for many years [9] and have, more recently, been considered for OCT applications [10]. The oper- ation of these devices relies on the Vernier effect between two sample gratings that leads to the single mode operation [9]. By varying the injection currents in these gratings, it is possible to sequentially select and tune each single mode over several nanometers. Wide bandwidth tuning is obtained with a sub- sequent tuning of a sequence of Vernier modes, thus the laser output is a sequence of highly coherent single mode sweeps sep- arated by transients. The coherence length of each mode could be estimated using the Schawlow-Townes approach [11] taking into account various noise sources. Coherence transfer in an akinetic swept source OCT laser with optical feedback While each mode has similar instantaneous linewidth, the hops between successive Vernier modes lead to random optical phase shifts that deteriorate the coherence of these lasers. arXiv:1910.07791v1 [physics.optic http://dx.doi.org/10.1364/ao.XX.XXXXXX http://dx.doi.org/10.1364/ao.XX.XXXXXX The development of Swept Source Optical Coherence Tomog- raphy (SS-OCT) technique [1] has increased the demand for ultra-fast, broadband and highly coherent swept sources [2]. Swept sources are lasers that optically sweep over a wide spec- trum range while displaying a small intensity modulation. The quality of OCT images depends on various parameters of the light sources including the spectral width, the relative intensity noise and the coherence length. This last parameter, which is in- versely proportional to the instantaneous linewidth, is the key in determining the imaging depth. While frequency swept sources originally relied on external cavity conﬁguration including an intra-cavity tunable element such as a rotating mirror [3], state- of-the-art sources include long cavity Fourier Domain Mode- Locked (FDML) Lasers [4], micro-electro-mechanical system (MEMS) based Vertical Cavity Surface Emitting Lasers (VC- SELs) [5], short cavity laser with MEMS tunable Fabry-Pérot ﬁlter [6, 7] and tunable Sampled Grating Distributed Bragg Re- ﬂector (SG-DBR) Lasers [8]. While the ﬁrst three technologies have demonstrated long coherence length that enable long imag- ing depth, SG-DBR lasers present the advantage of not including any mechanically movable parts. However, numerous mode hops across a single sweep deteriorate their coherence proper- ties. To improve the coherence, we use external optical feedback technique. External optical feedback has been used as an effec- tive tool to improve the coherence of continuous wave (cw) or tunable lasers, though under some parameters it can lead to the coherence deterioration [12]. Here we introduce the concept of optical feedback into a fast-sweeping laser using an example of akinetic conﬁguration, and derive the locking conditions for the subsequent modes. While in an akinetic laser each subse- quent single mode starts lasing from spontaneous emission, our method implies optical feedback to seed each lasing mode by a returned portion of the preceding lasing mode. In analogy with cw lasers, it is possible to derive Adler-like equation describing the locking conditions [13]. By introducing optical feedback, we show that it is possible to lock the phase of successive modes, and and maintain coherence over the mode hops. Let us consider the mode hopping event between the two subsequent modes 1 and 2 with complex amplitudes E1(t) and E2(t) correspondingly. Optics Letters 1 Optics Letters 1 Optics Letters Letter Coherence transfer in an akinetic swept source OCT laser with optical feedback We deﬁne t = 0 as the moment when mode 1 starts to decay and mode 2 starts to build up the lasing via relaxation oscillations. (b) Intensity of the time-delayed attenuated mode 1 given by γ2\|E1(t −Text)\|2 (thin line) and the box highlighting the period τgate when the gate is open (set by the modulator), and described by η(t)2\|E1(t −Text)\|2. During this period, the part of mode 1, reﬂected from the external mirror, is reinjected into the laser to seed mode 2. (c) and (d) visualise the switch of the values of the functions ρ1(t) and ρ2(t), correspondingly (see Eqs 1-2). Note, the ratio τgate/Text can be adjusted. (2) where the electric ﬁeld of each mode is En(t) = Rn exp(iφn(t)) with, here and below, n = 1, 2 staying for mode 1 or 2 respec- tively; dot corresponds to differentiation with respect to nor- malized time t; Rn is the amplitude of the electric ﬁeld and φn(t) = Ωn(t) + ξn is the phase, where Ωn(t) = ω0n + at is the angular frequency of each mode, which describes the lin- ear sweep starting from ω0n at a sweeping rate of a, ξn is arbi- trary initial phase; α is the linewidth enhancement factor; γN is the carrier decay rate and g0 is the pump parameter. The functions ρ1,2(t) describe the Vernier effect; ρ1(t) = κ1Θ(t), where Θ(t) is the Heaviside step function and κ1 is the decay rate of mode 1 when this mode becomes strongly damped as schematically explained in Fig. 1 (b). Similarly, ρ2(t) = κ2Θ(−t) (Fig. 1(c)). The term η(t)E1(t −Text) describes the time-gated optical feedback from mode 1 into mode 2, where η(t) = γΠ(t) is the time varying feedback level: γ is the feedback strength, Π(t) = Θ(t) −Θ(t −τgate) is the rectangular function with du- ration τgate; Text corresponds to the time delay of the light of mode 1 reinjected into mode 2. SG-DBR LASER M BS Lext OM Fig. 2. A schematic setup of the feedback. M - backreﬂecting mirror; BS - beam splitter; OM - optical modulator provides periodically opening window for the gated feedback; Lext corresponds to the optical path length of the external cavity and deﬁnes the delay time Text = 2Lext c , where c is the speed of light in the cavity. SG-DBR LASER M BS Lext OM Fig. 2. Coherence transfer in an akinetic swept source OCT laser with optical feedback A schematic setup of the feedback. M - backreﬂecting mirror; BS - beam splitter; OM - optical modulator provides periodically opening window for the gated feedback; Lext corresponds to the optical path length of the external cavity and deﬁnes the delay time Text = 2Lext c , where c is the speed of light in the cavity. Without optical feedback, i.e. if γ = 0, the temporal evolution of the optical phase of the two modes are independent since they are not coupled and, as a result, the laser loses its coher- ence at each mode hop. To illustrate this point, we numerically integrated Eqs. (1)-(3) for 100 different initial phase values (keep- ing the same value for the amplitude). In this case, the phase of the electric ﬁeld in the complex plane for time t = 20000 is randomly distributed on a circle with radius p g0 −1, as shown in Fig. 3(a), thus demonstrating the loss of coherence at each mode hop. When the time-gated feedback is applied to the same situation, all the transients of mode 2 converge towards the same value of the electric ﬁeld which is locked to mode 1 as shown by the green cross in Fig. 3(a) (and zoomed in Fig. 3(b)), demonstrating the coherence transfer from mode 1 to mode 2. where δΩ(t) = Ω2(t) −Ω1(t −Text). The two modes can only lock if the frequency difference between them follows the in- equality \|δΩ(t)\| < γ R1 R2 p 1 + α2. (5) (5) In order to retain the constant phase between the successive modes, it is important that the timing jitter of the modulator does not cause phase ﬂuctuations. To address this point, we should note that the phase difference δφ is constant when the two modes are locked, and their angular frequency difference is δΩ. If the switching time of the modulator is shorter than this frequency beating, then the phase ﬂuctuations will be insigniﬁcant. Coherence transfer in an akinetic swept source OCT laser with optical feedback The 1st mode is switched on for t ≤0 and This paper aims to investigate a means to improve the coher- 2 Optics Letters 2 Optics Letters 2 Letter Fig. 1. The time-gated feedback. (a) A schematic illustration (both axes are in arbitrary units) of a single mode hop event happening at time 0 between mode 1 (orange) and mode 2 (green). We deﬁne t = 0 as the moment when mode 1 starts to decay and mode 2 starts to build up the lasing via relaxation oscillations. (b) Intensity of the time-delayed attenuated mode 1 given by γ2\|E1(t −Text)\|2 (thin line) and the box highlighting the period τgate when the gate is open (set by the modulator), and described by η(t)2\|E1(t −Text)\|2. During this period, the part of mode 1, reﬂected from the external mirror, is reinjected into the laser to seed mode 2. (c) and (d) visualise the switch of the values of the functions ρ1(t) and ρ2(t), correspondingly (see Eqs 1-2). Note, the ratio τgate/Text can be adjusted. the 2nd mode is on for t > 0. First, we consider the time-gated synchronisation of two successive modes, where the feedback is turned on just before the mode hop as shown in Fig. 1(a). The part of the sweep emitted by mode 1 of duration τgate, just before this mode is switched off, is stored in an external cavity of length Lext in order to seed the second mode as illustrated in Fig. 1(a). Experimentally, that will require additional components in the external cavity, such as an optical amplitude modulator or a MEMS-actuated mirror, in order to control the feedback duration (Fig. 2). g The evolution of the complex ﬁeld amplitudes E1,2(t) and carrier density N(t) of the SG-DBR laser can be described by the following set of equations, with the time t normalised by the free running laser photon lifetime: ˙E1(t) = −ρ1(t)E1(t) + iΩ1(t)E1(t)+ 1 2 (1 + iα) (N(t) −1) E1(t), (1) ˙E2(t) = −ρ2(t)E2(t) + iΩ2(t)E2(t)+ 1 2 (1 + iα) (N(t) −1) E2(t) + η(t)E1(t −Text), (2) ) = −γN N(t) −g0 + N(t) \|E1(t)\|2 + \|E2(t)\|2 , (3) (1) Fig. 1. The time-gated feedback. (a) A schematic illustration (both axes are in arbitrary units) of a single mode hop event happening at time 0 between mode 1 (orange) and mode 2 (green). Coherence transfer in an akinetic swept source OCT laser with optical feedback The red dot near the origin accu- mulates 100 different initial conditions of the electric ﬁeld of mode 2, the initial conditions of mode 1 remains the same. (b) Zoom of the area in the dash square in (a). The parameters are Text = 200, γN = 0.001, g0 = 2, α = 5, κ1 = κ2 = 1, γ = 0.2, a = −0.002. For the simulation τgate = Text. t/Text t/Text 0 1 2 3 4 5 6 0.0 0.4 0.8 1.2 1.6 2.0 2.4 t/Text \|E\| 2 (arb. units) (d) 0 1 2 3 4 5 6 0.0 0.5 1.0 1.5 2.0 2.5 3.0 t/Text \|E\| 2 (arb. units) (e) Fig. 4. (a) Bifurcation diagram of the swept source laser with Fig. 4. (a) Bifurcation diagram of the swept source laser with continuous optical feedback demonstrating extrema of the intensity. The black (red) color corresponds to the decrease (increase) of the parameter ∆. The numbers I-IV denote the dynamical regimes, and corresponding intensity time traces are given for sliding frequency self-mixing regime I at ∆= 0.4 (b), sliding frequency mode-locking regime II at ∆= −0.12 (c), second harmonic mode-locked regime III at ∆= −0.06 (d), chaotic IV at ∆= 0.2 (e). The other parameters: Text = 100, γN = 0.01, g0 = 1.5, α = 2, γ = 0.1. Fig. 4. (a) Bifurcation diagram of the swept source laser with continuous optical feedback demonstrating extrema of the intensity. The black (red) color corresponds to the decrease (increase) of the parameter ∆. The numbers I-IV denote the dynamical regimes, and corresponding intensity time traces are given for sliding frequency self-mixing regime I at ∆= 0.4 (b), sliding frequency mode-locking regime II at ∆= −0.12 (c), second harmonic mode-locked regime III at ∆= −0.06 (d), chaotic IV at ∆= 0.2 (e). The other parameters: Text = 100, γN = 0.01, g0 = 1.5, α = 2, γ = 0.1. effect of optical feedback on the dynamics of semiconductor lasers has been the subject of numerous studies as it may either strongly improve or deteriorate the coherence properties of the laser depending on the parameters. In the case of a wavelength sweeping laser, the light fed back into the cavity is frequency shifted by ∆= aText with the instantaneous laser frequency ˙φn(t). Coherence transfer in an akinetic swept source OCT laser with optical feedback units) (d) 0 1 2 3 4 5 6 0.0 0.5 1.0 1.5 2.0 2.5 3.0 t/Text \|E\| 2 (arb. units) (e) Fig. 4. (a) Bifurcation diagram of the swept source laser wit continuous optical feedback demonstrating extrema of the intensity. The black (red) color corresponds to the decrease (increase) of the parameter ∆. The numbers I-IV denote the dynamical regimes, and corresponding intensity time trace are given for sliding frequency self-mixing regime I at ∆= (b), sliding frequency mode-locking regime II at ∆= −0.12 (c), second harmonic mode-locked regime III at ∆= −0.06 ( chaotic IV at ∆= 0.2 (e). The other parameters: Text = 100 γN = 0.01, g0 = 1.5, α = 2, γ = 0.1. γ √ 1 + α2. Since these modes may be stable or unstable depen ing on various parameters, the laser can potentially demonstr a wide range of behaviour including narrow linewidth em sion, mode-hopping, undamped relaxation oscillations or lo f ﬂ i -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 Re(E2) Im(E2) (a) 0.6 0.7 0.8 0.9 -0.8 -0.7 -0.6 -0.5 Re(E2) Im(E2) (b) Fig. 3. (a) Real and imaginary parts of the electric ﬁeld of mode 2 after t = 20000 calculated for 100 different initial conditions for three cases 1) free-running (the grey circle cor- responding to the randomly distributed phase and the blue crosses indicate 100 calculated solutions); 2) time-gated feed- back (green cross indicating the locking of the phase of mode 2 to mode 1); and 3) continuous feedback (the orange circle indicating the limit cycle). The red dot near the origin accu- mulates 100 different initial conditions of the electric ﬁeld of mode 2, the initial conditions of mode 1 remains the same. (b) Zoom of the area in the dash square in (a). The parameters are Text = 200, γN = 0.001, g0 = 2, α = 5, κ1 = κ2 = 1, γ = 0.2, a = −0.002. For the simulation τgate = Text. -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 (rad.) \|E\| 2 (arb. Coherence transfer in an akinetic swept source OCT laser with optical feedback The parameter ∆corresponds to the frequency shift be- tween the light in the cavity and the light reinjected into the cavity as a result of the feedback. As a result, the effect of optical feedback in a frequency swept source is closely related to the dynamics of frequency shifted feedback previously investigated in [14, 15] but the frequency shift can be of the order of a few GHz and, as a result, can be much larger than that obtained using an external modulator. In the case of a swept source, the main difference between using continuous instead of time-gated feedback is that continuous feedback may destabilise the laser, and deteriorate the coherence of each mode. γ √ 1 + α2. Since these modes may be stable or unstable depend- ing on various parameters, the laser can potentially demonstrate a wide range of behaviour including narrow linewidth emis- sion, mode-hopping, undamped relaxation oscillations or low frequency ﬂuctuations. In order to investigate various dynamical regimes that can be demonstrated by a swept source with constant feedback, we numerically integrated equations (6) and (7), and performed bifurcation analysis which revealed four possible outputs de- pending on parameter ∆which varies from −0.5 to 0.5 rad. Figure 4(a) shows the bifurcation diagram, and Figs. 4(b)-4(e) demonstrate simulated intensity time traces to illustrate each regime. At large values of \|∆\|, the laser operates in self-mixing regime [17], where the temporal evolution of the electric ﬁeld displays periodic oscillations with frequency corresponding to the frequency beating ∆between the emitted and re-injected light (see Region I in Fig. 4(a) and Fig. 4(b)). As ∆increases, the lasers enters regime II and emits periodic pulses corresponding to sliding frequency mode-locking (Fig. 4(c)) similar to those observed in [6, 18]. For higher negative values of ∆, the laser reachers region III which is similar to region II, but with the doubled frequency (Fig. 4(d)). For ∆= 0 and for small positive values of ∆the laser enters region IV where the above equations display a chaotic output characteristic of instabilities commonly observed in the Lang-Kobayashi equations [12] (Fig. 4(e)). To investigate this, we consider the dynamics of a single mode frequency swept source with constant continuous optical feedback. Coherence transfer in an akinetic swept source OCT laser with optical feedback -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 Re(E2) Im(E2) (a) Im(E2) -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 Re(E2) Im(E2) (a) 0.6 0.7 0.8 0.9 -0.8 -0.7 -0.6 -0.5 Re(E2) Im(E2) (b) -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 (rad.) \|E\| 2 (arb. units) (a) I II III IV I 0.6 0.7 0.8 0.9 -0.8 -0.7 -0.6 -0.5 Re(E2) (b) Re(E2) Fig. 3. (a) Real and imaginary parts of the electric ﬁeld of mode 2 after t = 20000 calculated for 100 different initial conditions for three cases 1) free-running (the grey circle cor- responding to the randomly distributed phase and the blue crosses indicate 100 calculated solutions); 2) time-gated feed- back (green cross indicating the locking of the phase of mode 2 to mode 1); and 3) continuous feedback (the orange circle indicating the limit cycle). The red dot near the origin accu- mulates 100 different initial conditions of the electric ﬁeld of mode 2, the initial conditions of mode 1 remains the same. (b) Zoom of the area in the dash square in (a). The parameters are Text = 200, γN = 0.001, g0 = 2, α = 5, κ1 = κ2 = 1, γ = 0.2, a = −0.002. For the simulation τgate = Text. Fig. 3. (a) Real and imaginary parts of the electric ﬁeld of mode 2 after t = 20000 calculated for 100 different initial conditions for three cases 1) free-running (the grey circle cor- responding to the randomly distributed phase and the blue crosses indicate 100 calculated solutions); 2) time-gated feed- back (green cross indicating the locking of the phase of mode 2 to mode 1); and 3) continuous feedback (the orange circle indicating the limit cycle). Coherence transfer in an akinetic swept source OCT laser with optical feedback To gain further insight on the locking dynamics, we can as- sume that the intensities of the mode \|E2(t)\|2 and \|E1(t −Text)\|2 are constant and derive an equation for the phase difference δφ(t) = φ2(t) −φ1(t): While the gated optical feedback can lock two consecutive modes, experimentally, it requires the incorporation of an optical modulator in the external cavity to switch on and off the optical feedback and, as an alternative, we consider the possibility of having continuous optical feedback in an SG-DBR laser. The δ ˙φ(t) = δΩ(t) −γ R1 R2 p 1 + α2 sin δφ(t), (4) Optics Letters 3 Optics Letters 3 Letter -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 Re(E2) Im(E2) (a) 0.6 0.7 0.8 0.9 -0.8 -0.7 -0.6 -0.5 Re(E2) Im(E2) (b) Fig. 3. (a) Real and imaginary parts of the electric ﬁeld of mode 2 after t = 20000 calculated for 100 different initial conditions for three cases 1) free-running (the grey circle cor- responding to the randomly distributed phase and the blue crosses indicate 100 calculated solutions); 2) time-gated feed- back (green cross indicating the locking of the phase of mode 2 to mode 1); and 3) continuous feedback (the orange circle indicating the limit cycle). The red dot near the origin accu- mulates 100 different initial conditions of the electric ﬁeld of mode 2, the initial conditions of mode 1 remains the same. (b) Zoom of the area in the dash square in (a). The parameters are Text = 200, γN = 0.001, g0 = 2, α = 5, κ1 = κ2 = 1, γ = 0.2, a = −0.002. For the simulation τgate = Text. -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 Re(E2) Im(E2) (a) 0.6 0.7 0.8 0.9 -0.8 -0.7 -0.6 -0.5 Re(E2) Im(E2) (b) Fig. 3. Coherence transfer in an akinetic swept source OCT laser with optical feedback (a) Real and imaginary parts of the electric ﬁeld of mode 2 after t = 20000 calculated for 100 different initial conditions for three cases 1) free-running (the grey circle cor- responding to the randomly distributed phase and the blue crosses indicate 100 calculated solutions); 2) time-gated feed- back (green cross indicating the locking of the phase of mode 2 to mode 1); and 3) continuous feedback (the orange circle indicating the limit cycle). The red dot near the origin accu- mulates 100 different initial conditions of the electric ﬁeld of mode 2, the initial conditions of mode 1 remains the same. (b) Zoom of the area in the dash square in (a). The parameters are Text = 200, γN = 0.001, g0 = 2, α = 5, κ1 = κ2 = 1, γ = 0.2, a = −0.002. For the simulation τgate = Text. effect of optical feedback on the dynamics of semiconductor lasers has been the subject of numerous studies as it may either strongly improve or deteriorate the coherence properties of the laser depending on the parameters. In the case of a wavelength sweeping laser, the light fed back into the cavity is frequency shifted by ∆= aText with the instantaneous laser frequency ˙φn(t). The parameter ∆corresponds to the frequency shift be- tween the light in the cavity and the light reinjected into the cavity as a result of the feedback. As a result, the effect of optical feedback in a frequency swept source is closely related to the dynamics of frequency shifted feedback previously investigated in [14, 15] but the frequency shift can be of the order of a few GHz and, as a result, can be much larger than that obtained using an external modulator. In the case of a swept source, the i diff b t i ti i t d f ti t d -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 (rad.) \|E\| 2 (arb. units) (a) I II III IV I 0 1 2 3 4 5 6 0.0 0.2 0.4 0.6 0.8 1.0 t/Text \|E\| 2 (arb. units) (b) 0 1 2 3 4 5 6 0.0 1.5 3.0 4.5 6.0 7.5 t/Text \|E\| 2 (arb. units) (c) 0 1 2 3 4 5 6 0.0 0.4 0.8 1.2 1.6 2.0 2.4 t/Text \|E\| 2 (arb. Coherence transfer in an akinetic swept source OCT laser with optical feedback units) (a) I II III IV I 0 1 2 3 4 5 6 0.0 0.2 0.4 0.6 0.8 1.0 t/Text \|E\| 2 (arb. units) (b) 0 1 2 3 4 5 6 0.0 1.5 3.0 4.5 6.0 7.5 t/Text \|E\| 2 (arb. units) (c) 0 1 2 3 4 5 6 0.0 0.4 0.8 1.2 1.6 2.0 2.4 t/Text \|E\| 2 (arb. units) (d) 0 1 2 3 4 5 6 0.0 0.5 1.0 1.5 2.0 2.5 3.0 t/Text \|E\| 2 (arb. units) (e) Fig. 4. (a) Bifurcation diagram of the swept source laser with i i l f db k d i f h -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 (rad.) \|E\| 2 (arb. units) (a) I II III IV I 0 1 2 3 4 5 6 0.0 0.2 0.4 0.6 0.8 1.0 t/Text \|E\| 2 (arb. units) (b) 0 1 2 3 4 5 6 0.0 1.5 3.0 4.5 6.0 7.5 t/Text \|E\| 2 (arb. units) (c) 0 1 2 3 4 5 6 0.0 0.4 0.8 1.2 1.6 2.0 2.4 t/Text \|E\| 2 (arb. units) (d) 0 1 2 3 4 5 6 0.0 0.5 1.0 1.5 2.0 2.5 3.0 t/Text \|E\| 2 (arb. units) (e) Fig. 4. (a) Bifurcation diagram of the swept source laser with continuous optical feedback demonstrating extrema of the intensity. The black (red) color corresponds to the decrease (increase) of the parameter ∆. The numbers I-IV denote the dynamical regimes, and corresponding intensity time traces are given for sliding frequency self-mixing regime I at ∆= 0.4 (b), sliding frequency mode-locking regime II at ∆= −0.12 (c), second harmonic mode-locked regime III at ∆= −0.06 (d), chaotic IV at ∆= 0.2 (e). The other parameters: Text = 100, γN = 0.01, g0 = 1.5, α = 2, γ = 0.1. REFERENCES Coldren, IEEE J. Quantum Electron. 29, 1824 (1993). 10. M. Salas, M. Augustin, F. Felberer, A. Wartak, M. Laslandes, L. Ginner, M. Niederleithner, J. Ensher, M. P. Minneman, R. A. Leitgeb et al., Biomed. Opt. Express 9, 1871 (2018). 11. A. L. Schawlow and C. H. Townes, Phys. Rev. 112, 1 The lasing regimes of the SG-DBR laser under the continu- ous feedback can be controlled by varying the frequency shift ∆and feedback strength γ, as demonstrated in the the two- dimensional map in Fig. 5. The black lines correspond to the relation \|∆\| = γ √ 1 + α2 indicating the frequency range within which the ECMs exist. Outside of this range, for \|∆\| > γ √ 1 + α2, the purple area corresponds to the sliding frequency self-mixing regime I, where the laser does not lock with the re-injected light. The green area corresponds to the non-periodic regimes III and IV and the localised red areas indicate the appearance of sliding frequency mode-locked regime II. Interestingly, as the major sliding frequency mode-locked regime can be obtained for the range of the feedback strength from 0.056 to 0.099 and negative frequency shifts, the analysis reveals the appearance of two ad- ditional small areas for the both positive and negative frequency shift values and smaller feedback strength. 12. G. Van Tartwijk and D. Lenstra, Quantum Semiclassical Opt. J. Eur. Opt. Soc. Part B 7, 87 (1995). 13. R. Adler, Proc. IRE 34, 351 (1946). 14. L. Yatsenko, B. Shore, and K. Bergmann, Opt. Commun. 236, 183 (2004). 15. S. Yun, D. Richardson, D. Culverhouse, and B. Kim, IEEE J. Sel. Top. Quantum Electron. 3, 1087 (1997). 16. R. Lang and K. Kobayashi, IEEE J. Quantum Electron. 16, 347 (1980). 17. M. Rudd, J. Phys. E: Sci. Instruments 1, 723 (1968). 17. M. Rudd, J. Phys. E: Sci. Instruments 1, 723 (1968). 18. T. Butler, D. Goulding, S. Slepneva, B. O’Shaughnessy, S. Hegarty, 18. T. Butler, D. Goulding, S. Slepneva, B. O’Shaughnessy, S. G. Huyet, and B. Kelleher, Opt. Express 27, 7307 (2019). G. Huyet, and B. Kelleher, Opt. Express 27, 7307 (2019) 19. S. Bégin, B. Burgoyne, V. Mercier, A. Villeneuve, R. Vallée, and D. Côté, Biomed. Opt. Express 2, 1296 (2011). 20. E. A. Avrutin and L. Zhang, The Eur. Phys. J. B 92, 137 (2019). 21. S. Slepneva, B. O’Shaughnessy, B. Kelleher, S. Hegarty, A. Vladimirov, H.-C. Coherence transfer in an akinetic swept source OCT laser with optical feedback The equations describing the evolution of this mode read ˙E(t) = iΩ(t) E(t) + 1 2 (1 + iα) (N(t) −1) E(t)+ +γE (t −Text) , (6) ˙N(t) = −γN N −g0(t) + N(t)\|E(t)\|2 . (7) ˙E(t) = iΩ(t) E(t) + 1 2 (1 + iα) (N(t) −1) E(t)+ ˙E(t) = iΩ(t) E(t) + 1 2 (1 + iα) (N(t) −1) E(t)+ +γE (t −Text) , (6) ˙N(t) = −γN N −g0(t) + N(t)\|E(t)\|2 . (7) (7) The frequency Ω(t) is assumed to be increasing linearly with time, i.e. Ω(t) = ω + at. For a = 0, these equations reduce to the well known Lang-Kobayashi equations [16]. In this regime, the external cavity modes are given by E(t) = R exp(iωt), where ω is a solution of ω = −γ p 1 + α2 sin (ωText + arctan α) . (8) (8) The observed regimes I, II and III are of high interest for non-linear optics applications including various swept-source based imaging modalities such as hyperspectral imaging [19] or From this equation, we note that the external cavity modes (ECMs) lie within the frequency range deﬁned by \|ω\| < 4 Letter Optics Letters SS-OCT. The lasers based on MEMS have been used for the latter technique [6]. Such devices operate in a pulsed regime with the repetition period corresponding to the cavity roundtrip time or half of the cavity roundtrip time, depending on the sweeping rate [18, 20]. The pulses are generated via the coherence trans- fer between the successive modes due to four-wave mixing as an interplay between the sweeping rate and the transmission bandwidth of the tunable ﬁlter. This results in undesired in- crease of the laser linewidth with the increase of the sweeping rate [21], however, the advantage of this laser is the possibility to implement a coherence revival scheme [22] for application in imaging. The frequency sweeping pulses can also be generated from mode-locked lasers [19] or FDML lasers applying the pulse compression [23] or master oscillator power ampliﬁer-based [24] methods. If compared to these methods of generation short pulses with sweeping frequency, the continuous optical feedback can be used for generation of different adjustable regimes, par- ticularly, a train of high-intensity sub-ns pulses with frequency variation between the pulses equal to ∆, which can be adjusted by changing either the sweeping rate a or the delay time Text. REFERENCES 1. S. R. Chinn, E. A. Swanson, and J. G. Fujimoto, Opt. Lett. 22, 340 (1997). 1. S. R. Chinn, E. A. Swanson, and J. G. Fujimoto, Opt. Lett. 22, 340 (1997). 0.00 0.02 0.04 0.06 0.08 0.10 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 Δ rad.) Fig. 5. The two-dimensional map of the dynamical regimes for the continuous feedback swept source. The black lines corre- spond to \|∆\| = γ √ 1 + α2 deﬁning the borders of the region within which the ECMs exist. The light green area indicates non-periodic output (regimes III and IV), the purple indicates sliding frequency self-mixing regime (regime I), and the red areas indicate sliding frequency mode locking (regime II). The other parameters are the same as in Fig. 4. 0.00 0.02 0.04 0.06 0.08 0.10 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 Δ rad.) 2. T. Klein and R. Huber, Biomed. Opt. Express 8, 828 (2017). 2. T. Klein and R. Huber, Biomed. Opt. Express 8, 828 (2017). 3. B. Johnson, W. Atia, M. Kuznetsov, B. D. Goldberg, P. Whitney, and D. C. Flanders, “Analysis of a spinning polygon wavelength swept laser,” (2015). ArXiv preprint arXiv:1501.07003. 4. R. Huber, M. Wojtkowski, and J. Fujimoto, Opt. Express 14, 3225 (2006). 5. D. D. John, C. B. Burgner, B. Potsaid, M. E. Robertson, B. K. Lee, W. J. Choi, A. E. Cable, J. G. Fujimoto, and V. Jayaraman, J. Light. Technol. 33, 3461 (2015). ( ) 6. B. Johnson, W. Atia, M. Kuznetsov, B. D. Goldberg, P. Whitney, and D. C. Flanders, Biomed. Opt. Express 8, 1045 (2017). Fig. 5. The two-dimensional map of the dynamical regimes for the continuous feedback swept source. The black lines corre- spond to \|∆\| = γ √ 1 + α2 deﬁning the borders of the region within which the ECMs exist. The light green area indicates non-periodic output (regimes III and IV), the purple indicates sliding frequency self-mixing regime (regime I), and the red areas indicate sliding frequency mode locking (regime II). The other parameters are the same as in Fig. 4. 7. B. Johnson, W. Atia, M. Kuznetsov, B. D. Goldberg, P. Whitney, and D. C. Flanders, Opt. express 26, 34909 (2018). 8. M. Bonesi, M. Minneman, J. Ensher, B. Zabihian, H. Sattmann, P. Boschert, E. Hoover, R. Leitgeb, M. Crawford, and W. Drexler, Opt. Express 22, 2632 (2014). 9. V. Jayaraman, Z.-M. Chuang, and L. A. FUNDING H2020-MSCA-IF-2017, ICOFAS project (800290); H2020-MSCA- RISE-2018 HALT; OPTIMAL project granted by the European Union by means of the Fond Européen de développement re- gional, FEDER; Government of Russian Federation (08-08). Coherence transfer in an akinetic swept source OCT laser with optical feedback can lead to the appearance of sliding frequency self-mixing and sliding-frequency mode locking similar to that observed in short cavity swept sources laser, which offers an opportunity of ex- ploiting schemes such as coherence revival [22]. The generation of sliding frequency mode locking delivering subnanosecond pulses is attractive for nonlinear optics applications [19]. While the power of the proposed technique has been demonstrated on the model of the multi-section semiconductor lasers, it can also be adapted to other swept source conﬁgurations exhibiting mode hops, or serve as a tool to couple several swept sources to increase the sweeping range with maintained coherence. REFERENCES Lyu, K. Karnowski, M. Wojtkowski, and G. Huyet, Opt. Express 22, 18177 (2014). 22. A.-H. Dhalla, D. Nankivil, and J. A. Izatt, Biomed. Opt. Express 3, 633 (2012). In conclusion, we have shown that it is possible to lock the phase of successive modes of a multi-section swept source semi- conductor laser using optical feedback. As a result of locking, the coherence of the laser does not deteriorate after the mode hops. In addition, we have demonstrated that optical feedback 23. C. M. Eigenwillig, W. Wieser, S. Todor, B. R. Biedermann, T. Klein, C Ji h k d R H b N t C 4 1848 (2013) 23. C. M. Eigenwillig, W. Wieser, S. Todor, B. R. Biedermann, T. Klein, C. Jirauschek, and R. Huber, Nat. Commun. 4, 1848 (2013). 24. S. Karpf and B. Jalali, Opt. Lett. 44, 1952 (2019).
https://openalex.org/W2123229312	https://eprints.soton.ac.uk/362985/1/Siddle%2520et%2520al.%2520-%25202009%2520-%2520MHC-linked%2520and%2520un-linked%2520class%2520I%2520genes%2520in%2520the%2520wallaby%25282%2529.pdf	English	null	MHC-linked and un-linked class I genes in the wallaby	BMC genomics	2,009	cc-by	12,576	BioMed Central BioMed Central BioMed Central Published: 14 July 2009 BMC Genomics 2009, 10:310 doi:10.1186/1471-2164-10-310 This article is available from: http://www.biomedcentral.com/1471-2164/10/310 This article is available from: http://www.biomedcentral.com/1 © 2009 Siddle et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2009 Siddle et al; licensee BioMed Central Ltd. ; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit BMC Genomics Open Access Open A Research article MHC-linked and un-linked class I genes in the wallaby Hannah V Siddle1, Janine E Deakin3, Penny Coggill2, Elizabeth Hart2, Yuanyuan Cheng1, Emily SW Wong1, Jennifer Harrow2, Stephan Beck4 and Katherine Belov1 Address: 1Faculty of Veterinary Science, University of Sydney, NSW 2006, Australia, 2Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton Hall, Hinxton, Cambridgeshire, CB10 1SA, UK, 3ARC Centre of Excellence for Kangaroo Genomics, Research School of Biological Sciences, Australian National University, Canberra, ACT 0200, Australia and 4UCL Cancer Institute, University College London, London WC1E 6BT, UK Email: Hannah V Siddle - h.siddle@usyd.edu.au; Janine E Deakin - deakin@rsbs.anu.edu.au; Penny Coggill - pcc@sanger.ac.uk; Elizabeth Hart - eah@sanger.ac.uk; Yuanyuan Cheng - y.cheng@usyd.edu.au; Emily SW Wong - e.wong@usyd.edu.au; Jennifer Harrow - jla1@sanger.ac.uk; Stephan Beck - s.beck@ucl.ac.uk; Katherine Belov - kbelov@vetsci.usyd.edu.au * Corresponding author Received: 22 October 2008 Accepted: 14 July 2009 Received: 22 October 2008 Accepted: 14 July 2009 Background mon ancestor ~80 million years ago and orthologous rela- tionships between their class I genes are not obvious, even where the function of the genes is homologous. g Major Histocompatibility Complex (MHC) class I anti- gens are responsible for the recognition of pathogenic peptides and form a complex gene family, which can vary in gene number and organization between different spe- cies. Although the function of class I genes varies, the structure of the functional molecule nearly always consists of an alpha chain divided into three extracellular domains (α1, α2 and α3), a transmembrane and cytoplasmic region and an associated β2 microglobulin [1]. MHC class Ia genes are found within the MHC region of all species studied to date, however, their organization within the MHC can vary between different species. In non-mammalian species, the MHC class I and class II genes are interspersed with the antigen processing genes (eg TAP) [20-23], which are responsible for transporting peptides from the cytoplasm into the endoplasmic reticu- lum, where they can be bound by class I molecules. It has been suggested that this ancestral organization of the MHC facilitated the co-evolution of advantageous class Ia and TAP haplotypes that conferred resistance to specific pathogens [20,21]. This organization may have provided a selective advantage to non-mammals with only one class Ia gene as duplication of class Ia would disrupt the co-evolution of TAP and class Ia alleles [21,24]. A single class Ia gene has been identified in the chicken MHC (B locus) [20] and the MHC of Xenopus [25]. However, mul- tiple class Ia genes have been described in detail within the quail MHC [7]. It has been suggested that there are multiple class Ia genes in other non-mammalian species, including the axolotl, but number and organization of genes has been estimated without genome sequencing [26]. Class I genes are classified according to function as classi- cal (class Ia) or non-classical (class Ib). Class Ia genes are responsible for pathogen recognition, and perform this function by binding endogenous foreign peptides and presenting them to cytotoxic T-cells on the surface of the cell. Class Ia molecules are ubiquitously expressed and the genes are highly polymorphic within the α1 and α2 domains, where peptide interaction occurs [2], consistent with their role in pathogen recognition. Background Although the number of class I genes found in different species can vary significantly, the number of expressed class Ia genes in eutherian mammals is reasonably consistent, varying between two to three [3] (the Rhesus macaque is a possi- ble exception [4]). Some non-mammals have only one highly expressed and polymorphic class Ia gene [5,6], while others have multiple class Ia genes [7]. Class Ib genes are related in sequence identity and molec- ular structure to the class Ia genes, but have lower expres- sion levels, tissue specific expression, low levels of polymorphism and often lack consensus residues impor- tant for peptide binding found in class Ia molecules [8]. Human class Ib genes (HLA-G, HLA-E, and HLA-F) do not play a prominent role in presenting antigens to T-cells, but have a variety of roles both related and unrelated to immune function. They interact with natural killer (NK) cells as part of the innate immune response [9,10] and are involved in regulation and suppression of the immune system [11,12]. The number of class Ib genes can vary sig- nificantly between species, even between eutherian mam- mals. Humans have three class Ib genes, while mice have over 30 [13]. Little is known about class Ib genes outside eutherian mammals, however, several class Ib sequences have been described in avians [14], amphibians [15] and one class Ib sequence has been described from a marsu- pial, the grey short-tailed opossum (Monodelphis domes- tica) (herein referred to as opossum) [16]. In the majority of eutherian species the class Ia genes are separated from the TAP genes by the class III region and are interspersed with the framework genes. The rat is an exception and has similarities with non-mammals, as the class Ia genes are linked to antigen processing genes [27]. The framework genes are found within the MHC of all mammals and are highly conserved in gene function and order. It is thought that the repositioning of class Ia genes away from the antigen processing genes and transporta- tion genes prevented the tight co-evolution of class Ia and TAP alleles, but also released constraints on the number of class Ia genes, allowing duplication of class Ia in eutherian mammals [21]. While the class Ia genes are found within the MHC region, class Ib genes have been found outside the MHC in a number of mammalian and non-mammalian species. Abstract Background: MHC class I antigens are encoded by a rapidly evolving gene family comprising classical and non-classical genes that are found in all vertebrates and involved in diverse immune functions. However, there is a fundamental difference between the organization of class I genes in mammals and non-mammals. Non-mammals have a single classical gene responsible for antigen presentation, which is linked to the antigen processing genes, including TAP. This organization allows co-evolution of advantageous class Ia/ TAP haplotypes. In contrast, mammals have multiple classical genes within the MHC, which are separated from the antigen processing genes by class III genes. It has been hypothesized that separation of classical class I genes from antigen processing genes in mammals allowed them to duplicate. We investigated this hypothesis by characterizing the class I genes of the tammar wallaby, a model marsupial that has a novel MHC organization, with class I genes located within the MHC and 10 other chromosomal locations. Results: Sequence analysis of 14 BACs containing 15 class I genes revealed that nine class I genes, including one to three classical class I, are not linked to the MHC but are scattered throughout the genome. Kangaroo Endogenous Retroviruses (KERVs) were identified flanking the MHC un-linked class I. The wallaby MHC contains four non-classical class I, interspersed with antigen processing genes. Clear orthologs of non-classical class I are conserved in distant marsupial lineages. Conclusion: We demonstrate that classical class I genes are not linked to antigen processing genes in the wallaby and provide evidence that retroviral elements were involved in their movement. The presence of retroviral elements most likely facilitated the formation of recombination hotspots and subsequent diversification of class I genes. The classical class I have moved away from antigen processing genes in eutherian mammals and the wallaby independently, but both lineages appear to have benefited from this loss of linkage by increasing the number of classical genes, perhaps enabling response to a wider range of pathogens. The discovery of non-classical orthologs between distantly related marsupial species is unusual for the rapidly evolving class I genes and may indicate an important marsupial specific function. Page 1 of 15 (page number not for citation purposes) BMC Genomics 2009, 10:310 http://www.biomedcentral.com/1471-2164/10/310 Page 2 of 15 (page number not for citation purposes) Background The open reading frames of the class I genes vary from 342 to 363 codons. ing scheme does not indicate orthology to other known marsupial class I genes, with the exception of Maeu-UM and Maeu-UK (see below). The class I genes have 48 – 88.4% amino acid identity to each other across the α1, α2 and α3 domains. The open reading frames of the class I genes vary from 342 to 363 codons. Previously, we reported the isolation (but not sequenc- ing) of nine tammar wallaby BACs containing class I genes that mapped to every autosome with the exception of chromosome 2, where the MHC class II and class III genes are located [32]. In this study four additional BACs containing class I genes were isolated and localized to the MHC region on chromosome 2. Six class I genes were dis- covered on the BACs that map to chromosome 2. These genes intersperse with antigen processing genes (PSMB8, PSMB9, TAP1B, TAP2B) and the class II gene (DMB). Nine class I genes with complete open reading frames were found on the BACs, which map outside the MHC to 9 chromosomal locations. The tammar wallaby is an Australian marsupial used as a model for studying the unique mode of marsupial repro- duction, where young are born after a short gestation period with no immune protection and undergo their development in a pouch. Marsupials are particularly use- ful for studying the evolution of immune genes in mam- mals as they last shared a common ancestor with eutherian mammals ~150 million years ago and fill an evolutionary gap in the vertebrate phylogeny between non-mammals and eutherian mammals [33]. A greater understanding of the immune genes of this species will allow comparisons to be made with the opossum, which last shared a common ancestor with the wallaby ~80 mil- lion years ago [34] and is the only marsupial for which the MHC has been fully annotated. The BACs which map outside the MHC also contain class I pseudogenes, identified due to frame shift mutations, absence of a start codon, or gene fragmentation, but not other genes that are usually encoded within the MHC. One possible exception is the presence two olfactory receptor genes found on MeKba_282L16 (chromosome 6p) along with Maeu-UB. Background A cluster of olfactory receptor genes is also found adjacent to the framework genes in eutherian mammals and the opossum [31,36], however, further analyses of these genes is required to determine the relationship of these olfactory receptors to the MHC- linked olfactory receptor genes in other species. Background In Xenopus and chicken, class Ib genes have been identified that are not linked to the MHC [15,28]. A more extreme case is that of the class I-like genes CD1, which are encoded outside the MHC in mammals and are very diver- gent in sequence and function to the class Ia and class Ib [29]. The relocation of class Ib genes away from the MHC in these examples is believed to have loosened the con- straints on their evolution allowing the genes to evolve independently, without influence of gene conversion events [30] and co-evolution of closely linked genes [24]. Class I genes evolve rapidly through gene duplication and divergence [17]. Due to their rapid evolution, class I genes have undergone species specific expansions and, unless the evolutionary relationship between species is very close (ie human and chimp), orthologous relationships between the class I genes of different species are difficult to detect [18,19]. Human and mouse last shared a com- Page 2 of 15 (page number not for citation purposes) BMC Genomics 2009, 10:310 http://www.biomedcentral.com/1471-2164/10/310 http://www.biomedcentral.com/1471-2164/10/310 http://www.biomedcentral.com/1471-2164/10/310 Studies have shown that the organization of the marsupial class I genes in the opossum (Monodelphis domestica) has similarity to both mammalian and non-mammalian pat- terns [31]. The class I are interspersed with the antigen processing rather than the framework genes, an organiza- tion similar to non-mammals. The opossum has three closely related class I genes that are polymorphic and highly expressed, however, only one class I gene, Modo- UA, has been classified as class Ia as the two closely related class I (Modo-UB &Modo-UC) are located outside the MHC and are not expressed at the same level as Modo-UA. The opossum also has six additional putative class Ib genes with unknown functions that are located within the MHC [31]. Class I genes do not localize to a single complex in two marsupials (the opossum and tammar wallaby (Mac- ropus eugenii) [31,32]. Two expressed class I genes (Modo- UB &Modo-UC) are located distal to the MHC at the end of the chromosome in the opossum, while in the tammar wallaby class I genes are MHC un-linked across six differ- ent chromosomes. ing scheme does not indicate orthology to other known marsupial class I genes, with the exception of Maeu-UM and Maeu-UK (see below). The class I genes have 48 – 88.4% amino acid identity to each other across the α1, α2 and α3 domains. There are two distinct groups of class I genes in the tammar wallaby The wallaby class I genes form two distinct groups, MHC- linked class I genes (Maeu-UE, UL, UK, UM, UO and UP) and MHC un-linked class I genes (Maeu-UA, UB, UC, UD, UF, UH, UI, UJ and UN). The MHC un-linked class I genes have greater amino acid identity to each other than to the six class I genes linked to the MHC, sharing on average 85% nucleotide identity with each other and 63% identity to the MHC-linked class I genes. There is one exception: Maeu-UP, located on chromosome 2, shares 72–75% nucleotide identity with the MHC un-linked class I genes and 61–67% identity to the MHC-linked class I genes. The MHC-linked class I genes share features of their amino acid sequence, including a deletion of varying size in the α1 domain between residues 59 and 66 (Additional file 2: Amino acid alignment of tammar wallaby class I genes, opossum class I genes and HLA-A) and a short intron between the α1 and α2 domains (data not shown), com- pared to the MHC un-linked class I genes. Here we report the sequencing of 14 tammar wallaby BACs containing 15 class I genes. Four BACs are located within the MHC proper on chromosome 2q while nine BACs localize to different chromosomes. We show that the class Ib genes remain linked to the MHC on chromo- some 2q and are interspersed with the class II and antigen processing genes, while the class Ia genes have MHC un- linked across the genome. Page 3 of 15 (page number not for citation purposes) Summary of sequenced BACs Fourteen BACs containing class I genes were sequenced and 15 class I genes with open reading frames have been annotated (summarized in Figure 1, Additional file 1 pro- vides a list of genes found on each BAC). The 15 class I genes described in this manuscript are named Maeu-UA through to Maeu-UP based on the nomenclature for class I genes proposed by Klein and colleagues [35]. The nam- Page 3 of 15 (page number not for citation purposes) Page 3 of 15 (page number not for citation purposes) BMC Genomics 2009, 10:310 http://www.biomedcentral.com/1471-2164/10/310 Schematic diagram of the genomic location and the annotated genes for each sequenced BAC Figure 1 Schematic diagram of the genomic location and the annotated genes for each sequenced BAC. The location of each sequenced BAC is indicated by purple stripes on chromosomes. The annotation of the sequenced BAC is shown next to its location. Red arrows indicate class I genes with open reading frames, blue arrows indicate non-class I genes with open read- ing frames, grey arrows indicate pseudogenes, green arrows indicate KERV sequences. A full list of genes and co-ordinates can be found in Additional file 1. Summary of sequenced BACs 5 7 3 4 6 1 2 Maeu-UP PSMB8 PSMB9 MHC Class I OR3 1 20000 40000 60000 80000 100000 120000 140000 158666 TAP2B MHC Class I PSMB8 TAP1B PSMB9 Maeu-UK MHC Class II (DM MEKBa_242G6 1 20000 40000 60000 80000 100000 120000 140000 160000 180806 Maeu-UM Maeu-UL Maeu-UE MEKBa_241L161 20000 40000 60000 80000 100000 120000 140000 160000 181574 Maeu-UO TAP2B MEKBa_146G20 1 20000 40000 60000 80000 100000 120000 140000 160000 172394 MEKBa_49O16 HNRPM MHC Class I MHC Class I MHC Class I MEKBa_192E16 1 20000 40000 60000 80000 100000 120000 140000 162330 Maeu-UF Maeu-UH MEKBa_211G21 1 20000 40000 60000 80000 100000 120000 140000 162599 Maeu-UD MHC Class I MEKBa_169B101 20000 40000 60000 80000 100000 120000140000 169595 MHC Class I Maeu-UN MEKBa_180C4 1 20000 40000 60000 80000 100000 120000140000 153855 Maeu-UB MHC Class I OR1 OR2 MEKBa_282L16 1 20000 40000 60000 80000 100000 120000 140000 160601 Maeu-UA MHC Class I MEKBa_248E8 1 20000 40000 60000 80000 100000 120000 140000 166457 Maeu-UI MHC Class I ARPP19 MEKBa_165K22 1 20000 40000 60000 80000 100000120000140000160000 186128 ARHGAP20 MHC Class I MHC Class I MEKBa_280H20 1 20000 40000 60000 80000 100000 120000 140000 159844 Maeu-UJ MHC Class I MEKBa_193K19 1 20000 40000 60000 80000 100000 120000 140000 160000 175272 Maeu-UC MEKBa_163M12 1 20000 40000 60000 80000 100000 120000 140000 161798 160000 GPR128 BMC Genomics 2009, 10:310 http://www.biomedcentral.com/1471-2164/10/310 1 2 ME ME ME Maeu-UJ MHC Class I MEKBa_193K19 1 20000 40000 60000 80000 100000 120000 140000 160000 175272 Maeu-UC MEKBa_163M12 1 20000 40000 60000 80000 100000 120000 140000 161798 2 4 5 7 6 Maeu-UD MHC Class I MEKBa_169B101 20000 40000 60000 80000 100000 120000140000 169595 MHC Class I Maeu-UN MEKBa_180C4 1 20000 40000 60000 80000 100000 120000140000 153855 Maeu-UB MHC Class I OR1 OR2 MEKBa_282L16 1 20000 40000 60000 80000 100000 120000 140000 16 Maeu-UA MHC Class I MEKBa_248E8 1 20000 40000 60000 80000 100000 120000 140000 166457 Maeu-UI MHC Class I ARPP19 MEKBa_165K22 1 20000 40000 60000 80000 100000120000140000160000 186128 ARHGAP20 MHC Class I MHC C MEKBa_280H20 1 20000 40000 60000 80000 100000 120000 140000 15 160000 GPR128 5 7 6 Maeu-UD MHC Class I MEKBa_169B101 20000 40000 60000 80000 100000 120000140000 169595 MHC Class I Maeu-UN MEKBa_180C4 1 20000 40000 60000 80000 100000 120000140000 153855 MEKBa_282L16 1 Maeu-UA MHC Class I MEKBa_248E8 1 20000 40000 60000 80000 100000 120000 140000 166457 Maeu-UI MHC Class I ARPP19 MEKBa_165K22 1 20000 40000 60000 80000 100000120000140000160000 186128 AR MEKBa_280H20 1 160000 GPR128 5 Schematic Figure 1 g g g q g Schematic diagram of the genomic location and the annotated genes for each sequenced BAC. The location of each sequenced BAC is indicated by purple stripes on chromosomes. The annotation of the sequenced BAC is shown next to its location. Red arrows indicate class I genes with open reading frames, blue arrows indicate non-class I genes with open read- ing frames, grey arrows indicate pseudogenes, green arrows indicate KERV sequences. A full list of genes and co-ordinates can be found in Additional file 1. wallaby (Macropus rufogriseus), brushtail possum (Tricho- surus vulpecula), Tasmanian devil (Sarcophilus harrisii) and tammar wallaby. Opossum class I sequences, including the Modo-UA (class Ia) and Modo-UG (class Ib) form a sis- ter clade (clade 2) to the MHC un-linked tammar wallaby class I genes. Interestingly, clade 3 contains the tammar wallaby MHC-linked class I genes (Maeu-UE, Maeu-UO, Maeu-UK, Maeu-UM and Maeu-UL), two class I sequences isolated from the brushtail possum(Trichosurus vulpecula) and the majority of the opossum class Ib sequences. This clade is basal to the opossum class Ia sequences and MHC un-linked tammar wallaby class I sequences. The MHC un-linked class I genes group together (clade 1), in a phylogenetic analysis, while MHC-linked class I genes on chromosome 2q form a separate and distinct clade (clade 3) (Figure 2). Again, Maeu-UP is an exception, occupying the basal position in clade 1. Although the position of Maeu-UP is not well supported using neigh- bour joining (Figure 2) or Bayesian analysis (Additional file 3: Phylogenetic tree showing divergence times for tam- mar wallaby class I genes and Additional file 4: Phyloge- netic tree produced for BEAST analysis) both methods produced trees with the same topology. Clade 1 also con- tains previously identified expressed class I transcripts from the Australian marsupials including the red-necked Page 4 of 15 (page number not for citation purposes) BMC Genomics 2009, 10:310 http://www.biomedcentral.com/1471-2164/10/310 ng phylogenetic analysis of the relationship between marsupial class I genes ning phylogenetic analysis of the relationship between marsupial class I genes. Bootstr provided. Estimated divergence times are indicated adjacent to the relevant node. The tammar wa u-I01 and Maeu-I02 are cDNA transcripts from Genbank. Schematic Figure 1 The phylogenetic tree is divided into t ncludes MHC un-linked wallaby class I genes and expressed class I transcripts from Australian mar sum class I genes only, including opossum class Ia (Modo-UA, UB and UC), clade 3 includes marsupi m both the American and Australian lineages. Maeu-UC _chr 1q Maeu-I01 Maeu-I02 Maeu-UB_chr 6p Maeu-UF_chr 4p Maeu-UJ_chr 1p Maeu-UI_chr 7q MaruUB01 Maeu-UD _chr 5p Maeu-UA_chr 7q Maeu-UH_chr 4p TruvUB01 Maeu-UN_chr 5q Saha-I01 Saha-I12 Maeu-UP_chr 2q Modo-UA Modo-UB Modo-UC Modo-UF Modo-UJ Modo-UG Maeu-UK_chr 2q Trvu19 Modo-UK Maeu-UM_chr 2q Modo-UM Modo-UE Modo-UL Maeu-UL_chr 2q Maeu-UE_chr 2q Maeu-UO _chr 2q Trvu20 M um u class I Feca class I HLA O ran-2-1 O ran-I-1 O ran-I-2 Gaga-I MIC A 100 100 100 100 75 53 99 99 100 99 74 100 99 100 60 99 99 61 100 67 99 74 99 100 100 95 56 74 48 90 46 68 99 48 67 35 82 29 15 20 13 0.1 Clade 1 Clade 2 Clade 3 87-141mya 80-129mya 65-107mya 100-160mya 59-97mya 142-207mya 51-85mya Clade 2 Maeu-UM_chr 2q Modo-UM Modo-UE Clade 3 Trvu20 Neighbour Figure 2 The MHC un-linked class I genes have characteristics of classical class I Nine MHC un-linked class I genes were found throughout the genome, three of these genes have characteristics of class Ia genes. Evidence of expression was found for seven of these genes, while two are either pseudogenes or have such low levels of expression that transcripts were not detected. A number of lines of evidence indicate that seven of these genes are expressed and that three are highly expressed and have a classical function. First, class I transcripts sequenced from blood and thymus samples identified from a number of individuals are most similar to MHC un-linked class I genes Maeu-UA (chr 7), Maeu- UB (chr 6), Maeu-UC (chr 1), Maeu-UD (chr 5), Maeu-UF (chr 4), Maeu-UH (chr 4) and Maeu-UN (chr 5) in a phy- logenetic tree (Additional file 5: Phylogenetic relationship between expressed class I transcripts and genomic class I genes). Between 7 and 13 sequences were isolated from every individual, indicating that at least seven class I genes are amplified, although assignment of sequences to loci was difficult due to high levels of sequence similarity and a level of polymorphism observed in the peptide binding region. The majority of transcripts sequenced from the blood and thymus samples were most similar to Maeu- UA, Maeu-UB and Maeu-UC. Class Ia genes have high lev- els of expression compared to class Ib genes and are more easily isolated from the transcriptome, thus the class I transcripts most readily isolated are most likely to be the class Ia genes. No transcripts showing high similarity to Maeu-UI and Maeu-UJ were identified suggesting that these loci may not be expressed. However, it cannot be ruled out that these genes are expressed at very low levels and not detected here. Transcripts similar to the class Ib genes on chromosome 2 are not amplified with these primers. Second, previously identified expressed class I genes isolated from Australian marsupials, including the red-necked wallaby, possum and Tasmanian devil, have a close relationship to the MHC un-linked wallaby class I Maeu-UK, Maeu-UM, Maeu-UE and Maeu-UO sequences were amplified from four wallabies of Kangaroo Island origin (I1, I2, I3 and I4. Again, no more than two alleles per gene were amplified from each individual (Table 1). The amplified alleles of Maeu-UK, Maeu-UM, Maeu-UE and Maeu-UO lacked variation at the peptide binding region with only a maximum of one non-synonymous substitution between the identified alleles of each gene. MHC-linked class I genes in the tammar wallaby appear marsupial specific MHC-linked class I genes in the tammar wallaby appear marsupial specific The topology of the phylogenetic tree clearly separates the opossum and Australian marsupial class I sequences of clades 1 and 2 from the class I sequences in clade 3. Rather than forming species specific clades, as is the norm of class I genes, the class I genes in clade 3 include class Ib genes of the tammar wallaby, brushtail possum and the opos- sum. Within this clade, two wallaby class I genes Maeu- UM and Maeu-UK are clearly orthologous to the opossum class Ib genes, Modo-UM (bootstrap of 100) and Modo-UK (bootstrap of 100). No orthologs for Modo-UE and Modo- UL were discovered in the wallaby, however, three closely related class Ib genes, Maeu-UE,UL and UO were identi- fied and cluster with Modo-UE and Modo-UL in clade 3. One of these genes, Maeu-UO, does have an orthologue in the brushtail possum. Six MHC-linked class I genes were identified, Maeu-UK, Maeu-UM, Maeu-UE, Maeu-UL, Maeu-UP and Maeu-UO. Four of these genes are expressed and have characteristics of class Ib genes, Maeu-UK, Maeu-UM, Maeu-UE and Maeu-UO. Gene specific primers were used to amplify each of these genes from the wallaby thymus and no more than two alleles was amplified for each primer set, sug- gesting the primers are gene specific. No expression was detected for Maeu-UP or Maeu-UL. Three of the MHC- linked class I genes were found to have tissue specific expression, Maeu-UM, Maeu-UE and Maeu-UL. Expression of Maeu-UM was found in wallaby lung, spleen and thy- mus, and transcripts of Maeu-UE and Maeu-UO were only detected in the thymus (Figure 3). Maeu-UK was widely expressed, found in all tissues examined, with the excep- tion of skin (liver, testes, lung, kidney, gut, skin spleen and thymus tissue (Figure 3). Neighbour Figure 2 g j g p y g y p p g g Neighbour-joining phylogenetic analysis of the relationship between marsupial class I genes. Bootstrap support for branches is provided. Estimated divergence times are indicated adjacent to the relevant node. The tammar wallaby class I sequences Maeu-I01 and Maeu-I02 are cDNA transcripts from Genbank. The phylogenetic tree is divided into the following clades; clade 1 includes MHC un-linked wallaby class I genes and expressed class I transcripts from Australian marsupials, clade 2 includes opossum class I genes only, including opossum class Ia (Modo-UA, UB and UC), clade 3 includes marsupial specific class I genes from both the American and Australian lineages. Page 5 of 15 (page number not for citation purposes) BMC Genomics 2009, 10:310 http://www.biomedcentral.com/1471-2164/10/310 Page 6 of 15 (page number not for citation purposes) The MHC un-linked class I genes have characteristics of classical class I Comparison of the level of polymorphism in these genes with the only other characterized marsupial class Ib, Modo-UG, revealed a similar level of polymorphism (Table 1). Expression profiles of MHC-linked class Ib genes Figure 3 Expression profiles of MHC-linked class Ib genes. A. Maeu-UM, B. Maeu-UE, C. Maeu-UK, D. Maeu-UO. The tissues tested are given as; Li – liver, T – testis, Lu – lung, K – kidney, G – gut, Sk – skin, Sp – spleen, Th – Thymus, N – negative, M – marker. The length of the amplified cDNA fragment is given next to the marker. A. B. C. D. Li T Lu K G Sk Sp Th N M 0.4Kb 0.3Kb 0.25Kb 0.3Kb A. Li T Lu K G Sk Sp Th N M 0.4Kb B. 0.3Kb C. 0.3Kb D. 0.25Kb D. p p g g Expression profiles of MHC-linked class Ib genes. A. Maeu-UM, B. Maeu-UE, C. Maeu-UK, D. Maeu-UO. The tissues tested are given as; Li – liver, T – testis, Lu – lung, K – kidney, G – gut, Sk – skin, Sp – spleen, Th – Thymus, N – negative, M – marker. The length of the amplified cDNA fragment is given next to the marker. Page 6 of 15 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/10/310 BMC Genomics 2009, 10:310 Table 1: Number of alleles for each Class Ib locus and number of polymorphic sites within the peptide binding region (PBR) Locus Number of alleles/ No. of individuals No. polymorphic sites within PBR Maeu-UM 3/4 1 Maeu-UE 2/4 0 Maeu-UK 3/4 1 Maeu-UO 2/4 0 Modo-UG 12/16 2 BACs. One full length KERV sequence was identified on chromosome 5p, adjacent to Maeu-UN. This KERV sequence had 90.6% nucleotide similarity to a previously described full length KERV (KERV-1) [39]. Numerous par- tial KERV sequences were identified at 1p, 3q, 4p, 5q and 6p and 7p (shown in Figure 1). The partial KERV sequences varied in length from 290 base pairs to 514 base pairs and shared between 67% and 99% nucleotide identity to the full length KERV on chromosome 5p. No significant matches to KERV were found within the BACs on chromosome 2. genes. The majority of these class I genes are thought to be class Ia genes, in particular Saha-I01 [37,38]. The MHC un-linked class I genes have characteristics of classical class I Finally, the promoter elements of the MHC un-linked class I genes are identifiable and highly similar in sequence identity to the promoter regions of class Ia genes in the opossum (Modo- UA) and eutherian mammals, while the promoters of the putative class Ib genes are more divergent (see detailed analysis below). Molecular dating of class I gene relocation Molecular dating was used to determine approximate tim- ing of the repositioning of class I genes away from the MHC in the tammar wallaby. The molecular dating per- formed in this study is treated as an estimate only as it does not take into account that MHC class I and class II genes evolve at a faster rate than neutral evolution would predict, due to balancing selection [40]. Maeu-UP is located within the MHC on chromosome 2 and is the most similar MHC-linked class I in sequence identity to the MHC un-linked class I genes. It is found at the base of clade 1, which contains putative classical class I sequences. Using a Bayesian approach for molecular dat- ing (see Methods), the MHC un-linked class I genes and Maeu-UP last shared a common ancestor ~86–140 mya. Maeu-UN on chromosome 5q3, the most basal MHC un- linked class I gene was the first class I gene to move away from chromosome 2, ~65–107 mya. The subsequent duplication of class I genes and their repositioning to dif- ferent chromosomal positions most likely occurred from this location with the most recent duplications and relo- cation occurring 15–38 mya. Promoter elements of class I genes Putative class I promoter elements were identified within 200 base pairs of the start codon for the class I genes and included TATA and CAAT sites, the S, X and Y regulatory motif, an interferon stimulated response element (ISRE) and an enhancer A site (Figure 4). The promoter elements of the MHC un-linked class I genes are highly similar to each other, sharing between 66–95% nucleotide identity in the 290 base pairs upstream of the gene start site. The promoter elements of the MHC un-linked class I are also similar to the regula- tory elements of Modo-UA, sharing 57–70% nucleotide iden- tity. However, Maeu-UJ is missing a TATA site and appears to have a truncated enhancer A element and Maeu-UH and Maeu-UI have no enhancer A element. These variations may account for the lack of evidence of transcription of Maeu-UJ and Maeu-UI. The upstream sequence of the class Ib, MHC- linked class I genes shared between 32–50% nucleotide identity with the MHC un-linked class I, 35–79% with one another and only 39–51% with the promoter sequence of Modo-UA, consistent with the more divergent nature of the coding sequence of these genes. However, it was still possible to identify the TATA, CAAT and SXY motifs in these genes, with the exception of Maeu-UK and Maeu-UP. Putative enhancer A and ISRE elements of the class Ib genes were identified with less confidence due to the lower sequence identity in the upstream sequence observed for these genes. Identification of Kangaroo Endogenous RetroViral (KERV) elements The organization of the Major Histocompatibility Com- plex of the wallaby is unique among mammals and pro- vides a model for understanding how genomic location affects gene function, co-expression and co-evolution. The Sequences with high similarity to Kangaroo Endogenous RetroViral (KERV) elements were identified on six of the Page 7 of 15 (page number not for citation purposes) Page 7 of 15 (page number not for citation purposes) BMC Genomics 2009, 10:310 http://www.biomedcentral.com/1471-2164/10/310 Promoter elements of MHC-linked and MHC-unlinked class I genes Figure 4 Promoter elements of MHC-linked and MHC-unlinked class I genes. 180 base pairs of sequence upstream from TATA box is shown for all wallaby class I genes, with the exception of Maeu-UK and Maeu-UP, for which promoter eleme could not be identified. The promoter elements for Modo-UA are also included. The boxed elements are, Enhancer A, an I stimulated response element (ISRE), S, X and Y motifs, a CAAT element and TATA box within 50 base pairs of the start s for each gene. http://www.biomedcentral.com/1471-2164/10/310 http://www.biomedcentral.com/1471-2164/10/310 BMC Genomics 2009, 10:310 BMC Genomics 2009, 10:310 rearrangement of genes. Three genes, ARHGAP20, GPR128 and HNRPM, located adjacent to MHC un-linked class I genes in the wallaby are in unexpected positions when compared to their homologues in the opossum genome and based on karyotype evolution of marsupials or gene mapping data (Table 2) [44,45]. These move- ments suggest these regions have been subject to rear- rangement in the wallaby. wallaby has 15 class I genes, 11 of which are transcribed. This figure is similar to the opossum, which has 11 class I genes, 9 of which are transcribed [31]. Of the 15 class I genes with open reading frames, three are putative class Ia genes (Maeu-UA (chr7), Maeu-UB (chr6) and Maeu-UC (chr1)), four are putative class Ib genes (Maeu-UM, Maeu- UK, Maeu-UE and Maeu-UO), four show no evidence of expression and the remaining genes appear to be expressed only at low levels and are not classified as class Ia or class Ib (Maeu-UD, Maeu-UF, Maeu-UH and Maeu- UN). The number of class Ia genes in the tammar wallaby is similar to eutherian species (usually 2 or 3). wallaby has 15 class I genes, 11 of which are transcribed. This figure is similar to the opossum, which has 11 class I genes, 9 of which are transcribed [31]. Of the 15 class I genes with open reading frames, three are putative class Ia genes (Maeu-UA (chr7), Maeu-UB (chr6) and Maeu-UC (chr1)), four are putative class Ib genes (Maeu-UM, Maeu- UK, Maeu-UE and Maeu-UO), four show no evidence of expression and the remaining genes appear to be expressed only at low levels and are not classified as class Ia or class Ib (Maeu-UD, Maeu-UF, Maeu-UH and Maeu- UN). The number of class Ia genes in the tammar wallaby is similar to eutherian species (usually 2 or 3). Retrotransposons can act as recombination hotspots in the genome via homologous recombination between ret- rotransposon sequences MHC un-linked on different chromosomes [46]. Phylogenetic analysis shows that wal- laby class I genes on different chromosomes may be more closely related to one another than to an adjacent class I (eg Maeu-UB-6q and Maeu-UF-4p). http://www.biomedcentral.com/1471-2164/10/310 A similar pattern emerges when the KERV fragments on different chromo- somes are compared and may indicate that the class I genes and KERVs are causing recombination hotspots within the genome, fueling class I gene diversification and polymorphism, and providing an advantage for the wal- laby MHC. The presence of numerous partial KERVs sug- gests these elements have not been recently active and that the original movement of a class I gene to chromosome 5 (perhaps by amplification of KERV and transposition) occurred early in the evolution of the Australian marsu- pial lineage, which is supported by the predicted date of movement, approximately 65 mya. It is likely that the tammar wallaby class Ia genes were originally located within the MHC on chromosome 2, interspersed with antigen processing genes. The first repo- sitioning of wallaby class I genes away from chromosome 2 led to the class I gene, Maeu-UN, at the telomeric end of chromosome 5q, given its basal position on the phyloge- netic tree (Figure 2). Molecular dating indicates that this repositioning occurred over 65 mya and that all the class Ia and class Ia-like genes trace back to this common ances- tor. Maeu-UP, located on chromosome 2, most likely gave rise to Maeu-UN as it is the class I gene most closely related to the MHC un-linked class I genes. We previously proposed that the Kangaroo Endogenous RetroViral ele- ments (KERV), found at the centromeres of all tammar wallaby chromosomes and near the telomere of chromo- some 5, may have played a role in the repositioning of class I genes by creating instability in the MHC region (which is located near a KERV-rich area at the centromere of chromosome 2) [32,41]. The results of this study con- firm the presence of partial KERV sequences located adja- cent to a number of the MHC un-linked class I genes (Figure 1) and provides support for the hypothesis that KERVs may have contributed to the duplication and movement of class I genes in the wallaby. All MHC un- linked class I genes described here are located at subtelo- meric or pericentric regions, which are known hotspots not only for segmental duplication [42], but also the insertion and retention of repeat sequences, illegitimate recombination and frequent rearrangements [43]. Promoter Figure 4 g g Promoter elements of MHC-linked and MHC-unlinked class I genes. 180 base pairs of sequence upstream from the TATA box is shown for all wallaby class I genes, with the exception of Maeu-UK and Maeu-UP, for which promoter elements could not be identified. The promoter elements for Modo-UA are also included. The boxed elements are, Enhancer A, an IFN stimulated response element (ISRE), S, X and Y motifs, a CAAT element and TATA box within 50 base pairs of the start site for each gene. Page 8 of 15 (page number not for citation purposes) Page 8 of 15 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/10/310 The conservation of orthologs, Modo-UM/Maeu-UM and Modo-UK/Maeu-UK, over such an extended period of time leads us to speculate that these genes may have a critical, marsupial-specific function. The marsupial mode of reproduction and development poses immunological challenges to the pouch young, which are born after a short gestation and are immuno-naïve at birth. We pro- pose that conserved marsupial class Ib molecules may play a role in the immunological protection of marsupial young. Maeu-UM is expressed in the lung as well as the spleen and thymus, and it is possible this molecule plays a role in the protection of exposed surfaces. Ongoing stud- ies in our laboratories will look into the function of these l l i f i l ary time suggests an advantage is gained in having class Ia genes organized in this way. It has been suggested that the repositioning of class Ia genes away from the antigen processing genes in mam- mals allowed the class Ia genes to duplicate [21]. We pro- pose that marsupials gained a similar advantage from moving class Ia genes outside the MHC. Some non-mam- mals have only one class Ia gene, which in species such as Xenopus [21], chicken [20] and nurse shark [22] is linked to the antigen processing genes within the MHC. This ancestral MHC organization has the advantage that TAP and class Ia alleles co-evolve [5]. For instance, the chicken TAP genes are polymorphic, with specific TAP alleles transporting peptides for specific class Ia alleles, providing an efficient response to certain pathogens [20]. Duplica- tion and subsequent diversification of such class Ia genes would therefore disrupt the advantageous linkage and resulting co-evolution between class Ia and TAP alleles [21,24]. Co-evolution of class I and TAP is not confined to non-mammals, but has been observed within the rat MHC as well [47]. However, in the majority of eutherian mammals the class Ia genes have moved away from the TAP genes to become interspersed with the framework genes of the MHC. The TAP genes are no longer polymor- phic, but have become promiscuous and can present a broader array of pathogenic peptides [24]. Marsupials and eutherian species, independently, found it advantageous to separate class Ia genes from antigen processing genes, resulting in differing class I organization between these lineages of mammals, but effectively the same outcome, multiple class Ia genes. http://www.biomedcentral.com/1471-2164/10/310 http://www.biomedcentral.com/1471-2164/10/310 http://www.biomedcentral.com/1471-2164/10/310 BMC Genomics 2009, 10:310 BMC Genomics 2009, 10:310 The MHC-linked class I genes in the tammar wallaby are non-classical class I genes, based on promoter elements, expression patterns and levels of polymorphism. The changes in promoter sequence of the class Ib genes in the tammar wallaby perhaps explains the variation in expres- sion patterns for these genes. Maeu-UM, UE, and UO have tissue-specific expression and are not polymorphic, with few substitutions in the PBR of different alleles. Maeu-UK has low levels of polymorphism and is expressed in all tis- sues. Maeu-UM and Maeu-UK are clear orthologs of the opossum class Ib genes, Modo-UM and Modo-UK, despite these species being separated by 80 million years of evo- lution and belonging to different Orders. An expressed transcript has been isolated from the brushtail possum (Trvu19) that is closely related to Maeu-UK and Modo-UK [49]. This transcript may represent a processed pseudog- ene as it has a frameshift mutation at the 5' end of the α1 domain [49]. However, expressed transcripts from the tammar wallaby show no evidence of this mutation and also include the leader peptide. Further, the frameshift mutation described in the possum sequence is very close to the forward primer site used to amplify this sequence. It is possible that the described mutation is a sequencing artifact, as conservation of a processed pseudogene in the possum is unlikely. As there is evidence that an ortholog of UK exists in the brushtail possum we predict all Austral- ian marsupials would share the UK class I gene. Orthology between class I genes of distantly related species is highly unusual for class I genes, which typically form species spe- cific clades due to their rapid evolution [50]. It has been proposed that MHC polymorphism can be passed to spe- cies as they evolve from a common ancestor [51], how- ever, no examples of class I orthology have been demonstrated between species belonging to different phy- logenetic orders [18,50]. The class I genes of human and mouse, separated by a similar evolutionary distance as the tammar wallaby and opossum, do not show clear orthol- ogy, despite some genes, such as HLA-E and H2-Qa1, shar- ing similar functions. It is believed their orthologous relationship is obscured by gene conversion [30]. http://www.biomedcentral.com/1471-2164/10/310 In the wallaby there is evidence of subtelomeric and pericentric The repositioning of class Ia genes out of the MHC in wal- laby ancestors over 65 million years ago indicates that class I genes are located outside the MHC in many other Australian species, including other macropods and the brushtail possum, as these species diverged less than 30 million years ago [34]. Previously we suggested that Modo- UB and Modo-UC were evolving towards class Ib genes due to their location outside the MHC [31]. Given that wallabies have non MHC-linked class Ia genes, it is feasi- ble that Modo-UB and UC are also classical given their close relationship to Modo-UA. The presence of class Ia genes, responsible for antigen presentation, outside the MHC of marsupials for an extended period of evolution- Page 9 of 15 (page number not for citation purposes) Table 2: Non-MHC genes on MHC un-linked Class I BACs and their predicted location in the wallaby Gene BAC location in wallaby Location in opossum Predicted location in wallaby HNRPM 3 3 4 [45] GPR128 5q (distal) 4 5q (proximal) [44] ARPP19 7 1 7 [45] ARHGAP20 6 4 5 [45] Table 2: Non-MHC genes on MHC un-linked Class I BACs and their predicted location in the wallaby Gene BAC location in wallaby Location in opossum Predicted location in wallaby HNRPM 3 3 4 [45] GPR128 5q (distal) 4 5q (proximal) [44] ARPP19 7 1 7 [45] ARHGAP20 6 4 5 [45] n MHC un-linked Class I BACs and their predicted location in the wallaby Table 2: Non-MHC genes on MHC un-linked Class I BACs and their predicted location in the wallaby http://www.biomedcentral.com/1471-2164/10/310 Page 10 of 15 (page number not for citation purposes) Phylogenetic and sequence analysis of class I genes The predicted, full length coding sequences of 15 class I genes were aligned with the following class I sequences from the NCBI database using ClustalW [53] with some manual adjustment: Tammar wallaby: Maeu-101, DQ304109; Maeu-102, DQ304110; Red-necked wal- laby: Maru UB01, L04952; Brushtail possum: Trvu UB01, AF359509; Trvu19, EU570828; Trvu20, EU570829; Tasmanian devil: Saha-I01, EF591089, Saha- http://www.biomedcentral.com/1471-2164/10/310 However, it should be noted that some non-mammalian species have multiple class Ia genes adjacent to TAP genes and may have diversified class I genes, while maintaining linkage with antigen processing genes [7]. The extensive duplication and dispersal of class I genes in the wallaby may have been assisted by their complete removal from the MHC and the constraints this region imposes. In eutherian mammals the class I genes are inter- spersed with the framework genes, which are highly con- served. The duplication and repositioning of class I genes in this region is believed to be restricted by these frame- work genes, as class I movement that disrupts these genes would be deleterious [48]. In the wallaby the class I genes have moved away from the MHC altogether and their duplication and repositioning is not restricted. Of the nine MHC un-linked class I genes, not all are expressed at the same level and three appear highly expressed and have evidence of polymorphism. Thus, there is still a restraint on the maximum number of class Ia genes due to the need to maintain a wide spectrum of T cells after thymic selec- tion. Page 10 of 15 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/10/310 BMC Genomics 2009, 10:310 I12, EF591100 & Saha-I13, EF591101; Opossum: Modo UA01, AF1255540, Modo UB01, AF522352, Modo UC01, AF522352, Modo-UF, Modo-UG, Modo- UJ, Modo-UE, Modo-UL, Modo-UM & Modo-UK can be found at http://bioinf.wehi.edu.au/opossum/seq/ Class_I.fa; Human: HLA-Cw1203, U06487, HLA- B0701, U21052 &; Mouse: Mumu H-2Db, U47325, Mumu H-2Dd U47326 & Mumu H-2Kb U47328; Platypus: Oran ClI, AY112715 (Oran-2-1), Oran-1-1, EU030443 (ABU86900.1), Oran-1-2,, EU030443 (ABU86901.1); Chicken: Gaga B-FIV, AF013491; Human: Hosa MICA, L14848. The nucleotide and amino acid identity between the sequences was calculated using Bioedit [54]. A neigh- bour joining tree was constructed with the nucleotide sequence of the α1, α2 and α3 domains (across 654 nucleotides) using Jukes-Cantor distance [40,55] with 1000 bootstraps in the Mega 4.0 software [56]. The phyl- ogenetic analysis was repeated with the α3 domain only and the α1 and α2 domains only to test for differences in relationships between different regions of the genes, but the topology of the tree was not altered. Conclusion The tammar wallaby has a novel MHC class I organiza- tion, which has successfully maintained functional, diverse class I genes. The class I genes do not follow con- ventional models of class I evolution as class Ib genes have remained linked to the MHC whereas the class Ia genes are not linked to the MHC but have maintained pol- ymorphism and high expression levels. This unique organization provides a model for studying the impor- tance of MHC gene clustering on gene function. Further- more, our analysis has shown that some marsupial class Ib genes are remarkably conserved within this highly adapted mammalian lineage. The important biological differences between marsupial and eutherian mammals in their mode of reproduction and the pathogenic pressures on their immune system can be used to provide insights into how class Ib function is adapted to the biology of a species. BAC selection and sequencing All BACs were confirmed to contain class I genes by southern blot analysis using a pre- viously described conserved class I exon 4 fragment as a probe [52]. This analysis was also used to determine which BACs were overlapping based on shared bands and redundant BACs were excluded from the sequencing proc- ess. In total, 14 BACs were selected, sequenced and anno- tated as previously described [36] and submitted under the following accession numbers to the Genbank data- base: MEKBa_248E8 [CU457750]; MEKBa_211G21 [CU302408]; MEKBa_242G6 [CU463018] MEKBa_146G20 [CU466525] MEKBa_49O16 [CU463996]; MEKBa_241L16 [CU463962]; MEKBa_282L16 [CU302415]; MEKBa_169B10 [CU302406]; MEKBa_180C4 [CU302373]; MEKBa_192K19 [CU302374]; MEKBa_165K22 [CU302372]; MEKBa_280H20 [CU302409]; MEKBa_192E16 [CU302407]; MEKBa_163M12 [CU302420]. Molecular dating of genes was accomplished using BEAST [57]. Calibration nodes were defined using divergence dates for tammar wallaby and opossum; brushtail possum and tammar wallaby; and block duplication dates of human HLA-C and HLA-B. The Yule process was used as tree prior. Three identical analyses were run to check for consistency between runs. Chain length was set to 10,000,000 and logged every 1,000 steps. Tracer [57] was used to analysis BEAST output. Trees were summarized using a burning value of 1,000. A maximum clade credi- bility summary tree was produced where node heights equaled mean heights. FigTree was used to produce the final tree. the following accession numbers to the Genbank data base: MEKBa_248E8 [CU457750]; MEKBa_211G21 [CU302408]; MEKBa_242G6 [CU463018] MEKBa_146G20 [CU466525] MEKBa_49O16 [CU463996]; MEKBa_241L16 [CU463962]; MEKBa_282L16 [CU302415]; MEKBa_169B10 [CU302406]; MEKBa_180C4 [CU302373]; MEKBa_192K19 [CU302374]; MEKBa_165K22 [CU302372]; MEKBa_280H20 [CU302409]; MEKBa_192E16 [CU302407]; MEKBa_163M12 [CU302420]. Promoter sequences were identified by performing a dot plot analysis [58] on the upstream nucleotide sequence (2000 bp) of Modo-UA and the wallaby class I genes to identify conserved regions of the sequence. Specific ele- ments were then identified manually by aligning the wal- laby class I genes to Modo-UA, mammalian (pig) and non- mammalian (duck) class I promoters using ClustalW. Nucleotide identity between promoter sequences were calculated as described above. KERV sequences were identified using BLAST (Basic Local Alignment Search Tool) with a previously identified full length KERV (KERV-1, Genbank-AF044909) sequence as the query. KERV sequences were extracted and aligned with KERV-1 using ClustalW. BAC selection and sequencing q g We have previously described the isolation and physical mapping of nine of the class I BACs described here from a 10× tammar wallaby BAC library (Arizona Genomics Institute) to chromosomes 1, 3, 4, 5, 6, and 7 [32]. An additional four class I BACs have now been isolated and physically mapped to chromosome 2q according to Deakin et al. (2007) [32]. All BACs were confirmed to contain class I genes by southern blot analysis using a pre- viously described conserved class I exon 4 fragment as a probe [52]. This analysis was also used to determine which BACs were overlapping based on shared bands and redundant BACs were excluded from the sequencing proc- ess. In total, 14 BACs were selected, sequenced and anno- tated as previously described [36] and submitted under the following accession numbers to the Genbank data- base: MEKBa_248E8 [CU457750]; MEKBa_211G21 [CU302408]; MEKBa_242G6 [CU463018] MEKBa_146G20 [CU466525] MEKBa_49O16 [CU463996]; MEKBa_241L16 [CU463962]; MEKBa_282L16 [CU302415]; MEKBa_169B10 [CU302406]; MEKBa_180C4 [CU302373]; MEKBa_192K19 [CU302374]; MEKBa_165K22 [CU302372]; MEKBa_280H20 [CU302409]; MEKBa_192E16 [CU302407]; MEKBa_163M12 [CU302420]. We have previously described the isolation and physical mapping of nine of the class I BACs described here from a 10× tammar wallaby BAC library (Arizona Genomics Institute) to chromosomes 1, 3, 4, 5, 6, and 7 [32]. An additional four class I BACs have now been isolated and physically mapped to chromosome 2q according to Deakin et al. (2007) [32]. All BACs were confirmed to contain class I genes by southern blot analysis using a pre- viously described conserved class I exon 4 fragment as a probe [52]. This analysis was also used to determine which BACs were overlapping based on shared bands and redundant BACs were excluded from the sequencing proc- ess. In total, 14 BACs were selected, sequenced and anno- tated as previously described [36] and submitted under the following accession numbers to the Genbank data- b M [C ] M G We have previously described the isolation and physical mapping of nine of the class I BACs described here from a 10× tammar wallaby BAC library (Arizona Genomics Institute) to chromosomes 1, 3, 4, 5, 6, and 7 [32]. An additional four class I BACs have now been isolated and physically mapped to chromosome 2q according to Deakin et al. (2007) [32]. Primer design Gene specific forward and reverse primers were designed to amplify the α1 and α2 domains of the MHC-linked class I genes, Maeu-UE (Primer set 1), Maeu- UK (Primer Page 11 of 15 (page number not for citation purposes) Page 11 of 15 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/10/310 BMC Genomics 2009, 10:310 http://www.biomedcentral.com/1471-2164/10/310 Second, to ensure all potential transcripts were isolated, the α3 reverse primer from primer set 6 and 5' GeneRacer primer (Invitrogen) were used to amplify expressed class I from the thymus sample of individual V with the follow- ing PCR conditions: Initial denaturation at 94.0°C for 3 min, followed by 29 cycles of 94.0°C for 30 s, 68°C for 30 s, and 72°C for 2 min, and a final extension at 72°C for 10 min. The resulting PCR products were cloned into the pGEM-T easy vector system (Promega) and 25 clones were sequenced in both directions. All sequences were quality checked in Sequencher 4.1.4 (GeneCodes) and all unique class I transcripts were compared to the genomic class I genes by constructing a neighbor joining tree with 1000 bootstraps in Mega 4.0. The resulting phylogenetic tree was used to determine class I transcripts highly similar to genomic class I genes and these class I transcripts have been submitted to Genbank with the accession numbers FJ238079 to FJ238088. set 2) Maeu-UO (Primer set 3) and Maeu-UM (Primer set 4) (Table 3). The forward primers were designed as close to the beginning of the α1 domain as possible and the reverse primers were designed as close to the end of the α2 domain as possible. Variation in the primer sites was nec- essary to maintain specificity for each gene. The nine MHC un-linked class I genes show a high level of nucle- otide identity, thus the design of locus specific primers for these genes was impossible. Instead, previously described tammar wallaby class I cDNA sequences isolated from the spleen [52] were used to design multi-locus primers that amplify the α1, α2 and α3 domains of the MHC un- linked class I genes (primer set 5). Expression studies on MHC un-linked class I genes RNA was extracted from the blood of four individuals (I– IV) (Tri-reagent BD, Sigma-Aldrich) and reverse tran- scribed (Superscript III, Invitrogen). RNA was extracted from the thoracic thymus of a fifth individual (V) and 5' RACE cDNA was made (GeneRacer, Invitrogen). Primer design To deter- mine which of the nine MHC un-linked class I genes are expressed two approaches were used. First, primer set 5 was used to amplify the α1, α2 and α3 domains of expressed class I genes from the blood of individuals I–IV using high fidelity taq polymerase (Platinum DNA taq polymerase high fidelity, Invitrogen) and the following PCR conditions: Initial denaturation at 94.0°C for 3 min, followed by 29 cycles of 94.0°C for 30 s, 60°C for 30 s, and 72°C for 2 min, and a final extension at 72°C for 10 min. Twenty-five clones were sequenced from individuals I–IV, a total of 125 clones. Expression studies on MHC-linked class I genes Expression studies on MHC-linked class I genes Gene specific primer sets (1–4), described above, were used to amplify Maeu-UE, Maeu-UO, Maeu-UP, Maeu-UM, and Maeu-UK from the thymus RACE cDNA of individual V, to determine if these genes are expressed. The PCR con- ditions for each gene showing evidence of expression were as follows: Maeu-UM – Initial denaturation at 94.0°C for 3 min, followed by 29 cycles of 94.0°C for 30 s, 63°C for 30 s, and 72°C for 1 min, and a final extension at 72°C for 10 min; Maeu-UK, Maeu-UE, Maeu-UO – Initial dena- turation at 94.0°C for 3 min, followed by 29 cycles of 94.0°C for 30 s, 60°C for 30 s, and 72°C for 1 min, and a Table 3: Primers used for class I gene amplification. Class I loci for which no expression was detected (Maeu-UP and Maeu-UL) were excluded. Class I locus Primers Maeu-UE α1F – ATG TGC CTG CAG AAA GTG TCT GCG Primer set 1 α2R – AT GGT TCA GGG CTC CTG AGT TCC Maeu-UK α1F – AGT AGT TAG AGA GAC GGA GCA CAC Primer set 2 α2R – GTA CTT CTG CAG CCA TTC AGT Maeu-UO α1F – CAA GAG ATA CCA GAT TAC TGG GA Primer set 4 α2R – CGT TCC CAG CGA TCC AAC TTA GA Maeu-UM α1F – GCG GGC CCA GAC TGG GGT TAG AG Primer set 4 α2R – ACG TTT AGG GCC ACG TTG TCC AAT MHC un-linked class I α1F – CACTCCATGAGGTATTTCGACA Primer set 5 α3R – GGCTCAGGCAGCCCCTCGTGC http://www.biomedcentral.com/1471-2164/10/310 BMC Genomics 2009, 10:310 approved final manuscript, with particular help from KB and JED. final extension at 72°C for 10 min. The amplified prod- ucts varied between 0.25 kb and 0.4 kb, depending on the primer set. The amplified products were cloned into the pGEM-T easy vector system (Promega) and six clones were sequenced in both the forward and reverse directions for each gene. No more than two alleles were amplified for each primer set. The sequences were quality checked in Sequencher 4.1.4 (GeneCodes) and aligned with the class I genes annotated on the BACs. All transcripts have been submitted to Genbank with the accession numbers FJ238064, FJ238067, FJ238071 and FJ238075. final extension at 72°C for 10 min. The amplified prod- ucts varied between 0.25 kb and 0.4 kb, depending on the primer set. Expression studies on MHC-linked class I genes The amplified products were cloned into the pGEM-T easy vector system (Promega) and six clones were sequenced in both the forward and reverse directions for each gene. No more than two alleles were amplified for each primer set. The sequences were quality checked in Sequencher 4.1.4 (GeneCodes) and aligned with the class I genes annotated on the BACs. All transcripts have been submitted to Genbank with the accession numbers FJ238064, FJ238067, FJ238071 and FJ238075. Polymorphism studies on MHC linked class I genes Polymorphism studies on MHC linked class I genes Primer sets 1–4 were used to amplify Maeu-UE, Maeu-UO, Maeu-UM and Maeu-UK from DNA of individuals I–V using the PCR conditions described above. Maeu-UL and Maeu-UP were excluded as expression was not detected for these genes (see below). The resulting fragment included exon 2, exon 3 and the intervening intron. The amplified fragments were cloned into a pGEM-T easy vector (Promega) and six clones from each individual were sequenced in both directions. The sequences were quality checked in Sequencher 4.1.4 (GeneCodes) before being imported into bioedit and compared for polymorphism. No more than two alleles were amplified for each gene. The unique alleles for each gene have been submitted to Genbank with the following accession numbers FJ238065, FJ238066, FJ238068, FJ238069, FJ238070, FJ23807, FJ238073, FJ238074, FJ238076 and FJ238077. Phylogenetic tree produced for BEAST analysis. Phylogenetic tree pro- duced for the BEAST analysis with posterior distribution values, 1 = 100%. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-310-S4.pdf] Additional file 3 Phylogenetic tree showing divergence times for tammar wallaby class I genes. Phylogenetic tree produced for the BEAST analysis with all diver- gence estimates and confidence belts in blue. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-310-S3.pdf] Additional file 2 Expression of Maeu-UE, Maeu-UO, Maeu-UM, and Maeu- UK was detected in the thymus. No evidence of expression was detected for Maeu-UL or Maeu-UP despite a range of PCR conditions trialed, so PCR conditions for these primer sets are not described here and further expression studies were not performed. The expression pattern of Maeu-UE, Maeu-UO, Maeu-UM, and Maeu-UK was then assessed in the spleen, testis, liver, lung, kidney and gut. RNA was extracted from the tissues of a single individual (Tri-reagent, Sigma-Aldrich) and reverse transcribed (Superscript III, Invitrogen). The RNA was not treated with DNase to enable the intron between exon 2 and exon 3 to be amplified and compared between different genes. Primer sets 1, 3, 4 and 5 were used to test for expression of each gene. Amino acid alignment of tammar wallaby class I genes, opossum class I genes and HLA-A. Amino acid alignment, the following features are marked, P – residue involved in peptide binding, – glycosylation site, c- cysteine residues forming disulphide bridges, a dash indicates a deletion in some tammar wallaby and opossum class Ib genes. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-310-S2.pdf] Additional file 5 Phylogenetic relationship between expressed class I transcripts and genomic class I genes. Neighbour joining phylogenetic tree comparing class I transcripts isolated from the tammar wallaby thymus and blood to genomic class I genes from BACs. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-310-S5.pdf] Acknowledgements This project was funded by an ARC grant to KB and SB and a Wellcome Trust grant (WT-084071) to SB. We thank Marilyn Renfree and Andrew Pask (University of Melbourne) and Cathy Herbert (University of Sydney) for wallaby samples, all members of the Sequencing Centre at the Well- come Sanger Institute and Rob Miller and Michelle Baker for sharing unpub- lished data and inspiring conversations. We also thank Anthony Papenfuss for his assistance creating Figure 1. HS is supported by a University of Syd- ney Postgraduate Award and received William and Catherine McIlrath Scholarship for travel to the Sanger Institute. PC, JH, EH and SB were sup- ported by the Wellcome Trust. Additional material Additional file 1 Table of all annotated genes. The co-ordinates of all genes and pseudo- genes annotated on each BAC. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-310-S1.xls] References 24. Kaufman J, Jacob J, Shaw I, Walker B, Milne S, Beck S, Salomonsen J: Gene organisation determines evolution of function in the chicken MHC. Immunol Rev 1999, 167:101-117. 1. Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC: Structure of the human class I histocompatibility antigen, HLA-A2. Nature 1987, 329:506-512. , 25. Ohta Y, Goetz W, Hossain MZ, Nonaka M, Flajnik MF: Ancestral 25. Ohta Y, Goetz W, Hossain MZ, Nonaka M, Flajnik MF: Ancestral organization of the MHC revealed in the amphibian Xeno- pus. J Immunol 2006, 176:3674-3685. organization of the MHC revealed in the amphibian Xeno- pus. J Immunol 2006, 176:3674-3685. g 2. Hughes A, Nei M: Pattern of nucleotide substitution at the major histocompatibility complex class I loci reveals over- dominant selection. Nature 1988, 335:1670170. 26. Sammut B, Du Pasquier L, Ducoroy P, Laurens V, Marcuz A, Tournefier A: Axolotl MHC architecture and polymorphism. Eur J Immunol 1999, 29:2897-2907. 3. Kelley J, Walter L, Trowsdale J: Comparative genomics of major histocompatibility complexes. Immunogenetics 2005, 56:683-695. J 27. Walter L, Tiemann C, Heine L, Gunther E: Genomic organization and sequence of the rat major histocompatibility complex class Ia gene RT1.Au. Immunogenetics 1995, 41:332. 4. Otting N, Heijmans CM, Noort RC, de Groot NG, Doxiadis GG, van Rood JJ, Watkins DI, Bontrop RE: Unparalleled complexity of the MHC class I region in rhesus macaques. Proc Natl Acad Sci USA 2005, 102:1626-1631. g g 28. Briles WE, Goto RM, Auffray C, Miller MM: A polymorphic system related to but genetically independent of the chicken major histocompatibility complex. Immunogenetics 1993, 37:408-414. 5. Kaufman J: Co-evolving genes in MHC haplotypes: the "rule" for nonmammalian vertebrates? Immunogenetics 1999, 50:228-236. p y p g 29. Jayawardena-Wolf J, Bendelac A: CD1 and lipid antigens: intrac- ellular pathways for antigen presentation. Curr Opin Immunol 2001, 13:109-113. 6. Flajnik MF, Kasahara M: Comparative genomics of the MHC: glimpses into the evolution of the adaptive immune system. Immunity 2001, 15:351-362. 30. Joly E, Rouillon V: The orthology of HLA-E and H2-Qa1 is hid- den by their concerted evolution with other MHC class I molecules. Biol Direct 2006, 1:2. y 7. Shiina T, Shimizu S, Hosomichi K, Kohara S, Watanabe S, Hanzawa K, Beck S, Kulski JK, Inoko H: Comparative genomic analysis of two avian (quail and chicken) MHC regions. J Immunol 2004, 172:6751-6763. 31. References Belov K, Deakin JE, Papenfuss AT, Baker ML, Melman SD, Siddle HV, Gouin N, Goode DL, Sargeant TJ, Robinson MD, et al.: Reconstruct- ing an ancestral mammalian immune supercomplex from a marsupial major histocompatibility complex. PLoS Biol 2006, 4:e46. 8. Stroynowski I, Lindahl KF: Antigen presentation by non-classical class I molecules. Curr Opin Immunol 1994, 6:38-44. 32. Deakin JE, Siddle HV, Cross JG, Belov K, Graves JA: Class I genes have split from the MHC in the tammar wallaby. Cytogenet Genome Res 2007, 116:205-211. p 9. Braud VM, Allan DS, O'Callaghan CA, Soderstrom K, D'Andrea A, Ogg GS, Lazetic S, Young NT, Bell JI, Phillips JH, et al.: HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 1998, 391:795-799. 33. Wakefield MJ, Graves JA: The kangaroo genome. Leaps and bounds in comparative genomics. EMBO Rep 2003, 4:143-147. 10. Lepin EJ, Bastin JM, Allan DS, Roncador G, Braud VM, Mason DY, Merwe PA van der, McMichael AJ, Bell JI, Powis SH, O'Callaghan CA: Functional characterization of HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors. Eur J Immunol 2000, 30:3552-3561. 34. Nilsson MA, Gullberg A, Spotorno AE, Arnason U, Janke A: Radia- tion of extant marsupials after the K/T boundary: evidence from complete mitochondrial genomes. J Mol Evol 2003, 57(Suppl 1):S3-12. ( pp ) 35. Klein J, Bontrop RE, Dawkins RL, Erlich HA, Gyllensten UB, Heise ER, Jones PP, Parham P, Wakeland EK, Watkins DI: Nomenclature for the major histocompatibility complexes of different species: a proposal. Immunogenetics 1990, 31:217-219. 11. Tripathi P, Naik S, Agrawal S: HLA-E and immunobiology of pregnancy. Tissue Antigens 2006, 67:207-213. p g y g , 12. Kovats S, Main EK, Librach C, Stubblebine M, Fisher SJ, DeMars R: A class I antigen, HLA-G, expressed in human trophoblasts. p g y g 12. Kovats S, Main EK, Librach C, Stubblebine M, Fisher SJ, DeMars R: A class I antigen, HLA-G, expressed in human trophoblasts. Science 1990, 248:220-223. p g g 12. Kovats S, Main EK, Librach C, Stubblebine M, Fisher SJ, DeMars R: A l I i HLA G d i h h bl class I antigen, HLA-G, expressed in human trophoblasts. Science 1990, 248:220-223. p p g 36. Stewart CA, Horton R, Allcock RJ, Ashurst JL, Atrazhev AM, Coggill P, Dunham I, Forbes S, Halls K, Howson JM, et al.: Complete MHC haplotype sequencing for common disease gene mapping. References Genome Res 2004, 14:1176-1187. 13. Kumanovics A, Fischer Lindahl K: Good copy, bad copy: choosing animal models for HLA-linked diseases. Curr Opin Genet Dev 2004, 14:258-263. 37. Siddle HV, Sanderson C, Belov K: Characterization of major his- tocompatibility complex class I and class II genes from the Tasmanian devil (Sarcophilus harrisii). Immunogenetics 2007, 59:753-60. 14. Moon DA, Veniamin SM, Parks-Dely JA, Magor KE: The MHC of the duck (Anas platyrhynchos) contains five differentially expressed class I genes. J Immunol 2005, 175:6702-6712. 15. Flajnik MF, Kasahara M, Shum BP, Salter-Cid L, Taylor E, Du Pasquier L: A novel type of class I gene organization in vertebrates: a large family of non-MHC-linked class I genes is expressed at the RNA level in the amphibian Xenopus. Embo J 1993, 12:4385-4396. 38. Lam MK, Belov K, Harrison GA, Cooper DW: An Mhc class I gene in the Australian brushtail possum (Trichosurus vulpecula). Immunogenetics 2001, 53:430-433. g 39. O'Neill RJ, O'Neill MJ, Graves JA: Undermethylation associated with retroelement activation and chromosome remodelling in an interspecific mammalian hybrid. Nature 1998, 393:68-72. 40. Piontkivska H, Nei M: Birth-and-death evolution in primate MHC class I genes: divergence time estimates. Mol Biol Evol 2003, 20:601-609. 16. Gouin N, Wright AM, Miska KB, Parra ZE, Samollow PB, Baker ML, Miller RD: Modo-UG, a marsupial nonclassical MHC class I locus. Immunogenetics 2006, 58:396-406. in an interspecific mammalian hybrid. Nature 1998, 393:68 72. 40. Piontkivska H, Nei M: Birth-and-death evolution in primate MHC class I genes: divergence time estimates. Mol Biol Evol 2003, 20:601-609. g 17. Nei M, Gu X, Sitnikova T: Evolution by the birth-and-death process in multigene families of the vertebrate immune sys- tem. Proc Natl Acad Sci USA 1997, 94:7799-7806. 41. Ferreri GC, Marzelli M, Rens W, O'Neill RJ: A centromere-specific retroviral element associated with breaks of synteny in macropodine marsupials. Cytogenet Genome Res 2004, 107:115-118. , 18. Rogers JH: Mouse histocompatibility-related genes are not conserved in other mammals. Embo J 1985, 4:749-753. J 19. Adams EJ, Parham P: Species-specific evolution of MHC class I genes in the higher primates. Immunol Rev 2001, 183:41-64. 42. Bailey JA, Yavor AM, Viggiano L, Misceo D, Horvath JE, Archidiacono N, Schwartz S, Rocchi M, Eichler EE: Human-specific duplication and mosaic transcripts: the recent paralogous structure of chromosome 22. Am J Hum Genet 2002, 70:83-100. g g p 20. http://www.biomedcentral.com/1471-2164/10/310 BMC Genomics 2009, 10:310 Authors' contributions KB and SB designed the project. HVS isolated BACs, car- ried out phylogenetic and sequence analysis of class I genes and promoters, carried out polymorphism and expression studies and drafted the manuscript. JED iso- lated BACs, carried out FISH and isolated KERV sequences. PC fingerprinted BACs. EH and JH annotated BACs. YC assisted with polymorphism studies. ESWW car- ried out molecular dating. All authors edited and Page 13 of 15 (page number not for citation purposes) Page 14 of 15 (page number not for citation purposes) References Joly E, Le Rolle AF, Gonzalez AL, Mehling B, Stevens J, Coadwell WJ, Hunig T, Howard JC, Butcher GW: Co-evolution of rat TAP transporters and MHC class I RT1-A molecules Curr Biol 1998 transporters and MHC class I RT1-A molecules. Curr Biol 1998, 8:169-172. 48. Amadou C: Evolution of the Mhc class I region: the framework hypothesis. Immunogenetics 1999, 49:362-367. 49. Holland OJ, Cowan PE, Gleeson DM, Chamley LW: Identification of novel major histocompatibility complex class I sequences in a marsupial, the brushtail possum (Trichosurus vulpec- ula). Immunogenetics 2008, 60:609-619. ) g 50. Hughes AL, Nei M: Evolution of the major histocompatibility complex: independent origin of nonclassical class I genes in different groups of mammals. Mol Biol Evol 1989, 6:559-579. g p 51. Figueroa F, Gunther E, Klein J: MHC polymorphism pre-dating speciation. Nature 1988, 335:265-267. 52. Siddle HV, Deakin JE, Baker ML, Miller RD, Belov K: Isolation of major histocompatibility complex Class I genes from the tammar wallaby (Macropus eugenii). Immunogenetics 2006, 58:487-493. 53. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22:4673-4680. g 54. Hall T: Bioedit: A user friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symposium Serials 1999, 41:95-98. y p 55. Kimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucle- otide sequences. J Mol Evol 1980, 16:111-120. 56. Kumar S, Tamura K, Nei M: MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 2004, 5:150-163. g f f 57. Drummond AJ, Rambaut A: BEAST: Bayesian evolutionary anal- ysis by sampling trees. BMC Evol Biol 2007, 7:214. 8 S i i i 58. Sonnhammer EL, Durbin R: A dot-matrix program with dynamic threshold control suited for genomic DNA and protein sequence analysis. Gene 1995, 167:GC1-10. References Kaufman J, Milne S, Gobel TW, Walker BA, Jacob JP, Auffray C, Zoorob R, Beck S: The chicken B locus is a minimal essential major histocompatibility complex. Nature 1999, 401:923-925. J 43. Warren WC, Hillier LW, Marshall Graves JA, Birney E, Ponting CP, Grutzner F, Belov K, Miller W, Clarke L, Chinwalla AT, et al.: Genome analysis of the platypus reveals unique signatures of evolution. Nature 2008, 455:256. j p y p 21. Nonaka M, Namikawa C, Kato Y, Sasaki M, Salter-Cid L, Flajnik MF: Major histocompatibility complex gene mapping in the amphibian Xenopus implies a primordial organization. Proc Natl Acad Sci USA 1997, 94:5789-5791. 44. Deakin JE, Koina E, Waters PD, Doherty R, Patel VS, Delbridge ML, Dobson B, Fong J, Hu Y, Hurk C van den, et al.: Physical map of two tammar wallaby chromosomes: a strategy for mapping in non-model mammals. Chromosome Res 2008, 16:1159-1175. 22. Ohta Y, McKinney EC, Criscitiello MF, Flajnik MF: Proteasome, transporter associated with antigen processing, and class I genes in the nurse shark Ginglymostoma cirratum: evidence for a stable class I region and MHC haplotype lineages. J Immunol 2002, 168:771-781. 45. Rens W, O'Brien PC, Yang F, Solanky N, Perelman P, Graphodatsky AS, Ferguson MW, Svartman M, De Leo AA, Graves JA, Ferguson- Smith MA: Karyotype relationships between distantly related 45. Rens W, O'Brien PC, Yang F, Solanky N, Perelman P, Graphodatsky AS, Ferguson MW, Svartman M, De Leo AA, Graves JA, Ferguson- Smith MA: Karyotype relationships between distantly related 23. Sambrook JG, Figueroa F, Beck S: A genome-wide survey of Major Histocompatibility Complex (MHC) genes and their paralogues in zebrafish. BMC Genomics 2005, 6:152. Page 14 of 15 (page number not for citation purposes) BMC Genomics 2009, 10:310 http://www.biomedcentral.com/1471-2164/10/310 http://www.biomedcentral.com/1471-2164/10/310 marsupials from South America and Australia. Chromosome Res 2001, 9:301-308. marsupials from South America and Australia. Chromosome Res 2001, 9:301-308. 46. Kulski JK, Gaudieri S, Martin A, Dawkins RL: Coevolution of PERB11 (MIC) and HLA class I genes with HERV-16 and ret- roelements by extended genomic duplication. J Mol Evol 1999, 49:84-97. 47. Joly E, Le Rolle AF, Gonzalez AL, Mehling B, Stevens J, Coadwell WJ, Hunig T, Howard JC, Butcher GW: Co-evolution of rat TAP transporters and MHC class I RT1-A molecules. Curr Biol 1998, 8:169-172. 47. References Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 15 of 15 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge Page 15 of 15 (page number not for citation purposes)
https://openalex.org/W1548054819	https://rbmfc.org.br/rbmfc/article/download/565/425	Portuguese	null	Atribuições do profissional da Educação Física no campo da Saúde	Revista Brasileira de Medicina de Família e Comunidade	2,012	cc-by	401	Rev bras med fam comunidade. Florianópolis, 2012 Jun; 7 Supl1: 30 Palavras-chave: Educação Física. Terapias Complementares. Saúde. Atribuições do profissional da Educação Física no campo da Saúde Atribuições do profissional da Educação Física no campo da Saúde Atribuições do profissional da Educação Física no campo da Saúde Julio Mizuno1, Henrique Luiz Monteiro1 1PG em Ciências da Motricidade, Instituto de Biociências, Unesp/RC. E-mails: julio_mizuno@ig.com.br, heu@fc.unesp.br Introdução: O processo de institucionalização das Racionalidades Médicas (RM) e das Práticas Integrativas e Complementares (PIC) em Saúde depende da participação conjunta de diversos profissionais, entre eles, o Educador Físico (EF), que pode atuar com práticas corporais e atividade física (PCAF), além da acupuntura. Objetivo: Descrever as atribuições e responsabilidades do EF no campo e área da saúde. Metodologia: Leitura e análise dos documentos: Estatuto do Conselho Federal de Educação Física (CONFEF,1998); Política Nacional de Promoção da Saúde (PNPS, 2006); Política Nacional de Práticas Integrativas e Complementares (PNPIC, 2006) e Diretrizes do Núcleo de apoio a Saúde da Família (DNASF, 2009). Resultados: Após a leitura dos documentos supracitados, apresentamos trechos e sínteses relacionados ao EF. Dentre as atribuições do Estatuto do CONFEF, observamos, “... relaxamento corporal, ioga,... e outras práticas corporais... que favoreçam o desenvolvimento da educação e da saúde, ..., visando à consecução do bem estar e da qualidade de vida, da consciência, da expressão, ..., da autonomia, da auto-estima, da cooperação, da integração, das relações sociais,...”. Em posterior resolução ficou reconhecida “... a possibilidade de utilização da Técnica de Acupuntura”. A PNPS propõe como ações na rede básica de saúde, “ofertar práticas corporais / atividade física”. A Medicina Tradicional Chinesa é indicada na PNPIC, que também sugere a acupuntura, liang gong, chi gong e tai-chi-chuan para prevenção de agravos e doenças, promoção e recuperação da saúde. A DNASF sugere que o EF seja responsável pelas práticas de ling gong, tai-chi-chuan e automassagem ou do-in, orientado na construção e fortalecimento da autonomia dos cidadãos, na busca da qualidade de vida. Conclusão: O EF é um dos protagonistas na institucionalização de RM e PIC no campo e área da saúde, destacando-se o potencial das PCAF no processo de prevenção de agravos, manutenção e restauração da saúde. Recomenda-se, entretanto, evitar os conteúdos técnico-pedagógicos e a ênfase na prática de exercícios físicos atrelados à performance humana e à avaliação antropométrica, valorizando a cultura corporal de movimento e o desenvolvimento integral do ser humano. Palavras-chave: Educação Física. Terapias Complementares. Saúde.
https://openalex.org/W4391913539	https://bmcplantbiol.biomedcentral.com/counter/pdf/10.1186/s12870-024-04785-3	English	null	Trichoderma cf. asperellum and plant-based titanium dioxide nanoparticles initiate morphological and biochemical modifications in Hordeum vulgare L. against Bipolaris sorokiniana	BMC plant biology	2,024	cc-by	15,908	© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. BMC Plant Biology BMC Plant Biology Metwally et al. BMC Plant Biology (2024) 24:118 https://doi.org/10.1186/s12870-024-04785-3 Open Access Trichoderma cf. asperellum and plant‑based titanium dioxide nanoparticles initiate morphological and biochemical modifications in Hordeum vulgare L. against Bipolaris sorokiniana Rabab A. Metwally1, Shereen A. Soliman1, Hanan Abdalla1 and Reda E. Abdelhameed1 Rabab A. Metwally1, Shereen A. Soliman1, Hanan Abdalla1 and Reda E. Abdelhameed1 Abstract Background Spot blotch is a serious foliar disease of barley (Hordeum vulgare L.) plants caused by Bipolaris soro- kiniana, which is a hemibiotrophic ascomycete that has a global impact on productivity. Some Trichoderma spp. is a promising candidate as a biocontrol agent as well as a plant growth stimulant. Also, the application of nanomate- rials in agriculture limits the use of harmful agrochemicals and helps improve the yield of different crops. The current study was carried out to evaluate the effectiveness of Trichoderma. cf. asperellum and the biosynthesized titanium dioxide nanoparticles (TiO2 NPs) to manage the spot blotch disease of barley caused by B. sorokiniana and to assess the plant’s innate defense response. Results Aloe vera L. aqueous leaf extract was used to biosynthesize TiO2 NPs by reducing TiCl4 salt into TiO2 NPs and the biosynthesized NPs were detected using SEM and TEM. It was confirmed that the NPs are anatase-crystalline phases and exist in sizes ranging from 10 to 25 nm. The T. cf. asperellum fungus was detected using morphological traits and rDNA ITS analysis. This fungus showed strong antagonistic activity against B. sorokiniana (57.07%). Addition- ally, T. cf. asperellum cultures that were 5 days old demonstrated the best antagonistic activity against the pathogen in cell-free culture filtrate. Also, B. sorokiniana was unable to grow on PDA supplemented with 25 and 50 mg/L of TiO2 NPs, and the diameter of the inhibitory zone increased with increasing TiO2 NPs concentration. In an in vivo assay, bar- ley plants treated with T. cf. asperellum or TiO2 NPs were used to evaluate their biocontrol efficiency against B. sorokini- ana, in which T. cf. asperellum and TiO2 NPs enhanced the growth of the plant without displaying disease symptoms. Furthermore, the physiological and biochemical parameters of barley plants treated with T. cf. asperellum or TiO2 NPs in response to B. sorokiniana treatment were quantitively estimated. Hence, T. cf. asperellum and TiO2 NPs improve the plant’s tolerance and reduce the growth inhibitory effect of B. sorokiniana. Conclusion Subsequently, T. cf. asperellum and TiO2 NPs were able to protect barley plants against B. sorokiniana via enhancement of chlorophyll content, improvement of plant health, and induction of the barley innate defense system. The present work emphasizes the major contribution of T. cf. asperellum and the biosynthesized TiO2 NPs Correspondence: Rabab A. Metwally rababmicro2009@gmail.com Full list of author information is available at the end of the article Background Barley (Hordeum vulgare L.) is the world’s fourth most- produced cereal [1, 2], coming after wheat, rice, and corn. According to estimates by Triticase et al. [3], 21% of barley production was used in the malting and brew- ing industries, 70% went towards animal feed, and only roughly 6% was consumed by humans.i Since they are considered symbiotic, opportunistic, and non-virulent, Trichoderma spp. have been used as biological control agents against plant pathogenic fungi instead of synthetic pesticides [23–25]. Their biological control strategies involve activating various processes, either indirectly through the competition for resources and space, the stimulation of plant growth and defense systems, or directly through mycoparasitism and anti- biosis [26, 27]. Some Trichoderma spp. is associated with numerous plants through endophytic associations and can colonize the root surface [28]. Because of this symbiotic association, the plant is efficiently protected from pathogens [29, 30], where Trichoderma causes the expression of genes involved in plants’ defensive mechanisms when it interacts with them [31, 32] and stimulates root development and plant growth [33–36]. For example, Morais et al. [27] investigated that Tricho- derma spp. was used to assess the antagonistic activ- ity against Colletotrichum truncatum, Lasiodiplodia theobromae, Sclerotium delphinii, and Macrophomina phaseolina. Also, T. viride and T. harzianum evidenced high efficiency against A. alternata and Drechslera hal- odes [37]. When employed to control R. solani and F. oxysporum f. sp. lycopersici in tomato plants, T. atrob- runneum and T. simmonsii significantly increased stem height and fresh weight in pathogen-treated tomato plants [38]. In agriculture, plant diseases contribute significantly to the depletion of natural resources and are thought to be a significant factor in global food production’s yearly decline [4, 5]. In particular, soil-borne pathogens pose a serious threat, with fungi being the most active [6]. Due to changes in agricultural practices over the past few years, the proliferation of various phytopathogenic fungi, including Fusarium spp, Rhizoctonia spp, Alternaria spp, Botrytis spp, and Helminthosporium spp. has been dam- aging to crops with significant economic losses [7–12]. Barley is an excellent example of such crops that are exposed to various fungal pathogens, among which Bipo- laris sorokiniana causes leaf blight (also known as spot blotch), black point, and other foliar and root diseases. p Spot blotch disease is one of the most serious diseases of barley and can reduce yield by more than 30% and have an impact on malting quality [13, 14]. Key points • T. cf. asperellum showed strong antagonistic activity against B. sorokiniana invitro. • T. cf. asperellum showed strong antagonistic activity against B. sorokiniana invitro. • Plant- based synthesis of TiO2 NPs (10- 25 nm) using Aloe vera L. aqueous leaf extract. • B. sorokiniana triggers morphological and biochemical changes in barley plants, causing spot blotch disease. • T. cf. asperellum or green synthesized TiO2 NPs positively increased the host plant’s tolerance against this disease by inducing of osmolytes and antioxidant defense-related enzyme production. K d A i i i i A i id i B l Bi l C ll f fil G h i • Plant- based synthesis of TiO2 NPs (10- 25 nm) using Aloe vera L. aqueous leaf extract. • B. sorokiniana triggers morphological and biochemical changes in barley plants, causing spot blo • B. sorokiniana triggers morphological and biochemical changes in barley plants, causing spot blotch disease. • T. cf. asperellum or green synthesized TiO2 NPs positively increased the host plant’s tolerance against this disease by inducing of osmolytes and antioxidant defense-related enzyme production. • T. cf. asperellum or green synthesized TiO2 NPs positively increased the host plant’s tolerance aga by inducing of osmolytes and antioxidant defense-related enzyme production. Keywords Antagonistic activity, Antioxidative enzymes, Barley, Biocontrol agent, Cell free filtrate, Green synthesis, Spot blotch disease, Trichoderma cf. asperellum plant development, combating soil-borne pathogens, and stimulating the plant’s immune system [20–22]. © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Metwally et al. BMC Plant Biology (2024) 24:118 Page 2 of 19 Metwally et al. BMC Plant Biology to the management of spot blotch disease in barley plants, and ultimately to the enhancement of barley plant quality and productivity. Key points • T. cf. asperellum showed strong antagonistic activity against B. sorokiniana invitro. • Plant- based synthesis of TiO2 NPs (10- 25 nm) using Aloe vera L. aqueous leaf extract. • B. sorokiniana triggers morphological and biochemical changes in barley plants, causing spot blotch disease. • T. cf. asperellum or green synthesized TiO2 NPs positively increased the host plant’s tolerance against this disease by inducing of osmolytes and antioxidant defense-related enzyme production. Keywords Antagonistic activity, Antioxidative enzymes, Barley, Biocontrol agent, Cell free filtrate, Green synthesis, Spot blotch disease, Trichoderma cf. asperellum to the management of spot blotch disease in barley plants, and ultimately to the enhancement of barley plant quality and productivity. Background It happens in warm, humid areas of the world. B. sorokiniana fungus may infect the plant’s coleoptiles, crowns, culms, leaves, and roots [15]. Small brown patches that are first caused by spot blotches grow into dark brown blotches [16]. A zone of yellow leaf tissue with different widths that separates leaf spots may cause the production of shriveled seeds and reduce yield [14, 17]. Management of spot blotch disease has relied on the application of fungicides, cultural prac- tices, disease-resistant cultivars, and biological control agents by beneficial microbes [18–20]. However, its man- agement by beneficial microorganisms is a promising bio- control strategy, as these beneficial microorganisms are essential for increasing nutrient availability, promoting Another way to enhance the defense machinery of plants is through the use of modern technologies, i.e. nanotechnology, which can be extremely helpful in addressing this problem by restricting the use of damag- ing agrochemicals and assisting in boosting the yield of different crops [39, 40]. Nanoparticles (NPs) are recog- nized as a plant growth stimulant that modifies physi- ological, biochemical, and physicochemical pathways [41, 42]. Recently, titanium dioxide nanoparticles (TiO2 Metwally et al. BMC Plant Biology (2024) 24:118 Metwally et al. BMC Plant Biology (2024) 24:118 Page 3 of 19 Fig. 1 Morphological and molecular characterization of (A) T. cf. asperellum (biocontrol agent): (a) colony on PDA, (b) vegetative mycelium microstructure showing conidiophore and phialides, (c) phylogenetic tree (B) B. sorokiniana (pathogen): (a) colony on PDA, (b) vegetative mycelium microstructure showing hyphae and conidia, (c) phylogenetic tree NPs), a type of metal oxide nanomaterial, have gained popularity as an environmentally friendly and clean pho- tocatalyst due to their optical qualities, chemical stabil- ity, and non-toxicity [43–45]. These NPs have powerful oxidizing properties that produce free radicals like super- oxide anion radicals, which inhibit the growth of micro- organisms. As a result, they can be used in the agriculture industry to protect plants and inactivate various patho- genic infections [46]. By taking advantage of the reducing qualities of plant secondary metabolites, green synthesis of NPs can advantageously synthesize functional NPs [45, 47]. These benefits include producing biologically active nanomaterials, using inexpensive reactants, and hav- ing an environmentally favorable synthesis process [40]. TiO2 NPs made from plants have the potential to decrease the severity of diseases and stimulate plant growth by quickly absorbing on the surface of the plant and pathogens [48]. In vitro antifungal assay T f ll In vitro antifungal assay T. cf. asperellum was isolated from the rhizosphere of plants grown in the Egyptian soil and showed an inhibitory effect against B. sorokiniana. The dual cul- ture test illustrated in Fig. 2A showed the inhibition zone between B. sorokiniana and T. cf. asperellum, where T. cf. asperellum exhibited strong antagonistic activity against B. sorokiniana (57.07%) that proved its potent activity as compared to B. sorokiniana (Fig. 2B). Also, Fig. 2 (C) showed the antagonistic effect of T. cf. asperellum cell-free culture filtrate against B. sorokini- ana. Figure 2 (D-F) showed the mycoparasitism of T asperellum against mycelium of B. sorokiniana, inten- sively vacuolated deformed mycelium of B. sorokini- ana and T. cf. asperellum and B. sorokiniana hyphal interaction. Background Currently, it is crucial to recog- nize the plant’s defense mechanism against fungal pathogens. Few scientific papers have discussed the function of TiO2 NPs in reducing disease severity and their effects on plant biochemistry and productivity in response to fungal stress [49]. Hence, in this study, we attempted to go deeply into investigating the impact of T. cf. asperellum and green synthesized TiO2 NPs on the growth of barley plants and the severity of spot blotch disease caused by B. sorokiniana. As well, their effects on biochemical analysis and the defense-related enzymes in barley plants were also evaluated. Fig. 1 Morphological and molecular characterization of (A) T. cf. asperellum (biocontrol agent): (a) colony on PDA, (b) vegetative mycelium microstructure showing conidiophore and phialides, (c) phylogenetic tree (B) B. sorokiniana (pathogen): (a) colony on PDA, (b) vegetative mycelium microstructure showing hyphae and conidia, (c) phylogenetic tree Fig. 1 Morphological and molecular characterization of (A) T. cf. asperellum (biocontrol agent): (a) colony on PDA, (b) vegetative mycelium microstructure showing conidiophore and phialides, (c) phylogenetic tree (B) B. sorokiniana (pathogen): (a) colony on PDA, (b) vegetative mycelium microstructure showing hyphae and conidia, (c) phylogenetic tree maximum composite likelihood approach was used to compute the evolutionary distances. Results Molecular identification of Trichoderma sp. and Bipolaris sp. The morphological identification (Fig. 1) of Tricho- derma sp. and Bipolaris sp. was verified through the use of ITS1/ITS4 primers in a molecular investigation of the ITS rDNA sequence (18S-28S rRNA), flanking ITS1 (5.8S rRNA), and ITS2 as T. cf. asperellum and B. sorokiniana. The retrieved ITS sequence was entered into NCBI GenBank as accession no.: OP108262 and OP714480 using the BLAST program, and in order to compare with other relevant strains, the sequence data was aligned. Phylogenetic analysis was performed using MEGA version 7 software, which stands for molecular evolutionary genetic analysis. Based on the ITS gene sequences, the phylogenetic tree displayed in Fig. 1c was constructed using the neighbor-joining method with 1000 bootstrap repetitions. Multiple sequence alignments were performed using the ClustalW pro- gramme in the MEGA7 software using the clos- est homologous sequences that were chosen. The Characterization of TiO2 NPs’ morphology and optics Characterization of TiO2 NPs’ morphology and optics Characterization of TiO2 NPs’ morphology and optics An early indicator of synthesis is the transformation of TiCl4 from a milky off-white to a reddish brown color after 4 h of stirring with A. vera aqueous extract. As shown in Fig. 3A and B, the SEM and TEM analysis of Page 4 of 19 Metwally et al. BMC Plant Biology (2024) 24:118 Fig. 2 Dual culture for the antagonistic effect evaluation of T. cf. asperellum against B. sorokiniana. A T. cf. asperellum and B. sorokiniana interaction, (B) B. sorokiniana control, (C) In vitro control of B. sorokiniana using cell-free culture filtrate of (150 µL) by well diffusion method after 5 days of incubation. D-F Mycoparasitism of T. cf. asperellum against mycelium of B. sorokiniana (D) intensively vacuolated deformed mycelium of B. sorokiniana, (E) and (F) T.cf. asperellum and B. sorokiniana hyphal interaction Fig. 2 Dual culture for the antagonistic effect evaluation of T. cf. asperellum against B. sorokiniana. A T. cf. asperellum and B. sorokiniana interaction, (B) B. sorokiniana control, (C) In vitro control of B. sorokiniana using cell-free culture filtrate of (150 µL) by well diffusion method after 5 days of incubation. D-F Mycoparasitism of T. cf. asperellum against mycelium of B. sorokiniana (D) intensively vacuolated deformed mycelium of B. sorokiniana, (E) and (F) T.cf. asperellum and B. sorokiniana hyphal interaction Fig. 3 SEM (A) and TEM (B) images of green synthesized TiO2 NPs from A. vera leaf aqueous extract. C In vitro control of B. sorokiniana using the green synthesized TiO2 NPs (25 and 50 mg/L) from A. vera leaf aqueous extract by well diffusion method after 5 days of incubation on these findings, a concentration of 50 mg/L showed a promising antifungal action against B. sorokiniana. Efficacy of T. cf. asperellum on growth attributes of barley against B. sorokiniana in response to TiO2 NPs g p 2 Infected barley plants with B. sorokiniana showed a decline in their morphological attributes (Fig. 4A-C). Figure 4(D) showed typical spot blotch symptoms on barley plant leaves challenged with B. sorokiniana. The barley height, shoot and root fresh weight (fwt), and dry weight (dwt) (Table 1) were significantly reduced com- pared to the healthy control. However, T. cf. asperellum significantly increased the barley growth parameters compared to the B. sorokiniana- infected plants and also showed a reduction in disease symptoms caused by B. sorokiniana. T. cf. asperellum increased barley height, shoot, and root fwt by 33.3, 20.7, and 34%, respectively. Generally, barley plants diseased with B. sorokiniana exhibited a significant decrease in spike fwt and dwt (Fig. 4C and Table 1) reaching 62.9 and 57.9% respec- tively, compared to control plants. Moreover, the data in Table 1 also revealed that the biosynthesized TiO2 NPs had a positive impact on the growth parameters of barley and improved both fwt and dwt. The percentage enhancement due to TiO2 NPs application of shoot fwt Fig. 3 SEM (A) and TEM (B) images of green synthesized TiO2 NPs from A. vera leaf aqueous extract. C In vitro control of B. sorokiniana using the green synthesized TiO2 NPs (25 and 50 mg/L) from A. vera leaf aqueous extract by well diffusion method after 5 days of incubation Fig. 3 SEM (A) and TEM (B) images of green synthesized TiO2 NPs from A. vera leaf aqueous extract. C In vitro control of B. sorokiniana using the green synthesized TiO2 NPs (25 and 50 mg/L) from A. vera leaf aqueous extract by well diffusion method after 5 days of incubation TiO2 NPs indicated that they are tetragonal in shape, with the majority of the nano-forms being found in the 10 to 25 nm size range. Antifungal activity of TiO2 NPs against B. sorokinianahf The well diffusion method was used to determine the effect of two different concentrations of green-synthesized TiO2 NPs against B. sorokiniana. The results are displayed in Fig. 3 (C). The results showed that on PDA agar plates treated with 25 and 50 mg/L of TiO2 NPs, B. sorokiniana was not able to grow and that the diameter of the inhibi- tion zone increased with TiO2 NPs concentration. Based Metwally et al. BMC Plant Biology (2024) 24:118 Page 5 of 19 Fig. 4 In vivo effect of T. cf. asperellum and TiO2 NPs against B. sorokiniana infection on barley plants (A), barley leaves (B), barley spikes (C) and (D) leaf spot blotch on barley leaves after 4 weeks from B. sorokiniana infection. T1, T2 and T3 refers to control, TiO2 NPs and T. cf. asperellum treated plants respectively Fig. 4 In vivo effect of T. cf. asperellum and TiO2 NPs against B. sorokiniana infection on barley plants (A), barley leaves (B), barley spikes (C) and (D) leaf spot blotch on barley leaves after 4 weeks from B. sorokiniana infection. T1, T2 and T3 refers to control, TiO2 NPs and T. cf. asperellum treated plants respectively Table 1 Effect of T. cf. asperellum and TiO2 NPs on the plant growth of barley infected with B. sorokiniana pathogen, and uninfected Values are the mean ± standard error of the mean (n = 5). Data analysis was done by using Duncan’s multiple range test at p ≤ 0.05. The same letters within a column are not significantly different Parameters Shoot height (cm) Shoot fwt (g) Root fwt (g) Shoot dwt (g) Root dwt (g) Spike fwt (g) Spike dwt (g) Treatments Control 30 ± 0.794b 13.97 ± 0.369b 1.130 ± 0.0299c 4.030 ± 0.106b 0.447 ± 0.012c 3.7 ± 0.098c 1.38 ± 0.036b B. sorokiniana 24 ± 0.635d 9.10 ± 0.241d 0.607 ± 0.016d 2.987 ± 0.079c 0.220 ± 0.0058e 1.37 ± 0.036f 0.580 ± 0.015d TiO2 NPs 38 ± 1.01a 15.90 ± 0.421a 1.407 ± 0.037b 4.729 ± 0.125a 0.662 ± 0.017b 4.1 ± 0.108b 1.428 ± 0.038ab TiO2 NPs + B. sorokiniana 27 ± 0.714c 11.83 ± 0.313c 1.060 ± 0.028c 3.927 ± 0.104b 0.376 ± 0.0099d 2.24 ± 0.059e 0.962 ± 0.025c T. cf. asperellum 40 ± 1.06a 16.87 ± 0.446a 1.517 ± 0.041a 4.967 ± 0.131a 0.753 ± 0.019a 4.5 ± 0.119a 1.498 ± 0.039a T. cf. asperellum + B. Antifungal activity of TiO2 NPs against B. sorokinianahf sorokiniana 28 ± 0.741bc 13.04 ± 0.345b 1.133 ± 0.029c 4.177 ± 0.111b 0.481 ± 0.0127c 2.89 ± 0.076d 1.051 ± 0.028c and dwt of B. sorokiniana- infected plants was 30 and 31.4 as compared to B. sorokiniana-infected ones. and dwt of B. sorokiniana- infected plants was 30 and 31.4 as compared to B. sorokiniana-infected ones. which revealed a severe deficiency in their contents of 26.7, 19.58, and 24.67%, respectively, in B. sorokini- ana pathogen-challenged plants compared to con- trols. Interestingly, treating diseased or healthy plants with T. cf. asperellum or TiO2 NPs led to a noticeable improvement in Chl pigments. When compared to healthy plants, T. cf. asperellum had the highest levels of Chl a, Chl b, and carotenoids (13.9, 8.56, and 42.7%, Evaluation of barley physio‑biochemical characteristics in response to T. cf. asperellum and TiO2 NPs application to B. sorokiniana stress Barley leaves were tested for the presence of pho- tosynthetic pigments (Chl a, Chl b, and total Chl), Metwally et al. BMC Plant Biology (2024) 24:118 Page 6 of 19 Table 2 Influence of T. cf. asperellum and TiO2 NPs on photosynthetic pigments (mg g−1 leaf fwt) of barley plants infected with B. sorokiniana pathogen and uninfected Table 2 Influence of T. cf. asperellum and TiO2 NPs on photosynthetic pigments (mg g−1 leaf fwt) of barley plants infected with B. sorokiniana pathogen, and uninfected Values are the mean ± standard error of the mean (n = 5). Data analysis was done by using Duncan’s multiple range test at p ≤ 0.05. The same letters within a column are not significantly different Parameters Treatments Chl a Chl b Total chlorophylls Carotenoids Total pigments Chl a/Chl b Control 1.450 ± 0.038b 0.572 ± 0.015b 2.022 ± 0.053b 0.737 ± 0.019c 2.759 ± 0.073c 2.534 ± 0.067a B. sorokiniana 1.063 ± 0.028d 0.460 ± 0.012c 1.523 ± 0.04d 0.716 ± 0.019c 2.240 ± 0.059d 2.312 ± 0.061b TiO2 NPs 1.501 ± 0.039b 0.581 ± 0.0153ab 2.082 ± 0.055b 0.905 ± 0.024b 2.987 ± 0.079b 2.583 ± 0.068a TiO2 NPs + B. sorokiniana 1.137 ± 0.031cd 0.441 ± 0.011c 1.578 ± 0.042cd 0.702 ± 0.018c 2.280 ± 0.06d 2.578 ± 0.068a T. cf. asperellum 1.652 ± 0.044a 0.621 ± 0.0164a 2.273 ± 0.06a 1.052 ± 0.027a 3.325 ± 0.087a 2.660 ± 0.07a T. cf. asperellum + B. sorokiniana 1.217 ± 0.032c 0.485 ± 0.013c 1.702 ± 0.045c 0.739 ± 0.0195c 2.441 ± 0.064d 2.507 ± 0.066ab Values are the mean ± standard error of the mean (n = 5). Data analysis was done by using Duncan’s multiple range test at p ≤ 0.05. The same letters within a column are not significantly different contents (6.194 ± 0.164 mg/g fwt and 132.09 ± 3.49 µmol/g fwt) as compared with the infected ones (4.854 ± 0.128 mg/g fwt and 77.28 ± 2.04 µmol/g fwt), and the same trend was reported for TiO2 NPs. respectively) in its photosynthetic pigment content (Table 2). Also, barley plants treated with TiO2 NPs also displayed improved levels of Chl a, Chl b, and total Chl. In plants under stress, the total Chl content increased in those exposed to TiO2 NPs from 1.523 to 2.082 mg/g leaf fwt. Reduction of oxidative burst and lipid peroxidationhi p p The findings presented in Fig. 6 (A and B) demonstrated that, in comparison to healthy plants, the B. sorokini- ana infection of barley plants resulted in a notable and elevated accumulation of H2O2 and MDA content (26.33 and 79.56%, respectively). It is important to note that T. cf. asperellum caused the least reduction in lipid peroxi- dation and H2O2 concentration. The application of T. cf. asperellum or TiO2 NPs retarded the values of H2O2 (27.8 and 12.5%) and lipid peroxidation (27.9 and 13.78%) as compared to healthy plants (Fig. 6). Moreover, the ele- vated concentration of H2O2 in the cellular system posi- tively and significantly correlates (Table 4, r = 0.949) with the oxidative changes affecting MDA content. In terms of total sugar contents (Fig. 5A), barley plants infected with B. sorokiniana showed a marked reduction in their amount (83.133 ± 2.199 mg/g dwt) as compared to uninfected plants (132.86 ± 3.52 mg/g dwt). Spraying T. cf. asperellum or TiO2 NPs had a notable and beneficial impact on total sugars in both healthy and B. sorokiniana-stressed plants (Fig. 5A, Table 3). Moreover, the data in Table 4 revealed a posi- tive Pearson’s correlation between total sugars and pig- ment fractions (Chl a ‘0.974’, Chl b ‘0.926’, and total pigments ‘0.983’). The results in Fig. 5B and C also revealed a rise in the total protein and proline con- tent in B. sorokiniana-infected plants compared to the healthy plants (negative control). However, barley plants treated with T. cf. asperellum and infected with B. sorokiniana showed further proliferation in their Fig. 5 Influence of T. cf. asperellum and TiO2 NPs on osmolytes: (A) total sugars, (B) protein and (C) proline of barley plants infected with B. sorokiniana pathogen, and uninfected. Data analysis was done by using Duncan’s multiple range test at p ≤ 0.05. The same letters are not significantly different and error bars reflect the standard error of the mean Fig. 5 Influence of T. cf. asperellum and TiO2 NPs on osmolytes: (A) total sugars, (B) protein and (C) proline of barley plants infected with B. sorokiniana pathogen, and uninfected. Data analysis was done by using Duncan’s multiple range test at p ≤ 0.05. The same letters are not significantly different and error bars reflect the standard error of the mean Metwally et al. Enhancing antioxidant and defense related enzymes activity POX, CAT, and PAL activities increased in barley plant leaves challenged with B. sorokiniana (42, 17, and 73%) as compared to untreated negative control plants. Remarka- bly, based on ANOVA results, barley plants sprayed with T. cf. asperellum or TiO2 NPs showed a significant increase in their activities over pathogen-challenged plants (Fig. 6C-E and Table 3). Furthermore, an increase in LOX activ- ity (Fig. 6F) was observed in barley plants infected with B. sorokiniana. Furthermore, under pathogen-challenged Reduction of oxidative burst and lipid peroxidationhi asperellum or TiO2 NPs slightly and non-significantly increased LOX levels in plant leaves when compared to B. sorokiniana-treated plants and untreated control plants. Reduction of oxidative burst and lipid peroxidationhi BMC Plant Biology (2024) 24:118 Page 7 of 19 Table 3 Analysis of variance (Two-way ANOVA) of the effect of T. cf. asperellum, TiO2 NPs, B. sorokiniana pathogen and their interactions on some morpho-biochemical parameters of barley plants fwt fresh weight, MDA malondialdehyde Significant at the p < 0.05; ns non-significant T. cf. asperellum TiO2 NPs B. sorokiniana T. cf. asperellum + B. sorokiniana B. sorokiniana + TiO2 NPs Shoot height * * * * * Root fwt * * * * ns Shoot fwt * * * * * Chl a * * * * * Chl b * * * * * H2O2 * * * * * MDA * * * * * Protein * * ns * * Total sugars * * * * * Proline * * * * * CAT * * * * * POX * ns * * * PAL * * * * * Table 3 Analysis of variance (Two-way ANOVA) of the effect of T. cf. asperellum, TiO2 NPs, B. sorokiniana pathogen and their interactions on some morpho-biochemical parameters of barley plants Table 4 Pearson’s correlation matrix between growth parameters of barley plants (shoot height, shoot fwt and root fwt)), photosynthetic pigments (Chl a, Chl b and total pigments) and other biochemical parameters (total sugars, MDA and H2O2). Each square indicates the Pearson’s correlation coefficient of a pair of parameters Table 4 Pearson’s correlation matrix between growth parameters of barley plants (shoot height, shoot fwt and root fwt)), photosynthetic pigments (Chl a, Chl b and total pigments) and other biochemical parameters (total sugars, MDA and H2O2). Each square indicates the Pearson’s correlation coefficient of a pair of parameters fwt fresh weight, MDA malondialdehyde * Correlation was significant at the p < 0.05 Correlation was significant at the p < 0.01 Measured parameters Shoot height Shoot fwt Root fwt Chl a Chl b Total pigments Total sugars MDA H2O2 Shoot height 1.000 Shoot fwt 0.951 1.000 Root fwt 0.921 0.981 1.000 Chl a 0.939 0.952 0.882 1.000 Chl b 0.896 0.889** 0.786* 0.981 1.000 Total pigments 0.964 0.935 0.867 0.987 0.972 1.000 Total sugars 0.964 0.956 0.917 0.974 0.926 0.983 1.000 MDA -0.844 -0.940 -0.939 -0.864 -0.782* -0.862 -0.888 1.000 H2O2 -0.887 -0.900 -0.898 -0.868 -0.783* -0.825 -0.930 0.949** 1.000 Measured parameters Shoot height Shoot fwt Root fwt Chl a Chl b Total pigm conditions, treatment with T. cf. Discussionh The hemibiotrophic ascomycete B. sorokiniana poses a major danger to the production of barley and other cereal crops. Numerous chemical fungicides have been applied, but since the disease is so sophisticated, the pathogens have evolved to become resistant to these chemicals [50]. Metwally et al. BMC Plant Biology (2024) 24:118 Metwally et al. BMC Plant Biology Page 8 of 19 Fig. 6 Effect of T. cf. asperellum and TiO2 NPs on stress markers: (A) H2O2, (B) Malondialdehyde (MDA), (C) POX, (D) CAT, (E) PAL and (F) LOX of barley infected with test B. sorokiniana pathogen, and uninfected. Data analysis was done by using Duncan’s multiple range test at p ≤ 0.05. The same letters are not significantly different and error bars reflect the standard error of the mean Fig. 6 Effect of T. cf. asperellum and TiO2 NPs on stress markers: (A) H2O2, (B) Malondialdehyde (MDA), (C) POX, (D) CAT, (E) PAL and (F) LOX of barley infected with test B. sorokiniana pathogen, and uninfected. Data analysis was done by using Duncan’s multiple range test at p ≤ 0.05. The same letters are not significantly different and error bars reflect the standard error of the mean Therefore, biological management may be a good choice for high quality and productivity in crop production and sustainable agriculture. Recently, biocontrol agents that employ bacteria and fungi have drawn a lot of interest as a secure and effective disease prevention strategy. To do this, scientists have looked into a variety of microor- ganisms, including fungi and bacteria like Chaetomium globosum, T. reesei, T. hamatum, T. harzianum and Bacil- lus subtilis TE3, to manage the spot blotch pathogen [51–53]. Therefore, biological management may be a good choice for high quality and productivity in crop production and sustainable agriculture. Recently, biocontrol agents that employ bacteria and fungi have drawn a lot of interest as a secure and effective disease prevention strategy. To do this, scientists have looked into a variety of microor- ganisms, including fungi and bacteria like Chaetomium globosum, T. reesei, T. hamatum, T. harzianum and Bacil- lus subtilis TE3, to manage the spot blotch pathogen [51–53]. quantities of osmotic solutes such as glycerol [55, 56]. Trichoderma attaches to the pathogen through cell-wall carbohydrates that bind to the lectins on the pathogen [57]. Discussionh More spe- cifically, anatase has the highest levels of photocatalytic and antibacterial activity. The anatase structure is capable of producing OH˙ radicals during a photocatalytic event, which can destroy the pathogen membranes. Using a leaf extract from Trigonella foenum-graecum, Gomathipriya and Subhapriya [62] biosynthesized TiO2 NPs, producing spherical NPs with diameters ranging from 20 to 90 nm. In this study, we evaluated the potential of synthetic TiO2 NPs as fungal pathogen biocontrol agents. T. cf. asperellum colonization of cucumber roots has been demonstrated to improve the availability of P and Fe to plants, resulting in appreciable increases in dwt, shoot length, and leaf area [73]. Additionally, auxins made by Trichoderma spp. can promote plant growth and root formation [74]. extract. Visual observation of this color change was in line with results presented in Satti et al. [49]. As previ- ously described in our study, according to XRD results, the synthesized TiO2 NPs were pure and formed of only the anatase crystalline phase [60]. Burda et al.’s study [61] confirmed that the two factors affecting TiO2 NPs’ physic- ochemical properties and consequently their antibacterial properties are their shape and crystal structure. More spe- cifically, anatase has the highest levels of photocatalytic and antibacterial activity. The anatase structure is capable of producing OH˙ radicals during a photocatalytic event, which can destroy the pathogen membranes. Using a leaf extract from Trigonella foenum-graecum, Gomathipriya and Subhapriya [62] biosynthesized TiO2 NPs, producing spherical NPs with diameters ranging from 20 to 90 nm. In this study, we evaluated the potential of synthetic TiO2 NPs as fungal pathogen biocontrol agents. T. cf. asperellum colonization of cucumber roots has been demonstrated to improve the availability of P and Fe to plants, resulting in appreciable increases in dwt, shoot length, and leaf area [73]. Additionally, auxins made by Trichoderma spp. can promote plant growth and root formation [74]. Under B. sorokiniana stress, Satti et al. [49] demon- strated that the biosynthesized TiO2 NPs improved the agro-morphological traits (fwt and dwt) and yield param- eters of wheat plants. Additionally, it was noted that applying TiO2 NPs improved Zea mays growth charac- teristics [75]. Mishra et al. Discussionh [76] state that TiO2 NPs may regulate the activity of N metabolism-related enzymes, which facilitates plant uptake of more nutrients as these NPs convert N2 into organic nitrogen in the form of pro- teins and chlorophyll pigments [77], which in turn raises plant biomass and dwt. A study by Jaberzadeh et al. [78] stated that the foliar application of TiO2 NPs to wheat plants shows a rise in starch and gluten contents, which in turn boosts yield due to increasing rubisco activ- ity. Also, Rizwan et al. [79] reported an increase in soy- bean yield after treating with TiO2 NPs because of the increased absorption of water by the plants.h The use of biosynthesized metal NPs to fight fungi that cause plant diseases has increased recently [63–66]. The results showed that B. sorokiniana was unable to grow on PDA agar plates that had been supplemented with 25 and 50 mg/L of TiO2 NPs; in addition, the diameter of the inhibitory zone increased with increasing TiO2 NPs con- centration. Our findings concur with those of Boxi et al. [67] and Irshad et al. [68] that hazardous plant pathogens such as F. solani, Venturia inaequalis, and Ustilago tritici were resistant to the strongest antifungal effects of TiO2 NPs. TiO2 NPs can induce cell damage in Pichia pasto- ris by impairing the ROS-associated scavenging system, which results in the accumulation of ROS [69]. Addi- tionally, these NPs have the ability to penetrate cells and destroy the fungal cell wall. However, their entry into the cell, diffusion, and endocytosis might cause the creation of ROS, which can impair the performance of numer- ous intracellular organs [64, 70, 71]. By causing oxidative stress, ROS appears to play a significant role in the anti- fungal response. Additionally, they can destroy all cellu- lar macromolecules, such as DNA and proteins [63]. NPs can also cause fungal death by disrupting cellular enzymes and interfering with the electron transfer chain [66]. p y p The physio-biochemical properties of the barley plants were examined in order to investigate the antifungal effects of T. cf. asperellum and the biosynthesized TiO2 NPs against spot blotch disease in barley plants. Photo- synthesis plays an essential role in plant productivity and takes place in green leaves [80]. A severe deficiency in the photosynthetic pigment contents in B. sorokiniana pathogen-challenged plants was detected. Discussionh The creation of cell wall-degrading enzymes and peptaibols [58] is the next stage, which makes it easier for Trichoderma hypha to enter the parasitized fungus’ lumen and for the cell-wall content to be assimilated. The one-step reaction, environmentally friendly reac- tants, and cost-effectiveness of the green NPs synthesis make it superior to the chemical and physical approaches. As a result, biocompatible active NPs with several biologi- cal and medicinal uses are created [40]. Plant secondary metabolites are important for reducing and stabilizing bulk materials in redox processes during the creation of NPs [59]. A. vera leaf aqueous extract was qualitatively assessed, as earlier mentioned in our previous study [60] and the results showed the presence of some phytochemi- cals such as phenolics, flavonoids, alkaloids, and tannins. In this study, TiO2 NPs were prepared by green synthesis at pH 9 using phytochemicals in A. vera extract to reduce the TiCl4 salt into TiO2 NPs. An initial sign of synthesis is the change of the milky off-white color of TiCl4 to a pink- ish brown color after 4 h of stirring with A. vera aqueous It is obvious that T. cf. asperellum showed an inhibitory effect against B. sorokiniana. Our results of the antifungal activity of T. cf. asperellum against B. sorokiniana agree with Yassin et al. [37], Matroudi et al. [54] and Mukher- jee et al. [55] who studied the efficacy of T. cf. asperel- lum against a wide range of soil-borne fungi due to the production of antifungal metabolites. Druzhinina et al. [26] and Morais et al. [27] confirmed that the mycopara- sitism and antibiosis attributes of some Trichoderma spp. in addition to competition for resources and space, are used by humans for biological control activities. Further- more, T. cf. asperellum induces morphological modifi- cations that allow it to penetrate the host and hold high Metwally et al. BMC Plant Biology (2024) 24:118 Metwally et al. BMC Plant Biology (2024) 24:118 Page 9 of 19 extract. Visual observation of this color change was in line with results presented in Satti et al. [49]. As previ- ously described in our study, according to XRD results, the synthesized TiO2 NPs were pure and formed of only the anatase crystalline phase [60]. Burda et al.’s study [61] confirmed that the two factors affecting TiO2 NPs’ physic- ochemical properties and consequently their antibacterial properties are their shape and crystal structure. Discussionh [85] noted that the photocatalytic capa- bility of TiO2 NPs, which degrade many pesticide types, might be important for protecting plants from diseases since they do not form poisonous or toxic chemicals, leading to a high pathogen disinfection capacity [86]. We hypothesized that the increase in Chl content would have a significant effect on the rate of photosynthesis, enhance the productivity of carbohydrates, and ultimately result in an increase in fwts and dwts.f In order to overcome the negative effects of spot blotch disease caused by B. sorokiniana on morphologi- cal and Chl content, barley plants activate dual defense, which is characterized by the enhanced accumulation of different osmolytes (sugars, proteins, and proline) and increased antioxidant and defense-related enzymes (POX, CAT, PAL, and LOX). Sugars participate in physi- ological processes related to plant growth and develop- ment and are also involved in the response to a number of stresses, acting as nutrient and metabolite signalling molecules [87, 88]. A sharp decrease in their amount was recorded in barley plants infected with B. sorokini- ana compared to uninfected, and with the applications of T. cf. asperellum or TiO2 NPs a significant and positive effect on total sugars was detected. These results agree with those of Khodakovskaya and Lahiani [84], in which TiO2 NPs produced more carbohydrates, thus promot- ing growth and photosynthesis rates in plants. Sugars act as osmotic agents, helping maintain plasma membrane integrity [87]. In addition, a study by Abdelhameed and Metwally [25] supported the idea that T. viride contrib- utes to the rise in sugar levels in onion plants. This might be explained by the enhancing effect of T. cf. asperellum and TiO2 NPs in raising Chl concentration (seen above), which had a favorable influence on photosynthetic rate and led to an increase in the production of soluble sug- ars, boosting fwt and dwt. Furthermore, Ze et al. [89] suggested that TiO2 NPs may enhance light harvesting complex II gene expression in chloroplasts, which is con- sistent with our finding that TiO2 NP supplementation results in a rise in the concentration of soluble sugars. Concerning the results of the total protein and pro- line content, there was an increase in their contents in B. sorokiniana-infected plants compared to the healthy plants. However, barley plants treated with T. cf. asperel- lum or TiO2 NPs and infected with B. sorokiniana showed further proliferation in their contents. Discussionh Our findings are consistent with Satti et al.’s [49] observa- tion that spot blotch stress led to significant increases in proline concentration in wheat plants. Furthermore, it was discovered that applying TiO2 NPs increased the proline content of the infected wheat plants. In the same respect, TiO2 NPs were proven to increase proline con- tent in broad bean plants under both normal and abiotic stress conditions by Abdel Latef et al. [91]. In addition to maintaining cell turgor or osmotic balance, accumulat- ing proline also stabilizes membranes to avoid electrolyte leakage and scavenges ROS to prevent protracted oxida- tive bursts in plants [92–94]. Lipids are crucial for pre- serving the structural integrity of cells. MDA is thought to be the most thiobarbituric acid-reacting molecule that demonstrates the extent of oxidative stress as a result of lipid peroxidation [93, 95, 96]. Infection of barley plants with B. sorokiniana induced a significant and high accu- mulation of H2O2 and MDA content. It is worth mention- ing that the application of TiO2 NPs or T. cf. asperellum reduced the amount of H2O2 and lipid peroxidation. Sim- ilar to this, Khalil et al. [72] reported that the application of T. viride controlled the production of H2O2 and lipid peroxidation, maintaining cell homeostasis in tomato plants that were not infected by the pathogen. In accord- ance with these results, Abdelrhim et al. [97] also showed an augmentation of H2O2 and lipid peroxidation in wheat plants challenged with R. solani and the role of SiO2 NPs in reducing their contents and inducing pathogen dis- ease resistance. As well, Abdalla et al. [60] showed a great reduction in the amounts of H2O2 and lipid peroxidation in soybean plants treated with TiO2 NPs. Moreover, the elevated concentration of H2O2 in the cellular system positively correlates with the oxidative changes affect- ing MDA content, as it is formed by the reaction of ROS (H2O2 or/and O−2) with lipid molecules [98]. Soliman et al. [99] have reported that infection with A. alternata increased the amount of lipid peroxidation in the patho- gen-inoculated pepper leaf samples. Antioxidant enzymes have a crucial function in scav- enging ROS and preventing the oxidative stress that leads pigment fractions with TiO2 NPs application, where TiO2 NPs increase plant growth and photosynthesis rate by producing more carbohydrates. Additionally, Rodrìguez- González et al. Discussionh This might be due to the plant’s failure to capture sunlight and the breakdown of Chl pigments and thus photosynthesis will be diminished [81, 82]. It is interesting that a notice- able improvement in Chl pigments appeared with T. cf. asperellum or TiO2 NPs applications. Our results are consistent with Khalil et al. [72] whose work showed that T. viride and C. globosum foliar application, either alone or in combination, exhibited a considerable increase in Chl contents but a decrease in H2O2 and MDA. The findings of our study also support those of Aldinary et al. [83], who found that the use of fungal endophytes increases the efficiency of photosynthesis due to numer- ous changes in the chloroplasts and contents of carotene and Chl, as Trichoderma increases gene expression regu- lating Chl biosynthesis, light-harvesting complex pro- teins, or Calvin cycle components. Similarly, Satti et al. [49] found that the spot blotch in wheat plants caused by B. sorokiniana resulted in a significant decline in all pigment fractions. Nevertheless, with the application of 40 mg/L TiO2 NPs, an improvement in these pigment fractions appeared. Morteza et al. [75] and Khodakovs- kaya and Lahiani [84] showed a significant increase in all In a field experiment, the antifungal properties of T. cf. asperellum and TiO2 NPs against the biotic stress of the spot blotch disease were investigated. The morphological traits of B. sorokiniana-infected barley plants decreased. However, adding biosynthesized TiO2 NPs or T. cf. asperellum, a fungus that promotes plant growth, greatly improved the growth parameters for barley plants. Our results are in agreement with Khalil et al. [72] results that the foliar application of T. viride and C. globosum either alone or in combination increased tomato fwt and dwt of shoots and roots as compared to control. Also, Abdelhameed and Metwally [25] reported that T. viride enhanced onion plant growth under normal conditions. Metwally et al. BMC Plant Biology (2024) 24:118 Page 10 of 19 Page 10 of 19 affect cucumber at the macromolecular level. Also, TiO2 NPs transform N2 into organic nitrogen in the form of proteins, which eventually results in an increase in pro- tein content. Moreover, T. viride caused a substantial increase in protein content in onion plants [35]. Similar to this, it has been noted that proline content increased in A. solani-infected eggplant and tomato [12, 90]. Discussionh Our find- ings are consistent with those of Khodakovskaya and Lahiani [84], who found significant differences in the amounts of amide and carbohydrates in cucumber plants treated with TiO2 NPs, suggesting that TiO2 NPs can Antioxidant enzymes have a crucial function in scav- enging ROS and preventing the oxidative stress that leads to harmful effects on many sensitive molecules [100– 103]. In general, POX, CAT, and PAL activities increased in barley plant leaves infected with B. sorokiniana and there was a further increase in their activities with T. cf. asperellum or TiO2 NPs application. It is known that B. sorokiniana stimulates cereals to activate a variety of Metwally et al. BMC Plant Biology (2024) 24:118 Page 11 of 19 Page 11 of 19 secondary metabolic pathways [104]. Bagy et al. [105] reported that POX, which catalyzes H2O2 breakdown, is also implicated in lignification and suberization pro- cesses, which reduce pathogenesis and aid in infection prevention. Also, Kaur et al. [14] and Singla et al. [106] stated that PAL, the primary enzyme that connects pri- mary to secondary metabolism, has been linked to the activation of responses against pathogenic fungi in barley. It is also involved in the synthesis of plant secondary anti- microbial substances that are essential for plant disease resistance and plays an essential role in the biosynthe- sis of lignin precursors [107]. Furthermore, Khalil et al. [72] revealed a substantial enhancement of CAT enzyme activity with T. viride and C. globosum applications. According to Naz et al. [108], a botanical chemical for- mulation caused cereals to become resistant to the fun- gus B. sorokiniana by activating POX and PAL enzymes. Similarly, wheat developed stress tolerance against this fungus by upregulating defense-related enzymes, includ- ing CAT, ascorbate peroxidase, PAL, and POX [52]. Moreover, Abdelrhim et al. [97] showed that the activ- ity of PAL was intensified as SiO2 NPs were applied in wheat plants against R. solani. Kaur et al. [107], Ferrer et al. [109], Król et al. [110] and Abdelhameed et al. [111] reported that PAL and POX enzymes might serve as markers of induced resistance to fungal diseases. Related study by Metwally and Abdelhamed [112] revealed that NPs could likely boost POX and CAT activities, which directly contribute to overcoming various stresses. 31°21′13.1"E), showing typical spot blotch disease symptoms, was collected in paper bags. Discussionh Isolation and purification of fungal pathogens were done accord- ing to Kumar et al. [117]. The infected wheat and bar- ley plants’ leaves were chopped into small pieces and surface sterilized in a 5% sodium hypochloride solu- tion for 5 min, then rinsed in sterile distilled water. After drying in a sterile filter, the plant tissues were incubated for 6 days at 27°C in a potato dextrose agar (PDA) medium. To obtain pure cultures of the patho- gen, colonies of the fungus that appeared were trans- ferred to fresh PDA plates. Isolation of biocontrol agent (Trichoderma sp.) In the Egyptian soil of Minia Al-Qamh, El-Sharkia Gov- ernorate (30°31′25.4"N 31°21′13.1"E), Trichoderma sp. was isolated from barley plant rhizospheres using a serial dilution plate method. One mL of the suspension from each dilution was added aseptically into sterile Petri dishes filled with Rose Bengal medium. Three days later, Trichoderma colonies were selected and grown on PDA media. Pure isolates were made by inoculating the fun- gal colonies and letting them grow for seven days at 28°C. Trichoderma sp. stock culture was kept viable on PDA slants. Molecular identification of isolated fungih Molecular identification of isolated fungi The pure isolates of Trichoderma sp. and Bipolaris sp. were sub-cultured on a PDA medium and grown for 5 days at 27°C, and their genomic DNA was isolated using the CTAB technique [120]. The cell walls of fun- gal mycelia were broken down in the presence of liquid N2 by grinding. After that, the CTAB extraction buffer was added, and the mixture was incubated at 65°C before being purified using phenol, chloroform, and isoamyl alcohol (25:24:1). Cold isopropanol was used to precipitate the genomic DNA, which was then washed twice with cold 70% ethyl alcohol. Finally, the DNA was dissolved in 50 μL of sterilized distilled water. Identification of fungal inoculants Morphological identification of isolated fungi Also, an increase in LOX activity was observed in barley plants infected with B. sorokiniana. Furthermore, treat- ment with T. cf. asperellum or TiO2 NPs increased LOX levels non-significantly. In the same trend, Ohta et al. [113] found an increase in LOX activity in rice leaves after infec- tion with rice blast fungus, which is correlated with plant resistance against pathogens. As well, Nandini et al. [114] showed an increase in LOX and POX activities in pearl mil- let plants infected with downy mildew disease using crude proteins extracted from six different Trichoderma spp. This was attributed to the function of LOX in the establishment of the hypersensitive response, a form of programmed cell death that serves as an active defense mechanism [115, 116]. A necrotic lesion develops as a result of the rapid death of plant cells in the area surrounding the infection site, which stops the pathogen from spreading and causes more damage to the surviving plant organ. By contrasting macroscopic characteristics on agar plates with those of microscopic aspects like hyphal branching pattern and conidial shape, the morphological charac- teristics of hyphae of Trichoderma sp. and Bipolaris sp., were confirmed [118, 119] respectively. Figure 1 illus- trates the morphological growth of both fungal inocu- lants of Trichoderma sp. (Fig. 1A [a and b]) and Bipolaris sp. (Fig. 1B [a and b]) on PDA plates. Antagonistic effects of Trichoderma sp. culture filtrate (Antibiosis) Trichoderma sp. was grown on PDA plates for 7 days at 28°C. A fungal disc of 5 mm in diam. of Trichoderma sp. mycelial growth was transferred from the periphery of mycelial growth and inoculated on Erlenmeyer flasks (250 mL) containing 100 mL of PDB. The flasks were then incubated in a shaking incubator at 150 rpm at 28°C. Mycelial growth was harvested on the 5th day of incu- bation. The culture was filtered through Whatman filter paper no. 1 and re-filtered through a Millipore syringe fil- ter (0.22 μm). A Petri dish (9 cm in diameter) containing 25 mL of PDA was supplied with 1 mL of Bipolaris sp. spore sus- pension and allowed to harden. After that, three wells with a diameter of 10 mm were made using a sterile cork- borer on each agar plate. The wells were subsequently filled with a cell-free culture of Trichoderma sp. (150 µL) using a well-diffusion technique described by Soliman et al. [124] with slight modifications. The plates were then placed in a refrigerator for 5h before being incubated at 28°C for 5 days. At the end of the incubation period, the inhibition zones were assessed. Mycoparasitism i y y The ITS1 and ITS2 as well as the inverted 5.8S coding rDNA were amplified using the ITS1 and ITS4 prim- ers. In a total volume of 50 µL, each PCR reaction mix- ture comprised 5–10 ng of genomic DNA, 1 µM of each ITS1/ITS4 primer, 5 µL of a 10X reaction buffer (50 mM KCl, 50 mM Tris–HCl; pH 8.3, 0.1 mg/mL bovine serum albumin (BSA), 3 mM MgCl2, 200 µM each of dNTP, and 2.5 U of Taq DNA polymerase (Promega, Mannheim, Germany). The PCR technique includes 35 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and elongation at 72°C for 1 min. Before DNA sequencing, the PCR amplicon was resolved using an 8% agarose gel and purified using a specific PCR puri- fication kit (Accu Prep® PCR DNA Purification Kit, K-3034–1, Bioneer Corporation, South Korea). Mac- rogenInc, (South Korea) sequenced the purified PCR products. All inter-transcribed spacer sequencing work was also performed by MacrogenInc, (South Korea) and was carried out on both strands of the submitted DNA fragments [121]. The purified PCR products were sequenced using an ABI 377 DNA. Auto-sequencer (PerkinElmer, Applied Biosystems Div., Waltham, USA) based on the same primers mentioned before. For microscopy examination, dual culture for both path- ogenic and Trichoderma sp. was examined after 7 days of incubation, the regions where the hyphae of Tricho- derma sp. and Bipolaris sp. interacted (the interaction zones) were observed. Characteristics used for differen- tiating between the hyphae of Trichoderma sp. and Bipo- laris sp., were studied according to Rifai [117] and Wiese [118] using a light microscope (Leitz WETZLAR, Wet- zlar, Germany). Isolation of fungal pathogen (Bipolaris sp.) The pathogenic fungus from diseased leaves of wheat and barley plants grown in the Egyptian soil of Minia Al-Qamh, El-Sharkia Governorate (30°31′25.4"N Page 12 of 19 Page 12 of 19 Metwally et al. BMC Plant Biology (2024) 24:118 Metwally et al. BMC Plant Biology (2024) 24:118 Biosynthesis and characterization of TiO2 NP Biosynthesis and characterization of TiO2 NP In our latest work, TiO2 NPs were prepared by an eco- friendly green synthesis method using titanium tet- rachloride (TiCl4) solution and an aqueous extract of Aloe vera plant leaves, which were obtained from the Horticulture Department, Faculty of Agriculture, Zagazig University, Egypt after permission from Zaga- zig University. In brief, 100 mL of A. vera leaf extract was added dropwise to a 100 mL 1N TiCl4 solution in deionized water under continuous stirring. The pH of the mixture was adjusted to 9, and the stirring contin- ued at room temperature for 4h. The formed NPs were filtered, washed with double-distilled water, and finally dried at 100°C overnight. The obtained dry powder was further calcined at 400°C for 4h. The prepared NPs were characterized by UV–Visible spectrophotometry, In vitro evaluation of the antifungal activity of Trichoderma sp. against fungal pathogen Trichoderma sp. was employed in an in vitro antago- nistic assay to assess its biocontrol effects against the pathogenic fungus of barley by the dual culture tech- nique [122]. Mycelial growth discs of 5 mm diam. removed under aseptic conditions from the growing edge of a 5 day-old pure culture of Trichoderma sp. and Bipolaris sp. were transferred and placed on the oppo- site of a Petri dish (9 cm) containing 20 mL of PDA and were kept 6 cm apart from each other. The plates were incubated for 7 days at 28°C, and the treatments were replicated in triplicate. Control dishes were cultures of Bipolaris sp. without the presence of Trichoderma sp. The growth of the pathogen and Trichoderma sp. was observed constantly, and radial growth was recorded by measuring the mean colony diameter on the 5th day of inoculation. The percent of inhibition (PI) of the test phytopathogenic fungus (Bipolaris sp.) was calculated using the following formula: %PI = [(R1 −R2)/R1] × 100 R1 was the radial growth of the pathogen without Trichoderma sp., and R2 represents the radial growth of the pathogen inoculated with Trichoderma sp. [123]. Page 13 of 19 Page 13 of 19 Metwally et al. BMC Plant Biology (2024) 24:118 Metwally et al. BMC Plant Biology Fourier transform infrared (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) as latest described [60]. Fourier transform infrared (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) as latest described [60]. plants, a field experiment was carried out (Fig. 7). Barley (Hordeum vulgare L.) grains were acquired after permis- sion from the Department of Plant Breeding and Genet- ics, Agricultural Research Center, Giza, Egypt, and were planted in the sandy clay soil of Zagazig, El-Sharkia Gov- ernorate. After one month of planting, a spore suspen- sion of Trichoderma sp. was used as a foliar spray and drenched the soil near the stem region of Trichoderma sp.-treated plants. A concentration of 50 mg/L of TiO2 NPs was applied twice to the barley plant leaves, i.e. once before Bipolaris sp. application and then 7 days after inoculation. Ten days after Trichoderma sp. application, a spore suspension of Bipolaris sp. was used for infec- tion by directly spraying the suspension using an atom- izer on the leaves of the barley plants. Plants sprayed and irrigated with tap water only were used as a negative control. Six treatments were directed and arranged in a factorial design (2 × 3). Each treatment had 3 replicates. Four weeks after the Bipolaris sp. infection, disease symptoms appear. Bipolaris sp. was re-isolated from symptomatic tissues, and its identity was confirmed. The harvested samples were either immediately used or rap- idly stored and frozen at − 20°C for further studies. TiO2 NPs suspension preparation and in vitro assessment of its antifungal activity and growth inhibition against Bipolaris sp. Suspension of 50 mg/L of these NPs is prepared by dissolving NPs in distilled water and sonicating for 25–30 min using Elma (E15H Elmasonic) for equal distri- bution of NPs in water. The antifungal activity of green synthesized TiO2 NPs was carried out using the well diffusion method [125]. One mL of Bipolaris sp. spore suspension prepared from 5-day-old culture was added to 25 mL of PDA and poured in Petri dish. After that, three wells with a 10 mm diam. were made. The wells were filled with 150 µL of two different concentrations of TiO2 NPs (25 and 50 mg/L) individually in triplicate. The culture plates were incu- bated at 28°C for 5 days, and the zones of inhibition were observed. Measurements Growth traits PDA plates with 7-day-old pure cultures of Tricho- derma sp. and Bipolaris sp. were used. After that, sterile water was added to the cultures, and the mycelium was scraped gently with a sterile glass rod. The spore suspen- sion concentrations of Trichoderma sp. and Bipolaris sp. were adjusted to 2.5 × 107 and 1.7 × 107 conidia/ mL; respectively. Morphological traits of barley plants were recorded 4 weeks after Bipolaris sp. infection. The following charac- teristics were measured for each treatment: shoot height (cm), fresh (fwt) and dry (dwt) weight of shoot (g), root, and spike. Three barley plants were collected for each treatment, and they were gently washed with flowing water to remove soil debris. Measurement of physio‑biochemical indexes y Fresh leaves of barley plants from each treatment were taken separately in order to measure the levels of enzymes linked to plant defense, proline, soluble protein, Planting, growth conditions, and treatments To assess Trichoderma sp.’s and the biosynthesized TiO2 NPs’ capability for biocontrol against Bipolaris sp. on barley Fig. 7 Schematic representation of the experimental set up Fig. 7 Schematic representation of the experimental set up Metwally et al. BMC Plant Biology (2024) 24:118 Page 14 of 19 total carbohydrates, malondialdehyde (MDA), hydrogen peroxide (H2O2), and chlorophyll (Chl). of lipid peroxidation were expressed as nmol g−1 fwt of the MDA-TBA complex formed. The MDA equivalent was calculated using the following equation: The quantitative analysis of Chl was done according to the method of Lichtenthaler and Wellburn [126] after the extraction of 100 mg of fresh leaves with acetone, and the absorbance was read at 644, 663, and 452.5 nm wavelengths, and its content was calculated by the fol- lowing formula: MDA (nmol/mL) = [(A532 −A600)/155000]106 MDA (nmol/mL) = [(A532 −A600)/155000]106 To quantify the H2O2 content in barley, leaf samples were collected from infected and treated plants. The Chl a(mg g−1leaf fwt) = [12.7(OD663) −2.69(OD644)] × E.V/1000 × fwt Chl b(mg g−1leaf fwt) = [22.9(OD644) −4.68(OD663)] × E.V/1000 × fwt Carotenoids (mg g−1leaf fwt) = (4.2OD452.5)−(0.0264Chl. a+0.426Chl. b)×E.V/(1000×fwt) Carotenoids (mg g−1leaf fwt) = (4.2OD452.5)−(0.0264Chl. a+0.426Chl. b)×E.V/(1000×fwt) reagent ferrous oxidation-xylenol orange was employed [131]. In 0.1% TCA, a known fwt of barley leaves (250 mg) was homogenized. Half mL of 100 mM potassium phosphate buffer (pH 6.8) and 2 mL reagent (1 M KI w/v) were added to 0.5 mL of the barley leaf extract. The reaction was left for 1 h in darkness, and the absorbance was measured at 390 nm. *OD = Optical density, E.V = Extraction volume of sam- ple, fwt = Fresh weight of sample.h The total soluble protein content of both infected and healthy barley leaves was measured [127] using a Folin– Ciocalteu reagent at 700 nm using bovine serum albumin as a standard. Fwt of barley leaves (250 mg) were ground in potassium phosphate buffer (50 mM pH 7.0) and centri- fuged for 7 min at 6000 rpm (4°C) (MIKRO 200R Hettich Zentrifugen, Germany). Then 1 mL of the resultant super- natant was mixed with 5 mL of an alkaline copper solution. Plant defense‑related enzyme determinationh The concentrations of peroxidase (POX), catalase (CAT), phenylalanine ammonia-lyase (PAL), and lipox- ygenase (LOX) were measured in order to confirm the effect of T. cf. asperellum and the biosynthesized TiO2 NPs against spot blotch disease in barley plants. To analyze the activities of POX and CAT, fwt of barley leaves (100 mg) were ground in 25 mL of potassium phosphate buffer (50 mM pH 7.0) and centrifuged at 6000 rpm (4°C) for 20 min. The supernatant was col- lected to measure the activities of POX and CAT, according to Bergmeyer [132] and Aebi [133].h The Bates et al. [129] method was used to estimate the proline content in barley leaves. In brief, 250 mg fwt of barley leaves were extracted and centrifuged in 3% sulpho- salicylic acid. Then, 2 mL of filtrate was reacted with 2 mL of ninhydrin reagent and 2 mL of glacial acetic acid and then placed in a boiling water bath. Four mL of toluene were added, and the upper colored layer was separated, and its absorbance was read at 520 nm. Cell membrane decomposition of barley leaves was determined as the concentration of total 2-thiobarbituric acid (TBA) reac- tive ingredients [130]. To summarize, 250 mg of barley leaf tissues were extracted in 5 ml of 0.1% trichloroacetic acid (TCA). The supernatant (500 L), after being centrifuged for 10 min at 6000 rpm, was mixed with 2 mL of 20% TCA containing 0.5% TBA, incubated for 30 min at 95°C, and cooled on ice immediately. Absorbance at 532 and 600 nm was used for the calculation of the MDA equivalent. Based on the extinction coefficient of 155 mM−1 cm−1, the rates The activity of PAL was determined in barley plant leaves [134] using phenylalanine as the substrate and the absorbance was recorded at 290 nm. According to Axelord et al. [135], the LOX activity was assessed using linoleic acid as a substrate. Linoleic acid (10 μL) and Tween 20 (50 μL) are both included in the test buffer, which is 10 mL of 0.1 M phosphate buffer with a pH of 9.0. By adding 0.1 mL of enzyme extract to 1 mL of freshly made assay buffer, the assay process was started. LOX activity was determined by observing the absorbance at 234 nm. Measurement of physio‑biochemical indexes Total carbohydrate content was estimated by comparison with a glucose standard curve, as described by Dubois et al. [128]. Barley dried leaves (100 mg) were heated with 10 mL of 2.5N HCl in a boiling water bath for 3 h. The extract (0.1 mL) was then taken, and 1 mL of phenol was added. Sulfu- ric acid (2.5 mL) was added after 1 h of mixed properly, and the absorbance was measured at 490 nm. Availability of data and materials y The relevant datasets supporting the results of this article are included within the article and the [GenBank NCBI] at: https://www.ncbi.nlm.nih.gov/nuccore/OP108262.1/ T. cf. asperellum. https://www.ncbi.nlm.nih.gov/nuccore/OP714480.1/ B. sorokiniana. The relevant datasets supporting the results of this article are included within the article and the [GenBank NCBI] at: https://www.ncbi.nlm.nih.gov/nuccore/OP108262.1/ T. cf. asperellum. https://www.ncbi.nlm.nih.gov/nuccore/OP714480.1/ B. sorokiniana. https://www.ncbi.nlm.nih.gov/nuccore/OP108262.1/ T. cf. asperellum. https://www.ncbi.nlm.nih.gov/nuccore/OP714480.1/ B. sorokiniana. Funding Open access funding provided by The Science, Technology & Innovation Fund- ing Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). Statistical analysish The average and standard errors of 5 replicates (n = 5) are represented in the tables and graphed findings. The Metwally et al. BMC Plant Biology (2024) 24:118 Page 15 of 19 R.A.M. and R.E.A.; Review and editing: R.A.M., S.A.S. and R.E.A. All authors read and approved the final manuscript. R.A.M. and R.E.A.; Review and editing: R.A.M., S.A.S. and R.E.A. All authors read and approved the final manuscript. analysis of variance (ANOVA) was used to statistically confirm the results and by using Duncan’s multiple range test (p < 0.05), it was concluded that there was a significant difference between the control and treat- ment groups. SPSS® 18.0 was used to perform the cal- culations. Using SPSS, Pearson’s correlation coefficients (r) were carried out to understand the relationship between growth indices and different physio-biochem- ical parameters. Figures were assembled using Origin- Pro 8.5 for data analysis and graphing software. analysis of variance (ANOVA) was used to statistically confirm the results and by using Duncan’s multiple range test (p < 0.05), it was concluded that there was a significant difference between the control and treat- ment groups. SPSS® 18.0 was used to perform the cal- culations. Using SPSS, Pearson’s correlation coefficients (r) were carried out to understand the relationship between growth indices and different physio-biochem- ical parameters. Figures were assembled using Origin- Pro 8.5 for data analysis and graphing software. Received: 14 December 2022 Accepted: 31 January 2024 Received: 14 December 2022 Accepted: 31 January 2024 Received: 14 December 2022 Accepted: 31 January 2024 Competing interests The authors declare no competing interests. Consent for publication Not applicable. Consent for publication Not applicable. References 1. Blake T, Blake V, Bowman J, Abdel-Haleem H. Barley feed uses and qual- ity improvement. In: Ullrich SE, editor. Barley: production, improvement and uses. Hoboken: Wiley-Blackwell; 2011. p. 522–31. y 2. FAO. FAOSTAT-Agriculture. 2018. 3. Triticase C, Amicarelli V, Lamonaca E, Rana RL. Economic analysis of the barley market and related uses. In: Zerihun T, editor. Grasses as food and feed. New York: IntechOpen; 2018. 4. Liu J, Sui Y, Wisniewski M, Droby S, Liu Y. Review: Utilization of antago- nistic yeast to manage postharvest fungal diseases of fruits. Int J Food Microbiol. 2013;167:153–60. 5. Ghorbanpour M, Omidvari M, Abbaszadeh-Dahaji P, Omidvar R, Kariman K. Mechanisms underlying the protective effects of beneficial fungi against plant diseases. Biol Control. 2018;117:147–57. 6. Almeida FB, Rodrigues LM, Coelho C. The still underestimated problem of fungal diseases worldwide. Front Microbiol. 2019;10:214. 7. Escrivá L, Front G, Manyes L. In vivo toxicity studies of fusarium mycotoxins in the last decade: A review. Food Chem Toxicol. 2015;78:185–206. 8. Zhang DD, Wang YX, Chen YJ, Kong QZ, Gui JY, Li YN, Bao MY, Dai X. Identification and characterization of a pathogenicity-related gene VdCYP1 from Verticillium dahliae. Sci Rep. 2016;6:27979. 9. Krylov BV, Petruk IM, Glushko IN, Khaldeeva VE, Mokeeva LV, Bilanenko NE, Lebedin SY, Eremin AS, Nifantiev EN. Carbohydrate specificity of antibodies against phytopathogenic fungi of the Aspergillus genus. Appl Biochem Microbiol. 2018;54:522–7. Acknowledgements Acknowledgements We greatly appreciate the financial support from the Academy of Scientific Research and Technology, Egypt. Acknowledgements We greatly appreciate the financial support from the Academy of Scientific Research and Technology, Egypt. 10. Li H, Bian R, Liu Q, Yang L, Pang T, Salaipeth L, Andika BI, Kondo H, Sun L. Identification of a novel hypovirulence-inducing Hypovirus from Alternaria alternaria. Front Microbiol. 2019;10:1076. Experimental research and field studies on plants “All relevant institutional, national and international guidelines and legislation were compiled or adhered to in the production of this study”. 11. Tyškiewicz R, Nowak A, Ozimek E, Jaroszuk-Sciseł J, ´,. Trichoderma: The current status of its application in agriculture for the biocontrol of fungal phytopathogens and stimulation of plant growth. Int J Mol Sci. 2022;23:2329. Conclusion This article does not contain any studies with human participants or animals performed by any of the authors.” To achieve sustainable food production, agriculture must employ innovative strategies to decrease the use of agrochemicals. Consequently, NPs and fungal biological agents have been suggested as viable strategies with less environmental impact. Our research confirmed that B. sorokiniana causes spot blotch disease in barley plants, which results in morphological and physio-biochemical alterations. Nevertheless, on the host plant side, the application of T. cf. asperellum or green synthesized TiO2 NPs positively increased the host plant’s tolerance against this pathogen and played an important role in the activation of a complex defense system that comprises: (1) induction of osmolytes such as proline, protein, and soluble sugars. (2) An increase in antioxidant defense- related enzyme production. All these defense systems neutralize the destructive effects of ROS, decreasing H2O2 and lipid peroxidation and maintaining homeo- stasis within B. sorokiniana-challenged barley plants. So, the application of T. cf. asperellum or green synthe- sized TiO2 NPs could be considered as an alternative or eco-friendly approach to protect barley plants from spot blotch disease caused by B. sorokiniana. Further studies with the B. sorokiniana pathogen must be carried out in the field to explore the potential of T. cf. asperellum and green synthesized TiO2 NPs combined applications as a viable strategy, which might be evaluated in the future to obtain better crop yields with less environmental impact, however further studies will be necessary to gain a com- prehensive understanding of biological agent-NPs-plant interaction mechanisms. Authors contributions Conceptualization: H.A., R.E.A., R.A.M. and S.A.S.; Methodology: R.E.A., R.A.M., H.A. and S.A.S.; Formal analysis and investigation: R.A.M. and R.E.A.; Writing: Author details 1 1 Botany and Microbiology Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt. 12. Attia MS, Hashem AH, Badawy AA, Abdelaziz AM. Biocontrol of early blight disease of eggplant using endophytic Aspergillus terreus: Authors’ contributions Conceptualization: H.A., R.E.A., R.A.M. and S.A.S.; Methodology: R.E.A., R.A.M., H.A. and S.A.S.; Formal analysis and investigation: R.A.M. and R.E.A.; Writing: 12. Attia MS, Hashem AH, Badawy AA, Abdelaziz AM. Biocontrol of early blight disease of eggplant using endophytic Aspergillus terreus: Metwally et al. BMC Plant Biology (2024) 24:118 Page 16 of 19 31. De Palma M, Salzano M, Villano C, Aversano R, Lorito M, Ruocco M, Docimo T, Piccinelli AL, D’Agostino N, Tucci M. Transcriptome repro- gramming, epigenetic modifications and alternative splicing orches- trate the tomato root response to the beneficial fungus Trichoderma harzianum. Horticul Res. 2019;6:5. improving plant immunological, physiological and antifungal activi- ties. Bot Stud. 2022;63:26. 13. Wang R, Leng Y, Zhao M, Zhong S. Fine mapping of a dominant gene conferring resistance to spot blotch caused by a new pathotype of Bipolaris sorokiniana in barley. Theor Appl Genet. 2019;132:41–51. 32. Pimentel MF, Arnao E, Warner AJ, Subedi A, Rocha LF, Srour AY, Bond JP, Fakhoury AM. Trichoderma isolates inhibit Fusarium virguliforme growth, reduce root rot, and induce defense-related genes on soybean seedlings. Plant Dis. 2020;104:1949–59. 14. Kaur S, Bhardwaj RD, Kaur J, Kaur S. Induction of Defense-Related Enzymes and Pathogenesis-Related Proteins Imparts Resistance to Barley Genotypes Against Spot Blotch Disease. J Plant Growth Regul. 2021;41:682–96. 33. Metwally RA, Al-Amri SM. Individual and interactive role of Trichoderma viride and arbuscular mycorrhizal fungi on growth and pigment con- tent of onion plants. Lett Appl Microbiol. 2019;70:79–86. 15. Gupta PK, Chand R, Vasistha NK, Pandey SP, Kumar U, Mishra VK, Joshi AK. Spot blotch disease of wheat, the current status of research on genetics and breeding. Plant Pathol. 2018;67:508–31. 16. Singh UB, Sharma AK. Compatible salt-tolerant rhizosphere microbe- mediated induction of phenylpropanoid cascade and induced systemic responses against Bipolaris sorokinina (Sacc.) Shoemaker causing spot blotch disease in wheat (Triticum aestivum L.). Appl Soil Ecol. 2016;108:300–6. 34. Metwally RA. Arbuscular mycorrhizal fungi and Trichoderma viride coop- erative effect on biochemical, mineral content, and protein pattern of onion plants. J Basic Microbiol. 2020;60(8):712–21. https://doi.org/10. 1002/jobm.202000087. 35. Metwally RA, Soliman SA, Abdel Latef AA, Abdelhameed RE. The individual and interactive role of arbuscular mycorrhizal fungi and Trichoderma viride on growth, protein content, amino acids fractiona- tion, and phosphatases enzyme activities of onion plants amended with fish waste. Ecotoxicol Environ Saf. 2021;214:112072. 17. Daba SD, Horsley R, Brueggeman R, Chao S, Mohammadi M. Authors’ contributions Genome-wide association studies and candidate gene identification for leaf scald and net blotch in barley (Hordeum vulgare L.). Plant Dis. 2019;103:880–9. 18. Al-Sadi AM. Variation in resistance to spot blotch and the aggressive- ness of Bipolaris sorokiniana on barley and wheat cultivars. J Plant Pathol. 2016;98(1):97–103. i 36. Metwally RA, Abdelhameed RE, Soliman SA, Al-Badwy AH. Potential use of beneficial fungal microorganisms and C-phycocyanin extract for enhancing seed germination, seedling growth and biochemical traits of Solanum lycopersicum L. BMC Microbiol. 2022;22:108. 19. Rios JA, Perez CA, Debona D, Neto LC, Rios VS, Rodriguesa FA. Changes in gas exchange, chlorophyll a fluorescence and antioxidant metabolism within wheat leaves infected by Bipolaris sorokiniana. Ann Appl Biol. 2016;170:189–203. 37. Yassin MT, Mostafa AA, Al-Askar AA. In vitro antagonistic activity of Trichoderma spp. against fungal pathogens causing black point disease of wheat, J. Taibah Univ Sci. 2022;16:1:57–65. 38. Natsiopoulos D, Tziolias A, Lagogiannis I, Mantzoukas S, Eliopoulos PA. Growth-promoting and protective effect of Trichoderma atrobrunneum and T. simmonsii on tomato against Soil-Borne fungal pathogens. Crops. 2022;2:202–17. 20. Chen J, Zhou L, Din IU, Arafat Y, Li Q, Wang J, Wu T, Wu L, Wu H, Qin X, Pokhrel GR, Lin S, Lin W. Antagonistic activity of Trichoderma spp. against Fusarium oxysporum in rhizosphere of Radix pseudostel- lariae triggers the expression of host defense genes and improves its growth under long-term monoculture system. Front Microbiol. 2021;12:579920. 39. Hao Y, Fang P, Ma C, White JC, Xiang Z, Wang H, Zhang Z, Rui Y, Xing B. Engineered nanomaterials inhibit Podosphaera pannosa infection on rose leaves by regulating phytohormones. Environ Res. 2019;170:1–6. 21. Matarese F, Sarrocco S, Gruber S, SeidlSeibot V, Vannacci G. Biocontrol of Fusarium head blight: interactions between Trichoderma and mycotoxigenic Fusarium. Microbiol. 2012;158(1):98–106. y g g p y 40. Javed B, Mashwani ZUR. Phytosynthesis of colloidal nanosilver from Mentha longifolia and Mentha arvensis: Comparative morphological and optical characterization. Microsc Res Tech. 2020;83(11):1299–307. https://doi.org/10.1002/jemt.23518. y g 22. Walters DR, Ratsep J, Havis ND. Controlling crop diseases using induced resistance: challenges for the future. J Exp Bot. 2013;64(5):1263–80. 41. Abu-Elsaad NI, Abdelhameed RE. Copper ferrite nanoparticles as nutri- tive supplement for cucumber plants grown under hydroponic system J Plant Nutri. 2019;42:1645–59. 23. Berg G. Plant–microbe interactions promoting plant growth and health: perspectives for controlled use of microorganisms in agriculture. Appl Microbiol Biotechnol. 2009;84:11–8. 42. Abdelhameed RE, Abu-Elsaad NI, Abdel Latef AAH, Metwally RA. Authors’ contributions Tracking of zinc ferrite nanoparticle effects on pea (Pisum sativum L.) plant growth, pigments, mineral content and arbuscular mycorrhizal colonization. Plants (Basel). 2021;10(3):583. 24. Ferreira FV, Musumeci MA. Trichoderma as biological control agent: scope and prospects to improve efficacy. World J Microbiol Biotechnol. 2021;37:90. 25. Abdelhameed RE, Metwally RA. Assessment of beneficial fungal micro- organism’s bio-efficacy in stimulating morphological and physiologi- cal parameters of Allium cepa plants grown in soil amended with fish wastes. BMC Plant Biol. 2022;22:617. 43. Rodríguez-González V, Terashima C, Fujishima A. Applications of photocatalytic titanium dioxide based nanomaterials in sustainable agriculture. J Photochem Photobiol C Photochem Rev. 2019;40:49–67. 44. Waghmode MS, Gunjal AB, Mulla JA, et al. Studies on the titanium dioxide nanoparticles: biosynthesis, applications and remediation. SN Appl Sci. 2019;1:310. https://doi.org/10.1007/s42452-019-0337-3. 26. Druzhinina IS, Seidl-Seiboth V, Herrera-Estrella A, Horwitz BA, Kenerley CM, Monte E. Trichoderma: The genomics of opportunistic success. Nat Rev Microbiol. 2011;9:749. 45 Satti SH, Raja NI, Ikram M, Oraby HF, Mashwani ZUR, Mohamed AH, Singh A, Omar AA. Plant-based titanium dioxide nanoparticles trigger biochemical and proteome modifications in Triticum aestivum L under. biotic stress of Puccinia striiformis. Molecules. 2022;27:4274. 27. Morais EM, Silva AAR, Sousa FWAd, Azevedo IMBd, Silva HF, Santos AMG, Júnior JEAB, Carvalho CPd, Eberlin MN, Porcari AM, Silva Araújo FDd. Endophytic Trichoderma strains isolated from forest species of the Cerrado-Caatinga ecotone are potential biocontrol agents against crop pathogenic fungi. PLoS ONE. 2022;17(4):e0265824. 46. Ganesan S, Babu IG, Mahendran D, Arulselvi PI, Elangovan N, Geetha N, Venkatachalam P. Green engineering of titanium dioxide nanoparticles using Ageratina altissima (L.) King & HE Robines. medicinal plant aque- ous leaf extracts for enhanced photocatalytic activity. Ann Phyomedi- cine. 2016;5:69–75. 28. Inglis PW, Mello SC, Martins I, Silva JB, Macêdo K, Sifuentes DN, Valadares-Inglis MC. Trichoderma from Brazilian garlic and onion crop soils and description of two new species: Trichoderma azevedoi and Trichoderma peberdyi. PLoS ONE. 2020;15:e0228485. 47. Javed B, Nadhman A, Mashwani Z. Optimization, characterization and anti- microbial activity of silver nanoparticles against plant bacterial pathogens phyto-synthesized by Mentha longifolia. Mater Res Express. 2020;7:085406. y 29. Mukherjee PK, Hurley JF, Taylor JT, Puckhaber L, Lehner S, Druzhinina I, Schumacher R, Kenerley CM. Ferricrocin, the intracellular siderophore of Trichoderma virens, is involved in growth, conidiation, gliotoxin bio- synthesis and induction of systemic resistance in maize. Biochem Bioph Res Commun. 2018;505:606–11. 48. p g y 49. Satti SH, Raja NI, Javed B, Akram A, Z-u-R M, Ahmad MS, Ikram M. Titanium dioxide nanoparticles elicited agromorphological and Authors’ contributions Ikram M, Raja NI, Javed B, Mashwani Z-R, Hussain M, Hussain M, Ehsan M, Rafique N, Malik K, Sultana T, Akram A. Foliar applications of bio- fabricated selenium nanoparticles to improve the growth of wheat plants under drought stress. Green Process Synth. 2020;9:706–14. 30. Galletti S, Paris R, Cianchetta S. Selected isolates of Trichoderma gamsii induce different pathways of systemic resistance in maize upon Fusarium verticillioides challenge. Microbiol Res. 2020;233:126406. 49. Satti SH, Raja NI, Javed B, Akram A, Z-u-R M, Ahmad MS, Ikram M. Titanium dioxide nanoparticles elicited agromorphological and 49. Satti SH, Raja NI, Javed B, Akram A, Z-u-R M, Ahmad MS, Ikram M. Titanium dioxide nanoparticles elicited agromorphological and Page 17 of 19 Metwally et al. BMC Plant Biology (2024) 24:118 physicochemical modifications in wheat plants to control Bipolaris sorokiniana. PLoS ONE. 2021;16(2):e0246880. 69. Liu Z, Zhang M, Han X, Xu H, Zhang B, Yu Q, Li M. TiO2 nanoparticles cause cell damage independent of apoptosis and autophagy by impairing the ROS-scavenging system in Pichia pastoris. Chem Biol Interact. 2016;252:9–18. 50. Joshi AK, Chand R. Progress of researches done to understand host- pathogen relationship for spot blotch pathogen of wheat. J Wheat Res. 2011;3:1–7. 70. Safaei M, Taran M, Imani MM. Preparation, structural characterization, thermal properties and antifungal activity of alginate-CuO bionano- composite. Mater Sci Eng C. 2019;101:323–9. 51. Singh PK, He X, Sansaloni CP, Juliana P, Dreisigacker S, Duveiller E, Kumar U, Joshi AK, Singh RP. Resistance to spot blotch in two mapping populations of common wheat is controlled by multiple QTL of minor effects. Int J Mol Sci. 2018;19:4054. 71. Liao C, Li Y, Tjong SC. Visible-light active titanium dioxide nanomaterials with bactericidal properties”. J Nanomater. 2020;10(1):124. 52. Singh UB, Malviya D, Singh S, Kumar M, Sahu PK, Singh HV, Kumar S, Roy M, Imran M, Rai JP, Sharma AK, Saxena AK. Trichoderma harzianum-and methyl jasmonate-induced resistance to Bipolaris sorokiniana through enhanced phenylpropanoid activities in bread wheat (Triticum aesti- vum L.). Front Microbiol. 2019;10:1697. 72. Khalil MII, Youssef SA, Tartoura KA, Eldesoky AA. Comparative evaluation of physiological and biochemical alteration in tomatoplants infected by Alternaria alternata in response to Trichoderma viride and Chaetomium globosum application. Physiol Mol Plant Pathol. 2021;115:101671. globosum application. Physiol Mol Plant Pathol. 2021;115:101671. 73. Yedidia I, Srivastva AK, Kapulnik Y, Chet I. Effect of Trichoderma harzianum on microelement concentrations and increased growth of cucumber plants. Plant Soil. 2001;235:235–42. 53. Authors’ contributions Effect of metal and metal oxide nanopar- ticles on growth and physiology of globally important food crops: A critical review. J Hazard Mater. 2017;322:2–16. 60. Abdalla H, Adarosy MH, Hegazy HS, Abdelhameed RE. Potential of green synthesized titanium dioxide nanoparticles for enhancing seedling emergence, vigor and tolerance indices and DPPH free radical scavenging in two varieties of soybean under salinity stress. BMC Plant Biol. 2022;22:560. 80. El-Tayeb MA. Response of barley grains to the interactive effect of salin- ity and salicylic acid. Plant Growth Regul. 2005;45(3):215–24. 81. Sharma P, Jha AB, Dubey RS, Pessarakli M. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J Bot. 2012;2012:ID 217037. https://doi.org/ 10.1155/2012/217037. 61. Burda C, Chen X, Narayanan R, El-Sayed MA. Chemistry and properties of nanocrystals of different shapes. Chem Rev. 2005;105:1025–102. 62. Subhapriya S, Gomathipriya P. Green synthesis of titanium dioxide (TiO2) nanoparticles by Trigonella foenum-graecum extract and its antimicrobial properties. Microb Pathog. 2018;116:215–20. 82. Gámez-Arcas S, Baroja-Fernández E, García-Gómez P, Muñoz FJ, Almagro G, Bahaji A, Sánchez-López ÁM, Pozueta-Romero J. Action mechanisms of small microbial volatile compounds in plants. J Exp Bot. 2022;73(2):498–510. https://doi.org/10.1093/jxb/erab463. 63. Lakshmeesha TR, Murali M, Ansari MA, Udayashankar AC, Alzohairy MA, Almatroudi A, Alomary MN, Asiri SMM, Ashwini BS, Kalagatur NK, Nayak CS, Niranjana SR. Biofabrication of zinc oxide nanoparticles from _Melia azedarach_ and its potential in controlling soybean seed-borne phytopathogenic fungi. Saudi J Biol Sci. 2020;27(8):1923–30.f 83. Aldinary AM, Abdelaziz AM, Farrag AA, Attia MS. Biocontrol of tomato fusarium wilt disease by a new moringa endophytic aspergillus isolates. materials today: Proceedings. 2021;2021. 64. Ilkhechi NN, Mozammel M, Khosroushahi AY. Antifungal effects of ZnO, TiO2 and ZnO- TiO2 nanostructures on Aspergillus flavus”. Pestic Biochem Phys. 2021;176:104869. 84. Khodakovskaya MV, Lahiani MH. Nanoparticles and plants: From toxicity to activation of growth. In: Sahu SC, Casciano DA, editors. Handbook Nanotoxicology, Nanomedicine Stem Cell Use Toxicol. Wiley online Library; 2014. p. 121–30. https://doi.org/10.1002/97811 18856017.ch7. 65. Abdelaziz AM, Salem SS, Khalil AMA, El-Wakil DA, Fouda HM, Hashem AH. Potential of biosynthesized zinc oxide nanoparticles to control Fusarium wilt disease in eggplant (Solanum melongena) and promote plant growth. Biometals. 2022;35:601–16. 85. Rodrìguez-González V, Obrego´n S, Patro´n-Soberano OA, Terashima C, Fujishima A. An approach to the photocatalytic mechanism in the TiO2-nanomaterials microorganism interface for the control of infec- tious processes. Appl Catal B Environ. 2020;270:118853. 66. Authors’ contributions Villa-Rodríguez E, Parra-Cota F, Castro-Longoria E, López-Cervantes J, de los Santos-Villalobos S,. Bacillus subtilis TE3: A promising biologi- cal control agent against Bipolaris sorokiniana, the causal agent of spot blotch in wheat (Triticum turgidum L. subsp. durum). Biol Contr. 2019;132:135–43. 74. Contreras-Cornejo HA, Macı´as-Rodrı´guez L, Corte´ s-Penagos C, Lo´ pez-Bucio J,. Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in Arabidopsis. Plant Physiol. 2009;149:1579–92. 54. Matroudi S, Zamani MR, Motallebi M. Antagonistic effects of three spe- cies of Trichoderma sp. on Sclerotinia sclerotiorum, the causal agent of canola stem rot. Egypt J Biol. 2009;11:37–44. 75. Morteza E, Moaveni P, Farahani HA, Kiyani M. Study of photosynthetic pigments changes of maize (Zea mays L.) under nano Tio2 spraying at various growth stages. Springerplus. 2013;2:1–5. 55. Mukherjee M, Mukherjee PK, Horwitz BA, Zachow C, Berg G, Zeilinger S. Trichoderma-plant-pathogen interactions: Advances in genetics of biological control. Indian J Microbiol. 2012;53:522–9. 76. Mishra V, Mishra RK, Dikshit A, Pandey AC. Interactions of nanoparti- cles with plants: An emerging prospective in the agriculture industry. An emerging prospective in the agriculture industry. Emerg Technol Manag Crop Stress Toler Biol Tech. 2014;1:159–80. 56. McIntyre M, Nielsen J, Arnau J, van der Brink H, Hansen K, Madrid S (eds). Proceedings of the 7th European Conference on Fungal Genetics. Copenhagen, Denmark. 2004. 57. Moreno-Ruiz D, Lichius A, Turra D, Di Pietro A, Zeilinger S. Chemotro- pism assays for plant symbiosis and mycoparasitism related compound screening in Trichoderma atroviride. Front Microbiol. 2020;11:601251. 77. Yang F, Hong FS, You WJ, Liu C, Gao F, Wu C, Yang P. Influences of nano- anatase TiO2 on the nitrogen metabolism of growing spinach. Biol Trace Element Res. 2006;110:179–90. 58. Howell CR. Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis. 2003;87:4–10. 78. Jaberzadeh A, Moaveni P, Tohidi Moghadam HR, Zahedi H. Influence of bulk and nanoparticles titanium foliar application on some agronomic traits, seed gluten and starch contents of wheat subjected to water deficit stress. Not Bot Horti Agrobot Cluj-Napoca. 2013;41:201. p 59. Javed B. Synergistic effects of physicochemical parameters on bio- fabrication of mint silver nanoparticles: Structural evaluation and action against HCT116 colon cancer cells. Int J Nanomed. 2020;15:3621. 79. Rizwan M, Ali S, Qayyum MF, Ok YS, Adrees M, Ibrahim M, Zia-Ur- Rehman M, Farid M, Abbas F. Authors’ contributions Ghorbani F, Gorji P, Mobarakeh MS, Mozaffari HR, Masaeli R, Safaei M. Optimized synthesis of Xanthan gum/ZnO/TiO2 nanocomposite with high antifungal activity against pathogenic Candida albicans". J Nano- mater. 2022;2022:10. 86. Pelaez M, Nolan NT, Pillai SC, Seery MK, Falaras P, Kontos AG, Dunlop PSM, Hamilton JWJ, Byrne JA, O’Shea K, Entezari MH, Dionysiou DD. A review on the visible light active titanium dioxide photo- catalysts for environmental applications. Appl Catal B Environ. 2012;125:331–49. 67. Boxi SS, Mukherjee K, Paria S. Ag doped hollow TiO2 nanoparticles as an effective green fungicide against Fusarium solani and Venturia inaequa- lis phytopathogens. Nanotechnology. 2016;27(8):085103. 87. Kubeš M, Drážná N, Konrádová H, Lipavská H. Robust carbohydrate dynamics based on sucrose resynthesis in developing Norway spruce somatic embryos at variable sugar supply. In Vitro Cell Dev Biol Plant. 2014;50(1):45–57. 68. Irshad MA, Nawaz R, Zia ur Rehman M, Imran M, Ahmad J, Ahmad S, Inam A, Razzaq A, Rizwan M, Ali S,. Synthesis and characterization of titanium dioxide nanoparticles by chemical and green methods and their antifun- gal activities against wheat rust. Chemosphere. 2020;258:127352. Page 18 of 19 Page 18 of 19 Metwally et al. BMC Plant Biology (2024) 24:118 Metwally et al. BMC Plant Biology (2024) 24:118 88. Abdelhameed RE, Metwally RA. Mitigation of salt stress by dual applica- tion of arbuscular mycorrhizal fungi and salicylic acid. Agrochimica. 2018;62:353–66. barley genotypes against spot blotch disease. J Plant Growth Regul. 2021;41(2). https://doi.org/10.1007/s00344-021-10333-2. 108. Naz R, Nosheen A, Yasmin H, Bano A, Keyani R. Botanical-chemical formulations enhanced yield and protection against Bipolaris soro- kiniana in wheat by inducing the expression of pathogenesis-related proteins. PLoS ONE. 2018;13(4):e0196194. 89. Ze Y, Liu C, Wang L, Hong M, Hong F. The regulation of TiO2 nanopar- ticles on the expression of light-harvesting complex II and photo- synthesis of chloroplasts of Arabidopsis thaliana. Biol Trace Elem Res. 2011;143:1131–41. 109. Ferrer JL, Austin MB, Stewart C Jr, Noel JP. Structure and function of enzymes involved in the biosynthesis of phenylpropanoids. Plant Physiol Biochem. 2008;46:356–70. 90. Kumari M, Pandey S, Bhattacharya A, Mishra A, Nautiyal C. Protective role of biosynthesized silver nanoparticles against early blight disease in Solanum lycopersicum. Plant Physiol Biochem. 2017;121:216–25. 110. Król P, Igielski R, Pollmann S, Kȩpczyńska E. Priming of seeds with methyl jasmonate induced resistance to hemi-biotroph Fusarium oxysporum f. sp. lycopersici in tomato via 12-oxo-phytodienoic acid, salicylic acid, and flavonol accumulation. J Plant Physiol. 2015;179:122–32. 91. Authors’ contributions Abdel Latef AAH, Srivastava AK, El-sadek MSA, Kordrostami M, Tran LSP. Titanium dioxide nanoparticles improve growth and enhance toler- ance of broad bean plants under saline soil conditions. Land Degrad Develop. 2018;29:1065–73. 111. Abdelhameed R, Metwally R, Soliman S. Prospects of Bacillus amyloliquefaciens (MZ945930) mediated enhancement of Capsicum annuum L. plants under stress of Alternaria alternata in terms of physiological traits, thiol content, antioxidant defense, and phy- tohormones. J Plant Growth Regul. 2023. https://doi.org/10.1007/ s00344-023-11132-7. 92. Hayat S, Hayat Q, Alyemeni MN, Wani AS, Pichtel J, Ahmad A. Role of proline under changing environments: a review. Plant Signal Behav. 2012;7:1456–66. 93 Metwally RA, Azab HS, Al-Shannaf HM, Rabie GH. Prospective of mycor- rhiza and Beauvaria bassiana silica nanoparticles on Gossypium hirsu- tum L. plants as biocontrol agent against cotton leafworm. Spodoptera littoralis BMC Plant Biol. 2022;22(1):1–18. 112. Metwally RA, Abdelhamed RE. Co-application of arbuscular mycorrhizal fungi and nano-ZnFe2O4 improves primary metabolites, enzymes and NPK status of Pea (Pisum sativum L.) plants. J Plant Nutr. 2023. https:// doi.org/10.1080/01904167.2023.2280121. 94. Metwally RA, Soliman SA. Alleviation of the adverse effects of NaCl stress on tomato seedlings (Solanum lycopersicum L.) by Trichoderma viride through the antioxidative defense system. Bot Stud. 2023;64:4. 113. Ohta H, Shida K, Peng Y, Furusawa I, Shishiyama J, Aibara S, Morita Y. A Lipoxygenase Pathway Is Activated in Rice after infection with the rice blast fungus Magnaporthe grisea. Plant Physiol. 1991;97(1):94–8. 95. Mohamed HI, Akladious SA. Changes in antioxidants potential, second- ary metabolites and plant hormones induced by different fungicides treatment in cotton plants. Pest Biochem Physiol. 2017;142:117–22. y 96. Metwally RA, Abdelhameed RE. Impact of Ridomil, Bavistin and Agrothoate on arbuscular mycorrhizal fungal colonization, biochemical changes and potassium content of cucumber plants. Ecotoxicology. 2019;28:487–98. 114. Nandini B, Hariprasad P, Prakash HS, Geetha N. Trichoderma oligosaccharides priming mediates resistance responses in pearl millet against downy mildew pathogen. J Appl Biol Biotechnol. 2017;5(2):97–103. 97. Abdelrhim AS, Mazrou YSA, Nehela Y, Atallah OO, El-Ashmony RM, Dawood MFA. Silicon dioxide nanoparticles induce innate immune responses and activate antioxidant machinery in wheat against Rhizoc- tonia solani. Plants. 2021;10:2758. 115. Koch E, Meier BM, Eiben HG, Slusarenko A. A lipoxygenase from leaves of tomato (Lycopersicon esculentum Mill.) is induced in response to plant pathogenic pseudomonads. Plant Physiol. 1992;99:571–6. 116. Rusterucci C, Montillet JL, Agnel JP, Battesti C, Alonso B, Knoll A, Bes- soule JJ, Etienne P, Suty L, Blein JP, Triantaphylides C. Authors’ contributions Involvement of lipoxygenase-dependent production of fatty acid hydroperoxides in the development of the hypersensitive cell death induced by cryp- togein on tobacco leaves. J Biol Chem. 1999;274:36446–55. 98. Shimizu N, Hosogi N, Hyon GS, Jiang S, Inoue K, Park P. Reactive oxygen species (ROS) generation and ROS induced lipid peroxidation are asso- ciated with plant membrane modifications in host cells in response to AK-toxin from Alternaria alternata Japanese pear pathotype. J Gen Plant Pathol. 2006;72:6–15. 117. Kumar S, Kaushik N, Edrada-Ebel R, Ebel R, Proksch P. Isolation, charac- terization, and bioactivity of endophytic fungi of Tylophora indica. World J Microbiol Biotechnol. 2011;27:571–7. 99 Soliman SA, Abdelhameed RE, Metwally RA. In vivo and In vitro evalua- tion of the antifungal activity of the PGPR Bacillus amyloliquefaciens RaSh1 (MZ945930) against Alternaria alternata with growth promo- tion influences on Capsicum annuum L. plants. Microb Cell Fact. 2023;22:70. 118. Rifai MA. A revision of the genus Trichoderma. Mycol Pap. 1969;116:1–56. 119. Wiese MV. Compendium of Wheat Diseases (St. Paul, MN: APS Press; 1987. 100. Abdelhameed RE, Metwally RA. Alleviation of cadmium stress by arbus- cular mycorrhizal symbiosis. Int J Phytoremediation. 2019;21:663–71. 120. Wu ZH, Wang TH, Huang W, Qu YB. A simplified method for chro- mosome DNA preparation from filamentous Fungi. Mycosystema. 2001;20:575–7. 101. Nasrallah DA, Morsi MA, El-Sayed F, Metwally RA. Structural, optical and electrical properties of copper chloride filled polyvinyl chloride/polysty- rene blend and its antifungal properties against Aspergillus avenaceus and Aspergillus terreus. Compos Commun. 2020;22:100451. 121. Tamura K, Nei M, Kumar S. Prospects for inferring very large phylog- enies by using the neighbor-joining method. Proc Natl Acad Sci USA. 2004;101:11030–5. 102. Abdelhameed RE, Abdel Latef AAH, Shehata RS. Physiological responses of salinized fenugreek (Trigonella foenum-graecum L.) plants to foliar application of salicylic acid. Plants. 2021;10:657. 122. Campanile G, Ruscelli A, Luisi N. Antagonistic activity of endophytic fungi towards Diplodia corticola assessed by in vitro and in planta tests. Eur J Plant Pathol. 2007;117:237–46. 103. Metwally RA, Abdelhameed RE. Synergistic effect of arbuscular mycor- rhizal fungi in growth and physiology of salt-stressed Trigonella foenum- graecum plants. Biocatal Agric Biotechnol. 2018;16:538–44. 123. Ghildial A, Pandey A. Isolation of cold tolerant antifungal strains of Trichoderma sp. from glacier sites of Indian Himalayan region. Res J Microbiol. 2008;3(8):559–64. 104. Ishihara A, Kumeda R, Hayashi N, Yagi Y, Sakaguchi N, Kokubo Y, Ube N, Tebayashi SI, Ueno K. 127 Lowry OH, Rosbrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951;193:265–75. Authors’ contributions 2003;412:170–5. 135. Axelord B, Cheesbrough TM, Laakso S. Lipoxygenases in soybean. Meth- ods Enzymol. 1981;71:441–51. 135. Axelord B, Cheesbrough TM, Laakso S. Lipoxygenases in soybean. Meth- ods Enzymol. 1981;71:441–51. Authors’ contributions Induced accumulation of tyramine, serotonin, and related amines in response to Bipolaris sorokiniana infection in barley. Biosci Biotechnol Biochem. 2017;81(6):1090–8. 124 Soliman SA, Khaleil MM, Metwally RA. Evaluation of the antifungal activity of Bacillus amyloliquefaciens and B. velezensis and characteriza- tion of the bioactive secondary metabolites produced against plant pathogenic fungi. Biology. 2022;11:1390. 105. Bagy HMMK, Hassan EA, Nafady NA, Dawood MFA. Efficacy of arbuscu- lar mycorrhizal fungi and endophytic strain Epicoccum nigrum ASU11 as biocontrol agents against blackleg disease of potato caused by bacterial strain Pectobacterium carotovora subsp. atrosepticum PHY7. Biol Control. 2019;134:103–13. 125. Mohamed AA, Abu-Elghait M, Ahmed NE, Salem SS. Ecofriendly mycogenic synthesis of ZnO and CuO nanoparticles for in vitro anti- bacterial, antibiofilm, and antifungal applications. Biol Trace Elem Res. 2021;199:2788–99. 106. Singla P, Bhardwaj RD, Kaur S, Kaur J, Grewal SK. Metabolic adjustments during compatible interaction between barley genotypes and stripe rust pathogen. Plant Physiol Biochem. 2020;147:295–302. 126. Lichtenthaler HK, Wellburn AR. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Analysis. 1983;11(5):591–2. 107. Kaur S, Bhardwaj R, kaur J, kaur S. Induction of defense-related enzymes and pathogenesis-related proteins imparts resistance to 127 Lowry OH, Rosbrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951;193:265–75. Page 19 of 19 Metwally et al. BMC Plant Biology (2024) 24:118 Metwally et al. BMC Plant Biology (2024) 24:118 128. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Calorimetric method for determination of sugars and related substances. Anal Chem. 1956;28:350–6. 129. Bates LS, Waldren RP, Teare LD. Rapid determination of free proline for water-stress studies. Plant Soil. 1973;39:205–7. 130 Heath RL, Packer L. Photoperoxidation in isolated chloroplasts: I. kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys. 1968;125(1):189–98. 131. Alexieva V, Sergiev I, Mapelli S, Karanov E. The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant Cell Environ. 2001;24:1337–44. 132. Bergmeyer HU. Methods of Enzymatic Analysis 1. New York: Seacond es. Acadimic press; 1974. 132. Bergmeyer HU. Methods of Enzymatic Analysis 1. New York: Seacond es. Acadimic press; 1974. 133. Aebi H. Catalase. In: Bergmeyer H, editor. Methods of enzymatic analy- sis. Weinheim: Weinheim-Verlagchemie; 1983. p. 273–86. 134. Alunni S, Cipiciani A, Fiorini G, Ottavi L. Mechanisms of inhibition of phenylalanine ammonia lyase by phenol inhibitors and phenol / glycine synergistic inhibitors. Arch Bioch Biophy. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W2994619324	https://repositorium.sdum.uminho.pt/bitstream/1822/66512/1/Scraping_News_Sites%20_Social_Networks_Prejudice_Term_Analysis_Henriques_Araujo_Ermida_Dias.pdf	English	null	SCRAPING NEWS SITES AND SOCIAL NETWORKS FOR PREJUDICE TERM ANALYSIS	null	2,019	cc-by	5,832	KEYWORDS Information Extraction, Social Web, Web Scraping, Computer-Mediated Communication ABSTRACT Computer-Mediated Communication (CMC) has paved the way for new patterns of linguistic aggravation. Hidden behind the screen, anyone can comment on any other person's opinion using an offensive or injurious tone. Besides, types of prejudice such as homophobia, sexism, racism, xenophobia, anticlericalism, body/addiction shaming, among others, are easily found nowadays in social networks and other forms of interactive Web sites potentiated by Web 2.0. This increasing violence deserves further investigation from different academic perspectives, among which Sociolinguistics stands out. This paper is concerned with the design and development of a set of computer-based tools to collect articles and posts with the respective comment threads that can be used as sources to extract prejudice terms and allow different analyses to be conducted. These prejudice terms were devised using a sociolinguistic variable stratification approach. We will focus on the filters used to extract the relevant fields from the Web pages collected, and on the converters used to adapt formats to obtain a common format for information representation. We will also introduce the statistical analysis processor that explores the extracted data, in that format, to output a set of indicators. SCRAPING NEWS SITES AND SOCIAL NETWORKS FOR PREJUDICE TERM ANALYSIS Pedro Rangel Henriques, Cristiana Araújo, Isabel Ermida and Idalete Dias Universidade do Minho, Portugal 16th International Conference on Applied Computing 2019 16th International Conference on Applied Computing 2019 1. INTRODUCTION Our team's project deals with hate speech online, i.e., the expression of prejudice and discrimination on the Internet. It aims to identify recurrent words, idioms and constructions, as well as communicative routines and patterns of pragmatic interaction, that may serve as a basis for abuse detection. The object of analysis is online social platforms, such as Facebook, YouTube, Twitter, and Web journal comment boards, where participants often engage in expressing opinions that are ideology-laden and biased against a range of social groups and subjects. The project aims to build and annotate a comparable corpus of online texts, on a range of similar topics but in different languages, namely Portuguese and English. This contrastive analysis will hopefully allow us to assess the socio-cultural similarities and differences of the two linguistic contexts in an admittedly globalized society. Annotation of the corpus will begin by metadata, which will stratify the texts according to sociolinguistic variables such as age, gender, ethnicity, nationality and social class. This will pave the way for studies in language variation. Structural taggers will be inserted next, providing information about the morpho-syntactic organization of the texts, which may carry ideological intent. Finally, content taggers will help design a descriptive ontology of the corpora that will lend itself to various interpretive tasks. The results of this extended study will help refine informatics tools of control and detection on institutional levels, including automatic identification of situations of risk and abuse online. The research question we intend to answer in this paper can be stated as follows: How to integrate into a single corpus posts with comments, containing prejudice terms, extracted from different social networks and news sites? 179 ISBN: 978-989-8533-95-1 © 2019 In this context, the paper focuses on the software paraphernalia that is under development to support the extraction from multiple sources of hate speech sentences. In this context, the paper focuses on the software paraphernalia that is under development to support the extraction from multiple sources of hate speech sentences. The tools and techniques we have used may not be new, but the novelty and the strength of our work so far is that we have managed to integrate all available tools into a unified approach. Thus, we have succeeded in tackling the various problems which the different sources posed and can now choose the appropriate tool and extraction method for each different source. 1. INTRODUCTION Besides, we have managed to integrate the partial results into a global analysis tool that is automated and available through a simple Web interface. Additionally - and crucially - we have also started to successfully collect the metadata of each extraction. y Section 2 presents a set of keywords for text extraction, devised according to a sociolinguistic variable stratification method. Section 3 introduces the approach followed to execute the tasks involved in the extraction. Then Sections 4 and 5 discuss in detail the extractors of material from sources such as social networks and newspaper sites. Section 6 presents the system of detection and analysis of discursive prejudices. Finally, Section 7 presents the conclusions and future work. 2. SOCIOLINGUISTIC STRATIFICATION OF ONLINE PREJUDICE AND DISCRIMINATION: KEYWORDS FOR DATA EXTRACTION The project aims to analyze the linguistic forms and strategies of prejudice in Computer-Mediated Communication (CMC). As CMC is a phenomenon that brings together multiple manifestations, not only at the linguistic level (morphology, syntax, lexical semantics), but also (inter)actionally and pragmatically, it is important to cross it with the sociolinguistic variables it involves. With this caveat in mind, our team devised a keyword table for data extraction, at the very outset of the project. This table covers the following elements: (i) different types of prejudice; (ii) the corresponding sociolinguistic variables they target; and (iii) the various keywords and expressions which, in English and Portuguese, are deemed typical lexical signals of prejudice expression. Ten types of prejudice were considered: sexism, ageism, racism, nationalism, classism, homophobia, anticlericalism, ideological shaming, body shaming, and addiction shaming. The former five correspond to the following classic sociolinguistic variables, respectively: gender, age, race (and ethnicity), nationality, and social class (Labov, 2001). Prejudice types six to eight correspond to the following identity categories: sexual identity, religious identity, and ideological/political identity (Coupland, 2007). Finally, items nine and ten correspond to physical identity and behavioral identity. For each of these parameters a list of keywords, especially slurs, was developed, to help scrape the Web for texts that express prejudice. This approach will cater for discursive and pragmatic analyses, with a view to identifying emerging ideological communities (Lambert, 2007). The aim is to know what actors - both active and passive, victimizers and victimized - populate the virtual world of marginalization and conflict, and what their recurrent identities and attitudes are in terms of linguistic expression. The approach for extracting and analyzing comment boards is presented in Section 3. 3. EXTRACTION AND ANALYSIS - THE APPROACH As previously mentioned, the project aims to analyze verbal interaction in online comment boards. At this stage, we are extracting data from two types of online platforms: newspaper sites and social networks (YouTube and Facebook), in the Portuguese language. Before starting the extraction process, it is necessary to find news articles and publications that can trigger biased and discriminatory comments. Our approach to searching across platforms has been to use the keywords (see Section 2) in the platform search to find the publications that contain those keywords. After finding them we start the extraction. It is important to note that in addition to extracting comments we also extract the news articles, publications, audio-visual texts, etc., that triggered the thread of comments. As the goal of the project is to construct a corpus for linguists and other specialists to use in their research, we find it appropriate to keep the source text that gave rise to the verbal interaction. The news articles found in the newspaper sites will then be looked for in the respective newspaper Facebook page in order to extract the post and the comment thread from there. In general, we have discovered that the same news article has a greater number of comments on 180 16th International Conference on Applied Computing 2019 16th International Conference on Applied Computing 2019 the newspaper's Facebook page, compared to the newspaper site, in the case of Portuguese newspapers. In the newspaper “Correio da Manhã” (a Portuguese tabloid), we are only going to extract the comment threads on the newspaper's Facebook page because the contents of the comment threads on the newspaper site have recently stopped being accessible. Our extraction and comment analysis schema is shown in Figure 1. Figure 1. General Extraction Schema1 Figure 1. General Extraction Schema1 Figure 1 is a simplified schema that contains all the steps from the extraction to the analysis of the comments. Extractors will scrape web pages (Newspaper, Facebook, YouTube) and as a result we obtain a file, which can be in a range of formats, such as HTML, JSON, CSV, etc., depending on each extractor. Subsequently the extracted files will be converted into a standard format resorting to the File Converters. In this case, we have chosen JSON as the standard format. 3. EXTRACTION AND ANALYSIS - THE APPROACH Finally, comment threads in JSON format will be examined according to a Statistical Analysis to evaluate the Frequency of occurrence of the keywords which reveal prejudice and discrimination in the comments. The results of this analysis are saved in a CSV file. In the following sections we will explain all these processes in more detail. Figure 1. General Extraction Schema1 Figure 1. General Extraction Schema1 Figure 1 is a simplified schema that contains all the steps from the extraction to the analysis of the comments. Extractors will scrape web pages (Newspaper, Facebook, YouTube) and as a result we obtain a file, which can be in a range of formats, such as HTML, JSON, CSV, etc., depending on each extractor. Subsequently the extracted files will be converted into a standard format resorting to the File Converters. In this case, we have chosen JSON as the standard format. Finally, comment threads in JSON format will be examined according to a Statistical Analysis to evaluate the Frequency of occurrence of the keywords which reveal prejudice and discrimination in the comments. The results of this analysis are saved in a CSV file. In the following sections we will explain all these processes in more detail. Figure 1 is a simplified schema that contains all the steps from the extraction to the analysis of the comments. Extractors will scrape web pages (Newspaper, Facebook, YouTube) and as a result we obtain a file, which can be in a range of formats, such as HTML, JSON, CSV, etc., depending on each extractor. Subsequently the extracted files will be converted into a standard format resorting to the File Converters. In this case, we have chosen JSON as the standard format. Finally, comment threads in JSON format will be examined according to a Statistical Analysis to evaluate the Frequency of occurrence of the keywords which reveal prejudice and discrimination in the comments. The results of this analysis are saved in a CSV file. In the following sections we will explain all these processes in more detail. 1 All the icons of Figures 1, 2 and 5 were taken from the website: “The Noun Project” (https://thenounproject.com/). Accessed: 2019-04- 10. All the icons of Figures 1, 2 and 5 were taken from the website: “The Noun Project” (https://thenounproject.com/). Accessed: 2019-0 2 YouTube Comment Scraper: http://ytcomments.klostermann.ca/. 16th International Conference on Applied Computing 2019 16th International Conference on Applied Computing 2019 It is important to note that this application only extracts the comment thread and does not extract any information regarding the publication. Extraction on Facebook is done with the tool Netvizz3, a Facebook application that can be found by typing the name into the main Facebook search box. To extract the data from a page, it is only necessary to enter the page ID and date scope. The result of the extraction is a CSV file that in this case contains the comment thread and publication information (Rieder, 2013). p According to Rieder (2013), the fields extracted by Netvizz are: position - postnumber_commentnumber (e.g. 0_0 for the first comment on the first post); post_id - ID of the post; post_by - author of the post; post_text - text of the post; post_published - publishing date of the post; comment_id - ID of the comment; comment_by - author of the comment (column no longer in use, remains for compatibility); is_reply - whether the comment is a reply to another comment (in threaded conversations); comment_message - text of the comment; comment_published - publishing date of the comment; comment_like_count - number of likes on the comment; attachment_type - attachment type (eg: animated_image_share); attachment_url - attachment URL. As the results of Facebook extraction are obtained in CSV format, it is necessary to convert them into JSON format. The transformation of files extracted from Facebook in CSV format to JSON format is performed by a script in Python. For each line of the CSV file, the Converter forms a name/value pair, where the field name corresponds to its column name and value is the extraction result of the respective field. For example, “comment_message”: “Os casamentos de Sto António são para casais (homem/mulher) e não para pares (2 do mesmo sexo)” [“Saint Anthony’s weddings are for man/woman couples, not for same-sex pairs”], where “comment_message” is a column name and “Os casamentos de Sto António são para casais (homem/mulher) e não para pares (2 do mesmo sexo)” corresponds to the extraction result. The JSON generated by the converter is listed in Figure 4. g In Section 5 we will explain the extractors of newspaper sites in detail. Figure 4 Comment thread extracted from Facebook in JSON (fragment) Figure 4. Comment thread extracted from Facebook in JSON (fragment) 3 Netvizz: https://apps.facebook.com/netvizz 4. EXTRACTING POSTS/COMMENTS FROM ONLINE SOCIAL NETWORKS Extracting the posts and comment threads from Web social networks (YouTube and Facebook) is carried out using two freely available online tools. A detailed outline of the steps of this extraction is shown in Figure 2. 181 181 ISBN: 978-989-8533-95-1 © 2019 Figure 2. YouTube and Facebook Extraction Schema Figure 2. YouTube and Facebook Extraction Schema Figure 2. YouTube and Facebook Extraction Schema On YouTube the extraction takes place using the application YouTube Comment Scraper2. To start the extraction, it is necessary to enter the publication link and the application automatically extracts the thread with all comments in JSON and CSV format. In our case, the thread was extracted in JSON format, to standardize all extractions. In Figure 3, a fragment of a comment thread extracted from YouTube is exhibited. Figure 2. YouTube and Facebook Extraction Schema On YouTube the extraction takes place using the application YouTube Comment Scraper2. To start the extraction, it is necessary to enter the publication link and the application automatically extracts the thread with all comments in JSON and CSV format. In our case, the thread was extracted in JSON format, to standardize all extractions. In Figure 3, a fragment of a comment thread extracted from YouTube is exhibited. On YouTube the extraction takes place using the application YouTube Comment Scraper2. To start the extraction, it is necessary to enter the publication link and the application automatically extracts the thread with all comments in JSON and CSV format. In our case, the thread was extracted in JSON format, to standardize all extractions. In Figure 3, a fragment of a comment thread extracted from YouTube is exhibited. Figure 3. Comment thread extracted from YouTube (fragment) According to Klostermann (2015), the fields extracted in each comment thread are: id - ID of the comment; user - name or nickname of the author of the comment; date - publishing date of the comment; timestamp - time an event is logged by a computer, not the time of the event itself; commentText - text of the comment; likes - number of likes on the comment; hasReplies - whether the comment has replies; numberOfReplies - number of replies; replies - whether the comment is a reply to another comment (in threaded conversations). 182 4 Requests: https://2.python-requests.org/en/master/ 5 Beautiful Soup: https://www.crummy.com/software/BeautifulSoup/bs4/doc/ 6 Selenium: https://www.seleniumhq.org 7 WebDriver: https://www.seleniumhq.org/projects/webdriver/ 5. EXTRACTING ARTICLES/COMMENTS FROM NEWSPAPER WEB PAGES This type of scraping extracts exactly all the information we see when we click on “view page source”. Static scraping works using two Python packages: Requests4 to find web pages and Beautiful Soup5 to parse HTML pages. Dynamic scraping uses a real browser and allows the JavaScript on the page to run. Subsequently, it queries the DOM to extract the content we are looking for. There are also cases where it is necessary to automate the browser, that is, to simulate the steps a user takes to obtain the content needed. Dynamic scraping works by using the Selenium6 Python package to automate the browser and interactively interact with the DOM. Selenium requires a WebDriver7 (the browser is automated) for the Internet browser (Mitchell, 2015; Jarmul and Lawson, 2017). In our case the most adequate scraping technique is the dynamic one because the content of the comments Figure 5. Newspaper Extraction Schema Figure 5. Newspaper Extraction Schema Faced with this difficulty, it was necessary to understand the different types of scraping techniques avaliable and how we would apply them to carry out the extraction. Scraping can be either static or dynamic. Static scraping bypasses JavaScript and searches the server's Web pages without the help of a browser. This type of scraping extracts exactly all the information we see when we click on “view page source”. Static scraping works using two Python packages: Requests4 to find web pages and Beautiful Soup5 to parse HTML pages. Dynamic scraping uses a real browser and allows the JavaScript on the page to run. Subsequently, it queries the DOM to extract the content we are looking for. There are also cases where it is necessary to automate the browser, that is, to simulate the steps a user takes to obtain the content needed. Dynamic scraping works by using the Selenium6 Python package to automate the browser and interactively interact with the DOM. Selenium requires a WebDriver7 (the browser is automated) for the Internet browser (Mitchell, 2015; Jarmul and Lawson, 2017). In our case, the most adequate scraping technique is the dynamic one because the content of the comments comes from another source, as explained above. 5. EXTRACTING ARTICLES/COMMENTS FROM NEWSPAPER WEB PAGES The comment thread and article extraction from the newspaper web pages is performed by Python scripts to extract the article from the newspaper, and another script to extract comments related to the article. A de li f h f hi i i h i Fi 5 The comment thread and article extraction from the newspaper web pages is performed by Python scripts, one to extract the article from the newspaper, and another script to extract comments related to the article. A detailed outline of the steps of this extraction is shown in Figure 5. p g The newspaper sites are dynamic Web pages, that is, they assemble content that comes from different sources. They use scripts that perform functions such as: database information, article comments, links to other news, links to different topics/news categories, etc., and fill the page with content from different sources in the 183 ISBN: 978-989-8533-95-1 © 2019 visualization moment (i.e. when the page is requested). This means that the page content is not fully included in the original static page. visualization moment (i.e. when the page is requested). This means that the page content is not fully included in the original static page. An example of content that is integrated into the newspaper article page is the comment thread and, therefore, its extraction becomes more difficult. This is noticeable when we click on “inspect element”: on the newspaper page we can see we can see the HTML code with the comment thread, but when we click on “view page source” we cannot find the HTML code with the comment thread. The explanation for this omission is that “view page source” only shows the content that was found on the server. However, the final DOM (Document Object Model) that is rendered by the browser can be very different, because the JavaScript that is compiled when opening the page manipulates the DOM and integrates content that comes from the different sources. This leads us to see the HTML code with the comment thread when we click on “inspect element” (Prutsachainimmit and Nadee, 2018). Figure 5. Newspaper Extraction Schema Faced with this difficulty, it was necessary to understand the different types of scraping techniques avaliable and how we would apply them to carry out the extraction. Scraping can be either static or dynamic. Static scraping bypasses JavaScript and searches the server's Web pages without the help of a browser. Figure 7. Extraction of Newspaper Article (example) 5. EXTRACTING ARTICLES/COMMENTS FROM NEWSPAPER WEB PAGES Dynamic scraping will allow us to find the information that comes from another source, but we also use static scraping to extract the information we want after finding it with dynamic scraping.Figure 6 shows a fragment of the Newspaper Article extractor (in this example we are extracting news from the Portuguese newspaper “Sol”), which, as already mentioned, uses dynamic scraping (Selenium and WebDriver) and static scraping (Beautiful Soup). 184 16th International Conference on Applied Computing 2019 16th International Conference on Applied Computing 2019 16th International Conference on Applied Computing 2019 All the data we want to extract from the newspaper article is on the source page, but we have decided to use both types of scraping, so to make it applicable to most types of content. In lines 5-14 we use dynamic scraping to open and load the page, and close cookie windows, etc. that appear when the page loads; and we use static scraping, lines 15-29, to search for the elements and extract the information. The WebDriver we used was Geckodriver as it is the driver used for the connection to Firefox (line 5). The driver runs the browser and opens the link to the page we entered in the driver.get function (line 6). Subsequently, we look for the tag <div> whose id is article-full and store in the variable dados (lines 13 and 14). This tag contains all the HTML code with the information that we want to extract. Figure 6. Extractor of Newspaper Article (fragment) Figure 7. Extraction of Newspaper Article (example) Figure 6. Extractor of Newspaper Article (fragment) Then we use Beautiful Soup to parse this HTML and save the HTML code returned by the Selenium, already converted to Beautiful Soup, within the variable soup (line 15). The soup variable is the tree root parsed from the HTML page that will allow us to browse and search elements in the tree. Within this tree we will look only for the specific elements that we want to extract: title - title of the article; subtitle - subtitle of the article; date - date on which the article was published; shares - number of article shares in social networks (when applicable); article - text of the article. For example, to extract the title of the article we will look in the tree for <div> whose class is “large-9 medium-8 column artic_content” (which contains the header and body of the article) and store it in the variable header. Within the variable header we will look for the <header> tag, and within that tag search for the <h1> tag and save the text (title of the article) found in the <h1> tag in the variable titleN. This extractor stores information extracted in JSON format (see Figure 7). Figure 7. Extraction of Newspaper Article (example) 185 ISBN: 978-989-8533-95-1 © 2019 The comment thread extractor is shown in Figure 8 and, similarly to the previous extractor, it also uses dynamic and static scraping to perform the extraction. In this case, dynamic scraping plays a very important role because the comment thread is imported from an external source, that is, it is not in the source code of the main page. On the other hand, it is also important when there is an extensive list of comments, as it is necessary to simulate a user clicking on the button in order to see the remaining comments, otherwise the extractor only extracts the comments that are visible (open) in the HTML page at that time. The search and extraction logic of this extractor is very similar to the previous one, the main difference is that it opens the main page of the news article (line 6), then it will look for the url of the sub-page (in the <iframe> tag in the attribute src) that contains the comment thread (lines 9-11), and finally, opens it to access its information (line 12). Figure 6. Extractor of Newspaper Article (fragment) When the list of comments is extensive, a button is displayed to click and see the remaining comments. The simulation of that click as if it were a user is performed in lines 16-22. Finally, in lines 27-29 the list of the comment thread is searched and stored in the variable listComments. The result of this extraction is an extensive HTML code that contains all the comments. The next step is to filter the extracted information and convert it to JSON format. To achieve this, we constructed a Text Filter and JSON Converter, in Flex, which looks for the information that we want to extract and saves it in JSON format. This phase poses significant challenges: one being that HTML is very verbose and so it takes several steps to extract a simple field; another is the occurrence of responses to nested comments within each other, which makes it difficult to identify where responses end up, given various levels of nesting. Figure 8. Extractor of the Newspaper Comment Thread (fragment) Figure 8. Extractor of the Newspaper Comment Thread (fragment) JSON generated by the application has the same fields as JSON extracted on YouTube. Figure 9 shows the JSON result of the HTML extraction. Figure 9. Newspaper Comment Thread in JSON format (fragment of a comment) Figure 9. Newspaper Comment Thread in JSON format (fragment of a comment) Figure 9. Newspaper Comment Thread in JSON format (fragment of a comment) 186 16th International Conference on Applied Computing 2019 16th International Conference on Applied Computing 2019 16th International Conference on Applied Computing 2019 It is important to point out that this whole extraction process (newspaper article extractor, comment thread extractor and text filter and JSON converter) is specific to the newspaper “Sol”. This means that for each newspaper it is necessary to create new extractors and a new text filter and JSON Converter, since each newspaper has a different web page. p p p g In Section 6 the analysis of comments to identify discriminatory and prejudice discourse will be discusse 6. PREJUDICE DISCOURSE DETECTION AND ANALYSIS It is important to remember that the final objective of the extraction process previously presented is to collect a stream of comment threads to build a comparable linguistic corpus that will be studied mainly in terms of verbal interaction in CMC. After extracting the comments, it is necessary to analyze if the comment thread exhibits a prejudiced and discriminatory stance. To carry out this analysis, we have constructed a Python script that does the statistical analysis for the occurrence of keywords in the comments. This analysis performed by the Python script compares the comment text with the keywords table, which is in JSON format. The code fragment shown in Figure 10 aims to search for the keywords (presented in Section 2) in the comments. When it finds them, it creates a triple analysis with the keyword, the number of occurrences of the keyword and the total number of words that the comment has. Figure 10. Statistical analysis for the occurrence of keywords in the comments (fragment) Figure 10. Statistical analysis for the occurrence of keywords in the comments (fragment) This script in Python does the statistical analysis by: type of prejudice in a comment thread; frequency with which this concept appears in a particular comment; total number of comments that have keywords out of the total number of comments of the thread; which keywords are present in the comments and how often they appear in the comment thread; which type of prejudice is present in the comment thread; which is the most prevalent prejudice in a comment thread. The analysis that is performed for a file with a comment thread can also be performed for a directory that contains several files with comment threads. The output of the statistical analysis for the occurrence of keywords in the comments is exported to a CSV file. An example of this result is shown in Figure 11. This example corresponds to the JSON sample of YouTube extraction in Figure 3. Figure 11. Result of statistical analysis for the occurrence of keywords in the comments Figure 11. 7. CONCLUSION With the globalized rise in online communication, people worldwide are spending increasing amounts of time, attention, and energy on social networks (Facebook, YouTube, Twitter, Reddit, etc.). They navigate these platforms on a daily basis, and use them to socialize, connect, express opinions and, crucially, absorb opinions. Dialogue exchange with virtual strangers allows Internet surfers to discuss and argue about a variety of topics, some of which are often openly polemical, without ever coming face to face with one another. The anonymity and distance that characterize this interaction are partly to blame for a generalized willingness to offend and a rising propensity to discriminate, persecute and express prejudice. Our goal is to extract these interactions (comment threads) from various sources and analyze them. To examine the different types of discriminatory discourse we have created a table with keywords, categorized by type of prejudice and the respective sociolinguistic variables. To collect the dialogue texts and extract the comment threads from Facebook and YouTube, as well as to extract the comment threads from the newspaper page and the newspaper article, we are using two existing tools and creating two scripts. As the output format is not always the same, it is necessary to create scripts that convert the extraction outputs to JSON. Finally, as we intend to analyze the type of discourse present in the comments, we have created a script to perform several analyses, one of which is to identify the prejudiced keywords in the comments and to count how often they appear in each comment and in the thread in general. Our project is aligned with many others in the CMC Community (Cougnon et al., 2019; Pahor de Mati et al., 2019; Franza and Fišer, 2019; Beißwenger et al., 2019a; Beißwenger et al., 2019b; Lombart, 2019), projects conducting various studies related to social media discourse, in particular socially unacceptable discourse. In our case, we aim at integrating multiple sources and analyzing a broader group of social variables. As future work we intend to implement extractors for other Portuguese and English newspapers. Another goal is to extract Twitter and Instagram comments. We also aim to improve the extraction of YouTube comment threads, that is, to create a script to extract information about publication, similar to the extractor of the newspaper article. 6. PREJUDICE DISCOURSE DETECTION AND ANALYSIS Result of statistical analysis for the occurrence of keywords in the comments 187 ISBN: 978-989-8533-95-1 © 2019 Finally, we have built a Web interface that integrates: the statistical analysis module; another module to fill in missing metadata fields; another component that allows for the inclusion of new categories of prejudice types and sociolinguistic variables, and for the addition of new keywords in English and Portuguese to each sociolinguistic variable. Additionally, after performing the statistical analysis and populating the missing metadata, it still stores the resulting Json file in the database. Section 7 presents our conclusions and future work. ACKNOWLEDGEMENT This work has been supported by FCT - Fundação para a Ciência e Tecnologia within the Project Scope: PTDC/LLT-LIN/29304/2017. We are also indebted to our students, André Salgueiro, Bruno Carvalho and Fábio Araújo, for their valuable help in developing the statistical analysis and visualization components. We also wish to thank Daria Bębeniec for her careful reading of the previous versions of the paper and her helpful comments and suggestions. Beißwenger, M. and Fladrich, M. and Imo, W. and Ziegler, E., 2019a. Collecting and Analyzing a Corpus of WhatsApp Interactions Using the MoCoDa2 Web Interfaces. Proceedings of the 7th Conference on CMC and Social Media Corpora for the Humanities (CMC-Corpora2019). Cergy-Pontoise, France, Eds. Longhi, J. and Marinica, C., pp. 72. Beißwenger, M. and Herzberg, L. and Lüngen, H. and Wigham, C., 2019b. cmc-core: A basic schema for encoding CMC corpora in TEI. Proceedings of the 7th Conference on CMC and Social Media Corpora for the Humanities (CMC-Corpora2019). Cergy-Pontoise, France, Eds. Longhi, J. and Marinica, C., pp. 73. REFERENCES Beißwenger, M. and Fladrich, M. and Imo, W. and Ziegler, E., 2019a. Collecting and Analyzing a Corpus of WhatsApp Interactions Using the MoCoDa2 Web Interfaces. Proceedings of the 7th Conference on CMC and Social Media Corpora for the Humanities (CMC-Corpora2019). Cergy-Pontoise, France, Eds. Longhi, J. and Marinica, C., pp. 72. Beißwenger, M. and Herzberg, L. and Lüngen, H. and Wigham, C., 2019b. cmc-core: A basic schema for encoding CMC corpora in TEI. Proceedings of the 7th Conference on CMC and Social Media Corpora for the Humanities (CMC-Corpora2019). Cergy-Pontoise, France, Eds. Longhi, J. and Marinica, C., pp. 73. 188 16th International Conference on Applied Computing 2019 Cougnon, L.A. and Coppin, J. and Gutierrez Figueroa, V., 2019. A Mixed Quantitative-Qualitative Approach to Disagreement in Online News Comments on Social Networking Sites. Proceedings of the 7th Conference on CMC and Social Media Corpora for the Humanities (CMC-Corpora2019). Cergy-Pontoise, France, Eds. Longhi, J. and Marinica, C., pp. 31-35. Coupland, N., 2007. Style: Language Variation and Identity. Cambridge University Press. Franza, J. and Fišer, D., 2019. The lexical inventory of Slovene socially unacceptable discourse on Facebook. Proceedings of the 7th Conference on CMC and Social Media Corpora for the Humanities (CMC-Corpora2019). Cergy-Pontoise, France, Eds. Longhi, J. and Marinica, C., pp. 42-46. Jarmul, K. and Lawson, R., 2017. Python Web Scraping. Packt Publishing. Jarmul, K. and Lawson, R., 2017. Python Web Scraping. Packt Publishing. Klostermann, P., 2015. Youtube comment scraper. http://ytcomments.klostermann.ca/. (Accessed in: 2019-01-20). Labov, W., 2001. Principles of Linguistic Change: Social Factors, Vol. 2. Blackwell. Labov, W., 2001. Principles of Linguistic Change: Social Factors, Vol. 2. Blackwell. Lambert Graham, S., 2007. Disagreeing to agree: Conflict, (im)politeness and identity in a computer-mediated community, Journal of Pragmatics, Vol. 39, No. 4, pp. 742 – 759. Special Issue: Identity Perspectives on Face and (Im)Politeness. mbart, E., 2019. Text-based messages environment types. Proceedings of the 7th Conference on CMC and Social Med Corpora for the Humanities (CMC-Corpora2019). Cergy-Pontoise, France, Eds. Longhi, J. and Marinica, C., pp. 75 Mitchell, R., 2015. Web Scraping with Python: Collecting Data from the Modern Web. O’Reilly Media. Pahor de Maiti, K. and Fišer, D. and Ljubešić, N., 2019. How haters write: analysis of nonstandard language in online hate speech. Proceedings of the 7th Conference on CMC and Social Media Corpora for the Humanities (CMC-Corpora2019). Cergy-Pontoise, France, Eds. Longhi, J. and Marinica, C., pp. 36-41. Prutsachainimmit, K. and Nadee, W., 2018. REFERENCES Towards data extraction of dynamic content from javascript web applications, International Conference on Information Networking (ICOIN), pp. 750–754. Rieder, B., 2013. Studying facebook via data extraction: The netvizz application. Proceedings of the 5th Annual ACM Web Science Conference, WebSci ’13. New York, NY, USA, pp. 346–355, ACM. 189 189
https://openalex.org/W3196418384	https://www.scielo.br/j/rbf/a/vRMPvpRsnhTyKDcVmQkbdct/?lang=en&format=pdf	English	null	Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará	Revista brasileira de fruticultura	2,021	cc-by	9,327	Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará Paulo Manoel Pontes Lins1, Ismael de Jesus Matos Viegas2, Eric Victor de Oliveira Ferreira3 Abstract- Fertilization greatly affects coconut productivity; nevertheless, information on crop nutrition is relatively scarce for coconut in the state of Pará, Brazil. This study evaluated the effect of fertilization with nitrogen (N), phosphorus (P), potassium (K), and magnesium (Mg) on the nutritional status and coconut yield under the edaphoclimatic conditions of the municipality of Moju, Pará State (Brazil). The experiment was conducted for 10 years on a SOCOCO® Farm and used a randomized block design with three replications. Three levels of P, K, and Mg were used in presence and absence of N. Response to fertilization in the treatments was studied in terms of leaf concentration of macronutrients (N, P, K, Ca, Mg and S) and micronutrients (B and Cl). We also assessed the number of coconuts/plant/year, fresh albumen weight (FAW)/coconut and FAW/ ha/year. The results were subjected to the analysis of variance (p<0.05) and the means compared by the Tukey test. In general, N fertilization did not influence concentration of macronutrients and micronutrients in leaves. In turn, P, K and Mg fertilization influenced nutrition of coconut palm. P and K fertilizations, isolated or in combination, increased the production of coconut/plant and FAW/ha. From the 6th year of plant age, 54 kg/ha/year of P2O5 and 96 kg/ha/year of K2O are indicated for the coconut culture in the region of the current study. N fertilization did not influence production parameters and Mg fertilization increased production in the presence of K fertilization. Indexing Terms: Amazon, Cocos nucífera L., macronutrients. 1PhD in Agricultural Sciences, Agricultural Superintendent at the Reunidas Sococo Company. Santa Isabel do Pará- PA, Brazil, E-mail: pmplins@uol.com.br(ORCID 0000-0003-3900-7261) 2PhD in Soil and Plant Nutrition, Professor at the Universidade Federal Rural da Amazônia (UFRA)- Campus Capanema, Capanema- PA, Brazil. Email: matosviegas@hotmail.com(ORCID 0000-0001-7212-1977) 3PhD in Soil and Plant Nutrition, Professor at UFRA- Campus Capitão Poço, Capitão Poço-PA, Brazil. E-mail: ericsolos@yahoo.com.br(ORCID 0000-0003-0142-8466) Copyright: All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License. ISSN 0100-2945 ISSN 0100-2945 DOI: http://dx.doi.org /10.1590/0100-29452021113 Nutrição e produção de coqueiro cultivado com adubação mineral no estado do Pará Resumo- A adubação é a prática que tem maior impacto na produtividade do coqueiro; ainda assim, as informações sobre a nutrição da cultura são relativamente escassas no Estado do Pará. O presente estudo objetivou avaliar o efeito das adubações nitrogenada, fosfatada, potássica e magnesiana sobre o estado nutricional e a produção do coqueiro nas condições edafoclimáticas de Moju (PA). O experimento foi conduzido durante dez anos, na fazenda SOCOCO®, e utilizou-se do delineamento em blocos ao acaso, com três repetições, e três níveis de P, três níveis de K e três níveis de Mg, na presença e na ausência de N. A resposta à aplicação dos tratamentos foi estudada em termos de concentração foliar dos macronutrientes (N, P, K, Ca, Mg e S) e dos micronutrientes (B e Cl), e da produção do número de cocos/planta/ano, peso de albúmen fresco (PAF)/coco e PAF/ ha/ano. Os resultados foram submetidos à análise de variância (p<0,05); e as médias, comparadas pelo teste de Tukey. De forma geral, a aplicação de N não influenciou as concentrações foliares dos macronutrientes e dos micronutrientes estudados. Por sua vez, as adubações fosfatada, potássica e magnesiana influenciaram a nutrição das plantas de coqueiro. As adubações fosfatada e potássica, isoladamente ou em interação, aumentaram a produção de cocos/planta e o PAF/ha. A partir do 6º ano de idade das plantas, os níveis de 54 kg/ha/ano de P2O5 e 96 kg/ha/ano de K2O são indicados para o cultivo do coqueiro na região da presente pesquisa. A adubação nitrogenada não influenciou os parâmetros de produção, e a adubação magnesiana aumentou a produção na presença da adubação potássica. Corresponding author: pmplins@uol.com.br Received: September 02, 2020 Accepted: May 04, 2021 Corresponding author: pmplins@uol.com.br Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará Soils and plant nutrition Soils and plant nutrition Soils and plant nutrition Introduction Despite recommendations (LINS and VIEGAS, 2020), liming is not a common practice in coconut plantations in the state of Pará; however, Ca is already normally supplied via P sources (superphosphates or natural phosphate). Coconut palm (Cocos nucifera L.) is an important crop for food security and income generation, as well as the production of many products (LINS and VIEGAS, 2008); thus, coconut has become an important agricultural activity for the Brazilian economy (FRÓES JÚNIOR et al., 2019). Areas cropped with coconut palms reach roughly 12 million ha worldwide and Indonesia, the Philippines, and India account for 73% of global production (FAOSTAT, 2018), yielding income through the export of coconut copra and oils (BRAINER, 2018). The nutritional status of coconut trees affects its vegetative growth, volume, and production (MATIAS et al., 2006; FERREIRA NETO et al., 2014). An adequate nutritional balance can increase coconut production by up to 125% (SECRETARIA and MARAVILLA, 1997). Thus, nutritional monitoring with the leaf analysis is an efficient fertilization method of coconut crops (SALDANHA et al., 2017), as observed for the state of Pará (LINS and VIEGAS, 2020). However, results on mineral nutrition in coconut palm crops are not commonly reported in the scientific literature in Brazil (LINS and VIEGAS, 2008) and may also vary depending on the coconut genotype (giant or hybrid) (ROGNON, 1984), requiring specific studies under the conditions of each region. Coconut crops in Brazil cover roughly 216,000 ha. The northeastern region accounts for 74% of the national production, southeastern region responds for 13%, and the northern region accounts for 12% (IBGE, 2018). In the northern region, the state of Pará has the largest area cropped with coconut in the country, totaling 18,600 ha, accounting for 93% of production with an average yield 9.35 t/ha (IBGE, 2018). The municipality of Moju alone accounts for 43% of the production in Pará State, with the world’s largest continuous area of coconut cultivation (FRÓES JÚNIOR et al., 2019). Therefore, the state of Pará has great potential in the coconut farming due to its edaphoclimatic characteristics, availability of rural credit, and knowledge of the activity at the commercial level (FRÓES JÚNIOR et al., 2019). Introduction The objective was to evaluate the effects of N, P, K, and Mg fertilization on the nutritional status and coconut production; therefore, an experiment was carried out in the edaphoclimatic conditions of Moju, Pará State, to provide information on crop fertilization in the region. Corresponding author: pmplins@uol.com.br Received: September 02, 2020 Accepted: May 04, 2021 Copyright: All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License. Termos para Indexação: Amazônia, Cocos nucífera L., macronutrientes. Termos para Indexação: Amazônia, Cocos nucífera L., macronutrientes. 1 2 P. M. P. Lins et al. Material and Methods ( ) Most coconut palm farms in Pará still operate with low investments, reflecting in low yields (LINS and VIEGAS, 2008), despite a highly technical production system, due to the presence of large commercial groups (FRÓES JÚNIOR et al., 2019). Mineral fertilization is used to promote growth and increase crop yield of coconut palm; however, information on this practice is scarce for the conditions in Pará (LINS and VIEGAS, 2008). Although soil fertility is not a limiting factor for coconut palm, fertilization greatly affects its yield (SOBRAL, 1998; SILVA et al., 2006; SILVA et al., 2009). Coconut trees have continuous growth, demanding large amounts of soil nutrients (RIBEIRO et al., 2016) that must be replenished by fertilization. Studies carried out in Brazil have evaluated mainly the response of coconut palm to N and K fertilization (SILVA et al., 2006; SILVA et al., 2009; RIBEIRO et al., 2011), but not to P and Mg applications. The coconut tree demands large amounts of K and, over the years, K fertilization may induce Mg deficiency in plants by antagonism (LIMA et al., 2018), a symptom commonly observed in coconut plantations. Thus, the balance of K and Mg supply for the coconut tree is of great importance, also justifying studies on Mg fertilization. The experiment was carried out at the property of SOCOCO S/A Group, located in the municipality of Moju, Pará State, Brazil, (02º 07’ 00” S and 48º W). The soil is Yellow Oxisol with sandy to sandy-loam texture and with low chemical fertility (Table 1). The climate of the municipality in the Ami type (Köppen classification), characterized as tropical rainy, without seasonal variations, and average total rainfall of 2,500 mm annually. We also recorded annual rainfall and minimum and maximum temperatures (from 21 to 33 ºC) in the site during the experimental period (Figure 1). The average relative air humidity is 82% and constant annual solar illumination of 2,200 h of sunshine. Table 1. Chemical characterization and clay concentration of the soil (0-20 cm) of the site before the experiment installation. pH SOC Ca+2 Mg+2 P K+ V Clay (H2O) g kg-1 -- cmolc dm3-- mg dm-3 ----%--- ------ 4.4 10.4 0.12 0.07 8 16 21 16 SOC- Soil Organic Carbon (colorimetric method). Ca+2, Mg+2 and K+ extracted by ion exchange resin. P (Olsen method). V- Base saturation. Table 1. Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará Figure 1. Annual rainfall and minimum and maximum temperatures in the site during the experimental period. Figure 1. Annual rainfall and minimum and maximum temperatures in the site during the e Each experimental plot consisted of seven rows with eight plants each, excluding external rows from the evaluations. The statistical design was randomized blocks, with three replications and the treatments consisted of two levels of N (N0 and N1), three levels of P (P0, P1, and P2), three levels of K (K0, K1, and K2), and three levels of Mg (Mg0, Mg1, and Mg2) (Table 2). In the second semester of the year, site preparation included mechanized deforestation, burning, and swathing. In December of the same year, cover plant Pueraria phaseoloides was cultivated, sowing 10 kg of seeds/ha, as recommended by Lins and Viegas (2020). In March of the following year, coconut trees were planted at spacing 8.5 x 8.5 m in the hexagonal system (7.4 m between rows), 160 plants/ha, covering an area of 9.45 ha. The cultivar used was hybrid PB – 121, a cross between yellow dwarf varieties from Malaysia and giant from western Africa. We carried out usual cultural treatments, necessary for the good development of coconut trees, such as cleaning pruning, crowning, and recess, among others. able 2. Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3 (e-113) Material and Methods Chemical characterization and clay concentration of the soil (0-20 cm) of the site before the experiment installation. SOC- Soil Organic Carbon (colorimetric method). Ca+2, Mg+2 and K+ extracted by ion exchange resin. P (Olsen method). V- Base saturation. SOC- Soil Organic Carbon (colorimetric method). Ca+2, Mg+2 and K+ extracted by ion exchange resin. P (Olsen method). V- Base saturation. SOC- Soil Organic Carbon (colorimetric method). Ca+2, Mg+2 and K+ extracted by ion exchange resin. P (Olsen method). V- Base saturation. Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3: (e-113) Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará Results and Discussion At the beginning of the rainy season in the region (December), samples of leaflets to determine the concentration of nutrients in the leaf were collected annually during eight years (4th and 5th and from the 8th to the 13th year of plant age), following the recommendations of IRHO (Institut de Recherches pour les Huiles et Oléagineux). In the first four years, leaf 9 was chosen to collect leaflets. From the fifth year onward, when the palms had more than 25 leaves, leaflets of leaf 14 were collected, according to the indications for the culture (LINS and VIEGAS, 2008). The foliar analyses were performed by the IRHO laboratory in France. The N was determined by the Kjedahl method; P by colorimetry using the ammonium vanadomolybdate method; K, Ca, and Mg, by atomic absorption spectrophotometry; S was determined by atomic absorption spectrophotometry, indirect method, via barium; B was determined by the azomethine H method, and Cl was analyzed by titration with silver nitrate, according to the method indicated by IRHO (1980). In addition, in 10 years of harvest (from the 4th to the 13th year of plant age), production of coconut palms was evaluated in terms of the number of coconuts/plant/year, average fresh albumen weight (FAW/coconut) and FAW/ha/year. For the production variables, significant interactions are presented (P and K levels, and K and Mg levels) on the average of the last three years of evaluation. The results were subjected to the analysis of variance (p <0.05) and the means compared by the Tukey test using the SISVAR statistical program (FERREIRA, 2011). p ( ) For plants in production (> 3 years) with N leaf concentrations of 18-19 g kg-1, for 160 plants/ha, it is recommended to apply N at 72 kg/ha/year (LINS and VIEGAS, 2020). Even in the absence of N fertilization (N0), N concentration was satisfactory and usually above the critical level (20.1 g kg-1) established by DRIS for the nutrient in hybrid coconut in the region (SALDANHA et al., 2017). As recommended for the cultivation of coconut tree (LINS and VIEGAS, 2008), the use of legume Pueraria phaseoloides ensures a satisfactory N nutrition. Leguminous plants fix atmospheric N by association with bacteria of the genus Rhizobium, making it available for successive or intercropping crops, after senescence. The literature indicates a contribution of P. Results and Discussion Results and Discussion Urea (45% N) was used as N source, simple superphosphate (only in the first year; 18% P2O5 + 18% Ca + 10% S) and triple superphosphate- TSP (in the other years; 45% P2O5 + 14% Ca) as P source, KCl as K source (60% K2O), and Mg oxide as Mg source (55% MgO). The levels were initially defined according to the soil analysis and then according to plant ages, based on the leaf diagnosis. The fertilizer levels in the first two years of cultivation were divided into two applications (middle and end of year) and the levels were applied only once in the middle of the year, in the other years. The applications were carried out uniformly in the projection area of the leaf crown, within a 2-m radius from the stipe base. Before deploying the treatments, the experimental site was uniformly fertilized with 300 kg ha-1 of natural phosphate (Gafsa; 28% P2O5), due to the natural P deficiency of the soil (Table 1). In all the years studied, N concentration in the leaves of the coconut tree was not influenced by the fertilization of N, P, K, and Mg (Figure 2a). The supply of N (72 kg/ha/year) only increased leaf N concentration in two (9 and 10) of the eight years evaluated. In Rio Grande do Norte State (Brazil), for the three-year- old green dwarf coconut tree, leaf N concentrations increased linearly and proportionally to the amount of N applied (SILVA et al., 2009). However, in Ceará State (Brazil), higher N supply did not raise its levels in leaf 9 of green dwarf coconut tree in the fourth year in an Entisol (MATIAS et al., 2016). N application to the soil did not increase its leaf concentration in dwarf coconut, due to the competition between the anions (Cl- and NO3 -) for the absorption and transport sites in the plant (FERREIRA NETO et al., 2014). The response to nutrient fertilization was evaluated in terms of the concentration of macronutrients (N, P, K, Ca, Mg, and S) and micronutrients (B and Cl) at the average coconut tree leaves. Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará Levels of nutrients applied in the treatments according to plant ages N0 N1 P0 P1 P2 K0 K1 K2 Mg0 Mg1 Mg2 Age ------N------ ---------P2O5-------- -----------K2O------- ------------MgO------ Years ----------------------------------------kg/ha/year----------------------------------------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 36 58 72 72 72 72 72 72 72 72 72 72 72 13* 0 0 0 0 0 0 0 0 0 0 0 0 0 26* 28 11 36 36 54 54 54 43 43 43 54 54 54 39* 62 22 72 72 108 108 108 86 86 86 108 108 108 26 0 0 0 0 0 0 0 0 0 0 0 0 0 53 62 19 77 96 96 96 96 96 77 77 96 96 96 106 125 38 154 192 192 192 192 192 154 154 192 192 192 14 0 0 0 0 0 0 0 0 0 0 0 0 0 28 20 13 44 44 44 44 44 35 35 22 44 44 44 48 40 26 88 88 88 88 88 70 70 44 88 88 88 *In this year, the simple superphosphate (18 % P2O5) was used as P source. Table 2. Levels of nutrients applied in the treatments according to plant ages Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3 (e-113) 4 P. M. P. Lins et al. Results and Discussion Leaf concentrations (g/kg) of N (a), P (b), and K (c) in coconut trees at different ages submitted to fertilization with nitrogen, phosphorus, potassium, and magnesium. Means followed by the same letters in the columns are considered statistically equal by the Tukey test (p> 0.05). C.L. – Critical Level established for the region of the present study with the DRIS method (SALDANHA et al., 2017). (MALAVOLTA et al., 1997). Interactions occur in mineral nutrition of the coconut tree, such as antagonism between K and Ca and between K and Mg, which is more significant (LINS and VIEGAS, 2008). On the other hand, P fertilization increased leaf P concentrations in all years studied, with no significant differences between the P1 and P2 levels in four years (Figure 2b). The average concentration of 1.4 g kg-1 in P1 coincides with the critical level proposed (LINS et al., 2003) and is close to the level established for the region (1.5 g kg-1; SALDANHA et al., 2017), although such levels were not reached, in most cases (Figure 2b). According to Ferreira Neto et al. (2014), the coconut tree has little requirement for P; nevertheless, P is important for its action in the regeneration of triphosphoric acid, abundant in young organs and in the energy transport related reactions. On the other hand, in all years studied, K fertilization increased leaf K concentrations of coconut trees, although below the critical level of 17.2 g kg-1 established for hybrid coconut plants in the region (SALDANHA et al., 2017). In the absence of KCl (K0), leaf K concentration was very low (average of 7.8 g kg-1), with an average value almost doubled in K1, reaching 15.8 g kg-1 in K2. Over the years, leaf K concentration in the absence of KCl decreased, due to the depressive effects of TSP and MgO on K nutrition, mainly in the absence of P fertilization (Figure 2c). In green dwarf coconut palm, K concentrations also increased in the leaves (SILVA et al., 2009; RIBEIRO et al., 2016) and in the water and in fruit peel, due to K fertilization (RIBEIRO et al., 2011). Results and Discussion phaseoloides as an N supplier of 455 kg/ha in the second year (PEREZ, 1997), much higher than the largest N dose applied in our study (Table 2). For a production of 130 fruits/plant/year, 57 kg/ha of N are exported (OUVRIER, 1990), well below the contribution of P. phaseoloides. Lower C/N ratio of the residue, higher rainfall, and high temperatures observed in the region (Figure 1) favor mineralization and consequent release of N fixed by the legume to the coconut crop. p Nitrogen fertilization increased leaf P concentration only in the fifth year of the coconut tree (Figure 2b). Despite a strong synergism between the N and P concentration in palm trees, N fertilization has a favorable effect on the P concentration in the plant (SILVA et al., 2009); however, this fact was not observed in our study. The leaf P concentration was only influenced by K fertilization in four years (8, 9, 10 and 13) and by Mg fertilization in two years (9 and 13). A synergism between Mg and P is observed in which absorption of Mg is maximum in the presence of P. Magnesium acts on chloroplast phosphorylation, synthesis of the ATP molecule and is a substrate for ATPases in the plant (MALAVOLTA et al., 1997). Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3: (e-113) Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará 5 Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará Figure 2. Leaf concentrations (g/kg) of N (a), P (b), and K (c) in coconut trees at different ages submitted to fertilization with nitrogen, phosphorus, potassium, and magnesium. Means followed by the same letters in the columns are considered statistically equal by the Tukey test (p> 0.05). C.L. – Critical Level established for the region of the present study with the DRIS method (SALDANHA et al., 2017). 5 p p Figure 2. Leaf concentrations (g/kg) of N (a), P (b), and K (c) in coconut trees at different ages submitted to fertilization with nitrogen, phosphorus, potassium, and magnesium. Means followed by the same letters in the columns are considered statistically equal by the Tukey test (p> 0.05). C.L. – Critical Level established for the region of the present study with the DRIS method (SALDANHA et al., 2017). Figure 2. Results and Discussion In Rio Grande do Norte State, leaf K concentrations showed a high correlation with the number of fruits in the sixth year Nitrogen fertilization did not influence leaf K concentrations, whereas P (P1 and P2) and Mg applications decreased their concentrations in most years evaluated (years 9, 10, 11 and 13) (Figure 2c). The effect of P fertilization on the reduction of leaf K concentrations must be related to the presence of Ca in P source (TSP; 18% CaO), since the increase in Ca supply can decrease K absorption by antagonism Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3 (e-113) 6 P. M. P. Lins et al. of cultivation of green dwarf coconut tree (FERREIRA NETO et al., 2014). Silva et al. (2009) suggest a high extraction power of K by the coconut tree, although the literature reports varied values. For Lins (2000), K has the largest withdrawal in the coconut crop (193 kg/ha) and, for a production of 130 fruits/plant/year, Ouvrier (1990) reported an export of 110 kg/ha of K2O. However, values of K accumulation by the coconut tree can reach 248 kg/ha with 78% of this accumulation removed by the fruits (MAGAT, 2005), indicating the need for replacement by fertilization, especially in soils with low K availability (Table 1). Leaf Ca concentrations were not influenced by N fertilization (Figure 3a), although there are reports of a significant increase in Ca concentrations due to N fertilization (LINS and VIEGAS, 2008). P fertilization increased Ca concentrations in coconut plants (Figure 3a), similar to reports in the literature (LINS and VIEGAS, 2008), due to the presence of Ca in the TSP composition. In the absence of P fertilization, Ca nutrition is deficient (Figure 3a). The supply of 54 kg/ha/year de P2O5 (P1) raised the average leaf Ca concentrations to 4.3 g kg-1, above the critical level 4.1 g kg-1 established for Ca under the edaphoclimatic conditions of Moju, Pará State (SALDANHA et al., 2017). For Lins (2000), Ca leaf concentrations between 3 and 4 g kg-1 are satisfactory and the application of Ca fertilizers does not mean yield increase. Experiments with PB-121 hybrid from the Ivory Coast shows that P fertilization increased leaf Ca concentrations by 23.5% in four years (IRHO, 1989). Figure 3. Leaf concentrations (g/kg) of Ca (a), Mg (b), and S (c) in coconut trees at different ages submitted to fertilization with nitrogen, phosphorus, potassium, and magnesium. Results and Discussion Except for years 11 and 12, leaf B concentrations were lower than the critical levels of 20 mg kg-1 (LINS et al., 2003) or 15 mg kg-1 (SALDANHA et al., 2017) established for the hybrid coconut tree in the region, indicating its deficiency. Low B absorption by plants in the present study (Figure 4a) may be attributed to lower OM concentration in the soil, low clay concentration, acidic pH, and high rainfall (DECHEN et al., 2018), besides the absence of B fertilization (Table 1; Figure 1). Intense rains in tropical conditions, especially in sandy soils, increase B losses by leaching, decreasing its availability and absorption (ABREU et al., 2007). Leaves sampled four months after the first B application to the soil in a five-year-old green dwarf coconut tree showed an average B concentration of 40 mg kg-1 (PINHO et al., 2008), well above the levels verified in the present study (Figure 4a). These authors verified B redistribution in coconut plants, regardless of the application mode, a relevant fact since B is known to be immobile (DECHEN et al., 2018). Research indicates that B can be mobile in certain plants because they produce simple sugars, polyols, which bind with B and form the sugar-B complex, promoting its mobility (BROWN and HU, 1998). There was no consistency in the response of leaf Mg concentrations in relation to N fertilization in the years of evaluation, with a concentration decrease observed only in some years (4, 9, 11, and 13) (Figure 3b). Silva et al. (2009) observed a linear reduction in leaf Mg concentrations due to N fertilization in green dwarf coconut. Except for the eighth year, leaf Mg concentrations increased (up to 29%) in all other years, due to P application. In addition, in all the years evaluated, Mg fertilization increased leaf Mg concentrations of coconut plants. Without the application of MgO, the average leaf Mg concentration is 1.8 g kg-1, while its leaf concentration in the presence of Mg1 increased to 2.7 g kg-1 of Mg, rising to 3.1 g kg-1 of Mg in the presence of the second level of Mg (Mg2). In most cases, leaf Mg concentration was way above the critical level 1.2 g kg-1 established for the culture in the region (SALDANHA et al., 2017). Results and Discussion Means followed by the same letters in the columns are considered statistically equal by the Tukey test (p> 0.05). C.L. - Critical Level established for the region of the present study with the DRIS method (SALDANHA et al., 2017). Figure 3. Leaf concentrations (g/kg) of Ca (a), Mg (b), and S (c) in coconut trees at different ages submitted to fertilization with nitrogen, phosphorus, potassium, and magnesium. Means followed by the same letters in the columns are considered statistically equal by the Tukey test (p> 0.05). C.L. - Critical Level established for the region of the present study with the DRIS method (SALDANHA et al., 2017). Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3: (e-113) Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará coconut palms. Compared to Cl- or NO3 -, B is more adsorbed to soil components (ABREU et al., 2007). However, high Cl- concentrations in the soil solution due to higher KCl levels may have caused greater B desorption from the colloids with consequent greater leaching of this micronutrient, intensified by the high rainfall levels in the region (Figure 1), contributing to less absorption of B by plants (Figure 4a). According to the International Plant Problem Insights (1989), as oil palm is a large KCl consumer, high rates of K fertilizer induce B deficiency; therefore, the application of both fertilizers is recommended, mainly in tropical acidic soils, poor in organic matter (OM), main B source in the soil (ABREU et al., 2007). Conversely, K and Mg fertilization decreased leaf Ca concentrations (Figure 3a), due to the antagonism of competitive inhibition between the nutrients (MALAVOLTA et al., 1997) also verified in coconut (LINS and VIEGAS, 2008). P fertilization decreased leaf Ca concentration in coconut by up to 53%. In the absence of K application, Ca concentration was 4.5 g kg-1 (average of the last three years of evaluations). In K1, Ca concentration reduced to 3.3 g kg-1, while in K2 showed a decrease of 50% of Ca concentration in the leaves (Figure 3a). Ferreira Neto et al. (2014) report that, as K is monovalent and with a lower hydration degree, it has preferential absorption over bivalent cations, such as Ca and Mg. Results and Discussion Lins et al. 8 Figure 4. Leaf concentrations of B (a), in mg/kg, and Cl (b), in g/kg in coconut trees at different ages submitted to fertilization with nitrogen, phosphorus, potassium, and magnesium. Means followed by the same letters in the columns are considered statistically equal by the Tukey test (p> 0.05). C.L. - Critical Level of B by the DRIS method (SALDANHA et al., 2017) and of Cl (MAGAT, 1991) established for the region of the present study. Figure 4. Leaf concentrations of B (a), in mg/kg, and Cl (b), in g/kg in coconut trees at different ages submitted to fertilization with nitrogen, phosphorus, potassium, and magnesium. Means followed by the same letters in the columns are considered statistically equal by the Tukey test (p> 0.05). C.L. - Critical Level of B by the DRIS method (SALDANHA et al., 2017) and of Cl (MAGAT, 1991) established for the region of the present study. In all the years evaluated, P fertilization increased the number of coconuts/plant (Figure 5a) and FAW/ ha (except for years 5 and 12) (Figure 5c); however, P addition reduced FAW/coconut by 6% from in 9-year- old plants (Figure 5b). Coconut plants fertilized with P yielded, on average, 66.2 coconuts/plant/year (P1) and 67.6 coconuts/plant/year (P2). In the absence of P application (P0), production was only 52.7 coconuts/ plant/year, on average, 30% less than the plants under P fertilization. The production of treatments P1 and P2 showed no significant differences between each other. During 10 years of evaluations, average yield of P1 was 76.2 coconuts/plant/year, while P2 reached 78.1 coconuts/plant/year (Figure 5). Assessment in 10 years of production (from the 4th to the 13th year of plant age), the number of coconuts/plant, FAW/coconut and FAW/ha/year were not influenced by N fertilization in the form of urea (Figures 5a, 5b, and 5c). Soil cover with legume Pueraria phaseoloides, sown before the implantation of the coconut crop, ensured soil protection and N supply to the plants (Figure 2a), in soil with medium concentration of OM (Table 1) and high rainfall indexes of the region (Figure 1). Soil OM is the main N source for plants in the soil, accounting for up to 95% of total N in the soil (SILVA and MENDONÇA, 2007). Results and Discussion Conversely, except for the eighth year, KCl fertilization decreased leaf Mg concentration of coconut trees (Figure 3b) by up to 45%, again due to the antagonism between K and Mg already observed for the crop (LINS and VIEGAS, 2008). In the Brazilian Northeast, KCl fertilization also decreased leaf Mg concentration of five-year-old (RIBEIRO et al., 2016) and three-year-old (SILVA et al., 2009) green dwarf coconut trees. In most years evaluated, leaf Cl concentrations were significantly influenced only by K fertilization with an increase up to five fold (Figure 4b). In the eighth year, K1 (96 kg/ha of K2O) increased leaf Cl concentration by 90% and by 109% with K2 (192 kg/ha of K2O). KCl application increased leaf Cl concentration of coconut tree in Rio Grande do Norte State (SILVA et al., 2009; FERREIRA NETO et al., 2014) and in Rio de Janeiro State (RIBEIRO et al., 2016). Adequate Cl supply to the coconut tree is important due to the crop requirement and Cl supply may deficient mainly in crops far from the coast (SOBRAL, 2003). The estimate of Cl extraction by the coconut tree is 125 kg/ha, which makes it the second most exported nutrient, even higher than N (LINS, 2000). With the exception of year 11 and 12, in most other cases, leaf Cl concentration was below (Figure 4b) the critical level (5.0 g kg-1) established by Magat (1991), despite KCl applications (Table 2). In most years evaluated, leaf S concentrations were not influenced by N, P, K, and Mg fertilizations and were always above (Figure 3c) the critical level 1.3 g kg-1 reported for the region of the present study (SALDANHA et al., 2017), except for year 11. i In most cases, there was no significant effect of N, P, and Mg fertilizations on leaf B concentrations up to the 13 years of age of the plants. However, K fertilization decreased leaf B concentrations of coconut trees in most years evaluated (Figure 4a). In eight years of evaluation, KCl fertilization (K2) reduced leaf B concentration by 25% on average. Applications of high KCl levels decreased leaf B concentrations by 4.6 mg kg-1, which becomes a limiting factor in the absence of B-based fertilizers. Therefore, there is possibly an antagonism in the absorption between chloride (present in KCl) and boric acid or borate in Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3 (e-113) P. M. P. Results and Discussion Furthermore, N applied as urea to the soil surface can promote significant N losses due to ammonia volatilization (CANTARELLA, 2007) and reduce crop response to its application (Figure 5a). p y ( g ) Regarding coconut quality, assessed by means of FAW/coconut, P applications in the form of TSP did not influence fruit quality in the first five years of evaluations. However, P supply subsequently decreased fruit weight (Figure 5b). In five consecutive years, FAW/ coconut was reduced by 6.3% in P1 treatment and by 5.4% in P2 treatment. In eight of the ten years evaluated, P application significantly increased FAW/ha (Figure 5c). The average yield in the last three years (11, 12, and 13) of P0 treatment was 3.52 t/ha of FAW, whereas P1 (54 kg/ha/year of P2O5) had an increase of 1.20 t/ha of FAW, a 33.9% gain, production statistically similar to that obtained in P2 (108 kg/ha/year of P2O5) (Figures 6a). Thus, for economic reasons, the lowest P dose (54 kg/ha/year of P2O5) is recommended for the coconut crop in the region evaluated. In Pará State, there is a recommendation of 72 kg/ha/year of P2O5 for coconut Lack of response to N application in coconut production has also been recorded in several experiments in West Africa and in Indonesia (OLLAGNIER and WAHYUNI, 1984). In São Paulo State (Brazil), N fertilization had a negative effect on water volume, average fruit mass, and on the number of fruits/bunch of dwarf coconut trees (TEIXEIRA et al., 2005a). On the other hand, in the Brazilian Northeast, coconut trees showed response to N fertilization (SOBRAL, 2004; SILVA et al., 2006). These results differ from our findings, due to different edaphoclimatic conditions, management practices, and age of plants. Regarding genotypic differences, Lins (2000) reported a great similarity in the extraction of nutrients, considering the same yield, between the hybrid and the giant coconut cultivars. Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3: (e-113) Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará 9 palm in production (> 3 years, 160 plants/ha) with leaf P concentration between 1.1 and 1.2 g kg-1 (LINS and VIEGAS, 2020). The lowest P dose of fertilization recommended in the present study indicates a specific recommendation for the coconut crop under the edaphoclimatic conditions of the municipality of Moju, compared to the recommendation for the state of Pará (LINS and VIEGAS, 2020). Results and Discussion - Critical Level established for the region of the present study with the DRIS method (SALDANHA et al., 2017). Figure 6. Production of fresh albumen weight-FAW/ha and leaf P concentration in coconut trees (11, 12, and 13 years old) submitted to phosphorus fertilization (a) and FAW/ha and K leaf concentration in coconut trees (11, 12, and 13 years old) submitted to potassium fertilization (b). Means, comparing FAW between the levels of each nutrient in each year followed by the same letters in the columns are considered statistically equal by the Tukey test (p>0.05). C.L. - Critical Level established for the region of the present study with the DRIS method (SALDANHA et al., 2017). Throughout the evaluation period, K fertilization had a positive effect on production variables, except for the number of coconuts/plant/year at five, six, and eight years of age (Figure 5). In the last three years evaluated (11-13), plants that received 96 kg/ha/year of K2O had an average yield of 85.6 coconuts compared to 67.8 coconuts/plant produced in K0 (without K), while with the application of 192 kg/ha of K2O (K2) yielded 86.8 coconuts/plant, statistically equal to K1 (Figure 5a). In the Ivory Coast, K fertilization raised the yield of hybrid PB 121 from 52.3 to 99 coconuts/ plant/year and increased the albumen weight by 13.9% (MANCIOT et al., 1980). In the Brazilian Northeast, the green dwarf coconut tree responded to K fertilization and, in the seventh year, the yield was above 200 fruits/ plant for all levels applied (FERREIRA NETO et al., 2014), also indicating possible genotypic differences in production capacity compared to the hybrid used in the present study (Figure 5). According to Lins and Viegas (2008), the coconut crop potential can reach 80 to 200 fruits/plant/year. 6b), thus, the level 96 kg/ha/year of K2O is indicated. Although K application increased its leaf concentration, the critical K level was not reached (Figure 6b). This indicates that the levels tested were not sufficient to provide adequate K nutrition to coconut trees (Figure 6b) and thus possibly limited their productive potential. Future studies should investigate higher K levels for the crop in the region. In the state of Pará, for 160 plants/ha, the recommendation is to apply 192 kg/ha/year of K2O for coconut plants with leaf K concentrations between 11 and 12 g kg-1 (LINS and VIEGAS, 2020). Results and Discussion In São Paulo State, 120 kg/ha of P2O5 (P resin < 13 mg/dm3) are recommended for an expected yield above 30 t/ha (TEIXEIRA et al., 2005b). (KHAN et al., 1985). P fertilization increased leaf P concentration; nevertheless, the P critical level was only reached in the 12-year-old plants, in the last three years evaluated (Figure 6a). This fact indicates a greater potential of crop response to P application at levels higher than the maximum level evaluated in this study (108 kg/ha/year of P2O5). In addition, in weathered tropical soils, P has low natural availability due to its high adsorption to Fe and Al oxides and precipitation with Al ions in solution (NOVAIS et al., 2007) thus increasing the potential of crop response. Unlike the results obtained in the current study, P application did not influence fruit production of the dwarf coconut tree planted in São Paulo State (TEIXEIRA et al., 2005a). Phosphorus is ranked seventh in order of nutritional importance for the coconut tree (OUVRIER, 1990). The positive response to P fertilization in plant production due to the low natural fertility of the cultivated soil (Table 1), which provided leaf concentrations below P critical level, despite fertilizations (Figures 2b) and the fact that the coconut crop is considered unresponsive P fertilization Figure 5. Production of coconuts/plant/year (a), fresh albumen weight-FAW/coconut (b) and FAW/ha/year (c) of coconut trees at different ages submitted to nitrogen, phosphorus, potassium, and magnesium fertilization. Means followed by the same letters in the columns are considered statistically equal by the Tukey test (p> 0.05). Figure 5. Production of coconuts/plant/year (a), fresh albumen weight-FAW/coconut (b) and FAW/ha/year (c) of coconut trees at different ages submitted to nitrogen, phosphorus, potassium, and magnesium fertilization. Means followed by the same letters in the columns are considered statistically equal by the Tukey test (p> 0.05). Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3 (e-113) 10 P. M. P. Lins et al. Figure 6. Production of fresh albumen weight-FAW/ha and leaf P concentration in coconut trees (11, 12, and 13 years old) submitted to phosphorus fertilization (a) and FAW/ha and K leaf concentration in coconut trees (11, 12, and 13 years old) submitted to potassium fertilization (b). Means, comparing FAW between the levels of each nutrient in each year followed by the same letters in the columns are considered statistically equal by the Tukey test (p>0.05). C.L. Results and Discussion In turn, in São Paulo State, it is recommended 300 kg/ha/year of K2O (exchangeable K+< 1.6 mmolc/dm3) (TEIXEIRA et al., 2005b). The average of the last three years of evaluation showed a significant effect of the combination of P and K supply on the number of coconuts/plant/year (Figure 7a) and on FAW/ha (Figure 7b). In the absence of P (0 kg/ha/year of P2O5) and K fertilization (0 kg/ha/year of K2O), production was negligible. The absence of K fertilization limited the production, even with the maximum P level (108 kg/ha/year of P2O5). In addition, at the highest K level (192 kg/ha/year of K2O) without P supply, plant production decreased significantly (Figures 7a and 7b). These results could be explained mainly by low natural soil fertility in terms of P and K in the experimental site (Table 1), requiring fertilization with both nutrients. The low natural fertility of the soil (Table 1) reflected in leaf K concentrations below its critical level, despite fertilizations (Figure 2c) and thus contributed to the positive response to K application in crop yield (Figure 5). Considering the last three years of evaluation (11, 12, and 13), K fertilization provided greater FAW/ha; however, without difference between the applications of 96 and 192 kg/ha/year of K2O (Figure Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3: (e-113) Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará 11 In the 11th year of the plants, on average, the number of coconuts/plant remained at 87.6, rising to 92 coconuts in the 12th year, and then falling to 62.7 coconuts in the 13th year. The difference in yield observed between the years evaluated was attributed to the water deficit in the region during the 12th year (Figure 1), reflecting on the yield of the following year. According to IRHO (1989), yield, rainfall, and water deficiency have a good relationship. Rainfall and yield curves are similar, while water deficiency reduces yield in the following year (MANCIOT et al., 1980). High temperatures and increased rainfall accelerate the leaf emission rate, allowing greater yield of coconut fruit (RIBEIRO et al., 2016). Results and Discussion A study carried out in the same region of the present study reported K and P as the nutrients that most indicated limitations in coconut plantations, using the DRIS leaf diagnosis method (SALDANHA et al., 2017). In oil palm trees, K fertilization was more efficient in the production of bunches under P fertilization (PACHECO et al., 1984). The coconut tree extracts P and N from the soil at great amounts, contributing to the formation of roots and fruits, as well as to stem thickening (OUVRIER, 1990), providing greater responses in terms of fruit quality (SILVA et al., 2006). However, the application of high K levels should be reviewed, considering the low recovery of K applied (RIBEIRO et al., 2011). K supply increased the production of dwarf coconut palm cultivated in the state of São Paulo (TEIXEIRA et al., 2005a), while its application (346 kg/ha/year) in the Brazilian Northeast provided the highest average weight of fruits (2.23 kg) of the coconut tree (SILVA et al., 2006). Mg fertilization did not increase production variables during 10 years of evaluation (Figure 5). Throughout the experimental period, the average yield of coconuts/plant/year (Figure 5a), FAW/coconut (Figure 5b), and FAW/ha/year (except for the 12th year) (Figure 5c) were statistically equal between treatments. Fertilizations carried out with Mg along the years increased leaf Mg concentrations way above its critical level (Figure 3b), favoring the absence of positive response of plants under production to Mg application (Figure 5). In Pará State, the application of 24 kg/ha/ year of Mg is recommended considering 160 plants/ha for plants with leaf Mg concentrations of 1.4-1.8 g kg-1 (LINS and VIEGAS, 2020). ( , ) Although isolated Mg fertilization did not influence coconut production, it had a significant effect on FAW/ha under K fertilization (Figure 7c). In the absence of K application (0 kg/ha/year of K2O), Mg fertilization did not increase FAW/ha, whereas Mg supply (44 and 88 kg/ha/year of MgO) showed greater production with the K fertilization (96 and 192 kg/ha/year of K2O). K excess could affect, positively or negatively, the absorption of other cations by the plants, as they are competing directly for the same site of the carrier, such as the depressive effect of K on Mg (MEURER et al., 2018). Mg deficiency could be induced by excessive K fertilization, increasing the K/ Mg ratio. Results and Discussion However, in K-deficient soils, condition of the current study (Table 1), K addition as fertilizer can lead to greater Mg absorption, which is accompanied by an increase in DM production (LIMA et al., 2018). Mg effect on production increase could reach 40%; however, this is only possible under K fertilization (IRHO, 1989). Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3 (e-113) 12 2 P. M. P. Lins et al. Figure 7. Effects of combinations of phosphorus and potassium fertilizations on production of coconuts/plant/year a) and on production of fresh albumen weight-FAW/ha (b) and magnesium and potassium fertilizations on FAW/ha c) in coconut trees (11, 12, and 13 years old). Means, comparing the variables between the levels of each nutrient n each supply of other, followed by the same letters in the columns are considered statistically equal by the Tukey test (p>0.05). P. M. P. Lins et al. Figure 7. Effects of combinations of phosphorus and potassium fertilizations on production of coconuts/plant/year (a) and on production of fresh albumen weight-FAW/ha (b) and magnesium and potassium fertilizations on FAW/ha (c) in coconut trees (11, 12, and 13 years old). Means, comparing the variables between the levels of each nutrient in each supply of other, followed by the same letters in the columns are considered statistically equal by the Tukey test (p>0.05). Figure 7. Effects of combinations of phosphorus and potassium fertilizations on production of coconuts/plant/year (a) and on production of fresh albumen weight-FAW/ha (b) and magnesium and potassium fertilizations on FAW/ha (c) in coconut trees (11, 12, and 13 years old). Means, comparing the variables between the levels of each nutrient in each supply of other, followed by the same letters in the columns are considered statistically equal by the Tukey test (p>0.05). P and K applications increase the number of coconuts/plant and fresh albumen weight (FAW/ha/ year). In the production phase (from the 6th year of plant age), levels of 54 kg/ha/year of P2O5 and 96 kg/ ha/year of K2O are indicated for coconut cultivation in the region of Moju, Pará State, Brazil;l References ABREU, C.A.; LOPES, A.S.; SANTOS, G.C.G. Micronutrientes. In: NOVAIS, R.F.; ALVAREZ, V.H.; BARROS, N.F.; FONTES, R.L.F.; CANTARUTTI, R.B.; NEVES, J.C.L. Fertilidade do solo. Viçosa: SBCS, 2007. p.645-736. (2) IBGE - Instituto Brasileiro de Geografia e Estatística. Anuário estatístico brasileiro. 2018. Disponível em: http://www2.sidra.ibge.gov.br/bda/tabela/listabl. asp?c=1613&z=t&o=11. Acesso em: 15 abr. 2020. (5) BETTER CROPS INTERNATIONAL. International plant problems insights: potassium and boron deficiencies in oil palm. Norcross: Potash and Phosphate Institute, 1989. (2) KHAN, H.H.; BIDDAPPA, C.C.; JOSHI, O.P. A review of Indian work on phosphorus nutrition of coconut. Journal of Plantation Crops, Kasarogod, v.13, n.1, p. 11-21, 1985. (1) BRAINER, M.S.C.P. Produção de coco: o Nordeste é destaque nacional. Fortaleza: ETENE/ Banco do Nordeste, 2018. (Caderno Setorial, 61) (1) LIMA, E.; VITTI, G.C.; SANTOS, L.A.; CICARONE, F. Cálcio e magnésio. In: FERNANDES, M.S.; SOUZA, S.R.; SANTOS, L.A. Nutrição mineral de plantas. 2.ed. Viçosa: SBCS, 2018. p.465-490. (2) BROWN, P.H.; HU, H. Manejo do boro de acordo com a sua mobilidade nas diferentes culturas. Piracicaba: POTAFÓS, 1998. (Informações Agronômicas, 48) (1) LINS, P.M.P. Resposta do coqueiro a adubação com N, P, K, Mg nas condições edafoclimáticas de Moju- PA. 2000. Dissertação (Mestrado em Agronomia) – Faculdade de Ciências Agrárias do Pará, Belém, 2000. (4) CANTARELLA, H. Nitrogênio. In: NOVAIS, R.F.; ALVAREZ, V.H.; BARROS, N.F.; FONTES, R.L.F.; CANTARUTTI, R.B.; NEVES, J.C.L. Fertilidade do solo. Viçosa: SBCS, 2007. p.376-449. (2) LINS, P.M.P.; VIÉGAS, I.J.M. Adubação do coqueiro no Pará. Belém: Embrapa Amazônia Oriental, 2008. (Documentos, 350) (1) DECHEN, A.R.; NACHTIGALL, G.R.; CARMELLO, Q.A.C.; SANTOS, L.A.; SPERANDIO, M.V.L. Micronutrientes. In: FERNANDES, M.S.; SOUZA, S.R.; SANTOS, L.A. Nutrição mineral de plantas. 2.ed. Viçosa: SBCS, 2018. p.491-562. (2) LINS, P.M.P.; VIÉGAS, I.J.M. Coqueiro. In: BRASIL, E.C.; CRAVO, M.S.; VIÉGAS, I.J.M. Recomendações de calagem e adubação para o estado do Pará. 2.ed. Brasília: Embrapa, 2020. p.347-349. (2) FAOSTAT. Food and Agriculture Organization of the United Nations. World production. Rome, 2018. Disponível em: http://faostat.fao.org/site/342/default. aspx. Acesso em: 15 abr. 2020. (5) LINS, P.M.P; NETO, J.T.F.; MULLER, A.A. Avaliação de híbridos de coqueiro (Cocos nucifera L.) para produção de frutos e de albúmen sólido fresco. Revista Brasileira de Fruticultura, Jaboticabal, v.25, n.3, p.468-470, 2003. (1) FERREIRA NETO, M.; HOLANDA, J.S.; GHEYI, H.R.; FOLEGATTI, M.V.; DIAS, N.S. Atributos químicos do solo e estado nutricional de coqueiro anão fertigado com nitrogênio e potássio. Revista Caatinga, Mossoró, v.27, n.3, p.30-40, 2014. (1) MAGAT, S.S. Coconut. In: IFA - International Fertilizer Association. Conclusion Applications of N do not increase nutrient con centrations in the coconut leaf, while the application of P increases the P, Ca, and Mg concentrations and decreases the K concentrations. Potassium fertiliza tion increases K and Cl concentrations and decreases Ca, Mg, and B concentrations, while Mg fertilization increases Mg concentrations and decreases K and Ca concentrations in the leaves; Nitrogen fertilization does not influence yield and Mg supply increases production in the presence of K fertilization. The coconut trees were well nourished in most years in terms of N, Mg, and S concentrations, but not for P, K, Ca, B, and Cl concentrations; Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3: (e-113) Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará 13 IRHO - Institut de Recherches Pour les Huiles et Oléagineux. Rapport d’activité. Oléagineux, Paris, v.44, n.4, p.1- 22, 1989. (1) References World fertilizer use manual. 2005. Disponível em: http://www.fertilizer.org/ifa/Home- Page/LIBRARY/Our-selection2/World-Fertilizer-Use- Manual/by-type-of-crops. Acesso em: 10 abr. 2020. (5) FERREIRA, D.F. Sisvar: a computer statistical analysis system. Ciência e Agrotecnologia, Lavras, v.35, n.6, p.1039-1042, 2011. (1) MAGAT, S.S. Fertilizer recommendations for coconut based on soil and leaf analyses. Philippine Journal of Coconut Studies, Ithaca, v.16, n.2, p.25-29, 1991. (1) FRÓES JÚNIOR, P.S.M.; AVIZ, W.L.C.; REBELLO, F.K.; SANTOS, M.A.S. Sources of Growth and Spatial Concentration of Coconut Crop in the State of Pará, Brazilian Amazon. Journal of Agricultural Science, Ontario, v.11, n.2, p.159-168, 2019. (1) MALAVOLTA, E.; VITTI, G.C.; OLIVEIRA, S.A. Avaliação do estado nutricional das plantas: princípios e aplicações. 2.ed. Piracicaba: POTAFÓS, 1997. 319p. (2) Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3 (e-113) 14 P. M. P. Lins et al. RIBEIRO, G.; MONNERAT, P.H.; CAMPANHARO, M.; RABELLO, W.S. Adubação potássica aplicada na axila foliar e no solo em coqueiro anão verde. Revista Ceres, Viçosa, MG, v.63, n.1, p.68-75, 2016. (1) MANCIOT, R.; OLLAGNIER, M.; OCHS, R. Nutricion mineral y fertilizacion del cocotero en la mundo. Oléagineux, Paris, v.35, n.1, p.13-27, 1980. (1) MATIAS, S.S.R.; AQUINO, B.F.; FREITAS, J.A.D.; HERNANDEZ, F.F.F. Análise foliar de coqueiro anão em duas épocas diferentes em relação a doses de nitrogênio e potássio. Revista Ciência Agronômica, Fortaleza, v.37, n.3, p.264-269, 2006. (1) RIBEIRO, G.; MONNERAT, P.H.; CAMPANHARO, M.; RABELLO, W.S. Qualidade do fruto de coqueiro anão verde em resposta à adubação potássica. Revista Caatinga, Fortaleza, v.24, n.2, p.187-191, 2011. (1) ROGNON, F. Cocotier. In: MARTIN-PRÉVEL, P.; GAGNARD, J.; GAUTIER, P. L’analyse végétale dans le contrôle de l’alimentation des plantes tempérées et tropicales. Paris: Tec&Doc, 1984. p.447-57. (2) MEURER, E.J.; TIECHER, T.; MATTIELLO, L. Potássio. In: FERNANDES, M.S.; SOUZA, S.R.; SANTOS, L.A. Nutrição mineral de plantas. 2.ed. Viçosa: SBCS, 2018. p.429-464. (2) SALDANHA; E.C.M.; SILVA JUNIOR, M.L.; LINS, P.M.P.; FARIAS, S.C.C.; WADT, P.G.S. Nutritional diagnosis in hybrid coconut cultivated in northeastern Brazil through Diagnosis and Recommendation Integrated System (DRIS). Revista Brasileira de Fruticultura, Jaboticabal, v.39, n.1, p.1-9, 2017. (1) NOVAIS, R.F.; SMYTH, T.J.; NUNES, F.N. Fósforo. In: NOVAIS, R.F.; ALVAREZ, V.H.; BARROS, N.F.; FONTES, R.L.F.; CANTARUTTI, R.B.; NEVES, J.C.L. Fertilidade do solo. Viçosa: SBCS, 2007. p.471- 550. (2) OLLAGNIER, M.; WAHYUNI, M. Mineral nutricion and fertilization of the Malayan Dwarf x West African Tall (PB-121- MAWA) hibryd coconut. Oléagineux, Paris, v.39, n., p.8-9, 1984. (1) SECRETARIA, M.I.; MARAVILLA, J.N. References Response of hybrid coconut palms to application of manures and fertilizers from field-planting to full-bearing stage. Plantations Recherche Développement, Montpellier, v.4, n.2, p.126-138, 1997. (1) OUVRIER, M. Evolution de la composition minérale du cocotier hybride PB 121 au jeune âge. Oléagineux, Paris, v.45, n.2, p.69-80, 1990. (1) SILVA, I.R.; MENDONÇA, E.S. Matéria orgânica do solo. In: NOVAIS, R.F.; ALVAREZ, V.H.; BARROS, N.F.; FONTES, R.L.F.; CANTARUTTI, R.B.; NEVES, J.C.L. Fertilidade do solo. Viçosa: SBCS, 2007. p.275- 374. (2) PACHECO, A.R.; TAILLIEZ, B.J.; SOUZA, R.L.R. de; LIMA, E.J. de. Deficiências minerales de la palma aceitera (E. guineensis Jacq.) en la region de Belém do Pará, Brasil. In: MESA REDONDA LATINOAMERICANA SOBRE PALMA ACEITEIRA, 2., 1984, Belém. Anais [...]. Belém: Ministério da Agricultura, 1984. p.237-268. (3) SILVA, R.A.; CAVALCANTE, L.F.; HOLANDA, J.S.; PEREIRA, W.E.; MOURA, M.F.; FERREIRA NETO, M. Qualidade de frutos do coqueiro-anão verde fertirrigado com nitrogênio e potássio. Revista Brasileira de Fruticultura, Jaboticabal, v.28, n.2, p. 310-313, 2006. (1) PEREZ, P.N.L. Crescimento, concentração e conteúdo de nutrientes em Pueraria phaseoloides L. com dois anos a oito anos de idade em Latossolo Amarelo distrófico, Tailândia, Pará. 1997. Dissertação (Mestrado em Agronomia) - Faculdade de Ciências Agrárias do Pará, Belém, 1997. (4) SILVA, R.A.; CAVALCANTE, L.F.; PAES, R.A.; HOLANDA, J.S.; COMASSETTO, F. Avaliação do estado nutricional do coqueiro anão verde fertirrigado com nitrogênio e potássio. Revista Caatinga, Fortaleza, v.22, n.1, p.119-130, 2009. (1) PINHO, L.G.R.; MONNERAT, P.H; PIRES, A.A.; SANTOS, A.L.A. Absorção e redistribuição de boro em Coqueiro-anão-verde. Pesquisa Agropecuária Brasileira, Brasília, DF, v.43, n.12, p.1769-1775, 2008. (1) SOBRAL, L.F. Fertirrigação do coqueiro anão verde com N e K no platô de Neópolis. In: FERTBIO, 2004, Lages. Anais [...]. Lages: UDESC/SBCS, 2004. CD- ROM. (3) Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3: (e-113) Nutrition and production of coconut palm cultivated with mineral fertilization in the state of Pará 15 SOBRAL, L.F. Nutrição e adubação do coqueiro. In: FERREIRA, J.M.S.; WARWICK, D.R.N.; SIQUEIRA, L.A. A cultura do coqueiro no Brasil. 2.ed. Aracaju: Embrapa-CPATC, 1998. p.129-157. (2) TEIXEIRA, L.A.J.; BATAGLIA, O.C.; BUZETTI, S.; FURLANI JUNIOR, E.; ISEPON, J.S. Adubação com NPK em coqueiro-anão-verde (Cocos nucifera L.) rendimento e qualidade de frutos. Revista Brasileira de Fruticultura, Jaboticabal, v.27, n.1, p. 120-123, 2005a. (1) SOBRAL, L.F. Nutrição e adubação. In: FONTES, H.R.; RIBEIRO, F.E.; FERNANDES, M.F. Coco: produção e aspectos técnicos. Brasília: Embrapa, 2003. p.44-52. (2) TEIXEIRA, L.A.J.; BATAGLIA, O.C.; BUZETTI, S.; FURLANI JUNIOR, E. SOBRAL, L.F. Nutrição e adubação. In: FONTES, H.R.; RIBEIRO, F.E.; FERNANDES, M.F. Coco: produção e aspectos técnicos. Brasília: Embrapa, 2003. p.44-52. (2) SOBRAL, L.F. Nutrição e adubação do coqueiro. In: FERREIRA, J.M.S.; WARWICK, D.R.N.; SIQUEIRA, L.A. A cultura do coqueiro no Brasil. 2.ed. Aracaju: Embrapa-CPATC, 1998. p.129-157. (2) References Recomendação de adubação e calagem para coqueiro (Cocos nucifera L.) no estado de São Paulo - 1ª Aproximação. Revista Brasileira de Fruticultura, Jaboticabal, v.27, n.3, p.519-520, 2005b. (1) Rev. Bras. Frutic., Jaboticabal, 2021, v. 43, n. 3 (e-113)
https://openalex.org/W3083307158	https://zenodo.org/record/4010159/files/A%20study%20of%20fibre%20bundles%E2%80%99%20formation%20regularities%20during%20the%20impact%20interaction%20of%20spikes%20with%20raw%20cotton%20particles.pdf	English	null	A study of fibre bundles’ formation regularities during the impact interaction of spikes with raw cotton particles	Eastern-European journal of enterprise technologies	2,020	cc-by	9,103	A STUDY OF FIBRE BUNDLES’ FORMATION REGULARITIES DURING THE IMPACT INTERACTION OF SPIKES WITH RAW COTTON PARTICLES The conditions for imparting a rotational motion to the flyer were determined when the flyer was connected with the supplied layer of raw cotton. Turning the flyer against the axis will twist the fibre bundle that binds the particle to the rest of the cotton mass, and this is a prerequisite for the fibre to bundle. The value of the twist angle will depend on both the frictional force and the time of the force action, which together determine the energy spent on twisting. By piecewise linearization of the P(t) curve, the processes of shock loading and unloading of the flyer during interaction with the spike are described in good agreement with the experimental oscil- lograms of the process. The eccentric interaction of the spike with a particle of raw cot- ton was considered. The relationship of this interaction with the formation of soft defects in the fibre – bundles – was proven exper- imentally and theoretically. It is recommended to install spike roll- ers having a flat front face in the system of raw cotton pre-cleaning. The use of spikes of this shape of the front edge ensures a steady reduction in the number of bundles in the raw cotton fibre, which increases the quality of the fibre and reduces the amount of defects and impurities. F . V e l i e v Doctor of Technical Sciences, Professor Department of Technological Machines and Equipment of the Branch Azerbaijan State University of Economics (UNEC) Istiglaliyyat str., 6, Baku, Azerbaijan, AZ 1001 E-mail: fazil-uzbekr@mail.ru F . V e l i e v Doctor of Technical Sciences, Professor Department of Technological Machines and Equipment of the Branch Azerbaijan State University of Economics (UNEC) Istiglaliyyat str., 6, Baku, Azerbaijan, AZ 1001 E-mail: fazil-uzbekr@mail.ru As a result of the experimental and theoretical studies, data have been obtained to make it possible to organise the efficient operation of cleaning machines in the cotton ginning industry f g g g y Keywords: fine impurity cleaner, feed rollers, spike rollers, blade, number of bundles Received date 25.05.2020 Accepted date 21.07.2020 Published date 17.08.2020 Received date 25.05.2020 Accepted date 21.07.2020 Published date 17.08.2020 Copyright © 2020, F. Veliev This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0) Copyright © 2020, F. Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 4/1 ( 106 ) 2020 UDC 3326. 01 UDC 3326. 01 The article describes a study of the dynamics of interacting between loosening roller spikes and particles of raw cotton as well as the conditions for the raw cotton particles’ bundling. The process of movement of raw cotton particles was considered on the mesh sur- face of a fine impurity cleaner; a search was made for ways to inten- sify the process and to increase its stability. DOI: 10.15587/1729-4061.2020.209052 1. Introduction An important reserve for improving the process of clean- ing raw cotton from weed particles is a new direction in the technology of processing raw cotton – preparing raw cotton for cleaning processes by a directed change in its technolo gical properties. So far, the process has been analysed only descriptively, that is, the theory of the process used to be described by the Newto- nian model of impact. This impact makes it possible to estimate the kinematic characteristics of the process before and after the impact, as well as the value of the complex characteristic of the impact-impulse. The state of the product before and after cleaning has been experimentally studied, but it only indirectly characterises the correctness of the adopted process models. The section for preliminary cleaning of raw cotton from small impurities is essentially an element of the roller-type fine impurity cleaner built into a large impurity cleaner. Its main purpose is to remove fine impurities from the mass of raw cotton through a mesh surface, but at the same time it performs a number of auxiliary functions. For example, it increases the uniformity of the feeding of the machine, changes to some extent the technological characteristics of raw cotton, and separates coarse and fine impurities from cotton, which prepares their further removal. It is of great national economic importance to solve the problem of establishing a theory of the behaviour of raw cot- ton in the process of its impact and the use of the basic laws of mechanics in relation to the technological processes of the primary processing of cotton in order to select their most ra- tional parameters that ensure an improvement in the quality of fibres and seeds. A number of studies [1–7] highlighted some of the technological aspects of impurity extraction in this type of a cleaner. A number of new problems were identified, the study of which became possible due to the use of modern means of experimental research and processing of its results. A STUDY OF FIBRE BUNDLES’ FORMATION REGULARITIES DURING THE IMPACT INTERACTION OF SPIKES WITH RAW COTTON PARTICLES Veliev This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0) them is the impact of the spikes and slats, and due to the high relative speed of the spikes (7.85–11.5 m/s), the value of the impact force can be significant. 4. An experimental study of the impact of splitting on a particle of raw cotton Studies [6, 7] are also noteworthy. In particular, it is of interest to research auger cleaners, due to which it becomes possible to use special ‘pockets’ that help bring down the mo- notony of the process and revive the ability of raw cotton to release small impurities. The complexity of an experimental study of the processes of shock interaction of the working bodies of cotton ma- chines with the processed product lies in the transience in time. The process is carried out within 0.002–0.004 s, and its registration requires high-frequency converters of mechani- cal values into electrical ones. Work [8] discloses in-depth tests of the effect of surface roughness parameters of the working bodies of machines on the conditions of contact with fibrous material. It was shown that the class of roughness of the working surface of the per- forming body such as spikes cannot objectively characterise the conditions of interaction of the surface with the fibre. The parameters of the contact process – friction and capture of individual fibres by the surface – depend on the ratio of the pitch and height of the microroughness, the magnitude of the radii at the tops and grooves of the microrelief, and the presence of any submicron unevenness. Moreover, the practice of scientific research recommends the choice of such a natural frequency of the system that it is 5–10 times higher than the highest frequency from the ex- pansion of a real process into a harmonic Fourier series [14]. If this frequency is unknown, which is most often the case in practice, the choice of the frequency response of the measure- ment equipment is carried out by sequential enumeration of the options for the natural frequency of the elements of the strain-gauge system. The dynamic estimate of the error of such a system can be given by an approximate method [8]. Technological properties of raw cotton as a material for cleaning have not been disclosed in practice in order to control the cleaning processes and improve product quality [9]. Stu dies indicate that for many years the quality of cotton has been assessed organoleptically, but it is planned to replace subjective visual assessment with objective instrumental measurements. Searches were made for systems with a high natural fre- quency (2,500–3,500 Hz), easy to manufacture and suitable for the use of conventional strain gauges. 2. Literature review and problem statement It is proposed to consider raw cotton containing structu ral particles of a spherical shape with several flyers in the pro- cesses occurring in cleaners of large and small impurities [1]. After the feeding device, the particles of raw cotton enter the area of movement of the spikes and slats of spike rollers or spike-slatted rollers. The first technological impact on 72 Engineering technological systems: Reference for Chief Technology Specialist at an industrial enterprise damaged, which leads to an increase in the amount of defects in the fibre. Indeed, the conditions for such cotton bundles to form occur in the pre-cleaning section. On the basis of this model, an attempt is made to reveal the conditions to throw material on the serrated surface and fix it with a brush roller, taking into account the deformation of the raw cotton slices. While solv- ing the problems under consideration, it is necessary to pay special attention to the need for uniform supply to the clea ners, which is a problem that has not been largely resolved yet. In [11], the issue of increasing the efficiency of cleaning raw cotton from impurities was studied only for raw cotton of machine harvesting. The author did not consider the friction forces and the influence of the elastic characteristics of raw cotton under shock loads. As a result, the desired cleaning effect was not achieved. Therefore, it should be taken into account that the issues of the influence of the elastic cha racteristics of raw cotton on the formation of bundles in the fibre, as well as ensuring uniform supply of the cleaners of the feed rollers, remain unsolved. The results considered in [1] were supplemented by study [2], where elements of the process of transporting raw cotton were researched experimentally. The air velocity in the transporting channel affected the quality of the fibre, and thus the property of the fibre was damaged. It was found that the geometric parameters of the transporting channel had changed the physical characteristics of the fibres. At a minimum, the values of the impact force obtained in this work were an order of magnitude higher than the actual ones. 3. The aim and objectives of the study The aim of the study is to reveal the regularities of the formation of bundles of raw cotton fibres resulting from the impact interaction of the flyer on the grate surface. In study [3], it was noted that fibre characteristics consti- tute an important factor in determining yarn properties. Cotton, whose physical properties vary depending on the region of culti- vation, is still the main raw material used in the textile industry. Properties such as fibre length, thickness, strength and ripeness of raw cotton grown in different regions affect its processing. To achieve the aim, the following tasks were set and done: – to study experimentally the impact of the process of splitting on a particle of raw cotton; – to develop an approximate method for describing the process of shock loading and unloading of raw cotton particles; – to research the conditions for the formation of bundles of fibres from an eccentric impact of a spike on a cotton particle. – to develop an approximate method for describing the process of shock loading and unloading of raw cotton particles; – to develop an approximate method for describing the process of shock loading and unloading of raw cotton particles; The framework of the analysis prevents from dwelling in more detail on a number of studies [4, 5] and others. It is essen- tial to add, however, that many studies follow the path of in- tensifying processes, increasing their technological reliability, and in the direction of creating conditions that prevent a na tural decrease in the ability of raw cotton to release impurities. – to research the conditions for the formation of bundles of fibres from an eccentric impact of a spike on a cotton particle. 2. Literature review and problem statement A conclusion can be made that the problems of the influ- ence of the elastic characteristics of raw cotton and a study of the dynamics of the interaction of the spikes of a loosening roller with particles of raw cotton should still be considered. The process of fibre bundling conditions and ensuring uni- form feeding of fine impurity cleaners are the issues to be researched. As shown in studies [1, 2], subsequently and obviously from the elementary calculation, the energy balance of the shock process and the nature of the change in the energy consumed by the flyer from the spike were not confirmed experimentally. However, the experiments described in this work were performed incorrectly in the sense of choosing the frequency of natural vibrations of the system. Table 1 The parameters of the process of impact interac of a spike with particles of raw cotton The parameters of the process of impact interaction of a spike with particles of raw cotton Impact parame- ters Cotton particles with one flyer two flyers 8.2 m/s 10.5 m/s 12.8 m/s 8.2 m/s 10.5 m/s 12.8 m/s Impact force P, N 0.598 0.780 0.966 0.801 1.05 1.31 Load rise time t, s 0.0022 0.0023 0.0023 0.0024 0.0025 0.0026 Total impact time, s 0.026 0.03 0.037 0.041 0.049 0.052 Fig. 1. A tubular converter of the magnitude of the impact force into an electrical signal: 1 – a thin-walled tube; 2 – a rod; 3 – a clamp; 4 – a strain gauge For the tube simulating a grate with l = 0.08 m, J = = 1.4557∙10–9 m4, and m = 0.2450 kg/m, we had f = 2,979 Hz. At l = 0.04 m, the value was f = 11,900 Hz; however, the sensi- tivity of the system sharply decreased. Since the amplifier had the carrier frequency of 5,000 Hz, the bandwidth of 2,000 Hz, and the frequency range of natu- ral oscillations of the loops of 900–6,000 Hz, this system can be considered suitable for studying processes with a lower frequency of 300–600 Hz, that is, for shock peaks with time of sinusoidal peak at 0.0025–0.0035 s. In article [10], it was stated that the fixed time of the load increase when the flyer hit the grate was much shorter than the load fall time. The same phenomenon was noted in the study of the impact of a spike on particles of raw cotton. Fig. 2 shows the diagram of a unit for measuring the force of a splitting impact on a particle of raw cotton, and Fig. 3 shows the typical oscillogram of the process. 1 2 3 4 5 6 Fig. 2. 4. An experimental study of the impact of splitting on a particle of raw cotton These requirements are met by a thin-walled tubular sensor with a diameter of 10–20 mm and a wall thickness of 0.4–0.5 mm, which is brought up to 0.08–0.1 mm at the place where the strain gauge is glued (Fig. 1). The working length of the sensor and, consequently, its frequency are regulated based on the specified sensitivity of the system and the value of the output signal by a rod that can move along the axis of the tube. In [10], the ways of increasing the efficiency of cleaning raw cotton from small impurities by improving the spike-slat- ted rollers were researched. However, changing the profile of the mesh surface did not improve the effect of cleaning cotton from coarse impurities. Practical tests have shown that when the spike hits cotton at a speed of more than 12 m/s, seeds are 73 4/1 ( 106 ) 2020 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 P(t), N 0.9 0.001 0.003 0.008 t, s P 𝑡𝑡𝑛𝑛 𝑡𝑡𝑝𝑝 Fig. 3. A typical oscillogram of the process of a spike impact on cotton particles Due to the thinness of the sensor, low weight and, at the same time, high resilience, the sensor has a high natural fre- quency of transverse vibrations, determined for the first (lo west) frequency [12]: f EJ m =     1 85 2 . ,  (1) (1) where l is the length of the console part of the sensor; E is Young’s modulus, for steel 2.06∙1011 n/m; J is the moment of inertia of the section; and m is the mass per unit length of the tube. For a spike of the section of preliminary cleaning of cot- ton, the calculated parameters of the tubular element were l = 0.07 m, J = 1.688∙10–9 m4, and m = 0.1225 kg/m; the natural frequency was f = 2,549.3 Hz. Fig. 3. A typical oscillogram of the process of a spike impact on cotton particles 1 2 3 4 Fig. 1. A tubular converter of the magnitude of the impact force into an electrical signal: 1 – a thin-walled tube; 2 – a rod; 3 – a clamp; 4 – a strain gauge Table 1 shows the values of the impact force P of a spike on a single flyer, with the time of the load rise tn at different splitting speeds. 4. An experimental study of the impact of splitting on a particle of raw cotton The parameters of the impact on a particle consisting of two flyers are also given. Table 1 The diagram of the unit for studying the conditions of dynamic interaction of the spike with the cotton: 1 – a roller; 2 – a tubular spike with strain gauges; 3 – a particle of raw cotton; 4 – a clamp; 5 – a variable drive; 6 – a mesh surface The nature of this phenomenon, associated with the pro perties of a tubular element as a thin-walled shell, is the man- ifestation of the material microflow mechanism during its unloading (if the fibre length is l ≤ 12 mm, then the material microflow occurs). With shock unloading, this phenomenon is inherent in steel, but especially for thin-walled shells made of it [11] (if the fibre length is l ≥ 26 mm, it is the thin-walled shell that is considered during shock unloading of the spike against raw cotton particles). 4 Therefore, the total impact time can be considered equal to (2.2–2.3)∙tn, and the time of the load drop on the oscillogram can be attributed to the properties of the tubular element. The established series of values of the force of the split- ting impact of the spike on the raw cotton flyer helps describe the dynamic properties of flyers under impact. In this case, some change in the load rise time can be a consequence of the nonlinearity of the loading characteristics as a function of both the shape of the deformable cotton particle and the properties of cotton as a multicomponent material. Fig. 2. The diagram of the unit for studying the conditions of dynamic interaction of the spike with the cotton: 1 – a roller; 2 – a tubular spike with strain gauges; 3 – a particle of raw cotton; 4 – a clamp; 5 – a variable drive; 6 – a mesh surface 74 Engineering technological systems: Reference for Chief Technology Specialist at an industrial enterprise For the first loading section, (7) and (8) at y0 = 0 and δ1 = 0 will be simplified: 5. The processes of shock loading and unloading of raw cotton particles and approximate methods of their description t P y P V 1 1 1 1 0 1 = arcsin , (9) V V y P 1 0 2 1 2 1 2 = − . (10) (9) (9) The processes of convergence and retreat of a fibrous cotton particle relative to the surface of a spike or grate are described by a nonlinear differential equation (in the absence of friction): (10) m y t cyn d d 2 2 0 + = , (2) If the final value of yr (during loading) is unknown in the last, r-th section, then the condition for the completion of the loading process will be zero velocity and (2) the solution of which in the general case for n ≠ 1 presents significant difficulties. t P V y P r r r r r r = − ( ) − − 1 1 1 arctg δ , (11) (11) It is possible to analyse the impact process by replacing the sections of the curve with straight lines – chords or tangents. In turn, they deviate slightly at the final length of an i-th sec- tion from the curve P(y) when y changes from yi–1 to yi and for which the linear differential equation of motion is valid: and the value of yr is found by substituting (11) into (4). Relations (4)–(8) are similarly used for unloading a parti- cle, and the initial conditions of the process will be different – y0 = ymax (or ymax–yp) V0, and at yr = 0, the rebound velocity Vr will be found in the last section. m y t k y i i d d 2 2 0 + − ( ) = δ , (3) (3) The values of the impact parameters depend on ki, on δi, as well as on the method of approximating the Р(y) curve by a broken line. In the case of replacing a portion of the curve between P cy i i n − − = 1 1 and P cy i i n = with a chord (chord method), the latter is described by the equation of a straight line (Fig. 4): where ki is the coefficient of proportionality of the linear relationship; P = ki(y–δi) for this section; δi is the coordinate of the point of intersection of the straight line Р(y) with the y-axis. 5. The processes of shock loading and unloading of raw cotton particles and approximate methods of their description Solution (3) with initial conditions: t = 0, y = y(i–1) and d d y t Vi = −1 will be written for the i-th section as follows: P P P y y y y P i i i i i i = − − − ( )+ − − − − 1 1 1 1, (12) (12) y y p t V p p t i i i i i i i = − ( ) + + − − 1 1 δ δ cos sin , (4) y y p t V p p t i i i i i i i = − ( ) + + − − 1 1 δ δ cos sin , (4) d d y t V p t y p p t i i i i i i = − − ( ) − − 1 1 cos sin , δ (5) (4) reduced evenly by dividing уmax into r equal parts to the form: P cy r i i y cy r i i i i n n n n n n n n = − − ( )   − − ( )− − ( )   − − max max . 1 1 1 1 1 (13) d d y t V p t y p p t i i i i i i = − − ( ) − − 1 1 cos sin , δ (5) (13) r r     P=c𝑦𝑦𝑛𝑛 0 1 2 i–1 i r y P(𝑦𝑦𝑖𝑖) P(𝑦𝑦𝑖𝑖−1) δ𝑖𝑖 𝑦𝑦𝑖𝑖 𝑦𝑦𝑖𝑖−1 P Fig. 4. The graph for approximating a portion of the Р(y) curve by a straight line (chord method) where where P p k m i i = (6) (6) is the natural frequency. At the end of the period of move- ment corresponding to the i-th section, (4) and (5) take the values of уi and Vi, respectively. One of them is given by dividing the curve, and it serves to determine the time of movement ti; the other is obtained by substituting ti into one of the listed equations. 5. The processes of shock loading and unloading of raw cotton particles and approximate methods of their description The velocity V(t) (solid lines) and acceleration а(t) (dashed lines) of cotton particles relative to the spike during its impact: 1 – V0 = 8.2 m/s; 2 – 10.5 m/s; and 3 – 12.8 m/s Since ki, Рi, and δi are taken for a system without fric- (21) V e V p n b t y p V nb p n b nb t i i i i i i i i i i i = − − − − ( ) + − × × − − − − 1 2 2 1 2 1 2 2 cos si δ n , p n b t i i 2 2 −                 (22) 0 0.0008 0.0016 0.0024 t, s 1.0 0.6 0.2 0.4 0.8 P, N y, mm 20 16 12 8 4 1 2 3 Fig. 5. The design graphs of the loading process: y(t) (dashed lines) and Р(t) (solid lines) at 1 – V0 = 8.2 m/s; 2 – 10.5 m/s; and 3 – 12.8 m/s (22) j δ δ i i i i i i i i i y p V nb V nb y = − ( ) + + − ( ) − − − − arctg 1 2 1 1 1 . (23) (23) δ i i i i V nb y + ( ) − − 1 1 0 0.0008 0.0016 t, s a, m/s−2 V, m/s−1 8,000 6,000 4,000 2,000 1 2 3 2 4 6 8 10 12 Fig. 6. The velocity V(t) (solid lines) and acceleration а(t) (dashed lines) of cotton particles relative to the spike during its impact: 1 – V0 = 8.2 m/s; 2 – 10.5 m/s; and 3 – 12.8 m/s 0 0.0008 0.0016 t, s a, m/s−2 V, m/s−1 8,000 6,000 4,000 2,000 1 2 3 2 4 6 8 10 12 Fig. 5. The design graphs of the loading process: y(t) (dashed lines) and Р(t) (solid lines) at 1 – V0 = 8.2 m/s; 2 – 10.5 m/s; and 3 – 12.8 m/s The total calculated impact time for all variants of tn (re- spectively equal to 0.002285 s, 0.002241 s, and 0.002205 s) agrees well with the experimental values. 5. The processes of shock loading and unloading of raw cotton particles and approximate methods of their description 75 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 4/1 ( 106 ) 2020 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 4/1 ( 106 ) 2020 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 4/1 ( 106 ) 2020 δi i i i i i i y y y P P P = − − − − − − − 1 1 1 1 (14) contracting it, we bring (17) to a linear form by means of the substitution: δi i i i i i i y y y P P P = − − − − − − − 1 1 1 1 (14) contracting it, we bring (17) to a linear form by means of the substitution: δi i i i i i i y y y P P P = − − − − − − − 1 1 1 1 (14) contracting it, we bring (17) to a linear form by means of the substitution: δi i i i i i i y y y P P P = − − − − − − − 1 1 1 1 r when dividing ymax by r equal parts: δi n n n n y i i i i i i = − ( )− − ( )   − − ( )   max . 1 1 1 (15) c mn b b = 2 , (18) k m p i i = 2 (19) and making the following transformation: (18) or when dividing ymax by r equal parts: (19) (15) and making the following transformation: The value of the proportionality coefficient ki is respec- tively determined from the equality: d d d d 2 2 2 2 0 y t nb y t p y i i i i i + + = , (20) (20) K P P y y cy r i i i i i i i n n n n = − − = − − ( )   − − − − 1 1 1 1 1 max . (16) (16) where ki is determined from (16). Solving (20), we obtain for the i-th section the values of displacements and velocities: where ki is determined from (16). 5. The processes of shock loading and unloading of raw cotton particles and approximate methods of their description It is easier to divide the displacement yi into several linearized sections, that is, to get the values of yi and to de- termine Vi from the found ti: t p V p y V p y i i i i i i i i i = × × + + − ( ) +       − − − − − 1 1 1 1 1 2 2 1 2 1 2 2 1 arccos δ δi i i i i i i y V p y ( ) −       − ( ) + − ( )       − − − 2 1 1 2 2 1 2 1 1 δ δ , (7) V V p t y p t i i i i i i i i = − − ( ) − − 1 1 cos . δ (8) t p i i = × 1 p V p y V p y i i i i i i i i i × + + − ( ) +       − − − − − 1 1 1 1 2 2 1 2 1 2 2 1 arccos δ δi i i i i i i y V p y ( ) −       − ( ) + − ( )       − − − 2 1 1 2 2 1 2 1 1 δ δ , (7) V V p t y p t i i i i i i i i = − − ( ) − − 1 1 cos . δ (8) Fig. 4. The graph for approximating a portion of the Р(y) curve by a straight line (chord method) (8) The value of the segment cut off by the straight line from the oy axis is determined from the relation: The value of the segment cut off by the straight line from the oy axis is determined from the relation: The obtained value of Vi together with yi will serve as the initial conditions for the next (i+1)-th section. 5. The processes of shock loading and unloading of raw cotton particles and approximate methods of their description Solving (20), we obtain for the i-th section the values of displacements and velocities: In the calculated ratios with known c and for a given Pmax, it is not difficult to determine ymax. In the calculated ratios with known c and for a given Pmax, it is not difficult to determine ymax. ± − ( ) = − ( ) + − ( )+ − × × − − − − y e y nb y V p n b p i nb t i i i i i i i i i i δ δ δ 1 2 1 1 2 2 2 sin − + ( ) n b t i i 2 j , (21) Fig. 5 shows the calculated oscillograms for cases of a spike impact on a single flyer with velocities of 8.2 m/s (curves 1), 10.5 m/s (2), and 12.8 m/s (3). In Fig. 5, the solid lines show the laws of increase in the load Р(t), and the dashed lines show the laws of change y(t). ±( ) = ( ) + − × × − y e y p n b p i i i i i i δ δ 1 2 2 2 sin − + ( ) n b t i i 2 j , (21) V e V p n b t y p V nb p n b nb t i i i i i i i i i i i = − − − − ( ) + − × × − − − − 1 2 2 1 2 1 2 2 cos si δ n , p n b t i i 2 2 −                 (22) j δ δ i i i i i i i i i y p V nb V nb y = − ( ) + + − ( ) − − − − arctg 1 2 1 1 1 . (23) 0 0.0008 0.0016 t, s a, m/s−2 V, m/s−1 8,000 6,000 4,000 2,000 1 2 3 2 4 6 8 10 12 Fig. 6. 5. The processes of shock loading and unloading of raw cotton particles and approximate methods of their description The graph of the eccentric impact interaction of a spike with a particle of raw cotton π 8 π 4 3π 8 π 2 α 1 2 0.7 5 0.5 0 0 0.25 𝑃𝑃𝑦𝑦, P Fig. 9. The dependences of Р(t) (curve 1) and its component Рy(t) (curve 2) on α and e 𝑉𝑉0 01 0 𝛼𝛼 y x e 𝑃𝑃𝑦𝑦 𝑉𝑉𝑛𝑛 𝑉𝑉𝜏𝜏 𝑟𝑟𝑙𝑙 P F 𝑟𝑟𝑘𝑘 Fig. 8. The graph of the eccentric impact interaction of a spike with a particle of raw cotton𝑃 The processes of microfluidity of the sensor material, apparently, are well described by the model with viscous fric- tion at sufficiently large values of ε, and at ε→1 the curve of the fall of P turns into an exponential. 5. The processes of shock loading and unloading of raw cotton particles and approximate methods of their description It tends to decrease with an increase in the initial rate of the process V0, which, of course, cannot be detected experimentally due to the small variation in tn. In this sense (for n = 1.174), the computational model is close to linear. Fig. 6. The velocity V(t) (solid lines) and acceleration а(t) (dashed lines) of cotton particles relative to the spike during its impact: 1 – V0 = 8.2 m/s; 2 – 10.5 m/s; and 3 – 12.8 m/s Fig. 6 shows the dependences of the particle velocity V and its acceleration a on time t for the corresponding values of the initial velocity. They show a significant role in the process of separating fibrous material and impurities, in loosening raw cotton, and disclosing the effective open surfaces of structural cotton particles by their deformation and downsizing. Since ki, Рi, and δi are taken for a system without fric- tion, their definition is similar to the loading conditions and there is no need for additional calculations; only ac- cording to the accepted numbering during unloading, the i-th section acquires the values of the (r–i+1)-th loading section. The motion of the system with a nonlinear elastic element and viscous friction is described by the differential equation: m y t c y cy b n d d d dt 2 2 0 + + = , (17) (17) Here, in the first section, V0 = 0 y0 = ymax; in the last one at the end of the process, y = (ymax–ym) = 0 and Vi0 = V0m. In (21), the plus sign is taken for ji > 0 and minus is for ji < 0. where cb is the coefficient of viscous friction. Using the chord method, replacing the i-th section of the curve with a chord 76 76 Engineering technological systems: Reference for Chief Technology Specialist at an industrial enterprise P m n r r e r r c V k n k n n n n n n = − ( ) + ( ) −     + ( )       + + + 1 2 2 2 2 1 1 1 0 2 1. (26) Fig. 5. The processes of shock loading and unloading of raw cotton particles and approximate methods of their description 7 shows the curves of changes in the load P(t) and the particle velocity V(t) for n = 1.174, c = 105.04, and V0 = 12.8 m/s as the calculation results; the ratio of the time for the load rise and fall was tn/tp = 0.233. In this case, the ratio of the coefficient of viscous resistance nbi to the frequen- cy Pi found from equation (23) was ε = 0.749. (26) With n≈1, (26) is simplified and reduced to a linear de- pendence on V0 and cos α: 8 0 0.002 0.004 0.006 0.008 0.010 t, s 1 2 P(t), N 0.4 0.8 12 4 V, m/s−1 Fig. 7. The calculated curves for V = 12.8 m/s with tp/tn = 4.3 and α = 2.84: 1 – P(t ); 2 – V(t) P mcV 1 0 = cos . α P mcV 1 0 = cos . α (27) V, m/s−1 (27) From (26) and (27) it is obvious that the impact force P de- creases with increasing e and the corresponding increase in the angle α. The value of the impact force, recorded by the sensor and directed along the velocity vector V0, changes even more: P P y = cos , α (28) P P y = cos , α (28) taking the following form for nonlinear stiffness: taking the following form for nonlinear stiffness: P m n c V y n n n n n n n = + ( ) ( )           + + + + 1 2 1 1 1 2 1 4 1 cos , α (29) (29) Fig. 7. The calculated curves for V = 12.8 m/s with tp/tn = 4.3 and α = 2.84: 1 – P(t ); 2 – V(t) and at n = 1, and at n = 1, P mcV y = 0 2 cos . α (30) (30) Let us note, firstly, a good coincidence of the nature of the curve, P(t) obtained by calculation, with the oscillogram shown in Fig. 3 and removed when struck with a spike on a raw cotton flyer. The nature of the change in the functions of Р and Рy is seen in Fig. 9. in Fig. 9. 𝑉𝑉0 01 0 𝛼𝛼 y x e 𝑃𝑃𝑦𝑦 𝑉𝑉𝑛𝑛 𝑉𝑉𝜏𝜏 𝑟𝑟𝑙𝑙 P F 𝑟𝑟𝑘𝑘 Fig. 8. M ≤μPr. The dependence of the moment of rotation M0 (curve 1) and the energy E transferred to the rotation of the flyer on α and e during the impact M ≤μPr. (36) Between entangled fibres of a special nature of fric- tion, where the reaction of the surface to the inclination of a tangible body interacting with it is to move tangentially at the point of contact, the equal sign takes place when friction reaches a threshold value, which is possible only with a sig- nificant value of Vτ. which can be transformed by the substitution: V V V e r r r k n = = + 0 0 sin , α V V V e r r r k n = = + 0 0 sin , α and also the values of P from (26) or (27). For the nonlinear system, we have: Therefore, the function of Mo, in the case of equality in (31) monotonically decreases synchronously with Р, which decreas- es with increasing α and e. In real conditions, with α = 0, P is equal to zero, and only when the splitting threshold is reached, the spike takes the calculated value (curve 1, Fig. 10). E t t e r r c m n r r e r r n p k n n k n k n = + ( ) + + ( ) + ( ) −     + ( )         + μ 1 1 2 2 1 2   + + V n n 0 3 1 1 , (37) (37) π 8 π 4 3π 8 π 2 𝛼𝛼 E, M 1 2 0.75 0.50 0.25 Fig. 10. The dependence of the moment of rotation M0 (curve 1) and the energy E transferred to the rotation of the flyer on α and e during the impact π 8 π 4 3π 8 π 2 𝛼𝛼 E, M 1 2 0.75 0.50 0.25 and for linear, it is: E t t mcV n p = + ( ) μ α α 0 2 2 sin cos . (38) (38) The nature of the change in the energy consumed by the flyer from the spike where the rotational movement takes place is shown in Fig. 10 (curve 2). It is obvious that the greatest twisting of the fibrous bond of the raw cotton par- ticles should occur at α = p/4, which was fully confirmed in experiments carried out at ginneries. Fig. 10. 6. A study of the conditions for the formation of fibre bundles as a result of an eccentric impact of a spike on a cotton particle The previously considered axial impact of the spike on the raw cotton particles can be implemented in the presence of a round spike only in isolated cases. More often, the velo city vector of a spike of a radius rk (Fig. 8) forms with the centre of the cotton flyer adopted in this consideration such eccentricity e that it has the shape of a ball of a radius rl. In this case, the angle between the spike velocity vec- tor Vo and the velocity component Vn, directed from the centre of the spike to the centre of the spike, is: Fig. 8. The graph of the eccentric impact interaction of a spike with a particle of raw cotton𝑃 Fig. 8. The graph of the eccentric impact interaction of a spike with a particle of raw cotton𝑃 α = + arcsin , e r r k n (24) (24) π 8 π 4 3π 8 π 2 α 1 2 0.7 5 0.5 0 0 0.25 𝑃𝑃𝑦𝑦, P and the value of the indicated velocity component is: V V V r r e r r n k n k n = = + ( ) − + 0 0 2 2 cos . α (25) (25) The velocity Vn determines the character of the impact interaction of the cotton particles with the spike. Equa- tion (25), in the case of the nonlinear dependence of Р(у), can be used to find the relationship between the impact force and the splitting speed V0 as a function of the eccentricity e: Fig. 9. The dependences of Р(t) (curve 1) and its component Рy(t) (curve 2) on α and e 77 77 4/1 ( 106 ) 2020 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 These dependences coincide with the experimental values of Py, obtained at different values of e and given in Table 2. The maximum power transmitted by the spike will be determined as: W PV max = μ (33) Table 2 (33) The experimental values of the impact force Py, N at various values of e and V0 Disaxial value e, mm Velocity V0, m/s 8.2 10.5 12.8 0 0.586 0.780 0.966 6 0.549 0.716 0.892 12 0.476 0.549 0.736 The experimental values of the impact force Py, N at various values of e and V0 considering that Vτ changes insignificantly during the impact. 6. A study of the conditions for the formation of fibre bundles as a result of an eccentric impact of a spike on a cotton particle The average value of the transmitted power at a small change of α during the collision period (tn+tp) can be deter- mined as follows: W V t t P t t op r n p t t n p = + ( ) ( ) + ( ) ∫ μ 0 d , (34) (34) and the total energy transmitted to the flyer will be: In an ideal model, in the absence of friction, the eccentric impact would not have negative consequences – the overall value of the shock load would decrease, and free or cohesive particles would be reflected in the direction of the velocity V0. In practice, the situation is somewhat different: during an impact, friction arises between the colliding surfaces due to the tangential component of the velocity Vτ and forming a moment relative to the centre of the flyer’s gravity (with Amontons’ laws of friction): In an ideal model, in the absence of friction, the eccentric impact would not have negative consequences – the overall value of the shock load would decrease, and free or cohesive particles would be reflected in the direction of the velocity V0. E W t t V P t t op n p r t t n p = + ( ) ( ) + ( ) ∫ μ 0 d . (35) (35) Taking into account the previously researched character of the shock peak, with a sufficient degree of accuracy, the integral in the latter relations can be replaced by the function of the maximum value of the impact force P, after which (35) is transformed to: In practice, the situation is somewhat different: during an impact, friction arises between the colliding surfaces due to the tangential component of the velocity Vτ and forming a moment relative to the centre of the flyer’s gravity (with Amontons’ laws of friction): E V P t t r n p = + ( ) μ 2 , (36) M ≤μPr. (31) (31) M ≤μPr. 8. Conclusion whence it follows that the most dangerous for the process under consideration is the case of α = π/4, when the rota- tional energy received by a cotton particle from the spike is maximum and the torsional rigidity ck of the bonds is minimal. In this case, the maximum twist angle occurs in a time corresponding to a quarter of the full period of torsio nal vibrations of the system: 1. The impact interaction of the spikes of a cleaning roller with particles of raw cotton was researched experimentally. The rise time and the magnitude of the shock load have been determined to be 0.0022–0.0026 s and 0.60–1.30 n respectively when the splitting speed changed from 8.2 m/s to 12.8 m/s. / 2. The experimental data were used to describe the cha racteristics of the nonlinearity of the dynamic stiffness of the flyers upon impact, with the possibility of its approximation by a law of the form P = cyn. It has been found that with a small difference in the load rise and fall times, the nature of the load fall almost completely coincides with the growth curves, whereas the accelerations and velocities have only the opposite sign. If tn < tp, the values of the accelerations and velocities of the flyer decrease due to energy dissipation, which can be established by analysing the corresponding model with viscous friction. / 2. The experimental data were used to describe the cha racteristics of the nonlinearity of the dynamic stiffness of the flyers upon impact, with the possibility of its approximation by a law of the form P = cyn. It has been found that with a small difference in the load rise and fall times, the nature of the load fall almost completely coincides with the growth curves, whereas the accelerations and velocities have only the opposite sign. If tn < tp, the values of the accelerations and velocities of the flyer decrease due to energy dissipation, which can be established by analysing the corresponding model with viscous friction. j π max . = 2 J ck (41) j π max . 7. Discussion of the results of studying the process of fibre bundles’ formation during the impact interaction with spikes A general estimate of the value of the maximum twist angle of a fibre strand jmax is possible as a result of analysing the solution of the differential equation of the rotational motion of a raw cotton particle with a moment of inertia J: If the flyer, entrained in the rotational motion by the spike, has a tangential velocity at the point of contact close to Vτ, then the instantaneous value of the power transmitted to the flyer will be determined from the relation: motion of a raw cotton particle with a moment of inertia J: J t C P t r k l d d 2 2 j j μ + = ( ) , (39) (32) J t C P t r k l d d 2 2 j j μ + = ( ) , (39) W t P t V ( ) = ( ) μ . (32) W t P t V ( ) = ( ) μ . (32) 78 Engineering technological systems: Reference for Chief Technology Specialist at an industrial enterprise and from the equation of the energy balance of the system that follows from (39). Formulae (37) and (38) simplify the problem even more, reducing the calculation to the relation: according to the developed theory of their analysis and synthesis have not yet been implemented in production, but their application, as the results of this research show, has good prospects, and work in this direction continues. jmax , = 2E ck (40) jmax , = 2E ck (40) 8. Conclusion L., Samandarov, S. A., Libster, S. L. (1977). Potochnaya liniya pervichnoy pererabotki hlopka-syrtsa PLPH. Hlopkovaya promyshlennost’, 3, 1–3. 4. Sapon, A. L., Samandarov, S. A., Libster, S. L. (1977). Potochnaya liniya pervichnoy pererabotki hlopka-syrtsa PLPH. Hlopkovaya promyshlennost’, 3, 1–3. 5. Korabel’nikov, R. V., Ibrogimov, H. I. (2008). Kompleksnyy pokazatel’ vozdeystviya ochistitelya hlopka na hlopok-syrets v protsesse ochistki. Tehnologiya tekstil’noy promyshlennosti, 3, 35–38. 5. Korabel’nikov, R. V., Ibrogimov, H. I. (2008). Kompleksnyy pokazatel’ vozdeystviya ochistitelya hlopka na hlopok-syrets v protsesse ochistki. Tehnologiya tekstil’noy promyshlennosti, 3, 35–38. 6. William, Y. M., Stedronsky, L. V. (1999). Effect of variations in design of gin-saw teeth on lint guality u ginning efficiency. Washington, D. C., 25. 6. William, Y. M., Stedronsky, L. V. (1999). Effect of variations in design of gin-saw teeth on lint guality u ginning efficiency. Washington, D. C., 25. 7. Ibrogimov, H. I., Korabel’nikov, R. V. (2008). Modelirovanie protsessa razrezheniya sloya hlopka-syrtsa v pitatele ochistitelya melkogo sora. Izv. VUZov. Tehnologiya tekstil’noy promyshlennosti, 4, 34–38. 7. Ibrogimov, H. I., Korabel’nikov, R. V. (2008). Modelirovanie protsessa razrezheniya sloya hlopka-syrtsa v pitatele ochistitelya melkogo sora. Izv. VUZov. Tehnologiya tekstil’noy promyshlennosti, 4, 34–38. 8. Shaw, G. S., Franks, S. N. (1994). Cleaning and extracting. Hand book for cotton Gunners. Washington, 28–32. 9. Matusiak, M., Walawska, A. (2010). Important Aspects of Cotton Colour Measurement. FIBRES & TEXTILES in Eastern Europe 18 (3 (80)), 17–23. 9. Matusiak, M., Walawska, A. (2010). Important Aspects of Cotton Colour Measurement. FIBRES & TEXTILES in E 18 (3 (80)), 17–23. 10. Veliev, F. (2019). Construction of a theoretical method for evaluating the kinematic and geometric parameters of loosening rollers in the cleaners of raw cotton from large impurities. Eastern-European Journal of Enterprise Technologies, 6 (7 (102)), 71–79. doi: https://doi.org/10.15587/1729-4061.2019.183864 10. Veliev, F. (2019). Construction of a theoretical method for evaluating the kinematic and geometric parameters of loosening rollers in the cleaners of raw cotton from large impurities. Eastern-European Journal of Enterprise Technologies, 6 (7 (102)), 71–79. doi: https://doi.org/10.15587/1729-4061.2019.183864 11. Nuriyev, M. N., Seydaliyev, I. M. (2018). Development of methods to control quality of the structure of cross-wound packages. Eastern-European Journal of Enterprise Technologies, 5 (1 (95)), 61–70. doi: https://doi.org/10.15587/1729-4061.2018.143825 12. Anan’ev, I. V. (1996). Spravochnik po raschetu sobstvennyh kolebaniy uprugih sistem. Moscow, 276. 11. Nuriyev, M. N., Seydaliyev, I. M. (2018). Development of methods to control quality of the structure of cross-wound packages. Eastern-European Journal of Enterprise Technologies, 5 (1 (95)), 61–70. 8. Conclusion = 2 J ck (41) Laboratory tests of rollers of a serial design and with spikes having a flat front surface and sharply reducing the probability of an eccentric impact on the particles of raw cot- ton have shown a steady decrease in the number of bundles in the fibre of raw cotton. For raw materials processed in an experimental fine impurity cleaner, the cleaning effect changed from 0.430 % to 0.337 % with an overall decrease in the amount of defects and contamination by 1–13 %. 3. It has been established that the moment generated relative to the centre of gravity of the flyer creates conditions for imparting rotational motion to the flyer. In this case, its rotation against the axis will lead to twisting of the bundle of fibres connecting the particle with the rest of the cotton mass, and this is a prerequisite for the fibre bundle formation. 3. It has been established that the moment generated relative to the centre of gravity of the flyer creates conditions for imparting rotational motion to the flyer. In this case, its rotation against the axis will lead to twisting of the bundle of fibres connecting the particle with the rest of the cotton mass, and this is a prerequisite for the fibre bundle formation. Using flat side spikes in the fine impurity cleaner and optimising the surfaces of the fibre cleaner, as predicted by previous studies, have shown a significant decrease in the number of soft defects in the fibre. The studies of the new methods for cleaning raw cotton with optimal grate profiles References 1. Nesterov, G. P., Borodin, P. N., Belyalov, R. F. (1978). Novaya potochnaya liniya sushki i ochistki hlopka-syrtsa. Hlopkovaya prom’shlennost’, 1, 2–4. 2. Rudovsky, P. H., Nuriyev, M. N., Recebov, I. S. (2019). Preparation of Flax Roving for Spinning in Electro-Chemical Activated Solutions. Fibres and Textiles in Eastern Europe, 27 (6 (138)), 34–38. doi: https://doi.org/10.5604/01.3001.0013.4465 y, P. H., Nuriyev, M. N., Recebov, I. S. (2019). Preparation of Flax Roving for Spinning in Electro Chemical Activa s. Fibres and Textiles in Eastern Europe, 27 (6 (138)), 34–38. doi: https://doi.org/10.5604/01.3001.0013.4465 3. G naydin, G. K., Soydan, A. S., Palamut u, S. (2018). Evaluation of Cotton Fibre Properties in Compact Yarn Spinning Processes and Investigation of Fibre and Yarn Properties. Fibres and Textiles in Eastern Europe, 26 (3 (129)), 23–34. doi: https:// doi.org/10.5604/01.3001.0011.7299 4. Sapon, A. 8. Conclusion doi: https://doi.org/10.15587/1729-4061.2018.143825 11. Nuriyev, M. N., Seydaliyev, I. M. (2018). Development of methods to control quality of the structure of cross-wound packages. Eastern-European Journal of Enterprise Technologies, 5 (1 (95)), 61–70. doi: https://doi.org/10.15587/1729-4061.2018.143825 79
https://openalex.org/W2993056987	https://www.iiste.org/Journals/index.php/RHSS/article/download/49170/50798	English	null	Practices and Challenges of Financial Resource Utilization in Government Secondary Schools of Dawro Zone	Research on Humanities and Social Sciences	2,019	cc-by	13,245	Abstract Abstract This study was conducted in secondary schools of Dawro Zone, Southern Nations Nationality and People Regional Government. The purpose of the study was to investigate the Practices and Challenges of financial Resource Utilization. Descriptive survey design was employed to conduct the study. It was conducted in two woredas and one city administration. Two woredas were selected at random and city administration was taken based on availability. Ten schools were chosen at random. Then, availability technique of the non-random method was used to take sample respondents of Kebele Education and Training Board and Parent Student Teacher Association chairpersons where as teachers were selected at random. Principals, vice principals, supervisors and school financial workers were included by availability. The total sample size of the study was 176. Questionnaire, interview guide, and document analysis were used to collect data .Both quantitative and qualitative methods of data analysis were employed. In line with the nature of the basic questions and the data collected frequency of response, percentage, mean and standard deviation were employed to present and analyze data quantitatively. Besides, data obtained from the open ended items, and interview were analyzed qualitatively. As a result, the study revealed that government secondary schools of the Zone are largely dependent on governmental and non-governmental organization or donors for its sources of finance; there was lack of effectiveness of school budget utilizing; the stake holders’ participation was not well known and highest as much as the expected degree of participation; the absence of internal audit and limited amount of the coordination, follow up, monitor, control, and feedback of external audit offered to schools can reduce proper implementation of school finance and can put schools below its standards; also schools encounter a lot of challenges in utilization of financial resources. Some of them were mentioned here, for instance, the allocated budget was not as equivalent as the needs of schools, principal is over loaded or busy in accomplishment of many activities rather than budget process, untimely disbursement of school budget from government to schools were identified as challenges among others that created ineffective utilization of school finance. www.iiste.org www.iiste.org Research on Humanities and Social Sciences Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 Abstract Finally, based on findings and conclusion of the study recommendations were made on schools have to diversify the sources of finance through expands internal income by agricultural product, co-curricular clubs and any other activities; For effective school budget utilization schools should have utilize approved budget according to plan, need to utilize allocated budget for expected educational purpose; schools and concerning body need to mobilize, develop awareness through discussion, and design strategy that can lead to highest participation of school committee and other stake holders; to promote the practice of schools performing or implementing financial issue the recruitment of internal audit is advisable by woreda and city administration education office and the coordination, follow up, monitor, control, and feedback of internal audit is obligatory; besides, for effective and efficient budget utilization the needs of schools more or less will be satisfied if schools create or generate their own maximum internal income as much as possible; Principals need to manage their time and delegate activities in order to fully utilize time and follow budget process; the local government should disburse school budget at the right time. Keywords: Finance; Resource; Utilization; Secondary school and Zone DOI: 10.7176/RHSS/9-15-01 Publication date: August 31st 2019 Publication date: August 31st 2019 Practices and Challenges of Financial Resource Utilization in Government Secondary Schools of Dawro Zone Israel Eyasu1 Endale Berhanu2 1.Corresponding author: Wolaita Sodo 2.Co-author; Wolaita Sodo University 1. Background of the Study The criteria for grants distribution mainly based on the size of the student population, the number of teachers, and class size (MoE, 2002).The size of the student population is the most crucial factor when allocating the block grant (MoE, 2002). The school director and the finance unit workers are relatively familiar with the details of the block grant, much more so than other school- level actors, such as parents, teachers, and students. In contrast with the block grant, the size of the school grant to be allocated to each school is only based on enrollment. The number of teachers and the class size are not taken in to account. Similarly, in recent time, Department For International Development (DFID) budget has been allocating to government secondary schools based on the number of students who have scored two and above two points in Ethiopia general secondary school examination. The amount of budget to be allocated for female students is greater than those of male students (SREB, 2015). The transfer mechanism of all grants to each school are more or less similar in our country Ethiopia. It was learned that a key player in the process is Ministry of Finance and Economic Development (MoFED). All information on the schools must be passed on, through several echelons, up to this ministry which is responsible for releasing the funds back down to schools. Schools prepare and submit the total number of their students population to their sub-city or woreda education office, which consolidates the data from all the schools and submits them to regional education bureau. In turn, this education bureau combines the same school data and submits them to MoFED. Following this process, the grants which are to be allocated to each schools pass to reach the school account. According to Blue Book at the school level, the block grant budget is expected to cover recurrent expenditures, that is to say salaries and running costs (MoE, 2002). According to (MoE,2002) Schools have governmental source of finance(block grant budget) and non- governmental source of finance(school grant budget) in addition to generating its own internal sources of finance. 1. Background of the Study g y Education is a backbone for the overall development of any society, it contributes for economic growth, development of science and technology; it can help change society by improving and strengthening skills, values and communication skill(UNESCO,2011).Among the priority in the education system in Ethiopia is quality and internal efficiency(MoE,2002).Studies revealed that education is strongly influenced by the resources made available to support the process and by how these resources are managed effectively. Furthermore, disbursing all the committed resources and delivering educational services to students under good governance are key for meeting education challenges successfully despite limited resources(UNESCO,2011). The education goals and objectives can be achieved by means of using different resources as inputs. Among the resources as inputs, finance is a key issue to get and utilize all important inputs and to run the task of education at all levels in appropriate ways. This is due to the fact that among the resources (inputs), finance is more commonly used by schools through a process and mechanism of budgeting (Melaku,2000). y y g p g g ( ) Financial resources are significant resources often assumed to be a part of physical capital. It is ac Financial resources are significant resources often assumed to be a part of physical capital. It is actually the 1 Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 Research on Humanities and Social Sciences Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 www.iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 basis for the procurement, utilization and maintenance of all other types of resources. Without strong financial base it will be difficult to produce the right of goods and services in desirable quality and quantity (Chinyere,2010). basis for the procurement, utilization and maintenance of all other types of resources. Without strong financial base it will be difficult to produce the right of goods and services in desirable quality and quantity (Chinyere,2010). ( y , ) In developing countries like Ethiopia, the government is the main sources of finance for the expansion of education. However, the financial, labor and material contribution of the community as well as school’s internal income and the support from non-government organizations could have their own contributions. In line with this there are criteria and mechanisms for grants’ distribution. 1. Background of the Study Based on school grant budget guidelines, the school grant should be used to improve the provision of quality education, with a focus on General Education Quality Improvement Program (GEQIP) and School Improvement Program (SIP) related items: the teaching-learning process, the school environment, community empowerment, student achievement and schools’ capacity to manage change. According to this guide line, six items are excluded from the list of expenditures with the school grant: construction of new buildings (construction of toilets is however permitted), purchase of televisions and DVD players, payment of salaries and per diems, payment to individuals, and purchase of fuel or weapons (SREB, 2015). There is also another source of finance for secondary schools from development partners and the name of budget is Department For International Development(DFID) budget. Based on the guide line of Department For International Development (DFID) budget, DFID budget focuses on students’ results and learning achievement rather than other activities. This budget should not be used to purchase laptop computer, generator, television, and tape recorder (SRBoFEDO,2016). The necessary conditions will be created for educational and training institutions (schools) to generate their own income and to use it to strengthen the educational process (MoE, 2002). Decentralization was introduced in Ethiopia in the 1995 constitution. All the nine regional states and two city administrations have their respective regional education bureau (REBs), under which woreda education offices (WEOs) are organized. Below the woredas, the Kebele Education and Training Board (KETB) , and Parent Student Teacher Association(PSTA) are the educational planning and management government organs at the local community level. Also they have the responsibility to strengthen community school relationship, create conducive learning environment, decrease drop rate, increase community support to schools in terms of material, labor and finance(MoE, 2002). The planning of school finances usually begins with the drafting of a budget. Budgeting is an ongoing and dynamic process that is typically marked by regular phases, such as, planning, needs assessment and priority setting. Budgeting is a forward looking process which should be guided by the schools vision for the future and a realistic assessment of the risks (Clarke,2007).Therefore, building the implementation capacity of woredas’ and other stakeholders based on training needs identification through short term domestic and/or abroad training programs on educational management, supervision, finance, purchasing and major strategy are undertaking(MoE,2002). The effectiveness and efficiency of school can center on the quality of its financial management sound. 2. Statement of the Problem There is positive relationship between the efficient utilization of school finance and the success of education system. As suggested by MoE (1994), sufficient budget allocation and proper utilization of the education budget for the intended purpose is critical issue. There is a strong need to create transparency system in schools that clearly indicates the sources of finance and allocation as well as its utilization. It is possible to say that the financing of education must be efficient and appropriate to promote equity and quality of education. The misuse of finance can affect the quality of education in general and the involvement of school community, parent and donors in financial support to school in particular. It also affects the cooperativeness among teachers, students, school principals and PSTA. To tell the truth, school principals are expected to be appointed on the basis of experience, services and qualification related to the position, but many principals do not have the required qualification in the area of financial resource management, allocation, and utilization (MoE,1994). Lack of planning capacity in the Ethiopian education system is on top of the agenda of both the government and donor’s since 1997(Tesfaye, 2008). As indicated in ESDP-4 (2010), many offices that is education however, do not have the required capacity to exercise their responsibilities effectively. The challenges stated by Melaku (2000), was local community members who contribute to school which are not in the appropriate position to appraise the efficiency of the school principals and others in managing finance. The challenges which can affect efficient financial utilization practice include; shortage of adequate skills, knowledge, and efficiency and effectiveness of school principals and other stake holders who directly or indirectly involve in utilization of school finance. There is problem of adequate support in the area of school finance resource utilization from zonal or woreda education office. Other problems are also likely to result from: Lack of participation of concerned bodies in budget process, transparency, and lack of clear and consistent procedures to be followed to prepare budget at school level. Besides, absence or infrequent auditing is also there to strengthen the problem. These problems can affect students, teachers, community and government at large because they have no chance to get appropriate services. Many principals face disciplinary measures due to miss utilization of financial resources, carelessness in managing financial resources (Dash & Nena, 2008). 1. Background of the Study Financial management ensures that school resources are effectively used to achieve a school’s mission, and its school improvement plan (SIP) and school development plan (SDP). This shows that strong relationship exists between finance and educational programs, or the capability of finance is influencing educational programs of schools (Melaku, 2000).In addition to this, finance in the school is a critical issue to accomplish different tasks 2 Research on Humanities and Social Sciences www.iiste.org iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 which support in insuring quality of education and expand education (MoE, 2002). which support in insuring quality of education and expand education (MoE, 2002). To this end, the allocated budget for educational purpose needs to follow the rules and regulations of financial organizations when the budget is put in to action and utilization. For this reason, the researcher tried to examine the practice and challenges of financial resources utilization and to recommend more advantageous ways of utilizing important and scarce resources in government secondary schools of Dawro Zone. 2. Statement of the Problem Because of these reasons, the current condition at school level in the study area shows that financial utilization practice faces different challenges. Furthermore, detailed analysis has not undertaken in the study area so far. Therefore, the study focused on investigating currently existing practice and challenges of financial resources utilization in government secondary schools of Dawro Zone. Hence, to achieve the objective of the research, the study guided by the following basic questions. y f g q What are the sources of finance for government secondary schools in Dawro Zone? H i th ff ti f h l b d t tili d i t d h l 2. How is the effectiveness of school budget utilized in government secondary schools of Dawro Zone? To what extent stakeholders are participating in utilizing finance in government secondary schools of D one? To what extent do the financial resources utilization are audited and monitored in government seco hools of Dawro zone? 3.1 General Objective 3.1 General Objective 3.1 General Objective The general objective of the study is to investigate the practices and challenges affecting the utilization of financial resources at government secondary schools of Dawro zone. j The general objective of the study is to investigate the practices and challenges affecting the utilization of financial resources at government secondary schools of Dawro zone. 3. Objectives of the Study j y The objectives of this study were presented as general and specific objectives. The objectives of this study were presented as general and specific objectives. 3.2 Specific Objectives p j The study has the following specific objectives: The study has the following specific objectives: 1.To identify the sources of finance for government secondary schools of Dawro Zone. To describe the effectiveness of school budget utilized in government secondary schools of Dawro Zone. To examine whether stakeholders are participating in utilizing finance in government secondary scho 3.To examine whether stakeholders are participating in utilizing finance in government secondary schools of Dawro Zone. 4.To assess whether appropriate auditing and monitoring system exist regarding to the government secondary schools’ financial resource utilization in Dawro Zone. 4.To assess whether appropriate auditing and monitoring system exist regarding to the government secondary schools’ financial resource utilization in Dawro Zone. 3 Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 p y 4.2.1 Primary Sources Quantitative data were collected using a structured questionnaires having close ended from teachers, vice principals, principals, and supervisors. Qualitative data were collected by using key informants include PSTA, KETB chairpersons, and school finance personnel and through open ended questionnaire by using the respondents of close ended items. All the above mentioned people have adequate information because they are directly or indirectly involved in each and every aspects of financial activities and the researcher assumed that they could provide valuable information for the study. The primary data were gathered through questionnaires and interviews. 4.2.2 Secondary Source y Secondary data sources were obtained from document analysis. Document analysis included minutes, model 19(used when materials submitted to a store) and model 22(helps to withdraw materials from a store), financial reports like auditing, expenditure and revenue report, income and expenditure records. 4. Research Design and Methods This chapter deals with the effective flow of information that could manage the study and the nature of participants and how they were selected. It gives the chance for the researcher to discuss how the instruments of data collection were developed and employed. Finally, it states research design, data sources, population, sample size, sampling procedures, the method of analyzing data and ethical consideration. 4.2 Sources of Data The sources of data for this study were considered to be both primary and secondary sources. Primary and secondary data were collected from teachers, vice principals, principals, and supervisors, and documents respectively. 4.1 Research Design This study employed descriptive survey design to conduct the study, since descriptive survey is a design used to get information relating to the existing condition of an issue or to discuss what exists within the condition of the situation. So, descriptive survey research is used to investigate the practices and challenges of financial resource utilization in government secondary schools of Dawro Zone. In addition, this design helped to gather different kinds of data by using data gathering tools for instance questionnaire, interview, and document analysis. 4.3 Sample Size and Sampling Techniques 4.3.1 Sample Size p From the Zone, two woredas and one city administration were selected. From two woredas and one city administration 10 schools were selected. For quantitative data a total of one hundred fourty four respondents were selected. Among them one hundred twelve(30%) were teachers because Arya (1972) says a 10% to 30% of sample of the total population is suitable when the population is large, seventeen were vice-principals, ten were principals, and five were supervisors. For qualitative data thirty two respondents who included twelve schools’ financial workers, ten Kebele Education and Training Board (KETB) chairpersons, and ten Parent Student Teacher Associations (PSTAs) chairpersons were selected for in-depth interview questions. 4.3.2 Sampling Technique Out of five woredas, which have 38 secondary schools, two woredas were selected using simple random sampling techniques because according to Kothari (2008) it gives each element in the population an equal probability of getting in to the sample. One city administration (contains 2 secondary schools) is taken based on availability because Kothari (2008) proposes use of availability when population is manageable size so, it is small in number and no need of selection. Out of the two woredas (Mareka and Loma) eight government secondary schools (out of seventeen) are selected by using random sampling techniques which includes Gozo Bamushi, Loma Bale, Disa, Yalo Lala, Gessa Dalba, Mari, Gendo, and Waka secondary schools. On the other hand, two schools (Tercha secondary and Maremea secondary schools) were included from Tercha City Administration by means of availability, which is 100% of sampling size, because there are limited number of schools and are already available so, no need of sampling. Out of three 369 teachers 112 were selected using random sampling. Ten principals, seventeen vice principals, five supervisors, and twelve schools’ financial workers/personnel, ten PSTA and ten KETB chairpersons were included based on availability. 4.4.3 Documents Document reviews was made concerning revenue, expenditure, auditing reports, accounting books, handling and utilization system of school properties because document analysis helps to evaluate and compare the standard set with actual performance and to triangulate the data collected by using the other tools. 4.4 Data Collection Instruments 4.4 Data Collection Instruments In this study the major instruments were questionnaires, interview, and relevant documents including minutes, model 19 and model 22, financial reports like auditing and expenditure report, income and expenditure records. 4.4.1 Questionnaire The process of data collection was mainly through questionnaire because of its suitability for survey study that has large size of respondents (Tadewos, 2014). In the structured questionnaire, both close-ended and open-ended questions were prepared to be filled by the teachers, vice principals, principals, and supervisors, who could be considered as respondents. 4.4.2 Interview Guide Besides the questionnaire, face to face interview checklist was employed as an instrument for data collection and get appropriate data that may not be handled by questionnaire and also to counter check the information obtained from the open-ended form of the questionnaire which was prepared to exploit greater depth of response because the participants were encouraged to answer in their own words. So, an interview checklist was prepared and employed with key persons include PSTA and KETB chairpersons, and school financial workers/personnel. 4.4.3 Documents 4.3 Sample Size and Sampling Techniques 4.3.1 Sample Size 4 Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 Table-1: population, sample size and sampling techniques No Respondents Population Sample size % Sampling techniques Data collection approach M F T M F T 1 Teachers 305 64 369 92 20 112 30 Random Questionnaire 2 Vice principals 17 -- 17 17 -- 17 100 Availability Questionnaire 3 Principals 10 -- 10 10 -- 10 100 Availability Questionnaire 4 Supervisors 5 -- 5 5 -- 5 100 Availability Questionnaire 5 School financial workers 8 4 12 8 4 12 100 Availability Interview 6 PSTA chairpersons 10 -- 10 10 -- 10 100 Availability Interview 7 KETB chairpersons 10 -- 10 10 - 10 100 Availability Interview Total 365 68 433 152 24 176 40 As a result, problems related to the content, wording, length, and instructions were addressed by doing t sary amendment and adaptation accordingly. 4.5 Pilot Study Before the questionnaires were administered, the questions were tested through pilot survey. According to Mugenda and Mugenda (1999), reliability is the degree to which a research instrument yields consistent results. This pilot test sample is based on what Mugenda and Mugenda (2003) indicate that, normally the pilot test sample is between 1% and 10% depending on the sample size. So that the test was conducted in Gozo Shasho secondary schools of the study area which was not among the sampled schools. From this school sixteen teachers, one principal, and one vice principal, and totally eighteen participants were included in the pilot test. The internal consistency reliability estimate was calculated using Cronbach‟s alpha coefficient for the Likert scale items. Then the researcher determined the reliability coefficient of the instruments by using SPSS program version 20. The alpha coefficient ranges from 0.71 - 0.79. So, this indicates acceptable degree of the questionnaires. Supporting this, George and Mallery (2003) also stated that the Cronbach‟s alpha result > 0.9 excellent, alpha > 0.8 good, alpha > 0.7 acceptable, alpha < 0.6 is questionable, and alpha < 0.5 is poor. The purpose of the pilot test was to identify and check the questionnaires and were valid and reliable. With the pilot testing, useful feedbacks were obtained on whether the respondents understand the questions easily, feel comfortable in answering the questions. As a result, problems related to the content, wording, length, and instructions were addressed by doing the necessary amendment and adaptation accordingly. 5 .iiste.org Research on Humanities and Social Sciences www.iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 4.6 Data Collection Procedures The questionnaire and interview guides were pilot tested for reliability before administering questionnaire and conducting interview. Research participants were oriented about the objective of the study and told that they participate only if they freely agree to do so. During the interview, the researcher first introduced himself to interviewee by telling who he was, what he was going to do and how an interviewee was chosen before directly to the question. As regard to questionnaires, the questionnaire was developed in English and distributed to all participants to collect the necessary data. 4.7 Method of Data Analysis y The data gathered through primary and secondary methods were analyzed by using both mixed data analysis methods, i.e. quantitative and qualitative ways because a mixed approach can provide a fuller description and more complete explanation of the phenomenon being studied by providing more than one perspective on it and the results from one method can help develop or inform the other method. Quantitative ways of presenting the data collected in the course of questionnaire using Statistical Package for Social Science (SPSS) application version 20 and descriptive statistics such as: percentages mean and standard deviation were used. Qualitative method of data analysis was employed for feedbacks obtained through interview, open ended questionnaires; and secondary data sources, and the data collected were analyzed and interpreted in the form of narration. As presented in Table -2, from the points of view of quantitative data among 144, 21(14.6%) were found at the age of below 26 years, 118( 81.9%) were found at the age range of 26-50 years and 5(3.5%) were at the age of greater than 50 years. From the points of view of qualitative data out of 32 respondents of the interview, 2 (6.3%) were found at the of below 26 years, 27(84.4%) were at the age range of 26-50 and, 3(9.3%) were at the age of above 50 years old. This data shows that in both types of respondents the majority of the respondents were at the age range of 26-50 years. A d ib d i T bl 2 h d i l l l f h d f i i f d b 5. Discussions and Findings 5.1. Background Information of Participants As indicated in Table-2, out of 144 respondents of the questionnaire 124(86.1%) were males and the rest 20(13.9%) were female. From qualitative aspects, out of 32 key informants 28(87.5%) were male and only 4(12.5%) were female; based on the above data it was concluded that participation of females as employees, as leaders and as other stakeholders by far less than males’ participation in education system of secondary schools of Dawro Zone. Table-2 Respondents by sex, age and level of education (N=176) No Background Information Category Respondents of Questionnaire Key Informants Freq. % Freq. % 1 Sex Male 124 86.1 28 87.5 Female 20 139 4 12.5 Total 144 100.0 32 100.0 2 Age <26 21 14.6 2 6.3 26-50 118 81.9 27 84.4 >50 5 3.5 3 9.3 Total 144 100.0 32 100.0 3 Level of Education <Diploma 0 0.0 0 0.0 Diploma 0 0.0 20 62.5 BA/ Bsc 131 91.0 12 37.5 >BA/ Bsc 13 9.0 0 0.0 Total 144 100.0 32 100.0 Table-2 Respondents by sex, age and level of education (N=176) As presented in Table -2, from the points of view of quantitative data among 144, 21(14.6%) were found at the age of below 26 years, 118( 81.9%) were found at the age range of 26-50 years and 5(3.5%) were at the age of greater than 50 years. From the points of view of qualitative data out of 32 respondents of the interview, 2 (6.3%) were found at the of below 26 years, 27(84.4%) were at the age range of 26-50 and, 3(9.3%) were at the age of above 50 years old. This data shows that in both types of respondents the majority of the respondents were at the age range of 26-50 years. g g y As described in Table 2, the educational level of the respondents of questionnaire were found to be none below diploma and equivalent to diploma, 131(91.0%) BA/Bsc degree,13(9%) above BA/Bsc or the holders of second degree. Educational background of key informants for the interview were found to be none of them below diploma level, 20(62.5%) at diploma level, 12(37.5%) BA/Bsc degree and none of them above BA/Bsc As described in Table 2, the educational level of the respondents of questionnaire were found to be none below diploma and equivalent to diploma, 131(91.0%) BA/Bsc degree,13(9%) above BA/Bsc or the holders of second degree. As presented in Table -2, from the points of view of quantitative data among 144, 21(14.6%) were found 4.8 Ethical Consideration Research ethics refers to the moral values and principles guiding the research from its beginning to its completion and publication of results. The principle of voluntary participation requires that people not be coerced in to participating in research. Closely related to the notion of voluntary participation is the requirement of informed consent (William, 2005).The participants have the chance of assuring that any information or data collected from them is strictly confidential and that they have complete anonymity. 5. Discussions and Findings q y, p g , , portrayed in Table-4 item 2, it was requested respondents to state the degree of utilizing allocated budget Note: F = frequency, % = percentage, M = mean, SD = standard deviation 5. Discussions and Findings Educational background of key informants for the interview were found to be none of them below diploma level, 20(62.5%) at diploma level, 12(37.5%) BA/Bsc degree and none of them above BA/Bsc 6 Research on Humanities and Social Sciences www.iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 degree. According to this data the holders of master degree are few and majority of them were the holders of BA/Bsc degree. www.iiste.org degree. According to this data the holders of master degree are few and majority of them were the holders of BA/Bsc degree. degree. According to this data the holders of master degree are few and majority of them were the holders of BA/Bsc degree. Table-3 Respondents by field of specialization and service years No. Background Information Category Respondents Key informants F % F % 1 Field of Specialization Subject Specialization 111 77.1 0 0.0 School Leadership 11 7.6 0 0.0 EdPm 10 6.9 0 0.0 Others 12 8.3 32 100.0 Total 144 100.0 32 100.0 2 Service Years in Current Position 1-5 33 22.9 4 12.5 6-10 42 29.2 5 15.6 11-15 45 31.3 10 31.3 16-20 13 9.0 10 31.3 >20 11 7.6 3 9.3 Total 144 100.0 32 100.0 3 Total Service years 1-5 10 6.9 2 6.3 6-10 90 62.5 7 21.8 11-15 28 19.4 11 34.4 16-20 8 5.6 8 25 >20 8 5.6 4 12.5 Total 144 100.0 32 100.0 As portrayed in Table-3, from the total number of 144 respondents of the questionnaire 111(77.1%) were subject specialized, 10(6.9%) were specialized in educational planning and management (EdPM), 11(7.6%) were specialized in school leadership, and the rest 12(8.3%) studied other fields. In the case of key informants of interview questions 32(100%) studied other fields rather than specific subject, school leadership and EdPM. This data shows more than three fourth of respondents of questionnaire were specialized subject matter and all of key informants for the interview were specialized in other fields. p Based on the data shown in Table-3, from total number of 144 participants of quantitative data 33(22.9%) had served from range of 1-5 years, 42(29.2%) had served from a range of 6 -10years, 45(31.3%) had served from a range of 11-15years, 13(9%) had served from a range of 16-20years, and 11(7.6%) had served for more than 20 years in their current positions. Note: F = frequency, % = percentage, M = mean, SD = standard deviation As portrayed in Table 4 item 2 it was requested respondents to state the q y, p g , , As portrayed in Table-4 item 2, it was requested respondents to state the degree of utilizing allocated budget 5. Discussions and Findings M SD F % F % F % F % F % 1 Utilizing approved budget according to plan 12 8.3 13 9.0 16 11.1 69 47.9 34 23.6 2.30 1.17 2 Utilizing allocated budget for its intended purpose 4 2.8 5 3.5 16 11.1 39 27.1 80 55.6 1.70 0.98 3 Spending school finance by financial rules and regulations 3 2.1 5 3.5 8 5.6 93 64.6 35 24.3 1.94 0.79 4 Timely submission of financial report 17 11.8 19 13.2 26 18.1 71 49.3 11 7.6 2.72 1.15 Aggregated Mean 2.16 1.02 Note: F = frequency % = percentage M = mean SD = standard deviation y g g y y p y Table-4: Degree of effectiveness of budget utilizing (N=144) S l 7 Research on Humanities and Social Sciences www.iiste.org iste.org for expected educational purpose. The respondents were replied as disagree with mean and standard deviation value of 1.70 and 0.98 respectively. This data indicates that the deployment of school finance was not for expected educational objectives and this practice could be the reason for wastage of scarce resources. for expected educational purpose. The respondents were replied as disagree with mean and standard deviation value of 1.70 and 0.98 respectively. This data indicates that the deployment of school finance was not for expected educational objectives and this practice could be the reason for wastage of scarce resources. Again as described in table-4 item 3, a question was raised to respondents if spending of school finance by following financial rules and regulations. Majority of the respondents were responded as disagree with mean, and standard deviation value of 1.94 and 0.79 respectively. During interview most of PSTA and KETB chairpersons revealed that the schools did not fully follow financial rules and regulation when spending school finance becaus school committee and principals lack the knowledge of government financial rules and regulations, they had not submitted financial report on appropriate time, did not purchase goods and materials in a legal way. school committee and principals lack the knowledge of government financial rules and regulations, th had not submitted financial report on appropriate time, did not purchase goods and materials in a leg y Accordingly, the response to item shows that there is lack of following financial rules and regulation when utilizing school finance and it could be the cause for misuse, extravagancy and corruption of financial resources. 5. Discussions and Findings According to MoE (2002) the schools have responsibility to run the school finance by following proper finance rule and regulations. Item 4 of table-4 portrays the data obtained on timely submission of financial utilization report to concerned body. Consequently, the respondents were replied as uncertain mean and standard deviation value of 2.72 and 1.15 respectively. So, this data shows the secondary schools of Dawro Zone do not submit financial utilization report on expected time base; and this practice can be the problem for local government as well as for other stakeholders. Aggregated mean value is 2.16 which means the effectiveness of school budget utilizing found at lower level. According to an interview guide with PSTA and KETB chairpersons, and majority of them described as there was lack of effectiveness of school budget planning, allocating and utilizing f ff f g p g g g because of lack of budget transparency, lack of skilled man power to conduct effective ecause of lack of budget transparency, lack of skilled man power to conduct effective budget utilization, and instability (immediate change) of principals. udget utilization, and instability (immediate change) of principals. For open ended question “what criteria are to be considered to allocate school grant, block grant and DFID budget?” most of the respondents replied as the number of students who enrolled at all grade levels in case of block grant and school grant budget and the number of each students who has got 2 and above 2 points in grade 10th national examination in case of DFID budget were criteria to allocate school budget. Table-5: Possible sources of school finance (N=144) No. Response F % Rank 1 Budget allocated by the government 35 24.3 2 2 Student fee 12 8.3 5 3 contribution made by the local community 28 19.4 3 4 Donation given by non-governmental organizations 44 30.6 1 5 Internal income 25 17.4 4 Total 144 100 Note: F=frequency %=percentage 5. Discussions and Findings On the other hand from the total number of 32 participants of interview 4(12.5%) had served from a range of 1-5 years, 5 (15.6%) had served from a range of 6-10years, 10(31.3%) had served from a range of both 11-15years and 16-20years, 3(9.3%) had served for more than 20 years. So, this indicates majority of the respondents of questionnaire and interview guide had better work experience. As depicted in Table-3 where majority of the respondents of questionnaire and key informants had total service years ranging from 6-10years and 11-15years, respectively. Based on the data shown in Table-3, from total number of 144 participants of quantitative data 33(22.9%) had served from range of 1-5 years, 42(29.2%) had served from a range of 6 -10years, 45(31.3%) had served from a range of 11-15years, 13(9%) had served from a range of 16-20years, and 11(7.6%) had served for more than 20 years in their current positions. On the other hand from the total number of 32 participants of interview 4(12.5%) had served from a range of 1-5 years, 5 (15.6%) had served from a range of 6-10years, 10(31.3%) had served from a range of both 11-15years and 16-20years, 3(9.3%) had served for more than 20 years. So, this indicates majority of the respondents of questionnaire and interview guide had better work experience. As depicted in Table-3 where majority of the respondents of questionnaire and key informants had total service years ranging from 6-10years and 11-15years, respectively. y g g y y p y Table-4: Degree of effectiveness of budget utilizing (N=144) No. Item Scale Strongly agree Agree Uncerta- in Disagree St. Disag. Note: F=frequency, %=percentage q y p g As shown in table-5, concerning sources of school finance, the data indicated that 44(30.60%) of respondents replied that donation from non-governmental organization is the major sources of school finance. 35(24.3%) responded as budget allocated by government is another the second source of school finance. Whereas 28 (19.4%) indicated that contribution made by local community is also the source of school finance. School internal income took fourth place as sources of finance. During interview the key informants provided similar response with above. This data tells that schools are largely dependent on governmental and non- governmental organization for its sources of finance rather than generating their own internal income. Interview respondents were replied similar responses with the above. p p p Table-6: Responses to whether school generate internal income (N=144) No. Response F % 1 Yes 56 38.9 2 No 75 52.1 3 No, I don’t understand 13 9.0 Total 144 100 Note F = frequency % = percentage to whether school generate internal income (N=144) No. Response F % 1 Yes 56 38.9 2 No 75 52.1 3 No, I don’t understand 13 9.0 Total 144 100 Note F = frequency, % = percentage q y, p g As indicated in Table-6, concerning generating internal income, the data shows that 75(52.1%) of the respondents were replied as no or schools are not generating its internal income; 56(38.9%) were responded as yes to denote schools are generating internal income; whereas the rest 13(9.0%) had no idea about whether schools generate or not internal income. Based on this data, the researcher concluded the number of secondary schools which do not generate its own internal income are greater in number and attention of stakeholders’ is 8 Research on Humanities and Social Sciences www.iiste.org mandatory. Table-7: Responses to sources of internal income Responses Agricultural product Selling of grass From clubs Selling of trees Selling of fire wood Others Total F 42 39 9 19 12 23 144 % 29.2 27.1 6.3 13.2 8.3 16.0 100 N F f % mandatory. Note: F=frequency, %=percentage Table-7: Responses to sources of internal income q y, p g As shown in Table-7, the respondents were requested to state sources of internal income; accordingly, 42(29.2%) of the respondents indicated that agricultural products were the main source of internal income for secondary schools of the zone where as 39(27.1%) replied as selling of grass was another source of school internal income. 9(6.3%), 19(13.2%), 12(8.3%), 23(16%), responded as school clubs, selling of trees, selling of fire wood, and other sources were different sources for schools internal income. Based on the above data, agricultural product is the major and school clubs are the least sources of internal income for secondary schools. Table-8: The practice of managing internal income (N=144) No. Item Scale Strongly agree Agree Uncerta- in Disagree St. Disag. M SD F % F % F % F % F % 1 The school is generating its maximum amount of internal income 15 10.4 34 23.4 38 26.4 55 38.2 2 1.4 3.03 1.09 2 Use of internal income according to priority of the needs of school. 33 22.9 23 16.0 25 17.4 54 37.5 9 6.3 3.11 1.30 3 Report/Announce/ the amount of internal income to concerned body . 20 13.9 26 18.1 38 26.4 50 37.4 10 6.9 2.77 1.17 4 Use of receipt/ legal document/ when collecting internal income 89 61.8 21 14.6 19 13.2 6 4.2 9 6.3 4.21 1.20 5 Use of internal income by following the legal ground. 36 25 30 20.8 56 38.9 10 6.9 12 8.3 3.47 1.18 Aggregated Mean 3.31 1.18 Note: M=Mean, SD= Standard Deviation, F=frequency %= percentage As depicted in Table-8 item 1 was raised to respondents if the school is generating its maximum amount of internal income; accordingly, respondents were replied as uncertain having mean and standard deviation value of 3.03 and 1.09 respectively. So, this indicates that the respondents were not sure whether schools are collecting its high amount of internal income or not and it can be concluded that schools had not used highest effort as well as strategies to generate maximum amount of internal income which can help to reduce burden of educational cost from government and to cover their budget shortage. Note: F=frequency, %=percentage According to Table-8 item 2, with mean, and standard deviation value of 3.11, and 1.30 respectively, respondents were responded as uncertain to imply that they were not sure schools use internal income based on needs priority. Therefore; this practice can have the chance to damage quality of education. As portrayed in the Table-8 item 3 with mean and standard deviation value of 2.77, and 1.17 respectively, most of the respondents were responded as uncertain to describe that whether schools legalize or announce or not the collected amount of internal income. In connection with this, the scarce resources have the chance to be exposed to corruption and rent seeking practice. As shown in Table-8 item 4 with mean and standard deviation value of 4.21, and 1.20 respectively, majority of the respondents were replied as agree to denote that schools used legal document (receipt) when collecting internal income. To strengthen this data, the researcher was conducted document analysis in 10 sampled secondary schools to assure the presence of receipt. So, in all schools the receipts were available. In line with this, schools were doing their best and this habit helps to develop transparency of budget implementation as well as it is the indication of good management of internal income. As indicated in Table-8 item 5 with mean and standard deviation value of 3.47, and 1.18 respectively, most of the respondents were replied as uncertain to explain that schools do not use internal income by following legal ground. So, in contrast to above item 4, this situation can expose school finance to extravagancy, misuse, and corruption as well as leads principals other stakeholders to be accountable. Based on the aggregated mean value 9 of 3.31 it is uncertain to say the practice of managing internal income in good situation. Table-9: Response to the extent of school committee follow up school budget ( N=144) No Item Response Total Always Most of the time Sometimes Never 1 How often does school committee(PSTA&KETB) make follow up to determine proper utilization of school budget F 20 40 76 8 144 % 13.9 27.8 52.8 5.6 100 Note: F=frequency, %=percent Table-9 shows the data provided by the respondents on the extent to which school committee make follow up to determine proper utilization of school budget. Note: F=frequency, %=percentage So, the absence of internal audit can reduce proper implementation of school finance and can put schools below its standards Table-11: The extent of internal auditing activity accomplished in schools(N=144) Item Response Very frequently Frequently Rarely Not at all Total To what extent does internal auditing activity accomplished in your school F 3 6 46 89 144 % 2.1 4.2 31.9 61.8 100 N F f % audit can reduce proper implementation of school finance and can put schools below its sta Table-11: The extent of internal auditing activity accomplished in schools(N=144) The extent of internal auditing activity accomplished in schools(N=144) Table-11: The extent of internal auditing activity accomplished in schools(N=144) Item Response Very frequently Frequently Rarely Not at all Total To what extent does internal auditing activity accomplished in your school F 3 6 46 89 144 % 2.1 4.2 31.9 61.8 100 Note: F=frequency, %=percentage Accordingly, 20(13.95), 40(27.8%), 76(52.8%), and 8(5.6%) were agreed on the assumption that always, most of the time, sometimes and never respectively follow up utilization of school budget at school level. This data indicates that more than half of the respondents replied as sometimes school committee follows up school finance and they did not follow up for expected amount of time. q y p Table-10: Responses regarding internal audit in schools (N=144) No. Response F % 1 Yes 9 6.3 2 No 127 88.2 3 No, I don’t understand 8 5.6 Total 144 100 N t F f % t Note: F=frequency, %=percentage q y, p g As depicted in table-10, concerning the availability of internal audit in schools, out of 144 respondents the data shows that 9(6.3%) of the respondents responded as yes, 127(88.2%) responded as no and the remaining 8(5.6%) responded as no, I do not understand. The above data implies that, the secondary schools of the study area had on employed internal audit as more than three fourth of the respondents replied there were no internal audit in our schools. Also the researcher interviewed school financial workers and school committee (PSTA and KETB chairpersons) of key informants in connection with the presence or absence of internal audit, their responses supported the above data by responding as schools have no internal audit. So, the absence of internal audit can reduce proper implementation of school finance and can put schools below its standards 8(5.6%) responded as no, I do not understand. The above data implies that, the secondary schools of the study area had on employed internal audit as more than three fourth of the respondents replied there were no internal audit in our schools. Also the researcher interviewed school financial workers and school committee (PSTA and KETB chairpersons) of key informants in connection with the presence or absence of internal audit, their responses supported the above data by responding as schools have no internal audit. Note: F=frequency, %=percentage 9 6.3 13 9.0 24 16.7 40 27.8 58 40.3 2.13 1.21 N t M M SD St d d D i ti F f % t Table-12: The practice of auditing, monitoring and capacity building in school (N=144) Scale Note: M=Mean, SD= Standard Deviation, F=frequency %= percentage As shown in Table-12, with measuring of index of five point likert scale to assess the attitude of respondents towards the practice of auditing and capacity building task item 1 with mean and standard deviation value of 4.11, and 1.20 respectively the respondents indicated their agreement to the statement the principal follow up and controls school fees; whereas with frequency and percentage value of only 7and4.9 respectively least amount of respondents indicated their disagreement. To this end, in schools, mainly the responsibility of follow up and control of school fees was conducted by school principals. Item 2 of Table-12 with mean and standard deviation value of 2.53 and 1.22 respectively respondents replied as uncertain to the statement external audit offer timely professional support to schools. Therefore; this data clarifies that external audit may not have provided professional support to schools; so that the capacity of school workers and stakeholders may not have improved for better performance. Item 3 of Table-12 with mean and standard deviation value of 2.14 and 0.93 respectively the respondents were indicated their disagreement to the statement on timely follow-up, monitor, control and feedback by external audit. In addition to this, next to the frequency and percentage of the respondents of disagreement, the amount of frequency and percentage of respondents of strongly disagreement took the second rank. None of the respondents were replied as strongly agree. In connection with this, the researcher conducted interview with 32 key informants of school financial workers, parent student teacher association (PSTA) and kebele education and training board (KETB) chairpersons they were responded as there was limitation of timely follow up, monitor, control and feed back by external audit. Also they were asked the reason and replied as there was limitation of timely follow up, monitor, control and feed back by external audit. Also they were asked the reason and replied as it was due to limited number of external audit and budget as well as lack of it was due to limited number of external audit and budget as well as lack of commitment of external audit. Note: F=frequency, %=percentage q y, p g As presented in Table-11, the respondents were asked about the internal audit activities accomplishments carried out in schools. To this end, only 3(2.1%) and 6(4.2%) of the respondents reported that very frequently and frequently respectively internal auditing activities carried out in schools where as 46(31.9%) and 89(61.8%) were replied as rarely and not at all respectively it was carried out in schools. In line with this, the researcher conducted interview with 20 parent student teacher association (PSTA) and kebele education and training board (KETB) chairpersons and 8 schools’ chairpersons replied as p y From this point of view, except few respondents majority of respondents were reported it is clear that there were no internal audit activities conducted in secondary schools of study area and this situation can be the obstacle for the effective and efficient utilization of school finance. According to Bischoff and Mestry (2003) the school should established sound internal financial control to ensure the reliability accuracy of its financial transactions. 10 10 Table-12: The practice of auditing, monitoring and capacity building in school (N=144) No. Item Scale Strongly agree Agree Uncerta- in Disagree St. Disag. M SD F % F % F % F % F % 1 The principal follow-up and controls school fees. 80 55.6 26 18.1 22 15.3 7 4.9 9 6.3 4.11 1.20 2 External auditing offer timely professional support to schools. 16 11.1 18 12.5 16 11.1 71 49.3 23 16.0 2.53 1.22 3 Timely follow-up, monitor, control & feedback by external audit. 0 0.0 15 10.4 29 20.1 62 43.1 38 26.4 2.14 0.93 4 Adequate provision of training by woreda finance & economic development office to school accountant internal audit & principal. 10 6.9 19 13.2 20 13.9 39 27.1 56 38.9 2.22 1.28 5 The stakeholders (PSTA and KETB) get relevant training on financial control and utilization in the school. Note: F=frequency, %=percentage Accordingly, the coordination, follow up, monitor, control, and feedback of external audit offered to schools were very limited; so that the practice of schools performing or implementing financial issue by following government financial rules and regulations was getting worse. Town/Woreda finance and economic development office should audit the school finance twice a year at half and end of budget year and present its results for concerned bodies (MoE, 2002). Item 4 of Table-12 with mean and standard deviation value of 2.22 and 1.28 respectively the respondents disagreed to the statement that adequate provision of training by woreda finance and economic development office to school accountant, internal audit and principal where as the least amount of the respondents with frequency and percentage value of 10 and 6.9 respectively responded as strongly agreed to the same item above. Based on this data, adequate provision of training or capacity building practice by local government was not 11 Research on Humanities and Social Sciences www.iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 Research on Humanities and Social Sciences www.iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 implemented and this gap can create weakness of implementation capacity of school financial workers and principals. The similar direct question proposed to respondents to assess the provision of adequate training given by local government or any concerned body to school committee, the respondents responded as disagree with the mean and standard deviation value of 2.13 and 1.21 respectively where as with the frequency and percentage value of only 9 and 6.3 the respondents replied as strongly agree to show the limitation of adequate training by concerned bodies to stake holders or school committee(PSTA and KETB). Table-13:The extent of stakeholders’ participation in utilizing school finance(N=144) No. Item Scale Strongly agree Agree Uncerta- in Disagree St. Disag. M SD F % F % F % F % F % 1 The local community participate in contributing money, materials, & labor. 15 10.4 25 17.4 73 50.7 13 9.0 18 12.5 3.04 1.08 2 The school participate stakeholders in school annual budget plan (revenue & expenditure plan). Note: F=frequency, %=percentage In connection with this item the researcher made interview with school committee members about the extent of their participation and18 of them responded as 12 www.iiste.org Research on Humanities and Social Sciences sometimes we participate in school budget process and the participation was not maximum or highest because we were busy over personal plan and responsibility th th h l ’ ibilit So, the data indicates that there was low participation of school committee on budgetary issue and this situation is the manifestation of the absence of integration, cooperation, and common decision among schools and school committee. In addition, open ended questions forwarded to respondents. They responded to the statements what are the problems of KETB and PSTA in relation with fully carrying out its duties and responsibilities in budget process? and what do suggest on the cooperation and integration among PSTA, KETB and principals? as follows. KETB chairpersons have another government responsibilities to lead kebele as the cabinet, and they also have their own personal duties and task so that they were busy as well as lack commitment and awareness on budget issue. The same event is true for PSTA chairpersons except the case of cabinet. Finally, they suggested as to improve cooperation and integration among different stakeholders in budget process, awareness creation through continuous discussion and training to develop sense of ownership of schools and to improve knowledge and skills is mandatory. y Item 4 of Table-13 with mean and standard deviation value of 3.04, and 1.12 respectively most of the respondents were indicated their uncertainty to the statement the school regularly inform stakeholders on release (allocate) of school annual budget. Therefore; the data shows that respondents were not sure whether schools do or do not do the above responsibility and puts the situation in dilemma. Based on aggregated mean value of 2.94 the participation of stakeholders in utilizing school finance was uncertain or it was not well known. p p g Table-14: Causes for ineffective budget utilization (N=144) No. Item Scale Rank Strongly agree Agree Uncerta- in Disagree St. Disagree M SD F % F % F % F % F % 1 Principal is over loaded in accomplishment of many activities. 26 16.7 72 50 20 13.9 21 14.6 7 4.9 3.59 1.08 2 2 Principal lacks the skill to manage the budget . Note: F=frequency, %=percentage 16 11.1 18 12.5 86 59.7 15 10.4 9 6.3 3.11 0.95 3 There is high participation of stakeholders (PSTA&KETB) in budget process 15 10.4 23 16.0 20 13.9 59 41 27 18.8 2.58 1.25 4 The school regularly inform stakeholders on release (allocation) of school annual budget. 18 12.5 25 17.4 61 42.4 25 17.4 15 10.4 3.04 1.12 Aggregated Mean 2 94 1 1 Note: M=Mean, SD= Standard Deviation, F=frequency %= percentage q y p g As shown in Table -13, with measuring of index of five point likert scale to assess the attitude of respondents towards the extent of stakeholders’ participation in utilizing school finance item 1 with mean and standard deviation value of 3.04, and 1.08 respectively the respondents indicated their uncertainty to the statement on whether the local community participate in contributing money, materials, and labor where as with frequency and percentage value of only 13 and 9.0 respectively the least amount of indicated their disagreement. Concerning this, the researcher conducted interview with school accountant, parent student teacher association and kebele education and training board chairpersons and 30 of them responded as p In line with this, the quantitative data shows that the respondents were not decided whether stake holders were participating or not; on the other hand, qualitative data shows that their participation was low and it indicates that the stake holders’ participation was not known and highest as much as the expected degree of participation. Item 2 with mean and standard deviation value of 3.11, and 0.95 respectively most of the respondents indicated their uncertainty to the statement the school participate stakeholders in school annual budget plan (revenue and expenditure plan).This data indicates that respondents were not sure whether or not school invites stake holders to participate in budget revenue and expenditure plan as well as it is difficult to say or to decide school invites stake holders to discuss on budget plan. Item 3 of Table-13 with mean and standard deviation value of 2.58, and 1.25 respectively the respondents were replied as disagree to the statement there is high participation of stakeholders (PSTA&KETB) in budget process. Note: F=frequency, %=percentage 65 45.1 43 29.9 25 17.4 9 6.3 2 1.4 4.11 0.99 7 3 Lack of commitment of financial workers. 52 36.1 68 47.2 14 9.7 10 6.9 0 0.0 4.12 0.85 5 4 Lack of commitment of school committee 64 44.4 40 27.8 15 10.4 19 13.2 6 4.2 3.95 1.20 8 5 Untimely disbursement of budget to school 24 16.7 71 49.3 21 14.6 18 12.5 10 6.9 3.56 1.12 3 6 Absence of transparency in school. 68 47.2 45 31.3 20 13.9 6 4.2 5 3.5 4.14 1.03 6 7 Illegal purcha- se of goods & materials. 39 27.1 70 48.6 20 13.9 8 5.6 7 4.9 3.87 1.03 4 8 The school lacks needs’ prioritization. 32 22.2 60 41.7 27 18.8 25 17.4 0 0.0 3.68 1.00 9 9 Mismatch b/n the amount of allocated budget & the need of the school 77 53.5 29 20.1 18 12.5 10 6.9 10 6.9 4.06 1.25 1 Note: M=Mean, SD= Standard Deviation, F=frequency %= percentage Table-14 shows the data provided by the respondents on challenges that can hinder school budget utilization It 1 ith d t d d d i ti l f 3 59 d 1 08 ti l th d t li d t Note: M=Mean, SD= Standard Deviation, F=frequency %= percentage Table-14 shows the data provided by the respondents on challenges that can hinder school budget utilization. Item 1 with mean and standard deviation value of 3.59 and 1.08 respectively the respondents replied as agree to the statement principal is over loaded or busy in accomplishment of many activities rather than budget process where as the least number of respondents were responded as strongly disagree to this item. So, this data indicates , , q y p g Table-14 shows the data provided by the respondents on challenges that can hinder school budget utilization. Item 1 with mean and standard deviation value of 3.59 and 1.08 respectively the respondents replied as agree to the statement principal is over loaded or busy in accomplishment of many activities rather than budget process where as the least number of respondents were responded as strongly disagree to this item. So, this data indicates Table-14 shows the data provided by the respondents on challenges that can hinder school budget utilization. ote: M=Mean, SD= Standard Deviation, F=frequency %= percentage Note: F=frequency, %=percentage To this end, with mean and standard deviation value of 3.56 and 1.12 respectively the respondents indicated their agreement to the statement. So as, untimely disbursement of school budget from government to schools is another challenge that creates ineffective budget utilization. Melaku (2010) in this regard states that budget ensures that required resources will be available at the right time in the right amount to be able to complete proposed actions and accomplish planned objectives. Again direct question proposed to respondents to assess the degree of the level of transparency when utilizing school finance was that demands respondents to indicate whether absence or presence of transparency of budget. Accordingly, with mean and standard deviation value of 4.14 and 1.03 respectively respondents indicated their agreement to the statement. Therefore; it is possible to say that lack of transparency during implementation of school finance is challenge for improper utilization of school budget. According to UNESCO (2006) ensuring transparency and accountability in the conduct of both public and private affairs is a necessary in any society. In addition to above the direct question proposed to respondents to assess the situation of purchase of goods and materials was that demands respondents to express whether there is legal or illegal purchase of goods and materials. So as, with mean and standard deviation value of 3.87 and 1.03 respectively the respondents indicated their agreement to the statement. Therefore; the data clarifies that purchasing goods and materials without following financial rules and regulation is identified as another challenge or problem for ineffective budget utilization. As presented in Table-14 item 8, the researcher requested the respondents the extent of prioritizing needs of school when preparing budget plant. The respondents were agreed with mean and standard deviation value of 3.67, and 1.00 respectively replied as whereas none of the respondents replied as strongly disagree. This shows that lack of needs prioritization during budget plan at school level is also identified as challenges for ineffective implementation of secondary schools’ finance. As depicted in Table-14 item 9, the researcher requested the respondents the extent of matching between the amount of allocated budget and the needs of the school. Accordingly, the respondents strongly agreed with mean and standard deviation value of 4.06, and 1.25 respectively. Note: F=frequency, %=percentage Item 1 with mean and standard deviation value of 3.59 and 1.08 respectively the respondents replied as agree to the statement principal is over loaded or busy in accomplishment of many activities rather than budget process where as the least number of respondents were responded as strongly disagree to this item. So, this data indicates 13 Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 Research on Humanities and Social Sciences www.iiste.org iiste.org that one of the challenges for ineffective budget utilization is principals having a lot of activities to be accomplished rather than following budgetary process. In many educational institutions, one of the most important areas of administration that take much of the time and attention of managers and planners is the management of finance. Once decision made by higher authorities, it is the responsibility of the institutional managers and planners to see that the fund allocated is utilized according to the plan. To this effect, good system of financial planning or budgeting, accounting, and auditing should prevail in school management (Melaku, 2010: 44). ) Item 2 of Table-14 with mean and standard deviation value of 4.11and 0.99 respectively the respondents replied as agree to the statement principal lacks the skill to manage the budget process. The data shows that principals lack the skill to manage budget issue is the second challenge for ineffective utilization of school finance. Item 3 of Table-14 with mean and standard deviation value of 4.12, and 0.85 respectively the respondents replied as agree to the statement lack of commitment of financial workers to carry out their responsibility. To this end, lack of commitment of financial workers to perform their duties and responsibilities is another challenge that can be obstacle for effective and efficient implementation of school budget. Item 4 of Table-14 with mean and standard deviation value of 3.95, and 1.20 respectively the respondents responded as agree to the statement lack of commitment of school committee to mobilize financial resources. This data indicates that lack of commitment of school committee to mobilize financial resources is critical challenge for ineffective utilization of secondary schools under the study. The other direct question proposed to respondents to assess disbursement of budget to schools was that demands respondents to indicate whether timely or untimely disbursement of budget to schools. Note: F=frequency, %=percentage Therefore; this data shows that the allocated budget was not as equivalent as the needs of schools; to mean that schools’ need is higher than allocated budget because resource is scarce and this circumstance was identified as another critical challenge that can be obstacle for effective financial resource utilization in secondary schools of the zone. During the in-depth interview, the key informants mentioned the above similar challenges and were reported that lack of professional support, follow up, monitor and control of external audit; delay of financial report at school; immediate transfer (change) of principals in case of one secondary school because socialization may take time and this leads to delay of budget plan; absence of punctuality of school committee at time of budgetary decision; lack of stakeholders’ participation in budget process; absence of internal audit in schools; schools are largely dependent on governmental and non-governmental sources of finance rather than generating maximum amount of their internal income to cover educational expenditure and low level of budget transparency were another challenge for unwise utilization of finance in secondary schools of the study area. Moreover, from document analysis shortage of budget guidelines in some schools and absence of external audit feedback were identified as additional 14 Research on Humanities and Social Sciences www.iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 challenges for ineffective budget utilization in studied secondary schools of Dawro zone. challenges for ineffective budget utilization in studied secondary schools of Dawro zone. ingly, the major findings of the analysis made were organized here under. The absence of internal audit can reduce proper implementation of school finance and can put schoo below its standards. • The coordination, follow up, monitor, control, and feedback of external audit offered to schools were very limited; so that the practice of schools performing or implementing financial issue by following government financial rules and regulations was getting worse. • One of the challenges for ineffective budget utilization is principals having a lot of activities to be accomplished rather than following budgetary process. • Principals lack the skill to manage budget issue is also the challenge for ineffective utilization of school finance. • Lack of commitment of financial workers to perform their duties and responsibilities is another challenge that can be obstacle for effective and efficient implementation of school budget. • Lack of commitment of school committee to mobilize financial resources is critical challenge for ineffective utilization of secondary schools under the study. Untimely disbursement of school budget from government to schools is another challenge that creat ineffective budget utilization. • Purchasing goods and materials without following financial rules and regulation is identified as another challenge or problem for ineffective budget utilization at school level. • Lack of transparency during implementation of school finance is also a challenge for improper utilization of school budget. • Lack of needs prioritization during budget plan at school level is also identified as challenge for ineffective implementation of secondary schools’ finance. • The allocated budget was not as equivalent as the needs of schools; to mean that schools’ need is higher than allocated budget because resource is scarce and this circumstance was identified as another critical challenge that can be obstacle for effective financial resource utilization in secondary schools of Dawro Zone. ingly, the major findings of the analysis made were organized here under. • Schools are largely dependent on governmental and non-governmental organization or donors for its sources of finance rather than generating their own internal income. • Schools are largely dependent on governmental and non-governmental organization or donors for its sources of finance rather than generating their own internal income. g g • Schools did not utilize and spend its budget based on plan; so this situation could be the reason for inappropriate budget utilization. • Schools did not utilize and spend its budget based on plan; so this situation could be the reason for inappropriate budget utilization. • The deployment of school finance was not for expected educational objectives and this practice could be the reason for wastage of scarce resources. • The deployment of school finance was not for expected educational objectives and this practice could be the reason for wastage of scarce resources. • There was lack of following financial rules and regulation when utilizing school finance because, school committee and principals lack the knowledge of government financial rules and regulations, they have not submitted financial report on appropriate time, did not purchase goods and materials in a legal way. so, these practices could be the cause for misuse, wastage and corruption of financial resources. • There was lack of effectiveness of school budget utilizing because of lack of budget transparency skilled man power to conduct effective budget utilization, and instability (immediate change) of pr g g g p y, skilled man power to conduct effective budget utilization, and instability (immediate change) of principals. • The stake holders’ participation was not well known and highest as much as the expected degree of participation. p g , y ( g ) p p • The stake holders’ participation was not well known and highest as much as the expected degree of participation. • There was low participation of school committee on budgetary issue and this situation is the manifestation of the absence of integration, cooperation, and common decision among schools and school committee. . • The absence of internal audit can reduce proper implementation of school finance and can put schools below its standards. There was low participation of school committee on budgetary issue and this situation is the manifestati of the absence of integration, cooperation, and common decision among schools and school committee. 7. Recommendations Principals need to manage (budget) their time and delegate activities in order to fully utilize time an follow budget process. • The skill and awareness of principals to utilize and manage budget can be improved through continuous training provided by the collaboration of woreda (city administration) education office and finance office; also the principals need to do experience sharing with knowledgeable bodies. • The skill and awareness of principals to utilize and manage budget can be improved through continuous training provided by the collaboration of woreda (city administration) education office and finance office; also the principals need to do experience sharing with knowledgeable bodies. • Schools financial workers and school committee will be committed if principals and other staff members discuss together with them about the issue and then treat the problem accordingly; also principals should manage employees by following government rules and regulation. • Schools financial workers and school committee will be committed if principals and other staff members discuss together with them about the issue and then treat the problem accordingly; also principals should manage employees by following government rules and regulation. • The local government (woreda and city administration education office and zonal education department) should disburse school budget at the right time, so the necessary educational materials, equipment, and facilities will be available at early time base and normal teaching learning process can takes place. • The local government (woreda and city administration education office and zonal education department) should disburse school budget at the right time, so the necessary educational materials, equipment, and facilities will be available at early time base and normal teaching learning process can takes place. • Principals and other stake holders need to follow government financial rules and regulations when purchasing educational goods and materials unless and otherwise corruption and mischief can be created and can be the cause for legal punishment by the law. • To develop participation of community, stakeholders and to reach up on intended educational purpose the transparency of budget implementation is mandatory. So as, school principals and other stake holders should pay due attention to do this responsibility. • To develop participation of community, stakeholders and to reach up on intended educational purpose the transparency of budget implementation is mandatory. So as, school principals and other stake holders should pay due attention to do this responsibility. 7. Recommendations • During the preparation of school budget plan, schools need to improve their capacity to make necessary prioritization if this is not done the most crucial educational activities will have been delayed and the non- crucial activities can be implemented first. • During the preparation of school budget plan, schools need to improve their capacity to make necessary prioritization if this is not done the most crucial educational activities will have been delayed and the non- crucial activities can be implemented first. • The needs of schools more or less will be satisfied if schools create or generate their maximum internal income as much as possible rather than waiting for governmental and donors sources of finance. To this end, conducive teaching learning activities will be done and this can be the road map to attain expected educational purposes as well as outcomes. • The needs of schools more or less will be satisfied if schools create or generate their maximum internal income as much as possible rather than waiting for governmental and donors sources of finance. To this end, conducive teaching learning activities will be done and this can be the road map to attain expected educational purposes as well as outcomes. 7. Recommendations Based on the conclusions of the study the following recommendations have been suggested. the conclusions of the study the following recommendations have been suggested. • It would be essential for schools have to diversify the sources of finance through expands internal income by agricultural product, co-curricular clubs and by any other activities rather than waiting for government and non-governmental sources (block grant, school grant and DFID budget). So that shortage of finance will be minimized. • For effective school budget utilization schools should have utilize approved budget according to plan, need to utilize allocated budget for expected educational purpose, stability of principals in case of some schools, transparency of school budget, and submission of timely financial report to concerned body are very crucial points. • For effective school budget utilization schools should have utilize approved budget according to plan, need to utilize allocated budget for expected educational purpose, stability of principals in case of some schools, transparency of school budget, and submission of timely financial report to concerned body are very crucial points. p • Schools should follow and respect financial rules and regulations when utilizing school finance if not these practices could be the cause for misuse, wastage and corruption of financial resources. • Schools should follow and respect financial rules and regulations when utilizing school finance if not these practices could be the cause for misuse, wastage and corruption of financial resources. • To enhance the participation of school committee and other stakeholders, schools and concerning body 15 Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 www.iiste.org iste.org need to mobilize, develop awareness through discussion, and design strategy that can lead to highest participation. need to mobilize, develop awareness through discussion, and design strategy that can lead to highest participation. • The recruitment of internal audit is advisable by woreda and city administration education office for effective and efficient budget implementation. • The recruitment of internal audit is advisable by woreda and city administration education office for effective and efficient budget implementation. • To promote the practice of schools performing or implementing financial issue, the coordination, follow up, monitor, control, and feedback of eternal audit is obligatory. • To promote the practice of schools performing or implementing financial issue, the coordination, follow up, monitor, control, and feedback of eternal audit is obligatory. thesis, Addis Ababa University. UNESCO.2011. Policy and Practice: Financial Management in schools in the Mafeteng District of Lesotho. University of free state, Bloemfontein, South Africa, 9300 pp156. William, T. 2005. Research Methods, The Concise Knowledge Base. Cornell University, Atomic Dog Publishing. Research on Humanities and Social Sciences www.iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.15, 2019 References A 19 Arya, D. 1972. Introduction to Research in Education. New York: Random House Ink. Chinyere O. Agabii (2010). Prudential Approach to Resource Management in Nigerian Education: A Theoretical Perspective. Department of Educational Management University of Education, Port Harcourt, Nigeria. International Journal of Scientific Research in Education,October 2010, Vol. 3 (2), 94-106). Clarke, A. 2007. The handbook of School Management. Cape Town. Juta and Company. Dash,M. and Nena.2008. School Management. New Delhi. Printed at Nice Printing Press. ESDP-4.2010. Education Sector Development Policy. Addis Abeba: EMPDA. Evaluation and Policy Analysis, 19(4), 309-317. George F. and Mallery M. 2003. Quantitative method for estimating the reliability of data. Western Machinegun University. Kothari, C.R. 2008. Research Methodology and Techniques: New age International Publisher. Ansa Daryagani, New Delhi. Melaku Yimam.2000. School financial and properties management (unpublished teaching Material). Addis Ababa University. Melaku Yimam.2010. Reading on resource management in Education (unpublished teaching material). Addis Ababa University. y MoE .1994. Education and Training Policy, Addis Ababa, st. George Printing Press. g y g g MoE.2002. Organization of Education Management, Community Participation and Finance Guideline. Addis Ababa. Education Materials Production and Distribution Agency. Mugenda, A. and Mugenda, O. 2003. Research methods: Quantitative and qualitative approaches. Nairobi: African Centre for Technology Studies Press SRB FEDO 2016 S h l R M d U ili i G id li N 20/2016 H Z k i gy RBoFEDO.2016. School Resource Management and Utilization Guideline No.20/2016: Hawassa, Zakprin SREB.2015. Second Round General Education Quality Improvement Project Schoolgrant Budget G Hawassa: Ministry Education and Educational Development Collaborative Agency Tadewos Lera.2014.Financial Resource Utilization Challenges in Some Selected Government Primary Schools of Damot Pullasa Woreda in Southern Nations Nationalities and People’s Region University, Ethiopia. MA 16 iiste.org 17
https://openalex.org/W4206792899	https://rbc.inca.gov.br/revista/index.php/revista/article/download/2280/1422	English	null	Fatores Preditivos para Envolvimento de Linfonodos no Câncer de Mama	Revista Brasileira de Cancerologia	2,001	cc-by	4,608	Axillary lymph nodes metastases (ALM) are considered to be the most important prognostic factor for breast cancer. Data from 306 infiltrating ductal carcinomas consecutively diagnosed and treated over a period of 20 months were analised through a case-control study to assess predictive factors for the ocurrence of ALM. Bivariate analysis showed that some of variables were statistically associated (p 0.05) with ALM: age at menarche, surgical treatment, tumor size, skin involvement, nuclear pleomorphism (NP), and vascular invasion (VI). Uncon- ditional logistic regression reveled some independent predictive factors for ALM (p < 0.05): patient’s age, tumor size, NP, VI, and the interaction term tumor size and patient’s age, where tumor size was only predictive for ALM in patients 60 years. Age at diagnosis, tumor size, NP, and VI were independents predictors for ALM. C-erbB-2, cathepsin D, MIB-1, PCNA and p53, and hormonal receptors were not useful ALM predictors in bivariate and multivariate analysis. Key Key Key Key Key words: words: words: words: words: breast neoplasms; infiltrating duct carcinoma; biological tumor markers; neoplasms metastasis; lymph nodes. 1Serviço de Patologia Cirurgica, Hospital do Câncer, Instituto Nacional de Câncer, Praça Cruz Vermelha 23; 20230-310 Rio de Janeiro, RJ, Brazil. Enviar correspondência para A.L.A.E. Rua das Acácias, 101/704, Gávea; 22.451-060 Rio de Janeiro, RJ, Brazil. Fax: (21)239.1680; E-mail: alamaral@inca.org.br % * % * g pidemiologia e Métodos Quantitativos, Escola Nacional de Saúde Pública, Fundação Oswaldo Cruz (FIOCRUZ), Av. Leopoldo Bulhões 1480; 21041-2 iro, RJ, Brazil. A detecção precoce do câncer de mama (CM) é um fator de reconhecida importância no tratamento e prognóstico das pacientes, e as metástases para os linfonodos axilares (MLA) são os preditores mais importantes do seu prognóstico. A partir de um estudo caso-controle com 306 carcinomas ductais infiltrantes (CDI), foi construido um modelo preditivo da ocorrência de MLA em pacientes com CM (casos: CDI com MLA; controles: CDI sem MLA). Foram estudadas variáveis relacionadas às pacientes e ao tumor (características macro e microscópicas e marcadores tumorais). Na análise bivariada, algumas variáveis se associaram estatisticamente com o desfecho em questão, porém, na regressão logística não condicional, somente as seguintes variáveis foram fatores preditivos independentes: idade da paciente, tamanho do tumor, pleomorfismo nuclear, invasão vascular e/ou linfática (IV/ IL) e o termo de interação tamanho do tumor e idade da paciente, onde o tamanho do tumor só foi fator preditivo em pacientes com 60 anos ou mais. De acordo com os resultados deste estudo somente as variáveis idade da paciente, tamanho do tumor, pleomorfismo nuclear e IV/IL foram fatores preditivos para a ocorrência de MLA. Tanto os receptores hormonais quanto os marcadores tumorais estudados (c-erbB-2, catepsina D, MIB-1, PCNA e p53) não se mostraram importantes para predizer o desfecho em estudo, tanto na análise bivariada quanto na análise multivariada. Palavras-chave: Palavras-chave: Palavras-chave: Palavras-chave: Palavras-chave: neoplasias mamárias; carcinoma de ductos infiltrante; marcadores biológicos de tumor; metástase neoplásica; gânglios linfáticos. +B Revista Brasileira de Cancerologia, 2001, 47(4): 389-96 "#$#"#4 %&'#(++/5+!$#,#,# 1996, ware assessed. Respective slides review was carried out twice at the Surgical Pathol- ogy Service by two pathologists (authors ALAE and LMMCR) regardless consultation to any other patient data. Breast cancer is the most important can- cer among women in Brazil and a major pub- lic health issue. In 1999, 31.200 new cases and 7.300 deaths are expected in the coun- try, accounting for 23.2% of all cancer sites among women. Differently than what hap- pened in other industrialized countries, mam- mography is scarcely performed among the poorest population strata, and diagnosis is often established when the disease is already in an advanced stage. Thus, 398 malignant tumors were ascer- tained and 306 of them classified as IDC. The later were further stratified upon the pres- ence/absence of lymph nodes metastasis, thus yielding two groups: 162 IDC tumor patients with lymph nodes metastasis, henceforth named cases, and 144 IDC patients without lymph nodes metastasis (controls). Macroscopic and microscopic study char- acteristics were the following: type of surgi- cal specimen (quadrantectomy; segmentec- tomy; simple mastectomy; Patey mastectomy; radical mastectomy; other); tumor size (larg- est diameter in centimeters); formation of tubules ( > 75%; 10 to 74%; < 10%); nuclear pleomorphism (low; moderate; high); num- ber of mitoses per ten high-power fields - hpf (< 10/hpf; 11 to 20/hpf; > 20/hpf); histologi- cal grade as proposed by Bloom and Richardson18 and later modified by Elston and Ellis4 (well-differentiated or grade I; moder- ately differentiated or grade II; poorly differ- entiated or grade III); vascular and or lym- phatic invasion; multicentricity; skin involve- ment; total number of lymph nodes examined; total number of lymph nodes involved by neo- plasm; presence of perinodal fat infiltration, and surgical limits. Lymph node involvement at diagnosis is currently accepted as the most important prog- nostic factor to breast cancer evolution. Nev- ertheless, 20% to 30% of patients showing negative lymph nodes may further present disease recurrence and high mortality, thus deserving an intensive therapeutic approach usually not used in such conditions. Indeed, the ascertainment of predictive factors that allows identification of breast cancer cases of poor evolvement has become a challenge to reduce breast cancer mortality. Other prognostic factors such tumor size, histology, nuclear pleomorphism, vascular invasion and hormonal receptors have been used to predict tumor behavior either accord- ing to overall survival or local recurrence of breast cancer.13 On the other hand, debate still remains on the use of tumor markers as reli- able predictors for breast cancer prognosis.14-16 Taking into account that lymph node in- volvement is accepted as one of the best pre- dictors to breast cancer evolvement, and that infiltrating ductal carcinoma (IDC) is its most prevalent histological type and displays worst prognosis,6,10,17 this study was carried out to ascertain if selected breast cancer prognostic factors are also predictors for axillary lymph nodes metastasis. Recovery of antigen in the immunohis- tochemical preparations was performed by moist heat (pressure cooker), and the method of detection was the peroxidase-antiperoxidase reaction (PAP). The preparations were also analyzed at the same time by the two patholo- gists aforementioned. Assessment of stained neoplastic cells distribution was performed, and only those moderately or intensely stained were considered as positive; weakly stained cells were considered negative. The cut-off point used to separate positive and negative stains was 10% of stained cells (this criterion is used by various authors in the specialized literature.7,15,19 Other cut-off points were used to quantify cells staining were: (+) 10% to 25%; (++) 25% to 75%; (+++) > 75%. For estrogen receptors, progesterone receptors, MIB-1, PCNA, and p53, nuclear positivity was BD Revista Brasileira de Cancerologia, 2001, 47(4): 389-96 # All malignant breast tumors (incident cases) submitted to surgery with axillary lymph nodes dissection by the Mastology Ser- vice of the Cancer Hospital/Brazilian National Cancer Institute, in Rio de Janeiro, Brazil, from January 1, 1995, through August 31, BD Revista Brasileira de Cancerologia, 2001, 47(4): 389-96 6+20&'22789+002'2' considered. For c-erbB-2, positivity was searched in the cytoplasmatic membrane, and for cathepsin D it was the presence of stained granules in the cytoplasm of tumor cells. Finally, kappa coefficients were ascer- tained to evaluate reliability of nuclear and histological grade estimated in this study. Data obtained from patient files were the following: age at diagnosis; time between ini- tial symptoms and first consultation at the HC/ INCA; age at menarche; age at menopause for patients having reached menopause natu- rally or by surgery; number of pregnancies, births and spontaneous or induced abortions; age at first at-term delivery for patients hav- ing given childbirth; family history of breast cancer; parenthood degree according to fam- ily history of breast cancer (mother; daugh- ter; sister; grandmother; aunt; cousin; other); family history of other types of cancer; ana- tomical location of family cancer (ovary; en- dometrium; colon; other). B@ Revista Brasileira de Cancerologia, 2001, 47(4): 389-96 Histological grade reability among patholo- gists was ascertained, yielding to a kappa value of 0.83, standard error of 0.04. Similar pro- cedure was carried out with nuclear pleomor- phism identification, with kappa value of 0.90 and standard error of 0.05. Mean and median age among cases and controls was 57 yr. old (1st quartile Q1: 47 yr., 3rd quartile Q3: 68 yr). The mean tumor size was 4.5 cm among cases and 3.8 among con- trols (2 = 7,11, p=0.008). The total number of analyzed lymph nodes did not differ among cases and controls, ranging from 4 to 65 (mean 21, s=9.2, median 20, 1st quartile: 15 and 3rd quartile: 25). Databank organization was performed using the EPI-INFO software version 6.04 (U.S. Department of Health and Human Ser- vices and Public Health Service and Centers for Disease Control, USA). Bivariate analy- sis was carried out and included crude odds ratios (OR) ascertainment with 95% confi- dence intervals (CI), in order to estimate the degree of association between the study vari- ables and hormonal receptors status; chi- square test (2) of linear trend for ordinal vari- ables; chi square test of independence (2) to evaluate null hypothesis of the observed asso- ciations; Mantel-Haenszel OR (ORMH) and 95% confidence intervals, following stratifi- cation of selected variables. Further, uncon- ditional logistic regression was developed for the construction of parsimonious models in the determination of lymph nodes metastasis as outcome in patients with breast IDC us- ing EGRET software (Epidemiological Graph- ics, Estimation, and Testing, version 0.26.6, 1985-1991, SERC & CYTEL). Cases and controls showed similar pat- terns according to histological grade and mi- tosis number (Table 1). On the other hand, #@ & -",""1 % 2" 1@BB5B' #@ & -",""1 % 2" 1@BB5B' Independent variables tested in the multi- variate analysis (logistic regression) were cho- sen following the previous stages (bivariate and stratified analyses) as well as biological criteria evaluated by the authors. Potential confounders and variables showing significant p-values (at 5%) for presence of interaction were selected for logistic regression, as well as variables of biological interest. "#$#"#! %&'#(++/5+!$#,#,# #A5 "#Q"-&"- " < #1% 2" 1@BB5B' A cases showed higher proportions of tumors displaying less tubular structures (p=0.03), higher nuclear pleomorphism (p =0.09) and vascular invasion (p=0.0001). Bivariate analy- sis carried out with several variables and lymph nodes metastasis as outcome (Table 2) re- vealed statistically significant odds ratios (OR) for the following: surgery (mastectomy vs. minor interventions), OR 4.90 (95% C.I. 2.03-12.22); vascular invasion, OR 2.64 (95% C.I. 1.54-4.54); age at menarche, OR 2.16 (95% C.I. 1.08-4.34); skin involvement OR 1.85 (95% C.I. 1.04-3.33); and tumor size, OR 1.65 (95% C.I. 1.00-2.71). ( ) Stratified analysis with several variables was performed on the association between tu- mor size and lymph nodes metastasis aiming to control potential confounding (Table 3). Mantel Hanszel odds ratios (ORM-H) were ob- tained for the following variables: tumor p53, ORM-H 1.72 (95% C.I. 1.08-2.75); PCNA, ORM-H 1.67 (95% C.I. 1.05-2.67), nuclear pleomorphism ORM-H 1.54 (95% C.I. 0.96- 2.49); tubular formation, ORM-H 1.69 (95% C.I. 1.05-2.70); estrogen receptor, ORM-H 1.62 (95% C.I. 1.01-2.61); parity, ORM-H 1.57 (95% C.I. 0.98-2.52); age at menarche, ORM- H 1.59 (95% C.I. 0.99-2.58); breast side (right vs. left), ORM-H 1.62 (95% C.I. 1.02-2.59). Effect modification (interaction) in the afore- mentioned association was observed with the variables patient’s age (p=0.04) and menopause (pre vs. post), p=0.06 (Table 3). #5 "#Q"-&"- "- ! #< #1% 2" 1@BB5B' #5 "#Q"-&"- "- ! #< #1% 2" 1@BB5B' Finally, unconditional logistic regression was carried out and a parsimonious model including the variables nuclear pleomorphism, lymphatic invasion, patient’s age, tumor size and an interaction term between patient’s age and tumor size (likelihood ratio test = 24.66, 5 d.f., p<0.001) was chosen (Table 4). BA Revista Brasileira de Cancerologia, 2001, 47(4): 389-96 6+20&'22789+002'2' 6+20&'22789+002'2' Differential probability of lymph nodes metastasis identification among cases and con- trols was searched towards the distributions of reviewed lymph nodes (p=0.42). Thus, both groups did not differ according to total num- ber of reviewed lymph nodes, suggesting they had had similar probabilities to have identi- fied lymph nodes metastasis if present. dition has also been associated as an indepen- dent predictor for breast cancer evolution in terms of general survival, metastasis-free sur- vival and disease-free survival.5,22-25 Accordingly with the literature10,20,2 our data revealed an as- sociation between vascular invasion and axil- lary lymph nodes metastasis (p<0.0001). #&-F"" ", , "H1 ""1% 2" 1@BB5B' #&-F"" ", , "H1 ""1% 2" 1@BB5B' In some series, the majority of breast can- cer cases was diagnosed among women 45- 50 yr. old,9 but ours was older (median age at diagnosis 57 yr. old). As previously reported by Recht and Houihan,20 we also observed a weak and statistically non significant associa- tion between age at diagnosis and lymph nodes involvement. A statistically significant asso- ciation between age at menarche (11 yr. as a cut-off point) and lymph nodes involvement was also observed in our data (Table 2). Tumor size, the most important predictor of lymph nodes involvement in breast cancer, showed a statistically significant association with that outcome (p<0.05), and revealed the dramatic reality still observed in Brazil accord- ing to breast cancer medical care: median tumor size in our data was 4.0 cm. Despite several reports on the association of hormonal receptors with a better evolution in breast cancer,2,3,13 some divergent observa- tions have also been published.2,2 In accordance to the latter, our data does not support an as- sociation between hormonal receptors and absence of metastasis to axillary lymph nodes among breast cancer patients. Despite some debate,20 histological grade is also acknowledged as an important predic- tor for lymph nodes involvement in the lit- erature, and high grade tumors have been related to an increased frequency of lymph nodes metastasis and to a poor evolution.2,4,17 Usually, breast cancer patients without axillary lymph nodes involvement do not present metastasis and local recurrence; there- fore, they do not require adjuvant therapy. Nevertheless, some present a different evolu- tion, remaining a challenge their identifica- tion. B Revista Brasileira de Cancerologia, 2001, 47(4): 389-96 Selection and information bias are usu- ally a main flaw in case-control studies. Aim- ing to ascertain their influence in this investi- gation, we evaluated some potential key vari- ables which could influence our results. The first one was the time interval between initial symptoms and first consultation among cases and controls (p=0.51), which indicates absence of statistically significant differences among both groups according to this variable. 6+20&'22789+002'2' The use of acknowledged prognostic fac- tors for patients without lymph nodes involve- ment further presenting poor survival has of- fered a weak contribution, and the search of new ones has been stimulated. Among them, c-erbB-2,24,2 Cathepsin D,15 MIB-1,13,17 PCNA11,14 and p538 have been suggested. In our study, histological grade was ascer- tained towards a blind evaluation carried out by two pathologists following Bloom and Richardson18 procedures, further modified by Elston and Ellis.4 Reliability analysis revealed an inter-observer agreement of 83% (p<0.0001), being 90% for nuclear pleomorphism (p<0.0001). Any association between histologi- cal grade and metastasis presence was observed, but a borderline association between nuclear pleomorphism and metastatic lymph nodes in- volvement (p=0.07) was observed (Table 1). High c-erbB-2 expression has been re- ported in breast cancer, ranging between 15 to 64%.30,31 In our data, 34.6% of tumors revealed membrane positivity to c-erbB-2, and only 7.5% showed diffuse positivity. Accord- ingly to other authors, 19,32,33our study also did not find association between c-erbB-2 and axillary lymph nodes metastasis, which re- mains a controversial result not observed by some researchers.34,35 Vascular invasion has been reported in the literature ranging between 5% to 49%,5,22 and we observed this condition in 32.4% of pa- tients, being twice more frequent among cases than controls (vascular invasion was consid- ered as positive whenever reported by both pathologists, and negative otherwise). This con- B Revista Brasileira de Cancerologia, 2001, 47(4): 389-96 "#$#"#! %&'#(++/5+!$#,#,# morphism, vascular invasion and the inter- action term between tumor size and age. Highest odds ratio (Table 3) were observed for nuclear pleomorphism (moderate vs. low grade, OR 8.61, 95% C.I. 1.04-70.99, p<0.05, and high vs. low grade, OR 10.44, 95% C.I. 1.23-88.8, p<0.05). Increased cathepsin D expression was as- certained in this study by citoplasmatic immuno-reactivity of tumor cells as reported elsewhere.15,36 Similarly than reported by oth- ers,37,38 an association between cathepsin D expression and lymph nodes involvement was not observed in our data. Analyzing the inclusion of an interaction term between tumor size and age, we observe the latter modifies the effect on the associa- tion between tumor size and axillary lymph nodes metastasis. Therefore, that association among women 60 yr. or older diagnosed with breast cancer provides a moderate risk esti- mate (OR 2.37, 95% C.I. 1.11-5.03), mean- while no association is observed among younger women (OR 0.77, 95% C.I. 0.39- 1.50). In this study, the best predictive variables to axillary lymph nodes involvement in breast cancer were patient´s age, tumor size, nuclear pleomorphism and vascular invasion. Neither hormonal receptors nor selected tumor mark- ers (c-erbB-2, cathepsin D, MIB-1, PCNA and p53) were good predictors for that out- come in the studied sample. Mutated p53 gene is quite a common phe- nomena in several human tumors, particu- larly breast cancer, which accumulate anoma- lous p53 protein in tumor cell nucleus. The p53 nuclear immunoreactivity has been con- sidered an indirect indicator of p53 gene mutation, and has been detected in breast cancer ranging from 9% to 52%.8, 6+20&'22789+002'2' This model is in accordance with oth- ers previously reported in the literature.2,6,10,17 The rate of tumor cell proliferation is an important and sensitive prognostic marker of different cancer sites including breast cancer. Different methods, such as microscopic mor- phometry, flux citometry and immunohis- tochemistry, among others,39 and antigens such as PCNA, MIB-1 and Ki-6740 have been developed to evaluate this cellular activity. The IDC analyzed in this study revealed 59.5% of positivity to MIB-1 and 72.2% for PCNA, being both markers strongly as- sociated in this study (p<0.0001). A simi- lar observation was reported by Haerslev et al.11 In accordance to some reports14,41 and disaccord to others,11,42 statistically sig- nificant association between MIB-1 or PCNA with axillary metastasis was not ob- served in our study. B Revista Brasileira de Cancerologia, 2001, 47(4): 389-96 6+20&'22789+002'2' 5. Lauria R, Perrone F, Carlomagno C, et al. The prognostic value of lymphatic and blood vessel invasion in operable breast cancer. Cancer 1995;76:1772-8. 17. Pereira H, Pinder SE, Sibbering DM, et al. Patho- logical prognostic factors in breast cancer. IV: Should you be typer or a grader? A compara- tive study of two histological prognostic fea- tures in operable breast carcinoma. Histopathol- ogy 1995;27:219-26. 6. Pinder SE, Ellis IO, Elston CW. Prognostic fac- tors in primary breast carcinoma. J Clin Pathol 1995;48:981-3. 18. Bloom HJ, Richardson WW. Histological grad- ing and prognosis in breast cancer: a study of 1049 cases of wich 359 have been followed for 15 years. Br J Cancer 1957;11:359-77. 7. Rosen PP, Lesser ML, Arroyo CD, Cranor M, Borgen P, Norton L. p53 in node-negative breast carcinoma: an immunohistochemical study of epidemiologic risk factors, histologic features, and prognosis. J Clin Oncol 1995;13:821-30. 19. Keshgegian AA. ErbB-2 oncoprotein overexpression in breast carcinoma: inverse correla- tion with biochemically - and immunohisto- chemically - determined hormone receptors. Breast Cancer Res Treat 1995;35:201-10. 8. Barbareschi M. Prognostic value of the immu- nohistochemical expression of p53 in breast carcinoma. A review of the literature involving over 9.000 patients. Appl Immunohistochem 1996;4:106-16. 20. Recht A, Houlihan MJ. Axillary lymph nodes and breast cancer: a review. Cancer 1995;76:1491-512. 9. Chung M, Chang HR, Bland KI, Wanebo HJ. Younger women with breast carcinoma have a poorer prognosis than older women. Cancer 1996;77:97-103. 21.Nixon AJ, Schnitt SJ, Gelman R, et al. Rela- tionship of tumor grade to other pathologic features and to treatment outcome of patients with early stage breast carcinoma treated with breast-conserving therapy. Cancer 1996; 78:1426-31. 10.Elston CW, Ellis IO. Prognostic factors in breast cancer. Annual Meeting [Handout].United States and Canadian Academy of Pathology, 1996:1-73. 22.Weigand RA, Isenberg WM, Russo J, Brennan MJ, Rich MA. The Breast Cancer Prognostic Study Associates. Blood vessel invasion and axillary lymph node involvement as prognostic indicators for human breast cancer. Cancer 1982;50:962-9. 11.Haerslev T, Jacobsen GK, Zedeler K. Correla- tion of growth fraction by Ki-67 and prolifer- ating cell nuclear antigen (PCNA) immuno- histochemistry with histopathological param- eters and prognosis in primary breast carcino- mas. Breast Cancer Res Treat 1996;37:101-13. 23. Clemente CG, Boracchi P, Andreola S, Del Vecchio M, Veronesi P, Rilke FO. Peritumoral lymphatic invasion in patients with node-nega- tive mammary duct carcinoma. Cancer 1992;69:1396-403. 12.Rosai J. Breast. In: Juan Rosai, eds. In our study, 17.6% IDC displayed p53 positivity, and, differently than reported in the literature, a statistically non-significant inverse association between p53 and lymph nodes involvement was observed (OR 0.66, 95% C.I. 0.35-1.25, p=0.17). In a review carried out by Barbareschi,8 the association between p53 and lymph nodes involvement was just observed in one among eight stud- ies. Sirvente et al.,16 Wakasugi44 also did not find such association, while Chen et al.,35 Hanzal et al.45 found it. As a whole, such di- vergent results reinforce Barbareschi8 point- of-view on the need for further research to confirm whether p53 could be indeed con- sidered as an independent predictor to breast cancer evolution. 1. Brasil. Ministério da Saúde. Secretaria Nacional de Assistência à Saúde. Instituto Nacional de Câncer. Estimativa da incidência e mortalidade por câncer no Brasil para 1999. Rio de Janeiro: INCA, 1999:8-13. 1. Brasil. Ministério da Saúde. Secretaria Nacional de Assistência à Saúde. Instituto Nacional de Câncer. Estimativa da incidência e mortalidade por câncer no Brasil para 1999. Rio de Janeiro: INCA, 1999:8-13. 2. Weidner N. Selected problems in breast pathol- ogy. Prognostic factors in breast carcinoma: which factors are important? Seminars in Pa- thology [Handout]. San Diego, Califórnia: So- ciety of Pathologists, 1994:1-57. 3. Pertschuk LP, Feldman JG, Kim DS, et al. Ste- roid hormone receptor immunohistochemistry and amplification of c-myc protooncogene: re- lationship to disease-free survival in breast can- cer. Cancer 1993;71:162-71. 4. Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histo- logical grade in breast cancer: experience from a large study with long-term follow-up. Histo- pathology 1991;19:403-10. Following multivariate analysis, the pre- dictive variables included in our model were age at diagnosis, tumor size, nuclear pleo- 6+20&'22789+002'2' B Revista Brasileira de Cancerologia, 2001, 47(4): 389-96 6+20&'22789+002'2' Ackerman’s surgical pathology. 8th ed. St. Louis: Mosby, 1996:1590-660, cap.20, v.2. 13.Rosen PP. Breast Pathology. Philadelphia: Lippincott-Raven, 1997:275-93. 24. Fisher ER, Anderson S, Redmond C, Fisher B. Pathologic findings from the National Surgical Adjuvant Breast Project (Protocol B-06). 10- year pathologic and clinical prognostic discrimi- nants. Cancer 1993;71:2507-14. 14.Gasparini G, Meli S, Pozza F, Cazzavillan S, Bevilacqua P. PC-10 antibody to proliferating cell nuclear antigen (PCNA) is not related to prognosis in human breast carcinoma. Growth Regul 1992;2:145-50. 25. Fisher ER, Costantino J, Fisher B, Redmond C. Collaborating National Surgical Adjuvant Breast and Bowel Project Investigators. Pathologic find- ings from the National Surgical Adjuvant Breast Project (Protocol 4). Discriminants for 15-year survival. Cancer 1993;71:2141-50. 15.Isola J, Weitz S, Visakorpi T, et al. Cathepsin D expression detected by immunohistochemistry has independent prognostic value in axillary node-negative breast cancer. J Clin Oncol 1993;11:36-43. 26.Pinder SE, Ellis IO, Galea M, O‘Rouke S, Blamey RW, Elston CW. Pathological prognos- tic factors in breast cancer. III. Vascular inva- sion: relationship with recurrence and survival in a large study with long-term follow-up. His- topathology 1994;24:41-7. 16.Sirvent JJ, Salvadó MT, Santafé M, et al. p53 in breast cancer. Its relation to histological grade, lymph-node status, hormone receptors, cell- proliferation fraction (ki-67) and c-erbB-2. Immunohistochemical study of 153 cases. Histol Histopathol 1995;10:531-39. B Revista Brasileira de Cancerologia, 2001, 47(4): 389-96 "#$#"#! %&'#(++/5+!$#,#,# 27.Stierer M, Rosen H, Weber R, Hanak H, Spona J, Tücheler H. Imunohistochemical and bio- chemical measurement of estrogen and proges- terone receptors in primary breast cancer. Cor- relation of histopathology and prognostic fac- tors. Ann Surg 1993;218:13-21. 36.Kandalaft PL, Chang KL, Ahn CW, Traweek ST, Mehta P, Battifora H. Prognostic signifi- cance of immunohistochemical analysis of cathe- psin D in low-stage breast cancer. Cancer 1993;71:2756-63. 37.Têtu B, Brisson J, Côté C, Brisson S, Potvin D, Roberge N. Prognostic significance of cathep- sin D expression in node-positive breast carci- noma: an immunohistochemical study. Int J Cancer 1993;55:429-35. 28.Macgrogan G, Soubeyran I, De Mascarel I, et al. Immunohistochemical detection of progest- erone receptors in breast invasive ductal carci- nomas: a correlative study of 942 cases. Appl Immunohistochem 1996;4:219-27. 38.Aaltonen M, Lipponen P, Kosma VW, Aaltomaa S, Syrjänen K. Prognostic value of cathepsin D expression in female breast cancer. Anticancer Res 1995;15:1033-7. 29.De Potter CR, Schelfhout AM. The neu-pro- tein and breast cancer. Virchows Archiv 1995;426:107-15. 39.Hilsenbeck SG, Allred DG. B' Revista Brasileira de Cancerologia, 2001, 47(4): 389-96 6+20&'22789+002'2' Improved meth- ods of estimating mitotic activity in solid tumors [Editorial]. Hum Pathol 1992;23:601-2. 30.Allred DC, Clark GM, Molina R, et al. Overexpression of HER-2/neu and its relation- ship with other prognostic factors change dur- ing the progression of in situ to invasive breast cancer. Hum Pathol 1992;23:974-9. 40.Leong AS-Y, Lee AKC. Biological indices in the assesssment of breast cancer. J Clin Pathol. Clin Mol Pathol 1995;48:M221-38. 31.Querzoli P, Marchetti E, Fabris G, et al. Immu- nohistochemical expression of c-erbB-2 in hu- man breast cancer by monoclonal antibody: cor- relation with lymph node and ER status. Tumori 1996;76:461-4. 41.Pinder SE, Wencyk P, Sibbering DM, et al. Assessment of the new proliferation marker MIB-1 in breast carcinoma using image analysis: associations with other prognostic factors and survival. Br J Cancer 1995;71:146-9. 32.Pierce LJ, Merino MJ, D’angelo T, et al. Is c- erbB-2 a predictor for recurrent disease in early stage breast cancer? Int J Radiat Oncol Biol Phys 1994;28:395-403. 42.Querzoli P, Albonico G, Ferretti S, et al. MIB-1 proliferative activity in invasive breast cancer measured by image analysis. J Clin Pathol 1996;49:926-30. 33. Chariyalertsak S, Chariyalertsak S, Cheirsilpa A, Sugano K, Ohkura H. Immunohistochemical de- tection of c-erbB-2 oncoprotein in patients with breast cancer. J Med Assoc Thai 1996;79:715-21. 43.Nagai MA. Alterações genéticas em câncer de mama. Rev Bras Mastol 1995;5:31-41. 44.Wakasugi E, Kobayashi T, Tamaki Y, et al. p21 (waf1/Cip1) and p53 protein expression in breast cancer. Am J Clin Pathol 1997;107:684-91. 34.Têtu B, Brisson J. Prognostic significance of HER-2/neu oncoprotein expression in node- positive breast cancer. The influence of the pat- tern of immunostaining and adjuvant therapy. Cancer 1994;73:2359-65. 45.Hanzal E, Gitsch G, Kohlberger P, Dadak C, Miechowiecka N, Breitenecker G. Immunohis- tochemical detection of mutant p53-suppres- sor gene product in patients with breast cancer: influence on metastasis-free survival. Antican- cer Res 1992;12: 2325-9. 35.Chen YH, Cai JJ, Lu YD. The relation between p53 expression and lymph node metastasis of breast cancer. Chung Hua Chung Liu Tsa Chih 1994;16:266-8.
https://openalex.org/W2298294443	https://europepmc.org/articles/pmc4726218?pdf=render	English	null	Super Soft All-Ethylene Oxide Polymer Electrolyte for Safe All-Solid Lithium Batteries	Scientific reports	2,016	cc-by	12,294	Luca Porcarelli, Claudio Gerbaldi, Federico Bella & Jijeesh Ravi Nair Here we demonstrate that by regulating the mobility of classic −EO− based backbones, an innovative polymer electrolyte system can be architectured. This polymer electrolyte allows the construction of all solid lithium-based polymer cells having outstanding cycling behaviour in terms of rate capability and stability over a wide range of operating temperatures. Polymer electrolytes are obtained by UV-induced (co)polymerization, which promotes an effective interlinking between the polyethylene oxide (PEO) chains plasticized by tetraglyme at various lithium salt concentrations. The polymer networks exhibit sterling mechanical robustness, high flexibility, homogeneous and highly amorphous characteristics. Ambient temperature ionic conductivity values exceeding 0.1 mS cm−1 are obtained, along with a wide electrochemical stability window (>5 V vs. Li/Li+), excellent lithium ion transference number (>0.6) as well as interfacial stability. Moreover, the efficacious resistance to lithium dendrite nucleation and growth postulates the implementation of these polymer electrolytes in next generation of all-solid Li-metal batteries working at ambient conditions. Present energy storage and production devices are based on combustible organic solvents that carry the risks of leakage and related fire hazards. This forces the manufacturer to enclose battery components in heavier and peculiar packaging structures to meet the stringent safety requisites. Such heavy protective packaging reduces the overall amount of ready-to-use energy (energy density). An all-solid construction will certainly enhance the overall performance of any energy storage and conversion devices that can be thought of1. p y gy g g An ideal ion conducting polymer electrolyte for ambient temperature energy storage has been the dream pursued by many researchers2–4. An enhanced safety may be achieved by complete replacement of organic carbonate-based liquid electrolytes, thus leading to facile and leak-free fabrication, flexibility, compactness, reduced weight and laminated structures5. Scientific community has been focusing on polymer systems contain- ing –EO– moieties for an all-solid cell construction, and other polymers (e.g., PMMA, PVdF) in mostly hybrid/ gel configurations6,7. It is well known that polyethylene oxide (PEO) crystallizes at about 55 °C8,9 and ionic as well as segmental mobility are limited to its molten (amorphous) state at elevated temperatures. This impedes its widespread application into the global market10. However, at elevated temperatures such polymers lose their dimensional stability being in molten state, which leads to non-homogeneity inside the cell11. This facilitates the diffusion of Li+ ions through localized favourable paths, increasing both concentration gradients and defects that may create temperature deflections and short-circuits. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports GAME Lab, CHENERGY Group, Department of Applied Science and Technology – DISAT, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129-Torino, Italy. Correspondence and requests for materials should be addressed to C.G. (email: claudio.gerbaldi@polito.it) or J.R.N (email: jijeesh.nair@polito.it) Super Soft All-Ethylene Oxide Polymer Electrolyte for Safe All- Solid Lithium Batteries received: 27 July 2015 accepted: 21 December 2015 Published: 21 January 2016 Luca Porcarelli, Claudio Gerbaldi, Federico Bella & Jijeesh Ravi Nair Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 Results and Discussions Physical-chemical characterization. Figure 1 shows the materials used in the study along with the real aspect of ISPE formed after UV exposure (right top), which is almost transparent, tack free, flexible and easy to manage. On the right bottom side of Fig. 1, the possibility of interlinking of polymer chains is hypothesized, with the in situ oligomer formation and plausible grafting of TEGDME molecules onto the long PEO chains upon 6 min. of UV irradiation (intensity on the surface of the sample of 40 mW cm−2).i Before finalising to the formulations reported in this work, several tests were performed to understand the fundamental aspects of polymer electrolytes, to decide the quantity and type of photoinitiator, as well as the suita- ble molecular weight of PEO depending on the easiness in processing. MBP was selected as the photo-cross-linker with superior solubility and optimum cross-linking properties in quantities equal to 7.5 wt. % with respect to the total materials’ mass. Such studies are not included here being out of scope of the present discussion. The overall characteristics and composition of the different samples under study are listed in Table 1, including the glass transition temperature (Tg) values and the gel content (insoluble fraction after cross-linking) against the corre- sponding –EO– to Li ratio. High-resolution FESEM analysis at 50K magnification (Fig. 2A–D) shows the characteristics of a soft, cross-linked polymer electrolyte (namely PTL-1) with rather high degree of amorphous nature. The micrographs are well in agreement with the results obtained by Schulze et al., who used a one-pot synthetic strategy based on polymerization-induced phase separation to generate nanostructured polymer electrolytes that exhibited an unprecedented combination of high modulus and ionic conductivity38. In the present case, phase separation is not possible as the material is made of similar –EO– based backbones. Indeed, viscosity as well as polymeri- zation induced aggregation and-to a certain extent - rearrangement of the PEO chains could be possible. The photopolymerisation was carried out after keeping the highly viscous reactive mixture under stressful conditions (90 °C, pressed at 20 bar) between two Mylar foils39. In particular, micrographs clearly evidence a bag-like poly- mer matrix that incorporates an extremely soft material. The surface roughness is very clearly visible in second- ary electron mode. Moreover, the overall aspect and characteristics indicate the exceptional homogeneity of the sample, with no noticeable presence of pores or voids. Moreover, the images showed in Fig. Luca Porcarelli, Claudio Gerbaldi, Federico Bella & Jijeesh Ravi Nair Due to the excellent properties imparted by glymes in the liquid electrolyte, recently they have received plenty of attention for next-gen systems beyond Li-ion, such as lithium sulphur2–4 and lithium air rechargeable batteries26–28. g Free radical photopolymerisation is a low cost, solvent-free and energy saving technique very well established for many applications in an easily implemented and versatile fashion29–31. Photopolymerisation can be suitably adapted to the preparation of polymer electrolytes due to its eco-friendliness, which is a key aspect that influences the fate of large-scale polymer electrolyte manufacturing32. Moreover, UV-induced reaction on multifunctional monomers permits rapid in situ generation of intimate electrode/electrolyte interfaces, which currently represent a major striking point to be fixed in the field of electrochemical devices33. Thus, the whole electrolyte preparation can be carried out in the absence of solvent. Most of the systems referring to glyme-based electrolytes are either blended with thermoplastic materi- als or directly used in their liquid form. Little work34,35 has been devoted to study their possible implementa- tion in a self-standing, softly cross-linked thermoplastic polymer matrix. In the present work, we use a system based on PEO and tetraglyme, and we directly cross-link it in one-pot along with the supporting lithium salt under UV irradiation to retain the solid-like nature and dimensional stability. By concurrent exploitation of photo-induced cross-linking and in situ functionalization procedures, kinetically driven inhibition of the PEO chains crystallization is readily achievable at ambient conditions, leading to polymer electrolytes that possess solid-like properties without hampering ionic mobility. They are prepared by mixing PEO as the polymer matrix, bis[2-(2-methoxyethoxy)ethyl]ether (tetraglyme, TEGDME) as the active plasticizer, lithium bistrifluoromethane sulfonimide (LiTFSI) as the source of Li+ ions and 4-methyl benzophenone (MBP) as the light-induced hydrogen abstraction mediator (photoinitiator). Under UV excitation, MBP abstracts an acidic proton from a methylene group and generates a free radical chain36,37. This free radical can combine with another free radical belonging to the same chain or other –EO– chains to interlink themselves. Tetraglyme also possesses methylene groups that can undergo hydrogen abstraction and following inter-radical reactions to form oligomers, or bond to the adjacent PEO chains. The final interlinked solid polymer electrolyte (ISPE) films are mechanically robust, highly flexible, homogeneous and largely amorphous. They also exhibit excellent properties in terms of compatibility with the lithium metal electrode and suppression of hazardous dendrite growth. Luca Porcarelli, Claudio Gerbaldi, Federico Bella & Jijeesh Ravi Nair Attempts have been also made to reduce the crystallinity to retain the amorphicity by incorporating various ceramics/metal oxides (e.g., Al2O3, CeO2, ZrO2, TiO2)12, but the target remains unachieved so far. Another problem is the solvent-based preparation procedure, where complete and “concrete” solvent removal is a herculean task; indeed, in most cases the unavoidable traces of solvent per- sisting in the polymer matrix create various interfacial stability issues, even enabling thermal runaway reactions.t g p y y g y Four decades after the discovery of ionic conduction in polymer electrolytes by Fenton et al.13 and Armand et al.14, the topic remains red hot. Moreover, the all-solid-state Li-metal battery concept is still appealing due to assured high energy density15. A polymer electrolyte that is less reactive towards Li-metal and highly resistant to dendrite formation/penetration would potentially open up the possibility of Li-metal based accumulators to entry in the market. After decades of efforts, PEO matrix still struggles to meet the requirements of global market due to its low ambient temperature conductivity and inferior safety arising from non-uniform plating/stripping of Li+ ions, which often results in hazardous dendrite formation. GAME Lab, CHENERGY Group, Department of Applied Science and Technology – DISAT, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129-Torino, Italy. Correspondence and requests for materials should be addressed to C.G. (email: claudio.gerbaldi@polito.it) or J.R.N (email: jijeesh.nair@polito.it) Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 1 1 www.nature.com/scientificreports/ A compromise between the characteristics of an all-solid state16 and a gel-like17 polymer electrolyte might represent the vital knot that researchers need to accomplish the goal. In this direction, remarkable results are reported in literature with the addition of room temperature ionic liquids18 into thermoplastic19 or thermoset20 polymer matrix. However, the problems such as restricted Li+ ion diffusion, cost and rate capabilities become the hurdle towards the commercialisation of such materials. Thus, slowly and steadily the scientific community is moving towards the addition of noncarbonated high boiling, thermally stable organic solvents such as organic nitriles21, glymes22, etc. Moreover, in the case of thermoplastic polymers the separator size deformation with var- ying temperature is a tough issue, while thermosets are limited by unsuitable ionic conductivity and brittleness of the polymer matrix. p y Glymes (various lengths) are well known for complexing with metal-ions through their multiple ether-like oxygen atoms23. When lithium salt is dissolved in glyme-based solvents, they show promising ionic conductivity and Li+ ion transport properties24,25. Luca Porcarelli, Claudio Gerbaldi, Federico Bella & Jijeesh Ravi Nair The sum of these characteristics enlighten the striking prospects of the newly developed ISPE as electrolyte separators in both Li-ion and Li metal batteries conceived for high energy and/or power demanding applications, including hybrid vehicles and smart grid storage systems. Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 Results and Discussions 2(E,F) demonstrate that the obtained ISPE is stretchable and highly elastic (once the stress is released, it can go back to the previous Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 2 www.nature.com/scientificreports/ Figure 1. Sketched representation of ISPE preparation along with used materials, and plausible illustration (right bottom) of interconnected PEO chains with hypothesized branched clusters of tetraglyme oligomers; on the top right, the real aspect of a freshly prepared ISPE. Figure 1. Sketched representation of ISPE preparation along with used materials, and plausible illustration (right bottom) of interconnected PEO chains with hypothesized branched clusters of tetraglyme oligomers; on the top right, the real aspect of a freshly prepared ISPE. LiTFSIψ PEOψ TEGDMEψ Tg § Gelψ EO:Li PTL-1 10 41.3 41.2 − 34 42 ± 2 54:1 PTL-2 15 38.8 38.7 − 38 39 ± 2 35:1 PTL-3 20 36.3 36.2 − 44 37 ± 3 23:1 Table 1. Composition of ISPEs, along with their Tg and gel-content values. MBP content is 7.5 wt. % of the total weight of materials. Ψunits in wt. %. §unit in °C. Table 1. Composition of ISPEs, along with their Tg and gel-content values. MBP content is 7.5 wt. % of th total weight of materials. Ψunits in wt. %. §unit in °C. shape). These results all together confirm that the proposed electrolyte is soft, weakly cross-linked, flexible, and shape retaining at ambient conditions.hfi shape). These results all together confirm that the proposed electrolyte is soft, weakly cross-linked, flexible, and shape retaining at ambient conditions.hfi The differential scanning calorimetry (DSC) values are tabulated in Table 1, and the respective profiles are shown in Fig. 3A. Both the glass transition and the melting temperatures were determined for all the samples. Tg values vary between –34 and –44 °C. As expected, the amount of salt noticeably influences the Tg of the ISPEs prepared with 1:1 tetraglyme to PEO ratio. Generally, low Tg values account for a moderately enhanced segmental motion of the –EO‒ moieties in the polymer matrix which is facilitated by low crystallinity. Moreover, a noticeable change in the peak associated with the melting of the crystalline region is clearly evidenced: an increase in LiTFSI content reduces the intensity of the melting peak, which also results broadened. As noted in Table 1, an increase in salt content reduces the [EO]/[Li] ratio from 54:1–23:1. Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 Results and Discussions Thus, an increased amount of Li+ ions is available for the coordination with PEO chains, thus reducing the tendency of forming crystalline phases. Moreover, the cross-linking effect further reduces the mobility of the PEO main chains. Overall, a relationship is present between the phase transition temperature and the salt content: the higher is the salt content, the lower is the transition temperature. This is expected as for the typical coordination effect by lithium salt, which weakly decreases the Tg 40. Furthermore, ISPEs in all cases exhibit crystalline melting peaks near room temperature during the heating cycle of the DSC analysis. This may be attributed to low cross-linking density (7.7 × 103 mol m–3 for PTL-1, calculated at 0 °C), which allows the long enough –EO– chains to rearrange and crystallize in the matrix41. It is worth not- ing that the melting points of these newly elaborated polymer electrolytes are well below the typical PEO-based electrolytes when tested by means of DSC40,42. If one calculates the number of MBP molecules per –EO– moieties, the latter exceeds 46 ± 3 per each photoinitiator molecule. This ratio is sufficient to enforce the rearrangement of the –EO– moieties to form weak crystalline phases. One cannot neglect the effect of tetraglyme in diluting the number of cross-linking per area, as some of the initiator molecules are actively involved in real-time oligomer- ization and eventual branching processes. Thus, the effective cross-linking would be lower than the theoretical calculations from mole ratios. Overall, the average cross-linking length obtained in the present work suggests that cross-linking is not sufficient to prevent the PEO crystallization. It can be hypothesized that a higher degree of cross-linking may not be also favoured, as it may induce the low molecular weight tetraglyme and/or its oligomers to squeeze out of the system during thermal stresses. Thus, we selected an optimum cross-linking, which could assure a good mechanical integrity along with good plasticizer retention (leak free).hh The thermal gravimetric analysis (TGA) (Fig. 3b) shows three main weight losses for all the samples. The first one is associated to tetraglyme degradation/evaporation, the second one to PEO decomposition and the last one to lithium salt decomposition. The first dip before 100 °C indicates the loss of humidity that may be absorbed during the handling of the sample for testing. Results and Discussions Taking into account the experimental errors related to Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Micrographs showing the overall morphology of sample PTL-1: cross-section under secondary electron mode (A,B) and top view (C,D), at different magnifications; (E,F) shown the images of the sample PTL-1 (at 25 °C) under stretch and bend mode, demonstrating the mechanical integrity and excellent elasticity. Figure 2. Micrographs showing the overall morphology of sample PTL-1: cross-section under secondary electron mode (A,B) and top view (C,D), at different magnifications; (E,F) shown the images of the sample PTL-1 (at 25 °C) under stretch and bend mode, demonstrating the mechanical integrity and excellent elasticity. Figure 2. Micrographs showing the overall morphology of sample PTL-1: cross-section under secondary electron mode (A,B) and top view (C,D), at different magnifications; (E,F) shown the images of the sample PTL-1 (at 25 °C) under stretch and bend mode, demonstrating the mechanical integrity and excellent elasticity. the measurement and the sample preparation, the overall weight loss is consistent with the polymer electrolyte composition. According to the differential thermal analysis (shown in dotted lines), the weight loss occurs for all the samples above 190 °C, the major contributor being tetraglyme41. Higher amounts of LiTFSI reduces the ther- mal stability of the polymer matrix, which is clearly visible in the differential curves (shift of the peak from 218 to 195 °C). This could be the general trend observed for tetraglyme kind of plasticizers that show the tendency to decompose in reaction with fluorinated anions22. Thus, both DSC and TGA profiles show that no unusual phase changes or weight losses occur in the temperature range between 25 and 150 °C, which makes the material ther- mally stable and useful as a polymer electrolyte under standard operating conditions in real battery configuration. Dynamic mechanical thermal analysis (DMTA) was performed on sample PTL-1 and the resulting profiles (both modulus and loss modulus) are shown in Figure S1(A,B). It clearly indicates that the material has low Tg, which is in agreement with the DSC analysis. In addition, the calculated cross-linking density (7.7 × 103 mol m–3, calculated at 0 °C) was found to be comparatively lower than other known systems43. This is an indication that the number of cross-linking points between the PEO chains is lower than expected, which allows the long enough –EO– chains to rearrange and crystallize in the matrix. Results and Discussions Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 4 www.nature.com/scientificreports/ Figure 3. (a) Differential scanning calorimetry (DSC) curves of the ISPEs PTL-1 to PTL-3 that contains various amounts of LiTFSI salt. (b) Thermogravimetric analysis (TGA) of the same series of ISPEs along with related differential curves (dotted lines of same color code). Taking into account the experimental errors related to the measurement and the sample preparation, all weight losses are consistent with the polymer electrolyte compositions. Figure 3. (a) Differential scanning calorimetry (DSC) curves of the ISPEs PTL-1 to PTL-3 that contains various amounts of LiTFSI salt. (b) Thermogravimetric analysis (TGA) of the same series of ISPEs along with related differential curves (dotted lines of same color code). Taking into account the experimental errors related to the measurement and the sample preparation, all weight losses are consistent with the polymer electrolyte compositions. The cross-linking effects and related properties were studied by an indirect method where the ISPE was sub- jected to dissolution in tetrahydrofuran (THF). The solubilized fraction was analysed using size exclusion chro- matography/gel-permeation chromatography (SEC/GPC) to calculate the molecular weight of oligomers formed by the bonding between various tetraglyme molecules. We chose PTL-1 as the representative sample and per- formed the test to understand the molecular weight of the soluble fraction. It is worth noting that the results of insoluble fraction study are in agreement with the gel-content studies performed on similar samples. The soluble fraction in THF while testing showed a broad molecular weight distribution primary caused by the tetraglyme molecules. Molecules with various molecular weights ranging from 300 up to 41000 Da eluted from the column at different time intervals (Figure S2), which correspond to the higher homologous of tetraglyme such as dimers, trimers or oligomers. This is a direct indication that tetraglyme molecules are taking part in the polymerization reaction to form oligomers and remain active upon UV irradiation. In such a scenario, some branches that arise from the reactions between the radicals generated from –EO– of PEO and –EO– of tetraglyme might be formed. The oligomers formed by tetraglyme may not remain in the polymer matrix, which is clearly visible when the membrane was placed in the THF bath. Thus, during the insoluble fraction test, these oligomers may diffuse out of the system, which may account for the low cross-linking content of the cross-linked material. Results and Discussions Tensile tests were carried out on the PTL-1 sample accord- ing to ASTM Standard D638; the Young’s modulus was found to be 0.3 MPa and maximum force at break was found to be 1.5 MPa. The material can stretch (Figure S1C) very well under stress, as justified by the maximum strain (elongation) of around 17 mm before the membrane was broken. the measurement and the sample preparation, the overall weight loss is consistent with the polymer electrolyte composition. According to the differential thermal analysis (shown in dotted lines), the weight loss occurs for all the samples above 190 °C, the major contributor being tetraglyme41. Higher amounts of LiTFSI reduces the ther- mal stability of the polymer matrix, which is clearly visible in the differential curves (shift of the peak from 218 to 195 °C). This could be the general trend observed for tetraglyme kind of plasticizers that show the tendency to decompose in reaction with fluorinated anions22. Thus, both DSC and TGA profiles show that no unusual phase changes or weight losses occur in the temperature range between 25 and 150 °C, which makes the material ther- mally stable and useful as a polymer electrolyte under standard operating conditions in real battery configuration. D i h i l h l l i (DMTA) f d l PTL 1 d h l i fil y p y y p g yi g Dynamic mechanical thermal analysis (DMTA) was performed on sample PTL-1 and the resulting profiles (both modulus and loss modulus) are shown in Figure S1(A,B). It clearly indicates that the material has low Tg, which is in agreement with the DSC analysis. In addition, the calculated cross-linking density (7.7 × 103 mol m–3, calculated at 0 °C) was found to be comparatively lower than other known systems43. This is an indication that the number of cross-linking points between the PEO chains is lower than expected, which allows the long enough –EO– chains to rearrange and crystallize in the matrix. Tensile tests were carried out on the PTL-1 sample accord- ing to ASTM Standard D638; the Young’s modulus was found to be 0.3 MPa and maximum force at break was found to be 1.5 MPa. The material can stretch (Figure S1C) very well under stress, as justified by the maximum strain (elongation) of around 17 mm before the membrane was broken. Results and Discussions Thus, the gel content achieved for our samples is lower than the values obtained by Kim et al.36, who used PEO and room tem- perature ionic liquids (RTILs). RTILs may not take part in the chemical reaction, thus a higher insoluble content was obtained when they calculated the insoluble fraction with respect to the PEO content. Here, we propose the use of tetraglyme instead of RTIL, due to the presence of accessible protons in tetraglyme that can take part in the dehydrogenation reaction under UV irradiation in the presence of light sensitive ketyl species. FTIR studies (Figure S3 in ESI) were performed on PTL-1 sample with and without UV curing to unravel any drastic difference induced by the irradiation. The FTIR spectra are identical and no noticeable changes are observed, which confirms that the soft cross-linking strategy does not induce any drastic modification in the polymeric components as well as with respect to the TFSI− anion. Moreover, for comparison purposes, FTIR analysis of liquid TEGDME added with 10 wt. % of LiTFSI was performed and compared to the spectra of PTL-1. Clearly, the spectra of liquid electrolyte and PTL-1 are very different. In general, in an electrolyte system three kinds of ions can be present: free ions, free ions co-existing with ion pairs and aggregates44. PTL-1 analysed after UV-curing was almost absent with the peaks corresponding to aggregates (1236 and 1143 cm‒1) if compared to the LiTFSI/TEGDME system that contains the same wt. % of lithium salt. As a result, we assume that the electro- lyte gets enriched with free ions and neutral ion pairs, which can move faster in the polymer matrix. Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 5 www.nature.com/scientificreports/ Figure 4. (A) Arrhenius plot showing the ionic conductivity vs. temperature for ISPEs prepared with various LiTFSI content. (B–D) VTF fitting of the samples PTL-1 to PTL-3. Figure 4. (A) Arrhenius plot showing the ionic conductivity vs. temperature for ISPEs prepared with variou LiTFSI content. (B–D) VTF fitting of the samples PTL-1 to PTL-3. Electrochemical characterization. Electrochemical impedance spectroscopy (EIS) analysis was carried out between 0 and 85 °C. The Arrhenius plots for all samples are shown in Fig. 4A. The plots of PTL-1 to PTL-3 demonstrate the influence of the lithium salt when PEO to tetraglyme ratio is 1:1. Results and Discussions It is widely accepted that ionic conductivities exceeding 0.1 mS cm−1 at room temperature are necessary for an electrolyte to function in real battery configuration. Nevertheless, the crystalline domains of PEO-based polymers restrict the ionic mobility. Recently, Khurana et al.45 reported a cross-linked –EO– based polymer electrolyte showing an ionic conductivity higher than 0.1 mS cm−1 at 25 °C. In our work, we are able to achieve even improved ionic conductivities (0.40 mS cm−1 for PTL-3) for interlinked PEO-based solid polymer electrolytes. The conductivity values increase with an increase in the salt concentration, then reaching the maximum for PTL-3 where the − EO− to Li ratio is 23:1. However, the difference is not huge; indeed, all membranes demonstrate conductivity values ≥ 0.1 mS cm−1 at 25 °C. We decided not to increase the salt concentration further as for the ion pairing nature of TFSI-based salts, which strongly influences the ionic mobility, probably due to the saturation of the hopping sites46. It is worth mentioning that the sample retains good elastic and mechanical integrity under stress. After several days of con- ductivity tests, which were carried out under 10 N pressure, it was observed that the membrane retains its size and shape without any noticeable damages around the edges. Indeed, the thickness variation after the test was < 2%. This is an encouraging result as ISPEs are super soft, low Tg and highly plasticized. Further, it confirms that no leakage of tetraglyme from the polymer matrix occurs. g g y p y Generally, for polymer electrolytes the dependence of ionic conductivity upon the temperature is not straight- forward. The overall plot that starts from 0 to 85 °C does not exhibit a linear behaviour. Between 0 to 30 °C, the conductivity increases with Vogel–Tamman–Fulcher (VTF) dependence for all ISPEs. The same behaviour (see Fig. 4B–D) is observed above the melting point between 35 and 85 °C. The deflection around 30 °C reflects the phase transitions occurring due to the melting of crystalline regions or rearrangement of –EO– moieties, which is in agreement with the behaviour observed by DSC analysis (see Table 2).h g y y The mechanism of ionic conductivity in polymer electrolytes can be understood from activation energy (Ea) calculations. Ea was calculated by fitting the conductivity values with VTF equation22. The corresponding plots were used to determine the Ea of the electrolyte system. Results and Discussions If one considers that these numbers are obtained at 25 °C for a truly solid polymer electrolyte, the results are surprisingly encouraging.hffii y p y y , p g y g g The Li+ ion diffusion coefficient (DLi+) can fit very well with the previously measured ionic conductivity and transference number. DLi+ (see Table 2) was estimated using the method reported by Ma et al.56. Typical responses are noted as natural logarithm of potential (V) versus time (t) at 25 °C. The results are in good agreement with the corresponding tLi+ values. Moreover, PTL-1 shows the highest value (5.6 × 10−6 cm2 s−1), which is at least one order of magnitude higher than the literature reports for similar systems57–59. The presence of free TFSI− ions and neutral ion pairs, which can move faster due to the reduced solvent salt interactions, thereby increases the dis- order in the polymer matrix44. This result is in agreement with the conductivity, transport number and diffusion coefficient studies as well as supported by FTIR studies. fi pp y A deep understanding of the interfacial properties between the lithium metal electrode and the polymer electrolyte is necessary in order to provide more insight over the factors controlling the recharge ability of lithium-based polymer batteries. The PTL-1 sample was examined in terms of compatibility (interfacial stability) with the lithium metal electrode. Sample PTL-1 was selected for further characterizations due to the optimal characteristics exhibited during the previously discussed analyses. As shown in Fig. 5a, the PTL-1 based lithium symmetric cell shows stable resistance after few days of testing. Indeed, the resistance increases during the initial days of storage indicating an appropriate formation of a thin solid electrolyte interface (SEI) layer at the surface of the lithium metal electrode60. The resistance rapidly decreases and stabilizes at around 700 Ω cm−2 after about 6 days, accounting for the very stable interfacial characteristics of the sample. This behaviour is typical of most of the polymer electrolytes, and is clearly related to the initial formation of the SEI layer, its stabilisation and, then, the improved contact achieved with time at the interface between the lithium metal electrode and the polymer electrolyte61,62. y Electrochemical stability window (ESW) is a fundamental parameter that determines the durability and energy output of a lithium cell. Figure 5b shows the electrochemical stability of PTL-1 sample towards anodic oxidation and cathodic reduction reactions. Results and Discussions Noteworthy, Ea increases with an increase in the salt concentration, which is ascribed to the formation of ion pairs and increased viscosity of the polymer matrix. Thus, the high ambient temperature conductivity values of the PTL series of electrolytes are predominantly associated with high ionic mobility.h may be related to thermal transitions, which may include chain rearrangement, dielectric relaxations or melting of crystalline domains, formed by the PEO chains between two cross-linking points. Noteworthy, Ea increases with an increase in the salt concentration, which is ascribed to the formation of ion pairs and increased viscosity of the polymer matrix. Thus, the high ambient temperature conductivity values of the PTL series of electrolytes are predominantly associated with high ionic mobility.h p y g y The Li+ ion transference number (tLi+)49 was calculated using the method reported by Abraham et al.22 who have also considered the resistance changes occurring due to side reactions. An optimum tLi+ is necessary for the functioning of a polymer electrolyte in a Li-ion cell. Low tLi+ may induce the build-up of anion concentration gradients, which may lead to salt decomposition and precipitation. Low tLi+ may also induce dendrites growth in Li-metal cells, which is one of the major obstacles restricting the widespread intrusion of such batteries into the market 50–53. In the present system, sample PTL-1 (Figure S4) shows the highest transference number (0.55 ± 0.06, Table 2) at 25 °C. It is worth noting that at higher salt concentrations, tLi+ reduces to smaller values, which may be ascribed to the formation of anion pairs or aggregates. Overall, the transport number in the PTL series of samples is comparatively higher than the classical literature data on polymer electrolytes, but it is close to the data obtained for systems that contain tetraglyme as co-solvent54–56. The reasons for such a high number is the absence of ion pairs, or the presence of more free ions and neutral ion pairs. Moreover, the oligomerisation of tetraglyme moi- eties weakens the coordination between Li+ ions and O atom of TEGDME. This facilitates the movement of Li+ ions inside the polymer matrix leading to improved transference number values. It was previously demonstrated by Kriz et al.55 that tetraglyme can loosen the coordination of Li+ ions with –EO– units of PEO chains, resulting in improved ion mobility, and might also enable Li+ ions to decouple from ion pairs. Results and Discussions The VTF equation is believed to describe the conduction behaviour of highly concentrated liquid electrolytes and molten salts47. As listed in Table 2, Ea varies from 4.3 to 7.9 kJ mol‒1, when the data are fitted with VTF equation below the deflection point, in the Arrhenius plot of Figure 4A. When the curves are fitted above the deflection point (35–85 °C, Ea’), values ranging from 3.4–4.5 kJ mol‒1 are obtained. The values obtained are superior to the data reported in the recent literature48. Above 0 °C, the ionic conductivity increases with a VTF-like dependence for all the samples. The discontinuities around 30 °C Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 6 www.nature.com/scientificreports/ Name Ea/Ea′ # tLi + DLi+ $ σ* PTL-1 4.3/3.4 0.55 ± 0.06 5.6 × 10‒6 0.11 PTL-2 6.6/3.9 0.48 ± 0.02 1.2 × 10‒7 0.24 PTL-3 7.9/4.5 0.32 ± 0.08 2.1 × 10‒8 0.40 Table 2. Ionic conductivity (σ, at 25 °C) and related characteristics of ISPEs prepared with different salt content. Ea is the activation energy before deflection (0 to 30 °C). Ea′ is the activation energy after deflection (35 to 85 °C). tLi+ is the lithium transference number; DLi+ is the lithium diffusion coefficient. mS cm−1. #kJ mol−1. $cm2 s−1. Name Ea/Ea′ # tLi + DLi+ $ σ PTL-1 4.3/3.4 0.55 ± 0.06 5.6 × 10‒6 0.11 PTL-2 6.6/3.9 0.48 ± 0.02 1.2 × 10‒7 0.24 PTL-3 7.9/4.5 0.32 ± 0.08 2.1 × 10‒8 0.40 Table 2. Ionic conductivity (σ, at 25 °C) and related characteristics of ISPEs prepared with different salt content. Ea is the activation energy before deflection (0 to 30 °C). Ea′ is the activation energy after deflection (35 to 85 °C). tLi+ is the lithium transference number; DLi+ is the lithium diffusion coefficient. mS cm−1. #kJ mol−1. $cm2 s−1. Table 2. Ionic conductivity (σ, at 25 °C) and related characteristics of ISPEs prepared with different salt content. Ea is the activation energy before deflection (0 to 30 °C). Ea′ is the activation energy after deflection (35 to 85 °C). tLi+ is the lithium transference number; DLi+ is the lithium diffusion coefficient. mS cm−1. #kJ mol−1. $cm2 s−1. may be related to thermal transitions, which may include chain rearrangement, dielectric relaxations or melting of crystalline domains, formed by the PEO chains between two cross-linking points. Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. (a) 3D Nyquist plot representing the evolution of the interfacial resistance with time for sample PTL- 1, using the Li/PTL-1/Li cell configuration. (b) Electrochemical stability window (anodic and cathodic scan) of PTL-1. The tests were performed at 25 °C. Figure 5. (a) 3D Nyquist plot representing the evolution of the interfacial resistance with time for sample PTL- 1, using the Li/PTL-1/Li cell configuration. (b) Electrochemical stability window (anodic and cathodic scan) of PTL-1. The tests were performed at 25 °C. our lithium metal polymer batteries. Such a test is of utmost importance when very long-term ageing of lithium metal polymer cells is envisaged50. Measurements were performed at 0.1 and 0.3 mA cm−2 current densities at 25 °C (3 h Li-plating and 3 h Li-stripping). When the current density is increased from 0.1–0.3 mA cm−2, a large change in potential is observed due to the formation of a dendritic short circuit 50. The cycling results of PTL-1 at 0.1 and 0.3 mA cm−2 current density are shown in Figure S5. Indeed, rather than the total charge deposited within the time, the current rate of 0.3 mA cm−2 induced an over potential as demonstrated in Figure S5. Thus, ISPE can be safely used at 0.1 mA cm−2, which is assumed as a remarkable value for a solid electrolyte system operating at ambient temperature. Prolonged galvanostatic cycling tests were performed (Fig. 6) at 0.1 and 0.2 mA cm−2 with plating and/or stripping steps lasting for 30 min. This test assures the durability and safe operation of the ISPEs in lithium metal cells conceived for ambient temperature applications. The total charge carried during the plating / stripping process is not very high, however, one can hypothesise that this is a good indication towards pursuing this path for future studies. p To demonstrate its practical application, PTL-1 was assembled in a lab-scale all-solid-state Li-polymer cell, and galvanostatically cycled at ambient temperature. The cell was assembled by combining a Li-metal anode with an electrode/electrolyte composite prepared by in situ UV cross-linking the ISPE directly over the TiO2-based working electrode (details in experimental). One of the major drawbacks of typical truly solid polymer electro- lytes is the insufficient contact between the electrode active materials and the polymer matrix. Thus, the direct cross-linking step over the electrode surface is fundamental to obtain a good electrode/electrolyte interfacial adhesion. Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 Results and Discussions The test was performed at 25 °C. From the cathodic profile, the almost ideally reversible lithium plating and stripping processes are well evidenced. Overall, a wide electrochem- ical window is accessible for the electrolyte to be safely used between 0 to above 5.2 V vs. Li/Li+. Such a high anodic stability window can be explained by the simultaneous oxidative decomposition of both the TFSI− anions, tetraglyme moieties and PEO matrix in the high potential regions58. In particular, the presence of tetraglyme in the electrolyte matrix increases the overall oxidation stability54. Even though the CH2‒CH2‒O‒ chemical moiety is same for both PEO and tetraglyme, the difference may arise from the –CH3 end group of tetraglyme, which avoids the interaction between electrode surface and the –OH terminal groups of PEO. However, the oxidation stability is anyway superior than the pure PEO-based system, and this is an intriguing aspect of this electrolyte. This value is excellent when one envisages the application with high voltage cathode materials. g g g Many researchers developed polymer electrolytes that exhibited ionic conductivity values as high as 0.1 mS cm−1 at 25 °C57,63, but very few investigated the lithium dendrite nucleation and growth resistance in real cell configuration15. Inspired by the dendrite studies reported by Balsara et al.64 and Khurana et al.45 we performed galvanostatic lithium plating/stripping measurements in symmetric Li/PTL-1/Li cells to determine the lifetime of Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 7 www.nature.com/scientificreports/ This is a convincing indica- tion of the good interfacial contact between the electrodes and the electrolyte separator. Noteworthy, the cycling response of the all-solid polymer cell is clearly at the level of the corresponding TiO2/Li cell assembled with a liq- uid electrolyte made of 1M LiTFSI solution in tetraglyme (TEGDME) (Figure S6), which accounts for the almost full capacity delivery when using the all-solid-state configuration.h discharge cycles. These are typical of the biphasic Li+ extraction/insertion mechanism of crystalline TiO2 anatase, with a steep potential increase/decay at its end. The polarization is rather limited, which accounts for an efficient redox reaction kinetics, due to the limited internal resistance at the electrode/electrolyte interface as well as the limited cell over potential contributions. In general, the material shows good cycling stability, as for the good overlapping of the charge/discharge curves, accounting for a Coulombic efficiency close to 100%. Although the specific capacity obtained is lower if compared to the same anatase electrode material at same current density in liquid electrolyte, the polymer cell shows good capacity retention approaching 90%. This is a convincing indica- tion of the good interfacial contact between the electrodes and the electrolyte separator. Noteworthy, the cycling response of the all-solid polymer cell is clearly at the level of the corresponding TiO2/Li cell assembled with a liq- uid electrolyte made of 1M LiTFSI solution in tetraglyme (TEGDME) (Figure S6), which accounts for the almost full capacity delivery when using the all-solid-state configuration.h p y y gi g The PTL-1 also demonstrated the ability to be galvanostatically cycled at 0 and 25 °C in lab-scale Li cell com- prising a LiFePO4 cathode. Proof-of-concept charge/discharge profiles are shown in Figure S7, which clearly enlighten the possibility of designing an all-solid polymer battery system that functions at low temperature even with various electrode materials. The approach can be extended to other energy-related device applications like Na-ion batteries, dye-sensitized solar cells65 and supercapacitors, owing to its simple, scalable, economic and eco-friendly preparation method, and a great potential to serve as a light-designed cell component. www.nature.com/scientificreports/ The process enables us to obtain a stable and thin (≈ 30 μ m) polymer electrolyte film with uniform distribution over the electrode. The general aspects of the bare electrode, electrolyte and the final aspect of the TiO2-based electrode film are shown in Fig. 7 (a,b). The cross-sectional FESEM images (Fig. 7c) show an intimate contact achieved between the active materials and the polymer electrolyte. Particularly at higher magnifications, it can be clearly observed that the electrolyte layer creates a conformal coating by following the contours of the electrode particles. This leads to improved active materials utilisation at the interface between the electrode and the polymer electrolyte, which correspondingly improves the specific energy and power of the cell. The oriented cross-linked polymer electrolyte morphology is observable on top of the electrode (Fig. 7c) along with the opti- mum interface and interpenetration between the electrode active material particles and the electrolyte.hi y The response of the cell at 0.1 mA cm−2 is shown in Fig. 8 in terms of galvanostatic charge/discharge profiles and specific capacity vs. cycle number. The cell was prepared by just contacting a lithium metal foil at the pol- ymer side of the electrode/electrolyte composite. The constant current charge/discharge profiles shown in plot (A) reflect the good properties of the electrolyte system, showing rather flat potential plateaus during charge and Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 8 www.nature.com/scientificreports/ Figure 6. Potential vs. test time of lithium stripping and plating of a symmetrical lithium cell at various current rates (i.e., 0.05, 0.1, 0.2 mA cm‒2) at 20 °C. Figure 6. Potential vs. test time of lithium stripping and plating of a symmetrical lithium cell at various current rates (i.e., 0.05, 0.1, 0.2 mA cm‒2) at 20 °C. discharge cycles. These are typical of the biphasic Li+ extraction/insertion mechanism of crystalline TiO2 anatase, with a steep potential increase/decay at its end. The polarization is rather limited, which accounts for an efficient redox reaction kinetics, due to the limited internal resistance at the electrode/electrolyte interface as well as the limited cell over potential contributions. In general, the material shows good cycling stability, as for the good overlapping of the charge/discharge curves, accounting for a Coulombic efficiency close to 100%. Although the specific capacity obtained is lower if compared to the same anatase electrode material at same current density in liquid electrolyte, the polymer cell shows good capacity retention approaching 90%. Conclusionsht The super soft polymer electrolyte network was architectured from a thermoplastic polymer matrix of known molecular weight using the rapid and cost-effective in situ photopolymerisation technique. A multidisciplinary approach was adopted to understand the role of photopolymerisation in tailor making the integral and requisite properties of the resulting polymer electrolyte to achieve acceptable conductivity, ionic mobility and resilience towards dendrite-induced short circuit reactions. Significantly, the feasibility of using such novel electrolyte in real cell configuration at ambient temperature with various nanostructured electrodes was established by suit- ably adopting in situ polymerization directly over the electrode films. The obstacles related to hazardous den- drites and reactivity towards Li-metal were nullified, leading to the assembly of superior Li-ion and Li-metal cells conceived for applications that demand high energy and/or power, including smart-grid storage and electric-/ hybrid-electric vehicles. We anticipate that the proposed approach would lead to a rational designing to address the significant challenges of Li-ion polymer batteries. Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 9 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 7. (a) Photograph of bare TiO2 based electrode and electrolyte before UV curing. (b) Freshly prepared self-supporting multiphase electrode/electrolyte composite obtained by direct hot-pressing and in situ photopolymerisation of the polymer electrolyte over the TiO2-electrode film supported over copper foil. (c) Cross-sectional FESEM images showing the optimum interface achieved after UV curing. Figure 7. (a) Photograph of bare TiO2 based electrode and electrolyte before UV curing. (b) Freshly prepared self-supporting multiphase electrode/electrolyte composite obtained by direct hot-pressing and in situ photopolymerisation of the polymer electrolyte over the TiO2-electrode film supported over copper foil. (c) Cross-sectional FESEM images showing the optimum interface achieved after UV curing. Experimental Section Materials and Methods. p Materials and Methods. The reactive formulations were based on poly(ethylene oxide) (PEO, average Mw 100,000, Sigma-Aldrich), and an active plasticiser bis[2-(2-methoxyethoxy) ethyl]ether (tetraglyme, TEGDME, Sigma-Aldrich) along with bis(trifluoromethane) sulfonimide lithium salt (CF3SO2NLi-SO2CF3, LiTFSI, battery grade, Solvionic) as Li+ ions source. The photo-induced hydrogen abstraction facilitator was 4-methylbenzophe- none (MBP, Sigma-Aldrich).h g The preparation of interlinked polymer electrolyte network did not involve any solvents or long/tiring steps. The materials were grinded in appropriate proportions at 70 °C. After blending, the resulting mixture was hot-pressed at 90 °C for 15 min to obtain a homogeneous thin film. The film was later exposed to UV light for 6 min to reticulate the reactive species, thus obtaining the cross-linked ISPE of average thickness 90 ± 10 μ m. The solid and non-tacky film was peeled off from the substrate (Mylar sheet) and used for further characterizations. The procedure was carried out in a dry room (10 m2, R.H. < 2% ± 1 at 20 °C) produced by Soimar (Caluso, Italy). As the prepared membrane contains all the necessary components for a functional electrolyte, no additional steps were required for further characterization. Characterization of the interlinked solid polymer electrolytes. The insoluble fraction (gel content) was determined as follows: samples of known weight were kept in a stainless steel metal net, and subsequently extracted with acetonitrile (CH3CN, ACN, Sigma-Aldrich) to dissolve the non-cross-linked polymer chain com- ponents66. Extraction included 18 h of residence time for the solvents to remove soluble contents from the mem- brane at 25 °C. Instead of using CHCl3, ACN was used due to the solubility of the components used for membrane preparation. Moreover, CHCl3 is suspected for carcinogenic activity. The cross-linked (insoluble) fraction was then calculated by dividing the mass of the dry sample left over after the extraction by the mass of the original sample (relative error = ± 1%).hi The overall morphology was investigated by field emission scanning electron microscopy (FESEM) on a ZEISS Supra 40, equipped with an energy dispersive X-ray spectrometer (EDX). For cross-sectional morphology char- acterization, the samples were cracked after dipping in liquid nitrogen to avoid any change in the morphology. Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 10 www.nature.com/scientificreports/ Figure 8. (A) Representative charge/discharge profiles of a cell assembled with the configuration of Li/PTL-1/TiO2. The cycling test was performed at 20 °C at a current density of 0.1 mA cm−2. Experimental Section Materials and Methods. (B) Graph illustrating the specific capacity vs. number of cycles along with Coulombic efficiency. Figure 8. (A) Representative charge/discharge profiles of a cell assembled with the configuration of Li/PTL-1/TiO2. The cycling test was performed at 20 °C at a current density of 0.1 mA cm−2. (B) Graph illustrating the specific capacity vs. number of cycles along with Coulombic efficiency. Then, the samples were subjected to metallization by sputtering a very thin Cr layer (~10 nm) to minimize the effect of the electron beam irradiation.hf f The glass transition temperature (Tg) of the samples was evaluated by differential scanning calorimetry (DSC) with a METTLER DSC-30 (Greifensee, Switzerland) instrument. In a typical measurement, the electrolyte sam- ples were cooled from ambient temperature to –85 °C and then heated at 5 °C min−1 up to 120 °C. The Tg was calculated as the midpoint of the heat capacity change observed in the DSC trace during the transition from glassy to rubbery state. The thermal stability was tested by thermo-gravimetric analysis (TGA) using a TGA/SDTA-851 instrument from METTLER (Switzerland) over the temperature range between 25 and 600 °C under N2 flux at a heating rate of 10 °C min−1.h g The mechanical properties were determined by dynamic-mechanical thermal analysis (DMTA) on a MK III Rheometrics Scientific Instrument at 1.0 Hz frequency on tensile configuration at a heating rate of 5 °C min−1. The specimen size was 20 mm × 4 mm × 0.2 mm. The storage modulus and the loss factor were measured from ‒80 to 30 °C. The Fourier Transform Infrared (FT-IR, NICOLET-5700) spectroscopy (400–4000 cm−1, resolution 2 cm−1) was carried out at 25 °C, on extremely thin films sandwiched between gold plates with an orifice. Tensile tests were carried out according to ASTM Standard D638, using a Sintech 10/D instrument equipped with an electromechanical extensometer (clip gauge).f p g g Gel permeation chromatography (GPC) was performed to examine the effect of UV curing using an Agilent Technologies 1200 Series (USA) instrument. The instrument was equipped with a refractive index (RI) detector and two Waters Styragel columns (HT2 and HT4) conditioned at 35 °C. A sample of cross-linked polymer was weighted, transferred into a Whatman glass microfiber thimble and extracted with THF for 24 hours using a Soxhlet apparatus. After extraction, a small quantity of the soluble fraction was transferred to a vial and left open to evaporate THF. Experimental Section Materials and Methods. The obtained solid was then diluted with chromatography-grade THF and used for GPC measurement. Electrochemical characterization methods. The following procedures were performed in the inert atmosphere of a dry glove box (MBraun Labstar, O2 and H2O content < 1 ppm) filled with extra pure Ar 6.0. The ionic conductivity was determined by electrochemical impedance spectroscopy (EIS) analysis of cells assembled by sandwiching the ISPE between two stainless steel (SS-316) blocking electrodes (area 2.54 cm2). A PARSTAT-2273 potentiostat/galvanostat/F.R.A. (Frequency Response Analyzer) by Princeton Applied Research (USA) was used for measurements at various temperatures between 100 kHz and 1 Hz at the open circuit poten- tial (OCV). The bulk resistance of the samples was calculated from the impedance curve. Then, the ionic conduc- tivity was calculated based on the following equation: Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 11 www.nature.com/scientificreports/ σ = ⋅ l R A b σ = ⋅ R A b ( ) 1 ⋅ R A b where σ is the ionic conductivity (S cm−1), Rb the bulk resistance, l and A are the thickness and the area of the specimen, respectively. The tests were performed using a climatic chamber (UFE-400 Memmert GmbH, Germany). The bulk resistance was given by the high frequency intercept determined by analysing the impedance response using a fitting program provided by Electrochemistry Power Suite software (V 2.58). Samples were kept at 80 °C overnight and then tested from 0 to 85 °C at every 10 °C. Measurements were repeated at least three times. ISPEs were tested for compatibility (interfacial stability) with the Li-metal electrode by monitoring the evo- lution of the impedance response of a symmetrical non-blocking Li/ISPE/Li cell with time at 25 °C under OCV condition.h The Li-ion transference number ( + tLi ) was measured18,39,54 at 25 °C by combined AC impedance and DC polar- ization measurements of a Li/ISPE/Li cell, as explained by Watanabe67 and Bruce49. The cell was kept at 80 °C overnight to achieve an intimate contact, and a stable interface between the electrolyte and the electrodes. Successively, after retaining at 25 °C, a DC potential (Δ V = 10 mV) was applied until a steady current was obtained (generally 3–5 hours), and the initial (I0) and steady (Iss) currents that flow through the cell were meas- ured. Experimental Section Materials and Methods. The equation used is given below:, σ σ = − ⋅ − ( ) E R T T ln ln 1 3 a VFT 0 0 ( ) 3 where σ is the ionic conductivity, Ea VFT is equivalent to the activation energy, R is the gas constant, T is the exper- imental temperature and T0 is the temperature which is 50 °C below the Tg.hffi where σ is the ionic conductivity, Ea VFT is equivalent to the activation energy, R is the gas constant, T is the exper- imental temperature and T0 is the temperature which is 50 °C below the Tg.hffi 56 The salt diffusion coefficient of the ISPEs was estimated using the method proposed by Ma et al.56. In this case, the cells are polarized at 5 mV before the potential is interrupted. Once the potential is interrupted, the cell is kept at OCV until a stable state is achieved. Later, the profiles are plotted as the natural logarithm of potential (V) versus time (t). The DLi+ values were calculated from the slope of the linear curves using the following equation: π = − ⋅ ( ) + Slope D L 4 Li 2 2 π = − ⋅ + Slope D L Li 2 2 ( ) 4 where L is the thickness of the ISPE under study. Galvanostatic charge/discharge cycling tests were performed on Li metal cells of area 2.54 cm2 using both TiO2 (potential scan range 1–3 V vs. Li/Li+) and LiFePO4 (potential scan range 2.5–4 V vs. Li/Li+) based working elec- trodes with an ARBIN BT2000 battery tester. The working electrodes were prepared from a slurry that contains TiO2 (Hombicat-100) or LiFePO4 (Clariant-LP2), carbon black and PVdF in 70:20:10 weight ratio, respectively. The slurry was deposited over Cu (or Al) foil and later dried overnight (120 °C). The typical preparation for the electrode-electrolyte composites followed the procedure reported elsewhere69. In a typical preparation proce- dure, appropriate amounts of PEO, tetraglyme, LiTFSI and MBP were mixed at 70 °C and mechanically grinded to obtain a viscous paste-like mixture. This mixture was later deposited over a composite electrode film, and hot pressed (20 bar, 90 °C) for 15 min to obtain a uniform coating over the electrode surface. This setup was exposed to UV light for 6 min to obtain a cross-linked polymer electrolyte system. Experimental Section Materials and Methods. Simultaneously, impedance spectra were recorded (100 KHz and 0.1 Hz), with an oscillating potential of 10 mV, before and after DC polarization. Subsequently, the initial (R0) and final (Rss) bulk resistances of the elec- trolyte, and the initial (RC0) and final (RCss) charge transfer resistances (Ω) of the interfacial layers of the Li metal electrode/electrolyte were derived. Using these measured values, + tLi was calculated by following equation: = ⋅ ⋅(∆ − ⋅ ) ⋅ (∆ − ⋅ ) ( ) + t I R V I RC I R V I RC 2 Li ss ss ss ss o o o o ( ) 2 The electrochemical stability window (ESW) of the ISPEs was evaluated at 25 °C by linear sweep voltamme- try (LSV) in two electrodes cells using a CHI-660 electrochemical workstation. Separate LSV tests were carried out on each of the samples to determine the cathodic and anodic breakdown voltages. Cell configuration for anodic scan (OCV to 6 V vs. Li/Li+): SS-316 as the working electrode, Li-metal discs as both the counter and the reference electrodes, and ISPE as the electrolyte (area 2.54 cm2)68. Cell configuration for cathodic scan (OCV to − 0.2 V vs. Li/Li+): Cu disc as the working electrode, Li metal discs as the counter and the reference electrodes, ISPE as the electrolyte at a scan rate of 0.100 mV s−1. The current onset of the cell was associated with the decom- position potential of the electrolyte.h p p y The activation energy was calculated from conductivity values obtained at various temperature and the result- ing values are fitted with Vogel–Tamman–Fulcher (VTF) equation, which is typically used to describe the relation between viscosity and temperature near the Tg of the polymer matrix. Experimental Section Materials and Methods. Then, electrolyte/electrode disks (area 2.54 cm2) were cut from the sheet and dried under vacuum overnight at 40 °C prior to cell assembly. The electrode films used in this study were pressed prior to pre-polymer deposistion, which can retain the some pores/voids for the electrolyte components to accommodate while overall processing. The electrodes we used in the present case are not pressed/calendared. 1. Armand, M. & Tarascon, J. M. Building better batteries. Nature 451, 652–657 (2008). 2. Barghamadi, M. et al. Lithium-sulfur batteries-the solution is in the electrolyte, but is the electrolyte a solution? Energy Environ. Sci. 7, 3902–3920 (2014). , , J g , ( ) 2. Barghamadi, M. et al. Lithium-sulfur batteries-the solution is in the electrolyte, but is the electrolyte a solution? Energy Environ. Sci 7, 3902–3920 (2014). References 1. Armand, M. & Tarascon, J. M. Building better batteries. Nature 451, 652–657 (2008). g , Nano Lett. 15, 2671–2678 (2015). 16. Zhou, D. et al. In Situ Synthesis of a Hierarchical All-Solid-State Electrolyte Based on Nitrile Materials for High-Performance Lithium-Ion Batteries. Adv. Energy Mater. DOI: 10.1002/aenm.201500353 (2015). gy 17. Wang, Y. et al. A Particle-Controlled, High-Performance, Gum-Like Electrolyte for Safe and Flexible Energy Storage Devices Adv. Energy Mater. 5, Art. No. 201400463 (2015). g g Energy Mater. 5, Art. No. 201400463 (2015). gy 18. MacFarlane, D. R. et al. Energy applications of ionic liquids. Energy Environ. Sci. 7, 232–250 (2014). 18. MacFarlane, D. R. et al. Energy applications 19. Suleman, M. et al. Structural and electrochemical properties of succinonitrile-based gel polymer electrolytes: role of ionic liquid addition. J. Phys. Chem. B 117, 7436–7643 (2013). y 0. Nair, J. R. et al. Methacrylic-based solid polymer electrolyte membranes for lithium-based batteries by a rapid UV-curing process React. Funct. Polym. 71, 409–416 (2011). y , ( ) 21. Fan, L. Z. et al. Succinonitrile as a Versatile Additive for Polymer Electrolytes. Adv. Funct. Mater. 17, 2800–2807 (2007). y y 22. Abraham, K. M. et al. Highly Conductive PEO-like Polymer Electrolytes. Chem. Mater. 9, 1978–1988 (1997) 23. Tang, S. et al. Glymes as versatile solvents for chemical reactions and processes: from the laboratory to industry. RSC Adv. 4, 11251–11287 (2014). 4. Zhang, C. et al. Ionic Conductivity in the Solid Glyme Complexes [CH3O(CH2CH2O)nCH3]:LiAsF6 (n = 3,4). J. Am. Chem. Soc 129, 8700–8701 (2007). 25. Zhang, C. et al. Structure and Conductivity of Small-Molecule Electrolytes [CH3O(CH2CH2O)nCH3]:LiAsF6 (n = 8–12). Chem. Mater. 20, 4039–4044 (2008). 26. Park, M. S. et al. A highly reversible lithium metal anode. Sci. Rep. 4, 3815–3822 (2014). d l f l l d h h h h ( ) 26. Park, M. S. et al. A highly reversible lithium metal anode. Sci. Rep. 4, 3815–3822 (2014). 27 Mandai T et al Criteria for solvate ionic liquids Phys Chem Chem Phys 16 8761–8772 (2014) k, g y R p , ( ) 7. Mandai, T. et al. Criteria for solvate ionic liquids. Phys. Chem. Chem. Phys. 16, 8761–8772 (2014). g y p 27. Mandai, T. et al. Criteria for solvate ionic liquids. Phys. Chem. Chem. Phys. 16, 8761–8772 (2014). T. et al. Criteria for solvate ionic liquids. Phys. Chem. Chem. Phys. 1 8. Ueno, K. et al. Glyme-Lithium Salt Equimolar Molten Mixtures: Concentrated Solutions or Solvate Ionic Liquids? J. Phys. Chem. B 116, 11323–11331 (2012). 29. Shao, J. et al. g , Nano Lett. 15, 2671–2678 (2015). et al. A tetraethylene glycol dimethylether - lithium 42. Xue, Z. et al. Poly(ethylene oxide)-based electrolytes for lithium-ion batteries. J. Mater. Chem. A 3, 19218–19253 (2015). 42. Xue, Z. et al. Poly(ethylene oxide)-based electrolytes for lit y y y 43. Hill, L. W. Calculation of crosslink density in short chain networks. Prog. Org. Coat. 31, 235–243 (1997). y g g 44. Wen, S. J. et al. FTIR characterization of PEO + LiN(CF3SO2)2 electrolytes. J. Electroanal. Chem. 408, 113–117 (1996). 44. Wen, S. J. et al. FTIR characterization of PEO + LiN(CF3SO2)2 3 2 2 y 45. Khurana, R. et al. Suppression of Lithium Dendrite Growth Using Cross-Linked Polyethylene/Poly(ethylene oxide) Elec N A h f P i l Li hi M l P l B i J A Ch S 136 7395 7402 (2014) y 45. Khurana, R. et al. Suppression of Lithium Dendrite Growth Using Cross-Linked Polyethylene/Poly(ethyle New Approach for Practical Lithium-Metal Polymer Batteries. J. Am. Chem. Soc. 136, 7395–7402 (2014)l hurana, R. et al. Suppression of Lithium Dendrite Growth Using Cross-Linked Polyethylene/Poly(ethylene oxide) Electrolytes: A ew Approach for Practical Lithium-Metal Polymer Batteries. J. Am. Chem. Soc. 136, 7395–7402 (2014)l 45. Khurana, R. et al. Suppression of Lithium Dendrite Growth Using Cross Linked Polyethylene/Poly(ethylene oxide) Elect New Approach for Practical Lithium-Metal Polymer Batteries. J. Am. Chem. Soc. 136, 7395–7402 (2014)l 46. Lahiri, A. et al. LiTFSI in 1-butyl-1-methylpyrrolidinium bis(fluorosulfonyl)amide: a possible electrolyte for ionic liquid based lithium ion batteries. Phys. Chem. Chem. Phys. 17, 11161–11164 (2015).h 46. Lahiri, A. et al. LiTFSI in 1-butyl-1-methylpyrrolidinium bis(fluorosulfonyl)amide: a possib lithium ion batteries. Phys. Chem. Chem. Phys. 17, 11161–11164 (2015).h y y 47. Smedley, S. I. et al. The Interpretation of Conductivity in Liquids, Plenum Press, New York, 82 (1980). 48. Barteau, K. P. et al. Allyl Glycidyl Ether-Based Polymer Electrolytes for Room Temperature Lithium Batteries. Macromolecul 8988–8994 (2013) 49. Evans, J. et al. Electrochemical measurement of transference numbers in polymer electrolytes. Polymer 28, 2324–2328 (1987). J p y y y , ( ) 50. Boaretto, N. et al. Highly Conducting 3D-Hybrid Polymer Electrolytes for Lithium Batteries Based on Siloxane Networks and Cross- Linked Organic Polar Interphases. Chem. Mater. 26, 6339–6350 (2014). o, N. et al. Highly Conducting 3D-Hybrid Polymer Electrolytes for 50. Boaretto, N. et al. Highly Conducting 3D-Hybrid Polymer Elect g 51. Bouchet, R. et al. Single-ion BAB triblock copolymers as highly efficient electrolytes for lithium-metal batteries. Nat. Mater. References Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 12 g , Nano Lett. 15, 2671–2678 (2015). Visible light initiating systems for photopolymerization: status, development and challenges Polym. Chem. 5, 4195–4210 (2014). V. et al. Photopolymer Materials and Processes for Advanced Techn p y g 31. Vilela, F. et al. Conjugated porous polymers for energy applications. Energy Environ. Sci. 5, 7819–7832 (2012)fit 31. Vilela, F. et al. Conjugated porous polymers for energy applications. Energy Environ. Sci. 5, 7819–7832 (2012).fit 32. Griffini, G. et al. Multifunctional Luminescent Down-Shifting Fluoropolymer Coatings: A Straightforward Strategy to Improv UV-Light Harvesting Ability and Long-Term Outdoor Stability of Organic Dye-Sensitized Solar Cells. Adv. Energy Mater. 5, Ar 201401312 (2015). ( ) 33 Zhang, S et al Ionic liquid-based green processes for energy production Chem Soc Rev 43, 7838–7869 (2014) ( ) 33. Zhang, S. et al. Ionic liquid-based green processes for energy production. Chem. Soc. Rev. 43, 7838–7869 (2014). 34. Kitazawa, Y. et al. Gelation of Solvate Ionic Liquid by Self-Assembly of Block Copolymer and Characteri Electrolyte. Macromolecules 47, 6009–6016 (2014). y ( ) 35. Xu, K. Electrolytes and Interphases in Li-Ion Batteries and Beyond. Chem. Rev. 114, 11503–11618 (2014).l T. B. et al. Patternable Protein Resistant Surfaces for Multifunction . Stachowiak, T. B. et al. Patternable Protein Resistant Surfaces for M 36. Stachowiak, T. B. et al. Patternable Protein Resistant Surfaces for Multifunctional Microfluidic Devices via Surface Hydrophiliz of Porous Polymer Monoliths Using Photografting. Chem. Mater. 18, 5950–5957 (2006). t 37. Kim, G. T. et al. UV cross-linked, lithium-conducting ternary polymer electrolytes containing ionic liquids. J. Power Sources 195, 6130–6137 (2010). 38. Schulze, M. W. et al. High-Modulus, High-Conductivity Nanostructured Polymer Electrolyte Membranes via Polymerization- Induced Phase Separation. Nano Lett. 14, 122–126 (2014).h Induced Phase Separation. Nano Lett. 14, 122–126 (2014). p 9. Yoo, P. J. et al. Polymer Elasticity-Driven Wrinkling and Coarsening in High Temperature Buckling of Metal-Capped Polymer Thin Films. Phys. Rev. Lett. 93, art. No. 034301-1 (2004).i p 39. Yoo, P. J. et al. Polymer Elasticity-Driven Wrinklin Films. Phys. Rev. Lett. 93, art. No. 034301-1 (2004) 39. Yoo, P. J. et al. Polymer Elasticity-Driven Wrinkling and Co Films. Phys. Rev. Lett. 93, art. No. 034301-1 (2004) 40. Angulakhsmi, N. et al. Cycling profile of innovative nanochitin-incorporated poly (ethylene oxide) based electrolytes for lithium batteries. J. Power Sources 228, 294–299 (2013). 41. Lee, D. J. et al. A tetraethylene glycol dimethylether - lithium bis(oxalate)borate (TEGDME-LiBOB) electrolyte for advanced lithium ion batteries. Electrochem. Commun. 14, 43–46 (2012). 41. Lee, D. J. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Arora, P. et al. Battery Separators, Chem. Rev. 104, 4419–4462 (2004 4. Lee, H. et al. A review of recent developments in membrane separators for rechargeable lithium-ion batteries. Energy Environ. Sci. 7, 3857–3886 (2014). 5. Jin, L. et al. An organic ionic plastic crystal electrolyte for rate capability and stability of ambient temperature lithium batteries Energy Environ. Sci. 7, 3352–3361 (2014). gy ( ) 6. Stephan, A. M. Review on gel polymer electrolytes for lithium batteries. Eur. Polym. J. 42, 21–42 (2006). gy 6. Stephan, A. M. Review on gel polymer electrolytes for lithium batteries. Eur. Polym. J. 42, 21–42 (2006).l 7. Zhu, Y. et al. Composite of a nonwoven fabric with poly(vinylidene fluoride) as a gel membrane of high safety for lithium ion battery Energy Environ. Sci. 6, 618–624 (2013). p Energy Environ. Sci. 6, 618–624 (2013). gy 8. Staunton, E. et al. Structure and conductivity of the crystalline polymer electrolyte beta-PEO6:LiAsF6. J. Am. Chem. Soc. 127 12176–12177 (2005). ( ) 9. Wright, P. V. Developments in Polymer Electrolytes for Lithium Batteries. MRS Bull. 27, 597–602 (2002). 10. Berthier, C. et al. Microscopic investigation of ionic conductivity in alkali metal salts-poly(ethylene oxide) adducts. Solid State Ionics 11, 91–96 (1983). 1. Aetukuri, N. B. et al. Flexible Ion-Conducting Composite Membranes for Lithium Batteries. Adv. Energy Mater. doi: 10.1002 aenm.201500265 (2015). ( ) 12. Fergus, J. W. Ceramic and polymeric solid electrolytes for lithium-ion batteries. J. Power Sources 195, 4554–4569 (2010). 13. Fenton, D. E. et al. Complexes of alkali metal ions with poly(ethylene oxide). Polymer 14, 589 (1973). y y y 14. Armand, M. Polymer Electrolytes. Annu. Rev. Mater. Res. 16, 245–261 (1986). M. Polymer Electrolytes. Annu. Rev. Mater. Res. 16, 245–261 (1986 14. Armand, M. Polymer Electrolytes. Annu. Rev. Mater. Res. 16, 24 y y 5. Hovington, P. et al. New lithium metal polymer solid-state battery for an ultrahigh energy: nano C-LiFePO₄ versus nano Li1.2V₃O₈ Nano Lett. 15, 2671–2678 (2015). g , Nano Lett. 15, 2671–2678 (2015). Electrochemical properties of cross-linked polymer electrolyte by electron beam irradiation and applicatio lithium ion batteries. J. Power Sources 196, 4756–4761 (2011). q y ( ) 6. Ueno, M. et al. Electrochemical properties of cross-linked polymer electrolyte by electron beam irradiation and application to lithium ion batteries. J. Power Sources 196, 4756–4761 (2011). 7. Watanabe, M. et al. Estimation of Li+ transport number in polymer electrolytes by the combination of complex impedance and potentiostatic polarization measurements. Solid State Ionics 28–30, 911–917 (1988).l 67. Watanabe, M. et al. Estimation of Li+ transport number in polymer electrolytes by the combination of complex impedance and potentiostatic polarization measurements. Solid State Ionics 28–30, 911–917 (1988).l p p 8. Zhang, H. et al. Lithium bis(fluorosulfonyl)imide/poly(ethylene oxide) polymer electrolyte. Electrochim. Acta 133, 529–538 (2014) G b ld C l H h V O b d L h fil l ll h d l l b l N E 68. Zhang, H. et al. Lithium bis(fluorosulfonyl)imide/poly(ethylene oxide) polymer electrolyte. Electrochim. Acta 133, 529–538 (2014). 69. Gerbaldi, C. et al. High-rate V2O5-based Li-ion thin film polymer cell with outstanding long-term cyclability. Nano Energy 2, 1279–1286 (2013). g (l y ) p y( y ) p y y ( ) 9. Gerbaldi, C. et al. High-rate V2O5-based Li-ion thin film polymer cell with outstanding long-term cyclability. Nano Energy 2 1279–1286 (2013). Author Contributions L.P. did the experiments, J.R.N. & C.G. conceived and organised the work and wrote the manuscript, F.B. did thermal characterisation and reference management. All Authors reviewed the manuscript. .P. did the experiments, J.R.N. & C.G. conceived and organised the work and wrote the manuscript, F.B. did hermal characterisation and reference management. All Authors reviewed the manuscript. Additional Information Acknowledgements g inancial support from MARS-EV project, which is funded by the European Union Seventh Framework Program FP7/2007-2013) under grant agreement n° 609201, is gratefully acknowledged. g , Nano Lett. 15, 2671–2678 (2015). 12, 452− 457 (2013).i 51. Bouchet, R. et al. Single-ion BAB triblock copolymers as highly efficient electrolytes for lithium-metal batteries. Nat. Mater. 12, 452− 457 (2013).i 2. Chazalviel, J. N. Electrochemical aspects of the generation of ramified metallic electrodeposits. Phys. Rev. A 42 7355− 7367 (1990) Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 13 www.nature.com/scientificreports/ 3. Thomas, K. E. et al. Comparison of lithium-polymer cell performance with unity and nonunity transference numbers. J. Powe Sources 89, 132− 138 (2000). 53. Thomas, K. E. et al. Comparison of lithium-polymer cell performance with unity and nonunity transference numbers. J. Power Sources 89, 132− 138 (2000). 54 W H l I f P i b Li hi M l d C i P l El l f PEO Li(CF SO ) N 54. Wang, H. et al. Interface Properties between Lithium Metal and a Composite Polymer Electrolyte of PEO18Li(CF3SO2 Tetraethylene Glycol Dimethyl Ether. Membranes 3, 298–310 (2013). y y y 5. Kriz, J. et al. Nature and dynamics of lithium ion coordination in oligo(ethylene glycol) dimethacrylate-solvent systems: NMR Raman, and quantum mechanical study. J. Phys. Chem. A 103, 8505–8515 (1999).h q y y ( ) 6. Ma, Y. P. et al. The Measurement of a Complete Set of Transport Properties for a Concentrated Solid Polymer Electrolyte Solution. J Electrochem. Soc. 142, 1859–1868 (1995). , ( ) 57. Schaefer, J. L. et al. High Lithium Transference Number Electrolytes via Creation of 3-Dimensional, Charged, Nanoporous Networks from Dense Functionalized Nanoparticle Composites. Chem. Mater. 25, 834–839 (2013). ( ) 57. Schaefer, J. L. et al. High Lithium Transference Number Electrolytes via Creation of 3-Dimensional, Charged, Nanoporous Netw f D F i li d N i l C i Ch M t 25 834 839 (2013) g y from Dense Functionalized Nanoparticle Composites. Chem. Ma p p 58. Zhang, H. et al. Lithium bis(fluorosulfonyl)imide /poly(ethylene oxide) polymer electrolyte. Electrochim. Acta 133, 529–538 ( 59 Laoire C O et al Rechargeable Lithium/TEGDME-LiPF -O Battery J Electrochem Soc 158 A302–A308 (2011) 58. Zhang, H. et al. Lithium bis(fluorosulfonyl)imide /poly(ethylene oxide) polymer electrolyte. Electrochim. Acta 133, 52 58. Zhang, H. et al. Lithium bis(fluorosulfonyl)imide /poly(ethylene oxide) polymer electrolyte. Electrochim. Acta 133, 529–538 (2014). 59. Laoire, C. O. et al. Rechargeable Lithium/TEGDME-LiPF6-O2 Battery. J. Electrochem. Soc. 158, A302–A308 (2011). gl y y y y y 59. Laoire, C. O. et al. Rechargeable Lithium/TEGDME-LiPF6-O2 Battery. J. Electrochem. Soc. 158, A302–A308 (2011). Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 g , Nano Lett. 15, 2671–2678 (2015). g y 60. Peled, E. et al. The Electrochemical Behavior of Alkali and Alkaline Earth Metals in Nonaqueous Battery Systems-The Electrolyte Interphase Model. J. Electrochem. Soc. 126, 2047–2051 (1979). y . Croce, F. et al. Physical and Chemical Properties of Nanocomposit 61. Croce, F. et al. Physical and Chemical Properties of Nanocomposite Polymer Electrolytes. J. Phys. Chem. B 103, 10632–10638 (1999). 62. Appetecchi, G. B. et al. Transport and interfacial properties of composite polymer electrolytes. Electrochim. Acta 45, 1481–1490 (2000). 61. Croce, F. et al. Physical and Chemical Properties of Nanocomposite Polymer Electrolytes. J. Phys. Chem. B 103, 10632–10638 (1999). 61. Croce, F. et al. Physical and Chemical Properties of Nanocomposite Polymer Electrolytes. J. Phys. Chem. B 103, 10632–10638 (1 62. Appetecchi, G. B. et al. Transport and interfacial properties of composite polymer electrolytes. Electrochim. Acta 45, 1481– (2000). ( ) 63. Borghini, M. C. et al. Reliability of lithium batteries with crosslinked polymer electrolytes. Electrochim. Acta 41, 2369–2373 (1996). 63. Borghini, M. C. et al. Reliability of lithium batteries with crossl 63. Borghini, M. C. et al. Reliability of lithium batteries with crosslinked polymer electrolytes. Electrochim. Acta 41, 2369–2373 (1996). 64 Stone G M et al Resolution of the Modulus versus Adhesion Dilemma in Solid Polymer Electrolytes for Rechargeable Lithium 63. Borghini, M. C. et al. Reliability of lithium batteries with crosslinked polymer electrolytes. Electrochim. Acta 41, 2369–2373 (1996). 64. Stone, G. M. et al. Resolution of the Modulus versus Adhesion Dilemma in Solid Polymer Electrolytes for Rechargeable Lithium M t l B tt i J El t h S 159 A222 A227 (2012) 63. Borghini, M. C. et al. Reliability of lithium batteries with crosslinked polymer electrolytes. Electrochim. Acta 41, 2369–2373 (1 64. Stone, G. M. et al. Resolution of the Modulus versus Adhesion Dilemma in Solid Polymer Electrolytes for Rechargeable Lith 64. Stone, G. M. et al. Resolution of the Modulus versus Adhesion Dilemma in Solid Polymer Electrolytes for Rec Metal Batteries. J. Electrochem. Soc. 159, A222–A227 (2012). 65. Bella, F. et al. Aqueous dye-sensitized solar cells. Chem. Soc. Rev. 44, 3431–3473 (2015). t al. Aqueous dye-sensitized solar cells. Chem. Soc. Rev. 44, 3431–3 . Bella, F. et al. Aqueous dye-sensitized solar cells. Chem. Soc. Rev. 65. Bella, F. et al. Aqueous dye-sensitized solar cells. Chem. Soc. Rev. 44, 3431–3473 (2015). q y ( ) 66. Ueno, M. et al. Additional Information upplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Porcarelli, L. et al. Super Soft All-Ethylene Oxide Polymer Electrolyte for Safe All-Solid ithium Batteries. Sci. Rep. 6, 19892; doi: 10.1038/srep19892 (2016). How to cite this article: Porcarelli, L. et al. Super Soft All-Ethylene Oxide Polymer Electrolyte for Safe All-Solid Lithium Batteries. Sci. Rep. 6, 19892; doi: 10.1038/srep19892 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 6:19892 \| DOI: 10.1038/srep19892 14
https://openalex.org/W4362617959	https://figshare.com/articles/journal_contribution/Figure_S7_from_Rapid_Construction_of_Antitumor_T-cell_Receptor_Vectors_from_Frozen_Tumors_for_Engineered_T-cell_Therapy/22536040/1/files/39999331.pdf	English	null	Figure S2 from Rapid Construction of Antitumor T-cell Receptor Vectors from Frozen Tumors for Engineered T-cell Therapy	null	2,023	cc-by	345	Supplementary Figure S7 Recognition of Glycine-substituted peptides for HLA-Cw03-binding NY-ESO-1(92-100) epitope. HLA-Cw3+ NY- ESO-1-negative cells were pulsed with wild-type (WT) NY-ESO-1(92-100: LAMPFATPM), or one of its Glycine- substituted peptides at an indicated amino acid position for 1 hour and were co-cultured with the indicated TCR gene- transduced T cells (A, tumor #2-derived clone #4 and B, tumor #3-derived clone #5 in Figures S5 and S6) for 20 hours. IFN-g levels in the supernatant were determined by ELISA. Each bar shows mean IFN-g level and the standard deviation. Supplementary Figure S7 Recognition of Glycine-substituted peptides for HLA-Cw03-binding NY-ESO-1(92-100) epitope. HLA-Cw3+ NY- ESO-1-negative cells were pulsed with wild-type (WT) NY-ESO-1(92-100: LAMPFATPM), or one of its Glycine- substituted peptides at an indicated amino acid position for 1 hour and were co-cultured with the indicated TCR gene- transduced T cells (A, tumor #2-derived clone #4 and B, tumor #3-derived clone #5 in Figures S5 and S6) for 20 hours. IFN-g levels in the supernatant were determined by ELISA. Each bar shows mean IFN-g level and the standard deviation. Supplementary Figure S7 Recognition of Glycine-substituted peptides for HLA-Cw03-binding NY-ESO-1(92-100) epitope. HLA-Cw3+ NY- ESO-1-negative cells were pulsed with wild-type (WT) NY-ESO-1(92-100: LAMPFATPM), or one of its Glycine- substituted peptides at an indicated amino acid position for 1 hour and were co-cultured with the indicated TCR gene- transduced T cells (A, tumor #2-derived clone #4 and B, tumor #3-derived clone #5 in Figures S5 and S6) for 20 hours. IFN-g levels in the supernatant were determined by ELISA. Each bar shows mean IFN-g level and the standard deviation. Recognition of Glycine-substituted peptides for HLA-Cw03-binding NY-ESO-1(92-100) epitope. HLA-Cw3+ NY- ESO-1-negative cells were pulsed with wild-type (WT) NY-ESO-1(92-100: LAMPFATPM), or one of its Glycine- substituted peptides at an indicated amino acid position for 1 hour and were co-cultured with the indicated TCR gene- transduced T cells (A, tumor #2-derived clone #4 and B, tumor #3-derived clone #5 in Figures S5 and S6) for 20 hours. IFN-g levels in the supernatant were determined by ELISA. Each bar shows mean IFN-g level and the standard deviation.
https://openalex.org/W2053725275	https://www.frontiersin.org/articles/10.3389/fmicb.2014.00666/pdf	English	null	Multitasking SecB chaperones in bacteria	Frontiers in microbiology	2,014	cc-by	13,748	PROTEIN FOLDING AND TARGETING IN BACTERIA targeted post-translationally to the YidC insertase at the inner membrane (Dalbey et al., 2011), most integral membrane pro- teins as well as some presecretory proteins are targeted co- translationally by the ribosome-associated RNA-protein complex SRP (Saraogi and Shan, 2014). SRP binds to hydrophobic signal- anchor or signal sequence in nascent chains and targets them to the Sec translocon via interaction with its membrane receptor FtsY (Luirink and Sinning, 2004). The majority of presecretory proteins are translocated post-translationally either folded via the twin-arginine translocation (Tat) pathway or in a non-native state via the Sec pathway. The Tat system is known to translocate folded proteins or assembled protein complexes (up to 70 Å in diam- eter) through the cytoplasmic membrane. Tat substrate proteins possess an amino-terminal signal sequence with a conserved twin- arginine motif, which mediates post-translational targeting to the Tat translocon (Palmer and Berks, 2012; Patel et al., 2014). They are often assisted by speciﬁc cytosolic chaperones called redox enzyme maturation proteins (REMPs) and by the generic chaperones DnaK and GroEL, which likely prevent their degra- dation and premature export, and facilitate their assembly and functional interaction with the translocon (Castanie-Cornet et al., 2014). A major challenge for the cells is to ensure the proper fold- ing and targeting of newly synthesized proteins to the differ- ent cellular compartments. Indeed, ongoing protein synthe- sis in the crowded cellular environment offers a window of opportunities for non-native interactions, which may eventually lead to proteostasis breakdown (Kramer et al., 2009). There- fore, to cope with noxious off pathways in protein biogenesis, cells have evolved universally conserved molecular chaperones and targeting factors, which act co- and/or post-translationally to guide the precise partitioning, localization and folding of newly synthesized proteins (Kramer et al., 2009; Kim et al., 2013). In bacteria, the folding of newly synthesized proteins is mainly assisted by three highly conserved cytosolic chaperones, namely trigger factor (TF), DnaK/DnaJ/GrpE (DnaKJE), and GroEL/GroES (GroESL; Deuerling et al., 1999; Agashe et al., 2004; Kerner et al., 2005). The ribosome-bound TF is the ﬁrst chap- erone to interact co-translationally with most newly synthesized proteins (Valent et al., 1995). Although the majority of the cytoso- lic proteins can reach their native state following interaction with TF, a substantial amount of proteins (about 30%) need fur- ther co- and/or post-translational assistance by the downstream DnaKJE and GroESL chaperones (Bukau et al., 2000). Ambre Sala, Patricia Bordes and Pierre Genevaux* Laboratoire de Microbiologie et Génétique Moléculaire, Centre National de la Recherche Scientiﬁque, Université Paul Sabatier, Toulouse, France Laboratoire de Microbiologie et Génétique Moléculaire, Centre National de la Recherche Scientiﬁque, Université Paul Sabatier, obiologie et Génétique Moléculaire, Centre National de la Recherche Scientiﬁque, Université Paul Sabatier, Toulouse, France Protein export in bacteria is facilitated by the canonical SecB chaperone, which binds to unfolded precursor proteins, maintains them in a translocation competent state and speciﬁcally cooperates with the translocase motor SecA to ensure their proper targeting to the Sec translocon at the cytoplasmic membrane. Besides its key contribution to the Sec pathway, SecB chaperone tasking is critical for the secretion of the Sec-independent heme- binding protein HasA and actively contributes to the cellular network of chaperones that control general proteostasis in Escherichia coli, as judged by the signiﬁcant interplay found between SecB and the trigger factor, DnaK and GroEL chaperones. Although SecB is mainly a proteobacterial chaperone associated with the presence of an outer membrane and outer membrane proteins, secB-like genes are also found in Gram-positive bacteria as well as in certain phages and plasmids, thus suggesting alternative functions. In addition, a SecB-like protein is also present in the major human pathogen Mycobacterium tuberculosis where it speciﬁcally controls a stress-responsive toxin–antitoxin system. This review focuses on such very diverse chaperone functions of SecB, both in E. coli and in other unrelated bacteria. REVIEW ARTICLE published: 05 December 2014 doi: 10.3389/fmicb.2014.00666 REVIEW ARTICLE published: 05 December 2014 doi: 10.3389/fmicb.2014.00666 Reviewed by: y Claes von Wachenfeldt, Lund University, Sweden Julien Brillard, Institut National de la Recherche Agronomique, France Correspondence: Pierre Genevaux, Laboratoire de Microbiologie et Génétique Moléculaire, Centre National de la Recherche Scientiﬁque, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex 9, France e-mail: pierre.genevaux@ibcg. biotoul.fr Keywords: protein folding and targeting, SecA, DnaK, trigger factor, proteases, toxin–antitoxins www.frontiersin.org Edited by: Edited by: Salvador Ventura, Universitat Autònoma de Barcelona, Spain Edited by: Salvador Ventura, Universitat Autònoma de Barcelona, Spain Reviewed by: Claes von Wachenfeldt, Lund University, Sweden Julien Brillard, Institut National de la Recherche Agronomique, France Correspondence: Pierre Genevaux, Laboratoire de Microbiologie et Génétique Moléculaire, Centre National de la Recherche Scientiﬁque, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex 9, France e-mail: pierre.genevaux@ibcg. biotoul.fr Salvador Ventura, Universitat Autònoma de Barcelona, Spain pp Figure 1B). SecB is a homotetrameric chaperone of 69 kDa with a cellular con- centration estimated to be between 4 and 20 μM in Escherichia coli. SecB binds co- and/or post-translationally to non-native precur- sor proteins, maintaining them in competent state for delivery to the Sec translocon via a well-described interaction with its SecA partner (Randall and Hardy, 1995, 2002; Chatzi et al., 2013). SecB binds to non-native protein substrates with low speci- ﬁcity and high afﬁnity (Kd in the nanomolar range), generally in a one to one ratio of tetrameric chaperone to substrate (Ran- dall and Hardy, 1995). SecB binds to regions within the mature part of preprotein substrates and does not speciﬁcally recognize signal sequences (Gannon et al., 1989; Liu et al., 1989). Substrate selectivity by SecB is thought to occur via a kinetic partitioning between binding to the chaperone and folding, which is modu- lated by the afﬁnity and the folding rate of the substrate protein (Hardy and Randall, 1991). Seminal work performed on the SecB substrate preMBP revealed the appearance of proteolysis resistant conformation of preMBP in the absence of SecB, thus suggesting that binding to SecB prevents precursor proteins from acquir- ing a stable tertiary structure incompatible with Sec-dependent translocation(Collieret al.,1988). Asinglemoleculestudyrecently conﬁrmed that binding to SecB maintains preMBP in a molten globule-like state, preventing the formation of stable tertiary inter- actions (Bechtluft et al., 2007). SecB binding motif was identiﬁed by peptide scan of protein substrates as a nine amino acids-long segment enriched in aromatic and basic residues, with acidic residues strongly disfavored. Such motifs statistically occur every 20–30 amino acid residues in both exported and cytosolic pro- teins, thus suggesting low substrate speciﬁcity (Knoblauch et al., 1999). p ( y ) The E. coli secB gene was initially identiﬁed genetically by selecting for mutants that were defective in the export of a fusion protein composed of the N-terminal part of maltose- binding protein (MBP) preMBP (containing the signal sequence) and β-galactosidase (Kumamoto and Beckwith, 1983). Further experiments showed that secB mutations delayed or blocked the processing of a subset of preproteins and exhibited a synergistic effect with temperature-sensitive alleles of secA, thus revealing a role for SecB in export (Kumamoto and Beckwith, 1983, 1985). E. PROTEIN FOLDING AND TARGETING IN BACTERIA Forceful genetic and biochemical analyzes have demonstrated signiﬁcant overlap and cooperation between these three major chaperones, revealing a dynamic network of chaperones to control intracellu- lar proteostasis (Teter et al., 1999; Genevaux et al., 2004; Calloni et al., 2012). The Sec translocon is conserved in all three domains of life. Its core is composed of a heterotrimeric membrane complex SecYEG in bacteria and Sec61αβγ in eukaryotes (du Plessis et al., 2011). While translocation in the endoplasmic reticulum via the Sec translocon is mainly mediated co-translationally and thus energized by polypeptide chain elongation, Sec transloca- tion across the bacterial plasma membrane preferentially occurs post-translationally and energy is provided by the SecA ATPase motor component (Chatzi et al., 2013). In this case, SecA binds to presecretory proteins with mildly hydrophobic signal sequences, Targeting of newly synthesized proteins to the bacte- rial cytoplasmic membrane can occur either co- or post- translationally. While certain small membrane proteins are December 2014 \| Volume 5 \| Article 666 \| 1 www.frontiersin.org Sala et al. Multitasking SecB SecB monomer is composed of four stranded antiparallel β-sheets (the ﬁrst two strands being at opposite sides and connected by a cross over loop) and two α-helices separated by a helix con- necting loop (Figure 1A). SecB dimer is formed via interactions between strands β1 and helices α1 of two monomers. The tetramer forms by packing the helices α1 of four monomers in between the eight stranded antiparallel β-sheets formed by each dimer, mainly via polar interactions. A peptide binding groove was sug- gested from these structures, lying between the end of the cross over loop and strand β2 on one side, and the helix connecting loop and the helix α2 on the other side. This proposed substrate binding region likely contains two subsites: the aromatic, deep subsite 1, and the shallower hydrophobic subsite 2, as presented in Figure 1. Two peptide binding grooves are present on each side of the SecB tetramer, each potentially allowing the binding of ∼20 amino acids-long extended segments. The fact that SecB can bind long fragments of approximately 150 residues in pre- protein substrates (Khisty et al., 1995) suggests that these might wrap around the chaperone using several possible routes. PROTEIN FOLDING AND TARGETING IN BACTERIA Accord- ingly, electron paramagnetic resonance spectrometry analysis of spin labeled SecB variants in the presence of the physiologic SecB substrate galactose binding protein revealed that in addition to the proposed peptide binding groove, a much larger area of SecB appears to make contact with the substrate (Crane et al., 2006; Figure 1B). targets them to the Sec translocon at the inner membrane via a direct interaction with SecY, and subsequently drives the translocation process by successive cycles of ATP binding and hydrolysis. Productive interaction with Sec is inﬂuenced by the folding rate of the substrate and facilitated by cytosolic chaperones capa- ble of preventing premature folding, aggregation or degradation of precursor proteins (Randall and Hardy, 2002). Accordingly, all three main generic chaperone machines involved in de novo pro- tein folding, namely TF, DnaKJE and GroEL, have been shown to participate at different levels in this process (Castanie-Cornet et al., 2014). Remarkably, most proteobacteria also possess the chaperone SecB, which in addition to its generic chaperone func- tion has the ability to speciﬁcally interact with SecA to facilitate post-translational delivery of presecretory proteins to the Sec translocon (Bechtluft et al., 2010). This review describes the Sec- dependent and Sec-independent cellular functions of SecB, its interplay with other molecular chaperones as well as the dis- tribution of SecB homologs in very diverse bacteria. The role of the recently identiﬁed SecB-like proteins in the control of intracellular stress-responsive toxin–antitoxin (TA) systems is also discussed. Single mutation Single mutation Cs below 23◦C and Ts at 46◦C on LB agar plates(1); sensitive to copper, ethanol, cholate, low pH, dibucaine, triclosan, verapamil, and to several antibiotics, including bacitracin, novobiocin, amoxicillin, carbenicillin, tetracycline, cefaclor, glufosfomycin, ceftazidime, tunicamycin(2); partially resistant to phage U3(3); produces slightly bigger cells(4); and induces synthesis of heat-shock proteins(4,5). Mutation in secB with polar effect on the downstream gpsA gene inhibits growth on LB agar plates(6). Mutation in secB suppresses erythromycin and rifampin sensitivity of lptE mutants with increased outer-membrane permeability(7); enhances growth and export defects of secA51 mutation(4); exacerbates Lon, DnaJ(8) and TF(1) toxicity. Ts, Cs and export defect of secB mutation are suppressed by tig mutation(1); Cs is suppressed by lon mutation(8) and by overexpression of σ32(9), DnaK/DnaJ(1,10), GroEL/GroES(10,11), Rv1957(12), SmegB(13), and less efﬁciently by DnaJ259(8) and SecA(1). Export defect of secB mutations is partially suppressed by secA853-128 mutation(14). Synthetic lethal with dnaKdnaJ mutation(1) and possibly with mutations in forty-one additional genes, including groEL, the dsbC, lolA, and cpxP genes encoding periplasmic stress proteins and/or chaperones, as well as rplW encoding for L23, the main chaperone docking site at the ribosomal peptide exit(15). Likely presents positive or negative epistasis with eighty-nine additional mutations in cell envelope biogenesis genes(15). Protein localization loci Mutation in secB blocks the phenotypic effects of the prlC8 (mutation in opdA encoding the cytoplasmic Oligopeptidase A) suppressor of lamB signal sequence mutation(16); inhibits prlA4 (secY [F286Y, I408N]) mediated suppression of maltose-binding protein (MBP) signal sequence mutations(17,18) and prlA4 and prlZ1 mediated suppression of LamB signal sequence mutations(19,20). The prlA1001 (secY [I90N]) and prlA1024 (secY [I408N]) mutations suppress export deﬁcient maltose-binding protein in the absence of SecB(17); the prlF1 mutation in the antitoxin gene sohA of the SohA-YhaV toxin–antitoxin system suppresses SecB-dependent accumulation of LamB precursors(21). Overexpression Partially suppresses the Ts of the double dnaK tig mutant(22); affects expression of the cytoplasmic response regulator OmpR(23); prevents activation of the mycobacterial HigBA1 toxin–antitoxin system expressed in E. coli(12). Partially suppresses the Ts of the double dnaK tig mutant(22); affects expression of the cytoplasmic response regulator OmpR(23); prevents activation of the mycobacterial HigBA1 toxin–antitoxin system expressed in E. coli(12). the targeting of precursor proteins to the translocon (Fekkes et al., 1997). 1991; Powers and Randall, 1995; Baars et al., 2006; Marani et al., 2006). aPhenotypes associated with mutation or overexpression of SecB, and genetic interactions between secB and other mutations. Cs and Ts stand for cold- and temperature-sensitive phenotype, respectively. 1Ullers et al. (2007); 2Nichols et al. (2011); 3Kumamoto and Beckwith (1985); 4Baars et al. (2006); 5Wild et al. (1993); 6Shimizu et al. (1997); 7Grabowicz et al. (2013); 8Sakr et al. (2010); 9Altman et al. (1991); 10Castanie-Cornet et al. (2014); 11Danese et al. (1995); 12Bordes et al. (2011); 13Sala et al. (2013b); 14Mcfarland et al. (1993); 15Babu et al. (2011); 16Trun and Silhavy (1989); 17Francetic et al. (1993); 18Derman et al. (1993); 19Wei and Stader (1994); 20Trun et al. (1988); 21Snyder and Silhavy (1992); 22Ullers et al. (2004); 23Jin and Inouye (1995). www.frontiersin.org pp Figure 1B). coli secB mutant strains were initially shown to be defective for growth on rich Luria broth media agar plates, but it later appeared that this phenotype was due to a polar effect on the downstream gpsA gene encoding a glycerol-3-phosphate dehydro- genase involved in phospholipid biosynthesis (Kumamoto and Beckwith, 1985; Shimizu et al., 1997). Deletion of secB with- out apparent polarity on gpsA results in a strong cold-sensitive phenotype below 23◦C, a moderate temperature-sensitive pheno- type at temperatures above 45◦C and a hypersensitivity to several antibiotics (Ullers et al., 2007; Table 1). Most of the relevant phe- notypes associated with secB mutations or SecB overexpression are shown in Table 1. Genetic interactions between secB and the vari- ous protein localization locus (prl) mutations known to suppress the export defect of sequence signal deﬁcient precursors are also presented (Table 1). Several SecB dependent presecretory substrates have been iden- tiﬁed in E. coli by pulse chase experiments, sequence prediction or following analysis of protein aggregates that accumulate in the absence of the chaperone. This includes 25 presecretory proteins, namely CsgF,DegP,FhuA,FkpA,GBP,LamB,MBP,OmpA,OmpC, OmpF, OmpT, OmpX, OppA, PhoE, TolB, TolC, YagZ, YaiO, YbgF, YcgK, YfaZ, YgiW, YftM, YliI, and YncE (Hayashi and Wu, 1985; Kumamoto and Beckwith,1985; Kusters et al.,1989; Laminet et al., The crystal structures of SecB from Haemophilus inﬂuenza (Xu et al., 2000) and E. coli (Dekker et al., 2003) revealed that it forms a tetramer that assembles as a dimer of dimers (Figure 1A). December 2014 \| Volume 5 \| Article 666 \| 2 Frontiers in Microbiology \| Microbial Physiology and Metabolism Multitasking SecB Sala et al. Table 1 \| Most relevant phenotypes associated with mutations or overexpression of the E. coli SecB chaperone. SecB Phenotypesa Single mutation An additional contact site between the two proteins has been described, which consists of the C-terminal α-helices of SecB and the N-terminal part of SecA involved in dimerization and ribosome binding (Randall et al., 2004, 2005; Singh et al., 2014). Such interaction was proposed to triggerdissociationof theSecAdimer,thusallowingtheopeningof a peptide binding groove that would favor substrate transfer from SecB to SecA (Randall et al., 2005). These two surfaces of contact were conﬁrmed by spin-labeling analyzes of SecB upon SecA bind- ing (Crane et al., 2005). Interestingly, this work also showed that the surfaces of SecB that interact with the precursor and with SecA signiﬁcantly overlap, thus likely facilitating substrate transfer for translocation (Figure 1B; Crane et al., 2005). Efﬁcient transfer of the precursor protein from SecB to SecA requires both a correct This is consistent with a model in which one SecA dimer binds to one SecB tetramer (Randall et al., 2005). An additional contact site between the two proteins has been described, which consists of the C-terminal α-helices of SecB and the N-terminal part of SecA involved in dimerization and ribosome binding (Randall et al., 2004, 2005; Singh et al., 2014). Such interaction was proposed to triggerdissociationof theSecAdimer,thusallowingtheopeningof a peptide binding groove that would favor substrate transfer from SecB to SecA (Randall et al., 2005). These two surfaces of contact were conﬁrmed by spin-labeling analyzes of SecB upon SecA bind- ing (Crane et al., 2005). Interestingly, this work also showed that the surfaces of SecB that interact with the precursor and with SecA signiﬁcantly overlap, thus likely facilitating substrate transfer for translocation (Figure 1B; Crane et al., 2005). Efﬁcient transfer of the precursor protein from SecB to SecA requires both a correct interaction with SecB and the binding of the functional signal sequence to SecA, which has a strong afﬁnity for signal sequences (Fekkes et al., 1998). To date, the precise mechanism of substrate transfer remains unknown. Once the ATP-dependent transloca- tion process initiates, SecB is released from the Sec translocon and is now free to initiate a new cycle of binding to precursor proteins (Fekkes et al., 1997). The fact that SecB has the ability to stimu- late SecA ATPase activity suggests that it could contribute to the translocation initiation process as well (Miller et al., 2002). Single mutation Proteomic analyzes of protein aggregates that accumulate in a secB mutant also revealed the presence of a small number of aggregated cytosolic proteins (Baars et al., 2006; Sakr et al., 2010; see SecB Networking). Contact regions between SecB and SecA have been studied as well. SecB mutants with amino acid substitutions at positions D20, E24, L75, and E77 that were originally selected on the basis of their export defect (Gannon and Kumamoto, 1993; Kimsey et al., 1995), were later shown to be defective for binding to SecA (Woodbury et al., 2000). Accordingly, the crystal structures of SecB revealed that all these residues localize within the negatively charged sur- face formed by the β-sheets on both sides of the tetramer (Figure 1; Xu et al., 2000; Dekker et al., 2003). The main SecB binding site of SecA, which encompasses the last 22 C-terminal amino acid of SecA is highly enriched in basic residues and possesses a zinc bindingsiterequiredforafunctionalinteractionwithSecB(Fekkes et al., 1998, 1999). The structure of H. inﬂuenzae SecB in complex with the last 24 amino acids of SecA further established such a spe- ciﬁc binding occurring mainly through electrostatic interactions, with one SecA C-terminal peptide being bound to each β-sheet surface on both sides of a SecB tetramer (Zhou and Xu, 2003). As stated above, SecB directly targets presecretory proteins to the Sec pathway via its speciﬁc interaction with the peripheral ATPase SecA: the motor component of the Sec translocon (Hartl et al., 1990; Figure 2A). SecA is an essential cytosolic protein of 102 kDa with an estimated cellular concentration of ∼7 μM in E. coli (Kusters et al., 2011; Chatzi et al., 2013). SecA forms a homodimer in solution, is found either soluble or membrane- bound, and can speciﬁcally interact with translating ribosomes mainly via its N-terminal helix (Singh et al., 2014). SecB can inter- act with both membrane-bound and soluble SecA, albeit with a signiﬁcantly lower afﬁnity for soluble SecA (1.5 μM versus 30 nM Kds, respectively; den Blaauwen et al., 1997). Interaction between SecB and membrane-bound SecA is further increased in the pres- ence of precursor proteins (Kd of ∼10 nM), in order to facilitate December 2014 \| Volume 5 \| Article 666 \| 3 Multitasking SecB Sala et al. FIGURE 1 \|The SecB chaperone. Single mutation (A) Three-dimensional structure of an Escherichia coli SecB tetramer (1QYN) in front (left) and side (right) views displayed in ribbon and molecular surface using Chimera. Secondary structural elements are colored in both representations as follows: β-sheet 1 in red, β-sheet 2 in orange, β-sheet 3 in yellow, β-sheet 4 in salmon, α-helix 1 in dark blue and α-helix 2 in light blue. On the side view, the proposed subsites 1 (S1) and 2 (S2) of interaction with the substrate are indicated. (B) The primary amino acid sequence of SecB is annotated with the secondary structural elements on the top with colors corresponding to (A). Residues involved in the interaction with SecA or with the substrate are indicated below the sequence with white or black rectangles, respectively. Black asterisks indicate positions known to alter SecB function in export and red asterisks positions known to trigger aggregation of the protein.The C-terminal deletion 143–155 which alters the interaction with SecA is indicated. The residues predicted as being part of the subsites 1 and 2 of interaction with the substrate are highlighted in black and gray, respectively. FIGURE 1 \|The SecB chaperone. (A) Three-dimensional structure of an Escherichia coli SecB tetramer (1QYN) in front (left) and side (right) views displayed in ribbon and molecular surface using Chimera. Secondary structural elements are colored in both representations as follows: β-sheet 1 in red, β-sheet 2 in orange, β-sheet 3 in yellow, β-sheet 4 in salmon, α-helix 1 in dark blue and α-helix 2 in light blue. On the side view, the proposed subsites 1 (S1) and 2 (S2) of interaction with the substrate are indicated. (B) The primary amino acid sequence of SecB is annotated with the secondary structural elements on the top with colors corresponding to (A). Residues involved in the interaction with SecA or with the substrate are indicated below the sequence with white or black rectangles, respectively. Black asterisks indicate positions known to alter SecB function in export and red asterisks positions known to trigger aggregation of the protein.The C-terminal deletion 143–155 which alters the interaction with SecA is indicated. The residues predicted as being part of the subsites 1 and 2 of interaction with the substrate are highlighted in black and gray, respectively. This is consistent with a model in which one SecA dimer binds to one SecB tetramer (Randall et al., 2005). Single mutation In addition to the post-translational targeting of the SecB- precursor complex to the SecYEG-bound SecA, recent studies suggest that SecB might be directly recruited to the preformed cytosolic SecA-precursor complex prior to SecA interaction with the protein conducting channel SecYEG (Chatzi et al., 2013; Figure 2A). This model is supported by the recently described December 2014 \| Volume 5 \| Article 666 \| 4 Frontiers in Microbiology \| Microbial Physiology and Metabolism Multitasking SecB Sala et al. FIGURE 2 \| Multiple functions of SecB chaperones. (A) Proposed model for SecB-mediated protein targeting via the Sec pathway and the T1SS, and interplay between SecB and other generic chaperones. See text for details. Abbreviations for the chaperones and targeting factors presented are: trigger factor (TF), DnaK/DnaJ/GrpE (KJE), GroEL/GroES (ESL), SecB (B), SecA (A), type I secretion system (T1SS), SecYEG (Sec). IM stands for inner membrane. The T1SS and secretion signals are shown in red and purple, respectively. A black arrow indicates an interaction of the substrate with the chaperone or targeting factor and the black dashed arrow indicates a possible interaction of TF with T1SS substrates that was not yet investigated. (B) Proposed model for Rv1957 function in TA control. The different proteins are depicted as follows: toxin (T), antitoxin (A, blue triangle), Rv1957 chaperone (C), SecA1 (A, blue circle), SecYEG (Sec). IM stands for inner membrane. The signal sequence of presecretory proteins is showed in purple. The brackets indicate that it is not known yet whether the chaperone is part of the inactive complex. The red cross indicates that under certain stress conditions the chaperone could be recruited to rescue accumulating presecretory proteins. In this case, the chaperone would no longer be available to protect the antitoxin from degradation by proteases and to facilitate its interaction with the toxin, thus provoking toxin activation and bacterial growth inhibition until normal condition resume. (T1SS) substrate that is strictly dependent on SecB. The T1SS, which is widespread among Gram-negative bacteria directs the one step translocation of polypeptides across both the inner and outer membranes, directly to the extracellular medium (Dele- pelaire, 2004). It allows secretion of proteins of diverse sizes (19–800 kDa) and functions (toxins, lipases, heme-binding, or S- layer proteins), which are presumably transported in an unfolded state via a C-terminal uncleaved secretion signal (Delepelaire, 2004; Holland et al., 2005). Single mutation HasA of Serratia marcescens is secreted by an archetypal T1SS comprising an inner membrane ABC (ATP binding cassette) protein HasD, a periplasmic adaptor HasE, and an outer membrane channel-forming protein of the TolC family, named HasF (Letoffe et al., 1994). SecB interacts with nascent HasA early during synthesis and holds it in an unfolded conformation competent for productive interaction with theABC transporter HasD at the inner membrane (Figure 2A; Delepelaire and Wandersman, 1998; Debarbieux and Wandersman, 2001). In support of such antifolding activity of SecB, it has been shown that slow folding mutants of HasA are secreted independently of SecB (Wolff et al.,2003). Despite the fact that SecB allows a functional interaction between the N-terminal region of HasA and HasD, no direct interaction could be detected between SecB and the transporter (Sapriel et al., 2002, 2003; Wolff et al., 2003). Remarkably, point mutations in SecB that are known to affect its interaction with SecA (i.e., mutations D20A, E24A, L75R, and E77V; Figure 1B) exhibited very little or no effect on HasA secretion, thus indicating that SecB functions independently of SecA in this process. In contrast, SecB mutations affecting its oligomeric state and thus its chaperone function (mutations C76Y and Q80R; Kimsey et al., 1995; Muren et al., 1999) have a very strong effect on HasA secretion (Sapriel et al., 2003), sug- gesting that substrate binding by SecB is sufﬁcient in this case. To date, the use of SecB generic chaperone function by SecA- independent secretion systems has only been shown for HasDEF and it remains to be determined whether other systems require similar assistance by SecB, and to what extent such chaperone redeployment could affect proper functioning of the SecA/SecB cascade in vivo. FIGURE 2 \| Multiple functions of SecB chaperones. (A) Proposed model for SecB-mediated protein targeting via the Sec pathway and the T1SS, and interplay between SecB and other generic chaperones. See text for details. Abbreviations for the chaperones and targeting factors presented are: trigger factor (TF), DnaK/DnaJ/GrpE (KJE), GroEL/GroES (ESL), SecB (B), SecA (A), type I secretion system (T1SS), SecYEG (Sec). IM stands for inner membrane. The T1SS and secretion signals are shown in red and purple, respectively. A black arrow indicates an interaction of the substrate with the chaperone or targeting factor and the black dashed arrow indicates a possible interaction of TF with T1SS substrates that was not yet investigated. SecB NETWORKING Signiﬁcant interplay between SecB and other major cytosolic chaperones has been described (Castanie-Cornet et al., 2014; Figure 2A). The functional cooperation and/or overlap, as well as the strong genetic interactions observed between SecB, TF and DnaKJE suggest a key role for SecB as part of the chaperone net- work that orchestrates proper protein folding and targeting in E. coli. Albeit signiﬁcantly less studied, a discrete link between SecB and the chaperonin GroEL has also been shown in some cases. In this part, we describe the intricate relationship between SecB and these main chaperones and discuss how SecB chaperone tasking contributes to such proteostasis network. interaction of SecA with the L23 ribosomal protein platform for ribosome interacting factors at the ribosome exit tunnel (Huber et al., 2011; Singh et al., 2014). Together these data further high- light the multifaceted interaction between SecA and SecB, and its key contribution to the selective post-translational targeting of precursor proteins in E. coli. Single mutation (B) Proposed model for Rv1957 function in TA control. The different proteins are depicted as follows: toxin (T), antitoxin (A, blue triangle), Rv1957 chaperone (C), SecA1 (A, blue circle), SecYEG (Sec). IM stands for inner membrane. The signal sequence of presecretory proteins is showed in purple. The brackets indicate that it is not known yet whether the chaperone is part of the inactive complex. The red cross indicates that under certain stress conditions the chaperone could be recruited to rescue accumulating presecretory proteins. In this case, the chaperone would no longer be available to protect the antitoxin from degradation by proteases and to facilitate its interaction with the toxin, thus provoking toxin activation and bacterial growth inhibition until normal condition resume. FIGURE 2 \| Multiple functions of SecB chaperones. (A) Proposed model for SecB-mediated protein targeting via the Sec pathway and the T1SS, and interplay between SecB and other generic chaperones. See text for details. Abbreviations for the chaperones and targeting factors presented are: trigger factor (TF), DnaK/DnaJ/GrpE (KJE), GroEL/GroES (ESL), SecB (B), SecA (A), type I secretion system (T1SS), SecYEG (Sec). IM stands for inner membrane. The T1SS and secretion signals are shown in red and purple, respectively. A black arrow indicates an interaction of the substrate with the chaperone or targeting factor and the black dashed arrow indicates a possible interaction of TF with T1SS substrates that was not yet investigated. (B) Proposed model for Rv1957 function in TA control. The different proteins are depicted as follows: toxin (T), antitoxin (A, blue triangle), Rv1957 chaperone (C), SecA1 (A, blue circle), SecYEG (Sec). IM stands for inner membrane. The signal sequence of presecretory proteins is showed in purple. The brackets indicate that it is not known yet whether the chaperone is part of the inactive complex. The red cross indicates that under certain stress conditions the chaperone could be recruited to rescue accumulating presecretory proteins. In this case, the chaperone would no longer be available to protect the antitoxin from degradation by proteases and to facilitate its interaction with the toxin, thus provoking toxin activation and bacterial growth inhibition until normal condition resume. SecB AND THE RIBOSOME-BOUND TRIGGER FACTOR CHAPERONE TF can delay the folding of large proteins and exhibits unfold- ing activity (Agashe et al., 2004; Hoffmann et al., 2012; O’Brien et al., 2012), which may facilitate targeting of presecretory pro- teins to the Sec translocon, as observed for SecB. TF interacts with outer membrane proteins (OMPs) and several OMPs and periplasmic proteins are signiﬁcantly decreased in the absence of TF (Oh et al., 2011). Remarkably, a substantial number of these exported subtrates is shared between SecB and TF: this includes precursors of OmpA, OmpC, OmpF, LamB, PhoE, TolC, DegP, FkpA, OppA, Bla, and MBP (Castanie-Cornet et al., 2014). Yet, in contrast with SecB, a direct role for TF in stabilizing translo- cation competent precursors has only been shown for proOmpA and in this case, deletion of the tig gene encoding TF exhibited no signiﬁcant defect on proOmpA processing (Crooke and Wickner, 1987; Crooke et al., 1988a,b). Instead, tig mutation was shown to accelerate translocation of several known SecB substrates, namely OmpA, OmpC, and OmpF (Lill et al., 1988; Guthrie and Wickner, 1990; Lee and Bernstein, 2002; Genevaux et al., 2004; Ullers et al., 2007) and to fully suppress both cold-sensitive and temperature- sensitive phenotypes of a secB null strain (Table 1; Guthrie and Wickner, 1990; Lee and Bernstein, 2002; Genevaux et al., 2004; Ullers et al., 2007). These data suggest that ribosome-bound TF could facilitate post-translational targeting of precursors by main- taining them competent either for binding to membrane-bound SecA (Gouridis et al., 2009) or for transfer to SecB, DnaKJE, or GroESL (Figure 2A; see subsections below). The fact that both TF and SecA bind to L23 at the ribosomal polypeptide exit suggests that TF could either cooperate with SecA or prevent unproduc- tive SecA binding to precursors that ﬁrst need to transit via SecB (Figure 2; Karamyshev and Johnson, 2005; Huber et al., 2011; Singh et al., 2014). Although more work is needed to shed light on such possible interplays between SecA,TF and SecB,it is important to note that both secB and rplW (the gene encoding L23) muta- tions likely synergize in vivo, further supporting an important role for SecB in this process (Table 1). Most of our current knowledge concerning DnaKJE’s contri- bution to the Sec pathway originates from studies concerning secB mutants and/or SecB substrates (Castanie-Cornet et al., 2014). SecB AND THE RIBOSOME-BOUND TRIGGER FACTOR CHAPERONE SecB AND THE RIBOSOME-BOUND TRIGGER FACTOR CHAPERONE The TF chaperone interacts with most newly synthesized polypep- tides in E. coli (Valent et al., 1995). It is believed that about 70% of the E. coli cytosolic proteins interacting with TF reach their native state without further assistance (Deuerling et al.,1999; Teter Besides its chaperone function during protein export via the Sec pathway SecB is a key player in the secretion of the small Sec- independent HasA hemoprotein (19.3 kDa), which is part of the heme acquisition system of Serratia marcescens (Letoffe et al., 1994). So far, HasA is the only known type 1 secretion system December 2014 \| Volume 5 \| Article 666 \| 5 www.frontiersin.org Multitasking SecB Sala et al. et al., 1999). TF speciﬁcally binds to the ribosomal protein L23 in the vicinity of the polypeptide exit tunnel and cycles on and off the ribosome in an ATP-independent manner (Kramer et al., 2002; Ferbitz et al., 2004; Genevaux et al., 2004). Following release from the ribosome, TF can stay bound to elongating polypep- tides and facilitate substrate transfer to downstream chaperones or possibly to the Sec translocon (Crooke et al.,1988b; Kaiser et al., 2006; Raine et al., 2006; Hoffmann et al., 2010; Saio et al., 2014). TF can delay the folding of large proteins and exhibits unfold- ing activity (Agashe et al., 2004; Hoffmann et al., 2012; O’Brien et al., 2012), which may facilitate targeting of presecretory pro- teins to the Sec translocon, as observed for SecB. TF interacts with outer membrane proteins (OMPs) and several OMPs and periplasmic proteins are signiﬁcantly decreased in the absence of TF (Oh et al., 2011). Remarkably, a substantial number of these exported subtrates is shared between SecB and TF: this includes precursors of OmpA, OmpC, OmpF, LamB, PhoE, TolC, DegP, FkpA, OppA, Bla, and MBP (Castanie-Cornet et al., 2014). Yet, in contrast with SecB, a direct role for TF in stabilizing translo- cation competent precursors has only been shown for proOmpA and in this case, deletion of the tig gene encoding TF exhibited no signiﬁcant defect on proOmpA processing (Crooke and Wickner, 1987; Crooke et al., 1988a,b). SecB AND THE RIBOSOME-BOUND TRIGGER FACTOR CHAPERONE Indeed, it has been shown that export of the SecB substrates OmpA, OmpC, and OmpF strongly relies on DnaK when protein translocation is compromised (Qi et al., 2002), and that overex- pression of DnaKJ suppresses both the cold-sensitive phenotype of a secB null strain and the export defect of the SecB-dependent substrates LamB and MBP (Wild et al., 1992; Ullers et al., 2007; Castanie-Cornet et al., 2014). Although, export of both LamB and MBP is not affected by a dnaK mutation (Wild et al., 1992), deple- tion of DnaKJ in the absence of SecB showed a further decrease in the processing of these proteins and a robust accumulation of protein aggregates in the E. coli cytoplasm (Wild et al., 1992; Ullers et al., 2007). These aggregated proteins include known DnaK sub- strates and several OMPs (i.e., OmpA, OmpC, OmpX, and PhoE) previously known as SecB substrates (Ullers et al., 2007). Such a major overlap between these two chaperones is further supported by the fact that SecB substrates were recently identiﬁed as bona ﬁde DnaK interactors in vivo (Calloni et al., 2012). This includes the OMPs OmpA,OmpC,OmpF,OmpT and OmpX,and the periplas- mic proteins OppA and DegP. Accordingly, peptide binding scans revealed that SecB and DnaK share many potential binding sites in polypeptide substrates and could interact with similar regions within protein (Knoblauch et al., 1999). These data are in com- plete agreement with the fact that mutations in secB and dnaK (or dnaJ) exhibit synthetic lethality (Table 1), and that expression of DnaK is upregulated in the absence of SecB, and recipro- cally (Muller, 1996; Ullers et al., 2007). These data also suggest that both chaperones could work in concert to assist the post- translational translocation of certain Sec substrates (Sakr et al., 2010). The physical interaction recently found between SecB and DnaK in vivo is in agreement with such hypothesis (Calloni et al., 2012). SecB AND THE RIBOSOME-BOUND TRIGGER FACTOR CHAPERONE Instead, tig mutation was shown to accelerate translocation of several known SecB substrates, namely OmpA, OmpC, and OmpF (Lill et al., 1988; Guthrie and Wickner, 1990; Lee and Bernstein, 2002; Genevaux et al., 2004; Ullers et al., 2007) and to fully suppress both cold-sensitive and temperature- sensitive phenotypes of a secB null strain (Table 1; Guthrie and Wickner, 1990; Lee and Bernstein, 2002; Genevaux et al., 2004; Ullers et al., 2007). These data suggest that ribosome-bound TF could facilitate post-translational targeting of precursors by main- taining them competent either for binding to membrane-bound SecA (Gouridis et al., 2009) or for transfer to SecB, DnaKJE, or GroESL (Figure 2A; see subsections below). The fact that both TF and SecA bind to L23 at the ribosomal polypeptide exit suggests that TF could either cooperate with SecA or prevent unproduc- tive SecA binding to precursors that ﬁrst need to transit via SecB (Figure 2; Karamyshev and Johnson, 2005; Huber et al., 2011; Singh et al., 2014). Although more work is needed to shed light on such possible interplays between SecA,TF and SecB,it is important to note that both secB and rplW (the gene encoding L23) muta- tions likely synergize in vivo, further supporting an important role for SecB in this process (Table 1). polypeptide sequences accessible during de novo protein fold- ing, translocation through biological membranes, during stress or within native protein complexes (Rudiger et al., 1997). In agree- ment with such a variety of potential interactors, the recently described in vivo interactome of DnaK obtained in the presence of SecB revealed that DnaK interacts with more than six hundred E. coli proteins at 37◦C, including cytosolic (∼80%), inner mem- brane (∼11%), outer membrane (∼3%) and periplasmic proteins (∼3%; Calloni et al., 2012). et al., 1999). TF speciﬁcally binds to the ribosomal protein L23 in the vicinity of the polypeptide exit tunnel and cycles on and off the ribosome in an ATP-independent manner (Kramer et al., 2002; Ferbitz et al., 2004; Genevaux et al., 2004). Following release from the ribosome, TF can stay bound to elongating polypep- tides and facilitate substrate transfer to downstream chaperones or possibly to the Sec translocon (Crooke et al.,1988b; Kaiser et al., 2006; Raine et al., 2006; Hoffmann et al., 2010; Saio et al., 2014). SecB AND THE DnaKJE CHAPERONE MACHINE SecB AND THE TF/DnaK PATHWAY FOR CYTOSOLIC PROTEIN FOLDING In addition to protein export, a role for SecB in rescuing cytosolic protein folding has been proposed. Such a SecB func- tion has emerged from studies generally focusing on both TF and DnaK chaperones. Indeed, it has been shown that SecB overexpression efﬁciently rescues the severe growth defect of a chaperone-deﬁcient strain carrying both dnaK and tig mutations, and suppresses the DnaK/TF-dependent accumulation of aggre- gated cytosolic proteins (Ullers et al., 2004). In vitro cross-linking experiments further showed that SecB is indeed capable of inter- acting co- and/or post-translationally with nascent RpoB in the absence of both chaperones. Such a possible SecB function is further supported by the fact that (i) SecB has preference for The ATP-dependent chaperone DnaK of E. coli is a well- characterized member of the Hsp70 chaperone family. It is an abundant cytosolic chaperone expressed constitutively and induced in response to different stresses (Genevaux et al., 2007). The DnaK chaperone cycle is tightly regulated by essential co-chaperones: (i) the DnaJ (Hsp40) co-chaperone family mem- bers that stimulate DnaK’s weak ATP activity and facilitate substrate delivery to DnaK, and (ii) the nucleotide exchange factor GrpE, which mediates the dissociation of ADP and the subsequent binding of a new ATP that triggers substrate release from DnaK (Liberek et al., 1991; Harrison et al., 1997; Brehmer et al., 2001). DnaK preferentially interacts with short extended hydrophobic December 2014 \| Volume 5 \| Article 666 \| 6 Frontiers in Microbiology \| Microbial Physiology and Metabolism Multitasking SecB Sala et al. bacteria could make use of such SecB chaperone function for the targeting of their OMPs to the Sec translocon (Zuber et al., 2008; Mah et al., 2010). Previous work showed that Rv1957 can replace SecB export function in E. coli, partially restoring the processing of both proOmpA and preMBP, and complement the cold-sensitive phenotype of a secB mutant strain (Table 1). In vitro, Rv1957 also forms a tetramer in solution and efﬁciently prevents aggregation of the known E. coli SecB substrate proOmpC at a level compa- rable to that of SecB (Bordes et al., 2011). These results strongly suggest that Rv1957 could act as a bona ﬁde SecB chaperone to assist protein export in M. tuberculosis. SecB AND THE CHAPERONIN GroESL The third main molecular chaperone potentially linked to SecB in E. coli is the chaperonin GroESL. The ATP-dependent chaperonin GroEL is a well-characterized member of the Hsp60 chaperone family. Together with its co-chaperone GroES (Hsp10), it pro- vides both a protected environment and a functional assistance to polypeptides generally up to 60 kDa. GroEL forms a barrel-shaped complex composed of two heptameric rings assembled back-to- back (Saibil et al.,2013). The GroEL folding cavity can be closed by a seven GroES co-chaperone lid, which allows conﬁnement of the polypeptide. It is believed that GroESL interacts with more than 10% of the E. coli cytosolic proteins, including aggregation-prone proteins that are strictly chaperonin-dependent for their folding in vivo (Kerner et al., 2005). Although poorly investigated, a direct involvement of GroESL in the Sec pathway has been observed and several SecB substrates have been shown to interact with or to be processed by GroEL (Kusukawa et al., 1989; Lecker et al., 1989; Phillips and Silhavy, 1990). Remarkably, ﬁve known SecB substrates were recently iden- tiﬁed as GroEL interactors in vivo. These include three OMPs, namely OmpA, OmpC and OmpF, and two periplasmic proteins OppA and YncE (Watanabe et al., 1988; Kerner et al., 2005; Baars et al., 2006). In addition, GroEL was previously shown to interact withprePhoEandproOmpAinvitro,andtostabilizeproOmpAfor translocation (Kusukawa et al.,1989; Lecker et al.,1989). Although groESL mutations exhibit no apparent effect on proOmpA and proOmpF processing,overexpression of GroESL efﬁciently rescues the cold-sensitive phenotype of a secB null strain (unpublished data). The fact that endogenous SecB level also increases in strains with impaired GroESL is in agreement with such ﬁndings (Muller, 1996). Together these data suggest that GroESL may actively con- tribute to the Sec-dependent export process, perhaps rescuing SecB substrates under certain stresses or even cooperate with SecB to facilitate their transfer to SecA, as proposed for TF and DnaK. The hypothetic dual role of Rv1957 both as a generic chap- erone potentially assisting protein export and as a specialized chaperone controlling a bacterial growth tuning system raises the question of a possible link between these two functions under cer- tain conditions (Figure 2B). An attractive hypothesis is that in case of a compromised translocon accumulation of preproteins could compete with the antitoxin for Rv1957 binding, resulting in antitoxin degradation and subsequent toxin activation. In this model, the SecB-like chaperone would thus function as a molecu- lar sentinel to watch over protein export. SecB-LIKE CHAPERONES AND TOXIN–ANTITOXIN SYSTEMS SecB-LIKE CHAPERONES AND TOXIN–ANTITOXIN SYSTEMS As stated above, SecB is usually found in proteobacteria. Yet, some SecB-like sequences are also found in other taxonomic groups, including Gram-positive bacteria (Sala et al., 2013b). The major human pathogen M. tuberculosis also encodes a SecB-like protein, namely Rv1957, which shares 19% amino acid sequence identity withtheE.coli SecB.Thefactthatmycobacteriahaveawell-deﬁned and characteristic outer membrane, named the mycomembrane, with a signiﬁcant number of predicted OMPs suggests that these SecB AND THE DnaKJE CHAPERONE MACHINE unstructured stretches of polypeptides that are not speciﬁcally found in exported proteins (Knoblauch et al., 1999), (ii) SecB pre- vents luciferase aggregation and cooperates with DnaKJE in the refolding of luciferase in vitro (Knoblauch et al., 1999), and (iii) cytosolicproteinscanbeisolatedfromaggregatedproteinfractions in both secB and secB lon mutant strains (Baars et al., 2006; Sakr et al., 2010). More work is warranted to elucidate whether SecB indeed has cytosolic protein substrates in vivo. Of note, the SecB- like chaperone Rv1957 in Mycobacterium tuberculosis was shown to directly assist the folding of a cytosolic antitoxin, arguing for such possible SecB function in other bacteria (see part below). In contrast with E. coli SecB, the Rv1957 encoding gene is clustered together with genes that are part of a stress-responsive type II TA system related to the HigBA family (Host Inhibi- tion of Growth; Gupta, 2009; Ramage et al., 2009; Bordes et al., 2011; Sala et al., 2013a,b; Schuessler et al., 2013). Type II TA sys- tems are genetic modules composed of a stable toxin and a less stable antitoxin, which interact together to form a complex in which the toxin is inactive (Gerdes et al., 2005; Goeders and Van Melderen, 2014). Under speciﬁc stress condition, the antitoxin is degraded by activated proteases, provoking the release of the active toxin, which will then act on its intracellular targets. Tox- ins from type II TA generally target essential cellular functions, such as translation or replication, resulting in growth inhibition. Modulation of bacterial growth by TA systems in response to environmental insults likely favors adaptation to stress (Lewis, 2010; Yamaguchi and Inouye, 2011). Remarkably, the SecB-like chaperone Rv1957 from M. tuberculosis speciﬁcally controls the inhibition of the HigBA TA system (Figure 2B). Indeed, Rv1957 interacts directly with the HigA antitoxin and protects it from both aggregation and degradation by proteases, thus facilitat- ing its folding and subsequent interaction with the toxin. This chaperone function is necessary for the efﬁcient inhibition of the toxin by the antagonistic antitoxin (Bordes et al., 2011). Such a tripartite system, named TAC for toxin-antitoxin-chaperone, is the ﬁrst example of a TA system controlled by a molecular chaperone. SecB AND THE CHAPERONIN GroESL The fact that the presence of a secB open reading frame associated with TA modules is not unique to M. tuberculosis or to mycobacteria (Sala et al., 2013b) indicates that such a mechanism might be conserved (see part below). TAXONOMIC DISTRIBUTION OF SOLITARY AND TA-ASSOCIATED SecB It has been proposed that SecB appeared in the last common ancestor of α-, β-, and γ-proteobacteria and that its conser- vation is linked to the presence of an outer membrane, and December 2014 \| Volume 5 \| Article 666 \| 7 www.frontiersin.org www.frontiersin.org Sala et al. Multitasking SecB A subset of SecB genes, representing approximately 7.5% of the total number of SecB sequences (52/688),are associated with genes encoding TA systems (or in some cases antitoxin genes alone), as observed for the TAC system of M. tuberculosis. This suggests that these putative SecB chaperones might function in the spe- ciﬁc control of their cognate TA systems in a manner comparable to that of Rv1957 (Bordes et al., 2011; Sala et al., 2013b). When SecB sequences are present in groups outside of α-, β-, and γ- proteobacteria, they seem to preferentially associate with a TA system (63%, 44/70). In this case, the vast majority of the genomes (>90%) do not possess an additional copy of solitary SecB. Inter- estingly, TA systems associated with SecB sequences often belong thus an increased need in protein export (van der Sluis and Driessen, 2006). Nevertheless, analysis of the taxonomic reparti- tion of PF02556, the Pfam domain characterizing SecB sequences (http://pfam.sanger.ac.uk/) in a set of 1631 complete and cured bacterial genomes revealed the presence of this domain in seven groups outside proteobacteria. Noticeably,these groups are mainly composed of diderm bacteria, except from the Firmicutes phylum (Figure 3A) and in most cases, SecB sequences occur at low fre- quency when compared to the total number of genomes. This is in sharp contrast with α-, β-, and γ-proteobacteria, where most of the genomes contain at least one SecB sequence (Sala et al., 2013b; Figure 3A). thus an increased need in protein export (van der Sluis and Driessen, 2006). Nevertheless, analysis of the taxonomic reparti- tion of PF02556, the Pfam domain characterizing SecB sequences (http://pfam.sanger.ac.uk/) in a set of 1631 complete and cured bacterial genomes revealed the presence of this domain in seven groups outside proteobacteria. Noticeably,these groups are mainly composed of diderm bacteria, except from the Firmicutes phylum (Figure 3A) and in most cases, SecB sequences occur at low fre- quency when compared to the total number of genomes. This is in sharp contrast with α-, β-, and γ-proteobacteria, where most of the genomes contain at least one SecB sequence (Sala et al., 2013b; Figure 3A). CONCLUDING REMARKS to different families of toxins and/or antitoxins, strongly suggest- ing that the event of association of a SecB encoding gene with a TA module is a widespread mechanism that occurred several times during evolution (Sala et al., 2013b). The possible involvement of these SecB chaperones in Sec-dependent protein export remains to be determined. Extensive genetic and biochemical analyses of SecB chaperone tasking have undoubtedly revealed its key cellular roles as part of the network of generic chaperones that orchestrate proteostasis in E. coli. The fact that SecB binds its substrates in a non-native state and prevents their unproductive folding is in agreement with its major role in delivering translocation competent proteins to the inner membrane, as observed for a large number of Sec-dependent presecretory proteins, for the ABC transporter substrate HasA, and perhaps for other proteins whose secretion relies on speciﬁc secretion systems that lack dedicated chaperones. Further analysis on all the bacterial sequences available on the Pfam server for the PF02556 conserved domain (i.e., from both complete and in progress genomes) was performed to study the homology links between SecB sequences using a graph par- titioning approach. This revealed that solitary SecB sequences are grouped together in a highly connected core, reﬂecting a high level of conservation (Figure 3B). In this core, sev- eral SecB communities (corresponding to the different colors) are well-deﬁned and generally correspond to the taxonomy: the red family contains mainly α-proteobacterial sequences, the dark orange mainly γ-proteobacterial sequences and the light orange mainly β-proteobacterial sequences. Another clearly deﬁned group that emerges from this core, in yellow, con- tains mainly sequences from Streptococcus pneumoniae strains (158/196). The other communities within the core are poorly deﬁned and mainly correspond to other Firmicutes sequences. Most of the TAC (or AC) chaperones are grouped in eight different communities, which seem to have diverged from the solitary SecB core from distinct origins (Figure 3B). The light orange group within the core also contains four TA-associated SecB sequences from δ-proteobacteria, thus strongly suggest- ing a common evolutionary history between TAC chaperones and canonical solitary SecB (Sala et al., 2013b). Yet, in sharp contrast with solitary SecB, the groups of TAC chaperones do not follow the taxonomy and most of them are comprised in regions containing horizontal gene transfer signatures, as it is the case for classical TA systems (Makarova et al., 2009; Sala et al., 2013b). ACKNOWLEDGMENTS ) Paralogs of SecA, SecE, SecY, and SecG are found in Actinobac- teria and Firmicutes, either forming a parallel pathway with a dedicated translocon or exploiting the generic Sec translocon to export a speciﬁc set of substrates, as it is the case for SecA2 in mycobacteria (Rigel et al., 2009; Sullivan et al., 2012). Inter- estingly, among the 1631 complete bacterial genomes analyzed 26 of them are predicted to have more than one solitary SecB sequence (up to three in Acetobacter pasteurianus IFO 3283-01- 42C), eight genomes contain both a solitary and a TA associated SecB, one genome contains two solitary and one TA associ- ated SecB, and two genomes contain two TAC or AC. These additional SecB sequences could function as specialized chaper- ones for the control of TA systems or for the export of speciﬁc substrates, or as generic chaperone induced in response to cer- tain stress conditions. Interestingly, single deletion of secB1 or secB2 from Francisella tularensis subsp. novicida exhibits a reduced bioﬁlm formation, suggesting that both chaperone par- alogs participate in the secretion of speciﬁc factors important for the attachment to abiotic surfaces (Margolis et al., 2010). Remarkably, the double secB mutant was not viable, suggesting that in the case of F. tularensis, SecB chaperones have over- lapping functions essential for bacterial survival (Margolis et al., 2010). We thank Petra Langendijk and Olivera Francetic for insightful discussion and Gwennaele Fichant’s group for sharing their in- house bacterial genome database. This work was supported by a French MENRT fellowship and an FRM grant (FDT20140930836) to Ambre Sala and a mycoTAC ANR grant (ANR-13-BSV8-0010- 01) to Pierre Genevaux. TAXONOMIC DISTRIBUTION OF SOLITARY AND TA-ASSOCIATED SecB FIGURE 3 \| Distribution and conservation of SecB sequences in bacteria. FIGURE 3 \| Distribution and conservation of SecB sequences in bacteria. plus 33 sequences identiﬁed previously (Sala et al., 2013b) as TAC chaperones that are absent from the PF02556. A graph was built in which nodes (Circles) correspond to protein sequences and weighted edges represent the BLASTP log(e-value) obtained between a pair of proteins. In this case, the homology relationship was inferred when an e-value less than or equal to 10−5 was observed between two sequences. Partitioning of this graph into communities of highly connected nodes was performed using the MCL program, with an inﬂat parameter of 1.5. This resulted in 18 groups of SecB sequences, represented by the different colors assigned to the circles in the graph. The highly connected core containing almost all solitary SecB sequences discussed in the text is circled in gray. The three circles with a thick gray line indicate the solitary SecB sequences found outside of the SecB conservation core. MTBC stands for M. tuberculosis complex species. FIGURE 3 \| Distribution and conservation of SecB sequences in bacteria. (A) Taxonomic distribution of SecB sequences. A number of 1631 complete and cured bacterial genomes (local database) were analyzed for the presence of SecB sequence by searching for (i) the signature of the PF02556 domain using RPS-BLAST with as threshold an e-value of 10−5, a score of 35 and a query coverage of 40%, or (ii)TAC (or AC) chaperones potentially not identiﬁed by the ﬁrst request, using an approach described previously (Sala et al., 2013b). This approach allowed the identiﬁcation of 688 SecB sequences. For each taxonomic group containing SecB sequences, the total number of genomes is given in bold characters and represented by a black frame. The number of genomes containing one or more SecB sequences is given in blue and represented by blue rectangles, and the number of genomes containing TAC or AC systems is depicted in green. (B) MCL (Markov Clustering) analysis of the 1981 bacterial sequences contained in the PF02256 conserved domain December 2014 \| Volume 5 \| Article 666 \| 8 Frontiers in Microbiology \| Microbial Physiology and Metabolism Multitasking SecB Sala et al. CONCLUDING REMARKS y In contrast with the speciﬁc and well-described cooperative cascade between SecB and SecA during post-translational target- ing of Sec-dependent precursors, the interplay between SecB, TF and DnaK remains poorly understood, and there is a clear lack of knowledge about the substrates that are shared between the three chaperones in vivo. In addition, it is not known whether these chaperones actively cooperate to facilitate export of certain proteins, both under normal and stress conditions, and to what extent such cooperation inﬂuences early partitioning of newly syn- thesized proteins. Similarly, it remains to be determined whether some cytosolic proteins or protein complexes do require SecB for their folding and/or assembly, as it is the case for the SecB-like protein Rv1957 and its TA system in M. tuberculosis. These are truly open questions that need to be addressed. The relatively frequent association of SecB proteins with dif- ferent TA families is intriguing and may reveal interesting new SecB functions, perhaps reﬂecting a link between toxin activation and membrane jamming. In this respect, an important mecha- nistic issue will be to determine how TA systems have acquired such a unique addiction for SecB chaperones. Finally, the sporadic presence of solitary SecB-like proteins in monoderm bacteria also suggests novel SecB functions to be discovered. REFERENCES Tuning of chaperone activity of Hsp70 proteins by modulation of nucleotide exchange. Nat. Struct. Biol. 8, 427–432. doi: 10.1038/87588 Microbiol. 29, 1179–1190. doi: 10.1046/j.1365-2958.1998.00997.x Fekkes, P., Van Der Does, C., and Driessen, A. J. (1997). The molecular chaperone SecB is released from the carboxy-terminus of SecA during initiation of pre- cursor protein translocation. EMBO J. 16, 6105–6113. doi: 10.1093/emboj/16. 20.6105 Bukau, B., Deuerling, E., Pfund, C., and Craig, E. A. (2000). Getting newly syn- thesized proteins into shape. Cell 101, 119–122. doi: 10.1016/S0092-8674(00) 80806-5 Ferbitz, L., Maier, T., Patzelt, H., Bukau, B., Deuerling, E., and Ban, N. (2004). Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins. Nature 431, 590–596. doi: 10.1038/nature02899 Calloni, G., Chen, T., Schermann, S. M., Chang, H. C., Genevaux, P., Agostini, F., et al. (2012). DnaK functions as a central hub in the E. coli chaperone network. Cell Rep. 1, 251–264. doi: 10.1016/j.celrep.2011.12.007 Francetic, O., Hanson, M. P., and Kumamoto, C. A. (1993). PrlA suppression of defective export of maltose-binding protein in SecB mutants of Escherichia coli. J. Bacteriol. 175, 4036–4044. Castanie-Cornet, M. P., Bruel, N., and Genevaux, P. (2014). Chaperone network- ing facilitates protein targeting to the bacterial cytoplasmic membrane. Biochim. Biophys. Acta 1843, 1442–1456. doi: 10.1016/j.bbamcr.2013.11.007 Gannon, P. M., and Kumamoto, C. A. (1993). Mutations of the molecular chaper- one protein SecB which alter the interaction between Secb and maltose-binding protein. J. Biol. Chem. 268, 1590–1595. Chatzi, K. E., Sardis, M. F., Karamanou, S., and Economou, A. (2013). Breaking on through to the other side: protein export through the bacterial Sec system. Biochem. J. 449, 25–37. doi: 10.1042/BJ20121227 p Gannon, P. M., Li, P., and Kumamoto, C. A. (1989). The mature portion of Escherichia coli maltose-binding protein (MBP) determines the dependence of MBP on SecB for export. J. Bacteriol. 171, 813–818. Collier, D. N., Bankaitis, V. A., Weiss, J. B., and Bassford, P. J. Jr. (1988). The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein. Cell 53, 273–283. doi: 10.1016/0092-8674(88)90389-3 Genevaux, P., Georgopoulos, C., and Kelley, W. L. (2007). The Hsp70 chap- erone machines of Escherichia coli: a paradigm for the repartition of chap- erone functions. Mol. Microbiol. 66, 840–857. doi: 10.1111/j.1365-2958.2007. 05961.x Crane, J. M., Mao, C. F., Lilly, A. A., Smith, V. F., Suo, Y. Y., Hubbell, W. L., et al. (2005). REFERENCES Mapping of the docking of SecA onto the chaperone SecB by site-directed spin labeling: insight into the mechanism of ligand transfer during protein export. J. Mol. Biol. 353, 295–307. doi: 10.1016/j.jmb.2005.08.022 Genevaux, P., Keppel, F., Schwager, F., Langendijk-Genevaux, P. S., Hartl, F. U., and Georgopoulos, C. (2004). In vivo analysis of the overlapping functions of DnaK and trigger factor. EMBO Rep. 5, 195–200. doi: 10.1038/sj.embor.7400067 Crane, J. M., Suo, Y. Y., Lilly, A. A., Mao, C. F., Hubbell, W. L., and Randall, L. L. (2006). Sites of interaction of a precursor polypeptide on the export chaper- one SecB mapped by site-directed spin labeling. J. Mol. Biol. 363, 63–74. doi: 10.1016/j.jmb.2006.07.021 Gerdes, K., Christensen, S. K., and Lobner-Olesen, A. (2005). Prokaryotic toxin-antitoxin stress response loci. Nat. Rev. Microbiol. 3, 371–382. doi: 10.1038/nrmicro1147 Crooke, E., Brundage, L., Rice, M., and Wickner, W. (1988a). ProOmpA spon- taneously folds in a membrane assembly competent state which trigger factor stabilizes. EMBO J. 7, 1831–1835. Goeders, N., and Van Melderen, L. (2014). Toxin-antitoxin systems as multilevel interaction systems. Toxins (Basel) 6, 304–324. doi: 10.3390/toxins6010304 Crooke, E., Guthrie, B., Lecker, S., Lill, R., and Wickner, W. (1988b). ProOmpA is stabilized for membrane translocation by either puriﬁed E. coli trigger factor or canine signal recognition particle. Cell 54, 1003–1011. doi: 10.1016/0092- 8674(88)90115-8 Gouridis, G., Karamanou, S., Gelis, I., Kalodimos, C. G., and Economou, A. (2009). Signal peptides are allosteric activators of the protein translocase. Nature 462, 363–367. doi: 10.1038/nature08559 Grabowicz, M., Yeh, J., and Silhavy, T. J. (2013). Dominant negative lptE mutation that supports a role for LptE as a plug in the LptD barrel. J. Bacteriol. 195, 1327–1334. doi: 10.1128/JB.02142-12 Crooke, E., and Wickner, W. (1987). Trigger factor: a soluble protein that folds pro- OmpA into a membrane-assembly-competent form. Proc. Natl. Acad. Sci. U.S.A. 84, 5216–5220. doi: 10.1073/pnas.84.15.5216 Gupta, A. (2009). Killing activity and rescue function of genome-wide toxin- antitoxin loci of Mycobacterium tuberculosis. FEMS Microbiol. Lett. 290, 45–53. doi: 10.1111/j.1574-6968.2008.01400.x Dalbey, R. E., Wang, P., and Kuhn, A. (2011). Assembly of bacterial inner membrane proteins. Annu. Rev. Biochem. 80, 161–187. doi: 10.1146/annurev-biochem- 060409-092524 Guthrie, B., and Wickner, W. (1990). Trigger factor depletion or overproduction causes defective cell division but does not block protein export. J. Bacteriol. 172, 5555–5562. Danese, P. N., Murphy, C. K., and Silhavy, T. J. (1995). Multicopy suppression of cold-sensitive sec mutations in Escherichia coli. J. Bacteriol. 177, 4969–4973. Hardy, S. REFERENCES Agashe, V. R., Guha, S., Chang, H. C., Genevaux, P., Hayer-Hartl, M., Stemp, M., et al. (2004). Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed. Cell 117, 199–209. doi: 10.1016/S0092-8674(04)00299-5 Altman, E., Kumamoto, C. A., and Emr, S. D. (1991). Heat-shock proteins can substitute for SecB function during protein export in Escherichia coli. EMBO J. 10, 239–245. Baars, L., Ytterberg, A. J., Drew, D., Wagner, S., Thilo, C., Van Wijk, K. J., et al. (2006). Deﬁning the role of the Escherichia coli chaperone SecB using comparative proteomics. J. Biol. Chem. 281, 10024–10034. doi: 10.1074/jbc.M509929200 Babu, M., Diaz-Mejia, J. J., Vlasblom, J., Gagarinova, A., Phanse, S., Graham, C., et al. (2011). Genetic interaction maps in Escherichia coli reveal functional crosstalk among cell envelope biogenesis pathways. PLoS Genet. 7:e1002377. doi: 10.1371/journal.pgen.1002377 Bechtluft, P., Nouwen, N., Tans, S. J., and Driessen, A. J. M. (2010). SecB-a chaperone dedicated to protein translocation. Mol. Biosyst. 6, 620–627. doi: 10.1039/b915435c December 2014 \| Volume 5 \| Article 666 \| 9 www.frontiersin.org Multitasking SecB Sala et al. Bechtluft, P., Van Leeuwen, R. G. H., Tyreman, M., Tomkiewicz, D., Nouwen, N., Tepper, H. L., et al. (2007). Direct observation of chaperone-induced changes in a protein folding pathway. Science 318, 1458–1461. doi: 10.1126/science. 1144972 du Plessis, D. J., Nouwen, N., and Driessen, A. J. (2011). The Sec translocase. Biochim. Biophys. Acta 1808, 851–865. doi: 10.1016/j.bbamem.2010.08.016 Fekkes, P., De Wit, J. G., Boorsma, A., Friesen, R. H., and Driessen, A. J. (1999). Fekkes, P., De Wit, J. G., Boorsma, A., Friesen, R. H., and Driessen, A. J. (1999). Zinc stabilizes the SecB binding site of SecA. Biochemistry 38, 5111–5116. doi: 10.1021/bi982818r Zinc stabilizes the SecB binding site of SecA. Biochemistry 38, 5111–5116. doi: 10.1021/bi982818r Bordes, P., Cirinesi, A. M., Ummels, R., Sala, A., Sakr, S., Bitter, W., et al. (2011). SecB-like chaperone controls a toxin-antitoxin stress-responsive system in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A. 108, 8438–8443. doi: 10.1073/pnas.1101189108 Fekkes, P., De Wit, J. G., Van Der Wolk, J. P., Kimsey, H. H., Kumamoto, C. A., and Driessen, A. J. (1998). Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to SecA. Mol. Microbiol. 29, 1179–1190. doi: 10.1046/j.1365-2958.1998.00997.x Brehmer, D., Rudiger, S., Gassler, C. S., Klostermeier, D., Packschies, L., Reinstein, J., et al. (2001). REFERENCES doi: 10.1110/ps.051889506 Khisty,V. J., Munske, G. R., and Randall, L. L. (1995). Mapping of the binding frame for the chaperone SecB within a natural ligand, galactose-binding protein. J. Biol. Chem. 270, 25920–25927. doi: 10.1074/jbc.270.43.25920 Margolis, J. J., El-Etr, S., Joubert, L. M., Moore, E., Robison, R., Rasley, A., et al. (2010). Contributions of Francisella tularensis subsp. novicida chitinases and Sec secretion system to bioﬁlm formation on chitin. Appl. Environ. Microbiol. 76, 596–608. doi: 10.1128/AEM.02037-09 Kim, Y. E., Hipp, M. S., Bracher, A., Hayer-Hartl, M., and Hartl, F. U. (2013). Molecular chaperone functions in protein folding and proteostasis. Annu. Rev. Biochem. 82, 323–355. doi: 10.1146/annurev-biochem-060208-092442 Mcfarland, L., Francetic, O., and Kumamoto, C.A. (1993). A mutation of Escherichia coli SecA protein that partially compensates for the absence of SecB. J. Bacteriol. 175, 2255–2262. Kimsey, H. H., Dagarag, M. D., and Kumamoto, C. A. (1995). Diverse effects of mutation on the activity of the Escherichia coli export chaperone SecB. J. Biol. Chem. 270, 22831–22835. doi: 10.1074/jbc.270.39.22831 Miller, A., Wang, L., and Kendall, D. A. (2002). SecB modulates the nucleotide- bound state of SecA and stimulates ATPase activity. Biochemistry 41, 5325–5332. doi: 10.1021/bi025639p Knoblauch, N. T., Rudiger, S., Schonfeld, H. J., Driessen, A. J., Schneider-Mergener, J., and Bukau, B. (1999). Substrate speciﬁcity of the SecB chaperone. J. Biol. Chem. 274, 34219–34225. doi: 10.1074/jbc.274.48.34219 Muller, J. P. (1996). Inﬂuence of impaired chaperone or secretion function on SecB production in Escherichia coli. J. Bacteriol. 178, 6097–6104. Kramer, G., Boehringer, D., Ban, N., and Bukau, B. (2009). The ribosome as a plat- form for co-translational processing, folding and targeting of newly synthesized proteins. Nat. Struct. Mol. Biol. 16, 589–597. doi: 10.1038/nsmb.1614 Muren, E. M., Suciu, D., Topping, T. B., Kumamoto, C. A., and Randall, L. L. (1999). Mutational alterations in the homotetrameric chaperone SecB that impli- cate the structure as dimer of dimers. J. Biol. Chem. 274, 19397–19402. doi: 10.1074/jbc.274.27.19397 Kramer,G.,Rauch,T.,Rist,W.,Vorderwulbecke,S.,Patzelt,H.,Schulze-Specking,A., et al. (2002). L23 protein functions as a chaperone docking site on the ribosome. Nature 419, 171–174. doi: 10.1038/nature01047 Nichols, R. J., Sen, S., Choo, Y. J., Beltrao, P., Zietek, M., Chaba, R., et al. (2011). Phenotypic landscape of a bacterial cell. Cell 144, 143–156. doi: 10.1016/j.cell.2010.11.052 Kumamoto, C. A., and Beckwith, J. (1983). Mutations in a new gene, secB, cause defective protein localization in Escherichia coli. J. Bacteriol. 154, 253–260. O’Brien,E.P.,Christodoulou,J.,Vendruscolo,M.,andDobson,C.M.(2012). REFERENCES 88, 2874–2878. doi: 10.1073/pnas.88.7.2874 Holland, I. B., Schmitt, L., and Young, J. (2005). Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway (review). Mol. Membr. Biol. 22, 29–39. doi: 10.1080/09687860500042013 Lill, R., Crooke, E., Guthrie, B., and Wickner, W. (1988). The “trigger factor cycle” includes ribosomes, presecretory proteins, and the plasma membrane. Cell 54, 1013–1018. doi: 10.1016/0092-8674(88)90116-X Huber, D., Rajagopalan, N., Preissler, S., Rocco, M. A., Merz, F., Kramer, G., et al. (2011). SecA interacts with ribosomes in order to facilitate posttranslational translocation in bacteria. Mol. Cell 41, 343–353. doi: 10.1016/j.molcel.2010.12.028 Liu, G., Topping, T. B., and Randall, L. L. (1989). Physiological role during export for the retardation of folding by the leader peptide of maltose-binding protein. Liu, G., Topping, T. B., and Randall, L. L. (1989). Physiological role during export for the retardation of folding by the leader peptide of maltose-binding protein. Proc. Natl. Acad. Sci. U.S.A. 86, 9213–9217. doi: 10.1073/pnas.86.23.9213 Jin, T., and Inouye, M. (1995). Identiﬁcation of the genes in multicopy plasmids affecting ompC and ompF expression in Escherichia coli. FEMS Microbiol. Lett. 133, 225–231. doi: 10.1111/j.1574-6968.1995.tb07889.x Luirink, J., and Sinning, I. (2004). SRP-mediated protein targeting: struc- ture and function revisited. Biochim. Biophys. Acta 1694, 17–35. doi: 10.1016/j.bbamcr.2004.03.013 Kaiser, C. M., Chang, H. C., Agashe, V. R., Lakshmipathy, S. K., Etchells, S. A., Hayer-Hartl, M., et al. (2006). Real-time observation of trigger factor function on translating ribosomes. Nature 444, 455–460. doi: 10.1038/nature 05225 Mah, N., Perez-Iratxeta, C., and Andrade-Navarro, M. A. (2010). Outer membrane pore protein prediction in mycobacteria using genomic comparison. Microbiology 156, 2506–2515. doi: 10.1099/mic.0.040089-0 Makarova, K. S., Wolf, Y. I., and Koonin, E. V. (2009). Comprehensive comparative- genomic analysis of type 2 toxin-antitoxin systems and related mobile stress response systems in prokaryotes. Biol. Direct 4, 19. doi: 10.1186/1745- 6150-4-19 Karamyshev, A. L., and Johnson, A. E. (2005). Selective SecA association with signal sequences in ribosome-bound nascent chains: a potential role for SecA in ribo- some targeting to the bacterial membrane. J. Biol. Chem. 280, 37930–37940. doi: 10.1074/jbc.M509100200 Kerner, M. J., Naylor, D. J., Ishihama, Y., Maier, T., Chang, H. C., Stines, A. P., et al. (2005). Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 122, 209–220. doi: 10.1016/j.cell.2005.05.028 Marani, P.,Wagner, S., Baars, L., Genevaux, P., De Gier, J.W., Nilsson, I., et al. (2006). New Escherichia coli outer membrane proteins identiﬁed through prediction and experimental veriﬁcation. Protein Sci. 15, 884–889. REFERENCES J., and Randall, L. L. (1991). A kinetic partitioning model of selective binding of nonnative proteins by the bacterial chaperone SecB. Science 251, 439– 443. doi: 10.1126/science.1989077 Debarbieux, L., and Wandersman, C. (2001). Folded HasA inhibits its own secretion through its ABC exporter. EMBO J. 20, 4657–4663. doi: 10.1093/emboj/20.17.4657 Dekker, C., De Kruijff, B., and Gros, P. (2003). Crystal structure of SecB from Escherichia coli. J. Struct. Biol. 144, 313–319. doi: 10.1016/j.jsb.2003.09.012 Harrison, C. J., Hayer-Hartl, M., Di Liberto, M., Hartl, F., and Kuriyan, J. (1997). Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK. Science 276, 431–435. doi: 10.1126/science.276.5311.431 Delepelaire, P. (2004). Type I secretion in gram-negative bacteria. Biochim. Biophys. Acta 1694, 149–161. doi: 10.1016/j.bbamcr.2004.05.001 Acta 1694, 149–161. doi: 10.1016/j.bbamcr.2004.05.001 Delepelaire, P., and Wandersman, C. (1998). The SecB chaperone is involved in the secretion of the Serratia marcescens HasA protein through an ABC transporter. EMBO J. 17, 936–944. doi: 10.1093/emboj/17.4.936 Hartl, F. U., Lecker, S., Schiebel, E., Hendrick, J. P., and Wickner, W. (1990). The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane. Cell 63, 269–279. doi: 10.1016/0092-8674(90)90160-G den Blaauwen, T., Terpetschnig, E., Lakowicz, J. R., and Driessen, A. J. (1997). Interaction of SecB with soluble SecA. FEBS Lett. 416, 35–38. doi: 10.1016/S0014- 5793(97)01142-3 Hayashi, S., and Wu, H. C. (1985). Accumulation of prolipoprotein in Escherichia coli mutants defective in protein secretion. J. Bacteriol. 161, 949–954. Hoffmann, A., Becker, A. H., Zachmann-Brand, B., Deuerling, E., Bukau, B., and Kramer, G. (2012). Concerted action of the ribosome and the associated chaper- one trigger factor conﬁnes nascent polypeptide folding. Mol. Cell 48, 63–74. doi: 10.1016/j.molcel.2012.07.018 Derman, A. I., Puziss, J. W., Bassford, P. J. Jr., and Beckwith, J. (1993). A signal sequence is not required for protein export in prlA mutants of Escherichia coli. EMBO J. 12, 879–888. Deuerling, E., Schulze-Specking, A., Tomoyasu, T., Mogk, A., and Bukau, B. (1999). Trigger factor and DnaK cooperate in folding of newly synthesized proteins. Nature 400, 693–696. doi: 10.1038/23301 Hoffmann, A., Bukau, B., and Kramer, G. (2010). Structure and function of the molecular chaperone Trigger Factor. Biochim. Biophys. Acta 1803, 650–661. doi: 10.1016/j.bbamcr.2010.01.017 December 2014 \| Volume 5 \| Article 666 \| 10 Frontiers in Microbiology \| Microbial Physiology and Metabolism Sala et al. Multitasking SecB ATPase activity of DnaK. Proc. Natl. Acad. Sci. U.S.A. REFERENCES Trigger factor slows co-translational folding through kinetic trapping while sterically protecting the nascent chain from aberrant cytosolic interactions. J. Am. Chem. Soc. 134, 10920–10932. doi: 10.1021/ja302305u Kumamoto, C. A., and Beckwith, J. (1985). Evidence for speciﬁcity at an early step in protein export in Escherichia coli. J. Bacteriol. 163, 267–274. Kusters, I., Van Den Bogaart, G., Kedrov, A., Krasnikov, V., Fulyani, F., Pool- man, B., et al. (2011). Quaternary structure of SecA in solution and bound to SecYEG probed at the single molecule level. Structure 19, 430–439. doi: 10.1016/j.str.2010.12.016 j Oh, E., Becker, A. H., Sandikci, A., Huber, D., Chaba, R., Gloge, F., et al. (2011). Selectiveribosomeproﬁlingrevealsthecotranslationalchaperoneactionof trigger Oh, E., Becker, A. H., Sandikci, A., Huber, D., Chaba, R., Gloge, F., et al. (2011). Selectiveribosomeproﬁlingrevealsthecotranslationalchaperoneactionof trigger factor in vivo. Cell 147, 1295–1308. doi: 10.1016/j.cell.2011.10.044 Kusters, R., De Vrije, T., Breukink, E., and De Kruijff, B. (1989). SecB protein stabilizes a translocation-competent state of puriﬁed prePhoE protein. J. Biol. Chem. 264, 20827–20830. Palmer, T., and Berks, B. C. (2012). The twin-arginine translocation (Tat) protein export pathway. Nat. Rev. Microbiol. 10, 483–496. doi: 10.1038/nrmicro2814 Patel, R., Smith, S. M., and Robinson, C. (2014). Protein transport by the bacterial Tat pathway. Biochim. Biophys. Acta 1843, 1620–1628. doi: 10.1016/j.bbamcr.2014.02.013 Kusukawa, N., Yura, T., Ueguchi, C., Akiyama, Y., and Ito, K. (1989). Effects of mutations in heat-shock genes groES and groEL on protein export in Escherichia coli. EMBO J. 8, 3517–3521. Phillips, G. J., and Silhavy, T. J. (1990). Heat-shock proteins DnaK and GroEL facilitate export of LacZ hybrid proteins in E. coli. Nature 344, 882–884. doi: 10.1038/344882a0 Laminet, A. A., Kumamoto, C. A., and Pluckthun, A. (1991). Folding in vitro and transport in vivo of pre-Beta-Lactamase are SecB independent. Mol. Microbiol. 5, 117–122. doi: 10.1111/j.1365-2958.1991.tb01832.x Powers, E. L., and Randall, L. L. (1995). Export of periplasmic galactose-binding protein in Escherichia coli depends on the chaperone SecB. J. Bacteriol. 177, 1906– 1907. Lecker, S., Lill, R., Ziegelhoffer, T., Georgopoulos, C., Bassford, P. J., Kumamoto, C. A., et al. (1989). Three pure chaperone proteins of Escherichia coli SecB, trigger factor and groel-form soluble complexes with precursor proteins in vitro. EMBO J. 8, 2703–2709. Qi, H. Y., Hyndman, J. B., and Bernstein, H. D. (2002). DnaK promotes the selective export of outer membrane protein precursors in SecA-deﬁcient Escherichia coli. J. Biol. Chem. 277, 51077–51083. doi: 10.1074/jbc.M209238200 Lee, H. C., and Bernstein, H. D. (2002). REFERENCES S., Luirink, J., Harms, N., Schwager, F., Georgopoulos, C., and Genevaux, P. (2004). SecB is a bona ﬁde generalized chaperone in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 101, 7583–7588. doi: 10.1073/pnas.0402398101 Acad. Sci. U.S.A. 101, 7583–7588. doi: 10.1073/pnas.0402398101 Rigel, N. W., Gibbons, H. S., Mccann, J. R., Mcdonough, J. A., Kurtz, S., and Braunstein, M. (2009). The accessory SecA2 system of mycobacteria requires ATP binding and the canonical SecA1. J. Biol. Chem. 284, 9927–9936. doi: 10.1074/jbc.M900325200 Valent, Q. A., Kendall, D. A., High, S., Kusters, R., Oudega, B., and Luirink, J. (1995). Early events in preprotein recognition in E. coli: interaction of SRP and trigger factor with nascent polypeptides. EMBO J. 14, 5494–5505. Rudiger, S., Germeroth, L., Schneider-Mergener, J., and Bukau, B. (1997). Sub- strate speciﬁcity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 16, 1501–1507. doi: 10.1093/emboj/16.7.1501 van der Sluis, E. O., and Driessen, A. J. (2006). Stepwise evolution of the Sec machinery in Proteobacteria. Trends Microbiol. 14, 105–108. doi: 10.1016/j.tim.2006.01.009 Saibil, H. R., Fenton, W. A., Clare, D. K., and Horwich, A. L. (2013). Struc- ture and allostery of the chaperonin GroEL. J. Mol. Biol. 425, 1476–1487. doi: 10.1016/j.jmb.2012.11.028 Watanabe, T., Hayashi, S., and Wu, H. C. (1988). Synthesis and export of the outer membrane lipoprotein in Escherichia coli mutants defective in generalized protein export. J. Bacteriol. 170, 4001–4007. Wei, S. Q., and Stader, J. (1994). A new suppressor of a lamB signal sequence muta- tion, prlZ1, maps to 69 minutes on the Escherichia coli chromosome. J. Bacteriol. 176, 5704–5710. Saio, T., Guan, X., Rossi, P., Economou, A., and Kalodimos, C. G. (2014). Structural basis for protein antiaggregation activity of the trigger factor chaperone. Science 344:1250494. doi: 10.1126/science.1250494 Wild, J., Altman, E., Yura, T., and Gross, C. A. (1992). DnaK and DnaJ heat shock proteins participate in protein export in Escherichia coli. Genes Dev. 6, 1165–1172. doi: 10.1101/gad.6.7.1165 Sakr, S., Cirinesi, A. M., Ullers, R. S., Schwager, F., Georgopoulos, C., and Genevaux, P. (2010). Lon protease quality control of presecretory proteins in Escherichia coli and its dependence on the SecB and DnaJ (Hsp40) chaperones. J. Biol. Chem. 285, 23504–23512. doi: 10.1074/jbc.M110.133058 Wild, J., Walter, W. A., Gross, C. A., and Altman, E. (1993). Accumulation of secretory protein precursors in Escherichia coli induces the heat shock response. J. Bacteriol. 175, 3992–3997. Sala, A., Bordes, P., Fichant, G., and Genevaux, P. (2013a). REFERENCES Trigger factor retards protein export in Escherichia coli. J. Biol. Chem. 277, 43527–43535. doi: 10.1074/jbc.M205950200 Raine,A., Lovmar, M.,Wikberg, J., and Ehrenberg, M. (2006). Trigger factor binding to ribosomes with nascent peptide chains of varying lengths and sequences. J. Biol. Chem. 281, 28033–28038. doi: 10.1074/jbc.M605753200 Letoffe, S., Ghigo, J. M., and Wandersman, C. (1994). Secretion of the Serratia marcescens HasA protein by an ABC transporter. J. Bacteriol. 176, 5372–5377. Ramage, H. R., Connolly, L. E., and Cox, J. S. (2009). Comprehensive functional analysis of Mycobacterium tuberculosis toxin-antitoxin systems: implications for pathogenesis, stress responses, and evolution. PLoS Genet. 5:e1000767. doi: 10.1371/journal.pgen.1000767 Lewis, K. (2010). Persister cells. Annu. Rev. Microbiol. 64, 357–372. doi: 10.1146/annurev.micro.112408.134306 Liberek, K., Marszalek, J., Ang, D., Georgopoulos, C., and Zylicz, M. (1991). Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate December 2014 \| Volume 5 \| Article 666 \| 11 www.frontiersin.org Sala et al. Multitasking SecB Randall, L. L., Crane, J. M., Lilly, A. A., Liu, G. P., Mao, C. F., Patel, C. N., et al. (2005). Asymmetric binding between SecA and SecB two symmetric pro- teins: implications for function in export. J. Mol. Biol. 348, 479–489. doi: 10.1016/j.jmb.2005.02.036 Trun, N. J., and Silhavy, T. J. (1989). PrlC, a suppressor of signal sequence muta- tions in Escherichia coli, can direct the insertion of the signal sequence into the membrane. J. Mol. Biol. 205, 665–676. doi: 10.1016/0022-2836(89)90312-4 Trun, N. J., Stader, J., Lupas,A., Kumamoto, C., and Silhavy, T. J. (1988). Two cellular components, PrlA and SecB, that recognize different sequence determinants are required for efﬁcient protein export. J. Bacteriol. 170, 5928–5930. Randall, L. L., Crane, J. M., Liu, G., and Hardy, S. J. (2004). Sites of interaction between SecA and the chaperone SecB, two proteins involved in export. Protein Sci. 13, 1124–1133. doi: 10.1110/ps.03410104 Ullers, R. S., Ang, D., Schwager, F., Georgopoulos, C., and Genevaux, P. (2007). Trigger factor can antagonize both SecB and DnaK/DnaJ chaperone func- tions in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 104, 3101–3106. doi: 10.1073/pnas.0608232104 Randall, L. L., and Hardy, S. J. (1995). High selectivity with low speciﬁcity: how SecB has solved the paradox of chaperone binding. Trends Biochem. Sci. 20, 65–69. doi: 10.1016/S0968-0004(00)88959-8 Randall, L. L., and Hardy, S. J. (2002). SecB, one small chaperone in the complex milieu of the cell. Cell. Mol. Life Sci. 59, 1617–1623. doi: 10.1007/PL00012488 Ullers, R. REFERENCES “Toxin-antitoxin loci in Mycobacterium tuberculosis,” in Prokaryotic Toxin-Antitoxins, ed. K. Gerdes (Berlin: Springer), 295–314. Wolff, N., Sapriel, G., Bodenreider, C., Chaffotte, A., and Delepelaire, P. (2003). Antifolding activity of the SecB chaperone is essential for secretion of HasA, a quickly folding ABC pathway substrate. J. Biol. Chem. 278, 38247–38253. doi: 10.1074/jbc.M302322200 Sala, A., Calderon, V., Bordes, P., and Genevaux, P. (2013b). TAC from Mycobac- terium tuberculosis: a paradigm for stress-responsive toxin-antitoxin systems controlled by SecB-like chaperones. Cell Stress Chaperones 18, 129–135. doi: 10.1007/s12192-012-0396-5 Woodbury, R. L., Topping, T. B., Diamond, D. L., Suciu, D., Kumamoto, C. A., Hardy, S. J., et al. (2000). Complexes between protein export chaperone SecB and SecA – Evidence for separate sites on SecA providing binding energy and regulatory interactions. J. Biol. Chem. 275, 24191–24198. doi: 10.1074/jbc. M002885200 Sapriel, G.,Wandersman, C., and Delepelaire, P. (2002). The N terminus of the HasA protein and the SecB chaperone cooperate in the efﬁcient targeting and secretion of HasA via the ATP-binding cassette transporter. J. Biol. Chem. 277, 6726–6732. doi: 10.1074/jbc.M108632200 Xu,Z.,Knafels,J.D.,andYoshino,K.(2000). Crystalstructureof thebacterialprotein export chaperone SecB. Nat. Struct. Biol. 7, 1172–1177. doi: 10.1038/82040 Sapriel, G., Wandersman, C., and Delepelaire, P. (2003). The SecB chaperone is bifunctional in Serratia marcescens: SecB is involved in the Sec pathway and required for HasA secretion by the ABC transporter. J. Bacteriol. 185, 80–88. doi: 10.1128/JB.185.1.80-88.2003 Yamaguchi, Y., and Inouye, M. (2011). Regulation of growth and death in Escherichia coli by toxin-antitoxin systems. Nat. Rev. Microbiol. 9, 779–790. doi: 10.1038/nrmicro2651 Saraogi, I., and Shan, S. O. (2014). Co-translational protein targeting to the bacterial membrane. Biochim. Biophys. Acta 1843, 1433–1441. doi: 10.1016/j.bbamcr.2013.10.013 Zhou, J., and Xu, Z. (2003). Structural determinants of SecB recognition by SecA in bacterial protein translocation. Nat. Struct. Biol. 10, 942–947. doi: 10.1038/nsb980 Schuessler, D. L., Cortes, T., Fivian-Hughes, A. S., Lougheed, K. E., Harvey, E., Buxton, R. S., et al. (2013). Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA. Mol. Microbiol. 90, 195–207. doi: 10.1111/mmi.12358 Zuber, B., Chami, M., Houssin, C., Dubochet, J., Grifﬁths, G., and Daffe, M. (2008). Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state. J. Bacteriol. 190, 5672–5680. REFERENCES doi: 10.1128/JB.01919-07 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Shimizu, H., Nishiyama, K., and Tokuda, H. (1997). Expression of gpsA encod- ing biosynthetic sn-glycerol 3-phosphate dehydrogenase suppresses both the LB- phenotype of a secB null mutant and the cold-sensitive phenotype of a secG null mutant. Mol. Microbiol. 26, 1013–1021. doi: 10.1046/j.1365-2958.1997.6392003.x Singh, R., Kraft, C., Jaiswal, R., Sejwal, K., Kasaragod, V. B., Kuper, J., et al. (2014). Cryo-electron microscopic structure of SecA protein bound to the 70S ribosome. Shimizu, H., Nishiyama, K., and Tokuda, H. (1997). Expression of gpsA encod- ing biosynthetic sn-glycerol 3-phosphate dehydrogenase suppresses both the LB- phenotype of a secB null mutant and the cold-sensitive phenotype of a secG null mutant. Mol. Microbiol. 26, 1013–1021. doi: 10.1046/j.1365-2958.1997.6392003.x mutant. Mol. Microbiol. 26, 1013–1021. doi: 10.1046/j.1365-2958.1997.6392003.x Received: 25 September 2014; accepted: 17 November 2014; published online: 05 December 2014. Singh, R., Kraft, C., Jaiswal, R., Sejwal, K., Kasaragod, V. B., Kuper, J., et al. (2014). Cryo-electron microscopic structure of SecA protein bound to the 70S ribosome. J. Biol. Chem. 289, 7190–7199. Citation: Sala A, Bordes P and Genevaux P (2014) Multitasking SecB chaperones in bacteria. Front. Microbiol. 5:666. doi: 10.3389/fmicb.2014.00666 Snyder,W. B., and Silhavy, T. J. (1992). Enhanced export of beta-galactosidase fusion proteins in prlF mutants is Lon dependent. J. Bacteriol. 174, 5661–5668. This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology. Sullivan, J. T., Young, E. F., Mccann, J. R., and Braunstein, M. (2012). The Mycobac- terium tuberculosis SecA2 system subverts phagosome maturation to promote growth in macrophages. Infect. Immun. 80, 996–1006. doi: 10.1128/IAI.05987-11 Copyright © 2014 Sala, Bordes and Genevaux. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, dis- tribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Teter, S. A., Houry, W. A., Ang, D., Tradler, T., Rockabrand, D., Fischer, G., et al. (1999). Polypeptide ﬂux through bacterial Hsp70: DnaK cooperates with trigger factor in chaperoning nascent chains. Cell 97, 755–765. REFERENCES doi: 10.1016/S0092- 8674(00)80787-4 December 2014 \| Volume 5 \| Article 666 \| 12 Frontiers in Microbiology \| Microbial Physiology and Metabolism
https://openalex.org/W4248701455	https://europepmc.org/articles/pmc3928443?pdf=render	English	null	Correction: Differential Effect of Actomyosin Relaxation on the Dynamic Properties of Focal Adhesion Proteins	PloS one	2,014	cc-by	261	1. Lavelin I, Wolfenson H, Patla I, Henis YI, Medalia O, et al. (2013) Differential Effect of Actomyosin Relaxation on the Dynamic Properties of Focal Adhesion Proteins. PLoS ONE 8(9): e73549. doi:10.1371/journal.pone.0073549 Correction Correction: Differential Effect of Actomyosin Relaxation on the Dynamic Properties of Focal Adhesion Proteins The PLOS ONE Staff A funding organization and grant were incorrectly omitted from the Funding statement. The Funding statement should read: "This study was supported by the National Institutes of Health (NIH) Common Fund Nanomedicine Program (PN2 EY016586), an NoE Systems Microscopy Grant no. 258068, funded through the European Union’s Seventh Framework Programme (to BG), an ERC Starting Grant 243047 INCEL (to OM), and a Deutsche Forschungsgemeinschaft-Deutsch-Israelische Projektkooperation grant (KL 1948/1-1, GA 309/10-1) (to YIH). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript." Correction: Differential Effect of Actomyosin Relaxation on the Dynamic Properties of Focal Adhesion Proteins The PLOS ONE Staff PLOS ONE \| www.plosone.org Reference Citation: The PLOS ONE Staff (2014) Correction: Differential Effect of Actomyosin Relaxation on the Dynamic Properties of Focal Adhesion Proteins. PLoS ONE 9(2): e90269. doi:10.1371/journal.pone.0090269 Published February 18, 2014 Published February 18, 2014 Published February 18, 2014 Copyright: 2014 The PLOS ONE Staff. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org February 2014 \| Volume 9 \| Issue 2 \| e90269 1
https://openalex.org/W4372351219	https://www.nature.com/articles/s41420-023-01450-w.pdf	English	null	Silencing Notch4 promotes tumorigenesis and inhibits metastasis of triple-negative breast cancer via Nanog and Cdc42	Cell death discovery	2,023	cc-by	8,779	INTRODUCTION events, including neutropenia, fatigue, nausea, leukopenia, diar- rhea, alopecia, anemia, and vomiting, were reported in almost all breast cancer patients in PF-03084014 clinical trials [20–22]. Similar low efﬁcacy and adverse events were reported in the other pan- Notch inhibitor RO4929097 clinical trials with breast cancer patients [23–26]. These studies showed that pan-Notch inhibitors failed to prove clinical beneﬁts and led to adverse events in clinical trials with breast cancer. However, a phase I clinical trial of PF- 06650808, a Notch3-speciﬁc antibody–drug conjugate, has demon- strated a reasonable safety proﬁle and early efﬁcacy signs of antitumor activity in patients with advanced breast cancer [27]. In this study, we report that the knockdown of Notch4 in TNBC cells promotes tumorigenesis and inhibits metastasis. Mechanistically, the knockdown of Notch4 upregulated Nanog, a transcription factor required for maintaining the pluripotency of embryonic stem cells [28], via Hes3 to promote tumorigenesis. Unexpectedly, the knock- down of Notch4 inhibits metastasis through downregulation of Cdc42 expression, which rendered diffuse distribution around the nucleus of Vimentin location, but not the reduction of Vimentin expression to suppress EMT shift. Hence, our demonstration showed that silencing Notch4 in TNBC cells promotes tumorigenesis and inhibits metastasis, indicating targeting Notch4 possibly not being a potential therapeutic strategy for TNBC. events, including neutropenia, fatigue, nausea, leukopenia, diar- rhea, alopecia, anemia, and vomiting, were reported in almost all breast cancer patients in PF-03084014 clinical trials [20–22]. Similar low efﬁcacy and adverse events were reported in the other pan- Notch inhibitor RO4929097 clinical trials with breast cancer patients [23–26]. These studies showed that pan-Notch inhibitors failed to prove clinical beneﬁts and led to adverse events in clinical trials with breast cancer. However, a phase I clinical trial of PF- 06650808, a Notch3-speciﬁc antibody–drug conjugate, has demon- strated a reasonable safety proﬁle and early efﬁcacy signs of antitumor activity in patients with advanced breast cancer [27]. Notch signal is implicated in several cancers, and aberrant activation is linked to cancer initiation, metastasis, and resistance to cancer therapy [1]. Mounting evidence pointed out that individual Notch proteins had distinct activities and outcomes in the subtype of breast cancer. For example, while Notch3 signaling exhibits the oncogenic potential in a murine breast cancer model and promotes luminal breast cancer metastasis [2–4], over- expression of Notch3 inhibits proliferation and metastasis in TNBC and is associated with better survival in TNBC patients [5–11]. www.nature.com/cddiscovery ARTICLE OPEN Silencing Notch4 promotes tumorigenesis and inhibits metastasis of triple-negative breast cancer via Nanog and Cdc42 Yuan Tian 1,2,4, Peipei Zhang 1,2,4, Yajun Mou 1,2, Wenxiu Yang1, Junhong Zhang1, Qing Li 3 a © The Author(s) 2023 © The Author(s) 2023 Elucidation of individual Notch protein biology in speciﬁc cancer is crucial to develop safe, effective, and tumor-selective Notch- targeting therapeutic reagents for clinical use [1]. Here, we explored the Notch4 function in triple-negative breast cancer (TNBC). We found that silencing Notch4 enhanced tumorigenic ability in TNBC cells via upregulating Nanog expression, a pluripotency factor of embryonic stem cells. Intriguingly, silencing Notch4 in TNBC cells suppressed metastasis via downregulating Cdc42 expression, a key molecular for cell polarity formation. Notably, downregulation of Cdc42 expression affected Vimentin distribution, but not Vimentin expression to inhibit EMT shift. Collectively, our results show that silencing Notch4 enhances tumorigenesis and inhibits metastasis in TNBC, indicating that targeting Notch4 may not be a potential strategy for drug discovery in TNBC. Cell Death Discovery (2023) 9:148 ; https://doi.org/10.1038/s41420-023-01450-w Received: 5 March 2023 Revised: 18 April 2023 Accepted: 24 April 2023 1Department of Pathology, The Afﬁliated Hospital of Guizhou Medical University, 550004 Guiyang, Guizhou, China. 2Clinical Research Center, The Afﬁliated Hospital of Guizhou Medical University, 550004 Guiyang, Guizhou, China. 3Department of Orthopedics, The Afﬁliated Hospital of Guizhou Medical University, 550004 Guiyang, Guizhou, China. 4These authors contributed equally: Yuan Tian, Peipei Zhang. ✉email: douxw@gmc.edu.cn INTRODUCTION However, other researchers argued that the Notch3 signal path- way promoted the growth and metastasis of TNBC [12, 13]. In TNBC cells, Notch4 maintained cancer stemness and promoted metastasis, indicating that Notch4 could be a potential therapeutic target for TNBC treatment [14, 15]. In ER+ breast cancer, Notch4 increases breast cancer stem cell activation and endocrine therapy resistance, proposing that Notch4 inhibition will overcome endocrine therapy resistance and decrease recurrence in ER+ breast cancer [16, 17]. In this study, we report that the knockdown of Notch4 in TNBC cells promotes tumorigenesis and inhibits metastasis. Mechanistically, the knockdown of Notch4 upregulated Nanog, a transcription factor required for maintaining the pluripotency of embryonic stem cells [28], via Hes3 to promote tumorigenesis. Unexpectedly, the knock- down of Notch4 inhibits metastasis through downregulation of Cdc42 expression, which rendered diffuse distribution around the nucleus of Vimentin location, but not the reduction of Vimentin expression to suppress EMT shift. Hence, our demonstration showed that silencing Notch4 in TNBC cells promotes tumorigenesis and inhibits metastasis, indicating targeting Notch4 possibly not being a potential therapeutic strategy for TNBC. Elucidation of the activities and outcomes of individual Notch in breast cancer subtypes aid in developing a novel therapeutic strategy that targets Notch-driven cancers with efﬁcacy and diminishes adverse events caused by nonspeciﬁc inhibition of Notch signal [1, 18]. For example, γ- Secretase inhibitor (GSI) PF- 03084014, a pan-Notch inhibitor, was reported to have no signiﬁcant clinical efﬁcacy in phase I clinical trials in patients with breast cancer. Although GSIs are ineffective as a monotherapy, the preclinical ability of GSIs to reduce cancer stem cell proliferative capacity and curtail chemoresistance has led to an investigation of clinical combinatorial strategies [19]. But treatment-related adverse Received: 5 March 2023 Revised: 18 April 2023 Accepted: 24 April 2023 RESULTS Low Notch4 expression seemingly predicts the recurrence in TNBC patients with lymph node metastasis Based on previous research and our results, we found that Notch4 was expressed predominantly in TNBC cell lines MDA-MB-231 and Low Notch4 expression seemingly predicts the recu TNBC patients with lymph node metastasis p y p Based on previous research and our results, we found that Notch4 was expressed predominantly in TNBC cell lines MDA-MB-231 and Received: 5 March 2023 Revised: 18 April 2023 Accepted: 24 April 2023 Ofﬁcial journal of CDDpress g. 1 Role of Notch4 in TNBC. A Western blot analysis showed that Notch4 was dominantly expressed in TNBC cells. B, C, F, G OS/RFS in reast cancer and TNBC patients according to Notch4 expression in Kaplan–Meier plotter database. D, H OS/RFS in breast cancer and TNBC atients without lymph node metastasis according to Notch4 expression in Kaplan–Meier plotter database. E, I OS/RFS in breast cancer and NBC patients with lymph node metastasis according to Notch4 expression in Kaplan–Meier plotter database. Y. Tian et al. Y. Tian et al. 2 Fig. 1 Role of Notch4 in TNBC. A Western blot analysis showed that Notch4 was dominantly expressed in TNBC cells. B, C, F, G OS/RFS in breast cancer and TNBC patients according to Notch4 expression in Kaplan–Meier plotter database. D, H OS/RFS in breast cancer and TNBC patients without lymph node metastasis according to Notch4 expression in Kaplan–Meier plotter database. E, I OS/RFS in breast cancer and TNBC patients with lymph node metastasis according to Notch4 expression in Kaplan–Meier plotter database. associated with breast cancer recurrence in TNBC with lymph node metastasis. Hs578T but not in luminal breast cancer cell lines MCF-7 and T47D (Fig. 1A) [6, 14]. To investigate the potential role of Notch4 in TNBC, we evaluated the prognostic value of Notch4 mRNA in a large online gene chip database of breast tumors from 1880 patients for overall survival (OS) and 4934 patients for recurrence- free survival (RFS) in Kaplan–Meier plotter. We found that there is almost no correlation between Notch4 expression and OS/RFS in breast cancer and TNBC patients (Fig. 1B, C, F, G). Although higher expression of Notch4 mRNA is seemingly associated with poorer OS and RFS in breast cancer and TNBC patients without lymph node metastasis (Fig. RESULTS 1D, H), lower expression of Notch4 mRNA is seemingly correlated with poorer OS and RFS in breast cancer and TNBC patients with lymph node metastasis (Fig. 1E, I). These results seemingly suggested that lower expression of Notch4 is Hs578T but not in luminal breast cancer cell lines MCF-7 and T47D (Fig. 1A) [6, 14]. To investigate the potential role of Notch4 in TNBC, we evaluated the prognostic value of Notch4 mRNA in a large online gene chip database of breast tumors from 1880 patients for overall survival (OS) and 4934 patients for recurrence- free survival (RFS) in Kaplan–Meier plotter. We found that there is almost no correlation between Notch4 expression and OS/RFS in breast cancer and TNBC patients (Fig. 1B, C, F, G). Although higher expression of Notch4 mRNA is seemingly associated with poorer OS and RFS in breast cancer and TNBC patients without lymph node metastasis (Fig. 1D, H), lower expression of Notch4 mRNA is seemingly correlated with poorer OS and RFS in breast cancer and TNBC patients with lymph node metastasis (Fig. 1E, I). These results seemingly suggested that lower expression of Notch4 is Silencing Notch4 promotes tumorigenesis in TNBC cells Silencing Notch4 promotes tumorigenesis in TNBC cells Since low Notch4 expression is seemingly associated with TNBC recurrence with lymph node metastasis based on the Kaplan–Meier plotter, we explored whether the knockdown of Notch4 in the TNBC cell line promoted tumorigenesis in TNBC cells. We generated a lentivirus expressing Notch4 shRNA. The knockdown of Notch4 in TNBC MDA-MB-231 cells was conﬁrmed using real-time RT-PCR, western blot, and promoter reporter assay (Fig. 2A–C). To conﬁrm that Notch4 depletion promotes tumor- igenesis in TNBC cells, we assessed the number of clones by clone expansion assay after 10 days’ culture. The knockdown of Notch4 Cell Death Discovery (2023) 9:148 Y. Tian et al. 2 The knockdown of Notch4 of TNBC cells promotes tumorigenesis in vitro. A The knockdown of Notch4 in MDA-MB-231 cells was ﬁrmed by real-time PCR. *P < 0.01. B Notch4 knockdown in MDA-MB-231 cells was conﬁrmed by western blot analysis. C The knockdown Notch4 in MDA-MB-231 cells was conﬁrmed by promoter reporter assay. P < 0.05. D, E Notch4 knockdown in MDA-MB-231 cells increased ne formation. *P < 0.01. F Overexpressing Notch4 in M231shN4 cells by real-time PCR. P < 0.05. G Overexpressing Notch4 in M231shN4 s by western blot analysis. H, I Overexpressing Notch4 in M231shN4 cells suppressed clone formation. P < 0.05. Y. Tian et al. 3 3 Fig. 2 The knockdown of Notch4 of TNBC cells promotes tumorigenesis in vitro. A The knockdown of Notch4 in MDA-MB-231 cells was conﬁrmed by real-time PCR. P < 0.01. B Notch4 knockdown in MDA-MB-231 cells was conﬁrmed by western blot analysis. C The knockdown of Notch4 in MDA-MB-231 cells was conﬁrmed by promoter reporter assay. P < 0.05. D, E Notch4 knockdown in MDA-MB-231 cells increased clone formation. *P < 0.01. F Overexpressing Notch4 in M231shN4 cells by real-time PCR. P < 0.05. G Overexpressing Notch4 in M231shN4 cells by western blot analysis. H, I Overexpressing Notch4 in M231shN4 cells suppressed clone formation. P < 0.05. in TNBC cell MDAMB231 signiﬁcantly enhanced clone formation (Fig. 2D, E). Similar results were obtained in TNBC cells BT549 and Hs578T depleting Notch4 (Supplementary Fig. 2SA–SF). To further explore whether Notch4 inhibits tumorigenesis in TNBC, we overexpressed Notch4 in TNBC cell MDAMB231 with depleting Notch4. The overexpression of Notch4 was conﬁrmed by real-time RT-PCR and western blot. Silencing Notch4 promotes tumorigenesis in TNBC cells As expected, Notch4 overexpression in TNBC cell MDAMB231 with depleting Notch4 suppressed clone formation by clone expansion assay after 10 days’ culture. Thus, these results strongly supported the idea that the knockdown of Notch4 promotes the tumorigenic ability of TNBC cells in vitro. As expected, the knockdown of Notch4 expression in TNBC cells enhanced Vimentin expression, a mesenchymal status marker, and E-cadherin expression, an epithelial status marker, indicating that depletion of Notch4 expression in TNBC cells boosted breast cancer metastasis (Fig. 3A, B). However, the knockdown of Notch4 shifted the spindle-shaped morphology to cobblestone- like morphology, suggesting that Notch4 depletion inhibited breast cancer metastasis (Fig. 3D). Indeed, migration assay and invasion assay conﬁrmed that silencing Notch4 inhibited migra- tion and invasion of TNBC cells (Fig. 3E–H). Similar results were obtained in TNBC cells BT549 and Hs578T depleting Notch4 (Supplementary Fig. 3SA–SD). Vimentin is at the core of epithelial- mesenchymal transition and metastasis in cancer cells [33, 34]. In order to explore the paradoxical outcome of Notch4 knockdown on migration, we detected the location of Vimentin expression in Notch4-depleted TNBC cells. Surprisingly, the depletion of Notch4 in TNBC cells shifted the location of Vimentin location from clustered on one side by the nucleus to distributed diffusely around the nucleus, implying that Notch4 knockdown in TNBC Y. Tian et al. 5 Fig. 3 Notch4 depletion of TNBC cells suppresses migration in vitro. A The knockdown of Notch4 in TNBC cells enhanced E-cadherin and Vimentin expression by real-time PCR. P < 0.05. B Notch4 knockdown in TNBC cells enhanced Vimentin expression by West blot analysis. C Notch4 depletion in TNBC cells affects Vimentin location by immunoﬂuorescence staining. D Knocking down Notch4 in TNBC cells affects cellular morphology. E, F Notch4 depletion in TNBC cells suppressed migration assessed by migration assay. P < 0.01. G, H The knockdown of Notch4 in TNBC cells inhibited invasion assessed by invasion assay. P < 0.01. I Overexpressing Notch4 in M231shN4 cells rescued cellular morphology alteration. J Notch4 overexpression in M231shN4 cells rescued the expression of Vimentin location. K, L Overexpressing Notch4 enhanced migration of M231shN4 cells assessed by migration assay. P < 0.01. M, N Notch4 overexpression promoted the invasion of M231shN4 cells assessed by invasion assay. P < 0.01. restoration of Notch4 in M231/shN4C cells rescued HES3 gene downregulation in M231/shN4C cells (Fig. 4K). To further explore the role of Hes3 in the tumorigenesis of TNBC cells, we overexpressed the HES3 gene in TNBC M231shN4C cells (Fig. 4L, M). As expected, HES3 overexpression in M231shN4C cells boosted clone formation by clone expansion assay after 10 days of culture (Fig. 4N, O). To verify the regulation of Nanog by Hes3 in TNBC cells, we examined whether Hes3 overexpression in M231shN4C cells regulated Nanog expression by real-time PCR and promoter reporter assay. Real-time PCR revealed that Hes3 overexpression in M231shN4C cells repressed the expression of the Nanog gene in TNBC cells (Fig. 4L). Promoter reporter assay showed that HES3 overexpression in M231shN4C cells decreased Fireﬂy luciferase expression, indicating that Hes3 inhibited the expression of Nanog in TNBC cells (Fig. 4P). Since Nanog expression was signiﬁcantly regulated by Hes3, we examined whether Hes3 directly regulated Nanog expression using ChIP assay. As expected, Hes3 levels on the NANOG promoter were signiﬁcantly increased in M231shN4C/ HES3 cells compared with shN4C/myc cells (Fig. 4Q, R). Taken together, these results suggest that Hes3 mediated Notch4 regulating Nanog expression and tumorigenesis in TNBC cells. in vitro as assessed by migration assay and invasion assay (Fig. 3K–N). Moreover, overexpressing Notch4 shifted the location of Vimentin expression from uniformly around the nucleus to clustered on one side by the nucleus in part of TNBC M231shN4 cells (Fig. 3J). Y. Tian et al. Collectively, these results indicate that silencing Notch4 in TNBC cells suppresses breast cancer migration in vitro via the location of Vimentin expression. Silencing Notch4 upregulates Nanog expression to promote tumorigenesis of TNBC cells through Hes3 tu o ge es s o BC ce s t oug es3 As we know, accumulating evidence has demonstrated that TNBC cells harbor the highest percentage of cancer stem cells [35, 36]. Previously, Notch pathway activation was found to be not required in undifferentiated human embryonic stem cells (hESCs) but required for hESCs to initiate all three embryonic germ layers differentiation. Blockade of Notch activation repressed expression of molecular markers of three embryonic germ layers differentia- tion but strongly promoted expression of pluripotency transcrip- tion factors like Nanog [37]. Since the depletion of Notch4 promoted the tumorigenic ability of TNBC cells, we inferred that the pluripotency factors may play an important role in promoting the tumorigenic ability of TNBC cells depleting Notch4 [38, 39]. Interestingly, real-time PCR revealed that the depletion of Notch4 in TNBC cells did not affect pluripotency factor OCT4 expression or reduce the expression of pluripotency factors SOX2, KLF4, or C- myc, but strongly enhanced pluripotency factor NANOG expression (Fig. 4A). To further examine the regulation of Nanog by Notch4, we overexpressed Notch4 in TNBC cell M231shN4C. As expected, real-time PCR displayed that the overexpression of Notch4 in TNBC cells suppressed Nanog expression at the mRNA level (Fig. 4B). Previously, the expression of Nanog was found to be implicated in breast cancer tumorigenesis [20, 21]. To examine whether Nanog played an important role in the tumorigenesis of TNBC cells, we generated lentivirus overexpressing Nanog. The overexpression of Nanog in TNBC cells MDA-MB-231 was conﬁrmed by real-time PCR and western blot analysis (Fig. 4C, D). To verify that overexpressing Nanog promotes tumorigenesis in TNBC cells, a clone expansion assay was utilized to assess the clone formation efﬁciency. After 10 days of culture, we found that overexpressing Nanog in TNBC cell MDA-MB-231 signiﬁcantly increased clone formation ability (Fig. 4E, F). To further examine the role of Nanog in the tumorigenesis of TNBC cells, we knocked down Nanog expression by siRNA in TNBC cells M231/shN4C (Fig. 4G). As expected, the knockdown of Nanog expression in TNBC cells M231/shN4C signiﬁcantly reduced the clone formation efﬁciency (Fig. 4H, I). Collectively, these results demonstrate that upregulation of Nanog expression mediated tumorigenesis of Notch4 depletion in TNBC cells. Silencing Notch4 inhibiting metastasis in TNBC cells g g Since the knockdown of Notch4 promoted clone formation efﬁciency in TNBC cells and tumorigenicity was linked to epithelial-mesenchymal transition (EMT) and metastasis [29, 30], we explored whether Notch4 knockdown played a role in EMT and metastasis in TNBC cells. Partial EMT cells were found to be required for metastasis formation and cancer progression [31, 32]. Cell Death Discovery (2023) 9:148 Y. Tian et al. mentin location around the nucleus uniformly tastasis of breast cancer (Fig. 3C). In order to test Notch4 on metastasis, we overexpressed Notch4 N4 cells. As expected, overexpression of Notch4 in M231shN4C shifted cobblestone-like morpholo spindle-shaped morphology, suggesting that breast cancer metastasis (Fig. 3I). Consistently, Notch4 promoted migration and invasion of TN Y. Tian et al. 4 M231shN4C shifted cobblestone-like morphology to the slightly spindle-shaped morphology, suggesting that Notch4 enhanced breast cancer metastasis (Fig. 3I). Consistently, overexpression of Notch4 promoted migration and invasion of TNBC M231shN4 cells Cell Death Discovery (2023) 9:148 cells rendered Vimentin location around the nucleus uniformly and inhibited metastasis of breast cancer (Fig. 3C). In order to test the inﬂuence of Notch4 on metastasis, we overexpressed Notch4 in TNBC M231shN4 cells. As expected, overexpression of Notch4 in M231shN4C shifted cobblestone-like morphology to the slightly spindle-shaped morphology, suggesting that Notch4 enhanced breast cancer metastasis (Fig. 3I). Consistently, overexpression of Notch4 promoted migration and invasion of TNBC M231shN4 cells M231shN4C shifted cobblestone-like morphology to the slightly spindle-shaped morphology, suggesting that Notch4 enhanced breast cancer metastasis (Fig. 3I). Consistently, overexpression of Notch4 promoted migration and invasion of TNBC M231shN4 cells Cell Death Discovery (2023) 9:148 Cell Death Discovery (2023) 9:148 Silencing Notch4 controls Vimentin location to suppress metastasis of TNBC cells through Cdc42 Real-time PCR revealed that the Cell Death Discovery (2023) 9:148 Y. Tian et al. ig. 4 Silencing Notch4 upregulates Nanog expression to promote tumorigenesis of TNBC cells through Hes3. A Silencing Notch4 in TNBC ells affected mRNA expression of pluripotency factors by real-time PCR. P < 0.01. B Overexpression of Notch4 in M231shN4C cells inhibited Nanog expression by real-time PCR. P < 0.05, *P < 0.01. C Overexpression of NANOG in MDA-MB-231 cells was conﬁrmed by real-time PCR P < 0.01. D Overexpression of Nanog in MDA-MB-231 cells was conﬁrmed by western blot analysis. E, F Overexpression of Nanog in MDA MB-231 cells enhanced clone formation. *P < 0.01. G The knockdown of Nanog in M231shN4C using siRNA was conﬁrmed by real-time PCR P < 0.01. H, I Depletion of Nanog expression in M231/shN4C cells reduced clone formation. P < 0.01. J Silencing Notch4 in TNBC cell ffected mRNA expression of downstream target genes by real-time PCR. P < 0.01. K Overexpression of Notch4 in M231shN4C cell upregulated HES3 expression by real-time PCR. P < 0.01. L Overexpression of HES3 in M231shN4C cells inhibited Nanog expression by real ime PCR. P < 0.01. M Overexpression of Hes3/myc in M231shN4C cells was conﬁrmed using myc tag antibody by western blot analysis N, O Overexpression of Hes3 in M231shN4C cells reduced clone formation. P < 0.01. P Overexpression of HES3 in M231shN4C cell uppressed NANOG promoter activity by promoter reporter assay. Promoter activity was expressed as the ratio of ﬁreﬂy luciferase/Renilla uciferase. P < 0.05. Q, R Hes3 bound to NANOG promoter by ChIP analysis. ChIP assay was done in M231shN4C cells transfected with contro myc or fusion gene HES3-myc and NANOG promoter was ChIP-ed with anti-myc or IgG control. Y. Tian et al. 6 Fig. 4 Silencing Notch4 upregulates Nanog expression to promote tumorigenesis of TNBC cells through Hes3. A Silencing Notch4 in TNBC cells affected mRNA expression of pluripotency factors by real-time PCR. *P < 0.01. B Overexpression of Notch4 in M231shN4C cells inhibited Nanog expression by real-time PCR. P < 0.05, P < 0.01. C Overexpression of NANOG in MDA-MB-231 cells was conﬁrmed by real-time PCR. P < 0.01. D Overexpression of Nanog in MDA-MB-231 cells was conﬁrmed by western blot analysis. E, F Overexpression of Nanog in MDA- MB-231 cells enhanced clone formation. P < 0.01. Silencing Notch4 controls Vimentin location to suppress metastasis of TNBC cells through Cdc42 Previously research showed that cell polarity is essential for effective cell mobility, and Cdc42 plays a central role in cell polarity establishment by recruiting its effectors to form a crescent-shaped polar cap, which orchestrates rearrangements of the actin cytoskeleton [42–44]. They found that the polar cap number increases with Cdc42 cytoplasmic concentration, and deposition of Cdc42 to the cap further stimulates actin accumulation at this site. Therefore, we inferred that silencing Notch4 in TNBC cells may cause a decrease of polar cap via downregulating Cdc42 cytoplasmic concentration and inhibits cell mobility. Expectedly, the knockdown of Notch4 in TNBC cells signiﬁcantly reduced Cdc42 expression at the mRNA level and protein level (Fig. 5A, B). To further explore the molecular mechanisms underlying the regulation of Cdc42 expression by Notch4, a CDC42 promoter vector was created, and promoter reporter assays were performed. The results conﬁrmed that the depletion of Notch4 in TNBC cells suppressed CDC42 promoter activity, indicating that silencing Notch4 in TNBC cells reduced Cdc42 expression (Fig. 5C). In order to examine whether Notch4 regulates Cdc42 expression in TNBC cells, we performed ChIP assay to assess the binding of Notch4 at CDC42 gene promoter. Indeed, we found that Notch4 is bound to the promoter region of the CDC42 gene (Fig. 5D, E). Taken together, these results indicate that Notch4 regulates Cdc42 expression in TNBC cells. To examine whether Cdc42 played an important role in the migration of TNBC cells, we overexpressed Cdc42 in M231shN4C cells. The overexpression of Cdc42 in M231shN4C cells was conﬁrmed by real-time PCR and western blot analysis (Fig. 5F, G). Interestingly, Cdc42 overexpression in M231shN4C cells changed the cobblestone-like morphology to the slightly spindle-shaped morphology, indicating that Cdc42 overexpression in M231shN4C cells enhanced the mobility of breast cancer (Fig. 5H). As expected, Cdc42 overexpression increased migration and invasion of TNBC HES and HEY genes encode a large family of basic helix-loop- helix repressor genes that are Notch protein effectors and play central roles in maintaining progenitor cells in an undifferentiated state [40, 41]. Real-time PCR revealed that silencing Notch4 in TNBC cells strongly repressed the expression of HES3 gene (Fig. 4J). We did not detect the expression of HES2 or HES5 genes both in Notch4 depletion or control TNBC cells. To further examine the regulation of HES3 gene expression by Notch4, we overexpressed Notch4 in TNBC M231/shN4C cells. Cell Death Discovery (2023) 9:148 Silencing Notch4 controls Vimentin location to suppress metastasis of TNBC cells through Cdc42 G The knockdown of Nanog in M231shN4C using siRNA was conﬁrmed by real-time PCR. P < 0.01. H, I Depletion of Nanog expression in M231/shN4C cells reduced clone formation. P < 0.01. J Silencing Notch4 in TNBC cells affected mRNA expression of downstream target genes by real-time PCR. P < 0.01. K Overexpression of Notch4 in M231shN4C cells upregulated HES3 expression by real-time PCR. P < 0.01. L Overexpression of HES3 in M231shN4C cells inhibited Nanog expression by real- time PCR. P < 0.01. M Overexpression of Hes3/myc in M231shN4C cells was conﬁrmed using myc tag antibody by western blot analysis. N, O Overexpression of Hes3 in M231shN4C cells reduced clone formation. P < 0.01. P Overexpression of HES3 in M231shN4C cells suppressed NANOG promoter activity by promoter reporter assay. Promoter activity was expressed as the ratio of ﬁreﬂy luciferase/Renilla luciferase. P < 0.05. Q, R Hes3 bound to NANOG promoter by ChIP analysis. ChIP assay was done in M231shN4C cells transfected with control myc or fusion gene HES3-myc and NANOG promoter was ChIP-ed with anti-myc or IgG control. M231shN4 cells in vitro as assessed by migration assay and invasion assay (Fig. 5I–L). Moreover, Cdc42 overexpression altered the location of Vimentin expression from uniformly around the nucleus to clustered on one side by the nucleus in part of TNBC M231shN4 cells (Fig. 5M). The role of Vimentin was found to stabilize microtubule network organization and enhance cell M231shN4 cells in vitro as assessed by migration assay and invasion assay (Fig. 5I–L). Moreover, Cdc42 overexpression altered the location of Vimentin expression from uniformly around the nucleus to clustered on one side by the nucleus in part of TNBC M231shN4 cells (Fig. 5M). The role of Vimentin was found to stabilize microtubule network organization and enhance cell polarity in directed cell migration. The location of Vimentin in the cells is oriented in the direction of migration at the wound edge. The knockdown of Vimentin causes a defect of polarity and reduces migrating speed [45]. Vimentin ﬁbers were aligned with ﬁbroblast branching and migration direction in ﬁbroblasts and required for the anisotropic orientation of traction stresses, Cell Death Discovery (2023) 9:148 indicating that vimentin’s role in cell motility is to govern the alignment of traction stresses that permit single-cell migration [46]. Silencing Notch4 controls Vimentin location to suppress metastasis of TNBC cells through Cdc42 Since Cdc42 and Vimentin are all implicated in the formation Complementation assay and Fireﬂy Luciferase Complementat Imaging Assay to detect Cdc42-Vimentin interaction [47]. T fusion proteins at a low level were utilized in the Bimolecu Fig. 5 Silencing Notch4 controls Vimentin location to suppress metastasis of TNBC cells through Cdc42. A The knockdown of Notch4 TNBC cells inhibited Cdc42 expression by real-time PCR. P < 0.01. B The knockdown of Notch4 inhibited Cdc42 expression in TNBC cells western blot analysis. C Notch4 depletion in TNBC cells suppressed Cdc42 promoter activity by promoter reporter assay. Promoter activity w expressed as the ratio of ﬁreﬂy luciferase/Renilla luciferase. P < 0.01. D, E Notch4 bound to Cdc42 promoter by ChIP analysis. ChIP assay w done in MDA-MB-231 cells, and the Cdc42 promoter was ChIP-ed with anti-Notch4 or IgG control. F Overexpression of Cdc42 in M231shN cells by real-time PCR. P < 0.01. G Overexpression of Cdc42 in M231shN4C cells by western blot analysis. H Overexpression of Cdc42 M231shN4C cells changed the morphology from a cobblestone-like one to a slightly spindle-shaped one. I Overexpression of Cdc42 M231shN4C cells altered the location of Vimentin expression. J, K Overexpression of Cdc42 in M231shN4C cells suppressed migrat assessed by migration assay. P < 0.01. L, M Overexpression of Cdc42 in M231shN4C cells inhibited invasion assessed by invasion ass P < 0.01. M Overexpression of Cdc42 in M231shN4C cells altered the location of Vimentin expression. N The binding of Cdc42 to Vimen was conﬁrmed by the Bimolecular Fluorescence Complementation assay. O The binding of Cdc42 to Vimentin was conﬁrmed by Fire Luciferase Complementation Imaging Assay. Y. Tian et al. Y. Tian et al. 7 7 Fig. 5 Silencing Notch4 controls Vimentin location to suppress metastasis of TNBC cells through Cdc42. A The knockdown of Notch4 in TNBC cells inhibited Cdc42 expression by real-time PCR. P < 0.01. B The knockdown of Notch4 inhibited Cdc42 expression in TNBC cells by western blot analysis. C Notch4 depletion in TNBC cells suppressed Cdc42 promoter activity by promoter reporter assay. Promoter activity was expressed as the ratio of ﬁreﬂy luciferase/Renilla luciferase. P < 0.01. D, E Notch4 bound to Cdc42 promoter by ChIP analysis. ChIP assay was done in MDA-MB-231 cells, and the Cdc42 promoter was ChIP-ed with anti-Notch4 or IgG control. F Overexpression of Cdc42 in M231shN4C cells by real-time PCR. P < 0.01. DISCUSSION The Notch signaling cascade is an evolutionarily conserved pathway that plays a key role in cell fate decision during development [18]. Disorder of Notch signal pathway regulation can result in cancer occurrence and metastasis [49]. Evidence suggests that four Notch proteins have distinct activities and outcomes in tumorigenesis [50]. Previous research showed that Notch4 played a role in maintaining quiescent mesenchymal-like breast cancer stem cells and promoting epithelial-mesenchymal transition in TNBC cells, indicating that Notch4 is a potential therapeutic target for TNBC [14, 15]. In our study, we found that silencing Notch4 inhibited migration and invasion ability but promoted tumorigenic ability in TNBC cells. Interestingly, we found that silencing Notch4 upregulated pluripotency factor Nanog expression to enhance tumorigenesis in TNBC cells. More importantly, silencing Notch4 affected Vimentin distribution but not Vimentin expression to inhibit migration via Cdc42 expression in TNBC cells. Therefore, our study suggests that silencing Notch4 in TNBC cells inhibits migration ability and enhances tumorigen- esis ability via Cdc42 and Nanog, respectively. Increasing evidence proved that the Notch signal pathway plays a vital role in tumorigenesis and the progression of breast cancer. Therapeutic strategy for targeting Notch signal has been studied for decades, but clinical outcome fails to meet the expectation. GSIs, like AL101, PF-03084014, and RO4929097, have been developed as a treatment for triple-negative breast cancer in phase II clinical trials, but most of these studies were terminated since the serious adverse events and poorer outcomes [60]. The reasons for adverse events of GSIs may be due to the inhibition of all Notch proteins. Thereby, clarity of activity and function of each individual Notch protein in a subtype of breast cancer facilitates the development of Notch-targeting therapeutic agents with safety and efﬁcacy. Previous research showed that the knockdown of Notch4 in TNBCS suppressed cancer stem cell activity and migration, indicating that Notch4 is a potential target for tumor intervention [14]. Our results showed that silencing Notch4 suppressed migration but enhanced tumorigenic ability in TNBC cells, suggesting that Notch4 should not be a potential therapeutic target for TNBC. Previous research showed that high expression of Nanog was a poorer prognostic factor in the TNBC subtype [51]. GFP reporter to sort Nanog+ cells from TNBC cells signiﬁcantly increased cancer stem cell activity in vitro and tumorigenic ability in vivo [52]. Silencing Notch4 controls Vimentin location to suppress metastasis of TNBC cells through Cdc42 In order to elucidate the molecular mechanisms of Vimentin distributing concentrated on one side of the nucleus and regulating cell polarity formation, Cdc42 was found to be at the center of cell polarity formation in previous research [56]. Cdc42 protein was localized and clustered in the cytoplasm and promotes cell polarity formation, which is essential for cell motility and migration [42, 57, 58]. Our results showed that silencing Notch4 reduced Cdc42 expression and rendered diffuse distribution of Vimentin expression in TNBC cells. Rescued assay showed that overexpression of Notch4 upregulated Cdc42 expression and caused Vimentin localization in a bell shape on one side of the nucleus in M231shN4C cells. More importantly, Cdc42 overexpression in TNBC M231shN4 cells altered Vimentin expression from diffuse distribution around the nucleus to a concentrated location on one side by the nucleus, indicating that Cdc42 mediates Notch4 regulating Vimentin localization and polarity formation in TNBC cells. Considering that Cdc42 binds to the microtube to foster polarity establishment and the binding of Vimentin to the microtube, we inferred that Cdc42 possibly binds to Vimentin to promote the concentrated distribution of Vimentin location and polarity formation [45, 59]. As expected, the Bimolecular Fluorescence Complementation assay and Fireﬂy Luciferase Complementation Imaging Assay conﬁrmed that Cdc42 is bound to Vimentin in TNBC cells. Taken together, our results suggest that silencing Notch4 in TNBC cells decreased Cdc42 expression and rendered diffuse distribution around the nucleus of Vimentin expression, thereby leading to cell polarity disappearance and migration inhibition. Bimolecular Fluorescence Complementation assay, we observed the Venus ﬂuorescence in the group, including Cdc42 fusion protein and Vimentin fusion protein, under the ﬂuorescence microscope, indicating that the binding of Cdc42 to Vimentin occurred. As a control, we did not observe the Venus ﬂuorescence in the group including control vectors VN173 and VC155, indicating that 10 ng split ﬂuorescent protein fragments did not self-assemble by random collision and generate false-positive signals (Fig. 5N). Similarly, in the Fireﬂy Luciferase Complementa- tion Imaging Assay, we detected Bioluminescent imaging in the group transfecting with Cdc42 fusion protein and Vimentin fusion protein by IVIS Lumina III imaging system, conﬁrming the binding of Cdc42 protein to Vimentin. We also did not detect Biolumines- cent imaging in the control group transfecting with 10 ng control vector PCAG-IN-PD1-CLuc and 10 ng control vector pcDNA3.1- NLuc-dsRED for 3 days (Fig. 5O). Silencing Notch4 controls Vimentin location to suppress metastasis of TNBC cells through Cdc42 G Overexpression of Cdc42 in M231shN4C cells by western blot analysis. H Overexpression of Cdc42 in M231shN4C cells changed the morphology from a cobblestone-like one to a slightly spindle-shaped one. I Overexpression of Cdc42 in M231shN4C cells altered the location of Vimentin expression. J, K Overexpression of Cdc42 in M231shN4C cells suppressed migration assessed by migration assay. P < 0.01. L, M Overexpression of Cdc42 in M231shN4C cells inhibited invasion assessed by invasion assay.. P < 0.01. M Overexpression of Cdc42 in M231shN4C cells altered the location of Vimentin expression. N The binding of Cdc42 to Vimentin was conﬁrmed by the Bimolecular Fluorescence Complementation assay. O The binding of Cdc42 to Vimentin was conﬁrmed by Fireﬂy Luciferase Complementation Imaging Assay. Complementation assay and Fireﬂy Luciferase Complementation Imaging Assay to detect Cdc42-Vimentin interaction [47]. The fusion proteins at a low level were utilized in the Bimolecular Fluorescence Complementation assay to avoid self-assembly and remove the generation of false-positive signals. In each sample, 10 ng vector is the appropriate quantity to remove the false positive without affecting speciﬁc signals [48]. Indeed, in the indicating that vimentin’s role in cell motility is to govern the alignment of traction stresses that permit single-cell migration [46]. Since Cdc42 and Vimentin are all implicated in the formation of crescent-shaped polar cap of cell polarity and migration, we proposed that Cdc42 may bind to Vimentin and govern the location of Vimentin expression in TNBC cells. To test this possibility, we utilized the Bimolecular Fluorescence Cell Death Discovery (2023) 9:148 Y. Tian et al. 8 that silencing Notch4 in TNBC cells rendered Vimentin expression from concentrated distribution and bell-shaped location on one side of the nucleus to diffuse distribution around the nucleus. Rescued assay showed that overexpression of Notch4 in M231shN4C cells caused Vimentin expression from diffuse distributed around the nucleus to concentrated distribution and bell-shaped location on one side of the nucleus again. Combined with previous research, we inferred that concentrated distribution of intracellular Vimentin on one side of the nucleus induced cell polarity formation and possibly drag nucleus mobility that promotes cell migration, and silencing Notch4 results in diffuse distribution of intracellular Vimentin around the nucleus and inhibits cell migration in TNBC cells. Silencing Notch4 controls Vimentin location to suppress metastasis of TNBC cells through Cdc42 Taken together, our results suggest that Cdc42 protein mediated the function of silencing Notch4 in TNBC cells that inhibits Vimentin cluster formation and metastasis. DISCUSSION Cx26 complexed with the pluripotency transcription factor NANOG and focal adhesion kinase (FAK) promotes cancer stem cell self- renewal of TNBC cells but not luminal breast cancer cells [53]. These studies indicated that Nanog plays an important role in cancer stem cell activity in TNBC cells. Our results showed that silencing Notch4 upregulated Nanog expression and enhanced the tumorigenic ability of TNBC cells, consistent with previous ﬁndings of Nanog controlling cancer stem cell activity in TNBC cells. In the gland-like structures formed by human renal cell carcinoma cells, the intracellular localization of Vimentin is most concentrated at the basal pole, indicating cell polarity has a close relationship with the subcellular localization of Vimentin [54]. Vimentin ﬁbers were aligned with ﬁbroblast branching and migration direction, suggesting that the role of Vimentin in cell motility is to control the alignment of traction stresses that permit single-cell migration [46]. Vimentin interacting with microtubules contributes to polarity maintenance in migrating cells. Knockdown of Vimentin prevents cell polarization and migration properly during wound healing, indicating the role of Vimentin in maintaining cell polarization in general and migration in particular [45, 55]. These studies implied that Vimentin plays an important role in cell polarity formation and migration. Our results showed REFERENCES Phase I study of the gamma secretase inhibitor PF-03084014 in combination with docetaxel in patients with advanced triple-negative breast cancer. Oncotarget. 2017;8:2320–8. 5. Zhang X, Liu X, Luo J, Xiao W, Ye X, Chen M, et al. Notch3 inhibits epithelial- mesenchymal transition by activating Kibra-mediated Hippo/YAP signaling in breast cancer epithelial cells. Oncogenesis. 2016;5:e269. 5. Zhang X, Liu X, Luo J, Xiao W, Ye X, Chen M, et al. Notch3 inhibits epithelial- mesenchymal transition by activating Kibra-mediated Hippo/YAP signaling in breast cancer epithelial cells. Oncogenesis. 2016;5:e269. 22. Curigliano G, Aftimos PG, Dees EC, LoRusso P, Pegram MD, Awada A, et al. Phase I dose-ﬁnding study of the gamma secretase inhibitor PF-03084014 (PF-4014) in combination with docetaxel in patients (pts) with advanced triple-negative breast cancer (TNBC). J Clin Oncol. 2015;33:1068. 6. Dou XW, Liang YK, Lin HY, Wei XL, Zhang YQ, Bai JW, et al. Notch3 maintains luminal phenotype and suppresses tumorigenesis and metastasis of breast cancer via trans-activating estrogen receptor-alpha. Theranostics. 2017;7:4041–56. 6. Dou XW, Liang YK, Lin HY, Wei XL, Zhang YQ, Bai JW, et al. Notch3 maintains luminal phenotype and suppresses tumorigenesis and metastasis of breast cancer via trans-activating estrogen receptor-alpha. Theranostics. 2017;7:4041–56. 23. Means-Powell J, Mayer I, Ismail-Khan R, Del Valle L, Tonetti D, Abramson V, et al. A phase Ib dose escalation trial of RO4929097 (a γ-secretase inhibitor) in combi- nation with exemestane in patients with ER + metastatic breast cancer (MBC). Clin Breast Cancer. 2022;22:103–14. 7. Lin HY, Liang YK, Dou XW, Chen CF, Wei XL, Zeng D, et al. Notch3 inhibits epithelial-mesenchymal transition in breast cancer via a novel mechanism, upregulation of GATA-3 expression. Oncogenesis. 2018;7:59. 8. Liang YK, Lin HY, Dou XW, Chen M, Wei XL, Zhang YQ, et al. MiR-221/222 promote epithelial-mesenchymal transition by targeting Notch3 in breast cancer cell lines. NPJ Breast Cancer. 2018;4:20. 24. Sardesai S, Badawi M, Mrozek E, Morgan E, Phelps M, Stephens J, et al. A phase I study of an oral selective gamma secretase (GS) inhibitor RO4929097 in combi- nation with neoadjuvant paclitaxel and carboplatin in triple negative breast cancer. Invest New Drugs. 2020;38:1400–10. 9. Chen CF, Dou XW, Liang YK, Lin HY, Bai JW, Zhang XX, et al. Notch3 over- expression causes arrest of cell cycle progression by inducing Cdh1 expression in human breast cancer cells. Cell Cycle. 2016;15:432–40. 25. Means-Powell JA, Mayer IA, Ismail-Khan R, Del Valle L, Tonetti D, Abramson VG, et al. REFERENCES dependent cancer stem cell activity. Cell Rep. 2015;12:1968–77. 17. Harrison H, Farnie G, Howell SJ, Rock RE, Stylianou S, Brennan KR, et al. Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor. Cancer Res. 2010;70:709–18. 1. Majumder S, Crabtree JS, Golde TE, Minter LM, Osborne BA, Miele L. Targeting Notch in oncology: the path forward. Nat Rev Drug Discov. 2021;20:125–44. 1. Majumder S, Crabtree JS, Golde TE, Minter LM, Osborne BA, Miele L. Targeting Notch in oncology: the path forward. Nat Rev Drug Discov. 2021;20:125–44. 2. Hu C, Diévart A, Lupien M, Calvo E, Tremblay G, Jolicoeur P. Overexpression of activated murine Notch1 and Notch3 in transgenic mice blocks mammary gland development and induces mammary tumors. Am J Pathol. 2006;168:973–90. 2. Hu C, Diévart A, Lupien M, Calvo E, Tremblay G, Jolicoeur P. Overexpression of activated murine Notch1 and Notch3 in transgenic mice blocks mammary gland development and induces mammary tumors. Am J Pathol. 2006;168:973–90. 18. Nowell CS, Radtke F. Notch as a tumour suppressor. Nat Rev Cancer. 2017;17:145–59. 19. Zhang CC, Yan Z, Zong Q, Fang DD, Painter C, Zhang Q, et al. Synergistic effect of the gamma-secretase inhibitor PF-03084014 and docetaxel in breast cancer models. Stem Cells Transl Med. 2013;2:233–42. 3. Sansone P, Ceccarelli C, Berishaj M, Chang Q, Rajasekhar VK, Perna F, et al. Self- renewal of CD133(hi) cells by IL6/Notch3 signalling regulates endocrine resis- tance in metastatic breast cancer. Nat Commun. 2016;7:10442. 3. Sansone P, Ceccarelli C, Berishaj M, Chang Q, Rajasekhar VK, Perna F, et al. Self- renewal of CD133(hi) cells by IL6/Notch3 signalling regulates endocrine resis- tance in metastatic breast cancer. Nat Commun. 2016;7:10442. 20. Messersmith WA, Shapiro GI, Cleary JM, Jimeno A, Dasari A, Huang B, et al. A phase I, dose-ﬁnding study in patients with advanced solid malignancies of the oral gamma-secretase inhibitor PF-03084014. Clin Cancer Res. 2015;21:60–7. 4. Landor SKJ, Santio NM, Eccleshall WB, Paramonov VM, Gagliani EK, Hall D, et al. PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion. J Biol Chem. 2021;296:100593. 4. Landor SKJ, Santio NM, Eccleshall WB, Paramonov VM, Gagliani EK, Hall D, et al. PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion. J Biol Chem. 2021;296:100593. 21. Locatelli M, Aftimos P, Dees E, LoRusso P, Pegram M, Awada A, et al. CONCLUSION I l i In conclusion, our study demonstrated that silencing Notch4 in TNBC cells suppressed metastasis ability but promoted tumori- genic ability. Mechanistically, silencing Notch4 upregulated Nanog expression, a transcription factor required for maintaining the pluripotency of embryonic stem cells, via Hes3 to promote tumorigenesis. Unexpectedly, silencing Notch4 rendered diffuse Cell Death Discovery (2023) 9:148 Fig. 6 Proposed model of Notch4 knockdown affecting tumorigenesis and metastasis of TNBC cells. Silencing Notch4 in TNBC cells enhances Nanog expression via Hes3 to promote tumorigenesis and affects Vimentin distribution via Cdc42 expression to inhibit metastasis. Y. Tian et al. Y. Tian et al. 9 Fig. 6 Proposed model of Notch4 knockdown affecting tumorigenesis and metastasis of TNBC cells. Silencing Notch4 in TNBC cells enhances Nanog expression via Hes3 to promote tumorigenesis and affects Vimentin distribution via Cdc42 expression to inhibit metastasis. 13. Leontovich A, Jalalirad M, Salisbury J, Mills L, Haddox C, Schroeder M, et al. NOTCH3 expression is linked to breast cancer seeding and distant metastasis. Breast Cancer Res. 2018;20:105. distribution around the nucleus of Vimentin but did not decrease Vimentin expression to suppress metastasis in TNBC cells. Generally, upregulation of Vimentin expression and downregula- tion of E-cadherin expression are regarded as the key molecular events for EMT shift and metastasis. Notably, Cdc42, a key molecular for polarity formation and metastasis mediates Notch4 regulating Vimentin distribution (Fig. 6). Therefore, our demon- stration indicated Notch4 will not be a potential therapeutic target for TNBC. 14. Zhou L, Wang D, Sheng D, Xu J, Chen W, Qin Y, et al. NOTCH4 maintains quiescent mesenchymal-like breast cancer stem cells via transcriptionally acti- vating SLUG and GAS1 in triple-negative breast cancer. Theranostics. 2020;10:2405–21. 15. Nagamatsu I, Onishi H, Matsushita S, Kubo M, Kai M, Imaizumi A, et al. NOTCH4 is a potential therapeutic target for triple-negative breast cancer. Anticancer Res. 2014;34:69–80. 16. Simoes BM, O’Brien CS, Eyre R, Silva A, Yu L, Sarmiento-Castro A, et al. Anti- estrogen resistance in human breast tumors is driven by JAG1-NOTCH4- dependent cancer stem cell activity. Cell Rep. 2015;12:1968–77. Reprints and permission information is available at http://www.nature.com/ reprints Reprints and permission information is available at http://www.nature.com/ reprints 45. Gan Z, Ding L, Burckhardt CJ, Lowery J, Zaritsky A, Sitterley K, et al. Vimentin intermediate ﬁlaments template microtubule networks to enhance persistence in cell polarity and directed migration. Cell Syst. 2016;3:252–63.e258 Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 46. Costigliola N, Ding L, Burckhardt CJ, Han SJ, Gutierrez E, Mota A, et al. Vimentin ﬁbers orient traction stress. Proc Natl Acad Sci USA. 2017;114:5195–200. 47. Chen H, Zou Y, Shang Y, Lin H, Wang Y, Cai R, et al. Fireﬂy luciferase com- plementation imaging assay for protein-protein interactions in plants. Plant Physiol. 2008;146:368–76. 48. Mao S, Ying Y, Ma Z, Yang Y, Chen AK. A background assessable and correctable bimolecular ﬂuorescence complementation system for nanoscopic single- molecule imaging of intracellular protein-protein interactions. ACS Nano. 2021;15:14338–46. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http:// creativecommons.org/licenses/by/4.0/. 49. Bigas A, Espinosa L. The multiple usages of Notch signaling in development, cell differentiation and cancer. Curr Opin Cell Biol. 2018;55:1–7. 50. Ranganathan P, Weaver KL, Capobianco AJ. Notch signalling in solid tumours: a little bit of everything but not all the time. Nat Rev Cancer. 2011;11:338–51. 51. Nagata T, Shimada Y, Sekine S, Moriyama M, Hashimoto I, Matsui K, et al. KLF4 and NANOG are prognostic biomarkers for triple-negative breast cancer. Breast Cancer. 2017;24:326–35. 52. Thiagarajan PS, Hitomi M, Hale JS, Alvarado AG, Otvos B, Sinyuk M, et al. Development of a ﬂuorescent reporter system to delineate cancer stem cells in triple-negative breast cancer. REFERENCES A phase Ib dose escalation trial of RO4929097 (a gamma-secretase inhibitor) in combination with exemestane in patients with ER + metastatic breast cancer (MBC). Clin Breast Cancer. 2022;22:103–14. 10. Chen W, Zhang Y, Li R, Huang W, Wei X, Zeng D, et al. Notch3 transactivates glycogen synthase kinase-3-beta and inhibits epithelial-to-mesenchymal transi- tion in breast cancer cells. Cells. 2022;11:2872. 26. Tolcher AW, Messersmith WA, Mikulski SM, Papadopoulos KP, Kwak EL, Gibbon DG, et al. Phase I study of RO4929097, a gamma secretase inhibitor of Notch signaling, in patients with refractory metastatic or locally advanced solid tumors. J Clin Oncol. 2012;30:2348–53. 11. Zhang YQ, Liang YK, Wu Y, Chen M, Chen WL, Li RH, et al. Notch3 inhibits cell proliferation and tumorigenesis and predicts better prognosis in breast cancer through transactivating PTEN. Cell Death Dis. 2021;12:502. 12. Choy L, Hagenbeek TJ, Solon M, French D, Finkle D, Shelton A, et al. Constitutive NOTCH3 signaling promotes the growth of basal breast cancers. Cancer Res. 2017;77:1439–52. 27. Rosen LS, Wesolowski R, Baffa R, Liao KH, Hua SY, Gibson BL, et al. A phase I, dose- escalation study of PF-06650808, an anti-Notch3 antibody-drug conjugate, in Cell Death Discovery (2023) 9:148 Y. Tian et al. 10 patients with breast cancer and other advanced solid tumors. Invest New Drugs. 2020;38:120–30. 53. Thiagarajan PS, Sinyuk M, Turaga SM, Mulkearns-Hubert EE, Hale JS, Rao V, et al. Cx26 drives self-renewal in triple-negative breast cancer via interaction with NANOG and focal adhesion kinase. Nat Commun. 2018;9:578. patients with breast cancer and other advanced solid tumors. Invest New Drugs. 2020;38:120–30. 28. Pan G, Thomson JA. Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res. 2007;17:42–9. 54. Sasaki M, Sawada N, Chiba H, Miyao N, Tsukamoto T, Satoh M, et al. Cellular polarity correlates with vimentin distribution, but not to keratin, in human renal cell carcinoma cells in vitro. Cell Struct Funct. 1994;19:115–21. 29. Lambert AW, Weinberg RA. Linking EMT programmes to normal and neoplastic epithelial stem cells. Nat Rev Cancer. 2021;21:325–38. 55. Schaedel L, Lorenz C, Schepers AV, Klumpp S, Koster S. Vimentin intermediate ﬁlaments stabilize dynamic microtubules by direct interactions. Nat Commun. 2021;12:3799. 30. Brabletz S, Schuhwerk H, Brabletz T, Stemmler MP. Dynamic EMT: a multi-tool for tumor progression. EMBO J. 2021;40:e108647. 31. Luond F, Sugiyama N, Bill R, Bornes L, Hager C, Tang F, et al. REFERENCES Distinct con- tributions of partial and full EMT to breast cancer malignancy. Dev Cell. 2021;56:3203–21. e3211 56. Etienne-Manneville S. Cdc42-the centre of polarity. J Cell Sci. 2004;117:1291–300. 57. Woods B, Kuo C-C, Wu C-F, Zyla TR, Lew DJ. Polarity establishment requires localized activation of Cdc42. J Cell Biol. 2015;211:19–26. 32. Pal A, Barrett T, Paolini R, Parikh A, Puram S. Partial EMT in head and neck cancer biology: a spectrum instead of a switch. Oncogene. 2021;40:5049–65. 58. Fairn GD, Hermansson M, Somerharju P, Grinstein S. Phosphatidylserine is polarized and required for proper Cdc42 localization and for development of cell polarity. Nat Cell Biol. 2011;13:1424–30. 33. Usman S, Waseem NH, Nguyen TKN, Mohsin S, Jamal A, Teh MT, et al. Vimentin is 33. Usman S, Waseem NH, Nguyen TKN, Mohsin S, Jamal A, Teh MT, et al. Vimentin is at the heart of epithelial mesenchymal transition (EMT) mediated metastasis. Cancers. 2021;13:4985. at the heart of epithelial mesenchymal transition (EMT) mediated metastasis. Cancers. 2021;13:4985. 59. Tian L, Nelson DL, Stewart DM. Cdc42-interacting protein 4 mediates binding of the Wiskott-Aldrich syndrome protein to microtubules. J Biol Chem. 2000;275:7854–61. 34. Chen Z, Fang Z, Ma J. Regulatory mechanisms and clinical signiﬁcance of vimentin in breast cancer. Biomed Pharmacother. 2021;133:111068. 60. Zhou B, Lin W, Long Y, Yang Y, Zhang H, Wu K, et al. Notch signaling pathway: architecture, disease, and therapeutics. Signal Transduct Target Ther. 2022;7:95. 35. Khaled WT, Choon Lee S, Stingl J, Chen X, Raza Ali H, Rueda OM, et al. BCL11A is a triple-negative breast cancer gene with critical functions in stem and progenitor cells. Nat Commun. 2015;6:5987. 36. Balko JM, Schwarz LJ, Luo N, Estrada MV, Giltnane JM, Davila-Gonzalez D, et al. Triple-negative breast cancers with ampliﬁcation of JAK2 at the 9p24 locus demonstrate JAK2-speciﬁc dependence. Sci Transl Med. 2016;8:334ra353. ACKNOWLEDGEMENTS This research was supported by the National Natural Science Foundation of China (No. 81860516) and Guizhou Provincial Natural Science Foundation ZK[2023] Key 033. 37. Yu X, Zou J, Ye Z, Hammond H, Chen G, Tokunaga A, et al. Notch signaling activation in human embryonic stem cells is required for embryonic, but not trophoblastic, lineage commitment. Cell Stem Cell. 2008;2:461–71. 38. Lu X, Mazur S, Lin T, Appella E, Xu Y. The pluripotency factor nanog promotes breast cancer tumorigenesis and metastasis. Oncogene. 2014;33:2655–64. ADDITIONAL INFORMATION ADDITIONAL INFORMATION 42. Chiou JG, Balasubramanian MK, Lew DJ. Cell polarity in yeast. Annu Rev Cell Dev Biol. 2017;33:77–101. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41420-023-01450-w. 43. Mogilner A, Allard J, Wollman R. Cell polarity: quantitative modeling as a tool in cell biology. Science. 2012;336:175–9. 44. Ozbudak EM, Becskei A, van Oudenaarden A. A system of counteracting feedback loops regulates Cdc42p activity during spontaneous cell polarization. Dev Cell. 2005;9:565–71. Correspondence and requests for materials should be addressed to Xiaowei Dou. Correspondence and requests for materials should be addressed to Xiaowei Dou. AUTHOR CONTRIBUTIONS YT and PZ performed most of the experiments. YM performed migration and invasion assay. WY and JZ instructed the pathological study. QL instructed the clinical study. XD conceived/designed the experiments and wrote the manuscript. YT and PZ performed most of the experiments. YM performed migration and invasion assay. WY and JZ instructed the pathological study. QL instructed the clinical study. XD conceived/designed the experiments and wrote the manuscript. 39. Zhang C, Samanta D, Lu H, Bullen J, Zhang H, Chen I, et al. Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA. Proc Natl Acad Sci USA. 2016;113:E2047–56. 40. Kageyama R, Ohtsuka T, Kobayashi T. The Hes gene family: repressors and oscillators that orchestrate embryogenesis. Development. 2007;134:1243–51. COMPETING INTERESTS The authors declare no competing interests. 41. Fischer A, Gessler M. Delta-Notch-and then? Protein interactions and proposed modes of repression by Hes and Hey bHLH factors. Nucleic Acids Res. 2007;35:4583–96. Reprints and permission information is available at http://www.nature.com/ reprints Stem Cells. 2015;33:2114–25. © The Author(s) 2023 © The Author(s) 2023 Cell Death Discovery (2023) 9:148
https://openalex.org/W2021588838	https://europepmc.org/articles/pmc4062419?pdf=render	English	null	The Future of Large Old Trees in Urban Landscapes	PloS one	2,014	cc-by	11,070	Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Darren Le Roux was funded by an Australian Postgraduate Award from the Australian National University and a top-up scholarship from the Land Development Agency (ACT Government). The authors also received funding from The Environmental Decision Hub of the Australian Government’s National Environmental Research Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: darren_lrx@yahoo.com [15] and North America [16], tropical rainforest in South America [17], and agricultural land in Australia [18]. However, few studies have investigated the decline of large old trees in urban landscapes [19,20]. This is a major concern given the unprecedented rate of global urbanisation, one of the most rapid and destructive forms of land-use change [21,22]. Population growth and rising demand for urban living space invariably puts pressure on existing urban habitat that can be important for biodiversity [23,24,25]. However, a great deal of uncertainty remains about the future of habitat structures in urban landscapes, especially structures like large old trees that are known to limit some species [26,27]. Large old trees are especially vulnerable to removal in urban landscapes worldwide due to the potential safety risks posed to the public and infrastructure from falling branches or trees [20,28,29]. Therefore, obtaining information about the future availability of large old trees in urban landscapes is of high priority, especially for practitioners who are challenged by balancing urban growth and maintaining critical habitat for biodiversity over the long term. The Future of Large Old Trees in Urban Landscapes Darren S. Le Roux, Karen Ikin, David B. Lindenmayer, Adrian D he Fenner School of Environment and Society, the Australian National University, Canberra, Australia The Fenner School of Environment and Society, the Australian National University, Canberra, Australia Abstract Large old trees are disproportionate providers of structural elements (e.g. hollows, coarse woody debris), which are crucial habitat resources for many species. The decline of large old trees in modified landscapes is of global conservation concern. Once large old trees are removed, they are difficult to replace in the short term due to typically prolonged time periods needed for trees to mature (i.e. centuries). Few studies have investigated the decline of large old trees in urban landscapes. Using a simulation model, we predicted the future availability of native hollow-bearing trees (a surrogate for large old trees) in an expanding city in southeastern Australia. In urban greenspace, we predicted that the number of hollow-bearing trees is likely to decline by 87% over 300 years under existing management practices. Under a worst case scenario, hollow- bearing trees may be completely lost within 115 years. Conversely, we predicted that the number of hollow-bearing trees will likely remain stable in semi-natural nature reserves. Sensitivity analysis revealed that the number of hollow-bearing trees perpetuated in urban greenspace over the long term is most sensitive to the: (1) maximum standing life of trees; (2) number of regenerating seedlings ha21; and (3) rate of hollow formation. We tested the efficacy of alternative urban management strategies and found that the only way to arrest the decline of large old trees requires a collective management strategy that ensures: (1) trees remain standing for at least 40% longer than currently tolerated lifespans; (2) the number of seedlings established is increased by at least 60%; and (3) the formation of habitat structures provided by large old trees is accelerated by at least 30% (e.g. artificial structures) to compensate for short term deficits in habitat resources. Immediate implementation of these recommendations is needed to avert long term risk to urban biodiversity. Citation: Le Roux DS, Ikin K, Lindenmayer DB, Manning AD, Gibbons P (2014) The Future of Large Old Trees in Urban Landscapes. PLoS ONE 9(6): e99403. doi:10. 1371/journal pone 0099403 Editor: Edward Webb, National University of Singapore, Singapore Editor: Edward Webb, National University of Singapore, Singapore Received January 14, 2014; Accepted May 14, 2014; Published June 18, 2014 Received January 14, 2014; Accepted May 14, 2014; Published June 18, 2014 Copyright: 2014 Le Roux et al. June 2014 \| Volume 9 \| Issue 6 \| e99403 The Future of Large Old Trees in Urban Landscapes The Future of Large Old Trees in Urban Landscapes zones, creating a total of 10 strata. Our land-use types were: (1) nature reserves, which are designated semi-natural areas managed for conservation; and (2) urban greenspace, made up of publicly accessible parklands (60%), roadside margins (24%), remnant vegetation (9%), and sports grounds (7%). Urban greenspace accounted for 11% of the total urban environment in our study area. We divided our study landscape into five geographic zones to capture variability and avoid biasing sampling effort to areas with specific local or historical attributes (e.g. fire history). An equal number of fixed area plots (50620 m; 0.1 ha) were randomly allocated by land-use type (n = 100) and geographic zone (n = 40). This resulted in a total of 200 plots or 20 ha of sampled land from 28 reserves and 100 urban greenspaces. Plots were .250 m apart to minimise spatial dependence and allocated to greenspace $0.2 ha. zones, creating a total of 10 strata. Our land-use types were: (1) nature reserves, which are designated semi-natural areas managed for conservation; and (2) urban greenspace, made up of publicly accessible parklands (60%), roadside margins (24%), remnant vegetation (9%), and sports grounds (7%). Urban greenspace accounted for 11% of the total urban environment in our study area. We divided our study landscape into five geographic zones to capture variability and avoid biasing sampling effort to areas with specific local or historical attributes (e.g. fire history). An equal number of fixed area plots (50620 m; 0.1 ha) were randomly allocated by land-use type (n = 100) and geographic zone (n = 40). This resulted in a total of 200 plots or 20 ha of sampled land from 28 reserves and 100 urban greenspaces. Plots were .250 m apart to minimise spatial dependence and allocated to greenspace $0.2 ha. challenges, urban environments also provide opportunities for innovative tree management, community engagement, people-led conservation strategies, and biodiversity offsets, which may include public tree planting initiatives and artificial nest box projects [32]. In this study, we used a simulation model to predict the future availability of native hollow-bearing trees in a rapidly expanding urban landscape. We used hollow-bearing trees as a surrogate for large old trees and other associated habitat structures such as coarse woody debris [4,33,34]. 2.5. Simulation model We conducted our study in and around the city of Canberra, Australian Capital Territory (ACT), southeastern Australia (35u 179 35. 640 S; 149u 079 27. 360 E). Canberra is Australia’s eighth largest city covering an area of 810 km2. The city supports a population of 375,000 people, which is projected to double by 2056 [37]. Canberra is a highly planned city described as the ‘‘Bush Capital’’ because of the extensive suburban tree cover and 34 nature reserves flanking the urban boundary [38]. The city is situated in the ecologically diverse Southern Tablelands region west of the Great Dividing Range. Lowland box-gum Eucalyptus woodlands and grasslands once dominated the region [39]. Box- gum grassy woodlands are characterised by two dominant species, yellow box (Eucalyptus melliodora) and Blakely’s red gum (E. blakelyi) that occur in association with other eucalypt species, including apple box (E. bridgesiana), red box (E. polyanthemos), red stringybark (E. macrorhyncha), and scribbly gum (E. rossii). Extensive land clearance for stock grazing and urban development has led to a near 95% decline in intact box-gum grassy woodlands, which is now listed as a critically endangered ecological community [40]. What vegetation remains exists in semi-natural nature reserves or as highly modified isolated remnant patches and scattered paddock and urban trees [41,42]. The simulation model described in [12], tracks the mean DBH of trees, including hollow-bearing trees, in separate size cohorts over time. The model has pre-defined rates of tree mortality and recruitment applied at each time step. For this study, we ran separate simulations for native tree populations occurring in nature reserves and urban greenspace. Exotic trees were recorded only in the urban greenspace and accounted for 30% of all recorded trees. We excluded exotic trees from our analyses because only native trees were recorded with hollows in our study area. Simulation models for both land-use types were parame- terised with the following baseline data: the current number of native trees in existing stands sorted by DBH cohort; the predicted age and growth rate of trees; the frequency of regeneration events; the number of seedlings at each regeneration event; and the rate of tree mortality. There were five principle steps in our modelling process (summarised in Fig. 2.5. Simulation model 1 and described further in Summary S1): (1) We calculated the mean number of trees in 10 cm DBH size cohorts (ranging from 0.1–10 cm to .100 cm) for each native tree species and dead trees, using data collected in each land-use type (Table S1). (2) We used a generalised logistic regression model with a binomial distribution and logit link to establish a relationship between hollow occurrence (i.e. the presence of at least one hollow $2 cm; binary response) and tree size (i.e. DBH; explanatory variable). We also fitted tree species as an explanatory variable in our model. Based on correlations in hollow occurrence by DBH 2.4. Data collection We measured the diameter at breast height over bark (DBH; 1.3 m above ground) of every living and dead tree in each plot. We measured only the largest stem of multi-stemmed trees [43]. Trees with stems ,1.3 m above the ground were measured at the base of the stem. The number of naturally regenerating and planted seedlings #10 cm (DBH) were counted in each plot and formed the first size class of our tree population. We identified all living trees to species level. Each tree was inspected for hollows from all angles on the ground using binoculars (10625). One observer (DSL) completed this task to reduce multi-observer bias and maintain consistency in hollow identification [44]. Our objective was not to determine the absolute number of hollows but rather relative hollow occurrence per tree. We selected a minimum entrance size of 2 cm for hollows. This was because: (1) the full range of hollow-dependent vertebrate taxa, including marsupials, birds, and bats, would be accounted for; and (2) hollows smaller than 2 cm were difficult to reliably identify from the ground [45]. 2.1. Ethics statement This research was conducted under ethical approval (protocol number A2012/37; The Australian National University Ethics Committee). Vegetation surveys undertaken on nature reserves and public greenspace were approved by permit from the ACT Government, Territory and Municipal Services in compliance with the Nature Conservation Act 1980. Field studies did not involve endangered or protected species. The Future of Large Old Trees in Urban Landscapes This is because it is well established that as trees age and their size increases so too does the probability of hollow occurrence [5,35,36]. Our four main study objectives were to: (1) compare future trajectories in hollow-bearing trees in urban greenspace with semi-natural nature reserves under existing land management practices; (2) identify which variables can be manipulated to increase the number of hollow-bearing trees occurring in urban greenspace over the long term; (3) test the efficacy of multiple alternative tree management strategies aimed at mitigating the decline of hollow-bearing trees; and (4) formulate recommendations that can be widely applied by practitioners to better maintain and perpetuate large old trees and their associated habitat structures in urban landscapes. Given the widespread nature of this issue in urban landscapes, we anticipate that our findings will be relevant to urban practitioners globally. Introduction Large old trees have been defined as keystone ecological structures because, relative to their size, they are disproportionate providers of resources crucial to other species [1,2]. As trees mature, they begin to form a set of unique physical attributes or structural elements, including large volumes of coarse woody debris and litter, peeling bark, dead branches and hollows [3,4]. Habitat structures provided by large old trees take centuries to form and are typically not provided by younger trees [5]. For example, hollows in Eucalyptus typically begin to form in trees 120– 220 years old [6]. Hollows alone provide critical nesting resources for a diverse range of taxa worldwide, including invertebrates [7], reptiles [8], birds [9], and mammals [10]. Once large old trees are removed, they can be extremely difficult to replace in the short term because of the prolonged time period needed for trees to mature. This time lag can have serious ecological and management implications, particularly in modified landscapes where the rate of large old tree removal exceeds the rate of tree replacement [11,12–14]. Species that depend on large old trees for survival (e.g. hollow-dependent fauna) may face extinction in the short term without actions that reverse current patterns of tree decline [2]. Although there are parallels between urban landscapes and other modified environments (e.g. agricultural land), the manage- ment of trees in human-dominated urban settings poses a suite of unique and complex challenges. The key interacting drivers of tree loss in the urban matrix include: (1) urban sprawl and in-fill practices [30], (2) public safety policies that facilitate managed tree removal in existing greenspace to protect people and infrastructure [20], and (3) reduced tree regeneration [31]. Despite these Human activities such as land clearance, logging and livestock grazing are responsible for the decline of large old trees in a diverse range of ecosystems, including: conifer forests in Europe 1 June 2014 \| Volume 9 \| Issue 6 \| e99403 PLOS ONE \| www.plosone.org June 2014 \| Volume 9 \| Issue 6 \| e99403 June 2014 \| Volume 9 \| Issue 6 \| e99403 The Future of Large Old Trees in Urban Landscapes Figure 1. Simple schematic highlighting the five principle steps of our simulation model. doi:10.1371/journal.pone.0099403.g001 yellow box equivalent using the method described in [18,26]. To do this, we first calculated each DBH value as a proportion of the maximum DBH recorded for each tree species and then multiplied this value by the largest DBH recorded for yellow box in our study area (151 cm). Therefore, we assumed that all species had proportionally equal growth rates that were similar to that of yellow box. Although this approach is not ideal because it is unlikely to yield precise age estimates for each species, it currently is the most practicable solution available in the absence of age- DBH relationship data for other eucalypt species [26,48]. Therefore, our model had a degree of uncertainty related to tree growth rates, as these data likely differ for each species. However, a previous study [12] found that long-term predictions for mature trees is not sensitive to uncertainty in this variable and suggests that the focus should instead be on testing the effects of uncertainty for other parameters in the model. (4) We simulated tree regeneration in both land-use types to ensure that uncertainties associated with regeneration were reflected in our models. Tree regeneration is an event-driven process that can be sporadic and influenced by natural phenom- ena and/or anthropogenic factors such as climate, competition, and planting effort [31,49]. At each regeneration event, viable seedlings may or may not establish and survive over time. To simulate these uncertainties, the number of seedlings ha21 for each run of our model was drawn randomly from a Poisson distribution with the mean equal to the mean number of trees recorded in the 0–10 cm DBH cohort for each species group. For species group one and two in urban greenspace, the mean number of trees in the 0–10 cm DBH cohort was 11 and 13 seedlings ha21, respectively. For species group one and two in nature reserves, the mean number of trees in the 0–10 cm DBH cohort was 119 and 193 seedlings ha21, respectively. The time-step for each run of the model was equivalent to the average age of trees in the 0–10 cm DBH cohort for both land-use types, which was approximately 8 years. (5) Annual tree mortality was modelled in a density-dependent manner to reflect declines in the number of trees over successive DBH cohorts or as trees age. The Future of Large Old Trees in Urban Landscapes Therefore, we assumed that tree densities would naturally thin out over time due to factors such as competition among conspecifics [50]. To simulate this process, we calculated annual mortality for each DBH cohort using the equation: 1 - s (1/y), where s is the proportion of trees that survive from one cohort to the next, and y is the number of years it takes trees to progress from one cohort to the next by 10 cm DBH increments. However, in some urban greenspaces (e.g. roadside margins), density-dependent mortality may be less pronounced as tree survivability may instead be predominantly influenced by tree planting and protection efforts. Therefore, for urban greenspace, we also tested the mean annual mortality rate across all cohorts, which yielded similar model trajectories to density-dependent mortality. We decided to apply density-dependent mortality to both land-use types for consistency and because a majority of urban greenspace sampled constituted parklands and remnant vegetation where natural regeneration and density-dependent mortality may still occur. We set 500 years as the maximum age that living trees will remain standing in both land-use types. This is based on the only longevity estimate available for eucalypts in our study area [47]. It is reasonable to assume that for other eucalypt species this age would also be the upper limit of survivability. Therefore, model uncertainties pertaining to species longevity are likely to be over-estimated and based on a best-case longevity. We assumed that once trees died in urban greenspace, they no longer functioned as hollow-bearing trees into the next time step. This is based on local tree management policies that facilitate dead tree Figure 1. Simple schematic highlighting the five principle steps of our simulation model. doi:10.1371/journal.pone.0099403.g001 between individual species, we identified three distinct species groupings. Species group one included yellow box, apple box, brittle gum (E. mannifera), broad-leaved peppermint (E. dives), bundy (E. goniocalyx), mealy bundy (E. nortonii), brown barrel (E. fastigata), alpine ash (E. delegatensis), ribbon gum (E. viminalis), mountain gum (E. dalrympleana), candlebark (E. rubida) and ironbark (E. sideroxylon). Group two included Blakely’s red gum, red box, red stringybark and scribbly gum. Group three was dead trees. We found that species groups differed significantly (Wald statistic = 101.5; P,0.001) from each other (Table 1). The relationship between tree size and hollow presence was highly significant in our model (Wald statistic = 388.1; P,0.001). 2.3. Sampling design We confined our sampling effort to a single vegetation type: the predicted pre-European (pre-1750) extent of box-gum grassy woodland. Within this vegetation type, we stratified sampling according to two dominant land-use types and five geographic June 2014 \| Volume 9 \| Issue 6 \| e99403 PLOS ONE \| www.plosone.org 2 The Future of Large Old Trees in Urban Landscapes 2.6. The availability of hollow-bearing trees under existing management practices We used our simulation model, parameterised with those data detailed above, to predict the mean number of hollow-bearing trees ha21 occurring in nature reserves and urban greenspace over time under existing land management protocols. Simulations were undertaken over 300 years using a Monte Carlo simulation based on 300 runs of our model (i.e. the number of iterations required for relatively well-defined distributions). This approach relies on random sampling over multiple simulations to generate probabil- ities in a heuristic manner [53]. Therefore, for each run of our model, input data for several variables were drawn randomly from defined distributions. The number of recruits was drawn from a Poisson distribution (step 4 above). Annual mortality was drawn from a normal distribution, where negative values were converted to zero. The maximum standing life of living trees was held at 500 years for nature reserves. However, for urban greenspace, values were drawn from a uniform distribution between 60 years (the estimated minimum standing life of trees in our study area) [54] and 500 years (the estimated maximum standing life of trees in our study area). This range of lifespans reflects variation in current tree management practices in different types of urban greenspace. Variables held constant in our model were the period between regeneration events (8 years) and coefficients for the age-DBH (0.019) and DBH-hollow (1.413) relationships. We used linear regression to test the relative sensitivity of our response variable (i.e. the mean number of hollow-bearing trees ha21) against the explanatory variables that are the parameters in our simulation model. We natural log-transformed (ln (x+1)) our response to satisfy assumptions of normality. There were no significant interactions between explanatory variables and inter- action terms were dropped from the final additive model. We used stepwise regression to determine the model of best fit. Percentage variance accounted for by our final model was 40%. Due to the high number of replications used in simulation models, it is inappropriate to rely on conventional P-values to indicate statistical significance [56]. Instead, we used relative effect size, as indicated by variance ratios, to identify the most sensitive parameters in our model. Variance ratios were calculated as the mean square of each term change divided by the residual mean squares of the original maximal model (Table 2). Predictions are presented only for variables with the greatest relative effect sizes (i.e. The Future of Large Old Trees in Urban Landscapes The area under the receiver operating characteristic curve of our model was 0.92, indicating that the discriminating ability of our model was excellent [46]. For each species group, we derived separate model equations which took the form: Logit (Pr. Hollows) = 27.112+(0.086 x DBH) + (species group estimate). (3) We established a relationship between DBH and tree age using the following equation: Age = 0.026p6(DBH standardised/ 2)2, where DBH standardised is the yellow box equivalent diameter (3) We established a relationship between DBH and tree age using the following equation: Age = 0.026p6(DBH standardised/ 2)2, where DBH standardised is the yellow box equivalent diameter for each tree. Yellow box is the only tree species for which data exist to establish a relationship between age and DBH [47]. We scaled all DBH values for each tree species relative to that of a (3) We established a relationship between DBH and tree age using the following equation: Age = 0.026p6(DBH standardised/ 2)2, where DBH standardised is the yellow box equivalent diameter for each tree. Yellow box is the only tree species for which data exist to establish a relationship between age and DBH [47]. We scaled all DBH values for each tree species relative to that of a 2) , where DBH standardised is the yellow box equivalent diameter for each tree. Yellow box is the only tree species for which data exist to establish a relationship between age and DBH [47]. We scaled all DBH values for each tree species relative to that of a June 2014 \| Volume 9 \| Issue 6 \| e99403 June 2014 \| Volume 9 \| Issue 6 \| e99403 PLOS ONE \| www.plosone.org 3 The Future of Large Old Trees in Urban Landscapes Table 1. Generalised logistic regression model used to predict the proportion of hollow-bearing trees in each 10 cm DBH (diameter at breast height) cohort. Table 1. Generalised logistic regression model used to predict the proportion of hollow-bearing trees in each 10 cm DBH (diameter at breast height) cohort. (diameter at breast height) cohort. 2.6. The availability of hollow-bearing trees under existing management practices most ecologically important), where all other explanatory variables are held at their mean model values. The Future of Large Old Trees in Urban Landscapes Variables Coefficient Standard error Lower 95% confidence interval Upper 95% confidence interval P-value Intercept 27.112 0.335 27.769 26.456 ,0.001 Species group 1 0.000 - - - - Species group 2 1.413 0.274 0.876 1.949 ,0.001* Species group 3 3.861 0.383 3.110 4.613 ,0.001* DBH 0.086 0.004 0.077 0.095 ,0.001* Coefficients, standard errors, 95% confidence intervals, and P-values are presented with species group one held as the reference level. doi:10.1371/journal.pone.0099403.t001 removal on public land [51]. However, for nature reserves, we conservatively estimated that dead trees could remain standing for at least 50 years after initial mortality (i.e. 550 years in total), based on observations of the standing life of dead trees in Eucalyptus forests [52], however, we acknowledge the paucity of available data to support this estimate. species groups one and two, testing various regeneration targets); (3) period between regeneration events (range: 1–50 years, testing various regeneration schedules); (4) rate of annual mortality (range: 0.03–0.1 model coefficients, testing various feasible survivability outcomes); and (5) rate of hollow formation (range: 1.5–3.7 model coefficients, testing a range of hollow acceleration strategies above an observed existing rate (i.e. 1.4) up to a rate observed for dead trees (i.e. 3.8) in our study area, which we assumed indicated a maximal hollow formation rate for living trees). We fixed the coefficient for the DBH-age relationship at 0.019 assuming that this could not be changed appreciably. 2.7. Variables that can be manipulated to mitigate the decline of hollow-bearing trees We also simulated a series of alternative management strategies using our simulation model. We modelled the mean number of hollow-bearing trees ha21 occurring in urban greenspace over 300 years. Scenarios were based on either: (1) a management strategy that manipulates only a single variable up to the maximum value defined in our regression model described above, or (2) a combined management strategy that manipulates all three variables for a set of values that we deemed most practicable for urban landscapes given other socio-economic constraints. Vari- ables not manipulated were fixed at their mean values under existing management practices. In all simulated scenarios, management actions were assumed to take effect immediately. Statistical analyses were completed using GenStat (15th edition, VSN International Ltd, Hemel Hempstead, UK). We performed a sensitivity analysis, as described in [55], to identify which variables can be manipulated in urban greenspace to mitigate the decline of hollow-bearing trees. For this analysis, we also used a Monte Carlo simulation based on 300 runs of our model. We repeatedly populated each run of the model with data drawn randomly from uniform distributions for each variable. Where applicable, values were drawn from a wider range than observed under existing management practices to more broadly test a range of alternative management strategies. Variables that can be manipulated by management included: (1) maximum standing life of trees (range: 60–500 years for species groups one and two, based on longevity estimates for urban trees in our study area); (2) number of seedlings ha21 (range: 0–60 seedlings ha21 for June 2014 \| Volume 9 \| Issue 6 \| e99403 PLOS ONE \| www.plosone.org 4 The Future of Large Old Trees in Urban Landscapes Table 2. Linear regression model used to perform a sensitivity analysis of the mean number of hollow-bearing trees ha21 (ln (x+1) transformed) perpetuated in urban greenspace over 300 years. 3.2. Variables that can be manipulated to mitigate the decline of hollow-bearing trees Sensitivity analysis revealed that the mean number of hollow- bearing tree ha21 was most sensitive to: (1) the maximum standing life of trees; (2) the number of seedlings ha21; and (3) the rate of hollow formation (Table 2). The mean number of hollow-bearing trees ha21 was least sensitive to the period between regeneration events and annual mortality. We also did not identify meaningful interactions between maximum standing life and annual mortality, maximum standing life and the rate of hollow formation, and the number of seedlings ha21 and the period between regeneration events. Results We recorded a total of 4,865 trees belonging to 16 eucalypt species. Of those trees, 85% (4,111 trees) were recorded in nature reserves and 15% (754 trees) in urban greenspace. The key difference between tree populations in nature reserves and urban greenspace was the number of seedlings recorded in the 0.1– 10 cm DBH cohort (Fig. 2). In reserves, we recorded 315 seedlings ha21, which was 13 times the number recorded in urban greenspace, with 25 seedlings ha21. 2.7. Variables that can be manipulated to mitigate the decline of hollow-bearing trees Variables Mean Standard deviation Coefficient Standard error Variance ratio Intercept - - 0.602 0.204 - Maximum standing life (years) 274.10 88.04 0.004 0.0003 138.61 Number of seedlings ha21 31.03 12.09 0.009 0.002 13.81 Rate of hollow formation (coefficient) 2.59 0.17 0.151 0.042 11.04 Rate of annual mortality (coefficient) 0.06 0.02 21.290 1.450 0.31 Period between regeneration (years) 24.74 13.40 0.000 0.002 0.00 Means, standard deviations, coefficients, standard errors, and variance ratios, which indicate the relative importance or effect size of each model term, are presented for each explanatory variable used to parameterise our simulation model. doi:10.1371/journal.pone.0099403.t002 Table 2. Linear regression model used to perform a sensitivity analysis of the mean number of hollow-bearing trees ha21 (ln (x+1) transformed) perpetuated in urban greenspace over 300 years. Table 2. Linear regression model used to perform a sensitivity analysis of the mean number of hollow-bearing trees ha21 (ln (x+1) transformed) perpetuated in urban greenspace over 300 years. Means, standard deviations, coefficients, standard errors, and variance ratios, which indicate the relative importance or effect size of each model term, are presented for each explanatory variable used to parameterise our simulation model. doi:10.1371/journal.pone.0099403.t002 by highly variable standing lives that trees are permitted to reach in different urban greenspaces (i.e. 60–500 years old). Prediction intervals indicate that under a worst case scenario (i.e. lower 95% prediction interval) all hollow-bearing trees may be lost from urban greenspace within 115 years. Even under a best case scenario (i.e. upper 95% prediction interval) hollow-bearing trees steadily decline over time. 3.1. The availability of hollow-bearing trees under existing management practices The number of seedlings ha21 also contributed to the number of hollow-bearing trees perpetu- ated in urban greenspace over the long term, although relative to maximum standing life this contribution was smaller (variance ratio = 13.81). We predicted that for every 10 additional native seedlings ha21, the number of hollow-bearing trees would increase by 0.3 trees ha21 (10%; Fig. 4B). However, we predicted that to perpetuate hollow-bearing trees even marginally above existing levels will require at least 30 seedlings ha21 and all trees to remain standing for at least 200 years (Fig. 5A). Figure 4. The predicted relative number of hollow-bearing trees ha21 (mean ±95 prediction intervals) in urban green- space over 300 years for a range of values for variables with the greatest relative effect sizes derived from a sensitivity analysis. Variables include: the maximum standing life of trees (A); the number of seedlings ha21 (B); and the rate of hollow formation (represented by the coefficient for the probability of hollow occurrence; C). Predicted thresholds under existing management practices are provided for reference (solid circles). doi:10.1371/journal.pone.0099403.g004 3.2.3. Rate of hollow formation. Similarly, the rate of hollow formation also contributed to the number of hollow- bearing trees perpetuated in urban greenspace over the long term, although relative to maximum standing life this contribution was smaller (variance ratio = 11.04). We predicted that hollow-bearing trees would increase by 0.2 trees ha21 (8%) for every 0.5 increase in the rate of hollow formation (Fig. 4C). However, we predicted that to perpetuate hollow-bearing trees even marginally above existing levels will require accelerating hollow formation to a rate of 2.5 (i.e. a 44% increase above the observed mean rate) and all trees to remain standing for at least 200 years (Fig. 5B). observed mean rate), then the mean number of hollow-bearing trees in urban greenspace is predicted to initially increase to 9 trees ha21 in the short term, but decline by 92% to 0.46 tree ha21 over the long term. observed mean rate), then the mean number of hollow-bearing trees in urban greenspace is predicted to initially increase to 9 trees ha21 in the short term, but decline by 92% to 0.46 tree ha21 over the long term. 3.1. The availability of hollow-bearing trees under existing management practices In urban greenspace, we found that under existing management practices, the mean number of hollow-bearing trees ha21 is predicted to decline by 87% over 300 years from an initial recorded stand density of 5.74 trees ha21 to 0.76 trees ha21 (Fig. 3). Conversely, in nature reserves, hollow-bearing tree densities fluctuate around a relatively stable mean density of 13.4 trees ha21. Prediction intervals for urban greenspace were more variable around the mean than for nature reserves. This is driven Figure 2. Frequency distribution of median tree diameter cohorts for tree stands (all species) in nature reserves (open bars) and urban greenspace (solid bars). doi:10.1371/journal.pone.0099403.g002 Figure 2. Frequency distribution of median tree diameter cohorts for tree stands (all species) in nature reserves (open bars) and urban greenspace (solid bars). doi:10.1371/journal.pone.0099403.g002 June 2014 \| Volume 9 \| Issue 6 \| e99403 PLOS ONE \| www.plosone.org 5 The Future of Large Old Trees in Urban Landscapes Figure 4. The predicted relative number of hollow-bearing trees ha21 (mean ±95 prediction intervals) in urban green- space over 300 years for a range of values for variables with the greatest relative effect sizes derived from a sensitivity analysis. Variables include: the maximum standing life of trees (A); the number of seedlings ha21 (B); and the rate of hollow formation (represented by the coefficient for the probability of hollow occurrence; C). Predicted thresholds under existing management practices are provided for reference (solid circles). doi:10.1371/journal.pone.0099403.g004 Figure 3. Simulations predicting the relative number of hollow- bearing trees ha21 (mean ±95% prediction interval) over 300 years under existing management practices in nature reserves (1) and urban greenspace (2). doi:10.1371/journal.pone.0099403.g003 Figure 3 Simulations predicting the relative number of hollow- Figure 3. Simulations predicting the relative number of hollow- bearing trees ha21 (mean ±95% prediction interval) over 300 years under existing management practices in nature reserves (1) and urban greenspace (2). doi:10.1371/journal.pone.0099403.g003 3.2.1. Maximum standing life. The number of hollow- bearing trees perpetuated in urban greenspace over the long term was most sensitive to the maximum standing life of trees (variance ratio = 138.61). We predicted that hollow-bearing trees would increase in urban greenspace by approximately 0.8 trees ha21 (22%) for each additional 50 years that trees are permitted to remain standing (Fig. 4A). 1 3.2.2. Number of seedlings. 3.3. The availability of hollow-bearing trees under alternative management strategies 3.3.2. Combined management approach. In contrast, a combined management approach that manipulates all sensitive explanatory variables is predicted to increase the number of hollow-bearing trees ha21 over the long term (Fig. 6). To achieve this will require at least: (1) increasing the standing life of trees to 450 years (approximately 40% longer average lifespans); (2) increasing the number of seedlings to 60 seedlings ha21 (approximately 60% greater regeneration rate); and accelerating hollow formation up to a rate of 2.0 (approximately 30% greater hollow formation rate; see Table 3). Under this scenario, the density of hollow-bearing trees will initially need to be actively increased in the short term by accelerating hollow formation to achieve at least 7 hollow-bearing trees ha21. Over time, the 3.3.1. Isolated management approach. If tree standing life were maximised to 500 years and all other variables were unchanged (i.e. held at their mean values under existing management practices), then the mean number of hollow-bearing trees in urban greenspace is predicted to still decline by 64% over the long term, from an initial stand density of 5.74 trees ha21 to 2.09 trees ha21 (Fig. 6). If the number of seedlings ha21 were increased only to 60 seedlings ha21, then the mean number of hollow-bearing trees in urban greenspace is predicted to still decline by 53% over the long term, from an initial stand density of 5.74 trees ha21 to 2.68 trees ha21. If hollow formation were accelerated only to a rate of 3.7 (i.e. the maximum rate of hollow formation observed for living trees and a 62% increase above the June 2014 \| Volume 9 \| Issue 6 \| e99403 PLOS ONE \| www.plosone.org 6 The Future of Large Old Trees in Urban Landscapes Figure 5. The predicted relative mean number of hollow-bearing trees ha21 in urban greenspace over 300 years for a combination of values for: the maximum standing life of trees and the number of seedlings ha21 (A); and the maximum standing life of trees and the rate of hollow formation (represented by the coefficient for the probability of hollow occurrence; B). doi:10.1371/journal.pone.0099403.g005 Figure 5. 3.3. The availability of hollow-bearing trees under alternative management strategies The predicted relative mean number of hollow-bearing trees ha21 in urban greenspace over 300 years for a combination of values for: the maximum standing life of trees and the number of seedlings ha21 (A); and the maximum standing life of trees and the rate of hollow formation (represented by the coefficient for the probability of hollow occurrence; B). doi:10.1371/journal.pone.0099403.g005 density of hollow-bearing trees is predicted to first gradually decline before an increase occurs within 250 years. scenario, hollow-bearing trees may be entirely lost from urban greenspace within 115 years. Our analysis revealed that the decline of hollow-bearing trees in urban greenspace is most sensitive to: the maximum standing life of trees, the number of regenerating seedlings ha21, and the rate of hollow formation. To mitigate the decline of large old trees in urban greenspace over the long term, we recommend a management strategy that collective- ly: (1) maximises the standing life of trees, (2) increases tree regeneration rates, and (3) accelerates the formation of habitat structures provided by large old trees. These results, and the methods used, have important implications for ecologically sustainable urban development. Discussion Large old trees support unique habitat structures (e.g. hollows, coarse woody debris), which form over extensive time periods and cannot be provided by younger trees [5,6]. The decline of large old trees in modified landscapes is a global conservation issue that has serious implications for biodiversity [11]. To date, few studies have addressed this problem in urban landscapes, which is a growing concern given the unprecedented rates of urbanisation in cities worldwide [25]. Using a simulation model, we investigated the decline of large old trees in an urban landscape over centuries. We predicted that hollow-bearing trees (a surrogate for large old trees) will decline by 87% over 300 years in urban greenspace under existing management practices. Under a worst case 4.1. Existing management practices Our results provide further evidence that urban landscapes face a concerning future of large old tree decline, which is comparable June 2014 \| Volume 9 \| Issue 6 \| e99403 PLOS ONE \| www.plosone.org 7 The Future of Large Old Trees in Urban Landscapes Figure 6. The predicted relative mean number of hollow-bearing trees ha21 over 300 years under a series of alternative urban tree management scenarios (dashed lines). Simulated scenarios include: increasing the standing life of trees only up to 500 years; increasing the number of seedlings only up to 60 ha21; accelerating hollow formation only by 62% above the observed mean rate (as represented by the coefficient for the probability of hollow occurrence); and a combined management approach (i.e. our recommended management proposal), which manipulates all three variables simultaneously. Scenarios under existing management practices are provided for reference by solid black lines for nature reserves (1) and urban greenspace (2). doi:10.1371/journal.pone.0099403.g006 Figure 6. The predicted relative mean number of hollow-bearing trees ha21 over 300 years under a series of alternative urban tree management scenarios (dashed lines). Simulated scenarios include: increasing the standing life of trees only up to 500 years; increasing the number of seedlings only up to 60 ha21; accelerating hollow formation only by 62% above the observed mean rate (as represented by the coefficient for the probability of hollow occurrence); and a combined management approach (i.e. our recommended management proposal), which manipulates all three variables simultaneously. Scenarios under existing management practices are provided for reference by solid black lines for nature reserves (1) and urban greenspace (2). doi:10.1371/journal.pone.0099403.g006 own are unlikely to achieve biodiversity conservation targets [58]. In addition, many species rely on networks of multiple habitat trees that extend over large areas of the landscape, including urban habitats [59]. For these reasons, we strongly encourage manage- ment strategies that focus on arresting large old tree decline within the ‘working’ urban matrix. This means that a re-evaluation of existing management practices in urban landscapes is needed to address the underlying drivers of tree decline. with other highly impacted landscapes, including agricultural land [12,57] and production forests [15,52]. We argue that predicted declines in hollow-bearing trees in urban greenspace (Fig. 3) will not only negatively impact hollow-dependent fauna (e.g. 4.1. Existing management practices birds, bats, mammals and invertebrates), but also will impact a much wider range of plant and animal species that rely on large old trees and associated habitat structures (e.g. coarse woody debris, litter, peeling bark) for a range of purposes (e.g. foraging, spatial connectivity, epiphyte attachment). Ultimately, these species may face local extinction in urban landscapes. This is supported by recent research, which demonstrates that the removal of large old trees from existing urban habitats will likely impact animal populations and community assemblages [19,20]. 4.2. Alternative management strategies Large old trees are especially susceptible to removal in urban landscapes worldwide [20,28,29,60]. With this in mind, we have formulated a set of targeted recommendations, based on results from our analyses, which we anticipate to be relevant to practitioners in a wide range of urban landscapes where trees are maintained. Predictions under existing management practices also highlight the important role that nature reserves play in bridging resource gaps across urban landscapes. In contrast to urban greenspace, we predicted that nature reserves adjacent to urban areas provide a relatively stable supply of hollow-bearing trees over time. Therefore, maintaining and establishing nature reserves in urban environments will likely provide important habitat refuge for species over the long term. However, nature reserves only represent a small proportion of the urban landscape and on their 4.2.1. Maximise tree standing life. A major source of tree mortality in urban landscapes is due to managed tree removal [20]. This is facilitated by public safety policies and practices, which aim to minimise risk of injury to people and damage to property due to falling trees and branches. For example, in our study area it is estimated that by 2050, approximately 175,600 Table 3. Summarised values for each variable used to parameterise our simulation model under existing management practices for nature reserves and urban greenspace. Table 3. Summarised values for each variable used to parameterise our simulation model under existing management practices for nature reserves and urban greenspace. Variables Nature reserves Urban greenspace Urban management recommendations Maximum standing life (years) 500 60–500 450 (,40% increase) Number of seedlings ha21(all species) 315 25 60 (,60% increase) Rate of hollow formation 1.4 1.4 2.0 (,30% increase) Rate of annual mortality 0.03 0.06 - Period between regeneration (years) 8 8 - Relative values are derived from raw vegetation data or, where applicable, published estimates. Urban management recommendations, derived from a series of simulated alternative management strategies, are indicated for variables identified as being the most ecologically important from a sensitivity analysis. doi:10.1371/journal.pone.0099403.t003 Relative values are derived from raw vegetation data or, where applicable, published estimates. Urban management recommendations, derived from a series of simulated alternative management strategies, are indicated for variables identified as being the most ecologically important from a sensitivity analysis. doi:10 1371/journal pone 0099403 t003 Relative values are derived from raw vegetation data or, where applicable, published estimates. The Future of Large Old Trees in Urban Landscapes damage to roots due to road works); (3) creating safe zones or barriers that separate the public from potentially hazardous trees thereby minimising safety risks (e.g. landscaping around the base of the tree using shrubs); (4) physically re-enforcing the structural integrity of large, old trees (e.g. supporting frames, cables or poles); and (5) safely retaining dead trees wherever possible. However, our results indicated that management strategies that only maximise the standing life of trees will be insufficient at mitigating the decline of hollow-bearing trees over the long term (Fig. 6). Accelerating hollow formation in urban areas is commonly achieved by replicating hollow structures, such as installing artificial nest boxes [32]. However, in urban areas, there are limitations with artificial habitat structures, including: occupancy by pest species, poor rates of target species occupancy, and rapid rates of attrition through collapse and decay of materials [67]. It may also not be feasible or practicable to install and maintain artificial habitat structures in large enough numbers across extensive areas over centuries. Therefore, strategies that accelerate the formation of habitat structures by other means should also be explored [68]. Methods previously proposed for hollows include: tree ringbarking or girdling [69], canopy topping [70], controlled fire burns [71], and injecting trees with herbicides [72]. These strategies are also likely to accelerate the formation of other important habitat structures provided by large old trees, including dead branches and coarse woody debris. In urban landscapes, sub- lethal methods of accelerating habitat structure formation are most preferable to also avoid compromising public safety. This may involve only partially injuring trees by carving out hollows on trunks and some branches [73] and using more invasive methods on trees with large diameters that are structurally robust in order to also maximise tree standing life [35]. More research is still needed to investigate methods aimed at accelerating habitat structure formation, especially in urban landscapes. Nevertheless, our results highlight that management strategies based solely on accelerating hollow formation can be effective at increasing the density of hollow-bearing trees in the short-term, but not over the long term (Fig. 6). 4.2.2. Increase tree regeneration. We found that the rate of tree regeneration in urban greenspace (both natural and planted) was 13 times lower than in nature reserves (Fig. 2). A lack of young trees is a major contributing factor of large old tree decline in urban greenspace over the long term. The Future of Large Old Trees in Urban Landscapes street trees (24% of all trees in urban greenspace) will have reached their safe standing life (ranging from 60 to 100 years old) and are likely to be removed [54]. Consequently, large old trees, hollow- bearing trees, dead trees and decaying branches are most susceptible to targeted removal prior to reaching their full potential in terms of forming and providing suitable habitat. We found that the number of hollow-bearing trees perpetuated in urban greenspace over the long term was most sensitive to the maximum standing life of trees (Table 2; Fig. 4A). Increasing the standing life of all trees by 50 years is predicted to increase the number of hollow-bearing trees ha21 in urban greenspace by 22% over the long term. increasing public awareness of regenerating areas through signage, and enhancing local microclimates that favour seedling establish- ment and survival such as retaining litter and logs [31,66]. However, our results indicated that management strategies based solely on increasing tree regeneration will be insufficient at mitigating the decline of hollow-bearing trees over the long term (Fig. 6). 4.2.3. Accelerate the formation of habitat structures provided by large old trees. The formation of habitat structures such as hollows is a slow process more likely to occur in large old trees [35]. This is because trees with compromised structural integrity are more susceptible to wood decay resulting in the formation of hollows and other structures such as fallen logs and dead branches. Strategies promoting the formation of habitat structures by artificial means can bypass the time needed for these structures to form naturally. Our results indicate that the density of hollow-bearing trees could be increased in urban greenspace by accelerating hollow formation (Fig. 4C). Policymakers need to recognize the important habitat resources provided by large old trees and accordingly formulate or amend tree management protocols so that large old trees are afforded better protection. This may involve re-evaluating criteria used to guide tree felling decisions [29]. Practical strategies that maximise the safe standing life of trees should also be implemented. This may involve: (1) allowing trees to age more naturally in urban greenspace frequented less by members of the public and where risk to people and property is minimal (e.g. derelict land, areas along stormwater wetlands, and some parklands); (2) avoiding structural damage to trees (e.g. 4.2. Alternative management strategies Urban management recommendations, derived from a series of simulated alternative management strategies, are indicated for variables identified as being the most ecologically important from a sensitivity analysis. doi:10.1371/journal.pone.0099403.t003 June 2014 \| Volume 9 \| Issue 6 \| e99403 PLOS ONE \| www.plosone.org 8 The Future of Large Old Trees in Urban Landscapes The Future of Large Old Trees in Urban Landscapes Older trees that eventually die and are removed from any given landscape need to be replaced by younger trees, thereby perpetuating the formation of important habitat structures over multiple generations [18,61]. We predicted that increasing tree regeneration by 10 native seedlings ha21 would increase the number of hollow-bearing trees in urban greenspace by 10% over the long term (Fig. 4B). Tree regeneration in urban habitats is typically achieved through planting initiatives and encouraging natural regeneration. Increasing the number of planted trees through government and community initiatives should increase the number of young trees persisting in urban habitats [62]. However, in some urban greenspace (e.g. roadside margins and residential areas), tree planting can be logistically challenging as practitioners need to balance multiple socio-economic and ecological factors when implementing planting strategies, including: site location, public safety, aesthetics, land ownership, and existing vegetation [63]. Furthermore, reducing seedling mortality in urban habitats is also an important consideration that may require additional protection measures (e.g. tree guards, supporting posts) and costs [64]. In some urban greenspace (e.g. parklands, wetlands) it may be more cost-effective over the long term to promote natural regeneration. Natural regeneration in urban habitats is predominantly limited because of: unfavourable seedbed conditions (e.g. impervious surfaces, pollution, and nutrient runoff), increased competition from invasive plants, and increased mortality due to mowing and pedestrian traffic [31,65]. Strategies that promote natural regen- eration could involve: fencing-off areas with existing re-growth, 4.2.4. Our management proposal: A combined management approach is needed. Our results emphasise that a combination of different management approaches, aimed at improving multiple aspects of tree management and maintenance, are needed to perpetuate hollow-bearing trees in urban greenspace over the long term (Fig. 5). We propose a management strategy based on simultaneously manipulating all three explanatory variables discussed above, which were identified as being the most sensitive model parameters in our analyses. Under this scenario (Fig. 6), we predicted that the decline of hollow-bearing trees in urban greenspace can be arrested within 250 years if: (1) trees remain standing for at least 450 years ensuring that they reach their maximum habitat potential; (2) at least 60 seedlings ha21 are planted or naturally regenerated; and (3) hollow formation is accelerated to a rate of 2.0 in the short term by installing nest boxes and sub-lethally creating hollows by other methods. References 1. Manning AD, Fischer J, Lindenmayer DB (2006) Scattered trees are keystone structures - implications for conservation. Biological Conservation 132: 311–321. 15. Andersson R, O¨ stlund L (2004) Spatial patterns, density changes and implications on biodiversity for old trees in the boreal landscape of northern Sweden. Biological Conservation 118: 443–453. 1. Manning AD, Fischer J, Lindenmayer DB (2006) Scattered trees are keystone structures - implications for conservation. Biological Conservation 132: 311–321. 2. Lindenmayer DB, Laurance WF, Franklin JF, Likens GE, Banks SC, et al. (2013) New policies for old trees: averting a global crisis in a keystone ecological structure. Conservation Letters 00: 1–9. 16. Lutz JA, van Wagtendonk JW, Franklin JF (2009) Twentieth-century decline of large-diameter trees in Yosemite National Park, California, USA. Forest Ecology and Management 257: 2296–2307. 3. Lindenmayer DB, Wood JT (2010) Long-term patterns in the decay, collapse, and abundance of trees with hollows in the mountain ash (Eucalyptus regnans) forests of Victoria, southeastern Australia. Canadian Journal of Forest Research 40: 48–54. g 17. Laurance WF, Delamonica P, Laurance SG, Vasconcelos HL, Lovejoy TE (2000) Rainforest fragmentation kills big trees. Nature 404: 836–836. 18. Fischer J, Zerger A, Gibbons P, Stott J, Law B (2010) Tree decline and the future of Australian farmland biodiversity. PNAS 107: 19597–19602. 4. Goodburn JM, Lorimer CG (1998) Cavity trees and course woody debris in old- growth and managed northern hardwood forests in Wisconsin and Michigan. Canadian Journal of Forest Research 28: 427–438. 19. Stagoll K, Lindenmayer DB, Knight E, Fischer J, Manning AD (2012) Large trees are keystone structures in urban parks. Conservation Letters 0: 1–8. 5. Ranius T, Niklasson M, Berg N (2009) Development of tree hollows in pedunculate oak (Quercus robur). Forest Ecology and Management 257: 303– 310. 20. Carpaneto G, Mazziotta A, Coletti G, Luiselli L, Audisio P (2010) Conflict between insect conservation and public safety: the case study of a saproxylic beetle (Osmoderma eremita) in urban parks. Journal of Insect Conservation 14: 555–565. 6. Gibbons P, Lindenmayer DB (2002) Tree hollows and wildlife conservation in Australia. Victoria, Australia: CSIRO publishing. 21. Cohen JE (2003) Human Population: The Next Half Century. Science 302: 1172–1175. 7. Ranius T (2002) Influence of stand size and quality of tree hollows on saproxylic beetles in Sweden. Biological Conservation 103: 85–91. 22. Grimm NB, Faeth SH, Golubiewski NE, Redman CL, Wu J, et al. (2008) Global change and the ecology of cities. Conclusion We have quantified the decline of hollow-bearing trees in an urban landscape over centuries. We provided a novel assessment of the conservation implications associated with existing tree management practices and the efficacy of a range of alternative management strategies. It is evident from our results that existing urban tree management practices require urgent re-evaluation if hollow and tree-dependent biodiversity are to be maintained in The Future of Large Old Trees in Urban Landscapes recreation and conservation. We recognize that it may not be possible to retain all trees to their maximum biological age due to public safety risks. It may also not be practical or feasible to accelerate the formation of habitat structures artificially on a large enough scale over prolonged time periods. Instead, we attempt to balance socio-economic and biodiversity benefits by combining multiple tree management and maintenance approaches in an achievable manner. Future research should also aim to investigate alternative management scenarios from a more financial perspec- tive, which too would benefit practitioners (e.g. numbers of hollow- bearing trees gained per management dollar spent). However, even under our proposed management strategy, the density of hollow-bearing trees is predicted to first decline, or undergo a bottleneck, before increasing. This is because of an extinction debt or the time lag between implementing management actions and actually observing an increase in hollow-bearing trees. Delaying mitigation is anticipated to further exacerbate the effects of time lags and require more drastic measures at greater costs to reverse tree declines [26]. Immediate action will likely also reduce bottlenecks in urban plant and animal populations that depend on large old trees for survival. urban landscapes. We recommend that: (1) large old trees are afforded better protection and remain standing over longer time periods; (2) tree regeneration is actively improved so that large old trees lost over time are replaced by younger trees; and (3) the formation of habitat structures provided by large old trees is accelerated to compensate for short term deficits in resource availability. Immediate implementation of these recommendations is needed to arrest the decline of large old trees, avoid lag effects, and avert long term risk to biodiversity in urban landscapes. Acknowledgments We thank Dr. Peter Lane for his statistical support, the many private landowners and reserve managers who granted access to properties for vegetation surveys, and anonymous reviewers who improved earlier versions of this manuscript. References Science 319: 756–760. 8. Webb JK, Shine R (1997) Out on a limb: Conservation implications of tree- hollow use by a threatened snake species (Hoplocephalus bungaroides: Serpentes, Elapidae). Biological Conservation 81: 21–33. 23. Theobald DM, Miller JR, Hobbs NT (1997) Estimating the cumulative effects of development on wildlife habitat. Landscape and Urban Planning 39: 25–36. p p g 9. Newton I (1994) The role of nest sites in limiting the numbers of hole-nesting birds: A review. Biological Conservation 70: 265–276. 24. Blewett CM, Marzluff JM (2005) Effects of urban sprawl on snags and the abundance and productivity of cavity-nesting birds. The Condor 107: 678–693. 10. Lindenmayer DB, Cunningham RB, Tanton MT, Smith AP (1990) The conservation of arboreal marsupials in the montane ash forests of the Central Highlands of Victoria, south-east Australia: II. The loss of trees with hollows and its implications for the conservation of leadbeater’s possum Gymnobelideus leadbeateri McCoy (marsupialia: petauridae). Biological Conservation 54: 133– 145. 25. McDonald RI, Kareiva P, Forman RTT (2008) The implications of current and future urbanization for global protected areas and biodiversity conservation. Biological Conservation 141: 1695–1703. 26. Manning AD, Gibbons P, Fischer J, Oliver DL, Lindenmayer DB (2012) Hollow futures? Tree decline, lag effects and hollow-dependent species. Animal Conservation 16: 395–405. 11. Lindenmayer DB, Laurance WF, Franklin JF (2012) Global Decline in Large Old Trees. Science 338: 1305–1306. 27. Cockle KL, Martin K, Wesolowski T (2011) Woodpeckers, decay, and the future of cavity-nesting vertebrate communities worldwide. Frontiers in Ecology and the Environment 9: 377–382. 12. Gibbons P, Lindenmayer DB, Fischer J (2008) The future of scattered trees in agricultural landscapes. Conservation Biology 22: 1309–1319. 28. Nagendra H, Gopal D (2010) Street trees in Bangalore: Density, diversity, composition and distribution. Urban Forestry & Urban Greening 9: 129–137. 13. Sodhi NS, Koh LP, Clements R, Wanger TC, Hill JK, et al. (2010) Conserving Southeast Asian forest biodiversity in human-modified landscapes. Biological Conservation 143: 2375–2384. 29. Terho M (2009) An assessment of decay among urban Tilia, Betula, and Acer trees felled as hazardous. Urban Forestry & Urban Greening 8: 77–85. 30. Pauleit S, Ennos R, Golding Y (2005) Modeling the environmental impacts of urban land use and land cover change—a study in Merseyside, UK. Landscape and Urban Planning 71: 295–310. 14. Laurance WF, Laurance SG, Ferreira LV, Rankin-de Merona JM, Gascon C, et al. (1997) Biomass Collapse in Amazonian Forest Fragments. Science 278: 1117– 1118. Author Contributions Conceived and designed the experiments: DSL KI DBL ADM PG. Performed the experiments: DSL PG. Analyzed the data: DSL PG. Contributed reagents/materials/analysis tools: DSL PG. Wrote the paper: DSL KI DBL ADM PG. Conceived and designed the experiments: DSL KI DBL ADM PG. Performed the experiments: DSL PG. Analyzed the data: DSL PG. Contributed reagents/materials/analysis tools: DSL PG. Wrote the paper: DSL KI DBL ADM PG. The Future of Large Old Trees in Urban Landscapes Our proposal considers the complexities associated with managing urban greenspaces for multiple purposes, including June 2014 \| Volume 9 \| Issue 6 \| e99403 PLOS ONE \| www.plosone.org 9 The Future of Large Old Trees in Urban Landscapes Supporting Information Table S1 List of recorded tree species and diameter size class distributions. (DOCX) Summary S1 Description of the simulation model used for analyses. (DOCX) Summary S1 Description of the simulation model used for analyses. (DOCX) The Future of Large Old Trees in Urban Landscapes Banks JCG, Brack CL (2003) Canberra’s Urban Forest: Evolution and planning for future landscapes. Urban Forestry and Urban Greening 1: 151–160. 60. Stagoll K, Lindenmayer DB, Knight E, Fischer J, Manning AD (2012) Large trees are keystone structures in urban parks. Conservation Letters. p y g 39. ACT Government (2004) Woodlands for Wildlife: ACT Lowland Woodland Conservation Strategy. In:Environment ACT, Urban Services, editor. Canberra: Environment ACT. 61. Weinberg A, Gibbons P, Briggs SV, Bonser SP (2011) The extent and pattern of Eucalyptus regeneration in an agricultural landscape. Biological Conservation 144: 227–233. 40. Department of Environment and Heritage (2006) EPBC policy statement 3.5 - white box - yelllow box - Blakely’s red gum grassy woodlands and derived native grasslands listing. Canberra, Australia. 62. Pincetl S (2010) Implementing Municipal Tree Planting: Los Angeles Million- Tree Initiative. Environmental Management 45: 227–238. 63. Wu C, Xiao Q, McPherson EG (2008) A method for locating potential tree- planting sites in urban areas: A case study of Los Angeles, USA. Urban Forestry & Urban Greening 7: 65–76. g g , 41. Gibbons P, Boak M (2002) The value of paddock trees for regional conservation in an agricultural landscape. Ecological Management & Restoration 3: 205–210. 42. Manning AD, Lindenmayer DB, Barry SC (2004) The conservation implications of bird reprodcution in the agricultural "matrix": A case study of the vulnerable superb parrot of south-eastern Australia. Biological Conservation 120: 363–374. 64. Nowak DJ, Kuroda M, Crane DE (2004) Tree mortality rates and tree population projections in Baltimore, Maryland, USA. Urban Forestry & Urban Greening 2: 139–147. p p g 43. Gibbons P, Briggs SV, Ayers DA, Doyle S, Seddon J, et al. (2008) Rapidly quantifying reference conditions in modified landscapes. Biological Conservation 141: 2483–2493. 65. Tonnesen AS, Ebersole JJ (1997) Human trampling effects on regeneration and age structures of Pinus edulis and Juniperus monosperma. Western North American Naturalist 57: 55–56. 44. Rayner L, Ellis M, Taylor JE (2011) Double sampling to assess the accuracy of ground-based surveys of tree hollows in eucalypt woodlands. Austral Ecology 36: 252–260. 66. Pauleit S (2003) Urban street tree plantings: identifying the key requirements. Proceedings of the ICE - Municipal Engineer. pp. 43–50. 67. Lindenmayer DB, Welsh A, Donnelly C, Crane M, Michael D, et al. (2009) Are nest boxes a viable alternative source of cavities for hollow-dependent animals? Long-term monitoring of nest box occupancy, pest use and attrition. Biological Conservation 142: 33–42. 45. The Future of Large Old Trees in Urban Landscapes The Future of Large Old Trees in Urban Landscapes 31. Lehva¨virta S, Rita H (2002) Natural regeneration of trees in urban woodlands. Journal of Vegetation Science 13: 57–66. study on trees with hollows in south-eastern Australia. Forest Ecology and Management 260: 975–982. study on trees with hollows in south-eastern Australia. Forest Ecology and Management 260: 975–982. g 53. Mooney CZ (1997) Monte Carlo Simulation. California: Sage Pub 32. Harper MJ, McCarthy MA, van der Ree R (2005) The use of nest boxes in urban natural vegetation remnants by vertebrate fauna. Wildlife Research 32: 509–516. 54. ACT Government (2012) Canberra Tree Audit: Method Field Guidelines. In: Territory and Municipal Services, editor. Canberra: ACT Government. Territory and Municipal Services, editor. Canberra: ACT Governme 33. Thor G, Johansson P, Jo¨nsson M (2010) Lichen diversity and red-listed lichen species relationships with tree species and diameter in wooded meadows. Biodiversity and Conservation 19: 2307–2328. 55. McCarthy MA, Burgman MA, Ferson S (1995) Sensitivity analysis for models of population viability. Biological Conservation 73: 93–100. 56. White JW, Rassweiler A, Samhouri JF, Stier AC, White C (2013) Ecologists should not use statistical significance tests to interpret simulation model results. Oikos 123: 385–388. 34. Manning AD, Lindenmayer DB, Cunningham RB (2007) A study of coarse woody debris volumes in two box-gum grassy woodland reserves in the Australian Capital Territory. Ecological Management & Restoration 8: 221– 224. 57. Pulido FJ, Dı´az M, Hidalgo De Trucios SJ (2001) Size structure and regeneration of Spanish holm oak Quercus ilex forests and dehesas: Effects of agroforestry use on their long-term sustainability. Forest Ecology and Management 146: 1–13. 35. Gibbons P, Lindenmayer DB, Barry SC, Tanton MT (2000) Hollow formation in eucalypts from temperate forests in southeastern Australia. Pacific Conservation Biology 6: 218–228. 58. Rodrigues ASL, Andelman SJ, Bakarr MI, Boitani L, Brooks TM, et al. (2004) Effectiveness of the global protected area network in representing species diversity. Nature 428: 640–643. 36. Harper MJ, McCarthy MA, van der Ree R (2005) The abundance of hollow- bearing trees in urban dry sclerophyll forest and the effect of wind on hollow development. Biological Conservation 122: 181–192. y 59. Rhodes M, Wardell-Johnson GW, Rhodes MP, Raymond BEN (2006) Applying Network Analysis to the Conservation of Habitat Trees in Urban Environments: a Case Study from Brisbane, Australia p g 37. ACT Government (2011) Population and residential density in Canberra. Canberra: ACT Government. 38. References PLOS ONE \| www.plosone.org June 2014 \| Volume 9 \| Issue 6 \| e99403 June 2014 \| Volume 9 \| Issue 6 \| e99403 10 The Future of Large Old Trees in Urban Landscapes Gibbons P, Lindenmayer DB, Barry SC, Tanton MT (2002) Hollow selection by vertebrate fauna in forests of southeastern Australia and implications for forest management. Biological Conservation 103: 1–12. g g 46. Pearce J, Ferrier S (2000) Evaluating the predictive performance of habitat models developed using logistic regression. Ecological Modelling 133: 225–245. 68. Bull EL, Partridge AD (1986) Methods of Killing Trees for Use by Cavity Nesters. Wildlife Society Bulletin 14: 142–146. 47. Banks JCG (1997) Tree ages and ageing in yellow box. In: Dargavel J, editor. The coming of age Forest age and heritage values.Canberra, Australia: Environment Australia. pp. 17–28. 69. Shepherd KR (1957) Some aspects of ringbarking in Alpine ash forests. Australian Forestry 21: 70–75. 70. Hane ME, Kroll AJ, Johnson JR, Rochelle M, Arnett EB (2012) Experimental effects of structural enrichment on avian nest survival. Forest Ecology and Management 282: 167–174. pp 48. Fischer J, Stott J, Zerger A, Warren G, Sherren K, et al. (2009) Reversing a tree regeneration crisis in an endangered ecoregion. PNAS 106: 10386–10391. 49. Wellington AB, Noble IR (1985) Post-Fire Recruitment and Mortality in a Population of the Mallee Eucalyptus Incrassata in Semi-Arid, South-Eastern Australia. Journal of Ecology 73: 645–656. 71. Adkins MF (2006) A burning issue: using fire to accelerate tree hollow formation in Eucalyptus species. Australian Forestry 69: 107–113. J gy 50. Scho¨nau APG, Coetzee J (1989) Initial spacing, stand density and thinning in eucalypt plantations. Forest Ecology and Management 29: 245–266. 72. Whitford KR, Stoneman GL, Freeman IA, Reynolds MJ, Birmingham TC (1995) Mortality of Eucalyptus marginata (jarrah) and E. calophylla (marri) trees following stem injection: effects of herbicide, dose, season, and spacing of injections. Australian Forestry 58: 172–178. yp p gy g 51. ACT Government (2013) Management of Trees on Public Land. In: Territory and Municipal Services, editor. Canberra, ACT. and Municipal Services, editor. Canberra, ACT. 73. Carey AB, Sanderson HR (1981) Routing to Accelerate Tree-Cavity Formation. Wildlife Society Bulletin 9: 14–21. 52. Gibbons P, McElhinny C, Lindenmayer DB (2010) What strategies are effective for perpetuating structures provided by old trees in harvested forests? A case June 2014 \| Volume 9 \| Issue 6 \| e99403 PLOS ONE \| www.plosone.org 11
https://openalex.org/W2401561002	https://www.scielo.br/j/rbla/a/vhkkVFfJ5xk5gSwHHDM3Jsm/?lang=pt&format=pdf	Portuguese	null	Ficções de si: a escrita entre línguas-culturas	Revista Brasileira de Lingüística Aplicada	2,015	cc-by	11,557	http://dx.doi.org/10.1590/1984-639820159047 http://dx.doi.org/10.1590/1984-639820159047 marluza.rosa@gmail.com 1 Considerações iniciais Não posso mais escrever de maneira monolingüe. O que quero dizer é que deporto e desarrumo minha língua, não elaborando sínteses, mas sim através de aberturas lingüísticas que me permitem conceber as relações das línguas entre si em nossos dias, na superfície da terra – relações de dominação, de convivência, de absorção, de opressão, de erosão, de tangência, etc. – como em um imenso drama, em uma imensa tragédia de que minha língua não pode ficar isenta e salva. E, por conseguinte, não posso escrever minha língua de maneira monolingüe; escrevo-a na presença dessa tragédia, na presença desse drama (GLISSANT, 2001/2005, p. 49-50). Com essas palavras – que poeticamente descrevem o turbilhão de línguas em meio ao qual o sujeito contemporâneo se constitui –, Édouard Glissant, escritor e crítico literário, reflete sobre a escritura. Em meio a tais condições, como escrever, de onde escrever, em qual língua, já que o movimento entre línguas carrega em si os desarranjos de uma subjetividade deslocada? Como assumir como minha a língua do outro? Não se pode escrever de maneira monolíngue, reiteradamente, soa, por um lado, como o sintoma de um mal-estar, de uma falta de pertencimento: não estou mais em minha língua, ou melhor, não há mais (se se pode dizer que algum dia houve) minha língua, única, intocada, inalterável, mas todas as línguas que, na relação estabelecida entre si, fazem-se presentes em meu dizer. Por outro lado, essa repetição soa como uma palavra de ordem: não se deve escrever de maneira monolíngue, pois, como não sou imune às transformações desencadeadas pela mobilidade, não posso escrever de fora dessas “relações de dominação, de convivência, de absorção, de opressão, de erosão, de tangência”. p g É da constituição de uma escrita de si no movimento e no contato entre línguas que trata esta reflexão, desenvolvida a partir de uma releitura de nossa dissertação de mestrado, na qual tratamos dos efeitos de sentido constituídos acerca da noção de língua pelo sujeito pesquisador da linguagem (DA ROSA, 2009). Naquele estudo, partimos da conjetura de que os modos de designar a língua – tais como língua materna, língua estrangeira, língua de origem, língua de adoção, dentre outros –, colocados em funcionamento em artigos acadêmicos pertencentes ao domínio das ciências da linguagem, são também influenciados pela identificação do pesquisador com as línguas por entre as quais se desloca. Ficções de si: a escrita entre línguas-culturas Self-fictions: writing among languages-cultures Marluza T. da Rosa Universidade Estadual de Campinas Campinas - São Paulo / Brasil RESUMO: Neste artigo, propomo-nos a indagar sobre o papel da escrita no aprendizado de línguas estrangeiras e na construção de uma imagem de si em uma língua outra. Para tanto, analisamos excertos da produção textual de aprendizes de português como língua estrangeira, realizada durante o ano letivo de 2013-2014. Inseridos no domínio da Linguística Aplicada, ancoramos esta discussão no cruzamento entre os estudos do discurso, da psicanálise e da desconstrução, entendendo que a escrita na língua do outro não só (d)enuncia o imaginário acerca do estrangeiro, mas também funciona como espelho para uma constituição identitária múltipla e em constante transformação. Os fragmentos analisados permitem-nos afirmar que é possível escrever(-se) para além da estaticidade que, muitas vezes, impera nas atividades de escrita escolares/acadêmicas, o que contribui para a (re)inscrição de si, afetada pela presença constante do outro, enquanto multiplicidade irredutível a uma única língua-cultura. PALAVRAS-CHAVE: encontros linguísticos; escrita; ensino de línguas. ALAVRAS-CHAVE: encontros linguísticos; escrita; ensino de línguas. ABSTRACT: This paper aim to call into question the role of writing in the foreign language learning process and in the construction of ‘self’ in another language. To achieve this goal, we analyzed texts written by Portuguese learners, which were produced during the school year of 2013-2014. Inserted in the Applied Linguistics field, this discussion is grounded on the theoretical intersection among discourse, psychoanalysis, and deconstruction studies, and understands that writing in another language not only (de)(an)nounces representations of the foreigner, but also works as a mirror for a multiple identity construction, which is in constant transformation. The data allowed us to state that it is possible to write oneself beyond the stativity that often prevails in school / academic writing activities, which contributes to the (re)inscription of ‘self’, affected by the constant presence of the other, as an irreducible multiplicity to a single language-culture. KEYWORDS: linguistic encounters; writing; language learning. *marluza.rosa@gmail.com RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 81 RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 1 « Comment celui qui écrit donne-t-il forme à son désir d’écrire, à partir de sa propre existence et d’un nouage intime à la ou aux langues dans lesquelles il écrit ? ». As traduções de todas as citações que figuram em nota de rodapé são nossas. 1 Considerações iniciais Assim, defendemos que, pelo modo de designar a língua, torna-se possível tratar da formulação/circulação de um discurso RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 82 constituído em e por um entre-lugar, ou seja, um lugar intervalar onde as línguas se imbricam na constituição do sujeito (CORACINI, 2007). É em torno da possibilidade de refletirmos sobre a relação entre línguas-culturas como determinante nessa constituição que discorreremos neste texto. Para tanto, o gesto de leitura que aqui desenvolvemos remonta à problemática que concerne à relação do sujeito com a(s) língua(s), mas também ao envolvimento com a escrita em um ambiente de aprendizagem formal: espaço cerceador, já que se deve escrever e se inserir em uma ordem de discurso, mas, ao mesmo tempo, espaço que potencializa uma escrita de si, a qual, por sua vez, pode mobilizar aspectos da subjetividade. Tendo em vista, contudo, que a relação do sujeito com as línguas não se restringe tão somente ao âmbito escolar, no qual uma simples distinção entre língua materna e língua estrangeira bastaria para estabelecer limites entre as línguas, para colocar cada uma em seu compartimento, interessa-nos revisitar a pergunta já formulada por Prieur (2006a, p. 485): “como aquele que escreve dá forma a seu desejo de escrever a partir de sua própria existência e de uma amarração íntima à ou às línguas nas quais escreve?”1. Abordaremos, primeiramente, a questão da escrita, para, então, adentrarmos nos meandros dessa escrita de si. 2 De uma escritura rizomática entre línguas-culturas Para indagarmos sobre o papel da escrita no aprendizado de línguas estrangeiras e, mais pontualmente, na constituição de uma imagem de si em uma língua outra, não podemos nos abster de sinalizar o vínculo entre essa problemática e a noção de discurso, seja como feixe de práticas discursivas, como o define Foucault (1969/2009), seja como laço social, segundo Lacan (1974). Esse é um ponto incontornável, posto que o discurso, nesse viés, é marcado pela heterogeneidade, enquanto lugar de estranhamento, de conflito e de constituição de si mesmo com o (e como) outro. Tais considerações exercem um papel importante para esta discussão, pois, quando tratamos de contatos linguísticos, o que ressoa é esse modo de constituição subjetiva em um interstício, ou seja, em um entremeio. A noção de interlíngua tem sido constantemente utilizada nos contextos de ensino-aprendizagem para tratar dessa relação. Contudo, RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 83 devemos permanecer atentos para que essa forma de abordagem, apesar de aparentemente aberta à inter-relação linguística, não reduza o múltiplo ao dual, ao tratar de um intervalo que seria apenas um entre dois. A esse respeito, concordamos com Prieur (2006b) quando o autor destaca que todo ser falante é constitutivamente um sujeito multilíngue, inscrito em uma lógica plural de falares, dialetos, estilos familiares ou formais, o que nos impõe a contingência de pensar para além de uma ordem binária: não se trata de tentar se comunicar entre duas línguas, mas de tomar uma posição e produzir sentidos na relação com ao menos duas línguas. Tal perspectiva não só possibilita que se compreenda esse processo como não sendo meramente cognitivo, em que seria possível “dominar” conscientemente dois ou diversos sistemas, mas também permite que se analise a própria materialidade linguística, a partir de sua dimensão social, cultural, histórica e subjetiva. Sendo assim, defendemos que esse espaço entre, esse interstício, não consiste em uma “fase”, em que o aprendiz “ainda não se desprendeu da língua chamada materna” (DA ROSA, 2009, p. 44), fase em que esta exerceria uma “interferência” prejudicial na aprendizagem da língua estrangeira, nos moldes de uma perspectiva que, segundo Coracini (2007, p. 145), apontaria “para a possibilidade de numerar as línguas, estudando a aquisição de uma (ou mais línguas) como algo passível de ser descrito, analisado, objetificado, categorizado”. 2 De uma escritura rizomática entre línguas-culturas Defender a existência de uma (má) interferência linguística, além de escamotear aspectos singulares que apontam para o investimento subjetivo dos falantes, reduz o que se considera “linguístico” ao plano tão somente formal ou gramatical. Ora, essa visão, por um lado, não se sustentaria, pois falar uma língua não se restringe ao âmbito cognitivo, consciente e racionalmente calculado, tampouco consiste em uma relação polarizada, mas múltipla e inumerável, uma vez que estão em jogo aspectos sócio-histórico- culturais, como já mencionamos, e não sistemas linguísticos descarnados. Por outro lado, essa abordagem não sustentaria (ou limitaria em muito) “um pensamento da descontinuidade, da flutuação, da multiplicidade” (PRIEUR, 2006b, p. 115), em consonância com o momento contemporâneo. É por essa razão que afirmamos que a produção de sentidos, nesse processo, possui um caráter de rizoma, nos termos de Deleuze e Guattari (1980/1995), no qual as línguas e as culturas assumem valores pelas relações que estabelecem entre si; relações estas que tendem para várias direções, caracterizando uma cartografia, na qual espaço e tempo se suspendem ou se abrem ao questionamento. RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 84 É também nesse (du)elo entre línguas-culturas que se pode tomar a noção de identidade, posto que esta não se afirma apenas pela diferença – sou idêntico a mim mesmo por ser diferente do outro, externo a mim –, mas pela ida ao encontro desse outro. Identidade rizoma, segundo Glissant (2001/2005, p. 28), que nos coloca frente a um dilema: “como ser si mesmo sem fechar-se ao outro, e como abrir-se ao outro sem perder-se a si mesmo?”. O que marca uma possível resposta a esse impasse é a insistência em uma poética e em uma política da relação, no sentido de compreender que, se há uma identidade possível, ela só pode se dar a partir do outro e do caminho que se faz para encontrá-lo. Falar e escrever ou, de forma mais ampla e intrínseca, inserir-se na linguagem, implica estar impregnado pelos outros e pelo Outro (enquanto amplitude da cultura, da inscrição social, mas também do inconsciente). Desse modo, talvez seja mais profícuo pensarmos nessa simbiose identitária nos termos de uma relação ou de um encontro, tanto como convergência, tal como o encontro entre rios, quanto como “dupla-captura” (DELEUZE e PARNET, 1996/1998), e não de um contato linguístico. RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 2 De uma escritura rizomática entre línguas-culturas Além disso, pensar em uma relação, que envolve tanto atração quanto repulsão, permite evocar as relações de poder-saber enquanto constitutivas do sujeito sócio-histórico, tal como o desenvolve Foucault (1969/2009); poder este que é, ao mesmo tempo, cerceador e produtivo. Sendo assim, a escolha por pensar a questão identitária pelo viés da convergência e da dupla-captura pode potencializar a problematização de fenômenos “que vêm misturar estas fronteiras, aqueles que as ultrapassam deixando assim aparecer o seu artifício histórico, isto é, as relações de força que aí se concentram”, como o quer Derrida (1996/2001, p. 21). Essas relações permitem-nos remontar ao excerto de Glissant (2001/2005), tomado como mote deste estudo, dados seus desdobramentos “de dominação, de convivência, de absorção, de opressão, de erosão, de tangência”, tendendo para uma multiplicidade incontornável. Tal pluralidade se deve ao movimento que tem marcado nossa sociedade e que não se restringe apenas à facilidade de nos deslocarmos fisicamente, mas também à possibilidade de interagirmos constantemente, devido ao desenvolvimento de novas formas de comunicação, principalmente ligadas à Internet. Contudo, salientamos que esse movimento não é uma condição sine qua non de um pensamento plural, pois toda língua é marcada pela diversidade de falares, pelo atravessamento de outras línguas, o que leva Prieur (2006b, p. 111) a assegurar que “não é língua se não for língua-mista, RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 85 não é subjetividade se não for compósita”2. Heterogêneos, sujeito e língua são marcados pelo outro (presente enquanto alteridade/heterogeneidade), o que os torna irredutíveis ao um/uno. Efeitos dessa não-unidade podem ser apontados na relação que se estabelece com a escrita na/da língua estrangeira, pois, segundo Rocha (2005, p. 11), essas “transformações do espaço-tempo e das realidades culturais, bem como os deslocamentos e as errâncias tornam-se constitutivos de sua própria escrita”. p p Se explorarmos o que a autora designa como “própria escrita”, podemos pensar em uma escrita característica de alguém, mas também que caracteriza alguém, uma vez que, se o sujeito é constituído nas e pelas línguas por entre as quais transita, é também a escrita de si, a maneira de escrever e de se inscrever nas línguas, que será modificada. Nesse sentido, sua “própria escrita” já funciona como uma escritura, nas palavras de Coracini (2007, p. 127), “inscrição do sujeito (híbrido) numa língua que é, como ele, sempre híbrida, que é sempre do outro e sempre a sua”. 2 « il n’est pas de langue qui ne soit une langue-mixte, il n’est pas de subjectivité qui ne soit composite ». 3 Le deuil de l’origine. 4 Tradução adaptada de « parce que d’une langue à l’autre peuvent s’inventer des ‘ponts verbaux’, des liens, des articulations, ceux d’un réseau associatif opérant à travers mots, phénomènes, assonances ou homophonies. Il peut être création d’un ‘espace ouvert’, et d’une subjectivité ou d’une trans-subjectivité à vivre dans le passage, la traversée, et qui, dans l’angoisse ou la jouissance du changement de corps et de langue, voit ses frontières questionnées ». 2 De uma escritura rizomática entre línguas-culturas Nesse sentido, tanto a reflexão de Derrida (1996/2001) sobre sua própria condição à beira das línguas – já que a única língua que ousa chamar de sua é o francês, a língua do colonizador, do outro, portanto –, quanto seus diálogos com Roudinesco em torno da problemática da herança (DERRIDA e ROUDINESCO, 2001/2004), são esclarecedores, pois falar uma língua não diz respeito apenas ao âmbito linguístico. Falar uma língua, como uma herança, implica a não passividade, a decisão de, ao mesmo tempo, aceitar e transformar o que se herda, em uma busca constante por se apropriar do inapropriável. Em função disso, é que nunca se está em casa em uma língua, é-se sempre estrangeiro, “sempre exilado”, no dizer de Coracini (2007, p. 48), para quem, toda língua é estrangeira, na medida em que provoca em nós estranhamentos, e toda língua é materna, na medida em que nela nos inscrevemos, em que ela se faz ninho, lar, lugar de repouso e de aconchego; ou melhor, toda língua é materna e estrangeira ao mesmo tempo (CORACINI, 2007, p. 48). toda língua é estrangeira, na medida em que provoca em nós estranhamentos, e toda língua é materna, na medida em que nela nos inscrevemos, em que ela se faz ninho, lar, lugar de repouso e de aconchego; ou melhor, toda língua é materna e estrangeira ao mesmo tempo (CORACINI, 2007, p. 48). Em diversos estudos que têm se voltado para essa questão, a compreensão do desconforto e do mal-estar frente à(s) língua(s) tem ganhado mais ênfase do que a simples análise valorativa dos erros do 2 « il n’est pas de langue qui ne soit une langue-mixte, il n’est pas de subjectivité qui ne soit composite ». RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 86 aprendiz, principalmente a partir da ancoragem psicanalítica, desde O estranho freudiano (1919/1976) ao Estrangeiros para nós mesmos, de Kristeva (1988/1994), ou a O luto da origem3, de Robin (2003). Contudo, se toda língua é materna e estrangeira, a experiência de saber-poder decorrente do encontro com outras línguas pode não ser sempre vivenciada como um mal- estar paralisante, mas como uma possibilidade de outra ficção de si-mesmo ou da criação de outro si-mesmo. 2 De uma escritura rizomática entre línguas-culturas No âmbito da escritura, enquanto inscrição de si, mas também como risco de perda, de morte, de finitude, esses duplos possíveis podem ganhar contornos, pois de uma língua à outra podem-se inventar ‘pontes verbais’, laços, articulações de uma rede associativa que opera através de palavras, fonemas, assonâncias ou homofonias. Podem-se criar um ‘espaço aberto’ e uma subjetividade ou uma trans-subjetividade a serem vividos na passagem, na travessia, e que, na angústia ou no gozo da mudança de corpo e de língua, veem suas fronteiras questionadas4 (PRIEUR, 2006b, p. 116). Experimentaremos essa possibilidade de reinvenção com o auxílio da noção de identidade narrativa, formulada por Ricoeur (1990; 2008), a qual não se caracteriza pura e simplesmente pela criação de uma personagem para agir em uma história, mas pelo modo como essa personagem (se) significa no tempo e no enredo narrativo, bem como pela forma como buscamos compreender nossa própria subjetividade com base nas narrativas que nossa cultura oferece. Constituímo-nos, construindo fantasias de nós mesmos, de modo que, se nossa identidade está em constante transformação, esta se mostra em uma dialética, constitutiva da própria noção de identidade, entre idem (a mesmidade, o idêntico) e ipse (a alteridade, o outro de – ou que não seja – si mesmo), entre concordância e discordância, reunidas em uma narrativa que se configura como uma “síntese do heterogêneo”, nos termos de Ricoeur, para quem “o que chamamos de subjetividade não é nem uma RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 87 sequência incoerente de acontecimentos nem uma substancialidade imutável, inacessível ao devir”5 (RICOEUR, 2008, p. 274). Assim, mesmo que não sejamos os autores da nossa vida, somos os narradores de nossa história, sendo essa dimensão narrativa o que caracteriza uma vida, no sentido biológico do termo, como vida humana, ainda em conformidade com o autor. Consequentemente, se aceitarmos que é diante do outro, na relação com traços de pessoas, textos, autores, personagens com os quais nos identificamos que compreendemos e construímos a nós mesmos, é possível afirmarmos que um acontecimento análogo também tem lugar/acontece em outras línguas, que nos proporcionam o confronto com outras culturas e outras narrativas. 5 « ce que nous appelons la subjectivité n’est ni une suite incohérente d’événements ni une substantialité immuable inaccessible au devenir ». 6 « l’histoire d’une vie procède d’histoires non racontées et refoulées en direction d’histoires effectives que le sujet pourrait prendre en charge et tenir pour constitutives de son identité personnelle ». 7 A esse respeito, conferir Prieur (2006a) e Deleuze e Parnet (1996/1998). ce que ous appe o s a subject v té est u e su te co é e te d évé e e ts ni une substantialité immuable inaccessible au devenir ». 6 « l’histoire d’une vie procède d’histoires non racontées et refoulées en direction d’histoires effectives que le sujet pourrait prendre en charge et tenir pour constitutives de son identité personnelle ». 2 De uma escritura rizomática entre línguas-culturas Mais ainda, temos a possibilidade de mesclar essas culturas e essas narrativas de modo a criar esse espaço aberto, essa subjetividade a ser vivenciada na travessia, de que trata Prieur (2006b), desterritorializando- nos e reterritorializando-nos, criando um território nosso, nosso lugar, compreendendo nosso pequeno drama subjetivo e situando-nos nessa “imensa tragédia” de que não saímos indenes. g q Contudo, a noção de identidade narrativa implica ainda uma outra dimensão, uma outra cena, diríamos psicanaliticamente, pois, de acordo com Ricoeur (2008, p. 271), “a história de uma vida procede de histórias não contadas e recalcadas em direção a histórias efetivas que o sujeito poderia assumir e sustentar como constitutivas de sua identidade pessoal”6. Histórias efetivas que, talvez, sejam mais aceitáveis, mais de acordo com dada cultura, dada época de uma sociedade. Se o que o autor chama de identidade pessoal é aquela história contada, a partir da junção de acontecimentos diversos em um todo coerente, o que faríamos com as histórias não contadas ou mal contadas? É sabido que a língua estrangeira pode consistir em uma válvula de escape para estas, como já apontaram pesquisadores que se voltam para o estudo de grandes escritores, os quais só escreveram sobre determinados temas tabus, sobre determinados afetos, afetados pela/na “língua do outro”, tais como Kafka, Beckett, Joyce e Artaud7. O principal exemplo, dentre os 5 « ce que nous appelons la subjectivité n’est ni une suite incohérente d’événements ni une substantialité immuable inaccessible au devenir ». 6 « l’histoire d’une vie procède d’histoires non racontées et refoulées en direction d’histoires effectives que le sujet pourrait prendre en charge et tenir pour constitutives de son identité personnelle ». RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 88 nossos, são os heterônimos de Fernando Pessoa e seus poemas escritos nas línguas inglesa e francesa. Essa identidade narrativa se constitui, portanto, dialeticamente, no espaço intervalar entre uma tentativa de estabilização identitária e o constante movimento. Ela é um ponto de apoio subjetivo, uma tábua de salvação ou, então, uma linha de fuga. Todavia, não é evidente que se possa, por meio da escritura na e pela língua estrangeira, refletir sobre si mesmo ou se dizer. A própria escolha por uma ou outra língua não é dada conscientemente. RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 2 De uma escritura rizomática entre línguas-culturas Tampouco é evidente que tal compreensão narrativa de nós mesmos tenha lugar nem na chamada língua materna, pois estamos imersos e ocupados em viver e narrar essa ficção que é nossa realidade, já que ambas se imbricam, interpenetram-se, como pontua Coracini (2007). É fundamental destacar, nesse sentido, que, mesmo quando não pensamos construir uma personagem, mesmo quando tentamos falar do que acreditamos ser nós mesmos, em nossa mais pura essência, construímos uma personagem, produzimos uma ficção. Foi o que já afirmamos ao analisar um corpus bastante distinto do que será aqui discutido, a saber, o dizer de estudantes sobre seu processo de formação para a pesquisa científica, em que foi mobilizado um imaginário sobre a ciência e sobre o pesquisador ou o cientista (DA ROSA, 2013). Nesse caso, embora falando da ciência e do cientista, o que, supostamente, distanciava-se do eu (pois os pesquisadores em formação não se viam como pesquisadores nem como cientistas, acreditando estarem longe de uma Ciência com maiúscula, já que só estudavam e faziam pesquisa), era também uma ficção de si que se construía nessa relação. É o que nos permite assegurar, juntamente com Foucault (1979/2010, p. 34), que nossa identidade é apenas “uma máscara, é apenas uma paródia: o plural a habita, almas inumeráveis nela disputam; os sistemas se entrecruzam e se dominam uns aos outros”. Somos, portanto, jogos de máscara que se potencializam no encontro com novas línguas e outras culturas. E não poderia ser de outro modo, pois a imagem de si, supostamente una e estável, constitui-se como parte de uma fantasia. Porém, antes que se possa pensar no caráter descartável dessas faces ou na possibilidade de facilmente intercambiarmos umas e outras a nosso bel prazer, salientamos que elas nos constituem como “almas”, retomando o termo foucaultiano, o que não nos deixa esquecer de sua dimensão inapreensível. RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 89 O que o multilinguismo permite, assim, não é a troca de identidade, a aquisição de uma nova máscara, mas a incorporação, no sentido de fazer corpo, de outras marcas, traços diferentes, mas também semelhantes aos que já considerávamos “nossos”. 8 Na instituição em questão, a disciplina de língua portuguesa integra, por um lado, o quadro de cursos oferecidos pelo Departamento de Estudos Transversais, destinados a todos os estudantes interessados, e, por outro, a grade de disciplinas ofertadas pelo Departamento de Línguas, com foco na formação em Estudos Hispânicos. O fato de a língua portuguesa ser uma das línguas estrangeiras exigidas pelo curso (sendo as outras opções o Latim e o Catalão) faz com que a maioria dos alunos de português esteja em preparação para a atuação como professores de língua espanhola. 2 De uma escritura rizomática entre línguas-culturas Logo, como concebe Deleuze, em entrevista com Parnet, “o multilingüismo não é apenas a posse de vários sistemas, sendo cada um homogêneo em si mesmo; é, antes de tudo, a linha de fuga ou de variação que afeta cada sistema impedindo-o de ser homogêneo” (DELEUZE e PARNET, 1996/1998, p. 12). Consequentemente, é também o que impede a constituição homogênea da subjetividade. Leremos os excertos apresentados a seguir de modo a compreender esse dinamismo que marca a relação entre línguas-culturas. O que buscamos, nessa visada, não é formular conclusões a respeito dos quatro estudantes autores das narrativas analisadas, mas interpretá-las como uma possibilidade de reflexão sobre a constituição identitária, por meio da escritura, no encontro linguístico. 9 Os excertos são apresentados tal como foram escritos, sem a intervenção/correção da professora. Atribuímos apenas nomes fictícios para identificar cada um dos participantes. 3 A escrit(ur)a nos meandros do entre-lugar O objeto de análise deste estudo foi constituído a partir de uma atividade de produção escrita feita por estudantes universitários, falantes de francês e de espanhol, iniciantes no curso de português como língua estrangeira, em uma instituição francesa de ensino superior8, durante o ano letivo de 2013-2014. Nem todos os estudantes participantes do curso de português eram de nacionalidade francesa, mas todos eram falantes de francês. Com relação ao espanhol, alguns alunos provinham de famílias mistas – franco-colombiana, por exemplo – outros, nascidos em países hispânicos, fizeram sua formação básica em liceus franceses nesses países e estavam na França para cursarem o ensino superior. Acreditamos ser impossível precisar, apenas com base na nacionalidade ou no local de nascimento, qual(is) seria(m) a(s) língua(s) materna(s) dos participantes RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 90 e qual(is) língua(s) lhe(s) seria(m) estrangeira(s), tampouco é esse nosso objetivo. Outra pesquisa precisaria ser desenvolvida com esse propósito específico, pois, para além da descendência (nascimento pelo sangue) e da nacionalidade dos participantes (nascimento pelo solo), é do nascimento pela língua, segundo Derrida (1996/2001), que tratamos aqui. A proposta de escrita que desencadeou as narrativas tinha como título “(Auto)biografia” e incitava os estudantes a escrever sobre si mesmos ou sobre uma personagem fictícia. Em ambos os casos, deveria haver uma breve descrição dessa personagem, bem como uma ordem narrativa que compreendesse, passado, presente e futuro. Desse modo, salientamos que a produção escrita não se deu de forma totalmente livre, mas circunscrita à proposta apresentada, a qual visava que fossem “colocados em prática” os conteúdos programáticos trabalhados ao longo do ano. Algumas formalidades escolares, tais como esta, ainda se mantêm, cerceando e, ao mesmo tempo, constituindo tanto alunos quanto professores. Trata-se, portanto, de uma escrita inscrita na ordem do discurso escolar-acadêmico. Contudo, tal determinação não anula a emergência de traços subjetivos e de faíscas das línguas-culturas que se entrechocam nessa constituição, pois, apesar de, nos excertos aqui analisados, os estudantes optarem pela invenção de uma personagem, é de si que tratam. Essa (con)fusão é sugerida pelo próprio título da proposta de escrita, o que torna, talvez, mais consciente esse falar de si, mas que não invalida a reflexão sobre a constituição subjetiva no encontro linguístico, já que não acreditamos na suposta transparência da linguagem nem no domínio (maîtrise) das línguas, que possibilitariam dizer exatamente aquilo que se quer e ser lido da mesma forma. RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 3 A escrit(ur)a nos meandros do entre-lugar O primeiro dos quatro excertos escolhidos para esta análise foi extraído da narrativa de Maria9, a cuja personagem é dado o nome de Ana. R1) No deserto de Atacama, o deserto mais árido do mundo, nasceu uma criatura, ninguém sabe como ou por que, chamada Ana. Aqueles que a viram, disseram que era pretinha, alta, com olhos grandes e escuros. Depois de seu nascimento, ela caminhou o deserto todo, procurando encontrar gente com quem viver (e, sobretudo, algo para jantar). Quando finalmente encontrou um pequeno povoado, uma corajosa mulher a adotou. Até sua morte, Ana foi considerada a filha do medo, e a preferida da cinco irmãos (Maria). 9 Os excertos são apresentados tal como foram escritos, sem a intervenção/correção da professora. Atribuímos apenas nomes fictícios para identificar cada um dos participantes. RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 91 Nascida em um deserto, uma personagem vaga sozinha, até encontrar um povoado e alguém que a adote e a alimente. Embora imaginário, esse lugar é atrelado, pelo nome próprio, ao deserto de Atacama, no Chile, em uma possível tentativa de desterritorialização da narrativa, da personagem fictícia e, por que não, de si mesma. Enquanto aprendiz de língua portuguesa do Brasil, falante de espanhol latino-americano, nascida no México, mas vivendo na França, Maria reterritorializa sua história em uma América já distante e (re)inventada. Trata-se de um primeiro deslocamento, não em termos físicos, mas simbólicos, possibilitado pela escrita. p p Se considerássemos apenas os níveis lexical, sintático e gramatical, haveria poucas marcas de uma real imbricação linguística. Todavia, o aspecto que mais nos chama a atenção é o que atrela língua, cultura e identidade no cenário da história: um deserto, o “mais árido do mundo”, no qual nasce a personagem feminina. O tom literário da narrativa, assim como a evocação da aridez e da própria imagem do deserto, não permite que desconsideremos seu caráter metafórico, principalmente se tomarmos esta como uma das primeiras escrit(ur)as na língua estranha: o vazio, a dificuldade, a ausência de interlocutores, são indícios que apontam para além de uma ficção descarnada, pois marcam o processo de constituição (o nascimento) de uma identidade narrativa. O relato desse nascimento não se refere necessariamente ao encontro com a língua portuguesa, visto que Maria já havia vivenciado outros encontros, na condição de sujeito “bilíngue” ou, diríamos, multilíngue. 3 A escrit(ur)a nos meandros do entre-lugar É nesse sentido que o deserto imaginado e imaginário pode estar em qualquer espaço- tempo, dizendo da subjetividade de quem (se) escreve de uma maneira não monolíngue; mais, ainda, pode ser quem (se) escreve. g Notamos que não há uma razão evidente para o nascimento dessa “criatura” (“ninguém sabe como ou por que”) ou, talvez, haja, mas seja externa à narração: a demanda, vinda do outro/Outro, para que uma escrita se produza e para que a personagem seja criada. Nesse âmbito narrativo, podemos pensar tanto na construção de um narrador-personagem que conta sua própria história em terceira pessoa, quanto na disjunção entre um narrador onisciente (e criador) e a personagem (sua criatura, que pode não ser humana). Sendo assim, mais do que uma invenção de Maria, Ana, a personagem, poderia ser um de seus duplos, uma das combinações que a habitam, uma das possibilidades de ser si-mesma, sendo outra. É por essa razão também que entendemos que a referência ao deserto está situada menos física do que subjetivamente. Segundo Deleuze, no diálogo RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 92 com Parnet, “o deserto, a experimentação sobre si mesmo é nossa única identidade, nossa única chance para todas as combinações que nos habitam” (DELEUZE e PARNET, 1996/1998, p. 19). Apesar de, no seio da ficção, os traços físicos de Ana serem mobilizados (“pretinha, alta, com olhos grandes e escuros”), conferindo maior verossimilhança ao relato (e atendendo à solicitação da proposta de escrita), estes constroem novamente a imagem de um duplo, já que Ana é também o nome de uma colega de classe com as mesmas características físicas da personagem e de Maria. Assim, Deleuze continua: Nós somos desertos, mas povoados de tribos, de faunas e floras. Passamos nosso tempo a arrumar essas tribos, a dispô-las de outro modo, a eliminar algumas delas, a fazer prosperar outras. E todos esses povoados, todas essas multidões não impedem o deserto, que é nossa própria ascese; ao contrário, elas o habitam, passam por ele, sobre ele (DELEUZE e PARNET, 1996/1998, p. 19). Por meio das palavras do autor, é possível compreender melhor essa narrativa alegórica em torno do deserto (de si), bem como da busca constante, interminável, que move o sujeito em direção ao outro e à procura de um lugar (“ela caminhou o deserto todo, procurando encontrar gente com quem viver”). RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 3 A escrit(ur)a nos meandros do entre-lugar Não é insignificante o fato de a narrativa começar pela delimitação de um cenário inóspito ou pouco acolhedor, pois, nos termos de Prasse (1997, p. 71), o desejo das línguas estrangeiras, (da língua) do outro, está diretamente relacionado a essa busca, vista como uma “inquietação de não estar no lugar necessário, de não poder encontrar seu próprio lugar na língua materna”. A autora se refere, nessa passagem, à leitura psicanalítica lacaniana a propósito da designação língua materna, que se caracterizaria por um traço negativo, já que é nessa língua que se instituem os interditos, diferentemente das línguas estrangeiras, nas quais haveria uma liberdade maior. Contudo, como enfatiza Coracini (2007, p. 119), “não se pode defender a exclusividade de um espaço para o maternal ou para o estrangeiro”, pois nenhuma língua é pura. Todas as línguas, assim como todo sujeito, retomando os termos de Deleuze já citados, são habitados e atravessados por outros, por multidões. Nesse relato, entretanto, não é anódino o encontro, depois de tanto se errar, com uma mãe adotiva (“uma corajosa mulher a adotou”) e, se continuarmos a explorar a narração como gênese ficcional da inscrição em uma língua estranha, essa imagem pode nos remeter não à língua estrangeira RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 93 como língua sem interdições, mas à ilusão de um espaço de familiaridade finalmente achado, uma ilusão de pertencimento, de se sentir em casa, embora se saiba ser sempre “considerada a filha do medo”, o que aponta para um mal- estar, uma insegurança que não deixará de se fazer presente até sua morte. g q p O sintagma “filha do medo”, que não deixa de funcionar em consonância com a narrativa, principalmente por se tratar de uma “criatura”, que só foi adotada por uma mulher “corajosa”, poderia ser considerado um “erro” causado pela distração ou pela dita interferência de outra(s) língua(s). Poderíamos conjeturar que o que Maria “quis dizer” tenha sido que Ana foi considerada a filha do meio e a preferida dentre os cinco irmãos. Seria mais “lógico”, poderíamos argumentar. Contudo, parece-nos também mais difícil sustentar tal interpretação (afirmando que, em espanhol, meio escreve-se medio e medo, miedo, de modo que Maria poderia ter-se confundido, por exemplo. Em francês, peur e milieu não possuem nenhuma aproximação com o português). Para além do que se teria querido dizer, o que analisamos é o que se diz. 10 Nos estudos do discurso, permeados pela abordagem psicanalítica, “equívoco” e “equivocidade” são noções constituídas por esse cruzamento teórico. A afirmação de que um dizer é equívoco (e, por vezes, de que a própria língua é equívoca) não está atrelada a uma escolha e/ou a uma intencionalidade do falante/enunciador (ao fato de ter se enganado/se equivocado ou de ter escolhido falar/escrever de um modo e não de outro). A equivocidade diz respeito à possibilidade que um termo, uma expressão ou uma proposição possui de apontar para efeitos de sentido outros, diversos e até contraditórios (noções como “deslize” e “deriva” também são empregadas, menos como sinônimos de “erro” do que como “abertura a interpretações”). 3 A escrit(ur)a nos meandros do entre-lugar E o sintagma em questão, além de presentificar a equivocidade10 constitutiva de toda língua – enquanto possibilidade de mal-entendidos, de ambiguidades, de falhas, de lapsos, de produção e de desestabilização de sentidos –, também autoriza uma interpretação direcionada a traços do falar de si. Se, como argumenta Ricoeur (2008), uma história também é feita de histórias não contadas ou recalcadas, é significativo pensar que esse dizer (que aproxima familiar e estranho, filha e medo) tenha se produzido na língua dita estrangeira, em que, como colocamos anteriormente, determinados afetos, talvez silenciados na chamada língua materna, podem, enfim, mesmo que por um deslize, ter lugar. Embora não seja evidente que se possa falar de si na língua do outro, esta é uma possibilidade, já que em toda língua há um espaço-tempo para o materno e para o estrangeiro. Em outros termos e em conformidade com o pensamento de Deleuze e Guattari (1980/1995), RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 94 podemos dizer que há um devir-materna e um devir-estrangeira em toda língua. A esse respeito, vejamos o excerto seguinte, R2) No fundo do mar nasceu Vanda uma menina abandonada por o céu. Foi durante uma grande tempestade que saiu da espuma uma criança minúscula azul e vermelha. [...] Ela vivia sola em sua ilha [...] Cuando se cansava, deitava-se nas raízes dum arvore embalada por seus ramos e dormia até amanhecer. Umas vezes ficava acordada durante a noite toda conversando com as estrelas e com os espiritus dos outros planetas (Taís). Talvez, uma das formas de se reescrever o pensamento da multiplicidade, ou a impossibilidade de se escrever de maneira monolíngue, seja a célebre passagem de John Donne, que abre um dos textos de Hemingway (1940/1976): “nenhum homem é uma ilha isolada; cada homem é uma partícula do continente, uma parte da terra”. Na ficção de Taís, ecoam essas palavras, pois, apesar de a personagem viver sozinha, é de uma simbiose quase cósmica que se trata em sua gênese e em sua identificação com a natureza e os astros. RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 3 A escrit(ur)a nos meandros do entre-lugar Além disso, ressoa em sua poética a existência mítica de personagens da literatura argentina – o que aponta para a constituição de uma identidade desterritorializada, não-europeia –, como O gaúcho Martín Fierro, sujeito lírico do poema épico de mesmo nome, que afirma: Nasci como nasce o peixe nas profundezas do mar; [...] como essas aves tão belas, que saltam de rama em rama, no trevo preparo a cama e me cubro com as estrelas (MEIRELES, 1948, s. p.). Notamos várias semelhanças entre o poema e a narrativa de Taís, bem como entre esta e a história criada por Maria, no excerto anterior, tanto no que concerne ao cenário inicial, de vazio e abandono aparentes, quanto na constituição da personagem, que está só. A recorrência da temática da solidão é um elemento significativo para tratarmos de uma constituição subjetiva entre línguas-culturas, em que, ao mesmo tempo em que se está em um lugar, não se tem lugar. De modo análogo ao excerto formulado por Maria, em que o nascimento, ou a criação de uma identidade, dá-se em meio à aridez, RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 95 nesse excerto, a gênese ocorre “durante uma grande tempestade”, o que também não é irrelevante, como imagem metafórica, em se tratando do contato e, frequentemente, do conflito com a língua do outro. Além disso, a atmosfera narrativa que envolve as personagens é semelhante e obedece à mesma lógica: primeiramente, ambas estão sós (em meio ao deserto ou à tempestade), mas acabam por achar um lugar, ao qual, no entanto, não pertencem. Ana é uma “criatura” adotada por uma mulher em um “pequeno povoado”, diferente de seu local de nascimento, mas, talvez, ainda desértico. Já Vanda, apesar de ser acolhida em um cenário menos inóspito, pois encontra abrigo e aconchego em uma ilha, é uma menina sendo cuidada e ninada por uma árvore. Em ambas as histórias, permanecem características que nos possibilitam compreender a constituição do estrangeiro como “relacionada ao não-pertencimento e à estranheza, destacando o fato de a estrangeiridade concernir a uma dimensão da exterioridade, definida enquanto alteridade” (DA ROSA, 2009, p. 75). 3 A escrit(ur)a nos meandros do entre-lugar Não é difícil, percebermos, nesse caso, a associação entre a árvore e a mãe adotiva do primeiro relato, de modo que, se tomarmos o cenário como uma forma de remissão ao lugar e à língua do outro, em ambos os textos esse espaço é visivelmente distante e imaginário, representando a ilusão do estar em casa, utopia do estrangeiro enquanto ser errante. Ademais, a referência a um espaço físico “exótico” e isolado, tanto no primeiro excerto, quanto no segundo, aponta para a escrita como possibilidade de dar contornos e sentidos para essa constituição intervalar (sem lugar definido), além de funcionar como uma linha de fuga, analogamente à utopia do “novo mundo”, representado como o Eldorado ou o país da Cocanha na época das grandes navegações. Ficção, portanto, dos outros, de seu lugar, “da maneira como gozam os outros”, nos termos de Prasse (1997, p. 71), e desejo de estar nesse espaço-tempo. A esse respeito, em ambas as narrativas, além do isolamento espacial, há uma espécie de suspensão do tempo ou de retorno a um tempo primitivo, principalmente no caso da história de Taís, que remonta, em seu nascimento, também ao antigo mito indiano sobre a origem do mundo, que a explica pela condensação “das águas do mar primordial, batidas pelos deuses criadores” (GINZBURG, 1976/2006, p. 103). Essa busca pela origem, por um lado, pode ser interpretada como uma tentativa de retorno às raízes (que, inclusive, aparecem no dizer de Taís), o que remete ao mito da língua materna, à ilusão de um “doce falar natural” RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 96 (DECROSSE, 1989), primeiro e puro. Por outro lado, pode se referir à instauração de uma “nova origem” (também mítica, também ilusória, como sabemos ser toda origem), um outro nascimento, por meio do contato com outras línguas-culturas, que carregam consigo a possibilidade de criação de um outro lugar. Nesse sentido, segundo Prieur (2006a, p. 485), “a escritura aparece, então, como um espaço de tensão e de reencontro entre línguas diferentes, espaço no interior do qual o escritor vai achar ‘sua língua’, sua linha própria, única, de invenção e de criação”11.i Na ficção construída por Taís, outro aspecto relevante é o fato de que a personagem, algumas vezes, “ficava acordada durante a noite toda conversando com as estrelas e com os espiritus dos outros planetas”. Em que língua, perguntamos, ocorreria tal conversação? 11 « L’écriture apparaît alors comme un espace de tension et de rencontre entre des langues différentes, espace à l’intérieur duquel l’écrivain va trouver « sa langue », sa ligne propre, unique d’invention et de création » 12 « dans quelle langue lui avait-elle donc parle durant si longtemps, tant d’heures et tant d’années? [...] Elle avait dû inventer une langue [...] La langue de Gratok c’était la langue des rêves ». 3 A escrit(ur)a nos meandros do entre-lugar A história de uma menina só, que conversa com interlocutores peculiares, evoca Gratok, de Robin (1995), e o caso da pequena menina judia, que inventou uma língua para falar com seu ursinho de pelúcia (o próprio Gratok), uma vez que não podia falar o iídiche, porque era proibido, nem o francês, porque seu sotaque denunciaria sua estrangeiridade. Já ao fim de Gratok, a autora se pergunta, questão que acreditamos ser possível relacionar à língua de Taís ou de sua personagem: “em qual língua ela tinha lhe falado durante tanto tempo, tantas horas, tantos anos? […] Ela deve ter inventado uma língua [...] A língua de Gratok era a língua dos sonhos” 12 (ROBIN, 1995, p. 486-487). É, portanto, a possibilidade de criar uma língua própria, um idioma, diríamos, como Derrida (1996/2001), e outra fantasia de si mesmo e do outro que o contato com diferentes línguas potencializa. Contudo, como bem relembra Coracini (2007, p. 131), “só é possível falar de uma língua depois de ter criado ‘ficções’ adequadas, úteis, no jogo da necessidade e da impossibilidade de apropriação”. Vejamos a história criada por Lucia, RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 97 R3) Esta história é a história duma bolinha. Ninguém sabe de onde vem nem de onde saiu. Simplesmente, esteve aí um dia e nunca foi embora. A princípio, era uma bolinha pequenina, muito, muito pequenina. Nem se via a cor dela. Era um ponto, um pó que andava por aí. No início, a vila toda estava muito surpresa com aquela bolinha. Sobre tudo, porque era tão pequena. Mas com o tempo, se acostumaram a ela. Além do mais, começaram a amá-la. E amarela era sua cor. Amarela como areia, como um pedaço do sol, como a terra argilosa mato-grossense na que surgiu esta história. Mas disso, a gente se deu conta depois quando ela começou a crescer, claro. A primeira vez que se viu sua cor foi numa noite de março [...] Cri-cri, cristal: como o canto dos grilos e das rãs que orquestravam seu andar noturno. E a bolinha passou a chamar-se Cris (Lucia). A criação de uma personagem pouco verossímil e de uma narrativa que tende para o aspecto surrealista em alguns momentos, pode nos auxiliar a compreender esse (du)elo entre a necessidade e a impossibilidade de apropriação da língua, impossibilidade de construir uma narração de si mesmo. 3 A escrit(ur)a nos meandros do entre-lugar A personagem é apenas uma forma que aparece de repente, pequena e insignificante (“pequenina, muito, muito pequenina”), como um ponto ou um pó. Poderíamos dizer que pequena é também a estatura física de quem a descreveu, mas o que importa? Há, pela narrativa, a invenção de um corpo, assim como nos excertos anteriores (não nos esqueçamos de que Vanda, a personagem criada por Taís, era uma menina azul e vermelha e não sabemos se a criatura inventada por Maria era um ser humano). Em um primeiro momento, não lhe é dado um nome próprio, já que é só depois, no desenrolar da narrativa, que a bolinha será chamada de Cris, em um deslocamento (de pó, grão de areia a cristal) significativo, para pensarmos sobre a identidade narrativa, já que nesta, como pontuamos anteriormente, ancora-se a tentativa de compreendermos nossa subjetividade, espelhados nas narrativas que nossa cultura oferece. A história em questão traz vestígios de uma memória discursiva na qual habitam outras metamorfoses: a de um belo cisne que já fora patinho feio, a de uma princesa que era limpadora de chaminés, a de um sapo que se transforma em príncipe, por que não uma bolinha que passa a ser cristal? Diferentemente das narrativas comentadas anteriormente, nas quais as personagens já são nomeadas quando de seu nascimento, é a partir do contato com os outros (habitantes da “vila”, possivelmente) que a referida bolinha passa a ter nome. Contudo, é pela onomatopeia, pelo barulho, feito como uma lalação na língua e no lugar do outro, que esse nome lhe vem. Esse uso RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 98 da onomatopeia pode ser lido, nos termos de Prieur (2006b), como uma das formas de se estabelecer pontes, laços, associações com a outra língua por parte da estudante. Além disso, se concordarmos com Melman (1992) que “em toda fala há o canto”, que a língua estrangeira nos atrai pela sonoridade, pela música, sendo a musicalidade da língua (dita) materna “o que recusamos abandonar” ao falarmos uma língua estrangeira, podemos entender por que é só depois que Cris, orquestrada pelo “canto dos grilos e das rãs”, passa a existir (já que nomear-se é dar-se uma existência) nesse outro lugar. “Isto poderia querer dizer que um dos elementos que asseguram a identidade daquele que fala uma língua é ligado à música? Por que não?” (MELMAN, 1992, p. 52). RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 3 A escrit(ur)a nos meandros do entre-lugar g g q p Ainda em torno desse canto, destacamos o jogo significante (audível por um ouvido brasileiro não purista, embora não formulado na escrita), no fragmento: “começaram a amá-la. E amarela era sua cor. Amarela como areia”, no qual “amá-la”, gramaticalmente adequado, evoca a oralidade despreocupada de “amar ela”, condensado e repetido em “amarela”. É impossível explorarmos todos os efeitos de sentido que transbordam (d) o amor à cor amarela, causados pela brincadeira que saboreia o coaxar e o cricrilar da língua alheia como uma forma de trazê-la para si, de inventar uma ponte “na angústia e no gozo da mudança de corpo e de língua”, como enfatizou Prieur (2006b, p. 116) em passagem já citada. Porém, a narrativa pode ser explorada também como (im)possibilidade de o estranho (o intruso, o forasteiro) ser visto, aceito e valorizado pelo outro, pois é somente depois que “começaram a amá-la” que se percebe sua cor, bem como a analogia a um “pedaço de sol” e a um cristal. O deslocamento, então, de pó a pedaço de sol, de areia a cristal, é dado pela inserção nesse outro lugar, passado o estranhamento inicial causado pela aparição repentina da personagem. Esse estranhamento, como todo contato com um elemento externo, produz surpresa (que pode ser tanto positiva, quanto negativa), a qual vai sendo substituída pelo acostumar-se com e pelo amar. Segundo Freud (1919/1976, p. 277), “podemos dizer que aquilo que é novo pode tornar-se facilmente assustador e estranho; algumas novidades são assustadoras, mas de modo algum todas elas. Algo tem de ser acrescentado ao que é novo e não familiar, para torná-lo estranho”. Nesse caso, notamos que a personagem é feita da mesma “terra argilosa” da história e, portanto, pertence a ela como elemento estranho-interno, estabelecendo uma relação de extimidade, que é a que sustenta o lugar do estrangeiro. No que concerne à gênese da personagem, a memória a que se recorre é a do mito cristão-católico da criação do homem, vindo do pó, da terra RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 99 que, nesse caso, é situada em um Brasil imaginário, “mato-grossense”; um Brasil poetizado por Manoel de Barros (2000), talvez, com seus grilos e rãs: “o grilo feridava o silêncio”, diz o poeta, “como o canto dos grilos e das rãs que orquestravam seu andar noturno”, diz a poética narrada de Lucia. 13 « c’est l’auteur qui s’est lui-même déguisé en narrateur et qui porte le masque de ses multiples personnages et, parmi tous ceux-ci, celui de la voix narrative dominante qui raconte l’histoire que nous lisons ». 3 A escrit(ur)a nos meandros do entre-lugar Assim, concebendo a identidade como um jogo de máscaras, conforme dito anteriormente, podemos afirmar que, nessas narrativas que interpretamos como alegóricas, “é o próprio autor que se disfarçou de narrador e que carrega a máscara de seus múltiplos personagens e, entre todos estes, a da voz narrativa dominante que conta a história que lemos”13 (RICOEUR, 2008, p. 275). Como nas histórias anteriores, o tempo da narrativa de Lucia é suspenso, como o tempo mítico da criação, como o tempo-espaço do mito do estrangeiro. Para encerrarmos este breve percurso exploratório, observemos a escrita de Jean, a seguir. R4) Na floresta amazônica, num tempo imemorial [...] nasci o herói dessa historia. Esse herói extraordinário tinha a particularidade, sabe Deus quão esquisito, de ter uma forma dum menino ordinário. Não pode-se dizer que ele era bonito. Na sua cara, que era bem branca, dia a dia se ampliou uma nariz que já era fino e longe quando nasci. Essa proeminência era enquadrada por dois olhos minúsculos que pareciam como dois esferas pretas. Sua cabeça estava em cima dum corpo magro cujos membros se estendiam ate o ridículo. Quando ele tinha dez anos, foi recolhido pelo tribo dos Tupinambas. Porque era branco, lhe deram o nome do ultimo explorador que comeram, Oedipus. Desde esse momento, Oedipus tinha uma infância alegra e despreocupada. Lhe gostava correr na praia e pescar. Sua vida era ritmada por prazeres simples. Mas quando entrou na adolescencia, as coisas se complicaram. Porque seu fisico nao era incrível, para nao dizer mais, as meninas sistematicamente fugiram quando lhe viam (Jean). R4) Na floresta amazônica, num tempo imemorial [...] nasci o herói dessa historia. Esse herói extraordinário tinha a particularidade, sabe Deus quão esquisito, de ter uma forma dum menino ordinário. Não pode-se dizer que ele era bonito. Na sua cara, que era bem branca, dia a dia se ampliou uma nariz que já era fino e longe quando nasci. Essa proeminência era enquadrada por dois olhos minúsculos que pareciam como dois esferas pretas. Sua cabeça estava em cima dum corpo magro cujos membros se estendiam ate o ridículo. Quando ele tinha dez anos, foi recolhido pelo tribo dos Tupinambas. Porque era branco, lhe deram o nome do ultimo explorador que comeram, Oedipus. Desde esse momento, Oedipus tinha uma infância alegra e despreocupada. Lhe gostava correr na praia e pescar. Sua vida era ritmada por prazeres simples. 3 A escrit(ur)a nos meandros do entre-lugar Mas quando entrou na adolescencia, as coisas se complicaram. Porque seu fisico nao era incrível, para nao dizer mais, as meninas sistematicamente fugiram quando lhe viam (Jean). No que concerne ao plano linguístico, o excerto em questão é representativo da imbricação entre línguas, pois, na escrita em português, irrompem fagulhas de outras línguas, tanto no aspecto estrutural – como em “Lhe gostava correr na praia e pescar” –, quanto lexical – como em “ter a forma dum menino ordinário”, na qual ressoa o adjetivo ordinaire (comum), em francês, ou em “tinha uma infância alegra e despreocupada”, na qual 13 « c’est l’auteur qui s’est lui-même déguisé en narrateur et qui porte le masque de ses multiples personnages et, parmi tous ceux-ci, celui de la voix narrative dominante qui raconte l’histoire que nous lisons ». RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 100 o adjetivo, invariável em português, é grafado no feminino. Além disso, apontamos a escrita como representação fonética atrelada à oralidade do próprio português em “nasci o herói dessa historia”. Tais aspectos, contudo, não são os únicos indicativos desse encontro, pois, como defendemos, as línguas carregam consigo redes de imagens que, por sua vez, remontam a culturas, e é para tal encadeamento que atentamos. A narrativa de Jean é construída em tom mítico, como se pode perceber nas primeiras linhas, relatando o nascimento de um “herói extraordinário” que, contraditoriamente, assume a forma de um menino comum. Nesse relato, o que chama a atenção, assim como em R1 e R2, é a utilização do elemento estrangeiro, “exótico”, por um lado, na criação do cenário onde é inserida a personagem: seu pertencimento à floresta amazônica, o que pode estar ligado ao imaginário de Brasil, metonimicamente representado pela Amazônia ou pela floresta, de modo geral; por outro, na referência a sua adoção por uma tribo, os Tupinambás, e suas atividades de lazer, pescar e correr na praia (porque, afinal, não se pode falar do Brasil sem se falar das praias, mesmo que a história se passe na floresta). Apesar de não ter sido sugerido que a história devesse acontecer no Brasil ou em qualquer país específico, três, dos quatro estudantes, localizaram espacialmente sua história, o que indicia a tentativa de saída de um território e movimento em direção ao outro, ao estrangeiro. RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 3 A escrit(ur)a nos meandros do entre-lugar Em linhas gerais, a história da acolhida de um menino branco por uma tribo indígena (“foi recolhido pelo tribo dos Tupinambas”), também pode ser interpretada, como vimos fazendo, como uma metaforização do encontro e da inscrição em uma língua outra, estranha e, por que não, “selvagem”, o que se justifica pela alusão à antropofagia (“o nome do ultimo explorador que comeram”). Notemos, também, que o nome próprio dado à personagem é o mesmo de alguém que foi devorado, sinalizando que ela talvez viesse a ter o mesmo fim do explorador anterior: ser deglutida e, assim, incorporada à (ou ao corpo da) tribo. Imbricada a essa narrativa, há uma descrição física da personagem, a qual, pode-se dizer, possui traços marcadamente “europeus”: nariz fino e longo (“longe”), cara bem branca (cara pálida, que evoca também o contraste entre a pele do índio e a do branco nos filmes de faroeste). Atendendo também à solicitação de fornecer outras características que fossem além da descrição física, Jean acrescenta que o menino não era bonito, que seus membros se estendiam “até o ridículo” (em uma avaliação negativa do corpo considerado muito magro), que teve uma infância alegre RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 101 e despreocupada, cuja tranquilidade veio a ser perturbada na adolescência, pois, não sendo belo, não atraía as meninas, mas, pelo contrário, afastava-as. Trata-se, como vimos sinalizando, de uma identidade narrativa que se atrela a uma ficção de si, marcada, segundo Pietroluongo (2001), na imagem de um “estrangeiro de si”, a qual decorreria da transferência de um sentimento de estranheza, já vivenciado em uma língua, para a outra, chamada estrangeira. A autora argumenta que, no encontro com essa outra língua, “a experiência do não-saber, do tatear, do fragmento, da falha, da opacidade ganha toda sua consistência” (PIETROLUONGO, 2001, p. 197). Essa hipótese de um falar de si pela língua outra não indica que esse narrar- se é intencional ou proposital, mas sim que é pela opacidade, nessa esfera de incerteza diante da língua, seja materna, seja estrangeira, que os sentidos se constituem. Podemos experimentar esses efeitos pela análise da frase: “na sua cara, que era bem branca, dia a dia se ampliou uma nariz que já era fino e longe quando nasci”, na qual o verbo nascer está conjugado em primeira e não em terceira pessoa. RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 3 A escrit(ur)a nos meandros do entre-lugar Pode-se, não sem alguma razão, afirmar que se trata apenas de um erro gramatical, natural na escrita de um estudante iniciante, sem nenhuma influência para além de sua necessidade de correção. Contudo, o efeito de sentido que aí se produz, para além da distração e da falha na conjugação verbal, é o de um dizer que se configura, de forma subjacente, sempre em primeira pessoa. Isso não significa que a primeira pessoa seja a forma da manifestação subjetiva por excelência, ou seja, que o sujeito esteja presente se e somente se for utilizada a primeira pessoa, mas sim que a presença de um “eu”, ainda que equívoca, aponta para a construção de uma imagem de si que ganha contornos de/na personagem. g q g p g Essa descrição contrasta com o início da história, deslocando-a de um “tempo imemorial” para uma época e uma cultura/mentalidade que nos parecem contemporâneas, marcadas na avaliação do corpo como ligado aos problemas de autoafirmação na adolescência. Esse deslocamento denuncia a introdução de um elemento estranho, porque estrangeiro, no relato, o que coincide com a metáfora da inserção da/na língua estrangeira de que entendemos se tratar nesse dizer. Como se pode facilmente imaginar, essa pode ser uma descrição pessoal de Jean: branco, olhos pretos (“olhos minúsculos que pareciam como dois esferas pretas”), nariz fino, corpo magro. Porém, no enredo, estamos diante da construção (do nascimento na/pela língua) de um Édipo adolescente, vivendo na floresta amazônica, o que, por mais incongruente que possa parecer, além de adequado ao que foi solicitado RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 102 pela professora, é singular enquanto reinterpretação do mito (tanto o de Sófocles, quanto o do país estrangeiro) e do fato de a língua do outro ser lugar de muitas possibilidades e poucas interdições. Não é aleatória, no entanto, a escolha desse nome próprio (com sua grafia “original” em latim ou em francês, por ser intraduzível?), pois, mesmo no senso-comum, reconhece-se a sina de Édipo, que, ao fugir de seu destino, o de matar o pai e unir-se à mãe, acaba por encontrá-lo. 3 A escrit(ur)a nos meandros do entre-lugar Na abordagem psicanalítica, contudo, o mito de Édipo se refere menos à morte do pai do que à interdição da mãe, o que resulta no fato de a designação língua materna, por exemplo, não ser compreendida como a língua da mãe, mas, ao contrário, como “aquela na qual não se alcança a mãe (o conforto, a familiaridade), tal língua se configura como a língua do interdito e, por isso, língua do desejo” (DA ROSA, 2009, p. 69). p Assim, se tomada enquanto língua do desejo, inalcançável, toda língua é materna, inclusive a estrangeira e, analogamente, se concebida como língua que faz retornar o que era há muito tempo conhecido, mas esquecido (FREUD, 1919/1976), levando a pensar o sujeito em sua própria alteridade, toda língua é estrangeira, inclusive a chamada língua materna. É por essa razão que entendemos que o encontro com línguas estrangeiras potencializa não só (o retorno de) um estranhamento, mas a possibilidade de outra ficção de si, já que, de acordo com Kristeva (1988/1994), estranhamente, o estrangeiro habita em nós: ele é a face oculta da nossa identidade, o espaço que arruína a nossa morada [...] o estrangeiro começa quando surge a consciência de minha diferença e termina quando nos reconhecemos todos estrangeiros (KRISTEVA, 1988/1994, p. 09). RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 4 Considerações finais Nos excertos aqui apresentados, o lugar intervalar entre línguas- culturas resignifica a atividade de criação narrativa para além dos limites da escrita escolar, excedendo também uma imagem de si e/ou contribuindo para que esta assuma outros contornos. Entendemos, por meio da abordagem dessa escritura, que o papel da escrita, na confluência entre línguas, consiste em possibilitar uma prática criativa um pouco menos atrelada às amarras com as quais lidamos corriqueiramente. Contudo, como é possível depreendermos pelas condições de produção em que tal prática se deu, nunca escreveremos do lado fora desse grande texto que é a língua-cultura RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 103 que ousamos chamar de nossa. Apesar disso, podemos (nos) escrever a partir de um pensamento múltiplo, afetados pela presença de outros, deportando ou desarrumando a(s) língua(s). Essa é uma reflexão válida para qualquer prática de escrita, independentemente de estar ou não inserida em um contexto de discussão concernente ao contato linguístico propriamente dito. Escrever é experienciar e experimentar a língua, experimentar os sentidos, jogar/brincar com ambos, da forma mais livre que conseguirmos, pois, em simbiose com o pensamento “raiz única”, que admite apenas um sentido como possível e verdadeiro, funcionam uma língua e uma escrita engessadas. Sem dúvida, situar-se nas margens não parece ser uma tarefa fácil, mas, como nos indicam as narrativas apresentadas, também não deve ser impossível. p p Sendo assim, o gesto de leitura que desenvolvemos encaminhou- nos a remontar dizeres que se relacionam em torno da problemática que concerne à relação do sujeito com a(s) língua(s)-cultura(s). Tais dizeres auxiliaram-nos a refletir, ainda que brevemente, sobre o movimento que tem marcado a constituição da subjetividade contemporaneamente. Mobilidade e multiplicidade que nos toma e nos reconfigura, redefinindo nosso dizer em meio ao drama de que falava Glissant, no excerto introdutório a esta reflexão. Em meio a essas condições, a noção de interlíngua, como um estágio de aprendizagem a ser superado, parece não abarcar, em toda sua complexidade, o movimento de sujeitos e de sentidos. Pelo contrário, enquanto etapa, parece estar condenada a representar a estaticidade com que as línguas são forçadamente ensinadas (aprendidas?) nos ambientes de ensino. Como conter em uma etapa o fluir de sentidos que o ser/estar entre proporciona? Como falar de interferência, quando se está em um turbilhão de línguas, do qual “minha língua não pode ficar isenta e salva” (retomando novamente o dizer de Glissant)? 5 Referências bibliográficas BARROS, M. de. (2000). Ensaios Fotográficos. Rio de Janeiro: Record. CORACINI, M. J. (2007). A celebração do outro. Campinas: Mercado de Letras. DA ROSA, M. T. (2009). Entre uma língua e outra: desdobramentos das designações língua materna e língua estrangeira no discurso do sujeito pesquisador da linguagem. 2009. Dissertação (Mestrado em Letras) – Centro de Artes e Letras, Universidade Federal de Santa Maria, Santa Maria, 134 p. BARROS, M. de. (2000). Ensaios Fotográficos. Rio de Janeiro: Record. CORACINI, M. J. (2007). A celebração do outro. Campinas: Mercado de Letras. DA ROSA, M. T. (2009). Entre uma língua e outra: desdobramentos das designações língua materna e língua estrangeira no discurso do sujeito pesquisador da linguagem. 2009. Dissertação (Mestrado em Letras) – Centro de Artes e Letras, Universidade Federal de Santa Maria, Santa Maria, 134 p. BARROS, M. de. (2000). Ensaios Fotográficos. Rio de Janeiro: Record. ARROS, M. de. (2000). Ensaios Fotográficos. Rio de Janeiro: Record. BARROS, M. de. (2000). Ensaios Fotográficos. Rio de Janeiro: Record. CORACINI, M. J. (2007). A celebração do outro. Campinas: Mercado de Letras. DA ROSA, M. T. (2009). Entre uma língua e outra: desdobramentos das designações língua materna e língua estrangeira no discurso do sujeito pesquisador da linguagem. CORACINI, M. J. (2007). A celebração do outro. Campinas: Mercado de Letras. DA ROSA, M. T. (2009). Entre uma língua e outra: desdobramentos das designações língua materna e língua estrangeira no discurso do sujeito pesquisador da linguagem. 2009. Dissertação (Mestrado em Letras) – Centro de Artes e Letras, Universidade Federal de Santa Maria, Santa Maria, 134 p. DA ROSA, M. T. (2009). Entre uma língua e outra: desdobramentos das designações língua materna e língua estrangeira no discurso do sujeito pesquisador da linguagem. 2009. Dissertação (Mestrado em Letras) – Centro de Artes e Letras, Universidade Federal de Santa Maria, Santa Maria, 134 p. DA ROSA, M. T. (2013). O discurso universitário-científico na contemporaneidade: marcas e implicações na constituição identitária do pesquisador em formação. Tese (Doutorado em Linguística Aplicada) Instituto de Estudos da Linguagem, Universidade Estadual de Campinas, Campinas, 251p. DECROSSE, A. (1989). Um mito histórico, a língua materna. In: BOUTET, J.; VERMES, G. (Orgs.). Multilingüismo. Trad. Tania Alkmim. Campinas: Editora da Unicamp, p. 19-27. DELEUZE, G; GUATTARI, F. (1980/1995). Mil Platôs: capitalismo e esquizofrenia. v. 1. Trad. Aurélio Guerra Neto e Celia Costa. Rio de Janeiro: Editora 34. DELEUZE, G; PARNET, C. (1996/1998). Diálogos. Trad. Eloisa Ribeiro. 4 Considerações finais “Não posso escrever minha língua de maneira monolingüe”, como repete o autor, referido na abertura deste artigo, leva-nos a pensar que é preciso estar entre, escrever entre, significar entre. Afirmações que, por sua vez, remontam às palavras de Deleuze, em seus Diálogos: “É preciso ir mais longe: fazer com que o encontro com as relações penetre e corrompa tudo, mine o ser, faça-o vacilar. Substituir o E ao É. A e B” (DELEUZE e PARNET, 1996/1998, p. 70). Sendo assim, seguimos no rastro dessa relação, buscando substituir o e ao consagrado ou da lógica binária e opositiva, ao é do pensamento essencialista que fixa e define a identidade, procurando 104 RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 considerar os desdobramentos do mesmo como diferente (idem e ipse), a fim de compreendermos a estrangeiridade da língua materna, a multiplicidade das línguas estrangeiras, das línguas-mistas que povoam a indecidibilidade do ser e do estar entre ao-menos-dois. 5 Referências bibliográficas São Paulo: Editora Escuta. DERRIDA, J. (1996/2001). O monolinguismo do outro ou a prótese de origem. Trad. Fernanda Bernardo. Porto: Campo das Letras Editores S. A. DERRIDA, J. (1972/1991). Margens da filosofia. Trad. Joaquim Torres Costa e António Magalhães. Campinas: Papirus Editora. DERRIDA, J. ; ROUDINESCO, E. (2001/2004). De que amanhã... Trad. André Telles. Rio de Janeiro: Jorge Zahar. FOUCAULT, M. (1969/2009). A arqueologia do saber. Trad. Luiz Felipe Baeta Neves. Rio de Janeiro: Forense Universitária. FOUCAULT, M. (1979/2010). Microfísica do Poder. Organização e tradução de Roberto Machado. Rio de Janeiro: Edições Graal. FREUD, S. (1919/1976). O estranho. In: FREUD, S. Obras completas. Edição standard brasileira das obras psicológicas completas de Sigmund Freud. v. 17. Trad. J. Salomão. Rio de Janeiro: Imago, p. 85-125. RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 105 GLISSANT, E. (2001/2005). Introdução a uma poética da diversidade. Trad. Enilce Albergaria Rocha. Juiz de Fora: Editora UFJF. GINZBURG, C. (1976/2006). O queijo e os vermes. Trad. Maria Betânia Amoroso e José Paulo Paes. São Paulo: Companhia das Letras. HEMINGWAY, E. (1940/1976). Por quem os sinos dobram. Trad. Monteiro Lobato. São Paulo: Companhia Editora Nacional. KRISTEVA, J. (1988/1994). Estrangeiros para nós mesmos. Trad. Maria Carlota Gomes. Rio de Janeiro: Rocco. LACAN, J. (1974). Télévision. Paris: Seuil. LACAN, J. (1974). Télévision. Paris: Seuil. MEIRELES, C. Martin Fierro: explicação e protesto do homem desamparado. Correio da manhã. Rio de Janeiro, 07 de novembro de 1948. Disponível em: http://memoria.bn.br/DocReader/Hotpage/HotpageBN. aspx?bib=089842_05&pagfis=44216&pesq=&url=http://memoria.bn.br/ docreader#. Acesso em: 19 out. 2015. MELMAN, C. (1992). Imigrantes: incidências subjetivas das mudanças de língua e país. Trad. Rosane Pereira. São Paulo: Escuta. PIETROLUONGO, M. A. (2001). O estrangeiro de mim. Gragoatá. Niterói: EDUFF, n. 11, p.193-206. PRASSE, J. (1997). O desejo das línguas estrangeiras. Revista Internacional. Rio de Janeiro. Ano 1, n. 1, p. 63-73. PRIEUR, J-. M. (2006a). Des écrivains en contact de langues. Études de linguistique appliquée. n. 144, pp. 485-492. Disponível em: www.cairn.info/revue-ela-2006- 4-page-485.htm. Acesso em: 28 out. 2014. PRIEUR, J-. M. (2006b). Contact de langues et positions subjectives. Langage et société. n. 116, p. 111-118. RICOEUR, P. (1990). Soi-même comme un autre. Paris: Éditions du Seuil. PRIEUR, J-. M. (2008). La vie : un récit en quête de narrateur. In: RICOEUR, P. Écrits et conférences I: autour de la psychanalyse. Paris: Éditions du Seuil, p. 257-276. ROBIN, R. (2003). Le deuil de l’origine. Paris : Éditions Kimé. ROBIN, R. (1995). 5 Referências bibliográficas Gratok, langue de vie et langue de mort. Meta. v. 40, n. 3, p. 482-487. ROCHA, E. A. (2005). Prefácio. In: GLISSANT, E. Introdução a uma poética da diversidade. Trad. Enilce Albergaria Rocha. Juiz de Fora: Editora UFJF, p. 9-11. Data de submissão: 12/08/15. Data de aprovação: 11/11/15. Data de submissão: 12/08/15. Data de aprovação: 11/11/15. 106 RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106 RBLA, Belo Horizonte, v. 16, n. 1, p. 81-106
https://openalex.org/W4383737778	https://www.nature.com/articles/s41598-023-38374-5.pdf	English	null	Helicobacter pylori strains isolated from raw poultry meat: frequency and molecular characteristics	Scientific reports	2,023	cc-by	9,464	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Helicobacter pylori strains isolated from raw poultry meat: frequency and molecular characteristics Tohid Piri‑Gharaghie 1, Ghazal Ghajari 2, Shakiba Tolou‑Shikhzadeh‑Yazdi 3, Mona Aghassizadeh‑Sherbaf 4 & Sahar Khorsand‑Dehkordi 5 OPEN ICLE Even though Helicobacter pylori (H. pylori) is a serious pathogen, its origin is unknown. Poultry (Chicken, Turkey, Quebec, Goose, and Ostrich) are consumed as a regular protein source by a large number of people across the world; therefore, sanitary ways of delivering poultry for food are important for global health. As a result, we looked at the distribution of the pathogenicity cagA, vacA, babA2, oipA, and iceA in H. pylori isolates in poultry meat, as well as their antimicrobial resistance. Wilkins Chalgren anaerobic bacterial medium was used to cultivate 320 raw poultry specimens. Disk diffusion and Multiplex-PCR were used to investigate antimicrobial resistance and genotyping patterns, separately. H. pylori was found in 20 of 320 (6.25%) raw poultry samples. The highest incidence of H. pylori was found in chicken raw meat (15%), whereas the fewest was found in Goose and Quebec (0.00%). Resistance to ampicillin (85%), tetracycline (85%), and amoxicillin (75%) were greatest in H. pylori isolates. The percentage of H. pylori isolates with a MAR value of more than 0.2 was 17/20 (85%). The most prevalent genotypes discovered were VacA s1a (75%), m1a (75%), s2 (70%) and m2 (65%), and cagA (60%). The most typically discovered genotype patterns were s1am1a (45%), s2m1a (45%), and s2m2 (30%). BabA2, OipA + , and OipA− genotypes were found in 40%, 30%, and 30% of the population. In summary, the poultry flesh was polluted by H. pylori, with the babA2, vacA, and cagA genotypes being more prevalent. The simultaneous occurrence of vacA, cagA, iceA, oipA, and babA2 genotypes in antibiotic-resistant H. pylori bacteria implies a serious public health concern about raw poultry eating. In the future, researchers should look into H. pylori’s resistance to multiple antibacterial drugs in Iran. ACTED ARTIC Poultry (Chicken, Turkey, Quebec, Goose, and Ostrich) is an essential source of proteins for people1. Chickens are killed, skinned, and torn to pieces by hand in regulated slaughtering operations. The corpse is drained, the visceral contents are separated, and the liver, heart, and intestines are gathered during the evisceration process2. The discharge of digestive contents might contaminate these tissues. The corpses are cleaned with water after excoriation, which might be a major cause of bacterial infection3. Scientific Reports \| (2023) 13:11116 www.nature.com/scientificreports/ the stomach of a variety of mammals6. These microorganisms are classed as stomach or enterohepatic based on their preferred colonization location7. These two different types of germs are referred to as zoonotic bacteria8. The gastrointestinal Helicobacter colonizes the stomachs in particular; the enterohepatic Helicobacter family colonizes the proximal region of the digestive tract and the biliary duct especially9. Helicobacter pullorum (H. pullorum), a species of the enterohepatic Helicobacter family, was initially derived from the cecum of seemingly healthy domesticated birds10. In addition, poultry was infectedwith Helicobacter hepaticus (H.hepaticus), Helicobacter canis (H. canis), Helicobacter bilis (H. bilis), and Helicobacter cinaedi (H. cinaedi) too11. Helicobacter pylori (H. pylori) is an opportunistic pathogen linked to stomach cancer and intestinal perfo- ration in humans12. Information on the frequency and distribution of H. pylori is critical for controlling the disease’s distribution and identifying high-risk patients, especially in areas where gastritis and stomach cancer are uncommon13. While H. pylori isolates have been identified from a variety of meals, the relevance of animal- derived products in the development of H. pylori infection is unknown14. H. pylori pathogenesis is linked to virulence genes. H. pylori is evolutionarily changeable, according to studies15,16, and particular virulence genes are only found in certain groups. H. pylori has been derived from a variety of clinical specimens and identified using a multiplex polymerase chain reaction (Multiplex-PCR). Several virulence genes in H. pylori isolates have been reported, including Vacuolating cytotoxin A (VacA)17, cytotoxin-associated A (cagA)17, restriction endonuclease A (IceA)18, Outer inflammatory protein A (OipA)19, and blood-group antigen-binding adhesin (BabA2)19. These pathogenicity genes may have a role in the progression of H. pylori. The cagA gene was detected in around half of all H. pylori strains and is involved in intestinal mucosal inflammation, IL-8 generation, and stomach cancer etiology20. Furthermore, researchers discovered that the vacA gene is present overall in H. pylori strains and is involved in the development of stomach cancer and ulceration by destroying the mucous membrane. Different signaling domains and mid-regions make up the vacA genome, which is polymorphic. The s-region is divided into two types: s1 and s2, and the m-region is divided into two types: m1 and m2. The s1 variety is divided into s1a, s1b, and s1c subgroups, while the m1 variety is divided into m1a and m1b subgroups21. www.nature.com/scientificreports/ The operational state of oipA is controlled by a repair process dependent on CT dinucleotide repetitions, which affect the reading frame and hence determine whether the gene is functional or not22. In H. pylori collected from PUD and gastritis individuals, the iceA gene was discovered. The iceA gene has at least two variants, iceA1, and iceA2. Some research has found that iceA (iceA1/iceA2) is substantially related to digestive tract diseases, whereas others have found the opposite23. In H. pylori, the babA2 gene encodes a membrane protein that helps the bacteria attach to the stomach mucosa24. As a result, multiplex-PCR molecular genotyping of H. pylori is regarded an intense approach to detecting virulence. Among the most efficient ways for studying relationships between H. pylori strains from diverse samples is genotyping using the virulence genes (cagA, vacA, babA2, oipA, and iceA)25. D ARTICLE Medication is another important technique to minimize the transmission of bacteria in the community, given H. pylori’s surprising resistance to multiple antimicrobial drugs26. H. pylori’s resistance to antibacterial treatments varies by geography and tends to be rising over time in many areas27–30. Moreover, employing multiple antibiotic resistance (MAR) scores to identify pathogen sources is thought to be a cost-effective and efficient strategy. Krumperman (1983) looked into this index and found that a value of 0.2 implies a greater frequency of illness in areas where antibacterial drugs are commonly utilized31.h TED g y There have been no published enough investigations on the antibiotic resistance of H. pylori obtained from edible and non-edible raw poultry (Chicken, Turkey, Quebec, Goose, and Ostrich) in Iran. H. pylori’s importance and prevalence in Iran are yet unknown. Human H. pylori infection can be prevented and controlled by eating animal-derived products, particularly fowl (chicken, turkey, Quebec, goose, and ostrich). As a result, the recent study looked at the propagation of the cagA, vacA, babA2, oipA, and iceA pathogenicity genotypes in H. pylori isolates derived from the meat of broiler chickens, turkeys, Quebec goose, and ostriches in vitro, as well as their resistance to multiple antibiotics. RACTE Helicobacter pylori strains isolated from raw poultry meat: frequency and molecular characteristics Tohid Piri‑Gharaghie 1, Ghazal Ghajari 2, Shakiba Tolou‑Shikhzadeh‑Yazdi 3, Mona Aghassizadeh‑Sherbaf 4 & Sahar Khorsand‑Dehkordi 5 OPEN ICLE Poultry is consumed by millions of people around the world every day as a source of animal protein, hence sanitary techniques of delivering hens for food are critical to public healthcare4.l T p Helicobacter genera are Gram-negative helical coccoid flagellar bacteria that range in length from 2 to 4 μm and breadth from 0.5 to 1.0 μm5. Helicobacter may be quite harmful, and it has been found in the biliary tract and 1Biotechnology Research Center, Faculty of Basic Sciences, Islamic Azad University, East-Tehran Branch, Tehran, Iran. 2Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran. 3Department of Biology, Faculty of Sciences, Islamic Azad University, Mashhad Branch, Mashhad, Iran. 4Department of Biology, Faculty of Basic Sciences, Islamic Azad University, East-Tehran Branch, Tehran, Iran. 5Department of Biology, Faculty of Basic Sciences, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran. *email: tohidpirie@yahoo.com Scientific Reports \| (2023) 13:11116 \| https://doi.org/10.1038/s41598-023-38374-5 www.nature.com/scientificreports/ Methods S l i TRA Sample origin. From April to July 2020, 320 Poultry samples were randomly gathered from farms, retail shops, supermarkets, abattoirs…. etc. in the Shahrekord region, Iran, containing specimens of Chicken (n = 60), Turkey (n = 55), Quebec (n = 65), Goose (n = 65), and Ostrich (n = 75). All sample was stored in a specific sterile Ziploc bag that was water-resistant. For isolation and molecular characterization by Multiplex-PCR, samples were gathered from meat, livers, and gizzards, including the jejunum, cecum, and colon (Fig. 1). Till further analysis, all samples were kept at − 80 °C. A statement to confirm that all methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) (PLoS Bio 8(6), e1000412,2010). All studies were con- ducted per the National Institutes of Health’s Guide for the Clinical and Laboratory Standards Institute Animals (NIH Publications No. 8023). The university’s Ethics Committee approved them for Animal Care (Iran). The study was approved by the Ethics Committee of the Islamic Azad University of Shahrekord Branch in Iran (IR. IAU.SHK.REC). TR Helicobacter genus determination. Colony morphology, gram staining, and Biochemical analysis of H. pylori. H. pylori Specific Peptone (HPSP) agar media revealed normal Helicobacter colonies as distinct, round colonies with a width of 0.5–2 mm after 5–7 days of incubation. The gram-negative, S- or C-shaped bacteria were seen by transferring the colonies on slants and staining them with gram. It was discovered that rod and coccoid forms exist. Biochemical tests were performed on the purified cultures to confirm their identity 3. As a control strain, the H. pylori ATCC 700392 strain was used. The urease assay was used to quickly detect H. pylori. On a urea agar medium, a single colony of the investigated microorganism was streaked across the whole surface. For 18–24 h, the samples were incubated at 37 °C in the surrounding atmosphere. H. pylori, which generates cytochrome oxidase enzyme, was also biochemically identified using the oxidase assay. Utilizing oxidase testing https://doi.org/10.1038/s41598-023-38374-5 Scientific Reports \| (2023) 13:11116 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ w.nature.com/scientificreports/ Figure 1. Meat and gizzard samples were collected from chicken, turkey, Quebec, goose, and ostrich from Shahrekord farms. TRACTED ARTICLE E Figure 1. Meat and gizzard samples were collected from chicken, turkey, Quebec, goose, and ostrich from Shahrekord farms. TRA kits, all strains’ oxidase activities (blue/purple hue) were assessed (Sigma-Aldrich, USA). Furthermore, the col- lected isolates’ catalase test was performed using the drop method. Methods S l i TRA In a nutshell, a purified colony was treated with hydrogen peroxide (H2O2) before being put on a slide. The creation of oxygen bubbles was thought to be a good thing. TR Genotypical identification of H. pylori by 16S rRNA‑based PCR confirmation. The Helicobacter genus was iden- tified using 16S rRNA (Table 1). Lactofeed Biotech Group approved oligonucleotide sequences (Iran). Wilkins Chalgren anaerobe broth enhanced colonies were sub-cultured. Using a DNA extraction kit, genomic DNA was isolated from bacteria (Cinna-colon, Iran). The process was carried out according to the manufacturer’s instruc- tions. The extracted DNA’s quality (A260/A280) and quantity were then tested (NanoDrop, Thermo Scientific, Waltham, MA, USA). On a 2% agarose gel dyed with ethidium bromide (0.5 g/mL), the DNA’s veracity was evaluated (Thermo Fisher Scientific, St. Leon-Rot, Germany). A PCR thermal cycler (Eppendorf Co., Hamburg, Germany) was used to execute the polymerase chain reaction (PCR) according to the Piri-Gharaghie et al.32 protocol. H. pylori antibiotic sensitivity pattern. There seem to be no generally recognized standardized methods for checking H. pylori antibiotic susceptibilities, and therefore procedures shown in this research were focused on Ranjbar et al.5 and Performance Standards for Antibiotic Sensitivity Testing- Clinical and Laboratory Stand- https://doi.org/10.1038/s41598-023-38374-5 Scientific Reports \| (2023) 13:11116 \| www.nature.com/scientificreports/ ards Institute—30th ed CLSI supplement M100. To inoculate Muller Hinton agar plates, bacterial solutions were diluted to 0.5 Mcfarland (equal to 1–2 × 108 CFU/ml). The current study employed antibiotic discs with var- ied doses to investigate the in vitro susceptibility of H. pylori isolates to antimicrobial drugs routinely used to treat H. pylori. Antimicrobial discs (amoxicillin (10 μg), ampicillin (10 μg), metronidazole (5 μg), streptomycin (10 μg), cefsulodin (30 μg), erythromycin (5 μg), levofloxacin (5 μg), trimethoprim (25 μg), furazolidone (1 μg), clarithromycin (2 μg), rifampin (30 μg), tetracycline (30 μg), and spiramycin (100 μg) (Mast, UK) were used, and th l t i b t d t 35 °C f 16 18 h d bi diti Th t d d t h i d t Table 1. Oligonucleotide sequences, product length, and cycling conditions of H. pylori virulence genotypes. Methods S l i TRA Target gene Oligonucleotide sequence (5′—> 3′) Size (bp) Tm (°C) References 16S rRNA F: CTATGACGGGTATCCGGC 1026 53 This study R: ATTCCACCTACCTCTCCCA VacA s1a F: CTCTCGCTTTAGTAGGAGC 213 64 This study R: CTGCTTGAATGCGCCAAAC s1b F: AGCGCCATACCGCAAGAG 187 64 This study R: CTGCTTGAATGCGCCAAAC s1c F: CTCTCGCTTTAGTGGGGYT 213 64 This study R: CTGCTTGAATGCGCCAAAC s2 F: GCTAACACGCCAAATGATCC 199 64 This study R: CTGCTTGAATGCGCCAAAC m1a F: GGTCAAAATGCGGTCATGG 290 64 52 R: CCATTGGTACCTGTAGAAAC m1b F: GGCCCCAATGCAGTCATGGA 291 64 This study R: GCTGTTAGTGCCTAAAGAAGCAT m2 F: GGAGCCCCAGGAAACATTG 352 64 This study R: CATAACTAGCGCCTTGCA CagA CagA F: GATAACAGCCAAGCTTTTGAGG 300 56 52 R: CTGCAAAAGATTGTTTGGCAGA IceA IceA1 F: GTGTTTTTAACCAAAGTATC 247 56 This study R: CTATAGCCASTYTCTTTGCA IceA2 F: GTTGGGTATATCACAATTTAT 229/334 56 This study R: TTRCCCTATTTTCTAGTAGGT OipA F: GTTTTTGATGCATGGGATTT 401 56 This study R: GTGCATCTCTTATGGCTTT BabA2 F: CCAAACGAAACAAAAAGCGT 105–124 57 This study R: GCTTGTGTAAAAGCCGTCGT RACTED ARTICLE ards Institute—30th ed CLSI supplement M100. To inoculate Muller Hinton agar plates, bacterial solutions were diluted to 0.5 Mcfarland (equal to 1–2 × 108 CFU/ml). The current study employed antibiotic discs with var- ied doses to investigate the in vitro susceptibility of H. pylori isolates to antimicrobial drugs routinely used to treat H. pylori. Antimicrobial discs (amoxicillin (10 μg), ampicillin (10 μg), metronidazole (5 μg), streptomycin (10 μg), cefsulodin (30 μg), erythromycin (5 μg), levofloxacin (5 μg), trimethoprim (25 μg), furazolidone (1 μg), clarithromycin (2 μg), rifampin (30 μg), tetracycline (30 μg), and spiramycin (100 μg) (Mast, UK) were used, and the plates were incubated at 35 °C for 16–18 h under anaerobic condition. The standard technique was used to assess and analyze the inhibition zone induced by each antibiotic. H. pylori ATCC 26695 and ATCC 43,504 were used as quality management isolates. The following formula was used to calculate the MAR index of each strain: MAR i d N b f i i bi l d hi h h b i i TRACT ards Institute—30th ed CLSI supplement M100. To inoculate Muller Hinton agar plates, bacterial solutions were diluted to 0.5 Mcfarland (equal to 1–2 × 108 CFU/ml). The current study employed antibiotic discs with var- ied doses to investigate the in vitro susceptibility of H. pylori isolates to antimicrobial drugs routinely used to treat H. pylori. Methods S l i TRA Antimicrobial discs (amoxicillin (10 μg), ampicillin (10 μg), metronidazole (5 μg), streptomycin (10 μg), cefsulodin (30 μg), erythromycin (5 μg), levofloxacin (5 μg), trimethoprim (25 μg), furazolidone (1 μg), clarithromycin (2 μg), rifampin (30 μg), tetracycline (30 μg), and spiramycin (100 μg) (Mast, UK) were used, and the plates were incubated at 35 °C for 16–18 h under anaerobic condition. The standard technique was used to assess and analyze the inhibition zone induced by each antibiotic. H. pylori ATCC 26695 and ATCC 43,504 were used as quality management isolates. The following formula was used to calculate the MAR index of each strain: MAR inde Number of antimicrobial drugs to hich the bacterium is TRACT MAR index = Number of antimicrobial drugs to which the bacterium is resistant/Total number of antimicrobial drugs MAR index = Number of antimicrobial drugs to which the bacterium is R de Nu be o a t c ob a d ugs to w c t e ba resistant/Total number of antimicrobial drugs Genotyping analysis. Multiplex-PCR was used to determine the prevalence of the cagA, vacA, babA2, oipA, and iceA alleles19–22. The primers and PCR conditions used to genotype the cagA, vacA, babA2, oipA, and iceA alleles are listed in Table 1. Initially, all specimens were subjected to pre-tests to determine the best reac- tion time, temperature, and volume. In all PCR operations, a programmed DNA thermo-cycler was employed. Positively and negatively controlled were PCR-grade water and H. pylori standard strains (ATCC 43504), respec- tively. The total volume of 25 µl consisted of 5 µl of deoxy-nucleoside triphosphate mix, 2.5 µl of 10X PCR buffer, 0.25 µl of the primer, and 1 µl of the DNA template, was performed. Ethidium bromide (Sigma, USA) has been used to dye ten microliters of PCR product electrophoresed in a 2 percent agarose gel in 1X TBE buffer at 80 V for 30 min. The UVI doc gel documentation devices (Grade GB004, Jencons PLC, London, UK) were used for image processing. Analytical statistics. The IBM Statistical Package for the Social Sciences (SPSS) software, version 20.0 for Windows, was used to conduct the statistical study. The information was given in the form of a mean, standard https://doi.org/10.1038/s41598-023-38374-5 Scientific Reports \| (2023) 13:11116 \| www.nature.com/scientificreports/ deviation, or percentage. For categorical variables, the Chi-squared test was utilized. At < 0.05, the P value was significant. Ethical approval. Methods S l i TRA The study was conducted according to the National Academy of Sciences guide for the care and use of laboratory animals and in compliance with best practices of veterinary care. A statement to con- firm that all methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). Ethical approval. The study was conducted according to the National Academy of Sciences guide for the care and use of laboratory animals and in compliance with best practices of veterinary care. A statement to con- firm that all methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). Results H li b Helicobacter spp. prevalence in poultry based on morphological and biochemical analysis. In 320 cases of poultry flesh, the presence of H. pylori was evaluated. Table 2 shows the prevalence of Helicobacter spp. in poultry flesh. The urease, oxidase, and catalase assays were used to rapidly diagnose Helicobacter spp. The 20 positive Helicobacter spp. were detected by urease, oxidase, and catalase assays after 4 h of incubation, respec- tively, by a purple hue, a blue/purple color, and the generation of oxygen bubbles. Helicobacter spp. were found in 20 of 320 (6.25%) poultry meat specimens. According to findings, 9 (15.00%) Chicken specimens, 7 (12.72%) Turkey specimens, 0 (0%) Quebec specimens, 0 (0%) Goose specimens, and 4 (5.33%) Ostrich specimens were all infected with Helicobacter spp. CLE H. pylori was identified via PCR amplification of the 16SrRNA. The 16SrRNA gene PCR amplifica- tion was used to confirm all of the strains. The electrophoretically displayed PCR results from 20 Helicobacter spp. identified from 320 poultry flesh specimens. H. pylori was recognized as Helicobacter spp. with a 1026-bp PCR product of 16S rRNA in 20/20 (100%). According to PCR results, all 20 (100%) isolates belonged to H. pylori (Fig. 2). The largest incidence of H. pylori bacteria was found in chicken (15.00%) and turkey (12.72%) meat RTICL Table 2. Prevalence of H. pylori in different types of raw poultry meat samples. Raw meat samples No samples collected N (%) of H. pylori-positive samples H. pylori 16SrRNA PCR confirmation (%) Chicken 60 9 (15.00) 9 (15.00) Turkey 55 7 (12.72) 7 (12.72) Quebec 65 0 0 Goose 65 0 0 Ostrich 75 4 (5.33) 4 (5.33) Total 320 20 (6.25) 20 (6.25) ED ART Table 2. Prevalence of H. pylori in different types of raw poultry meat samples. ED Figure 2. Gel electrophoresis for the DNA products of each gene. 1: 16SrRNA, 2: VacA s1a, 3: VacA s1c, 4: VacA s1b, 5: VacA s2, 6: VacA m1a, 7: VacA m1b, 8: VacA m2, 9: CagA, 10: IceA1, 11: IceA2, 12: OipA, 13: BabA2, NC: negative control for PCR, M: 100 bp DNA ladder. RETRACTE Figure 2. Gel electrophoresis for the DNA products of each gene. Table 4. Antimicrobial resistance profile of H. pylori strains (n = 20). www.nature.com/scientificreports/ specimens, while the fewest were found in Quebec and goose (0.00%). Between the specimens and the frequency of H. pylori isolates, a significant statistical variation (P < 0.05) was found. H. pylori sensitivity to antibiotics and the MAR index. Antimicrobial resistance profiles of H. pylori isolates recovered from various kinds of specimens collected are depicted in Table 3. Antimicrobial resistance was found to be most common in H. pylori isolates ampicillin (85%), tetracycline (85%), and amoxicillin (75%). H. pylori isolates also had the lowest rate of resistance to furazolidone (5%), spiramycin (30%), cefsulodin (30%), and levofloxacin (30%). Furthermore, resistance to metronidazole (50%) and streptomycin (50%) was com- mon, as was 40% resistance to erythromycin, rifampin, and 35% resistance to trimethoprim and clarithromycin. Results showed that 17/20 (85%) of the H. pylori isolates obtained from poultry samples were resistant to at least three antibiotics. In fact, these isolates showed the Multi-Drag Resistant (MDR) phenotype. There was a statisti- cal difference between the specimens and antimicrobial resistance incidence (P < 0.05).hl E f p The MAR index of 20 H. pylori isolates in poultry flesh is shown in Table 4. All H. pylori isolates had an average MAR index of 0.622. 17 of the 20 H. pylori isolates tested positive for antibiotic resistance (MDR phenotype), with MAR indexes varying from 0.230 to 1. Strains No. 1 and 2 were highly resistant to all antibacterial agents (MAR index of 1.0), whereas strains Nos. 3–5 were resistant to 12 of the 13 tested antibiotics (MAR index of 0.923). The MAR index for strains 6–7 was 0.846. The MAR scores for Nos. 8–17 ranged from 0.23 to 0.769. Nos. 19 and 20 had the lowest MAR score (0.076). The percentage of H. pylori isolates with a MAR value of more than 0.2 was 17/20 (85%); the frequency with a MAR value of less than 0.2 was 3/20 (15%). As a result, H. pylori is extremely resistant to numerous antibacterial drugs that have been evaluated and have large MAR index values. TICLE extremely resistant to numerous antibacterial drugs that have been evaluated and have large MAR index values. Table 3. Antibiotic resistance pattern of H. pylori strains isolated from different types of raw poultry meat samples. www.nature.com/scientificreports/ AM10: ampicillin (10 μg), Met5: metronidazole (5 μg), ER5: erythromycin (5 μg), CLR2: clarithromycin (2 μg), AMX10: amoxicillin (10 μg), Tet30: tetracycline (30 μg), Lev5: levofloxacin (5 μg), S10: streptomycin (10 μg), RIF30: rifampin (30 μg), Cef30: cefsulodin (30 μg), TRP25: trimethoprim (25 μg), FZL1: furazolidone (1 μg) and Spi100: spiramycin (100 μg). Type of raw meat samples (N of H. pylori strains) N (%) isolates resistant to each antibiotic AM10a Met5 ER5 CLR2 AMX 10 Tet30 Lev5 S10 RIF30 Cef30 TRP25 FZL1 Spi100 Chicken (9) 8 (88.88) 6 (66.66) 4 (44.44) 4 (44.44) 7 (77.77) 7 (77.77) 3 (33.33) 2 (22.22) 3 (33.33) 2 (22.22) 3 (33.33) 2 (22.22) 3 (33.33) Turkey (7) 5 (71.42) 2 (28.57) 3 (42.85) 2 (28.57) 5 (71.42) 7 (100) 2 (28.57) 7 (100) 4 (57.14) 4 (57.14) 3 (42.85) 3 (42.85) 3 (42.85) Ostrich (4) 4 (100) 2 (50) 1 (25) 1 (25) 3 (75) 3 (75) 1 (25) 1 (25) 1 (25) – 1 (25) – – Total (20) 17 (85) 10 (50) 8 (40) 7 (35) 15 (75) 17 (85) 6 (30) 10 (50) 8 (40) 6 (30) 7 (35) 5 (25) 6 (30) Table 4. Antimicrobial resistance profile of H pylori strains (n=20) No. Results H li b 1: 16SrRNA, 2: VacA s1a, 3: VacA s1c, 4: VacA s1b, 5: VacA s2, 6: VacA m1a, 7: VacA m1b, 8: VacA m2, 9: CagA, 10: IceA1, 11: IceA2, 12: OipA, 13: BabA2, NC: negative control for PCR, M: 100 bp DNA ladder. RETRACTE Figure 2. Gel electrophoresis for the DNA products of each gene. 1: 16SrRNA, 2: VacA s1a, 3: VacA s1c, 4: VacA s1b, 5: VacA s2, 6: VacA m1a, 7: VacA m1b, 8: VacA m2, 9: CagA, 10: IceA1, 11: IceA2, 12: OipA, 13: BabA2, NC: negative control for PCR, M: 100 bp DNA ladder. Scientific Reports \| (2023) 13:11116 \| https://doi.org/10.1038/s41598-023-38374-5 www.nature.com/scientificreports/ Scientific Reports \| (2023) 13:11116 \| www.nature.com/scientificreports/ Antimicrobial resistance profile MAR index 1 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5, S10, RIF30, Cef30, TRP25, FZL1, Spi100 1 2 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5, S10, RIF30, Cef30, TRP25, FZL1, Spi100 1 3 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5, S10, RIF30, Cef30, TRP25, FZL1 0.923 4 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5, S10, RIF30, Cef30, TRP25, FZL1 0.923 5 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5, S10, RIF30, Cef30, TRP25, FZL1 0.923 6 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5, S10, RIF30, Cef30, TRP25 0.846 7 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5, S10, RIF30, Cef30, TRP25 0.846 8 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5, S10, RIF30, Cef30 0.769 9 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5, S10, RIF30, Cef30 0.769 10 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5, S10, RIF30, Cef30 0.769 11 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5, S10, RIF30 0.692 12 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5, S10 0.615 13 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5, S10 0.615 14 AM10, Met5, ER5, CLR2, AMX10, Tet30, Lev5 0.538 15 AM10, Met5, ER5, CLR2, AMX10 0.384 16 AM10, Met5, ER5, CLR2 0.307 17 AM10, Met5, ER5 0.230 18 AM10, Met5 0.153 19 AM10 0.076 20 AM10 0.076 Average 0.622 RETRACTED ARTIC extremely resistant to numerous antibacterial drugs that have been evaluated and have large MAR index values. Table 3. Antibiotic resistance pattern of H. pylori strains isolated from different types of raw poultry meat samples. AM10: ampicillin (10 μg), Met5: metronidazole (5 μg), ER5: erythromycin (5 μg), CLR2: clarithromycin (2 μg), AMX10: amoxicillin (10 μg), Tet30: tetracycline (30 μg), Lev5: levofloxacin (5 μg), S10: streptomycin (10 μg), RIF30: rifampin (30 μg), Cef30: cefsulodin (30 μg), TRP25: trimethoprim (25 μg), FZL1: furazolidone (1 μg) and Spi100: spiramycin (100 μg). Type of raw meat samples (N of H. Discussion Too far, there is little indication that poultry is a major reservoir for the H. pylori bacteria prevalent in people. As a result, the H. pylori bacteria separated from commercial broiler flesh in this investigation are likely to have been acquired during shooting and/or processing. Since men are the bacterium’s primary natural host, butcher employees were most probably the principal cause of H. pylori infection in our specimens collected. In the present study, 20 (6.25 percent) H. pylori strains were discovered in 320 commercial poultry samples, indicating that this bacterium poses a risk to humans. Even though the root cause of such a result is unknown, cross-contamination of poultry meat is considered to be a significant source of H. pylori infection in the poultry meat industry. The three primary operations that may increase the incidence of H. pylori infection include cutting, keeping, and shipping poultry meat. Ranjbar et al. discovered that H. pylori can survive in water in a separate study5,32. As a result, an additional cause for the occurrence of H. pylori in the poultry sample obtained is the use of polluted water in the meat industry. Furthermore, contaminated slaughtering personnel and equipment, like blades, may contribute to a higher prevalence of this pathogen33. Generally, our findings are similar to those of Meng et al. (2008), who used Multiplex-PCR to analyze 11 fresh chicken specimens (total chicken including skin) and discovered that 4 (36%) were H. pylori-positive, although our ratios (6.25%) were significantly smaller. H. pylori is also a foodborne organism that may be transferred to humans, according to these investigators34. El Dairouty et al. (2016) reported that 5% of ground beef, raw bird, and sandwich meat specimens tested positive for H. pylori35. Genomic approaches have subsequently been employed by several studies to discover the various genotypes TED ARTIC Too far, there is little indication that poultry is a major reservoir for the H. pylori bacteria prevalent in people. As a result, the H. pylori bacteria separated from commercial broiler flesh in this investigation are likely to have been acquired during shooting and/or processing. Since men are the bacterium’s primary natural host, butcher employees were most probably the principal cause of H. pylori infection in our specimens collected. In the present study, 20 (6.25 percent) H. pylori strains were discovered in 320 commercial poultry samples, indicating that this bacterium poses a risk to humans. Discussion Even though the root cause of such a result is unknown, cross-contamination of poultry meat is considered to be a significant source of H. pylori infection in the poultry meat industry. The three primary operations that may increase the incidence of H. pylori infection include cutting, keeping, and shipping poultry meat. Ranjbar et al. discovered that H. pylori can survive in water in a separate study5,32. As a result, an additional cause for the occurrence of H. pylori in the poultry sample obtained is the use of polluted water in the meat industry. Furthermore, contaminated slaughtering personnel and equipment, like blades, may contribute to a higher prevalence of this pathogen33. Generally, our findings are similar to those of Meng et al. (2008), who used Multiplex-PCR to analyze 11 fresh chicken specimens (total chicken including skin) and discovered that 4 (36%) were H. pylori-positive, although our ratios (6.25%) were significantly smaller. H. pylori is also a foodborne organism that may be transferred to humans, according to these investigators34. El Dairouty et al. (2016) reported that 5% of ground beef, raw bird, and sandwich meat specimens tested positive for H. pylori35. Genomic approaches have subsequently been employed by several studies to discover the various genotypes of H. pylori, which also are strongly connected to its distribution. Multiplex-PCR is a commonly used test for genotyping and identifying homologous genes in H. pylori isolates obtained from clinical specimens35,36. The 16S rRNA gene was employed as a reference gene in this investigation. The frequency of this gene was 100 percent, indicating that the 16S rRNA gene is a good candidate for identifying different H. pylori isolate. Similar find- ings were achieved by Piri-Gharaghie and El Dairouty et al.32,35. The 16S rRNA genetic code is a unique gene for recognizing bacterial species recovered from specimens, according to these scientists, when contrasted to the other reference genes. H. pylori’s proliferation and cell wall formation is dependent on the 16S rRNA gene. As a result, this gene has indeed been widely used to diagnose H. pylori infections36. Our research looks at the incidence of the virulence genes IceA, babA2, OipA, vacA, and cagA. In H. pylori isolates collected from edible and non-edible tissues from the poultry meat industry, the IceA (27.5 percent), babA2 (40 percent), OipA (25 percent), vacA (48.57 percent), and cagA (60 percent) genes were all found. www.nature.com/scientificreports/ pylori strains) N (%) isolates resistant to each antibiotic AM10a Met5 ER5 CLR2 AMX 10 Tet30 Lev5 S10 RIF30 Cef30 TRP25 FZL1 Spi100 Chicken (9) 8 (88.88) 6 (66.66) 4 (44.44) 4 (44.44) 7 (77.77) 7 (77.77) 3 (33.33) 2 (22.22) 3 (33.33) 2 (22.22) 3 (33.33) 2 (22.22) 3 (33.33) Turkey (7) 5 (71.42) 2 (28.57) 3 (42.85) 2 (28.57) 5 (71.42) 7 (100) 2 (28.57) 7 (100) 4 (57.14) 4 (57.14) 3 (42.85) 3 (42.85) 3 (42.85) Ostrich (4) 4 (100) 2 (50) 1 (25) 1 (25) 3 (75) 3 (75) 1 (25) 1 (25) 1 (25) – 1 (25) – – Total (20) 17 (85) 10 (50) 8 (40) 7 (35) 15 (75) 17 (85) 6 (30) 10 (50) 8 (40) 6 (30) 7 (35) 5 (25) 6 (30) TED ARTIC RTIC meat samples. AM10: ampicillin (10 μg), Met5: metronidazole (5 μg), ER5: erythromycin (5 μg), CLR2: clarithromycin (2 μg), AMX10: amoxicillin (10 μg), Tet30: tetracycline (30 μg), Lev5: levofloxacin (5 μg), S10: streptomycin (10 μg), RIF30: rifampin (30 μg), Cef30: cefsulodin (30 μg), TRP25: trimethoprim (25 μg), FZL1: furazolidone (1 μg) and Spi100: spiramycin (100 μg). TED https://doi.org/10.1038/s41598-023-38374-5 Scientific Reports \| (2023) 13:11116 \| www.nature.com/scientificreports/ Genotype distribution among H. pylori isolates obtained from various kinds of poultry sam‑ ples. The genotype distribution of H. pylori isolates recovered from various kinds of poultry specimens is shown in Table 5. The most commonly found genotypes among the H. pylori bacteria isolated from various sorts of poultry specimens were VacA s1a (75%), m1a (75%), s2 (70%), and m2 (65%), and cagA (60%). The H. pylori isolates identified from several sorts of poultry samples with the lowest frequency were VacA s1c (5%) and IceA2 (15%). VacAs1b, VacAm1b, and OipA genes were also found in 25% of Helicobacter pylori strains from various poultry specimens. IceA1 and BabA2 genes were distributed in 40% of the population. Between the kinds of specimens and the incidence of genotypes, there was a statistical difference (P < 0.05). H. pylori strains’ genotyping patterns. The genotyping frequency of H. pylori isolates recovered from varying sorts of poultry specimens is shown in Table 6. The most commonly found genotyping patterns of the vacA alleles of H. pylori bacteria originating from various kinds of poultry fresh meat specimens were s1am1a (45%), s2m1a (45%), and s2 m2 (30%). www.nature.com/scientificreports/ BabA2, OipA + , and OipA− genotypes were distributed 40 percent, 30 percent, and 30 percent, respectively. we discovered that iceA1/iceA2 genotyping was present in 10% of H. pylori isolates. Among the diverse genotyping profiles of H. pylori isolates, S1cm1b (0%), S1 cm2 (5%), S1cm1a (5%), and CagA + (5%) exhibited the lowest frequency. The distribution of other genotypes including s1am1b (15%), s1 am2 (25%), s1bm1a (15%), s1bm1b (10%), s1bm2 (10%), s2m1b (15%), CagA− (25%), BabA2 + (25%) and IceA1/IceA2 (10%) were moderate. ICLE RETRACT Discussion As a result, certain virulence genes, notably cagA, were found in larger numbers in commercial poultry flesh, which is regarded as ready-to-eat human food. The major impediments of H. pylori in the human digestive system are thought to be increased by these genotypes. Bibi et al. earlier hypothesized a relationship between the existence of the H. pylori babA2/cagA + / vacAs1 genotype and the prevalence of gastroenteritis, stomach carcinoma, and ulcerative colitis25. In H. pylori isolates recovered from clinical specimens of human and animal populations, a high incidence of vacA, cagA, TRACTED ARTIC Table 5. Distribution of genotypes amongst the H. pylori strains isolated from different types of raw poultry meat samples. Type of raw meat samples (N of H. pylori strains) N (%) isolates harbor each genotype VacA CagA IceA OipA BabA2 s1a s1b s1c s2 m1a m1b m2 IceA1 IceA2 Chicken (9) 7 (77.77) 3 (33.33) 1 (11.11) 6 (66.66) 7 (77.77) 3 (33.33) 6 (66.66) 6 (66.66) 4 (44.44) 2 (22.22) 3 (33.33) 4 (44.44) Turkey (7) 6 (85.71) 2 (28.57) – 6 (85.71) 6 (85.71) 1 (14.28) 5 (71.42) 5 (71.42) 3 (42.85) 1 (14.28) 2 (28.57) 3 (42.85) Ostrich (4) 2 (50) – – 2 (50) 2 (50) 1 (25) 2 (50) 1 (25) 1 (25) – – 1 (25) Total (20) 15 (75) 5 (25) 1 (5) 14 (70) 15 (75) 5 (25) 13 (65) 12 (60) 8 (40) 3 (15) 5 (25) 8 (40) R Table 5. Distribution of genotypes amongst the H. pylori strains isolated from different types of raw poultry meat samples. https://doi.org/10.1038/s41598-023-38374-5 Scientific Reports \| (2023) 13:11116 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ . Genotyping pattern of H. pylori strains isolated from different types of raw poultry meat samples. www.nature.com/scientificreports/ iceA1, oipA, and babA2 genotypes has also been described25–38. Moreover, H. pylori isolates recovered from vary- ing sorts of dietary specimens have shown a significant frequency of these genes39,40. Previous studies have linked the H. pylori genotypes vacA, cagA, iceA, oipA, and babA2 to interleukin-8 and cytotoxin exudation, attachment to gastric epithelial cells, increase in the frequency of inflammatory impact, vacuolization, apoptosis process in gastric epithelial cells, stomach ulcers ulceration, increased intense neutrophilic incursion38–42. Consumption of fresh poultry meat infected with virulent isolates of H. pylori would increase duodenum ulcers, gastric epithelium shrinkage, and stomach carcinoma because the H. pylori strain in this experiment carried the vacA, cagA, iceA, oipA, and babA2 genes. Furthermore, certain H. pylori strains tested positive for multiple genotypes at the same time, indicating that they are more harmful43. g y Another noteworthy result in the ongoing investigation is the high prevalence of antibiotic resistance among H. pylori isolates. H. pylori bacteria showed significant resistance to antimicrobials ampicillin (85%), tetracycline (85%), and amoxicillin (75%) in this study. Similar findings were found by Mousavi et al. (2014). These researchers discovered that H. pylori bacteria in meat were resistant to ampicillin (84.4%), tetracycline (76.6%), erythromycin (70.5%), and metronidazole (70.5%)44. In addition, previous researchers found that H. pylori in food products showed high levels of resistance to amoxicillin, metronidazole, ampicillin, and oxytetracycline. Furthermore, epidemiologic studies in different countries found that H. pylori isolate in healthcare specimens had a high level of resistance to antimicrobials like metronidazole, ampicillin, tetracycline, and amoxicillin which is consistent with our results39–45. According to with MAR index, 85 percent of the H. pylori isolates tested positive for 3 or more antibiotic medicines employed in the study, indicating a large chance of infection in poultry. Antibiotic resistance may have become more common as a result of the nonselective use of such antibacterial medicines, according to our findings. Many researchers have looked at the incidence of H. pylori resistance to multiple antibiotics, but some of them have run into problems, notably with the number of isolates studied 46,47. Antimicrobial resistance testing revealed that H. pylori were transmitted from infectious poultry samples to meat. The lesser resistance of H. Conclusions H. pylori infection in humans could be spread through raw poultry flesh. This analysis revealed that poultry is another reservoir of pathogenic H. pylori isolates. As a result, slaughterhouses and butchering sanitary measures are critical in reducing the risk of H. pylori infection from poultry meat spreading to humans. Furthermore, the H. pylori isolates showed great resistance to ampicillin (85%), tetracycline (85%), and amoxicillin (75%), as well as having high MAR index values. on the other hand, H. pylori demonstrated lower resistance to metronidazole (50%) and streptomycin (50%) as well as erythromycin (40%), rifampin (40%), trimethoprim (35%), and clarithromycin (35%); hence, we propose utilizing these antibiotic drugs in Iran to combat H. pylori. Also, Our research looks at the incidence of the virulence genes IceA, babA2, OipA, vacA, and cagA. In H. pylori isolates collected from edible and non-edible tissues from the poultry meat industry, the IceA (27.5 percent), babA2 (40 percent), OipA (25 percent), vacA (48.57 percent), and cagA (60 percent) genes were all found. As a result, certain virulence genes, notably cagA, were found in larger numbers in commercial poultry flesh, which is regarded as ready-to-eat human food. The major impediments of H. pylori in the human digestive system are thought to be increased by these genotypes. TRACTED www.nature.com/scientificreports/ pylori isolates to metronidazole (50%) and streptomycin (50%) was common, as was 40% resistance to erythromycin (40%), rifampin (40%), trimethoprim (35%), and clarithromycin (35%) was also discovered in our investigation, which might be attributed to the antibiotic medications being prescribed less often. There has been some speculation about a link between virulence genes and antibiotic resistance. According to research done in Ireland in 2009, the lack of cagA could be a potential risk for acquiring metronidazole sensitivity48. Other research has linked clarithromycin susceptibility changes to the less pathogenic vacA genotypes49. Some other studies identified a link between cagE and vacA S1 and clarithromycin and metronidazole susceptibility50, whereas others reported no link between cagA or vacA and susceptibility51–53. As a result, it’s crucial to determine whether there’s a link between the existence of pathogenic indicators and antimicrobial resistance within H. pylori isolates. D ARTICLE are specime oxicillin whi ates tested p n in poultry. tibacterial m TICL a availability R y The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. TR Received: 2 February 2023; Accepted: 7 July 2023 T Discussion at Genotyping pattern (%) s1am1a s1am1b s1 am2 s1bm1a s1bm1b s1bm2 s1cm1a s1cm1b s1 cm2 s2m1a s2m1b s2 m2 CagA + CagA− IceA1/ IceA2 OipA + OipA− BabA2 + BabA2- 5 (55.55) 2 (22.22) 4 (44.44) 2 (22.22) 1 (11.11) 2 (22.22) 1 (11.11) – 1 (11.11) 4 (44.44) 2 (22.22) 3 (33.33) 6 (66.66) 3 (33.33) 1 (11.11) 3 (33.33) 6 (66.66) 4 (44.44) 5 (55.55) 7) 3 (42.85) 1 (14.28) 1 (14.28) 1 (14.28) 1 (14.28) - – – – 3 (42.85) 1 (14.28) 2 (28.57) 3 (42.85) 2 (28.57) 1 (14.28) 2 (28.571) – 1 (14.28) 2 (28.57) (4) 1 (25) – – – – – – – – 2 (50) – 1 (25) 1 (25) – – 1 (25) – – 1 (25) 0) 9 (45) 3 (15) 5 (25) 3 (15) 2 (10) 2 (10) 1 (5) – 1 (5) 9 (45) 3 (15) 6 (30) 10 (5) 5 (25) 2 (10) 6 (30) 6 (30) 5 (25) 8 (40) RTICLE Table 6. Genotyping pattern of H. pylori strains isolated from different types of raw poultry meat samples. Type of raw meat samples (N of H. pylori strains) Genotyping pattern (%) s1am1a s1am1b s1 am2 s1bm1a s1bm1b s1bm2 s1cm1a s1cm1b s1 cm2 s2m1a s2m1b s2 m2 CagA + CagA− IceA1/ IceA2 OipA + OipA− BabA2 + BabA2- Chicken (9) 5 (55.55) 2 (22.22) 4 (44.44) 2 (22.22) 1 (11.11) 2 (22.22) 1 (11.11) – 1 (11.11) 4 (44.44) 2 (22.22) 3 (33.33) 6 (66.66) 3 (33.33) 1 (11.11) 3 (33.33) 6 (66.66) 4 (44.44) 5 (55.55) Turkey (7) 3 (42.85) 1 (14.28) 1 (14.28) 1 (14.28) 1 (14.28) - – – – 3 (42.85) 1 (14.28) 2 (28.57) 3 (42.85) 2 (28.57) 1 (14.28) 2 (28.571) – 1 (14.28) 2 (28.57) Ostrich (4) 1 (25) – – – – – – – – 2 (50) – 1 (25) 1 (25) – – 1 (25) – – 1 (25) Total (20) 9 (45) 3 (15) 5 (25) 3 (15) 2 (10) 2 (10) 1 (5) – 1 (5) 9 (45) 3 (15) 6 (30) 10 (5) 5 (25) 2 (10) 6 (30) 6 (30) 5 (25) 8 (40) TICLE Scientific Reports \| (2023) 13:11116 \| https://doi.org/10.1038/s41598-023-38374-5 RETRACTED ARTI https://doi.org/10.1038/s41598-023-38374-5 Scientific Reports \| (2023) 13:11116 \| https://doi.org/10.1038/s41598-023-38374-5 www.nature.com/scientificreports/ g y g py ( ) 8. Piri Gharaghie, T. & Hajimohammadi, S. Comparison of anti-candida effects of aqueous, ethanolic extracts and essential oil of E. angustifolia with fluconazole on the growth of clinical strains of Candida. New Cell. Mol. Biol. J. 11(43), 25–38 (2021). www.nature.com/scientificreports/ Prevalence of Helicobacter pylori vacA, cagA, cagE, oipA, iceA, babA2 and babB genotypes in Iranian dyspeptic patients. Microb. Pathog. 105, 226–230 (2017). 39 Kh ji L B i h if G & Al i I G i f h H li b l i i l f d di i l d i d TR 9. Khaji, L., Banisharif, G. & Alavi, I. Genotyping of the Helicobacter pylori isolates of raw meat and traditional dairy products Microbiol. Res. 8(2), 43–46 (2017). TR 40. Hemmatinezhad, B., Momtaz, H. & Rahimi, E. VacA, cagA, iceA and oipA genotypes status and antimicrobial resistance properties of Helicobacter pylori isolated from various types of ready to eat foods. Ann. Clin. Microbiol. 15(1), 1–9 (2016). k h h d f l b l l d f b l T 1. Torkan, S. & Shahreza, M. H. VacA, CagA, IceA and OipA genotype status of Helicobacter pylori isolated from biopsy samples from Iranian dogs. Trop. J. Pharm. Res. 15(2), 377–384 (2016). g p 2. Podzorski, R. P., Podzorski, D. S., Wuerth, A. & Tolia, V. Analysis of the vacA, cagA, cagE, iceA, and babA2 genes in Helicobacter pylori from sixty-one pediatric patients from the Midwestern United States. Diagn. Microbiol. Infect. 46(2), 83–88 (2003).h 43. Alexander, S. M. et al. Helicobacter pylori in human stomach: The inconsistencies in clinical outcomes and the probable causes. Front. Microbiol. 2277, 713955 (2021). 44. Souod, N., Kargar, M., Doosti, A., Ranjbar, R. & Sarshar, M. Genetic analysis of cagA and vacA genes in Helicobacter pylori isolates and their relationship with gastroduodenal diseases in the west of Iran. Iran. Red Crescent Med. J. 15(5), 371 (2013). 5. Mousavi, S. & Dehkordi, F. S. Virulence factors and antibiotic resistance of Helicobacter pylori isolated from raw meat and unpas- teurized dairy products in Iran. JVATiTD 20, 1–7 (2015). 46. Yahaghi, E. et al. Helicobacter pylori in vegetables and salads: Genotyping and antimicrobial resistance properties. Biomed Res. Int. 2014, 757941 (2014). 47. Li, J., Deng, J., Wang, Z., Li, H. & Wan, C. Antibiotic resistance of Helicobacter pylori strains isolated from pediatric patients in Southwest China. Front. Microbiol. 11, 621791 (2021). 8. Sabbagh, P. et al. Diagnostic methods for Helicobacter pylori infection: Ideals, options, and limitations. EJCMID 38(1), 55–66 (2019). 49. Taneike, I. et al. www.nature.com/scientificreports/ Ansari H, Doosti A, Kargar M, Bijanzadeh M, Jafarinya M. Antimicrobial resistant determination and prokaryotic express D A 28. Ansari H, Doosti A, Kargar M, Bijanzadeh M, Jafarinya M. Antimicrobial resistant determination and smpA gene of Acinetobacter baumannii isolated from admitted patients. Jundishapur J.Microbiol. 10( D A 28. Ansari H, Doosti A, Kargar M, Bijanzadeh M, Jafarinya M. Antimicrobial resistant determination and prokaryotic express smpA gene of Acinetobacter baumannii isolated from admitted patients. Jundishapur J.Microbiol. 10(11), (2017). 29 Kh d i F P i F H i i E Akb i M & S f i H G H li b t l i i I A t ti i th ti D A 29. Khademi, F., Poursina, F., Hosseini, E., Akbari, M. & Safaei, H. G. Helicobacter pylori in Iran: A systematic review on the antibiotic resistance. IJBMS 18(1), 2 (2015).h ED 30. Alexander, S. M. et al. Helicobacter pylori in human stomach: The inconsistencies in clinical outcomes and the probable causes. Front. Microbiol. 12, 2277 (2021). TED 31. Krumperman, P. H. Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamin of foods. Appl. Environ. Microbiol. 46(1), 165–170 (1983).hf TE 32. Tohid, G. P. & Shandiz, S. A. S. The inhibitory effects of silver nanoparticles on bap gene expression in antibiotic-resistant Aciento‑ bacter bumanni isolates using real-time PCR. JIUMS 26(4), 175–85 (2018). CTE g 3. Gilani, A., Razavilar, V., Rokni, N. & Rahimi, E. VacA and cagA genotypes of Helicobacter pylori isolated from raw meat in Isfahan province, Iran. Vet. Res. Forum. 8, 1–75 (2017). CT 4. Meng, X., Zhang, H., Law, J., Tsang, R. & Tsang, T. Detection of Helicobacter pylori from food sources by a novel multiplex PCR assay. J. Food Saf. 28(4), 609–619 (2008). ACT y f 35. El Dairouty, R. K. et al. Helicobacter pylori and its interrelations with other foodborne pathogenic bacteria in Egyptian meat and some meat products. Curr. Sci. Int. 5(2), 139–146 (2016). AC 6. Talimkhani A, Mashak Z. Prevalence and genotyping of Helicobacter pylori isolated from meat, meat and vegetable in Iran. Jundis hapur J.Microbiol. 10(11), (2017). RA p J ( ), ( ) 37. Momtaz, H., Dabiri, H., Souod, N. & Gholami, M. Study of Helicobacter pylori genotype status in cows, sheep, goats and human beings. BMC Gastroenterol. 14(1), 1–7 (2014). TRA 38. Dabiri, H. et al. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 9. Zarinnezhad, A., Shahhoseini, M. H. & Piri, G. T. Evaluating the relative frequency of fungal infections in the serum of patients with multiple sclerosis and healthy subjects using PCR. BJM 10(37), 37–50 (2021). p y j g 0. Javed, S., Gul, F., Javed, K. & Bokhari, H. Helicobacter pullorum: An emerging zoonotic pathogen. Front. Microbiol. 8, 604 (2017) p g g p g 1. Hamada, M. et al. Helicobacter pylori in a poultry slaughterhouse: Prevalence, genotyping and antibiotic resistance pattern. Saud J. Biol. Sci. 25(6), 1072–1078 (2018).ffi J. Biol. Sci. 25(6), 1072 1078 (2018). 12. Chehelgerdi, M. & Doosti, A. Effect of the cagW-based gene vaccine on the immunologic properties of BALB/c mouse: An efficient candidate for Helicobacter pylori DNA vaccine J Nanobiotechnol 18(1) 1 6 (2020) 12. Chehelgerdi, M. & Doosti, A. Effect of the cagW-based gene vaccine on the immunologic properties of BALB/c mouse: An ef candidate for Helicobacter pylori DNA vaccine. J. Nanobiotechnol. 18(1), 1–6 (2020).l y 13. Sepulveda, A. R. Helicobacter, inflammation, and gastric cancer. Curr. Pathobiol. Rep. 1(1), 9–18 (2013). Ö 13. Sepulveda, A. R. Helicobacter, inflammation, and gastric cancer. Curr. Pathobiol. Rep. 1(1), 9–18 (2013). 14 Öztekin M Yılmaz B Ağagündüz D & Capasso R Overview of Helicobacter pylori infection: Clinical feature l 14. Öztekin, M., Yılmaz, B., Ağagündüz, D. & Capasso, R. Overview of Helicobacter pylori infection: Clinical features, treatment nutritional aspects. Diseases 9(4), 66 (2021). 15. Kishk, R. M. et al. Genotyping of Helicobacter pylori virulence genes caga and vaca: Regional and national study. Int. J. Microbiol. https://doi.org/10.1155/2021/5540560 (2021). E p g 6. Syam, A. F. et al. Helicobacter pylori in the Indonesian Malay’s descendants might be imported from other ethnicities. Gut Pathog 13(1), 1 (2021). E ( ) ( ) 17. Mahmoudi Vashian, Z. & Doosti, A. Cloning and gene expression of ureG gene as a DNA vaccine candidate against Helicobacter pylori. J. GUMS. 26(102), 20–29 (2017). LE py ( ) ( ) 18. Kisiala, M. et al. Restriction endonucleases that cleave RNA/DNA heteroduplexes bind dsDNA in A-like conformation. Nuclei Acids Res. 48(12), 6954–6969 (2020).l LE 9. Rizzato, C. et al. Risk of advanced gastric precancerous lesions in Helicobacter pylori infected subjects is influenced by ABO blood group and cagA status. IJC 133(2), 315–322 (2013). CL g p g 20. Keikha, M. & Karbalaei, M. References 1. Oz, F. & Celik, T. Proximate composition, color and nutritional profile of raw and cooked goose meat with different methods. J Food Process. Preserv. 39(6), 2442–2454 (2015). ( ), ( ) 2. Piri Gharaghie, T., Doosti, A. & Mirzaei, S. A. Prevalence and antibiotic resistance pattern of Acinetobacter spp. infections in Shahrekord medical centers. Dev. Biol. 13(4), 35–46 (2021). 2. Piri Gharaghie, T., Doosti, A. & Mirzaei, S. A. Prevalence and antibiotic resistance pattern of Acinetobacter spp. infections in Shahrekord medical centers. Dev. Biol. 13(4), 35–46 (2021). ( ) ( ) 3. Ricci, C., Holton, J. & Vaira, D. Diagnosis of Helicobacter pylori: Invasive and non-invasive tests. Best Pract. Res. Clin. Gastroenterol 21(2), 299–313 (2007). T l S ll l l d f d A Gl b d S ( ) 4. Wong, J. T. et al. Small scale poultry and food security in resource poor settings: A review. Glob. Food Sec. 15, 43 52 (2017). 5. Ranjbar, R., Farsani, F. Y. & Dehkordi, F. S. Phenotypic analysis of antibiotic resistance and genotypic study of the vacA, cagA, iceA, oipA and babA genotypes of the Helicobacter pylori strains isolated from raw meat. Antimicrob. Resist. Infect. Control. 7(1), 1–4 (2018). g y g py ( ) 8. Piri Gharaghie, T. & Hajimohammadi, S. Comparison of anti-candida effects of aqueous, ethanolic extracts and essential oil of E. angustifolia with fluconazole on the growth of clinical strains of Candida. New Cell. Mol. Biol. J. 11(43), 25–38 (2021). https://doi.org/10.1038/s41598-023-38374-5 Scientific Reports \| (2023) 13:11116 \| www.nature.com/scientificreports/ EPIYA motifs of Helicobacter pylori cagA genotypes and gastrointestinal diseases in the Iranian population: A systematic review and meta-analysis. NMNI 41, 100865 (2021). ICL p p y y ( ) 21. Ghorbani, F., Gheisari, E. & Dehkordi, F. S. Genotyping of vacA alleles of Helicobacter pylori strains recovered from some Iranian food items. Trop. J. Pharm. Res. 15(8), 1631–1636 (2016). TIC yp g food items. Trop. J. Pharm. Res. 15(8), 1631–1636 (2016). 22. Farzi, N., Yadegar, A., Aghdaei, H. A., Yamaoka, Y. & Zali, M. R. Genetic diversity and functional analysis of oipA gene in associa- tion with other virulence factors among Helicobacter pylori isolates from Iranian patients with different gastric diseases. Infect. Genet. Evol. 60, 26–34 (2018). RTIC ( ) 3. Abu-Taleb, A. M. et al. Prevalence of Helicobacter pylori cagA and iceA genes and their association with gastrointestinal diseases Int. J. Microbiol. https://doi.org/10.1155/2018/4809093 (2018).t RT g 24. Doohan, D., Rezkitha, Y. A., Waskito, L. A., Yamaoka, Y. & Miftahussurur, M. Helicobacter pylori BabA–SabA key roles in the adherence phase: The synergic mechanism for successful colonization and disease development. Toxins 13(7), 485 (2021). 25 Bibi F et al Detection and genotyping of Helicobacter pylori among gastric ulcer and cancer patients from Saudi Arabia PJMHS ART 24. Doohan, D., Rezkitha, Y. A., Waskito, L. A., Yamaoka, Y. & Miftahussurur, M. Helicobacter pylori BabA–SabA key roles in the adherence phase: The synergic mechanism for successful colonization and disease development. Toxins 13(7), 485 (2021). 25. Bibi, F. et al. Detection and genotyping of Helicobacter pylori among gastric ulcer and cancer patients from Saudi Arabia. PJMHS 33(2), 320 (2017). ART adherence phase: The synergic mechanism for successful colonization and disease development. Toxins 13(7), 485 (2021). 25. Bibi, F. et al. Detection and genotyping of Helicobacter pylori among gastric ulcer and cancer patients from Saudi Arabia. PJMHS 33(2), 320 (2017). AR ( ) ( ) 26. Cardos, I. A., Zaha, D. C., Sindhu, R. K. & Cavalu, S. Revisiting therapeutic strategies for H. pylori treatment in the conte antibiotic resistance: Focus on alternative and complementary therapies. Molecules 26(19), 6078 (2021). d l b l d d ( ) ( ) D AR p y p 27. Mégraud, F. H pylori antibiotic resistance: Prevalence, importance, and advances in testing. Gut 53(9), 1374–1384 (2004). D A 27. Mégraud, F. H pylori antibiotic resistance: Prevalence, importance, and advances in testing. Gut 53(9), 1374–1384 (2004). 28. 3 3 3 4 4 4 4 4 RETR www.nature.com/scientificreports/ www.nature.com/scientificreports/ 50. Boyanova, L., Markovska, R., Yordanov, D., Gergova, G. & Mitov, I. Clarithromycin resistance mutations in Helicobacter pylori in association with virulence factors and antibiotic susceptibility of the strains. Microb. Drug Resist. 22(3), 227–232 (2016). 50. Boyanova, L., Markovska, R., Yordanov, D., Gergova, G. & Mitov, I. Clarithromycin resistance mutations in Helicobacter pylori in association with virulence factors and antibiotic susceptibility of the strains. Microb. Drug Resist. 22(3), 227–232 (2016). J ( ) ( ) 52. van Doorn, L. J. et al. Accurate prediction of macrolide resistance in Helicobacter pylori by a PCR line probe assay for detection of mutations in the 23S rRNA gene: multicenter validation study. Antimicrob. Agents Chemother. 45(5), 1500–1504 (2001). 52. van Doorn, L. J. et al. Accurate prediction of macrolide resistance in Helicobacter pylori by a PCR line probe assay for detection of mutations in the 23S rRNA gene: multicenter validation study. Antimicrob. Agents Chemother. 45(5), 1500–1504 (2001). g y g ( ), ( ) Hou, P. et al. Helicobacter pylori vacA genotypes and cagA status and their relationship to associated diseases. World J. Gastroen‑ erol. 6(4), 605 (2000). 53. Hou, P. et al. Helicobacter pylori vacA genotypes and cagA status and their relationship to associated diseases. World J. Gastroen‑ terol. 6(4), 605 (2000). g authors would like to thank the staff members of the Biotechnology Research Center of the Islamic Azad ersity of East-Tehran Branch in Iran. E g The authors would like to thank the staff members of the Biotechnology Research Center of the Islamic Azad University of East-Tehran Branch in Iran. E g The authors would like to thank the staff members of the Biotechnology Research Center of the Islamic Azad University of East-Tehran Branch in Iran. E University of East-Tehran Branch in Iran. Author contributions T.P.G and G.G.: wrote the main draft of the manuscript, M.A.S, and S.T.S.Y: prepared tables, S.KH.D.: sample collection. The authors read and approved the final manuscript. Competing interests The authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to T.P.-G. Reprints and permissions information is available at www.nature.com/reprints. RTICLE Competing interests h p g The authors declare no competing interests. www.nature.com/scientificreports/ Analysis of drug resistance and virulence-factor genotype of Irish Helicobacter pylori strains: Is there any relation- ship between resistance to metronidazole and cagA status. AP&T 30(7), 784–90 (2009). https://doi.org/10.1038/s41598-023-38374-5 Scientific Reports \| (2023) 13:11116 \| Author contributions T.P.G and G.G.: wrote the main draft of the manuscript, M.A.S, and S.T.S.Y: prepared tables, S.KH.D.: sampl collection. The authors read and approved the final manuscript. LE RETRACT Additional information Reprints and permissions information is available at www.nature.com/reprints. RTI Reprints and permissions information is available at www.nature.com/reprints. RTI Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. 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https://openalex.org/W4315619515	https://curis.ku.dk/ws/files/203406694/emmm.201808931.full.pdf	English	null	Enriched environment enhances β‐adrenergic signaling to prevent microglia inflammation by amyloid‐β	EMBO molecular medicine	2,023	cc-by	14,408	Citation for published version (APA): Xu, H., Rajsombath, M. M., Weikop, P., & Selkoe, D. J. (2018). Enriched environment enhances -adrenergic signaling to prevent microglia inflammation by amyloid-. EMBO Molecular Medicine, 10(9), [e8931]. https://doi.org/10.15252/emmm.201808931 university of copenhagen university of copenhagen Enriched environment enhances -adrenergic signaling to prevent microglia inflammation by amyloid- Xu, Huixin; Rajsombath, Molly M; Weikop, Pia; Selkoe, Dennis J university of copenhagen Download date: 24. Oct. 2024 Xu, Huixin; Rajsombath, Molly M; Weikop, Pia; Selkoe, Dennis J Published in: EMBO Molecular Medicine Published in: EMBO Molecular Medicine DOI: 10.15252/emmm.201808931 Document version Publisher's PDF, also known as Version of record Document version Publisher's PDF, also known as Version of record Document version Publisher's PDF, also known as Version of record Enriched environment enhances -adrenergic signaling to prevent microglia inflammation by amyloid- Xu, Huixin; Rajsombath, Molly M; Weikop, Pia; Selkoe, Dennis J Abstract may still leave many elderly humans underserved. Population stud- ies increasingly suggest that lifelong experiences such as education, challenging occupation, exercise, and socialization may all improve cognitive reserve and provide some protection against the develop- ment of AD later in life (Nithianantharajah & Hannan, 2009; Then et al, 2015). Environmental enrichment (EE) is a rodent behavioral paradigm that can model the cognitive benefits to humans associated with intellectual activity and exercise. We recently discovered EE’s anti- inflammatory protection of brain microglia against soluble oligo- mers of human amyloid b-protein (oAb). Mechanistically, we report that the key factor in microglial protection by EE is chronically enhanced b-adrenergic signaling. Quantifying microglial morphol- ogy and inflammatory RNA profiles revealed that mice in standard housing (SH) fed the b-adrenergic agonist isoproterenol experi- enced similar protection of microglia against oAb-induced inflammation as did mice in EE. Conversely, mice in EE fed the b- adrenergic antagonist propranolol lost microglial protection against oAb. Mice lacking b1/b2-adrenergic receptors showed no protection of microglia by EE. In SH mice, quantification of nore- pinephrine in hippocampus and interstitial fluid showed that oAb disrupted norepinephrine homeostasis, and microglial-specific analysis of b2-adrenergic receptors indicated a decreased receptor level. Both features were rescued by EE. Thus, enhanced b-adre- nergic signaling at the ligand and receptor levels mediates potent benefits of EE on microglial inflammation induced by human Ab oligomers in vivo. Environmental enrichment (EE) is an animal housing paradigm aimed at understanding the biological mechanisms that build cogni- tive reserve (Nithianantharajah & Hannan, 2006; Pang & Hannan, 2013; Hannan, 2014), thereby providing insights for developing non-pharmaceutical therapeutic approaches to AD. In rodents, EE has been shown to ameliorate multiple AD-like neuronal pheno- types, including synaptic loss, impaired synaptic plasticity, decreased neurogenesis, and altered cognition (Lazarov et al, 2005; Cracchiolo et al, 2007; Herring et al, 2009; Li et al, 2013). Environ- mental enrichment provides resistance to neuronal/synaptic toxicity from exposure to soluble oligomers of amyloid b-protein (oAb), the most genetically and biologically well-validated pathogenic factor in AD (Selkoe & Hardy, 2016). A major feature of AD pathobiology that has barely been studied with regard to EE is the brain’s innate immune system. Changes in microglia contribute importantly to the AD phenotype both in human studies and in animal models (Zhang et al, 2013). Document license: CC BY Document license: CC BY Citation for published version (APA): Xu, H., Rajsombath, M. M., Weikop, P., & Selkoe, D. J. (2018). Enriched environment enhances -adrenergic signaling to prevent microglia inflammation by amyloid-. EMBO Molecular Medicine, 10(9), [e8931]. https://doi.org/10.15252/emmm.201808931 Download date: 24. Oct. 2024 Published online: August 9, 2018 Research Article Abstract Genome- wide association studies (GWAS) had identified over 20 AD-asso- ciated gene variants by 2016, and a majority were expressed by microglia (Villegas-Llerena et al, 2016). We recently showed for the first time that EE confers extensive anti-inflammatory effects on microglia in mice examining very early events in oAb toxicity (Xu et al, 2016). Elucidating the molecular mechanisms by which EE dampens oAb-induced microglial inflammation could not only support behavioral approaches but also identify key signaling paths that might provide new pharmacological targets to slow or prevent AD. Keywords Alzheimer’s disease; environmental enrichment; microglia; neuroinflammation; b-adrenergic signaling DOI 10.15252/emmm.201808931 \| Received 29 January 2018 \| Revised 12 July 2018 \| Accepted 16 July 2018 EMBO Mol Med (2018) e8931 Keywords Alzheimer’s disease; environmental enrichment; microglia; neuroinflammation; b-adrenergic signaling DOI 10.15252/emmm.201808931 \| Received 29 January 2018 \| Revised 12 July 2018 \| Accepted 16 July 2018 EMBO Mol Med (2018) e8931 Enriched environment enhances b-adrenergic signaling to prevent microglia inflammation by amyloid-b Huixin Xu1 , Molly M Rajsombath1, Pia Weikop2 & Dennis J Selkoe1,* 1 Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital & Harvard Medical School, Boston, MA, USA 2 Center for Translational Neuromedicine, University of Copenhagen, Copenhagen, Denmark Corresponding author. Tel: +1 617 525 5200; E-mail: dselkoe@bwh.harvard.edu EE prevents Ab microgliosis via b-AR Huixin Xu et al EE prevents Ab microgliosis via b-AR Huixin Xu et al reported to connect EE with NE in the DG of healthy mice, the NE system has been found to regulate various features of cognitive activity (Sara, 2015) that are strongly enhanced by EE. A previous study from our laboratory found that EE activates b2-adrenergic receptors (b2-AR) in neuronal synapses as an early part of its neuro- protective effect against oAb, suggesting links that b2-AR signaling has with both EE and oAb toxicity (Li et al, 2013). Microglia express b1- and b2-adrenergic receptors (Mori et al, 2002; Tanaka et al, 2002), and their activation by exogenous agonists causes changes in microglia morphology and suppression of the pro-inflammatory cascade induced by lipopolysaccharide stimulation (Hetier et al, 1991; Dello Russo et al, 2004; Markus et al, 2010; Qian et al, 2011). Furthermore, NE was shown to suppress microglial inflammation and increase phagocytosis of synthetic Ab42 fibrils in cell culture, while NE deficiency in APP transgenic mice enhanced brain tissue cytokine levels and exacerbated AD-like cytopathology (Heneka et al, 2002, 2006, 2010; Kalinin et al, 2007; Kong et al, 2010). between SH mice on isoproterenol and on plain water, although the exact amount of water consumed by individual mouse was untrace- able which may contribute to some variations among mice of the same treatment group. Also, no significant differences in activity level and body weight were observed. We first analyzed microglia morphology in SH mice fed isopro- terenol or not and then microinjected i.c.v with oAb-rich soluble cortical extracts (ADTBS) prepared from clinically and neuropatho- logically typical AD patients (Shankar et al, 2008). TBS extracts prepared identically from age-matched normal human cortex lack- ing oAb (Fig EV1) while containing similar basal levels of cytokines (Appendix Fig S1) served as the control injectates (designated Control-TBS). After 48 h (Xu et al, 2016), we immunostained contralateral hemisphere cryosections for P2ry12 and CD68 as microglial markers. The contralateral hemisphere was exposed to equal amounts of oAb as the ipsilateral hemisphere by the i.c.v. injections (Appendix Fig S2) and was therefore studied to avoid any confounding tissue injury effects from the ipsilateral injection site. EE prevents Ab microgliosis via b-AR Huixin Xu et al As described previously (Xu et al, 2016), we performed Z-stack confocal microscopy in the dentate gyrus (DG), the region with the most robust EE benefits and strongest morphological responses toward oAb shown by our previous work, and compressed the stacking images into one 2D image for comprehensive visualization of all microglial processes. We quantified microglia for their cell density (#microglia/mm2), circularity, solidity, %CD68 reactivity per microglia (defined as the percentile of P2ry12+ pixels of each microglia that are also CD68+), and #branches per microglia using ImageJ for unbiased morphometry. In the untreated SH mice exposed to oAb in vivo, we observed significantly decreased micro- glial density (vs. Control-TBS injections), increased microglial circu- larity and solidity, decreased #branches/microglia, and increased % CD68/microglia, all of which indicate a more inflammatory micro- glial state (Kettenmann et al, 2011). These results are entirely consistent with our recent report of i.c.v oAb effects in SH mice (Xu et al, 2016). In contrast, SH mice fed with isoproterenol for 4– 6 weeks showed significantly lower inflammatory changes in their microglial morphology on all the aforementioned measurements, as shown both by the original confocal images and Imaris 3D-recon- structed individual microglia cells (Fig 1A and C). Indeed, the microglial morphometry of the isoproterenol-fed SH mice exposed to oAb yielded similar results to those of plain water-fed EE mice exposed to oAb (Xu et al, 2016), indicating that isoproterenol treat- ment has microglial immunosuppressive capacity toward oAb simi- lar to that of EE. Isoproterenol-fed vs. untreated SH mice showed no significant differences in microglial morphology after injection of Control-TBS extracts (Fig 1B and C). Thus, the ADTBS-induced dif- ferences in microglial morphology described above are associated with oAb. Images with all channels displayed individually are included in Appendix Fig S3. Here, we report multiple lines of evidence that b-adrenergic signaling drives the anti-inflammatory benefits of EE on microglia early in the in vivo response to oAb isolated directly from human (AD) brain. By combining selective housing with neuropharmaco- logical treatments, we first show that a b-adrenergic receptor agonist mimics EE’s anti-inflammatory effects in wild-type mice housed in standard cages (SH) and exposed to intracerebroventric- ular (i.c.v) microinjections of human oAb. Conversely, an antago- nist to b-adrenergic receptors largely blocks such benefits of EE. Mice with germline knockouts of both b1- and b2-AR also lost EE’s anti-inflammatory protection of microglia, in accord with the mice fed propranolol. EE prevents Ab microgliosis via b-AR Huixin Xu et al Further, we find that, in SH mice, oAb significantly increases norepinephrine level in the dentate gyrus and decreases it in brain interstitial fluid, and it downregulates microglial b1/b2AR levels. We conclude that environmental enrichment upregulates hippocampal b-adrenergic signaling to provide robust protection of microglia against the inflammatory effects of human oAb oligomers. 2 of 17 EMBO Molecular Medicine e8931 \| 2018 ª 2018 The Authors Introduction The steady aging of the human population and a resultant rise in the prevalence of Alzheimer’s disease (AD) have made AD among the most common and feared diagnoses in medicine. Pharmaceuti- cal approaches have had very limited success to date, and even when a safe and effective disease-modifying agent is approved, the cost of its chronic administration and its complex delivery logistics Norepinephrine (NE) and b-adrenergic signaling are centrally involved in neuronal activity in cognition and, more recently, in microglial immune function. NE is released by noradrenergic neurons that innervate many brain regions, including the dentate gyrus (DG) where we observed the most robust influences of EE on microglia (Xu et al, 2016). Although no direct evidence has been EMBO Molecular Medicine e8931 \| 2018 1 of 17 ª 2018 The Authors. Published under the terms of the CC BY 4.0 license EMBO Molecular Medicine Published online: August 9, 2018 EMBO Molecular Medicine Published online: August 9, 2018 Prolonged oral administration of isoproterenol to SH mice prevents human oAb-induced microglia inflammation in vivo, mimicking the protection by EE In control SH mice, ADTBS caused significant decrease in microglial density; increase in circularity, solidity, and %CD68; and decrease in branching complexity in comparison with control-TBS (P < 0.05; P < 0.01; *P < 0.0001, N = 6) Isoproterenol treatment significantly rescued all above-mentioned morphological features (P < 0.05; P < 0.01; *P < 0.0001, N = 6). All quantitative data ar presented as mean SD. Exact P values are listed in Appendix Table S1. All statistical analysis were performed using multiple t-test (unpaired, do not assume equal SDs) with Holm-Sidak method to determine significance. A B B A C C Figure 1. Isoproterenol treatment successfully neutralizes oAb-induced microglia morphology change in SH mice. A, B Representative images from SH mice treated with isoproterenol vs. water and injected with ADTBS and control-TBS. When injected with ADTBS, SH mice treated with isoproterenol have microglia with significantly less inflammatory morphological features comparing to their water control. When injected with control-TBS, SH mice under both treatments showed minimal microglia inflammatory features. Red: P2ry12; green: CD68; blue: DAPI. Scale bar = 100 lm. Corresponding Imaris 3D-reconstructed images featuring two cells per condition are presented under each panel. The spatial relation between each two cells may be adjusted to fit in the frame with maximum magnification. Scale bar = 50 lm. C Quantification analysis of SH microglia morphology under different treatments. In control SH mice, ADTBS caused significant decrease in microglial density; increase in circularity, solidity, and %CD68; and decrease in branching complexity in comparison with control-TBS (P < 0.05; P < 0.01; *P < 0.0001, N = 6). A, B Representative images from SH mice treated with isoproterenol vs. water and injected with ADTBS and control-TBS. When injected with ADTBS, SH mice treated with isoproterenol have microglia with significantly less inflammatory morphological features comparing to their water control. When injected with control-TBS, SH mice under both treatments showed minimal microglia inflammatory features. Red: P2ry12; green: CD68; blue: DAPI. Scale bar = 100 lm. Corresponding Imaris 3D-reconstructed images featuring two cells per condition are presented under each panel. The spatial relation between each two cells may be adjusted to fit in the frame with maximum magnification. Scale bar = 50 lm. C Quantification analysis of SH microglia morphology under different treatments. Prolonged oral administration of isoproterenol to SH mice prevents human oAb-induced microglia inflammation in vivo, mimicking the protection by EE Microglia express significant levels of b1/b2AR, suggesting these receptors have functional roles in microglial biology. To examine the mechanism underlying EE’s strongly protective effect against the in vivo microglial reaction to oAb that we recently reported (Xu et al, 2016), namely EE significantly rescues microglial morphologi- cal changes and inflammatory RNA profile shifts induced by expo- sure to oAb, we asked whether prolonged activation of b1/b2AR signaling could mimic the immunomodulatory effects of EE on microglia. To achieve constant in vivo stimulation of b1/b2AR with- out causing stress to the animals, we administered isoproterenol, a non-selective b1/b2AR agonist, to 4-weeks SH mice continuously in their daily drinking water (0.1 g/l) for 4–6 weeks, the same period we have used for EE exposure. The compound is tasteless, and non- transparent water bottles were used for isoproterenol solution and plain water. No difference in water consumption was observed Next, we performed mRNA expression profiling by NanoString on microglia isolated by gradient fractionation and fluorescent-acti- vated cell sorting (FACS) after the i.c.v. injection of ADTBS. To provide a baseline for normalization and cross-comparison with minimal risk of identifying genes responding to patient-to-patient differences in the brain extract instead of to oAb, an aliquot of the same patient’s ADTBS extract immunodepleted (ID) of its Ab by the 4G8 monoclonal antibody was i.c.v. injected into littermate mice within each study. An Abx-42 ELISA developed in our laboratory 2 of 17 EMBO Molecular Medicine e8931 \| 2018 ª 2018 The Authors Published online: August 9, 2018 Huixin Xu et al EE prevents Ab microgliosis via b-AR EMBO Molecular Medicine A C B Figure 1. Isoproterenol treatment successfully neutralizes oAb-induced microglia morphology change in SH mice. A, B Representative images from SH mice treated with isoproterenol vs. water and injected with ADTBS and control-TBS. When injected with ADTBS, SH mice treated with isoproterenol have microglia with significantly less inflammatory morphological features comparing to their water control. When injected with control-TBS, SH mice under both treatments showed minimal microglia inflammatory features. Red: P2ry12; green: CD68; blue: DAPI. Scale bar = 100 lm. Corresponding Imar 3D-reconstructed images featuring two cells per condition are presented under each panel. The spatial relation between each two cells may be adjusted to fit in the frame with maximum magnification. Scale bar = 50 lm. C Quantification analysis of SH microglia morphology under different treatments. ª 2018 The Authors EE prevents Ab microgliosis via b-AR Huixin Xu et al EE prevents Ab microgliosis via b-AR Huixin Xu et al FACS-isolated 48 h after the i.c.v injections using CD11b+/ CD45Med/Ly6CLo/Ly6GLo as sorting markers, and their RNA samples were analyzed by NanoString nCounter Mouse-inflammatory (V2) CodeSet containing 249 inflammatory genes and six housekeeping genes. Only genes with higher-than-background expression were included in further analysis. Each individual microglial sample from the ADTBS-injected group was normalized to the average of the whole control-injected group (ID-ADTBS) for each of the two treat- ments (SH water and SH isoproterenol). Ratios were presented in the heat map after conversion to Log2 format. Positive values (red) indicate a relative increase in an mRNA due to oAb exposure, while negative ones (blue) indicate relative suppression by oAb. We found that untreated SH mice had significantly altered inflammatory gene mRNA profiles upon oAb exposure, while in striking contrast, isoproterenol-fed SH mice had a largely unaltered and even slightly immunosuppressive mRNA profile (Fig 2A), similar to what we have observed in EE mice maintained on regular drinking water (Xu et al, 2016). Twenty-nine genes that were significantly altered by oAb exposure in water-fed SH mice were significantly neutralized by the isoproterenol feeding (analyzed using the ADTBS/ID-ADTBS ratio; results after Holm–Sidak correction; Fig 2B). An additional 19 genes showed significant neutralization by isoproterenol only before the Holm–Sidak correction (Fig 2C). Raw NanoString values prior to their normalization are found in the Dataset EV1. No significant dif- ferences were observed between isoproterenol-treated and control SH mice that had no oAb injection, which is consistent with our previous observations in the EE vs. SH paradigm (Xu et al, 2016). were similar to those of SH mice on all of the aforementioned measures of individual microglial morphology, with decreased microglial density, increased microglial circularity and solidity, markedly decreased #branches/microglia, and much more increased %CD68 than untreated EE mice, again as shown both by the original confocal images and Imaris 3D-reconstructed microglia cells (Fig 3A and C). These data show that propranolol-fed EE mice have micro- glial morphologies corresponding to a more inflammatory state, i.e., a more “SH-like” state, upon oAb injection than do water-fed EE mice. Again, propranolol-treated vs. untreated EE mice showed no difference in microglia morphology upon injection of Control-TBS extract (Fig 3B and C), suggesting that the ADTBS-induced differences in morphology represent a response to oAb. Images with all channels displayed individually are included in Appendix Fig S4. EE prevents Ab microgliosis via b-AR Huixin Xu et al Next, mRNA profiling of FACS-purified microglia after ADTBS vs. oAb immunodepleted (ID)-TBS injection was performed on the NanoString nCounter Mouse-inflammatory (V2) CodeSet. The resul- tant heat map (Fig 4A) includes all genes with higher-than-back- ground expression and was generated following the same rules as those in Fig 2A (red indicates relative increase in expression by oAb over the ID control, while blue indicates relative suppression). Side- by-side comparison of the propranolol-fed and untreated EE mice revealed significantly altered inflammatory mRNA profiles upon oAb exposure by propranolol (Fig 4A). Indeed, 13 genes whose expression levels were successfully neutralized by EE upon oAb exposure were now significantly stimulated or suppressed in the propranolol-treated EE mice (analyzed using ADTBS/ID-ADTBS ratio; results after Holm–Sidak correction; Fig 4B), suggesting a more pro-inflammatory expression state. In addition, 19 other genes also fit into this category before but not after Holm–Sidak correction (Fig 4C). Raw NanoString values prior to normalization are included in the Dataset EV2. Like what we observed in isopro- terenol- vs. water-treated SH mice and in SH vs. EE mice, propra- nolol alone did not induce any significant microglial inflammatory gene profile shift. In summary, our results so far demonstrate that prolonged oral exposure to isoproterenol in SH mice can successfully protect against human oAb-induced microglial inflammation in vivo. The microglial responses to oAb in isoproterenol-treated SH mice are more “EE-like”, strongly suggesting that norepinephrine signaling helps mediate EE’s anti-inflammatory benefit for microglia. Prolonged oral exposure of EE mice to propranolol significantly diminishes the protective benefits of EE on microglia In summary, propranolol significantly blocked EE’s immunomod- ulatory benefits to microglia upon oAb exposure, rendering the EE microglia more “SH-like”. Collectively, our data strongly suggest a quantitatively important and indispensable involvement of nora- drenergic signaling in mediating the EE-microglia effects. To assess further whether b1/b2AR signaling is required for EE’s anti-inflammatory benefits, we fed EE mice for 4–6 weeks with propranolol (0.4 g/l in the drinking water), a non-selective bAR antagonist shown to block EE’s benefits for neuronal function in previous work on oAb (Li et al, 2013). No sign of decreased physi- cal activity, body weight, and food/water intake was observed in the propranolol-treated EE mice. Similar to the case of isoproterenol treatment, the exact amount of water consumed by individual mouse was untraceable which may contribute to some variations among mice of the same treatment group. In the two NanoString profile studies in SH isoproterenol vs. water paradigm and EE propranolol vs. water paradigm, we high- lighted Ccl2, Ccl3, Ccl4, Tnf, and Cxcl10 because they were among the most significantly altered genes under both paradigms and are well studied for their biological functions in inflammation, making them reliable and robust representatives to evaluate microglial inflammatory status in this and future studies. To confirm their increase in response to oAb stimulation at the protein level, we quantified the levels of CCL2, CCL3, CCL4, and CXCL10 in ADTBS- vs. ID-ADTBS-injected brain tissues by ELISA and observed signifi- cant increases in all four cytokines upon ADTBS exposure (Fig EV2). TNFa was not detectable at the protein level with our 1 pg/ml detection limit. Using the same analytical system as described in the prior section, we first analyzed microglial morphology from the EE mice fed propranolol or not and then injected i.c.v. with oAb-rich AD- TBS. As expected, water-fed EE mice showed prevention of oAb- induced effects on microglial morphology in the contralateral hippocampus DG area, as shown both in the original confocal images and in Imaris 3D-reconstructed microglia cells (Fig 3A and C), as evidenced by minimal changes in cell density (#microglia/ mm2), circularity, solidity, and branching complexity, as well as a modest %CD68 increase. 4 of 17 EMBO Molecular Medicine e8931 \| 2018 Prolonged oral administration of isoproterenol to SH mice prevents human oAb-induced microglia inflammation in vivo, mimicking the protection by EE In control SH mice, ADTBS caused significant decrease in microglial density; increase in circularity, solidity, and %CD68; and decrease in branching complexity in comparison with control-TBS (P < 0.05; P < 0.01; *P < 0.0001, N = 6). Isoproterenol treatment significantly rescued all above-mentioned morphological features (P < 0.05; P < 0.01; *P < 0.0001, N = 6). All quantitative data are presented as mean SD. Exact P values are listed in Appendix Table S1. All statistical analysis were performed using multiple t-test (unpaired, do not assume equal SDs) with Holm-Sidak method to determine significance. 3D reconstructed images featuring two cells per condition are presented under each panel. The spatial relation between each two cells may be adjusted to fit in the frame with maximum magnification. Scale bar = 50 lm. C Quantification analysis of SH microglia morphology under different treatments. In control SH mice, ADTBS caused significant decrease in microglial density; increase in circularity, solidity, and %CD68; and decrease in branching complexity in comparison with control-TBS (P < 0.05; P < 0.01; *P < 0.0001, N = 6). Isoproterenol treatment significantly rescued all above-mentioned morphological features (P < 0.05; P < 0.01; *P < 0.0001, N = 6). All quantitative data are presented as mean SD. Exact P values are listed in Appendix Table S1. All statistical analysis were performed using multiple t-test (unpaired, do not assume equal SDs) with Holm-Sidak method to determine significance. (Yang et al, 2013) indicated that ID-ADTBS had < 4% of the total Ab42 in the original ADTBS (Fig EV1) while having no significant differences in endogenous human cytokines (Appendix Fig S2), indicating the inflammatory differences we observed are Ab- induced. The microglia from a 3 mm cubic block of the contralateral hemisphere directly symmetrical to the ipsilateral injection site were EMBO Molecular Medicine e8931 \| 2018 3 of 17 EMBO Molecular Medicine Published online: August 9, 2018 EMBO Molecular Medicine Published online: August 9, 2018 ª 2018 The Authors Prolonged oral exposure of EE mice to propranolol significantly diminishes the protective benefits of EE on microglia In contrast, propranolol-fed EE mice showed significant morphological changes upon oAb exposure that We then attempted to confirm our findings by using a more puri- fied oAb preparation from human brain that lacks small molecules (e.g., glutamate and drugs), the brain levels of which could vary from patient to patient. For this confirmatory step, we adopted a ª 2018 The Authors Published online: August 9, 2018 Huixin Xu et al EE prevents Ab microgliosis via b-AR g EMBO Molecular Medicine A A A B C Figure 2. Isoproterenol treatment significantly rescues oAb-induced microglial inflammatory gene profile shift in SH mice. A Heat map of all microglial inflammatory genes with expression level above background cutoff by NanoString nCounter analysis. All data are presented by Log2 (ADTBS/ID ratio). B, C Forty-seven genes are significantly rescued by isoproterenol treatment among 140 genes actively expressed by microglia. Twenty-nine of them are significant (P < 0.01) with Holm–Sidak correction for multi-comparison (B), and 19 of them are significant (P < 0.01) without correction (C). N = 6. All data are presented as mean SD. Exact P values are listed in Appendix Table S2. C B C B gure 2. Isoproterenol treatment significantly rescues oAb-induced microglial inflammatory gene profile shift in SH mice. nflammatory genes with expression level above background cutoff by NanoString nCounter analysis. All data are presented by A Heat map of all microglial inflammatory genes with expression level above background cutoff by NanoString nCounter analysis. All data are presented by Log2 (ADTBS/ID ratio). B, C Forty-seven genes are significantly rescued by isoproterenol treatment among 140 genes actively expressed by microglia. Twenty-nine of them are significant (P < 0.01) with Holm–Sidak correction for multi-comparison (B), and 19 of them are significant (P < 0.01) without correction (C). N = 6. All data are presented as mean SD. Exact P values are listed in Appendix Table S2. Log2 (ADTBS/ID ratio). B, C Forty-seven genes are significantly rescued by isoproterenol treatment among 140 genes actively expressed by microglia. Twenty-nine of them are significant (P < 0.01) with Holm–Sidak correction for multi-comparison (B), and 19 of them are significant (P < 0.01) without correction (C). N = 6. All data are presented as mean SD. Exact P values are listed in Appendix Table S2. Prolonged oral exposure of EE mice to propranolol significantly diminishes the protective benefits of EE on microglia EMBO Molecular Medicine e8931 \| 2018 5 of 17 ª 2018 The Authors EMBO Molecular Medicine Published online: August 9, 2018 EE prevents Ab microgliosis via b-AR Huixin Xu et al A C B A B A B B A C Figure 3. Propranolol treatment significantly diminishes EE’s immunosuppressive effects on microglia against oAb-induced morphology change. A, B Representative images from EE mice treated with propranolol vs. water and injected with TBS extract from ADTBS and control-TBS. When injected with ADTBS, EE mice treated with propranolol have microglia with significant inflammatory morphological features, while their water controls showed much more physiologica microglia. When injected with control-TBS, EE mice under both treatments showed minimal microglia inflammatory features. Red: P2ry12; green: CD68; blue: DAPI. Scale bar = 100 lm. Corresponding Imaris 3D-reconstructed images featuring two cells per condition are presented under each panel. The spatial relation between each two cells may be adjusted to fit in the frame with maximum magnification. Scale bar = 50 lm. C Quantification analysis of EE microglia morphology under different treatments. Propranolol-treated EE mice had significantly decreased microglial density, increased microglia circularity, solidity, and %CD68, while decreased branching complexity after oAb injection comparing to their water-treated controls (P < 0.05 P < 0.01; P < 0.001; *** P < 0.0001, N = 6). All quantitative data are presented as mean SD. Exact P values are listed in Appendix Table S3. All statistical analysis were performed using multiple t-test (unpaired, do not assume equal SDs) with Holm-Sidak method to determine significance. C C C Figure 3. Propranolol treatment significantly diminishes EE’s immunosuppressive effects on microglia against oAb-induced morphology change. gure 3. Propranolol treatment significantly diminishes EE’s immunosuppressive effects on microglia against oAb-induced m treatment significantly diminishes EE’s immunosuppressive effects on microglia against oAb-induced morphology change. C Quantification analysis of EE microglia morphology under different treatments. Propranolol-treated EE mice had significantly decreased microglial density, increased microglia circularity, solidity, and %CD68, while decreased branching complexity after oAb injection comparing to their water-treated controls (P < 0.05; P < 0.01; P < 0.001; ** P < 0.0001, N = 6). All quantitative data are presented as mean SD. Exact P values are listed in Appendix Table S3. All statistical analysis were performed using multiple t-test (unpaired, do not assume equal SDs) with Holm-Sidak method to determine significance. Prolonged oral exposure of EE mice to propranolol significantly diminishes the protective benefits of EE on microglia 6 of 17 EMBO Molecular Medicine e8931 \| 2018 ª 2018 The Authors Published online: August 9, 2018 Huixin Xu et al EE prevents Ab microgliosis via b-AR EMBO Molecular Medicine A A B C Figure 4. ª 2018 The Authors EMBO Molecular Medicine e8931 \| 2018 7 of B C Figure 4. C B B C C EMBO Molecular Medicine e8931 \| 2018 7 of 17 EMBO Molecular Medicine e8931 \| 2018 7 of 17 ª 2018 The Authors EMBO Molecular Medicine Published online: August 9, 2018 EE prevents Ab microgliosis via b-AR Huixin Xu et al EE prevents Ab microgliosis via b-AR Huixin Xu et al Figure 4. Propranolol treatment significantly diminishes EE’s immunosuppressive effects on microglia against oAb-induced microglial inflammatory gene profile shift. A Heat map of all microglial inflammatory genes with expression level above background cutoff by NanoString nCounter analysis. All data are presented by Log2 (ADTBS/ID ratio). B, C Thirty-two genes are significantly altered by propranolol treatment among 160 genes actively expressed by microglia. Thirteen of them are significant (P < 0.01) with Holm–Sidak correction for multi-comparison (B), and 19 of them are significant (P < 0.01) without correction (C). N = 6. All data are presented as mean SD. Figure 4. Propranolol treatment significantly diminishes EE’s immunosuppressive effects on microglia against oAb-induced microglial inflammatory gene profile shift. Figure 4. Propranolol treatment significantly diminishes EE’s immunosuppressive effects on microglia against oAb-induc profile shift. A Heat map of all microglial inflammatory genes with expression level above background cutoff by NanoString nCounter analysis. All data are presented by Log2 (ADTBS/ID ratio). B, C Thirty-two genes are significantly altered by propranolol treatment among 160 genes actively expressed by microglia. Thirteen of them are significant (P < 0.01) with Holm–Sidak correction for multi-comparison (B), and 19 of them are significant (P < 0.01) without correction (C). N = 6. All data are presented as mean SD. Exact P values are listed in Appendix Table S4. (ADTBS/ID ratio). B, C Thirty-two genes are significantly altered by propranolol treatment among 160 genes actively expressed by microglia. Thirteen of them are significant (P < 0.01) with Holm–Sidak correction for multi-comparison (B), and 19 of them are significant (P < 0.01) without correction (C). N = 6. All data are presented as mean SD. Exact P values are listed in Appendix Table S4. Prolonged oral exposure of EE mice to propranolol significantly diminishes the protective benefits of EE on microglia branching complexity, and increased CD68+ lysosomal alteration) upon ADTBS exposure vs. control-TBS in the DG of Adrb1/2KO SH mice. In stark contrast to our earlier findings in wild-type mice, no quantifiable difference between SH and EE environments was observed in Adrb1/2KO mice for all aforementioned morphological parameters upon in vivo oAb exposure, as shown both by the original confocal images and by Imaris 3D-reconstructed microglia cells, indi- cating a complete loss of EE’s protection against microglial inflamma- tion (Fig 6A and B). Images with all channels displayed individually are included in Appendix Fig S4. Similar morphological changes were also observed in CA of hippocampus (Fig EV5), but without obvious changes in microglial density, which is also consistent with that of WT SH mice as reported in our previous work (Xu et al, 2016). previously published oAb purification protocol that relies on using size-exclusion chromatography (SEC) to enrich oAb in the “void volume” fraction during a quantitative removal of small molecules, followed by 37°C incubation of that fraction to dissociate high MW Ab oligomers to lower MW oAb comprising dimers and moderately larger oligomers, which we have found to have particularly high bioactivity on microglia and neurons in vivo (Yang et al, 2017). To ensure the injected material had a physiological background, all incubated oAb-rich solutions were lyophilized and reconstituted in sterile phosphate-buffered saline (PBS), pH 7.4, before in vivo injec- tions. The reconstituted samples were quantified using an Abx-42 ELISA. Subsequent adjustment of the reconstitution volume was done so that each mouse received ~4 pg of human Abx-42 in vivo, a dose we found to induce significant increases of key cytokines in SH mice by 24 h after i.c.v. injection. Selective immunodepletion of Ab successfully removed the bioactivity of this void volume-derived oAb, rendering it indistinguishable from blank vehicle (PBS; Fig EV3). Next, we performed i.c.v. injections of the SEC-purified human brain low MW oAb fractions (vs. PBS) into EE vs. SH Adrb1/2KO mice and analyzed the expression levels of the top five cytokines (Ccl2, Ccl3, Ccl4, Cxcl10, and Tnf) by qPCR. oAb now produced indistinguishable pro-inflammatory responses in both SH and EE KO mice (Fig 6C). Collectively, the morphometry and expression analy- ses indicate that the lifelong absence of b1/b2AR leads to almost complete loss of the beneficial modulation of microglial phenotype by EE, consistent with our earlier observations in EE mice chroni- cally treated with propranolol. Quantification of norepinephrine in mice reveals disrupted NE signaling in SH but not EE mice We quantified norepinephrine (NE) in the DG of wild-type mice kept in SH or EE. Knowledge of NE properties in the mouse dentate gyrus during EE is lacking. We first measured NE levels in dissected whole DG tissue. The DG was rapidly dissected on ice and snap-frozen until analysis by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The results showed that when the mice were injected i.c.v. with the SEC-purified oAb (vs. PBS), a small (~20%) but highly significant increase in DG NE level of SH mice was observed at 24 h, whereas no detectable change in NE occurred in EE mice (Fig 7A). Prolonged oral exposure of EE mice to propranolol significantly diminishes the protective benefits of EE on microglia Using this SEC-isolated, low MW Ab oligomer preparation and its vehicle serving as the negative control, we observed microglial acti- vation patterns consistent with those obtained from the in vivo stud- ies of oral isoproterenol or propranolol treatment in SH mice or EE mice using the whole brain TBS extracts. Specifically, transcript levels of the five cytokines highlighted by NanoString in both our prior treatment paradigms (above) were quantified by qPCR. Isopro- terenol-treated SH mice showed significant prevention of microglial inflammation upon this SEC-purified oAb injection, whereas propra- nolol-treated EE mice lost their protection (Fig 5A and B). Note that with this purified material, we observed much more robust activa- tion of all five genes than with the unfractionated ADTBS, possibly due to the enrichment and the reduced influence of other brain- derived molecules. Further NanoString analysis on those samples confirmed that the SEC-purified oAb induces consistent gene profile patterns like ADTBS vs. ID-ADTBS (Fig EV4). 8 of 17 EMBO Molecular Medicine e8931 \| 2018 ª 2018 The Authors b1/b2AR germline knockout mice fail to gain EE-mediated protection of microglia against oAb SEC-purified AD brain extract can recapitulate the phenotypes observed using straight AD brain extract in SH and EE mice orally treated with isoproterenol or propranolol. A In SH mice, five top cytokines highlighted by NanoString screening are significantly increased by injection of SEC separated oAb vs. PBS, measured by qPCR. Isoproterenol treatment successfully neutralizes such inflammatory responses. B In EE mice treated with propranolol, the same five top cytokines are significantly increased by injection of SEC separated oAb vs. PBS, measured by qPCR. Control EE mice showed complete rescue consistent with previous studies. Data information: All data are presented as mean SD. P < 0.01; P < 0.001; P < 0.0001; *P < 0.00001. N = 6. Exact P values are listed in Appendix Table S5. All statistical analysis were performed using multiple t-test (unpaired, do not assume equal SDs) with Holm-Sidak method to determine significance. xtract can recapitulate the phenotypes observed using straight AD brain extract in SH and EE mice orally treated with ol, the same five top cytokines are significantly increased by injection of SEC separated oAb vs. PBS, measured by qPCR. Control EE sistent with previous studies. Data information: All data are presented as mean SD. P < 0.01; P < 0.001; P < 0.0001; **P < 0.00001. N = 6. Exact P values are listed in Appendix Table S5. All statistical analysis were performed using multiple t-test (unpaired, do not assume equal SDs) with Holm-Sidak method to determine significance. Data information: All data are presented as mean SD. P < 0.01; P < 0.001; P < 0.0001; **P < 0.00001. N = 6. Exact P values are listed in Appendix Table S5. All statistical analysis were performed using multiple t-test (unpaired, do not assume equal SDs) with Holm-Sidak method to determine significance. mouse from the same housing environment (SH or EE), and both these mice were attached simultaneously to a single shared micro- dialysis perfusion pump. All oAb-injected mice that had been living in either SH or EE housing had their NE levels normalized to the average NE level of all PBS-injected mice from that same housing state, thereby generating a percentage. Mice with any bleeding noticeable at the microdialysis surgical site prior to probe insertion were excluded. Strikingly, we found that, in SH mice, i.c.v. ª 2018 The Authors EMBO Molecular Medicine e8931 \| 2018 9 of 17 b1/b2AR germline knockout mice fail to gain EE-mediated protection of microglia against oAb Next, we sampled interstitial fluid (ISF) from the DG by in vivo microdialysis to specifically quantify extracellular NE levels. The mice underwent implantation of a microdialysis guide cannula into the hippocampal dentate gyrus contralateral to the side of the i.c.v. injection of purified human oAb or PBS. ISF samples were collected 24 h after injection at 1-h intervals using a push-only system with a 1-mm-long, 6 kDa molecular weight cutoff (MWCO) dialysis membrane and 1 ll/min perfusion of artificial CSF (Fig 7B). ISF samples were collected at 4°C and then snap-frozen until later anal- ysis by HPLC-ECD. The room was kept quiet with minimal activities during ISF sampling to avoid confounding effects on norepinephrine production. To minimize any technical differences during microdial- ysis, each oAb-injected mouse was paired with a PBS-injected To confirm the apparently critical role of functional noradrenergic receptor signaling in mediating the EE effects against microglial oAb toxicity, we acquired from Jackson Laboratory mice with a germline global b1/b2AR deletion (homozygous null for both Adrb1 and Adrb2 genes, referred to here as Adrb1/2KO). The mice were bred in-house and exposed to EE starting at 4 weeks of age for a total of 4–6 weeks, identical to the EE paradigm we conducted above using wild-type mice. At the end of EE, the mice were given ADTBS or control-TBS by i.c.v. microinjection. Brains were harvested and analyzed for microglial morphology as before. We observed statistically significant signs of inflammation (de- creased microglial density, increased circularity/solidity, decreased 8 of 17 EMBO Molecular Medicine e8931 \| 2018 ª 2018 The Authors Huixin Xu et al EE prevents Ab microgliosis via b-AR EMBO Molecular Medicine Published online: August 9, 2018 Huixin Xu et al EE prevents Ab microgliosis via b-AR Published online: August 9, 2018 Published online: August 9, 2018 EMBO Molecular Medicine A B Figure 5. SEC-purified AD brain extract can recapitulate the phenotypes observed using straight AD brain extract in SH and EE mice orally treated with soproterenol or propranolol. A In SH mice, five top cytokines highlighted by NanoString screening are significantly increased by injection of SEC separated oAb vs. PBS, measured by qPCR. Isoproterenol treatment s ccessf ll ne trali es s ch inflammator responses A A B B Figure 5. SEC-purified AD brain extract can recapitulate the phenotypes observed using straight AD brain extract in SH and EE mice orally treated with isoproterenol or propranolol. Figure 5. rmline knockout of b1 and b2AR (Adrb1/2 KO) leads to loss of EE protection on microglia with acute oAb exposure. Pro-inflammatory microglial activities are believed to produce various detrimental consequences in the brain and help promote neurodegeneration. One very recent study demonstrated that inflammatory microglia promote Ab aggregation in vivo by releasing ASC (apoptosis-associated speck-like protein containing CARD) specks into extracellular space, offering yet another strong piece of evidence for the destructive power of microglial inflammation (Venegas et al, 2017). The recent identification of microglial gene variants contributing to late-onset AD in a GWAS study of 85,000+ subjects further highlighted the importance of microglia in AD pathogenesis (Sims et al, 2017). The molecular mechanisms driving EE’s immunomodulation of microglia, once identified, could inform the development of synergistic behavioral and pharmacological approaches to downregulate microglial inflammation and neutralize its damage, but, until the current study, the responsible biological mechanism remained known. Lastly, we tested whether the cAMP/PKA pathway, a common signaling pathway utilized by b1/b2AR, mediates the downstream effects. We inhibited cAMP and PKA pathways by one cAMP inhi- bitor (cAMP-Rp, 50 lM) and two PKA inhibitors (KT5720, 100 nM; PKA inhibitor fragment (6-22) amide (marked as PI (6-22)), 100 nM) in primary microglia cultures treated with oAb and isopro- terenol (100 lM). All doses were chosen to be within fivefold of the compound’s IC50 without any cytotoxicity. The cells were harvested after 4 h. Because our culturing system (20,000 cells/well in 150 ll culture media) does not yield sufficient amount of cytokines at protein level to be detected by ELISA, we analyzed the treated primary microglia by qPCR for the expression of the five cytokines highlighted by our previous NanoString analysis (Ccl2, Ccl3, Ccl4, Cxcl10, and Tnf). We found that 10 pg of oAb induced significant increases of all five cytokines by 4 h in the primary cultures, and isoproterenol had significant rescue effects. cAMP-Rp significantly diminished the anti-inflammatory effects of isoproterenol in Tnf expression but not as much for the other four cytokines. Both PKA inhibitors failed to reverse any isoproterenol effects (Fig 7F). The findings suggest that the downstream signaling of b1/b2AR’s anti- inflammatory effects on oAb-stimulated microglia is only partially cAMP-dependent and not PKA-independent. Here, we have identified b-adrenergic signaling as the apparent key mediator of EE’s highly consistent benefit of preventing oAb- induced microglial inflammation in the living brain. rmline knockout of b1 and b2AR (Adrb1/2 KO) leads to loss of EE protection on microglia with acute oAb exposure. Corresponding Imaris 3D-reconstructed images featuring two cells per condition are presented under each panel. The spatial relation between each two cells may be adjusted to fit in the frame with maximum magnification. Scale bar = 50 lm. B Quantification analysis of microglia morphology in DG. Both SH and EE Adrb1/2KO mice had significantly decreased microglial density, increased microglia circularity, solidity, and %CD68, while decreased branching complexity after oAb injection comparing to their controls (P < 0.05; P < 0.01; P < 0.001, N = 5). Exact P values are listed in Appendix Table S6. p p y j g l B Quantification analysis of microglia morphology in DG. Both SH and EE Adrb1/2KO mice had significantly decreased microglial density, increased microglia circularity, solidity, and %CD68, while decreased branching complexity after oAb injection comparing to their controls (P < 0.05; P < 0.01; P < 0.001, N = 5). Exact P values are listed in Appendix Table S6. p p y j g l B Quantification analysis of microglia morphology in DG. Both SH and EE Adrb1/2KO mice had significantly decreased microglial density, increased microglia circularity, solidity, and %CD68, while decreased branching complexity after oAb injection comparing to their controls (P < 0.05; P < 0.01; P < 0.001, N = 5). Exact P values are listed in Appendix Table S6. pp C qPCR analysis of SH and EE Adrb1/2KO mice microglia after i.c.v. injection of SEC separated oAb vs. PBS. Both SH and EE mice showed increase in cytokine expression with oAb injection. N = 4. No significant difference between SH and EE is observed. C qPCR analysis of SH and EE Adrb1/2KO mice microglia after i.c.v. injection of SEC separated oAb vs. PBS. Both SH and EE mice showed increase in cytokine expression with oAb injection. N = 4. No significant difference between SH and EE is observed. e data are presented as mean SD. All statistical analysis were performed using multiple t-test (unpaired, do not assume equal SDs) rmine significance. Data information: All quantitative data are presented as mean SD. All statistical analysis were performed using multiple t-test (un with Holm-Sidak method to determine significance. Data information: All quantitative data are presented as mean SD. All statistical analysis were performed using multiple t-test (unpaired, do not assume equal SDs) with Holm-Sidak method to determine significance. rmline knockout of b1 and b2AR (Adrb1/2 KO) leads to loss of EE protection on microglia with acute oAb exposure. Our findings from a combined comparison of housing paradigms and pharmaco- logical treatments indicate that prolonged daily exposure to isopro- terenol successfully prevented SH mice from developing microglial inflammation upon exposure of oAb, while prolonged exposure of EE mice to propranolol significantly diminished EE’s protective effects and allowed much greater microglial inflammation from oAb even though the EE mice treated with propranolol still maintained their daily activity and had a body habitus indistinguishable from their untreated littermates. These data clearly demonstrate that a very substantial part of EE’s immunosuppression on microglia is driven via b1/b2AR signaling. Blocking b1/b2AR signaling prevented acquisition of microglial immuno-protection. None of our drug treatments or the EE paradigm alone induced any significant microglial inflammatory gene profile changes when oAb was not present, indicating that the regulation of microglia via b1/b2AR signaling is more relevant to microglial inflammatory status than to physiological status. Importantly, these pharmacological results were confirmed through genetic manipulation when we observed a consistent loss of EE benefits on oAb-exposed microglia in mice completely lacking functional b1/b2ARs. While a microglia-specific Adrb KO mouse line is not yet available, the recently identified microglia-specific marker, Sall1, and the successful downregulation of other microglial genes via Sall1-Cre (Buttgereit et al, 2016) indi- cate that future research could potentially develop mice with selec- tive deletion of microglial b-ARs in order to further dissect the functions of b-adrenergic signaling in the EE-mediated downregula- tion of microglial inflammation. rmline knockout of b1 and b2AR (Adrb1/2 KO) leads to loss of EE protection on microglia with acute oAb exposure. A Representative images from SH and EE Adrb1/2KO mice injected with ADTBS and control-TBS. When injected with ADTBS, both SH and EE mice have microglia with significant inflammatory morphological features in DG and CA. When injected with control-TBS, both SH and EE mice showed minimal microglia inflammatory features. Red: P2ry12; green: CD68; blue: DAPI. Scale bar = 100 lm. Corresponding Imaris 3D-reconstructed images featuring two cells per condition are presented under each panel. The spatial relation between each two cells may be adjusted to fit in the frame with maximum magnification. Scale bar = 50 lm. B Quantification analysis of microglia morphology in DG. Both SH and EE Adrb1/2KO mice had significantly decreased microglial density, increased microglia circularity, solidity, and %CD68, while decreased branching complexity after oAb injection comparing to their controls (P < 0.05; P < 0.01; P < 0.001, N = 5). Exact P values are listed in Appendix Table S6. A Representative images from SH and EE Adrb1/2KO mice injected with ADTBS and control-TBS. When injected with ADTBS, both SH and EE mice have microglia with significant inflammatory morphological features in DG and CA. When injected with control-TBS, both SH and EE mice showed minimal microglia inflammatory features. Red: P2ry12; green: CD68; blue: DAPI. Scale bar = 100 lm. Corresponding Imaris 3D-reconstructed images featuring two cells per condition are presented under each panel. The spatial relation between each two cells may be adjusted to fit in the frame with maximum magnification. Scale bar = 50 lm. B Q tifi ti l i f i li h l i DG B th SH d EE Ad b1/2KO i h d i ifi tl d d i li l d it i d i li i l it A Representative images from SH and EE Adrb1/2KO mice injected with ADTBS and control-TBS. When injected with ADTBS, both SH and EE mice have microglia with significant inflammatory morphological features in DG and CA. When injected with control-TBS, both SH and EE mice showed minimal microglia inflammatory features. Red: P2ry12; green: CD68; blue: DAPI. Scale bar = 100 lm. Corresponding Imaris 3D-reconstructed images featuring two cells per condition are presented under each panel. The spatial relation between each two cells may be adjusted to fit in the frame with maximum magnification. Scale bar = 50 lm. features. Red: P2ry12; green: CD68; blue: DAPI. Scale bar = 100 lm. ª 2018 The Authors EMBO Molecular Medicine e8931 \| 2018 11 of 17 Figure 6. Germline knockout of b1 and b2AR (Adrb1/2 KO) leads to loss of EE protection on microglia with acute Figure 6. Germline knockout of b1 and b2AR (Adrb1/2 KO) leads to loss of EE protection on microglia with acute oAb exp b1/b2AR germline knockout mice fail to gain EE-mediated protection of microglia against oAb human oAb induced a significant ~50% decrease in extracellular (ISF) NE level, but in EE mice, no effect of oAb was observed (Fig 7C). from four contralateral brain blocks as described above, we were able to show that oAb injection caused a complete loss of the b2AR band by Western blot in SH mice but not in EE mice (Fig 7E; raw blot images in Appendix Fig S6), confirming our findings at the RNA level. No b1AR antibody was found to perform under these conditions, but because of the similarities between b1AR and b2AR, we speculate that b1AR should follow the same type of change as b2AR. Collectively, these data point to a substantially disrupted NE signaling system in SH mice exposed to human oAb, as shown by increased total tissue [NE] but lowered free extracellular [NE] and decreased availability of b1/b2AR in the dentate gyrus. In striking contrast, EE mice maintained stable NE levels in both whole tissue and ISF and unchanged b1- and b2-AR transcript levels upon oAb injection, indicating that under environmental enrichment, unal- tered noradrenergic homeostasis is associated with the prevention of microglial inflammation. Furthermore, qPCR analysis of the post-i.c.v. injection contralat- eral microglia indicates that both b1-AR and b2-AR transcription levels decreased ~50% in SH mice after oAb injection but did not change at all in EE mice (Fig 7D). We were able to identify one b2AR antibody of a specific lot that successfully detects microglial b2AR despite the very low protein yields. When pooling microglia harvested EMBO Molecular Medicine e8931 \| 2018 9 of 17 ª 2018 The Authors EMBO Molecular Medicine Published online: August 9, 2018 EE prevents Ab microgliosis via b-AR Huixin Xu et al A B C A A A A B C Figure 6. 10 of 17 EMBO Molecular Medicine e8931 \| 2018 ª 2018 The A B C Figure 6. 10 of 17 EMBO Molecular Medicine e8931 \| 2018 ª 2018 T B B C Figure 6. C C 10 of 17 EMBO Molecular Medicine e8931 \| 2018 10 of 17 EMBO Molecular Medicine e8931 \| 2018 ª 2018 The Authors Huixin Xu et al EE prevents Ab microgliosis via b-AR Published online: August 9, 2018 Huixin Xu et al EE prevents Ab microgliosis via b-AR Published online: August 9, 2018 Huixin Xu et al EE prevents Ab microgliosis via b-AR Published online: August 9, 2018 EMBO Molecular Medicine Discussion Mounting evidence in humans suggests potential cognitive benefits detectable in old age of intellectually challenging and physically active lifestyles. With the widespread availability of disease-modifying drugs for Alzheimer’s disease still years away, understanding the biological mechanisms underlying lifestyle benefits could lead to valuable addi- tional approaches to lessen the global crisis of age-related dementia and could simultaneously enable discoveries of new pharmacological targets other than the key pathological proteins currently being targeted in AD clinical trials. Environmental enrichment is a labora- tory adaptation to model effects of human lifestyle. It has been studied in rodent models for its potential benefit in ameliorating neuropatho- logical features resembling those of AD. We recently documented potent immunomodulatory effects of EE in protecting microglia from the early phase of oAb-induced inflammation in vivo, thereby expand- ing EE’s known benefits from purely neuronal to the realm of innate immunity (Xu et al, 2016). One other study offered support for EE- microglia effects by reporting enhanced microglial clearance of Ab plaques by EE in 5 × FAD mice, revealing another feature of EE’s modulation of microglia when the cells are chronically exposed to excessive levels of oAb (Ziegler-Waldkirch et al, 2018). EMBO Molecular Medicine e8931 \| 2018 11 of 17 ª 2018 The Authors EMBO Molecular Medicine Published online: August 9, 2018 EE prevents Ab microgliosis via b-AR Huixin Xu et al A D F E B C B C A C A B D D E E F Figure 7. Direct quantification of norepinephrine (NE) showed disrupted NE homeostasis by oAb in SH mice but not in EE mice. e 7. Direct quantification of norepinephrine (NE) showed disrupted NE homeostasis by oAb in SH mice but not in EE mice. Whole tissue NE quantification of the DG shows significant increase in NE level in SH mice injected with oAb vs. PBS. EE mice showe NE level with oAb injection vs. PBS. P = 0.0083, N = 6. g ( ) D qPCR analysis of microglial Adrb1 and Adrb2 RNA level showed significant decrease in both Adrb1 and Adrb2 transcription in SH mice after oAb injection. EE fully rescued the phenotype. **P < 0.0001 (Adrb1 P = 4.1E-05; Adrb2 P = 8.7E-07). N = 6. p yp ( ) E Western blots of b2AR in SH and EE mice +/ oAb injection. Discussion Taken together, our data demonstrate for the first time that EE has a direct impact on the NE dynamics in hippocampal tissue and that EE suppresses excessive microglial inflammation in the early phase of oAb expo- sure by maintaining the strength of b-adrenergic signaling in micro- glia. We speculate that oAb toxicity may impair the NE release/ reuptake machinery in SH mice, leading to increased trapping of NE inside cells and a significant decrease in the adjacent extracellular space, and hence a loss of anti-inflammatory NE signal. The distur- bance of NE homeostasis, together with a lowered b-AR level which exacerbates the loss of NE signal for microglia, renders the micro- environment in the SH hippocampus exposed to oAb as pro-inflam- matory. Whether the loss of b1/b2AR is a result of disrupted membrane receptor recycling could not be determined due to techni- cal limitations in antibody sensitivity and protein amounts. However, our previous study in neurons successfully demonstrated increased b2AR internalization induced by oAb (Li et al, 2013), suggesting it is possible that in microglia the dynamics of b1/b2AR follows the same pattern. In this work, we solely used oAb isolated directly from human (AD) brain tissue. Various sources of Ab oligomers have been used in past studies, the vast majority of them being synthetic. Whether synthetic oAb assemblies faithfully represent the pathological protein conformers accumulating in the human disease is major concern. Saline extracts of brain tissues from confirmed AD patients provide the most clinically relevant source of Ab oligomers. More- over, human oAb is highly bioactive, perhaps ~100-fold more so than synthetic oAb preps such as ADDLs when tested side-by-side on wild-type differentiated rat neurons (Jin et al, 2011). When administered i.c.v at picomolar doses to wild-type mice, oAb-rich AD brain extracts provide the most disease-relevant model for the early oAb toxicity encountered near the onset of the AD process. Using wild-type mice avoids confounding developmental issues from overexpression of pathogenic Ab from embryonic stages on, as seen in APP transgenic mouse lines, and may serve as better models of the earliest features of sporadic human AD, providing more trans- lational relevance to early disease mechanisms in humans than do APP-overexpressing transgenic models. Discussion A few reports have suggested that b2AR activation can increase Ab and tau pathology via regulating the internalization of surface APP molecules and/or c-secretase activity, thereby increas- ing Ab production, leading to a recommendation of using b- blockers such as propranolol in the clinical management of AD (Ni et al, 2006; Dobarro et al, 2013; Wang et al, 2013). However, more recent studies suggest that b-adrenergic signaling is instead protec- tive (Heneka et al, 2010; Branca et al, 2014; Liu et al, 2015). The fact that locus coeruleus (LC) neurons develop tangles and die out in AD, leading to reduced NE signaling in humans (Matthews et al, 2002), is potentially relevant to our work. Indeed, lesioning LC in mice and rats increases Ab cytopathology (Kalinin et al, 2007; Heneka et al, 2010; Jardanhazi-Kurutz et al, 2011). Moreover, one b1AR agonist was recently found to decrease overall brain inflammation and lessen AD pathology in an aggressive AD mouse model (Ardestani et al, 2017), providing strong support that b- adrenergic signaling plays a positive role in helping control AD- related brain injury. In addition to those studies utilizing exoge- nous agonist to the NE signaling, our discovery emphasizes that EE provides anti-inflammatory protection on microglia via endoge- nously enhanced b-adrenergic signaling independent of any phar- macological manipulation. The long established comprehensive benefits of EE to brain health, both in laboratory models and in human studies, strongly support the positive role of b-adrenergic signaling. In combination with earlier reports of decreased NE signaling in AD brain, our study highlights the potential impor- tance of avoiding chronic beta-blockers in MCI and AD subjects. In this regard, a recent study conducted in > 10,000 nursing home denizens showed a strong correlation between usage of b-blockers and functional decline, and this negative effect was more obvious in residents with moderate or severe cognitive deficits (Steinman et al, 2017). to oAb-impaired NE regulatory machinery in SH mice, a phenotype that was again successfully neutralized by EE. Besides reduced NE availability, we also observed strong decreases in the transcription levels of both adrenergic receptors and the protein level of b2AR from oAb in SH mice, whereas minimal and insignificant changes occurred in EE mice. A decrease in the mRNA level of both these receptors has been reported in microglia stimulated with LPS (Gyoneva & Traynelis, 2013), connecting a pro-inflammatory state to the decrease in microglial b1/b2AR expression. Discussion Each sample contained microglia isolated from four mice following the same protocol as that of NanoString analysis. g y F qPCR analysis of primary microglia culture treated with oAb, isoproterenol, cAMP-Rp, KT5720, and PKA inhibitor fragment (6-22) amides (PI (6-22)) showed significant increase in cytokine expression by oAb and robust rescue effects by isoproterenol. cAMP-Rp reversed isoproterenol protection in Tnf but not others. Both KT5720 and PKA inhibitor fragment (6-22) amides did not affect isoproterenol. P < 0.05, *P < 0.01. N = 8. Exact P values are listed in Appendix Table S7. F qPCR analysis of primary microglia culture treated with oAb, isoproterenol, cAMP-Rp, KT5720, and PKA inhibitor fragment (6-22) amides (PI (6-22)) showed significant increase in cytokine expression by oAb and robust rescue effects by isoproterenol. cAMP-Rp reversed isoproterenol protection in Tnf but not others. Both KT5720 and PKA inhibitor fragment (6-22) amides did not affect isoproterenol. P < 0.05, **P < 0.01. N = 8. Exact P values are listed in Appendix Table S7. Data information: All quantitative data are presented as mean SD. All statistical analysis were performed using multiple t-test (unpaired, do not assume equal SDs) with Holm-Sidak method to determine significance. Data information: All quantitative data are presented as mean SD. All statistical analysis were performed using multiple t-test (unpaired, do not assume equal SDs) with Holm-Sidak method to determine significance. embedded within the DG revealed that ISF NE levels, which repre- sent the portion of total NE with impact on biological signaling, dropped by 50% in SH mice in response to oAb. The increase in tissue NE and decrease in free extracellular NE in DG together point Next, we complemented these mechanistic findings by quantify- ing brain and ISF NE levels. Direct quantification of NE in whole DG tissue showed a small but significant increase in [NE] in SH mice upon exposure to oAb. In contrast, microdialysis via a short probe 12 of 17 EMBO Molecular Medicine e8931 \| 2018 ª 2018 The Authors ª 2018 The Authors Huixin Xu et al EE prevents Ab microgliosis via b-AR Published online: August 9, 2018 Huixin Xu et al EE prevents Ab microgliosis via b-AR Published online: August 9, 2018 EMBO Molecular Medicine stage fright. ª 2018 The Authors EMBO Molecular Medicine e8931 \| 2018 13 of 17 Stereotactic intracerebroventricular (i.c.v.) injection The Harvard Medical School Standard Committee on Animals and BWH IACUC approved all experiments involving mice in this study. All mice were male. Animals were housed in a temperature- controlled room on a 12-h light/12-h dark cycle and had free access to food and water. BL6/129 mice were purchased from Taconic. Mice with germline Adrb1/2 knockout were purchased from Jack- son Laboratory (Stock No: 003810) and bred in-house. All mice used in the experiments were 8- to 10-weeks old. The lateral ventricle was located by stereotactic coordinates of bregma2.5 mm, midline+3.1 mm, and dura3.7 mm in wild-type mice, and bregma2.4 mm, midline+3.0 mm, and dura2.7 mm in Adrb1/2 KO mice. Through a Hamilton syringe (25 ll) in a stereo- tactic holder, 4 ll of injectate was slowly delivered over 5 min. When this was combined with microdialysis, the ventricle coordi- nates were changed to bregma2.5 mm, midline3.1 mm, and dura3.7 mm in wild-type mice to accommodate the microdialysis probe placement. Microscopy and image analysis All images were acquired by Zeiss LSM710 confocal microscope using 20× objective. A Z-stack was collected for each field of view to capture all details in microglial processes. Microglia morphological features were quantified by ImageJ particle analysis and Skeleton analysis. Imaris reconstruction was performed using Imaris software with the same Z-stacks. Detailed methods regarding environmental enrichment setup, mouse brain tissue staining and microglial morphology analysis, microglia isolation and RNA analysis, and human brain extract preparation and analysis are reported by previous publications (Yang et al, 2013, 2017; Xu et al, 2016). Methods unique to this study are listed below: In vivo microdialysis These mice had an intracerebral guide cannula implanted using coordinates for right hippocampal dentate gyrus placement (bregma1.9 mm, midline1.1 mm, and dura1.5 mm). After inserting probes with 6 kDa molecular weight cutoff (MWCO) membranes (CMA), artificial CSF (in mM: 1.3 CaCl2, 1.2 MgSO4, 3 KCl, 0.4 KH2PO4, 25 NaHCO3, and 122 NaCl, pH 7.35, filtered and degassed) was perfused at flow rates 1.0 ll/min with an infusion syringe pump (Stoelting). Microdialysates were collected in a frac- tion collector at 4°C every hour. Mice were housed in a Raturn cage system (Bioanalytical Systems), which allowed normal movements and activity. All ISF samples were snap-frozen and stored at 80°C until further analysis. Antibodies The following antibodies were used for this study: P2ry12 (1:500), a generous gift from Dr. Oleg Butovsky; CD68 (1:200), Abcam ab53444, RRID:AB_869007; 4G8 (20 lg/reaction), Biolegend RRID: AB_10175149; Anti-CD11b (1:100), BD Biosciences Cat# 552850 RRID:AB_394491; anti-CD45 (1:200), eBioscience Cat# 17-0451-82 162 RRID:AB_469392; anti-Ly6C (1:400), eBioscience Cat# 12-5932- 82 RRID:AB_10804510; anti-Ly6G (1:100), Biolegend 163 Cat# 127605 RRID: AB_1236488; GAPDH (1:5,000), Sigma-Aldrich G9545 RRID: AB 796208; b2AR (1:500), Abcam ab182136 (lot: GR302897- 20) RRID: AB not available. Discussion Moreover, we establish here a model for systematically investigating Propranolol has long been used in the clinic for medical condi- tions such as high blood pressure, certain arrhythmias, and even EMBO Molecular Medicine e8931 \| 2018 13 of 17 EMBO Molecular Medicine Published online: August 9, 2018 EMBO Molecular Medicine Published online: August 9, 2018 EE prevents Ab microgliosis via b-AR Huixin Xu et al EE prevents Ab microgliosis via b-AR Huixin Xu et al consumption of individual animals cannot be determined and there- fore may add variation to the study. signaling pathways and their functions on microglia in AD—using human brain-derived oAb on non-genetically manipulated, wild- type mice. Our identification of NE signaling as a principal mediator between EE and microglia suggests that increased neuronal activity from EE stimulation may modulate microglial behavior through interactions of neurotransmitters with their cognate receptors expressed on microglia, providing evidence that CNS neurotransmit- ters can modulate not just neurons but also innate immune cells. NE signaling was previously identified as a major mediator of EE’s protection of neuronal function following human oAb exposure (Li et al, 2013). Our data suggest the existence of common cellular events triggered by EE that benefit both neurons and microglia via the same signaling pathway but with different manifestations. The beneficial role of enhanced b-adrenergic signaling identified here could apply to other neurological diseases, providing new insights into the biological underpinnings of EE and how to maximize its benefits for the brain. Mouse cytokine quantification at protein level Mice were injected i.c.v. with ADTBS or ID-ADTBS. The brain tissue was harvested following the same method as that for Nano- String analysis. The brain blocks were homogenized in TBS with protease inhibitor at 1:2 ratio (1 mg wet tissue: 2 ml homogeniza- tion buffer). The brain TBS homogenates were centrifuged at 50,000 g for 1 h at 4 degree to remove all non-water-soluble frac- tions. The supernatant fractions were analyzed by mouse cytokine ELISA kit from Meso Scale Discovery (K15069L). For each sample, the loading volume was adjusted based on total protein concentra- tion determined by BCA protein assay so each ELISA reaction received same amount of total protein. Materials and Methods All human brain extracts were analyzed by LEGNEDplex TM Human Inflammation Panel to cover major cytokines that may be present in human brains. Discussion Further, we adapted a previ- ously published SEC purification protocol (Yang et al, 2017) to produce much purer oAb solutions that were quantified, lyophi- lized, and reconstituted in a buffer of choice to the desired concen- tration for in vivo studies. Using this highly enriched and cleaner preparation, we could reproduce the same phenomena we had observed with cruder ADTBS extracts using different brains, with the quantitative differences among treatment groups accentuated. We believe this approach has facilitated consistent preparations and usage of endogenous human oAb for future research here and else- where. On the other hand, any potential benefit of long-term b-agonist usage (e.g., in treating asthma) for cognitive function in the elderly is not well documented. A recent epidemiology study identified b- agonist usage as a protective factor against Parkinson’s disease (Mittal et al, 2017). Similar studies that focus on AD and dementia would be of great value, for although active lifestyle and constant intellectual challenge are highly recommended in the middle-aged and elderly populations to potentially lessen AD and dementia, in practice, such prolonged behavioral modification will not be accessi- ble to all for physical and economic reasons. Our study identifies b- adrenergic signaling as the principal cellular event that is chroni- cally enhanced by an enriched environment to prevent aberrant microglial inflammation occurring from oAb in AD. Further clinical investigation and epidemiological studies of people who are prescribed b-agonists for chronic use could indicate whether such treatment leads to lower incidence and less rapid progression of mild cognitive impairment (MCI) or AD symptoms. Although a life- style modification approach has many superior aspects over phar- macological agents both physiologically and financially, such studies could be important for paving the road toward developing a safe b-agonist as a therapeutic for AD when lifestyle enrichment is not possible for certain patients. In summary, our multifaceted findings reveal that b-adrenergic signaling is a major, if not dominant, mediator of the immunosup- pressive effects of EE on oAb-induced microglial inflammation. Results Results Here, we provide evidence that enhanced b-adrenergic signaling is the key behind EE’s anti-inflammatory benefits on microglia under Ab assault. We show that oral treatment of b-adrenergic receptor (b-AR) agonist mimics EE’s benefits on microglia in mice housed in standard housing (SH), while oral treatment of b-AR antagonist to environmen- tal enriched mice diminished EE’s protection. Genetically removing b- AR also blocks the mice from gaining microglial benefits from EE. Further, we demonstrate that SH mice experienced disrupted b-adre- nergic signaling with Ab exposure shown by decreased extracellular norepinephrine and decreased level of microglial b-AR. EE maintains the b-adrenergic signaling strength in mice with Ab exposure, there- fore keeping brain microglia at a less inflammatory state. Impact p The identification of b-adrenergic pathway activation as one major molecular event mediating EE’s effects on microglia connects outside environment to brain innate immune system by neurotransmitter signaling. The finding might open up new research into the neuro- microglia communication via neurotransmitters and have clinical implications regarding usage of b-agonists and b-antagonists to treat other diseases and their potential cognitive impact in the population vulnerable to AD. Further research might decide whether the same mechanism can benefit other neurological disorders where aberrantly activated microglia play pivotal roles in disease progression. Preparation of primary microglia and treatment Primary microglia cultures were prepared from P0 mouse pups following a published protocol (Lian et al, 2016). Briefly, the cortices of the brains were harvested and digested by trypsin. The cells were plated in poly-D-lysine (PDL)-coated T-75 flasks as mixed glial cultures and let grow till confluent astrocyte layers form. The microglia were isolated by tapping the flasks vigorously. Purified microglia were seeded into PDL-coated 96-well plates at 20,000 cells/well density and let recover overnight. The cells were treated with 10 pg of SEC-purified oAb, 100 lM isoproterenol, 50 lM cAMP-Rp (Tocris 1337), 100 nM KT5720 (Tocris 1288), and 100 nM PKA inhibitor fragment (6-22) amide (Tocris 1904). Cells were harvested after 4 h of incubation at 37°C and lysed for RNA analysis. cohorts to eliminate cohort-specific bias. The initial sample sizes were larger than calculated to compensate for any possible exclu- sion of data points, which was minimal. Any animal with significant bleeding during/after surgery was excluded from the study to avoid confounding from excessive tissue injury. For all experiments, the animals were assigned to different groups by random selection and the animal caregiver was not the investigator. Sample processing and data analysis were carried out by investigators blinded to the intervention. Heat maps were generated using the heatmap.2 func- tion in the gplots package in R. Oral administration of propranolol and isoproterenol Mice were orally exposed to isoproterenol or propranolol through the daily drinking water for 4–6 weeks. Isoproterenol was adminis- tered at 0.1 g/l, and propranolol was administered at 0.4 g/l. Colored, light-proof water bottles were used in all groups. Total water consumption of each cage was recorded. Note that the water 14 of 17 EMBO Molecular Medicine e8931 \| 2018 ª 2018 The Authors Huixin Xu et al EE prevents Ab microgliosis via b-AR Published online: August 9, 2018 Huixin Xu et al EE prevents Ab microgliosis via b-AR Published online: August 9, 2018 EMBO Molecular Medicine Statistical analysis and heat maps All quantification results were analyzed using multiple t-test (un- paired, do not assume equal SDs). Statistical significance was deter- mined using the Holm–Sidak method, with alpha = 0.05. All data were analyzed by Shapiro–Wilk test first to determine normality (all passed). N indicates total number of mice under each condition. The sample sizes were determined by power analysis based on pre- specified effect and variation estimated from preliminary studies. Each experiment included animals from two or more independent Expanded View for this article is available online. Problem Alzheimer’s disease (AD) is affecting a rapidly growing population globally. A pharmacological solution still faces multiple challenges, but lifelong experiences such as education, challenging occupation, exercise, and socialization are shown to provide protection against the development of AD later in life. Environmental enrichment (EE), a laboratory model to study such lifestyle benefits, has extensive anti- inflammatory effects on brain microglia whose malfunction under amyloid-b (Ab) challenge is tightly connected to the progression of AD both in animal models and human subjects. Identifying the molecular mechanisms that drive EE’s protective effects on microglia against Ab is critical for offering new insights into therapeutic development and gaining knowledge on the communication and regulation between the environment and brain innate immune system to benefit future research on brain health. The paper explained All mouse brain samples were snap-frozen upon dissection and analyzed at the Neurochemistry Core Facility at Vanderbilt Univer- sity by Dr. Ginger Milne. All interstitial fluid (ISF) samples from in vivo microdialysis were analyzed at the Center for Translational Neuromedicine (University of Copenhagen, Denmark) by Dr. Pia Weikop. All the ISF samples were processed on Prodigy 3 lm ODS- 3 C18 column (2 mm × 100 mm, particle size 3 lm, Phenomenex). The mobile phase consisted of 55 mM sodium acetate, 1 mM octanesulfonic acid, 0.1 mM Na2EDTA, and 8% acetonitrile, adjusted to pH 3.2 with 0.1 M acetic acid, and was degassed using an online degasser. Seven ll of the samples was injected, and the flow rate was 0.15 ml/min. Electrochemical detection was accom- plished using an amperometric detector (Antec Decade, Antec, Leiden, NL) with a glassy carbon electrode set at 0.8 V, with a Ag/ AgCl electrode as reference electrode. The output was recorded on a computer program LC Solution from Shimadzu, which was also used to calculate the peak areas. ª 2018 The Authors Western blot of microglial b2AR Brain microglia were purified as described by Xu et al (2016). Microglia from four mice were pooled and lysed in 40 ll of RIPA lysis buffer and treated for 30 min at 37°C with 2 units of DNase (TURBO, Invitrogen AM2239) to remove DNA released during lysing. The samples were then mixed with 4X SDS and incubated at room temperature for 10 min and boiled at 65°C for 5 min. The Western blots were developed using Pierce TM Fast Western Blot Kits, SuperSignal TM West Femto. GAPDH was used as loading control. References Ardestani PM, Evans AK, Yi B, Nguyen T, Coutellier L, Shamloo M (2017) Modulation of neuroinflammation and pathology in the 5XFAD mouse model of Alzheimer’s disease using a biased and selective beta-1 adrenergic receptor partial agonist. Neuropharmacology 116: 371 – 386 Kalinin S, Gavrilyuk V, Polak PE, Vasser R, Zhao J, Heneka MT, Feinstein DL (2007) Noradrenaline deficiency in brain increases beta-amyloid plaque burden in an animal model of Alzheimer’s disease. Neurobiol Aging 28: 1206 – 1214 Branca C, Wisely EV, Hartman LK, Caccamo A, Oddo S (2014) Administration of a selective beta2 adrenergic receptor antagonist exacerbates neuropathology and cognitive deficits in a mouse model of Alzheimer’s disease. Neurobiol Aging 35: 2726 – 2735 Kettenmann H, Hanisch U-K, Noda M, Verkhratsky A (2011) Physiology of microglia. Physiol Rev 91: 461 – 553 Kong Y, Ruan L, Qian L, Liu X, Le Y (2010) Norepinephrine promotes microglia to uptake and degrade amyloid beta peptide through upregulation of mouse formyl peptide receptor 2 and induction of insulin-degrading enzyme. J Neurosci 30: 11848 – 11857 Buttgereit A, Lelios I, Yu X, Vrohlings M, Krakoski NR, Gautier EL, Nishinakamura R, Becher B, Greter M (2016) Sall1 is a transcriptional regulator defining microglia identity and function. Nat Immunol 17: 1397 – 1406 Lazarov O, Robinson J, Tang YP, Hairston IS, Korade-Mirnics Z, Lee VM, Hersh LB, Sapolsky RM, Mirnics K, Sisodia SS (2005) Environmental enrichment reduces Abeta levels and amyloid deposition in transgenic mice. Cell 120: 701 – 713 Cracchiolo JR, Mori T, Nazian SJ, Tan J, Potter H, Arendash GW (2007) Enhanced cognitive activity–over and above social or physical activity–is required to protect Alzheimer’s mice against cognitive impairment, reduce Abeta deposition, and increase synaptic immunoreactivity. Neurobiol Learn Mem 88: 277 – 294 Li S, Jin M, Zhang D, Yang T, Koeglsperger T, Fu H, Selkoe DJ (2013) Environmental novelty activates beta2-adrenergic signaling to prevent the impairment of hippocampal LTP by Abeta oligomers. Neuron 77: 929 – 941 Dello Russo C, Boullerne AI, Gavrilyuk V, Feinstein DL (2004) Inhibition of microglial inflammatory responses by norepinephrine: effects on nitric oxide and interleukin-1b production. J Neuroinflammation 1: 9 the impairment of hippocampal LTP by Abeta oligomers. Neuron 77: 929 – 941 Lian H, Roy E, Zheng H (2016) Protocol for primary microglial culture preparation. Bio Protoc 6: e1989 Dobarro M, Gerenu G, Ramirez MJ (2013) Propranolol reduces cognitive deficits, amyloid and tau pathology in Alzheimer’s transgenic mice. Conflict of interest The authors declare that they have no conflict of interest. Jin M, Shepardson N, Yang T, Chen G, Walsh D, Selkoe DJ (2011) Soluble amyloid beta-protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration. Proc Natl Acad Sci USA 108: 5819 – 5824 EE prevents Ab microgliosis via b-AR Huixin Xu et al EE prevents Ab microgliosis via b-AR Huixin Xu et al Author contributions Hetier E, Ayala J, Bousseau A, Prochiantz A (1991) Modulation of interleukin-1 and tumor necrosis factor expression by b-adrenergic agonists in mouse ameboid microglial cells. Exp Brain Res 86: 407 – 413 HX and DJS designed the experiment. HX, MMR, and PW conducted the experi- ments. HX and DJS interpreted the data and prepared the manuscript. ameboid microglial cells. Exp Brain Res 86: 407 – 413 Jardanhazi-Kurutz D, Kummer MP, Terwel D, Vogel K, Thiele A, Heneka MT (2011) Distinct adrenergic system changes and neuroinflammation in response to induced locus ceruleus degeneration in APP/PS1 transgenic mice. Neuroscience 176: 396 – 407 References Int J Neuropsychopharmacol 16: 2245 – 2257 Liu X, Ye K, Weinshenker D (2015) Norepinephrine protects against amyloid- beta toxicity via TrkB. J Alzheimers Dis 44: 251 – 260 Gyoneva S, Traynelis SF (2013) Norepinephrine modulates the motility of resting and activated microglia via different adrenergic receptors. J Biol Chem 288: 15291 – 15302 Markus T, Hansson SR, Cronberg T, Cilio C, Wieloch T, Ley D (2010) beta- Adrenoceptor activation depresses brain inflammation and is neuroprotective in lipopolysaccharide-induced sensitization to oxygen- glucose deprivation in organotypic hippocampal slices. J Neuroinflammation 7: 94 Hannan AJ (2014) Environmental enrichment and brain repair: harnessing the therapeutic effects of cognitive stimulation and physical activity to enhance experience-dependent plasticity. Neuropathol Appl Neurobiol 40: 13 – 25 Matthews KL, Chen CPLH, Esiri MM, Keene J, Minger SL, Francis PT (2002) Noradrenergic changes, aggressive behavior, and cognition in patients with dementia. Biol Psychiat 51: 407 – 416 Heneka MT, Galea E, Gavriluyk V, Dumitrescu-Ozimek L, Daeschner J, O’Banion MK, Weinberg G, Klockgether T, Feinstein DL (2002) Noradrenergic depletion potentiates beta-amyloid-induced cortical inflammation: implications for Alzheimer’s disease. J Neurosci 22: 2434 – 2442 Mittal S, Bjørnevik K, Im DS, Flierl A, Dong X, Locascio JJ, Abo KM, Long E, Jin M, Xu B et al (2017) b2-Adrenoreceptor is a regulator of the a-synuclein gene driving risk of Parkinson’s disease. Science 357: 891 Mori K, Ozaki E, Zhang B, Yang L, Yokoyama A, Takeda I, Maeda N, Sakanaka M, Tanaka J (2002) Effects of norepinephrine on rat cultured microglial cells that express a1, a2, b1 and b2 adrenergic receptors. Neuropharmacology 43: 1026 – 1034 Heneka MT, Ramanathan M, Jacobs AH, Dumitrescu-Ozimek L, Bilkei-Gorzo A, Debeir T, Sastre M, Galldiks N, Zimmer A, Hoehn M et al (2006) Locus ceruleus degeneration promotes Alzheimer pathogenesis in amyloid precursor protein 23 transgenic mice. J Neurosci 26: 1343 – 1354 Ni Y, Zhao X, Bao G, Zou L, Teng L, Wang Z, Song M, Xiong J, Bai Y, Pei G (2006) Activation of beta2-adrenergic receptor stimulates gamma- secretase activity and accelerates amyloid plaque formation. Nat Med 12: 1390 – 1396 Heneka MT, Nadrigny F, Regen T, Martinez-Hernandez A, Dumitrescu-Ozimek L, Terwel D, Jardanhazi-Kurutz D, Walter J, Kirchhoff F, Hanisch UK et al (2010) Locus ceruleus controls Alzheimer’s disease pathology by modulating microglial functions through norepinephrine. Acknowledgments We thank Dr. Ginger Milne at Vanderbilt University Neurochemistry Core for performing analysis on tissue NE level. We thank Dr. Oleg Butovsky for providing P2ry12 antibody. The study was supported by NIH grant AG006173 (to DJS). EMBO Molecular Medicine e8931 \| 2018 15 of 17 EMBO Molecular Medicine Published online: August 9, 2018 References Proc Natl Acad Sci USA 107: 6058 – 6063 Nithianantharajah J, Hannan AJ (2006) Enriched environments, experience- dependent plasticity and disorders of the nervous system. Nat Rev Neurosci 7: 697 – 709 Nithianantharajah J, Hannan AJ (2006) Enriched environments, experience- dependent plasticity and disorders of the nervous system. Nat Rev Neurosci 7: 697 – 709 Herring A, Ambree O, Tomm M, Habermann H, Sachser N, Paulus W, Keyvani K (2009) Environmental enrichment enhances cellular plasticity in transgenic mice with Alzheimer-like pathology. Exp Neurol 216: 184 – 192 Nithianantharajah J, Hannan AJ (2009) The neurobiology of brain and cognitive reserve: mental and physical activity as modulators of brain disorders. Prog Neurobiol 89: 369 – 382 Nithianantharajah J, Hannan AJ (2009) The neurobiology of brain and cognitive reserve: mental and physical activity as modulators of brain disorders. Prog Neurobiol 89: 369 – 382 transgenic mice with Alzheimer-like pathology. Exp Neurol 216: 184 – 192 16 of 17 EMBO Molecular Medicine e8931 \| 2018 ª 2018 The Authors Huixin Xu et al EE prevents Ab microgliosis via b-AR Published online: August 9, 2018 EMBO Molecular Medicine Microglia-derived ASC specks cross-seed amyloid-beta in Alzheimer’s disease. Nature 552: 355 – 361 Pang TY, Hannan AJ (2013) Enhancement of cognitive function in models of brain disease through environmental enrichment and physical activity. Neuropharmacology 64: 515 – 528 Villegas-Llerena C, Phillips A, Garcia-Reitboeck P, Hardy J, Pocock JM (2016) Microglial genes regulating neuroinflammation in the progression of Villegas-Llerena C, Phillips A, Garcia-Reitboeck P, Hardy J, Pocock JM (2016) Microglial genes regulating neuroinflammation in the progression of Alzheimer’s disease. Curr Opin Neurobiol 36: 74 – 81 Paxinos G, Franklin KBL (2012) The mouse brain in stereotaxic coordinates, 4th edn. San Diego, CA: Academic Press Alzheimer’s disease. Curr Opin Neurobiol 36: 74 – 81 Qian L, Wu HM, Chen SH, Zhang D, Ali SF, Peterson L, Wilson B, Lu RB, Hong JS, Flood PM (2011) beta2-adrenergic receptor activation prevents rodent dopaminergic neurotoxicity by inhibiting microglia via a novel signaling pathway. J Immunol 186: 4443 – 4454 Wang D, Fu Q, Zhou Y, Xu B, Shi Q, Igwe B, Matt L, Hell JW, Wisely EV, Oddo S et al (2013) beta2 adrenergic receptor, protein kinase A (PKA) and c-Jun N-terminal kinase (JNK) signaling pathways mediate tau pathology in Alzheimer disease models. References J Biol Chem 288: 10298 – 10307 Xu H, Gelyana E, Rajsombath M, Yang T, Li S, Selkoe D (2016) Environmental enrichment potently prevents microglia-mediated neuroinflammation by human amyloid beta-protein oligomers. J Neurosci 36: 9041 – 9056 Sara SJ (2015) Locus Coeruleus in time with the making of memories. Curr Opin Neurobiol 35: 87 – 94 Sara SJ (2015) Locus Coeruleus in time with the making of memories. Curr Opin Neurobiol 35: 87 – 94 Selkoe DJ, Hardy J (2016) The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol Med 8: 595 – 608 Selkoe DJ, Hardy J (2016) The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol Med 8: 595 – 608 Selkoe DJ, Hardy J (2016) The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol Med 8: 595 – 608 Yang T, Hong S, O’Malley T, Sperling RA, Walsh DM, Selkoe DJ (2013) New ELISAs with high specificity for soluble oligomers of amyloid beta-protein detect natural Abeta oligomers in human brain but not CSF. Alzheimers Dement 9: 99 – 112 Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE, Smith I, Brett FM, Farrell MA, Rowan MJ, Lemere CA et al (2008) Amyloid-beta protein dimers isolated directly from Alzheimer’s brains impair synaptic plasticity and memory. Nat Med 14: 837 – 842 Yang T, Li S, Xu H, Walsh DM, Selkoe DJ (2017) Large soluble oligomers of amyloid beta-protein from alzheimer brain are far less neuroactive than the smaller oligomers to which they dissociate. J Neurosci 37: 152 – 163 Sims R, van der Lee SJ, Naj AC, Bellenguez C, Badarinarayan N, Jakobsdottir J, Kunkle BW, Boland A, Raybould R, Bis JC et al (2017) Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer’s disease. Nat Genet 49: 1373 – 1384 Zhang B, Gaiteri C, Bodea LG, Wang Z, McElwee J, Podtelezhnikov AA, Zhang C, Xie T, Tran L, Dobrin R et al (2013) Integrated systems approach identifies genetic nodes and networks in late-onset Alzheimer’s disease. Cell 153: 707 – 720 mmunity in Alzheimer’s disease. Nat Genet 49: 1373 – 1384 Steinman MA, Zullo AR, Lee Y, Daiello LA, Boscardin WJ, Dore DD, Gan S, Fung K, Lee SJ, Komaiko KD et al (2017) Association of beta-blockers with functional outcomes, death, and rehospitalization in older nursing home residents after acute myocardial infarction. EMBO Molecular Medicine e8931 \| 2018 17 of 17 References JAMA Intern Med 177: 254 – 262 Ziegler-Waldkirch S, d’Errico P, Sauer JF, Erny D, Savanthrapadian S, Loreth D, Katzmarski N, Blank T, Bartos M, Prinz M et al (2018) Seed-induced Abeta deposition is modulated by microglia under environmental enrichment in a mouse model of Alzheimer’s disease. EMBO J 37: 167 – 182 Tanaka KF, Kashima H, Suzuki H, Ono K, Sawada M (2002) Existence of functional beta1- and beta2-adrenergic receptors on microglia. J Neurosci Res 70: 232 – 237 License: This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduc- tion in any medium, provided the original work is properly cited. Then FS, Luck T, Luppa M, König H-H, Angermeyer MC, Riedel-Heller SG (2015) Differential effects of enriched environment at work on cognitive decline in old age. Neurology 84: 2169 – 2176 Venegas C, Kumar S, Franklin BS, Dierkes T, Brinkschulte R, Tejera D, Vieira-Saecker A, Schwartz S, Santarelli F, Kummer MP et al (2017) ª 2018 The Authors
W1997083552.txt	https://bmccancer.biomedcentral.com/counter/pdf/10.1186/1471-2407-13-163	en		Evaluation of the prognostic role of centromere 17 gain and HER2/topoisomerase II alpha gene status and protein expression in patients with breast cancer treated with anthracycline-containing adjuvant chemotherapy: pooled analysis of two Hellenic Cooperative Oncology Group (HeCOG) phase III trials	BMC cancer	2,013	cc-by	10,803	Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 RESEARCH ARTICLE Open Access Evaluation of the prognostic role of centromere 17 gain and HER2/topoisomerase II alpha gene status and protein expression in patients with breast cancer treated with anthracycline-containing adjuvant chemotherapy: pooled analysis of two Hellenic Cooperative Oncology Group (HeCOG) phase III trials George Fountzilas1†, Urania Dafni2†, Mattheos Bobos3, Vassiliki Kotoula4, Anna Batistatou5, Ioannis Xanthakis1, Christos Papadimitriou6, Ioannis Kostopoulos4, Triantafillia Koletsa4, Eleftheria Tsolaki3, Despina Televantou3, Eleni Timotheadou1, Angelos Koutras7, George Klouvas8, Epaminontas Samantas9, Nikolaos Pisanidis10, Charisios Karanikiotis11, Ioanna Sfakianaki1, Nicholas Pavlidis12, Helen Gogas13, Helena Linardou14, Konstantine T Kalogeras1,15, Dimitrios Pectasides16 and Meletios A Dimopoulos6 Abstract Background: The HER2 gene has been established as a valid biological marker for the treatment of breast cancer patients with trastuzumab and probably other agents, such as paclitaxel and anthracyclines. The TOP2A gene has been associated with response to anthracyclines. Limited information exists on the relationship of HER2/TOP2A gene status in the presence of centromere 17 (CEP17) gain with outcome of patients treated with anthracyclinecontaining adjuvant chemotherapy. Methods: Formalin-fixed paraffin-embedded tumor tissue samples from 1031 patients with high-risk operable breast cancer, enrolled in two consecutive phase III trials, were assessed in a central laboratory by fluorescence in situ hybridization for HER2/TOP2A gene amplification and CEP17 gain (CEP17 probe). Amplification of HER2 and TOP2A were defined as a gene/CEP17 ratio of >2.2 and ≥2.0, respectively, or gene copy number higher than 6. Additionally, HER2, TopoIIa, ER/PgR and Ki67 protein expression was assessed by immunohistochemistry (IHC) and patients were classified according to their IHC phenotype. Treatment consisted of epirubicin-based adjuvant chemotherapy followed by hormonal therapy and radiation, as indicated. (Continued on next page) Correspondence: fountzil@auth.gr † Equal contributors 1 Department of Medical Oncology, “Papageorgiou” Hospital, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece Full list of author information is available at the end of the article © 2013 Fountzilas et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 Page 2 of 16 (Continued from previous page) Results: HER2 amplification was found in 23.7% of the patients and TOP2A amplification in 10.1%. In total, 41.8% of HER2-amplified tumors demonstrated TOP2A co-amplification. The median (range) of HER2, TOP2A and CEP17 gain was 2.55 (0.70-45.15), 2.20 (0.70-26.15) and 2.00 (0.70-26.55), respectively. Forty percent of the tumors had CEP17 gain (51% of those with HER2 amplification). Adjusting for treatment groups in the Cox model, HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with time to relapse or time to death. Conclusion: HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with outcome in high-risk breast cancer patients treated with anthracycline-based adjuvant chemotherapy. Trial registration: Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12611000506998 and ACTRN12609001036202 Keywords: HER2, TOP2A, TopoIIa, Prognostic factors, Predictive factors, Adjuvant chemotherapy, Anthracyclines, Taxanes, Breast cancer Background Breast cancer is the most frequent non-skin malignancy and the second leading cause of cancer death in American and European women [1,2]. Adjuvant chemotherapy is administered to most patients with high-risk operable breast cancer, since it prolongs disease-free survival (DFS) and overall survival (OS) [3]. Anthracyclines and taxanes are considered to be two of the most efficient drugs in this setting [4,5]. Despite intensive clinical research devoted to the role of adjuvant chemotherapy, the majority of patients do not benefit from its use and a small but considerable percentage of them suffer from long-term life-threatening toxicities, such as acute leukemia, myelodysplasia or irreversible congestive heart failure [6,7]. To select candidate patients for such aggressive treatments, robust prognostic markers in human breast cancer are needed. Investigators intensively evaluate wellestablished oncogenes or chromosome aberrations, using large tumor repositories, in an effort to widen their knowledge on the molecular mechanisms, gene interrelationships or gene function underlying breast cancer. It has long been established that breast cancer is often characterized by gains or losses of specific chromosomes, leading to activation of oncogenes or inactivation of tumor suppressor genes [8]. Chromosome 17 is the second most gene-dense chromosome in the human genome, housing important genes for breast cancer pathophysiology, such as BRCA1, HER2, RAD51C, RARA, TOP2A and TP53 [9]. Changes of chromosome 17 copy number (aneusomy) are extremely frequent in breast cancer [10]. These chromosome aberrations (reviewed in ref. [11]) are tightly linked to important cell functions, such as proliferation, apoptosis, angiogenesis and motility. Increased numbers of centromere 17 copies are seen in 10% to 50% of breast tumors [12-14], depending on the criteria used, and this is more common in tumors with HER2 gene amplification. However, it has to be stated that an increase in chromosome 17 signals seen with fluorescence in situ hybridization (FISH) does not always correspond to true polysomy of the whole chromosome, but may rather represent a focal pericentromeric gain or partial polysomy [15]. Other abnormalities of chromosome 17 include losses and gains of genetic material in both the p and q arms, focal copy number gains and losses and other structural rearrangements [15,16]. Indeed, recent studies using different techniques, such as comparative genomic hybridization (CGH) [17,18], multiplex ligation-dependent probe amplification (MLPA) [19], single nucleotide polymorphism arrays (SNP arrays) [15], or FISH using alternative chromosome 17 reference genes (RARA, TP53, SMS) [20] suggested that true chromosome 17 polysomy is a rare event in breast cancer. In fact, in most of the cases, polysomy, as detected by FISH or chromogen in situ hybridization (CISH), actually reflects a gain or amplification in the pericentromeric region of the chromosome [21]. For these reasons the term “CEP17 gain” instead of “chromosome 17 polysomy”, is used here, referring to its detection by the centromere 17 enumeration probe (CEP17 probe). CEP17 gain has been incriminated for the inconsistencies seen in cases with HER2 gene amplification defined by absolute gene copy numbers, versus gene amplification defined by the ratio of HER2 gene copy number to CEP17. Misclassification of HER2 gene status based on dual-color FISH assays, due to CEP17 gain, may have important therapeutic implications since a number of patients considered being HER2-negative by the second definition could be denied trastuzumab. Importantly, recently published data from retrospectively assessed (although prospectively collected) tumors, by triple color FISH, from 1762 patients who participated Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 in the National Epirubicin Adjuvant Trial (NEAT/BR9601) suggested that CEP17 duplication was associated with increased relapse-free and overall survival in patients treated with an anthracycline compared to CMF [22]. The HER2 oncogene is located on the long arm of chromosome 17 (17q12) [23]. HER2 amplification and/ or protein overexpression has been identified in 15% to 25% of invasive breast tumors [24] and is associated with worse prognosis [25]. HER2 gene amplification has been shown to predict benefit from the use of several chemotherapeutic agents, including anthracyclines and paclitaxel [26,27]. Notably, a meta-analysis provided compelling evidence that the use of anthracyclines benefits exclusively those patients with HER2 amplification [28]. However, other investigators could not confirm these data [29,30], suggesting that other genes, also located on chromosome 17, may regulate anthracycline responsiveness [26]. One such gene is the topoisomerase II alpha gene (TOP2A), which is located ~700 kb telomerically to HER2 and encodes the alpha isozyme of the human topoisomerase II. In general, topoisomerases are responsible for transcription, replication and chromosome condensation and segregation during cell division [31,32]. TOP2A in particular is considered a molecular target for anthracyclines and other chemotherapeutic agents [33,34]. The TOP2A gene is amplified in 30%-40% of the tumors with HER2 gene amplification, while deletions are frequently observed [35]. TOP2A gene amplification [36] and, perhaps, topoisomerase II alpha (TopoIIa) protein overexpression [37] may benefit high-risk breast cancer patients treated with anthracyclines. Information regarding the interaction of HER2/TOP2A gene status in the presence of CEP17 gain with the outcome of breast cancer patients is limited. This urged us to investigate the prognostic role of HER2 and TopoIIa protein expression, as well as HER2 and TOP2A gene status along with CEP17 gain in a large cohort of breast cancer patients. This is a prospective-retrospective study as described by Simon [38], performed in the context of two randomized, consecutively conducted, phase III trials (HE10/97 and HE10/00) with epirubicin-based adjuvant chemotherapy with or without paclitaxel [39-42]. Methods Clinical studies The HE10/97 trial [39] was a randomized phase III trial (ACTRN12611000506998) in patients with high-risk node-negative or intermediate/high-risk node-positive operable breast cancer, comparing four cycles of epirubicin (E) followed by four cycles of intensified CMF (E-CMF) with three cycles of E, followed by three cycles of paclitaxel (T, TaxolW, Bristol Myers-Squibb, Princeton, NJ) followed by three cycles of intensified CMF (E-T-CMF). Page 3 of 16 All cycles were given every two weeks with G-CSF support. Dose intensity of all drugs in both treatment arms was identical, but cumulative doses and duration of chemotherapy period differed. Totally, 595 eligible patients entered the study in a period of 3.5 years (1997–2000). The HE10/00 trial [40,41] was a randomized phase III trial (ACTRN12609001036202), in which patients were treated with E-T-CMF (exactly as in the HE10/97 trial) or with four cycles of epirubicin/paclitaxel (ET) combination (given on the same day) every three weeks followed by three cycles of intensified CMF every two weeks (ET-CMF). By study design, the cumulative doses and the chemotherapy duration were identical in the two arms but dose intensity of epirubicin and paclitaxel was double in the E-T-CMF arm. A total of 1086 eligible patients with node-positive operable breast cancer were accrued in a period of 5 years (2000–2005). HER2-positive patients received trastuzumab upon relapse, as previously described [43]. Treatment schedules for the two studies, baseline characteristics and clinical outcomes of both trials have already been described in detail [39-42]. Primary tumor diameter, axillary nodal status and tumor grade were obtained from the pathology report. Clinical protocols were approved by local regulatory authorities, while the present translational research protocol was approved by the Bioethics Committee of the Aristotle University of Thessaloniki School of Medicine. All patients signed a study-specific written informed consent before randomization, which in addition to giving consent for the trial allowed the use of biological material for future research purposes. Tissue microarray (TMA) construction Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 1031 patients (61.3% of 1681 randomized patients) were collected from both trials, retrospectively in the first (HE10/97) and prospectively in the second (HE10/00). The REMARK diagram [44] for the study is shown in Figure 1. Hematoxylin-eosin stained sections from the tissue blocks were reviewed by two experienced breast cancer pathologists (M.B. and D.T.) and the most representative tumor areas were marked for the construction of the TMA blocks with the use of a manual arrayer (Model I, Beecher Instruments, San Prairie, WI), as previously described [45,46]. Each case was represented by 2 tissue cores, 1.5 mm in diameter, obtained from the most representative areas of primary invasive tumors or in some cases (9.6%) from synchronous axillary lymph node metastases and re-embedded in 51 microarray blocks. Each TMA block contained 38 to 66 tissue cores from the original tumor tissue blocks, while cores from various neoplastic, non-neoplastic and reactive tissues were also included, serving as controls for Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 HE10/97 trial 595 eligible patients 367 FFPE tumor blocks retrospectively collected • • 58 blocks excluded • 26 In situ/no tumor • 32 Inadequate material Page 4 of 16 • • HE10/00 trial 1086 eligible patients 895 FFPE tumor blocks prospectively collected 117 blocks excluded • 47 In situ/no tumor • 70 Inadequate material 309 blocks with appropriate/adequate material 778 blocks with appropriate/adequate material 291 blocks in HE10/97 plus 740 blocks in HE10/00 A total of 1031 tumor tissue samples were evaluated for HER2 and TOP2A gene amplification and CEP17 gain by FISH In addition, 1014 tumor tissue samples were evaluated by IHC for HER2 protein expression and 953 for TopoIIa protein expression Figure 1 REMARK diagram. slide-based assays. Cases not represented, damaged or inadequate on the TMA sections were re-cut from the original blocks and these sections were used for protein and gene analysis. Histological grade was evaluated according to the Scarff, Bloom and Richardson system. described [47]. TopoIIa protein expression was evaluated using the KiS1 monoclonal antibody (Dako), as previously described [48] with slight modifications (antibody dilution: 1:200; detection system: Envision™, Dako). Interpretation of the IHC results Immunohistochemistry (IHC) Immunohistochemical labeling was performed according to standard protocols on serial 2.5 μm thick sections from the original blocks or the TMA blocks. All cases were also stained for vimentin (clone V9, Dako, Glostrup, Denmark) and cytokeratin 8/18 (clone 5D3, Novocastra™, Leica Biosystems, Newcastle, U.K), which were used as control stains for tissue immunoreactivity and fixation, as well as identification of tumor cells. Tissue samples negative for the above antibodies were excluded from the study. To assure optimal reactivity, immunostaining was applied 7 to 10 days after sectioning at the Laboratory of Molecular Oncology of the Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine. The staining procedures for HER2 (A0485 polyclonal antibody, Dako), estrogen receptor (ER, clone 6 F11, Novocastra™, Leica Biosystems), progesterone receptor (PgR, clone 1A6, Novocastra™, Leica Biosystems) and Ki67 (clone MIB-1, Dako) were performed using a Bond Max™ autostainer (Leica Microsystems, Wetzlar, Germany), as previously The evaluation of all IHC sections was done by two experienced breast cancer pathologists (M.B. and A.B.), blinded as to the patients’ clinical characteristics and survival data, according to existing established criteria, as previously described [43]. Briefly, HER2 protein expression was scored in a scale from 0 to 3+, the latter corresponding to uniform, intense membrane staining in >30% invasive tumor cells [49]; ER and PgR were evaluated using the Histoscore method (max score: 400) and were considered positive if staining was present in ≥1% of tumor cell nuclei [50]; for Ki67, the expression was defined as low (<14%) or high (≥14%) based on the percentage of stained/unstained nuclei from the tumor areas [51]; and, for TopoIIa immunostaining, a tumor was considered to be positive if moderate to intense nuclear staining was detected in >5% of tumor cells [52]. The mean percentage of stained cells from the two cores was calculated, while in cases with different intensities, the higher intensity score obtained from the two cores was used. If one of the tissue cores was lost or damaged the overall score was determined Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 from the remaining one. When whole tissue sections were used, the entire tumor area was evaluated. Fluorescence in situ hybridization (FISH) TMA sections or whole sections (5 μm thick) were cut for FISH analysis, using the ZytoLightW SPEC HER2/TOP2A/ CEP17 triple-color probe kit (ZytoVision, Bremerhaven, Germany). The FISH was performed according to the manufacturer’s protocol with minor modifications. Four carcinoma cell lines (MDA-MB-231, MDA-MB-175, MDA-MB-453 and SK-BR-3) from the Oracle HER2 Control Slide (Leica Biosystems), with a known HER2 gene status, were also used as a control for the FISH assays and analyzed for HER2 and TOP2A genomic status. For all probes, sequential (5 planes at 1.0 μm) digital images were captured using the Plan Apo VC 100x/1.40 oil objective (Nikon, Japan) using specific filters for each probe. The resulting images were reconstructed using specifically developed software for cytogenetics (XCytoGen, ALPHELYS, Plaisir, France). Processed sections were considered eligible for FISH evaluation according to the ASCO/CAP criteria [49]. For the evaluation of HER2/TOP2A/CEP17 status, non-overlapping nuclei from the invasive part of the tumor were randomly selected, according to morphological criteria using DAPI staining, and scored (M.B and E.T). The virtual slides of HER2, ER or PgR stains, created as previously described [47], were used for selecting the invasive part of the tumor in each TMA. Twenty tumor nuclei were counted according to Press et al. [53]. The HER2 gene was considered to be amplified when the HER2/CEP17 ratio was >2.2 [49], or the mean HER2 copy number was >6 [54] and deleted when the ratio was <0.8. The TOP2A gene was considered to be amplified when the TOP2A/CEP17 ratio was ≥2.0 and deleted when the ratio was <0.8 [36]. Cases with ≥3 CEP17 hybridization signals detected in >30% of counted nuclei were classified as CEP17 gain [55]. Re-classification of CEP17 gain was performed for the current analysis in comparison to the previous report [56]. In cases with ratios at or near the cut-off (1.8-2.2 for amplifications and 0.7-0.9 for deletions), additional 20 or 40 nuclei were counted and the ratio was recalculated. In cases with a borderline ratio at 60 nuclei, additional FISH assays were performed in whole sections [57]. In addition, tumors were classified according to the number of gene copies as normal (≤4 copies), low gain (4–6 copies) or high gain (>6 copies) tumors. The first category included tumors with possible gene losses, diploid, or with replicated DNA; the second, tumors with possible polysomy for the gene of interest; and, the third, tumors with unequivocal gene amplification. All primary image data of the TMA and whole tumor sections have been digitally scanned and made publicly available at: Page 5 of 16 http://www.hecog-images.gr/HER2/TOP2A/CEN17/ FISH_HE10/97_HE10/00. Statistical analysis Categorical data are presented as numbers and corresponding percentages, while continuous data are presented as median and range values. The Fisher’s exact or Pearson chi-square tests were used for group comparison of categorical data, while for continuous data the Mann– Whitney test was used. DFS was defined as the time interval from study entry to first locoregional recurrence, first distant metastasis, contralateral breast cancer, secondary neoplasm, death from the disease, or death from any cause, whichever occurred first [58]. OS was measured from study entry until death from any cause. Surviving patients were censored at the date of last contact. Kaplan-Meier curves and log-rank tests were used for comparing time to event distributions. Cox proportional hazard regression analyses, adjusted for treatment, were performed for the examined markers, as well as for the combination of HER2/TOP2A gene status to assess prognostic significance on DFS and OS. In multivariate analysis, a backward selection procedure with p > 0.10 as a removal criterion based on the likelihood ratio test was performed to identify significant clinicopathological variables among the following: age (≥50 vs. <50), treatment group (E-CMF vs. ET-CMF vs. E-T-CMF), menopausal status (postmenopausal vs. premenopausal), histological grade (III-undifferentiated vs. I-II), Ki67 protein expression (high vs. low), tumor size (>5 cm vs. 2 to 5 cm vs. <2 cm), number of positive axillary nodes (≥4 vs. 0–3), ER/PgR status (positive vs. negative), adjuvant hormonotherapy (yes vs. missing vs. no) and type of operation (breast-conserving surgery vs. modified radical mastectomy). Treatment group and the examined markers were included in the final model, in order to examine whether they added independent prognostic information to the model containing the significant clinicopathological parameters. The results of this study are presented according to reporting recommendations for tumor marker prognostic studies [44]. No adjustments for multiple comparisons were done. Statistical analyses were performed using the following statistical software: SPSS for Windows (version 15.0, IBM Corporation, Armonk, NY) and SAS for Windows (version 9.3, SAS Institute Inc., Cary, NC). Results A total of 1031 patients with available FFPE tumor tissue blocks were included in the analysis at a median followup of 106 months (updated in March 2012). Results at median follow-up of 92 months were presented at the 2011 San Antonio Breast Cancer Symposium [56]. Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 Page 6 of 16 Selected patient and tumor characteristics are presented in Table 1. The majority of the patients were postmenopausal (53.1%), had ≥4 positive nodes (60.4%) and tumors of ductal histology (77.6%), while approximately half of the patients had tumors of high grade (50.2%). The basic clinicopathological characteristics were similar between patients with and without available tissue blocks in each study, except for the number of positive nodes, radiotherapy treatment and histological grade (above II) (Additional file 1: Table S1). Patients with available tissue blocks had a higher incidence of ≥4 positive nodes (p = 0.022 and p = 0.027 for HE10/97 and HE10/00, respectively). This fact was probably reflected in the corresponding more frequent use of radiotherapy treatment (p = 0.024 and p = 0.021), while higher histological grade was more frequent in patients with available blocks only in the HE10/00 trial (p = 0.026). Table 1 Selected patient and tumor characteristics Representative FISH images for HER2, TOP2A and CEP17 are shown in Figure 2. The distribution of centrally assessed tumor markers by FISH and IHC are presented in Table 2. Cases with HER2 deletions (n = 27, 2.6%) were grouped together with HER2 normal tumors, for analysis purposes. Amplification, classified according to gene/CEP17 ratios, was found for HER2 in 23.7% and for TOP2A in 10.1% of the tumors. The incidence of amplified tumors was lower when amplification was considered according to the cut-off of >6 copies for each gene (Table 2). Ten cases were equivocal for HER2 (with HER2/CEP17 ratios between 1.8-2.2 and ≤6 gene copies) and they were also included in the HER2 normal tumors, for analysis purposes. CEP17 gain was seen in approximately 40% of tumors (Table 2). Histograms of the distribution of HER2, TOP2A and CEP17 copy numbers are presented in Figure 3. Examining the association of markers with clinicopathological parameters, CEP17 gene gain was found to be associated only with postmenopausal status (48.1% in no gain vs. 60.6% in gain, p < 0.001) (Additional file 1: Table S2). HER2 gene amplification was associated with higher histological grade (45% in non-amplified vs. 66% in amplified, p < 0.001), ductal carcinoma (75% in nonamplified vs. 87% in amplified, p < 0.001), negative receptor status (16% in non-amplified vs. 44% in amplified, p < 0.001) and high Ki67 (64% in non-amplified vs. 79% in amplified, p < 0.001), while TOP2A amplification was associated with higher histological grade (58% in deleted vs. 49% in non-amplified vs. 62% in amplified, p = 0.023) and negative receptor status (37% in deleted vs. 20% in non-amplified vs. 40% in amplified, p < 0.001). Overall, 24% of the patients had a HER2-positive status, based on either HER2 gene/CEP17 ratio of >2.2 or gene copy number of >6 or an IHC score of 3+. Interestingly, 27 tumors with HER2 IHC scores of 0 (7 cases) or 1+ (20 cases), were found to be amplified either by gene gain >6 (n = 3) or FISH ratio >2.2 (n = 24) (Additional file 1: Table S3). In addition, among cases with HER2 IHC scores of 0 or 1+, there were 17 tumors (2.4%) with HER2 deletion. It is worth noting that among 204 cases with HER2/CEP17 ratio >2.2 (i.e., amplified by ratio criteria), 184 (90%) also had >6 HER2 gene copies (i.e., amplified by gene copy criteria). N = 1031 Age in years Median (range) 52.5 (22–79) Number of positive nodes Median (range) 4 (0–43) N (%) Randomization group E-T-CMF 504 (48.9) E-CMF 157 (15.2) ET-CMF 370 (35.9) <50 426 (41.3) ≥50 605 (58.7) Age Menopausal status Premenopausal 484 (46.9) Postmenopausal 547 (53.1) Type of surgery MRM Breast-conserving 706 (68.5) 325 (31.5) Tumor size (cm) ≤2 320 (31.0) 2.1-5 584 (56.6) >5 127 (12.3) Number of positive nodes 0-3 408 (39.6) ≥4 623 (60.4) Histological grade I-II 513 (49.8) III-Undifferentiated 518 (50.2) Histology type Ductal 800 (77.6) Lobular 105 (10.2) Mixed 73 (7.1) Other 53 (5.1) Radiotherapy 782 (75.8) Hormonal therapy 799 (77.5) MRM, modified radical mastectomy; N, number. Incidence and associations between examined biological markers Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 Page 7 of 16 Figure 2 Representative FISH images in invasive breast carcinoma (IBC) cases, using the HER2/TOP2A/CEP17 triple-color probes. In the first four panels (A-D) an IBC case is shown with normal status of the HER2 gene (A), TOP2A gene (B) and CEP17 (C). An IBC case (E-H) showing simultaneous amplification of the HER2 and TOP2A genes (E-F), as well as CEP17 gain (G). The third IBC case presented in panels (I-L) showed amplification of the HER2 gene (I), normal status of the TOP2A gene (J) and CEP17 gain (K). In the last case, co-amplification of the HER2 (M) and TOP2A genes (N) was found in tumor cells, accompanied by high-level CEP17 gain (O). The last panel for each case (panels D, H, L and P) depicts a merged image of the three-colored probes. Magnification x1000. CEP17, centromere 17 enumeration probe. Tumors with CEP17 gain were also HER2 amplified in about one third of the cases (N = 120), while they were TOP2A amplified in 15% of the cases (N = 59) (Table 3). Among 244 HER2 amplified tumors, 51% had CEP17 gain. Similar percentages were observed for CEP17 gain in TOP2A amplified (58%) and deleted tumors (65%). Overall, tumors with low HER2 or TOP2A copy numbers had CEP17 gain in 37% and 36%, respectively (Table 3). In addition, among 827 tumors with HER2/ CEP17 ratio ≤2.2, 327 (40%) had CEP17 gain. Among 10 equivocal cases with HER2/CEP17 ratios between 1.8-2.2 there was only one case with CEP17 gain. The distribution of TOP2A and CEP17 by breast cancer tumor subtypes is presented in Table 4. Among 126 triplenegative breast cancer (TNBC) tumors, no amplifications of TOP2A were found. CEP17 gain was more frequent in Luminal-HER2 and HER2-enriched tumors. Associations of TOP2A gene status and TopoIIa protein expression are shown in detail (Additional file 1: Table S4). TOP2A deletions did not result in lower TopoIIa expression. Among 953 cases with paired TOP2A gene status and protein expression data, there were 28 tumors with TOP2A gene deletion and simultaneous protein expression. No association was found between TopoIIa protein expression and TOP2A gene amplification (p = 0.22). Significant associations were observed between CEP17 gene status and HER2 protein expression, as well as TopoIIa protein expression (Additional file 1: Table S5). More specifically, CEP17 gain was more frequent in HER2 2+ and 3+ tumors and in tumors expressing TopoIIa. Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 Page 8 of 16 Table 2 Distribution of centrally assessed tumor markers by FISH and IHC N (%) FISH CEP17 status (n = 1031) Median (range) 2.00 (0.70-26.55) No gain 620 (60.1) Gain 411 (39.9) HER2 (gene copies) (n = 1031) Median (range) Low normal-replicated (≤4) 2.55 (0.70-45.15) 742 (72.0) Low gain (4–6) 65 (6.3) High gain (>6) 224 (21.7) HER2 gene status (n = 1031) Non-amplified1 787 (76.3) Amplified2 244 (23.7) TOP2A (gene copies) (n = 1031) Median (range) Low normal-replicated (≤4) 2.15 (0.70-26.15) 875 (84.9) Low gain (4–6) 77 (7.5) High gain (>6) 79 (7.7) TOP2A gene status (n = 1031) Deleted IHC 52 (5.0) Non-amplified 875 (84.9) Amplified3 104 (10.1) HER2 (n = 1014) 0 319 (31.5) 1+ 379 (37.4) 2+ 171 (16.9) 3+ 145 (14.3) TopoIIa (n = 953) Negative 441 (46.3) Positive 512 (53.7) ER (n = 1018) Negative 272 (26.7) Positive 746 (73.3) PgR (n = 1024) Negative 335 (32.7) Positive 689 (67.3) Ki67 (n = 1000) Low 322 (32.2) High 678 (67.8) 27 cases (2.6%) had a deletion with a HER2/CEP17 ratio <0.8. 204 cases (83.6%) with HER2/CEP17 ratio >2.2 and 40 cases (16.4%) with HER2 gene gain (>6 copies). 3 64 cases (61.5%) with TOP2A/CEP17 ratio ≥2.0 and 40 cases (38.5%) with TOP2A gene gain (>6 copies). 1 2 In total, 42% of HER2 amplified tumors demonstrated TOP2A co-amplification (Additional file 1: Table S6). Among the HER2 non-amplified cases, 28 deletions (3.6%) and only two amplifications of the TOP2A gene were identified. Associations of examined markers with prognosis DFS and OS did not differ significantly between treatment groups. At a median follow-up of 106 months (range 0.1-167), the 5-year DFS rates were 75%, 69% and 75%, while the OS rates were 88%, 81% and 86%, for the E-T-CMF, E-CMF and ET-CMF groups, respectively (Additional file 1: Table S7). HER2 amplification, TOP2A amplification, TopoIIa protein expression, CEP17 gain and HER2/TOP2A coamplification were not associated with either relapse or death (Figures 4, 5, 6 and 7). Similarly, when examining combined TOP2A gene pathology (deletion and amplification) vs. normal TOP2A, no effect on patient outcome was observed. This did not change when adjusting for treatment group in the Cox regression model. HER2 and TOP2A gene copy numbers (amplified vs. low gain vs. low-normal-replicated) were also not associated with DFS or OS. Stratifying by CEP17 status, differences in outcome by HER2 gene status (amplified vs. nonamplified tumors) and by TOP2A gene status (amplified vs. deleted vs. non-amplified tumors) were examined. No such differences were observed for either DFS or OS. The predictive role of all examined markers for paclitaxel treatment were also evaluated, performing Cox model analysis with interaction terms of each gene with treatment (paclitaxel vs. no paclitaxel). None of the markers tested was predictive for paclitaxel treatment. Multivariate analyses for the examined biological markers, in the presence of significant clinical parameters and treatment group, are presented by forest plots (Figure 8). Clinicopathological factors associated with increased risk for both relapse and death were tumor size of more than 5 cm (p = 0.009 for DFS and p = 0.001 for OS) and four or more positive nodes (p < 0.001 for both DFS and OS). Hormonal therapy was associated with improved DFS and OS (p = 0.028 and p = 0.002, respectively), while breast-conserving surgery was associated with improved DFS only (p = 0.011) and high histological grade with poor OS only (p = 0.039). No association was found with DFS or OS for any of the examined chromosome 17 markers. Finally, none of the examined markers were associated with either DFS or OS in the context of univariate or multivariate analyses, when excluding the lymph node samples. Discussion In the present study we investigated the prognostic role of CEP17 gain in relation to HER2 and TOP2A gene Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 Page 9 of 16 A B 800 Frequency Frequency 600 400 200 600 400 200 0 0 0 5 10 15 20 25 30 35 40 45 50 55 0 5 10 15 20 25 30 35 40 45 50 55 TOP2A gene copies HER2 gene copies C 1.000 Frequency 800 600 400 200 0 0 5 10 15 20 25 30 35 40 45 50 55 CEP17 copies Figure 3 Distribution of HER2, TOP2A and CEP17 copies (A, B and C). Red line represents 6 gene copies (for A and B) and 3 copies for C. status and protein expression in 1031 patients with operable breast cancer. All these patients were treated with epirubicin-based adjuvant chemotherapy in the context of two consecutively conducted phase III trials [39-41]. In a previous study published by our group for the Table 3 CEP17 status according to HER2 and TOP2A gene copy number and amplification status CEP17 status No gain Gain N (%) N (%) HER2 gene copies p <0.001 ≤4 506 (68.2) 236 (31.8) 4-6 10 (15.4) 55 (84.6) >6 104 (46.4) 120 (53.6) TOP2A gene copies <0.001 ≤4 587 (67.1) 288 (32.9) 4-6 13 (16.9) 64 (83.1) >6 20 (25.3) 59 (74.7) HER2 gene status <0.001 Non-amplified 500 (63.5) 287 (36.5) Amplified 120 (49.2) 124 (50.8) Deleted 18 (34.6) 34 (65.4) Non-amplified 558 (63.8) 317 (36.2) Amplified 44 (42.3) 60 (57.7) TOP2A gene status <0.001 HE10/00 and HE10/97 cohorts [42], patients with either luminal B, luminal-HER2 or HER2-enriched tumors performed worse than those with luminal A tumors, while patients with triple-negative tumors had the worst outcome. In addition, it was observed that the HER2-enriched subtype was predictive of response to paclitaxel-containing treatments. These prognostic and predictive HER2-related effects were breast cancer subtype specific and were not maintained in the present study. An earlier observation reported for this cohort, of HER2 amplification being predictive for OS benefit from adjuvant treatment with paclitaxel [56], was not confirmed in the current analysis with updated follow-up. In both analyses however, the ability to detect any predictive impact of HER2/TOP2A amplification or CEP17 gain in the presence of taxanes was limited (only 1 of the 4 trial arms did not include taxanes). The present results concerning HER2 are in line with reports on the prognostic value of this marker [59,60]. A recent metaanalysis suggests that patients with both HER2 amplified and non-amplified tumors may benefit from anthracyclines [61,62]. This could not be investigated in the current study, since all patients had been treated with anthracyclines. Among HER2 amplified tumors, 42% exhibited TOP2A co-amplification, which is within the reported range of 35%-50% for this genomic alteration [26,29,63,64]. TOP2A deletions were more common in HER2 amplified tumors, comprising approximately 10% of the HER2 amplified cases. TOP2A gene pathology (amplification, deletion and combinations of both) has been reported as a favorable Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 Page 10 of 16 Table 4 HER2, TOP2A and CEP17 status and TopoIIa protein expression according to breast cancer subtypes defined by immunohistochemistry Luminal A Luminal B Luminal-HER2 HER2-enriched TNBC N (%) N (%) N (%) N (%) N (%) 242 (100.0) 386 (100.0) 3 (2.2) 1 (0.9) 126 (100.0) 0 0 135 (97.8) 107 (99.1) 0 FISH HER2 gene status Non-amplified Amplified TOP2A gene status Deleted Non-amplified Amplified 5 (2.1) 15 (3.9) 12 (8.7) 11 (10.2) 8 (6.3) 236 (97.5) 371 (96.1) 65 (47.1) 55 (50.9) 118 (93.7) 1 (0.4) 0 61 (44.2) 42 (38.9) 0 CEP17 status No gain 149 (61.6) 234 (60.6) 70 (50.7) 50 (46.3) 93 (73.8) Gain 93 (38.4) 152 (39.4) 68 (49.3) 58 (53.7) 33 (26.2) Negative 165 (73.7) 130 (35.2) 48 (36.9) 43 (41.7) 49 (42.6) Positive 59 (26.3) 239 (64.8) 82 (63.1) 60 (58.3) 66 (57.4) IHC TopoIIa TNBC, triple-negative breast cancer. Patients were classified as: luminal A (ER-positive and/or PgR-positive, HER2-negative, Ki67low); luminal B (ER-positive and/or PgR-positive, HER2-negative, Ki67high); luminal-HER2 (ER-positive and/or PgR-positive, HER2-positive); HER2-enriched (ER-negative, PgR-negative, HER2-positive); and TNBC (ER-negative, PgR-negative, HER2-negative). prognostic and predictive marker in adjuvant-treated breast cancer patients [36,65]. However, in the present study we did not observe any association between patient outcome and TOP2A amplification, deletion, or both, in accordance with the recent meta-analysis mentioned above [62]. The clinical importance of CEP17 gain, as detected by FISH, in human breast cancer remains a controversial issue. From the biological perspective, CEP17 gain and chromosome 17 polysomy do not represent the same situation, since the first one corresponds to the fluorescent signals of a 5.6 kb region, while the second reflects aberrant numbers of the whole chromosome, which should be demonstrated with spectral karyotyping (SKY) or other cytogenetic approaches. The CEP17 FISH probe detects the alpha-satellite repeat region at the centromere of chromosome 17, at 17p11.1-17q11.1. The specificity of CEP17 remains undetermined, while this probe and a centromeric probe detecting additional neighboring regions on 17p11.2-12 yield different results concerning Figure 4 Kaplan-Meier curves for DFS and OS according to HER2 gene status (log-rank test p-values). Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 Page 11 of 16 Figure 5 Kaplan-Meier curves for DFS and OS according to TOP2A gene status (log-rank test p-values). chromosome 17 status and, therefore, different HER2 gene amplification status, when the latter is assessed as HER2/ CEP17 ratio of >2.2 [66]. This may reflect the presence of the probed satellite repeats outside the centromeric region, which happen during evolution [67] and probably during cancer clone evolution, as well. Another problem for assessing low copy gains is that during DNA synthesis and in the G2/M phases, the targeted regions will appear double (three to four copies instead of two, taking into account the nuclear truncation effect during paraffin block sectioning). The cut-off used in the present study for the classification of CEP17 gains has been shown to correct for the maximally four centromeric signals that would be expected in this situation [55]. With this cut-off, we detected CEP17 gain in approximately 40% of all carcinomas examined, which is in the range of published results when using the FISH method in all-type breast carcinoma series (10-50%) [12-14,54,68,69]. Measurement of CEP17 probe signals reflects the condition of the corresponding centromeric area and can by no means reflect gains of the entire chromosome 17 or “chromosome 17 polysomy”, as often reported in the literature. In the same treatment settings, depending on how CEP17 signals are classified and interpreted, and also depending on the drugs administered, the effect of CEP17 status on patient outcome may vary. Thus, in the adjuvant setting, by using the same FISH probe, duplication of the CEP17 region of chromosome 17 seems to be predictive of benefit from anthracyclines [22,70] or of borderline association with clinical response to the same drugs [71]. Furthermore, CEP17 gain in the absence of HER2/TOP2A amplification has been reported by one recent study to be an unfavorable prognostic marker [72]; however, no prognostic value was identified for CEP17 gain in other studies [55,64,69], which is in line with our present findings. Figure 6 Kaplan-Meier curves for DFS and OS according to CEP17 gain (log-rank test p-values). Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 Page 12 of 16 Figure 7 Kaplan-Meier curves for DFS and OS according to HER2/TOP2A co-amplification (log-rank test p-values). CEP17 appears to be related with disease prognosis when this marker is combined with HER2 status. Whether “polysomy” 17 drives HER2 amplification or the opposite is true, as was recently suggested [54,73], remains unanswered; it is, however, noteworthy that “polysomy” 17 is rarely observed in circulating tumor cells from patients with metastatic breast cancer and when present, it corresponds to HER2-negative primary tumors [74]. In the absence of HER2 amplification, CEP17 “polysomy” has been reported to confer a more favorable prognosis [75] or to be associated with aggravating prognostic markers [76]. In addition to these contradictory results, herein we did not observe any interaction between CEP17 and HER2 status. Methodological differences in the assessment of Figure 8 Multivariate analyses presented by forest plots in terms of DFS (A) and OS (B). Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 these parameters and cohort-fitted results should account for the diversity of data regarding the role of CEP17/HER2 status on adjuvant-treated breast cancer patients. Although more than half of the tumors with CEP17 gain were not HER2 or TOP2A amplified, we did observe a higher than double incidence of CEP17 gain in tumors with aberrant HER2 and TOP2A genes (amplification, deletion, and, especially, high copy gains) in comparison to tumors with a normal status of these genes. These data are in line with “polysomy” 17 correlating with multiple copies of HER2 but not with HER2 amplification [77], while they further justify the higher incidence of CEP17 gain in the luminal-HER2 and HER2-enriched subtypes, as described in this study. Equivocal HER2 IHC findings were observed in cases with chromosome 17 “polysomy” and correspondingly increased HER2 gene copy numbers [54,78]. In the present study, the incidence of CEP17 gains was strongly related to HER2 IHC grades, but it did not contribute to the further assessment of HER2 IHC 2+ cases. With respect to FISH equivocal cases, herein we used very stringent criteria involving both gene/CEP17 ratios and gene copy numbers. Increased CEP17 ratios might result in false negative HER2/CEP17 ratios of ≤2.2; on the other hand, their coincidence with increased HER2 copies is not equivalent to HER2-positive disease [79]. The few (n = 10, <0.01% of the entire tumor series) FISH equivocal tumors for HER2 gene status were HER2 IHC 0 to 2+. CEP17 gain was observed in only one such case. Hence, at least in the present series, CEP17 gain did not aid further in the classification of equivocal HER2 gene status cases. In addition, we observed that most of the tumors with low-gain of HER2 and/or TOP2A copies indeed had CEP17 gains as well. However, in order to evaluate the impact of these concomitant alterations on patient outcome, larger patient series with this sub-category of tumors would be needed. Conclusions The present combined chromosome 17 marker analysis by FISH represents one of the largest of its type in early highrisk breast cancer. With the cut-offs used for the characterization of CEP17 gain, as well as HER2 and TOP2A gene amplification, these chromosome 17 markers, individually or in conjunction, did not appear to be related with patient outcome. Additional file Additional file 1: Table S1. Comparison of basic clinicopathological characteristics between patients with and without available tissue material (paraffin blocks) per study. Table S2. Association of CEP17, HER2 and TOP2A markers with basic clinicopathological parameters. Table S3. Distribution of HER2 gene status according to HER2 protein expression. Page 13 of 16 Table S4. Distribution of TOP2A gene status according to TopoIIa protein expression. Table S5. Distribution of CEP17 status according to HER2 and TopoIIa protein expression. Table S6. Association of HER2 and TOP2A gene status assessed by FISH. Table S7. Survival data for the total study population and according to randomization group. Abbreviations CGH: Comparative genomic hybridization; ANZCTR: Australian New Zealand Clinical Trials Registry; BRCA1: Breast cancer type 1 susceptibility protein; CEP17: Centromere 17 enumeration probe; CISH: Chromogen in situ hybridization; CMF: Cyclophosphamide, Methotrexate, Fluorouracil; DFS: Diseasefree survival; DNA: Deoxyribonucleic acid; E: Epirubicin; ER: Estrogen receptor; FFPE: Formalin-fixed, paraffin-embedded; FISH: Fluorescence in situ hybridization; G-CSF: Granulocyte-colony stimulating factor; HeCOG: Hellenic Cooperative Oncology Group; HER2: Human epidermal growth factor receptor 2; HR: Hazard ratio; HT: Hormonal therapy; IHC: Immunohistochemistry; Ki67: Antigen Ki67; MLPA: Multiplex ligation-dependent probe amplification; OS: Overall survival; PgR: Progesterone receptor; RAD51C: RAD51 homolog C; RARA: Retinoic acid receptor, alpha; REMARK: Reporting Recommendations for Tumor Marker Prognostic Studies; RNA: Ribonucleic acid; SMS: Spermine synthase; SNP: Single nucleotide polymorphism; T: Taxol (Paclitaxel); TMA: Tissue microarray; TNBC: Triple-negative breast cancer; TOP2A: Topoisomerase II alpha (gene expression); TopoIIa: Topoisomerase II alpha (protein expression); TP53: Tumor protein 53. Competing interests On behalf of the Hellenic Foundation for Cancer Research, Athens, Greece, the senior author (GF) has pending patent applications with Siemens Healthcare Diagnostics, Tarrytown, NY. The rest of the authors declare that they have no competing interests. Authors’ contributions GF conceived of the study, participated in its design and coordination, contributed to the analysis and interpretation of data and drafted the manuscript. UD conceived of the study, participated in its design, contributed to the analysis and interpretation of data and drafted the manuscript. MB carried out the IHC and FISH analysis, contributed to the analysis and interpretation of data and drafted the manuscript. VK conceived of the study, participated in its design, carried out the molecular studies, contributed to the analysis and interpretation of data and drafted the manuscript. AB carried out the immunoassays and contributed to the analysis and interpretation of data. IX participated in the acquisition of data and contributed to the collection of the tumor tissue samples analyzed in the study. CP participated in the acquisition of data and contributed to the collection of the tumor tissue samples analyzed in the study. TK carried out the molecular studies and contributed to the analysis and interpretation of data. ET carried out the FISH assays and contributed to the analysis and interpretation of data. DT carried out the immunoassays and contributed to the analysis and interpretation of data. ET participated in the acquisition of data and contributed to the collection of the tumor tissue samples analyzed in the study. AK participated in the acquisition of data and contributed to the collection of the tumor tissue samples analyzed in the study. GK participated in the acquisition of data. ES participated in the acquisition of data and contributed to the collection of the tumor tissue samples analyzed in the study. NP participated in the acquisition of data and contributed to the collection of the tumor tissue samples analyzed in the study. CK participated in the acquisition of data and contributed to the collection of the tumor tissue samples analyzed in the study. IS participated in the acquisition of data. NP participated in the acquisition of data and contributed to the collection of the tumor tissue samples analyzed in the study. HG participated in the acquisition of data and contributed to the collection of the tumor tissue samples analyzed in the study. HL participated in the acquisition of data and contributed to the collection of the tumor tissue samples analyzed in the study. KTK conceived of the study, participated in its design, contributed to the analysis and interpretation of data and drafted the manuscript. DP conceived of the study, participated in its design and contributed to the analysis and interpretation of data. MAD conceived of the study, participated in its design, contributed to the analysis and interpretation of data and drafted the manuscript. All authors read and approved the final manuscript. Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 Acknowledgements Presented in part at the 34th Annual San Antonio Breast Cancer Symposium, December 6–10, 2011. The authors are indebted to all patients and their families for their trust and participation in the HE10/97 and HE10/00 trials and for the provision of biological material for research purposes. The authors also wish to thank all HeCOG personnel (data managers, research assistants and monitors) for their dedication, M. Moschoni for data coordination, T. Spinari for collection of FFPE tissue blocks and S. Dallidou for secretarial assistance. Supported by an internal Hellenic Cooperative Oncology Group (HeCOG) translational research grant (HE TRANS_BR). Author details Department of Medical Oncology, “Papageorgiou” Hospital, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece. 2 Laboratory of Biostatistics, University of Athens School of Nursing, Athens, Greece. 3Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece. 4Department of Pathology, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece. 5Department of Pathology, Ioannina University Hospital, Ioannina, Greece. 6Department of Clinical Therapeutics, “Alexandra” Hospital, University of Athens School of Medicine, Athens, Greece. 7Division of Oncology, Department of Medicine, University Hospital, University of Patras Medical School, Patras, Greece. 8Second Department of Medical Oncology, “Metropolitan” Hospital, Athens, Greece. 9Third Department of Medical Oncology, “Agii Anargiri”, Cancer Hospital, Athens, Greece. 10Department of Medical Oncology, IKA Hospital, Thessaloniki, Greece. 11Department of Medical Oncology, 424 Army General Hospital, Thessaloniki, Greece. 12Department of Medical Oncology, Ioannina University Hospital, Ioannina, Greece. 13First Department of Medicine, “Laiko” General Hospital, University of Athens School of Medicine, Athens, Greece. 14First Department of Medical Oncology, “Metropolitan” Hospital, Athens, Greece. 15 Translational Research Section, Hellenic Cooperative Oncology Group, Data Office, Athens, Greece. 16Oncology Section, Second Department of Internal Medicine, “Hippokration” Hospital, Athens, Greece. 1 Page 14 of 16 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Received: 21 November 2012 Accepted: 20 March 2013 Published: 28 March 2013 References 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ: Cancer statistics. CA Cancer J Clin 2009, 59(4):225–249. 2. La Vecchia C, Bosetti C, Lucchini F, Bertuccio P, Negri E, Boyle P, Levi F: Cancer mortality in Europe, 2000–2004, and an overview of trends since 1975. Ann Oncol 2010, 21(6):1323–1360. 3. Early Breast Cancer Trialists' Collaborative Group (EBCTCG): Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet 2005, 365(9472):1687–1717. 4. Levine MN, Whelan T: Adjuvant chemotherapy for breast cancer–30 years later. N Engl J Med 2006, 355(18):1920–1922. 5. Smith I, Chua S: Medical treatment of early breast cancer. III: chemotherapy. Bmj 2006, 332(7534):161–162. 6. Doyle JJ, Neugut AI, Jacobson JS, Grann VR, Hershman DL: Chemotherapy and cardiotoxicity in older breast cancer patients: a population-based study. J Clin Oncol 2005, 23(34):8597–8605. 7. Hershman D, Neugut AI, Jacobson JS, Wang J, Tsai WY, McBride R, Bennett CL, Grann VR: Acute myeloid leukemia or myelodysplastic syndrome following use of granulocyte colony-stimulating factors during breast cancer adjuvant chemotherapy. J Natl Cancer Inst 2007, 99(3):196–205. 8. Thompson F, Emerson J, Dalton W, Yang JM, McGee D, Villar H, Knox S, Massey K, Weinstein R, Bhattacharyya A, et al: Clonal chromosome abnormalities in human breast carcinomas. I. Twenty-eight cases with primary disease. Genes Chromosomes Cancer 1993, 7(4):185–193. 9. Zody MC, Garber M, Adams DJ, Sharpe T, Harrow J, Lupski JR, Nicholson C, Searle SM, Wilming L, Young SK, et al: DNA sequence of human chromosome 17 and analysis of rearrangement in the human lineage. Nature 2006, 440(7087):1045–1049. 10. Botti C, Pescatore B, Mottolese M, Sciarretta F, Greco C, Di Filippo F, Gandolfo GM, Cavaliere F, Bovani R, Varanese A, et al: Incidence of 21. 22. 23. 24. 25. 26. 27. 28. 29. chromosomes 1 and 17 aneusomy in breast cancer and adjacent tissue: an interphase cytogenetic study. J Am Coll Surg 2000, 190(5):530–539. Reinholz MM, Bruzek AK, Visscher DW, Lingle WL, Schroeder MJ, Perez EA, Jenkins RB: Breast cancer and aneusomy 17: implications for carcinogenesis and therapeutic response. Lancet Oncol 2009, 10(3):267–277. Salido M, Tusquets I, Corominas JM, Suarez M, Espinet B, Corzo C, Bellet M, Fabregat X, Serrano S, Sole F: Polysomy of chromosome 17 in breast cancer tumors showing an overexpression of ERBB2: a study of 175 cases using fluorescence in situ hybridization and immunohistochemistry. Breast Cancer Res 2005, 7(2):26. Dal Lago L, Durbecq V, Desmedt C, Salgado R, Verjat T, Lespagnard L, Ma Y, Veys I, Di Leo A, Sotiriou C, et al: Correction for chromosome-17 is critical for the determination of true Her-2/neu gene amplification status in breast cancer. Mol Cancer Ther 2006, 5(10):2572–2579. Hyun CL, Lee HE, Kim KS, Kim SW, Kim JH, Choe G, Park SY: The effect of chromosome 17 polysomy on HER-2/neu status in breast cancer. J Clin Pathol 2008, 61(3):317–321. Vranic S, Teruya B, Repertinger S, Ulmer P, Hagenkord J, Gatalica Z: Assessment of HER2 gene status in breast carcinomas with polysomy of chromosome 17. Cancer 2011, 117(1):48–53. Yeh IT, Martin MA, Robetorye RS, Bolla AR, McCaskill C, Shah RK, Gorre ME, Mohammed MS, Gunn SR: Clinical validation of an array CGH test for HER2 status in breast cancer reveals that polysomy 17 is a rare event. Mod Pathol 2009, 22(9):1169–1175. Viale G: Be precise! The need to consider the mechanisms for CEP17 copy number changes in breast cancer. J Pathol 2009, 219(1):1–2. Marchio C, Lambros MB, Gugliotta P, Di Cantogno LV, Botta C, Pasini B, Tan DS, Mackay A, Fenwick K, Tamber N, et al: Does chromosome 17 centromere copy number predict polysomy in breast cancer? A fluorescence in situ hybridization and microarray-based CGH analysis. J Pathol 2009, 219(1):16–24. Moelans CB, de Weger RA, van Diest PJ: Absence of chromosome 17 polysomy in breast cancer: analysis by CEP17 chromogenic in situ hybridization and multiplex ligation-dependent probe amplification. Breast Cancer Res Treat 2010, 120(1):1–7. Tse CH, Hwang HC, Goldstein LC, Kandalaft PL, Wiley JC, Kussick SJ, Gown AM: Determining true HER2 gene status in breast cancers with polysomy by using alternative chromosome 17 reference genes: implications for anti-HER2 targeted therapy. J Clin Oncol 2011, 29(31):4168–4174. Moelans CB, Reis-Filho JS, van Diest PJ: Implications of rarity of chromosome 17 polysomy in breast cancer. Lancet Oncol 2011, 12(12):1087–1089. Bartlett JM, Munro AF, Dunn JA, McConkey C, Jordan S, Twelves CJ, Cameron DA, Thomas J, Campbell FM, Rea DW, et al: Predictive markers of anthracycline benefit: a prospectively planned analysis of the UK National Epirubicin Adjuvant Trial (NEAT/BR9601). Lancet Oncol 2010, 11(3):266–274. Pauletti G, Godolphin W, Press MF, Slamon DJ: Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization. Oncogene 1996, 13(1):63–72. Tanner M, Jarvinen P, Isola J: Amplification of HER-2/neu and topoisomerase IIalpha in primary and metastatic breast cancer. Cancer Res 2001, 61(14):5345–5348. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL: Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987, 235(4785):177–182. Jarvinen TA, Liu ET: Simultaneous amplification of HER-2 (ERBB2) and topoisomerase IIalpha (TOP2A) genes–molecular basis for combination chemotherapy in cancer. Curr Cancer Drug Targets 2006, 6(7):579–602. Hayes DF, Thor AD, Dressler LG, Weaver D, Edgerton S, Cowan D, Broadwater G, Goldstein LJ, Martino S, Ingle JN, et al: HER2 and response to paclitaxel in node-positive breast cancer. N Engl J Med 2007, 357(15):1496–1506. Gennari A, Sormani MP, Pronzato P, Puntoni M, Colozza M, Pfeffer U, Bruzzi P: HER2 status and efficacy of adjuvant anthracyclines in early breast cancer: a pooled analysis of randomized trials. J Natl Cancer Inst 2008, 100(1):14–20. Di Leo A, Larsimont D, Gancberg D, Jarvinen T, Beauduin M, Vindevoghel A, Michel J, Focan CH, Ries F, Gobert PH, et al: HER-2 and topo-isomerase IIalpha as predictive markers in a population of node-positive breast cancer patients randomly treated with adjuvant CMF or epirubicin plus cyclophosphamide. Ann Oncol 2001, 12(8):1081–1089. Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 30. Konecny GE, Thomssen C, Luck HJ, Untch M, Wang HJ, Kuhn W, Eidtmann H, du Bois A, Olbricht S, Steinfeld D, et al: Her-2/neu gene amplification and response to paclitaxel in patients with metastatic breast cancer. J Natl Cancer Inst 2004, 96(15):1141–1151. 31. Kellner U, Sehested M, Jensen PB, Gieseler F, Rudolph P: Culprit and victim – DNA topoisomerase II. Lancet Oncol 2002, 3(4):235–243. 32. Beser AR, Tuzlali S, Guzey D, Dolek Guler S, Hacihanefioglu S, Dalay N: HER2, TOP2A and chromosome 17 alterations in breast cancer. Pathol Oncol Res 2007, 13(3):180–185. 33. Champoux JJ: DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 2001, 70:369–413. 34. Ju BG, Lunyak VV, Perissi V, Garcia-Bassets I, Rose DW, Glass CK, Rosenfeld MG: A topoisomerase IIbeta-mediated dsDNA break required for regulated transcription. Science 2006, 312(5781):1798–1802. 35. Jarvinen TA, Tanner M, Barlund M, Borg A, Isola J: Characterization of topoisomerase II alpha gene amplification and deletion in breast cancer. Genes Chromosomes Cancer 1999, 26(2):142–150. 36. Knoop AS, Knudsen H, Balslev E, Rasmussen BB, Overgaard J, Nielsen KV, Schonau A, Gunnarsdottir K, Olsen KE, Mouridsen H, et al: Retrospective analysis of topoisomerase IIa amplifications and deletions as predictive markers in primary breast cancer patients randomly assigned to cyclophosphamide, methotrexate, and fluorouracil or cyclophosphamide, epirubicin, and fluorouracil: Danish Breast Cancer Cooperative Group. J Clin Oncol 2005, 23(30):7483–7490. 37. Schindlbeck C, Mayr D, Olivier C, Rack B, Engelstaedter V, Jueckstock J, Jenderek C, Andergassen U, Jeschke U, Friese K: Topoisomerase IIalpha expression rather than gene amplification predicts responsiveness of adjuvant anthracycline-based chemotherapy in women with primary breast cancer. Cancer Res Clin Oncol 2010, 136(7):1029–1037. 38. Simon RM, Paik S, Hayes DF: Use of archived specimens in evaluation of prognostic and predictive biomarkers. J Natl Cancer Inst 2009, 101(21):1446–1452. 39. Fountzilas G, Skarlos D, Dafni U, Gogas H, Briasoulis E, Pectasides D, Papadimitriou C, Markopoulos C, Polychronis A, Kalofonos HP, et al: Postoperative dose-dense sequential chemotherapy with epirubicin, followed by CMF with or without paclitaxel, in patients with high-risk operable breast cancer: a randomized phase III study conducted by the Hellenic Cooperative Oncology Group. Ann Oncol 2005, 16(11):1762–1771. 40. Fountzilas G, Dafni U, Gogas H, Linardou H, Kalofonos HP, Briasoulis E, Pectasides D, Samantas E, Bafaloukos D, Stathopoulos GP, et al: Postoperative dose-dense sequential chemotherapy with epirubicin, paclitaxel and CMF in patients with high-risk breast cancer: safety analysis of the Hellenic Cooperative Oncology Group randomized phase III trial HE 10/00. Ann Oncol 2008, 19(5):853–860. 41. Gogas H, Dafni U, Karina M, Papadimitriou C, Batistatou A, Bobos M, Kalofonos HP, Eleftheraki AG, Timotheadou E, Bafaloukos D, et al: Postoperative dose-dense sequential versus concomitant administration of epirubicin and paclitaxel in patients with node-positive breast cancer: 5-year results of the Hellenic Cooperative Oncology Group HE 10/00 phase III Trial. Breast Cancer Res Treatment 2012, 132(2):609–619. 42. Fountzilas G, Dafni U, Bobos M, Batistatou A, Kotoula V, Trihia H, MalamouMitsi V, Miliaras S, Chrisafi S, Papadopoulos S, et al: Differential response of immunohistochemically defined breast cancer subtypes to anthracycline-based adjuvant chemotherapy with or without paclitaxel. PLoS One 2012, 7(6):e37946. 43. Razis E, Bobos M, Kotoula V, Eleftheraki AG, Kalofonos HP, Pavlakis K, Papakostas P, Aravantinos G, Rigakos G, Efstratiou I, et al: Evaluation of the association of PIK3CA mutations and PTEN loss with efficacy of trastuzumab therapy in metastatic breast cancer. Breast Cancer Research Treat 2011, 128(2):447–456. 44. McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM: REporting recommendations for tumor MARKer prognostic studies (REMARK). Breast Cancer Res Treat 2006, 100(2):229–235. 45. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G, Kallioniemi OP: Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 1998, 4(7):844–847. 46. Skacel M, Skilton B, Pettay JD, Tubbs RR: Tissue microarrays: a powerful tool for high-throughput analysis of clinical specimens: a review of the Page 15 of 16 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. method with validation data. Appl Immunohistochem Mol Morphol 2002, 10(1):1–6. Fountzilas G, Kourea HP, Bobos M, Televantou D, Kotoula V, Papadimitriou C, Papazisis KT, Timotheadou E, Efstratiou I, Koutras A, et al: Paclitaxel and bevacizumab as first line combined treatment in patients with metastatic breast cancer: the Hellenic Cooperative Oncology Group experience with biological marker evaluation. Anticancer Res 2011, 31(9):3007–3018. Christodoulou C, Kostopoulos I, Kalofonos HP, Lianos E, Bobos M, Briasoulis E, Gogas H, Razis E, Skarlos DV, Fountzilas G: Trastuzumab combined with pegylated liposomal doxorubicin in patients with metastatic breast cancer. phase II Study of the Hellenic Cooperative Oncology Group (HeCOG) with biomarker evaluation. Oncology 2009, 76(4):275–285. Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, Dowsett M, Fitzgibbons PL, Hanna WM, Langer A, et al: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 2007, 25(1):118–145. Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, Fitzgibbons PL, Francis G, Goldstein NS, Hayes M, et al: American society of clinical oncology/college of American pathologists guideline recommendations for immunohistochemical testing of Estrogen and progesterone receptors in breast cancer. J Clin Oncol 2010, 28(16):2784–2795. Cheang MC, Chia SK, Voduc D, Gao D, Leung S, Snider J, Watson M, Davies S, Bernard PS, Parker JS, et al: Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer. J Natl Cancer Inst 2009, 101(10):736–750. Bhargava R, Lal P, Chen B: HER-2/neu and topoisomerase IIa gene amplification and protein expression in invasive breast carcinomas: chromogenic in situ hybridization and immunohistochemical analyses. Am J Clin Pathol 2005, 123(6):889–895. Press MF, Sauter G, Buyse M, Bernstein L, Guzman R, Santiago A, Villalobos IE, Eiermann W, Pienkowski T, Martin M, et al: Alteration of topoisomerase II-alpha gene in human breast cancer: association with responsiveness to anthracycline-based chemotherapy. J Clin Oncol 2011, 29(7):859–867. Vanden Bempt I, Van Loo P, Drijkoningen M, Neven P, Smeets A, Christiaens MR, Paridaens R, De Wolf-Peeters C: Polysomy 17 in breast cancer: clinicopathologic significance and impact on HER-2 testing. J Clin Oncol 2008, 26(30):4869–4874. Perez EA, Reinholz MM, Hillman DW, Tenner KS, Schroeder MJ, Davidson NE, Martino S, Sledge GW, Harris LN, Gralow JR, et al: HER2 and chromosome 17 effect on patient outcome in the N9831 adjuvant trastuzumab trial. J Clin Oncol 2010, 28(28):4307–4315. Dafni U, Bobos M, Tsolaki E, Batistatou A, Koletsa F, Televantou D, Gogas H, Linardou H, Pectasides D, Kalogeras KT, Galani E, Koutras A, Papadimitriou CA, Fountzilas G: Effect of HER2/Topoisomerase II alpha (TOP2A) Gene Status or Protein Expression and Chromosome 17 (CEP17) Polysomy on the Outcome of Breast Cancer Patients Treated with AnthracyclineContaining Dose-Dense Sequential Adjuvant Chemotherapy with or without Paclitaxel. A pooled analysis of two Hellenic Cooperative Oncology Group (HeCOG) phase III trials. Cancer Res 2011, 71(24 Suppl): Abstract nr PD05–02. Sauter G, Lee J, Bartlett JM, Slamon DJ, Press MF: Guidelines for human epidermal growth factor receptor 2 testing: biologic and methodologic considerations. J Clin Oncol 2009, 27(8):1323–1333. Hudis CA, Barlow WE, Costantino JP, Gray RJ, Pritchard KI, Chapman JA, Sparano JA, Hunsberger S, Enos RA, Gelber RD, et al: Proposal for standardized definitions for efficacy end points in adjuvant breast cancer trials: the STEEP system. J Clin Oncol 2007, 25(15):2127–2132. Clark GM, McGuire WL: Follow-up study of HER-2/neu amplification in primary breast cancer. Cancer Res 1991, 51(3):944–948. Allred DC, Clark GM, Tandon AK, Molina R, Tormey DC, Osborne CK, Gilchrist KW, Mansour EG, Abeloff M, Eudey L, et al: HER-2/neu in node-negative breast cancer: prognostic significance of overexpression influenced by the presence of in situ carcinoma. J Clin Oncol 1992, 10(4):599–605. Bonnefoi HR: Anthracyclines, HER2, and TOP2A: the verdict. Lancet Oncol 2011, 12(12):1084–1085. Di Leo A, Desmedt C, Bartlett JM, Piette F, Ejlertsen B, Pritchard KI, Larsimont D, Poole C, Isola J, Earl H, et al: HER2 and TOP2A as predictive markers for anthracycline-containing chemotherapy regimens as adjuvant treatment Fountzilas et al. BMC Cancer 2013, 13:163 http://www.biomedcentral.com/1471-2407/13/163 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. of breast cancer: a meta-analysis of individual patient data. Lancet Oncol 2011, 12(12):1134–1142. Hicks DG, Tubbs RR: Assessment of the HER2 status in breast cancer by fluorescence in situ hybridization: a technical review with interpretive guidelines. Hum Pathol 2005, 36(3):250–261. Nielsen KV, Ejlertsen B, Moller S, Jensen MB, Balslev E, Muller S, Knoop A, Mouridsen HT: Lack of independent prognostic and predictive value of centromere 17 copy number changes in breast cancer patients with known HER2 and TOP2A status. Mol Oncol 2012, 6(1):88–97. O’Malley FP, Chia S, Tu D, Shepherd LE, Levine MN, Bramwell VH, Andrulis IL, Pritchard KI: Topoisomerase II alpha and responsiveness of breast cancer to adjuvant chemotherapy. J Natl Cancer Inst 2009, 101(9):644–650. Varga Z, Tubbs RR, Wang Z, Sun Y, Noske A, Kradolfer D, Bosshard G, Jochum W, Moch H, Ohlschlegel C: Co-amplification of the HER2 gene and chromosome 17 centromere: a potential diagnostic pitfall in HER2 testing in breast cancer. Breast Cancer Res Treat 2012, 132(3):925–935. She X, Horvath JE, Jiang Z, Liu G, Furey TS, Christ L, Clark R, Graves T, Gulden CL, Alkan C, et al: The structure and evolution of centromeric transition regions within the human genome. Nature 2004, 430(7002):857–864. Baehner FL, Achacoso N, Maddala T, Shak S, Quesenberry CP Jr, Goldstein LC, Gown AM, Habel LA: Human epidermal growth factor receptor 2 assessment in a case–control study: comparison of fluorescence in situ hybridization and quantitative reverse transcription polymerase chain reaction performed by central laboratories. J Clin Oncol 2010, 28(28):4300–4306. Zaczek A, Markiewicz A, Supernat A, Bednarz-Knoll N, Brandt B, Seroczynska B, Skokowski J, Szade J, Czapiewski P, Biernat W, et al: Prognostic Value of TOP2A Gene Amplification and Chromosome 17 Polysomy in Early Breast Cancer. Pathol Oncol Res 2012, 18(4):885–894. Munro AF, Cameron DA, Bartlett JM: Targeting anthracyclines in early breast cancer: new candidate predictive biomarkers emerge. Oncogene 2010, 29(38):5231–5240. Pritchard KI, Munro A, O’Malley FP, Tu D, Li X, Levine MN, Shepherd L, Chia S, Bartlett JM: Chromosome 17 centromere (CEP17) duplication as a predictor of anthracycline response: evidence from the NCIC Clinical Trials Group (NCIC CTG) MA.5 Trial. Breast Cancer Res Treat 2012, 131(2):541–551. Kim A, Shin HC, Bae YK, Kim MK, Kang SH, Lee SJ, Lee EH: Multiplication of Chromosome 17 Centromere Is Associated with Prognosis in Patients with Invasive Breast Cancers Exhibiting Normal HER2 and TOP2A Status. J Breast Cancer 2012, 15(1):24–33. Vanden Bempt I, Drijkoningen M, De Wolf-Peeters C: The complexity of genotypic alterations underlying HER2-positive breast cancer: an explanation for its clinical heterogeneity. Curr Opin Oncol 2007, 19(6):552–557. Hayashi N, Nakamura S, Yagata H, Shimoda Y, Ota H, Hortobagyi GN, Cristofanilli M, Ueno NT: Chromosome 17 polysomy in circulating tumor cells in patients with metastatic breast cancer: a case series. Int J Clin Oncol 2011, 16(5):596–600. Torrisi R, Rotmensz N, Bagnardi V, Viale G, Curto BD, Dell’orto P, Veronesi P, Luini A, D’Alessandro C, Cardillo A, et al: HER2 status in early breast cancer: relevance of cell staining patterns, gene amplification and polysomy 17. Eur J Cancer 2007, 43(16):2339–2344. Krishnamurti U, Hammers JL, Atem FD, Storto PD, Silverman JF: Poor prognostic significance of unamplified chromosome 17 polysomy in invasive breast carcinoma. Mod Pathol 2009, 22(8):1044–1048. Watters AD, Going JJ, Cooke TG, Bartlett JM: Chromosome 17 aneusomy is associated with poor prognostic factors in invasive breast carcinoma. Breast Cancer Res Treat 2003, 77(2):109–114. Bose S, Mohammed M, Shintaku P, Rao PN: Her-2/neu gene amplification in low to moderately expressing breast cancers: possible role of chromosome 17/Her-2/neu polysomy. Breast J 2001, 7(5):337–344. Ross JS: Human epidermal growth factor receptor 2 testing in 2010: does chromosome 17 centromere copy number make any difference? J Clin Oncol 2010, 28(28):4293–4295. doi:10.1186/1471-2407-13-163 Cite this article as: Fountzilas et al.: Evaluation of the prognostic role of centromere 17 gain and HER2/topoisomerase II alpha gene status and protein expression in patients with breast cancer treated with anthracycline-containing adjuvant chemotherapy: pooled analysis of two Hellenic Cooperative Oncology Group (HeCOG) phase III trials. BMC Cancer 2013 13:163. Page 16 of 16 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W3199611646	https://link.springer.com/content/pdf/10.1007/s00404-021-06246-w.pdf	English	null	Peripartal pain perception and pain therapy: introduction and validation of a questionnaire as a quality instrument	Archives of gynecology and obstetrics	2,021	cc-by	7,811	* A. Linzbach aissa.linzbach@med.uni-jena.de Abstract Keywords Labor pain · Questionnaire · Pain measurement · Pain therapy · Vaginal birth · NRS—numeric rating scale Keywords Labor pain · Questionnaire · Pain measurement · Pain therapy · Vaginal birth · NRS—num Peripartal pain perception and pain therapy: introduction and validation of a questionnaire as a quality instrument A. Linzbach1 · D. Nitschke1 · J. Rothaug1 · M. Komann2 · C. Weinmann2 · E. Schleußner1 · W J. Jimenez Cruz3 · U. Schneider1 Received: 14 July 2020 / Accepted: 2 September 2021 / Published online: 20 September 2021 © The Author(s) 2021 1 Department of Obstetrics, Division of Prenatal Diagnostics and Fetal Physiology, University Hospital Jena, Am Klinikum 1, 07747 Jena, Germany Archives of Gynecology and Obstetrics (2022) 305:1409–1419 https://doi.org/10.1007/s00404-021-06246-w Archives of Gynecology and Obstetrics (2022) 305:1409–1419 https://doi.org/10.1007/s00404-021-06246-w MATERNAL-FETAL MEDICINE 2 Department of Anesthesiology and Intensive Care Medicine, Section Pain Therapy, University Hospital Jena, Jena, Germany Abstract Background Labor pain is difficult to measure. The aim of this proof-of-concept study is to implement and test a question- naire assessing pain sensation during and after vaginal deliveries. Its key aspect is a highly standardized survey of patient- reported outcome (PRO) by staff not involved in routine care. f Methods Between January and November 2015 339 women were assessed 24–48 h after spontaneous or operative-vaginal delivery of a singleton. German language skills were a prerequisite to participate. The test–retest reliability was calculated in 38 women 24–36 and 48–72 h postpartum between July and October 2017. Primiparae after spontaneous delivery and multiparae with no history of operative deliveries were compared in a subgroup analysis. Results Maximum labor pain and post-partum pain were reported a median of 9 [8–10] and 4 [3–6]. Higher ratings were associated with younger age, higher gestational ages, infant’s biometrics, and the duration of laboring. y g g , g g g , , g Only regional analgesia tended to reduce pain perception (NRS 8 vs. 9). Higher-degree injuries were associated with less pain postpartum. The questionnaire proved to be reliable in most aspects (Cronbach's α > 0.6 for 19/21 questions) and showed an acceptable content and criterion validity (Cohen correlation > ± 0.3, interrelation between items). Conclusion Labor is a very painful experience, irrespective of previous obstetric history. Ratings indicate inadequateness of treatment except for patients receiving preventive postoperative pain management. Systematic postpartum pain assessment, h i ill di i Adj ill b d i l kill d ifi i ff i Only regional analgesia tended to reduce pain perception (NRS 8 vs. 9). Higher-degree injuries were associated with less pain postpartum. The questionnaire proved to be reliable in most aspects (Cronbach's α > 0.6 for 19/21 questions) and showed an acceptable content and criterion validity (Cohen correlation > ± 0.3, interrelation between items). Conclusion Labor is a very painful experience, irrespective of previous obstetric history. Ratings indicate inadequateness of treatment except for patients receiving preventive postoperative pain management. Systematic postpartum pain assessment, hence, is still a pending issue. Adjustments will be made concerning language skills and specific questions on effectiveness of analgesia otherwise good reliability and validity of the questionnaire were proven. * A. Linzbach aissa.linzbach@med.uni-jena.de 1 Department of Obstetrics, Division of Prenatal Diagnostics and Fetal Physiology, University Hospital Jena, Am Klinikum 1, 07747 Jena, Germany 2 Department of Anesthesiology and Intensive Care Medicine, Section Pain Therapy, University Hospital Jena, Jena, Germany 3 Department of Obstetrics and Prenatal Medicine, University Hospital Bonn, Bonn, Germany Methods This study is a prospective, non-randomized, unblinded sur- vey study on subjective pain experience during and after spontaneous or operative-vaginal delivery (outcome ques- tionnaire) and its association with demographic and obstet- ric aspects (process questionnaire). The questionnaire were modified based on the original QUIPS template and will be integrated into the QUIPS project after adjustment. The local ethics committee of Jena University Hospital has approved the study. y The observation period covered the months from January to November 2015 at the Department of Obstetrics at Jena University Hospital. Staff not involved in peripartum care visited the maternity ward on 3–4 days per week. For Re- Test validation, a second survey period was performed from July 2017 to October 2017 (see Statistics section for details). Eligible women were visited 24–48 h after vaginal deliv- ery of a singleton, consented, and the questionnaire was handed to them and re-collected after 30 min. Since this study was conceived as a validation study of a questionnaire in German language, lack of German language skills had to be treated as an exclusion criterion. The observation period covered the months from January to November 2015 at the Department of Obstetrics at Jena University Hospital. Staff not involved in peripartum care visited the maternity ward on 3–4 days per week. For Re- Test validation, a second survey period was performed from July 2017 to October 2017 (see Statistics section for details). Eligible women were visited 24–48 h after vaginal deliv- ery of a singleton, consented, and the questionnaire was handed to them and re-collected after 30 min. Since this study was conceived as a validation study of a questionnaire in German language, lack of German language skills had to be treated as an exclusion criterion. Therefore, pain is difficult to measure. Improvement of labor-pain management is hardly achievable by conducting prospective, randomized controlled trials. Supplementary tools are required to adjust interventions to the needs of the individual based on robust epidemiological data and semi- quantitative measures. The event progression and pain therapy data were col- lected from the birth register and the patient files of mother and newborn in the maternity ward. Introduction multi-dimensionally. It incorporates understanding the ori- gin of labor pain stimuli and the mechanisms of pain modu- lation, but also the consideration of individual psychological aspects including suffering and respectability to consolation [1]. Labor and giving birth are physiological processes, but also fundamentally painful experiences. Pain experience during childbirth depends on the interaction of multiple physiologic and psychosocial factors and should be assessed The major pain causing mechanisms during labor, espe- cially during the progression of the presenting part of the fetus through the birth canal, are ischaemia of the pelvic organs and stretching of the pelvic floor [2, 3]. Cervical, myometrial, and peritoneal nociceptors in the uterus are stimulated during stage I of labor when the lower uterine segment and the cervix are stretched. Low velocity C-fibres mediate visceral, throbbing, colic-like pain to the segments Th10-S5 of the spinal cord, accompanied by vegetative phe- nomena. In the second stage, the stretching perineal fascia, perineal skin, and subcutaneous tissue lead to the perception of sharp somatic, well localized pain via S2-4. (0123 1 3456789) 3 Archives of Gynecology and Obstetrics (2022) 305:1409–1419 1410 Labor pain is individual and subject to a variety of com- plex influences based on personal expectations, previous experiences (obstetric and non-obstetric) and foreseeable and non-foreseeable obstetric aspects. Nowadays, expect- ing women tend to link the prospect of giving birth naturally with a desire for maximum medical safety. Medical staff on the other hand may underestimate the relevance of perinatal pain management by prejudging the physiological nature of the process [4, 5]. key aspect of QUIPS is a highly standardized assessment of PRO by staff not involved in routine care, thus aiming to reduce questioning bias. The objective of this study is to implement a question- naire for the PRO assessment of peripartum pain sensation during and after vaginal, both spontaneous and operative- vaginal, deliveries. The questionnaire that is based on the established QUIPS template, is used in a monocentric proof- of-concept study to test its applicability, reliability, and validity. The results aim to uncover deficits in the current quality standards of the peripartum pain management in our department. Plausibility of the results is verified according to anticipated interrelations between solid obstetric data and the results of the survey. Several concepts are available for pain management dur- ing labor. Water baths, acupuncture or massages tend to pos- itively influence the birth experience [6]. Introduction Close care during childbirth has proven to be particularly important. This not only leads to a reduction in surgical interventions, but also to a reduced need for pain relievers [7]. Pharmacological interventions range from non-opioids with spasmolytic effect (i.e., butylscopolamine via different routes of administra- tion, sometimes in combination with paracetamol), inhala- tion agents (nitric oxide), opioids (meptazinol, pethidine, fentanyl, remifentanil, alfentanil) to regional analgesia. An epidural analgesia (synonymous peridural analgesia, PDA) is considered the gold-standard to relieve labor pain [8]. A PDA has proven most effective in reducing labor pain in randomized controlled trials [9, 10]. However, a PDA is an invasive procedure, requiring skilled staff and continu- ous monitoring, and is associated with rare but potentially severe risks. In addition, the use of an epidural analgesia should be incorporated into a multidimensional manage- ment. Although, epidural analgesia was previously thought to cause prolongation of labor, a higher rate of malpresen- tation and obstruction of labor, more recent studies could demonstrate that it did not increase the rates of operative- vaginal deliveries [10]. Taking into account the modern care strategies for pain control under labor, it seems that both appropriate choice of analgesic tools, depending on stage and progression of labor, and participation of the women in the decision-making process are decisive in the experience of pain [11]. 3 Methods In addition to demo- graphic data, these data provide details on the course of labor, delivery, birth injury, and the pain therapy adminis- tered during and after birth.f The QUIPS project (Quality-Improvement-in-Postoper- ative-Pain-Management) is an initiative conducted by the Jena University Hospital and has been implemented for many years for internal and cross-clinic quality management of pain therapy based on the collection of patient-reported outcomes (PRO) and processed parameters in the context of surgical interventions. In the obstetric context, QUIPS was used in patients after cesarean section [12], demonstrating inadequate pain management in the majority of women. One With the aim to differentiate co-variates and to check the plausibility of the results the subgroups of primiparae after spontaneous delivery [primiparae] and multiparae after spontaneous delivery with no history of operative deliver- ies [multiparae] were compared in a subgroup analysis (see Fig. 1 for details). The following co-variates were studied: maternal age, gestational age at birth, duration of birth, body 3 Archives of Gynecology and Obstetrics (2022) 305:1409–1419 1411 Fig. 1 Flowchart of the study collective. Hx = history (anamnesis) Fig. 1 Flowchart of the study collective. Hx = history (anamnesis) metrics of the newborn, induction of labor, and method of induction. categorical variables. A two-way ANOVA was additionally carried out to check for a significant interaction between the induction of labor and the gestational week. The data collected in 2015 were included in the descriptive and exploratory analyses, as well as a test for validity. To test the questionnaire for reliability, a second survey was carried out with 38 women 24–36 h and 48–72 h postpartum using a test–retest under the same conditions. A sufficient correla- tion was assumed for Cronbach's alpha > 0.6. The content validity (questionnaires created by experts from various disciplines for items already validated from other question- naires with similar content) and the criterion validity were tested using plausible relationships between process and result items. Correlation analyses were carried out to check for criterion validity. The correlation coefficient Spearman Rho was used for the investigation of two ordinally distrib- uted data, Kendall's Tau for the correlation of ordinally with binary-distributed data, and Phi for two binary items. The correlation coefficients were rated according to Cohen's effect size. A sufficiently high or medium correlation was assumed at ≥ 0.3. The (outcome) questionnaire consists of three parts. Methods The first part contains questions about pain and pain therapy dur- ing childbirth (questions 1–6). The second part focuses on pain and pain-related impairments, as well as pain therapy after childbirth (questions 7–13). The last section focuses on the general attitude towards pain therapy, perceived quality of care, as well as non-medicinal methods for pain relief (questions 14–19). The women were also asked to assess the extent and satisfaction with midwifery care during childbirth (questions 20 and 21). Pain intensity and satisfaction with pain therapy, as well as the care provided by the hospital staff were assessed using a numerical rating scale (NRS). Satisfaction and pain intensity was assessed using a 11-step numerical rating scale (NRS) with 0—‘very dissatisfied’ to 10—‘very satisfied’ and 0—‘no pain’ to 10—‘worst pain imaginable’ [13]. The complete questionnaire in German language is provided in the Supplements. Statistical analysis The statistical evaluation was carried out using SPSS ver- sion 21 (SPSS Inc. Chicago, IL). Statistical significance was assumed at a p value of < 0.05, trends at a p value < 0.1. The quantitative variables were evaluated descriptively (median and quartile range) and group differences between groups were assessed using the Mann–Whitney U test or the Student’s T test depending on normal distribution of the variables. Cross tables and chi-squared tests were used for Study population Figure 1 depicts the composition of the study population. From 1376 deliveries during the study period, the survey finally included 339 patients who are further treated as the 1 3 Archives of Gynecology and Obstetrics (2022) 305:1409–1419 1412 the indication threshold for systematic pain management. In general, there were no differences between primiparae and multiparae for perception of pain during labor and postpar- tum. 48.7% of the primiparae and 20.9% of the multiparae reported the labor pain as ‘too long’ or even ‘unbearably long’ and about one-third wished to have asked for more analgesics. In contrast, the personal involvement into deci- sion-making and the satisfaction with the pain regimen used were high. We observed a positive relation between the severity of pain and the request for analgesics, which does not mirror in the median NRS values (Kendells Tau, total p = 0.035; primiparae p = 0.056; multiparae p = 0.455). The questionnaire in use did not explicitly ask about pain relief by analgesics during labor (see “Discussion”). Nevertheless, women under epidural analgesia experienced less pain dur- ing labor, than through all other analgesic administrations (p = 0.088; Fig. 2). ‘total’ study cohort (see Table 1). The age of the participants ranged from 18 to 42 years. A total of 35 operative-vaginal deliveries were observed, 27 (15.7%) in 172 primiparae and 8 (2.3%) in 167 multiparous women (p < 0.001). For sub- group comparisons between primiparae and multiparae all histories of operative deliveries (current and previous) were excluded. The collective of subgroups, therefore, consisted of 145 primiparae and 129 multiparae (Fig. 1; Table 1). The mean age of all patients was 30.4 (± 4.5) years; 28.5 (± 4.4) years in the primiparae group and 31.7 (± 3.8) years in the multiparae group. Significant differences between the two subgroups were found with regard to age, the duration of birth, the frequency of operative-vaginal deliveries and obstetric injuries, the request for analgesics during childbirth and the size of the infant (Table 1). Overall, 132 (38.9%) patients during labor and 198 (58.4%) postpartum did not request or receive analgesic therapy. In the subgroups the respective proportions were 24.8%/65.5% (primiparae) and 62.0%/58.9% (multiparae). Table 3 and Fig. 3 give an overview on the interrelation between obstetric factors and perception of pain during labor and postpartum in primiparae who delivered spontaneously. Study population Gestational age, duration of birth, and neonatal biometric factors are positively correlated to intrapartum pain (Table 3; illustration in Fig. 4). Our survey shows that giving birth is retrospectively per- ceived as a very painful experience (Median 9 [8–10] on the NRS) (Table 2). A median NRS of 4 [3–6] as in the post- partum period is traditionally considered as being beyond Table 1 Characterization of the study population 1 Non-opioid analgesics: Butylscopolamine p.o./i.v./i.m./supp., Paracetamol supp. 2 Opioids: Fentanyl i.v., Pethidine i.m., Meptazinol i.m. 3 No. for postpartum non-opioid request/use Mean ± standard deviation (SD), Median [25;75]. Items Total N (%) Primiparae Multiparae p value N 339 145 129 Age [years] 30.4 ± 4.46 Y 28.5 ± 4.42 Y 31.7 ± 3.81 Y < 0.001 Duration of birth [hours] 5.15 [3.15;7.83] h 6.23 [3.93;8.38] h 4.08 [2.23;6.23] h < 0.001 Induction of labor 139 (41%) 68 (46.9%) 39 (30.2%) 0.005 Prostaglandins 123 (88.5%) 48 (70.5%) 25 (64.1%) Balloon catheter 35 (25.2%) 17 (25.0%) 8 (20.5%) Others 16 (11.6%) 3 (4.5%) 6 (15.4%) Documented obstetric injuries 276 (81.4%) 138 (95.2%) 81 (62.8%) < 0.001 Episiotomy 51 (15%) 24 (17.4%) 3 (3.7%) Perineal tear II 69 (20.4%) 35 (25.4%) 18 (22.2%) Perineal tear III/cervical laceration/high vaginal tear 13 (3.8%) 4 (2.9%) 2 (2.5%) Pain management during childbirth* 202 (59.6%) 106 (73.1%) 47 (36.4%) < 0.001 Non opioids1 122/339 (36%) Opioids2 162/339 (47.8%) Nitrous oxide 21/339 (6.2%) PDA 47/339 (13.9%) Pudendus block 8/339 (2.4%) Analgesics postpartum3 132 (38.9%) 44 (30.3%) 48 (37.2%) 0.251 Birth weight newborn [gram] 3415 ± 513 g 3293 ± 544 g 3513 ± 495 g 0.001 Length newborn [centimeter] 52 ± 3 cm 51 ± 3 cm 52 ± 3 cm 0.045 Head circumference newborn [centimeter] 35 ± 2 cm 34 ± 2 cm 35 ± 2 cm 0.022 1 Non-opioid analgesics: Butylscopolamine p.o./i.v./i.m./supp., Paracetamol supp. 1 Archives of Gynecology and Obstetrics (2022) 305:1409–1419 1413 Archives of Gynecology and Obstetrics (2022) 305:1409–1419 1413 Archives of Gynecology and Obstetrics (2022) 305:1409–1419 Table 2 Questionnaire results Figures are given as median (med.) and interquartiles [25;75] or percentages, were applicable (%). Discussion Although childbirth is known to be one of the most pain- ful experiences, there is no validated instrument in German language designed specifically for assessing pain during or after vaginal birth. Our international data research revealed hardly any studies that examined questionnaires about pain during vaginal birth [14]. To our knowledge, this is the first prospective, non-randomized, unblinded survey on subjec- tive pain experience during and after vaginal delivery (out- come questionnaire) and its association with demographic and obstetric aspects (process questionnaire). As a first step, and to homogenize the testing population for validation purposes, the questionnaire is kept in German. Excluding patients after operative deliveries from the subgroup analy- sis, we aimed to reduce confounders for the evaluation of pain, since women after instrumental birth were expected to have different levels of suffering. Pain levels were estimated on a numeric rating scale (NRS) from 0 (no pain) to 10 (most severe pain imaginable). The boxplots depict medians with quartile ranges. Satisfaction with pain management was acceptable over- all and similar between subgroups. In the correlations analy- sis, we observed a significant inversed correlation between satisfaction and wish for more analgesics and duration of labor. On the other hand, satisfaction with midwifery care and involvement in pain management significantly cor- related with higher overall satisfaction levels (Table 4). Exemplary data in Table 4 illustrate the validation process using Cohen’s correlation. All correlations were statistically significant and showed an average strength of the correla- tion > 0.3 (r). ff Women often hesitate to request pain medication because they are afraid of possible side effects on the process of labor itself, the child’s health or the possibility of breast feeding [15, 16]. Knowledge and attitude of healthcare providers have a relevant influence on pain management during labor [17]. Nevertheless, there is a risk of reduced function, chron- ification of pain, and an augmentation of mental disorders in the puerperium related to pain experience [18, 19]. There- fore, a comprehensive education regarding the options of peripartum pain therapy is indispensable. On the other hand, an effective and easy-to-use tool for peripartal pain assess- ment based on PROs is needed to assess the quality of pain management in obstetrics. In addition to a detailed medical history, the women should be asked about fears and desires regarding labor to adapt the pain management accordingly. Study population N given for each item separately: number of subjects who did answer the respective question Item Total N = 339 Primiparae N = 145 Multiparae N = 129 p value N Med [25;75] N Med [25;75] N Med [25;75] Maximum pain during labor 338 9 [8;10] 144 9 [9;10] 129 9 [8;10] 0.852 Duration of maximum pain 334 145 127 < 0.001 None Bearably long Too long Unbearably long 7 204 101 22 2.1% 61.1% 30.2% 6.6% 3 72 57 13 2.1% 49.7% 39.7% 9.0% 3 97 19 8 2.4% 75.2% 14.7% 6.2% Involvement in decision making 316 9 [7;10] 138 9 [6.75;10] 115 9 [6;10] 0.443 Satisfaction with pain managment 294 8 [5;9.25] 130 8 [5;9.25] 106 8 [5.75;9.25] 0.595 Wish to have received more analgesics 325 107 (32.9%) 141 51 (36.2%) 123 40 (32.5%) 0.604 Maximum pain postpartum 338 4 [3;6] 145 4 [2.5;5] 128 4 [3;6] 0.267 Satisfaction with postpartum pain management 289 9 [7;10] 124 9 [7;10] 106 9 [7;10] 0.892 Satisfaction with midwifery care 315 10 [9;10] 134 10 [9;10] 120 10 [9;10] 0.813 Figures are given as median (med.) and interquartiles [25;75] or percentages, were applicable (%). N given for each item separately: number of subjects who did answer the respective question As during labor, higher pain intensity was related to an increased request for analgesic in the postpartum period (median 4 [3;6] vs. 4 [2;5], p = 0.014). Interestingly, higher-degree of perineal tears was associated with less pain in the postpartum period (p = 0.056; Fig. 4). Reliability In the subsample of 38 subjects surveyed between July and October 2017 test–retest reliability was assessed using Cronbach’s alpha. Only 2 out of 21 items of the question- naire were below Cronbach’s alpha < 0.6. (Question 8: Are you affected by the pain—When caring for the child? Yes / No (Cronbach's alpha 0.4), Question 18: Have you used or received non-medicinal methods for pain relief? Yes relaxation (Cronbach's alpha 0.5)). On average, correla- tions were > 0.9. Thus, the questionnaire achieved a high test–retest reliability and, apart from two items, proved to be reliable (figures given in the Supplements). 1 3 Archives of Gynecology and Obstetrics (2022) 305:1409–1419 1414 1414 Archives of Gynecology and Obstetrics (2022) 305:1409–1419 Fig. 2 Maximum pain during labor in relation to pain man- agement (study cohort N = 339). Pain levels were estimated on a numeric rating scale (NRS) from 0 (no pain) to 10 (most severe pain imaginable). The boxplots depict medians with quartile ranges Table 3 Labor pain and obstetric factors in primiparae (N = 145) Item Maximum pain during labor Maximum pain postpartum Correlation p value Correlation p value Maternal age – 0.022 0.792 – 0.193 0.02 Gestational age 0.167 0.045 0.032 0.7 Duration of labor 0.197 0.018 – 0.095 0.256 Neonatal weight 0.304 < 0.001 Neonatal length 0.325 < 0.001 Neonatal head Circ 0.334 < 0.001 Median [25/75] Median [25/75] Induction of labor 9 [8;10] vs. 9 [8;10] 0.075 4 [2;5] vs. 3 [2.5;5.5] 0.83 Prostaglandin vs. Bal- loon ripening 9 [8;10] vs. 10 [7.5;10] 0.942 4 [3;6] vs. 3 [1;4.5] 0.032 Table 3 Labor pain and obstetric factors in primiparae (N = 145) overall satisfaction with intrapartum care, though, remains contradictory and may indicate that these standards are not directly applicable particularly to labor pain. The problem of ceiling effects of birth pain has to be considered as well. Differentiation in the upper range is difficult due to the very high maximum pain values, especially during childbirth.l Very high levels of pain intensity during birth have been described previously in the literature [1]. Table 3 Labor pain and obstetric factors in primiparae (N = 145) Reliability The high pain rat- ings of a median of 9 on a numeric rating scale (NRS) from 0 (no pain) to 10 (most severe pain imaginable) during child- birth and a median of 4 after childbirth in this study confirm these findings and underline the requirement to introduce a standardized pain survey policy that is specifically adapted to vaginal deliveries. According to current studies, a cut- off value of 4 on the NRS is a tolerable pain threshold for acute and postoperative pain [20]. Although these cut-off values might not apply to all types of painful procedures, a pain intensity level of 9 indicates inadequate treatment. The The personal obstetric history showed no influence on the maximum pain levels recorded in this study. However, the analysis did not examine the number of vaginal deliveries in relation to the maximum pain. It is, therefore, not possible to say whether a certain number of vaginal deliveries are associated with reduced pain severity. The gestational age, as 3 3 Archives of Gynecology and Obstetrics (2022) 305:1409–1419 1415 Fig. 3 Relation between pain perception during labor, gestational age, and duration of labor (primiparae, N = 145). Pain levels were estimated on a numeric rating scale (NRS) from 0 (no pain) to 10 (most severe pain imaginable). The boxplots depict medians with quartile ranges Fig. 3 Relation between pain perception during labor, gestational age, and duration of labor (primiparae, N = 145). Pain levels were estimated on a numeric rating scale (NRS) from 0 (no pain) to 10 (most severe pain imaginable). The boxplots depict medians with quartile ranges well as the infant's dimensions, had the expected significant influence on the maximum pain in the group of primiparae. It is surprising that this effect could not be demonstrated in the case of multiparae compared to primiparae, although the children of the multiparae were significantly larger and heavier in comparison. The infant’s biometrics should, there- fore, be included in future studies on pain management. Women with balloon induction reported significantly less pain compared to women after prostaglandin induction. As a limitation, it must be mentioned that the analysis was carried out with a small number of cases. In addition, the prosta- glandins were not further classified according to the type of prostaglandin, dosage or the way of application. Currently, Fig. 4 Postpartum pain experi- ence and severity of perineal lacerations (total, N = 339). Fig. 4 Postpartum pain experi- ence and severity of perineal lacerations (total, N = 339). Pain levels were estimated on a numeric rating scale (NRS) from 0 (no pain) to 10 (most severe pain imaginable). The boxplots depict medians with quartile ranges Reliability Pain levels were estimated on a numeric rating scale (NRS) from 0 (no pain) to 10 (most severe pain imaginable). The boxplots depict medians with quartile ranges Fig. 4 Postpartum pain experi- ence and severity of perineal lacerations (total, N = 339). Pain levels were estimated on a numeric rating scale (NRS) from 0 (no pain) to 10 (most severe pain imaginable). The boxplots depict medians with quartile ranges g. 4 Postpartum pain experi- ce and severity of perineal cerations (total, N = 339). ain levels were estimated on numeric rating scale (NRS) om 0 (no pain) to 10 (most vere pain imaginable). The oxplots depict medians with uartile ranges well as the infant's dimensions, had the expected significant influence on the maximum pain in the group of primiparae. It is surprising that this effect could not be demonstrated in the case of multiparae compared to primiparae, although the children of the multiparae were significantly larger and heavier in comparison. The infant’s biometrics should, there- fore, be included in future studies on pain management. well as the infant's dimensions, had the expected significant influence on the maximum pain in the group of primiparae. It is surprising that this effect could not be demonstrated in the case of multiparae compared to primiparae, although the children of the multiparae were significantly larger and heavier in comparison. The infant’s biometrics should, there- fore, be included in future studies on pain management. Women with balloon induction reported significantly less pain compared to women after prostaglandin induction. As a limitation, it must be mentioned that the analysis was carried out with a small number of cases. In addition, the prosta- glandins were not further classified according to the type of prostaglandin, dosage or the way of application. Currently, 1 3 Archives of Gynecology and Obstetrics (2022) 305:1409–1419 1416 Table 4 Validation process Cross cor- relation Duration of labor Involvement in pain man- agement Request for more analgesics Satisfac- tion with midwifery care Satisfaction with pain manage- ment – 0.363 0.545 – 0.423 0.63 Table 4 Validation process Cross cor- relation Duration of labor Involvement in pain man- agement Request for more analgesics Satisfac- tion with midwifery care Satisfaction with pain manage- ment – 0.363 0.545 – 0.423 0.63 of the QUIPS questionnaire after cesarean section, this study group developed a version for vaginal delivery. Quality criteria of the questionnaire The test–retest reliability always depends on the time inter- val between the two measurements. Because measurement was only possible at a short interval, there is a risk of mem- ory effects and an artificially increased test–retest reliabil- ity[30]. The difficulty in pain assessment is that pain and memory of pain is a dynamic trait. The relevant correlation of 0.6 was missed only for the item “difficulties with car- ing for the child” due to pain after birth. The information changed exclusively from "impairment" to "no impairment". This may be explained by the fact that after a time interval of more than 48 h, the patients were more experienced in dealing with the newborn. The improved care of the child through reduced pain in the course is also possible. All other assessed items showed robust reliability. The lack of a suit- able external criterion of quality hindered the procedure of external validation to be carried out. At the moment, no questionnaire to determine construct validity is available. This only left the verification of content and criterion valid- ity. The questionnaire was created by experts from various disciplines based on an already validated questionnaire that has been established for years for postoperative pain [31]. Care was taken to ensure that the questionnaire has a multidimensional approach in describing the quality of the results. In addition to maximum pain, functional effects of pain, psychological aspects, and patient satisfaction were taken into account. This multidimensional approach coin- cides with the demands made by other experts to describe the quality of pain therapy [32–35]. The selection of the measurement method for satisfaction and maximum pain, using NRS, has already been validated in other studies [28, 29]. Overall, it can be assumed that the questionnaire covers all facets of pain and the associated restrictions and thus has good content validity. With a median of 5 for maximum postpartum pain, women with an episiotomy reported significant higher pain scores compared to all other perineal or vaginal traumas. This correlates with previous studies, which also showed that patients who had an episiotomy indicated more pain after the episiotomy, as well as in the puerperium, com- pared to a second degree perineal tear [22, 23]. Women with higher-grade injuries reported less pain in the postpartum period. Reliability The questionnaire uses NRS to assess maximum pain and satisfaction with the care provided by the midwife and pain therapy. In comparison to other one-dimensional scales, the NRS shows the best results in terms of sensitivity, error rate, handling, and acceptance[28, 29]. The questionnaire not only records the impairment in mood, but also differentiates between the individual feelings (e.g., sadness, fear, exces- sive demands, etc.). Overall, the questionnaire is a versatile, easy-to-use and a well-accepted survey tool for interviewers and patients (response rate 80.1%), that can easily be inte- grated into everyday clinical practice[25–27]. it is questionable why the method of induction may have an impact on pain intensity after birth and not during birth. Regional analgesia such as epidural (synonymously used peridural analgesia – PDA) tended to subjectively reduce the pain felt during childbirth. This result reflects the superior- ity of regional analgesia, which has already been proven in several randomized studies, especially in comparison to opioid therapy [9, 21]. Nevertheless, the questionnaire failed to differentiate whether pharmacological interventions other than PDA resulted in pain relief since there was no specific prior-posterior question addressing this issue. Therefore, one must not jump to the premature conclusion that interventions other than PDA may be of no use in labor pain management. Quality criteria of the questionnaire This seemingly contradictory result is likely to be associated to the circumstance that these patients were pref- erably treated under general or epidural/spinal analgesia and preventive postoperative pain management was applied. It underlines the importance of a systematic approach to post- partum pain relief. Special attention with regard to effective pain therapy should, therefore, be given to patients after an episiotomy. Another group requiring more attention are par- ticularly young primiparae as they represent a risk group for increased postoperative pain [24], even after vaginal delivery. With the aim of improving quality in postoperative pain therapy, the QUIPS project has been established for many years and is used in various disciplines. Several studies showed that the questionnaire is a useful tool for quality improvement and can be used in routine clinical practice [25–27]. Multicenter use of this tool allows benchmarking and opens the possibility to identify best clinical practice and learn from each other, resulting in better care for patients. Therefore, based on the experience earned in the application The criterion validity was checked against theoretical cri- teria, which should demonstrate the plausibility of the results. The correlation analyses carried out show that the results are conclusive and as expected. In validation studies about chronic The correlation analyses carried out show that the results are conclusive and as expected. In validation studies about chronic 1 3 3 Archives of Gynecology and Obstetrics (2022) 305:1409–1419 1417 management. Therefore, systematic assessment of pain per- ception in the maternity ward is a pending issue. We present a questionnaire of acceptable validity specifically designed to direct pain management sub partu and postpartum. It may be useful to assess department-specific changes of standards of procedure and propel standardized comparison between departments as a means for applied clinical science. In result to this validation study, we incorporated a question specifi- cally asking the effect of pharmacologic intervention on labor pain perception. To additionally address women of a different cultural background, which do not have sufficient German language skills, the validated version of this ques- tionnaire will be translated and may serve as an instrument to compare cultural differences of labor pain perception as well. Even severe pain sub partu does not necessarily lead to dissatisfaction with intrapartum care. pain, weak to medium correlations were reported with regard to an external criterion[36–39]. The other significant relation- ships further support the plausibility of the results. Limitations Studies have shown that the pain memory is not exact and strongly context-dependent [29]. The results can also be influ- enced by a memory distortion effect called “recall bias” [40, 41]. The questionnaire on pain during childbirth was carried out retrospectively, the questionnaire on pain after childbirth in turn relate to the current condition and is, therefore, not, or less affected by memory effects. Pain intensity and pain memory after trauma or after surgery decrease over time. This effect has also been observed for labor pain[42]. Due to the design of the survey, no statement can be made about the further development or duration of the pain after birth. It cannot be ruled out, but is unlikely to be a major setback, that the patients were exposed to a peak of pain at the time of the interview. The risk of divergent answers was minimized by the independent responses of the patients and a survey by personnel not involved in woman’s care [43]. An influence of the “Hawthorne effect” or the presence of relatives on the indication of pain intensity cannot be ruled out with certainty [44, 45]. The study has so far been carried out monocentrically at only one German hospital (Jena University Hospital) and is, therefore, not a representative cross-section of German hospi- tals. Additionally, it has so far only been possible to interview German-speaking patients. Therefore, there are some limits to the generalizability, especially regarding the influence of the cultural background in dealing with pain [46]. However, it is planned to provide versions in several other languages, as it was done in PAIN OUT, the international part of the QUIPS project. Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00404-021-06246-w. Acknowledgements We would like to thank everyone who was involved in this study, and we would particularly like to thank the par- ticipants in this study. We particularly like to acknowledge Dr Anna Multhaup for her revision of the manuscript as a native speaker. Author contributions AL: Manuscript writing. DN: Formal analysis and investigation, data analysis, and manuscript writing. JR: Project development, conception and study design, and methodology. MK: Specialist advice. CW: Specialist advice. ES: Supervision and spe- cialist advice. WM: Specialist advice. JJC: Project development and conception and study design. US: Project development, conception and study design, and writing—review and editing. Funding Open Access funding enabled and organized by Projekt DEAL. Limitations Availability of data and material The raw/processed data required to reproduce these findings cannot be shared at this time due to legal or ethical reasons. The questionnaire will be available on the QUIPS platform at http://www.quips-projekt.de/. Code availability Not applicable. Quality criteria of the questionnaire In sum- mary, it can be concluded that the questionnaire allows state- ments about maximum pain and its associated restrictions with sufficient reliability. fi Another possibility would be the use of external criteria to assess pain intensity or functional restrictions, e.g., in the form of medical staff notes. Furthermore, the pain-related functional restrictions could be verified with a variety of activities. In the best case, several validity criteria are used for each item to identify the best possible external criterion. Declarations Conflict of interest The authors declare that they have no conflict of interest. Ethics approval The authors confirm that the study was approved by the appropriate institutional and/or national research ethics committee (ethics committee of Jena University Hospital, Ethics approval number: 2722–12/09) and certify that the study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. References 1. Lowe N (2002) The nature of labor pain. Am J Obstet Gynecol 186(5):S16–S24. https://doi.org/10.1067/mob.2002.121427 19. Fahey JO (2017) Best practices in management of postpartum pain. J Perin Neon Nurs 31(2):126–136. https://doi.org/10.1097/ JPN.0000000000000241 2. Heesen M, Veeser M (2012) Analgesia in obstetrics. Geburtsh Frauenheilk 72(07):596–601. https://doi.org/10.1055/s-0031- 1298444l 20. Gerbershagen HJ, Rothaug J, Kalkman CJ, Meissner W (2011) Determination of moderate-to-severe postoperative pain on the numeric rating scale: a cut-off point analysis applying four dif- ferent methods. Br J Anaesth 107(4):619–626. https://doi.org/10. 1093/bja/aer195 3. Kranke P (2018) Die geburtshilfliche Anästhesie. Springer Ü l 4. Gross MM (2003) Die Geburtserfahrung - eine Übersichtsar- beit. Geburtsh Frauenheilk 63:321–325. https://doi.org/10. 1055/s-2003-39242 21. Philipsen T, Jensen N-H (1989) Epidural block or parenteral pethi- dine as analgesic in labour; a randomized study concerning pro- gress in labour and instrumental deliveries. Eur J Obstet Gynecol Reprod Biol 30(1):27–33. https://doi.org/10.1016/0028-2243(89) 90090-7 5. Junge C, von Soest T, Weidner K, Seidler A, Eberhard-Gran M, Garthus-Niegel S (2018) Labor pain in women with and without severe fear of childbirth: A population-based, longitudinal study. Birth 45(4):469–477. https://doi.org/10.1111/birt.12349 6. Jones L, Othman M, Dowswell T, et al (2012) Pain management for women in labour: an overview of systematic reviews. Cochrane Pregnancy and Childbirth Group, ed. Cochrane Database of Sys- tematic Reviews. Published online March 14, 2012. https://doi. org/10.1002/14651858.CD009234.pub2 22. Andrews V, Thakar R, Sultan AH, Jones PW (2008) Evaluation of postpartum perineal pain and dyspareunia—A prospective study. Eur J Obstet Gynecol Reprod Biol 137(2):152–156. https://doi. org/10.1016/j.ejogrb.2007.06.005 23. Langer B, Minetti A (2006) Immediate and long term com- plications of episiotomy. J Gynecol Obstet Biol Reprod. 35(1 Suppl):159–5167 7. Knape N, Schnepp W, Krahl A, zu Sayn-Wittgenstein F (2013) Die Effektivität der Eins-zu-eins-Betreuung während der Geburt. Eine Literaturübersicht. Z Geburtshilfe Neonatol. 217(05):161–172. https://doi.org/10.1055/s-0033-1355382 24. Caumo W, Schmidt AP, Schneider CN et al (2002) Preopera- tive predictors of moderate to intense acute postoperative pain in patients undergoing abdominal surgery. Acta Anaesthesiol Scand 46(10):1265–1271. https://doi.org/10.1034/j.1399-6576.2002. 461015.x 8. Reynolds F (2011) Labour analgesia and the baby: good news is no news. Int J Obstet Anesth 20(1):38–50. https://doi.org/10. 1016/j.ijoa.2010.08.004fi 9. Jain S, Arya VK, Gopalan S, Jain V (2003) Analgesic efficacy of intramuscular opioids versus epidural analgesia in labor. Int J Gynecol Obstet 83(1):19–27. https://doi.org/10.1016/S0020- 7292(03)00201-7 25. Jiménez Cruz J, Diebolder H, Dogan A et al (2014) Combination of pre-emptive port-site and intraoperative intraperitoneal ropiv- acaine for reduction of postoperative pain: a prospective cohort study. Conclusion Giving birth is a severely painful process; a phenomenon requiring more attention of care-givers. Even when reporting overall satisfaction with their situation, a relevant number of women, if asked, report significant subjective pain levels on NRS that would trigger medical pain relief in a postopera- tive ward. The reported pain levels indicate inadequate pain Informed consent Informed consent was obtained from all individual participants included in the study. 1 3 Archives of Gynecology and Obstetrics (2022) 305:1409–1419 1418 15. Lindholm A, Hildingsson I (2015) Women’s preferences and received pain relief in childbirth – A prospective longitudinal study in a northern region of Sweden. Sexual Reprod Healthcare 6(2):74–81. https://doi.org/10.1016/j.srhc.2014.10.001 Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 16. van der Hulst LAM, van Teijlingen ER, Bonsel GJ, Eskes M, Bleker OP (2004) Does a pregnant woman’s intended place of birth influence her attitudes toward and occurrence of obstet- ric interventions? Birth 31(1):28–33. https://doi.org/10.1111/j. 0730-7659.2004.0271.x 17. McCauley M, Actis Danna V, Mrema D, van den Broek N (2018) “We know it’s labour pain, so we don’t do anything”: healthcare provider’s knowledge and attitudes regarding the provision of pain relief during labour and after childbirth. BMC Pregnancy Childbirth 18(1):444. https://doi.org/10.1186/ s12884-018-2076-7 18. ACOG Committee Opinion No (2018) 742 Summary: Postpartum Pain Management. Obstet Gynecol 132(1):252–253. https://doi. org/10.1097/AOG.0000000000002711 References Eur J Obstet Gynecol Reprod Biol 179:11–16. https://doi. org/10.1016/j.ejogrb.2014.05.001 10. Anim-Somuah M, Smyth RM, Cyna AM, Cuthbert A (2018) Epi- dural versus non-epidural or no analgesia for pain management in labour. Cochrane Pregnancy and Childbirth Group, ed. Cochrane Database of Systematic Reviews. Published online May 21, 2018. https://doi.org/10.1002/14651858.CD000331.pub4 26. Meissner W, Mescha S, Rothaug J, et al (2008) Quality Improve- ment in Postoperative Pain Management – Results From the QUIPS Project. Deutsches Aerzteblatt Online. Published online December 12, 2008. https://doi.org/10.3238/arztebl.2008.0865 11. Nanji JA, Carvalho B (2020) Pain management during labor and vaginal birth. Best Pract Res Clin Obstet Gynaecol 67:100–112. https://doi.org/10.1016/j.bpobgyn.2020.03.002 27. Poller K, Volk GF, Wittekindt C, Meissner W, Guntinas-Lichius O (2011) Verbesserung der Schmerztherapie nach Tonsillektomie bei Erwachsenen durch Schmerzmessung mit QUIPS („Qualitäts- verbesserung in der postoperativen Schmerztherapie”). Laryngo- Rhino-Otol 90(02):82–89. https://doi.org/10.1055/s-0030-12698 48 12. Marcus H, Gerbershagen HJ, Peelen LM et al (2015) Quality of pain treatment after caesarean section: Results of a multicentre cohort study: Post-operative pain after caesarean section. EJP 19(7):929–939. https://doi.org/10.1002/ejp.619 28. Aubrun F, Paqueron X, Langeron O, Coriat P, Riou B (2005) What pain scales do nurses use in the postanaesthesia care unit? Eur J Anaesthesiol 20(9):745–749. https://doi.org/10.1017/S026502150 3001212 13. Breivik EK, Björnsson GA, Skovlund E (2000) A comparison of pain rating scales by sampling from clinical trial data. Clin J Pain 16(1):22–28. https://doi.org/10.1097/00002508-20000 3000-00005 29. Breivik H, Borchgrevink PC, Allen SM et al (2008) Assessment of pain. Br J Anaesth 101(1):17–24. https://doi.org/10.1093/bja/ aen103 14. Niven C, Gijsbers K (1984) A study of labour pain using the MCGILL pain questionnaire. Soc Sci Med 19(12):1347–1351. https://doi.org/10.1016/0277-9536(84)90023-6 1 3 1419 Archives of Gynecology and Obstetrics (2022) 305:1409–1419 30. Moosbrugger H, Kelava A (2012) Testtheorie und Fragebogen- konstruktion: mit 66 Abbildungen und 41 Tabellen. 2., aktualisi- erte und überarbeitete Auflage. Springer 39. Rosemann T, Szecsenyi J (2007) Cultural adaptation and valida- tion of a German version of the Arthritis Impact Measurement Scales (AIMS2). Osteoarthr Cartil 15(10):1128–1133. https://doi. org/10.1016/j.joca.2007.03.021 l 31. Rothaug J, Weiss T, Meissner W (2012) Externe Validität der schmerzbedingten Funktionsbeeinträchtigung: Messen wir, was wir messen wollen? Schmerz 26(4):396–401. https://doi.org/10. 1007/s00482-012-1154-4 40. Apfelbaum JL, Chen C, Mehta Gan SSTJ (2003) Postoperative pain experience: results from a national survey suggest postopera- tive pain continues to be undermanaged. Anesth Analg 97(2):534– 540. https://doi.org/10.1213/01.ANE.0000068822.10113.9E 32. Gordon DB, Pellino TA, Miaskowski C et al (2002) A 10-year review of quality improvement monitoring in pain management: Recommendations for standardized outcome measures. References Pain Manag Nurs 3(4):116–130. https://doi.org/10.1053/jpmn.2002. 127570 41. Breme K, Altmeppen J, Taeger K (2000) Patientenkontrollierte Analgesie. Der Schmerz 14(3):137–145. https://doi.org/10.1007/ s004820070039 42. Waldenström U, Schytt E (2009) A longitudinal study of women’s memory of labour pain-from 2 months to 5 years after the birth. BJOG Int J Obstet Gynaecol 116(4):577–583. https://doi.org/10. 1111/j.1471-0528.2008.02020.x 33. Gordon DB, Dahl JL, Miaskowski C et al (2005) American pain society recommendations for improving the quality of acute and cancer pain management: American pain society quality of care task force. Arch Intern Med 165(14):1574. https://doi.org/10. 1001/archinte.165.14.1574 43. Sloman R, Rosen G, Rom M, Shir Y (2005) Nurses’ assessment of pain in surgical patients. J Adv Nurs 52(2):125–132. https:// doi.org/10.1111/j.1365-2648.2005.03573.x 34. Kehlet H (1997) Multimodal approach to control postoperative pathophysiology and rehabilitation. Br J Anaesth 78(5):606–617. https://doi.org/10.1093/bja/78.5.606 44. Gruber T, Sharma A, Daneschvar H, Estfan B (2003) The Haw- thorne effect in the assessment of pain by house staff. Am J Hosp Palliat Care 20(3):231–234. https://doi.org/10.1177/1049909103 02000314 p g j 35. Kehlet H (2004) Effect of postoperative pain treatment on outcome?current status and future strategies. Langenbecks Arch Surg. https://doi.org/10.1007/s00423-004-0460-4 45. Block AR, Kremer EF, Gaylor M (1980) Behavioral treatment of chronic pain: The spouse as a discriminative cue for pain behav- ior. Pain 9(2):243–252. https://doi.org/10.1016/0304-3959(80) 90011-1 36. Grøvle L, Haugen AJ, Keller A, Natvig B, Brox JI, Grotle M (2008) Reliability, validity, and responsiveness of the norwegian versions of the maine-seattle back questionnaire and the sciatica bothersomeness and frequency indices. Spine 33(21):2347–2353. https://doi.org/10.1097/BRS.0b013e31818047d6 46. Green CR, Anderson KO, Baker TA et al (2003) The Unequal Bur- den of Pain: Confronting Racial and Ethnic Disparities in Pain. Pain Med 4(3):277–294. https://doi.org/10.1046/j.1526-4637. 2003.03034.x 37. McCarthy M, Jonasson O, Chang C-H et al (2005) Assess- ment of patient functional status after surgery. J Am Coll Surg 201(2):171–178. https://doi.org/10.1016/j.jamcollsurg.2005.03. 035 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 38. Roos M, Klässbo LS (1999) Lohmander EM (1999) WOMAC Osteoarthritis Index: Reliability, validity, and responsiveness in patients with arthroscopically assessed osteoarthritis. Scand J Rheumatol. 28(4):210–215. https://doi.org/10.1080/0300974995 0155562 1 3 1 3 1 3 1 3
https://openalex.org/W4386708342	https://www.nature.com/articles/s41467-023-41334-2.pdf	English	null	Variation in spatial dependencies across the cortical mantle discriminates the functional behaviour of primary and association cortex	Nature communications	2,023	cc-by	11,606	Variation in spatial dependencies across the cortical mantle discriminates the functional behaviour of primary and association cortex Robert Leech 1 , Reinder Vos De Wael2, František Váša 1, Ting Xu 3, R. Austin Benn4, Robert Scholz 5, Rodrigo M. Braga6, Michael P. Milham3, Jessica Royer2, Boris C. Bernhardt 2, Emily J. H. Jones7, Elizabeth Jefferies8, Daniel S. Margulies4 & Jonathan Smallwood 9 Robert Leech 1 , Reinder Vos De Wael2, František Váša 1, Ting Xu 3, R. Austin Benn4, Robert Scholz 5, Rodrigo M. Braga6, Michael P. Milham3, Jessica Royer2, Boris C. Bernhardt 2, Emily J. H. Jones7, Elizabeth Jefferies8, Daniel S. Margulies4 & Jonathan Smallwood 9 Recent theories of cortical organisation suggest features of function emerge from the spatial arrangement of brain regions. For example, association cortex is located furthest from systems involved in action and perception. Associa- tion cortex is also ‘interdigitated’ with adjacent regions having different pat- terns of functional connectivity. It is assumed that topographic properties, such as distance between regions, constrains their functions, however, we lack a formal description of how this occurs. Here we use variograms, a quantiﬁ- cation of spatial autocorrelation, to proﬁle how function changes with the distance between cortical regions. We ﬁnd function changes with distance more gradually within sensory-motor cortex than association cortex. Impor- tantly, systems within the same type of cortex (e.g., fronto-parietal and default mode networks) have similar proﬁles. Primary and association cortex, there- fore, are differentiated by how function changes over space, emphasising the value of topographical features of a region when estimating its contribution to cognition and behaviour. Contemporary evidence suggests that local topographical properties inﬂuence a region’s function in a complicated, inter- dependent manner. For example, neural systems concerned with sensation and movement, such as the visual or motor cortex, are spatially distant from each other, yet both of these systems tend to be relatively spatially contiguous, and both contain topographic fea- tures resembling maps, either of the external environment or how the organism engages with the outside world7–10. Other systems, such as the default mode or frontoparietal networks, are located in regions of association cortex, are spatially adjacent to one another, both are spatially distributed across cortex; yet functionally these One of the most important discoveries in human neuroscience is that brain topography plays an important role in determining how a region contributes to cognition and behaviour1. Article https://doi.org/10.1038/s41467-023-41334-2 1Centre for Neuroimaging Science, King’s College London, London, UK. 2McConnell Brain Imaging Centre, McGill University, Montreal, QC, Canada. 3Center for the Developing Brain, Child Mind Institute, New York, USA. 4Integrative Neuroscience and Cognition Center (UMR 8002), Centre National de la Recherche Scientiﬁque (CNRS) and Université de Paris, Paris, France. 5Max Planck School of Cognition, Leipzig, Germany. 6Neurology, Interdepartmental Neuroscience Program, Northwestern University, Evanston, IL, USA. 7Centre for Brain and Cognitive Development, Birkbeck College, University of London, London, UK. 8Department of Psychology, University of York, York, UK. 9Department of Psychology, Queens University, Kingston, ON, Canada. e-mail: robert.leech@kcl.ac.uk Variation in spatial dependencies across the cortical mantle discriminates the functional behaviour of primary and association cortex These topographic features can shape a region’s function in many ways including: (i) through the inﬂuence of neighbouring neural systems that make up the local environment within which a speciﬁc region is embedded2, (ii) the physical location of the network on the cortical mantle with respect to core cortical landmarks3, (iii) and more abstracttopographical features such as the degree to which functional activity within a network is spatially distributed across the cortical mantle2,4, or, instead is limited to adjacent regions, often within a single cortical lobe5,6. 1Centre for Neuroimaging Science, King’s College London, London, UK. 2McConnell Brain Imaging Centre, McGill University, Montreal, QC, Canada. 3Center for the Developing Brain, Child Mind Institute, New York, USA. 4Integrative Neuroscience and Cognition Center (UMR 8002), Centre National de la Recherche Scientiﬁque (CNRS) and Université de Paris, Paris, France. 5Max Planck School of Cognition, Leipzig, Germany. 6Neurology, Interdepartmental Neuroscience Program, Northwestern University, Evanston, IL, USA. 7Centre for Brain and Cognitive Development, Birkbeck College, University of London, London, UK. 8Department of Psychology, University of York, York, UK. 9Department of Psychology, Queens University, Kingston, ON, Canada. e-mail: robert.leech@kcl.ac.uk Nature Communications\| (2023) 14:5656 Nature Communications\| (2023) 14:5656 1 https://doi.org/10.1038/s41467-023-41334-2 Article important to understand how the balance of meso and macro scale inﬂuences combine in order to understand how topography inﬂuences function within a given brain region. Our study set out to understand meso and macro scale changes in the inﬂuence of topographical fea- tures on brain function by examining whether there are regional dif- ferences in the way distance impacts functional connectivity. systems appear to serve different, often opposing roles in human cognition11. Topography is also important for understanding mac- roscale brain function, because systems that tend to be more spa- tially discontinuous (e.g., the default mode network) tend to be more distant from sensory and motor systems where spatial discontinuity is an exception rather than the norm (e.g. sensorimotor or visual cortex)3. In contemporary neuroscience, macroscale topographical features provide a useful heuristic for understanding the involve- ment of frontoparietal and default mode networks in cognition. These networks are hypothesised to be at the transmodal apex regions of a broad sensory-fugal hierarchy, allowing oversight across broad areas of cortex12. In contrast, mesoscale features of topo- graphy, such as the retinotopic maps located within sensory cortex, are thought to explain aspects of how the visual system represents and extracts features of the environment from retinal input8. Variation in spatial dependencies across the cortical mantle discriminates the functional behaviour of primary and association cortex In order to establish how distance between regions inﬂuences their similarity in function, we calculated for each cortical surface vertex how the similarity of its activity changes with all other vertices as a function of the distance between them; quantifying the local rate of change of similarity across the cortex. This is a simpliﬁed version of the empirical variogram14, as illustrated schematically in the upper panel of Fig. 1. Spatial variograms are expected to show that similarity in function declines with distance until it reaches an asymptote, the distance after which there is no longer a spatial dependency between vertices. The empirical variogram can be summarised by ﬁtting an exponential function which in turn can be described by two values capturing how similarity changes with distance for each vertex: the effective range and the sill. The sill is the height (i.e., degree of dis- similarity between two regions) and the range is asymptote (i.e., the spatial distance between the two regions). Heterogeneity in the spatial variogram across regions can be used to quantify the different ways topography inﬂuences function in different cortical locations. For example, in regions where function is more inﬂuenced by the local neighbourhood, the spatial variogram shows a relatively shallow decline in similarity with distance. In contrast, in regions where func- tion is relatively distinct from the local environment, the variogram should increase more rapidly with the distance. This approach allows for the presence of variable spatial dependencies across the cortex, in contrast to accounts that imply a homogeneous spatial relationship, e.g., a single exponential distance rule15. Topography at both macro and mesoscale is, therefore, a key principle of brain organisation and is crucial for understanding brain function both within speciﬁc systems and across the cortex as a whole. Our study set out to formally examine how the meso and macro scale perspectives can be combined to formally understand the relationship between topography and brain function. The distance between regions, calculated as the geodesic distance between two vertices, provides one metric to understand how topography inﬂuences func- tion. This measure has been used to describe macroscale features of cortical topography, for example, highlighting that systems like the default mode and frontal-parietal cortex are distant from both systems concerned with sensory input and motor output systems13. Variation in spatial dependencies across the cortical mantle discriminates the functional behaviour of primary and association cortex However, a given location on the cortical mantle may be inﬂuenced by local topographical features as well, such as the features of the local neighbourhood in which the region is situated, or, whether the system is part of a distributed or localised network. Accordingly, it is Fig. 1 \| Calculation of variograms. Top Panel. Schematic illustration of how spatial variograms can be used to characterise how functional connectivity changes as distance increases between brain regions. Bottom Left. Whole-brain variograms of functional connectivity can be calculated by comparing how the distance along the cortical surface is related to the average similarity in brain activity between regions. Bottom Right. Whole brain variograms are shown for the left and right cortices and can be seen to be broadly similar. The thick lines/dots are the mean across parti- cipants, and the ﬁlled area is the standard error of the mean. The dashed lines are the estimated location of the sill (asymptotic correlation between vertices) and range (distance in mm between vertices at which the asymptote is reached). N t C i ti \| (2023)14 5656 2 Bottom Right. Whole brain variograms are shown for the left and right cortices and can be seen to be broadly similar. The thick lines/dots are the mean across parti- cipants, and the ﬁlled area is the standard error of the mean. The dashed lines are the estimated location of the sill (asymptotic correlation between vertices) and range (distance in mm between vertices at which the asymptote is reached). Fig. 1 \| Calculation of variograms. Top Panel. Schematic illustration of how spatial variograms can be used to characterise how functional connectivity changes as distance increases between brain regions. Bottom Left. Whole-brain variograms of functional connectivity can be calculated by comparing how the distance along the cortical surface is related to the average similarity in brain activity between regions. Nature Communications\| (2023) 14:5656 2 Article https://doi.org/10.1038/s41467-023-41334-2 The lower panel of Fig. 2 shows that regions closer to the transmodal end of the principal gradient tend to be regions where the variograms tend to have a relatively high sill and short effective distance (i.e., regions where dissimilarity shows a relatively rapid increase), in gen- eral. Regional variation in spatial dependency across the cortex Regional variation in spatial dependency across the cortex The whole-brain variograms establish that in humans, distance leads to an increase in dissimilarity in neural function that is asymptotic exponential in nature and that these measurements are broadly con- sistent within an individual over time. This aligns with descriptions of spatial similarity previously reported in humans and non-human pri- mates e.g., refs. 15–18. By computing variograms, we are able to go beyond a single description of spatial dependency in each region of the brain, and this therefore allows us to capture regional differences in spatial dependencies (see also Supplementary Fig. 2, for random models with homogeneous spatial dependency structures to contrast with the empirical results). To understand whether there are sys- tematic differences in how distance leads to changes in neural function across different brain regions, we calculated separate variograms for each vertex across the cortex. The middle panel in Fig. 2 summarises how the two metrics (sill and effective distance) vary across the cortex. It can be seen that sill (reﬂecting the spatial dissimilarity in functional connectivity across the cortex) ranges between 0.25 and 0.5, and that in some regions the dissimilarity continues to increase to the max- imum range of our measurements (150 mm). Having established that heterogeneity in spatial dependencies capture important features of brain organisation in humans, we next sought to understand whether this generalises to non-human primates. To this end, we repeated this analysis in a sample of macaques (using homologue networks, see Methods for details). This analysis identiﬁed that the network proﬁle of each species is broadly similar. For example, in both species the limbic network has the highest sills and the shortest effective distances, and the visual system provides the clearest exam- ple of the opposite proﬁle (low sills and longer effective distance). We note that some regions within the limbic network have been reported to have signal dropout and related issues in the Human Connectome Project dataset19 and so should be interpreted with caution. Whole-brain spatial dependency We ﬁrst quantiﬁed the spatial dependency between functional con- nectivity and distance by calculating whole-brain variograms assessing how functional connectivity (Pearson’s correlation) varies with dis- tance along the cortical surface for each hemisphere (Fig. 1, lower panel). We used resting state fMRI data from 51 participants from the Human Connectome Project. We took two scans on the same data for each individual allowing us to calculate the reliability of these metrics within an individual. Averaging these vertex-wise variograms across the whole cortex, the global variogram, reveals an initially steep rise (rapidly increasing dissimilarity with distance (Fig. 1). This is followed by a continuous increase up to the measured limit (all vertices included distances up to 150 mm, which was the maximum distance present for all vertices (see Supplementary Figure 1 for vertex distance distribu- tions and higher upper measurement limits). The variograms for the left and right hemispheres show a similar pattern (see left hand panel). The landscape of these variograms can be formally understood by comparing the observed rate of change in function with distance with different mathematical growth functions (e.g., exponential, gaussian, sinusoidal and power-law). It can be seen in Fig. 1 that the whole brain variogram of the human is most similar to an exponential relationship. For the purposes of our analyses, we extracted the two para- meters used to ﬁt the theoretical function to the empirical variograms: (i) the sill, which is the height the variogram reaches at 95% of its asymptote and reﬂects the approximate point at which there is no longer a relationship between space and functional connectivity (i.e., that vertex’ baseline average correlation level with other vertices); and (ii) the range which is where the sill occurs. These are both dis- played in the top panel of Fig. 2. Importantly comparing the variogram calculated for each of the participants from separate resting state scans on the same day shows a high degree of correspondence both in terms of the sill (the average difference in correlation between ver- tices) and the distance (i.e., rho > 0.73; Fig. 2 top panel). We ﬁrst quantiﬁed the spatial dependency between functional con- nectivity and distance by calculating whole-brain variograms assessing how functional connectivity (Pearson’s correlation) varies with dis- tance along the cortical surface for each hemisphere (Fig. 1, lower panel). We used resting state fMRI data from 51 participants from the Human Connectome Project. Variation in spatial dependencies across the cortical mantle discriminates the functional behaviour of primary and association cortex In contrast, regions closer to the unimodal end of the principal gradient tend to have a relatively lower sill and a longer effective dis- tance (i.e., regions that show a slower rate of decline in function with increasing distance). This analysis provides preliminary support that two broad types of cortex (primary and association cortex) can be discriminated based on how activity varies with distance. Spin per- mutation tests (Fig. 2, bottom, right) as well as generative null models based on randomisation or randomisation followed by smoothing with a homogeneous function (Supplementary ﬁg. 2) show that these rela- tionships are unlikely to be due to chance. Relationship between spatial dependency and cortical organisation Having highlighted the features that whole brain variograms have, we next considered how this varied across the cortex. To this end, we examined how the distribution of the sill and the effective range varies across the principal gradient of change in functional connectivity3. This gradient can be derived by application of dimensionality reduc- tion techniques to functional connectivity data3, and recapitulates foundational features of the sensory-transmodal cortical hierarchy1. Whole-brain spatial dependency We took two scans on the same data for each individual allowing us to calculate the reliability of these metrics within an individual. Averaging these vertex-wise variograms across the whole cortex, the global variogram, reveals an initially steep rise (rapidly increasing dissimilarity with distance (Fig. 1). This is followed by a continuous increase up to the measured limit (all vertices included distances up to 150 mm, which was the maximum distance present for all vertices (see Supplementary Figure 1 for vertex distance distribu- tions and higher upper measurement limits). The variograms for the left and right hemispheres show a similar pattern (see left hand panel). The landscape of these variograms can be formally understood by comparing the observed rate of change in function with distance with different mathematical growth functions (e.g., exponential, gaussian, sinusoidal and power-law). It can be seen in Fig. 1 that the whole brain variogram of the human is most similar to an exponential relationship. The principal gradient provides an organising principle for mac- roscale features of brain function, including large-scale brain networks (see ref. 3). Next, we examined how the large-scale networks that span the principal gradient, focusing on a well-deﬁned set of canonical resting state networks from Yeo and colleagues4. Figure 3 (upper panel) shows the average empirical variogram for each network while the lower panel shows the average sill and effective distance of each network. Regions making up the limbic network (Cream) have the highest sill and the shortest effective distance, a pattern that is also seen in the transmodal networks (Default mode, Red; Fronto-parietal network, Orange) but to a lesser degree. Regions that make up unimodal cortex (Visual network, Purple; Motor cortex, Blue) show the reverse proﬁle with variograms with small sills and relatively long effective distance. Finally, the two attention networks (Dorsal and Ventral) show intermediate proﬁles both having moderate sills and effective ranges. These two systems are distinguished from each other because the Dorsal attention network has a longer effective distance and a short sill, and so is more similar to the unimodal systems, whereas the ventral attention network shows the opposite proﬁle. Whole-brain spatial dependency For the purposes of our analyses, we extracted the two para- meters used to ﬁt the theoretical function to the empirical variograms: (i) the sill, which is the height the variogram reaches at 95% of its asymptote and reﬂects the approximate point at which there is no longer a relationship between space and functional connectivity (i.e., that vertex’ baseline average correlation level with other vertices); and (ii) the range which is where the sill occurs. These are both dis- played in the top panel of Fig. 2. Importantly comparing the variogram calculated for each of the participants from separate resting state scans on the same day shows a high degree of correspondence both in terms of the sill (the average difference in correlation between ver- tices) and the distance (i.e., rho > 0.73; Fig. 2 top panel). This network analysis contrasts with a comparison between broad features of brain organisation such as the principal gradient. In parti- cular, while there are clear differences between networks in terms of their variogram proﬁle, networks embedded in similar types of cortex show relatively high similarity. In particular, both the default mode network and the frontoparietal network, embedded within association cortex, show similar proﬁles. Likewise, the variograms of visual and motor systems, which are both embedded in primary cortex, are also similar. To quantify this apparent similarity we randomly permuted the location of the Yeo networks (by rotating them on the sphere) to generate null models and compared the difference in the Range and Sill parameters. This analysis showed the only signiﬁcant differences were between different types of cortex (see Fig. 4), e.g., between visual and default mode networks. Nature Communications\| (2023) 14:5656 Results Whole-brain spatial dependency Clustering variation in spatial dependency D The regional distribution of the range (the distance of the sill) and (E) the sill (the height of the generalises to out-of-sample data. Comparing each individual partici- pant’s empirical variograms across scans showed within-cluster corre- lations (cluster variograms from scan 1 correlated with cluster variograms from scan 2) substantially higher than across clusters. independent test of this idea, we performed hierarchical clustering on the binned data from the vertex-wise variograms and display the results coloured by different canonical networks. The top panel of Fig. 5 pre- sents the dendrogram produced by this analysis. Clustering vertices based on their variogram proﬁles gives rise to two groups, one pre- dominantly encompassing the unimodal systems (primary sensor- imotor networks as well as parts of the dorsal attention network) and the other corresponding to limbic and transmodal systems, as well as the ventral attention network. This analysis, therefore, highlights a broad dissociation of cortex into two classes based on their variograms: one class of regions where the variograms have low sills and long con- nectivity and a second class of regions with higher sills and shorter effective distances. We also assessed how consistent these results were for individuals’ variograms across different scans (Fig. 5C), to ensure the cluster structure was not a consequence of group averaging and independent test of this idea, we performed hierarchical clustering on the binned data from the vertex-wise variograms and display the results coloured by different canonical networks. The top panel of Fig. 5 pre- sents the dendrogram produced by this analysis. Clustering vertices based on their variogram proﬁles gives rise to two groups, one pre- dominantly encompassing the unimodal systems (primary sensor- imotor networks as well as parts of the dorsal attention network) and the other corresponding to limbic and transmodal systems, as well as the ventral attention network. This analysis, therefore, highlights a broad dissociation of cortex into two classes based on their variograms: one class of regions where the variograms have low sills and long con- nectivity and a second class of regions with higher sills and shorter effective distances. We also assessed how consistent these results were for individuals’ variograms across different scans (Fig. Clustering variation in spatial dependency The variograms stratiﬁed by resting-state network suggest that there may be a small set of spatial dependency proﬁles that characterise a larger number of networks, and that these likely correspond to the difference between association and primary cortex. To provide an Nature Communications\| (2023) 14:5656 3 Article https://doi.org/10.1038/s41467-023-41334-2 g. 2 \| Distribution of the sill and effective distance of variograms across the ortex. A Variograms can be formally described through comparison of the bserved rate of change between similarity in brain activity and distance with fferent mathematical growth functions. We observe that the whole-brain vario- ram has most similarity to an exponential function. B Variograms can be char- cterised by two numbers, the partial sill (the height of the curve at 95% of its symptote) and the effective range (the distance of the sill). C Both the sill and the nge of the whole brain variogram show reasonable similarity when measured ithin the same individual in two scans on the same day (>0.73). D The regional stribution of the range (the distance of the sill) and (E) the sill (the height of the variogram at 95% of its asymptote) across the vertices of the human cortex. It ca be seen that the sill varies from 0.25 and 0.5 across the cortex and that in some regions the range can be as long as 15 cm. The relationship between the F distribution of the principal gradient of intrinsic connectivity and (G) variogram at each vertex (as described by each vertex’s partial sill and effective distance). H Spin permutation tests to assess the signiﬁcance of the correlation between th principal gradient and theoretical variogram parameters (the range and the sill) The true values are depicted by the dashed lines and the histogram displays the distribution of correlations from the permuted maps. variogram at 95% of its asymptote) across the vertices of the human cortex. It can be seen that the sill varies from 0.25 and 0.5 across the cortex and that in some regions the range can be as long as 15 cm. The relationship between the F distribution of the principal gradient of intrinsic connectivity and (G) variograms at each vertex (as described by each vertex’s partial sill and effective distance). H Spin permutation tests to assess the signiﬁcance of the correlation between the principal gradient and theoretical variogram parameters (the range and the sill). Clustering variation in spatial dependency The true values are depicted by the dashed lines and the histogram displays the distribution of correlations from the permuted maps. Fig. 2 \| Distribution of the sill and effective distance of variograms across the cortex. A Variograms can be formally described through comparison of the observed rate of change between similarity in brain activity and distance with different mathematical growth functions. We observe that the whole-brain vario- gram has most similarity to an exponential function. B Variograms can be char- acterised by two numbers, the partial sill (the height of the curve at 95% of its asymptote) and the effective range (the distance of the sill). C Both the sill and the range of the whole brain variogram show reasonable similarity when measured within the same individual in two scans on the same day (>0.73). D The regional distribution of the range (the distance of the sill) and (E) the sill (the height of the variogram at 95% of its asymptote) across the vertices of the human cortex. It can be seen that the sill varies from 0.25 and 0.5 across the cortex and that in some regions the range can be as long as 15 cm. The relationship between the F distribution of the principal gradient of intrinsic connectivity and (G) variograms at each vertex (as described by each vertex’s partial sill and effective distance). H Spin permutation tests to assess the signiﬁcance of the correlation between the principal gradient and theoretical variogram parameters (the range and the sill). The true values are depicted by the dashed lines and the histogram displays the distribution of correlations from the permuted maps. Fig. 2 \| Distribution of the sill and effective distance of variograms across the cortex. A Variograms can be formally described through comparison of the observed rate of change between similarity in brain activity and distance with different mathematical growth functions. We observe that the whole-brain vario- gram has most similarity to an exponential function. B Variograms can be char- acterised by two numbers, the partial sill (the height of the curve at 95% of its asymptote) and the effective range (the distance of the sill). C Both the sill and the range of the whole brain variogram show reasonable similarity when measured within the same individual in two scans on the same day (>0.73). https://doi.org/10.1038/s41467-023-41334-2 https://doi.org/10.1038/s41467-023-41334-2 Fig. 3 \| Variograms calculated for each canonical resting state network (Yeo, Krienen et al.47) in humans and in homologue networks in macaques. The middle panel shows the mean variogram (FC dissimilarity by distance along the cortex) calculated across all vertices for each Yeo network in the human Human Connectome Project data; the ﬁlled areas are the standard errors of the mean across vertices. Below is a similar analysis with fMRI data averaged from 14 awake Macaque monkey as a comparison. Data to recreate the variograms in Fig. 3 is available in source data ﬁle. Fig. 3 \| Variograms calculated for each canonical resting state network (Yeo, Krienen et al.47) in humans and in homologue networks in macaques. The middle panel shows the mean variogram (FC dissimilarity by distance along the cortex) calculated across all vertices for each Yeo network in the human Human Connectome Project data; the ﬁlled areas are the standard errors of the mean across vertices. Below is a similar analysis with fMRI data averaged from 14 awake Macaque monkey as a comparison. Data to recreate the variograms in Fig. 3 is available in source data ﬁle. Connectome Project data; the ﬁlled areas are the standard errors of the mean across vertices. Below is a similar analysis with fMRI data averaged from 14 awake Macaque monkey as a comparison. Data to recreate the variograms in Fig. 3 is available in source data ﬁle. Fig. 4 \| Permutation tests to assess the difference in average Sill and Range between networks. The results from spin permutation tests comparing the dif- ference in the differences between the range and sill between each pair of canonical resting state networks (in the human); network pairs with signiﬁcant differences (FDR-corrected, α < 0.1) are indicated with an asterisk. shows how brain regions related to different cognitive states differ in terms of their proﬁle of spatial dependencies. In general, more exter- nally focused tasks (e.g., labelled “visual” or “motor”) showed slower decrease in similarity with distance and a lower sill; whereas cognitive tasks associated with more abstract functions (such as “emotion”, “social”, “memory”), were associated with the opposite pattern with shorter ranges and higher sills. We subsequently clustered the tasks according to their sills/ranges to allow us to easily visualise the varia- bility in the variograms associated with each task (the red/blue colours in Fig. 5, panels A–E). https://doi.org/10.1038/s41467-023-41334-2 This allowed us to create a composite task acti- vation map for each cluster and plot the associated variograms showing the different spatial dependency proﬁles. Fig. 4 \| Permutation tests to assess the difference in average Sill and Range between networks. The results from spin permutation tests comparing the dif- ference in the differences between the range and sill between each pair of canonical resting state networks (in the human); network pairs with signiﬁcant differences (FDR-corrected, α < 0.1) are indicated with an asterisk. Nature Communications\| (2023) 14:5656 5 resting state networks (in the human); network pairs with signiﬁcant differences (FDR-corrected, α < 0.1) are indicated with an asterisk. Fig. 4 \| Permutation tests to assess the difference in average Sill and Range between networks. The results from spin permutation tests comparing the dif- ference in the differences between the range and sill between each pair of canonical resting state networks (in the human); network pairs with signiﬁcant differences (FDR-corrected, α < 0.1) are indicated with an asterisk. Fig. 4 \| Permutation tests to assess the difference in average Sill and Range between networks. The results from spin permutation tests comparing the dif- ference in the differences between the range and sill between each pair of canonical resting state networks (in the human); network pairs with signiﬁcant differences (FDR-corrected, α < 0.1) are indicated with an asterisk. resting state networks (in the human); network pairs with signiﬁcant differences (FDR-corrected, α < 0.1) are indicated with an asterisk. shorter ranges and higher sills. We subsequently clustered the tasks according to their sills/ranges to allow us to easily visualise the varia- bility in the variograms associated with each task (the red/blue colours in Fig. 5, panels A–E). This allowed us to create a composite task acti- vation map for each cluster and plot the associated variograms showing the different spatial dependency proﬁles. shorter ranges and higher sills. We subsequently clustered the tasks according to their sills/ranges to allow us to easily visualise the varia- bility in the variograms associated with each task (the red/blue colours in Fig. 5, panels A–E). This allowed us to create a composite task acti- vation map for each cluster and plot the associated variograms showing the different spatial dependency proﬁles. shows how brain regions related to different cognitive states differ in terms of their proﬁle of spatial dependencies. Clustering variation in spatial dependency 5C), to ensure the cluster structure was not a consequence of group averaging and Our analysis highlights that variograms vary between primary and association cortex, but do not separate large-scale networks such as the default mode and fronto-parietal cortex, even though these have contrasting behaviour at rest20 and have differing functional proﬁles. Our next analysis, therefore, examined how the variograms vary with meta-analytic descriptions of function. To this end, we averaged vertex-wise estimates of the range and sill parameters for responsive vertices (deﬁned as those with an estimated evoked BOLD response greater than threshold) in 24 topic maps generated by data mining the neuroimaging-related literature21 and discovering brain maps associated with them from an automated meta-analysis22. Figure 5 Nature Communications\| (2023) 14:5656 4 Article https://doi.org/10.1038/s41467-023-41334-2 Broadly, transmodal regions were lustered together in a separate cluster (red) to unimodal sensorimotor regions (blue). C Correlation of empirical variograms across vertices are consistent within each cluster within individuals and across different MR scans; bars are the standard error of the mean. D Average empirical variograms for each of the clusters within individuals reveals that one cluster exhibits more dramatic change in functional similarity with distance (shaded areas are the standard error of the mean). E The range and sill for each vertex, coloured by the cluster label for the left and right hemispheres. F the ranges and sills calculated across vertices activated by different cognitive processes (taken from a large automatic meta-analysis); These are over- layed on vertices coloured by their cluster membership from E. Fig. 5 \| Clustering vertices based on empirical variograms. Left A: clustering vertices based on empirical variograms. The dendograms, are coloured by the Yeo network that each vertex belongs to, displaying the tree structure of the similarity between variograms; the number for each column is the index of the representative vertex. B The dendogram was used to cluster the data into two clusters (coloured red and blue) for the left and right hemispheres. The order of the clustering was arbitrary across hemispheres and has been coloured based on approximate simi- larity between the left and right hemispheres. Broadly, transmodal regions were clustered together in a separate cluster (red) to unimodal sensorimotor regions Fig. 5 \| Clustering vertices based on empirical variograms. Left A: clustering (blue). C Correlation of empirical variograms across vertices are consistent within each cluster within individuals and across different MR scans; bars are the standard error of the mean. D Average empirical variograms for each of the clusters within individuals reveals that one cluster exhibits more dramatic change in functional similarity with distance (shaded areas are the standard error of the mean). E The range and sill for each vertex, coloured by the cluster label for the left and right hemispheres. F the ranges and sills calculated across vertices activated by different cognitive processes (taken from a large automatic meta-analysis); These are over- layed on vertices coloured by their cluster membership from E. Fig. 5 \| Clustering vertices based on empirical variograms. Left A: clustering vertices based on empirical variograms. https://doi.org/10.1038/s41467-023-41334-2 Importantly, while these broad types of cortex differed substantially in terms of their spatial dependencies, networks located within similar types of cortex were generally similar to each other, an observation which is important because these sys- tems are often thought to have contrasting functional and cognitive associations. These differences between unimodal and association cortex in humans were broadly similar to those seen in macaques Fig. 5 \| Clustering vertices based on empirical variograms. Left A: clustering vertices based on empirical variograms. The dendograms, are coloured by the Yeo network that each vertex belongs to, displaying the tree structure of the similarity between variograms; the number for each column is the index of the representative vertex. B The dendogram was used to cluster the data into two clusters (coloured red and blue) for the left and right hemispheres. The order of the clustering was arbitrary across hemispheres and has been coloured based on approximate simi- larity between the left and right hemispheres. Broadly, transmodal regions were clustered together in a separate cluster (red) to unimodal sensorimotor regions (blue). C Correlation of empirical variograms across vertices are consistent within each cluster within individuals and across different MR scans; bars are the standard error of the mean. D Average empirical variograms for each of the clusters within individuals reveals that one cluster exhibits more dramatic change in functional similarity with distance (shaded areas are the standard error of the mean). E The range and sill for each vertex, coloured by the cluster label for the left and right hemispheres. F the ranges and sills calculated across vertices activated by different cognitive processes (taken from a large automatic meta-analysis); These are over- layed on vertices coloured by their cluster membership from E. Article https://doi.org/10.1038/s41467-023-41334-2 ig. 5 \| Clustering vertices based on empirical variograms. Left A: clustering ertices based on empirical variograms. The dendograms, are coloured by the Yeo etwork that each vertex belongs to, displaying the tree structure of the similarity etween variograms; the number for each column is the index of the representative ertex. B The dendogram was used to cluster the data into two clusters (coloured ed and blue) for the left and right hemispheres. The order of the clustering was rbitrary across hemispheres and has been coloured based on approximate simi- arity between the left and right hemispheres. https://doi.org/10.1038/s41467-023-41334-2 The dendograms, are coloured by the Yeo network that each vertex belongs to, displaying the tree structure of the similarity between variograms; the number for each column is the index of the representative vertex. B The dendogram was used to cluster the data into two clusters (coloured red and blue) for the left and right hemispheres. The order of the clustering was arbitrary across hemispheres and has been coloured based on approximate simi- larity between the left and right hemispheres. Broadly, transmodal regions were clustered together in a separate cluster (red) to unimodal sensorimotor regions Relationship between spatial dependency and intracortical myelin This ﬁnding suggests a more complex relationship between functional connectivity and distance along the cortex than has typically been reported. For example, multiple previous studies have deﬁned a homogeneous cortex- or brain-wide relationship between function and distance (such as a single exponential distance rule, e.g., refs. 15,17,18,23, although23 noted that a single spatial relationship was inadequate to fully explain patterns of brain activity). The regional variability that we observed, reﬂects known functional divisions of brain function. Notably, the observed differences in spatial depen- dence proﬁle recapitulated the distinction between primary sensor- imotor and transmodal association cortex. In primary sensorimotor cortices, including visual and somatosensory cortex, we found that increasing distance is associated with a gradual change in function. In contrast, in association cortex we found that function changed with distance at a much faster rate. Importantly, while these broad types of cortex differed substantially in terms of their spatial dependencies, networks located within similar types of cortex were generally similar to each other, an observation which is important because these sys- tems are often thought to have contrasting functional and cognitive associations. These differences between unimodal and association cortex in humans were broadly similar to those seen in macaques suggesting that they are conserved across the primate nervous system. We found that these changes in how distance impacts functional var- iation are likely to be at least partly related to differences in micro- structure, as we found differences between association and unimodal Our ﬁnal analysis examined how microstructural features of different regions of the cortex correspond to the observed differences in spatial dependency proﬁles across cortex. Given its role in signal propagation, we examined whether myelination is linked to the shape of the vario- grams for different vertices. Figure 6 depicts the spatial distribution of estimated cortical myelin. We split vertices into deciles based on their levels of cortical myelination and plotted separate variograms for each decile. A clear separation emerges, with more highly myelinated ver- tices displaying, on average, longer distance spatial dependencies, and lower sills. This is made more explicit by plotting the range and the sill per vertex (Fig. 6) coloured by the level of myelination (warm colours indicating higher myelination). https://doi.org/10.1038/s41467-023-41334-2 In general, more exter- nally focused tasks (e.g., labelled “visual” or “motor”) showed slower decrease in similarity with distance and a lower sill; whereas cognitive tasks associated with more abstract functions (such as “emotion”, “social”, “memory”), were associated with the opposite pattern with Nature Communications\| (2023) 14:5656 5 Article https://doi.org/10.1038/s41467-023-41334-2 Relationship between spatial dependency and intracortical myelin Our ﬁnal analysis examined how microstructural features of different regions of the cortex correspond to the observed differences in spatial dependency proﬁles across cortex. Given its role in signal propagation, we examined whether myelination is linked to the shape of the vario- grams for different vertices. Figure 6 depicts the spatial distribution of estimated cortical myelin. We split vertices into deciles based on their levels of cortical myelination and plotted separate variograms for each decile. A clear separation emerges, with more highly myelinated ver- tices displaying, on average, longer distance spatial dependencies, and lower sills. This is made more explicit by plotting the range and the sill per vertex (Fig. 6) coloured by the level of myelination (warm colours indicating higher myelination). Discussion Given emerging evidence of the importance of topography in the mammalian cortex3,12, our study set out to understand how the dis- tance between regions relates to their functional similarity. In parti- cular, we examined whether this proﬁle of spatial dependence is heterogeneous varying across different cortical regions Our analysis This ﬁnding suggests a more complex relationship between functional connectivity and distance along the cortex than has typically been reported. For example, multiple previous studies have deﬁned a homogeneous cortex- or brain-wide relationship between function and distance (such as a single exponential distance rule, e.g., refs. 15,17,18,23, although23 noted that a single spatial relationship was inadequate to fully explain patterns of brain activity). The regional variability that we observed, reﬂects known functional divisions of brain function. Notably, the observed differences in spatial depen- dence proﬁle recapitulated the distinction between primary sensor- imotor and transmodal association cortex. In primary sensorimotor cortices, including visual and somatosensory cortex, we found that increasing distance is associated with a gradual change in function. In contrast, in association cortex we found that function changed with distance at a much faster rate. Nature Communications\| (2023) 14:5656 Discussion Given emerging evidence of the importance of topography in the mammalian cortex3,12, our study set out to understand how the dis- tance between regions relates to their functional similarity. In parti- cular, we examined whether this proﬁle of spatial dependence is heterogeneous, varying across different cortical regions. Our analysis ﬁrst established whole brain variograms are reasonably consistent across hemispheres, individuals, and within individuals measured in different scans on the same day. When we examined these on a regional basis, we observed substantial differences across the cortex. Nature Communications\| (2023) 14:5656 6 Article https://doi.org/10.1038/s41467-023-41334-2 Fig. 6 \| Variograms vary with intracortical myelin. A The empirical variograms between functional connectivity and distance split into deciles based on vertices’ myelin value (pink-greener colours correspond to higher-myelin content; shaded area is the standard error of the mean across individuals). Individual average esti- mated intracortical myelin for the two clusters. B The estimated range and sill for each vertex, coloured by estimated myelin. The inset brain is the average dis- tribution of estimated cortical myelin (from the HCP group average dataset). C the average estimated myelin distribution from the lateral and medial surfaces. D spin permutation tests comparing the spatial distribution of myelin with the range and sill parameters; the true correlations are depicted by the dashed lines. Fig. 6 \| Variograms vary with intracortical myelin. A The empirical variograms between functional connectivity and distance split into deciles based on vertices’ myelin value (pink-greener colours correspond to higher-myelin content; shaded area is the standard error of the mean across individuals). Individual average esti- mated intracortical myelin for the two clusters. B The estimated range and sill for each vertex, coloured by estimated myelin. The inset brain is the average dis- tribution of estimated cortical myelin (from the HCP group average dataset). C the average estimated myelin distribution from the lateral and medial surfaces. D spin permutation tests comparing the spatial distribution of myelin with the range and sill parameters; the true correlations are depicted by the dashed lines. cortex similar to those seen when exploring variation in intracortical microstructure approximated by the ratio of T1w/T2w image intensity a known proxy for intracortical myeloarchitecture24. systems terminate within close proximity of one another. Discussion This topo- graphic system could form the basis of an architecture that is hypo- thesised to explain why both the fronto-parietal34 and default mode networks12 contribute to multiple different forms of behaviour in a relatively abstract manner. These more complex, interdigitated pat- terns of function are captured empirically by the variograms which show rapid functional changes as a function of distance in each of the large-scale networks in association cortex. Importantly, our analysis suggests that both the fronto-parietal and default mode network share similar variogram proﬁles, suggesting that this is likely to explain simi- larities in their function rather than their differences. These results have implications for understanding how topographic differences inﬂuence cortical function. First, our data provides support for an organisation of unimodal cortex that supports the progressive elaboration of encoded stimulus features25. Our analysis established that both sensorimotor cortex and visual cortex are situated within regions in which the changes in function over distance are some of the most gra- dual when the cortex is viewed as a whole. When contrasted with asso- ciation cortex, this pattern is consistent with the view that sensory regions have a spatial organisation in which adjacent regions encode progressively complex features of the information extracted from sen- sory signals and that these compressed signals form the basis of signal processing for the next stage in the hierarchy e.g. ref. 26. This pattern of progressive change is assumed to be important in regions of primary cortex, such as visual cortex, and is captured empirically by the vario- grams in these regions which show relatively small steady changes in functional properties as the distance between two regions increases. Our study provides insights into the important observation that the default mode network, a brain system located at the maximal dis- tance from primary landmarks like the calcarine sulcus, also has a functional proﬁle which is one of the most unique in the mammalian nervous system3. Our analysis suggests regions of cortex where the default mode network is located combine two unique topographic properties that together explain why the distance between these sys- tems and the primary sensorimotor landmarks corresponds to the primary dimension of functional differentiation with the whole brain connectivity space3. Discussion Our analysis suggests that the increasing distance from primary landmarks in sensory cortex, and regions of the DMN would ﬁrst lead to increasing differences in functional similarity through the slow progressive changes in function with distance that emerge in primary cortex. In conjunction, with these gradual changes, our study suggests that the cortex where the DMN is where function changes most rapidly with increasing spatial distance. Thus, the observation that the distance between the DMN and sensory cortex corresponds to the greatest differentiation in function (i.e. the princi- ple gradient of functional connectivity3) is inevitable because this dis- tance combines (i) the progressive changes in function within primary sensorimotor cortex, and (ii) the complex interdigitated structure seen within the DMN6. Based on our analysis of T1w/T2w images it is possible that microstructural differences, such as myelin content, may be an important feature in distinguishing these types of cortex, an important question for future research to explore with more detailed anatomical techniques (e.g. ref. 35, than those used in the current investigation. Our study also provides insight into theoretical perspectives on how neural processing occurs in regions of association cortex. For example, contemporary work highlights that regions of association cortex can have relatively unique features both in terms of the functions they support, and in their observed neural properties (for a similar argument see12). For example, both the fronto-parietal and default mode networks are implicated in cognition in a relatively abstract manner, highlighted by their involvement in a wide range of tasks which despite being superﬁcially different may draw on similar underlying cognitive operations. For example, situations that have superﬁcially different features, such as the Stroop27 or working memory28, but show a common reliance on executive control, tend to activate the fronto- parietal network, as well as other task positive systems29. Similarly, the default mode network is often observed as contributing to situations when information from memory may be important for organising cognition, such as during mental time travel30, memory processes that rely on semantic31 or episodic knowledge32. Our analysis suggests that both of these large-scale systems are situated in regions of cortex where there are fairly rapid changes in functional similarity with increasing distance. These rapid changes in function over relatively short distances are likely to reﬂect the interdigitated nature of these systems6,33. Discussion These perspectives assume that a general property of associative cortex may be a topographic organisation in which relatively different functional Although our study highlights how different types of cortex can be understood through the emergence of functional differentiation across space, it also raises a number of important questions for future research into how topography shapes function. First, although our study shows that association and unimodal cortex systematically vary Nature Communications\| (2023) 14:5656 7 Article https://doi.org/10.1038/s41467-023-41334-2 in how function changes across the surface of the brain, this metric does not discriminate between systems that are known to be dis- tinctive in their functions. For example, although the variograms for both the fronto-parietal and default mode networks are similar, the situations in which these systems contribute to cognition are different. Likewise, the variograms in motor and visual cortex are similar, yet these systems have clear functional differences. It is likely that the different roles that these systems play in cognition may arise, not from the general way that function changes with space in these areas of cortex, but in terms of the speciﬁc location that these systems inhabit within the broader cortical landscape. In this way, our study highlights the more abstract properties that distinguish association and unim- odal cortex, but do not provide a concrete explanation for how these systems contribute to cognition and behaviour in a distinctive manner. Group averaged data from 14 macaque monkeys (two female) was used from the Newcastle cohort. Surface geodesic distance and homologous regions to the human data were taken from ref. 39. The vertex-wise map of cortical myelin was the group-average map taken from the Human Connectome Project 900-subject release; it is released in the Conte32k surface space and reduced to the same 10,000 vertices as the fMRI data. Similarly, the Yeo cortical parcellation4 in Conte32k surface space was taken from the same HCP 900 data release and was also reduced to 10,000 vertices. The 50 Neurosynth data-derived topic maps were downloaded in MNI152 2 mm space and then projected onto the mid-thickness Conte32k surface using the Connectome Workbench40 and then reduced to the same 10,000 vertices. Topics that were not related to cognitive tasks/ states were removed, leaving 24 topics. y g Second, our study does not constrain accounts of why association and unimodal cortex have differences in the spatial differentiation that we observe. Geodesic distance Pairwise geodesic distance was calculated along the cortical surface between all vertices (excluding the medial wall) using the Connectome Workbench tools, as implemented through the BrainSmash toolbox41. This was done on each hemisphere’s mid-thickness Conte32k surface reduced to 10,000 vertices prior to calculating the distances. The resulting vertex-wise distance matrices were used in all subsequent analyses. Methods The research presented here complies with relevant ethical regulations (King’s College London College Research Ethics Committee) govern- ing reanalysis of existing data. Functional connectivity The functional connectivity afﬁnity matrix was ﬁrst calculated between all 10,000 vertices for each individual fMRI scan using Pearson’s cor- relation between the BOLD time series. For group-average results, the correlation coefﬁcients were subsequently Fisher transformed and then for each vertex, averaged across subjects before applying an inverse Fisher transform, resulting in values between −1 and 1 for each edge of the functional connectivity matrix. Using a bounded similarity metric (0 = no similarity, 1/−1 identical) aids comparison across indi- viduals/vertices and facilitates interpretation for the resulting empiri- cal variograms. Empirical variograms The empirical variogram was calculated by quantifying how functional connectivity decreases in similarity as distance increases. To do this, all distances between pairs of vertices were collapsed into 20 equally spaced bins. Subsequently, dissimilarity matrix was created from the functional connectivity (1- Pearson’s correlation coefﬁcient) between pairs of vertices. These values were formed into equally spaced bins using a Gaussian smoothing function (following the approach set out in refs. 41,42). This resulted in a whole-cortex empirical variogram. For vertex-wise variograms, the same approach was taken but repeated for every row of the functional connectivity/distance matrix separately, resulting in a simpliﬁed form of the empirical variogram for each vertex. The empirical variogram captures the rate of change of (dis)similarity along the cortical surface, either globally or locally for each vertex. Discussion Our analysis highlights that microstructural differences, via a proxy of intracortical myelination, systematically trackdifferences in the empirical variograms. However, there are likely to be multiple microstructural features that track these differences, and these microstructural properties may also vary as a consequence of experi- ence. Therefore, it is important for future work to examine the different genetic and experiential changes that inﬂuence how function varies as a function of distance in both primary and association cortex to fully understand the inﬂuences that determine this fundamental feature of cortical organisation. One possibility is that the high degree of spatial heterogeneity within association cortex may result from the long- distance connections that link speciﬁc regions36. By extrapolation, these long-distance connections may provide a clue into how regions within these areas of cortex are able to serve distinct cognitive func- tions. Understanding how the broad changes in the parameters cap- tured by the variograms relate to long-distance connections is an important question for future research to address. In addition, from a methodological perspective, it is important for future work to under- stand how data analysis decisions (such as smoothing) impact variation in spatial autocorrelation as well as their consequences for quantifying large-scale cortical organisation37 and making statistical inferences. Imaging data The data used in this study are available from the Human Connectome Project (https://www.humanconnectome.org/study/hcp-young-adult/ document/extensively-processed-fmri-data-documentation), the PRI- MatE Data and Resource Exchange (https://fcon-1000.projects.nitrc. org/indi/indiPRIME.html), and Neurosynth https://neurosynth.org/ analyses/topics/). y p The majority of the analyses were performed on 51 participants’ resting state fMRI from the Human Connectome Project’s minimally pre-processed dataset (34 female); this involved registration to a common MNI152 template, minimal spatial smoothing and extensive ﬁltering for slow drifts, motion and other nuisance signals estimated using independent components analysis38. The 4D fMRI datasets for each participant were projected onto the Conte32k surface and the number of faces reduced resulting in 10,000 remaining vertices (using Matlab’s reducepatch command). Two resting-state runs (with opposite phase encoding direction, left-to-right and right-to-left, from the same scanning session) were taken from each participant. No further pre-processing was performed on the data. Since we were not focused on across-participant or within-participant variability, and for computational efﬁciency, we focused only on two scans from a subset of the whole Human Connectome Project dataset. Cognitive tasks From the Neurosynth 50 data-derived topics dataset22, those that did not refer to cognitive or behavioural states were removed, leaving: cognitive, inhibition, motor, numerical, action, conﬂict, spatial, emo- tion, empathy, decision, pain, memory, language, semantic, face, imagery, visual, eye movement, motion, attention, auditory, reward, social and working memory. The corresponding map for each topic was thresholded (absolute value z > 10, although qualitatively similar results were observed for other thresholds) and binarized, resulting in a vertex-wise mask of values that were strongly implicated for that topic (other thresholds produced qualitatively similar results). For each topic, the range and sill (taken from the theoretical variogram from the group average functional connectivity analysis) for each vertex within each mask were averaged together. Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Low-dimensional embedding of functional connectivity The principal connectivity gradient was calculated using the Brain- space toolbox44. This involved taking the group-average functional connectivity afﬁnity matrix and performing non-linear dimensionality reduction using the Laplacian Eigenmaps approach, separately for each hemisphere. Theoretical variogram It is common practice to ﬁt a function to empirical variograms, this is typically used prior to spatial regression; however, in our case, it allows us to compactly summarise the shape of the empirical variogram with a small number of parameters, facilitating comparisons across datasets and vertices, and aggregation across multiple vertices. For the repor- ted analyses we used an exponential function. This is motivated by a range of prior studies suggesting exponential relationships between distance and various neural measures (e.g., ref. 43). We also performed a similar ﬁt for three other theoretical models (a Gaussian, a power- law model, and a periodic model which allows for non-monotonic functions), with qualitatively similar results. Empirical variograms were trimmed to bins between 2 and 19 (to remove bins with few sampled distances). Subsequently, non-linear least squares was used to Nature Communications\| (2023) 14:5656 8 Article https://doi.org/10.1038/s41467-023-41334-2 of the empirical functional connectivity matrix from which the vario- grams were generated: full random permutation, Mantel permutation (that preserved row and column structure), or Mantel permutation followed by spatial smoothing matched to the empirical variogram and then resampling (similar to the approach taken by41 but applied to the functional connectivity matrix). All three approaches enforce an approximately homogeneous spatial dependency across the brain, although in the case of the randomisation and Mantel randomisation the spatial dependency is destroyed. For approach 3, a smoothing kernel was chosen iteratively to maximise the overlap with the mea- sured empirical variogram; thereby approximately capturing the whole- brain spatial dependency but with an homogeneous spatial relation- ship. Each model was recalculated 1000 times, and the sill and the range for each vertex calculated. The true sill and range parameters could then be compared to the equivalent null model parameters. estimate the sill and range parameters. Given that the distribution of pairwise distances varies across vertices (see Supplementary Fig. 1, left), for the main analyses we restricted the maximum distance to be 150 mm for calculating bins. However, qualitatively similar results were obtained by varying the upper distance limit (see Supplementary Fig. 1, right). estimate the sill and range parameters. Given that the distribution of pairwise distances varies across vertices (see Supplementary Fig. 1, left), for the main analyses we restricted the maximum distance to be 150 mm for calculating bins. However, qualitatively similar results were obtained by varying the upper distance limit (see Supplementary Fig. 1, right). Data availability The data used in this study are available from the Human Connectome Project (https://www.humanconnectome.org/study/hcp-young-adult/ document/extensively-processed-fmri-data-documentation), the PRI- MatE Data and Resource Exchange (https://fcon-1000.projects.nitrc. org/indi/indiPRIME.html) and Neurosynth https://neurosynth.org/ analyses/topics/). Data to recreate the variograms in Fig. 3 is avail- able in source data ﬁle. Source data are provided with this paper. Myelin 4. Thomas Yeo, B. T. et al. The organization of the human cerebral cortex estimated by intrinsic functional connectivity. J. Neurophy- siol. 106, 1125–1165 (2011). The estimated intracortical myelin maps derived from the ratio of T1 and T2 weighted MR images24 from the Human Connectome Project were split into deciles based on their estimated myelin level. The empirical variograms of vertices within each decile were averaged. In addition, the estimated average myelin value for each of the clusters (see above) were calculated. 5. Braga, R. M., Sharp, D. J., Leeson, C., Wise, R. J. S. & Leech, R. Echoes of the brain within default mode, association, and heteromodal cortices. J. Neurosci. 33, 14031–14039 (2013). 5. Braga, R. M., Sharp, D. J., Leeson, C., Wise, R. J. S. & Leech, R. Echoes of the brain within default mode, association, and heteromodal cortices. J. Neurosci. 33, 14031–14039 (2013). 6. Braga, R. M. & Buckner, R. L. Parallel interdigitated distributed networks within the individual estimated by intrinsic functional connectivity. Neuron 95, 457–471.e5 (2017). 6. Braga, R. M. & Buckner, R. L. Parallel interdigitated distributed networks within the individual estimated by intrinsic functional connectivity. Neuron 95, 457–471.e5 (2017). References 1. Mesulam, M. M. From sensation to cognition. Brain 121, 1013–1052 (1998). 2. Felleman, D. J. & Van, D. C. Essen, distributed hierarchical proces- sing in the primate cerebral cortex. Cereb. Cortex 1, 1–47 (1991). 3. Margulies, D. S. et al. Situating the default-mode network along a principal gradient of macroscale cortical organization. Proc. Natl Acad. Sci. USA 113, 12574–12579 (2016). Code availability Python code to reproduce the analyses and ﬁgures is available at https://github.com/ActiveNeuroImaging/BrainVariograms.git. Clustering Agglomerative hierarchical clustering, with ward linkage and the Eucli- dean distance metric was applied simultaneously to all the vertex-wise variograms separately for each cortical hemisphere. Subsequently, SciPy’s fcluster command was used to ﬂatten the hierarchy into two clusters. To assess the robustness of the resulting clusters each vertex’s variogram was correlated with all other variograms calculated in a separate fMRI run within the same individual. The correlation scores were Fisher transformed and then subsequently averaged both within and across clusters. Nature Communications\| (2023) 14:5656 Null models We used spin permutation tests to assess the strength of correlations between theoretical variogram parameters with the principal gradient and estimated myelin spatial maps. A 1000 permutations of randomly rotated data were generated for the spatial maps using ref. 45 and permutation correlation values were then compared to the true value, resulting in a p-value. We also applied a similar approach to spinning the Yeo7 parcellation on the sphere 1000 times, and then calculating the difference in estimated range and sill between each of the Yeo7 networks; this resulted in a distribution of random differences for the sill and the parameter against which the true difference scores could be assessed. 7. Tootell, R. B. H., Silverman, M. S., Switkes, E. & De Valois, R. L. Deoxyglucose analysis of retinotopic organization in primate striate cortex. Science 218, 902–904 (1982). 8. Sereno, M. I. et al. Borders of multiple visual areas in humans revealed by functional magnetic resonance imaging. Science 268, 889–893 (1995). 9. Sereno, M. I. & Huang, R.-S. A human parietal face area contains aligned head-centered visual and tactile maps. Nat. Neurosci. 9, 1337–1343 (2006). 10. Formisano, E. et al. Mirror-symmetric tonotopic maps in human primary auditory cortex. Neuron 40, 859–869 (2003). We also used generative null models (Supplementary Fig. 2)46 both to generate alternate statistics but also to illustrate the difference between homogeneous spatial dependency structure and the observed heterogeneous structure. To this end, three generative null models were applied to a downsampled (for computational efﬁciency) version 11. Raichle, M. E. The Brain’s default mode network. Annu. Rev. Neurosci. 38, 433–447 (2015). 12. Smallwood, J. et al. The default mode network in cognition: a topographical perspective. Nat. Rev. Neurosci. 22, 503–513 (2021). Nature Communications\| (2023) 14:5656 9 Article https://doi.org/10.1038/s41467-023-41334-2 38. Salimi-Khorshidi, G. et al. Automatic denoising of functional MRI data: combining independent component analysis and hierarchical fusion of classiﬁers. NeuroImage 90, 449–468 (2014). 13. Oligschläger, S. et al. Gradients of connectivity distance are anchored in primary cortex. Brain Struct. Funct. 222, 2173–2182 (2017). 14. Cressie, N. A. C. Statistics for Spatial Data (J. Wiley, 2022). 39. Xu, T. et al. Interindividual variability of functional connectivity in awake and anesthetized Rhesus Macaque Monkeys. Biol. Psychiatry Cogn. Neurosci. Neuroimaging 4, 543–553 (2019). 15. Ercsey-Ravasz, M. et al. A predictive network model of cerebral cortical connectivity based on a distance rule. Neuron 80, 184–197 (2013). 40. Marcus, D. S. et al. Null models Human Connectome Project informatics: quality control, database services, and data visualization. NeuroImage 80, 202–219 (2013). 16. Leopold, D. A., Murayama, Y. & Logothetis, N. K. Very slow activity ﬂuctuations in monkey visual cortex: implications for functional brain imaging. Cereb. Cortex 13, 422–433 (2003). 41. Burt, J. B., Helmer, M., Shinn, M., Anticevic, A. & Murray, J. D. Gen- erative modeling of brain maps with spatial autocorrelation. Neu- roImage 220, 117038 (2020). 17. Mišić, B. et al. The functional connectivity landscape of the human brain. PLoS ONE 9, e111007 (2014). 18. Song, H. F., Kennedy, H. & Wang, X.-J. Spatial embedding of structural similarity in the cerebral cortex. Proc. Natl Acad. Sci. USA 111, 16580–16585 (2014). 42. Viladomat, J., Mazumder, R., McInturff, A., McCauley, D. J. & Hastie, T. Assessing the signiﬁcance of global and local correlations under spatial autocorrelation: a nonparametric approach. Biometrics 70, 409–418 (2014). 19. Smith, S. M. et al. Resting-state fMRI in the Human Connectome Project. Neuroimage 80, 144–168 (2013). 43. M. Shinn, et al. Spatial and temporal autocorrelation weave com- plexity in brain networks. bioRxiv https://doi.org/10.1101/2021.06. 01.446561 (2022). 20. Fox, M. D. et al. The human brain is intrinsically organized into dynamic, anticorrelated functional networks. Proc. Natl Acad. Sci. USA 102, 9673–9678 (2005). 44. Vos de Wael, R. et al. BrainSpace: a toolbox for the analysis of macroscale gradients in neuroimaging and connectomics datasets. Commun. Biol. 3, 1–10 (2020). 21. Poldrack, R. A. et al. Discovering relations between mind, brain, and mental disorders using topic mapping. PLoS Comput. Biol. 8, e1002707 (2012). 45. Markello, R. D. & Misic, B. Comparing spatial null models for brain maps. NeuroImage 236, 118052 (2021). 22. Yarkoni, T., Poldrack, R. A., Nichols, T. E., Van Essen, D. C. & Wager, T. D. Large-scale automated synthesis of human functional neu- roimaging data. Nat. Methods 8, 665–670 (2011). 46. Váša, F. & Mišić, B. Null models in network neuroscience. Nat. Rev. Neurosci. 23, 493–504 (2022). 23. Choi, H. & Mihalas, S. Synchronization dependent on spatial structures of a mesoscopic whole-brain network. PLoS Comput. Biol. 15, e1006978 (2019). 47. Yeo, B. T. T. et al. The organization of the human cerebral cortex estimated by intrinsic functional connectivity. J Neurophysiol. 106, 1125–1165 (2011). 24. Glasser, M. F. & Essen, D. C. V. Mapping human cortical areas in vivo based on myelin content as revealed by T1- and T2-weighted MRI. J. Neurosci. 31, 11597–11616 (2011). Competing interests 31. Ralph, M. A. L., Jefferies, E., Patterson, K. & Rogers, T. T. The neural and computational bases of semantic cognition. Nat. Rev. Neurosci. 18, 42–55 (2017). 32. Buckner, R. L. & DiNicola, L. M. The brain’s default network: updated anatomy, physiology and evolving insights. Nat. Rev. Neurosci. 20, 593–608 (2019). 33. Goldman-Rakic, P. S. & Schwartz, M. L. Interdigitation of con- tralateral and ipsilateral columnar projections to frontal association cortex in primates. Science 216, 755–757 (1982). Correspondence and requests for materials should be addressed to Robert Leech. Correspondence and requests for materials should be addressed to Robert Leech. 34. Duncan, J. An adaptive coding model of neural function in pre- frontal cortex. Nat. Rev. Neurosci. 2, 820–829 (2001). Peer review information Nature Communications thanks Enrico Amico, Janine Bijsterbosch and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review ﬁle is avail- able. Peer review information Nature Communications thanks Enrico Amico, Janine Bijsterbosch and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review ﬁle is avail- able. 35. Bajada, C. J., Schreiber, J. & Caspers, S. Fiber length proﬁling: a novel approach to structural brain organization. NeuroImage 186, 164–173 (2019). 36. Bullmore, E. & Sporns, O. Complex brain networks: graph theore- tical analysis of structural and functional systems. Nat. Rev. Neu- rosci. 10, 186–198 (2009). Acknowledgements Acknowledgements R.L. was funded by the Medical Research Council (Ref: MR/R005370/1), Wellcome/EPSRC Centre for Medical Engineering (Ref: WT 203148/Z/16/ Z), Simons Foundation (SFG640710) and support from the Data to Early Diagnosis and Precision Medicine Industrial Strategy Challenge Fund, UK Research and Innovation (UKRI). The authors would also like to acknowl- edge support from the Data to Early Diagnosis and Precision Medicine Industrial Strategy Challenge Fund, UK Research and Innovation (UKRI). R.L. was funded by the Medical Research Council (Ref: MR/R005370/1), Wellcome/EPSRC Centre for Medical Engineering (Ref: WT 203148/Z/16/ 25. Quartz, S. R. The constructivist brain. Trends Cogn. Sci. 3, 48–57 (1999). 26. Freeman, J. & Simoncelli, E. P. Metamers of the ventral stream. Nat. Neurosci. 14, 1195–1201 (2011). 27. Stroop, J. R. Studies of interference in serial verbal reactions. J. Exp. Psychol. 18, 643–662 (1935). 28. Baddeley, A. Working memory. Science 255, 556–559 (1992). Author contributions 29. Duncan, J. The multiple-demand (MD) system of the primate brain: mental programs for intelligent behaviour. Trends Cogn. Sci. 14, 172–179 (2010). R.L. and J.S. designed the study; R.L., R.W.V.D.W., F.V., T.X., R.A.B., R.S., R.M.B., M.P.M., J.R., B.C.B., E.J.H.J.,E.J., D.S.M. and J.S. contributed tothe analysis of the data and the manuscript. 30. Schacter, D. L. & Addis, D. R. The cognitive neuroscience of con- structive memory: remembering the past and imagining the future. Philos. Trans. R. Soc. B: Biol. Sci. 362, 773–786 (2007). Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-41334-2. Competing interests The authors declare no competing interests. Nature Communications\| (2023) 14:5656 Reprints and permissions information is available at 37. Watson, D. M. & Andrews, T. J. Connectopic mapping techniques do not reﬂect functional gradients in the brain. NeuroImage 277, 120228 (2023). Publisher’s note Springer Nature remains neutral with regard to jur- isdictional claims in published maps and institutional afﬁliations. 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https://openalex.org/W2024884332	https://europepmc.org/articles/pmc2888785?pdf=render	English	null	Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR	Infectious agents and cancer	2,010	cc-by	5,069	* Correspondence: rlopez@ipicyt.edu.mx 1 División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, Camino de la Presa San José 2055, 78216 San Luis Potosí S.L.P., México Full list of author information is available at the end of the article METHODOLOGY Open Access Open Access BioMed Central © 2010 Hernández-Arteaga and López-Revilla; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided the original work is properly cited. Hernández-Arteaga and López-Revilla Infectious Agents and Cancer 2010, 5:9 http://www.infectagentscancer.com/content/5/1/9 Hernández-Arteaga and López-Revilla Infectious Agents and Cancer 2010, 5:9 http://www.infectagentscancer.com/content/5/1/9 Hernández-Arteaga and López-Revilla Infectious Agents and Cancer 2010, 5:9 http://www.infectagentscancer.com/content/5/1/9 Background Invasive cervical cancer develops in women with persis- tent infection by high-risk human papillomavirus (HR- HPV), among which HPV16 and HPV18 are the most fre- quent types [1], HPV16 being the most prevalent and associated with nearly half of the invasive cervical cancer cases in the world [2,3]. In this work we developed an ultrasensitive method to quantify E6-HPV16 oncogene sequences in two steps: 1) preamplification of the E6-1 (645 bp) sequence through 15 cycles of direct conventional PCR, and 2) amplifica- tion of the E6-2 (237 bp) sequence through nested qPCR in the presence of EvaGreen. Preamplification signifi- cantly increased the sensitivity and the method con- firmed that the SiHa cell line contains only one copy of the HPV16 genome per cell. Molecular methods to quantify the number of HPV16 genome copies serve to determine the viral load and the progression of HPV16 neoplastic lesions. Hybrid capture [4] or "nested" PCR [5] are used for molecular diagnosis of HPV infection, whereas qPCR is used to assess viral load and integration to the cellular genome [6,7]. Abstract Background: We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR (qPCR) in the presence of the DNA intercalating agent EvaGreen. Results: Amplification mixtures calibrated with a known number of pHV101 copies carrying a 645 base pair (bp)-long insert of the human papillomavirus type 16 (HPV16) E6 oncogene were used to generate the E6-1 amplicon of 645 bp by conventional PCR and then the E6-2 amplicon of 237 bp by nested qPCR. Direct and nested qPCR mixtures for E6-2 amplification corresponding to 2.5 × 102-2.5 × 106 initial pHV101 copies had threshold cycle (Ct) values in the ranges of 18.7-29.0 and 10.0-25.0, respectively. The Ct of qPCR mixtures prepared with 1/50 volumes of preamplified mixtures containing 50 ng of DNA of the SiHa cell line (derived from an invasive cervical cancer with one HPV16 genome per cell) was 19.9. Thermal fluorescence extinction profiles of E6-2 amplicons generated from pHV101 and SiHa DNA were identical, with a peak at 85.5°C. Conclusions: Our method based on conventional preamplification for 15 cycles increased 10,750 times the sensitivity of nested qPCR for the quantitation of the E6 viral oncogene and confirmed that the SiHa cell line contains one E6- HPV16 copy per cell. cific and more sensitive in qPCR mixtures containing the DNA intercalating agent EvaGreen [10]. Results Conventional nested PCR is more sensitive than con- ventional "direct" PCR to detect HPV16 E6 oncogene sequences [8,9] whose quantitation is reproducible, spe- * Correspondence: rlopez@ipicyt.edu.mx Preamplification and nested qPCR To increase qPCR sensitivity E6-1 was first "preamplified" in conventional PCR mixtures and E6-2 was then "ampli- fied" in nested qPCR mixtures containing EvaGreen, the LCRS/E7AS primer pair, and 1/50 volume of the corre- sponding preamplified mixtures. The families of type curves required to calibrate the number of E6 copies and to quantify them in problem samples could be generated from nested qPCR mixtures derived from two types of preamplification mixtures: a) "complete" mixtures, prepared with serial logarithmic dilutions of the pHV101 template molecules before pre- amplification, and b) "minimum" mixtures, prepared with serial logarithmic dilutions from a single preamplified mixture containing the maximum number of template molecules used in complete mixtures. Typical Ct values of nested qPCR: 10.80 for 2.5 × 106, 14.35 for 2.5 × 105, 17.71 for 2.5 × 104, 21.62 for 2.5 × 103 and 25.02 for 2.5 × 102 initial pHV101 copies in the pre- amplification mixtures (Table 1) were consistent (ΔCt range: 3.3-3.9) and reproducible (R2 = 1.000), whereas the negative control mixture devoid of template DNA had a Ct = 27 (Fig. 1B). Figure 1 Family of E6-2 amplification curves obtained by direct and nested qPCR. RFU, relative fluorescence units. Threshold (thick horizontal line): 50 RFU. (A) Family of curves obtained in direct qPCR mixtures with pHV101 dilutions and primers for E6-2. Curve 1, 2.5 × 106 pHV101 copies. Curve 2, 2.5 × 105 copies. Curve 3, 2.5 × 104 copies. Curve 4, 2.5 × 103 copies. Curve 5, Blank (without DNA). (B) Family of curves obtained by nested qPCR with E6-2 primers and 1/50 volume of mixtures preamplified 15 cycles with pHV101 and E6-1 primers. Curve 1, 2.5 × 106 pHV101 copies. Curve 2, 2.5 × 105 copies. Curve 3, 2.5 × 104 copies. Curve 4, 2.5 × 103 copies. Curve 5, 2.5 × 102 copies. Curve 6, blank (without DNA). Figure 1 Family of E6-2 amplification curves obtained by direct and nested qPCR. RFU, relative fluorescence units. Threshold (thick horizontal line): 50 RFU. (A) Family of curves obtained in direct qPCR mixtures with pHV101 dilutions and primers for E6-2. Curve 1, 2.5 × 106 pHV101 copies. Curve 2, 2.5 × 105 copies. Curve 3, 2.5 × 104 copies. Curve 4, 2.5 × 103 copies. Curve 5, Blank (without DNA). 2,500 copies of the E6 oncogene can be quantified by direct qPCR (Fig. 1A). 2,500 copies of the E6 oncogene can be quantified by direct qPCR (Fig. 1A). 2,500 copies of the E6 oncogene can be quantified by direct qPCR (Fig. 1A). Since labor, time and the amount of reagents are signif- icantly reduced using minimum mixtures, we compared the results from nested qPCR mixtures prepared with 1/ 50 volume samples from preamplified mixtures which contained pHV101 molecules in the range of 2.5 × 102-to 2.5 × 106 copies with those prepared using serial logarith- mic dilutions from a single mixture containing 2.5 × 106 pHV101 molecules that had been preamplified. We also added 50 ng of "carrier" normal human blood DNA to both complete and minimum positive preamplification mixtures (same amount as SiHa DNA in problem mix- tures) and found that all of them generate equivalent nested qPCR amplification results. E6-2 amplification by direct qPCR E6-2 amplification from pHV101 in direct qPCR mix- tures containing the pU1M/pU2R primer pair and 2.5 × 102 to 2.5 × 106 pHV101 copies in the presence of EvaGreen yielded a consistent family of type curves with Ct values between 18.70 and 29.00, ΔCt values in the 3.1- 3.8 range, and lack of fluorescence increase in mixtures devoid of DNA. These experiments indicated that at least Page 2 of 7 Page 2 of 7 Hernández-Arteaga and López-Revilla Infectious Agents and Cancer 2010, 5:9 http://www.infectagentscancer.com/content/5/1/9 Hernández-Arteaga and López-Revilla Infectious Agents and Cancer 2010, 5:9 http://www.infectagentscancer.com/content/5/1/9 E6 copies per SiHa cell The sensitivity and reliability of the nested qPCR method was tested by determining the HPV16 genome load through quantitation of E6 sequences present in the SiHa line, known to contain only one copy of the HPV16 genome per cell [11]. The following PCR mixtures containing the LCRS/ E7AS primer pair and the DNA templates indicated were incubated for 15 cycles under conditions to generate ("preamplify") the E6-1 amplicon: a) positive controls with 2.5 × 106-2.5 × 102 copies of pHV101 and 50 ng of "carrier" normal human blood DNA; b) negative controls with 50 ng of "carrier" DNA; c) problem samples with 50 ng of SiHa DNA; and d) blanks, without DNA. Nested qPCR mixtures containing EvaGreen, the pU1M/pU2R primer pair and 1/50 volume from each preamplified mixture were incubated for 35 cycles. Preamplification and nested qPCR (B) Family of curves obtained by nested qPCR with E6-2 primers and 1/50 volume of mixtures preamplified 15 cycles with pHV101 and E6-1 primers. Curve 1, 2.5 × 106 pHV101 copies. Curve 2, 2.5 × 105 copies. Curve 3, 2.5 × 104 copies. Curve 4, 2.5 × 103 copies. Curve 5, 2.5 × 102 copies. Curve 6, blank (without DNA). Hernández-Arteaga and López-Revilla Infectious Agents and Cancer 2010, 5:9 http://www.infectagentscancer.com/content/5/1/9 Page 3 of 7 Table 1: Ct values in direct and nested qPCR E6-2 amplicon mixtures with a variable number of pHV101 copies pHV101 copies Ct ΔCt Direct qPCR Nested qPCR 2.5 × 102 --- 25.02 --- 2.5 × 103 29.00 21.62 7.40 2.5 × 104 25.90 17.71 8.10 2.5 × 105 22.10 14.35 7.75 2.5 × 106 18.70 10.80 7.90 Ct values in direct and nested qPCR E6-2 amplicon mixtures with a variable number of pHV101 copies initial pHV101 copy number. On the other hand, the Ct value (19.9) and E6 copy number obtained from a typical preamplification mixture containing 50 ng of SiHa cell DNA (equivalent to 6.05 × 103 HPV16 genome copies) had the magnitudes expected (i.e., one E6 copy per 7.1 pg of DNA, equivalent to the genomic mass of a human dip- loid cell) (Fig. 2A). The average decrease in Ct values of nested qPCR resulting from preamplification for 15 cycles was 7.78 (Table 1), corresponding to a 215-fold sensitivity increase. Multiplying this factor by the 50-fold dilution factor of preamplified samples in nested reactions implies that under the conditions used the overall sensitivity of nested qPCR increased 10,750 times. The thermal denaturation ("fluorescence extinction") profiles of nested qPCR products generated from pHV101 and SiHa DNA were identical, with a major peak around 85.5°C whose magnitude was proportional to the initial number of pHV101 copies, and clearly different from the secondary peak at 78.5°C which appeared in the negative controls containing just the forward primer of the second pair (Fig. 2B). Discussion Our group has already confirmed that detection of HPV infection is more sensitive by nested conventional PCR through the successive synthesis of the E6-1 and E6-2 amplicons used in this work [9], and that quantitation of E6 oncogene sequences by qPCR in the presence of EvaGreen is reproducible and specific [10]. The PCR mixtures required to calibrate and quantify the number of E6 copies present in problem samples were prepared using serial logarithmic dilutions of pHV101, a pGEM construct containing a 645 bp-insert whose sequence corresponds to the E6-HPV16 ORF [10]. In nested qPCR mixtures for E6-2 containing 1/50 vol- umes of serial logarithmic dilutions from preamplified mixtures with 2.5 × 106 to 2.5 × 102 pHV101 molecules the Ct values ranged from 10.8 to 25.0 cycles (R2 = 1.000; slope = -3.57), and the ΔCt values were consistent (range: 3.3-3.9). The Ct values of nested qPCR mixtures prepared without preamplified samples were 27.0 and 28.0, whereas the basal fluorescence decreased slightly in pre- amplification mixtures containing SiHa DNA with the pU1M primer only (Fig. 2A). To increase amplification sensitivity, E6-1 was "pream- plified" for 15 conventional PCR cycles and then E6-2 was "amplified" by 35 "nested" qPCR cycles in the presence of normal human DNA using, as source of template, 1/50 volume of either preamplified mixtures prepared with serial logarithmic dilutions of pHV101 or with serial log- arithmic dilutions of a single preamplified mixture pre- pared with the highest number of pHV101 copies. Direct The family of type curves obtained from nested qPCR mixtures for E6-2 amplification was consistent with the Hernández-Arteaga and López-Revilla Infectious Agents and Cancer 2010, 5:9 http://www.infectagentscancer.com/content/5/1/9 Hernández-Arteaga and López-Revilla Infectious Agents and Cancer 2010, 5:9 http://www.infectagentscancer.com/content/5/1/9 Page 4 of 7 PCR ld tif littl 2 500 E6 HPV16 l ft l 27 i PCR i t d ith l Figure 2 Thermal denaturation (fluorescence extinction) profiles of the E6-2 amplicons generated from pHV101 and SiHa DNA. RFU, relative fluorescence units. (A) Family of curves obtained with serially diluted pHV101 or 50 ng of SiHa DNA. Threshold (thick horizontal line): 50 RFU. Curve 1, 2.5 × 106 pHV101 copies. Curve 2, 2.5 × 105 pHV101 copies. Curve 3, 2.5 × 104 pHV101 copies. Curve 4, 2.5 × 103 pHV101 copies. Curve 5, 2.5 × 102pHV101 copies. Curve 6, Blank (without DNA). Curve 7, normal human blood DNA. Discussion Use of the SiHa cell line, originally isolated from an invasive cervical cancer caused by HPV16 and known to contain only one viral genome per cell [11], confirmed Non-specific fluorescence appears to depend on the interaction of the two E6-2 primers because it increased Hernández-Arteaga and López-Revilla Infectious Agents and Cancer 2010, 5:9 http://www.infectagentscancer.com/content/5/1/9 Hernández-Arteaga and López-Revilla Infectious Agents and Cancer 2010, 5:9 http://www.infectagentscancer.com/content/5/1/9 Page 5 of 7 65 559 7155 A 544 858 LCR E6 E7 A 26 670 LCRS E7AS E6-1 645 bp 655 409 pU1M pU2R E6-2 237 bp 544 858 7904/1 1000 p p p 26 AAGGGCGTAA CCGAAATCGG TTGAACCGAA ACCGGTTAGT ATAAAAGCAG ACATTTTATG 86 CACCAAAAGA GAACTGCAAT TTTCAGGACC CACAGGAGCG ACCCAGAAAG TTACCACAGT 146 TATGCACAGA GCTGCAAACA ACTATACATG ATATAATATT AGAATGTGTG TACTGCAAGC 206 AACAGTTACT GCGACGTGAG GTATATGACT TTGCTTTTCG GGATTTATGC ATAGTATATA B 206 AACAGTTACT GCGACGTGAG GTATATGACT TTGCTTTTCG GGATTTATGC ATAGTATATA 266 GAGATGGGAA TCCATATGCT GTATGTGATA AATGTTTAAA GTTTTATTCT AAAATTAGTG 326 AGTATAGACA TTATTGTTAT AGTTTGTATG GAACAACATT AGAACAGCAA TACAACAAAC 386 CGTTGTGTGA TTTGTTAATT AGGTGTATTA ACTGTCAAAA GCCCACTGTGT CCTGAAGAAC 446 AAGCAAAGAC ATCTGGACAA AAAGCAAAGA TTCCATAATA TAAGGGGTCG GTGGACCGGT 506 GATGTATGTC TTGTTGCAGA TCATCAAGAA CACGTAGAGA AACCCAGCTG TAATCATGCT 566 ATGGAGATAC ACCTACATTG CATGAATATA TGTTAGATTT GCAACCAGAG ACAACTGATC 566 ATGGAGATAC ACCTACATTG CATGAATATA TGTTAGATTT GCAACCAGAG ACAACTGATC 626 CTACTGTTAT GAGCAATTAA ATGACAGCTC AGAGGAGGAG GATGA 65 559 7155 A 544 858 LCR E6 E7 A 26 670 LCRS E7AS E6-1 645 bp 655 409 pU1M pU2R E6-2 237 bp 544 858 7904/1 1000 p p p 26 AAGGGCGTAA CCGAAATCGG TTGAACCGAA ACCGGTTAGT ATAAAAGCAG ACATTTTATG 86 CACCAAAAGA GAACTGCAAT TTTCAGGACC CACAGGAGCG ACCCAGAAAG TTACCACAGT 146 TATGCACAGA GCTGCAAACA ACTATACATG ATATAATATT AGAATGTGTG TACTGCAAGC 206 AACAGTTACT GCGACGTGAG GTATATGACT TTGCTTTTCG GGATTTATGC ATAGTATATA B 206 AACAGTTACT GCGACGTGAG GTATATGACT TTGCTTTTCG GGATTTATGC ATAGTATATA 266 GAGATGGGAA TCCATATGCT GTATGTGATA AATGTTTAAA GTTTTATTCT AAAATTAGTG 326 AGTATAGACA TTATTGTTAT AGTTTGTATG GAACAACATT AGAACAGCAA TACAACAAAC 386 CGTTGTGTGA TTTGTTAATT AGGTGTATTA ACTGTCAAAA GCCCACTGTGT CCTGAAGAAC 446 AAGCAAAGAC ATCTGGACAA AAAGCAAAGA TTCCATAATA TAAGGGGTCG GTGGACCGGT 506 GATGTATGTC TTGTTGCAGA TCATCAAGAA CACGTAGAGA AACCCAGCTG TAATCATGCT 566 ATGGAGATAC ACCTACATTG CATGAATATA TGTTAGATTT GCAACCAGAG ACAACTGATC 566 ATGGAGATAC ACCTACATTG CATGAATATA TGTTAGATTT GCAACCAGAG ACAACTGATC 626 CTACTGTTAT GAGCAATTAA ATGACAGCTC AGAGGAGGAG GATGA 65 559 7155 A 544 858 LCR E6 E7 A 26 670 LCRS E7AS E6-1 645 bp 655 409 pU1M pU2R E6-2 237 bp 544 858 7904/1 1000 p p p A 26 AAGGGCGTAA CCGAAATCGG TTGAACCGAA ACCGGTTAGT ATAAAAGCAG ACATTTTATG 86 CACCAAAAGA GAACTGCAAT TTTCAGGACC CACAGGAGCG ACCCAGAAAG TTACCACAGT 146 TATGCACAGA GCTGCAAACA ACTATACATG ATATAATATT AGAATGTGTG TACTGCAAGC 206 AACAGTTACT GCGACGTGAG GTATATGACT TTGCTTTTCG GGATTTATGC ATAGTATATA B 206 AACAGTTACT GCGACGTGAG GTATATGACT TTGCTTTTCG GGATTTATGC ATAGTATATA 266 GAGATGGGAA TCCATATGCT GTATGTGATA AATGTTTAAA GTTTTATTCT AAAATTAGTG 326 AGTATAGACA TTATTGTTAT AGTTTGTATG GAACAACATT AGAACAGCAA TACAACAAAC 386 CGTTGTGTGA TTTGTTAATT AGGTGTATTA ACTGTCAAAA GCCCACTGTGT CCTGAAGAAC 446 AAGCAAAGAC ATCTGGACAA AAAGCAAAGA TTCCATAATA TAAGGGGTCG GTGGACCGGT 506 GATGTATGTC TTGTTGCAGA TCATCAAGAA CACGTAGAGA AACCCAGCTG TAATCATGCT 566 ATGGAGATAC ACCTACATTG CATGAATATA TGTTAGATTT GCAACCAGAG ACAACTGATC 566 ATGGAGATAC ACCTACATTG CATGAATATA TGTTAGATTT GCAACCAGAG ACAACTGATC 626 CTACTGTTAT GAGCAATTAA ATGACAGCTC AGAGGAGGAG GATGA 26 AAGGGCGTAA CCGAAATCGG TTGAACCGAA ACCGGTTAGT ATAAAAGCAG ACATTTTATG 86 CACCAAAAGA GAACTGCAAT TTTCAGGACC CACAGGAGCG ACCCAGAAAG TTACCACAGT 146 TATGCACAGA GCTGCAAACA ACTATACATG ATATAATATT AGAATGTGTG TACTGCAAGC 206 AACAGTTACT GCGACGTGAG GTATATGACT TTGCTTTTCG GGATTTATGC ATAGTATATA B 206 AACAGTTACT GCGACGTGAG GTATATGACT TTGCTTTTCG GGATTTATGC ATAGTATATA 266 GAGATGGGAA TCCATATGCT GTATGTGATA AATGTTTAAA GTTTTATTCT AAAATTAGTG 326 AGTATAGACA TTATTGTTAT AGTTTGTATG GAACAACATT AGAACAGCAA TACAACAAAC 386 CGTTGTGTGA TTTGTTAATT AGGTGTATTA ACTGTCAAAA GCCCACTGTGT CCTGAAGAAC 446 AAGCAAAGAC ATCTGGACAA AAAGCAAAGA TTCCATAATA TAAGGGGTCG GTGGACCGGT 506 GATGTATGTC TTGTTGCAGA TCATCAAGAA CACGTAGAGA AACCCAGCTG TAATCATGCT 566 ATGGAGATAC ACCTACATTG CATGAATATA TGTTAGATTT GCAACCAGAG ACAACTGATC 566 ATGGAGATAC ACCTACATTG CATGAATATA TGTTAGATTT GCAACCAGAG ACAACTGATC 626 CTACTGTTAT GAGCAATTAA ATGACAGCTC AGAGGAGGAG GATGA B Figure 3 Sequences of the E6-1 and E6-2 amplicons generated from pHV101. Discussion Curve 8, 50 ng of SiHa cell DNA with the pU1M/pU1R primer pair. Curve 9, 50 ng of SiHa cell DNA with the pU1M forward primer only. (B) Thermal fluorescence extinction profiles of nested qPCR products. Profiles of pHV101 and SiHa DNA mixtures with both primers (peaks at 85.5°C), mixtures without DNA or with normal human blood DNA with both primers (peaks at 78.5°C), and SiHa DNA with the pU1M primer only (nearly horizontal line close to the baseline). Figure 2 Thermal denaturation (fluorescence extinction) profiles of the E6-2 amplicons generated from pHV101 and SiHa DNA. RFU, relative fluorescence units. (A) Family of curves obtained with serially diluted pHV101 or 50 ng of SiHa DNA. Threshold (thick horizontal line): 50 RFU. Curve 1, 2.5 × 106 pHV101 copies. Curve 2, 2.5 × 105 pHV101 copies. Curve 3, 2.5 × 104 pHV101 copies. Curve 4, 2.5 × 103 pHV101 copies. Curve 5, 2.5 × 102pHV101 copies. Curve 6, Blank (without DNA). Curve 7, normal human blood DNA. Curve 8, 50 ng of SiHa cell DNA with the pU1M/pU1R primer pair. Curve 9, 50 ng of SiHa cell DNA with the pU1M forward primer only. (B) Thermal fluorescence extinction profiles of nested qPCR products. Profiles of pHV101 and SiHa DNA mixtures with both primers (peaks at 85.5°C), mixtures without DNA or with normal human blood DNA with both primers (peaks at 78.5°C), and SiHa DNA with the pU1M primer only (nearly horizontal line close to the baseline). PCR could quantify as little as 2,500 E6-HPV16 mole- cules, whereas nested qPCR was around 11,000 times more sensitive, a value close to that expected for 15 pre- amplification cycles which would increase the initial number of E6 template copies by a factor of 213 = 8,192. after cycle 27 in qPCR mixtures prepared with samples from preamplified mixtures containing normal human DNA or HPV16 DNA, but not from those containing SiHa DNA and the E6-2 forward primer only. E6-1 and E6-2 amplification was maximized in 50 μL conventional PCR mixtures containing 2.5 × 106 pHV101 copies under the following conditions: 3 mM MgCl2, 0.05 mM of each deoxynucleoside triphosphate (dNTP), 0.15 μM of each of the forward and reverse primers (Table 2), 30 U/ml Taq polymerase (Invitrogen), and annealing temperature of 57°C. The nested qPCR method may also be used in the future to quantify viral load as well as viral and cellular transcripts with high sensitivity and reliability. Conclusions Our method, based on conventional PCR preamplifica- tion for 15 cycles increased 10,750 times the sensitivity of nested qPCR for quantitation of the E6 oncogene, and confirmed that the SiHa cell line contains only one E6- HPV16 copy per cell. PCR products were analyzed by electrophoresis in sodium borate-1.5% agarose gels [12] for 2 h at 90 V, fol- lowed by ethidium bromide staining; they were visualized by ultraviolet light transillumination and their digital images recorded in a ChemiDoc EQ photodocumenter (BioRad, Hercules, CA) using the Quantity One (BioRad) software. Discussion (A) Diagram of the HPV16 long control region (LCR) and the E6 and E7 genes, whose sequences overlap partially. The E6-1 amplicon (645 bp) is generated first by direct PCR with the LCRS/E7AS primer pair and then the E6-2 (237 bp) amplicon is generated by nested PCR with the pU1M/pU2R primer pair. The scale in the lower part of the figure indicates the posi- tions of LCR, the E6 and E7 oncogenes and the E6-1 and E6-2 amplicon sequences on the HPV16 genome. (B) The E6-645 insert sequence corresponds to the cloned E6-1 amplicon, spans nucleotides 26-671 (645 bp) of the HPV16 genome and is flanked by the LCRS and E7AS primers (white letters on black background) at the 5' and 3' ends, respectively; it contains 38% GC and is 99% identical to the E6-HPV16 ORF [10]. The fragment corresponding to the E2-237 amplicon (in italics) spans nucleotides 419-656 (237 bp) of the HPV16 genome and is flanked by the pU1M and pU2R primers (white italics on black background), respectively; its sequence contains 41% GC and is identical to the corresponding ORF sequence of the E6-HPV16 onco- gene. The three gray italic letters on black background correspond to the overlapping bases of the pU2R and E7AS primers. Table 2: Oligonucleotide primers Pair Primer Sequence (5'T3') Amplicon 1 LCRS (Forward) AAGGGAGTAACCGAAAACGGT E6-1 (645 bp) E7AS (Reverse) TCATCCTCCTCCTCTGAG 2 pU1M (Forward) TGTCAAAAACCGTTGTGTCC E6-2 (237 bp) pU2R (Reverse) GAGCTGTCGCTTAATTGCTC Table 2: Oligonucleotide primers Page 6 of 7 Hernández-Arteaga and López-Revilla Infectious Agents and Cancer 2010, 5:9 http://www.infectagentscancer.com/content/5/1/9 Figure 4 Complete and minimum E6-1 preamplification mixtures used to perform E6-2 nested qPCR amplification. (A) Complete preampli- fication series. Successive stages: 1) preparation of E6-1 preamplification mixture containing; 2) "preamplification" by conventional PCR; 3) E6-2 ampli- fication in "nested" qPCR mixtures containing EvaGreen and 1/50 volume of the E6-1 preamplified mixture. Tubes 1, a, b, c and d: positive control preamplification mixtures with serial logarithmic dilutions of pHV101 in the range of 2.5 × 106-2.5 × 102 molecules per tube. Tube 2: Blank preamplifi- cation (without DNA). Tube 3: Problem preamplification (50 ng of SiHa DNA). Tubes 4 and 5: Negative preamplification controls (50 ng "carrier" normal human blood DNA). Asterisks indicate preamplified mixtures. (B) Minimum preamplification series. Figure 4 Complete and minimum E6-1 preamplification mixtures used to perform E6-2 nested qPCR amplification. (A) Complete preampli- fication series. Successive stages: 1) preparation of E6-1 preamplification mixture containing; 2) "preamplification" by conventional PCR; 3) E6-2 ampli- fication in "nested" qPCR mixtures containing EvaGreen and 1/50 volume of the E6-1 preamplified mixture. Tubes 1, a, b, c and d: positive control preamplification mixtures with serial logarithmic dilutions of pHV101 in the range of 2.5 × 106-2.5 × 102 molecules per tube. Tube 2: Blank preamplifi- cation (without DNA). Tube 3: Problem preamplification (50 ng of SiHa DNA). Tubes 4 and 5: Negative preamplification controls (50 ng "carrier" normal human blood DNA). Asterisks indicate preamplified mixtures. (B) Minimum preamplification series. Successive stages: 1) preparation of E6-1 pream- plification mixture including only the positive control ("calibration") with the highest pHV101 content; 2a) E6-1 "preamplification" by conventional PCR; 2b) serial logarithmic dilutions of the preamplified calibration mixture; 3) amplification of E6-2 in nested qPCR mixtures containing EvaGreen, the E6- 2 primers and 1/50 volume of E6-1 preamplified mixtures. Tube 1: Positive control amplification mixture with 2.5 × 106 pHV101 molecules. Tube 2: Blank preamplification mixture (without DNA). Tube 3: Problem preamplification mixture (50 ng of SiHa DNA). Tubes 4 and 5: Negative preamplifica- tion controls (50 ng "carrier" normal human blood DNA). Tubes a, b, c, and d: serial logarithmic dilutions from the preamplified positive control mixture used to prepare nested qPCR mixtures equivalent to those preamplified with 2.5 × 105-2.5 × 102 pHV101 molecules. Asterisks of numbered tubes in- dicate preamplified mixtures. conventional direct PCR and then the E6-2 (237 bp) amplicon through conventional nested PCR (Fig. 3) [10]. E6-1 and E6-2 amplification was maximized in 50 μL conventional PCR mixtures containing 2.5 × 106 pHV101 copies under the following conditions: 3 mM MgCl2, 0.05 mM of each deoxynucleoside triphosphate (dNTP), 0.15 μM of each of the forward and reverse primers (Table 2), 30 U/ml Taq polymerase (Invitrogen), and annealing temperature of 57°C. conventional direct PCR and then the E6-2 (237 bp) amplicon through conventional nested PCR (Fig. 3) [10]. the sensitivity and reliability of our nested qPCR method, since preamplified mixtures containing 50 ng of SiHa DNA generated Ct values corresponding to one copy of the E6-HPV16 oncogene per human diploid genome. Discussion Successive stages: 1) preparation of E6-1 pream- plification mixture including only the positive control ("calibration") with the highest pHV101 content; 2a) E6-1 "preamplification" by conventional PCR; 2b) serial logarithmic dilutions of the preamplified calibration mixture; 3) amplification of E6-2 in nested qPCR mixtures containing EvaGreen, the E6- 2 primers and 1/50 volume of E6-1 preamplified mixtures. Tube 1: Positive control amplification mixture with 2.5 × 106 pHV101 molecules. Tube 2: Blank preamplification mixture (without DNA). Tube 3: Problem preamplification mixture (50 ng of SiHa DNA). Tubes 4 and 5: Negative preamplifica- tion controls (50 ng "carrier" normal human blood DNA). Tubes a, b, c, and d: serial logarithmic dilutions from the preamplified positive control mixture used to prepare nested qPCR mixtures equivalent to those preamplified with 2.5 × 105-2.5 × 102 pHV101 molecules. Asterisks of numbered tubes in- dicate preamplified mixtures. Author Details División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, Camino de la Presa San José 2055, 78216 San Luis Potosí S.L.P., México Preamplification and calibration of nested qPCR E-645, the pHV101 insert sequence spanning the E6- HPV16 open reading frame (ORF), was used as the initial template to generate the E6-1 (645 bp) amplicon through Calculation of the number of E6-HPV16 copies is based on the size of pHV101 (3,645 bp) and the average molecu- lar weight of a deoxynucleotide pair (650 Da). We esti- Page 7 of 7 Hernández-Arteaga and López-Revilla Infectious Agents and Cancer 2010, 5:9 5:9 2. Bosch FX: Epidemiology of human papillomavirus infections: new options for cervical cancer prevention. Salud Publica Mex 2003, 45(Suppl 3):S326-339. mated that 1 ng of pHV101 contains 2.5 × 108 molecules with the formula of Staroscik [13]: number of copies = ((amount in ng) * 6.022 × 1023))/((length in bp) * 109 * 650). 3. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Munoz N: Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999, 189(1):12-19. The E6-1 positive control mixtures required to generate the family type curves contained the LCRS/E7AS primer pair, 50 ng of "carrier" normal human DNA and serial log- arithmic dilutions of pHV101; they were preamplified for 15 cycles (denaturation at 94°C for 15 sec, annealing at 57°C for 1 min and extension at 72°C for 1 min). Problem preamplification mixtures contained 50 ng of SiHa cell DNA. Negative controls were a blank (i.e., without DNA) and another one with 50 ng of carrier normal human blood DNA. 4. Gravitt PE, Burk RD, Lorincz A, Herrero R, Hildesheim A, Sherman ME, Bratti MC, Rodriguez AC, Helzlsouer KJ, Schiffman M: A comparison between real-time polymerase chain reaction and hybrid capture 2 for human papillomavirus DNA quantitation. Cancer Epidemiol Biomarkers Prev 2003, 12(6):477-484. 5. Sotlar K, Diemer D, Dethleffs A, Hack Y, Stubner A, Vollmer N, Menton S, Menton M, Dietz K, Wallwiener D, et al.: Detection and typing of human papillomavirus by e6 nested multiplex PCR. J Clin Microbiol 2004, 42(7):3176-3184. 6. Hubbard RA: Human papillomavirus testing methods. Arch Pathol Lab Med 2003, 127(8):940-945. 6. Hubbard RA: Human papillomavirus testing methods. Arch Pathol Lab Med 2003, 127(8):940-945. 7. Mayrand MH, Duarte-Franco E, Rodrigues I, Walter SD, Hanley J, Ferenczy A, Ratnam S, Coutlee F, Franco EL: Human papillomavirus DNA versus Papanicolaou screening tests for cervical cancer. N Engl J Med 2007, 357(16):1579-1588. 7. Authors' contributions RLR i d h d RLR conceived the study and obtained the funds to carry it out. SHA per- formed the bioinformatics analyses and the molecular studies. Both authors drafted the manuscript. Competing interests Th h d l h Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Acknowledgements This work was partially supported by the Fondo Mixto CONACYT-SLP (grant FMSLP-C01-2006-62319) and a scholarship to SHA (No. 176112) from the Con- sejo Nacional de Ciencia y Tecnología (Mexico). Preamplification and calibration of nested qPCR Proc Soc Exp Biol Med 1970, 135(2):543-545. 11. Friedl F, Kimura I, Osato T, Ito Y: Studies on a new human cell line (SiHa) derived from carcinoma of uterus. I. Its establishment and morphology. Proc Soc Exp Biol Med 1970, 135(2):543-545. 12. Brody JR, Kern SE: Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques 2004, 36(2):214-216. 13. Calculator for determining the number of copies of a template [http:// www.uri.edu/research/gsc/resources/cndna.html] doi: 10.1186/1750-9378-5-9 Cite this article as: Hernández-Arteaga and López-Revilla, Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR Infectious Agents and Cancer 2010, 5:9 Preamplification and calibration of nested qPCR Mayrand MH, Duarte-Franco E, Rodrigues I, Walter SD, Hanley J, Ferenczy A, Ratnam S, Coutlee F, Franco EL: Human papillomavirus DNA versus Papanicolaou screening tests for cervical cancer. N Engl J Med 2007, 357(16):1579-1588. Positive control preamplification mixtures were pre- pared either a) as five "complete" PCR mixtures contain- ing serial logarithmic dilutions of pHV101 to attain 2.5 × 106-2.5 × 102 pHV101 copies/mixture (Fig. 4A), or b) as a single "minimum" PCR mixture containing 2.5 × 106 pHV101 copies (Fig. 4B); 1/50 volumes from both types of mixtures were added to prepare nested qPCR amplifi- cation mixtures. 8. Noda T, Sasagawa T, Dong Y, Fuse H, Namiki M, Inoue M: Detection of human papillomavirus (HPV) DNA in archival specimens of benign prostatic hyperplasia and prostatic cancer using a highly sensitive nested PCR method. Urol Res 1998, 26(3):165-169. 8. Noda T, Sasagawa T, Dong Y, Fuse H, Namiki M, Inoue M: Detection of human papillomavirus (HPV) DNA in archival specimens of benign prostatic hyperplasia and prostatic cancer using a highly sensitive nested PCR method. Urol Res 1998, 26(3):165-169. 9. Lopez-Revilla R, Martinez-Contreras LA, Sanchez-Garza M: Prevalence of high-risk human papillomavirus types in Mexican women with cervical intraepithelial neoplasia and invasive carcinoma. Infect Agent Cancer 2008, 3:3. 9. Lopez-Revilla R, Martinez-Contreras LA, Sanchez-Garza M: Prevalence of high-risk human papillomavirus types in Mexican women with cervical intraepithelial neoplasia and invasive carcinoma. Infect Agent Cancer 2008, 3:3. qPCR mixtures optimized to amplify E6-2 with the pU1M/pU2R primer pair in the presence of EvaGreen (Biotium, Hayward, CA) were incubated with the same thermocycler program. To control nested amplification and to determine the number of E6 copies in problem samples, additional mixtures containing 1/50 volume of preamplified positive, negative and problem mixtures were prepared; in these experiments an additional nega- tive control mixture was used which contained preampli- fied SiHa DNA and the forward (pU1M) but not the reverse (pU1R) primer (Fig. 4). 10. Hernandez-Arteaga S, Lopez-Revilla R: Quantitation of human papillomavirus type 16 E6 oncogene sequences by real-time or quantitative PCR with EvaGreen. Anal Biochem 2008, 380(1):131-133. 10. Hernandez-Arteaga S, Lopez-Revilla R: Quantitation of human papillomavirus type 16 E6 oncogene sequences by real-time or quantitative PCR with EvaGreen. Anal Biochem 2008, 380(1):131-133. 11. Friedl F, Kimura I, Osato T, Ito Y: Studies on a new human cell line (SiHa) derived from carcinoma of uterus. I. Its establishment and morphology. References 1. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ: Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003, 348(6):518-527. 1. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ: Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003, 348(6):518-527.
https://openalex.org/W1973213730	https://europepmc.org/articles/pmc3509707?pdf=render	English	null	Shiga Toxins and the Pathophysiology of Hemolytic Uremic Syndrome in Humans and Animals	Toxins	2,012	cc-by	12,740	Toxins 2012, 4, 1261-1287; doi:10.3390/toxins4111261 Toxins 2012, 4, 1261-1287; doi:10.3390/toxins4111261 toxins ISSN 2072-6651 www.mdpi.com/journal/toxins OPEN ACCESS toxins ISSN 2072-6651 www.mdpi.com/journal/toxins OPEN ACCESS 1. Introduction It has been only thirty years since Shiga toxin-producing Escherichia coli was recognized as a human pathogen after the first outbreak investigated as hemorrhagic colitis in 47 patients in Oregon and Michigan [1]. This E. coli O157:H7 strain was then considered to be rare, but the 1993 multistate outbreak from undercooked hamburgers at a fast-food chain gained national attention for this newly emerging pathogen [2]. By 2002 there were 350 outbreaks (≥2 cases with same exposure source) reported to the CDC in 49 states, sickening 8598 people with a 17.4% hospitalization rate [3]. Contaminated ground beef and fresh produce account for about 75% of the infections, with the remainder being person-to-person, waterborne or animal contact sources and the vast majority (92%) occurring seasonally from May to November [3]. Enterohemorrhagic E. coli (EHEC) are not invasive, so bacteremia is rare, but they secrete ribosome inactivating Shiga-like toxins (Stx1 and Stx2 with variants) which are responsible for much of the organ damage [4,5], and Stx2 is more frequently associated with severe disease. Several recent reviews discuss the molecular basis for bacterial success and toxin biochemistry [6–8]. Bacterial colonization of the intestine with attaching and effacing lesions support type III secretion of toxins into the vasculature [6]. Micropinocytosis may contribute as toxin antigen is observed in patient gut epithelial cells in the absence of A/E lesions [9]. The very young and the elderly are most susceptible to complications and death from EHEC infection. This bacterial infection is the leading cause of acute kidney failure in otherwise healthy children in the US with an average infection rate of 1/100,000. Children less than five years of age are more likely to develop complications requiring hospitalization and kidney dialysis, and the elderly >60 years are more likely to die regardless of the clinical complications [10]. This emerging pathogen has become a global problem as well. The 1996 O157:H7 outbreak in thousands in Sakai City, Japan was purportedly from contaminated bean sprouts distributed in elementary school lunches [11]. The summer 2011 outbreak sourced to bean sprouts in Germany left over 4000 infected [12] and sent a clear warning through the EHEC research and clinical infectious disease communities that more virulent strains are emerging. This O104:H4 strain was a combination of an enteroaggregative E. Chad L. Mayer †, Caitlin S. Leibowitz †, Shinichiro Kurosawa and Deborah J. Stearns-Kurosawa * Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, MA 02118, USA; E-Mails: cmayer@bu.edu (C.L.M.); csleibow@bu.edu (C.S.L.); kurosawa@bu.edu (S.K.) † These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: dstearns@bu.edu; Tel.: +1-617-414-7092; Fax: +1-617-414-7073. Received: 17 September 2012; in revised form: 1 November 2012 / Accepted: 2 November 2012 / Published: 8 November 2012 Received: 17 September 2012; in revised form: 1 November 2012 / Accepted: 2 November 2012 / Published: 8 November 2012 Abstract: Food-borne diseases are estimated at 76 million illnesses and 5000 deaths every year in the United States with the greatest burden on young children, the elderly and immunocompromised populations. The impact of efficient food distribution systems and a truly global food supply ensures that outbreaks, previously sporadic and contained locally, are far more widespread and emerging pathogens have far more frequent infection opportunities. Enterohemorrhagic E. coli is an emerging food- and water-borne pathogen family whose Shiga-like toxins induce painful hemorrhagic colitis with potentially lethal complications of hemolytic uremic syndrome (HUS). The clinical manifestations of Shiga toxin-induced HUS overlap with other related syndromes yet molecular mechanisms differ considerably. As discussed herein, understanding these differences and the novel properties of the toxins is imperative for clinical management decisions, design of appropriate animal models, and choices of adjunctive therapeutics. The emergence of new strains with rapidly aggressive virulence makes clinical and research initiatives in this field a high public health priority. Keywords: Enterohemorrhagic E. coli; Shiga toxins; hemolytic uremic syndrome; animal models Toxins 2012, 4 Toxins 2012, 4 1262 Toxins 2012, 4 1263 Toxins 2012, 4 multi-state prospective study of 259 US children with HUS as a complication of EHEC O157:H7 infection demonstrated unequivocally that exposure to antibiotics of any class in the first week of illness was independently associated with development of HUS and tripled the risk [18]. For the O157:H7 strain, antibiotics increase toxin production due to location of stx genes within antibiotic-inducible resident lambdoid prophages [19]. The recent outbreak in Germany with the Stx2-producing O104:H4 strain was notable for its high HUS rate at 22%, occurring overwhelmingly in adults (88%) and most of these being young women [12,15,20]. In this outbreak, use of antibiotics at one hospital center was beneficial, significantly reducing intestinal bacterial colonization duration and improving clinical outcome [21]. Unlike the more common O157:H7 strain, antibiotics reduced Stx secretion from this strain in vitro [22]. Argentina has the dubious distinction of having the highest incidence of pediatric diarrhea-associated HUS (13.9/100,000 population) and ~20% of Argentina’s pediatric kidney transplants are the result of EHEC infections [23]. Relative to other infectious disease outbreaks, the death rate from EHEC infection is fairly low at around 3%–5%, but morbidity associated with kidney injury is significant. About 12% of patients with diarrhea-associated HUS progress to end stage renal failure within 4 years and about 25% have long term renal impairment [24,25], putting them at high risk for other clinical complications as eventually encounter pregnancy, high blood pressure, diabetes, cardiovascular disease and other complications of aging. Because of the importance of HUS complications, considerable clinical and basic research has focused on this aspect of EHEC infections to define the onset of HUS and to identify cellular dysfunctions that contribute to the pathology. The intent is to identify clinical patterns and molecular biomarkers that identify those at highest risk in order to prevent or mitigate HUS. Not all cases of HUS are due to EHEC infections, and the similarities and distinctions between EHEC-induced HUS and related syndromes of atypical HUS and thrombotic thrombocytopenic purpura can delay diagnosis and complicate clinical management of patients. Identifying effective non-antibiotic adjunctive therapeutics depends on understanding the molecular and pathophysiological mechanisms that contribute to organ injury, and in this respect, development of appropriate and representative animal models is critical. Both of these aspects are discussed below. 1. Introduction coli strain, normally characterized by intestinal mucosa adherence to cause self-limiting diarrhea [13], that picked up the stx2 gene, thereby acquiring high virulence and causing severe clinical consequences, particularly acute kidney injury from hemolytic uremic syndrome and severe neurological abnormalities [14,15]. Karmali et al. was the first to make the association between Stx, diarrheal E. coli infection and the idiopathic hemolytic uremic syndrome of infancy and childhood [16,17]. This critical link provided the first molecular explanation for the clinical observations, thereby opening new routes for differential diagnosis and pathogen-specific treatment modalities. Hemolytic uremic syndrome (HUS) is a serious clinical complication of EHEC infection and the severity of a public outbreak is often discussed in terms of the HUS rate. HUS is a clinical composite of thrombocytopenia, hemolytic anemia and thrombotic microangiopathy that contributes to acute kidney injury, often requiring dialysis and can progress to acute renal failure and death. Epidemiology studies have shown that HUS typically develops in about 5%–15% of patients, but this varies between bacterial strains and geographic location. Treatment is supportive, no toxin-specific therapies are available and antibiotics are usually contraindicated, depending on serotype. Recently, a large 2. Distinguishing the Thrombotic Microangiopathy of EHEC-Related HUS Hemolytic uremic syndrome (HUS) is a clinical syndrome comprising the triad of thrombocytopenia, thrombotic microangiopathy, and hemolytic anemia, and is frequently associated with acute kidney injury. Pathogenic challenge that results in endothelial injury or dysfunction shifts the hemostatic balance toward a pro-thrombotic environment, resulting in thrombotic microangiopathy with inappropriate deposition of clots in microvascular beds causing tissue ischemia. The kidney is a particular target during EHEC infection in part because the toxin receptors are expressed at higher density on glomerular endothelial cells [26] and toxin activities induce endothelial expression of adhesion molecules that support interactions with activated platelets and leukocytes [27] which contribute significantly to clot formation [28]. Development of a thrombotic microangiopathy can arise as a complication of bacterial infections like EHEC, from genetic mutations in complement regulatory pathways (atypical HUS, aHUS), from deficiencies of regulatory ADAMTS13 enzyme (thrombotic thrombocytopenic purpura, TTP) or even Toxins 2012, 4 Toxins 2012, 4 Toxins 2012, 4 1264 secondarily as a consequence of autoimmune diseases such as systemic lupus erythematosus. These pathways to HUS have different etiologies but frequently overlapping clinical presentation (Figure 1). Figure 1. Thrombotic Microangiopathy can result from different molecular pathways. Hemolytic uremic syndrome (HUS), atypical HUS (aHUS) and thrombotic thrombocytopenic purpura (TTP) have shared clinical manifestations, but differing molecular etiologies. EHEC-related HUS initiated by bacterial Stx injures endothelial cells by inducing endoplasmic reticulum (ER) stress responses and transcription events which include generation of inflammatory cytokines and chemokines. Endothelial injury and a pro-thrombotic environment in aHUS results from genetic mutations in complement pathway members and aberrant activation (complement factors H, IB, 3: CFH, CFI, CFB, C3). Coagulopathy during TTP results from inherited or immune-acquired deficiency in a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), needed to cleave vonWillebrand Factor (VWF) released from endothelial cells to prevent accumulation of prothrombotic ultra-large VWF (UL-VWF) oligomers. Thrombotic microangiopathy is a shared consequence and is related to, but distinct from, disseminated intravascular coagulation (DIC), a consumptive coagulopathy. The coagulopathy of HUS is distinguished from DIC by several parameters, including a normal or elevated fibrinogen level and normal or slightly elevated clotting times because coagulation factors are not consumed (Figure 2). Markers of fibrinolysis (fibrin degradation products, D-dimer) are only modestly increased in HUS relative to DIC, but measures of microangiopathic hemolytic anemia (MAHA) such as schistocytes are markedly increased. Additionally, the renal failure of DIC is attributable to acute tubular necrosis, whereas that associated with HUS is predominately a thrombotic microangiopathy of glomerular vasculature, with or without tubular necrosis. Thrombotic microangiopathy is a shared consequence and is related to, but distinct from, disseminated intravascular coagulation (DIC), a consumptive coagulopathy. The coagulopathy of HUS is distinguished from DIC by several parameters, including a normal or elevated fibrinogen level and normal or slightly elevated clotting times because coagulation factors are not consumed (Figure 2). Markers of fibrinolysis (fibrin degradation products, D-dimer) are only modestly increased in HUS relative to DIC, but measures of microangiopathic hemolytic anemia (MAHA) such as schistocytes are markedly increased. Additionally, the renal failure of DIC is attributable to acute tubular necrosis, whereas that associated with HUS is predominately a thrombotic microangiopathy of glomerular vasculature, with or without tubular necrosis. 1265 3. Hemolytic Uremic Syndrome (EHEC and Stx) Over 90% of HUS cases are associated with EHEC infection, also referred to as D + HUS, to indicate that there was diarrhea before the development of HUS. HUS from infection with other pathogens, usually Streptococcus pneumoniae (SP-HUS) which exposes the Thomsen-Freidenreich antigen with autoimmune consequences, results in longer hospitalization and long-term kidney injury [31,32]. A diarrheal prodrome can be observed in non-EHEC HUS cases, albeit rarely [33]. Patients usually develop a prodromal hemorrhagic colitis after EHEC contaminated food or water has been ingested [34], with HUS developing after approximately 7–10 days [35]. It is widely acknowledged that the Shiga-like toxins produced by the enterohemorrhagic E. coli are the main virulence factors driving the organ damage in patients and animal models. EHEC secrete Shiga-like toxin-1 (Stx1) and/or Shiga-like toxin 2 (Stx2), and there are multiple Stx2 variants [36]. The two major toxins are structurally similar with a shared AB5 domain structure, but only 56% amino acid homology. Stx are ribosome-inactivating toxins, similar to Shiga toxin from Shigella dysenteriae serotype-1 [37] and ricin from castor beans. The enzymatic A subunit and a cell binding B subunit that organizes into pentamers recognize a globotriaosylceramide (Gb3) membrane receptor on cells, particularly glomerular endothelial cells [38]. Internalized toxin-receptor complexes undergo retrograde transport to the endoplasmic reticulum via the Golgi apparatus where the A subunit N-glycosidase activity removes an adenine from 28S ribosomal RNA to inhibit protein synthesis [39]. A recent study shows Stx1 intracellular trafficking is mediated by Golgi protein GPP130 and disruption of this interaction leads to lysosomal degradation of Stx1 and protection of mice from lethal toxemia [40]. The toxins also induce inflammation in patients [41] and animal models [42], and endoplasmic reticulum stress-induced transcriptional events are stimulated in susceptible cells [43,44]. The Shiga toxins target Gb3-rich tubular epithelium, as well as the glomerular endothelial cells, with cellular apoptosis, necrosis and thrombotic microangiopathy contributing to the acute kidney failure seen in patients [45]. In patients, the renal pathology as a consequence of EHEC infection includes cortical necrosis, glomerular thromboses and congestion with widened subendothelial space, endothelial cell swelling, neutrophilia, and occasional mesangiolysis [46,47]. Toxins 2012, 4 Murine models of EHEC toxemia do not develop thrombocytopenia and, therefore, do not present with HUS. Recently these Stx models have included bacterial endotoxin (Gram negative lipopolysaccharide, LPS), tumor necrosis factor-α (TNFα), adenosine diphosphate (ADP) or similar along with the toxin to induce platelet loss in an effort to replicate full-spectrum HUS [29]. While thrombocytopenia is observed in the mice after LPS + Stx, it is a consequence of pro-inflammatory priming, resulting in endotoxin-induced DIC [30], not Stx-induced HUS, with different coagulation mechanisms. Figure 2. Thrombocytopenia is common to HUS and DIC, but these are clinically distinct entities. Platelet loss occurs with both HUS and DIC, but markers of coagulopathy differ and highlight mechanistic differences. DIC is a consequence of lipopolysaccharide (LPS) in experimental systems (e.g., murine models) or clinically as a complication of underlying diseases such as bacterial sepsis or malignancy. DIC is a consumptive coagulopathy characterized by decreasing fibrinogen and coagulation factors with marked up-regulation of fibrinolysis markers such as fibrin degradation products (FDP) and D-dimer. In contrast, HUS does not consume coagulation factors and fibrinogen levels are often increased in EHEC patients. Moderate levels of fibrinolysis are present, but markers of microangiopathic hemolytic anemia (MAHA), such as schistocytes, are markedly elevated. Toxins 2012, 4 Toxins 2012, 4 Toxins 2012, 4 1266 3. Hemolytic Uremic Syndrome (EHEC and Stx) In the baboon model of Stx challenge which presents with all manifestations of HUS [48], glomerular injury after Stx1 and Stx2 challenge featured prominent HUS-like capillary wall changes, including thickening of the glomerular basement membrane and double contouring with prominent thrombus formation, and severe endothelial injury with diffuse cell swelling and focal endothelial denudation (Figure 3). Because this is an animal model allowing comparisons between toxins, some differences emerged with glomerular endothelial injury predominating after Stx1, whereas mesangiolysis and eosinophilic infiltration accompanied the glomerular injury in the Stx2-challenged animals [49]. Endothelial activation by the Shiga-like toxins induces an inflammatory milieu that is a strong contributor to the thrombi formation in the microvasculature during development of HUS. Stx2 upregulates chemokines monocyte chemotactic protein-1 (MCP-1, CCL2) and IL-8 (CCL8) [27,42] and Stx1 increases expression of cellular adhesion molecules ICAM-1, VCAM-1, and E-selectin on endothelial cells [50]. In human monocytes, EHEC infection and Shiga toxins induces inflammatory cytokines and chemokines including IL-1β, IL-6, IL-8, and TNFα [51]. In the GI of patients, the colon is severely affected, showing edema and hemorrhage, consistent with the hemorrhagic colitis that is Toxins 2012, 4 Loss of thrombomodulin Toxins 2012, 4 1268 expression, or other members of this pathway, correlates with a pro-coagulant environment [61] and circulating soluble thrombomodulin levels are accepted measures of endothelial injury [62]. However, endothelial and leukocyte activation are probably only part of what induces the formation of microthrombi during D + HUS. Studies have also looked at whether platelet activation might contribute to microthrombi genesis. Plasma from patients with D + HUS has shown evidence of platelet activation and degranulation [63]. Stx1 can activate endothelial cells, especially ones that express more Gb3 receptor due to TNFα or LPS priming, to cause increased adhesion of platelets [64] and inhibition of platelet cluster degradation by the inhibition of ADAMTS13 [65]. Collectively, endothelial, platelet and neutrophil activation all contribute to the formation of microthrombi that then becomes a major driving force of D + HUS pathophysiology. Cytokines can disrupt the anti-thrombotic properties of the microvascular endothelial cells and increase cell-surface Gb3 receptor expression [53]. Obrig et al. hypothesized previously that the sensitivity of endothelial cells to Shiga toxins depended greatly on their source and levels of Gb3 expression, and found that while TNFα and lipopolysaccharide (LPS) could increase sensitivity of human umbilical vein endothelial cells to Shiga toxins, they had no effect on renal microvascular endothelial cell Gb3 expression or Stx sensitivity [26]. It has been shown that the numbers of polymorphonuclear leukocytes present at the beginning of D + HUS is predictive of outcome, but not in cases of atypical HUS [66], a related but distinct syndrome (see below). Stx induces an inflammatory and chemotactic environment, and recruited neutrophils may transport the toxins in the blood [67], which could explain how the toxin passes from the gut to the kidney without bacteremia or invasion by the EHEC. Whether this occurs in patients is debated [68] because Stx antigen is sometimes [67,69,70], but not always [71] detected on the surface of circulating neutrophils. Close proximity of neutrophils and their enzymes to renal endothelial cells can also alter the surface availability of some proteins, changing the functionality of the endothelium. It is well known that neutrophil elastase cleaves endothelial-expressed thrombomodulin [72], a necessary cofactor of the protein C anticoagulant pathway [59]. Toxins 2012, 4 1267 Toxins 2012, 4 Toxins 2012, 4 often seen preceding HUS [52]. Intestinal leukocytosis is more prominent with D + HUS compared to non-EHEC HUS [53], and with Stx in the intestinal epithelial cells [9], there is likely production of pro-inflammatory and chemotactic mediators (IL-1, IL-6, IL-8, TNFα) from epithelial cells and resident macrophages [54–56]. Together, this group of chemokines and adhesion molecules serves to recruit and activate neutrophils, contributing to increased tissue damage. Figure 3. Histopathology of renal injury in nonhuman primates after challenge with Stx1 or Stx2. Light microscopy evaluation of kidney sections from baboons challenged i.v. with different doses of Stx1 or Stx2 reveal dose-dependent kidney injury. (A–C) Comparison of Stx1 challenges reveals deteriorating glomerular structure, increasing tubular edema and increasing hemorrhage with increasing toxin dose. Clinically, the 10 ng/kg Stx1 dose induced mild and transient effects in the animals, with unremarkable pathology at the light microscopy level. In contrast, a 100 ng/kg Stx1 challenge was lethal within 3–4 days accompanied by fulminant HUS, acute renal failure and systemic inflammatory responses; (D) The 50ng/kg Stx2 dose is comparatively severe and 100% lethal by day 5 post-challenge, also with HUS and renal failure. Both toxins elicited leukocyte infiltration and interstitial hemorrhage. Hematoxylin and eosin staining of paraffin embedded tissue sections; 40× magnification. Normally endothelial cells present non-thrombogenic surfaces, and both Shiga toxins and inflammatory mediators can shift the endothelium to a pro-thrombotic state, contributing to thrombus formation [57]. For example, Stx2 causes about a 15% decrease in thrombomodulin surface antigen expression of human glomerular endothelial cells in vitro, which is further potentiated by TNFα [58], although whether this amount of antigen loss significantly impacted function was not determined. Thrombomodulin is an essential cofactor of the protein C anticoagulant pathway, an assembly of molecules on the endothelium that maintains blood fluidity [59,60]. Loss of thrombomodulin Normally endothelial cells present non-thrombogenic surfaces, and both Shiga toxins and inflammatory mediators can shift the endothelium to a pro-thrombotic state, contributing to thrombus formation [57]. For example, Stx2 causes about a 15% decrease in thrombomodulin surface antigen expression of human glomerular endothelial cells in vitro, which is further potentiated by TNFα [58], although whether this amount of antigen loss significantly impacted function was not determined. Thrombomodulin is an essential cofactor of the protein C anticoagulant pathway, an assembly of molecules on the endothelium that maintains blood fluidity [59,60]. Toxins 2012, 4 A potential role for the complement pathway during development of Stx-induced HUS has recently gained renewed interest. Complement is an ancient non-specific opsonic defense mechanism dating to pre-Cambrian ancestors [78], and is now a highly regulated mammalian innate immune strategy contributing to host defense or tissue injury depending on the context [79]. There is some evidence that complement is activated during EHEC infection. During acute phase EHEC-HUS, complement factor C3 or C9 is detected on platelet-leukocyte complexes from patients, and Stx can induce ex vivo release of complement-bearing microparticles from platelets or monocytes [80]. Using purified components, Stx2 was shown to activate complement via the alternative pathway and slow complement factor H (CFH) regulatory activity [81]. A recent study showed Stx-induced C3b interactions with P-selectin to promote a pro-thrombotic surface on dermal microvascular endothelial cells activated with human serum and/or blood under shear stress [82]. These observations, along with C3 deposition on glomeruli from mice challenged with Stx2 + LPS [82], suggest a role for complement in the pathophysiology of endothelial dysfunction during HUS. However, patient data has less apparent clarity. In a small patient study, increased levels of alternative pathway members Bb and SC5b-9 were observed early in D + HUS patients, but there was no correlation with clinical parameters or outcome [83]. There also was no comparison with non-EHEC renal patients so it is difficult to evaluate a specific relationship to Stx-HUS as opposed to a generalized host defense response. In addition, despite apparent success in three severely ill infants [84], treatment of 67 patients with Eculizumab (Soliris®, Alexion Pharmaceuticals), the anti-C5 inhibitor antibody treatment for atypical HUS [85], during the 2011 outbreak of Stx2-producing EHEC 0104:H4 in Germany was not effective and likely worsened patient outcome [21]. This was a severely ill cohort with other co-treatments so efficacy interpretation is debated [86,87], but this is likely the patient population that such a treatment would target. Given the fundamental contribution of the complement pathway to pathogenesis of many diseases, it could potentially contribute to disease severity, but whether it actually initiates HUS during this bacterial infection remains to be determined. Toxins 2012, 4 If this proteolysis is incomplete, and removes only the amino-terminal lectin domain of thrombomodulin, this may contribute to organ injury and thrombotic microangiopathy, as suggested in murine models of Stx2 challenge with endotoxin priming [73]. It is interesting to note that while this information helps to explain the forces driving disease in the kidney and other organs, they do not go as far in explaining the neurological signs that are often seen accompanying HUS. In the recent 2011 German outbreak of EHEC infection, the rate of HUS was unusually high and the neurologic complications were severe, from aphasia to epileptic seizures. Notably, there were no histologically observed brain microthrombi, vessel occlusions or evidence of ischemia [74]. How then did the patients develop neurological symptoms, and was Stx to blame? One study in rats has shown that Stx1 effect on astrocytes could indirectly affect the blood-brain barrier [75]. During the German outbreak, immunoadsorption of IgG helped attenuate the neurological complications of Stx2-producing E. coli O104:H4 [76]. They did not find evidence that the antibodies were directed against ADAMTS13, loss of which contributes to a related thrombotic microangiopathy (see below), and instead hypothesized that the antibodies were against Stx itself and were crosslinking receptors, causing the release of larger von Willebrand Factor multimers [77] to indirectly inhibit ADAMTS13 [65] and increasing risk of thrombus formation. Further investigation will show whether this interesting hypothesis is true. 1269 5. Thrombotic Thrombocytopenic Purpura A third syndrome closely related to aHUS is thrombotic thrombocytopenic purpura (TTP), a thrombotic microangiopathy defined by a “pentad” consisting of the triad of HUS with accompanying fever and neurological manifestations [99]. Many HUS patients develop neurological symptoms and TTP patients can have kidney failure, so in many cases the diagnosis is given as HUS/TTP. A major risk factor for the development of TTP is a deficiency in the plasma von Willebrand factor-cleaving protease ADAMTS13, due to either a genetic etiology or autoimmune antibodies against the protease [99]. The resultant high levels of ultra-large vWF multimers in the circulation lead to increased platelet-containing thrombi. Although HUS, aHUS, and TTP are closely related diseases and often difficult to distinguish in the clinic, they do have separate causes as well as distinct outcomes. Because aHUS and TTP are due to autoantibodies, deficiencies or mutations of plasma factors, plasma exchange can be used to treat both. In addition, for aHUS, kidney or combined kidney-liver transplants have also shown to be beneficial when supplemented with plasma exchanges [100]. Recent studies have shown that the distinctions between TTP and D + HUS with respect to clinical signs and pathophysiology may be less defined based on the report by Motto et al. that Stx can trigger TTP in ADAMTS13-deficient mice although absence of ADAMTX13 alone is insufficient [77]. In addition, Stx1 or Stx2 causes the release of large vWF multimers from human endothelial cells, while also impairing ADAMTS13 activity, and LPS was shown to have minimal contribution [65]. Huang et al. later showed that the Stx B subunit was sufficient to induce vWF secretion and thrombotic microangiopathy in ADAMTS13−/− mice back-bred for toxin susceptibility [77], something which had been shown for Shiga toxins earlier [101]. Toxins 2012, 4 Atypical HUS can be sporadic or familial, and is associated with dysregulation of complement typically characterized either by over-activation or compromised regulators of the alternative pathway of complement. Ordinarily, complement factor H (CFH), along with complement factor I (CFI) and membrane cofactor protein (MCP) accelerates the decay of C3 convertase, inhibiting complement activation. Thrombomodulin, an endothelial cell surface membrane protein usually associated with anti-coagulant function [62], aids this enzymatic complex in its activity. Mutations in thrombomodulin [93] as well as CFH [94], CFI [90], and MCP [95] are all implicated as risk factors for development of aHUS, along with mutations in complement factor B [96] and C3 [97]. In addition, autoantibodies against CFH have also been demonstrated [89]. While infection can be a possible trigger for the manifestation of aHUS, in general there is much less evidence of leukocytosis and inflammation in patients with aHUS than in those with D + HUS [53]. Yet, there may be overlap and at least one patient with EHEC-HUS has been shown to also have a mutation in MCP that would confer risk of developing aHUS [98], emphasizing how the lines between these syndromes can be blurred. 4. Atypical Hemolytic Uremic Syndrome (Non-EHEC) HUS not due to enteropathogenic bacterial infection is known as atypical HUS (aHUS) and there are many triggers besides infection, with the underlying cause being genetic deficiencies in regulators of the alternative pathway of complement [88]. In this case, the alternative pathway of complement is a fundamental initiator of disease. aHUS leads to recurring bouts of HUS, and can lead to stroke, heart complications and end-stage renal failure, although how quickly this progresses depends on the nature of mutation in the complement pathway members [89]. Histologically, aHUS is indistinguishable from D + HUS in the kidney. A majority (60%–70%) of patients with cofactor H (CFH) mutations progressed to end-stage renal failure within a year of aHUS symptoms [90]. Many children experience their first bout of aHUS before age 6 months, which can help lead a differential diagnosis towards aHUS instead of D + HUS [91]. Treatment with Eculizumab (Soliris®), a humanized monoclonal antibody against complement C5, has had success in aHUS patients because it blocks the rampant, dysregulated activation of C5 thereby preventing formation of inflammatory C5a and the cytotoxic terminal attack complex C5b-9 [85,92]. 1270 6. Animal Models Though the first recorded US outbreak of enterohemorrhagic Escherichia coli occurred in 1982, a complete picture of how EHEC elicits disease in humans remains to be elucidated. EHEC infections are typically unpredictable common source outbreaks, so there are no endemic patient populations for systemic study of disease pathogenesis. The development of animal models has thus been necessary in 1271 Toxins 2012, 4 Toxins 2012, 4 7. Gnotobiotic Piglet Models One of the earliest animal models of EHEC-mediated HUS was the gnotobiotic piglet. This animal had previously been used to study other human enteric infections [115]. Piglets were infected with ~1 × 1010 CFU E. coli O157:H7 twenty-four hours after delivery and were euthanized and necropsied on either day three or day five. Ulcers and mononuclear cell infiltration were observed in the cecums of the animals euthanized on day five. Only minor focal lesions associated with bacterial antigen were observed in the kidney, and no hematological symptoms of hemolytic uremic syndrome were described [116]. Gnotobiotic piglets were again used as an animal model for EHEC infection by Gunzer et al. in 2002, and in these experiments the course of disease resembled that of human EHEC infection in the GI tract as well as other organs. Renal histopathology changes included surface petechiae and diffuse glomerular endothelial swelling with mild to moderate tubular damage. Notably, renal thrombotic microangiopathy was observed in most animals. Limitations to the model can be found in the clinical chemistry and hematologic findings. Though individual animals had elevated serum creatinine levels, the averages were not statistically significant between the experimental groups and the control animals. Furthermore, thrombocytopenia was not observed, nor were schistocytes in peripheral blood smears [103]. Use of gnotobiotic piglets as a model for EHEC is thus useful in studies that focus on neurological or renal changes, but does not accurately mimic the full spectrum of HUS symptoms observed in humans. Toxins 2012, 4 Toxins 2012, 4 order to study toxin-specific mechanisms that govern the observed disease process in humans. Animals may receive purified toxin(s) or bacteria, but despite the numerous animal models tried, none of them completely replicate the EHEC infection and HUS observed in patients. However, they do fulfill an important role in that they allow for the investigation of one or more aspects of pathogenesis of EHEC disease and HUS [102]. A brief summary of animal model features and limitations are shown in Table 1 with representative references. Table 1. Animal models. Model Pre-treatment Animal Features Limitations Reference EHEC none Gnotobiotic piglet Focal renal lesions, renal thrombotic microangiopathy Normal serum creatinine; no thrombocytopenia [103] EHEC streptomycin CD-1 mice Bacterial colonization; loose stools, anorexia, lethargy No disease unless host- adapted strain used; no glomerular damage, coagulopathy or thrombocytopenia [104,105] EHEC streptomycin and mitomycin C ICR mice Weakness, weight loss, microhemorrhages in brain and spinal cord, high BUN Serum creatinine normal, unremarkable kidney pathology [106] EHEC None or TNFα Germ free IQI mice Anorexia, renal tubular necrosis, thrombocytopenia, leukocytosis No glomerular histopathology, inflammation only with TNFα pre-treatment [107] EHEC 24 hour fast C3H/HeJ mice Gastrointestinal, neurologic and systemic symptoms, renal inflammation and necrosis, fragmented red blood cells Kidney function and platelets not measure [108] EHEC none Newly weaned BALB/c mice Renal damage, high urea concentrations, colon pathology Thrombotic microangiopathy not evaluated [109] EHEC Protein calorie malnutrition C57Bl/6J mice Systemic and neurologic symptoms, increased BUN, mild renal tubular degeneration Normal serum creatinine, normal glomeruli, no significant platelet changes [110] EHEC Host-adapted bacteria Dutch Belted rabbits Diarrhea, lethargy, anorexia, dehydration glomerular thrombi and renal congestion No consistent thrombocytopenia [111] Stx1 none baboon Thrombocytopenia, schistocytosis and hemolytic anemia, renal failure, GI injury, lesions in CNS, systemic inflammation Non-bacterial [42,48] Stx2 none baboon Thrombocytopenia, leukocytosis, acute renal failure, schistocytosis, hemolytic uremia, glomerular thrombotic microangiopathy, systemic inflammation Non-bacterial [42,112] Stx2 none C57Bl/6J mice Increased plasma BUN and creatinine, hemolysis, neutrophilia Non-bacterial, no thrombocytopenia [113] Stx2+LPS none C57Bl/6J mice Neutrophilia, thrombocytopenia, hemolysis, increased BUN and creatinine, renal histopathology Non-bacterial, LPS effect depends on timing of administration, consumptive coagulopathy [29] EHEC culture supernatant none Sprague- Dawley rats Increased BUN and creatinine, thrombocytopenia, hemolytic anemia and leukocytosis, renal histologic changes, watery diarrhea Non-bacterial, crude bacterial supernatants rather than purified toxin(s). [114] Table 1. Animal models. Toxins 2012, 4 Toxins 2012, 4 1272 Toxins 2012, 4 It had been demonstrated previously that addition of mitomycin C to in vitro EHEC cultures resulted in induction of phage expression and increased toxin production [118,119]. Young male ICR mice were pre-treated with streptomycin and mitomycin, then infected with EHEC strain E32511/HSC (Stx2c variant) pre-selected to be resistant to both antibiotics. The animals developed weakness and weight loss, but not diarrhea [106]. Microhemorrhages were observed in the brain and spinal cord, as were slight degeneration of tubules in the kidney. Blood urea nitrogen was significantly higher in experimental mice than in controls, suggesting renal injury, but serum creatinine levels did not change and light microscopy kidney pathology examinations were unremarkable. Acute encephalopathy with loss of blood brain barrier function, brain endothelial cell edema, and demyelination were predominant histologic features. Toxin antigen was detected in affected neurons, but it was not clear whether the neurologic symptoms were from primary neuronal lesions or secondary to hypoxia from thrombotic microangiopathy. The rationale behind antibiotic pretreatment of mice can be extended to include germ-free mouse models of EHEC infection. Isogai et al. showed that infection with a high dose (2 × 109 CFU) of E. coli O157:H7 was lethal within 7 days of inoculation for germ-free IQI mice. The mice became anorexic and urine retention was associated with renal tubular necrosis [107]. Interestingly, thrombocytopenia and leukocytosis were observed in infected animals. In order to elucidate a role for pro-inflammatory TNFα in EHEC pathogenesis, the authors included a group of mice pretreated with the cytokine. The addition of TNFα nearly doubled the lethality from 60% to 100% with the addition of neurologic symptoms. Furthermore, cytokine-treated mice demonstrated histopathologic changes in the kidney including proliferation of glomerular mesangial cells and microthrombosis, and increased cytokine responses in serum, kidney and brains. Based on these experiments, the authors concluded that the toxins produced by EHEC and/or bacterial endotoxin, induce production of TNFα to have an additive effect EHEC pathogenesis [107]. Priming of cells with pro-inflammatory mediators, especially TNFα, is well known to enhance subsequent cellular responses to pathogenic challenge, and this occurs both in vitro and in vivo [120]. Similarly, TNFα induced by bacterial endotoxin enhances subsequent Stx cytotoxicity in rat astrocytes and releases chemokines that may attract PMNs to contribute to neurological injury and loss of endothelial barrier function [121,122]. 8. Murine EHEC Models Relative to humans, mice are fairly resistant to EHEC infection, so murine models include stressors intended to increase susceptibility to intestinal bacterial colonization and infection. These approaches include pre-treatment with antibiotics, germ-free environments, and protein calorie malnutrition diets [117]. Antibiotics added to the animals’ drinking water reduces or alters the normal intestinal facultative bacteria, increasing the likelihood of EHEC intestinal colonization by decreasing bacterial competition. An early study used streptomycin to study the colonization abilities of several E. coli isolates [104] and Wadolkowski et al. adopted streptomycin pre-treatment to develop a murine model of EHEC infection [105]. Streptomycin-treated male CD-1 mice were infected with either wild-type E. coli O157:H7, a mutant strain that did not contain the plasmid encoding fimbriae, or both. Animals demonstrated colonization of the bacteria in all three conditions, though disease symptoms were not observed. However, mice inoculated with the mutant strain that had been previously recovered from the feces of one of the co-infection animals demonstrated symptoms of infection including loose stools, anorexia and lethargy between days 4–10 post-inoculation, with lethality following within a few days. Colonization without infection was the outcome unless the bacteria strain was host-adapted. The colons of these mice did not show gross or histological lesions, indicating that the animals did not die of hemorrhagic colitis. Rather, death appeared to be due to acute necrosis of proximal convoluted tubules, a lesion which is characteristic of toxin exposure in mice. However, the mice did not show evidence of glomerular damage, thrombotic microangiopathy, or thrombocytopenia [105]. 1273 9. Rabbit EHEC Model Dutch Belted rabbits orally infected with 5–9 × 108 CFU of host-adapted EHEC O103 (Stx1+ Stx2-) or patient-derived O157:H7 (Stx1+ Stx2+) developed renal vascular lesions and glomerular lesions containing erythrocyte fragments [111]. These animals also demonstrated diarrhea, lethargy, anorexia, dehydration and weight loss with a low hematocrit observed in five of six infected animals. Similar to that seen in humans, glomerular, perivascular and tubular injury was observed in all animals after challenge with either strain, as was glomerular endothelial edema, mesangial deposits and fibrin thrombi occluding capillary lumens. Lack of changes in creatinine suggests renal injury rather than renal insufficiency, and although mild to moderate thrombocytopenia was observed in only 3 of 30 experimental animals, glomerular thrombi and renal congestion suggest the presence of coagulopathy if not a full thrombotic microangiopathy. Intestinal edema and vascular lesions with colonic inflammation with leukocyte infiltration was observed, as was surface-adherent E. coli in the cecal mucosa and crypt lumens. This animal model is a reasonable mimic of the human renal and intestinal lesions induced by EHEC and its toxins, although lack of consistent thrombocytopenia limits this model with respect to recapitulating human HUS. Toxins 2012, 4 Newly weaned BALB/c mice showed increased susceptibility to EHEC infection and Stx-mediated renal damage without pretreatment to alter commensal flora [109]. Between 22% and 27% of infected young animals developed high urea concentrations in the serum and all of these mice died. Mortality was associated with increased circulating neutrophils, followed by a significant decrease in all circulating leukocytes shortly before death. Colon pathology included leukocyte infiltration, and the kidney showed focal cortical necrosis with tubular epithelial swelling, although thrombotic microangiopathy was not evaluated. To initiate EHEC colonization and infection, murine models require much higher bacteria doses compared to humans (murine ~109 CFU versus 10–100 organisms for humans). The observation that a subset of children infected with EHEC had an unbalanced diet prior to infection inspired Kurioka et al. to hypothesize that protein calorie malnourished (PCM) mice would contract disease from lower doses of EHEC than conventional mice. Indeed, the minimal infectious dose of E. coli in the PCM mice was lower at 2 × 106 CFU. [110]. The infected PCM mice developed systemic and neurologic symptoms, no gastrointestinal symptoms were observed. BUN levels, but not creatinine, increased and acute kidney injury was minimal with mild renal tubular degeneration and normal glomeruli. Significant platelet changes were not observed [110]. The advantage of the PCM murine model is the lower inoculum dose with neurologic complications, but the animals do not appear to develop HUS. Toxins 2012, 4 Though pre-treatment with antibiotics or the use of germ-free animals increases the likelihood of gut EHEC colonization and infection, it does not accurately depict the circumstance when normal gut flora is present as is the case with patients. To address this, alternative models were developed. C3H/HeJ mice have a TLR4 mutation, rendering them moderately immunocompromised and thus more susceptible to infection. These mice were fasted for 24 h prior to infection, and challenged with 1 × 109 CFU/mL EHEC expressing Stx2 by oral gavage [108]. They developed gastrointestinal symptoms including loose, watery stools, neurological symptoms including ataxia and convulsions, and systemic symptoms including lethargy, anorexia and shivering with ~40% mortality. Histologic examination of colon tissue demonstrated inflammatory infiltrates and necrotic foci. In the kidneys, they observed proliferation of glomerular mesangial cells, diffuse interstitial inflammation and necrosis of tubular cells. In addition, the authors noted fragmented red blood cells in the sick mice. Blood samples were not analyzed for markers of kidney function or for platelet numbers, so a full description of HUS pathology was not available [108]. 1274 Toxins 2012, 4 injury to the GI tract. Lesions in the central nervous system were observed by electron microscopy. Stearns-Kurosawa et al. expanded the baboon model to include dose-response studies of both toxins for comparative purposes [42]. Stx2-induced renal injury and mortality was delayed 2–3 days compared to Stx1 challenge, and animals were ultimately more sensitive to Stx2, succumbing at lower doses (50 ng/kg for Stx2 versus 100 ng/kg Stx1). Both toxins elicited a systemic inflammatory response, but levels of cytokines and chemokines were elevated earlier and to higher levels after Stx1 challenge. Care was taken to ensure low endotoxin contamination of the toxins, and TNFα was not detectable in either plasma or urine from any of the animals. If pro-inflammatory priming is a necessary event for Stx toxicity, then it may be more complicated than production of TNFα. Unlike other animal models, the development of thrombocytopenia was consistently observed and was toxin dose-dependent. Loss of platelets was more gradual in Stx2-challenged animals as compared to Stx1, however anemia development was comparable and long-lasting [112]. Challenge with either toxin resulted in renal failure, increased plasma blood urea nitrogen (BUN) and creatinine, and deteriorating urinalysis profiles. The baboons could be rescued from lethal Stx2 challenge with a custom synthetic peptide designed to counter Stx2 activity within cells [112]. Renal function and urine output was preserved even when the peptide was administered 24 h after the otherwise lethal Stx2 challenge. Thus, both Stx1 and Stx2 are capable of eliciting HUS in baboons, but the timing and magnitude of the responses differ. 10. Nonhuman Primate Toxin Models of HUS Several animal models examine the effects of purified Shiga toxin, in the absence of bacteria, with variable success in mimicking human HUS symptoms. A descriptive study of a baboon model of Shiga toxin 1 responses observed renal failure with death in 57 h or less, and the animals presented, for the first time, with all the classic clinical symptoms of HUS [48]. Animals in the low-dose Shiga toxin 1 group (50–200 ng/kg i.v.; n = 8) demonstrated thrombocytopenia, schistocytosis and hemolytic anemia, renal injury with glomerular endothelial injury and an inflammatory profile, renal failure, and 1275 Toxins 2012, 4 Priming of the Stx system in this way with LPS, TNFα, ADP or similar potentiates toxin chemotactic and inflammatory responses in vitro and in vivo [29,117], but the relevance to disease pathogenesis in patients is not clear. In experimental settings, addition of LPS priming to Stx2 challenge in mice will induce thrombocytopenia, but this etiology is a consumptive disseminated intravascular coagulation [124,125] rather than HUS and they have different coagulation profiles. This differentiation was discussed by Karmali et al. decades ago [17]. Additionally, the notion that compromised intestinal barrier function allows bacterial translocation or LPS leakage from commensal or pathogenic bacteria is far from established. This theory has been discussed with variable levels of support since the 1940s with respect to bacterial sepsis mechanisms and is proven to be poorly related to the patient condition, particularly for endotoxemia [126]. Some patients with EHEC O157:H7 infection have antibodies (usually IgM or IgA) against the bacterial LPS serotype [127,128] but detection in healthy control sera as well as cross-reactivity with other LPS serotypes adds complexity [129], and there is no prospective evidence showing these antibodies are protective or prognostic. In non-EHEC bacterial sepsis, circulating E. coli LPS is detectable in patients, but targeted neutralization of Gram negative LPS has consistently failed to improve 28 day all-cause mortality [130,131]. The current thinking is that the primary contribution of the intestine during these infections comes from host cellular injury with resultant host-derived mediators to propagate local and systemic inflammation effects [132,133]. Rats were studied as a Shiga toxemia model by Zotta et al. in 2008 to evaluate their ability to mimic development of HUS. Adult male Sprague-Dawley rats were challenged intraperitoneally with increasing volumes of filtered EHEC culture supernatant containing Stx2, but low endotoxin, and disease progression was monitored for 48 h. At an estimated toxin dose of 20 μg Stx2/kg body weight, all animals developed increased BUN and creatinine, thrombocytopenia (decreased from 84 × 106/μL to 33 × 106/μL), hemolytic anemia and leukocytosis [114], clinical markers that correlate with disease progression in patients [2,18]. Histological observations included necrotic glomerular areas, tubular injury, and thrombotic microangiopathy. Watery diarrhea due to colonic mucosa damage was also observed, but hemorrhagic colitis was not a feature. This methodology provides a small animal model of HUS that effectively reproduces the pathology of human HUS. Toxins 2012, 4 However, this model has not been used frequently, likely because it used crude bacterial supernatants rather than purified toxin. Bacterial virulence factors other than Stx may contribute to disease pathophysiology [134] and the observation that crude bacterial supernatants, but not purified Stx, can induce HUS in rodents is consistent with this. These presumed non-Stx virulence factors bear further investigation. 11. Rodent Models of Stx Challenge Although the pathology of human HUS can be mimicked in nonhuman primates through the administration of Stx alone, a small animal model of HUS would provide many benefits and be accessible to more investigators. C67BL/6J mice, when given only multiple sub-lethal doses of Stx2 and no pre-treatments, develop some but not all aspects of human HUS [113]. Significantly increased plasma BUN and creatinine levels and proteinuria reflect glomerular renal injury. Hemolysis, neutrophilia and lymphocytopenia were observed. Histologically, the kidneys showed fibrin (ogen) deposition in the glomerular capillary loops and swollen subendothelial zones containing flocculent material [113]. Thrombocytopenia, however, was not observed and lack of this HUS symptom appears to be common to all Stx murine models unless co-treated with an accessory pro-inflammatory mediator. Modeling HUS using mice challenged with EHEC toxins has been described using co-administration of Gram negative endotoxin (a.k.a., lipopolysaccharide, LPS) and Stx2 [123], a model that has been used recently by multiple investigators. This approach was based on the hypothesis that LPS from the commensal flora of the gut could potentially contribute to the pathogenesis of HUS. However, LPS had either a synergistic or protective effect, depending on the timing of administration. Keepers et al. found that when C57BL/6 mice were given low sublethal doses of both LPS and Stx2 concurrently, they exhibited all signs of clinical HUS [29]. Neutrophilia, thrombocytopenia, red cell hemolysis and increases in serum creatinine and BUN were all observed. In the kidney, glomerular fibrin deposition, microthrombi formation and glomerular ultrastructure changes were demonstrated by histology and electron microscopy [29]. 1276 Toxins 2012, 4 Toxins 2012, 4 Toxins 2012, 4 report patient physiological responses to clinical intervention will ultimately prove to be of greatest value. report patient physiological responses to clinical intervention will ultimately prove to be of greatest value. Acknowledgements The authors gratefully acknowledge Joel Henderson, (Department of Pathology and Laboratory Medicine, Boston University School of Medicine) for his renal pathology expertise. Support was provided by NIH/NIAID U01AI075386 (S.K.), The Mallory Research Fund from the Department of Pathology and Laboratory Medicine at Boston University School of Medicine (C.L.) and NIH Cardiovascular Biology training grant T32 HL007969 (K. Ravid, PI) in support of C.M. Appendix Expanded post-hoc analysis data presented at the November 2012 meeting of the American Society of Nephrology by Dr. Rolf Stahl (University Hospital Hamburg-Eppendorf, Hamburg, Germany) provided very promising and strong evidence for efficacy of Eculizumab (anti-complement C5; Alexion) in their severely ill patient cohort during the recent EHEC O104:H4 outbreak. Data showing more rapid recovery of platelet counts with resolution of acute kidney injury and neurological symptoms compared with a clinically comparable cohort not treated with antibody was presented and is under peer review for publication. This gives renewed optimism that targeting complement pathways may be beneficial for patients with EHEC infection. Conflicts of Interest The authors declare no conflict of interest. 12. Conclusions In the pursuit of effective translational medicine for EHEC infection, animal models that accurately mimic the pathogenesis of EHEC-induced human HUS are crucial if we are to fully elucidate the mechanisms involved. A plethora of such models have been spurned by this need, however none encapsulate the pathogenesis of EHEC infection and Shiga toxins-induced human HUS in its entirety. Nonetheless, each model provides certain insights, and may ultimately help the scientific community progress in its understanding of this disease. From these models and patient studies, identification of biomarkers that discriminate related clinical syndromes, quantify the early onset or risk of HUS, and 1277 Toxins 2012, 4 Toxins 2012, 4 1278 6. Savage, P.J.; Campellone, K.G.; Leong, J.M. Interaction of enterohemorrhagic Escherichia coli (EHEC) with mammalian cells: Cell adhesion, type iii secretion, and actin pedestal formation. Curr. Protoc. Microbiol. 2007, doi:10.1002/9780471729259.mc05a01s05. 7. Farfan, M.J.; Torres, A.G. Molecular mechanisms that mediate colonization of Shiga toxin-producing Escherichia coli strains. Infect. Immun. 2012, 80, 903–913. 8. Melton-Celsa, A.; Mohawk, K.; Teel, L.; O’Brien, A. Pathogenesis of Shiga-toxin producing Escherichia coli. Curr. Top Microbiol. Immunol. 2012, 357, 67–103. 9. Malyukova, I.; Murray, K.F.; Zhu, C.; Boedeker, E.; Kane, A.; Patterson, K.; Peterson, J.R.; Donowitz, M.; Kovbasnjuk, O. Macropinocytosis in Shiga toxin 1 uptake by human intestinal epithelial cells and transcellular transcytosis. Am. J. Physiol. Gastrointest. Liver Physiol. 2009, 296, G78–G92. 10. Gould, L.H.; Demma, L.; Jones, T.F.; Hurd, S.; Vugia, D.J.; Smith, K.; Shiferaw, B.; Segler, S.; Palmer, A.; Zansky, S.; et al. Hemolytic uremic syndrome and death in persons with Escherichia coli O157:H7 infection, foodborne diseases active surveillance network sites, 2000–2006. Clin. Infect. Dis. 2009, 49, 1480–1485. 11. Fukushima, H.; Hashizume, T.; Morita, Y.; Tanaka, J.; Azuma, K.; Mizumoto, Y.; Kaneno, M.; Matsuura, M.; Konma, K.; Kitani, T. Clinical experiences in Sakai City hospital during the massive outbreak of enterohemorrhagic Escherichia coli O157 infections in Sakai City, 1996. Pediatr. Int. 1999, 41, 213–217. 12. Frank, C.; Faber, M.; Askar, M.; Bernard, H.; Fruth, A.; Gilsdorf, A.; Hohle, M.; Karch, H.; Krause, G.; Prager, R.; et al. Large and ongoing outbreak of haemolytic uraemic syndrome, Germany, May 2011. Euro. Surveill. 2011, 16, 1–3. 13. Pan, D.; Das, A.; Liu, D.; Veazey, R.S.; Pahar, B. Isolation and characterization of intestinal epithelial cells from normal and SIV-infected rhesus macaques. PLoS One 2012, 7, e30247. 14. Bielaszewska, M.; Mellmann, A.; Zhang, W.; Köck, R.; Fruth, A.; Bauwens, A.; Peters, G.; Karch, H. Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: A microbiological study. Lancet Infect. Dis. 2011, 11, 671–676. 15. Frank, C.; Werber, D.; Cramer, J.P.; Askar, M.; Faber, M.; an der Heiden, M.; Bernard, H.; Fruth, A.; Prager, R.; Spode, A.; et al. Epidemic profile of Shiga-toxin-producing Wscherichia coli O104:H4 outbreak in Germany—Preliminary report. N. Engl. J. Med. 2011, 365,1771–1780. 16. Karmali, M.A.; Petric, M.; Lim, C.; Fleming, P.C.; Steele, B.T. Escherichia coli cytotoxin, haemolytic-uraemic syndrome, and haemorrhagic colitis. Lancet 1983, 2, 1299–1300. 17. References 1. Riley, L.W.; Remis, R.S.; Helgerson, S.D.; McGee, H.B.; Wells, J.G.; Davis, B.R.; Hebert, R.J.; Olcott, E.S.; Johnson, L.M.; Hargrett, N.T.; et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 1983, 308, 681–685. 2. Bell, B.P.; Goldoft, M.; Griffin, P.M.; Davis, M.A.; Gordon, D.C.; Tarr, P.I.; Bartleson, C.A.; Lewis, J.H.; Barrett, T.J.; Wells, J.G.; et al. A multistate outbreak of Escherichia coli O157: H7-associated bloody diarrhea and hemolytic uremic syndrome from hamburgers. The Washington experience. J. Am. Med. Assoc. 1994, 272, 1349–1353. 3. Rangel, J.M.; Sparling, P.H.; Crowe, C.; Griffin, P.M.; Swerdlow, D.L. Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982–2002. Emerg. Infect. Dis. 2005, 11, 603–609. 3. Rangel, J.M.; Sparling, P.H.; Crowe, C.; Griffin, P.M.; Swerdlow, D.L. Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982–2002. Emerg. Infect. Dis. 2005, 11, 603–609. 4. Schmidt, H.; Geitz, C.; Tarr, P.I.; Frosch, M.; Karch, H. Non-O157:H7 pathogenic Shiga toxin-producing Escherichia coli: Phenotypic and genetic profiling of virulence traits and evidence for clonality. J. Infect. Dis. 1999, 179, 115–123. 5. Beutin, L.; Zimmermann, S.; Gleier, K. Human infections with Shiga toxin-producing Escherichia coli other than serogroup O157 in germany. Emerg. Infect. Dis. 1998, 4, 635–639. Toxins 2012, 4 Karmali, M.A.; Petric, M.; Lim, C.; Fleming, P.C.; Arbus, G.S.; Lior, H. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J. Infect. Dis. 1985, 151, 775–782. 18. Wong, C.S.; Mooney, J.C.; Brandt, J.R.; Staples, A.O.; Jelacic, S.; Boster, D.R.; Watkins, S.L.; Tarr, P.I. Risk factors for the hemolytic uremic syndrome in children infected with Escherichia coli O157:H7: A multivariable analysis. Clin. Infect. Dis. 2012, 55, 33–41. Toxins 2012, 4 1279 19. Kimmitt, P.T.; Harwood, C.R.; Barer, M.R. Toxin gene expression by Shiga toxin-producing Escherichia coli: The role of antibiotics and the bacterial SOS response. Emerg. Infect. Dis. 2000, 6, 458–465. 19. Kimmitt, P.T.; Harwood, C.R.; Barer, M.R. Toxin gene expression by Shiga toxin-producing Escherichia coli: The role of antibiotics and the bacterial SOS response. Emerg. Infect. Dis. 2000, 6, 458–465. 20. Buchholz, U.; Bernard, H.; Werber, D.; Bohmer, M.M.; Remschmidt, C.; Wilking, H.; Deleré, Y.; an der Heiden, M.; Adlhoch, C.; Dreesman, J.; et al. German outbreak of Escherichia coli O104:H4 associated with sprouts. N. Engl. J. Med. 2011, 365, 1763–1770. 21. Menne, J.; Nitschke, M.; Stingele, R.; Abu-Tair, M.; Beneke, J.; Bramstedt, J.; Bremer, J.P.; Brunkhorst, R.; Busch, V.; Dengler, R.; et al. Validation of treatment strategies for enterohaemorrhagic Escherichia coli O104:H4 induced haemolytic uraemic syndrome: Case-control study. Br. Med. J. 2012, 345, e4565. 22. Corogeanu, D.; Willmes, R.; Wolke, M.; Plum, G.; Utermohlen, O.; Kronke, M. Therapeutic concentrations of antibiotics inhibit Shiga toxin release from enterohemorrhagic E. coli O104:H4 from the 2011 German outbreak. BMC Microbiol. 2012, 12, 160. 23. Rivero, M.A.; Passucci, J.A.; Rodriguez, E.M.; Signorini, M.L.; Tarabla, H.D.; Parma, A.E. Factors associated with sporadic verotoxigenic Escherichia coli infection in children with diarrhea from the central eastern area of Argentina. Foodborne Pathog. Dis. 2011, 8, 901–906. 24. Garg, A.X.; Suri, R.S.; Barrowman, N.; Rehman, F.; Matsell, D.; Rosas-Arellano, M.P.; Salvadori, M.; Haynes, R.B.; Clark, W.F. Long-term renal prognosis of diarrhea-associated hemolytic uremic syndrome: A systematic review, meta-analysis, and meta-regression. JAMA 2003, 290, 1360–1370. 25. Oakes, R.S.; Kirkham, J.K.; Nelson, R.D.; Siegler, R.L. Duration of oliguria and anuria as predictors of chronic renal-related sequelae in post-diarrheal hemolytic uremic syndrome. Pediatr. Nephrol. 2008, 23, 1303–1308. 26. Obrig, T.G.; Louise, C.B.; Lingwood, C.A.; Boyd, B.; Barley-Maloney, L.; Daniel, T.O. Endothelial heterogeneity in Shiga toxin receptors and responses. J. Biol. Chem. 1993, 268, 15484–15488. 27. Toxins 2012, 4 Zoja, C.; Angioletti, S.; Donadelli, R.; Zanchi, C.; Tomasoni, S.; Binda, E.; Imberti, B.; te Loo, M.; Monnens, L.; Remuzzi, G.; et al. Shiga toxin-2 triggers endothelial leukocyte adhesion and transmigration via NF-κb dependent up-regulation of IL-8 and MCP-1. Kidney Int. 2002, 62, 846–856. 28. Reitsma, P.H.; Versteeg, H.H.; Middeldorp, S. Mechanistic view of risk factors for venous thromboembolism. Arterioscler. Thromb. Vasc. Biol. 2012, 32, 563–568. 29. Keepers, T.R.; Psotka, M.A.; Gross, L.K.; Obrig, T.G. A murine model of HUS: Shiga toxin with lipopolysaccharide mimics the renal damage and physiologic response of human disease. J. Am. Soc. Nephrol. 2006, 17, 3404–3414. 30. Camerer, E.; Cornelissen, I.; Kataoka, H.; Duong, D.N.; Zheng, Y.-W.; Coughlin, S.R. Roles of protease-activated receptors in a mouse model of endotoxemia. Blood 2006, 107, 3912–3921. 31. Constantinescu, A.R.; Bitzan, M.; Weiss, L.S.; Christen, E.; Kaplan, B.S.; Cnaan, A.; Trachtman, H. Non-enteropathic hemolytic uremic syndrome: Causes and short-term course. Am. J. Kidney Dis. 2004, 43, 976–982. Toxins 2012, 4 1280 32. Banerjee, R.; Hersh, A.L.; Newland, J.; Beekmann, S.E.; Polgreen, P.M.; Bender, J.; Shaw, J.; Copelovitch, L.; Kaplan, B.S.; Shah, S.S.; et al. Streptococcus pneumoniae-associated hemolytic uremic syndrome among children in North America. Pediatr. Infect. Dis. J. 2011, 30, 736–739. 33. Noris, M.; Remuzzi, G. Hemolytic uremic syndrome. J. Am. Soc. Nephrol. 2005, 1 .; Remuzzi, G. Hemolytic uremic syndrome. J. Am. Soc. Nephrol. 2005, 16, 1035–1050. 34. Obrig, T.G. Escherichia coli Shiga toxin mechanisms of action in renal disease. Toxins 2010, 2, 2769–2794. 35. Karch, H.; Tarr, P.I.; Bielaszewska, M. Enterohaemorrhagic Escherichia coli in human medicine. Int. J. Med. Microbiol. 2005, 295, 405–418. 36. Paton, J.C.; Paton, A.W. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 1998, 11, 450–479. 37. O’Brien, A.O.; Lively, T.A.; Chen, M.E.; Rothman, S.W.; Formal, S.B. Escherichia coli O157:H7 strains associated with haemorrhagic colitis in the United States produce a shigella dysenteriae 1 (Shiga) like cytotoxin. Lancet 1983, 1, 702. 38. Okuda, T.; Tokuda, N.; Numata, S.; Ito, M.; Ohta, M.; Kawamura, K.; Wiels, J.; Urano, T.; Tajima, O.; Furukawa, K.; et al. Targeted disruption of Gb3/CD77 synthase gene resulted in the complete deletion of globo-series glycosphingolipids and loss of sensitivity to verotoxins. J. Biol. Chem. 2006, 281, 10230–10235. 39. Saxena, S.; O’Brien, A.; Ackerman, E. Shiga toxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of 28S RNA when microinjected into Xenopus oocytes. J. Biol. Toxins 2012, 4 Toxins 2012, 4 47. Chaisri, U.; Nagata, M.; Kurazono, H.; Horie, H.; Tongtawe, P.; Hayashi, H.; Watanabe, T.; Tapchaisri, P.; Chongsa-nguan, M.; Chaicumpa, W. Localization of Shiga toxins of enterohaemorrhagic Escherichia coli in kidneys of paediatric and geriatric patients with fatal haemolytic uraemic syndrome. Microb. Pathog. 2001, 31, 59–67. 48. Taylor, F.B., Jr.; Tesh, V.L.; DeBault, L.; Li, A.; Chang, A.C.; Kosanke, S.D.; Pysher, T.J.; Siegler, R.L. Characterization of the baboon responses to Shiga-like toxin: Descriptive study of a new primate model of toxic responses to Stx-1. Am. J. Pathol. 1999, 154, 1285–1299. 49. Stearns-Kurosawa, D.J.; Oh, S.-Y.; Cherla, R.P.; Lee, M.-S.; Tesh, V.L.; Papin, J.; Henderson, J.; Kurosawa, S. Distinct renal pathology and a chemotactic phenotype after enterohemorrhagic E. coli Shiga toxins. 2012, submitted for publication. 50. Morigi, M.; Micheletti, G.; Figliuzzi, M.; Imberti, B.; Karmali, M.A.; Remuzzi, A.; Remuzzi, G.; Zoja, C. Verotoxin-1 promotes leukocyte adhesion to cultured endothelial cells under physiologic flow conditions. Blood 1995, 86, 4553–4558. 51. Van Setten, P.A.; Monnens, L.A.; Verstraten, R.G.; van den Heuvel, L.P.; van Hinsbergh, V.W. Effects of verocytotoxin-1 on nonadherent human monocytes: Binding characteristics, protein synthesis, and induction of cytokine release. Blood 1996, 88, 174–183. 52. Richardson, S.E.; Karmali, M.A.; Becker, L.E.; Smith, C.R. The histopathology of the hemolytic uremic syndrome associated with verocytotoxin-producing Escherichia coli infections. Hum. Pathol. 1988, 19, 1102–1108. 53. King, A.J. Acute inflammation in the pathogenesis of hemolytic-uremic syndrome. Kidney Int. 2002, 61, 1553–1564. 54. Thorpe, C.M.; Hurley, B.P.; Lincicome, L.L.; Jacewicz, M.S.; Keusch, G.T.; Acheson, D.W. Shiga toxins stimulate secretion of interleukin-8 from intestinal epithelial cells. Infect. Immun. 1999, 67, 5985–5993. 55. Tesh, V.L.; Ramegowda, B.; Samuel, J.E. Purified Shiga-like toxins induce expression of proinflammatory cytokines from murine peritoneal macrophages. Infect. Immun. 1994, 62, 5085–5094. 56. Thorpe, C.M.; Smith, W.E.; Hurley, B.P.; Acheson, D.W.K. Shiga toxins induce, superinduce, and stabilize a variety of C-X-C chemokine mRNAs in intestinal epithelial cells, resulting in increased chemokine expression. Infect. Immun. 2001, 69, 6140–6147. 57. Zoja, C.; Buelli, S.; Morigi, M. Shiga toxin-associated hemolytic uremic syndrome: Pathophysiology of endothelial dysfunction. Pediatr. Nephrol. 2010, 25, 2231–2240. 58. Fernandez, G.C.; te Loo, M.W.; van der Velden, T.J.; van der Heuvel, L.P.; Palermo, M.S.; Monnens, L.L. Decrease of thrombomodulin contributes to the procoagulant state of endothelium in hemolytic uremic syndrome. Pediatr. Nephrol. 2003, 18, 1066–1068. 59. Weiler, H. Regulation of inflammation by the protein c system. Crit. Care Med. 2010, 38, S18–S25. Toxins 2012, 4 Chem. 1989, 264, 596–601. 40. Mukhopadhyay, S.; Linstedt, A.D. Manganese blocks intracellular trafficking of Shiga toxin and protects against Shiga toxicosis. Science 2012, 335, 332–335. 41. Proulx, F.; Turgeon, J.P.; Litalien, C.; Mariscalco, M.M.; Robitaille, P.; Seidman, E. Inflammatory mediators in Escherichia coli O157:H7 hemorrhagic colitis and hemolytic-uremic syndrome. Pediatr. Infect. Dis. J. 1998, 17, 899–904. 42. Stearns-Kurosawa, D.J.; Collins, V.; Freeman, S.; Tesh, V.L.; Kurosawa, S. Distinct physiologic and inflammatory responses elicited in baboons after challenge with Shiga toxin type 1 or 2 from enterohemorrhagic Escherichia coli. Infect. Immun. 2010, 78, 2497–2504. 43. Smith, W.E.; Kane, A.V.; Campbell, S.T.; Acheson, D.W.K.; Cochran, B.H.; Thorpe, C.M. Shiga toxin 1 triggers a ribotoxic stress response leading to p38 and JNK activation and induction of apoptosis in intestinal epithelial cells. Infect. Immun. 2003, 71, 1497–1504. 44. Lee, S.-Y.; Lee, M.-S.; Cherla, R.P.; Tesh, V.L. Shiga toxin 1 induces apoptosis through the endoplasmic reticulum stress response in human monocytic cells. Cell. Microbiol. 2008, 10, 770–780. 45. Karpman, D.; Håkansson, A.; Perez, M.T.; Isaksson, C.; Carlemalm, E.; Caprioli, A.; Svanborg, C. Apoptosis of renal cortical cells in the hemolytic-uremic syndrome: In vivo and in vitro studies. Infect. Immun. 1998, 66, 636–644. 46. Inward, C.D.; Howie, A.J.; Fitzpatrick, M.M.; Rafaat, F.; Milford, D.V.; Taylor, C.M. Renal histopathology in fatal cases of diarrhoea-associated haemolytic uraemic syndrome. Pediatr. Nephrol 1997, 11, 556–559. 1281 Toxins 2012, 4 Toxins 2012, 4 1282 61. Lin, S.M.; Wang, Y.M.; Lin, H.C.; Lee, K.Y.; Huang, C.D.; Liu, C.Y.; Wang, C.H.; Kuo, H.P. Serum thrombomodulin level relates to the clinical course of disseminated intravascular coagulation, multiorgan dysfunction syndrome, and mortality in patients with sepsis. Crit. Care Med. 2008, 36, 683–689. 61. Lin, S.M.; Wang, Y.M.; Lin, H.C.; Lee, K.Y.; Huang, C.D.; Liu, C.Y.; Wang, C.H.; Kuo, H.P. Serum thrombomodulin level relates to the clinical course of disseminated intravascular coagulation, multiorgan dysfunction syndrome, and mortality in patients with sepsis. Crit. Care Med. 2008, 36, 683–689. 62. Kurosawa, S.; Stearns-Kurosawa, D.J.; Kinasewitz, G.T. Soluble thrombomodulin: A sign of bad times. Crit. Care Med. 2008, 36, 985–987. 63. Karpman, D.; Manea, M.; Vaziri-Sani, F.; Stahl, A.L.; Kristoffersson, A.C. Platelet activation in hemolytic uremic syndrome. Semin. Thromb. Hemost. 2006, 32, 128–145. 64. Morigi, M.; Galbusera, M.; Binda, E.; Imberti, B.; Gastoldi, S.; Remuzzi, A.; Zoja, C.; Remuzzi, G. Verotoxin-1-induced up-regulation of adhesive molecules renders microvascular endothelial cells thrombogenic at high shear stress. Blood 2001, 98, 1828–1835. 65. Nolasco, L.H.; Turner, N.A.; Bernardo, A.; Tao, Z.; Cleary, T.G.; Dong, J.; Moake, J.L. Hemolytic uremic syndrome-associated Shiga toxins promote endothelial-cell secretion and impair ADAMTS13 cleavage of unusually large von willebrand factor multimers. Blood 2005, 106, 4199–4209. 66. Walters, M.D.; Matthei, I.U.; Kay, R.; Dillon, M.J.; Barratt, T.M. The polymorphonuclear leucocyte count in childhood haemolytic uraemic syndrome. Pediatr. Nephrol. 1989, 3, 130–134. 67. Te Loo, D.M.; Monnens, L.A.; der Velden, T.J.; Vermeer, M.A.; Preyers, F.; Demacker, P.N.; van den Heuvel, L.P.; van Hinsbergh, V.W. Binding and transfer of verocytotoxin by polymorphonuclear leukocytes in hemolytic uremic syndrome. Blood 2000, 95, 3396–3402. 68. Brigotti, M. The interactions of human neutrophils with Shiga toxins and related plant toxins: Danger or safety? Toxins 2012, 4, 157–190. 69. Tazzari, P.L.; Ricci, F.; Carnicelli, D.; Caprioli, A.; Tozzi, A.E.; Rizzoni, G.; Conte, R.; Brigotti, M. Flow cytometry detection of Shiga toxins in the blood from children with hemolytic uremic syndrome. Cytometry B Clin. Cytom. 2004, 61, 40–44. 70. Brigotti, M.; Tazzari, P.L.; Ravanelli, E.; Carnicelli, D.; Rocchi, L.; Arfilli, V.; Scavia, G.; Minelli, F.; Ricci, F.; Pagliaro, P.; et al. Clinical relevance of Shiga toxin concentrations in the blood of patients with hemolytic uremic syndrome. Pediatr. Infect. Dis. J. 2011, 30, 486–490. 71. Geelen, J.M.; van der Velden, T.J.A.M.; te Loo, D.M.W.M.; Boerman, O.C.; van den Heuvel, L.P.W.J.; Monnens, L.A.H. Toxins 2012, 4 60. Esmon, C.T. The interactions between inflammation and coagulation. Br. J. Haematol. 2005, 131, 417–430. Toxins 2012, 4 Toxins 2012, 4 75. Landoni, V.I.; Schierloh, P.; de Campos Nebel, M.; Fernandez, G.C.; Calatayud, C.; Lapponi, M.J.; Isturiz, M.A. Shiga toxin 1 induces on lipopolysaccharide-treated astrocytes the release of tumor necrosis factor-alpha that alter brain-like endothelium integrity. PLoS Pathog. 2012, 8, e1002632. 75. Landoni, V.I.; Schierloh, P.; de Campos Nebel, M.; Fernandez, G.C.; Calatayud, C.; Lapponi, M.J.; Isturiz, M.A. Shiga toxin 1 induces on lipopolysaccharide-treated astrocytes the release of tumor necrosis factor-alpha that alter brain-like endothelium integrity. PLoS Pathog. 2012, 8, e1002632. 76. Greinacher, A.; Friesecke, S.; Abel, P.; Dressel, A.; Stracke, S.; Fiene, M.; Ernst, F.; Selleng, K.; Weissenborn, K.; Schmidt, B.M.; et al. Treatment of severe neurological deficits with igg depletion through immunoadsorption in patients with Escherichia coli O104:H4-associated haemolytic uraemic syndrome: A prospective trial. Lancet 2011, 378, 1166–1173. 77. Huang, J.; Motto, D.G.; Bundle, D.R.; Sadler, J.E. Shiga toxin B subunits induce vWF secretion by human endothelial cells and thrombotic microangiopathy in ADAMTS13-deficient mice. Blood 2010, 116, 3653–3659. 78. Zhu, Y.; Thangamani, S.; Ho, B.; Ding, J.L. The ancient origin of the complement system. EMBO J. 2005, 24, 382–394. Sarma, J.V.; Ward, P.A. The complement system. Cell Tissue Res. 2011, 343, 227–235. 80. Stahl, A.L.; Sartz, L.; Karpman, D. Complement activation on platelet-leukocyte complexes and microparticles in enterohemorrhagic Escherichia coli-induced hemolytic uremic syndrome. Blood 2011, 117, 5503–5513. 81. Orth, D.; Khan, A.B.; Naim, A.; Grif, K.; Brockmeyer, J.; Karch, H.; Joannidis, M.; Clark, S.J.; Day, A.J.; Fidanzi, S.; et al. Shiga toxin activates complement and binds factor H: Evidence for an active role of complement in hemolytic uremic syndrome. J. Immunol. 2009, 182, 6394–6400. 82. Morigi, M.; Galbusera, M.; Gastoldi, S.; Locatelli, M.; Buelli, S.; Pezzotta, A.; Pagani, C.; Noris, M.; Gobbi, M.; Stravalaci, M.; et al. Alternative pathway activation of complement by Shiga toxin promotes exuberant C3a formation that triggers microvascular thrombosis. J. Immunol. 2011, 187, 172–180. 83. Thurman, J.M.; Marians, R.; Emlen, W.; Wood, S.; Smith, C.; Akana, H.; Holers, V.M.; Lesser, M.; Kline, M.; Hoffman, C.; et al. Alternative pathway of complement in children with diarrhea-associated hemolytic uremic syndrome. Clin. J. Am. Soc. Nephrol. 2009, 4, 1920–1924. 84. Lapeyraque, A.L.; Malina, M.; Fremeaux-Bacchi, V.; Boppel, T.; Kirschfink, M.; Oualha, M.; Proulx, F.; Clermont, M.J.; Le Deist, F.; Niaudet, P.; et al. Eculizumab in severe Shiga-toxin-associated HUS. N. Engl. J. Med. 2011, 364, 2561–2563. 85. Toxins 2012, 4 Lack of specific binding of Shiga-like toxin (verocytotoxin) and non-specific interaction of Shiga-like toxin 2 antibody with human polymorphonuclear leucocytes. Nephrol. Dial. Transplant. 2007, 22, 749–755. 72. Abe, H.; Okajima, K.; Okabe, H.; Takatsuki, K.; Binder, B.R. Granulocyte proteases and hydrogen peroxide synergistically inactivate thrombomodulin of endothelial cells in vitro. J. Lab. Clin. Med. 1994, 123, 874–881. 73. Zoja, C.; Locatelli, M.; Pagani, C.; Corna, D.; Zanchi, C.; Isermann, B.; Remuzzi, G.; Conway, E.M.; Noris, M. Lack of the lectin-like domain of thrombomodulin worsens Shiga toxin-associated hemolytic uremic syndrome in mice. J. Immunol. 2012. 74. Magnus, T.; Rother, J.; Simova, O.; Meier-Cillien, M.; Repenthin, J.; Moller, F.; Gbadamosi, J.; Panzer, U.; Wengenroth, M.; Hagel, C.; et al. The neurological syndrome in adults during the 2011 northern German E. coli serotype O104:H4 outbreak. Brain 2012, 135, 1850–1859. 1283 Toxins 2012, 4 Nurnberger, J.; Philipp, T.; Witzke, O.; Opazo Saez, A.; Vester, U.; Baba, H.A.; Kribben, A.; Zimmerhackl, L.B.; Janecke, A.R.; Nagel, M.; et al. Eculizumab for atypical hemolytic-uremic syndrome. N. Engl. J. Med. 2009, 360, 542–544. 86. Artunc, F. Treating Shiga toxin induced haemolytic uraemic syndrome. Br. J. Haematol. 2012, 345, e4598. 87. Ruggenenti, P.; Remuzzi, G. Thrombotic microangiopathy: E. coli O104:H4 German outbreak: A missed opportunity. Nat. Rev. Nephrol. 2012, 8, 558–560. 88. Noris, M.; Caprioli, J.; Bresin, E.; Mossali, C.; Pianetti, G.; Gamba, S.; Daina, E.; Fenili, C.; Castelletti, F.; Sorosina, A.; et al. Relative role of genetic complement abnormalities in sporadic and familial aHUS and their impact on clinical phenotype. Clin. J. Am. Soc. Nephrol. 2010, 5, 1844–1859. Toxins 2012, 4 1284 89. Kavanagh, D.; Goodship, T. Genetics and complement in atypical HUS. Pediatr. Nephrol. 2010, 25, 2431–2442. 90. Caprioli, J.; Noris, M.; Brioschi, S.; Pianetti, G.; Castelletti, F.; Bettinaglio, P.; Mele, C.; Bresin, E.; Cassis, L.; Gamba, S.; et al. Genetics of HUS: The impact of MCP, CFH, and IF mutations on clinical presentation, response to treatment, and outcome. Blood 2006, 108, 1267–1279. 91. Loirat, C.; Fremeaux-Bacchi, V. Atypical hemolytic uremic syndrome. Orphanet J. Rare Dis. 2011, 6, 60. 92. Gruppo, R.A.; Rother, R.P. Eculizumab for congenital atypical hemolytic-uremic syndrome. N. Engl. J. Med. 2009, 360, 544–546. 93. Delvaeye, M.; Noris, M.; de Vriese, A.; Esmon, C.T.; Esmon, N.L.; Ferrell, G.; Del-Favero, J.; Plaisance, S.; Claes, B.; Lambrechts, D.; et al. Thrombomodulin mutations in atypical hemolytic-uremic syndrome. N. Engl. J. Med. 2009, 361, 345–357. 94. Warwicker, P.; Goodship, T.H.; Donne, R.L.; Pirson, Y.; Nicholls, A.; Ward, R.M.; Turnpenny, P.; Goodship, J.A. Genetic studies into inherited and sporadic hemolytic uremic syndrome. Kidney Int. 1998, 53, 836–844. 95. Richards, A.; Kathryn Liszewski, M.; Kavanagh, D.; Fang, C.J.; Moulton, E.; Fremeaux-Bacchi, V.; Remuzzi, G.; Noris, M.; Goodship, T.H.; Atkinson, J.P. Implications of the initial mutations in membrane cofactor protein (MCP; CD46) leading to atypical hemolytic uremic syndrome. Mol. Immunol. 2007, 44, 111–122. 96. Goicoechea de Jorge, E.; Harris, C.L.; Esparza-Gordillo, J.; Carreras, L.; Arranz, E.A.; Garrido, C.A.; Lopez-Trascasa, M.; Sanchez-Corral, P.; Morgan, B.P.; Rodriguez de Cordoba, S. Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome. Proc. Natl. Acad. Sci. USA 2007, 104, 240–245. 97. 102. Melton-Celsa, A.R.; O’Brien, A.D. Animal models for STEC-mediated disease. Methods Mol. Med. 2003, 73, 291–305. Toxins 2012, 4 Fremeaux-Bacchi, V.; Miller, E.C.; Liszewski, M.K.; Strain, L.; Blouin, J.; Brown, A.L.; Moghal, N.; Kaplan, B.S.; Weiss, R.A.; Lhotta, K.; et al. Mutations in complement C3 predispose to development of atypical hemolytic uremic syndrome. Blood 2008, 112, 4948–4952. 98. Fang, C.J.; Fremeaux-Bacchi, V.; Liszewski, M.K.; Pianetti, G.; Noris, M.; Goodship, T.H.; Atkinson, J.P. Membrane cofactor protein mutations in atypical hemolytic uremic syndrome (ahus), fatal Stx-HUS, C3 glomerulonephritis, and the HELLP syndrome. Blood 2008, 111, 624–632. 99. Tsai, H.M. Pathophysiology of thrombotic thrombocytopenic purpura. Int. J. Hematol. 2010, 91, 1–19. 100. Saland, J.M.; Ruggenenti, P.; Remuzzi, G. Liver-kidney transplantation to cure atypical hemolytic uremic syndrome. J. Am. Soc. Nephrol. 2009, 20, 940–949. 101. Motto, D.G.; Chauhan, A.K.; Zhu, G.; Homeister, J.; Lamb, C.B.; Desch, K.C.; Zhang, W.; Tsai, H.M.; Wagner, D.D.; Ginsburg, D. Shiga toxin triggers thrombotic thrombocytopenic purpura in genetically susceptible ADAMTS13-deficient mice. J. Clin. Invest. 2005, 115, 2752–2761. 102. Melton-Celsa, A.R.; O’Brien, A.D. Animal models for STEC-mediated disease. Methods Mol. Med. 2003, 73, 291–305. 1285 Toxins 2012, 4 103. Gunzer, F.; Hennig-Pauka, I.; Waldmann, K.-H.; Sandhoff, R.; Grone, H.-J.; Kreipe, H.-H.; Matussek, A.; Mengel, M. Gnotobiotic piglets develop thrombotic microangiopathy after oral infection with enterohemorrhagic Escherichia coli. Am. J. Clin. Pathol. 2002, 118, 364–375. 103. Gunzer, F.; Hennig-Pauka, I.; Waldmann, K.-H.; Sandhoff, R.; Grone, H.-J.; Kreipe, H.-H.; Matussek, A.; Mengel, M. Gnotobiotic piglets develop thrombotic microangiopathy after oral infection with enterohemorrhagic Escherichia coli. Am. J. Clin. Pathol. 2002, 118, 364–375. 104. Myhal, M.L.; Laux, D.C.; Cohen, P.S. Relative colonizing abilities of human fecal and K 12 strains of Escherichia coli in the large intestines of streptomycin-treated mice. Eur. J. Clin. Microbiol. 1982, 1, 186–192. 105. Wadolkowski, E.A.; Burris, J.A.; O’Brien, A.D. Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7. Infect. Immun. 1990, 58, 2438–2445. 106. Fujii, J.; Kita, T.; Yoshida, S.; Takeda, T.; Kobayashi, H.; Tanaka, N.; Ohsato, K.; Mizuguchi, Y. Direct evidence of neuron impairment by oral infection with verotoxin-producing Escherichia coli O157:H- in mitomycin-treated mice. Infect. Immun. 1994, 62, 3447–3453. 107. Isogai, E.; Isogai, H.; Kimura, K.; Hayashi, S.; Kubota, T.; Fujii, N.; Takeshi, K. Role of tumor necrosis factor alpha in gnotobiotic mice infected with an Escherichia coli O157:H7 strain. Infect. Immun. 1998, 66, 197–202. 108. Karpman, D.; Connell, H.; Svensson, M.; Scheutz, F.; Alm, P.; Svanborg, C. Toxins 2012, 4 1286 Toxins 2012, 4 117. Mohawk, K.L.; O’Brien, A.D. Mouse models of Escherichia coli O157:H7 infection and shiga toxin injection. J. Biomed. Biotechnol. 2011, 2011, 258185. 118. Al-Jumaili, I.; Burke, D.A.; Scotland, S.M.; al-Mardini, H.; Record, C.O. A method of enhancing verocytotoxin production by Escherichia coli. FEMS Microbiol. Lett. 1992, 72, 121–125. 119. MacLeod, D.L.; Gyles, C.L. Effects of culture conditions on yield of Shiga-like toxin-II from Escherichia coli. Can. J. Microbiol. 1989, 35, 623–629. 120. Lomas-Neira, J.; Perl, M.; Venet, F.; Chung, C.S.; Ayala, A. The role and source of tumor necrosis factor-alpha in hemorrhage-induced priming for septic lung injury. Shock 2012, 37, 611–620. 121. Landoni, V.I.; de Campos-Nebel, M.; Schierloh, P.; Calatayud, C.; Fernandez, G.C.; Ramos, M.V.; Rearte, B.; Palermo, M.S.; Isturiz, M.A. Shiga toxin 1-induced inflammatory response in lipopolysaccharide-sensitized astrocytes is mediated by endogenous tumor necrosis factor alpha. Infect. Immun. 2010, 78, 1193–1201. 122. Landoni, V.I.; Schierloh, P.; de Campos Nebel, M.; Fernández, G.C.; Calatayud, C.; Lapponi, M.J.; Isturiz, M.A. Shiga toxin 1 induces on lipopolysaccharide-treated astrocytes the release of tumor necrosis factor-alpha that alter brain-like endothelium integrity. PLoS Pathog. 2012, 8, e1002632. 123. Barrett, T.J.; Potter, M.E.; Wachsmuth, I.K. Bacterial endotoxin both enhances and inhibits the toxicity of Shiga-like toxin II in rabbits and mice. Infect. Immun. 1989, 57, 3434–3437. 124. Taylor, F.B.; Toh, C.H.; Hoots, W.K.; Wada, H.; Levi, M. Towards definition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation. On behalf of the scientific subcommittee on disseminated intravascular coagulation (DIC) of the International Society on Thrombosis and Haemostasis (ISTH). Thromb. Haemost. 2001, 86, 1327–1330. 125. Stearns-Kurosawa, D.J.; Osuchowski, M.F.; Valentine, C.; Kurosawa, S.; Remick, D.G. The pathogenesis of sepsis. Annu. Rev. Pathol. 2011, 6, 19–48. 126. Remick, D.G.; Ward, P.A. Evaluation of endotoxin models for the study of sepsis. Shock 2005, 24, 7–11. 127. Chart, H.; Smith, H.R.; Scotland, S.M.; Rowe, B.; Milford, D.V.; Taylor, C.M. Serological identification of Escherichia coli O157:H7 infection in haemolytic uraemic syndrome. Lancet 1991, 337, 138–140. 128. Bitzan, M.; Moebius, E.; Ludwig, K.; Muller-Wiefel, D.E.; Heesemann, J.; Karch, H. High incidence of serum antibodies to Escherichia coli O157 lipopolysaccharide in children with hemolytic-uremic syndrome. J. Pediatr. 1991, 119, 380–385. 129. Chart, H.; Jenkins, C. The serodiagnosis of infections caused by verocytotoxin-producing Escherichia coli. J. Appl. Microbiol. 1999, 86, 731–740. 130. Toxins 2012, 4 The role of lipopolysaccharide and Shiga-like toxin in a mouse model of Escherichia coli O157:H7 infection. J. Infect. Dis. 1997, 175, 611–620. 109. Brando, R.J.; Miliwebsky, E.; Bentancor, L.; Deza, N.; Baschkier, A.; Ramos, M.V.; Fernandez, G.C.; Meiss, R.; Rivas, M.; Palermo, M.S. Renal damage and death in weaned mice after oral infection with Shiga toxin 2-producing Escherichia coli strains. Clin. Exp. Immunol. 2008, 153, 297–306. 110. Kurioka, T.; Yunou, Y.; Kita, E. Enhancement of susceptibility to Shiga toxin-producing Escherichia coli O157:H7 by protein calorie malnutrition in mice. Infect. Immun. 1998, 66, 1726–1734. 111. Garcia, A.; Bosques, C.J.; Wishnok, J.S.; Feng, Y.; Karalius, B.J.; Butterton, J.R.; Schauer, D.B.; Rogers, A.B.; Fox, J.G. Renal injury is a consistent finding in Dutch Belted rabbits experimentally infected with enterohemorrhagic Escherichia coli. J. Infect. Dis. 2006, 193, 1125–1134. 112. Stearns-Kurosawa, D.J.; Collins, V.; Freeman, S.; Debord, D.; Nishikawa, K.; Oh, S.Y.; Leibowitz, C.S.; Kurosawa, S. Rescue from lethal Shiga toxin 2-induced renal failure with a cell-permeable peptide. Pediatr. Nephrol. 2011, 26, 2031–2039. 113. Sauter, K.A.; Melton-Celsa, A.R.; Larkin, K.; Troxell, M.L.; O’Brien, A.D.; Magun, B.E. Mouse model of hemolytic-uremic syndrome caused by endotoxin-free Shiga toxin 2 (Stx2) and protection from lethal outcome by anti-stx2 antibody. Infect. Immun. 2008, 76, 4469–4478. 114. Zotta, E.; Lago, N.; Ochoa, F.; Repetto, H.A.; Ibarra, C. Development of an experimental hemolytic uremic syndrome in rats. Pediatr. Nephrol. 2008, 23, 559–567. 115. Moon, H.W.; Whipp, S.C.; Argenzio, R.A.; Levine, M.M.; Giannella, R.A. Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect. Immun. 1983, 41, 1340–1351. 116. Tzipori, S.; Wachsmuth, I.K.; Chapman, C.; Birden, R.; Brittingham, J.; Jackson, C.; Hogg, J. The pathogenesis of hemorrhagic colitis caused by Escherichia coli O157:H7 in gnotobiotic piglets. J. Infect. Dis. 1986, 154, 712–716. Toxins 2012, 4 Ziegler, E.J.; Fisher, C.J., Jr.; Sprung, C.L.; Straube, R.C.; Sadoff, J.C.; Foulke, G.E.; Wortel, C.H.; Fink, M.P.; Dellinger, R.P.; Teng, N.N.; et al. Treatment of gram-negative bacteremia and septic shock with HA-1a human monoclonal antibody against endotoxin. A randomized, double-blind, placebo-controlled trial. The HA-1a sepsis study group. N. Engl. J. Med. 1991, 324, 429–436. Toxins 2012, 4 1287 Toxins 2012, 4 131. Angus, D.C.; Birmingham, M.C.; Balk, R.A.; Scannon, P.J.; Collins, D.; Kruse, J.A.; Graham, D.R.; Dedhia, H.V.; Homann, S.; MacIntyre, N. E5 murine monoclonal antiendotoxin antibody in gram-negative sepsis: A randomized controlled trial. E5 study investigators. J. Am. Med. Asso. 2000, 283, 1723–1730. 132. Deitch, E.A. Gut-origin sepsis: Evolution of a concept. Surgeon 2012, doi:10.1016/j.surge.2012.03.003. 132. Deitch, E.A. Gut-origin sepsis: Evolution of a concept. Surgeon 2012, doi:10.1016/j.surge.2012.03.003. 133. Matzinger, P. The danger model: A renewed sense of self. Science 2002, 296, 301–305. 133. Matzinger, P. The danger model: A renewed sense of self. Science 2002, 296, 301–305. 134. Wolfson, J.J.; May, K.L.; Thorpe, C.M.; Jandhyala, D.M.; Paton, J.C.; Paton, A.W. Subtilase cytotoxin activates PERK, IRE1 and ATF6 endoplasmic reticulum stress-signalling pathways. Cell Microbiol. 2008, 10, 1775–1786. 134. Wolfson, J.J.; May, K.L.; Thorpe, C.M.; Jandhyala, D.M.; Paton, J.C.; Paton, A.W. Subtilase cytotoxin activates PERK, IRE1 and ATF6 endoplasmic reticulum stress-signalling pathways. Cell Microbiol. 2008, 10, 1775–1786. © 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W2140325231	https://europepmc.org/articles/pmc3597259?pdf=render	English	null	Soil acidity, ecological stoichiometry and allometric scaling in grassland food webs	Global change biology	2,009	cc-by	8,112	Abstract The factors regulating the structure of food webs are a central focus of community and ecosystem ecology, as trophic interactions among species have important impacts on nutrient storage and cycling in many ecosystems. For soil invertebrates in grassland ecosystems in the Netherlands, the site-speciﬁc slopes of the faunal biomass to organism body mass relationships reﬂected basic biochemical and biogeochemical processes associated with soil acidity and soil C : N : P stoichiometry. That is, the higher the phosphorus availability in the soil, the higher, on average, the slope of the faunal biomass size spectrum (i.e., the higher the biomass of large-bodied invertebrates relative to the biomass of small invertebrates). While other factors may also be involved, these results are consistent with the growth rate hypothesis from biological stoichiometry that relates phosphorus demands to ribosomal RNA and protein production. Thus our data represent the ﬁrst time that ecosystem phosphorus availability has been associated with allometry in soil food webs (supporting information available online). Our results have broad implications, as soil invertebrates of different size have different effects on soil processes. Keywords: allometry, biological stoichiometry, biomass-size spectra, carbon, land use change, mass– abundance relationships, nitrogen, phosphorus, soil food webs Received 15 December 2008; revised version received 14 February 2009 and accepted 16 February 2009 potential deterministic extinction of P-rich herbivores (Perez-Moreno & Read, 2001; Sterner & Elser, 2002). Many of these ideas have been extensively explored in aquatic systems (Blanco et al., 1998; Sterner & Schulz, 1998; Gamble et al., 2006; Eyto & Irvine, 2007). Soil acidity, ecological stoichiometry and allometric scaling in grassland food webs C H R I S T I A N M U L D E R * and J A M E S J . E L S E R w Department of Ecology, RIVM, 9 Antonie van Leeuwenhoeklaan, Bilthoven, 3720 BA, The Netherlands, wSchool of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA C H R I S T I A N M U L D E R and J A M E S J . E L S E R w *Department of Ecology, RIVM, 9 Antonie van Leeuwenhoeklaan, Bilthoven, 3720 BA, The Netherlands, wSchool of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA Global Change Biology (2009) 15, 2730–2738, doi: 10.1111/j.1365-2486.2009.01899.x Introduction Here we investigate if the distribution of biomass across body-size classes of all occurring soil invertebrates (henceforth, the faunal biomass spectrum) and their microbial resources are associated with nu- trient concentrations and soil C : N : P ratios. In P-deﬁ- cient sites, bacteria are expected to grow quite slowly and thus to have reduced P content due to lower RNA concentrations (Elser et al., 2000a, 2003; Gillooly et al., 2005), potentially imposing stoichiometric food quality constraints and impairing the development of higher trophic levels. The numerical abundance of bacteria reﬂects abiotic conditions and is positively correlated with soil pH (Mulder et al., 2005a, b). To evaluate the possible impacts of soil conditions on the structure of soil food webs we examined the relationships between soil pH and C : N : P ratios (in mass units) and the regression slopes of both the faunal biomass spectra (which plot log biomass by body–mass categories as a function of log body mass) and the mass–abundance planes (which plot log numerical abundance as a func- tion of log body mass) for a large number of grassland soils encompassing broad ecological gradients in the Netherlands. Introduction Biological stoichiometry (Elser et al., 1996, 2000a,b; Ster- ner & Elser, 2002) is the study of the balance of energy and multiple chemical elements in living systems. A central idea of biological stoichiometry is the growth rate hypothesis (GRH), which proposes that variation in C : N : P ratios among organisms reﬂects differential allo- cation to P-rich ribosomal RNA in support of rapid growth rate. Thus, fast-growing taxa have low C : P and N : P biomass ratios, making them more susceptible to P-based stoichiometric food quality constraints (Elser et al., 2000b; Sterner & Elser, 2002). As a result, ecosystem conditions that produce organic matter with high C : P and N : P ratios are likely to result in inefﬁcient trophic transfer, reduced biomass of upper trophic levels, and Body size relations are important for understanding ecological processes (Peters, 1983; Calder, 1984; Mulder, 2006; Damuth, 2007), but ecological effects of C : P and N : P stoichiometric imbalance have not been widely considered for the differently sized soil invertebrates that make up belowground food webs. In aquatic habitats, in fact, allometric associations of nutrient con- tent with abundance and biomass have already been established (e.g., Sheldon et al., 1972; Blanco et al., 1998). In contrast, in terrestrial ecosystems the connections between nutrient stoichiometry and size scaling are only now beginning to be considered (Schade et al., 2003; Kerkhoff et al., 2005; Meehan, 2006; Enquist et al., 2007; Martinson et al., 2008; Reuman et al., 2009). Furthermore, the size spectra of different clades in terrestrial ecosystems have traditionally been studied separately, often from an entomological perspective Correspondence: Christian Mulder, tel. 1 31 30 2 743 148, fax 1 31 30 2 744 413, e-mail: christian.mulder@rivm.nl Re-use of this article is permitted in accordance with the Creative Commons Deed Attribution 2.5, which does not permit commer- cial exploitation. r 2009 Blackwell Publishing Ltd 2730 S O I L p H , S T O I C H I O M E T R Y A N D A L L O M E T R I C S C A L I N G 2731 2731 Managed grassland Abandoned grassland Site U 231 Site I 251 (Siemann et al., 1999), obscuring community-wide allo- metric patterns. Sites and organisms Sites and organisms One recent study showed that grasslands under organic management might exhibit a departure from power law behaviour due to organic fertilizer inputs (Reuman et al., 2008). To avoid such effects, we selected, from all the locations monitored by the RIVM (Bilthoven, the Netherlands), 12 of the organic grasslands under low- intensive, bio-dynamic management and 10 ex-organic farms that were abandoned for at least a decade (Table S1). In contrast to soil acidity and P, which typically decrease following land abandonment (Fig. 1), soil nutrient ratios are expected to increase after abandon- ment (Knops & Tilman, 2000). Environmental gradients were chosen to obtain a certain overlap in all the investigated abiotic correlates (Table 1). Table 1 Soil C : N : P stoichiometry and abiotic properties Main abiotic properties Abandoned Managed Carbon (g kg soil dry mass1) 54.0 23.7 37.9 17.1 Nitrogen (g kg soil dry mass1) 2.9 1.0 2.5 0.7 Phosphorus (g kg soil dry mass1) 0.6 0.3 0.9 0.4 Carbon-to-phosphorus ratio 98.0 58.4 41.1 8.8 Carbon-to-nitrogen ratio 18.5 4.7 14.7 2.3 Nitrogen-to-phosphorus ratio 5.2 2.2 2.8 0.7 Soil organic matter (%) 9.3 4.1 6.5 2.9 Soil acidity (pH in KCl) 4.5 0.3 5.3 0.4 Means SE for 10 abandoned grasslands (mature meadows) and 12 biomanaged grasslands under organic regime (com- plete data online in Table S1). Table 1 Soil C : N : P stoichiometry and abiotic properties Main abiotic properties Abandoned Managed Table 1 Soil C : N : P stoichiometry and abiotic properties For this study, upper horizons (top 10 cm) of the 22 Pleistocene sandy soils selected across the Netherlands were randomly sampled between 2002 and 2005. Hor- izons have similar bulk density (1.20rb 0.29 SD) and organic matter content (7.78% 3.69 SD). Soil acidity of oven-dried soil samples was measured in 1 M potassium chloride solution (Table S1). Soils were analysed, at least in triplicate, after preliminary wet digestion. Ntot and Ptot (henceforth N and P) were determined by colori- metric analysis. Soil P content was also measured as phosphate (Pw) by using aqueous extraction at water-to- Means SE for 10 abandoned grasslands (mature meadows) and 12 biomanaged grasslands under organic regime (com- plete data online in Table S1). r 2009 Blackwell Publishing Ltd, Global Change Biology, 15, 2730–2738 Materials and methods Fig. 1 The development of mature grasslands is a primary con- cern of many restoration efforts in the Netherlands. We investi- gated both managed grasslands (average soil pH 5.3 and C: N:P ratio 41.1:2.8 :1 in mass units) as abandoned grasslands (average pH 4.5 and C :N:P ratio 98: 5.2 :1 in mass units). Holcus lanatus, Lolium perenne, and Dactylis glomerata characterise the studied grasslands together with Alopecurus geniculatus and Poa trivialis. Fig. 1 The development of mature grasslands is a primary con- cern of many restoration efforts in the Netherlands. We investi- gated both managed grasslands (average soil pH 5.3 and C: N:P ratio 41.1:2.8 :1 in mass units) as abandoned grasslands (average pH 4.5 and C :N:P ratio 98: 5.2 :1 in mass units). Holcus lanatus, Lolium perenne, and Dactylis glomerata characterise the studied grasslands together with Alopecurus geniculatus and Poa trivialis. Allometric scaling Descriptors derived from allometric patterns require a brief explanation. If the species or genera of a local community are plotted as points in a plane with abscis- sa (horizontal axis) log(M), and with ordinate log(N), then the points have been found to fall approximately along a straight line with negative slope. This mass– abundance slope is the coefﬁcient a in the linear model log(N) 5 a log(M) 1 b ﬁtted to data from a single soil community food web. The data can comprise organisms belonging to different kingdoms or other taxonomic groups. We computed the mass–abundance slopes for all soil invertebrates (A, from Animalia), and added two further aggregated points, one for Fungi (F, resulting in a scatter of all A 1 F points) and one for Eubacteria (E, resulting in a A 1 E 1 F scatter of all recovered soil eukaryotes and bacterial cells). Because the biomass of a taxon is its numerical abundance times its body mass, B 5 NM, and because increasing M is associated with increasing trophic height (Mulder et al., 2005a, 2008; Reuman et al., 2008), the mass–abundance slopes for A, A 1 F, and A 1 E 1 F are supposed to indicate how (faunal) biomass and population density change for (faunal) taxa of increasing trophic height. Mass–abun- dance relationships reveal how the size structure of a community food web interacts with its trophic structure and biomass distribution (White et al., 2007). If the resulting mass–abundance slope is exactly 1, then the trend is for all taxa to have equal biomass. All identiﬁed genera (Table 2) fell into 22 trophic guilds. Trophic links among guilds (from resource to consu- mer) were inferred from published literature (Table S2). p y g The soil invertebrates (Table S2) were measured as follows. For worm-like animals (nematodes and enchy- traeids), the body length and width of at least 150 nematodes were measured to the nearest 5 mm with an eyepiece micrometer; all enchytraeids were measured individually, including juveniles and resting stages. The soil nematode samples were collected randomly across the investigated site. In each of the 22 locations, a composite sample was obtained by mixing 320 soil cores (diameter 2.3 cm 10 cm) in a plastic container, and approximately 500 g of wet soil was collected in glass jars. 2732 C . M U L D E R & J . J . E L S E R Table 2 Faunal web structure, soil microbiology and biodi- versity distribution Main biotic properties Abandoned Managed Total number of potential trophic links (linkages between genera) 710 159 706 225 Biodiversity of nematodes (Nematoda) 28 3 25 5 Biodiversity of soil mites (Acarina) 16 3 16 5 Biodiversity of springtails (Insecta) 6 3 10 3 Biodiversity of enchytraeids (Oligochaeta) 6 1 5 1 Fungal-to-bacterial ratio (mg C mg1 C) 0.15 0.08 0.26 0.31 Means SE for 10 abandoned grasslands (mature meadows) and 12 biomanaged grasslands under organic regime (com- plete data online in Table S2). were made on mass slides. Nematodes were identiﬁed by light microscopy at 400–600 (Mulder et al., 2003, 2005b). Microarthropods were extracted from the soil by placing six discs of the soil sample in a Tullgren funnel (Siepel & Van de Bund, 1988; Ro¨mbke et al., 2006). The temperature in the upper part of the funnel was set at 30 1C and kept at 5 1C in the lower part. The organisms moved downwards to escape the heat, dropped through a funnel and were collected into a bottle containing 70% ethanol. The total extraction time was 1 week. For each sample, 70 individuals were counted and identiﬁed at 200–1000 via a gel-based subsampling method (Jagers op Akkerhuis et al., 2008). Extracted arthropods were divided in body-size classes to estimate the correspond- ing individual body mass (M in microgram of dry weight). All values were averaged over the life-stages of all the counted individuals N (Mulder et al., 2005a, b). These body-mass averages at genus level offer the best available combination of high environmental informa- tion and low noise (Mulder et al., 2006, 2008). Table 2 Faunal web structure, soil microbiology and biodi- versity distribution Means SE for 10 abandoned grasslands (mature meadows) and 12 biomanaged grasslands under organic regime (com- plete data online in Table S2). organic matter (SOM) using SOM 5 Corg 1.724 (the traditional Van Bemmelen conversion factor). The total abundance of bacterial cells was determined in duplicate by ﬂuorescent staining [5-(4, 6-dichloro- triazin-2-yl) aminoﬂuorescein] and combined micro- scopy and automatic image analysis. 2732 C . M U L D E R & J . J . E L S E R For the conversion of bacterial cell volume (mm3) to dry bio- mass (mg) we used the biovolume-to-carbon factor of Van Veen & Paul (1979), assuming a bacterial carbon content of 50% (Herbert, 1976; Mulder et al., 2005a; Table 2). Hyphae were viewed with ﬂuorescent staining and direct microscopy at 2500 and the total hyphal length was estimated by the line intercept method. The myce- lium was inferred from the length of the counted branches, assuming a hyphal diameter of 2.5 mm (Table S2). All fungal measurements used direct microscopy and palynological treatments (Mulder et al., 2005b). r 2009 Blackwell Publishing Ltd, Global Change Biology, 15, 2730–2738 Sites and organisms soil ratio of 60 : 1 by volume, after 22 h of pre-equilibra- tion with soil with water and 1 h of gentle shaking before ﬁltration (Sissingh, 1971). Average values of C (dry combustion) were obtained by conversion of soil r 2009 Blackwell Publishing Ltd, Global Change Biology, 15, 2730–2738 2732 C . M U L D E R & J . J . E L S E R Allometric scaling Nematodes were extracted, within 1 week, from 100g of wet soil using the funnel elutriation comple- mented with sieving and cottonwood extraction after crawling through a cotton ﬁlter over a 2-day period. Two clean suspensions in 10 mL water were screened with a stereoscope to count the individuals; the total of nema- todes was estimated by counting 10% of the extracted animals twice. Permanent mounts in 4% formaldehyde r 2009 Blackwell Publishing Ltd, Global Change Biology, 15, 2730–2738 S O I L p H , S T O I C H I O M E T R Y A N D A L L O M E T R I C S C A L I N G 2733 It was assumed that every taxon in a resource guild was trophically linked to every taxon in a consumer guild. P becomes curtailed under lower soil pH (Bohn et al., 1985), as in our abandoned grasslands. Merging the classic allometric formula log(N) 5 a log(M) 1 b with log(B) 5 log(M) 1 log(N), we ob- tain log(B) 5 log(M) 1 a log(M) 1 b 5 (1 1 a) log(M) 1 b (e.g., Cyr et al., 1997; Mulder et al., 2008) Theoreti- cally, thus, the slope of a faunal biomass spectrum is 1 plus the slope of the relation between log(N) and log(M) for all the occurring animals; e.g., a zero slope of the biomass spectrum (constant biomass across trophic levels) corresponds to a slope of 1 in a plot of log(N) in bins as a function of log(M) (Rossberg et al., 2008). To compute the faunal biomass spectrum slopes using bins of constant linear width, the range from the smallest log(M) to the largest log(M) occurring in any of the grasslands was divided into 10 equal bins. For each bin, the log of the total faunal biomass was computed. All values for bins containing fauna (bins with zero obser- vations are excluded) were regressed against the log(M) at the centre of each bin. To test whether binning introduces a bias towards small or large organisms as suggested by White et al. (2008), we compared (binned) biomass spectrum slopes with three (unbinned) mass– abundance slopes. Computations used SAS version 9.1.3, PC-ORD version 4.20 and the EXCEL Visual Basic optimiza- tion toolbox. We set 1% signiﬁcance to detect violations. Allometric scaling Soil acidity of abandoned grasslands was almost seven times that of managed grasslands (pH averaged 4.48 0.28 and 5.32 0.36 SD, respectively). Soil pH contributes strongly to the explanation of the allometric variation within a pH range of 3.8–6.0 (Fig. 2 and Table S1). We tested the resulting correlation between soil pH and the biomass spectrum slope by adding 10 locations belonging to a completely different ecosystem, namely dry heathlands with very low soil pH (3.15 0.23 SD), to extend the investigated pH range down to 2.9. We obtained for this recalculated linear regression a slope of 0.375 0.025 SE, which is statistically indistinguish- able from the original slope of 0.351 0.046 SE with a 99% CI. Managed grasslands supported invertebrate commu- nities in which biomass increased with the central log(M) values of bins in biomass spectra. Steep faunal biomass slopes were associated here with high popula- tion densities of enchytraeids that fall at the upper end of the range of body sizes (Table S2). In contrast, abandoned grasslands supported invertebrate commu- nities in which biomass increased less with increasing central log(M) values of bins in biomass spectra. Shal- low faunal biomass slopes were associated with higher R2 = 0.7482 0 0.2 0.4 0.6 0.8 1 3.5 4 4.5 5 5.5 6 6.5 Soil pH Faunal biomass spectrum slope Abandoned Managed (liming) P < 0.00001 R2 = 0.7482 0 0.2 0.4 0.6 0.8 1 3.5 4 4.5 5 5.5 6 6.5 Soil pH Faunal biomass spectrum slope Abandoned Managed (liming) P < 0.00001 Fig. 2 The slope of the biomass spectrum of soil invertebrates as predicted by soil acidity (pH in KCl). We combined the body masses of all individuals whose size fell within an equal interval of body size and plotted, separately for each grassland, the log summed biomass of all animal taxa within log(M) bins against bin centres. In this way, the slopes of the linear regression of log summed biomass become a function of the bin centres on a log(M) scale. Pearson’s correlation coefﬁcients show a signiﬁcant correlation only between the soil pH and the logarithm of C : P (P 5 0.0010). Managed grasslands are squares and abandoned grasslands are diamonds. r 2009 Blackwell Publishing Ltd, Global Change Biology, 15, 2730–2738 Results and discussion There were signiﬁcant differences in the concentrations of soil macronutrients. The C and N concentrations were closely correlated (Table S1) and highest, on average, in the abandoned grasslands (i.e., not fertilized for at least one decade) and lowest, on average, in the managed grasslands (Table 1). Total soil P contents, on the contrary, were highest in the managed grass- lands, due to inputs of organic fertilizers, and lowest in the abandoned grasslands (939 400 vs. 627 277 SD mg P kg1). Coefﬁcients of variation (100 SD/ mean) of total soil P were 43% and 44% for managed and abandoned grasslands, respectively. Soil P content was uncorrelated with soil pH, in contrast to the phos- phate content after water extraction [Pearson’s correla- tion coefﬁcients 0.024 for P and pH (P40.05) and 0.53 for Pw and pH (P 5 0.0115), respectively]. Phosphate content after water extraction seemed to reﬂect the maximal possible concentration of phosphorus in the thin layer of water around soil particles (35.3 9.1 vs. 19.6 24.6 SD mg L1 in managed and abandoned grasslands, respectively). Biologically available P is thought to increase within a pH range of 5–6 (Chapin & Eviner, 2003; Sims & Pierzynski, 2005; Cleveland & Liptzin, 2007), as in our managed grasslands where fertilization and liming occurred regularly. In contrast, Fig. 2 The slope of the biomass spectrum of soil invertebrates as predicted by soil acidity (pH in KCl). We combined the body masses of all individuals whose size fell within an equal interval of body size and plotted, separately for each grassland, the log summed biomass of all animal taxa within log(M) bins against bin centres. In this way, the slopes of the linear regression of log summed biomass become a function of the bin centres on a log(M) scale. Pearson’s correlation coefﬁcients show a signiﬁcant correlation only between the soil pH and the logarithm of C : P (P 5 0.0010). Managed grasslands are squares and abandoned grasslands are diamonds. r 2009 Blackwell Publishing Ltd, Global Change Biology, 15, 2730–2738 2734 C . M U L D E R & J . J . Results and discussion E L S E R R2 = 0.4917 R2 = 0.3341 R2 = 0.2880 0 0.2 0.4 0.6 0.8 1 R2 = 0.4198 R2 = 0.3296 R 2 = 0.1808 –1.1 –0.9 –0.7 –0.5 –0.3 –0.1 R2 = 0.5344 R2 = 0.3871 R2 = 0.2756 –0.9 –0.7 –0.5 –0.3 –0.1 R2 = 0.3410 R2 = 0.3634 R 2 = 0.054 –1.2 –1 –0.8 –0.6 –0.4 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 P = 0.01 P = 0.005 P < 0.05 P = 0.0003 P = 0.001 P = 0.005 P = 0.01 P = 0.002 P = 0.0001 n.s. P = 0.004 P = 0.003 Log (C : N) Log (N : P) Log (C : P) Mass-abundance slope (A+E+F) Mass-abundance slope (A+F) Mass-abundance slope (A) Biomass spectrum slope (A) Fig. 3 Different allometric scalings and soil nutrient ratios. Arranged according to a decreasing coefﬁcient of variation in our (slopes rescaled in degrees, not shown), from top to bottom: faunal biomass spectrum slope (A 5 Animalia), faunal mass – abun slope (A 5 Animalia), eukaryotes’ mass – abundance slope (A 1 F 5Animalia and Fungi), and mass – abundance slope of the com community food web (A 1 E 1 F 5Animalia, Eubacteria, and Fungi). The logarithm of the C : P ratio equals the sum of the log C : N log N : P ratios and is the best sole predictor for allometric scaling after pH. Symbols as in Fig. 2. 2734 C . M U L D E R & J . J . E L S E R R2 = 0.2880 0 0.2 0.4 0.6 0.8 1 R 2 = 0.1808 –1.1 –0.9 –0.7 –0.5 –0.3 –0.1 R2 = 0.2756 –0.9 –0.7 –0.5 –0.3 –0.1 R 2 = 0.054 –1.2 –1 –0.8 –0.6 –0.4 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 P = 0.01 P < 0.05 P = 0.01 n.s. Results and discussion r 2009 Blackwell Publishing Ltd, Global Change Biology, 15, 2730–2738 S O I L p H , S T O I C H I O M E T R Y A N D A L L O M E T R I C S C A L I N G 2735 lated with the logarithm of the soil C : P ratio (Pearson’s correlation coefﬁcient 0.7013, P 5 0.0003), but less with the logarithms of the N : P and C:N ratios (P 5 0.0049 and P 5 0.0099, respectively). As a matter of fact, in all cases of allometric scaling (n 5 22, four kinds of lump- ing, resulting in 88 investigated cases comprehending two ecosystem types), the logarithm of the C : P ratio was the best sole predictor (Fig. 3, right column). (microarthropod populations). Equivalently, the less the limitation by either nitrogen or carbon relative to phos- phorus, or the greater the phosphorus limitation, the more rapidly the population density of invertebrate genera decreases as the average body mass of the invertebrate genera increases. All the negative trends along the environmental correlates of Fig. 3 indicate thus (i) that faunal biomass declined with increased body size within log(M) bins, and (ii) that faunal population density decreased with increased body size. Hence differences in soil P availability are reﬂected within our grasslands (and possibly in other biomes as well) in the body-size distribution of the biomass and density of the soil microfauna and mesofauna, since stoichiometric theory predicts that soil fauna with high- er P demands would suffer a competitive disadvantage in lower P soils due to poorer stoichiometric food quality. The GRH (Elser et al., 2000a, 2003) suggests that high P content and low C : P ratio in biomass reﬂect increased allocation to P-rich ribosomal RNA, which in turn enables increased protein synthesis, increased growth rates of individuals, and increased population growth rates. Empirical investigations support the GRH in varied contexts (e.g., Elser et al., 2000a, 2003). The GRH provides a natural context for interpretation of at least some of our observations. Agricultural soils with higher P content, as a result of edaphic conditions, land use and history (in our case, increased P availability due to fertilization and liming effects on pH), have a twofold lower average soil C : P ratio than abandoned grass- lands (Table 1). Results and discussion p g g For further interpretation of mass–abundance slopes, derived from unlumped and unbinned points with speciﬁc numerical abundance and body–mass average, one brief example may be useful. Let us imagine a simple food web consisting of four genera, namely one nematode, one mite, one springtail, and one enchy- traeid. Let their respective dry weights be 0.1, 1, 10, and 100 mg on average (extensive empirical data in Table S2). After log-transformation, their log(M) will become 1, 0, 1, and 2. Seen that in a plane with abscissa log(M) and with ordinate log(N) most populations have been found to fall approximately along a straight line with negative slope, we assume for ease of computation that the popu- lation densities of these four genera are equal to 10000, 1000, 100, and 10 individuals per square meter, respec- tively. After log-transformation, their log(N) will become 4, 3, 2, and 1. Their speciﬁc log(B) equals log(M)1 log(N)51145 013511 252115 3. Hence, these four populations will keep a dry biomass of 1mgm2 and, if plotted in a log(M)log(N) plane, the result- ing linear regression slope will be exactly equal to 1. Our mass–abundance slopes were always negative (1oao0), as expected from the positive biomass spec- trum slope. The question which arises here is: how do slopes derived from such mass–abundance relationships reﬂect soil resource limitation? As recently stated by Urabe & Waki (2009, p. 529), ‘herbivores tend to increase the ingestion rate when food with low nutrient content relative to C is supplied’. Many larger-bodied soil herbivores are further known for a certain combination of intrinsic and microbial sources of cellulolysis in their diet (e.g., Douglas, 2009, p. 42), and most growth and reproduction rates have been observed to decline as a function of body mass (Hendriks & Mulder, 2008; Beardall et al., 2009). We suggest that increase in P availability relaxes stoichio- metric food quality constraints on small-bodied pri- mary consumers (here, microbivores), which increases trophic transfer efﬁciency, and supports larger-bodied consumers (including herbivores) also at higher trophic levels. That is, soils richer in the key nutrient P have organic matter that is of higher quality for invertebrates, allowing the maintenance of food webs with increased abundances of larger taxa. Results and discussion Log (C : N) Mass-abundance slope (A+E+F) Mass-abundance slope (A+F) Mass-abundance slope (A) Biomass spectrum slope (A) Fig 3 Different allometric scalings and soil nutrie R2 = 0.3871 R2 = 0.3634 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 P = 0.002 P = 0.003 Log (N : P) R2 = 0.3410 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 P = 0.004 Log (C : P) R2 = 0.3634 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 P = 0.003 Log (N : P) Log (C : P) Log (C : N) Log (N : P) Fig. 3 Different allometric scalings and soil nutrient ratios. Arranged according to a decreasing coefﬁcient of variation in our sites (slopes rescaled in degrees, not shown), from top to bottom: faunal biomass spectrum slope (A 5 Animalia), faunal mass – abundance slope (A 5 Animalia), eukaryotes’ mass – abundance slope (A 1 F 5Animalia and Fungi), and mass – abundance slope of the complete community food web (A 1 E 1 F 5Animalia, Eubacteria, and Fungi). The logarithm of the C : P ratio equals the sum of the log C : N and log N : P ratios and is the best sole predictor for allometric scaling after pH. Symbols as in Fig. 2. Plotting biomass spectrum slopes and mass–abun- dance slopes against the logarithms of the soil nutrient ratios revealed strong relationships between nutrient ratios and faunal biomass size spectra (Fig. 3). The interpretation of the biomass spectrum slope is intui- tive: negative trends indicate that faunal biomass under nutrient deﬁciency declined with increased body size. Biomass spectrum slopes were most signiﬁcantly corre- population densities of nematodes that fall at the lower end of the size range, indicating that biomass within log(M) bins increased with average body size but increased less rapidly on average than after application of manure and lime. Similar associations of soil pH with different taxa and communities have been recorded recently (Mulder et al., 2003, 2005b; Fierer & Jackson, 2006). r 2009 Blackwell Publishing Ltd, Global Change Biology, 15, 2730–2738 References Our 10 abandoned grasslands (lower soil pH) have a higher concentration of [H 1]-ions than grass- lands managed with lime, in which the cation exchange capacity increases. Furthermore, our data are correla- tive in nature. Thus, the hypothesis that stoichiometric imbalance imposed by P-limitation alters soil biomass spectra is in need of experimental test. Such a test would involve quantifying biomass spectra in experi- mental plots subjected to different levels of nutrient enrichment. Such experiments seem particularly timely given the recent indication that P-limitation of primary production may be more widespread in terrestrial eco- systems than previously appreciated (Elser et al., 2007). Bohn H, McNeal B, O’Connor G (1985) Soil Chemistry, 2nd edn. Wiley, New York, NY. Calder WA (1984) Size, Function, and Life History. Harvard Uni- versity Press, Cambridge, MA. Chapin FS III, Eviner VT (2003) Biogeochemistry of terrestrial net primary production. Treatise on Geochemistry, 8, 215–247. Cleveland CC, Liptzin D (2007) C : N : P stoichiometry in soil: is there a ‘‘Redﬁeld ratio’’ for the microbial biomass? Biogeochem- istry, 85, 235–252. Cyr H, Downing JA, Peters RH (1997) Density-body size relation- ships in local aquatic communities. Oikos, 79, 333–346. Damuth J (2007) A macroevolutionary explanation for energy equivalence in the scaling of body size and population density. American Naturalist, 169, 621–631. Douglas AE (2009) The microbial dimension in insect nutritional ecology. Functional Ecology, 23, 38–47. Elser JJ, Acharya K, Kyle M et al. (2003) Growth rate – stoichio- metry couplings in diverse biota. Ecology Letters, 6, 936–943. In summary, faunal biomass spectrum slopes were strongly associated with liming practice, soil nutrient conditions (as reﬂected by phosphate), and organic fertilizers (as reﬂected by the soil C : P ratio). Previous studies on faunal biomass size spectra in aquatic eco- systems reported lower slopes in systems subjected to heavy ﬁshing and hypothesized that ﬁshing decreased the slope of a biomass spectrum by preferentially re- moving larger organisms (Pope et al., 1988; Jennings, 2005), a ‘top down’ explanation. Our data showing that soil P availability is associated with increasingly posi- tive slopes of the faunal biomass size spectrum in soil systems represent the ﬁrst observation from soil food webs for a ‘bottom up’ effect on soil food-web size structure. The GRH, with or without increased trophic transfer efﬁciencies, provides a natural interpretation of why (artiﬁcially) P-enriched systems have food webs in Elser JJ, Bracken MES, Cleland EE et al. References Beardall J, Allen D, Bragg J et al. (2009) Allometry and stoichio- metry of unicellular, colonial and multicellular phytoplankton. New Phytologist, 181, 295–309. Blanco JM, Quin˜ones RA, Guerrero F, Rodrı´guez J (1998) The use of biomass spectra and allometric relations to estimate respira- tion of planktonic communities. Journal of Plankton Research, 20, 887–900. y, , growth rates, and RNA demands of soil fauna. The association of the faunal biomass spectrum to soil acidity is more than a chemical reaction to a relative concentration of [H 1]-ions since soil pH has a strong impact on the nutrient mobility, adsorption, and pre- cipitation. Our 10 abandoned grasslands (lower soil pH) have a higher concentration of [H 1]-ions than grass- lands managed with lime, in which the cation exchange capacity increases. Furthermore, our data are correla- tive in nature. Thus, the hypothesis that stoichiometric imbalance imposed by P-limitation alters soil biomass spectra is in need of experimental test. Such a test would involve quantifying biomass spectra in experi- mental plots subjected to different levels of nutrient enrichment. Such experiments seem particularly timely given the recent indication that P-limitation of primary production may be more widespread in terrestrial eco- systems than previously appreciated (Elser et al., 2007). In summary, faunal biomass spectrum slopes were strongly associated with liming practice, soil nutrient conditions (as reﬂected by phosphate), and organic fertilizers (as reﬂected by the soil C : P ratio). Previous studies on faunal biomass size spectra in aquatic eco- systems reported lower slopes in systems subjected to heavy ﬁshing and hypothesized that ﬁshing decreased the slope of a biomass spectrum by preferentially re- moving larger organisms (Pope et al., 1988; Jennings, 2005), a ‘top down’ explanation. Our data showing that soil P availability is associated with increasingly posi- tive slopes of the faunal biomass size spectrum in soil systems represent the ﬁrst observation from soil food webs for a ‘bottom up’ effect on soil food-web size structure. The GRH, with or without increased trophic transfer efﬁciencies, provides a natural interpretation of why (artiﬁcially) P-enriched systems have food webs in The association of the faunal biomass spectrum to soil acidity is more than a chemical reaction to a relative concentration of [H 1]-ions since soil pH has a strong impact on the nutrient mobility, adsorption, and pre- cipitation. Conclusions Alterations in soil C : N : P ratios, such as those driven by direct anthropogenic inﬂuences like nitrogen deposi- tion, liming, and fertilizer application (Hunt & Wall, 2002; Wardle, 2002; Mulder et al., 2005b, 2008; Persson et al., 2008) and/or those driven by climatic changes like increased atmospheric CO2, global warming and tor- rential rainfall (Wardle et al., 1998; Hunt & Wall, 2002; Voigt et al., 2003, 2007; Sardans & Pen˜uelas, 2007; Urabe & Waki, 2009), may affect the rates at which soil biota carry out ecosystem services by affecting the faunal biomass distribution. However, these ideas require further investigation and we require more data on the C : N : P stoichiometry, threshold elemental ratios, growth rates, and RNA demands of soil fauna. Acknowledgements This research was supported by the RIVM Directorate (grant EIA-S607001) and by the NSF (grant DEB-0516494). We thank L. Posthuma, A. M. Breure, and J. Bloem for assistance and pre- cious support, and J. E. Cohen, H. A. Den Hollander, D. C. Reuman, and D. De Zwart for conversations and discussions. Results and discussion As a result, biomass spectra in P-rich soils have steep positive slopes while relatively inefﬁcient food webs in stoichiometrically imbalanced low-P soils support mostly small-bodied fauna and thus produce shallow (and theoretically even negative) bio- mass spectrum slopes. The extent to which low-P soils The linear regressions of log(N) as function of log(M) account for nearly 50% of the variation in log(N) of organisms in most grasslands, but linear regressions may account for only 30% of the variation in some managed grasslands. The expected numerical abun- dances of ubiquitous smallest animals (nematodes) vary more than the numerical abundances of the larger animals (mites, springtails, and enchytraeids; see Table S2). Numerical abundances of smaller soil invertebrates differ up to four orders of magnitude from those of larger invertebrates. Moreover, the body–mass ranges of soil invertebrates reﬂect dominant feeding habits, since opposite functional types coming out of taxo- nomic inventories can be recognized: 55% of the microarthropods graze, browse or ingest fungal re- mains and 40% of nonparasitic nematodes graze on bacterial cells (Mulder et al., 2005b and Table S2). Therefore the allometric scaling strongly suggests that the microfauna (nematode populations) copes better with P-limited conditions than the mesofauna r 2009 Blackwell Publishing Ltd, Global Change Biology, 15, 2730–2738 C . M U L D E R & J . J . E L S E R 2736 which biomass increases with consumer body size while biomass declines with body size in P-limited habitats. These insights potentially connect biogeo- chemistry with the allometric relationships between biomass, abundance, and body size. support the population density of the small-bodied fauna is shown by the more negative mass–abundance slopes. S O I L p H , S T O I C H I O M E T R Y A N D A L L O M E T R I C S C A L I N G 2737 Hunt HW, Wall DH (2002) Modelling the effects of loss of soil bio- diversity on ecosystem function. Global Change Biology, 8, 33–50. Jagers op Akkerhuis GAJM, Dimmers WJ, Van Vliet PCJ, Goed- hart GFP, Martakis GFP, De Goede RGM (2008) Evaluating the use of gel-based sub-sampling for assessing responses of terrestrial micro-arthropods (Collembola and Acari) to differ- ent slurry applications and organic matter contents. Applied Soil Ecology, 38, 239–248. Reuman DC, Cohen JE, Mulder C (2009) Human and environ- mental factors inﬂuence soil faunal abundance-mass allometry and structure. Advances in Ecological Research, 41, 45–85. Reuman DC, Mulder C, Raffaelli D, Cohen JE (2008) Three allometric relations of population density to body mass: theoretical integration and empirical tests in 149 food webs. Ecology Letters, 11, 1216–1228. Jennings S (2005) Size-based analyses of aquatic food webs. In: Aquatic Food Webs – An Ecosystem Approach (eds Belgrano A, Scharler UM, Dunne J, Ulanowicz RE), pp. 86–97. Oxford University Press, Oxford, UK. Ro¨mbke J, Sousa J-P, Schouten T, Riepert F (2006) Monitoring of soil organisms: a set of standardized ﬁeld methods proposed by ISO. European Journal of Soil Biology, 42, S61–S64. Kerkhoff AJ, Enquist BJ, Elser JJ, Fagan WF (2005) Plant allome- try, stoichiometry and the temperature-dependence of primary productivity. Global Ecology and Biogeography, 14, 585–598. Rossberg AG, Ishii R, Amemiya T, Itoh K (2008) The top–down mechanism for body-mass abundance scaling. Ecology, 89, 567–580. Knops JMH, Tilman D (2000) Dynamics of soil nitrogen and carbon accumulation for 61 years after agricultural abandon- ment. Ecology, 81, 99–98. Sardans J, Pen˜uelas J (2007) Drought changes phosphorus and potassium accumulation patterns in an evergreen Mediterra- nean forest. Functional Ecology, 21, 191–201. Martinson HM, Schneider K, Gilbert J, Hines JE, Hamba PA, Fagan WF (2008) Detritivory: stoichiometry of a neglected trophic level. Ecological Research, 23, 487–491. Schade JD, Kyle M, Hobbie SE, Fagan WF, Elser JJ (2003) Stoichiometric tracking of soil nutrients by a desert insect herbivore. Ecology Letters, 6, 96–101. Meehan TD (2006) Energy use and animal abundance in litter and soil communities. Ecology, 87, 1650–1658. Sheldon RW, Prakash A, Sutcliffe WHJ (1972) The size distribu- tion of particles in the ocean. Limnology and Oceanography, 17, 327–340. Mulder C (2006) Driving forces from soil invertebrates to eco- system functioning: the allometric perspective. Naturwis- senschaften, 93, 467–479. Siemann E, Tilman D, Haarstad J (1999) Abundance, diversity and body size: patterns from a grassland arthropod commu- nity. S O I L p H , S T O I C H I O M E T R Y A N D A L L O M E T R I C S C A L I N G 2737 S O I L p H , S T O I C H I O M E T R Y A N D A L L O M E T R I C S C A L I N G 2737 Eyto E, Irvine K (2007) Assessing the status of shallow lakes using an additive model of biomass size spectra. Aquatic Conservation: Marine and Freshwater Ecosystems, 17, 724–736. agro-environments in the Netherlands. Ecological Complexity, 3, 219–230. Mulder C, Den Hollander HA, Hendriks AJ (2008) Above- ground herbivory shapes the biomass distribution and ﬂux of soil invertebrates. Public Library of Science ONE, 3 e3573, 1–7. Fierer N, Jackson RB (2006) The diversity and biogeography of soil bacterial communities. Proceedings of the National Academy of Science USA, 103, 626–631. Gamble AE, Lloyd R, Aiken J, Johannsson OE, Mills EL (2006) Using zooplankton biomass size spectra to assess ecological change in a well-studied freshwater lake ecosystem: Oneida Lake, New York. Canadian Journal of Fisheries and Aquatic Sciences, 63, 2687–2699. Mulder C, Van Wijnen HJ, Van Wezel AP (2005b) Numerical abundance and biodiversity of below-ground taxocenes along a pH gradient across the Netherlands. Journal of Biogeography, 32, 1775–1790. Perez-Moreno J, Read DJ (2001) Nutrient transfer from soil nematodes to plants: a direct pathway provided by the mycor- rhizal mycelial network. Plant, Cell and Environment, 24, 1219–1226. Gillooly JF, Allen AP, Brown JH et al. (2005) The metabolic basis of whole-organism RNA and phosphorus content. Proceedings of the National Academy of Science USA, 102, 11923–11927. Hendriks AJ, Mulder C (2008) Scaling of offspring number and mass to plant and animal size: model and meta-analysis. Oecologia, 155, 705–716. Persson J, Vrede T, Holmgren S (2008) Responses in zooplankton populations to food quality and quantity changes after whole lake nutrient enrichment of an oligotrophic sub-alpine reser- voir. Aquatic Sciences, 70, 142–155. Herbert D (1976) Stoichiometric aspects of microbial growth. In: Continuous Culture 6: Application and New Fields (eds Dean ACR, Ellwood DC, Evans CGT, Melling J), pp. 1–30. Ellis Horwood, Chichester, UK. Peters RH (1983) The Ecological Implications of Body Size. Cam- bridge University Press, Cambridge, UK. Pope JG, Stokes TK, Murawski SA, Idoine SI (1988) A compar- ison of ﬁsh size composition in the North Sea and on Georges Bank. In: Ecodynamics (eds Wolff W, Soeder C, Drepper F), pp. 146–152. Springer, New York, NY. References (2007) Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems. Ecology Letters, 10, 1135–1142. Elser JJ, Dobberfuhl D, MacKay NA, Schampel J (1996) Organism size, life history, and N : P stoichiometry: towards a uniﬁed view of cellular and ecosystem processes. BioScience, 46, 674–684. Elser JJ, Fagan WF, Denno RF et al. (2000a) Nutritional con- straints in terrestrial and freshwater food webs. Nature, 408, 578–580. Elser JJ, Sterner RW, Gorokhova E et al. (2000b) Biological stoi- chiometry from genes to ecosystems. Ecology Letters, 3, 540–550. Enquist BJ, Kerkhoff AJ, Huxman TE, Economo EP (2007) Adaptive differences in plant physiology and ecosystem para- doxes: insights from metabolic scaling theory. Global Change Biology, 13, 591–609. r 2009 Blackwell Publishing Ltd, Global Change Biology, 15, 2730–2738 2738 C . M U L D E R & J . J . E L S E R 2738 White EP, Enquist BJ, Green JL (2008) On estimating the exponent of power-law frequency distributions. Ecology, 89, 905–912. Sterner RW, Elser JJ (2002) Ecological Stoichiometry: The Biology of Elements from Molecules to the Biosphere. Princeton University Press, Princeton, NJ. Sterner RW, Schulz KL (1998) Zooplankton nutrition: re- cent progress and a reality check. Aquatic Ecology, 32, 261– 279. White EP, Ernest SKM, Kerkhoff AJ, Enquist BJ (2007) Relation- ships between body size and abundance in ecology. Trends in Ecology and Evolution, 22, 323–330. Urabe J, Waki N (2009) Mitigation of adverse effects of rising CO2 on a planktonic herbivore by mixed algal diets. Global Change Biology, 15, 523–531. Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Soil and faunal properties of the investigated grasslands. A cluster dendrogram of the environmental correlates is given in the second worksheet. Table S2. Complete inventory of multitrophic interactions of soil food webs. All links consistent with literature-derived guilds are included. One summary listing all papers from which guilds were derived is given in the ﬁrst worksheet. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Soil and faunal properties of the investigated grasslands. A cluster dendrogram of the environmental correlates is given in the second worksheet. Table S2. Complete inventory of multitrophic interactions of soil food webs. All links consistent with literature-derived guilds are included. One summary listing all papers from which guilds were derived is given in the ﬁrst worksheet. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Van Veen JA, Paul EA (1979) Conversion of biovolume measure- ments of soil organisms, grown under various moisture ten- sions, to biomass and their nutrient content. Applied and Environmental Microbiology, 37, 686–692. r 2009 Blackwell Publishing Ltd, Global Change Biology, 15, 2730–2738 S O I L p H , S T O I C H I O M E T R Y A N D A L L O M E T R I C S C A L I N G 2737 Journal of Animal Ecology, 68, 824–835. Mulder C, Cohen JE, Seta¨la¨ H, Bloem J, Breure AM (2005a) Bacterial traits, organism mass, and numerical abundance in the detrital soil food web of Dutch agricultural grasslands. Ecology Letters, 8, 80–90. Siepel H, Van de Bund CF (1988) The inﬂuence of management practices on the microarthropod community of grassland. Pedobiologia, 31, 339–354. Mulder C, De Zwart D, Van Wijnen HJ, Schouten AJ, Breure AM (2003) Observational and simulated evidence of ecological shifts within the soil nematode community of agroecosystems under conventional and organic farming. Functional Ecology, 17, 516–525. Sims JT, Pierzynski GM (2005) Chemistry of phosphorus in soils. In: Chemical Processes in Soils (eds Tabatabai MA, Sparks DL), pp. 151–192. Soil Science Society of America, Madison, WI. Sissingh HA (1971) Analytical technique of the Pw method, used for the assessment of the phosphate status of arable soils in the Netherlands. Plant and Soil, 34, 483–486. Mulder C, Den Hollander H, Schouten T, Rutgers M (2006) Allometry, biocomplexity, and web topology of hundred r 2009 Blackwell Publishing Ltd, Global Change Biology, 15, 2730–2738 2738 C . M U L D E R & J . J . E L S E R Supporting Information Additional Supporting Information may be found in the online version of this article: Voigt W, Perner J, Davis AJ et al. (2003) Trophic levels are differentially sensitive to climate. Ecology, 84, 2444– 2453. Table S1. Soil and faunal properties of the investigated grasslands. A cluster dendrogram of the environmental correlates is given in the second worksheet. Voigt W, Perner J, Jones TH (2007) Using functional groups to investigate community response to environmental changes: two grassland case studies. Global Change Biology, 13, 1710–1721. Table S2. Complete inventory of multitrophic interactions of soil food webs. All links consistent with literature-derived guilds are included. One summary listing all papers from which guilds were derived is given in the ﬁrst worksheet. Wardle DA (2002) Communities and Ecosystems: Linking the Above- ground and Belowground Components. Princeton University Press, Princeton, NJ. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Wardle DA, Verhoef HA, Clarholm M (1998) Trophic relation- ships in the soil microfood-web: predicting the responses to a changing global environment. Global Change Biology, 4, 713– 727.
https://openalex.org/W4283030292	https://cris.unibo.it/bitstream/11585/920693/3/jamaoncol-e221981-s001.pdf	English	null	Association of High Tumor Mutation Burden in Non–Small Cell Lung Cancers With Increased Immune Infiltration and Improved Clinical Outcomes of PD-L1 Blockade Across PD-L1 Expression Levels	JAMA oncology	2,022	cc-by	9,668	Supplemental Online Content Ricciuti B, Wang X, Alessi JV, et al. Association of high tumor mutation burden in non–small cell lung cancers with increased immune infiltration and improved clinical outcomes of PD-L1 blockade across PD-L1 expression levels. JAMA Oncology. Published online June 16, 2022. doi:10.1001/jamaoncol.2022.1981 eAppendix. Supplemental Methods eFigure 1. Statistical Approach for the Determination and Validation of Tumor Mutational Burden Optimal Cut-Point in this Study eFigure 1. Statistical Approach for the Determination and Validation of Tumor Mutational Burden Optimal Cut-Point in this Study eFigure 2. (A) Tumor Mutational Burden of 3591 NSCLCs Which Underwent Next-Generation Sequencing at the Dana-Farber Cancer Institute, Correlation Between TMB With (B) Tobacco History, (C) Number of Tobacco Pack- Years, (D) Tumor Histology, and (E) NSCLC Stage at the Time of Next-Generation Sequencing eFigure 2. (A) Tumor Mutational Burden of 3591 NSCLCs Which Underwent Next-Generation Sequencing at the Dana-Farber Cancer Institute, Correlation Between TMB With (B) Tobacco History, (C) Number of Tobacco Pack- Years, (D) Tumor Histology, and (E) NSCLC Stage at the Time of Next-Generation Sequencing eFigure 2. (A) Tumor Mutational Burden of 3591 NSCLCs Which Underwent Next-Generation Sequencing at the Dana-Farber Cancer Institute, Correlation Between TMB With (B) Tobacco History, (C) Number of Tobacco Pack- Years, (D) Tumor Histology, and (E) NSCLC Stage at the Time of Next-Generation Sequencing eFigure 3. (A) TMB Distributions According to NSCLC Genotype and (B) Q Values for Pairwise Comparisons of Tumor Genotype in Comparison to One Another (Benjamini-Hochberg Procedure) eFigure 4. Box Plot Showing the Distribution of TMB Among Patients With Non-Small Cell Lung Cancer Who Experienced a Complete/Partial Response, Stable Disease, and Progressive Disease as Best Response to PD-(L)1 Inhibition in the MSKCC, DFCI, and SU2C/Mark Foundation Cohorts eFigure 4. Box Plot Showing the Distribution of TMB Among Patients With Non-Small Cell Lung Cancer Who Experienced a Complete/Partial Response, Stable Disease, and Progressive Disease as Best Response to PD-(L)1 Inhibition in the MSKCC, DFCI, and SU2C/Mark Foundation Cohorts eFigure 5. Normalization and Standardization of TMB Distributions Bring the Next-Generation Sequencing (MSK- IMPACT and DFCI OncoPanel) and WES Cohort (SU2C/Mark Foundation) Distributions Into Alignment eFigure 5. Normalization and Standardization of TMB Distributions Bring the Next-Generation Sequencing (MSK- IMPACT and DFCI OncoPanel) and WES Cohort (SU2C/Mark Foundation) Distributions Into Alignment eFigure 6. (A) Unbiased Regression Tree Modeling the Objective Response to PD-(L)1 Blockade as Function of Tumor Mutational Burden Identified an Optimal Threshold of 19.0 Mutations/Megabase That Discriminates Responders Versus Nonresponders in the MSKCC Discovery Cohort, (B) Objective Response Rate, (C) Progression-Free, and (D) Overall Survival to Immunotherapy in Patients With TMB-High (>19 mut/Mb) vs TMB-Low (≤19 mut/Mb) NSCLC in the MSKCC Discovery Cohort eFigure 7. eAppendix. Supplemental Methods (A) Objective Response Rate, (B) Progression-Free, and (C) Overall Survival to Immunotherapy in the DFCI Cohort According to TMB-High (>19.3 mut/Mb/TMB Z-Score >1.16) Versus TMB-Low TMB (≤19.3 mut/Mb/TMB Z-Score ≤1.16), (D) Objective Response Rate, (E) Progression-Free, and (F) Overall Survival to Immunotherapy According to TMB-High (>16.0 mut/Mb/TMB Z-Score >1.16) Versus TMB-Low TMB (≤16.0 mut/Mb/TMB Z-Score ≤1.16) Versus Low Harmonized TMB eFigure 8. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Memorial Sloan Kettering Cancer Center Cohort eFigure 8. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Memorial Sloan Kettering Cancer Center Cohort eFigure 9. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Dana-Farber Cancer Institute Cohort eFigure 9. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Dana-Farber Cancer Institute Cohort eFigure 10. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Stand Up 2 Cancer/Mark Foundation Cohort eFigure 10. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Stand Up 2 Cancer/Mark Foundation Cohort eFigure 11. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Memorial Sloan Kettering Cancer Center Cohort eFigure 12. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Dana-Farber Cancer Institute Cohort © 2022 Ricciuti B et al. JAMA Oncology. eFigure 13. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Stand Up 2 Cancer/Mark Foundation Cohort eFigure 14. (A) Objective Response Rate, (B) Progression-Free Survival, and (C) Overall Survival in Patients With High Versus Low Harmonized TMB in the Pooled Cohort of NSCLCs Treated With PD-(L)1 Blockade From DFCI, MSKCC, and the SU2C/Mark Foundation Dataset, After Excluding EGFR and ALK Positive Cases eFigure 15. Objective Response Rate by Increasing TMB Percentiles Thresholds (Upper Panel), and in Each TMB Decile (Lower Panel) in the Combined Cohort eFigure 16. Overall Survival in Patients at DFCI and MSKCC With Advanced Non-Small Cell Lung Cancer Who Never Received Immunotherapy According to TMB Levels eFigure 17. (A) Response Rate, (B) Progression-Free, and (C) Overall Survival to PD-(L)1 Inhibition According to TMB Levels Among Non-Small Cell Lung Cancers With a PD-L1 TPS <1% eFigure 18. eAppendix. Supplemental Methods Percentage of Tumor, Immune, and Total Cells With PD-L1 Expression Among NSCLC Samples With Low (N = 384) and High (N = 44) TMB Which Also Underwent Multiplexed Immunofluorescence at the DFCI eFigure 19. Linear Correlation Between TMB and CD8+, PD-1+, CD8+ PD-1+, and Foxp3+Cells Intratumorally (A) and at the Tumor-Stroma Interface (B) Among 428 NSCLCs at DFCI Which Underwent Multiplexed Immunofluorescence eFigure 20. Linear Correlation Between TMB and (A) Total CD8+, PD-1+, CD8+ PD-1+, and Foxp3+ Cells, and (B) Linear Correlation Between Tumoral, Immune, and total PD-L1+ Cells Among 428 NSCLCs at DFCI Which Underwent Multiplexed Immunofluorescence eFigure 21. Multiplexed Immunofluorescence for CD8, PD-1, Foxp3, PD-L1, in Three Index Cases With High TMB (A) and Three Index Cases With Low TMB (B) eFigure 22. Deconvolution of RNAseq Data From the NSCLC TCGA Dataset (N=998) Into Tumor-Associated Immune Cells, Showing Cell Types That Are Significantly Enriched in NSCLCs With High vs Low TMB eFigure 23. OncoPrint Plot Showing the Top 20 Mutated Genes in 3168 Nonsquamous NSCLCs With High and Low TMB in the DFCI Genomic Cohort eFigure 24. OncoPrint Plot Showing the Top 20 Mutated Genes in 409 Squamous NSCLCs With High and Low TMB in the DFCI Genomic Cohort eFigure 25. Volcano Plot Showing Gene Mutations Enriched in TMB High Versus TMB Low (A) Nonsquamous (N=3168) and (B) Squamous (N=409) Non-Small Cell Lung Cancers in the DFCI Genomic Cohort eFigure 26. Comutation Patterns Among Lung Non-Squamous Carcinomas With (A) High TMB (N=365) and (B) Low TMB (N=2803) in the DFCI Genomic Cohort eFigure 27. Comutation Patterns Among Lung Squamous Carcinomas With (A) High TMB (N=39) and (B) Low TMB (N=370) in the DFCI Genomic Cohort eFigure 28. Volcano Plot Showing Gene Mutations Enriched in TMB High Versus TMB Low Nonsquamous NSCLC Among 915 Samples Which Underwent NGS at MSKCC eFigure 29. (A) Boxplot Showing Overall Distribution of Nucleotide Conversions and Stacked Barplot Showing Fraction of Conversions in Each NSCLC Sample With TMB High in the DFCI Genomic Cohort (N=404) and (B) Boxplot Showing Overall Distribution of Nucleotide Conversions and Stacked Barplot Showing Fraction of Conversions in Each NSCLC Sample With TMB Low in the DFCI Genomic Cohort (N=3173) eFigure 30. eAppendix. Supplemental Methods (A) Progression-Free and Overall Survival to PD-(L)1 Blockade Among Patients With High (≥1) Versus Low (<1) Transversion/Transition Ratio Among Patients With TMB High and (B) Progression-Free and Overall Survival to PD-(L)1 Blockade Among Patients With High (≥1) Versus Low (<1) Transversion/Transition Ratio Among Patients With TMB Low © 2022 Ricciuti B et al. JAMA Oncology. eFigure 31. Gene Set Enrichment Analysis Showing Prioritized Pathways Upregulated in TMB High Versus TMB Low NSCLC in (A) Lung Adenocarcinoma, and (B) Lung Squamous Carcinoma in the TCGA Cohort eTable 1. Clinicopathologic and Genomic Characteristics of the 3591 NSCLCs Which Underwent Next-Generation Sequencing at the Dana-Farber Cancer Institute eTable 2. Characteristics of Patients With NSCLC Treated With Immune Checkpoint Inhibitors at Memorial Sloan Kettering Cancer Center (MSKCC), Dana-Farber Cancer Institute (DFCI), and Stand Up To Cancer Foundation (SU2C)/Mark Foundation Dataset eTable 3. Tumor Mutational Burden (TMB) Values (in Mutations per Megabase, mut/Mb) at the Memorial Sloan Kettering Cancer Center (MSKCC), Dana-Farber Cancer Institute (DFCI), and Stand up To Cancer/Mark Foundation (SU2C) Cohorts Which Correspond With the Harmonized TMB Z-Score of 1.16 eTable 4. Adjusted Odds Ratio for Response and Adjusted Hazard Ratio for Progression-Free and Overall Survival to PD-(L)1 Inhibition in the MSKCC Cohort After Multiple Imputation to Account for PD-L1 Missingness eTable 5. Adjusted Odds Ratio for Response and Adjusted Hazard Ratio for Progression-Free and Overall Survival to PD-(L)1 Inhibition in the DFCI Cohort After Multiple Imputation to Account for PD-L1 Missingness eTable 6. Adjusted Odds Ratios for Response and Adjusted Hazard Ratio for Progression-Free and Overall Survival to PD-(L)1 Inhibition in the SU2C/Mark Foundation Cohort After Multiple Imputation to Account for PD-L1 Missingness eTable 7. Impact of TMB High Versus Low on Objective Response, Progression-Free, and Overall Survival in a Meta-analysis of the MSKCC and DFCI Cohorts eTable 7. Impact of TMB High Versus Low on Objective Response, Progression-Free, and Overall Survival in a Meta-analysis of the MSKCC and DFCI Cohorts © 2022 Ricciuti B et al. JAMA Oncology. This supplemental material has been provided by the authors to give readers additional information about their work. Memorial Sloan Kettering Cancer Center cohort g Patients at the Memorial Sloan Kettering Cancer Center were included if they had advanced NSCLC which underwent tumor next generation sequencing and if they had also consented to institutional review board-approved protocols. Stand Up to Cancer/Mark Foundation Patients included in the Stand Up to Cancer/Mark Foundation cohort were enrolled if they had advanced NSCLC which was treated with PD-1/PD-L1 inhibitors. Stand Up to Cancer/Mark Foundation Patients included in the Stand Up to Cancer/Mark Foundation cohort were enrolled if they had advanced NSCLC which was treated with PD-1/PD-L1 inhibitors. DFCI OncoPanel and MSK-IMPACT Tumor mutational burden (TMB), defined as the number of somatic, coding, base substitution, and indel mutations per megabase (Mb) of genome examined, was determined using the OncoPanel (Dana-Faber) and MSK-IMPACT (MSKCC) NGS platforms, as previously described1,2. DFCI mutation counts were divided by the number of bases covered in each OncoPanel version: v1, 0.753334 Mb; v2, 0.826167 Mb; and v3, 1.315078 Mb. For MSKCC samples, the mutation count was divided by 0.896665, 1.016478, and 1.139322 Mb for the 341-, 410-, and 468-gene panels, respectively. Tumor genomic profiling and somatic variant calling in the DFCI and MSKCC cohorts Tumor genomic profiling and somatic variants were performed using clinically validated bioinformatics pipelines1,2. Sequence reads were aligned to reference sequence b37 edition from the Human Genome Reference Consortium using bwa (http://bio- bwa.sourceforge.net/bwa.shtml), and further processed using Picard (version 1.90, http://broadinstitute.github.io/picard/) to remove duplicates and Genome Analysis Toolkit (GATK) to perform localized realignment around indel sites. Single nucleotide variants were called using MuTect v1.1.4, insertions and deletions were called using GATK Indelocator, and variants were annotated using Oncotator. In the DFCI cohort, to filter out potential germline variants, the standard pipeline removed SNPs present at >0.1% in Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP) (URL: http://evs.gs.washington.edu/EVS/), present in dbSNP, or present in an in-house panel of normals, but rescues those also present in the COSMIC database. For this study, variants were further filtered by removing variants present at >0.1% in the gnomAD v.2.1.1 database or were annotated as Benign or Likely Benign in the ClinVar database (PMID: 32461654, 29165669). In the MSKCC cohort, patient-matched normal DNA was used to filter out germline variants, as previously described. eReferences This supplemental material has been provided by the authors to give readers additional information about their work. Dana-Farber Cancer Institute cohort Patients at the Dana-Farber Cancer Institute who consented to institutional review board-approved protocols DF/HCC 02-180, 11-104, 13-364, and/or 17-000 which allowed for conducting translational research and tumor next-generation sequencing, respectively, were included. Programmed death ligand 1 immunohistochemistry The PD-L1 tumor proportion score (TPS) was determined by immunohistochemistry using validated anti-PD-L1 antibodies: E1L3N (Cell Signaling Technology, Danvers, MA), 22C3 (Dako North America Inc, Carpinteria, CA), 28-8 (Epitomics Inc, Burlingame, CA), according to local institutional practice. Cell subset analysis from the TCGA dataset To perform cell type enrichment analyses, RNA sequencing data from the LUAD and LUSC TCGA cohort were deconvoluted to estimate cell subsets using the xCell package. xCell estimates the abundance scores of 64 cell types, including adaptive and innate immune cells, hematopoietic progenitors, epithelial cells, and extracellular matrix cells, based on single sample gene set enrichment analysis (ssGSEA) data4. Gene expression values (RSEM V2) were converted into Z-scores and used to compute cell type enrichment scores with the xCellAnalysis function. Statistical significance of differential cell type enrichment between cohorts was estimated with Wilcox Rank Sum test. Cell subtypes examined included: aDC, Adipocytes, Astrocytes, B-cells, Basophils, CD4+ memory T-cells, CD4+ naive T-cells, CD4+ T-cells, CD4+ Tcm, CD4+ Tem, CD8+ naive T-cells, CD8+ T-cells, CD8+ Tcm, CD8+ Tem, cDC, Chondrocytes, Class-switched memory B-cells, CLP, CMP, DC, Endothelial cells, Eosinophils, Epithelial cells, Erythrocytes, Fibroblasts, GMP, Hepatocytes, HSC, iDC, Keratinocytes, Endothelial cells, Macrophages, Macrophages M1, Macrophages M2, Mast cells, Megakaryocytes, Melanocytes, Memory B- cells, MEP, Mesangial cells, Monocytes, MPP, MSC, Endothelial cells, Myocytes, naive B-cells, Neurons, Neutrophils, NK cells, NKT, Osteoblast, pDC, Pericytes, Plasma cells, Platelets, Preadipocytes, pro B-cells, Sebocytes, Skeletal muscle, Smooth muscle, Tgd cells, Th1 cells, Th2 cells, Tregs. Stand Up to Cancer cohort whole exome sequencing DNA was extracted from FFPE tumor specimens and either matched normal whole blood or in cases where this was unavailable, adjacent normal FFPE specimens. Extraction was performed using the Qiagen AllPrep DNA/RNA Mini Kit (cat# 80204). A single aliquot of 150-500 ng input DNA in 100 μl TE buffer was used for library generation. Library preparation was performed using the Kapa HyperPrep kit, and quantification was performed using PicoGreen. Adapter ligation was performed © 2022 Ricciuti B et al. JAMA Oncology. © 2022 Ricciuti B et al. JAMA Oncology. using the TruSeq DNA exome kit from Illumina per manufacturer’s instructions. Sequencing of pooled libraries was performed using a HiSeq2500 with 76 bp paired end reads. Mean target coverage for tumor and normal samples were 150X and 80X, respectively. Tumor mutational burden was defined as the number of non-synonymous base substitutions, indel mutations per megabase of genome examined, using an exome size of 35.8 Mb. using the TruSeq DNA exome kit from Illumina per manufacturer’s instructions. Sequencing of pooled libraries was performed using a HiSeq2500 with 76 bp paired end reads. Mean target coverage for tumor and normal samples were 150X and 80X, respectively. Tumor mutational burden was defined as the number of non-synonymous base substitutions, indel mutations per megabase of genome examined, using an exome size of 35.8 Mb. Multiplexed immunofluorescence (ImmunoProfile) Multiplexed immunofluorescence (mIF) was performed on samples from the Dana-Farber Cancer Institute by staining 5-micron formalin-fixed, paraffin-embedded whole tissue sections with standard, primary antibodies sequentially and paired with a unique fluorochrome followed by staining with nuclear counterstain/4′,6-diamidino-2-phenylindole (DAPI), as previously described5. All samples were stained for PD-L1 (clone E1L3N), PD-1 [clone EPR4877(2)], CD8 (clone 4B11), FOXP3 (clone D608R), Cytokeratin (clone AE1/AE3), and DAPI (nuclear counterstain). Each sample had a single slide stained and scanned at 20x resolution by a Vectra Polaris imaging platform. Regions of Interest (ROIs) were defined for each image, and only these regions were used for quantitative image analysis currently. Within each ROI, InForm Image Analysis software (PerkinElmer/Akoya) was run to phenotype and score cells based on biomarker expression. A custom script quantified the number/percentage of cells which are positive for relevant biomarkers in specific tissue regions. Each ROI was divided into one or more of these defined regions: intra-tumoral (IT), which was defined as the region of the slide consisting of tumor beyond the tumor-stroma interface; tumor-stroma interface (TSI), which was defined as the region within 40 microns to either side of the defined border between tumor and stroma; and total (IT + TSI). Cell count was calculated per ROI and averaged (unweighted) across ROIs, reported as count per millimeter squared +/- standard error. Statistical significance of differential cell type enrichment between groups was estimated with Wilcox Rank Sum test. Tumor mutational burden normalization across different platforms TMB distributions were harmonized between the two platforms by applying a normal transformation followed by standardization to Z-scores, as previously described3. Briefly, power transformations were first used to normalize cohort-specific TMB distributions; second, Tukey’s ladder of powers in the rcompanion package was used to identify the optimal transformation coefficient. Third, the normalized distributions were then standardized into z scores by subtracting the transformed distribution mean and dividing by the standard deviation. Statistical analysis Clinicopathologic data and immunotherapy response data were abstracted from the electronic medical record. Overall response rate was determined by a blinded radiologist using Response Evaluation Criteria In Solid Tumors, version (RECIST) 1.1. Progression-free survival was determined from the start date of immunotherapy until the date of disease progression or death, and overall survival was calculated from the date of diagnosis of advanced NSCLC until the date of death. All p-values are two- sided and confidence intervals are at the 95% level. Overall survival among patients who never received PD-(L)1 inhibition was calculated from the date of the start of systemic therapy for advanced disease, other than immunotherapy. TMB comparisons were computed using the Mann-Whitney U test or the Kruskal-Wallis test, when appropriate. Linear correlations were evaluated using Spearman’s test, and categorical variables were evaluated using Fisher’s exact test. Event-time distributions were estimated using Kaplan-Meier methodology. Log-rank tests were used to test for differences in event-time distributions, and Cox proportional hazards models were fitted to obtain estimates of hazard ratios in univariate and multivariate models. The proportional hazards assumption was assessed with Schoenfeld residuals. All P-values are 2-sided and confidence intervals are at the 95% level, with significance pre-defined to be at <0.05. Multiple comparison correction was performed using the Benjamini- Hochberg procedure. Missing values were handled using inverse probability weighting (IPW) and multiple imputation approaches using R package MICE, as previously described. All statistical analyses were performed using R version 3.6.3. Clinicopathologic data and immunotherapy response data were abstracted from the electronic medical record. Overall response rate was determined by a blinded radiologist using Response Evaluation Criteria In Solid Tumors, version (RECIST) 1.1. Gene expression analysis © 2022 Ricciuti B et al. JAMA Oncology. Gene expression data were downloaded from the Firehose website (TCGA Firehose Legacy version) while somatic mutation data were downloaded from cBioPortal website (cbioportal.org). The RSEM V2 values were used to represent gene expression and genes with counts less than 10 were filtered out. Gene expression profiles were analyzed according to TMB categories. Median expression within each group was used to estimate expression fold-change (FC) to minimize the possible impact of outlier samples. Gene differential expression analyses across TMB subgroups were conducted using R package DESeq2. P- values were corrected for multiple hypothesis testing via false discovery rate (FDR) adjustment. Fold-change threshold of an absolute value greater than 1.5 and FDR adjusted P-value threshold less than 0.1 were utilized to identify differentially expressed genes. Pathway enrichment analyses were conducted separately for up- and down-regulated genes using Molecular Signatures Database (MSigDB) collections. absolute value greater than 1.5 and FDR adjusted P-value threshold less than 0.1 were utilized to identify differentially expressed genes. Pathway enrichment analyses were conducted separately for up- and down-regulated genes using Molecular Signatures Database (MSigDB) collections. © 2022 Ricciuti B et al. JAMA Oncology. TMB cut-off identification and validation To identify and validate TMB thresholds associated with immunotherapy efficacy, an unbiased recursive partitioning algorithm was used to investigate an optimal grouping of TMB with respect to the objective response rate to immune checkpoint inhibition in a discovery cohort comprised of patients from the MSKCC cohort, using the partykit function in R, as previously described6. A 10-fold cross-validation method was used to train and measure the performance of the model using the caret function in R, as previously described7. The threshold identified was validated in two independent cohorts of patients treated with PD-(L)1 blockade in the DFCI and SU2C/Mark Foundation cohorts, following TMB harmonization across platforms, as described above and as previously described3. As PD-L1 tumor proportion score (TPS) is an important predictor for ICI efficacy, we applied both Inverse probability weighting (IPW) and multiple imputation approaches using R package MICE to address the potential selection bias arising from the PD-L1 TPS missingness. Variables used for multiple imputation and to calculate the weights for PD-L1 TPS missingness included sex, age, ECOG performance status, histology, smoking status, and line of therapy for ICI. IPW and multiple imputation were conducted separately in each cohort, and the multivariable analyses results were pooled based on 5 repeated complete imputed datasets. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 1 © 2022 Ricciuti B et al. JAMA Oncology. eFigure 1 g eFigure 1. Statistical Approach for the Determination and Validation of Tumor Mutational Bu Optimal Cut-Point in this Study. MSKCC, Memorial Sloan Kettering Cancer Center; DFCI, Da Farber Cancer Institute; SU2C, Stand Up To Cancer/Mark Foundation; ORR, objective respo eFigure 1. Statistical Approach for the Determination and Validation of Tumor Mutational Burden Optimal Cut-Point in this Study. MSKCC, Memorial Sloan Kettering Cancer Center; DFCI, Dana- Farber Cancer Institute; SU2C, Stand Up To Cancer/Mark Foundation; ORR, objective response rate; PFS, progression-free survival; OS, overall survival. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 2 © 2022 Ricciuti B et al. JAMA Oncology. eFigure 2 eFigure 2. (A) Tumor Mutational Burden of 3591 NSCLCs Which Underwent Next-Generation Sequencing at the Dana-Farber Cancer Institute, Correlation Between TMB With (B) tobacco History (C) Number of Tobacco Pack Years (D) Tumor Histology and (E) NSCLC Stage at th eFigure 2. (A) Tumor Mutational Burden of 3591 NSCLCs Which Underwent Next-Generation Sequencing at the Dana-Farber Cancer Institute, Correlation Between TMB With (B) tobacco History, (C) Number of Tobacco Pack-Years, (D) Tumor Histology, and (E) NSCLC Stage at the Time of Next-Generation Sequencing. eFigure 2. (A) Tumor Mutational Burden of 3591 NSCLCs Which Underwent Next-Generation eFigure 2. (A) Tumor Mutational Burden of 3591 NSCLCs Which Underwent Next-Generation Sequencing at the Dana-Farber Cancer Institute, Correlation Between TMB With (B) tobacco History, (C) Number of Tobacco Pack-Years, (D) Tumor Histology, and (E) NSCLC Stage at the Time of Next-Generation Sequencing. eFigure 2. (A) Tumor Mutational Burden of 3591 NSCLCs Which Underwent Next-Generation Sequencing at the Dana-Farber Cancer Institute, Correlation Between TMB With (B) tobacco History, (C) Number of Tobacco Pack-Years, (D) Tumor Histology, and (E) NSCLC Stage at the Time of Next-Generation Sequencing. eFigure 3 eFigure 3. (A) TMB Distributions According to NSCLC Genotype and (B) Q Values for Pairwise Comparisons of Tumor Genotype in Comparison to One Another (Benjamini- Hochberg Procedure). Samples with concurrent mutations in ≥2 driver mutations were excluded from these panels. eFigure 3 eFigure 3 eFigure 3. (A) TMB Distributions According to NSCLC Genotype and (B) Q eFigure 3. (A) TMB Distributions According to NSCLC Genotype and (B) Q Values for eFigure 3. (A) TMB Distributions According to NSCLC Genotype and (B) Q Values for Pairwise Comparisons of Tumor Genotype in Comparison to One Another (Benjamini- Hochberg Procedure). Samples with concurrent mutations in ≥2 driver mutations were l d d f th l Pairwise Comparisons of Tumor Genotype in Comparison to One Another (Benjamini- Hochberg Procedure). Samples with concurrent mutations in ≥2 driver mutations were excluded from these panels. eFigure 4 eFigure 4. Box Plot Showing the Distribution of TMB Among Patients With Non-Small eFigure 4. Box Plot Showing the Distribution of TMB Among Patients With Non-Small eFigure 4. Box Plot Showing the Distribution of TMB Among Patients Wit Cell Lung Cancer Who Experienced a Complete/Partial Response, Stable Disease, and Progressive Disease as Best Response to PD-(L)1 Inhibition in the MSKCC, DFCI, and SU2C/Mark Foundation Cohorts. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 5 eFigure 5. Normalization and Standardization of TMB Distributions Bring the Next- Generation Sequencing (MSK-IMPACT and DFCI OncoPanel) and WES Cohort (SU2C/Mark Foundation) Distributions Into Alignment. The left side shows the kernel density plot of unadjusted TMB values in each cohort, and the right side shows the transformed density plot of TMB z-scores that demonstrate high overlap. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 6 eFigure 6. (A) Unbiased Regression Tree Modeling the Objective Response to PD-(L)1 Blockade as Function of Tumor Mutational Burden Identified an Optimal Threshold of eFigure 6. (A) Unbiased Regression Tree Modeling the Objective Response to PD-(L)1 Blockade as Function of Tumor Mutational Burden Identified an Optimal Threshold of 19.0 Mutations/Megabase That Discriminates Responders Versus Nonresponders in the MSKCC Discovery Cohort, (B) Objective Response Rate, (C) Progression-Free, and (D) eFigure 6. (A) Unbiased Regression Tree Modeling the Objective Response to PD-(L)1 Blockade as Function of Tumor Mutational Burden Identified an Optimal Threshold of eFigure 6. (A) Unbiased Regression Tree Modeling the Objective Response to PD-(L)1 Blockade as Function of Tumor Mutational Burden Identified an Optimal Threshold of 19.0 Mutations/Megabase That Discriminates Responders Versus Nonresponders in the MSKCC Discovery Cohort, (B) Objective Response Rate, (C) Progression-Free, and (D) Overall Survival to Immunotherapy in Patients With TMB-High (>19 mut/Mb) vs TMB- Low (≤19 mut/Mb) NSCLC in the MSKCC Discovery Cohort. eFigure 6. © 2022 Ricciuti B et al. JAMA Oncology. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 8. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Memorial Sloan Kettering Cancer Center Cohort. eFigure 3 (A) Unbiased Regression Tree Modeling the Objective Response to PD-(L)1 Blockade as Function of Tumor Mutational Burden Identified an Optimal Threshold of 19.0 Mutations/Megabase That Discriminates Responders Versus Nonresponders in the MSKCC Discovery Cohort, (B) Objective Response Rate, (C) Progression-Free, and (D) Overall Survival to Immunotherapy in Patients With TMB-High (>19 mut/Mb) vs TMB- Low (≤19 mut/Mb) NSCLC in the MSKCC Discovery Cohort. eFigure 6. (A) Unbiased Regression Tree Modeling the Objective Response to PD-(L)1 eFigure 6. (A) Unbiased Regression Tree Modeling the Objective Response to PD-(L)1 Blockade as Function of Tumor Mutational Burden Identified an Optimal Threshold of 19.0 Mutations/Megabase That Discriminates Responders Versus Nonresponders in the MSKCC Discovery Cohort, (B) Objective Response Rate, (C) Progression-Free, and (D) Overall Survival to Immunotherapy in Patients With TMB-High (>19 mut/Mb) vs TMB- Low (≤19 mut/Mb) NSCLC in the MSKCC Discovery Cohort. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 7 eFigure 7. (A) Objective Response Rate, (B) Progression-Free, and (C) Overall Survival to Immunotherapy in the DFCI Cohort According to TMB-High (>19.3 mut/Mb/TMB Z-Score >1.16) Versus TMB-Low TMB (≤19.3 mut/Mb/TMB Z-Score ≤1.16), (D) Objective Response Rate, (E) Progression-Free, and (F) Overall Survival to Immunotherapy According to TMB-High (>16.0 mut/Mb/TMB Z-Score >1.16) Versus TMB-Low TMB (≤16.0 mut/Mb/TMB Z-Score ≤1.16) Versus Low Harmonized TMB. eFigure 7. (A) Objective Response Rate, (B) Progression-Free, and (C) Overall eFigure 7. (A) Objective Response Rate, (B) Progression-Free, and (C) Overall eFigure 7. (A) Objective Response Rate, (B) Progression Free, and (C) Overall Survival to Immunotherapy in the DFCI Cohort According to TMB-High (>19.3 mut/Mb/TMB Z-Score >1.16) Versus TMB-Low TMB (≤19.3 mut/Mb/TMB Z-Score ≤1.16), (D) Objective Response Rate, (E) Progression-Free, and (F) Overall Survival to Immunotherapy According to TMB-High (>16.0 mut/Mb/TMB Z-Score >1.16) Versus TMB-Low TMB (≤16.0 mut/Mb/TMB Z-Score ≤1.16) Versus Low Harmonized TMB. Survival to Immunotherapy in the DFCI Cohort According to TMB-High (>19.3 mut/Mb/TMB Z-Score >1.16) Versus TMB-Low TMB (≤19.3 mut/Mb/TMB Z-Score ≤1.16), (D) Objective Response Rate, (E) Progression-Free, and (F) Overall Survival to Immunotherapy According to TMB-High (>16.0 mut/Mb/TMB Z-Score >1.16) Versus TMB-Low TMB (≤16.0 mut/Mb/TMB Z-Score ≤1.16) Versus Low Harmonized TMB. © 2022 Ricciuti B et al. JAMA Oncology. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 8 eFigure 8 eFigure 8. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Memorial Sloan Kettering Cancer Center Cohort. eFigure 8. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Memorial Sloan Kettering Cancer Center Cohort. Memorial Sloan Kettering Cancer Center Cohort. eFigure 9. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Dana- Farber Cancer Institute Cohort. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 9 eFigure 9 eFigure 9. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Dana- Farber Cancer Institute Cohort. eFigure 9. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Dana- Farber Cancer Institute Cohort. eFigure 9. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Dana- Farber Cancer Institute Cohort. eFigure 9. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Dana- Farber Cancer Institute Cohort. eFigure 10 © 2022 Ricciuti B et al. JAMA Oncology. eFigure 10 eFigure 10 eFigure 10. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Stand Up 2 Cancer/Mark Foundation Cohort. ure 10. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the d Up 2 Cancer/Mark Foundation Cohort. eFigure 10. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Stand Up 2 Cancer/Mark Foundation Cohort. eFigure 10. Multivariable Analysis for Response, Progression-Free, and Overall Survival to PD-(L)1 Blockade in the Stand Up 2 Cancer/Mark Foundation Cohort. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 11. eFigure 11. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression- Free, and Overall Survival to PD-(L)1 Blockade in the Memorial Sloan Kettering Cancer Center Cohort. ure 11. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression- , and Overall Survival to PD-(L)1 Blockade in the Memorial Sloan Kettering Cancer Center Cohort. eFigure 11. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression- Free, and Overall Survival to PD-(L)1 Blockade in the Memorial Sloan Kettering Cancer Center Cohort. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 12. eFigure 12. re 12. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression- and Overall Survival to PD-(L)1 Blockade in the Dana-Farber Cancer Institute Cohort. eFigure 12. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression- Free, and Overall Survival to PD-(L)1 Blockade in the Dana-Farber Cancer Institute Cohort. eFigure 12. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression- Free, and Overall Survival to PD-(L)1 Blockade in the Dana-Farber Cancer Institute Cohort. eFigure 12. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression- Free, and Overall Survival to PD-(L)1 Blockade in the Dana-Farber Cancer Institute Cohort. eFigure 12. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression- Free, and Overall Survival to PD-(L)1 Blockade in the Dana-Farber Cancer Institute Cohort. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 13. eFigure 13. ure 13. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression- and Overall Survival to PD-(L)1 Blockade in the Stand Up 2 Cancer/Mark Foundation Cohort. eFigure 13. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression- Free, and Overall Survival to PD-(L)1 Blockade in the Stand Up 2 Cancer/Mark Foundation Cohort. eFigure 13. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression- Free, and Overall Survival to PD-(L)1 Blockade in the Stand Up 2 Cancer/Mark Foundation Cohort. eFigure 13. Multivariable Analysis With Inverse Probability Weighting for PD-L1 Expression for Response, Progression- Free, and Overall Survival to PD-(L)1 Blockade in the Stand Up 2 Cancer/Mark Foundation Cohort. eFigure 14 eFigure 14 eFigure 14. (A) Objective Response Rate, (B) Progression-Free Survival, and (C) Overall Survival in Patients With High Versus Low Harmonized TMB in the Pooled Cohort of NSCLCs Treated With PD-(L)1 Blockade From DFCI, MSKCC, and the SU2C/Mark Foundation Dataset, After Excluding EGFR and ALK Positive Cases. (D) Objective response rate, (E) progression-free survival, and (F) overall survival in patients with high versus low harmonized TMB in the pooled cohort of NSCLCs treated with PD-(L)1 blockade from DFCI, MSKCC, and the SU2C/Mark Foundation dataset, after excluding never smokers. WT, wild type. eFigure 14. (A) Objective Response Rate, (B) Progression-Free Survival, and (C) eFigure 14. (A) Objective Response Rate, (B) Progression-Free Survival, and (C) Overall Survival in Patients With High Versus Low Harmonized TMB in the Pooled Cohort of NSCLCs Treated With PD-(L)1 Blockade From DFCI, MSKCC, and the SU2C/Mark Foundation Dataset, After Excluding EGFR and ALK Positive Cases. (D) Objective response rate, (E) progression-free survival, and (F) overall survival in patients with high versus low harmonized TMB in the pooled cohort of NSCLCs treated with PD-(L)1 blockade from DFCI, MSKCC, and the SU2C/Mark Foundation dataset, after excluding never smokers. WT, wild type. Overall Survival in Patients With High Versus Low Harmonized TMB in the Pooled Cohort of NSCLCs Treated With PD-(L)1 Blockade From DFCI, MSKCC, and the SU2C/Mark Foundation Dataset, After Excluding EGFR and ALK Positive Cases. (D) Objective response rate, (E) progression-free survival, and (F) overall survival in patients with high versus low harmonized TMB in the pooled cohort of NSCLCs treated with PD-(L)1 blockade from DFCI, MSKCC, and the SU2C/Mark Foundation dataset, after excluding never smokers. WT, wild type. © 2022 Ricciuti B et al. JAMA Oncology. © 2022 Ricciuti B et al. JAMA Oncology. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 13. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 15 eFigure 15. (A) Objective Response Rate by Increasing TMB Percentiles Thresholds eFigure 15 eFigure 15. (A) Objective Response Rate by Increasing TMB Percentiles Thresholds (Upper Panel), and in Each TMB Decile (Lower Panel) in the Combined Cohort. P values are comparing each decile with the lowest decile of TMB (0-9th). (B) Forest plot for progression-free and (C) overall survival to PD-(L)1 blockade according to increasing TMB thresholds in the pooled cohort (MSKCC + DFCI + SU2C, N = 1552). (D) Forest plot for progression-free and (E) overall survival to PD-(L)1 blockade in each TMB decile versus the lowest decile, as reference, in the pooled cohort (MSKCC + DFCI + SU2C, N = 1552). © 2022 Ricciuti B et al. JAMA Oncology. eFigure 16 eFigure 16. Overall Survival in Patients at DFCI and MSKCC With Advanced Non- Small Cell Lung Cancer Who Never Received Immunotherapy According to TMB Levels. eFigure 16. Overall Survival in Patients at DFCI and MSKCC With Advance eFigure 16. Overall Survival in Patients at DFCI and MSKCC With Advanced Non- Small Cell Lung Cancer Who Never Received Immunotherapy According to TMB Levels. eFigure 16. Overall Survival in Patients at DFCI and MSKCC With Advanced Non- Small Cell Lung Cancer Who Never Received Immunotherapy According to TMB Levels. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 17 eFigure 17 eFigure 17. (A) Response Rate, (B) Progression-Free, and (C) Overall Survival eFigure 17 eFigure 17. (A) Response Rate, (B) Progression-Free, and (C) Overall Survival to PD- (L)1 Inhibition According to TMB Levels Among Non-Small Cell Lung Cancers With a PD-L1 TPS <1%. (D) Response rate, (E) progression-free, and (F) overall survival to PD-(L)1 inhibition according to TMB levels among non-small cell lung cancers with a PD-L1 TPS of 1-49%. (G) Response rate, (H) progression-free, and (I) overall survival to PD-(L)1 inhibition according to TMB levels among non-small cell lung cancers with a PD-L1 TPS ≥50%. (L)1 Inhibition According to TMB Levels Among Non-Small Cell Lung Cancers With a PD-L1 TPS <1%. (D) Response rate, (E) progression-free, and (F) overall survival to PD-(L)1 inhibition according to TMB levels among non-small cell lung cancers with a PD-L1 TPS of 1-49%. (G) Response rate, (H) progression-free, and (I) overall survival to PD-(L)1 inhibition according to TMB levels among non-small cell lung cancers with a PD-L1 TPS ≥50%. © 2022 Ricciuti B et al. JAMA Oncology. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 13. © 2022 Ricciuti B et al. JAMA Oncology. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 18 eFigure 18. Percentage of Tumor, Immune, and Total Cells With PD-L1 Expression Among NSCLC Samples With Low (N = 384) and High (N = 44) TMB Which Also Underwent Multiplexed Immunofluorescence at the DFCI. eFigure 18. Percentage of Tumor, Immune, and Total Cells With PD-L1 Expression Among NSCLC Samples With Low (N = 384) and High (N = 44) TMB Which Also Underwent Multiplexed Immunofluorescence at the DFCI. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 19 eFigure 19. Linear Correlation Between TMB and CD8+, PD-1+, CD8+ PD-1+, and Foxp3+Cells Intratumorally (A) and at the Tumor-Stroma Interface (B) Among 428 NSCLCs at DFCI Which Underwent Multiplexed Immunofluorescence. eFigure 19. Linear Correlation Between TMB and CD8+, PD-1+, CD8+ PD-1+, and Foxp3+Cells Intratumorally (A) and at the Tumor-Stroma Interface (B) Among 428 NSCLCs at DFCI Which Underwent Multiplexed Immunofluorescence. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 20 eFigure 20. Linear Correlation Between TMB and (A) Total CD8+, PD-1+, CD8+ PD-1+, and Foxp3+ Cells, and (B) Linear Correlation Between Tumoral, Immune, and total PD- L1+ Cells Among 428 NSCLCs at DFCI Which Underwent Multiplexed Immunofluorescence. eFigure 20. Linear Correlation Between TMB and (A) Total CD8+, PD-1+, CD8+ PD-1+, and Foxp3+ Cells, and (B) Linear Correlation Between Tumoral, Immune, and total PD- L1+ Cells Among 428 NSCLCs at DFCI Which Underwent Multiplexed Immunofluorescence. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 21 eFigure 21. Multiplexed Immunofluorescence for CD8, PD-1, Foxp3, PD-L1, in Three Index Cases With High TMB (A) and Three Index Cases With Low TMB (B). eFigure 21. Multiplexed Immunofluorescence for CD8, PD-1, Foxp3, PD-L1, in Three Index Cases With High TMB (A) and Three Index Cases With Low TMB (B). © 2022 Ricciuti B et al. JAMA Oncology. eFigure 22 eFigure 22. Deconvolution of RNAseq Data From the NSCLC TCGA Dataset (N=998) Into Tumor-Associated Immune Cells, Showing Cell Types That Are Significantly Enriched in NSCLCs With High vs Low TMB. eFigure 22 eFigure 22 eFigure 22. Deconvolution of RNAseq Data From the NSCLC TCGA Dataset (N=998) Into Tumor-Associated Immune Cells, Showing Cell Types That Are Significantly Enriched in NSCLCs With High vs Low TMB. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 23 eFigure 23. © 2022 Ricciuti B et al. JAMA Oncology. © 2022 Ricciuti B et al. JAMA Oncology. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 13. OncoPrint Plot Showing the Top 20 Mutated Genes in 3168 Nonsquamous NSCLCs With High and Low TMB in the DFCI Genomic Cohort. eFigure 23 eFigure 23 eFigure 23 eFigure 23. OncoPrint Plot Showing the Top 20 Mutated Genes in 3168 Nonsquamous NSCLCs With High and Low TMB in the DFCI Genomic Cohort. eFigure 24 eFigure 24 eFigure 24. OncoPrint Plot Showing the Top 20 Mutated Genes in 409 Squamous NSCLCs With High and Low TMB in the DFCI Genomic Cohort. eFigure 24. OncoPrint Plot Showing the Top 20 Mutated Genes in 409 Squamous NSCLCs With High and Low TMB in the DFCI Genomic Cohort. eFigure 25 eFigure 25. Volcano Plot Showing Gene Mutations Enriched in TMB High Versus TMB Low (A) Nonsquamous (N=3168) and (B) Squamous (N=409) Non-Small Cell Lung Cancers in the DFCI Genomic Cohort. eFigure 25. Volcano Plot Showing Gene Mutations Enriched in TMB High Versus TMB Low (A) Nonsquamous (N=3168) and (B) Squamous (N=409) Non-Small Cell Lung Cancers in the DFCI Genomic Cohort. Cancers in the DFCI Genomic Cohort. eFigure 26 eFigure 26 eFigure 26. Comutation Patterns Among Lung Non-Squamous Carcinomas With (A) High TMB (N=365) and (B) Low TMB (N=2803) in the DFCI Genomic Cohort. Five random samplings of 365 cases in the TMB low group confirmed significant co- occurrence of KRAS/STK11 and KRAS/KEAP1 mutation, indicating that the lack of co- mutation in these genes in the TMB high group is not influenced by the sample size. eFigure 26. Comutation Patterns Among Lung Non-Squamous Carcinomas With (A) High TMB (N=365) and (B) Low TMB (N=2803) in the DFCI Genomic Cohort. Five random samplings of 365 cases in the TMB low group confirmed significant co- occurrence of KRAS/STK11 and KRAS/KEAP1 mutation, indicating that the lack of co- mutation in these genes in the TMB high group is not influenced by the sample size. eFigure 26. Comutation Patterns Among Lung Non-Squamous Carcinomas With (A) High TMB (N=365) and (B) Low TMB (N=2803) in the DFCI Genomic Cohort. Five random samplings of 365 cases in the TMB low group confirmed significant co- occurrence of KRAS/STK11 and KRAS/KEAP1 mutation, indicating that the lack of co- mutation in these genes in the TMB high group is not influenced by the sample size. eFigure 26. Comutation Patterns Among Lung Non-Squamous Carcinomas With (A) High TMB (N=365) and (B) Low TMB (N=2803) in the DFCI Genomic Cohort. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 13. Five random samplings of 365 cases in the TMB low group confirmed significant co- occurrence of KRAS/STK11 and KRAS/KEAP1 mutation, indicating that the lack of co- mutation in these genes in the TMB high group is not influenced by the sample size. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 27 eFigure 27. Comutation Patterns Among Lung Squamous Carcinomas With (A) High TMB (N=39) and (B) Low TMB (N=370) in the DFCI Genomic Cohort. eFigure 27. Comutation Patterns Among Lung Squamous Carcinomas With (A) High TMB (N=39) and (B) Low TMB (N=370) in the DFCI Genomic Cohort. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 28 eFigure 28. (A) Volcano Plot Showing Gene Mutations Enriched in TMB High Versus TMB Low Nonsquamous NSCLC Among 915 Samples Which Underwent NGS at MSKCC. Co-mutation patterns among lung non-squamous carcinomas with (B) high TMB and (C) low TMB in the MSKCC genomic cohort. eFigure 28 eFigure 28 eFigure 28. (A) Volcano Plot Showing Gene Mutations Enriched in TMB High Versus TMB Low Nonsquamous NSCLC Among 915 Samples Which Underwent NGS at MSKCC. Co-mutation patterns among lung non-squamous carcinomas with (B) high TMB and (C) low TMB in the MSKCC genomic cohort. eFigure 28. (A) Volcano Plot Showing Gene Mutations Enriched in TMB High Versus TMB Low Nonsquamous NSCLC Among 915 Samples Which Underwent NGS at MSKCC. Co-mutation patterns among lung non-squamous carcinomas with (B) high TMB and (C) low TMB in the MSKCC genomic cohort. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 29 eFigure 29 eFigure 29. (A) Boxplot Showing Overall Distribution of Nucleotide Conv Stacked Barplot Showing Fraction of Conversions in Each NSCLC Samp High in the DFCI Genomic Cohort (N=404) and (B) Boxplot Showing Ov eFigure 29. (A) Boxplot Showing Overall Distribution of Nucleotide Conversions and Stacked Barplot Showing Fraction of Conversions in Each NSCLC Sample With TMB High in the DFCI Genomic Cohort (N=404) and (B) Boxplot Showing Overall Distribution of Nucleotide Conversions and Stacked Barplot Showing Fraction of Conversions in Each NSCLC Sample With TMB Low in the DFCI Genomic Cohort (N=3173). eFigure 29. (A) Boxplot Showing Overall Distribution of Nucleotide Conv eFigure 29. eFigure 13. (A) Boxplot Showing Overall Distribution of Nucleotide Conversions and Stacked Barplot Showing Fraction of Conversions in Each NSCLC Sample With TMB High in the DFCI Genomic Cohort (N=404) and (B) Boxplot Showing Overall Distribution of Nucleotide Conversions and Stacked Barplot Showing Fraction of Conversions in Each NSCLC Sample With TMB Low in the DFCI Genomic Cohort (N=3173). © 2022 Ricciuti B et al. JAMA Oncology. eFigure 30 eFigure 30 eFigure 30. (A) Progression-Free and Overall Survival to PD-(L)1 Blockade Among Patients With High (≥1) Versus Low (<1) Transversion/Transition Ratio Among Patients With TMB High and (B) Progression-Free and Overall Survival to PD-(L)1 Blockade Among Patients With High (≥1) Versus Low (<1) Transversion/Transition Ratio Among Patients With TMB Low. eFigure 30. (A) Progression-Free and Overall Survival to PD-(L)1 Blockade Among Patients With High (≥1) Versus Low (<1) Transversion/Transition Ratio Among Patients eFigure 30. (A) Progression-Free and Overall Survival to PD-(L)1 Blockade Among Patients With High (≥1) Versus Low (<1) Transversion/Transition Ratio Among Patients With TMB High and (B) Progression-Free and Overall Survival to PD-(L)1 Blockade Among Patients With High (≥1) Versus Low (<1) Transversion/Transition Ratio Among Patients With TMB Low. © 2022 Ricciuti B et al. JAMA Oncology. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 31 eFigure 31. Gene Set Enrichment Analysis Showing Prioritized Pathways Upregulated in TMB High Versus TMB Low NSCLC in (A) Lung Adenocarcinoma, and (B) Lung Squamous Carcinoma in the TCGA Cohort. eFigure 31. Gene Set Enrichment Analysis Showing Prioritized Pathways Upregulated in TMB High Versus TMB Low NSCLC in (A) Lung Adenocarcinoma, and (B) Lung Squamous Carcinoma in the TCGA Cohort. eFigure 31. Gene Set Enrichment Analysis Showing Prioritized Pathways Upregulated in TMB High Versus TMB Low NSCLC in (A) Lung Adenocarcinoma, and (B) Lung Squamous Carcinoma in the TCGA Cohort. © 2022 Ricciuti B et al. JAMA Oncology. eTable 1. Clinicopathologic and Genomic Characteristics of the 3591 NSCLCs Which Underwent Next-Generation Sequencing at the Dana-Farber Cancer Institute. Clinical Characteristic N = 3591 (%) Age, median (range) 66 (18-99) Sex Female Male 2111 (58.8) 1480 (41.2) Smoking Status Never Former Current Unknown 774 (21.7) 2078 (58.1) 723 (20.2) 16 Histology Nonsquamous Squamous 3181 (88.6) 410 (11.4) Stage at NGS I II III IV 862 (24.0) 280 (7.8) 522 (14.5) 1927 (53.7) PD-L1 TPS <1% 1-49% ≥50% Not assessed 575 (34.3) 607 (36.1) 498 (29.6) 1911 Tumor purity (%), median (range) 40 (20-100) Genotype No known driver KRAS EGFR MET BRAF ALK HER2 RET ROS1 1242 (34.6) 1141 (31.8) 666 (18.5) 170 (4.7) 154 (4.3) 84 (2.3) 70 (2.0) 38 (1.1) 26 (0.7) PD-L1, programmed death ligand 1 TPS tumor proportion score eTable 1. Clinicopathologic and Genomic Chara Underwent Next-Generation Sequencing at the Clinical Characteristic N = 3591 (%) Age, median (range) 66 (18-99) Sex Female Male 2111 (58.8) 1480 (41.2) Smoking Status Never Former Current Unknown 774 (21.7) 2078 (58.1) 723 (20.2) 16 Histology Nonsquamous Squamous 3181 (88.6) 410 (11.4) Stage at NGS I II III IV 862 (24.0) 280 (7.8) 522 (14.5) 1927 (53.7) PD-L1 TPS <1% 1-49% ≥50% Not assessed 575 (34.3) 607 (36.1) 498 (29.6) 1911 Tumor purity (%), median (range) 40 (20-100) Genotype No known driver KRAS EGFR MET BRAF ALK HER2 RET ROS1 1242 (34.6) 1141 (31.8) 666 (18.5) 170 (4.7) 154 (4.3) 84 (2.3) 70 (2.0) 38 (1.1) 26 (0.7) PD-L1, programmed death ligand 1 TPS, tumor proportion score NGS, next generation sequencing eTable 1. Clinicopathologic and Genomic Characteristics of the 3591 NSCLCs Which Underwent Next-Generation Sequencing at the Dana-Farber Cancer Institute. eTable 1. Clinicopathologic and Genomic Characteristics of the 3591 NSCLCs Which Underwent Next-Generation Sequencing at the Dana-Farber Cancer Institute. eTable 2. © 2022 Ricciuti B et al. JAMA Oncology. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 31 Characteristics of Patients With NSCLC Treated With Immune Checkpoint Inhibitors at Memorial Sloan Kettering Cancer Center (MSKCC), Dana-Farber Cancer Institute (DFCI), and Stand Up To Cancer Foundation (SU2C)/Mark Foundation Dataset. Clinical Characteristic MSKCC N = 672 (%) DFCI N = 714 (%) SU2C N = 166 (%) Age, median (range) 67 (22-92) 66 (25-92) 63.5 (39-86) Sex Male Female 317 (47.2) 355 (52.8) 325 (45.5) 389 (54.5) 80 (48.2) 86 (51.8) Smoking status Current/Former Never 569 (84.7) 103 (15.3) 605 (84.7) 109 (15.3) 148 (89.2) 18 (10.8) Histology Non-squamous Squamous 586 (87.2) 86 (12.8) 629 (88.1) 85 (11.9) 132 (79.5) 34 (20.5) Oncogenic driver mutation KRAS EGFR Other None identified 234 (34.8) 67 (10.0) 48 (7.1) 323 (48.1) 249 (34.9) 71 (9.9) 85 (11.9) 309 (43.3) 36 (21.7) 5 (3.0) 6 (3.6) 119 (71.7) ECOG performance status 0-1 ≥2 Not available 622 (92.6) 50 (7.4) 563 (79.6) 144 (20.4) 7 0 (0.0%) 0 (0.0%) 166 Line of therapy 1st ≥2nd 216 (32.1) 456 (67.9) 244 (34.2) 470 (65.8) 71 (42.8) 95 (57.2) PD-L1 expression <1% 1-49% ≥50% Not assessed 169 (40.9) 78 (18.9) 166 (40.2) 259 87 (17.0) 171 (33.4) 254 (49.6) 202 25 (26.0) 38 (39.6) 33 (34.4) 70 ECOG, Eastern Cooperative Oncology Group NSCLC NOS, non-small cell lung cancer not otherwise specified PD-L1, programmed death ligand 1 TMB i l b d © 2022 Ricciuti B et al. JAMA Oncology. eTable 3. Tumor Mutational Burden (TMB) Values (in Mutations per Megabase, mut/Mb) at the Memorial Sloan Kettering Cancer Center (MSKCC), Dana-Farber Cancer Institute (DFCI), and Stand up To Cancer/Mark Foundation (SU2C) Cohorts Which Correspond With the Harmonized TMB Z-Score of 1.16. NGS, next-generation sequencing; WES, whole-exome sequencing MSKCC (NGS) DFCI (NGS) SU2C (WES) TMB (mut/Mb) 19.0 19.3 16.0 Percentile within cohort 89th 90th 88th eTable 3. Tumor Mutational Burden (TMB) Values (in Mutations per Megabase, mut/Mb) at the Memorial Sloan Kettering Cancer Center (MSKCC), Dana-Farber Cancer Institute (DFCI), and Stand up To Cancer/Mark Foundation (SU2C) Cohorts Which Correspond With the Harmonized TMB Z-Score of 1.16. eTable 3. Tumor Mutational Burden (TMB) Values (in Mutations per Megabase, mut/Mb) at the Memorial Sloan Kettering Cancer Center (MSKCC), Dana-Farber Cancer Institute (DFCI), and Stand up To Cancer/Mark Foundation (SU2C) Cohorts Which Correspond With the Harmonized TMB Z-Score of 1.16. © 2022 Ricciuti B et al. JAMA Oncology. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 31 NGS, next-generation sequencing; WES, whole-exome sequencing MSKCC (NGS) DFCI (NGS) SU2C (WES) TMB (mut/Mb) 19.0 19.3 16.0 Percentile within cohort 89th 90th 88th MSKCC (NGS) DFCI (NGS) SU2C (WES) TMB (mut/Mb) 19.0 19.3 16.0 Percentile within cohort 89th 90th 88th NGS, next-generation sequencing; WES, whole-exome sequencing eTable 4. Adjusted Odds Ratio for Response and Adjusted Hazard Ratio for Progression-Free and Overall Survival to PD-(L)1 Inhibition in the MSKCC Cohort After Multiple Imputation to Account for PD-L1 Missingness. eTable 4. Adjusted Odds Ratio for Response and Adjusted Hazard Ratio for Progression-Free and Overall Survival to PD-(L)1 Inhibition in the MSKCC Cohort After Multiple Imputation to Account for PD-L1 Missingness. Objective response Variable Odds Ratio [95% CI] P value Age 0.99 [0.97-1.01] 0.54 Sex (male vs female) 0.82 [0.53-1.24] 0.35 Smoking history (ever vs never) 2.10 [1.04-4.21] 0.036 Histology (squamous vs non-squamous) 1.27 [0.70-2.31] 0.42 ECOG PS (0-1 vs ≥2) 1.55 [0.61-3.94] 0.35 Line of ICI (1st vs ≥2nd) 0.71 [0.45-1.12] 0.14 PD-L1 TPS (≥50% vs <1%) 4.01 [2.33-6.89] <0.0001 PD-L1 TPS (1-49% vs <1%) 1.95 [1.01-3.79] 0.049 TMB (high vs low) 3.08 [1.74-5.43] 0.0001 Progression-free survival Variable Hazard Ratio [95% CI] P value Age 1.00 [0.99-1.01] 0.45 Sex (male vs female) 1.11 [0.94-1.31] 0.21 Smoking history (ever vs never) 0.69 [0.55-0.87] 0.001 Histology (squamous vs non-squamous) 1.04 [0.81-1.33] 0.73 ECOG PS (0-1 vs ≥2) 0.88 [0.65-1.19] 0.41 Line of ICI (1st vs ≥2nd) 1.24 [1.02-1.50] 0.03 PD-L1 TPS (≥50% vs <1%) 0.69 [0.56-0.84] <0.001 PD-L1 TPS (1-49% vs <1%) 0.92 [0.69-1.21] 0.57 TMB (high vs low) 0.40 [0.29-0.55] 0.0001 Overall survival Variable Hazard Ratio [95% CI] P value Age 1.01 [1.00-1.02] 0.005 Sex (male vs female) 1.10 [0.91-1.33] 0.29 Smoking history (ever vs never) 0.94 [0.73-1.20] 0.63 Histology (squamous vs non-squamous) 1.23 [0.95-1.60] 0.11 ECOG PS (0-1 vs ≥2) 0.73 [0.53-1.00] 0.05 Line of ICI (1st vs ≥2nd) 1.31 [1.06-1.62] 0.01 PD-L1 TPS (≥50% vs <1% 0.69 [0.54-0.87] 0.002 PD-L1 TPS (1-49% vs <1%) 0.88 [0.67-1.17] 0.41 TMB (high vs low) 0.51 [0.35-0.71] 0.0001 © 2022 Ricciuti B et al. JAMA Oncology. eTable 5. Adjusted Odds Ratio for Response and Adjusted Hazard Ratio for Progression-Free and Overall Survival to PD-(L)1 Inhibition in the DFCI Cohort After Multiple Imputation to Account for PD-L1 Missingness. eTable 5. eFigure 31 Adjusted Odds Ratio for Response and Adjusted Hazard Ratio for Progression-Free and Overall Survival to PD-(L)1 Inhibition in the DFCI Cohort After Multiple Imputation to Account for PD-L1 Missingness. Objective response Variable Odds Ratio [95% CI] P value Age 1.01 [0.99-1.03] 0.16 Sex (male vs female) 0.72 [0.50-1.06] 0.09 Smoking history (ever vs never) 2.09 [1.11-3.94] 0.02 Histology (squamous vs non-squamous) 1.39 [0.79-2.44] 0.24 ECOG PS (0-1 vs ≥2) 3.07 [1.71-5.51] <0.001 Line of ICI (1st vs ≥2nd) 0.84 [0.54-1.29] 0.43 PD-L1 TPS (≥50% vs <1%) 1.97 [1.03-3.76] 0.04 PD-L1 TPS (1-49% vs <1%) 0.99 [0.51-1.95] 0.99 TMB (high vs low) 3.20 [1.85-5.52] <0.0001 Progression-free survival Variable Hazard Ratio [95% CI] P value Age 0.99 [0.98-1.00] 0.07 Sex (male vs female) 1.22 [1.04-1.44] 0.01 Smoking history (ever vs never) 0.74 [0.59-0.92] 0.009 Histology (squamous vs non-squamous) 0.93 [0.73-1.19] 0.59 ECOG PS (0-1 vs ≥2) 0.51 [0.42-0.63] <0.0001 Line of ICI (1st vs ≥2nd) 1.14 [0.93-1.39] 0.20 PD-L1 TPS (≥50% vs <1%) 0.63 [0.44-0.92] 0.036 PD-L1 TPS (1-49% vs <1%) 0.91 [0.69-1.20] 0.52 TMB (high vs low) 0.48 [0.36-0.65] <0.0001 Overall survival Variable Hazard Ratio [95% CI] P value Age 1.00 [0.99-1.01] 0.80 Sex (male vs female) 1.29 [1.06-1.51] 0.01 Smoking history (ever vs never) 0.87 [0.69-1.11] 0.29 Histology (squamous vs non-squamous) 1.01 [0.77-1.32] 0.92 ECOG PS (0-1 vs ≥2) 0.37 [0.29-0.45] <0.0001 Line of ICI (1st vs ≥2nd) 1.37 [1.08-1.73] 0.01 PD-L1 TPS (≥50% vs <1%) 0.73 [0.48-1.12] 0.19 PD-L1 TPS (1-49% vs <1%) 1.04 [0.76-1.43] 0.78 TMB (high vs low) 0.51 [0.37-0.72] 0.0001 © 2022 Ricciuti B et al. JAMA Oncology. eTable 6. Adjusted Odds Ratios for Response and Adjusted Hazard Ratio for Progression-Free and Overall Survival to PD-(L)1 Inhibition in the SU2C/Mark Foundation Cohort After Multiple Imputation to Account for PD-L1 Missingness. eTable 6. Adjusted Odds Ratios for Response and Adjusted Hazard Ratio for Progression-Free and Overall Survival to PD-(L)1 Inhibition in the SU2C/Mark Foundation Cohort After Multiple Imputation to Account for PD-L1 Missingness. eFigure 31 Objective response Variable Odds Ratio [95% CI] P value Age 1.02 [0.98-1.06] 0.28 Sex (male vs female) 0.78 [0.37-1.63] 0.51 Smoking history (ever vs never) 1.13 [0.34-3.72] 0.84 Histology (squamous vs non-squamous) 2.01 [0.77-5.26] 0.15 Line of ICI (1st vs ≥2nd) 0.48 [0.23-1.01] 0.06 PD-L1 TPS (≥50% vs <1%) 2.94 [0.94-9.18] 0.07 PD-L1 TPS (1-49% vs <1%) 1.34 [0.49-3.64] 0.56 TMB (high vs low) 25.8 [5.21-128.02] 0.0001 Progression-free survival Variable Hazard Ratio [95% CI] P value Age 0.99 [0.97-1.01] 0.48 Sex (male vs female) 1.22 [0.83-1.78] 0.29 Smoking history (never vs ever) 1.20 [0.67-2.15] 0.54 Histology (squamous vs non-squamous) 0.99 [0.60-1.63] 0.97 Line of ICI (1st vs ≥2nd) 1.47 [0.99-2.19] 0.06 PD-L1 TPS (≥50% vs <1%) 0.61 [0.29-1.28] 0.22 PD-L1 TPS (1-49% vs <1%) 0.74 [0.44-1.26] 0.28 TMB (high vs low) 0.15 [0.06-0.35] <0.0001 Overall survival Variable Hazard Ratio [95% CI] P value Age 0.99 [0.97-1.02] 0.79 Sex (male vs female) 1.13 [0.72-1.76] 0.59 Smoking history (never vs ever) 1.92 [0.93-3.97] 0.08 Histology (squamous vs non-squamous) 0.86 [0.48-1.56] 0.63 Line of ICI (1st vs ≥2nd) 1.72 [1.06-2.78] 0.03 PD-L1 TPS (≥50% vs <1%) 0.56 [0.28-1.10] 0.11 PD-L1 TPS (1-49% vs <1%) 0.71 [0.40-1.23] 0.23 TMB (high vs low) 0.13 [0.04-0.43] 0.001 © 2022 Ricciuti B et al. JAMA Oncology. eTable 7. Impact of TMB High Versus Low on Objective Response, Progression-Free, and Overall Survival in a Meta-analysis of the MSKCC and DFCI Cohorts. SU2C cohort was excluded as ECOG PS was not available for this cohort. © 2022 Ricciuti B et al. JAMA Oncology. © 2022 Ricciuti B et al. JAMA Oncology. eFigure 31 Objective response (TMB high versus TMB low) Model Adjusted odds Ratio [95% CI] P value Complete dataset 2.90 [1.78-4.70] <0.0001 Inverse probability weighting 2.93 [1.97-4.37] <0.0001 Multiple imputation 3.14 [2.12-4.66] <0.0001 Progression-free survival (TMB high versus TMB low) Model Adjusted Hazard Ratio [95% CI] P value Complete dataset 0.47 [0.36-0.61] <0.0001 Inverse probability weighting 0.47 [0.35-0.62] <0.0001 Multiple imputation 0.44 [0.36-0.55] <0.0001 Overall survival (TMB high versus TMB low) Model Adjusted Hazard Ratio [95% CI] P value Complete dataset 0.59 [0.44-0.79] 0.0005 Inverse probability weighting 0.59 [0.43-0.82] 0.0014 Multiple imputation 0.51 [0.40-0.65] <0.0001 Objective response (TMB high versus TMB low) Model Adjusted odds Ratio [95% CI] P value Complete dataset 2.90 [1.78-4.70] <0.0001 Inverse probability weighting 2.93 [1.97-4.37] <0.0001 Multiple imputation 3.14 [2.12-4.66] <0.0001 Progression-free survival (TMB high versus TMB low) Model Adjusted Hazard Ratio [95% CI] P value Complete dataset 0.47 [0.36-0.61] <0.0001 Inverse probability weighting 0.47 [0.35-0.62] <0.0001 Multiple imputation 0.44 [0.36-0.55] <0.0001 Overall survival (TMB high versus TMB low) Model Adjusted Hazard Ratio [95% CI] P value Complete dataset 0.59 [0.44-0.79] 0.0005 Inverse probability weighting 0.59 [0.43-0.82] 0.0014 Multiple imputation 0.51 [0.40-0.65] <0.0001 Objective response (TMB high versus TMB low) eReferences eReferences eReferences 1. Garcia, E. P. et al. Validation of OncoPanel: A Targeted Next-Generation Sequencing Detection of Somatic Variants in Cancer. Arch. Pathol. Lab. Med. 141, 751–758 (2017 2. Cheng, D. T. et al. Memorial sloan kettering-integrated mutation profiling of actionab targets (MSK-IMPACT): A hybridization capture-based next-generation sequencing c for solid tumor molecular oncology. J. Mol. Diagnostics (2015) doi:10.1016/j.jmoldx. 3. Vokes, N. I. et al. Harmonization of Tumor Mutational Burden Quantification and Ass Response to Immune Checkpoint Blockade in Non–Small-Cell Lung Cancer. JCO Pre (2019) doi:10.1200/po.19.00171. 4. Aran, D., Hu, Z. & Butte, A. J. xCell: Digitally portraying the tissue cellular heterogen landscape. Genome Biol. (2017) doi:10.1186/s13059-017-1349-1. 5. Bogusz, A. M. et al. Quantitative Immunofluorescence Reveals the Signature of Activ Receptor Signaling in Diffuse Large B-cell Lymphoma. Clin. Cancer Res. 18, 6122–6 6. Hothorn, T., Hornik, K. & Zeileis, A. Unbiased recursive partitioning: A conditional in framework. J. Comput. Graph. Stat. (2006) doi:10.1198/106186006X133933. 7. Kuhn, M. Building Predictive Models in R Using the caret Package. J. Stat. Softw. 28, 1. Garcia, E. P. et al. Validation of OncoPanel: A Targeted Next-Generation Sequencing Assay for the Detection of Somatic Variants in Cancer. Arch. Pathol. Lab. Med. 141, 751–758 (2017). 2. Cheng, D. T. et al. Memorial sloan kettering-integrated mutation profiling of actionable cancer targets (MSK-IMPACT): A hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology. J. Mol. Diagnostics (2015) doi:10.1016/j.jmoldx.2014.12.006. 3. Vokes, N. I. et al. Harmonization of Tumor Mutational Burden Quantification and Association With Response to Immune Checkpoint Blockade in Non–Small-Cell Lung Cancer. JCO Precis. Oncol. (2019) doi:10.1200/po.19.00171. 4. Aran, D., Hu, Z. & Butte, A. J. xCell: Digitally portraying the tissue cellular heterogeneity landscape. Genome Biol. (2017) doi:10.1186/s13059-017-1349-1. 4. Aran, D., Hu, Z. & Butte, A. J. xCell: Digitally portraying the tissue ce landscape. Genome Biol. (2017) doi:10.1186/s13059-017-1349-1. p 5. Bogusz, A. M. et al. Quantitative Immunofluorescence Reveals the Signature of Active B-cell Receptor Signaling in Diffuse Large B-cell Lymphoma. Clin. Cancer Res. 18, 6122–6135 (2012). 6. Hothorn, T., Hornik, K. & Zeileis, A. Unbiased recursive partitioning: A conditional inference framework. J. Comput. Graph. Stat. (2006) doi:10.1198/106186006X133933. p p 7. Kuhn, M. Building Predictive Models in R Using the caret Package. J. Stat. Softw. 28, 1–26 (2008).
https://openalex.org/W3168747565	https://revistas.ucm.es/index.php/ARTE/article/download/75914/4564456557373	es		Ready made: desdibujando el pensamiento	Arteterapia	2,021	cc-by	83	Ready made: desdibujando el pensamiento Técnica 2020 [dibujo en lápiz sobre goma de borrar]. Reflexión Objeto de borrar anulado por un dibujo. Dibujo automático, dibujo imborrable, objeto dibujo: poema objeto. Autora Eva C. Mesas Escobar. Bellas Artes y Arteterapeuta por la Universidad de Murcia. Miembro de MURARTT (Asociación profesional de arteterapeutas de la Región de Murcia). Arteterapia: papeles de arteterapia y educación artística para la integración social. Monográfico: Las miradas del arte y el arteterapia en tiempos de la Covid19. ISSN-e: 1988-8309 https://dx.doi.org/10.5209/arte.75914
https://openalex.org/W2579567829	https://rua.ua.es/dspace/bitstream/10045/61754/1/CultCuid_46_08.pdf	Spanish; Castilian	null	Cuidados enfermeros en la España del siglo XVII: el manual de enfermería de Simón López	Cultura de los cuidados	2,016	cc-by	4,438	Cultura de los Cuidados Cultura de los Cuidados Cuidados enfermeros en la España del siglo XVII: el manual de enfermería de Simón López Nursing care during seventeenth century in Spain: Nursing manual belonging to Simon Lopez Cuidados de enfermagem no século XVII na Espanha: manual de enfermagem Simon Lopez Sonia Sánchez Aragó Enfermera en Unidad de Cirugía del Hospital Nuestra Señora de Gracia, Máster en bioética y bioderecho. Cómo citar este artículo en edición digital: Sánchez Aragó, S. (2016). Cuidados enfermeros en la España del siglo XVII: el manual de enfermería de Simón López. Cultura de los Cuidados (Edición digital), 20( 46). Disponible en: < http://dx.doi.org/10.14198/cuid.2016.46.08> Correspondencia: Calle Perpetuo Socorro 4,5A. CP.50006. Zaragoza. Correo electrónico: sonits_@hotmail.com Recibido: 08/02/2016; Aceptado: 22/08/2016 Correspondencia: Calle Perpetuo Socorro 4,5A. CP.50006. Zaragoza. Correo electrónico: sonits_@hotmail.com Recibido: 08/02/2016; Aceptado: 22/08/2016 Correspondencia: Calle Perpetuo Socorro 4,5A. CP.50006. Zaragoza. Correo electrónico: sonits_@hotmail.com Recibido: 08/02/2016; Aceptado: 22/08/2016 and roles played these caregivers , recognition and social acceptance of trade among others. Methods: To carry out this work has used the work Directory Nurses , whose full title is Direct Nurses and architect of charity to cure diseases of the body. Results: For its purpose and content, the work Directory Nurses , Simon Lopez, is a key to know the Spanish Nursing seventeenth cen tury. Conclusions: The work offers a compre hensive view of the person taking into account the biological , psychological and social sphere, along with the human aspect of care. ABSTRACT Keywords: history of nursing, cares, nurs ing, health. Objective: Throughout the different his torical periods , care has been studied nursing. Speaking of the history of Spanish Nursing, it is generally accepted by most researchers as a key date institutionalization and profession alization of the last century , and more specifi cally that of 1857. In this case , it is intended to expose the origins of a trade, as was the nurse or care was exercised in hospitals in the seven teenth century in Spain , see how were the tasks 3er Cuatrimestre 2016 • Año XX - N.° 46 Cultura de los Cuidados Cultura de los Cuidados de Enfermeros y artífice de obras de caridad para curar las enfermedades del cuerpo. Neste caso, pretende-se expor o origens de um comércio, como era a enfermeira ou o cuida do foi exercido em hospitais no século XVII na Espanha, ver como eram as tarefas e papéis desempenhados esses cuidadores, o reconhe cimento ea aceitação social do comércio, entre outros. Neste caso, pretende-se expor o origens de um comércio, como era a enfermeira ou o cuida do foi exercido em hospitais no século XVII na Espanha, ver como eram as tarefas e papéis desempenhados esses cuidadores, o reconhe cimento ea aceitação social do comércio, entre outros. Resultados: Por su finalidad y contenido, la obra Directorio de Enfermeros, de Simón López, es una pieza clave para conocer la En fermería español del siglo XVII. Conclusión: La obra ofrece una visión in tegral de la persona teniendo en cuenta la esfe ra biológica, psicológica y social, junto con la vertiente humana de los cuidados. Metodologia: Para realizar este trabalho utilizou os enfermeiros Diretório de trabalho, cujo título completo é Nurses directos e arqui teto da caridade para curar doenças do corpo Palabras clave: historia de enfermería, cui dados, enfermería, salud. Resultados: Para seu propósito e conteú do, as enfermeiras Diretório trabalho, Simon Lopez, é a chave para conhecer a Enfermagem do século XVII espanhol. INTRODUCCIÓN A lo largo de las distintas épocas históri cas, el cuidado ha sido objeto de estudio de la enfermería, ha evolucionado a través del saber filosófico, los avances científicos y el desarro llo, dando lugar a un cambio en los roles de nuestra disciplina (Siles González,2004). Conclusão: O trabalho oferece uma visão abrangente da pessoa tendo em conta a esfe ra biológica, psicológica e social, juntamente com o lado humano de cuidados. Palavras-chave: história da enfermagem , cuidado, nutrição , saúde. Al hablar de la historia de la Enferme ría española, se suele aceptar por la mayoría de los investigadores como fecha clave de su institucionalización y profesionalización la de mediados del siglo pasado, y más concreta mente la del año 1857.Es en ese año cuando se promulgó la Ley de Bases para la Instruc ción Pública, conocida como Ley Moyano por el nombre del ministro que la auspició. Esta ley estableció la creación de los denominados Practicantes, que agrupaban a los anteriores, más Callistas, Dentistas (hasta 1877) y asisten tes a partos (Gonzalez,2000). RESUMO Objectivo: Ao longo dos vários períodos históricos, o cuidado tem sido estudado enfer magem. Falando da história da Enfermagem espanhola, é geralmente aceite pela maioria dos pesquisadores como uma institucionaliza ção chave de data e profissionalização do sécu lo passado, e mais especificamente a de 1857. 3er Cuatrimestre 2016 • Año XX - N.° 46 86 3er Cuatrimestre 2016 • Año XX - N.° 46 Cultura de los Cuidados Cultura de los Cuidados rar, en la medida de sus posibilidades, las nue vas técnicas y conocimientos que el desarrollo científico de la época proporcionaba (García Martínez, 2004). cuyo título completo es Directo de Enfermeros y artífice de obras de caridad para curar las en fermedades del cuerpo (Hernández Cortina, 2004). Los datos sobre el autor de la obra son esca sos. Por las referencias que se han encontrado, es una persona castellana de origen, enfermero y barbero de profesión que trabajó ejerciendo la enfermería durante más de dos décadas en distintos hospitales castellanos (Burgos More no,2009). Se deduce que es un hombre culto, y que dominaba los clásicos en latín y castellano y los principales autores de su época; ya que en su única obra cita a unos cincuenta (García- Carpintero Blas, 2007). El siglo XVII fue testigo de los primero intentos de explicar la mecánica del cuerpo Humano (Hernández Martín,1996). Jerome Fabricus 1619 fue el primer embriólogo el de sarrollo de método clínico fue contribución de Thomas Sydeham 1624, propuso nuevos métodos en el cuidado de los enfermos, es el propulsor de sustituir el ambiente cargado de la habitación por aire fresco, se descubrió también la utilidad de la autopsia. (Martínez, 2004) El primer borrador se fechó en 1651 y en cuanto a la fecha exacta de su terminación, en sus páginas prologales figura la de 1668. Se di vide en ocho tratados y 150 capítulos, consta de un total de 565 páginas de texto (junto con otras 22 de índices y aprobaciones) y segura mente quedó manuscrita a causa de la censura (López, 1668). Durante los siglos XVII y XVIII causaron estragos, hubo brotes devastadores e tifus, pes te bubónica, el estilo de vida urbano empezaba a favorecer estas enfermedades, la higiene era deficiente, la carencia de instalaciones sanita rias, la depuración de las aguas residuales era inadecuada y no había leyes de salud públi ca (López, 2001).Todos los acontecimientos acaecidos en las diversas épocas han forjado la historia de la profesión enfermera (Hernández Martín, 1997). Los títulos de los tratados son los siguien tes: El Tratado 1 (capítulos 1 a 9) se titula: “De las casos contingentes, en el qual se incluien to dos quantos géneros de unturas ay y se pueden hacer en todo género de enfermedades con el Méthodo que combiene, según el buen orden de la Medicina”. Cultura de los Cuidados En segundo lugar, el Tratado 2 (capítulos 20 a 33) titulado “De otros casos con tingentes en muchas enfermedades menudas, con algunos remedios en los principios de ellas”. En tercer lugar, el Tratado 3 (capítulos 34 a 40) se titula: “De cómo se han de dar los xaraues y asistir a las sangrías y hechar los clisteres y supositorios o calas”. Por otro lado, el Tratado 4 (capítulos 41 a 56) se titula “De las fluxiones de vientre con las disenterías propias y impropias”. En quinto lugar, el Tratado 5 (capítulos 57 a 108) se titula “De las fiebres agudas y de las ter 3er Cuatrimestre 2016 • Año XX - N.° 46 RESUMEN Objetivo: A lo largo de las distintas épocas históricas, el cuidado ha sido objeto de estu dio de la enfermería. Al hablar de la historia de la Enfermería española, se suele aceptar por la mayoría de los investigadores como fecha clave de su institucionalización y profesionali zación la de mediados del siglo pasado, y más concretamente la del año 1857. En este caso, se pretende exponer los orígenes de un oficio, como era el de enfermero o cuidados que se ejercía en los hospitales en el siglo XVII en España, ver cómo eran las tareas y funciones que desempeñaban estos cuidadores, el reco nocimiento y aceptación social del oficio entre otros. A medida que nos remontamos en el tiem po, las complicaciones son mayores, debido fundamentalmente, a la escasez de documen tación generada y conservada hasta nuestros días (García Martínez,1998). Desde comien zos del siglo XVI, los hospitales españoles no dejaron de evolucionar, intentando incorpo Metodología: Para llevar a cabo el presen te trabajo se ha utilizado la obra Directorio de Enfermeros, cuyo título completo es Directo 87 Cultura de los Cuidados Cultura de los Cuidados sarrollado un problema de salud, así como las que se encuentran en peligro de desarrollarlo. Enfermería juega un papel fundamental en el fomento de la salud así como en la prevención. La obra ha sido concebida, fundamentalmen te, para la formación del enfermero, tanto el que se iniciaba en el oficio como el que ejercía la profesión. Ya en la obra Directorio de enfer meros encontramos descritas actividades en caminadas a cubrir las necesidades básicas del ser humano. La función asistencial es la más desarrollada en la obra de las funciones que se llevaban a cabo por parte de enfermería, aun que también se halla plenamente desarrollada a lo largo del material la actividad docente, con el fin de proporcionar a los enfermeros los conocimientos teóricos y prácticos imprescin dibles para el ejercicio de la profesión (García Martinez,1993). cianas y quartanas, con el regimiento de ellas, y cómo nos emos de regir en tiempo de peste y preuenciones para ella”. En sexto lugar, el Tra tado 6 (capítulos 109 a 137) se titula “De otras enfermedades, assí crónicas como ereditarias y accidentales”. En penúltimo lugar, el Tratado 7 (capítulos 138 a 148) se titula “De la calidad del agua y quál es la mexor, y de todo género de aguas cogidas que beuen los enfermos y el modo de cogerlas con su peso y medida”. Por último el Tratado 8 comprende los capítulos 149 y 150 y se titula “De la calidad de los mantenimientos simples que nos sirben para nuestra conserua ción” (López, 1668). Además, lleva incorporada tres aproba ciones, todas ellas de insignes médicos de la época, como la de D. Juan Lázaro Gutiérrez (catedrático de propiedad de Medicina en la Real Universidad de Valladolid), el D. Geromo Pardo (catedrático de Methodo Medendi en la Real Universidad de Valladolidad) y el Dr. Juan de Río Noriega, médico de la ciudad de Rioseco y catedrático en de la Universidad de Salamanca (López,1668). Virginia A. Henderson definía la profesión enfermera, con las siguientes palabras: «Creo que la enfermera es, y debe ser legalmente, un profesional independiente capaz de hacer juicios independientes mientras no haga diag nósticos, prescriba tratamientos o emita pro nósticos, porque éstas son las funciones del médico. Pero la enfermera es la mayor auto ridad en los cuidados básicos de enfermería. Cultura de los Cuidados Quizá deba explicar que por cuidados básicos de enfermería me refiero a ayudar a los pa cientes en las siguientes actividades o darles los conocimientos necesarios para que puedan llevarlas a cabo sin ayuda [se nombran 14 acti vidades]» (García Martínez,1993). En la obra de Simón López, escrita tres siglos antes, se tie ne interés en formar al enfermero, pero tam bién al paciente, con la finalidad de que este se valiese por sí mismo existiendo una relación con la definición de la autora Virginia Hen derson. Por ello, se han agrupado las distintas necesidades del ser humano en grupos con el Esta obra constituye un precedente en los textos modernos de enfermería, que junto al manual realizado por la Mínima Congrega ción de los Hermanos Enfermeros Pobres titu lado: Instrucción de enfermeros, para aplicar los remedios a todo género de enfermedades, y acudir a muchos accidentes que sobrevienen en ausencia de los Médicos, aunque más co nocido como “enfermeros obregones”, y en su segunda edición (1625), ampliada por el Her mano Andrés Fernández, constituyen las obras clásicas sobre la enfermería del siglo XVII es pañol (López, 1668). MÉTODO Para poder adentrarse en la esfera del co nocimiento del pasado de cualquier fenómeno son cada vez más utilizados los métodos etno históricos (Hernández Cortina,2004).En este caso, se pretende exponer los orígenes de un oficio, como era el de enfermero o cuidados que se ejercía en los hospitales en el siglo XVII en España, ver cómo eran las tareas y funcio nes que desempeñaban estos cuidadores, el reconocimiento y aceptación social del oficio entre otros (Burgos Moreno, 2009). Para llevar a cabo el presente trabajo se ha utilizado la obra Directorio de Enfermeros, 3er Cuatrimestre 2016 • Año XX - N.° 46 88 3er Cuatrimestre 2016 • Año XX - N.° 46 Cultura de los Cuidados Cultura de los Cuidados fin de comprender las actividades, tareas y téc nicas enfermeras que realizaba el enfermero de la época (López, 1668). En lo que respecta a la respiración, se des cribe patología respiratoria tales como asma, pleuresía, taudardillo entre otras. Para tratar las dolencias eran muy empleados los sahume rios que consistía en producir humo de una sustancia aromática que se quema y poseía una finalidad broncodilatadora y expectorante (Siles González, 2007). También se hablaba del ambiente que debía proporcionar el enfermo, recogido en los manuales de hoy en día. Así en la obra de Simón López se expresaba: “El apo sento para estos enfermos de dolor de costado ha de ser abrigado; mayormente en imbierno hanse de bestir las paredes o, por lo menos, la alcoba, si la hubiere, con algunos paramentos y, el suelo, con esteras. Siempre ha de hauer un brasero de lumbre de brasa de leña o de carbón muy bien ençendido (porque el tufo tapa la res piración)” (López, 1668). porque con esto no abre la boca tanto y puede mejor paladear.” (García Martínez, 1993). Con lo que respecta al patrón de elimi nación, Simón López propone una serie de medidas consistentes en la administración de enemas, calas o clisteres para esos casos. En el texto se nombra la estangurria que consiste en la dificultad para orinar (López,1668). Para ello, se formulan diversas formas de actuar, como la ingesta de bellota por sus propieda des diuréticas, así como la toma de hojas de rábano cocidas en caldo de puchero. Los ca pítulos 37,38 y 39 recogen la composición de los clisteres y la forma de administrarlos (Siles González, 2007). La alimentación y dietética ocupan un lu gar primordial en la formación del enfermero. En la obra se expone la importancia de los ali mentos en la mejora del proceso del enferme dad, para ello, se establecen las características de los alimentos y se insiste en la creación de hábitos saludables. Por ejemplo, se explica a los enfermeros la forma de fabricar un embu do de latón y se da importancia que el paciente mantenga una postura adecuada al darle de comer. 3er Cuatrimestre 2016 • Año XX - N.° 46 DESARROLLO La práctica de la enfermería abarca tanto el cuidado de las personas sanas como las han de 89 Cultura de los Cuidados Cultura de los Cuidados taban ya presentes en esta obra del siglo XVII (García Martínez, 1993). También se hace hincapié en la obra de las necesidades psicológicas y espirituales. En la obra se considera la esfera biológica, espiritual y social de la persona. Por ello, el enfermero debe hablar con el enfermo, alegrarle y ani marle, ponerle música y en definitiva ganarse la confianza del enfermo (López, 1668). Con respecto a la necesidad de termorre gulación encontramos medidas encaminadas a mantener la temperatura corporal. En el ca pítulo 28 titulado “del sudor, quál es bueno o malo y cómo se conocerá y qual es el que deue guardar y quál no” se expresa: “Muchas oca siones se le ofreçerán al enfermero con enfer mos de calenturas malignas donde suele aber sudor, en los días judiçiarios, y porque tenga alguna notiçia quando les biniere estos sudo res para abisar al Médico y bea lo que se ha de haçer” (López, 1668). Por otro lado, la obra también apuesta por enseñar al enfermo, es decir, por el autocuida do. En la página 216 de la obra se muestra este aspecto: “Y si esto puede hacer el enfermo, le enseñará el enfermero cómo lo ha de hacer, te niendo cuidado de renouarle a menudo las dos escudillas o basos de agua fría...” . En otras pá ginas también se expresa este concepto de au tocuidado, así en la página 267: “«... y enseñar al enfermo cómo ha de tomar los lamedores y cómo ha de acer los gargarismos y otras cosas que aora se dirán que le tocan, de todo lo qual ya dexo dicho arto en sus lugares donde podrá recurrir, por no me detener aquí más»” (López, 1668). En la necesidad de higiene, se recalca en la obra, que su carencia o deficiencia provocan y favorecen la propagación de las enfermedades. Por ello, entre las actividades que debe realizar los enfermeros se destaca bañar al paciente, cambiar la ropa y mantener limpios los apo sentos. Además se recalca en la importancia de aplicar medidas de higiene y desinfección durante las epidemias, así como el lavado de manos (López, 1668). Por último, también se encuentran en la obra consejos y recomendaciones dirigidos a la comunidad con respecto a la adopción de medidas higiénicas y medioambientales (Ló pez, 1668). Cultura de los Cuidados En siglo XVII las epidemias estaban muy presentes, por lo tanto también se expre sa en la obra medidas para evitar la propaga ción de enfermedades infecciosas, entre ellas destaca la purificación del aire, cubrir con cal los cadáveres (García Martínez, 2004). La obra establece un amplia relación de enfermedades y afecciones, medidas preventivas y curativas, que nos proporcionan una visión de este oficio en este siglo. (García Martínez, 1993) En estrecha relación, se encuentra la segu ridad física, ya que puede poner en peligro la integridad del enfermo si no se tiene en con sideración. En el capítulo 66 se recalcan las medidas de seguridad que deben ser emplea das por el enfermero ante un enfermo deliran te (Siles González,2007). Así se expresa en la obra: “Han suçedido y suçeden oy tantas des gracias y fatales fines con enfermos frenéticos, por no cuidar con diligengia y atençión a su asistençia, que fuera nunca acabar el aberlos de referir. Y, no hay duda, de que si preguntaran en cada comunidad o hospital algunos casos lastimosos de frenéticos, que todos tendrían arto que deçir de enfermos que se desgraçiaron por falta de asistençia y ignorançia de los enfer meros o assistentes” (López, 1668). 3er Cuatrimestre 2016 • Año XX - N.° 46 Cultura de los Cuidados El autor explica a los enfermeros, de forma clara y detallada, la forma de fabricar el embudo de latón y la postura del paciente para darle de comer: “El tamaño del embudo será como seys o siete dedos de trauessía con copa y todo y de largo; el cañón de la copa para abajo no ha de ser derecho, sino algo arquea do, al modo de una cornucopia; la punta deste embudo no ha de quedar redonda sino balada, La necesidad de sueño y descanso tam bién se encuentra en esta obra. El papel del enfermero se encaminaba hacia la vigilancia, a conseguir un ambiente óptimo que facilitase el sueño. También se indica cómo administrar medicación hipnótica e inductora del sueño, preparada fundamentalmente a base de plan tas como por ejemplo, la papaver somniferum. El capítulo 68 está completamente dedicado al descanso de los enfermos frenéticos. También el empleo de musicoterapia, la creación de un ambiente relajado y la presencia de flores es 3er Cuatrimestre 2016 • Año XX - N.° 46 90 BIBLIOGRAFÍA En la obra de Simón López, se aprecia un interés por delimitar el trabajo de los profesio nales que ejercen en el hospital en esa época (barbero, boticario, médico) insistiendo sobre todo en la labor de enfermería (López, 2001). Por eso, ya se encuentran reflejadas las cuatro funciones que en la actualidad se le reconoce a la profesión enfermera: docencia, asistencia, investigación y administración, siendo las dos primeras, con gran diferencia, las más amplia mente desarrolladas (Hernández Martín, 1997). - Burgos Moreno, M., y Paravic Klijn, T. (2009). Enfermería como profesión. Revista cubana de enfermería, 25(1-2). Disponible en: http:// scielo.sld.cu/scielo.php?script=sci_arttext&pid =S0864-03192009000100010. Fecha de acceso 21 de abril de 2016. - Burgos Moreno, M., y Paravic Klijn, T. (2009). Enfermería como profesión. Revista cubana de enfermería, 25(1-2). Disponible en: http:// scielo.sld.cu/scielo.php?script=sci_arttext&pid =S0864-03192009000100010. Fecha de acceso 21 de abril de 2016. - García-Carpintero Blas, E. (2007). Reflexión del papel de la enfermería a lo largo de la historia. Enfermería Global, 6(2),1-6. - García-Carpintero Blas, E. (2007). Reflexión del papel de la enfermería a lo largo de la historia. Enfermería Global, 6(2),1-6. - García Martínez A.C., García Martínez M.J., Hernández Martín F., Pérez Melero A. y Pinar García M.E. (1993). Aproximación a la Enfermería española de los siglos XVI- XVII. Presentación y análisis de la obra Instrucción de enfermeros, de Andrés Fernández, 1625. Madrid: Edición a cargo del Consejo General de Colegios de Diplomados en Enfermería.110-232. - García Martínez A.C., García Martínez M.J., Hernández Martín F., Pérez Melero A. y Pinar García M.E. (1993). Aproximación a la Enfermería española de los siglos XVI- XVII. Presentación y análisis de la obra Instrucción de enfermeros, de Andrés Fernández, 1625. Madrid: Edición a cargo del Consejo General de Colegios de Diplomados en Enfermería.110-232. A lo largo de la obra, se intenta explicar las técnicas y las tareas llevadas a cabo por enfer mería de forma razonada y haciendo fácil su compresión mediante la utilización de diver sos recursos didácticos tales como compara ciones y ejemplos (Hernández Martín, 1996). Cultura de los Cuidados Cultura de los Cuidados administrar medicación pauta explícitamente por el médico (Hernández Martín, 1997). Así se ha encontrado una correlación entre la de finición de enfermería de Virgina Henderson y las necesidades básicas expresadas por ésta, con los contenido de la obra expuestos tres si glos antes. En definitiva, la obra ofrece una vi sión integral de la persona teniendo en cuenta la esfera biológica, psicológica y social, junto con la vertiente humana de los cuidados (Her nández Cortina, 2004). los cuidados dispensados a los enfermos du rante su estancia en estos centros y al personal sanitario que los proporcionaba, las enferme ras y enfermeros, han sido escasos, aun sabien do que la esencia de estas instituciones y su razón de ser fundamental es precisamente ésa: proporcionar cuidados de salud y curar (Siles González, 2000). Por su finalidad y contenido, consideramos a Directorio de Enfermeros, de Simón López, una obra clave para conocer la Enfermería es pañol del siglo XVII (García Martínez,1993). CONCLUSIONES De todos los estudios realizados sobre los hospitales fundados entre fines de la Edad Me dia y toda la Edad Moderna, los dedicados a 3er Cuatrimestre 2016 • Año XX - N.° 46 91 3er Cuatrimestre 2016 • Año XX - N.° 46 BIBLIOGRAFÍA El trabajo del enfermero de la época queda expuesto a lo largo de la obra de Simón López, especialmente en el prólogo, en las que desta can las siguientes funciones y tareas: ejecutar las prescripciones ordenadas por el médico, proveer lo necesario a la hora de realizar curas, mantener unas condiciones óptimas de higie ne y limpieza, asistir al enfermo en ausencia del médico o en situaciones de urgencia, ob servar la evolución del enfermo e informar al médico de los acontecimientos, dar de comer al enfermero en las condiciones adecuadas y - García Martínez, M.J., y García Martínez, A.C.(1998). La enseñanza de la Enfermería en la España del siglo XVII. Cultura de los Cuidados, 2(3),15-23. - García Martínez, M.J., y García Martínez, A.C.(1998). La enseñanza de la Enfermería en la España del siglo XVII. Cultura de los Cuidados, 2(3),15-23. - García Martínez, M.J.( 2004). Cuidados enfermeros en la España del siglo XVII. Hacia la búsqueda de una identidad profesional. Gazeta de Antropología, 20, 22. - García Martínez, M. J. (2004). El método en la enfermería: una mirada desde la historia (siglo XVII). Metas de enfermería, 7(10), 58-64. - Hernández Martín, F.J. (1996). Historia de la Enfermería en España (desde la Antigüedad hasta nuestros días). Madrid: Editorial Síntesis. - Hernández Martín, F.J. (1996). Historia de la Enfermería en España (desde la Antigüedad hasta nuestros días). Madrid: Editorial Síntesis. 3er Cuatrimestre 2016 • Año XX - N.° 46 92 Cultura de los Cuidados Cultura de los Cuidados - Hernández Martín, F., Gallego Lastra, R. D., Alcaraz González, S., González Ruiz, J. M. (1997). La enfermería en la historia. Un análisis desde la perspectiva profesional. Cultura de los cuidados, 2(3), 21-35. J. (2001). Directorio de enfermeros y artífice de obras de caridad para curar las enfermedades del cuerpo. Madrid: Enfermundi. - Hernández Martín, F., Gallego Lastra, R. D., Alcaraz González, S., González Ruiz, J. M. (1997). La enfermería en la historia. Un análisis desde la perspectiva profesional. Cultura de los cuidados, 2(3), 21-35. - Siles González, J.(2004). La construcción social de la Historia de la Enfermería. Index de enfermería, 47(13),7-10. - Hernández Cortina, A., Guardado de la Paz, C. (2004). La enfermería como disciplina profesional holística. Revista cubana de enfermería, 20(2), 1-1. - Siles González, J. (2000). Antropología, historia y enfermería. Cultura de los cuidados: Revista de enfermería y humanidades, 4(7-8),5-7. - López, S. (1668). Directorio de enfermeros y artífice de obras de caridad para curar las enfermedades del cuerpo Signatura:Manuscrito Ms 259. Biblioteca Universitaria de Salamanca. - López, S. (1668). Directorio de enfermeros y artífice de obras de caridad para curar las enfermedades del cuerpo Signatura:Manuscrito Ms 259. Biblioteca Universitaria de Salamanca. - Siles González, J., Solano Ruiz, M. D. C. (2007). El origen fenomenológico del “cuidado” y la importancia del concepto de tiempo en la historia de la enfermería. Cultura de los cuidados, 11(21), 19-27. - López, S., García Martínez, A. C. y García Martínez, M. - López, S., García Martínez, A. C. y García Martínez, M. 3er Cuatrimestre 2016 • Año XX - N.° 46 3er Cuatrimestre 2016 • Año XX - N.° 46 93
https://openalex.org/W2048732556	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0022303&type=printable	English	null	Immuno-Therapy with Anti-CTLA4 Antibodies in Tolerized and Non-Tolerized Mouse Tumor Models	PloS one	2,011	cc-by	10,478	Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution L restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by NIH grant R01CA080192 (to A.L.); National Science Council Grant NSC96-2628-B001-003-MY3 (t Postdoctoral Training Grant (to I.B.); and Swedish Research Council Postdoctoral Grant 524-2009-539 (to J.P.). The funders had no collection and analysis, decision to publish, or preparation of the manuscript. supported by NIH grant R01CA080192 (to A.L.); National Science Council Grant NSC96-2628-B001-003-MY3 (to S.R.); Deutsche Krebshilfe nt (to I.B.); and Swedish Research Council Postdoctoral Grant 524-2009-539 (to J.P.). The funders had no role in study design, data ecision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lieber00@u.washington.edu non-small lung breast cancer, and pancreatic cancer [5]. Two fully humanized monoclonal antibodies, ipilimumab (MDX-010, Me- darex) and tremelimumab (CP-675,206, Pfizer), have been investigated in cancer [6,7]. A Phase III trial of tremelimumab has been halted after it failed to demonstrate superior therapeutic activity over standard chemotherapy in advanced melanoma patients. The discrepancy in pre-clinical and clinical studies with anti-CLTA4 antibodies requires more mechanistic studies in adequate pre-clinical models. A potential mechanism by which anti-CTLA4 may provide an antitumor response is through depletion of regulatory T-cells (Tregs), as Tregs have constitutive expression of CTLA4 and are known to have suppressive activity. Alternatively, CTLA4 blockade may activate effector T-cells allowing them to be more resistant to Treg suppression. Recent studies indicate that anti-CTLA4 induce immune responses mainly by direct activation of effector T-cells rather than by affecting Tregs [8,9]. Jonas Persson1, Ines Beyer1, Roma Yumul1, ZongYi Li1, Hans-Peter Kiem2, Steve Roffler3, Andre´ Lieber1,4* 1 Department of Medicine, University of Washington, Seattle, Washington, United States of America, 2 Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 3 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, 4 Department of Pathology University of Washington, Washington, United States of America Abstract Monoclonal antibodies specific for cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA4) are a novel form of cancer immunotherapy. While preclinical studies in mouse tumor models have shown anti-tumor efficacy of anti-CTLA4 injection or expression, anti-CTLA4 treatment in patients with advanced cancers had disappointing therapeutic benefit. These discrepancies have to be addressed in more adequate pre-clinical models. We employed two tumor models. The first model is based on C57Bl/6 mice and syngeneic TC-1 tumors expressing HPV16 E6/E7. In this model, the HPV antigens are neo- antigens, against which no central tolerance exists. The second model involves mice transgenic for the proto-oncogen neu and syngeneic mouse mammary carcinoma (MMC) cells. In this model tolerance to Neu involves both central and peripheral mechanisms. Anti-CTLA4 delivery as a protein or expression from gene-modified tumor cells were therapeutically efficacious in the non-tolerized TC-1 tumor model, but had no effect in the MMC-model. We also used the two tumor models to test an immuno-gene therapy approach for anti-CTLA4. Recently, we used an approach based on hematopoietic stem cells (HSC) to deliver the relaxin gene to tumors and showed that this approach facilitates pre-existing anti-tumor T-cells to control tumor growth in the MMC tumor model. However, unexpectedly, when used for anti-CTLA4 gene delivery in this study, the HSC- based approach was therapeutically detrimental in both the TC-1 and MMC models. Anti-CTLA4 expression in these models resulted in an increase in the number of intratumoral CD1d+ NKT cells and in the expression of TGF-b1. At the same time, levels of pro-inflammatory cytokines and chemokines, which potentially can support anti-tumor T-cell responses, were lower in tumors of mice that received anti-CTLA4-HSC therapy. The differences in outcomes between the tolerized and non- tolerized models also provide a potential explanation for the low efficacy of CTLA4 blockage approaches in cancer immunotherapy trials. Citation: Persson J, Beyer I, Yumul R, Li Z, Kiem H-P, et al. (2011) Immuno-Therapy with Anti-CTLA4 Antibodies in Tolerized and Non-Tolerized Mouse Tumor Models. PLoS ONE 6(7): e22303. doi:10.1371/journal.pone.0022303 Editor: Eric J. Kremer, French National Centre for Scientific Research, France Citation: Persson J, Beyer I, Yumul R, Li Z, Kiem H-P, et al. (2011) Immuno-Therapy with Anti-CTLA4 Antibodies in Tolerized and Non-Tolerized Mouse Tumor Models. PLoS ONE 6(7): e22303. doi:10.1371/journal.pone.0022303 ved March 9, 2011; Accepted June 23, 2011; Published July 14, pyright: 2011 Persson et al. Cells Mouse mammary carcinoma (MMC) cells were established from a spontaneous tumor in a neu-tg mouse [21]. TC-1 cells were from the American Type Culture Collection (ATCC) Culture conditions for MMC and TC-1 cells were RPMI-1640 medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine (Gln), 100 U/ml penicillin (P), and 100 mg/ml streptomycin (S). To obtain mouse HSCs, donor mice were injected with 5-FU (150 mg/kg) i.v. two days before bone marrow isolation. Bone marrow cells were cultured for three days in IMDM, 18%FBS, 5% mIL-3 (BD Biosciences, San Jose, CA), 100 U/ml mIL-6, 50 U/ ml SCF (PeproTech, Rocky Hill, NJ), P/S, and Gln. Non- adherent cells were collected and incubated with lentivirus vectors at an MOI of 2 genomes/cell on retronectin-coated plates for two days. Animal studies All experimental procedures involving animals were conducted in accordance with the institutional guideline set forth by the University of Washington. Neu-transgenic (neu-tg) mice [FVB/N- Tg(MMTVneu)202 Mul] were obtained from the Jackson Labo- ratory (Bar Harbor, ME). These mice harbor non-mutated, non- activated rat neu under control of the mouse mammary tumor virus (MMTV) promoter. The neu transgene is expressed at low levels in normal mammary epithelium, salivary gland, and lung. Until the age of 8 months ,35% of female neu-tg mice spontaneously develop mammary carcinomas that display high Neu-expression levels. CTLs specific for the immunodominant H-2 Dq/ RNEU420–429 epitope can be detected in neu-tg mice using the corresponding tetramer [22]. For HSC transplantation, a total of 16106 of whole bone marrow cells or lineage cell depleted bone marrow cells from 5-FU treated mice were transplanted into lethally irradiated (1050 cGy) female neu-tg mice. Six weeks after bone marrow transplantation, the mice received 56105 MMC or 56104 TC-1 cells via subcutaneous injection. Tumors were measured every other day and tumor volume was calculated as the product of length x width x width. For survival studies tumor sizes $500 mm3 were considered the experimental endpoint. Animals with skin surface ulcerations were excluded from experiments and sacrificed immediately. For delivery of anti-CTLA4 immunotherapy, we used three different approaches; i) systemic application of a monoclonal antibody against murine CTLA4 (4F10), ii) intratumoral expres- sion of a secreted form of this antibody from genetically modified tumor cells, iii) expression of the anti-CTLA4 antibody after gene delivery using a stem cell based approach. The central findings from our studies are i) anti-CTLA4 therapy is inefficient in the tolerized MMC model and ii) in both tumor models, anti-CTLA4 expression mediated by the HSC delivery approach not only failed to exert anti-tumor effects, but increased the rate of tumor growth. Our data suggests that the latter involves an increase in intratumoral CD1d+ NKT cells, production of IFNb1, as well as suppression of cytokines and chemokines that are involved in mediating anti-tumor immune responses. Our findings shed light on the complexity of immune regulation, specifically in the context of anti-CTLA4 therapy. Anti-CTLA4 antibodies Monoclonal antibodies against mouse CTLA4 were purified from the supernatant of UC10-F10-11 hybridoma cells (ATCC) as described previously [16]. Introduction Tolerance against TAA has to be considered in tumor models that are used to delineate the anti- tumor mechanisms of anti-CTLA4 antibodies. This is accom- plished in our second animal model, based on neu-transgenic (neu- tg) mice. These mice overexpress the rat protooncogene Neu and develop spontaneous mammary tumors between 4 and 8 months of age [14,15]. Mouse mammary carcinoma cells (MMC) are a transplantable carcinoma line derived from a spontaneous mammary tumor from neu-tg mice. The neu-tg/MMC model has significant biologic and pathologic similarity to human neu- associated estrogen receptor-negative breast cancer. MMC tumors are resistant to doxorubicin, hormone therapy, and Neu-specific mAbs. The tumor antigen repertoire in MMC-tumor bearing mice appears to be predictive for human breast cancer antigens. Importantly, neu-tg mice mimic central/peripheral tolerance to an endogenous tumor antigen that is seen in cancer patients. In this context, Neu-targeted vaccines, which raise strong CD8-T cell responses to a dominant peptide (RNEU420-429) in (non- tolerized) WT FVB/N mice and protect them from a neu- expressing tumor challenge, fail to do so in neu-transgenic mice. The latter suggests significant differences between tolerized and non-tolerized tumor models, which have to be considered in testing the effect of new immunotherapy agents. based on human papillomavirus (HPV)-16 E6/E7–expressing TC- 1 tumors. In this model, the HPV antigens represent neo-antigens against which no central tolerance mechanisms exit in mice. Most studies on the mechanisms of immune-activation by CTLA4- blocking antibodies have been performed in such ‘‘non-tolerized’’ models [10,11,12,13]. In humans, however, most tumor-associated antigens (TAAs) are non-mutated self-antigens, which are overexpressed or re-expressed on cancer cells. Several mechanisms of central and peripheral tolerance therefore exist against self- TAAs that blunt T-cell responses. Tolerance against TAA has to be considered in tumor models that are used to delineate the anti- tumor mechanisms of anti-CTLA4 antibodies. This is accom- plished in our second animal model, based on neu-transgenic (neu- tg) mice. These mice overexpress the rat protooncogene Neu and develop spontaneous mammary tumors between 4 and 8 months of age [14,15]. Mouse mammary carcinoma cells (MMC) are a transplantable carcinoma line derived from a spontaneous mammary tumor from neu-tg mice. The neu-tg/MMC model has significant biologic and pathologic similarity to human neu- associated estrogen receptor-negative breast cancer. MMC tumors are resistant to doxorubicin, hormone therapy, and Neu-specific mAbs. The tumor antigen repertoire in MMC-tumor bearing mice appears to be predictive for human breast cancer antigens. Introduction Importantly, neu-tg mice mimic central/peripheral tolerance to an endogenous tumor antigen that is seen in cancer patients. In this context, Neu-targeted vaccines, which raise strong CD8-T cell responses to a dominant peptide (RNEU420-429) in (non- tolerized) WT FVB/N mice and protect them from a neu- expressing tumor challenge, fail to do so in neu-transgenic mice. The latter suggests significant differences between tolerized and non-tolerized tumor models, which have to be considered in testing the effect of new immunotherapy agents. epitope and platelet-derived growth factor receptor transmem- brane domain. A stop codon was introduced immediately after the myc epitope to allow secretion of the antibody. The cDNA fragment coding the hinge-CH2-CH3 of human IgG1 was inserted between the scFv and myc epitope. The entire anti-CTLA4 cassette was then transferred into pLVPT-rTRKRAB [18] to generate pLVaCTLA4. To create the construct for the insulated vector (I-LV-aCTLA4), the aCTLA-4, IRES, tetR-KRAB and WPRE from pLVaCTLA4 was transferred into pLenti-cHS-PGK [19] containing a 0.4 kb fragment of the chicken HS4 insulator within the 39 LTR. VSV-G pseudotyped viruses were generated as described earlier [20]. Genome titers were measured by qPCR and ranged from 1-56107 genomes/ml. PLoS ONE \| www.plosone.org Introduction non-small lung breast cancer, and pancreatic cancer [5]. Two fully humanized monoclonal antibodies, ipilimumab (MDX-010, Me- darex) and tremelimumab (CP-675,206, Pfizer), have been investigated in cancer [6,7]. A Phase III trial of tremelimumab has been halted after it failed to demonstrate superior therapeutic activity over standard chemotherapy in advanced melanoma patients. The discrepancy in pre-clinical and clinical studies with anti-CLTA4 antibodies requires more mechanistic studies in adequate pre-clinical models. A potential mechanism by which anti-CTLA4 may provide an antitumor response is through depletion of regulatory T-cells (Tregs), as Tregs have constitutive expression of CTLA4 and are known to have suppressive activity. Alternatively, CTLA4 blockade may activate effector T-cells allowing them to be more resistant to Treg suppression. Recent studies indicate that anti-CTLA4 induce immune responses mainly by direct activation of effector T-cells rather than by affecting Tregs [8,9]. Activation of T-cells requires recognition of antigens presented in complex with CD80 and CD86. These costimulatory molecules interact with CD28, which is constitutively expressed on T cells and triggers T-cell activation. Once activated, T-cells transiently up-regulate cytotoxic T lymphocyte–associated antigen 4 (CTLA4) on their cell surface. CTLA4 shares structural features with the costimulatory receptor CD28 and reciprocally targets the same costimulatory molecules (CD80/86) on the antigen-presenting cell, but with higher affinity. This results in inhibition of T-cell proliferation and IL-2 production. Blocking CTLA4 with anti- CTLA4 antibodies enhances effector T-cell responses and can induce T-cell mediated rejection of certain tumors in mouse models [1,2,3,4]. Monoclonal antibodies specific for cytotoxic T lymphocyte-associated antigen 4 (CTLA4) are a form of experimental immunotherapy for treatment of patients with advanced cancers, including melanoma, prostate cancer, renal cell carcinoma, non-Hodgkin’s lymphoma, colorectal carcinoma, In this study, we used two tumor models that assess anti-CTLA4 antibody therapy. The first is a murine cervical cancer model PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e22303 PLoS ONE \| www.plosone.org 1 Anti-CTLA4 Antibody Therapy based on human papillomavirus (HPV)-16 E6/E7–expressing TC- 1 tumors. In this model, the HPV antigens represent neo-antigens against which no central tolerance mechanisms exit in mice. Most studies on the mechanisms of immune-activation by CTLA4- blocking antibodies have been performed in such ‘‘non-tolerized’’ models [10,11,12,13]. In humans, however, most tumor-associated antigens (TAAs) are non-mutated self-antigens, which are overexpressed or re-expressed on cancer cells. Several mechanisms of central and peripheral tolerance therefore exist against self- TAAs that blunt T-cell responses. Mouse cytokine array Pieces of tumor were homogenized in Complete Lysis Buffer M (Roche) using TissueRuptor (Qiagen). The total protein concen- tration was determined using the Protein Assay reagent from Bio Rad. For each sample, 100 mg were assayed and the Cytokine Array Panel A (R&D Systems) was performed according to manufacturer’s suggestions. For the development of the assay, ECL Plus (GE Healthcare) was used together with Amersham Hyperfilm ECL (GE Healthcare). The developed films were scanned and intensities were quantified with SigmaGel (Jandel Scientific, San Rafael, CA). Lentivirus vectors Two-fold serial dilutions of the culture medium were incubated for 45 minutes in 96-well microtiter plates previously coated with 500 ng/well recombinant CTLA4 protein [16]. After washing, the wells were incubated with biotin-labeled anti-HA antibody (Roche, Mannheim Germany), followed by streptavidin-HRP (Jackson ImmunoResearch Laboratories) and finally 1-StepTM Ultra TMB-ELISA (Thermo Scientific, Rockford, IL) substrate for The anti-CTLA4 scFv was cloned from total RNA isolated from UC10-F10-11 hybridoma cells (ATCC). Leucine residues at positions 43 and 89 in the 4F10 variable region light chain sequence were mutated to methionine and glutamine, respectively, to increase scFv expression [17]. The anti-CTLA4 scFv was inserted into pHook (Invitrogen, Carlsbad, CA) immediately after the Vk leader sequence and HA epitope tag and before the myc July 2011 \| Volume 6 \| Issue 7 \| e22303 2 Anti-CTLA4 Antibody Therapy Anti-CTLA4 Antibody Therapy 30 min at room temperature. The absorbance of wells (405 nm) was measured with a microplate reader. Anti-CTLA4 mRNA was equalized to levels of GAPDH mRNA measured in parallel in each sample. Ct values were calculated using the Sequence Detection System software (Applied Biosys- tems). The difference between the number of PCR cycles required for the two samples to reach a certain fluorescence signal shows how much of the mRNA of interest is present in the two samples relative to each other. Each cycle difference is equal to a fold difference of 2. qRT-PCR TGF-b1 Pieces of tumor were homogenized using TissueRuptor (Qiagen) and total RNA was purified with miRQURY RNA Isolation Kit (Exiqon, Woburn, MA). RNA concentration was measured on a NanoDrop ND-1000 (Thermo Scientific). Generation of cDNA was done with QuantiTect Reverse Transcription Kit (Qiagen) and the qPCR reaction was run, in triplicates, on a 7900HT Fast Real-Time PCR System (Applied Biosystems/Life Technologies) using the SensiMix SYBR Kit (Quantace, London, UK). Anti-CTLA4 mRNA mRNA isolation from MMC-Rlx cells and qRT-PCR was performed as described recently [23]. cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen). For PCR the SYBR Kit (Bioline, Taunton, MA) and the following primers were used (Q ) Primers (synthesized by Integrated DNA Technologies): mTGFB1 fw 59-GGCTACCATGCCAACTTCTG -39 mTGFB1 rev 59-CGCACAATCATGTTGGACA -39 mTGFB2 fw 59-TGAGGTGTGAATGCAAGGAG-39 aCTLA4, fw 59- ACC CCT CAC AAT CAC TGT CC -39 aCTLA4, rev 59- CAC CTG CAG GAA GAA CTG GT -39 Figure 1. Effect of anti-CTLA4 antibody injection on MMC and TC-1 tumor growth. A) C57Bl/6 mice with subcutaneous TC-1 tumors we injected with 100 mg anti-CTLA4 and control IgG antibody intraperitoneally (i.p.) every other day. Treatment was started when tumor reached volume of 50 mm3. Tumor volumes were measured thrice a week. Each line represents an individual animal. p,0.05 for time points after day 16. neu-transgenic mice with subcutaneous MMC tumors were treated as described in A). The difference between the two groups was not significa doi:10.1371/journal.pone.0022303.g001 PLoS ONE \| www.plosone.org 3 July 2011 \| Volume 6 \| Issue 7 \| e223 Figure 1. Effect of anti-CTLA4 antibody injection on MMC and TC-1 tumor growth. A) C57Bl/6 mice with subcutaneous TC-1 tumors were injected with 100 mg anti-CTLA4 and control IgG antibody intraperitoneally (i.p.) every other day. Treatment was started when tumor reached a volume of 50 mm3. Tumor volumes were measured thrice a week. Each line represents an individual animal. p,0.05 for time points after day 16. B) neu-transgenic mice with subcutaneous MMC tumors were treated as described in A). The difference between the two groups was not significant. doi:10.1371/journal.pone.0022303.g001 July 2011 \| Volume 6 \| Issue 7 \| e22303 July 2011 \| Volume 6 \| Issue 7 \| e22303 PLoS ONE \| www.plosone.org 3 Anti-CTLA4 Antibody Therapy Figure 2. Lentivirus vectors expressing anti-CTLA4. A) Structure of integrated provirus genomes. The vectors contain the gene for the monoclonal antibody 4F10 (ATCC: UC10-4F10-11) specific to mouse CTLA4. The anti-CTLA4 gene contains an immunoglobulin Vk signal peptide, an HA epitope, the anti–CTLA4 scFv, the hinge, CH2 and CH3 domains of human IgG1, and a myc epitope. The anti-CTLA4 gene is under the control of a PLoS ONE \| www.plosone.org 4 July 2011 \| Volume 6 \| Issue 7 \| e22303 Figure 2. Lentivirus vectors expressing anti-CTLA4. A) Structure of integrated provirus genomes. Immunohistochemistry for mouse tissue and organs y g Tumors were embedded in Optimal Cutting Temperature (OCT) medium and frozen at 280uC. Sections were cut at a thickness of 8 mm and fixed in methanol:aceton (1:1 v/v) at 220uC for 10 min. Nonspecific binding was blocked by 2% non- fat dry milk in PBS for 20 min at RT. Primary antibodies were incubated at RT for 1 h. We used anti-CD1d-biotin (clone 1B1, BD Biosciences) and anti-NK1.1-FITC (clone PK136, BD Biosciences) antibodies. For histological assessment of autoimmune disease, mouse tissues and organs (heart, lung, brain, stomach, mesenterium, liver, kidney, muscle, skin) were fixed in 10% formalin and processed for hematoxilin and eosin staining. All samples were examined by two experienced pathologists for typical inflammation signs in a blind fashion. Immunohistochemistry for IgG on kidney sections was performed as described for tumor sections using a polyclonal, HRP-labeled, anti mouse IgG antibody (eBiosciences). To test our therapy approaches, we generated improved versions of lentivirus vectors. These vectors are self-inactivating (SIN), i.e. contain a deletion within the 39LTR, which abolishes the LTR promoter activity (Fig. 2A). Because it had been shown that a chromatin insulator derived from the chicken globin locus mTGFB2rev 59-CAGTGAAGTGGAAGGGGAAA-39 mTGFB3fw 59-GCCATTTCCCTCCTACCCTA-39 mTGFB3 rev 59-CATCCATGATTCCCCAAAAA -39 Statistical significance of in vivo data was analyzed by Kaplan- Meier survival curves and logrank test (GraphPad Prism Version 4). Statistical significance of in vitro data was calculated by two- sided Student’s t-test (Microsoft Excel). P values,0.05 were considered statistically significant. JMP statistical package was used to perform power analysis and determine the minimal number of animals per group. Using parameters of alpha = 0.05; power = 80%; effect size = 50% (80% chance of observing a difference of 50% in tumor size at a level of significance of 0.05), we arrived at a minimal group size of 5 for a comparison of two groups. Therefore, all experiments were performed at least once with 5 animals per group and, if required, repeated with additional animals until significance was achieved. PCR was carried out as follows: after an initial 10-minute enzyme activation step at 95uC, 40 amplification cycles were run, each consisting of 95uC for 15 s and 60uC for 1 min. Lastly, a final elongation step was performed for two minutes at 60uC. Data was collected initially and after every incubation at 60uC. Anti-TGF-b mRNA was equalized to levels of GAPDH mRNA measured in parallel in each sample. Anti-CTLA4 mRNA B) Evaluation of anti-CTLA4 expression on protein level for clones derived after transduction of MMC cells with LV-aCTLA4 (upper panel) or I-LV-aCTLA4 (lower panel). Solid bars: Dox induced expression. Clones were treated with Dox and 24 h later anti-CTLA4 was measured by ELISA in culture supernatants. Empty bars (to the right side of solid bars): non-induced expression levels: supernatant from clones w/o Dox treatment were analyzed by ELISA. (Note, that these bars are not visible for LV-aCTLA4). Anti-CTLA4 levels in the corresponding populations of transduced cells (Pop) are shown on the right. C) Fold change of anti- CTLA4 mRNA levels after culture with or without doxycycline for 24 h measured. Clones that expressed the highest levels of anti-CTLA4 protein were included in this analysis. mRNA was isolated and subjected to qRT-PCR for GAPDH and anti-CTLA4 mRNA. Shown is the fold difference of GAPDH normalized anti-CTLA4 mRNA levels with and without Dox induction. Standard deviation was less than 10%. doi:10.1371/journal.pone.0022303.g002 mTGFB2rev 59-CAGTGAAGTGGAAGGGGAAA-39 mTGFB3fw 59-GCCATTTCCCTCCTACCCTA-39 mTGFB3 rev 59-CATCCATGATTCCCCAAAAA -39 PCR was carried out as follows: after an initial 10-minute enzyme activation step at 95uC, 40 amplification cycles were run, each consisting of 95uC for 15 s and 60uC for 1 min. Lastly, a final elongation step was performed for two minutes at 60uC. Data was collected initially and after every incubation at 60uC. Anti-TGF-b mRNA was equalized to levels of GAPDH mRNA measured in parallel in each sample. Analysis of TILs Three weeks after tumor cell transplantation, mice were sacrificed and tumors and spleen were harvested. Isolated MMC tumors were minced and filtered through a 70-mm cell strainer. Tumor infiltrating lymphocytes (TILs) were then isolated from tumor cells/erythrocytes by centrifugation in a Ficoll gradient. TILs as well as splenocytes from tumor-bearing mice were used for analysis of Neu-specific T-cells using a Neu-tetramer assay. The PE- labeled H-2Dq/RNEU420–429 (H-2D(q)PDSLRDLSVF) tetra- mer was obtained from the National Institute of Allergy and Infectious Diseases MHC Tetramer Core Facility (Atlanta, GA). Flow cytometry was performed with the following monoclonal antibodies (final concentration 5 mg/ml): anti-FoxP3-PE (clone FHK16s, eBiosciences, San Diego, CA), anti-CD4-PE, anti-CD8- PE, anti-CD8-FITC, anti-CD25-FITC (clone 7D4) (all BD Biosciences), anti-CD25-FITC (clone PC61.5; eBiosciences), and NK1.1-FITC (clone PK136, BD Biosciences), anti CD1d-PE (clone 1B1, BD Biosciences). All samples were treated with Fc- block (anti–CD16/CD32, BD Biosciences). Corresponding isotope controls yielded no significant staining. Expression of anti-CTLA4 antibody from tumor cells Expression of anti-CTLA4 antibody from tumor cells While systemic anti-CTLA4 administration is technically straightforward, it is cost-extensive and also bears the risk of inducing auto-immune responses [16]. These problems can, in part, be addressed by gene therapy approaches resulting in intratumoral expression of genes encoding anti-CTLA4 antibod- ies. Expression of anti-CTLA4 antibodies inside the tumor has advantages over systemic administration. Presumably, at sites where the TAA levels are elevated, such as in the tumor microenvironment, peripheral tolerizing mechanisms must be enhanced relative to other tissues. To be most effective, the concentration of immune-stimulatory molecules should therefore be high in the tumor environment. Recently, an immunostimu- latory effect of intratumoral expression of a gene encoding a secreted form of the anti-CTLA4 antibody has been shown in a model for autoimmune diabetes [26]. Anti-CTLA4 mRNA The vectors contain the gene for the monoclonal antibody 4F10 (ATCC: UC10-4F10-11) specific to mouse CTLA4. The anti-CTLA4 gene contains an immunoglobulin Vk signal peptide, an HA epitope, the anti–CTLA4 scFv, the hinge, CH2 and CH3 domains of human IgG1, and a myc epitope. The anti-CTLA4 gene is under the control of a PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e22303 4 Anti-CTLA4 Antibody Therapy Anti-CTLA4 Antibody Therapy tTR-KRAB system [18]. tRT-KRAB bound to tet-operator sequences represses promoters in the vicinity of 3-4 kb. Addition of Dox releases this repression. The vector also contains a central polypurine tract (cPPT) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRC). In the insulated vector version (I-LV-aCTLA4), a 0.4 kb cHS4 insulator element [43] is inserted into the 398 bp U3 promoter/enhancer deletion (U3D). Upon proviral integration into host genome, the U3 region containing the cHS4 is copied over to the 59 LTR. B) Evaluation of anti-CTLA4 expression on protein level for clones derived after transduction of MMC cells with LV-aCTLA4 (upper panel) or I-LV-aCTLA4 (lower panel). Solid bars: Dox induced expression. Clones were treated with Dox and 24 h later anti-CTLA4 was measured by ELISA in culture supernatants. Empty bars (to the right side of solid bars): non-induced expression levels: supernatant from clones w/o Dox treatment were analyzed by ELISA. (Note, that these bars are not visible for LV-aCTLA4). Anti-CTLA4 levels in the corresponding populations of transduced cells (Pop) are shown on the right. C) Fold change of anti- CTLA4 mRNA levels after culture with or without doxycycline for 24 h measured. Clones that expressed the highest levels of anti-CTLA4 protein were included in this analysis. mRNA was isolated and subjected to qRT-PCR for GAPDH and anti-CTLA4 mRNA. Shown is the fold difference of GAPDH normalized anti-CTLA4 mRNA levels with and without Dox induction. Standard deviation was less than 10%. doi:10.1371/journal.pone.0022303.g002 tTR-KRAB system [18]. tRT-KRAB bound to tet-operator sequences represses promoters in the vicinity of 3-4 kb. Addition of Dox releases this repression. The vector also contains a central polypurine tract (cPPT) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRC). In the insulated vector version (I-LV-aCTLA4), a 0.4 kb cHS4 insulator element [43] is inserted into the 398 bp U3 promoter/enhancer deletion (U3D). Upon proviral integration into host genome, the U3 region containing the cHS4 is copied over to the 59 LTR. Systemic application of anti-CTLA4 antibody Systemic application of anti-CTLA4 antibody Clinically, antiCTLA4 antibodies are administered systemically. For our studies in mouse models, we used a monoclonal antibody against murine CTLA4 (4F10). Systemic administration of this antibody has been shown before to trigger tumor-destructive immune responses in several mouse tumor models [24,25]. As in those studies, we started injecting anti-CTLA4 or control antibody when tumors reached a volume of 50 mm3. Injections were repeated every other day. While in the TC-1 tumor model, anti- CTLA4 injection significantly delayed tumor growth, it had no therapeutic effect in the MMC tumor model (Figs. 1A, B). These studies indicate that anti-CTLA acts differently in tolerized and non-tolerized tumor models. Blood analysis Mouse blood was analyzed using a HemaVet 950FS machine. PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e22303 5 Anti-CTLA4 Antibody Therapy Figure 3. Growth and analysis of tumors derived from tumor cells transduced with LV-aCTLA4. A and B) MMC-aCTLA4 cells were inje and Dox treatment was started when tumors reached a volume of 50 mm3. Kaplan-Meier survival studies (cut-off is 1000 mm3). Dox was deliv with i.p. injection (A) or drinking water (B). N = 5. C) Flow cytometry of tumor infiltrating leukocytes and splenocytes in the MMC-aCTLA4 mode day 30, tumors, tumor-draining lymph nodes and spleens were harvested. The percentages of Neu specific CD8+ T-cells were measured by tetra assay. Shown are the average percentages of marked cells in all TILs, lymph node cells and splenocytes. Standard deviations were less than N = 3. D) TC-aCTLA4 cells were injected and Dox treatment was started when tumors reached a volume of 50 mm3. Kaplan-Meier survival studies off is 1000 mm3). Dox was delivered with drinking water. N = 5. doi:10.1371/journal.pone.0022303.g003 Figure 3. Growth and analysis of tumors derived from tumor cells transduced with LV-aCTLA4. A and B) MMC-aCTLA4 cells were injected and Dox treatment was started when tumors reached a volume of 50 mm3. Kaplan-Meier survival studies (cut-off is 1000 mm3). Dox was delivered with i.p. injection (A) or drinking water (B). N = 5. C) Flow cytometry of tumor infiltrating leukocytes and splenocytes in the MMC-aCTLA4 model. At day 30, tumors, tumor-draining lymph nodes and spleens were harvested. The percentages of Neu specific CD8+ T-cells were measured by tetramer assay. Shown are the average percentages of marked cells in all TILs, lymph node cells and splenocytes. Standard deviations were less than 10%. N = 3. D) TC-aCTLA4 cells were injected and Dox treatment was started when tumors reached a volume of 50 mm3. Kaplan-Meier survival studies (cut- off is 1000 mm3). Dox was delivered with drinking water. N = 5. doi:10.1371/journal.pone.0022303.g003 Figure 3. Growth and analysis of tumors derived from tumor cells transduced with LV-aCTLA4. A and B) MMC-aCTLA4 cells were injected and Dox treatment was started when tumors reached a volume of 50 mm3. Kaplan-Meier survival studies (cut-off is 1000 mm3). Dox was delivered with i.p. injection (A) or drinking water (B). N = 5. C) Flow cytometry of tumor infiltrating leukocytes and splenocytes in the MMC-aCTLA4 model. Blood analysis At day 30, tumors, tumor-draining lymph nodes and spleens were harvested. The percentages of Neu specific CD8+ T-cells were measured by tetramer assay. Shown are the average percentages of marked cells in all TILs, lymph node cells and splenocytes. Standard deviations were less than 10%. N = 3. D) TC-aCTLA4 cells were injected and Dox treatment was started when tumors reached a volume of 50 mm3. Kaplan-Meier survival studies (cut- off is 1000 mm3). Dox was delivered with drinking water. N = 5. doi:10.1371/journal.pone.0022303.g003 July 2011 \| Volume 6 \| Issue 7 \| e22303 PLoS ONE \| www.plosone.org 6 Anti-CTLA4 Antibody Therapy PLoS ONE \| www.plosone.org 7 July 2011 \| Volume 6 \| Issue 7 \| e223 PLoS ONE \| www.plosone.org 7 July 2011 \| Volume 6 \| Issue 7 \| e22303 PLoS ONE \| www.plosone.org Anti-CTLA4 Antibody Therapy Figure 4. Therapy study with LV-aCTLA4/I-LV-aCTLA4 transduced HSCs and induction of transgene expression by Dox in drinking water. A) Scheme of the experiment: A total of 56105 LV-transduced mouse HSCs were transplanted into lethally irradiated neu-tg mice via tail injection. Six weeks after HSCs engraftment, MMC tumors were established via injection of 56105 MMC cells subcutaneously. Selected groups of mice received Dox in drinking water. B) Therapy study with mice that were transplanted with LV-aCTLA4 or I-LV-aCTLA4 transduced mouse HSCs; Tx(LV- aCTLA4, and Tx(I-LV-aCTLA4), respectively. Dox (0.2 mg/ml) was added to drinking water of selected groups starting at day 1 after MMC cell implantation. Each line represents an individual animal. C) Survival of MMC tumor bearing mice. The day tumors reached a volume of 900 mm3 represented the endpoint in Kaplan-Meier survival studies. N.5. doi:10.1371/journal.pone.0022303.g004 aCTLA4) by inserting the 0.4 kb cHS4 into the 39 LTR. In the integrated I-LV-aCTLA4 provirus DNA, the transgene cassette is therefore flanked by two HS4 insulators (Fig. 2A, lower panel). A corresponding vector without cHS4 insulators was called LV- aCTLA4) by inserting the 0.4 kb cHS4 into the 39 LTR. In the integrated I-LV-aCTLA4 provirus DNA, the transgene cassette is therefore flanked by two HS4 insulators (Fig. 2A, lower panel). A corresponding vector without cHS4 insulators was called LV- control region DNase I hypersensitivity 4 region (cHS4) protects retrovirus vectors from chromosomal position effects of integration and from silencing, particularly in HSCs and their progeny [27,28], we constructed an ‘‘insulated’’ SIN vector (I-LV- Figure 5. HSC based delivery of anti-CTLA4 gene y g Because viral gene transfer to epithelial tumors is inefficient, we employed a new stem cell based approach to deliver the anti- CTLA4 gene to the tumor [20]. Both types of tumors have epithelial features including various intercellular junctions and extracellular matrix surrounding tumor nests. These physical barriers limit the efficacy of gene delivery using virus-based vectors [29,30]. Because of this, stem cell based gene delivery approaches have been evaluated. In this context, the tropism of mesenchymal stem cells (MSC) for tumors has been exploited to deliver anti- tumor cytokine genes using ex vivo gene-modified MSCs [31]. Furthermore, based on the finding that monocytes/macrophages have the ability to migrate within tissues, even in hypoxic microenvironments, genetically modified monocytes/macrophag- es or progenitors have been used to delivery therapeutic genes to tumors [32]. We have previously developed an approach based on hematopoietic stem cells (HSCs) for in vivo gene delivery [20]. This approach is based on the fact that tumor cells secrete a number of chemokines that actively mobilize myeloid progenitors from the bone marrow and recruit them to the tumor stroma, where they differentiate into tumor-associated macrophages (TAMs). TAMs are critical for tumor survival as they produce factors that trigger/ support tumor growth, neoangiogenesis, immune escape and stroma development. Our approach involves the ex vivo transduc- tion of bone marrow derived HSCs with lentivirus vectors that express the transgene under control of a Doxycyline (Dox)- inducible transcription cassette, and the transplantation of these cells into myelo-conditioned recipients, where they engraft in the bone marrow and provide a long-term source of genetically modified cells that will home to tumors. This approach allows for efficient transgene delivery to the center of tumors. For example, in mice transplanted with HSCs transduced with a GFP expressing lentivirus vector, ,5% of all cells in MMC tumors were GFP positive, whereby most of the transgene expressing cells were TAMs [20,33]. In a recent study, we also showed that the inducible intratumoral expression of the peptide hormone relaxin after the transplantation of mouse HSCs transduced with a relaxin-expressing lentivirus vector, delayed tumor growth in the MMC-tumor model [20]. Here we used this ‘‘Trojan Horse’’ approach to deliver the anti-CTLA4 antibody gene to TC-1 and MMC tumors. For therapy studies in vivo, we used an MMC cell clone that stably expressed anti-CTLA4 under Dox control (clone 4 generated from LV-aCTLA4 transduced MMC cells; see Fig. 2B). Blood analysis This safety feature was built in to control potential side effects of anti-CTLA4 by expressing it only transiently. To functionally validate the vectors and assess the impact of the cHS4 insulators on position effects of integration, we infected MMC cells at an MOI of 1 cfu/cell and established clonal cultures by limited dilution. Anti-CTLA4 protein levels were measured in supernatants of the population (w/o subcloning) and 20 clones with and without Dox induction (Fig. 2B). There was no significant difference in induced anti-CTLA4 protein levels between I-LV-aCTLA4 transduced MMC cell clones and clones that were transduced with the non-insulated vector. As a more sensitive means to measure anti-CTLA4 expression we used qRT- PCR (Fig.2C). The increase in anti-CTLA4 mRNA levels upon Dox treatment was also not significantly greater in I-LV-aCTLA4 transduced clones compared to LV-aCTLA4 clones (p = 0.06). Overall, these data show that the inclusion of chromatin insulators into lentivirus vectors did not improve Dox mediated regulation of transgene expression. Blood analysis Therapy study with I-LV-aCTLA4 transduced HSCs and induction of transgene expression by Dox by intraperitoneal injection. Treatment scheme was as described in Fig.2. Mice received an i.p. injection of PBS or Dox (0.5 mg/mouse in 500 ml PBS) starting at day 7 after MMC cell transplantation and then every other day. A) Tumor volumes, B) Kaplan-Meier survival study (cut-off volume was 700 mm3). N.5. doi:10.1371/journal.pone.0022303.g005 Figure 5. Therapy study with I-LV-aCTLA4 transduced HSCs and induction of transgene expression by Dox by intraperitoneal injection. Treatment scheme was as described in Fig.2. Mice received an i.p. injection of PBS or Dox (0.5 mg/mouse in 500 ml PBS) starting at day 7 after MMC cell transplantation and then every other day. A) Tumor volumes, B) Kaplan-Meier survival study (cut-off volume was 700 mm3). N.5. doi:10.1371/journal.pone.0022303.g005 July 2011 \| Volume 6 \| Issue 7 \| e22303 PLoS ONE \| www.plosone.org 8 Anti-CTLA4 Antibody Therapy aCTLA4. The anti-CTLA4 antibody expression cassette can be activated by the addition of Dox, and Dox withdrawal ceases anti- CTLA4 antibody expression. This safety feature was built in to control potential side effects of anti-CTLA4 by expressing it only transiently. To functionally validate the vectors and assess the impact of the cHS4 insulators on position effects of integration, we infected MMC cells at an MOI of 1 cfu/cell and established clonal cultures by limited dilution. Anti-CTLA4 protein levels were measured in supernatants of the population (w/o subcloning) and 20 clones with and without Dox induction (Fig. 2B). There was no significant difference in induced anti-CTLA4 protein levels between I-LV-aCTLA4 transduced MMC cell clones and clones that were transduced with the non-insulated vector. As a more sensitive means to measure anti-CTLA4 expression we used qRT- PCR (Fig.2C). The increase in anti-CTLA4 mRNA levels upon Dox treatment was also not significantly greater in I-LV-aCTLA4 transduced clones compared to LV-aCTLA4 clones (p = 0.06). Overall, these data show that the inclusion of chromatin insulators into lentivirus vectors did not improve Dox mediated regulation of transgene expression. T cells [16]. Again these studies suggest that anti-CTLA4 has no therapeutic effect in tolerized tumor models, although it appears to increase the number of intratumoral effector T-cells. aCTLA4. The anti-CTLA4 antibody expression cassette can be activated by the addition of Dox, and Dox withdrawal ceases anti- CTLA4 antibody expression. HSC based delivery of anti-CTLA4 gene A) Tumors infiltrating leukocytes and splenocytes were subjected to flow cytometry for Cd1d, CD4, CD25, and NK1.1. N = 3. B) Tumors were sectioned and stained with antibodies against CD1d (red) and CD4 (green) (upper panel) or CD1d (red) and NK1.1 (green) (lower panel). Representative sections are shown. The scale bar is 40 mm. C) Flow cytometry analysis of splenocytes. N = 3. D) Immunofluorescence analysis of spleen sections. upper panel: CD1d (red) and CD4 (green); lower panel: CD1d (red) and NK1.1 (green). doi:10.1371/journal.pone.0022303.g007 Figure 7. Analysis of immune cells in tumors and spleens of mice treated with I-LV-aCTLA4 transduced HSCs and induction of transgene expression by Dox by intraperitoneal injection. At day 28, tumors and spleens from Tx(I-LV-aCTLA4) and Tx(I-LV-aCTLA4)+Dox mice were harvested and analyzed. A) Tumors infiltrating leukocytes and splenocytes were subjected to flow cytometry for Cd1d, CD4, CD25, and NK1.1. N = 3. B) Tumors were sectioned and stained with antibodies against CD1d (red) and CD4 (green) (upper panel) or CD1d (red) and NK1.1 (green) (lower panel). Representative sections are shown. The scale bar is 40 mm. C) Flow cytometry analysis of splenocytes. N = 3. D) Immunofluorescence analysis of spleen sections. upper panel: CD1d (red) and CD4 (green); lower panel: CD1d (red) and NK1.1 (green). doi:10.1371/journal.pone.0022303.g007 As a source of HSCs, we used bone marrow cells from mice that were injected intravenously with 5-FU (150 mg/kg) two days prior to the collection of bone marrow. Bone marrow cells were cultured for three days and non-adherent cells (enriched for HSCs and primitive progenitors) were mock-transduced or transduced with LV-aCLTA4 or I-LV-aCTLA4 at an MOI of 1 cfu/cell. An aliquot of transduced HSCs was used to confirm successful transduction and anti-CTLA4 expression by qRT-PCR on cells cultured for 2 days. The rest of the cells were transplanted into lethally irradiated neu-tg mice. Six weeks later, subsequent to the bone marrow engraftment of genetically modified cells, mice were subcutaneously injected with MMC cells and Dox was given in drinking water to selected groups of animals (for a schematic of the experiment see (Fig. 4A). Mice were followed for 35 days. Animals were sacrificed before the end of the observation period if the tumors reached a volume of 1,000 mm3 or ulcerated. In explanted tumors, anti-CTLA4 mRNA was measured by qRT-PCR. HSC based delivery of anti-CTLA4 gene The outcome of studies in the TC-1 tumor model was similar. subset expressing both TCR and NK cell receptors. Most NKT cells are restricted by the MHC class I–like molecule CD1d. In the mouse, most CD1d+ NKT cells are CD4+. An involvement of NKT in mediating tolerance to self-antigens and suppressing auto-immune inflammatory reactions has been reported in experimental and human autoimmune diseases [36]. A potential pathway that leads to immunosuppression involves the secretion of IL-13 by NKT cells and subsequent activation of Gr- 1+CD11b+ myeloid suppressor cells, which in turn produce TGF-b1 [37]. In agreement with earlier studies [16], we found less CD4/CD25+ Tregs in I-LV-aCTLA+Dox tumors and spleen than in I-LV-aCTLA tumors (Figs. 7A and C). Importantly, however, both flow cytometry analysis of TILs (Fig.7A) and immunofluorescence analysis of tumor section (Fig.7B) showed significantly more CD1d+ cells in TILs of mice where anti-CTLA4 production was induced by Dox than in Tx(LV-aCTLA4) mice without Dox treatment and control mice that received mock transplantation (p = 0.026). The majority of CD1d+ cells in Tx(I-LV-aCTLA+Dox) tumors were CD4 cells (Fig.7A). Flow cytometry data were supported by costaining of tumor sections for CD1d and CD4 or NK.1.1 (Fig.7B). Notably, there was no significant difference in the composition and percentages of MMC tumor infiltrating leukocytes in Tx(I-LV- aCTLA) mice (without Dox induction) and mice that did not receive a bone marrow transplantation. Analysis of splenocytes of treated animals also showed a significant difference in the percentage of CD1d+ of I-LV-aCTLA+Dox and I-LV-aCTLA animals (Fig.7C, D). Figure 7 shows data from studies in the MMC tumor model. The outcome of studies in the TC-1 tumor model was similar. To further elucidate mechanisms of failure of HSC-based anti- CTLA4 therapy, we analyzed the expression of cytokines and chemokines in tumors. As TGF-b1 potentially mediates the immunosuppressive effect of CD11d+ NKT cells, we measured TGF-b1 mRNA by qRT-PCR in TC1 and TC1-aCTLA4+Dox tumors (Fig.8A). We found 49+/25-fold higher concentrations of TGF-b1 mRNA in TC1-aCTLA4+Dox tumors than in TC-1 tumors. RNA levels of TGF-b2 and TGF-b3, i.e. cytokines that are not involved in immunosuppression did not differ between the two groups. Other cyto- and chemokines were analyzed by commercial proteome arrays. (These arrays did not cover TGF-b). The outcome of these studies was consistent for the TC-1 and MMC tumor models. HSC based delivery of anti-CTLA4 gene In mice that received LV-aCTLA4 transduced HSCs, tumor anti-CTLA4 mRNA levels were 36-fold (+/25) higher in Dox-treated mice compared to mice that received drinking water without Dox. There was no significant difference in anti-CTLA4 levels between LV-aCTLA4 and I-LV-aCTLA4 (Fig. 4B). Tumor volumes of individual mice and Kaplan-Meier survival studies are shown in Fig.4C. Unexpectedly, Dox treatment, i.e. induction of anti- CTLA4 expression in tumors, shortened the survival of mice (Tx(I- LV-aCTLA4) + Dox vs Tx(I-LV-aCTLA4): p = 0.029 and Tx(LV- aCTLA4) +Dox vs Tx(LV-aCTLA4): p = 0.095). Dox treatment of mock-transplanted mice did not affect MMC tumor growth [20]. To consolidate these findings, we performed a second experiment where Dox was given intraperitoneally to better control its delivery to mice (Fig. 5). As seen before, Dox-induced anti-CTLA expression did not exert therapeutic effects and shortened the life-span of animals. subset expressing both TCR and NK cell receptors. Most NKT cells are restricted by the MHC class I–like molecule CD1d. In the mouse, most CD1d+ NKT cells are CD4+. An involvement of NKT in mediating tolerance to self-antigens and suppressing auto-immune inflammatory reactions has been reported in experimental and human autoimmune diseases [36]. A potential pathway that leads to immunosuppression involves the secretion of IL-13 by NKT cells and subsequent activation of Gr- 1+CD11b+ myeloid suppressor cells, which in turn produce TGF-b1 [37]. In agreement with earlier studies [16], we found less CD4/CD25+ Tregs in I-LV-aCTLA+Dox tumors and spleen than in I-LV-aCTLA tumors (Figs. 7A and C). Importantly, however, both flow cytometry analysis of TILs (Fig.7A) and immunofluorescence analysis of tumor section (Fig.7B) showed significantly more CD1d+ cells in TILs of mice where anti-CTLA4 production was induced by Dox than in Tx(LV-aCTLA4) mice without Dox treatment and control mice that received mock transplantation (p = 0.026). The majority of CD1d+ cells in Tx(I-LV-aCTLA+Dox) tumors were CD4 cells (Fig.7A). Flow cytometry data were supported by costaining of tumor sections for CD1d and CD4 or NK.1.1 (Fig.7B). Notably, there was no significant difference in the composition and percentages of MMC tumor infiltrating leukocytes in Tx(I-LV- aCTLA) mice (without Dox induction) and mice that did not receive a bone marrow transplantation. Analysis of splenocytes of treated animals also showed a significant difference in the percentage of CD1d+ of I-LV-aCTLA+Dox and I-LV-aCTLA animals (Fig.7C, D). Figure 7 shows data from studies in the MMC tumor model. HSC based delivery of anti-CTLA4 gene MMC-aCTLA4 cells were injected into neu-tg mice. When tumors reached a volume of 50 mm3, Dox was given either intraperitoneally (Fig. 3A) or in drinking water (Fig. 3B) to half of the mice. Induction of anti-CTLA4 expression in vivo was confirmed on the mRNA level by qRT-PCR and protein level by ELISA with tumor lysates (data not shown). Dox induction of anti-CTLA4 expression in MMC-aCTLA4 cells in vivo did not prolong survival in both models. Flow cytometry analysis revealed significantly higher percentage of Neu-specific CD8 cells in the tumor and tumor-infiltrating lymph nodes in the MMC- aCTLA4+Dox group compared to the corresponding group that did not receive Dox (Fig. 3C). Interestingly, Dox induced anti- CTLA4 expression appeared to increase the percentage of NK cells in the tumor and spleen. There was no significant difference in the number of CD4/CD25 cells, i.e Tregs. For studies with the TC-1 model, we selected a TC1-aCTLA4 cell clone that produced similar anti-CTLA4 levels as the MMC- aCTLA4 clone upon Dox induction. In contrast to the study in the MMC model, anti-CTLA4 expression from TC-1 cells resulted in a significant delay in tumor growth (Fig. 3D). This is in agreement with an earlier study, in which we also showed that the anti-tumor effect is mediated by an increase of tumor-infiltrating IFNc-producing CD8+ Figure 6. HSC-based anti-CTLA4 gene therapy in the TC-1 tumor model. Mice were treated as described in Fig. 4A. The experiment was terminated at day 18. A) Tumors were excised and measured. N = 5. B) Representative excised tumors. doi:10.1371/journal.pone.0022303.g006 Figure 6. HSC-based anti-CTLA4 gene therapy in the TC-1 tumor model. Mice were treated as described in Fig. 4A. The experiment was terminated at day 18. A) Tumors were excised and measured. N = 5. B) Representative excised tumors. doi:10.1371/journal.pone.0022303.g006 PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e22303 9 Anti-CTLA4 Antibody Therapy Anti-CTLA4 Antibody Therapy PLoS ONE \| www.plosone.org 10 July 2011 \| Volume 6 \| Issue 7 \| e22303 PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e22303 PLoS ONE \| www.plosone.org 10 Anti-CTLA4 Antibody Therapy Figure 7. Analysis of immune cells in tumors and spleens of mice treated with I-LV-aCTLA4 transduced HSCs and induction of transgene expression by Dox by intraperitoneal injection. At day 28, tumors and spleens from Tx(I-LV-aCTLA4) and Tx(I-LV-aCTLA4)+Dox mice were harvested and analyzed. PLoS ONE \| www.plosone.org Discussion Increased understanding of immune-regulatory mechanisms is required for the development of new immunotherapy agents that can modulate these signaling pathways and potentially break tumor tolerance. In this study, we report two findings: i) systemic delivery of anti-CTLA4 antibodies or intratumoral expression has different effects in non-tolerized and tolerized mouse tumor models and ii) HSC-mediated anti-CTLA4 expression triggers immunosuppressive mechanisms, which facilitate tumor progres- sion. There is an emerging picture that the same mechanisms that prevent autoimmunity also inhibit anti-tumor immune responses. The central problem in cancer immunotherapy is that most TAAs are non-mutated self-antigens that have triggered both central and peripheral tolerance. It is therefore important to test new immunotherapy approaches in mouse models, in which tolerance against the inoculated tumor and specific TAAs exist. Central tolerance is established by selection in the thymus: T-cells bearing T-cell receptors with high affinity for self-antigen are eliminated through apoptosis [38]. Additionally, peripheral T-cell tolerance is required to suppress the remaining auto-reactive T-cells in the periphery. It remains in question, why expression of anti-CTLA4 from TC-1 cells had a positive therapeutic effect, while anti-CTLA4 expression after HSC transplantation into C57Bl/6 mice did not. Progeny of transplanted HSCs not only home to tumors and differentiate into TAMs, but also reconstitute spleen, thymus, and, potentially, macrophages in other tissues [20]. This implies that anti-CTLA4 is also expressed in non-tumoral tissue, which could have accounted for the unexpected outcome described above. Based on recent evidence that TAMs have a unique gene expression signature that distinguishes it from other tissue macrophages [42], we are currently working on TAM-specific expression systems to increase the tumor-specificity of transgene expression. One goal of this study was to evaluate the anti-tumor effect of anti-CTLA4 in a mouse model that resembled key features of breast cancer in patients, most importantly tolerance to a TAA (Neu) and the presence of Neu-reactive T-cells. Anti-CTLA4 delivery as a protein or expression from gene-modified tumor cells were therapeutically efficacious in the non-tolerized TC-1 tumor model, but had no effect in the MMC-model, in spite of the fact that anti-CTLA4 expression from MMC tumors increased the number of Neu-specific T-cells. Neu-tg mice are tolerant to Neu. It has been discussed that in this model, subpopulations of high- avidity Neu-specific T cells are deleted centrally, whereas T-cells with lower avidity can leave the thymus but are subject to peripheral mechanisms of tolerance [22,39]. 1. Leach DR, Krummel MF, Allison JP (1996) Enhancement of antitumor immunity by CTLA-4 blockade. Science 271: 1734–1736. Discussion This implies that tolerance to Neu involves both central and peripheral mechanisms. This finding suggests that the central tolerance mechanism must be overcome to enable intra-tumoral Neu-specific T-cells to kill tumor cells. Overall, these findings suggest that stem cell based delivery methods, particularly for immuno-stimulatory genes, must ensure homing of stem cells to tumors or exclusive expression within tumors. The differences in outcomes between the tolerized and non-tolerized models also provide a potential explanation for the low efficacy of CTLA4 blockage approaches in cancer immuno- therapy trials. The model involving ex vivo transduced tumor cells is clinically not relevant. We therefore assessed an approach that would allow in vivo delivery of the anti-CTLA4 gene to the tumor. While viral gene delivery to epithelial tumors is inefficient after systemic application, recently a number of stem cell-based approaches have shown more promise. Our stem cell gene delivery approach is based on the ex vivo modification of HSCs, which home to the tumor after transplantation and deliver therapeutic transgenes to HSC based delivery of anti-CTLA4 gene Anti-TGF–b RNA was equalized to levels of GAPDH mRNA measured in parallel in each sample. Shown is the fold increase in Tx(I-LV-aCTLA4)+Dox vs Tx(Mock) groups in the TC-1 model. N = 3. The outcome in the MMC model was similar. B–C) cytokine proteome array. Tumor lysates were pooled and analyzed for the indicated cytokines using the Proteome Profiler from R&D Systems. B) Assembled images of dot blots for key cytokines from MMC and TC-1 tumors. C) (TC1) and D) (MMC). The plots were scanned and signal intensity was measured. BLC = CXCL13, I-309 = CCL1, eotaxin = CCL11, IP-10 = CXCL10, JE = CCL2, MIG = CXCL9, MIP1a = CCL3, MIP1b = CCL4, RANTES = CCL5, SDF-1 = CXCL12, TARC = CCL-17. The array includes positive controls provided by the manufacturer. doi:10.1371/journal.pone.0022303.g008 the tumor stroma. For HSC transduction, we used insulated SIN lentivirus vector with Dox-inducible transgene expression. In recent studies, we used the HSC-based approach to deliver the relaxin gene to tumors [20,40]. In the MMC tumor model we showed that this approach facilitates pre-existing anti-tumor T- cells to control tumor growth. Furthermore, in xenograft models with Her2/neu positive breast cancer cells, HSC-mediated relaxin expression improved the anti-tumor efficacy of trastuzumab/ Herceptin [33]. However, unexpectedly, when used for anti- CTLA4 gene delivery in this study, the HSC-gene approach was therapeutically detrimental in both the TC-1 and MMC- models. Anti-CTLA4 expression in these models triggered at least two reactions. It increased the percentage of CD1d+ NKT cells in tumors. These cells can theoretically activate myeloid suppressor cells, which in turn, produce TGF-b1. In support of this, we found ,50-fold higher TGF-b1 mRNA levels in tumors of mice that received anti-CTLA4-HSC gene therapy. Additionally, HSC- based anti-CTLA4 gene delivery resulted in lower intratumoral levels of cytokines (e.g. IL-1b) and chemokines (e.g. MIG, MIP-1, RANTES) that stimulate immune responses. Proinflammatory cytokines such as IL-1b create a milieu in the tumor that is supportive for the activation of T- effector cells. Furthermore, a number of cells of the immune system, including pre-cDCs, show positive chemotaxis to CCL3 in a dose-dependent manner [41]. tumor immune responses and attraction of T-cells in anti-CTLA4 expressing tumors. 2. Egen JG, Kuhns MS, Allison JP (2002) CTLA-4: new insights into its biological function and use in tumor immunotherapy. Nat Immunol 3: 611–618. Author Contributions Conceived and designed the experiments: JP SR AL. Performed the experiments: JO IB RY ZY. Analyzed the data: JP IB SR AL. Contributed reagents/materials/analysis tools: SR H-PK. Wrote the paper: AL. HSC based delivery of anti-CTLA4 gene In both models, HSC-mediated anti- CTLA4 expression resulted in a marked decrease of pro- inflammatory cyto- and chemokines in tumors, including IL-1b, MIG, MIP-1a, MIP-1b, and RANTES (Figs. 8B-D). In the TC-1 model, so far, injection of anti-CTLA4 or expression from TC-1 cells significantly delayed tumor growth. However, when we employed the HSC-based approach for in vivo expression of anti-CTLA4, we found a marked stimulation of TC- 1 tumor growth upon Dox induction of anti-CTLA4 expression. Furthermore, tumor growth in the Tx(I-LV-aCTLA4 +Dox) group was more invasive involving subcutaneous muscle tissues. Because of this, it was impossible to measure tumor volumes over time. We therefore show the tumor volumes at the end of the observation period (day 35) (Fig. 6A). Representative explanted tumors are show in Fig.6B. In summary, when the anti-CTLA4 gene was delivered using the HSC-based approach, it stimulated tumor growth in both tumor models. Notably, in all therapy studies, there were no signs of auto- immune responses such as changes in fur color or presence of inflammatory infiltrates on tissue sections of liver, lung or colon. Furthermore, immunohistochemistry staining for IgG complexes on kidney sections did not reveal abnormalities. Blood cell counts were normal in all groups of both models. Mechanism of failure of HSC-based anti-CTLA4 therapy To understand why anti-CTLA4 in these models did not suppress tumor growth, we performed flow cytometry and immunofluorescence analyses of immune cells in the spleen and the tumors. In addition to standard analyses for CD4 T-cells, NK cells, and Tregs, we also searched for changes in potential immunosuppressive cells. Among the latter is a specialized subset of NKT cells [10,34,35]. NKT cells are a unique T-cell In summary, anti-CTLA4 expression from HSC progeny increases the percentage of CD4+/CD1d+ cells in tumors, which correlates with increased production of TGF-b1. Additionally, we found less cytokines that are involved in the activation of anti- PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e22303 11 Anti-CTLA4 Antibody Therapy PLoS ONE \| www.plosone.org 12 July 2011 \| Volume 6 \| Issue 7 \| e22303 July 2011 \| Volume 6 \| Issue 7 \| e22303 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org 12 Anti-CTLA4 Antibody Therapy Figure 8. Analysis of intratumoral cytokine and chemokine expression. At day 28, TC-1 and MMC tumors from Tx(Mock) and Tx(I-LV- aCTLA4)+Dox were harvested. A) qRT-PCR for TGF-b RNA. References 2. Egen JG, Kuhns MS, Allison JP (2002) CTLA-4: new insights into its biological function and use in tumor immunotherapy. Nat Immunol 3: 611–618. PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e22303 PLoS ONE \| www.plosone.org 13 Anti-CTLA4 Antibody Therapy Anti-CTLA4 Antibody Therapy 3. Hurwitz AA, Foster BA, Kwon ED, Truong T, Choi EM, et al. (2000) Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade. Cancer Res 60: 2444–2448. 22. Knutson KL, Dang Y, Lu H, Lukas J, Almand B, et al. (2006) IL-2 immunotoxin therapy modulates tumor-associated regulatory T cells and leads to lasting immune-mediated rejection of breast cancers in neu-transgenic mice. J Immunol 177: 84–91. 4. van Elsas A, Hurwitz AA, Allison JP (1999) Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA- 4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation. J Exp Med 190: 355–366. 23. Stone D, Liu Y, Li ZY, Strauss R, Finn EE, et al. (2008) Biodistribution and safety profile of recombinant adeno-associated virus serotype 6 vectors following intravenous delivery. J Virol 82: 7711–7715. 24. Read S, Greenwald R, Izcue A, Robinson N, Mandelbrot D, et al. (2006) Blockade of CTLA-4 on CD4+CD25+ regulatory T cells abrogates their function in vivo. J Immunol 177: 4376–4383. by autoimmune depigmentation. J Exp Med 190: 355–366. 5. Agarwala SS, Ribas A (2010) Current experience with CTLA4-blocking monoclonal antibodies for the treatment of solid tumors. J Immunother 33: 557–569. 25. Quezada SA, Peggs KS, Curran MA, Allison JP (2006) CTLA4 blockade and GM-CSF combination immunotherapy alters the intratumor balance of effector and regulatory T cells. J Clin Invest 116: 1935–1945. 6. Camacho LH, Antonia S, Sosman J, Kirkwood JM, Gajewski TF, et al. (2009) Phase I/II trial of tremelimumab in patients with metastatic melanoma. J Clin Oncol 27: 1075–1081. 26. Shieh SJ, Chou FC, Yu PN, Lin WC, Chang DM, et al. (2009) Transgenic expression of single-chain anti-CTLA-4 Fv on beta cells protects nonobese diabetic mice from autoimmune diabetes. J Immunol 183: 2277–2285. 7. Attia P, Phan GQ, Maker AV, Robinson MR, Quezado MM, et al. (2005) Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4. J Clin Oncol 23: 6043–6053. 27. References Nat Rev Immunol 4: 231–237. 14. Lu H, Knutson KL, Gad E, Disis ML (2006) The tumor antigen repertoire identified in tumor-bearing neu transgenic mice predicts human tumor antigens. Cancer Res 66: 9754–9761. 35. Hegde S, Fox L, Wang X, Gumperz JE (2010) Autoreactive natural killer T cells: promoting immune protection and immune tolerance through varied interac- tions with myeloid antigen-presenting cells. Immunology 130: 471–483. 15. Knutson KL, Almand B, Dang Y, Disis ML (2004) Neu antigen-negative variants can be generated aftaer neu-specific antibody therapy in neu transgenic mice. Cancer Res 64: 1146–1151. 36. Teige A, Bockermann R, Hasan M, Olofsson KE, Liu Y, et al. (2010) CD1d- dependent NKT cells play a protective role in acute and chronic arthritis models by ameliorating antigen-specific Th1 responses. J Immunol 185: 345–356. 16. Tuve S, Chen BM, Liu Y, Cheng TL, Toure P, et al. (2007) Combination of tumor site-located CTL-associated antigen-4 blockade and systemic regulatory T-cell depletion induces tumor-destructive immune responses. Cancer Res 67: 5929–5939. 37. Terabe M, Berzofsky JA (2008) The role of NKT cells in tumor immunity. Adv Cancer Res 101: 277–348. 38. Li MO, Flavell RA (2008) TGF-beta: a master of all T cell trades. Cell 134: 392–404. 17. Griffin MD, Holman PO, Tang Q, Ashourian N, Korthauer U, et al. (2001) Development and applications of surface-linked single chain antibodies against T-cell antigens. J Immunol Methods 248: 77–90. 39. Tuve S, Liu Y, Tragoolpua K, Jacobs JD, Yumul RC, et al. (2009) In situ adenovirus vaccination engages T effector cells against cancer. Vaccine 27: 4225–4239. g 18. Szulc J, Wiznerowicz M, Sauvain MO, Trono D, Aebischer P (2006) A versatile tool for conditional gene expression and knockdown. Nat Methods 3: 109–116. 40. Beyer I, Li Z, Persson J, Liu Y, van Rensburg R, et al. (2010) Controlled Extracellular Matrix Degradation in Breast Cancer Tumors Improves Therapy by Trastuzumab. Mol Ther. 19. Malik P, Arumugam PI, Yee JK, Puthenveetil G (2005) Successful correction of the human Cooley’s anemia beta-thalassemia major phenotype using a lentiviral vector flanked by the chicken hypersensitive site 4 chromatin insulator. Ann N Y Acad Sci 1054: 238–249. y 41. Diao J, Zhao J, Winter E, Cattral MS (2010) Recruitment and differentiation of conventional dendritic cell precursors in tumors. J Immunol 184: 1261–1267. 42. References Emery DW, Yannaki E, Tubb J, Stamatoyannopoulos G (2000) A chromatin insulator protects retrovirus vectors from chromosomal position effects. Proc Natl Acad Sci U S A 97: 9150–9155. 8. Khan S, Burt DJ, Ralph C, Thistlethwaite FC, Hawkins RE, et al. (2011) Tremelimumab (anti-CTLA4) mediates immune responses mainly by direct activation of T effector cells rather than by affecting T regulatory cells. Clin Immunol 138: 85–96. 28. Li CL, Emery DW (2008) The cHS4 chromatin insulator reduces gammare- troviral vector silencing by epigenetic modifications of integrated provirus. Gene Ther 15: 49–53. 29. Strauss R, Sova P, Liu Y, Li Z-Y, Tuve S, et al. (2009) Epithelial phenotype of ovarian cancer mediates resistance to oncolytic adenoviruses. Cancer Research 15: 5115–5125. 9. Kavanagh B, O’Brien S, Lee D, Hou Y, Weinberg V, et al. (2008) CTLA4 blockade expands FoxP3+ regulatory and activated effector CD4+ T cells in a dose-dependent fashion. Blood 112: 1175–1183. 30. Strauss R, Lieber A (2009) Anatomical and physical barriers to tumor targeting with oncolytic adenoviruses in vivo. Curr Opin Mol Ther 11: 513–522. 10. Yamaguchi T, Sakaguchi S (2006) Regulatory T cells in immune surveillance and treatment of cancer. Semin Cancer Biol 16: 115–123. 31. Klopp AH, Gupta A, Spaeth E, Andreeff M, Marini F, 3rd (2011) Concise review: Dissecting a discrepancy in the literature: do mesenchymal stem cells support or suppress tumor growth? Stem Cells 29: 11–19. 11. Ko K, Yamazaki S, Nakamura K, Nishioka T, Hirota K, et al. (2005) Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor- infiltrating Foxp3+CD25+CD4+ regulatory T cells. J Exp Med 202: 885–891. pp pp g 32. De Palma M, Naldini L (2009) Tie2-expressing monocytes (TEMs): novel targets and vehicles of anticancer therapy? Biochim Biophys Acta 1796: 5–10. 12. Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakaguchi S (2002) Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self- tolerance. Nature Immunol 3: 135–142. 33. Beyer I, Li Z, Persson J, Liu Y, van Rensburg R, et al. (2011) Controlled extracellular matrix degradation in breast cancer tumors improves therapy by trastuzumab. Mol Ther 19: 479–489. 13. Shimizu J, Yamazaki S, Sakaguchi M (1999) Induction of tumor immunity by removing CD25+CD4+ T cells: a acommon basis between tumor immunity and autoimmunity. J Immunol 163: 5211–5218. 34. Godfrey DI, MacDonald HR, Kronenberg M, Smyth MJ, Van Kaer L (2004) NKT cells: what’s in a name? References Stearman RS, Dwyer-Nield L, Grady MC, Malkinson AM, Geraci MW (2008) A macrophage gene expression signature defines a field effect in the lung tumor microenvironment. Cancer Res 68: 34–43. 20. Li Z, Liu Y, Tuve S, Xun Y, Fan X, et al. (2009) Toward a stem cell gene therapy for breast cancer. Blood 113: 5423–5433. 43. Aker M, Tubb J, Groth AC, Bukovsky AA, Bell AC, et al. (2007) Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects. Hum Gene Ther 18: 333–343. 21. Knutson KL, Lu H, Stone B, Reiman JM, Behrens MD, et al. (2006) Immunoediting of cancers may lead to epithelial to mesenchymal transition. J Immunol 177: 1526–1533. PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e22303 14
https://openalex.org/W2888919601	https://inria.hal.science/hal-02125757/document	English	null	ForenCity: A Playground for Self-Motivated Learning in Computer Forensics	IFIP advances in information and communication technology	2,018	cc-by	5,358	University of Johannesburg, Johannesburg, South Africa {fblauw,wsleung}@uj.ac.za Abstract. Striking a balance between theory and practice in computer forensics education is considered essential to producing successful grad- uates with the necessary skills to take on cybersecurity challenges in the workplace. Adequately incorporating both such aspects can be particu- larly challenging, especially in courses or modules oered within a short time-frame. In such situations, preparing the students will require that they are incentivized to actively engage with the extensive background learning material and remain current on latest developments to correctly grasp the theoretical underpinning of the subject. In this paper, we de- scribe the development of an adventure game to make the learning of applicable theory attractive and relevant. ForenCity takes the form of a web-based scavenger hunt in which students must apply their knowledge of computer forensics and correctly process digital evidence and progress through the game. Keywords: Game-based learning · Self-motivated learning · Computer forensics education. ForenCity: A Playground for Self-Motivated Learning in Computer Forensics Frans F. Blauw[0000−0001−5866−8335] and Wai Sze Leung[0000−0002−9015−6329] Frans F. Blauw[0000−0001−5866−8335] and Wai Sze Leung[0000−0002−9015−6329 1 Introduction A signicant challenge that educators encounter when teaching computer foren- sics is being able to cover the subject matter adequately. Much debate exists on the topic of what content should go into a computer or digital forensics cur- riculum, with numerous research eorts dedicated to the topic [1, 2]. Critics of training-based courses argue that teaching the subject as a series of steps to be followed in a laboratory produces graduates lacking the theoretical underpinning of the tasks involved [2]. Similarly, academics placing too much emphasis on the- oretical knowledge may result in digital investigators sorely lacking concerning practical experience [3]. As cyberthreats grow increasingly sophisticated [4], attempts to achieve a cybsersecure society may depend on educators being able to produce profession- als suciently skilled in identifying, collecting, preserving, and analyzing digital artifacts [5]. Understanding how cybercriminals operate is essential for informing cybersecurity experts in their tasks [5]. As a branch of computer science, computer forensics naturally requires an understanding of computer systems, their underlying technology, and how these F.F. Blauw and W.S. Leung 2 technologies work [2]. This prerequisite knowledge, which not all students may necessarily possess must be met as part of the curriculum. However, in cases where the oering is limited to a short time frame, covering this knowledge may be a luxury that is ill-aorded, prompting students to acquire this knowledge elsewhere either in other modules or by engaging in self-study. Success in com- puter forensics can, therefore, depend on how educators nd ways to encourage their students in actively taking charge of their learning. One potential and popular strategy is to adopt a game-based approach and deliver the content so that students learn abstract concepts and explore digi- tal forensic processes and technologies in a much more interactive manner [6]. Such an approach represents an attractive option as the learning process allows students to overcome dierent challenges [7]. A second approach (not unique to computer forensics), is the adoption of blended learning [8] where the best of both physical and digital worlds are com- bined to deliver education services and grant students greater control over their own studying [9]. The leveraging of mobile technology in several initiatives have demonstrated that the combination of both strategies above can be quite successful. 1 Introduction In one case, a teacher was able to take their class on a virtual tour of Africa [10] while another oered users an interactive, guided tour of the less popular points of interest on campus [11]. Inspired by such cases, we propose the development of ForenCity, a mobile adventure game in which players must draw on cross-disciplinary techniques and knowledge to investigate a case. While systems for developing mobile adventures (such as ARIS) already exist, the need to develop our own arose from two short- comings, namely (i) the need to promote variations and encourage independent problem solving amongst students, (ii) who primarily owned Android smart de- vices. This paper thus reports specically on our process of designing and develop- ing such a game, including the game engine. Section 2 describes specic require- ments for the use of games in achieving active learning, leading to details of the design of our new game engine in Section 3. Section 4 details the implementation of our game system while Section 5 describes how a particular game oering was presented using ForenCity. Section 6 discusses plans for future implementation improvements to the system while Section 7 concludes the paper. 3.1 Design Inuence First released in May 1984, the Adventure Game Interpreter (AGI) was a high- level game engine built by Sierra On-Line to build adventure games for series such as King's Quest, Police Quest, Space Quest, and Leisure Suit Larry [13]. Following in 1987, LucasArts developed the Script Creation Utility for Ma- niac Mansion (SCUMM) to ease the development of Maniac Mansion, and later the rst of the Monkey Island series [14]. In 1997, Chris Jones released Adventure Game Studio (AGS) that provides the tools to make your own adventure, for free![15]. All these engines (AGI, SCUMM, and AGS) use locations, characters, items, dialogue, and a basic custom scripting as building blocks to create an adventure. For the ForenCity Engine, we took the idea of building blocks to create our adventures. Each adventure is broken up into several scenes, each with their description and requirements to complete. Individual players traverse from scene to scene to complete their adventure. 2 Designing an Eective Learning Tool While research is careful to acknowledge that attempts to accurately quantify the ecacy of games in learning remain immature at best, the overwhelming view regards games in a favorable light, subject to a number of guidelines [12]: Promote social learning and team-teaching permit students to work (and learn) together. Promote social learning and team-teaching permit students to work (and learn) together. Feedback that is benecial to the attainment of success provide appropriate hints/clues to ensure that students can progress. 3 ForenCity: A Playground for Self-Motivated Learning in Computer Forensics Balance between playability and learning outcomes ensure that the game is equally fun to take part in while activities align with appropriate assessment opportunities. Balance between playability and learning outcomes ensure that the game is equally fun to take part in while activities align with appropriate assessment opportunities. With these requirements in mind, the following section describes the design of the ForenCity Engine that enables us to present to our Computer Forensics students with an opportunity that encourages further self-study and to apply their theoretical knowledge practically. 3 ForenCity Engine Design ForenCity consists of two distinct modules: the ForenCity Game Client and ForenCity Maker. ForenCity Maker is the administrative side of the ForenCity Engine to build and manage a ForenCity-based game. The player will interact with the ForenCity Game Client. A basic interface provides the player with feedback as they progress through their adventure. F.F. Blauw and W.S. Leung Based on the scene description, the player can be presented with scene in- formation, describing their whereabouts and progress. If a le is available to be downloaded, the player has access to a Download link. Likewise, if a YouTube video is available, the video will be loaded. The player now has the option to scan the scene. When a player scans the scene, the game engine will determine their current location (based on their GPS coordinates). If the location matches the required location as set up by the creator, the engine will display a success message. Otherwise, a nothing found message is displayed. If a follow-up question is required after completing a scene, the client dis- plays the question, prompting the player to answer it. Otherwise, players will automatically progress to the following scene. Once a player reaches the end of their adventure, they will be presented with a success message. 3.2 Basic Gameplay Activity First, a game creator must create several scenes using the ForenCity Maker. Each scene will contain a basic description as well as requirements that players must meet to progress to the following scene. Only players registered on the system will be able to participate in the game. Section 4.1 discusses the ForenCity Maker in greater detail. The player can now load the ForenCity Game Client by entering the ForenCity URL for the specic adventure. They will be presented with a login page where they can log in with credentials provided to them. Once logged in, the ForenCity Engine will see at which scene the player currently is and load the appropriate scene description. 4 4 ForenCity Engine Implementation ForenCity Game Engine Backend The ForenCity Game Engine (both the Game Client and Maker) is built using PHP with a JSON datastore. PHP is a widely-used web development scripting language. Many Linux web hosts provide PHP for dynamic websites, with such packages often available on the cheapest packages. Relational databases (such as MySQL) often come at a premium on web- hosts, and so we decided to store the engine's data in JSON (JavaScript Object Notation) les. JSON is a lightweight format (as opposed to XML) and PHP has built-in JSON parsing features. Using JSON comes at the price of not having relational data to easily produce information. However, due to the linear nature of an adventure, it was not seen as a problem. HTML5 markup was used to render client-side pages. HTML5 includes basic layout structuring but also gave us access to the device's GPS information (this is discussed later). ForenCity Game Client The ForenCity Game Client is built as a mobile- rst web application to allow cross compatibility among many devices. The only requirement is a modern smartphone with an HTML5 compatible web browser, camera, and GPS capabilities. jQuery Mobile 1.4.5 was used to create the user interface for the Game Client. Even though newer technologies (such as Bootstrap) are available, we believed that jQuery Mobile gave us a more comfortable all-in-one package that imme- diately produces a web app that looks complete and familiar to a user. ForenCity Maker ForenCity Maker's only requirement is a modern web browser. Bootstrap 3.3.7 was used to create the user interface that renders on both desk- top and mobile devices [16]. The navigation bar and menu give easy access to all modules in the Maker. 5 ForenCity: A Playground for Self-Motivated Learning in Computer Forensics 5 4.1 ForenCity Maker The Maker allows a game creator to manage an adventure using three modules: Game Manager, Scene Creator, and Player Manager. Game Manager The Game Manager provides basic administrative functional- ity for the Game Engine. From this module, a game creator can enable or disable the adventure, set the adventure name, set the default GPS radius, and many more options. Scene Creator The Scene Creator allows the game creator to create dierent scenes. Each scene has a scene description that is composed of a set of elements such as the scene's ID, its name, the description shown to the player as well as all clue data and requirements. Scene Variables Scene variables allow the creator to personalise the adventure's experience for each player. For instance, names and adjectives can be person- alised in the scene's description. The YouTube video and GPS coordinates re- quirement can also be set individually for each player. When a scene loads for a player, variables will be replaced by individual values as dened in the player description and set in the Player Manager. Player Manager Each player in the game has a set of elements including their login username and password, their name how they are addressed by the game as well as their unique variables. Player Variables For each variable that has been dened in a scene description by the creator, the actual value of the variable can be set for each player. ForenCity Maker provides a simple interface that displays all players and their respective variable denitions to allow a creator to quickly change them. 4.2 Download Implementation Some scenes allow the user to download a le. The le can be specic to the scene or the particular player. Regardless of the le to be downloaded, the lename of the le can be set in the scene description. Due to the stateless nature of HTTP, a download link can easily be shared between users. We overcame this by generating a unique download link for each player. The link contains a key which consisting of the player's ID and the scene for which the download is available. This key is encrypted using Aaron Francis's Urlcrypt module [17] that produces URL-friendly encrypted strings. When a player selects the download link, the Game Engine will decrypt the URL key. If it is a valid download, the le will be served to the player. F.F. Blauw and W.S. Leung 6 4.3 GPS Scene Requirement An adventure relies heavily on a player's GPS coordinates. Using a browser- based web application, we made use of HTML5's geolocation component [18]. The geolocation component uses the mobile device's location services to obtain the current GPS coordinates. GPS coordinates are then loaded into form inputs and sent to the server. Browser Requirements Apart from having location services on the device, the user must allow the site to access their location. When rst loading a site that requires geolocating, the browser will prompt the user for permission. A site requesting location information must be loaded via HTTPS. Otherwise, the browser will not even prompt the user for permission. Obtaining GPS coordinates HTML5 gives two methods to request the cur- rent location of a device. Obtaining GPS coordinates HTML5 gives two methods to request the cur- rent location of a device. The rst is navigator.geolocation.getCurrentPosition. This method simply asks the device for its current location. The device can then provide the GPS coordinates. However, GPS modules are normally not always running on mo- bile devices (in order to save power). As such, a once-o location request could potentially be quite inaccurate. The second available method is navigator.geolocation.watchPosition. The device is now continually polled for GPS coordinates. As the GPS modules are actively running, the accuracy will be improved over a brief period. This does use more battery power, but the requests are only fullled if the site is currently active. During development, we initially made use of the rst method in order to save power, but found that the accuracy was out far too often, and then opted for the second method. Using GPS coordinates Using simple JavaScript, the GPS coordinates are loaded into two hidden form elds (latitude and longitude). This location is then submitted along with the form to the server. The ForenCity Game Engine now received the player's current location ac- cording to their mobile device. If the requirement for the player's current scene is a GPS location, the Game Engine will determine if the player's location matches the location requirement. However, this check is not as simple as seeing if the GPS coordinates of the player directly match the GPS coordinates of the scene requirement. GPS coordinates on consumer devices can often be o by several meters (especially if attempted indoors). The Game Engine compensates for this by rst calculating the distance between the player's submitted coordinates and the scene's requirement coordinates. If this distance is within a preset maximum radius, the game engine will accept the GPS coordinates. This maximum distance can be set on an adventure-wide level, but can also be overridden on a per-scene 7 ForenCity: A Playground for Self-Motivated Learning in Computer Forensics basis. This way, indoor scenes can have a more relaxed distance than outdoor scenes as GPS coordinates might be more inaccurate under roof or concrete. g A scene using GPS coordinates is shown as part of the ForenCity Require- ments checking in Figure 1. 4.4 QR Code Scene Requirement Some scenes need more than GPS coordinates, such as requiring the user to scan a QR code. In these cases, the player will not be presented with the option to Scan for Clues. The scene description should give an indication that something more is required to complete the scene. QR codes can merely contain more information for the player to progress or can be used as proof that a player observed (and handled) a physical item in the real world. Once a player scans a ForenCity QR code, the ForenCity Engine will rst determine if a valid player is logged in. If not, the player will be presented with the login screen. Since some scenes may require GPS coordinates in addition to a QR code being scanned, the player will be presented with the option to examine this new clue once the scene has been loaded. This allows the client to submit GPS coordinates to the server as well. If the QR code is valid for the player's current scene, the Game Engine will perform a GPS check and allow the player to progress. If the QR code is not valid for the scene, the player will be presented with a normal nothing found description. QR Code Generation The ForenCity Maker provides a QR Code generator that generates a standard URL encoded QR Code. The URL consists of the current adventure's base URL as well as a key consisting of the scene's ID in an encrypted form, once again encrypted using Urlcrypt. 4.5 Miscellaneous Functionality The ForenCity Engine captures the IP Address and User Agent of the player when they log in and then compares it with every page load. If either does not match, the session is destroyed. Session Hijacking A player attempting to hijack another player's au- thenticated session will have their session automatically destroyed. This can happen if a player shares their cookie with another player. The ForenCity Engine captures the IP Address and User Agent of the player when they log in and then compares it with every page load. If either does not match, the session is destroyed. y Download Link Sharing Every download link is unique to the player (this enables each player to work with evidence les containing variations unique to that player). If the player shares the link with a fellow player, the ForenCity Engine will not allow the le to be downloaded. Follow-up Question Hijacking A player could share the form where the follow-up question is asked with a fellow player, in order to potentially bypass an actual GPS or QR code check. Every Follow-up Question form has a unique key for every player. If the key is not present for the correct player, the answer will be rejected. Follow-up Question Hijacking A player could share the form where the follow-up question is asked with a fellow player, in order to potentially bypass an actual GPS or QR code check. Every Follow-up Question form has a unique key for every player. If the key is not present for the correct player, the answer will be rejected. Having described the various features of ForenCity, the following section will now describe a particular case set up for a group of students. 4.5 Miscellaneous Functionality Logging As ForenCity is rst and foremost an implementation for a computer forensics game, it would obviously require that all actions performed by the player are logged. Such information enables us to monitor how each student progresses through the game to unlock the next part of the game. To achieve this, a log le is created that shows exactly what they are attempting. Each log entry includes the current date and time, the player's current IP address, the player's current GPS coordinates and the action that was performed: Player logs in and out. Player loads a scene. Player downloads a le. Player reaches a GPS or QR goal and progresses. Player is presented with and answers a follow-up question. Player attempts but fails, to progress to the next scene. Cheat attempt. F.F. Blauw and W.S. Leung 8 Player ForenCity Game Engine Verify GPS coordinates [GPS required] Display "nothing found" error [invalid GPS] Display Follow-up question [follow-up required] Progress Player to next Scene Answer follow-up question [no follow-up required] App automatically submits current GPS coordinates [no GPS required] Display "scene complete" message Display "investigate evidence" page Select "Investigate Further" [QR matches scene] [QR does not match scene] Authenticate Player Redirect to Login page [valid player] [not logged in] [valid GPS] Calculate distance Load ForenCity Load player's current scene Wait for QR Code [QR Code Required] Wait for Player interaction [No QR Required] Load scene requirements Select "Search for Clues" Scan QR Code Fig. 1. ForenCity Requirement checking Authenticate Player Select "Investigate Further" [no GPS required] App automatically submits current GPS coordinates [QR does not match scene] Display "nothing found" error Verify GPS coordinates [invalid GPS] Display "scene complete" message Answer follow-up question Display Follow-up question Fig. 1. ForenCity Requirement checking 9 ForenCity: A Playground for Self-Motivated Learning in Computer Forensics Anti-Cheating Several anti-cheat features have been implemented. All cheat attempts are also logged. Anti-Cheating Several anti-cheat features have been implemented. All cheat attempts are also logged. Anti-Cheating Several anti-cheat features have been implemented. All cheat attempts are also logged. Session Hijacking A player attempting to hijack another player's au- thenticated session will have their session automatically destroyed. This can happen if a player shares their cookie with another player. 5 Solving for a Kidnapping In 2017, we deputized 15 students as investigators, tasking them to investigate the kidnapping of a mining magnate's young daughter. The case is, in essence, a race against time (although students were given the space of a week to conclude drafting a nal report on how they reached the end goal). This section showcases select scenes from the game, detailing the activities taking place, and expected outcomes from the students. Scene 1: Police Headquarters (Debrieng) Scene 2: Gotham Heights (The First Witness) • Description: Investigators arriving at the location of the coordinates will be greeted by a cranky caretaker who complains about a âsus- picious individual with a crying kid.â Being very alert, he not only gives the investigator a description of the van that the suspect drives (the name of a business is given) , but also provides a USB that the suspicious individual had dropped in a hurry. y • Expectations: To receive the USB, the student downloads a le of the image. A cursory scan of the USB reveals a single le, the same photograph of Gugu attached in the ransom email. However, by making use of appropriate forensic tools, the student will recover a deleted le: a password-protected PDF. Students may attempt to crack the password with appropriate tools. However, it is expected that the student should rather pursue the other clue about the van as the business refers to a popularly known business that can be physically found on the university campus. Scenes 3 and 4 cut for brevity Scene 5: The Bulgarian Consultant • Description: The investigator makes their way up the building, arriving at the fourth oor where they are greeted by the Bulgarian Information Broker who is rumored to have many connections and holds the right an- swers. She gestures towards a code on her oce window before returning to her work. • Expectations: Being in a building where it will be dicult to ascertain the student's position in terms of oor level, a QR code is provided as the next clue. We had previously made arrangements with our faculty's librarian to play the role of the Bulgarian Information Broker. The student scans this QR code to obtain another set of coordinates that will lead them to an Internet café. Scenes 6 and 7 cut for brevity Scenes 6 and 7 cut for brevity Scene 8: The Locker f y Scene 8: The Locker Scene 8: The Locker • Description: After questioning the witness in the previous scene, the investigator approaches the locker where the suspect is believed to have been loitering about previously. Some answers have been uncovered but there may be further clues that could cement the case for the investigator. Scene 1: Police Headquarters (Debrieng) • Description: Investigators view a video in which Detective Sergeant Tango debriefs them on their latest case: the kidnapping of Gugu, the daughter of a mining magnate. The only clue available at present is an email that the perpetrator(s) sent to Gugu's father from a disposable temporary email address. Attached is a photograph of Gugu along with the demand for 888 Bitcoin, to be credited into a Bitcoin wallet. • Expectations: The student must analyze the email for clues. The stu- dent should notice (in their nal report) that the email is from a throw- away address. The attached photograph, however, contains metadata that includes coordinates suggesting Gugu's last known location (when the photograph was taken). If the student is not familiar with how GPS coordinates work, they will now need to research how to decipher the values to visit the location physically. F.F. Blauw and W.S. Leung 10 Scene 2: Gotham Heights (The First Witness) Scene 2: Gotham Heights (The First Witness) 6 Future Implementation Following with our inuences (Section 3.1), we want ForenCity Engine to have more than only scenes. As indicated in Section 2, we plan to create a more non- linear adventure where players will have the opportunity to explore all scenes at any time and interact with characters, interactive objects, and an inventory system. Non-Linear Gameplay the current ForenCity Game Engine only allows for a linear story to be told. However, in real-life, dierent investigators' reasoning might lead them to dierent clues rst. As such, we want most scenes to be available from the start of the adventure, but giving (or remov- ing) information as dierent tasks are completed. Dierent scenes might also change depending on the time of day. Non-Linear Gameplay the current ForenCity Game Engine only allows for a linear story to be told. However, in real-life, dierent investigators' reasoning might lead them to dierent clues rst. As such, we want most scenes to be available from the start of the adventure, but giving (or remov- ing) information as dierent tasks are completed. Dierent scenes might also change depending on the time of day. Non-Linear Gameplay the current ForenCity Game Engine only allows for a linear story to be told. However, in real-life, dierent investigators' reasoning might lead them to dierent clues rst. As such, we want most scenes to be available from the start of the adventure, but giving (or remov- ing) information as dierent tasks are completed. Dierent scenes might also change depending on the time of day. Characters Forensic investigation is not only limited to inanimate objects. Often interaction (and interrogation) with other people will be required. We want to develop a character component that will include a complete dialogue tree system. Players will have the opportunity to meet with these virtual characters and have interactions with them. Depending on their dialogue choices, characters will either reveal or withhold vital information. Interactive Objects Forensic investigate will require interaction with objects at a scene. Similar to Characters described above, we want to im- plement a component where the player can interact with virtual objects to reveal more information. Interactions can be limited to something simple such as switching a light on or o. More complicated interactions can in- clude performing a search and nding les on a computer. Scene 2: Gotham Heights (The First Witness) Unfortunately, the locker is secured with a padlock and they are only in possession of the rst three digits of the four-digit combination. It is also now, that Detective Sergeant Tango calls, ordering the investigator to nalize their investigator and put together a report. g g • Expectations: The student may attempt to open the locker in one of two ways: since only one digit is missing, it is possible to apply brute force and test out all ten possible combinations. Alternatively, a physical clue in the previous step may yield indented writing of the combination written on a notepad. The game is set such that a variety of choices made throughout the game may yield the same end result. However, the majority of marks awarded will come from the student's ability to 11 ForenCity: A Playground for Self-Motivated Learning in Computer Forensics correctly motivate their rationale for carrying out a certain action, as detailed in their nal report. 6 Future Implementation Inventory Often one piece of evidence will lead to more and dierent clues. As the player progresses through their adventure, they should be able to pick up items along the way. Items can then be used alongside other items, objects or even characters. 7 Conclusion As seen in our discussion on the various features of ForenCity, we have developed a platform that enables educators to create engaging and customized problem- based assessments in the form of an adventure game that takes students beyond the physical classroom. Based on participants' feedback, we enjoyed a rather positive and enthusiastic response from students excited to draw on their com- puter science and computer forensics knowledge in order to unlock the next clue and reveal how the next chapter in the mystery would unfold. In ForenCity, we were able to create an environment that augmented physical items and spaces on our institution's campus with virtual characters and props to F.F. Blauw and W.S. Leung 12 guide each student through a police investigation that developed as they engaged with both the physical and virtual props around them. Restricted only by our imagination and storytelling skills, ForenCity could potentially be used to assess students on a variety of other cybersecurity skills. Beyond implementing the extra features in ForenCity, we look forward to expanding ForenCity's storyline, inviting students in other subjects to test their mettle while getting to apply and experience the skills and knowledge they have acquired thus far. References 1. Irons, A.D., Stephens, P., Ferguson, R.I.: Digital Investigation as a distinct disci- pline: A pedagogic perspective. Digital Investigation 6, pp.8290 (2009). 2. Lang, A., Bashir, M., Campbell, R., Destefano, L.: Developing a new digital forensics curriculum. Digital Investigation 11, pp. S76S84 (2014). ( ) 3. Willems, C., Meinel, C.: Online assessment for hands-on cyber security training in a virtual lab. In: Proceedings of the 2012 IEEE Global Engineering Education Conference (EDUCON), pp. 110. IEEE, Princeton, NJ, USA (2012). ( ), pp , , , ( ) 4. Six Cyber Threats to Really Worry About in 2018, https://www.technologyreview.com/s/609641/ ( ), pp , , , ( ) 4. Six Cyber Threats to Really Worry About in 2018, https://www.technologyreview.com/s/609641/ six-cyber-threats-to-really-worry-about-in-2018/. ( ) ( ) 4. Six Cyber Threats to Really Worry About in 2018, https://www.technologyreview.com/s/609641/ six-cyber-threats-to-really-worry-about-in-2018/. 2018, https://www.technologyreview.com/s/609641/ six-cyber-threats-to-really-worry-about-in-2018/. 5. Cyber Security Awareness Month: Cyber Security vs. Cyber Forensics, http://www.stevenson.edu/online/blog-news-events/ cyber-security-vs-cyber-forensics. 6. Pan, Y., Schwartz, D., Mishra, S.: Gamied digital forensics course modules for undergraduates. In: 2015 IEEE Integrated STEM Education Conference, pp. 100 105. IEEE, Princeton, NJ, USA (2015) 7. What is Game-based learning? https://www.game-learn.com/ what-is-game-based-learning/. 8. Blended learning is the future, https://mg.co.za/article/ 2017-03-17-00-blended-learning-is-the-future. 9. Graham, C.R.: Blended Learning Systems: Denition, Current Trends, and Future Directions. In: Handbook of blended learning: Global perspectives, local designs, pp. 321. Pfeier Publishing, San Francisco, CA, USA (2006) 10. ARIS: A Field Day Lab Experiment, https://fielddaylab.org/make/aris/. 11. Garay-Cortes, J., Uribe-Quevedo, A.: Location-based augmented reality game to engage students in discovering institutional landmarks. In: 2016 7th International Conference on Information, Intelligence, Systems Applications (IISA), pp. 14. IEEE, Princeton, NJ, USA (2016) , , , ( ) 12. de Freitas, S.: Are Games Eective Learning Tools? A Review of Educational Games. Educational Technology & Society, 21(2), pp.7484 (2018). 13. Adventure Game Interpreter, https://en.wikipedia.org/wiki/Adventure_ Game_Interpreter. 14. Script Creation Utility for Maniac Mansion (SCUMM), https://en.wikipedia. org/wiki/SCUMM. 15. Adventure Game Studio, http://www.adventuregamestudio.co.uk/. 16. Bootstrap, https://getbootstrap.com/. 17. Urlcrypt, https://github.com/cheerful/URLcrypt. 18. HTML5 Geolocation, https://www.w3schools.com/Html/html5_geolocation. asp.
https://openalex.org/W4281556188	https://www.researchsquare.com/article/rs-1677553/latest.pdf	English	null	Peer researchers’ experiences of a co-produced research project on supported decision-making	Research Square (Research Square)	2,022	cc-by	8,842	Peer researchers’ experiences of a co-produced research project on supported decision-making Paul Webb Praxis Care David Falls Praxis Care Fionnuala Keenan Praxis Care Barbara Norris Mencap Aine Owens Mencap Gavin Davidson (  g.davidson@qub.ac.uk ) Queen's University Belfast Rosalie Edge Mencap Berni Kelly Queen's University Belfast Aisling McLaughlin Ulster University Lorna Montgomery Queen's University Belfast Christine Mulvenna Mencap Rebecca Shea Irvine University of Michigan License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Conclusions The peer researchers’ involvement challenged assumptions about the inability of people with intellectual disabilities and/or mental health problems to participate proactively in a research project whilst also highlighting the importance of training for all team members. Background Making decisions about your own life is a key aspect of independence, freedom, human rights and social justice. There are disabled people who, without support, would be assessed as incapable of making certain decisions but with the appropriate support are capable of making those decisions and so to not provide that support infringes their rights, undermines their autonomy and reinforces their exclusion from society. However, there is limited research evidence available about disabled people’s experiences of the range of approaches provided to support decision- making. This article will explore the experiences of four peer researchers who co-produced a research project on how people have, or have not been, supported to make their own decisions. Two of the peer researchers have experience of mental health problems and two are people with intellectual disabilities. Making decisions about your own life is a key aspect of indep justice. There are disabled people who, without support, would certain decisions but with the appropriate support are capable provide that support infringes their rights, undermines their au society. However, there is limited research evidence available a range of approaches provided to support decision- making. Th four peer researchers who co-produced a research project on h supported to make their own decisions. Two of the peer resea problems and two are people with intellectual disabilities. Results The peer researchers reported a number of positive effects of being involved in the research project which included improvements in skills and self-confidence. Research Article Page 1/18 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/18 Methods The peer researchers interviewed 21 people with mental health problems and 20 people with intellectual disabilities to gain an in-depth understanding of their experiences and preferences for how decision- making should be supported. Peer researcher experiences at each stage of the study from design to analysis were explored using data collected from the peer researchers via blogs written at early stages of the study, discussions at team meetings as the fieldwork progressed and at a final workshop at the end of the study which gave the peer researchers the opportunity to focus on their overall reflections of being a peer researcher. The article also discusses motivations to undertake the peer research role, the process of co-production and the challenges negotiated during the study. Plain English Summary Making your own decisions is important. There are disabled people who need support to make some decisions. Supporting disabled people to make decisions is needed so that they are not excluded from society. However, the evidence is limited on disabled people’s experiences of how best to support decision-making. This article will explore the experiences of four researchers with mental health problems Page 3/18 Page 3/18 or intellectual disabilities who worked with other researchers to conduct a project on how people have, or have not been, supported to make their own decisions. These researchers are called peer researchers. or intellectual disabilities who worked with other researchers to conduct a project on how people have, or have not been, supported to make their own decisions. These researchers are called peer researchers. The peer researchers interviewed 21 people with mental health problems and 20 people with intellectual disabilities. These interviews were conducted in order to gain a detailed understanding of the experiences and preferences for how decision-making should be supported. Blogs written by the peer researchers, discussions in team meetings and a workshop at the end of the study enabled the peer researchers to reflect on their experiences. The peer researchers reported a number of positive effects of being involved in the research project which included improvements in skills and self-confidence. The peer researchers’ involvement challenged assumptions about the inability of people with intellectual disabilities and/or mental health problems to participate in a research project whilst also highlighting the importance of training for all team members. Background This study was undertaken to build on the emerging body of literature on the views of people with mental health problems and intellectual disabilities on supported decision-making and to contribute to an understanding of how best to implement supported decision-making under the new legislation in Northern Ireland. Given the focus of the research on participation in decisions, it was important that the study enabled a participatory approach to involving people with mental health problems and intellectual disabilities in the research process (Hoole & Morgan, 2011). From the outset, therefore, a co-production approach was adopted involving four people with mental health problems or intellectual disabilities as peer researchers. The involvement of service users in policy and practice is now a common practice supported by local policy in NI, such as, the Health and Social Care (Reform) Act NI (2009) which provides a legal obligation for health and social care organisations to involve service users and carers in health and social care services; and globally under Articles 29 and 30 of the UNCRPD on the rights of disabled people to participate in public and political life. Alongside these policies that highlight service user involvement, there has been a strong emphasis on participatory research in both mental health and disability research over recent decades (Boxall & Beresford, 2013). Levels of service user involvement in research have ranged from consultation or advisory roles to active participation in the design and conduct of the study (Bigby, 2014). Within this context, a growing number of studies relating to mental health and disability have involved people with mental health and intellectual disabilities as peer researchers (Garcia Eriarte et al., 2014; Kavanagh et al., 2012). These studies have shown how peer research can add considerable value to the research in terms of authenticity, relevance and use of personal experience. However, these studies have also identified significant challenges related to resourcing, recruiting and supporting peer researchers (Bigby, 2014; Garcia Eriarte et al., 2014). It is within this context that the peer research methodology reported here was developed. Whilst other literature on peer research has sought to examine the benefits and challenges of peer research, this article will provide a more detailed focus on the actual experience of the peer researchers as they navigated their involvement in the study, from the early stages of recruitment and training to the latter stages of dissemination and impact. Background The research was conducted in Northern Ireland (NI) where the Mental Capacity Act (NI) became statute law in May 2016, and was partially implemented in 2020. In contrast to other countries, when fully implemented, this law will replace rather than be in parallel to a mental health law. This is a unique and progressive development and a core principle of the new Act is that people are "not to be treated as unable to make a decision...unless all practicable help and support to enable the person to make a decision about the matter have been given without success" (Article 1(4)). This law is supported at an international level by the principles of the United Nations Convention on the Rights of Persons with Disabilities (UNCRPD), ratified by the UK in 2009. Under Article 12 of this Convention, State Parties must provide a range of formal and informal supports to disabled people to assist them to fully exercise their legal capacity. Page 4/18 Whilst these requirements are to be welcomed, there is limited evidence available about disabled people’s experiences of supported decision-making to inform the implementation of the legislation in practice. Whilst there is a growing body of literature on legal aspects of supported decision-making (Carney, 2015), models of supported decision-making (Bigby et al., 2017) and relationships with supporters of decision- making (Watson & Joseph, 2015), there is a more limited range of research focused on the views and experiences of people with mental health problems and intellectual disabilities (Knight et al., 2018; People First, 2017; Webb et al., 2020). In NI ‘intellectual disabilities’ are usually referred to as ‘learning disabilities’ but, as the audience for this article is international, intellectual disabilities is used. It is clear that people with mental health problems and intellectual disabilities want to be involved in decisions affecting their lives and appreciate support from trusted people to reach decisions but are often restricted in exercising their own decisions by disempowering, paternalistic attitudes and practices (McDaid & Delaney, 2011; People First, 2017). Although a range of supported decision-making approaches have been developed, as decisions become more complex, support can diminish (Harding & Tascioglu, 2018). Background This exploration of the journey of the peer researchers as the research process unfolded will highlight what helped to make it a positive experience but also how to enhance approaches to peer research with people with mental health and intellectual disabilities in future studies. Throughout the article draws on data collected from the peer researchers based on blogs written at early stages of the study, discussions at team meetings as the fieldwork progressed and a final workshop at the end of the study focused on their overall reflections on being a peer researcher. Methods The aim of the peer research approach was to ensure that the study design, method, data collection and analysis was informed by the expertise of those with lived experience of mental health problems or intellectual disabilities (Biziewska and Johnston, 2010). It was expected that this level of co-production Page 5/18 Page 5/18 would improve the accessibility of the interview approach, encourage participant engagement, provide deeper insight from a service user perspective, and add to the impact of dissemination. The team were also hopeful that it would have some benefits for the peer researchers in terms of training, experience and empowerment (Kelly et al., 2017). would improve the accessibility of the interview approach, encourage participant engagement, provide deeper insight from a service user perspective, and add to the impact of dissemination. The team were also hopeful that it would have some benefits for the peer researchers in terms of training, experience and empowerment (Kelly et al., 2017). Four peer researchers were recruited through an open recruitment process led by Praxis Care and Mencap (partnering voluntary sector organisations who support people with mental health problems and intellectual disabilities). A two-day peer researcher training programme was delivered by academic researchers on the team from Queen’s University. Training focused on building the peer researchers’ capacity and confidence covering: roles and responsibilities; research methods; interviewing skills; research ethics; self-care; analysis; and report writing. The training also included role-plays and reflection to help prepare for a range of potential scenarios during fieldwork. Semi-structured interviews were used to find out about people's experiences of supported decision-making and peer researchers collaborated with the wider team to devise appropriate interview questions, including an adapted accessible version of the interview schedule. P 6/18 Praxis Care and Mencap led on the purposive selection of forty-one participants ensuring a range of experiences and a broad demographic profile (e.g. gender, age). Participants were invited onto the study by phone/email/talking directly with a staff member familiar with the study about the level of involvement expected. With consent, participants took part in an interview with a peer researcher. Peer researchers and participants were matched based on experience of mental health problems or intellectual disability. The lead in each organisation played an important role in this process and also ensured that peer researchers were informed about the communication needs and preferences of participants prior to interviews. Beginnings: recruitment, training and preparing The motivating drivers for the peer researchers to undertake the role were twofold: to use their personal and professional experience to enable people with mental health problems and intellectual disabilities to have a voice; and to develop their own experience and skills: Wanting to help people... be more open, to help them understand that there’s help out there too… The thing that motivated me is probably speaking up for people with learning disability in my local area… I haven’t really spoke much but I have gotten better at speaking about things now. To make sure other people had that opportunity to be supported, to do what’s best for them… because I’d been in a position where I was supported to make a decision when the decision could very easily have been taken out of my hands. For one peer researcher the project offered her the first experience of paid employment and for another it provided a route back into paid employment: For one peer researcher the project offered her the first experience of paid employment and for another it provided a route back into paid employment: The other motivation was to get back into… employment that was in a very safe and controlled environment, you know rather than being dropped into a big corporation like I was before so really to build up your confidence to get me back to employment. The other motivation was to get back into… employment that was in a very safe and controlled environment, you know rather than being dropped into a big corporation like I was before so really to build up your confidence to get me back to employment. Peer researchers were particularly encouraged to apply for the position when they saw that the essential criteria for the post included experience of mental ill health or learning disability: Peer researchers were particularly encouraged to apply for the position when they saw that the essential criteria for the post included experience of mental ill health or learning disability: On the job advertisement, it said that you must have a current or past mental ill health and I was like, YES! I thought they’re actually going to see it as an asset instead of a disadvantage for the job. So that was like “Oh that sounds like the type of people I want to work for”. Methods The interview was structured around three key areas: experiences of making decisions, approaches to support and ideas for future support. Prompt sheets were used as visual aids to support the interview process, visually presenting questions and possible responses in a clear, concise and accessible format. Peer researchers were accompanied to interviews by another researcher on the team who provided transport and support, if needed. Prior to starting the interviews, the accompanying researcher read through the ‘participant information sheet’ with the participant reminding them of the purpose of the study and what was expected before written consent was given. Easy read versions of the information sheet and consent form were developed in partnership with the peer researchers. The peer researcher then led the interview with support, if necessary, from the accompanying researcher. All participants provided consent for the interviews to be audio recorded. These were transcribed and anonymised for analysis. An initial coding frame was developed by a peer researcher and an academic researcher based on a thematic analysis of a sample of transcriptions, independently identifying codes and then discussing them, using the qualitative data software NVivo. This coding framework was then shared with the other peer researchers and the wider research team who made amendments/additions. Three of the peer researchers continued to work with the wider research team to identify recommendations from the findings, develop easy read versions of the final report and to disseminate the findings to a range of audiences including presentations at a launch event and a participant workshop where they presented the findings to all those who were interviewed. recommendations from the findings, develop easy read versions of the final report and to disseminate the findings to a range of audiences including presentations at a launch event and a participant workshop where they presented the findings to all those who were interviewed. Page 6/18 Page 6/18 Page 6/18 Beginnings: recruitment, training and preparing The peer researchers also explained that the inclusive approach to training was important as it showed respect for the contribution of the peer researchers and allayed fears of tokenism: Before the training I’d been a bit unsure about what the level of involvement was going to be, whether it was going to be a just token thing, you know to tick the box to get funding… which thankfully it hasn’t been. It’s been the complete opposite of it… The training wasn’t kind of something that was forced on us. It was a dialogue and you could see that the rest of the team were listening to what I said so it was quite a nice. It really was a team… so it was really good. The training was also a learning opportunity for the academic team members who were challenged to deliver the content in a way that was accessible for everyone. One of the peer researchers explained how they helped the academic researchers to adapt the pace of their approach to make it more accessible: It was good to get to know the people from Queen’s… the last time we saw the people from Queen’s, they went backwards and forwards, backwards and forwards, very very fast and one of us was just saying “Will you calm down and explain to < name removed > and < name removed > sort of thing?”… So… we could relate to their topic. Beginnings: recruitment, training and preparing On the job advertisement, it said that you must have a current or past mental ill health and I was like, YES! I thought they’re actually going to see it as an asset instead of a disadvantage for the job. So that was like “Oh that sounds like the type of people I want to work for”. Another peer researcher also explained that the level of support provided by the research team was also an important factor in her decision to apply for the role: Another peer researcher also explained that the level of support provided by the research team was also an important factor in her decision to apply for the role: I knew I’d be getting support where a regular job could be a challenge. I knew I’d be getting support where a regular job could be a challenge. From the outset of the project, the peer researchers had a range of hopes and fears related to the role. They hoped to develop their research skills and confidence but also to have a positive impact on the accessible design of the study and its overall impact: One of my expectations would be to… help those ones who we were interviewing because… the ones who we interview might not be able to understand you. Page 7/18 I would expect to get some sort of hope out of doing it. From interacting with more people… being taken out of my comfort zone quite a bit, so I went in with the expectation, the hope really that there’ll be Page 7/18 positive outcomes. Peer researchers were also anxious about what their role would be but these anxieties were diminished by the training programme. One peer researcher wrote the following blog about her experience of the two-day peer researcher training programme: I thought the training was very interesting and very well explained... We now have a better understanding of the role we will be taking up... and the roles the rest of the team... I really did enjoy it and because it was interactive... and everyone had an input. I felt really relaxed during the training and everyone in the team is nice and friendly... I learnt more about the project and I now know what some of the big words mean. I really feel positive about the role now training is finished. Co-production: designing and conducting interviews With regard to the semi-structured interview schedules which the peer researchers helped to design, one peer researcher was concerned about the wording of a question and helped the team to re-phrase the question in a more sensitive manner: There was one of the questions at the training I kind of vetoed and was like ‘I’m not asking that’ because that could traumatise people a wee bit and it could then impact me as well… I thought “Oh dear if you ask some people that, with mental health - that could just be a can of worms”… So the way the question was put was very, very different then. But it was nice to have that level of involvement and be able to air my concerns and be listened to. Page 8/18 Similarly, another peer researcher described developing shorter, more accessible questions for participants with intellectual disabilities and allowing more time for these interviews: Similarly, another peer researcher described developing shorter, more accessible questions for participants with intellectual disabilities and allowing more time for these interviews: Page 8/18 Page 8/18 We did come up with questions people would be willing to answer but not to bombard them with too many as well and not to make them too long so they don’t get bored. Prompt sheets were also used during interviews where needed. The prompt sheets included the visual presentation of questions together with clear response options. This level of accessibility was important for participants but also for the peer researchers, especially those with intellectual disabilities: Reading and listening [was important] in interviews because some people ask you to repeat some questions so you couldn’t really go too far down the page. So it was a good because the pages were easy read, you didn’t really get lost in them… They were just the right length for some people. Throughout the interviews, peer researchers found they had to adapt their approach to the individual circumstances of the individual and their environment and, in some cases, this was challenging. Peer researchers recalled feeling initially unsure how to address unexpected disruptions to interviews, for example, coping with background noise. In some cases, they found it frustrating when participants had forgotten about the interview and it had to be re-scheduled or when participants focused on issues not relevant to the core interview questions, as two peer researchers explained: Some interviews were a bit challenging. Co-production: designing and conducting interviews There wasn’t any harming stories but there were some difficult stories. She [accompanying researcher] always made sure at the end of the interview, was everything ok and I was always ok. There wasn’t any harming stories but there were some difficult stories. The peer researchers developed confidence in their skills and ability to respond to fieldwork challenges as they gained more experience of interviewing: The peer researchers developed confidence in their skills and ability to respond to fieldwork challenges as they gained more experience of interviewing: I think there was some challenges along the way but you know working through them has led more positives. They were just temporary stumbling blocks but I’ve learnt then, as the interviews were going along, I learnt to take in the knowledge from the previous interviews and adapt a wee bit. I think there was some challenges along the way but you know working through them has led more positives. They were just temporary stumbling blocks but I’ve learnt then, as the interviews were going along, I learnt to take in the knowledge from the previous interviews and adapt a wee bit. The peer researchers considered their personal experiences of mental health problems/intellectual disabilities to be a major benefit in the interview process. They found that, when they explained to participants that they were employed as peer researchers who had personal experience of mental health problems or disability, they established a connection with participants that enabled an open channel of communication and instilled a sense of hope for some participants: Having the experience of being supported to make a decision, I felt gave me a pretty good understanding of what we were setting out to do… At some of the interviews, as soon as I mentioned my background, that I wasn’t an academic… I was a peer with mental health issues, with two people in particular, that just broke down so many barriers because they kind of opened up more because I wasn’t an authority figure… and they kind of could understand that I could see where they were coming from more. Co-production: designing and conducting interviews I remember one person who… was very talkative about his mum in any answer... It was a bit challenging… but I still managed ok… There was one where the person was just looking to show photos… but they still answered those questions really well. It was kind of like they were trying to avoid a topic that you were trying to talk about… On a couple of our interviews when we got to the place the people forgot… change could happen on the day. I used to be bad with changes but I’m getting there. It was also challenging to hear personal stories, however, peer researchers emphasised the importance of listening to these narratives: It was also challenging to hear personal stories, however, peer researchers emphasised the importance of listening to these narratives: People kept telling you their personal life. Sometimes it could be very difficult for them but you have to show them that you’re listening. People kept telling you their personal life. Sometimes it could be very difficult for them but you have to show them that you’re listening. In most cases, peer researchers had some prior information about the person’s communication preferences, however, there were situations where peer researchers felt they would have benefitted from further advance information about the needs and preferences of the participant: In most cases, peer researchers had some prior information about the person’s communication preferences, however, there were situations where peer researchers felt they would have benefitted from further advance information about the needs and preferences of the participant: I must admit that’s what I found tough. Obviously people are entitled to their privacy but having a slight indication of what you were maybe going to be facing would have been helpful [to] know how to set the tone of interviews… If someone… was able to say like something like “Don’t maintain eye contact”… wee things that might have helped a bit… or even if the staff could say “Oh so and so is having a good day or a bad day today”. Page 9/18 Given these challenges, debriefing with the accompanying researcher was helpful although not always necessary: Given these challenges, debriefing with the accompanying researcher was helpful although not always necessary: She [accompanying researcher] always made sure at the end of the interview, was everything ok and I was always ok. Co-production: designing and conducting interviews So I think that helped in those cases… They knew that someone like me could relate to them maybe that bit more… They were almost more interested in me and how I’d got to the stage where I was at… The last person I interviewed in particular was like “Oh that gives me some hope now that in a year’s time maybe I can be you know, further on”. This personal experience was augmented by the peer researchers’ additional experience of having a social network comprising other individuals with mental health problems or intellectual disability which widened their knowledge of the salient issues for this service user group and the skills required to engage them in the research process: Most of my friends in my local area and the ones I’ve done research for which involve the community and mostly people with learning disabilities so that helped too. Page 10/18 Critics of peer research have previously highlighted concerns that peer researchers may not have the skills to deal with ethical dilemmas that arise during fieldwork, including participant distress (Bigby, 2014). In contrast, the peer researchers in this study were acutely aware of the risk of emotional harm to the participant and themselves. Peer researchers suggested that their own personal experiences equipped them with key skills for identifying distress and intervening early to offer a break or access to support, if necessary. This was apparent in the re-designing of questions so they were worded more sensitively but Page 10/18 also in the ways in which peer researchers recognised and responded to potential signs of uneasiness during interviews: also in the ways in which peer researchers recognised and responded to potential signs of uneasiness during interviews: It was knowing when not to push it because you could sense people getting agitated at times… I just knew some tell-tale signs that I would have had, that they had, so I think having that knowledge of mental health helped to know when not to… Maybe I was more cautious going into it…. that there could be anxiety there of talking to someone new and people could get a bit worked up about things so I took quite a softly approach. Given the fieldwork challenges, regular team meetings were held to report on study progress. Co-production: designing and conducting interviews Staff from the partnering organisations also met with the peer researchers to debrief/offer support and to ensure consistent communication and dialogue with peer researchers and address self-care needs: The continuing meetings were good… so we’ve been kept in the loop so throughout it all… It’s about the support that’s been in place as well. You wouldn’t get a lot of places from the very start just even talking about self-care plans… Our experience has been seen as assets but there has be recognition that you may still need support so that’s been quite good. Endings: Data Analysis and Ongoing Co-Production Endings: Data Analysis and Ongoing Co-Production Analysis involved the development of a coding frame and subsequent thematic analysis within NVivo. The coding frame was developed by a peer researcher and an academic researcher using a sub-set of transcripts. Amendments and/or additions to the coding frame were then made by the other members of the research team including peer researchers. Involving each peer researcher in different ways at this stage of analysis demonstrated an ongoing commitment to facilitating co-production and sharing ownership of the project at later stages of the research process: We met and read through what you already analysed... It was in themes and if there was ever anything missing we told the coordinator… We thought that would have been the best way of doing it. Some previous peer research studies have only involved peer researchers in the data collection process with data analysis being undertaken by academics with analytical expertise. In this study, the involvement of peer researchers in the data analysis was critical as they could identify themes in the transcripts that the academic researcher may not have observed. One peer researcher, who co-worked on the initial data analysis with an academic, commented on this process and the use of multiple peer research experience to inform data analysis: For the coding framework we might have seen themes in places where other people wouldn’t have… knowing some of the lingo… might help identify some themes. And I think having so many different perspectives involved… helped quite a bit as well because obviously if I was looking at transcripts I would only really understand probably more the mental health ones and the learning disability transcripts I wouldn’t pick up a lot of things in so I think it has been helpful having people with these backgrounds involved too. Endings: Data Analysis and Ongoing Co-Production Page 11/18 Page 11/18 This level of involvement in analysis was greatly appreciated by peer researchers who had previously been involved in research but not given full credit for the outputs from the study: This level of involvement in analysis was greatly appreciated by peer researchers who had previously been involved in research but not given full credit for the outputs from the study: My dealings with academics… previously was that they would never let anyone take credit for anything whereas the credit is actively being directed to us so that was completely mind blowing… It was good because you felt really valued then, you know that you’re trusted to be able to, you know, talk on their behalf… I just wasn’t used to a lot of academics sharing credit… I’m still sort of pleasantly surprised at how inclusive it was and the level of involvement we had… We’ve been incredibly involved with it and pretty much every decision that’s been made we’ve had input… It has made you more committed to the project and more interested and you’ve bought into it more so you want to see the outcomes and see it through… It’s been very empowering by being that involved and getting the responsibility and trusted with the responsibility to do the job. It is very inclusive and empowering to our needs but also to each other, which you wouldn’t have in many other jobs. Three peer researchers also maintained their involvement with the study through to project completion and were therefore heavily involved with the development of recommendations, an easy read final report and with dissemination. Involvement at these later stages of the study were important to the peer researchers as it gave them an opportunity to have a voice at stakeholder events and to deliver impactful messages from the study to policy makers and service providers. At the end of the study, the peer researchers participated in a workshop facilitated by one of the academic researchers to focus on reflections on their overall experience and next steps. The peer researchers were very positive about the interpersonal and presentational skills they had developed during the study. Indeed, based on their experience, two peer researchers had been offered positions on advisory groups and another had secured a research post: It was good and interesting. Discussion The breadth and quality of data collected is evidence of the success of the peer research approach which facilitated the collection of rich data that provides insight into the experience of supported decision- making. In alignment with the principles of supported decision-making, the experience of the peer researchers challenges assumptions about the people with disabilities and demonstrates their capacity to fully participate at each stage of the research process, with appropriate training and support. However, the peer research experience also shows that genuine participation does not always mean everyone is participating in the same way and at the same level at every stage. At times, peer researchers were leading, at other times advising, contributing ideas and critiquing themes. Indeed, allowing for changing roles and different ways to participate empowered the peer researchers and ensured their inclusion at all stages of the research process in a way that was responsive to their preferences and skill set (Ellis, 2018). Roles in the research process differed depending on the preferences and strengths of the individual peer researcher and factors such as their mental health/intellectual disabilities and the number of hours they were permitted to work (without affecting disability benefit/other employment). For example, the team decided that one peer researcher with experience of mental health problems would work in partnership with an academic researcher on the initial analysis of data as he had prior experience of analysing research data and the two peer researchers with intellectual disabilities did not feel it played to their strengths to read transcripts and analyse dense text. Instead, they preferred to comment on the initial code book and suggest additions or changes to emerging themes. In this way, they provided an internal validation of the identified themes based on their own personal experiences and experiences of the interviews that they had carried out. The fourth peer researcher also contributed her views on the code book but ended her participation in the study at this point before dissemination activities began due to other personal commitments. The flexibility to respond to individual needs and circumstances is important in peer research and reflects the more unpredictable nature of co-production with each team member contributing at a pace and level that is appropriate at various stages of the project. Endings: Data Analysis and Ongoing Co-Production I learned new skills of different ways of talking and approaching people… There’s another opportunity that I’ve been given I probably wouldn’t have got if I didn’t do this. It’s a service user forum for another area. Other longer term benefits from being involved in the study included the establishment of new social and community networks, being empowered by the sense of achievement of successfully completing the peer research project, and feeling a sense of contribution to a project that may have a valuable impact on people’s lives. Peer researchers also noted that the project had given them a new perspective on their own personal experience of mental ill health and disability and encouraged them to see their experiences as an asset of value to a range of projects: It was a new perspective of self… Before the project started I guess I was just someone with mental health issues and that was a disadvantage whereas this involvement has been an asset because I’ve been able Page 12/18 Page 12/18 to use it in a positive way. So being able to kind of just get that more positive outlook on myself has been quite a big benefit from it. to use it in a positive way. So being able to kind of just get that more positive outlook on myself has been quite a big benefit from it. Discussion Utilising the expertise of all team members was a key feature of the study that allowed each member to feel they were making a valued contribution to the process. Service users brought personal experience, previous related skills and a growing confidence in research skills. Partnering organisations contributed much to the recruitment and coordination of the study, whilst also supporting individual peer researchers and participants. Academics contributed their research expertise in terms of study design, training and supporting peer researchers and assisting with analysis and write-up of findings. Engagement in the peer research process was also beneficial to the academics and partnering organisations involved in terms of Page 13/18 Page 13/18 developing greater insight into service user perspectives and shared learning about co-production. The inter-dependence of the research team, drawing on a combined skill set, greatly strengthened the study. developing greater insight into service user perspectives and shared learning about co-production. The inter-dependence of the research team, drawing on a combined skill set, greatly strengthened the study. At the peer research workshop, the team reflected on the recommendations they would have for other researchers considering a peer research approach to mental health or disability studies. The peer researchers emphasised that they would strongly encourage other people with mental health problems and intellectual disabilities to consider a peer research role and would like to raise awareness of the benefits of peer research and the process involved: It’s getting the word out to people about what peer research involves and the benefits of it could maybe encourage more people to get involved in peer research because I think from a mental health point of view it’s kind of quite therapeutic being involved in it. It’s getting the word out to people about what peer research involves and the benefits of it could maybe encourage more people to get involved in peer research because I think from a mental health point of view it’s kind of quite therapeutic being involved in it. I would say for future researchers don’t let your mental health or learning disability put you off... If you believe in it, you can do it. I would say for future researchers don’t let your mental health or learning disability put you off... If you believe in it, you can do it. Consent for publication Participants provided informed consent before completing the study. Discussion Our experience of co-production was grounded in a mutual commitment to Page 14/18 researching the lived experience of people with mental health problems or intellectual disabilities to develop recommendations for improving approaches to supported decision-making. The benefits for everyone involved were clearly evidenced, and the opportunity for peer researchers to make a difference was concisely captured by a peer researcher who said: researching the lived experience of people with mental health problems or intellectual disabilities to develop recommendations for improving approaches to supported decision-making. The benefits for everyone involved were clearly evidenced, and the opportunity for peer researchers to make a difference was concisely captured by a peer researcher who said: It was being involved in something and feeling useful and that you’re actually contributing something has been quite good because you know the outcome of the project is going to have real impact on people’s lives. It was being involved in something and feeling useful and that you’re actually contributing something has been quite good because you know the outcome of the project is going to have real impact on people’s lives. Conclusions The experience of the peer researchers challenged traditional assumptions about the inability of people with intellectual disabilities and/or mental health problems to participate proactively as part of a research team. The peer researchers reported a number of benefits to peer research which included improvements in skills and confidence as well as a sense of empowerment. They also reported appreciating how their experiences as people with mental health problems and/or intellectual disabilities were valued. The peer researchers’ experience with mental health problems and/or intellectual disabilities also helped establish a rapport with participants which meant that they could elicit high quality responses during each interview. Flexible roles within the team meant that the preferences and skills sets of peer researchers were fully utilised. Training for all team members was essential as were regular meetings and de-briefing with peer researchers after each interview. Effective, multi-agency partnership working which included peer researchers, academics and staff working in services was seen as essential to the success of this co-produced research project. Ethics approval and consent to participate The research was reviewed and approved by the School of Social Sciences, Education and Social Work Research Ethics Committee at Queen’s University Belfast. All methods were carried out in accordance with the approved protocol and written consent was obtained from all study participants. Discussion However, peer researchers also highlighted that future projects must provide robust training and support for peer researchers to prepare them for the challenges of fieldwork: However, peer researchers also highlighted that future projects must provide robust training and support for peer researchers to prepare them for the challenges of fieldwork: The training made us more aware what we would dealing with so I would say if someone was going for a similar project they would need to do some training to be prepared more as there are some interviews that can be overwhelming and challenging. The training made us more aware what we would dealing with so I would say if someone was going for a similar project they would need to do some training to be prepared more as there are some interviews that can be overwhelming and challenging. Indeed, academics also require training on co-production, accessibility and how best to support peer researchers with mental health problems or intellectual disabilities. Working in partnership with peer researchers requires a commitment to sharing power and a flexible approach to the design and development of the research process. These values and skills are relevant to both peer researchers and academics involved in co-produced research. Peer researchers particularly emphasised the importance of being involved at all stages of the research from the design of research questions to fieldwork, data analysis and dissemination. This level of involvement helps to avoid tokenistic approaches to co-production and ensures that peer researchers have full opportunity to influence the research and contribute fully to the study. For this study, the peer researchers were recruited after the funding for the study had been secured. It would be ideal if peer researchers could also be involved in the initial application process, and there are now an increasing number of people with experience as peer researchers who would be able to do this, but how that would be funded remains a challenge. Our study benefitted greatly from the involvement of voluntary organisations who assisted with participant recruitment and matching, the coordination of fieldwork and support for peer researchers. Future peer research studies would benefit from firm partnerships with agencies who have strong networks in the community and who have experience of supporting people with mental health problems or intellectual disabilities. Availability of data and materials The full dataset is not available as, even when anonymised, there is the potential for people to be identifiable given the relatively small number of people and services involved. Competing interests Competing interests Page 15/18 Authors' contributions GD and RSI conducted the literature review. D, AMcL and LM delivered a two-day peer researcher training programme. PW, DF, FK, BN, AO, GD, RE, BK, AMcL, LM, CM and RSI designed the interview schedule, information sheets and consent forms, the coding frame, contributed to the analysis and helped to produce the standard and easy read versions of the report. RE, CM and PW recruited participants to the study and supported peer researchers during interviews with participants and at a number of dissemination events. BK collected blog, team meeting and workshop data and performed the analysis for this article. GD, BK and PW wrote the article which was reviewed by the team members prior to submission. PW drafted the GRIPP. GD finalised. BK collected blog, team meeting and workshop data and performed the analysis for this article. The remaining authors declare that they have no competing interests. The remaining authors declare that they have no competing interests. Funding This research was funded as part of the Disability Research on Independent Living & Learning (DRILL) Programme. DRILL was fully funded by the Big Lottery Fund and delivered in partnership by Disability Action, Disability Rights UK, Disability Wales and Inclusion Scotland. The DRILL Programme was led by disabled people and funded co-produced research and pilot projects focused on exploring how disabled people can live as full citizens and take part socially, economically and politically. Page 15/18 GD is the Praxis Chair of Social Care at Queen's University Belfast and this post is part funded by Praxis Care. GD is the Praxis Chair of Social Care at Queen's University Belfast and this post is part funded by Praxis Care. References 1. Boxall, K. & Beresford, P. Service user research in social work and disability studies in the United Kingdom. Disability & Society. 2013; 28(5), 587-600. 2. Bigby, C. Conceptualizing Inclusive Research with People with Learning Disability. Journal of Applied Research in Learning Disabilities. 2014; 27, 3-12. 2. Bigby, C. Conceptualizing Inclusive Research with People with Learning Disability. Journal of Applied Research in Learning Disabilities. 2014; 27, 3-12. 3. Bigby, C., Douglas, J., Carney, T., Then, S. N., Wiesel, I., & Smith, E. Delivering Decision-making Support to People with Cognitive Disability— What has been learned from pilot programs in Australia from 2010–2015? Australian Journal of Social Issues. 2017; 52: 222–240. 3. Bigby, C., Douglas, J., Carney, T., Then, S. N., Wiesel, I., & Smith, E. Delivering Decision-making Support to People with Cognitive Disability— What has been learned from pilot programs in Australia from 2010–2015? Australian Journal of Social Issues. 2017; 52: 222–240. 4. Biziewska, D. & Johnston, G. Peer Research. 2010. https://www.seemescotland.org/media/7368/peer-research-in-mental-health-paper.pdf Accessed 19 07 21. 5. Carney, T. Supporting People with Cognitive Disabilities with Decision-making: Any Australian Law Reform Contributions? Research and Practice in Learning and Developmental Disabilities. 2015; 2(1), 6-16. 6. Ellis, L. Making decisions together? Exploring the decision making process in an inclusive research project, Disability & Society. 2018; 33 (3), 454-475. 7. Garcia Iriarte, E., O'Brien, P. & Chadwick, D. Involving people with learning disabilities within research teams: Lessons learned from an Irish experience, Journal of Policy and Practice in Learning Disabilities. 2014; 11 (2), 149-157. 8. Harding, R., & Tascioglu, E. Supported Decision-Making from Theory to Practice: Implementing the Right to Enjoy Legal Capacity, Societies. 2018; 8 (25), doi:10.3390/soc8020025. 8. Harding, R., & Tascioglu, E. Supported Decision-Making from Theory to Practice: Implementing the Right to Enjoy Legal Capacity, Societies. 2018; 8 (25), doi:10.3390/soc8020025. 9. Hoole, L., & Morgan, S. ‘It’s only right that we get involved’: service‐user perspectives on involvement in learning disability services. British Journal of Learning Disabilities. 2011; 39(1), 5-10. 9. Hoole, L., & Morgan, S. ‘It’s only right that we get involved’: service‐user perspectives on involvement in learning disability services. British Journal of Learning Disabilities. 2011; 39(1), 5-10. 10. Kavanagh, D., Daly, M., Harper, M., Davidson, G. & Campbell, J. (2012) Mental health service users and carers as researchers: reflections on a qualitative study of citizens' experiences of compulsory mental health laws in NI, In Goodson, L. & Phillimore, P. Acknowledgements The project was informed at key points throughout the research process by an International Advisory Group which included specialist disabled and non-disabled researchers with expertise in this area. Advisory group members advised on issues such as the existing research, the interview schedule, data analysis and dissemination. The International Advisory Group members were: Dr Anna Arstein-Kerslake, Senior Lecturer, Melbourne Law School and Convenor of the Disability Research Initiative, University of Melbourne, Associate Professor Lisa Brophy, from the Centre for Mental Health, Melbourne School of Population and Global Health, University of Melbourne and is also Mind Australia’s Principal Research Fellow, Dr Nancy Hansen, Director of the Interdisciplinary Master’s Program in Disability Studies at the University of Manitoba, Margaret Kelly, Director of Mencap NI, Keith Lynch, People First Scotland (who had recently completed directly relevant research in Scotland about supported decision-making), Taryn Page 16/18 Page 16/18 McKeen, Mental Health and Capacity Unit, Department of Health (previously Tomas Adell, Mental Health and Capacity Unit, Department of Health), Professor Richard O’Reilly, a Professor of Psychiatry at Western University in London, Ontario and at the Northern Ontario School of Medicine. Professor O’Reilly has researched extensively in the area of mental health law and Professor Michael Schwartz, Director of the Disability Rights Clinic in the Office of Clinical Legal Education at Syracuse University College of Law, in New York State, where he supervises students in disability advocacy and teaches clinical skills and disability law. References (Eds.) Community research: from theory to method. Bristol: Policy Press; 2012. p. 235-252. 11. Kelly, B. Friel, S., Smith, D., Pinkerton, J., McShane, T. & Gilligan, E. More than we expected! A guide to peer research with young people. Belfast: QUB; 2017. 11. Kelly, B. Friel, S., Smith, D., Pinkerton, J., McShane, T. & Gilligan, E. More than we expected! A guide to peer research with young people. Belfast: QUB; 2017. Page 17/18 Page 17/18 12. Knight, F., Kokanović, R., Ridge, D., Brophy, L., Hill, N., Johnston-Ataata, K., & Herrman, H. Supported Decision-Making: The Expectations Held by People With Experience of Mental Illness. Qualitative Health Research. 2018; 28(6), 1002-1015. 13. McDaid, S. and Delaney, S. A social approach to decision-making capacity: exploring research with people with experience of mental health treatment. Disability & Society. 2011; 26(6), 729-742. 13. McDaid, S. and Delaney, S. A social approach to decision-making capacity: exploring research with people with experience of mental health treatment. Disability & Society. 2011; 26(6), 729-742. 14. People First (Scotland) Does it matter? Decision-making by people with learning disabilities. Glasgow: Animate Consulting; 2017. 14. People First (Scotland) Does it matter? Decision-making by people with learning disabilities. Glasgow: Animate Consulting; 2017. 15. Watson, J., & Joseph, R. People with severe or profound learning disabilities leading lives they prefer through supported decision-making: Listening to those rarely heard. A guide for supporters. A training package developed by Scope. Melbourne: Scope; 2015. 15. Watson, J., & Joseph, R. People with severe or profound learning disabilities leading lives they prefer through supported decision-making: Listening to those rarely heard. A guide for supporters. A training package developed by Scope. Melbourne: Scope; 2015. 16. Webb, P., Davidson, G., Edge, R., Falls, D., Keenan, F., Kelly, B., McLaughlin, A., Montgomery, L., Mulvenna, C., Norris, B., Owens, B., Shea Irvine, R. Service users’ experiences and views of support for decision making. Health Soc Care Community. 2020; 00:1-10. 16. Webb, P., Davidson, G., Edge, R., Falls, D., Keenan, F., Kelly, B., McLaughlin, A., Montgomery, L., Mulvenna, C., Norris, B., Owens, B., Shea Irvine, R. Service users’ experiences and views of support for decision making. Health Soc Care Community. 2020; 00:1-10. GRIPP2SFv1.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. GRIPP2SFv1.docx Page 18/18 Page 18/18

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